Occupational and Environmental Medicine 1997;54:854-860

Assessment of potential damage to DNA in urineof coke oven workers: an assay of unscheduledDNA synthesis

F Roos, A Renier, J Ettlinger, Y Iwatsubo, M Letourneux, JM Haguenoer, M C Jaurand,J C Pairon

Institut National de laSante et de laRecherche Medicale(INSERM) Unit6 139,IM 3, Faculte deM6decine, 8 rue duGeneral Sarrail, 94010Cr6teil cedex, FranceF RoosA RenierY IwatsuboM C JaurandJ C Pairon

Service M6dical duTravail, Sollac,Dunkerque, FranceJ Ettlinger

InstitutInteruniversitaire deMedecine du Travailde Paris, Dle de France,Paris, FranceY IwatsuboJ C Pairon

Institut de M6decinedu Travail de BasseNormandie, Caen,FranceM Letourneux

Institut de Medecinedu Travail du Nord,Lille, FranceJM Haguenoer

Correspondence to:Dr JC Pairon, INSERMUnite 139 IM3,Faculty de Medecine,8 rue du General Sarrail,94010 Criteil cedex, France.Tel: 0033 149 81 36 56;Fax: 0033 149 81 35 33

AbstractObjectives-A study was conducted incoke oven workers to evaluate the biologi-cal consequences of the exposure of theseworkers, particularly production ofpoten-tial genotoxic factors.Methods-60 coke oven workers and 40controls were recruited in the same ironand steel works. Exposure to polycyclicaromatic hydrocarbons (PAHs) was as-

sessed by job and measurement of1-hydroxypyrene (1OHP) in urine sam-

ples. An unscheduled DNA synthesis assay

was performed on rat pleural mesothelialcells used as a test system to evaluate theeffect of the workers' filtered urine on theDNA repair capacity of rat cells todetermine whether DNA damaging agentsare present in the urine ofthese workers.Results-Urinary concentrations of

1OHP ranged from 0.06 to 24.2 (mean(SD) 2.1 (3.6)) jImolimol creatinine inexposed coke oven workers, and from 0.01to 0.9 in controls (0.12 (0.15)). These highconcentrations in coke oven workers re-flected recent exposure to PAHs and werein agreement with the assessment ofexpo-sure by job. No significant difference wasfound between coke oven workers andcontrols in the DNA repair level of ratcells treated with urine samples. However,the rat cell repair capacity decreased withincreasing 1OHP concentrations in theexposed population (r= -0.28, P< 0.05).Conclusions-As high concentrations of1OHP were found in the urine of some

workers, a more stringent control ofexpo-sures to PAHs in the workplace is re-

quired. Exposure to PAHs was notassociated with a clear cut modification ofthe urinary excretion of DNA damagingfactors in this test, as shown by theabsence of increased unscheduled DNAsynthesis in rat cells. However, impair-

ment of some repair mechanisms byurinary constituents is suspected.

(Occup Environ Med 1997;54:854-860)

Keywords: unscheduled DNA synthesis; polycyclic aro-

matic hydrocarbons; occupational exposure

Coke production entails exposure ofworkers tohigh environmental concentrations of airbornepollutants including polycyclic aromatic hydro-

carbons (PAHs). Both epidemiological andexperimental evidence indicate that PAHs arecarcinogenic in animals and possibly also inhumans. Several PAHs-for example, diben-z(a,h)anthracene, benzo(a)pyrene-have beenshown to induce tumours in numerous testspecies.' Exposure to PAHs has long beenassociated with an excess of various cancers inhumans, mainly skin cancers and scrotalcancers, particularly among chimney sweeps.' 2An increased risk of lung cancer has beenreported among workers involved in variousindustries such as chimney sweeping, alu-minium refining, coal gasification, coke pro-duction, and in iron and steel foundries.3"These compounds are produced during in-complete combustion of natural or syntheticorganic materials; they are lipid soluble and canbe absorbed into the body through all of theusual routes. Skin and lung routes are predomi-nant in occupational exposure."6 The level ofexposure to PAHs in the workplace may beevaluated by atmospheric measurement ofvarious PAHs or by measurement of PAHmetabolites in urine.7 8 Thus, the hydroxylatedmetabolite of pyrene in urine (1-hydroxy-pyrene, 1OHP) is considered to be a good bio-logical indicator of exposure to PAHs whichhas the advantage of taking into account thevarious routes of penetration into the body.

