Female rasH2 Mice Research Articles

Carcinogenicity and testicular toxicity of 2-bromopropane in a 26-week inhalation study using the rasH2 mouse model

2-Bromopropane (2-BP) is a colorless liquid at room temperature and is used in closed systems in factories, mainly as an intermediate for medicines, pesticides, and other chemicals. However, the carcinogenicity of 2-BP is still unknown. The CByB6F1-Tg(HRAS)2Jic (rasH2) transgenic mouse model has been established as an alternative to long-term studies (1.5 years—lifetime) to detect carcinogenicity in as short a time as six months. We performed a 26-week inhalation exposure study of 2-BP using the rasH2 mouse model. Male and female rasH2 mice were exposed to 0, 67, 200, or 600 ppm of 2-BP for 6 h/day, 5 days/week for 26 weeks. All tissues and blood were collected and subjected to biological and histopathological analyses. The results showed a concentration-dependent increase in lung tumor development in male and female rasH2 mice exposed by inhalation to 2-BP, which was significant by Peto’s and Poly-3 trend tests. Furthermore, in male rasH2 mice, 2-BP was found to be a testicular toxin. This study is the first to demonstrate that 2-BP is carcinogenic in male and female mice and a testicular toxin in male mice using the rasH2 mouse model.

No evidence for carcinogenicity of titanium dioxide nanoparticles in 26-week inhalation study in rasH2 mouse model

With the rapid development of alternative methods based on the spirit of animal welfare, the publications of animal studies evaluating endpoints such as cancer have been extremely reduced. We performed a 26-week inhalation exposure studies of titanium dioxide nanoparticles (TiO2 NPs) using CByB6F1-Tg(HRAS)2Jic (rasH2) mice model for detecting carcinogenicity. Male and female rasH2 mice were exposed to 2, 8 or 32 mg/m3 of TiO2 NPs for 6 h/day, 5 days/week for 26 weeks. All tissues and blood were collected and subjected to biological and histopathological analyses. TiO2 NPs exposure induced deposition of particles in lungs in a dose-dependent manner in each exposure group. Exposure to TiO2 NPs, as well as other organs, did not increase the incidence of lung tumors in any group, and pulmonary fibrosis and pre-neoplastic lesions were not observed in all groups. Finally, the cell proliferative activity of alveolar epithelial type 2 cells was examined, and it was not increased by exposure to TiO2 NPs. This is the first report showing the lack of pulmonary fibrogenicity and carcinogenicity (no evidence of carcinogenic activity) of TiO2 NPs in 26-week inhalation study in rasH2 mice exposed up to 32 mg/m3, which is considered to be a high concentration.

Alpha-Glycosyl Isoquercitrin (AGIQ) and its lack of carcinogenicity in rasH2 mice

alpha-Glycosyl Isoquercitrin (AGIQ), is used in Japan as a food additive and was granted generally recognized as safe (GRAS) status in 2005 (FEMA) and 2007 (FDA). The safety and toxicity information for AGIQ is sparse and therefore, the carcinogenicity potential of AGIQ was examined in the CByB6F1-Tg(HRAS)2Jic (rasH2) model. One hundred female and male rasH2 mice, each, were allocated to one of four designated dose groups; 0 (control)%, 1.5%, 3.0% or 5.0% AGIQ. Animals were administered the diets for six months and an additional 10 females and 10 males, each, were administered a positive control, N-methyl-N-nitrosourea (MNU). Body weights and clinical observations were collected. A full screen necropsy, organ weights, clinical chemistry, urinalysis and histopathology were performed. The positive control animals elicited appropriate responses specific to this strain (rasH2) of mice. There were statistically significant sporadic non-dose-dependent changes in clinical chemistries without corresponding pathological correlation. No microscopic AGIQ-related findings were noted; the range of pathology observations were all considered background findings, either specific to rasH2 mice or common to inbred strains of mice. Therefore, under the study conditions, the no-observed-adverse-effect level (NOAEL) was determined to be more than 5.0% (7215.4 mg/kg BW/day in male mice and 14685.5 mg/kg/day in female mice).

Carcinogenicity of intermediate frequency magnetic field in Tg.rasH2 mice.

