Chemico-Biological Interactions 148 (2004) 69–92

DNA adducts in rats and mice following exposure to[4-14C]-1,2-epoxy-3-butene and to [2,3-14C]-1,3-butadiene

Peter J. Boogaard∗, Kees P. de Kloe, Ewan D. Booth1, William P. Watson1

Shell Laboratory for Molecular Toxicology, Shell Research and Technology Centre, Amsterdam,Shell International Chemicals B.V., Amsterdam, The Netherlands

Received 12 December 2003; received in revised form 28 February 2004; accepted 29 February 2004

Abstract

1,3-Butadiene (BD) is a major industrial chemical and a rodent carcinogen, with mice being much more susceptible thanrats. Oxidative metabolism of BD, leading to the DNA-reactive epoxides 1,2-epoxy-3-butene (BMO), 1,2-epoxy-3,4-butanediol(EBD) and 1,2:3,4-diepoxybutane (DEB), is greater in mice than rats. In the present study the DNA adduct profiles in liver andlungs of rats and mice were determined following exposure to BMO and to BD since these profiles may provide qualitative andquantitative information on the DNA-reactive metabolites in target tissues. Adducts detected in vivo were identified by compari-son with the products formed from the reaction of the individual epoxides with 2′-deoxyguanosine (dG). In rats and mice exposedto [4-14C]-BMO (1–50 mg/kg, i.p.), DNA adduct profiles were similar in liver and lung withN7-(2-hydroxy-3-butenyl)guanine(G1) andN7-(1-(hydroxymethyl)-2-propenyl)guanine (G2) as major adducts andN7-2,3,4-trihydroxybutylguanine (G4) as mi-nor adduct. In rats and mice exposed to 200 ppm [2,3-14C]-BD by nose-only inhalation for 6 h,G4 was the major adductin liver, lung and testes whileG1 andG2 were only minor adducts. AnotherN7-trihydroxybutylguanine adduct (G3), whichcould not unambiguously be identified but is either another isomer ofN7-2,3,4-trihydroxybutylguanine or, more likely,N7-(1-hydroxymethyl-2,3-dihydroxypropyl)guanine, was present at low concentrations in liver and lung DNA of mice, but absent inrats. The evidence indicates that the major DNA adduct formed in liver, lung and testes following in vivo exposure to BD isG4,which is formed from EBD, and not from DEB.© 2004 Elsevier Ireland Ltd. All rights reserved.

Keywords:1,3-Butadiene; 1,2-Epoxy-3-butene; 1,2:3,4-Diepoxybutane; 1,2-Dihydroxy-3-butene; 3,4-Epoxy-1,2-butanediol; DNA adducts;Inhalation exposure;N7-(1-(Hydroxymethyl)-2-propenyl)guanine;N7-(2-Hydroxy-3-butenyl)guanine;N7-(2,3,4-Trihydroxybutyl)guanine;N7-(1-(Hydroxymethyl)-2,3-dihydroxypropyl)guanine; Carcinogenicity; Rat; Mouse; Liver; Lung; Testis

Abbreviations:A1, N3-(2-hydroxy-3-butenyl)adenine;A2, N3-(1-(hydroxymethyl)-2-propenyl)adenine; BD, 1,3-butadiene; BMO, 1,2-epoxy-3-butene; DEB, 1,2:3,4-diepoxybutane; DEPT, distortionless enhancement by polarisation transfer; DHB, 1,2-dihydroxy-3-butene; EBD, 3,4-epoxy-1,2-butanediol;G1, N7-(1-(hydroxymethyl)-2-propenyl)guanine;G2, N7-(2-hydroxy-3-butenyl)guanine;G3, N7-(trihydroxybutyl)guanine;G4, N7-(2,3,4-trihydroxybutyl)guanine; HEPES,N-[2-hydroxyethyl]piperazine-N′-[2-ethanesulfonic acid]; HMBC,heteronuclear multiple bond correlation; HMQC, heteronuclear multiple quantum coherence; LCS, liquid scintillation counting; MOPS,3-[N-morpholino]propane sulfonic acid; SBR, styrene–butadiene rubber

∗ Corresponding author. Present address: Shell Health Services, Shell International B.V., P.O. Box 162, 2501 AN The Hague, TheNetherlands. Tel.:+31-70-377-2123; fax:+31-70-377-2840.

E-mail address:Peter.Boogaard@Shell.com (P.J. Boogaard).1 Present address: Syngenta Central Toxicology Laboratory, Alderley Park, Macclesfield 8K10 4TJ, UK.

0009-2797/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved.doi:10.1016/j.cbi.2004.02.002

70 P.J. Boogaard et al. / Chemico-Biological Interactions 148 (2004) 69–92

1. Introduction

1,3-Butadiene (BD) is an important industrialmonomer that is used in high volumes in the manu-facture of styrene–butadiene rubber (SBR) and a widevariety of other synthetic rubbers and resins. Occupa-tional exposure to BD may occur during the produc-tion of BD monomer itself and during the productionof BD-based polymers and derivatives. In addition,the general population may be exposed to BD sinceBD is a ubiquitous environmental pollutant found incigarette smoke and automotive exhausts. However,ambient air levels are extremely low, e.g. the runningannual average in the UK is well below 1 ppb[1].

Epidemiological studies suggest that workers in theSBR industry may have an increased incidence of lym-phatic and hematopoietic cancers (reviewed in[2]).However, these retrospective cohort studies are dif-ficult to interpret. A more recent study reported anapparent association between occupational exposureto BD in the SBR industry[3] whilst there was nosuch association in operators exposed to BD monomeralone[4,5]. In a recent re-evaluation the InternationalAgency for the Research on Cancer (IARC) decidedto maintain its classification as a 2A carcinogen, basedon limited evidencein humans for the carcinogenicityof BD [6,7]. In contrast, both the USA National Toxi-cology Programme[8] and the European Commission[9] have recently classified BD as a human carcino-gen. Despite the fact that IARC considered that thereis sufficient evidencefor the carcinogenicity of BD inanimals, the use of animal data to support the humanrisk assessment of BD is confounded by the large vari-ation in the response that has been observed betweenexperimental species. In female B6C3F1 mice lungtumours were observed after exposure to 6 ppm BD,and hemangiosarcomas of the heart after exposure to20 ppm. Induction even occurred after not more than13 weeks of exposure to 625 ppm BD. In contrast,tumours in Sprague–Dawley rats were only seen af-ter exposure to concentrations of more than 1000 ppm[2]. In addition to the fact that mice are 2–3 ordersof magnitude more susceptible to BD-induced cancerthan rats, the pattern of tumour types differs highly inmice and rats even at the highest concentrations.

These observations may be due to species differ-ences in the metabolism of BD. As depicted inFig. 1,BD is metabolised to the reactive epoxides 1,2-epoxy-

O

O

O

O

OH

BMO

DEB

EBD

BD

Cyt P450

Cyt P450

Cyt P450

Epoxide hydrolase

Epoxide hydrolase

OH

OH

OH

DHB

Fig. 1. Oxidative metabolism and hydrolysis of 1,3-butadi-ene (BD) and its epoxides 1,2-epoxy-3-butene (BMO), 1,2-epoxy-3,4-butanediol (EBD), 1,2-dihydroxy-3-butene (DHB) and1,2:3,4-diepoxybutane (DEB). BMO, EBD and DEB are genotoxic.

3-butene (butadiene monoepoxide, BMO), 1,2-epoxy-3,4-butanediol (EBD) and 1,2:3,4-diepoxybutane(butadiene diepoxide, DEB)[10–13], all highly muta-genic compounds in vitro[14]. The enormous differ-ence in cancer susceptibility between rats and micewould be explained by the differences in the balancebetween the rates of formation and subsequent detox-ification of these epoxides by hydrolysis and GSHconjugation [2,15–18]. Comparative data from invitro studies indicate that human produce much lessBMO and DEB than rodents and have a higher abilityfor metabolic detoxification[17–19]. Consequently,the human susceptibility towards BD-induced canceris expected to be significantly lower than that of themouse. However, these results do not explain whymice developed tumours at exposure concentrationsas low as 6 ppm, while tumours were not found in ratsbelow 8000 ppm, even though BMO accumulating inthe rat at this dose level exceeds that in the mouse at6 ppm[2]. It is widely hypothesised that mice and ratsdiffer in their metabolism of BD only quantitativelyand not qualitatively. However, we recently found thatthe metabolic fate of inhaled BD in mice differed notonly quantitatively but also qualitatively from that inrats[20]. Nevertheless, the differences found gave noclear indication for species-dependent differences inDNA-reactive metabolites. The present study inves-

P.J. Boogaard et al. / Chemico-Biological Interactions 148 (2004) 69–92 71

tigates the DNA adduct profiles in liver and lungs ofrats and mice following exposure to BMO by i.p. in-jection and to BD by inhalation since these profilesprovide qualitative and quantitative information onthe DNA-reactive metabolites in target tissues. BMOand BD with a high specific activity (>20 Ci/mol)were synthesised to allow the desired limit of detec-tion for individual adducts of approximately 1 adductper 108 nucleotides in a 1-mg DNA sample.

2. Materials and methods

2.1. Safety

1,3-Butadiene (CAS no. 106-99-0) is highlyflammable, toxic and a known animal carcinogen.All work with the chemical should be conducted in awell-ventilated fume hood while wearing protectiveclothing.

2.2. Radiochemicals

[4-14C]-1,2-Epoxy-3-butene (14C-BMO) was syn-thesised from [14C]-methyltriphenylphosphoniumiodide and racemic glycidaldehyde, using the Wittigreaction as described previously[21]. The specificradioactivity was 23 Ci/mol. GC analysis showed achemical purity of 75% with benzene as the major im-purity. For treatment of animals14C-BMO was dilutedwith unlabelled BMO (Aldrich) in dimethylsulfoxide:the chemical purity of the administered14C BMO was>94%[21]. The radiochemical purity was greater than99% as determined by GC analysis with radiodetection(RAGA 90, Raytek Scientific, Straubenhardt, Ger-many) and HPLC analysis with radiodetection (Ra-mona 90, Raytek Scientific). [2,3-14C]-1,3-Butadiene(14C-BD) was custom-synthesised from 1,2-dibromo-[1,2-14C]-ethane by Amersham International (Amer-sham, UK) as described previously[20]. The specificradioactivity was 21 Ci/mol. The radiochemical puritywas 98.7% as determined by GC with radiodetection.

