(CANCER RESEARCH 53. 3493-3498. August I. 19931

Chemoprevention of Colon Carcinogenesis by Organosulfur Compounds1

Bandaru S. Reddy,2 Chinthalapally V. Rao, Abraham Rivenson, and Gary Kelloff

Division of Nutritional Carcinogenesis ¡B.S. R., C. V. R.¡and Division of Pathology and Toxicology ¡A.R.¡,American Health Foundation. Valhalla. New York I05V5. andChemoprevention Branch. Division of Cancer Control and Prevention. National Cancer Institute. Bethesda. Maryland 20892 [G. K.I

ABSTRACT

It has been reported that several naturally occurring and related synthetic organosulfur compounds exert chemopreventive effects in severaltarget organs in rodent models. The chemopreventive actions of 40 and80% maximum tolerated doses (MTD) of organosulfur compounds,namely anethole trithione, diali) I disulfide, W-acelylcysteine, and taurine,administered in AIN-76A diet, on azoxymethane (AOM)-induced neopla

sia were investigated in male F344 rats. Also, the effects of these agents onthe activities of phase II enzymes, namely glutathione S-lransferase (GST),NAD(P)H-dependent quinone reducíase, and UDP-glucuronosyl trans-

ferase, in the liver and colonie mucosa and tumors were assessed. TheMTD levels of anethole trithione, diali) I disillude. jV-acetylcysteine, and

taurine were determined in male F344 rats and found to be 250,250,1500,and 1500 ppm, respectively. At 5 weeks of age, animals were fed the controldiet (AIN-76A) or experimental diets containing 40 or 80% MTD levels of

each test agent. All animals in each group, except those allotted for vehicle(saline) treatment, were administered AOM s.c. at a dose rate of 15 mg/kgbody weight once weekly for 2 weeks. All animals were necropsied duringweek 52 after the second AOM injection. Colonie mucosa! and tumor andliver enzyme activities were measured in animals fed 80% MTD levels ofeach test agent. Colon tumors were subjected to histopathological evaluation and classified as invasive or noninvasive adenocarcinomas. Colontumor incidence (percentage of animals with tumors) and tumor multiplicity (tumors/animal) were compared among various dietary groups.The results indicated that administration of 200 ppm (80% MTD) anethole trithione significantly inhibited the incidence and multiplicity of bothinvasive and noninvasive adenocarcinomas, whereas feeding of 100 ppm(40% MTD) anethole trithione or 100 (40% MTD) or 200 ppm (80%MTD) diali) I disulfide suppressed only invasive adenocarcinomas of thecolon. Although diets containing W-acelylcysleine and taurine inhibitedcolon tumor multiplicity, the effect was somewhat marginal. GST, N VI)-(P)H-dependent quinone reducíase,and UDP-glucuronosyl transferase ac

tivities in colonie mucosa and tumor and liver were significantly elevatedin animals fed anethole trithione or diali) Idisulfide, compared to those fedthe control diet. ¿V-Acetylcysteine and taurine slightly but significantly

increased only the GST activity in the liver. Although other mechanismsare not excluded, inhibition of AOM-induced colon carcinogenesis by

anethole trithione and diali) I disulfide may be associated, in part, withincreased activities of phase II enzymes such as GST, NAD(P)H-dependentquinone reducíase, and UDP-glucuronosyl transferase in Ihe liver and

colon.

INTRODUCTION

Cancer of the colon is a major public health problem in the UnitedStates and Canada, as well as other western countries (1). Although alarge volume of data derived from epidemiological and laboratorystudies suggest that dietary factors play an important role in theetiology of colon cancer (2-5), there is also increasing evidence that

minor compounds present in the diet and their related synthetic analogues reduce the risk of colon cancer development in rodent models(6, 7). Naturally occurring substances and their synthetic analoguesthat have been intensely investigated previously for their chemopre-

Received 1/28/93; accepted 5/26/93.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby marked advertisement in accordance with18 U.S.C. Section 1734 solely to indicate this fact.

' Supported by USPHS Contract NOI-CN-85095-05 and Grant CA-17613 from the

National Cancer Institute.2 To whom requests for reprints should be addressed, at Division of Nutritional Car

cinogenesis, American Health Foundation, One Dana Road, Valhalla, NY 10595.

ventive properties include inorganic and organic selenium salts, phenolic antioxidants, inositol-6-phosphate, calcium salts, vitamin D,

arachidonic acid cascade inhibitors, and difluoromethylornithine, tocite a few (6-17).

