Activated oncogenes in B6C3F1 mouse liver tumors: implications for risk assessment.

The validity of mouse liver tumor end points in assessing the potential hazards of chemical exposure to humans is a controversial but important issue, since liver neoplasia in mice is the most frequent tumor target tissue end point in 2-year carcinogenicity studies. The ability to distinguish between promotion of background tumors versus a genotoxic mechanism of tumor initiation by chemical treatment would aid in the interpretation of rodent carcinogenesis data. Activated oncogenes in chemically induced and spontaneously occurring mouse liver tumors were examined and compared as one approach to determine the mechanism by which chemical treatment caused an increased incidence of mouse liver tumors. Data suggest that furan and furfural caused an increased incidence in mouse liver tumors at least in part by induction of novel weakly activating point mutations in ras genes even though both chemicals did not induce mutations in Salmonella assays. In addition to ras oncogenes, two activated raf genes and four non-ras transforming genes were detected. The B6C3F1 mouse liver may thus provide a sensitive assay system to detect various classes of proto-oncogenes that are susceptible to activation by carcinogenic insult. As illustrated with mouse liver tumors, analysis of activated oncogenes in spontaneously occurring and chemically induced rodent tumors will provide information at a molecular level to aid in the use of rodent carcinogenesis data for risk assessment.

Activation of the K-ras protooncogene in lung tumors from rats and mice chronically exposed to tetranitromethane.

Dominant transforming genes were detected in lung tumors from Fischer 344 rats and C57BL/6 X C3H F1 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. Experiments showed that there was a GC----AT transition in the second base of the 12th codon of the K-ras 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 allele with 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 sequence around the 12th codon indicated that the normal allele was also present in the majority of the tumors, suggesting that the loss of normal allele is not necessary for the development of neoplasia. One rat lung tumor had no normal allele 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-ras oncogene and stimulation of cell proliferation by its irritant properties.

Formation and persistence of benzo(a)pyrene metabolite-DNA adducts.

Benzo(a)pyrene (BP) and other polycyclic aromatic hydrocarbons (PAH) are ubiquitous environmental pollutants and are suspected to be carcinogenic in man. The in vivo formation of BP metabolite-DNA adducts has been characterized in a variety of target and nontarget tissues of mice and rabbits. Tissues included were lung, liver, forestomach, colon, kidney, muscle, and brain. The major adduct identified in each tissue was the (+)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydro-BP (BPDEI)-deoxyguanosine adduct. A 7 beta, 8 alpha-dihydroxy-9 beta,10 beta-epoxy-7,8,9,10-tetrahydro-BP (BPDEII)-deoxyguanosine adduct, a (-)-BPDEI-deoxyguanosine adduct, and an unidentified adduct were also observed. The adduct levels are unexpectedly similar in all the tissues examined from the same BP-treated animal. For example, the BPDEI-DNA adduct levels in muscle and brain of mice were approximately 50% of those in lung and liver at each oral BP dose used. We have also examined adduct levels formed in vivo in several cell types of lung and liver. Macrophages, type II cells, and Clara cells from lung and hepatocytes and nonpparenchymal cells from liver were isolated from BP-treated rabbits. BPDEI-deoxyguanosine adduct was observed in each cell type and, moreover, the levels were similar in various cell types. These and previous results strongly suggest that DNA in many human tissues is continuously damaged from known exposure of humans to BP and other PAH. Moreover, DNA adducts formed from BP are persistent in lung and brain.(ABSTRACT TRUNCATED AT 250 WORDS)

The role of oncogenes in chemical carcinogenesis.

Proto-oncogenes are cellular genes that are expressed during normal growth and developmental processes. Altered versions of normal proto-oncogenes have been implicated in the development of human neoplasia. In this report, we show the detection of activated proto-oncogenes in various spontaneous and chemically induced rodent tumors. The majority of activated proto-oncogenes found in these tumors are members of the ras gene family and have been activated by a point mutation. Characterization of the activating mutation may be useful in determining whether this proto-oncogene was activated by direct interaction of the chemical with the DNA. Comparison of activating lesions in spontaneous versus chemically induced tumors should be helpful in determining whether the chemical acts via a genotoxic or a nongenotoxic mechanism. All of this information may be helpful in the assessment of potential carcinogenic hazards of human exposure to chemicals.

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.

Ubiquitous binding of benzo[a]pyrene metabolites to DNA and protein in tissues of the mouse and rabbit.

The in vivo formation of benzo[alpha]pyrene (BP) metabolite-DNA adducts in several tissues of mice and rabbits was examined. Included were tissues with widely divergent xenobiotic metabolizing capabilities such as liver and brain. The major adduct identified in each tissue was the (+)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydro-BP (BPDEI)-deoxyguanosine adduct. A 7 beta,8 alpha-dihydroxy-9 beta,10 beta-epoxy-7,8,9,10-tetrahydro-BP (BPDEII)-deoxyguanosine adduct, a (-)-BPDEI-deoxyguanosine adduct and an unidentified adduct were also observed. These adducts were present in all of the tissues of the mice and in the lungs of the rabbits; only BPDEI and BPDEII were seen in the rest of the rabbit tissues. In all of the tissues studied, the DNA adduct levels were unexpectedly similar. For example, the BPDEI-DNA adduct levels in muscle and brain of mice were approx. 50% of those in lung and liver at each oral BP dose examined. After an i.v. dose of BP in rabbits, the BPDEI adduct levels in lung were three times those in brain or liver and twice those in muscle. The binding of BP metabolites to protein was also determined in these tissues. The tissue-to-tissue variation in protein binding levels of BP metabolites was greater than that for BPDEI-DNA adducts. There are several possible explanations for the in vivo binding of BP metabolites to DNA and protein of various tissues. First, oxidative metabolism of BP in each of the examined tissues might account for the observed binding. Second, reactive metabolites could be formed in tissues such as liver and lung and be transported to cells in tissues such as muscle and brain where they bind to DNA and protein. In any case, the tissue-to-tissue variations in protein and DNA binding of BP-derived radioactivity do not correlate with differences in cytochrome P-450 activity.

