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Inhibition by methionine of pancreatic carcinogenesis in hamsters after initiation with N-nitrosobis(2-oxopropyl) amine
Cancer Letters 152 (2000) 163±167
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Inhibition by methionine of pancreatic carcinogenesis in hamsters after initiation with N-nitrosobis(2-oxopropyl) amine Fumio Furukawa a,*, Akiyoshi Nishikawa a, In-Seon Lee b, Hwa-Young Son b, Hideaki Nakamura b, Makoto Miyauchi b, Michihito Takahashi b, Masao Hirose b b
a Division of Pathology, National Institute of Health Sciences, 1-18-1 Kamlyoga, Setagaya-ku, Tokyo 158-8501, Japan Department of Food Science and Technology, School of Science, Keimyung University, 1000 Shindang-Dong, Dalseo-ku, Taegu 700-200, South Korea
Received 17 November 1999; received in revised form 15 December 1999; accepted 16 December 1999
Abstract The modifying effects of dietary l-methionine in the post-initiation phase of pancreatic carcinogenesis were investigated in hamsters treated with N-nitrosobis(2-oxopropyl)amine (BOP). Groups consisting of 20 and 30 animals, respectively, were given BOP subcutaneously, once a week ®ve times at a dose of 10 mg/kg body wt. and then continuously fed diet supplemented with 2% (group 1) or 0% (group 2) methionine (weeks 5±32). After ®ve subcutaneous injections of saline, group 3 animals were similarly fed diet supplemented with 2% methionine for the same period. The incidence of pancreatic ductal adenocarcinomas was signi®cantly lower in group 1 (36.8%, P , 0:05) than in group 2 (71.4%). Multiplicity of adenocarcinomas was also signi®cantly lowered (0.52 and 1.28/hamster, P , 0:05). Similarly, total numbers of combined adenocarcinomas and dysplastic lesions were signi®cantly decreased in group 1 (2.05, P , 0:05) as compared with group 2 (3.67). Methionine enhanced atrophic change of pancreatic acinar cells in hamsters given BOP, indicating that the inhibitory effects on the post-initiation stage of BOP-induced pancreatic carcinogenesis in hamsters could be generally linked to suppression of growth. q 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Methionine; Pancreatic carcinogenesis; Hamster; N-Nitrosobis(2-oxopropyl)amine
1. Introduction It is well known that diet is important not only as a source of environmental risk factors for cancer development but also for chemoprevention [1]. In rodents, prolonged intake of a methionine-de®cient diet, even without exposure to any known carcinogen, has been shown to result in the development of liver tumors [2], whereas diets supplemented with choline and methionine prevent against or at least diminish the carcinogenic effects of some chemicals [3] and prolonged the * Corresponding author.
survival of AKR mice, which spontaneously develop leukemia [4]. It has been proposed that methionine plays a critical role in cell growth because it is a precursor of S-adenosylmethionine, which is the primary methyl-group donor in a great variety of methylation reactions [5] and the precursor of the aminopropyl moieties of spermidine and spermine [6]. Effects appear to be largely due to inhibition of DNA synthesis although increased phenotypic reversion of preneoplastic liver lesions has been reported [7±10]. N-Nitrosobis(2-oxopropyl)amine (BOP) induces lung, pancreatic, liver and kidney tumors in hamsters
0304-3835/00/$ - see front matter q 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0304-383 5(99)00448-6
164
F. Furukawa et al. / Cancer Letters 152 (2000) 163±167
[11,12]. The hamster model is considered to have particular advantages for assessing modi®cation effects of chemicals on pancreatic carcinogenicity because of the histological and biological similarities of the induced lesions to those observed in man [11,12]. Since little is known about the effects of methionine on experimental pancreatic carcinogenesis, in the present study we determined the outcome of dietary supplementation in Syrian hamsters initiated with BOP.
