Tumors In B6C3F1 Mice Research Articles

Pentachlorophenol (but not Phenobarbital) Promotes Intrahepatic Biliary Cysts Induced by Diethylnitrosamine to Cholangio Cystic Neoplasms in B6C3F1 Mice Possibly Due to Oxidative Stress

Pentachlorophenol (but not Phenobarbital) Promotes Intrahepatic Biliary Cysts Induced by Diethylnitrosamine to Cholangio Cystic Neoplasms in B6C3F1 Mice Possibly Due to Oxidative Stress

Pentachlorophenol (but not phenobarbital) promotes intrahepatic biliary cysts induced by diethylnitrosamine to cholangio cystic neoplasms in B6C3F1 mice possibly due to oxidative stress.

Administration of diethylnitrosamine (DEN) to B6C3F1 mice at low dose (20 ppm) in drinking water for long duration resulted in formation of multifocal cystic biliary lesions in the liver. To investigate the potential of the lesions to be promoted to neoplasias by chemicals, we examined the effects of 2 different types of hepatocarcinogenesis promoters, pentachlorophenol (PCP) and phenobarbital (PB) in B6C3F1 mice. Two weeks' exposure to PCP at a concentration of 600 ppm in the diet increased 8-oxodeoxyguanosine (8-oxodG) levels in liver nuclear DNA, and cell proliferation quantified by bromodeoxyuridine (BrdU) incorporation in epithelial cells of intrahepatic bile ducts as well as hepatocytes. In mice initiated with DEN at 20 ppm in the drinking water for the first 13 weeks followed, after a 4-week recovery interval, by PCP at a concentration of 600 ppm in the diet for 25 weeks, cystic atypical hyperplasias, cholangiomas, and cholangiocarcinomas were present at statistically significant higher incidences. In contrast, neoplasia did not occur in animals treated with 500 ppm PB, and there were no elevations in 8-oxodG levels or increases in the proliferation of biliary epithelium, although proliferation was increased in hepatocytes. These findings suggest that oxidative stress due to PCP might exert a promoting action on the biliary cystic lesions produced by DEN.

Mechanisms of 2-butoxyethanol carcinogenicity: studies on Syrian Hamster Embryo (SHE) cell transformation.

Previous studies showed that 2-butoxyethanol increased liver tumors in B6C3F1 mice following chronic exposure. While the mechanism of 2-butoxyethanol-induced liver carcinogenicity has not been defined, 2-butoxyethanol has been shown to induce hemolysis in rodents via 2-butoxyacetic acid, the major metabolite of 2-butoxyethanol. This toxic effect, coupled with the observation that continued treatment with 2-butoxyethanol results in hemosiderin deposition in the liver, has led to our hypothesis that liver carcinogenicity by 2-butoxyethnaol is mediated via oxidative stress (iron catalyzed) and Kupffer cell activation. The present study used Syrian Hamster Embryo (SHE) cell transformation, a surrogate in vitro model for carcinogenesis in vivo, to examine whether 2-butoxyethanol, 2-butoxyacetic acid, or iron (ferrous sulfate) produced cell transformation. SHE cells were treated with either 2-butoxyethanol (0.5-20 mM), 2-butoxyacetic acid (0.5-20 mM), or ferrous sulfate (0.5-75 microg/ml) for 7 days. 2-Butoxyethanol and 2-butoxyacetic acid did not induce cellular transformation. In contrast, treatment with ferrous sulfate (2.5 and 5.0 microg/ml) increased morphological transformation. Cotreatment of ferrous sulfate with the antioxidants alpha-tocopherol (vitamin E) or (-)-epigallocatechin-3-gallate (EGCG) prevented ferrous sulfate-induced transformation, suggesting the involvement of oxidative stress in SHE cell transformation. The level of oxidative DNA damage (OH8dG) increased following ferrous sulfate treatment in SHE cells; additionally, using single cell gel electrophoresis (comet assay), ferrous sulfate treatment produced an increase in DNA damage. Both DNA lesions were decreased by cotreatment of ferrous sulfate with antioxidants. These data support our proposal that iron, produced indirectly through hemolysis, and not 2-butoxyethanol or its metabolite 2-butoxyacetic acid, is responsible for the observed carcinogenicity of 2-butoxyethanol.

