Latest Publications

@article{smerdova_inflammatory_2013,

title = {Inflammatory mediators accelerate metabolism of benzo[a]pyrene in rat alveolar type II cells: the role of enhanced cytochrome P450 1B1 expression.},

author = {Lenka Smerdová and Jiří Neča and Jana Svobodová and Jan Topinka and Jana Schmuczerová and Alois Kozubík and Miroslav Machala and Jan Vondráček},

doi = {10.1016/j.tox.2013.09.001},

issn = {1879-3185 0300-483X},

year  = {2013},

date = {2013-12-01},

journal = {Toxicology},

volume = {314},

number = {1},

pages = {30–38},

abstract = {Long-term deregulated inflammation represents one of the key factors contributing to lung cancer etiology. Previously, we have observed that tumor necrosis factor-α (TNF-α), a major pro-inflammatory cytokine, enhances genotoxicity of benzo[a]pyrene (B[a]P), a highly carcinogenic polycyclic aromatic hydrocarbon, in rat lung epithelial RLE-6TN cells, a model of alveolar type II cells. Therefore, we analyzed B[a]P metabolism in RLE-6TN cells under inflammatory conditions, simulated using either recombinant TNF-α, or a mixture of inflammatory mediators derived from activated alveolar macrophage cell line. Inflammatory conditions significantly accelerated BaP metabolism, as evidenced by decreased levels of both parent B[a]P and its metabolites. TNF-α altered production of the metabolites associated with dihydrodiol-epoxide and radical cation pathways of B[a]P metabolism, especially B[a]P-dihydrodiols, and B[a]P-diones. We then evaluated the role of cytochrome P450 1B1 (CYP1B1), which is strongly up-regulated in cells treated with B[a]P under inflammatory conditions, in the observed effects. The siRNA-mediated CYP1B1 knock-down increased levels of B[a]P and reduced formation of stable DNA adducts, thus confirming the essential role of CYP1B1 in B[a]P metabolism under inflammatory conditions. TNF-α also reduced expression of aldo-keto reductase 1C14, which may compete with CYP1B1 for B[a]P-7,8-dihydrodiol and divert it from the formation of ultimate B[a]P dihydrodiol epoxide. Together, the present data suggests that the CYP1B1-catalyzed metabolism of polycyclic aromatic hydrocarbons might contribute to their enhanced bioactivation and genotoxic effects under inflammatory conditions.},

note = {Place: Ireland},

keywords = {Animals, Aryl Hydrocarbon Hydroxylases/*biosynthesis/genetics, ATP Binding Cassette Transporter, Benzo(a)pyrene/*metabolism, Blotting, Cell Line, Conditioned, Culture Media, CYP1B1, Cytochrome P-450 CYP1B1, Cytokines/metabolism, DNA adducts, Inflammation, Inflammation Mediators/*pharmacology, metabolism, Oxidoreductases Acting on Aldehyde or Oxo Group Donors/biosynthesis/genetics, Polycyclic aromatic hydrocarbons, Pulmonary Alveoli/cytology/drug effects/*metabolism, Rats, Real-Time Polymerase Chain Reaction, RNA, Small Interfering, Subfamily B/biosynthesis/genetics, Tandem Mass Spectrometry, Transfection, Western},

pubstate = {published},

tppubtype = {article}

}

@article{umannova_benzopyrene_2011,

title = {Benzo[a]pyrene and tumor necrosis factor-α coordinately increase genotoxic damage and the production of proinflammatory mediators in alveolar epithelial type II cells.},

author = {Lenka Umannová and Miroslav Machala and Jan Topinka and Jana Schmuczerová and Pavel Krčmář and Jiří Neča and Klára Šujanová and Alois Kozubík and Jan Vondráček},

