Cytochrome P450 3A forms in rainbow trout (Oncorhynchus mykiss)

Rainbow trout (Oncorhynchus mykiss) has been used as a model species for toxicology and cancer study due to its sensitivity to chemicals. Previous purified trout LMC5 P450 showed 6β-hydroxylation activity of steroids and cross-reacted with antibodies raised against human CYP3A4 on the SDS-PAGE. These findings suggest that rainbow trout P450 systems include CYP3A forms. Therefore, study was conducted to isolate and to characterize the CYP3A forms in rainbow trout.; A new CYP3A form containing 518 amino acids was cloned and assigned to CYP3A27 by the Cytochrome P450 Nomenclature Committee to represent the first CYP3A subfamily member characterized in aquatic species. The major expression site for CYP3A27 mRNA was intestinal ceca and liver. Southern blot analysis demonstrated that multiple CYP3A27-related genes exist in rainbow trout.; To examine the cellular localization and the expression pattern of CYP3A27, immunohistochemical (IH) studies were performed. The relative distribution of CYP3A27 mRNA and protein along the trout gastrointestinal tract analyzed by Northern and Western blots were supportive of the IH observations. These results confirmed that the rainbow trout possesses a similar tissue distribution pattern of CYP3A expression to that observed in mammals.; In Western blot analysis, the CYP3A27 protein expressed in baculovirus system comigrated with the purified LMC5 P450 and was recognized by LMC5 IgG. The expressed CYP3A27 protein showed catalytic activities against progesterone, nifedipine, and testosterone. These results demonstrated not only that trout CYP3A27 has the capability to metabolize steroid hormones, but also that trout have a system to support CYP3A-catalyzed first-pass metabolism of xenobiotics in the intestine as reported for mammalian CYP3A forms.; A second new CYP3A subfamily member was isolated in intestinal ceca and assigned as CYP3A45 by the Cytochrome P450 Nomenclature Committee. Major expression sites of CYP3A45 mRNA were found in the intestinal ceca and proximal descending intestine rather than in liver. The Western blot result showed that the expressed CYP3A45 protein in insect cells comigrated with purified LMC5 P450 and was recognized with LMC5 IgG on the SDS-PAGE.; Results obtained from this study contribute to a further understanding of trout CYP systems and provide a basis for studying xenobiotic metabolism and comparative toxicology in that species.