Low frequency of H-ras mutations in hepatocellular adenomas and carcinomas and in hepatoblastomas from B6C3F1 mice exposed to oxazepam in the diet

Oxazepam has been the subject of recent toxicological and carcinogenesisstudies because it is a commonly prescribed tranquilizer andhas been shown to cause tumors in rodents. In this study, maleand female B6C3F1 mice receIved 0,125, 2500 or 5000 p.p.m. oxazepamin the diet for up to 2 years. Hepatocellular adenomas and carcinomas,as well as hepatoblastomas, which developed in these mice, wereexamined for the presence of activated ms proto-oncogenes. DNAwas Isolated from 20 or more tumors from each exposure groupand analyzed by oligonudeotide hybridiza tion, single-strandedconformation polymorphism analysis and direct sequencing ofPCR-ampllfied H-ms gene fragments for codon 61 mutations. Thirteenof 37 (35%) hepatocellular adenomas and carcinomas from the125 p.p.m. exposure group had mutations in codon 61, while mutationswere detected in only 2 of 25 or 8% of the liver tumors fromthe 2500 p.p.m. exposure group and none of the 22 tumors fromthe 5000 p.p.m. group. This compares to 63% of 126 historIcalcontrol liver tumors and 55% of 20 liver tumors from unexposedB6C3F1 mice in this study. In addition, 12 hepatoblastomas fromthe two high dose groups were examined for H-ras mutations atcodon 61, but none were detected. No tumor DNAs from any ofthe exposure groups tested had mutations In codons 12, 13 or117 of the H-ras gene or codons 12 or 13 of the K-ras gene,the other known hotspots for ras activation in mouse liver tumors.These results, together with those from the National ToxicologyProgram study showing no evidence of cytotoxicity or genotoxicityby oxazepam, suggest that oxazepam preferentially promotes cellsthat have activating lesions other than ras.