Experimental model of gastric carcinogenesis with N-methyl-N-nitrosourea for F344 rats and C3H mices is valid for Wistar rats?

Tarso, Lissandro; Meyer, Fabíola Schons; Cioato, Marta Giotti; Meurer, Luíse; Schirmer, Carlos Cauduro

doi:10.1590/S0102-67202011000100012

Abstracts

BACKGROUND: The N-methyl-N-nitrosourea (MNU) is a direct acting carcinogen, inducing tumors in several species in a variety of organs, including stomach of rats. Treatment of MNU in the drinking water for 25-42 weeks selectively induced glandular gastric carcinoma in F344 rats and C3H mice. AIM: To establish an experimental model for selective MNU induction of glandular stomach cancer in Wistar rats. METHODS: A total of 48 males eight-week-old Wistar rats were used in the present study. MNU (Sigma-Aldrich) was dissolved in DMSO and provided as the drinking water ad libitum for a period ranging from 16 to 70 weeks. After 16 weeks, four rats were randomly selected and killed. After every six weeks four animals were killed until 70 weeks. RESULTS: Survival rate was higher than 90%. It had the induction of two adenocarcinomas, one squamous cell carcinoma and one sarcoma. The incidence of gastric adenocarcinoma was 4.5% (0.5 to 15). CONCLUSIONS: The experimental model of gastric carcinogenesis in Wistar rats, using MNU dissolved in water, showed not practice viability in this study due to the low rate of gastric adenocarcinoma.

Wistar Rats; Tumor markers, biological; Stomach neoplasms

INTRODUÇÃO: O N-metil-N-nitrosourea (MNU) tem ação cancerígena direta, induzindo tumores em várias espécies em uma variedade de órgãos, incluindo o estômago de ratos. Tratamento do MNU na água de beber por 25-42 semanas, seletivamente, induz carcinoma gástrico glandular de ratos F344 e camundongos C3H. OBJETIVO: Estabelecer um modelo experimental para indução seletiva de câncer no estômago glandular de ratos Wistar com MNU. MÉTODOS: Um total de 48 ratos Wistar machos com oito semanas, foram utilizados no presente estudo. MNU (Sigma-Aldrich) foi dissolvido em DMSO e liberada água potável ad libitum por um período variando de 16 a 70 semanas. Após 16 semanas, quatro ratos foram selecionados aleatoriamente e mortos. Depois, de seis em seis semanas, quatro animais também foram mortos até 70 semanas. RESULTADOS: A taxa de sobrevivência foi superior a 90%. Ocorreu a indução de dois adenocarcinomas, um carcinoma espinocelular e um sarcoma. A incidência de adenocarcinoma gástrico foi de 4,5% (0,5 a 15). CONCLUSÕES: O modelo experimental de carcinogênese gástrica em ratos Wistar, utilizando MNU dissolvido na água, não mostrou viabilidade prática neste estudo, devido à baixa taxa de adenocarcinoma gástrico que ocorreu.

Ratos Wistar; Marcadores biológicos de tumor; Neoplasias gástricas

ORIGINAL ARTICLE

Correspondência

ABSTRACT

BACKGROUND: The N-methyl-N-nitrosourea (MNU) is a direct acting carcinogen, inducing tumors in several species in a variety of organs, including stomach of rats. Treatment of MNU in the drinking water for 25-42 weeks selectively induced glandular gastric carcinoma in F344 rats and C3H mice.

AIM: To establish an experimental model for selective MNU induction of glandular stomach cancer in Wistar rats.

METHODS: A total of 48 males eight-week-old Wistar rats were used in the present study. MNU (Sigma-Aldrich) was dissolved in DMSO and provided as the drinking water ad libitum for a period ranging from 16 to 70 weeks. After 16 weeks, four rats were randomly selected and killed. After every six weeks four animals were killed until 70 weeks.

RESULTS: Survival rate was higher than 90%. It had the induction of two adenocarcinomas, one squamous cell carcinoma and one sarcoma. The incidence of gastric adenocarcinoma was 4.5% (0.5 to 15).

CONCLUSIONS: The experimental model of gastric carcinogenesis in Wistar rats, using MNU dissolved in water, showed not practice viability in this study due to the low rate of gastric adenocarcinoma.

Headings: Wistar Rats. Tumor markers, biological. Stomach neoplasms.

