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Sodium dichloroacetate (DCA) is an investigational drug that shows promise in the treatment of acquired and congenital mitochondrial diseases, including myocardial ischemia and failure. DCA increases glucose utilization and decreases lactate production, so it may also have clinical utility in reducing lactic acidosis during labor. In the current study, we tested the ability of DCA to cross the placenta and be measured in fetal blood after intravenous administration to pregnant ewes during late gestation and labor. Sustained administration of DCA to the mother over 72 hours achieved pharmacologically active levels of DCA in the fetus and decreased fetal plasma lactate concentrations. Multicompartmental pharmacokinetics modeling indicated that drug metabolism in the fetal and maternal compartments is best described by the DCA inhibiting lactate production in both compartments, consistent with our finding that the hepatic expression of the DCA-metabolizing enzyme glutathione transferase zeta1 was decreased in the ewes and their fetuses exposed to the drug. We provide the first evidence that DCA can cross the placental compartment to enter the fetal circulation and inhibit its own hepatic metabolism in the fetus, leading to increased DCA concentrations and decreased fetal plasma lactate concentrations during its parenteral administration to the mother. SIGNIFICANCE STATEMENT: This study was the first to administer sodium dichloroacetate (DCA) to pregnant animals (sheep). It showed that DCA administered to the mother can cross the placental barrier and achieve concentrations in fetus sufficient to decrease fetal lactate concentrations. Consistent with findings reported in other species, DCA-mediated inhibition of glutathione transferase zeta1 was also observed in ewes, resulting in reduced metabolism of DCA after prolonged administration.

Balancing high energy-consuming danger resistance and low energy supply of disease tolerance is a universal survival principle that often fails during sepsis. Our research supports the concept that sepsis phosphorylates and deactivates mitochondrial pyruvate dehydrogenase complex control over the tricarboxylic cycle and the electron transport chain. StimulatIng mitochondrial energetics in septic mice and human sepsis cell models can be achieved by inhibiting pyruvate dehydrogenase kinases with the pyruvate structural analog dichloroacetate. Stimulating the pyruvate dehydrogenase complex by dichloroacetate reverses a disruption in the tricarboxylic cycle that induces itaconate, a key mediator of the disease tolerance pathway. Dichloroacetate treatment increases mitochondrial respiration and ATP synthesis, decreases oxidant stress, overcomes metabolic paralysis, regenerates tissue, organ, and innate and adaptive immune cells, and doubles the survival rate in a murine model of sepsis.

We reviewed the status of interventional clinical trials for primary mitochondrial diseases. Using national and international search engines, we found 48 randomized controlled trials (RCTs) registered as of May 15, 2019. Consilience between lay and professional mitochondrial disease communities to engage in RCTs has increased, as has progress in developing new disease and treatment biomarkers and potential therapies. The continued advancement of general knowledge of mitochondrial biology has fostered appreciation for the fundamental role mitochondria play in the etiopathology of other rare and common illnesses, emphasizing the therapeutic potential of mitochondrially-targeted small molecules for an increasing spectrum of human diseases.

Previous studies in our laboratory have suggested that the increase in stillbirth in pregnancies complicated by chronic maternal stress or hypercortisolemia is associated with cardiac dysfunction in late stages of labor and delivery. Transcriptomics analysis of the overly represented differentially expressed genes in the fetal heart of hypercortisolemic ewes indicated involvement of mitochondrial function. Sodium dichloroacetate (DCA) has been used to improve mitochondrial function in several disease states. We hypothesized that administration of DCA to laboring ewes would improve both cardiac mitochondrial activity and cardiac function in their fetuses. Four groups of ewes and their fetuses were studied: control, cortisol-infused (1 g/kg/day from 115 to term; CORT), DCA-treated (over 24 h), and DCA + CORT-treated; oxytocin was delivered starting 48 h before the DCA treatment. DCA significantly decreased cardiac lactate, alanine, and glucose/glucose-6-phosphate and increased acetylcarnitine/isobutyryl-carnitine. DCA increased mitochondrial activity, increasing oxidative phosphorylation ( PCI, PCI + II) per tissue weight or per unit of citrate synthase. DCA also decreased the duration of the QRS, attenuating the prolongation of the QRS observed in CORT fetuses. The effect to reduce QRS duration with DCA treatment correlated with increased glycerophosphocholine and serine and decreased phosphorylcholine after DCA treatment. There were negative correlations of acetylcarnitine/isobutyryl-carnitine to both heart rate (HR) and mean arterial pressure (MAP). These results suggest that improvements in mitochondrial respiration with DCA produced changes in the cardiac lipid metabolism that favor improved conduction in the heart. DCA may therefore be an effective treatment of fetal cardiac metabolic disturbances in labor that can contribute to impairments of fetal cardiac conduction.

