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CLONING CYP 2 D 21 AND CYP 3 A 22 CDNAS FROM LIVER OF MINIATURE PIGS

To compare the identity of the primary structure of drug-metabolizing cytochrome P450 between miniature pigs and humans, two cDNA clones, coding for miniature pig CYP2D21 and CYP3A22, were isolated. The deduced amino acid sequences of CYP2D21 and CYP3A22 were 78.3 and 75.0% identical to human CYP2D6 and CYP3A4, respectively. These values were nearly the same as those of bovine, dog, and some rodent isoforms, and 12.2 to 18.4% lower than those of nonhuman primates such as cynomolgus monkeys, Japanese monkey, and marmosets. These data indicate that miniature pig P450s are genetically not so close as monkey P450s to human P450s as previously expected. The recombinant CYP2D21 enzyme, however, showed bufuralol 1 -hydroxylase activity, suggesting that miniature pig CYP2D21 is capable of metabolizing some of the same substrates associated with human CYP2D6 despite its low identity to human counterparts. The miniature pig is small in size ( 50 kg body weight in adult animals) and has been developed through selective breeding so as to override the shortcomings arising from the large body size of domestic pigs (Khan, 1984). The miniature pig is becoming a popular alternative to traditional nonrodent species in pharmacological and toxicological studies. Porcine hepatocytes continue to find increasing application in bioartificial liver devices (Watanabe et al., 1997). These uses are, in part, based on findings that many anatomical, physiological, and biochemical properties of domestic and miniature pigs are close to those of humans (Swindle and Smith, 1998). However, it has been well recognized that qualitative and quantitative species differences in drug-metabolizing systems are present among animal species including man (Caldwell, 1981). Thus, information on the primary structure of drug-metabolizing enzymes of experimental animal species and information on the identity of those enzymes between human and experimental animal species is important to explain species differences in drug-metabolizing systems and to predict drug metabolism in the human body. Genetic distance of some porcine genes to human orthologs has been clarified. Regarding porcine CYP genes, some forms of P450 concerned with endogenous metabolism such as CYP5A (accession no. L131128), CYP11B1 (D38590), CYP19 (U37311), CYP21 (M83939), and CYP51 (AB042975) have been isolated from domestic pigs. These forms shared amino acid sequence identities ranging from 73 to 95% to human orthologous genes. cDNA cloning of the entire coding sequence of two drug-metabolizing P450s, namely CYP2D25 and CYP3A29, from domestic pigs has been reported (Postlind et al., 1997; Nissen et al., 1998). Additionally, the partial sequences of cDNAs for five drug-metabolizing CYP2Cs (2C32, 2C33, 2C34, 2C35 and 2C42) from domestic pigs have been reported (Zaphiropoulos et al., 1995; Nissen et al., 1998). However, study of cDNA cloning from miniature pig has not yet been reported. The aims of the present study were to demonstrate in miniature pigs the primary structure of drug-metabolizing P450s, especially CYP3A and CYP2D, which are responsible for the metabolism of many prescribed drugs, and to compare these with those of human orthologs. Materials and Methods Animals. Two 24-week-old male Göttingen miniature pigs of about 15 kg in body weight were used in this study. Livers were stored at 80°C until use for the preparation of RNA and microsomes. All experiments were performed in accordance with the Principles of Laboratory Animal Care (National Institutes of Health publication 85-23, revised 1985). Construction and Screening of the Miniature Pig Liver cDNA Library. Poly(A) RNA was prepared from the liver of an untreated male miniature pig by oligo(dT)-cellulose column chromatography. A cDNA library was constructed as described previously (Sakuma et al., 1994). Approximately 5 10 plaques were screened by plaque hybridization using the cDNA fragments of human CYP2D6 and cynomolgus monkey CYP3A8 as probes. Expression of CYP2D21 in Yeast Cells and Analysis of Bufuralol Hydroxylase Activity. An expression plasmid for CYP2D21 was constructed with the pAAH5 yeast expression vector. The resulting expression plasmid, pAAMS2D, contains native coding, six bases of 5 -noncoding sequences, and 83 bases of 3 -noncoding sequences. Transformation of the yeast, Saccharomyces cerevisiae AH22, cultivation of the recombinant yeasts, preparation of microsomal fractions, and determination of P450 contents were carried out as described previously (Sakuma et al., 1994). Measurement of the bufuralol 1 -hydroxylase activity was carried out as described previously (Yokoi et al., 1996). The kinetic parameters of bufuralol 1 -hydroxylation of microsomes from the recombinant yeasts or miniature pig livers were calculated from Lineweaver-Burk plots. Results and Discussion To clarify the identity of the primary structure of P450 between miniature pigs and humans, two cDNA clones containing the entire coding sequence of CYP2D or CYP3A were isolated from a cDNA This work was supported in part by a grant-in-aid for Scientific Research from the Ministry of Education, Sciences, Culture and Sports of Japan. 1 Abbreviations used are: P450, cytochrome P450; bp, base pairs; SRS, substrate recognition site. Address correspondence to: Dr. Tsutomu Sakuma, Department of Toxicology, Faculty of Pharmaceutical Sciences, Toyama Medical and Pharmaceutical University, 2630 Sugitani, Toyama, Toyama 930-0194, Japan. E-mail: tsakuma@ms.toyama-mpu.ac.jp 0090-9556/04/3204-376–378$20.00 DRUG METABOLISM AND DISPOSITION Vol. 32, No. 4 Copyright © 2004 by The American Society for Pharmacology and Experimental Therapeutics 1275/1138603 DMD 32:376–378, 2004 Printed in U.S.A. 376 at A PE T Jornals on Sptem er 0, 2017 dm d.aspurnals.org D ow nladed from