Genome-Wide DNA Methylation in Policemen Working in Cities Differing by Major Sources of Air Pollution.

DNA methylation is the most studied epigenetic mechanism that regulates gene expression, and it can serve as a useful biomarker of prior environmental exposure and future health outcomes. This study focused on DNA methylation profiles in a human cohort, comprising 125 nonsmoking city policemen (sampled twice), living and working in three localities (Prague, Ostrava and Ceske Budejovice) of the Czech Republic, who spent the majority of their working time outdoors. The main characterization of the localities, differing by major sources of air pollution, was defined by the stationary air pollution monitoring of PM2.5, B[a]P and NO2. DNA methylation was analyzed by a genome-wide microarray method. No season-specific DNA methylation pattern was discovered; however, we identified 13,643 differentially methylated CpG loci (DML) for a comparison between the Prague and Ostrava groups. The most significant DML was cg10123377 (log2FC = -1.92, p = 8.30 × 10-4) and loci annotated to RPTOR (total 20 CpG loci). We also found two hypomethylated loci annotated to the DNA repair gene XRCC5. Groups of DML annotated to the same gene were linked to diabetes mellitus (KCNQ1), respiratory diseases (PTPRN2), the dopaminergic system of the brain and neurodegenerative diseases (NR4A2). The most significant possibly affected pathway was Axon guidance, with 86 potentially deregulated genes near DML. The cluster of gene sets that could be affected by DNA methylation in the Ostrava groups mainly includes the neuronal functions and biological processes of cell junctions and adhesion assembly. The study demonstrates that the differences in the type of air pollution between localities can affect a unique change in DNA methylation profiles across the human genome.

The processes associated with lipid peroxidation in human embryonic lung fibroblasts, treated with polycyclic aromatic hydrocarbons and organic extract from particulate matter.

Polycyclic aromatic hydrocarbons (PAHs) may cause lipid peroxidation via reactive oxygen species generation. 15-F2t-isoprostane (IsoP), an oxidative stress marker, is formed from arachidonic acid (AA) by a free-radical induced oxidation. AA may also be converted to prostaglandins (PG) by prostaglandin-endoperoxide synthase (PTGS) induced by NF-κB. We treated human embryonic lung fibroblasts (HEL12469) with benzo[a]pyrene (B[a]P), 3-nitrobenzanthrone (3-NBA) and extractable organic matter (EOM) from ambient air particulate matter <2.5 µm for 4 and 24 h. B[a]P and 3-NBA induced expression of PAH metabolising, but not antioxidant enzymes. The concentrations of IsoP decreased, whereas the levels of AA tended to increase. Although the activity of NF-κB was not detected, the tested compounds affected the expression of prostaglandin-endoperoxide synthase 2 (PTGS2). The levels of prostaglandin E2 (PGE2) decreased following exposure to B[a]P, whereas 3-NBA exposure tended to increase PGE2 concentration. A distinct response was observed after EOM exposure: expression of PAH-metabolising enzymes was induced, IsoP levels increased after 24-h treatment but AA concentration was not affected. The activity of NF-κB increased after both exposure periods, and a significant induction of PTGS2 expression was found following 4-h treatment. Similarly to PAHs, the EOM exposure was associated with a decrease of PGE2 levels. In summary, exposure to PAHs with low pro-oxidant potential results in a decrease of IsoP levels implying 'antioxidant' properties. For such compounds, IsoP may not be a suitable marker of lipid peroxidation.

Butyrate alters expression of cytochrome P450 1A1 and metabolism of benzo[a]pyrene via its histone deacetylase activity in colon epithelial cell models.

