Development and application of a real-time quantitative PCR assay for determining CYP1A transcripts in three genera of salmonids

Abstract

The expression of CYP1A (cytochrome P450 1A) can be induced by a large array of aromatic and organic compounds in teleost fishes. We developed a real-time quantitative PCR assay useful for measuring β-naphthoflavone (BNF) induction of liver CYP1A mRNA in four salmonid species. First, to obtain necessary information for the design of a cRNA standard, full-length CYP1A cDNA sequences were determined for two Salvelinus species, lake trout ( S. namaycush) and brook trout ( S. fontinalis). Each cDNA was found to share the same characteristics with known CYP1A sequences of Atlantic salmon ( Salmo salar) and rainbow trout ( Oncorhynchus mykiss): a start codon, conserved heme-binding region, putative poly-adenylation signal, stop codon, relatively long 3′-untranslated region (UTR; >1 kb), and a protein length of 523 amino acid residues. The brook trout and lake trout CYP1A cDNA’s were 2636 and 2672 base pairs (bp) in length and shared greater than 97% coding region sequence identity with Atlantic salmon and rainbow trout CYP1A’s. Next, using the generated sequence information, we developed a CYP1A-specific real-time quantitative PCR assay. Primers and a fluorescent-labeled probe were designed from a 68 bp region that was found to be conserved among salmonid CYP1A genes. The assay was designed to allow for simultaneous comparison of CYP1A expression among each experimental group. Finally, groups ( n=4–8) of hatchery-raised Atlantic salmon, brook trout, lake trout, and rainbow trout were given an intraperitoneal injection of a corn oil control, 25 mg kg −1 BNF, or 50 mg kg −1 BNF and sacrificed after 48 h. Liver tissue was collected and CYP1A mRNA levels were estimated. In all species, BNF treated fish showed 1.8–3.0 orders of magnitude higher CYP1A than control fish. The CYP1A induction levels were not different in fish treated with both dosages. Mean base levels of CYP1A expression ranged from 7.24×10 6 (rainbow trout) to 1.05×10 7 (brook trout) transcripts μg −1 total RNA. Mean induced levels of CYP1A expression ranged from 1.07×10 8 (lake trout) to 1.05×10 9 (brook trout) trancripts μg −1 total RNA.