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An understanding of the basic mechanisms responsible for the pathogenesis of liver neoplasms is needed in order to develop better therapeutic strategies. The present study utilized a pharmacogenetic mouse model to assess the role of cytochrome P4501A1 (Cyp1a1) in modulating genetic damage to oncogenic and tumor suppressor loci following in utero exposure to the polycyclic aromatic hydrocarbon, 3-methylcholanthrene (MC). Analysis of the Ha-ras, Kiras, INK4a and p53 genes was carried out with lysates from paraffin-embedded liver tissue from transplacentallytreated mice. The lysates were subjected to DNA amplification by the PCR technique followed by allele-specific oligonucleotide hybridization screening and SSCP analysis. All of the 26 neoplasms screened (23 hepatocellular carcinomas, two hepatocellular adenomas and one sarcoma) exhibited a GGC→CGC (GLY 13 →ARG 13 ) transversion at the Ki-ras gene locus. None of the tumors had Ki-ras mutations at codon 12 of exon 1. Approximately 12% (3/ 26) of the liver tumors exhibited point mutations in exon 1o f theINK4a gene, with each of the three tumors exhibiting two point mutations. Analysis of exon 2 of the INK4a gene showed the presence of a CCG→CTG (PRO 73 →LEU 73 ) transition in two of the 26 neoplasms. No mutations were found in exons 1 or 2 of the Ha-ras gene, or in exons 5‐8 of the p53 gene. Analysis of tumor RNAs showed overexpression of Ha-ras, cip1 and c-jun in ~38% of the liver tumor samples. The results of this study suggest that mutagenic damage to oncogenes and tumor suppressor genes may be critical factors in mediating transplacentallyinduced liver tumorigenesis. The fact that Ki-ras mutations were found in all of the tumors suggests that mutation at this gene locus may be an early event in liver tumor pathogenesis, while mutation in tumor suppressor genes may occur later during tumor progression. These combined results are consistent with the pathogenesis of cancer in humans. *Abbreviations: ASO, allele-specific oligonucleotide hybridization; cdk, cyclin-dependent kinase; Cyp1a1, cytochrome P4501A1; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MC, 3-methylcholanthrene; PAH, polycyclic aromatic hydrocarbon; PCR, polymerase chain reaction; Rb, retinoblastoma; SSCP, single strand conformation polymorphism; SDS, sodium dodecylsulfate; 13 SSC, 0.15 M NaCl/0.015 M sodium citrate (pH 7.0); BPB, bromophenol blue; 13 TBE, 100 mM Tris‐HCL/90 mM boric acid/ 1 mM EDTA.

A pharmacogenetic mouse model was utilized to determine the role of Cyp1a1 expression on the formation of Ki-ras mutations in lung tumors following transplacental exposure to polycyclic aromatic hydrocarbons (PAHs). A backcross between Ah responsive male B6D2F1 mice and nonresponsive female DBA mice resulted in a litter in which both responsive and nonresponsive fetuses resided in the same nonresponsive maternal environment. Pregnant mothers received a single i.p. injection of either 10 or 30 mg/kg of 3-methylcholanthrene (MC) or olive oil vehicle on day 17 of gestation. At the higher dose of MC, the responsive offspring of both sexes had significantly (P0.05) higher incidences of lung tumors than their nonresponsive littermates. The male responsive mice also exhibited a significantly increased liver tumor incidence over the nonresponsive mice at the P0.05 level. Administration of 10 mg/kg of MC caused a very low incidence of lung tumors and did not result in the appearance of macroscopically visible liver tumors. Exons 1 and 2 of the Ki-ras gene were amplified from paraffin-embedded tissue samples. The PCR products were screened by allele-specific oligonucleotide hybridization (ASO). Thirteen of 16 lung tumors (81%) screened exhibited point mutations in the 12th or 13th codon, including seven tumors that contained GGT--GTT (GLY12--VAL12) transversions, four which exhibited GGT--TGT (GLY12--CYS12) transversions, and two which contained GGC--CGC (GLY13--ARG13) transversions. None of the tumors had mutations at codon 61. The results obtained by ASO were confirmed by cloning and sequence analysis of the PCR products from four of these tumors. Within the subset of 16 tumors examined in this study, the same types of mutations in the Ki-ras gene were generally present in both responsive and nonresponsive mice, although G--C transversions were found in two tumors from a single responsive female mouse. Interestingly, while both males and females exhibited the GGT--GTT mutations at codon 12, the GGT--TGT transversion was only found in male mice. These results are consistent with a key role for Cyp1a1 in modulating individual susceptibility to cancer formation through the formation of reactive intermediates that bind to DNA and result in activating mutations in key regulatory molecules.