Characterization of Phase I biotransformation enzymes in coho salmon (Oncorhynchus kisutch)

Abstract

Wild stocks of Pacific salmon in the Northwestern United States have declined in recent years, and the major factors contributing to these losses include water pollution and loss of habitat. In salmon, sublethal chemical exposures may impact critical behaviors (such as homing, feeding, predator-avoidance) that are important for species survival. Therefore, understanding the potential for these species to biotransform organic compounds within sensitive target tissues such as liver, gills and olfactory region can help estimate or predict their susceptibility to pollutants. In this study, we used real-time quantitative polymerase chain reaction (Q-PCR), Western blotting, and catalytic assays to characterize the expression of Phase I biotransformation enzymes in coho salmon (Oncorhynchus kisutch), a sensitive species in the Pacific Northwest. Gene expression analysis using Q-PCR assays developed for coho genes revealed the presence of the predominant cytochrome P450 mRNAs (CYP1A, CYP2K1, CYP2M1, CYP3A27) in the olfactory rosettes and provided quantitative mRNA expression levels in coho liver and gills. Q-PCR analysis revealed relatively high expression of the major CYP isoforms in the liver and olfactory rosettes, which was generally confirmed by Western blotting. Extrahepatic CYP expression was generally higher in the olfactory rosettes as compared to the gills. Catalytic studies demonstrated functional CYP1A-dependent ethoxyresorufin-O-deethylase, CYP2-dependent pentoxyresorufin-O-dealkylase, CYP2K1-dependent testosterone 16beta-hydroxylase, and CYP3A27-dependent testosterone 6beta-hydroxylase activities in liver, but not at detectable levels in gills. In contrast, flavin-containing monooxygenase (FMO)-dependent thiourea S-oxidase activity was readily observed in the gills and was substantially higher than that observed in liver. Collectively, the results of this study suggest that the olfactory rosettes are important sites of extrahepatic biotransformation in coho salmon, and that tissue specific-differences in Phase I metabolism may lead to contrasting tissue-specific biotransformation capabilities in this species.

Fig. 1

Quantitative PCR data in CYP isoforms of juvenile coho salmon (mean ± SEM; N=6) normalized to the expression of housekeeping gene β-actin. Differential tissue expression of CYP isoforms was seen for the constitutive CYP2K1 (b), CYP2M1 (c), and CYP3A27 (d), but not for the inducible CYP1A (a). Significant differences (P<0.05) are represented by a = relative to liver, b = relative to gills, and c = relative to olfactory rosettes. ND = not determined.

Fig. 1

Quantitative PCR data in CYP isoforms of juvenile coho salmon (mean ± SEM; N=6) normalized to the expression of housekeeping gene β-actin. Differential tissue expression of CYP isoforms was seen for the constitutive CYP2K1 (b), CYP2M1 (c), and CYP3A27 (d), but not for the inducible CYP1A (a). Significant differences (P<0.05) are represented by a = relative to liver, b = relative to gills, and c = relative to olfactory rosettes. ND = not determined.

Fig. 2

a) Western blots of CYP2K1-, CYP2M1, and CYP3A27-like proteins in microsomes from juvenile coho salmon tissues. CYP1A and FMO1 were not detected by immunoblotting (not shown). Protein load: 40 μg/lane. OR = olfactory rosette; L = liver. Molecular weights of bands are shown by arrows; b) Relative optical density of CYP-like isoforms detected by Western blotting illustrating tissue-specific differences in isoform expression.