Butyrate, a short-chain fatty acid produced by fermentation of dietary fiber, is an important regulator of colonic epithelium homeostasis. In this study, we investigated the impact of this histone deacetylase (HDAC) inhibitor on expression/activity of cytochrome P450 family 1 (CYP1) and on metabolism of carcinogenic polycyclic aromatic hydrocarbon, benzo[a]pyrene (BaP), in colon epithelial cells. Sodium butyrate (NaBt) strongly potentiated the BaP-induced expression of CYP1A1 in human colon carcinoma HCT116 cells. It also co-stimulated the 7-ethoxyresorufin-O-deethylase (EROD) activity induced by the 2,3,7,8-tetrachlorodibenzo-p-dioxin, a prototypical ligand of the aryl hydrocarbon receptor. Up-regulation of CYP1A1 expression/activity corresponded with an enhanced metabolism of BaP and formation of covalent DNA adducts. NaBt significantly potentiated CYP1A1 induction and/or metabolic activation of BaP also in other human colon cell models, colon adenoma AA/C1 cells, colon carcinoma HT-29 cells, or in NCM460D cell line derived from normal colon mucosa. Our results suggest that the effects of NaBt were due to its impact on histone acetylation, because additional HDAC inhibitors (trichostatin A and suberanilohydroxamic acid) likewise increased both the induction of EROD activity and formation of covalent DNA adducts. NaBt-induced acetylation of histone H3 (at Lys14) and histone H4 (at Lys16), two histone modifications modulated during activation of CYP1A1 transcription, and it reduced binding of HDAC1 to the enhancer region of CYP1A1 gene. This in vitro study suggests that butyrate, through modulation of histone acetylation, may potentiate induction of CYP1A1 expression, which might in turn alter the metabolism of BaP within colon epithelial cells.

Oxidative stress in newborns by different modes of delivery.

OBJECTIVES: The aim of our study is to investigate the impact of the type of delivery - vaginal vs. cesarean section on oxidative damage determined as the lipid peroxidation (15-F2t-isoprostane (15-F2t-IsoP) in the cord blood of newborns and venous blood from mothers in two localities with different levels of air pollution: Ceske Budejovice (CB), a locality with a clean air, and Karvina, a locality with high air pollution. RESUTLS: In Karvina, the concentration of PM2.5 was higher than in CB in the summer 2013 (mean±SD: 20.41±6.28 vs. 9.45±3.62 µg/m3, p<0.001) and in the winter 2014 (mean±SD: 53.67±19.76 vs. 27.96±12.34 µg/m3, p<0.001). Similarly, the concentration of B[a]P was higher in Karvina than in CB in the summer 2013 (mean±SD: 1.16±0.91 vs. 0.16±0.26 ng/m3, p<0.001) and in the winter 2014 (5.36±3.64 vs. 1.45±1.19 ng/m3, p<0.001). Delivery procedures differed by the type of anesthesia; at the Cesarean section in CB was used general anesthesia in 73.8% vs. 20.8% in Karvina (p<0.001), epidural anesthesia in CB in 26.2% vs. 77.1% in Karvina (p<0.001), at vaginal delivery was local anesthesia used in CB in 58.9% vs. 14.1% in Karvina (p<0.001). In CB was oxidative stress higher after vaginal delivery (101.7±31.0 pg 15-F2t-isoP/ml plasma) vs. Cesarean section (83.9±26.9 pg 15-F2t-isoP/ml plasma, p<0.001), no difference between the type of delivery was observed in Karvina. CONCLUSION: No difference between the types of delivery was observed in mothers in CB as well as in Karvina. Oxidative stress in newborns in Karvina was significantly affected by the concentrations of PM2.5 and B[a]P in the polluted air.

Toxic Effects of the Major Components of Diesel Exhaust in Human Alveolar Basal Epithelial Cells (A549).

