Mechanisms of Inhibition of Chemical Toxicity and Carcinogenesis by Diallyl Sulfide (DAS) and Related Compounds From Garlic

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Recent Advances on the Nutritional Effects Associated withthe Use of Garlic as a Supplement

Mechanisms of Inhibition of Chemical Toxicity and Carcinogenesis by

Diallyl Sulde (DAS) and Related Compounds from Garlic1,2

Chung S. Yang, 3 Saranjit K. Chhabra, Jun-Yan Hong and Theresa J. SmithLaboratory for Cancer Research, College of Pharmacy, Rutgers, The State University of New Jersey,Piscataway, NJ 08854-8020

ABSTRACT Diallyl sulde (DAS) is a avor compound derived from garlic and is sequentially converted to diallylsulfoxide (DASO) and diallyl sulfone (DASO 2 ) by cytochrome P 450 2E1 (CYP2E1). These compounds have beenshown to reduce the incidence of a multitude of chemically induced tumors in animal models. The impediment ofphase I activation of these carcinogens is hypothesized to be accountable for the reduction in tumor incidence.Indeed, DAS, DASO and DASO 2 are competitive inhibitors of CYP2E1. DASO 2 , in addition, is a suicide inhibitor ofCYP2E1. These compounds have been shown to reduce carbon tetrachloride-, N-nitrosodimethylamine- and

acetaminophen-induced toxicity in rodents. All three chemicals are substrates for CYP2E1. The protective effectwas observed when the organosulfur compounds were given before, during or soon after chemical treatment. DASand DASO 2 inhibited the bioactivation of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and related lungtumorigenesis in A/J mice. Because CYP2E1 does not play a key role in NNK activation, the inhibition of other CYPenzymes active in NNK metabolism is likely. DAS also has been shown to induce other CYP and phase II enzymesas well as decrease hepatic catalase activity. All of these effects are observed at concentrations much higher thanwhat is normally ingested by humans. The biological activities of garlic and its related compounds at lowerconcentrations that mimic human consumption remain to be studied further. J. Nutr. 131: 1041S1045S, 2001.

KEY WORDS: garlic diallyl sulde cancer chemoprevention cytochrome P 450 acetaminophen

Garlic ( Allium sativum) is a bulbous root with a strong tasteand smell. It is used widely in culinary preparations and as afolk medicine. Epidemiologic studies in China and Italy indi-cate that frequent consumption of garlic and other alliumvegetables may be associated with decreased gastric cancerincidence (Buiatti et al.1989, You 1989). Animal studies haveshown inhibition of chemically induced skin (Belman 1983,Sadhna and Rao 1988, Singh and Shukla 1998a and 1998b),cervical (Hussain et al. 1990), forestomach (Sparnins et al.1988), lung (Sparnins et al. 1988), colon (Wargovich 1987)and esophageal tumors (Wargovich et al. 1988) with garlic orrelated compounds. These and other biological activities of garlic have been reviewed (Fukushima et al. 1997, Lau et al.1990, Milner 1996, Wargovich et al. 1996).

One of the constituents of garlic, diallyl sulde (DAS),4which is responsible in part for its strong taste and odor, hasbeen shown by our laboratory and others to inhibit selectivelyas well as induce certain P450 enzymes (Brady et al. 19881991a and 1991b, Yang et al.1994a), and thus may explainsome of the anticarcinogenic actions of garlic. The inhibitoryactions and mechanisms of DAS and other biologically activeconstituents of garlic will be discussed in this paper.

Metabolism of DAS

Alliin ( S-allylcysteine sulfoxide), the prime organosulfurcomponent of garlic, is an unstable compound, which forms amultitude of breakdown organosulfur products through theaction of the enzyme allinase, cooking or metabolism in ani-mals (Block 1985).

DAS, a lipophilic thioether, is one of these compounds,

found exclusively in garlic. It can further undergo extensive1

Presented at the conference Recent Advances on the Nutritional Benets Accompanying the Use of Garlic as a Supplement held November 1517, 1998in Newport Beach,CA. Theconference wassupported by educational grantsfromPennsylvania State University, Wakunaga of America, Ltd. and the NationalCancer Institute. The proceedings of this conference are published as a supple-ment to The Journal of Nutrition . Guest editors: John Milner, The PennsylvaniaState University, University Park, PA and Richard Rivlin, Weill Medical College ofCornell University and Memorial Sloan-Kettering Cancer Center, New York, NY.