Several studies indicate that organosulfur compounds present incruciferous vegetables (brussels sprouts, cauliflower, and cabbage),such as benzylisothiocyanate and dithiolethiones, and in Allium species (onion and garlic), such as diallyl disulfide and diallyl sulfide andseveral of their substituted compounds, have a role in cancer inhibition (6, 7, 9). Diallyl disulfide is a major organosulfur compoundpresent in garlic (18). Pioneering studies by Wattenberg and Bueding(19) and subsequent studies from several laboratories demonstratedthat oltipraz [5-(2-pyrazinyl)-4-methyl-l,2-dithiole-3-thione], a sub

stituted dithiolethione, inhibits chemically induced carcinogenesis inseveral target organs and AFB,'-induced hepatotoxicity in rats (20-

23). Experiments conducted in our laboratory indicate that oltiprazinhibits chemically induced colon carcinogenesis when administeredduring the initiation and postinitiation stages (24). Anethole trithione,a substituted dithiolethione. has not been tested as a potential chemopreventive agent in colon cancer models (Fig. 1).

Organosulfur compounds present in garlic and onions, such as allylsulfide, allyl disulfide, and allyl methyl di- and trisulfides, and garlic

and onion oils have been found to inhibit carcinogenesis at severalorgan sites (25-30). Allyl methyl trisulfide, diallyl sulfide, and diallyl

trisulfide administered p.o. prior to carcinogen treatment inhibitedbenzo[i/]pyrene-induced and nitrosodiethylamine-induced forestom-

ach tumors in mice (25, 26), with the most potent compound beingdiallyl disulfide (Fig. 1). Diallyl sulfide administered prior to carcinogen treatment inhibited DMH-induced colon carcinogenesis andjV-nitrosomethylbenzylamine-induced esophageal cancer in rats (27,

28). The inhibitory effects of these compounds may be due to inhibition of carcinogen activation through suppression of oxidative metabolism of carcinogen (31 ). Administration of diallyl disulfide duringthe postinitiation phase inhibited DMH-induced colon carcinogenesis

in rats (29), but this inhibition of colon carcinogenesis was associatedwith the retardation of body weight gain.

Several other organosulfur compounds that are formed endoge-nously in the body, including taurine (2-aminoethanesulfonate) andJV-acetylcysteine (Fig. 1), have been shown to ameliorate toxicityinduced by a variety of chemicals in various organs (32-34). N-A.ce-tylcysteine has been shown to inhibit cyclophosphamide-induced tox

icity such as hemorrhagic cystitis (34). Oral administration of taurinepartially protected against lipid perxoidation induced by toxic doses oftaumustin (32) and isoprenaline (33). The beneficial effect of taurinemay be due, in part, to inhibition of lipid peroxidation and of deterioration of membrane phospholipids (32). Although these observationsdo not necessarily provide evidence that these compounds act aschemopreventive agents, they raise the possibility that these agentsmay possess tumor-inhibitory properties.

Although the mechanism of action of many of the chemopreventiveagents is poorly understood, it appears that these compounds mayinhibit carcinogenesis by blocking the formation of or trapping ultimate carcinogen electrophiles and/or by decreasing activation of car-

1The abbreviations used are: AFB,. aflatoxin B,; DMH. 1.2-dimethylhydrazine; AOM.

azoxymelhane; GST. gluatathione 5-transferase: NAD(P)H:QR, NAD(P)H:quinonereducíase;UDP-GT. UDP-glucuronosyl transferase; MTD. maximum tolerated dose.

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ORGANOSULFUR COMPOUNDS AND COLON CANCER INHIBITION

CHgO

ANETHOLE TRITHIONE

CH2=CH-CH2-S-S-CH2-CH=CH2

DIALLYL DISULFIDE

HS-CH2-CH-COOH

HN-COCHg

N-ACETYLCYSTEINE

H2N-CH2-CH2-S03H

TAURINEFig. 1. Structures of organosulfur compounds.

cinogens and by suppressing the effect of promoters in the target organ(6). Several organosulfur compounds inhibit carcinogenesis by increasing the metabolism, detoxification, and elimination of carcinogens through phase 1 and phase II enzymes (6, 35). For example,oltipraz and dithiolethione induce liver GST and NAD(P)H:QR activities in rodents (23-35). Earlier studies from our laboratory demonstrated that the colon tumor-inhibitory effect of oltipraz was associated with an increase in GST, NAD(P)H:QR, and UDP-GT activities

of the colon (36). It has been suggested that the protecive effects ofthese organosulfur compounds may be accounted for, at least in part,by their ability to induce GST and other phase II enzymes (6, 35).