Evaluation of the early consequences ofhuman exposure to carcinogens in the work-place is an important issue. This has led to thedevelopment of various assays designed toidentify early biological responses.9 Recently,many studies have tried to evaluate the biologi-cally effective dose by measuring various mark-ers as surrogates for the target tissue dose-namely, urinary d-glucaric acid as an index ofhepatic enzyme activity," " measurement ofsister chromatid exchanges in peripherallymphocytes,"-" and binding of PAH elec-trophilic metabolites to DNA in nucleatedblood cells or to blood proteins." 15-20 Most ofthese studies were performed in coke ovenworkers,'0 12-14 1819 graphite electrode plantworkers,'0 12 14 workers exposed to PAHs frombitumen fumes,"1 and primary aluminiumplant and foundry workers.'5 20 These studiesshowed contradictory results. Some of themdid not show any difference between biologicaldata obtained in subjects exposed to PAHs andin controls -for example, d-glucaric acid,'""sister chromatid exchanges in peripherallymphocytes," antibody to benzo(a)pyrene

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DNA adducts,'9 and benzo(a)pyrene-albuminadducts.'5 Other studies reported a significantdifference between exposed subjects andcontrols for some markers-for example,sister chromatid exchanges in peripherallymphocytes,'1 12 "4benzo(a)pyrene-diol-epoxide(BPDE) adducts to haemoglobin,'2 PAH-DNAadducts,13 17 20 and anti-BPDE((±)-r-7,t-8-dihydroxy-t-9, 1 0-oxy-7,8,9, 1 0-tetrahydroben-zo(a) pyrene)-DNA adducts." A few authorsused urinary mutagenicity-for example, theAmes test, modified nucleosides-to investi-gate whether body fluids contain factors with a

genotoxic potential as a result of occupationalexposure.'0 12 21 In these studies, the urinarymutagenic activity was more likely dependenton smoking habits than on occupational expo-

sure. Nevertheless, a significant effect of occu-pational exposure was also shown with suchassays when tobacco smoking and diet were

taken into account.In the present work, we performed an

unscheduled DNA synthesis assay on rat pleu-ral mesothelial cells used as a test system, toevaluate the DNA damaging potency of urineof coke oven workers. Rat pleural mesothelialcells have been shown to be able to metabolisePAH, indicating P-450 metabolic activity ofthese cells.22-24 The unscheduled DNA synthe-sis assay has been previously performed withthe same cell type in chromium workers andcoal miners.2526 In this assay, growth arrestedrat cells showed a basal repair rate correspond-ing to the repair of spontaneously occurringlesions. An enhancement of DNA repairindicates the presence of genotoxic agents pro-

ducing DNA damage. This repair can bedetected by an increase in the amount of radi-olabelled nucleoside incorporated into theDNA. By contrast, an impairment of repairmechanisms is associated with a decrease in[3H] methyl thymidine ([3H]dThd) incorpora-tion into DNA.

Materials and methodsSUBJECTSUrine samples were collected from 60 malecoke oven workers (group 1) and 40 male con-

trols recruited in the same steel foundry inBasse Normandie, France. Each subject was

asked to fill in a questionnaire providing detailson tobacco smoking, alcohol habits, drug con-

sumption, medical history, and work condi-tions. For 40 of the 60 coke oven workers, a

second urine sample was collected threemonths later, after the end ofexposure once thefactory was closed. These subjects are includedin group 2. Two sets of data were obtained inthis group; one provided by urine samples col-lected during the working period (group 2a),the other one supplied by samples collectedafter the end of exposure (group 2b). No sub-ject had any known active disease at the time ofthe study. Subjects receiving chemotherapy,antibiotics, or known metabolic inducers were

excluded. Blue collar controls with the same

mean age (± 2 years) were selected on the basisofthe absence ofnotable previous occupationalexposure to carcinogens, as assessed by the

occupational questionnaire. They had similarsmoking habits to those of subjects of group 2.