(group 1) or 0% (group 2) methionine, in the postinitiation stage (weeks 5±32). After ®ve subcutaneous injections of saline, group 3 consisting of 20 animals was similarly fed diet supplemented with 2% methionine during weeks 5±32. The hamsters were observed daily and weighed once every 4 weeks. At the end of week 32, all surviving animals were killed and autopsied for histopathological examination. Moribund or dead animals were also completely autopsied. 2.3. Histological examination
2. Materials and methods 2.1. Animals and chemicals A total of 70 female Syrian hamsters (Japan SLC, Inc., Shizuoka, Japan), 6-weeks-old and weighing about 80 g at the commencement, were used in this experiment. The animals were housed, ®ve per polycarbonate cage, in an air-conditioned room at 23^28C; 60 ^ 5% humidity under a daily cycle of alternating 12-h period of light and darkness. Oriental MF powder diet (Oriental Yeast Co., Ltd., Tokyo, Japan) and tap water were available ad libitum. BOP was obtained from Nacalai Tesque (Kyoto, Japan) and l-methionine (food additive grade) from Kyowa Hakko Kogyo Co. Ltd. (Tokyo) 2.2. Experimental protocol As shown in Fig. 1, groups 1 and 2, consisting of 20 and 30 hamsters, were given BOP subcutaneously once a week for ®ve times at a dose of 10 mg/kg body wt. After this initiation treatment, the animals were continuously fed diet supplemented with 2%
Fig. 1. Experimental design
At autopsy, the pancreas was carefully examined macroscopically, and then ®xed in 10% phosphatebuffered formalin for conventional embedding, and microscopic observation of sections stained with hematoxylin and eosin. Proliferative lesions were diagnosed histopathologically and counted in representative sections. The results were statistically analyzed by analysis of variance (ANOVA) and the Fisher's exact probability test. 3. Results Body weights of animals fed the control and methionine diets were comparable in all the groups. Final body weight was signi®cantly lower in group 1 (P , 0:01) than in group 2. Histologic typing of pancreatic tumors was made as described previously [11,12]. Dysplastic lesions with moderate epithelial atypia were considered to be preneoplastic. Adenocarcinomas were mostly well or moderately differentiated with frequent invasion into the surrounding tissues. Table 1 summarizes quantitative data for pancreatic proliferative lesions. The incidence of pancreatic adenocarcinomas was signi®cantly lower in group 1 (36.8%, P , 0:05) than in group 2 (71.4%). The incidence of dysplastic lesions were also lower but this was not statistically signi®cant. Average numbers of adenocarcinomas per hamster were signi®cantly lower in group 1 (0.52 ^ 0.77, P , 0:05) than in group 2 (1.28 ^ 1.21). Total numbers of combined adenocarcinomas and dysplastic lesions were also signi®cantly decreased in group 1 (2.05 ^ 1.64, P , 0:05) as compared with the group 2 value (3.67 ^ 2.14). As shown in Table 2, the grade of fatty change of
F. Furukawa et al. / Cancer Letters 152 (2000) 163±167
165
Table 1 Incidences and multiplicity of pancreatic proliferative lesions Group
1 (BOP/methionine) 2 (BOP) 3 (Methionine)
Effective no. of animals
19 28 20
No. of animals with (%)
No. of lesions/animal (mean ^ SD)
Adc a
Dys b
Total
Adc
Dys
Total
7 (36.8) c 20 (71.4) 0
13 (68.4) 25 (89.3) 0
16 (84.2) 27 (89.2) 0
0.52 ^ 0.77 c 1.28 ^ 1.21 0
1.52 ^ 1.57 2.39 ^ 1.47 0
2.05 ^ 1.64 c 3.67 ^ 2.14 0
a
Adc, adenocarcinoma. Dys, dysplastic lesion. c P , 0:05 compared with the BOP alone group. b
pancreatic exocrine tissues was signi®cantly (P , 0:01) greater in group 1 (Fig. 2) than in group 2 (Fig. 3). 4. Discussion The present experiment clearly demonstrated for the ®rst time to our knowledge, that dietary methionine supplementation can inhibit the development of hamster pancreas cancers after BOP initiation, although some possible in¯uence of calorie restriction or toxicity cannot be completely excluded because of lack of food intake data. Methionine is utilized for protein synthesis and is converted to S-adenosylmethionine, a primary methyl donor in the body [13] and a precursor in polyamine synthesis [14]. Some carcinogens may interfere with enzymatic methylation of DNA, and thus bring about oncogene activation [15]. Such methylation is an important component of control and may serve as a silencing mechanism for gene function [15]. In