Clearly, the influence of organosulfur compounds on carcinogenesis is complex and may depend on the specific compounds, the organsite, and the carcinogen administered. The present study was designedto investigate the modulating effects of organosulfur compounds,namely diallyl disulfide, anethole trithione, iV-acetylcysteine, and tau

rine, in colon carcinogenesis. Dose selection of these compounds wasbased on MTD values determined in the current study. In addition, theeffect of these compounds on colonie mucosal GST, NAD(P)H:QR,and UDP-GT activities was measured. The major goal of this preclin-

ical study was to determine which of these organosulfur compoundscould be used as effective chemopreventive agents in colon carcinogenesis.

MATERIALS AND METHODS

Animals, Diets, and Organosulfur Compounds. Weanling male inbredF344 rats were obtained from Charles River Breeding Laboratories (Kingston,NY). Diallyl disulfide, taurine, and /V-acetylcysteine were purchased fromSigma Chemical Co. (St. Louis, MO). AOM (CAS 25843^15-2) was purchased from Ash-Stevens (Detroit, MI). All ingredients of the semipurifieddiets were obtained from Dyets, Inc. (Bethlehem, PA) and stored at 4°Cprior

to formulation of the experimental diets.A total of 636 male F344 rats were used in this study to determine MTD

values and efficacy of organosulfur compounds. Male F344 rats received atweaning were maintained in isolation for 10 days and had access to modified

AIN-76A (control) diet (Table 1) (37). The animals were then randomly di

vided by weight into control and experimental dietary groups and transferredto an animal holding room. They were housed in plastic cages with wood chipbedding and filter tops (3 rats/cage) under controlled conditions of a 12-hlight/12-h dark cycle, 50% humidity, and 21°Ctemperature. Animals were

allowed free access to food and water at all times, and food cups werereplenished with fresh diet three times weekly. Cages were changed weekly.Experimental diets were prepared by mixing test agents with modified A1N-

76A diet (Table 1). All control and experimental diets were prepared weekly inour laboratory and stored in a cold room in airtight plastic containers filled withnitrogen.

Determination of Maximum Tolerated Doses of Test Agents. The experimental protocols were as described previously (38). At 35 days of age. groupsof male F344 rats (12/group) were fed the modified AIN-76A diet containing

the various levels of chemopreventive agents. Anethole trithione and diallyldisulfide were each tested at dose levels of 62.5, 125, 250, 500, and 1000 ppm,whereas iV-acetylcysteine and taurine were each tested at concentrations of

125, 250, 1000, 1500, and 2000 ppm in the diet. Body weights were recordedtwice weekly for 6 weeks. At the end of 6 weeks, all animals were sacrificed,and colon, small intestine, stomach, liver, and kidney were examined grosslyunder a dissection microscope for any abnormalities. The MTD is defined asthe highest dose that causes no more than 10% weight decrement, compared tothe appropriate control diet group, and does not produce mortality or anyexternal signs of toxicity that would be predicted to shorten the natural lifespan of the animal. These external signs of toxicity include roughened coat,ill-kept appearance, chromodacryorrhea, rhinitis, and prostration, to cite a few.

Based on the observation that dietary anethole trithione or diallyl disulfide atdose levels of 500 and 1000 ppm and dietary jV-acetylcysteine or taurine at

2000 ppm decreased the body weights by >10%, compared to the control diet(data not shown), the MTDs of diallyl disulfide, /V-acetylcysteine, anethole

trithione. and taurine are 250, 1500, 250, and 1500 ppm, respectively.Experimental Procedure for Efficacy Study. The experiment was de

signed to determine the efficacy of 40 and 80% MTD of each test agent,administered in the modified AIN-76A diet (Table 1), on AOM-induced colon

carcinogenesis in male F344 rats. The 40 and 80% MTD levels, respectively,of test agents were as follows: diallyl disulfide, 100 and 200 ppm; W-acetyl-

