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Metabolism of the Nonylphenol Isomer [Ring-U-14c]-4-(3prime5prime-Dimethyl-3prime-Heptyl)-Phenol by CellSuspension Cultures of Agrostemma githago andSoybeanBurkhard Schmidt a Hildegard Patti a Gregor Hommes a amp Ingolf Schuphan aa Department of Biology V (Ecology Ecotoxicology Ecochemistry) RWTH AachenUniversity Aachen GermanyPublished online 30 Mar 2012

To cite this article Burkhard Schmidt Hildegard Patti Gregor Hommes amp Ingolf Schuphan (2004) Metabolism of theNonylphenol Isomer [Ring-U-14c]-4-(3prime5prime-Dimethyl-3prime-Heptyl)-Phenol by Cell Suspension Cultures of Agrostemma githagoand Soybean Journal of Environmental Science and Health Part B Pesticides Food Contaminants and AgriculturalWastes 394 533-549 DOI 101081PFC-200026776

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JOURNAL OF ENVIRONMENTAL SCIENCE AND HEALTH

Part BmdashPesticides Food Contaminants and Agricultural Wastes

Vol B39 No 4 pp 533ndash549 2004

Metabolism of the Nonylphenol Isomer

[Ring-U-14c]-4-(3050-Dimethyl-30-Heptyl)-Phenol by

Cell Suspension Cultures of Agrostemma githagoand Soybean

Burkhard Schmidt Hildegard Patti Gregor Hommes

and Ingolf Schuphan

Department of Biology V (Ecology Ecotoxicology Ecochemistry) RWTH

Aachen University Aachen Germany

ABSTRACT

The biotransformation of the nonylphenol isomer [ring-U-14C]-4-(3050-dimethyl-

30-heptyl)-phenol (4-353-NP consisting of two diastereomers) was studied in

soybean and Agrostemma githago cell suspension cultures With the A githago

cells a batch two-liquid-phase system (mediumn-hexadecane 2001 vv) was used

in order to produce higher concentrations and amounts of 4-353-NP metabolites

for their identification 4-353-NP was applied via the n-hexadecane phase Initial

concentrations of [14C]-4-353-NP were 1mgL1 (soybean) and 5 and 10mgL1

(A githago) After 2 (soybean) and 7 days (A githago) of incubation the applied

4-353-NP was transformed almost completely by both plant species to four types

of products glycosides of parent 4-353-NP glycosides of primary 4-353-NP

metabolites nonextractable residues and unknown possibly polymeric materials

detected in the media The latter two products emerged especially in soybean

cultures Portions of primary metabolites amounted to 19ndash22 (soybean) and

Correspondence Burkhard Schmidt Department of Biology V (Ecology Ecotoxicology

Ecochemistry) RWTH Aachen University Worringerweg 1 Aachen 52056 Germany

Fax +49-241-8022182 E-mail burkhardschmidtpostrwth-aachende

533

DOI 101081LESB-200026776 0360-1234 (Print) 1532-4109 (Online)

Copyright amp 2004 by Marcel Dekker Inc wwwdekkercom

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21ndash42 of applied 14C (A githago) After liberation from their glycosides the

primary 4-353-NP metabolites formed by A githago were isolated by HPLC and

examined by GC-EIMS as trimethylsilyl derivatives In the chromatograms

eight peaks were detected which due to their mass spectra could be traced back to

4-353-NP Seven of the compounds were side-chain monohydroxylated 4-353-NP

metabolites while the remaining was a (side-chain) carboxylic acid derivative

Unequivocal identification of the sites of hydroxylationoxidation of all transfor-

mation products was not possible The main primary metabolites produced by

A githago were supposed to be four diastereomers of 60-hydroxy-4-353-NP

(about 80 of all products identified) It was concluded that plants contribute to

the environmental degradation of the xenoestrogen nonylphenol the toxicological

properties of side-chain hydroxylated nonylphenols remain to be examined

Key Words Plant cell culture Agrostemma githago Soybean Metabolism

Nonylphenol isomer 4-(3050-Dimethyl-30-heptyl)-phenol Two-liquid-phase

system Metabolic profiling Hydroxylated nonylphenol metabolites

INTRODUCTION

Alkylphenol polyethoxylates (APEs) belong to the class of nonionic surfactantsand are used world-wide as detergents emulsifiers solubilizers wetting agents anddispersants in both industry (about 85 of the market) and households APEs serveas cleaning products as well as industrial process aids Applications of APEs includeresin plastic and elastomer manufacturing paper and pulp production industrialcleaners pesticide formulations cold cleaners for cars and household cleaners[12]

APEs derived from nonylphenol are designated as nonylphenol polyethoxylates(NPEs) and comprise about 80 of the total market volume[1] Nonylphenol issynthesized industrially by alkylation of phenol with nonene The product containsabout 90 p-nonylphenol besides prevailingly o-nonylphenol and op-di-nonylphe-nols As a result of the method utilized to manufacture nonene (propylene trimer)nonylphenol consists of a complex mixture of predominantly branched-chain C9

derivatives of phenol Recently 22 p-nonylphenol isomers have been identified in theindustrial product[34] and characterization of predominant structures (MS NMR)after separation of commercial nonylphenol has been achieved[5]

Because of their wide use especially in aqueous solutions NPEs are dischargedinto municipal and industrial waste waters which enter sewage treatment plantsDuring treatment a complex biodegradation occurs which leads to the formation ofa number of metabolites with shortened ethoxy chains and ultimately nonylphenolNonylphenol itself is considerably lipophilic and a comparatively stable substanceThus nonylphenol and partially also its preceding metabolic residues were shownto be released with effluents of treatment plants to surface waters additionallynonylphenol was detected in sewage sludge[1246ndash10] Besides the use of NPEs inpesticide formulations sewage sludge frequently applied to agricultural soils andeffluents occasionally used in irrigation may constitute a source of contamination ofterrestrial plants with NPEs and nonylphenol These facts raise the question ofuptake and metabolic fate of nonylphenol in plants particularly those used for foodproduction[1112] Nonylphenol was recently reported to be present in foodstuffs[13]

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The world-wide interest in the (environmental) fate of nonylphenol originatesfrom its proven estrogenic activity the compound is considered as xenoestrogenor as an endocrine disrupter[781314] Consequently nonylphenol may affect bothhuman health and ecosystems Regarding human health the risk associated withlow-level concentrations of nonylphenol in foodstuffs however is discussedcontroversially[1315] Early research on the environmental and metabolic fate ofnonylphenol eg in plant tissues[1112] was mainly performed with the straight-chain 4-n-nonylphenol due to the unavailability of individual isomers of theindustrial productmdashespecially in 14C-labelled form However 4-n-nonylphenolappears to be no or only a trace constituent of industrial nonylphenol Thusincreasing efforts in the last years aimed at the synthetic preparation of definedbranched-chain nonylphenol isomers (preferentially also 14C-labelled) and the studyof their environmental fate and effects[516ndash20] Up to now data on the metabolism ofbranched-chain nonylphenols in plant tissues are not at hand

Plant cell cultures are often used to examine the metabolism of xenobiotics inplant tissues[2122] The studies executed prior or parallel to experiments with intactplants render possible the rapid identification of metabolites of interest So recentlywe studied the metabolism of 4-n-nonylphenol in plant cell suspension cultures andpresented the results as a poster[23] The studies were performed according to astandardized procedure[21] with 1mgL1 of the 14C-labelled xenobiotic (applied inmethanol) and 2 days of incubation using a one-liquid-phase system Since GC-EIMSidentification of the primary metabolites of 4-n-nonylphenol was not sufficientlyclear-cut we intended to produce higher concentrations and amounts of the metabolicproducts The low aqueous solubility of nonylphenols in general however raised thequestion of an alternative mode of application Recently we thus could demonstratethat considerable amounts of 4-n-nonylphenol dissolved in an n-hexadecane layerwere absorbed and metabolized by suspended cultivated cells of Agrostemma githago(corn cockle) in a satisfactory manner the n-hexadexane layer representing theorganic phase of a two-liquid-phase system affected plant cell growth only little[24]

In the present article we report data on the metabolism of the 14C-labellednonylphenol isomer 4-(3050-dimethyl-30-heptyl)-phenol (Fig 1) by cell suspensioncultures of soybean and A githago The isomer (as diastereomeric mixture) wassynthesized according to a published procedure[16] Due to GC-EI (retention timesmass spectra) the isomer is an important constituent of industrially producednonylphenol The preliminary metabolism experiment with the soybean suspensionswas performed as conventional one-liquid-phase study[21] In order to producehigher amounts and concentrations of the primary metabolites of 4-(3050-dimethyl-30-heptyl)-phenol for their identification (GC-EIMS) the subsequent study withA githago suspension cultures was executed as two-liquid-phase experiment using ann-hexadecane layer as organic phase as described before for 4-n-nonylphenol[24]

Figure 1 Chemical structure of 4(3050-dimethyl-30-heptyl)-phenol (4-353-NP)

Metabolism of the Nonylphenol Isomer 535

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MATERIALS AND METHODS

Chemicals

The nonylphenol isomer [ring-U-14C]-4-(3050-dimethyl-30-heptyl)-phenol (4-353-NP) shown in Fig 1 was synthesized as described[16] the specific activity of thecompound was 298MBqmmol1 its radiochemical purity 969 Nonlabelled4-353-NP was synthesized accordingly Both 14C-labelled and nonlabelled 4-353-NPconsisted of a diastereomeric mixture (47555245 GC-EIMS see below) Forthe metabolism studies the specific activity of the radiochemical was adjustedappropriately by addition of nonlabelled 4-353-NP n-Hexadecane was supplied byAldrich (Deisenhofen Germany)

Plant Cell Cultures and Treatments

Cell suspension cultures of Agrostemma githago L were initiated and grown in20mL of MS medium contained in 100mL Erlenmeyer flasks as described[2125]

Three days after subcultivation three of the cultures were treated with 100 mg(28 kBq) and three were treated with 200 mg (28 kBq) of [ring-U-14C]-4-353-NP perassay With all assays the radiochemical was dissolved in 100 mL of n-hexadecanewhich constituted the organic part of the two-liquid-phase system (mediumn-hexadecane 2001 vv) The assays were incubated for 7 days until the end ofthe subcultivation interval of the cultures Soybean (Glycine max L Merr cvMandarin) cell suspension cultures utilized in the preliminary conventional one-liquid-phase study were maintained in B5 medium as described[2126] Two daysbefore the end of the subcultivation interval the cultures (5 parallels) were treatedwith 20 mg (1mgL1 30 kBq) of [ring-U-14C]-4-353-NP per assay and wereincubated for 48 h

Extraction of Assays

The assays (A githago) were individually transferred to centrifuge tubes and10mL of n-hexane was added After centrifugation (15min 5000 g) the organicphase containing the n-hexadecane layer was removed Remaining aqueous phases(media) were separated from the cells by suction filtration The cells were suspendedin chloroformmethanol 12 (vv) stored at 20C for 16 h and were extractedby means of sonication (Bandelin Berlin Germany) Insoluble cell debris wereseparated from the cell extracts by suction filtration and washed with chloroformmethanolwater 1208 (vvv) Air-dried filter papers with adhering cell debris weresubjected to combustion analysis (Biological Oxidizer OX 500 ZinsserHarveyInstruments Frankfurt Germany) The individual cell extracts were examined byTLC corresponding parallels were combined and concentrated After examinationby TLC media were discarded Excepting the separation of the n-hexadecanephase the assays of the experiment with soybean cells were similarly extracted andexamined

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Hydrolytic Treatments

Concentrated and combined cell extracts (100 and 200 mg assays) derivedfrom the study with A githago were refluxed in 40mL of 2M HClmethanol 11 (vv)for 2 h The methanol portion of the reaction mixture was evaporated and theremaining HCl phase was extracted with diethyl ether Recoveries of 14C in theextracts were 45 and 46 with the 100 and 200 mg assays respectively based on 14Cfound in corresponding combined cell extracts Remaining HCl phases wereconcentrated and hydrolyzed for 2 h using 2M HCl at reflux (recoveries 25 and17 respectively) All diethyl ether extracts (hydrolysates) obtained from thehydrolytic treatments were analyzed by TLC and HPLC Subsequently theradioactivity contained in the first hydrolysate derived from the 200 mg assays wasseparated by preparative HPLC and isolated fractions were examined by GC-EIMSThe combined cell extracts of the experiment with soybean cells were similarilytreated The hydrolysates (total recovery 86) however were only subjected byTLC and HPLC analyses

Analytical and Chromatographic Procedures

Liquid scintillation counting (LSC) was performed using a LS 5000 TD analyzer(Beckmann Munchen) and LumasafeTM Plus and CarbomaxTM Plus (Canberra-Packard Dreieich Germany) scintillation cocktails

Thin-layer chromatography (TLC) was performed on silica gel plates SIL G-25UV254 (025mm Macherey-Nagel Duren Germany) The solvent systems used wereas follows A n-hexanediethyl etheracetic acid 50501 (vvv) B n-hexanediethyletheracetic acid 20801 (vvv) C ethyl acetateiso-propanol 4060 (vv) D ethylacetateiso-propanolH2O 652412 (vvv) Separated 14C peaks were located andquantified by means of a Tracemaster 40 radiochromatogram scanner (BertholdWildbad Germany) nonlabelled 4-353-NP was visualized under UV at 254 nm

High-performance liquid chromatography (HPLC) was executed on a SystemGold Personal chromatograph (Beckman Munchen Germany) consisting ofProgrammable Solvent Module 126 Diode Array Detector Modul 168 andRadioisotope Detector 171 The latter was equipped with a 2420 quartz cell(silanized glass 20ndash30 mm internal diameter 55mm cell volume 037mL RaytestStraubenhardt Germany) All HPLC analyses were performed on a reversed phasecolumn (CC 2504 Nucleosil 100-5 C18 HD Macherey-Nagel Duren Germany) at20C using a flow of 1mLmin1 Nonlabelled 4-353-NP was detected at 280 nmElution was performed with solvents A (01 acetic acid in water vv) and B (01acetic acid in acetonitrile vv) as follows AB (6535 vv) for 5min then linear30min gradient to 100 B isocratic B (100) for 5min and return to initialconditions in 5min All analyses were terminated by washing the column with AB(6535 vv) for 5min

For GC-EIMS analysis (gas chromatography-electron impact mass spectrom-etry) samples were concentrated using an N2 stream and derivatized with 100 mL ofN-methyl-N-trimethyl-silyltrifluoroacetamide (MSTFA Fluka Buchs Switzerland)at 70C for 30min GC-EIMS was executed with a Hewlett-Packard 5890 Series II

Metabolism of the Nonylphenol Isomer 537

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gas chromatograph (Agilent Waldbronn Germany) equipped with a FS-SE-54-NB-05 column (25m 025mm film thickness 046 mm CS Chromatographie ServiceLangerwehe Germany) and carrier gas helium injection volume was 1 mL The gaschromatograph was connected to a Hewlett-Packard 5971 A MSD (mass selectivedetector) which was operated in scan mode (mass rangemz 50ndash480) with an electronenergy of 70 eV Temperature programming was isothermal 85C for 5min 85ndash280Cat 10Cmin1 and isothermal for 3min injector and interface temperatures were250ndash280C respectively

RESULTS AND DISCUSSION

Distribution of Radioactivity

The distributions of 14C found after termination of the metabolism experimentsare shown in Table 1 Concerning the preliminary study with soybean the majorityof 14C was present in the media (357of applied radioactivity) while 299 and 203were detected in cell extracts and insoluble cell debris respectively In contrastmore than 60 of applied 14C were found in the cell extracts derived from the two-liquid-phase study performed with Agrostemma githago cells Portions in mediaand nonextractable residues were below 20 only traces of 14C were left in then-hexadecane phase Individual analyses with TLC (systems A and B) demonstratedthat in the media and cell extracts of the soybean assays free 4-353-NP was only

Table 1 Distribution of radioactivity in Agrostemma githago cell culture afters 7 days

of incubation with 100 mg (5mgL1 28 kBq 3 parallels) or 200 mg (10mgL1 28 kBq

3 parallels) of [ring-U-14C]-4-353-NP per assay in two-liquid-phase system and soybean cell

culture (5 parallels) after 2 days of incubation with 20mg (1mgL1 30 kBq) of [ring-U-14C]-

4-353-NP per assay in conventional one-liquid-phase system (mean values are displayed)

Soybean A githago A githago

20mg assays 100mg assays 200 mg assays

Fraction of applied 14C of applied 14C of applied 14C

n-Hexadecane phase mdash 24 28

Medium 357 151 17414C-residues on filter papera mdashb 51 35

Cell extract 299 639 603

Nonextractable residues 203 118 141

Recovered radioactivity 859 983 981

Recovered 4-353-NPc 18 00 00

Primary metabolitesd 19ndash22 28ndash42 21ndash33

aFilter papers used to separate cells and mediabFilter papers were not examinedcPortions were determined from results of TLC analysis of cell extracts and mediadPortions were estimated from results of TLC and HPLC analyses of hydrolysates obtained

from hydrolytic treatments of combined cell extracts (single values are shown)

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present in trace amounts free 4-353-NP however was completely absent in theassays with the A githago cells The chromatographic behavior (TLC systems AndashD)of the radioactivity contained in all cell extracts pointed to glycosides as metabolicproducts of 4-353-NP With all TLC systems used the radioactivity associated withthe media proved to be preponderantly immobile Extraction of these 14C portionswith organic solvents such as ethyl acetate was not successful the same held true forhydrolysis procedures in order to cleave possible glycosidic linkages Additionallydirect HPLC analysis was not practicable since the necessary concentration of themedia resulted in highly viscous samples

The results obtained with the A githago cells were comparable to data publishedrecently on the biotransformation of 4-n-nonylphenol by the same cell cultureusing the same two-liquid-phase system[24] Concerning the intended identification ofsolublemetabolites uptake of 4-353-NP from the n-hexadecane layer by theA githagocells was considered satisfactory with both the 100 and 200 mg assays The same heldfor recoveries of 14C noticeable portions of soluble 14C in cell extracts and completedisappearance (turnover) of the applied 4-353-NP The different distribution ofradioactivity observed in the soybean experiment was referred to the conventionalone-liquid-phase system used but additionally also to the plant species[23] With bothplant species examined formation of glycosides from the phenolic xenobiotic orpossible primary transformation products was expected according to data publishedon 4-n-nonylphenol[11122324] The chemical nature of the radioactivity present inthe media especially of the soybean cells remained unknown Recently we found14C materials with similar (chromatographic) behavior in the media of plantcell suspensions (including soybean) after application of 14C-bisphenol A andsuspected polymerized products of the compound[27] Polymeres (or copolymers)may also have been formed from 4-353-NP

Hydrolysis Experiments Isolation and Identification of Metabolites

The combined cell extracts were subjected to two consecutive hydrolysissteps (2M HCl-methanol reflux 2M HCl reflux) in order to release the expected14C-aglycons from their corresponding glycosides In the case of soybean 86 of theradioactivity introduced into these experiments could be extracted (diethyl ether)from the reaction mixture With A githago recoveries in the hydrolysates amountedto totals of 70 (100 mg assays) and 63 (200 mg assays) All hydrolysates wereexamined using TLC and HPLC The results of TLC analysis of the samples(hydrolysis steps 1 and 2) derived from the A githogo study (Fig 2) demonstratedthat in three of the samples noticeable portions of 4-353-NP (Rf about 09) werepresent whereas in all samples 14C peaks at Rf about 05 and Rf 00 were detectedHPLC examination of the hydrolysates prevailingly confirmed these data radio-chromatograms of the samples derived from hydrolysis step 1 are shown in Fig 3In addition to a peak originating from 4-353-NP (Rt 310min) further 14C peaksappeared at Rt 150ndash200min Since the 14C peaks with Rf about 05 (TLC) and Rt

150ndash200min (HPLC) were not detected prior to hydrolysis the correspondingcompounds were regarded as primary metabolites of 4-353-NP Both 4-353-NP andprimary metabolites of 4-353-NP were thus liberated from their glycosides by acid

Metabolism of the Nonylphenol Isomer 539

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treatment Similar results were obtained from the soybean experiment (data notshown) Estimated from TLC and HPLC analysis the portions of primarymetabolites of 4-353-NP were between 19 and 42 of applied 14C (Table 1)depending on the method of calculation Low amounts of primary 4-353-NPmetabolites were detected with soybean this fact additionally supported theutilization of A githago cells in two-liquid-phase system for production andidentification of these transformation products

Figure 2 TLC radiochromatograms (solvent system B) of the samples derived from the

hydrolysis experiments A hydrolysis step 1 100 mg assays B hydrolysis step 1 200mg assays

C hydrolysis step 2 100 mg assays D hydrolysis step 2 200mg assays

Figure 3 HPLC radiochromatograms of samples derived from the hydrolysis experiments

A hydrolysis step 1 100mg assays B hydrolysis step 1 200 mg assays

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The radioactivity contained in the hydrolysate of the first hydrolytic step derivedfrom the 200 mg assays was separated by HPLC (using multiple runs) Two fractionswere isolated fraction 1 corresponding with the 14C peaks at Rt 150ndash200min andfraction 2 (4-353-NP Rt 310min) Total yields recovered after concentration ofHPLC eluates were 19 and 7mg with fraction 1 and fraction 2 respectively Bothfractions were derivatized with MSTFA (trimethylsilyl) and subjected to GC-EIMSanalyses the corresponding chromatograms are shown in Fig 4 The chromatogramof fraction 2 exhibited two peaks (IX and X Rt 1620ndash1640min) which accordingto their retention times and mass spectra (Table 2) were identified with the twodiastereomers of parent 4-353-NP While the [Mthorn] peak was observed at mz 292further prominent fragments emerged at mz 263 (-C2H5) 221 (-C5H11 base peak)193 179 and 73 (trimethylsilyl) According to data published on branched side-chain 4-nonylphenol isomers and 4-n-nonylphenol derivatives[2371216] the MSfragmentation scheme of 4-353-NP shown in Fig 5 is proposed The chemicalstructure of the fragment with mz 179 (trimethylsilyl derivative of hydroxyltropylium ion with mz 107) was deduced from recent literature data[3]

In the GC-EIMS chromatogram obtained from fraction 2 eight peaks weredetected (Rt 1860ndash2020min) which by means of their mass spectra could be tracedback to structures related to 4-353-NP (Fig 4) Seven of these compounds (IndashVII)exhibited an [Mthorn] at mz 380 and prominent fragments at mz 261 221 (base peak)

Figure 4 GC-EIMS chromatograms of the samples obtained from HPLC separation

(cf Fig 3) of the first hydrolysate derived from the 200 mg assays A fraction containing the

primary metabolites of 4-353-NP (peaks I and IV appeared as shoulders of II and V

respectively) B fraction containing nonmetabolized 4-353-NP

Metabolism of the Nonylphenol Isomer 541

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Table 2 Mass spectra of side-chain monohydroxylated metabolites (IndashVII) of 4-353-NP a

carboxylic acid metabolite (VII) of 4-353-NP and of 4-353-NP diastereomers (IX X) after

trimethylsilyl derivatization Compounds were detected using GC-EIMS analysis and were

contained in fractions A and B (obtained by HPLC separation) of a cell extract hydrolysate

(first hydrolysis step 200mg assays)

Peak

Rt EIMS

IsomeraPortionb

(min) mz (abundance in ) ()

Fraction 1

Ic 1865 380 (2) 365 (1) 351 (8) 275 (1) 261 (33)

233 (1) 221 (100) 206 (3) 193 (6) 191 (5)

179 (5) 151 (2) 117 (33) 73 (43)

unknown 214c

II 1870 380 (3) 365 (1) 351 (7) 275 (1) 261 (34)

233 (2) 221 (100) 206 (3) 193 (7) 191 (8)

179 (8) 163 (2) 151 (3) 117 (29) 73 (49)

60-OH-

4-353-NP

214c

III 1874 380 (2) 365 (1) 351 (5) 275 (1) 261 (34)

233 (1) 221 (100) 206 (4) 193 (6) 191 (6)

179 (8) 163 (2) 151 (3) 117 (32) 73 (50)

60-OH-

4-353-NP

175

IVd 1892 380 (2) 365 (1) 351 (6) 275 (1) 261 (36)

233 (2) 221 (100) 206 (4) 193 (7) 191 (6)

179 (8) 163 (2) 151 (3) 117 (27) 73 (52)

60-OH-

4-353-NP

473d

V 1896 380 (2) 365 (1) 351 (6) 275 (1) 261 (37)

233 (1) 221 (100) 206 (3) 193 (7) 191 (6)

179 (8) 163 (2) 151 (3) 117 (26) 73 (45)

60-OH-

4-353-NP

473d

VI 1925 380 (2) 365 (1) 351 (5) 336 (1) 309 (6) 295

(1) 261 (9) 233 (4) 221 (100) 206 (2) 193

(31) 191 (7) 179 (12) 163 (2) 151 (4) 147

(7) 117 (4) 103 (11) 73 (60)

10-OH-

4-353-NP

52

VII 1947 380 (2) 365 (1) 351 (5) 295 (2) 261 (8) 253

(3) 233 (3) 221 (100) 207 (4) 193 (21) 191

(6) 179 (10) 163 (6) 151 (2) 117 (3) 103

(4) 73 (47)

20-OH-

4-353-NP

59

VIII 2019 394 (2) 379 (4) 365 (11) 247 (2) 313 (4)

275 (2) 247 (4) 233 (33) 221 (100) 207 (2)

193 (11) 191 (4) 179 (8) 173 (9) 131 (3)

116 (3) 97 (6) 73 (40)

70-COOH-

4-353-NP

26

Fraction 2

IX 1629 292 (5) 277 (3) 263 (18) 221 (100) 207 (7)

193 (55) 179 (14) 163 (3) 151 (6) 135 (2)

129 (3) 115 (4) 103 (3) 91 (3) 73 (34)

4-353-NP 468

X 1638 292 (5) 277 (3) 263 (19) 221 (100) 207 (8)

193 (55) 179 (13) 163 (2) 151 (6) 135 (2)

129 (4) 115 (3) 103 (1) 91 (3) 73 (31)

4-353-NP 532

aStructures of metabolites IndashVIII of 4-353-NP are proposed according to EIMS fragmentation

patternsbCalculated from peak areas total area of peaks IndashVIII and IXndashX respectively corresponds

with 100cPeak I appeared as shoulder of II separate integration of peaks I and II was not possibledPeak IV appeared as shoulder of V separate integration of peaks IV and V was not possible

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179 and 73 complete mass spectra are listed in Table 2 The [Mthorn] of compoundsIndashVII pointed to mono-hydroxylated derivatives of 4-353-NP while all patterns offragmentation and their similarity with that of 4-353-NP (especially peaks at mz 221and 179) indicated that hydroxylation had exclusively occurred at the aliphatic side-chain Compound VIII had an [Mthorn] at mz 394 and prominent fragments at mz 221(base peak) 179 and 73 These findings pointed a (side-chain) carboxylic acidderivative of 4-353-NP With both fraction 1 and fraction 2 GC-EIMS peakscorresponding with possible ring-hydroxylated or side-chain olefinic products[24]

were not detected The hydrolysate derived from the soybean experiment wassimilarly separated by HPLC a fraction corresponding with primary 4-353-NPmetabolites was collected and examined by GC-EIMS after silylation Due tolow yield no distinct peaks which could be related to parent 4-353-NP were foundin the corresponding chromatograms as such However using the GC-EIMSfindings of the A githago study a trace was identified at Rt 1905min the massspectrum of which corresponded with compounds IV or V We concluded that theA githago and soybean cells at least demonstrated certain similarities concerningthe biotransformation of 4-353-NP

Though the GC-EIMS data unequivocally proved that 4-353-NP was metabo-lized by A githago cell suspensions at the aliphatic side-chain to monohydroxylatedproducts and a carboxylic acid derivative the sites of hydroxylationoxidation partlyremained unclear Compounds IIndashV emerged in the chromatograms as two pairs ofpeaks (IIIII and IVV) and exhibited noticeably similar mass spectra (Table 2)Besides [Mthorn] at mz 380 and fragments observed with all other monohydroxylatedmetabolites (mz 365 351 261 221 179 73) a prominent fragment of compoundsIIndashV hadmz 117 with 26ndash32 abundance According to data published on side-chain

Figure 5 Proposed MS fragmentation of 4(3050-dimethyl-30-heptyl)-phenol

Metabolism of the Nonylphenol Isomer 543

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monohydroxylated derivatives of both 4-n-nonylphenol[1224] and model side-chainisomers 2- 3- and 4-(4-hydroxyphenol)-nonane[4] fragment mz 117 was referred to[H3CCHOSi(CH3)3

thorn] This fragment is characteristic of trimethylsilyl derivatizedhydroxyl functions at secondary carbon atoms adjacent to terminal CH3

groups[4122428] Due to abundances (34ndash37) of fragments mz 261 (-HOSi(CH3)3-C2H5)

[2429] it was supposed that compounds IIndashV were 60-hydroxy derivatives of4-353-NP Applied 4-353-NP consisted of two diastereomers (each a couple ofenatiomers) hydroxylation at position 60 consequently introduced a third chiral centerwhich resulted in four diastereomers An MS fragmentation scheme for IIndashV isproposed in Fig 6 Quantitative evaluation of the chromatogram showed thatcompounds IIndashV were the main primary metabolites in A githago cells amountingto about 80 of the products identified

The mass spectrum of compound VI (Table 2) differed from those of productsIIndashV in particular regarding three fragments mz 309 (abundance 6) 295 (1) and103 (11) According to previous results presented on 4-n-nonylphenol[24] thefragment mz 103 was supposed to arise from [H2COSi(CH3)3

thorn] pointing tohydroxylation at a primary carbon[2428] Furthermore fragments mz 309 and 295indicated loss of C5H11 and C6H13 respectively from [Mthorn] (mz 380) with retentionof two silylated hydroxy functions at the remaining fragment ion It was thusassumed that compound VI was a 4-353-NP metabolite hydroxylated at position 10

of the side-chain Due to a similar line of argumentation (presence of fragments mz117 and 295) compound VII possibly consisted of the 20-hydroxy derivative of4-353-NP The structure of compound I remained unknown mainly becausemetabolite I was detected as shoulder of II resulting in large overlapping of its mass

Figure 6 Proposed MS fragmentation of the 6-hydroxy metabolites of 4(3050-dimethyl-

30-heptyl)-phenol

544 Schmidt et al

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spectrum with that of II Due to its [Mthorn] of mz 394 compound VIII was identifiedwith a carboxylic acid metabolite of 4-353-NP since formation of a hydroxyderivative with an additional carbonyl function was regarded unlikely The massspectrum observed with VIII supported this assumption Fragments showingsimilarity to compounds IndashVII were mz 379 (-CH3) and 365 (-C2H5) whereasfragments mz 131 and 116 were supposed to consist of frac12CH2COOSiethCH3THORN

thorn3 and

frac12CH2COOSiethCH3THORNthorn2 respectively which pointed to a carboxyl function at positions

10 or 70 A characteristic feature of the fragmentation of trimethylsilyl derivatizedfatty acids is separation of CH3 (15) and HOSi(CH3)2 (75) consecutively[30]

With VIII this would result in fragment mz 304 which was only present in thebackground However ions resulting from successive fragmentation of mz 304were observed mz 275 (-HCO -29) 247 (-C2H428) 233 (-CH214) and finally221 This sequence was regarded as prove that 4-353-NP was oxidized to itscarboxylic acid at position 70 of the molecule It should be mentioned that I VI VIIand VIII were only minor metabolites formed from 4-353-NP by the A githago cellsuspensions (Table 2)

Metabolism of 4-353-NP by Agrostemma githagoPlant Cell Suspension Cultures

The nonylphenol isomer 4-353-NP consisting of two diastereomers wastransformed by theAgrostemma githago suspension cultures to four types of productsglycosides of parent 4-353-NP glycosides of primary 4-353-NP metabolitesnonextractable residues and unknown possibly polymeric materials detected in themedia Similar products emerged in the soybean cultures The present investigationconcentrated on the primary metabolites of 4-353-NP These were produced in the Agithago suspensions using a two-liquid-phase system The primary products identifiedby GC-EIMS after liberation from their glycosides were one side-chain carboxylicacid and seven side-chain monohydroxylated metabolites Due to their mass spectrathe main products were four diastereomers of 60-hydroxy-4-353-NP

As yet data on the biotransformation of 4-353-NP are mainly not at handConcerning plant tissues (cell cultures plants) side-chain hydroxylated metaboliteswere reported on 4-n-nonylphenol[11122324] With A githago cells the main productwas 4-(80-hydroxy-n-nonyl)-phenol[24] ie 4-n-nonylphenol was metabolized bysubterminal oxidation corresponding with the main products observed with 4-353-NP in the present study Side-chain hydroxylated metabolites were also formed fromp-tert-octylphenol in barley plants[31] Experiments with model nonylphenols 2- 3-and 4-(4-hydroxyphenyl)-nonane demonstrated that in rainbow trout all isomerswere transformed to monohydroxy products with the site of oxidation at carbon 8(subterminal) of the nonyl residue The metabolites emerged as glucuronic acidconjugates while glucuronides of the parent nonylphenols were also formed[4] In asimilar study both side-chain and ring hydroxylated metabolites of 4-n-nonylphenolwere detected as glucuronic acid conjugates[9] Recently data on the metabolism ofthe nonylphenol isomer 4(3060-dimethyl-30-heptyl)-phenol in pond snails to a (ringhydroxylated) catechol derivative (with unmodified side-chain) were reported[1920]

Contrasting with 4-353-NP of the present study the isomer examined possessed

Metabolism of the Nonylphenol Isomer 545

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a branching at subterminal position 60 of the side-chain besides that at carbon 30 Thecatechol was found conjugated to glucuronic acid[1920] As technical productnonylphenol appears to be completely degradated in soil[1] though isomer 4-(3060-dimethyl-30-heptyl)-phenol proved to be resistant in sedimentwater systems[18] Upto now the routes of nonylphenolrsquos microbial degradation are not known in detailDue to experiments with an isolated bacterial strain initial oxidation at the aromaticmoiety leads to degradation of the phenyl ring while side-chains remain asnonanols[3233] one of the studies was executed with radio-labelled 4-353-NP[33]

The present data on the metabolism of 4-353-NP by A githago cell suspensionsagree with those reported in the literature on 4-n-nonylphenol in plant tissues andmodel isomers in rainbow trout In contrast with trout the capacity of the A githagocells for hydroxylation of the side-chain appeared to be broader ie the nonyl residuewas hydroxylated at different sites of the aliphatic chain The enzymes responsible forside-chain hydroxylation of 4-353-NP in A githago are not known we suspectedcytochromes P450 In rats and humans isozyme CYP2B2 was thought to catalyzepredominantly the hydroxylation of nonylphenol isomers[10] After expression inyeast human CYP2B6 and CYP2C19 were shown to metabolize nonylphenol (pluralbranched chains) while 4-n-nonylphenol was transformed by CYP1A2 CYP2B6 andCYP2C19 ring and side-chain hydroxlated products were identified with branchedchain nonylphenols and 4-n-nonylphenol[34] We assume that plants in general havethe capacity to transform nonylphenol isomers to side-chain hydroxylated productsand thus contribute to the environmental degradation of the xenoestrogen If cropplants are contaminated with nonylphenol both nonylphenol and its side-chainhydroxylated products are expected to be present in plant tissues as glycosideconjugates Xenobiotic glycosides are known to be cleaved in the gastrointestinal tractwith release of possibly toxic aglycons Data on the toxicological properties of side-chain hydroxylated nonylphenols are not available Our future research will focus onthe metabolism of nonylphenol isomers with different branching patterns on theidentification of the enzymes responsible for the metabolism of nonylphenols and onthe (eco-) toxicology of side-chain hydroxylated nonylphenol isomers

ACKNOWLEDGMENTS

For the synthesis of [ring-U-14C]-4(3050-dimethyl-30-heptyl)-phenol the authorswish to thank R Vinken and the lsquolsquoAachen Graduate College Elimination ofEndocrine-Disrupting Substances from Waste-Waterrsquorsquo (AGEESA) funded by theDeutsche Forschungsgemeinschaft

REFERENCES

1 Staples CA Williams JB Blessing RL Varineau PT Measuring thebiodegradability of nonylphenol ether carboxylates octylphenol ether carboxyl-ates and nonylphenol Chemosphere 1999 38 (9) 2029ndash2039

2 Thiele B Gunther K Schwuger MJ Alkylphenol ethoxylates trace analysisand environmental behaviour Chem Rev 1997 97 (8) 3247ndash3272

546 Schmidt et al

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3 Wheeler TF Heim JR LaTorre MR Janes AB Mass spectral

characterization of p-nonylphenol isomers using high-resolution capillary

GC-MS J Chromatogr Sci 1997 35 (1) 19ndash304 Meldahl AC Nithipatikom K Lech JJ Metabolism of several 14C-

nonylphenol isomers by rainbow trout (Oncorhynchus mykiss) in vivo and

in vitro Metabolites Xenobiotica 1996 26 (11) 1167ndash11805 Kim Y-S Katase T Sekine S Inoue T Makino M Uchiyama T

Fujimoto Y Yamashita N Variation in estrogenic activity among fractions

of a commercial nonylphenol by high performance liquid chromatography

Chemosphere 2004 54 (8) 1127ndash11346 Giger W Stephanou E Schaffner C Persistent organic chemicals in sewage

effluents 1 Identification of nonylphenols and nonylphenolethoxylates by

capillary gas chromatographymass spectrometry Chemosphere 1981 10 (11ndash1)

1253ndash12637 Giger W Brunner PH Schaffner C 4-Nonylphenol in sewage sludge

accumulation of toxic metabolites from nonionic surfactants Science 1984

225 (4662) 623ndash6258 Jobst H Chlorophenols an nonylphenols in sewage sludges 1 Occurrence in

sewage sludges of Western German treatment plants from 1987ndash1989 Acta

Hydrochim Hydrobiol 1995 23 (1) 20ndash259 Coldham NG Sivapathasundaram SDM Ashfield LA Pottinger TG

Biotransformation tissue distribution and persistence of 4-nonylphenol

residues in juvenile rainbow trout (Oncorhynchus mykiss) Drug Metab

Dispos 1998 26 (4) 347ndash35410 Lee PC Marquardt M Lech JJ Metabolism of nonylphenol by rat and

human microsomes Toxicol Lett 1998 99 (7) 117ndash12611 Bokern M Harms HH Toxicity and metabolism of 4-n-nonylphenol in cell

suspension cultures of different plant species Environ Sci Technol 1997

31 (7) 1849ndash185412 Bokern M Nimtz M Harms HH Metabolites of 4-n-nonylphenol in wheat

cell suspension cultures J Agric Food Chem 1996 44 (4) 1123ndash112713 Guenther K Heinke V Thiele B Kleist E Prast H Raecker T

Endocrine disrupting nonylphenols are ubiquitous in food Environ Sci

Technol 2002 36 (8) 1676ndash168014 Soto AM Justicia H Wray JW Sonnenschein C p-Nonylphenol an

estrogenic xenobiotic released from lsquolsquomodifiedrsquorsquo polystyrene Environ Health

Perspect 1991 92 167ndash17315 Degen DH Bolt HM Comments on lsquolsquoendocrine disrupting nonylphenols

are ubiquitous in foodrsquorsquo Envirn Sci Technol 2003 37 (11) 2622ndash262316 Vinken R Schmidt B Schaffer A Synthesis of tertiary 14C-labelled

nonylphenol isomers J Label Compd Radiopharm 2002 45 (14) 1253ndash126317 Lalah JO Lenoir D Henkelmann B Hertkorn N Gunther K

Schramm K-W Kettrup A Synthesis of ring-14C-labelled 4(3060-dimethyl-

30-heptyl)-phenol J Label Compd Radiopharm 2001 44 (6) 459ndash46318 Lalah JO Schramm K-W Henkelmann B Lenoir D Behechti A

Gunther K Kettrup A The dissipation distribution and fate of a branched

Metabolism of the Nonylphenol Isomer 547

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14C-nonylphenol isomer in lake watersediment systems Environ Pollut 2003

122 (2) 195ndash20319 Lalah JO Schramm K-W Severin GF Lenoir D Henkelmann B

Behechti A Gunther K Kettrup A In vivo metabolism and organ

distribution of a branched 14C-nonylphenol isomer in pond snails Lymnaea

stagnalis L Aquat Toxicol 2003 62 (4) 305ndash31920 Lalah JO Behechti A Severin GF Lenoir D Gunther K Kettrup A

Schramm K-W The Bioaccumulation and fate of a branched 14C-

p-nonylphenol isomer in Lymnaea stagnalis L Environ Toxicol Chem 2003

22 (7) 1428ndash143621 Schmidt B Metabolic profiling using plant cell suspension cultures In

Pesticide Transformation in Plants and Microorganisms Hall JC Hoagland

RE Zablotowicz RM Eds American Chemical Society Washington DC

2001 40ndash5622 Komoszliga D Langebartels C Sandermann H Metabolic processes for

organic chemicals in plants In Plant ContaminationmdashModeling and Simulation

of Organic Chemical Processes Trapp S Mc Farlane JC Eds Lewis

Publishers Boca Raton 1995 69ndash10323 Schmidt B Schuphan I Metabolism of the Xenoestrogens 4-n-nonylphenol

and bisphenol A in plant cell suspension cultures In Book of Abstracts 10th

IUPAC International Congress on the Chemistry of Crop Protection Basel

August 4ndash9 IUPAC Basel 2002 Vol 2 3424 Schmidt B Patti H Niewersch C Schuphan I Biotransformation of [ring-

U-14C]4-n-nonylphenol by Agrostemma githago cell culture in a two-liquid-

phase system Biotechnol Lett 2003 25 (16) 1375ndash138125 Murashige T Skoog F Revised medium for rapid growth and bio assay with

tobacco tissue cultures Physiol Plant 1962 15 473ndash49726 Gamborg OL Miller RA Ojima K Nutrient requirements of suspension

cultures of soybean root cells Exp Cell Res 1968 50 151ndash15827 Schmidt B Schuphan I Metabolism of the environmental estrogen bisphenol

A by plant cell suspension cultures Chemosphere 2002 49 (1) 51ndash5928 Diekman J Thomson JB Djerassi C Mass spectrometry and stereo-

chemical problems CLV Electron impact induced fragmentations and

rearrangements of some trimethylsilyl ethers of aliphatic glycols and related

compounds J Org Chem 1968 33 (6) 2271ndash228429 Diekman J Thomson JB Djerassi C Mass spectrometry and stereo-

chemical problems CXLI Electron impact induced fragmentations and

rearrangements of trimethylsilyl ethers amines and sulfides J Org Chem

1967 32 (12) 3904ndash391930 Odham G Fatty acids In Biochemical Applications of Mass Spectrometry

Waller GR Dermer OC Eds Wiley-Interscience New York 1980 First

Supplementary Volume 153ndash17131 Stolzenberg GE Olson PA Zaylskie RG Mansager ER Behavior and

fate of ethoxylated alkylphenol nonionic surfactant in barley plants J Agric

Food Chem 1982 30 (4) 637ndash644

548 Schmidt et al

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32 Tanghe T Dhooge W Verstraete W Isolation of a bacterial strain able todegrade branched nonylphenol Appl Environ Microbiol 1999 65 (2)746ndash751

33 Corvini PFX Vinken R Hommes G Schmidt B Dohmann MDegradation of the radioactive and non-labelled branched 4(3050-dimethyl-30-heptyl)-phenol nonylphenol isomer by Sphingomonas TTNP3 Biodegradation2003 15 (1) 1ndash10

34 Inui H Shiota N Motoi Y Ido Y Inoue T Kodama T Ohkawa YOhkawa H Metabolism of herbicides and other chemicals in humancytochrome P450 species and in transgenic potato plants co-expressinghuman CYP1A1 CYP2B6 and CYP2C19 J Pestic Sci 2001 26 (1) 28ndash40

Received February 13 2004

Metabolism of the Nonylphenol Isomer 549

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21ndash42 of applied 14C (A githago) After liberation from their glycosides the

primary 4-353-NP metabolites formed by A githago were isolated by HPLC and

examined by GC-EIMS as trimethylsilyl derivatives In the chromatograms

eight peaks were detected which due to their mass spectra could be traced back to

4-353-NP Seven of the compounds were side-chain monohydroxylated 4-353-NP

metabolites while the remaining was a (side-chain) carboxylic acid derivative

Unequivocal identification of the sites of hydroxylationoxidation of all transfor-

mation products was not possible The main primary metabolites produced by

A githago were supposed to be four diastereomers of 60-hydroxy-4-353-NP

(about 80 of all products identified) It was concluded that plants contribute to

the environmental degradation of the xenoestrogen nonylphenol the toxicological

properties of side-chain hydroxylated nonylphenols remain to be examined

Key Words Plant cell culture Agrostemma githago Soybean Metabolism

Nonylphenol isomer 4-(3050-Dimethyl-30-heptyl)-phenol Two-liquid-phase

system Metabolic profiling Hydroxylated nonylphenol metabolites

INTRODUCTION

Alkylphenol polyethoxylates (APEs) belong to the class of nonionic surfactantsand are used world-wide as detergents emulsifiers solubilizers wetting agents anddispersants in both industry (about 85 of the market) and households APEs serveas cleaning products as well as industrial process aids Applications of APEs includeresin plastic and elastomer manufacturing paper and pulp production industrialcleaners pesticide formulations cold cleaners for cars and household cleaners[12]

APEs derived from nonylphenol are designated as nonylphenol polyethoxylates(NPEs) and comprise about 80 of the total market volume[1] Nonylphenol issynthesized industrially by alkylation of phenol with nonene The product containsabout 90 p-nonylphenol besides prevailingly o-nonylphenol and op-di-nonylphe-nols As a result of the method utilized to manufacture nonene (propylene trimer)nonylphenol consists of a complex mixture of predominantly branched-chain C9

derivatives of phenol Recently 22 p-nonylphenol isomers have been identified in theindustrial product[34] and characterization of predominant structures (MS NMR)after separation of commercial nonylphenol has been achieved[5]

Because of their wide use especially in aqueous solutions NPEs are dischargedinto municipal and industrial waste waters which enter sewage treatment plantsDuring treatment a complex biodegradation occurs which leads to the formation ofa number of metabolites with shortened ethoxy chains and ultimately nonylphenolNonylphenol itself is considerably lipophilic and a comparatively stable substanceThus nonylphenol and partially also its preceding metabolic residues were shownto be released with effluents of treatment plants to surface waters additionallynonylphenol was detected in sewage sludge[1246ndash10] Besides the use of NPEs inpesticide formulations sewage sludge frequently applied to agricultural soils andeffluents occasionally used in irrigation may constitute a source of contamination ofterrestrial plants with NPEs and nonylphenol These facts raise the question ofuptake and metabolic fate of nonylphenol in plants particularly those used for foodproduction[1112] Nonylphenol was recently reported to be present in foodstuffs[13]

534 Schmidt et al

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The world-wide interest in the (environmental) fate of nonylphenol originatesfrom its proven estrogenic activity the compound is considered as xenoestrogenor as an endocrine disrupter[781314] Consequently nonylphenol may affect bothhuman health and ecosystems Regarding human health the risk associated withlow-level concentrations of nonylphenol in foodstuffs however is discussedcontroversially[1315] Early research on the environmental and metabolic fate ofnonylphenol eg in plant tissues[1112] was mainly performed with the straight-chain 4-n-nonylphenol due to the unavailability of individual isomers of theindustrial productmdashespecially in 14C-labelled form However 4-n-nonylphenolappears to be no or only a trace constituent of industrial nonylphenol Thusincreasing efforts in the last years aimed at the synthetic preparation of definedbranched-chain nonylphenol isomers (preferentially also 14C-labelled) and the studyof their environmental fate and effects[516ndash20] Up to now data on the metabolism ofbranched-chain nonylphenols in plant tissues are not at hand

Plant cell cultures are often used to examine the metabolism of xenobiotics inplant tissues[2122] The studies executed prior or parallel to experiments with intactplants render possible the rapid identification of metabolites of interest So recentlywe studied the metabolism of 4-n-nonylphenol in plant cell suspension cultures andpresented the results as a poster[23] The studies were performed according to astandardized procedure[21] with 1mgL1 of the 14C-labelled xenobiotic (applied inmethanol) and 2 days of incubation using a one-liquid-phase system Since GC-EIMSidentification of the primary metabolites of 4-n-nonylphenol was not sufficientlyclear-cut we intended to produce higher concentrations and amounts of the metabolicproducts The low aqueous solubility of nonylphenols in general however raised thequestion of an alternative mode of application Recently we thus could demonstratethat considerable amounts of 4-n-nonylphenol dissolved in an n-hexadecane layerwere absorbed and metabolized by suspended cultivated cells of Agrostemma githago(corn cockle) in a satisfactory manner the n-hexadexane layer representing theorganic phase of a two-liquid-phase system affected plant cell growth only little[24]

In the present article we report data on the metabolism of the 14C-labellednonylphenol isomer 4-(3050-dimethyl-30-heptyl)-phenol (Fig 1) by cell suspensioncultures of soybean and A githago The isomer (as diastereomeric mixture) wassynthesized according to a published procedure[16] Due to GC-EI (retention timesmass spectra) the isomer is an important constituent of industrially producednonylphenol The preliminary metabolism experiment with the soybean suspensionswas performed as conventional one-liquid-phase study[21] In order to producehigher amounts and concentrations of the primary metabolites of 4-(3050-dimethyl-30-heptyl)-phenol for their identification (GC-EIMS) the subsequent study withA githago suspension cultures was executed as two-liquid-phase experiment using ann-hexadecane layer as organic phase as described before for 4-n-nonylphenol[24]

Figure 1 Chemical structure of 4(3050-dimethyl-30-heptyl)-phenol (4-353-NP)

Metabolism of the Nonylphenol Isomer 535

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MATERIALS AND METHODS

Chemicals

The nonylphenol isomer [ring-U-14C]-4-(3050-dimethyl-30-heptyl)-phenol (4-353-NP) shown in Fig 1 was synthesized as described[16] the specific activity of thecompound was 298MBqmmol1 its radiochemical purity 969 Nonlabelled4-353-NP was synthesized accordingly Both 14C-labelled and nonlabelled 4-353-NPconsisted of a diastereomeric mixture (47555245 GC-EIMS see below) Forthe metabolism studies the specific activity of the radiochemical was adjustedappropriately by addition of nonlabelled 4-353-NP n-Hexadecane was supplied byAldrich (Deisenhofen Germany)

