metabolism of diallyl disulfide by human liver microsomal ...

0090-9556/99/2707-0835–841$02.00/0 

DRUG METABOLISM AND DISPOSITION Vol. 27, No. 7 

Copyright © 1999 by The American Society for Pharmacology and Experimental Therapeutics Printed in U.S.A. 

ABSTRACT: 

METABOLISM OF DIALLYL DISULFIDE BY HUMAN LIVER MICROSOMAL 

CYTOCHROMES P-450 AND FLAVIN-CONTAINING MONOOXYGENASES 

CAROLINE TEYSSIER, LUCIEN GUENOT, MARC SUSCHETET, AND MARIE-HÉLÈNE SIESS 

Unité de Toxicologie Nutritionnelle, Institut National de la Recherche Agronomique, 21034 Dijon Cedex, France 

The metabolism of diallyl disulfide (DADS), a garlic sulfur compound, 

was investigated in human liver microsomes. Diallyl thiosulfinate 

(allicin) was the only metabolite observed and its formation 

followed Michaelis-Menten kinetics with a K m � 0.61 � 0.2 mM 

and a V max � 18.5 � 4.2 nmol/min/mg protein, respectively (mean � 

S.E.M., n � 4). Both flavin-containing monooxygenase and the 

cytochrome P-450 monooxygenases (CYP) were involved in DADS 

oxidation, but the contribution of CYP was predominant. The cytochrome 

P-450 isoforms involved in this metabolism were investigated 

using selective chemical inhibitors, microsomes from cells 

expressing recombinant CYP isoenzymes, and studying the correlation 

of the rate of DADS oxidation with specific monooxygenase 

activities of human liver microsomes. Diethyldithiocarbamate and 

The chemistry of the sulfur-containing compounds of garlic (Allium 

sativum) is complex. Fresh garlic contains allicin (S-allylcysteine 

sulfoxide) from which many molecules are formed when a garlic 

clove is crushed. Among the transformation products, ajoene, sulfides, 

and allicin (diallyl thiosulfinate or DADSO; Fig. 1) 1 were noticed for 

their medicinal properties such as antimicrobial, antifungal, antitumoral, 

antithrombotic, hypotensive, hypoglycemic, and hypolipemic 

properties (see Lin, 1989, for review). 

Diallyl disulfide (DADS) is one of the major volatile degradative 

compounds of garlic formed from DADSO (Block, 1992; Augusti, 

1996). It was found in the breath of human subjects who ate dry or 

fresh garlic (Cai et al., 1995). Many studies on animals showed its 

protective effects against chemically induced toxicity and against 

carcinogenesis (Ip et al., 1992; Reddy et al., 1993). The modulation of 

the metabolism of carcinogens by DADS was considered one of the 

possible mechanisms of its protective effect against the occurrence of 

cancer (Reddy et al., 1993). Previous studies in our laboratory dem- 

This work was supported by the Conseil Régional de Bourgogne and was not 

previously presented. 

1 Abbreviations used are: DADSO, allicin; COH, coumarin 7-hydroxylase; CYP, 

cytochrome P-450 monooxygenase; DADS, diallyl disulfide; DADSO 2, diallyl thiosulfonate; 

DOD, dextromethorphan O-demethylase; ECOD, 7-ethoxycoumarin 

deethylase; EROD, ethoxyresorufin O-deethylase; FMO, flavin-containing monooxygenase; 

LAH, laurate hydroxylase; MpH, mephenytoin hydroxylase; MMO, 

methimazole oxidase; NfO, nifedipine oxidase; PNPH, p-nitrophenol hydroxylase; 

TDH, tolbutamide hydroxylase. 

Send reprint requests to: Dr. Caroline Teyssier, Unité de Toxicologie Nutritionnelle, 

INRA, 17 rue Sully, 21034 Dijon Cedex, France. E-mail: teyssier@ 

dijon.inra.fr 

(Received May 12, 1998; accepted April 13, 1999) 

This paper is available online at http://www.dmd.org 

835 

tranylcypromine inhibited allicin formation, whereas other specific 

inhibitors had low or no effect. Most of the different human microsomes 

from cells expressing CYP were able to catalyze this reaction, 

but CYP2E1 showed the highest affinity with a substantial 

activity. Furthermore, allicin formation by human liver microsomes 

was correlated with p-nitrophenol hydroxylase activity, a marker of 

CYP2E1, and tolbutamide hydroxylase activity, a marker of 

CYP2C9. Among these approaches only CYP2E1 was identified in 

each case, which suggested that DADS is preferentially metabolized 

to allicin by CYP2E1 in human liver. However the minor participation 

of other CYP forms and flavin-containing monooxygenases 

is likely. 

onstrated that the administration of DADS to rats modified the quantity 

and the activity of several hepatic drug metabolism enzymes 

(Haber et al., 1994; 1995). DADS produced an enhancement of the 

microsomal level of cytochrome P-450 monooxygenase (CYP)2B1/2 

and of the activities of UDP glucuronyltransferases and of glutathione 

S-transferases. It decreased the nitrosodimethylamine demethylase 

activity and the level of CYP2E1. In addition, there are many reports 

about the chemical analysis and biological activities of sulfur-containing 

compounds of garlic. However, few studies have dealt with the 

metabolism and pharmacokinetics of garlic constituents. One study 

performed in mice with radioactive DADS indicated that the uptake of 

radioactivity was highest in the liver at 90 min after i.p. injection 

(Pushpendran et al., 1980). In metabolic studies using perfused rat 

liver, DADS appeared to be converted to allyl mercaptan (Egen- 

Schwind et al., 1992). This information and previous results concerning 

the ability of DADS to modulate drug-metabolizing enzymes led 

us to hypothesize that DADS could be metabolized by cytochrome 

P-450 enzymes in the liver. 

