DNA adduct formation by 7H‐dibenzo[c,g]carbazole and its tissue‐ and organ‐specific derivatives in Chinese hamster V79 cell lines stably expressing cytochrome P450 enzymes

Abstract

The cytochrome P4501A subfamily (CYP1A) is involved in the metabolic activation of 7H-dibenzo[c,g]carbazole (DBC) and its tissue- and organ-specific derivatives, N-methyldibenzo[c,g]carbazole (MeDBC)and 5,9-dimethyldibenzo[c,g]carbazole (diMeDBC). In this study, we have evaluated the relationship between the tissue specificity and (32)P-postlabeled adduct patterns produced by these compounds by using a panel of Chinese hamster V79 cell lines stably expressing human CYP1A1 and CYP1A2 and/or N-acetyltransferase. Treatment of the parental cell lines V79MZ and V79NH, which are devoid of any CYP activity, with DBC and its derivatives did not result in detectable adducts. The highest DNA adduct levels were found in CYP1A1-expressing V79MZh1A1 cells after DBC and MeDBC treatment (24.5 +/- 7.2 and 16.2 +/- 3.6 adducts/10(8) nucleotides, respectively). Exposure of this cell line to DBC resulted in five distinct spots, while six spots with different chromatographic mobilities were detected in MeDBC-treated cells. DiMeDBC produced only very low levels of DNA adducts in V79MZh1A1 cells. DBC and MeDBC formed relatively low levels of DNA adducts in CYP1A2-expressing V79MZh1A2 cells (0.7 +/- 0.2 and 2.1 +/- 1.2 adducts/10(8) nucleotides, respectively). DBC formed three weak spots and MeDBC five spots in V79MZh1A2 cells, and all the spots had different chromatographic mobilities. In contrast, diMeDBC did not induce any DNA adducts in these cells, although diMeDBC induced a significant dose-dependent increase in micronucleus frequency under similar treatment conditions (r = 0.76; P < 0.001). The significant increase in DNA damage in the Comet assay following incubation of exposed cells with a repair-specific endonuclease (Fpg protein) suggests that base modifications such as 8-oxodG or Fapy-adducts might be responsible for the genotoxicity of diMeDBC in V79MZh1A2 cells. The similarities between the DNA adduct patterns produced by DBC and MeDBC in V79MZh1A1 and V79MZh1A2 cells suggest that biotransformation mediated via CYP1A1 and CYP1A2 might depend on a PAH-type pathway involving the aromatic ring system.

Genotoxicity of 7H-dibenzo[c,g]carbazole and its tissue-specific derivatives in human hepatoma HepG2 cells is related to CYP1A1/1A2 expression

“…A significant level of Fpg-sensitive sites detected in DiMeDBC-treated cells suggest that either oxidative DNA damage or unstable DNA adducts formed by one-electron oxidation [Chen et al, 1997] might underlie the biological activity of DiMeDBC in human hepatoma cells. Our results are in line with previous ones determined in the rat progenitor WB-F344 cells [Valovicova et al, 2009] and in the Chinese hamster V79MZh1A2 cells [Gabelova et al, 2004]. A significant level of Fpg-sensitive sites in HepG2 cells exposed to DiMeDBC reported also Lazarova and Slamenova, [2004].…”

“…A significant level of Fpg-sensitive sites in HepG2 cells exposed to DiMeDBC reported also Lazarova and Slamenova, [2004]. As a potent aryl hydrocarbon receptor (AhR)-agonist [Vondracek et al, 2006] DiMeDBC increased significantly the expression of AhR-mediated CYP1A1/2 mRNA in HepG2 cells though it is poorly metabolized by these enzymes [Gabelova et al, 2004]. We suppose that DiMeDBC/CYP1A1-mediated uncoupling caused generation of reactive oxygen species (ROS) in a similar manner as in the case of the halogenated hydrocarbons [Shertzer et al, 2004].…”

“…Consistent with these results, DiMeDBC and mainly N-MeDBC increased substantially the CYP1A1/2 mRNA levels in HepG2 cells. Previous in vitro studies convincingly demonstrated that human CYP1A1 and CYP1A2 are involved in metabolism of DBC and its tissue-specific derivatives [Gabelova et al, 1998[Gabelova et al, , 2000[Gabelova et al, , 2004Farkasova et al, 2001;Shertzer et al, 2007]. The CYP1A1/2-mediated N-MeDBC and DBC activation resulted in DNA breakage, increased frequency of micronuclei, DNA-adduct formation, and induction of gene mutations.…”

“…Because CYP1 family of enzymes were shown to be involved in DBC metabolism [Gabelova et al, 2002[Gabelova et al, , 2004Vondracek et al, 2006], the qRT-PCR technique was used to measure the levels of CYP1A1/2 and CYP1B1 mRNAs expression in human hepatoma cells exposed to DBC and its derivatives for 24 hr. The aim of these experiments was to understand the mechanisms underlying N-MeDBC and DiMeDBC genotoxicity in HepG2 cells.…”

Ultraviolet A radiation potentiates the cytotoxic and genotoxic effects of 7H‐dibenzo[c,g]carbazole and its methyl derivatives

“…More than a 100‐fold decrease in cell viability was found in HaCaT cells treated with these carcinogens and irradiated with UVA, compared with almost negligible cytotoxicity in the dark. The most striking evidence for UVA‐enhanced cytotoxicity of carcinogens was for DiMeDBC, as this hepatocarcinogen on its own exerts no cytotoxic or genotoxic activity in HaCaT cells [Valovicova et al, ] due to the very low expression of the cytochrome CYP1A2, which is involved in DiMeDBC metabolism [Gabelova et al, ; Valovicova et al, ]. Moreover, a linear correlation has been found between the susceptibility of the DBC derivatives to undergo photoexcitation (expressed by the HOMO/LUMO molecular orbital energy difference E gap ) and the observed cytotoxic potencies (characterized by the IC 50 values).…”