Publications by authors named "Kateřina Pěnčíková"

23 Publications

  • Page 1 of 1

Environmental six-ring polycyclic aromatic hydrocarbons are potent inducers of the AhR-dependent signaling in human cells.

Environ Pollut 2020 Nov 3;266(Pt 2):115125. Epub 2020 Jul 3.

Department of Chemistry and Toxicology, Veterinary Research Institute, Hudcova 70, 62100 Brno, Czech Republic.

The toxicities of many environmental polycyclic aromatic hydrocarbons (PAHs), in particular those of high-molecular-weight PAHs (with MW higher than 300), remain poorly characterized. The objective of this study was to evaluate the ability of selected environmentally relevant PAHs with MW 302 (MW302 PAHs) to activate the aryl hydrocarbon receptor (AhR), since this represents a major toxic mode of action of PAHs. A large number of the evaluated compounds exhibited strong AhR-mediated activities, in particular in human models. The studied MW302 PAHs also significantly contributed to the overall calculated AhR activities of complex environmental mixtures, including both defined standard reference materials and collected diesel exhaust particles. The high AhR-mediated activities of representative MW302 PAHs, e.g. naphtho[1,2-k]fluoranthene, corresponded with the modulation of expression of relevant AhR target genes in a human lung cell model, or with the AhR-dependent suppression of cell cycle progression/proliferation in estrogen-sensitive cells. This was in a marked contrast with the limited genotoxicity of the same compound(s). Given the substantial levels of the AhR-activating MW302 PAHs in combustion particles, it seems important to continue to investigate the toxic modes of action of this large group of PAHs associated with airborne particulate matter.
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http://dx.doi.org/10.1016/j.envpol.2020.115125DOI Listing
November 2020

Gadolinium labelled nanoliposomes as the platform for MRI theranostics: in vitro safety study in liver cells and macrophages.

Sci Rep 2020 03 16;10(1):4780. Epub 2020 Mar 16.

Veterinary Research Institute, Brno, Czech Republic.

Gadolinium (Gd)-based contrast agents are extensively used for magnetic resonance imaging (MRI). Liposomes are potential nanocarrier-based biocompatible platforms for development of new generations of MRI diagnostics. Liposomes with Gd-complexes (Gd-lip) co-encapsulated with thrombolytic agents can serve both for imaging and treatment of various pathological states including stroke. In this study, we evaluated nanosafety of Gd-lip containing PE-DTPA chelating Gd prepared by lipid film hydration method. We detected no cytotoxicity of Gd-lip in human liver cells including cancer HepG2, progenitor (non-differentiated) HepaRG, and differentiated HepaRG cells. Furthermore, no potential side effects of Gd-lip were found using a complex system including general biomarkers of toxicity, such as induction of early response genes, oxidative, heat shock and endoplasmic reticulum stress, DNA damage responses, induction of xenobiotic metabolizing enzymes, and changes in sphingolipid metabolism in differentiated HepaRG. Moreover, Gd-lip did not show pro-inflammatory effects, as assessed in an assay based on activation of inflammasome NLRP3 in a model of human macrophages, and release of eicosanoids from HepaRG cells. In conclusion, this in vitro study indicates potential in vivo safety of Gd-lip with respect to hepatotoxicity and immunopathology caused by inflammation.
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http://dx.doi.org/10.1038/s41598-020-60284-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7075985PMC
March 2020

Modulation of endocrine nuclear receptor activities by polyaromatic compounds present in fractionated extracts of diesel exhaust particles.

Sci Total Environ 2019 Aug 27;677:626-636. Epub 2019 Apr 27.

Department of Chemistry and Toxicology, Veterinary Research Institute, Hudcova 70, 62100 Brno, Czech Republic. Electronic address:

Organic pollutants associated with diesel exhaust particles (DEP), such as polycyclic aromatic hydrocarbons (PAHs) and their derivatives, may negatively impact human health. However, a comprehensive overview of their effects on endocrine nuclear receptor activities is still missing. Here, we evaluated the effects of extracts and chromatographic fractions (fractionated according to increasing polarity) of two standard reference materials derived from distinct types of diesel engines (SRM 2975, SRM 1650b), on activation of androgen receptor (AR), estrogen receptor alpha (ERα), peroxisome proliferator-activated receptor γ (PPARγ), glucocorticoid receptor (GR) and thyroid receptor α (TRα), using human cell-based reporter gene assays. Neither DEP standard modulated AR or GR activities. Crude extracts and fractions of SRM 1650b and SRM 2975 suppressed ERα-mediated activity in the ER-CALUX™ assay; however, this effect could be partly linked to their cytotoxicity in this cell line. We observed that only SRM 2975 extract and its fractions were partial PPARγ inducers, while SRM 1650b extract was not active towards this receptor. Importantly, we found that both extracts and polar fractions of SRM activated TRα and significantly potentiated the activity of endogenous TRα ligand, triiodothyronine. Based on a detailed chemical analysis of both extracts and their polar fractions, we identified several oxygenated PAH derivatives, that were present at relatively high levels in the analyzed DEP standards, including 3-nitrobenzanthrone (3-NBA), anthracene-9,10-dione, phenanthrene-9,10-dione, 9H-fluoren-9-one or benzo[a]anthracene-7,12-dione, to activate TRα activity. Nevertheless, these compounds provided only a minor contribution to the overall TRα activity identified in polar fractions. This suggests that yet unidentified polar polyaromatic compounds associated with DEP may, apart from their known impact on the aryl hydrocarbon receptor or steroid signaling, deregulate activities of additional nuclear receptors, in particular of TRα. This illustrates the need to better characterize endocrine disrupting activities of DEP.
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http://dx.doi.org/10.1016/j.scitotenv.2019.04.390DOI Listing
August 2019

Aryl Hydrocarbon Receptor-Dependent Metabolism Plays a Significant Role in Estrogen-Like Effects of Polycyclic Aromatic Hydrocarbons on Cell Proliferation.