cysteine, 600 and 1200 ppm; anethole trithione, 100 and 200 ppm; and taurine,600 and 1200 ppm. Beginning at the fifth week of age, male F344 rats wererandomized by weight into various groups and fed one of the diets containing0, 40, or 80% MTD levels of each chemopreventive agent (Table 1). After 2weeks on control or experimental diets, groups of animals intended for carcinogen treatment (36 animals/group) received AOM s.c. once weekly for 2weeks at a dose rate of 15 mg/kg body weight/week. Animals intended forvehicle treatment (12 animals/group) received an equal volume of normalsaline. All animals were fed their respective test agents until the termination ofthe study. Body weights were recorded every 2 weeks for the first 10 weeks andthen every 4-6 weeks. The experiment was terminated at 52 weeks after thesecond AOM treatment. Both vehicle-treated and AOM-treated animals were

sacrificed by CO2 euthanasia as scheduled. Following laparotomy. the entire

Table 1 Percentage composition of experimental semipurified diets

IngredientsCasein»i.-MethionineCorn

starchDextroseCom

oilAlphacelMineral

mix.AINVitaminmix. AINrevisedCholine

bitartrateAnetholetrithioneDiallyldisulfideN-AcetylcysteineTaurineCompositionControl

diet"2(1.00.352.013.05.05.03.51.00.20000(*)Experimentaldiet*20.00.352.013.05.05.03.51.00.2200

or 100ppm200or 100ppm1

200 or 600ppm1200 or 600 ppm

" Adopted from American Institute of Nutrition Reference Diet AIN-76A with mod-

ificaton of the source of carbohydrate (38).h Test agents at 40 and 80^ MTD levels were added to the diets at the expense of corn

starch.

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ORGANOSULFUR COMPOUNDS AND COLON CANCER INHIBITION

stomach, small intestine, and large intestine were resected and opened longitudinally, and the contents were flushed with normal saline. Using a dissectionmicroscope, tumors were noted grossly for their location, number, and size. Allother organs, including kidney and liver, were also grossly examined under themicroscope. In addition, colon tumors with a diameter of >0.5 cm were cutapproximately into two halves; one portion of the tumor was used for enzymeassays and the other for histopathology. Colonie mucosa was scraped fromeach animal for biochemical analysis according to our previously describedmethod (24). Portions of colonie tumors and colonie mucosa intended forbiochemical determinations were quickly frozen in liquid nitrogen until anal

yses.For histopathological confirmation, tumors were fixed in 10% buffered

formalin, embedded in paraffin blocks, and processed by routine histologicalmethods using hematoxylin and eosin stains. The stained sections were examined histologically for tumor types, using histological criteria described pre

viously (14).Biochemical Study. Liver and colonie mucosal and tumor samples were

homogenized in 4 volumes (w/v) of 50 MTris buffer (pH 7.4) containing 0.25M sucrose, with a Polytron homogenizer. Homogenates were centrifuged at105,000 X g; the resulting supernatant was used for GST and NAD(P)H:QRassays and the membrane fraction was used for assay of UDP-GT activity. GST

activity was assayed according to the method of Habig et al. (39), usingl-chloro-2,4-dinitrobenzene as substrate. Dicoumarol-inhibited NAD(P)H:QR

activity was assayed according to the modified method of Benson et al. (40),using 2.6-dichloroindophenol as final electron acceptor. UDP-GT activity wasassayed according to the method of Temple et ai. (41 ). using /7-nitrophenol assubstrate. Protein was determined by the Bio-Rad method.

Statistical Analysis. Tumor incidence is expressed as percentage of animals with tumors and was compared between the groups by x~ test- Tumor

multiplicity is expressed as mean number of tumors/animal and was comparedby the Student's t test. Body weights and enzyme values for the control and

experimental groups were analyzed by analysis of variance. Differences wereconsidered statistically significant at P < 0.05.

RESULTS

The body weights of animals fed the chemopre venti ve agents werecomparable to those of the animals fed the control diet in AOM- andsaline-treated groups, except that, at termination, the animals fed 200

ppm (80% MTD) diallyl disulfide showed slightly but significantlylower (about 5%) body weights than did those fed the control diet