2.3. Other chemicals

Unless otherwise stated, chemicals were of thehighest available purity. Chemicals used for DNAextraction were of molecular biology grade wherever

available. Solvents used for HPLC were HPLC-grade, all solvents used for other purposes were an-alytical grade. (2S,3S/2R,3R)-1,2:3,4-Diepoxybutane(±-DEB; CAS no. 298-18-0) and (R,S)-1,2-epoxy-3-butene (CAS no. 930-22-3) were purchased fromSigma–Aldrich Chemie (Zwijndrecht, The Nether-lands). 1,2:3,4-Diepoxybutane (mixed isomers,rac-DEB; CAS no. 1464-53-5) was bought from AcrosChimica (Geel, Belgium). (R,S)-1,2-Dihydroxy-3-butene ((R,S)-DHB; CAS no. 497-06-3) and (S)-1,2-dihydroxy-3-butene ((S)-DHB; CAS no. 62214-39-5)were obtained from Fluka Chemie (Zwijndrecht,The Netherlands).N3-(2-Hydroxy-3-butenyl)adenine(A1, see Fig. 2) and N3-(1-(hydroxymethyl)-2-propenyl)adenine (A2) were a gift of Dr. J.A. Swen-berg (UNC, Chapel Hill, USA). The preparation andcharacterisation of these compounds was describedby Tretyakova et al.[22,23]. (2R,3S/2S,3R)-1,2:3,4-Diepoxybutane (meso-DEB, CAS no. 564-00-1)and (R)-1,2-dihydroxy-3-butene ((R)-DHB; CAS no.86106-09-4) were custom-synthesised by Dr. DapingZhang (University of Newcastle upon Tyne, UK).

2.4. Synthesis of reference standards

2.4.1. Guanine adducts of BMOGuanine adducts of BMO (G1 andG2, seeFig. 2)

were prepared according to Citti et al.[24], with somemodifications. Briefly, 1 mmol (283 mg) guanosinewas suspended in 5 ml glacial acetic acid by heatingto 60◦C in a block heater and a 10-fold molar excessof BMO (805�l, 701 mg) was added. This mixturewas stirred during 5 h at 60◦C (block heater). Thereaction mixture was subsequently diluted with anequal volume of acetone and 4 volumes of diethylether. The crude adduct precipitated and, after decant-ing of the solvent, was dissolved in approximately2 ml of purified water. The aqueous mixture wasacidified by addition of 0.25 ml concentrated HCland hydrolysed during 5 h at 80◦C in a block heater.The reaction mixture was neutralised by addition of1 N KOH while stirring. The adducts precipitated andwere collected by centrifugation (5 min, 2000× g).The precipitate was washed with alkalinised water,collected by centrifugation and dissolved in a smallvolume of 0.1 N HCl. The adducts were purified byHPLC (System B). There were four peaks: guanine,as identified by co-chromatography, an unknown im-

72 P.J. Boogaard et al. / Chemico-Biological Interactions 148 (2004) 69–92

A1:

N3-(2-hydroxy-3-butenyl)

adenine

A2:

N3-(1-hydroxymethyl-

2-propenyl)adenine

N N

NN

NH2

HO

NN

NN

NH2

HO

G1:N7-(2-hydroxy-3-butenyl)

guanine

G2:N7-(1-hydroxymethyl-2-

propenyl)guanine

NN

NNNH2

OOH

HH

OH

O

H2

NN

NNN

NN

NNNH2

O

OH

H

G4: N7-2,3,4-trihydroxybutyl

guanine

OH

OH

H2

O

OH

H

OH

OHNN

NNN

G3 (?): N7-(1-(hydroxymethyl)-2,3- dihydroxypropyl)guanine

1

23

4

567

89

10 1112 13

Fig. 2. Chemical structures of the purine adducts of the reactiveepoxides of 1,3-butadiene.

purity, and two isomeric guanine adducts, which werecollected and lyophilised (Modulyo, Edwards, Craw-ley, UK). The identities of the two guanine adductswere confirmed as:N7-(2-hydroxy-3-butenyl)guanine(G1) andN7-(1-(hydroxymethyl)-2-propenyl)guanine(G2) by LC–MS, UV, and1H NMR analysis.1HNMR (360 MHz in d6-DMSO): G1: δH 4.05 + 4.24(2H, ddd; Gua-N7-CH2–CHOH–CH=CH2), 4.33 (1H,m; Gua-N7-CH2–CHOH–CH=CH2), 5.06+5.17 (2H,ddd; cis- and trans-Gua-N7-CH2–CHOH–CH=CH2),5.43 (1H, d; Gua-N7-CH2–CHOH–CH=CH2), 5.83(1H, dd; Gua-N7-CH2–CHOH–CH=CH2), 6.10 (2H,s; exocyclic NH2), 7.76 (1H, s; C8-H), 10.95 (1H,

s; N1-H), and G2: δ 3.74 + 3.91 (2H, ddd; Gua-N7-CH(CH=CH2)–CH2OH), 5.08 (1H, br, t; Gua-N7-CH(CH=CH2)–CH2OH), 5.18 (1H, m; Gua-N7-CH(CH=CH2)–CH2OH), 5.1 + 5.2 (2H, dd; cis-and trans-Gua-N7-CH(CH=CH2)–CH2OH), 6.06(2H, s; exocyclic NH2), 6.20 (1H, m; Gua-N7-CH(CH=CH2)–CH2OH), 7.95 (1H, s; C8-H), 10.7(1H, s; N1-H). UV analysis (dissolved in 20 mMaqueous Tris buffer, pH 7.8):λmax 245 nm (shoulder),λmin 261 nm andλmax 284 nm for bothG1 and G2.The purity was greater than 96% by HPLC (SystemA, see below).

2.4.2. Guanine adducts of DEBGuanine adducts of the various DEB isomers

were prepared as follows 0.21 mmol (57 mg) 2′-deoxyguanosine monohydrate and 2.0 mmol DEB(163�l) were dissolved in 10 ml 20 mM ammo-nium formate buffer pH 5.0 in a 50 ml vial. Thevial was sealed and the mixture stirred for 66 h at37◦C (heating block). The reaction mixture wascooled down to room temperature and extracted twicewith 10 ml dichloromethane. The reaction mixturewas analysed by HPLC (System A) and two peakseluted. According to the UV spectrum, recordedin the elution buffer (λmax 253 nm (shoulder),λmin260 nm andλmax 287 nm), the minor peak was aN7-guanine adduct. LC–MS analysis confirmed thisadduct (G3) to be a N7-(trihydroxybutyl)guanineadduct (m/z 278 (M + Na)+, 256 (M + H)+, and152 (guaninium)). The adduct was purified bysemi-preparative HPLC (System B) but insufficientamounts were available for an unambiguous char-acterisation by13C NMR. From the available evi-dence obtained by LC–MS and1H NMR, the adductcould be identified as either a diastereomer ofN7-(2,3,4-trihydroxybutyl)guanine or its regioisomerN7-(1-hydroxymethyl-2,3-dihydroxypropyl)guanine.1H NMR (400 MHz in d6-DMSO): δH 2.42 + 2.62(2H, m; Gua-N7-C2H3OH–CHOH–CH2OH), 2.88(1H, br, s; Gua-N7-CH2–CHOH–CHOH–CH2OH orGua-N7-CH(CH2OH)–CHOH–CH2OH), 3.6 (1H, br,s; Gua-N7-C2H3OH–CHOH–CH2OH), 4.12 + 4.29(2H, m; Gua-N7-CH2–CHOH–CHOH–CH2OH orGua-N7-CH(CH2OH)–CHOH–CH2OH), 5.45 (br, s;Gua-N7-C3H4(OH)2–CH2OH), 6.1 (2H, s; exocyclicNH2), 7.8 (1H, s; C8-H), 10.9 (1H, br, s; N1-H). Thepurity was greater than 97% by HPLC (System A).

P.J. Boogaard et al. / Chemico-Biological Interactions 148 (2004) 69–92 73

2′-Deoxyguanosine monohydrate (50 mg, 0.175mmol) was dissolved in water (9 ml) by heatingto 50◦C, cooled to room temperature and thenmeso-DEB (5�l, 0.068 mmol) was added. The re-action mixture was then stirred at room temper-ature with the exclusion of light. After 18 h thereaction mixture was subjected to neutral thermalhydrolysis (90◦C, 90 min) to afford a clear solu-tion, which was then centrifuged and analysed byHPLC (System E). This system was also used forthe semi-preparative isolation of the desired reac-tion product G4. The purified isolated adductG4was identified as a single diastereomer ofN7-(2,3,4-trihydroxybutyl)guanine: UV,λmax (H2O) 253 nm(shoulder),λmin 260 nm andλmax 282 nm;1H NMR(400 MHz in d6-DMSO): δH 3.31 (1H, m, Gua-N7-CH2–CHOH–CHOH–CH2OH), 3.36+ 3.58 (2H, 2×dd, a and b Gua-N7-CH2–CHOH– CHOH–CH2OH),3.67 (1H, m, Gua-N7-CH2–CHOH–CHOH–CH2OH),4.10+4.45 (2H, 2×dd, a and b Gua-N7-CH2–CHOH–CHOH–CH2OH), 6.50 (2H, s, exocyclic NH2), 7.8(1H, s; C8-H); 13C NMR (100.6 MHz in d6-DMSO):δ 50.56 (C-10), 64.42 (C-13), 71.82 (C-12), 74.93 (C-11), 109.69 (C-6), 145.70 (C-5), 154.70 (C-2), 156.4(C-6), 161.21 (C-4).

2.4.3. Reaction of EBD isomers with2′-deoxyguanosine (Fig. 3)

The various stereomers of EBD were preparedfrom the corresponding DHB stereomers by treatmentwith 3-chloroperbenzoic acid as follows: (R)-, (S)- or(R,S)-DHB were dissolved in dry dichloromethaneand chilled to 0◦C on an ice-bath. A 1.15-fold mo-lar excess of 3-chloroperbenzoic acid was addedand the mixture was stirred overnight. The pre-cipitated 3-chlorobenzoic acid was filtered off andthe dichloromethane evaporated. The residue wastaken up in purified water and the undissolved 3-chloroperbenzoic acid was filtered off. The aqueoussolutions of the formed EBD isomers (concentrationof 40 mM) were used directly for subsequent reactionwith 1.25-fold molar excess of 2′-deoxyguanosine.After 90 h at room temperature, the mixtures weresubjected to neutral thermal hydrolysis at 95◦C for2 h. After cooling to 4◦C, the reaction mixtureswere centrifuged and the clear supernatants, contain-ing the guanine adducts, were analysed by HPLC(System A).

2.4.4. Reaction of calf thymus DNA with14C-BMOCalf thymus DNA was dried overnight under vac-

uum and 10.9 mg of the dried DNA was dissolvedin 3.0 ml 20 mM Tris–HCl buffer, pH 7.2. Of thissolution, 2.5 ml was heated for 10 min at 100◦C in ashaking waterbath. The resulting single stranded DNAsolution was cooled rapidly to room temperature byimmersion in melting ice. An aliquot of 1.9 ml of thesingle stranded DNA solution (approximately 5 mgDNA) was transferred to a conical flask and 6.1 mg14C-BMO (specific activity: 23 Ci/mol) was added.The mixture was heated at 37◦C for 90 h in a shakingwaterbath. Subsequently, the DNA solution was ex-tracted three times with 8 ml water-saturated diethylether. The excess diethyl ether was evaporated witha gentle flow of nitrogen, and the DNA solution wastransferred to a Centricon-10 filter (Amicon, Etten-Leur, The Netherlands) and centrifuged for 90 min at5000× g at 10◦C. The retentate was allowed to re-dissolve in 1 ml water at 5◦C overnight. The solutionwas adjusted in a volumetric flask to 2 ml with water.The DNA content of the solution was determined byUV spectroscopy (see below).

