Cytogenetic Endpoints Research Articles

616 Assessment of genotoxic exposure in turkish coke oven workers by cytogenetic endpoints

616 Assessment of genotoxic exposure in turkish coke oven workers by cytogenetic endpoints

What do human micronuclei contain?

As micronuclei (MN) derive from chromosomal fragments and whole chromosomes lagging behind in anaphase, the MN assay can be used to show both clastogenic and aneugenic effects. The distinction between these phenomena is important, since the exposure studied often induces only one type of MN. This particularly concerns the use of MN as a biomarker of genotoxic exposure and effects, where differences in MN frequencies between exposed subjects and referents are expected to be small. A specific analysis of the induced type of MN may considerably improve the sensitivity of detecting the exposure effect. MN harbouring chromosomes can be distinguished from those harbouring acentric fragments by the presence of a centromere. The proportion of centromere-positive MN in human lymphocytes increases with age, which primarily reflects an age-dependent micronucleation of the X and Y chromosomes. The X chromosome especially tends to lag behind in female lymphocyte anaphase, being micronucleated more efficiently than autosomes. There is some evidence for an enhanced prevalence of fragments from chromosome 9 in spontaneous human lymphocyte MN and from chromosomes 1, 9 or 16 in MN induced in vitro by some clastogens; the breakage appears to occur in the heterochromatic block of these chromosomes. Although there are indications that centromere identification can improve the detection of clastogenic effects in humans in vivo, smokers have not shown an increase in centromere-negative MN in their cultured lymphocytes, although smoking is known to produce chromosomal aberrations. This may suggest that fragment-containing MN and chromosomal aberrations cover partly different phenomena. Understanding the mechanistic origin and contents of MN is essential for the proper use of this cytogenetic end-point in biomarker studies, genotoxicity testing and risk assessment.

Genotoxicity of pesticides: a review of human biomonitoring studies.

Pesticides constitute a heterogeneous category of chemicals specifically designed for the control of pests, weeds or plant diseases. Pesticides have been considered potential chemical mutagens: experimental data revealed that various agrochemical ingredients possess mutagenic properties inducing mutations, chromosomal alterations or DNA damage. Biological monitoring provides a useful tool to estimate the genetic risk deriving from an integrated exposure to a complex mixture of chemicals. Studies available in scientific literature have essentially focused on cytogenetic end-points to evaluate the potential genotoxicity of pesticides in occupationally exposed populations, including pesticide manufacturing workers, pesticide applicators, floriculturists and farm workers. A positive association between occupational exposure to complex pesticide mixtures and the presence of chromosomal aberrations (CA), sister-chromatid exchanges (SCE) and micronuclei (MN) has been detected in the majority of the studies, although a number of these failed to detect cytogenetic damage. Conflicting results from cytogenetic studies reflect the heterogeneity of the groups studied with regard to chemicals used and exposure conditions. Genetic damage associated with pesticides occurs in human populations subject to high exposure levels due to intensive use, misuse or failure of control measures. The majority of studies on cytogenetic biomarkers in pesticide-exposed workers have indicated some dose-dependent effects, with increasing duration or intensity of exposure. Chromosomal damage induced by pesticides appears to have been transient in acute or discontinuous exposure, but cumulative in continuous exposure to complex agrochemical mixtures. Data available at present on the effect of genetic polymorphism on susceptibility to pesticides does not allow any conclusion.

