polycyclic aromatic hydrocarbon-dna adducts in white blood ... · aromatic hydrocarbon (pah)-dna...

Vol. 4, 69-77, January/February 1995 Cancer Epidemiology, Biomarkers & Prevention 69

Polycyclic Aromatic Hydrocarbon-DNA Adducts in White BloodCell DNA and 1-Hydroxypyrene in the Urine from Aluminum

Workers: Relation with Job Category and SynergisticEffect of Smoking’1

Frederik Jan van Schooten, Frans J. Jongeneelen,Michel J. X. Hillebrand, Flora E. van Leeuwen,Aart J. A. de Looff, Andr#{233}P. G. Dijkmans,Joost G. M. van Rooij, Leo den Engelse,and Erik Kriek2Department of Health Risk Analysis and Toxicology, University of

Limburg, P.O. Box 616, 6200 MD Maastricht IF. J. v. 5.1; Indus ToxConsult, 6503 CB Nijmegen IF. I. J.� I. G. M. v. RE; Division of MolecularCarcinogenesis EM. I. x. H., L. d. E., E. K.l; Department of Epidemiology

IF. E. v. LI; Netherlands Cancer Institute, 121 Plesmanlaan 1066 CXAmsterdam; Occupational Health Service Zeeland, 4382 ND Vlissingen

IA. J. A. d. LI; and Pechiney Nederland NV, 4380 AA Vlissingen, the

Netherlands IA. P. C. D.I

AbstractWe examined a group of 1 05 workers from a primaryaluminum plant for the presence of polycyclicaromatic hydrocarbon (PAH)-DNA adducts in theirWBC and 1 -hydroxypyrene in their urine. Workerswere recruited from five job categories with differentPAH exposure: the anode factory; the bake oven; andthe electrolysis and the pot-relining departments.Unexposed workers from the foundry departmentserved as the control group. The exposure to PAH wasmeasured by personal monitoring, and the averagePAH concentrations in the work atmosphere rangedfrom 0.4 pg/m3 in the foundry to 150 pg/m3 in thepot-relining department. The average exposure tobenzo(a)pyrene was under the Swedish exposure limitof 5 pg/m3.

The internal dose of pyrene was measured utilizingthe 1 -hydroxypyrene concentration in pre- and postshifturine samples. High exposure to PAH in the workatmosphere was associated with increasedconcentrations of 1 -hydroxypyrene in the urine. Theaverage increase in concentration of 1 -hydroxypyreneranged from 0.2 pmol/mol creatinine in the controlgroup to 5.9 pmol/mol creatinine in the pot-reliningdepartment; an accumulation of 1 -hydroxypyrene over a5-day working period was observed. A good correlationwas found between PAH exposure and the concentrationof 1-hydroxypyrene in the urine on a group level(r, = 0.90; P = 0.02).

PAH-DNA adduds were determined by 32P-postlabeling analysis (nuclease P1 enrichmentprocedure). In 93% of the workers, PAH-DNA addudswere deteded and workers with a high PAH exposurehad significantly higher PAH-DNA adduct values thandid those with a low PAH exposure. A good correlationwas found between PAH exposure and the average PAH-DNA adduct values in blood on a group level (r, = 0.90;P = 0.02), but not on the level of individual workers. Ahighly significant correlation was found between theaverage PAH-DNA addud values and the concentrationof 1 -hydroxypyrene in the urine at the end of day 5 forsmokers (r, = 0.66; P < 0.001 ; n = 23), but fornonsmokers this relationship was only marginallysignificant (1; = 0.25; P = 0.089; n = 31).

Multiple regression analysis clearly showed theeffect of smoking on DNA adduct values in WBCs(P = 0.10), as well as on 1-hydroxypyreneconcentration in the urine (P < 0.001 ). Higher DNAadduct values in smokers were found only in those jobgroups with high PAH exposure. Higherconcentrations of 1 -hydroxypyrene in smokers werefound in all groups, but the difference betweensmokers and nonsmokers was most pronounced inthose groups with the highest PAH exposure. Thestrong effect of smoking in the exposed groupssuggests a synergistic effect of smoking incombination with occupational PAH exposure on theformation of PAH-DNA adducts and 1 -hydroxypyrenein the urine.

Introdudion

Aluminum workers are exposed to coal tar fumes and dustsduring manufacturing of the anodes, which are made froma mixture of coke and coal tan pitch. Epidemiobogical sun-veys in occupationally exposed populations, such as cokeoven and iron foundry workers, roofers, and primary alu-minum plant workers, indicate that bong-term exposure tocoal tar-derived products is associated with certain cancers(1 , 2). Coal tar pitch fumes and dusts contain PAHs, ‘ manyof which have shown carcinogenic activity in experimentalanimals and are suspected carcinogens in humans. PAHsform a well-studied class of compounds, and extensiveknowledge exists about their metabolism (3). The metabo-

Received 6/28/94; revised 10/6/94; accepted 10/6/94.1 Supported by Grant PCT99.2050 from the Dutch Program Committee for

Toxicological Research and by Pechiney Nederland NV.2 To whom correspondence should be addressed.

3 The abbreviations used are: PAH, polycyclic aromatic hydrocarbon; B(a)P,

benzo(a)pyrene; BPDE, (±)frans-7,8-dihydroxy-anti-9,i 0-epoxy-7,8,9, 1 0-tet-

rahydrobenzolalpyrene; r�, Spearman’s rank correlation coefficient.

