Dichloroacetate-induced peripheral neuropathy

CHAPTER EIGHT

Dichloroacetate-induced peripheral neuropathy Peter W. Stacpoolea,b,*, Christopher J. Martyniukc, Margaret O. Jamesd, Nigel A. Calcutte a

Department of Medicine, College of Medicine, University of Florida, Gainesville, FL, United States Department of Biochemistry and Molecular Biology, College of Medicine, University of Florida, Gainesville, FL, United States c Department of Physiological Sciences, Center for Environmental and Human Toxicology, College of Veterinary Medicine, University of Florida, Gainesville, FL, United States d Department of Medicinal Chemistry, College of Pharmacy, University of Florida, Gainesville, FL, United States e Department of Pathology, University of California San Diego, La Jolla, CA, United States *Corresponding author: e-mail address: [email protected] b

Contents 1. 2. 3. 4.

Introduction Dichloroacetate pharmacokinetics and metabolism Dichloroacetate pharmacodynamics and clinical utility Dichloroacetate toxicity and neuropathy 4.1 Environmental toxin 4.2 Drug-induced peripheral neuropathy 4.3 Animal models of DCA-induced neuropathy 4.4 Mechanisms of dichloroacetate-induced neuropathy 5. Future avenues of potential research 6. Summary References Further reading

212 213 214 218 218 219 220 224 228 232 233 238

Abstract 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 10–50 mg/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

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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.

1. Introduction The xenobiotic dichloroacetate (DCA) holds an almost unique position at the interface between environmental science and allopathic medicine, being viewed as both a population health hazard and as a drug of intriguing clinical potential. DCA is a minor metabolite of certain halogenated chemicals designated significant health hazards by the Superfund Program (i.e., toxic chemicals found at specific hazardous waste sites in the USA; IARC Monograph #106, 2014), such as trichloroethylene. However, its major environmental source is as a by-product of water chlorination and, therefore, is consumed by most Americans at a dose of approximately 1–4 μg/kg/day. The ubiquity of DCA in our biosphere is further emphasized by its detection in fog and rain (Stacpoole, 2011). The developmental history of DCA as a medicinal agent is more circuitous (Stacpoole, 1989). In the 1950s, the compound diisopropylammonium dichloroacetate (DIPA) was used in the synthesis of methylated derivatives of a purportedly naturally occurring B vitamin (pangamic acid; dgluconodimethylaminoacetate). Anecdotal clinical reports appeared claiming efficacy in various metabolic and cardiovascular disorders from pharmaceutical mixtures of pangamic acid and DIPA. In 1970, DCA was identified as the metabolically active moiety of DIPA (Stacpoole & Felts, 1970) and it has been used thereafter almost exclusively as the sodium salt. DCA has been used to treat a variety of diseases (see below) but remains an investigational drug that is, thus far, not FDA approved to treat any condition.

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2. Dichloroacetate pharmacokinetics and metabolism DCA is the only drug in clinical use metabolized by the zeta1 family isoform of glutathione transferase (GSTZ1) in a reaction that requires, but does not consume, glutathione ( James et al., 2017). GSTZ1 dehalogenates DCA to the naturally occurring molecule glyoxylate. Thereafter, glyoxylate undergoes several biotransformation reactions that yield oxalate, CO2 and various urinary glycine conjugates as end products. Trace amounts of monochloroacetate are also formed in blood by an unknown monodehalogenation mechanism ( James et al., 2017). GSTZ protein is expressed primarily in liver cytosol, although much lower expression is present in hepatic mitochondria and in several other tissues. Cytoplasmic and mitochondrial GSTZ1 expression is virtually absent in human fetal liver and increases postnatally to plateau in early adulthood (Li, Gu, James, et al., 2012; Zhong, James, Smeltz, et al., 2018). DCA is a mechanism-based (suicide) inhibitor of GSTZ1. Thus, repeated dosing results in marked suppression of enzyme protein and activity, which leads to a significant decrease in plasma half-life and an increase in plasma DCA levels. However, with chronic administration, plasma DCA trough (dosing interval) levels do not rise continuously, but achieve a new, stable, plateau (Abdelmalak, Lew, & Ramezani, et al., 2013). This phenomenon, presumably, reflects a new equilibrium between GSTZ1 inactivation and new enzyme synthesis. Hepatic GSTZ1 is also known as maleylacetoacetate isomerase (MAAI) and catalyzes the penultimate step in the catabolism of the amino acids phenylalanine and tyrosine (Fig. 1) ( James et al., 2017). Inhibition of GSTZ1/MAAI by DCA leads to accumulation of the products maleylacetoacetate (MAA) and maleylacetone (MA), the latter of which increases in the urine of subjects chronically treated with DCA. In addition, DCA doses employed clinically increase the urinary level of succinylacetone, leading to urinary accumulation of the heme precursor delta-aminolevulinate (δ-ALA) (Shroads, Langaee, Coats, et al., 2012). MAA, MA and δ-ALA are reactive molecules capable of forming adducts with proteins and promoting oxidative stress ( James & Stacpoole, 2016). Urinary MA and δ-ALA levels have been monitored in clinical trials of DCA and have been shown to increase (Stacpoole, Kerr, Barnes, et al., 2006), but their toxicological potential in DCA-exposed subjects is unknown.

