Antimutagenicity of Murdannia loriformis in the Salmonella mutation assay and its inhibitory effects on azoxymethane-induced DNA methylation and aberrant crypt focus formation in male F344 rats Yaowarate Intiyot, Takemi Kinouchi, Keiko Kataoka, Hideki Arimochi Tomomi Kuwahara, Usanee Vinitketkumnuen and Yoshinari Ohnishi Department of Bacteriology, The University of Tokushima School of Medicine, Tokushima, Japan; and †Department of Biochemistry, Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand Abstract:An80%ethanol extract of Murdannia loriformis, a Thai medicinal plant, was examined for antimutagenic activity and cancer chemopreventive activity. In the Salmonella mutation assay, the extract showed antimutagenicity against2-amino-3-methylimidazo [4,5-f]quinoline, 2-amino-3,4-dimethylimidazo[4,5-f]quinoline, 2-amino-3,8-dimethylimidazo [4,5-f]quinoxaline, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine, 2-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole, 3-amino-1-methyl-5H-pyrido[4,3-b]indole, 2-amino-6-methyldipyrido [1,2-a:3',2'-d] imidazole, 2-aminodipyrido[1,2-a:3',2'-d]imidazole, 2-aminoanthracene, 2-(2-furyl)-3-(5-nitro-2-furyl) acrylamide, N-methyl-N'-nitro-N-nitrosoguanidine and methylazoxymethanol acetate and reduced their mutagenicities to 31.4~67.9% at the dose of 10 mg/plate. However, it did not inhibit the mutagenicities of 2-amino-9H-pyrido[2,3-b]indole, 2-amino-3-methyl-9H-pyrido[2,3-b]indole, benzo[a]pyrene, N-ethyl-N'-nitro-N-nitrosoguanidine and 1-nitropyrene. The extract it self showed no mutagenicity. The chemopreventive activity of M. loriformis was examined using azoxymethane (AOM)-induced aberrant crypt focus (ACF) formation in the colon of F344 rats. The extract at doses of 0.1-1.0g/kg wt significantly inhibited ACF formation in the initiation stage (21-51%), although it was more effective at a lower dose. In the post-initiation stage, the extract also tended to inhibit ACF formation (12-27%) and significantly decreased the number of larger ACFs that have more than 3 aberrant crypts per focus. The extract inhibited the formation of O6-methylguanine and N7-methylguanine in the colonic mucosa and muscular layers but not or increased in the liver. These results indicate that M. loriformis extract has antimutagenic activity toward various known mutagens and that it inhibits AOM-induced ACF formation both in the initiation and post-initiation stages in the rat colon. J. Med. Invest. 49:25-34, 2002 Keywords:Murdannia loriformis;antimutagenicity;azoxymethane-induced aberrant crypt foci;O6-methylguanine INTRODUCTION Environmental factors, especially food, have been epidemiologically demonstrated to be closely associated with human colorectal cancer (1-3). While complete removal of causative agents of cancer may not always be possible, chemoprevention of cancer using antimutagens and anticarcinogens present in foods and in the environment has been suggested to be most effective method to prevent human cancer. Large numbers of antimutagens and anticarcinogens exist in natural products, especially in the plant kingdom (4). These antimutagens and anticarcinogens may inhibit one or more stages of carcinogenesis and prevent or delay cancer development (5). Geographic variation in colon cancer may also be attributable to differences in consumption of protective factors in the diet such as fiber, antioxidants and antiprolifelative agents. Various kinds of Thai medicinal plants have been shown to be antimutagenic and anticarcinogenic. Lemon grass, for example, has been shown to be antimutagenic against various mutagens (6) and to inhibit azoxymethane (AOM)-induced DNA adduct formation and aberrant crypt focus (ACF) formation in the rat colon (7). Roselle and bitter melon have been shown to inhibit colon carcinogen-induced ACF formation in the rat colon (8, 9). Tong Tak (10), Acanthus ebracteatus Vahl., Plumbago indica Linn. and Rhinacanthus nasuthus Kurz. (11) have also been found to be antimutagenic. Murdannia loriformis (Hassk.) Rolla Rao et Kammathy, a Thai medicinal plant, is a traditional drug in Thailand used for relief of bronchitis and cancer (12). Previous studies have shown that M. loriformis extract inhibits the mutagenicity of some kinds of mutagens in the Salmonella mutation assay and induces DT-diaphorase activity in a murine hepatoma cell line (Hepa 1c1c 7) (13). In addition, an active glycosphingolipid isolated from M. lo riformis exerted cytotoxicity against human colon carcinoma and a human breast cancer cell line (14). However, in vivo effects of M. loriformis extract have not been reported. ACFs are an early neoplastic lesions in the colonic mucosa and were first observed