A Preliminary Study to Assess Possible Chromosomal Damage Among Users of Digital Mobile Phones

In a preliminary study to examine possible lymphocyte chromosomal damage, we have tested two cytogenetic endpoints, namely, chromosomal aberrations (CA) and sister chromatid exchange frequencies (SCE), in 24 mobile phone users (12 nonsmoker–nonalcoholic subjects and 12 smoker–alcoholics), who used digital mobile phones for at least 2 years, employing Gaussian Minimum Shift Keying modulations with uplink frequencies at 935–960 MHz. and downlinks at 890–915 MHz. For comparison, the control study group included another 24 individuals, matched according to their age, sex, drinking and smoking habits, as well as similar health status, working habits, and professional careers; but did not use mobile phones. Blood samples of 12 mobile users (6 smoker–alcoholic and 6 nonsmoker–nonalcoholic) and 12 controls (identical to mobile users in every respect) were further treated with a known mutagen Mitomycin‐C (MMC) to find out comutagenic/synergistic effect. A complete blood picture for each individual was assessed with an automatic particle cell counter.There was a significant increase (P < 0.05) in dicentric chromosomes among mobile users who were smoker–alcoholic as compared to nonsmoker–nonalcoholic; the same held true for controls of both types. After MMC treatment, there was a significant increase in dicentrics (P < 0.05) and ring chromosomes (P < 0.001) in both smoker–alcoholic and nonsmoker–nonalcoholic mobile users when compared with the controls. Although SCEs showed a significant increase among mobile users, no change in cell cycle progression was noted. The hematological picture showed only minor variations between mobile users and controls.

Possible transient adaptive response to mitomycin C in peripheral lymphocytes from thyroid cancer patients after iodine-131 therapy.

Our study attempted to assess the possible induction and persistence of an adaptive response in lymphocytes of thyroidectomized thyroid cancer patients treated with 131I (2,590 MBq, corresponding to whole body doses in the range of 200-300 mGy), to a testing dose of mitomycin C (MMC) in vitro. The cytogenetic endpoint studied was the induction of micronuclei in cytokinesis-blocked peripheral blood lymphocytes, immediately before treatment and 1, 6 and 24 months after therapy. One month after therapy, induction of micronucleated cytokinesis-blocked lymphocytes ( per thousand ) by MMC was lower (34.6 +/- 7.7) than before therapy (52.1 +/- 5.0). In 7 of 11 patients this reduction was significant. However, at 6 months, induction of micronuclei was markedly higher (133.1 +/- 13.6). This significant increase was observed regardless of the decrease at 1 month. At 24 months, the frequency of micronucleated cells decreased (84.8 +/- 5.5), but remained higher than before treatment. The results obtained 1 month after therapy could reflect adaptation due to radiation, or a higher rate of early apoptosis or cell death, with bone marrow suppression, visible as a lower response in vitro towards MMC. At 6 months, recovery of the lymphocyte population may occur, and higher responses to MMC in vitro could reflect higher chromosomal instability in the previously irradiated stem cells with a concomitant disappearance of adaptation, whereas at 24 months the results show a tendency to return to pretherapy values.

Combined treatment with 4-(N-methyl-N-nitrosamino)-1- (3-pyridyl)-1-butanone and dibutyl phthalate enhances ozone-induced genotoxicity in B6C3F1 mice.

Potential toxicological interactions of 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and/or dibutyl phthalate (DBP) with ozone were investigated. Male and female B6C3F1 mice were exposed to ozone (0.5 p.p.m.), NNK (1.0 mg/kg), DBP (5000 p.p.m.) and different combinations of these toxicants 6 h/day for 16, 32 and 52 weeks. Two cytogenetic end-points, determined by the chromosomal aberration (CA) and supravital micronucleus (SMN) assays, were investigated in vivo. Our results show that all treated groups of both sexes showed genotoxic effects when compared with the control group. Additive and/or synergistic responses were observed in the CA assay for all test periods when mice of both sexes were exposed to ozone and NNK, ozone and DBP and the combination of ozone, NNK and DBP. In the SMN assay, additive interactions were noted for both sexes in the 16 and 32 week studies, similar to the results with the CA assay. All combination groups of both sexes showed synergistic interactions in the 52 week study. The results indicate that combined exposure to ozone, NNK and DBP in both sexes of mice has enhanced genotoxic effects compared with exposure to ozone alone.

