Superfund Research Program
Aquatic Biomarkers in Site Characterization and Remediation
Project Leader: David E. Hinton (Duke University)
Grant Number: P42ES004699
Funding Period: 1995 - 2005
Final Progress Reports
Year: 2004 1999
Two full-length CYP3A forms have been cloned and characterized in medaka (Oryzias latipes). Comprising over 60% of total hepatic P450 content in fish liver and possessing a role in the metabolism of endogenous gonadal steroids, xenobiotics and carcinogens, CYP3A is of extreme importance to our Superfund goals. The two forms share structural features specific to the cytochrome P450 superfamily and a high degree of sequence identity to the CYP3A subfamily. Assigned as CYP3A38 and CYP3A40 by the cytochrome P450 nomenclature committee, analysis of CYP3A38 and CYP3A40 and other teleost and mammalian CYP3A gene sequences demonstrate a unique clustering of the medaka genes with that of trout and killifish independent and that these are separate from mammalian sequences, suggesting an early lineage of this subfamily. We have partially characterized cytochrome P450 system as a function of development in medaka embryos and demonstrated a differential temporal expression profile for each of the CYP3A genes. Whereas CYP3A40 is constitutively expressed after fertilization and continues throughout adulthood, CYP3A38 is tightly repressed during embryonic development with expression occurring only after hatching. Consistent with this expression pattern is a highly reduced ability of medaka embryos to metabolize CYP3A substrates. Total P450 content is additionally highly reduced compared to adult liver and suggests that developing teleost embryos may be more susceptible/sensitive to a variety of xenobiotics. Finally, to address functionality, CYP3A38 has been cloned into an eukaryotic expression vector and transiently expressed in mammalian cells. Activity of heterologous CYP3A38 enzyme resulted in the 6b and 16b hydroxylation of testosterone. However, the ratio of metabolite formation heavily favored the formation of the 16b-OH metabolite. This is a novel role for this enzyme compared to mammalian CYP3A forms. Studies are currently in progress to further characterize the functional activity of both CYP3As 38 and 40.