Pharmacologic or genetic ablation of maleylacetoacetate isomerase increases levels of toxic tyrosine catabolites in rodents
Introduction
Animals catabolize phenylalanine and tyrosine by a multienzyme pathway that leads to formation of fumarate and acetoacetate (Fig. 1). Loss of function mutations in the terminal enzyme of this pathway, fumarylacetoacetate hydrolase, is the cause of hereditary tyrosinemia type I [1]. Absence of hydrolase activity leads to accumulation of intermediates of phenylalanine/tyrosine metabolism, such as maleylacetoacetate (MAA) and fumarylacetoacetate (FAA), which are considered to be hepato-toxins, and their ketone derivatives. Hereditary tyrosinemia type I is also associated with accumulation of succinylacetone (SA). SA inhibits an early step in heme synthesis, resulting in buildup of the heme precursor delta-aminolevulinate (δ-ALA). These perturbations in heme metabolism are considered to be responsible for the neuropathic complication of hereditary tyrosinemia type I.
The xenobiotic DCA is a by-product of water chlorination and thus is a ubiquitous environmental contaminant [2]. However, it is also an investigational drug for the treatment of diseases of mitochondrial energetics, including congenital forms of lactic acidosis (CLA) [3], [4], [5]. DCA facilitates both cellular lactate removal and energy production by activating carbon flux through the mitochondrial pyruvate dehydrogenase (PDH) complex, mainly by inhibiting PDH-kinase and maintaining PDH in its unphosphorylated, catalytically active state [3]. DCA is hepato-carcinogenic in rodents at doses 10,000-fold higher than environmental levels [6], [7], [8], [9], [10], [11]. Although clinical trials do not show evidence of carcinogenicity in humans [12], [13], reversible peripheral neuropathy and mild hepato-toxicity may be common side effects of chronic DCA exposure in humans, at doses of 25 mg/kg/day or greater [3], [14].2
DCA is biotransformed to glyoxylate in the liver by the cytosolic enzyme glutathione transferase zeta 1 (GSTZ1) [15]. GSTZ1 is identical to MAAI, the penultimate enzyme in the phenylalanine/tyrosine catabolic pathway [16]. We reported earlier that DCA exposure decreases MAAI activity in rat hepatic cytosol, and that rats chronically exposed to DCA accumulate significant amounts of maleylacetone (MA) in their urine [17], [18]. Recent studies from two groups have shown that a combination of DCA and GSH irreversibly inactivates rodent and human MAAI in vitro[15], [18], suggesting that enzyme inactivation and subsequent degradation of the modified protein may decrease both protein expression and MAAI activity in vivo. These discoveries have relevance for understanding both DCA toxicology and the unusual kinetics of DCA biotransformation [3], [19].
The exact molecular mechanism for DCA-induced depletion of MAAI protein and activity in vivo remains uncertain, although it is possible that irreversible modification by the drug may play a central role. DCA has been shown to affect gene expression of multiple cellular proteins, including maleic enzyme [20], HMG-CoA reductase [21], [22], stearoyl-CoA desaturase, alpha-1 protease inhibitor, cytochrome b5, and carboxylesterase [23]. We therefore investigated the steady-state transcript levels of MAAI mRNA to determine whether there was a genetic basis for drug-induced MAAI protein depletion. Furthermore, it is not known whether blockade of MAAI diverts DCA into alternate pathways of biotransformation. Studies in humans exposed to []DCA indicate that the compound may undergo reductive dehalogenation to []monochloroacetate [24]. We therefore used the recently developed MAAI-KO mouse model to test the hypothesis that genetic ablation of MAAI prevents DCA biotransformation and leads to accumulation of potentially toxic metabolites of tyrosine, similar to or greater than that seen with pharmacologic ablation of the enzyme.
Section snippets
Chemicals
Na[1,2-]DCA was purchased from Cambridge Isotope Laboratories, and unlabeled Na-DCA from TCI America. All other chemicals used were of the purest grade available from Sigma-Aldrich Corp., or Fisher Scientific.
[]DCA, specific activity 55.5 mCi/mmol, was purchased from American Radiolabeled Chemicals. MA and fumarylacetone (FA) were prepared by the method of Fowler and Seltzer [25]. MA was generated within 2 days of use by hydrolysis of its synthetic precursor,
Statistical analyses
Means and standard deviations were calculated using Microsoft Excel software (Microsoft). Statistical tests of significance (one-way ANOVA) were performed utilizing MINITAB release 13.0 statistical software (Minitab, Inc.).
DCA depletes MAAI protein in rat liver cytosol
SD rats were exposed to various doses of DCA by oral gavage for 1 or 5 days and steady-state levels of MAAI in liver cytosol were measured by Western blotting (Fig. 2A). A 1-day exposure to 200 mg/kg DCA caused a 2.5-fold reduction in protein level compared to control (one-way ANOVA, P<0.0001; data not shown). Repeated dosing for 5 days decreased enzyme levels for all doses (one-way ANOVA, P<0.02 for the 4 mg dose, P<0.0001 for the 12.5, 50, and 200 mg doses; data not shown). A time- and
Discussion
Repeated treatment with DCA significantly prolongs its plasma elimination half-life in both rodents [17] and humans [19], [30]. In vitro studies with hepatic cytosol derived from DCA-treated rats have shown that DCA inhibits MAAI activity in both SD [17] and Fischer 344 rats [31], and a single 1 g/kg dose completely eliminates enzyme activity [18]. This effect on MAAI activity appears to be directly correlated with DCA-induced loss of immunoreactive MAAI protein in liver cytosol of male Fischer
Acknowledgements
This work was supported by ES 07355 and ES 07375 to Peter W. Stacpoole and DK 48252 to Markus Grompe from the National Institutes of Health. We thank A. Aller and C. Caputo for technical and editorial assistance, respectively.
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These authors contributed equally to this work.