The flavin-containing monooxygenases (FMOs) play a role in 

xenobiotic and drug metabolism. These enzymes have been characterized 

in several mammalian species, including human. Among the 

identified isoenzymes, FMO3 is the predominant form in adult human 

liver. The FMOs are able to metabolize a wide variety of xenobiotics 

including thiols, sulfides, and disulfides but the involvement of such 

enzymes in the metabolism of garlic compounds has not been reported. 

In this study we investigated the in vitro metabolism of DADS in 

presence of human liver microsomes. We identified one metabolite 

formed and determined the kinetic parameters of the reaction. The 

contribution of both CYP and FMO were shown. Then we identified 

Downloaded from 

dmd.aspetjournals.org by guest on November 24, 2012

836 TEYSSIER ET AL. 

FIG. 1.Structures of DADS and DADSO. 

the CYP isoenzymes mainly involved in the reaction using several 

approaches: 1) effect of selective chemical inhibitors on DADS oxidation, 

2) determination of DADS oxidation by microsomes from 

cells expressing recombinant CYP isoenzymes, and 3) study of the 

correlation of DADS oxidation with marker activities of CYP isoenzymes 

in different human liver microsomes. 

Experimental Procedures 

Materials. DADS (purity of 80%) was obtained from Aldrich Chemical Co. 

(Strasbourg, France). The other 20% were identified by HPLC as diallyl 

sulfide and diallyl trisulfide. DADS was used without purification for the 

inhibition assays. DADS was further purified by vacuum distillation to a level 

of 95% for the determination of kinetic parameters and the study of the 

involvement of FMOs. DADSO was donated by Professor Auger, Institut de 

Recherche sur la Biologie de l’Insecte, University F. Rabelais of Tours 

CYP 

EROD a 

1A2 

COH b 

2A6 

TDH b 

2C9 

TABLE 1 

Biochemical characteristics of human liver samples 

MpH a 

2C19 

(France). It was synthesized from the corresponding disulfide according to the 

method of Ferary (1996). 5,5-Dithiobis(2-nitrobenzoic acid), �-naphthoflavone, 

chlorzoxazone, coumarin, dextromethorphan, diethyldithiocarbamate, 

NADPH, nifedipine, orphenadrine, quinidine, sulfaphenazole, tolbutamide, 

tranylcypromine, and troleandomycin were purchased from Sigma-Aldrich 

Chimie (Saint Quentin Fallavier, France). Dextrorphan and S-(�)-mephenytoin 

were purchased from Ultrafine Chemicals (Salford, England). 1-Aminobenzotriazole 

was donated by Dr. Cabanne, Laboratoire de Phytopharmacie, 

Institut National de la Recherche Agronomique (Dijon, France). [1- 14 C]Lauric 

acid and [ 14 C] S-(�)-mephenytoin were purchased from Amersham Pharmacia 

Biotech (Les Ulis, France). Microsomes from B-lymphoblastoid cell lines 

expressing individual human CYPs or baculovirus-infected insect cells expressing 

individual human FMOs were purchased from Gentest (Woburn, 

MA). All other chemicals and reagents used were of the highest commercial 

quality available. 

Human Liver Samples. Human liver samples were provided by Professor 

Favre, Département de Chirurgie Digestive Thoracique et Cancérologique of 

the General Hospital of Dijon, France. The protocol was approved by a local 

ethics committee. The used tissues came from patients undergoing liver resection 

for various clinical reasons. The samples were frozen in liquid nitrogen 

and stored at �80°C until use for microsome preparation as described below. 

Biochemical characteristics of the microsomes are mentioned in Table 1. 

Preparation of Microsomal Fractions and Determination of Protein and 

Total CYP Contents. The microsomes were prepared as described previously 

(Haber et al., 1994). Microsomal proteins were quantified by the method of 

Bradford (1976) using bovine serum albumin fraction V as standard. Cytochrome 

P-450 was assayed according to Omura and Sato (1964). 