Point mutations of K- ras and H- ras genes in forestomach neoplasms from control B6C3F1 mice and following exposure to 1,3-butadiene, isoprene or chloroprene for up to 2-years

1,3 Butadiene (BD), isoprene (IP) and chloroprene (CP) are structural analogs. There were significantly increased incidences of forestomach neoplasms in B6C3F1 mice exposed to BD, IP or CP by inhalation for up to 2-years. The present study was designed to characterize genetic alterations in K- and H- ras proto-oncogenes in a total of 52 spontaneous and chemically induced forestomach neoplasms. ras mutations were identified by restriction fragment length polymorphism, single strand conformational polymorphism analysis, and cycle sequencing of PCR-amplified DNA isolated from paraffin-embedded forestomach neoplasms. A higher frequency of K- and H- ras mutations was identified in BD-, IP- and CP-induced forestomach neoplasms (83, 70 and 57%, respectively, or combined 31/41, 76%) when compared to spontaneous forestomach neoplasms (4/11, 36%). Also a high frequency of H- ras codon 61 CAA→CTA transversions (10/41, 24%) was detected in chemically induced forestomach neoplasms, but none were present in the spontaneous forestomach neoplasms examined. Furthermore, an increased frequency (treated 13/41, 32% versus untreated 1/11, 9%) of GGC→CGC transversion at K- ras codon 13 was seen in BD-, and IP-induced forestomach neoplasms, similar to the predominant K- ras mutation pattern observed in BD-induced mouse lung neoplasms. These data suggest that the epoxide intermediates of the structurally related chemicals (BD, IP, and CP) may cause DNA damage in K- ras and H- ras proto-oncogenes of B6C3F1 mice following inhalation exposure and that mutational activation of these genes may be critical events in the pathogenesis of forestomach neoplasms induced in the B6C3F1 mouse.

Nine cases of granular cell tumors in B6C3F1 mice.

The histological characteristics of 9 cases of granular cell tumors (GCTs) observed in B6C3F1 mice were examined to determine their cellular origin. Seven of the 9 cases were found in the uterus and other 2 cases were in the subcutaneous tissue. Tumor cells had abundant granules in the cytoplasm which were stained with PAS and were resistant to diastase treatment. Ultrastructurally, the granules were identified as lysosomes. The cell surface had cytoplasmic processus showing interdigitation with adjacent cells. A character feature of the tumor cells was the presence of a desmosome-like structure on their cell surface but no basal lamina was demonstrated. Although GCTs have been considered to be derived from Schwann cells on the basis of their ultrastructural features and S-100 protein-immunopositive findings, the absence of basal lamina in the present cases may raise a controversy as to their origin.

Correlation between Bcl-2 overexpression and H-ras mutation in naturally occurring hepatocellular proliferative lesions of the B6C3F1 mouse.

The correlation between the mutation at codon 61 of the H-ras gene and the expression of the Bcl-2 protein was investigated in naturally occurring hepatocellular proliferative lesions in B6C3F1 mice. Specimens of histologically diagnosed neoplastic or preneoplastic lesions of the liver, obtained from the control mice used for 2-year carcinogenicity studies, were examined by immunohistochemical techniques. All of 25 lesions confirmed to be hepatocellular carcinomas stained positive for the Bcl-2 protein. Three of 12 foci of cellular alterations, as well as 24 of 42 hepatocellular adenomas, stained weakly positive. Bcl-2 protein was expressed to a greater degree in hepatocellular carcinomas as opposed to adenomas and confirmed by Western blot analysis. Seven of 18 hepatocellular adenomas that stained positive for Bcl-2 and three of 16 hepatocellular adenomas that stained negative had a mutation at codon 61 of the H-ras gene. Overexpression of Bcl-2 protein is likely to enhance the malignant turnover of the neoplastic cells, following a mutation at codon 61 of the H-ras gene particularly. These findings suggest that Bcl-2 overexpression and the mutation at codon 61 of the H-ras gene may be critical factors in the development of naturally occurring hepatocellular tumors in B6C3F1 mice.