2.5. Exposures

2.5.1. BMO studyMale B6C3F1 mice (20–30 g) and male Sprague–

Dawley rats (200–300 g) obtained from Charles River(Manston, Kent, UK) were given a single dose of14C-BMO in DMSO by i.p. injection as describedpreviously [21]. Male animals were used to allowdirect comparison with our previous work[20,21].The studies were carried out according to the guide-lines of the UK Home Office Licence for Animals(Scientific Procedures) Act 1986.14C-BMO was di-luted with unlabelled BMO in DMSO as the dosevehicle. Several batches of14C-BMO were preparedin this manner each with a target specific activityof 1 Ci/mol. BMO was shown to be stable with nolosses when stored frozen in the DMSO formulationfor up to 21 days. Target dose levels were 1, 5, 20and 50 mg/kg14C-BMO equivalent to 0.014, 0.071,0.286 and 0.714 mmol/kg, respectively, and includedthose previously reported for unlabelled BMO[31].The 1, 5 and 50 mg/kg14C-BMO dose groups con-sisted of three rats or five mice per dose level. The20 mg/kg14C-BMO group consisted of six rats or 25

74P.J.

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he

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s1

48

(20

04

)6

9–

92

2

2R,3R-EBD

O

OH

HOH

HO

OH

HOH

H

2S,3R-EBD

H

S-DHB

HO

OH

H

HHO

OH

OH

RH

HHO

OH

OH

RH

HHO

OH

RH

HHO

OH

OH

RR

OH

OH

HOH

HR

OH

OH

HOH

HR

OH

OH

HOH

HR

OH

OH

HOH

H

2

2R,3S-EBD

H

HOH

OH

OO

OH

OHH

H

2S,3S-EBD

H

R-DHB

OH

OH

OH

2 2

1111

2S,3R-G4 2R,3R-G3 2R,3R-G4 2S,3R-G3 2S,3S-G4 2R,3S-G3 2R,3S-G4 2R,3S-G3

O

H

O

H

S-BMO R-BMO

BD

Fig. 3. Stereochemical metabolism of 1,3-butadiene (BD) to its DNA reactive epoxides and the subsequent reactions with theN7 of guanine.

P.J. Boogaard et al. / Chemico-Biological Interactions 148 (2004) 69–92 75

mice. After dosing, rats were maintained individuallyand mice in groups of five in metabolism cages infume hoods for 48 h. Animals had free access to food(LAD2) and water. At the end of the 48 h holding pe-riod the animals were sacrificed by cervical disloca-tion, liver and lung were harvested, frozen in liquidnitrogen, and stored at−80◦C until DNA isolation.

2.5.2. BD studyTwelve male Sprague–Dawley rats (400 g, 12

weeks) and 50 male B6C3F1 mice (30 g, 12 weeks),purchased from Charles River (Raleigh, NC) and ac-climatised for approximately 14 days, were exposedon separate occasions to 200 ppm14C-BD for 6 husing a modified Cannon nose-only exposure sys-tem as described previously[20]. A concentrationof 200 ppm was chosen since this level is within therange for the development of tumours in male micebut below the no observed effect level of 1000 ppm inthe rat. Animals were supplied food (NIH 07 diet) anddeionised water ad libitum, except during exposureto 14C-BD, and maintained on a 12:12 h light–darkcycle at a temperature of 22± 2 ◦C and relative hu-midity of 55± 5%. This study was approved by theInstitutional Animal Care and Use Committee andwas performed in accordance with the declarationof Helsinki and the Guide for the Care and Use ofLaboratory Animals as adopted and promulgated bythe USA National Institutes of Health. The inhalationexposures were short term and were performed in theabsence of food and water.

The BD exposure atmosphere was continuouslymonitored before entering the exposure system byan on-line IR spectrophotometer (MIRAN, Foxboro,MA) calibrated by a closed loop technique with unla-belled butadiene. The atmosphere was also analysedusing a gas chromatograph (Model 5880, Hewlett-Packard) equipped with a flame ionisation (FID) anda radioactivity detector (GC-RAM, In/Us, Fairfield,NJ). Exposure concentrations were within 90% of thetarget concentration of 200 ppm during the course ofthe exposure (averaging 201± 21 ppm for rats and201± 13 ppm for mice) and 95% of the target 6 hexposure time (5 h and 46 min for rats and 5 h and43 min for mice)[20].

Seven rats and six groups of five mice were sac-rificed immediately after exposure by administrationof a lethal dose of sodium pentobarbital and hep-

arin. Following confirmed death, liver, lung and testeswere harvested, frozen in liquid nitrogen, and storedat −80◦C until isolation of DNA. In addition, threerats and one group of five mice were transferred togas-tight metabolism cages immediately after the 6 hof exposure. Urine and faeces were collected oversolid CO2 for 0–6, 6–24 and 24–42 h following ex-posure. Radioactivity in samples was quantified byscintillation counting and oxygen combustion as de-scribed below. The expired air from each cage wastrapped on charcoal filters to determine exhaled or-ganic volatiles (14C-BD and14C-metabolites) and sub-sequently through a potassium hydroxide solution forthe determination of14C-CO2. Traps for volatiles and14C-CO2 were changed at intervals 1, 3, 6, 24 and 42 hafter the 6 h exposure period and analysed as describedpreviously[20]. The other two rats and three groupsof five mice were transferred to normal metabolismcages and kept for 42 h. At the end of the 42 h pe-riod, all animals were sacrificed, liver, lungs and testeswere removed, frozen in liquid nitrogen, and stored at−80◦C until DNA isolation.

2.6. DNA isolation

DNA was extracted from selected organs using threedifferent methods. For all methods lung tissue wascooled in liquid nitrogen and pulverised using a ham-mer mill (6700 Freezer/Mill, Glen Creston Inc., Stan-more, UK) and liver was minced with scissors priorto homogenising.

The first method was based on the solvent extractiontechnique described by Beach and Gupta[25]. Fol-lowing precipitation, the DNA was dried by lyophili-sation.

The second method started with processing the tis-sues with a Braun homogeniser with 20–25 strokesat 700 rpm in an aqueous buffer of 0.8 M guani-dinium hydrochloride with RNAse A (EC 3.1.27.5;Type III-A, from bovine pancreas; 54 U/ml) and T1(EC 3.1.27.3; from Aspergillus oryzae; 13.8 U/ml),0.03 M Tris base, 0.03 M EDTA, 5% (w/v) Tween-20and 0.5% (w/v) Triton X-100, adjusted with sodiumhydroxide to pH 8.0 (approximately 50 ml/g tissue).After addition of 1 ml proteinase K (EC 3.4.21.14;from Tritirachium album) solution (400 U/ml) pergram of tissue, the homogenised suspension wasincubated for 5 h at 37◦C in a shaking waterbath.

76 P.J. Boogaard et al. / Chemico-Biological Interactions 148 (2004) 69–92

Following the incubation, the suspension was storedovernight at−80◦C. The next day, the suspensionwas thawed, anion exchange columns (Qiagen 500/Ggenomic tip; Qiagen, Hilden, Germany) were washedwith 10 ml equilibration buffer (0.75 M sodium chlo-ride in 15% aqueous ethanol with 0.15% (w/v) TritonX-100, buffered with 50 mM MOPS, adjusted to pH7.0 with sodium hydroxide) and 20 ml of the sus-pension was eluted through the column by gravity.The columns were subsequently washed twice with15 ml wash buffer (1.0 M sodium chloride in 15%aqueous ethanol, buffered with 50 mM MOPS, ad-justed to pH 7.0 with sodium hydroxide) at roomtemperature. Finally, the DNA was eluted from thecolumns by washing with 15 ml elution buffer (1.25 Msodium chloride in 15% aqueous ethanol, bufferedwith 50 mM Tris adjusted to pH 8.5) at 37◦C. TheDNA was then precipitated by addition of half a vol-ume of isopropanol and collected by centrifugation at7800× g and 4◦C for 15 min. The pellet was washedwith ice-cold 70% (w/v) ethanol and centrifugedonce more at 7800× g and 4◦C for 15 min. Thepellet was then lyophilised. The third method startedwith isolation of relatively large amounts of DNAusing an automated nucleic acid extractor (AppliedBiosystems 340A; Applied Biosystems, Foster City,CA, USA) as described previously[26]. The isolatedDNA was then further purified using a modification ofthe hydroxyapatite column chromatographic methodpublished by Beland and co-workers[27]. Hydroxya-patite gel was suspended in 0.12 M sodium phosphatebuffer pH 6.8, shaken for 15 min in a boiling waterbath and subsequently allowed to cool down to roomtemperature and settle. The settled gel was used topack an open-end 50 cm× 1.5 cm chromatographycolumn by gravity. After equilibration of the gelwith 0.12 M phosphate buffer pH 6.8, the columnwas loaded with the isolated DNA and subsequentlyeluted with 0.12 M phosphate buffer, while the eluatewas scanned atλ = 254 nm (Uvicord S 2138 UVdetector, LKB Bromma, Stockholm, Sweden). Thecolumn was eluted with 0.12 M phosphate buffer untilthe UV signal returned to baseline level and subse-quently the double stranded DNA was eluted with0.48 M phosphate buffer collected and concentratedby ultrafiltration using a Centriprep-10 filter (Ami-con) for 15 min at 3000× g. The retained DNA wascollected and lyophilised.

For quantification, regardless the method of isola-tion, the lyophilised DNA was dissolved in purifiedwater and the UV absorbance atλ = 230, 260 and280 nm was determined. The absorbance at 260 nmwas used to estimate the concentration of DNA as-suming that a concentration of 1 mg/ml would resultin an absorbance of 20.4. Purity of the DNA prepara-tion was checked by the ratio of absorbances at 260and 280 nm, which should be in the range of 1.8–2.0.An aliquot of the purified DNA solution was used todetermine the total radioactivity by LSC. Adduct lev-els were expressed per 108 nucleosides assuming that1 mg of DNA equals 1.85× 108 nucleotides.

2.7. Analysis

The DNA solution was heated to 95–100◦C for1 h in a Thermomixer 5437 (Eppendorf, Hamburg,Germany) and the released adducts were isolated bymicro-ultrafiltration through a filter with a molec-ular weight cut-off of 3000 Da (Centricon-3 filters(Amicon) for large and Microcon-3 filters (Ami-con) for small volumes). The remaining depurinatedDNA was washed from the filter and lyophilised.The dry residue was reconstituted in a small vol-ume of purified water (approximately 5 mg DNA/ml).The radioactivity in an aliquot of this solutionwas determined by LSC. In principle, neutral ther-mal hydrolysis does not release DNA base adductsthat are covalently bound at positions that do notcause a charge in the aromatic system, e.g.O6-or N2-adducts in guanine. Therefore, the reconsti-tuted, depurinated DNA was diluted with purifiedwater to a concentration of about 1 mg/ml and hy-drolysed enzymatically by subsequent treatmentwith an equal volume of nuclease P1 suspension(EC 3.1.30.1; from Penicillium citrinum; dissolvedin 100 mM bis–tris buffer, pH 6.5 with 0.25 mMZnCl2 and 2 mM MgCl2 and a final concentration50 U/ml) for 4 h at 37◦C in a shaking water bathand a mixture and alkaline phosphatase (EC 3.1.3.1;Type III, from Escherichia coli; final concentration:6.0 U/ml) and acid phosphatase (EC 3.1.3.2; TypeI, from wheat germ; final concentration: 0.4 U/ml)overnight at 37◦C. The released nucleosides wereseparated from the enzymes by micro-ultrafiltration(Centricon-3). The filtrates with released adductsor nucleosides were lyophilised, and the residue

P.J. Boogaard et al. / Chemico-Biological Interactions 148 (2004) 69–92 77

was dissolved in a solution of non-labelled refer-ence standards in the HPLC buffer and analysedby HPLC with on-line UV and radioactivity detec-tion while fraction were collected for subsequentLSC.

