RADIATION RESEARCH 158, (2002) /02 $ by Radiation Research Society. All rights of reproduction in any form reserved.

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1 RADIATION RESEARCH 58, (22) /2 $5. 22 by Rdition Reserch Society. All rights of reproduction in ny form reserved. Three Somtic Genetic Biomrkers nd Covrites in Rdition-Exposed Russin Clenup Workers of the Chernobyl Nucler Rector 6 3 Yers fter Exposure Irene M. Jones,, Hether Glick, Pul Kto, Richrd G. Lnglois, Mortimer L. Mendelsohn, Glori A. Murphy, Pvel Pleshnov, b Mrilyn J. Rmsey, Cynthi B. Thoms, Jmes D. Tucker, Ludmil Turev, c Irin Vorobtsov d nd Dvid O. Nelson Biology nd Biotechnology Reserch Progrm, Lwrence Livermore Ntionl Lbortory, Livermore, Cliforni; b Applied Ecology Reserch Lbortory, Ministry of Helth of Russi, Moscow, Russi; c Tul Dignostic Center, Tul, Russi; nd d Centrl Reserch Institute for Roentgenology nd Rdiology, Ministry of Helth of Russi, St. Petersburg, Russi Jones, I. M., Glick, H., Kto, P., Lnglois, R. G., Mendelsohn, M. L., Murphy, G. A., Pleshnov, P., Rmsey, M. J., Thoms, C. B., Tucker, J. D., Turev, L., Vorobstov, I. nd Nelson, D. O. Three Somtic Genetic Biomrkers nd Covrites in Rdition-Exposed Russin Clenup Workers of the Chernobyl Nucler Rector 6 3 Yers fter Exposure. Rdit. Res. 58, (22). Three somtic muttion ssys were evluted in men exposed to low-dose, whole-body, ionizing rdition. Blood smples were obtined between 992 nd 999 from 625 Russin Chernobyl clenup workers nd 82 Russin controls. The ssys were chromosome trnsloctions in lymphocytes detected by FISH, hypoxnthine phosphoribosyltrnsferse (HPRT) mutnt frequency in lymphocytes by cloning, nd flow cytometic ssy for glycophorin A (GPA) vrint frequency of both deletion (N/Ø) nd recombintion (N/N) events detected in erythrocytes. Over 3 exposure nd lifestyle covrites were vilble from questionnires. Among the covrites evluted, some incresed (e.g. ge, smoking) nd others decresed (e.g. dte of smple) biomrker responses t mgnitude comprble to Chernobyl exposure. When djusted for covrites, exposure t Chernobyl ws sttisticlly significnt fctor for trnsloction frequency (increse of 3%, 95% CI of % 53%, P.2) nd HPRT mutnt frequency (increse of 4%, 95% CI of 9% 66%, P.), but not for either GPA ssy. The estimted verge dose for the clenup workers bsed on the verge increse in trnsloctions ws 9.5 cgy. Trnsloction nlysis is the preferred biomrker for low-dose rdition dosimetry given its sensitivity, reltively few covrites, nd dose response dt. Bsed on this estimted dose, the risk of exposure-relted cncer is expected to be low. 22 by Rdition Reserch Society INTRODUCTION A criticl element in ssessing the potentil for helth risk fter exposure to rdition is n estimte of the dose nd the biologicl effectiveness of the exposure. This need is especilly difficult to meet when the exposure is low, time hs pssed since the exposure, nd physicl dosimetry is limited or unvilble. The exposure of hundreds of thousnds of people during the continment nd clenup effort fter the Chernobyl nucler power ccident poses chllenge nd n opportunity. This report presents n extensive comprison of the bility of three somtic genetic biomrkers to detect nd estimte effects of rdition exposure nd other covrites in Russin Chernobyl clenup workers. Exposure to rdition cuses mny types of dmge to the genetic mteril. Filure of cells to repir this dmge completely nd ccurtely results in chromosome nd gene ltertions. The extent of these consequences is relted to the dose, to the individul s inherited cpcity to repir ech type of dmge, nd to the error-prone nture of some repir processes. An underlying ssumption of somtic genetic biomrker studies is tht risk of cncer rises with incresing genetic ltertions in somtic cells. Prospective studies ssociting frequency of chromosoml ltertions in peripherl blood lymphocytes with cncer risk hve recently provided support for this ssumption ( 4). Biomrkers tht monitor the frequency of somtic cell gene muttions nd chromosome berrtions, the two mjor types of genetic ltertions ssocited with progression towrd cncer, hve been developed nd used in number of popultion studies. Severl excellent overviews of the genetic ltertions ssyed, the cell types used, nd exmples of ssy usge re vilble (5 9). Author to whom correspondence should be ddressed t Biology nd Biotechnology Reserch Progrm, Lwrence Livermore Ntionl Lbortory, L-44, P.O. Box 88, Livermore, CA 9455; e-mil: jones2@llnl.gov. 424

2 SOMATIC BIOMARKERS IN CHERNOBYL CLEANUP WORKERS 425 A brief summry of studies of somtic genetic biomrkers in rdition-exposed popultions plces the current study in context. Studies of tomic bomb survivors provided dt on responses mesured decdes fter exposure. The vilbility of reconstructed doses for tomic bomb survivors hs been invluble for the interprettion of biomrker studies. Extensive studies of chromosome berrtions, by rnge of methodologies pplied over the yers, indicte tht this end point hs chrcteristic dose response nd high sensitivity s reporter, even decdes fter exposure (, ). Assys of HLA, T-cell receptor nd hypoxnthine phosphopribosyltrnsferse (HPRT) muttion in tomic bomb survivors lymphocytes tended to hve low signl when mesured decdes fter exposure [reviewed in refs. (8, 2)]. The ssy for glycophorin A (GPA) vrint cell frequency using erythrocytes detected dose response in tomic bomb survivors (3, 4) similr to tht seen fter more recent exposures [cf. Chernobyl (5); Goiâni 37 Cs ccident (6)], but interindividul vrition in tomic bomb survivor GPA vrint frequency ws substntil. Although the dt sets for the specific gene muttion ssys in tomic bomb survivors re limited, they hve provided proof-ofprinciple nd highlight the limittions of these ssys when substntil time hs pssed since n cute exposure. Studies of biomrkers in people exposed to rdition s result of the Chernobyl nucler power plnt ccident in April 986 provide contrst to the tomic bomb survivor studies. An estimted 6, to 8, people were involved in the clenup. Accepting tht the registered doses for the 9, clenup workers with confirmed doses hve mny shortcomings, those people who were t Chernobyl in 986 nd 987 re estimted to hve received men whole-body exposure of 6 cgy nd 9 cgy, respectively (7). The dose estimtes re bsed only on externl exposure, with 3 I (for those t Chernobyl in the erliest times), 34 Cs nd primrily 37 Cs being the key rdionuclides. The erliest biomrker studies emphsizing the more highly exposed individuls lrgely confirmed