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70 PAH-DNAAdducts

lism of PAHs results mainly in the formation of more water-soluble derivatives that are excreted in the urine, but some-times a part ofthe parent compound becomes activated intobiochemically reactive forms. For example, metabolic ad-tivation of B(a)P results in the formation of dihydnodiol

epoxides, which are the ultimate reactive forms capable ofbinding to nucleic acids (4). Noncancinogenic PAHs such aspyrene do not yield metabobites that form DNA adducts (5),but they are excreted in the urine as conjugated metabo-lites. DNA adduct formation is thought to be involved inmutation and neoplastic transformation of target organs

(6, 7).To improve safety at the workplace and to identify

subpopulations of workers that are at an increased risk forcancer, it is essential to monitor human exposure tocarcinogenic and mutagenic agents. Monitoring of hu-man exposure in workplaces traditionally consistedmainly of measuring the concentration of certain chem-icals in ambient air. Biological monitoring of workers islikely to bead to a much more reliable risk assessment.Monitoring human urine for i-hydnoxypyrene, a metab-olite of pyrene, is one way of biological monitoring andhas been shown to be a reliable indicator for PAH expo-sure in several occupational settings (8, 9). Furthermore,it has been suggested that determination of the extent ofbinding of PAH to DNA in humans may serve as abiomanker of exposure to genotoxic PAH. Therefore, sen-sitive detection methods have been optimized [enzymeimmunoassays (1 0, 1 1 ); ‘2P-postlabeling assays (1 2); andfluonometnic assays (1 3, 1 4)1 . Several studies have shownthat levels of PAH-DNA adducts are significantly in-creased in peripheral WBC of occupationally exposedcoke-oven workers, foundry workers, and roofers (i 5). Arecent study of workers employed in two aluminum pro-

duction plants showed increased WBC adduct levels inone plant, but not in the other, compared with those ofcontrol subjects (1 6, 1 7). Although the effect of smokingon adduct levels in WBC from healthy non-occupation-ably exposed individuals has not been clarified yet (18-20), aromatic-DNA adduct levels in other human tissues,such as lung (21-23) and placenta (24), have been asso-ciated with smoking. These uncertainties on the effect ofsmoking on adduct levels in WBC are also encounteredin studies with occupationally exposed workers (25, 26).

In this investigation we examined a group of 105 alu-minum workers from five job categories as a model popu-lation for testing the applicability of two biomonitoningtechniques to assess occupational exposure to PAH. Theworkers were examined for i -hydroxypyrene in urine andfor PAH-DNA adducts in WBC by 32P-postlabeling assay forthe following purposes: (a) to determine whether 1 -hy-droxypyrene in urine and PAH-DNA adduct bevels in WBCare rebated to external PAH exposure in different job date-gonies; (b) to examine whether urinary 1 -hydroxypyneneexcretion and PAH-DNA adduct levels depend on otherfactors, e.g., smoking and the use of protective devices; and(c) to see if PAH-DNA adduct levels in WBC correlate withthe urinary excretion of i-hydroxypyrene.

MethodsChoice of Subjeds, Sample Collection, and QuestionnaireData. Aluminum is produced by electrolytic reduction ofalumina. Differences between the electrolysis processes es-sentialby depend on the type of anodes used. These anodes

can be either self-baking anodes in the Soderberg process,or prebaked in ovens before being used. Our study popu-lation consisted of 105 workers employed in an aluminumproduction plant operating with the prebake anode process.In this type of aluminum plant, anodes are produced in the

anode production departments composed of a paste-man-ufactuning plant and a bake oven. Significant amounts ofPAH are released in these departments. The studied workerswere recruited from four job categories with different PAHexposure: the anode factory (mixing and shaping of the

anode) (n = 16); the bake oven (baking of the anode)

(n = 20); the pot-relining department (n = 1 3); and theelectrolysis department (n = 24). As a control group, 32workers were chosen from the foundry department of the

same aluminum plant. The average ± SD age of all partic-ipants was 41 .0 ± 8.3 years, and their ages ranged from 24

to 58 years. A trained interviewer conducted inquiriesabout smoking habits, occupational history, alcohol con-

sumption, medication, and use of protective equipment(gloves and face masks).

Sampling of blood, urine, and ambient air took placefrom May to October 1 989. Blood samples were collectedat days 1 , 3, and 5 of a 5-day working period. Samples werecoded, placed on ice, and transported immediately to theNetherlands Cancer Institute. Total WBC were isolated onthe same day. Urine samples were collected from eachworker; 1 sample at the beginning and 1 sample at the endof an 8-h working period on 5 consecutive working days.The urine samples were immediately frozen at -20#{176}C.Onthe same 5 days, exposure levels of PAHs in the breathingzone were determined by personal air sampling. Duringworking hours, the particulate PAHs were collected on ateflon filter using a personal air sampling pump (Dupont deNemours Inc., models P4000 and 52500) with a constantflow of 2.0 liters/mm through a teflon membrane filter(diameter, 25 mm; pore size, 0.5 pm; Millipore FHLP). Thesamples were designed according to International Stan-dards Organization TC146 standard. The PAHs with three

or more aromatic rings are mainly retained on the filterabsorbed to particulate matter, while PAHs of lower mo-lecular weight in the vapor phase are not retained. Aftersampling, the filters were stored in the dark at -20#{176}Cuntilanalysis.

