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Epidemiologic studies indicate that prolonged exposure to high pollution levels is associated with increased risk of cancer, especially lung cancer. However, under conditions of moderate or low air pollution, epidemiologic evidence does not permit reliable conclusions. Biomarker-based population studies may serve as complementary tools providing a better understanding of the relative contribution of ambient atmospheric pollution to the overall genotoxic burden suffered by city dwellers. However, past efforts to apply biomarkers to studies of low levels exposure to urban air pollution have given inconclusive results, partly because of the absence of adequate data on personal exposure, covering a time-window which is appropriate for the biomarkers being examined, as well as a battery of biomarkers reflecting different stages of the carcinogenic process. In the present paper, the potential of biomarker-based population studies to aid the assessment of the genotoxic and carcinogenic effects of urban air pollution is reviewed by reference to the achievements and limitations of earlier reported studies. The design and methodology adopted in a recently completed large-scale population study, carried out in the context of the European Union Environment and Climate Programme, known by the short name of AULIS project, is discussed and descriptive statistics of the main findings of the project are presented. These findings indicate that for cohorts suffering moderate-to-low exposures to airborne particulate-bound polycyclic aromatic hydrocarbons (PAHs), no simple correlation with biomarkers of genotoxicity existed and suggest that additional factors made a significant contribution to the overall genotoxic burden.

The relationship between intrauterine growth retardation (IUGR) and exposure to particulate matter [less than/equal to] 10 microm (PM(10)) and particulate matter [less than and equal to] 2.5 microm (PM(2.5))( )in early pregnancy was recently studied in the highly polluted district of Teplice (Northern Bohemia). From this observation rose the question about the possible role of the carcinogenic fraction of polycyclic aromatic hydrocarbons (c-PAHs), which are usually bound to fine particles. The impact of c-PAHs and fine particles on IUGR was analyzed in Teplice and in Prachatice, a region with similarly high c-PAH but low particle levels. All European, single live births occurring in a 4-year period in Teplice (n = 3,378) and Prachatice (n = 1,505) were included. Detailed personal data were obtained via questionnaires and medical records. Mean PM(10), PM(2.5,) and c-PAHs levels during the 9 gestational months (GM) were estimated for each mother. Adjusted odds ratios (AORs) of IUGR for three levels of c-PAHs (low, medium, and high) and for continuous data were estimated after adjustment for a range of covariates using logistic regression models. In the present 4-year sample from Teplice, previously published results about increasing IUGR risk after exposure to particles in the first GM were fully confirmed, but no such effects were found in Prachatice. The AOR of IUGR for fetuses from Teplice exposed to medium levels of c-PAHs in the first GM was 1.60 [confidence interval (CI), 1.06-2. 15], and to high levels 2.15 (CI, 27-3.63). An exposure-response relationship was established by analyzing the continuous data. For each 10 ng increase of c-PAHs in the first GM, the AOR was 1.22 (CI, 1.07-1.39). About the same relationship was observed in Prachatice in spite of the low particle levels. The results prove that exposure to c-PAHs in early gestation may influence fetal growth. The particulate matter-IUGR association observed earlier may be at least partly explained by the presence of c-PAHs on particle surfaces.

It is envisaged that flow cytometric analysis of lymphocyte subsets in cord blood may be used as a biomarker for effects on the immune system of exposure to environmental factors. In order to investigate the possible application of this parameter, we first studied the effects of other factors that may influence the outcome of subset analysis in cord blood. FACS analysis was performed in 112 pairs of umbilical cord blood and of peripheral maternal blood sampled at labour. Whereas in maternal blood no statistically significant effects of medication during labour on T lymphocyte numbers and NK cells were found, in oxytocin- and in oxytocin and prostaglandin-treated mothers B cell numbers showed a statistically significant increase. In cord blood, the course of labour and/or medication during labour were identified as the most important factors determining distribution of major lymphocyte subsets. In cord blood after deliveries without medication or after neuroplegic analgesia (NPA), the mean percentage of cord blood T lymphocytes (CD3 + ) was highest (59%) and that of NK lymphocytes (CD3 ± /CD16 + 56 + ) lowest (20%). The mean percentage of T lymphocytes was significantly lower (52%) and that of NK lymphocytes higher (28%) in cord blood where deliveries were done under NPA in combination with infusion of oxytocin. The combination of NPA with oxytocin and induction of labour by prostaglandin E2 led to a further reduction of T lymphocytes and an increase of NK cells (39% and 38% respectively). The changes in ratio of T and NK lymphocytes were due both to decreasing absolute counts of T lymphocytes and increasing counts of NK lymphocytes. Thus, the effects of labour and/or medication during labour must be taken into account when this parameter is applied as a potential biomarker of effects of environmental factors on the immune system.