Toxicol Sci 2018 10;165(2):447-461

Department of Cytokinetics, Institute of Biophysics of the Czech Academy of Sciences, 61265 Brno, Czech Republic.

Polycyclic aromatic hydrocarbons (PAHs) are widespread environmental contaminants that interact in a complex manner with both the aryl hydrocarbon receptor (AhR) and estrogen receptors (ER). Their potential endocrine-disrupting activities may depend on both inhibitory AhR-ER cross-talk and on AhR-dependent metabolic production of estrogenic PAH metabolites. Here, we analyzed the impact of AhR on estrogen-like effects of PAHs, such as benzo[a]pyrene (BaP), in particular, on control of cell cycle progression/cell proliferation. Using AhR knockout variant of estrogen-sensitive human breast cancer MCF-7 cells (MCF-7 AhRKO cells), we observed that the AhR-dependent control of cytochrome P450 family 1 (CYP1) expression played a major role in formation of estrogenic BaP metabolites, most notably 3-OH-BaP, which contributed to the ER-dependent induction of cell cycle progression/cell proliferation. Both BaP metabolism and the BaP-induced S-phase transition/cell proliferation were inhibited in MCF-7 AhRKO cells, whereas these cells remained sensitive towards both endogenous estrogen 17β-estradiol or hydroxylated BaP metabolites. BaP was found to increase the activity of ER-dependent luciferase reporter gene in wild-type MCF-7 cells; however, unlike its hydroxylated metabolite, BaP failed to stimulate luciferase activity in MCF-7 AhRKO cells. Similarly, estrogen-like effects of other known estrogenic PAHs, such as benz[a]anthracene or 3-methylcholanthrene, were diminished in MCF-7 AhRKO cells. Ectopic expression of human CYP1A1 and CYP1B1 enzymes partly restored both BaP metabolism and its effects on cell proliferation. Taken together, our data suggest that the AhR-dependent metabolism of PAHs contributes significantly to the impact of PAHs on cell proliferation in estrogen-sensitive cells.
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http://dx.doi.org/10.1093/toxsci/kfy153DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6154273PMC
October 2018

Adaptive changes in global gene expression profile of lung carcinoma A549 cells acutely exposed to distinct types of AhR ligands.

Toxicol Lett 2018 Aug 25;292:162-174. Epub 2018 Apr 25.

Department of Chemistry and Toxicology, Veterinary Research Institute, Brno, Czech Republic. Electronic address:

Exposure to persistent ligands of aryl hydrocarbon receptor (AhR) has been found to cause lung cancer in experimental animals, and lung adenocarcinomas are often associated with enhanced AhR expression and aberrant AhR activation. In order to better understand the action of toxic AhR ligands in lung epithelial cells, we performed global gene expression profiling and analyze TCDD-induced changes in A549 transcriptome, both sensitive and non-sensitive to CH223191 co-treatment. Comparison of our data with results from previously reported microarray and ChIP-seq experiments enabled us to identify candidate genes, which expression status reflects exposure of lung cancer cells to TCDD, and to predict processes, pathways (e.g. ER stress, Wnt/β-cat, IFNɣ, EGFR/Erbb1), putative TFs (e.g. STAT, AP1, E2F1, TCF4), which may be implicated in adaptive response of lung cells to TCDD-induced AhR activation. Importantly, TCDD-like expression fingerprint of selected genes was observed also in A549 cells exposed acutely to both toxic (benzo[a]pyrene, benzo[k]fluoranthene) and endogenous AhR ligands (2-(1H-Indol-3-ylcarbonyl)-4-thiazolecarboxylic acid methyl ester and 6-formylindolo[3,2-b]carbazole). Overall, our results suggest novel cellular candidates, which could help to improve monitoring of AhR-dependent transcriptional activity during acute exposure of lung cells to distinct types of environmental pollutants.
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http://dx.doi.org/10.1016/j.toxlet.2018.04.024DOI Listing
August 2018

In vitro profiling of toxic effects of prominent environmental lower-chlorinated PCB congeners linked with endocrine disruption and tumor promotion.

Environ Pollut 2018 Jun 15;237:473-486. Epub 2018 Mar 15.

