title: Mechanisms for epigallocatechin gallate induced inhibition of drug metabolizing enzymes in rat liver microsomes.
abstract: Epigallocatechin gallate (EGCG) inhibits drug metabolizing enzymes by unknown mechanisms. Here we examined if the inhibition is due to covalent-binding of EGCG to the enzymes or formation of protein aggregates. EGCG was incubated with rat liver microsomes at 1-100μM for 30min. The EGCG-binding proteins were affinity purified using m-aminophenylboronic acid agarose and probed with antibodies against glyceraldehyde-3-phosphate dehydrogenase (GAPDH), actin, cytochrome P450 (CYP) 1A1, CYP1A2, CYP2B1/2, CYP2E1, CYP3A, catechol-O-methyltransferase (COMT) and microsomal glutathione transferase 1 (MGST1). All but actin and soluble COMT were positively detected at ≥1μM EGCG, indicating EGCG selectively bound to a subset of proteins including membrane-bound COMT. The binding correlated well with inhibition of CYP activities, except for CYP2E1 whose activity was unaffected despite evident binding. The antioxidant enzyme MGST1, but not cytosolic GSTs, was remarkably inhibited, providing novel evidence supporting the pro-oxidative effects of EGCG. When microsomes incubated with EGCG were probed on Western blots, all but the actin and CYP2E1 antibodies showed a significant reduction in binding at ≥1μM EGCG, suggesting that a fraction of the indicated proteins formed aggregates that likely contributed to the inhibitory effects of EGCG but were not recognizable by antibodies against the intact proteins. This raised the possibility that previous reports on EGCG regulating protein expression using GAPDH as a reference should be revisited for accuracy. Remarkable protein aggregate formation in EGCG-treated microsomes was also observed by analyzing Coomassie Blue-stained SDS-PAGE gels. EGCG effects were partially abolished in the presence of 1mM glutathione, suggesting they are particularly relevant to the in vivo conditions when glutathione is depleted by toxicant insults.
authors.affiliation: Division of Systems Biology, National Center for Toxicological Research, Food and Drug Administration, 3900 NCTR Road, Jefferson, AR 72079, USA.
authors.forename: Zuquan
authors.initials: Z
authors.lastname: Weng
authors.forename: James
authors.initials: J
authors.lastname: Greenhaw
authors.forename: William F
authors.initials: WF
authors.lastname: Salminen
authors.forename: Qiang
authors.initials: Q
authors.lastname: Shi
mesh_terms: D000818:Animals; D000975:Antioxidants / Q000494:pharmacology*; D015153:Blotting, Western; D002392:Catechin / Q000031:analogs & derivatives* / Q000494:pharmacology; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D003600:Cytosol / Q000187:drug effects / Q000201:enzymology; D004305:Dose-Response Relationship, Drug; D004336:Drug Antagonism; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005978:Glutathione / Q000494:pharmacology; D005982:Glutathione Transferase / Q000037:antagonists & inhibitors / Q000378:metabolism; D008297:Male; D008862:Microsomes, Liver / Q000187:drug effects* / Q000201:enzymology; D011485:Protein Binding; D051381:Rats; D017207:Rats, Sprague-Dawley
chemical_list: D000975:Antioxidants; D065607:Cytochrome P-450 Enzyme Inhibitors; D004791:Enzyme Inhibitors; D002392:Catechin; D003577:Cytochrome P-450 Enzyme System; C045651:epigallocatechin gallate; C122107:microsomal glutathione S-transferase-I; D005982:Glutathione Transferase; D005978:Glutathione