Susceptibilities of p53 Knockout and rasH2 Transgenic Mice to Urethane-Induced Lung Carcinogenesis are Inherited from their Original Strains

Abstract

In the present study, susceptibility of CB6F1 mice carrying the human prototype c-Ha-ras gene (rasH2 mice) and p53 gene knockout mice (p53 (+/−) mice) to urethane-induced lung carcinogenesis was compared under the same experimental conditions. Both strains were administered 500 ppm urethane in their drinking water for 3 weeks. At week 26, lung adenocarcinomas and adenomas were observed in 53% and 100% of rasH2 mice, respectively, and lung adenomas were observed in 67% of rasH2 littermate (non-Tg) mice. However, lung tumors were not observed in either p53 (+/−) or p53 (+/+) mice. Peliosis hepatis and hepatic hemangiomas were observed in 27% and 67% of p53 (+/−) mice, but only in 6.7% and 6.7% of the rasH2 animals, respectively. Under the same experimental conditions, BALB/c mice, the strain of origin of the rasH2 mice, developed lung adenomas at an incidence of 93%, whereas none of the C57BL/6 original strain for p53 (+/−) mice developed lung tumors. Peliosis hepatis was observed in 40% of the C57BL/6 mice, but not in BALB/c mice; hepatic and splenic hemangiomas were not observed in these animals. These results indicate that organ susceptibility of rasH2 and p53 (+/−) mice is inherited from their strains of origin, the rasH2 and BALB/c lines being much more sensitive to the induction of pulmonary carcinogenesis.

Introduction

Urethane ([carbonyl-(14)C]ethyl carbamate (NH₂COCO C₂H₅)) is classified as a genotoxic-mitogenic carcinogen, producing histologic types of tumors in rats and mice (Tannenbaum, 1964; Mirvish, 1968). In mice, lung adenomas and adenocarcinomas are induced at high incidences (IARC, 1974). The background strains for rasH2 mice, BALB/c and C57BL/6 (Saitoh et al., 1990), carry the human c-Ha-ras proto-oncogene and is a promising animal model for the development of a rapid carcinogenicity testing system due to their high susceptibility to genotoxic carcinogens, especially those targeting the pulmonary system (Ogawa et al., 1996; Yamamoto et al., 1996; Mori et al., 2000, 2001; Tomisawa et al., 2003).

Similarly, p53 tumor suppressor gene knockout mice (p53 (+/−)mice), derived from C57BL/6 mice, are considered to have advantages for in vivo short- or medium-term bioassays (Harvey et al., 1993). However, it has been reported that whereas C57BL/6 mice are highly susceptible to hepatic vascular carcinogenesis, susceptibility to urethane-induced lung carcinogenesis is low (Carmichael et al., 2000). Thus, there are clear differences between rasH2 and p53 (+/−) mice in this regard. However, no data are available for direct comparisons of susceptibility under the same experimental conditions. Therefore, the induction of pulmonary neoplasms in the 2 strains was evaluated to determine whether organ susceptibility depends on the strain of origin (Mitsumori, 2002). Differences in sensitivity to hepatocarcinogenesis between p53 (+/−) mice (C57BL/6 background) and p53 (+/−) CBA (back-crossed with CBA female mice) have also been reported (Uehara et al., 2002). Therefore, the yield of hepatic tumors was also assessed.

Materials and Methods

Test Chemical and Route of Exposure

Urethane (purity > 99%) was obtained from Wako Pure Chemical Industries, Ltd. (Osaka, Japan) and administered orally by addition to drinking water at a concentration of 500 ppm. Dose formulations were prepared twice a week and supplied in 150 ml plastic water bottles.