Absence of correlation between telomerase activity and hepatic neoplasia in B6C3F1 mice

Telomeres are the physical ends of eukaryotic chromosomes, which maintain chromosome stability and are progressively shortened with aging in somatic cells. The enzyme telomerase elongates telometric DNA and while not usually detectable in human somatic cells is expressed in most human tumors. The present study was conducted to determine if telomerase activity is a marker for spontaneous hepatic neoplastic changes in B6C3F1 mice, a strain frequently used in rodent carcinogenicity studies. Telomerase activity was generally higher in microscopically normal liver tissue from 8-week-old compared to aged mice (110-week-old); however, telomerase activity was not consistently increased in hepatocellular adenomas and carcinomas. It is proposed that, while elevated telomerase activity may modulate human tumor development, modulation of telomerase activity is not a feature of hepatic tumors in B6C3F1 mice and therefore is unlikely to have utility as a molecular marker for hepatic neoplasia in this mouse strain.

Expression of surfactant protein mRNA in normal and neoplastic lung of B6C3F1 mice as demonstrated by in situ hybridization.

The localization of surfactant protein (SP), A, B, C, and D mRNAs was examined in B6C3F1 mice in the normal lung, and in a range of spontaneous proliferative lung lesions using nonisotopic in situ hybridization (ISH). The aim was to develop diagnostic markers, and if possible, throw further light on the histogenesis of these lesions. Tissues from 21 animals were examined, the lesions studied were: 4 alveolar epithelial hyperplasias, 12 alveolar/bronchiolar (A/B) adenomas, and 5 A/B carcinomas. Lung metastases of hepatocellular carcinoma (HCC) were used as controls. In the nonneoplastic lung, staining for SP A, B, and C mRNA was observed in normal and hyperplastic type II cells but not in the bronchiolar epithelium. SP mRNAs were present in all lung tumors, with SPs A, B, and C being coexpressed in 10/12 (83%) of adenomas and 4/ 5 (80%) of carcinomas in both solid and tubulopapillary areas. No signals for SP D mRNA were noted in normal or neoplastic lung. Additionally, no staining for any SP transcript was observed in the HCC metastases examined. In summary, ISH for SP A, B, or C mRNA was a helpful aid in the diagnosis of proliferative lesions of the murine lung, enabling differentiation from hepatocellular metastases. Furthermore, this work provides strong support for the proposal that spontaneous lung tumors in B6C3F1 mice are of alveolar, not bronchiolar origin, and consistently show type II cell differentiation. We suggest that such tumors should be referred to as alveolar adenomas and carcinomas.

Effect of dichloroacetic acid and trichloroacetic acid on DNA methylation in liver and tumors of female B6C3F1 mice.

Dichloroacetic acid (DCA) and trichloroacetic acid (TCA) are found in drinking water and are metabolites of trichloroethylene. They are carcinogenic and promote liver tumors in B6C3F1 mice. Hypomethylation of DNA is a proposed nongenotoxic mechanism involved in carcinogenesis and tumor promotion. We determined the effect of DCA and TCA on the level of DNA methylation in mouse liver and tumors. Female B6C3F1 mice 15 days of age were administered 25 mg/kg N-methyl-N-nitrosourea and at 6 weeks started to receive 25 mmol/liter of either DCA or TCA in their drinking water until euthanized 44 weeks later. Other animals not administered MNU were euthanized after 11 days of exposure to either DCA or TCA. DNA was isolated from liver and tumors, and after hydrolysis 5-methylcytosine (5MeC) and the four bases were separated and quantitated by HPLC. In animals exposed to either DCA or TCA for 11 days but not 44 weeks, the level of 5MeC in DNA was decreased in the liver. 5MeC was also decreased in liver tumors from animals exposed to either chloroacetic acid. The level of 5MeC in TCA-promoted carcinomas appeared to be less than in adenomas. Termination of exposure to DCA, but not to TCA, resulted in an increase in the level of 5MeC in adenomas to the level found in noninvolved liver. Thus, hypomethylated DNA was found in DCA and TCA promoted liver tumors and the difference in the response of DNA methylation to termination of exposure appeared to support the hypothesis of different mechanisms for their carcinogenic activity.

