[Mechanism of mono-hydroxylation of daidzein in human liver microsomes].
OBJECTIVE
To identify the cytochrome P450 (CYP) isoform (s) involved in daidzein mono-hydroxylated metabolites using human liver microsomes.


METHODS
Kinetic analysis of the rates of formation 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), was performed using human liver microsomes (HLM) and recombinant enzymes at substrate concentrations ranging from 0.5 to 400 micromol x L(-1). 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 one-component enzyme kinetic model. The mean Km (micromol x L(-1) ) and V(max) (micromol x g(-1) x min(-1)) values were 27 +/- 10 and 4. 8 +/- 2.1, 54 +/- 22 and 2.3 +/- 1.0, 51 +/- 29 and 2.2 +/- 0.8, 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) apparently inhibited the formation of mono-hydroxylated metabolites, The IC50 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 mol x L(-1), respectively. The IC50 of estrogen for the formation of 7,3',4'-THI, 6,7,4'-THI and 7,8,4'-THI were 51, 60 and 64 mol x L(-1) respectively. The IC50 of MAB-1A2 for the formation of the mono-hydroxylated products was 1 mol x L(-1), 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
The in vitro studies indicated that daidzein mono-hydroxylated products were principally metabolized by CYP1A2 in human.
Peng
Wen-Xing
WX
Department of Clinical Pharmacy, Second Xiang Ya Hospital, Central South University, Changsha 410011, China. pwx.csu@163.com
Li
Huan-De
HD
Zhou
Hong-Hao
HH
D019388:Cytochrome P-450 CYP1A2 / Q000378:metabolism; D065609:Cytochrome P-450 CYP1A2 Inhibitors; D004967:Estrogens / Q000494:pharmacology; D006801:Humans; D006900:Hydroxylation; D007529:Isoflavones / Q000378:metabolism*; D008862:Microsomes, Liver / Q000378:metabolism*; D013806:Theophylline / Q000031:analogs & derivatives* / Q000494:pharmacology
D065609:Cytochrome P-450 CYP1A2 Inhibitors; D004967:Estrogens; D007529:Isoflavones; C004742:daidzein; D013806:Theophylline; C050131:furafylline; D019388:Cytochrome P-450 CYP1A2