Effects of flavonoid phytochemicals on cortisol production and on activities of steroidogenic enzymes in human adrenocortical H295R cells.
Inhibitory effects of flavonoid phytochemicals, flavones, flavonols and isoflavones on cortisol production were examined in human adrenal H295R cells stimulated with di-buthylyl cAMP. In addition, the inhibitory effects of these chemicals on the activity of P450scc, 3beta-HSD type II (3beta-HSD II), P450c17, P450c21 and P45011beta, steroidogenic enzymes involved in cortisol biosynthesis, were examined in the same cells. Exposure to 12.5 microM of the flavonoids 6-hydroxyflavone, 4'-hydroxyflavone, apigenin, daidzein, genistein and formononetin significantly decreased cortisol production (by 6.3, 69.6, 47.5, 26.6, 13.8 and 11.3%, respectively), and biochanin A significantly decreased cortisol production (by 47.3%) at a concentration of 25 microM without any significant cytotoxic effects or changes in cell number. Daidzin, the 7-glucoside of daidzein, did not alter cortisol production by H295R cells at concentrations over 10 microg/ml (24 microM). Daidzein-induced reduction of cortisol production by H295R cells was not inhibited by the estrogen receptor antagonist ICI 182,780. The flavonoids 6-hydroxyflavone, daidzein, genistein, biochanin A and formononetin strongly and significantly inhibited microsomal 3beta-HSD II activity at concentrations from 1 to 25 microM, and I(50) values were estimated to be 1.3, 2, 1, 0.5 and 2.7 microM, respectively. In addition, these flavonoids significantly inhibited microsomal P450c21 activity at 12.5 and/or 25 microM. In addition, 6-hydroxyflavone inhibited activity of microsomal P450c17 and mitochondrial P45011beta at 12.5 and/or 25 microM. Results of Lineweaver-Burk's plot analysis indicate that daidzein is a competitive inhibitor of the activity of 3beta-HSD II and P450c21. K(m) and V(max) values of 3beta-HSD II for DHEA were estimated to be 6.6 microM and 328pmol/minmg protein, respectively. K(m) and V(max) values of P450c21 for progesterone were estimated to be 2.8 microM and 16pmol/minmg protein, respectively. K(i) values of 3beta-HSD II and P450c21 for daidzein were estimated to be 2.9 and 33.3 microM, respectively.
Ohno
Shuji
S
Department of Biochemistry, Faculty of Pharmaceutical Sciences, Hoshi University, 2-4-41 Ebara, Shinagawa-ku, Tokyo 142-8501, Japan.
Shinoda
Satoshi
S
Toyoshima
Satoshi
S
Nakazawa
Hiroyuki
H
Makino
Tsunehisa
T
Nakajin
Shizuo
S
D015096:3-Hydroxysteroid Dehydrogenases / Q000037:antagonists & inhibitors / Q000378:metabolism; D000302:Adrenal Cortex / Q000166:cytology / Q000187:drug effects* / Q000201:enzymology; D002460:Cell Line; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D004958:Estradiol / Q000031:analogs & derivatives* / Q000494:pharmacology; D020847:Estrogen Receptor Modulators / Q000494:pharmacology; D005419:Flavonoids / Q000494:pharmacology*; D000077267:Fulvestrant; D006801:Humans; D006854:Hydrocortisone / Q000096:biosynthesis*; D007529:Isoflavones / Q000494:pharmacology; D007700:Kinetics
D065607:Cytochrome P-450 Enzyme Inhibitors; D020847:Estrogen Receptor Modulators; D005419:Flavonoids; D007529:Isoflavones; D000077267:Fulvestrant; C013908:daidzin; D004958:Estradiol; D003577:Cytochrome P-450 Enzyme System; D015096:3-Hydroxysteroid Dehydrogenases; D006854:Hydrocortisone