(CANCER RESEARCH 48, 5937-5940, November 1, 1988]

Effect of Diallyl Sulfide on Rat Liver Microsomal Nitrosamine Metabolism andOther Monooxygenase Activities1

John F. Brady, Dechun Li,2 Hiroyuki Ishizaki, and Chung S. Yang3

Department of Biochemistry, University of Medicine and Dentistry of New Jersey, Newark, New Jersey 07103, and Department of Chemical Biology and Pharmacognosy,College of Pharmacy, Rutgers university, Piscataway, New Jersey 08S55

ABSTRACT

It has been reported that p.o. administration of dialiyl sulfide (DAS),a naturally occurring component of garlic (Allium sativum), inhibits 1,2-dimethylhydrazine-induced colon and liver cancer in rodents. A possiblemechanism for this protective effect is inhibition of hepatic activation ofthe procarcinogen. The effect of DAS on P450IIE1, an isozyme ofcytochrome P-450 which is active in the oxidative metabolism of di-methylhydrazine, was conveniently assayed in the present study by determination of A'-dimothylnitrnsamine demethylase (NDMAd) activity at1 HIMA'-dimethylnitrosamine in Sprague-Dawley rat liver microsomal

incubations. DAS was found to be a competitive inhibitor of NDMAd, incontrast to the irreversible inactivation of NDMAd produced by carbontetrachloride incubated under similar conditions. The inhibition by DASof the demethylation of several substrates was selective. The thioetherwas most potent against .'V-dimcthylnitrosaminc, less effective against ,V-nitrosomethylbenzylamine, and essentially ineffective against benzphet-amine and ethylmorphine. Microsomes prepared at 3 h after DASadministration (200 mg/kg in corn oil intragastrically) showed moderateinhibition (<30% inhibition compared to control microsomes) of severaldemethylase activities; however, microsomes prepared 18 h posttreatmentshowed a marked decrease (about 80% inhibition compared to controls)in NDMAd activity, minor effects on other demethylase activities, and a6-fold increase in pentoxyresorufin dealkylation. These trends at 18 hagreed with immunoblot analyses which showed suppression in the levelof P450IIE1 and an elevation in P450IIB1. The selective inhibition ofP4SOIIE1 activity and suppression of its level in microsomes may contribute to the reported chemoprotective effects of DAS.

INTRODUCTION

The identification and exploitation of dietary an t¡carcinogensmay substantially contribute to the prevention of cancer inhumans. The salutary properties of garlic (Allium sativum), awidely consumed herb, have long been recognized in folklore(3) and are a current subject of scientific investigation (4-7).Diallyl sulfide [(CH2=CHCH2)2S], a component of garlic oil,has recently been shown to possess potent inhibitory activitytoward the induction of colon (8, 9) and liver (10) cancer by1,2-dimethylhydrazine (CH3NHNHCH3) and toward esopha-geal cancer induced by NMBA4'5 in laboratory animals. The

bioactivation of dimethylhydrazine involves the sequential hepatic oxidation of azomethane (CH3N=NCH3) to azoxy-methane [CH3N=N(O)CH3] to methylazoxymethanol[HOCH2N=N(O)CH3] (11). Since the virtual absence of this

Received 4/5/88; revised 6/15/88; accepted 7/29/88.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 inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1Supported by NIH Grants ES-03938 and CA-37137.2On leave from the Department of Biochemistry, Tianjin College of Tradi

tional Chinese Medicine, Tianjin, China.3To whom requests for reprints should be addressed, at Rutgers University.4The abbreviations used are: NMBA, Ar-nitrosomethylbenzylamine; DAS,

diallyl sulfide; NDMA, .V-nitrosodiniethylaminc; P-450, cytochrome P-450. Thenomenclature of the P-450 isozymes follows the convention in Ref. l. IsozymeP450IIE1, encoded by the rat gene P450HE1, has also been referred to as P-450«by our laboratory and as P-450j by Ryan et al. (2).

5M. J. Wargovich, C. Woods, V. W. S. Eng, L. C. Stephens, and K. N. Gray.Complete inhibition of A'-nitrosomethylbenzylamine-induced esophageal cancer

in rats by the naturally occurring thioether, diallyl sulfide, submitted for publication.

last catalytic activity in colon mucosa has been demonstrated(11) and probable routes for the transport and exposure of thereactive metabolites to the colon exist (11, 12), a potentialmechanism for the anticarcinogenic property of DAS is inhibition of the initial hepatic conversions. Other sulfur-containingcompounds have been shown to block azomethane ./V-oxidationin vivo (13).