2 Supported by National Institutes of Health research grants ES03938 andES05693.

3 To whom correspondence should be addressed.E-mail: [email protected].

4 Abbreviations used: AFB 1 , aatoxin B 1 ; APAP, acetaminophen; BP, benzo-(a)pyrene; CYP, cytochrome P 450 ; DADS, diallyl disulde; DAS, diallyl sulde;DASO, diallyl sulfoxide; DASO 2 , diallyl sulfone; DATS, diallyl trisulde; DMH,1,2-dimethylhydrazine; EROD, 7-ethoxyresorun O-deethylase; FGH, fresh garlichomogenate; GPx, glutathione peroxidase; GSH, glutathione; GST, glutathioneS -transferase; IC 50 , 50% inhibitory concentration; IQ, 2-amino-3-methyl-imi-dazo[4,5-f]quinoline; NDMA, N-nitrosodimethylamine; NNK, 4-(methylnitro-samino)-1-(3-pyridyl)-1-butanone; NQO, NAD(P)H: quinone oxidorectuase;PROD, 7-pentoxyresorun O-dealkylase; UDPG, uridine diphosphate glucose.

0022-3166/01 $3.00 2001 American Society for Nutritional Sciences.

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oxidation at three positions, i.e., the sulfur atom, the allyliccarbon and the terminal double bonds. Cytochrome P 450(CYP) enzyme-mediated oxidation at the sulfur atom of DASproduces diallyl sulfoxide (DASO) and diallyl sulfone(DASO 2 ), sequentially (Brady et al. 1991a). The avin-de-pendent monoxygenase can also catalyze these conversions(unpublished results). These metabolites are further convertedto epoxide intermediates (Jin and Baillie 1997).

Jin and Baillie (1997) identied 10 glutathione (GSH)conjugates in the bile of rats dosed with DAS (200 mg/kg). Nine of these were found in the bile of rats treated with DASO(226 mg/kg) and six in those treated with DASO 2 (256 mg/kg). Identication of the structures of these conjugates indi-cated that DAS undergoes extensive oxidation at variouspositions (the sulfur atom, the allylic carbon and the terminaldouble bonds) in the molecule. Six metabolites were GSHconjugates of epoxides of DAS, DASO and DASO2 . DASOand DASO 2 are more likely to undergo oxidation at theterminal double bonds, giving rise to epoxides that are subse-quently conjugated with GSH. It has also been shown thatDASO 2 is not reduced into DASO, which is not reduced intoDAS. Further, these observations suggest that oxidation at thesulfur atom is favored over the other positions, and that this isconsistent with the competitive CYP2E1-inhibitory propertiesof DAS and DASO.

CYP2E1-mediated sequential oxidation of DAS to DASOand DASO 2 has been demonstrated, but other enzymes mayalso catalyze the oxidation. The oxidation of the terminaldouble bonds of DASO2 by CYP2E1 is the key event leadingto the autocatalytic destruction of the enzyme, rst observedby Brady et al. (1991a), whereas formation of other electro-philic species such as allyl sulnic acid and acrolein may alsoplay a role in vivo (Jin and Baillie 1997).

Effects of DAS on CYP and conjugation enzymes

Effect on CYP and phase I activation. Our laboratory hasshown that a single oral dose of 200 mg/kg of DAS to ratsproduced a signicant increase in hepatic pentoxyresorundealkylase (PROD) activity, which reached a plateau of 100-fold at 2448 h (Brady et al.1988, Yang et al. 1994a). Subse-quent studies showed that this was due to an induction of CYP2B1 at the transcriptional level (Pan et al. 1993a and1993b). The activity of hepatic N-nitrosodimethylamine(NDMA) demethylase (an activity manifested by CYP2E1)decreased after DAS administration in a time-dependent man-ner (lowest at 15 h and returned to normal after 2 d). DASO2administration caused a rapid decrease in NDMA demethylaseactivity, observable after 10 min and reduced to only 25% of the control at 2 h (Brady et al. 1988 and 1991b). A single dose

of DAS caused only slight changes in total CYP and NADPH-cytochrome c reductase activity and slightly decreased 16 -testosterone hydroxylase activity (due to CYP3A) in liver(Brady et al. 1991b). A slight decrease in 7-ethoxyresorunO-deethylase (EROD) activity (due to CYP1A2) was observedin mice fed DAS (200 mg/kg) or DASO2 (50 mg/kg) (Yang etal. 1994a). DASO 2 was ineffective in the inactivation of PROD and benzphetamine demethylase activity (Brady etal.1991b) but did slightly inhibit (1 mmol/L) activities of CYP3A and 1A with 50% inhibitory concentration (IC 50 )values of 5 mmol/L (Lin et al.1996).

