Latest Publications

@article{trilecova_toxic_2011,

title = {Toxic effects of methylated benzo[a]pyrenes in rat liver stem-like cells.},

author = {Lenka Trilecová and Simona Krčková and Soňa Marvanová and Kateřina Pĕnčíková and Pavel Krčmář and Jiří Neča and Petra Hulinková and Lenka Pálková and Miroslav Ciganek and Alena Milcová and Jan Topinka and Jan Vondráček and Miroslav Machala},

doi = {10.1021/tx200049x},

issn = {1520-5010 0893-228X},

year  = {2011},

date = {2011-06-01},

journal = {Chemical research in toxicology},

volume = {24},

number = {6},

pages = {866–876},

abstract = {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.},

note = {Place: United States},

keywords = {Animals, Apoptosis/drug effects, Aryl Hydrocarbon/*metabolism, Benzo(a)pyrene/*chemistry/*toxicity, Cell Cycle/drug effects, Cell Line, Checkpoint Kinase 1, DNA Adducts/metabolism, Epithelial Cells/drug effects/metabolism, Gene Expression Regulation/drug effects, Liver/*cytology, Methylation, Mutagens/*chemistry/*toxicity, Oxidative Stress/drug effects, Protein Kinases/metabolism, Rats, Receptors, Stem Cells/drug effects/metabolism, Tumor, Tumor Suppressor Protein p53/metabolism},

pubstate = {published},

tppubtype = {article}

}

@article{topinka_dna_2008,

title = {DNA adducts formation and induction of apoptosis in rat liver epithelial 'stem-like' cells exposed to carcinogenic polycyclic aromatic hydrocarbons.},

author = {Jan Topinka and Sona Marvanová and Jan Vondrácek and Oksana Sevastyanova and Zuzana Nováková and Pavel Krcmár and Katerina Pencíková and Miroslav Machala},

doi = {10.1016/j.mrfmmm.2007.09.004},

issn = {0027-5107},

year  = {2008},

date = {2008-02-01},

journal = {Mutation research},

volume = {638},

number = {1-2},

pages = {122–132},

abstract = {The bipotent liver progenitor cells, so called oval cells, may participate at the early stages of hepatocarcinogenesis induced by chemical carcinogens. Unlike in mature parenchymal cells, little is known about formation of DNA adducts and other genotoxic events in oval cells. In the present study, we employed spontaneously immortalized rat liver WB-F344 cell line, which is an established in vitro model of oval cells, in order to study genotoxic effects of selected carcinogenic polycyclic aromatic hydrocarbons (PAHs). With exception of dibenzo[a,l]pyrene, and partly also benzo[g]chrysene and benz[a]anthracene, all other PAHs under the study induced high levels of CYP1A1 and CYP1B1 mRNA. In contrast, we observed distinct genotoxic and cytotoxic potencies of PAHs. Dibenzo[a,l]pyrene, and to a lesser extent also benzo[a]pyrene, benzo[g]chrysene and dibenzo[a,e]pyrene, formed high levels of DNA adducts. This was accompanied with accumulation of Ser-15 phosphorylated form of p53 protein and induction of apoptosis. Contrary to that, benz[a]anthracene, chrysene, benzo[b]fluoranthene and dibenzo[a,h]anthracene induced only low amounts of DNA adducts formation and minimal apoptosis, without exerting significant effects on p53 phosphorylation. Finally, we studied effects of 2,4,3',5'-tetramethoxystilbene and fluoranthene, inhibitors of CYP1B1 activity, which plays a central role in metabolic activation of dibenzo[a,l]pyrene. In a dose-dependent manner, both compounds inhibited apoptosis induced by dibenzo[a,l]pyrene, suggesting that it interferes with the metabolic activation of the latter one. The present data show that in model cell line sharing phenotypic properties with oval cells, PAHs can be efficiently metabolized to form ultimate genotoxic metabolites. Liver progenitor cells could be thus susceptible to this type of genotoxic insult, which makes WB-F344 cell line a useful tool for studies of genotoxic effects of organic contaminants in liver cells. Our results also suggest that, unlike in mature hepatocytes, CYP1B1 might be a primary enzyme responsible for formation of DNA adducts in liver progenitor cells.},

note = {Place: Netherlands},

keywords = {*Apoptosis, Animals, Aryl Hydrocarbon Hydroxylases/genetics, Cells, Cultured, Cytochrome P-450 CYP1A1/genetics, Cytochrome P-450 CYP1B1, DNA Adducts/*metabolism, Inbred F344, Liver/*cytology, Messenger/analysis, Polycyclic Aromatic Hydrocarbons/*pharmacology, Rats, RNA, Stem Cells/*metabolism},

pubstate = {published},

tppubtype = {article}

}

@article{vondracek_aryl_2005,

title = {Aryl hydrocarbon receptor-activating polychlorinated biphenyls and their hydroxylated metabolites induce cell proliferation in contact-inhibited rat liver epithelial cells.},

author = {Jan Vondrácek and Miroslav Machala and Vítezslav Bryja and Katerina Chramostová and Pavel Krcmár and Cornelia Dietrich and Ales Hampl and Alois Kozubík},

doi = {10.1093/toxsci/kfi009},

issn = {1096-6080 1096-0929},

year  = {2005},

date = {2005-01-01},

journal = {Toxicological sciences : an official journal of the Society of Toxicology},