2.8. HPLC

The pH of the elution buffer greatly influenced theseparation of the free and adducted purines, especiallythe separation from adenine and its BMO adducts washighly dependent on pH. Several gradients were testedusing buffers with pH values varying from 3.8 to 7.0.Optimum separation was found using 50 mM ammo-nium formate buffer pH 4.0 for 8 min and then a lin-ear gradient with acetonitrile as modifier from 0 upto 8.5% from 8 to 50 min (System A). Under theseconditions typical retention times (based on UV) ofguanine, adenine,G4, A1, G3, A2, G1 and G2 of9.2, 14.0, 16.7, 22.3, 23.7, 27.3, 31.2 and 33.5 minwere found, respectively. For semi-preparative HPLCslightly different HPLC systems were used (Systems Band C).

2.8.1. System AApparatus: HP1100 liquid chromatograph equipped

with an on-line degasser, a variable UV detector setat λ = 284 nm, an autoinjector (Hewlett-Packard,Amstelveen, The Netherlands) and a FC204 fractioncollector (Gilson Medical Electronics, Villiers-le-Bel,France).Column: Beckman Ultrasphere (Fullerton,CA, USA), 3�m ODS, 250 mm× 4.6 mm. Elu-ent: 50 mM ammonium formate buffer pH 4.0 (A)and acetonitrile (B).Flow: 1.0 ml/min. Gradient:0–8 min: 100% A, 8–50 min linear increase to 8.5%B, 50–70 min 100% B.

2.8.2. System BApparatus: HP1050 liquid chromatograph equipped

with an on-line degasser, a variable UV detector setat λ = 284 nm (Hewlett-Packard), a Rheodyne in-jection port with 250�l injection loop, and a LKB2212 Helirac fraction collector.Column: HiChromNucleosil-120, ODS 5�m, 250 mm× 10 mm. Elu-ent: 20 mM ammonium formate buffer pH 4.2 (A)and 80% aqueous acetonitrile (B).Flow: 4.0 ml/min.Gradient: Isocratic 85% A, 15% B.

2.8.3. System CApparatus: HP1100 liquid chromatograph with on-

line degasser, an autoinjector (Hewlett-Packard) andon-line UV scanning over the rangeλ = 200–300 nm(Rapiscan SA 6508 multiple wavelength detector,Severn Analytical, Shefford, UK).Column: BeckmanUltrasphere (Fullerton, CA, USA), 3�m ODS, 250mm × 10.0 mm. Eluent: Purified water (A) and ace-tonitrile (B). Flow: 1.0 ml/min. Gradient: 0–30 min,linear increase from 6 to 50% B; 30–50 min 50% B.

2.8.4. System DApparatus: HP1100 liquid chromatograph equipped

with an on-line degasser, a variable UV detector setatλ = 284 nm, an autoinjector (Hewlett-Packard) anda FC204 fraction collector.Column: Beckman Ultra-sphere (Fullerton, CA, USA), 3�m ODS, 250 mm×10.0 mm. Eluent: Purified water (A) and acetonitrile(B). Flow: 1.0 ml/min. Gradient: 0–8 min: 100% A,8–30 min linear increase to 0.5% B, 35–40 min linearincrease to 50% B, 40–45 min 50% B.

2.8.5. System EApparatus: Shimadzu liquid chromatography sys-

tem comprising a LC-10 controller, solvent mixerand DGU-14A on-line solvent degasser, a Hewlett-Packard series 1050 variable UV detector set atλ = 284 nm, and a Rheodyne 7725i injector.Column:Beckman Ultrasphere (Fullerton, CA, USA), 5�mODS, 250 mm× 4.6 mm. Eluent: 0.050 M ammo-nium formate, pH 4 (A) and acetonitrile (B).Flow:1.0 ml/min.Gradient: 0–8 min: 100% A, 50 min 8.5%B, 70 min 100% B, 80 min 0% B, 86 min 0% B. Datawere acquired and analysed using Ramona 90 V11.4software.

2.9. LC–MS

LC–MS analyses were performed using a HP1050 liquid chromatograph with UV detection (λ =254 nm) coupled to a Micromass Quattro quadrupolemass spectrometer. Electrospray was used as theLC–MS interfacing and ionisation technique (pos-itive ions). The scan range was 100–1500 Da with0.5 scan/s. The chromatographic conditions (column,eluent, flow and gradient) were identical to those ofSystem A.

78 P.J. Boogaard et al. / Chemico-Biological Interactions 148 (2004) 69–92

2.10. Liquid scintillation counting (LSC)

Radioactivity in solutions was measured us-ing TriCarb 2200 CA liquid scintillation counters(Canberra-Packard, Groningen, The Netherlands) inantistatic scintillation vials with 10 volumes of Ul-tima Gold scintillation cocktail (Canberra-Packard).The machine was calibrated using a commercial14C internal standard kit for organic solvents (Wal-lac, Turku, Finland). The calibration was checkeddaily by counting a set of quenched standards com-mercially prepared in sealed glass vials. Countingefficiency was determined using the spectral indexof the internal standard (SIE) and cpm values wereautomatically transformed to dpm. Samples werecorrected for background. Individual samples werecounted for at least 60 min, samples with low ac-tivity were counted for up to 300 min each in orderto obtain a relative standard deviation of less than0.05. Statistical derivation of means was carried outby Microsoft Excel and are quoted throughout asaverage± S.E.

2.11. NMR

1H NMR spectra of synthetic reference chemicalsexceptG4 were measured on an Inova 400 MHz spec-trometer (Varian, Houten, The Netherlands) or a WH360 MHz spectrometer (Bruker, Wormer, The Nether-lands).13C NMR spectra of these reference chemi-cals were measured on a AC 250 MHz spectrometer(Bruker). Spectra were calibrated to TMS as an inter-nal standard at 298.1 K.

The optical purity of the (S)- and (R)-DHB, dis-solved at a concentration of approximately 0.15 M indry d3-acetonitrile, was determined using the opticalshift reagent europium III tris[3-(heptafluoropropyl-hydroxymethylene)-(+)-camphorate] in a concentra-tion of approximately 0.015 M according to Wilson[28] using the Inova 400 MHz machine.

All 1H and 13C NMR (fully decoupled), DEPT(distortionless enhancement by polarisation transfer),HMQC (heteronuclear multiple quantum coherence),and HMBC (heteronuclear multiple bond correlation)NMR experiments for the structure elucidation ofG4were performed on a DPX 400 spectrometer (Bruker),fitted with a QNP probe (400 MHz, d6-DMSO) us-ing a standard water suppression programme. Spectra

were acquired and processed using Xwin NMR Ver-sion 2.6 (Bruker).

2.12. UV

UV spectra were recorded using a Lambda 16 spec-trophotometer (Perkin–Elmer, Nieuwerkerk aan denIJssel, The Netherlands).

3. Results

3.1. Reaction of BMO with guanosine

The UV data of the adducts of guanosine with BMOmatched exactly with the data reported by Citti et al.[24]. Based on their UV and NMR spectra and avail-able literature data[23,24,29]the two isolated adductswere identified asN7-(2-hydroxy-3-butenyl)guanine(G2) andN7-(1-(hydroxymethyl)-2-propenyl)guanine(G2).

3.2. Reaction of EBD isomers with2′-deoxyguanosine

The optical purities of theS- and R-isomers ofDHB were investigated with NMR by determinationof the effect of the optical shift reagent europium(III) tris[3-(heptafluoropropylhydroxymethylene)-(+)-camphorate] on the unpaired vinyl proton. Alarger downfield shift was observed with (R)-DHBcompared with (S)-DHB, but both spectra showedeight sharp peaks without any sign of shoulders. Thespectrum of a mixture of (S)- and (R)-DHB showedeight pairs of peaks, indicating that both compoundswere virtually optically pure[28].

EBD was prepared from the DHB isomers by epox-idation of the double bound using 3-chloroperbenzoicacid. When the epoxidation products of (R,S)-DHBwere allowed to react with 2′-deoxyguanine and sub-sequently analysed by HPLC a complex peak with sev-eral shoulders and a retention time similar to that ofG4 (∼17 min) and a single peak with the same reten-tion time asG3 (∼24 min) were observed. Co-elutionexperiments with the reference standards and LC–MSanalyses confirmed that the peak were indeedG3 andG4. Using a very shallow HPLC gradient (System D)the complex peak at∼17 min (G4) could be resolved

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into 4 distinct peaks: two closely eluting peaks at re-tention times of 21.1 and 21.9 min and two distinctpeaks at 23.8 and 26.4 min. LC–MS analysis showedthat the mass spectra of these four peaks were iden-tical with three major signals atm/z 278 (M+ Na)+,m/z 256 (M+ H)+, andm/z 152 (guanine ion). Sincethe starting materials, (R)- and (S)-DHB, were opti-cally pure and epoxidation of the double bond using3-chloroperbenzoic acid proceeds with retention of theconfiguration on the adjacent carbon atom (C3), it wasexpected that only two stereo-isomers ofG4 would beformed (Fig. 3). However, in both cases theG4 peakcould be resolved by HPLC (System D) into four con-secutively eluting peaks, two of which were relativelylarge and the two other were much smaller, indicatingthat some racemisation at C3 had occurred.

3.3. Reaction of DEB isomers with2′-deoxyguanosine

HPLC analyses of the reaction mixtures of DEBwith 2′-deoxyguanosine showed a peak with aretention time similar to that ofG4 (∼17 min)and a peak with the same retention time asG3(∼24 min). The UV data of the reaction productsindicated that this second adduct eluting from theHPLC column was aN7-guanine adduct; LC–MSand 1H NMR analysis confirmed that this prod-uct was a N7-(trihydroxybutyl)guanine (G3), butthe product could not be unambiguously be identi-fied and could represent either a specific diastere-omer of N7-(2,3,4-trihydroxybutyl)guanine or, morelikely, its regioisomer N7-(1-hydroxymethyl-2,3-dihydroxypropyl)guanine. There were significantdifferences between the various DEB isomers in thecomposition of the other peak (G4). In the reac-tion mixtures with rac-DEB and with ±-DEB theG4 peak showed shoulders. In the reaction mixturewith meso-DEB, the G4 peak was a single peakand LC–MS analysis showed that the molecular ion(M + H)+ of the G4 peak had am/z of 256. Suffi-cient amounts of the compound representing theG4peak were isolated by semi-preparative HPLC (Sys-tem E). The isolated compound had a UV spectrumindicative of a substitutedN7 adduct, and was char-acterised by the1H NMR spectrum. In order to fullyassign the spectrum ofG4 unambiguously13C and13C–1H NMR correlation spectra were also recorded

Table 1Short range HMQC, and long range HMBC correlations observedfor adductG4

Carbon δ (ppm) HMQC HMBC

C-2 154.70C-4 161.21 H-8C-5 109.69 H-8, H-10a;H-10bC-6 156.40C-8 145.70 H-8 H-10a;H-10bC-10 50.56 H-10a;H-10b H-8C-11 74.93 H-11 H-13a;H-13bC-12 71.82 H-12 H-10a;H-13a;H-13bC-13 64.42 H-13a;H-13b

The carbon atom numbering is given inFig. 2.