In studies assessing the competitive inhibition of p-nitro-phenol hydroxylase activity (mainly CYP2E1 mediated) andsuicide inhibition of CYP2E1 in rat liver microsomes, the K i

value for DASO2 was 188 mol/L and the maximal rate of inactivation was 0.32 min 1 . DAS and DASO are also com-

petitive inhibitors of CYP2E1-catalyzed reactions, but are noteffective suicide inhibitors (Brady et al. 1991a).

The effects of repeated treatment with DAS on drug-metabolizing enzymes were studied in our laboratory (unpub-lished results). Changes in total CYP content was observedonly with treatments of 50 mg/kg DAS for 8 d (1.5-foldincrease) and 200 mg/kg for 29 d ( twofold decrease). Cyto-chrome b5 content and NADPH-cytochrome c reductase ac-tivity increased signicantly in rat liver in a dose- and time-dependent manner, with DAS treatment (unpublishedresults), e.g., a dose of 200 mg/kg DAS for 29 d resulted iincreases from 4.74 to 6.1 nmol/mg for cytochrome b5 andfrom 302 to 609 U/mg for NADPH-cytochrome c reductaseactivity. Activity of NDMA demethylase was found to de-crease in rat lung and kidney microsomes ( 1.5-fold in lungand 1.5- to 2.5-fold in kidney). PROD activity in the rat lungdecreased 4- to 10-fold with DAS. Pulmonary EROD de-creased 1.2- to 1.4-fold with high doses of DAS (unpublishedresults). Srivastava et al. (1997) also found a decrease inEROD activity in mouse lung with DAS and a small butsignicant increase in liver.

Chronic intragastric administration of DAS [50 and 200mg/(kg d)] to rats over a period of 4 wk resulted in a decreasof hepatic NDMA demethylase activity and CYP2E1 level aswell as the appearance of DASO (45 g/mL), DASO2 (11.7

g/mL) and elevated acetone levels (2.02.8 g/mL for thelower dose and 3.43.9 g/mL for the higher dose) in theblood (Chen et al. 1994). These observations were consisten tover time and did not display a cumulative effect. A singleacute dose of DAS (50 or 200 mg/kg) also resulted in a six- andninefold higher blood acetone level, which returned to norma lafter 48 and 12 h, respectively. Acetone, which is one of theketone bodies produced in the metabolism of fatty acids, is asubstrate of CYP2E1. CYP2E1 inactivation blocks the oxida-tion of acetone to acetol and methylglyoxal and leads to theelevation of acetone in blood. These observations are consis-

tent with the competitive inhibition of CYP2E1 by DAS,DASO and DASO 2 . Effect on glutathione S -transferase (GST) and phase II

detoxication. DAS has also been shown to induce GST- in mouse liver and forestomach (Hu et al. 1996a, Hu andSingh 1997) and GST- and - in rat liver (Dragnev et al.1995), glutathione peroxidase and glutathione reductase(Maurya and Singh 1991). However, the induction of thedetoxication enzymes is a rather slow process and, in manycases, low in magnitude (Maurya and Singh 1991, Sparnins etal.1988). Sheen et al. (1996) showed signicant decrease inGST, glutathione reductase, and glutathione peroxidase(GPx) activities in cultured rat hepatocytes treated with 5mmol/L DAS. We found no change in GPx or superoxide

dismutase activities in DAS-treated (50 or 200 mg/kg for 8 or29 d) rat liver, kidney, lung, and brain (Chen et al. 1999).Treatment of rats with 50 or 200 mg/kg DAS for 8 d

resulted in signicant reduction of liver catalase activity (55and 95%, respectively) and a synchronous decrease in catalaseprotein (Chen et al. 1999). No change occurred in kidney,lung and brain catalase, but a slight decrease of heart catalaseactivity was observed. Garlic homogenates but not DASO2also reduced catalase activity in mouse liver. DAS or DASO2given in vitro did not affect catalase activity.

