5,9-Dimethyldibenzo[c,g]carbazole (DMDBC) is a synthetic derivative of the environmental pollutant 7H-dibenzo[c,g]carbazole. DMDBC is a potent genotoxic carcinogen specific for mouse liver. Using the MutaMouse lacZ transgenic mouse model and a positive selection assay, we measured lacZ mutant frequency (MF) in the liver 28 days after a single s.c. administration of DMDBC at 3, 10, 30, 90 or 180 mg/kg. MF remained low at 3 and 10 mg/kg, but increased markedly from 30 mg/kg onwards. To investigate the reason for this non-linear response, we examined mechanisms potentially involved in mutation induction in the liver. Genotoxic effects such as DNA adduct formation were detected in 32P-post-labelling studies. Liver sections were examined for microscopic changes and cell proliferation. These parameters, and MF, were studied 2, 4, 7, 14, 21 and 28 days after a single s.c. administration of 10 or 90 mg/kg DMDBC. At 10 mg/kg, a dose found to double the MF on day 28, DNA adducts reached a level of 200-600 adducts per 10(8) nucleotides from day 4 to day 28. No changes in histology or cell proliferation were detected at this low dose. At 90 mg/kg, MF increased gradually from day 7 to day 28 (maximum 44-fold). The DNA adduct level ranged from 400 to 4500 adducts per 10(8) nucleotides on day 2, then stabilized at approximately 400 adducts per 10(8) nucleotides on day 4. An early cytotoxic effect was detected microscopically in centrilobular hepatocytes, and was followed by liver cell proliferation. These data suggest that the marked increase in MF in MutaMouse liver after treatment in vivo with DMDBC at 90 mg/kg may be explained by the induction of replicative DNA synthesis due to a cytotoxic effect, allowing the fixation of persistent DNA adducts into mutations.

The purpose of this work was to investigate the impact of cell proliferation on liver mutagenesis. The genotoxic hepatocarcinogen 5, 9-dimethyldibenzo[c,g]carbazole (DMDBC) was administered to lacZ transgenic MutaTMMice at a non-hepatotoxic dose of 10 mg/kg, which induces only a slight increase in the liver lacZ mutant frequency (MF). To determine if cell proliferation stimuli enhanced DMDBC mutagenicity, MF was analyzed in mice first receiving DMDBC 10 mg/kg, then approximately 2 weeks later, either carbon tetrachloride (CCl4, a cytotoxic agent inducing regenerative cell proliferation) or phenobarbital (PB, a mitogenic agent inducing direct hyperplasia). In preliminary studies, the extent of cell proliferation induced by CCl4, PB and DMDBC was determined in non-transgenic CD2F1 mice by means of 5-bromodeoxyuridine labeling. The labeling index was significantly increased after CCl4 and PB, while no change was detected with DMDBC. MF was then determined in MutaTMMice 28 days after initial DMDBC treatment. No increase in MF was detected in mice receiving CCl4 or PB alone. A 2- to 3-fold increase in MF was detected in mice treated with 10 mg/kg DMDBC alone. In contrast, MF was markedly increased in mice receiving DMDBC followed by proliferative treatment (15-fold with CCl4 and 25-fold with PB). These results demonstrate that expression of DMDBC-induced mutations in mouse liver largely depends on the induction of cell proliferation (by a cytotoxic or mitogenic stimulus) and illustrate that MutaTMMouse is a valuable tool to investigate the early events of liver carcinogenesis.

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