Cloning, tissue expression, and inducibility of CYP 3A79 from sea bass (Dicentrarchus labrax)

Abstract

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.

Steroid catabolism in marine and freshwater fish

“…This is especially true for progesterone, which does not contain a hydroxyl group and therefore requires phase I metabolism (hydroxylation or keto reduction) in order to undergo conjugation and elimination. Studies in fish with 17-␤-estradiol, progesterone and testosterone show that steroid catabolism through hydroxylation is mainly carried out by microsomal P450s that are normally expressed in liver [9][10][11][12][13][14][15][16][17][18]. This is similar to findings with mammalian species, which have been studied more extensively [5,19].…”

“…Compared with mammalian species, much less is known about the P450 forms in fish that catalyze specific positions of hydroxylation of steroids. Some similarities between fish and mammals have been found in positional metabolism of testosterone, where the 6␤ position is hydroxylated by CYP3A family P450s in those fish and mammals that have been studied to date [11,[14][15][16][17], and of 17-␤-estradiol, where the 2-and 4-positions are hydroxylated by CYP1 family members [10,12,18]. In contrast, while human CYP3A4 readily hydroxylated 17-␤-estradiol at the 2-position, zebrafish CYP3A65 produced only small amounts of 2-hydroxy-17-␤-estradiol, compared with other zebrafish CYPs [12].…”

The cytochrome P450 genes of channel catfish: Their involvement in disease defense responses as revealed by meta-analysis of RNA-Seq data sets

“…Currently, all 19 vertebrate CYP families have been identified in teleost fish, including rainbow trout ( Oncorhynchus mykiss ) (37-44), Japanese pufferfish ( Fugu rubripes ) (45, 46), zebrafish ( Danio rario ) (47-56), Atlantic salmon ( Salmo salar ) (57-60), European seabass ( Dicentrarchus labrax ) (61-63), largemouth bass ( Micropterus salmoides ) (64), medaka ( Oryzias latipes ) (65-71), common carp ( Cyprinus carpio ) (72-74), mummichog ( Fundulus heteroclitus ) (75-78), three-spined stickleback ( Gasterosteus aculeatus ) (70), gilthead seabream ( Sparus aurata ) (79), fathead minnow ( Pimephales promelas ) (80, 81), half-smooth tongue sole ( Cynoglossus semilaevis ) (82, 83). By far, with the accomplishment of whole genome sequence (84, 85), CYP genes in Japanese pufferfish (45) and zebrafish (55) have been analyzed at the whole genome level, revealing 54 CYP s (update to 61 CYP s, Table 3) and 94 CYP s are presented in the Japanese pufferfish and zebrafish, respectively.…”

Transcriptome-Based Identification and Molecular Evolution of the Cytochrome P450 Genes and Expression Profiling under Dimethoate Treatment in Amur Stickleback (Pungitius sinensis)

“…CYP3 and CYP4 were assigned to Group III. The CYP3 family shows testosterone hydroxylase activity, and the enzyme activity of some members is induced by treatment with dexamethasone, dehydroepiandrosterone (DHEA), and other compounds [58,59]. The CYP4 family catalyzes the hydroxylation of fatty acids, and some peroxisome proliferating agent can induce its transcription [60].…”