Cloning and sequence analysis of benzo-a-pyreneinducible cytochrome P450 1A in Nile tilapia (Oreochromis niloticus)

Polycyclic aromatic hydrocarbons (PAHs), dioxins, dibenzofurans and polychlorinated biphenyls (PCBs) present in polluted environment induce cytochrome P4501A (CYP1A) isozyme in fish which inturn results in a marked increased production of carcinogenic metabolites. The induction of hepatic CYP1A in fish by certain classes of chemicals has been suggested as an early warning system, a “mostsensitive biological response” for assessing environmental contamination conditions. This has implications for human fish consumption as well as for the health status of aquatic organisms.Considering the importance of Oreochromis niloticus fish as a laboratory animal, the common CYP1A sequence was determined from cDNA and genomic DNA after intraperitoneal injection with benzo-apyrene (BaP). The full-length cDNA was 2530 bp long and contained an open reading frame of 1566 bp encoding a protein of 521 amino acids and a stop codon. The sequence exhibited 5' and 3' noncodingregions of 134 and 830 bp, respectively. The deduced amino acid sequence of O. niloticus CYP1A shows similarities of 80.5, 79.3, 79.1, 77.8, 77.6, 74.3, 72.4, 77.2, 71.8, 70.7 and 50.8% with Europeanflounder CYP1A, scup CYP1A, killifish CYP1A, butterfly fish CYP1A, European sea bass CYP1A, rainbow trout CYP1A, Japanese eel CYP1A, toad fish CYP1A, European eel CYP1A, red sea bream CYP1A and common carp CYP1A, respectively. The phylogenetic tree based on the amino acid sequences clearly shows tilapia CYP1A and killifish CYP1A to be more closely related to each other than to the other CYP1A subfamilies. Sequence analysis of 3727 bp of genomic DNA showed that the clone obtained was the structural gene of CYP1A which consists of seven exons and six introns, the initiation codon was not found in the first exon but in the second one as was reported for the CYP1A genes of fish and mammals

Development and application of a real-time quantitative PCR assay for determining expression of benzo-apyrene- Inducible cytochrome P450 1A in Nile tilapia (Oreochromis niloticus)

Cytochrome P4501A’s (CYP1A) constitute a ubiquitous family of proteins associated with the detoxification of organic compounds such as PCB (polychlorinated biphenyl), PAH (polyaromatic hydrocarbons) and dioxin. These compounds are documented to induce the CYP1A gene in a variety of tissues of many fish species. Consequently, changes in CYP1A gene expression have been used as a biomarker for contaminant exposure in fish populations using a variety of techniques. Of all of thesemethods, quantitative PCR appears to be the most sensitive. It has been used to assess impact of environmental pollution in marine ecosystems using different fish models. Subsequently, for measuring benzo-a-pyrene (BaP) induction of CYP1A mRNA in different organs of tilapia (Oreochromis niloticus), ribosomal protein large P0-like protein (RPLP0-like protein) and -actin genes as internal controls were selected based on previous studies to assess their expression variability. Real-time polymerase chainreaction (real-time PCR) analysis of liver, intestine, gills and kidney revealed a distinct induced expression in liver and intestine (127.1 and 79.3 in liver, 26 and 56.1 in intestine using RPLP0 and -actin genes respectively as internal controls) with no detectable expression in the other organs studied