Food Phytochemicals for Cancer Prevention I - American Chemical

Chapter 12

Chemoprevention of Colon Cancer by Thiol and Other Organosulfur Compounds Bandaru S. Reddy and Chinthalapally V. Rao

Downloaded by FUDAN UNIV on March 3, 2017 | http://pubs.acs.org Publication Date: December 20, 1993 | doi: 10.1021/bk-1994-0546.ch012

Division of Nutritional Carcinogenesis, American Health Foundation, Valhalla, NY 10595

Epidemiological studies suggest that consumption of garlic and cruciferous vegetables rich in organosulfur compounds is associated with a reduced risk for cancer development including cancer of the colon in man. We used laboratory animal models to test isolated organosulfur compounds and their substituted analogues as chemopreventive agents. The effect of two dose levels of dietary oltipraz [5-(2-pyrazinyl)-4-methyl-1,2-dithiole-3-thione], a substituted dithiolethione, anethole trithione (ATT), and diallyl disulfide (DDS) on azoxymethane (AOM)-induced colon carcinogenesis was studied in male F344 rats. The maximum tolerated doses (MTD) of oltipraz, ATT or DDS added to a semipurified diet were found to be 500, 250 or 250 ppm, respectively. Dietary oltipraz, ATT, and DDS at levels of 40% M T D and 80% M T D were tested as inhibitors of A O M induced colon cancer. The results demonstrated that these dietary organosulfur compounds significantly inhibited colon carcinogenesis in a dose-dependent manner.

A role for nutrition in the etiology of cancer is increasingly apparent. Cancers of several major sites, including colon, stomach, breast, endometrium and prostate, are directly or indirectly associated with dietary factors. In the United States and other Western countries, colorectal cancer is one of the leading causes of cancer deaths (1). Although several epidemiological and experimental studies have demonstrated that diets high in total fat and low in fiber are generally associated with increased risk for colon cancer (2-6), the etiology of colon cancer is multifactorial and complex in that it may arise from the combined actions of environmental factors — such as low levels of a number of, as yet unidentified, genotoxic agents — and endogenous formation of tumorigenic substances. Strategies for cancer prevention involving reduction or elimination of human exposure to these environmental factors may not always be possible. An alternative approach with a potential for more immediate impact is to identify safe and effective agents which prevent the

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endogenous formation or enhance detoxification of carcinogens and/or inhibit tumor promotion and progression (7-9). Epidemiological studies also suggest that generous consumption of fruits and vegetables is associated with a reduced risk for development of several types of cancer including cancer of the colon in humans (2,10,11). Although the nature of constituents of these fruits and vegetables responsible for reduced risk has not been fully elucidated, it is apparent that the foodstuffs are a source of naturally occurring anticarcinogenic agents that hinder the formation of carcinogens from precursors in the body or that act protectively to lessen or eliminate the effects of carcinogens and tumor promoters (8). Green and yellow vegetables, including cabbage, brussels sprouts and other cruciferous vegetables contain several organosulfur compounds including isothiocyanates and dithiolethiones (12,13). Among dithiolethiones, oltipraz, a sub stituted form of dithiolthione, [5-(2-pyrazinyl)-4-methyl-l,2-dithiole-3-thione], has been used in man as an antischistosomal drug (14). Recent work has also focused on organosulfur compounds found in garlic (Allium species) namely diallyl sulfide, diallyl disulfide and diallyl trisulfide (8). In animal models, Belman (75) demonstrated that garlic oil inhibited mouse skin tumorigenesis. Several of these agents have been tested in short term screening and long term efficacy studies in laboratory animals for their chemopreventive properties (9,16). The purpose of this chapter is to provide an overview of the inhibitory effect of dietary organosulfur compounds — namely oltipraz, anethole thrithione, and diallyl disulfide — in colon carcinogenesis. The focus will be on the results thus far generated in our laboratory. The structures of oltipraz anethole thrithione and diallyl disulfide are shown in Figure 1.