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Fig. 1 Pathway of phenylalanine and tyrosine catabolism. Maleylacetoacetate, maleylacetone and DCA are substrates for maleylacetoacetate isomerase/glutathione transferase ζ 1 (MAAI; GSTZ1).

3. Dichloroacetate pharmacodynamics and clinical utility The sites and mechanisms of action of DCA continue to be actively investigated. Currently, the most well established dynamic actions are on carbohydrate and lipid metabolism and on cellular bioenergetics (Table 1). DCA is a noncompetitive inhibitor of HMG-CoA-reductase, the ratelimiting enzyme of cholesterogenesis. By an unknown mechanism, DCA also inhibits hepatic triglyceride synthesis (Stacpoole, Harwood, & Varmado, 1983). These actions account for the lipid and lipoprotein-lowering effect of DCA in patients with diabetes mellitus (Stacpoole et al., 1978) and the rare condition homozygous familial hypercholesterolemia (Moore et al., 1979),

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Table 1 Pharmacological properties of dichloroacetate. Property Mechanism Ref.

Increased OXPHOS and bioenergetics

# PDK " PDC

Stacpoole (1989); Whitehouse and Randle (1973)

Decreased blood glucose in fasting or diabetes

# PDK " PDC

Stacpoole, Moore, and Kornhauser (1978)

Decreased blood and CSF lactate

# PDK " PDC

Stacpoole, Nagaraja, and Hutson (2003)

Decreased blood total and LDL cholesterol

# HMG CoA reductase ! # synthesis

Harwood, Bridge, and Stacpoole (1987); Moore, Swift, et al. (1979)

Decreased blood triglycerides # Hepatic TG and VLDL cholesterol synthesis ! # VLDL synthesis

Stacpoole et al. (1978)

Reversal of Warburg effect in # PDK " PDC cancer, PAH and other proliferative conditions

Kankotia and Stacpoole (2014)

CSF, cerebrospinal fluid; HMG CoA, hydroxymethylglutaryl coenzyme A; LDL, low-density lipoprotein; OXPHOS, oxidative phosphorylation; PAH, pulmonary arterial hypertension; PDC, pyruvate dehydrogenase complex; PDK, pyruvate dehydrogenase kinase; TG, triglyceride; VLDL, very low-density lipoprotein. From James, M. O., & Stacpoole, P. W. (2016). Pharmacogenetic considerations with dichloroacetate dosing. Pharmacogenetics, 17, 743–753.

for which it has received FDA Orphan Product designation. However, the most widely studied site and mechanism of action, which has potentially the most far-reaching therapeutic applications, is by regulating the mitochondrial pyruvate dehydrogenase complex (PDC)/pyruvate dehydrogenase kinase (PDK) axis. PDC is a gatekeeper enzyme that controls the aerobic oxidation of glucose. It links glucose-derived pyruvate in the cytoplasm to the mitochondrial tricarboxylic acid (TCA) cycle by irreversibly decarboxylating pyruvate to acetyl CoA (Fig. 2). Consequently, it facilitates the mitochondrial oxidation of pyruvate and molecules, such as lactate and alanine, in equilibrium with pyruvate. As a result of its action on PDC, the drug accelerates the oxidative removal of lactate, rendering DCA as the most potent lactate-lowering agent in clinical use (Table 2). A major mechanism regulating the activity of PDC is reversible phosphorylation. Four human isoforms of PDK (PDK 1–4) phosphorylate and render PDC inactive, while two human isoforms of pyruvate dehydrogenase