Department of Chemistry and Toxicology, Veterinary Research Institute, Hudcova 70, 62100 Brno, Czech Republic. Electronic address:

The mechanisms contributing to toxic effects of airborne lower-chlorinated PCB congeners (LC-PCBs) remain poorly characterized. We evaluated in vitro toxicities of environmental LC-PCBs found in both indoor and outdoor air (PCB 4, 8, 11, 18, 28 and 31), and selected hydroxylated metabolites of PCB 8, 11 and 18, using reporter gene assays, as well as other functional cellular bioassays. We focused on processes linked with endocrine disruption, tumor promotion and/or regulation of transcription factors controlling metabolism of both endogenous compounds and xenobiotics. The tested LC-PCBs were found to be mostly efficient anti-androgenic (within nanomolar - micromolar range) and estrogenic (at micromolar concentrations) compounds, as well as inhibitors of gap junctional intercellular communication (GJIC) at micromolar concentrations. PCB 8, 28 and 31 were found to partially inhibit the aryl hydrocarbon receptor (AhR)-mediated activity. The tested LC-PCBs were also partial constitutive androstane receptor (CAR) and pregnane X receptor (PXR) agonists, with PCB 4, 8 and 18 being the most active compounds. They were inactive towards other nuclear receptors, such as vitamin D receptor, thyroid receptor α, glucocorticoid receptor or peroxisome proliferator-activated receptor γ. We found that only PCB 8 contributed to generation of oxidative stress, while all tested LC-PCBs induced arachidonic acid release (albeit without further modulations of arachidonic acid metabolism) in human lung epithelial cells. Importantly, estrogenic effects of hydroxylated (OH-PCB) metabolites of LC-PCBs (4-OH-PCB 8, 4-OH-PCB 11 and 4'-OH-PCB 18) were higher than those of the parent PCBs, while their other toxic effects were only slightly altered or suppressed. This suggested that metabolism may alter toxicity profiles of LC-PCBs in a receptor-specific manner. In summary, anti-androgenic and estrogenic activities, acute inhibition of GJIC and suppression of the AhR-mediated activity were found to be the most relevant modes of action of airborne LC-PCBs, although they partially affected also additional cellular targets.
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http://dx.doi.org/10.1016/j.envpol.2018.02.067DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5908724PMC
June 2018

Atropisomers of 2,2',3,3',6,6'-hexachlorobiphenyl (PCB 136) exhibit stereoselective effects on activation of nuclear receptors in vitro.

Environ Sci Pollut Res Int 2018 Jun 9;25(17):16411-16419. Epub 2017 Nov 9.

Department of Chemistry and Toxicology, Veterinary Research Institute, 62100, Brno, Czech Republic.

PCB 136 is an environmentally relevant chiral PCB congener, which has been found in vivo to be present in form of rotational isomers (atropisomers). Its atropselective biotransformation or neurotoxic effects linked with sensitization of ryanodine receptor suggest that it might interact also with other intracellular receptors in a stereospecific manner. However, possible atropselective effects of PCB 136 on nuclear receptor transactivation remain unknown. Therefore, in this study, atropselective effects of PCB 136 on nuclear receptors controlling endocrine signaling and/or expression of xenobiotic and steroid hormone catabolism were investigated. PCB136 atropisomers were found to exert differential effects on estrogen receptor (ER) activation; (+)-PCB 136 was estrogenic, while (-)-PCB 136 was antiestrogenic. In contrast, inhibition of androgen receptor (AR) activity was not stereospecific. Both PCB136 stereoisomers induced the constitutive androgen receptor (CAR)-dependent gene expression; however, no significant stereospecificity of PCB 136 atropisomers was observed. PCB136 was a partial inducer of the pregnane X receptor (PXR)-dependent gene expression. Here, (-)-PCB 136 was a significantly more potent inducer of PXR activity than (+)-PCB 136. Taken together, the present results indicate that at least two nuclear receptors participating in endocrine regulation or metabolism, ER and PXR, could be regulated in an atropselective manner by chiral PCB 136. The enantioselective enrichment of PCB atropisomers in animal and human tissues may thus have significant consequences for endocrine-disrupting effects of chiral ortho-substituted PCB congeners.
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http://dx.doi.org/10.1007/s11356-017-0683-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5943194PMC
June 2018

Comparative Analysis of Toxic Responses of Organic Extracts from Diesel and Selected Alternative Fuels Engine Emissions in Human Lung BEAS-2B Cells.

Int J Mol Sci 2016 Nov 3;17(11). Epub 2016 Nov 3.

Department of Genetic Toxicology and Nanotoxicology, Institute of Experimental Medicine AS CR, Videnska 1083, 142 20 Prague, Czech Republic.

This study used toxicogenomics to identify the complex biological response of human lung BEAS-2B cells treated with organic components of particulate matter in the exhaust of a diesel engine. First, we characterized particles from standard diesel (B0), biodiesel (methylesters of rapeseed oil) in its neat form (B100) and 30% by volume blend with diesel fuel (B30), and neat hydrotreated vegetable oil (NEXBTL100). The concentration of polycyclic aromatic hydrocarbons (PAHs) and their derivatives in organic extracts was the lowest for NEXBTL100 and higher for biodiesel. We further analyzed global gene expression changes in BEAS-2B cells following 4 h and 24 h treatment with extracts. The concentrations of 50 µg extract/mL induced a similar molecular response. The common processes induced after 4 h treatment included antioxidant defense, metabolism of xenobiotics and lipids, suppression of pro-apoptotic stimuli, or induction of plasminogen activating cascade; 24 h treatment affected fewer processes, particularly those involved in detoxification of xenobiotics, including PAHs. The majority of distinctively deregulated genes detected after both 4 h and 24 h treatment were induced by NEXBTL100; the deregulated genes included, e.g., those involved in antioxidant defense and cell cycle regulation and proliferation. B100 extract, with the highest PAH concentrations, additionally affected several cell cycle regulatory genes and p38 signaling.
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http://dx.doi.org/10.3390/ijms17111833DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5133834PMC
November 2016

Assessment of the aryl hydrocarbon receptor-mediated activities of polycyclic aromatic hydrocarbons in a human cell-based reporter gene assay.