(Table 1). 13C NMR and 13C NMR DEPT spectradetermined the resonances of the carbon atoms in thepurine and differentiated CH and CH2 positions inthe 2,3,4-trihydroxy butyl side chain moiety (Fig. 4).13C–1H NMR correlation spectra, HMQC to deter-mine CH correlations via one-bond couplings andHMBC to determine CH correlations via long-range

Fig. 4. 13C NMR spectra of theG4 guanine adduct: normal (top)and DEPT (bottom). SeeSection 2for chemical shift assignments.

80 P.J. Boogaard et al. / Chemico-Biological Interactions 148 (2004) 69–92

Fig. 5. 2D NMR spectra of theG4 guanine adduct. HMBC NMR spectrum (top) and HMQC NMR spectrum (bottom) of the side chainregion only (seeTable 1for assignments).

couplings, unambiguously assigned the connectivityof the side chain to the purine moiety (Fig. 5). Fromthe long range couplings observed in the HMBC spec-tra, C-4-H-8, C-5-H-8;H-10a;H-10b, C-8-H-10a;10b,C-10-H-8. C-11-H-13a;H-13b, C-12-H-10a;H-13a;H-13b, the connectivity of the side chain was shownto be via the CH2 adducted to theN7 residue of thepurine (Fig. 5, top panel). Direct couplings were ob-served in the HMQC spectrum between; C-8-H-8,C-10-H-10a;H-10b, C-11-H-11, C-12-H-12, C-13-H-13a;H-13b which confirmed the presence of the three

substituted carbons in the side chain moiety (Fig. 5,bottom panel). Thus, the structure ofG4 was proven tobe a single diastereomer ofN7-2,3,4-trihydroxybutylguanine.

3.4. Formation of14C-BMO-derived adducts in calfthymus DNA

The reaction of14C-BMO with single-strand calfthymus DNA resulted in a total binding of 27�Ci/mgDNA. Following neutral thermal hydrolysis and sub-

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Fig. 6. HPLC profile following neutral thermal hydrolysis of single strand calf thymus DNA treated with excess14C-BMO. Arrow headspoint to peaks co-eluting with the reference standards.

sequent ultrafiltration, approximately 94% of the to-tal radioactivity in the DNA solution was readily fil-tered and approximately 5% of the radioactivity wasretained on the filter. Less than 1% of the radioactiv-ity could not be recovered and was probably stuck tothe filter.

HPLC analysis of the ultrafiltrate showed two peaks,clearly visible in both UV and with radio-detection(Fig. 6). These two peaks co-eluted with the syntheticstandards ofG1 and G2 and represented approxi-mately equal amounts of radioactivity (ratio 49:51).A rather large amount of radioactivity (up to 10% ofthe total) eluted between 15 and 30 min as a series ofsmall but relatively broad peaks. Monitoring by UVshowed two small, distinct peaks exactly co-elutingwith A1 andA2. These two peaks (based on the UVsignal atλ = 284 nm) had a ratio of 34:66, and wereonly 6.7% of the UV signal for the guanine adducts.Enzymatic hydrolysis and subsequent HPLC analysisof the depurinated DNA retained on the filter showed

that there were no detectable levels of adducts presentin the depurinated DNA.

3.5. Adducts in DNA isolated from14C-BMOexposed animals

Mild signs of toxicity, salivation and hunched andunkempt appearance, were observed in rats soon afterreceiving doses of BMO of≥20 mg/kg. These effectsdisappeared approximately 60–90 min after dosing. Incontrast, mice showed no signs of toxicity after receiv-ing equivalent doses of BMO. Both mice and rats ap-peared healthy and were eating and drinking normallyduring the 48-h holding period in metabolism cages.For the BMO study, the doses actually administeredwere based on assay of the dose solutions before andafter the completion of dosing. Rats received 1, 5, 18or 51 mg/kg of14C-BMO with specific activity of 1.3,1.1, 0.8 and 1.0 Ci/mol, respectively. Mice received 1,5, 21 or 54 mg/kg of14C-BMO with specific activity

82 P.J. Boogaard et al. / Chemico-Biological Interactions 148 (2004) 69–92

Table 2Average hepatic adduct levels (±S.E.) in rats and mice killed 48 h following i.p. administration of14C-BMO

Dose Mouse (n = 1) Rat (n = 3)

54 mg/kg 21 mg/kg 51 mg/kg 18 mg/kg 5 mg/kg

AdductG1 + G2 600 368 2100± 100b 857 ± 291 76± 28G3 NDa 28 NDa 21 ± 12 4 ± 4G4 NDa 50 NDa 101 ± 25 16± 5

Values are expressed as adducts/108 nucleotides.a ND: not detectable, due to analytical interference (see results section); in the mouse analytical interference also prevented the detection

of adducts at the 5 mg/kg level.b n = 2.

of 1.3, 1.1, 0.8 and 1.0 Ci/mol, respectively. Standardphenol/chloroform extraction resulted in DNA with asmall but significant protein contamination. The twoguanine adducts,G1 and G2, could be identified inthe highest dose group (50 mg/kg) of both rats andmice: the adduct levels were 20 and 22 adducts/106

nucleotides in rats and 6 adducts/106 nucleotides inmice (sum ofG1 andG2). However, the impurities inthe DNA prevented the detection of any BMO adductsof adenine or DEB adducts of guanine and also madeit difficult to quantify G1 andG2 in the lowest dosegroups. In order to remove these impurities, an alter-native method for DNA isolation was devised usingspecially designed anion exchange columns. Usingthis method, very pure DNA was obtained withoutany of the contaminants, and per mg of DNA, a de-tection limit for individual adducts of 20 adducts per108 nucleotides was obtained. Adduct levels in bothrat and mouse lung were too low to be quantifieddue to the limited amount of DNA that could be iso-lated from lungs, despite the fact that lung tissue waspooled from 15 mice. The adduct profiles in liver werevery similar in rats and mice, but significantly higheradduct levels were seen in rats than in mice (Fig. 7).The profiles of hepatic DNA adducts in rats and micewere also very similar to the profile observed in calfthymus DNA treated with14C-BMO (Fig. 6). Themajor adducts were theN7-guanine adducts of BMO,G1 and G2, which were formed in equal amounts.In addition, small amounts ofG3, barely detectablein some cases, and ofG4 were found. In rats dosedwith 18 and 5 mg BMO per kg body weight, the av-erage levels ofG2 + G2 were 857± 291 and 76± 28adducts/108 nucleotides, respectively, the levels ofG3 were 21± 12 and 4± 4 adducts/108 nucleotides,

respectively, and the levels ofG4 were 101± 25 and16±5 adducts/108 nucleotides, respectively (Table 2).Adduct levels in rats dosed with 1 mg BMO/kg bodyweight were below the limit of detection. In mice,adducts were below the detection limit in both the1 and 5 mg/kg dose groups. In the mice dosed with21 mg BMO/kg, the average concentration ofG1+G2was 368 adducts/108 nucleotides, and ofG3 andG428 and 50 adducts/108 nucleotides, respectively. En-zymatic hydrolysis and subsequent HPLC analysis ofthe depurinated DNA that had resulted from the neu-tral thermal hydrolysis gave no indications for otheradducts than the depurination products.

3.6. Adducts in DNA isolated from animals exposedto 14C-BD by nose-only inhalation

No signs of toxicity were observed in any of theanimals immediately after the14C-BD exposure. Ani-mals were damp, either from urine or humidity of theair, but otherwise appeared well. All animals appearedhealthy and were eating and drinking normally dur-ing the 42-h holding period in metabolism cages. Thetotal uptake of radioactivity by the animals from theBD inhalation study was calculated by summation ofthe radioactivity in urine, faeces, organic volatiles and14C-CO2 collected during and after exposure, and theradioactivity remaining in tissues and carcasses 42 hafter exposure as described previously[20]. Volatilesexhaled during exposure were trapped, but their na-ture could not be determined due to the high amountof 14C-BD present from the exposure atmosphere. Thecalculated uptake was 1.52 ± 0.12 mCi per rat and0.23±0.01 mCi per mouse. Based on the average bodyweight of rats (0.4 kg) and mice (0.03 kg), the uptake

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Fig. 7. Typical HPLC profile of DNA adducts released by neutral thermal hydrolysis from hepatic DNA isolated from rats (top) andmice (bottom) 48 h after i.p. administration of [4-14C]-1,2-epoxy-3-butene (specific radioactivity: 0.8 Ci/mol). The dose was 18 mg/kg bodyweight for rats and or 21 mg/kg body weight for mice. Arrows point to peaks co-eluting with the reference standards.

was 3.80 and 7.51 mCi/kg, equivalent to doses of14C-BD of 10 and 21 mg/kg for rats and mice, respectively.Thus, when exposed to a similar concentration of14C-BD, the uptake by mice was approximately twice theuptake by rats. Based on minute volumes of 250 and45 ml/min for rats and mice respectively, the radioac-tivity in air inhaled during the 6 h inhalation expo-sure to 200 ppm14C-BD was 14.63 mCi for rats and2.63 mCi for mice. Thus, the radioactivity retained andprocessed accounted for 10 and 9% of the total ra-

dioactivity inhaled from the exposure atmosphere perrat and per mouse, respectively.

DNA was isolated, using the anion exchangecolumns, from liver, lung and testes of the exposedanimals. Tissues were analysed for individual rats; formice tissues were pooled from five animals for liversand from 15 animals for lungs and testes before DNAextraction to lower the detection limit. Significantlyhigher levels of DNA adducts were seen in the mousecompared to the rat. Qualitatively, the same hepatic

84 P.J. Boogaard et al. / Chemico-Biological Interactions 148 (2004) 69–92

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Fig. 8. Typical HPLC profile of DNA adducts released by neutral thermal hydrolysis from hepatic DNA isolated from rats (top) and mice(bottom) 42 h after cessation of 6 h nose-only inhalation exposure to 200 ppm [2,3-14C]-1,3-butadiene (specific radioactivity: 21 Ci/mol).Arrows point to peaks co-eluting with the reference standards.