Animals, Housing, and Clinical Observations

Male p53 (+/−) and wild-type p53 (+/+) mice were purchased from Taconic (Germantown, NY, USA); CB6F1 rasH2 and wild-type (non-Tg) mice were obtained from the Central Institute for Experimental Animals (Kawasaki, Japan) at 6 weeks of age. BALB/c and C57BL/6 mice of the parent strains were purchased from Charles River Japan Inc. (Atsugi, Japan) at 6 weeks of age. All were housed individually in polycarbonate cages (125W × 200D × 110H mm) in an animal room maintained at 22 ± 3° with relative humidity of 55 ± 15%; 10 air changes per hour, and a 12-hour light/dark cycle. Basal pellet diet (CE-2, CLEA Japan, Inc.) and tap water or urethane-containing water were provided ad libitum. After an 11- to 13-day acclimatization period, clinically normal mice were selected for the present study at 7 weeks of age. All animals were handled in accordance with the Guidelines for Animal Experimentation by the Japanese Association for Laboratory Animal Science (1987). Mice were observed daily for clinical signs and mortality. Body weights and food consumption were recorded once a week throughout the study period, and average water consumption was measured weekly during the 3-week treatment period. Average urethane intake was calculated using the following calculation; urethane intake (mg/kg/day) = (water intake (g/day) × urethane concentration (ppm))/body weight (g).

Experimental Design

The experimental protocol is shown in Figure 1. Fifteen mice of each strain were given urethane in the drinking water ad libitum for 3 weeks and then maintained on tap water for 23 weeks. Control mice (15 males of each strain) were given tap water ad libitum throughout the experimental period. At 26 weeks, all mice were sacrificed by exsanguination from the abdominal aorta under light ether anesthesia and subjected to a complete necropsy.

Pathological Examination

At necropsy, all major organs were examined and weighed. All grossly visible lung nodules were recorded, and the lungs and other organs were fixed in 10% neutral-buffered formalin and embedded in paraffin, sectioned at 4–6 microns, and stained with hematoxylin and eosin (H&E) for histopathological examination.

Statistical Analysis

The following statistical methods (Gad and Weil, 1994) were used to determine the significance of differences in the results between the 2 groups. The Student’s t-test or Aspin-Welch test was used to determine the significance of body weight, food consumption, water intake, and organ weight differences between treated and control mice. The Fisher’s exact test was used to determine the significance of the incidence of histopathological lesions. Significance was concluded at p < 0.05 or p < 0.01.

Results

Clinical Signs, Mortality, Body Weight, Organ Weight, and Urethane Intake

RasH2 mice treated with urethane were hypoactive, emaciated, and exhibited piloerection and abnormal gait. These clinical signs gradually disappeared after cessation of urethane treatment. Clinical signs of toxicity were not observed in p53 (+/−) mice treated with urethane. Sixty percent (9/15) of the rasH2 mice given urethane died during the study. Cause of death was considered to be hemorrhage from lung lesions. Mean body weights of rasH2 mice treated with urethane were significantly less than ( p < 0.01) those of the other strains at 20 and 26 weeks (Figure 2). Average urethane consumption by p53 (+/−) mice was less than in the nontreatment group, but the difference was not statistically significant (Figure 3).

There was no mortality, no clinical signs of toxicity, and no significant differences in body weight in BALB/c or C57BL/6 mice regardless of the urethane treatment (Figure 2). In urethane-treated p53 (+/−) mice, the absolute splenic and heart weights were significantly greater ( p < 0.01) and the relative liver and salivary weights were significantly less ( p < 0.01 or p < 0.05) than those of the controls (Table 1). In urethane-treated C57BL/6 mice, the absolute and relative pituitary gland weights were significantly less ( p < 0.05), the absolute testes weights were significantly greater ( p < 0.05) than those of the controls. In urethane-treated BALB/c mice, the relative pituitary weights were significantly less ( p < 0.05) than those of the controls.

Pathological Examination

The results of necropsy are shown in Table 2. The numbers and mean numbers of lung nodules in urethane-treated mice were significantly greater than those in the controls. Microscopically, alveolar hyperplasia, adenomas, and adenocarcinomas were identified. Hyperplasia and adenoma were observed in all urethane-treated rasH2, non-Tg, and BALB/c mice. Adenocarcinomas were significantly increased only in rasH2 mice, and lung tumors were not observed in p53 (+/−), p53 (+/+), or C57BL/6 mice treated with urethane. These findings indicate that rasH2 and BALB/c mice are susceptible to pulmonary carcinogenesis induction by urethane. Morphologically, most of the adenomas in urethane-treated rasH2, non-Tg, and BALB/c mice were composed of cuboidal to columnar epithelial cells arranged in papillary or glandular patterns. Adenocarcinoma in rasH2 mice treated with urethane exhibited papillary, glandular, or solid patterns. Tumor cells were cuboidal to columnar in shape, and pleomorphic and anaplastic cells were also observed in some adenocarcinomas (Figure 4).

