Cloning Cyp2d21 and Cyp3a22 Cdnas From Liver of Miniature Pigs

Sakuma, Tsutomu; Shimojima, Tsukasa; Miwa, Kiyoshi; Kamataki, Tetsuya

doi:10.1124/dmd.32.4.376

Cited by 28 publications

(14 citation statements)

References 14 publications

(14 reference statements)

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“…[38][39][40][41] These CYPs exhibit amino acid sequence identities of 75-80% with the respective human orthologs, and the homologies are comparable to the results for UGT1A1 in this study. To confirm whether minipigs can be a useful experimental animal in studies on safety evaluation and biotransformation for drug development, further studies are required to clarify the mechanism of comprehensive CYP-UGT function in minipigs as well as the enzymatic properties of individual minipig CYP and UGT isoforms.…”

Section: Discussionsupporting

confidence: 69%

cDNA Cloning and Functional Analysis of Minipig Uridine Diphosphate-Glucuronosyltransferase 1A1

Miyake

Mayumi

Jinno

et al. 2013

Biological & Pharmaceutical Bulletin

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Uridine diphosphate (UDP)-glucuronosyltransferase 1A1 (UGT1A1) plays important roles in the glucuronidation of various drugs and endogenous substances. Minipigs have been used as experimental animals in pharmacological and toxicological studies, because many of their physiological characteristics are similar to those of humans. In this study, the similarities and differences in enzymatic properties of UGT1A1 between humans and minipigs were precisely identified. Minipig UGT1A1 (mpUGT1A1) cDNA was firstly cloned by the rapid amplification of cDNA ends (RACE) method, and the corresponding protein as well as human UGT1A1 (hUGT1A1) enzyme was expressed in insect cells. Then the kinetics of estradiol at 3-hydroxy position (E-3OH) and 7-ethyl-10-hydroxycamptothecin (SN-38) glucuronidation by recombinant UGT1A1s as well as human and minipig liver microsomes were analyzed. The homology between mpUGT1A1 and hUGT1A1 at the amino acid level was 80.9%. E-3OH and SN-38 glucuronidation by recombinant hUGT1A1 and mpUGT1A1 showed allosteric sigmoidal kinetics. The CL max value (29.1 µL/min/mg protein) for E-3OH glucuronidation of mpUGT1A1 was significantly higher (1.4-fold) than that of hUGT1A1, whereas the CL max value (0.83 µL/min/mg protein) for SN-38 glucuronidation was significantly lower (27%) than that of hUGT1A1; however, the kinetic models and parameter levels for E-3OH and SN-38 glucuronidation by human and minipig liver microsomes did not parallel those in the respective species. These findings suggest that the enzymatic properties of UGT1A1 are considerably different between humans and minipigs. The information on species differences in UGT1A1 function gained in this study should help with in vivo extrapolation of xenobiotic metabolism and toxicity.Key words uridine diphosphate-glucuronosyltransferase (UGT); UGT1A1; minipig; estradiol at 3-hydroxy position; 7-ethyl-10-hydroxycamptothecin Glucuronidation is important for the detoxification and elimination of various lipophilic endogenous substances (e.g. bile acids, bilirubin and steroids) and xenobiotics (e.g. drugs, environmental chemicals and dietary constituents) in mammals.1) This reaction is catalyzed by a multigenic family of uridine diphosphate (UDP)-glucuronosyltransferases (UGTs) that are localized in the endoplasmic reticulum membrane. 2-4)The human UGT superfamily comprises two families (UGT1 and UGT2) and three subfamilies (UGT1A, UGT2A, and UGT2B).2) The UGT1A gene is localized on chromosome 2q37 and codes proteins with unique N-terminal domains and identical C-terminal domains, which are formed from the alternate mRNA splicing of unique first exons with common exons 2-5. In contrast, UGT2A and UGT2B genes are clustered on chromosome 4q13 and individual UGT proteins are coded by unique genes with six exons. 5,6) In addition, each UGT has been demonstrated to exhibit unique substrate and tissue specificities. 3,4,7) UGT1A1 is expressed in the liver, small intestine and colon, and is exclusively involved in the metabolism of bilirubin. 4,8,9) UGT1A1 has b...

