Differences in DNA damage and repair produced by systemic, hepatocarcinogenic and sarcomagenic dibenzocarbazole derivatives in a model of rat liver progenitor cells

Laboratory of Mutagenesis and Carcinogenesis, Cancer Reserach Institute, SAS, Vlárska 7, 833 91 Bratislava, Slovakia.
Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis (Impact Factor: 3.68). 07/2009; 665(1-2):51-60. DOI: 10.1016/j.mrfmmm.2009.02.014
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Liver progenitor (oval) cells are a potential target cell population for hepatocarcinogens. Our recent study showed that the liver carcinogens 7H-dibenzo[c,g]carbazole (DBC) and 5,9-dimethyldibenzo[c,g]carbazole (DiMeDBC), but not the sarcomagen N-methyldibenzo[c,g]carbazole (N-MeDBC), induced several cellular events associated with tumor promotion in WB-F344 cells, an in vitro model of liver oval cells [J. Vondracek, L. Svihalkova-Sindlerova, K. Pencikova, P. Krcmar, Z. Andrysik, K. Chramostova, S. Marvanova, Z. Valovicova, A. Kozubik, A. Gabelova, M. Machala, 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. Fundam. Mol. Mech. Mutagen. 596 (2006) 43-56]. In this study, we focused on the genotoxic effects generated by these dibenzocarbazoles in WB-F344 cells to better understand the cellular and molecular mechanisms involved in hepatocarcinogenesis. Lower IC(50) values determined for DBC and DiMeDBC, as compared with N-MeDBC, indicated a higher sensitivity of WB-F344 cells towards hepatocarcinogens. Accordingly, DBC produced a dose-dependent DNA-adduct formation resulting in substantial inhibition of DNA replication and transcription. In contrast, DNA-adduct number detected in DiMeDBC-exposed cells was almost negligible, whereas N-MeDBC produced a low level of DNA adducts. Although all dibenzocarbazoles significantly increased the level of strand breaks (p<0.05) and micronuclei (p<0.001) after 2-h treatment, differences in the kinetics of strand break rejoining were found. The strand break level in DiMeDBC- and N-MeDBC-exposed cells returned to near the background level within 24h after treatment, whereas a relatively high DNA damage level was detected in DBC-treated cells up to 48h after exposure. Additional breaks detected after incubation of DiMeDBC-exposed WB-F344 cells with a repair-specific endonuclease, along with a nearly 3-fold higher level of reactive oxygen species found in these cells as compared with control, suggest a possible role of oxidative stress in DiMeDBC genotoxicity. We demonstrated qualitative differences in the DNA damage profiles produced by hepatocarcinogens DBC and DiMeDBC in WB-F344 cells. Different lesions may trigger distinct cellular pathways involved in hepatocarcinogenesis. The low amount of DNA damage, together with an efficient repair, may explain the lack of hepatocarcinogenicity of N-MeDBC.

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Available from: Alena Gábelová, Oct 02, 2015
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    • "Micronucleus formation was scored at two sampling times, 24 h and 48 h, after a 2-h exposure of the cells to the dibenzocarbazoles, B[a]P, or the vehicle (DMSO) according to the protocol described previously [23]. We analyzed 2 × 10 3 cells per dish with an Olympus BX51 fluorescence microscope. "
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    ABSTRACT: 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.
    Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis 03/2012; 743(1-2):91-8. DOI:10.1016/j.mrgentox.2011.12.030 · 3.68 Impact Factor
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    • "Confluent HepG2 cells were exposed for 24 hr to carcinogenic compounds or to 0.1% DMSO, as vehicle control. Protein p53 and histone H2AX phosphorylation were determined in whole-cell lysates prepared by harvesting the cells in lysis buffer (1% sodium dodecyl sulfate (SDS), TRIS, 10% glycerol, protease inhibitor cocktail) as described [Valovicova et al., 2009] In brief, total protein concentrations were determined using DC Protein Assay (BioRad, Hercules, CA). For western blot analysis, equal amounts of total protein were subjected to 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE), electrotransferred onto Hybond-P, immunodetected using appropriate primary and secondary antibodies, and visualized with ECL1Plus reagent, according to manufacturer's instructions. "
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    ABSTRACT: The goal of this study was to investigate the genotoxicity of 7H-dibenzo[c,g]carbazole (DBC), a ubiquitous environmental pollutant, and its methyl derivatives, 5,9-dimethylDBC (DiMeDBC), a strict hepatocarcinogen, and N-methylDBC (N-MeDBC), a specific sarcomagen in human hepatoma HepG2 cells, and to infer potential mechanisms underlying the biological activity of particular carcinogen. All dibenzocarbazoles, regardless the tissue specificity, induced significant DNA strand break levels and micronuclei in HepG2 cells; though a mitotic spindle dysfunction rather than a chromosome breakage was implicated in N-MeDBC-mediated micronucleus formation. While DBC and N-MeDBC produced stable DNA adducts followed with p53 protein phosphorylation at Ser-15, DiMeDBC failed. A significant increase in DNA strand breaks following incubation of exposed cells with a repair-specific endonuclease (Fpg protein) suggested that either oxidative DNA damage or unstable DNA-adducts might underlie DiMeDBC genotoxicity in human hepatoma cells. DiMeDBC and N-MeDBC increased substantially also the amount of CYP1A1/2 expression in HepG2 cells. Pretreatment of cells with substances affecting AhR-mediated CYP1A family of enzymes expression; however, diminished DiMeDBC and N-MeDBC genotoxicity. Our data clearly demonstrated differences in the mechanisms involved in the biological activity of DiMeDBC and N-MeDBC in human hepatoma cells; the genotoxicity of these DBC derivatives is closely related to CYP1A1/2 expression.
    Environmental and Molecular Mutagenesis 10/2011; 52(8):636-45. DOI:10.1002/em.20664 · 2.63 Impact Factor
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    • "Despite relatively high levels of 6-TG r mutations induced by particular dibenzocarbazoles in these cell lines (Gabelova et al., 2002), no nucleotide variations in the coding region of the Hprt gene were detected. Despite a similar chemical structure, differences in the biological activity of DiMeDBC and DBC were found in vivo and in vitro (Valero et al., 1985; Valovicova et al., 2009; Vondracek et al., 2006). In V79MZh3A4 cells, DBC was less cytotoxic (IC 50 23.3 "
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    ABSTRACT: 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.
    Toxicology and Applied Pharmacology 07/2011; 255(3):307-15. DOI:10.1016/j.taap.2011.06.027 · 3.71 Impact Factor
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