Although the likelihood of exposure to leaking intermediate frequency magnetic fields (MFs) from electronic devices, such as induction‐heating and wireless power transfer systems, has increased, biological data assessing the health risks associated with human exposure remain insufficient. We examined the carcinogenicity of a 20 kHz MF, a typical frequency produced by induction‐heating cookers, using a transgenic rasH2 mouse model. Twenty‐five male and female CByB6F1‐Tg(HRAS)2Jic mice were exposed to a 0.20 mT, 20 kHz MF (22 h/day) or sham‐exposed for 26 weeks. As a positive control, 10 male and female rasH2 mice from the same batch were administered a single intraperitoneal injection of 75 mg/kg N‐methyl‐N‐nitrosourea. A blinded histopathological evaluation was performed, and the same experiments were conducted twice, independently, to confirm the reproducibility of the results. Histopathological examination revealed that spontaneous neoplastic lesions, such as splenic hemangiosarcomas and gastric squamous cell papillomas, were less (1–3 per group) in the MF‐ and sham‐exposed groups. The frequency of the neoplastic lesions was not significantly different between the groups. Eight to ten mice in each positive‐control group exhibited malignant lymphoma. The outcomes were consistent between duplicated experiments, which indicates lack of carcinogenicity of 20 kHz MF in the rasH2 mouse model. Bioelectromagnetics. © 2019 The Authors. Bioelectromagnetics Published by Wiley Periodicals, Inc.

Lung Tumor Induction by 26-week Dermal Application of 1,2-Dichloroethane in CB6F1-Tg rasH2 Mice.

Short-term alternatives to traditional 2-year carcinogenic studies in rodents are being actively pursued. Recently, a 26-week short-term carcinogenicity study using CB6F1-Tg rasH2@Jcl (rasH2) mice has become a worldwide standard for the evaluation of chemical carcinogenesis. However, an acceptable short-term carcinogenic study model for dermally applied products is still lacking. To investigate the suitability of using the rasH2 mouse to test carcinogenic potential, 1,2-dichloroethane (1,2-DCE) was dermally applied to rasH2 mice: 1,2-DCE is a known carcinogen that causes lung bronchiolo-alveolar adenomas and adenocarcinomas when administered topically, orally, or by inhalation exposure; 1,2-DCE at a dose level of 126 mg/mouse in 200 μl acetone or acetone alone (vehicle control) was applied to the dorsal skin of 10 mice of each sex 3 times a week for 26 weeks. As a positive control, 10 mice of each sex received a single intraperitoneal injection of 75 mg/kg of N-methyl- N-nitrosourea. Bronchiolo-alveolar adenomas and adenocarcinomas were significantly increased in 1,2-DCE-treated rasH2 mice of both sexes, and bronchiolo-alveolar hyperplasias were significantly increased in female mice. Overall, almost all mice of each sex developed adenomas and/or adenocarcinomas with 100% of female rasH2 mice developing bronchiolo-alveolar adenocarcinomas.

The rasH2 Mouse Model for Assessing Carcinogenic Potential of Pharmaceuticals

A factor limiting widespread use of the transgenic rasH2 mouse model for carcinogenicity testing of pharmaceuticals is the paucity of published data on actual drug candidates in rasH2 mice. This report addresses this gap by highlighting rasH2 mouse study data for 10 pharmaceutical candidates. These results were compared with findings in the 2-year studies in Sprague-Dawley rats for the same 10 compounds. In the 6-month rasH2 studies, only 2 of the 10 compounds tested positive for carcinogenicity and these correlated with positive findings in the companion 2-year rat studies. One compound, sunitinib, produced gastroduodenal carcinoma in both sexes and increased hemangiosarcoma in spleen and uterus in female rasH2 mice; in rats it produced gastroduodenal carcinoma and increased pheochromocytoma (males only). The second compound, bazedoxifene, produced ovarian granulosa cell neoplasms in rasH2 mice and rats, and renal tubular neoplasms associated with increased chronic progressive nephropathy only in rats. The higher percentage of carcinogenicity positive rat bioassays could be attributed to rat-specific phenomena with little or low relevance to man. Thus, this article confirms previous reports that rasH2 mice develop rodent-specific neoplasms less frequently than rats and positive findings, when present, are accompanied by similar positive results in the rat.

