Synthesis and analysis of DNA adducts of arylamines

Atylamines and nitroarenes are very important environmental and occupational pollutants. Genotoxic effects of arylamines are believed to be initiated by the formation of DNA adducts. DNA adducts of arylamines have been found in experimental animals and in exposed humans, and are predominantly formed with the carbon 8 of 2'-deoxyguanosine. Reference standards are necessary to develop methods for the quantification of DNA-adducts. Therefore, we have synthesized the 2'-deoxyguanosin-8-yI adducts of 2-methylaniline, 2-chloroaniline, 4-chloroaniline, 2,4dimethylaniline, and 2,6-dimethylaniline. The products were characterized by ¹H-NMR, ¹³C-NMR, MS and UV. The corresponding 2'-deoxyguanosine-3' -monophosphate adducts were synthesized for the quantification of DNA adducts by the ³²P-postlabelling technique. A GC-MS method was developed for the analysis of the new adducts as an alternative to the ³²P-postlabelling. DNA was spiked with the synthesized adducts and treated with 0.3 m NaOH overnight at 110 °C in the presence of a deuterated internal standard. We observed up to 80% recovery from about 1 adduct in 10⁸ to 1 in 10⁵ nucleotides.     

In vitro genotoxicity of PAH mixtures and organic extract from urban air particles part I: acellular assay

Acellular assay of calf thymus DNA ± rat liver microsomal S9 fraction coupled with ³²P-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/10⁸ 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/10⁸ 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/10⁸ nucleotides, for Kosice from 22 to 85 and for Sofia from 6 to 144 adducts/10⁸ 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 ³²P-postlabelling.     

P-Postlabelling and mass spectrometric methods for analysis of bulky, polyaromatic carcinogen—DNA adducts in humans

There has been significant recent progress toward the development of human carcinogen—DNA adduct biomonitoring methods. ³²P-Postlabelling is a technique which has found wide application in human studies. ³²P-Postlabelling involves enzymatic preparation and labelling of DNA samples, followed by chromatographic separation of carcinogen—nucleotide adducts from unadducted nucleotides. Thin-layer ion-exchange and high-performance liquid chromatography (HPLC) have been utilized. This paper critically reviews ³²P-postlabelling methods for analysis of bulky, polyaromatic carcinogen—DNA adducts and details a strategy to optimize this technique for monitoring human samples. Development of a human carcinogen biomonitoring method requires that the biomarker meet certain criteria: that the biomarker be responsive to exposures known to increase human cancer risk, to reductions in those exposures, and to the influence of metabolic differences. In addition, reliable samples must be available by non-invasive means. The ability of ³²P-postlabelling to meet these criteria is traced in the literature and discussed. Identification of specific carcinogen—DNA adducts is a difficult task due to the low (femtomole) levels in human target tissues. Because co-chromatography in thin-layer chromatography (TLC) is generally not considered to be proof of chemical identity, both synchronous fluorescence and HPLC in conjunction with ³²P-postlabelling and TLC are used to confirm the identity of specific carcinogen-DNA adducts in human samples. Mass spectrometry is a highly specific method, the sensitivity of which has been improved to the point which may allow its use to confirm the identity of carcinogen—DNA adducts isolated by ³²P-postlabelling and other methods. The literature relating to the use of mass spectral techniques in carcinogen—DNA adduct analysis is reviewed.     

Combination of high-performance liquid chromatography and thin-layer chromatography separation of five adducted nucleotides isolated from liver resulting from intraperitoneal administration with 7H-dibenzo[c,g]carbazole to mice

7H-Dibenzo[c,g]carbazole, DBC, is a potent environmental liver carcinogen. Liver DNA from mice treated with DBC exhibited seven distinct DBC-DNA adducts as detected by ³²P-postlabeling using multidimensional TLC. To improve quantitation and chemically characterize the adducts, DNA samples were hydrlyzed, ³²P-postlabeled and the adducts were separated from the unadducted normal nucleotides on TLC using a D1 solvent, 0.65 M sodium phosphate (pH 6.8). Adducts were eluted from the TLC plates with 4.0 M pyridinium formate, concentrated, resuspended in 50% aqueous methanol and injected onto the HPLC; five individual adduct peaks were resolved and collected by this method. This approach will prove useful to decrease analysis time and improve chemical characterization of tightly clustered DNA adducts generated in vivo.     