Measurement of PAH in Air Samples. The particulatePAH collected on the teflon filters were extracted ultra-sonically for 30 mm with acetonitnile (1 .5 ml). Clearsupennatants of the extracts were used to analyze PAHsby HPLC equipped with a Vydac C18 column and afluorescence detector. Elution was performed at a flowrate of 1 .0 mI/mm with a gradient of water/acetonitnile,starting with a 1 :1 mixture which changed to pure ace-tonitnile. Standard PAH mixtures (EEC Bureau of Stan-dards, Brussels, Belgium) were used as reference materi-abs. The air samples were analyzed for 1 2 different PAHs:phenanthrene; anthracene; fluoranthene; pyrene; ben-z(a)anthracene; chrysene; benz[b]fluoranthene; benz[k-Ifluoranthene; benzo(a)pyrene; d i benz(a, h)anthracene;indeno[1 ,2,3-c,d]pyrene; and benz[g, h, i]perylene.

Determination of 1 -Hydroxypyrene in Urine. The mea-surement of urinary 1 -hydroxypyrene excretion was as per-formed by the method ofJongeneelen eta!. (8) and will onlybe summarized here. The method includes enzymatic hy-drolysis ofthe conjugated metabolites by a mixture of gluc-uronidase and sulfatase and clean up of the samples by



a GM, geometric mean.

“Sum of 12 PAHs.

Cancer Epidemiology, Biomarkers & Prevention 71

Sep-Pack C18 (Waters, Milford, MA). Analysis by HPLC

allows the determination of the sum of free and conjugated1 -hydroxypyrene in the urine (nmol/liters). Chromatogra-

phy was performed on a LiChrosorb PR-i 8 column (Merck,Darmstadt, Germany) with a linear gradient of methanol inwater from 46 to 96% methanol (flow rate, 0.8 mI/mm) in35 mm. 1 -Hydroxypyrene was detected by fluorometry

(excitation, 242 nm; emission, 388 nm). The reproducibility(coefficient of variation = 1 1 .6%) of the assay was deter-mined by repeated analysis of 30 randomly selected urinesamples. Concentrations of 1 -hydroxypyrene were ex-pressed as pmol/mol creatinine to correct for volume dif-

ferences. Since correction with extremely low on high cre-atinine values was considered unreliable, urine sampleswith creatinine values beyond the range of 4-34 mmol/litenwere excluded from analysis.

DNA Isolation. Peripheral blood samples (50 ml) werewithdrawn by venapunction into tubes containing EDTA.

WBC were collected by centnifugation after incubation with

0.1 2 M NH4CI to lyse the erythrocytes. DNA was isolated bytreatment with pancreatic RNase, proteinase K, and extrac-

tion with phenol, chloroform:isoamyl alcohol (24:1), anddiethyl ether. DNA was precipitated from the aqueousphase with cold ethanol and dissolved in 2 ms’i Tnis-HCI, pH7.4 (2.0 mg/mb), for 32P-postlabeling analysis. Coded DNAsamples were stored at -20#{176}C.

DNA Adduct Determination by 32P-Postlabeling Assay.DNA samples were analyzed essentially as described byReddy and Randerath (27). Five pg DNA was digested for

3.5 h at 37#{176}Cwith 250-mU micrococcal nuclease (SigmaChemical Co., St. Louis, MO) and 8-mU spleen phos-phodiesterase (Cooper Biomedical Corp., Malvern, UK)in a total volume of 5 p1 containing 10 mtvt sodiumsuccinate and 10 mM CaCI2, pH 6. Samples were thendigested further with 2 pg nuclease P1 (Boehninger,Mannheim, Germany) in a total volume of 7.5 Pb’ con-taming 0.1 mM ZnCI2 and 0.06 M sodium acetate (pH 5)for 1 h at 37#{176}C.After the addition of 2.5 p1 0.5 M Tnisbase, the DNA digest was labeled with [y-32P]ATP (50pCi) using 2.5 units of T4 pobynucleotide kinase (Boehr-inger). The reaction was terminated with 40 mtvt potatoapyrase (Sigma) after 30 mm. The [y-32P]ATP was syn-thesized in our laboratory using carrier-free 32P-or-thophosphate (Amersham, Buckinghamshire, U K). Thespecific activity was determined as described and wasgenerally around 3000 Ci/mmol. Purification and reso-lution of 32P-labeled adducts were carried out by TLC onpolyethyleneimine-cellulose sheets (Macherey-Nagel,D#{252}ren,Germany). Chromatography was done accordingto a published method (27) using the following solvents:Dl , 1 .0 M sodium phosphate (pH 6.0); D2, 3.5 si lithiumformate-8.5 M urea (pH 3.5); D3, 0.8 M LiCI-0.5 M Tnis-HCI-8.5 M urea, (pH 8.0); and D4, 1 .7 M sodium phos-phate, (pH 6.0).

