Assessing the Mechanism of Metabolism-Dependent Valproic Acid-Induced in Vitro Cytotoxicity

Abstract

This study was designed to distinguish and evaluate the contribution of reactive metabolite and reactive oxygen species as the mechanism of metabolism-dependent valproic acid-induced in vitro cytotoxicity. The involvement of reactive oxygen species in the mechanism of in vitro cytotoxicity was examined by the addition of a series of antioxidant enzymes and iron chelators to the reaction mixture. Addition of catalase to the reaction mixture resulted in a complete prevention of valproic acid-induced cytotoxicity. Co-incubation of a cell impermeable iron chelator deferoxamine did not effect cytotoxicity, whereas 1,10-phenanthroline, a chelator with the ability to traverse cell membranes at low concentrations, afforded significant protection against valproic acid-induced cytotoxicity. A possible inhibitory effect of catalase and 1,10-phenanthroline on the microsomal metabolism of valproic acid was disproved by the quantification of valproic acid metabolites in the presence and absence of these compounds. To assess the specificity of the mechanism of in vitro valproic acid-induced cytotoxicity, prevention of in vitro acetaminophen-induced cytotoxicity by antioxidant enzymes and iron chelators was also evaluated. Addition of catalase to the reaction mixture in the presence of acetaminophen resulted in a moderate reduction in the level of but a lack of complete protection of cytotoxicity. Addition of 1,10-phenanthroline to the reaction mixture in the presence of acetaminophen did not result in a detectable change in acetaminophen-induced cytotoxicity. These data suggest the involvement of reactive oxygen species in the mechanism of toxicity of valproic acid and perhaps reactive metabolites as the major cause of cytotoxicity in the case of acetaminophen in the in vitro model investigated. Inhibition of poly(ADP-ribose) polymerase activity by various antagonists resulted in complete prevention of valproic acid-induced in vitro cytotoxicity. The cytoprotective effects of known poly(ADP-ribose) polymerase antagonists implicate poly(ADP-ribose) polymerase in the mechanism of in vitro metabolism-dependent valproic acid-induced cytotoxicity under these conditions. These results further point to nuclear DNA as the intracellular site of insult by the generated oxygen radicals. Overall, the data obtained support the hypothesis that the metabolism-dependent valproic acid-induced in vitro cytotoxicity is the result of generation of hydrogen peroxide in the medium that can readily cross cell membranes and subsequently interact intracellularly with iron to produce the highly reactive hydroxyl free radicals.

Valproic Acid–Induced Gene Expression through Production of Reactive Oxygen Species

“…2A, VPA treatment increased DCF fluorescence in a time-dependent manner, indicating increased ROS production in these cells. The data are consistent with reports in other cell systems that VPA induces the production of ROS (20)(21)(22). Other HDAC inhibitors that are of interest as cancer chemotherapeutic agents, i.e., SAHA (23), MS-275 (24,25), and sodium butyrate (26) have also been reported to increase the production of ROS, although in some cases, ROS production occurred only in transformed cells (25).…”

“…Furthermore, recent reports that VPA induces production of ROS (20)(21)(22) do not explore how the resultant elevated level of ROS influences expression of any gene. Our study clearly provides mechanistic insight into the disposition of VPA-or other HDAC inhibitor-induced ROS production vis-à-vis gene expression; it provides a link between ROS-induced activation of redox-sensitive transcription factors (typified by Nrf2) and their subsequent interaction with the ARE on specific gene promoters.…”

“…VPA (20)(21)(22) and other class I/II HDAC inhibitors, i.e., suberoylanilide hydroxamic acid (SAHA; ref. 23), MS-275 (24,25), and sodium butyrate (26), have been reported to elevate cellular levels of reactive oxygen species (ROS).…”

Valproic Acid Increases Conservative Homologous Recombination Frequency and Reactive Oxygen Species Formation: A Potential Mechanism for Valproic Acid-Induced Neural Tube Defects

“…Our results demonstrate that preincubation with PEG-catalase before the administration of valproic acid significantly decreased the production of ROS, although ROS levels did not decrease to baseline levels. In a previous study using in vitro human lymphocyte preparations, catalase significantly protected against valproic acid-induced cytotoxicity; however, similar to findings in the present study, cell death levels were not completely attenuated to baseline levels (Tabatabaei and Abbott, 1999). Given that increased DCF fluorescence can result because of exposure to various ROS and reactive nitrogen species, DCF is considered a general marker for these species (Li et al, 2002).…”

“…For example, previous research has shown that catalase, which detoxifies hydrogen peroxide, prevented valproic acid-induced lymphocyte toxicity and that 1,10-phenanthroline, an iron chelator, also decreased valproic acid-mediated cytotoxicity in vitro (Tabatabaei and Abbott, 1999). These results suggest that the production of hydrogen peroxide and the succeeding iron-catalyzed formation of hydroxyl radicals may be the specific ROS that mediates valproic acid-induced toxicity (Tabatabaei and Abbott, 1999). In addition, valproic acid has been shown to inhibit cardiomyocyte differentiation of embryoid bodies derived from murine pluripotent embryonic stem cells through an increase in ROS (Na et al, 2003).…”