FUNCTIONAL CHARACTERIZATION OF ZEBRAFISH CYTOCHROME P450 1 AND 3A GENES USING HETEROLOGOUSLY EXPRESSED PROTEINS

Abstract

<p>Cytochrome P4501 (CYP1) and CYP3A enzymes are hepatic proteins critical for the metabolism of many endogenous and exogenous compounds. CYPl and CYP3A proteins are well characterized in mammals, but aside from CYP1A, little is known of the function of the other CYPls and CYP3A in non-mammalian vertebrates. In this study, zebra-fish CYP1A, CYP1Bl, CYP1Cl, CYP1C2, CYP1Dl and CYP3A65 have been heterologously cloned, expressed and purified. The catalytic activity of each CYP was tested using 11 synthetic mammalian CYP substrates. The catalytic activity and regioselectivity of each CYP to metabolize 17β-estradiol (E₂), an endogenous steroid, and benzo[a]pyrene (BaP), a model exogenous compound were also tested. The major CYPs that metabolized E2 were CYP1A and CYP1Cl, and to a lesser extent CYP1C2. Overall CYP1A has the broadest specificity and the highest activity for most substrates. CYP1Cl and CYP1C2 have similar specificity and rates of metabolism for most substrates. Zebrafish CYP1Bl is similar to mammalian CYP1Bl in both relative activity to CYP1A and substrate specificity. CYP1Dl, the most recently identified vertebrate CYP1, poorly metabolized all substrates except for BaP. The role of CYP1Dl is currently unknown, but given the metabolic differences seen here compared to the other CYPls, CYP1Dl may have a function unlike that of the other CYPis. Zebrafish CYP3A65 had a low activity for typical mammalian CYP3A substrates suggesting it could also have a different substrate profile than mammalian CYP3As. This is the first study to analyze the activity of the complete CYPl and CYP3A profile of a non-mammalian vertebrate and will be important in determining the functions of these proteins.</p>