DNA adducts were found as in animals exposed toBMO, with exception ofG3 which was not observedin rats exposed to BD (Figs. 8 and 9). Quantitatively,there was a large difference (Table 3). The most strik-ing difference was the concentration ofG4 whichwas the major adduct in liver, lung and testes of bothrats and mice. In the testes of animals killed imme-diately after exposure,G4 was the only detectableadduct at a concentration of 73 and 5 adducts/108

nucleotides in mouse and rat (n = 1), respectively.In mice, the concentration ofG4 was 90± 11 and102± 4 adducts/108 nucleotides in liver and 139 and80 ± 4 adducts/108 nucleotides in lung immediatelyafter exposure and 42 h later, respectively. In rats theconcentrations ofG4 were, like in testes, an order ofmagnitude lower: 13± 1 and 10± 2 adducts/108 nu-cleotides in liver and 11± 1 and 13± 0.2 adducts/108

nucleotides in lung immediately after exposure and

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G1 G2G3

Fig. 9. Typical HPLC profile of DNA adducts released by neutral thermal hydrolysis from pulmonary DNA isolated from rats (top) and mice(bottom) 42 h after cessation of 6 h nose-only inhalation exposure to 200 ppm [2,3-14C]-1,3-butadiene (specific radioactivity: 21 Ci/mol).Arrows point to peaks co-eluting with the reference standards.

42 h later, respectively. The concentration ofG3,which was only observed in mice, was significantlyhigher in liver (20± 3 and 25± 2 adducts/108

nucleotides at 0 and 42 h after cessation of expo-sure, respectively) than in lung (12 and 4.3 ± 0.1adducts/108 nucleotides at 0 and 42 h after cessationof exposure, respectively). The concentration of theN7-guanine adducts of BMO,G1 + G2, were sim-ilar in rat lung and liver (1.6 ± 0.5 and 3.6 ± 1.2adducts/108 nucleotides in lung and 1.9 ± 0.5 and

1.9±0.2 adducts/108 nucleotides in liver, at 0 and 42 hafter cessation of exposure, respectively). In mice,the levels ofG1 + G2 tended to be slightly higher(7 and 3.4 ± 1.3 adducts/108 nucleotides in lung and8 ± 2 and 21± 9 adducts/108 nucleotides in liver, at0 and 42 h after cessation of exposure, respectively).Enzymatic hydrolysis of the DNA did not give otheradducts than those observed following neutral thermalhydrolysis at the detection limit of ca. 1 adduct/108

nucleotides.

86 P.J. Boogaard et al. / Chemico-Biological Interactions 148 (2004) 69–92

Table 3Average DNA adduct levels (±S.E.) in rats and mice killed 42 hafter cessation of nose-only exposure to 200 ppm14C-BD for 6 h

Adduct Mousea Rat

6 h 42 h 6 h 42 h

Liver (n = 6) (n = 4) (n = 7) (n = 4)G1 + G2 8 ± 2 21 ± 9 1.9 ± 0.5 1.9± 0.2G3 20 ± 3 25 ± 2 NDb NDG4 90 ± 11 102± 4 13 ± 1 10 ± 2

Lung (n = 1) (n = 2) (n = 3) (n = 2)G1 + G2 7 3.4 ± 1.3 1.6± 0.5 3.6± 1.2G3 12 4.3± 0.1 ND NDG4 139 80± 4 11 ± 1 13 ± 0.2

Values are expressed as adducts/108 nucleotides.a Values for mouse tissues are based on DNA isolated from

pooled tissue samples (5 mice/sample for liver and 15 mice/samplefor lungs).

b ND: not detectable (below limit of detection).

Using the hydroxyapatite method for isolation ofDNA, large amounts of DNA (∼25 mg) were isolatedfrom mouse liver DNA. The DNA was depurinatedby neutral thermal hydrolysis, the released adductswere collected by ultrafiltration and analysed by mul-tiple injections on HPLC (System A). The fractionscorresponding toG4 and G3 were collected fromall runs, lyophilised, each dissolved in a small vol-ume of purified water and analysed by LC–MS. Thefraction corresponding toG3 co-eluted with the ref-erence standard forG3 and showed an identical massspectrum. Similarly, the fraction corresponding toG4co-eluted with the compound isolated from the reac-tion of meso-DEB and guanosine that was identifiedas N7-2,3,4-trihydroxybutylguanine and showed anidentical mass spectrum.

3.7. Covalent binding index (CBI)

From the total amount of radioactivity bound toDNA covalent binding indices (CBIs) were calculatedas defined by Lutz[30]. Following i.p. administrationof BMO, at a dose level of 20 mg/kg, the CBI in liverwas 45 for rats and 22 for mice, at 42 h after cessa-tion of exposure. At a dose level of 5 mg BMO per kg,the CBI was 23 for rats (no data for mice available).Following inhalation exposure to 200 ppm BD for 6 h,the CBIs were much lower but about 5 times higherin mice than in rats. The hepatic CBI was 0.94 and

0.82 for rats and 4.0 and 5.0 for mice, at 0 and 42 h af-ter cessation of exposure, respectively. The pulmonaryCBI was 0.76 and 1.0 for rats and 5.1 and 2.7 for mice,at 0 and 42 h after cessation of exposure, respectively.

4. Discussion

The main objective of the present studies was toinvestigate the DNA binding of BD and its major pri-mary metabolite BMO following in vivo exposures toeach chemical.

The hepatic DNA adduct profiles following expo-sure to14C-BMO were different from those follow-ing exposure to14C-BD in both rats and in mice, butsimilar to the profile obtained by in vitro treatment ofcalf thymus DNA with14C-BMO. G1 and G2 werethe major adducts and their levels were significantlyhigher in rat liver than in mouse liver, reflecting themuch higher capacity of the mouse to detoxify BMOthrough hydrolysis and GSH conjugation[17,18]. Pul-monary adduct levels were below the limit of detec-tion which indicates that the hepatic detoxification ofBMO is rapid and efficient. In both species, the for-mation of G1 and G2 was proportionally related tothe amount of14C-BMO administered. In addition,in liver G4 and G3 were present, the latter at levelsjust above the limit of detection. The formation ofG3andG4 following administration of BMO implies thatBMO is either epoxidised to DEB or hydrolysed toDHB with subsequent oxidation to EBD. DNA adductprofiles were different following exposure to14C-BD:G4 was by far the most important adduct whileG1andG2, at levels that are an order of magnitude lowerthan forG4, were only minor adducts. In addition,G3was not detected in rat tissues, but present in mouseboth liver and lung and in significantly higher concen-trations thanG1 andG2.

Several in vitro studies on the DNA binding ofBMO and DEB have been performed and a wide va-riety of purine adducts was reported[22,29,32–38].However, only few of the potentially formed adductswere actually found in animals exposed to BD in vivo.The major DNA adducts observed after in vivo expo-sure to BD were invariably theN7-guanine adductsof BMO (G1, G2), and aN7-trihydroxybutyl adductof guanine (G3 and/or G4), while in some studiesvery low levels of A1, A2, and the corresponding

P.J. Boogaard et al. / Chemico-Biological Interactions 148 (2004) 69–92 87

N6-adenine adducts were detected following multipledays of exposure[39–43]. A single study has beenreported in which animals were exposed to [1,4-14C]BD in a closed exposure system at an initial con-centration of 500 ppm. In this study, again, onlyG1,G2, and a singleN7-trihydroxybutyl guanine adductwere reported as the major adducts[44]. G4, themajor adduct in the present study, was unambigu-ously identified asN7-(1,2,3-trihydroxy)butyl gua-nine. However, to the best of our knowledge anotherN7-trihydroxybutyl adduct of guanine with signifi-cantly different chromatographic properties (G3) wasnever reported[45]. This is not surprising since inall these studies non-labelled BD was used and, as aconsequence, only adducts that were expected or forwhich reference standards had been prepared wereobserved and could be quantified. In our studies,G3andG4 were not formed when BMO was allowed toreact with guanosine. When the commercially avail-able rac-DEB or ±-DEB was allowed to react withguanosine, bothG3 andG4 were formed.

Our previous studies on the use of urinary mercap-turic acids and haemoglobin adducts as biomarkersof exposure to BD, indicated that EBD, rather thanBMO or DEB, is the crucial reactive metabolite ofBD [46]. These biomarker results led to a series ofexperiments in which the products resulting from thereaction of EBD with 2′-deoxyguanosine were stud-ied. Mixtures of (2S,3S)- and (2R,3S)-EBD and of(2S,3R)- and (2R,3R)-EBD were prepared from (R)-and (S)-DHB, respectively (seeFig. 3) and reactedwith 2′-deoxyguanosine. These reactions resulted,indeed, in the formation ofG4 as a major adductwith only very small amounts ofG3. G4 was formedfrom EBD as a mixture of four diastereomers butwe were unable to obtain complete base-line sep-aration for all four isomers in our HPLC systems.G3 was also formed from EBD, but only as a minoradduct. Sincemeso-DEB has a plane of symmetry,G4 was formed as a single isomer whenmeso-DEBwas allowed to react with guanosine. Using semi-preparative HPLC, sufficient amounts of this isomercould be isolated to record NMR spectra which al-lowedG4 to be identified asN7-2,3,4-trihydroxybutylguanine. However, we were unable to characterisethe other N7-trihydroxybutyl guanine adduct,G3,with certainty. Based on LC–MS and1H NMRdata, G3 is either a stereoisomer ofG4 or, more

likely, its regioisomer, N7-(1-(hydroxymethyl)-2,3-dihydroxypropyl)guanine. The latter structure seemsmore likely based on the very different chromato-graphic behaviour ofG3 compared toG4. G3 has notbeen reported by other investigators[45,47].

In both rat and mouse testes,G4 was the only de-tectable adduct (Fig. 10). In the rat, the concentrationof G4 in testes was lower than in liver and lung.Based on the concentrations ofG1, G2 and G4 inliver and lung, it is expected that the levels ofG1 andG2 in testes would be below the limit of detection. Inmouse testes the concentration ofG4 was comparableto that measured in liver and lung. Based on the con-centrations ofG1, G2, andG3 in liver and lung it wastherefore expected that these adducts would be mea-surable in testes as well. The absence of adducts otherthanG4 in mouse testes may be indicative of differ-ences in local metabolism of BD and/or its epoxides,differences in DNA repair in the testes, or differencesin accessibility of testicular DNA. Interestingly, otherinvestigators also reported quantitative and qualitativedifferences in DNA adducts of BMO in mouse lungand testis following 5 days of exposure to 500 ppmBD. In the testes the total DNA adduct level was sig-nificantly higher than in the lungs, but in the lungs fourdifferent isomers were measured, while in the testisonly two of those four isomers were detected, whichwas explained by differences in DNA repair[40]. Inmice, following exposure to [1,2-14C]-ethylene oxide,very similar results were found with testicular levelsof N7-(2-hydroxyethyl)guanine being 60% lower thanin brain, lung, liver, spleen and kidneys[26].

The present finding thatG4 is the major adductin liver, kidneys, and testes of rats and mice exposedto BD by inhalation seems to confirm the conclusionfrom recent biomarkers work that EBD, and not BMOor DEB, is the most prominent DNA reactive metabo-lite of BD available for macromolecular binding invivo [43,45,46]. The results are also in accordancewith our previous findings on the metabolism of BMOin humans, rats and mice following exposure to BD,where we found that the proportion of BMO that un-dergoes further oxidation to DEB was of the rank or-der mouse> rat � human[20,21,46]. Even if DEBwas formed, it would rapidly be hydrolysed to EBD,especially in humans and rats[17], or be detoxifiedby conjugation with GSH[16]. Hence, the extent towhich DEB, if at all, would be available to react with

88 P.J. Boogaard et al. / Chemico-Biological Interactions 148 (2004) 69–92

0

10

20

30

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44

Time (min)

Ad

du

cts/

10 8 n

ucl

eoti

des

G4

0

10

20

30

40

50

60

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44

Time (min)

Ad

du

cts/

10 8 n

ucl

eoti

des

G4

Fig. 10. Typical HPLC profile of DNA adducts released by neutral thermal hydrolysis from testicular DNA isolated from rats (top) and mice(bottom) 42 h after cessation of 6 h nose-only inhalation exposure to 200 ppm [2,3-14C]-1,3-butadiene (specific radioactivity: 21 Ci/mol).Arrows point to peaks co-eluting with the reference standards.