Dramatic metabolic reprogramming between an anabolic resistance and catabolic tol-erance state occurs within the immune system in response to systemic infection with the sepsis syndrome. While metabolic tissues such as the liver are subject to end-organ damage during sepsis and are the primary cause of sepsis death, how their metabolic and energy reprogramming during sepsis state ensures survival is unclear. Employing comprehensive metabolomic screening, targeted lipidomic screening, and transcriptional profiling in a mouse model of septic shock, we show that hepatocyte li-pid metabolism, mitochondrial TCA energetics, and redox balance are significantly re-programed after cecal ligation and puncture (CLP). We identify increases in TCA cycle metabolites citrate, cis-aconitate, and itaconate with reduced fumarate and triglyceride accumulation in septic hepatocytes. Transcription analysis of liver tissue supports and extends the hepatocyte findings. Strikingly, the administration of the pyruvate dehy-drogenase kinase (PDK) inhibitor dichloroacetate (DCA) reverses dysregulated hepatocyte metabolism and mitochondrial dysfunction. Our data indicate sepsis pro-motes hepatic metabolic dysfunction. Furthermore, our data indicate that targeting the mitochondrial PDC/PDK energy homeostat rebalances transcriptional and metabolic manifestations of sepsis within the liver. ### Competing Interest Statement The authors have declared no competing interest.

Dichloroacetate (DCA) 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, glutathione transferase zeta 1 (GSTZ1), which is also critical in the detoxification of maleylacetoacetate and maleylacetone. GSTZ1 (-/-) knockout mice show upregulation of glutathione transferases (GSTs) and antioxidant enzymes as well as an increase in the ratio of oxidized glutathione (GSSG) to reduced glutathione (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 alpha, mu, pi, and omega class GSTs, NAD(P)H dehydrogenase [quinone] 1 (NQO1), gamma-glutamylcysteine ligase complex (GCLC), and glutathione synthetase (GSS). GSH and GSSG levels were measured by liquid chromatography-tandem mass spectrometry. Enzyme activity was measured in hepatic cytosol using 1-chloro-2,4-dinitrobenzene, 1,2-dichloro-4-nitrobenzene, and 2,6-dichloroindophenol as substrates. In comparison with 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. SIGNIFICANCE STATEMENT: Treatment with the investigational drug, dichloroacetate (DCA), results in loss of glutathione transferase zeta 1 (GSTZ1) and subsequent increases in body burden of the electrophilic tyrosine metabolites, maleylacetoacetate and maleylacetone. Loss of GSTZ1 in genetically modified mice is associated with induction of glutathione transferases and alteration of the ratio of oxidized to reduced glutathione. Therefore, we determined whether pharmacological depletion of GSTZ1 through repeat administration of DCA produced similar changes in the liver, which could affect responses to other drugs and toxicants.

Glutathione transferase zeta 1 (GSTZ1), expressed in liver and several extrahepatic tissues, catalyzes dechlorination of dichloroacetate (DCA) to glyoxylate. DCA inactivates GSTZ1, leading to autoinhibition of its metabolism. DCA is an investigational drug for treating several congenital and acquired disorders of mitochondrial energy metabolism, including cancer. The main adverse effect of DCA, reversible peripheral neuropathy, is more common in adults treated long-term than in children, who metabolize DCA more quickly after multiple doses. One dose of DCA to Sprague Dawley rats reduced GSTZ1 expression and activity more in liver than in extrahepatic tissues; however, the effects of multiple doses of DCA that mimic its therapeutic use have not been studied. Here, we examined the expression and activity of GSTZ1 in cytosol and mitochondria of liver, kidney, heart, and brain 24 hours after completion of 8-day oral dosing of 100 mg/kg per day sodium DCA to juvenile and adult Sprague Dawley rats. Activity was measured with DCA and with 1,2-epoxy-3-(4-nitrophenoxy)propane (EPNPP), reported to be a GSTZ1-selective substrate. In DCA-treated rats, liver retained higher expression and activity of GSTZ1 with DCA than other tissues, irrespective of rodent age. DCA-treated juvenile rats retained more GSTZ1 activity with DCA than adults. Consistent with this finding, there was less measurable DCA in tissues of juvenile than adult rats. DCA-treated rats retained activity with EPNPP, despite losing over 98% of GSTZ1 protein. These data provide insight into the differences between children and adults in DCA elimination under a therapeutic regimen and confirm that the liver contributes more to DCA metabolism than other tissues. SIGNIFICANCE STATEMENT: Dichloroacetate (DCA) is one of few drugs exhibiting higher clearance from children than adults, after repeated doses, for reasons that are unclear. We hypothesized that juveniles retain more glutathione transferase zeta 1 (GSTZ1) than adults in tissues after multiple DCA doses and found this was the case for liver and kidney, with rat as a model to assess GSTZ1 protein expression and activity with DCA. Although 1,2-epoxy-3-(4-nitrophenoxy)propane was reported to be a selective GSTZ1 substrate, its activity was not reduced in concert with GSTZ1 protein.