Environ Pollut 2017 Jan 29;220(Pt A):307-316. Epub 2016 Sep 29.

Department of Chemistry and Toxicology, Veterinary Research Institute, Hudcova 70, 62100 Brno, Czech Republic. Electronic address:

Activation of the aryl hydrocarbon receptor (AhR)-mediated activity is one of key events in toxicity of polycyclic aromatic hydrocarbons (PAHs). Although various classes of AhR ligands may differentially activate human and rodent AhR, there is presently a lack of data on the human AhR-inducing relative potencies (REPs) of PAHs. Here, we focused on estimation of the AhR-mediated activities of a large set of environmental PAHs in human gene reporter AZ-AhR cell line, with an aim to develop the human AhR-based REP values with potential implications for risk assessment of PAHs. The previously identified weakly active PAHs mostly failed to activate the AhR in human cells. The order for REPs of individual PAHs in human cells largely corresponded with the available data from rodent-based experimental systems; nevertheless, we identified differences up to one order of magnitude in REP values of PAHs between human and rodent cells. Higher REP values were found in human cells for some important environmental contaminants or suspected carcinogens, such as indeno[1,2,3-cd]pyrene, benz[a]anthracene or benzo[b]fluoranthene, while lower REP values were determined for methyl-substituted PAHs. Our results also indicate that a different rate of metabolism for individual PAHs in human vs. rodent cells may affect estimation of REP values in human cell-based assay, and potentially alter toxicity of some compounds, such as benzofluoranthenes, in humans. We applied the AZ-AhR assay to evaluation of the AhR-mediated activity of complex mixtures of organic compounds associated with diesel exhaust particles, and we identified the polar compounds present in these mixtures as being particularly highly active in human cells, as compared with rodent cells. The present data suggest that differences may exist between the AhR-mediated potencies of PAHs in human and rodent cells, and that the AhR-mediated effects of polar PAH derivatives and metabolites in human cell models deserve further attention.
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http://dx.doi.org/10.1016/j.envpol.2016.09.064DOI Listing
January 2017

Day-to-day variability of toxic events induced by organic compounds bound to size segregated atmospheric aerosol.

Environ Pollut 2015 Jul 26;202:135-45. Epub 2015 Mar 26.

Department of Chemistry and Toxicology, Veterinary Research Institute, Hudcova 70, 62100 Brno, Czech Republic. Electronic address:

This study quantified the temporal variability of concentration of carcinogenic polycyclic aromatic hydrocarbons (c-PAHs), genotoxicity, oxidative DNA damage and dioxin-like activity of the extractable organic matter (EOM) of atmospheric aerosol particles of aerodynamic diameter (dae, μm) coarse (1 < dae < 10), upper- (0.5 < dae < 1) and lower-accumulation (0.17 < dae < 0.5) and ultrafine (<0.17) fractions. The upper accumulation fraction formed most of the aerosol mass for 22 of the 26 study days and contained ∼44% of total c-PAHs, while the ultrafine fraction contained only ∼11%. DNA adduct levels suggested a crucial contribution of c-PAHs bound to the upper accumulation fraction. The dioxin-like activity was also driven primarily by c-PAH concentrations. In contrast, oxidative DNA damage was not related to c-PAHs, as a negative correlation with c-PAHs was observed. These results suggest that genotoxicity and dioxin-like activity are the major toxic effects of organic compounds bound to size segregated aerosol, while oxidative DNA damage is not induced by EOM.
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http://dx.doi.org/10.1016/j.envpol.2015.03.024DOI Listing
July 2015

Consensus toxicity factors for polychlorinated dibenzo-p-dioxins, dibenzofurans, and biphenyls combining in silico models and extensive in vitro screening of AhR-mediated effects in human and rodent cells.

Chem Res Toxicol 2015 Apr 13;28(4):641-50. Epub 2015 Feb 13.

†Department of Chemistry, Umeå University, SE-901 87 Umeå, Sweden.

