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Activation of Nrf2 at Critical Windows of Development Alters Protein S-Glutathionylation in the Zebrafish Embryo (Danio rerio)

Abstract
Perturbation of cellular redox homeostasis to a more oxidized state has been linked to adverse human health effects such as diabetes and cancer. However, the impact of altering the regulation of redox homeostasis during development is not fully understood. Specifically, this project investigates the role of the Nrf2 antioxidant response pathway and its effect on glutathione (GSH; cellular redox buffer) at critical windows of development. To explore this, we used zebrafish embryos (Danio rerio) as a model due to the function of GSH and the Nrf2 being conserved among vertebrates. We exposed zebrafish embryos to three Nrf2 activators: two antioxidant enhancing molecules: sulforaphane (SFN; 40 µM) and Dimethyl fumarate (DFM; 7µM) as well as the pro-oxidant tert-Butylhydroquinone (tBHQ; 1µM) for 6 hours at critical windows development: 24, 48, 72 hours post fertilization (hpf). Following exposure, we visualized Nrf2 protein levels and glutathionylation rates using immunohistochemistry and confocal imaging. We found that changes in Nrf2 expression were dependent on the tissue type with there being significant changes in Nrf2 when looking at the pancreatic beta cells. Also in the beta cells, exposure to SFN, tBHQ, and DMF were found to increase Nrf2 translocation into the nucleus. Most notably, all three Nrf2 activators significantly altered glutathionylation levels depending on the time-point the zebrafish were exposed. SFN and tBHQ were also found to significantly increase glutathionylation at 48 and 72hpf, but led to a significant decrease at 96hpf while DMF increased glutathionylation at all three time-points. Interestingly, there was little correlation between Nrf2 protein levels and glutathionylation, but zebrafish with a mutated Nrf2 did have significantly different glutathionylation rates than the wild type fish. This suggests that oxidative stress is not the sole regulator of glutathionylation and instead Nrf2 may also be regulating glutathionylation through GSH storage. My data indicate that the effects of Nrf2 activation on Nrf2 levels and glutathionylation depend on the timing of exposure to the perturbing chemicals and the tissue type. Finding these windows of development where redox homeostasis is most sensitive in humans can allow for possible preventative and/or protective measures to oxidative stress during development.
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