Cytochrome P450 monooxygenases are heme-containing enzymes that insert oxygen into a wide range of xenobiotics, such as pollutants, pesticides, and drugs, promoting their clearance from the body. Although they provide a vital defense system against environmental toxins, P450s may also catalyze the formation of products that are more toxic or carcinogenic than their precursors. P450s perform essential roles in development and homeostasis as well, including steroid hormone synthesis. It is unclear how these defensive and developmental roles of the P450 system are coordinated and regulated; to address this problem in vivo, a genetic analysis was initiated in the model organism Drosophila melanogaster. P450s that reside on the endoplasmic reticulum (ER) generally require a redox partner, Cytochrome P450 Reductase (CPR). Most animals have dozens of P450 genes, but CPR is often encoded by a single gene, providing a unique """"""""Achilles' heel"""""""" for genetically inactivating the ER P450 system. The principal investigator's lab generated an allelic series of mutations in the Drosophila CPR gene (Cpr), including 6 novel lethal alleles. Flies were constructed which conditionally express a Cpr cDNA; this transgene can rescue the lethal mutations, and it causes visible phenotypes when overexpressed. These genetic tools provide the means to elucidate the numerous functions of the highly pleiotropic Cpr gene.
3 aims are proposed: I) Characterize embryonic and larval phenotypes associated with Cpr, to identify its developmental functions. II) Develop an assay system to determine the degree to which metabolism of a compound depends on CPR. The sensitivity (or resistance) of the mutants to several xenobiotics and mixtures will be tested. A method for generating CPR-deficient adults will be established, to facilitate compound testing and analysis of adult Cpr phenotypes. Ill) Test for feedback induction of the P450s. Toxin and drug interactions are often caused by P450 induction. It was recently hypothesized that vertebrates have a large-scale system to overexpress P450s when CPR is compromised. P450 mRNAs will be quantified in Cpr mutants to determine if this system exists and can be investigated in Drosophila. ? ? ?
