Emmanuelle Germain, Joelle Chevalier, Marlène Steib, Marie-Hélène Siess* and Caroline Teyssier *

1. *** 3 CYP) pmoles 2 / min / pmoles 1 DADSO ( 0 control MC PB AC DX CL 1A 2B 2E1 3A 4A Figure 2: Effect of CYP inducers on DADS oxidation by rat liver microsomes. Value marked with asterisks differ significantly (Dunnett’s test) from the corresponding control value (p0.05). AMSO2 allyl methyl sulfone AMSO allyl methyl sulfoxide AMS allyl methyl sulfide DADS AM allyl mercaptan GST CYP 2B1/2 CYP 2E1 FMO AGS allyl glutathionyl sulfide DADSO In vitro and ex vivo metabolism of diallyl disulfide, a characteristic sulfur compound of garlic, in rat. Emmanuelle Germain, Joelle Chevalier, Marlène Steib, Marie-Hélène Siess* and Caroline Teyssier* Institut National de la Recherche Agronomique / ENSBANA, UMR Toxicologie Alimentaire, 17 rue Sully, 21065 Dijon cedex, France INTRODUCTION The chemistry of the sulfur-containing compounds of garlic (Allium sativum) is complex. When a garlic clove is crushed, many sulphur molecules like allicin (DADSO) are formed from its precursor alliin. Diallyl disulfide (DADS), one of the major volatile compounds of garlic formed from DADSO, is known for its medicinal properties such as antimicrobial and antifungical properties. Many studies in animals have shown its protective effects against chemically-induced carcinogenesis. The modulation of the metabolism of carcinogens by DADS was considered as one of the possible mechanisms of its protection against the cancer occurrence. However, little is known about the metabolism and pharmacokinetic of this garlic constituent. In this study, we investigated ex vivo metabolism of DADS in an isolated perfused rat liver. To understand the metabolic pathway between the metabolites found, we undertook in vitro studies with rat subcellular fractions. METHODS Ex vivo metabolism : Perfusion of isolated rat liver was performed as described by Teyssier and Siess (2000). Briefly, liver was perfused with DADS (1 mM) and the level of DADS in the perfusate was periodically measured by HPLC. At the end of the experiment, sulphur compounds were extracted from liver and the extract was analysed with GC-MS (DB 1701 capillary column, electron impact at 70eV). In vitro metabolism : Monooxygenase incubations: microsomes corresponding to 300 pmol of CYP (induced or non-induced), 1.5 mM substrate (DADS, DADSO or AM), 1 mM NADPH, 50 mM Tris-HCl pH 7 for 30 min at 37°C, in presence or absence of CYP inhibitor. Glutathion transferase (GST) incubations: 0.75 mg of rat cytosolic protein, 1.5 mM substrate and 5 mM GSH in 50 mM Tris pH 7 for 30 min at 37°C. For each incubation: after protein precipitation, the supernatant was analysed by HPLC with an Interstil ODS-3 column. The quantification was made at 254 nm. All results are means of four replicate experiments  SEM. RESULTS-DISCUSSION Table 1: Single dose pharmacokinetic parameters for DADS in an isolated perfused rat liver. kel (min-1) 0.13 ± 0.03 AUC0- (min.mmol/l) 4.77 ± 0.80 t1/2 (min) 6.09 ± 1.26 Clearance (ml/min) 34.22 ± 5.6 Volume of distribution (ml) 311.9 ± 0.08 DADS Oxidation by Rat Liver Microsomes • Figure 4: Proposed scheme for the metabolism of DADS. • Dotted arrows represent hypothetical routes. The width of the arrows symbolizes the importance of the route. Figure 3: Inhibition of DADS oxidation by chemical CYP inhibitors in rat liver microsomes. Values are the percentage of control (without inhibitor). • When isolated rat liver was perfused with DADS, the kinetical parameters indicate a rapid disappearance of DADS (table 1) . Different metabolites were obtained : AM appeared in the perfusate and in the liver tissues, while AM, AMS, AMSO, AMSO2 and AGS were detected in the liver tissue only (figure 1). DADSO was not detected in the perfusate or in the liver tissues. • In the presence of rat monooxygenases, DADS is oxidized to DADSO. The reaction followed Michaelis-Menten kinetics with an apparent Km = 0.86  0.1 mM and an apparent Vmax = 0.85  0.2 nmol/min/nmol CYP. The effects of CYP induction on the oxidation of DADS indicate the involvement of the isoforme CYP2B1/2 (figure 2) whereas the CYP2B1/2 and CYP2E1 specific inhibitors decreased the formation of DADSO (figures 3). In presence of rat GST, DADS is transformed into AGS following Michaelis-Menten kinetics with apparent Km = 9.03±2.6 mM and Vmax = 0.25±0.04 nmol/min/mg protein. CONCLUSION The in vitro metabolism of DADS was investigated with rat subcellular fractions. DADSO is formed by monooxygenases, and conjugation with glutathione takes place with DADS and DADSO to produce AGS. To link the compounds found in the perfused liver, we hypothesise that AM would be methylated to AMS and then oxidated to AMSO and AMSO2 (figure 4). The isoforms of involved CYP are CYP2E1 and CYP2B in rat whereas in human CYP2E1 was the only one (Teyssier et al. 1999). The pharmacokinetic parameters in a perfused liver indicated a little storage of DADS and its rapid disappearance is this tissue. This could explain the absence of DADSO in the output of the perfused liver. Garlic is widely used in the folk medicine. In light of above discussion, we can suppose that the beneficial health properties of garlic are not only due to DADS but also due to its other metabolites. REFERENCES Teyssier C and Siess MH (2000) Metabolism of dipropyl disulfide by rat liver phase I and phase II enzymes and by isolated perfused rat liver. Drug Metabolism and Disposition 28:648-654. Teyssier C, Guenot L, Suschetet M and Siess MH (1999) Metabolism of diallyl disulfide by human liver microsomal cytochromes P- 450 and flavin-containing monooxygenases. Drug Metabolism and Disposition 27:835-841. These studies were supported by Grant from the European Community