| BackgroundIsoflavones, one of phytoestrogens, including daidzein, genistein, glycitein and their conjugates, have been identified in fruits, vegetables, and whole grains commonly consumed by humans. Epidemiological studies suggest that consumption of soy protein-containing isoflavones lowers the risk of certain cancers, including prostate and breast and perhaps colon cancer. Genistein and daidzein inhibit the proliferation of different types of cancer cells, normal 3T3 cells, and normal colonic mucosa in tissue culture. The exact mechanism for this effect is not yet elucidated, but many hypotheses have been suggested. Soy isoflavones activate estrogen receptors, inhibit the activity of growth-promoting steroid hormones by inhibiting the enzymes progesterone 5a-reductase and 17 B-hydroxysteroid dehydrogenase, have antioxidant activities, inhibit protein-tyrosine kinase-mediated signal transduction, inhibit DNA topoisomerase (ATP- hydrolyzing), inhibit transforming growth factor Brmediated signal transduction, and inhibit angiogenesis. Although these disparate effects of isoflavone, it is possible that the administration of large doses might be associated with some toxicity.Previous studies have shown that only 7-30% of daidzein could elimination unchanged from urine and <10% from feces after oral administration, indicating that this chemical undergoes extensive biotransformation in vivo. In vitro studies with liver microsomes from both rats and human demonstrated that daidzein was readily metabolized to mono-hydroxylated products (7,3',4'-THI, 6,7,4'-THI and 7,8,4'- Till) and dihydroxylation products. It is report rat liver micromes and human recombinant CYP isozyme metabilzed genistein. However, the drug metabolizing-enzyme that involves in the biotransformation of daidzein in human remains to be unclear. Two distinct drug-metabolizing isozyme of the P450 family, namely CYP1A2 and CYP3A4 are known to be primarycontributors to the metabolism of estrogen. It is reported that CYP1A2 mediated flavones metabolism. It has been demonstrated that exogenous estrogen could inhibit CYP1A2 mediated caffeine metabolism. So, it is worthwhile to investigate that daidzein whether or not influence drug metabolic enzyme.Part IEvidence for the involvement of human liver microsome CYP1A2 in themono-hydroxylation of daidzeinObjective: The present study was designed to identify the cytochrome P450 (CYP) isozymes that involved in mono-hydroxylation of daidzein.Methods: Kinetic analysis for the formation of mono-hydroxylated metabolites of daidzein, including 7,8,4'-trihydroxyisoflavone (7,8,4'-THI), 7,3',4'-trihydroxyisofIavone (7,3',4'-THI) and 6,7,4'-trihydroxyisoflavone (6,7,4'-THI), were performed with human liver microsomes (HLM) and recombinant enzymes at substrate concentrations range of l-400u mol.L-1. Selective inhibitors or substrates of various P450 isozymes were also applied to screen the enzyme responsible for mono-hydroxylated metabolism of daidzein.Results: The data of kinetics was consistent with the one-enzyme Michaelis-Menten equation. Mean Km(munol.L-1) and Vmax (mol.g-1.min-1) values (+SD) were 26.86 ( 10.45) and 4.76 ( 2.07 ) for formation of 7,8,4'-THI, 53.83 ( 22.25) and 2.29 ( 1.04 ) for 7,3 ',4'-THI, 51.48 ( 29.32 ) and 2.21( 0.82 ) for formation of 6,7,4'-TIII, respectively. Both the selective inhibitor ftirafylline and monoclonal antibody of CYP1A2 substantially inhibited the formation of the mono-hydroxylated products, while other selective inhibitors of P450 isozymes or probe substrates, including coumarin (CYP2D6), sulphaphenzole (CYP2C9/10), omeprazole (CYP2C19), quinidine(CYP2D6), diethyldithio-carbamate (CYP2E1), troleandomycin (CYP3A4) and keteconazole (CYP3A4), were found to have little or no inhibitory effect on this reaction. These results were also confirmed by studies with recombinant enzymes.Conclusion: CYP1A2 is the primary P450 isozyme that responsible for the mono-hydroxylation of daidzein in human liv...
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