Acellular assay of calf thymus DNA+/-rat liver microsomal S9 fraction coupled with (32)P-postlabelling was used to study the genotoxic potential of organic compounds bound onto PM10 particles collected in three European cities-Prague (CZ), Kosice (SK) and Sofia (BG) during summer and winter periods. B[a]P alone induced DNA adduct levels ranging from 4.8 to 768 adducts/10(8) nucleotides in the concentration dependent manner. However, a mixture of 8 c-PAHs with equimolar doses of B[a]P induced 3.7-757 adducts/10(8) nucleotides, thus suggesting the inhibition of DNA adduct forming activity by interaction among various PAHs. Comparison of DNA adduct levels induced by various EOMs indicates higher variability among seasons than among localities. DNA adduct levels for Prague collection site varied from 19 to 166 adducts/10(8) nucleotides, for Kosice from 22 to 85 and for Sofia from 6 to 144 adducts/10(8) nucleotides. Bioactivation with S9 microsomal fraction caused 2- to 7-fold increase in DNA adduct levels compared to -S9 samples, suggesting a crucial role of indirectly acting genotoxic EOM components, such as PAHs. We have demonstrated for the first time a significant positive correlation between B[a]P content in EOMs and total DNA adduct levels detected in the EOM treated samples (R=0.83; p=0.04). These results suggest that B[a]P content in EOM is an important factor for the total genotoxic potential of EOM and/or B[a]P is a good indicator of the presence of other genotoxic compounds causing DNA adducts. Even stronger correlation between the content of genotoxic compounds in EOMs and total DNA adduct levels detected (R=0.94; p=0.005) was found when eight c-PAHs were taken into the consideration. Our findings support a hypothesis that a relatively limited number of EOM components is responsible for a major part of its genotoxicity detectable as DNA adducts by (32)P-postlabelling.

Principal aims of this study were at first, to find a relevant human derived cell line to investigate the genotoxic potential of PAH-containing complex mixtures and second, to use this cell system for the analysis of DNA adduct forming activity of organic compounds bound onto PM10 particles. Particles were collected by high volume air samplers during summer and winter periods in three European cities (Prague, Kosice, and Sofia), representing different levels of air pollution. The genotoxic potential of extractable organic matter (EOM) was compared with the genotoxic potential of individual carcinogenic polycyclic aromatic hydrocarbons (c-PAHs) as well as their artificial mixtures. Metabolically competent human hepatoma HepG2 cells, confluent cultures of human diploid lung fibroblasts (HEL), and the human monocytic leukemia cell line THP-1 were used as models. DNA adducts were analyzed by (32)P-postlabeling. The total DNA adduct levels induced in HepG2 cells after exposure to EOMs were higher than in HEL cells treated under the same conditions (15-190 versus 2-15adducts/10(8) nucleotides, in HepG2 and HEL cells, respectively). THP-1 cells exhibited the lowest DNA adduct forming activity induced by EOMs (1.5-3.7adducts/10(8) nucleotides). A direct correlation between total DNA adduct levels and c-PAH content in EOM was found for all EOMs in HepG2 cells incubated with 50microg EOM/ml (R=0.88; p=0.0192). This correlation was even slightly stronger when B[a]P content in EOMs and B[a]P-like adduct spots were analyzed (R=0.90; p=0.016). As THP-1 cells possess a limited metabolic capacity for most c-PAHs to form DNA reactive intermediates and are also more susceptible to toxic effects of PAHs and various EOM components, this cell line seemed to be an inappropriate system for genotoxicity studies of PAH-containing complex mixtures. The seasonal variability of genotoxic potential of extracts was stronger than variability among the three localities studied. In HepG2 cells, the highest DNA adduct levels were induced by EOM collected in Prague in the winter period, followed by Sofia and Kosice. However, in the summer sampling period, the order was quite opposite: KosiceSofiaPrague. When the EOM content per m(3) of air was taken into consideration in order to compare real exposures of humans to genotoxic compounds in all three localities, extracts from respirable dust particles collected in Sofia exhibited the highest genotoxicity regardless of the sampling period. The results indicate that most of DNA adducts detected in cells incubated with EOMs have their origin in low concentrations of c-PAHs representing 0.03-0.17% of EOM total mass. Finally, our results suggest that HepG2 cells have a metabolic capacity for PAHs similar to human hepatocytes and represent therefore the best in vitro model for investigating the genotoxic potential of complex mixtures containing PAHs among the three cell lines tested in this study.

