Functional differences in the cytochrome P450 1 family enzymes from zebrafish (Danio rerio) using heterologously expressed proteins

Abstract

Mammalian cytochrome P450 1 (CYP1) genes are well characterized, but in other vertebrates only the functions of CYP1A genes have been well studied. We determined the catalytic activity of zebrafish CYP1A, CYP1B1, CYP1C1, CYP1C2, and CYP1D1 proteins using 11 fluorometric substrates and benzo[a]pyrene (BaP). The resorufin-based substrates, 7-ethoxyresorufin, 7-methoxyresorufin, and 7-benzyloxyresorufin, were well metabolized by all CYP1s except CYP1D1. CYP1A metabolized nearly all substrates tested, although rates for non-resorufin substrates were typically lower than resorufin-based substrates. Zebrafish CYP1s did not metabolize 7-benzyloxyquinoline, 3-[2-(N,N-diethyl-N-methylamino)ethyl]-7-methoxy-4-methylcoumarin or 7-methoxy-4-(aminomethyl)-coumarin. CYP1B1 and CYP1C2 had the highest rates of BaP metabolism. 3-Hydroxy-BaP was a prominent metabolite for all but CYP1D1. CYP1A showed broad specificity and had the highest metabolic rates for nearly all substrates. CYP1C1 and CYP1C2 had similar substrate specificity. CYP1D1 had very low activities for all substrates except BaP, and a different regioselectivity for BaP, suggesting that CYP1D1 function may be different from other CYP1s.

Figure 1

Metabolism of alkoxyresorufins by expressed zebrafish CYP1s. Dealkylation was detected by quantification of the fluorescent metabolite resorufin (see materials and methods, and Table 1 for full details). Results are Mean ± SEM, n=2. PR metabolism by CYP1B1 and MR metabolism by CYP1D1 were below the limits of detection.

Figure 2

Metabolism of trifluoromethylcoumarins by expressed zebrafish CYP1s. Dealkylation was detected by quantification of the fluorescent metabolite HFC from either BFC or MFC (see materials and methods, and Table 1 for full details). Results are Mean ± SEM, n=2. MFC metabolism by CYP1B1, and MFC and BFC metabolism by CYP1D1 were below the limits of detection.

Figure 3

Metabolism of CEC and DBF by zebrafish. Metabolism was quantified by detection of the fluorescent metabolite CHC or fluorescein for CEC and DBF, respectively (see materials and methods, and Table 1 for full details). Results are Mean ± SEM, n=2. Metabolism of DBF by CYP1C1 and CYP1C2 and CEC metabolism by CYP1D1 were below the limits of detection.