Mixed Inhibition of P450 3A4 as a Chemoprotective Mechanism against Aflatoxin B₁-Induced Cytotoxicity with cis-Terpenones

Abstract

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.

Antimalarial activity of a cis-terpenone

“…OHCT, like artemisinin, rapidly kills the early ring stage in vitro , suggesting that it could be used in combination with a slow-acting drug, such as chloroquine. The previous demonstrations that OHCT inhibits human liver cytochrome P450 3A4 [ 8 , 9 ] further suggests that it may reduce metabolism of anti-malarial compounds, such as artemisinin, that are metabolized by this enzyme [ 11 ], thus increasing the half-life and rendering them more effective. The inhibition of human liver cytochrome P450 3A4 by OHCT was shown to be mixed competitive inhibition indicating that OHCT was binding at a site different from the active site [ 9 ].…”

“…Synthesis of HCT and OHCT was described previously [ 7 , 8 ]. It was previously shown that OHCT protects human liver cells against aflatoxin and inhibits activity of liver microsomal cytochrome P450 3A4 [ 9 ]. This enzyme not only activates toxins, such as aflatoxin [ 10 ], but also contributes to the degradation of anti-malarials, such as artemisinin, thus suggesting that OHCT might increase the half-life of current anti-malarials [ 11 ].…”

Design, Synthesis, and In Vitro and In Vivo Biological Studies of a 3′-Deoxythymidine Conjugate that Potentially Kills Cancer Cells Selectively

“…Electrospray ionization mass spectroscopy (ESI-MS) analysis was carried out with a Thermo Fisher TSQ Quantum Max Triple Stage Quadrupole mass spectrometer (MA, USA). Compound 1 was obtained as previously reported [23].…”

“…Doxorubicin was used as a comparison at a final concentration of 0.05, 0.1, 0.2, 0.5, 1.0 or 2.0 µM. After 72 h, MTT assay was carried out as reported [23]. The cell viability of each treatment was plotted with GraphPad Prism program, and IC 50 values were obtained using sigmoidal dose-response analysis provided by the software (version 4.00, GraphPad Software, CA, USA).…”

“…The purpose of adding the quinoxaline moiety into dT-QX structure was to block cellular removal of thymidine analogs [16]–[18] via DNA intercalation in the DNA synthesis of cancer cells, because quinoxaline moiety is a known DNA intercalator [22]. In addition, the quinoxaline structure can be conveniently synthesized by one step condensation of a diketone compound 1 [23] and an ortho-phenylenediamine (Figure 1). More importantly, the quinoxaline structure is highly amendable for chemical modifications with a variety of substituents for advanced structure activity study to optimize the potential biological activity.…”