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Fig. 2 Role of the pyruvate dehydrogenase complex in intermediary metabolism and site of action of DCA. PDC-PO4 is the inactive phosphorylated form of pyruvate dehydrogenase complex; PDC is the active unphosphorylated form. PDK is pyruvate dehydrogenase kinase; PDP is pyruvate dehydrogenase phosphatase; e- represents transfer of an electron. From James, M. O., Jahn, S. C., Zhong, G., Smeltz, M. G., Hu, Z., & Stacpoole, P. W. (2017). Therapeutic applications of dichloroacetate and the role of glutathionine transferase zeta 1. Pharmacology & Therapeutics, 170, 166–180.

phosphatase (PDP 1 and 2) restore enzyme activity. DCA is the prototypic inhibitor of PDKs, thereby maintaining PDC in its unphosphorylated, catalytically active form ( James et al., 2017; Stacpoole, 1989) and stimulating a rapidly growing pharmaceutical effort in generating new PDK inhibitors (Stacpoole, 2017). Oral and intravenous doses of the drug in humans typically range from 10 to 50 mg/kg/day. PDC is a critical homeostat for sustaining adequate energy stores through oxidative phosphorylation (OXPHOS). Loss of function mutations in the complex give rise to congenital PDC deficiency, a devastating condition associated with lactic acidosis and progressive neurological and neuromuscular degeneration (Patel, O’Brien, Subramony, et al., 2012). A multicenter, placebo-controlled randomized controlled trial is in progress to test the ability of chronic, oral DCA to decrease morbidity in this disorder (Stacpoole, Shuster, Thompson, et al., 2018).

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Table 2 Conditions in which the lactate-lowering effect of DCA has been demonstrated. Condition Animals Humans

Congenital lactic acidosis

+

+

Malaria

+

+

Diabetes mellitus

+

+

Hypoxia

+

+

Hypotension

+

+

Heart failure

+

+

Endotoxemia

+

+

Bacterial infection

+

Cancer

+

+

Liver failure

+

+

Renal failure

+

Stroke

+

Catecholamine excess

+

Exercise

+

+

Metabolic flexibility may be defined as the ability of a cell to switch between metabolic pathways required to maintain normal energy status and physiological functions. As general rules, carbohydrate metabolism is the predominant cellular resource for bioenergy and biomass. Quiescent (fully differentiated) cells are oxidative, generating the energy (ATP) required to sustain normal cellular functions through OXPHOS (Stacpoole et al., 2006). In contrast, proliferating cells rely proportionately more on glycolysis for both energetic and biomass needs. A hallmark of chronic tissue injury (e.g., inflammation and fibrosis) is hypoxia-driven neoplasia and a loss of metabolic flexibility. Such diseases are often manifested at the level of the PDC/PDK axis, whereby pathological upregulation of one or more PDKs results in inhibition of PDC, lactate accumulation and depressed OXPHOS. Coincidently, cells adopt an increased rate of glycolysis, despite adequate tissue oxygenation, a phenomenon known as aerobic glycolysis, or the Warburg effect (Kankotia & Stacpoole, 2014). In preclinical models of pulmonary arterial hypertension (PAH), DCA stimulates aerobic glucose oxidation and apoptosis of the proliferating