Extremely weak tumor-promoting effect of troglitazone on splenic hemangiosarcomas in rasH2 mice induced by urethane

To examine the tumor-promoting effect of troglitazone (TRG), a novel thiazolidinedione insulin-sensitizing agent, on splenic hemangiosarcomas in rasH2 mice, histopathological and molecular analyses were performed in the spleen of female rasH2 mice fed a diet containing 6,000 or 0 ppm TRG for 16 weeks after 1,000 or 0 mg/kg urethane (UR) initiation. Histopathologically, splenic hemangiosarcomas were observed in the UR-alone and UR + TRG groups, but there was no significant difference in the incidence of splenic hemangiosarcomas between the UR-alone and UR+TRG groups. There were increasing tendencies in the number of positive cells for anti-PCNA antibody and gene expression in the UR + TRG group, but such a change was not statistically significant as compared to that in the UR-alone group. The gene expressions of VEGF, VEGFR1, VEGFC, VEGFR2 and Tie2 related to angiogenesis; c-fos related to MAPK cascade activation; and cyclin D1 related to cell cycle in the UR-alone and UR + TRG groups were significantly higher than those in the untreated control group. However, only the Tie2 gene in the UR + TRG group was significantly increased as compared to that in the UR-alone group. These results suggest that the vascular tumor-promoting activity of TRG in rasH2 mice is extremely low in the present experimental condition and a part of the gene related to angiogenesis probably contributes to the promotion of splenic hemangiosarcomas in rasH2 mice given TRG.

Carcinogenic susceptibility of rasH2 mice to troglitazone

To evaluate the carcinogenicity of troglitazone in rasH2 mice, 7-week-old male and female rasH2 mice were fed a diet containing 0, 3,000 or 6,000 ppm troglitazone for 26 weeks. An increased tendency in the incidence of vascular tumors was observed in females of the 6,000 ppm group. The preliminary analysis using a high-density oligonucleotide microarray on a splenic hemangiosarcoma of a high dose female that could be obtained as a fresh sample showed that several genes related to the ras/MAPK pathway activation, angiogenesis, cell cycle and cell multiplication were up-regulated. In addition, most of the genes up-regulated were confirmed by the reverse transcriptase-polymerase chain reaction (RT-PCR). These results may suggest that the carcinogenic susceptibility of rasH2 mice to troglitazone is relatively low and up-regulations of the ras/MAPK pathway and angiogenesis-related genes are probably involved in the production of splenic hemangiosarcomas in rasH2 mice given troglitazone.

EXAMINATION OF PERCUTANEOUS APPLICATION IN A 26-WEEK CARCINOGENICITY TEST IN CB6F1-TG rasH2 MICE

We examined the possibility of expanding applications of rasH2 mice, which are genetically manipulated mice for short-term carcinogenicity tests, to percutaneous application. A 26-week short-term carcinogenicity study was performed on a total of 300 mice including 75 male and female rasH2 mice each, and 75 male and female non-Tg mice each from the same litter as the rasH2 mice divided into untreated group, an ethanol group, a white Vaseline group, an acetone group, and a phorbol 12-myristate 13-acetate (TPA) group. Only shaving of dorsal skin was performed on the untreated mice. As a positive control, TPA was administered percutaneously at a dose of 2.5 microg/kg and 3 times/week for 26 weeks based on the protocol for Tg.AC mice in the ILSI/HESI international validation study. In the ethanol, white Vaseline, and acetone groups, no tumorous changes were observed on the skin at the administration site. In the TPA group, nodular changes at the administration site were observed from seven weeks after the start of administration in rasH2 mice, and the incidence in males and females was 50.0% (7/14) and 53.3% (8/15), respectively. In a pathological examination, nodules in 21.4% (3/14) of males and 46.7% (7/15) of females were diagnosed as skin papilloma or keratoacanthoma, and the rest as squamous cell hyperplasia. In the non-Tg mice, no nodules or tumorigenic changes were observed at the administration site. These findings show that percutaneous application in rasH2 mice is possible in 26-week carcinogenicity tests.

A 26-Week Carcinogenicity Study of 2-Amino-3-Methylimidazo[4,5-f]Quinoline in rasH2 Mice

To evaluate the carcinogenic susceptibility of rasH2 mice to 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 7-week-old rasH2 mice and their wild-type littermates (non-Tg mice) of both the sexes were fed a diet containing 0 or 300 ppm IQ for 26 weeks. Microscopical examinations revealed that the proliferative lesions of the forestomach, including squamous cell hyperplasias, papillomas, and carcinomas, were frequently encountered in male and female rasH2 mice fed with IQ. In non-Tg mice, no significant differences in the incidence of forestomach lesions were observed between the 0 ppm and 300 ppm groups. Histopathological changes such as periportal hepatocellular hypertrophy and oval cell proliferation in the liver were more apparent in female rasH2 and non-Tg mice than in males, and the incidence of hepatocellular altered foci significantly increased in female rasH2 mice in the 300 ppm group as compared to that in the 0 ppm group. These results suggest that the carcinogenic potential of IQ can be detected in rasH2 mice by a 26-week, short-term carcinogenicity test.

