Oncogene activation in spontaneous and chemically induced rodent tumors: implications for risk analysis.

The validity of rodent tumor end points in assessing the potential hazards of chemical exposure to humans is a somewhat controversial but very important issue since most chemicals are classified as potentially hazardous to humans on the basis of long-term carcinogenesis studies in rodents. The ability to distinguish between genotoxic, cytotoxic, or receptor-mediated promotion effects of chemical treatment would aid in the interpretation of rodent carcinogenesis data. Activated oncogenes in spontaneously occurring and chemically induced rodent tumors were examined and compared as one approach to determine the mechanism by which chemical treatment caused an increased incidence of rodent tumors. Different patterns of activated oncogenes were found not only in spontaneous versus chemically induced mouse liver tumors but also in a variety of spontaneous rat tumors versus chemically induced rat lung tumors. In the absence of cytotoxic effects, it could be argued that the chemicals in question activated protooncogenes by a direct genotoxic mechanism. These results provided a basis for the analysis of activated oncogenes in spontaneous and chemically induced rodent tumors to provide information at a molecular level to aid in the extrapolation of rodent carcinogenesis data to human risk assessment.


Introduction
Several approaches have been used to identify environmental agents posing significant carcinogenic hazards to man. These include in vitro assays for mutagenesis and in vivo tumorigenicity assays. However, most chemicals are classified as potentially hazardous to humans on the basis of long-term carcinogenesis studies in rodents. While these rodent carcinogenesis studies are designed to mimic the route of human exposure in the environment or workplace, the dose of a given chemical is usually higher than that which actually occurs in human exposure. The extrapolation of rodent carcinogenic data to human risk is therefore complicated by the higher doses of chemicals that are employed in the rodent bioassays, especially in the absence of information regarding the mechanism(s) of tumor induction. Another com-*This paper was presented at the conference on  plicating factor in the interpretation of rodent carcinogenic data is the appearance of speciesand strainspecific spontaneously occurring tumors in rodents that are used in the bioassays.
Increasing evidence suggests that a small set of cellular genes appear to be targets for genetic alterations that contribute to the neoplastic transformation of cells. These genes, termed protooncogenes, were initially discovered as the transduced oncogenes of acute transforming retroviruses (1). Recent studies have established that protooncogenes can also be activated as oncogenes by mechanisms independent of retroviral involvement. These mechanisms include point mutations or gross DNA rearrangements such as translocations or gene amplifications (1,2). Protooncogenes appear to play a crucial role in normal cellular growth and/or differentiation since they are highly conserved in nature, being detected in species as divergent as yeast, Drosophila, and humans.
The activation of protooncogenes in spontaneous and chemically induced tumors has been studied in great detail during the past several years. The number of protooncogenes that must be activated in the mul- tistep process of carcinogenesis is unclear at present although the coincerte(d expression of at least two oncogeines, ras an(l myc, are neededl for the transformation of a primary cell in vitro (3). Also, in addlition to the activation of protooncogenes, the loss of specific regulatory functioils such as tumnor suppressor genes may be a (listiinct step inl neoplastic transformation (4). Investigatioins in ro(lent mo(lels for chemical carciinogeniesis imply that oncogenes are activated by carciilogein treatimient ancl that this activation process is an early evenit in tumor in(luction (5-7). These finidiings suggest that one approach to (letermine the Im echanIismil(s) by which a chenmical causes an increased incidence of tumliors in a rodent bioassay is to compare oncogeine activation in sponitaneous and cheimiically in(lucedl tumuors. If the activated oncogenes (letected in chemically indlucedl tumors were different from those foundl in spontaneous tumors, then one coul(l argue that the chemical activated the protooncogene by a genotoxic imiechanism. Alternatively, the (letectioni of similar activatiing lesions in oncogeines of spontaneous andl chemically inducecd tumnors could imlply that the chemical was increasing the spontaneous tumor incidence by a mnechanism such as cytotoxicity or receptor-ime(licated promotion. This paper will discuss the cletection of activated oncogeines in spontaneous and chemically induced tumnors of the B6C3F1 mouse and the Fischer 344/N rat, two species used extensively in National 'oxicology Program/National Caincer Institute (NTP/NCI) carcinogenesis stu(lies. I'he implications of activated oncogenes in these rodent tumors will also be discussed in terms of the extrapolatioin of rodent carcinogenic data to human risk assessimient Activated Oncogenes in Liver Tumors of the B6C3F1 Mouse DNA transfectioin analysis of a series of spontaneous (8,9) andl chemically induced (10) liver tumors showed oncogene activation in both beinign and malignant tumors. Southern blot analysis indicated a high incidlence of Ha-ras gene activation in both the spontaneous (9) and chemically induced (10) liver tumors. Activated Ki-ras genes were detected in the chemically indlucedl liver tumors but not in the spontaneous liver tumors (9,10). Also, raf oncogenes andl non-ras oncogenes were (letected in both the spontaneous an(l cheimically indluce(d liver tumors (9,10).
Oligonucleotide analysis indicated that all of the Haras oncogeines detected in spontaneous liver tumors were activated by point mutations in codon 61 whereas the Ha-ras oncogenes detected in the chemically induced liver tumors were activated by poinlt mutation at a number of different codons (10). The detection and analysis of activated oncogenes in mouse liver tumors is summarized in Table 1.

