ABSTRACTc-Ki-ras and N-ras oncogenes have been characterized in aflatoxin B1-induced hepatocellular carcinomas. Detection of different protooncogene and oncogene sequences and estimation of their frequency distribution were accomplished by polymerase chain reaction, cloning, and plaque screening methods. Two c-Ki-ras oncogene sequences were identified in DNA from liver tumors that contained nucleotide changes absent in DNA from livers of untreated control rats. Sequence changes involving G-C to T-A or G-C to A-T nucleotide substitutions in codon 12 were scored in three of eight tumor-bearing animals. Distributions of c-Ki-ras sequences in tumors and normal liver DNA indicated that the observed nucleotide changes were consistent with those expected to result from direct mutagenesis of the germ-line protooncogene by aflatoxin B1. N-ras oncogene sequences were identified in DNA from two of eight tumors. Three N-ras gene regions were identified, one of which was shown to be associated with an oncogene containing a putative activating amino acid residing at codon 13. All three N-ras sequences, including the region detected in N-ras oncogenes, were present at similar frequencies in DNA samples from control livers as well as liver tumors. The presence of a potential germ-line oncogene may be related to the sensitivity of the Fischer rat strain to liver carcinogenesis by aflatoxin B1 and other chemical carcinogens.The carcinogenic properties of the aflatoxins have been extensively studied, and much information has been produced concerning various aspects of their mechanisms of action, occurrence in foods, and possible involvement as human cancer risk factors (1). Aflatoxin B1 (AFB1), in particular, has been shown to be a powerful carcinogen for the liver of many experimental animals, including the rat (2). Studies of the metabolism of AFB1 have revealed activation to its electrophilic DNA binding form through an epoxidation pathway (3). Furthermore, activation and DNA binding of AFB1 produces identical DNA-chemical adduct spectra in both man and rat including the major DNA adduct form, 2,3-dihydro-2-(N7-guanyl)-3-hydroxy-AFB1 (4).Previous work by many other investigators has suggested (5) that the activation of c-ras genes by specific single-base mutations may be one of several steps involved in the transformation of normal cells to malignancy. In addition, studies of in vitro mutagenesis of c-ras protooncogenes have shown that many mutations within codons 12, 13, 59, and 61 of the c-Ha-ras and N-ras genes may lead to oncoproteins with similar properties with respect to their potential to transform NIH 3T3 cells. In contrast, c-ras mutations that arise in DNA of tumors of animals after exposure to specific chemicals often result in a more restricted set of nucleotide substitutions at these positions. This narrower range of mutations could be related to characteristics of binding of chemicals to DNA and to subsequent DNA repair of specific lesions. For instance, it has been suggested that differences in the spectra of mu...

Activated c-Ki-ras genes in liver tumors from rats exposed to the potent hepatocarcinogen aflatoxin B1 were analyzed to determine the nature of their activation by characterization of two c-Ki-ras alleles present in tumor-derived NIH 3T3 mouse transformants. Using selective hybridization of synthetic oligonucleotides to transformant DNA, we have determined that a single G-C to A-T base transition in either the first or second position of the 12th codon is associated with activation of the gene. Such mutations would lead to amino acid substitutions of aspartate or serine for glycine in the mutant proteins. To confirm these findings, we applied a technique for direct sequence analysis of a 90-base-pair region of the rat c-Ki-ras gene produced by primer-directed enzymatic amplification. Findings produced by this approach, which provides a convenient method to characterize mutations in multiple alleles without the necessity to clone individual genes, confirmed the presence and identity of the 12th codon mutations in the activated oncogene, as initially determined by the oligonucleotide hybridization technique.

xthA-Escherichia coli, which are missing a major cellular apurinic/apyrimidinic (AP) endonuclease, are 5-to 10-fold more sensitive than xthA+ bacteria to mutagenesis by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) under conditions that induce the "adaptive response." The xthA--dependent mutations are also dependent on SOS mutagenic processing and consist of both transversion and transition base substitutions. When MNNG-adapted xthA-bacteria are challenged with a high dose of MNNG, more xthA-dependent SOS-dependent mutations are induced, and transversions are enhanced relative to transitions. The mutations induced by challenge are eliminated in xthA-alkA-bacteria, which are also deficient for 3-methyladenine glycosylase II activity. These data are consistent with the hypothesis that AP sites, at least some of which are produced by glycosylase activity, are mutagenic intermediates following cellular DNA alkylation.Apurinic and apyrimidinic (AP) sites cause errors in DNA replication in vitro and are mutagenic when present in transforming DNA (1, 2). In addition to being a probable cause of spontaneous mutations (3, 4), AP sites have been implicated as a common mutagenic lesion created by a number of DNA-damaging agents (reviewed in ref.

Oligonucleotide-directed mutagenesis of the beta-lactamase gene, bla, on pBR322 was used to change the codon for the active-site serine 70, AGC, to CGC, coding for arginine. Escherichia coli cells carrying the mutant plasmid, pGD104, were sensitive to ampicillin, indicating that the arginine-containing enzyme is inactive. We characterized the reversion of the mutant bla gene by a number of mutagens and in different genetic backgrounds and demonstrated that full ampicillin resistance can be restored only by a G-C-to-T-A transversion occurring at the first base of the codon. Thus, reversion of the mutant bla gene is diagnostic for G-C-to-T-A transversions, and bacteria carrying pGD104 can be used as test strains to detect the occurrence of this mutation.

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