contrast, demethylation may be a necessary, but not always suf®cient, condition for enhanced transcription [15]. While DNA hypomethylation has been observed in many cancer cells and tumors [15], it is possible that oncogenic transformation may be prevented or even reversed by dietary excess methionine [15]. Several reports have provided evidence that methionine and/or methionine-related metabolites such as choline and folates may reduce the incidence of colorectal cancer and that `feeding of large amounts of choline and/or methionine may change oncogenic cells back to normal' [16], in good agreement with our present results. A high methionine diet, through its effects on the formation of S-adenosylmethionine, is effective in inhibiting hepatocarcinogenesis in the rat [7,8,17]. An increase in cell loss by apoptosis in preneoplastic hepatocytes is also reported with Sadenosylmethionine [9], a naturally occurring, nontoxic and non-mutagenic compound, which enters liver cell [18]. The biochemical and molecular mechanisms underlying the chemopreventive effects of S-adenosylmethionine are not yet completely
Table 2 Effects of l-methionine on body weight and fatty changes of the exocrine pancreas Group
1 (BOP/methionine) 2 (BOP) 3 (Methionine) a
Effective no. of animals
19 28 20
1, Slight. 11, Marked. c P , 0:01 compared with the BOP alone group. d P , 0:01 compared with the methionine alone group. b
Final body weight (g)
172 ^ 16 c 192 ^ 14 175 ^ 14
No. of animals with fatty changes 1a
11 b
Total
3 (16) 10 (36) 19 (95)
16 (84) d 10 (36) 1 (5)
19 (100) 20 (72) 20 (100)
166
F. Furukawa et al. / Cancer Letters 152 (2000) 163±167
Fig. 2. Photomicrograph illustrating markedly fatty changes of the exocrine pancreas in a group 1 hamster. (£ 40)
understood but at the phenomenological level they appear related to its ability to induce apoptosis and remodeling in hepatic nodules [19]. At the molecular level, S-adenosylmethionine is an agent, which can methylate some growth-related genes and inhibit their expression [20,21], including examples related to apoptosis and remodeling [19]. Therefore, it is likely that methionine and related precursors (e.g. betaine and choline) may also mediate apoptosis by increasing S-adenosylmethionine synthesis. Such mechanisms could be involved in the inhibition of BOP-induced pancreatic carcinogenesis in hamsters by methionine. In a previous study in which Chinese hamsters were given daily intraperitoneal injections of l-methionine, pancreatic acinar cells exhibited loss of basophilia and induction of necrotic changes and subsequently the acinar architecture was distorted [22]. Atrophy of
acini was noted with replacement by ®brous and adipose tissues, goblet cells occurred in the ducts, and islet b-cells often showed decreased granulation with some necrosis [22]. Similarly, the present study revealed atrophy of pancreas acini and replacement to some extent by fatty tissues. Atrophic changes found in the exocrine pancreas were more severe in the BOP followed by methionine-treated group than in the BOP alone group in the present experiment, indicating that the nitrosamine may initiate acinar cell damage that is subsequently enhanced by methionine. Previously, we showed that soybean trypsin inhibitor protected against pancreatic acinar cell damage and inhibited pancreatic carcinogenesis [23,24]. However, since pancreatic ducts do not appear to be directly in¯uenced by methionine, there may be no direct association with the observed inhibition of pancreatic proliferative lesions and acinar cell change. In conclusion, our results indicate that dietary methionine modulates pancreatic carcinogenesis in hamsters receiving BOP when given in the post-initiation phase. Acknowledgements This work was supported by a Grant-in-Aid for Cancer Research from the Ministry of Health and Welfare of Japan, and in part by a grant (HS-5226) for Comprehensive Research Project on Health Sciences Focusing on Drug Innovation from the Japan Health Sciences Foundation. References
Fig. 3. Photomicrograph illustrating slightly fatty changes of the exocrine pancreas in a group 2 hamster (£ 40).
