István Vincze

Impact of metabolic genotypes on levels of biomarkers of genotoxic exposure.

Phase I and Phase II xenobiotic-metabolising enzyme families are involved in the metabolic activation and detoxification of various classes of environmental carcinogens. Particular genetic polymorphisms of these enzymes have been shown to influence individual cancer risk. A brief overview is presented about recent research of the relationship between metabolic genotypes and internal dose, biologically effective dose and cytogenetic effects of complex and specific genotoxic exposures of human study populations, and we report our new results from two molecular epidemiological studies. We investigated the effects of multiple interactions among CYP1A1 Ile462Val, CYP1A1 MspI, CYP1B1 Leu432Val, CYP2C9 Arg144Cys, CYP2C9 Ile359Leu, NQO1 Pro189Ser, GSTM1 gene deletion and GSTP1 Ile105Val genotypes on the levels of carcinogen-DNA adducts determined by (32)P-postlabelling and PAH-DNA immunoassay in peripheral blood lymphocytes from workers occupationally exposed to polycyclic aromatic hydrocarbons in aluminium plants, and in bronchial tissue from smoking lung patients. A statistically significant positive linear correlation was observed between white blood cell aromatic DNA adduct and urinary 1-hydroxypyrene (1-OHPY) levels from potroom workers with GSTM1 null genotype (P=0.011). Our results suggest interactions between GSTM1 and GSTP1 alleles in modulation of urinary 1-OHPY levels and white blood cell DNA adduct levels in the PAH-exposed workers. Interactions between GSTM1 and GSTP1 alleles, in association with particular genotype combinations of CYPs, were also recognised in bronchial aromatic DNA adduct levels of smoking lung patients. The impact of single metabolic genotypes and their combinations on biomarkers of exposure was usually weak, if any, in both our studies and reports of the literature. The effect of special metabolic gene interactions may be better recognised if the compared groups of individuals are stratified for multiple potential modulators of the observable biomarker end-point, and/or if chemical structure-specific biomarker methods are applied.

Biomonitoring of human genotoxicity induced by complex occupational exposures

Sensitivity, specificity and correlations among several biomarkers for monitoring occupational exposure to complex mixtures of genotoxic agents were assessed in occupational environments in Hungarian study populations. The studies have been focused on DNA adduct formation, urinary metabolites, mutations and micronuclei induced by exposures to complex organic mixtures. In two Hungarian aluminium plants, increased DNA adduct and 1-hydroxypyrene (1-OH-PY) levels were observed in workers as compared to controls. However, no association between the biomarker levels was evident on an individual basis. In Hungarian garage mechanics, DNA adduct determinations did not show increased genotoxic exposure as compared to the controls. However, ambient air measurements, significantly enhanced 1-OH-PY levels, and slightly enhanced frequency of micronuclei indicated increased polycyclic aromatic hydrocarbon (PAH) exposure in the garages, as compared to the general environment. In a Hungarian vulcanizing plant, DNA adduct determinations and 1-OH-PY did not show significantly elevated exposure levels as compared to controls. The glycophorin A (GPA) somatic mutation assay was also negative for this occupational exposure. The results support previous observations of a lack of correlation between DNA adducts detectable by 32P-postlabelling and those measured by the PAH-DNA immunoassay in the same DNA sample. These studies also demonstrate a lack of close correlation between levels of DNA adducts and urinary 1-OH-PY in the same individual.

32P-postlabelling detection of aromatic DNA adducts in peripheral blood lymphocytes from aluminium production plant workers.

Aluminium production plant workers are exposed to a great number of airborne polycyclic aromatic hydrocarbons and epidemiological studies suggest that these workers are at increased risk of lung and bladder cancer. Blood samples from 46 workers at 2 primary aluminium plants and from 29 occupationally unexposed control individuals were analysed. DNA was isolated from the peripheral blood lymphocytes and aromatic DNA adducts were detected by 32P-postlabelling assay using the nuclease P1 digestion procedure for the enrichment of the adducts. The total levels of DNA adducts of exposed individuals varied from the detection limit of about 0.5 adducts/10(8) nucleotides up to 7.1 adducts/10(8) nucleotides and control adduct levels were up to 2.42 adducts/10(8) nucleotides. There was no significant difference between the mean adduct levels of the control group and of the individuals of one plant. However, the mean DNA adduct level obtained from workers of the second plant was significantly higher than that of the controls (p less than 0.001) and of the first plant (p less than 0.01), respectively. This difference can be attributed to differences in the design of technology and different levels of exposure at the 2 plants. The results of this study encourage further investigations of the use of peripheral white blood cells as marker cells and of 32P-postlabelling analysis for monitoring occupational exposure to mixtures of environmental carcinogenic pollutants.

Biomonitoring of genotoxic exposure in aluminium plant workers by determination of DNA adducts in human peripheral blood lymphocytes

A longitudinal human biomonitoring study has been performed in two Hungarian primary aluminium production plants that operated Söderberg cells. Carcinogen-DNA adducts have been determined by 32P-postlabelling and competitive enzyme-linked immunosorbent assay in peripheral blood lymphocytes from potroom workers and occupationally unexposed control individuals. Blood samples were collected on three occasions; the first two occasions were 1 year apart during normal operation, and the last samples were taken 6 months after close-down of aluminium production. Assays of the first set of samples demonstrated no significant difference between the control group and workers in Plant I. Workers in Plant II had significantly higher DNA adduct levels than individuals in the control group and workers in Plant I. One year later a significant elevation of DNA adducts was detected in Plant I so that values approached those seen in Plant II, which remained unchanged. In the last sample set there was no difference between former potroom workers and occupationally unexposed individuals. The results suggest that carcinogen-DNA adducts are a useful biomarker for monitoring occupational genotoxic exposure to polycyclic aromatic hydrocarbons, and that the findings can contribute to improved health risk assessment.

32P-Postlabelling analysis of DNA adducts of benzo[a]pyrene formed in complex metabolic activation systems in vitro

The influence of cytochrome P-450-linked monooxygenase, epoxide hydrolase, UDP-glucuronyltransferase and glutathione S-transferases on the metabolic activation of benzo[a]pyrene (BP) was studied by incubating BP with preparations of rat-liver microsomal and cytosolic fractions in the presence of exogenous DNA. 32P-Postlabelling analysis of the DNA revealed the presence of covalently bound adducts formed by BP, which were visualised as radioactive spots on autoradiographs of thin-layer chromatograms. The effects on the adduct profile of adding different combinations of 1,2-epoxy-3,3,3-trichloropropane (TCPO), UDP-glucoronic acid (UDPGA) and glutathione (GSH) to the incubation mixture were determined. As many as 14 different DNA adducts were resolved and quantitated on the chromatograms, the numbers and quantities of which varied within a large range depending on the incubation conditions. The influence of the enzyme inhibitor and cofactors on the adduct patterns reflects the complex effects of simultaneous enzyme interactions on the metabolic activation of BP.

Geographical Aspects of Mortality and Morbidity Data in Hungary: A GIS Analysis

Within the framework of the National Environmental Health Action Programme of Hungary (NEHAP) (Pinter 1996) a health environmental geographic information system (HEGIS) has been developed in the National Institute of Environmental Health. The aim of this system was to evaluate the association between health and environmental data, in order to track spatial and temporal changes and thus to provide a basis for planning of health risk reduction and intervention programmes (Gordon and Womersley 1997).