in colon carcinogen-treated mice and rats (15). They are morphologically distinguishable from normal crypts by their larger size and the more elliptical shape of the luminal opening, with thicker lining of epithelial cells. These lesions have also been observed in the human colon (16). AOM is a typical colon carcinogen, and AOM-induced ACFs in the rat colon have been used to identify chemopreventive agents for colon cancer (17). DNA adduct formation is also recognized as one of the common properties of most potent carcinogens and is the basis of current strategies in molecular epidemiology and biomonitoring (18). AOM-induced DNA adducts are N7-methylguanine (N7-meG) and O6-methyguanine(O6-meG). N7-MeG is quantitatively the major alkylation product (19), but the level and persistence of O6-meG in the target tissue is more closely correlated with the carcinogenicity (20). In the present study, we investigated the inhibitory effects of M. loriformis extract on AOM-induced DNA adduct formation and ACF formation in the rat colon. MATERIALS AND METHODS 1) Chemicals All heterocyclic amines (HCAs), including 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3,4-dimethylimidazo[4,5-b]quinoline (MeIQ), 3-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), 2-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1), 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2), 2-amino-6-methyldipyrido[1,2-a:3',2'-d]imidazole (Glu-P-1), 2-aminodipyrido[1,2-a:3',2'-d]imidazole (Glu-P-2), 2-amino-9H-pyrido[2,3-b] indole (Aα C), and 2-amino-3-methyl-9H-pyrido[2,3-b] indole (MeAα C) were kindly provided by Dr.K. Wakabayashi, National Cancer Center Research Institute. O6-MeG was also a generous gift from Drs. K. Ishizaki and M. Ikenaga, Kyoto University. Aflatoxin B1 (AFB1), benzo[a]pyrene (B[a]P), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG), methylazoxymethanol acetate (MAM acetate), AOM, N7-meG were purchased from Sigma Chemical Co., St. Louis, MO. 1-Nitropyr ene (1-NP) was obtained from Aldrich Chemical Co., Inc., Milwaukee, Wis. 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide (AF-2), 2-aminoanthracene (2-AA) and other chemicals, reagent grade or higher, were from Wako Pure Chemical Industries, Ltd., Osaka, Japan. 2) Extraction of Murdannia loriformis M. loriformis was obtained from the garden at the Research Institute for Health Science, Chiang Mai University, Chiang Mai, Thailand. The whole fresh plant was washed with tap water, cut into small pieces, freeze-dried, and ground to a fine powder. The dry powder (100 g) was extracted with 1 liter of 80%ethanol for 24h by stirring at room temperature. Then the extarct was filtered through a paper filter by suction, and the residue was extracted again with 80%ethanol. The filtrates were combined and evaporated to dryness in a rotary evaporator under reduced pressure at 50°C. The dried residue was used as M. loriformis extract that is abbreviated to ML in Tables and Figures. The extract was weighed, dissolved in 25% dimethylsulfoxide (DMSO), and kept at 4°Cuntil used. The yield of the extract was 0.12% from fresh plant and 1.2% from freeze-dried powder of the plant. For the Salmonella mutation assay, the extract was sterilized by filtration with a disposable filter unit (cellulose acetate membran e, pore size of 0.45 µm, Advantec) before being used. 3) Mutagenicity test The mutagenicity and antimutagenicity of M. loriformis extract were assayed using Salmonella typhimurium strains TA98 and TA100 according to the procedure of Maron and Ames (21) with the modification of preincubation (22) in the presence or absence of 9,000×g supernatant fraction (S9)prepared from the liver of rats treated with drug-metabolizing enzyme inducers, benzoflavone and phenobarbital. The number of spontaneous revertants was 24±6 in strain TA98 without S9, 37±10 in strain TA98 with S9, 112±26 in strain TA100 without S9, and 105±18 in strain TA100 with S9, and they were subtracted in all data except for Table1. 4) Animals Four-week-old male F344 rats (80-100 g) were purchased from SLC Japan (Hamamatsu, Japan). Animals were housed in plastic cages with sawdust bedding, maintained for3days and given food and water ad libitum. The room in which the rats were kept, in the Institute of Animal Experimentation, The University of Tokushima School of Medicine, was controlled at a temperature of 23±2°C, humid-ity of55±10%, and a13-h light/11-h dark cycle. 