INTRODUCTION

Since the first report of experimental production of adenocarcinomas of the glandular stomach in rats with N-methyl-N'-nitro-N-nitrosoguanidina (MNNG)¹⁶, many other mammals have been shown to be susceptible to this carcinogen, including hamsters, ferrets and dogs^2,3. The agent N-methyl-N-nitrosourea (MNU) is a direct acting carcinogen, inducing tumors in several species in a variety of organs, including the central nervous system, stomach, intestine, Kidney, and Skin^1,4,9,10.

The mouse and rat are particularly useful species for carcinogenesis studies because of the availability of a number of transgenic, mutant, and chimeric strains. Treatment of MNU in the drinking water for 25-42 weeks selectively induced glandular gastric carcinoma in F344 rats and C3H mice^{5,8,11,13,14,18,21}. These animal models have been widely used not only for investigating the pathogenesis of gastric carcinogenesis but also for identifying possible tumor promoters and chemopreventive agents^11,14.

The aims of the present study were establishing an experimental model for selective MNU induction of glandular stomach cancer in Wistar rats and describe the time to get a level of depth tumor (T).

METHODS

Experimental design

A total of 48 males eight-week-old Wistar rats were house in plastic cages (three rats/cage) on hard ward chips in air-conditioned room with a 12 h light/12 h dark cycle. Rats were allowed two weeks for acclimation before starting of experiments. MNU (Sigma-Aldrich) was dissolved in dimethyl sulfoxide (DMSO) and the resulting stock solution was stored in a cool, and dark place. The solution was diluted to 200 ppm with tap water just before using (three times a week), and provided as the drinking water ad libitum from light-shielded bottles to prevent photolysis for 16 to 70 weeks. Regular chow pellets (Nuvilab CR-1 - Nuvital S/A) were available ad libitum. At 16 weeks, four rats were randomly selected and killed, from each six weeks four animals were killed until 70 weeks. The study protocol was approved by the Research Ethics Committee of the Research and Postgraduate Group of Hospital de Clínicas de Porto Alegre, Porto Alegre, RS, Brazil.

Exclusion criteria

Animals that survived less than 16 weeks were excluded in the effective numbers. Necropsies were performed on all animals which died.

Histopathological analysis

The excised stomachs were fixed in buffered formalin, cut into about eight strips, and routinely processed for embedding in paraffin. Tissue sections were stained with HE for histopathological assessment of lesion development. Neoplastic lesions were classified as adenocarcinomas, squamous carcinoma and sarcomas.

Statistical analysis

The confidence interval for proportions using the Binomial Method was applied for calculated of gastric adenocarcinomas incidence. Student's t test was performed for means of quantitative variables.

RESULTS

A total of 48 rats were used. Four animals died before the week 16 and were undergone to autopsy. These deaths were caused by pneumonia, and they were excluded from the effective numbers. Survival rates were higher than 90%. The average initial body weight was 346 ± 33 g. The weekly body weight gain during follow-up was lower in rats that developed cancer than those who did not (2.1g x 3.2 g, p=0.07). A total of 44 animals was killed from weeks 16 to 70. Characteristics of the effective number of rats are summarized in Table 1.

Thumbnail

Gross and microscopic findings

From weeks 52 to 70, diffuse inflammatory infiltration in the gastric mucosa was found (Figure 1). Intestinal metaplasia was not found in the stomach of any rat in this experiment. At 64 weeks, one extensive lesion in the anterior wall of the stomach was found and histopathological analysis revealed to be a sarcoma. At 70 weeks, one adrenal tumor was found. In the forestomach, only one squamous carcinoma was found at week 22. At week 52 and 70, two well differentiated adenocarcinomas were observed in the glandular stomach. The tumors invaded the muscularis proper superficially (T2a) (Figure 2). The incidence of gastric adenocarcinoma was 4.5% (0.5 to 15). Histopathological findings for the stomach are summarized in Table 2.

Thumbnail

DISCUSSION

It was not able to confirm that MNU in drinking water can induce a high incidence of carcinomas in the Wistar rat glandular stomach. Only two adenocarcinomas developed in the animals receiving 200 ppm MNU. Earlier studies indicate that cancer of the glandular stomach can be produced in F344 rats and C3H mice by the administration of MNU, a widely used mutagen. When MNU is administered in a drinking water for 25-42 weeks, selectively induced glandular gastric carcinoma in F344 rats and C3H mouse^{5,8,11,13,14,18,21}.