We investigated the toxicity of benzo[a]pyrene (B[a]P), 1-nitropyrene (1-NP) and 3-nitrobenzanthrone (3-NBA) in A549 cells. Cells were treated for 4 h and 24 h with: B[a]P (0.1 and 1 µM), 1-NP (1 and 10 µM) and 3-NBA (0.5 and 5 µM). Bulky DNA adducts, lipid peroxidation, DNA and protein oxidation and mRNA expression of CYP1A1, CYP1B1, NQO1, POR, AKR1C2 and COX2 were analyzed. Bulky DNA adducts were induced after both treatment periods; the effect of 1-NP was weak. 3-NBA induced high levels of bulky DNA adducts even after 4-h treatment, suggesting rapid metabolic activation. Oxidative DNA damage was not affected. 1-NP caused protein oxidation and weak induction of lipid peroxidation after 4-h incubation. 3-NBA induced lipid peroxidation after 24-h treatment. Unlike B[a]P, induction of the aryl hydrocarbon receptor, measured as mRNA expression levels of CYP1A1 and CYP1B1, was low after treatment with polycyclic aromatic hydrocarbon (PAH) nitro-derivatives. All test compounds induced mRNA expression of NQO1, POR, and AKR1C2 after 24-h treatment. AKR1C2 expression indicates involvement of processes associated with reactive oxygen species generation. This was supported further by COX2 expression induced by 24-h treatment with 1-NP. In summary, 3-NBA was the most potent genotoxicant, whereas 1-NP exhibited the strongest oxidative properties.

Impact of Air Pollution to Genome of Newborns.

The Northern Moravia Region is the most polluted region in the Czech Republic by particulate matter (PM2.5) and carcinogenic polycyclic aromatic hydrocarbons (c-PAHs) as benzo[a]pyrene (B[a]P) by heavy industry and local heating. This specific situation was used to study the impact of air pollution on newborns in the exposed Karviná district and control district of Ceské Budejovice. Biological material from newborns and mothers was collected in summer and winter seasons. This project is highly detailed, analyzing the concentrations of PAHs in ambient air and diet, in human breast milk, in the urine of mothers and newborns, using biomarkers of genetic damage as DNA adducts and gene expression analysis, biomarkers of oxidative stress as 8-oxodG adducts and lipid peroxidation (15-F2t-isoprostane immunoassay). All 400 children, for whom the biomarker data at delivery were obtained, will be followed for morbidity up to 2 years of age. The Northern Moravia Region seems to be to be a model area for studying the long-term impact of human health exposure to c-PAHs. Our observations will indicate possible genetic and oxidative damage in newborns, which may significantly affect their morbidity.

Inhibition of ß-catenin signalling promotes DNA damage elicited by benzo[a]pyrene in a model of human colon cancer cells via CYP1 deregulation.

Deregulation of Wnt/ß-catenin signalling plays an important role in the pathogenesis of colorectal cancer. Interestingly, this pathway has been recently implicated in transcriptional control of cytochrome P450 (CYP) family 1 enzymes, which are responsible for bioactivation of a number of dietary carcinogens. In the present study, we investigated the impact of inhibition of Wnt/ß-catenin pathway on metabolism and genotoxicity of benzo[a]pyrene (BaP), a highly mutagenic polycyclic aromatic hydrocarbon and an efficient ligand of the aryl hydrocarbon receptor, which is known as a primary regulator of CYP1 expression, in cellular models derived from colorectal tumours. We observed that a synthetic inhibitor of ß-catenin, JW74, significantly increased formation of BaP-induced DNA adducts in both colorectal adenoma and carcinoma-derived cell lines. Using the short interfering RNA (siRNA) targeting ß-catenin, we then found that ß-catenin knockdown in HCT116 colon carcinoma cells significantly enhanced formation of covalent DNA adducts by BaP and histone H2AX phosphorylation, as detected by (32)P-postlabelling technique and immunocytochemistry, respectively, and it also induced expression of DNA damage response genes, such as CDKN1A or DDB2. The increased formation of DNA adducts formed by BaP upon ß-catenin knockdown corresponded with enhanced production of major BaP metabolites, as well as with an increased expression/activity of CYP1 enzymes. Finally, using siRNA-mediated knockdown of CYP1A1, we confirmed that this enzyme plays a major role in formation of BaP-induced DNA adducts in HCT116 cells. Taken together, the present results indicated that the siRNA-mediated inhibition of ß-catenin signalling, which is aberrantly activated in a majority of colorectal cancers, modulated genotoxicity of dietary carcinogen BaP in colon cell model in vitro, via a mechanism involving up-regulation of CYP1 expression and activity.

Analysis of biomarkers in a Czech population exposed to heavy air pollution. Part I: bulky DNA adducts.

