辣椒素香草环在P450催化下代谢产生活性中间体

1、 Reactive Intermediates Produced from the Metabolism of theVanilloid Ring of Capsaicinoids by P450 Enzymes Speaker :Contents Introduction Materials and methods Results Discussion Conclusion2Introduction Capsaicinoids are the pungent compounds in hot peppers.Capsaicin (trans)represents 6070% of all c

2、apsaicinoids in natural sources.OHOHNO3(trans-8-methyl-N-vanillyl-6-nonenamide)IntroductionPharmacodynamics studies Several studies have demonstrated that capsaicinoids may be chemopreventive. Paradoxically, capsaicinoids were also reported to act as Cocarcinogens，and increased rates of liver and st

3、omach cancer. These results are consistent with previous reports of genotoxicity for capsaicinoids following S9 bioactivation.45Introduction objectives Characterized the formation of reactive intermediates from capsaicinoids by P450 enzymes.Characterized the way of the GSH addition to the intermedia

4、tes .6Materials and methods ?VivoVitroCapsaicin+GSH+P450Capsaicin+BME+P450Capsaicin+HRPG1-G9 G1*-G9*B1-B9DimerLC/MS2 1H NMR gHSQC7Materials and methods HRP HRP is a glycoprotein, because its content is very high in horseradish, so named. Its composed of several isozymes, based on iron porphyrins, mo

5、lecular weight is 40000, isoelectric point is PH3 9, hydrogen peroxide can catalytic the polymerization reactions of phenol and phenylamine and their substituendums when H2O2 existed. Usually used as organization chemical tracers in optical and electron microscope. +AH +A HRP + A + P +2H2OS + HRP +

6、H2O2 8Mechanism of action： + AH + H2O2 Materials and methodsMetabolic Incubations (P450) Incubations (250 L) in phosphatebuffered saline, pH 7.2, contained 50 pmol P450 (human liver microsomes) , 2.5 mM NADPH, 2.5 mM GSH, capsaicin, typically at 25 or 100M. Reactions were performed at 37 C,5min ,and

7、 P450 turnover was initiated by the addition of NADPH (2.5 mM).Reactions were terminated by adding 150 L of methanol containing 0.1 ng/L internal standard.Samples were clarified by centrifugation at 21,000g for 5 min, the supernatant was transferred to a clean tube,dried in a vacuum centrifuge, and

8、stored frozen until assayed by LC/MS, as described below.9Materials and methodsMetabolic Incubations (HRP) Samples were prepared in 50 mM sodium acetate, pH 5.5, at room temperature and initiated by the addition of 500 M H2O2. Reactions using HRP were processed as described above. incubation time wa

9、s 10 min. For the determination of Km and the relative proportions of conjugate formation samples were prepared using substrate concentrations of 0.5, 1, 2.5, 10,25, 50, 100, 250, and 500 M.10Materials and methodsSynthesis and NMR Analysis of Conjugate B6 (G6) Capsaicin (2 mg) was dissolved in 2 mL

10、of dry acetonitrile and heated to 60 C.Silver oxide powder (150 mg) was added to the solution, and the reaction was constantly mixed at 60 C for 7.5 min. The mixture was filtered and collected in a tube containing 200 L of BME. The solution was mixed, dried under N2, reconstituted in 200 L of 70% me

11、thanol/water. BME B6-conjugate was enriched by HPLC，and was analysised with NMR.11Materials and methodsSynthesis of N-(Trimethoxybenzyl)nonanamide Analogues N-(2,3,4-trimethoxybenzyl)nonanamide N-(3,4,5-trimethoxybenzyl)nonanamide N-(2,4,5-trimethoxybenzyl)-nonanamideNH2OCH3OCH3OCH3+CLONHOCH3OCH3OCH