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vascular endothelial and pulmonary smooth muscle cells, resulting in decreased right heart strain and increased survival. An open label trial of oral DCA in adults with idiopathic PAH showed treatment was associated with improvement in cardiac function and physical endurance (Michelakis, Gurtu, Webster, et al., 2017). DCA also reverses Warburg metabolism in a diverse assortment of human xenografts of tumor-bearing animals, resulting in decreased tumor burden and increased survival (Kankotia & Stacpoole, 2014; Stacpoole, 2017). Several small, phase I trials of chronic, oral DCA in adults with recurrent brain or other solid tumors have been published (Stacpoole, 2017). Beneficial effects on cardiac function have been reported from short term DCA administration in patients with coronary artery disease or severe heart failure, in keeping with preclinical studies in which DCA increased cardiac PDC, glucose oxidation and mechanical function in ischemic hearts (Bersin & Stacpoole, 1997) or in hearts from rats following hemorrhagic shock (Subramani, Lu, Warren, et al., 2017). Coronary stent failure is also a Warburgmediated process involving luminal narrowing from glycolysis-driven coronary vascular endothelial cell proliferation that is reversed by genetic or DCA knockdown of vascular PDK (Deuse, Hua, Wang, et al., 2014). Most recently, a single intraperitoneal DCA dose of 25 mg/kg was found to reverse the immunometabolic paralysis of circulating immune cells and vital end organs in a murine model of severe sepsis, also by inhibiting PDK expression and PDC phosphorylation, and resulting in a marked increase in host survival (McCall, Zabalawic, Liu, et al., 2018). Severe sepsis is the most common cause of in-hospital mortality in the developed world (Swiger, Deutschmano, Seymour, et al., 2016) and lacks any FDA-approved mechanism-based therapies, thus relying on general supportive care with antibiotics, fluids and vasopressor drugs. In summary, the pathobiology of many acquired diseases encompasses pathological perturbation of the PDC/PDK axis, leading to impairment of mitochondrial OXPHOS and ATP generation. Although still an investigational drug, much current translational and clinical research suggests a wide therapeutic window exists for DCA, provided it can be administered with adequate tolerability and safety.

4. Dichloroacetate toxicity and neuropathy 4.1 Environmental toxin As noted above, DCA is one of five haloacetic acid contaminants of drinking and swimming pool water that has undergone chlorination. The maximal

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amount of the combined five haloacetic acids allowed in drinking water by the U.S. Environmental Protection Agency (EPA) is 60 μg/L and EPA health guidelines suggest a maximal DCA concentration of 0.7 μg/L (0.7 parts per billion)—a level identified as producing a one in a million lifetime risk of cancer (IARC Monograph #106, 2014). DCA levels in tap water can vary dramatically among water suppliers, with concentration ranges of 3–18 μg/L reported in England (Zhang, Collins, & Graham, 2010), 1–45 μg/L in Australia (Simpson and Hayes, 1998), 10–13μg/L in Beijing, China (Wei et al., 2010) and approaching as high as 100 μg/L in certain water districts in the US (EWG Tap Water database, 2015). There are no epidemiological reports of DCA–induced cancer in humans due to exposure via drinking water. Concern for potential carcinogenic effects of DCA comes from preclinical toxicity studies in rodents and dogs, where liver enlargement, glycogen accumulation, adenomas and carcinomas are described (EPA report, 2003). However, it should be noted that toxicity studies in rodents and dogs have used bolus or repeat doses of DCA that exceed levels in tap water by orders of magnitude (0.5–5.0 g/L: reviewed in IARC Monograph #106, 2014) and DCA is not listed as a carcinogen by the US EPA or the European Union. While DCA-induced carcinogenicity is unlikely to arise from environmental exposure, the possibility that low level DCA exposure disrupts cellular metabolic processes to hasten onset or increase severity of pathological processes arising from other metabolic disorders has not been discounted.

4.2 Drug-induced peripheral neuropathy The growing number of clinical studies using DCA as an investigational drug to lower lactate levels (Table 2) and treat diverse clinical conditions has caused patients to be exposed to high doses of DCA that may be repeated frequently over periods of weeks to years. For example, exploratory studies used 50 mg/kg/day DCA for 4 months to treat hypercholesterolemia (Moore et al., 1979), 12.5 mg/kg twice daily for 6 months to treat congenital lactic acidosis (Stacpoole et al., 2006) and a dose escalation from 12.5 to 25 mg/kg twice daily for 15 months in patients with glioblastoma (Michelakis et al., 2010). While there was no evidence of hematologic, hepatic, renal or cardiac toxicity associated with long term DCA use in the later study, a dose de-escalation program was required, due to the onset of undefined peripheral neuropathy that resolved following dose stabilization at 6.25 mg/kg twice daily. Distal symmetrical peripheral neuropathy is widely reported as a side effect of DCA treatment. The initial report of a subject exposed to