Microsomal Metabolism of DADS. The reaction medium described below 

was the same for each experiment. This reaction medium consisted of human 

liver microsomes corresponding to 300 pmol of CYP, 1 mM DADS, 1 mM 

DOD b 

2D6 

PNPH b 

2E1 

(�-1)LAH b 

2E1 

KS1 100 4.3 0.09 66.1 0.58 1.3 3.95 2.2 17.0 

K12 18 2.9 0.15 nd nd 3.9 6.05 2.9 5.8 

K17 36 2.7 0.43 0 2.72 2.1 6.41 1.0 24.3 

KS21 33 3.6 0.33 249 0.14 1.4 5.44 2.1 13.0 

K25 75 7.3 0.28 nd nd 1.8 7.04 1.8 19.8 

K26 45 4.5 0.37 nd 0.15 2.2 8.75 2.4 19.1 

KS27 22 2.1 0.08 162 0.14 0.6 2.47 0.6 7.7 

KS28 32 8.0 0.27 nd 0.03 1.9 5.49 2.7 15.4 

KT28 95 6.9 0.35 nd 0.06 2.2 6.18 4.5 15.7 

KS29 67 6.3 0.21 23.9 0.21 1.1 3.42 1.8 10.0 

KS30 81 2.6 0.28 95.1 0.35 1.4 5.90 2.0 12.8 

KT30 62 1.8 0.46 21.5 0.34 1.3 4.69 2.5 11.2 

K32 138 4.0 0.31 328 0.06 2.3 7.97 3.4 18.0 

K33 147 5.2 0.22 nd 0.35 1.7 4.54 3.7 15.7 

KT34 244 5.1 0.61 197 1.11 2.4 2.68 4.6 14.8 

KS38 45 6.8 0.03 nd 0.48 2.4 1.77 1.7 14.3 

K40 146 3.2 0.15 15.6 0.46 nd 3.89 1.8 12.2 

KS41 69 4.6 0.25 nd nd 1.8 3.73 2.2 17.9 

K44 100 4.1 0.17 149 0.02 2.4 2.28 2.4 13.7 

KS47 85 3.4 0.58 0.00 0.64 2.3 4.94 0.8 10.4 

KS48 100 3.7 0.33 175 0.67 3.3 6.21 0.9 19.8 

K49 20 5.0 0.49 17.1 0.89 2.7 4.39 0.8 13.5 

K50 36 6.0 0.36 294 0.18 1.5 3.14 0.5 11.1 

K51 99 4.7 0.78 91.4 1.00 3.0 12.85 1.7 22.8 

K52 114 4.7 0.79 354 nd 2.0 6.00 0.9 15.3 

K53 43 2.3 0.63 135 0.45 1.2 5.30 0.9 17.7 

K55 nd nd 0.68 4.3 0.42 nd nd nd nd 


KS57 nd nd 0.52 0.0 0.79 nd nd nd nd 



KS63 nd nd 0.94 18.9 nd nd nd nd nd 

KT63 nd nd 0.76 39.3 0.24 nd nd nd nd 

a pmol/min/nmol CYP 

b nmol/min/nmol CYP 

nd, not determined 

NfO b 

3A4 

(�)LAH b 

4A 



DIALLYL DISULFIDE METABOLISM BY HUMAN LIVER MONOOXYGENASES 

NADPH, 50 mM Tris-HCl pH 7 in a total volume of 500 �l. After 30 min at 

37°C, the reaction was stopped by adding 320 �l of acetonitrile. After 15 min 

of protein precipitation, the mixture was centrifuged at 10,500g for 10 min and 

40 �l of the supernatant was analyzed by HPLC. The same protocol was 

applied with 25 pmol for each cDNA-expressed human CYP microsome. In 

case of cDNA-expressed human FMO3 microsomes, 100 �g of protein was 

added in a total volume of 125 �l. Many DADS concentrations were used to 

determine the kinetic parameters. 

Thermal inactivation of microsomes was performed by a preincubation of 

microsomes in Tris-HCl buffer for 10 min at 37°C in the absence of NADPH, 

or in the presence of NADPH for control microsomes. Then DADS and 

NAPDH were added subsequently to start the incubation for 30 min. 

Inhibition of DADS Metabolism. Inhibitors were added to the incubation 

mixtures before initiation of the reaction. Only with the mechanism-based 

inhibitors such as diethyldithiocarbamate or aminobenzotriazole, microsomes 

were preincubated for 10 min at 37°C before the addition of DADS. Nonhydrosoluble 

inhibitors were dissolved in 100% ethanol and the following volumes 

of ethanol were added to the incubation medium (total volume, 500 �l): 

0.2 �l for sulfaphenazole and orphenadrine, 0.3 �l for coumarin, 0.4 �l for 

diethyldithiocarbamate, 0.5 �l for aminobenzotriazole and quinidine, 0.8 �l 

for �-naphthoflavone, and 2.5 �l for nifedipine. The human samples KS1, 

K12, K25, KS28, K33, K40, KS41, and KS63 were used for the inhibitory 

experiments. 

HPLC Analysis. HPLC analysis was carried out using a Waters (Saint 

Quentin-en-Yrelines, France) system equipped with a model 600 pump, a 

model 717 auto sampler, a model 996 photodiode array UV detector, and a GL 

Sciences Inc. (Tokyo, Japan). Interstil ODS-3 column (4.6 � 150 mm). The 

flow rate was 0.6 ml/min and the solvent-isocratic program was 30:70 (v/v, 

acetonitrile/water) for 20 min. The spectrum from 190 to 300 nm was used to 

detect DADS and its metabolites. The quantification was made at 254 nm. Data 

were processed by Waters Millenium software. 

Determination of Kinetic Constants. K m and V max were determined with 

a range of substrate concentrations of 0 to 7.5 mM with human liver microsomes 

and FMO cDNA-expressed microsomes, and 0 to 5 mM with CYP 

cDNA-expressed microsomes. The values were estimated by fitting the 

Michaelis-Menten equation using a nonlinear regression program of SAS 

Software (Cary, NC). The human samples KS1, KS30, K33, and K40 were 

used in this experiment. The apparent V max of FMO3 cDNA-expressed microsomes 

was determined considering that 100 �g of protein corresponded to 

approximately 100 pmol of enzyme. This consideration was based on the 

specific activity of recent lots of FMO3 from Gentest. 

Enzyme Assays. Detailed information for these assays are given Table 2. 

The coumarin 7-hydroxylase (COH) activity was determined according 

to Maurice et al. (1991); dextromethorphan O-demethylase (DOD) according 

to Bourrié et al. (1996); 7-ethoxycoumarin deethylase (ECOD) according to 

Ullrich and Weber (1972); ethoxyresorufin O-deethylase (EROD) according to 

Burke et al. (1985); laurate hydroxylase (LAH) according to Parker and Orton 

(1980) with few modifications; mephenytoin hydroxylase (MpH) according to 

Meier et al. (1985); methimazole oxidase (MMO) according to Dixit and 

Roche (1984); nifedipine oxidase (NfO) according to Guengerich et al. (1986); 

p-nitrophenol hydroxylase (PNPH) according to Tassaneeyakul et al. (1993); 

and tolbutamide hydroxylase (TDH) according to Bourrié et al. (1996). 

