Dichloroacetate (DCA) is an investigational drug for treatment of children and adults with genetic or acquired mitochondrial diseases and cancer. The pharmacokinetics and dynamics of DCA vary by subject age. For example, the chronic use of DCA in adults is associated with the development of reversible peripheral neuropathy, a side effect that is seldom observed in children, who metabolize DCA more quickly. The dechlorination of DCA to its inactive metabolite, glyoxylate, is catalyzed by maleylacetoacetate isomerase/glutathione transferase zeta 1 (MAAI/GSTZ1). GSTZ1 is also critical in tyrosine catabolism through trans‐isomerization of maleylacetoacetate (MAA) and maleylacetone (MA). However, DCA causes mechanism‐based inactivation of GSTZ1, resulting in inhibition of both DCA metabolism and isomerization of MAA and MA. The resulting tissue accumulation of DCA, MAA, and MA may be a mechanism of toxicity. Studies in GSTZ1(−/−) mice showed induction of antioxidant enzymes and GSTs A1/A2, M1 and M5, as well as an increase in the GSSG:GSH ratio, suggesting that GSTZ1 deficiency causes oxidative stress. In this study, we determined if loss of the expression and activity of GSTZ1 in rat tissues following multiple DCA dosages that mimicked therapy resulted in similar effects. Adult (52 wk) female Sprague Dawley rats (8 per group) and young (4 wk) male and female Sprague Dawley rats (4 per group) were dosed with sodium DCA (100 mg/kg) or sodium acetate (100 mg/kg) by oral gavage daily for 8 consecutive days. On the 9th day, rats were euthanized, livers were removed and cytosolic and mitochondrial subcellular fractions were prepared. Protein expression was assessed by Western blot using antibodies to A, M, P, T and O‐class GSTs and to NAD(P)H quinone dehydrogenase 1 (NQO1), gamma‐glutamylcysteine ligase complex (GCLC), and glutathione synthetase (GSS). GSH and GSSG levels were measured by LC/MS/MS. Enzyme activity was measured in hepatic cytosol using 1‐chloro‐2,4‐dinitrobenzene (CDNB), 1,2‐dichloro‐4‐nitrobenzene (DCNB), and 2,6‐dichloroindophenol (DCPIP) as substrates. In comparison to acetate‐treated controls, GST activities with CDNB and DCNB were higher in DCA‐treated rats irrespective of age (p < 0.05), as was the relative expression of GSTA1/A2 (p < 0.01), whereas DCA‐treated adults displayed higher levels of GSTM1 (p < 0.05) and GSTO1 (p < 0.05). Changes in the expression of P and T‐class GSTs following multiple doses of DCA were not observed for either group. NQO1 expression and activity were higher in juveniles after DCA dosing (p < 0.05). GSH concentrations were increased by DCA in adults but the GSH:GSSG ratio was not changed. Levels of GCLC and GSS were higher and lower, respectively, in adults treated with DCA. We conclude that DCA‐mediated depletion of GSTZ1 causes oxidative stress and promotes the induction of antioxidant enzymes that may vary between age groups. These findings will be important when interpreting outcomes of ongoing clinical trials of DCA in children and adults. Support or Funding Informati...

Dichloroacetate is an investigational drug that is used in the treatment of various congenital and acquired disorders of energy metabolism. Although DCA is generally well-tolerated, some patients experience peripheral neuropathy, a side effect more common in adults than children. Repetitive DCA dosing causes downregulation of its metabolizing enzyme, GSTZ1, which is also critical in the detoxification of maleylacetoacetate and maleylacetone. GSTZ1 (-/-) knockout mice show upregulation of GSTs and antioxidant enzymes as well as an increase in GSSG:GSH, suggesting GSTZ1 deficiency causes oxidative stress. We hypothesized that DCA-mediated depletion of GSTZ1 causes oxidative stress and used the rat to examine induction of GSTs and antioxidant enzymes after repeated DCA exposure. We determined the expression of A, M, P, and O-class GSTs, NAD(P)H quinone dehydrogenase 1 (NQO1), gamma-glutamylcysteine ligase complex (GCLC), and glutathione synthetase (GSS). GSH and GSSG levels were measured by LC/MS/MS. Enzyme activity was measured in hepatic cytosol using 1-chloro-2,4dinitrobenzene (CDNB), 1,2-dichloro-4-nitrobenzene (DCNB), and 2,6-dichloroindophenol (DCPIP) as substrates. In comparison to acetate-treated controls, DCA dosing increased the relative expression of GSTA1/A2 irrespective of rodent age, whereas only adults displayed higher levels of GSTM1 and GSTO1. NQO1 expression and activity were higher in juveniles after DCA dosing. GSH concentrations were increased by DCA in adults but the GSH:GSSG ratio was not changed. Levels of GCLC and GSS were higher and lower, respectively, in adults treated with DCA. We conclude that DCA-mediated depletion of GSTZ1 causes oxidative stress and promotes the induction of antioxidant enzymes that may vary between age groups.