doi = {10.1016/j.toxlet.2011.06.029},

issn = {1879-3169 0378-4274},

year  = {2011},

date = {2011-10-01},

journal = {Toxicology letters},

volume = {206},

number = {2},

pages = {121–129},

abstract = {Alveolar type II epithelial (AEII) cells regulate lung inflammatory response and, simultaneously, they are a target of environmental carcinogenic factors. We employed an in vitro model of rat AEII cells, the RLE-6TN cell line, in order to analyze the interactive effects of tumor necrosis factor-α (TNF-α), a cytokine which plays a key role in the initiation of inflammatory responses in the lung, and benzo[a]pyrene (BaP), a highly carcinogenic polycyclic aromatic hydrocarbon. TNF-α strongly augmented the formation of stable BaP diol epoxide-DNA adducts in AEII cells, which was associated with enhanced p53-Ser15 phosphorylation and decreased cell survival. The increased genotoxicity of BaP was associated with altered expression of cytochrome P450 (CYP) enzymes involved in its bioactivation, a simultaneous suppression of CYP1A1 and enhancement of CYP1B1 expression. Importantly, BaP and TNF-α acted synergistically to upregulate key inflammatory regulators in AEII cells, including the expression of inducible NO synthase and cyclooxygenase-2 (COX-2), and enhanced prostaglandin E2 production and expression of proinflammatory cytokines, such as TNF-α, interleukin-1β and interleukin-6. We observed that BaP and TNF-α together strongly activated p38 kinase, a principal regulator of inflammatory response. SB202190, a specific p38 inhibitor, prevented induction of both COX-2 and proinflammatory cytokines, thus confirming that p38 activity was crucial for the observed inflammatory reaction. Taken together, our data demonstrated, for the first time, that a proinflammatory cytokine and an environmental PAH may interact to potentiate both DNA damage and the inflammatory response in AEII cells, which may occur through coordinated upregulation of p38 activity.},

note = {Place: Netherlands},

keywords = {Alveolar Epithelial Cells/*drug effects/immunology/*metabolism, Animals, Apoptosis/drug effects, Aryl Hydrocarbon Hydroxylases/genetics/metabolism, Benzo(a)pyrene/metabolism/*toxicity, Carcinogens, Cell Line, Cell Proliferation/drug effects, Cytochrome P-450 CYP1A1/genetics/metabolism, Cytochrome P-450 CYP1B1, DNA Adducts/*metabolism, Environmental/toxicity, Enzyme Activation/drug effects, Gene Expression Regulation/drug effects, Inflammation Mediators/*metabolism, Messenger/metabolism, Mutagens/*toxicity, p38 Mitogen-Activated Protein Kinases/antagonists & inhibitors/metabolism, Phosphorylation/drug effects, Post-Translational/drug effects, Protein Kinase Inhibitors/pharmacology, Protein Processing, Rats, RNA, Tumor Necrosis Factor-alpha/*metabolism, Tumor Suppressor Protein p53/metabolism},

pubstate = {published},

tppubtype = {article}

}

@article{prochazkova_interplay_2011,

title = {The interplay of the aryl hydrocarbon receptor and β-catenin alters both AhR-dependent transcription and Wnt/β-catenin signaling in liver progenitors.},

author = {Jirina Procházková and Markéta Kabátková and Vítezslav Bryja and Lenka Umannová and Ondrej Bernatík and Alois Kozubík and Miroslav Machala and Jan Vondrácek},

doi = {10.1093/toxsci/kfr129},

issn = {1096-0929},

year  = {2011},

date = {2011-08-01},

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

volume = {122},

number = {2},

pages = {349–360},

abstract = {β-catenin is a key integrator of cadherin-mediated cell-cell adhesion and transcriptional regulation through the Wnt/β-catenin pathway, which plays an important role in liver biology. Using a model of contact-inhibited liver progenitor cells, we examined the interactions of Wnt/β-catenin signaling with the aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor, which mediates the toxicity of dioxin-like compounds, including their effects on development and hepatocarcinogenesis. We found that AhR and Wnt/β-catenin cooperated in the induction of AhR transcriptional targets, such as Cyp1a1 and Cyp1b1. However, simultaneously, the activation of AhR led to a decrease of dephosphorylated active β-catenin pool, as well as to hypophosphorylation of Dishevelled, participating in regulation of Wnt signaling. A sustained AhR activation by its model ligand, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), led to a downregulation of a number of Wnt/β-catenin pathway target genes. TCDD also induced a switch in cytokeratin expression, where downregulation of cytokeratins 14 and 19 was accompanied with an increased cytokeratin 8 expression. Together with a downregulation of additional markers associated with stem-like phenotype, this indicated that the AhR activation interfered with differentiation of liver progenitors. The downregulation of β-catenin was also related to a reduced cell adhesion, disruption of E-cadherin-mediated cell-cell junctions and an increased G1-S transition in liver progenitor cell line. In conclusion, although β-catenin augmented the expression of selected AhR target genes, the persistent AhR activation may lead to downregulation of Wnt/β-catenin signaling, thus altering differentiation and/or proliferative status of liver progenitor cells.},

note = {Place: United States},

keywords = {Animals, Aryl Hydrocarbon Hydroxylases/genetics/metabolism, Aryl Hydrocarbon/genetics/*metabolism, beta Catenin/genetics/*metabolism, Cadherins/genetics, Cell Adhesion, Cell Differentiation, Cell Line, Cytochrome P-450 CYP1A1/genetics/metabolism, Cytochrome P-450 CYP1B1, Down-Regulation/drug effects, Hepatocytes/drug effects, Inbred F344, Liver/*drug effects, Polychlorinated Dibenzodioxins/toxicity, Rats, Receptors, Wnt Proteins/genetics/*metabolism, Wnt Signaling Pathway},