[1] Z. Madar, I. Zusman, The role of dietary factors in preventive of chemically-induced cancers (Review), Int. J. Oncol. 11 (1997) 1141±-114. [2] A. Ghoshal, E. Farber, The induction of liver cancer by a dietary de®ciency of choline and methionine without added carcinogens, Carcinogenesis 5 (1984) 1367±-1370. [3] R.M. Hoffman, Altered methionine metabolism, DNA methylation and oncogene expression in carcinogenesis. A review and synthesis, Biochim. Biophys. Acta 738 (1984) 49±-87. [4] E. Wainfan, M. Dizik, M. Kilkenny, J.P. O'Callaghan, Prolonged survival of female AKR mice fed diets supplemented with choline and methionine, Carcinogenesis 11 (1990) 361±363. [5] A.J.L. Cooper, Biochemistry of sulfur-containing amino acids, Annu. Rev. Biochem. 52 (1989) 187±-222.
F. Furukawa et al. / Cancer Letters 152 (2000) 163±167 [6] A.E. Pegg, Recent advances in the biochemistry of polyamines in eukaryotes, Biochem. J. 234 (1986) 249±-262. [7] R. Garcea, R. Pascale, L. Daino, S. Franssetto, P. Cozzolino, M.E. Ruggiu, M.G. Vannini, L. Gaspa, F. Feo, Variation of ornithine decarboxylase activity and S-adenosyl-l-methionine and 5 0 -methyl-thioadenosine contents during the development of diethylnitrosamine-induced liver hyperplastic nodules and hepatocellular carcinomas, Carcinogenesis 8 (1987) 653±658. [8] R.M. Pascale, V. Marras, M.M. Simile, L. Daino, G. Pinna, S. Bennati, M. Carta, M.A. Seddaiu, G. Massarelli, F. Feo, Chemoprevention of rat liver carcinogenesis by S-adenosyll-methionine: a long-term study, Cancer Res. 52 (1992) 4979±-4986. [9] R. Gracea, L. Daino, R.M. Pascale, M.M. Simile, M. Puddu, S. Frassetto, P. Cozzolino, M.A. Seddaiu, L. Gaspa, F. Feo, Inhibition of promotion and persistent nodule growth by S-adenosyl-l-methionine in rat liver carcinogenesis: role of remodeling and apoptosis, Cancer Res. 49 (1989) 1850± 1856. [10] R. Pascale, M.M. Simile, M.E. Ruggiu, M.A. Seddaiu, G. Satta, M.J. Sequenza, L. Daino, M.G. Vannini, P. Lai, F. Feo, Reversal by 5-azacytidine of the S-adenosyl-l-methionine-induced inhibition of the development of putative preneoplastic foci in rat liver carcinogenesis, Cancer Lett. 56 (1991) 259±265. [11] F. Furukawa, A. Nishikawa, T. Imazawa, K. Kasahara, M. Takahashi, Enhancing effects of quinacrine on development of hepatopancreatic lesions in N-nitrosobis (2-oxopropyl) amine-initiated hamsters, Jpn. J. Cancer Res. 89 (1998) 131±136. [12] F. Furukawa, A. Nishikawa, T. Enami, M. Mitsui, T. Imazawa, Z. Tanakamaru, H.-C. Kim, K. Kasahara, M. Takahashi, Promotional effects of 4-(methylnitrosamino)-1-(3pyridyl)-1-butanol (NNAL) on N-nitrosobis (2-oxopropyl) amine-initiated carcinogenesis in hamsters, Food Chem. Toxicol. 35 (1997) 387±392. [13] A.J.L. Cooper, Biochemistry of sulfur-containing amino acids, Annu. Rev. Biochem. 52 (1983) 187±222. [14] J. JaÈnne, H. PoÈsoÈ, A. Raina, Polyamines in rapid growth and cancer, Biochim. Biophys. Acta 473 (1978) 241±293. [15] J.V.D. Westhuyzen, Methionine metabolism and cancer, Nutr. Cancer 7 (1985) 179±183.
