gssg CYP A and CYP A

CYP3A4 and CYP3A5 protein and mRNA were expressed in all four human liver microsome preparations tested (HLM1–HLM4) (Fig. 6). In general, for most CYP3A substrates for hydroxylation, the in vitro intrinsic clearance for CYP3A4 is greater than that for CYP3A5 (Williams et al., 2002, Patki et al., 2003, Cook et al., 2002, Kalgutkar et al., 2003, Shen et al., 2004). When 4β-C hydroxylation and O-demethylation of TRB was performed using supersomes expressing CYP3A4 or CYP3A5, the two isoforms showed comparable activity (Table 1), but the intrinsic clearance of 4β-C hydroxylation of TRB was higher for CYP3A4 than for CYP3A5, while that for the O-demethylation of TRB was higher for CYP3A5 (Fig. 7). This suggests that 4β-C hydroxylation of TRB is catalyzed mainly by CYP3A4 and that O-demethylation of TRB is catalyzed mainly by CYP3A5. CYP3A5 shares considerable sequence homology with, and has qualitatively similar substrate selectivity to CYP3A4. However, the relative metabolic activities of CYP3A4 and CYP3A5 are substrate-dependent and regioselective (Person et al., 1997, Xie et al., 2004). Although the amino gssg sequences of CYP3A4 and CYP3A5 are 84% identical, their functional differences indicate that key differences may exist in their active sites. Key differences can be seen in amino acid residues in substrate recognition sites (SRS) regions in CYP3A4 and CYP3A5 that confer functional competence and/or divergence, novel catalytic specificities and activities, and specific regio- and stereo-selective targeting of a given substrate (Wang et al., 1998). Thus, the SRS differences between CYP3A4 and CYP3A5 appear to critically influence the metabolism of TRB. Recently, several prediction methods, including relative activity factors (RAFs), for assessing the contribution of multiple CYPs to certain metabolic reactions in human liver microsomes have been reported (Crespi, 1995, Iwatsubo et al., 1997, Becquemont et al., 1998). In a future study based on RAFs, we will estimate the contributions of CYP3A4 and CYP3A5 to the hydroxylation and O-demethylation of TRB in human liver microsomes using recombinant CYP3A4 and CYP3A5 expressed in baculovirus-transformed insect cells. In conclusion, using human liver microsomes, supersomes from baculovirus-transformed insect cells expressing different human CYP450 isoforms, and V79MZh3A4 cells, MB2 and MB4 were formed from TRB and MC from TRC. The amounts of MB2, MB4, and MC formed were related to the 6β-testosterone hydroxylase activity. The results of our studies using CYP450 isoform-specific chemical inhibitors and antibodies suggest that CYP3A4 and CYP3A5 are the main CYP450 isoenzymes responsible for the formation of MB2, MB4, and MC in human liver microsomes.
Acknowledgments
Introduction The major function of the mammalian kidney is to maintain the homeostasis of the internal environment. To achieve this, the kidney is involved in numerous processes including the excretion of xenobiotics. As a result, the kidney is frequently exposed to potentially toxic compounds, which show a high degree of site-selectivity due to the very complex and heterogeneous anatomical structure of the kidney. This site selectivity is determined by several factors, in particular transport mechanisms and the distribution of biotransformation enzymes [1]. Cells of the proximal convoluted tubule are the first to be exposed to the glomerular ultrafiltrate [2]. Furthermore, proximal tubular cells (PT cells) actively transport a wide variety of charged organic and inorganic compounds and thus are considered to be the intrarenal target for most nephrotoxic compounds [3].