Previous work has shown that hepatic levels of human glutathione transferase zeta 1 (GSTZ1) protein, involved in tyrosine catabolism and responsible for metabolism of the investigational drug dichloroacetate, increase in cytosol after birth before reaching a plateau around age 7. However, the mechanism regulating this change of expression is still unknown, and previous studies showed that GSTZ1 mRNA levels did not correlate with GSTZ1 protein expression. In this study, we addressed the hypothesis that microRNAs (miRNAs) could regulate expression of GSTZ1. We obtained liver samples from donors aged less than 1 year or older than 13 years and isolated total RNA for use in a microarray to identify miRNAs that were downregulated in the livers of adults compared with children. From a total of 2578 human miRNAs tested, 63 miRNAs were more than 2-fold down-regulated in adults, of which miR-376c-3p was predicted to bind to the 3' untranslated region of GSTZ1 mRNA. There was an inverse correlation of miR-376c-3p and GSTZ1 protein expression in the liver samples. Using cell culture, we confirmed that miR-376c-3p could downregulate GSTZ1 protein expression. Our findings suggest that miR-376c-3p prevents production of GSTZ1 through inhibition of translation. These experiments further our understanding of GSTZ1 regulation. Furthermore, our array results provide a database resource for future studies on mechanisms regulating human hepatic developmental expression. SIGNIFICANCE STATEMENT: Hepatic glutathione transferase zeta 1 (GSTZ1) is responsible for metabolism of the tyrosine catabolite maleylacetoacetate as well as the investigational drug dichloroacetate. Through examination of microRNA (miRNA) expression in liver from infants and adults and studies in cells, we showed that expression of GSTZ1 is controlled by miRNA. This finding has application to the dosing regimen of the drug dichloroacetate. The miRNA expression profiles are provided and will prove useful for future studies of drug-metabolizing enzymes in infants and adults.

Biotransformation of dichloroacetate (DCA) to glyoxylate by hepatic glutathione transferase zeta 1 (GSTZ1) is considered the principal determinant of the rate of plasma clearance of the drug. However, several other organismal and subcellular factors are also known to influence DCA metabolism. We utilized a female rat model to study these poorly understood processes. Rats aged 4 weeks (young) and 42 to 52 weeks (adult) were used to model children and adults, respectively. Hepatic chloride concentrations, which influence the rate of GSTZ1 inactivation by DCA, were lower in rat than in human tissues and rats did not show the age dependence previously seen in humans. We found GSTZ1 expression and activity in rat brain, heart, and kidney cell-free homogenates that were age-dependent. GSTZ1 expression in brain was higher in young rats than adult rats, whereas cardiac and renal GSTZ1 expression levels were higher in adult than young rats. GSTZ1 activity with DCA could not be measured accurately in kidney cell-free homogenates due to rapid depletion of glutathione by γ-glutamyl transpeptidase. Following oral administration of DCA, 100 mg/kg, to rats, GSTZ1 expression and activity were reduced in all rat tissues, but chloride concentrations were not affected. Together, these data extend our understanding of factors that determine the in vivo kinetics of DCA.