Carcinogen DNA adducts and the risk of colon cancer: case-control study

Colorectal cancer represents 8.5% of all tumours at the King Faisal Specialist Hospital & Research Centre. Environmental and dietary carcinogens such as polycyclic aromatic hydrocarbons (PAHs) and heterocyclic amines (HCAs) have long been suspected to play a prominent role in colon cancer aetiology. We designed a case-control study to test the hypothesis of whether or not the presence of DNA adducts can play a role in the aetiology of colon cancer. DNA adducts were measured in 24 cancerous and 20 non-cancerous tissue samples of newly diagnosed colon cancer patients by ³²P-post-labelling technique. Normal tissue from 19 hospital patients served as controls. The mean levels of adducts per 10¹⁰ nucleotides in cancerous and non-cancerous tissue were 151.75±217.27 and 114.81±186.10, respectively; however, only adducts in cancerous tissue were significantly higher than controls (32.78±57.51 per 10¹⁰ nucleotides) with p-values of 0.017. No BPDE-DNA adducts were found. No relationship was found between urinary cotinine as a marker of tobacco smoke and 1-hydroxypyrene as an indicator of an individual's internal dose of PAHs and DNA adducts. In a logistic regression model, only adducts in cancerous tissue were associated with the subsequent risk of colon cancer, with an odds ratio of 3.587 (95% confidence interval 0.833-15.448) after adjustment for age and the duration of living in the current region, but of a borderline significance (p=0.086). Although it is difficult to arrive at a definite conclusion from a small dataset, our preliminary results suggest the potential role of DNA adducts in the colon carcinogenesis process. Additional studies with larger sample sizes are needed to confirm our preliminary finding. It is also important to identify the structural characterization of these unknown DNA adducts in order to have a better understanding of whether or not environmental carcinogens play a role in the aetiology of colon cancer.     

PAH-DNA adducts in environmentally exposed population in relation to metabolic and DNA repair gene polymorphisms

Epidemiologic studies indicate that prolonged exposure to particulate air pollution may be associated with increased risk of cardiovascular diseases and cancer in general population. These effects may be attributable to polycyclic aromatic hydrocarbons (PAHs) adsorbed to respirable air particles. It is expected that metabolic and DNA repair gene polymorphisms may modulate individual susceptibility to PAH exposure. This study investigates relationships between exposure to PAHs, polymorphisms of these genes and DNA adducts in group of occupationally exposed policemen (EXP, N = 53, males, aged 22–50 years) working outdoors in the downtown area of Prague and in matched “unexposed” controls (CON, N = 52). Personal exposure to eight carcinogenic PAHs (c-PAHs) was evaluated by personal samplers during working shift prior to collection of biological samples. Bulky-aromatic DNA adducts were analyzed in lymphocytes by ³²P-postlabeling assay. Polymorphisms of metabolizing (GSTM1, GSTP1, GSTT1, EPHX1, CYP1A1-MspI) and DNA repair (XRCC1, XPD) genes were determined by PCR-based RFLP assays. As potential modifiers and/or cofounders, urinary cotinine levels were analyzed by radioimmunoassay, plasma levels of vitamins A, C, E and folates by HPLC, cholesterol and triglycerides using commercial kits. During the sampling period ambient particulate air pollution was as follows: PM10 32–55 μg/m³, PM2.5 27–38 μg/m³, c-PAHs 18–22 ng/m³; personal exposure to c-PAHs: 9.7 ng/m³ versus 5.8 ng/m³ (P < 0.01) for EXP and CON groups, respectively. The total DNA adduct levels did not significantly differ between EXP and CON groups (0.92 ± 0.28 adducts/10⁸ nucleotides versus 0.82 ± 0.23 adducts/10⁸ nucleotides, P = 0.065), whereas the level of the B[a]P-“like” adduct was significantly higher in exposed group (0.122 ± 0.036 adducts/10⁸ nucleotides versus 0.099 ± 0.035 adducts/10⁸ nucleotides, P = 0.003). A significant difference in both the total (P < 0.05) and the B[a]P-“like” DNA adducts (P < 0.01) between smokers and nonsmokers within both groups was observed. A significant positive association between DNA adduct and cotinine levels (r = 0.368, P < 0.001) and negative association between DNA adduct and vitamin C levels (r = −0.290, P = 0.004) was found. The results of multivariate regression analysis showed smoking, vitamin C, polymorphisms of XPD repair gene in exon 23 and GSTM1 gene as significant predictors for total DNA adduct levels. Exposure to ambient air pollution, smoking, and polymorphisms of XPD repair gene in exon 6 were significant predictors for B[a]P-“like” DNA adduct. To sum up, this study suggests that polymorphisms of DNA repair genes involved in nucleotide excision repair may modify aromatic DNA adduct levels and may be useful biomarkers to identify individuals susceptible to DNA damage resulting from c-PAHs exposure.     

