Mixed inhibition of P450 3A4 as a chemoprotective mechanism against aflatoxin B1-induced cytotoxicity with cis-terpenones.
We recently reported the protective effect of 2-hydroxy- cis-terpenone (HCT) against aflatoxin B 1 (AFB1)-induced cytotoxicity in human HepG2 liver cells ( Zhou et al. Chem. Res. Toxicol. 2006, 19, 1415-1419 ); however, the mechanism was not clear. In this paper, the chemoprotective mechanism was investigated with liver microsomes and purified P450 3A4 enzyme. HCT showed effective inhibition of the metabolic conversion of AFB1 in liver microsomes at 40 microM, and more importantly, the inhibition of the carcinogenic exo-AFB1-epoxide formation from AFB1. Further study indicated the direct inhibition of purified P450 3A4 enzyme activity by HCT with an IC 50 value of 20 microM. Under aqueous conditions, HCT was slowly converted to an oxidized product OHCT, which exhibits similar inhibitory effects on both P450 3A4 and the metabolic conversion and carcinogenic activation of AFB1 with liver microsomes as those of HCT. Enzyme mechanism studies revealed that OHCT acted as a mixed inhibitor of P450 3A4 with K i and K i' at 17.6 +/- 5.6 and 7.6 +/- 1.5 microM, respectively. Finally, OHCT showed no cytotoxicity at 60 microM in HepG2 liver cells and effective chemoprotection at 40 and 60 microM against AFB1 (2 microM) induced cytotoxicity. In contrast, ketoconazole alone exhibited 20% cell mortality at 20 microM, while chemoprotection with ketoconazole against 2 microM AFB1 in HepG2 was observed at 10 and 20 microM, which was much higher than the 1 microM concentration used in the inhibitory assays of P450 3A4 activity and AFB1 metabolism with liver microsomes.
Zhou
Qibing
Q
Department of Chemistry, Virginia Commonwealth University, 1001 West Main Street, Richmond, Virginia 23284-2006, USA. qzhou@vcu.edu
Zhang
Lin
L
Zuniga
Miguel A
MA
Tombes
Robert M
RM
Stewart
Jennifer K
JK
D016604:Aflatoxin B1 / Q000037:antagonists & inhibitors* / Q000633:toxicity*; D000465:Algorithms; D016588:Anticarcinogenic Agents / Q000494:pharmacology*; D002273:Carcinogens / Q000037:antagonists & inhibitors* / Q000633:toxicity*; D002478:Cells, Cultured; D051544:Cytochrome P-450 CYP3A; D065692:Cytochrome P-450 CYP3A Inhibitors; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005978:Glutathione / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D066298:In Vitro Techniques; D007202:Indicators and Reagents; D007654:Ketoconazole / Q000633:toxicity; D007700:Kinetics; D008862:Microsomes, Liver / Q000187:drug effects / Q000201:enzymology; D010616:Phenanthrenes / Q000494:pharmacology*
C529147:4b,5,6,7,8,8a-hexahydro-2-hydroxy-4b,8,8-trimethylphenanthren-9(10H)-one; D016588:Anticarcinogenic Agents; D002273:Carcinogens; D065692:Cytochrome P-450 CYP3A Inhibitors; D004791:Enzyme Inhibitors; D007202:Indicators and Reagents; D010616:Phenanthrenes; D016604:Aflatoxin B1; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human; D005978:Glutathione; D007654:Ketoconazole