As recently demonstrated using microsomal (14) and purifiedenzyme systems,6 oxidation of azoxymethane to methylazoxy

methanol is catalyzed by cytochrome P450IIE1. This isozymeis present constitutively in mammalian species (15-17) and isinducible by acetone or isoniazid treatment, fasting, diabetes,and numerous other factors (see Ref. 18). It is active in themetabolism of various small organic compounds such asNDMA, acetone, ethanol, carbon tetrachloride, and /i-pentane.An interaction between P450IIE1 and DAS was consideredlikely since this compound bears structural similarity to diethylether, an inhibitor of P450IIE1 -mediated reactions in vivo andin vitro and a high-affinity substrate for this isozyme (see Ref.19).

In order to elucidate the mechanism for its anticarcinogenicproperties, the effect of DAS on selected rat hepatic microsomalmonooxygenase parameters was determined.

MATERIALS AND METHODS

Materials. Diallyl sulfide was obtained from Aldrich Chemical Co.(Milwaukee, WI). Corn oil was a product of Mazóla,CPC International,Inc. (Englewood Cliffs, NJ). yV-Nitrosomethylbenzylamine (NMBA)was from Ash Stevens, Inc. (Detroit, MI). p-Nitroanisole was from theEastman Kodak Co. (Rochester, NY). Aminopyrine and ethylmorphinewere obtained from Sterling-Winthrop Research Institute (Rensselaer,NJ) and Mallinckrodt Chemical Works (St. Louis, MO), respectively.Other chemicals were obtained from sources indicated previously (19).

Animals and Microsomes. Male Sprague-Dawley rats (90-100 ginitial body weight) were maintained as described (19). The acetone andphénobarbitaltreatments were conducted using previous methods (19).Diallyl sulfide was given intragastrically at a dose of 200 mg/kg bodyweight in corn oil (1 ml/100 g body weight). Animals were sacrificedbetween 9 and 10 a.m. for preparation of microsomes (18).

Metabolic Assays and Analyses. The methods for determination ofP-450 concentration, NADPH/P-450 reducíaseactivity, and immunoblot analysis have been described (19). A previous method (18) for thedetermination of microsomal demethylase activity was used. The incubation volume was 0.5 ml unless otherwise indicated. DAS apparentlydissolves in water at a concentration of 2 HIM.For the inhibition studies,DAS was added from a fresh aqueous solution containing a 10-foldhigher concentration than in the final incubation. Preliminary analysisfor DAS utilized a Hewlett Packard 5710A gas Chromatograph withflame ionization detection and a 5% Carbowax 20M, 60/80 Carbopack-B column from Supelco (Bellefonte, PA). Statistical analyses wereperformed using Student's t test.

RESULTS

In Vitro Inhibition of Microsomal Monooxygenase Activities.DAS was a potent inhibitor of the NDMA demethylase activity

6 O. S. Sohn, H. Ishizaki, C. S. Yang, and E. S. Fiala, manuscript in prepara

tion.

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EFFECT OF DAS ON MICROSOMAL MONOOXYGENASE ACTIVITIES

displayed by acetone-induced rat liver microsomes. In incubations containing varied concentrations of substrate, an apparentKm of 25.8 ±2.3 (SD) MM(n = 3) was observed. DAS exhibitedcompetitive inhibition with an apparent K, of 26.8 ±7.2 ¿iM(n= 3) (Fig. 1).

In order to assess the selectivity of the inhibitory actions ofDAS, its effect on monooxygenase activities associated withcontrol and phenobarbital-induced microsomes was determined(Table 1). In both types of microsomes, DAS was a potentinhibitor of NDMA demethylation, a moderate inhibitor ofNMBA demethylation, and a weak inhibitor of benzphetamineand ethylmorphine demethylations. These results demonstratedthat the inhibition by DAS was not a general effect on allmixed-function oxidase activities but was somewhat selective.

Influence of DAS and Carbon Tetrachloride on NDMA De-methylase and on the Stability of Cytochrome P-450. The possibility of a metabolism-dependent inactivation of NDMA de-methylase by DAS was investigated by determination of thetime dependency of the inhibition and by comparison with theeffects of a previously characterized (20) irreversible inhibitor,carbon tetrachloride (Table 2). A 20-min preincubation, lackinginhibitors, in the absence or presence of NADPH resulted in aslight decrease in the NDMA demethylation determined in asubsequent 10-min incubation. Preincubation in the presence

1.5

O>E

\cE

\oEc

0.5

10 20 30

Fig. 1. Lineweaver-Burk plot showing the inhibition of NDMA demethylaseactivity by diallyl sulfide. The incubations contained acetone-induced microsomes(0.13 mg/ml) and NDMA (0.04 to 0.6 HIM)either with (•)or without (•)0.2mm diallyl sulfide in a final volume of 0.25 ml. The V„„was 6.31 HCHO/min/mg. Each data point represents the average from duplicate incubations.