12、3O12Materials and methodsAnalysis of GSH Conjugates in Mouse Liver Mice were treated i.p. with 10.0 mg/kg capsaicin (in 50% ethanol). 30 min later , lethal injection (100 mg/kg pentobarbital), and their livers were excised. The livers were washed, homogenized in 5 volumes of 50% methanol/water, and

13、centrifuged at 2,500g for 10 min and again at 100,000g for 60 min. The supernatant was then lyophilized. The residue was reconstituted with 50% methanol/water and again clarified by centrifugation at 20,000g for 5 min . Precursor-to-product ion transitions:m/z 597468, 611308, 611482, and 627498.13Ma

14、terials and methodsAnalysis of N-Acetylcysteine Conjugates in Mouse Mice were treated p.o. or i.v. with 2.5 mg/kg capsaicin, and urine was collected at 24 (n = 5), 48 (n = 2), and 96 h (n = 2) and pooled for each time point. Urine (1.5 mL) was fortified with internal standard (nonivamide) at 100 ng/

15、mL, diluted 2 in phosphatebuffered saline, pH 7.2, clarified by centrifugation, and loaded onto a 3mL Hypersep C18 solid-phase extraction column. The analytes eluted using methanol. The extracts were dried under N2 and reconstituted in mobile phase for LC/MS analysis.14Results(GSH Conjugates)1571.4R

16、esults (G1、G2)16Results (G1、G2)17Results (G1、G2) The formation from capsaicinoid analogues lacking the 4-OH vanilloid ring constituent (i.e., N-benzylnonanamide )was diminished. Analogues with either the exact or similar vanilloid ring structure (i.e., nonivamide) was unchanged. N-(3,4-dihydroxybenz

17、yl) nonanmide and N-(4-hydroxybenzyl)-nonanamide was increased .18O HOHNOResults (B1、B2)19Results (B1、B2) Using nonivamide as the substrate, the fragment ion pattern for B1 was unchanged,despite a 12 amu shift in the molecular ion m/z 368. However, for B2 of nonivamide, the alkyl fragment ions m/z17

18、0, 153, and 135 observed with capsaicin were appropriatelyshifted by 12 amu.NHOHOCH3Ononivamide2021Results (G3)22Results (G3) Formation of G3 from nonivamide was comparable to that of capsaicin. But absent for all analogues lacking a 4-OH moiety (i.e., N-benzylnonanamide). G3 formation from N-(4-hyd

19、roxybenzyl) nonanamide was greater. A quinone methide intermediate being the source of G3. 23Results (B3)2425Results (G4-G6)26Results (B6)2728Results (G7-G9)29Results (G7-G9)30Aromatic hydroxylation of capsaicin was previously shown to be catalyzed byCYP1A2 and 2C19 (M5), and 2B6, 2C8, ,2E1 (M7), wh

20、ile CYP3A4 ,1A1, 2E1, 2C8, 2D6, and 2B6 (M8). G7 and G8 were produced mainly by CYP1A2, suggesting these conjugates were derived from M5.Results (G7-G9)31Results(G7-G9) Hydration of quinone methide intermediates versus P450-catalyzed hydroxyl. Incubations were performed in 50% v/v H218O. Incorporati

21、on of 18O from was not observed. P450-catalyzed oxygenation.CLOCL2-chloronicotinoylchloride32Results(B7)33Results(B8、B9)3435Results(dimer)36Results(dimer)37Results(dimer)3839Assessment of GSH Conjugate Formation In Vivo40Discussion The formation of metabolites via sequential metabolism of capsaicin

22、by P450 enzymes to produce alkyl dehydrogenated and vanilloid ring-modified products (i.e., the G* adducts) was novel. The metabolites diminish activation of TRPV1 but readily form toxic electrophiles. Specific enzyme/substrate interactions play vital roles in the initial bioactivation of the substrates and in determining how GSH interacts with electrophiles during the catalytic process.41Discussion One hypothesis is that capsaicinoids attenuate procarcinogen bioactivation since capsaicinoids appear to inhibit P450 enzymes. Alternate hypotheses suggest