The health of human populations living in industrial regions is negatively affected by exposure to environmental air pollutants. In this study, we investigated the impact of air pollution on a cohort of subjects living in Ostrava, a heavily polluted industrial region and compared it with a cohort of individuals from the relatively clean capital city of Prague. This study consisted of three sampling periods differing in the concentrations of major air pollutants (winter 2009, summer 2009 and winter 2010). During all sampling periods, the study subjects from Ostrava region were exposed to significantly higher concentrations of benzo[a]pyrene (B[a]P) and benzene than the subjects in Prague as measured by personal monitors. Pollution by B[a]P, particulate matter of aerodynamic diameter <2.5 µm (PM2.5) and benzene in the Ostrava region measured by stationary monitors was also higher than in Prague, with the exception of PM2.5 in summer 2009 when concentration of the pollutant was significantly elevated in Prague. To evaluate DNA damage in subjects from both locations we determined the levels of bulky DNA adducts in peripheral blood lymphocytes using the (32)P-postlabeling method. Despite higher B[a]P air pollution in the Ostrava region during all sampling periods, the levels of B[a]P-like DNA adducts per 10(8) nucleotides were significantly higher in the Ostrava subjects only in winter 2009 (mean ± SD: 0.21 ± 0.06 versus 0.28 ± 0.08 adducts/10(8) nucleotides, P < 0.001 for Prague and Ostrava subjects, respectively; P < 0.001). During the other two sampling periods, the levels of B[a]P-like DNA adducts were significantly higher in the Prague subjects (P < 0.001). Multivariate analyses conducted among subjects from Ostrava and Prague separately during all sampling periods revealed that exposure to B[a]P and PM2.5 significantly increased levels of B[a]P-like DNA adducts in the Ostrava subjects, but not in subjects from Prague.

Ultrafine particles are not major carriers of carcinogenic PAHs and their genotoxicity in size-segregated aerosols.

Some studies suggest that genotoxic effects of combustion-related aerosols are induced by carcinogenic polycyclic aromatic hydrocarbons (c-PAHs) and their derivatives, which are part of the organic fraction of the particulate matter (PM) in ambient air. The proportion of the organic fraction in PM is known to vary with particle size. The ultrafine fraction is hypothesized to be the most important carrier of c-PAHs, since it possesses the highest specific surface area of PM. To test this hypothesis, the distribution of c-PAHs in organic extracts (EOMs) was compared for four size fractions of ambient-air aerosols: coarse (1

Effects of various environments on epigenetic settings and chromosomal damage.

Air pollution is a dominant environmental exposure factor with significant health consequences. Unexpectedly, research in a heavily polluted region of the Czech Republic, with traditional heavy industry, revealed repeatedly the lowest frequency of micronuclei in the season with the highest concentrations of air pollutants including carcinogenic benzo[a]pyrene (B[a]P). Molecular findings have been collected for more than 10 years from various locations of the Czech Republic, with differing quality of ambient air. Preliminary conclusions have suggested adaptation of the population from the polluted locality (Ostrava, Moravian-Silesian Region (MSR)) to chronic air pollution exposure. In this study we utilize the previous findings and, for the first time, investigate micronuclei (MN) frequency by type: (i) centromere positive (CEN+) MN, representing chromosomal losses, and (ii) centromere negative (CEN-) MN representing chromosomal breaks. As previous results indicated differences between populations in the expression of XRCC5, a gene involved in the non-homologous end-joining (NHEJ) repair pathway, possible variations in epigenetic settings in this gene were also investigated. This new research was conducted in two seasons in the groups from two localities with different air quality levels (Ostrava (OS) and Prague (PG)). The obtained new results show significantly lower frequencies of chromosomal breaks in the OS subjects, related to the highest air pollution levels (p < 0.001). In contrast, chromosomal losses were comparable between both groups. In addition, significantly lower DNA methylation was found in 14.3% of the analyzed CpG loci of XRCC5 in the population from OS. In conclusion, the epigenetic adaptation (hypomethylation) in XRCC5 involved in the NHEJ repair pathway in the population from the polluted region, was suggested as a reason for the reduced level of chromosomal breaks. Further research is needed to explore the additional mechanisms, including genetic adaptation.