DNA was expected to be very small. In contrast, theconcentrations of EBD available might be significantto form DNA adducts. In a NADPH-activated rat hep-atic microsomal system, BD was readily metabolisedto BMO, DHB and EBD. The concentration of DHBwas about two times and the concentration of EBD wasabout 10 times higher than the concentration of BMO,but DEB was not detected, suggesting that EBD, incontrast to DEB, is a very poor substrate for micro-

somal epoxide hydrolase[11,17]. The fact that onlyminute quantities of erythritol, which would resultfrom epoxide hydrolase catalysed hydrolysis of EBD,were found when DEB was incubated with rodent mi-crosomes, supports the assumption that the enzymecatalysed hydrolysis of EBD is limited or virtually ab-sent [17]. G3 was present in mouse liver and lung,but could not be detected in any of the rat tissues.Although concentrations ofG4 in the rat were about

P.J. Boogaard et al. / Chemico-Biological Interactions 148 (2004) 69–92 89

an order of magnitude lower than in the mouse,G3would have been detectable if the ratioG3:G4 inrats had been comparable to that in mice. Assumingthat G3 originates from DEB, the absence ofG3 inrat tissues following exposure to 200 ppm BD for 6 hmay be due to the much higher epoxide hydrolaseactivity towards DEB in rat tissues than mouse tis-sues[17] and its presence, theoretically, might indi-cate the potential of formation of DNA cross-links inmice, since strong evidence of the formation ofN7-(2-hydroxy-3,4-epoxybutyl)guanine, as an intermedi-ate in the formation ofG3 from the reaction of DEBwith guanosine, was recently reported[22,54,55]. Asthis intermediate has still an epoxy group, it may re-act with another nucleophilic centre in the DNA andform crosslinks, which are known to be highly muta-genic lesions[48]. The exclusive presence ofG3 inmouse tissues would therefore provide an explanationfor the observed carcinogenicity of BD in mice at lowexposure concentrations. However, DNA cross-links,which would have depurinated upon neutral thermalhydrolysis, were not observed in the present studynor are there any reports that confirm the existenceof DNA cross-links in vivo caused by DEB. More-over, several studies showed that the concentrations ofDEB in blood and lung tissue of rats and mice follow-ing inhalation exposure to 12 ppm DEB for 6 h weresignificantly higher than following inhalation expo-sure to 1250 ppm BD for 6 h[12,51,52]. Nevertheless,only local toxicity in the upper respiratory tract of ratsand mice was observed after inhalation exposure to5 ppm DEB for 6 weeks, 5 days per week and 6 h perday, and a carcinogenic response was observed onlyin the rat[53]. This carcinogenic response in the ratwas seen in the upper respiratory tract, the site of thetoxic lesions, and is most likely related to cytotoxi-city rather than genotoxicity. In vitro studies on themutagenicity rank the potency of the epoxide metabo-lites of BD in the order: DEB� BMO > EBD [14].This apparent discrepancy is best explained by thedifferences between in vitro and in vivo model sys-tems. We have previously shown that in vivo DEB israpidly detoxified by epoxide hydrolase catalysed hy-drolysis and glutathione-S-transferase catalysed con-jugation with GSH in vivo[49]. In competent in vitromodels, DEB was rapidly inactivated as well[15–17].However, the human TK6 lymphoblastoid cell modeldoes not translate very well to the in vivo situation

since the glutathione-S-transferase activity in this cellline is very low [50] and, although there is no pub-lished information pertaining to the epoxide hydrolaseactivity in the human TK6 cells, the results of the mu-tagenicity studies imply that it is also very low or evenabsent[14]. TheG3 detected in mouse tissues exposedto BD by inhalation might also be originating directlyfrom EBD, because when EBD was allowed to reactwith guanosine in vitro,G3 was also formed as a mi-nor adduct. The ratio betweenG4 andG3 seemed alsoto be determined by the stereochemistry of the epox-ides since the ratiosG4:G3 differed for the formationof G4 from the reaction of guanosine with the epox-idation products of (S)-DHB and the correspondingreaction with the epoxidation products of (R)-DHB.A distinct stereospecific preference was also observedfor G1 andG2 when (R)- and (S)-BMO were allowedto react with 2′-deoxyguanosine monophosphate invitro, and in vivo following inhalation exposure to BD[39,40,56]. Enantioselective preference for the forma-tion of specific BD metabolites has also been observedin other studies and may have an enzymatic basis ordepend on differences in chemical reactivity. A smallstereoselective preference for the oxidation of BD to(S)-BMO over (R)-BMO in mouse liver microsomeswas recently reported[57], which is in line with thefindings by Wistuba et al. on a wide variety of oxiranes[58]. cDNA-Expressed human cytochrome P450 2E1catalysed oxidation of racemic BMO led to almost twotimes higher concentrations ofmeso-DEB than of±-DEB [59]. Together, the available experimental dataindicate that that the metabolism of BD to its epoxidesand their subsequent reaction with the bases in DNAis not only determined by species differences but alsoto a great extent by stereochemical factors.

The total binding to DNA following inhalation ex-posure to14C-BD, as expressed by the CBI values, waslow compared to known genotoxicants[30] and sug-gests that other factors than genotoxicity play a rolein the carcinogenicity of BD as well.G4 will likelylead, like otherN7-guanine adducts, to the formationof apurinic sites which are not very mutagenic. In ad-dition, its concentration in mice was only about one or-der in magnitude larger than in rats. Hence, these factsseem unable to explain the much higher susceptibilityof mice and also suggests that differences in promo-tional pressure in species and organs in combinationwith G3 play an important role in the carcinogenic-

90 P.J. Boogaard et al. / Chemico-Biological Interactions 148 (2004) 69–92

ity of BD. Finally, the present study showed thatG3and G4 originate from EBD, either through DEB orthrough DHB. Both the concentrations of DHB (fromBMO) and DEB will be much less in humans than ratsand much less in rats than in mice[46]. Hence it is ex-pected that the formation ofG3 and/orG4 in humanswill be less than in rats and much less than in mice.

References

[1] EPAc QS, A Recommendation for a UK Air Quality Standardfor 1,3-Butadiene, HMSO, London, UK, 1994.

[2] M.W. Himmelstein, J.F. Acquavella, L. Recio, M.A.Medinsky, J.A. Bond, Toxicology and epidemiology of 1,3-butadiene, Crit. Rev. Toxicol. 27 (1997) 1–108.

[3] E. Delzell, M. Macaluso, N. Sathiakumar, R. Matthews,Leukemia and exposure to 1,3-butadiene, styrene anddimethyldithiocarbamate among workers in the syntheticrubber industry, Chem. Biol. Interact. 135/136 (2001) 515–534.

[4] B.J. Divine, C.M. Hartman, Mortality update of butadieneproduction workers, Toxicology 113 (1996) 169–181.

[5] E. Delzell, N. Sathiakumar, H. Hovinga, M. Macaluso, J.Julian, R. Larson, P. Cole, D.C. Muir, A follow-up study ofsynthetic rubber workers, Toxicology 113 (1996) 182–189.

[6] IARC occupational exposures to mists and vapours fromstrong inorganic acids and other industrial chemicals, IARCMonogr. 54 (1994) 237–285.

[7] IARC re-evaluation of some organic chemicals, hydrazineand hydrogen peroxide. IARC Monogr. 71 (1999) 109–226.

[8] National Toxicology Program, The 9th Report onCarcinogens, Department of Health and Human Services, USPublic Health Services, NTP, Research Triangle Park, NC,USA, 2000.

[9] European Commission, Commission Directive 2001/59/EC of6 August 2001 adapting to technical progress for the 28thtime Council Directive 67/548/EEC on the approximation ofthe laws, regulations and administrative provisions relating tothe classification and labelling of dangerous substances, 2001.

[10] E. Malvoisin, M. Roberfroid, Hepatic microsomal metabolismof 1,3-butadiene, Xenobiotica 12 (1982) 137–144.

[11] X. Cheng, J.A. Ruth, A simplified methodology forquantitation of butadiene metabolites. Application to the studyof 1,3-butadiene metabolism by rat liver microsomes, DrugMet. Dispos. 21 (1993) 121–124.

[12] M.W. Himmelstein, M.J. Turner, B. Asgharian, J.A. Bond,Comparison of blood concentrations of 1,3-butadiene andbutadiene epoxides in mice and rats exposed to 1,3-butadieneby inhalation, Carcinogenesis 15 (1994) 1479–1486.

[13] J.R. Thornton-Manning, A.R. Dahl, W.E. Bechtold,W.C. Griffith, R.F. Henderson, Disposition of butadienemonoepoxide and butadiene diepoxide in various tissues ofrats and mice following a low-level inhalation exposure to1,3-butadiene, Carcinogenesis 16 (1995) 1723–1731.

[14] J.E. Cochrane, T.R. Skopek, Mutagenicity of butadieneand its epoxide metabolites: I. Mutagenic potential1,2-epoxybutene, 1,2,3,4-diepoxybutane and 3,4-epoxy-1,2-butanediol in cultured human lymphoblasts, Carcinogenesis15 (1994) 713–717.

[15] P.J. Boogaard, S.C.J. Sumner, M.J. Turner, J.A. Bond,Hepatic and pulmonary glutathione conjugation of 1:2,3:4-diepoxybutane in human, rat, and mouse in vitro, Toxicology113 (1996) 297–299.

[16] P.J. Boogaard, S.C. J Sumner, J.A. Bond, Glutathioneconjugation of 1:2,3:4-diepoxybutane in human liver and ratand mouse liver and lung in vitro, Toxicol. Appl. Pharmacol.136 (1996) 307–316.

[17] P.J. Boogaard, J.A. Bond, The role of hydrolysis in thedetoxification of 1,2:3,4-diepoxybutane by human, rat, andmouse liver and lung in vitro, Toxicol. Appl. Pharmacol. 141(1996) 617–627.

[18] G.A. Csanády, F.P. Guengerich, J.A. Bond, Comparison ofthe biotransformation of 1,3-butadiene and its metabolite,butadiene monoepoxide, by hepatic and pulmonary tissuesfrom humans, rats and mice, Carcinogenesis 13 (1992) 143–1153.

[19] J.A. Bond, M.W. Himmelstein, M. Seaton, P.J. Boogaard,M.A. Medinsky, Metabolism of butadiene by mice, rats, andhumans: a comparison of physiologically based toxicokineticpredictions and experimental data, Toxicology 113 (1996)48–54.