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Section: Discussionsupporting

confidence: 69%

cDNA Cloning and Functional Analysis of Minipig Uridine Diphosphate-Glucuronosyltransferase 1A1

Miyake

Mayumi

Jinno

et al. 2013

Biological & Pharmaceutical Bulletin

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“…The nucleotide sequences are 81.5–83.5% homologous to the human CYP3A4 genes, whereas the amino acid sequences for these isoforms show 75–77.8% homology. CYP3A22 and CYP3A29 v5 are considered the CYP3A4 homologues in minipigs, with a 75% and 75.3% identity of amino acid sequences in Göttingen minipigs compared with human CYP3A4, respectively . Additionally, porcine CYP3A enzymes present similar biotransformation properties to the human homologues .…”

mentioning

confidence: 99%

Ontogeny of CYP3A and P‐Glycoprotein in the Liver and the Small Intestine of the Göttingen Minipig: An Immunohistochemical Evaluation

Peer

Verbueken

Saad

et al. 2013

Basic Clin Pharma Tox

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Despite the increasing use of the minipig as a non-rodent species in general and juvenile toxicity studies, knowledge on their biotransformation processes and their ontogeny is scarce. Such data are prerequisite for the correct interpretation of nonclinical studies in this species. Therefore, the aim of our investigation was to immunohistochemically document the presence of the drug transporter P-glycoprotein (Pgp) and the metabolizing cytochrome P450 (CYP) 3A subfamily in the livers (n = 115) and the small intestines (n = 74) of foetal, neonatal, juvenile and adult G€ ottingen minipigs. Pgp was expressed in the liver in all age groups, whereas its presence in the jejunum was detected from 86 days of gestation onwards. Low expression of CYP3A was detected in the jejunums and livers from foetal and neonatal piglets. During postnatal development, the immunoreactivity for CYP3A increased in both organs. A centrilobular pattern, with a more intense staining for CYP3A of the hepatocytes surrounding the central vein, was noticed in the postnatal livers. In conclusion, the presented data suggest that the intestinal and hepatic ontogeny of P-glycoprotein and CYP3A in minipigs corresponds to that in man, in which a similar spatio-temporal expression has been reported.

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“…It presents a high quantity in the liver and is involved in oxidative metabolism of an estimated 60% exogenous compounds [1,2]. However, only four of CYP3A enzymes (CYP3As) in pig were reported (CYP3A22, CYP3A29, CYP3A39, and partial sequence of CYP3A46) [3,4]. The studies suggested that at least two proteins in the livers of pig could be recognized by antibodies against human CYP3A4.…”

Section: Introductionmentioning

confidence: 97%

Transcriptional Profile of CYP3As and Functional Expression of CYP3A29 from Piglets

Yao

Liu

Dai

et al. 2009

2009 3rd International Conference on Bioinformatics and Biomedical Engineering

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Expression profile of CYP3A individuals from piglets and characterization of CYP3A29 were investigated. Real-Time PCR showed that the liver and small intestines present the highest expression levels of CYP3A29, a human CYP3A4 homolog. The CYP3A29 was functional expressed in Bac-to-Bac baculovirus expression system together with NADPH p450 reductase (NPR) and cytochrome b5. For nifedipine oxidation (NOD) activity, recombinant CYP3A29 system showed similar enzyme kinetic parameters (Km=14.1 ～ 25.3 μM) to human recombinant CYP3A4, and a little variant to liver microsomes (Km=29.6 ～ 45.2 μM) from piglets. The NOD activity of recombinant CYP3A29 was strongly inhibited by ketoconazole (KET) and troleandomycin (TAO), and was slightly changed by inhibitors for other p450 enzymes from human. These results provided in detailed information of the characterizations of CYP3A29. We conclude from these results that caution should be hold when specific inhibitors or probes for human p450 enzymes are employed to investigate the veterinary drug metabolism.

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Cloning Cyp2d21 and Cyp3a22 Cdnas From Liver of Miniature Pigs

Cited by 28 publications

References 14 publications

cDNA Cloning and Functional Analysis of Minipig Uridine Diphosphate-Glucuronosyltransferase 1A1

cDNA Cloning and Functional Analysis of Minipig Uridine Diphosphate-Glucuronosyltransferase 1A1

Ontogeny of CYP3A and P‐Glycoprotein in the Liver and the Small Intestine of the Göttingen Minipig: An Immunohistochemical Evaluation

Transcriptional Profile of CYP3As and Functional Expression of CYP3A29 from Piglets

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