USE OF IC TAGS IN SHORT-TERM CARCINOGENICITY STUDY ON CB6F1 TGrasH2 MICE

We studied the effect of IC tags, subcutaneously implanted animal identification tools, on rasH2 mice. A 26-week short-term carcinogenicity study was performed on a total of 299 mice including 75 male and female rasH2 mice each, and 74 male and 75 female non-Tg mice from the same litter as the rasH2 mice divided into a non-IC tag group, the IC-tag group, acetone group, TPA group and MNU group (all of the animals except for those in the non-IC tag group) had IC tags implanted subcutaneously in their backs. The administration methods of the positive control drugs TPA (2.5 micro g/kg, 3 times/week, percutaneously) and MNU (75 mg/kg, single intraperitoneal injection) were based on the protocol of the ILSI/HESI international collaborative study. The results showed no differences in the tumorigenic incidence and organs developing tumors between the IC tag and non-IC tag groups in both rasH2 and non-Tg mice. In the positive control MNU group, the tumorigenic incidence and organs developing tumors were the same as the background data and no promotion of carcinogenesis was observed. In all IC tag groups including the TPA group and MNU group, a fibrous capsule was formed around the IC tags subcutaneously, but no inflammatory changes or neoplastic changes were observed. From these findings, it was concluded that the IC tag has no effect on a 26-week carcinogenicity test of rasH2 mice under the conditions of the present study.

Evaluation of the Carcinogenic Potential of Clofibrate in the rasH2 Mouse

The purpose of the study was to support of the International Life Sciences Institute (ILSI) alternative carcinogenicity models initiative to evaluate the carcinogenic potential of the nongenotoxic carcinogen, clofibrate, a peroxisome proliferator-activated receptor (PPAR) alpha agonist, following oral administration to rasH2 mice. Peroxisome proliferators are one of the most widely studied of the nongenotoxic carcinogens and have diverse industrial and therapeutic uses (Gonzalez et al. J. Nat. Cancer Inst. 90: 1702-1709, 1998); however, the nongenotoxic mechanism of carcinogenicity is currently unknown. Male mice were administered doses of clofibrate at 50, 100, or 200 mg/kg/day and female mice were administered doses of 50, 150, or 250 mg/kg/day by oral gavage at 10 ml/kg for 27 weeks. In addition, rasH2 male and female mice were treated with N-nitroso-N-methylurea (NMU). Nontransgenic male and female mice were treated with 200 and 250 mg/kg/day, respectively, of clofibrate. The NMU-treated mice were given a single intraperitoneal dose of 75 mg/kg, which was followed by a 90-day observation period; all others were sacrificed after 6 months of daily dosing. Hepatocellular neoplasms were observed in clofibrate-treated rasH2 male mice after 6 months of treatment but not in nontransgenic males or females. Clofibrate treatment (250 mg/kg/day) of female rasH2 mice was associated with a slight increase in the incidence of various neoplasms (harderian gland, lungs, skin, spleen, tail, thymus, and uterus) compared with untreated transgenic mice and with similarly treated nontransgenic mice. Non-neoplastic changes were found in the liver of transgenic and nontransgenic mice of both sexes and in the kidneys of male mice. NMU produced findings are consistent with previous studies. The data suggest that the rasH2 mice are a good model for testing epigenetic carcinogens in a shorter timeframe than conventional mouse carcinogenicity bioassays.

The mouse rasH2/BHT model as an in vivo rapid assay for lung carcinogens.

We have demonstrated the utility of a 9–week in vivo two‐stage assay for lung cancer initiating agents, using transgenic mice carrying the human prototype c‐Ha‐ras gene (rasH2 mice) and butylhydroxytoluene (BHT) as a potent lung promoter (rasH2/BHT model). In the present study, to ascertain appropriate conditions for BHT administration in this model, the effects of exposure on proliferation of alveolar type II cells in male rasH2 mice were examined. Additionally, use of BHT was validated for promotion of urethane (UR) carcinogenesis in male and female rasH2 mice. In a time‐course study of a single intragastric administration of BHT at a dose of 400 mg/kg, increased bromodeoxyuridine‐labeling index (LI) reached a maximum 3 days after treatment and was still observed after 7 days. In a dose‐response study, effects were dose‐dependent, the dose of 400 mg/kg causing eight‐fold elevation as compared to the control. With repeated administration, whereas the LI was increased dramatically at first, effects gradually diminished with further exposure, and finally six BHT treatments failed to induce cell proliferation. In a two‐stage model using UR as the initiator, although up to five consecutive doses of BHT were able to exert continued enhancing effects in terms of adenoma yield, no increment was evident with further treatments. The data overall indicate that a rasH2/BHT model with five weekly administrations of BHT at a dose of 400 mg/kg is most efficacious.