Consensus toxicity factors (CTFs) were developed as a novel approach to establish toxicity factors for risk assessment of dioxin-like compounds (DLCs). Eighteen polychlorinated dibenzo-p-dioxins, dibenzofurans (PCDD/Fs), and biphenyls (PCBs) with assigned World Health Organization toxic equivalency factors (WHO-TEFs) and two additional PCBs were screened in 17 human and rodent bioassays to assess their induction of aryl hydrocarbon receptor-related responses. For each bioassay and compound, relative effect potency values (REPs) compared to 2,3,7,8-tetrachlorodibenzo-p-dioxin were calculated and analyzed. The responses in the human and rodent cell bioassays generally differed. Most notably, the human cell models responded only weakly to PCBs, with 3,3',4,4',5-pentachlorobiphenyl (PCB126) being the only PCB that frequently evoked sufficiently strong responses in human cells to permit us to calculate REP values. Calculated REPs for PCB126 were more than 30 times lower than the WHO-TEF value for PCB126. CTFs were calculated using score and loading vectors from a principal component analysis to establish the ranking of the compounds and, by rescaling, also to provide numerical differences between the different congeners corresponding to the TEF scheme. The CTFs were based on rat and human bioassay data and indicated a significant deviation for PCBs but also for certain PCDD/Fs from the WHO-TEF values. The human CTFs for 2,3,4,7,8-pentachlorodibenzofuran, 1,2,3,4,7,8-hexachlorodibenzofuran, 1,2,3,4,6,7,8-heptachlorodibenzo-p-dioxin, and 1,2,3,4,7,8,9-heptachlorodibenzofuran were up to 10 times greater than their WHO-TEF values. Quantitative structure-activity relationship models were used to predict CTFs for untested WHO-TEF compounds, suggesting that the WHO-TEF value for 1,2,3,7,8-pentachlorodibenzofuran could be underestimated by an order of magnitude for both human and rodent models. Our results indicate that the CTF approach provides a powerful tool for condensing data from batteries of screening tests using compounds with similar mechanisms of action, which can be used to improve risk assessment of DLCs.
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http://dx.doi.org/10.1021/tx500434jDOI Listing
April 2015

The aryl hydrocarbon receptor-mediated and genotoxic effects of fractionated extract of standard reference diesel exhaust particle material in pulmonary, liver and prostate cells.

Toxicol In Vitro 2015 Apr 10;29(3):438-48. Epub 2014 Dec 10.

Department of Chemistry and Toxicology, Veterinary Research Institute, Hudcova 70, 62100 Brno, Czech Republic. Electronic address:

Diesel exhaust particles (DEP) and the associated complex mixtures of organic pollutants, such as polycyclic aromatic hydrocarbons (PAHs), or their derivatives, have been suggested to exert deleterious effects on human health. We used a set of defined cellular models representing liver, lung and prostate tissues, in order to compare non-genotoxic and genotoxic effects of crude and fractionated extract of a standard reference DEP material - SRM 1650b. We focused on the aryl hydrocarbon receptor (AhR)-mediated activity, modulation of cell proliferation, formation of DNA adducts, oxidative DNA damage, and induction of DNA damage responses, including evaluation of apoptosis, and phosphorylation of p53 tumor suppressor and checkpoint kinases (Chk). Both PAHs and the polar aromatic compounds contributed to the AhR-mediated activity of DEP-associated organic pollutants. The principal identified AhR agonists included benzo[k]fluoranthene, indeno[1,2,3-c,d]pyrene, chrysene and several non-priority PAHs, including benzochrysenes and methylated PAHs. In contrast to PAHs, polar compounds contributed more significantly to overall formation of DNA adducts associated with phosphorylation of p53, Chk1 or Chk2, and partly with apoptosis. Therefore, more attention should be paid to identification of DEP-associated polar organic compounds, contributing to the AhR activation and cytotoxic/genotoxic effects of complex airborne mixtures of organic contaminants produced by diesel engines.
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http://dx.doi.org/10.1016/j.tiv.2014.12.002DOI Listing
April 2015

Interactive effects of inflammatory cytokine and abundant low-molecular-weight PAHs on inhibition of gap junctional intercellular communication, disruption of cell proliferation control, and the AhR-dependent transcription.

Toxicol Lett 2015 Jan 28;232(1):113-21. Epub 2014 Sep 28.

Department of Cytokinetics, Institute of Biophysics AS CR, Královopolská 135, 61265 Brno, Czech Republic. Electronic address:

Polycyclic aromatic hydrocarbons (PAHs) with lower molecular weight exhibit lesser genotoxicity and carcinogenicity than highly carcinogenic PAHs with a higher number of benzene rings. Nevertheless, they elicit specific effects linked with tumor promotion, such as acute inhibition of gap junctional intercellular communication (GJIC). Although inflammatory reaction may alter bioactivation and toxicity of carcinogenic PAHs, little is known about the impact of pro-inflammatory cytokines on toxic effects of the low-molecular-weight PAHs. Here, we investigated the impact of a pro-inflammatory cytokine, tumor necrosis factor-α (TNF-α), on the effects associated with tumor promotion and with induction of the aryl hydrocarbon receptor (AhR)-dependent gene expression in rat liver epithelial cells. We found that a prolonged incubation with TNF-α induced a down-regulation of GJIC, associated with reduced expression of connexin 43 (Cx43), a major connexin isoform found in liver epithelial cells. The Cx43 down-regulation was partly mediated by the activity of the mitogen-activated protein (MAP) p38 kinase. Independently of GJIC modulation, or p38 activation, TNF-α potentiated the AhR-dependent proliferative effect of a model low-molecular-weight PAH, fluoranthene, on contact-inhibited cells. In contrast, this pro-inflammatory cytokine repressed the fluoranthene-induced expression of a majority of model AhR gene targets, such as Cyp1a1, Ahrr or Tiparp. The results of the present study indicate that inflammatory reaction may differentially modulate various toxic effects of low-molecular-weight PAHs; the exposure to pro-inflammatory cytokines may both strengthen (inhibition of GJIC, disruption of contact inhibition) and repress (expression of a majority of AhR-dependent genes) their impact on toxic endpoints associated with carcinogenesis.
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http://dx.doi.org/10.1016/j.toxlet.2014.09.023DOI Listing
January 2015

In vitro and in silico derived relative effect potencies of ah-receptor-mediated effects by PCDD/Fs and PCBs in rat, mouse, and guinea pig CALUX cell lines.