Correlation Analysis. To investigate the involvement of different CYP 

isoenzymes in DADS oxidation, marker activities of several isoenzymes 

(CYP1A2, CYP2A6, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4, and 

CYP4A) were measured for at least 24 samples of human liver microsomes. 

The enzyme assays were performed as described above. Calculations of the 

correlation coefficient between the rate of transformation of DADS and the 

marker CYP activities were made with the SAS system (Cary, NC) with the 

following equation: r � covariance (x,y) / (variance (x) � variance (y)) 1/2 . 

Results 

Identification of Metabolite. When DADS was incubated with 

human liver microsomes and NADPH, only one peak was detected by 

HPLC (Fig. 2). This peak was identified as DADSO by comparing its 

retention time in different HPLC gradients and its absorption spectrum 

with that of synthesized DADSO. No other metabolite was 

detected for various incubation times of DADS. When either NADPH 

or microsomes were omitted, no DADSO was detected. 

Kinetics of Reaction. The formation of DADSO was linear over a 

period of 45 min. An incubation time of 30 min was therefore 

routinely used. A concentration of 1 mM NADPH was optimal for the 

reaction. The kinetic of formation of DADSO by liver microsomes 

was consistent with the Michaelis-Menten equation. The apparent Km was 0.61 � 0.2 mM and the apparent Vmax was 18.5 � 4.2 nmol/ 

min/mg protein. Values are means � S.E.M. for four samples. 

Contribution of FMOs to DADS Oxidation. To evaluate the 

respective roles of FMOs and CYP in the oxidation of DADS, we 

initiated incubations of DADS in the presence of : 1) 1-aminobenzotriazole, 

a suicide inhibitor of CYPs (De Montellano and Mathews, 

1981; Fig. 3 shows the inhibition of DADS oxidation by this inhibi- 

TABLE 2 

Incubation conditions for the determination of marker enzyme activities in human liver microsomes 

COH DOD ECOD EROD LAH MpH MMO NfO PNPH TDH 

Buffer A B C D E B F G H B 

NADPH (mM) 1 1 0.05 0.25 1.5 1 0.1 1 1 1 

Microsomal proteins 

(mg/ml) 

0.28 1 0.13 1 0.125 2 0.3 2 1 2 

Substrate (mM) 0.1 0.05 1 0.001 0.13 0.2 1 0.2 0.1 2 

Total volume (ml) 0.36 0.5 2 0.4 0.5 0.5 1 0.5 0.5 1 

Temperature (°C) 37 37 30 30 37 37 37 37 37 37 

Incubation time (min) 3 20 4 3 15 30 5 10 20 30 

Stop reagent (�l) HCl 1N HCl 1N HCl 1N AcN/TCA 

20%, 50/50 

Perchloric acid 0.15 M H3PO4 HPLC separation Yes Yes Yes Yes Yes Yes 

UV detection (nm) 412 254 340 230 

Radiometric detection 14C 14C Fluorometric 

detection 

Excitation (nm) 

380 280 380 522 

Emission (nm) 450 312 450 586 

A: 50 mM sodium phosphate buffer (pH 7.4) 

B: 100 mM potassium phosphate buffer (pH 7.4) 

C: 100 mM Tris-HCl buffer (pH 7.7), 1 mM EDTA 

D: 100 mM Tris-HCl buffer (pH 7.7), 25 mM MgCl 2 

E: 66 mM Tris-HCl buffer (pH 7.4) 

F: 0.1 M tricine (pH 8.4), 1 mM EDTA, 0.06 mM 5,5�-dithiobis(2-nitrobenzoate), 6 mM potassium phosphate (pH 8.4), 0.02 mM dithiothreitol 

G: 50 mM potassium phosphate buffer (pH 7.4), 3 mM MgCl 2 

H: 100 mM sodium phosphate buffer (pH 6.8) 

837 




FIG. 2.HPLC profile of an incubation of DADS with microsomes and NADPH, 

obtained with UV detection at 254 nm. 

If DADSO2 has been formed, its elution would be between the peak of NADPH 

(AU � 0.03) and the peak of DADSO. 

FIG. 3.Effects of 1-aminobenzotriazole on DADS oxidase activity. 

Values are mean of four samples � S.E.M. and are expressed relative to control 

(without inhibitor). 

TABLE 3 

Effect of thermal treatment of microsomes on few activities 

Values are the means of five determinations � S.E.M. 

CYP DADS a Oxidase MMO b 

ECOD b 

10 min 37°C 20.0 � 6.3 40.2 � 9.6 0.25 � 0.0 

Control 23.0 � 7.4 194 � 50.1 0.25 � 0.0 

a pmol/min/pmol CYP 

b nmol/min/mg protein 

tor), 2) microsomes containing irreversibly inactivated FMOs (this FMO 

inactivation is produced by heating the microsomes, Grothusen et al., 

1996); the results indicated that FMO inactivation by heat treatment 

caused a 13% decrease of DADS oxidation (Table 3); it was verified that 

this thermal treatment inactivated the FMOs by measure of MMO activity 

although residual MMO activity was observed after 10 min heating; 

this was due to the CYP ability to metabolize the methimazole (results not 

shown and Poulsen et al., 1974); we checked also that thermal inactivation 

did not modify the ECOD activity; this reaction is often used as a 

standard marker for CYP-mediated reactions as several forms of CYP are 

involved in this activity), and 3) microsomes prepared from baculovirusinfected 

insect cell lines expressing the human FMO3 (in this medium, 

the same product DADSO was obtained as with human liver microsomes; 

the kinetic parameters of the reaction were calculated: K m � 

10.15 mM and V max � 41.9 pmol/min/pmol FMO3). 