Effect of Dichloroacetic Acid and Trichloroacetic Acid on DNA Methylation in Liver and Tumors of Female B6C3F1 Mice

Dichloroacetic acid (DCA) and trichloroacetic acid (TCA) are found in drinking water and are metabolites of trichloroethylene. They are carcinogenic and promote liver tumors in B6C3F1 mice. Hypomethylation of DNA is a proposed nongenotoxic mechanism involved in carcinogenesis and tumor promotion. We determined the effect of DCA and TCA on the level of DNA methylation in mouse liver and tumors. Female B6C3F1 mice 15 days of age were administered 25 mg/kg N-methyl-N-nitrosourea and at 6 weeks started to receive 25 mmol/liter of either DCA or TCA in their drinking water until euthanized 44 weeks later. Other animals not administered MNU were euthanized after 11 days of exposure to either DCA or TCA. DNA was isolated from liver and tumors, and after hydrolysis 5-methylcytosine (5MeC) and the four bases were separated and quantitated by HPLC. In animals exposed to either DCA or TCA for 11 days but not 44 weeks, the level of 5MeC in DNA was decreased in the liver. 5MeC was also decreased in liver tumors from animals exposed to either chloroacetic acid. The level of 5MeC in TCA-promoted carcinomas appeared to be less than in adenomas. Termination of exposure to DCA, but not to TCA, resulted in an increase in the level of 5MeC in adenomas to the level found in noninvolved liver. Thus, hypomethylated DNA was found in DCA and TCA promoted liver tumors and the difference in the response of DNA methylation to termination of exposure appeared to support the hypothesis of different mechanisms for their carcinogenic activity.

Induction of mouse cytochrome P450 2B enzymes by amine metabolites of musk xylene: contribution of microsomal enzyme induction to the hepatocarcinogenicity of musk xylene.

Musk xylene (MX) is a synthetic nitromusk perfume ingredient that, although uniformly negative in genotoxicity testing, causes liver tumors in B6C3F1 mice. MX is also capable of inducing cytochrome P450 enzymes in a manner similar to that of phenobarbital (PB), which suggests that epigenetic mechanisms may be involved in the carcinogenic response. At the same time, MX is metabolized in vivo by nitroreduction, a reaction catalyzed by intestinal flora that yields aromatic amine metabolites. These amine metabolites are also capable of inactivating CYP2B10, the major cytochrome P450 enzyme induced by MX treatment. In the study reported here, the monoamine metabolites of MX, o- and p-NH2-MX, were evaluated for their potential to induce CYP2B10 and CYP1A2 mRNAs. Northern blot analyses indicated that both amines markedly induced CYP2B10 mRNA, whereas CYP1A2 mRNA, the enzyme implicated in the bioactivation of aromatic amines and frequently induced by aromatic amines, was induced only slightly, a response that was not different from that seen with PB. Induction of CYP2B10 mRNA suggested that the amine metabolites may contribute to the enzyme induction profile seen with MX treatment. To test this hypothesis, mice were treated with broad-spectrum antibiotics (neomycin, tetracycline, and bacitracin) to eliminate the intestinal flora and prevent formation of o- and p-NH2-MX. In antibiotic-treated mice treated with MX (200 mg/kg) for 4 d, no evidence of microsomal enzyme induction was observed, including no increases in liver weight, total cytochrome P450 content, or CYP2B protein levels. These results indicate that the amine metabolites of MX are responsible for the enzyme induction seen after MX administration. Thus, the biochemical and molecular effects of amine metabolites of MX are markedly different from those of other aromatic amines but very similar to those of PB. Therefore, it appears that MX is a non-genotoxic chemical that may cause mouse liver tumors in a manner analogous to that of PB.

Assessment of the mutagenicity of dichloroacetic acid in lacI transgenic B6C3F1 mouse liver.

