WP 6 Cancer General objectives To elucidate the
WP 6 : Cancer General objectives Ø To elucidate the mechanisms of action of garlic and sulphur compounds on different biological events involved in carcinogenesis: inhibition of bio-activation and/or stimulation of detoxification of carcinogens prevention of genotoxicity inhibition of the initiation of liver carcinogenesis influence on apoptosis in tumour and normal cells To investigate the metabolism of garlic sulfur compounds WP 6 , UMR de Toxicologie Alimentaire INRA
WP 6 : Bioavailability and metabolic fate of sulfur compounds Caroline Teyssier, Emmanuelle Germain, Joëlle Chevalier Milestones : Ø In vitro metabolism of DADS and allicine by human and rat subcellular fractions (Year 1) In vitro Ø Study of the bioavailability of DADS in rat by measuring the concentrations of the metabolites in blood and main organs (Year 2) In vivo Ø Study of the metabolism of DADS by an isolated perfused rar liver (additional objective, year 2) Ex vivo WP 6 , UMR de Toxicologie Alimentaire INRA
WP 6 : In vivo metabolism of DADS, OBJECTIVES Ø to identify the metabolites formed from DADS after a single oral administration Ø to investigate the absorption , distribution and bioavailability of DADS and its metabolites Ø to determine the pharmacokinetic parameters of DADS and its metabolites WP 6 , UMR de Toxicologie Alimentaire INRA
WP 6 : Ex vivo and in vitro metabolism of DADS OBJECTIVES Ex vivo : liver perfusion To better understand the role of liver in the metabolism of DADS : Øidentification of metabolites formed from DADS Ødetermination of pharmacokinetic parameters of DADS and its metabolites In vitro : metabolism of by rat and human liver subcellular fractions To validate the hypothetical pathway for the metabolism of DADS : Øconfirmation of the different transformations (oxidation, conjugation. . ) Øidentification of the enzymes involved in these transformation WP 6 , UMR de Toxicologie Alimentaire INRA
In vivo metabolism of DADS EXPERIMENTAL Sample analysis DADS i. g. 200 mg/kg Stomach Liver ühomogenization, p-cymene (internal standard) 4 protein precipitation (TCA, 30%) 4 Extraction with CH 2 CL 2 (3 times) Urine Plasma Identification: GC-MS analysis Quantification : Selected Ion Monitoring WP 6 , UMR de Toxicologie Alimentaire INRA
In vivo metabolism of DADS in the rat RESULTS Identification of metabolites in stomach, liver plasma and urine SH Allylmercaptan AM Allylmethylsulfide AMS Allylmethylsulfoxide AMSO Allylmethylsulfone AMSO 2 WP 6 , UMR de Toxicologie Alimentaire INRA
In vivo metabolism of DADS in the rat DADS AM 100 AMS Compounds (mg. g-1) 80 AMSO 2 60 40 20 0 0 5 10 15 days Time course of DADS and its metabolites in stomach after a single oral administration WP 6 , UMR de Toxicologie Alimentaire INRA
In vivo metabolism of DADS in the rat 60 AMS Compounds (mg. g-1) AMSO 2/10 40 20 0 0 5 10 15 days Time course of DADS metabolites in liver after a single oral administration WP 6 , UMR de Toxicologie Alimentaire INRA
In vivo metabolism of DADS in the rat AMS 15 AMSO/10 Compounds (mg. g-1) AMSO 2/10 10 5 0 0 5 10 15 days Time course of DADS metabolites in plasma after a single oral administration WP 6 , UMR de Toxicologie Alimentaire INRA
In vivo metabolism of DADS in the rat AMS 15 AMSO Compounds (mg. g-1) AMSO 2 10 5 0 0 5 days 10 15 Time course of DADS metabolites in urine after a single oral administration WP 6 , UMR de Toxicologie Alimentaire INRA
