The chemistry and biology of 7H-dibenzo[c,g]carbazole: synthesis and characterization of selected derivatives, metabolism in rat liver preparations and mutagenesis mediated by cultured rat hepatocytes.

Authentic stable standards of 7H-dibenzo[c,g]carbazole (DBC), a potent environmental carcinogen, were synthesized in order to study the compound's metabolism and mutagenesis in whole cell systems. Complete characterization of 2-OH-DBC, 3-OH-DBC, 4-OH-DBC, 13c-OH-DBC and N-methyl-DBC was accomplished by UV, IR, fluorescence and high resolution NMR spectra, and by high resolution mass spectrometric procedures. Metabolites of DBC were isolated and separated by HPLC from extracts of rat liver microsomal incubations and the medium of primary cultures of rat liver cells. Identification of metabolites was accomplished by comparisons between the authentic standards and isolated metabolites by UV and fluorescence spectroscopy, mass spectral analyses, and by co-chromatographic techniques. 2-OH-DBC and 3-OH-DBC were found in all rat liver preparations as well as three other unidentified phenols. 4-OH-DBC, 13c-OH-DBC or N-methyl-DBC were not isolated under any conditions. The rates of appearance of DBC metabolites in cultures of rat liver cells were compared to those for benzo[a]pyrene (BaP) at 10, 25, 50 and 100 microM substrate. At 25 microM substrate or greater, DBC metabolites appeared in the culture medium at significantly faster rates than those of BaP. At 100 microM substrate, DBC metabolites appeared at a rate approximately 4-times the rate observed for BaP. When the mutagenic potential of DBC was compared to that of BaP under identical conditions in a co-cultivation system of rat liver cells and an epithelial cell line, DBC was found to produce significantly higher rates of mutagenesis than BaP at concentrations of 0.4, 4.0 and 40.0 microM in the culture medium. The mutagenic potential of DBC was compared to that of several derivatives of the parent compound. 3-OH-DBC, 13c-OH-DBC and N-methyl-DBC were found to be mutagenic in the co-cultivation system at 40 microM, with mutation frequencies of 4.4 +/- 0.8, 8.0 +/- 3.1 and 12.9 +/- 5.4 mutants per 10(5) survivors, respectively. The parent compound induced 8.0 +/- 2.8 mutants per 10(5) survivors at the same concentration. 2-OH-DBC and 4-OH-DBC were not mutagenic under the same conditions. The studies have shown that metabolism of 7H-DBC leads predominantly to phenols in rat liver cells. The results of the mutagenesis experiments indicate that, of the derivatives studied, those associated by induction to the nitrogen are mutagenic. The latter studies suggest that the nitrogen is involved in the activation of the parent compound through inductive mechanisms.

Mutagenicity of cyanate, a decomposition product of MNU.

Knox reported that the short-term effects of the carcinogen methylnitrosourea (MNU) were due to the formation of its decomposition product, the cyanate ion. He showed that cell survival and DNA synthesis decreased as the concentration of MNU and the cyanate ion (NCO-) increased in the medium. Further, the product of MNU decomposition comigrated with NCO- when added to his chromatographic test system. However, Knox did not study the mutagenicity of MNU or its breakdown products. We compared the mutagenicity of MNU and potassium cyanate (KNCO) in mammalian cells. Our results demonstrate that, although it is toxic to cells, KNCO does not induce ouabain-resistant mutants in cultured Chinese hamster cells (V79).

Studies of cytogenetic effects of sodium arsenicals on mammalian cells in vitro.

The cytotoxic and cytogenetic effect of sodium arsenite and sodium arsenate on Chinese hamster ovary (CHO) cells is reported. Chromosome aberrations were induced with both arsenic compounds. Trivalent arsenic was more clastogenic than pentavalent arsenic. Sodium arsenite was also shown to produce increased sister chromatid exchange in CHO cells and increased chromosome breakage in human lymphocytes.

Enhancement of recovery of chemical carcinogen-induced ouabain-resistant mutants in Chinese hamster cells by the tumor-promoting agent, 12-o-tetradecanoyl-phorbol-13-acetate.

The effects of a tumor-promoting agent on the frequency of mutation to ouabain resistance and survival of Chinese hamster cells treated with a chemical carcinogen have been investigated. 12-O-Tetradecanoyl-phorbol-13-acetate (TPA) significantly enhanced the mutation frequency induced by the carcinogen, methylazoxymethanol acetate (MAM), without having similar effects on cytotoxicity, at concentrations of 2 micrograms/ml or less. The observed degree of enhancement of mutagenesis increased with promoter concentration up to the point where the latter exhibited frank toxicity. Exposure of the cells to the promoter for a period of 2 or 6 h was found ineffective, but subsequently a significant enhancement was found after a 27-h exposure time. The maximum effect occurred after a 5-day exposure, with a increase in the mutation frequency of 250%. Treatment of cells with TPA alone resulted in no enhancement of spontaneous mutation rates, nor did treatment of cells prior to addition of carcinogen-induced mutagenesis. In contrast, TPA was found to be effective when applied as late as 6 weeks following carcinogen treatment. These results are consistent with the hypothesis that TPA owes its promoting activity toward chemically-induced mutagenesis and carcinogenesis to its ability to enhance expression of latent somatic genetic modifications by epigenetic mechanisms. They do not support mechanisms involving TPA-induced inhibition of DNA-repair replication, or mutagenic activity of TPA per se. The notably similar qualitative response to TPA of several parameters in mouse-skin tumorigenesis and Chinese hamster cell mutagenesis suggest that the mechanism of action of the promoter is similar in the 2 diverse biological systems.

Effect of alkane tumor-promoting agents on chemically induced mutagenesis in cultured V79 Chinese hamster cells.

Linear alkanes of specific chain length between 6 and 16 carbon atoms, an aryl derivative of dodecane, and a phorbol diester were tested in a cell culture system for relative ability to enhance mutagenesis induced by a chemical carcinogen, methylazoxymethanol acetate (MAM). Mutation frequencies at the ouabain-resistance locus were measured. Results indicated an excellent correlation between the relative activities of the above compounds in enhancing mutagenesis in the in vitro culture system and their tumor-promoting activities in mouse skin. None of the compounds tested showed mutagenic activity per se, further lending support to the theory that promoters act via derepression of latent carcinogen-induced damage to the genome.