pubstate = {published},

tppubtype = {article}

}

@article{umannova_tumor_2008,

title = {Tumor necrosis factor-alpha potentiates genotoxic effects of benzo[a]pyrene in rat liver epithelial cells through upregulation of cytochrome P450 1B1 expression.},

author = {Lenka Umannová and Miroslav Machala and Jan Topinka and Zuzana Nováková and Alena Milcová and Alois Kozubík and Jan Vondrácek},

doi = {10.1016/j.mrfmmm.2008.02.001},

issn = {0027-5107},

year  = {2008},

date = {2008-04-01},

journal = {Mutation research},

volume = {640},

number = {1-2},

pages = {162–169},

abstract = {Benzo[a]pyrene (BaP) is a ubiquitous environmental pollutant, which may contribute to the development of human cancer. The ultimate carcinogenic BaP metabolite produced by cytochrome P450 enzymes (CYP), such as CYP1A1 and CYP1B1, anti-BaP-7,8-diol-9,10-epoxide, binds covalently to DNA and causes mutations. The levels of various CYP isoforms can be significantly modulated under inflammatory conditions. As the chronic inflammation is known to contribute to carcinogenesis, we investigated interactions of a major proinflammatory cytokine, tumor necrosis factor-alpha (TNF-alpha), and BaP in regulation of the expression of CYP1A1/1B1 and induction of DNA damage in rat liver epithelial WB-F344 cells. TNF-alpha enhanced induction of CYP1B1, while it simultaneously suppressed the BaP-induced CYP1A1 expression. The observed deregulation of CYP1 induction was found to be associated with a significantly enhanced formation of DNA adducts. The elevated DNA damage corresponded with increased phosphorylation of p53 tumor suppressor at Ser-15 residue, enhanced accumulation of cells in the S-phase of cell cycle and potentiation of BaP-induced apoptosis. Inhibition of CYP1B1 by fluoranthene significantly decreased both the formation of DNA adducts and the induction of apoptosis in WB-F344 cells treated with BaP and TNF-alpha, thus suggesting that this isoform might be responsible for genotoxic effects of BaP in nonparenchymal liver cells. Our results seem to indicate that inflammatory conditions might enhance genotoxic effects of carcinogenic polycyclic aromatic hydrocarbons through upregulation of CYP1B1 expression.},

note = {Place: Netherlands},

keywords = {Animals, Aryl Hydrocarbon Hydroxylases/*metabolism, Benzo(a)pyrene/*toxicity, Cell Line, Cytochrome P-450 CYP1B1, Drug Synergism, Epithelial Cells/drug effects/enzymology, Liver/*drug effects/*enzymology, Male, Rats, Tumor Necrosis Factor-alpha/*pharmacology, Up-Regulation},

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{umannova_tumor_2007,

title = {Tumor necrosis factor-alpha modulates effects of aryl hydrocarbon receptor ligands on cell proliferation and expression of cytochrome P450 enzymes in rat liver "stem-like" cells.},

author = {Lenka Umannová and Jirina Zatloukalová and Miroslav Machala and Pavel Krcmár and Zuzana Májková and Bernhard Hennig and Alois Kozubík and Jan Vondrácek},

doi = {10.1093/toxsci/kfm149},

issn = {1096-6080 1096-0929},

year  = {2007},

date = {2007-09-01},

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

volume = {99},

number = {1},

pages = {79–89},

abstract = {Various liver diseases lead to an extensive inflammatory response and release of a number of proinflammatory cytokines, such as tumor necrosis factor-alpha (TNF-alpha). This cytokine is known to play a major role in liver regeneration as well as in carcinogenesis. We investigated possible interactions of TNF-alpha with ligands of the aryl hydrocarbon receptor (AhR) and known liver carcinogens, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and coplanar 3,3',4,4',5-pentachlorobiphenyl (PCB 126). These compounds have been previously found to disrupt cell cycle control in contact-inhibited rat liver WB-F344 cells, an in vitro model of adult liver progenitor cells. TNF-alpha itself had no significant effect on the proliferation/apoptosis ratio in the WB-F344 cell line. However, it significantly potentiated proliferative effects of low picomolar range doses of both TCDD and PCB 126, leading to an increase in cell numbers, as well as an increased percentage of cells entering the S-phase of the cell cycle. The combination of TNF-alpha with low concentrations of AhR ligands increased both messenger RNA (mRNA) and protein levels of cyclin A, a principle cyclin involved in disruption of contact inhibition. TNF-alpha temporarily inhibited AhR-dependent induction of cytochrome P450 1A1 (CYP1A1). In contrast, TNF-alpha significantly enhanced induction of CYP1B1 at both mRNA and protein levels, by a mechanism, which was independent of nuclear factor-kappaB activation. These results suggest that TNF-alpha can significantly amplify effects of AhR ligands on deregulation of cell proliferation control, as well as on expression of CYP1B1, which is involved in metabolic activation of a number of mutagenic compounds.},