Table 1 Effect of diallyl sulfide on monooxygenase activities associated withcontrol and phenobarbital-induced rat liver microsomes

The incubation mixtures contained control microsomes (0.43 mg/ml) or phenobarbital-induced microsomes (0.29 mg/ml) and 1 TDMconcentrations of theindicated substrate. Values are the average (with percentage of uninhibited rate)from two incubations.

Activity (nmol HCHO/min/mg) atfollowing diallyl sulfide concentration

SubstrateControl

microsomesNDMANMBABenzphetamine

EthylmorphinePhenobarbital-inducedmicrosomes

NDMANMBABenzphetamineEthylmorphine0

mM1.88

0.575.898.803.05

2.8624.5229.620.05

mM1.05(55.9)

0.53 (93.2)5.80 (98.5)8.70(98.8)1.89(62.0)

2.55 (89.2)23.47 (95.7)27.03(91.3)1.0

mM<0.05

0.16(27.3)5.30 (89.9)8.11(92.2)0.27

(8.9)1.20(41.9)

18.72(76.3)23.94 (80.8)

Table 2 Influence of incubation conditions on the inhibition by DAS or carbontetrachloride (CT) of the NDMA demethylase activity associated with

acetone-induced microsomes

In experiment 1, microsomes (0.32 mg/ml) were incubated for 10 min in thepresence of the NADPH-generating system, 1 mM NDMA, and in the absence orpresence of inhibitor (0.2 mm). In experiments 2 and 3, the microsomes werepreincubated (for 20 min) in the absence or presence of the NADPH-generatingsystem, followed by the addition of NDMA (1 mM) (plus NADPH-generatingsystem in experiment 2) for a 10-min incubation; the inhibitors were present at0.20 mM during the preincubation. Values are the mean ±SD from threeincubations. The data in each column were submitted to an analysis of varianceand tested for significant differences using the Student-Newman Keuls test.

Activity(nmol HCHO/min/nmol P-450)

ConditionsNo inhibitorDASCT

1. No preincubation2. 20-min preincubation

without NADPH3. 20-min preincubation

with NADPH

7.63 ±0.24 2.73 ±0.05 4.36 ±0.066.86 ±0.05" 3.95 ±0.17°'* 5.28 ±0.12°

7.05 ±0.07" 6.87 ±0.03°'* 5.29 ±0.31°

" Significantly different from the values in Line l, P < 0.05.* Significantly different from each other, P < 0.05.

Table 3 Effect of diallyl sulfide or carbon tetrachloride on the stability ofcytochrome P-450

Hepatic microsomes from acetone-treated rats were incubated at 37'C for 40

min in the presence of the compounds shown. The reaction was terminated bycooling to ice temperature. Values are the mean ±SD from three incubations.

IncubationconditionsWithout

incubationWith NADPH-generating system

+ Diallyl sulfide (1 mM)+ Carbon tetrachloride (1 mM)jiM

P-4501.30

±0.030.63 ±0.020.54 ±0.080.23 ±0.02%100.0

48.841.717.7

of either DAS or carbon tetrachloride, but in the absence ofNADPH, resulted in a moderate reduction in the inhibitionthat had been observed without preincubation. The inhibitionpersisted when NADPH was present during the preincubationwith carbon tetrachloride but was essentially eliminated whenDAS was preincubated under similar conditions. These observations confirmed an irreversible inactivation by carbon tetrachloride and were consistent with the competitive inhibition byDAS described above. The possibility that DAS was convertedto a noninhibitory metabolite during the preincubation wassupported by a preliminary gas Chromatographie analysis whichrevealed a >90% decrease in the concentration of DAS whenNADPH was present (not shown).

This contrast between the two inhibitors was also apparentwhen their effects on the binding of CO to dithionite-reducedmicrosomes, after a 40-min incubation, were compared (Table

3). The decrease in CO binding during 40 min in the controlwas probably due to lipid peroxidation (21). The presence ofDAS during the incubation had no effect on the CO bindingcompared to the control, while carbon tetrachloride gave apronounced decrease in measurable P-450.