Capillary zone electrophoresis with on-line blotting for separation and detection of 32P-postlabelled DNA adducts

A new technique for the detection of ³²P-postlabelled DNA adducts separated by capillary electrophoresis was developed. By direct transfer from the capillary outlet to a positively charged moving filter paper, eluted radioactive peaks can be quantified using a phosphor imaging detector. With this method it is possible to separate DNA adducts from different carcinogens after ³²P-postlabelling of the modified and unmodified nucleotides with high sensitivity approaching 1 adduct per 10⁹ nucleotides.     

Biological significance of DNA adducts investigated by simultaneous analysis of different endpoints of genotoxicity in L5178Y mouse lymphoma cells treated with methyl methanesulfonate

The biological significance of DNA adducts is under continuous discussion because analytical developments allow determination of adducts at ever lower levels. Central questions refer to the biological consequences of adducts and to the relationship between background DNA damage and exposure-related increments. These questions were addressed by measuring the two DNA adducts 7-methylguanine (7-mG) and O⁶-methyl-2′-deoxyguanosine (O⁶-mdGuo) by LC–MS/MS in parallel to two biological endpoints of genotoxicity (comet assay and in vitro micronucleus test), using large batches of L5178Y mouse lymphoma cells treated with methyl methanesulfonate (MMS). The background level of 7-mG was 1440 adducts per 10⁹ nucleotides while O⁶-mdGuo was almost 50-fold lower (32 adducts per 10⁹ nucleotides). In the comet assay and the micronucleus test, background was in the usual range seen with smaller batches of cells (2.1% Tail DNA and 12 micronuclei-containing cells per 1000 binucleated cells, respectively). For the comparison of the four endpoints for dose-related increments above background in the low-response region we assumed linearity at low dose and used the concept of the “doubling dose”, i.e., we estimated the concentration of MMS necessary to double the background measures. Doubling doses of 4.3 and 8.7 μM MMS were deduced for 7-mG and O⁶-mdGuo, respectively. For doubling the background measures in the comet assay and the micronucleus test, 5 to 15-fold higher concentrations of MMS were necessary (45 and 66 μM, respectively). This means that the contribution of an increase in DNA methylation to biological endpoints of genotoxicity is overestimated. For xenobiotics that generate adducts without background, the difference is even more pronounced because the dose–response curve starts at zero and the limit of detection of an increase is not affected by background variation. Consequences for the question of thresholds in dose–response relationships and for the setting of tolerable exposure levels are discussed.     

DNA adducts in human placenta as biomarkers for environmental pollution, analysed by the 32P-HPLC method

During pregnancy, mothers are exposed to complex chemical mixtures, such as air pollution and smoke from incomplete combustion. In this study DNA adducts were measured in human placentas from 29 mothers. Environmental exposure and several possible biomarkers in relation to levels of DNA adducts were measured. Placental aromatic and bulky DNA adducts were measured with the ³²     

Use of the photo-micronucleus assay in Chinese hamster V79 cells to study photochemical genotoxicity

2-Methoxyaniline (o-anisidine) is a urinary bladder carcinogen in both mice and rats. Since the urinary bladder contains substantial peroxidase activity, we investigated the metabolism of this carcinogen by prostaglandin H synthase (PHS), a prominent enzyme in the urinary bladder, and lactoperoxidase as model mammalian peroxidases. Horseradish peroxidase (HRP)-mediated oxidation of o-anisidine was also determined and compared with the reactions catalyzed by mammalian peroxidases. All three peroxidases oxidized o-anisidine via a radical mechanism. Using HPLC combined with electrospray tandem mass spectrometry, we determined that peroxidases oxidized o-anisidine to a diimine metabolite, which subsequently hydrolyzed to form a quinone imine. Two additional metabolites were identified as a dimer linked by an azo bond and another metabolite consisting of three methoxybenzene rings, which exact structure has not been identified as yet. Using [¹⁴C]-labeled o-anisidine, we observed substantial peroxidase-dependent covalent binding of o-anisidine to DNA, tRNA and polydeoxynucleotides [poly(dX)]. The ³²P-postlabeling assay (a standard procedure and enrichment of adducts by digestion with nuclease P1 or by extraction into 1-butanol prior to ³²P-labeling) was employed as the second method to detect and quantitate binding of o-anisidine to DNA. Using these versions of the ³²P-postlabeling technique we did not observe any DNA adducts derived from o-anisidine. The o-anisidine-DNA adducts became detectable only when DNA modified by o-anisidine was digested using three times higher concentrations of micrococcal nuclease and spleen phosphodiesterase (MN/SPD). We found deoxyguanosine to be the target for o-anisidine binding in DNA using poly(dX) and deoxyguanosine 3′-monophosphate (dGp). A diimine metabolite of o-anisidine is the reactive species forming adducts in dGp. The results strongly indicate that peroxidases play an important role in o-anisidine metabolism to reactive species, which might be responsible for its genotoxicity, and its carcinogenicity to the urinary bladder in rodents. The limitation of the ³²P-postlabeling technique to analyze DNA adducts derived from o-anisidine as a means to estimate its genotoxicity is discussed.     