The chromatograms were visualized by autoradiogra-phy at -80#{176}Cusing intensifying screens. Adducts were ex-cised from the chromatograms and radioactivity was mea-sured by Cerenkov counting. Carcinogen-DNA adductbevels were calculated by determining the relative adductlabeling, which is the ratio of count rates of adduct nude-otides to count rates of total (adduct plus normal) nucleoti-des. The specific radioactivity of [y32PIATP, determined bylabeling a known amount ofdAp (deoxyadenosine 3’-phos-phate), was used for relative adduct-labeling calculations

Table 1 Average concentrations of pyrene, benzo)a)pyrene, and total

PAH of five different job categories )n = number of measurements)

Pyrene Benzo(a)pyrene Total PAH”

Department n GM” GM GM

)range)(pg/m’) )range)(pg/rn) )rangehpg/rn’)

Bake oven 22 1.5 (0.5-22.7) 0.35 (0.1-14.4) 8.7 (3.0-107)

Anodefactory 40 5.6 (0.3-318) 1.51 (0.1-11.6) 23 (1.1-854)

Pot-relmning 41 32.3 (0.15-223) 1.05 (<0.02-9) 150 (0.9-1709)

Electrolysis 23 0.1 2 (0.03-0.7) 0.03 (<0.02-0.2) 1 .0 (0.3-4.6)

Foundry 16 0.04 (<0.02-0.2) 0.02 (<0.02-0.06) 0.4 (0.1-3.8)

(27). In each experiment a [3HJBPDE-DNA standard with amodification level of i adduct/i07 nucleotides was in-cluded and recoveries between 80 and 1 00% were usuallyobtained. The reproducibility of the assay (coefficient ofvariation, -20%) was determined by repeated assays of the[3HIBPDE-DNA standard.

Statistical Methods. Correlation coeffic ients were calcu-bated with the use of Spearman’s rank correlation analysis.For these analyses the urinary excretion of 1 -hydnoxypyreneof a worker was defined as the difference between the post-and the preshift samples (the increase oven 5 working days).For blood samples the average value of 3 days was used; forair measurements the average of the exposures of 5 workingdays was used. Differences in 1 -hydroxypynene concentra-tion in the urine and average DNA adduct levels in blood

samples among groups were compared by Student’s t test.Multiple regression analysis was carried out with the

level of DNA adducts or 1-hydroxypyrene as the dependentvariable with the SPSS/PC+ computer software package.Independent variables included: smoking habits (yes or no),number of cigarettes smoked per day during the last week,job category (5 levels), age, number of years employed bythe plant, and use of protective equipment (yes or no). Thenumber of workers engaged in each analysis varies

somewhat due to missing data.

Results

Air Monitoring. The average exposure to total PAHs,pyrene, and B(a)P of individual aluminum workers is pre-sented in Table i for each ofthe four job categories and thecontrol group. Large differences in exposure to PAH wereobserved between the various categories. The highestconcentrations of PAH and pynene were measured in thepot-relining department. The highest concentration of B(a)Pwas detected in the anode factory.

The Spearman’s rank correlation coefficient betweenpyrene and total PAH concentration in the work atmo-sphere of 20 workers was r� = 0.98 (average of 5 con-secutive working days). This highly significant conrela-tion was found at three work sites: the bake oven (r� =

0.90; n = 5); the anode factory (r� = 0.98; n = 8); and thepot-relining department (r5 = 0.96; n = 7). A good come-lation was also found between B(a)P and total PAHs inthe anode factory (r� = 0.97; n = 8) and for the bake oven(r5 = 0.69; P < 0.001 ; n = 5). The correlation betweenB(a)P and total PAH in the pot-nelining department (r� =

0.43; P= 0.Oi ; n = 7) was bower than thatfon pynene andtotal PAH but still significant.

A typical PAH profile found in the anode factory is



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� �L�i�’1 i � 1T�IDay 1 Day 2 Day 3 Day 4 Day 5

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Fig. 2. Average assay values of 1 -hydroxypyrene (pmol/mol creatinine) in

the urine of five bake oven workers at the beginning and at the end of an 8-hworkshift during 5 consecutive working days. Columns, mean; bars, SD.

FA PYR BA CHA BFA BaP BPL IP

Polyaromatic hydrocarbon

Fig. 1. Average PAH profile obtained by personal sampling during an 8-hworking shift of aluminum workers engaged in the anode factory. Com-

pounds with four or more aromatic rings are illustrated. FA, fluoranthene;PYR, pyrene; BA, benzlalanthracene; CHR, chrysene; BFA, benzolb, I, kI-fluoranthene; B)a)P, benzo)a)pyrene; BPL, benzolg, h, :]perylene; and IP,indenoli,2,3-c,dlpyrene. Columns, mean; bars, SD.

Table 2 Average (± SD) assay values of PAH-DNA adducts in WBC from

smoking and nonsmoking aluminum workers (n = number of workers

72 PAH-DNAAdducts

13

� 10E0)

3

0

assayed)

Department �

‘2P-postlabeling analysis )adducts/i 0” nucleotides)

n Smokers n Nonsmokers

High exposure

Bake oven

Anode factory

Pot-relining

All

6 48.5 ± 40.9 10

1 34.7 5

5 58.3 ± 67.7 2

12 51.5±49.8” 17

30.1 ± 42.1

26.2 ± 1 5.0

47.3 ± 39.1

31.0± 34.5

Low exposure

Electrolysis

Foundry

All

5 6.4 ± 6.3 9

7 7.2 ± 6.4 7

12 6.9 ± 6.1 16

12.8 ± 10.0

7.4 ± 9.6

10.5 ± 9.9

“ Value was compa red with nonsmokers (P = 0.233).

illustrated in Fig. 1 , showing that pynene is the most abun-dant polyaromatic hydrocarbon. Similar profiles werefound at other worksites with high PAH exposure. Althoughlarge fluctuations were found in the absolute total PAHconcentration on different days, the relative amounts ofeach of the 1 2 individual PAHs remained fairly constant.