In vivo metabolism of DADS in the rat Table : Pharmacokinetic parameters of DADS and its metabolites t 1/2 (hr) AUC (hr. mmol. l-1) DADS AM AMSO 2 <1 4. 4 6. 8 7. 2 8. 6 <0. 1 0. 32 0. 33 23. 7 116. 8 WP 6 , UMR de Toxicologie Alimentaire INRA
In vivo metabolism of DADS in the rat CONCLUSION Ø DADS is absorbed and transformed into AM, AMSO and AMSO 2 which are detected throughout the follow-up period. Ø The highest amounts of metabolites are measured 2 -3 days after DADS application. Ø AMSO 2 is the most abundant and the most persistent of these compounds in all analysed tissues. Ø The levels of all the sulfur compounds rapidly decline within the first week and disappear during the second one. Ø AMSO and AMSO 2 are significantly excreted in urine. ØFurther studies to assess the effect of AMSO and AMSO 2 WP 6 , UMR de Toxicologie Alimentaire INRA
Metabolism of DADS by isolated perfused rat liver EXPERIMENTAL reservoir liver collection of bile WP 6 , UMR de Toxicologie Alimentaire INRA
Metabolism of DADS by isolated perfused rat liver Metabolites identified in the output SH Allylmercaptan AM Allylmethylsulfide AMS S glutathione Allylmethylsulfoxide AMSO Allylmethylsulfone AMSO 2 Allylglutathione sulfide AGS WP 6 , UMR de Toxicologie Alimentaire INRA
Metabolism of DADS by isolated perfused rat liver Perfusate Liver tissue Bile DADS AM AMS AMSO 2 AMSO 2 AGS AGS nd ? WP 6 , UMR de Toxicologie Alimentaire INRA
Metabolism of DADS by isolated perfused rat liver CONCLUSION Ø Confirmation of the metabolites identified in the in vivo protocol Ø Pharmacokinetic parameters indicate a very rapid transformation of DADS WP 6 , UMR de Toxicologie Alimentaire INRA
WP 6 : In vitro metabolism of DADS OBJECTIVES : In vitro : metabolism of by rat and human liver subcellular fractions To validate the hypothetical pathway for the metabolism of DADS : Øconfirmation of the different transformations (oxidation, conjugation. . ) Øidentification of the enzymes involved in these transformation WP 6 , UMR de Toxicologie Alimentaire INRA
In vitro metabolism of DADS and its metabolites by subcellular fractions EXPERIMENTAL DADS AM cofactors AMS. . . ultracentrifugation HPLC-UV GC-MS microsomes cytosols subcellular fractions rat or human WP 6 , UMR de Toxicologie Alimentaire INRA
In vitro metabolism of DADS and its metabolites by subcellular fractions Oxidation : microsomes CYP, Flavine monoxygenase (FMO) CYP 2 B, 2 E 1 FMO WP 6 , UMR de Toxicologie Alimentaire INRA not shown
In vitro metabolism of DADS and its metabolites by subcellular fractions Methylation : microsomes S-methyl transferase not shown SH AM AMS WP 6 , UMR de Toxicologie Alimentaire INRA
In vitro metabolism of DADS and its metabolites by subcellular fractions Conjugation : cytosols glutathione S-transferase SH Allylmercaptan GST + GSH S glutathione DADS Non enzymatic + GSH S glutathione DADSO WP 6 , UMR de Toxicologie Alimentaire INRA Allylglutathione sulfide
CONCLUSION : Proposed scheme for the metabolism of DADS AMSO 2 allyl methyl sulfone Compounds obtained in in vitro Compounds obtained in vivo AMSO allyl methyl sulfoxide MT AMS allyl methyl sulfide DADS SH CYP 2 B 1/2 CYP 2 E 1 FMO AM allyl mercaptan GST S glutathione AGS allyl glutathionyl sulfide DADSO WP 6 , UMR de Toxicologie Alimentaire INRA
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