Effect of Human CYP Inhibitors on DADS Oxidation. To further 

assess which CYP isoenzymes are involved in DADS oxidation, 

the effects of the following chemical inhibitors were determined: 

�-naphthoflavone (specific of CYP1A), chlorzoxazone (CYP2E1; 

Amet et al., 1995), coumarin (CYP2A6; Harris et al., 1994), diethyldithiocarbamate 

(CYP2E1; Guengerich et al., 1991), mephenytoin 

(CYP2C19; Harris et al., 1994), nifedipine (CYP3A4; Harris et al., 

1994), orphenadrine (CYP2B6; Chang et al., 1993), quinidine 

(CYP2D6; Harris et al., 1994), sulfaphenazole (CYP2C9; Baldwin et 

al., 1995), tolbutamide (CYP2C9; Harris et al., 1994), tranylcypromine 

(CYP2C19; Postlind et al., 1998), and troleandomycin 

(CYP3A4; Rodrigues, 1994). 

The results are presented in Fig. 4. The strongest inhibitions were 

observed in the presence of diethyldithiocarbamate, followed by tranylcypromine. 

A slighter inhibition rate appeared with coumarin, 

chlorzoxazone, mephenytoin, nifedipine, and sulfaphenazole. Orphenadrine, 

tolbutamide, and troleandomycin had almost no effect on 

the DADSO formation, and �-naphthoflavone and quinidine had no 

effect at all. To verify the selectivity of some of the inhibitors used, 

we measured PNPH activity, a marker of CYP2E1, in the presence of 

30 �M tranylcypromine, or of 22 or 200 �M coumarin. The result is 

shown in Table 4. Tranylcypromine and coumarin, described as 

selective inhibitors of CYP2C19 and CYP2A6 respectively, were also 

inhibitors of PNPH activity. These results suggest that tranylcypromine 

and coumarin can inhibit other CYPs, at least CYP2E1. 

Metabolism by Microsomes Containing cDNA-Expressed 

CYPs. The abilities of different human CYP enzymes to catalyze the 

S-oxidation of DADS to DADSO were investigated in many microsomes 

derived from cells expressing CYP DNA. CYP2A6, CYP2C8, 

CYP2C9, CYP2C19, CYP2D6, and CYP2E1 catalyzed the S-oxidation 

of DADS. CYP2D6, CYP2C19, and CYP2E1 were the most 

active enzymes. We can observe in Fig. 5, that every CYP isoform 

followed a Michaelis-Menten kinetic except CYP2E1. For this latter 

enzyme, the DADS oxidase activity decreased quickly as soon as a 

certain amount of DADSO was produced. This could be explained by 

the CYP2E1 mechanism-based inhibitor property of DADSO. No 

detectable amounts of DADSO were found when using microsomes 

expressing CYP1A2, CYP3A4, or CYP4A11, or when using microsomes 

from B-lymphoblastoid cells transfected with an empty vector. 

The kinetic parameters were determined for CYP2A6, CYP2C8, 

CYP2C9, CYP2C19, CYP2D6, and CYP2E1 (Table 5). CYP2E1 

exhibited the smallest K m whereas CYP2D6, CYP2C19, and CYP2E1 

had close V max values. The intrinsic clearance (V max/K m ratio) was 

2010 for CYP2E1 whereas it reached the maximum value of 108 for 

the other CYPs (Table 5). 

Correlation Study between DADS Oxidation and Specific CYP 

Activities. The correlation between DADS oxidation and CYP marker 

activities was analyzed with a panel of at least 24 individual samples 

of human liver microsomes. The characteristic transformations by 

individual CYPs were EROD for CYP1A2, COH for CYP2A6, TDH 

for CYP2C9, MpH for CYP2C19, DOD for CYP2D6, (�-1)LAH and 

PNPH for CYP2E1, NfO for CYP3A4, and (�)LAH for CYP4A. 

These results are shown in Table 6. The best correlations (r) were 

found with PNPH, TDH, and (�-1)LAH activities, whereas the correlations 

with EROD, COH, DOD, NfO, MpH, and (�)LAH were 

very low. 




FIG. 4.Inhibition of DADS oxidation by chemical CYP inhibitors in human liver microsomes. 

Values are the percentage of control (without inhibitor). They are expressed as means � S.E.M. of four human samples. 

TABLE 4 

Inhibitory effects of various compounds on PNPH activity 

Values are the percentages of control activity (without inhibitor) and are means � S.E.M. 

of four human samples (KS27, KS30, KS44, and KS52). 

Compound PNPH Activity 

Tranylcypromine, 30 �M 21.3 � 1.0 

Coumarin, 22 �M 39.0 � 1.5 

Coumarin, 200 �M 34.6 � 1.8 

FIG. 5.Michaelis-Menten plot for the oxidation of DADS by human 

cDNA-expressed CYP microsomes. 

Values are means of duplicate. 

Discussion 

We have studied the metabolism of DADS in the presence of 

human liver microsomes and we have observed the appearance of 

DADSO formed by the oxidation of one sulfur atom. Both DADS and 

TABLE 5 

Kinetic parameters of DADS oxidation by cDNA-expressed human CYP or 

FMO microsomes 

Values are the means of two determinations. 