We observed signicantly increased GST activity in ratliver, brain and kidney (1.3- to 2.8-fold in liver at 200 mg/kgfor 8 d and 50 and 200 mg/kg for 29 d; 1.3-fold in brain at 5and 200 mg/kg for 8 d and 200 mg/kg for 29 d; 1.2-fold in

kidney at 50 mg/kg for 8 d) with DAS treatment (unpublishedresults). Rat sulfotransferase activity increased marginally, but

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signicantly, in liver, lung and brain at the higher doses (1.2-to 1.5-fold). Decreased sulfotransferase activity was observedin kidney at all doses and treatment times (1.3- to 3.2-fold)and heart (1.3-fold at 50 mg/kg for 29 d). Uridine diphosphateglucose (UDPG)-transferase activity was found to increase 8-to 12-fold in liver at all doses and time periods, twofold inkidney and threefold in brain (200 mg/kg DAS for 8 d). NAD(P)H: quinone oxidoreductase (NQO) activity increasedsignicantly with DAS treatment in rat liver ( twofold for200 mg/kg for 29 d), kidney (1.2- to 1.6-fold for 200 mg/kg for8 d and 50 and 200 mg/kg for 29 d), lung ( 1.2 to 1.9-fold for200 mg/kg for 8 d and 50 and 200 mg/kg for 29 d), heart( 1.4-fold for 200 mg/kg DAS for 8 and 29 d), and brain( 1.2- to 1.4-fold for all doses) (unpublished results).

Singh et al. (1998) showed that treatment of mice withdiallyl disulde (DADS) and diallyl trisulde (DATS) [potentinhibitors of benzo(a)pyrene-induced forestomach tumors] re-sulted in a signicant increase (2.4- and 1.5-fold, respectively)in forestomach NQO activity. These were much more potentinducers of NQO activity than DAS, which is a poor inhibitorof BP-induced forestomach tumors. In mouse lung, DAS,which is a strong BP-induced pulmonary tumor inhibitor,induced NQO 3.2-fold. DAS and DADS were shown recentlyto inhibit, in a dose-dependent manner, the activity of N-acetyltransferase (NAT) in a human colon adenocarcinomacell line (Chen et al. 1998).

Effect on chemically induced hepatotoxicity. CCl 4 and NDMA are activated by CYP2E1 and produce hepatotox-icity. DAS (1.75 mmol/kg), when administered to ratsbefore NDMA (75 mg/kg) or CCl4 (1 mg/kg), effectivelydiminished the elevation of serum transaminases (gluta-mate-pyruvate transaminase and glutamate-oxaloacetatetransaminase) (Brady et al. 1991b). This likely broughtresults from the inhibition of CYP2E1.

Acetaminophen (APAP) is a leading analgesic and antipy-retic drug used in the United States. Overdose is known to

cause hepatotoxicity and nephrotoxicity in humans and ro-dents. Although 90% of APAP is converted into sulfate andglucouronide conjugates and excreted in the urine, a smallportion is metabolized by CYP2E1 and 3A4 to N-acetyl- p-benzoquinoneimine (Patten et al. 1993). This can arylatecritical cell proteins and cause toxicity. Our laboratory hasinvestigated the action of organosulfur compounds of garlicagainst APAP-induced hepatotoxicity in detail and foundprotective effects.

DAS (50 mg/kg) administered 3 h before APAP (0.75 g/kg)signicantly protected Fischer rats from hepatotoxicity (Hu etal. 1996b). DASO 2 (25 mg/kg) given 1 h before, immediatelyafter or 20 min after APAP, completely protected mice fromdevelopment of hepatotoxicity (Lin et al. 1996). This protec-

tive effect was observed with doses as low as 5 mg/kg given 1 hbefore treatment and was dose and time dependent (Yang etal. 1994a). DASO 2 (0.1 mol/L) inhibited the rate of APAPoxidation to N-acetyl- p-benzoquinoneimine-glutathione 6075%, primarily due to the inhibition of CYP2E1 (IC50 0.11mmol/L) and partly by inhibition of CYP3A4 and CYP1A2(Lin et al. 1996, Yang et al. 1993).