PAH-DNA Adduds in WBC. Fifty-four (95%) of the 57blood samples tested showed PAH-DNA adduct levelsabove the detection limit (Table 2). All 43 workers of thebake oven, anode factory, pot-relining and electrolysis de-partments, and i 1 (79%) of 1 4 workers from the controlgroup (foundry) showed adduct levels above the detectionlimit. Lange intra- and intenindividual differences betweenadduct values were found and it was not possible to detectan increase in DNA adduct levels oven a 5-day workingperiod. Therefore, the average adduct levels pen worker of2 or 3 days of a 5-day working period were calculated. Theaverage adduct level ofthe highly exposed groups, the bakeoven, anode factory, and the pot-nelining department, 40.8± 4i .6 adducts/1 0” nucleotides (n = 29), was significantly

enhanced compared with that of the control group, 7.3 ±

7.8 adducts/i 0” nucleotides (n = 1 4; P < 0.0001 ) (Table 2).No significant differences in DNA adduct levels were found

between workers from the three highly exposed groups.Adduct levels of workers from the electrolysis departmentand from the control group did not differ significantly.

Among the i 02 workers questioned, 47 (46%) werecurrent smokers, whereas 55 (54%) were nonsmokers.Appreciable differences in smoking habits between thevarious departments were noted. In the bake oven and inthe anode factory, 35 and 25%, respectively, ofthe work-ens were smokers, whereas the pot-relining workers had60%, the electrolysis workers had 54%, and the controlgroup had 53% smokers. The number of cigarettessmoked was comparable; the average number of ciga-nettes smoked/day was i 7.

In the total exposed population, the average PAH-DNAadduct value of smokers (29.2 adducts/1 08 nucleotides; n =

24) was 86% higher than that of nonsmokers (1 5.7 adducts/1 08 nucleotides; n = 32; P = 0.08). Although a differencewas found, it was not statistically significant, which may bedue to the large SD and the small size of the groups. Norelationship was noted between the PAH-DNA adduct 1ev-

els and the number of cigarettes smoked during the lastweek, and theme was also no association with the amountsmoked during the last 24 h. Adduct levels in smokers wereincreased only in the exposed groups (Table 2).

1 -Hydroxypyrene in the Urine. The 1-hydroxypyrene con-centration in the urine ranged between 0.1 7 and 26.9 pmol/mol creatinine. To illustrate the time course of 1 -hydroxy-pyrene excretion in urine oven a 5-day working period, theaverage 1 -hydroxypynene concentrations in preshift urinesamples and in postshift urine samples of five bake ovenworkers are presented in Fig. 2. An increase in urinary1 -hydroxypyrene concentration was found during one shift(from 8 am to 4 pm), as well as in the course of a 5-dayworking period. A decrease was observed between two

shifts, but high values did not drop tothe pneshift level of theday before. This is in agreement with the reported elimina-tion half-life of 1 -hydroxypyrene in the range of 6-35 h (8).

A significant effect of smoking on the excretion of1 -hydroxypyrene was observed for all job groups, but thedifference in 1 -hydroxypyrene excretion between smokersand nonsmokers was the largest in the three departmentswith high PAH exposure (Table 3). This difference in 1 -hy-droxypynene concentration was found in preshift urine sam-



120

96

72

48

24

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FOU ELO BAK APF BRS

FACTORY

I � Adducts PAH

12.00

pIes, as well as in postshift urine samples. In preshift urinesamples of day i , the average 1 -hydroxypynene concentra-tion of smokers (0.66 pmol/mol creatinine; n = 44) was 89%higher than that of nonsmokers (0.35 pmol/mol creatinine;n = 5i ; P = 0.0002). The increase of i -hydroxypyreneconcentration oven a 5-day working period in workers em-pboyed in the anode production and bake oven departmentswas higher in smokers than in nonsmokers.

A significant relationship was noted between the num-ben of cigarettes smoked during the last week and the1 -hydroxypyrene concentrations in preshift, as well as inpostshift urine samples (P= 0.005). The association with thenumber of cigarettes smoked during the last week wasmuch stronger than the one with the amount smoked duringthe past 24 h. Urine samples of workers in the control groupdid not show a significant increase in 1 -hydroxypyreneconcentration oven a 5-day working period.

Relationships between the Various Parameters. The nela-tionship between the air concentration of PAH and theaverage PAH-DNA adduct values of days 1 , 3, and 5 wasexamined for the total exposed population. At the level ofthe individual workers no significant correlations (all are<0.20) were found between PAH-DNA adducts in bloodand the air concentrations of PAH, pynene, on B(a)P sepa-rateby, and those PAHs that are known to form diol epoxide-DNA adducts. However, good correlations (r = 0.90; P =

0.02) were found between PAH exposure and PAH-DNAadducts in blood, and 1 -hydroxypynene in the urine at thelevel of job category (Fig. 3).