Genotoxic, cytotoxic and ontogenetic effects of tri- n-butyltin on the marine worm, Platynereisdumerilii (Polychaeta: Nereidae)

The genotoxic, cytotoxic and ontogenetic (embryo-larval) or developmental effects of tri- n-butyltin (TBT), were investigated in Platynereis dumerilii. Following the determination of maximum tolerated dose with regard to ontogenetic effects and mortality, early life stages of P. dumerilii were exposed to a range of TBT concentrations. Subsequently, the embryo-larvae were analysed for evidence of genotoxicity and cytotoxicity. Genotoxicity was assessed using cytogenetic endpoints that included the frequency of sister chromatid exchanges and chromosomal aberrations from metaphase spreads. Cytotoxicity was evaluated by determining the proliferative rate index of the growing embryo-larval cells using 5-bromodeoxyuridine labelling of the chromosomes or fluorescence plus Giemsa staining technique. TBT-exposed embryo-larvae of P. dumerilii exhibited sensitivity similar to that of other invertebrates, indicating that P. dumerilii is a suitable ecotoxicity test species. The results also suggested dose-dependent effects for genotoxic and cytotoxic end points in relation to TBT exposure. The present study highlights the need to elucidate the relative importance of direct genotoxic and indirect effects through production of genotoxic hormonal derivatives.

Chromosomal damage in peripheral blood lymphocytes of traffic policemen and taxi drivers exposed to urban air pollution

Urban air contains a diversity of chemical compounds, some of which are genotoxins. An increased risk of cancer has also been reported in occupations with heavy exposure to traffic-related pollution. The aim of this study was to assess the cytogenetic effects of urban air pollution by analyzing the chromosomal aberration (CA) frequencies in lymphocytes and to estimate the polycyclic aromatic hydrocarbons (PAHs) exposure by measuring urinary 1-hydroxypyrene (1-OHP) levels. A total of 15 traffic policemen and 17 taxi drivers working in the city of Ankara were the exposed groups and 23 healthy men working in the office departments were the control group. The overall mean±S.D. values of 1-OHP excretions of traffic policemen, taxi drivers and control subjects were 0.59±0.40 μmol/ mol creatinine, 0.32±0.25 μmol/mol creatinine and 0.57±0.36 μmol/mol creatinine, respectively. Urinary 1-OHP levels of nonsmoking policemen were significantly greater than those of nonsmoking control subjects ( p<0.05). The overall mean±S.D. values for CA frequencies (%) from policemen, taxi drivers and control group were 1.29±1.59, 1.81±1.79, and 0.26±0.73, respectively. There was a significantly greater frequency of CAs in exposed groups relative to the matched control population (p<0.05; p<0.01) . Age, sex and smoking habits have not influenced the cytogenetic end-point in this study. Our results demonstrate that occupational exposure to urban air pollutants leads to a significant induction of cytogenetic damage in peripheral lymphocytes of traffic policemen and taxi drivers.

Studies on The Genotoxicity of An Organophosphorous Pesticide Baytex-1000

Baytex-1000 is an organophorous pesticide. It is widely used both as a larvicide and insecticide in the Na- tional Malaria Eradication Programme in India. Malaria con- trol workers are occupationally exposed to Baytex. Genotoxicity of this pesticide was studied by assaying Mitotic Index (MI), Chromosome Aberrations (CA), Sister Chromatid Exchanges (SCE) and Satellite Associations (SA), in short term lympho- cyte cultures. Peripheral blood samples were obtained from 60 malaria control workers and equal number of matched controls. MI increased upto exposure period of 5 years and declined there- after. CA in controls comprised acentric fragment, chromatid gaps and breaks only, whereas in exposed samples, dicentrics, rings, chromosomal gaps, breaks and translocations; chroma- tid gaps, breaks and isochromatid exchanges were recorded. The frequencies of CA and SCE were significantly increased by smoking and alcohol consumption (P<0.5) and depicted posi- tive correlation with period of exposure. D-G-type SA outnum- bered 2D-2G types. The mean frequencies of various param- eters in controls and workers exposed to Baytex respectively were: MI (4.29 & 6.43), CA (0.933 & 4.200), SCE (4.32 & 7.28) and SA (5.11 & 16.5). The differences were statistically significant (P < 0.05). Time and dose-yield effects of Baytex on micronuclei induction in mouse test system were also signifi- cant (p-0.05). All the cytogenetic end points investigated con- clusively show that Baytex-1000 is a highly genotoxic pesti- cide. The mechanism of its action has been described.