Enzyme K m V max V max/K m 

mM pmol/min/pmol enzyme 

CYP1A2 nd nd 

CYP2A6 0.33 20.9 63.3 

CYP2C8 2.03 3.8 1.87 

CYP2C9 0.50 5.2 10.4 

CYP2C19 1.71 96.3 56.3 

CYP2D6 1.12 121 108.0 

CYP2E1 0.03 60.3 2010 

CYP3A4 nd nd 

CYP4A1 nd nd 

Control nd nd 

FMO3 10.15 41.9 4.13 

nd, not determined in absence of any activity. 

839 

DADSO are natural compounds found in crushed garlic but the latter 

has been considered as the most important biologically active compound 

of garlic (Reuter, 1995; Augusti, 1996). Among several sulfides 

from Allium studied, DADS exhibited in vivo the strongest 

anticarcinogenic properties (Ip et al., 1992; Haber-Mignard et al., 

1996) and protected against carcinogen-induced DNA strand breaks 

(Le Bon et al., 1997). These effects were attributed to in vivo modulation 

of hepatic drug-metabolizing enzymes by DADS (Haber et al., 

1994). Knowing the in vitro DADS metabolism, we suppose that the 

in vivo effects of DADS are in fact due to DADSO. 

In this study, we have observed the oxidation of DADS to sulfoxide. 

The in vivo metabolisms of diallyl sulfide (DAS; Brady et al., 

1991, Chen et al., 1994) and of dipropyl sulfide (Nickson and Mitchell, 

1994), two sulfur compounds from Allium, implicate a first step of 

oxidation to form a sulfoxide and a second one to form a sulfone. With 

DADS two steps of oxidation were not excluded, even if formation of 

diallyl thiosulfonate (DADSO 2) was not observed in the incubation 

medium. In fact we have characterized DADSO as a mechanismbased 

inactivator of CYP2E1 (results not published). This should 

mean that DADSO could be oxidized to a reactive species (possibly 




TABLE 6 

Correlation between DADS oxidation and CYP isoform specific activities in a 

panel of human liver microsomes 

Enzyme Activity CYP 

Correlation 

Coefficient (r) 

EROD 1A2 0.07 

COH 2A6 0.06 

TDH 2C9 0.52 a 

MpH 2C19 0.05 

DOD 2D6 0.05 

PNPH 2E1 0.63 b 

(�-1)LAH 2E1 0.40 c 

NfO 3A4 0.19 

(�)LAH 4A 0.07 

a p � .001, n � 33 

b p � .001, n � 25 

c p � .05, n � 26 

DADSO 2) that should interact with the enzyme and should not be 

released from the active site of the enzyme. 

Egen-Schwind et al. (1992) have studied the metabolism of 

DADSO in a perfused rat liver. They observed that while passing 

through the liver, DADSO was metabolized to DADS. The discrepancy 

could be explained by the models that were used in both studies. 

They did not add NADPH during the liver perfusion. Due to the 

sensitivity of DADSO to temperature, a dismutation of DADSO in 

DADS could be possible. We observed the formation of DADS when 

DADSO was incubated with microsomes at 37°C without NADPH. In 

addition, Jin and Baillie (1997) studied the metabolism of DAS in rat. 

They proposed that the reduction of allyl sulfoxide to DAS was 

impossible with respect to the glutathione conjugates observed with 

rat fed with DAS or allyl sulfoxide. 

Flavin or CYPs are the only enzymes present in microsomes that 

can catalyze NADPH- and oxygen-dependent oxidation of xenobiotics. 

The inhibition of CYP by 1-aminobenzotriazole, a suicide inhibitor, 

as well as the irreversible inactivation of FMO by heating 

induced a decrease of the rate of DADS oxidation. Moreover, the 

DADS oxidation was observed with microsomes prepared from cells 

expressing human FMO3. These results suggest a contribution of both 

CYP and FMO-containing monooxygenases. Some results allow the 

evaluation of the FMO implication in this oxidation: its K m is much 

higher than the one obtained for FMOs associated with CYP in human 

microsomes (10.15 versus 0.61 mM). The similar comparison made 

with cDNA-expressed isoenzymes gave 10.15 mM for FMO3 and 

0.03 mM for CYP2E1. When the incubation of microsomes and 

DADS was made in the presence of inhibited CYP, the DADS oxidase 

activity is very low, whereas when the FMOs were inactivated this 

activity was slightly decreased. Each of these results suggests that 

FMOs are less active than CYP in the metabolism of DADS. Nevertheless, 

to our knowledge, this is the first study that describes the 

implication of FMOs in the oxidation of sulfur compounds issued 

from garlic. 

Three approaches have been developed to identify the CYP isoenzymes 

involved in DADS oxidation. They were based on: 1) the use 

of cDNA-expressed CYP isoenzymes, 2) the use of selective chemical 

inhibitors, and 3) the study of the correlation of DADS oxidation 

activity with marker activities of CYP isoenzymes. Several pieces of 

evidence indicate that CYP2E1 is the major cytochrome P-450 responsible 

for the metabolic process: 1) the rate of formation of 

DADSO was substantially inhibited by the CYP2E1 inhibitors diethyldithiocarbamate 

and chlorzoxazone, 2) PNPH and (�-1)LAH activities 

(two marker activities of CYP2E1) in 25 individual human liver 

microsomes exhibited the best correlation with the formation of 

DADSO, and 3) with a K m of 0.03 mM and a V max/K m ratio of 2010 

calculated with cDNA-expressed CYP2E1, this isoenzyme exhibited 

the highest affinity and the highest intrinsic clearance among the 

isoforms tested. Even if the quantity of CYP2E1 in human microsomes 

was evaluated to 7% of whole CYP versus 18% for CYP2C and 

29% for CYP3A (Shimada et al., 1994), the relative involvement of 

CYP2E1 is predominant. 

Nevertheless, our results suggest the involvement of other CYPs. 