(Fig. 2). Saline-treated animals weighed more than those treated with

AOM in all groups (data not shown).Tumor Data. In the present study, >90% of colon tumors were

adenocarcinomas and the remainder were benign adenomas. Sincethere were no differences in the incidence or multiplicity of colonadenomas between the control and experimental groups. Table 2 summarizes only the results for adenocarcinomas. Adenocarcinomas wereclassified as invasive or noninvasive type based on invasion intomuscularis mucosa. Tumors invading beyond the submucosa intomuscularis mucosa were classified as invasive carcinomas, whereasthose that had developed exophytically with the roots above the muscularis mucosa were termed as noninvasive carcinomas. Daily administration of 200 ppm anethole trithione significantly inhibited theincidence and multiplicity of both invasive and noninvasive adenocarcinomas of the colon, whereas anethole trithione at 100 ppm suppressed the incidence and multiplicity of invasive adenocarcinomas.The results also indicate that daily feeding of 100 or 200 ppm diallyldisulfide significantly reduced the incidence and multiplicity of invasive colon adenocarcinomas but had no effect on noninvasive adenocarcinomas. Diets containing 600 or 1200 ppm W-acetylcysteine or

taurine also decreased slightly the incidence of invasive adenocarcinomas of the colon, but the difference did not reach statistical significance (P > 0.05). The multiplicity of invasive adenocarcinomas wassignificantly inhibited in animals fed 600 or 1200 ppm taurine, but/V-acetylcysteine had no effect on the multiplicity of invasive adeno

carcinomas.Biochemical Data. The activities of GST, NAD(P)H:QR, and

UDP-GT were analyzed in liver and colonie mucosa and tumors ofAOM-treated animals fed the control diet and diets containing 80%MTD each of anethole trithione, diallyl disulfide, /V-acetylcysteine,and taurine and in liver and colonie mucosa of saline-treated animals

(Table 3). In general, AOM treatment had no measurable effect onthese enzyme activities in liver and colonie mucosa. Long term administration of anethole trithione and diallyl disulfide significantlyincreased the activities of GST, NAD(P)H:QR, and UDP-GT in liver

and colonie mucosa and tumors, compared to control diet (P <0.01-0.0001). The degree of enhancement of enzyme activities was

more pronounced in animals fed anethole trithione, compared to those

Fig. 2. Body weights of AOM-treated maleF344 rats fed the control diet and experimentaldiets containing 40 and 80% MTD levels of anethole trithione (ATT) (100 and 200 ppm), diallyldisulfide (DSS) (100 and 200 ppm), W-acetylcys-teine (NAS) (600 and 1200 ppm), and taurine( TAU ) (600 and 1200 ppm). The number of animalsin each group was 36. There were no significantdifferences in body weights among the dietarygroups ( P > 0.05) except that, at week 52. 2(X)ppmdiallyl disulfide slightly but significantly decreasedthe body weight (P < 0.05).

M

E

30»

•¿�D

OCD

500 r

375

250

125

~H~ Control diet

-•- ATT, 200ppm

~*- ATT. lOOppm

* DSS, 200ppm

-*•- DSS, lOOppm

-•- NAS,1200ppm

-* - NAS, eOOppm

-•- TAU, 1200ppm

-»- TAU, eOOppm

8 16 24 36

Weeks on experimental diets

52

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ORGANOSULFUR COMPOUNDS AND COLON CANCER INHIBITION

Table 2 Effect of organosulfur compounds on AOM-induced colon adenocarcinomas in male F344 raïs

ChemopreventiveagentstestedControl

dietAnetholetrithione. 2(K)ppmAnetholetrithione, 100ppmDiallyl

disulfide, 200ppmDiallyldisulfide. I(X)ppmW-Acetylcysteine,

1200ppmW-Acetylcysteine.600ppmTaurine,

1200ppmTaurine,600 ppm(9Invasive"348.3'\lf5.5'11'19221716Incidence

-' of animals withtumors)Noninvasive''53yf47585342444761Total'7842'58646461646166Invasive0.63

±0.18''0.11±0.06«0.28

±0.14*0.11±0.07«0.30

±0.17"0.41

±0.170.41±0.150.25±0.11*0.24

±0.09*Multiplicity

(tumors/animal)Noninvasive1

.03 ±0.200.53±0.15*(0.72

±0.170.94±0.121.03±0.190.69±0.110.66

±0.150.75±0.161.03±0.17Total.66

±0.21).64±O.I5*.00±O.I8/1.05

±0.12*.33

±0.22.10±0.21/l.08±O.I9A.00±O.I7*.27

±0.18" Invasive adenocurcinomas include those with invasion into the muscularis mucosa of colon.'' Noninvasive adenocarcinomas include those that do not invade into muscularis mucosa.' Total tumors represent invasive and noninvasive adenocarcinomas.d Mean ±SD.*"Significantly different from control diet group by x2 test, P < 0.01.