A highly significant correlation was found between the

average PAH-DNA adduct values (days 1 , 3, and 5) and theconcentration of 1 -hydroxypyrene in the urine at the end ofday 5 for smokers (r5 = 0.66; P< 0.001 ; n = 23) (Fig. 4), butfor nonsmokers this relationship was not significant (r., =

0.25; P= 0.089; n = 31).Table 4 shows the results of multiple regression models

predicting DNA adduct levels in WBC and 1-hydroxy-pyrene in the urine in the whole study population and theexposed workers. Only smoking and job category wereboth significantly associated with PAH-DNA adduct levelsand excretion of 1 -hydroxypyrene in the urine. Table 2 alsoshows that within the highly exposed groups (anodefactory, bake oven, and pot relining), job category is notsignificantly associated with adduct bevels.

No significant relationship was found between

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400

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200 .E

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FOU ELO BAK APF

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ii �j 1-HO-pyr � PAH

Fig. 3. A, average PAH-DNA adduct values and the average PAH ( 1 2

compounds) concentrations in the work atmosphere; B, average 1 -hydroxy-pyrene ( 1-HO-pyrene) concentrations in the urine )pmol/mol creatinine) at

the end of a S-day working period and the average PAH ) 1 2 compounds)concentrations in the work atmosphere of the hake oven )BAK), the anodefactory )APF), and the pot-relining (BRS), electrolysis )ELO). and toundry)FOU) departments. Columns, mean; bars, SD.

PAH-DNA adduct levels in blood on with i -hydnoxypyrenein the urine and any of the other factors examined, i.e., thenumber of years employed at the plant, the use of protectiveequipment for hands and face, on the consumption ofalcohol.

2.40

BRS


Table 3 Average )± SD) assay values of 1 -hydroxypyrene (pmol/mol creatinine) in the urine of smoking and nonsmoking aluminuni workers at thebeginning and at the end of a S-day working period (number of workers assayed is given between parentheses)

DepartmentSmokers Nonsmokers

---�--� � - - -___Preshift PostshiftPreshift Postshift

High exposure

Bake oven

Anode factory

Pot-relining

All

1 .00 ± 0.72 (6)

1 .34 ± 0.52 (2)

0.81 ± 0.48 (6)

0.97 ± 0.47 (14)’

8.43 ± 4.08 (6)

4.84 ± 3.64 (2)

6.72 ± 4.25 (6)

7.19 ± 4.00 (14)”

0.58 ± 0.31 (10)

0.41 ± 0.1 8 (4)

0.28 ± 0.1 7 (4)

0.47 ± 0.28 (18)

3.65 ± 2.1 1 (9)

3.25 ± 1 .69 (5)

6.20 ± 8.44 (2)

3.84 ± 2.99 (16)

Low exposure

Electrolysis

Foundry

All

0.58 ± 0.31 (1 3)

0.47 ± 0.37 (1 6)

0.52 ± 0.34 (29)’

0.88 ± 0.42 (1 3)

0.62 ± 0.31 (1 7)

0.74 ± 0.38 (30)”

0.30 ± 0.1 8 (1 3)

0.35 ± 0.32 (1 4)

0.33 ± 0.26 (27)

0.48 ± 0.27 (9)

0.47 ± 0.20 (15)

0.47 ± 0.22 (24)

.‘ Value was compared with preshift nonsmokers (P = 0.009).

“ Value was compared with end of shift nonsmokers (P = 0.01 7).‘ Value was compared with preshift nonsmokers (P = 0.019).

(I Value was compared with end of shift nonsmokers (P = 0.003).

ci

z

0

‘I)

0

V

0

.(zaI



C)

z

0

U)

0

VVCU

zaI

a.

120

003691215

1-HO-pymene in urine (pmol/mol croat.)

Fig. 4. Correlation between the average PAH-DNA adduct values (days 1,

3, and 5) and the average 1 -hydroxypyrene ) 1-HO-pyrene) concentrations in

the urine from smokers at the end of a 5-day working period (Spearman rankcorrelation coefficient #{231}= 0.66; P< 0.001 ; n = 23). Nuc., nucleotide; creat.,

creatinine.

Discussion

Studies evaluating exposure of workers to PAH in alumi-num production plants are limited. Older data refer mainlyto exposures in Sodenbeng plants (28, 29), and it is difficultto compare these data with those from studies in modernprebaked smelters. Recently, Tjoe Ny et a!. (30) reportedextremely high air sampling data and urinary 1-hydroxy-pyrene concentrations in Soderberg plant workers. Tolos et

a!. (31 ) screened 1 8 workers, all of who worked in theanode bake area of an aluminum production plant, bypersonal sampling and found levels of 9.5-94.6 pg ofPAHs/m’ and 1 .2-7.4 pg of pyrene/m3. In our study, 20workers were monitored individually during a 5-day work-ing period, and in the bake oven area, concentrations vary-ing between 3-107 pg of PAHs/m3 and 0.5-22.7 pg ofpyrene/m3 were determined (Table 1 ). The PAH concentra-tions are in good agreement with those ofTolos eta!. (31)but we found higher pynene concentrations. Workers in theanode factory and pot-relining department had the highestexposure to PAH, pyrene, and B(a)P (Table 2). For thetotal-exposed population, the average concentration ofB(a)P was below the Swedish limit of 5 pg/m3, but theaverage concentrationof total PAH was above the Norwe-gian limit of 40 pgjm3. Although appreciable changes inabsolute PAH concentrations were observed, the ratio be-tween pyrene on B(a)P to total PAH remained relatively

constant for all exposed groups. Pyrene was the most abun-dant aromatic hydrocarbon among the 1 2 PAHs measuredin all workplaces. Its relative abundance ranged from 9.3 to35.6% of total PAHs, and the concentration of pyrene washighly correlated with the total amount of PAH in air (r,=

0.90-0.98). Jongeneelen et a!. (9) reported a similar highcorrelation for coke ovens (r� = 0.88). Tolos et a!. (3 1 ) for analuminum plant and Buchet et a!. (32) for a graphite ebec-trode manufacturing plant also found high correlations forpyrene and total PAHs (r = 0.82 and 0.83, respectively).

