metabolism of diallyl disulfide by human liver microsomal ...
metabolism of diallyl disulfide by human liver microsomal ... metabolism of diallyl disulfide by human liver microsomal ...
840 TEYSSIER ET AL. 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 Downloaded from dmd.aspetjournals.org by guest on November 24, 2012
DIALLYL DISULFIDE METABOLISM BY HUMAN LIVER MONOOXYGENASES 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. Downloaded from dmd.aspetjournals.org by guest on November 24, 2012
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DIALLYL DISULFIDE METABOLISM BY HUMAN LIVER MONOOXYGENASES<br />
and changes in its activity in female mouse <strong>liver</strong> with nutritional and diurnal conditions. Arch<br />
Biochem Biophys 233:50–63.<br />
Draper AJ, Madan A and Parkinson A (1997) Inhibition <strong>of</strong> coumarin 7-hydroxylase activity in<br />
<strong>human</strong> <strong>liver</strong> microsomes. Arch Biochem Biophys 341:47–61.<br />
Egen-Schwind C, Eckard R, Jekat FW and Winterh<strong>of</strong>f H (1992) Pharmacokinetics <strong>of</strong> vinyldithiins,<br />
transformation products <strong>of</strong> allicin. Planta Med 58:8–13.<br />
Ferary S (1996) Analyse des composés soufrés émis par quelques espèces végétales dans les<br />
relations plantes-insectes. P.h.D. thesis l’Université de Tours, France.<br />
Grothusen A, Hardt J, Brautigam L, Lang D and Bocker R (1996) A convenient method to<br />
discriminate between cytochrome P450 enzymes and flavin-containing monooxygenases in<br />
<strong>human</strong> <strong>liver</strong> microsomes. Arch Toxicol 71:64–71.<br />
Guengerich FP, Kim DH and Iwasaki M (1991) Role <strong>of</strong> <strong>human</strong> cytochrome P-450 IIE1 in the<br />
oxidation <strong>of</strong> many low molecular weight cancer suspects. Chem Res Toxicol 4:168–179.<br />
Guengerich FP, Martin MV, Beaune PH, Kremers P, Wolff T and Waxman DJ (1986) Characterization<br />
<strong>of</strong> rat and <strong>human</strong> <strong>liver</strong> <strong>microsomal</strong> cytochrome P-450 forms involved in nifedipine<br />
oxidation, a prototype for genetic polymorphism in oxidative drug <strong>metabolism</strong>. J Biol Chem<br />
261:5051–5060.<br />
Haber D, Siess MH, Canivenc-Lavier MC, Le Bon AM and Suschetet M (1995) Differential<br />
effects <strong>of</strong> dietary <strong>diallyl</strong> sulfide and <strong>diallyl</strong> <strong>disulfide</strong> on rat intestinal and hepatic drugmetabolizing<br />
enzymes. J Toxicol Environ Health 44:423–434.<br />
Haber D, Siess MH, De Waziers I, Beaune P and Suschetet M (1994) Modification <strong>of</strong> hepatic<br />
drug-metabolizing enzymes in rat fed naturally occurring allyl sulphides. Xenobiotica 24:169–<br />
182.<br />
Haber-Mignard D, Suschetet M, Berges R, Astorg P and Siess MH (1996) Inhibition <strong>of</strong> aflatoxin<br />
B1- and N-nitrosodiethylamine-induced <strong>liver</strong> preneoplastic foci in rats fed naturally occurring<br />
allyl sulfides. Nutr Cancer 25:61–70.<br />
Harris JW, Rahman A, Kim BR, Guengerich FP and Collins JM (1994) Metabolism <strong>of</strong> Taxol <strong>by</strong><br />
<strong>human</strong> hepatic microsomes and <strong>liver</strong> slices: Participation <strong>of</strong> cytochrome P450 3A4 and an<br />
unknown P450 enzyme. Cancer Res 54:4026–4035.<br />
Ip C, Lisk DJ and Stoewsand GS (1992) Mammary cancer prevention <strong>by</strong> regular garlic and<br />
selenium-enriched garlic. Nutr Cancer 17:279–286.<br />
Jin L and Baillie TA (1997) Metabolism <strong>of</strong> the chemoprotective agent <strong>diallyl</strong> sulfide to<br />
glutathione conjugates in rats. Chem Res Toxicol 10:318–327.<br />