5) Analysis of aberrant crypt foci After quarantine for one week, rats were divided into eight groups for ACF determination for the initiation stage as shown in Fig.1 (a). Rats in groups1through 5 were subcutaneously injected with AOM (15 mg/kg body weight) once a week for two weeks. Groups 6, 7 and 8 were injected with saline as vehicle controls. Groups 2, 3 and 4 were given M. loriformis extract (0.1, 0.5 or 1.0 g/kg body weight, respectively) once a day by intragastric gavage. Groups1and6received 25% DMSO as control. Group 5 was given1.0 g/kg M. loriformis extract for only 1 week and on the next day they received the first AOM injection. All of the rats were sacrificed by cervical dislocation under anesthesia with lethal doses of diethyl ether. The protocol for the post-initiation stage is shown in Fig.1 (b). The rats were divided into six groups. Two weeks after the second AOM injection, rats began to be given M. loriformis extracts (1.0 or 0.1g/kg body weight) by intragastric gavage for12weeks. Animals were carefully observed daily and weighed weekly. The large intestines of sacrificed rats were removed, expanded with 10% formalin in a phosphate buffered saline solution (pH7.4) on ice for 15 min, and then cut open longitudinally along the main axis. The intestines were cut into three portions:the first is the rectum to 2cm from the anus, and the remaining colon was divided into two parts called the proximal and distal colon. Then each segment was placed between two pieces of filter paper, fixed in 10% buffered formalin for 24h, and stained with 0.2% methylene blue in saline. ACF was examined under a microscope at a magnification of 40×according to the procedure of Bird (15). 6) Analysis of DNA adducts Rats were treated and sacrified 12 h after the second AOM injection as described in Fig.1 (c). The perfused liver and colon were immediately removed. The colon was cut open longitudinally and washed with saline to remove colonic contents. Then the colon was laid flat on a glass plate and the mucosa was scraped off with a glass slide. The livers, colonic mucosa and colonic muscle layers were kept at -80°C until analysis of DNA adducts. DNA was isolated by phenol extraction following treatment with RNaset A, T1 and proteinase K (23). The two step procedure for detection of DNA adducts was based on the method of Beranek et al.(24). Purified DNA was dissolved in 10mM sodium cacodylate (pH7.0)at the concentration of 5mg/ml and was subjected to neutral thermal hydrolysis by heating at 100°C for 30 min to release N7-meG. The partially apurinic DNA was precipitated from the neutral thermal hydrolysate by addition of 0.1 volume of cold 1N HCl and was collected by centrifugation at 3,000rpm for 20 min at 0°C. The pellet was suspended in 50 mM Bis-Tris-1 mM MgCl2 (pH 6.5) and completely hydrolyzed in 0.1 volume of 1N HCl at 70°C for 30min (acid hydrolysis). Methylated bases in neutral thermal hydrolysates (N7-meG) and acid hydrolysates (O6-meG) were analyzed by high-performance liquid chromatography (HPLC) using a Chemcosorb7-SCX cation exchange column (4.6×250 mm, Chemco Scientific Co., Ltd., Osaka, Japan) and 4 mM ammonium formate (pH 3.0) as the mobile phase at a flow rate of 1.0 ml/min. Elution of the fluorescing base was monitored at286nm as an excitation wavelength and at 365nm as an emission wavelength. Authentic N7-meG and O6-meG were used as standards. 7) Statistical analysis The data were analyzed by one-way analysis of variance. RESULTS 1) Antimutagenicity of M. loriformis M. loriformis extract was not mutagenic for Salmonella typhimurium strains TA98 and TA100 in either the presence or absence of S9 mix (Table 1). Antimutagenic activities of the extract against various known mutagens are shown in Table 2. The extract showed antimutagenicity against tested heterocyclic amines, IQ, MeIQ, MeIQx, PhIP, Trp-P-1, Trp-P-2, Glu-P-1 and Glu-P-2, in dose-dependent manner except for AαC and MeAαC. About 40-60% of their mutagenicities was decreased at 10 mg/plate. The extract also decreased the mutagenicities of 2-AA, AF-2, MNNG and MAM, and the mutagenicity of MAM was decreased to 36.7% even at the lowest dose (0.31mg/plate). The extract had no effect on the mutagenicities of B[a]P, ENNG and1-NP. 2) Inhibitory effect of M. loriformis extract on AOM-induced DNA methylation The inhibitory effects of M. loriformis extract on AOM-induced DNA methylation are shown in Table 3. Methylated DNA adducts were not detected in the solvent control group or in the extract treated-group. The extract inhibited the formation of O6-meG by about 10-20% and 30-60% in the colonic mucosa and muscular layers of the AOM-treated rats, respectively, but not in a dose-dependent manner, and a significant decrease was observed only in the muscular layers of rats treated with AOM and 1.0 g/kg of the extract. N7-MeG was not decreased by continuous feeding. Pretreatment at a dose of 1.0 g/kg for 1 week before AOM administration was most effective, but the level of O6-meG in the colonic mucosa was not significantly lower than that in the AOM-treated rats. In the pretreatment group, the extract also significantly inhibited the formation of N7-meG both in the colonic mucosa and muscular layer. Methylguanines in the liver tended to be increased by the extract. The levels of O6-meG and N7-meG in the liver o f pretreatment group (group 4) were increased by 2.2 and 1.5 fold, respectively, as compared with those of the AOM-treated group. 