Maekava, et al.⁸ examined the effects of MNU in the drinking water at 100 ppm in F344 rats. After 42 weeks, 18% of rats had adenocarcinoma gastric. Tatematsu, et al.¹⁸ induced glandular stomach cancer in C3H mice with MNU in the drinking water at 120 ppm for 30 weeks and had 40% of adenocarcinomas after 54 weeks.12 Hirota used MNU at 400 ppm in F344 rats for 25 weeks and had 100% of invasive adenocarcinomas.9 It was used here an intermediate concentration of MNU (200 ppm), because studies with lower concentrations showed a low incidence of adenocarcinoma, and higher concentrations have developed advanced tumors. The aim was to obtain early lesions in order to describe the time to get a level of depth tumor (T).

Lee⁷ examined the effects of concomitant administration of dimethylitaconate (DMI) and showed a significant increase of adenocarcinoma incidence, which was 95% compared with 12% MNU alone group (p<0.005). There is evidence that sodium chloride administration enhances the carcinogenic effects of MMNG on the rat stomach¹⁷. Maybe the addition of promoting substance with MNU could have increased our incidence of gastric adenocarcinoma.

Tatsuta, et al.¹⁹ examined the effects of a very-low-protein on the incidence of gastric cancer induced by MNNU in Wistar rats. Administration p.o. of a very-low-protein diet resulted in a significant increase in the incidence of gastric cancers. The mechanism of this diet is not known, but at least three possible ways may be considered.

One is immunomodulation by dietary protein. Dietary components may influence the development of neoplasias by effects on the host immune mechanisms. In particular, the level and type of fat have been shown to modulate immune responsiveness^12,20. Another possible mechanism is an effect on the secretions of various hormones, including growth hormone, thyroid hormones, gastrin, and somatostatin^6,15. Growth hormone and thyroid hormones are closely related to the growth of gastrointestinal mucosa. A third possibility, is an increase in activity of the sympathetic nervous system. Diet influences the activity of the sympathetic nervous system in experimental animals.

There is evidence of neural involvement in the control of cell proliferation²². In this study, it was used a normal protein diet.

It is well known that MNU is a multipotent carcinogen and the carcinogenic action of MNU varies with the strain of rat used and the route of administration⁸. In addition, it has been demonstrated that the organ specificity of N-nitroso compounds given orally is influenced not only by chemical structure of the test compounds or by the strain of rats used, but also by the dose level of the test compounds⁸. In this experiment, it was also shown that the organ specificity of MNU was influenced by the strain of rats and/or by the dose level. Further studies are needed to explain this phenomenon.

CONCLUSIONS

The experimental model of carcinogenesis in Wistar rats, using a 200 ppm of MNU dissolved in water, showed not practice viability in this study, due to the low rate of development of adenocarcinomas.

ACKNOWLEDGMENTS

Acknowledgements to the financial support granted by Fundo de Incentivo à Pesquisa e Eventos (FIPE) of Hospital de Clínicas de Porto Alegre, Porto alegre, RS, Brazil.

