一、七種人類細胞色素P450 3A4受質抑制土震素代謝反應之研究;二、以RAF值探討細胞色素P450 3A於大鼠及人體肝臟微粒體中土震素代謝的角色

Abstract

1. Inhibition Study of Territrem 4β-C hydroxylation and O-demethylation by Seven Human Cytochrome P450 3A4 substrates Territrems are the structure related tremogenic mycotoxins, which are produced by Aspergillus terreus 23-1. Previous research indicated that MA1 can be produced from TRA hydroxylation, and TRB can produce MB2 and MB4 by 4β-C hydroxylation and O-demethylation. These reactions are mainly catalyzed by P450 3A4 in liver. Structure of CYP3A4 contains great active site, and also capable of binding of more than two substrates. Interactions between substrates cause the kinetics of CYP3A4 become atypical. In addition, some amino acids in CYP3A4 active site plays an important role in substrate binding. To understand the relations of the location of TRA and TRB metabolism, this experience uses cell line V79MZh3A4 which expressing human CYP3A4. We provide TRA and TRB in time to metabolize and analyze the metabolite by HPLC. Double reciprocal plot facilitates understanding of types of inhibition. Results shows that in the process of TRA producing MA1 and TRB producing MB2, TRB and TRA are mutually mixed-type inhibition. In the process of TRB producing MB4, TRB and TRA are un-competitive inhibition. To delve more into the combinational location of TRB on CYP3A4, select seven kinds of CYP3A4 substrates to metabolize with TRB at the same time, in order to indirectly infer the location of TRB on CYP3A4. The results presents that in the metabolizing process from TRB to MB2, inhibition occurs with testosterone and un-inhibition occurs with budesonide. Also, in the metabolizing process of TRB to MB4, un-competitive inhibition occurs with testosterone, etoposide and mixed-type inhibition occurs with budesonide. Inferring from above, combination of TRA and TRB on CYP3A4 have different location, but one combination of another might change the structural location, causing declines affinity of CYP3A4. Additionally, by predicting the competitive locations of TRB and testosterone, we suggest that TRB can form a hydrogen bond with serine-119 in CYP 3A4 active site. But above inhibitors locate differently, and they only interact with each other. About the modes of TRB and substrate, it still remains unknown. Moreover, our experiment observed many atypical characteristics of CYP3A4, proving the complexity of CYP3A4. 2.Application of the Relative Activity Factor to Approach the Role of Cytochrome P450 3A to the Metabolism of Territrems by Wistar Rats and Human Liver Microsomes Cytochrome P450 is the most crucial oxidative enzyme system responsible for metabolizing over 90% endogenous and xenobiotics in human. Its prevalent characteristics of substrate specificity often result in interactions between substrates and, moreover, two or more CYP enzymes often contribute to the metabolism of a single drug. Due to above reasons, it’s important to determine the relative contribution of each CYP to net metabolism of the drug.. The concept of RAF (relative activity factor) was first proposed by Crespi at 1995. The RAF approach has been applied successfully to estimate CYP isoforms contribution to drug metabolism. According to the previous research in our Laboratory, during metabolism of TRB to MB2, CYP3A4 was major involving enzyme, containing 97.1%: during metabolism of TRB to MB4, CYP3A4 was the major, containing 64.3%. To clarify further the relative contributions of each CYP isoforms to the metabolism of TRA and TRB, we use rP450s and different ages and genders of human, and Wistar rats, and calculate the RAF. Experiment results indicated that during metabolism of TRA to MA1 in rat and human liver microsomes, CYP3A2 (84.7%) and CYP3A4 (85.9%) are major involving enzyme. During metabolism of TRB to MB2 and MB4 in rat liver microsomes, CYP3A1 (92%) and CYP3A2 (65.2%) are major involving enzyme. Furthermore, the results indicated that comparing the sex and age differences, 8-week-old male rats presented best results in the process of metabolism. But for human bodies, there is no obvious correlation between age and sex. Many factors might explain this, including the chemical structure of TRA and TRB, the mRNA and protein expression level of CYP3A1/2 in rats, the expression level and activity of CYP3A4/5 in different human bodies, amino acid sequence homology between CYP3A1/2 and CYP3A4/5, and the substrate binding sites in CYPs.