Detection of DNA adducts using a quantitative long PCR technique and the fluorogenic 5′ nuclease assay (TaqMan)

The detection of DNA adducts is an important component in assessing the mutagenic potential of exogenous and endogenous compounds. Here, we report an in vitro quantitative long PCR (XL-PCR) assay to measure DNA adducts in human genomic DNA based on their ability to block and inhibit PCR amplification. Human genomic DNA was exposed to test compounds and then a target sequence was amplified by XL-PCR. The amplified sequence was then quantified using fluorogenic 5′ nuclease PCR (TaqMan^®) and normalized to a solvent-treated control. The extent of DNA adduction was determined based on the reduction in amplification of the target sequence in the treated sample. A 17.7 kb β-globin fragment was chosen as the target sequence for these studies, since preliminary experiments revealed a two-fold increased sensitivity of this target compared to a 10.4 kb HPRT fragment for detecting hydrogen peroxide-induced DNA damage. Validation of the XL-PCR assay with various compounds demonstrated the versatility of the assay for detecting a wide range of adducts formed by direct acting or S9-activated mutagens. The same DNA samples were also analyzed using ³²P-postlabeling techniques (thin-layer chromatography or high-performance liquid chromatography) to confirm the presence of DNA adducts and estimate their levels. Whereas ³²P-postlabeling with nuclease P₁ enrichment was more sensitive for detecting bulky adducts induced by the compounds benzo[a]pyrene, dimethylbenzanthracene, 3-methylindole, indole 3-carbinol, or 2-acetylaminofluorene, the XL-PCR procedure was more sensitive for detecting smaller or labile DNA adducts formed by the compounds methyl methanesulfonate, diethyl nitrosamine, ethylnitrosourea, diepoxybutane, ICR-191, styrene oxide, or aflatoxin B₁. Compounds not expected to form adducts in DNA, such as clofibrate, phenobarbital, chloroform or acetone, did not produce a positive response in the XL-PCR assay. Thus, quantitative XL-PCR provides a rapid, high-throughput assay for detecting DNA damage that complements the existing ³²P-postlabeling assay with nuclease P₁ enrichment.     

DNA damage and repair in gastric cancer--a correlation with the hOGG1 and RAD51 genes polymorphisms

Aristolochic acid (AA) is a potent nephrotoxin and carcinogen and is the causative factor for Chinese herb nephropathy. AA has been associated with the development of urothelial cancer in humans, and kidney and forestomach tumors in rodents. To investigate the molecular mechanisms responsible for the tumorigenicity of AA, we determined the DNA adduct formation and mutagenicity of AA in the liver (nontarget tissue) and kidney (target tissue) of Big Blue rats. Groups of six male rats were gavaged with 0, 0.1, 1.0 and 10.0 mg AA/kg body weight five times/week for 3 months. The rats were sacrificed 1 day after the final treatment, and the livers and kidneys were isolated. DNA adduct formation was analyzed by ³²P-postlabeling and mutant frequency (MF) was determined using the λ Select-cII Mutation Detection System. Three major adducts (7-[deoxyadenosin-N⁶-yl]-aristolactam I, 7-[deoxyadenosin-N⁶-yl]-aristolactam II and 7-[deoxyguanosin-N²-yl]-aristolactam I) were identified. There were strong linear dose-responses for AA-induced DNA adducts in treated rats, ranging from 25 to 1967 adducts/10⁸ nucleotides in liver and 95–4598 adducts/10⁸ nucleotides in kidney. A similar trend of dose-responses for mutation induction also was found, the MFs ranging from 37 to 666 × 10⁻⁶ in liver compared with the MFs of 78–1319 × 10⁻⁶ that we previously reported for the kidneys of AA-treated rats. Overall, kidneys had at least two-fold higher levels of DNA adducts and MF than livers. Sequence analysis of the cII mutants revealed that there was a statistically significant difference between the mutation spectra in both kidney and liver of AA-treated and control rats, but there was no significant difference between the mutation spectra in AA-treated livers and kidneys. A:T → T:A transversion was the predominant mutation in AA-treated rats; whereas G:C → A:T transition was the main type of mutation in control rats. These results indicate that the AA treatment that eventually results in kidney tumors in rats also results in significant increases in DNA adduct formation and cII MF in kidney. Although the same treatment does not produce tumors in rat liver, it does induce DNA adducts and mutations in this tissue, albeit at lower levels than in kidney.     

