Valproic Acid Promotes Acetylation of Superoxide Dismutase-2 During Neurogenesis. Valproic acid (VPA) is a known developmental toxicant associated with a high prevalence of neural tube defects (NTD). The mechanism of VPA-induced NTD is unclear, but oxidative stress may be implicated. To understand how embryotoxic oxidative stress may occur, we measured superoxide dismutase (SOD) activity following VPA treatment in the embryonic pluripotent P19 mouse carcinoma cell line. In undifferentiated P19 cultures treated with VPA (5 mM), dichlorofluorescein fluorescence increased 15% compared to untreated controls over 20 min, indicating a modest, yet statistically significant increase in ROS generation. Undifferentiated P19 cells were treated with VPA for 6 h, after which total SOD and mitochondrial SOD (SOD2) activities were measured. VPA treatment decreased total SOD activity by approximately 20% but SOD2 activity was undetectable; but this was not a consequence of changes to SOD (SOD1 or SOD2) protein concentrations. Interestingly, glutathione redox state increased from -262 mV to -245 mV after a 6 h treatment with VPA, indicating significant oxidation of the cellular redox environment. Measurement of mitochondrial superoxide levels showed an increase following VPA treatments. While it is unlikely that VPA works directly as an oxidant, these data suggest that VPA may promote oxidative stress through an alternative means, such as via the inhibition of SOD activity and thus, allow for an increase in ROS. Importantly, VPA is a known deacetylase inhibitor, and SOD2 function is regulated by acetylation. As such, we evaluated the acetylation state of SOD2 to determine potential disruption via acetylation. Treated undifferentiated P19 cells showed a significant increase in SOD2 acetylation. However, in fully differentiated P19-derived neurons, cells showed no such SOD2 acetylation. Additionally, pretreatment with dithiole-3-thione (D3T), a Nrf2 activator of the antioxidant response, attenuated VPA-induced mitochondrial ROS production and SOD2 acetylation and improved SOD2 activity, suggesting Nrf2 as a potential means to reduce VPA-mediated oxidative stress. To evaluate the effects in the embryo proper, gestational day 8 mouse embryos were treated with VPA in culture for 6 h. Similar to P19 cells, VPA-treated neurulating embryos showed significant SOD2 acetylation and a concomitant decrease in total SOD activity. These data support a similar consequence of VPA-induced oxidative stress in embryos as is demonstrated in our cellular model. Since no SOD2 acetylation is observed in differentiated neurons and VPAinduced SOD2 acetylation occurs more prevalently in undifferentiated/differentiating cells, these data purport means by which VPA preferentially induces oxidative stress in developing systems.