Dichloroacetate (DCA) has been the focus of research by both environmental toxicologists and biomedical scientists for over 50 years. As a product of water chlorination and a metabolite of certain industrial chemicals, DCA is ubiquitous in our biosphere at low μg/kg body weight daily exposure levels without obvious adverse effects in humans. As an investigational drug for numerous congenital and acquired diseases, DCA is administered orally or parenterally, usually at doses of 10-50mg/kg per day. As a therapeutic, its principal mechanism of action is to inhibit pyruvate dehydrogenase kinase (PDK). In turn, PDK inhibits the key mitochondrial energy homeostat, pyruvate dehydrogenase complex (PDC), by reversible phosphorylation. By blocking PDK, DCA activates PDC and, consequently, the mitochondrial respiratory chain and ATP synthesis. A reversible sensory/motor peripheral neuropathy is the clinically limiting adverse effect of chronic DCA exposure and experimental data implicate the Schwann cell as a toxicological target. It has been postulated that stimulation of PDC and respiratory chain activity by DCA in normally glycolytic Schwann cells causes uncompensated oxidative stress from increased reactive oxygen species production. Additionally, the metabolism of DCA interferes with the catabolism of the amino acids phenylalanine and tyrosine and with heme synthesis, resulting in accumulation of reactive molecules capable of forming adducts with DNA and proteins and also resulting in oxidative stress. Preliminary evidence in rodent models of peripheral neuropathy suggest that DCA-induced neurotoxicity may be mitigated by naturally occurring antioxidants and by a specific class of muscarinic receptor antagonists. These findings generate a number of testable hypotheses regarding the etiology and treatment of DCA peripheral neuropathy.

Dichloroacetate (DCA) has been the focus of research by both environmental toxicologists and biomedical scientists for over 50 years. As a product of water chlorination and a metabolite of certain industrial chemicals, DCA is ubiquitous in our biosphere at low μg/kg body weight daily exposure levels without obvious adverse effects in humans. As an investigational drug for numerous congenital and acquired diseases, DCA is administered orally or parenterally, usually at doses of 10-50mg/kg per day. As a therapeutic, its principal mechanism of action is to inhibit pyruvate dehydrogenase kinase (PDK). In turn, PDK inhibits the key mitochondrial energy homeostat, pyruvate dehydrogenase complex (PDC), by reversible phosphorylation. By blocking PDK, DCA activates PDC and, consequently, the mitochondrial respiratory chain and ATP synthesis. A reversible sensory/motor peripheral neuropathy is the clinically limiting adverse effect of chronic DCA exposure and experimental data implicate the Schwann cell as a toxicological target. It has been postulated that stimulation of PDC and respiratory chain activity by DCA in normally glycolytic Schwann cells causes uncompensated oxidative stress from increased reactive oxygen species production. Additionally, the metabolism of DCA interferes with the catabolism of the amino acids phenylalanine and tyrosine and with heme synthesis, resulting in accumulation of reactive molecules capable of forming adducts with DNA and proteins and also resulting in oxidative stress. Preliminary evidence in rodent models of peripheral neuropathy suggest that DCA-induced neurotoxicity may be mitigated by naturally occurring antioxidants and by a specific class of muscarinic receptor antagonists. These findings generate a number of testable hypotheses regarding the etiology and treatment of DCA peripheral neuropathy.

The transsulfuration pathway (TS) acts in sulfur amino acid metabolism by contributing to the regulation of cellular homocysteine, cysteine production, and the generation of H2S for signaling functions. Regulation of TS pathway kinetics involves stimulation of cystathionine β-synthase (CBS) by S-adenosylmethionine (SAM) and oxidants such as H2O2, and by Michaelis-Menten principles whereby substrate concentrations affect reaction rates. Although pyridoxal phosphate (PLP) serves as coenzyme for both CBS and cystathionine γ-lyase (CSE), CSE exhibits much greater loss of activity than CBS during PLP insufficiency. Thus, cellular and plasma cystathionine concentrations increase in vitamin B6 deficiency mainly due to the bottleneck caused by reduced CSE activity. Because of the increase in cystathionine, the canonical production of cysteine (homocysteine → cystathionine → cysteine) is largely maintained even during vitamin B6 deficiency. Typical whole body transsulfuration flux in humans is 3-7 μmol/h per kg body weight. The in vivo kinetics of H2S production via side reactions of CBS and CSE in humans are unknown but they have been reported for cultured HepG2 cells. In these studies, cells exhibit a pronounced reduction in H2S production capacity and rates of lanthionine and homolanthionine synthesis in deficiency. In humans, plasma concentrations of lanthionine and homolanthionine exhibit little or no mean change due to 4-wk vitamin B6 restriction, nor do they respond to pyridoxine supplementation of subjects in chronically low-vitamin B6 status. Wide individual variation in responses of the H2S biomarkers to such perturbations of human vitamin B6 status suggests that the resulting modulation of H2S production may have physiological consequences in a subset of people. Supported by NIH grant DK072398. This paper refers to data from studies registered at clinicaltrials.gov as NCT01128244 and NCT00877812.