Studies on The Genotoxicity of An Organophosphorous Pesticide Baytex-1000

Baytex-1000 is an organophorous pesticide. It is widely used both as a larvicide and insecticide in the Na- tional Malaria Eradication Programme in India. Malaria con- trol workers are occupationally exposed to Baytex. Genotoxicity of this pesticide was studied by assaying Mitotic Index (MI), Chromosome Aberrations (CA), Sister Chromatid Exchanges (SCE) and Satellite Associations (SA), in short term lympho- cyte cultures. Peripheral blood samples were obtained from 60 malaria control workers and equal number of matched controls. MI increased upto exposure period of 5 years and declined there- after. CA in controls comprised acentric fragment, chromatid gaps and breaks only, whereas in exposed samples, dicentrics, rings, chromosomal gaps, breaks and translocations; chroma- tid gaps, breaks and isochromatid exchanges were recorded. The frequencies of CA and SCE were significantly increased by smoking and alcohol consumption (P<0.5) and depicted posi- tive correlation with period of exposure. D-G-type SA outnum- bered 2D-2G types. The mean frequencies of various param- eters in controls and workers exposed to Baytex respectively were: MI (4.29 & 6.43), CA (0.933 & 4.200), SCE (4.32 & 7.28) and SA (5.11 & 16.5). The differences were statistically significant (P < 0.05). Time and dose-yield effects of Baytex on micronuclei induction in mouse test system were also signifi- cant (p-0.05). All the cytogenetic end points investigated con- clusively show that Baytex-1000 is a highly genotoxic pesti- cide. The mechanism of its action has been described.

Effect of the Route of Administration on the Induction of Cytogenetic Damage and DNA Adducts in Peripheral Blood Lymphocytes of Rats and Mice by Polycyclic Aromatic Hydrocarbons

Experiments were designed to investigate how the route of exposure to polycyclic aromatic hydrocarbons (PAHs) in mice and rats affects the induction of cytogenetic end points and DNA adduction. Both mice and rats were exposed to 100 mg/kg of benz[ a ]anthracene (B[ a ]A), benzo[ b ]fluoranthene (B[ b ]F), benzo[ a ]pyrene (B[ a ]P), or chrysene (Chr) by gavage or by intraperitoneal injection (i.p.). Peripheral blood was removed by cardiac puncture 7 days after PAH administration. Blood samples were analyzed in parallel for sister chromatid exchange (SCE) frequency, the frequency of micronuclei in cytochalasin B-induced binucleate cells (MN bn ), and DNA adduction using 32P-postlabeling. The i.p. route of exposure produced both the highest levels of cytogenetic damage and DNA adducts for each PAH. The mouse was more sensitive than the rat to PAH exposure as measured by SCE induction and the total amount of DNA adducts/ w g DNA.

Effect of the Route of Administration on the Induction of Cytogenetic Damage and DNA Adducts in Peripheral Blood Lymphocytes of Rats and Mice by Polycyclic Aromatic Hydrocarbons

Experiments were designed to investigate how the route of exposure to polycyclic aromatic hydrocarbons (PAHs) in mice and rats affects the induction of cytogenetic end points and DNA adduction. Both mice and rats were exposed to 100 mg/kg of benz[ a ]anthracene (B[ a ]A), benzo[ b ]fluoranthene (B[ b ]F), benzo[ a ]pyrene (B[ a ]P), or chrysene (Chr) by gavage or by intraperitoneal injection (i.p.). Peripheral blood was removed by cardiac puncture 7 days after PAH administration. Blood samples were analyzed in parallel for sister chromatid exchange (SCE) frequency, the frequency of micronuclei in cytochalasin B-induced binucleate cells (MN bn ), and DNA adduction using 32P-postlabeling. The i.p. route of exposure produced both the highest levels of cytogenetic damage and DNA adducts for each PAH. The mouse was more sensitive than the rat to PAH exposure as measured by SCE induction and the total amount of DNA adducts/ w g DNA.

