Lack of Urinary Bladder Carcinogenicity of Sodium L-Ascorbate in Human c-Ha- ras Proto-Oncogene Transgenic Rats

Abstract

Sodium L-ascorbate (Na-AsA) is widely known to be a tumor promoter of rat bladder carcinogenesis but tests negative in standard 2-year bioassays. In the present study, bladder-cancer-susceptible transgenic rats designated Hras128 were used to further examine the tumorigenicity of Na-AsA. A total of 40 7-week-old male transgenic (Tg) and 42 littermate nontransgenic (Non-tg) rats were divided into 4 groups and given powdered MF diet with or without 5% Na-AsA for 57 weeks. Tg rats showed significantly short survival compared with Non-tg, independent of Na-AsA treatment. Tg rats treated with Na-AsA showed a slightly higher incidence of carcinoma (29.6%) as compared to those without Na-AsA treatment (15.4%), but this was without statistical significance. Moreover, the total bladder tumor incidences, including papillomas, did not differ statistically (with Na-AsA, 37.0%; without Na-AsA, 30.8%). No bladder tumor was detected in Non-tg rats. Various kinds of other lesions in various organs were noted in Tg rats treated with or without Na-AsA treatment, but no intergroup differences were evident. In conclusion, Na-AsA did not show tumorigenicity in highly bladder-cancer-susceptible transgenic Hras128 rats. These results suggest that Na-AsA is a pure promoter but not a complete carcinogen in rats.

Introduction

Sodium L-ascorbate (Na-AsA), like other sodium salts of saccharin (glutamate and bicarbonate), produces mild superficial urothelial cytotoxicity and regeneration (Fukushima et al., 1983, 1990; Cohen et al., 1995, 1998) when fed at high doses to rats. Na-AsA exerts a promotional effect on rat 2-stage urinary bladder carcinogenesis initiated by genotoxic bladder carcinogens such as N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) (Fukushima et al., 1983, 1990; Chen et al., 1999a, 1999b), but demonstrated no tumorigenicity for rat urinary bladder in standard 2-year carcinogenicity tests (Ellwein and Cohen, 1988, 1990; Cohen and Ellwein, 1990; Cohen et al., 1995, 1998). However, Cohen et al. (1998) did demonstrate the carcinogenic potential of Na-AsA in male rats using a 2-generation bioassay, which suggests that the period of Na-AsA treatment and/or the age of the rat at the start of the study may influence the carcinogenic potential of Na-AsA. The purpose of the present study, therefore, was to further evaluate the carcinogenicity of Na-AsA using highly bladder-cancer-susceptible animals.

Various kinds of transgenic and knockout mice are now established as providing good animal models for analysis of gene functions, especially those relevant to carcinogenesis. Some of these animals exhibit high susceptibility to chemical carcinogens in a tissue-specific manner, and now they are receiving much attention with regard to potential application in carcinogenicity tests for risk assessment.

Asamoto et al. (2000) have established a transgenic rat carrying 3 copies of the human c-Ha-ras proto-oncogene with its own promoter region, designated Hras128, which is highly susceptible to induction of tumors in mammary tissue by N-methyl-N-nitrosourea (MNU) and 7,12-dimrthylbenz[a]anthracene (DBMA) (Asamoto et al., 2000; Tsuda et al., 2001; Fukamachi et al., 2004), and also in the esophagus by N-nitrosomethylbenzylamine (Asamoto et al., 2002) and in the skin by DMBA treatment with or without 12-O-tetradecanoylphorbol 13-acetate (TPA) promotion (Park et al., 2004). Moreover, this strain shows remarkable susceptibility to bladder carcinogenesis induced by BBN (Ota et al., 2000).

In the present study, to further assess the tumorigenicity of Na-AsA in the rat urinary bladder, we used this species of cancer-susceptible transgenic rat in a long-term carcinogenicity test.