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50 mg/kg/day DCA described tingling in the hands and feet, decreased deep tendon reflexes and loss of muscle strength that was most prominent in distal extremities and accompanied by distal muscle denervation and large fiber conduction slowing in posterior tibial nerves, with complete loss of responses in the peroneal and sural nerves (Moore et al., 1979). Similar indices of peripheral neuropathy were reported in other case reports and small studies (Abdelmalak et al., 2013; Brandsma et al., 2010; Chu et al., 2015; Kurlemann et al., 1995; Saitoh et al., 1998). A larger study of children and young adults with diverse mitochondrial diseases and initially treated with 25 mg/kg DCA twice daily for 12 months reported electrophysiological indices of sensory and motor neuropathy that were particularly notable in the distal motor nerves of older subjects (Spruijt et al., 2001), while a study of 30 adult subjects given 25 mg/kg/day DCA to treat patients with mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes (MELAS) was discontinued due to onset of more severe indices of peripheral neuropathy, including distal limb paresthesias, numbness and pain, accompanied by large fiber conduction slowing and diminished action potential amplitude (Kaufmann et al., 2006). In all studies, peripheral neuropathy generally resolved over 6–18 months following dose de-escalation or discontinuation, although in some cases it persisted. DCA therefore appears to induce a dose-dependent, largely reversible, distal peripheral neuropathy that afflicts large and small fibers of motor and sensory nerves and causes both sensory loss and pain. This places it alongside a variety of drugs, such as the chemotherapeutics paclitaxel, platins, vinka alkaloids and bortezomib, and antiretrovirals, in which optimal therapeutic drug dose and duration of treatment can be limited by peripheral neuropathy ( Jones et al., 2019). Understanding the pathogenesis of DCA-induced neuropathy may identify sites for intervention and facilitate development of adjuvants that allow optimal therapeutic doses of DCA by preventing the unwanted side effect of peripheral neuropathy.

4.3 Animal models of DCA-induced neuropathy 4.3.1 CNS pathology Neuropathy associated with DCA exposure has been modeled in laboratory animals. DCA given to dogs (50–100 mg/kg/day) or rats (125–2000 mg/kg/ day) caused dose-dependent limb paralysis and white matter damage in the spinal cord and brain (Katz et al., 1981). It should be noted that these doses are in excess of those used in humans, allowing for an allosteric conversion factor of 6 in rats and 2 in dogs (Freireich et al., 1966). In contrast, daily oral

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gavage of DCA at doses ranging from simple replication of the upper human clinical dose to above the rat-equivalent dose (50-500 mg/kg/day) did not induce locomotor dysfunction and were without discernable pathological effect on white or gray matter after 4 months of treatment, although there was a loss of the spinal H reflex at the highest dose (Calcutt et al., 2009). Thus, while DCA readily penetrates the CNS and impacts brain glucose metabolism (Abemayor, Kovachich, & Haugaard, 1984; Kuroda et al., 1984; Itoh et al., 2003) it does not cause overt degeneration of neurons or glia within the dose range commonly used in humans. 4.3.2 Axonal pathology As described above, peripheral neuropathy in humans treated with DCA presents with both negative (numbness) and positive (tingling/pain) symptoms accompanied by reduced large fiber action potential amplitude and velocity in distal sensory and motor nerves. To our knowledge there are no reports of whether these functional disorders are accompanied by nerve structural pathology in any fiber type, although electromyographical findings have been interpreted as reflecting a predominantly distal axonopathy. In rats, an initial report of reduced tibial nerve diameter (Yount et al., 1982) was extended by detailed functional and morphometric analysis following 4 months of daily dosing with 50–500 mg/kg DCA. DCA caused dose and duration dependent sciatic nerve MNCV and SNCV slowing in juvenile rats that was accompanied at study end by dose dependent reductions in axonal caliber of myelinated fibers in the sciatic, sural and tibial nerves in the absence of overt nerve fiber loss or myelin pathology (Calcutt et al., 2009). Adult rats developed earlier MNCV and SNCV slowing following DCA treatment that was also accompanied by reduced mean axonal diameter attributable to a shift toward smaller fibers and not either fiber loss or reduced myelin thickness/g ratio. Thus, rats exhibit functional and structural indices of early large fiber sensorimotor axonopathy that may mirror the human condition. 4.3.3 Pain The manifestations of neuropathy most likely to provoke DCA dose reduction or discontinuation include weakness, numbness and tingling/pain. Evaluating chronic spontaneous pain in laboratory animals currently relies on measuring modifications to normal behavior patterns (reviewed in Tappe-Theodor and Kuner, 2014) and has not yet been applied to DCAinduced neuropathy. There is an early description of touch-induced