URINE SAMPLESUrine samples were collected in polyethyleneflasks at the end of the workshift, after at leastfive consecutive days of work for group 1 sub-jects and during routine occupational medicalsurvey for group 2b subjects and controls. Thesamples were frozen at - 20'C and kept in thedark until analysis.

EVALUATION OF EXPOSURE TO PAHSTwo methods were used: an assessment ofexposure by job by the occupational physician;and measurement of urinary 1-hydroxypyrene(1OHP).To assess PAH exposure by job, the occupa-

tional physician used a standardised question-naire. Each worker was asked to list his succes-sive jobs throughout his working life, withspecific attention being paid to the tasksperformed during the week preceding urinecollection. This allowed the level of exposure toPAHs to be evaluated, based on concentrationsof atmospheric PAHs registered between 1986and 1990. Atmospheric measurements ofPAHs were performed by gas chromatographyafter collection of individual dynamic samples(during a half shift of four hours) and staticsamples. This study showed that subjectsworking on top or near the door of the oven(atmospheric benzo(a)pyrene concentrationgenerally >10 jg/m3) presented a high level ofexposure (class 1), subjects working on the sideof the coke oven (atmospheric benzo(a)pyreneconcentration generally 1-10 jg/m3) wereexposed to intermediate levels (class 2) andsubjects with no direct contact with the ovenarea (atmospheric benzo(a)pyrene level <1gg/m') were considered to be exposed to lowlevels (class 3). Two exposure indices wereestablished for each subject: the "last dayexposure", corresponding to the class of expo-sure of the last workshift preceding urinecollection; the "week index", obtained by add-ing the individual scores of all workshifts of theweek for a given subject (individual scores ofclass 1, class 2, and class 3 were 3, 2, and 1,respectively; the week index for a given cokeoven worker therefore ranged from 5-15).Urinary 1OHP was determined by an adap-

tation of the method ofJongeneelen et al.7 Thetechnique included enzymatic hydrolysis of theconjugates with a mixture of glucuronidase andsulphatase followed by chromatography onoctadecyl silica cartridges (Bond-Elut 500 mg,Analytichem, Harbor City, CA, USA) per-formed with a Vac Elut SPS 24 from Varianpreviously activated with methanol and dis-tilled water. Samples were then extracted withmethanol, dried with an evaporator, and thenresuspended in 200 gl acetonitrile. A secondchromatography was performed on a Nucleosil10 C18 column with an isocratic elution(distilled water: acetonitrile; 60:40 v/v) at aflow rate of 1 ml/min. The high performanceliquid chromatography system was 9000 Var-ian including two 9010 Varian pumps. Theretention time of 1OHP was found to be six

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minutes. 1-Hydroxypyrene concentrations weremeasured by fluorimetry (excitation 240 nm,emission 390 nm, 821-FP JASCO spectrofluor-ometer). The procedure was regularly cali-brated with aliquots of 0.1 gg/l to 20.0 jig/l of1OHP in the elution medium (Aldrich, Paris).An external standard was regularly chromato-graphed to assess the validity of the procedure.The detection limit was 0.1 jig/l and the resultswere standardised to urinary creatinine.