^Significantly different from control diet group by \2 test, P < 0.05.* Significantly different from control diet group by Student's < test, P < 0.01.* Significantly different from control diet group by Student's I test. P < 0.05.

fed diallyl disulfide. In contrast, W-acetylcysteine and taurine had no

significant effect on these enzyme activities in liver and colonie mucosa and tumors, except that the GST activity was found to be significantly elevated in the livers of animals fed N-acetylcysteine or

taurine.

DISCUSSION

The purpose of this investigation, which is a part of a large-scale

study on the efficacy of several naturally occurring and related synthetic agents as inhibitors of colon carcinogenesis, was to elucidate thechemopreventive properties of organosulfur compounds in an established colon cancer model. Our findings in relation to the colontumor-inhibitory effect of anethole trithione are noteworthy because

100 and 200 ppm levels of this agent significantly suppressed invasiveadenocarcinomas of the colon, which are the most severe form ofcarcinomas. In addition, 200 ppm anethole trithione inhibited lesssevere noninvasive adenocarcinomas of the colon. Recent studiesfrom our laboratory have demonstrated that oltipraz, a substituteddithiolethione, administered at both the initiation and postinitiationstages of carcinogenesis inhibits invasive and noninvasive adenocarcinomas of the colon (36). Indeed, the observations of our current and

previous studies with colon cancer (36) and the results of earlierstudies with mammary gland, liver, pancreas, forestomach, bladder,and skin cancer models (15, 23, 35) collectively suggest that thesubstituted dithiolethiones appear to possess chemopreventive properties against several classes of carcinogens in many target organs.Diallyl disulfide has been shown to be effective in the prevention ofcarcinogenesis in a number of animal models including DMH-inducedcolon carcinogenesis (29) and diethylnitrosamine-induced forestom

ach and lung tumor development (25). In the present study, 100 and200 ppm diallyl disulfide significantly inhibited AOM-induced invasive adenocarcinomas of the colon. In contrast, the effects of N-ace-

tylcysteine and taurine in modulating colon tumor inhibition weresomewhat marginal. Collectively, the results of current and earlierstudies (36) indicate that, (a) of all organosulfur compounds tested,oltipraz and anethole trithione are the most effective chemopreventive agents in reducing the risk of colon cancer, followed by diallyldisulfide, and (b) the dithiolethiole nucleus of substituted dithiolethiones may be a contributing factor to colon cancer chemopreventive activity.

Although the mechanisms by which anethole trithione and diallyldisulfide exert their inhibitory effects in AOM-induced colon carcino-

Table 3 Effect of organosulfur compounds on liver and colonie mucosal and Iunior enzyme activities

Liver Colon

Mucosa Tumors

GST NAD<P)H:QR UDP-GT GST NAD(P)H:QR UDP-GT GST NALXP)H:QR UDP-GT((imol/mg/minl" (nmol/mg/min) (nmol/mg/min) l/imol/mg/minl (nmol/mg/min) (nmol/mg/min) (/¿mol/mg/min) (^mol/mg/min) (nmol/mg/min)

AOM-treatedControldietAnethole

trithione.200ppmDiallyl

disulfide.200ppmW-Acetylcysteine.1200

ppmTaurine,1200ppmSaline-treatedControl

dietAnetholetrithione.200

ppmDiallyldisulfide.200

ppmA/-Acetylcysteine,1200

ppmTaurine.1200 ppm720

±114*6196±906'2662

±372''1076

±135'953

±120'678

±1285476±1156''2553

±296'1103±

173'988

±102'158

±34889±127''277

±33rf194

±24219±42176

±36889±127r264

±43d185

±43181

±443.3

±0.610.9±1.7'5.8

±0.7'4.0

±0.73.3

±0.53.0

±0.610.0±1.5r5.4

±0.6r3.6

±0.52.9

±0.5372

±502010±262''!089±

174'353

±36415

±32336

±641894±106'1098

±114''358

±50414

±67450

±572702±381''660

±49^450

±55440

±41438

±642424±283'649

±49"'449

±58448

±391.7

±0.36 209 ±44 336 ±383.6±0.6'' 1302±171' 2501±421'2.0

±0.2 960 ±117' 642±54'1.6

±0.3 347 ±50"' 478 ±821.5

±0.3 276 ±70 410±531.6

±0.33.9±0.4'2.0

±0.41.9

±0.21.5

±0.31.8

±0.23.7±O.(f2.5

±0.4'1.