Chem Res Toxicol 2014 Jul 18;27(7):1120-32. Epub 2014 Jun 18.

Department of Chemistry, Umeå University , SE-901 87 Umeå, Sweden.

For a better understanding of species-specific relative effect potencies (REPs), responses of dioxin-like compounds (DLCs) were assessed. REPs were calculated using chemical-activated luciferase gene expression assays (CALUX) derived from guinea pig, rat, and mouse cell lines. Almost all 20 congeners tested in the rodent cell lines were partial agonists and less efficacious than 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). For this reason, REPs were calculated for each congener using concentrations at which 20% of the maximal TCDD response was reached (REP20TCDD). REP20TCDD values obtained for PCDD/Fs were comparable with their toxic equivalency factors assigned by the World Health Organization (WHO-TEF), while those for PCBs were in general lower than the WHO-TEF values. Moreover, the guinea pig cell line was the most sensitive as indicated by the 20% effect concentrations of TCDD of 1.5, 5.6, and 11.0 pM for guinea pig, rat, and mouse cells, respectively. A similar response pattern was observed using multivariate statistical analysis between the three CALUX assays and the WHO-TEFs. The mouse assay showed minor deviation due to higher relative induction potential for 2,3,7,8-tetrachlorodibenzofuran and 2,3,4,6,7,8-hexachlorodibenzofuran and lower for 1,2,3,4,6,7,8-heptachlorodibenzofuran and 3,3',4,4',5-pentachlorobiphenyl (PCB126). 2,3,7,8-Tetrachlorodibenzofuran was more than two times more potent in the mouse assay as compared with that of rat and guinea pig cells, while measured REP20TCDD for PCB126 was lower in mouse cells (0.05) as compared with that of the guinea pig (0.2) and rat (0.07). In order to provide REP20TCDD values for all WHO-TEF assigned compounds, quantitative structure-activity relationship (QSAR) models were developed. The QSAR models showed that specific electronic properties and molecular surface characteristics play important roles in the AhR-mediated response. In silico derived REP20TCDD values were generally consistent with the WHO-TEFs with a few exceptions. The QSAR models indicated that, e.g., 1,2,3,7,8-pentachlorodibenzofuran and 1,2,3,7,8,9-hexachlorodibenzofuran were more potent than given by their assigned WHO-TEF values, and the non-ortho PCB 81 was predicted, based on the guinea-pig model, to be 1 order of magnitude above its WHO-TEF value. By combining in vitro and in silico approaches, REPs were established for all WHO-TEF assigned compounds (except OCDD), which will provide future guidance in testing AhR-mediated responses of DLCs and to increase our understanding of species variation in AhR-mediated effects.
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http://dx.doi.org/10.1021/tx5001255DOI Listing
July 2014

TGF-β1 signaling plays a dominant role in the crosstalk between TGF-β1 and the aryl hydrocarbon receptor ligand in prostate epithelial cells.

Cell Signal 2012 Aug 25;24(8):1665-76. Epub 2012 Apr 25.

Department of Cytokinetics, Institute of Biophysics, Academy of Sciences of the Czech Republic, Brno, Czech Republic.

Crosstalk between the aryl hydrocarbon receptor (AhR) and transforming growth factor-β1 (TGF-β1) signaling has been observed in various experimental models. However, both molecular mechanism underlying this crosstalk and tissue-specific context of this interaction are still only partially understood. In a model of human non-tumorigenic prostate epithelial cells BPH-1, derived from the benign prostatic hyperplasia, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) persistently activates the AhR signaling pathway and induces expression of xenobiotic metabolizing enzymes, such as CYP1A1 or CYP1B1. Here we demonstrate that TGF-β1 suppresses the AhR-mediated gene expression through multiple mechanisms, involving inhibition of AhR expression and down-regulation of nuclear AhR, via a SMAD4-dependent pathway. In contrast, TCDD-induced AhR signaling does not affect either TGF-β1-regulated gene expression or epithelial-to-mesenchymal transition. These observations suggest that, in the context of prostate epithelium, TGF-β1 signaling plays a dominant role in the crosstalk with AhR signaling pathway. Given the importance of TGF-β1 signaling in regulation of prostate epithelial tissue homeostasis, as well as the recently revealed role of AhR in prostate development and tumorigenesis, the above findings contribute to our understanding of the mechanisms underlying the crosstalk between the two signaling pathways in the prostate-specific context.
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http://dx.doi.org/10.1016/j.cellsig.2012.04.008DOI Listing
August 2012

Activation of the aryl hydrocarbon receptor is the major toxic mode of action of an organic extract of a reference urban dust particulate matter mixture: the role of polycyclic aromatic hydrocarbons.

Mutat Res 2011 Sep 5;714(1-2):53-62. Epub 2011 Jul 5.

Department of Cytokinetics, Institute of Biophysics AS CR, Královopolská 135, 61265 Brno, Czech Republic.

