Chapter 11
Citrus Limonoids Obacunone and Limonin Inhibit the Development of a Precursor Lesion, Aberrant Crypt Foci, for Colon Cancer in Rats 1
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Takuji Tanaka , Hiroyuki Kohno , Kunihiro Kawabata , Shiro Honjo , Masaki Miyake , and Keiji Wada 1
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1st Department of Pathology, Kanazawa Medical University, Uchinada, Ishikawa 920-0293, Japan lst Department of Pathology, Gifu University School of Medicine, 40 Tsukasa-machi, Gifu 500-8705, Japan Faculty of Pharmaceutical Sciences, Hokkaido Iryo University, Ishikari-Tobetsu, Hokkaido 061-0293, Japan
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The typical bitter limonoids obacunone and limonin occurred widely in citrus have been reported to shorten the sleeping time in mice and limonin has been shown to inhibit forestomach and buccal pouch carcinogenesis in rodents. Their ability to prevent azoxymethane (AOM)-induced aberrant crypt foci (ACF) and to induce detoxification (phase II) enzymes, such as glutathione S-transferase (GST) and quinone reductase (QR) was investigated using male F344 rats. Obacunone or limonin was administered continuously in the diet starting one week prior to the first of two weekly 20 mg/kg s.c. injections of AOM and until the rats were killed four weeks later ("initiation" feeding). In addition, rats were fed the diet containing each chemical for four weeks, starting two weeks after the second AOM exposure ("postinitiation" feeding). At a concentration of 0.02% or 0.05% of obacunone and limonin diets caused significant reduction (55-65% by "initiation" feeding and 28-42% by "postinitiation" feeding) in the yield of ACF. The ability of both compounds when administered during "initiation" or "postinitiation" stage to reduce the proliferating cell nuclear antigen (PCNA)-labeling index in the crypts and to increase GST and QR activities in the liver and colonic mucosa correlated well with the prevention of ACF. These results indicate that the citrus limonoids obacunone and limonin are possible chemopreventive agents against colon carcinogenesis. © 2000
American Chemical Society
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146 Colon cancer is the third most malignant neoplasm in the world (7) and the second leading cause of cancer deaths in the USA. In Japan, colon cancer incidence has been increasing, being the third leading cause of cancer deaths. It is well known that dietary factors can modulate the development of certain types of human cancer, including colon cancer (2). Also, epidemiological data suggest that ingestion of some constituents from vegetables and fruits may contribute to reduction of cancer incidence in humans (3). Since an inverse relationship between the intake of fruits/vegetables and human colon cancer has been suggested, primary prevention, including chemoprevention utilizing the active compounds in edible plants is important for reducing this malignancy. Among possible chemopreventers for cancer development, certain inducers of phase II detoxifying enzymes including glutathione S-transferase (GST) and quinone reductase (QR) are considered to be promising chemopreventive agents against cancer (4). Our search for effective cancer chemopreventive compounds in edible plants revealed that several compounds able to induce phase II drugmetabolizing enzymes exert inhibitory effects on chemically induced colon carcinogenesis (5-7). Limonoids are a group of triterpene derivatives present in the Rutaceae and Meliaceaefamilies. Limonoids including obacunone (Figure 1) and limonin (Figure 1) are also found in citrus seeds (8), commercial citrus juices (9), and Philodendron amurense (Kihada)(70). For example, commercial orange juices contain an average of 320 ppm of total limonoid glucosides: the major glucoside in citrus juices is limonin 17-0-P-D-glucopyranoside, being over 50% of the total limonoid glucosides in the juices, followed by other limonoid glucosides including obacunone (77). Limonoids including obacunone and limonin are considered to be responsible for delayedbitterness in citrus juices and processed products. Among 38 limonoid aglycones, 23 neutral and 15 acidic, reported to occur in citrus and its hybrids (77), obacunone and limonin could enhance glutathione S-transferase (GST) activity in various organs of mice (72), including liver (72-75). Limonin was reported to enhance small intestinal GST activity (14). Obacunone and limonin have been shown to shorten the sleeping time induced by α-chloralose and urethane (75) or anesthetics (76). Limited data are available on the cancer preventive properties of limonin in rodent models. Limonin inhibits 7,12dimethylbenz[a]anthracene (DMBA)-induced hamster buccal pouch carcinogenesis (77). Also, limonin 17-P-D-glucopyranoside could inhibit the development of DMBA-inducedbuccal pouch tumors in hamsters (18). To our knowledge, there are no studies indicating that the dietary administration of limonin and obacunone has been tested in a colon cancer model or in any other carcinogenesis model other than forestomach, buccal pouch, lung, and skin (79). Early preneoplastic lesions, aberrant crypt foci (ACF), for colon carcinoma have consistendy been observed in the colon of rats exposed to colon carcinogens (20) and are present in the colonic mucosa of patients with colon cancer (27). Thus, ACF are putative precursor lesions from which adenoma and adenocarcinoma may develop (22). There is evidence that several inhibitors in laboratory animals suggesting that A C F induction can be used to evaluate novel agents for their potential chemopreventive properties against colon carcinoma (23).