UNSCHEDULED DNA SYNTHESIS ASSAYRat pleural mesothelial cells were cultured in24 well cluster culture dishes (Costar).24 Eightythousand cells were seeded per well incomplete medium. The cells grew as a monol-ayer and reached confluence after four days ofincubation; the medium was then replaced withRoswell Park Memorial Institute (RPMI)medium containing 1% fetal calf cells (5 mM,hydroxy urea (HU) 100 U/ml penicillin, and 50jg/ml streptomycin. The cells were incubatedfor 24 hours at 370C in a humidifiedatmosphere of 5% CO2 in air then incubatedfor 24 hours in RPMI medium containing 1%fetal calf cells (5 mM HU) and 4 iCi/ml['H]dThd (specific activity: 50 to 60 Ci/mmol),with or without urine. A laboratory reference(an RPMI medium containing 500 gg/mlcrocidolite fibres) was used to check theresponse of rat cells. Crocidolite fibres producea considerable enhancement ofDNA repair inrat cells due to DNA damage.'4 The urinesamples were previously filtered through Ami-con membrane filters YM 10 according toEmerit et at!7 and the volume added to eachwell was adjusted to initial urinary creatinine(maximum volume 20 il). The cells were thenwashed three times with phosphate bufferedsaline. Acid soluble material was removed byrinsing the cell layer with 10% cold trichloro-acetic acid for 10 minutes, followed by incuba-tion in a mixture of 0.2 M NaOH and 1%sodium dodecyl sulphate. Aliquots (200 ptl)were placed in vials and scintillation fluid(Ultima Gold MV, Packard) was added.Radioactivity was determined on a BeckmanLS 6000SC apparatus. The DNA cell contentwas assessed by fluorometric assay."8 Fluores-cence was measured with a Millipore Cytofluor2350. The cell repair capacity was evaluatedfrom the ratio of [3H]dThd incorporation(dpm)/,ug DNA in each well. Results of theunscheduled DNA synthesis assay were ex-pressed as the ratio of the repair capacity of ratcells treated with urine to that ofuntreated cellsin RPMI medium.

MTT ASSAYThe purpose of this test was to control theviability of rat cells after treatment with urinesamples. The 3-[4,5-dimethylthiazol-2-YL]-2,5-diphenyltetrazolium bromide (MT) isreduced into formazan by mitochondrial dehy-drogenases. This reduction can only occur inactive cells. The quantity of formazan isdirectly proportional to the number of viablecells.'9 Rat pleural mesothelial cells werecultured in 96 well cluster culture dishes (Cos-tar) in complete medium. After confluence, the

cells were treated with the filtered urinesamples, or crocidolite, or cultured in RPMImedium, as in the unscheduled DNA synthesisassay. Five wells were used for each treatment.The cells were incubated for 24 hours underthe same conditions as for the unscheduledDNA synthesis assay. To each well MTT solu-tion (40 ,tl, 2 mg/ml) was then added. Afterthree hours of incubation (37°C in a humidi-fied atmosphere, 5% CO2), the supernatantwas carefully removed and the formazancrystals were dissolved by addition of 200 ,uldimethyl sulphoxide. The cluster culture disheswere agitated for two minutes with a multidishagitator and the optical density was measuredby spectrophotometry at 540 nm. The meanoptical density of the five replicate wells wascalculated for each treatment. Results wereexpressed as the ratio of the mean obtained foreach group to the mean obtained for samples inthe same dish in RPMI medium (opticaldensity in urine treated rat cells/ optical densityin untreated rat cells).

DETERMINATION OF COTININE IN URINE SAMPLESCotinine is a metabolite of nicotine, which canbe used to evaluate the last two days ofindividual nicotine intake in smokers, takinginto account various parameters, such astobacco strength, duration of inhalation, fil-tered or unfiltered cigarettes. Cotinine wasevaluated by an automated colorimetricmethod on COBAS-BIO with 1% barbituricacid derived from the manual method of Peachet al."0 Results were expressed in relation tourinary creatinine.

STATISTICAL ANALYSIS OF RESULTSThe relation between PAH exposure and cellrepair capacity was examined by two methods:comparison of the ['H]dThd incorporation /DNA ratio between exposure groups; and cor-relation of this ratio with urinary 1OHP.Three types of comparisons were carried out

between exposure groups: coke oven workersduring exposure versus controls (group 1 vcontrols); coke oven workers after the end ofexposure versus controls (group 2b v controls);coke oven workers during exposure versus cokeoven workers after cessation of exposure(groups 2a v 2b). Coke oven workers and con-trols were compared by x' test and Student's ttest for univariate analysis. A paired t test wasused to examine the variables of coke ovenworkers before and after the end of exposure.