Plant Cell Cultures and Treatments

Cell suspension cultures of Agrostemma githago L were initiated and grown in20mL of MS medium contained in 100mL Erlenmeyer flasks as described[2125]

Three days after subcultivation three of the cultures were treated with 100 mg(28 kBq) and three were treated with 200 mg (28 kBq) of [ring-U-14C]-4-353-NP perassay With all assays the radiochemical was dissolved in 100 mL of n-hexadecanewhich constituted the organic part of the two-liquid-phase system (mediumn-hexadecane 2001 vv) The assays were incubated for 7 days until the end ofthe subcultivation interval of the cultures Soybean (Glycine max L Merr cvMandarin) cell suspension cultures utilized in the preliminary conventional one-liquid-phase study were maintained in B5 medium as described[2126] Two daysbefore the end of the subcultivation interval the cultures (5 parallels) were treatedwith 20 mg (1mgL1 30 kBq) of [ring-U-14C]-4-353-NP per assay and wereincubated for 48 h

Extraction of Assays

The assays (A githago) were individually transferred to centrifuge tubes and10mL of n-hexane was added After centrifugation (15min 5000 g) the organicphase containing the n-hexadecane layer was removed Remaining aqueous phases(media) were separated from the cells by suction filtration The cells were suspendedin chloroformmethanol 12 (vv) stored at 20C for 16 h and were extractedby means of sonication (Bandelin Berlin Germany) Insoluble cell debris wereseparated from the cell extracts by suction filtration and washed with chloroformmethanolwater 1208 (vvv) Air-dried filter papers with adhering cell debris weresubjected to combustion analysis (Biological Oxidizer OX 500 ZinsserHarveyInstruments Frankfurt Germany) The individual cell extracts were examined byTLC corresponding parallels were combined and concentrated After examinationby TLC media were discarded Excepting the separation of the n-hexadecanephase the assays of the experiment with soybean cells were similarly extracted andexamined

536 Schmidt et al

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Hydrolytic Treatments

Concentrated and combined cell extracts (100 and 200 mg assays) derivedfrom the study with A githago were refluxed in 40mL of 2M HClmethanol 11 (vv)for 2 h The methanol portion of the reaction mixture was evaporated and theremaining HCl phase was extracted with diethyl ether Recoveries of 14C in theextracts were 45 and 46 with the 100 and 200 mg assays respectively based on 14Cfound in corresponding combined cell extracts Remaining HCl phases wereconcentrated and hydrolyzed for 2 h using 2M HCl at reflux (recoveries 25 and17 respectively) All diethyl ether extracts (hydrolysates) obtained from thehydrolytic treatments were analyzed by TLC and HPLC Subsequently theradioactivity contained in the first hydrolysate derived from the 200 mg assays wasseparated by preparative HPLC and isolated fractions were examined by GC-EIMSThe combined cell extracts of the experiment with soybean cells were similarilytreated The hydrolysates (total recovery 86) however were only subjected byTLC and HPLC analyses

Analytical and Chromatographic Procedures

Liquid scintillation counting (LSC) was performed using a LS 5000 TD analyzer(Beckmann Munchen) and LumasafeTM Plus and CarbomaxTM Plus (Canberra-Packard Dreieich Germany) scintillation cocktails

Thin-layer chromatography (TLC) was performed on silica gel plates SIL G-25UV254 (025mm Macherey-Nagel Duren Germany) The solvent systems used wereas follows A n-hexanediethyl etheracetic acid 50501 (vvv) B n-hexanediethyletheracetic acid 20801 (vvv) C ethyl acetateiso-propanol 4060 (vv) D ethylacetateiso-propanolH2O 652412 (vvv) Separated 14C peaks were located andquantified by means of a Tracemaster 40 radiochromatogram scanner (BertholdWildbad Germany) nonlabelled 4-353-NP was visualized under UV at 254 nm

High-performance liquid chromatography (HPLC) was executed on a SystemGold Personal chromatograph (Beckman Munchen Germany) consisting ofProgrammable Solvent Module 126 Diode Array Detector Modul 168 andRadioisotope Detector 171 The latter was equipped with a 2420 quartz cell(silanized glass 20ndash30 mm internal diameter 55mm cell volume 037mL RaytestStraubenhardt Germany) All HPLC analyses were performed on a reversed phasecolumn (CC 2504 Nucleosil 100-5 C18 HD Macherey-Nagel Duren Germany) at20C using a flow of 1mLmin1 Nonlabelled 4-353-NP was detected at 280 nmElution was performed with solvents A (01 acetic acid in water vv) and B (01acetic acid in acetonitrile vv) as follows AB (6535 vv) for 5min then linear30min gradient to 100 B isocratic B (100) for 5min and return to initialconditions in 5min All analyses were terminated by washing the column with AB(6535 vv) for 5min

For GC-EIMS analysis (gas chromatography-electron impact mass spectrom-etry) samples were concentrated using an N2 stream and derivatized with 100 mL ofN-methyl-N-trimethyl-silyltrifluoroacetamide (MSTFA Fluka Buchs Switzerland)at 70C for 30min GC-EIMS was executed with a Hewlett-Packard 5890 Series II

Metabolism of the Nonylphenol Isomer 537

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gas chromatograph (Agilent Waldbronn Germany) equipped with a FS-SE-54-NB-05 column (25m 025mm film thickness 046 mm CS Chromatographie ServiceLangerwehe Germany) and carrier gas helium injection volume was 1 mL The gaschromatograph was connected to a Hewlett-Packard 5971 A MSD (mass selectivedetector) which was operated in scan mode (mass rangemz 50ndash480) with an electronenergy of 70 eV Temperature programming was isothermal 85C for 5min 85ndash280Cat 10Cmin1 and isothermal for 3min injector and interface temperatures were250ndash280C respectively

RESULTS AND DISCUSSION

Distribution of Radioactivity

The distributions of 14C found after termination of the metabolism experimentsare shown in Table 1 Concerning the preliminary study with soybean the majorityof 14C was present in the media (357of applied radioactivity) while 299 and 203were detected in cell extracts and insoluble cell debris respectively In contrastmore than 60 of applied 14C were found in the cell extracts derived from the two-liquid-phase study performed with Agrostemma githago cells Portions in mediaand nonextractable residues were below 20 only traces of 14C were left in then-hexadecane phase Individual analyses with TLC (systems A and B) demonstratedthat in the media and cell extracts of the soybean assays free 4-353-NP was only

Table 1 Distribution of radioactivity in Agrostemma githago cell culture afters 7 days

of incubation with 100 mg (5mgL1 28 kBq 3 parallels) or 200 mg (10mgL1 28 kBq

3 parallels) of [ring-U-14C]-4-353-NP per assay in two-liquid-phase system and soybean cell

culture (5 parallels) after 2 days of incubation with 20mg (1mgL1 30 kBq) of [ring-U-14C]-

4-353-NP per assay in conventional one-liquid-phase system (mean values are displayed)

Soybean A githago A githago

20mg assays 100mg assays 200 mg assays

Fraction of applied 14C of applied 14C of applied 14C

n-Hexadecane phase mdash 24 28

Medium 357 151 17414C-residues on filter papera mdashb 51 35

Cell extract 299 639 603

Nonextractable residues 203 118 141

Recovered radioactivity 859 983 981

Recovered 4-353-NPc 18 00 00

Primary metabolitesd 19ndash22 28ndash42 21ndash33

aFilter papers used to separate cells and mediabFilter papers were not examinedcPortions were determined from results of TLC analysis of cell extracts and mediadPortions were estimated from results of TLC and HPLC analyses of hydrolysates obtained

from hydrolytic treatments of combined cell extracts (single values are shown)

538 Schmidt et al

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present in trace amounts free 4-353-NP however was completely absent in theassays with the A githago cells The chromatographic behavior (TLC systems AndashD)of the radioactivity contained in all cell extracts pointed to glycosides as metabolicproducts of 4-353-NP With all TLC systems used the radioactivity associated withthe media proved to be preponderantly immobile Extraction of these 14C portionswith organic solvents such as ethyl acetate was not successful the same held true forhydrolysis procedures in order to cleave possible glycosidic linkages Additionallydirect HPLC analysis was not practicable since the necessary concentration of themedia resulted in highly viscous samples

The results obtained with the A githago cells were comparable to data publishedrecently on the biotransformation of 4-n-nonylphenol by the same cell cultureusing the same two-liquid-phase system[24] Concerning the intended identification ofsolublemetabolites uptake of 4-353-NP from the n-hexadecane layer by theA githagocells was considered satisfactory with both the 100 and 200 mg assays The same heldfor recoveries of 14C noticeable portions of soluble 14C in cell extracts and completedisappearance (turnover) of the applied 4-353-NP The different distribution ofradioactivity observed in the soybean experiment was referred to the conventionalone-liquid-phase system used but additionally also to the plant species[23] With bothplant species examined formation of glycosides from the phenolic xenobiotic orpossible primary transformation products was expected according to data publishedon 4-n-nonylphenol[11122324] The chemical nature of the radioactivity present inthe media especially of the soybean cells remained unknown Recently we found14C materials with similar (chromatographic) behavior in the media of plantcell suspensions (including soybean) after application of 14C-bisphenol A andsuspected polymerized products of the compound[27] Polymeres (or copolymers)may also have been formed from 4-353-NP

Hydrolysis Experiments Isolation and Identification of Metabolites

The combined cell extracts were subjected to two consecutive hydrolysissteps (2M HCl-methanol reflux 2M HCl reflux) in order to release the expected14C-aglycons from their corresponding glycosides In the case of soybean 86 of theradioactivity introduced into these experiments could be extracted (diethyl ether)from the reaction mixture With A githago recoveries in the hydrolysates amountedto totals of 70 (100 mg assays) and 63 (200 mg assays) All hydrolysates wereexamined using TLC and HPLC The results of TLC analysis of the samples(hydrolysis steps 1 and 2) derived from the A githogo study (Fig 2) demonstratedthat in three of the samples noticeable portions of 4-353-NP (Rf about 09) werepresent whereas in all samples 14C peaks at Rf about 05 and Rf 00 were detectedHPLC examination of the hydrolysates prevailingly confirmed these data radio-chromatograms of the samples derived from hydrolysis step 1 are shown in Fig 3In addition to a peak originating from 4-353-NP (Rt 310min) further 14C peaksappeared at Rt 150ndash200min Since the 14C peaks with Rf about 05 (TLC) and Rt

150ndash200min (HPLC) were not detected prior to hydrolysis the correspondingcompounds were regarded as primary metabolites of 4-353-NP Both 4-353-NP andprimary metabolites of 4-353-NP were thus liberated from their glycosides by acid

Metabolism of the Nonylphenol Isomer 539

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treatment Similar results were obtained from the soybean experiment (data notshown) Estimated from TLC and HPLC analysis the portions of primarymetabolites of 4-353-NP were between 19 and 42 of applied 14C (Table 1)depending on the method of calculation Low amounts of primary 4-353-NPmetabolites were detected with soybean this fact additionally supported theutilization of A githago cells in two-liquid-phase system for production andidentification of these transformation products

Figure 2 TLC radiochromatograms (solvent system B) of the samples derived from the

hydrolysis experiments A hydrolysis step 1 100 mg assays B hydrolysis step 1 200mg assays

C hydrolysis step 2 100 mg assays D hydrolysis step 2 200mg assays

Figure 3 HPLC radiochromatograms of samples derived from the hydrolysis experiments

A hydrolysis step 1 100mg assays B hydrolysis step 1 200 mg assays

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The radioactivity contained in the hydrolysate of the first hydrolytic step derivedfrom the 200 mg assays was separated by HPLC (using multiple runs) Two fractionswere isolated fraction 1 corresponding with the 14C peaks at Rt 150ndash200min andfraction 2 (4-353-NP Rt 310min) Total yields recovered after concentration ofHPLC eluates were 19 and 7mg with fraction 1 and fraction 2 respectively Bothfractions were derivatized with MSTFA (trimethylsilyl) and subjected to GC-EIMSanalyses the corresponding chromatograms are shown in Fig 4 The chromatogramof fraction 2 exhibited two peaks (IX and X Rt 1620ndash1640min) which accordingto their retention times and mass spectra (Table 2) were identified with the twodiastereomers of parent 4-353-NP While the [Mthorn] peak was observed at mz 292further prominent fragments emerged at mz 263 (-C2H5) 221 (-C5H11 base peak)193 179 and 73 (trimethylsilyl) According to data published on branched side-chain 4-nonylphenol isomers and 4-n-nonylphenol derivatives[2371216] the MSfragmentation scheme of 4-353-NP shown in Fig 5 is proposed The chemicalstructure of the fragment with mz 179 (trimethylsilyl derivative of hydroxyltropylium ion with mz 107) was deduced from recent literature data[3]

In the GC-EIMS chromatogram obtained from fraction 2 eight peaks weredetected (Rt 1860ndash2020min) which by means of their mass spectra could be tracedback to structures related to 4-353-NP (Fig 4) Seven of these compounds (IndashVII)exhibited an [Mthorn] at mz 380 and prominent fragments at mz 261 221 (base peak)

Figure 4 GC-EIMS chromatograms of the samples obtained from HPLC separation

(cf Fig 3) of the first hydrolysate derived from the 200 mg assays A fraction containing the

primary metabolites of 4-353-NP (peaks I and IV appeared as shoulders of II and V

respectively) B fraction containing nonmetabolized 4-353-NP

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Table 2 Mass spectra of side-chain monohydroxylated metabolites (IndashVII) of 4-353-NP a

carboxylic acid metabolite (VII) of 4-353-NP and of 4-353-NP diastereomers (IX X) after

trimethylsilyl derivatization Compounds were detected using GC-EIMS analysis and were

contained in fractions A and B (obtained by HPLC separation) of a cell extract hydrolysate

(first hydrolysis step 200mg assays)

Peak

Rt EIMS

IsomeraPortionb

(min) mz (abundance in ) ()

Fraction 1

Ic 1865 380 (2) 365 (1) 351 (8) 275 (1) 261 (33)

233 (1) 221 (100) 206 (3) 193 (6) 191 (5)

179 (5) 151 (2) 117 (33) 73 (43)

unknown 214c

II 1870 380 (3) 365 (1) 351 (7) 275 (1) 261 (34)

233 (2) 221 (100) 206 (3) 193 (7) 191 (8)

179 (8) 163 (2) 151 (3) 117 (29) 73 (49)

60-OH-

4-353-NP

214c

III 1874 380 (2) 365 (1) 351 (5) 275 (1) 261 (34)

233 (1) 221 (100) 206 (4) 193 (6) 191 (6)

179 (8) 163 (2) 151 (3) 117 (32) 73 (50)

60-OH-

4-353-NP

175

IVd 1892 380 (2) 365 (1) 351 (6) 275 (1) 261 (36)

233 (2) 221 (100) 206 (4) 193 (7) 191 (6)

179 (8) 163 (2) 151 (3) 117 (27) 73 (52)

60-OH-

4-353-NP

473d

V 1896 380 (2) 365 (1) 351 (6) 275 (1) 261 (37)

233 (1) 221 (100) 206 (3) 193 (7) 191 (6)

179 (8) 163 (2) 151 (3) 117 (26) 73 (45)

60-OH-

4-353-NP

473d

VI 1925 380 (2) 365 (1) 351 (5) 336 (1) 309 (6) 295

(1) 261 (9) 233 (4) 221 (100) 206 (2) 193

(31) 191 (7) 179 (12) 163 (2) 151 (4) 147

(7) 117 (4) 103 (11) 73 (60)

10-OH-

4-353-NP

52

VII 1947 380 (2) 365 (1) 351 (5) 295 (2) 261 (8) 253

(3) 233 (3) 221 (100) 207 (4) 193 (21) 191

(6) 179 (10) 163 (6) 151 (2) 117 (3) 103

(4) 73 (47)

20-OH-

4-353-NP

59

VIII 2019 394 (2) 379 (4) 365 (11) 247 (2) 313 (4)

275 (2) 247 (4) 233 (33) 221 (100) 207 (2)

193 (11) 191 (4) 179 (8) 173 (9) 131 (3)

116 (3) 97 (6) 73 (40)

70-COOH-

4-353-NP

26

Fraction 2

IX 1629 292 (5) 277 (3) 263 (18) 221 (100) 207 (7)

193 (55) 179 (14) 163 (3) 151 (6) 135 (2)

129 (3) 115 (4) 103 (3) 91 (3) 73 (34)

4-353-NP 468

X 1638 292 (5) 277 (3) 263 (19) 221 (100) 207 (8)

193 (55) 179 (13) 163 (2) 151 (6) 135 (2)

129 (4) 115 (3) 103 (1) 91 (3) 73 (31)

4-353-NP 532

aStructures of metabolites IndashVIII of 4-353-NP are proposed according to EIMS fragmentation

patternsbCalculated from peak areas total area of peaks IndashVIII and IXndashX respectively corresponds

with 100cPeak I appeared as shoulder of II separate integration of peaks I and II was not possibledPeak IV appeared as shoulder of V separate integration of peaks IV and V was not possible

542 Schmidt et al

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179 and 73 complete mass spectra are listed in Table 2 The [Mthorn] of compoundsIndashVII pointed to mono-hydroxylated derivatives of 4-353-NP while all patterns offragmentation and their similarity with that of 4-353-NP (especially peaks at mz 221and 179) indicated that hydroxylation had exclusively occurred at the aliphatic side-chain Compound VIII had an [Mthorn] at mz 394 and prominent fragments at mz 221(base peak) 179 and 73 These findings pointed a (side-chain) carboxylic acidderivative of 4-353-NP With both fraction 1 and fraction 2 GC-EIMS peakscorresponding with possible ring-hydroxylated or side-chain olefinic products[24]

were not detected The hydrolysate derived from the soybean experiment wassimilarly separated by HPLC a fraction corresponding with primary 4-353-NPmetabolites was collected and examined by GC-EIMS after silylation Due tolow yield no distinct peaks which could be related to parent 4-353-NP were foundin the corresponding chromatograms as such However using the GC-EIMSfindings of the A githago study a trace was identified at Rt 1905min the massspectrum of which corresponded with compounds IV or V We concluded that theA githago and soybean cells at least demonstrated certain similarities concerningthe biotransformation of 4-353-NP

Though the GC-EIMS data unequivocally proved that 4-353-NP was metabo-lized by A githago cell suspensions at the aliphatic side-chain to monohydroxylatedproducts and a carboxylic acid derivative the sites of hydroxylationoxidation partlyremained unclear Compounds IIndashV emerged in the chromatograms as two pairs ofpeaks (IIIII and IVV) and exhibited noticeably similar mass spectra (Table 2)Besides [Mthorn] at mz 380 and fragments observed with all other monohydroxylatedmetabolites (mz 365 351 261 221 179 73) a prominent fragment of compoundsIIndashV hadmz 117 with 26ndash32 abundance According to data published on side-chain

Figure 5 Proposed MS fragmentation of 4(3050-dimethyl-30-heptyl)-phenol

Metabolism of the Nonylphenol Isomer 543

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monohydroxylated derivatives of both 4-n-nonylphenol[1224] and model side-chainisomers 2- 3- and 4-(4-hydroxyphenol)-nonane[4] fragment mz 117 was referred to[H3CCHOSi(CH3)3

thorn] This fragment is characteristic of trimethylsilyl derivatizedhydroxyl functions at secondary carbon atoms adjacent to terminal CH3

groups[4122428] Due to abundances (34ndash37) of fragments mz 261 (-HOSi(CH3)3-C2H5)

[2429] it was supposed that compounds IIndashV were 60-hydroxy derivatives of4-353-NP Applied 4-353-NP consisted of two diastereomers (each a couple ofenatiomers) hydroxylation at position 60 consequently introduced a third chiral centerwhich resulted in four diastereomers An MS fragmentation scheme for IIndashV isproposed in Fig 6 Quantitative evaluation of the chromatogram showed thatcompounds IIndashV were the main primary metabolites in A githago cells amountingto about 80 of the products identified

The mass spectrum of compound VI (Table 2) differed from those of productsIIndashV in particular regarding three fragments mz 309 (abundance 6) 295 (1) and103 (11) According to previous results presented on 4-n-nonylphenol[24] thefragment mz 103 was supposed to arise from [H2COSi(CH3)3

thorn] pointing tohydroxylation at a primary carbon[2428] Furthermore fragments mz 309 and 295indicated loss of C5H11 and C6H13 respectively from [Mthorn] (mz 380) with retentionof two silylated hydroxy functions at the remaining fragment ion It was thusassumed that compound VI was a 4-353-NP metabolite hydroxylated at position 10

of the side-chain Due to a similar line of argumentation (presence of fragments mz117 and 295) compound VII possibly consisted of the 20-hydroxy derivative of4-353-NP The structure of compound I remained unknown mainly becausemetabolite I was detected as shoulder of II resulting in large overlapping of its mass

Figure 6 Proposed MS fragmentation of the 6-hydroxy metabolites of 4(3050-dimethyl-

30-heptyl)-phenol

544 Schmidt et al

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spectrum with that of II Due to its [Mthorn] of mz 394 compound VIII was identifiedwith a carboxylic acid metabolite of 4-353-NP since formation of a hydroxyderivative with an additional carbonyl function was regarded unlikely The massspectrum observed with VIII supported this assumption Fragments showingsimilarity to compounds IndashVII were mz 379 (-CH3) and 365 (-C2H5) whereasfragments mz 131 and 116 were supposed to consist of frac12CH2COOSiethCH3THORN

thorn3 and

frac12CH2COOSiethCH3THORNthorn2 respectively which pointed to a carboxyl function at positions

10 or 70 A characteristic feature of the fragmentation of trimethylsilyl derivatizedfatty acids is separation of CH3 (15) and HOSi(CH3)2 (75) consecutively[30]

With VIII this would result in fragment mz 304 which was only present in thebackground However ions resulting from successive fragmentation of mz 304were observed mz 275 (-HCO -29) 247 (-C2H428) 233 (-CH214) and finally221 This sequence was regarded as prove that 4-353-NP was oxidized to itscarboxylic acid at position 70 of the molecule It should be mentioned that I VI VIIand VIII were only minor metabolites formed from 4-353-NP by the A githago cellsuspensions (Table 2)

Metabolism of 4-353-NP by Agrostemma githagoPlant Cell Suspension Cultures

The nonylphenol isomer 4-353-NP consisting of two diastereomers wastransformed by theAgrostemma githago suspension cultures to four types of productsglycosides of parent 4-353-NP glycosides of primary 4-353-NP metabolitesnonextractable residues and unknown possibly polymeric materials detected in themedia Similar products emerged in the soybean cultures The present investigationconcentrated on the primary metabolites of 4-353-NP These were produced in the Agithago suspensions using a two-liquid-phase system The primary products identifiedby GC-EIMS after liberation from their glycosides were one side-chain carboxylicacid and seven side-chain monohydroxylated metabolites Due to their mass spectrathe main products were four diastereomers of 60-hydroxy-4-353-NP

As yet data on the biotransformation of 4-353-NP are mainly not at handConcerning plant tissues (cell cultures plants) side-chain hydroxylated metaboliteswere reported on 4-n-nonylphenol[11122324] With A githago cells the main productwas 4-(80-hydroxy-n-nonyl)-phenol[24] ie 4-n-nonylphenol was metabolized bysubterminal oxidation corresponding with the main products observed with 4-353-NP in the present study Side-chain hydroxylated metabolites were also formed fromp-tert-octylphenol in barley plants[31] Experiments with model nonylphenols 2- 3-and 4-(4-hydroxyphenyl)-nonane demonstrated that in rainbow trout all isomerswere transformed to monohydroxy products with the site of oxidation at carbon 8(subterminal) of the nonyl residue The metabolites emerged as glucuronic acidconjugates while glucuronides of the parent nonylphenols were also formed[4] In asimilar study both side-chain and ring hydroxylated metabolites of 4-n-nonylphenolwere detected as glucuronic acid conjugates[9] Recently data on the metabolism ofthe nonylphenol isomer 4(3060-dimethyl-30-heptyl)-phenol in pond snails to a (ringhydroxylated) catechol derivative (with unmodified side-chain) were reported[1920]

Contrasting with 4-353-NP of the present study the isomer examined possessed

Metabolism of the Nonylphenol Isomer 545

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a branching at subterminal position 60 of the side-chain besides that at carbon 30 Thecatechol was found conjugated to glucuronic acid[1920] As technical productnonylphenol appears to be completely degradated in soil[1] though isomer 4-(3060-dimethyl-30-heptyl)-phenol proved to be resistant in sedimentwater systems[18] Upto now the routes of nonylphenolrsquos microbial degradation are not known in detailDue to experiments with an isolated bacterial strain initial oxidation at the aromaticmoiety leads to degradation of the phenyl ring while side-chains remain asnonanols[3233] one of the studies was executed with radio-labelled 4-353-NP[33]

The present data on the metabolism of 4-353-NP by A githago cell suspensionsagree with those reported in the literature on 4-n-nonylphenol in plant tissues andmodel isomers in rainbow trout In contrast with trout the capacity of the A githagocells for hydroxylation of the side-chain appeared to be broader ie the nonyl residuewas hydroxylated at different sites of the aliphatic chain The enzymes responsible forside-chain hydroxylation of 4-353-NP in A githago are not known we suspectedcytochromes P450 In rats and humans isozyme CYP2B2 was thought to catalyzepredominantly the hydroxylation of nonylphenol isomers[10] After expression inyeast human CYP2B6 and CYP2C19 were shown to metabolize nonylphenol (pluralbranched chains) while 4-n-nonylphenol was transformed by CYP1A2 CYP2B6 andCYP2C19 ring and side-chain hydroxlated products were identified with branchedchain nonylphenols and 4-n-nonylphenol[34] We assume that plants in general havethe capacity to transform nonylphenol isomers to side-chain hydroxylated productsand thus contribute to the environmental degradation of the xenoestrogen If cropplants are contaminated with nonylphenol both nonylphenol and its side-chainhydroxylated products are expected to be present in plant tissues as glycosideconjugates Xenobiotic glycosides are known to be cleaved in the gastrointestinal tractwith release of possibly toxic aglycons Data on the toxicological properties of side-chain hydroxylated nonylphenols are not available Our future research will focus onthe metabolism of nonylphenol isomers with different branching patterns on theidentification of the enzymes responsible for the metabolism of nonylphenols and onthe (eco-) toxicology of side-chain hydroxylated nonylphenol isomers

ACKNOWLEDGMENTS

For the synthesis of [ring-U-14C]-4(3050-dimethyl-30-heptyl)-phenol the authorswish to thank R Vinken and the lsquolsquoAachen Graduate College Elimination ofEndocrine-Disrupting Substances from Waste-Waterrsquorsquo (AGEESA) funded by theDeutsche Forschungsgemeinschaft

REFERENCES

1 Staples CA Williams JB Blessing RL Varineau PT Measuring thebiodegradability of nonylphenol ether carboxylates octylphenol ether carboxyl-ates and nonylphenol Chemosphere 1999 38 (9) 2029ndash2039

2 Thiele B Gunther K Schwuger MJ Alkylphenol ethoxylates trace analysisand environmental behaviour Chem Rev 1997 97 (8) 3247ndash3272

546 Schmidt et al

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3 Wheeler TF Heim JR LaTorre MR Janes AB Mass spectral

characterization of p-nonylphenol isomers using high-resolution capillary

GC-MS J Chromatogr Sci 1997 35 (1) 19ndash304 Meldahl AC Nithipatikom K Lech JJ Metabolism of several 14C-

nonylphenol isomers by rainbow trout (Oncorhynchus mykiss) in vivo and

in vitro Metabolites Xenobiotica 1996 26 (11) 1167ndash11805 Kim Y-S Katase T Sekine S Inoue T Makino M Uchiyama T

Fujimoto Y Yamashita N Variation in estrogenic activity among fractions

of a commercial nonylphenol by high performance liquid chromatography

Chemosphere 2004 54 (8) 1127ndash11346 Giger W Stephanou E Schaffner C Persistent organic chemicals in sewage

effluents 1 Identification of nonylphenols and nonylphenolethoxylates by

capillary gas chromatographymass spectrometry Chemosphere 1981 10 (11ndash1)

1253ndash12637 Giger W Brunner PH Schaffner C 4-Nonylphenol in sewage sludge

accumulation of toxic metabolites from nonionic surfactants Science 1984

225 (4662) 623ndash6258 Jobst H Chlorophenols an nonylphenols in sewage sludges 1 Occurrence in

sewage sludges of Western German treatment plants from 1987ndash1989 Acta

Hydrochim Hydrobiol 1995 23 (1) 20ndash259 Coldham NG Sivapathasundaram SDM Ashfield LA Pottinger TG

Biotransformation tissue distribution and persistence of 4-nonylphenol

residues in juvenile rainbow trout (Oncorhynchus mykiss) Drug Metab

Dispos 1998 26 (4) 347ndash35410 Lee PC Marquardt M Lech JJ Metabolism of nonylphenol by rat and

human microsomes Toxicol Lett 1998 99 (7) 117ndash12611 Bokern M Harms HH Toxicity and metabolism of 4-n-nonylphenol in cell

suspension cultures of different plant species Environ Sci Technol 1997

31 (7) 1849ndash185412 Bokern M Nimtz M Harms HH Metabolites of 4-n-nonylphenol in wheat

cell suspension cultures J Agric Food Chem 1996 44 (4) 1123ndash112713 Guenther K Heinke V Thiele B Kleist E Prast H Raecker T

Endocrine disrupting nonylphenols are ubiquitous in food Environ Sci

Technol 2002 36 (8) 1676ndash168014 Soto AM Justicia H Wray JW Sonnenschein C p-Nonylphenol an

estrogenic xenobiotic released from lsquolsquomodifiedrsquorsquo polystyrene Environ Health

Perspect 1991 92 167ndash17315 Degen DH Bolt HM Comments on lsquolsquoendocrine disrupting nonylphenols

are ubiquitous in foodrsquorsquo Envirn Sci Technol 2003 37 (11) 2622ndash262316 Vinken R Schmidt B Schaffer A Synthesis of tertiary 14C-labelled

nonylphenol isomers J Label Compd Radiopharm 2002 45 (14) 1253ndash126317 Lalah JO Lenoir D Henkelmann B Hertkorn N Gunther K

Schramm K-W Kettrup A Synthesis of ring-14C-labelled 4(3060-dimethyl-

30-heptyl)-phenol J Label Compd Radiopharm 2001 44 (6) 459ndash46318 Lalah JO Schramm K-W Henkelmann B Lenoir D Behechti A

Gunther K Kettrup A The dissipation distribution and fate of a branched

Metabolism of the Nonylphenol Isomer 547

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14C-nonylphenol isomer in lake watersediment systems Environ Pollut 2003

122 (2) 195ndash20319 Lalah JO Schramm K-W Severin GF Lenoir D Henkelmann B

Behechti A Gunther K Kettrup A In vivo metabolism and organ

distribution of a branched 14C-nonylphenol isomer in pond snails Lymnaea

stagnalis L Aquat Toxicol 2003 62 (4) 305ndash31920 Lalah JO Behechti A Severin GF Lenoir D Gunther K Kettrup A

Schramm K-W The Bioaccumulation and fate of a branched 14C-

p-nonylphenol isomer in Lymnaea stagnalis L Environ Toxicol Chem 2003

22 (7) 1428ndash143621 Schmidt B Metabolic profiling using plant cell suspension cultures In

Pesticide Transformation in Plants and Microorganisms Hall JC Hoagland

RE Zablotowicz RM Eds American Chemical Society Washington DC

2001 40ndash5622 Komoszliga D Langebartels C Sandermann H Metabolic processes for

organic chemicals in plants In Plant ContaminationmdashModeling and Simulation

of Organic Chemical Processes Trapp S Mc Farlane JC Eds Lewis

Publishers Boca Raton 1995 69ndash10323 Schmidt B Schuphan I Metabolism of the Xenoestrogens 4-n-nonylphenol

and bisphenol A in plant cell suspension cultures In Book of Abstracts 10th

IUPAC International Congress on the Chemistry of Crop Protection Basel

August 4ndash9 IUPAC Basel 2002 Vol 2 3424 Schmidt B Patti H Niewersch C Schuphan I Biotransformation of [ring-

U-14C]4-n-nonylphenol by Agrostemma githago cell culture in a two-liquid-

phase system Biotechnol Lett 2003 25 (16) 1375ndash138125 Murashige T Skoog F Revised medium for rapid growth and bio assay with

tobacco tissue cultures Physiol Plant 1962 15 473ndash49726 Gamborg OL Miller RA Ojima K Nutrient requirements of suspension

cultures of soybean root cells Exp Cell Res 1968 50 151ndash15827 Schmidt B Schuphan I Metabolism of the environmental estrogen bisphenol

A by plant cell suspension cultures Chemosphere 2002 49 (1) 51ndash5928 Diekman J Thomson JB Djerassi C Mass spectrometry and stereo-

chemical problems CLV Electron impact induced fragmentations and

rearrangements of some trimethylsilyl ethers of aliphatic glycols and related

compounds J Org Chem 1968 33 (6) 2271ndash228429 Diekman J Thomson JB Djerassi C Mass spectrometry and stereo-

chemical problems CXLI Electron impact induced fragmentations and

rearrangements of trimethylsilyl ethers amines and sulfides J Org Chem

1967 32 (12) 3904ndash391930 Odham G Fatty acids In Biochemical Applications of Mass Spectrometry

Waller GR Dermer OC Eds Wiley-Interscience New York 1980 First

Supplementary Volume 153ndash17131 Stolzenberg GE Olson PA Zaylskie RG Mansager ER Behavior and

fate of ethoxylated alkylphenol nonionic surfactant in barley plants J Agric

Food Chem 1982 30 (4) 637ndash644

548 Schmidt et al

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32 Tanghe T Dhooge W Verstraete W Isolation of a bacterial strain able todegrade branched nonylphenol Appl Environ Microbiol 1999 65 (2)746ndash751

33 Corvini PFX Vinken R Hommes G Schmidt B Dohmann MDegradation of the radioactive and non-labelled branched 4(3050-dimethyl-30-heptyl)-phenol nonylphenol isomer by Sphingomonas TTNP3 Biodegradation2003 15 (1) 1ndash10

34 Inui H Shiota N Motoi Y Ido Y Inoue T Kodama T Ohkawa YOhkawa H Metabolism of herbicides and other chemicals in humancytochrome P450 species and in transgenic potato plants co-expressinghuman CYP1A1 CYP2B6 and CYP2C19 J Pestic Sci 2001 26 (1) 28ndash40

Received February 13 2004

Metabolism of the Nonylphenol Isomer 549

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The world-wide interest in the (environmental) fate of nonylphenol originatesfrom its proven estrogenic activity the compound is considered as xenoestrogenor as an endocrine disrupter[781314] Consequently nonylphenol may affect bothhuman health and ecosystems Regarding human health the risk associated withlow-level concentrations of nonylphenol in foodstuffs however is discussedcontroversially[1315] Early research on the environmental and metabolic fate ofnonylphenol eg in plant tissues[1112] was mainly performed with the straight-chain 4-n-nonylphenol due to the unavailability of individual isomers of theindustrial productmdashespecially in 14C-labelled form However 4-n-nonylphenolappears to be no or only a trace constituent of industrial nonylphenol Thusincreasing efforts in the last years aimed at the synthetic preparation of definedbranched-chain nonylphenol isomers (preferentially also 14C-labelled) and the studyof their environmental fate and effects[516ndash20] Up to now data on the metabolism ofbranched-chain nonylphenols in plant tissues are not at hand

Plant cell cultures are often used to examine the metabolism of xenobiotics inplant tissues[2122] The studies executed prior or parallel to experiments with intactplants render possible the rapid identification of metabolites of interest So recentlywe studied the metabolism of 4-n-nonylphenol in plant cell suspension cultures andpresented the results as a poster[23] The studies were performed according to astandardized procedure[21] with 1mgL1 of the 14C-labelled xenobiotic (applied inmethanol) and 2 days of incubation using a one-liquid-phase system Since GC-EIMSidentification of the primary metabolites of 4-n-nonylphenol was not sufficientlyclear-cut we intended to produce higher concentrations and amounts of the metabolicproducts The low aqueous solubility of nonylphenols in general however raised thequestion of an alternative mode of application Recently we thus could demonstratethat considerable amounts of 4-n-nonylphenol dissolved in an n-hexadecane layerwere absorbed and metabolized by suspended cultivated cells of Agrostemma githago(corn cockle) in a satisfactory manner the n-hexadexane layer representing theorganic phase of a two-liquid-phase system affected plant cell growth only little[24]

In the present article we report data on the metabolism of the 14C-labellednonylphenol isomer 4-(3050-dimethyl-30-heptyl)-phenol (Fig 1) by cell suspensioncultures of soybean and A githago The isomer (as diastereomeric mixture) wassynthesized according to a published procedure[16] Due to GC-EI (retention timesmass spectra) the isomer is an important constituent of industrially producednonylphenol The preliminary metabolism experiment with the soybean suspensionswas performed as conventional one-liquid-phase study[21] In order to producehigher amounts and concentrations of the primary metabolites of 4-(3050-dimethyl-30-heptyl)-phenol for their identification (GC-EIMS) the subsequent study withA githago suspension cultures was executed as two-liquid-phase experiment using ann-hexadecane layer as organic phase as described before for 4-n-nonylphenol[24]

Figure 1 Chemical structure of 4(3050-dimethyl-30-heptyl)-phenol (4-353-NP)

Metabolism of the Nonylphenol Isomer 535

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MATERIALS AND METHODS

Chemicals

The nonylphenol isomer [ring-U-14C]-4-(3050-dimethyl-30-heptyl)-phenol (4-353-NP) shown in Fig 1 was synthesized as described[16] the specific activity of thecompound was 298MBqmmol1 its radiochemical purity 969 Nonlabelled4-353-NP was synthesized accordingly Both 14C-labelled and nonlabelled 4-353-NPconsisted of a diastereomeric mixture (47555245 GC-EIMS see below) Forthe metabolism studies the specific activity of the radiochemical was adjustedappropriately by addition of nonlabelled 4-353-NP n-Hexadecane was supplied byAldrich (Deisenhofen Germany)

Plant Cell Cultures and Treatments

Cell suspension cultures of Agrostemma githago L were initiated and grown in20mL of MS medium contained in 100mL Erlenmeyer flasks as described[2125]

Three days after subcultivation three of the cultures were treated with 100 mg(28 kBq) and three were treated with 200 mg (28 kBq) of [ring-U-14C]-4-353-NP perassay With all assays the radiochemical was dissolved in 100 mL of n-hexadecanewhich constituted the organic part of the two-liquid-phase system (mediumn-hexadecane 2001 vv) The assays were incubated for 7 days until the end ofthe subcultivation interval of the cultures Soybean (Glycine max L Merr cvMandarin) cell suspension cultures utilized in the preliminary conventional one-liquid-phase study were maintained in B5 medium as described[2126] Two daysbefore the end of the subcultivation interval the cultures (5 parallels) were treatedwith 20 mg (1mgL1 30 kBq) of [ring-U-14C]-4-353-NP per assay and wereincubated for 48 h

Extraction of Assays

The assays (A githago) were individually transferred to centrifuge tubes and10mL of n-hexane was added After centrifugation (15min 5000 g) the organicphase containing the n-hexadecane layer was removed Remaining aqueous phases(media) were separated from the cells by suction filtration The cells were suspendedin chloroformmethanol 12 (vv) stored at 20C for 16 h and were extractedby means of sonication (Bandelin Berlin Germany) Insoluble cell debris wereseparated from the cell extracts by suction filtration and washed with chloroformmethanolwater 1208 (vvv) Air-dried filter papers with adhering cell debris weresubjected to combustion analysis (Biological Oxidizer OX 500 ZinsserHarveyInstruments Frankfurt Germany) The individual cell extracts were examined byTLC corresponding parallels were combined and concentrated After examinationby TLC media were discarded Excepting the separation of the n-hexadecanephase the assays of the experiment with soybean cells were similarly extracted andexamined

536 Schmidt et al

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Hydrolytic Treatments

Concentrated and combined cell extracts (100 and 200 mg assays) derivedfrom the study with A githago were refluxed in 40mL of 2M HClmethanol 11 (vv)for 2 h The methanol portion of the reaction mixture was evaporated and theremaining HCl phase was extracted with diethyl ether Recoveries of 14C in theextracts were 45 and 46 with the 100 and 200 mg assays respectively based on 14Cfound in corresponding combined cell extracts Remaining HCl phases wereconcentrated and hydrolyzed for 2 h using 2M HCl at reflux (recoveries 25 and17 respectively) All diethyl ether extracts (hydrolysates) obtained from thehydrolytic treatments were analyzed by TLC and HPLC Subsequently theradioactivity contained in the first hydrolysate derived from the 200 mg assays wasseparated by preparative HPLC and isolated fractions were examined by GC-EIMSThe combined cell extracts of the experiment with soybean cells were similarilytreated The hydrolysates (total recovery 86) however were only subjected byTLC and HPLC analyses

Analytical and Chromatographic Procedures

Liquid scintillation counting (LSC) was performed using a LS 5000 TD analyzer(Beckmann Munchen) and LumasafeTM Plus and CarbomaxTM Plus (Canberra-Packard Dreieich Germany) scintillation cocktails

Thin-layer chromatography (TLC) was performed on silica gel plates SIL G-25UV254 (025mm Macherey-Nagel Duren Germany) The solvent systems used wereas follows A n-hexanediethyl etheracetic acid 50501 (vvv) B n-hexanediethyletheracetic acid 20801 (vvv) C ethyl acetateiso-propanol 4060 (vv) D ethylacetateiso-propanolH2O 652412 (vvv) Separated 14C peaks were located andquantified by means of a Tracemaster 40 radiochromatogram scanner (BertholdWildbad Germany) nonlabelled 4-353-NP was visualized under UV at 254 nm

High-performance liquid chromatography (HPLC) was executed on a SystemGold Personal chromatograph (Beckman Munchen Germany) consisting ofProgrammable Solvent Module 126 Diode Array Detector Modul 168 andRadioisotope Detector 171 The latter was equipped with a 2420 quartz cell(silanized glass 20ndash30 mm internal diameter 55mm cell volume 037mL RaytestStraubenhardt Germany) All HPLC analyses were performed on a reversed phasecolumn (CC 2504 Nucleosil 100-5 C18 HD Macherey-Nagel Duren Germany) at20C using a flow of 1mLmin1 Nonlabelled 4-353-NP was detected at 280 nmElution was performed with solvents A (01 acetic acid in water vv) and B (01acetic acid in acetonitrile vv) as follows AB (6535 vv) for 5min then linear30min gradient to 100 B isocratic B (100) for 5min and return to initialconditions in 5min All analyses were terminated by washing the column with AB(6535 vv) for 5min

For GC-EIMS analysis (gas chromatography-electron impact mass spectrom-etry) samples were concentrated using an N2 stream and derivatized with 100 mL ofN-methyl-N-trimethyl-silyltrifluoroacetamide (MSTFA Fluka Buchs Switzerland)at 70C for 30min GC-EIMS was executed with a Hewlett-Packard 5890 Series II

Metabolism of the Nonylphenol Isomer 537

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gas chromatograph (Agilent Waldbronn Germany) equipped with a FS-SE-54-NB-05 column (25m 025mm film thickness 046 mm CS Chromatographie ServiceLangerwehe Germany) and carrier gas helium injection volume was 1 mL The gaschromatograph was connected to a Hewlett-Packard 5971 A MSD (mass selectivedetector) which was operated in scan mode (mass rangemz 50ndash480) with an electronenergy of 70 eV Temperature programming was isothermal 85C for 5min 85ndash280Cat 10Cmin1 and isothermal for 3min injector and interface temperatures were250ndash280C respectively

RESULTS AND DISCUSSION

Distribution of Radioactivity

The distributions of 14C found after termination of the metabolism experimentsare shown in Table 1 Concerning the preliminary study with soybean the majorityof 14C was present in the media (357of applied radioactivity) while 299 and 203were detected in cell extracts and insoluble cell debris respectively In contrastmore than 60 of applied 14C were found in the cell extracts derived from the two-liquid-phase study performed with Agrostemma githago cells Portions in mediaand nonextractable residues were below 20 only traces of 14C were left in then-hexadecane phase Individual analyses with TLC (systems A and B) demonstratedthat in the media and cell extracts of the soybean assays free 4-353-NP was only

Table 1 Distribution of radioactivity in Agrostemma githago cell culture afters 7 days

of incubation with 100 mg (5mgL1 28 kBq 3 parallels) or 200 mg (10mgL1 28 kBq

3 parallels) of [ring-U-14C]-4-353-NP per assay in two-liquid-phase system and soybean cell

culture (5 parallels) after 2 days of incubation with 20mg (1mgL1 30 kBq) of [ring-U-14C]-

4-353-NP per assay in conventional one-liquid-phase system (mean values are displayed)

Soybean A githago A githago

20mg assays 100mg assays 200 mg assays

Fraction of applied 14C of applied 14C of applied 14C

n-Hexadecane phase mdash 24 28

Medium 357 151 17414C-residues on filter papera mdashb 51 35

Cell extract 299 639 603

Nonextractable residues 203 118 141

Recovered radioactivity 859 983 981

Recovered 4-353-NPc 18 00 00

Primary metabolitesd 19ndash22 28ndash42 21ndash33

aFilter papers used to separate cells and mediabFilter papers were not examinedcPortions were determined from results of TLC analysis of cell extracts and mediadPortions were estimated from results of TLC and HPLC analyses of hydrolysates obtained

from hydrolytic treatments of combined cell extracts (single values are shown)

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present in trace amounts free 4-353-NP however was completely absent in theassays with the A githago cells The chromatographic behavior (TLC systems AndashD)of the radioactivity contained in all cell extracts pointed to glycosides as metabolicproducts of 4-353-NP With all TLC systems used the radioactivity associated withthe media proved to be preponderantly immobile Extraction of these 14C portionswith organic solvents such as ethyl acetate was not successful the same held true forhydrolysis procedures in order to cleave possible glycosidic linkages Additionallydirect HPLC analysis was not practicable since the necessary concentration of themedia resulted in highly viscous samples

The results obtained with the A githago cells were comparable to data publishedrecently on the biotransformation of 4-n-nonylphenol by the same cell cultureusing the same two-liquid-phase system[24] Concerning the intended identification ofsolublemetabolites uptake of 4-353-NP from the n-hexadecane layer by theA githagocells was considered satisfactory with both the 100 and 200 mg assays The same heldfor recoveries of 14C noticeable portions of soluble 14C in cell extracts and completedisappearance (turnover) of the applied 4-353-NP The different distribution ofradioactivity observed in the soybean experiment was referred to the conventionalone-liquid-phase system used but additionally also to the plant species[23] With bothplant species examined formation of glycosides from the phenolic xenobiotic orpossible primary transformation products was expected according to data publishedon 4-n-nonylphenol[11122324] The chemical nature of the radioactivity present inthe media especially of the soybean cells remained unknown Recently we found14C materials with similar (chromatographic) behavior in the media of plantcell suspensions (including soybean) after application of 14C-bisphenol A andsuspected polymerized products of the compound[27] Polymeres (or copolymers)may also have been formed from 4-353-NP

Hydrolysis Experiments Isolation and Identification of Metabolites

The combined cell extracts were subjected to two consecutive hydrolysissteps (2M HCl-methanol reflux 2M HCl reflux) in order to release the expected14C-aglycons from their corresponding glycosides In the case of soybean 86 of theradioactivity introduced into these experiments could be extracted (diethyl ether)from the reaction mixture With A githago recoveries in the hydrolysates amountedto totals of 70 (100 mg assays) and 63 (200 mg assays) All hydrolysates wereexamined using TLC and HPLC The results of TLC analysis of the samples(hydrolysis steps 1 and 2) derived from the A githogo study (Fig 2) demonstratedthat in three of the samples noticeable portions of 4-353-NP (Rf about 09) werepresent whereas in all samples 14C peaks at Rf about 05 and Rf 00 were detectedHPLC examination of the hydrolysates prevailingly confirmed these data radio-chromatograms of the samples derived from hydrolysis step 1 are shown in Fig 3In addition to a peak originating from 4-353-NP (Rt 310min) further 14C peaksappeared at Rt 150ndash200min Since the 14C peaks with Rf about 05 (TLC) and Rt

150ndash200min (HPLC) were not detected prior to hydrolysis the correspondingcompounds were regarded as primary metabolites of 4-353-NP Both 4-353-NP andprimary metabolites of 4-353-NP were thus liberated from their glycosides by acid

Metabolism of the Nonylphenol Isomer 539

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treatment Similar results were obtained from the soybean experiment (data notshown) Estimated from TLC and HPLC analysis the portions of primarymetabolites of 4-353-NP were between 19 and 42 of applied 14C (Table 1)depending on the method of calculation Low amounts of primary 4-353-NPmetabolites were detected with soybean this fact additionally supported theutilization of A githago cells in two-liquid-phase system for production andidentification of these transformation products

Figure 2 TLC radiochromatograms (solvent system B) of the samples derived from the

hydrolysis experiments A hydrolysis step 1 100 mg assays B hydrolysis step 1 200mg assays

C hydrolysis step 2 100 mg assays D hydrolysis step 2 200mg assays

Figure 3 HPLC radiochromatograms of samples derived from the hydrolysis experiments

