title: 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.
authors.affiliation: Department of Environmental Sciences, University of California, Riverside, CA 92521, USA. matsuoaline@aol.com
authors.forename: Aline Y O
authors.initials: AY
authors.lastname: Matsuo
authors.forename: Evan P
authors.initials: EP
authors.lastname: Gallagher
authors.forename: Mary
authors.initials: M
authors.lastname: Trute
authors.forename: Patricia L
authors.initials: PL
authors.lastname: Stapleton
authors.forename: Ramon
authors.initials: R
authors.lastname: Levado
authors.forename: Daniel
authors.initials: D
authors.lastname: Schlenk
mesh_terms: D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases / Q000235:genetics / Q000378:metabolism; D001711:Biotransformation / Q000235:genetics; D015153:Blotting, Western; D019363:Cytochrome P-450 CYP1A1 / Q000235:genetics / Q000378:metabolism; D019362:Cytochrome P-450 CYP2B1 / Q000235:genetics / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D029941:Fish Proteins / Q000235:genetics / Q000378:metabolism*; D015971:Gene Expression Regulation, Enzymologic; D005880:Gills / Q000201:enzymology; D008099:Liver / Q000201:enzymology; D050216:Metabolic Detoxication, Phase I / Q000235:genetics; D008861:Microsomes / Q000201:enzymology; D009833:Olfactory Pathways / Q000201:enzymology; D017684:Oncorhynchus kisutch / Q000235:genetics / Q000378:metabolism*; D010078:Oxazines / Q000378:metabolism; D010105:Oxygenases / Q000235:genetics / Q000378:metabolism; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000032:analysis; D013250:Steroid Hydroxylases / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D013739:Testosterone / Q000378:metabolism