Dichloroacetate (DCA), commonly used to treat metabolic disorders, is under investigation as an anti-cancer therapy due to its ability to reverse the Warburg effect and induce apoptosis in tumor cells. While DCA’s mechanism of action is well-studied, other factors that influence its potential as a cancer treatment have not been thoroughly investigated. Here we show that expression of glutathione transferase zeta 1 (GSTZ1), the enzyme responsible for conversion of DCA to its inactive metabolite, glyoxylate, is downregulated in liver cancer and upregulated in some breast cancers, leading to abnormal expression of the protein. The cellular concentration of chloride, an ion that influences the stability of GSTZ1 in the presence of DCA, was also found to be abnormal in tumors, with consistently higher concentrations in hepatocellular carcinoma than in surrounding non-tumor tissue. Finally, results from experiments employing two- and three-dimensional cultures of HepG2 cells, parental and transduced to express GSTZ1, demonstrate that high levels of GSTZ1 expression confers resistance to the effect of high concentrations of DCA on cell viability. These results may have important clinical implications in determining intratumoral metabolism of DCA and, consequently, appropriate oral dosing.

ObjectiveTo describe clinical, biochemical, and genetic features of participants with mitochondrial diseases (MtDs) enrolled in the North American Mitochondrial Disease Consortium (NAMDC) Registry.MethodsThis cross-sectional, multicenter, retrospective database analysis evaluates the phenotypic and molecular characteristics of participants enrolled in the NAMDC Registry from September 2011 to December 2018. The NAMDC is a network of 17 centers with expertise in MtDs and includes both adult and pediatric specialists.ResultsOne thousand four hundred ten of 1,553 participants had sufficient clinical data for analysis. For this study, we included only participants with molecular genetic diagnoses (n = 666). Age at onset ranged from infancy to adulthood. The most common diagnosis was multisystemic disorder (113 participants), and only a minority of participants were diagnosed with a classical mitochondrial syndrome. The most frequent classical syndromes were Leigh syndrome (97 individuals) and mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (71 individuals). Pathogenic variants in the mitochondrial DNA were more frequently observed (414 participants) than pathogenic nuclear gene variants (252 participants). Pathogenic variants in 65 nuclear genes were identified, with POLG1 and PDHA1 being the most commonly affected. Pathogenic variants in 38 genes were reported only in single participants.ConclusionsThe NAMDC Registry data confirm the high variability of clinical, biochemical, and genetic features of participants with MtDs. This study serves as an important resource for future enhancement of MtD research and clinical care by providing the first comprehensive description of participant with MtD in North America.

Glutathione transferase zeta1 (GSTZ1) catalyzes glutathione (GSH)-dependent dechlorination of dichloroacetate (DCA), an investigational drug with therapeutic potential in metabolic disorders and cancer. GSTZ1 is expressed in both hepatic cytosol and mitochondria. Here, we examined the ontogeny and characterized the properties of human mitochondrial GSTZ1. GSTZ1 expression and activity with DCA were determined in 103 human hepatic mitochondrial samples prepared from livers of donors aged 1 day to 84 years. DNA from each sample was genotyped for three common GSTZ1 functional single nucleotide polymorphisms. Expression of mitochondrial GSTZ1 protein increased in an age-dependent manner to a plateau after age 21 years. Activity with DCA correlated with expression, after taking into account the somewhat higher activity of samples that were homo- or heterozygous for GSTZ1A. In samples from livers with the GSTZ1C variant, apparent enzyme kinetic constants for DCA and GSH were similar for mitochondria and cytosol after correcting for the loss of GSH observed in mitochondrial incubations. In the presence of 38 mM chloride, mitochondrial GSTZ1 exhibited shorter half-lives of inactivation compared with the cytosolic enzyme (P = 0.017). GSTZ1 protein isolated from mitochondria was shown by mass spectrometry to be identical to cytosolic GSTZ1 protein in the covered primary protein sequence. In summary, we report age-related development in the expression and activity of human hepatic mitochondrial GSTZ1 does not have the same pattern as that reported for cytosolic GSTZ1. Some properties of cytosolic and mitochondrial GSTZ1 differed, but these were not related to differences in amino acid sequence or post-translationally modified residues.