3) Inhibitory effect of M. loriformis extract on AOM-induced ACF formation The body weights of rats in the control and M. loriformis-treated groups were not significantly different in either the initiation or the post-initiation stage during the experiments. No abnormality was seen with the naked eye in any group both in the initiation and post-initiation stages. The effects of M. loriformis extract on AOM-induced ACF formation in the initia-tion stage are shown in Table4. Pretreatment with the extract only for 1week before AOM injection at a dose of 1.0g/kg most effectively inhibited the ACF formation, and the number of ACFs significantly (p<0.0001) decreased to 44%of that in the positive control group (25% DMSO+AOM). When the extract was administered throughout the experimental period at doses of 1.0, 0.5 and 0.1g/kg, it inhibited ACF formation by about 21%, 34%and 39%, respectively. The number of ACFs was significantly decreased only at 0.5 g/kg wt (p<0.005) and at 0.1 g/kg wt(p<0.0005). However, the extract was more effective at a lower dose. Multiplicity of ACF (crypt/fo cus) was decreased by M. loriformis treatment only in the rectum. In the post-initiation stage, the extract did not significantly decrease ACF formation either in the colon or rectum (Table 5). However, it significantly inhibited the formation of larger ACFs (more than3 crypts/focus) by 27.0% in the total colon at 0.1 g/kg. Multiplicity of ACF was significantly decreased by treatment with the extract both at 0.1 and 1.0g/kg. 4) Correlation between the number of ACFs and DNA adduct level A significant correlation between the number of ACFs and the level of O6-meG in the colonic mucosa was observed (r=0.808, p=0.0382, Fig.2), but not between the number of ACFs and the level of N7-meG in the colonic mucosa (data not shown). DISCUSSION Colorectal cancer has a high mortality rate in Western countries and this may also soon be the case in Asian countries because of rapid Westernization of life style. However, the mortality rate of colorectal cancer in Thailand is still low compared with that in Japan (25, 26). This may be partially due to greater consumption of spices, herbs and medicinal plants in Thailand. Many kinds of chemical compounds present in natural dietary products have been shown to be protective against chemically induced toxicity and carcinogenesis (4, 27-29). Some Thai medicinal plants have been reported to be antimutagenic (6, 8-11, 13) and to inhibit AOM-induced ACF formation in the rat colon (7-9). M. loriformis is a Thai medicinal plant that has been used for relief of bronchitis and cancer (12). In the present study, we examined 80% ethanol extract of M. loriformis for its antimutagenic activity and inhibitory effects on AOM-induced ACFs formation. Antimutagenicity of M. loriformis against various known mutagens was measured by the Salmonella mutation assay in both strains TA98 and TA100 in the presence or absence of S9 mix. The extract inhibited the mutagenicity of tested heterocyclic amines except for AαC derivatives. These results are similar to those of a previous report (13). Since M. loriformis inhibited almost all of the HCA-induced mutations, it may possibly inhibit the metabolic activation of HCAs by cytochrome P450 1A2. Factors affecting the metabolic activation and DNA adduct formation of HCAs were reviewed by Kato (30). Unsaturated fatty acids such as oleic and linoleic acids inhibited the mutagenicity of Trp-P-2 mainly through the inhibition of hepatic mixed function oxidase (31), and microsomal lipids or lecithin diminished the amount of N-OH Trp-P-2 without inhibiting the metabolism of Trp-P-2. Compound lipids such as glycosphingolipid in M. loriformis possibly inhibit P450 1A2-mediated activation or interact with activated H CAs. Mutagenicity of methylating agents was also inhibited, especially in the case of MAM, whose mutagenicity was reduced by about 70%with maximum inhibition at the lowest dose. The interactions of some components of M. loriformis with MAM or its ultimate form might contribute to the decrease of mutagenicity. Since M. loriformis extract efficiently inhibited MAM-induced mutation (Table 2), we examined whether the extract inhibits AOM-induced DNA methylation and ACF formation in the rat colon. Formation of ACF in the rat colon in the initiation stage was inhibited by feeding rats the extract, while the formation of O6-meG in the rat colon was weakly inhibited by M. loriformis treatment. Since the level of DNA adducts correlated well with the number of ACFs in the colon (Fig.2), inhibition of ACF formation was thought to be partially due to the decrease in the level of adducts. AOM was oxidatively metabolized to MAM by P450 2E1 and transported to the colon via the blood stream (32). Alternatively, MAM was conjugated with glucuronic acid and transported via bile to the colon (33). Conjugated MAM is hydrolyzed by bacterial β-glucuronidase to free MAM in the intestine (34), and released MAM is further metabolized and methylates DNA in the colon (35). Although N7-meG is a major adduct, O6-meG in the target tissue is more closely correlated with carcinogenicity than is N7-meG because thymine can be incorporated opposite O6-meG resulting in GC→AT transition mutations(19), and such alterations are frequently observed in tumor-associated genes (36). In the present study, M. loriformis extract inhibited AOM-induced ACF formation partially due to the decrease of O6-meG level. Although mechanisms of inhibition of DNA adducts formation by the ex-tract are still unclear, current evidence suggests that the components of M. loriformis can induce chemoprotective enzymes such as glutathione S-transferase, UDP-glucuronyl transferase and DT-diaphorase (37). However, the level of both O6-meG and N7-meG in the liver were rather increased by the extract. Modification of AOM metabolism in the liver and lowered distribution of the metabolites to the colon might contribute to the decrease of O6-meG in the colon. Other possible mechanisms of inhibition of ACF formation are enhanced DNA repair or modification of cell proliferation as do curcumin and other plant components (38). The number of ACFs was more decreased at a lower dose in the continuously fed group and most strongly inhibited in the pretreatment group (Table 4). It is not known why a lower dose was more effective in inhibition of ACF formation. In the post-initiation stage, multiplicity of ACF in the total colon was significantly decreased by the extract (Table 5). The number of ACFs that have more than 3 crypts/focus, which is highly correlated with tumor formation (17), was also significantly decreased. These results suggest that M. loriformis inhibits the growth of ACFs. Recent studies have shown that dietary sphingolipids inhibit dimethylhydrazine-induced colon cancer in CF1 mice (39-41), presumably because they are digested to the lipid backbones ceramide and sphingosine that inhibit cell growth and induce differentiation and apoptosis (42). Glycosphingolipids, constituents of M. loriformis, could contribute to the inhibition of ACF formation by the above mechanisms. We have investigated antimutagenicity of Thai medicinal plants and their inhibitory effects on AOM-induced ACF formation. Among tested plants, M. loriformis and roselle (8) decreased the mutagenicity of heterocyclic amines such as PhIP and IQ, which are colon carcinogens in rodents. Lemon grass was the strongest inhibitor against the ACF formation in the initiation stage by enhancing detoxification enzyme activity (7). Inhibitory effect of M. loriformis on the ACF formation was similar to those of roselle (8) and bitter melon (9). In conclusion, M. loriformis extract showed antimutagenicity against various known mutagens and inhibited the formation of AOM-induced ACF and DNA adduct formation in the rat colon. Chemopreventive agents have been classified into three categories based on the time period that the agents exert their inhibitory activity in animal carcinogenesis models:inhibitors of carcinogen formation, blocking agents that are inhibitors of tumor initiation, and suppressing agents that are inhibitors of tumor promotion and/or progression (5). M. loriformis extract may act as either a blocking or suppressing agent. However, Yamada et al. (43) currently suggested that β-catenine-accumulated crypts, which are independent of ACFs, are truly premalignant lesions for colon cancer. Usefulness of M. loriformis extract for colon cancer chemoprevention should be further investigated. ACKNOWLEDGEMENTS We thank Drs. K. Ishizaki and M. 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Address correspondence and reprint requests to Prof. Yoshinari Ohnishi, Department of Bacteriology, The University of Tokushima School of Medicine, Kuramoto-cho, Tokushima 770-8503, Japan and Fax:+81-88-633-7069.