Ν

Γ

C H

Oltipraz

3

0 ^ ^

Anethole trithione

H C = C - C - S - S - C - C = CH Η H2 H2 Η 2

2

Diallyl disulfide

Figure 1. Chemical structures of organosulfur compounds

Animal Models for Colon Cancer A variety of compounds, namely, 1,2-dimethylhydrazine (DMH), azoxymethane (AOM), methylazoxymethanol acetate ( M A M ) , 3,2'-dimethyl-4-aminobiphenyl ( D M A B ) , methylnitrosourea (MNU), and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), that are carcinogenic to the colon have been used in a number of animal models to study the effect of dietary constituents on tumorigenesis at this site (77, 18). These carcinogens produce both benign adenomas and adenocarcinomas similar to those observed in humans. There is a consensus in the literature that, both morphologically and clinically, the colon tumors induced in rats by these agents

Huang et al.; Food Phytochemicals for Cancer Prevention I ACS Symposium Series; American Chemical Society: Washington, DC, 1993.


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FOOD PHYTOCHEMICALS I: FRUITS AND VEGETABLES

resemble relevant neoplasms in man. The spreading of rat colon adenocarcinomas via lymphogenic region to regional and distal lymph nodes by implantation in peritoneum and hematogenous dissemination in lungs and other viscera also bear much resemblance to human phenomenon. Additionally, these rodent models have been used for over two decades as unique tools for systematic studies of risk factors for colon cancer observed in the human setting and for determining whether or not suspected etiologic factors can be reproduced under controlled laboratory conditions. Thus, as is described here, a number of major elements observed in humans could not have been established without careful, deliberate investigations carried out in laboratory animals. In all our studies presented here, colon cancer was initiated with A O M in male F344 rats to evaluate potential chemopreventive properties of organosulfur compounds. Beginning at 5 weeks of age, groups of male F344 rats were fed a modified AIN-76A (control) diet and experimental diets containing 40 and 80% of the maximum tolerated doses (MTD) of organosulfur compounds (Table I). After two weeks on the diets, groups of animals intended for carcinogen treatment received 15 mg/kg body weight A O M subcutaneously once per week for 2 weeks or one injection of 30 mg/kg body weight. The experiments were terminated 52 weeks after the A O M treatment. Colon tumors were subjected to histopathologic evaluation by routine procedures (18).

Table I. Percentage Composition of Experimental Semi-purified Diets Ingredients Casein DL-Methionine Corn starch Dextrose Corn oil Alphacel Mineral mix, AIN Vitamin mix, AIN revised Choline bitartrate Chemopreventive agents a

b

Control diet

a

20.0 0.3 52.0 13.0 5.0 5.0 3.5 1.0 0.2 0

Experimental diet

b

20.0 0.3 52.0 13.0 5.0 5.0 3.5 1.0 0.2 40 and 80% M T D

Adapted from American Institute of Nutrition Reference Diet (AIN-76A) with modification of the source of carbohydrate. Chemopreventive agents were added to the diets at the expense of corn starch.

Maximum Tolerated Doses of Organosulfur Compounds Prior to efficacy studies of organosulfur compounds, the maximum tolerated dose (MTD) of each test agent added to AIN-76A semipurified diet was determined in male F344 rats. At 5 weeks of age, groups of male F344 rats were fed the AIN-76A diet (control) and experimental diets containing 5 concentrations of each test agent. Body weights were recorded twice weekly for 10 weeks. At the end of 10 weeks, all animals were sacrificed and the organs were examined grossly for any


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abnormalities. Based on body weights and clinical signs of toxicity, the MTDs of test agents were calculated. The M T D is defined as the highest dose of the agent in the diet that causes no more than a 10% body weight decrement as compared to the appropriate control diet group and does not produce mortality or any clinical signs of toxicity that would be predicted to shorten the natural life span of the animal. The MTDs of organosulfur compounds were: anethole trithione, 250 ppm; oltipraz, 500 ppm; and diallyl disulfide, 250 ppm.


