Cloning, tissue expression, and inducibility of CYP 3A79 from sea bass (Dicentrarchus labrax).
Multiple members of the CYP3A subfamily have been identified and intensively studied in mammals as they represent prominent CYP enzymes involved in drug metabolism. Also in fish, some CYP3A genes have been identified by cDNA cloning and immunological techniques, but relatively little is known about their function, distribution, and inducibility. In this study, a novel CYP3A, designated as CYP3A79 was isolated from adult male sea bass, an economically valuable species in fisheries. The sea bass CYP3A79 that was cloned contained an open-reading frame of 1512 bp that encoded a 504 amino acid protein and shared a high-sequence identity with medaka, killifish, and trout CYP3As. Interestingly, CYP3A79 also shares five of six substrate recognition sites (SRS) with the SRS of other fish CYP3As, suggesting an evolutionary conservation of the function of these enzymes. In this fish, we also investigated the expression of CYP3A79 and its susceptibility to induction by various compounds including clotrimazole and dehydroepiandrosterone, two strong ligands of zebrafish PXR. The expression of CYP3A79 mRNA was detected by RT-PCR only in the intestine and liver. The immunoblot analysis by antitrout CYP3A27 confirmed the presence of a CYP3A-like protein in the microsomes of these tissues, but, in addition, a immunoreactive protein with this antibody was also observed in the heart microsomes, suggesting the presence of other CYP3A isoforms in this fish. Accordingly, the southern blot analysis of genomic DNA indicated that multiple CYP 3As may be present in sea bass. All attempts to induce 6beta-testosterone hydroxylase, as a marker of CYP3A79, by dexametasone, 17beta-estradiol, pregnenolone 16alpha-carbonitrile, corticosterone, clotrimazole, and dehydroepiandrosterone failed. On the contrary, the administration of 17beta-estradiol, pregnenolone 16alpha-carbonitrile, and corticosterone strongly inhibited this activity and, in parallel, reduced the expression of CYP3A79 transcript. Thus, the sea bass CYP3A79 appears to be resistant to induction, suggesting that this enzyme and likely other CYP3As are regulated differently compared to those of mammals.
Vaccaro
E
E
Laboratory of Pharmacogenetic and Drug Metabolism, Istituto di Fisiologia Clinica, CNR, via Moruzzi 1 56100 Pisa, Italy.
Salvetti
A
A
Del Carratore
R
R
Nencioni
S
S
Longo
V
V
Gervasi
P G
PG
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001492:Bass / Q000235:genetics* / Q000378:metabolism*; D015139:Blotting, Southern; D015153:Blotting, Western; D003001:Cloning, Molecular; D051544:Cytochrome P-450 CYP3A / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D018076:DNA, Complementary / Q000235:genetics; D004790:Enzyme Induction; D020869:Gene Expression Profiling; D016678:Genome / Q000235:genetics; D008099:Liver / Q000201:enzymology; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology; D008969:Molecular Sequence Data; D010089:Oxidoreductases, N-Demethylating / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid
D018076:DNA, Complementary; D012333:RNA, Messenger; D051544:Cytochrome P-450 CYP3A; D010089:Oxidoreductases, N-Demethylating