Many isoenzymes, such as CYP2A6, CYP2C9, CYP2C19, CYP2D6, 

and CYP2E1 were able to oxidize DADS to DADSO. Their K m values 

showed that in a competitive context like in human microsomes, only 

CYP2E1 would be involved. The apparent velocity of CYP2E1 was 

not the highest one. We demonstrated that DADSO was a mechanismbased 

inhibitor of CYP2E1 (Martin, Teyssier and Siess, publication in 

preparation). This means that the more DADSO is produced, more 

CYP2E1 is inhibited. This observation may serve as an explanation 

for the low V max of CYP2E1. 

In the inhibitory experiments, tranylcypromine, an inhibitor described 

as being specific of CYP2C19 (Postlind et al., 1998), strongly 

inhibited DADS oxidase activity, suggesting the participation of this 

isoenzyme. However, the specificity of this inhibitor should be reconsidered 

because Draper et al. (1997) recently demonstrated that this 

molecule is among the strongest inhibitors of the coumarin hydroxylase 

activity (CYP2A6 activity marker), and we showed the PNPH 

activity inhibition by tranylcypromine. Therefore, CYP2C19 does not 

seem to be involved in DADS oxidation. 

DADS has a chemical structure similar to DAS; they differ only by 

one sulfur atom. These two molecules are metabolized by CYP2E1 

and produce a mechanism-based inhibitor of CYP2E1. The metabolism 

of DADS and its pathway is one more similarity between these 

two molecules issued of garlic. 

In conclusion, the oxidation of DADS to DADSO in human liver 

microsomes is mainly mediated by CYPs but also by FMOs. Among 

the CYP isoenzymes, CYP2E1 seems to be the most involved isoenzyme 

even if a few other isoenzymes are also able to catalyze this 

reaction. 

Acknowledgments. We thank Jacques Auger for providing 

DADSO and Marie-France Vernevaut for her technical assistance. 

References 

Amet Y, Berthou F, Baird S, Dreano Y, Bail JP and Menez JF (1995) Validation of the 

(omega-1)-hydroxylation of lauric acid as an in vitro substrate probe for human liver CYP2E1. 

Biochem Pharmacol 50:1775–1782. 

Augusti KT (1996) Therapeutic values of onion (Allium cepa L) and garlic (Allium sativum L). 

Indian J Exp Biol 34:634–640. 

Baldwin SJ, Bloomer JC, Smith GJ, Ayrton AD, Clarke SE and Chenery RJ (1995) Ketoconazole 

and sulphaphenazole as the respective selective inhibitors of P4503A and 2C9. Xenobiotica 

25:261–270. 

Block E (1992) The organosulfur chemistry of the genus Allium. Implications for the organic 

chemistry of sulfur. Angew Chem Int Ed Eng 31:1135–1178. 

Bourrié M, Meunier V, Berger Y and Fabre G (1996) Cytochrome P450 isoform inhibitors as a 

tool for the investigation of metabolic reactions catalyzed by human liver microsomes. 

J Pharmacol Exp Ther 277:321–332. 

Brady JF, Ishizaki H, Fukuto JM, Lin MC, Fadel A, Gapac JM and Yang CS (1991) Inhibition 

of cytochrome P-450 2E1 by diallyl sulfide and its metabolites. Chem Res Toxicol 4:642–647. 

Burke MD, Thompson S, Elcombe CR, Halpert J, Haaparanta T and Mayer RT (1985) Ethoxy-, 

pentoxy- and benzyloxyphenoxazones and homologues: A series of substrates to distinguish 

between different induced cytochromes P-450. Biochem Pharmacol 34:3337–3345. 

Cai Y, Baer-Dubowska W, Ashwood-Smith MJ, Ceska D, Tachibana S and Digiovanni J (1995) 

Mechanism based inactivation of hepatic ethoxyresorufin O-dealkylation activity by naturally 

occurring coumarin. Chem Res Toxicol 9:729–736. 

Chang T, Weber C, Crespi C and Waxman D (1993) Differential activation of cyclophosphamide 

and ifosphamide by cytochromes P450 2B and 3A in human liver microsomes. Cancer Res 

53:5629–5637. 

Chen L, Lee M, Hong JY, Huang W, Wang E and Yang CS (1994) Relationship between 

cytochrome P450 2E1 and acetone catabolism in rats as studied with diallyl sulfide as an 

inhibitor. Biochem Pharmacol 48:2199–2205. 

De Montellano O and Mathews JM (1981) Autocatalytic alkylation of the cytochrome P-450 

prosthetic haem group by 1-aminobenzotriazole. Isolation of an NN-bridged benzyneprotoporphyrin 

IX adduct. Biochem J 195:761–764. 

Dixit A and Roche TE (1984) Spectrophotometric assay of the flavin-containing monooxygenase 




and changes in its activity in female mouse liver with nutritional and diurnal conditions. Arch 

Biochem Biophys 233:50–63. 

Draper AJ, Madan A and Parkinson A (1997) Inhibition of coumarin 7-hydroxylase activity in 

human liver microsomes. Arch Biochem Biophys 341:47–61. 

Egen-Schwind C, Eckard R, Jekat FW and Winterhoff H (1992) Pharmacokinetics of vinyldithiins, 

transformation products of allicin. Planta Med 58:8–13. 

Ferary S (1996) Analyse des composés soufrés émis par quelques espèces végétales dans les 

relations plantes-insectes. P.h.D. thesis l’Université de Tours, France. 

Grothusen A, Hardt J, Brautigam L, Lang D and Bocker R (1996) A convenient method to 

discriminate between cytochrome P450 enzymes and flavin-containing monooxygenases in 

human liver microsomes. Arch Toxicol 71:64–71. 