Assessment of chemotherapy‐induced DNA damage in peripheral blood leukocytes of cancer patients using the alkaline comet assay

The alkaline comet assay was employed to assess the pre- and post-treatment levels of in vivo DNA damage in peripheral blood leukocytes of cancer patients. During the study all patients were given antineoplastic drugs, mainly as polychemotherapy. To quantify the DNA damage, two different comet parameters were evaluated: the tail length and the tail moment. Our results indicate marked interindividual variations between baseline DNA damage in peripheral blood leukocytes recorded among cancer patients prior to the chemotherapy. After intravenous administration of various antineoplastic drugs, a significantly increased level of DNA damage in all cancer patients compared to their pre-treatment values was recorded The highest level of DNA damage was seen following administration of 5-fluorouracil, adriamycin, and cisplatin (FAP protocol). The results indicate that administration of antineoplastic drugs in standard protocols is accompanied by significant DNA damage in peripheral blood leukocytes. In order to diminish the potential risks of developing second neoplasms, a continuous biomonitoring of cancer patients after the ending of chemotherapy becomes important. Despite their limitations, present results confirm the usefulness of the alkaline comet assay as a sensitive biomarker of exposure that enables rapid and simple detection of primary DNA damage in peripheral blood leukocytes of cancer patients. Together with standard cytogenetic endpoints, the comet assay provides a powerful technique for the routine detection of critical DNA lesions produced after administration of antineoplastic drugs in the clinical settings.

DNA damage and repair in human leukocytes exposed to styrene-7,8-oxide measured by the comet assay

Styrene-7,8-oxide (SO) is produced by cytochrome p450 monooxygenases as the main mammalian metabolite of styrene, an important industrial chemical present at high concentrations in the ambient air of fiberglass-reinforced plastic plants. Previous studies have shown positive results for SO in the induction of several cytogenetic endpoints in vitro. In this work we have evaluated, by means of the comet assay, the potential of SO to act as a DNA damaging agent in human peripheral leukocytes and the ability of white blood cells to repair the DNA damage induced by this compound. Our results show that SO induces DNA damage at concentrations higher than 50 μM in a dose-dependent manner, and that the lesions produced by SO are efficiently removed within a few hours after the end of treatment.

A systematic review of cytogenetic studies conducted in human populations exposed to cadmium compounds.

Exposure to cadmium fumes or dusts has been associated with an increased risk of lung cancer and the characterisation of the genotoxic potential of cadmium compounds is, among other possible mechanisms, an important element in the assessment of the carcinogenic hazard of the element. While there is some evidence that in experimental systems, cadmium compounds may exert genotoxic effects, the results of the epidemiological studies having examined cytogenetic endpoints in humans exposed to cadmium appear conflicting. Therefore, a systematic review was undertaken to assess whether a cytogenetic effect of cadmium exposure is supported by the studies with the strongest design. The relevant literature was identified through several databases and assessed with a check-list by two reviewers. Causes of heterogeneity between studies were looked for. Results were extracted and the strength of the evidence was evaluated with causality criteria. No studies met the criteria for being considered as very convincing. Several factors were identified that could explain contradictory findings (small sample size, selection bias, insufficient characterisation of exposure, lack of consideration of confounders) but their actual impact could not be conclusively assessed with the published information. Importantly, it should be recognised that the absence of a clear mechanism for the cytogenetic action of cadmium compounds did not allow to select the most appropriate endpoint to be examined. No clear association between cadmium exposure and cytogenetic endpoint appeared but no definite conclusion can be drawn from the existing studies in humans. Future research efforts should mainly focus on experimental studies to understand how cadmium compounds could produce genotoxic/carcinogenic effects, in order to target the most relevant endpoint to be examined in humans.

