No abstract

The major phenobarbital-inducible rat hepatic cytochromes P-450, CYP2B1 and CYP2B2, are the paradigmatic members of a cytochrome P-450 gene subfamily that contains at least seven additional members. Specific oligonucleotide probes for these genomic members of the CYP2B subfamily were used to assess their tissue-specific expression. In Northern-blot analysis a probe specific to gene 4 (which is designated now as CYP2B12) hybridized to a single mRNA present in the preputial gland, an organ which is used as a model for sebaceous glands, but did not hybridize to mRNA isolated from the liver or from five other tissues of untreated or Aroclor 1254-treated rats. The cDNA sequence for the CYP2B12 RNA was determined from overlapping cDNA clones and contained a long open reading frame of 1476 bp. The nucleotide sequence of the CYP2B12 cDNA was 85% similar to the sequence of the CYP2B1 cDNA in its coding region and was different from any CYP2B cDNA characterized until now. The cDNA-derived primary structure of the CYP2B12 protein contains a signal sequence for its insertion into the endoplasmic reticulum and the putative haem-binding site characteristic of cytochromes P-450. A part of the potential haem pocket of CYP2B12 was identical with a similar structure in a bacterial protocatechuate dioxygenase. In immunoblot analysis of preputial-gland microsomes, antibodies against CYP2B1 recognized a single abundant protein with a lower apparent molecular mass than that of CYP2B1. Our results demonstrate that the CYP2B12 protein has the potential to be enzymically active and are the first demonstration that a member of the CYP2B subfamily is expressed exclusively and at high levels in an extrahepatic organ.

We have studied the expression of three P-450 gene subfamilies in hepatic and extrahepatic tissues using the sensitive RNAse A protection assay. Members of the P4501A subfamily, which encodes the major methylcholanthrene-inducible cytochromes P-450, were found to be not expressed in extrahepatic tissues of untreated animals, raising the question whether these P-450 play a role in the metabolism of carcinogens in unexposed individuals. In contrast, members of the P45011B family, some of which encode the major phenobarbital-inducible cytochromes P-450, were found to be expressed in some extrahepatic tissues of untreated rats and here most notably in the lung and in sebaceous glands. Members of the P450IIC family, which encode some constitutively expressed cytochromes P-450, were found to be expressed exclusively in the liver.

We have studied the expression of three P-450 gene subfamilies in hepatic and extrahepatic tissues using the sensitive RNAse A protection assay. Members of the P4501A subfamily, which encodes the major methylcholanthrene-inducible cytochromes P-450, were found to be not expressed in extrahepatic tissues of untreated animals, raising the question whether these P-450 play a role in the metabolism of carcinogens in unexposed individuals. In contrast, members of the P45011B family, some of which encode the major phenobarbital-inducible cytochromes P-450, were found to be expressed in some extrahepatic tissues of untreated rats and here most notably in the lung and in sebaceous glands. Members of the P450IIC family, which encode some constitutively expressed cytochromes P-450, were found to be expressed exclusively in the liver.

Cytochromes P450 metabolize a wide variety of endogenous and exogenous compounds. The genes coding for cytochromes P450 can be classified into various gene families according to their sequence homology (eg. CYP1A, CYP2B, CYP3A etc.). Most of these gene families contain several closely related members. The CYP2B family contains members which encode the major phenobarbital inducible hepatic cytochromes P450. Within the P450IIB family we have detected the expression of a novel Ρ450ΠΒ gene by oligonucleotide hybridization to mRNA of the preputial gland which is a large sebaceous gland being highly active in the metabolism of steroids. This gene was not expressed in the liver or five other tissues tested. By immunoblotting analysis of microsomal protein from the preputial gland, we detected high levels of a protein that reacted with anti-CYPIIBl antibodies The expression of this protein was age and sex independent. Moreover immunohistochemistry revealed that the corresponding P450IEB protein is also localized in the meibomian gland of the eyelids which secretes a protective lipid into the tear film. The Ρ450ΠΒ protein was also found to be expressed in the hair shafts. The full-length cDNA of this Ρ450ΠΒ protein was isolated and sequenced. Based on this sequence the P450 was designated as CYP2B12. The sequence of CYP2B12 revealed that the cDNA derived primary structure of the corresponding protein contained the signal sequence for insertion of proteins into the membrane of the ER and a Cys residue which is part of the heme binding site (see Fig. 1). Recently a putative heme pocket has been identified in some cytochromes P450 (Refs 1,2,3)). In this functionally important structure CYP2B12 shows major differences to other cytochromes P450 (see Fig. 1). However in this region CYP2B12 displays a complete identity to the ß-subunit of the bacterial protocatechuate oxygenase which is also a hemo-protein. To our knowledge this is the first time that such a close similarity has been shown for a dioxygenase and a cytochrome P450. The presence of this specific cytochrome P450 isoform in hair follicles might explain the phenomenon of allopecia (loss of hair) observed after treatment of cancer patients with cyclophosphamide which is known to be metabolized by members of the CYP2B family. We are currently in the process of expressing this cDNA in cell cultures 80 Brought to you by | University of Glasgow Library Authenticated Download Date | 6/25/15 11:12 PM