Materials and Methods

Animals

The Hras128 rat line (Asamoto et al., 2000) was generated by injecting copies of the human c-Ha-ras proto-oncogene into pronuclei of fertilized rat oocytes from Sprague–Dawley rats obtained from Clea Japan, Inc. (Tokyo, Japan). Subsequent matings were carried out between Tg and Non-tg Sprague–Dawley rats to maintain rat heterozygotes for the transgene (Clea Japan Inc., Tokyo). All the rats subjected to this study were confirmed for the genotype at Clea Japan Inc. (Tokyo, Japan).

Experimental Protocol

A total of 40 7-week-old male Tg rats were divided into 2 groups. Twenty-seven (group 1) and 13 (group 2) rats were given a powdered MF diet (Oriental Yeast Co., Ltd., Tokyo) with or without 5% Na-AsA (Wako Pure Chemical Industries, Tokyo Japan), respectively. Similarly, a total of 42 7-week-old male Non-tg rats were divided into 2 groups, and 30 (group 3) and 12 (group 4) animals were given a diet with or without 5% Na-AsA, respectively.

Animals were housed in groups of 2 to 3 in plastic cages with hard wood chips for bedding in an animal room with a 12-hour light, 12-hour dark cycle at a temperature of 22 ± 2°C and a humidity of 55 ± 5%. Body weights were measured weekly, and food consumption and water intake were calculated biweekly during the course of the experiment. Animals were carefully monitored until being sacrificed at final week 57 of the experimental period. Those becoming moribund during the course of the study, or when tumors appeared externally, were sacificed under ether anesthesia for autopsy and macroscopic and microscopic examinations.

All the animal experimental procedures used were approved by the Institutional Animal Care and Use Committee of Osaka City University Medical School.

Histological Examinations

All the animals were sacrificed under ether anesthesia at 57 weeks from the beginning of the experiment. Major organs including skin/subcutaneous tissue, mammary gland, forestomach, glandular stomach, small and large intestine, liver, pancreas, lung, kidney, testis, prostate, thymus, spleen, and urinary bladder were macroscopically examined. Any tumors apparent in those various organs were collected. Tissues were fixed in 10% phosphate-buffered formalin, embedded in paraffin, and sectioned for routine hematoxylin and eosin staining for histopathological analysis. Urinary bladder lesions were classified based on the WHO International Classification of Rodent Tumors (Kunze and Chowaniec, 1990).

Statistical Analysis

Surviving curves were created using the Kaplan–Meier method, and the statistical significance of differences was calculated by the log-rank test. Variations in incidences of tumors between the different treatments or animal types were evaluated with the nonparametric Fisher’s exact probability test or the χ² test. All the calculations for statistical analysis were performed using the Statview SE+ Graphics, version 5.0 (Abacus Concepts, Berkeley, CA).

Results

Survival Rates

Tg rats harbored various kinds of spontaneous tumors in various organs when the experiment was terminated after 57 weeks. Survival curves for all groups are shown in Figure 1. As a whole, Tg rats showed a short lifetime, regardless of 5% Na-AsA treatment (groups 1 and 2), compared with their littermate Non-tg rats (groups 3 and 4), but no significant differences were noted between group 1 and group 2 (p = 0.4001). About half of group 1 had died or been sacrificed due to the weight loss and/or tumor latency, and group 1’s survival curve was significantly different from that of group 3 (p < 0.0001). The survival curves for groups 3 and 4 were similar. A significant difference was also evident between groups 2 and 4 (p < 0.05). No animal died throughout the study in group 4, and 2 animals in group 3 died from pneumonia and fibrosarcoma of subcutaneous tissue with lung metastasis, respectively.

Tumor Incidences

Table 1 summarizes data for incidences of bladder tumors. All the lesions were transitional cell papillomas or carcinomas (Figure. 2A, B), and no bladder tumors occurred in either groups 3 or 4. No difference in the combined overall incidence of transitional cell carcinomas and papillomas was detected between groups 1 (37.0%) and 2 (30.8%). There was a tendency for Tg rats treated with Na-AsA to show a higher incidence of carcinoma (29.6%), compared to those without Na-AsA (15.4%), but no statistical difference was noted. Simple hyperplasia was detected in all the rats treated with Na-AsA, regardless of genotype.