Limited understanding of the mechanisms responsible for life-threatening organ and immune failure hampers scientists' ability to design sepsis treatments. Pyruvate dehydrogenase kinase 1 (PDK1) is persistently expressed in immune-tolerant monocytes of septic mice and humans and deactivates mitochondrial pyruvate dehydrogenase complex (PDC), the gate-keeping enzyme for glucose oxidation. Here, we show that targeting PDK with its prototypic inhibitor dichloroacetate (DCA) reactivates PDC; increases mitochondrial oxidative bioenergetics in isolated hepatocytes and splenocytes; promotes vascular, immune, and organ homeostasis; accelerates bacterial clearance; and increases survival. These results indicate that the PDC/PDK axis is a druggable mitochondrial target for promoting immunometabolic and organ homeostasis during sepsis.

Background: Low chronic vitamin B-6 status can occur in a subset of women who use oral contraceptives (OCs) with uncertain metabolic consequences. An insufficiency of cellular pyridoxal 5′-phosphate (PLP), which is the coenzyme form of vitamin B-6, may impair many metabolic processes including one-carbon and tryptophan metabolism. Objective: We investigated the effects of vitamin B-6 supplementation on the in vivo kinetics of one-carbon metabolism and the concentration of one-carbon and tryptophan metabolites in vitamin B-6–deficient OC users. Design: A primed, constant infusion of [13C5]methionine, [3-13C]serine, and [2H3]leucine was performed on 10 OC users (20–40 y old; plasma PLP concentrations

We recently reported that, in a concentration-dependent manner, chloride protects hepatic glutathione transferase zeta 1 from inactivation by dichloroacetate, an investigational drug used in treating various acquired and congenital metabolic diseases. Despite the importance of chloride ions in normal physiology, and decades of study of chloride transport across membranes, the literature lacks information on chloride concentrations in animal tissues other than blood. In this study we measured chloride concentrations in human liver samples from male and female donors aged 1 day to 84 years (n = 97). Because glutathione transferase zeta 1 is present in cytosol and, to a lesser extent, in mitochondria, we measured chloride in these fractions by high-performance liquid chromatography analysis following conversion of the free chloride to pentafluorobenzylchloride. We found that chloride concentration decreased with age in hepatic cytosol but increased in liver mitochondria. In addition, chloride concentrations in cytosol, (105.2 ± 62.4 mM; range: 24.7–365.7 mM) were strikingly higher than those in mitochondria (4.2 ± 3.8 mM; range 0.9–22.2 mM). These results suggest a possible explanation for clinical observations seen in patients treated with dichloroacetate, whereby children metabolize the drug more rapidly than adults following repeated doses, and also provide information that may influence our understanding of normal liver physiology.

We developed an Observer-Reported Outcome (ObsRO) survey instrument to be applied in a multicenter, placebo-controlled, crossover randomized controlled trial of dichloroacetate in children with pyruvate dehydrogenase complex deficiency. The instrument quantifies a subject’s at-home level of functionality, as reported by a parent/caregiver, who were instrumental in providing the clinical descriptors and domains that formed the instrument’s content. Feasibility testing of the ObsRO tool showed it to be easy to use and comprehensive in capturing the major clinical functional limitations of affected children and requires less than five minutes for a parent/caregiver to complete daily.

The purpose of this statement is to provide consensus-based recommendations for optimal management and care for patients with primary mitochondrial disease. This statement is intended for physicians who are engaged in the diagnosis and management of these patients. Working group members were appointed by the Mitochondrial Medicine Society. The panel included members with several different areas of expertise. The panel members utilized surveys and the Delphi method to reach consensus. We anticipate that this statement will need to be updated as the field continues to evolve. Consensus-based recommendations are provided for the routine care and management of patients with primary genetic mitochondrial disease.

We reviewed the anti-cancer effects of DCA, an orphan drug long used as an investigational treatment for various acquired and congenital disorders of mitochondrial intermediary metabolism. Inhibition by DCA of mitochondrial pyruvate dehydrogenase kinases and subsequent reactivation of the pyruvate dehydrogenase complex and oxidative phosphorylation is the common mechanism accounting for the drug's anti-neoplastic effects. At least two fundamental changes in tumor metabolism are induced by DCA that antagonize tumor growth, metastases and survival: the first is the redirection of glucose metabolism from glycolysis to oxidation (reversal of the Warburg effect), leading to inhibition of proliferation and induction of caspase-mediated apoptosis. These effects have been replicated in both human cancer cell lines and in tumor implants of diverse germ line origin. The second fundamental change is the oxidative removal of lactate, via pyruvate, and the co-incident buffering of hydrogen ions by dehydrogenases located in the mitochondrial matrix. Preclinical studies demonstrate that DCA has additive or synergistic effects when used in combination with standard agents designed to modify tumor oxidative stress, vascular remodeling, DNA integrity or immunity. These findings and limited clinical results suggest that potentially fruitful areas for additional clinical trials include 1) adult and pediatric high grade astrocytomas; 2) BRAF-mutant cancers, such as melanoma, perhaps combined with other pro-oxidants; 3) tumors in which resistance to standard platinum-class drugs alone may be overcome with combination therapy; and 4) tumors of endodermal origin, in which extensive experimental research has demonstrated significant anti-proliferative, pro-apoptotic effects of DCA, leading to improved host survival.