A hydrolysis step 1 100mg assays B hydrolysis step 1 200 mg assays

540 Schmidt et al

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The radioactivity contained in the hydrolysate of the first hydrolytic step derivedfrom the 200 mg assays was separated by HPLC (using multiple runs) Two fractionswere isolated fraction 1 corresponding with the 14C peaks at Rt 150ndash200min andfraction 2 (4-353-NP Rt 310min) Total yields recovered after concentration ofHPLC eluates were 19 and 7mg with fraction 1 and fraction 2 respectively Bothfractions were derivatized with MSTFA (trimethylsilyl) and subjected to GC-EIMSanalyses the corresponding chromatograms are shown in Fig 4 The chromatogramof fraction 2 exhibited two peaks (IX and X Rt 1620ndash1640min) which accordingto their retention times and mass spectra (Table 2) were identified with the twodiastereomers of parent 4-353-NP While the [Mthorn] peak was observed at mz 292further prominent fragments emerged at mz 263 (-C2H5) 221 (-C5H11 base peak)193 179 and 73 (trimethylsilyl) According to data published on branched side-chain 4-nonylphenol isomers and 4-n-nonylphenol derivatives[2371216] the MSfragmentation scheme of 4-353-NP shown in Fig 5 is proposed The chemicalstructure of the fragment with mz 179 (trimethylsilyl derivative of hydroxyltropylium ion with mz 107) was deduced from recent literature data[3]

In the GC-EIMS chromatogram obtained from fraction 2 eight peaks weredetected (Rt 1860ndash2020min) which by means of their mass spectra could be tracedback to structures related to 4-353-NP (Fig 4) Seven of these compounds (IndashVII)exhibited an [Mthorn] at mz 380 and prominent fragments at mz 261 221 (base peak)

Figure 4 GC-EIMS chromatograms of the samples obtained from HPLC separation

(cf Fig 3) of the first hydrolysate derived from the 200 mg assays A fraction containing the

primary metabolites of 4-353-NP (peaks I and IV appeared as shoulders of II and V

respectively) B fraction containing nonmetabolized 4-353-NP

Metabolism of the Nonylphenol Isomer 541

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Table 2 Mass spectra of side-chain monohydroxylated metabolites (IndashVII) of 4-353-NP a

carboxylic acid metabolite (VII) of 4-353-NP and of 4-353-NP diastereomers (IX X) after

trimethylsilyl derivatization Compounds were detected using GC-EIMS analysis and were

contained in fractions A and B (obtained by HPLC separation) of a cell extract hydrolysate

(first hydrolysis step 200mg assays)

Peak

Rt EIMS

IsomeraPortionb

(min) mz (abundance in ) ()

Fraction 1

Ic 1865 380 (2) 365 (1) 351 (8) 275 (1) 261 (33)

233 (1) 221 (100) 206 (3) 193 (6) 191 (5)

179 (5) 151 (2) 117 (33) 73 (43)

unknown 214c

II 1870 380 (3) 365 (1) 351 (7) 275 (1) 261 (34)

233 (2) 221 (100) 206 (3) 193 (7) 191 (8)

179 (8) 163 (2) 151 (3) 117 (29) 73 (49)

60-OH-

4-353-NP

214c

III 1874 380 (2) 365 (1) 351 (5) 275 (1) 261 (34)

233 (1) 221 (100) 206 (4) 193 (6) 191 (6)

179 (8) 163 (2) 151 (3) 117 (32) 73 (50)

60-OH-

4-353-NP

175

IVd 1892 380 (2) 365 (1) 351 (6) 275 (1) 261 (36)

233 (2) 221 (100) 206 (4) 193 (7) 191 (6)

179 (8) 163 (2) 151 (3) 117 (27) 73 (52)

60-OH-

4-353-NP

473d

V 1896 380 (2) 365 (1) 351 (6) 275 (1) 261 (37)

233 (1) 221 (100) 206 (3) 193 (7) 191 (6)

179 (8) 163 (2) 151 (3) 117 (26) 73 (45)

60-OH-

4-353-NP

473d

VI 1925 380 (2) 365 (1) 351 (5) 336 (1) 309 (6) 295

(1) 261 (9) 233 (4) 221 (100) 206 (2) 193

(31) 191 (7) 179 (12) 163 (2) 151 (4) 147

(7) 117 (4) 103 (11) 73 (60)

10-OH-

4-353-NP

52

VII 1947 380 (2) 365 (1) 351 (5) 295 (2) 261 (8) 253

(3) 233 (3) 221 (100) 207 (4) 193 (21) 191

(6) 179 (10) 163 (6) 151 (2) 117 (3) 103

(4) 73 (47)

20-OH-

4-353-NP

59

VIII 2019 394 (2) 379 (4) 365 (11) 247 (2) 313 (4)

275 (2) 247 (4) 233 (33) 221 (100) 207 (2)

193 (11) 191 (4) 179 (8) 173 (9) 131 (3)

116 (3) 97 (6) 73 (40)

70-COOH-

4-353-NP

26

Fraction 2

IX 1629 292 (5) 277 (3) 263 (18) 221 (100) 207 (7)

193 (55) 179 (14) 163 (3) 151 (6) 135 (2)

129 (3) 115 (4) 103 (3) 91 (3) 73 (34)

4-353-NP 468

X 1638 292 (5) 277 (3) 263 (19) 221 (100) 207 (8)

193 (55) 179 (13) 163 (2) 151 (6) 135 (2)

129 (4) 115 (3) 103 (1) 91 (3) 73 (31)

4-353-NP 532

aStructures of metabolites IndashVIII of 4-353-NP are proposed according to EIMS fragmentation

patternsbCalculated from peak areas total area of peaks IndashVIII and IXndashX respectively corresponds

with 100cPeak I appeared as shoulder of II separate integration of peaks I and II was not possibledPeak IV appeared as shoulder of V separate integration of peaks IV and V was not possible

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179 and 73 complete mass spectra are listed in Table 2 The [Mthorn] of compoundsIndashVII pointed to mono-hydroxylated derivatives of 4-353-NP while all patterns offragmentation and their similarity with that of 4-353-NP (especially peaks at mz 221and 179) indicated that hydroxylation had exclusively occurred at the aliphatic side-chain Compound VIII had an [Mthorn] at mz 394 and prominent fragments at mz 221(base peak) 179 and 73 These findings pointed a (side-chain) carboxylic acidderivative of 4-353-NP With both fraction 1 and fraction 2 GC-EIMS peakscorresponding with possible ring-hydroxylated or side-chain olefinic products[24]

were not detected The hydrolysate derived from the soybean experiment wassimilarly separated by HPLC a fraction corresponding with primary 4-353-NPmetabolites was collected and examined by GC-EIMS after silylation Due tolow yield no distinct peaks which could be related to parent 4-353-NP were foundin the corresponding chromatograms as such However using the GC-EIMSfindings of the A githago study a trace was identified at Rt 1905min the massspectrum of which corresponded with compounds IV or V We concluded that theA githago and soybean cells at least demonstrated certain similarities concerningthe biotransformation of 4-353-NP

Though the GC-EIMS data unequivocally proved that 4-353-NP was metabo-lized by A githago cell suspensions at the aliphatic side-chain to monohydroxylatedproducts and a carboxylic acid derivative the sites of hydroxylationoxidation partlyremained unclear Compounds IIndashV emerged in the chromatograms as two pairs ofpeaks (IIIII and IVV) and exhibited noticeably similar mass spectra (Table 2)Besides [Mthorn] at mz 380 and fragments observed with all other monohydroxylatedmetabolites (mz 365 351 261 221 179 73) a prominent fragment of compoundsIIndashV hadmz 117 with 26ndash32 abundance According to data published on side-chain

Figure 5 Proposed MS fragmentation of 4(3050-dimethyl-30-heptyl)-phenol

Metabolism of the Nonylphenol Isomer 543

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monohydroxylated derivatives of both 4-n-nonylphenol[1224] and model side-chainisomers 2- 3- and 4-(4-hydroxyphenol)-nonane[4] fragment mz 117 was referred to[H3CCHOSi(CH3)3

thorn] This fragment is characteristic of trimethylsilyl derivatizedhydroxyl functions at secondary carbon atoms adjacent to terminal CH3

groups[4122428] Due to abundances (34ndash37) of fragments mz 261 (-HOSi(CH3)3-C2H5)

[2429] it was supposed that compounds IIndashV were 60-hydroxy derivatives of4-353-NP Applied 4-353-NP consisted of two diastereomers (each a couple ofenatiomers) hydroxylation at position 60 consequently introduced a third chiral centerwhich resulted in four diastereomers An MS fragmentation scheme for IIndashV isproposed in Fig 6 Quantitative evaluation of the chromatogram showed thatcompounds IIndashV were the main primary metabolites in A githago cells amountingto about 80 of the products identified

The mass spectrum of compound VI (Table 2) differed from those of productsIIndashV in particular regarding three fragments mz 309 (abundance 6) 295 (1) and103 (11) According to previous results presented on 4-n-nonylphenol[24] thefragment mz 103 was supposed to arise from [H2COSi(CH3)3

thorn] pointing tohydroxylation at a primary carbon[2428] Furthermore fragments mz 309 and 295indicated loss of C5H11 and C6H13 respectively from [Mthorn] (mz 380) with retentionof two silylated hydroxy functions at the remaining fragment ion It was thusassumed that compound VI was a 4-353-NP metabolite hydroxylated at position 10

of the side-chain Due to a similar line of argumentation (presence of fragments mz117 and 295) compound VII possibly consisted of the 20-hydroxy derivative of4-353-NP The structure of compound I remained unknown mainly becausemetabolite I was detected as shoulder of II resulting in large overlapping of its mass

Figure 6 Proposed MS fragmentation of the 6-hydroxy metabolites of 4(3050-dimethyl-

30-heptyl)-phenol

544 Schmidt et al

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spectrum with that of II Due to its [Mthorn] of mz 394 compound VIII was identifiedwith a carboxylic acid metabolite of 4-353-NP since formation of a hydroxyderivative with an additional carbonyl function was regarded unlikely The massspectrum observed with VIII supported this assumption Fragments showingsimilarity to compounds IndashVII were mz 379 (-CH3) and 365 (-C2H5) whereasfragments mz 131 and 116 were supposed to consist of frac12CH2COOSiethCH3THORN

thorn3 and

frac12CH2COOSiethCH3THORNthorn2 respectively which pointed to a carboxyl function at positions

10 or 70 A characteristic feature of the fragmentation of trimethylsilyl derivatizedfatty acids is separation of CH3 (15) and HOSi(CH3)2 (75) consecutively[30]

With VIII this would result in fragment mz 304 which was only present in thebackground However ions resulting from successive fragmentation of mz 304were observed mz 275 (-HCO -29) 247 (-C2H428) 233 (-CH214) and finally221 This sequence was regarded as prove that 4-353-NP was oxidized to itscarboxylic acid at position 70 of the molecule It should be mentioned that I VI VIIand VIII were only minor metabolites formed from 4-353-NP by the A githago cellsuspensions (Table 2)

Metabolism of 4-353-NP by Agrostemma githagoPlant Cell Suspension Cultures

The nonylphenol isomer 4-353-NP consisting of two diastereomers wastransformed by theAgrostemma githago suspension cultures to four types of productsglycosides of parent 4-353-NP glycosides of primary 4-353-NP metabolitesnonextractable residues and unknown possibly polymeric materials detected in themedia Similar products emerged in the soybean cultures The present investigationconcentrated on the primary metabolites of 4-353-NP These were produced in the Agithago suspensions using a two-liquid-phase system The primary products identifiedby GC-EIMS after liberation from their glycosides were one side-chain carboxylicacid and seven side-chain monohydroxylated metabolites Due to their mass spectrathe main products were four diastereomers of 60-hydroxy-4-353-NP

As yet data on the biotransformation of 4-353-NP are mainly not at handConcerning plant tissues (cell cultures plants) side-chain hydroxylated metaboliteswere reported on 4-n-nonylphenol[11122324] With A githago cells the main productwas 4-(80-hydroxy-n-nonyl)-phenol[24] ie 4-n-nonylphenol was metabolized bysubterminal oxidation corresponding with the main products observed with 4-353-NP in the present study Side-chain hydroxylated metabolites were also formed fromp-tert-octylphenol in barley plants[31] Experiments with model nonylphenols 2- 3-and 4-(4-hydroxyphenyl)-nonane demonstrated that in rainbow trout all isomerswere transformed to monohydroxy products with the site of oxidation at carbon 8(subterminal) of the nonyl residue The metabolites emerged as glucuronic acidconjugates while glucuronides of the parent nonylphenols were also formed[4] In asimilar study both side-chain and ring hydroxylated metabolites of 4-n-nonylphenolwere detected as glucuronic acid conjugates[9] Recently data on the metabolism ofthe nonylphenol isomer 4(3060-dimethyl-30-heptyl)-phenol in pond snails to a (ringhydroxylated) catechol derivative (with unmodified side-chain) were reported[1920]

Contrasting with 4-353-NP of the present study the isomer examined possessed

Metabolism of the Nonylphenol Isomer 545

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a branching at subterminal position 60 of the side-chain besides that at carbon 30 Thecatechol was found conjugated to glucuronic acid[1920] As technical productnonylphenol appears to be completely degradated in soil[1] though isomer 4-(3060-dimethyl-30-heptyl)-phenol proved to be resistant in sedimentwater systems[18] Upto now the routes of nonylphenolrsquos microbial degradation are not known in detailDue to experiments with an isolated bacterial strain initial oxidation at the aromaticmoiety leads to degradation of the phenyl ring while side-chains remain asnonanols[3233] one of the studies was executed with radio-labelled 4-353-NP[33]

The present data on the metabolism of 4-353-NP by A githago cell suspensionsagree with those reported in the literature on 4-n-nonylphenol in plant tissues andmodel isomers in rainbow trout In contrast with trout the capacity of the A githagocells for hydroxylation of the side-chain appeared to be broader ie the nonyl residuewas hydroxylated at different sites of the aliphatic chain The enzymes responsible forside-chain hydroxylation of 4-353-NP in A githago are not known we suspectedcytochromes P450 In rats and humans isozyme CYP2B2 was thought to catalyzepredominantly the hydroxylation of nonylphenol isomers[10] After expression inyeast human CYP2B6 and CYP2C19 were shown to metabolize nonylphenol (pluralbranched chains) while 4-n-nonylphenol was transformed by CYP1A2 CYP2B6 andCYP2C19 ring and side-chain hydroxlated products were identified with branchedchain nonylphenols and 4-n-nonylphenol[34] We assume that plants in general havethe capacity to transform nonylphenol isomers to side-chain hydroxylated productsand thus contribute to the environmental degradation of the xenoestrogen If cropplants are contaminated with nonylphenol both nonylphenol and its side-chainhydroxylated products are expected to be present in plant tissues as glycosideconjugates Xenobiotic glycosides are known to be cleaved in the gastrointestinal tractwith release of possibly toxic aglycons Data on the toxicological properties of side-chain hydroxylated nonylphenols are not available Our future research will focus onthe metabolism of nonylphenol isomers with different branching patterns on theidentification of the enzymes responsible for the metabolism of nonylphenols and onthe (eco-) toxicology of side-chain hydroxylated nonylphenol isomers

ACKNOWLEDGMENTS

For the synthesis of [ring-U-14C]-4(3050-dimethyl-30-heptyl)-phenol the authorswish to thank R Vinken and the lsquolsquoAachen Graduate College Elimination ofEndocrine-Disrupting Substances from Waste-Waterrsquorsquo (AGEESA) funded by theDeutsche Forschungsgemeinschaft

REFERENCES

1 Staples CA Williams JB Blessing RL Varineau PT Measuring thebiodegradability of nonylphenol ether carboxylates octylphenol ether carboxyl-ates and nonylphenol Chemosphere 1999 38 (9) 2029ndash2039

2 Thiele B Gunther K Schwuger MJ Alkylphenol ethoxylates trace analysisand environmental behaviour Chem Rev 1997 97 (8) 3247ndash3272

546 Schmidt et al

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3 Wheeler TF Heim JR LaTorre MR Janes AB Mass spectral

characterization of p-nonylphenol isomers using high-resolution capillary

GC-MS J Chromatogr Sci 1997 35 (1) 19ndash304 Meldahl AC Nithipatikom K Lech JJ Metabolism of several 14C-

nonylphenol isomers by rainbow trout (Oncorhynchus mykiss) in vivo and

in vitro Metabolites Xenobiotica 1996 26 (11) 1167ndash11805 Kim Y-S Katase T Sekine S Inoue T Makino M Uchiyama T

Fujimoto Y Yamashita N Variation in estrogenic activity among fractions

of a commercial nonylphenol by high performance liquid chromatography

Chemosphere 2004 54 (8) 1127ndash11346 Giger W Stephanou E Schaffner C Persistent organic chemicals in sewage

effluents 1 Identification of nonylphenols and nonylphenolethoxylates by

capillary gas chromatographymass spectrometry Chemosphere 1981 10 (11ndash1)

1253ndash12637 Giger W Brunner PH Schaffner C 4-Nonylphenol in sewage sludge

accumulation of toxic metabolites from nonionic surfactants Science 1984

225 (4662) 623ndash6258 Jobst H Chlorophenols an nonylphenols in sewage sludges 1 Occurrence in

sewage sludges of Western German treatment plants from 1987ndash1989 Acta

Hydrochim Hydrobiol 1995 23 (1) 20ndash259 Coldham NG Sivapathasundaram SDM Ashfield LA Pottinger TG

Biotransformation tissue distribution and persistence of 4-nonylphenol

residues in juvenile rainbow trout (Oncorhynchus mykiss) Drug Metab

Dispos 1998 26 (4) 347ndash35410 Lee PC Marquardt M Lech JJ Metabolism of nonylphenol by rat and

human microsomes Toxicol Lett 1998 99 (7) 117ndash12611 Bokern M Harms HH Toxicity and metabolism of 4-n-nonylphenol in cell

suspension cultures of different plant species Environ Sci Technol 1997

31 (7) 1849ndash185412 Bokern M Nimtz M Harms HH Metabolites of 4-n-nonylphenol in wheat

cell suspension cultures J Agric Food Chem 1996 44 (4) 1123ndash112713 Guenther K Heinke V Thiele B Kleist E Prast H Raecker T

Endocrine disrupting nonylphenols are ubiquitous in food Environ Sci

Technol 2002 36 (8) 1676ndash168014 Soto AM Justicia H Wray JW Sonnenschein C p-Nonylphenol an

estrogenic xenobiotic released from lsquolsquomodifiedrsquorsquo polystyrene Environ Health

Perspect 1991 92 167ndash17315 Degen DH Bolt HM Comments on lsquolsquoendocrine disrupting nonylphenols

are ubiquitous in foodrsquorsquo Envirn Sci Technol 2003 37 (11) 2622ndash262316 Vinken R Schmidt B Schaffer A Synthesis of tertiary 14C-labelled

nonylphenol isomers J Label Compd Radiopharm 2002 45 (14) 1253ndash126317 Lalah JO Lenoir D Henkelmann B Hertkorn N Gunther K

Schramm K-W Kettrup A Synthesis of ring-14C-labelled 4(3060-dimethyl-

30-heptyl)-phenol J Label Compd Radiopharm 2001 44 (6) 459ndash46318 Lalah JO Schramm K-W Henkelmann B Lenoir D Behechti A

Gunther K Kettrup A The dissipation distribution and fate of a branched

Metabolism of the Nonylphenol Isomer 547

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14C-nonylphenol isomer in lake watersediment systems Environ Pollut 2003

122 (2) 195ndash20319 Lalah JO Schramm K-W Severin GF Lenoir D Henkelmann B

Behechti A Gunther K Kettrup A In vivo metabolism and organ

distribution of a branched 14C-nonylphenol isomer in pond snails Lymnaea

stagnalis L Aquat Toxicol 2003 62 (4) 305ndash31920 Lalah JO Behechti A Severin GF Lenoir D Gunther K Kettrup A

Schramm K-W The Bioaccumulation and fate of a branched 14C-

p-nonylphenol isomer in Lymnaea stagnalis L Environ Toxicol Chem 2003

22 (7) 1428ndash143621 Schmidt B Metabolic profiling using plant cell suspension cultures In

Pesticide Transformation in Plants and Microorganisms Hall JC Hoagland

RE Zablotowicz RM Eds American Chemical Society Washington DC

2001 40ndash5622 Komoszliga D Langebartels C Sandermann H Metabolic processes for

organic chemicals in plants In Plant ContaminationmdashModeling and Simulation

of Organic Chemical Processes Trapp S Mc Farlane JC Eds Lewis

Publishers Boca Raton 1995 69ndash10323 Schmidt B Schuphan I Metabolism of the Xenoestrogens 4-n-nonylphenol

and bisphenol A in plant cell suspension cultures In Book of Abstracts 10th

IUPAC International Congress on the Chemistry of Crop Protection Basel

August 4ndash9 IUPAC Basel 2002 Vol 2 3424 Schmidt B Patti H Niewersch C Schuphan I Biotransformation of [ring-

U-14C]4-n-nonylphenol by Agrostemma githago cell culture in a two-liquid-

phase system Biotechnol Lett 2003 25 (16) 1375ndash138125 Murashige T Skoog F Revised medium for rapid growth and bio assay with

tobacco tissue cultures Physiol Plant 1962 15 473ndash49726 Gamborg OL Miller RA Ojima K Nutrient requirements of suspension

cultures of soybean root cells Exp Cell Res 1968 50 151ndash15827 Schmidt B Schuphan I Metabolism of the environmental estrogen bisphenol

A by plant cell suspension cultures Chemosphere 2002 49 (1) 51ndash5928 Diekman J Thomson JB Djerassi C Mass spectrometry and stereo-

chemical problems CLV Electron impact induced fragmentations and

rearrangements of some trimethylsilyl ethers of aliphatic glycols and related

compounds J Org Chem 1968 33 (6) 2271ndash228429 Diekman J Thomson JB Djerassi C Mass spectrometry and stereo-

chemical problems CXLI Electron impact induced fragmentations and

rearrangements of trimethylsilyl ethers amines and sulfides J Org Chem

1967 32 (12) 3904ndash391930 Odham G Fatty acids In Biochemical Applications of Mass Spectrometry

Waller GR Dermer OC Eds Wiley-Interscience New York 1980 First

Supplementary Volume 153ndash17131 Stolzenberg GE Olson PA Zaylskie RG Mansager ER Behavior and

fate of ethoxylated alkylphenol nonionic surfactant in barley plants J Agric

Food Chem 1982 30 (4) 637ndash644

548 Schmidt et al

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32 Tanghe T Dhooge W Verstraete W Isolation of a bacterial strain able todegrade branched nonylphenol Appl Environ Microbiol 1999 65 (2)746ndash751

33 Corvini PFX Vinken R Hommes G Schmidt B Dohmann MDegradation of the radioactive and non-labelled branched 4(3050-dimethyl-30-heptyl)-phenol nonylphenol isomer by Sphingomonas TTNP3 Biodegradation2003 15 (1) 1ndash10

34 Inui H Shiota N Motoi Y Ido Y Inoue T Kodama T Ohkawa YOhkawa H Metabolism of herbicides and other chemicals in humancytochrome P450 species and in transgenic potato plants co-expressinghuman CYP1A1 CYP2B6 and CYP2C19 J Pestic Sci 2001 26 (1) 28ndash40

Received February 13 2004

Metabolism of the Nonylphenol Isomer 549

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MATERIALS AND METHODS

Chemicals

The nonylphenol isomer [ring-U-14C]-4-(3050-dimethyl-30-heptyl)-phenol (4-353-NP) shown in Fig 1 was synthesized as described[16] the specific activity of thecompound was 298MBqmmol1 its radiochemical purity 969 Nonlabelled4-353-NP was synthesized accordingly Both 14C-labelled and nonlabelled 4-353-NPconsisted of a diastereomeric mixture (47555245 GC-EIMS see below) Forthe metabolism studies the specific activity of the radiochemical was adjustedappropriately by addition of nonlabelled 4-353-NP n-Hexadecane was supplied byAldrich (Deisenhofen Germany)

Plant Cell Cultures and Treatments

Cell suspension cultures of Agrostemma githago L were initiated and grown in20mL of MS medium contained in 100mL Erlenmeyer flasks as described[2125]

Three days after subcultivation three of the cultures were treated with 100 mg(28 kBq) and three were treated with 200 mg (28 kBq) of [ring-U-14C]-4-353-NP perassay With all assays the radiochemical was dissolved in 100 mL of n-hexadecanewhich constituted the organic part of the two-liquid-phase system (mediumn-hexadecane 2001 vv) The assays were incubated for 7 days until the end ofthe subcultivation interval of the cultures Soybean (Glycine max L Merr cvMandarin) cell suspension cultures utilized in the preliminary conventional one-liquid-phase study were maintained in B5 medium as described[2126] Two daysbefore the end of the subcultivation interval the cultures (5 parallels) were treatedwith 20 mg (1mgL1 30 kBq) of [ring-U-14C]-4-353-NP per assay and wereincubated for 48 h

Extraction of Assays

The assays (A githago) were individually transferred to centrifuge tubes and10mL of n-hexane was added After centrifugation (15min 5000 g) the organicphase containing the n-hexadecane layer was removed Remaining aqueous phases(media) were separated from the cells by suction filtration The cells were suspendedin chloroformmethanol 12 (vv) stored at 20C for 16 h and were extractedby means of sonication (Bandelin Berlin Germany) Insoluble cell debris wereseparated from the cell extracts by suction filtration and washed with chloroformmethanolwater 1208 (vvv) Air-dried filter papers with adhering cell debris weresubjected to combustion analysis (Biological Oxidizer OX 500 ZinsserHarveyInstruments Frankfurt Germany) The individual cell extracts were examined byTLC corresponding parallels were combined and concentrated After examinationby TLC media were discarded Excepting the separation of the n-hexadecanephase the assays of the experiment with soybean cells were similarly extracted andexamined

536 Schmidt et al

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Hydrolytic Treatments

Concentrated and combined cell extracts (100 and 200 mg assays) derivedfrom the study with A githago were refluxed in 40mL of 2M HClmethanol 11 (vv)for 2 h The methanol portion of the reaction mixture was evaporated and theremaining HCl phase was extracted with diethyl ether Recoveries of 14C in theextracts were 45 and 46 with the 100 and 200 mg assays respectively based on 14Cfound in corresponding combined cell extracts Remaining HCl phases wereconcentrated and hydrolyzed for 2 h using 2M HCl at reflux (recoveries 25 and17 respectively) All diethyl ether extracts (hydrolysates) obtained from thehydrolytic treatments were analyzed by TLC and HPLC Subsequently theradioactivity contained in the first hydrolysate derived from the 200 mg assays wasseparated by preparative HPLC and isolated fractions were examined by GC-EIMSThe combined cell extracts of the experiment with soybean cells were similarilytreated The hydrolysates (total recovery 86) however were only subjected byTLC and HPLC analyses

Analytical and Chromatographic Procedures

Liquid scintillation counting (LSC) was performed using a LS 5000 TD analyzer(Beckmann Munchen) and LumasafeTM Plus and CarbomaxTM Plus (Canberra-Packard Dreieich Germany) scintillation cocktails

Thin-layer chromatography (TLC) was performed on silica gel plates SIL G-25UV254 (025mm Macherey-Nagel Duren Germany) The solvent systems used wereas follows A n-hexanediethyl etheracetic acid 50501 (vvv) B n-hexanediethyletheracetic acid 20801 (vvv) C ethyl acetateiso-propanol 4060 (vv) D ethylacetateiso-propanolH2O 652412 (vvv) Separated 14C peaks were located andquantified by means of a Tracemaster 40 radiochromatogram scanner (BertholdWildbad Germany) nonlabelled 4-353-NP was visualized under UV at 254 nm

High-performance liquid chromatography (HPLC) was executed on a SystemGold Personal chromatograph (Beckman Munchen Germany) consisting ofProgrammable Solvent Module 126 Diode Array Detector Modul 168 andRadioisotope Detector 171 The latter was equipped with a 2420 quartz cell(silanized glass 20ndash30 mm internal diameter 55mm cell volume 037mL RaytestStraubenhardt Germany) All HPLC analyses were performed on a reversed phasecolumn (CC 2504 Nucleosil 100-5 C18 HD Macherey-Nagel Duren Germany) at20C using a flow of 1mLmin1 Nonlabelled 4-353-NP was detected at 280 nmElution was performed with solvents A (01 acetic acid in water vv) and B (01acetic acid in acetonitrile vv) as follows AB (6535 vv) for 5min then linear30min gradient to 100 B isocratic B (100) for 5min and return to initialconditions in 5min All analyses were terminated by washing the column with AB(6535 vv) for 5min

For GC-EIMS analysis (gas chromatography-electron impact mass spectrom-etry) samples were concentrated using an N2 stream and derivatized with 100 mL ofN-methyl-N-trimethyl-silyltrifluoroacetamide (MSTFA Fluka Buchs Switzerland)at 70C for 30min GC-EIMS was executed with a Hewlett-Packard 5890 Series II

Metabolism of the Nonylphenol Isomer 537

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gas chromatograph (Agilent Waldbronn Germany) equipped with a FS-SE-54-NB-05 column (25m 025mm film thickness 046 mm CS Chromatographie ServiceLangerwehe Germany) and carrier gas helium injection volume was 1 mL The gaschromatograph was connected to a Hewlett-Packard 5971 A MSD (mass selectivedetector) which was operated in scan mode (mass rangemz 50ndash480) with an electronenergy of 70 eV Temperature programming was isothermal 85C for 5min 85ndash280Cat 10Cmin1 and isothermal for 3min injector and interface temperatures were250ndash280C respectively

RESULTS AND DISCUSSION

Distribution of Radioactivity

The distributions of 14C found after termination of the metabolism experimentsare shown in Table 1 Concerning the preliminary study with soybean the majorityof 14C was present in the media (357of applied radioactivity) while 299 and 203were detected in cell extracts and insoluble cell debris respectively In contrastmore than 60 of applied 14C were found in the cell extracts derived from the two-liquid-phase study performed with Agrostemma githago cells Portions in mediaand nonextractable residues were below 20 only traces of 14C were left in then-hexadecane phase Individual analyses with TLC (systems A and B) demonstratedthat in the media and cell extracts of the soybean assays free 4-353-NP was only

Table 1 Distribution of radioactivity in Agrostemma githago cell culture afters 7 days

of incubation with 100 mg (5mgL1 28 kBq 3 parallels) or 200 mg (10mgL1 28 kBq

3 parallels) of [ring-U-14C]-4-353-NP per assay in two-liquid-phase system and soybean cell

culture (5 parallels) after 2 days of incubation with 20mg (1mgL1 30 kBq) of [ring-U-14C]-

4-353-NP per assay in conventional one-liquid-phase system (mean values are displayed)

Soybean A githago A githago

20mg assays 100mg assays 200 mg assays

Fraction of applied 14C of applied 14C of applied 14C

n-Hexadecane phase mdash 24 28

Medium 357 151 17414C-residues on filter papera mdashb 51 35

Cell extract 299 639 603

Nonextractable residues 203 118 141

Recovered radioactivity 859 983 981

Recovered 4-353-NPc 18 00 00

Primary metabolitesd 19ndash22 28ndash42 21ndash33

aFilter papers used to separate cells and mediabFilter papers were not examinedcPortions were determined from results of TLC analysis of cell extracts and mediadPortions were estimated from results of TLC and HPLC analyses of hydrolysates obtained

from hydrolytic treatments of combined cell extracts (single values are shown)

538 Schmidt et al

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present in trace amounts free 4-353-NP however was completely absent in theassays with the A githago cells The chromatographic behavior (TLC systems AndashD)of the radioactivity contained in all cell extracts pointed to glycosides as metabolicproducts of 4-353-NP With all TLC systems used the radioactivity associated withthe media proved to be preponderantly immobile Extraction of these 14C portionswith organic solvents such as ethyl acetate was not successful the same held true forhydrolysis procedures in order to cleave possible glycosidic linkages Additionallydirect HPLC analysis was not practicable since the necessary concentration of themedia resulted in highly viscous samples

The results obtained with the A githago cells were comparable to data publishedrecently on the biotransformation of 4-n-nonylphenol by the same cell cultureusing the same two-liquid-phase system[24] Concerning the intended identification ofsolublemetabolites uptake of 4-353-NP from the n-hexadecane layer by theA githagocells was considered satisfactory with both the 100 and 200 mg assays The same heldfor recoveries of 14C noticeable portions of soluble 14C in cell extracts and completedisappearance (turnover) of the applied 4-353-NP The different distribution ofradioactivity observed in the soybean experiment was referred to the conventionalone-liquid-phase system used but additionally also to the plant species[23] With bothplant species examined formation of glycosides from the phenolic xenobiotic orpossible primary transformation products was expected according to data publishedon 4-n-nonylphenol[11122324] The chemical nature of the radioactivity present inthe media especially of the soybean cells remained unknown Recently we found14C materials with similar (chromatographic) behavior in the media of plantcell suspensions (including soybean) after application of 14C-bisphenol A andsuspected polymerized products of the compound[27] Polymeres (or copolymers)may also have been formed from 4-353-NP

Hydrolysis Experiments Isolation and Identification of Metabolites

The combined cell extracts were subjected to two consecutive hydrolysissteps (2M HCl-methanol reflux 2M HCl reflux) in order to release the expected14C-aglycons from their corresponding glycosides In the case of soybean 86 of theradioactivity introduced into these experiments could be extracted (diethyl ether)from the reaction mixture With A githago recoveries in the hydrolysates amountedto totals of 70 (100 mg assays) and 63 (200 mg assays) All hydrolysates wereexamined using TLC and HPLC The results of TLC analysis of the samples(hydrolysis steps 1 and 2) derived from the A githogo study (Fig 2) demonstratedthat in three of the samples noticeable portions of 4-353-NP (Rf about 09) werepresent whereas in all samples 14C peaks at Rf about 05 and Rf 00 were detectedHPLC examination of the hydrolysates prevailingly confirmed these data radio-chromatograms of the samples derived from hydrolysis step 1 are shown in Fig 3In addition to a peak originating from 4-353-NP (Rt 310min) further 14C peaksappeared at Rt 150ndash200min Since the 14C peaks with Rf about 05 (TLC) and Rt

150ndash200min (HPLC) were not detected prior to hydrolysis the correspondingcompounds were regarded as primary metabolites of 4-353-NP Both 4-353-NP andprimary metabolites of 4-353-NP were thus liberated from their glycosides by acid

Metabolism of the Nonylphenol Isomer 539

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treatment Similar results were obtained from the soybean experiment (data notshown) Estimated from TLC and HPLC analysis the portions of primarymetabolites of 4-353-NP were between 19 and 42 of applied 14C (Table 1)depending on the method of calculation Low amounts of primary 4-353-NPmetabolites were detected with soybean this fact additionally supported theutilization of A githago cells in two-liquid-phase system for production andidentification of these transformation products

Figure 2 TLC radiochromatograms (solvent system B) of the samples derived from the

hydrolysis experiments A hydrolysis step 1 100 mg assays B hydrolysis step 1 200mg assays

C hydrolysis step 2 100 mg assays D hydrolysis step 2 200mg assays

Figure 3 HPLC radiochromatograms of samples derived from the hydrolysis experiments

A hydrolysis step 1 100mg assays B hydrolysis step 1 200 mg assays

540 Schmidt et al

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The radioactivity contained in the hydrolysate of the first hydrolytic step derivedfrom the 200 mg assays was separated by HPLC (using multiple runs) Two fractionswere isolated fraction 1 corresponding with the 14C peaks at Rt 150ndash200min andfraction 2 (4-353-NP Rt 310min) Total yields recovered after concentration ofHPLC eluates were 19 and 7mg with fraction 1 and fraction 2 respectively Bothfractions were derivatized with MSTFA (trimethylsilyl) and subjected to GC-EIMSanalyses the corresponding chromatograms are shown in Fig 4 The chromatogramof fraction 2 exhibited two peaks (IX and X Rt 1620ndash1640min) which accordingto their retention times and mass spectra (Table 2) were identified with the twodiastereomers of parent 4-353-NP While the [Mthorn] peak was observed at mz 292further prominent fragments emerged at mz 263 (-C2H5) 221 (-C5H11 base peak)193 179 and 73 (trimethylsilyl) According to data published on branched side-chain 4-nonylphenol isomers and 4-n-nonylphenol derivatives[2371216] the MSfragmentation scheme of 4-353-NP shown in Fig 5 is proposed The chemicalstructure of the fragment with mz 179 (trimethylsilyl derivative of hydroxyltropylium ion with mz 107) was deduced from recent literature data[3]

In the GC-EIMS chromatogram obtained from fraction 2 eight peaks weredetected (Rt 1860ndash2020min) which by means of their mass spectra could be tracedback to structures related to 4-353-NP (Fig 4) Seven of these compounds (IndashVII)exhibited an [Mthorn] at mz 380 and prominent fragments at mz 261 221 (base peak)

Figure 4 GC-EIMS chromatograms of the samples obtained from HPLC separation

(cf Fig 3) of the first hydrolysate derived from the 200 mg assays A fraction containing the

primary metabolites of 4-353-NP (peaks I and IV appeared as shoulders of II and V

respectively) B fraction containing nonmetabolized 4-353-NP

Metabolism of the Nonylphenol Isomer 541

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Table 2 Mass spectra of side-chain monohydroxylated metabolites (IndashVII) of 4-353-NP a

carboxylic acid metabolite (VII) of 4-353-NP and of 4-353-NP diastereomers (IX X) after

trimethylsilyl derivatization Compounds were detected using GC-EIMS analysis and were

contained in fractions A and B (obtained by HPLC separation) of a cell extract hydrolysate

(first hydrolysis step 200mg assays)

Peak

Rt EIMS

IsomeraPortionb

(min) mz (abundance in ) ()

Fraction 1

Ic 1865 380 (2) 365 (1) 351 (8) 275 (1) 261 (33)

233 (1) 221 (100) 206 (3) 193 (6) 191 (5)

179 (5) 151 (2) 117 (33) 73 (43)

unknown 214c

II 1870 380 (3) 365 (1) 351 (7) 275 (1) 261 (34)

233 (2) 221 (100) 206 (3) 193 (7) 191 (8)

179 (8) 163 (2) 151 (3) 117 (29) 73 (49)

60-OH-

4-353-NP

214c

III 1874 380 (2) 365 (1) 351 (5) 275 (1) 261 (34)

233 (1) 221 (100) 206 (4) 193 (6) 191 (6)

179 (8) 163 (2) 151 (3) 117 (32) 73 (50)

60-OH-

4-353-NP

175

IVd 1892 380 (2) 365 (1) 351 (6) 275 (1) 261 (36)

233 (2) 221 (100) 206 (4) 193 (7) 191 (6)

179 (8) 163 (2) 151 (3) 117 (27) 73 (52)

60-OH-

4-353-NP

473d

V 1896 380 (2) 365 (1) 351 (6) 275 (1) 261 (37)

233 (1) 221 (100) 206 (3) 193 (7) 191 (6)

179 (8) 163 (2) 151 (3) 117 (26) 73 (45)

60-OH-

4-353-NP

473d

VI 1925 380 (2) 365 (1) 351 (5) 336 (1) 309 (6) 295

(1) 261 (9) 233 (4) 221 (100) 206 (2) 193

(31) 191 (7) 179 (12) 163 (2) 151 (4) 147

(7) 117 (4) 103 (11) 73 (60)

10-OH-

4-353-NP

52

VII 1947 380 (2) 365 (1) 351 (5) 295 (2) 261 (8) 253

(3) 233 (3) 221 (100) 207 (4) 193 (21) 191

(6) 179 (10) 163 (6) 151 (2) 117 (3) 103

(4) 73 (47)

20-OH-

4-353-NP

59

VIII 2019 394 (2) 379 (4) 365 (11) 247 (2) 313 (4)

275 (2) 247 (4) 233 (33) 221 (100) 207 (2)

193 (11) 191 (4) 179 (8) 173 (9) 131 (3)

116 (3) 97 (6) 73 (40)

70-COOH-

4-353-NP

26

Fraction 2

IX 1629 292 (5) 277 (3) 263 (18) 221 (100) 207 (7)

193 (55) 179 (14) 163 (3) 151 (6) 135 (2)

129 (3) 115 (4) 103 (3) 91 (3) 73 (34)

4-353-NP 468

X 1638 292 (5) 277 (3) 263 (19) 221 (100) 207 (8)

193 (55) 179 (13) 163 (2) 151 (6) 135 (2)

129 (4) 115 (3) 103 (1) 91 (3) 73 (31)

4-353-NP 532

aStructures of metabolites IndashVIII of 4-353-NP are proposed according to EIMS fragmentation

patternsbCalculated from peak areas total area of peaks IndashVIII and IXndashX respectively corresponds

with 100cPeak I appeared as shoulder of II separate integration of peaks I and II was not possibledPeak IV appeared as shoulder of V separate integration of peaks IV and V was not possible

542 Schmidt et al

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179 and 73 complete mass spectra are listed in Table 2 The [Mthorn] of compoundsIndashVII pointed to mono-hydroxylated derivatives of 4-353-NP while all patterns offragmentation and their similarity with that of 4-353-NP (especially peaks at mz 221and 179) indicated that hydroxylation had exclusively occurred at the aliphatic side-chain Compound VIII had an [Mthorn] at mz 394 and prominent fragments at mz 221(base peak) 179 and 73 These findings pointed a (side-chain) carboxylic acidderivative of 4-353-NP With both fraction 1 and fraction 2 GC-EIMS peakscorresponding with possible ring-hydroxylated or side-chain olefinic products[24]

were not detected The hydrolysate derived from the soybean experiment wassimilarly separated by HPLC a fraction corresponding with primary 4-353-NPmetabolites was collected and examined by GC-EIMS after silylation Due tolow yield no distinct peaks which could be related to parent 4-353-NP were foundin the corresponding chromatograms as such However using the GC-EIMSfindings of the A githago study a trace was identified at Rt 1905min the massspectrum of which corresponded with compounds IV or V We concluded that theA githago and soybean cells at least demonstrated certain similarities concerningthe biotransformation of 4-353-NP

Though the GC-EIMS data unequivocally proved that 4-353-NP was metabo-lized by A githago cell suspensions at the aliphatic side-chain to monohydroxylatedproducts and a carboxylic acid derivative the sites of hydroxylationoxidation partlyremained unclear Compounds IIndashV emerged in the chromatograms as two pairs ofpeaks (IIIII and IVV) and exhibited noticeably similar mass spectra (Table 2)Besides [Mthorn] at mz 380 and fragments observed with all other monohydroxylatedmetabolites (mz 365 351 261 221 179 73) a prominent fragment of compoundsIIndashV hadmz 117 with 26ndash32 abundance According to data published on side-chain

Figure 5 Proposed MS fragmentation of 4(3050-dimethyl-30-heptyl)-phenol

Metabolism of the Nonylphenol Isomer 543

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monohydroxylated derivatives of both 4-n-nonylphenol[1224] and model side-chainisomers 2- 3- and 4-(4-hydroxyphenol)-nonane[4] fragment mz 117 was referred to[H3CCHOSi(CH3)3

thorn] This fragment is characteristic of trimethylsilyl derivatizedhydroxyl functions at secondary carbon atoms adjacent to terminal CH3

groups[4122428] Due to abundances (34ndash37) of fragments mz 261 (-HOSi(CH3)3-C2H5)

[2429] it was supposed that compounds IIndashV were 60-hydroxy derivatives of4-353-NP Applied 4-353-NP consisted of two diastereomers (each a couple ofenatiomers) hydroxylation at position 60 consequently introduced a third chiral centerwhich resulted in four diastereomers An MS fragmentation scheme for IIndashV isproposed in Fig 6 Quantitative evaluation of the chromatogram showed thatcompounds IIndashV were the main primary metabolites in A githago cells amountingto about 80 of the products identified

The mass spectrum of compound VI (Table 2) differed from those of productsIIndashV in particular regarding three fragments mz 309 (abundance 6) 295 (1) and103 (11) According to previous results presented on 4-n-nonylphenol[24] thefragment mz 103 was supposed to arise from [H2COSi(CH3)3

thorn] pointing tohydroxylation at a primary carbon[2428] Furthermore fragments mz 309 and 295indicated loss of C5H11 and C6H13 respectively from [Mthorn] (mz 380) with retentionof two silylated hydroxy functions at the remaining fragment ion It was thusassumed that compound VI was a 4-353-NP metabolite hydroxylated at position 10

of the side-chain Due to a similar line of argumentation (presence of fragments mz117 and 295) compound VII possibly consisted of the 20-hydroxy derivative of4-353-NP The structure of compound I remained unknown mainly becausemetabolite I was detected as shoulder of II resulting in large overlapping of its mass

Figure 6 Proposed MS fragmentation of the 6-hydroxy metabolites of 4(3050-dimethyl-

30-heptyl)-phenol

544 Schmidt et al

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ORDER REPRINTS

spectrum with that of II Due to its [Mthorn] of mz 394 compound VIII was identifiedwith a carboxylic acid metabolite of 4-353-NP since formation of a hydroxyderivative with an additional carbonyl function was regarded unlikely The massspectrum observed with VIII supported this assumption Fragments showingsimilarity to compounds IndashVII were mz 379 (-CH3) and 365 (-C2H5) whereasfragments mz 131 and 116 were supposed to consist of frac12CH2COOSiethCH3THORN

thorn3 and

frac12CH2COOSiethCH3THORNthorn2 respectively which pointed to a carboxyl function at positions

10 or 70 A characteristic feature of the fragmentation of trimethylsilyl derivatizedfatty acids is separation of CH3 (15) and HOSi(CH3)2 (75) consecutively[30]

With VIII this would result in fragment mz 304 which was only present in thebackground However ions resulting from successive fragmentation of mz 304were observed mz 275 (-HCO -29) 247 (-C2H428) 233 (-CH214) and finally221 This sequence was regarded as prove that 4-353-NP was oxidized to itscarboxylic acid at position 70 of the molecule It should be mentioned that I VI VIIand VIII were only minor metabolites formed from 4-353-NP by the A githago cellsuspensions (Table 2)

Metabolism of 4-353-NP by Agrostemma githagoPlant Cell Suspension Cultures

The nonylphenol isomer 4-353-NP consisting of two diastereomers wastransformed by theAgrostemma githago suspension cultures to four types of productsglycosides of parent 4-353-NP glycosides of primary 4-353-NP metabolitesnonextractable residues and unknown possibly polymeric materials detected in themedia Similar products emerged in the soybean cultures The present investigationconcentrated on the primary metabolites of 4-353-NP These were produced in the Agithago suspensions using a two-liquid-phase system The primary products identifiedby GC-EIMS after liberation from their glycosides were one side-chain carboxylicacid and seven side-chain monohydroxylated metabolites Due to their mass spectrathe main products were four diastereomers of 60-hydroxy-4-353-NP

As yet data on the biotransformation of 4-353-NP are mainly not at handConcerning plant tissues (cell cultures plants) side-chain hydroxylated metaboliteswere reported on 4-n-nonylphenol[11122324] With A githago cells the main productwas 4-(80-hydroxy-n-nonyl)-phenol[24] ie 4-n-nonylphenol was metabolized bysubterminal oxidation corresponding with the main products observed with 4-353-NP in the present study Side-chain hydroxylated metabolites were also formed fromp-tert-octylphenol in barley plants[31] Experiments with model nonylphenols 2- 3-and 4-(4-hydroxyphenyl)-nonane demonstrated that in rainbow trout all isomerswere transformed to monohydroxy products with the site of oxidation at carbon 8(subterminal) of the nonyl residue The metabolites emerged as glucuronic acidconjugates while glucuronides of the parent nonylphenols were also formed[4] In asimilar study both side-chain and ring hydroxylated metabolites of 4-n-nonylphenolwere detected as glucuronic acid conjugates[9] Recently data on the metabolism ofthe nonylphenol isomer 4(3060-dimethyl-30-heptyl)-phenol in pond snails to a (ringhydroxylated) catechol derivative (with unmodified side-chain) were reported[1920]

Contrasting with 4-353-NP of the present study the isomer examined possessed

Metabolism of the Nonylphenol Isomer 545

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a branching at subterminal position 60 of the side-chain besides that at carbon 30 Thecatechol was found conjugated to glucuronic acid[1920] As technical productnonylphenol appears to be completely degradated in soil[1] though isomer 4-(3060-dimethyl-30-heptyl)-phenol proved to be resistant in sedimentwater systems[18] Upto now the routes of nonylphenolrsquos microbial degradation are not known in detailDue to experiments with an isolated bacterial strain initial oxidation at the aromaticmoiety leads to degradation of the phenyl ring while side-chains remain asnonanols[3233] one of the studies was executed with radio-labelled 4-353-NP[33]

The present data on the metabolism of 4-353-NP by A githago cell suspensionsagree with those reported in the literature on 4-n-nonylphenol in plant tissues andmodel isomers in rainbow trout In contrast with trout the capacity of the A githagocells for hydroxylation of the side-chain appeared to be broader ie the nonyl residuewas hydroxylated at different sites of the aliphatic chain The enzymes responsible forside-chain hydroxylation of 4-353-NP in A githago are not known we suspectedcytochromes P450 In rats and humans isozyme CYP2B2 was thought to catalyzepredominantly the hydroxylation of nonylphenol isomers[10] After expression inyeast human CYP2B6 and CYP2C19 were shown to metabolize nonylphenol (pluralbranched chains) while 4-n-nonylphenol was transformed by CYP1A2 CYP2B6 andCYP2C19 ring and side-chain hydroxlated products were identified with branchedchain nonylphenols and 4-n-nonylphenol[34] We assume that plants in general havethe capacity to transform nonylphenol isomers to side-chain hydroxylated productsand thus contribute to the environmental degradation of the xenoestrogen If cropplants are contaminated with nonylphenol both nonylphenol and its side-chainhydroxylated products are expected to be present in plant tissues as glycosideconjugates Xenobiotic glycosides are known to be cleaved in the gastrointestinal tractwith release of possibly toxic aglycons Data on the toxicological properties of side-chain hydroxylated nonylphenols are not available Our future research will focus onthe metabolism of nonylphenol isomers with different branching patterns on theidentification of the enzymes responsible for the metabolism of nonylphenols and onthe (eco-) toxicology of side-chain hydroxylated nonylphenol isomers

ACKNOWLEDGMENTS

For the synthesis of [ring-U-14C]-4(3050-dimethyl-30-heptyl)-phenol the authorswish to thank R Vinken and the lsquolsquoAachen Graduate College Elimination ofEndocrine-Disrupting Substances from Waste-Waterrsquorsquo (AGEESA) funded by theDeutsche Forschungsgemeinschaft