Rapid induction of uterine endometrial proliferative lesions in transgenic mice carrying a human prototype c-Ha- ras gene (rasH2 mice) given a single intraperitoneal injection of N-ethyl- N-nitrosourea

In our previous study, uterine endometrial stromal sarcomas and atypical hyperplasias of the endometrial glands were induced in heterozygous p53 deficient mice (p53 (+/−) mice) of the CBA strain given a single dose of N-ethyl- N-nitrosourea (ENU). In order to clarify whether uterine tumors can be induced in transgenic mice carrying a human prototype c-Ha- ras gene (rasH2 mice) that are very susceptible to genotoxic carcinogens, rasH2 mice and their wild-type littermates received an intraperitoneal injection of 120 or 0 mg/kg body weight of ENU followed by no further treatment for 22 weeks. Eighteen and 94% of ENU-treated rasH2 mice had uterine endometrial adenocarcinomas and atypical hyperplasias, respectively. Other malignant and benign tumors such as lung alveolar/bronchiolar adenomas and carcinomas, forestomach squamous cell papillomas and carcinomas, splenic hemangiomas/sarcomas, skin papillomas, malignant lymphomas and harderian gland adenomas were also observed in ENU-treated rasH2 mice. The result in the present study suggests that female rasH2 mice are very susceptible to uterine carcinogenesis, providing a useful model for ENU-induced uterine epithelial tumors.

Modifying Effects of Genistein, 4-Nonylphenol and Methoxychlor on <i>N</i>-ethyl-<i>N</i>-nitrosourea-induced Uterine Carcinogenesis in RasH2 Transgenic Mice Harboring a Human Prototype C-Ha-<i>ras</i> Gene

In our previous study, uterine endometrial atypical hyperplasias and adenocarcinomas were induced in female transgenic mice harboring human proto type c-Ha-ras gene (rasH2 mice) within 22 weeks by a single intraperitoneal injection of N-ethyl-N-nitrosourea (ENU). In order to clarify the modifying effects of genistein (GE), 4-nonylphenol (NP), and methoxychlor (MXC) on uterine endometrial carcinogenesis, female rasH2 mice received an intraperitoneal injection of 120 mg/kg body weight (bw) of ENU, followed by no further treatment, diet containing 250 ppm GE, diet containing 250 ppm NP, or diet containing 1,000 ppm MXC for 24 weeks. Uterine proliferative lesions that were observed in treated groups were composed of endometrial hyperplasias, atypical hyperplasias of the endometrium, and adenocarcinomas. The incidence of adenocarcinomas in the ENU alone, ENU+GE, ENU+NP, and ENU+MXC groups was 55.6, 57.1, 47.1, and 0%, respectively, that in the ENU+MXC group being significantly depressed as compared to the ENU alone group (p<0.05). The incidence of atypical hyperplasias in the ENU+MXC group was also decreased. The results in the present study suggest that 1,000 ppm MXC, but not GE and NP, shows an inhibitory effect on the development of uterine adenocarcinomas in rasH2 mice initiated with ENU.

Butylhydroxytoluene (BHT) increases susceptibility of transgenic rasH2 mice to lung carcinogenesis.

Transgenic mice carrying the human prototype c-Ha-ras gene (rasH2 mice) are highly susceptible to lung carcinogens. In order to investigate the possibility of developing a rapid in vivo assay for lung carcinogens, we examined whether the tumor-promoting activity of butylhydroxytoluene (BHT) is efficacious in rasH2 mice. rasH2 mice and wild littermates of both genders were pre-treated with carcinogens [urethane (UR), 4-nitroquinoline 1-oxide (4NQO) or diethylnitrosamine (DEN)], and, one day later, given a 400 mg/kg dose of BHT. Six weeks after the initiation treatment, evidence of carcinogenicity could be detected in male and female rasH2 mice that had received UR doses of > or = 250 mg/kg and > or = 125 mg/kg, respectively, prior to exposure to BHT, whereas only 500 mg/kg of UR was sufficient to induce tumors in female rasH2 mice given the carcinogen alone. The carcinogenicity of 15 mg/kg of 4NQO could be detected after 9 weeks in male rasH2 mice given the carcinogen followed by BHT. Similarly, the carcinogenicity of 60 mg/kg of DEN could be detected after 9 weeks and 6 weeks, respectively, in male and female rasH2 mice given the carcinogen followed by BHT. No carcinogenicity could be demonstrated through the experimental period with doses of 4NQO or DEN given alone. These results indicate that BHT administration increases the susceptibility of rasH2 mice to lung carcinogens, and suggest that the use of BHT in rasH2 mice might lead to the establishment of a rapid in vivo assay for lung carcinogens.