Le Bon AM, Roy C, Dupont C and Suschetet M (1997) In vivo antigenotoxic effects <strong>of</strong> dietary<br />
allyl sulfides in the rat. Cancer Lett 114:131–134.<br />
841<br />
Lin RI (1989) Garlic in Nutrition and Medicine. Nutrition International Co., Irvine, CA.<br />
Maurice M, Emiliani S, Dalet BI, Derancourt J and Lange R (1991) Isolation and characterization<br />
<strong>of</strong> a cytochrome P450 <strong>of</strong> the IIA subfamily from <strong>human</strong> <strong>liver</strong> microsomes. Eur J Biochem<br />
200:511–517.<br />
Meier UT, Kronbach T and Meyer UA (1985) Assay <strong>of</strong> mephenytoin <strong>metabolism</strong> in <strong>human</strong> <strong>liver</strong><br />
microsomes <strong>by</strong> high-performance liquid chromatography. Anal Biochem 151:286–291.<br />
Nickson RM and Mitchell SC (1994) Fate <strong>of</strong> dipropyl sulphide and dipropyl sulphoxide in rat.<br />
Xenobiotica 24:157–168.<br />
Parker GL and Orton TC (1980) Induction <strong>by</strong> oxyisobutyrates <strong>of</strong> hepatic and kidney <strong>microsomal</strong><br />
cytochrome P-450 with specificity towards hydroxylation <strong>of</strong> fatty acids, in Biochem Biophys<br />
and Regulation <strong>of</strong> Cytochrome P-450 (Gustafsson JA, Carlstedt-Duke J, Mode A and Rafter<br />
J eds) p 373–377, Elsevier, Amsterdam.<br />
Postlind H, Danielson, Lindgren A and Andersson SHG (1998) Tolterodine, a new muscarinic<br />
receptor antagonist, is metabolized <strong>by</strong> cytochromes P450 2D6 and 3A in <strong>human</strong> <strong>liver</strong><br />
microsomes. Drug Metab Dispos 26:289–293.<br />
Poulsen LL, Hyslop RM and Ziegler DM (1974) S-oxygenation <strong>of</strong> N-substituted thioureas<br />
catalyzed <strong>by</strong> the pig <strong>liver</strong> <strong>microsomal</strong> FAD-containing monooxygenases. Arch Biochem<br />
Biophys 198:78–88.<br />
Pushpendran CK, Devasagayam TP, Chintalwar GJ, Banerji A and Eapen J (1980) The metabolic<br />
fate <strong>of</strong> [35S]-<strong>diallyl</strong> disulphide in mice. Experientia 36:1000–1001.<br />
Reddy BS, Rao CV, Rivenson A and Kell<strong>of</strong>f G (1993) Chemoprevention <strong>of</strong> colon carcinogenesis<br />
<strong>by</strong> organosulfur compounds. Cancer Res 53:3493–3498.<br />
Reuter HD (1995) Allium sativum and allium ursinum: Part 2. Pharmacology and medicinal<br />
application. Phytomedicine 2:73–91.<br />
Rodrigues AD (1994) Use <strong>of</strong> in vitro <strong>human</strong> <strong>metabolism</strong> studies in drug development. An<br />
industrial perspective. Biochem Pharmacol 48:2147–2156.<br />
Shimada T, Yamazaki H, Mimura M, Inui Y and Guengerich FP (1994) Interindividual variations<br />
in <strong>human</strong> <strong>liver</strong> cytochrome P-450 enzymes involved in the oxidation <strong>of</strong> drugs, carcinogens and<br />
toxic chemicals: Studies with <strong>liver</strong> microsomes <strong>of</strong> 30 Japanese and 30 Caucasians. J Pharmacol<br />
Exp Ther 270:414–423.<br />
Tassaneeyakul W, Veronese ME, Birkett DJ, Gonzalez FJ and Miners JO (1993) Validation <strong>of</strong><br />
4-nitrophenol as an in vitro substrate probe for <strong>human</strong> <strong>liver</strong> CYP2E1 using cDNA expression<br />
and <strong>microsomal</strong> kinetic techniques. Biochem Pharmacol 46:1975–1981.<br />
Ullrich V and Weber P (1972) The O-dealkylation <strong>of</strong> 7-ethoxycoumarin <strong>by</strong> <strong>liver</strong> microsomes. A<br />
direct fluorometric test. Hoppe-Seyler’s Z Physiol Chem 353:1171–1177.<br />
Downloaded from<br />
dmd.aspetjournals.org <strong>by</strong> guest on November 24, 2012
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