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WP 6 : Cancer General objectives

WP 6 : Cancer General objectives. 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

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WP 6 : Cancer General objectives

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  1. WP 6 : Cancer General objectives • 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

  2. WP6 : Bioavailability and metabolic fate of sulfur compounds Caroline Teyssier, Emmanuelle Germain, Joëlle Chevalier Milestones : • In vitro metabolism of DADS and allicine by human and rat subcellular fractions (Year 1) In vitro • Study of the bioavailability of DADS in rat by measuring the concentrations of the metabolites in blood and main organs (Year 2) In vivo • 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

  3. WP6 :In vivometabolism of DADS,OBJECTIVES • to identify the metabolites formed from DADS after a single oral administration • to investigate the absorption , distribution and bioavailability of DADS and its metabolites • to determine the pharmacokinetic parameters of DADS and its metabolites WP 6 , UMR de Toxicologie Alimentaire INRA

  4. WP6 :Ex vivo and in vitrometabolism of DADS OBJECTIVES In vitro : metabolism of by rat and human liver subcellular fractions Ex vivo : liver perfusion • To validate the hypothetical pathway for the metabolism of DADS : • confirmation of the different transformations (oxidation, conjugation..) • identification of the enzymes involved in these transformation • To better understand the role of liver in the metabolism of DADS : • identification of metabolites formed from DADS • determination of pharmacokinetic parameters of DADS and its metabolites WP 6 , UMR de Toxicologie Alimentaire INRA

  5. In vivometabolism of DADS EXPERIMENTAL Sampleanalysis DADS i.g. 200 mg/kg Stomach • homogenization, p-cymene (internal standard) • protein precipitation (TCA, 30%) • Extraction with CH2CL2 (3 times) Liver Urine Identification: GC-MSanalysis Quantification : Selected Ion Monitoring Plasma WP 6 , UMR de Toxicologie Alimentaire INRA

  6. SH In vivo metabolism of DADS in the rat RESULTS Identification of metabolites in stomach, liver plasma and urine Allylmercaptan AM Allylmethylsulfide AMS Allylmethylsulfoxide AMSO Allylmethylsulfone AMSO2 WP 6 , UMR de Toxicologie Alimentaire INRA

  7. DADS AM AMS 100 AMSO 80 Compounds (mg.g-1) AMSO2 60 40 20 0 0 5 10 15 days In vivo metabolism of DADS in the rat Time course of DADS and its metabolites in stomach after a single oral administration WP 6 , UMR de Toxicologie Alimentaire INRA

  8. 60 40 Compounds (mg.g-1) 20 0 0 5 10 15 days AMS AMSO AMSO2/10 In vivo metabolism of DADS in the rat Time course of DADS metabolites in liver after a single oral administration WP 6 , UMR de Toxicologie Alimentaire INRA

  9. 15 10 Compounds (mg.g-1) 5 0 0 5 10 15 days AMS AMSO/10 AMSO2/10 In vivo metabolism of DADS in the rat Time course of DADS metabolites in plasma after a single oral administration WP 6 , UMR de Toxicologie Alimentaire INRA

  10. 15 10 Compounds (mg.g-1) 5 0 0 5 10 15 days AMS AMSO AMSO2 In vivo metabolism of DADS in the rat Time course of DADS metabolites in urine after a single oral administration WP 6 , UMR de Toxicologie Alimentaire INRA

  11. DADS AM AMS AMSO AMSO2 <1 4.4 6.8 7.2 8.6 t1/2 (hr) <0.1 0.32 0.33 23.7 116.8 AUC (hr.mmol.l-1) In vivo metabolism of DADS in the rat Table : Pharmacokinetic parameters of DADS and its metabolites WP 6 , UMR de Toxicologie Alimentaire INRA

  12. In vivo metabolism of DADS in the rat CONCLUSION • DADS is absorbed and transformed into AM, AMS, AMSO and AMSO2 which are detected throughout the follow-up period. • The highest amounts of metabolites are measured 2-3 days after DADS application. • AMSO2 is the most abundant and the most persistent of these compounds in all analysed tissues. • The levels of all the sulfur compounds rapidly decline within the first week and disappear during the second one. • AMSO and AMSO2 are significantly excreted in urine. • Further studies to assess the effect of AMSO and AMSO2 WP 6 , UMR de Toxicologie Alimentaire INRA

  13. Metabolism of DADS by isolated perfused rat liver EXPERIMENTAL reservoir liver collection of bile WP 6 , UMR de Toxicologie Alimentaire INRA

  14. SH S glutathione Metabolism of DADS by isolated perfused rat liver Metabolites identified in the output Allylmercaptan AM Allylmethylsulfide AMS Allylmethylsulfoxide AMSO Allylmethylsulfone AMSO2 Allylglutathione sulfide AGS WP 6 , UMR de Toxicologie Alimentaire INRA

  15. Metabolism of DADS by isolated perfused rat liver Perfusate Liver tissue Bile DADS AM AMS AMSO AMSO2 AGS DADS AM AMS AMSO AMSO2 AGS DADS AM AGS nd ? WP 6 , UMR de Toxicologie Alimentaire INRA

  16. Metabolism of DADS by isolated perfused rat liver CONCLUSION • Confirmation of the metabolites identified in the in vivo protocol • Pharmacokinetic parameters indicate a very rapid transformation of DADS WP 6 , UMR de Toxicologie Alimentaire INRA

  17. WP6 :In vitrometabolism of DADS OBJECTIVES : In vitro : metabolism of by rat and human liver subcellular fractions • To validate the hypothetical pathway for the metabolism of DADS : • confirmation of the different transformations (oxidation, conjugation..) • identification of the enzymes involved in these transformation WP 6 , UMR de Toxicologie Alimentaire INRA

  18. In vitro metabolism of DADS and its metabolites by subcellular fractions EXPERIMENTAL DADS AM AMS... cofactors ultracentrifugation HPLC-UV GC-MS microsomes cytosols subcellular fractions rat or human WP 6 , UMR de Toxicologie Alimentaire INRA

  19. In vitro metabolism of DADS and its metabolites by subcellular fractions Oxidation : microsomes CYP, Flavine monoxygenase (FMO) CYP 2B, 2E1 FMO not shown WP 6 , UMR de Toxicologie Alimentaire INRA

  20. SH In vitro metabolism of DADS and its metabolites by subcellular fractions Methylation : microsomes S-methyl transferase not shown AM AMS WP 6 , UMR de Toxicologie Alimentaire INRA

  21. SH S S glutathione glutathione In vitro metabolism of DADS and its metabolites by subcellular fractions Conjugation : cytosols glutathione S-transferase Allylmercaptan GST + GSH Allylglutathione sulfide DADS Non enzymatic Allylglutathione sulfide + GSH DADSO WP 6 , UMR de Toxicologie Alimentaire INRA

  22. Compounds obtained in in vitro Compounds obtained in vivo MT CYP 2B1/2 CYP 2E1 FMO CONCLUSION : Proposed scheme for the metabolism of DADS AMSO2 allyl methyl sulfone AMSO allyl methyl sulfoxide AMS allyl methyl sulfide DADS SH AM allyl mercaptan GST S glutathione AGS allyl glutathionyl sulfide DADSO WP 6 , UMR de Toxicologie Alimentaire INRA

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