Metal Nanoparticles with Antimicrobial Properties: The Toxicity Response in Mouse Mesenchymal Stem Cells.

Some metal nanoparticles (NP) are characterized by antimicrobial properties with the potential to be used as alternative antibiotics. However, NP may negatively impact human organism, including mesenchymal stem cells (MSC), a cell population contributing to tissue growth and regeneration. To address these issues, we investigated the toxic effects of selected NP (Ag, ZnO, and CuO) in mouse MSC. MSC were treated with various doses of NP for 4 h, 24 h, and 48 h and multiple endpoints were analyzed. Reactive oxygen species were generated after 48 h CuO NP exposure. Lipid peroxidation was induced after 4 h and 24 h treatment, regardless of NP and/or tested dose. DNA fragmentation and oxidation induced by Ag NP showed dose responses for all the periods. For other NP, the effects were observed for shorter exposure times. The impact on the frequency of micronuclei was weak. All the tested NP increased the sensitivity of MSC to apoptosis. The cell cycle was most affected after 24 h, particularly for Ag NP treatment. In summary, the tested NP induced numerous adverse changes in MSC. These results should be taken into consideration when planning the use of NP in medical applications where MSC are involved.

The Genotoxicity of Organic Extracts from Particulate Emissions Produced by Neat Gasoline (E0) and a Gasoline-Ethanol Blend (E15) in BEAS-2B Cells.

Emissions from modern gasoline engines represent an environmental and health risk. In this study, we aimed to compare the toxicity of organic compound mixtures extracted from particulate matter (PM extracts) produced by neat gasoline (E0) and a blend containing 15% ethanol (E15), which is offered as an alternative to non-renewable fossil fuels. Human lung BEAS-2B cells were exposed to PM extracts, and biomarkers of genotoxicity, such as DNA damage evaluated by comet assay, micronuclei formation, levels of phosphorylated histone H2AX, the expression of genes relevant to the DNA damage response, and exposure to polycyclic aromatic hydrocarbons (PAHs), were determined. Results showed that both PM extracts significantly increased the level of oxidized DNA lesions. The E0 extract exhibited a more pronounced effect, possibly due to the higher content of nitrated PAHs. Other endpoints were not substantially affected by any of the PM extracts. Gene expression analysis revealed mild but coordinated induction of genes related to DNA damage response, and a strong induction of PAH-inducible genes, indicating activation of the aryl hydrocarbon receptor (AhR). Our data suggest that the addition of ethanol into the gasoline diminished the oxidative DNA damage, but no effect on other genotoxicity biomarkers was observed. Activated AhR may play an important role in the toxicity of gasoline PM emissions.

Influence of immunization with non-genotoxic PAH-KLH conjugates on the resistance of organisms exposed to benzo(a)pyrene.

Polycyclic aromatic hydrocarbons (PAH) are recognized as common environmental pollutants released into the environment from many natural as well as man-made sources, and some have been classified as potent carcinogens. The main representative of the carcinogenic PAH is benzo(a)pyrene (B(a)P) which is known to induce genotoxic effects in vitro and in vivo, detected as PAH-DNA adducts. Long-term PAH exposure may be accompanied by an immunological response with the formation of antibodies against PAH as well as against PAH-DNA adducts. This paper describes the use of four PAH-keyhole-limpet haemocyanin (KLH) conjugates for the induction of specific and cross-reactive anti-PAH antibodies and focuses on the potential protective effects of anti-PAH antibodies produced after immunization of mice. In the in vitro experiments with HepG-2 cells, the genotoxicity of the PAH-KLH conjugates and the neutralizing effect of induced anti-PAH antibodies were evaluated. The titer of specific anti-PAH antibodies in sera and the amounts of DNA adducts in liver homogenates from immunized mice were investigated in vivo. The results show that anti-PAH antibodies of class IgG were induced during immunization. All the PAH-KLH conjugates tested were non-genotoxic and did not induce detectable DNA adducts in HepG2 cells or in the liver of immunized mice. The results show that only B(a)P-specific and B(a)P cross-reactive antibodies are able to neutralize B(a)P or its activated metabolites, which was revealed by a sudden decrease in the titer of anti-B(a)P antibodies in mouse sera after exposure to B(a)P. Furthermore, the anti-B(a)P antibodies produced by immunization were effective in reducing the amount of DNA adducts in mouse livers after intraperitoneal (i.p.) exposure to B(a)P. The results suggest that immunization with PAH-KLH conjugates can protect organisms against the adverse effects of carcinogenic PAH.