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148 The present study was designed to evaluate the inhibitory activities of obacunone and limonin on azoxymethan (AOM)-induced ACF formation in the rat colon. The major goal of this study was to determine whether these natural agents are conceivably effective chemopreventive agents in pre-clinical efficacy studies and, eventually in human clinical trials.
Materials and Methods Animals, Diets, Carcinogen and Test Compounds. A O M was obtained from Sigma Chemicals (St. Louis, MO). Obacunone (99.9% pure) and limonin (99.9% pure) were isolated from the barks of Philodendron amurense (Kihada). Male F344 rats aged4 weeks were purchased from Japan SLC Inc. (Hamamatsu, Japan). The rats were held in quarantine for 1 week and had access to powdered basal diet, CE-2 (CLEA Japan, Inc. Tokyo, Japan). They were randomly distributed by wt into various dietary groups and were transferred to an animal holding room where they were housed in plastic cages, three rats/cage, under controlled conditions of a 12-h light/ 12-h dark cycle, 50% relative humidity and 21°C room temperature. Experimental diets were prepared by mixing obacunone (0.02% and 0.05% w/w) or limonin (0.02% and 0.05% w/w) with a basal diet, CE-2 and the test compounds in diets were quite stable. Experimental Procedure. At 5 weeks of age, groups of rats were fed the basal diet, CE-2 or experimental diets containing obacunone (0.02% or 0.05%) and limonin (0.02% or 0.05%), as shown in Figure 2. At 6 weeks of age, all animals except those given experimental diets alone and the untreated rats received A O M s.c. once weekly for 2 weeks at a dose of 20 mg/kg body wt per week. Animals intended for vehicle treatment were given an equal volume of normal saline. The animals given A O M and fed the experimental diet for 4 weeks after the start were sacrificed at week 4. The rats given A O M and basal diet were fed the experimental diets for 4 weeks, starting week 4 and sacrificed at week 8 when they were 13 weeks of age. The other groups consisted of rats given the experimental diets alone or untreated rats. At each sacrifice point (week 4 and 8), animals were killed by C 0 euthanasia and their colons were removed,flushedwith normal saline, opened from cecum to anus, and fixed flat between two pieces of filter paper in 10% buffered formalin. After staining with 0.2% methylene blue for 30 sec, ACF were observed through a light microscope, counted and recorded(6). 2
PCNΑ-labeling Index. Formalin-fixed colonic tissue in the distal 2 cm from anus was cut out from each colon andembeddedin paraffin. Serial cross-sections of 3 mm each were cut parallel to the mucosal surface and mounted onto gelatin-coated glass slides. The paraffin was removed with xylene and the tissue sections placed in 2N HC1 at 37°C for 30 min. Endogenous peroxidase was quenched by placing the tissue sections in 3% H 0 (Sigma Chemical Co.) for 30 min. Anti-PCNA 2
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^ Azoxymethane (20 mg/kg bw), sc I
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IJiUJjijijIjIl 0.05% obakunone or limonin in diet Figure 2. Experimental protocol.
150 antibody (Dako Co., Kyoto, Japan) was used with the avidin-biotin complex method. The immunohistochemical staining was performed according to the method described in our previous study (24). The number of PCNA-labeled and unlabeled cryptal cells in the cross-sections of the crypts in an A C F and of "normalappearing" crypts were recorded from the mouth to the base of crypts. The total number of cells in each crypt was calculated by adding the number of labeled and unlabeled cells. The PCNA-labeling index was determined by dividing the number of PCNA-positive cells by the total number of cells in a cryptx 100. GST and Q R Activities in Liver and C o l o n . At sacrifice, livers and colonic mucosa removed at each sacrifice time point were used for measuring GST andQR activities according to a standard procedure (6). Statistics. All results were expressed as the means ± SD and were analyzed by Student's Mest or Welch's ί-test. Differences were considered statistically significant at p 0.05% obacunone Group 8, AOM -> 0.02% limonin; Group 9, AOM -> 0.05% limonin; Group 10, 0.05% obacunone; Group 11, 0.05% limonin; and Group 12, untreated, a, p
