| Cytochrome P450's represent the single most important enzyme family involved with detoxification of xenobiotics. Induction of specific P450 isozymes in insects is often observed after treatment with a variety of inorganic chemicals (Scott 1999), and induction of the P450 system can have important consequences to the ability of insects to tolerate exposure to pesticides. Studies performed in our lab have measured the effect of atrazine exposure on cytochrome P450-dependent monooxygenase activity and have found increased activity as a result of atrazine exposure (10 ppm) in midge larvae (Chironomus tentans). Identification of specific atrazine-inducible genes enhances sensitivity of detection and provides insight into potential consequences of exposure.; Research results presented in this thesis identified a CYP4 gene with an open reading frame of 1,680 bp (accession number AY880065). Northern blot analysis employing a fragment of 1,200 bp from the CYP4 gene as a probe confirmed over-expression in C. tentans exposed to atrazine (10 ppm). Additionally, expression of CYP4 was present in all five insect stages but was highest in late instar larvae.; In a second set of experiments, proteomics were used to identify a set of fourteen proteins from C. tentans third instar larvae exposed to atrazine that exhibited differences in expression. The cytochrome P450 previously identified was not identified by these studies.; The effect of atrazine exposure on cytochrome P450-dependent monooxygenase activity was also measured in adults of the fathead minnow (Pimephales promelas) and increased activity was found in males exposed to atrazine (2000 ppb). Additionally, proteomics were used to identify a set of four proteins from microsomes prepared from adult livers of P. promelas that showed differences in expression in midges exposed to atrazine.; These results provide important methods for understanding pesticide toxicity in non-target organisms using molecular and proteomics techniques. The study of the affected proteins by atrazine in these species will contribute to the identification of target genes and provide a means to better predict the effects of exposure. |