REFERENCES

1 Staples CA Williams JB Blessing RL Varineau PT Measuring thebiodegradability of nonylphenol ether carboxylates octylphenol ether carboxyl-ates and nonylphenol Chemosphere 1999 38 (9) 2029ndash2039

2 Thiele B Gunther K Schwuger MJ Alkylphenol ethoxylates trace analysisand environmental behaviour Chem Rev 1997 97 (8) 3247ndash3272

546 Schmidt et al

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3 Wheeler TF Heim JR LaTorre MR Janes AB Mass spectral

characterization of p-nonylphenol isomers using high-resolution capillary

GC-MS J Chromatogr Sci 1997 35 (1) 19ndash304 Meldahl AC Nithipatikom K Lech JJ Metabolism of several 14C-

nonylphenol isomers by rainbow trout (Oncorhynchus mykiss) in vivo and

in vitro Metabolites Xenobiotica 1996 26 (11) 1167ndash11805 Kim Y-S Katase T Sekine S Inoue T Makino M Uchiyama T

Fujimoto Y Yamashita N Variation in estrogenic activity among fractions

of a commercial nonylphenol by high performance liquid chromatography

Chemosphere 2004 54 (8) 1127ndash11346 Giger W Stephanou E Schaffner C Persistent organic chemicals in sewage

effluents 1 Identification of nonylphenols and nonylphenolethoxylates by

capillary gas chromatographymass spectrometry Chemosphere 1981 10 (11ndash1)

1253ndash12637 Giger W Brunner PH Schaffner C 4-Nonylphenol in sewage sludge

accumulation of toxic metabolites from nonionic surfactants Science 1984

225 (4662) 623ndash6258 Jobst H Chlorophenols an nonylphenols in sewage sludges 1 Occurrence in

sewage sludges of Western German treatment plants from 1987ndash1989 Acta

Hydrochim Hydrobiol 1995 23 (1) 20ndash259 Coldham NG Sivapathasundaram SDM Ashfield LA Pottinger TG

Biotransformation tissue distribution and persistence of 4-nonylphenol

residues in juvenile rainbow trout (Oncorhynchus mykiss) Drug Metab

Dispos 1998 26 (4) 347ndash35410 Lee PC Marquardt M Lech JJ Metabolism of nonylphenol by rat and

human microsomes Toxicol Lett 1998 99 (7) 117ndash12611 Bokern M Harms HH Toxicity and metabolism of 4-n-nonylphenol in cell

suspension cultures of different plant species Environ Sci Technol 1997

31 (7) 1849ndash185412 Bokern M Nimtz M Harms HH Metabolites of 4-n-nonylphenol in wheat

cell suspension cultures J Agric Food Chem 1996 44 (4) 1123ndash112713 Guenther K Heinke V Thiele B Kleist E Prast H Raecker T

Endocrine disrupting nonylphenols are ubiquitous in food Environ Sci

Technol 2002 36 (8) 1676ndash168014 Soto AM Justicia H Wray JW Sonnenschein C p-Nonylphenol an

estrogenic xenobiotic released from lsquolsquomodifiedrsquorsquo polystyrene Environ Health

Perspect 1991 92 167ndash17315 Degen DH Bolt HM Comments on lsquolsquoendocrine disrupting nonylphenols

are ubiquitous in foodrsquorsquo Envirn Sci Technol 2003 37 (11) 2622ndash262316 Vinken R Schmidt B Schaffer A Synthesis of tertiary 14C-labelled

nonylphenol isomers J Label Compd Radiopharm 2002 45 (14) 1253ndash126317 Lalah JO Lenoir D Henkelmann B Hertkorn N Gunther K

Schramm K-W Kettrup A Synthesis of ring-14C-labelled 4(3060-dimethyl-

30-heptyl)-phenol J Label Compd Radiopharm 2001 44 (6) 459ndash46318 Lalah JO Schramm K-W Henkelmann B Lenoir D Behechti A

Gunther K Kettrup A The dissipation distribution and fate of a branched

Metabolism of the Nonylphenol Isomer 547

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14C-nonylphenol isomer in lake watersediment systems Environ Pollut 2003

122 (2) 195ndash20319 Lalah JO Schramm K-W Severin GF Lenoir D Henkelmann B

Behechti A Gunther K Kettrup A In vivo metabolism and organ

distribution of a branched 14C-nonylphenol isomer in pond snails Lymnaea

stagnalis L Aquat Toxicol 2003 62 (4) 305ndash31920 Lalah JO Behechti A Severin GF Lenoir D Gunther K Kettrup A

Schramm K-W The Bioaccumulation and fate of a branched 14C-

p-nonylphenol isomer in Lymnaea stagnalis L Environ Toxicol Chem 2003

22 (7) 1428ndash143621 Schmidt B Metabolic profiling using plant cell suspension cultures In

Pesticide Transformation in Plants and Microorganisms Hall JC Hoagland

RE Zablotowicz RM Eds American Chemical Society Washington DC

2001 40ndash5622 Komoszliga D Langebartels C Sandermann H Metabolic processes for

organic chemicals in plants In Plant ContaminationmdashModeling and Simulation

of Organic Chemical Processes Trapp S Mc Farlane JC Eds Lewis

Publishers Boca Raton 1995 69ndash10323 Schmidt B Schuphan I Metabolism of the Xenoestrogens 4-n-nonylphenol

and bisphenol A in plant cell suspension cultures In Book of Abstracts 10th

IUPAC International Congress on the Chemistry of Crop Protection Basel

August 4ndash9 IUPAC Basel 2002 Vol 2 3424 Schmidt B Patti H Niewersch C Schuphan I Biotransformation of [ring-

U-14C]4-n-nonylphenol by Agrostemma githago cell culture in a two-liquid-

phase system Biotechnol Lett 2003 25 (16) 1375ndash138125 Murashige T Skoog F Revised medium for rapid growth and bio assay with

tobacco tissue cultures Physiol Plant 1962 15 473ndash49726 Gamborg OL Miller RA Ojima K Nutrient requirements of suspension

cultures of soybean root cells Exp Cell Res 1968 50 151ndash15827 Schmidt B Schuphan I Metabolism of the environmental estrogen bisphenol

A by plant cell suspension cultures Chemosphere 2002 49 (1) 51ndash5928 Diekman J Thomson JB Djerassi C Mass spectrometry and stereo-

chemical problems CLV Electron impact induced fragmentations and

rearrangements of some trimethylsilyl ethers of aliphatic glycols and related

compounds J Org Chem 1968 33 (6) 2271ndash228429 Diekman J Thomson JB Djerassi C Mass spectrometry and stereo-

chemical problems CXLI Electron impact induced fragmentations and

rearrangements of trimethylsilyl ethers amines and sulfides J Org Chem

1967 32 (12) 3904ndash391930 Odham G Fatty acids In Biochemical Applications of Mass Spectrometry

Waller GR Dermer OC Eds Wiley-Interscience New York 1980 First

Supplementary Volume 153ndash17131 Stolzenberg GE Olson PA Zaylskie RG Mansager ER Behavior and

fate of ethoxylated alkylphenol nonionic surfactant in barley plants J Agric

Food Chem 1982 30 (4) 637ndash644

548 Schmidt et al

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32 Tanghe T Dhooge W Verstraete W Isolation of a bacterial strain able todegrade branched nonylphenol Appl Environ Microbiol 1999 65 (2)746ndash751

33 Corvini PFX Vinken R Hommes G Schmidt B Dohmann MDegradation of the radioactive and non-labelled branched 4(3050-dimethyl-30-heptyl)-phenol nonylphenol isomer by Sphingomonas TTNP3 Biodegradation2003 15 (1) 1ndash10

34 Inui H Shiota N Motoi Y Ido Y Inoue T Kodama T Ohkawa YOhkawa H Metabolism of herbicides and other chemicals in humancytochrome P450 species and in transgenic potato plants co-expressinghuman CYP1A1 CYP2B6 and CYP2C19 J Pestic Sci 2001 26 (1) 28ndash40

Received February 13 2004

Metabolism of the Nonylphenol Isomer 549

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Hydrolytic Treatments

Concentrated and combined cell extracts (100 and 200 mg assays) derivedfrom the study with A githago were refluxed in 40mL of 2M HClmethanol 11 (vv)for 2 h The methanol portion of the reaction mixture was evaporated and theremaining HCl phase was extracted with diethyl ether Recoveries of 14C in theextracts were 45 and 46 with the 100 and 200 mg assays respectively based on 14Cfound in corresponding combined cell extracts Remaining HCl phases wereconcentrated and hydrolyzed for 2 h using 2M HCl at reflux (recoveries 25 and17 respectively) All diethyl ether extracts (hydrolysates) obtained from thehydrolytic treatments were analyzed by TLC and HPLC Subsequently theradioactivity contained in the first hydrolysate derived from the 200 mg assays wasseparated by preparative HPLC and isolated fractions were examined by GC-EIMSThe combined cell extracts of the experiment with soybean cells were similarilytreated The hydrolysates (total recovery 86) however were only subjected byTLC and HPLC analyses

Analytical and Chromatographic Procedures

Liquid scintillation counting (LSC) was performed using a LS 5000 TD analyzer(Beckmann Munchen) and LumasafeTM Plus and CarbomaxTM Plus (Canberra-Packard Dreieich Germany) scintillation cocktails

Thin-layer chromatography (TLC) was performed on silica gel plates SIL G-25UV254 (025mm Macherey-Nagel Duren Germany) The solvent systems used wereas follows A n-hexanediethyl etheracetic acid 50501 (vvv) B n-hexanediethyletheracetic acid 20801 (vvv) C ethyl acetateiso-propanol 4060 (vv) D ethylacetateiso-propanolH2O 652412 (vvv) Separated 14C peaks were located andquantified by means of a Tracemaster 40 radiochromatogram scanner (BertholdWildbad Germany) nonlabelled 4-353-NP was visualized under UV at 254 nm

High-performance liquid chromatography (HPLC) was executed on a SystemGold Personal chromatograph (Beckman Munchen Germany) consisting ofProgrammable Solvent Module 126 Diode Array Detector Modul 168 andRadioisotope Detector 171 The latter was equipped with a 2420 quartz cell(silanized glass 20ndash30 mm internal diameter 55mm cell volume 037mL RaytestStraubenhardt Germany) All HPLC analyses were performed on a reversed phasecolumn (CC 2504 Nucleosil 100-5 C18 HD Macherey-Nagel Duren Germany) at20C using a flow of 1mLmin1 Nonlabelled 4-353-NP was detected at 280 nmElution was performed with solvents A (01 acetic acid in water vv) and B (01acetic acid in acetonitrile vv) as follows AB (6535 vv) for 5min then linear30min gradient to 100 B isocratic B (100) for 5min and return to initialconditions in 5min All analyses were terminated by washing the column with AB(6535 vv) for 5min

For GC-EIMS analysis (gas chromatography-electron impact mass spectrom-etry) samples were concentrated using an N2 stream and derivatized with 100 mL ofN-methyl-N-trimethyl-silyltrifluoroacetamide (MSTFA Fluka Buchs Switzerland)at 70C for 30min GC-EIMS was executed with a Hewlett-Packard 5890 Series II

Metabolism of the Nonylphenol Isomer 537

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gas chromatograph (Agilent Waldbronn Germany) equipped with a FS-SE-54-NB-05 column (25m 025mm film thickness 046 mm CS Chromatographie ServiceLangerwehe Germany) and carrier gas helium injection volume was 1 mL The gaschromatograph was connected to a Hewlett-Packard 5971 A MSD (mass selectivedetector) which was operated in scan mode (mass rangemz 50ndash480) with an electronenergy of 70 eV Temperature programming was isothermal 85C for 5min 85ndash280Cat 10Cmin1 and isothermal for 3min injector and interface temperatures were250ndash280C respectively

RESULTS AND DISCUSSION

Distribution of Radioactivity

The distributions of 14C found after termination of the metabolism experimentsare shown in Table 1 Concerning the preliminary study with soybean the majorityof 14C was present in the media (357of applied radioactivity) while 299 and 203were detected in cell extracts and insoluble cell debris respectively In contrastmore than 60 of applied 14C were found in the cell extracts derived from the two-liquid-phase study performed with Agrostemma githago cells Portions in mediaand nonextractable residues were below 20 only traces of 14C were left in then-hexadecane phase Individual analyses with TLC (systems A and B) demonstratedthat in the media and cell extracts of the soybean assays free 4-353-NP was only

Table 1 Distribution of radioactivity in Agrostemma githago cell culture afters 7 days

of incubation with 100 mg (5mgL1 28 kBq 3 parallels) or 200 mg (10mgL1 28 kBq

3 parallels) of [ring-U-14C]-4-353-NP per assay in two-liquid-phase system and soybean cell

culture (5 parallels) after 2 days of incubation with 20mg (1mgL1 30 kBq) of [ring-U-14C]-

4-353-NP per assay in conventional one-liquid-phase system (mean values are displayed)

Soybean A githago A githago

20mg assays 100mg assays 200 mg assays

Fraction of applied 14C of applied 14C of applied 14C

n-Hexadecane phase mdash 24 28

Medium 357 151 17414C-residues on filter papera mdashb 51 35

Cell extract 299 639 603

Nonextractable residues 203 118 141

Recovered radioactivity 859 983 981

Recovered 4-353-NPc 18 00 00

Primary metabolitesd 19ndash22 28ndash42 21ndash33

aFilter papers used to separate cells and mediabFilter papers were not examinedcPortions were determined from results of TLC analysis of cell extracts and mediadPortions were estimated from results of TLC and HPLC analyses of hydrolysates obtained

from hydrolytic treatments of combined cell extracts (single values are shown)

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present in trace amounts free 4-353-NP however was completely absent in theassays with the A githago cells The chromatographic behavior (TLC systems AndashD)of the radioactivity contained in all cell extracts pointed to glycosides as metabolicproducts of 4-353-NP With all TLC systems used the radioactivity associated withthe media proved to be preponderantly immobile Extraction of these 14C portionswith organic solvents such as ethyl acetate was not successful the same held true forhydrolysis procedures in order to cleave possible glycosidic linkages Additionallydirect HPLC analysis was not practicable since the necessary concentration of themedia resulted in highly viscous samples

The results obtained with the A githago cells were comparable to data publishedrecently on the biotransformation of 4-n-nonylphenol by the same cell cultureusing the same two-liquid-phase system[24] Concerning the intended identification ofsolublemetabolites uptake of 4-353-NP from the n-hexadecane layer by theA githagocells was considered satisfactory with both the 100 and 200 mg assays The same heldfor recoveries of 14C noticeable portions of soluble 14C in cell extracts and completedisappearance (turnover) of the applied 4-353-NP The different distribution ofradioactivity observed in the soybean experiment was referred to the conventionalone-liquid-phase system used but additionally also to the plant species[23] With bothplant species examined formation of glycosides from the phenolic xenobiotic orpossible primary transformation products was expected according to data publishedon 4-n-nonylphenol[11122324] The chemical nature of the radioactivity present inthe media especially of the soybean cells remained unknown Recently we found14C materials with similar (chromatographic) behavior in the media of plantcell suspensions (including soybean) after application of 14C-bisphenol A andsuspected polymerized products of the compound[27] Polymeres (or copolymers)may also have been formed from 4-353-NP

Hydrolysis Experiments Isolation and Identification of Metabolites

The combined cell extracts were subjected to two consecutive hydrolysissteps (2M HCl-methanol reflux 2M HCl reflux) in order to release the expected14C-aglycons from their corresponding glycosides In the case of soybean 86 of theradioactivity introduced into these experiments could be extracted (diethyl ether)from the reaction mixture With A githago recoveries in the hydrolysates amountedto totals of 70 (100 mg assays) and 63 (200 mg assays) All hydrolysates wereexamined using TLC and HPLC The results of TLC analysis of the samples(hydrolysis steps 1 and 2) derived from the A githogo study (Fig 2) demonstratedthat in three of the samples noticeable portions of 4-353-NP (Rf about 09) werepresent whereas in all samples 14C peaks at Rf about 05 and Rf 00 were detectedHPLC examination of the hydrolysates prevailingly confirmed these data radio-chromatograms of the samples derived from hydrolysis step 1 are shown in Fig 3In addition to a peak originating from 4-353-NP (Rt 310min) further 14C peaksappeared at Rt 150ndash200min Since the 14C peaks with Rf about 05 (TLC) and Rt

150ndash200min (HPLC) were not detected prior to hydrolysis the correspondingcompounds were regarded as primary metabolites of 4-353-NP Both 4-353-NP andprimary metabolites of 4-353-NP were thus liberated from their glycosides by acid

Metabolism of the Nonylphenol Isomer 539

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treatment Similar results were obtained from the soybean experiment (data notshown) Estimated from TLC and HPLC analysis the portions of primarymetabolites of 4-353-NP were between 19 and 42 of applied 14C (Table 1)depending on the method of calculation Low amounts of primary 4-353-NPmetabolites were detected with soybean this fact additionally supported theutilization of A githago cells in two-liquid-phase system for production andidentification of these transformation products

Figure 2 TLC radiochromatograms (solvent system B) of the samples derived from the

hydrolysis experiments A hydrolysis step 1 100 mg assays B hydrolysis step 1 200mg assays

C hydrolysis step 2 100 mg assays D hydrolysis step 2 200mg assays

Figure 3 HPLC radiochromatograms of samples derived from the hydrolysis experiments

A hydrolysis step 1 100mg assays B hydrolysis step 1 200 mg assays

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The radioactivity contained in the hydrolysate of the first hydrolytic step derivedfrom the 200 mg assays was separated by HPLC (using multiple runs) Two fractionswere isolated fraction 1 corresponding with the 14C peaks at Rt 150ndash200min andfraction 2 (4-353-NP Rt 310min) Total yields recovered after concentration ofHPLC eluates were 19 and 7mg with fraction 1 and fraction 2 respectively Bothfractions were derivatized with MSTFA (trimethylsilyl) and subjected to GC-EIMSanalyses the corresponding chromatograms are shown in Fig 4 The chromatogramof fraction 2 exhibited two peaks (IX and X Rt 1620ndash1640min) which accordingto their retention times and mass spectra (Table 2) were identified with the twodiastereomers of parent 4-353-NP While the [Mthorn] peak was observed at mz 292further prominent fragments emerged at mz 263 (-C2H5) 221 (-C5H11 base peak)193 179 and 73 (trimethylsilyl) According to data published on branched side-chain 4-nonylphenol isomers and 4-n-nonylphenol derivatives[2371216] the MSfragmentation scheme of 4-353-NP shown in Fig 5 is proposed The chemicalstructure of the fragment with mz 179 (trimethylsilyl derivative of hydroxyltropylium ion with mz 107) was deduced from recent literature data[3]

In the GC-EIMS chromatogram obtained from fraction 2 eight peaks weredetected (Rt 1860ndash2020min) which by means of their mass spectra could be tracedback to structures related to 4-353-NP (Fig 4) Seven of these compounds (IndashVII)exhibited an [Mthorn] at mz 380 and prominent fragments at mz 261 221 (base peak)

Figure 4 GC-EIMS chromatograms of the samples obtained from HPLC separation

(cf Fig 3) of the first hydrolysate derived from the 200 mg assays A fraction containing the

primary metabolites of 4-353-NP (peaks I and IV appeared as shoulders of II and V

respectively) B fraction containing nonmetabolized 4-353-NP

Metabolism of the Nonylphenol Isomer 541

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Table 2 Mass spectra of side-chain monohydroxylated metabolites (IndashVII) of 4-353-NP a

carboxylic acid metabolite (VII) of 4-353-NP and of 4-353-NP diastereomers (IX X) after

trimethylsilyl derivatization Compounds were detected using GC-EIMS analysis and were

contained in fractions A and B (obtained by HPLC separation) of a cell extract hydrolysate

(first hydrolysis step 200mg assays)

Peak

Rt EIMS

IsomeraPortionb

(min) mz (abundance in ) ()

Fraction 1

Ic 1865 380 (2) 365 (1) 351 (8) 275 (1) 261 (33)

233 (1) 221 (100) 206 (3) 193 (6) 191 (5)

179 (5) 151 (2) 117 (33) 73 (43)

unknown 214c

II 1870 380 (3) 365 (1) 351 (7) 275 (1) 261 (34)

233 (2) 221 (100) 206 (3) 193 (7) 191 (8)

179 (8) 163 (2) 151 (3) 117 (29) 73 (49)

60-OH-

4-353-NP

214c

III 1874 380 (2) 365 (1) 351 (5) 275 (1) 261 (34)

233 (1) 221 (100) 206 (4) 193 (6) 191 (6)

179 (8) 163 (2) 151 (3) 117 (32) 73 (50)

60-OH-

4-353-NP

175

IVd 1892 380 (2) 365 (1) 351 (6) 275 (1) 261 (36)

233 (2) 221 (100) 206 (4) 193 (7) 191 (6)

179 (8) 163 (2) 151 (3) 117 (27) 73 (52)

60-OH-

4-353-NP

473d

V 1896 380 (2) 365 (1) 351 (6) 275 (1) 261 (37)

233 (1) 221 (100) 206 (3) 193 (7) 191 (6)

179 (8) 163 (2) 151 (3) 117 (26) 73 (45)

60-OH-

4-353-NP

473d

VI 1925 380 (2) 365 (1) 351 (5) 336 (1) 309 (6) 295

(1) 261 (9) 233 (4) 221 (100) 206 (2) 193

(31) 191 (7) 179 (12) 163 (2) 151 (4) 147

(7) 117 (4) 103 (11) 73 (60)

10-OH-

4-353-NP

52

VII 1947 380 (2) 365 (1) 351 (5) 295 (2) 261 (8) 253

(3) 233 (3) 221 (100) 207 (4) 193 (21) 191

(6) 179 (10) 163 (6) 151 (2) 117 (3) 103

(4) 73 (47)

20-OH-

4-353-NP

59

VIII 2019 394 (2) 379 (4) 365 (11) 247 (2) 313 (4)

275 (2) 247 (4) 233 (33) 221 (100) 207 (2)

193 (11) 191 (4) 179 (8) 173 (9) 131 (3)

116 (3) 97 (6) 73 (40)

70-COOH-

4-353-NP

26

Fraction 2

IX 1629 292 (5) 277 (3) 263 (18) 221 (100) 207 (7)

193 (55) 179 (14) 163 (3) 151 (6) 135 (2)

129 (3) 115 (4) 103 (3) 91 (3) 73 (34)

4-353-NP 468

X 1638 292 (5) 277 (3) 263 (19) 221 (100) 207 (8)

193 (55) 179 (13) 163 (2) 151 (6) 135 (2)

129 (4) 115 (3) 103 (1) 91 (3) 73 (31)

4-353-NP 532

aStructures of metabolites IndashVIII of 4-353-NP are proposed according to EIMS fragmentation

patternsbCalculated from peak areas total area of peaks IndashVIII and IXndashX respectively corresponds

with 100cPeak I appeared as shoulder of II separate integration of peaks I and II was not possibledPeak IV appeared as shoulder of V separate integration of peaks IV and V was not possible

542 Schmidt et al

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179 and 73 complete mass spectra are listed in Table 2 The [Mthorn] of compoundsIndashVII pointed to mono-hydroxylated derivatives of 4-353-NP while all patterns offragmentation and their similarity with that of 4-353-NP (especially peaks at mz 221and 179) indicated that hydroxylation had exclusively occurred at the aliphatic side-chain Compound VIII had an [Mthorn] at mz 394 and prominent fragments at mz 221(base peak) 179 and 73 These findings pointed a (side-chain) carboxylic acidderivative of 4-353-NP With both fraction 1 and fraction 2 GC-EIMS peakscorresponding with possible ring-hydroxylated or side-chain olefinic products[24]

were not detected The hydrolysate derived from the soybean experiment wassimilarly separated by HPLC a fraction corresponding with primary 4-353-NPmetabolites was collected and examined by GC-EIMS after silylation Due tolow yield no distinct peaks which could be related to parent 4-353-NP were foundin the corresponding chromatograms as such However using the GC-EIMSfindings of the A githago study a trace was identified at Rt 1905min the massspectrum of which corresponded with compounds IV or V We concluded that theA githago and soybean cells at least demonstrated certain similarities concerningthe biotransformation of 4-353-NP

Though the GC-EIMS data unequivocally proved that 4-353-NP was metabo-lized by A githago cell suspensions at the aliphatic side-chain to monohydroxylatedproducts and a carboxylic acid derivative the sites of hydroxylationoxidation partlyremained unclear Compounds IIndashV emerged in the chromatograms as two pairs ofpeaks (IIIII and IVV) and exhibited noticeably similar mass spectra (Table 2)Besides [Mthorn] at mz 380 and fragments observed with all other monohydroxylatedmetabolites (mz 365 351 261 221 179 73) a prominent fragment of compoundsIIndashV hadmz 117 with 26ndash32 abundance According to data published on side-chain

Figure 5 Proposed MS fragmentation of 4(3050-dimethyl-30-heptyl)-phenol

Metabolism of the Nonylphenol Isomer 543

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monohydroxylated derivatives of both 4-n-nonylphenol[1224] and model side-chainisomers 2- 3- and 4-(4-hydroxyphenol)-nonane[4] fragment mz 117 was referred to[H3CCHOSi(CH3)3

thorn] This fragment is characteristic of trimethylsilyl derivatizedhydroxyl functions at secondary carbon atoms adjacent to terminal CH3

groups[4122428] Due to abundances (34ndash37) of fragments mz 261 (-HOSi(CH3)3-C2H5)

[2429] it was supposed that compounds IIndashV were 60-hydroxy derivatives of4-353-NP Applied 4-353-NP consisted of two diastereomers (each a couple ofenatiomers) hydroxylation at position 60 consequently introduced a third chiral centerwhich resulted in four diastereomers An MS fragmentation scheme for IIndashV isproposed in Fig 6 Quantitative evaluation of the chromatogram showed thatcompounds IIndashV were the main primary metabolites in A githago cells amountingto about 80 of the products identified

The mass spectrum of compound VI (Table 2) differed from those of productsIIndashV in particular regarding three fragments mz 309 (abundance 6) 295 (1) and103 (11) According to previous results presented on 4-n-nonylphenol[24] thefragment mz 103 was supposed to arise from [H2COSi(CH3)3

thorn] pointing tohydroxylation at a primary carbon[2428] Furthermore fragments mz 309 and 295indicated loss of C5H11 and C6H13 respectively from [Mthorn] (mz 380) with retentionof two silylated hydroxy functions at the remaining fragment ion It was thusassumed that compound VI was a 4-353-NP metabolite hydroxylated at position 10

of the side-chain Due to a similar line of argumentation (presence of fragments mz117 and 295) compound VII possibly consisted of the 20-hydroxy derivative of4-353-NP The structure of compound I remained unknown mainly becausemetabolite I was detected as shoulder of II resulting in large overlapping of its mass

Figure 6 Proposed MS fragmentation of the 6-hydroxy metabolites of 4(3050-dimethyl-

30-heptyl)-phenol

544 Schmidt et al

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spectrum with that of II Due to its [Mthorn] of mz 394 compound VIII was identifiedwith a carboxylic acid metabolite of 4-353-NP since formation of a hydroxyderivative with an additional carbonyl function was regarded unlikely The massspectrum observed with VIII supported this assumption Fragments showingsimilarity to compounds IndashVII were mz 379 (-CH3) and 365 (-C2H5) whereasfragments mz 131 and 116 were supposed to consist of frac12CH2COOSiethCH3THORN

thorn3 and

frac12CH2COOSiethCH3THORNthorn2 respectively which pointed to a carboxyl function at positions

10 or 70 A characteristic feature of the fragmentation of trimethylsilyl derivatizedfatty acids is separation of CH3 (15) and HOSi(CH3)2 (75) consecutively[30]

With VIII this would result in fragment mz 304 which was only present in thebackground However ions resulting from successive fragmentation of mz 304were observed mz 275 (-HCO -29) 247 (-C2H428) 233 (-CH214) and finally221 This sequence was regarded as prove that 4-353-NP was oxidized to itscarboxylic acid at position 70 of the molecule It should be mentioned that I VI VIIand VIII were only minor metabolites formed from 4-353-NP by the A githago cellsuspensions (Table 2)

Metabolism of 4-353-NP by Agrostemma githagoPlant Cell Suspension Cultures

The nonylphenol isomer 4-353-NP consisting of two diastereomers wastransformed by theAgrostemma githago suspension cultures to four types of productsglycosides of parent 4-353-NP glycosides of primary 4-353-NP metabolitesnonextractable residues and unknown possibly polymeric materials detected in themedia Similar products emerged in the soybean cultures The present investigationconcentrated on the primary metabolites of 4-353-NP These were produced in the Agithago suspensions using a two-liquid-phase system The primary products identifiedby GC-EIMS after liberation from their glycosides were one side-chain carboxylicacid and seven side-chain monohydroxylated metabolites Due to their mass spectrathe main products were four diastereomers of 60-hydroxy-4-353-NP

As yet data on the biotransformation of 4-353-NP are mainly not at handConcerning plant tissues (cell cultures plants) side-chain hydroxylated metaboliteswere reported on 4-n-nonylphenol[11122324] With A githago cells the main productwas 4-(80-hydroxy-n-nonyl)-phenol[24] ie 4-n-nonylphenol was metabolized bysubterminal oxidation corresponding with the main products observed with 4-353-NP in the present study Side-chain hydroxylated metabolites were also formed fromp-tert-octylphenol in barley plants[31] Experiments with model nonylphenols 2- 3-and 4-(4-hydroxyphenyl)-nonane demonstrated that in rainbow trout all isomerswere transformed to monohydroxy products with the site of oxidation at carbon 8(subterminal) of the nonyl residue The metabolites emerged as glucuronic acidconjugates while glucuronides of the parent nonylphenols were also formed[4] In asimilar study both side-chain and ring hydroxylated metabolites of 4-n-nonylphenolwere detected as glucuronic acid conjugates[9] Recently data on the metabolism ofthe nonylphenol isomer 4(3060-dimethyl-30-heptyl)-phenol in pond snails to a (ringhydroxylated) catechol derivative (with unmodified side-chain) were reported[1920]

Contrasting with 4-353-NP of the present study the isomer examined possessed

Metabolism of the Nonylphenol Isomer 545

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a branching at subterminal position 60 of the side-chain besides that at carbon 30 Thecatechol was found conjugated to glucuronic acid[1920] As technical productnonylphenol appears to be completely degradated in soil[1] though isomer 4-(3060-dimethyl-30-heptyl)-phenol proved to be resistant in sedimentwater systems[18] Upto now the routes of nonylphenolrsquos microbial degradation are not known in detailDue to experiments with an isolated bacterial strain initial oxidation at the aromaticmoiety leads to degradation of the phenyl ring while side-chains remain asnonanols[3233] one of the studies was executed with radio-labelled 4-353-NP[33]

The present data on the metabolism of 4-353-NP by A githago cell suspensionsagree with those reported in the literature on 4-n-nonylphenol in plant tissues andmodel isomers in rainbow trout In contrast with trout the capacity of the A githagocells for hydroxylation of the side-chain appeared to be broader ie the nonyl residuewas hydroxylated at different sites of the aliphatic chain The enzymes responsible forside-chain hydroxylation of 4-353-NP in A githago are not known we suspectedcytochromes P450 In rats and humans isozyme CYP2B2 was thought to catalyzepredominantly the hydroxylation of nonylphenol isomers[10] After expression inyeast human CYP2B6 and CYP2C19 were shown to metabolize nonylphenol (pluralbranched chains) while 4-n-nonylphenol was transformed by CYP1A2 CYP2B6 andCYP2C19 ring and side-chain hydroxlated products were identified with branchedchain nonylphenols and 4-n-nonylphenol[34] We assume that plants in general havethe capacity to transform nonylphenol isomers to side-chain hydroxylated productsand thus contribute to the environmental degradation of the xenoestrogen If cropplants are contaminated with nonylphenol both nonylphenol and its side-chainhydroxylated products are expected to be present in plant tissues as glycosideconjugates Xenobiotic glycosides are known to be cleaved in the gastrointestinal tractwith release of possibly toxic aglycons Data on the toxicological properties of side-chain hydroxylated nonylphenols are not available Our future research will focus onthe metabolism of nonylphenol isomers with different branching patterns on theidentification of the enzymes responsible for the metabolism of nonylphenols and onthe (eco-) toxicology of side-chain hydroxylated nonylphenol isomers

ACKNOWLEDGMENTS

For the synthesis of [ring-U-14C]-4(3050-dimethyl-30-heptyl)-phenol the authorswish to thank R Vinken and the lsquolsquoAachen Graduate College Elimination ofEndocrine-Disrupting Substances from Waste-Waterrsquorsquo (AGEESA) funded by theDeutsche Forschungsgemeinschaft

REFERENCES

1 Staples CA Williams JB Blessing RL Varineau PT Measuring thebiodegradability of nonylphenol ether carboxylates octylphenol ether carboxyl-ates and nonylphenol Chemosphere 1999 38 (9) 2029ndash2039

2 Thiele B Gunther K Schwuger MJ Alkylphenol ethoxylates trace analysisand environmental behaviour Chem Rev 1997 97 (8) 3247ndash3272

546 Schmidt et al

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3 Wheeler TF Heim JR LaTorre MR Janes AB Mass spectral

characterization of p-nonylphenol isomers using high-resolution capillary

GC-MS J Chromatogr Sci 1997 35 (1) 19ndash304 Meldahl AC Nithipatikom K Lech JJ Metabolism of several 14C-

nonylphenol isomers by rainbow trout (Oncorhynchus mykiss) in vivo and

in vitro Metabolites Xenobiotica 1996 26 (11) 1167ndash11805 Kim Y-S Katase T Sekine S Inoue T Makino M Uchiyama T

Fujimoto Y Yamashita N Variation in estrogenic activity among fractions

of a commercial nonylphenol by high performance liquid chromatography

Chemosphere 2004 54 (8) 1127ndash11346 Giger W Stephanou E Schaffner C Persistent organic chemicals in sewage

effluents 1 Identification of nonylphenols and nonylphenolethoxylates by

capillary gas chromatographymass spectrometry Chemosphere 1981 10 (11ndash1)

1253ndash12637 Giger W Brunner PH Schaffner C 4-Nonylphenol in sewage sludge

accumulation of toxic metabolites from nonionic surfactants Science 1984

225 (4662) 623ndash6258 Jobst H Chlorophenols an nonylphenols in sewage sludges 1 Occurrence in

sewage sludges of Western German treatment plants from 1987ndash1989 Acta

Hydrochim Hydrobiol 1995 23 (1) 20ndash259 Coldham NG Sivapathasundaram SDM Ashfield LA Pottinger TG

Biotransformation tissue distribution and persistence of 4-nonylphenol

residues in juvenile rainbow trout (Oncorhynchus mykiss) Drug Metab

Dispos 1998 26 (4) 347ndash35410 Lee PC Marquardt M Lech JJ Metabolism of nonylphenol by rat and

human microsomes Toxicol Lett 1998 99 (7) 117ndash12611 Bokern M Harms HH Toxicity and metabolism of 4-n-nonylphenol in cell

suspension cultures of different plant species Environ Sci Technol 1997

31 (7) 1849ndash185412 Bokern M Nimtz M Harms HH Metabolites of 4-n-nonylphenol in wheat

cell suspension cultures J Agric Food Chem 1996 44 (4) 1123ndash112713 Guenther K Heinke V Thiele B Kleist E Prast H Raecker T

Endocrine disrupting nonylphenols are ubiquitous in food Environ Sci

Technol 2002 36 (8) 1676ndash168014 Soto AM Justicia H Wray JW Sonnenschein C p-Nonylphenol an

estrogenic xenobiotic released from lsquolsquomodifiedrsquorsquo polystyrene Environ Health

Perspect 1991 92 167ndash17315 Degen DH Bolt HM Comments on lsquolsquoendocrine disrupting nonylphenols

are ubiquitous in foodrsquorsquo Envirn Sci Technol 2003 37 (11) 2622ndash262316 Vinken R Schmidt B Schaffer A Synthesis of tertiary 14C-labelled

nonylphenol isomers J Label Compd Radiopharm 2002 45 (14) 1253ndash126317 Lalah JO Lenoir D Henkelmann B Hertkorn N Gunther K

Schramm K-W Kettrup A Synthesis of ring-14C-labelled 4(3060-dimethyl-

30-heptyl)-phenol J Label Compd Radiopharm 2001 44 (6) 459ndash46318 Lalah JO Schramm K-W Henkelmann B Lenoir D Behechti A

Gunther K Kettrup A The dissipation distribution and fate of a branched

Metabolism of the Nonylphenol Isomer 547

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14C-nonylphenol isomer in lake watersediment systems Environ Pollut 2003

122 (2) 195ndash20319 Lalah JO Schramm K-W Severin GF Lenoir D Henkelmann B

Behechti A Gunther K Kettrup A In vivo metabolism and organ

distribution of a branched 14C-nonylphenol isomer in pond snails Lymnaea

stagnalis L Aquat Toxicol 2003 62 (4) 305ndash31920 Lalah JO Behechti A Severin GF Lenoir D Gunther K Kettrup A

Schramm K-W The Bioaccumulation and fate of a branched 14C-

p-nonylphenol isomer in Lymnaea stagnalis L Environ Toxicol Chem 2003

22 (7) 1428ndash143621 Schmidt B Metabolic profiling using plant cell suspension cultures In

Pesticide Transformation in Plants and Microorganisms Hall JC Hoagland

RE Zablotowicz RM Eds American Chemical Society Washington DC

2001 40ndash5622 Komoszliga D Langebartels C Sandermann H Metabolic processes for

organic chemicals in plants In Plant ContaminationmdashModeling and Simulation

of Organic Chemical Processes Trapp S Mc Farlane JC Eds Lewis

Publishers Boca Raton 1995 69ndash10323 Schmidt B Schuphan I Metabolism of the Xenoestrogens 4-n-nonylphenol

and bisphenol A in plant cell suspension cultures In Book of Abstracts 10th

IUPAC International Congress on the Chemistry of Crop Protection Basel

August 4ndash9 IUPAC Basel 2002 Vol 2 3424 Schmidt B Patti H Niewersch C Schuphan I Biotransformation of [ring-

U-14C]4-n-nonylphenol by Agrostemma githago cell culture in a two-liquid-

phase system Biotechnol Lett 2003 25 (16) 1375ndash138125 Murashige T Skoog F Revised medium for rapid growth and bio assay with

tobacco tissue cultures Physiol Plant 1962 15 473ndash49726 Gamborg OL Miller RA Ojima K Nutrient requirements of suspension

cultures of soybean root cells Exp Cell Res 1968 50 151ndash15827 Schmidt B Schuphan I Metabolism of the environmental estrogen bisphenol

A by plant cell suspension cultures Chemosphere 2002 49 (1) 51ndash5928 Diekman J Thomson JB Djerassi C Mass spectrometry and stereo-

chemical problems CLV Electron impact induced fragmentations and

rearrangements of some trimethylsilyl ethers of aliphatic glycols and related

compounds J Org Chem 1968 33 (6) 2271ndash228429 Diekman J Thomson JB Djerassi C Mass spectrometry and stereo-

chemical problems CXLI Electron impact induced fragmentations and

rearrangements of trimethylsilyl ethers amines and sulfides J Org Chem

1967 32 (12) 3904ndash391930 Odham G Fatty acids In Biochemical Applications of Mass Spectrometry

Waller GR Dermer OC Eds Wiley-Interscience New York 1980 First

Supplementary Volume 153ndash17131 Stolzenberg GE Olson PA Zaylskie RG Mansager ER Behavior and

fate of ethoxylated alkylphenol nonionic surfactant in barley plants J Agric

Food Chem 1982 30 (4) 637ndash644

548 Schmidt et al

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32 Tanghe T Dhooge W Verstraete W Isolation of a bacterial strain able todegrade branched nonylphenol Appl Environ Microbiol 1999 65 (2)746ndash751

33 Corvini PFX Vinken R Hommes G Schmidt B Dohmann MDegradation of the radioactive and non-labelled branched 4(3050-dimethyl-30-heptyl)-phenol nonylphenol isomer by Sphingomonas TTNP3 Biodegradation2003 15 (1) 1ndash10

34 Inui H Shiota N Motoi Y Ido Y Inoue T Kodama T Ohkawa YOhkawa H Metabolism of herbicides and other chemicals in humancytochrome P450 species and in transgenic potato plants co-expressinghuman CYP1A1 CYP2B6 and CYP2C19 J Pestic Sci 2001 26 (1) 28ndash40

Received February 13 2004

Metabolism of the Nonylphenol Isomer 549

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gas chromatograph (Agilent Waldbronn Germany) equipped with a FS-SE-54-NB-05 column (25m 025mm film thickness 046 mm CS Chromatographie ServiceLangerwehe Germany) and carrier gas helium injection volume was 1 mL The gaschromatograph was connected to a Hewlett-Packard 5971 A MSD (mass selectivedetector) which was operated in scan mode (mass rangemz 50ndash480) with an electronenergy of 70 eV Temperature programming was isothermal 85C for 5min 85ndash280Cat 10Cmin1 and isothermal for 3min injector and interface temperatures were250ndash280C respectively

RESULTS AND DISCUSSION

Distribution of Radioactivity

The distributions of 14C found after termination of the metabolism experimentsare shown in Table 1 Concerning the preliminary study with soybean the majorityof 14C was present in the media (357of applied radioactivity) while 299 and 203were detected in cell extracts and insoluble cell debris respectively In contrastmore than 60 of applied 14C were found in the cell extracts derived from the two-liquid-phase study performed with Agrostemma githago cells Portions in mediaand nonextractable residues were below 20 only traces of 14C were left in then-hexadecane phase Individual analyses with TLC (systems A and B) demonstratedthat in the media and cell extracts of the soybean assays free 4-353-NP was only

Table 1 Distribution of radioactivity in Agrostemma githago cell culture afters 7 days

of incubation with 100 mg (5mgL1 28 kBq 3 parallels) or 200 mg (10mgL1 28 kBq

3 parallels) of [ring-U-14C]-4-353-NP per assay in two-liquid-phase system and soybean cell

culture (5 parallels) after 2 days of incubation with 20mg (1mgL1 30 kBq) of [ring-U-14C]-

4-353-NP per assay in conventional one-liquid-phase system (mean values are displayed)

Soybean A githago A githago

20mg assays 100mg assays 200 mg assays

Fraction of applied 14C of applied 14C of applied 14C

n-Hexadecane phase mdash 24 28

Medium 357 151 17414C-residues on filter papera mdashb 51 35

Cell extract 299 639 603

Nonextractable residues 203 118 141

Recovered radioactivity 859 983 981

Recovered 4-353-NPc 18 00 00

Primary metabolitesd 19ndash22 28ndash42 21ndash33

aFilter papers used to separate cells and mediabFilter papers were not examinedcPortions were determined from results of TLC analysis of cell extracts and mediadPortions were estimated from results of TLC and HPLC analyses of hydrolysates obtained

from hydrolytic treatments of combined cell extracts (single values are shown)

538 Schmidt et al

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present in trace amounts free 4-353-NP however was completely absent in theassays with the A githago cells The chromatographic behavior (TLC systems AndashD)of the radioactivity contained in all cell extracts pointed to glycosides as metabolicproducts of 4-353-NP With all TLC systems used the radioactivity associated withthe media proved to be preponderantly immobile Extraction of these 14C portionswith organic solvents such as ethyl acetate was not successful the same held true forhydrolysis procedures in order to cleave possible glycosidic linkages Additionallydirect HPLC analysis was not practicable since the necessary concentration of themedia resulted in highly viscous samples

The results obtained with the A githago cells were comparable to data publishedrecently on the biotransformation of 4-n-nonylphenol by the same cell cultureusing the same two-liquid-phase system[24] Concerning the intended identification ofsolublemetabolites uptake of 4-353-NP from the n-hexadecane layer by theA githagocells was considered satisfactory with both the 100 and 200 mg assays The same heldfor recoveries of 14C noticeable portions of soluble 14C in cell extracts and completedisappearance (turnover) of the applied 4-353-NP The different distribution ofradioactivity observed in the soybean experiment was referred to the conventionalone-liquid-phase system used but additionally also to the plant species[23] With bothplant species examined formation of glycosides from the phenolic xenobiotic orpossible primary transformation products was expected according to data publishedon 4-n-nonylphenol[11122324] The chemical nature of the radioactivity present inthe media especially of the soybean cells remained unknown Recently we found14C materials with similar (chromatographic) behavior in the media of plantcell suspensions (including soybean) after application of 14C-bisphenol A andsuspected polymerized products of the compound[27] Polymeres (or copolymers)may also have been formed from 4-353-NP

Hydrolysis Experiments Isolation and Identification of Metabolites

The combined cell extracts were subjected to two consecutive hydrolysissteps (2M HCl-methanol reflux 2M HCl reflux) in order to release the expected14C-aglycons from their corresponding glycosides In the case of soybean 86 of theradioactivity introduced into these experiments could be extracted (diethyl ether)from the reaction mixture With A githago recoveries in the hydrolysates amountedto totals of 70 (100 mg assays) and 63 (200 mg assays) All hydrolysates wereexamined using TLC and HPLC The results of TLC analysis of the samples(hydrolysis steps 1 and 2) derived from the A githogo study (Fig 2) demonstratedthat in three of the samples noticeable portions of 4-353-NP (Rf about 09) werepresent whereas in all samples 14C peaks at Rf about 05 and Rf 00 were detectedHPLC examination of the hydrolysates prevailingly confirmed these data radio-chromatograms of the samples derived from hydrolysis step 1 are shown in Fig 3In addition to a peak originating from 4-353-NP (Rt 310min) further 14C peaksappeared at Rt 150ndash200min Since the 14C peaks with Rf about 05 (TLC) and Rt

150ndash200min (HPLC) were not detected prior to hydrolysis the correspondingcompounds were regarded as primary metabolites of 4-353-NP Both 4-353-NP andprimary metabolites of 4-353-NP were thus liberated from their glycosides by acid

Metabolism of the Nonylphenol Isomer 539

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treatment Similar results were obtained from the soybean experiment (data notshown) Estimated from TLC and HPLC analysis the portions of primarymetabolites of 4-353-NP were between 19 and 42 of applied 14C (Table 1)depending on the method of calculation Low amounts of primary 4-353-NPmetabolites were detected with soybean this fact additionally supported theutilization of A githago cells in two-liquid-phase system for production andidentification of these transformation products

Figure 2 TLC radiochromatograms (solvent system B) of the samples derived from the

hydrolysis experiments A hydrolysis step 1 100 mg assays B hydrolysis step 1 200mg assays

C hydrolysis step 2 100 mg assays D hydrolysis step 2 200mg assays

Figure 3 HPLC radiochromatograms of samples derived from the hydrolysis experiments

A hydrolysis step 1 100mg assays B hydrolysis step 1 200 mg assays

540 Schmidt et al

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The radioactivity contained in the hydrolysate of the first hydrolytic step derivedfrom the 200 mg assays was separated by HPLC (using multiple runs) Two fractionswere isolated fraction 1 corresponding with the 14C peaks at Rt 150ndash200min andfraction 2 (4-353-NP Rt 310min) Total yields recovered after concentration ofHPLC eluates were 19 and 7mg with fraction 1 and fraction 2 respectively Bothfractions were derivatized with MSTFA (trimethylsilyl) and subjected to GC-EIMSanalyses the corresponding chromatograms are shown in Fig 4 The chromatogramof fraction 2 exhibited two peaks (IX and X Rt 1620ndash1640min) which accordingto their retention times and mass spectra (Table 2) were identified with the twodiastereomers of parent 4-353-NP While the [Mthorn] peak was observed at mz 292further prominent fragments emerged at mz 263 (-C2H5) 221 (-C5H11 base peak)193 179 and 73 (trimethylsilyl) According to data published on branched side-chain 4-nonylphenol isomers and 4-n-nonylphenol derivatives[2371216] the MSfragmentation scheme of 4-353-NP shown in Fig 5 is proposed The chemicalstructure of the fragment with mz 179 (trimethylsilyl derivative of hydroxyltropylium ion with mz 107) was deduced from recent literature data[3]

In the GC-EIMS chromatogram obtained from fraction 2 eight peaks weredetected (Rt 1860ndash2020min) which by means of their mass spectra could be tracedback to structures related to 4-353-NP (Fig 4) Seven of these compounds (IndashVII)exhibited an [Mthorn] at mz 380 and prominent fragments at mz 261 221 (base peak)

Figure 4 GC-EIMS chromatograms of the samples obtained from HPLC separation

(cf Fig 3) of the first hydrolysate derived from the 200 mg assays A fraction containing the

primary metabolites of 4-353-NP (peaks I and IV appeared as shoulders of II and V

respectively) B fraction containing nonmetabolized 4-353-NP

Metabolism of the Nonylphenol Isomer 541

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Table 2 Mass spectra of side-chain monohydroxylated metabolites (IndashVII) of 4-353-NP a

carboxylic acid metabolite (VII) of 4-353-NP and of 4-353-NP diastereomers (IX X) after

trimethylsilyl derivatization Compounds were detected using GC-EIMS analysis and were

contained in fractions A and B (obtained by HPLC separation) of a cell extract hydrolysate

(first hydrolysis step 200mg assays)

Peak

Rt EIMS

IsomeraPortionb

(min) mz (abundance in ) ()

Fraction 1

Ic 1865 380 (2) 365 (1) 351 (8) 275 (1) 261 (33)

233 (1) 221 (100) 206 (3) 193 (6) 191 (5)

179 (5) 151 (2) 117 (33) 73 (43)

unknown 214c

II 1870 380 (3) 365 (1) 351 (7) 275 (1) 261 (34)

233 (2) 221 (100) 206 (3) 193 (7) 191 (8)

179 (8) 163 (2) 151 (3) 117 (29) 73 (49)

60-OH-

4-353-NP

214c

III 1874 380 (2) 365 (1) 351 (5) 275 (1) 261 (34)

233 (1) 221 (100) 206 (4) 193 (6) 191 (6)

179 (8) 163 (2) 151 (3) 117 (32) 73 (50)

60-OH-

4-353-NP

175

IVd 1892 380 (2) 365 (1) 351 (6) 275 (1) 261 (36)

233 (2) 221 (100) 206 (4) 193 (7) 191 (6)