the reltionship between biomrker responses, physicl dosimetry, nd clinicl sttus [mong mny others, see refs. (5, 8, 9)]. Recent studies of workers whose work ssignments were intended to limit exposure to 25 cgy or less hve ssessed the biologicl effect of low-dose, low-dose-rte exposure, nd the relibility of vilble dosimetry. Over 7 clenup workers from Estoni nd Ltvi nd 5 locl controls were ssyed for GPA vrint frequency in n effort to prescreen the popultion for sufficient exposure to wrrnt epidemiologicl studies of cncer incidence (2). The GPA results were interpreted to be consistent with n exposure tht did not gretly exceed cgy. Estimtes of doses bsed on physicl dosimetry in this popultion rnged from.2 to 28 cgy. Chromosome trnsloction studies by fluorescence in situ hybridiztion (FISH) of 8 Estonin clenup workers in this popultion found no increse in trnsloctions (2), ruling out n exposure of cgy or more nd suggesting tht the dosimetry vilble my hve overestimted exposures for these clenup workers. In contrst, FISH trnsloction study of 34 Russin clenup workers estimted n exposure of 25 cgy, compred to 26 cgy s their documented dose (22). The difference between these two studies most likely is due to the heterogeneity of the exposures ssocited with different work ssignments nd selection of group with higher exposures in the ltter study. It should be noted tht the distribution of registered doses suggests tht reltively few clenup workers received doses in excess of 25 cgy (7). In n erlier report (23), we described initil results of our studies of Chernobyl clenup workers. This dt set reveled sttisticlly significnt increses in the trnsloction frequency nd HPRT mutnt frequency in the exposed subjects, trnsloctions hving the lrger increse. There ws no increse in GPA vrint frequency. Using n in vitro cytogenetic dose response curve, the verge dose to the clenup workers studied ws estimted to be 9 cgy. Dt were limited for comprisons of the biomrkers within the sme subject, or over time. Key questions tht remined were: Wht is the reltive sensitivity of these somtic genetic biomrkers for detecting exposures in popultions with low-dose, low-dose-rte rdition exposures? As time psses fter exposure, re biomrker responses ffected? Wht is the ccurcy of n estimte of exposure for the popultion? Are there optiml wys to use these ssys? The results presented here extend through 999 the study begun in 99. The gol ws to ccumulte dditionl dt using ech biomrker over longer period, to enhnce comprisons of the biomrkers, nd to study the effect of pssge of time on results, nd thereby gin more insight into the nswers to these key questions. MATERIALS AND METHODS Recruitment of Subjects nd Blood Smples The recruitment of subjects, obtining of smples, nd completion of questionnires were reviewed by Institutionl Review Bords t ll institutions involved nd by the Office for Protection from Reserch Risk of the U.S. Public Helth Service. All subjects gve informed consent prior to prticiption in this study. Subjects from Russi. All individuls were mles 8 yers of ge or older. Subjects were primrily selected t helth cre clinics in St. Petersburg or Tul, Russi. In St. Petersburg there were four clinics: t the Centrl Reserch Institute for Roentgenology nd Rdiology, Ministry of Helth of Russi; t the Consulting Dignostic Center of St. Petersburg Peditric Institute; t the Division of Occuptionl Pthology in Territoril Medicl Unit N 2; nd t the St. Petersburg Institute of Pulmonology, Ministry of Helth of Russi. In Tul the selection site ws the Tul Institute of New Medicl Technologies. Some subjects were recruited from clinics in Moscow. Individuls from the exposed clenup worker popultion ttended one of these clinics periodiclly to monitor their helth sttus. Individuls included in the control popultion were selected from friends nd reltives of the clenup workers nd other ttendees of the clinics, with the gol of group mtching the clenup worker popultion with respect to ge, sex nd lifestyle. All subjects donted venous blood smples of 4 ml nd were sked to complete questionnire (see below). Blood smples were collected into cid citrte dextrose nticogulnt nd kept t low temperture (4 5 C) during short-term stor-

3 426 JONES ET AL. ge nd shipment. All smples were shipped to the Lwrence Livermore Ntionl Lbortory (LLNL) by courier service s rpidly s possible fter collection. Shipping usully took bout 3 dys. Visul inspections of smples showed vrition in their condition but no obvious ptterns over time. Subjects from Livermore. Blood smples for the in vitro dose response studies were obtined from helthy, locl mle subjects, ge 2 to 46. Informtion on ge, smoking history, diet nd lifestyle fctors ws obtined with questionnire. Smples were irrdited nd cultures estblished the sme dy s venipuncture, s described previously (24). Questionnire Ech donor ws sked to complete questionnire. The questionnire ws printed in Russin nd informtion nd instruction were provided in tht lnguge. For those subjects enrolled in the study prior to Mrch 997, the questionnire ws 2 pges long, s described previously (23). Subjects enrolled fter tht dte were sked to complete 6-pge questionnire designed to cpture similr informtion in more user-friendly mnner, with fewer free text responses requiring trnsltion nd recoding. Included were mritl sttus, current occuption nd employment sttus, potentil occuptionl exposure to crcinogens nd rdition, medicl history including medictions nd dentl nd medicl rdition exposures, smoking history, nd cffeine nd lcohol consumption. In ddition, for clenup workers, officil dose estimte, time(s) t Chernobyl, site of work, nd work ssignment when t Chernobyl were queried. Some selfreported dose estimtes were bsed on thermoluminescence detectors (TLDs); most were bsed on interviews conducted by the Russins. It is unknown which were bsed on TLDs nd which were bsed on interview informtion. Copies of the originl Russin questionnires (either on pper or on computer diskettes) were sent to LLNL where they were trnslted onto n identicl form in English. All informtion provided in the questionnires ws entered into computerized dtbse (Sybse). Dt from the first phse of the study were exported from the erlier dtbse, with recoding s necessry to bring dt on ll subjects into the sme dt fields. Hndling of Biomrker Assys On rrivl t LLNL, blood smples were divided into liquots for ech ssy, depending on the smple volume. In generl the volume of blood required for ech ssy ws s follows: