Sensitivity of different endpoints for in vitro measurement of genotoxicity of extractable organic matter associated with ambient airborne particles (PM10).

Sensitivity and correlations among three endpoints were evaluated to assess the genotoxic potential of organic complex mixtures in vitro. This study was focused on DNA adduct formation, DNA single strand break induction and tumour suppressor p53 protein up-regulation produced by extractable organic matter (EOM) absorbed on respirable particulate matter PM(10) (particulate matter<10microm) collected in three European cities (Prague, Sofia, Kosice) during winter and summer period. To compare the sensitivity of particular endpoints for in vitro measurement of complex mixture genotoxicity, the metabolically competent human hepatoma cell line Hep G2 was treated with equivalent EOM concentration of 50microg/ml. Cell exposure to EOMs resulted in significant DNA adduct formation and DNA strand break induction, however, a lack of protein p53 up-regulation over the steady-state level was found. While the maximum of DNA strand breaks was determined after 2h cell exposure to EOMs, 24h treatment interval was optimal for DNA adduct determination. No substantial location- and season-related differences in EOM genotoxicity were detected using DNA strand break assessment. In agreement with these results no significant variation in DNA adduct levels were found in relation to the locality and season except for the monitoring site in Prague. The Prague EOM sample collected during summer period produced nearly three-fold lower DNA adduct level in comparison to the winter EOM sample. Comparable results were obtained when the ambient air genotoxicity, based on the concentration of carcinogenic PAHs in cubic meter of air (ng c-PAHs/m(3)), was elicited using either DNA adduct or strand break determination. In general, at least six-fold higher genotoxicity of the winter air in comparison to the summer air was estimated by each particular endpoint. Moreover, the genotoxic potential of winter air revealed by DNA adduct assessment and DNA strand break measurement increased in the same order: Kosice<

Assessment of oxidative DNA damage formation by organic complex mixtures from airborne particles PM(10).

The free radical generating activity of airborne particulate matter (PM(10)) has been proposed as a primary mechanism in biological activity of ambient air pollution. In an effort to determine the impact of the complex mixtures of extractable organic matter (EOM) from airborne particles on oxidative damage to DNA, the level of 8-oxo-2'-deoxyguanosine (8-oxodG), the most prevalent and stable oxidative lesion, was measured in the human metabolically competent cell line Hep G2. Cultured cells were exposed to equivalent EOM concentrations (5-150microg/ml) and oxidative DNA damage was analyzed using a modified single cell gel electrophoresis (SCGE), which involves the incubation of whole cell DNA with repair specific DNA endonuclease, which cleaves oxidized DNA at the sites of 8-oxodG. EOMs were extracted from PM(10) collected daily (24h intervals) in three European cities: Prague (Czech Republic, two monitoring sites, Libus and Smíchov), Kosice (Slovak Republic) and Sofia (Bulgaria) during 3-month sampling periods in the winter and summer seasons. No substantial time- and dose-dependent increase of oxidative DNA lesions was detected in EOM-treated cells with the exception of the EOM collected at the monitoring site Kosice, summer sampling. In this case, 2h cell exposure to EOM resulted in a slight but significant increase of oxidative DNA damage at three from total of six concentrations. The mean 8-oxodG values at these concentrations ranged from 15.3 to 26.1 per 10(6) nucleotides with a value 3.5 per 10(6) nucleotides in untreated cells. B[a]P, the positive control, induced a variable but insignificant increase of oxidative DNA damage in Hep G2 cell (approximately 1.6-fold increase over control value). Based on these data we believe that EOM samples extracted from airborne particle PM(10) play probably only a marginal role in oxidative stress generation and oxidative lesion formation to DNA. However, adsorbed organic compounds can undergo various interactions (additive or synergistic) with other PM components or physical factors (UV-A radiation) and in this way they might enhance/multiply the adverse health effects of air pollution.

The XPD 751Gln allele is associated with an increased risk for esophageal adenocarcinoma: a population-based case-control study in Sweden.

