Evidence for the involvement of human liver microsomes CYP1A2 in the mono-hydroxylation of daidzein.
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.
Peng
Wen Xing
WX
Department of Clinical Pharmacy of the Second Xiangya Hospital, Central South University, Changsha, China.
Wang
Lian Sheng
LS
Li
Huan De
HD
Abd El-Aty
A M
AM
Chen
Guo Lin
GL
Zhou
Hong Hao
HH
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
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