9±0.31.8

±0.3

"GST activity is expressed as jimol l-chloro-2,4-dinitrobenzene conjugated/mg protein/min, NAD(P)H:QR activity is expressed as ^tmol 2,6-dichloroindophenol reduced/mgprotein/min, and UDP-GT activity is expressed as nmol p-nitrophenol conjugated/mg protein/min.

* Values are mean ±SD (n = 6).' Significantly different from the control group by Student's t test, P < O.OXX)!.'' Significantly different from the control diet group by Student's i test, P < 0.001.*'Significantly different from the control diet group by Student's / test, P < 0.01.

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genesis remain to be elucidated, the inhibition of tumorigenesis bythese agents can proceed through several mechanisms, as discussed byWattenberg (6) and Talalay (35) for other chemopreventive agents. Atenable possibility is that these agents alter the metabolism of carcinogens in the target organs. The metabolic activation of AOM to areactive species capable of alkylating DNA occurs through the hy-

droxylation of AOM to methylazoxymethanol in the liver, and themetabolism of methylazoxymethanol to a highly reactive electrophile,namely methyldiazonium ion, which can methylate cellular nucleo-

philes including DNA occurs in both liver and colon (42, 43). Severalcolon tumor inhibitors, such as pyrazole, disulfiram or its metabolites,oltipraz, and benzylselenocyanate, have been shown to alter AOMmetabolism and/or DNA adduci formation in the colon, thereby inhibiting carcinogenicity (24, 44-46). The mechanistic studies carriedout by Kensler et al. (23) on the inhibition of AFB, -induced liver

tumorigenicity by oltipraz demonstrated that this agent reduced thebinding of AFB, to DNA in male F344 rats. It is, therefore, likely thatanethole trithione and diallyl disulfide might increase the rate ofdetoxification of AOM in liver and colon, thereby decreasing theformation of carcinogen metabolites and reducing AOM-induced co

lon carcinogenesis by these agents.Another possible mechanism of the colon tumor-inhibitory action

of these agents is through an increase in the levels of detoxifyingenzymes in the liver and colon. Substituted dithiolethiones such asoltipraz and anethole dithione have been shown to increase glu-

tathione levels, GST activity, and other detoxifying enzyme activities(23, 35, 47). It was also reported that, in AFB,-induced liver carcino

genesis, modifications of AFB, metabolism and disposition producedby alterations in the activities of both phase I and phase II enzymes aremajor components of the chemopreventive activity of dithiolethionesin this model (23). Our recent study demonstrated that dietary oltiprazinduced colonie and liver GST, NAD(P)H:QR, and UDP-GT activities

(37). As observed in the present study, administration of anetholetrithione strikingly increased the activities of GST, NAD(P)H:QR, andUDP-GT in the liver and colonie mucosa and tumors of animalstreated with AOM and the liver and colonie mucosa of saline-treated

animals. Although diallyl disulfide significantly increased the activities of GST and NAD(P)H:Q in liver and colonie mucosa and tumors,the degree of enhancement of these enzyme activities was more profound in anethole trithione-fed animals, compared to animals fed

diallyl disulfide. This observation is supported by the finding in thecurrent study that the degree of colon tumor inhibition for both invasive and noninvasive adenocarcinomas is more pronounced in anethole trithione-fed animals than in animals fed diallyl disulfide. Taken

together, these observations corroborate the inhibitory effect of anethole trithione and diallyl disulfide on invasive and/or noninvasiveadenocarcinomas of the colon.

In conclusion, the results of this study demonstrate that dietaryanethole trithione and diallyl disulfide inhibit colon carcinogenesisand that the degree of inhibition is more pronounced with anetholetrithione than diallyl disulfide. Although the exact mechanisms involved in their protective effects against colon carcinogenesis are notclearly understood at present, the results for colonie mucosal and liverGST, NAD(P)H:QR, and UDP-GT activities should provide a stimu

lus to design additional studies on the mechanisms of colon tumorinhibition by anethole trithione and diallyl disulfide.

ACKNOWLEDGMENTS

We thank Donna Virgil for preparation of the manuscript and the staff

of the Research Animal Facility and Histology Facility for their technical

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