Dichloroacetic acid (DCA) is a chlorination byproduct found in finished drinking water. When administered in drinking water this chemical has been shown to produce hepatocellular adenomas and carcinomas in B6C3F1 mice over the animal's lifetime. In this study, we investigated whether mutant frequencies were increased in mouse liver using treatment protocols that yielded significant tumor induction. DCA was administered continuously at either 1.0 or 3.5 g/l in drinking water to male transgenic B6C3F1 mice harboring the bacterial lacI gene. Groups of five or six animals were killed at 4, 10 or 60 weeks and livers removed. At both 4 and 10 weeks of treatment, there was no significant difference in mutant frequency between the treated and control animals at either dose level. At 60 weeks, mice treated with 1.0 g/l DCA showed a 1.3-fold increase in mutant frequency over concurrent controls (P = 0.05). Mice treated with 3.5 g/l DCA for 60 weeks had a 2.3-fold increase in mutant frequency over the concurrent controls (P = 0.002). The mutation spectrum recovered from mice treated with 3.5 g/l DCA for 60 weeks contained G:C-->A:T transitions (32.79%) and G:C-->T:A transversions (21.31%). In contrast, G:C-->A:T transitions comprised 53.19% of the recovered mutants among control animals. Although only 19.15% of mutations among the controls were at T:A sites, 32.79% of the mutations from DCA-treated animals were at T:A sites. This is consistent with the previous observation that the proportion of mutations at T:A sites in codon 61 of the H-ras gene was increased in DCA-induced liver tumors in B6C3F1 mice. The present study demonstrates DCA-associated mutagenicity in the mouse liver under conditions in which DCA produces hepatic tumors.

Electrospray analysis of biological samples for trace amounts of trichloroacetic acid, dichloroacetic acid, and monochloroacetic acid.

Trichloroethylene (TCE) has been identified as a widespread groundwater contaminant. Trichloroacetic acid (TCA) and dichloroacetic acid (DCA) are toxicologically relevant metabolites of TCE that produce tumors in B6C3F1 mice. A sensitive method for measuring these metabolites in plasma has been developed to obtain pharmacokinetic data from TCE exposure. This is particularly important because DCA is more potent at producing hepatoproliferative lesions than TCA. At present, it is unclear whether DCA is produced by humans. Existing gas chromatographic methods cannot detect DCA at low nanogram-per-milliliter levels. A Finnigan TSQ 700 mass spectrometer (MS) with electrospray ionization was used to measure TCA, DCA, and monochloroacetic acid (MCA) in plasma. The MS was operated in negative ion tandem MS mode. The limit of detection for TCA and DCA was 4 ng/ml, and the limit of detection for MCA was 25 ng/mL. Plasma samples from human subjects exposed to 100 ppm TCE for 4 h contained TCA at concentrations as high as 10 micrograms/mL. DCA concentrations were less than 5 ng/mL, and MCA was not detected (less than 25 ng/mL).

Analysis of Ha-ras Gene in the B6C3F1 Mouse Liver by Non-RI PCR-SSCP.

The codon 61 of the activated Ha-ras gene is detected most frequently among mutations of proto-oncogenes in spontaneously-occurring hepatocellular tumors in B6C3F1 mice. In the present study, the ras mutations and their patterns in normal liver tissue, foci of cellular alteration (FCA), and hepatocellular tumor from untreated mice were analyzed using the non-RI PCR-SSCP method. The Ha-ras gene with mutated codon 61 was detected in 1.6% (2/122) from normal liver tissues, 16.7% (6/36) from spontaneous FCA tissues, 30.4% (48/158) from spontaneous hepatocellular tumor tissues, and 7.7% (3/39) from normal portion in spontaneous hepatocellular tumor tissues. Furthermore, mutation in the Ha-ras gene was compared between spontaneous and induced hepatocellular proliferative lesions including hepatocellular tumor from archival materials at our Center. Although no clear difference was found between genotoxic carcinogen and non-genotoxic carcinogens in the incidence of Ha-ras gene mutation in the induced tumors, the incidence of Ha-ras gene mutation in FCA, which was induced by genotoxic carcinogen, was higher than that in FCA induced by non-genotoxic carcinogens. These data suggested that to analyze the incidence of Ha-ras gene mutation in proliferative lesions in the livers of B6C3F1 mice, especially in the FCA is useful in evaluation of hepatocarcinogenicity of the chemicals.

