title: Evidence for the involvement of human liver microsomes CYP1A2 in the mono-hydroxylation of daidzein.
abstract: BACKGROUND
  In vitro studies with rats and human liver microsomes (HLM) demonstrated that daidzein is readily metabolized to mono-hydroxylated compounds. In this study, daidzein mono-hydroxylated metabolites was investigated using human liver microsomes to identify the cytochrome P450 (CYP) isoform(s) involved in this metabolic pathway.
  
  
  METHODS
  Kinetic analysis for the formation rates of mono-hydroxylated metabolites of daidzein, including 7,8,4'-trihydroxyisoflavone (7,8,4'-THI), 7,3,4'-trihydroxyisoflavone (7,3,4'-THI) and 6,7,4'-trihydroxyisoflavone (6,7,4'-THI), were performed using human liver microsomes (HLM) and recombinant enzymes at substrate concentrations ranging from 0.5 to 400 micromol/l. Nine selective inhibitors or substrate probes specific for different CYP isoforms were applied for screening the isoform(s) responsible for mono-hydroxylated metabolism of daidzein.
  
  
  RESULTS
  Michaelis-Menten kinetic parameters were best fitted to a one-component enzyme kinetic model. The mean K(m) (micromol/l) and V(max) (micromol/g min) values (+/-S.D.) were 26.86 (10.45) and 4.76 (2.07), 53.83 (22.25) and 2.29 (1.04), 51.48 (29.32) and 2.21(0.82), for the formation rates of 7,8,4'-THI, 7,3',4'-THI and 6,7,4'-THI, respectively. Furafylline, the CYP1A2-specific inhibitor, estrogen and monoclonal antibody raised against human CYP1A2 (MAB-1A2) substantially inhibited the formation rates of mono-hydroxylated metabolites. The IC(50) of Fur for the formation of 7,3',4'-THI, 6,7,4'-THI and 7,8,4'-THI was 1.0, 0.9 and 0.8 micromol/l, respectively. The IC(50) of estrogen for the formation of 7,3',4'-THI, 6,7,4'-THI and 7,8,4'-THI was 51, 60 and 64 micromol/l, respectively. The IC(50) of MAB-1A2 for the formation of the mono-hydroxylated products was 1 micromol/l, but neither other selective inhibitor nor substrate probes, including coumarin (CYP2D6), sulphaphenzole (CYP2C9/10), omeprazole (CYP2C19), quinidine (CYP2D6), diethyldithiocarbamate (CYP2E1), troleandomycin (CYP3A4) and keteconazole (CYP3A4), did so with human liver microsomes.
  
  
  CONCLUSIONS
  Daidzein mono-hydroxylated products are principally metabolized by CYP1A2 in human.
authors.affiliation: Department of Clinical Pharmacy of the Second Xiangya Hospital, Central South University, Changsha, China.
authors.forename: Wen Xing
authors.initials: WX
authors.lastname: Peng
authors.forename: Lian Sheng
authors.initials: LS
authors.lastname: Wang
authors.forename: Huan De
authors.initials: HD
authors.lastname: Li
authors.forename: A M
authors.initials: AM
authors.lastname: Abd El-Aty
authors.forename: Guo Lin
authors.initials: GL
authors.lastname: Chen
authors.forename: Hong Hao
authors.initials: HH
authors.lastname: Zhou
mesh_terms: D000465:Algorithms; D001711:Biotransformation; D002851:Chromatography, High Pressure Liquid; D019388:Cytochrome P-450 CYP1A2 / Q000378:metabolism*; D065609:Cytochrome P-450 CYP1A2 Inhibitors; D018076:DNA, Complementary / Q000096:biosynthesis / Q000235:genetics; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004968:Estrogens, Non-Steroidal / Q000378:metabolism*; D006801:Humans; D006900:Hydroxylation; D066298:In Vitro Techniques; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000378:metabolism; D007529:Isoflavones / Q000378:metabolism*; D007700:Kinetics; D008862:Microsomes, Liver / Q000187:drug effects / Q000201:enzymology*; D011994:Recombinant Proteins / Q000494:pharmacology; D021241:Spectrometry, Mass, Electrospray Ionization
chemical_list: D065609:Cytochrome P-450 CYP1A2 Inhibitors; D018076:DNA, Complementary; D004791:Enzyme Inhibitors; D004968:Estrogens, Non-Steroidal; D007527:Isoenzymes; D007529:Isoflavones; D011994:Recombinant Proteins; C004742:daidzein; D019388:Cytochrome P-450 CYP1A2