No evidence for chromosomal instability in radiation workers with in vivo exposure to plutonium.

The availability of cultured lymphocyte preparations from radiation workers with internal deposits of plutonium provided the opportunity to examine whether irradiation of bone marrow cells had induced a transmissible genomic instability manifesting as an increase in de novo chromosome aberrations in descendant cells in the peripheral blood. The men were originally classified as having more than 20% of the maximum permissible body burdens of plutonium, and recent red bone marrow dose calculations provided individual cumulative estimates at the time of sampling ranging up to 1.8 Sv. The initial sampling occurred approximately 10 years after the main major intake, and samples were subsequently taken during three further periods over the following 20 years. Control samples were available from three of the four sampling times. Chromosome analysis of solid Giemsa-stained material revealed no significant differences either in comparisons between the total group of plutonium workers and controls for comparable periods or when the comparisons were restricted to a group of plutonium workers with initial red bone marrow plutonium doses greater than 0.25 Sv. However, the frequencies of cells containing chromatid exchanges, chromatid breaks, and chromosome and chromatid gaps decreased significantly over the study period for both the plutonium workers as a whole and the controls, and a similar fluctuating pattern was seen when sequential samples from groups of the same individuals were examined. Cells with dicentrics, centric rings and excess acentric fragments remained at similar frequencies throughout the study period. There was therefore no evidence from the study of blood lymphocytes for the induction of persistent transmissible genomic instability in the bone marrow of radiation workers with internal deposits of plutonium. The work has, however, confirmed the need for appropriate controls when conducting studies of cytogenetic end points of instability.

Chromosomal aberrations, sister chromatid exchanges and micronuclei induced by pentoxifylline in in vitro cultivated Chinese hamster cells (V79) and human blood lymphocytes

Pentoxifylline (PTX) is a methylxanthine widely used in clinical practice. The mechanism of PTX effects on cellular and molecular level have not been fully explained yet. The present study was carried out to investigate the cytogenetic effect of this drug using cultured Chinese hamster V79 cells and human blood lymphocytes in vitro. The occurrence of chromosomal aberrations (CA), sister chromatid exchanges (SCE) and micronuclei (MN) was observed after the treatment of cells by different concentrations (0.002–2.0 mg/ml) of PTX. In exposed V79 cells and lymphocytes as well, the dose-dependent increases of the above mentioned cytogenetic endpoints were found. The statistically significant increase has appeared at lower PTX concentrations in human lymphocytes than in V79 cells in all the investigated parameters. Our results show that, the applied concentrations of PTX has the clastogenic effect on in vitro cultured V79 cells and human lymphocytes. These findings are notable because of the frequent use of this drug and may serve as preliminary data to the further detailed examination of PTX action on molecular level.

Analysis of adaptive response to bleomycin and mitomycin C

Genetic instability resulting from the disturbances in various mechanisms of DNA-repair is the characteristic feature of cancer cells. One of the possibilities to evaluate the effectiveness of DNA-repair system is the adaptive response (AR) analysis. The AR is a phenomenon by which cells exposed to low, non-genotoxic doses of a mutagen become significantly resistant to a subsequent higher dose of the same or another genotoxic agent. Generally, it is postulated that AR is related to a reduction of damage by the induction of free radical detoxification and/or DNA-repair systems. The existence of various DNA-repair mechanisms poses the question whether there are differences in AR induced by chemicals causing DNA-damage that requires different pathways for its repair. In this paper we present the study on the AR induced by two chemical mutagens, bleomycin (BLM) and mitomycin C (MMC), which differ in their action on DNA. BLM is a radiomimetic agent causing mainly single-strand breaks (SSB) and double-strand breaks (DSB) and, thus, inducing chromosomal aberrations (CA). MMC is a potent bifunctional mutagen acting as an alkylating agent, causing DNA cross-links and inducing sister chromatid exchanges (SCEs). The protective effect induced by low doses of tested chemicals was analysed in whole blood human lymphocytes using cytogenetic endpoints (CA for BLM and SCE for MMC, respectively) as a measure of chromosomal instability. There was a significant difference between the protective effects induced by BLM and MMC in the lymphocytes of the same group of donors. The pre-treatment with a low dose of BLM-induced almost 50% decrease in the frequency of CA induced by challenging dose (CD), while the protective effect of MMC was below 20%. The higher AR induced by BLM may be related to the repair processing of BLM-induced DNA-damages. There was also a variability in ARs among individuals, which may reflect the differences in individual DNA-repair capacity.