Influence of DAS Treatment on Microsomal and Soluble Monooxygenase Activities. In previous in vivo studies (8-10), DASusually was administered intragastrically 1-3 h prior to exposure to procarcinogen. The foregoing in vitro studies demonstrated that DAS acted as a competitive inhibitor of certainmonooxygenase activities. The possibility that administeredDAS p.o. also exerts either an inductive or suppressive effecton these activities was also examined. A slight inhibition ofNDMA demethylase and other oxidative activities was observedin microsomes and in the postmitochondrial supernatant fraction (S-9) obtained 3 h after the treatment (Table 4). Thepercentage of inhibition in both the S-9 fraction and in microsomes was not diminished at higher NDMA concentrations.These observations were not consistent with competitive inhi-

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EFFECT OF DAS ON MICROSOMAL MONOOXYGENASEACTIVITIES

Table 4 Effect ofi-h or 18-h corn oil (C.O.) or DAS pretreatment on microsomal and 5-9 fraction monooxygenase activities'

TreatmentMicrosomesC.O.

(3h)DAS(3h)C.O.(18h)DAS(18h)S-9

fractionC.O.(3h)DAS(3 h)nmol

HCHO/min/mgNDMA(1

mM)2.31

±0.731.72±0.243.30

±0.280.71±0.04C0.47

±0.170.23±0.05CNDMA(0.2

mM)2.07

±0.661.48±0.212.92

±0.250.53±0.02'0.34

±0.160.20±0.06NMBA1.14

+0.260.98±0.141.53±0.161.06

+ 0.16Benzphetamine6.27

±1.015.60±1.087.35±1.068.77±0.63Ethylmorphine8.40

±1.017.45±1.6112.79±1.315.76+ 0.47Cp-Nitroanisole2.78

±0.881.98+0.273.56±0.032.08±O.OTAminopyrine3.93

±1.223.12±0.835.11

±0.433.97+ 0.177-Pentoxyresorufin

O-dealkylation*(pmol

resorufin/min/mg)20.6

±2.811.8±1.012.5

±1.574.3±21.Ie

" Substrates were present at 1 mM unless otherwise indicated. Values are the mean ±SD from four separate microsomal preparations.* For comparison, the rate by microsomes from phenobarbital-induced rats was 1350 pmol resorufin/min/mg. The substrate concentration was 10 IÕM.' Values are significantly different from those of the corresponding control, P < 0.002.

Fig. 2. Immunoblot analysis of hepatic microsomes from control and diallyl sulfide-treated rats. Microsomes were subjected tosodium dodecyl sulfate-polyacrylamide gelelectrophoresis followed by immunoblot analysis. Lanes a-e are immunoblots using anti-P450IIE1 IgG; Lanes f-j are immunoblots using anti-P4501IBl IgG. Lanes b and c (alsocorresponding to Lanes g and A, respectively)contained microsomal proteins from two separate control rats. Lanes d and e (also corresponding to Lanes i and j, respectively) contained microsomal proteins from two separatediallyl sulfide-treated rats. Purified P450IIE1(0.13 jig) was applied to Lane A. Lane /contained 0.8 Mgof purified P450IIB1. Lanes b-eand g-j contained 5.3 ng microsomal proteineach. abcdefghijbition by residual DAS in the tissue preparations, as had beendemonstrated previously in similar experiments using diethylether (20), but suggested that irreversible inactivation or loss ofthe NDMA demethylase had occurred. Further evidence forthis possibility was obtained in studies using an 18-h pretreatment, which led to a selective and marked decrease in NDMAdemethylase compared to the other activities measured. Unexpectedly, a slight elevation in benzphetamine demethylationand a 6-fold increase in pentoxyresorufin dealkylation wereobserved, indicating induction of P450IIB1 isozymes (22).Binding of CO to reduced microsomes was unchanged butreducíase activity increased by 40% (data not shown). Nochange in the wavelength of maximal absorbance in the CO-binding spectrum (450 nm) was detected.

Immunoblot Analyses. The levels of P450IIE1 (Fig. 2, Lanesa-e) and P450IIB1 (Lanes f-j), as determined in immunoblotanalyses of microsomes from 18-h DAS-treated rats, exhibitedtrends that were similar to the measured catalytic activities.From densitometry measurements, the average intensity of thebands in Lanes d and e (DAS treated) was 30% that of Lanes band c (control). The average intensity of the bands in Lanes iand j (DAS treated) was approximately 3-fold higher than inLanes g and h (control). Thus, suppression of the microsomalactivity and also the amount of immunoreactive protein associated with P450IIE1 did not indicate a generalized depletionin P-450, since the opposite effects on P450IIB1 were observed.