volume = {83},

number = {1},

pages = {53–63},

abstract = {Polychlorinated biphenyls (PCBs) exhibit tumor-promoting effects in experimental animals. We investigated effects of six model PCB congeners and hydroxylated PCB metabolites on proliferation of contact-inhibited rat liver epithelial WB-F344 cells. The 'dioxin-like' PCB congeners, PCB 126, PCB 105, and 4'-OH-PCB 79, a metabolite of the planar PCB 77 congener, induced cell proliferation in a concentration-dependent manner. In contrast, the 'non-dioxin-like' compounds that are not aryl hydrocarbon receptor (AhR) agonists, PCB 47, PCB 153, and 4-OH-PCB 187, an abundant noncoplanar PCB metabolite, had no effect on cell proliferation at concentrations up to 10 muM. The concentrations of dioxin-like PCBs leading to cell proliferation corresponded with the levels inducing the expression of cytochrome P450 1A1 mRNA, suggesting that the release from contact inhibition was associated with AhR activation. The effects of PCB 126 and PCB 153 on expression of proteins controlling G0/G1-S-phase transition and S-phase progression were compared. Only PCB 126 was found to upregulate cyclin A and D2 protein levels, and to increase both total cyclin-dependent kinase 2 (cdk2) and cyclin A/cdk2 complex activities. Despite the observed upregulation of cyclin D2, no increase in cdk4 activity was observed. The expression of cdk inhibitor p27Kip1 was not affected by either PCB 126 or PCB 153. These results suggest that dioxin-like PCBs can induce cell proliferation of contact-inhibited rat liver epithelial cells by increasing cyclin A protein levels, a process that then leads to upregulation of cyclin A/cdk2 activity and initiation of DNA replication. This mechanism could be involved in tumor-promoting effects of dioxin-like PCBs.},

note = {Place: United States},

keywords = {Animals, Aryl Hydrocarbon/*metabolism, Cell Line, Cell Proliferation/*drug effects, Cyclin A/biosynthesis, Cyclin D2, Cyclin-Dependent Kinases/biosynthesis, Cyclins/biosynthesis, Dose-Response Relationship, Drug, Epithelial Cells/*drug effects/enzymology/metabolism, Hydroxylation, Liver/*cytology, Polychlorinated Biphenyls/metabolism/*toxicity, Rats, Receptors, Stem Cells/*drug effects/enzymology/metabolism, Up-Regulation},

pubstate = {published},

tppubtype = {article}

}

@article{chramostova_polycyclic_2004,

title = {Polycyclic aromatic hydrocarbons modulate cell proliferation in rat hepatic epithelial stem-like WB-F344 cells.},

author = {Katerina Chramostová and Jan Vondrácek and Lenka Sindlerová and Borivoj Vojtesek and Alois Kozubík and Miroslav Machala},

doi = {10.1016/j.taap.2003.12.008},

issn = {0041-008X},

year  = {2004},

date = {2004-04-01},

journal = {Toxicology and applied pharmacology},

volume = {196},

number = {1},

pages = {136–148},

abstract = {Although many polycyclic aromatic hydrocarbons (PAHs) are recognized as potent mutagens and carcinogens, relatively little is known about their role in the tumor promotion. It is known that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) can induce release of rat hepatic oval epithelial cells from contact inhibition by a mechanism possibly involving the aryl hydrocarbon receptor (AhR) activation. Many PAHs are AhR ligands and are known to act as transient inducers of AhR-mediated activity. In this study, effects of 19 selected PAHs on proliferation of confluent rat liver epithelial WB-F344 cells were investigated. Non-mutagens that are weak activators or nonactivators of AhR-mediated activity had no effect on cell proliferation. Relatively strong or moderate AhR ligands with low mutagenic potencies, such as benzofluoranthenes, benz[a]anthracene, and chrysene, were found to increase cell numbers, which corresponded to an increased percentage of cells entering S-phase. Strong mutagens, including benzo[a]pyrene and dibenzo[a,l]pyrene, increased a percentage of cells in S-phase without inducing a concomitant increase in cell numbers. The treatment with mutagenic PAHs was associated with an increased DNA synthesis and induction of cell death, which corresponded with the activation of p53 tumor suppressor. Apoptosis was blocked by pifithrin-alpha, the chemical inhibitor of p53. Both weakly and strongly mutagenic PAHs known as AhR ligands were found to induce significant increase of cytochrome P4501A activity, suggesting a presence of functional AhR. The results of the present study seem to suggest that a release from contact inhibition could be a part of tumor promoting effects of AhR-activating PAHs; however, the genotoxic effects of some PAHs associated with p53 activation might interfere with this process.},

note = {Place: United States},

keywords = {Animals, Apoptosis/drug effects, Aryl Hydrocarbon/metabolism, Cell Division/drug effects, Cells, Cultured, Cytochrome P-450 CYP1A1/metabolism, Dose-Response Relationship, Drug, Epithelial Cells/*drug effects/enzymology/metabolism, Liver/*cytology, Mutagens/*toxicity, Polycyclic Aromatic Hydrocarbons/*toxicity, Rats, Receptors, Stem Cells/*drug effects/enzymology/metabolism, Tumor Suppressor Protein p53/biosynthesis},

pubstate = {published},

tppubtype = {article}

}