cytogenetics, 3 ml; HPRT, ml with few exceptions of 6 to ml; glycophorin A, ml, N/M blood type only. Priority ws given to cytogenetic over HPRT nlysis when blood volume ws limiting. Ech lbortory tht performed the biossys hd ongoing qulity control to ensure comprbility of results over time. To prevent bis, ll ssys were scored with smple clssifiction (clenup worker or control) blinded. Chromosome Aberrtion Assys Lymphocyte cultures. Whole blood (.8 ml) ws dded to ml of RPMI 64 medium (Gibco/BRL) supplemented with 5% fetl bovine serum, IU/ml penicillin, g/ml streptomycin, % sodium heprin, 2% phytohemgglutinin nd 2 mm L-glutmine. Multiple cultures were prepred for ech subject. Cultures were incubted t 37 C in 95% ir/5% CO 2 environment for 52 h, the lst 4 h with. g/ml Colcemid. Cells were hrvested by swelling in.75 M KCl, then fixed minimum of three times in 3: (v/v) methnol:glcil cetic cid. Well-spred metphse cells, with little overlying cytoplsm, were prepred for chromosome pinting s hs been described in detil (25). Slides were stored t 2 C in seled plstic bgs in the presence of desiccnt nd N 2 gs until needed for hybridiztion. Slides mde from smples received up until 995 were hybridized with Spectrum Ornge-conjugted chromosome specific DNA probes (Gibco/BRL) for chromosomes, 2 nd 4 simultneously ccording to the mnufcturer s instructions with minor modifictions, essentilly s described previously (26, 27). Slides mde from smples received in 996 nd therefter were hybridized with whole-chromosome pinting probes (Cytocell, Ltd) for six chromosomes simultneously:, 2 nd 4 were lbeled in red, nd 3, 5 nd 6 were lbeled in green (28). All slides were mounted in DAPI ntifde solution (29). Cell scoring. All cells were visulized with dul- or triple-bnd pss filters (Vysis, Inc.) for one- or two-color pinting, respectively, which llowed simultneous viewing of the pinted nd the DAPI-counterstined chromosomes. Metphse cells were considered scorble if they met the following criteri: () The cells ppered to be intct, (b) the centromeres of the chromosomes were morphologiclly detectble s primry constrictions, (c) the centromeres from ll pinted chromosomes were present, nd (d) the lbel ws sufficiently bright to detect exchnges between pinted nd unpinted chromosomes or between chromosomes lbeled in different colors. Every bnorml metphse cell ws photogrphed with Kodk Ektchrome 4 film or recorded with CCD-bsed imge cpture system. These imges provide permnent record of ech berrnt cell nd were used to resolve mbiguous berrtions. All berrtions were initilly scored ccording to the Protocol for Aberrtion Identifiction nd menclture Terminology (PAINT) (3). For sttisticl nlysis ll trnsloctions were subsequently rectegorized so tht reciprocl nd nonreciprocl trnsloctions were ech counted s single trnsloction. This pproch is bsed on the rtionle tht the low doses nd low dose rtes encountered by the clenup workers generlly did not result in complex chromosome rerrngements involving multiple color junctions which re often ssocited with nonreciprocl exchnges. There is lso evidence tht trnsloctions tht pper nonreciprocl t the cytologicl level usully contin telomere signls (3), suggesting tht mny of these exchnges my ctully be reciprocl t the moleculr level. In this study, trnsloction frequencies re reported s the number of trnsloctions per cells scored, with the number of cells scored converted to genome equivlents (see below). Assys for generting in vitro dose response curves. To estimte rdition doses to exposed subjects, we conducted in vitro dose response studies. Lymphocytes from four helthy U.S. donors were exposed to 37 Cs source in vitro t dose rtes of 5 to 6 cgy per minute for doses rnging from 5 cgy to 4 Gy. For most dt sets, doses from to Gy (in steps of 2 cgy) were emphsized. The number of cell equivlents scored rnged from 367 t dose to 67 t the highest dose. The cute exposures in this clibrtion study re not expected to model the chronic exposures experienced by the clenup workers. However, in the bsence of specific knowledge bout the dose rte for ech worker, nd becuse of uncertinties bout the mgnitude of the dose-rte reduction effect, the dose estimtes reported here re bsed on the cute dose response curve generted from these dt. Conversion of metphse cells scored to whole-genome equivlents. The frction of ll trnsloctions detected by hybridiztion (F h ) ws determined from the frction of the genome pinted in ech color nd ws compred to the frction observble by G-bnding (F b ) using the eqution Fh p 2pq q 2pr 2qr r, where p nd q represent the frction of the genome pinted red (chromosomes, 2 nd 4) nd green (chromosomes 3, 5 nd 6), nd r represents the unpinted frction of the genome. The terms p 2 nd q 2 represent chromosome exchnges between two chromosomes pinted in the sme color, nd the term r 2 represents exchnges between two unpinted chromosomes. The remining terms represent exchnges between chromosomes lbeled in different colors. Thus the frction of ll chromosome exchnges detected is 2pq 2pr 2qr. For single-color pinting, ll terms involving q drop out, nd the eqution reduces to 2 2 Fh p 2pr r. When chromosomes 6 re pinted in two colors, 56% of the exchnges re detected; when chromosomes, 2 nd 4 re pinted, 34.4% re detected (32). In this study, pproximtely 5 metphse cells were scored per subject. This is equivlent to 84 (5.56) metphse cells if the full genome were scored (defined s cell equivlents) when

4 SOMATIC BIOMARKERS IN CHERNOBYL CLEANUP WORKERS 427 six chromosome pirs were pinted, or 5 cell equivlents (5.344) when three chromosome pirs were pinted. HPRT Mutnt Frequency Assys Lymphocytes were seprted nd the HPRT mutnt frequency ssys were performed s described previously (33). Mononucler cells were isolted from 6- to 4-ml liquots of blood smples using Lymphocyte Seprtion Medium (ICN Biomedicls INC, Auror, OH). The isolted cells in most smples were cultured immeditely. Mononucler cells of some smples were cryopreserved for up to 6 months prior to culture. Cryopreservtion ws in RPMI 64 medium supplemented with 8% dimethylsulfoxide nd 2% fetl bovine serum nd ws performed in controlled freezing chmber. Cells then were stored in liquid nitrogen. Cells were cultured t 37 C in5%co 2 for up to 4 h t 6 cells per milliliter with the mitogen phytohemgglutinin (PHA; g/ml; type HA6, Murex Biotech, Kent, Englnd), then counted nd plted in roundbottomed wells with 5% (v/v) LAK superntnt (lymphokine-ctivted killer cell superntnt contining 5, U/ml humn