167
[16] R.M. Hoffman, Altered methionine metabolism, DNA methylation and oncogene expression in carcinogenesis. A review and synthesis, Biochim. Biophys. Acta 738 (1984) 49±87. [17] F. Feo, R. Garcea, L. Daino, R. Pascale, L. Pirisi, S. Frassetto, M.E. Ruggiu, Early stimulation of polyamine biosynthesis during promotion by phenobarbital of diethylnitrosamineinduced rat liver carcinogenesis. The effects of variations of the S-adenosyl-l-methionine cellular pool, Carcinogenesis 6 (1985) 1713±1720. [18] R.M. Pascale, M.M. Simile, F. Feo, Genomic abnormalities in hepatocarcinogenesis. Implications for a chemopreventive strategy, Anticancer Res. 13 (1993) 1341±1356. [19] R.M. Pascale, M.M. Simile, M.R. De. Miglio, A. Nufris, L. Daino, M.A. Seddaiu, P.M. Rao, S. Rajalakshmi, D.S.R. Sarma, F. Feo, Chemoprevention by S-adenosyl-l-methionine of rat liver carcinogenesis initiated by 1,2-dimethylhydrazine and promoted by orotic acid , Carcinogenesis 16 (1995) 427± 430. [20] R. Garcea, L. Daino, R.M. Pascale, M.M. Simile, M. Puddu, M.E. Ruggiu, M.A. Seddaiu, G. Satta, M.J. Sequenza, F. Feo, Protooncogene methylation and expression in regenerating and preneoplastic liver nodules induced in the rat by diethylnitrosamine: effect of variations of S-adenosylmethionine: Sadenosylhomocysteine ratio, Carcinogenesis 10 (1989) 1183± 1192. [21] M.M. Simile, R.M. Pascale, M.R. De Miglio, A. Nufris, L. Daino, M.A. Seddaiu, L. Gaspa, F. Feo, Correlation between S-adenosyl-l-methionine content and production of c-myc, cHa-ras, and c-Ki-ras mRNA transcripts in the early stages of rat liver carcinogenesis, Cancer Lett. 79 (1994) 9±16. [22] L. Boquist, The effects of excess methionine on the pancreas, Lab. Invest. 21 (1969) 96±104. [23] F. Furukawa, K. Imaida, H. Okamiya, K. Shinoda, M. Sato, T. Imazawa, Y. Hayashi, M. Takahashi, Inhibitory effects of soybean trypsin inhibitor on induction of pancreatic neoplastic lesions in hamsters by N-nitrosobis (2-oxopropyl) amine, Carcinogenesis 12 (1991) 2123±2125. [24] F. Furukawa, A. Nishikawa, K. Imaida, M. Mitsui, T. Enami, Y. Hayashi, M. Takahashi, Inhibitory effects of crude soybean trypsin inhibitor on pancreatic ductal carcinogenesis in hamsters after initiation with N-nitrosobis (2-oxopropyl) amine, Carcinogenesis 13 (1992) 2133±2135.