Evaluation of transgenic mouse bioassays for identifying carcinogens and noncarcinogens

Data supporting the use of transgenic lines to identify carcinogens and noncarcinogens are thus far based on a limited number of chemicals for which there are also long-term bioassay results in rats and/or mice. Six chemicals have been tested in the heterozygous p53-deficient mice and 13 in the Tg · AC line. The results show that the p53 def responds rapidly to mutagenic carcinogens and the Tg · AC responds rapidly to both mutagenic and nonmutagenic carcinogens. Neither transgenic line responded to the noncarcinogens that were tested. The p53 def line failed to respond to two nonmutagenic carcinogens ( N-methyloacrylamide and reserpine), the Tg · AC line failed to respond to ethyl acrylate, a nonmutagenic chemical that induced tumors of the forestomach when administered by gavage, and to triethanolamine that caused an increase in hepatocellular tumors in B6C3F1 mice via skin painting. Both of the latter chemicals are examples of highly specific responses related to either route of administration or to strain susceptibility. Further efforts to evaluate the range of chemicals to which these transgenic lines respond are currently in progress.

Conversion of trichloroacetic acid to dichloroacetic acid in biological samples.

Trichloroethylene (TCE) has been identified as an environmental contaminant in groundwater. Trichloroacetic acid (TCA), dichloroacetic acid (DCA), chloral hydrate (CH), trichloroethanol (TCOH), and trichloroethanol glucuronide (TCOG) have been identified as metabolites of TCE. Studies have shown that TCA and DCA are toxicologically significant metabolites that can induce liver tumors in B6C3F1 mice. Methods for the analysis of these metabolites are important for conducting pharmacokinetic studies. In this study, TCA and DCA were derivatized to their methyl esters by dimethyl sulfate under acidic conditions and analyzed by gas chromatography with electron capture detection. In developing a method for esterifying TCA and DCA, the conversion of TCA to DCA was observed in freshly drawn blood upon the addition of acid. After blood was drawn from the animals, the amount of TCA converted to DCA by the addition of acid decreased with time. This conversion could be prevented by freezing blood samples overnight prior to the addition of acid. Further experiments demonstrated that this activity could be restored by the addition of dithionite to inactivate blood samples or the addition of dithionite to methemoglobin prior to the addition of acid. The results reported here show that reduced hemoglobin may be involved in the acid-catalyzed conversion of TCA to DCA.

Hepatocarcinogenicity of chlordane in B6C3F1 and B6D2F1 male mice: evidence for regression in B6C3F1 mice and carcinogenesis independent of ras proto-oncogene activation.

Logistic regression analysis of age-specific prevalences for neoplastic and non-neoplastic liver lesions was used to examine treatment responses for B6C3F1 and B6D2F1 male mice continuously exposed to chlordane (55 p.p.m.) and to determine whether neoplasms were dependent on continuous exposure in the B6C3F1 mice. In order to determine if ras oncogene activation plays a role in the carcinogenicity of chlordane and whether the activation is dependent on genetic background, liver tumors from chlordane-treated B6C3F1 and B6D2F1 mice were analyzed for the presence of activating mutations in the ras oncogene. The overall liver tumor prevalence at terminal killing was nearly 100% for both strains; however, the age-specific prevalence increased more rapidly in B6C3F1 mice than in B6D2F1 mice. Tumor-bearing B6C3F1 mice had an average of two or more tumors per liver than B6D2F1 mice at their respective terminal killings (5.4 versus 3.3). When chlordane exposure was discontinued for a group of B6C3F1 mice ('stop' group) at 491 days of age, overall tumor multiplicity significantly decreased by 30% from an average of 4.4 per tumor-bearing-animal at 525 days to 3.1 at terminal killing (568 days). Over the same time period the prevalence of hepatocellular carcinomas significantly decreased from 80 to 54% and adenomas from 100 to 93% by terminal killing in B6C3F1 'stop-group' mice. Chlordane induced diffuse hepatocellular centrilobular hypertrophy, frequent multinucleate hepatocytes, toxic change and hepatoproliferative lesions composed predominantly of acidophilic hepatocytes in nearly 100% of both the B6C3F1 and B6D2F1 mice. The development of histological evidence of toxicity closely paralleled the temporal development of hepatocellular neoplasia and decreased in severity when the tumor burden was maximal. No H- or K-ras mutations were detected in the chlordane-induced hepatocellular tumors in B6C3F1 mice (15 adenomas and 15 carcinomas) or B6D2F1 mice (10 adenomas and 10 carcinomas). In conclusion, chlordane induced liver tumors in both B6C3F1 and B6D2F1 male mice by mechanisms independent of ras oncogene activation and 30% of both benign and malignant liver tumors in the B6C3F1 mice regressed after exposure was discontinued.

