Expression of CYP1A1 in livers and gonads of Pacific salmon: quantitation of mRNA levels by RT-cPCR

Abstract

A reverse-transcriptase-competitive-polymerase-chain-reaction (RT-cPCR) assay was developed to accurately quantitate CYP1A1 mRNA levels in various tissues of Pacific salmon. Juvenile chinook salmon were injected intraperitoneally with the polynuclear aromatic hydrocarbon β-naphthoflavone (BNF), and levels of CYP1A1 mRNA were measured in livers and gonads over a 4-week period using RT-cPCR. There was a significant increase in hepatic CYP1A1 mRNA levels in the fish injected with BNF over the 28-day experiment, from undetectable levels (< 0.83 × 10 −3 fmol/μg RNA) at time zero to a mean level of 133.56 × 10 −3 fmol/ μg RNA, an increase of at least 160-fold. Although low levels of CYP1A1 expression were detected in the livers of control fish, hepatic CYP1A1 mRNA levels in BNF-treated juvenile chinook were always very significantly elevated above control values throughout the 4-week experiment. CYP1A1 mRNA levels were also significantly elevated (to a maximum of 18-fold above the assay detection limit) in the gonads of BNF-treated fish, whereas no detectable levels were observed in the gonads of control fish at any time. To examine the response of gonadal tissue in more detail, ovarian follicles isolated from maturing coho salmon were exposed to various doses of BNF or 20-methylcholanthrene (20-MC) in vitro, and CYP1A1 mRNA levels in the follicular cells quantitated. Exposure to either BNF or 20-MC for 18 h in vitro induced CYP1A1 in coho ovarian follicles. A dose response for both PAHs was obtained (between 50.0 nM and 5.0 μM) and revealed a greater sensitivity of ovarian follicle cells to induction by 20-MC. This study shows that RT-cPCR can be used to detect and quantify CYP1A1 gene expression in various tissues of fish in response to xenobiotic exposure. The induction of CYP1A1 in the ovaries and testes of juvenile chinook salmon, and in isolated ovarian follicles of mature coho salmon, following exposure to PAHs indicates that xenobiotics may impact on reproductive processes directly at the gonadal level.