Mutation Research/Genetic Toxicology and Environmental Mutagenesis
Genotoxicity of 7H-dibenzo[c,g]carbazole and its methyl derivatives in human keratinocytes
Introduction
Tissue specificity is a hallmark of many chemical carcinogens. While the liver and the urinary bladder, for example, are the primary target organs of aromatic amines, the polycyclic aromatic hydrocarbons (PAHs) have a strong local activity in the lung and the skin. One reason for such a strict specificity could be the chemical structure of the particular compounds, which affects their mode of biotransformation. Differences between tissues in the expression of drug-metabolizing enzymes may therefore greatly contribute to the tissue-specificity of chemical carcinogens.
Recent studies have convincingly demonstrated that 7H-dibenzo[c,g]carbazole (DBC) (Fig. 1), a ubiquitous environmental pollutant, is a potent multi-site and multi-species carcinogen [1], [2]. DBC induces tumors at the site of application (e.g., the skin), as well as at distant sites, mainly in the liver [3]. In contrast, its synthetic methyl derivatives 5,9-dimethyldibenzo[c,g]carbazole (DiMeDBC) and N-methylDBC (N-MeDBC) show a specific tropism to liver and skin, respectively [3], [4].
In vivo experiments have revealed a clear correlation between the DNA-binding capacity of tissue-specific DBC derivatives and their ability to induce tumors in a particular tissue. In mouse skin, the parent compound DBC and the sarcomagen N-MeDBC induce tumors, DNA adducts and gene mutations; in contrast, the strict hepatocarcinogen DiMeDBC is devoid of any biological activity in the skin [3], [4], [5]. The rodent epidermis constitutively expresses low levels of cytochrome P450 1a1 (CYP1a1) enzyme which is highly inducible by many procarcinogens [6], [7]. A substantial increase in CYP1b1 mRNA has been found in mouse skin after exposure to dioxin [7]. Previous in vitro studies have indicated that DBC and N-MeDBC are metabolized by both CYP1A1 and CYP1A2, whereas DiMeDBC is the substrate only for CYP1A2 [8], [9], [10], [11]. Another recent in vitro study has shown that CYP3A4, the most abundant enzyme in the liver, may also contribute to bio-activation of DiMeDBC [12]. Since CYP1A1 is not involved in biotransformation of DiMeDBC and neither CYP1A2 nor CYP3A4 are expressed in the mouse epidermis, this may be the reason why DiMeDBC lacks any carcinogenic activity in mouse skin.
The International Agency for Research on Cancer (IARC) [13], [14] lists DBC as a possible human carcinogen (Group 2B); the extent of human exposure to this compound is, however, still largely unknown. As an inherent component of various complex environmental mixtures including cigarette smoke, DBC may hence pose a potential hazard for human health. The skin, the largest organ of the human body, is a major site of exposure to many chemical compounds including carcinogens. Because DBC is metabolized by the human CYP1A family of enzymes [10] and since the main metabolites are phenols, as in the case of metabolism in mouse and rat [11], we evaluated the genotoxic potential of DBC and its tissue-specific derivatives DiMeDBC and N-MeDBC in immortalized human keratinocytes HaCaT [15], a relevant surrogate model of human skin [16]. Many studies have shown that CYP1A1 is strongly and preferentially up-regulated in human skin, in cultured keratinocytes or in HaCaT cells [17], [18], [19]; moreover, the induction of CYP1B1 after exposure to UV radiation has also been observed in these cells [20]. In contrast, the expression/inducibility of CYP1A2 is often low or negligible in human keratinocytes or HaCaT cells [21], [22]; therefore, the CYP1A1 enzyme seems to be the primary CYP1A isoform present and inducible in human skin. Various endpoints, including formation of DNA strand-breaks, micronuclei and DNA adducts were analyzed as biomarkers of biological activity of particular tissue-specific DBC derivatives in HaCaT cells. In addition, phosphorylation of histone H2AX and p53 protein were determined, in order to obtain comprehensive information about the biological effects of DBC, N-MeDBC and DiMeDBC in the immortalised human keratinocytes. Based on the data obtained, potential cellular and molecular mechanisms involved in the biological activity of DBC, N-MeDBC and DiMeDBC were inferred.
Section snippets
Chemicals and reagens
DBC (CAS No. 194-59-2), DiMeDBC and N-MeDBC were kindly provided by Prof. Francois Périn (Institute Curie, France). Benzo[a]pyrene (B[a]P, CAS No. 50-32-8), dibenzo[a,l]pyrene (DB[a,l]P, CAS No. 189-55-9), 4′,6′-diamidino-2-phenylindole (DAPI, CAS No. 28718-90-3), spleen phosphodiesterase, RNases A and T1, proteinase K, micrococcal nuclease, and nuclease P1 were purchased from Sigma (Lambda Life, Slovakia and Deisenhofen, Germany); T4 polynucleotide kinase was from USB (Cleveland, OH); γ-32
Cytotoxicity of DBC and its derivatives
The viability of HaCaT cells exposed to DBC and its tissue-specific derivatives after both short (2 h) and long (24 h) treatment is shown in Fig. 2. Based on the dose–response curves, the IC50 values were calculated. No cytotoxicity was found in HaCaT cells exposed to dibenzocarbazoles or B[a]P (as a model genotoxin) for 2 h (IC50 > 100 μM). In contrast, significant variations in cell survival were detected after the 24-h exposure. While DBC was the most cytotoxic compound (IC50 33.1 μM), no
Discussion
Human skin is daily exposed to many environmental toxic and carcinogenic compounds. Interactions of xenobiotics and the skin are therefore mainly influenced by the metabolic capacity of the epidermis. There is increasing evidence that various xenobiotic/drug-metabolizing enzymes that are present in the liver are also expressed in the skin, albeit at substantially lower levels. A majority of studies have suggested that rodent and human epidermis as well as cultured keratinocytes constitutively
Funding
This study was supported by the VEGA grant 2/6063/26, by the Czech Ministry of Agriculture (grant No. MZE0002716202) and by the Czech Ministry of Education (grant No. 2B08005). This publication is the result of the project implementation: TRANSMED, ITMS: 26240120008, supported by the Research & Development Operational Program funded by the ERDF.
Conflict of interests statement
The authors declare that there are no conflicts of interests.
Acknowledgement
The authors express their appreciation to Prof. F. Périn, Department of Genotoxicity and Carcinogenicity, Institute Curie, France, who kindly offered DBC and its tissue specific derivatives and Prof. N. Fusenig, German Cancer Research Center, Heidelberg, Germany, for providing us with human keratinocytes (HaCaT cells). The authors wish to thank Mrs. A. Vokáliková for excellent technical assistance.
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