Many of the toxic and carcinogenic effects of urban air pollution have been linked to polycyclic aromatic hydrocarbons (PAHs) adsorbed to airborne particulate matter (PM). The carcinogenic properties of PAHs in complex organic mixtures derived from PM have been chiefly attributed to their mutagenicity. Nevertheless, PAHs are also potent activators of the aryl hydrocarbon receptor (AhR), which may contribute to their nongenotoxic effects, including tumor promotion. As the genotoxicity of carcinogenic PAHs in complex mixtures derived from urban PM is often inhibited by other mixture constituents, the AhR-mediated activity of urban PM extracts might significantly contribute to the carcinogenic activity of such mixtures. In the present study, we used an organic extract of the urban dust standard reference material, SRM1649a, as a model mixture to study a range of toxic effects related to DNA damage and AhR activation. Both the organic extract and its neutral aromatic fraction formed a low number of DNA adducts per nucleotide in the liver epithelial WB-F344 cells model, without inducing DNA damage response, such as tumor suppressor p53 activation and apoptosis. In contrast, we found that this extract, as well as its neutral and polar fractions, were potent inducers of a range of AhR-mediated responses, including induction of the AhR-mediated transcription, such as cytochrome P450 1A1/1B1 expression, and the AhR-dependent cell proliferation. Importantly, these toxic events occurred at doses one order of magnitude lower than DNA damage. The AhR-mediated activity of the neutral fraction was linked to PAHs and their derivatives, as polychlorinated dibenzo-p-dioxins, dibenzofurans and biphenyls were only minor contributors to the overall AhR-mediated activity. Taken together, our data suggest that more attention should be paid to the AhR-dependent nongenotoxic events elicited by urban PM constituents, especially PAHs and their derivatives.
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http://dx.doi.org/10.1016/j.mrfmmm.2011.06.011DOI Listing
September 2011

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

Chem Res Toxicol 2011 Jun 3;24(6):866-76. Epub 2011 Jun 3.

Department of Chemistry and Toxicology, Veterinary Research Institute, Hudcova 70, 62100 Brno, Czech Republic.

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.
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http://dx.doi.org/10.1021/tx200049xDOI Listing
June 2011

In vitro toxicity profiling of ultrapure non-dioxin-like polychlorinated biphenyl congeners and their relative toxic contribution to PCB mixtures in humans.

Toxicol Sci 2011 May 25;121(1):88-100. Epub 2011 Feb 25.

Institute for Environmental Studies (IVM), VU University Amsterdam, 1081 HV Amsterdam, The Netherlands.

The toxic equivalency concept used for the risk assessment of polychlorinated biphenyls (PCBs) is based on the aryl hydrocarbon receptor (AhR)-mediated toxicity of coplanar dioxin-like (DL) PCBs. Most PCBs in the environment, however, are non-dioxin-like (NDL) PCBs that cannot adopt a coplanar structure required for AhR activation. For NDL-PCBs, no generally accepted risk concept is available because their toxicity is insufficiently characterized. Here, we systematically determined in vitro toxicity profiles for 24 PCBs regarding 10 different mechanisms of action. Prior to testing, NDL-PCB standards were purified to remove traces of DL compounds. All NDL-PCBs antagonized androgen receptor activation and inhibited gap junctional intercellular communication (GJIC). Lower chlorinated NDL-PCBs were weak estrogen receptor (ER) agonists, whereas higher chlorinated NDL-PCBs were weak ER antagonists. Several NDL-PCBs inhibited estradiol-sulfotransferase activity and bound to transthyretin (TTR) but with much weaker potencies than reported for hydroxylated PCB metabolites. AhR-mediated expression of uridine-glucuronyl transferase isozyme UGT1A6 was induced by DL-PCBs only. Hierarchical cluster analysis of the toxicity profiles yielded three separate clusters of NDL-PCBs and a fourth cluster of reference DL-PCBs. Due to small differences in relative potency among congeners, the highly abundant indicator PCBs 28, 52, 101, 118, 138, 153, and 180 also contributed most to the antiandrogenic, (anti)estrogenic, antithyroidal, tumor-promoting, and neurotoxic potencies calculated for PCB mixtures reported in human samples, whereas the most potent AhR-activating DL-PCB-126 contributed at maximum 0.2% to any of these calculated potencies. PCB-168 is recommended as an additional indicator congener, given its relatively high abundance and antiandrogenic, TTR-binding, and GJIC-inhibiting potencies.
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http://dx.doi.org/10.1093/toxsci/kfr043DOI Listing
May 2011

Polar compounds dominate in vitro effects of sediment extracts.

Environ Sci Technol 2011 Mar 24;45(6):2384-90. Epub 2011 Feb 24.

Helmholtz Centre for Environmental Research - UFZ, Leipzig, Germany.

