In order to better understand the molecular events in murine hepatocarcinogenesis, the frequency and types of mutations in the murine H-ras proto-oncogene isolated from 184 independent, spontaneously occurring hepatic lesions were determined. Hepatocellular foci, hyperplasias, adenomas and carcinomas were obtained from archival samples of control male (134 samples) and female (50 samples) B6C3F1 mice used in oncogenicity studies that were conducted at Lilly Research Laboratories from 1979 to 1986. The 61st codon region of the H-ras oncogene from these sections was amplified using the polymerase chain reaction. Mutation frequencies were determined by restriction fragment length polymorphism analysis. The types of mutations were characterized by allele-specific oligonucleotide hybridization and confirmed by DNA sequencing. Forty-two per cent of the carcinomas, 44% of the adenomas, 42% of the hyperplasias and 29% of the foci contained mutations at the 61 codon. The mutation spectra for the carcinomas, adenomas and hyperplasias consisted of mostly CAA-AAA transversions, followed by CAA-CGA transitions, followed by CAA-CTA transversions. These results demonstrate that: (i) the frequency of spontaneous mutations in the H-ras 61st codon is equivalent in murine hyperplasias, adenomas and carcinomas, and (ii) sex was not a determining factor in either the mutation frequency or mutation spectrum for the spontaneous lesions. If these lesions represent successive stages in the carcinogenic process, then these results suggest that mutations in the 61st codon of H-ras are early events in spontaneous murine hepatocarcinogenesis.

The effects of the antihistamine methapyrilene (MP) on DNA synthesis in rats and mice were investigated. Previous studies have demonstrated a dose response for tumor induction in the rat but no carcinogenic effect in the mouse. To study the role of DNA synthesis in MP carcinogenesis, rats and mice were administered MP at doses of 0, 62.5, 125, 250 or 1000 p.p.m. in the diet for a period of 1-12 weeks. Bromodeoxyuridine was administered continuously using an osmotic minipump during the last week of treatment to provide an index of DNA synthesis. Results demonstrated that in the rat 250 and 1000 p.p.m. MP increased DNA synthesis in a dose-dependent manner that correlated with the tumor response in previous oncogenic studies. MP at 62.5 p.p.m. did not increase DNA synthesis, indicating a no effect level for cell proliferation and suggesting a no effect level for carcinogenicity by this compound in the rat. MP did not induce DNA synthesis in mice after exposure to 1000 p.p.m. for 12 weeks, nor did it induce changes in serum chemistries or liver histopathology suggestive of overt toxicity as was seen in the rat at 1000 p.p.m. The correlations between labeling index and tumorigenicity in the rat and mouse strongly support a role of cell proliferation in the carcinogenic mechanism of MP.

There is a growing concern that the rodent bioassay may not always serve as an appropriate model to assess the carcinogenic risk for humans exposed to certain compounds. Mechanistic research that examines the effects of a compound in rodent and man could help in the interpretation of bioassay results. This paper reports a novel use of two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) technology to assess similarities and differences in the response of rodents and humans to the rat-specific hepatocarcinogen, methapyrilene (MP). A sequential examination of rodent and human hepatic proteins was conducted following in vivo exposure of rats and mice and in vitro exposure of rat, mouse, and human hepatocytes to MP. Results showed that covalent modifications observed in rats and mice in vivo were duplicated both qualitatively and quantitatively in the corresponding in vitro systems and that these modifications correlated with carcinogenic susceptibility. Covalent modifications in human hepatocytes were minimal despite exposure to concentrations of MP that were 6-fold higher than those used in rodent hepatocytes. These studies suggest that in the case of MP the rat is not the most appropriate model for assessing the human situation. Furthermore, these data show that in vitro-in vivo comparisons based on 2-D PAGE may provide adjunctive information for extrapolating rodent toxicity/bioassay results to human risk assessment.

Hepatocellular tumors were induced in 15 day old male B6C3F1 mice following a single exposure to N-nitrosodiethylamine (DEN; 5 mg/kg, i.p.). Tumors were collected at 38 and 65 weeks to compare the frequencies and types of mutations in the 61st codon of the H-ras oncogene. The 61st codon was amplified using the polymerase chain reaction (PCR). Allele-specific oligonucleotide (ASO) probes were used to determine the frequency and types of mutations present in these tumors. Forty-nine nodular hepatic lesions were obtained from seven animals at the 38 week timepoint. Five of these samples (10%) had mutations at the 61st codon with one CAA-AAA, one CAA-CGA and three CAA-CTA. Thirty-six nodular hepatic lesions were obtained from six animals at the 65 week timepoint. Ten of these samples (28%) had mutations at the 61st codon with one CAA-AAA, five CAA-CGA and four CAA-CTA. These data indicate that DEN-induced mutations at the 61st codon of the mouse H-ras oncogene (i) are an infrequent event, (ii) have different frequencies at the 38 and 65 week timepoints and (iii) are different from the types of mutations seen in spontaneous lesions.

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