interleukin 2, serum-free medium nd ny fctors present in the superntnt fter the 3 4-dy ctivtion of peripherl blood monocytes), 5% T-Stim Culture Supplement (Collbortive Biomedicl Products, Bedford, MA), reduced PHA (. g/ml), irrdited lymphoblstoid feeder cells (2, vible irrdited TK-6 or 9x-C4 cells per well; irrdited with 5 Gy 37 Cs delivered t 4.2 Gy per min), nd -mercptoethnol (5 mm), with or without thiogunine ( g/ml) selection, essentilly s per O Neill et l. (34, 35). Throughout this work the bsl medium ws RPMI 64 supplemented with 5% fetl bovine serum, 2% HL- (Bio-Whittker, Wlkersville, MD), penicillin ( U/ml) nd streptomycin ( g/ml). Donor cells were plted t nd 2 or 2, 5 nd cells/well in the bsence of thiogunine (96 wells for ech cell density) to determine the nonselective cloning efficiency (CE), nd t 4 cells/well in 96 wells nd t 2 4 cells/well using the rest of the lymphocytes in the presence of thiogunine to determine the cloning efficiency in the presence of thiogunine (mutnt efficiency). Mutnt frequency ws clculted from individul cloning efficiency nd mutnt efficiency vlues s described in the Sttisticl Methods below. The number of pltes t ech cell density with thiogunine ws dependent on the number of cells vilble for nlysis. Pltes were incubted t 37 C in 95% ir/5% CO 2 nd scored for growth in individul wells using n inverted microscope 5 8 dys of incubtion. Assys of Glycophorin A Vrint Frequency Since the GPA vrint frequency ssy cn be performed only on individuls with heterozygous N/M blood type (36), ech smple ws immunotyped using commercil ntiser (Ortho Dignostics, Rritn, NJ). This blood type comprises 5% of the humn popultion. The BR6 version of the GPA ssy ws performed s described previously (36). Briefly, blood smples were treted using sodium dodecyl sulfte (SDS) to produce sphericl cells;. ml whole blood ws mixed with. ml of Isolyte-S multi-electrolyte solution (Kendll McGrw Lbortories, Irvine, CA) contining 5 g/ml SDS nd mg/ml bovine serum lbumin (BSA). After min, the sphered cells were dded to ml fixtive solution [9.7 ml Isolyte S,.3 ml formlin (37% formldehyde), g/ml SDS] nd held in fixtive overnight t room temperture. These fixed cells were rinsed twice with buffer [ mm sodium phosphte (ph 7.2),.5 M NCl, 5 mg/ml BSA,.% nidet P-4, nd g/ml NN 3 ], then refrigerted in this buffer until stining. Fixed cells were immunolbeled with fluorescein-lbeled monoclonl ntibody BRIC 57, specific for the N-form of GPA, nd biotinylted 6A7, specific for the M-form of GPA, followed by streptvidin-phycoerythrin (Cltg Lbortories, South Sn Frncisco, CA). All cells were then counterstined with propidium iodide. A FACScn flow cytometer (Becton Dickinson, Mountin View, CA) ws used to determine the frequency of vrint erythrocytes. Anlysis ws limited to erythrocyte singlets bsed on forwrd sctter nd log side sctter distributions. Two vrint cell phenotypes were mesured simultneously in the GPA ssy; hemizygous phenotype cells (N/Ø) lck expression of the M llele nd express the N llele normlly, nd homozygous phenotype cells (N/N) lck expression of the M llele nd express the N llele t twice the heterozygote level. The GPA ssy ws not ffected by blood trnsit times up to 2 weeks becuse cell vibility is not required (36). Duplicte fixtions nd nlyses were performed on ech smple with totl of 5 6 erythrocytes mesured for ech nlysis. Men vlues of these duplicte nlyses were used to determine the frequencies of N/Ø nd N/N vrint cells per million erythrocytes. Sttisticl Methods All nlyses were performed using S-PLUS (37).. Significnce testing All P vlues re two-sided. Tests of homogeneity of discrete vribles cross multiple groups were performed using 2 tests. Tests of homogeneity of continuous vribles cross two groups were performed using Wilcoxon rnk-sum test, while tests for continuous vribles cross more thn two groups were performed using Kruskl-Wllce test. P vlues were clculted when ny cell in tble hd count less thn five, or when ny group hd fewer thn five unique vlues. Tests of significnce for regression coefficients re symptotic tests using stndrd errors derived from the observed informtion mtrix. 2. Generl pproch to the nlysis of biomrker dt Ech of the three biomrkers required slightly different type of model to describe its reltionship with covrites of interest. For chromosome berrtion ssys, the outcome is the number of trnsloctions observed upon viewing some number of metphse cells. Becuse of the discrete nture of trnsloctions nd the presence of vrition beyond wht would be expected using Poisson models, negtive binomil mximum likelihood models were used to nlyze trnsloction frequencies. For HPRT mutnt frequencies, the outcome to be nlyzed is mximum-likelihood estimte of log mutnt frequency, s well s n estimte of the precision with which the mutnt frequency ws estimted. For this outcome, weighted liner models were used on the log-trnsformed mutnt frequencies obtined by mximum likelihood. Finlly, for GPA vrint frequencies, where no estimte of reltive precision ws vilble, ordinry liner models were used on log-trnsformed vrint frequencies. Two nlyses were performed for ech biomrker. The first nlysis ttempted to determine the overll verge effect ssocited with being clenup worker. This nlysis did not involve ny vribles specific to being clenup worker, e.g., the job performed t Chernobyl or the length of time in the 3-km zone. For ech biomrker, the result of this nlysis ws n estimte of the verge increse in the biomrker due to being t Chernobyl. The second nlysis for ech biomrker ws performed for clenup workers only. This nlysis ttempted to further dissect ny observed verge increse nd discover if there were ny Chernobyl-specific exposure vribles tht were ssocited with biomrker levels. The result of this nlysis ws set of exposure vribles tht further prtition the vrition seen in clenup worker biomrker vlues. For both nlyses, djustments for covrites were lso produced. For the first nlysis, the reltionship between ech individul biomrker nd being t Chernobyl ws ssessed by the following procedure. First, bckwrds elimintion procedure ws used on n initil set of vribles to remove vribles tht did not ffect the outcome from further nlysis. The initil set of vribles included most of the vribles from the questionnire. Exceptions were due to intentionl redundncy in the questionnire informtion solicited for the smoking questions, which were recoded to produce more orthogonl set of questions. At ech stge of this procedure, likelihood rtio tests were performed on ech vrible currently in the model to compre the current model with one tht eliminted tht vrible. The vrible with the lrgest P vlue ws eliminted. The bckwrds elimintion procedure ws terminted when the P vlues for ll vribles tested were less thn.5, producing finl mrginl model for ech outcome. Next, for HPRT nd GPA outcomes, outliers mong the observtions