179 (8) 163 (2) 151 (3) 117 (27) 73 (52)

60-OH-

4-353-NP

473d

V 1896 380 (2) 365 (1) 351 (6) 275 (1) 261 (37)

233 (1) 221 (100) 206 (3) 193 (7) 191 (6)

179 (8) 163 (2) 151 (3) 117 (26) 73 (45)

60-OH-

4-353-NP

473d

VI 1925 380 (2) 365 (1) 351 (5) 336 (1) 309 (6) 295

(1) 261 (9) 233 (4) 221 (100) 206 (2) 193

(31) 191 (7) 179 (12) 163 (2) 151 (4) 147

(7) 117 (4) 103 (11) 73 (60)

10-OH-

4-353-NP

52

VII 1947 380 (2) 365 (1) 351 (5) 295 (2) 261 (8) 253

(3) 233 (3) 221 (100) 207 (4) 193 (21) 191

(6) 179 (10) 163 (6) 151 (2) 117 (3) 103

(4) 73 (47)

20-OH-

4-353-NP

59

VIII 2019 394 (2) 379 (4) 365 (11) 247 (2) 313 (4)

275 (2) 247 (4) 233 (33) 221 (100) 207 (2)

193 (11) 191 (4) 179 (8) 173 (9) 131 (3)

116 (3) 97 (6) 73 (40)

70-COOH-

4-353-NP

26

Fraction 2

IX 1629 292 (5) 277 (3) 263 (18) 221 (100) 207 (7)

193 (55) 179 (14) 163 (3) 151 (6) 135 (2)

129 (3) 115 (4) 103 (3) 91 (3) 73 (34)

4-353-NP 468

X 1638 292 (5) 277 (3) 263 (19) 221 (100) 207 (8)

193 (55) 179 (13) 163 (2) 151 (6) 135 (2)

129 (4) 115 (3) 103 (1) 91 (3) 73 (31)

4-353-NP 532

aStructures of metabolites IndashVIII of 4-353-NP are proposed according to EIMS fragmentation

patternsbCalculated from peak areas total area of peaks IndashVIII and IXndashX respectively corresponds

with 100cPeak I appeared as shoulder of II separate integration of peaks I and II was not possibledPeak IV appeared as shoulder of V separate integration of peaks IV and V was not possible

542 Schmidt et al

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179 and 73 complete mass spectra are listed in Table 2 The [Mthorn] of compoundsIndashVII pointed to mono-hydroxylated derivatives of 4-353-NP while all patterns offragmentation and their similarity with that of 4-353-NP (especially peaks at mz 221and 179) indicated that hydroxylation had exclusively occurred at the aliphatic side-chain Compound VIII had an [Mthorn] at mz 394 and prominent fragments at mz 221(base peak) 179 and 73 These findings pointed a (side-chain) carboxylic acidderivative of 4-353-NP With both fraction 1 and fraction 2 GC-EIMS peakscorresponding with possible ring-hydroxylated or side-chain olefinic products[24]

were not detected The hydrolysate derived from the soybean experiment wassimilarly separated by HPLC a fraction corresponding with primary 4-353-NPmetabolites was collected and examined by GC-EIMS after silylation Due tolow yield no distinct peaks which could be related to parent 4-353-NP were foundin the corresponding chromatograms as such However using the GC-EIMSfindings of the A githago study a trace was identified at Rt 1905min the massspectrum of which corresponded with compounds IV or V We concluded that theA githago and soybean cells at least demonstrated certain similarities concerningthe biotransformation of 4-353-NP

Though the GC-EIMS data unequivocally proved that 4-353-NP was metabo-lized by A githago cell suspensions at the aliphatic side-chain to monohydroxylatedproducts and a carboxylic acid derivative the sites of hydroxylationoxidation partlyremained unclear Compounds IIndashV emerged in the chromatograms as two pairs ofpeaks (IIIII and IVV) and exhibited noticeably similar mass spectra (Table 2)Besides [Mthorn] at mz 380 and fragments observed with all other monohydroxylatedmetabolites (mz 365 351 261 221 179 73) a prominent fragment of compoundsIIndashV hadmz 117 with 26ndash32 abundance According to data published on side-chain

Figure 5 Proposed MS fragmentation of 4(3050-dimethyl-30-heptyl)-phenol

Metabolism of the Nonylphenol Isomer 543

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monohydroxylated derivatives of both 4-n-nonylphenol[1224] and model side-chainisomers 2- 3- and 4-(4-hydroxyphenol)-nonane[4] fragment mz 117 was referred to[H3CCHOSi(CH3)3

thorn] This fragment is characteristic of trimethylsilyl derivatizedhydroxyl functions at secondary carbon atoms adjacent to terminal CH3

groups[4122428] Due to abundances (34ndash37) of fragments mz 261 (-HOSi(CH3)3-C2H5)

[2429] it was supposed that compounds IIndashV were 60-hydroxy derivatives of4-353-NP Applied 4-353-NP consisted of two diastereomers (each a couple ofenatiomers) hydroxylation at position 60 consequently introduced a third chiral centerwhich resulted in four diastereomers An MS fragmentation scheme for IIndashV isproposed in Fig 6 Quantitative evaluation of the chromatogram showed thatcompounds IIndashV were the main primary metabolites in A githago cells amountingto about 80 of the products identified

The mass spectrum of compound VI (Table 2) differed from those of productsIIndashV in particular regarding three fragments mz 309 (abundance 6) 295 (1) and103 (11) According to previous results presented on 4-n-nonylphenol[24] thefragment mz 103 was supposed to arise from [H2COSi(CH3)3

thorn] pointing tohydroxylation at a primary carbon[2428] Furthermore fragments mz 309 and 295indicated loss of C5H11 and C6H13 respectively from [Mthorn] (mz 380) with retentionof two silylated hydroxy functions at the remaining fragment ion It was thusassumed that compound VI was a 4-353-NP metabolite hydroxylated at position 10

of the side-chain Due to a similar line of argumentation (presence of fragments mz117 and 295) compound VII possibly consisted of the 20-hydroxy derivative of4-353-NP The structure of compound I remained unknown mainly becausemetabolite I was detected as shoulder of II resulting in large overlapping of its mass

Figure 6 Proposed MS fragmentation of the 6-hydroxy metabolites of 4(3050-dimethyl-

30-heptyl)-phenol

544 Schmidt et al

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spectrum with that of II Due to its [Mthorn] of mz 394 compound VIII was identifiedwith a carboxylic acid metabolite of 4-353-NP since formation of a hydroxyderivative with an additional carbonyl function was regarded unlikely The massspectrum observed with VIII supported this assumption Fragments showingsimilarity to compounds IndashVII were mz 379 (-CH3) and 365 (-C2H5) whereasfragments mz 131 and 116 were supposed to consist of frac12CH2COOSiethCH3THORN

thorn3 and

frac12CH2COOSiethCH3THORNthorn2 respectively which pointed to a carboxyl function at positions

10 or 70 A characteristic feature of the fragmentation of trimethylsilyl derivatizedfatty acids is separation of CH3 (15) and HOSi(CH3)2 (75) consecutively[30]

With VIII this would result in fragment mz 304 which was only present in thebackground However ions resulting from successive fragmentation of mz 304were observed mz 275 (-HCO -29) 247 (-C2H428) 233 (-CH214) and finally221 This sequence was regarded as prove that 4-353-NP was oxidized to itscarboxylic acid at position 70 of the molecule It should be mentioned that I VI VIIand VIII were only minor metabolites formed from 4-353-NP by the A githago cellsuspensions (Table 2)

Metabolism of 4-353-NP by Agrostemma githagoPlant Cell Suspension Cultures

The nonylphenol isomer 4-353-NP consisting of two diastereomers wastransformed by theAgrostemma githago suspension cultures to four types of productsglycosides of parent 4-353-NP glycosides of primary 4-353-NP metabolitesnonextractable residues and unknown possibly polymeric materials detected in themedia Similar products emerged in the soybean cultures The present investigationconcentrated on the primary metabolites of 4-353-NP These were produced in the Agithago suspensions using a two-liquid-phase system The primary products identifiedby GC-EIMS after liberation from their glycosides were one side-chain carboxylicacid and seven side-chain monohydroxylated metabolites Due to their mass spectrathe main products were four diastereomers of 60-hydroxy-4-353-NP

As yet data on the biotransformation of 4-353-NP are mainly not at handConcerning plant tissues (cell cultures plants) side-chain hydroxylated metaboliteswere reported on 4-n-nonylphenol[11122324] With A githago cells the main productwas 4-(80-hydroxy-n-nonyl)-phenol[24] ie 4-n-nonylphenol was metabolized bysubterminal oxidation corresponding with the main products observed with 4-353-NP in the present study Side-chain hydroxylated metabolites were also formed fromp-tert-octylphenol in barley plants[31] Experiments with model nonylphenols 2- 3-and 4-(4-hydroxyphenyl)-nonane demonstrated that in rainbow trout all isomerswere transformed to monohydroxy products with the site of oxidation at carbon 8(subterminal) of the nonyl residue The metabolites emerged as glucuronic acidconjugates while glucuronides of the parent nonylphenols were also formed[4] In asimilar study both side-chain and ring hydroxylated metabolites of 4-n-nonylphenolwere detected as glucuronic acid conjugates[9] Recently data on the metabolism ofthe nonylphenol isomer 4(3060-dimethyl-30-heptyl)-phenol in pond snails to a (ringhydroxylated) catechol derivative (with unmodified side-chain) were reported[1920]

Contrasting with 4-353-NP of the present study the isomer examined possessed

Metabolism of the Nonylphenol Isomer 545

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a branching at subterminal position 60 of the side-chain besides that at carbon 30 Thecatechol was found conjugated to glucuronic acid[1920] As technical productnonylphenol appears to be completely degradated in soil[1] though isomer 4-(3060-dimethyl-30-heptyl)-phenol proved to be resistant in sedimentwater systems[18] Upto now the routes of nonylphenolrsquos microbial degradation are not known in detailDue to experiments with an isolated bacterial strain initial oxidation at the aromaticmoiety leads to degradation of the phenyl ring while side-chains remain asnonanols[3233] one of the studies was executed with radio-labelled 4-353-NP[33]

The present data on the metabolism of 4-353-NP by A githago cell suspensionsagree with those reported in the literature on 4-n-nonylphenol in plant tissues andmodel isomers in rainbow trout In contrast with trout the capacity of the A githagocells for hydroxylation of the side-chain appeared to be broader ie the nonyl residuewas hydroxylated at different sites of the aliphatic chain The enzymes responsible forside-chain hydroxylation of 4-353-NP in A githago are not known we suspectedcytochromes P450 In rats and humans isozyme CYP2B2 was thought to catalyzepredominantly the hydroxylation of nonylphenol isomers[10] After expression inyeast human CYP2B6 and CYP2C19 were shown to metabolize nonylphenol (pluralbranched chains) while 4-n-nonylphenol was transformed by CYP1A2 CYP2B6 andCYP2C19 ring and side-chain hydroxlated products were identified with branchedchain nonylphenols and 4-n-nonylphenol[34] We assume that plants in general havethe capacity to transform nonylphenol isomers to side-chain hydroxylated productsand thus contribute to the environmental degradation of the xenoestrogen If cropplants are contaminated with nonylphenol both nonylphenol and its side-chainhydroxylated products are expected to be present in plant tissues as glycosideconjugates Xenobiotic glycosides are known to be cleaved in the gastrointestinal tractwith release of possibly toxic aglycons Data on the toxicological properties of side-chain hydroxylated nonylphenols are not available Our future research will focus onthe metabolism of nonylphenol isomers with different branching patterns on theidentification of the enzymes responsible for the metabolism of nonylphenols and onthe (eco-) toxicology of side-chain hydroxylated nonylphenol isomers

ACKNOWLEDGMENTS

For the synthesis of [ring-U-14C]-4(3050-dimethyl-30-heptyl)-phenol the authorswish to thank R Vinken and the lsquolsquoAachen Graduate College Elimination ofEndocrine-Disrupting Substances from Waste-Waterrsquorsquo (AGEESA) funded by theDeutsche Forschungsgemeinschaft

REFERENCES

1 Staples CA Williams JB Blessing RL Varineau PT Measuring thebiodegradability of nonylphenol ether carboxylates octylphenol ether carboxyl-ates and nonylphenol Chemosphere 1999 38 (9) 2029ndash2039

2 Thiele B Gunther K Schwuger MJ Alkylphenol ethoxylates trace analysisand environmental behaviour Chem Rev 1997 97 (8) 3247ndash3272

546 Schmidt et al

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3 Wheeler TF Heim JR LaTorre MR Janes AB Mass spectral

characterization of p-nonylphenol isomers using high-resolution capillary

GC-MS J Chromatogr Sci 1997 35 (1) 19ndash304 Meldahl AC Nithipatikom K Lech JJ Metabolism of several 14C-

nonylphenol isomers by rainbow trout (Oncorhynchus mykiss) in vivo and

in vitro Metabolites Xenobiotica 1996 26 (11) 1167ndash11805 Kim Y-S Katase T Sekine S Inoue T Makino M Uchiyama T

Fujimoto Y Yamashita N Variation in estrogenic activity among fractions

of a commercial nonylphenol by high performance liquid chromatography

Chemosphere 2004 54 (8) 1127ndash11346 Giger W Stephanou E Schaffner C Persistent organic chemicals in sewage

effluents 1 Identification of nonylphenols and nonylphenolethoxylates by

capillary gas chromatographymass spectrometry Chemosphere 1981 10 (11ndash1)

1253ndash12637 Giger W Brunner PH Schaffner C 4-Nonylphenol in sewage sludge

accumulation of toxic metabolites from nonionic surfactants Science 1984

225 (4662) 623ndash6258 Jobst H Chlorophenols an nonylphenols in sewage sludges 1 Occurrence in

sewage sludges of Western German treatment plants from 1987ndash1989 Acta

Hydrochim Hydrobiol 1995 23 (1) 20ndash259 Coldham NG Sivapathasundaram SDM Ashfield LA Pottinger TG

Biotransformation tissue distribution and persistence of 4-nonylphenol

residues in juvenile rainbow trout (Oncorhynchus mykiss) Drug Metab

Dispos 1998 26 (4) 347ndash35410 Lee PC Marquardt M Lech JJ Metabolism of nonylphenol by rat and

human microsomes Toxicol Lett 1998 99 (7) 117ndash12611 Bokern M Harms HH Toxicity and metabolism of 4-n-nonylphenol in cell

suspension cultures of different plant species Environ Sci Technol 1997

31 (7) 1849ndash185412 Bokern M Nimtz M Harms HH Metabolites of 4-n-nonylphenol in wheat

cell suspension cultures J Agric Food Chem 1996 44 (4) 1123ndash112713 Guenther K Heinke V Thiele B Kleist E Prast H Raecker T

Endocrine disrupting nonylphenols are ubiquitous in food Environ Sci

Technol 2002 36 (8) 1676ndash168014 Soto AM Justicia H Wray JW Sonnenschein C p-Nonylphenol an

estrogenic xenobiotic released from lsquolsquomodifiedrsquorsquo polystyrene Environ Health

Perspect 1991 92 167ndash17315 Degen DH Bolt HM Comments on lsquolsquoendocrine disrupting nonylphenols

are ubiquitous in foodrsquorsquo Envirn Sci Technol 2003 37 (11) 2622ndash262316 Vinken R Schmidt B Schaffer A Synthesis of tertiary 14C-labelled

nonylphenol isomers J Label Compd Radiopharm 2002 45 (14) 1253ndash126317 Lalah JO Lenoir D Henkelmann B Hertkorn N Gunther K

Schramm K-W Kettrup A Synthesis of ring-14C-labelled 4(3060-dimethyl-

30-heptyl)-phenol J Label Compd Radiopharm 2001 44 (6) 459ndash46318 Lalah JO Schramm K-W Henkelmann B Lenoir D Behechti A

Gunther K Kettrup A The dissipation distribution and fate of a branched

Metabolism of the Nonylphenol Isomer 547

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14C-nonylphenol isomer in lake watersediment systems Environ Pollut 2003

122 (2) 195ndash20319 Lalah JO Schramm K-W Severin GF Lenoir D Henkelmann B

Behechti A Gunther K Kettrup A In vivo metabolism and organ

distribution of a branched 14C-nonylphenol isomer in pond snails Lymnaea

stagnalis L Aquat Toxicol 2003 62 (4) 305ndash31920 Lalah JO Behechti A Severin GF Lenoir D Gunther K Kettrup A

Schramm K-W The Bioaccumulation and fate of a branched 14C-

p-nonylphenol isomer in Lymnaea stagnalis L Environ Toxicol Chem 2003

22 (7) 1428ndash143621 Schmidt B Metabolic profiling using plant cell suspension cultures In

Pesticide Transformation in Plants and Microorganisms Hall JC Hoagland

RE Zablotowicz RM Eds American Chemical Society Washington DC

2001 40ndash5622 Komoszliga D Langebartels C Sandermann H Metabolic processes for

organic chemicals in plants In Plant ContaminationmdashModeling and Simulation

of Organic Chemical Processes Trapp S Mc Farlane JC Eds Lewis

Publishers Boca Raton 1995 69ndash10323 Schmidt B Schuphan I Metabolism of the Xenoestrogens 4-n-nonylphenol

and bisphenol A in plant cell suspension cultures In Book of Abstracts 10th

IUPAC International Congress on the Chemistry of Crop Protection Basel

August 4ndash9 IUPAC Basel 2002 Vol 2 3424 Schmidt B Patti H Niewersch C Schuphan I Biotransformation of [ring-

U-14C]4-n-nonylphenol by Agrostemma githago cell culture in a two-liquid-

phase system Biotechnol Lett 2003 25 (16) 1375ndash138125 Murashige T Skoog F Revised medium for rapid growth and bio assay with

tobacco tissue cultures Physiol Plant 1962 15 473ndash49726 Gamborg OL Miller RA Ojima K Nutrient requirements of suspension

cultures of soybean root cells Exp Cell Res 1968 50 151ndash15827 Schmidt B Schuphan I Metabolism of the environmental estrogen bisphenol

A by plant cell suspension cultures Chemosphere 2002 49 (1) 51ndash5928 Diekman J Thomson JB Djerassi C Mass spectrometry and stereo-

chemical problems CLV Electron impact induced fragmentations and

rearrangements of some trimethylsilyl ethers of aliphatic glycols and related

compounds J Org Chem 1968 33 (6) 2271ndash228429 Diekman J Thomson JB Djerassi C Mass spectrometry and stereo-

chemical problems CXLI Electron impact induced fragmentations and

rearrangements of trimethylsilyl ethers amines and sulfides J Org Chem

1967 32 (12) 3904ndash391930 Odham G Fatty acids In Biochemical Applications of Mass Spectrometry

Waller GR Dermer OC Eds Wiley-Interscience New York 1980 First

Supplementary Volume 153ndash17131 Stolzenberg GE Olson PA Zaylskie RG Mansager ER Behavior and

fate of ethoxylated alkylphenol nonionic surfactant in barley plants J Agric

Food Chem 1982 30 (4) 637ndash644

548 Schmidt et al

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32 Tanghe T Dhooge W Verstraete W Isolation of a bacterial strain able todegrade branched nonylphenol Appl Environ Microbiol 1999 65 (2)746ndash751

33 Corvini PFX Vinken R Hommes G Schmidt B Dohmann MDegradation of the radioactive and non-labelled branched 4(3050-dimethyl-30-heptyl)-phenol nonylphenol isomer by Sphingomonas TTNP3 Biodegradation2003 15 (1) 1ndash10

34 Inui H Shiota N Motoi Y Ido Y Inoue T Kodama T Ohkawa YOhkawa H Metabolism of herbicides and other chemicals in humancytochrome P450 species and in transgenic potato plants co-expressinghuman CYP1A1 CYP2B6 and CYP2C19 J Pestic Sci 2001 26 (1) 28ndash40

Received February 13 2004

Metabolism of the Nonylphenol Isomer 549

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present in trace amounts free 4-353-NP however was completely absent in theassays with the A githago cells The chromatographic behavior (TLC systems AndashD)of the radioactivity contained in all cell extracts pointed to glycosides as metabolicproducts of 4-353-NP With all TLC systems used the radioactivity associated withthe media proved to be preponderantly immobile Extraction of these 14C portionswith organic solvents such as ethyl acetate was not successful the same held true forhydrolysis procedures in order to cleave possible glycosidic linkages Additionallydirect HPLC analysis was not practicable since the necessary concentration of themedia resulted in highly viscous samples

The results obtained with the A githago cells were comparable to data publishedrecently on the biotransformation of 4-n-nonylphenol by the same cell cultureusing the same two-liquid-phase system[24] Concerning the intended identification ofsolublemetabolites uptake of 4-353-NP from the n-hexadecane layer by theA githagocells was considered satisfactory with both the 100 and 200 mg assays The same heldfor recoveries of 14C noticeable portions of soluble 14C in cell extracts and completedisappearance (turnover) of the applied 4-353-NP The different distribution ofradioactivity observed in the soybean experiment was referred to the conventionalone-liquid-phase system used but additionally also to the plant species[23] With bothplant species examined formation of glycosides from the phenolic xenobiotic orpossible primary transformation products was expected according to data publishedon 4-n-nonylphenol[11122324] The chemical nature of the radioactivity present inthe media especially of the soybean cells remained unknown Recently we found14C materials with similar (chromatographic) behavior in the media of plantcell suspensions (including soybean) after application of 14C-bisphenol A andsuspected polymerized products of the compound[27] Polymeres (or copolymers)may also have been formed from 4-353-NP

Hydrolysis Experiments Isolation and Identification of Metabolites

The combined cell extracts were subjected to two consecutive hydrolysissteps (2M HCl-methanol reflux 2M HCl reflux) in order to release the expected14C-aglycons from their corresponding glycosides In the case of soybean 86 of theradioactivity introduced into these experiments could be extracted (diethyl ether)from the reaction mixture With A githago recoveries in the hydrolysates amountedto totals of 70 (100 mg assays) and 63 (200 mg assays) All hydrolysates wereexamined using TLC and HPLC The results of TLC analysis of the samples(hydrolysis steps 1 and 2) derived from the A githogo study (Fig 2) demonstratedthat in three of the samples noticeable portions of 4-353-NP (Rf about 09) werepresent whereas in all samples 14C peaks at Rf about 05 and Rf 00 were detectedHPLC examination of the hydrolysates prevailingly confirmed these data radio-chromatograms of the samples derived from hydrolysis step 1 are shown in Fig 3In addition to a peak originating from 4-353-NP (Rt 310min) further 14C peaksappeared at Rt 150ndash200min Since the 14C peaks with Rf about 05 (TLC) and Rt

150ndash200min (HPLC) were not detected prior to hydrolysis the correspondingcompounds were regarded as primary metabolites of 4-353-NP Both 4-353-NP andprimary metabolites of 4-353-NP were thus liberated from their glycosides by acid

Metabolism of the Nonylphenol Isomer 539

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treatment Similar results were obtained from the soybean experiment (data notshown) Estimated from TLC and HPLC analysis the portions of primarymetabolites of 4-353-NP were between 19 and 42 of applied 14C (Table 1)depending on the method of calculation Low amounts of primary 4-353-NPmetabolites were detected with soybean this fact additionally supported theutilization of A githago cells in two-liquid-phase system for production andidentification of these transformation products

Figure 2 TLC radiochromatograms (solvent system B) of the samples derived from the

hydrolysis experiments A hydrolysis step 1 100 mg assays B hydrolysis step 1 200mg assays

C hydrolysis step 2 100 mg assays D hydrolysis step 2 200mg assays

Figure 3 HPLC radiochromatograms of samples derived from the hydrolysis experiments

A hydrolysis step 1 100mg assays B hydrolysis step 1 200 mg assays

540 Schmidt et al

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The radioactivity contained in the hydrolysate of the first hydrolytic step derivedfrom the 200 mg assays was separated by HPLC (using multiple runs) Two fractionswere isolated fraction 1 corresponding with the 14C peaks at Rt 150ndash200min andfraction 2 (4-353-NP Rt 310min) Total yields recovered after concentration ofHPLC eluates were 19 and 7mg with fraction 1 and fraction 2 respectively Bothfractions were derivatized with MSTFA (trimethylsilyl) and subjected to GC-EIMSanalyses the corresponding chromatograms are shown in Fig 4 The chromatogramof fraction 2 exhibited two peaks (IX and X Rt 1620ndash1640min) which accordingto their retention times and mass spectra (Table 2) were identified with the twodiastereomers of parent 4-353-NP While the [Mthorn] peak was observed at mz 292further prominent fragments emerged at mz 263 (-C2H5) 221 (-C5H11 base peak)193 179 and 73 (trimethylsilyl) According to data published on branched side-chain 4-nonylphenol isomers and 4-n-nonylphenol derivatives[2371216] the MSfragmentation scheme of 4-353-NP shown in Fig 5 is proposed The chemicalstructure of the fragment with mz 179 (trimethylsilyl derivative of hydroxyltropylium ion with mz 107) was deduced from recent literature data[3]

In the GC-EIMS chromatogram obtained from fraction 2 eight peaks weredetected (Rt 1860ndash2020min) which by means of their mass spectra could be tracedback to structures related to 4-353-NP (Fig 4) Seven of these compounds (IndashVII)exhibited an [Mthorn] at mz 380 and prominent fragments at mz 261 221 (base peak)

Figure 4 GC-EIMS chromatograms of the samples obtained from HPLC separation

(cf Fig 3) of the first hydrolysate derived from the 200 mg assays A fraction containing the

primary metabolites of 4-353-NP (peaks I and IV appeared as shoulders of II and V

respectively) B fraction containing nonmetabolized 4-353-NP

Metabolism of the Nonylphenol Isomer 541

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Table 2 Mass spectra of side-chain monohydroxylated metabolites (IndashVII) of 4-353-NP a

carboxylic acid metabolite (VII) of 4-353-NP and of 4-353-NP diastereomers (IX X) after

trimethylsilyl derivatization Compounds were detected using GC-EIMS analysis and were

contained in fractions A and B (obtained by HPLC separation) of a cell extract hydrolysate

(first hydrolysis step 200mg assays)

Peak

Rt EIMS

IsomeraPortionb

(min) mz (abundance in ) ()

Fraction 1

Ic 1865 380 (2) 365 (1) 351 (8) 275 (1) 261 (33)

233 (1) 221 (100) 206 (3) 193 (6) 191 (5)

179 (5) 151 (2) 117 (33) 73 (43)

unknown 214c

II 1870 380 (3) 365 (1) 351 (7) 275 (1) 261 (34)

233 (2) 221 (100) 206 (3) 193 (7) 191 (8)

179 (8) 163 (2) 151 (3) 117 (29) 73 (49)

60-OH-

4-353-NP

214c

III 1874 380 (2) 365 (1) 351 (5) 275 (1) 261 (34)

233 (1) 221 (100) 206 (4) 193 (6) 191 (6)

179 (8) 163 (2) 151 (3) 117 (32) 73 (50)

60-OH-

4-353-NP

175

IVd 1892 380 (2) 365 (1) 351 (6) 275 (1) 261 (36)

233 (2) 221 (100) 206 (4) 193 (7) 191 (6)

179 (8) 163 (2) 151 (3) 117 (27) 73 (52)

60-OH-

4-353-NP

473d

V 1896 380 (2) 365 (1) 351 (6) 275 (1) 261 (37)

233 (1) 221 (100) 206 (3) 193 (7) 191 (6)

179 (8) 163 (2) 151 (3) 117 (26) 73 (45)

60-OH-

4-353-NP

473d

VI 1925 380 (2) 365 (1) 351 (5) 336 (1) 309 (6) 295

(1) 261 (9) 233 (4) 221 (100) 206 (2) 193

(31) 191 (7) 179 (12) 163 (2) 151 (4) 147

(7) 117 (4) 103 (11) 73 (60)

10-OH-

4-353-NP

52

VII 1947 380 (2) 365 (1) 351 (5) 295 (2) 261 (8) 253

(3) 233 (3) 221 (100) 207 (4) 193 (21) 191

(6) 179 (10) 163 (6) 151 (2) 117 (3) 103

(4) 73 (47)

20-OH-

4-353-NP

59

VIII 2019 394 (2) 379 (4) 365 (11) 247 (2) 313 (4)

275 (2) 247 (4) 233 (33) 221 (100) 207 (2)

193 (11) 191 (4) 179 (8) 173 (9) 131 (3)

116 (3) 97 (6) 73 (40)

70-COOH-

4-353-NP

26

Fraction 2

IX 1629 292 (5) 277 (3) 263 (18) 221 (100) 207 (7)

193 (55) 179 (14) 163 (3) 151 (6) 135 (2)

129 (3) 115 (4) 103 (3) 91 (3) 73 (34)

4-353-NP 468

X 1638 292 (5) 277 (3) 263 (19) 221 (100) 207 (8)

193 (55) 179 (13) 163 (2) 151 (6) 135 (2)

129 (4) 115 (3) 103 (1) 91 (3) 73 (31)

4-353-NP 532

aStructures of metabolites IndashVIII of 4-353-NP are proposed according to EIMS fragmentation

patternsbCalculated from peak areas total area of peaks IndashVIII and IXndashX respectively corresponds

with 100cPeak I appeared as shoulder of II separate integration of peaks I and II was not possibledPeak IV appeared as shoulder of V separate integration of peaks IV and V was not possible

542 Schmidt et al

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179 and 73 complete mass spectra are listed in Table 2 The [Mthorn] of compoundsIndashVII pointed to mono-hydroxylated derivatives of 4-353-NP while all patterns offragmentation and their similarity with that of 4-353-NP (especially peaks at mz 221and 179) indicated that hydroxylation had exclusively occurred at the aliphatic side-chain Compound VIII had an [Mthorn] at mz 394 and prominent fragments at mz 221(base peak) 179 and 73 These findings pointed a (side-chain) carboxylic acidderivative of 4-353-NP With both fraction 1 and fraction 2 GC-EIMS peakscorresponding with possible ring-hydroxylated or side-chain olefinic products[24]

were not detected The hydrolysate derived from the soybean experiment wassimilarly separated by HPLC a fraction corresponding with primary 4-353-NPmetabolites was collected and examined by GC-EIMS after silylation Due tolow yield no distinct peaks which could be related to parent 4-353-NP were foundin the corresponding chromatograms as such However using the GC-EIMSfindings of the A githago study a trace was identified at Rt 1905min the massspectrum of which corresponded with compounds IV or V We concluded that theA githago and soybean cells at least demonstrated certain similarities concerningthe biotransformation of 4-353-NP

Though the GC-EIMS data unequivocally proved that 4-353-NP was metabo-lized by A githago cell suspensions at the aliphatic side-chain to monohydroxylatedproducts and a carboxylic acid derivative the sites of hydroxylationoxidation partlyremained unclear Compounds IIndashV emerged in the chromatograms as two pairs ofpeaks (IIIII and IVV) and exhibited noticeably similar mass spectra (Table 2)Besides [Mthorn] at mz 380 and fragments observed with all other monohydroxylatedmetabolites (mz 365 351 261 221 179 73) a prominent fragment of compoundsIIndashV hadmz 117 with 26ndash32 abundance According to data published on side-chain

Figure 5 Proposed MS fragmentation of 4(3050-dimethyl-30-heptyl)-phenol

Metabolism of the Nonylphenol Isomer 543

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monohydroxylated derivatives of both 4-n-nonylphenol[1224] and model side-chainisomers 2- 3- and 4-(4-hydroxyphenol)-nonane[4] fragment mz 117 was referred to[H3CCHOSi(CH3)3

thorn] This fragment is characteristic of trimethylsilyl derivatizedhydroxyl functions at secondary carbon atoms adjacent to terminal CH3

groups[4122428] Due to abundances (34ndash37) of fragments mz 261 (-HOSi(CH3)3-C2H5)

[2429] it was supposed that compounds IIndashV were 60-hydroxy derivatives of4-353-NP Applied 4-353-NP consisted of two diastereomers (each a couple ofenatiomers) hydroxylation at position 60 consequently introduced a third chiral centerwhich resulted in four diastereomers An MS fragmentation scheme for IIndashV isproposed in Fig 6 Quantitative evaluation of the chromatogram showed thatcompounds IIndashV were the main primary metabolites in A githago cells amountingto about 80 of the products identified

The mass spectrum of compound VI (Table 2) differed from those of productsIIndashV in particular regarding three fragments mz 309 (abundance 6) 295 (1) and103 (11) According to previous results presented on 4-n-nonylphenol[24] thefragment mz 103 was supposed to arise from [H2COSi(CH3)3

thorn] pointing tohydroxylation at a primary carbon[2428] Furthermore fragments mz 309 and 295indicated loss of C5H11 and C6H13 respectively from [Mthorn] (mz 380) with retentionof two silylated hydroxy functions at the remaining fragment ion It was thusassumed that compound VI was a 4-353-NP metabolite hydroxylated at position 10

of the side-chain Due to a similar line of argumentation (presence of fragments mz117 and 295) compound VII possibly consisted of the 20-hydroxy derivative of4-353-NP The structure of compound I remained unknown mainly becausemetabolite I was detected as shoulder of II resulting in large overlapping of its mass

Figure 6 Proposed MS fragmentation of the 6-hydroxy metabolites of 4(3050-dimethyl-

30-heptyl)-phenol

544 Schmidt et al

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spectrum with that of II Due to its [Mthorn] of mz 394 compound VIII was identifiedwith a carboxylic acid metabolite of 4-353-NP since formation of a hydroxyderivative with an additional carbonyl function was regarded unlikely The massspectrum observed with VIII supported this assumption Fragments showingsimilarity to compounds IndashVII were mz 379 (-CH3) and 365 (-C2H5) whereasfragments mz 131 and 116 were supposed to consist of frac12CH2COOSiethCH3THORN

thorn3 and

frac12CH2COOSiethCH3THORNthorn2 respectively which pointed to a carboxyl function at positions

10 or 70 A characteristic feature of the fragmentation of trimethylsilyl derivatizedfatty acids is separation of CH3 (15) and HOSi(CH3)2 (75) consecutively[30]

With VIII this would result in fragment mz 304 which was only present in thebackground However ions resulting from successive fragmentation of mz 304were observed mz 275 (-HCO -29) 247 (-C2H428) 233 (-CH214) and finally221 This sequence was regarded as prove that 4-353-NP was oxidized to itscarboxylic acid at position 70 of the molecule It should be mentioned that I VI VIIand VIII were only minor metabolites formed from 4-353-NP by the A githago cellsuspensions (Table 2)

Metabolism of 4-353-NP by Agrostemma githagoPlant Cell Suspension Cultures

The nonylphenol isomer 4-353-NP consisting of two diastereomers wastransformed by theAgrostemma githago suspension cultures to four types of productsglycosides of parent 4-353-NP glycosides of primary 4-353-NP metabolitesnonextractable residues and unknown possibly polymeric materials detected in themedia Similar products emerged in the soybean cultures The present investigationconcentrated on the primary metabolites of 4-353-NP These were produced in the Agithago suspensions using a two-liquid-phase system The primary products identifiedby GC-EIMS after liberation from their glycosides were one side-chain carboxylicacid and seven side-chain monohydroxylated metabolites Due to their mass spectrathe main products were four diastereomers of 60-hydroxy-4-353-NP

As yet data on the biotransformation of 4-353-NP are mainly not at handConcerning plant tissues (cell cultures plants) side-chain hydroxylated metaboliteswere reported on 4-n-nonylphenol[11122324] With A githago cells the main productwas 4-(80-hydroxy-n-nonyl)-phenol[24] ie 4-n-nonylphenol was metabolized bysubterminal oxidation corresponding with the main products observed with 4-353-NP in the present study Side-chain hydroxylated metabolites were also formed fromp-tert-octylphenol in barley plants[31] Experiments with model nonylphenols 2- 3-and 4-(4-hydroxyphenyl)-nonane demonstrated that in rainbow trout all isomerswere transformed to monohydroxy products with the site of oxidation at carbon 8(subterminal) of the nonyl residue The metabolites emerged as glucuronic acidconjugates while glucuronides of the parent nonylphenols were also formed[4] In asimilar study both side-chain and ring hydroxylated metabolites of 4-n-nonylphenolwere detected as glucuronic acid conjugates[9] Recently data on the metabolism ofthe nonylphenol isomer 4(3060-dimethyl-30-heptyl)-phenol in pond snails to a (ringhydroxylated) catechol derivative (with unmodified side-chain) were reported[1920]

Contrasting with 4-353-NP of the present study the isomer examined possessed

Metabolism of the Nonylphenol Isomer 545

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a branching at subterminal position 60 of the side-chain besides that at carbon 30 Thecatechol was found conjugated to glucuronic acid[1920] As technical productnonylphenol appears to be completely degradated in soil[1] though isomer 4-(3060-dimethyl-30-heptyl)-phenol proved to be resistant in sedimentwater systems[18] Upto now the routes of nonylphenolrsquos microbial degradation are not known in detailDue to experiments with an isolated bacterial strain initial oxidation at the aromaticmoiety leads to degradation of the phenyl ring while side-chains remain asnonanols[3233] one of the studies was executed with radio-labelled 4-353-NP[33]

The present data on the metabolism of 4-353-NP by A githago cell suspensionsagree with those reported in the literature on 4-n-nonylphenol in plant tissues andmodel isomers in rainbow trout In contrast with trout the capacity of the A githagocells for hydroxylation of the side-chain appeared to be broader ie the nonyl residuewas hydroxylated at different sites of the aliphatic chain The enzymes responsible forside-chain hydroxylation of 4-353-NP in A githago are not known we suspectedcytochromes P450 In rats and humans isozyme CYP2B2 was thought to catalyzepredominantly the hydroxylation of nonylphenol isomers[10] After expression inyeast human CYP2B6 and CYP2C19 were shown to metabolize nonylphenol (pluralbranched chains) while 4-n-nonylphenol was transformed by CYP1A2 CYP2B6 andCYP2C19 ring and side-chain hydroxlated products were identified with branchedchain nonylphenols and 4-n-nonylphenol[34] We assume that plants in general havethe capacity to transform nonylphenol isomers to side-chain hydroxylated productsand thus contribute to the environmental degradation of the xenoestrogen If cropplants are contaminated with nonylphenol both nonylphenol and its side-chainhydroxylated products are expected to be present in plant tissues as glycosideconjugates Xenobiotic glycosides are known to be cleaved in the gastrointestinal tractwith release of possibly toxic aglycons Data on the toxicological properties of side-chain hydroxylated nonylphenols are not available Our future research will focus onthe metabolism of nonylphenol isomers with different branching patterns on theidentification of the enzymes responsible for the metabolism of nonylphenols and onthe (eco-) toxicology of side-chain hydroxylated nonylphenol isomers

ACKNOWLEDGMENTS

For the synthesis of [ring-U-14C]-4(3050-dimethyl-30-heptyl)-phenol the authorswish to thank R Vinken and the lsquolsquoAachen Graduate College Elimination ofEndocrine-Disrupting Substances from Waste-Waterrsquorsquo (AGEESA) funded by theDeutsche Forschungsgemeinschaft

REFERENCES

1 Staples CA Williams JB Blessing RL Varineau PT Measuring thebiodegradability of nonylphenol ether carboxylates octylphenol ether carboxyl-ates and nonylphenol Chemosphere 1999 38 (9) 2029ndash2039

2 Thiele B Gunther K Schwuger MJ Alkylphenol ethoxylates trace analysisand environmental behaviour Chem Rev 1997 97 (8) 3247ndash3272

546 Schmidt et al

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3 Wheeler TF Heim JR LaTorre MR Janes AB Mass spectral

characterization of p-nonylphenol isomers using high-resolution capillary

GC-MS J Chromatogr Sci 1997 35 (1) 19ndash304 Meldahl AC Nithipatikom K Lech JJ Metabolism of several 14C-

nonylphenol isomers by rainbow trout (Oncorhynchus mykiss) in vivo and

in vitro Metabolites Xenobiotica 1996 26 (11) 1167ndash11805 Kim Y-S Katase T Sekine S Inoue T Makino M Uchiyama T

Fujimoto Y Yamashita N Variation in estrogenic activity among fractions

of a commercial nonylphenol by high performance liquid chromatography

Chemosphere 2004 54 (8) 1127ndash11346 Giger W Stephanou E Schaffner C Persistent organic chemicals in sewage

effluents 1 Identification of nonylphenols and nonylphenolethoxylates by

capillary gas chromatographymass spectrometry Chemosphere 1981 10 (11ndash1)

1253ndash12637 Giger W Brunner PH Schaffner C 4-Nonylphenol in sewage sludge

accumulation of toxic metabolites from nonionic surfactants Science 1984

225 (4662) 623ndash6258 Jobst H Chlorophenols an nonylphenols in sewage sludges 1 Occurrence in

sewage sludges of Western German treatment plants from 1987ndash1989 Acta

Hydrochim Hydrobiol 1995 23 (1) 20ndash259 Coldham NG Sivapathasundaram SDM Ashfield LA Pottinger TG

Biotransformation tissue distribution and persistence of 4-nonylphenol

residues in juvenile rainbow trout (Oncorhynchus mykiss) Drug Metab

Dispos 1998 26 (4) 347ndash35410 Lee PC Marquardt M Lech JJ Metabolism of nonylphenol by rat and

human microsomes Toxicol Lett 1998 99 (7) 117ndash12611 Bokern M Harms HH Toxicity and metabolism of 4-n-nonylphenol in cell

suspension cultures of different plant species Environ Sci Technol 1997

31 (7) 1849ndash185412 Bokern M Nimtz M Harms HH Metabolites of 4-n-nonylphenol in wheat

cell suspension cultures J Agric Food Chem 1996 44 (4) 1123ndash112713 Guenther K Heinke V Thiele B Kleist E Prast H Raecker T

Endocrine disrupting nonylphenols are ubiquitous in food Environ Sci

Technol 2002 36 (8) 1676ndash168014 Soto AM Justicia H Wray JW Sonnenschein C p-Nonylphenol an

estrogenic xenobiotic released from lsquolsquomodifiedrsquorsquo polystyrene Environ Health

Perspect 1991 92 167ndash17315 Degen DH Bolt HM Comments on lsquolsquoendocrine disrupting nonylphenols

are ubiquitous in foodrsquorsquo Envirn Sci Technol 2003 37 (11) 2622ndash262316 Vinken R Schmidt B Schaffer A Synthesis of tertiary 14C-labelled

nonylphenol isomers J Label Compd Radiopharm 2002 45 (14) 1253ndash126317 Lalah JO Lenoir D Henkelmann B Hertkorn N Gunther K

Schramm K-W Kettrup A Synthesis of ring-14C-labelled 4(3060-dimethyl-

30-heptyl)-phenol J Label Compd Radiopharm 2001 44 (6) 459ndash46318 Lalah JO Schramm K-W Henkelmann B Lenoir D Behechti A

Gunther K Kettrup A The dissipation distribution and fate of a branched

Metabolism of the Nonylphenol Isomer 547

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14C-nonylphenol isomer in lake watersediment systems Environ Pollut 2003

122 (2) 195ndash20319 Lalah JO Schramm K-W Severin GF Lenoir D Henkelmann B

Behechti A Gunther K Kettrup A In vivo metabolism and organ

distribution of a branched 14C-nonylphenol isomer in pond snails Lymnaea

stagnalis L Aquat Toxicol 2003 62 (4) 305ndash31920 Lalah JO Behechti A Severin GF Lenoir D Gunther K Kettrup A

Schramm K-W The Bioaccumulation and fate of a branched 14C-

p-nonylphenol isomer in Lymnaea stagnalis L Environ Toxicol Chem 2003

22 (7) 1428ndash143621 Schmidt B Metabolic profiling using plant cell suspension cultures In

Pesticide Transformation in Plants and Microorganisms Hall JC Hoagland

RE Zablotowicz RM Eds American Chemical Society Washington DC

2001 40ndash5622 Komoszliga D Langebartels C Sandermann H Metabolic processes for

organic chemicals in plants In Plant ContaminationmdashModeling and Simulation

of Organic Chemical Processes Trapp S Mc Farlane JC Eds Lewis

Publishers Boca Raton 1995 69ndash10323 Schmidt B Schuphan I Metabolism of the Xenoestrogens 4-n-nonylphenol

and bisphenol A in plant cell suspension cultures In Book of Abstracts 10th

IUPAC International Congress on the Chemistry of Crop Protection Basel

August 4ndash9 IUPAC Basel 2002 Vol 2 3424 Schmidt B Patti H Niewersch C Schuphan I Biotransformation of [ring-

U-14C]4-n-nonylphenol by Agrostemma githago cell culture in a two-liquid-

phase system Biotechnol Lett 2003 25 (16) 1375ndash138125 Murashige T Skoog F Revised medium for rapid growth and bio assay with

tobacco tissue cultures Physiol Plant 1962 15 473ndash49726 Gamborg OL Miller RA Ojima K Nutrient requirements of suspension

cultures of soybean root cells Exp Cell Res 1968 50 151ndash15827 Schmidt B Schuphan I Metabolism of the environmental estrogen bisphenol

A by plant cell suspension cultures Chemosphere 2002 49 (1) 51ndash5928 Diekman J Thomson JB Djerassi C Mass spectrometry and stereo-

chemical problems CLV Electron impact induced fragmentations and

rearrangements of some trimethylsilyl ethers of aliphatic glycols and related

compounds J Org Chem 1968 33 (6) 2271ndash228429 Diekman J Thomson JB Djerassi C Mass spectrometry and stereo-

chemical problems CXLI Electron impact induced fragmentations and

rearrangements of trimethylsilyl ethers amines and sulfides J Org Chem

1967 32 (12) 3904ndash391930 Odham G Fatty acids In Biochemical Applications of Mass Spectrometry

Waller GR Dermer OC Eds Wiley-Interscience New York 1980 First

Supplementary Volume 153ndash17131 Stolzenberg GE Olson PA Zaylskie RG Mansager ER Behavior and

fate of ethoxylated alkylphenol nonionic surfactant in barley plants J Agric

Food Chem 1982 30 (4) 637ndash644

548 Schmidt et al

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32 Tanghe T Dhooge W Verstraete W Isolation of a bacterial strain able todegrade branched nonylphenol Appl Environ Microbiol 1999 65 (2)746ndash751

33 Corvini PFX Vinken R Hommes G Schmidt B Dohmann MDegradation of the radioactive and non-labelled branched 4(3050-dimethyl-30-heptyl)-phenol nonylphenol isomer by Sphingomonas TTNP3 Biodegradation2003 15 (1) 1ndash10

34 Inui H Shiota N Motoi Y Ido Y Inoue T Kodama T Ohkawa YOhkawa H Metabolism of herbicides and other chemicals in humancytochrome P450 species and in transgenic potato plants co-expressinghuman CYP1A1 CYP2B6 and CYP2C19 J Pestic Sci 2001 26 (1) 28ndash40

Received February 13 2004

Metabolism of the Nonylphenol Isomer 549

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treatment Similar results were obtained from the soybean experiment (data notshown) Estimated from TLC and HPLC analysis the portions of primarymetabolites of 4-353-NP were between 19 and 42 of applied 14C (Table 1)depending on the method of calculation Low amounts of primary 4-353-NPmetabolites were detected with soybean this fact additionally supported theutilization of A githago cells in two-liquid-phase system for production andidentification of these transformation products

Figure 2 TLC radiochromatograms (solvent system B) of the samples derived from the

hydrolysis experiments A hydrolysis step 1 100 mg assays B hydrolysis step 1 200mg assays

C hydrolysis step 2 100 mg assays D hydrolysis step 2 200mg assays

Figure 3 HPLC radiochromatograms of samples derived from the hydrolysis experiments

A hydrolysis step 1 100mg assays B hydrolysis step 1 200 mg assays

540 Schmidt et al

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The radioactivity contained in the hydrolysate of the first hydrolytic step derivedfrom the 200 mg assays was separated by HPLC (using multiple runs) Two fractionswere isolated fraction 1 corresponding with the 14C peaks at Rt 150ndash200min andfraction 2 (4-353-NP Rt 310min) Total yields recovered after concentration ofHPLC eluates were 19 and 7mg with fraction 1 and fraction 2 respectively Bothfractions were derivatized with MSTFA (trimethylsilyl) and subjected to GC-EIMSanalyses the corresponding chromatograms are shown in Fig 4 The chromatogramof fraction 2 exhibited two peaks (IX and X Rt 1620ndash1640min) which accordingto their retention times and mass spectra (Table 2) were identified with the twodiastereomers of parent 4-353-NP While the [Mthorn] peak was observed at mz 292further prominent fragments emerged at mz 263 (-C2H5) 221 (-C5H11 base peak)193 179 and 73 (trimethylsilyl) According to data published on branched side-chain 4-nonylphenol isomers and 4-n-nonylphenol derivatives[2371216] the MSfragmentation scheme of 4-353-NP shown in Fig 5 is proposed The chemicalstructure of the fragment with mz 179 (trimethylsilyl derivative of hydroxyltropylium ion with mz 107) was deduced from recent literature data[3]

In the GC-EIMS chromatogram obtained from fraction 2 eight peaks weredetected (Rt 1860ndash2020min) which by means of their mass spectra could be tracedback to structures related to 4-353-NP (Fig 4) Seven of these compounds (IndashVII)exhibited an [Mthorn] at mz 380 and prominent fragments at mz 261 221 (base peak)

Figure 4 GC-EIMS chromatograms of the samples obtained from HPLC separation

(cf Fig 3) of the first hydrolysate derived from the 200 mg assays A fraction containing the

primary metabolites of 4-353-NP (peaks I and IV appeared as shoulders of II and V

respectively) B fraction containing nonmetabolized 4-353-NP

Metabolism of the Nonylphenol Isomer 541

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Table 2 Mass spectra of side-chain monohydroxylated metabolites (IndashVII) of 4-353-NP a

carboxylic acid metabolite (VII) of 4-353-NP and of 4-353-NP diastereomers (IX X) after

trimethylsilyl derivatization Compounds were detected using GC-EIMS analysis and were

contained in fractions A and B (obtained by HPLC separation) of a cell extract hydrolysate

(first hydrolysis step 200mg assays)

Peak

Rt EIMS

IsomeraPortionb

(min) mz (abundance in ) ()