DNA adduct formation from quaternary benzo[c]phenanthridine alkaloids sanguinarine and chelerythrine as revealed by the P-postlabeling technique

Using the ³²P-postlabeling assay, we investigated the ability of quaternary benzo[c]phenanthridine alkaloids, sanguinarine, chelerythrine and fagaronine, to form DNA adducts in vitro. Two enhanced versions of the assay (enrichment by nuclease P1 and 1-butanol extraction) were utilized in the study. Hepatic microsomes of rats pre-treated with β-naphthoflavone or those of uninduced rats, used as metabolic activators, were incubated in the presence of calf thymus DNA and the alkaloids, with NADPH used as a cofactor. Under these conditions sanguinarine and chelerythrine, but not fagaronine, formed DNA adducts detectable by ³²P-postlabeling. DNA adduct formation by both alkaloids was found to be concentration dependent. When analyzing different atomic and bond indices of the C₁₁---C₁₂ bond (ring B) in alkaloid molecules we found that fagaronine behaved differently from sanguinarine and chelerythrine. While sanguinarine and chelerythrine showed a preference for electrophilic attack indicating higher potential to be activated by cytochrome P450, fagaronine exhibited a tendency for nucleophilic attack. Our results demonstrate that sanguinarine and chelerythrine are metabolized by hepatic microsomes to species, which generate DNA adducts.     

Smoking-reIated DNA adducts and genetic polymorphism for metabolic enzymes in human lymphocytes

Smoking-related aromatic DNA adducts in lymphocytes were measured from smokers (n = 76), ex-smokers (n = 25) and non-smokers (n = 56) by the ³²P-postlabelling method, to clarify whether a genetic polymorphism for metabolic enzymes could explain the inter-individual variation of DNA adduct levels. Adduct levels were compared with respect to smoking status and polymorphic genotypes of cytochrome P4501A1 (CYP1A1) and glutathione S-transferase M1 (GTSM1). The mean adduct level (1.24 per 10⁸ nucleotides) in smokers was significantly higher than that (0.85 per 10⁸) in non-smokers. Although we expected higher adduct levels in the CYP1A1 variant or GSTM1 null subjects, the adduct level in 'GSN1 nulls' was significantly lower than that in 'GSTM1 presents' among smokers. DNA adduct levels had significant positive correlations with smoking indices such as number of cigarettes or smoking years in all subjects. In smokers only, however, no correlation was found, because there were negative correlations between adduct levels and smoking dose in GSTM1 null genotypes. CYP1A1 genotypes had no effects on adduct levels.     

Polycyclic aromatic hydrocarbon-DNA adducts in humans: relevance as biomarkers for exposure and cancer risk

The methodology applied for DNA adducts in humans has become more reliable in recent years, allowing to detect even background carcinogenic adduct levels in environmentally exposed persons. Particularly, combinations of the various methods now allow the elucidation of specific adduct structures with detection limits of 1 adduct in 10⁸ unmodified nucleotides or even lower. The quantification of polycyclic aromatic hydrocarbon-DNA (PAH-DNA) adducts in human tissues and cells has been achieved with a number of highly sensitive techniques: immunoassays and immunocytochemistry using polyclonal or monoclonal antisera specific for DNA adducts or modified DNA, the ³²P-postlabelling assay, and adduct identification using physicochemical instrumentation. The results summarized in this review show that PAH-DNA adducts have been detected in a variety of human tissues, including target organs of PAH- and tobacco-associated cancers. Although dosimetry has not always been precise, a large number of data now clearly show that lowering exposure to carcinogenic PAH results in decreasing PAH-DNA adduct levels. In most studies, however, bulk DNA of a certain tissue or cell type has been examined, and there were relatively few studies in which mutations as a consequence of DNA damage at specific genes have been investigated. Promising as these biomarker studies seem for epidemiology and health surveillance, future biomonitoring and molecular epidemiological studies should be directed to combine several endpoint measurements: i.e., adduct formation (preferably at specific sites), mutational spectra in cancer-relevant genes, and genetic markers of (cancer) susceptibility in a number of cancer-predisposing genes.     