5 428 JONES ET AL. with respect to this finl mrginl model were tested for using the method described in Hdi nd Simonoff (38). Method of the Hdi nd Simonoff procedure ws used to obtin the initil clen set of observtions. For chromosome berrtions, no outlier-detection lgorithm ws vilble, so the distribution of crude berrtion frequencies ws exmined for obviously influentil observtions. One observtion hd vlue two orders of mgnitude lrger thn the rest of the dt set nd ws eliminted. Finlly, regression ws performed for ech outcome to estimte the overll verge effect due to being clenup worker. This regression used the finl set of observtions nd the finl set of vribles for ech outcome. The second nlysis on Chernobyl clenup workers ws done in similr fshion. Only Chernobyl workers not eliminted s outliers in the first nlysis were used. First, bckwrds elimintion procedure ws performed on n initil set of vribles. These vribles included ll of the covrites identified s significnt in the first nlysis, long with ll of the vribles ssocited with being clenup worker. The sme likelihood rtio pproch to vrible elimintion ws used. After the vrible elimintion step, regression ws performed on the remining vribles. outlier detection ws performed on the second nlysis. 3. Regressions with trnsloctions s n outcome Regressions involving trnsloctions s n outcome were performed using n extended form of negtive-binomil mximum generlized liner model in which the dispersion term is lso estimted by mximum likelihood (39). Tht is, the number of trnsloctions counted in ech subject ws ssumed to follow negtive binomil distribution with n unknown common dispersion term. For biomrker dt, the logrithm of the underlying trnsloction frequency per cell scored ws ssumed to be liner function of covrites. For dose response dt, the frequency itself ws ssumed to be qudrtic function of dose, but with different bckground level for ech subject. For negtive binomil models, the percentge of vrition ccounted for by model ws estimted by fitting corresponding liner model to the squre-root-trnsformed outcome nd using the resulting R Computing HPRT mutnt frequencies The methods nd rtionle for dt nlysis hve been described in detil (4). Briefly, for ech study subject, the nturl logrithms of cloning efficiency nd mutnt frequency were estimted directly from the counts of positive nd negtive wells by the method of mximum likelihood, using binomil generlized liner model with complementry log-log link. Stndrd errors (SEs) were estimted using the symptotic stndrd errors provided s by-product of the fit. Tht is, the stndrd errors re the squre root of the digonl elements of the inverse of the Hessin mtrix t the mximum likelihood estimte. The distributions of cloning efficiency nd mutnt frequency were skewed, s is common with these kinds of dt. Consequently, the nturl logrithm of mutnt frequency ws used in regressions to reduce the effect of skewness. In ddition, the precision of the estimtes of log mutnt frequency differed gretly mong study subjects, due in prt to reduced number of cells plted under selective conditions nd/or low cloning efficiency. To ccount for differences in precision, estimtes of the reltionships between log mutnt frequency outcomes nd predictors of interest (nd their interctions) were clculted using weighted liner regression. The weight used for ny given subject ws the inverse of the vrince of the estimted mutnt frequency for tht subject, estimted by the squre of /SE, where SE corresponds to the stndrd error in estimting the mutnt frequency for tht subject. 5. Estimting verge dose The dose response reltionship ws estimted by regression on in vitro dose response dt, using negtive-binomil mximum likelihood regression with n identity link, s described bove. Insufficient dt were vilble to estimte the vrition in nd between individuls. Consequently, common dose response curve ws ssumed for ll subjects. Stndrd errors used for nd were those obtined s by-product of the fit. Tht is, the stndrd errors re the squre root of the digonl elements of the inverse of the Hessin mtrix t the mximum likelihood estimte. The increse in trnsloction frequency for ny specific clenup worker due to being t Chernobyl ws estimted s follows. First, the worker s predicted trnsloction frequency R ws clculted from the finl mrginl model for trnsloctions nd tht individul s ge, smoking sttus, nd retirement sttus (covrites found to be significnt in the popultion). The devince residuls from the mrginl fit were exmined using quntile-quntile plot, nd they did not indicte ny unusul devition from norml distribution (dt not shown). Next, the worker s predicted trnsloction frequency R were tht person to hve been control ws lso clculted from the finl mrginl model. The increse due to being t Chernobyl ws then estimted s R R. The stndrd error of the increse ws estimted by dding the squre of the stndrd error for R to the squre of the prediction error for R nd tking the squre root of the resulting sum. The verge increse over ll of the clenup workers in the smple ws estimted by verging the individul R R vlues. The stndrd error of the verge increse ws found by summing the squres of the individul stndrd errors, dividing by the squre of the number of clenup workers, nd tking the squre root. The verge dose ws then clculted by solving for dose in the eqution 2 Averge increse (dose) (dose). Finlly, the stndrd error for the estimted dose ws clculted by the Delt Method (i.e. Tylor expnsion), incorporting the error estimtes for the verge increse,, nd. RESULTS The Study Subjects The study smple is presented in Tble. Only smples obtined fter August 24, 992 re included. On this dte consistent use of questionnire nd of smple cquisition nd hndling prctices for studies with multiple end points were instituted. The lst smples included were received on December 2, 999. Subjects were excluded if they hd hd chemotherpy, rdiotherpy or blood trnsfusion in the pst yer. Subjects recruited t clinics in three cities in Russi entered the study from 992 to 999 with the receipt of the first blood smple they provided (Tble 2). Anlyses of the dt for ech biomrker were performed to determine whether the smpling dte ffected the vlue of the biomrker (see below). The potentil for differences between subjects from different cities ws ssessed for ech biomrker (see below). This report contins results tht extend the report of Moore et l. (23) to include 7 more control subjects nd 49 more clenup workers who were enrolled in the study over n dditionl period of pproximtely 4 yers. Generl Demogrphic Informtion For this report, informtion collected by the originl questionnire (23) nd the revised questionnire (see the Mterils nd Methods) ws used. Although the mjority of controls nd clenup workers provided questionnires (78 nd 79%, respectively), not ll questions were nswered by ll subjects. As result, differences between the groups my not hve been detected. For merging of informtion from the two questionnires nd for nlyticl purposes, summry ttributes were developed, e.g. coding medicl rdition exposure other thn rdiotherpy s ny X