Fresh garlic homogenates (5 g/kg) given to mice before orimmediately after APAP treatment (0.2 g/kg) reduced hepa-totoxicity as measured by serum levels of alanine aminotrans-ferase and lactate dehydrogenase (Wang et al. 1996). Of theorganosulfur compounds tested, DAS (0.2), DASO (0.2) andDASO 2 (0.2 mmol/kg) were the most potent when given 1 hbefore APAP. Diallyl disulde had little protective effect.

Dimethyl sulfoxide was protective at higher doses (2.0 mol/kg). Thus, substitution of the allyl group decreased potency.

This decrease was also seen with substitution of the thiol groupwith allyl, methyl or ethyl groups. Acetylation of the aminogroup slightly increased potency (Wang et al. 1996).

Inhibition of carcinogenesis

Effect on NNK-induced lung tumors in mice. NNK is apotent tobacco carcinogen believed to be important in theetiology of human oral cancer in tobacco chewers and lungcancer in cigarette smokers (Hecht and Hoffmann 1988). Ourlaboratory demonstrated the decrease of NNK activation inmouse lung and rat nasal mucosa microsomes after an oral doseof DAS (200 mg/kg) (Hong et al. 1991 and 1992). The

-methylene oxidation pathway, which leads to the genera-tion of a methylating agent and keto aldehyde, was decreasedby 7080% in mouse lung and liver microsomes. This wasmore pronounced in rat nasal microsomes compared withliver. Because NNK activation is not mediated by CYP2E1,other mechanisms must be responsible for the reduced activa-tion and must be elucidated.

In vitro studies demonstrate a dose-dependent inhibition of DAS (0.52 mmol/L) on NNK oxidative metabolism in mouselung (Hong et al. 1994). This suggests that the decreasedmetabolic activation could be contributed to in part by thedirect inhibition by DAS or its metabolites on NNK-metab-olizing enzyme activity(ies).

We demonstrated the inhibition of NNK metabolism byDAS using the A/J mouse lung carcinogenesis model, whichdevelops 100% tumors after a single dose of NNK (Hong et al.1992). When DAS was given at a dose of 200 mg/kg 3 d beforeand 2 h before a single dose of NNK (2 mg), there was asignicant reduction in tumor incidence of 60% (from 100 to38%) and tumor multiplicty of 90% (from 7.2 1.1 to 0.6

0.2) (Hong et al. 1992). When given as a single dose (100mg/kg) 2 h before NNK, DASO2 reduced the tumor incidenceby 50% and tumor multiplicity by 91%. A lower dose o

DASO 2 (20 mg/kg) caused a 38% reduction of tumor multi-plicity but not of tumor incidence.Surprisingly, a 3-d treatment with fresh garlic homogenate

(FGH) (5 g/kg) before a single NNK dose signicantly in-creased the tumor multiplicity in mice (unpublished results).This experiment was repeated twice and increases of 88 and52% were observed, respectively. A 7-d treatment with FGHgiven 1 wk after NNK did not enhance tumor incidence ormultiplicity. Neither did fresh garlic juice (5 g/kg) when givenfor 3 d before NNK nor a daily dose of 1 g/kg FGH given beforor after NNK or 3% FGH in the drinking water for 9 wk(started 1 wk before NNK) (unpublished results). This suggeststhat FGH contains compound(s) that enhance lung tumori-genicity of NNK in the early initiation stage. Preliminary

experiments indicated that FGH may have enhanced thehyperproliferation of bronchiolar cells after NNK treatment.Lung and liver microsomes from mice treated with FGH (1and 5 g/kg) for 3 d demonstrated a dose-dependent inhibitionin rates of formation of keto aldehyde and keto alcohol ( -oxidation pathway of NNK). In mice receiving the higher 5g/kg dose of FGH, a signicant reduction in NNK-inducedDNA methylation in liver was observed, consistent with low-ered rates of -hydroxylation. In lung, however, a 30% in-crease in DNA methylation was observed 48 h after NNKadministration, and this may contribute to enhanced carcino-genesis (unpublished observations).

DAS has been shown to inhibit BP-induced forestomachand pulmonary adenoma (Sparnins et al. 1988), 1,2-di-

methylhydrazine (DMH)-induced colon tumors (Sumiyoshiand Wargovich 1990, Wargovich 1987), diethylnitro-

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samine-induced hepatocellular adenomas (Pereira 1995), N-nitrosomethylbenzylamine-induced esophageal tumors(Wargovich et al. 1988 and 1992), aatoxin B 1 (AFB1 )-and NDEA-induced liver preneoplastic foci (Haber-Mig-nard et al. 1996), 2-amino-3-methyl-imidazo[4,5-f]quino-line (IQ)-induced hepatocarcinogenesis initiation (Tsudaet al. 1994), aristolochic acid-induced forestomach tumors(Hadjiolov et al. 1993) in rats, and dimethylbenzanthra-cene-induced buccal pouch and forestomach tumors inhamsters (Nagabhushan et al. 1992).