Dichloroacetate (DCA) has several therapeutic applications based on its pharmacological property of inhibiting pyruvate dehydrogenase kinase. DCA has been used to treat inherited mitochondrial disorders that result in lactic acidosis, as well as pulmonary hypertension and several different solid tumors, the latter through its ability to reverse the Warburg effect in cancer cells and restore aerobic glycolysis. The main clinically limiting toxicity is reversible peripheral neuropathy. Although administration of high doses to rodents can result in liver cancer, there is no evidence that DCA is a human carcinogen. In all studied species, including humans, DCA has the interesting property of inhibiting its own metabolism upon repeat dosing, resulting in alteration of its pharmacokinetics. The first step in DCA metabolism is conversion to glyoxylate catalyzed by glutathione transferase zeta 1 (GSTZ1), for which DCA is a mechanism-based inactivator. The rate of GSTZ1 inactivation by DCA is influenced by age, GSTZ1 haplotype and cellular concentrations of chloride. The effect of DCA on its own metabolism complicates the selection of an effective dose with minimal side effects.

The mitochondrial pyruvate dehydrogenase complex (PDC) irreversibly decarboxylates pyruvate to acetyl coenzyme A, thereby linking glycolysis to the tricarboxylic acid cycle and defining a critical step in cellular bioenergetics. Inhibition of PDC activity by pyruvate dehydrogenase kinase (PDK)-mediated phosphorylation has been associated with the pathobiology of many disorders of metabolic integration, including cancer. Consequently, the PDC/PDK axis has long been a therapeutic target. The most common underlying mechanism accounting for PDC inhibition in these conditions is post-transcriptional upregulation of one or more PDK isoforms, leading to phosphorylation of the E1α subunit of PDC. Such perturbations of the PDC/PDK axis induce a "glycolytic shift," whereby affected cells favor adenosine triphosphate production by glycolysis over mitochondrial oxidative phosphorylation and cellular proliferation over cellular quiescence. Dichloroacetate is the prototypic xenobiotic inhibitor of PDK, thereby maintaining PDC in its unphosphorylated, catalytically active form. However, recent interest in the therapeutic targeting of the PDC/PDK axis for the treatment of cancer has yielded a new generation of small molecule PDK inhibitors. Ongoing investigations of the central role of PDC in cellular energy metabolism and its regulation by pharmacological effectors of PDKs promise to open multiple exciting vistas into the biochemical understanding and treatment of cancer and other diseases.

The investigational drug dichloroacetate (DCA) is a metabolic regulator that has been successfully used to treat acquired and congenital metabolic diseases and, recently, solid tumors. Its clinical use has revealed challenges in selecting appropriate doses. Chronic administration of DCA leads to inhibition of DCA metabolism and potential accumulation to levels that result in side effects. This is because conversion of DCA to glyoxylate is catalyzed by one enzyme, glutathione transferase zeta 1 (GSTZ1-1), which is inactivated by DCA. SNPs in the GSTZ1 gene result in expression of polymorphic variants of the enzyme that differ in activity and rates of inactivation by DCA under physiological conditions: these properties lead to considerable variation between people in the pharmacokinetics of DCA.