The relative abundance of B(a)P ranged from 0.i-9.7%of total PAH. Despite its lower relative proportion, B(a)Pwas also webb correlated with the total amount of PAHs

(C. 0.69-0.97). These results demonstrate that pynene andB(a)P are appropriate indicators for mixtures of PAHs.

74 PAH-DNA Adducts

During the past 8 years several studies have been+ reported in which PAH-DNA adducts in WBCs from occu-

+ pationally exposed workers in coke oven plants (25, 26,33-35) and foundries (36-38) have been determined by

+ 32P-postlabeling analysis and/or immunoassays, but very

few studies in aluminum plants have been described.In this extensive study of a primary aluminum produc-

tion plant, we analyzed DNA from WBC from 57 workers,including 14 workers from the foundry department of thesame plant as controls. We found a high correlation be-tween PAH-DNA adduct levels and exposure groups, de-fined as job categories (r� = 0.90; P= 0.02), as illustrated in

________________________________________ Fig. 3. When comparing our results with those of anotherrecent aluminum plant study (16, 17), we found 15-20times higher adduct values. Schoket et a!. (1 7) found ahigher average DNA adduct level in peripheral blood lym-phocyte samples from only one of the factories, comparedwith a local control group, but job categories, PAH con-

centrations in air, and excretion of 1 -hydroxypyrene werenot reported.

The results of two recent coke oven studies (25, 35)

also showed a significant relationship between PAH-DNAadduct values and PAH exposure by job category.

In our study smokers had consistently higher PAH-DNA adduct values than nonsmokers in the highly exposedgroups (P= 0.08), but no effect of smoking was found in the

electrolysis department and in the control group (Table 2).No correlation at the individual level was found betweenPAH-DNA adducts in blood and the concentration of PAHson B(a)P in the air, but a good correlation was found at thegroup bevel (Fig. 3). There was no relationship between

adducts and the number of cigarettes smoked per day.These results differ only slightly from those of our previousstudy in coke oven workers (26). In that study, smokingcontrols had higher adduct levels than nonsmoking con-trols, but this difference was not statistically significant(P = 0.07). Only in the control group was a significantrelationship noted between adduct levels and the numberof cigarettes smoked/day (r = 0.33; P = 0.03).

In several previous studies on occupational PAH ex-posune the effect of smoking has been examined, but a cleanrelationship was not always found. In their study on coke

oven workers, #{248}vrebo et a!. (25) found a significant come-lation between smoking and adduct levels only in the con-

trol group, when measured by 32P-postlabebing (P = 0.02),but not by ultrasensitive enzyme radioimmunoassay.Schoket eta!. (1 7) reported that neither 32P-postbabeling norELISA gave detectable differences in adduct levels betweensmokers and nonsmokers. Two other studies of foundryworkers showed no differences in adduct levels betweensmokers and nonsmokers (36, 37).

It must be emphasized, however, that direct compani-son of DNA adduct values between different studies shouldbe done with caution because many variables may influ-ence the results. First, the PAH profile varies among alumi-num plants depending on the process used. Secondly, the

bioavailability of PAH, their absorption to particulate mat-ten, and the size of the particles will influence the uptake.The relative amounts of the various subpopulations of WBCand differences in their capacity to metabolize PAH will bean important factor in the formation of DNA adducts inindividuals. Together with individual differences in DNArepair capacity, these parameters will determine the ubti-mate level of DNA adducts measured in a particular tissueat a certain time. The 32P-postlabeling analysis itself may be




Table 4 Multiple regression analysis with average PAH-DNA adduct levels in blood and average 1 -hydroxypyrene concentrations in the urine at day 5 as

dependent variables’

VariableLogIPAH-DNA adductsl” LogIl -Hydroxypyrenel’

Regression coefficient (± SE) P-value Regression coefficient (± SE) P-value

Bake oven’�’ 0.84 (± 0.24) 0.001 0.76 )± 0.14) <0.0001

Anode factory’� 0.71 (± 0.32) 0.03 0.66 (± 0.16) 0.0001

Pot relining” 0.93 (± 0.25) 0.0005 0.80 (± 0.1 3) <0.0001No. of cigarettes smoked last weeke -0.02 (± 0.02) 0.20 0.01 (± 0.004) 0.004Smoked cigarettes last week (yes)1 0.39 (± 0.3) 0.20 0.21 (± 0.06) 0.002

Face unprotected5

Always -0.28 (± 0.25) 0.26 0.09 1± 0.1 3) 0.46

Often 0.08 (± 0.44) 0.85 -0.13 (± 0.21) 0.52

Sometimes 0.03 (± 0.22) 0.90 0.10)± 0.13) 0.43

Hands unprotected”

Always -0.12 (± 0.32) 0.71 -0.07 (± 0.14) 0.63

Often 0.20 (± 0.35) 0.58 0.19 (± 0.19) 0.32

Sometimes 0.19 (± 0.25) 0.45 0.07 (± 0.12) 0.54

Lunch in restaurant’

Always 0.07 (± 0.22) 0.74 0.13 (± 0.10) 0.23

Sometimes 0.34 (± 0.39) 0.40 0.02 (± 0.16) 0.89

a R2 = 0.45 (experimental variability).