Genotoxicity of 7H-dibenzo[c,g]carbazole and its methyl derivatives in human keratinocytes.

Differences between tissues in the expression of drug-metabolizing enzymes may substantially contribute to tissue-specificity of chemical carcinogens. To verify this hypothesis, the spontaneously immortalized human keratinocytes HaCaT were used, in order to evaluate the genotoxic potential of 7H-dibenzo[c,g]carbazole (DBC), a known hepatocarcinogen and sarcomagen, and its synthetic tissue-specific derivatives, 5,9-dimethyl-DBC (DiMeDBC) and N-methyl-DBC (N-MeDBC), which manifest specific tropism to the liver and skin, respectively. HaCaT cells mainly express cytochrome P4501A1 (CYP1A1), which is involved in metabolism of DBC and N-MeDBC, but not DiMeDBC [10]. Both DBC and the sarcomagen N-MeDBC induced significant levels of DNA strand-breaks, micronuclei, and DNA adducts followed by the phosphorylation of the p53 protein and histone H2AX in HaCaT cells. In contrast, the specific hepatocarcinogen DiMeDBC was devoid of any significant genotoxic activity in this cell line. Our study demonstrates that the absence of drug-metabolizing enzyme(s) involved in DiMeDBC metabolism may contribute substantially to the tissue-specific genotoxicity of this hepatocarcinogen.

A possible link between cognitive development in 5 years old children and prenatal oxidative stress.

BACKGROUND: To study the impact of oxidative damage associated with particulate matter< 2.5 µm (PM2.5) during prenatal period on the cognitive development in five years old children. METHODS: Two cohorts of children aged five years, born in the years 2013 and 2014, were studied for their cognitive development in the polluted district Karvina and the control district Ceske Budejovice. Exposure to PM2.5 in the ambient air was measured for each mother during the 3rd trimester of pregnancy. Oxidative damage was determined from the level of biomarkers at delivery in mothers´ and newborns´ urine as 8-oxo-7,8-dihydro-2´-deoxyguanosine (8-oxodG) and in plasma as 15-F2t-isoprostane levels (15-F2t-IsoP). The Bender Visual Motor Gestalt Test (BG test) and the Raven Colored Progressive Matrices (RCPM test) were used as psychological cognitive tests. RESULTS: Average concentrations of PM2.5 ± SD in the 3rd trimester of mothers´ pregnancies were 37.7 ± 14.7 µg/m3 and 17.1 ± 4.8 µg/m3 in Karvina and Ceske Budejovice, respectively (p < 0.001). The maternal level of 15-F2t-IsoP in plasma at the time of delivery was significantly associated with the results of the RCPM test (p < 0.05) and the BG test (p < 0.05) in five years old children. CONCLUSIONS: Lipid peroxidation in maternal plasma at the time of delivery has an adverse effect on the results of psychological cognitive tests in five years old children.

Oxidative Stress and Antioxidant Response in Populations of the Czech Republic Exposed to Various Levels of Environmental Pollutants.