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Inhibition by methionine of pancreatic carcinogenesis in hamsters after initiation with N-nitrosobis(2-oxopropyl) amine Fumio Furukawa a,*, Akiyoshi Nishikawa a, In-Seon Lee b, Hwa-Young Son b, Hideaki Nakamura b, Makoto Miyauchi b, Michihito Takahashi b, Masao Hirose b b
a Division of Pathology, National Institute of Health Sciences, 1-18-1 Kamlyoga, Setagaya-ku, Tokyo 158-8501, Japan Department of Food Science and Technology, School of Science, Keimyung University, 1000 Shindang-Dong, Dalseo-ku, Taegu 700-200, South Korea
Received 17 November 1999; received in revised form 15 December 1999; accepted 16 December 1999
Abstract The modifying effects of dietary l-methionine in the post-initiation phase of pancreatic carcinogenesis were investigated in hamsters treated with N-nitrosobis(2-oxopropyl)amine (BOP). Groups consisting of 20 and 30 animals, respectively, were given BOP subcutaneously, once a week ®ve times at a dose of 10 mg/kg body wt. and then continuously fed diet supplemented with 2% (group 1) or 0% (group 2) methionine (weeks 5±32). After ®ve subcutaneous injections of saline, group 3 animals were similarly fed diet supplemented with 2% methionine for the same period. The incidence of pancreatic ductal adenocarcinomas was signi®cantly lower in group 1 (36.8%, P , 0:05) than in group 2 (71.4%). Multiplicity of adenocarcinomas was also signi®cantly lowered (0.52 and 1.28/hamster, P , 0:05). Similarly, total numbers of combined adenocarcinomas and dysplastic lesions were signi®cantly decreased in group 1 (2.05, P , 0:05) as compared with group 2 (3.67). Methionine enhanced atrophic change of pancreatic acinar cells in hamsters given BOP, indicating that the inhibitory effects on the post-initiation stage of BOP-induced pancreatic carcinogenesis in hamsters could be generally linked to suppression of growth. q 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Methionine; Pancreatic carcinogenesis; Hamster; N-Nitrosobis(2-oxopropyl)amine
1. Introduction It is well known that diet is important not only as a source of environmental risk factors for cancer development but also for chemoprevention [1]. In rodents, prolonged intake of a methionine-de®cient diet, even without exposure to any known carcinogen, has been shown to result in the development of liver tumors [2], whereas diets supplemented with choline and methionine prevent against or at least diminish the carcinogenic effects of some chemicals [3] and prolonged the * Corresponding author.
survival of AKR mice, which spontaneously develop leukemia [4]. It has been proposed that methionine plays a critical role in cell growth because it is a precursor of S-adenosylmethionine, which is the primary methyl-group donor in a great variety of methylation reactions [5] and the precursor of the aminopropyl moieties of spermidine and spermine [6]. Effects appear to be largely due to inhibition of DNA synthesis although increased phenotypic reversion of preneoplastic liver lesions has been reported [7±10]. N-Nitrosobis(2-oxopropyl)amine (BOP) induces lung, pancreatic, liver and kidney tumors in hamsters
0304-3835/00/$ - see front matter q 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0304-383 5(99)00448-6
164
F. Furukawa et al. / Cancer Letters 152 (2000) 163±167
[11,12]. The hamster model is considered to have particular advantages for assessing modi®cation effects of chemicals on pancreatic carcinogenicity because of the histological and biological similarities of the induced lesions to those observed in man [11,12]. Since little is known about the effects of methionine on experimental pancreatic carcinogenesis, in the present study we determined the outcome of dietary supplementation in Syrian hamsters initiated with BOP.