Sediment extracts from three polluted sites of the river Elbe basin were fractionated using a novel online fractionation procedure. Resulting fractions were screened for mutagenic, aryl hydrocarbon receptor (AhR)-mediated, transthyretin (TTR)-binding, and estrogenic activities and their potency to inhibit gap junctional intercellular communication (GJIC) to compare toxicity patterns and identify priority fractions. Additionally, more than 200 compounds and compound classes were identified using GC-MS/MS, LC-MS/MS, and HPLC-DAD methods. For all investigated end points, major activities were found in polar fractions, which are defined here as fractions containing dominantly compounds with at least one polar functional group. Nonpolar PAH fractions contributed to mutagenic and AhR-mediated activities while inhibition of GJIC and estrogenic and TTR-binding activities were exclusively observed in the polar fractions. Known mutagens in polar fractions included nitro- and dinitro-PAHs, azaarenes, and keto-PAHs, while parent and monomethylated PAHs such as benzo[a]pyrene and benzofluoranthenes were identified in nonpolar fractions. Additionally, for one sample, high AhR-mediated activities were determined in one fraction characterized by PCDD/Fs, PCBs, and PCNs. Estrone, 17β-estradiol, 9H-benz[de]anthracen-7-one, and 4-nonylphenol were identified as possible estrogenic and TTR-binding compounds. Thus, not only nonpolar compounds such as PAHs, PCBs, and PCDD/Fs but also the less characterized and investigated more polar substances should be considered as potent mutagenic, estrogenic, AhR-inducing, TTR-binding, and GJIC-inhibiting components for future studies.
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http://dx.doi.org/10.1021/es103381yDOI Listing
March 2011

Role of aryl hydrocarbon receptor in modulation of the expression of the hypoxia marker carbonic anhydrase IX.

Biochem J 2009 Apr;419(2):419-25

Institute of Virology, Centre of Molecular Medicine, Slovak Academy of Sciences, Dubravska cesta 9, 845 05 Bratislava, Slovak Republic.

Tumour-associated expression of CA IX (carbonic anhydrase IX) is to a major extent regulated by HIF-1 (hypoxia-inducible factor-1) which is important for transcriptional activation and consists of the oxygen-regulated subunit HIF-1alpha and the partner factor ARNT [AhR (aryl hydrocarbon receptor) nuclear translocator]. We have previously observed that HIF-1alpha competes with the AhR for interaction with ARNT under conditions when both conditionally regulated factors are activated. We have therefore investigated whether TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin)-induced activation of the AhR pathway might interfere with CA IX expression. The results from the present study suggest that TCDD treatment reduces hypoxic induction of both CA IX mRNA and protein expression. Moreover, the transcriptional activity of the CA9 promoter was significantly reduced by expression of CAAhR (constitutively active AhR), which activates transcription in a ligand-independent manner. Finally, we found that ARNT is critical for both hypoxic induction and the TCDD-mediated inhibition of CA9 expression.
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http://dx.doi.org/10.1042/BJ20080952DOI Listing
April 2009

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

Mutat Res 2008 Feb 16;638(1-2):122-32. Epub 2007 Sep 16.

Laboratory of Genetic Ecotoxicology, Institute of Experimental Medicine, AS CR, 142 20 Prague, Czech Republic.

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.
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http://dx.doi.org/10.1016/j.mrfmmm.2007.09.004DOI Listing
February 2008

Concentrations of methylated naphthalenes, anthracenes, and phenanthrenes occurring in Czech river sediments and their effects on toxic events associated with carcinogenesis in rat liver cell lines.

Environ Toxicol Chem 2007 Nov;26(11):2308-16

Institute of Biophysics, Academy of Sciences of the Czech Republic, 61265 Brno, Czech Republic.

Alkylated polycyclic aromatic hydrocarbons (PAHs) are important environmental pollutants. In the present study, we determined levels of monomethylated naphthalenes (MeNap), phenanthrenes (MePhe), and anthracenes (MeAnt) in Czech river sediments. The levels of MePhe generally were lower than the concentrations of phenanthrene. In contrast, both MeNap and MeAnt were found at levels higher than their respective parent compounds in the majority of sampling sites. We then investigated their aryl hydrocarbon receptor (AhR)-mediated activity, accumulation of phosphorylated p53 protein, induction of expression of cytochrome P450 1A1 (CYP1A1), inhibition of gap junctional intercellular communication (GJIC), and effects on cell proliferation in rat liver cell models to evaluate the relative importance of these toxicity mechanisms of low-molecular-weight methylated PAHs. Methylated phenanthrene and anthracene compounds were weak inducers of AhR-mediated activity as determined both in a reporter gene assay system and by detection of the endogenous gene (Cyp1a1) induction. 2-Methylphenanthrene was the most potent AhR ligand. Contribution of MeAnt and MePhe to overall AhR-inducing potencies should be taken into account in PAH-contaminated environments. Nevertheless, their effects on AhR were not sufficient to modulate cell proliferation in a normal rat liver progenitor cell model system. These PAHs only had a marginal effect on p53 phosphorylation at high doses of 1-, 3-, and 9-MePhe as well as 1 MeAnt. On the other hand, both 2- and 9-MeAnt as well as all the MePhe under study were efficient inhibitors of GJIC, suggesting that these compounds might act as tumor promoters. In summary, inhibition of GJIC and partial activation of AhR seem to be the most prominent toxic effects of the methylated PAHs in the present study.
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http://dx.doi.org/10.1897/07-161R.1DOI Listing
November 2007

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.

Mutat Res 2006 Apr 9;596(1-2):43-56. Epub 2006 Jan 9.

Laboratory of Cytokinetics, Institute of Biophysics, Academy of Sciences of the Czech Republic, Královopolská 135, 612 65 Brno, Czech Republic.

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.
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http://dx.doi.org/10.1016/j.mrfmmm.2005.11.005DOI Listing
April 2006