[20] K.A. Richardson, M.M.C.G. Peters, B.A. Wong, H.J.J.J.Megens, P.A. Van Elburg, E.D. Booth, P.J. Boogaard, J.A.Bond, M.A. Medinsky, W.P. Watson, N.J. Van Sittert,Quantitative and qualitative differences in the metabolismof 14C-1,3-butadiene in rats and mice: relevance for cancersusceptibility, Toxicol. Sci. 49 (1999) 186–201.

[21] K.A. Richardson, M.M.C.G. Peters, H.J.J.J. Megens, P.A.Van Elburg, B.T. Golding, P.J. Boogaard, W.P. Watson, N.J.Van Sittert, Identification of novel metabolites of butadienemonoepoxide in rats and mice, Chem. Res. Toxicol. 11 (1998)1543–1555.

[22] N.Y. Tretyakova, Y.-P. Lin, P.B. Upton, R. Sangaiah, J.A.Swenberg, Macromolecular adducts of butadiene, Toxicology113 (1996) 70–76.

[23] N.Y. Tretyakova, Y.-P. Lin, R. Sangaiah, P.B. Upton, J.A.Swenberg, Identification and quantitation of DNA adductsfrom calf thymus DNA exposed to 3,4-epoxy-1-butene,Carcinogenesis 18 (1997) 137–147.

[24] L. Citti, P.G. Gervasi, G. Turchi, G. Bellucci, R. Bianchini,The reaction of 3,4-epoxy-1-butene with deoxyguanosine andDNA in vitro: synthesis and characterisation of the mainadducts, Carcinogenesis 5 (1984) 47–52.

[25] A.C. Beach, R.C. Gupta, Human biomonitoring and the32P-postlabeling assay, Carcinogenesis 7 (1992) 1053–1074.

[26] D. Potter, H.C.A. Brandt, R.W. Loose, R.A.J. Priston, S.A.Wright, W.P. Watson, Studies on the dermal and systemicbioavailability of polycyclic aromatic hydrocarbons in highviscosity oil products, Arch. Toxicol. 73 (1999) 129–140.

P.J. Boogaard et al. / Chemico-Biological Interactions 148 (2004) 69–92 91

[27] F.A. Beland, K.L. Dooley, D.A. Casciano, Rapid isolationof carcinogen-bound DNA and RNA by hydroxyapatitechromatography, J. Chromatogr. 174 (1979) 177–186.

[28] J. Wilson, Ph.D. Thesis, University of Newcastle upon Tyne,UK, 1999.

[29] I. Neagu, P. Koivistu, C. Neagu, R. Kostiainen, K. Stenby, K.Peltonen, Butadiene monoxide and deoxyguanosine alkylationproducts at theN7-position, Carcinogenesis 16 (1995) 1809–1813.

[30] W.K. Lutz, In vivo covalent binding of organic chemicals toDNA as a quantitative indicator in the process of chemicalcarcinogenesis, Mutat. Res. 65 (1979) 289–356.

[31] A.A. Elfarra, J.E. Sharer, R.J. Duescher, Synthesisand characterisation ofN-acetyl-l-cysteine-S-conjugates ofbutadiene monoxide and their detection and quantitation inurine of rats and mice given butadiene monoxide, Chem. Res.Toxicol. 8 (1995) 68–76.

[32] P. Koivisto, R. Kostiainen, I. Kilpeläinen, K. Steinby, K.Peltonen, Preparation, characterisation and32P-postlabeling ofbutadiene monoepoxideN6-adenine adducts, Carcinogenesis16 (1995) 2999–3007.

[33] R.R. Selzer, A.A. Elfarra, Synthesis and biochemicalcharacterisation ofN1-, N2-, and N7-guanosine adducts ofbutadiene monoxide, Chem. Res. Toxicol. 9 (1996) 126–132.

[34] R.R. Selzer, A.A. Elfarra, Characterisation ofN1- and N6-adenosine adducts andN1-inosine adducts formed by thereaction of butadiene monoxide with adenosine: evidence forthe N1-adenosine adducts as major initial products, Chem.Res. Toxicol. 9 (1996) 875–881.

[35] R.R. Selzer, A.A. Elfarra, In vitro reactions ofbutadiene monoxide with single- and double-strandedDNA: characterisation and quantitation of several purineand pyrimidine adducts, Carcinogenesis 20 (1999a) 285–292.

[36] R.R. Selzer, A.A. Elfarra, Chemical modification ofdeoxycytidine at different sites yields adducts of differentstabilities: characterisation ofN3- and O2-deoxycytidineand N3-deoxyuridine adducts of butadiene monoxide, Arch.Biochem. Biophys. 343 (1999) 63–72.

[37] N.Y. Tretyakova, R. Sangaiah, T.-Y. Yen, J.A. Swenberg,Synthesis, characterisation, and in vitro quantitation ofN-7-guanine adducts of diepoxybutane, Chem. Res. Toxicol. 10(1997) 779–785.

[38] N.Y. Tretyakova, R. Sangaiah, T.-Y. Yen, J.A. Swenberg,Adenine adducts with diepoxybutane: isolation and analysisin exposed calf thymus DNA, Chem. Res. Toxicol. 10 (1997)1171–1179.

[39] P. Koivisto, I.-D. Adler, F. Pacchierotti, K. Peltonen, Regio-and stereospecific DNA adduct formation in mouse lung atN6

andN7 position of adenine and after 1,3-butadiene inhalationexposure, Biomarkers 3 (1998) 385–397.

[40] P. Koivisto, I.-D. Adler, F. Pacchierotti, K. Peltonen, DNAadducts in mouse testis and lung after inhalation exposure to1,3-butadiene, Mutat. Res. 397 (1998) 3–10.

[41] N.Y. Tretyakova, S.-Y. Chiang, V.E. Walker, J.A. Swenberg,Quantitative analysis of 1,3-butadiene-induced DNA adductsin vivo and in vivo using liquid chromatography electrospray

ionisation tandem mass spectrometry, J. Mass Spectrom. 33(1998) 363–376.

[42] T. Oe, S.J. Kambouris, V.E. Walker, Q. Meng, L. Recio, S.Wherli, A.K. Chaudhary, I.A. Blair, Persistence ofN7-(2,3,4-trihydroxybutyl)guanine adducts in the livers of mice andrats exposed to 1,3-butadiene, Chem. Res. Toxicol. 12 (1999)247–257.

[43] H. Koc, N.Y. Tretyakova, V.E. Walker, R.F. Henderson,J.A. Swenberg, Molecular dosimetry ofN-7 Guanine adductformation in mice and rats exposed to 1,3-butadiene, Chem.Res. Toxicol. 12 (1999) 566–574.

[44] H.M. Bolt, B. Jelitto, Biological formation of the1,3-butadiene DNA adducts 7-N-(2-hydroxy-3-buten-1-yl)guanine, 7-N-(2-hydroxy-3-buten-2-yl)guanine and adducts7-N-(2,3,4-trihydroxy-butyl)guanine, Toxicology 113 (1996)328–330.

[45] P.J. Boogaard, K.P. De Kloe, K.A. Richardson, M.M.C.G.Peters, P.A. Van Elburg, W.P. Watson, N.J. Van Sittert, DNAadduct profiles differ after 1,3-butadiene or 1,2-epoxy-3-butene and between rats and mice, Toxicol. Sci. 42 (1998)S83.

[46] N.J. Van Sittert, H.J.J.J. Megens, W.P. Watson, P.J. Boogaard,Biomarkers of exposure to 1,3-butadiene as basis forcancer risk assessment, Toxicol. Sci. 56 (2000) 189–202.

[47] P.J. Boogaard, N.J. van Sittert, W.P. Watson, K.P. DeKloe, A novel DNA adduct, originating from 1,2-epoxy-3,4-butanediol, is the major DNA adduct after exposure to [2,3-14C]-1,3-butadiene, but not after exposure to [4-14C]-1,2-epoxy-3-butene, Chem. Biol. Interact. 135/136 (2001) 687–693.

[48] J.T. Millard, M.M. White, Diepoxybutane cross-links DNAat 5′-GNC sequences, Biochemistry 32 (1993) 2120–2124.

[49] J.L. Valentine, P.J. Boogaard, L.M. Sweeney, M.J. Turner, J.A.Bond, M.A. Medinsky, Disposition of butadiene epoxides inSprague–Dawley rats, Chem.-Biol. Interact. 104 (1997) 103–115.

[50] D.B. McGregor, I. Edwards, C.R. Wolf, L.M. Forrester, W.J.Caspary, Endogenous xenobiotic enzyme levels in mammaliancells, Mutat. Res. 261 (1991) 29–39.

[51] R.F. Henderson, F.F. Hahn, J.M. Benson, E.B. Barr, W.E.Bechtold, D.G. Burt, A.D. Dahl, Dosimetry and acute toxicityof inhaled butadiene diepoxide in rats and mice, Toxicol. Sci.51 (1999) 146–152.

[52] M.W. Himmelstein, B. Asgharian, J.A. Bond, Highconcentrations of butadiene epoxides in livers and lungs ofmice compared to rats exposed to 1,3-butadiene, Toxicol.Appl. Pharmacol. 132 (1995) 281–288.

[53] R.F. Henderson, F.F. Hahn, J.M. Benson, E.B. Barr, S.A.Belinsky, M.G. Ménache, J.M. Benson, Carcinogenicity ofinhaled butadiene diepoxide in female B6C3F1 mice andSprague–Dawley rats, Toxicol. Sci. 52 (1999) 33–44.

[54] X.Y. Zhang, A.A. Elfarra, Identification and characterisationof a series of nucleoside adducts formed by the reactionof 2′-deoxyguanosine and 1,2,3,4-diepoxybutane underphysiological conditions, Chem. Res. Toxicol. 16 (2003)1606–1615.

92 P.J. Boogaard et al. / Chemico-Biological Interactions 148 (2004) 69–92

[55] S. Park, N. Tretyakova, Structural characterisation of themajor DNA–DNA cross-link of 1,2,3,4-diepoxybutane, Chem.Res. Toxicol. 17 (2004) 129–139.

[56] P. Koivisto, M. Sorsa, F. Pacchierotti, K. Peltonen,32P-Postlabelling/HPLC assay reveals an enantioselective adductformation inN7 guanine residues in vivo after 1,3-butadieneinhalation exposure, Carcinogenesis 18 (1997) 439–443.

[57] J.L. Nieusma, D.J. Claffey, C. Maniglier-Poulet, T. Imiolczyk,D. Ross, J.A. Ruth, Stereochemical aspects of 1,3-butadienemetabolism and toxicity in rat and mouse liver microsomesand freshly isolated rat hepatocytes, Chem. Res. Toxicol. 10(1997) 450–456.

[58] D. Wistuba, H.P. Nowotny, O. Trager, V. Schurig, CytochromeP-450-catalyzed asymmetric epoxidation of simple prochiraland chiral aliphatic alkenes: species dependence and effectof enzyme induction on enantioselective oxirane formation,Chirality 1 (1989) 127–136.

[59] R.J. Krause, A.A. Elfarra, Oxidation of butadienemonoxide to meso- and (±)-diepoxybutane by cDNAexpressed human cytochrome P450s and by mouse, rat,and human liver microsomes: evidence for preferentialhydration of meso-diepoxybutane in rat and human livermicrosomes, Arch. Biochem. Biophys. 337 (1997) 176–184.