2-Acetylaminofluorene and 2-aminofluorene are among the most intensively studied of all chemical mutagens and carcinogens. Fundamental research findings concerning the metabolism of 2-acetylaminofluorene to electrophilic derivatives, the interaction of these derivatives with DNA, and the carcinogenic and mutagenic responses that are associated with the resulting DNA damage have formed the foundation upon which much of genetic toxicity testing is based. The parent compounds and their proximate and ultimate mutagenic and carcinogenic derivatives have been evaluated in a variety of prokaryotic and eukaryotic assays for mutagenesis and DNA damage. The reactive derivatives are active in virtually all systems, while 2-acetylaminofluorene and 2-aminofluorene are active in most systems that provide adequate metabolic activation. Knowledge of the structures of the DNA adducts formed by 2-acetylaminofluorene and 2-aminofluorene, the effects of the adducts on DNA conformation and synthesis, adduct distribution in tissues, cells and DNA, and adduct repair have been used to develop hypotheses to understand the genotoxic and carcinogenic effects of these compounds. Molecular analysis of mutations produced in cell-free, bacterial, in vitro mammalian, and intact animal systems have recently been used to extend these hypotheses.

Genetic toxicity of 2-acetylaminofluorene, 2-aminofluorene and some of their metabolites and model metabolites

Mutations in the ras proto-oncogene: clues to etiology and molecular pathogenesis of mouse liver tumors.

Molecular aspects of chemical carcinogenesis: The roles of oncogenes and tumour suppressor genes

Genetic alterations in cancer knowledge system: analysis of gene mutations in mouse and human liver and lung tumors.

Previous studies have shown that a high proportion (5/6) of human liver angiosarcomas (ASL) associated with exposure to vinyl chloride (VC) contains a GC-->AT mutation at the Ki-ras codon 13. This mutation, however, has not been found in 5 ASL or 2 hepatocellular carcinomas (HCC) induced in rats by VC. These 2 HCC did contain a mutation at codon 61 of the Ha-ras gene. In order to extend this study and further explore the mechanisms of tumour induction, an additional 6 ASL and 6 HCC induced in rats by VC were analysed for ras gene point mutations, as well as 10 rat and 10 murine ASL induced by vinyl fluoride (VF), and 5 ASL, 6 Kupffer cell sarcomas, 4 HCC and 2 cholangiocellular carcinomas induced by Thorotrast in rats. Tumour DNA was analysed by PCR-SSCP and direct sequencing. None of the rodent ASL contained a mutation at codon 13 of the Ki-ras gene showing that the ras gene mutational pattern is species-specific. The CAA-->CTA mutation, previously found at codon 61 of the Ha-ras gene in rat HCC, was observed in 5 further VC-induced HCC but was not detected in the Thorotrast-induced HCC, suggesting carcinogen-specificity. This mutation was also absent in VC-induced ASL, which supports the cell-specificity of the ras mutational pattern in chemically induced tumours. No predominant mutation was detected in VF- and Thorotrast-induced tumours. Thus, a given mutation in a tumour may be carcinogen-specific but also depend on the species and the cell type.

Ras gene mutations in vinyl chloride-induced liver tumours are carcinogen-specific but vary with cell type and species.

The high incidence and profile of ras gene mutations reported in spontaneous and chemically induced liver tumours of the B6C3F1 mouse provides a potential means of determining in vivo genotoxicity and its relevance to carcinogenicity. We analysed spontaneous and chemically induced [with 4-amino-biphenyl (ABP), 2-acetylaminofluorene (AAF) and diethylnitrosamine (DEN)] hepatocellular tumours of the C57Bl/10J mouse for H-ras, K-ras and N-ras gene mutations to see if mutational analysis of the ras genes could be useful for such a determination in this strain. Regions of DNA spanning codons 12, 13 and 61 of the ras genes were amplified from formalin fixed liver tumour sections using the polymerase chain reaction. Mutations were detected using allele specific oligonucleotide probing and confirmed by sequencing. We have found that there are few ras mutations in either spontaneous or chemically induced liver tumours in the C57Bl/10J mouse. Out of 25 spontaneous tumours two contained an A to T transversion and one contained an A to G transition in base 2 of H-ras codon 61 and two contained a G to A transition in base 2 of K-ras codon 13 (the K-ras mutations were only faintly detectable and may be present in a subpopulation of the tumour cells). In the case of the 18 ABP induced tumours one contained a C to A transversion in base 1 of H-ras codon 61, and one contained an A to T transversion in base 2 of H-ras codon 61 and one contained a G to C transversion in base 1 of K-ras codon 13. One C to A transversion in base 1 of H-ras codon 61 was detected out of eight AAF induced tumours. Of the 25 DEN induced tumours, one contained an A to G transition and one contained an A to C transversion in base 2 of H-ras codon 61. The data indicate that at least in hepatocellular tumours of the C57Bl/10J strain and using chronic dosing regimes the ras genes do not represent markers for in vivo genotoxic activity.

Katy J. Hardaker

Papers

Point mutation analysis of ras genes in spontaneous and chemically induced C57Bl/10J mouse liver tumours.