Mechanisms behind the strong associations of esophageal adenocarcinoma risk with gastroesophageal reflux (GOR) and body mass remain to be defined. In a nationwide population-based case-control study, we examined associations of polymorphisms in the DNA repair genes XPD, XPC, XRCC1 and XRCC3 with risk of esophageal adenocarcinoma, squamous-cell carcinoma (SCC) and gastric cardia adenocarcinoma, and paid special attention to possible interactions with symptomatic reflux or body mass. We collected blood samples from 96, 81 and 126 interviewed incident cases of esophageal adenocarcinoma, esophageal SCC and gastric cardia adenocarcinoma, respectively, and 472 randomly selected controls, frequency-matched with regard to age and sex. DNA was extracted and polymorphisms in XPD codon 751 (Lys-->Gln), codon 312 (Asp-->Asn), C insertion in intron 10 of XPD, XPC codon 939 (Lys-->Gln), XRCC1 codon 399 (Arg-->Gln) and XRCC3 codon 241 (Thr-->Met) were examined using PCR-RFLP. Odds ratios (ORs) derived from multivariate logistic regression with adjustments for potential confounding factors estimated relative risks. XPD codon 751 Lys/Gln and Gln/Gln genotypes, compared with Lys/Lys genotype, were both associated with a more than doubled risk for esophageal adenocarcinoma (OR=2.4; 95% CI=1.4-4.4; OR=2.7, 95% CI=1.3-5.9). The combined effects of these genotypes and symptomatic GOR or body mass showed borderline significant deviation from additivity. Excess risks for esophageal SCC were also noted for XPD 751Gln variant genotypes. Other studied variants were not found to be related to the three tumors. Our study suggests that XPD 751Gln allele is a potential genetic marker for susceptibility to esophageal adenocarcinoma.

Effect of dietary intake of vitamin A or E on the level of DNA damage, chromosomal aberrations, and micronuclei induced in freshly isolated rat hepatocytes by different carcinogens.

Hepatocytes freshly isolated from male Wistar rats fed a common diet or a vitamin A- or vitamin E-supplemented diet (each for 21, 28, or 41 days) were assayed for sensitivity to DNA breakage and cytogenetic changes induced by carcinogens. Different indirectly acting carcinogens were assayed. N-nitrosomorpholine (NMOR) was the only agent that induced DNA breaks, chromosomal aberrations, and micronuclei in all experiments. Benzo[a]pyrene (B[a]p) and dimethyldibenzo [c,g]carbazole (diMeDBC) induced only DNA breaks in all experiments. Occasionally, B[a]P induced chromosomal aberrations and micronuclei, and diMeDBC induced micronuclei, but not chromosomal aberrations. These results demonstrated that the tested carcinogens assayed at concentrations highly effective in a hypoxanthine phosphoribosyltransferase/V79 system significantly increased DNA damage, while cytogenetic changes were less frequent. In hepatocytes from rats fed vitamin A, a reduction in the severity of all three end points was observed after NMOR treatment. After B[a]P treatment, we found a reduction in DNA breaks and chromosomal aberrations; after treatment with diMeDBC, we observed a reduction in DNA breaks. Treatment with vitamin E was less effective: it reduced DNA strand breaks induced by B[a]P and partially reduced those induced by diMeDBC and NMOR and the level of micronuclei induced by NMOR and B[a]P. Both vitamins reduced the level of DNA strand breaks induced by the oxidative effect of a visible light-excited photosensitizer.

Mutagenicity of 7H-dibenzo[c,g]carbazole and its tissue specific derivatives in genetically engineered Chinese hamster V79 cell lines stably expressing cytochrome P450.

Genetically engineered Chinese hamster V79 cell lines with stable expression of human cytochrome P4501A1 and 1A2 were used to characterize the particular form of P450 enzymes capable of activating 7H-dibenzo[c,g]carbazole (DBC) and its tissue- and organ-specific derivatives, N-methylDBC (N-MeDBC) and 5,9-dimethylDBC (diMeDBC). In addition, a V79 cell line with co-expression of CYP1A2 together with a phase II enzyme, N-acetyltransferase was utilized to study the role of an entire metabolic activation system in biotransformation of these carbazoles. The rise of 6-thioguanine resistant (6-TG(r)) mutations was followed as a marker of biological activity of these agents. None of the carbazoles elevated significantly the frequency of mutations in the parental V79MZ cell line lacking any cytochrome P450 (CYP) activity or in the V79NH cells expressing N-acetyltransferase activity. A variable, however, increase of mutations was found in the cell lines expressing CYP activity. Both DBC, a potent liver and skin carcinogen, and N-MeDBC, a specific sarcomagen, increased significantly (P<0.001) the frequency of 6-TG(r) mutations in V79MZh1A1 cells, expressing the human CYP1A1; in contrast, a strict hepatocarcinogen diMeDBC was devoid of any activity. All carbazoles elevated significantly the level of mutations in the V79MZh1A2 cell line expressing the human CYP1A2, N-MeDBC was most efficient. Co-expression of CYP1A2 together with NAT activity significantly reduced or totally eliminated the mutagenicity of all carbazoles. These data confirm that CYP1A1 is explicitly involved in the activation of sarcomagenic DBC derivatives, whereas CYP1A2 is included in biotransformation of all DBC derivatives. Reactive intermediates formed due to CYP1A2 activation are substrate for conjugation reactions mediated by N-acetyltransferase.