Effect of Cyp2e1 Deletion on Methylglyoxal Detoxification Through the Nrf2 Pathway - PROJECT SUMMARY Cytochrome P450 2E1 (CYP2E1, Cyp2e1 in mice) is a well-conserved mammalian enzyme known for metabolizing both exogenous and endogenous substances, often resulting in the formation of electrophilic, toxic metabolites. For example, Cyp2e1 metabolism of acetone is known to contribute to the generation of endogenous methylglyoxal (MGO). While Cyp2e1 metabolism of substrates can produce toxic metabolites, it has also been proposed that Cyp2e1 can generate reactive oxygen species (ROS), particularly superoxide, through uncoupling of its catalytic cycle. One important protein that acts as a sensor of ROS is the transcription factor Nrf2, which plays a critical role in activating antioxidant gene transcription. A gene important for MGO detoxification Glyoxalase 1 (Glo1) is known to be under the control of Nrf2 and downregulated in Cyp2e1 KO mice Based on these findings, I hypothesize that Cyp2e1 deletion impairs MGO detoxification via the Nrf2 pathway. To test this hypothesis, I will be primarily working in the Hartman lab under the supervision of Drs. Hartman and Rockey. I will use both pharmacological and genetic approaches in primary hepatocytes and bulk liver tissue. In Aim 1, I will examine how Cyp2e1 deletion affects the enzymes and metabolites involved in MGO detoxification, using genetic and pharmacological strategies to rescue the phenotype in vivo. For genetic rescue, mice will be treated with either an AAV8-pTBG-Cyp2e1 construct to restore Cyp2e1 expression, or an AAV8-pTBG-Glo1 construct to elevate Glo1 expression to wild-type levels. For pharmacological rescue, mice will be treated with NAC to assess whether glutathione (GSH) acts as a limiting factor in MGO detoxification. A final strategy will combine both the AAV8-pTBG-Glo1 construct and NAC treatment to determine whether the dual approach enhances rescue efficiency. To assess the impact of these rescue strategies, I will use analytical techniques to quantify levels of enzymes and metabolites within the pathway. In Aim 2, I will investigate the oxidative stress-responsive Nrf2 pathway in vitro. First, I will evaluate the sensitivity of wild-type (WT) and knockout (KO) hepatocytes to an acute oxidative challenge with hydrogen peroxide. Next, I will assess Nrf2 expression and its downstream target genes using qPCR and western blot analysis. Finally, I will perform two rescue experiments in hepatocytes, either restoring Cyp2e1 or introducing constitutively active Nrf2. Collectively, this study will offer novel insights into the endogenous role of Cyp2e1 activity, and these findings may have broader implications for diseases and complications beyond the liver. In addition to its scientific impact, the proposed project will provide a valuable training platform for my thesis work and to support progression toward my future career goals. The proposed training plan will support my development in critical thinking, project planning, experimental techniques, science communication, teaching and mentoring, as well as ethics and responsible conduct of research.
