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  • Recently a sandwich cultured hepatocyte model has been


    Recently, a sandwich-cultured hepatocyte model has been proved to be a valuable in vitro system that maintains specific hepatic cytomorphology and function relevant to drug metabolism, disposition and toxicity, and thus, closely resembles the in vivo setting. This hepatocyte model was recognized as a reliable toxicological method to determine mechanisms of drug-induced toxicity, identify biomarkers and predict chemical toxicity. Therefore, based on the previous in vivo findings, we hypothesized that the relationship between TP and CYP3A underlying the mechanism of TP-induced liver injury may be very complicated. Therefore, the aim of this study was initially to evaluate the inhibitory effects of TP on rat CYP3A in vitro. The study was further focused on the effect of CYP3A modulators on metabolism and toxicity of TP. The time-dependent CYP3A inhibition by TP was investigated using an IC50 shift assay. The enzyme kinetics was characterized in rat liver microsomes (RLM). A study based on sandwich-cultured rat hepatocytes (SCRH) was carried out to confirm the results from liver microsomes. An inducer and inhibitor of CYP3A were then used to reveal the role of CYP3A enzyme in the TP toxicity and to clarify the risk of drug–drug interaction.
    Materials and methods
    Discussion An important feature of CYP W123 synthesis is that they may be easily induced or inhibited. Many drugs commonly used in the clinic have the potential for CYP enzyme induction or inhibition. Therefore, when two or more drugs are administered simultaneously, this may lead to the metabolism based drug interactions. Liver microsomes and cultured primary hepatocytes are commonly used in vitro to investigate potential drug–drug interactions (Gonzalez, 1989, Guengerich, 1992). It is particularly important for the drug with a narrow therapeutic index, such as TP, because it is often co-administered with other drugs. The toxicity caused by TP has created extensive attention for many years. Recently it was discovered that CYP3A is involved in the metabolism of TP, and therefore CYP3A may be associated with TP-induced liver injury (Ye et al., 2010, Xue et al., 2011). However, the mechanism underlying the CYP3A enzyme mediated drug interaction of TP has not been reported, especially the correlation of CYP3A activity with the drug toxicity in the presence of CYP3A inducer and inhibitor has not been investigated at the cellular level. In the present study, we revealed, for the first time, the time-dependent inhibition of CYP3A activity by TP in RLM and hepatocytes. The inhibitory activity of TP on rat CYP3A without pre-incubation was relatively weak, with an IC50 value of 190.8μM. However, a much stronger inhibitory effect was observed when TP was pre-incubated in the presence of NADPH. The IC50 shift and value (105.1μM) indicated that time-dependent inhibition could be a contributor towards CYP3A inhibition by TP observed in rats (Yao et al., 2010), although the IC50 value was more than 50μM. In the SCRH study, MTT assay also showed that TP had no significant effect on cell viability with the IC50 value of 163.9μM. However, the CYP3A enzyme activity was significantly reduced when the hepatocytes were incubated with TP for 24h at the TP concentration more than 5μM (Fig. 3). The western blotting assay also showed that the protein expression of CYP3A was significantly reduced by TP (10μM) at 24h (Fig. 4). Thus, the inhibitory effect of W123 synthesis TP on CYP3A enzyme activity was a time-dependent process. This was in consistent with the in vivo results that the CYP3A enzyme activity was inhibited by TP following a continuous oral administration for two weeks (Yao et al., 2010). The above findings indicate that the inhibition of TP on rat CYP3A involves a weak and time-dependent mechanism. In order to find appropriate concentrations of CYP3A inducer and inhibitor that did not cause hepatotoxicity or interfere with TP-induced hepatotoxicity, the MTT assay was first used to screen the non-cytotoxic concentrations of inducer and inhibitor. After treatment with the CYP3A modulators (DEX and Keto) in the SCRH model, the CYP3A enzyme activity was induced by DEX and inhibited by Keto. When co-incubated with DEX, the CYP3A activity was induced more than 3.5-fold (Fig. 5), the t1/2 of TP was reduced and CLint increased (Fig. 6 and Fig. 7). Consequently, the releases of biochemical indictors (ALT, AST and LDH) and the MDA formation were decreased (Table 1). The Keto had an opposite effect on CYP3A enzyme activity, t1/2 and CLint of TP. As the result, the biochemical parameters were significantly increased. Our results were similar to the findings of the protective effect of Xiaoyaosan on TP toxicity (Cai, 2012). Xiaoyaosan increased the metabolic clearance rate of TP by the induction of CYP3A activity, thereby inhibiting downstream damage pathways to protect the liver. The influence of TP on cell morphology illustrated the TP induced hepatotoxicity. When co-incubated with the CYP3A inducer or inhibitor, the inducer protected cell morphology through accelerated metabolism, and the inhibitor enhanced the toxic effect of TP by CYP3A inhibition (Fig. 8). The present study revealed the CYP3A based detoxification mechanism of TP. The degree of the TP-induced hepatotoxicity was associated with the CYP 3A mediated metabolism. These data provide very important information for avoiding TP related drug interactions and guiding the safe clinical applications.