Fraction 1

Ic 1865 380 (2) 365 (1) 351 (8) 275 (1) 261 (33)

233 (1) 221 (100) 206 (3) 193 (6) 191 (5)

179 (5) 151 (2) 117 (33) 73 (43)

unknown 214c

II 1870 380 (3) 365 (1) 351 (7) 275 (1) 261 (34)

233 (2) 221 (100) 206 (3) 193 (7) 191 (8)

179 (8) 163 (2) 151 (3) 117 (29) 73 (49)

60-OH-

4-353-NP

214c

III 1874 380 (2) 365 (1) 351 (5) 275 (1) 261 (34)

233 (1) 221 (100) 206 (4) 193 (6) 191 (6)

179 (8) 163 (2) 151 (3) 117 (32) 73 (50)

60-OH-

4-353-NP

175

IVd 1892 380 (2) 365 (1) 351 (6) 275 (1) 261 (36)

233 (2) 221 (100) 206 (4) 193 (7) 191 (6)

179 (8) 163 (2) 151 (3) 117 (27) 73 (52)

60-OH-

4-353-NP

473d

V 1896 380 (2) 365 (1) 351 (6) 275 (1) 261 (37)

233 (1) 221 (100) 206 (3) 193 (7) 191 (6)

179 (8) 163 (2) 151 (3) 117 (26) 73 (45)

60-OH-

4-353-NP

473d

VI 1925 380 (2) 365 (1) 351 (5) 336 (1) 309 (6) 295

(1) 261 (9) 233 (4) 221 (100) 206 (2) 193

(31) 191 (7) 179 (12) 163 (2) 151 (4) 147

(7) 117 (4) 103 (11) 73 (60)

10-OH-

4-353-NP

52

VII 1947 380 (2) 365 (1) 351 (5) 295 (2) 261 (8) 253

(3) 233 (3) 221 (100) 207 (4) 193 (21) 191

(6) 179 (10) 163 (6) 151 (2) 117 (3) 103

(4) 73 (47)

20-OH-

4-353-NP

59

VIII 2019 394 (2) 379 (4) 365 (11) 247 (2) 313 (4)

275 (2) 247 (4) 233 (33) 221 (100) 207 (2)

193 (11) 191 (4) 179 (8) 173 (9) 131 (3)

116 (3) 97 (6) 73 (40)

70-COOH-

4-353-NP

26

Fraction 2

IX 1629 292 (5) 277 (3) 263 (18) 221 (100) 207 (7)

193 (55) 179 (14) 163 (3) 151 (6) 135 (2)

129 (3) 115 (4) 103 (3) 91 (3) 73 (34)

4-353-NP 468

X 1638 292 (5) 277 (3) 263 (19) 221 (100) 207 (8)

193 (55) 179 (13) 163 (2) 151 (6) 135 (2)

129 (4) 115 (3) 103 (1) 91 (3) 73 (31)

4-353-NP 532

aStructures of metabolites IndashVIII of 4-353-NP are proposed according to EIMS fragmentation

patternsbCalculated from peak areas total area of peaks IndashVIII and IXndashX respectively corresponds

with 100cPeak I appeared as shoulder of II separate integration of peaks I and II was not possibledPeak IV appeared as shoulder of V separate integration of peaks IV and V was not possible

542 Schmidt et al

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179 and 73 complete mass spectra are listed in Table 2 The [Mthorn] of compoundsIndashVII pointed to mono-hydroxylated derivatives of 4-353-NP while all patterns offragmentation and their similarity with that of 4-353-NP (especially peaks at mz 221and 179) indicated that hydroxylation had exclusively occurred at the aliphatic side-chain Compound VIII had an [Mthorn] at mz 394 and prominent fragments at mz 221(base peak) 179 and 73 These findings pointed a (side-chain) carboxylic acidderivative of 4-353-NP With both fraction 1 and fraction 2 GC-EIMS peakscorresponding with possible ring-hydroxylated or side-chain olefinic products[24]

were not detected The hydrolysate derived from the soybean experiment wassimilarly separated by HPLC a fraction corresponding with primary 4-353-NPmetabolites was collected and examined by GC-EIMS after silylation Due tolow yield no distinct peaks which could be related to parent 4-353-NP were foundin the corresponding chromatograms as such However using the GC-EIMSfindings of the A githago study a trace was identified at Rt 1905min the massspectrum of which corresponded with compounds IV or V We concluded that theA githago and soybean cells at least demonstrated certain similarities concerningthe biotransformation of 4-353-NP

Though the GC-EIMS data unequivocally proved that 4-353-NP was metabo-lized by A githago cell suspensions at the aliphatic side-chain to monohydroxylatedproducts and a carboxylic acid derivative the sites of hydroxylationoxidation partlyremained unclear Compounds IIndashV emerged in the chromatograms as two pairs ofpeaks (IIIII and IVV) and exhibited noticeably similar mass spectra (Table 2)Besides [Mthorn] at mz 380 and fragments observed with all other monohydroxylatedmetabolites (mz 365 351 261 221 179 73) a prominent fragment of compoundsIIndashV hadmz 117 with 26ndash32 abundance According to data published on side-chain

Figure 5 Proposed MS fragmentation of 4(3050-dimethyl-30-heptyl)-phenol

Metabolism of the Nonylphenol Isomer 543

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monohydroxylated derivatives of both 4-n-nonylphenol[1224] and model side-chainisomers 2- 3- and 4-(4-hydroxyphenol)-nonane[4] fragment mz 117 was referred to[H3CCHOSi(CH3)3

thorn] This fragment is characteristic of trimethylsilyl derivatizedhydroxyl functions at secondary carbon atoms adjacent to terminal CH3

groups[4122428] Due to abundances (34ndash37) of fragments mz 261 (-HOSi(CH3)3-C2H5)

[2429] it was supposed that compounds IIndashV were 60-hydroxy derivatives of4-353-NP Applied 4-353-NP consisted of two diastereomers (each a couple ofenatiomers) hydroxylation at position 60 consequently introduced a third chiral centerwhich resulted in four diastereomers An MS fragmentation scheme for IIndashV isproposed in Fig 6 Quantitative evaluation of the chromatogram showed thatcompounds IIndashV were the main primary metabolites in A githago cells amountingto about 80 of the products identified

The mass spectrum of compound VI (Table 2) differed from those of productsIIndashV in particular regarding three fragments mz 309 (abundance 6) 295 (1) and103 (11) According to previous results presented on 4-n-nonylphenol[24] thefragment mz 103 was supposed to arise from [H2COSi(CH3)3

thorn] pointing tohydroxylation at a primary carbon[2428] Furthermore fragments mz 309 and 295indicated loss of C5H11 and C6H13 respectively from [Mthorn] (mz 380) with retentionof two silylated hydroxy functions at the remaining fragment ion It was thusassumed that compound VI was a 4-353-NP metabolite hydroxylated at position 10

of the side-chain Due to a similar line of argumentation (presence of fragments mz117 and 295) compound VII possibly consisted of the 20-hydroxy derivative of4-353-NP The structure of compound I remained unknown mainly becausemetabolite I was detected as shoulder of II resulting in large overlapping of its mass

Figure 6 Proposed MS fragmentation of the 6-hydroxy metabolites of 4(3050-dimethyl-

30-heptyl)-phenol

544 Schmidt et al

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spectrum with that of II Due to its [Mthorn] of mz 394 compound VIII was identifiedwith a carboxylic acid metabolite of 4-353-NP since formation of a hydroxyderivative with an additional carbonyl function was regarded unlikely The massspectrum observed with VIII supported this assumption Fragments showingsimilarity to compounds IndashVII were mz 379 (-CH3) and 365 (-C2H5) whereasfragments mz 131 and 116 were supposed to consist of frac12CH2COOSiethCH3THORN

thorn3 and

frac12CH2COOSiethCH3THORNthorn2 respectively which pointed to a carboxyl function at positions

10 or 70 A characteristic feature of the fragmentation of trimethylsilyl derivatizedfatty acids is separation of CH3 (15) and HOSi(CH3)2 (75) consecutively[30]

With VIII this would result in fragment mz 304 which was only present in thebackground However ions resulting from successive fragmentation of mz 304were observed mz 275 (-HCO -29) 247 (-C2H428) 233 (-CH214) and finally221 This sequence was regarded as prove that 4-353-NP was oxidized to itscarboxylic acid at position 70 of the molecule It should be mentioned that I VI VIIand VIII were only minor metabolites formed from 4-353-NP by the A githago cellsuspensions (Table 2)

Metabolism of 4-353-NP by Agrostemma githagoPlant Cell Suspension Cultures

The nonylphenol isomer 4-353-NP consisting of two diastereomers wastransformed by theAgrostemma githago suspension cultures to four types of productsglycosides of parent 4-353-NP glycosides of primary 4-353-NP metabolitesnonextractable residues and unknown possibly polymeric materials detected in themedia Similar products emerged in the soybean cultures The present investigationconcentrated on the primary metabolites of 4-353-NP These were produced in the Agithago suspensions using a two-liquid-phase system The primary products identifiedby GC-EIMS after liberation from their glycosides were one side-chain carboxylicacid and seven side-chain monohydroxylated metabolites Due to their mass spectrathe main products were four diastereomers of 60-hydroxy-4-353-NP

As yet data on the biotransformation of 4-353-NP are mainly not at handConcerning plant tissues (cell cultures plants) side-chain hydroxylated metaboliteswere reported on 4-n-nonylphenol[11122324] With A githago cells the main productwas 4-(80-hydroxy-n-nonyl)-phenol[24] ie 4-n-nonylphenol was metabolized bysubterminal oxidation corresponding with the main products observed with 4-353-NP in the present study Side-chain hydroxylated metabolites were also formed fromp-tert-octylphenol in barley plants[31] Experiments with model nonylphenols 2- 3-and 4-(4-hydroxyphenyl)-nonane demonstrated that in rainbow trout all isomerswere transformed to monohydroxy products with the site of oxidation at carbon 8(subterminal) of the nonyl residue The metabolites emerged as glucuronic acidconjugates while glucuronides of the parent nonylphenols were also formed[4] In asimilar study both side-chain and ring hydroxylated metabolites of 4-n-nonylphenolwere detected as glucuronic acid conjugates[9] Recently data on the metabolism ofthe nonylphenol isomer 4(3060-dimethyl-30-heptyl)-phenol in pond snails to a (ringhydroxylated) catechol derivative (with unmodified side-chain) were reported[1920]

Contrasting with 4-353-NP of the present study the isomer examined possessed

Metabolism of the Nonylphenol Isomer 545

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a branching at subterminal position 60 of the side-chain besides that at carbon 30 Thecatechol was found conjugated to glucuronic acid[1920] As technical productnonylphenol appears to be completely degradated in soil[1] though isomer 4-(3060-dimethyl-30-heptyl)-phenol proved to be resistant in sedimentwater systems[18] Upto now the routes of nonylphenolrsquos microbial degradation are not known in detailDue to experiments with an isolated bacterial strain initial oxidation at the aromaticmoiety leads to degradation of the phenyl ring while side-chains remain asnonanols[3233] one of the studies was executed with radio-labelled 4-353-NP[33]

The present data on the metabolism of 4-353-NP by A githago cell suspensionsagree with those reported in the literature on 4-n-nonylphenol in plant tissues andmodel isomers in rainbow trout In contrast with trout the capacity of the A githagocells for hydroxylation of the side-chain appeared to be broader ie the nonyl residuewas hydroxylated at different sites of the aliphatic chain The enzymes responsible forside-chain hydroxylation of 4-353-NP in A githago are not known we suspectedcytochromes P450 In rats and humans isozyme CYP2B2 was thought to catalyzepredominantly the hydroxylation of nonylphenol isomers[10] After expression inyeast human CYP2B6 and CYP2C19 were shown to metabolize nonylphenol (pluralbranched chains) while 4-n-nonylphenol was transformed by CYP1A2 CYP2B6 andCYP2C19 ring and side-chain hydroxlated products were identified with branchedchain nonylphenols and 4-n-nonylphenol[34] We assume that plants in general havethe capacity to transform nonylphenol isomers to side-chain hydroxylated productsand thus contribute to the environmental degradation of the xenoestrogen If cropplants are contaminated with nonylphenol both nonylphenol and its side-chainhydroxylated products are expected to be present in plant tissues as glycosideconjugates Xenobiotic glycosides are known to be cleaved in the gastrointestinal tractwith release of possibly toxic aglycons Data on the toxicological properties of side-chain hydroxylated nonylphenols are not available Our future research will focus onthe metabolism of nonylphenol isomers with different branching patterns on theidentification of the enzymes responsible for the metabolism of nonylphenols and onthe (eco-) toxicology of side-chain hydroxylated nonylphenol isomers

ACKNOWLEDGMENTS

For the synthesis of [ring-U-14C]-4(3050-dimethyl-30-heptyl)-phenol the authorswish to thank R Vinken and the lsquolsquoAachen Graduate College Elimination ofEndocrine-Disrupting Substances from Waste-Waterrsquorsquo (AGEESA) funded by theDeutsche Forschungsgemeinschaft

REFERENCES

1 Staples CA Williams JB Blessing RL Varineau PT Measuring thebiodegradability of nonylphenol ether carboxylates octylphenol ether carboxyl-ates and nonylphenol Chemosphere 1999 38 (9) 2029ndash2039

2 Thiele B Gunther K Schwuger MJ Alkylphenol ethoxylates trace analysisand environmental behaviour Chem Rev 1997 97 (8) 3247ndash3272

546 Schmidt et al

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ORDER REPRINTS

3 Wheeler TF Heim JR LaTorre MR Janes AB Mass spectral

characterization of p-nonylphenol isomers using high-resolution capillary

GC-MS J Chromatogr Sci 1997 35 (1) 19ndash304 Meldahl AC Nithipatikom K Lech JJ Metabolism of several 14C-

nonylphenol isomers by rainbow trout (Oncorhynchus mykiss) in vivo and

in vitro Metabolites Xenobiotica 1996 26 (11) 1167ndash11805 Kim Y-S Katase T Sekine S Inoue T Makino M Uchiyama T

Fujimoto Y Yamashita N Variation in estrogenic activity among fractions

of a commercial nonylphenol by high performance liquid chromatography

Chemosphere 2004 54 (8) 1127ndash11346 Giger W Stephanou E Schaffner C Persistent organic chemicals in sewage

effluents 1 Identification of nonylphenols and nonylphenolethoxylates by

capillary gas chromatographymass spectrometry Chemosphere 1981 10 (11ndash1)

1253ndash12637 Giger W Brunner PH Schaffner C 4-Nonylphenol in sewage sludge

accumulation of toxic metabolites from nonionic surfactants Science 1984

225 (4662) 623ndash6258 Jobst H Chlorophenols an nonylphenols in sewage sludges 1 Occurrence in

sewage sludges of Western German treatment plants from 1987ndash1989 Acta

Hydrochim Hydrobiol 1995 23 (1) 20ndash259 Coldham NG Sivapathasundaram SDM Ashfield LA Pottinger TG

Biotransformation tissue distribution and persistence of 4-nonylphenol

residues in juvenile rainbow trout (Oncorhynchus mykiss) Drug Metab

Dispos 1998 26 (4) 347ndash35410 Lee PC Marquardt M Lech JJ Metabolism of nonylphenol by rat and

human microsomes Toxicol Lett 1998 99 (7) 117ndash12611 Bokern M Harms HH Toxicity and metabolism of 4-n-nonylphenol in cell

suspension cultures of different plant species Environ Sci Technol 1997

31 (7) 1849ndash185412 Bokern M Nimtz M Harms HH Metabolites of 4-n-nonylphenol in wheat

cell suspension cultures J Agric Food Chem 1996 44 (4) 1123ndash112713 Guenther K Heinke V Thiele B Kleist E Prast H Raecker T

Endocrine disrupting nonylphenols are ubiquitous in food Environ Sci

Technol 2002 36 (8) 1676ndash168014 Soto AM Justicia H Wray JW Sonnenschein C p-Nonylphenol an

estrogenic xenobiotic released from lsquolsquomodifiedrsquorsquo polystyrene Environ Health

Perspect 1991 92 167ndash17315 Degen DH Bolt HM Comments on lsquolsquoendocrine disrupting nonylphenols

are ubiquitous in foodrsquorsquo Envirn Sci Technol 2003 37 (11) 2622ndash262316 Vinken R Schmidt B Schaffer A Synthesis of tertiary 14C-labelled

nonylphenol isomers J Label Compd Radiopharm 2002 45 (14) 1253ndash126317 Lalah JO Lenoir D Henkelmann B Hertkorn N Gunther K

Schramm K-W Kettrup A Synthesis of ring-14C-labelled 4(3060-dimethyl-

30-heptyl)-phenol J Label Compd Radiopharm 2001 44 (6) 459ndash46318 Lalah JO Schramm K-W Henkelmann B Lenoir D Behechti A

Gunther K Kettrup A The dissipation distribution and fate of a branched

Metabolism of the Nonylphenol Isomer 547

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14C-nonylphenol isomer in lake watersediment systems Environ Pollut 2003

122 (2) 195ndash20319 Lalah JO Schramm K-W Severin GF Lenoir D Henkelmann B

Behechti A Gunther K Kettrup A In vivo metabolism and organ

distribution of a branched 14C-nonylphenol isomer in pond snails Lymnaea

stagnalis L Aquat Toxicol 2003 62 (4) 305ndash31920 Lalah JO Behechti A Severin GF Lenoir D Gunther K Kettrup A

Schramm K-W The Bioaccumulation and fate of a branched 14C-

p-nonylphenol isomer in Lymnaea stagnalis L Environ Toxicol Chem 2003

22 (7) 1428ndash143621 Schmidt B Metabolic profiling using plant cell suspension cultures In

Pesticide Transformation in Plants and Microorganisms Hall JC Hoagland

RE Zablotowicz RM Eds American Chemical Society Washington DC

2001 40ndash5622 Komoszliga D Langebartels C Sandermann H Metabolic processes for

organic chemicals in plants In Plant ContaminationmdashModeling and Simulation

of Organic Chemical Processes Trapp S Mc Farlane JC Eds Lewis

Publishers Boca Raton 1995 69ndash10323 Schmidt B Schuphan I Metabolism of the Xenoestrogens 4-n-nonylphenol

and bisphenol A in plant cell suspension cultures In Book of Abstracts 10th

IUPAC International Congress on the Chemistry of Crop Protection Basel

August 4ndash9 IUPAC Basel 2002 Vol 2 3424 Schmidt B Patti H Niewersch C Schuphan I Biotransformation of [ring-

U-14C]4-n-nonylphenol by Agrostemma githago cell culture in a two-liquid-

phase system Biotechnol Lett 2003 25 (16) 1375ndash138125 Murashige T Skoog F Revised medium for rapid growth and bio assay with

tobacco tissue cultures Physiol Plant 1962 15 473ndash49726 Gamborg OL Miller RA Ojima K Nutrient requirements of suspension

cultures of soybean root cells Exp Cell Res 1968 50 151ndash15827 Schmidt B Schuphan I Metabolism of the environmental estrogen bisphenol

A by plant cell suspension cultures Chemosphere 2002 49 (1) 51ndash5928 Diekman J Thomson JB Djerassi C Mass spectrometry and stereo-

chemical problems CLV Electron impact induced fragmentations and

rearrangements of some trimethylsilyl ethers of aliphatic glycols and related

compounds J Org Chem 1968 33 (6) 2271ndash228429 Diekman J Thomson JB Djerassi C Mass spectrometry and stereo-

chemical problems CXLI Electron impact induced fragmentations and

rearrangements of trimethylsilyl ethers amines and sulfides J Org Chem

1967 32 (12) 3904ndash391930 Odham G Fatty acids In Biochemical Applications of Mass Spectrometry

Waller GR Dermer OC Eds Wiley-Interscience New York 1980 First

Supplementary Volume 153ndash17131 Stolzenberg GE Olson PA Zaylskie RG Mansager ER Behavior and

fate of ethoxylated alkylphenol nonionic surfactant in barley plants J Agric

Food Chem 1982 30 (4) 637ndash644

548 Schmidt et al

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32 Tanghe T Dhooge W Verstraete W Isolation of a bacterial strain able todegrade branched nonylphenol Appl Environ Microbiol 1999 65 (2)746ndash751

33 Corvini PFX Vinken R Hommes G Schmidt B Dohmann MDegradation of the radioactive and non-labelled branched 4(3050-dimethyl-30-heptyl)-phenol nonylphenol isomer by Sphingomonas TTNP3 Biodegradation2003 15 (1) 1ndash10

34 Inui H Shiota N Motoi Y Ido Y Inoue T Kodama T Ohkawa YOhkawa H Metabolism of herbicides and other chemicals in humancytochrome P450 species and in transgenic potato plants co-expressinghuman CYP1A1 CYP2B6 and CYP2C19 J Pestic Sci 2001 26 (1) 28ndash40

Received February 13 2004

Metabolism of the Nonylphenol Isomer 549

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Request PermissionOrder Reprints

Reprints of this article can also be ordered at

httpwwwdekkercomservletproductDOI101081PFC200026776

Request Permission or Order Reprints Instantly

Interested in copying and sharing this article In most cases US Copyright Law requires that you get permission from the articlersquos rightsholder before using copyrighted content

All information and materials found in this article including but not limited to text trademarks patents logos graphics and images (the Materials) are the copyrighted works and other forms of intellectual property of Marcel Dekker Inc or its licensors All rights not expressly granted are reserved

Get permission to lawfully reproduce and distribute the Materials or order reprints quickly and painlessly Simply click on the Request Permission Order Reprints link below and follow the instructions Visit the US Copyright Office for information on Fair Use limitations of US copyright law Please refer to The Association of American Publishersrsquo (AAP) website for guidelines on Fair Use in the Classroom

The Materials are for your personal use only and cannot be reformatted reposted resold or distributed by electronic means or otherwise without permission from Marcel Dekker Inc Marcel Dekker Inc grants you the limited right to display the Materials only on your personal computer or personal wireless device and to copy and download single copies of such Materials provided that any copyright trademark or other notice appearing on such Materials is also retained by displayed copied or downloaded as part of the Materials and is not removed or obscured and provided you do not edit modify alter or enhance the Materials Please refer to our Website User Agreement for more details

Dow

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The radioactivity contained in the hydrolysate of the first hydrolytic step derivedfrom the 200 mg assays was separated by HPLC (using multiple runs) Two fractionswere isolated fraction 1 corresponding with the 14C peaks at Rt 150ndash200min andfraction 2 (4-353-NP Rt 310min) Total yields recovered after concentration ofHPLC eluates were 19 and 7mg with fraction 1 and fraction 2 respectively Bothfractions were derivatized with MSTFA (trimethylsilyl) and subjected to GC-EIMSanalyses the corresponding chromatograms are shown in Fig 4 The chromatogramof fraction 2 exhibited two peaks (IX and X Rt 1620ndash1640min) which accordingto their retention times and mass spectra (Table 2) were identified with the twodiastereomers of parent 4-353-NP While the [Mthorn] peak was observed at mz 292further prominent fragments emerged at mz 263 (-C2H5) 221 (-C5H11 base peak)193 179 and 73 (trimethylsilyl) According to data published on branched side-chain 4-nonylphenol isomers and 4-n-nonylphenol derivatives[2371216] the MSfragmentation scheme of 4-353-NP shown in Fig 5 is proposed The chemicalstructure of the fragment with mz 179 (trimethylsilyl derivative of hydroxyltropylium ion with mz 107) was deduced from recent literature data[3]

In the GC-EIMS chromatogram obtained from fraction 2 eight peaks weredetected (Rt 1860ndash2020min) which by means of their mass spectra could be tracedback to structures related to 4-353-NP (Fig 4) Seven of these compounds (IndashVII)exhibited an [Mthorn] at mz 380 and prominent fragments at mz 261 221 (base peak)

Figure 4 GC-EIMS chromatograms of the samples obtained from HPLC separation

(cf Fig 3) of the first hydrolysate derived from the 200 mg assays A fraction containing the

primary metabolites of 4-353-NP (peaks I and IV appeared as shoulders of II and V

respectively) B fraction containing nonmetabolized 4-353-NP

Metabolism of the Nonylphenol Isomer 541

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Table 2 Mass spectra of side-chain monohydroxylated metabolites (IndashVII) of 4-353-NP a

carboxylic acid metabolite (VII) of 4-353-NP and of 4-353-NP diastereomers (IX X) after

trimethylsilyl derivatization Compounds were detected using GC-EIMS analysis and were

contained in fractions A and B (obtained by HPLC separation) of a cell extract hydrolysate

(first hydrolysis step 200mg assays)

Peak

Rt EIMS

IsomeraPortionb

(min) mz (abundance in ) ()

Fraction 1

Ic 1865 380 (2) 365 (1) 351 (8) 275 (1) 261 (33)

233 (1) 221 (100) 206 (3) 193 (6) 191 (5)

179 (5) 151 (2) 117 (33) 73 (43)

unknown 214c

II 1870 380 (3) 365 (1) 351 (7) 275 (1) 261 (34)

233 (2) 221 (100) 206 (3) 193 (7) 191 (8)

179 (8) 163 (2) 151 (3) 117 (29) 73 (49)

60-OH-

4-353-NP

214c

III 1874 380 (2) 365 (1) 351 (5) 275 (1) 261 (34)

233 (1) 221 (100) 206 (4) 193 (6) 191 (6)

179 (8) 163 (2) 151 (3) 117 (32) 73 (50)

60-OH-

4-353-NP

175

IVd 1892 380 (2) 365 (1) 351 (6) 275 (1) 261 (36)

233 (2) 221 (100) 206 (4) 193 (7) 191 (6)

179 (8) 163 (2) 151 (3) 117 (27) 73 (52)

60-OH-

4-353-NP

473d

V 1896 380 (2) 365 (1) 351 (6) 275 (1) 261 (37)

233 (1) 221 (100) 206 (3) 193 (7) 191 (6)

179 (8) 163 (2) 151 (3) 117 (26) 73 (45)

60-OH-

4-353-NP

473d

VI 1925 380 (2) 365 (1) 351 (5) 336 (1) 309 (6) 295

(1) 261 (9) 233 (4) 221 (100) 206 (2) 193

(31) 191 (7) 179 (12) 163 (2) 151 (4) 147

(7) 117 (4) 103 (11) 73 (60)

10-OH-

4-353-NP

52

VII 1947 380 (2) 365 (1) 351 (5) 295 (2) 261 (8) 253

(3) 233 (3) 221 (100) 207 (4) 193 (21) 191

(6) 179 (10) 163 (6) 151 (2) 117 (3) 103

(4) 73 (47)

20-OH-

4-353-NP

59

VIII 2019 394 (2) 379 (4) 365 (11) 247 (2) 313 (4)

275 (2) 247 (4) 233 (33) 221 (100) 207 (2)

193 (11) 191 (4) 179 (8) 173 (9) 131 (3)

116 (3) 97 (6) 73 (40)

70-COOH-

4-353-NP

26

Fraction 2

IX 1629 292 (5) 277 (3) 263 (18) 221 (100) 207 (7)

193 (55) 179 (14) 163 (3) 151 (6) 135 (2)

129 (3) 115 (4) 103 (3) 91 (3) 73 (34)

4-353-NP 468

X 1638 292 (5) 277 (3) 263 (19) 221 (100) 207 (8)

193 (55) 179 (13) 163 (2) 151 (6) 135 (2)

129 (4) 115 (3) 103 (1) 91 (3) 73 (31)

4-353-NP 532

aStructures of metabolites IndashVIII of 4-353-NP are proposed according to EIMS fragmentation

patternsbCalculated from peak areas total area of peaks IndashVIII and IXndashX respectively corresponds

with 100cPeak I appeared as shoulder of II separate integration of peaks I and II was not possibledPeak IV appeared as shoulder of V separate integration of peaks IV and V was not possible

542 Schmidt et al

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179 and 73 complete mass spectra are listed in Table 2 The [Mthorn] of compoundsIndashVII pointed to mono-hydroxylated derivatives of 4-353-NP while all patterns offragmentation and their similarity with that of 4-353-NP (especially peaks at mz 221and 179) indicated that hydroxylation had exclusively occurred at the aliphatic side-chain Compound VIII had an [Mthorn] at mz 394 and prominent fragments at mz 221(base peak) 179 and 73 These findings pointed a (side-chain) carboxylic acidderivative of 4-353-NP With both fraction 1 and fraction 2 GC-EIMS peakscorresponding with possible ring-hydroxylated or side-chain olefinic products[24]

were not detected The hydrolysate derived from the soybean experiment wassimilarly separated by HPLC a fraction corresponding with primary 4-353-NPmetabolites was collected and examined by GC-EIMS after silylation Due tolow yield no distinct peaks which could be related to parent 4-353-NP were foundin the corresponding chromatograms as such However using the GC-EIMSfindings of the A githago study a trace was identified at Rt 1905min the massspectrum of which corresponded with compounds IV or V We concluded that theA githago and soybean cells at least demonstrated certain similarities concerningthe biotransformation of 4-353-NP

Though the GC-EIMS data unequivocally proved that 4-353-NP was metabo-lized by A githago cell suspensions at the aliphatic side-chain to monohydroxylatedproducts and a carboxylic acid derivative the sites of hydroxylationoxidation partlyremained unclear Compounds IIndashV emerged in the chromatograms as two pairs ofpeaks (IIIII and IVV) and exhibited noticeably similar mass spectra (Table 2)Besides [Mthorn] at mz 380 and fragments observed with all other monohydroxylatedmetabolites (mz 365 351 261 221 179 73) a prominent fragment of compoundsIIndashV hadmz 117 with 26ndash32 abundance According to data published on side-chain

Figure 5 Proposed MS fragmentation of 4(3050-dimethyl-30-heptyl)-phenol

Metabolism of the Nonylphenol Isomer 543

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monohydroxylated derivatives of both 4-n-nonylphenol[1224] and model side-chainisomers 2- 3- and 4-(4-hydroxyphenol)-nonane[4] fragment mz 117 was referred to[H3CCHOSi(CH3)3

thorn] This fragment is characteristic of trimethylsilyl derivatizedhydroxyl functions at secondary carbon atoms adjacent to terminal CH3

groups[4122428] Due to abundances (34ndash37) of fragments mz 261 (-HOSi(CH3)3-C2H5)

[2429] it was supposed that compounds IIndashV were 60-hydroxy derivatives of4-353-NP Applied 4-353-NP consisted of two diastereomers (each a couple ofenatiomers) hydroxylation at position 60 consequently introduced a third chiral centerwhich resulted in four diastereomers An MS fragmentation scheme for IIndashV isproposed in Fig 6 Quantitative evaluation of the chromatogram showed thatcompounds IIndashV were the main primary metabolites in A githago cells amountingto about 80 of the products identified

The mass spectrum of compound VI (Table 2) differed from those of productsIIndashV in particular regarding three fragments mz 309 (abundance 6) 295 (1) and103 (11) According to previous results presented on 4-n-nonylphenol[24] thefragment mz 103 was supposed to arise from [H2COSi(CH3)3

thorn] pointing tohydroxylation at a primary carbon[2428] Furthermore fragments mz 309 and 295indicated loss of C5H11 and C6H13 respectively from [Mthorn] (mz 380) with retentionof two silylated hydroxy functions at the remaining fragment ion It was thusassumed that compound VI was a 4-353-NP metabolite hydroxylated at position 10

of the side-chain Due to a similar line of argumentation (presence of fragments mz117 and 295) compound VII possibly consisted of the 20-hydroxy derivative of4-353-NP The structure of compound I remained unknown mainly becausemetabolite I was detected as shoulder of II resulting in large overlapping of its mass

Figure 6 Proposed MS fragmentation of the 6-hydroxy metabolites of 4(3050-dimethyl-

30-heptyl)-phenol

544 Schmidt et al

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spectrum with that of II Due to its [Mthorn] of mz 394 compound VIII was identifiedwith a carboxylic acid metabolite of 4-353-NP since formation of a hydroxyderivative with an additional carbonyl function was regarded unlikely The massspectrum observed with VIII supported this assumption Fragments showingsimilarity to compounds IndashVII were mz 379 (-CH3) and 365 (-C2H5) whereasfragments mz 131 and 116 were supposed to consist of frac12CH2COOSiethCH3THORN

thorn3 and

frac12CH2COOSiethCH3THORNthorn2 respectively which pointed to a carboxyl function at positions

10 or 70 A characteristic feature of the fragmentation of trimethylsilyl derivatizedfatty acids is separation of CH3 (15) and HOSi(CH3)2 (75) consecutively[30]

With VIII this would result in fragment mz 304 which was only present in thebackground However ions resulting from successive fragmentation of mz 304were observed mz 275 (-HCO -29) 247 (-C2H428) 233 (-CH214) and finally221 This sequence was regarded as prove that 4-353-NP was oxidized to itscarboxylic acid at position 70 of the molecule It should be mentioned that I VI VIIand VIII were only minor metabolites formed from 4-353-NP by the A githago cellsuspensions (Table 2)

Metabolism of 4-353-NP by Agrostemma githagoPlant Cell Suspension Cultures

The nonylphenol isomer 4-353-NP consisting of two diastereomers wastransformed by theAgrostemma githago suspension cultures to four types of productsglycosides of parent 4-353-NP glycosides of primary 4-353-NP metabolitesnonextractable residues and unknown possibly polymeric materials detected in themedia Similar products emerged in the soybean cultures The present investigationconcentrated on the primary metabolites of 4-353-NP These were produced in the Agithago suspensions using a two-liquid-phase system The primary products identifiedby GC-EIMS after liberation from their glycosides were one side-chain carboxylicacid and seven side-chain monohydroxylated metabolites Due to their mass spectrathe main products were four diastereomers of 60-hydroxy-4-353-NP

As yet data on the biotransformation of 4-353-NP are mainly not at handConcerning plant tissues (cell cultures plants) side-chain hydroxylated metaboliteswere reported on 4-n-nonylphenol[11122324] With A githago cells the main productwas 4-(80-hydroxy-n-nonyl)-phenol[24] ie 4-n-nonylphenol was metabolized bysubterminal oxidation corresponding with the main products observed with 4-353-NP in the present study Side-chain hydroxylated metabolites were also formed fromp-tert-octylphenol in barley plants[31] Experiments with model nonylphenols 2- 3-and 4-(4-hydroxyphenyl)-nonane demonstrated that in rainbow trout all isomerswere transformed to monohydroxy products with the site of oxidation at carbon 8(subterminal) of the nonyl residue The metabolites emerged as glucuronic acidconjugates while glucuronides of the parent nonylphenols were also formed[4] In asimilar study both side-chain and ring hydroxylated metabolites of 4-n-nonylphenolwere detected as glucuronic acid conjugates[9] Recently data on the metabolism ofthe nonylphenol isomer 4(3060-dimethyl-30-heptyl)-phenol in pond snails to a (ringhydroxylated) catechol derivative (with unmodified side-chain) were reported[1920]

Contrasting with 4-353-NP of the present study the isomer examined possessed

Metabolism of the Nonylphenol Isomer 545

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a branching at subterminal position 60 of the side-chain besides that at carbon 30 Thecatechol was found conjugated to glucuronic acid[1920] As technical productnonylphenol appears to be completely degradated in soil[1] though isomer 4-(3060-dimethyl-30-heptyl)-phenol proved to be resistant in sedimentwater systems[18] Upto now the routes of nonylphenolrsquos microbial degradation are not known in detailDue to experiments with an isolated bacterial strain initial oxidation at the aromaticmoiety leads to degradation of the phenyl ring while side-chains remain asnonanols[3233] one of the studies was executed with radio-labelled 4-353-NP[33]

The present data on the metabolism of 4-353-NP by A githago cell suspensionsagree with those reported in the literature on 4-n-nonylphenol in plant tissues andmodel isomers in rainbow trout In contrast with trout the capacity of the A githagocells for hydroxylation of the side-chain appeared to be broader ie the nonyl residuewas hydroxylated at different sites of the aliphatic chain The enzymes responsible forside-chain hydroxylation of 4-353-NP in A githago are not known we suspectedcytochromes P450 In rats and humans isozyme CYP2B2 was thought to catalyzepredominantly the hydroxylation of nonylphenol isomers[10] After expression inyeast human CYP2B6 and CYP2C19 were shown to metabolize nonylphenol (pluralbranched chains) while 4-n-nonylphenol was transformed by CYP1A2 CYP2B6 andCYP2C19 ring and side-chain hydroxlated products were identified with branchedchain nonylphenols and 4-n-nonylphenol[34] We assume that plants in general havethe capacity to transform nonylphenol isomers to side-chain hydroxylated productsand thus contribute to the environmental degradation of the xenoestrogen If cropplants are contaminated with nonylphenol both nonylphenol and its side-chainhydroxylated products are expected to be present in plant tissues as glycosideconjugates Xenobiotic glycosides are known to be cleaved in the gastrointestinal tractwith release of possibly toxic aglycons Data on the toxicological properties of side-chain hydroxylated nonylphenols are not available Our future research will focus onthe metabolism of nonylphenol isomers with different branching patterns on theidentification of the enzymes responsible for the metabolism of nonylphenols and onthe (eco-) toxicology of side-chain hydroxylated nonylphenol isomers

ACKNOWLEDGMENTS

For the synthesis of [ring-U-14C]-4(3050-dimethyl-30-heptyl)-phenol the authorswish to thank R Vinken and the lsquolsquoAachen Graduate College Elimination ofEndocrine-Disrupting Substances from Waste-Waterrsquorsquo (AGEESA) funded by theDeutsche Forschungsgemeinschaft

REFERENCES

1 Staples CA Williams JB Blessing RL Varineau PT Measuring thebiodegradability of nonylphenol ether carboxylates octylphenol ether carboxyl-ates and nonylphenol Chemosphere 1999 38 (9) 2029ndash2039

2 Thiele B Gunther K Schwuger MJ Alkylphenol ethoxylates trace analysisand environmental behaviour Chem Rev 1997 97 (8) 3247ndash3272

546 Schmidt et al

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3 Wheeler TF Heim JR LaTorre MR Janes AB Mass spectral

characterization of p-nonylphenol isomers using high-resolution capillary

GC-MS J Chromatogr Sci 1997 35 (1) 19ndash304 Meldahl AC Nithipatikom K Lech JJ Metabolism of several 14C-

nonylphenol isomers by rainbow trout (Oncorhynchus mykiss) in vivo and

in vitro Metabolites Xenobiotica 1996 26 (11) 1167ndash11805 Kim Y-S Katase T Sekine S Inoue T Makino M Uchiyama T

Fujimoto Y Yamashita N Variation in estrogenic activity among fractions

of a commercial nonylphenol by high performance liquid chromatography

Chemosphere 2004 54 (8) 1127ndash11346 Giger W Stephanou E Schaffner C Persistent organic chemicals in sewage

effluents 1 Identification of nonylphenols and nonylphenolethoxylates by

capillary gas chromatographymass spectrometry Chemosphere 1981 10 (11ndash1)

1253ndash12637 Giger W Brunner PH Schaffner C 4-Nonylphenol in sewage sludge

accumulation of toxic metabolites from nonionic surfactants Science 1984

225 (4662) 623ndash6258 Jobst H Chlorophenols an nonylphenols in sewage sludges 1 Occurrence in

sewage sludges of Western German treatment plants from 1987ndash1989 Acta

Hydrochim Hydrobiol 1995 23 (1) 20ndash259 Coldham NG Sivapathasundaram SDM Ashfield LA Pottinger TG

Biotransformation tissue distribution and persistence of 4-nonylphenol

residues in juvenile rainbow trout (Oncorhynchus mykiss) Drug Metab

Dispos 1998 26 (4) 347ndash35410 Lee PC Marquardt M Lech JJ Metabolism of nonylphenol by rat and

human microsomes Toxicol Lett 1998 99 (7) 117ndash12611 Bokern M Harms HH Toxicity and metabolism of 4-n-nonylphenol in cell

suspension cultures of different plant species Environ Sci Technol 1997

31 (7) 1849ndash185412 Bokern M Nimtz M Harms HH Metabolites of 4-n-nonylphenol in wheat

cell suspension cultures J Agric Food Chem 1996 44 (4) 1123ndash112713 Guenther K Heinke V Thiele B Kleist E Prast H Raecker T

Endocrine disrupting nonylphenols are ubiquitous in food Environ Sci

Technol 2002 36 (8) 1676ndash168014 Soto AM Justicia H Wray JW Sonnenschein C p-Nonylphenol an

estrogenic xenobiotic released from lsquolsquomodifiedrsquorsquo polystyrene Environ Health

Perspect 1991 92 167ndash17315 Degen DH Bolt HM Comments on lsquolsquoendocrine disrupting nonylphenols

are ubiquitous in foodrsquorsquo Envirn Sci Technol 2003 37 (11) 2622ndash262316 Vinken R Schmidt B Schaffer A Synthesis of tertiary 14C-labelled

nonylphenol isomers J Label Compd Radiopharm 2002 45 (14) 1253ndash126317 Lalah JO Lenoir D Henkelmann B Hertkorn N Gunther K

Schramm K-W Kettrup A Synthesis of ring-14C-labelled 4(3060-dimethyl-

30-heptyl)-phenol J Label Compd Radiopharm 2001 44 (6) 459ndash46318 Lalah JO Schramm K-W Henkelmann B Lenoir D Behechti A

Gunther K Kettrup A The dissipation distribution and fate of a branched

Metabolism of the Nonylphenol Isomer 547

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14C-nonylphenol isomer in lake watersediment systems Environ Pollut 2003

122 (2) 195ndash20319 Lalah JO Schramm K-W Severin GF Lenoir D Henkelmann B

Behechti A Gunther K Kettrup A In vivo metabolism and organ

distribution of a branched 14C-nonylphenol isomer in pond snails Lymnaea

stagnalis L Aquat Toxicol 2003 62 (4) 305ndash31920 Lalah JO Behechti A Severin GF Lenoir D Gunther K Kettrup A

Schramm K-W The Bioaccumulation and fate of a branched 14C-

p-nonylphenol isomer in Lymnaea stagnalis L Environ Toxicol Chem 2003

22 (7) 1428ndash143621 Schmidt B Metabolic profiling using plant cell suspension cultures In

Pesticide Transformation in Plants and Microorganisms Hall JC Hoagland

RE Zablotowicz RM Eds American Chemical Society Washington DC

2001 40ndash5622 Komoszliga D Langebartels C Sandermann H Metabolic processes for

organic chemicals in plants In Plant ContaminationmdashModeling and Simulation

of Organic Chemical Processes Trapp S Mc Farlane JC Eds Lewis

Publishers Boca Raton 1995 69ndash10323 Schmidt B Schuphan I Metabolism of the Xenoestrogens 4-n-nonylphenol

and bisphenol A in plant cell suspension cultures In Book of Abstracts 10th

IUPAC International Congress on the Chemistry of Crop Protection Basel

August 4ndash9 IUPAC Basel 2002 Vol 2 3424 Schmidt B Patti H Niewersch C Schuphan I Biotransformation of [ring-

U-14C]4-n-nonylphenol by Agrostemma githago cell culture in a two-liquid-

phase system Biotechnol Lett 2003 25 (16) 1375ndash138125 Murashige T Skoog F Revised medium for rapid growth and bio assay with

tobacco tissue cultures Physiol Plant 1962 15 473ndash49726 Gamborg OL Miller RA Ojima K Nutrient requirements of suspension

cultures of soybean root cells Exp Cell Res 1968 50 151ndash15827 Schmidt B Schuphan I Metabolism of the environmental estrogen bisphenol

A by plant cell suspension cultures Chemosphere 2002 49 (1) 51ndash5928 Diekman J Thomson JB Djerassi C Mass spectrometry and stereo-

chemical problems CLV Electron impact induced fragmentations and

rearrangements of some trimethylsilyl ethers of aliphatic glycols and related

compounds J Org Chem 1968 33 (6) 2271ndash228429 Diekman J Thomson JB Djerassi C Mass spectrometry and stereo-

chemical problems CXLI Electron impact induced fragmentations and

rearrangements of trimethylsilyl ethers amines and sulfides J Org Chem

1967 32 (12) 3904ndash391930 Odham G Fatty acids In Biochemical Applications of Mass Spectrometry

Waller GR Dermer OC Eds Wiley-Interscience New York 1980 First

Supplementary Volume 153ndash17131 Stolzenberg GE Olson PA Zaylskie RG Mansager ER Behavior and

fate of ethoxylated alkylphenol nonionic surfactant in barley plants J Agric

Food Chem 1982 30 (4) 637ndash644

548 Schmidt et al

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32 Tanghe T Dhooge W Verstraete W Isolation of a bacterial strain able todegrade branched nonylphenol Appl Environ Microbiol 1999 65 (2)746ndash751

33 Corvini PFX Vinken R Hommes G Schmidt B Dohmann MDegradation of the radioactive and non-labelled branched 4(3050-dimethyl-30-heptyl)-phenol nonylphenol isomer by Sphingomonas TTNP3 Biodegradation2003 15 (1) 1ndash10

34 Inui H Shiota N Motoi Y Ido Y Inoue T Kodama T Ohkawa YOhkawa H Metabolism of herbicides and other chemicals in humancytochrome P450 species and in transgenic potato plants co-expressinghuman CYP1A1 CYP2B6 and CYP2C19 J Pestic Sci 2001 26 (1) 28ndash40

Received February 13 2004

Metabolism of the Nonylphenol Isomer 549

Dow

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ober

201

4

Request PermissionOrder Reprints

Reprints of this article can also be ordered at

httpwwwdekkercomservletproductDOI101081PFC200026776

Request Permission or Order Reprints Instantly

Interested in copying and sharing this article In most cases US Copyright Law requires that you get permission from the articlersquos rightsholder before using copyrighted content

All information and materials found in this article including but not limited to text trademarks patents logos graphics and images (the Materials) are the copyrighted works and other forms of intellectual property of Marcel Dekker Inc or its licensors All rights not expressly granted are reserved

Get permission to lawfully reproduce and distribute the Materials or order reprints quickly and painlessly Simply click on the Request Permission Order Reprints link below and follow the instructions Visit the US Copyright Office for information on Fair Use limitations of US copyright law Please refer to The Association of American Publishersrsquo (AAP) website for guidelines on Fair Use in the Classroom

The Materials are for your personal use only and cannot be reformatted reposted resold or distributed by electronic means or otherwise without permission from Marcel Dekker Inc Marcel Dekker Inc grants you the limited right to display the Materials only on your personal computer or personal wireless device and to copy and download single copies of such Materials provided that any copyright trademark or other notice appearing on such Materials is also retained by displayed copied or downloaded as part of the Materials and is not removed or obscured and provided you do not edit modify alter or enhance the Materials Please refer to our Website User Agreement for more details

Dow

nloa

ded

by [

Am

sG

iron

aba

rri L

ib]

at 2

357

07

Oct

ober

201

4

ORDER REPRINTS

Table 2 Mass spectra of side-chain monohydroxylated metabolites (IndashVII) of 4-353-NP a

carboxylic acid metabolite (VII) of 4-353-NP and of 4-353-NP diastereomers (IX X) after

trimethylsilyl derivatization Compounds were detected using GC-EIMS analysis and were

contained in fractions A and B (obtained by HPLC separation) of a cell extract hydrolysate

(first hydrolysis step 200mg assays)

Peak

Rt EIMS

IsomeraPortionb

(min) mz (abundance in ) ()

Fraction 1

Ic 1865 380 (2) 365 (1) 351 (8) 275 (1) 261 (33)

233 (1) 221 (100) 206 (3) 193 (6) 191 (5)

179 (5) 151 (2) 117 (33) 73 (43)

unknown 214c

II 1870 380 (3) 365 (1) 351 (7) 275 (1) 261 (34)

233 (2) 221 (100) 206 (3) 193 (7) 191 (8)

179 (8) 163 (2) 151 (3) 117 (29) 73 (49)

60-OH-

4-353-NP

214c

III 1874 380 (2) 365 (1) 351 (5) 275 (1) 261 (34)

233 (1) 221 (100) 206 (4) 193 (6) 191 (6)

179 (8) 163 (2) 151 (3) 117 (32) 73 (50)

60-OH-

4-353-NP

175

IVd 1892 380 (2) 365 (1) 351 (6) 275 (1) 261 (36)

233 (2) 221 (100) 206 (4) 193 (7) 191 (6)

179 (8) 163 (2) 151 (3) 117 (27) 73 (52)

60-OH-

4-353-NP

473d

V 1896 380 (2) 365 (1) 351 (6) 275 (1) 261 (37)

233 (1) 221 (100) 206 (3) 193 (7) 191 (6)

179 (8) 163 (2) 151 (3) 117 (26) 73 (45)

60-OH-

4-353-NP

473d

VI 1925 380 (2) 365 (1) 351 (5) 336 (1) 309 (6) 295

(1) 261 (9) 233 (4) 221 (100) 206 (2) 193

(31) 191 (7) 179 (12) 163 (2) 151 (4) 147

(7) 117 (4) 103 (11) 73 (60)

10-OH-

4-353-NP

52

VII 1947 380 (2) 365 (1) 351 (5) 295 (2) 261 (8) 253

(3) 233 (3) 221 (100) 207 (4) 193 (21) 191

(6) 179 (10) 163 (6) 151 (2) 117 (3) 103

(4) 73 (47)

20-OH-

4-353-NP

59

VIII 2019 394 (2) 379 (4) 365 (11) 247 (2) 313 (4)

275 (2) 247 (4) 233 (33) 221 (100) 207 (2)

193 (11) 191 (4) 179 (8) 173 (9) 131 (3)

116 (3) 97 (6) 73 (40)

70-COOH-

4-353-NP

26

Fraction 2

IX 1629 292 (5) 277 (3) 263 (18) 221 (100) 207 (7)

193 (55) 179 (14) 163 (3) 151 (6) 135 (2)

129 (3) 115 (4) 103 (3) 91 (3) 73 (34)

4-353-NP 468

X 1638 292 (5) 277 (3) 263 (19) 221 (100) 207 (8)

193 (55) 179 (13) 163 (2) 151 (6) 135 (2)

129 (4) 115 (3) 103 (1) 91 (3) 73 (31)