6 SOMATIC BIOMARKERS IN CHERNOBYL CLEANUP WORKERS 429 Study group ttributes TABLE The Study Smple Controls (N 87) Resons for exclusion from study: Acquired before August 24, 992 Blood trnsfusion nswer Rdition tretment nswer Chemotherpy nswer Finl study subjects Questionnire vilble c ,b Exposure sttus Clenup workers (N 744) b Overll (N 93) b te. The initil ll-mle study smple nd numbers of subjects tht did not meet the criteri for inclusion re presented. Three individuls whose smples were received in 2 were excluded. b Individuls not included in the finl study set include those with ny of the four exclusion ttributes. c Absence of questionnire ws not used s n exclusion criterion. rys, yes/no, to summrize vriety of such exposures. Summry vribles for lcohol consumption nd smoking were developed in ddition to detiled informtion. Vitmin usge ws coded yes/no. Dt were coded s no nswer when questionnire response ws not provided, enbling detection of vrition due to the nonresponding subjects. This prctice hs the dvntge of incresing power by including more subjects, but it hs the potentil to introduce bis if nonresponders nd responders re not rndomly distributed. The bove procedure for dt with missing vlues ws pplied consistently throughout the nlyses. Some of the primry chrcteristics of controls nd clenup workers who provided questionnire re presented in Tble 3. Controls nd clenup workers differed significntly in 8 of these 7 ttributes. In ddition to the vribles in Tble 3, ll informtion vilble for controls nd clenup workers from the questionnires on employment sttus, occuptionl exposures, medicines tken, nd diet ws nlyzed to identify which of these frctions ffected the biomrker outcomes. Results of the univrite nlyses for the three biomrkers for 34 potentil risk fctors used to identify potentil covrites re not presented. Exposure Informtion for Clenup Workers Chrcteristics of individul exposure histories t Chernobyl re presented in Tble 4 s function of the first yer t Chernobyl. Dt on self-reported ( officil ) dose, length of time worked (longer in lter yers presumbly due to lower dose rtes), nd working in block 4 (the site of the dmged rector; fewer clenup workers t Chernobyl in lter yers) re consistent with lower exposure rtes for those t Chernobyl in the lter yers. Age when first t Chernobyl ws firly constnt. There is slight decline in the time elpsed between first work t Chernobyl nd providing smple for those who were t Chernobyl in the lter yers. However, this is reltively smll group. Due to the preferentil recruitment of subjects who were first t Chernobyl in 986, there re few subjects who first worked t Chernobyl in yers 989 nd lter. The Person correltion between the dte entered into study nd the yers elpsed since first t Chernobyl ws.94. Occuption t Chernobyl ws summrized in nine ctegories (listed in Yer entered study Controls (N 6) 5 Moscow (N 36) TABLE 2 Subject Entry into Study over Time by Exposure Sttus nd Clinic Site Clenup workers (N 3) Yer of first smple from subject. Controls (N 29) St. Petersburg (N 46) Clenup workers (N 332) Controls (N 45) Tul (N 294) Clenup workers (N 249) Exposure sttus (ll cities) Controls (8) Clenup workers (6) Totl (79)

7 43 JONES ET AL. TABLE 3 Chrcteristics of Those Controls nd Clenup Workers Studied for whom Questionnire ws Avilble Chrcteristics Controls (N 4) Number (%) 4 (22%) 485 (78%) Age Medin Mritl sttus Divorced Mrried Never mrried nswer Smoking sttus Never smoked Ex-smoker Current smoker nswer Smoked cigrettes or popirossi thing Cigrettes only Popirossi only Cigrettes nd popirossi nswer Yers smoked cigrettes Medin nswer Pck-yers of cigrettes Medin nswer Yers smoked popirossi b Medin nswer Pck-yers of popirossi b Medin nswer Currently employed nswer Retired 8 (3.%) 9 (65.%) 28 (2.%) 3 (2.%) 35 (25.%) 2 (5.%) 8 (58.%) 3 (2.%) 35 (25.%) 7 (5.%) 3 (9.3%) 8 (3.%) 3 (2.%) (7.%) 4 (29.%) (.7%) Clenup workers (N 485) P vlue c 54 (.%) 422 (87.%) 3 (.62%) 6 (.2%) 6 (24.%) 59 (2.%) 296 (6.%) 4 (2.9%) 6 (24.%) 266 (55.%) 25 (5.2%) 54 (.%) 24 (4.9%) (53.%) 222 (46.%) 8 (.6%) nswer 8 (5.7%) 44 (3.%) 88 (63.%) 37 (7.6%) 9 (39.%) 257 (53.%) Beer consumed (bottles/week) ne 24 (7.%) 5 (22.%) or2 3or4 More thn 4 nswer Wine consumed (bottles/month) ne or2 3or4 More thn 4 nswer Other lcohol (bottles/month) ne or2 3or4 More thn 4 nswer 9 (6.4%) 6 (4.3%) 6 (4.3%) 95 (68.%) 29 (2.%) 3 (9.3%) 3 (2.%) (.7%) 94 (67.%) 5 (.%) 25 (8.%) 4 (2.9%) (.7%) 95 (68.%) 42 (8.7%) 32 (6.6%) 6 (3.3%) 29 (6.%) 28 (26.%) 49 (.%) 7 (.4%) 3 (.62%) 298 (6.%) 69 (4.%) 9 (22.%) 5 (3.%) 5 (.%) 287 (59.%) Chrcteristics Coffee consumed (cups/dy) ne or2 3or4 More thn 4 nswer Vitmins tken regulrly nswer Tke nerve medicines nswer Exposure to chemicls or solvents nswer Any dignostic X rys d nswer TABLE 3 Continued Controls (N 4) 3 (9.3%) 72 (5.%) 8 (5.7%) 5 (3.6%) 42 (3.%) (7.%) 39 (28.%) (.7%) 7 (84.%) 23 (6.%) (%) 45 (32.%) 7 (2.%) 78 (56.%) 2 (8.6%) 28 (9.%) (.%) Clenup workers (N 485) P vlue c 59 (2.%) 258 (53.%) 3 (6.2%) 7 (3.5%) 2 (25.%) 299 (62.%) 7 (35.%) 5 (3.%) 337 (69.%) 47 (3.%) (.2%) 23 (47.%) 5 (%) 25 (42%) 3 (27.%) 33 (68.