DISCUSSION

Previous studies (8-10) have demonstrated that DAS blocksthe induction of carcinogenic responses in liver and colon which

develop subsequent to dimethylhydrazine administration in rodents. The present findings suggest that a plausible mechanismis the selective inhibition of cytochrome P450IIE1, which isinvolved in the initial hepatic activation of the procarcinogen(14). This isozyme activity is conveniently assayed in microsomes by monitoring the demethylation at 1 mM NDMA (18).

Two modes of inhibition by DAS of NDMA demethylaseactivity were evident. The first type, observed in incubationsusing acetone-induced microsomes, was competitive inhibition.The low apparent K\ suggested that DAS has a high affinity forP450IIE1 which would be in agreement with previous studiesusing structurally analogous compounds such as diethyl ether(Km 13 /¿M)and pentane (Km 9 MM)(see Ref. 19). The presenceof a small hydrophobic binding pocket at the active site hasbeen proposed7 and would accommodate DAS and similar

compounds. As expected, the observed inhibition was selectivetoward NDMA metabolism, as compared with the oxidation ofsubstrates catalyzed by other P-450 isozymes (Table 1).

The possibility of irreversible inactivation was consideredsince substrates containing carbon-carbon double bonds areoften involved in the destruction of P-450 (23). Furthermore, apotential product of the P-450-dependent oxidation of a compound such as DAS is the sulfhydryl-reactive reagent, acrolein(OCHCH=CH2) (23, 24). However, no evidence for an irreversible inhibition by DAS was observed. The effect of varyingthe concentration of DAS was not determined but could influence the observed inhibition (24). Preliminary experimentsusing gas Chromatographie analysis showed that initial levelsof DAS decreased in incubations containing NADPH. Two

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microsomal enzyme systems, a flavin-containing monooxygen-ase and P-450, oxidize sulfur-containing compounds (25-27).Determination of the products and enzyme specificity of DASmetabolism will require further investigation.

A second mode of inhibition of NDMA demethylase wasobserved after DAS administration in vivo. The selectivesuppression in the activity and in the immunologically determined levels of P450IIE1 could substantially contribute to thereported chemoprotective properties of DAS. The inactivationwas time dependent, suggesting a requirement for metabolismand redistribution of this thioether. The in vivo metabolic fateof the compound is unknown but may be examined by usingradiolabeled substrate (28). The decrease in the level ofP450IIE1 could also be due to an inhibition in the productionof this isozyme.

DAS potentially affects monooxygenase activities in nonhe-patic tissues. The reported elimination of NMBA-inducedesophageal cancer by DAS5 may be due to a local inhibition ofmicrosomal isozymes of P-450 present in the mucosa thatcatalyze the activation of NMBA to alkylating agents (29, 30).

The increase of P450IIB1 observed in the present in vivostudy was unexpected, but along with the minimal changeobserved in P-450 content, supported the notion that DAS isnot a general suppressor of monooxygenase activities. Thislevel of elevation of P450IIB1 was also observed after administration of diethyl ether (19). The mechanism of induction bythese, and by the potent prototypic agent phénobarbital,is notknown but may involve hormonal changes (31).

Doses of DAS used previously and in the present study werequite high (up to 200 mg/kg body weight) considering theestimate that <1 mg is present in a clove of garlic (9). Theoctanohwater partition ratio for DAS has not been reported butis 89 for the structurally similar compound diethyl sulfide (32).DAS might partition into lipid-rich environments such as microsomal membranes, increasing its local concentration andprolonging its effect. Thus moderate levels of DAS consumedin a normal diet may nonetheless inhibit the activation of lowlevels of some types of environmentally encountered procarcin-ogens.

Further understanding of the biochemical properties of DASand related compounds will aid in the search for other naturallyoccurring and synthetic anticarcinogenic agents.

ACKNOWLEDGMENTS

The authors would like to thank Shu M. Ning and Mao-Jung Leefor expert technical assistance, Dr. David Wade for the preparation ofpurified P4501IB1, and Harold L. Newmark and Dr. Michael J. War-govich for helpful discussions.

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1988;48:5937-5940. Cancer Res   John F. Brady, Dechun Li, Hiroyuki Ishizaki, et al.   Metabolism and Other Monooxygenase ActivitiesEffect of Diallyl Sulfide on Rat Liver Microsomal Nitrosamine

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