4-353-NP 532

aStructures of metabolites IndashVIII of 4-353-NP are proposed according to EIMS fragmentation

patternsbCalculated from peak areas total area of peaks IndashVIII and IXndashX respectively corresponds

with 100cPeak I appeared as shoulder of II separate integration of peaks I and II was not possibledPeak IV appeared as shoulder of V separate integration of peaks IV and V was not possible

542 Schmidt et al

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179 and 73 complete mass spectra are listed in Table 2 The [Mthorn] of compoundsIndashVII pointed to mono-hydroxylated derivatives of 4-353-NP while all patterns offragmentation and their similarity with that of 4-353-NP (especially peaks at mz 221and 179) indicated that hydroxylation had exclusively occurred at the aliphatic side-chain Compound VIII had an [Mthorn] at mz 394 and prominent fragments at mz 221(base peak) 179 and 73 These findings pointed a (side-chain) carboxylic acidderivative of 4-353-NP With both fraction 1 and fraction 2 GC-EIMS peakscorresponding with possible ring-hydroxylated or side-chain olefinic products[24]

were not detected The hydrolysate derived from the soybean experiment wassimilarly separated by HPLC a fraction corresponding with primary 4-353-NPmetabolites was collected and examined by GC-EIMS after silylation Due tolow yield no distinct peaks which could be related to parent 4-353-NP were foundin the corresponding chromatograms as such However using the GC-EIMSfindings of the A githago study a trace was identified at Rt 1905min the massspectrum of which corresponded with compounds IV or V We concluded that theA githago and soybean cells at least demonstrated certain similarities concerningthe biotransformation of 4-353-NP

Though the GC-EIMS data unequivocally proved that 4-353-NP was metabo-lized by A githago cell suspensions at the aliphatic side-chain to monohydroxylatedproducts and a carboxylic acid derivative the sites of hydroxylationoxidation partlyremained unclear Compounds IIndashV emerged in the chromatograms as two pairs ofpeaks (IIIII and IVV) and exhibited noticeably similar mass spectra (Table 2)Besides [Mthorn] at mz 380 and fragments observed with all other monohydroxylatedmetabolites (mz 365 351 261 221 179 73) a prominent fragment of compoundsIIndashV hadmz 117 with 26ndash32 abundance According to data published on side-chain

Figure 5 Proposed MS fragmentation of 4(3050-dimethyl-30-heptyl)-phenol

Metabolism of the Nonylphenol Isomer 543

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monohydroxylated derivatives of both 4-n-nonylphenol[1224] and model side-chainisomers 2- 3- and 4-(4-hydroxyphenol)-nonane[4] fragment mz 117 was referred to[H3CCHOSi(CH3)3

thorn] This fragment is characteristic of trimethylsilyl derivatizedhydroxyl functions at secondary carbon atoms adjacent to terminal CH3

groups[4122428] Due to abundances (34ndash37) of fragments mz 261 (-HOSi(CH3)3-C2H5)

[2429] it was supposed that compounds IIndashV were 60-hydroxy derivatives of4-353-NP Applied 4-353-NP consisted of two diastereomers (each a couple ofenatiomers) hydroxylation at position 60 consequently introduced a third chiral centerwhich resulted in four diastereomers An MS fragmentation scheme for IIndashV isproposed in Fig 6 Quantitative evaluation of the chromatogram showed thatcompounds IIndashV were the main primary metabolites in A githago cells amountingto about 80 of the products identified

The mass spectrum of compound VI (Table 2) differed from those of productsIIndashV in particular regarding three fragments mz 309 (abundance 6) 295 (1) and103 (11) According to previous results presented on 4-n-nonylphenol[24] thefragment mz 103 was supposed to arise from [H2COSi(CH3)3

thorn] pointing tohydroxylation at a primary carbon[2428] Furthermore fragments mz 309 and 295indicated loss of C5H11 and C6H13 respectively from [Mthorn] (mz 380) with retentionof two silylated hydroxy functions at the remaining fragment ion It was thusassumed that compound VI was a 4-353-NP metabolite hydroxylated at position 10

of the side-chain Due to a similar line of argumentation (presence of fragments mz117 and 295) compound VII possibly consisted of the 20-hydroxy derivative of4-353-NP The structure of compound I remained unknown mainly becausemetabolite I was detected as shoulder of II resulting in large overlapping of its mass

Figure 6 Proposed MS fragmentation of the 6-hydroxy metabolites of 4(3050-dimethyl-

30-heptyl)-phenol

544 Schmidt et al

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ORDER REPRINTS

spectrum with that of II Due to its [Mthorn] of mz 394 compound VIII was identifiedwith a carboxylic acid metabolite of 4-353-NP since formation of a hydroxyderivative with an additional carbonyl function was regarded unlikely The massspectrum observed with VIII supported this assumption Fragments showingsimilarity to compounds IndashVII were mz 379 (-CH3) and 365 (-C2H5) whereasfragments mz 131 and 116 were supposed to consist of frac12CH2COOSiethCH3THORN

thorn3 and

frac12CH2COOSiethCH3THORNthorn2 respectively which pointed to a carboxyl function at positions

10 or 70 A characteristic feature of the fragmentation of trimethylsilyl derivatizedfatty acids is separation of CH3 (15) and HOSi(CH3)2 (75) consecutively[30]

With VIII this would result in fragment mz 304 which was only present in thebackground However ions resulting from successive fragmentation of mz 304were observed mz 275 (-HCO -29) 247 (-C2H428) 233 (-CH214) and finally221 This sequence was regarded as prove that 4-353-NP was oxidized to itscarboxylic acid at position 70 of the molecule It should be mentioned that I VI VIIand VIII were only minor metabolites formed from 4-353-NP by the A githago cellsuspensions (Table 2)

Metabolism of 4-353-NP by Agrostemma githagoPlant Cell Suspension Cultures

The nonylphenol isomer 4-353-NP consisting of two diastereomers wastransformed by theAgrostemma githago suspension cultures to four types of productsglycosides of parent 4-353-NP glycosides of primary 4-353-NP metabolitesnonextractable residues and unknown possibly polymeric materials detected in themedia Similar products emerged in the soybean cultures The present investigationconcentrated on the primary metabolites of 4-353-NP These were produced in the Agithago suspensions using a two-liquid-phase system The primary products identifiedby GC-EIMS after liberation from their glycosides were one side-chain carboxylicacid and seven side-chain monohydroxylated metabolites Due to their mass spectrathe main products were four diastereomers of 60-hydroxy-4-353-NP

As yet data on the biotransformation of 4-353-NP are mainly not at handConcerning plant tissues (cell cultures plants) side-chain hydroxylated metaboliteswere reported on 4-n-nonylphenol[11122324] With A githago cells the main productwas 4-(80-hydroxy-n-nonyl)-phenol[24] ie 4-n-nonylphenol was metabolized bysubterminal oxidation corresponding with the main products observed with 4-353-NP in the present study Side-chain hydroxylated metabolites were also formed fromp-tert-octylphenol in barley plants[31] Experiments with model nonylphenols 2- 3-and 4-(4-hydroxyphenyl)-nonane demonstrated that in rainbow trout all isomerswere transformed to monohydroxy products with the site of oxidation at carbon 8(subterminal) of the nonyl residue The metabolites emerged as glucuronic acidconjugates while glucuronides of the parent nonylphenols were also formed[4] In asimilar study both side-chain and ring hydroxylated metabolites of 4-n-nonylphenolwere detected as glucuronic acid conjugates[9] Recently data on the metabolism ofthe nonylphenol isomer 4(3060-dimethyl-30-heptyl)-phenol in pond snails to a (ringhydroxylated) catechol derivative (with unmodified side-chain) were reported[1920]

Contrasting with 4-353-NP of the present study the isomer examined possessed

Metabolism of the Nonylphenol Isomer 545

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a branching at subterminal position 60 of the side-chain besides that at carbon 30 Thecatechol was found conjugated to glucuronic acid[1920] As technical productnonylphenol appears to be completely degradated in soil[1] though isomer 4-(3060-dimethyl-30-heptyl)-phenol proved to be resistant in sedimentwater systems[18] Upto now the routes of nonylphenolrsquos microbial degradation are not known in detailDue to experiments with an isolated bacterial strain initial oxidation at the aromaticmoiety leads to degradation of the phenyl ring while side-chains remain asnonanols[3233] one of the studies was executed with radio-labelled 4-353-NP[33]

The present data on the metabolism of 4-353-NP by A githago cell suspensionsagree with those reported in the literature on 4-n-nonylphenol in plant tissues andmodel isomers in rainbow trout In contrast with trout the capacity of the A githagocells for hydroxylation of the side-chain appeared to be broader ie the nonyl residuewas hydroxylated at different sites of the aliphatic chain The enzymes responsible forside-chain hydroxylation of 4-353-NP in A githago are not known we suspectedcytochromes P450 In rats and humans isozyme CYP2B2 was thought to catalyzepredominantly the hydroxylation of nonylphenol isomers[10] After expression inyeast human CYP2B6 and CYP2C19 were shown to metabolize nonylphenol (pluralbranched chains) while 4-n-nonylphenol was transformed by CYP1A2 CYP2B6 andCYP2C19 ring and side-chain hydroxlated products were identified with branchedchain nonylphenols and 4-n-nonylphenol[34] We assume that plants in general havethe capacity to transform nonylphenol isomers to side-chain hydroxylated productsand thus contribute to the environmental degradation of the xenoestrogen If cropplants are contaminated with nonylphenol both nonylphenol and its side-chainhydroxylated products are expected to be present in plant tissues as glycosideconjugates Xenobiotic glycosides are known to be cleaved in the gastrointestinal tractwith release of possibly toxic aglycons Data on the toxicological properties of side-chain hydroxylated nonylphenols are not available Our future research will focus onthe metabolism of nonylphenol isomers with different branching patterns on theidentification of the enzymes responsible for the metabolism of nonylphenols and onthe (eco-) toxicology of side-chain hydroxylated nonylphenol isomers

ACKNOWLEDGMENTS

For the synthesis of [ring-U-14C]-4(3050-dimethyl-30-heptyl)-phenol the authorswish to thank R Vinken and the lsquolsquoAachen Graduate College Elimination ofEndocrine-Disrupting Substances from Waste-Waterrsquorsquo (AGEESA) funded by theDeutsche Forschungsgemeinschaft

REFERENCES

1 Staples CA Williams JB Blessing RL Varineau PT Measuring thebiodegradability of nonylphenol ether carboxylates octylphenol ether carboxyl-ates and nonylphenol Chemosphere 1999 38 (9) 2029ndash2039

2 Thiele B Gunther K Schwuger MJ Alkylphenol ethoxylates trace analysisand environmental behaviour Chem Rev 1997 97 (8) 3247ndash3272

546 Schmidt et al

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3 Wheeler TF Heim JR LaTorre MR Janes AB Mass spectral

characterization of p-nonylphenol isomers using high-resolution capillary

GC-MS J Chromatogr Sci 1997 35 (1) 19ndash304 Meldahl AC Nithipatikom K Lech JJ Metabolism of several 14C-

nonylphenol isomers by rainbow trout (Oncorhynchus mykiss) in vivo and

in vitro Metabolites Xenobiotica 1996 26 (11) 1167ndash11805 Kim Y-S Katase T Sekine S Inoue T Makino M Uchiyama T

Fujimoto Y Yamashita N Variation in estrogenic activity among fractions

of a commercial nonylphenol by high performance liquid chromatography

Chemosphere 2004 54 (8) 1127ndash11346 Giger W Stephanou E Schaffner C Persistent organic chemicals in sewage

effluents 1 Identification of nonylphenols and nonylphenolethoxylates by

capillary gas chromatographymass spectrometry Chemosphere 1981 10 (11ndash1)

1253ndash12637 Giger W Brunner PH Schaffner C 4-Nonylphenol in sewage sludge

accumulation of toxic metabolites from nonionic surfactants Science 1984

225 (4662) 623ndash6258 Jobst H Chlorophenols an nonylphenols in sewage sludges 1 Occurrence in

sewage sludges of Western German treatment plants from 1987ndash1989 Acta

Hydrochim Hydrobiol 1995 23 (1) 20ndash259 Coldham NG Sivapathasundaram SDM Ashfield LA Pottinger TG

Biotransformation tissue distribution and persistence of 4-nonylphenol

residues in juvenile rainbow trout (Oncorhynchus mykiss) Drug Metab

Dispos 1998 26 (4) 347ndash35410 Lee PC Marquardt M Lech JJ Metabolism of nonylphenol by rat and

human microsomes Toxicol Lett 1998 99 (7) 117ndash12611 Bokern M Harms HH Toxicity and metabolism of 4-n-nonylphenol in cell

suspension cultures of different plant species Environ Sci Technol 1997

31 (7) 1849ndash185412 Bokern M Nimtz M Harms HH Metabolites of 4-n-nonylphenol in wheat

cell suspension cultures J Agric Food Chem 1996 44 (4) 1123ndash112713 Guenther K Heinke V Thiele B Kleist E Prast H Raecker T

Endocrine disrupting nonylphenols are ubiquitous in food Environ Sci

Technol 2002 36 (8) 1676ndash168014 Soto AM Justicia H Wray JW Sonnenschein C p-Nonylphenol an

estrogenic xenobiotic released from lsquolsquomodifiedrsquorsquo polystyrene Environ Health

Perspect 1991 92 167ndash17315 Degen DH Bolt HM Comments on lsquolsquoendocrine disrupting nonylphenols

are ubiquitous in foodrsquorsquo Envirn Sci Technol 2003 37 (11) 2622ndash262316 Vinken R Schmidt B Schaffer A Synthesis of tertiary 14C-labelled

nonylphenol isomers J Label Compd Radiopharm 2002 45 (14) 1253ndash126317 Lalah JO Lenoir D Henkelmann B Hertkorn N Gunther K

Schramm K-W Kettrup A Synthesis of ring-14C-labelled 4(3060-dimethyl-

30-heptyl)-phenol J Label Compd Radiopharm 2001 44 (6) 459ndash46318 Lalah JO Schramm K-W Henkelmann B Lenoir D Behechti A

Gunther K Kettrup A The dissipation distribution and fate of a branched

Metabolism of the Nonylphenol Isomer 547

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14C-nonylphenol isomer in lake watersediment systems Environ Pollut 2003

122 (2) 195ndash20319 Lalah JO Schramm K-W Severin GF Lenoir D Henkelmann B

Behechti A Gunther K Kettrup A In vivo metabolism and organ

distribution of a branched 14C-nonylphenol isomer in pond snails Lymnaea

stagnalis L Aquat Toxicol 2003 62 (4) 305ndash31920 Lalah JO Behechti A Severin GF Lenoir D Gunther K Kettrup A

Schramm K-W The Bioaccumulation and fate of a branched 14C-

p-nonylphenol isomer in Lymnaea stagnalis L Environ Toxicol Chem 2003

22 (7) 1428ndash143621 Schmidt B Metabolic profiling using plant cell suspension cultures In

Pesticide Transformation in Plants and Microorganisms Hall JC Hoagland

RE Zablotowicz RM Eds American Chemical Society Washington DC

2001 40ndash5622 Komoszliga D Langebartels C Sandermann H Metabolic processes for

organic chemicals in plants In Plant ContaminationmdashModeling and Simulation

of Organic Chemical Processes Trapp S Mc Farlane JC Eds Lewis

Publishers Boca Raton 1995 69ndash10323 Schmidt B Schuphan I Metabolism of the Xenoestrogens 4-n-nonylphenol

and bisphenol A in plant cell suspension cultures In Book of Abstracts 10th

IUPAC International Congress on the Chemistry of Crop Protection Basel

August 4ndash9 IUPAC Basel 2002 Vol 2 3424 Schmidt B Patti H Niewersch C Schuphan I Biotransformation of [ring-

U-14C]4-n-nonylphenol by Agrostemma githago cell culture in a two-liquid-

phase system Biotechnol Lett 2003 25 (16) 1375ndash138125 Murashige T Skoog F Revised medium for rapid growth and bio assay with

tobacco tissue cultures Physiol Plant 1962 15 473ndash49726 Gamborg OL Miller RA Ojima K Nutrient requirements of suspension

cultures of soybean root cells Exp Cell Res 1968 50 151ndash15827 Schmidt B Schuphan I Metabolism of the environmental estrogen bisphenol

A by plant cell suspension cultures Chemosphere 2002 49 (1) 51ndash5928 Diekman J Thomson JB Djerassi C Mass spectrometry and stereo-

chemical problems CLV Electron impact induced fragmentations and

rearrangements of some trimethylsilyl ethers of aliphatic glycols and related

compounds J Org Chem 1968 33 (6) 2271ndash228429 Diekman J Thomson JB Djerassi C Mass spectrometry and stereo-

chemical problems CXLI Electron impact induced fragmentations and

rearrangements of trimethylsilyl ethers amines and sulfides J Org Chem

1967 32 (12) 3904ndash391930 Odham G Fatty acids In Biochemical Applications of Mass Spectrometry

Waller GR Dermer OC Eds Wiley-Interscience New York 1980 First

Supplementary Volume 153ndash17131 Stolzenberg GE Olson PA Zaylskie RG Mansager ER Behavior and

fate of ethoxylated alkylphenol nonionic surfactant in barley plants J Agric

Food Chem 1982 30 (4) 637ndash644

548 Schmidt et al

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32 Tanghe T Dhooge W Verstraete W Isolation of a bacterial strain able todegrade branched nonylphenol Appl Environ Microbiol 1999 65 (2)746ndash751

33 Corvini PFX Vinken R Hommes G Schmidt B Dohmann MDegradation of the radioactive and non-labelled branched 4(3050-dimethyl-30-heptyl)-phenol nonylphenol isomer by Sphingomonas TTNP3 Biodegradation2003 15 (1) 1ndash10

34 Inui H Shiota N Motoi Y Ido Y Inoue T Kodama T Ohkawa YOhkawa H Metabolism of herbicides and other chemicals in humancytochrome P450 species and in transgenic potato plants co-expressinghuman CYP1A1 CYP2B6 and CYP2C19 J Pestic Sci 2001 26 (1) 28ndash40

Received February 13 2004

Metabolism of the Nonylphenol Isomer 549

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179 and 73 complete mass spectra are listed in Table 2 The [Mthorn] of compoundsIndashVII pointed to mono-hydroxylated derivatives of 4-353-NP while all patterns offragmentation and their similarity with that of 4-353-NP (especially peaks at mz 221and 179) indicated that hydroxylation had exclusively occurred at the aliphatic side-chain Compound VIII had an [Mthorn] at mz 394 and prominent fragments at mz 221(base peak) 179 and 73 These findings pointed a (side-chain) carboxylic acidderivative of 4-353-NP With both fraction 1 and fraction 2 GC-EIMS peakscorresponding with possible ring-hydroxylated or side-chain olefinic products[24]

were not detected The hydrolysate derived from the soybean experiment wassimilarly separated by HPLC a fraction corresponding with primary 4-353-NPmetabolites was collected and examined by GC-EIMS after silylation Due tolow yield no distinct peaks which could be related to parent 4-353-NP were foundin the corresponding chromatograms as such However using the GC-EIMSfindings of the A githago study a trace was identified at Rt 1905min the massspectrum of which corresponded with compounds IV or V We concluded that theA githago and soybean cells at least demonstrated certain similarities concerningthe biotransformation of 4-353-NP

Though the GC-EIMS data unequivocally proved that 4-353-NP was metabo-lized by A githago cell suspensions at the aliphatic side-chain to monohydroxylatedproducts and a carboxylic acid derivative the sites of hydroxylationoxidation partlyremained unclear Compounds IIndashV emerged in the chromatograms as two pairs ofpeaks (IIIII and IVV) and exhibited noticeably similar mass spectra (Table 2)Besides [Mthorn] at mz 380 and fragments observed with all other monohydroxylatedmetabolites (mz 365 351 261 221 179 73) a prominent fragment of compoundsIIndashV hadmz 117 with 26ndash32 abundance According to data published on side-chain

Figure 5 Proposed MS fragmentation of 4(3050-dimethyl-30-heptyl)-phenol

Metabolism of the Nonylphenol Isomer 543

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monohydroxylated derivatives of both 4-n-nonylphenol[1224] and model side-chainisomers 2- 3- and 4-(4-hydroxyphenol)-nonane[4] fragment mz 117 was referred to[H3CCHOSi(CH3)3

thorn] This fragment is characteristic of trimethylsilyl derivatizedhydroxyl functions at secondary carbon atoms adjacent to terminal CH3

groups[4122428] Due to abundances (34ndash37) of fragments mz 261 (-HOSi(CH3)3-C2H5)

[2429] it was supposed that compounds IIndashV were 60-hydroxy derivatives of4-353-NP Applied 4-353-NP consisted of two diastereomers (each a couple ofenatiomers) hydroxylation at position 60 consequently introduced a third chiral centerwhich resulted in four diastereomers An MS fragmentation scheme for IIndashV isproposed in Fig 6 Quantitative evaluation of the chromatogram showed thatcompounds IIndashV were the main primary metabolites in A githago cells amountingto about 80 of the products identified

The mass spectrum of compound VI (Table 2) differed from those of productsIIndashV in particular regarding three fragments mz 309 (abundance 6) 295 (1) and103 (11) According to previous results presented on 4-n-nonylphenol[24] thefragment mz 103 was supposed to arise from [H2COSi(CH3)3

thorn] pointing tohydroxylation at a primary carbon[2428] Furthermore fragments mz 309 and 295indicated loss of C5H11 and C6H13 respectively from [Mthorn] (mz 380) with retentionof two silylated hydroxy functions at the remaining fragment ion It was thusassumed that compound VI was a 4-353-NP metabolite hydroxylated at position 10

of the side-chain Due to a similar line of argumentation (presence of fragments mz117 and 295) compound VII possibly consisted of the 20-hydroxy derivative of4-353-NP The structure of compound I remained unknown mainly becausemetabolite I was detected as shoulder of II resulting in large overlapping of its mass

Figure 6 Proposed MS fragmentation of the 6-hydroxy metabolites of 4(3050-dimethyl-

30-heptyl)-phenol

544 Schmidt et al

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spectrum with that of II Due to its [Mthorn] of mz 394 compound VIII was identifiedwith a carboxylic acid metabolite of 4-353-NP since formation of a hydroxyderivative with an additional carbonyl function was regarded unlikely The massspectrum observed with VIII supported this assumption Fragments showingsimilarity to compounds IndashVII were mz 379 (-CH3) and 365 (-C2H5) whereasfragments mz 131 and 116 were supposed to consist of frac12CH2COOSiethCH3THORN

thorn3 and

frac12CH2COOSiethCH3THORNthorn2 respectively which pointed to a carboxyl function at positions

10 or 70 A characteristic feature of the fragmentation of trimethylsilyl derivatizedfatty acids is separation of CH3 (15) and HOSi(CH3)2 (75) consecutively[30]

With VIII this would result in fragment mz 304 which was only present in thebackground However ions resulting from successive fragmentation of mz 304were observed mz 275 (-HCO -29) 247 (-C2H428) 233 (-CH214) and finally221 This sequence was regarded as prove that 4-353-NP was oxidized to itscarboxylic acid at position 70 of the molecule It should be mentioned that I VI VIIand VIII were only minor metabolites formed from 4-353-NP by the A githago cellsuspensions (Table 2)

Metabolism of 4-353-NP by Agrostemma githagoPlant Cell Suspension Cultures

The nonylphenol isomer 4-353-NP consisting of two diastereomers wastransformed by theAgrostemma githago suspension cultures to four types of productsglycosides of parent 4-353-NP glycosides of primary 4-353-NP metabolitesnonextractable residues and unknown possibly polymeric materials detected in themedia Similar products emerged in the soybean cultures The present investigationconcentrated on the primary metabolites of 4-353-NP These were produced in the Agithago suspensions using a two-liquid-phase system The primary products identifiedby GC-EIMS after liberation from their glycosides were one side-chain carboxylicacid and seven side-chain monohydroxylated metabolites Due to their mass spectrathe main products were four diastereomers of 60-hydroxy-4-353-NP

As yet data on the biotransformation of 4-353-NP are mainly not at handConcerning plant tissues (cell cultures plants) side-chain hydroxylated metaboliteswere reported on 4-n-nonylphenol[11122324] With A githago cells the main productwas 4-(80-hydroxy-n-nonyl)-phenol[24] ie 4-n-nonylphenol was metabolized bysubterminal oxidation corresponding with the main products observed with 4-353-NP in the present study Side-chain hydroxylated metabolites were also formed fromp-tert-octylphenol in barley plants[31] Experiments with model nonylphenols 2- 3-and 4-(4-hydroxyphenyl)-nonane demonstrated that in rainbow trout all isomerswere transformed to monohydroxy products with the site of oxidation at carbon 8(subterminal) of the nonyl residue The metabolites emerged as glucuronic acidconjugates while glucuronides of the parent nonylphenols were also formed[4] In asimilar study both side-chain and ring hydroxylated metabolites of 4-n-nonylphenolwere detected as glucuronic acid conjugates[9] Recently data on the metabolism ofthe nonylphenol isomer 4(3060-dimethyl-30-heptyl)-phenol in pond snails to a (ringhydroxylated) catechol derivative (with unmodified side-chain) were reported[1920]

Contrasting with 4-353-NP of the present study the isomer examined possessed

Metabolism of the Nonylphenol Isomer 545

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a branching at subterminal position 60 of the side-chain besides that at carbon 30 Thecatechol was found conjugated to glucuronic acid[1920] As technical productnonylphenol appears to be completely degradated in soil[1] though isomer 4-(3060-dimethyl-30-heptyl)-phenol proved to be resistant in sedimentwater systems[18] Upto now the routes of nonylphenolrsquos microbial degradation are not known in detailDue to experiments with an isolated bacterial strain initial oxidation at the aromaticmoiety leads to degradation of the phenyl ring while side-chains remain asnonanols[3233] one of the studies was executed with radio-labelled 4-353-NP[33]

The present data on the metabolism of 4-353-NP by A githago cell suspensionsagree with those reported in the literature on 4-n-nonylphenol in plant tissues andmodel isomers in rainbow trout In contrast with trout the capacity of the A githagocells for hydroxylation of the side-chain appeared to be broader ie the nonyl residuewas hydroxylated at different sites of the aliphatic chain The enzymes responsible forside-chain hydroxylation of 4-353-NP in A githago are not known we suspectedcytochromes P450 In rats and humans isozyme CYP2B2 was thought to catalyzepredominantly the hydroxylation of nonylphenol isomers[10] After expression inyeast human CYP2B6 and CYP2C19 were shown to metabolize nonylphenol (pluralbranched chains) while 4-n-nonylphenol was transformed by CYP1A2 CYP2B6 andCYP2C19 ring and side-chain hydroxlated products were identified with branchedchain nonylphenols and 4-n-nonylphenol[34] We assume that plants in general havethe capacity to transform nonylphenol isomers to side-chain hydroxylated productsand thus contribute to the environmental degradation of the xenoestrogen If cropplants are contaminated with nonylphenol both nonylphenol and its side-chainhydroxylated products are expected to be present in plant tissues as glycosideconjugates Xenobiotic glycosides are known to be cleaved in the gastrointestinal tractwith release of possibly toxic aglycons Data on the toxicological properties of side-chain hydroxylated nonylphenols are not available Our future research will focus onthe metabolism of nonylphenol isomers with different branching patterns on theidentification of the enzymes responsible for the metabolism of nonylphenols and onthe (eco-) toxicology of side-chain hydroxylated nonylphenol isomers

ACKNOWLEDGMENTS

For the synthesis of [ring-U-14C]-4(3050-dimethyl-30-heptyl)-phenol the authorswish to thank R Vinken and the lsquolsquoAachen Graduate College Elimination ofEndocrine-Disrupting Substances from Waste-Waterrsquorsquo (AGEESA) funded by theDeutsche Forschungsgemeinschaft

REFERENCES

1 Staples CA Williams JB Blessing RL Varineau PT Measuring thebiodegradability of nonylphenol ether carboxylates octylphenol ether carboxyl-ates and nonylphenol Chemosphere 1999 38 (9) 2029ndash2039

2 Thiele B Gunther K Schwuger MJ Alkylphenol ethoxylates trace analysisand environmental behaviour Chem Rev 1997 97 (8) 3247ndash3272

546 Schmidt et al

Dow

nloa

ded

by [

Am

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iron

aba

rri L

ib]

at 2

357

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Oct

ober

201

4

ORDER REPRINTS

3 Wheeler TF Heim JR LaTorre MR Janes AB Mass spectral

characterization of p-nonylphenol isomers using high-resolution capillary

GC-MS J Chromatogr Sci 1997 35 (1) 19ndash304 Meldahl AC Nithipatikom K Lech JJ Metabolism of several 14C-

nonylphenol isomers by rainbow trout (Oncorhynchus mykiss) in vivo and

in vitro Metabolites Xenobiotica 1996 26 (11) 1167ndash11805 Kim Y-S Katase T Sekine S Inoue T Makino M Uchiyama T

Fujimoto Y Yamashita N Variation in estrogenic activity among fractions

of a commercial nonylphenol by high performance liquid chromatography

Chemosphere 2004 54 (8) 1127ndash11346 Giger W Stephanou E Schaffner C Persistent organic chemicals in sewage

effluents 1 Identification of nonylphenols and nonylphenolethoxylates by

capillary gas chromatographymass spectrometry Chemosphere 1981 10 (11ndash1)

1253ndash12637 Giger W Brunner PH Schaffner C 4-Nonylphenol in sewage sludge

accumulation of toxic metabolites from nonionic surfactants Science 1984

225 (4662) 623ndash6258 Jobst H Chlorophenols an nonylphenols in sewage sludges 1 Occurrence in

sewage sludges of Western German treatment plants from 1987ndash1989 Acta

Hydrochim Hydrobiol 1995 23 (1) 20ndash259 Coldham NG Sivapathasundaram SDM Ashfield LA Pottinger TG

Biotransformation tissue distribution and persistence of 4-nonylphenol

residues in juvenile rainbow trout (Oncorhynchus mykiss) Drug Metab

Dispos 1998 26 (4) 347ndash35410 Lee PC Marquardt M Lech JJ Metabolism of nonylphenol by rat and

human microsomes Toxicol Lett 1998 99 (7) 117ndash12611 Bokern M Harms HH Toxicity and metabolism of 4-n-nonylphenol in cell

suspension cultures of different plant species Environ Sci Technol 1997

31 (7) 1849ndash185412 Bokern M Nimtz M Harms HH Metabolites of 4-n-nonylphenol in wheat

cell suspension cultures J Agric Food Chem 1996 44 (4) 1123ndash112713 Guenther K Heinke V Thiele B Kleist E Prast H Raecker T

Endocrine disrupting nonylphenols are ubiquitous in food Environ Sci

Technol 2002 36 (8) 1676ndash168014 Soto AM Justicia H Wray JW Sonnenschein C p-Nonylphenol an

estrogenic xenobiotic released from lsquolsquomodifiedrsquorsquo polystyrene Environ Health

Perspect 1991 92 167ndash17315 Degen DH Bolt HM Comments on lsquolsquoendocrine disrupting nonylphenols

are ubiquitous in foodrsquorsquo Envirn Sci Technol 2003 37 (11) 2622ndash262316 Vinken R Schmidt B Schaffer A Synthesis of tertiary 14C-labelled

nonylphenol isomers J Label Compd Radiopharm 2002 45 (14) 1253ndash126317 Lalah JO Lenoir D Henkelmann B Hertkorn N Gunther K

Schramm K-W Kettrup A Synthesis of ring-14C-labelled 4(3060-dimethyl-

30-heptyl)-phenol J Label Compd Radiopharm 2001 44 (6) 459ndash46318 Lalah JO Schramm K-W Henkelmann B Lenoir D Behechti A

Gunther K Kettrup A The dissipation distribution and fate of a branched

Metabolism of the Nonylphenol Isomer 547

Dow

nloa

ded

by [

Am

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iron

aba

rri L

ib]

at 2

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ober

201

4

ORDER REPRINTS

14C-nonylphenol isomer in lake watersediment systems Environ Pollut 2003

122 (2) 195ndash20319 Lalah JO Schramm K-W Severin GF Lenoir D Henkelmann B

Behechti A Gunther K Kettrup A In vivo metabolism and organ

distribution of a branched 14C-nonylphenol isomer in pond snails Lymnaea

stagnalis L Aquat Toxicol 2003 62 (4) 305ndash31920 Lalah JO Behechti A Severin GF Lenoir D Gunther K Kettrup A

Schramm K-W The Bioaccumulation and fate of a branched 14C-

p-nonylphenol isomer in Lymnaea stagnalis L Environ Toxicol Chem 2003

22 (7) 1428ndash143621 Schmidt B Metabolic profiling using plant cell suspension cultures In

Pesticide Transformation in Plants and Microorganisms Hall JC Hoagland

RE Zablotowicz RM Eds American Chemical Society Washington DC

2001 40ndash5622 Komoszliga D Langebartels C Sandermann H Metabolic processes for

organic chemicals in plants In Plant ContaminationmdashModeling and Simulation

of Organic Chemical Processes Trapp S Mc Farlane JC Eds Lewis

Publishers Boca Raton 1995 69ndash10323 Schmidt B Schuphan I Metabolism of the Xenoestrogens 4-n-nonylphenol

and bisphenol A in plant cell suspension cultures In Book of Abstracts 10th

IUPAC International Congress on the Chemistry of Crop Protection Basel

August 4ndash9 IUPAC Basel 2002 Vol 2 3424 Schmidt B Patti H Niewersch C Schuphan I Biotransformation of [ring-

U-14C]4-n-nonylphenol by Agrostemma githago cell culture in a two-liquid-

phase system Biotechnol Lett 2003 25 (16) 1375ndash138125 Murashige T Skoog F Revised medium for rapid growth and bio assay with

tobacco tissue cultures Physiol Plant 1962 15 473ndash49726 Gamborg OL Miller RA Ojima K Nutrient requirements of suspension

cultures of soybean root cells Exp Cell Res 1968 50 151ndash15827 Schmidt B Schuphan I Metabolism of the environmental estrogen bisphenol

A by plant cell suspension cultures Chemosphere 2002 49 (1) 51ndash5928 Diekman J Thomson JB Djerassi C Mass spectrometry and stereo-

chemical problems CLV Electron impact induced fragmentations and

rearrangements of some trimethylsilyl ethers of aliphatic glycols and related

compounds J Org Chem 1968 33 (6) 2271ndash228429 Diekman J Thomson JB Djerassi C Mass spectrometry and stereo-

chemical problems CXLI Electron impact induced fragmentations and

rearrangements of trimethylsilyl ethers amines and sulfides J Org Chem

1967 32 (12) 3904ndash391930 Odham G Fatty acids In Biochemical Applications of Mass Spectrometry

Waller GR Dermer OC Eds Wiley-Interscience New York 1980 First

Supplementary Volume 153ndash17131 Stolzenberg GE Olson PA Zaylskie RG Mansager ER Behavior and

fate of ethoxylated alkylphenol nonionic surfactant in barley plants J Agric

Food Chem 1982 30 (4) 637ndash644

548 Schmidt et al

Dow

nloa

ded

by [

Am

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iron

aba

rri L

ib]

at 2

357

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ober

201

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ORDER REPRINTS

32 Tanghe T Dhooge W Verstraete W Isolation of a bacterial strain able todegrade branched nonylphenol Appl Environ Microbiol 1999 65 (2)746ndash751

33 Corvini PFX Vinken R Hommes G Schmidt B Dohmann MDegradation of the radioactive and non-labelled branched 4(3050-dimethyl-30-heptyl)-phenol nonylphenol isomer by Sphingomonas TTNP3 Biodegradation2003 15 (1) 1ndash10

34 Inui H Shiota N Motoi Y Ido Y Inoue T Kodama T Ohkawa YOhkawa H Metabolism of herbicides and other chemicals in humancytochrome P450 species and in transgenic potato plants co-expressinghuman CYP1A1 CYP2B6 and CYP2C19 J Pestic Sci 2001 26 (1) 28ndash40

Received February 13 2004

Metabolism of the Nonylphenol Isomer 549

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Request PermissionOrder Reprints

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ORDER REPRINTS

monohydroxylated derivatives of both 4-n-nonylphenol[1224] and model side-chainisomers 2- 3- and 4-(4-hydroxyphenol)-nonane[4] fragment mz 117 was referred to[H3CCHOSi(CH3)3

thorn] This fragment is characteristic of trimethylsilyl derivatizedhydroxyl functions at secondary carbon atoms adjacent to terminal CH3

groups[4122428] Due to abundances (34ndash37) of fragments mz 261 (-HOSi(CH3)3-C2H5)

[2429] it was supposed that compounds IIndashV were 60-hydroxy derivatives of4-353-NP Applied 4-353-NP consisted of two diastereomers (each a couple ofenatiomers) hydroxylation at position 60 consequently introduced a third chiral centerwhich resulted in four diastereomers An MS fragmentation scheme for IIndashV isproposed in Fig 6 Quantitative evaluation of the chromatogram showed thatcompounds IIndashV were the main primary metabolites in A githago cells amountingto about 80 of the products identified

The mass spectrum of compound VI (Table 2) differed from those of productsIIndashV in particular regarding three fragments mz 309 (abundance 6) 295 (1) and103 (11) According to previous results presented on 4-n-nonylphenol[24] thefragment mz 103 was supposed to arise from [H2COSi(CH3)3

thorn] pointing tohydroxylation at a primary carbon[2428] Furthermore fragments mz 309 and 295indicated loss of C5H11 and C6H13 respectively from [Mthorn] (mz 380) with retentionof two silylated hydroxy functions at the remaining fragment ion It was thusassumed that compound VI was a 4-353-NP metabolite hydroxylated at position 10

of the side-chain Due to a similar line of argumentation (presence of fragments mz117 and 295) compound VII possibly consisted of the 20-hydroxy derivative of4-353-NP The structure of compound I remained unknown mainly becausemetabolite I was detected as shoulder of II resulting in large overlapping of its mass

Figure 6 Proposed MS fragmentation of the 6-hydroxy metabolites of 4(3050-dimethyl-

30-heptyl)-phenol

544 Schmidt et al

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ORDER REPRINTS

spectrum with that of II Due to its [Mthorn] of mz 394 compound VIII was identifiedwith a carboxylic acid metabolite of 4-353-NP since formation of a hydroxyderivative with an additional carbonyl function was regarded unlikely The massspectrum observed with VIII supported this assumption Fragments showingsimilarity to compounds IndashVII were mz 379 (-CH3) and 365 (-C2H5) whereasfragments mz 131 and 116 were supposed to consist of frac12CH2COOSiethCH3THORN

thorn3 and

frac12CH2COOSiethCH3THORNthorn2 respectively which pointed to a carboxyl function at positions

10 or 70 A characteristic feature of the fragmentation of trimethylsilyl derivatizedfatty acids is separation of CH3 (15) and HOSi(CH3)2 (75) consecutively[30]

With VIII this would result in fragment mz 304 which was only present in thebackground However ions resulting from successive fragmentation of mz 304were observed mz 275 (-HCO -29) 247 (-C2H428) 233 (-CH214) and finally221 This sequence was regarded as prove that 4-353-NP was oxidized to itscarboxylic acid at position 70 of the molecule It should be mentioned that I VI VIIand VIII were only minor metabolites formed from 4-353-NP by the A githago cellsuspensions (Table 2)

Metabolism of 4-353-NP by Agrostemma githagoPlant Cell Suspension Cultures

The nonylphenol isomer 4-353-NP consisting of two diastereomers wastransformed by theAgrostemma githago suspension cultures to four types of productsglycosides of parent 4-353-NP glycosides of primary 4-353-NP metabolitesnonextractable residues and unknown possibly polymeric materials detected in themedia Similar products emerged in the soybean cultures The present investigationconcentrated on the primary metabolites of 4-353-NP These were produced in the Agithago suspensions using a two-liquid-phase system The primary products identifiedby GC-EIMS after liberation from their glycosides were one side-chain carboxylicacid and seven side-chain monohydroxylated metabolites Due to their mass spectrathe main products were four diastereomers of 60-hydroxy-4-353-NP

As yet data on the biotransformation of 4-353-NP are mainly not at handConcerning plant tissues (cell cultures plants) side-chain hydroxylated metaboliteswere reported on 4-n-nonylphenol[11122324] With A githago cells the main productwas 4-(80-hydroxy-n-nonyl)-phenol[24] ie 4-n-nonylphenol was metabolized bysubterminal oxidation corresponding with the main products observed with 4-353-NP in the present study Side-chain hydroxylated metabolites were also formed fromp-tert-octylphenol in barley plants[31] Experiments with model nonylphenols 2- 3-and 4-(4-hydroxyphenyl)-nonane demonstrated that in rainbow trout all isomerswere transformed to monohydroxy products with the site of oxidation at carbon 8(subterminal) of the nonyl residue The metabolites emerged as glucuronic acidconjugates while glucuronides of the parent nonylphenols were also formed[4] In asimilar study both side-chain and ring hydroxylated metabolites of 4-n-nonylphenolwere detected as glucuronic acid conjugates[9] Recently data on the metabolism ofthe nonylphenol isomer 4(3060-dimethyl-30-heptyl)-phenol in pond snails to a (ringhydroxylated) catechol derivative (with unmodified side-chain) were reported[1920]

Contrasting with 4-353-NP of the present study the isomer examined possessed

Metabolism of the Nonylphenol Isomer 545

Dow

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by [

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ORDER REPRINTS

a branching at subterminal position 60 of the side-chain besides that at carbon 30 Thecatechol was found conjugated to glucuronic acid[1920] As technical productnonylphenol appears to be completely degradated in soil[1] though isomer 4-(3060-dimethyl-30-heptyl)-phenol proved to be resistant in sedimentwater systems[18] Upto now the routes of nonylphenolrsquos microbial degradation are not known in detailDue to experiments with an isolated bacterial strain initial oxidation at the aromaticmoiety leads to degradation of the phenyl ring while side-chains remain asnonanols[3233] one of the studies was executed with radio-labelled 4-353-NP[33]

The present data on the metabolism of 4-353-NP by A githago cell suspensionsagree with those reported in the literature on 4-n-nonylphenol in plant tissues andmodel isomers in rainbow trout In contrast with trout the capacity of the A githagocells for hydroxylation of the side-chain appeared to be broader ie the nonyl residuewas hydroxylated at different sites of the aliphatic chain The enzymes responsible forside-chain hydroxylation of 4-353-NP in A githago are not known we suspectedcytochromes P450 In rats and humans isozyme CYP2B2 was thought to catalyzepredominantly the hydroxylation of nonylphenol isomers[10] After expression inyeast human CYP2B6 and CYP2C19 were shown to metabolize nonylphenol (pluralbranched chains) while 4-n-nonylphenol was transformed by CYP1A2 CYP2B6 andCYP2C19 ring and side-chain hydroxlated products were identified with branchedchain nonylphenols and 4-n-nonylphenol[34] We assume that plants in general havethe capacity to transform nonylphenol isomers to side-chain hydroxylated productsand thus contribute to the environmental degradation of the xenoestrogen If cropplants are contaminated with nonylphenol both nonylphenol and its side-chainhydroxylated products are expected to be present in plant tissues as glycosideconjugates Xenobiotic glycosides are known to be cleaved in the gastrointestinal tractwith release of possibly toxic aglycons Data on the toxicological properties of side-chain hydroxylated nonylphenols are not available Our future research will focus onthe metabolism of nonylphenol isomers with different branching patterns on theidentification of the enzymes responsible for the metabolism of nonylphenols and onthe (eco-) toxicology of side-chain hydroxylated nonylphenol isomers

ACKNOWLEDGMENTS

For the synthesis of [ring-U-14C]-4(3050-dimethyl-30-heptyl)-phenol the authorswish to thank R Vinken and the lsquolsquoAachen Graduate College Elimination ofEndocrine-Disrupting Substances from Waste-Waterrsquorsquo (AGEESA) funded by theDeutsche Forschungsgemeinschaft

REFERENCES

1 Staples CA Williams JB Blessing RL Varineau PT Measuring thebiodegradability of nonylphenol ether carboxylates octylphenol ether carboxyl-ates and nonylphenol Chemosphere 1999 38 (9) 2029ndash2039

2 Thiele B Gunther K Schwuger MJ Alkylphenol ethoxylates trace analysisand environmental behaviour Chem Rev 1997 97 (8) 3247ndash3272

546 Schmidt et al

Dow

nloa

ded

by [

Am

sG

iron

aba

rri L

ib]

at 2

357

07

Oct

ober

201

4

ORDER REPRINTS

3 Wheeler TF Heim JR LaTorre MR Janes AB Mass spectral

characterization of p-nonylphenol isomers using high-resolution capillary

GC-MS J Chromatogr Sci 1997 35 (1) 19ndash304 Meldahl AC Nithipatikom K Lech JJ Metabolism of several 14C-

nonylphenol isomers by rainbow trout (Oncorhynchus mykiss) in vivo and

in vitro Metabolites Xenobiotica 1996 26 (11) 1167ndash11805 Kim Y-S Katase T Sekine S Inoue T Makino M Uchiyama T

Fujimoto Y Yamashita N Variation in estrogenic activity among fractions

of a commercial nonylphenol by high performance liquid chromatography

Chemosphere 2004 54 (8) 1127ndash11346 Giger W Stephanou E Schaffner C Persistent organic chemicals in sewage

effluents 1 Identification of nonylphenols and nonylphenolethoxylates by

capillary gas chromatographymass spectrometry Chemosphere 1981 10 (11ndash1)

1253ndash12637 Giger W Brunner PH Schaffner C 4-Nonylphenol in sewage sludge

accumulation of toxic metabolites from nonionic surfactants Science 1984

225 (4662) 623ndash6258 Jobst H Chlorophenols an nonylphenols in sewage sludges 1 Occurrence in

sewage sludges of Western German treatment plants from 1987ndash1989 Acta

Hydrochim Hydrobiol 1995 23 (1) 20ndash259 Coldham NG Sivapathasundaram SDM Ashfield LA Pottinger TG

Biotransformation tissue distribution and persistence of 4-nonylphenol

residues in juvenile rainbow trout (Oncorhynchus mykiss) Drug Metab

Dispos 1998 26 (4) 347ndash35410 Lee PC Marquardt M Lech JJ Metabolism of nonylphenol by rat and

human microsomes Toxicol Lett 1998 99 (7) 117ndash12611 Bokern M Harms HH Toxicity and metabolism of 4-n-nonylphenol in cell

suspension cultures of different plant species Environ Sci Technol 1997

31 (7) 1849ndash185412 Bokern M Nimtz M Harms HH Metabolites of 4-n-nonylphenol in wheat

cell suspension cultures J Agric Food Chem 1996 44 (4) 1123ndash112713 Guenther K Heinke V Thiele B Kleist E Prast H Raecker T

Endocrine disrupting nonylphenols are ubiquitous in food Environ Sci

Technol 2002 36 (8) 1676ndash168014 Soto AM Justicia H Wray JW Sonnenschein C p-Nonylphenol an

estrogenic xenobiotic released from lsquolsquomodifiedrsquorsquo polystyrene Environ Health

Perspect 1991 92 167ndash17315 Degen DH Bolt HM Comments on lsquolsquoendocrine disrupting nonylphenols

are ubiquitous in foodrsquorsquo Envirn Sci Technol 2003 37 (11) 2622ndash262316 Vinken R Schmidt B Schaffer A Synthesis of tertiary 14C-labelled

nonylphenol isomers J Label Compd Radiopharm 2002 45 (14) 1253ndash126317 Lalah JO Lenoir D Henkelmann B Hertkorn N Gunther K

Schramm K-W Kettrup A Synthesis of ring-14C-labelled 4(3060-dimethyl-

30-heptyl)-phenol J Label Compd Radiopharm 2001 44 (6) 459ndash46318 Lalah JO Schramm K-W Henkelmann B Lenoir D Behechti A

Gunther K Kettrup A The dissipation distribution and fate of a branched

Metabolism of the Nonylphenol Isomer 547

Dow

nloa

ded

by [

Am

sG

iron

aba

rri L

ib]

at 2

357

07

Oct

ober

201

4

ORDER REPRINTS

14C-nonylphenol isomer in lake watersediment systems Environ Pollut 2003

122 (2) 195ndash20319 Lalah JO Schramm K-W Severin GF Lenoir D Henkelmann B

Behechti A Gunther K Kettrup A In vivo metabolism and organ

distribution of a branched 14C-nonylphenol isomer in pond snails Lymnaea

stagnalis L Aquat Toxicol 2003 62 (4) 305ndash31920 Lalah JO Behechti A Severin GF Lenoir D Gunther K Kettrup A

Schramm K-W The Bioaccumulation and fate of a branched 14C-

p-nonylphenol isomer in Lymnaea stagnalis L Environ Toxicol Chem 2003

22 (7) 1428ndash143621 Schmidt B Metabolic profiling using plant cell suspension cultures In

Pesticide Transformation in Plants and Microorganisms Hall JC Hoagland

RE Zablotowicz RM Eds American Chemical Society Washington DC

2001 40ndash5622 Komoszliga D Langebartels C Sandermann H Metabolic processes for

organic chemicals in plants In Plant ContaminationmdashModeling and Simulation

of Organic Chemical Processes Trapp S Mc Farlane JC Eds Lewis

Publishers Boca Raton 1995 69ndash10323 Schmidt B Schuphan I Metabolism of the Xenoestrogens 4-n-nonylphenol

and bisphenol A in plant cell suspension cultures In Book of Abstracts 10th

IUPAC International Congress on the Chemistry of Crop Protection Basel

August 4ndash9 IUPAC Basel 2002 Vol 2 3424 Schmidt B Patti H Niewersch C Schuphan I Biotransformation of [ring-

U-14C]4-n-nonylphenol by Agrostemma githago cell culture in a two-liquid-

phase system Biotechnol Lett 2003 25 (16) 1375ndash138125 Murashige T Skoog F Revised medium for rapid growth and bio assay with

tobacco tissue cultures Physiol Plant 1962 15 473ndash49726 Gamborg OL Miller RA Ojima K Nutrient requirements of suspension

cultures of soybean root cells Exp Cell Res 1968 50 151ndash15827 Schmidt B Schuphan I Metabolism of the environmental estrogen bisphenol