Lack of modifying effects of eugenol on development of lung proliferative lesions induced by urethane in transgenic mice carrying the human prototype c-Ha-ras gene.

To investigate the modifying effects of eugenol (EUG), a component of cigarette smoke, on lung carcinogenesis, male and female transgenic mice carrying the human prototype c-Ha-ras gene (rasH2 mice) were given a single intraperitoneal injection of 250 mg/kg urethane (UR) or saline, followed by a diet containing 6,000 ppm EUG or basal diet for 26 weeks. Their non-transgenic CB6F1 littermates (non-Tg mice) were also treated in the same manner. In both male and female rasH2 mice, alveolar/bronchiolar hyperplasias, adenomas and carcinomas were observed in all UR-treated groups. However, there were no significant intergroup differences in the incidences and multiplicities of these lesions between the UR alone and UR + EUG groups. In non-Tg mice, alveolar/bronchiolar hyperplasias, adenomas or carcinomas were sporadically observed in UR-treated groups of both sexes, with no significant differences in the incidences and multiplicities between the UR alone and UR + EUG groups. There were no intergroup differences between them in the PCNA-positive ratios of adenomas or carcinomas and the areas of adenomas or carcinomas to the whole lung area examined. The present results suggest that the EUG treatment does not exert modifying effects on lung carcinogenesis induced by UR in both male and female rasH2 mice and non-Tg mice.

Hepatic drug metabolizing enzymes induced by clofibrate in rasH2 mice

Hepatic drug metabolizing enzyme activities were determined, after treatment with clofibrate, in transgenic mice carrying human c-Ha-ras (rasH2 mice). Changes in the drug metabolizing enzyme activities in these mice by gene integration were also evaluated. Male and female rasH2 mice (Tg) and the litter mates not carrying the gene (non-Tg) received orally 500 mg/kg of clofibrate or the vehicle for 12 consecutive days. Liver homogenate and microsomes were prepared and the contents and activities of cytochrome P450 (CYP), cytochrome b5 content and enzyme activities related to peroxisome proliferation were determined. Relative liver weights, CYP4A and activities of catalase and carnitine palmitoyl transferase increased to the same extent in Tg and non-Tg mice treated with clofibrate. In Tg and non-Tg groups that received vehicle, contents and activities of CYP and cytchrome b5 contents were comparable. It was concluded that gene integration did not alter drug metabolizing enzymes and responses to clofibrate.

Carcinogen dose-dependent variation in the transgene mutation spectrum in urethane-induced lung tumors in transgenic mice carrying the human prototype c-Ha- ras gene

Urethane-induced lung tumors and their genetic changes were investigated in transgenic (Tg) mice carrying a human prototype c-Ha- ras gene (rasH2 mice). Male and female rasH2 mice and non-transgenic (non-Tg) littermates were injected intraperitoneally with 1000 mg/kg of urethane once or three times at 2-day intervals. Hyperplasias and adenomas of the lung were observed in all animals of each group from week 10, and carcinomas were observed in male and female rasH2 mice of the triple injection group from week 10 and female non-Tg mice of the single injection group at 15/20 weeks. The multiplicities of lung proliferative lesions including hyperplasias, adenomas and carcinomas, in treated rasH2 mice were significantly higher than those in treated non-Tg mice. CAG to CTG transversions were observed in the c-Ha- ras gene in these lung proliferative lesions of rasH2 mice of the single injection group at high incidence (male: 58.3%, female: 62.5%), but no mutations of the mouse c-Ki- ras gene were evident in either rasH2 or non-Tg mice. In the triple injection group, transgene mutations were detected at a relatively low incidence, and mouse c-Ki- ras gene mutations(CAA to CGA) were observed in both rasH2 and non-Tg mice. These results suggest that the variation of the lesions induced by different doses of urethane was not the cause of the variation of the mutation spectrum and mutations of both transgene and mouse c-K- ras gene are not principal genetic events in urethane-induced lung proliferative lesions in rasH2 mice.