DAS has been shown by others to inhibit AFB1-, vinyl car-bamate- and NDMA-induced mutagenesis (Haber-Mignard et al.1996, LeBon et al. 1997, Surh et al. 1995, Tadi et al. 1991a and1991b), and DNA binding and adduct formation (Ludeke et al.1992, Tadi et al. 1991a and 1991b). DAS, when added to boiledpork juice, was found to inhibit the formation of three heterocy-clic amines, i.e., IQ, 2-amino-3,4-dimethylimidazo[4,5-f]quinoxa-line and 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (Tsai et al.1996).

Although the exact mechanism(s) for inhibition of carci-nogenesis and toxicity by garlic and its compounds remainvague, many hypotheses have been proposed. The ability of DAS (and DASO 2 ) to suppress carcinogen-activating P450(CYP2E1), induce phase II GST and scavenge ultimate car-cinogenic species may all contribute, singly or in combination,to the reduction of chemical insult.

The inhibitory activity of DAS against DMH- (Hayes et al.1987) and NNK-induced tumorigenesis is probably due to inhi-bition of the bioactivation of these carcinogens. In the case of DMH, inactivation and inhibition of CYP2E1 is the most likelymechanism, preventing the oxidation of the rst intermediate of DMH, azoxymethane, into methylazoxymethanol, and then tothe hypothetical intermediate, methylazoxyformaldehyde, whichis further converted into the methylating species, methyldiazo-nium hydroxide (Bertram 1989). Based on this, allyl mercaptan,diallyl disulde and dipropyl sulde, which are not effective

CYP2E1 inhibitors, as expected, were not effective in the inhi-bition of tumorigenesis (Yang et al. 1994b). Diallyl disulde andallyl mercaptan, however, were found to be effective in inhibiting NDEA-induced forestomach tumors in mice (Wattenberg et al.1989), whereas DAS was not. Apparently this is through mech-anisms independent of CYP2E1.

Miscellaneous

In addition to the above, garlic has also been shown to haveantibacterial (Cellini et al. 1996, Farbman et al. 1993, Feld-berg et al. 1988), hypoglycemic (Chang and Johnson 1980, Jain and Vyas 1975, Sheela et al. 1995), hypolipdemic (Adlerand Holub 1997, Mathew et al. 1996, Yeh and Yeh 1994) and

antiatherosclerotic (Ide and Lau 1997, Munday et al. 1999,Orekhov and Tertov 1997) properties.

Conclusions and remarks

Garlic and its related compounds (DAS, DASO andDASO 2) have inhibitory effects on chemical carcinogenesisand mutagenesis. The ability of these compounds to compet-itively inhibit a major carcinogen activating enzyme, CYP2E1,is a viable mechanism in systems in which CYP2E1 substratesare used as the carcinogens. These compounds may inhibit theactivation of other carcinogens at low efciency. The induc-tion of GST and phase II enzymes may also play a role. Othermechanisms should be explored.

Although a reduction of hepatic injury is observed withDAS, the inhibition of hepatic activation enzymes, and con-

sequently rst-pass clearance, may increase the blood level othe unmetabolized carcinogen and in turn increase exposure of extrahepatic, downstream organs to the carcinogen (Andersonet al. 1995). Thus, the levels of dietary compounds used toinhibit carcinogenesis must be studied and analyzed carefully.

Additional studies on the chemopreventive nature of thesecompounds on human cancers are warranted. A major con-cern, which must be addressed when extrapolating animalndings to humans, is the dosage of the agent studied. Thelevels of DAS and DASO2 used in many studies are muchhigher than what is normally consumed by humans. This willhave to be taken into consideration in the design of humanepidemiologic studies and in evaluating the role of garlic andits related compounds as chemopreventives.

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Mechanisms of Inhibition of Chemical Toxicity and Carcinogenesis by Diallyl Sulfide (DAS) and Related Compounds From Garlic