Similar comparisons were carried out for theMTT test.

Relations between urinary 1OHP and otherbiological variables were examined by Pear-son's correlation coefficients and adjustmentfor tobacco smoking was made with urinarycotinine by multiple linear regression.

All statistical analyses were performed withthe SAS package.'

ResultsTable 1 shows the characteristics of thesubjects included in the study (age, smokinghabits) and the urinary cotinine and creatinineof these subjects. Coke oven workers and con-

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Table 1 Sociodemographic characteristics of the study population and biological indices of tobacco smoking

Coke oven workers Controls

Variables Group 1 (n=60) Group 2a (n=40) Group 2b (n=40) (n=40)

Age (y):Mean (SD) 40.9 (8.6) 43.2 (6.3) 41.7 (8.8)Median (range) 43 (19-51) 44.5 (28-51) 44.5 (21-51)

Smoking habits:Current smokers (n (%)) 30 (50) 16 (40) 18 (45)Former smokers (n (%)) 16 (26.7) 13 (32.5) 11 (27.5)Non-smokers (n (%)) 14 (23.3) 11 (27.5) 11 (27.5)

Urinary creatinine (mmol/1):Mean (SD) 14.1 (6.7) 13.8 (6.7) 16.5(8.9) 11.7 (0.6)Median(range) 13.9 (1.2-32.4) 13.5 (3.5-32.4) 15.9(5.8-45.1) 11.3 (2.7-24.7)

Urinary cotinine (mmol/mol creatinine):Mean (SD) 2.7 (3.1) 2.0 (2.5) 2.0(2.6) 3.2 (4.3)Median (range) 1.1 (0-10.9) 0.5 (0-8.8) 0.5(0-8.6) 0.9 (0-18.3)

trols were similar for age, smoking, and alcoholhabits. Twenty per cent of subjects consumeddrugs. No significant difference was foundbetween coke oven workers and controls forurinary variables, except in group 2b, in whichurinary creatinine was higher than in controls.The urinary cotinine concentration was con-

sidered to reflect current smoking consump-tion, whereas 1OHP was considered to berepresentative of exposure to PAHs. Coke ovenworkers had higher urinary 1OHP concentra-tions than controls, even after adjustment forurinary cotinine (fig 1). After three monthswithout occupational exposure, the urinary1OHP concentrations were similar in cokeoven workers and in controls. There was a goodagreement between urinary 1OHP values andPAH exposure levels as defined by theassessment of exposure by job. Coke ovenworkers with high exposure (class 1) during thelast shift before urine collection had higher1OHP concentrations than less exposed sub-jects (class 2 and class 3 subjects), even aftertaking urinary cotinine concentration intoaccount (median values, mean (SD) 3.7 (5.6(6.5)), 1.3 (1.7 (1.2)) and 0.6 (1 (0.8)) pmol1OHP/mol creatinine for class 1, class 2, andclass 3 subjects, respectively; P<0.001). Theurinary 1OHP value was strongly correlatedwith the week index (r=0.52; P=0.001), evenwhen smoking habits were taken into account.

Table 2 shows the results of DNA repaircapacity and viability (MTT test) of rat cellstreated with urinary filtrates. No significantdifference was found for dpm/DNA valuesbetween group 1 workers and controls, orbetween coke oven workers before (group 2a)and after the end of exposure (group 2b)regardless of smoking status (fig 2). Similarresults were found when analysis was restrictedto comparison between subjects with the high-est level of exposure and controls. This lack ofsignificant change was not due to a dysfunctionof rat cells used as a test system, as ['H]dThdincorporation was higher in the cells treatedwith crocidolite (mean (SD) = 8420 (751)dpm/ng) than in the untreated cells (mean(SD) = 7687 (1079) dpm/pg) (P<0.001), asexpected.The viability of rat cells treated with urine

samples from coke oven workers was slightlyaltered (although not significantly; P=0.08) incomparison with urine from controls. Thisphenomenon was found both for samples

collected during working activity and afterthree months without occupational exposure.Viability of rat cells treated with crocidolite wasdecreased to 89.9% compared with untreatedrat cells (P<0.00 1).The correlation between DNA repair capac-

ity of rat cells and individual biologicalvariables showed a reduction of incorporationof ['H]dThd which depended on the urinary1OHP concentration in G1 workers (r= - 0.28,P= 0.05). This reduction cannot be attributedto a loss of cell viability, as the viability of ratcells was not significantly modified by urinetreatment. Moreover, crocidolite reduced the