Activated Oncogenes in Tumors of the Fischer 344/N Rat
In contrast to the results obtained with the spontaneous mouse liver tumors, oncogenes detectable by transfection analysis were absent in 9 malignant and 20 benign spontaneous nonliver tumors of the Fischer 344/N rat. These tumors included 8 mammary adenomas or fibroadenomas, 5 testicular interstitial cell adenomas, 4 subcutaneous fibromas or fibroadenomas, 3 mononuclear cell leukemias, 2 fibrosarcomas, and a single adrenal pheochromocytoma, mammary adlenocarcinoma, pancreatic acinar adenoma, pancreatic islet cell adenoma, pituitary adenoma, splenic hemangiosarcoma, and prostatic adenocarcinoma (9). I)NA transfection analysis of a series of rat luing tumors, generated in a recent long-term carcinogenesis stu(ly conducted by NTP, indlicated that 18 of 19 tumors contained detectable oncogenes. Southern blot and oligonucleotide analysis indicated that all of the rat lung tumors contained an activated Ki-ras gene with a GGT --(GAT mutation in codon 12 (1]). The detection of activatecd oncogenes in rat tumors is sutimmarize(l in ITable 2.

Interpretation of Rodent Carcinogenesis Data and Its Implications for Human Risk Assessment
The regulation of humain exposure to a chemical, basedl oIn long-term carcinogenesis studlies in rodents, shouldl take into account whether the chemiiical in questioin is mutagenic, cytotoxic, or has a receptorme(liate(d mechaniismii of promiiotion. Oincogene analysis of tumliors from sponitaneous origin and( froIm longtermii carcinogenesis stu(lies can be useful in several ways. Investigationi of oncogene activationi in tuimlors will allow an analysis of the mechanism of tumor formation at a molecular level. For instance, the finding of activating mutations in different codons of the Haras gene in chemically induced liver tumors versus finding activating mutations in only one codon of the Ha-ras gene in spontaneous liver tumors could, in the absence of any cytotoxic effects, be used to argue that the chemical itself activated the Ha-ras protooncogene by a genotoxic event. Likewise, different patterns of oncogene activation in spontaneous versus chemically induced rat tumors would also be helpful in the classification of a rodent carcinogen as an initiator, promoter, or cytotoxic chemical. This type of analysis might be of particular importance for compounds which are negative for mutagenicity on short-term tests but which are positive for carcinogenicity in long-term bioassays. Knowledge of the mechanisms by which chemicals induce tumors in long-term rodent bioassays may remove some of the uncertainty of lowdose and species-to-species extrapolation of human risk from rodent carcinogenesis data.