Detection and identification of activated oncogenes in spontaneously occurring benign and malignant hepatocellular tumors of the B6C3F1 mouse.

Species- and strain-specific spontaneously occurring tumors have been observed in rodents maintained under normal laboratory conditions. Elucidation of the molecular mechanisms associated with the development of these spontaneous tumors may provide a better understanding of tumor development associated with exposure to chemical carcinogens. In view of the high frequencies of oncogene activation shown in rodent tumors induced by known chemical carcinogens, we have investigated oncogene activation in spontaneous tumors of the B6C3F1 mouse and Fischer 344/N rat by DNA transfection techniques. A marked difference in the presence of activated oncogenes in spontaneous rat tumors versus spontaneous mouse liver tumors was observed in this study. All rat tumors tested failed to yield activated oncogenes (0/29), whereas 30% (3/10) of mouse hepatocellular adenomas and 77% (10/13) of hepatocellular carcinomas scored positive by DNA transfection. These transforming genes were identified as an activated Ha-ras gene in all the adenoma transfectants and in 8 of the 10 carcinoma transfectants. The two remaining hepatocellular carcinomas contained transforming genes that appear not to be members of the known ras gene family. The B6C3F1 mouse liver system might provide a very sensitive assay not only for assessing the potential of a chemical to activate a cellular proto-oncogene, but also for detecting various classes of proto-oncogenes that are susceptible to mutational activation.

Activating mutations of the c-Ha-ras protooncogene in chemically induced hepatomas of the male B6C3 F1 mouse.

Activated c-Ha-ras protooncogenes have recently been identified in the DNA of some spontaneous hepatic tumors found in 2-year-old B6C3 F1 mice. Activation of c-Ha-ras has now been demonstrated in DNA from well-differentiated hepatomas initiated by a single dose of carcinogen given to male B6C3 F1 mice at 12 days of age. DNA from each of 25 hepatomas, induced by N-hydroxy-2-acetylaminofluorene, vinyl carbamate, or 1'-hydroxy-2',3'-dehydroestragole, containing transforming activity in the NIH 3T3 transfection assay. Southern analysis of NIH 3T3 cells transformed by DNA from 24 of these hepatomas revealed amplified and/or rear-ranged restriction fragments homologous to a Ha-ras probe. The other tumor contained an activated Ki-ras gene. Immunoprecipitation and NaDodSO4/PAGE analysis of p21 ras proteins in NIH 3T3 transformants derived from a majority of the hepatomas suggested that the activating mutations were localized in the 61st codon of the c-Ha-ras gene. Creation of a new Xba I restriction site by an AT----TA transversion at the second position of codon 61 was detected in DNA from primary tumors and NIH 3T3 cells transformed by DNA from 6 of 7 vinyl carbamate- and 5 of 10 1'-hydroxy-2',3'-dehydroestragole-induced hepatomas. Selective oligonucleotide hybridization demonstrated a CG----AT transversion at the first position of the 61st codon in NIH 3T3 transformants derived from 7 of 7 N-hydroxy-2-acetylaminofluorene-induced hepatomas. By the same criterion, an AT----GC transition at the second position of codon 61 was the activating mutation in 1 of 7 vinyl carbamate- and 5 of 10 1'-hydroxy-2',3'-dehydroestragole-induced tumors. Thus, c-Ha-ras activation is apparently an early event in B6C3 F1 mouse hepatocarcinogenesis that results directly from reaction of ultimate chemical carcinogens with this gene in vivo.

Detection of activated proto-oncogenes in N-nitrosodiethylamine-induced liver tumors: a comparison between B6C3F1 mice and Fischer 344 rats.

DNA from B6C3F1 mouse and Fischer 344 rat liver tumors induced by N-nitrosodiethylamine (DEN) were examined for the ability to induce morphological transformation of NIH3T3 cells. DNAs from 14 of 33 of the mouse liver tumors induced by a single injection of DEN at 12 or 15 days of age were positive in this assay while DNA from only one of 28 DEN-induced rat liver tumors was active. Southern blot analysis of the NIH3T3 transformants derived from the mouse liver tumors revealed amplified and/or rearranged restriction fragments homologous to the H-ras proto-oncogene. DNA from two independent foci induced by the rat tumor DNA did not hybridize to probes for members of the ras gene family or c-raf. Activating mutations in the H-ras genes from the DEN-induced mouse liver tumors were characterized by selective oligonucleotide hybridization and the detection of a new XbaI restriction site by Southern blot analysis. In activated H-ras genes from the DEN-induced mouse liver tumor DNA, seven of 14 had a CG----AT transversion at the first base of the 61st codon, three of 14 had an AT----GC transition and four of 14 had the AT----TA transversion at the second base of codon 61. This spectrum of mutations is very similar to that recently observed in activated H-ras genes found in spontaneously occurring B6C3F1 mouse liver tumors. Taken together, the data suggest that the DEN-induced rat and mouse liver carcinogenesis may involve genetic targets other than or in addition to the H-ras gene.