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Figure 1 Determination of urinary IOHPI Each opencircle represents the individual value of urinary 1OHP of agiven subject. Black horizontal bar represents median foreach group of workers. 1OHP = 1 hydroxypyrene. G1=60coke oven workers, urine collection during the workingperiod; G2=a second urine sample was collected threemonths after closure of the factory in 40 of the 60 coke ovenworkers; G2a=samples collected during the working period;G2b=samples collected after the end of exposure; C=bluecollar controls. *p<O. 05; ***p<O. 001.

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Figure 2 DNA repair in rat pleural mesothelial cellstreated with urinefrom coke oven workers or blue collarcontrols according to smoking habits. Results of theunscheduled DNA synthesis assay were expressed as theratio of the repair capacity of rat cells treated with urinefrom coke oven workers or controls to that of untreated cells

(RPMI medium control). Gl =60 coke oven workers, urinecollection during the working period; G2=a second urinesample was collected three months after closure of thefactory in 40 of the 60 coke oven workers; G2a=samplescollected during the working period; G2b=samples collectedafter the end of exposure; C=blue collar controls;CS=current smokers; FS=former smokers; NS=non-smokers. There was no significant difference for dpmlDNAvalues between groups of workers, whatever the smokingstatus.

cell viability but enhanced DNA repair. Bycontrast, there was no correlation betweenDNA repair capacity of rat cells and urinarycotinine (r= - 0.20, NS), or between DNArepair capacity and results of viability of ratcells (r= - 0.06, NS) in group 1 subjects. TheDNA repair capacity of rat cells was not corre-

lated with any of these variables in group 2bsubjects or in controls.No correlation between rat cells viability and

urinary cotinine or 1OHP concentrations was

found in any of the groups.

DiscussionThis study was designed to assess whether uri-nary filtrates from subjects with various levelsof occupational exposure to PAH were able toinduce DNA damage or modify DNA repairprocesses in rat cells used as a test system. Anenhancement of DNA repair would indicatethat the urine filtrates contain clastogenic mol-ecules; a decrease in DNA repair would suggestthe presence of compounds able to impairrepair processes.Exposure to PAHs was evaluated according

to the levels of an internal dose indicator,urinary 1OHP, and by an assessment of expo-sure by job by the occupational physician.1-Hydroxypyrene is a non-mutagenic metabo-

lite which can be found in the urine of humansexposed to PAHs. Its concentration in biologi-cal fluids cannot be used to evaluate the carci-nogenic risk for exposed workers, which prob-ably depends on the various PAH profiles ofpitch and tar volatiles polluting the work envi-ronment. However, because of the very goodcorrelation between PAH concentrations in thework environment and urinary 1OHPconcentrations,.2.34 this metabolite has beenproposed as a biological indicator of occupa-

tional exposure to PAHs.8 35 Moreover, thismetabolite shows exposure to pyrene inde-pendently of the route of exposure.32 36 Thevalues of urinary 1OHP found in the presentstudy were in the range 0.01-24.2 jmol/molcreatinine and are consistent with thosereported by other authors in workers withsimilar occupations.8 12 21 32 Moreover, a fairlygood correlation was found between 1OHPand the week exposure index, confirming thatthis variable is a good marker of recentexposure. The urinary 1OHP concentrationswere also lower three months after the end ofoccupational exposure and were similar tocontrol concentrations. This is in agreementwith the half life of this metabolite, which isabout 18 hours.8 The urinary 1OHP concen-