(group 1) or 0% (group 2) methionine, in the postinitiation stage (weeks 5±32). After ®ve subcutaneous injections of saline, group 3 consisting of 20 animals was similarly fed diet supplemented with 2% methionine during weeks 5±32. The hamsters were observed daily and weighed once every 4 weeks. At the end of week 32, all surviving animals were killed and autopsied for histopathological examination. Moribund or dead animals were also completely autopsied. 2.3. Histological examination
2. Materials and methods 2.1. Animals and chemicals A total of 70 female Syrian hamsters (Japan SLC, Inc., Shizuoka, Japan), 6-weeks-old and weighing about 80 g at the commencement, were used in this experiment. The animals were housed, ®ve per polycarbonate cage, in an air-conditioned room at 23^28C; 60 ^ 5% humidity under a daily cycle of alternating 12-h period of light and darkness. Oriental MF powder diet (Oriental Yeast Co., Ltd., Tokyo, Japan) and tap water were available ad libitum. BOP was obtained from Nacalai Tesque (Kyoto, Japan) and l-methionine (food additive grade) from Kyowa Hakko Kogyo Co. Ltd. (Tokyo) 2.2. Experimental protocol As shown in Fig. 1, groups 1 and 2, consisting of 20 and 30 hamsters, were given BOP subcutaneously once a week for ®ve times at a dose of 10 mg/kg body wt. After this initiation treatment, the animals were continuously fed diet supplemented with 2%
Fig. 1. Experimental design
At autopsy, the pancreas was carefully examined macroscopically, and then ®xed in 10% phosphatebuffered formalin for conventional embedding, and microscopic observation of sections stained with hematoxylin and eosin. Proliferative lesions were diagnosed histopathologically and counted in representative sections. The results were statistically analyzed by analysis of variance (ANOVA) and the Fisher's exact probability test. 3. Results Body weights of animals fed the control and methionine diets were comparable in all the groups. Final body weight was signi®cantly lower in group 1 (P , 0:01) than in group 2. Histologic typing of pancreatic tumors was made as described previously [11,12]. Dysplastic lesions with moderate epithelial atypia were considered to be preneoplastic. Adenocarcinomas were mostly well or moderately differentiated with frequent invasion into the surrounding tissues. Table 1 summarizes quantitative data for pancreatic proliferative lesions. The incidence of pancreatic adenocarcinomas was signi®cantly lower in group 1 (36.8%, P , 0:05) than in group 2 (71.4%). The incidence of dysplastic lesions were also lower but this was not statistically signi®cant. Average numbers of adenocarcinomas per hamster were signi®cantly lower in group 1 (0.52 ^ 0.77, P , 0:05) than in group 2 (1.28 ^ 1.21). Total numbers of combined adenocarcinomas and dysplastic lesions were also signi®cantly decreased in group 1 (2.05 ^ 1.64, P , 0:05) as compared with the group 2 value (3.67 ^ 2.14). As shown in Table 2, the grade of fatty change of
F. Furukawa et al. / Cancer Letters 152 (2000) 163±167
165
Table 1 Incidences and multiplicity of pancreatic proliferative lesions Group
1 (BOP/methionine) 2 (BOP) 3 (Methionine)
Effective no. of animals
19 28 20
No. of animals with (%)
No. of lesions/animal (mean ^ SD)
Adc a
Dys b
Total
Adc
Dys
Total
7 (36.8) c 20 (71.4) 0
13 (68.4) 25 (89.3) 0
16 (84.2) 27 (89.2) 0
0.52 ^ 0.77 c 1.28 ^ 1.21 0
1.52 ^ 1.57 2.39 ^ 1.47 0
2.05 ^ 1.64 c 3.67 ^ 2.14 0
a
Adc, adenocarcinoma. Dys, dysplastic lesion. c P , 0:05 compared with the BOP alone group. b
pancreatic exocrine tissues was signi®cantly (P , 0:01) greater in group 1 (Fig. 2) than in group 2 (Fig. 3). 4. Discussion The present experiment clearly demonstrated for the ®rst time to our knowledge, that dietary methionine supplementation can inhibit the development of hamster pancreas cancers after BOP initiation, although some possible in¯uence of calorie restriction or toxicity cannot be completely excluded because of lack of food intake data. Methionine is utilized for protein synthesis and is converted to S-adenosylmethionine, a primary methyl donor in the body [13] and a precursor in polyamine synthesis [14]. Some carcinogens may interfere with enzymatic methylation of DNA, and thus bring about oncogene activation [15]. Such methylation is an important component of control and may serve as a silencing mechanism for gene function [15]. In