BACKGROUND/OBJECTIVES The zeta-1 family isoform of GST biotransforms the investigational drug dichloroacetate (DCA) and certain other halogenated carboxylic acids. Haplotype variability in GSTZ1 influences the kinetics and, possibly, the toxicity of DCA. DCA metabolism correlates with expression of the GSTZ1 protein, so it is important to document variables that affect expression. Following up on a limited previous study, we tested the hypothesis that a coding single nucleotide polymorphism (SNP), the lysine (K) amino acid (E32>K) in GSTZ1 haplotypes linked to a promoter region SNP results in lower hepatic expression of GSTZ1. MATERIALS AND METHODS The influence of K carrier and non-K carrier haplotypes on GSTZ1 expression was determined by analyzing 78 liver samples from individuals aged 7-84 years of various racial and ethnic backgrounds. GSTZ1 expression data were analyzed on the basis of the presence or absence of lysine 32. RESULTS GSTZ1 protein expression differed significantly between K carrier and non-K carrier haplotypes (P=0.001) in Whites, but not in African-Americans (P=0.277). We attribute this difference in GSTZ1 expression among K carrier haplotypes in Whites to the linkage disequilibrium between the K or A allele from the G>A SNP (rs7975), within the promoter G>A-1002 SNP (rs7160195) A allele. There is no linkage disequilibrium between these two polymorphisms in African-Americans. CONCLUSION We conclude that the lower expression of GSTZ1 in Whites who possess the K carrier haplotype results in lower enzymatic activity and slower metabolism of DCA, compared with those who possess the non-K carrier haplotype. These results further define safe, genetics-based dosing regimens for chronic DCA administration.

Background Chloral hydrate (CH), a sedative and metabolite of the environmental contaminant trichloroethylene, is metabolized to trichloroacetic acid, trichloroethanol, and possibly dichloroacetate (DCA). DCA is further metabolized by glutathione transferase zeta 1 (GSTZ1), which is identical to maleylacetoacetate isomerase (MAAI), the penultimate enzyme in tyrosine catabolism. DCA inhibits its own metabolism through depletion/inactivation of GSTZ1/MAAI with repeated exposure, resulting in lower plasma clearance of the drug and the accumulation of the urinary biomarker maleylacetone (MA), a metabolite of tyrosine. It is unknown if GSTZ1/MAAI may participate in the metabolism of CH or any of its metabolites and, therefore, affect tyrosine catabolism. Stable isotopes were utilized to determine the biotransformation of CH, the kinetics of its major metabolites, and the influence, if any, of GSTZ1/MAAI. Methods Eight healthy volunteers (ages 21-40 years) received a dose of 1 g of CH (clinical dose) or 1.5 μg/kg (environmental) for five consecutive days. Plasma and urinary samples were analyzed by gas chromatography-mass spectrometry. Results Plasma DCA (1.2-2.4 μg/mL), metabolized from CH, was measured on the fifth day of the 1 g/day CH dosage but was undetectable in plasma at environmentally relevant doses. Pharmacokinetic measurements from CH metabolites did not differ between slow and fast GSTZ1 haplotypes. Urinary MA levels increased from undetectable to 0.2-0.7 μg/g creatinine with repeated CH clinical dose exposure. Kinetic modeling of a clinical dose of 25 mg/kg DCA administered after 5 days of 1 g/day CH closely resembled DCA kinetics obtained in previously naive individuals. Conclusions These data indicate that the amount of DCA produced from clinically relevant doses of CH, although insufficient to alter DCA kinetics, is sufficient to inhibit MAAI and tyrosine catabolism, as evidenced by the accumulation of urinary MA.

Dichloroacetate (DCA), a small molecule commonly used to treat metabolic disorders, is under investigation as an anti-cancer therapy due to its ability to reverse the Warburg effect and induce apoptosis. While DCA's mechanism of action is well-studied, other factors that influence its success as a cancer treatment have not been thoroughly studied. Here we present data showing that expression of glutathione transferase zeta 1 (GSTZ1), the enzyme responsible for the metabolism of DCA, is often misregulated in cancer, resulting in expression in tissues where there generally is none and a lack of expression in sites that have a high level of basal expression. Expression of GSTZ1 in a tumor would be expected to reduce intratumoral DCA concentrations. As we see a disconnect between GSTZ1 mRNA and protein expression in these tumors, control of expression is likely through miRNA. We also report that chloride concentrations, which strongly impact the stability of GSTZ1 in the presence of DCA, may be abnormal in tumors, further influencing DCA metabolism, concentration, and treatment success. Both two- and three-dimensional cell culture of cells expressing or not expressing GSTZ1 provide a proof of principle that GSTZ1 expression confers a level of resistance to DCA. These results have important clinical implications as GSTZ1 expression and chloride concentration could be used as markers to predict outcomes of DCA anticancer therapy. Citation Format: Stephan C. Jahn, Mohamed H. Solaymen, Ryan J. Lorenzo, Taimour Langaee, Peter W. Stacpoole, Margaret O. James. Chemoresistance through the intratumoral metabolism of dichloroacetate. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr A62.