tration is considered to be influenced by smok-ing habits only in the case of low occupationalexposure to PAHs.8 36-38 Thus, as some of theexposed workers had a low exposure during theweek preceding collection of the urine sampleand as smoking habit is a confounding factor,we selected controls with similar smoking hab-its. This allowed evaluation of the true effect ofoccupational exposure on the DNA repaircapacity of rat cells treated with urine fromthese workers. The effect of diet, particularlyfried or grilled meat, may affect the urinaryexcretion of 1OHP.'6 39 Although eating behav-iour was not controlled in our study, results of1OHP were consistent with the evaluation ofthe level of exposure of the last workshift, as

assessed by job by the occupational physician.We therefore consider that diet had a minimalinfluence in our population compared with thatof occupation.The DNA repair assay has been previously

performed on rat cells with concentrated urinesamples from subjects exposed to suspected or

known genotoxic agents.25 26 A clastogenicactivity was shown in rat cells treated withurine from chromium workers and patientsreceiving radiotherapy with or without chemo-therapy for cancer.25 Emerit et a!27 40 41 showedthe presence of clastogenic factors in variousbiological samples (plasma, synovial fluid, cer-

ebrospinal fluid) from patients with congenital

Table 2 Mean (SD) values ofDNA repair and viability of rat cells treated with urinefiltrates

Coke oven workersGroup I Group 2a Group 2b Controls

Variable (n=60) (n=40) (n=40) (n=40)

DNA repair:['IH]dMd incorporation (dpm) 0.89 (0.10) 0.89 (0.10) 0.90 (0.09) 0.91 (0.11)DNA (lsg) 0.99 (0.07) 1.0 (0.07) 0.98 (0.07) 0.97 (0.07)[H]dTMd incorporation/DNA (dpm/4Lg DNA) 0.90 (0.13) 0.90 (0.13) 0.92 (0.13) 0.95 (0.14)

Viability (MIT test) OD (540 nm) 0.94 (0.12) 0.93 (0.14) 0.92 (0.12) 0.99 (0.13)

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breakage syndromes such as ataxia telangiecta-sia and Bloom's syndrome, as well as chronicinflammatory diseases such as rheumatoidarthritis, systemic lupus erythematosus, andprogressive systemic sclerosis. Most of theclastogenic factors isolated to date have amolecular weight lower than 10 000 Da-suchas 4-hydroxynonenal (150 Da) or inosinetriphosphate (388 Da). Our urine filtrationprocedure allowed collection of these factors inour filtrates. Incorporation of ['H]dThd wassignificantly enhanced in cells treated with cro-cidolite, compared with untreated cells, attest-ing that our test system responded accordingly.Results obtained with urine samples from cokeoven workers were unexpected, as the resultsobtained by Pilliere et at25 with urine samplesfrom chromium workers suggested that thisurine enhanced DNA repair in rat cellsindicated the presence of clastogenic factors inurine as in other fluids.40 By contrast, in ourstudy, we found a tendency for less incorpora-tion of ['H]dThd in the cells treated with urinesamples from exposed coke oven workers when1OHP was increased. These results cannot beexplained by smoking habits (as assessed byurinary cotinine), or by urinary concentration,as the volume was adjusted in each wellaccording to the initial concentration. Animpairment of cell viability is also unlikely.The different results obtained for DNA

repair capacity of rat cells treated with urinefrom chromium workers and from coke ovenworkers suggests that subjects exposed todifferent types of carcinogens excrete differentcompounds in urine. Urine samples frompopulations exposed to chromium derivativesmight therefore enhance DNA repair of ratcells as a result of DNA breakage, whereasother urine samples might decrease the cell'sability to repair DNA. This tendency was sug-gested by the results obtained by Knudsen etal.42 These authors showed that unscheduledDNA synthesis in peripheral lymphocytesinduced by N-acetoxy-N-acetylaminofluorenewas decreased in welders compared with a ref-erence population. Exposure to mutagenic orcarcinogenic agents may therefore lead to theformation of factors inhibiting DNA repair, aneffect that might account for the accumulationof errors in DNA integrity.Although the biological response found in

the DNA repair assay performed on rat cellstreated with urine filtrates from coke ovenworkers was only moderate, urinary 1OHPconcentrations indicated that exposure toPAHs was excessive in this population. Urinefrom coke oven workers also exhibited someinhibitory effect on the DNA repair capacity ofrat cells. More stringent control of exposure istherefore required in the workplace.