Incidences of tumor-bearing rats in groups 1, 2, 3, and 4 were 70.3, 69.2, 20.0, and 25.0%, respectively (Table 2). The Tg rats showed significantly high incidences of tumors compared with Non-tg rats, both with and without Na-AsA treatment. However, there was no statistical difference between groups 1 and 2. A summary of tumors generated in organs other than the bladder is given in Table 3. Independent of the Na-AsA treatment, transgenic rats exhibited various kinds of malignant tumors in various organs, without significant differences between groups 1 and 2.

Discussion

In 1998, Cohen et al. (1998) examined the tumorigenicity of Na-AsA in male rats using a 2-generation bioassay that involved feeding to the male and female parental rats for 4 weeks before mating, feeding the dams during gestation and lactation, and then feeding the weaned male F₁ generation rats for the remainder of their lifetime. In that experiment, high-dose (5.0 and 7.0%) treatment of Na-AsA demonstrated an increase in urinary bladder urothelial papillary, nodular hyperplasia, and even papillomas and carcinomas with dose dependence. This implies the possibility of bladder cancer production on the part of this chemical, but no positive cancer induction has been observed thus far in standard, 1-generation-carcinogenicity tests using rodent models.

In the present study, a reexamination of the rat bladder carcinogenesis of the Na-AsA of Hras128 rat, a highly susceptible strain, provided no evidence of cancer induction. The nonsignificant tendency for an increase in the number of malignant tumors in Tg rats suggests the possibility of a weak promotion effect after spontaneous initiation.

Transgenic mice carrying human c-Ha-ras proto-oncogenes (rasH2) are highly susceptible to various carcinogens and have been demonstrated to be more predictive of human response than the classical cancer test methods (Mitsumori, 2003). This animal model is now regarded as a useful tool for evaluating chemical carcinogenicity, in accordance with the recommendation of the International Conference on Harmonization of Technical Requirements of Pharmaceuticals for Human Use (ICH). However, Na-AsA is not known to exert an enhancing influence on mice bladder carcinogenesis after BBN treatment (Tamano et al., 1993). Therefore, we employed a rat model to evaluate the carcinogenicity of Na-AsA.

In rodent urinary bladder carcinomas, those induced by BBN with and/or without Na-AsA in rats are mostly of the superficial type, while the invasive type is more frequent in mice. Moreover, almost all of the bladder cancers in rats are TCCs, whereas in mice most are squamous cell carcinomas. In the human bladder, TCCs are a major histological type, and this is classified as a “superficial type.” They are associated with frequent recurrence and invasion to the muscle layer, resulting in the poor prognosis. However, the reasons for such recurrence and invasive characteristics have not yet been elucidated in detail. We used a rat transgenic animal model representing the superficial type to reexamine the carcinogenic potential of Na-AsA, to match the histological type of human bladder carcinomas.

Ota et al. (2000) examined the susceptibility of bladder carcinogenesis in the same Tg rats using the potent genotoxic carcinogen, BBN, and revealed a high incidence of bladder carcinomas. Also, the Hras128 rat was found to exhibit greater tumor progression as compared to Non-tg counterparts. Mutations of both the exogenous and endogenous c-Ha-ras gene are rare, which may indicate that enhanced tumor development was not due primarily to mutations occurring in the transgene. The authors concluded that the overexpression of total c-Ha-ras protein, due to the expression of integrated genes, plays a major role in rat bladder carcinogenesis. Taking these findings into account, the molecular target of Na-AsA in enhancing bladder cancer production may differ from the c-Ha-ras overexpression pathway.

In conclusion, the rat bladder promoter Na-AsA did not show tumorigenicity, even when administered to highly bladder-cancer-susceptible Hras128 rats. However, the tendency for an increase of malignancy in Tg rats’ bladder tumors suggests that Na-AsA may have promotional effects. Taking the previous reports into account, these results suggest that Na-AsA is a pure promoter but not a complete carcinogen in rats.