Inhibitory Effect of Anethole trithione and Oltipraz on Colon Carcinogenesis Oltipraz and other dithiolethiones have been shown to have a broad spectrum of anticarcinogenic actions against several types of carcinogens in a variety of organs (79). A pioneering study by Wattenberg and Bueding (20) indicated that oral administration of oltipraz inhibited benzo[a]pyrene (B[a]P)-, diethylnitrosamine (DEN)-, and uracil mustard-induced pulmonary and/or forestomach tumors in mice. Oltipraz has also been shown to protect against hepatotoxicity induced by acetaminophen, carbon tetrachloride (27), and aflatoxin B i (22). Roebuck et al (23) have shown that oltipraz inhibits aflatoxin-induced liver cancer in F344 rats. Because of the low toxicity and inhibitory effect of oltipraz against diverse carcinogens, we evaluated the efficacy of this compound and another substituted dithiole thione, namely anethole trithione, in colon carcinogenesis. In these studies, experimental diets containing 40 and 80% M T D levels of oltipraz or anethole trithione were fed 2 weeks before and during carcinogen treatment and until termination of study at 52 weeks. The body weights of animals fed the control diet and experimental diets containing oltipraz and anethole trithione were comparable. The results of our study, which are summarized in Table II, demonstrate that feeding 200 (40% MTD) and 400 ppm (80% MTD) oltipraz significantly inhibited the incidence of AOM-induced colon adenocarcinomas. Anethole trithione at 100 (40% MTD) and 200 ppm (80% MTD) in the diet also suppressed the incidence and multiplicity of colon adenocarcinomas in a dose-dependent manner (Table II).

Table II. Effect of Organosulfur Compounds on AOM-induced Colon Tumor Incidence in Male F344 Rats Organosulfur compound tested

Colon adenocarcinoma incidence (%)

a

Experiment l Control diet Oltipraz (200 ppm) Experiment 2 Control diet Anethole trithione (100 ppm) (200 ppm) Diallyl disulfide (100 ppm) (200 ppm)

72 47*

b

33 11* 8* 11* 6*

a

Invasive and noninvasive colon adenocarcinomas. Invasive colon adenocarcinomas. * Significantly different from its respective control diet group, ρ < 0.05. b


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We also measured serum Table III. Effect of dietary oltipraz oltipraz levels at different time points on serum oltipraz levels in male F344 rats during the period of the study (Table III). Serum oltipraz levels were Amount of oltipraz in diet maintained throughout the study. The Weeks on 200 ppm 400 ppm animals fed 80% M T D (400 ppm) of oltipraz diet oltipraz showed increased levels of 277±43 426 ± 55 1 serum oltipraz as compared to those 505 ± 47 331+33 8 fed 40% M T D (200 ppm) of oltipraz. 304 ± 58 536 ± 7 1 16 In order to understand the 584 ± 43 24 352 + 43 mechanism by which dietary oltipraz exerts its inhibitory action on A O M - Nanograms oltipraz per ml serum (mean induced colon carcinogenesis, we value ± SD; n=9). assessed the effect of this agent on hepatic and colonic mucosal activities of glutathione ^-transferase (GST) and tyrosine protein kinase (TPK) and on the hepatic and colonic D N A methylation in rats treated with A O M (24). The results show that dietary oltipraz significantly enhanced hepatic and colonic GST activity which is accompanied by a decrease in D N A methylation (7-methylguanine and 0 -methylguanine) in the colon and liver (Table IV). Although the significance of 7-methylguanine in tumor formation has been questioned, the formation and persistence of 0 -methylguanine in the D N A of target tissue is closely correlated with carcinogenicity (25,26). Many chemopreventive agents including several phenolic antioxidants have been shown to inhibit carcinogenesis by modulating the activities of detoxifying enzymes that are involved in the metabolism of carcinogens (27,28). It has also been reported that oltipraz elevates several enzymes such as epoxide hydrolase and NADP(H)quinone reductase (27) that are involved in the inactivation of electrophilic species. There is a possibility that oltipraz alters the metabolism of carcinogen in the target tissue. A O M is first metabolized to methylazoxymethanol (MAM), a proximate carcinogen, in the liver by the microsomal mixed function oxidases, and M A M can be further metabolized in the liver to a DNA-alkylating species, presumably the methyldiazonium ion, which is considered to be an ultimate carcinogen (29,30). Additional studies have shown that the M A M that is not metabolized in the liver is carried to colon by circulation and activated to methyldiazonium ion (31) which can methylate colonic cellular nucleophiles, including DNA. For example, several colon tumor inhibitors have been shown to interact with A O M metabolism and to inhibit D N A alkylation and carcinogenicity (30, 32). Therefore, the possibility exists that dietary oltipraz alters the metabolism of A O M in the liver and colon mucosa thereby inhibiting colon tumor formation. It is also possible that increased GST activity in the liver and colon of animals fed the oltipraz decreases the level of D N A adduct formation by modulating A O M metabolism. Dietary oltipraz significantly inhibited colonic mucosal and liver tyrosine protein kinase (TPK) activity (Table IV). Evidence from various studies indicates that the phosphorylation of proteins at tyrosine residues plays an important role in the regulation of cellular growth and differentiation (33). TPK activity is associated with cellular receptors for epidermal growth factor, platelet-derived growth factors, insulin receptor (34) and several oncogenes (33). Increased expression of T P K activity may be responsible for unlimited growth (35). Increased levels of T P K a