We are indebted to the coke oven workers who agreed toparticipate in the study, to Anne Philibert and Chantal Cordierfor their helpful contribution in the collection of biological sam-ples, to Francois Bureau for his contribution to themeasurement of creatinine, to Gaspard Gohin for his participa-tion in preparation of urine samples, to Marie-Claudine Jacque-mont for her contribution to measurement of urinary 1OHP,and to Dr Claude Philippon for urinary cotinine measurement.This work was supported by a grant from the Service des EtudesMedicales from EDF-GDF, Paris, France.

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3 International Agency for Research on Cancer. Polynucleararomatic compounds, industrial exposures in aluminiumproduction, coal gasification, coke production, and ironand steel foundries. IARC Monogr Eval Carcinog Risks Hum1984;34:101-31.

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5 Van Rooij JGM, Van Lieshout EMA, Bodeliere-Bade MM,Jongeneelen FJ. Effect of the reduction of skin contamina-tion on the internal dose of creosote workers exposed topolycyclic aromatic hydrocarbons. Scand J Work EnvironHealth 1993;19:200-7.

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14 Van Hummelen P, Gennart JP, Buchet JP, Lauwerys R,Kirsch-Volders M. Biological markers in PAH exposedworkers and controls. Mutat Res 1993;300:231-9.

15 Omland 0, Sherson D, Hansen AM, Sigsgaard T, AutrupH, Overgaard E. Exposure of iron foundry workers to poly-cyclic aromatic hydrocarbons: benzo(a)pyrene-albuminadducts and 1-hydroxypyrene as biomarkers for exposure.Occup Environ Med 1994;51:513-8.

16 Ferreira Jr M, Tas S, Dell'Omo M, Goormans G, Buchet JP,Lauwerys R. Determinants of benzo(a)pyrenediol epoxideadducts to haemoglobin in workers exposed to polycyclicaromatic hydrocarbons. Occup Environ Med 1994;51:451-5.

17 Perera FP, Dickey C, Santella R, O'Neill JP, Albertini RJ,Ottman R, et al. Carcinogen-DNA adducts and gene muta-tion in foundry workers with low-level exposure to polycy-clic aromatic hydrocarbons. Carcinogenesis 1994;15:2905-10.

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23 Buard A, Beaune PH, Renier A, Jaurand MC, Bignon J,Laurent P. Expression of cytochrome P450 in rat pleuralmesothelial cells in secondary cultures J Cell Physiol 1994;160:176-84.

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All manuscripts submitted to Occup EnvironMed should conform to the uniform require-ments for manuscripts submitted to biomedi-cal journals (known as the Vancouver style.)

Occup Environ Med, together with manyother international biomedical journals, hasagreed to accept articles prepared in accord-ance with the Vancouver style. The style(described in full in the JAMA [1]) is intendedto standardise requirements for authors, and isthe same as in this issue.

References should be numbered consecu-tively in the order in which they are first men-tioned in the text by Arabic numerals on theline in square brackets on each occasionthe reference is cited (Manson[1] confirmedother reports [2] [3] [4] [5]). In future ref-erences to papers submitted to Occup EnvironMed should include: the names of all

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1 International Committee of Medical Journal Editors.Uniform requirements for manuscripts submitted tobiomed journals. JAA'A 1993;269:2282-6.

2 Soter NA, Wasserman SI, Austen KF. Cold urticaria:release into the circulation of histmaine and eosinophilchemotactic factor of anaphylaxis during cold challenge.N Engl _t Med 1976;294:687-90.

3 Weinstein L, Swartz MN. Pathogenic properties of invad-ing micro-organisms. In: Sodeman WA Jr, Sodeman WA,eds. Pathologic physiology, mechanisms of disease. Philadel-phia: W B Saunders, 1974:457-72.

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