Inversion of genetic predisposition to carcinogenesis in liver of two lines of mice selected for resistance (Car-R) or susceptibility (Car-S) to skin carcinogenesis.

Two lines of mice, one resistant (Car-R) and one susceptible (Car-S) to skin carcinogenesis, were produced by bi-directional selective breeding. To see whether the characteristics of susceptibility or resistance to tumorigenesis were also expressed in the liver and lung, the two lines were submitted comparatively to treatment with 5,9-dimethyl dibenzo[c,g]carbazole (DiMeDBC), a potent hepatocarcinogenic derivative of the ubiquitous heterocyclic carcinogenic pollutant, 7H-dibenzo[c,g]-carbazole (DBC). An inversion of genetic predisposition to carcinogenesis in liver was observed. Car-R animals displayed rapid tumorigenesis in 100% of cases while Car-S mice were remarkably less sensitive, showing a 4-fold lower mean tumor multiplicity and a 4-month longer latency time. In parallel adduct formation by DiMeDBC and DBC in liver DNA was analyzed by the 32P-postlabeling method, showing a remarkably higher level in Car-R mice than in Car-S animals. These data indicate that tissue-specific sensibility in carcinogenesis may involve gene expression at various levels.

Inhibition of 5,9-dimethyldibenzo[c,g]carbazole-DNA adduct formation in mouse liver by pretreatment with cytochrome P4501A inducers in vivo.

Mice of the XVIInc/Z and DBA/2N strains, which are responsive and nonresponsive, respectively, to the aryl hydrocarbon (Ah) receptor, were treated with the hepatocarcinogen 5,9-dimethyldibenzo[c,g]carbazole and their livers were examined by nuclease P1-enhanced 32P-postlabeling for the levels of DNA adducts formed. Pretreatment at the doses usually reported in the literature with the cytochrome P4501A (CYP1A) inducers 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), beta-naphthoflavone (BNF), and isosafrole modulated DNA adduction. In XVIInc/Z mice, DNA adduction was totally inhibited by TCDD (a CYP1A1/1A2 inducer), BNF (a CYP1A1/1A2 inducer), and isosafrole (a CYP1A2 inducer). In DBA/2N mice, in which DNA adduction was also inhibited by TCDD, about 25% of the DNA adduct levels persisted after pretreatment with BNF (not a CYP1A1/1A2 inducer in this strain) or isosafrole (a CYP1A2 inducer in this strain). The increase (in all cases less than twofold) in the levels of the phase-II drug-metabolizing enzymes glutathione S-transferase and uridine diphospho-glucuronyltransferase after treatment with inducers cannot explain the total disappearance of DNA adducts. Assays of 5-bromo-2'-deoxyuridine incorporation did not show any induction of DNA synthesis which could explain the decrease in adducts. These results suggest that in vivo 1) increases in CYP1A enzymes by inducers are not correlated with enhanced levels of certain DNA adducts; and 2) phase-II drug metabolizing enzymes are not the main cellular protection pathway for detoxification. An additional mechanism, perhaps also induced by the Ah receptor but highly dependent on the dose of inducer, could be involved in parallel to multidrug resistance (mdr); further experiments are needed to identify this process used by the cell to enhance its protection against toxic or genotoxic effects.

Tissue-specific activities of methylated dibenzo[c,g]carbazoles in mice: carcinogenicity, DNA adduct formation, and CYP1A induction in liver and skin.

The potent multitissue carcinogen 7H-dibenzo[c,g]carbazole and nine methylated derivatives, synthesized on the basis of the positions in the parent compound that are involved in metabolism, were tested for their ability to induce sarcomas and hepatic tumors in XVIInc/Z mice. In addition, the capacity of these compounds to induce DNA adducts in skin and liver was investigated by (32)P-postlabeling analysis after their topical administration. Induction by these compounds of cytochromes P450 of the 1A family in liver and skin was investigated and correlated to their carcinogenic potential. A clear correlation was found between the tissue specificity of DNA binding and the capacity of each compound to induce skin or liver tumors. In contrast, no direct relationship was observed between the capacity of the compounds to induce cytochromes 1A1/1A2 and the tissue specificity of carcinogenesis or DNA binding in liver or skin. The results are discussed with respect to the positions of methyl groups in the 7H-dibenzo[c,g]carbazole molecule.

Kinetics of DNA adduct formation and removal in mouse hepatocytes following in vivo exposure to 5,9-dimethyldibenzo[c,g]carbazole.

5,9-Dimethyldibenzo[c,g]carbazole (DMDBC), a potent mouse hepatocarcinogen, has been shown to induce a non-linear increase in mutant frequency in the liver of the transgenic MutaMouse. To gain insight into the mechanisms underlying the mutagenicity of DMDBC in vivo, DNA damage formation and removal were monitored in mouse hepatocytes over 4-144 h after a single skin application of 10 or 90 mg/kg DMDBC. DNA adducts were measured by (32)P-post-labeling. DNA repair was assessed by: (i) the unscheduled DNA synthesis (UDS) assay, which measures [(3)H]thymidine incorporation into hepatocyte DNA undergoing excision repair; (ii) the Comet assay, which detects DNA strand breaks transiently produced between the incision and rejoining steps of the excision repair process. A plateau of approximately 400 DNA adducts/10(8) nucleotides was reached 24 h after treatment with 10 mg/kg and remained unchanged until 144 h. UDS activity was significantly induced at 15 and 24 h, while no DNA strand breaks were observed at any sampling time. These results suggest that DNA repair mechanisms were efficiently induced and the formation of a high degree of DNA damage was avoided at this dose level. Following exposure to 90 mg/kg DMDBC, the number of DNA adducts increased sharply to a maximum at 24 h ( approximately 8000/10(8) nucleotides) and then declined to approximately 500/10(8) nucleotides at 144 h. UDS activity was markedly induced from 15 to 72 h. Low levels of DNA strand breaks were observed at 24 and 48 h. The formation of large numbers of DNA adducts and the emergence of DNA strand breaks despite a strong initial induction of UDS activity suggested that DNA repair mechanisms were saturated at this dose level. This phenomenon could partly account for the non-linear induction of gene mutations previously reported in the liver of the transgenic MutaMouse.