REFERENCES

1. Alexandrov VA. Uterine, vaginal and mammary tumours induced bynitrosoureas in pregnant rats. Nature 1969; 222:1064-1065.
2. Fox JG, Wishnok JS, Murphy JC, Tannenbaum SR, Correa P. MNNGinduced gastric carcinoma in ferrets infected with Helicobacter mustelae. Carcinogenesis 1993; 14:1957-1961.
3. Fujimura S, Kogure K, Oboshi S, Sugimura T. Production of tumorsin glandular stomach of hamsters by N-methyl-N´-nitro-N nitrosoguanidina. Cancer Res 1970; 30:1444-1448.
4. Graffi A, Hoffmann F, Schutt M. N-Methyl-N-nitrosourea as a strong topical carcinogen when painted on skin of rodents. Nature 1967; 214:611.
5. Hirota N, Aonuma T, Yamada S, Kawai T, Saito K, Yokoyama T. Selective induction of glandular stomach carcinoma in F344 rats by N-Methyl-N-nitrosurea. Jpn J Cancer Res 1987; 78 (7):634-638.
6. Kaptein EM, Fisler JS, Duda MJ, Nicoloff JT, Drenick EJ. Relationship between the changes in serum thyroid hormone levels and protein status during prolonged protein supplemented caloric deprivation. Clin Endocrinol 1985; 22:1-15.
7. Lee YS and Jang J. Potent promoting activity of dimethylitaconate on gastric carcinogenesis induced by N-methylnitrosourea. Cancer Lett 1994; 85(2):177-184.
8. Maekawa A, Matsuoka C, Onodera H, Tanigawa H, Furuta K, Ogiu T, Mitsumori K, Hayashi Y. Organ-specific carcinogenicity of N-Methyl-N-Nitrosurea in F344 and ACI/N rats. J Cancer Res Clin Oncol 1985; 109:178-182.
9. Magee PN, Barnes JM. Carcinogenic nitroso compounds. Adv Cancer Res 1967; 10:163-246.
10. Narisawa T, Wong CQ, Maronpot RR, Weisburger JH. Large bowel carcinogenesis in mice and rats by several intrarectal doses of methylnitrosourea and negative effect of nitrite plus methylurea. Cancer Res 1976; 36:505-510.
11. Ohgaki H, Ludeke BI, Meier I, Kleihues P, Lutz WK, Schlatter C. DNA methylation in the digestive tract of F344 rats during chronic exposure to N-methyl-N-nitrosurea. J Cancer Res Clin Oncol 1991; 117(1):13-18.
12. Olson LM, Clinton SK, Everitt JI, Johnston PV, Visek WJ.. Lymphocyte activation, cell-mediated cytotoxicity and their relationship to dietary fa-enhanced mammary tumorogenisis in C3H/OUJ mice. J. Nutr 1987; 117:955-963.
13. Shimizu M, Suzui M, Moriwaki H, Mori H, Yoshimi N.. No involvement of b-catenin gene mutation in gastric carcinomas induced by N-methyl-N-nitrosourea in male F344 rats. Cancer Letters 2003; 195:147-152.
14. Shimizu M, Yoshimi N, Yamada Y, Matsunaga K, Kawabata K, Hara A, Moriwaki H, Mori H.. Suppressive effects of clorogenic acid on N-methyl-N-nitrosurea-induced glandular stomach carcinogenesis in male F433 rats. J Toxicol Sci 1999; 24:433-439.
15. Sommervile BA, Harvey S. The effect of low levels of dietary protein and calcium o growth rate, growth hormone, and vitamin D metabolism in the chicken. Gen Comp Endocrinol 1988, 71:93-96.
16. Sugimura T and Sugimura S. Tumor production in glandular stomach of rats by N-methyl-N´-nitro-N-nitrosoguanidina. Nature 1967; 216:943-944.
17. Takahashi M, Kokubo T, Furukawa F, Kurokawa Y, Tatematsu M, Hayashi Y. Effect of high salt diet on rat gastric carcinogenesis induced by N-methyl-N-nitro-N-nitrosoguanidine. Carcinogenesis 1982; 3:1419-1422.
18. Tatematsu M, Yamamoto M, Iwata H, Fukami H, Yuasa H, Tezuka N, Masui T, Nakanishi H. Induction of glandular stomach cancers in C3H mice treated with N-methyl-N-nitrosourea in the drinking water. Jpn J Cancer Res 1993; 84:1258-1264.
19. Tatsuta M, Iishi H, Baba M, Uehara H, Nakaizumi A, Taniguchi H.. Enhanced induction of gastric carcinogenisis by N-Methyl-N´ nitro-N-nitrosoguanidine in Wistar rats fed a low-protein diet. Cancer Research 1991; 51:3493-3496.
20. Thomas IK and Erickon KL. Lipid modulation of mammary tumor cell cytolysis: direct influence of dietary fats on the effector component of cell-mediated cytotoxicity. J Natl Cancer Inst 1885; 74:675-680.
21. Yamachika T, Nakanishi H, Inada K, Tsukamoto T, Shimizu N,Kobayashi K, Fukushima S, Tatematsu M. N-Methyl-N-Nitrosurea concentration-dependent, rather than total intake-dependent, induction of adenocarcinoma in the glandular stomach of BALB/c mice. Jpn J Cacer Res 1998; (89):385-391.
22. Young JB, Kaufman LN, Saville ME, Landsberg L. Increased sympathetic nervous system activity in rats fed a low-protein diet. Am J Physiol 1985; 248:R627-R637.