Purification and recovery of bulky hydrophobic DNA adducts.

For many years (32)P postlabeling has detected DNA adducts at very low levels and yet has not been able to identify unknown adducts. Mass spectrometry offers substantially improved identification powers, albeit at some loss in detection limits. With this ultimate utilization of mass spectrometry in mind, the current research presents a new method to quantitatively purify bulky hydrophobic DNA adducts at levels that are pertinent to ongoing DNA adduct research in human health and environmental fields. This method was demonstrated with benzo[a]pyrene adducts. Purification was accomplished with the use of small columns (7.5-mm frits) with an 11 mg bed of polystyrene-divinlybenzene beads which retained the adducts while permitting the nonadducted nucleotides to be washed out with water. Subsequently, the adducts were eluted with 50% MeOH and the sample was reduced in volume in an evacuated centrifuge. Purification was demonstrated at adduct levels ranging from 4 adducts in 10(6) nonadducted nucleotides to 4 in 10(8). For these levels, analyses by capillary electrophoresis with sample stacking and UV detection determined that recoveries ranged from 91 to 54%, respectively. The adduct quantities isolated should be sufficient to allow the use of current MS capabilities that are linked on-line to separation methodologies such as capillary electrophoresis, capillary electrochromatography, and high-pressure liquid chromatography.     

Lymphocyte DNA adducts and polymorphism in the DNA repair enzyme XPD

The effect of genetic polymorphism of DNA repair enzyme on the DNA adduct levels was evaluated in this study. We explored the relationship between polymorphism in the nucleotide excision repair enzyme XPD and DNA adduct levels in lymphocytes. Lymphocyte DNA adducts were measured by a ³²     

Evidence for the DNA binding and adduct formation of estrone and 17β-estradiol after dimethyldioxirane activation

Estrogens, used widely from hormone replacement therapy to cancer treatment, are themselves carcinogenic, causing uterine and breast cancers. However, the mechanism of their carcinogenic action is still not known. Recently, we found that estrone (E₁) and 17β-estradiol (E₂) could be activated by the versatile epoxide-forming oxidant dimethyldioxirane (DMDO), resulting in the inhibition of rat liver nuclear and nucleolar RNA synthesis in a dose-dependent manner in vitro. Since epoxidation is often required for the activation of chemical carcinogens, we proposed that estrogen epoxidation is the underlying mechanism for the initiation of estrogen carcinogenesis (Carcinogenesis 17 (1996) 1957–1961). It is known that initiation requires the binding of a carcinogen to DNA with the formation of DNA adducts. One of the critical tests of our hypothesis is therefore to determine whether E₁ and E₂ after activation are able to bind DNA. This paper reports that after DMDO activation, [³H]E₁ and [³H]E₂ were able to bind to both A-T and G-C containing DNAs. Furthermore, the formation of E₁–DNA and E₂–DNA adducts was detected by ³²P-postlabeling analysis.     

Benzoapyrene-induced DNA damage in Mytilus galloprovincialis: measurement of bulky DNA adducts and DNA oxidative damage in terms of 8-oxo-7,8-dihydro-2´-deoxyguanosine formation

Bulky DNA adducts and 8-oxo-7,8-dihydro-2´-deoxyguanosine (8-oxodGuo) were measured in gill DNA of benzo[a]pyrene (B[a]P)-exposed mussels (50 mg kg^-1 dw day^-1), respectively by the ³²P-post-labelling technique and high performance liquid chromatography coupled to electrochemical detection assay. A time-course study was performed for both biomarkers and their potential use for marine biomonitoring discussed for the sentinel species studied. In gills, B[a]P-related DNA adducts were positively correlated with B[a     

In vitro genotoxicity of PAH mixtures and organic extract from urban air particles part II: human cell lines

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 ³²P-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 2–15 adducts/10⁸ nucleotides, in HepG2 and HEL cells, respectively). THP-1 cells exhibited the lowest DNA adduct forming activity induced by EOMs (1.5–3.7 adducts/10⁸ 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 50 μg 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: Kosice > Sofia > Prague. When the EOM content per m³ 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.