The incidences of peliosis hepatis were significantly increased in urethane-treated p53 (+/−) and C57BL/6 mice (Table 3). Hemangiomas were significantly increased in the liver and spleen of p53 (+/−) and rasH2 mice, respectively (Table 3). With the exception of the rasH2 mice, neither hepatic nor splenic hemangiomas were observed in the untreated controls.

Malignant lymphomas of the thymus and a squamous cell carcinoma of the forestomach were observed in a urethane-treated p53 (+/−) mouse and a rasH2 mouse, respectively. Comparison of susceptibilities among p53 (+/−), rasH2, C57BL/6, and BALB/c mice with regard to urethane-induced lesions are summarized in Table 4.

Discussion

Ogawa and colleagues earlier reported high incidences of urethane-induced lung adenomas and adenocarcinomas in rasH2 mice given a dose of 500 ppm in the drinking water for 21 days (Ogawa et al., 1996). In contrast, Carmichael et al. (2000) found low incidences of urethane-induced lung adenomas in p53 (+/−mice receiving 100 mg/kg/day by gavage for 180 days. Our present results with the 2 strains under the same experimental conditions confirmed that p53 (+/−) mice have low susceptibility, whereas the rasH2 strain is highly sensitive to urethane-induced lung carcinogenesis. It is also well established that BALB/c mice are susceptible to lung tumorigenesis, whereas C57BL/6 is a resistant strain (Della Porta et al., 1967; Malkinson, 1989, 1998). In this experiment, lung adenomas were observed in 93% of BALB/c mice, but in none of the C57BL/6 mice after the treatment of urethane.

Two types of p53 knockout mice originating from different strains, with a C57BL/6 background and from CBA, have been reported to demonstrate differing sensitivity to hepatocarcinogenesis and tumorigenesis in other organs (Mitsumori, 2002; Uehara et al., 2002). Mitsumori noted that the biological characteristics of genetically modified animals may be inherited from their original strain (Mitsumori, 2002), and Carmichael et al. described high susceptibility of p53 (+/−) mice (C57BL/6 background) to urethane-induced vascular tissue carcinogenesis. In the present study, C57BL/6 mice developed hemangioma-like lesions, peliosis hepatis, in the liver after urethane treatment. The p53 (+/−) mice also showed a high susceptibility to urethane-induced liver vascular tumorigenesis. Peliosis hepatis in mice can probably be regarded as an early stage of hemangioendothelioma (Trainin, 1963; Frith et al., 1994). Thus, these findings indicate that both p53 (+/−) and C57BL/6 mice possess a similar susceptibility to liver lesion induction by urethane. These results provide further support for the conclusion that organ susceptibility of rasH2 and p53 (+/−) mice is inherited from their strains of origin and that rasH2 mice are more appropriate for assessment of pulmonary carcinogenic potential.

Under the present conditions, lung adenomas were observed in both rasH2 mice and non-Tg littermates, but lung adenocarcinomas were present only in the transgenic mice; by contrast, splenic hemangiomas were found in 67% of rasH2 mice but not in either BALB/c or C57BL/6 animals. These results suggest that the c-Ha-ras transgene plays an important role in progression of lung adenomas to adenocarcinomas and in development of splenic hemangiomas in rasH2 mice. Results also suggest that heterozygous knockout of the p53 gene increases the sensitivity to vascular tumorigenesis in the liver but not in the spleen.

Splenic hemangiomas were observed in only rasH2 mice in this experiment. Mitsumori et al. (1998) reported that splenic hemangiomas in untreated rasH2 mice were observed spontaneously. Saitoh et al. (1990) observed the high incidence of point mutations of transgenes in hemangiosarcoma that spontaneously occurred in rasH2 mice. These findings might suggest the rasH2 mice develop a high susceptibility to spleen tumorigenesis by c-Ha-ras transgene.

In conclusion, the present findings indicate that the susceptibilities of p53 (+/−) and rasH2 mice to urethane-induced lung and liver tumorigenesis are to a large extent inherited from their strains of origin.