“ Adducts/1 08 nucleotides.C pmol/mol creatinine.d Reference category: electrolysis and foundry departments.e Reference category: no cigarettes smoked.

I Reference category: not smoked during last week.

S Reference category: face never unprotected.

h Reference category: hands never unprotected.

, Reference category: never lunch in restaurant.

subject to appreciable variation, as was demonstrated in arecent intenbaboratory trial (39).

The concentrations of urinary 1 -hydnoxypyrene in thesame groups of workers showed an increase of 1 -hydroxy-pynene during the day and a decrease overnight. A generalincrease was observed during a 5-day working period, atthe end of which a difference in 1 -hydroxypyrene concen-tration was found of more than 1 5 times over that seen incontrols. This is in good agreement with a 1 7-fold differencereported by Tobos et a!. (31 ). A strong correlation was foundbetween 1-hydroxypyrene in the urine at day 5 and theaverage PAH concentration of the five job categories(r� = 0.90; P= 0.02) (Fig. 3). On an individual basis, how-even, correlations between 1 -hydroxypyrene in the urineand PAH in the work atmosphere of the three highlyexposed groups were bow (<0.20).

Smokers had significantly higher 1 -hydroxypyreneconcentrations in the urine than did nonsmokers at thebeginning as well as at the end of a 5-day working period(P < 0.001). This difference was found in all groups, but itwas most pronounced in those departments with high PAHexposure. A clean relationship was found between 1 -hy-droxypyrene in the urine and the number of cigarettessmoked pen day (P = 0.005). No relationship was foundbetween adduct levels or 1 -hydroxypyrene concentrationsand the use of protective materials for hands and/or face. Itis qu ite Ii kely, however, that protective materials weremostly used by workers with the highest PAH exposure,which renders it impossible to evaluate their effectivity in agroup of workers with a large variation in degree of expo-sure. Other factors such as the number of years employed atthe plant on alcohol consumption did not have anyinfluence on either PAH-DNA adduct values or on1 -hydnoxypynene concentrations.

The effect of smoking in occupationally exposedaluminum workers may be explained by a synergisticeffect between the ambient PAH and those in cigarettesmoke. One explanation for the apparent higher PAH-DNA adduct formation in smokers is that the intake of aPAH mixture can result in enhanced DNA adduct fonma-tion from specific carcinogens within the mixture, e.g.,other components of the mixture may serve to enhancethe metabolic pathway involved in forming specific ne-active intermediates. Results of in vitro experiments withhamster embryo cell cultures by Smolanek et a!. (40)suggested that pretreatment of B(a)P induces a specificisozyme of cytochnome P-450, CYP1A1, increasing theproportion of B(a)P metabolized to the most reactivemetabolite, anti-BPDE. In a similar fashion, the inductionof CYP1A1 may also increase the proportion of PAHconverted to phenobic metabolites. CuIp and Beland (41)showed that total-DNA binding was greaten in coal tar-fed mice than in mice fed B(a)P alone, and the adductbevels decreased in the following order: lung > liver >

fonestomach. Hughes and Phillips (42) found that a mix-tune of dibenzo[a,e]pyrene and B(a)P resulted in DNAbinding 65% higher than that expected from the bindinglevels of the PAH when applied separately. On the otherhand, when d ibenzo[a, 11pynene, d i benzo[a,elpynene,and B(a)P were applied together to mouse skin, the totalbinding was 35% lower than expected (42). These resultsindicate that there are tissue-specific differences in theactivation of PAH components in a mixture whencompared to a specific compound contained within themixture.

We also studied skin contamination in a small group ofworkers from the anode paste factory (n = 8), the bake oven(n = 5), and the pot-relining department (n = 7) and the



76 PAH-DNA Adducts

1 7. Schoket, B., Phillips, D. H., Poirier, M. C., and Vincze, I. DNA adductsin peripheral blood lymphocytes from aluminum production plant workers

results are reported elsewhere (43). The dermal exposure ofpyrene and B(a)P was monitored at three sites, forearm,neck, and upper leg, using exposure pads as pseudoabsonb-ing skin. The daily dermal dose was almost 5 times higherthan the daily inhaled dose, 395 and 83 pg pyrene, respec-tively, pen 8-h working period. The correlation betweenurinary 1 -hydroxypyrene and pynene in air was equal to orlower than the correlation between 1 -hydroxypyrene andpynene on skin pads. We concluded that denmal exposurecontributes substantially to the body burden of pyrene andthat dermal absorption may be an important modifyingfactor of the relation between air concentration and biomar-kens of PAH. The scale of sampling of dermal exposure wastoo small to be a factor in the statistical analysis of thisstudy.

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1995;4:69-77. Cancer Epidemiol Biomarkers Prev F J van Schooten, F J Jongeneelen, M J Hillebrand, et al. smoking.workers: relation with job category and synergistic effect ofcell DNA and 1-hydroxypyrene in the urine from aluminum Polycyclic aromatic hydrocarbon-DNA adducts in white blood

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