GdR Phycotox. J. Alarcan, E. Dubreil, L. Le Hégarat, D. Hurtaud-Pessel, V. Fessard

Transcription

1 GdR Phycotox In vitro investigation on the human metabolism of lipophilic phycotoxins PT-2 and SP-1 using liquid chromatography hyphenated with high resolution Orbitrap mass spectrometry J. Alarcan, E. Dubreil, L. Le Hégarat, D. Hurtaud-Pessel, V. Fessard Villefranche sur mer 15th and 16th of March,

2 2 possibilities : Why studying metabolism? xenobiotic I/II enzyme xenobiotic R xenobiotic I/II enzyme xenobiotic R Detoxification Metabolic bioactivation Need to know the fate of phycotoxins and identify metabolites to fully apprehend toxicity 2

3 How to perform in vitro metabolism? Biological tool Analytical tool Biological model Features Analytical technique Features Enzyme recombinant S9 liver fraction Onetargeted reaction both phase I &II Mass spectrometry Spectroscopy Quantification / Structural elucidation Quantification Liver microsomes Mainly phase I NMR Full structural elucidation Hepatic cell line All phases S9 liver fraction mass spectrometry 3

4 Reminds on metabolism xenobiotic excretion Phase I xenobiotic - OH xenobiotic Phase II Phase II xenobiotic - R excretion Phase II: SULT GSH METHYL UGT 4 * cell scheme from Servier Medical Art

5 Published data Autors Toxin Reaction Biological model Results Kittler et al PT 2 hydroxylation glucuronidation rat S9 5 phase I metabolites No phase II metabolites GSH conjugation Joseph P. M. Hui et al SP 1 hydroxylation glucuronidation HLM 9 phase I metabolites No phase II metabolites Scarce information Lack certain phase II reactions 5

6 Methodology SP-1 PT-2 OH Phase I inactivated S9 active S9 SULT MET GSH UGT Phase II 3h incubation 1 ml MeOH centrifugation Injection of supernatant LTQ orbitrap 6

7 Methodology 1) Dosage of parent compound 2) Seek of metabolites via MetWorks software Toxin ESI Adduct Mass RT (min) PT 2 + [M] NH4+ 876,51 8,48 SP 1 + [M] H+ 692,45 5,74 Full scan mode analysis Bank of metabolic reactions based on mass shifts From parent compound mass, screening of reactions via bank data SP1 Y = * R^2 = W: Equal Area ugml Multi-toxins calibration 0, 5, 10, 25, 50, 75, 100 µg/l 7

8 SP-1 8

9 SP-1 Rat S9 liver fraction 100 inactivated S9 active S9 80 % Recovery OH SULT METHYL GSH UGT Reaction OH SULT METHYL GSH UGT Metabolite detected via MW 9

10 SP-1 Rat S9 liver fraction 100 inactivated S9 active S9 80 % Recovery OH SULT METHYL GSH UGT Reaction OH SULT METHYL GSH UGT Metabolite detected via MW 10

11 SP-1 Rat S9 liver fraction Human S9 liver fraction 100 inactivated S9 active S9 100 inactivated S9 active S % Recovery % Recovery OH SULT METHYL GSH UGT 0 OH SULT METHYL GSH UGT Reaction Metabolite detected via MW Reaction Metabolite detected via MW OH OH SULT SULT METHYL METHYL GSH GSH UGT UGT 11

12 SP-1 Rat S9 liver fraction Human S9 liver fraction 100 inactivated S9 active S9 100 inactivated S9 active S % Recovery % Recovery OH SULT METHYL GSH UGT 0 OH SULT METHYL GSH UGT Reaction Metabolite detected via MW Reaction Metabolite detected via MW OH OH SULT SULT METHYL METHYL GSH GSH UGT UGT 12

13 SP-1 active S9 Rat S9 SP ,99 SP ,99 13

14 SP-1 inactivated S9 Rat S9 14

15 PT-2 15

16 PT-2 Rat S9 liver fraction 200 inactivated S9 active S % Recovery OH SULT METHYL GSH UGT Reaction OH SULT METHYL GSH UGT Metabolite detected via MW 16

17 PT-2 Rat S9 liver fraction 200 inactivated S9 active S % Recovery OH SULT METHYL GSH UGT Reaction OH SULT METHYL GSH UGT Metabolite detected via MW 17

18 PT-2 Rat S9 liver fraction Human S9 liver fraction 200 inactivated S9 active S inactivated S9 active S % Recovery % Recovery OH SULT METHYL GSH UGT 0 OH SULT METHYL GSH UGT Reaction Metabolite detected via MW Reaction Metabolite detected via MW OH OH SULT SULT METHYL METHYL GSH GSH UGT UGT 18

19 PT-2 Rat S9 liver fraction Human S9 liver fraction 200 inactivated S9 active S inactivated S9 active S % Recovery % Recovery OH SULT METHYL GSH UGT 0 OH SULT METHYL GSH UGT Reaction Metabolite detected via MW Reaction Metabolite detected via MW OH OH SULT SULT METHYL METHYL GSH GSH UGT UGT 19

20 PT-2 active S9 Rat S9 PT ,99 PT ,99 20

21 PT-2 inactivated S9 Rat S9 21

22 Positive controls 22

23 Reaction Positif control Result OH Diclofenac SULT Apigenin METHYL Epinephrine Phenol GSH MCY LR UGT 4 methylumbelliferone 23

24 Conclusions 24

25 Conclusions Use of LC/HRMS is adapted for metabolism survey S9 fraction easy-to-use tool, worth for first examination Comparison man-rat qualitative but not quantitative

26 Prospects Seek explanations for recoveries 100 inactivated S9 active S9 80 % Recovery Due to protein binding Due to ionization Due to co factors 0 OH SULT METHYL GSH UGT

27 Prospects Elucidate metabolites structures through fragmentation assays Frag-SP-OH-MS6-inj05 #392 RT: 5.66 AV: 1 NL: 2.17E1 F: ITMS + c ESI Full ms @cid @cid @cid @cid @cid35.00 [ ] Relative Abundance m/z

28 Prospects Experiment screening of phase I & II altogether thanks to hepatic cell lines / S9 fractions DSP DSP Phase I DSP-OH Phase II HepaRG cells Phase II DSP- SULT/UGT/ GSH/MET DSP- SULT/UGT/ GSH/MET 28

29 Acknowledgement Toxicology unit Valérie Fessard Ludovic Le Hégarat Marine Biotoxins RNL Ronel Biré Frédéric Hommet Analytical Chemistry unit Dominique Hurtaud-Pessel Estelle Dubreil Jean Michel Delmas Food Safety Dept Alfonso Lampen Stefanie Hessel 29

30 Thanks for your attention 30