Expression Analysis Of Antioxidant Genes In Mutant And Wild Type Drosophila Melanogaster

Acetaminophen (APAP) is a common pain reliever found in over the counter medicine. APAP used in excess can be responsible for liver failure due to the metabolite NAPQI (Husenoivic 2017). It is estimated that approximately 50% of liver transplants in the U.S. are due APAP overdose (Bender et al. 2004). NAPQI is usually processed through the GSH antioxidant pathway. Without this pathway there would be an accumulation of NAPQI which would cause cellular toxicity (James et al. 2015). In addition, in vertebrates there is also evidence for involvement of the Superoxide Dismutase (SOD) and Catalase (Cat) pathways in preventing APAP toxicity. In our previous studies with Drosophila we found increased levels of GSH/GSSG in wildtype flies exposed to APAP, but we did not see increased levels of SOD or Cat activity suggesting that in flies GSH is the primary agent for detoxifying NAPQI. Dose response studies in Drosophila indicate that flies that carry mutations in the SOD or CAT genes are less sensitive to APAP in comparison to wild type or the Rosy mutant flies (Missirlis, Phillips, & Jäckle, 2001, Kimble et al., unpublished data) . Rosy is a mutation that affects uric acid synthesis, another known antioxidant. We will be testing for expression levels of the genes that are responsible for GSH synthesis (GCLc and GCLm), and GSSG recycling (GCLc and GCLm). We will use GPDH and B-tubulin (B-tub) as controls to compare expression levels of the gene among all specimen. We will be isolating RNA from larval stages of Drosophila. The RNA will be used in a real-time polymerase chain reaction (RT PCR) to determine the level of expression of the GCL & TRXr genes. We expect that the SOD and Cat mutants will show increased levels of genes involved in GSH synthesis and or recycling relative to the wild type or rosy mutants under control conditions (in the absence of APAP). If time permits, we will also look at the level of expression of the genes in the same strains of flies exposed to APAP