contrast, demethylation may be a necessary, but not always suf®cient, condition for enhanced transcription [15]. While DNA hypomethylation has been observed in many cancer cells and tumors [15], it is possible that oncogenic transformation may be prevented or even reversed by dietary excess methionine [15]. Several reports have provided evidence that methionine and/or methionine-related metabolites such as choline and folates may reduce the incidence of colorectal cancer and that `feeding of large amounts of choline and/or methionine may change oncogenic cells back to normal' [16], in good agreement with our present results. A high methionine diet, through its effects on the formation of S-adenosylmethionine, is effective in inhibiting hepatocarcinogenesis in the rat [7,8,17]. An increase in cell loss by apoptosis in preneoplastic hepatocytes is also reported with Sadenosylmethionine [9], a naturally occurring, nontoxic and non-mutagenic compound, which enters liver cell [18]. The biochemical and molecular mechanisms underlying the chemopreventive effects of S-adenosylmethionine are not yet completely
Table 2 Effects of l-methionine on body weight and fatty changes of the exocrine pancreas Group
1 (BOP/methionine) 2 (BOP) 3 (Methionine) a
Effective no. of animals
19 28 20
1, Slight. 11, Marked. c P , 0:01 compared with the BOP alone group. d P , 0:01 compared with the methionine alone group. b
Final body weight (g)
172 ^ 16 c 192 ^ 14 175 ^ 14
No. of animals with fatty changes 1a
11 b
Total
3 (16) 10 (36) 19 (95)
16 (84) d 10 (36) 1 (5)
19 (100) 20 (72) 20 (100)
166
F. Furukawa et al. / Cancer Letters 152 (2000) 163±167
Fig. 2. Photomicrograph illustrating markedly fatty changes of the exocrine pancreas in a group 1 hamster. (£ 40)
understood but at the phenomenological level they appear related to its ability to induce apoptosis and remodeling in hepatic nodules [19]. At the molecular level, S-adenosylmethionine is an agent, which can methylate some growth-related genes and inhibit their expression [20,21], including examples related to apoptosis and remodeling [19]. Therefore, it is likely that methionine and related precursors (e.g. betaine and choline) may also mediate apoptosis by increasing S-adenosylmethionine synthesis. Such mechanisms could be involved in the inhibition of BOP-induced pancreatic carcinogenesis in hamsters by methionine. In a previous study in which Chinese hamsters were given daily intraperitoneal injections of l-methionine, pancreatic acinar cells exhibited loss of basophilia and induction of necrotic changes and subsequently the acinar architecture was distorted [22]. Atrophy of
acini was noted with replacement by ®brous and adipose tissues, goblet cells occurred in the ducts, and islet b-cells often showed decreased granulation with some necrosis [22]. Similarly, the present study revealed atrophy of pancreas acini and replacement to some extent by fatty tissues. Atrophic changes found in the exocrine pancreas were more severe in the BOP followed by methionine-treated group than in the BOP alone group in the present experiment, indicating that the nitrosamine may initiate acinar cell damage that is subsequently enhanced by methionine. Previously, we showed that soybean trypsin inhibitor protected against pancreatic acinar cell damage and inhibited pancreatic carcinogenesis [23,24]. However, since pancreatic ducts do not appear to be directly in¯uenced by methionine, there may be no direct association with the observed inhibition of pancreatic proliferative lesions and acinar cell change. In conclusion, our results indicate that dietary methionine modulates pancreatic carcinogenesis in hamsters receiving BOP when given in the post-initiation phase. Acknowledgements This work was supported by a Grant-in-Aid for Cancer Research from the Ministry of Health and Welfare of Japan, and in part by a grant (HS-5226) for Comprehensive Research Project on Health Sciences Focusing on Drug Innovation from the Japan Health Sciences Foundation. References
Fig. 3. Photomicrograph illustrating slightly fatty changes of the exocrine pancreas in a group 2 hamster (£ 40).
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