%) 23 (4.7%) Among the 49 self-reported cigrette smokers only. b Among the self-reported popirossi smokers only. c P vlues reflect differences between chrcteristics of controls nd clenup workers. nswer individuls were included. P vlues were clculted using non-prmetric rnk tests for homogeneity between controls nd clenup workers. d Includes ll who hd ny dignostic X rys, dentl, bone, chest, specil. Tble 8, discussed below). The first yer t Chernobyl nd dte first in 3-km zone (n re of restricted ccess surrounding the rector) re synonymous. Self-reported dose did not vry systemticlly s function of dte entered in the study (Tble 5), suggesting consistency in recruitment over the spn of the study. Biomrker Assys Performed Tble 6 summrizes the ssys performed. When blood volume ws limited, priority ws given to trnsloction nlysis over HPRT. Trnsloction nlyses were completed on ll possible smples; bout one qurter of cultures did not provide sufficient metphses to complete trnsloction frequency nlyses. Culture filures tended to occur for ll smples of given shipment, suggesting tht problems encountered in trnsit were likely cuse. These filures showed no pttern over time. HPRT nlyses of clenup worker smples were performed preferentilly on GPA heterozygotes. GPA ssys were performed on essentilly ll N/M heterozygotes, i.e. 58% of the smples received. The.2-fold enrichment over the expected 5% occurrence of N/M heterozygotes mong the clenup workers ws the

8 SOMATIC BIOMARKERS IN CHERNOBYL CLEANUP WORKERS 43 Chrcteristics TABLE 4 Summry of Exposure History of Clenup Workers by First Yer t Chernobyl First yer t Chernobyl nd 99 Unknown All clenup workers (N 6) Number of clenup workers Self-reported dose (cgy) Minimum 25 percentile Medin 75 percentile Mximum Dys in 3-km zone b Minimum 25 percentile Medin 75 percentile Mximum Worked in Block 4 c Age t Chernobyl (yers) d Minimum 25 percentile Medin 75 percentile Mximum Yers between work t Chernobyl nd smple e Minimum 25 percentile Medin 75 percentile Mximum Informtion not vilble for 259 clenup workers. b Informtion not vilble for 49 clenup workers. c Informtion not vilble for 53 clenup workers. d Informtion not vilble for 49 clenup workers. e Informtion not vilble for 49 clenup workers. result of occsionl prescreening of subjects for blood type in Russi. Anlysis of Trnsloction Results Five fctors were identified s contributing to the best model for chromosome trnsloction frequencies for controls nd clenup workers in the sense tht removing ny one of them reduced the fit of the model in sttisticlly significnt wy. Together these fctors ccount for pproximtely 28% of the vrition in trnsloction frequencies. The percentge increses in trnsloction frequency for these fctors re given in Tble 7. Three fctors tht cn be esily understood s ffecting trnsloction frequency, exposure sttus, i.e. whether person ws control or clenup worker, smoking history, nd ge, were sttisticlly significnt, s hs been noted in other studies [for exmple, refs. (4 43) nd references cited therein]. The other two vribles re less esily understood reltive to genetic dmge. For both controls nd clenup workers, trnsloction frequencies for smples obtined in 998 were 5% lower thn smples obtined in 995. When the other yers were tested reltive to 995 using multiple testing procedure (Sidk s method), only the 998 vlues were sttisticlly significntly different from those for 995 t the.5 level. explntion is vilble for the lower vlues in 998. It does not correlte with ny known difference in methodology (pinting of six chromosomes rther thn three begn in 996) or ny known difference in the subjects studied. The 998 effect my be rndom finding or my be surrogte for otherwise unscertined differences in lifestyle or environmentl exposures of the subjects tht resulted in lower genetic dmge, such s ltered diet, reduced pollution, or immunologicl/helth sttus. An nlysis of trnsloction frequency s function of the yer of the smple, djusted for other covrites, detected trend over time to lower vlues (P.). There is no evidence tht the trend differs for clenup workers nd controls. Additionl nlyses were performed to ssess the impct

9 432 JONES ET AL. 992 (N 29) Self-reported dose, cgy Minimum percentile.5 Medin percentile 24. Mximum (N 47) TABLE 5 Self-Reported Dose by Yer Smple Acquired 994 (N 9) Yer smple cquired 995 (N 7) (N 97) (N 2) (N 22) (N 54) nswer, no P vlue for 997 dose being different ws All (N 6) of clenup workers exposure histories on trnsloction frequencies. When covrites identified in the first nlysis of trnsloction results (Tble 7) nd the five Chernobyl exposure vribles listed in Tble 4 plus the first yer n individul worked t Chernobyl fter the ccident were tested for inclusion in best-fit model for clenup workers lone, four vribles were identified, one of which ws n exposure-relted risk fctor. The percentge increses in trnsloction frequency for these vribles from the regression nlysis re given in Tble 8. These four vribles ccount for pproximtely 25% of the vrition in trnsloction frequencies of clenup workers. The primry new result in this nlysis is the ssocition of trnsloction frequency with occuption t Chernobyl. Reltive to those with unknown tsks t Chernobyl, those who were either construction workers or mechnics hd trnsloction frequencies incresed by 6%. Anlysis of HPRT Mutnt Frequency Results Nine fctors were identified s contributing to the best model for HPRT mutnt frequencies of controls nd clen- TABLE 6 Number of Assys Completed for ech Biomrker nd Combintion of Biomrkers Biomrker combintion Ech biomrker Trnsloctions HPRT mutnt frequency GPA vrint frequency Exposure sttus Controls (N 8) Clenup workers (N 6) Overll (N 79) Two biomrkers Trnsloctions nd HPRT mutnt frequency Trnsloctions nd GPA vrint frequency HPRT mutnt frequency nd GPA vrint frequency All three biomrkers All instnces in which two or more biomrkers were mesured. up workers. The percentge increses in HPRT mutnt frequency for these fctors re given in Tble 9. These nine vribles ccount for pproximtely 26% of the vrition in HPRT mutnt frequencies (multiple R 2 for the regression). Clenup worker sttus nd ge were significnt fctors, s in previous studies (7, 23, 33, 44). The other seven vribles re less esily understood reltive to genetic dmge. There ws downwrd trend with incresed wine consumption (P. for ny differences) but n upwrd trend with incresed consumption of lcohol other thn beer TABLE 7 Percentge Increse in Trnsloction Frequency for Importnt Risk Fctors, Adjusting for Other Covrites Risk fctors Vlue 95% confidence intervl Low High P vlue (Intercept) Exposure sttus Control Clenup workers 29.9%.3% 53.%.2 Age (per yer) 4.% 3.2% 4.8%. Yer of smple Smoking sttus n-smoker Ex-smoker Current smoker nswer Retired nswer 26.7% 3.2% 2.% 22.5% 25.9% 5.3% 26.5% % 22.5% 3.3% 3.4% 42.% 64.7% 48.9% % 65.4% 5.6% 55.2% 5.4% 32.8% 5.5% Chosen s reference due to being the yer with the lrgest number of smples.

