[CANCER RESEARCH 47, 3212-3219, June 15, 1987] Activation of the K-ras Protooncogene in Lung Tumors from Rats and Mice Chronically Exposed to Tetranitromethane S. Jill Stowers,1 Paul L. Glover, Steven H. Reynolds, Lawrence R. Boone, Robert R. Maronpot, and Marshall W. Anderson Laboratory of Biochemical Risk Analysis, Biometry and Risk Assessment Program fS. J. S., S. H. R., M. W. A.], Cellular and Genetic Toxicology Branch [P. L. G., L. R. B.I. and Chemical Pathology Branch [R. R. M.], Toxicology Research and Testing Program, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709; and Toxicology Program, North Carolina Slate University, Raleigh, North Carolina 27650 fS. J. S.J ABSTRACT Dominant transforming genes were detected in lung tumors from Fischer 344 rats and C57BL/6 x C3H I , mice chronically exposed by inhalation to tetranitromethane, a highly volatile compound used in several industrial processes. The rat lung neoplasms were classified as adenocarcinomas, squamous cell carcinomas (epidermoid carcinomas), or adenosquamous carcinomas. The mouse lung tumors were classified as papillary adenocarcinomas or adenomas. In both species, the tumors were morphologically similar to lung tumors in humans. The transfection assay using NIH/3T3 mouse fibroblasts detected transforming genes in 74% (14 of 19) of the rat lung tumors and in 100% (4 of 4) of the mouse lung tumors. Southern blot analysis indicated that transforming gene was an activated K-ras protooncogene in both species. The first exon of the k- ras gene in normal DNA and in DNA from two cell lines transformed by tumor DNA was compared by cloning and sequencing the gene. Experi ments showed that there was a (.(—»Al transition in the second base of the 12th codon of the k-ru.v oncogene in the two transfectant DNAs. Oligonucleotide hybridization indicated that all of the rat and mouse transfectants had this activating lesion. Additional tumor DNA was then tested for the presence of a mutated alÃ-elewith the GC—»AT transition. All of the rat tumors tested and all of the mouse tumors tested had this mutation present. Hybridization using the normal oligonucleotide se quence around the 12th codon indicated that the normal alÃ-elewas also present in the majority of the tumors, suggesting that the loss of normal alÃ-ele is not necessary for the development of neoplasia. One rat lung tumor had no normal alÃ-ele present, possibly suggesting that this tumor could have been in a more advanced stage than the other tumors. This is the first study to detect activated protooncogenes in rodent tumors induced under conditions which mimic human exposure to a chemical in the workplace. Tetranitromethane may exert its carcinogenic action by both activation of the K-ra\ oncogene and stimulation of cell proliferation by its irritant properties. INTRODUCTION Recent studies suggest that the activation of protooncogenes by genetic alterations may play a role in leading a cell to neoplastic development. These genetic alterations include gross chromosomal rearrangements, amplification of genes, and point mutations. Oncogenes that have been shown to acquire transforming activity by point mutation in their coding se quence include members of the ras oncogene family, the H-ras, K-ras, and the N-ras (1-13) and the neu oncogene (14). The activation of the raÃ-family of genes usually occurs via a point mutation at the 12th, 13th, or 61st codons in human tumors and tumor cell lines (1-13). Studiesina variety of animal model systems have shown that specific activation of a protooncogene by point mutation can be caused by chemical or physical insult (1-8). Animal model systems for carcinogenesis have provided a good means to study protooncogene activation in tumor devel- Received 11/24/86; revised 3/23/87; accepted 3/25/87. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1To whom requests for reprints should be addressed. opment. The H-ras protooncogene has reproducibly been found activated in rat mammary carcinomas induced by a single injection of yV-methyl-./V-nitrosourea given during sexual devel opment (3). The H-ras protooncogene has also been found activated in mouse skin papillomas and carcinomas induced by DMBA2 followed by phorbol ester (12-0-tetradecanoylphorbol- 13-acetate) promotion (1, 2, 7). In both models, the H-ras protooncogene was found to be activated in 90-100% of all of the tumors examined. Other studies have found K-ras and N- ras activation in X-ray- or JV-methyl-7V-nitrosourea-induced mouse thymomas and in rat mesenchymal kidney tumors in duced by treatment with methyl(methoxy-methyi)nitrosamine (4, 5, 15). One conclusion from these studies is that exposure to carcinogens either by relatively high single or multiple doses causes changes in the DNA resulting in activation of oncogenes. However, no studies have examined protooncogene activation in tumors that develop after long term, chronic exposure to chemicals. The identification of chemicals as potential human carcinogens is often made on the basis of long term rodent bioassays which are designed to consider route of human ex posure and concentrations similar to those present in the envi ronment, workplace or home. In a recent bioassay conducted by the National Toxicology Program, chronic exposure to the industrial chemical TNM induced a high incidence of primary lung tumors in Fischer 344 rats and C57BL/6 x C3H F, (hereafter called B6C3F,) mice.3 TNM is a highly volatile compound used as a reagent in industrial nitrosating processes, as an oxidant in rocket fuel, and as an explosive when mixed with toluene (tetranitro- toluene). Because of its irritant properties, TNM has also been proposed as a war gas. The threshold limit for occupational exposure to TNM based on its irritant properties has been set at 1 ppm. In the bioassay, groups of 50 male and 50 female Fischer 344 rats or B6C3F, mice were exposed to TNM by inhalation for 6 h a day, 5 days a week for 2 years. The rats were exposed to 0, 2, and 5 ppm of TNM while the mice were exposed to 0, 0.5, and 1 ppm. Based on histomorphological examination, the TNM-induced primary lung tumors were ad enomas, adenocarcinomas, squamous cell carcinomas, and ad enosquamous carcinomas in rats and papillary adenomas and adenocarcinomas in mice. These tumors were morphologically similar to primary lung tumors in humans. The purpose of this study was to identify and characterize any activated oncogenes that might be present in lung tumors from rats and mice after chronic exposure to TNM. MATERIALS AND METHODS Lung Tumor Generation. Two-year toxicity and carcinogenicity stud ies of TNM were performed under National Toxicology Program 2The abbreviations used are: DMBA, 7,12-dimethylbenzanthracene; TNM, tetranitromethane. 3The National Toxicology Program has not yet completed its evaluation of the data collected during the studies with TNM. Therefore, the apparent associ ation of TNM exposure with lung tumors in rats or mice should be considered preliminary, pending approval of the National Toxicology Program Technical Report on TNM by the National Toxicology Program Board of Scientific Coun selors Technical Reports Review Subcommittee. 3212 Research. on December 2, 2021. © 1987 American Association for Cancer cancerres.aacrjournals.org Downloaded from