Cloning and tissues expression of the pig CYP1B1 and CYP2J34

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Abstract

Cytochrome P450 (CYP) 1B1 and CYP2J have been studied in various mammals, but not in pig.

The sequences encoding pig CYP1B1 and CYP2J34 were isolated from liver cDNA by RACE and sequenced. The open reading frames of pig CYP1B1 showed a higher sequence homology to bovine 1B1 (89%) than to dog 1B1 (88%) or to human 1B1 (85%). On the other hand, the coding sequence of pig CYP2J34 showed a similar homology (83–85%) to CYP2J of these species. From the substrate recognition sites (SRS 1–6) analysis of the deduced proteins, it was found that the porcine CYP1B1, unlike CYP2J34, completely shared the six SRS with the bovine counterpart. RT-PCR analysis of CYP1B1 and CYP2J34 expression in ten porcine tissues revealed that CYP1B1 was principally expressed in adrenal gland, whereas CYP2J34 was predominantly expressed in small intestine. These results further support the pig as an useful model for human.

Introduction

Cytochrome P450 (CYP) enzymes are a superfamily of heme-containing monooxygenases that catalyse the oxidation of a vast number of both endogenous and xenobiotic compounds (Nelson et al., 1996, Guengerich, 2005).

In human, 57 functional CYP genes have been identified to date (Nelson et al., 2004). A similar level of information is still lacking for pig, although this species shows many physiological characteristics that are close to those of humans (Swindle and Smith, 1998).

However, the use of pig is increasing as an alternative of non-rodent species in metabolic and toxicological studies (Skaanild, 2006) and to supply hepatocytes-based bio-artificial livers for patients waiting for liver transplantation (Gerlach, 1996, Desille et al., 1999). Up to now, a wide number of CYP genes, responsible for drug metabolism such as those belonging to 1–3 families, have been identified and cloned in pig (Nissen et al., 1998, Kojima and Morozumi, 2004, Sakuma et al., 2004, Anzenbacherova et al., 2005, Messina et al., 2008) but other essential CYPs such as the CYP1B1 and CYP2J remain to be fully investigated.

CYP1B1 enzymes are important because they were found very active in catalysing the bioactivation of many pro-carcinogenic polycyclic hydrocarbons and are constitutively expressed mainly in various steroidogenic and steroid-responsive tissues including testes, adrenal glands, breast and prostate (Chun and Kim, 2003). Also the CYP2J enzymes are of interest, as they were found to be active in the oxidation of endogenous substrates such as arachidonic acid producing metabolites particularly relevant in the cardiovascular system and are expressed primarily in the extra-hepatic tissues of mammals, including heart, intestine and kidney (Scarborough et al., 1999).

In the current study, we determined the open reading frame of two cDNAs encoding the porcine CYP1B1 and CYP2J34 and examined, by RT-PCR, their expression levels in several organs of domestic pig.

Section snippets

Chemicals

All chemicals and reagents were of analytical grade. ThermoScript III RT, Gene Racer Kit, Platinum Pfx Taq Polymerase were from Invitrogen Life Technologies (Carlsbad, USA). Master Mix PCR, pGEM-T easy vector, Wizard® Plus SV MiniPrepsDNA Purification System were purchased from Promega (Madison, USA).

Animals

To perform this study we used three male castrated Large White per Landrace hybrid domestic pigs (30–35 kg body weight) aged about 3 months. The animals were housed at least for 10 days in floored

Results and discussion

In the present study, we have isolated and sequenced, using a PCR approach followed by RACE, two coding sequence cDNAs encoding porcine enzymes belonging to subfamilies CYP1B and CYP2J, which are particularly important in the metabolism of endogenous compounds beside the exogenous ones. These isolated CYP sequences submitted to Dr. D. Nelson (http://drnelson.uthsc.edu/CytochromeP450.html) were named porcine CYP1B1 and CYP2J34.

The nucleotide sequence of CYP1B1 possesses an open reading frame of

Conclusions

The availability of the coding sequences of porcine CYP1B1 and CYP2J34 will make possible to express these genes in a heterologous system yielding the corresponding functional enzymes and thereby to study their substrate specificity. This will extend our understanding of pig as a model of drug metabolism in human.

Acknowledgements

The authors acknowledge Dr. Salvini Mariangela from Dipartimento di Biologia delle Piante Agrarie Sezione di Genetica Università di Pisa, for phylogenetic analysis.

References (17)

  • O. Gotoh

    Substrate recognition sites in cytochrome P450 family 2 (CYP2) proteins inferred from comparative analysis of amino acid and coding nucleotide sequences

    Journal of Biological Chemistry

    (1992)
  • E. Anzenbacherova et al.

    Model systems based on experimental animals for studies on drug metabolism in man: (Mini) pig cytochrome P450 3A29 and 2E1

    Basic & Clinical Pharmacology & Toxicology

    (2005)
  • I. Bieche et al.

    Reverse transcriptase-PCR quantification of mRNA levels from cytochrome CYP1, CYP2 and CYP3 families in 22 different tissues

    Pharmacogenetics and Genomics

    (2007)
  • Y.-J. Chun et al.

    Discovery of cytochrome P4501B1 inhibitors as new promising anti-cancer agents

    Medical Research Review

    (2003)
  • M. Desille et al.

    Detoxifying activity in pig livers and hepatocytes intended for xenotherapy

    Transplantation

    (1999)
  • J.C. Gerlach

    Development of a hybrid liver support system. A review

    International Journal of Artificial Organs

    (1996)
  • F.P. Guengerich

    Human cytochrome P450 enzymes

  • M. Kojima et al.

    Cloning of six full-length cDNA encoding pig cytochrome P450 enzymes and gene expression of these enzymes in the liver and kidney

    Journal of Health Science

    (2004)
There are more references available in the full text version of this article.

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