10 SOMATIC BIOMARKERS IN CHERNOBYL CLEANUP WORKERS 433 TABLE 8 Percentge Increse in Trnsloction Frequency for Importnt Exposure Risk Fctors mong Clenup Workers, Adjusting for Other Covrites Risk fctors (Intercept) Age (per yer) Yer of smple Smoking sttus n-smoker Ex-smoker Current smoker nswer Chernobyl job Unknown Administrtor Construction worker Digger/scrcophgus work Driver Decontmintion worker Mechnic Medicl/rdition specilist Other b Vlue % 6.4%.98% 6.5% 2.4% 2.3% 45.2% 9.% % confidence intervl Low High P vlue % 8.3% 3.% 7.% 8.3% 37.9% 57.8% 64.7% % 66.% 47.8% 36.7% 58.2% 2.2% 29.% 64.7% Chosen s reference due to being the yer with the lrgest number of smples. b Includes smll numbers of multiple other tsks. TABLE 9 Percentge Increse in HPRT Mutnt Frequencies for Importnt Risk Fctors, Adjusting for Other Covrites Risk fctors Vlue 95% confidence intervl Low High P vlue (Intercept) Exposure sttus Control Clenup worker Yer of smple %.5% 29.4% 22.5% 28.7% 23.% 7.2% 63.2% 32.5% 44.8% 29.% 43.% 9.7% % 47.4% 45.7% 25.%.% 3.2% 8.9% 53.5% Age (per yer) Retired nswer Exposure to chemicls or solvents nswer Tke nerve medicines nswer Wine (bottles/month) ne More thn 4 nswer Other lcohol (bottles/month) ne More thn 4 nswer Clinic city St. Petersburg Tul Moscow Chosen s reference due to being the yer with the lrgest number of smples. or wine (P.5 for ny differences), nd there were sttisticlly significnt differences (P.3) between clinic cities. For exposure to chemicls or solvents nd for wine consumption, the ctegory of people for whom informtion ws not vilble hd sttisticlly significntly different mutnt frequency effects thn those reporting no exposure. Interestingly, no smoking effect ws detected with HPRT mutnt frequency in this dt set, unlike erlier studies of this nd other popultions (7, 33, 4, 44). An nlysis of HPRT mutnt frequency s function of the yer of the smpling, djusted for other covrites, detected no trend over time (P.4). Additionl nlyses were performed to ssess the impct of clenup workers exposure histories on HPRT mutnt frequency. When covrites identified in the first nlysis of HPRT mutnt frequency (bove, Tble ) nd the Chernobyl exposure vribles in Tble 4 were tested for inclusion in best-fit model for clenup workers lone, two exposure vribles were identified s sttisticlly significnt: first yer t Chernobyl nd yers between first yer t Chernobyl (P.2) nd dte of blood smpling (P.3). Unexpectedly, those who were t Chernobyl first in 987 or 988 hd sttisticlly significntly incresed HPRT mutnt frequency reltive to those there first in 986. This difference, whose cuse is unknown, my ccount for the pprent increse in HPRT mutnt frequency with time

11 434 JONES ET AL. TABLE Percentge Increse in HPRT Mutnt Frequencies for Importnt Exposure Risk Fctors mong Clenup Workers, Adjusting for Other Covrites Risk fctors Vlue 95% confidence intervl Low High P vlue (Intercept) Yer of smple % 4.2% 37.% 4.7% 46.3% 47.2% 39.% 7.4% 42.3% 56.4% 94.8% 59.3% 9.% % 63.9% 68.9% 68.% 3.7% 2.%.4% 5.6% Age (per yer) 2.5%.5% 3.4%. Retired nswer Wine (bottles/month) ne or2 3or4 More thn 4 nswer 55.5% 38.3% 9.3% 48.2% 38.5% 4.% Other lcohol (bottles/month) ne More thn 4 nswer Clinic city St. Petersburg Tul Moscow First yer t Chernobyl % 82.5% 27.9% 3.% 3.4% 4.9% 77.8% 99% 58.6% 6.% 3.%.8% 7.7% 85.8% 7.4%.4% 38.3% 22.9% 38.3% 9.8% 43.7% 7.5% 2.6% 38.% 5.5% 33.7% 4.% 9.4% 54.2% 89.2% 32.6% 45.% 27% 332% 5.8% 42.% 33.2% 33.4% 6% 548% 48% 6.4% Yers between work t Chernobyl nd smple (per yer) 5.8%.6% 3.8%.3 Chosen s reference due to being the yer with the lrgest number of smples. TABLE Percentge Increse in GPA N/Ø Vrint Frequency for Importnt Risk Fctors, Adjusting for other Covrites Risk fctors Vlue 95% confidence intervl Low High P vlue (Intercept) Yer of smple Coffee (cups/dy) ne or2 3or4 4 nswer Wine (bottles/month) ne or2 3or4 More thn 4 nswer 47.2% 28.4% 2.2% 8.% 9.7% 6.9% 38.8% 9.% 39.3% 33.% 8.8% 5.6% 294% 2.6% 7.% 5.3% 46.6% 36.5% 2.7% 26.5% 32.5% 23.% 33.4% 54.6% 56.3% 33.6% 5.6% 98.% 39.% 8.5% 6% 3.8% 2.% 33.7%.9% 2.4% 23.%.6% 8.4% % 32.% 683% 42% 25.4% Chosen s reference due to being the yer with the lrgest number of smples. elpsed since exposure. When considering clenup workers only, ll of the covrites identified for both controls nd clenup workers (Tble 9) remined significnt with the exception of tking nerve medicines. The eight vribles ccount for pproximtely 28% of the vrition in HPRT mutnt frequencies in clenup workers (multiple R 2 for the regression). As bove, the yer of the smple ffected the HPRT mutnt frequency of clenup workers, with estimtes of ll of the yers in the study being lower thn the reference yer of 995. These results suggest tht for some unknown reson clenup workers smpled in 995 hd higher HPRT mutnt frequencies. In comprison, trnsloction frequencies were reduced only for clenup workers smpled in 998 (Tble 8). Anlysis of GPA N/Ø Vrint Frequency Results For GPA N/Ø vrint frequency, exposure sttus (control compred to clenup worker) ws not significnt fctor, unlike trnsloctions nd HPRT. Three fctors were identified in the best-fit model for GPA N/Ø vrint frequency. The percentge increses in GPA N/Ø vrint frequency for the three vribles re given in Tble. These three vribles ccount for pproximtely 5% of the vrition in GPA N/Ø vrint frequency (multiple R 2 for the regression). There ws consistently lower GPA N/Ø vrint frequency for ll levels of coffee consumption nd consistently higher vrint frequency for ll levels of wine consumption (P.6 nd P.4 for ny differences, respectively). As with trnsloction frequencies nd HPRT mutnt frequency, GPA N/Ø vrint frequency vried from yer to yer in sttisticlly significnt wy (P. for ny differences). An nlysis of GPA N/Ø vrint frequency s function of the yer of the smple, djusted for other covrites, detected trend over time to lower vlues (P.3).