Molecular Cloning and Expression of Cytochrome P450 1C1 in Japanese Eel ( Anguilla Japonica )

Abstract

Cytochrome P450 (CYP) enzymes constitute a multigene family of many endogenous and xenobiotic substances. The CYP1 family is of particular interest in environmental toxicology because its members are dominant in the metabolism of polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), and aryl amines. A new cDNA of the CYP1C subfamily encoding CYP1C1 was isolated from Japanese eel (Anguilla japonica) liver after intraperitoneal injection with βnaphthoflavone (BNF). The full-length cDNA obtained (3508 bp) contained a 5' noncoding region of 355 bp, an open reading frame of 1581 bp coding for 526 amino acids, a stop codon, and a 3' noncoding region of 1572 bp. The predicted molecular weight of the protein was approximately 59.33 kDa. The deduced amino acid sequence of Japanese eel CYP1C1 had the lower similarity of 70% with that of killifish CYP1C1 while the higher similarity (79 and 81%) was observed with that of rainbow trout CYP1C2 and -1C1 sequences respectively. It exhibited similarities of 71% with that of Indian medaka CYP1C1 and zebrafish CYP1C2. Also the similarity of 74% was registered with the sequence of three-spined stickleback fish CYP1C1, 1C2 and carp CYP1C2. It showed similarity of 77% with that of Nile tilapia CYP1C1, scup CYP1C1 and scup CYP1C2. The phylogenetic tree showed the newly identified Japanese eel CYP1C1 sequence to be clustered with rainbow trout CYP1C1 and -1C2. Japanese eel CYP1C1 was aligned with the CYP1 sequences and has been deposited in the Gen Bank / EMBL data bank with the accession number AY444748. Quantitative real-time polymerase chain reaction (QRT-PCR) analysis of liver, kidney, intestine and gills revealed a distinct induced expression in all organs studied (283.33, 579.35, 20.96 and 3642.32 respectively).