a

6

6



1.1 ±0.08 7.1 ±0.20***

Colon Control Oltipraz 1567 ± 2 6 284 ± 2 3 * * *

6343 ± 6 6 7 2185 ±228***

7 -Methy lguanine (μπιοΐ/ηιοί guanine)

6

232 ± 24 157 ± 1 1 *

728 ± 63 306 ±32***

0 -Methylguanine (μιτιοΐ/mol guanine)

220 ± 1 3 158 ± 8 * *

333 ± 1 5 146± 14***

32

Tyrosine protein kinase (pmol P/mg protein/min)

A l l values are the mean ± SE from 6 rats. Asterisks indicate significant difference from respective control, *: ρ < 0.05; **: ρ < 0.01; ***: ρ < 0.001.)

4.9 ±0.17 22.7 ± 1.70***

Glutathione ^-transferase (μπιοΐ/mg protein/min)

Liver Control Oltipraz

Experimental group

Table IV. Effect of Dietary Oltipraz on AOM-induced Hepatic and Colonic Mucosal Enzymes and DNA Methylation in Male F344 Rats


170


activity have been found in neoplastic tissue (33,34). In the present study, A O M induced TPK activity in the cytosol and membrane fractions of liver and colonic mucosa was significantly inhibited by oltipraz. The mechanism of TPK inhibition by oltipraz in unclear. There is, however, a possibility that the inhibition by oltipraz might be similar to that of Genistein- and Erbstatin-like specific T P K inhibitors. These agents inhibit TPK activity by altering D N A topoisomerase II activity and by acting at the level of the ATP site (36,37).


Inhibitory Effect of Diallyl Disulfide on Colon Carcinogenesis Investigations were carried out to determine the chemopreventive properties of diallyl disulfide on AOM-induced colon carcinogenesis in male F344 rats. The experimental protocols were as described above. A l l animals were fed 40 (100 ppm) and 80% (200 ppm) M T D of this agent in AIN-76A diet starting 2 weeks before and during carcinogen administration and until termination of the study at 52 weeks after carcinogen treatment. The body weights of animals fed the experimental diets containing 100 and 200 ppm of diallyl disulfide were comparable to those fed the control diet. Diallyl disulfide at 100 and 200 ppm in the diet significantly inhibited the incidence and multiplicity of colon adenocarcinomas (unpublished observations; Table II). Experiments are in progress to determine the mechanism(s) by which diallyl disulfide inhibits colon carcinogenesis. Summary and Conclusions In conclusion, the results of our studies demonstrate that several organosulfur compounds — namely oltipraz, anethole trithione and diallyl disulfide — inhibit colon carcinogenesis in a laboratory animal model. The combined observations from several laboratories indicate a broad spectrum of chemopreventive properties of oltipraz and diallyl disulfide in several cancer models. The inhibition of colon carcinogenesis by dietary oltipraz is associated with an increase in liver and colonic GST activity and reduced formation of D N A adducts. In addition, dietary oltipraz modulates liver and colonic TPK activity that has been shown to play a role in the regulation of cellular growth and differentiation. We believe that the organosulfur compounds have great potential as chemopreventive agents for colon cancer. Acknowledgments We thank Donna Virgil for preparation of the manuscript. This work was supported by USPHS Grant CA-17613 and Contracts NO1-CN-85095-01 and NO1-CN-8509505 from the National Cancer Institute. Literature Cited 1. Boring, C. C.; Squires, T. S.; Tong, T. CA-A Cancer J. Clinicians, 1992, 42, 19-38. 2. Committee on Diet, Nutrition, and Cancer, National Research Council Diet, Nutrition and Cancer, National Academy Press: Washington, DC, 1982; pp 358-370.



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