note = {Place: United States},

keywords = {Animals, Aryl Hydrocarbon Hydroxylases/genetics/*metabolism, Aryl Hydrocarbon/*drug effects/metabolism, Carcinogens/metabolism/toxicity, Cell Proliferation/drug effects, Cells, Cultured, Cytochrome P-450 CYP1A1/genetics/metabolism, Cytochrome P-450 CYP1B1, Dose-Response Relationship, Drug, Drug Combinations, Drug Interactions, Enzymologic/*drug effects, Epithelial Cells/drug effects/enzymology, Gene Expression Regulation, Inbred F344, Ligands, Liver/cytology, Polychlorinated Biphenyls/metabolism/*toxicity, Polychlorinated Dibenzodioxins/metabolism/*toxicity, Rats, Receptors, Stem Cells/*drug effects/enzymology, Tumor Necrosis Factor-alpha/*pharmacology},

pubstate = {published},

tppubtype = {article}

}

@article{vondracek_7h-dibenzocgcarbazole_2006,

title = {7H-Dibenzo[c,g]carbazole and 5,9-dimethyldibenzo[c,g]carbazole exert multiple toxic events contributing to tumor promotion in rat liver epithelial 'stem-like' cells.},

author = {Jan Vondrácek and Lenka Svihálková-Sindlerová and Katerina Pencíková and Pavel Krcmár and Zdenek Andrysík and Katerina Chramostová and Sona Marvanová and Zuzana Valovicová and Alois Kozubík and Alena Gábelová and Miroslav Machala},

doi = {10.1016/j.mrfmmm.2005.11.005},

issn = {0027-5107},

year  = {2006},

date = {2006-04-01},

journal = {Mutation research},

volume = {596},

number = {1-2},

pages = {43–56},

abstract = {Immature liver progenitor cells have been suggested to be an important target of hepatotoxins and hepatocarcinogens. The goal of the present study was to assess the impact of 7H-dibenzo[c,g]carbazole (DBC) and its tissue-specific carcinogenic N-methyl (N-MeDBC) and 5,9-dimethyl (DiMeDBC) derivatives on rat liver epithelial WB-F344 cells, in vitro model of liver progenitor cells. We investigated the cellular events associated with both tumor initiation and promotion, such as activation of aryl hydrocarbon receptor (AhR), changes in expression of enzymes involved in metabolic activation of DBC and its derivatives, effects on cell cycle, cell proliferation/apoptosis and inhibition of gap junctional intercellular communication (GJIC). N-MeDBC, a tissue-specific sarcomagen, was only a weak inhibitor of GJIC or inducer of AhR-mediated activity, and it did not affect either cell proliferation or apoptosis. DBC was efficient GJIC inhibitor, while DiMeDBC manifested the strongest AhR inducing activity. Accordingly, DiMeDBC was also the most potent inducer of cytochrome P450 1A1 (CYP1A1) and CYP1A2 expression among the three compounds tested. Both DBC and DiMeDBC induced expression of CYP1B1 and aldo-keto reductase 1C9 (AKR1C9). N-MeDBC failed to significantly upregulate CYP1A1/2 and it only moderately increased CYP1B1 or AKR1C9. Only the potent liver carcinogens, DBC and DiMeDBC, caused a significant increase of p53 phosphorylation at Ser15, an increased accumulation of cells in S-phase and apoptosis at micromolar concentrations. In addition, DiMeDBC was found to stimulate cell proliferation of contact-inhibited WB-F344 cells at 1 microM concentration, which is a mode of action that might further contribute to its hepatocarcinogenicity. The present data seem to suggest that the AhR activation, induction of enzymes involved in metabolic activation, inhibition of GJIC or stimulation of cell proliferation might all contribute to the hepatocarcinogenic effects of DBC and DiMeDBC.},

note = {Place: Netherlands},

keywords = {Animals, Aryl Hydrocarbon Hydroxylases/genetics, Base Sequence, Carbazoles/*toxicity, Carcinogens/*toxicity, Cell Death/drug effects, Cytochrome P-450 CYP1A1/genetics, Cytochrome P-450 CYP1A2/genetics, Cytochrome P-450 CYP1B1, DNA Primers, Epithelial Cells/drug effects/*pathology, Inbred F344, Liver/*cytology/drug effects, Methylation, Molecular Structure, Mutagens, Rats, Reverse Transcriptase Polymerase Chain Reaction},

pubstate = {published},

tppubtype = {article}

}