Micronuclei in peripheral blood lymphocytes and buccal epithelial cells of Polish farmers exposed to pesticides

In this biomonitoring study, we investigated whether an occupational exposure to a complex mixture of chemical pesticides produced a significant increase of micronuclei (MN) in both peripheral blood lymphocytes and buccal cells. Forty-nine male workers exposed to pesticides, from an agricultural area of Małopolska Region in Southern Poland, together with 50 men from the same area without indication of exposure to pesticides that served as controls, were used in this investigation. No statistically significant differences in the frequencies of cytogenetic damage were detected between exposed and control individuals, for either type of cells. The multiple linear regression analysis in the case of lymphocytes indicated that the studied cytogenetic endpoints were inversely influenced by alcohol; whilst a negative binomial regression, in the case of buccal cells, indicated that the MN values were directly influenced by the ingestion of red meat. An inverse negative relationship between the cytokinesis-block proliferation index and age, and a significant increase of miscarriages due to the exposure to pesticides were also observed.

Biomarkers for assessing occupational exposures to 1,3-butadiene

The overall objective of this study was to evaluate a continuum of biomarkers in blood and urine for their sensitivities as indicators of low level occupational exposures to 1,3 butadiene (BD). The study design was largely cross-sectional, with biological samples collected within a short timeframe. Personal 8-h BD exposure measures were made on several occasions over a 60-day period for each potentially exposed worker in order provide maximum accuracy for this independent variable and to accommodate the different expression intervals of the several biomarkers. Co-exposures to styrene, toluene and benzene were also measured. The study included 24 BD monomer production workers (mean BD exposure=0.642 mg/m 3), 34 polymerization workers (mean BD exposure=1.794 mg/m 3) and 25 controls (mean BD exposure=0.023 mg/m 3). The several biomarkers were measured by a consortium of investigators at different locations in the US and Europe. These biomarkers included: (1) metabolic genotypes (CYP2E1, EH, GST M1, GST T1, ADH2, ADH3), determined in Prague and Burlington, VT; (2) urinary M1 and M2 metabolites (1,2-dihydroxy-4-[ N-acetylcysteinyl]-butane and 1-hydroxy-2-[ N-acetylcysteinyl]-3-butene, respectively), determined in Albuquerque, NM and Leiden; (3) hemoglobin adducts ( N-[2-dihydroxy-3-butenyl]valine=HBVal and N-[2,3,4-trihydroxybutyl]valine=THBVal), determined in Amsterdam and Chapel Hill, NC, respectively; (4) HPRT mutations determined by autoradiographic assay in Galveston, TX, with slides re-read in Burlington, VT; (6) hypoxanthine-guanine phosphoribosyltransferase (HPRT) mutations determined by cloning assay in Leiden with mutational spectra characterized in Burlington, VT; (7) sister chromatid exchanges and chromosome aberrations determined by standard methods and FISH analysis in Prague. Urinary M1 and M2 metabolites and HBVal and THBVal hemoglobin adducts were all significantly correlated with BD exposure levels, with adducts being the most highly associated. No significant relationships were observed between BD exposures and HPRT mutations or any of the cytogenetic endpoints, regardless of method of assay.