Model Systems Based on Experimental Animals for Studies on Drug Metabolism in Man: (Mini)Pig Cytochromes P450 3A29 and 2E1

Abstract

A quest for an optimal experimental animal for drug metabolism studies in the man leads to inter-species comparisons of liver microsomal cytochrome P450 (CYP) enzymes from rat, rabbit, dog, as well as from the pig (minipig). The rat CYP enzymes seem not to be real models as they exhibit different substrate specificity as well as altered modes of regulation and induction when compared to the human counterparts (Souček & Gut 1992; Tabb et al. 2004). On the other hand, pig or minipig CYPs have been proven to be a good model of human metabolism either at the cellular, subcellular or molecular level (Olsen et al.1997; Anzenbacher et al. 1998; Skaanild & Friis 1997; Souček et al. 2001). The pig CYP3A29 mRNA has been sequenced (Nissen et al. 1998, PubMed accession No. Z93099). Similarly, the pig CYP2E1 mRNA primary sequence is accessible (PubMed accession No. P79383). However, the enzymes have not been purified to-date and hence there are no data on their substrate specificities available. This work is the first to describe the preliminary results on the isolation and properties of the purified CYP enzymes of the minipig CYP3A29 and 2E1. Chemicals. All reagents and chemicals were purchased from Sigma Aldrich (Prague, Czech Republic) if not stated otherwise. DEAE Sephacel was product of Pharmacia Biotech (Uppsala, Sweden). Polyclonal rat anti-CYP3A4 and -CYP2E1 IgG were purchased from Daichi Pure Chemicals (Tokyo, Japan). Chemiluminiscence kit for Western blotting (Immun Star) was from Bio Rad (Hercules, CA, USA) and the membrane from Amersham Biosciences (Little Chalfont, GB, U.K.). Purification of minipig enzymes. Liver microsomal fractions were prepared from Goettingen minipigs (Research Institute of Veterinary Medicine, Brno, Czech Republic, 25–30 kg body weight, male castrates, N=5, age 6 months) treated in accordance with ethical standards given by EC and Czech directives on protection of animals and approved by the local ethical committee. Microsomal fractions were obtained by standard methods and the isolation of purified enzymes from cholate-solubilised microsomes was performed in general by the method of Guengerich & Martin (1998). First, the solubilisate was applied onto a octyl-Sepharose column (where the NADPH:cytochrome P450 reductase was eluted). Subsequently, DEAE-Sephacel was used to separate the CYP3A-containing fractions. CYP2E1 was present in fractions from octyl-Sepharose eluted with higher concentrations of nonionic detergent. The details of the isolation procedure were described previously (Souček et al. 2001). cDNA sequencing. cDNA was sequenced using a ABI Prism 310 DNA Sequencer (Applied Biosystems, Foster City, CA, USA) and a BigDye terminator kit according to producer's instructions. Activity assays. Nifedipine oxidase activity of CYP3A enzymes was determined using the method of Guengerich et al. (1986). Chlorzoxazone 6-hydroxylating activity was determined according to Lucas et al. (1996). Reconstituted systems were prepared as described previously (Souček et al. 2001). To analyze the data from enzyme kinetics, a Sigma Plot 8.0 (SPSS, Chicago, IL, USA) has been used. The minipig CYP3A29 enzyme was purified to electrophoretic homogeneity. The enzyme exhibited a prototypic activities of human CYP3A4, nifedipine hydroxylation and testosterone 6β-hydroxylation (table 1). The minipig enzyme hence had both typical activities comparable to its human counterpart CYP3A4. Minipig CYP3A cDNA clone was subjected to sequencing. The respective primers were placed at the pCW 5′-end, 3′-end, and at approximately one third of the CYP3A coding sequence. Sequence was compared to the human CYP3A4 and available pig CYP3A29. Minipig CYP3A sequence was submitted to GenEMBL under number AF424780. Minipig and pig CYP3A29 differ only in eight amino acid residues. Six changes are conservative, hence do not influence the character of the protein, two are however in favour of less basic residue (in positions 299 and 485 there is proline present in the minipig enzyme instead of glutamine of the pig protein). This enzyme, the pig or minipig orthologue of human CYP3A4, exhibits 76% sequence identity with CYP3A4 (only the monkey CYP3A8 scores better with 85%). The substrate recognition sites (SRS) are well conserved with the most important residues for substrate specificity identical (F102, A117, V101, T103, S119, N206, L210, D217) which apparently is in line with similar properties of human and minipig enzyme. Minipig CYP2E1 (accepted in GenEMBL under accession No. AY581116) exhibits even higher sequence identity with the human orthologue (80%) than CYP3A29. Here, differences between minipig and human protein are localized in the close vicinity of the SRS 1 (and SRS 2) where two (H92, N95) and one (K194) residues are present instead of two aspartic and one glutamic acids in the human enzyme. The structure of the substrate binding site is well conserved which is reflected in the fact that the chlorzoxazone (a prototypic substrate of human CYP2E1) is metabolized also by the minipig or pig CYP2E1 (table 2). The results indicate the relative similarity of CYP2E1 activities in various systems. The human cytochrome P450 systems can hence be successfully modelled by pig or minipig cytochromes P450 in microsomal and reconstituted systems. The authors thank the Grant Agency of Czech Republic (203/02/ 1152) and COST project B.15.50 (Czech Ministry of Education, OC B15.50) for support.