We aimed to identify the variables that modify levels of oxidatively damaged DNA and lipid peroxidation in subjects living in diverse localities of the Czech Republic (a rural area, a metropolitan locality, and an industrial region). The sampling of a total of 126 policemen was conducted twice in two sampling seasons. Personal characteristics, concentrations of particulate matter of aerodynamic diameter <2.5 µm and benzo[a]pyrene in the ambient air, activities of antioxidant mechanisms (superoxide dismutase, catalase, glutathione peroxidase, and antioxidant capacity), levels of pro-inflammatory cytokines (TNF-α, IL-1ß, and IL-6), concentrations of persistent organic pollutants in blood plasma, and urinary levels of polycyclic aromatic hydrocarbon metabolites were investigated as parameters potentially affecting the markers of DNA oxidation (8-oxo-7,8-dihydro-2'-deoxyguanosine) and lipid peroxidation (15-F2t-isoprostane). The levels of oxidative stress markers mostly differed between the localities in the individual sampling seasons. Multivariate linear regression analysis revealed IL-6, a pro-inflammatory cytokine, as a factor with the most pronounced effects on oxidative stress parameters. The role of other variables, including environmental pollutants, was minor. In conclusion, our study showed that oxidative damage to macromolecules was affected by processes related to inflammation; however, we did not identify a specific environmental factor responsible for the pro-inflammatory response in the organism.

The role of human cytochrome P4503A4 in biotransformation of tissue-specific derivatives of 7H-dibenzo[c,g]carbazole.

The environmental pollutant 7H-dibenzo[c,g]carbazole (DBC) and its derivative, 5,9-dimethylDBC (DiMeDBC), produced significant and dose-dependent levels of micronuclei followed by a substantial increase in the frequency of apoptotic cells in the V79MZh3A4 cell line stably expressing the human cytochrome P450 (hCYP) 3A4. In contrast, neither micronuclei nor apoptosis were found in cells exposed to the sarcomagenic carcinogen, N-methylDBC (N-MeDBC). A slight but significant level of gene mutations and DNA adducts detected in V79MZh3A4 cells treated with N-MeDBC, only at the highest concentration (30µM), revealed that this sarcomagenic carcinogen was also metabolized by hCYP3A4. Surprisingly, DBC increased the frequency of 6-thioguanine resistant (6-TG(r)) mutations only at the highest concentration (30µM), while DiMeDBC failed to increase the frequency of these mutations. The resistance to 6-thioguanine is caused by the mutations in the hypoxanthine-guanine phosphoribosyltransferase (Hprt) gene. The molecular analysis of the coding region of Hprt gene showed a deletion of the entire exon 8 in DiMeDBC-induced 6-TG(r) mutants, while no changes in the nucleotide sequences were identified in 6-TG(r) mutants produced by DBC and N-MeDBC. Based on our results, we suggest that hCYP3A4 is involved in the metabolism of DBC and its tissue-specific derivatives. While hCYP3A4 probably plays an important role in biotransformation of the liver carcinogens, DBC and DiMeDBC, it might only have a marginal function in N-MeDBC metabolism.

Toxic effects of methylated benzo[a]pyrenes in rat liver stem-like cells.

The methylated benzo[a]pyrenes (MeBaPs) are present at significant levels in the environment, especially in the sediments contaminated by petrogenic PAHs. However, the existing data on their toxic effects in vitro and/or in vivo are still largely incomplete. Transcription factor AhR plays a key role in the metabolic activation of PAHs to genotoxic metabolites, but the AhR activation may also contribute to the tumor promoting effects of PAHs. In this study, the AhR-mediated activity of five selected MeBaP isomers was estimated in the DR-CALUX reporter gene assay performed in rat hepatoma cells. Detection of other effects, including induction of CYP1A1, CYP1B1, and AKR1C9 mRNAs, DNA adduct formation, production of reactive oxygen species, oxidation of deoxyguanosine, and cell cycle modulation and apoptosis, was performed in the rat liver epithelial WB-F344 cell line, a model of liver progenitor cells. We identified 1-MeBaP as the most potent inducer of AhR activation, stable DNA adduct formation, checkpoint kinase 1 and p53 phosphorylation, and apoptosis. These effects suggest that 1-MeBaP is a potent genotoxin eliciting a typical sequence of events ascribed to carcinogenic PAHs: induction of CYP1 enzymes, formation of high levels of DNA adducts, activation of DNA damage responses (including p53 phosphorylation), and cell death. In contrast, 10-MeBaP, representing BaP isomers substituted with the methyl group in the angular ring, elicited only low levels DNA adduct formation and apoptosis. Other MeBaPs under study also elicited strong apoptotic responses associated with DNA adduct formation as the prevalent mode of toxic action of these compounds in liver cells. MeBaPs induced a weak production of ROS, which did not lead to significant oxidative DNA damage. Importantly, 1-MeBaP and 3-MeBaP were found to be potent AhR agonists, one order of magnitude more potent than BaP, thus suggesting that the AhR-dependent modulations of gene expression, deregulation of cell survival mechanisms, and further nongenotoxic effects associated with AhR activation may further contribute to their tumor promotion and carcinogenicity.

