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
Source: PubMed


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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    • "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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    • "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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    • "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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