Increased frequency of K-ras mutations in lung neoplasms from female B6C3F1 mice exposed to ozone for 24 or 30 months.

The National Toxicology Program recently completed long-term ozone inhalation studies in B6C3F1 mice and F344/N rats. Mice and rats were exposed to 0, 0.5 or 1.0 p.p.m. ozone by inhalation for 24 or 30 months. There was an increased incidence of lung neoplasms in B6C3F1 mice. However, there was no evidence of carcinogenicity in F344/N rats. The objectives of this study were to (i) evaluate benign and malignant lung neoplasms from B6C3F1 mice for mutations in the K-ras gene at codons 12, 13 and 61, (ii) determine if the frequency and spectra of K-ras mutations were unique for ozone-induced lung neoplasms, (iii) determine if specific K-ras mutations were associated with the size and morphological patterns of lung neoplasms or ozone exposure concentrations and (iv) screen lung neoplasms by immunohistochemical methods for the p53 protein. K-ras mutations were detected by single-strand conformation analysis and identified by direct sequencing of polymerase chain reaction-amplified DNA isolated from formalin-fixed, paraffin-embedded neoplasms. K-ras mutations were detected in 73% of ozone-induced neoplasms, as compared with 33% of lung neoplasms from controls. The predominant mutations consisted of A-->T transversions at codon 61 (8/19) and G-->T transversions at codon 12 (7/19). Specific K-ras mutations in lung neoplasms were not associated with various morphological patterns. Our data suggests that ozone may cause direct and/or indirect DNA damage in the K-ras proto-oncogene of B6C3F1 mice.

Ras oncogene activation during hepatocarcinogenesis in B6C3F1 male mice by dichloroacetic and trichloroacetic acids.

Dichloroacetic (DCA) and trichloroacetic (TCA) acids, two major by-products formed during chlorine disinfection of drinking water, increase the incidence of tumors in B6C3F1 mice by 6- and 3-fold respectively. In order to understand better the mechanism by which these two compounds induce liver tumors, the incidence and spectrum of mutations in the K- and H-ras proto-oncogenes in these tumors were analyzed. DNA from spontaneous, DCA- and TCA-induced liver tumor from B6C3F1 male mice was evaluated for point mutations in exons 1, 2 and 3 of the two genes by single-stranded conformation polymorphism. Results demonstrated a similar incidence of mutations for exon 2 of H-ras in spontaneous carcinomas (58%), and in carcinomas induced by DCA 3.5 g/l (50%), 1.0 g/l (48%) and TCA 4.5 g/l (45%). Only four showed mutations in the other exons of Hras or in K-ras. Sequence analysis of spontaneous tumor samples with second exon H-ras mutations revealed a change in codon 61 from CAA to AAA in 80% and CAA to CGA in 20% of tumors. In contrast, tumors with H-ras mutations from DCA-treated mice revealed a H-61 change from CAA to AAA in 21% at 3.5 g/l and 16% at 1.0 g/l. CAA to CGA was observed in 50% of tumors from mice given DCA 3.5 or 1.0 g/l, and CAA to CTA was present in 29% and 34% of the two dosage groups respectively. Interestingly, TCA showed the same mutational spectrum as the spontaneous liver tumors. The data indicates that induction of liver carcinoma by DCA and TCA involves activation of the H-ras proto-oncogene at a frequency similar to that observed in spontaneous tumors. However, the mechanism(s) for including hepatocellular carcinoma does not appear to be identical for DCA and TCA.

Morphology of spontaneous Harderian gland tumors in aged B6C3F1 mice.

Spontaneous Harderian gland tumors in B6C3F1 mice were found in 18 (3.1%) (one male had bilateral tumors) of 589 males and 18 (3.0%) of 609 females. The tumor-bearing mice ranged in age from 73 to 109 weeks. These tumors comprised 32 adenomas (86.5%), four adenocarcinomas (10.8%) and one pleomorphic tumor (2.7%); the adenomas were classified, based on the growth patterns, into 17 papillary, nine cystic papillary, five acinar and one cystic types. Two adenocarcinomas grew papillary and invaded surrounding tissues, and the other two grew acinously. The pleomorphic tumor consisted of pleomorphic cells some of which had a keratin-positive epithelial feature and acini lined by atypical epithelial cells.