Testing Strategies of the In Vitro Micronucleus Assay for the Genotoxicity Assessment of Nanomaterials in BEAS-2B Cells.

The evaluation of the frequency of micronuclei (MN) is a broadly utilised approach in in vitro toxicity testing. Nevertheless, the specific properties of nanomaterials (NMs) give rise to concerns regarding the optimal methodological variants of the MN assay. In bronchial epithelial cells (BEAS-2B), we tested the genotoxicity of five types of NMs (TiO2: NM101, NM103; SiO2: NM200; Ag: NM300K, NM302) using four variants of MN protocols, differing in the time of exposure and the application of cytochalasin-B combined with the simultaneous and delayed co-treatment with NMs. Using transmission electron microscopy, we evaluated the impact of cytochalasin-B on the transport of NMs into the cells. To assess the behaviour of NMs in a culture media for individual testing conditions, we used dynamic light scattering measurement. The presence of NMs in the cells, their intracellular aggregation and dispersion properties were comparable when tests with or without cytochalasin-B were performed. The genotoxic potential of various TiO2 and Ag particles differed (NM101 < NM103 and NM302 < NM300K, respectively). The application of cytochalasin-B tended to increase the percentage of aberrant cells. In conclusion, the comparison of the testing strategies revealed that the level of DNA damage induced by NMs is affected by the selected methodological approach. This fact should be considered in the interpretation of the results of genotoxicity tests.

Genotoxicant exposure, activation of the aryl hydrocarbon receptor, and lipid peroxidation in cultured human alveolar type II A549 cells.

The aryl hydrocarbon receptor (AhR) transcription factor is activated by polycyclic aromatic hydrocarbons (PAH) and other ligands. Activated AhR binds to dioxin responsive elements (DRE) and initiates transcription of target genes, including the gene encoding prostaglandin endoperoxide synthase 2 (PTGS-2), which is also activated by the transcription factor NF-ĸB. PTGS-2 catalyzes the conversion of arachidonic acid (AA) into prostaglandins, thromboxanes or isoprostanes. 15-F2t-Isoprostane (IsoP), regarded as a universal marker of lipid peroxidation, is also induced by PAH exposure. We investigated the processes associated with lipid peroxidation in human alveolar basal epithelial cells (A549) exposed for 4 h or 24 h to model PAH (benzo[a]pyrene, BaP; 3-nitrobenzanthrone, 3-NBA) and organic extracts from ambient air particulate matter (EOM), collected in two seasons in a polluted locality. Both EOM induced the expression of CYP1A1 and CYP1B1; 24 h treatment significantly reduced PTGS-2 expression. IsoP levels decreased after both exposure periods, while the concentration of AA was not affected. The effects induced by BaP were similar to EOM except for increased IsoP levels after 4 h exposure and elevated AA concentration after 24 h treatment. In contrast, 3-NBA treatment did not induce CYP expression, had a weak effect on PTGS-2 expression, and, similar to BaP, induced IsoP levels after 4 h exposure and AA levels after 24 h treatment. All tested compounds induced the activity of NF-ĸB after the longer exposure period. In summary, our data suggest that EOM, and partly BaP, reduce lipid peroxidation by a mechanism that involves AhR-dependent inhibition of PTGS-2 expression. The effect of 3-NBA on IsoP levels is probably mediated by a different mechanism independent of AhR activation.