Activation of the c-Ki-ras oncogene in aflatoxin B1-induced hepatocellular carcinoma and adenoma in the rat: detection by denaturing gradient gel electrophoresis.

Abstract

Sequence alterations in the exon 1 region of the rat c-Ki-ras gene were studied in DNA isolated from aflatoxin B1 (AFBl)-induced rat liver carcinomas and precursor lesions appearing 56 weeks after administration of the carcinogen. To detect the mutations with high sensitivity, DNA samples were analyzed by using polymerase chain reaction (PCR) amplification in conjunction with aflele-specific oligonucleotide (ASO) hybridization together with a modified PCR-G+C clamp-denaturing gradient gel electrophoresis (DGGE) method. Mutations in the Ki-ras gene were present in all adenomas and carcinomas examined. The predominant mutation observed was a G-C-to-A-T base transition in codon 12 (GGT to GAT). Also present, but at low frequency, was a G-C-to-T-A base transversion in the same codon (GGT to TGT). In addition, 20% of the samples contained a G-C-to-T-A transversion in the second base position of codon 12 (GGT to GTT), a mutation not previously observed in AFB1-induced rat liver tumors. These results confirm and extend our previous findings that Ki-ras mutation is a prevalent event in hepatocellular carcinogenesis induced in Fischer 344 rats by AFB1. The modified DGGE method described is applicable to the screening of multiple mutations in neoplastic lesions with high fidelity and sensitivity.Aflatoxins have been implicated by epidemiologic studies as a causative factor for hepatocellular carcinoma in humans (1). Metabolic studies of aflatoxin B1 (AFB1) have shown that its active form, AFB1 8,9-epoxide, is highly mutagenic and carcinogenic for the liver in rats and other experimental animals, with mutagenicity correlating with carcinogenicity (2, 3). AFB1 binds exclusively to guanine residues of DNA, both in vivo and in vitro (4). The major DNA adduct formed by the binding of AFB1 at the N7 position of guanine is highly unstable and depurinates readily or is converted into the more stable, open-ring formamidopyrimidine adduct (1). The presence of DNA adducts in the genome may contribute to genetic alterations in loci involved in neoplastic development. Previous work by our group and others on AFB1-induced liver carcinogenesis in rodents has demonstrated that activation of cellular ras (c-ras) genes by specific base substitutions occurs at a high frequency in tumor DNA (5, 6).In human tumors as well as in animal tumor models, c-ras genes have been shown to be mutated, the frequency of mutation varying widely among different tumor types. Accumulating evidence suggests that in many human neoplasms, RAS oncogenes may play a role in the early stages of carcinogenesis (7-10). Presently, little information is available regarding the frequency of c-ras oncogene activation during the early stages of liver tumorigenesis in humans or in experimental animal models. Previous studies of AFB1-induced hepatocellular carcinomas in Fischer 344 rats showed activating mutations in codon 12 ofthe c-Ki-ras gene, consisting of single-nucleotide changes (6). To develop a more complete understanding of the role of ras oncogenes in live...

Fish models for environmental carcinogenesis: the rainbow trout.

“…The number of mutant alleles observed is too small at present to know if the DMBA mutational spectrum is protocol dependent. Among (Table 4) and rats (156) show frequent ras mutation, yet this has not been reported to occur in hepatocellular carcinoma from AFB1-exposed humans. Mutant Ha-ras alleles have, however, been reported in human cholangiocellular carcinoma (157).…”

A one-step DGGE scanning method for detection of mutations in the K-, N-, and H-ras oncogenes: Mutations at codons 12, 13 and 61 are rare in B-cell non-Hodgkin's lymphoma

“…In previously described DGGE-based assays for the detection of ras mutations, several experimental conditions were required to scan all the relevant codons (Ridanpää and Husgafvel-Pursiainen, 1993;Abrams et al, 1993;Soman and Wogan, 1993). Moreover, a PCR/DGGE-based method for detection of mutations in the H-ras genes has, to our knowledge, not been described previously.…”

“…The incidence of ras gene mutations, however, varies considerably among different types of cancer, and the profile of ras oncogene activation is often specific for each tumor type. For example, K-ras mutations are the predominant ras mutation found in pancreatic cancer (Soman and Wogan, 1993;Smit et al, 1988); N-ras mutations predominate in acute myeloid leukemia (Ahuja et al, 1990); and H-ras mutations, which are generally rare, are the ras mutations most frequently found in bladder cancer (Burchill et al, 1994). These are, however, only general patterns, and the ras genes preferentially mutated in the different cancers are rarely exclusively mutated.…”

The genetics of human cancer: implications for ecotoxicology.

“…Trout contain K-ras mutations in a majority of the tumors (14) and rats contain K-ras or N-ras mutations in a small subset of liver tumors (15,16). Using more sensitive detection methods, K-ras mutations have been detected in the majority of samples representing early and late-stage hepatocellular adenomas and carcinomas obtained after treatment of rats with aflatoxin B, (17). In contrast, other hepatocarcinogen-induced rat liver tumors (18) and primary hepatocellular carcinoma samples obtained from humans contain few ras mutations even in those samples derived from individuals at geographic risk for mycotoxin exposure (19,20).…”