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vocalization in two rats treated with 2000 mg/kg/day DCA prior to death (Katz et al., 1981). This effect was not evident in rats given sub-lethal amounts of DCA; both young and adult rats treated with 50–500 mg/kg/day DCA for 8–16 weeks displayed allodynia (withdrawal response to a normally non-painful stimulus), when tested with von Frey filaments that apply graded light touch pressure to the plantar surface of the hind paw (Calcutt et al., 2009). This test has been equated to pain evoked by wearing of socks or the touch of bed sheets at night in patients with a variety of peripheral neuropathies. Pain-associated behavior can be equated to altered neuronal activity in peripheral nerves and/or the spinal cord by measuring the paw flinching response to the painful stimulus of paw formalin injection. In an unpublished study (Fig. 3), we treated adult female Sprague-Dawley rats with 500 mg/kg DCA for 8 weeks before injecting 50 μL of 0.5% formalin into the dorsum of one hind paw. Control rats displayed the widely reported biphasic paw flinching response to formalin injection (WheelerAceto, Porreca, & Cowan, 1990) with an initial period of flinching (phase1) that resolved after 10 min and then re-emerged over the subsequent 50 min (phase 2). DCA-treated rats displayed significantly increased paw flinching frequency during phase 1 and phase 2. This is consistent with other models of neuropathic pain, such as diabetic rats (Malmberg, Yaksh, & Calcutt, 1993). As phase 1 of the formalin test is considered to reflect primary afferent responses to the formalin injection whereas phase 2 reflects ongoing primary afferent input that is amplified at the spinal cord level (Dickenson and Sullivan, 1987), it appears that hyperalgesia in DCA-treated rats may involve enhanced primary afferent activity.

900

Control DCA

25

800

Control

700

DCA

600

20 AUC

Number of flinches/minute

p<0.001 30

15 10

500 400 300 200

5

p<0.02

100

0 0

10

20

30

40

Time (minutes)

50

60

0

1

2 Formalin test phase

Fig. 3 DCA-induced hyperalgesia. Paw formalin-evoked flinching (50 μL, 0.5%) in adult female Sprague-Dawley rats after receiving daily DCA (500 mg/kg) or vehicle by oral gavage. Data are mean  SEM of N ¼ 6–8/group shown as flinching time course (left panel) and AUC for phases 1 and 2 (right panel). Statistical comparison by two tailed unpaired test (Mixcoatl-Zecuatl, Liu, & Calcutt, unpublished data).

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4.3.4 Sensory loss In peripheral neuropathies, such as those caused by chemotherapeutic agents or diabetes, spontaneous or stimulus evoked pain is frequently accompanied by loss of sensation in the same limb. DCA-treated rats show a similar sensory phenotype, with both paw allodynia to light touch and hypoalgesia to heat being present (Calcutt et al., 2009). Loss of sensory functions mediated by small sensory fibers is commonly associated with the loss of epidermal innervation; however, the loss of sensation preceding the loss of these intra-epidermal nerve fibers (IENF) in diabetic mice suggests an initial neurochemical dysfunction (Beiswenger, Calcutt, & Mizisin, 2008). Heat hypoalgesia in rats treated with 500 mg/kg/day DCA for 4 months was not accompanied by loss of IENF and, indeed, IENF values were increased. However, shorter durations of DCA exposure induced both heat hypoalgesia and loss of IENF in paw skin of mice (Calcutt et al., 2017). One plausible explanation for these apparently diverse findings is that DCA-induced heat hypoalgesia results from biochemical and structural disorders of primary afferents, while long term studies also detect regenerative and collateral sprouting. In unpublished studies (Fig. 4), we have also found that adult female Swiss Webster mice given 1000mg/kg/day (equivalent to 500 mg/kg/day in rat) DCA for 4 weeks develop significant loss of small sensory nerves in the cornea, as measured by corneal confocal microscopy (Chen et al., 2013). As corneal nerve loss was restricted to distal portions of nerve in the subbasal nerve plexus and did not extend to the more proximal corneal stroma, 60

p<0.05

Occupancy (%)

50

control DCA

40 30 20 10 0

SBNP

stroma

Fig. 4 DCA-induced sensory nerve loss: Representative image of sensory nerves (white arrows) in the corneal sub-basal nerve plexus captured by corneal confocal microscopy (left panel) and quantification of nerve occupancy in the corneal sub-basal nerve plexus and stroma of control and DCA-treated (1000 mg/kg/day for 4 weeks) adult female Swiss Webster mice. Data are mean  SEM of N ¼ 8–9/group. Statistical comparison by two tailed unpaired test (Frizzi & Calcut, unpublished data).