Guengerich FP, Kim DH and Iwasaki M (1991) Role of human cytochrome P-450 IIE1 in the 

oxidation of many low molecular weight cancer suspects. Chem Res Toxicol 4:168–179. 

Guengerich FP, Martin MV, Beaune PH, Kremers P, Wolff T and Waxman DJ (1986) Characterization 

of rat and human liver microsomal cytochrome P-450 forms involved in nifedipine 

oxidation, a prototype for genetic polymorphism in oxidative drug metabolism. J Biol Chem 

261:5051–5060. 

Haber D, Siess MH, Canivenc-Lavier MC, Le Bon AM and Suschetet M (1995) Differential 

effects of dietary diallyl sulfide and diallyl disulfide on rat intestinal and hepatic drugmetabolizing 

enzymes. J Toxicol Environ Health 44:423–434. 

Haber D, Siess MH, De Waziers I, Beaune P and Suschetet M (1994) Modification of hepatic 

drug-metabolizing enzymes in rat fed naturally occurring allyl sulphides. Xenobiotica 24:169– 

182. 

Haber-Mignard D, Suschetet M, Berges R, Astorg P and Siess MH (1996) Inhibition of aflatoxin 

B1- and N-nitrosodiethylamine-induced liver preneoplastic foci in rats fed naturally occurring 

allyl sulfides. Nutr Cancer 25:61–70. 

Harris JW, Rahman A, Kim BR, Guengerich FP and Collins JM (1994) Metabolism of Taxol by 

human hepatic microsomes and liver slices: Participation of cytochrome P450 3A4 and an 

unknown P450 enzyme. Cancer Res 54:4026–4035. 

Ip C, Lisk DJ and Stoewsand GS (1992) Mammary cancer prevention by regular garlic and 

selenium-enriched garlic. Nutr Cancer 17:279–286. 

Jin L and Baillie TA (1997) Metabolism of the chemoprotective agent diallyl sulfide to 

glutathione conjugates in rats. Chem Res Toxicol 10:318–327. 

Le Bon AM, Roy C, Dupont C and Suschetet M (1997) In vivo antigenotoxic effects of dietary 

allyl sulfides in the rat. Cancer Lett 114:131–134. 

841 

Lin RI (1989) Garlic in Nutrition and Medicine. Nutrition International Co., Irvine, CA. 

Maurice M, Emiliani S, Dalet BI, Derancourt J and Lange R (1991) Isolation and characterization 

of a cytochrome P450 of the IIA subfamily from human liver microsomes. Eur J Biochem 

200:511–517. 

Meier UT, Kronbach T and Meyer UA (1985) Assay of mephenytoin metabolism in human liver 

microsomes by high-performance liquid chromatography. Anal Biochem 151:286–291. 

Nickson RM and Mitchell SC (1994) Fate of dipropyl sulphide and dipropyl sulphoxide in rat. 

Xenobiotica 24:157–168. 

Parker GL and Orton TC (1980) Induction by oxyisobutyrates of hepatic and kidney microsomal 

cytochrome P-450 with specificity towards hydroxylation of fatty acids, in Biochem Biophys 

and Regulation of Cytochrome P-450 (Gustafsson JA, Carlstedt-Duke J, Mode A and Rafter 

J eds) p 373–377, Elsevier, Amsterdam. 

Postlind H, Danielson, Lindgren A and Andersson SHG (1998) Tolterodine, a new muscarinic 

receptor antagonist, is metabolized by cytochromes P450 2D6 and 3A in human liver 

microsomes. Drug Metab Dispos 26:289–293. 

Poulsen LL, Hyslop RM and Ziegler DM (1974) S-oxygenation of N-substituted thioureas 

catalyzed by the pig liver microsomal FAD-containing monooxygenases. Arch Biochem 

Biophys 198:78–88. 

Pushpendran CK, Devasagayam TP, Chintalwar GJ, Banerji A and Eapen J (1980) The metabolic 

fate of [35S]-diallyl disulphide in mice. Experientia 36:1000–1001. 

Reddy BS, Rao CV, Rivenson A and Kelloff G (1993) Chemoprevention of colon carcinogenesis 

by organosulfur compounds. Cancer Res 53:3493–3498. 

Reuter HD (1995) Allium sativum and allium ursinum: Part 2. Pharmacology and medicinal 

application. Phytomedicine 2:73–91. 

Rodrigues AD (1994) Use of in vitro human metabolism studies in drug development. An 

industrial perspective. Biochem Pharmacol 48:2147–2156. 

Shimada T, Yamazaki H, Mimura M, Inui Y and Guengerich FP (1994) Interindividual variations 

in human liver cytochrome P-450 enzymes involved in the oxidation of drugs, carcinogens and 

toxic chemicals: Studies with liver microsomes of 30 Japanese and 30 Caucasians. J Pharmacol 

Exp Ther 270:414–423. 

Tassaneeyakul W, Veronese ME, Birkett DJ, Gonzalez FJ and Miners JO (1993) Validation of 

4-nitrophenol as an in vitro substrate probe for human liver CYP2E1 using cDNA expression 

and microsomal kinetic techniques. Biochem Pharmacol 46:1975–1981. 

Ullrich V and Weber P (1972) The O-dealkylation of 7-ethoxycoumarin by liver microsomes. A 

direct fluorometric test. Hoppe-Seyler’s Z Physiol Chem 353:1171–1177.

metabolism of diallyl disulfide by human liver microsomal ...

You also want an ePaper? Increase the reach of your titles

Delete template?

Save as template?