A by plant cell suspension cultures Chemosphere 2002 49 (1) 51ndash5928 Diekman J Thomson JB Djerassi C Mass spectrometry and stereo-

chemical problems CLV Electron impact induced fragmentations and

rearrangements of some trimethylsilyl ethers of aliphatic glycols and related

compounds J Org Chem 1968 33 (6) 2271ndash228429 Diekman J Thomson JB Djerassi C Mass spectrometry and stereo-

chemical problems CXLI Electron impact induced fragmentations and

rearrangements of trimethylsilyl ethers amines and sulfides J Org Chem

1967 32 (12) 3904ndash391930 Odham G Fatty acids In Biochemical Applications of Mass Spectrometry

Waller GR Dermer OC Eds Wiley-Interscience New York 1980 First

Supplementary Volume 153ndash17131 Stolzenberg GE Olson PA Zaylskie RG Mansager ER Behavior and

fate of ethoxylated alkylphenol nonionic surfactant in barley plants J Agric

Food Chem 1982 30 (4) 637ndash644

548 Schmidt et al

Dow

nloa

ded

by [

Am

sG

iron

aba

rri L

ib]

at 2

357

07

Oct

ober

201

4

ORDER REPRINTS

32 Tanghe T Dhooge W Verstraete W Isolation of a bacterial strain able todegrade branched nonylphenol Appl Environ Microbiol 1999 65 (2)746ndash751

33 Corvini PFX Vinken R Hommes G Schmidt B Dohmann MDegradation of the radioactive and non-labelled branched 4(3050-dimethyl-30-heptyl)-phenol nonylphenol isomer by Sphingomonas TTNP3 Biodegradation2003 15 (1) 1ndash10

34 Inui H Shiota N Motoi Y Ido Y Inoue T Kodama T Ohkawa YOhkawa H Metabolism of herbicides and other chemicals in humancytochrome P450 species and in transgenic potato plants co-expressinghuman CYP1A1 CYP2B6 and CYP2C19 J Pestic Sci 2001 26 (1) 28ndash40

Received February 13 2004

Metabolism of the Nonylphenol Isomer 549

Dow

nloa

ded

by [

Am

sG

iron

aba

rri L

ib]

at 2

357

07

Oct

ober

201

4

Request PermissionOrder Reprints

Reprints of this article can also be ordered at

httpwwwdekkercomservletproductDOI101081PFC200026776

Request Permission or Order Reprints Instantly

Interested in copying and sharing this article In most cases US Copyright Law requires that you get permission from the articlersquos rightsholder before using copyrighted content

All information and materials found in this article including but not limited to text trademarks patents logos graphics and images (the Materials) are the copyrighted works and other forms of intellectual property of Marcel Dekker Inc or its licensors All rights not expressly granted are reserved

Get permission to lawfully reproduce and distribute the Materials or order reprints quickly and painlessly Simply click on the Request Permission Order Reprints link below and follow the instructions Visit the US Copyright Office for information on Fair Use limitations of US copyright law Please refer to The Association of American Publishersrsquo (AAP) website for guidelines on Fair Use in the Classroom

The Materials are for your personal use only and cannot be reformatted reposted resold or distributed by electronic means or otherwise without permission from Marcel Dekker Inc Marcel Dekker Inc grants you the limited right to display the Materials only on your personal computer or personal wireless device and to copy and download single copies of such Materials provided that any copyright trademark or other notice appearing on such Materials is also retained by displayed copied or downloaded as part of the Materials and is not removed or obscured and provided you do not edit modify alter or enhance the Materials Please refer to our Website User Agreement for more details

Dow

nloa

ded

by [

Am

sG

iron

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rri L

ib]

at 2

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Oct

ober

201

4

ORDER REPRINTS

spectrum with that of II Due to its [Mthorn] of mz 394 compound VIII was identifiedwith a carboxylic acid metabolite of 4-353-NP since formation of a hydroxyderivative with an additional carbonyl function was regarded unlikely The massspectrum observed with VIII supported this assumption Fragments showingsimilarity to compounds IndashVII were mz 379 (-CH3) and 365 (-C2H5) whereasfragments mz 131 and 116 were supposed to consist of frac12CH2COOSiethCH3THORN

thorn3 and

frac12CH2COOSiethCH3THORNthorn2 respectively which pointed to a carboxyl function at positions

10 or 70 A characteristic feature of the fragmentation of trimethylsilyl derivatizedfatty acids is separation of CH3 (15) and HOSi(CH3)2 (75) consecutively[30]

With VIII this would result in fragment mz 304 which was only present in thebackground However ions resulting from successive fragmentation of mz 304were observed mz 275 (-HCO -29) 247 (-C2H428) 233 (-CH214) and finally221 This sequence was regarded as prove that 4-353-NP was oxidized to itscarboxylic acid at position 70 of the molecule It should be mentioned that I VI VIIand VIII were only minor metabolites formed from 4-353-NP by the A githago cellsuspensions (Table 2)

Metabolism of 4-353-NP by Agrostemma githagoPlant Cell Suspension Cultures

The nonylphenol isomer 4-353-NP consisting of two diastereomers wastransformed by theAgrostemma githago suspension cultures to four types of productsglycosides of parent 4-353-NP glycosides of primary 4-353-NP metabolitesnonextractable residues and unknown possibly polymeric materials detected in themedia Similar products emerged in the soybean cultures The present investigationconcentrated on the primary metabolites of 4-353-NP These were produced in the Agithago suspensions using a two-liquid-phase system The primary products identifiedby GC-EIMS after liberation from their glycosides were one side-chain carboxylicacid and seven side-chain monohydroxylated metabolites Due to their mass spectrathe main products were four diastereomers of 60-hydroxy-4-353-NP

As yet data on the biotransformation of 4-353-NP are mainly not at handConcerning plant tissues (cell cultures plants) side-chain hydroxylated metaboliteswere reported on 4-n-nonylphenol[11122324] With A githago cells the main productwas 4-(80-hydroxy-n-nonyl)-phenol[24] ie 4-n-nonylphenol was metabolized bysubterminal oxidation corresponding with the main products observed with 4-353-NP in the present study Side-chain hydroxylated metabolites were also formed fromp-tert-octylphenol in barley plants[31] Experiments with model nonylphenols 2- 3-and 4-(4-hydroxyphenyl)-nonane demonstrated that in rainbow trout all isomerswere transformed to monohydroxy products with the site of oxidation at carbon 8(subterminal) of the nonyl residue The metabolites emerged as glucuronic acidconjugates while glucuronides of the parent nonylphenols were also formed[4] In asimilar study both side-chain and ring hydroxylated metabolites of 4-n-nonylphenolwere detected as glucuronic acid conjugates[9] Recently data on the metabolism ofthe nonylphenol isomer 4(3060-dimethyl-30-heptyl)-phenol in pond snails to a (ringhydroxylated) catechol derivative (with unmodified side-chain) were reported[1920]

Contrasting with 4-353-NP of the present study the isomer examined possessed

Metabolism of the Nonylphenol Isomer 545

Dow

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ded

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at 2

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ober

201

4

ORDER REPRINTS

a branching at subterminal position 60 of the side-chain besides that at carbon 30 Thecatechol was found conjugated to glucuronic acid[1920] As technical productnonylphenol appears to be completely degradated in soil[1] though isomer 4-(3060-dimethyl-30-heptyl)-phenol proved to be resistant in sedimentwater systems[18] Upto now the routes of nonylphenolrsquos microbial degradation are not known in detailDue to experiments with an isolated bacterial strain initial oxidation at the aromaticmoiety leads to degradation of the phenyl ring while side-chains remain asnonanols[3233] one of the studies was executed with radio-labelled 4-353-NP[33]

The present data on the metabolism of 4-353-NP by A githago cell suspensionsagree with those reported in the literature on 4-n-nonylphenol in plant tissues andmodel isomers in rainbow trout In contrast with trout the capacity of the A githagocells for hydroxylation of the side-chain appeared to be broader ie the nonyl residuewas hydroxylated at different sites of the aliphatic chain The enzymes responsible forside-chain hydroxylation of 4-353-NP in A githago are not known we suspectedcytochromes P450 In rats and humans isozyme CYP2B2 was thought to catalyzepredominantly the hydroxylation of nonylphenol isomers[10] After expression inyeast human CYP2B6 and CYP2C19 were shown to metabolize nonylphenol (pluralbranched chains) while 4-n-nonylphenol was transformed by CYP1A2 CYP2B6 andCYP2C19 ring and side-chain hydroxlated products were identified with branchedchain nonylphenols and 4-n-nonylphenol[34] We assume that plants in general havethe capacity to transform nonylphenol isomers to side-chain hydroxylated productsand thus contribute to the environmental degradation of the xenoestrogen If cropplants are contaminated with nonylphenol both nonylphenol and its side-chainhydroxylated products are expected to be present in plant tissues as glycosideconjugates Xenobiotic glycosides are known to be cleaved in the gastrointestinal tractwith release of possibly toxic aglycons Data on the toxicological properties of side-chain hydroxylated nonylphenols are not available Our future research will focus onthe metabolism of nonylphenol isomers with different branching patterns on theidentification of the enzymes responsible for the metabolism of nonylphenols and onthe (eco-) toxicology of side-chain hydroxylated nonylphenol isomers

ACKNOWLEDGMENTS

For the synthesis of [ring-U-14C]-4(3050-dimethyl-30-heptyl)-phenol the authorswish to thank R Vinken and the lsquolsquoAachen Graduate College Elimination ofEndocrine-Disrupting Substances from Waste-Waterrsquorsquo (AGEESA) funded by theDeutsche Forschungsgemeinschaft

REFERENCES

1 Staples CA Williams JB Blessing RL Varineau PT Measuring thebiodegradability of nonylphenol ether carboxylates octylphenol ether carboxyl-ates and nonylphenol Chemosphere 1999 38 (9) 2029ndash2039

2 Thiele B Gunther K Schwuger MJ Alkylphenol ethoxylates trace analysisand environmental behaviour Chem Rev 1997 97 (8) 3247ndash3272

546 Schmidt et al

Dow

nloa

ded

by [

Am

sG

iron

aba

rri L

ib]

at 2

357

07

Oct

ober

201

4

ORDER REPRINTS

3 Wheeler TF Heim JR LaTorre MR Janes AB Mass spectral

characterization of p-nonylphenol isomers using high-resolution capillary

GC-MS J Chromatogr Sci 1997 35 (1) 19ndash304 Meldahl AC Nithipatikom K Lech JJ Metabolism of several 14C-

nonylphenol isomers by rainbow trout (Oncorhynchus mykiss) in vivo and

in vitro Metabolites Xenobiotica 1996 26 (11) 1167ndash11805 Kim Y-S Katase T Sekine S Inoue T Makino M Uchiyama T

Fujimoto Y Yamashita N Variation in estrogenic activity among fractions

of a commercial nonylphenol by high performance liquid chromatography

Chemosphere 2004 54 (8) 1127ndash11346 Giger W Stephanou E Schaffner C Persistent organic chemicals in sewage

effluents 1 Identification of nonylphenols and nonylphenolethoxylates by

capillary gas chromatographymass spectrometry Chemosphere 1981 10 (11ndash1)

1253ndash12637 Giger W Brunner PH Schaffner C 4-Nonylphenol in sewage sludge

accumulation of toxic metabolites from nonionic surfactants Science 1984

225 (4662) 623ndash6258 Jobst H Chlorophenols an nonylphenols in sewage sludges 1 Occurrence in

sewage sludges of Western German treatment plants from 1987ndash1989 Acta

Hydrochim Hydrobiol 1995 23 (1) 20ndash259 Coldham NG Sivapathasundaram SDM Ashfield LA Pottinger TG

Biotransformation tissue distribution and persistence of 4-nonylphenol

residues in juvenile rainbow trout (Oncorhynchus mykiss) Drug Metab

Dispos 1998 26 (4) 347ndash35410 Lee PC Marquardt M Lech JJ Metabolism of nonylphenol by rat and

human microsomes Toxicol Lett 1998 99 (7) 117ndash12611 Bokern M Harms HH Toxicity and metabolism of 4-n-nonylphenol in cell

suspension cultures of different plant species Environ Sci Technol 1997

31 (7) 1849ndash185412 Bokern M Nimtz M Harms HH Metabolites of 4-n-nonylphenol in wheat

cell suspension cultures J Agric Food Chem 1996 44 (4) 1123ndash112713 Guenther K Heinke V Thiele B Kleist E Prast H Raecker T

Endocrine disrupting nonylphenols are ubiquitous in food Environ Sci

Technol 2002 36 (8) 1676ndash168014 Soto AM Justicia H Wray JW Sonnenschein C p-Nonylphenol an

estrogenic xenobiotic released from lsquolsquomodifiedrsquorsquo polystyrene Environ Health

Perspect 1991 92 167ndash17315 Degen DH Bolt HM Comments on lsquolsquoendocrine disrupting nonylphenols

are ubiquitous in foodrsquorsquo Envirn Sci Technol 2003 37 (11) 2622ndash262316 Vinken R Schmidt B Schaffer A Synthesis of tertiary 14C-labelled

nonylphenol isomers J Label Compd Radiopharm 2002 45 (14) 1253ndash126317 Lalah JO Lenoir D Henkelmann B Hertkorn N Gunther K

Schramm K-W Kettrup A Synthesis of ring-14C-labelled 4(3060-dimethyl-

30-heptyl)-phenol J Label Compd Radiopharm 2001 44 (6) 459ndash46318 Lalah JO Schramm K-W Henkelmann B Lenoir D Behechti A

Gunther K Kettrup A The dissipation distribution and fate of a branched

Metabolism of the Nonylphenol Isomer 547

Dow

nloa

ded

by [

Am

sG

iron

aba

rri L

ib]

at 2

357

07

Oct

ober

201

4

ORDER REPRINTS

14C-nonylphenol isomer in lake watersediment systems Environ Pollut 2003

122 (2) 195ndash20319 Lalah JO Schramm K-W Severin GF Lenoir D Henkelmann B

Behechti A Gunther K Kettrup A In vivo metabolism and organ

distribution of a branched 14C-nonylphenol isomer in pond snails Lymnaea

stagnalis L Aquat Toxicol 2003 62 (4) 305ndash31920 Lalah JO Behechti A Severin GF Lenoir D Gunther K Kettrup A

Schramm K-W The Bioaccumulation and fate of a branched 14C-

p-nonylphenol isomer in Lymnaea stagnalis L Environ Toxicol Chem 2003

22 (7) 1428ndash143621 Schmidt B Metabolic profiling using plant cell suspension cultures In

Pesticide Transformation in Plants and Microorganisms Hall JC Hoagland

RE Zablotowicz RM Eds American Chemical Society Washington DC

2001 40ndash5622 Komoszliga D Langebartels C Sandermann H Metabolic processes for

organic chemicals in plants In Plant ContaminationmdashModeling and Simulation

of Organic Chemical Processes Trapp S Mc Farlane JC Eds Lewis

Publishers Boca Raton 1995 69ndash10323 Schmidt B Schuphan I Metabolism of the Xenoestrogens 4-n-nonylphenol

and bisphenol A in plant cell suspension cultures In Book of Abstracts 10th

IUPAC International Congress on the Chemistry of Crop Protection Basel

August 4ndash9 IUPAC Basel 2002 Vol 2 3424 Schmidt B Patti H Niewersch C Schuphan I Biotransformation of [ring-

U-14C]4-n-nonylphenol by Agrostemma githago cell culture in a two-liquid-

phase system Biotechnol Lett 2003 25 (16) 1375ndash138125 Murashige T Skoog F Revised medium for rapid growth and bio assay with

tobacco tissue cultures Physiol Plant 1962 15 473ndash49726 Gamborg OL Miller RA Ojima K Nutrient requirements of suspension

cultures of soybean root cells Exp Cell Res 1968 50 151ndash15827 Schmidt B Schuphan I Metabolism of the environmental estrogen bisphenol

A by plant cell suspension cultures Chemosphere 2002 49 (1) 51ndash5928 Diekman J Thomson JB Djerassi C Mass spectrometry and stereo-

chemical problems CLV Electron impact induced fragmentations and

rearrangements of some trimethylsilyl ethers of aliphatic glycols and related

compounds J Org Chem 1968 33 (6) 2271ndash228429 Diekman J Thomson JB Djerassi C Mass spectrometry and stereo-

chemical problems CXLI Electron impact induced fragmentations and

rearrangements of trimethylsilyl ethers amines and sulfides J Org Chem

1967 32 (12) 3904ndash391930 Odham G Fatty acids In Biochemical Applications of Mass Spectrometry

Waller GR Dermer OC Eds Wiley-Interscience New York 1980 First

Supplementary Volume 153ndash17131 Stolzenberg GE Olson PA Zaylskie RG Mansager ER Behavior and

fate of ethoxylated alkylphenol nonionic surfactant in barley plants J Agric

Food Chem 1982 30 (4) 637ndash644

548 Schmidt et al

Dow

nloa

ded

by [

Am

sG

iron

aba

rri L

ib]

at 2

357

07

Oct

ober

201

4

ORDER REPRINTS

32 Tanghe T Dhooge W Verstraete W Isolation of a bacterial strain able todegrade branched nonylphenol Appl Environ Microbiol 1999 65 (2)746ndash751

33 Corvini PFX Vinken R Hommes G Schmidt B Dohmann MDegradation of the radioactive and non-labelled branched 4(3050-dimethyl-30-heptyl)-phenol nonylphenol isomer by Sphingomonas TTNP3 Biodegradation2003 15 (1) 1ndash10

34 Inui H Shiota N Motoi Y Ido Y Inoue T Kodama T Ohkawa YOhkawa H Metabolism of herbicides and other chemicals in humancytochrome P450 species and in transgenic potato plants co-expressinghuman CYP1A1 CYP2B6 and CYP2C19 J Pestic Sci 2001 26 (1) 28ndash40

Received February 13 2004

Metabolism of the Nonylphenol Isomer 549

Dow

nloa

ded

by [

Am

sG

iron

aba

rri L

ib]

at 2

357

07

Oct

ober

201

4

Request PermissionOrder Reprints

Reprints of this article can also be ordered at

httpwwwdekkercomservletproductDOI101081PFC200026776

Request Permission or Order Reprints Instantly

Interested in copying and sharing this article In most cases US Copyright Law requires that you get permission from the articlersquos rightsholder before using copyrighted content

All information and materials found in this article including but not limited to text trademarks patents logos graphics and images (the Materials) are the copyrighted works and other forms of intellectual property of Marcel Dekker Inc or its licensors All rights not expressly granted are reserved

Get permission to lawfully reproduce and distribute the Materials or order reprints quickly and painlessly Simply click on the Request Permission Order Reprints link below and follow the instructions Visit the US Copyright Office for information on Fair Use limitations of US copyright law Please refer to The Association of American Publishersrsquo (AAP) website for guidelines on Fair Use in the Classroom

The Materials are for your personal use only and cannot be reformatted reposted resold or distributed by electronic means or otherwise without permission from Marcel Dekker Inc Marcel Dekker Inc grants you the limited right to display the Materials only on your personal computer or personal wireless device and to copy and download single copies of such Materials provided that any copyright trademark or other notice appearing on such Materials is also retained by displayed copied or downloaded as part of the Materials and is not removed or obscured and provided you do not edit modify alter or enhance the Materials Please refer to our Website User Agreement for more details

Dow

nloa

ded

by [

Am

sG

iron

aba

rri L

ib]

at 2

357

07

Oct

ober

201

4

ORDER REPRINTS

a branching at subterminal position 60 of the side-chain besides that at carbon 30 Thecatechol was found conjugated to glucuronic acid[1920] As technical productnonylphenol appears to be completely degradated in soil[1] though isomer 4-(3060-dimethyl-30-heptyl)-phenol proved to be resistant in sedimentwater systems[18] Upto now the routes of nonylphenolrsquos microbial degradation are not known in detailDue to experiments with an isolated bacterial strain initial oxidation at the aromaticmoiety leads to degradation of the phenyl ring while side-chains remain asnonanols[3233] one of the studies was executed with radio-labelled 4-353-NP[33]

The present data on the metabolism of 4-353-NP by A githago cell suspensionsagree with those reported in the literature on 4-n-nonylphenol in plant tissues andmodel isomers in rainbow trout In contrast with trout the capacity of the A githagocells for hydroxylation of the side-chain appeared to be broader ie the nonyl residuewas hydroxylated at different sites of the aliphatic chain The enzymes responsible forside-chain hydroxylation of 4-353-NP in A githago are not known we suspectedcytochromes P450 In rats and humans isozyme CYP2B2 was thought to catalyzepredominantly the hydroxylation of nonylphenol isomers[10] After expression inyeast human CYP2B6 and CYP2C19 were shown to metabolize nonylphenol (pluralbranched chains) while 4-n-nonylphenol was transformed by CYP1A2 CYP2B6 andCYP2C19 ring and side-chain hydroxlated products were identified with branchedchain nonylphenols and 4-n-nonylphenol[34] We assume that plants in general havethe capacity to transform nonylphenol isomers to side-chain hydroxylated productsand thus contribute to the environmental degradation of the xenoestrogen If cropplants are contaminated with nonylphenol both nonylphenol and its side-chainhydroxylated products are expected to be present in plant tissues as glycosideconjugates Xenobiotic glycosides are known to be cleaved in the gastrointestinal tractwith release of possibly toxic aglycons Data on the toxicological properties of side-chain hydroxylated nonylphenols are not available Our future research will focus onthe metabolism of nonylphenol isomers with different branching patterns on theidentification of the enzymes responsible for the metabolism of nonylphenols and onthe (eco-) toxicology of side-chain hydroxylated nonylphenol isomers

ACKNOWLEDGMENTS

For the synthesis of [ring-U-14C]-4(3050-dimethyl-30-heptyl)-phenol the authorswish to thank R Vinken and the lsquolsquoAachen Graduate College Elimination ofEndocrine-Disrupting Substances from Waste-Waterrsquorsquo (AGEESA) funded by theDeutsche Forschungsgemeinschaft

REFERENCES

1 Staples CA Williams JB Blessing RL Varineau PT Measuring thebiodegradability of nonylphenol ether carboxylates octylphenol ether carboxyl-ates and nonylphenol Chemosphere 1999 38 (9) 2029ndash2039

2 Thiele B Gunther K Schwuger MJ Alkylphenol ethoxylates trace analysisand environmental behaviour Chem Rev 1997 97 (8) 3247ndash3272

546 Schmidt et al

Dow

nloa

ded

by [

Am

sG

iron

aba

rri L

ib]

at 2

357

07

Oct

ober

201

4

ORDER REPRINTS

3 Wheeler TF Heim JR LaTorre MR Janes AB Mass spectral

characterization of p-nonylphenol isomers using high-resolution capillary

GC-MS J Chromatogr Sci 1997 35 (1) 19ndash304 Meldahl AC Nithipatikom K Lech JJ Metabolism of several 14C-

nonylphenol isomers by rainbow trout (Oncorhynchus mykiss) in vivo and

in vitro Metabolites Xenobiotica 1996 26 (11) 1167ndash11805 Kim Y-S Katase T Sekine S Inoue T Makino M Uchiyama T

Fujimoto Y Yamashita N Variation in estrogenic activity among fractions

of a commercial nonylphenol by high performance liquid chromatography

Chemosphere 2004 54 (8) 1127ndash11346 Giger W Stephanou E Schaffner C Persistent organic chemicals in sewage

effluents 1 Identification of nonylphenols and nonylphenolethoxylates by

capillary gas chromatographymass spectrometry Chemosphere 1981 10 (11ndash1)

1253ndash12637 Giger W Brunner PH Schaffner C 4-Nonylphenol in sewage sludge

accumulation of toxic metabolites from nonionic surfactants Science 1984

225 (4662) 623ndash6258 Jobst H Chlorophenols an nonylphenols in sewage sludges 1 Occurrence in

sewage sludges of Western German treatment plants from 1987ndash1989 Acta

Hydrochim Hydrobiol 1995 23 (1) 20ndash259 Coldham NG Sivapathasundaram SDM Ashfield LA Pottinger TG

Biotransformation tissue distribution and persistence of 4-nonylphenol

residues in juvenile rainbow trout (Oncorhynchus mykiss) Drug Metab

Dispos 1998 26 (4) 347ndash35410 Lee PC Marquardt M Lech JJ Metabolism of nonylphenol by rat and

human microsomes Toxicol Lett 1998 99 (7) 117ndash12611 Bokern M Harms HH Toxicity and metabolism of 4-n-nonylphenol in cell

suspension cultures of different plant species Environ Sci Technol 1997

31 (7) 1849ndash185412 Bokern M Nimtz M Harms HH Metabolites of 4-n-nonylphenol in wheat

cell suspension cultures J Agric Food Chem 1996 44 (4) 1123ndash112713 Guenther K Heinke V Thiele B Kleist E Prast H Raecker T

Endocrine disrupting nonylphenols are ubiquitous in food Environ Sci

Technol 2002 36 (8) 1676ndash168014 Soto AM Justicia H Wray JW Sonnenschein C p-Nonylphenol an

estrogenic xenobiotic released from lsquolsquomodifiedrsquorsquo polystyrene Environ Health

Perspect 1991 92 167ndash17315 Degen DH Bolt HM Comments on lsquolsquoendocrine disrupting nonylphenols

are ubiquitous in foodrsquorsquo Envirn Sci Technol 2003 37 (11) 2622ndash262316 Vinken R Schmidt B Schaffer A Synthesis of tertiary 14C-labelled

nonylphenol isomers J Label Compd Radiopharm 2002 45 (14) 1253ndash126317 Lalah JO Lenoir D Henkelmann B Hertkorn N Gunther K

Schramm K-W Kettrup A Synthesis of ring-14C-labelled 4(3060-dimethyl-

30-heptyl)-phenol J Label Compd Radiopharm 2001 44 (6) 459ndash46318 Lalah JO Schramm K-W Henkelmann B Lenoir D Behechti A

Gunther K Kettrup A The dissipation distribution and fate of a branched

Metabolism of the Nonylphenol Isomer 547

Dow

nloa

ded

by [

Am

sG

iron

aba

rri L

ib]

at 2

357

07

Oct

ober

201

4

ORDER REPRINTS

14C-nonylphenol isomer in lake watersediment systems Environ Pollut 2003

122 (2) 195ndash20319 Lalah JO Schramm K-W Severin GF Lenoir D Henkelmann B

Behechti A Gunther K Kettrup A In vivo metabolism and organ

distribution of a branched 14C-nonylphenol isomer in pond snails Lymnaea

stagnalis L Aquat Toxicol 2003 62 (4) 305ndash31920 Lalah JO Behechti A Severin GF Lenoir D Gunther K Kettrup A

Schramm K-W The Bioaccumulation and fate of a branched 14C-

p-nonylphenol isomer in Lymnaea stagnalis L Environ Toxicol Chem 2003

22 (7) 1428ndash143621 Schmidt B Metabolic profiling using plant cell suspension cultures In

Pesticide Transformation in Plants and Microorganisms Hall JC Hoagland

RE Zablotowicz RM Eds American Chemical Society Washington DC

2001 40ndash5622 Komoszliga D Langebartels C Sandermann H Metabolic processes for

organic chemicals in plants In Plant ContaminationmdashModeling and Simulation

of Organic Chemical Processes Trapp S Mc Farlane JC Eds Lewis

Publishers Boca Raton 1995 69ndash10323 Schmidt B Schuphan I Metabolism of the Xenoestrogens 4-n-nonylphenol

and bisphenol A in plant cell suspension cultures In Book of Abstracts 10th

IUPAC International Congress on the Chemistry of Crop Protection Basel

August 4ndash9 IUPAC Basel 2002 Vol 2 3424 Schmidt B Patti H Niewersch C Schuphan I Biotransformation of [ring-

U-14C]4-n-nonylphenol by Agrostemma githago cell culture in a two-liquid-

phase system Biotechnol Lett 2003 25 (16) 1375ndash138125 Murashige T Skoog F Revised medium for rapid growth and bio assay with

tobacco tissue cultures Physiol Plant 1962 15 473ndash49726 Gamborg OL Miller RA Ojima K Nutrient requirements of suspension

cultures of soybean root cells Exp Cell Res 1968 50 151ndash15827 Schmidt B Schuphan I Metabolism of the environmental estrogen bisphenol

A by plant cell suspension cultures Chemosphere 2002 49 (1) 51ndash5928 Diekman J Thomson JB Djerassi C Mass spectrometry and stereo-

chemical problems CLV Electron impact induced fragmentations and

rearrangements of some trimethylsilyl ethers of aliphatic glycols and related

compounds J Org Chem 1968 33 (6) 2271ndash228429 Diekman J Thomson JB Djerassi C Mass spectrometry and stereo-

chemical problems CXLI Electron impact induced fragmentations and

rearrangements of trimethylsilyl ethers amines and sulfides J Org Chem

1967 32 (12) 3904ndash391930 Odham G Fatty acids In Biochemical Applications of Mass Spectrometry

Waller GR Dermer OC Eds Wiley-Interscience New York 1980 First

Supplementary Volume 153ndash17131 Stolzenberg GE Olson PA Zaylskie RG Mansager ER Behavior and

fate of ethoxylated alkylphenol nonionic surfactant in barley plants J Agric

Food Chem 1982 30 (4) 637ndash644

548 Schmidt et al

Dow

nloa

ded

by [

Am

sG

iron

aba

rri L

ib]

at 2

357

07

Oct

ober

201

4

ORDER REPRINTS

32 Tanghe T Dhooge W Verstraete W Isolation of a bacterial strain able todegrade branched nonylphenol Appl Environ Microbiol 1999 65 (2)746ndash751

33 Corvini PFX Vinken R Hommes G Schmidt B Dohmann MDegradation of the radioactive and non-labelled branched 4(3050-dimethyl-30-heptyl)-phenol nonylphenol isomer by Sphingomonas TTNP3 Biodegradation2003 15 (1) 1ndash10

34 Inui H Shiota N Motoi Y Ido Y Inoue T Kodama T Ohkawa YOhkawa H Metabolism of herbicides and other chemicals in humancytochrome P450 species and in transgenic potato plants co-expressinghuman CYP1A1 CYP2B6 and CYP2C19 J Pestic Sci 2001 26 (1) 28ndash40

Received February 13 2004

Metabolism of the Nonylphenol Isomer 549

Dow

nloa

ded

by [

Am

sG

iron

aba

rri L

ib]

at 2

357

07

Oct

ober

201

4

Request PermissionOrder Reprints

Reprints of this article can also be ordered at

httpwwwdekkercomservletproductDOI101081PFC200026776

Request Permission or Order Reprints Instantly

Interested in copying and sharing this article In most cases US Copyright Law requires that you get permission from the articlersquos rightsholder before using copyrighted content

All information and materials found in this article including but not limited to text trademarks patents logos graphics and images (the Materials) are the copyrighted works and other forms of intellectual property of Marcel Dekker Inc or its licensors All rights not expressly granted are reserved

Get permission to lawfully reproduce and distribute the Materials or order reprints quickly and painlessly Simply click on the Request Permission Order Reprints link below and follow the instructions Visit the US Copyright Office for information on Fair Use limitations of US copyright law Please refer to The Association of American Publishersrsquo (AAP) website for guidelines on Fair Use in the Classroom

The Materials are for your personal use only and cannot be reformatted reposted resold or distributed by electronic means or otherwise without permission from Marcel Dekker Inc Marcel Dekker Inc grants you the limited right to display the Materials only on your personal computer or personal wireless device and to copy and download single copies of such Materials provided that any copyright trademark or other notice appearing on such Materials is also retained by displayed copied or downloaded as part of the Materials and is not removed or obscured and provided you do not edit modify alter or enhance the Materials Please refer to our Website User Agreement for more details

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ORDER REPRINTS

3 Wheeler TF Heim JR LaTorre MR Janes AB Mass spectral

characterization of p-nonylphenol isomers using high-resolution capillary

GC-MS J Chromatogr Sci 1997 35 (1) 19ndash304 Meldahl AC Nithipatikom K Lech JJ Metabolism of several 14C-

nonylphenol isomers by rainbow trout (Oncorhynchus mykiss) in vivo and

in vitro Metabolites Xenobiotica 1996 26 (11) 1167ndash11805 Kim Y-S Katase T Sekine S Inoue T Makino M Uchiyama T

Fujimoto Y Yamashita N Variation in estrogenic activity among fractions

of a commercial nonylphenol by high performance liquid chromatography

Chemosphere 2004 54 (8) 1127ndash11346 Giger W Stephanou E Schaffner C Persistent organic chemicals in sewage

effluents 1 Identification of nonylphenols and nonylphenolethoxylates by

capillary gas chromatographymass spectrometry Chemosphere 1981 10 (11ndash1)

1253ndash12637 Giger W Brunner PH Schaffner C 4-Nonylphenol in sewage sludge

accumulation of toxic metabolites from nonionic surfactants Science 1984

225 (4662) 623ndash6258 Jobst H Chlorophenols an nonylphenols in sewage sludges 1 Occurrence in

sewage sludges of Western German treatment plants from 1987ndash1989 Acta

Hydrochim Hydrobiol 1995 23 (1) 20ndash259 Coldham NG Sivapathasundaram SDM Ashfield LA Pottinger TG

Biotransformation tissue distribution and persistence of 4-nonylphenol

residues in juvenile rainbow trout (Oncorhynchus mykiss) Drug Metab

Dispos 1998 26 (4) 347ndash35410 Lee PC Marquardt M Lech JJ Metabolism of nonylphenol by rat and

human microsomes Toxicol Lett 1998 99 (7) 117ndash12611 Bokern M Harms HH Toxicity and metabolism of 4-n-nonylphenol in cell

suspension cultures of different plant species Environ Sci Technol 1997

31 (7) 1849ndash185412 Bokern M Nimtz M Harms HH Metabolites of 4-n-nonylphenol in wheat

cell suspension cultures J Agric Food Chem 1996 44 (4) 1123ndash112713 Guenther K Heinke V Thiele B Kleist E Prast H Raecker T

Endocrine disrupting nonylphenols are ubiquitous in food Environ Sci

Technol 2002 36 (8) 1676ndash168014 Soto AM Justicia H Wray JW Sonnenschein C p-Nonylphenol an

estrogenic xenobiotic released from lsquolsquomodifiedrsquorsquo polystyrene Environ Health

Perspect 1991 92 167ndash17315 Degen DH Bolt HM Comments on lsquolsquoendocrine disrupting nonylphenols

are ubiquitous in foodrsquorsquo Envirn Sci Technol 2003 37 (11) 2622ndash262316 Vinken R Schmidt B Schaffer A Synthesis of tertiary 14C-labelled

nonylphenol isomers J Label Compd Radiopharm 2002 45 (14) 1253ndash126317 Lalah JO Lenoir D Henkelmann B Hertkorn N Gunther K

Schramm K-W Kettrup A Synthesis of ring-14C-labelled 4(3060-dimethyl-

30-heptyl)-phenol J Label Compd Radiopharm 2001 44 (6) 459ndash46318 Lalah JO Schramm K-W Henkelmann B Lenoir D Behechti A

Gunther K Kettrup A The dissipation distribution and fate of a branched

Metabolism of the Nonylphenol Isomer 547

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14C-nonylphenol isomer in lake watersediment systems Environ Pollut 2003

122 (2) 195ndash20319 Lalah JO Schramm K-W Severin GF Lenoir D Henkelmann B

Behechti A Gunther K Kettrup A In vivo metabolism and organ

distribution of a branched 14C-nonylphenol isomer in pond snails Lymnaea

stagnalis L Aquat Toxicol 2003 62 (4) 305ndash31920 Lalah JO Behechti A Severin GF Lenoir D Gunther K Kettrup A

Schramm K-W The Bioaccumulation and fate of a branched 14C-

p-nonylphenol isomer in Lymnaea stagnalis L Environ Toxicol Chem 2003

22 (7) 1428ndash143621 Schmidt B Metabolic profiling using plant cell suspension cultures In

Pesticide Transformation in Plants and Microorganisms Hall JC Hoagland

RE Zablotowicz RM Eds American Chemical Society Washington DC

2001 40ndash5622 Komoszliga D Langebartels C Sandermann H Metabolic processes for

organic chemicals in plants In Plant ContaminationmdashModeling and Simulation

of Organic Chemical Processes Trapp S Mc Farlane JC Eds Lewis

Publishers Boca Raton 1995 69ndash10323 Schmidt B Schuphan I Metabolism of the Xenoestrogens 4-n-nonylphenol

and bisphenol A in plant cell suspension cultures In Book of Abstracts 10th

IUPAC International Congress on the Chemistry of Crop Protection Basel

August 4ndash9 IUPAC Basel 2002 Vol 2 3424 Schmidt B Patti H Niewersch C Schuphan I Biotransformation of [ring-

U-14C]4-n-nonylphenol by Agrostemma githago cell culture in a two-liquid-

phase system Biotechnol Lett 2003 25 (16) 1375ndash138125 Murashige T Skoog F Revised medium for rapid growth and bio assay with

tobacco tissue cultures Physiol Plant 1962 15 473ndash49726 Gamborg OL Miller RA Ojima K Nutrient requirements of suspension

cultures of soybean root cells Exp Cell Res 1968 50 151ndash15827 Schmidt B Schuphan I Metabolism of the environmental estrogen bisphenol

A by plant cell suspension cultures Chemosphere 2002 49 (1) 51ndash5928 Diekman J Thomson JB Djerassi C Mass spectrometry and stereo-

chemical problems CLV Electron impact induced fragmentations and

rearrangements of some trimethylsilyl ethers of aliphatic glycols and related

compounds J Org Chem 1968 33 (6) 2271ndash228429 Diekman J Thomson JB Djerassi C Mass spectrometry and stereo-

chemical problems CXLI Electron impact induced fragmentations and

rearrangements of trimethylsilyl ethers amines and sulfides J Org Chem

1967 32 (12) 3904ndash391930 Odham G Fatty acids In Biochemical Applications of Mass Spectrometry

Waller GR Dermer OC Eds Wiley-Interscience New York 1980 First

Supplementary Volume 153ndash17131 Stolzenberg GE Olson PA Zaylskie RG Mansager ER Behavior and

fate of ethoxylated alkylphenol nonionic surfactant in barley plants J Agric

Food Chem 1982 30 (4) 637ndash644

548 Schmidt et al

Dow

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ded

by [

Am

sG

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aba

rri L

ib]

at 2

357

07

Oct

ober

201

4

ORDER REPRINTS

32 Tanghe T Dhooge W Verstraete W Isolation of a bacterial strain able todegrade branched nonylphenol Appl Environ Microbiol 1999 65 (2)746ndash751

33 Corvini PFX Vinken R Hommes G Schmidt B Dohmann MDegradation of the radioactive and non-labelled branched 4(3050-dimethyl-30-heptyl)-phenol nonylphenol isomer by Sphingomonas TTNP3 Biodegradation2003 15 (1) 1ndash10

34 Inui H Shiota N Motoi Y Ido Y Inoue T Kodama T Ohkawa YOhkawa H Metabolism of herbicides and other chemicals in humancytochrome P450 species and in transgenic potato plants co-expressinghuman CYP1A1 CYP2B6 and CYP2C19 J Pestic Sci 2001 26 (1) 28ndash40

Received February 13 2004

Metabolism of the Nonylphenol Isomer 549

Dow

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at 2

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ober

201

4

Request PermissionOrder Reprints

Reprints of this article can also be ordered at

httpwwwdekkercomservletproductDOI101081PFC200026776

Request Permission or Order Reprints Instantly

Interested in copying and sharing this article In most cases US Copyright Law requires that you get permission from the articlersquos rightsholder before using copyrighted content

All information and materials found in this article including but not limited to text trademarks patents logos graphics and images (the Materials) are the copyrighted works and other forms of intellectual property of Marcel Dekker Inc or its licensors All rights not expressly granted are reserved

Get permission to lawfully reproduce and distribute the Materials or order reprints quickly and painlessly Simply click on the Request Permission Order Reprints link below and follow the instructions Visit the US Copyright Office for information on Fair Use limitations of US copyright law Please refer to The Association of American Publishersrsquo (AAP) website for guidelines on Fair Use in the Classroom

The Materials are for your personal use only and cannot be reformatted reposted resold or distributed by electronic means or otherwise without permission from Marcel Dekker Inc Marcel Dekker Inc grants you the limited right to display the Materials only on your personal computer or personal wireless device and to copy and download single copies of such Materials provided that any copyright trademark or other notice appearing on such Materials is also retained by displayed copied or downloaded as part of the Materials and is not removed or obscured and provided you do not edit modify alter or enhance the Materials Please refer to our Website User Agreement for more details

Dow

nloa

ded

by [

Am

sG

iron

aba

rri L

ib]

at 2

357

07

Oct

ober

201

4

ORDER REPRINTS

14C-nonylphenol isomer in lake watersediment systems Environ Pollut 2003

122 (2) 195ndash20319 Lalah JO Schramm K-W Severin GF Lenoir D Henkelmann B

Behechti A Gunther K Kettrup A In vivo metabolism and organ

distribution of a branched 14C-nonylphenol isomer in pond snails Lymnaea

stagnalis L Aquat Toxicol 2003 62 (4) 305ndash31920 Lalah JO Behechti A Severin GF Lenoir D Gunther K Kettrup A

Schramm K-W The Bioaccumulation and fate of a branched 14C-

p-nonylphenol isomer in Lymnaea stagnalis L Environ Toxicol Chem 2003

22 (7) 1428ndash143621 Schmidt B Metabolic profiling using plant cell suspension cultures In

Pesticide Transformation in Plants and Microorganisms Hall JC Hoagland

RE Zablotowicz RM Eds American Chemical Society Washington DC

2001 40ndash5622 Komoszliga D Langebartels C Sandermann H Metabolic processes for

organic chemicals in plants In Plant ContaminationmdashModeling and Simulation

of Organic Chemical Processes Trapp S Mc Farlane JC Eds Lewis

Publishers Boca Raton 1995 69ndash10323 Schmidt B Schuphan I Metabolism of the Xenoestrogens 4-n-nonylphenol

and bisphenol A in plant cell suspension cultures In Book of Abstracts 10th

IUPAC International Congress on the Chemistry of Crop Protection Basel

August 4ndash9 IUPAC Basel 2002 Vol 2 3424 Schmidt B Patti H Niewersch C Schuphan I Biotransformation of [ring-

U-14C]4-n-nonylphenol by Agrostemma githago cell culture in a two-liquid-

phase system Biotechnol Lett 2003 25 (16) 1375ndash138125 Murashige T Skoog F Revised medium for rapid growth and bio assay with

tobacco tissue cultures Physiol Plant 1962 15 473ndash49726 Gamborg OL Miller RA Ojima K Nutrient requirements of suspension

cultures of soybean root cells Exp Cell Res 1968 50 151ndash15827 Schmidt B Schuphan I Metabolism of the environmental estrogen bisphenol

A by plant cell suspension cultures Chemosphere 2002 49 (1) 51ndash5928 Diekman J Thomson JB Djerassi C Mass spectrometry and stereo-

chemical problems CLV Electron impact induced fragmentations and

rearrangements of some trimethylsilyl ethers of aliphatic glycols and related

compounds J Org Chem 1968 33 (6) 2271ndash228429 Diekman J Thomson JB Djerassi C Mass spectrometry and stereo-

chemical problems CXLI Electron impact induced fragmentations and

rearrangements of trimethylsilyl ethers amines and sulfides J Org Chem

1967 32 (12) 3904ndash391930 Odham G Fatty acids In Biochemical Applications of Mass Spectrometry

Waller GR Dermer OC Eds Wiley-Interscience New York 1980 First

Supplementary Volume 153ndash17131 Stolzenberg GE Olson PA Zaylskie RG Mansager ER Behavior and

fate of ethoxylated alkylphenol nonionic surfactant in barley plants J Agric

Food Chem 1982 30 (4) 637ndash644

548 Schmidt et al

Dow

nloa

ded

by [

Am

sG

iron

aba

rri L

ib]

at 2

357

07

Oct

ober

201

4

ORDER REPRINTS

32 Tanghe T Dhooge W Verstraete W Isolation of a bacterial strain able todegrade branched nonylphenol Appl Environ Microbiol 1999 65 (2)746ndash751

33 Corvini PFX Vinken R Hommes G Schmidt B Dohmann MDegradation of the radioactive and non-labelled branched 4(3050-dimethyl-30-heptyl)-phenol nonylphenol isomer by Sphingomonas TTNP3 Biodegradation2003 15 (1) 1ndash10

34 Inui H Shiota N Motoi Y Ido Y Inoue T Kodama T Ohkawa YOhkawa H Metabolism of herbicides and other chemicals in humancytochrome P450 species and in transgenic potato plants co-expressinghuman CYP1A1 CYP2B6 and CYP2C19 J Pestic Sci 2001 26 (1) 28ndash40

Received February 13 2004

Metabolism of the Nonylphenol Isomer 549

Dow

nloa

ded

by [

Am

sG

iron

aba

rri L

ib]

at 2

357

07

Oct

ober

201

4

Request PermissionOrder Reprints

Reprints of this article can also be ordered at

httpwwwdekkercomservletproductDOI101081PFC200026776

Request Permission or Order Reprints Instantly

Interested in copying and sharing this article In most cases US Copyright Law requires that you get permission from the articlersquos rightsholder before using copyrighted content

All information and materials found in this article including but not limited to text trademarks patents logos graphics and images (the Materials) are the copyrighted works and other forms of intellectual property of Marcel Dekker Inc or its licensors All rights not expressly granted are reserved

Get permission to lawfully reproduce and distribute the Materials or order reprints quickly and painlessly Simply click on the Request Permission Order Reprints link below and follow the instructions Visit the US Copyright Office for information on Fair Use limitations of US copyright law Please refer to The Association of American Publishersrsquo (AAP) website for guidelines on Fair Use in the Classroom

The Materials are for your personal use only and cannot be reformatted reposted resold or distributed by electronic means or otherwise without permission from Marcel Dekker Inc Marcel Dekker Inc grants you the limited right to display the Materials only on your personal computer or personal wireless device and to copy and download single copies of such Materials provided that any copyright trademark or other notice appearing on such Materials is also retained by displayed copied or downloaded as part of the Materials and is not removed or obscured and provided you do not edit modify alter or enhance the Materials Please refer to our Website User Agreement for more details

Dow

nloa

ded

by [

Am

sG

iron

aba

rri L

ib]

at 2

357

07

Oct

ober

201

4

ORDER REPRINTS

32 Tanghe T Dhooge W Verstraete W Isolation of a bacterial strain able todegrade branched nonylphenol Appl Environ Microbiol 1999 65 (2)746ndash751

33 Corvini PFX Vinken R Hommes G Schmidt B Dohmann MDegradation of the radioactive and non-labelled branched 4(3050-dimethyl-30-heptyl)-phenol nonylphenol isomer by Sphingomonas TTNP3 Biodegradation2003 15 (1) 1ndash10

34 Inui H Shiota N Motoi Y Ido Y Inoue T Kodama T Ohkawa YOhkawa H Metabolism of herbicides and other chemicals in humancytochrome P450 species and in transgenic potato plants co-expressinghuman CYP1A1 CYP2B6 and CYP2C19 J Pestic Sci 2001 26 (1) 28ndash40

Received February 13 2004

Metabolism of the Nonylphenol Isomer 549

Dow

nloa

ded

by [

Am

sG

iron

aba

rri L

ib]

at 2

357

07

Oct

ober

201

4

Request PermissionOrder Reprints

Reprints of this article can also be ordered at

httpwwwdekkercomservletproductDOI101081PFC200026776

Request Permission or Order Reprints Instantly

Interested in copying and sharing this article In most cases US Copyright Law requires that you get permission from the articlersquos rightsholder before using copyrighted content

All information and materials found in this article including but not limited to text trademarks patents logos graphics and images (the Materials) are the copyrighted works and other forms of intellectual property of Marcel Dekker Inc or its licensors All rights not expressly granted are reserved

Get permission to lawfully reproduce and distribute the Materials or order reprints quickly and painlessly Simply click on the Request Permission Order Reprints link below and follow the instructions Visit the US Copyright Office for information on Fair Use limitations of US copyright law Please refer to The Association of American Publishersrsquo (AAP) website for guidelines on Fair Use in the Classroom

The Materials are for your personal use only and cannot be reformatted reposted resold or distributed by electronic means or otherwise without permission from Marcel Dekker Inc Marcel Dekker Inc grants you the limited right to display the Materials only on your personal computer or personal wireless device and to copy and download single copies of such Materials provided that any copyright trademark or other notice appearing on such Materials is also retained by displayed copied or downloaded as part of the Materials and is not removed or obscured and provided you do not edit modify alter or enhance the Materials Please refer to our Website User Agreement for more details

Dow

nloa

ded

by [

Am

sG

iron

aba

rri L

ib]

at 2

357

07

Oct

ober

201

4

Request PermissionOrder Reprints

Reprints of this article can also be ordered at

httpwwwdekkercomservletproductDOI101081PFC200026776

Request Permission or Order Reprints Instantly

Interested in copying and sharing this article In most cases US Copyright Law requires that you get permission from the articlersquos rightsholder before using copyrighted content

All information and materials found in this article including but not limited to text trademarks patents logos graphics and images (the Materials) are the copyrighted works and other forms of intellectual property of Marcel Dekker Inc or its licensors All rights not expressly granted are reserved

Get permission to lawfully reproduce and distribute the Materials or order reprints quickly and painlessly Simply click on the Request Permission Order Reprints link below and follow the instructions Visit the US Copyright Office for information on Fair Use limitations of US copyright law Please refer to The Association of American Publishersrsquo (AAP) website for guidelines on Fair Use in the Classroom

The Materials are for your personal use only and cannot be reformatted reposted resold or distributed by electronic means or otherwise without permission from Marcel Dekker Inc Marcel Dekker Inc grants you the limited right to display the Materials only on your personal computer or personal wireless device and to copy and download single copies of such Materials provided that any copyright trademark or other notice appearing on such Materials is also retained by displayed copied or downloaded as part of the Materials and is not removed or obscured and provided you do not edit modify alter or enhance the Materials Please refer to our Website User Agreement for more details

Dow

nloa

ded

by [

Am

sG

iron

aba

rri L

ib]

at 2

357

07

Oct

ober

201

4