It has been hypothesized that mutational events may be involved in the atherogenetic process and that at least a portion of atherosclerotic plaques may be the results of monoclonal proliferation of a single mutated smooth muscle cell (SMC). Therefore, atherosclerosis may be similar to carcinogenesis and may have an environmental etiology. We have analyzed bulky-aromatic DNA adducts in human thoracic aortas from male subjects, aged between 30-60 years, who died suddenly or accidentally, and who had been examined by autopsy within 24 h after death. We found significantly (P < 0.001) higher DNA adduct levels in the samples from subjects with frequent atherosclerotic changes in the whole body ("Cases", N = 76) compared with those having few atherosclerotic changes ("Controls", N = 57). We also observed a significantly elevated weight of heart and plasma levels of total and LDL cholesterol in "Cases" vs "Controls". Significant differences in DNA adduct levels between smokers and nonsmokers were observed in "Controls" only. Multivariate linear regression analyses with age-adjusted data confirmed a significant influence of LDL cholesterol (P < 0.001), vitamin A (P < 0.01), smoking behavior (P < 0.05; evaluated as plasma cotinine levels) and NAT2 genotypes (P < 0.05) on bulky-aromatic DNA adduct levels. The induction of DNA adducts suggests that alterations at the DNA level may contribute to the development of atherosclerosis. Furthermore, atherogenesis and carcinogenesis may share a similar etiology, i.e. genotoxic action of environmental chemicals.

The modulation of 1,3-butadiene (BD)-induced DNA adducts by occupational exposure, glutathione S-transferase (GST) genotypes, single-strand breaks, and cytogenetic end points was studied in 15 workers and 11 controls. The exposed group consisted of 8 smokers and 7 nonsmokers, whereas the control group consisted of 7 nonsmokers and 4 smokers. Among all subjects, the adduct levels in workers lacking GSTM1 were significantly higher than in those who were GSTM1 positive (P = 0.026), and individuals with combined GSTM1(−) and GSTT1(+) genotype had elevated level of adducts compared to that of persons with GSTM1(+) and GSTT1(+) (P = 0.049). The increase in BD–DNA adduct levels in all subjects was significantly related to BD exposure and GSTM1 genotype (linear multiple regression analysis, P = 0.001; P = 0.035). The results suggest that DNA adducts serve as a sensitive and specific biomarker, integrating exposure and host metabolic capacity, although the data are limited to a small number of subjects. Environ. Mol. Mutagen. 37:226–230, 2001. © 2001 Wiley-Liss, Inc.

Objectives: To assess in venous cord blood the distribution of major lymphocyte subsets according to pH and medications used during labor. Design and methods: Venous cord blood was sampled immediately after labor from 70 newborns (35 males and 35 females) delivered vaginally. Lymphocytes were immunophenotyped by flow cytometry and pH was measured using the AVL 900 automated blood gas analysis system. Data on birth weight, gestational age at delivery, length of labor, presence of stained amniotic fluid, medications used during labor, maternal risk factors, age and parity were collected. Results: The percentage of T lymphocytes decreased while the percentage of NK lymphocytes increased with decreasing pH over the whole range of pH values. The proportions of T and NK lymphocytes were associated with the administration of neuroplegics, spasmolytics or dihydroergotoxin in the first stage of labor. Conclusions: Cord blood pH and labor-associated variables should be taken into account to adequately interpret the profile of major lymphocyte subsets as a marker of the effect of different prenatal factors on the immune system of neonates.

1,3-Butadiene (BD) is an important chemical widely used in the synthetic rubber industry. Hemoglobin adducts of two of its reactive metabolites have been already investigated as possible parameters for exposure assessment. In this study hemoglobin adducts of epoxybutene (EB) were analyzed in blood samples from 17 workers in a BD monomer production unit and 19 controls in a heat production unit of a petrochemical plant near Prague, Czech Republic. BD exposure was determined by personal air sampling. The median level of exposure was 440 microg/m3 (range11-17 mg/m3) for the exposed workers and6 microg/m3 (5-150 microg/m3) for the controls. The adduct N-(2-hydroxy-3-butenyl)valine (HBVal) formed by the reaction of the N-terminal valine of globin with carbon-1 of EB was measured. The N-alkylated amino acid was analyzed by gas chromatography/mass spectrometry (GC/MS) after degradation by the modified Edman procedure. Using published methods problems arose with high background levels, especially in the negative ion chemical ionization (NCI) mode. In the present study a limit of detection of 0.2 pmol/g globin was achieved by using 400 mg globin, a variation in extraction solvents, an additional purification step and a widely extended GC temperature program. The median hemoglobin adduct level of the Czech BD monomer production workers (0.7 pmol/g globin; n = 17) was significantly higher than that of the controls (0.2 pmol/g globin; n = 19; P0.05). Smoking controls showed higher hemoglobin adduct levels than nonsmoking controls (P0.1) and significantly higher BD exposure levels (P0.01).

It has long been hypothesized that schizophrenia may be related to a specific injury of genetic material. There is some evidence of chromosomal abnormalities (DeLisi et al. 1988). Another study examined the repair defects seen in schizophrenic patients when their lymphocytes were treated with 4-nitroquinoline or gamma-radiation (Zasukhina 1987). However, no significant DNA-repair defect was observed by Magin et al. (1991).