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these data suggest that DCA induces a distal degenerative neuropathy in small sensory fibers of the mouse. Potential explanations for the apparently divergent impact of DCA on small fiber density in target organs between rat and mouse include inter-species differences in DCA neurotoxicity and differences in the dose and duration of DCA exposure (500 mg/kg/day for 16 weeks in the rat study, 1000 mg/kg/day for 4 or 8 weeks in the mouse studies). To our knowledge, the impact of DCA on sensory nerve density in skin or cornea in human subjects has not been reported.

4.4 Mechanisms of dichloroacetate-induced neuropathy Clinical descriptions of DCA-induced peripheral neuropathy have emphasized that it is more common and severe in adults than children (Kaufmann et al., 2006; Stacpoole et al., 2008). Interestingly, age-related shifts in expression and activity of GSTZ1, the enzyme that biotransforms DCA to glyoxylate (Fig. 1) have been described (Shroads, Guo, Dixit, et al., 2008). In liver, cytosolic GSTZ1 expression (the dominant cellular fraction) reaches adult levels at around 7 years of age, while the mitochondrial form only reaches maximal expression and activity at around 21 years of age (Li et al., 2012; Zhong et al., 2018). It is therefore plausible that the neurotoxicity of DCA is associated with the attainment of maximal mitochondrial GSTZ1 activity and/or maximal ratio of mitochondrial: cytosolic GSTZ1 activity, although this association has not yet been explored. Another possible explanation of the effect of age is that liver to body weight ratios are higher in children than adults, such that DCA clearance is faster, even after knockdown of GSTZ1. This explanation is supported by the finding that DCA clearance is reduced less in children than adults following 6 months DCA treatment (Shroads et al., 2008). Because DCA slows its own clearance by inhibiting GSTZ1 activity and reducing protein levels (Cornett et al., 1999; James et al., 1998; Shroads et al., 2008) repeated exposure to DCA results in accumulation of DCA. There is also accumulation of maleylacetoacetate (MAA), the endogenous substrate of GSTZ1 and of precursors and alternative metabolic products, such as malelyacetone (MA) and succinylacetoacetate, which is decarboxylated to succinylacetone, an inhibitor of a proximal step in heme synthesis (Fig. 1). It is possible that accumulation of MAA, MA and other intermediates of tyrosine metabolism may be directly neurotoxic, due to their capacity for protein modification by adduct formation (Lopachin and Decaprio, 2005). The heme precursor delta-aminolevulinate (δ-ALA) is another potential

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neurotoxin and is associated with neuropathy in patients with acute porphyria (Flugel and Druschky, 1977; Meyer, Schuurmans, & Lindberg, 1998). δ-ALA appears in the urine of DCA-treated subjects (Stacpoole et al., 2006) and produces neuropathy when given directly to mice (Sima et al., 1981) and humans (Sylantiev et al., 2005) or when levels are increased in mice secondary to disruption of δ-ALA clearance mechanisms (Lindberg et al., 1996; 1999). When exposed to myelinating co-cultures of Schwann cells and sensory neurons, δ-ALA reduced expression of multiple myelin proteins and lipids, increased indices of oxidative damage and suppressed expression of mitochondrial respiratory chain proteins by Schwann cells, but not neurons (Felitsyn et al., 2008). Schwann cell toxicity by δ-ALA is consistent with reports of myelin damage in the CNS during high dose DCA toxicity studies (Katz et al., 1981). However, frank demyelination is not a feature of rodent models of DCA-induced neuropathy when given in its therapeutic dose range (Calcutt et al., 2009). The neuropathy phenotype associated with excess δ-ALA tends to be predominantly primary axonal motor and autonomic in nature (Meyer et al. 1998), in contrast to the distal sensorimotor neuropathy observed in patients receiving therapeutic doses of DCA. The therapeutic use of DCA for mitochondrial disorders is based on its role as a PDK inhibitor to enhance flow of glycolysis-derived pyruvate into mitochondrial OXPHOS and thus increase ATP production by surviving mitochondria (Fig. 2). Such mitochondrial overdrive, while effective in improving overall tissue energetic status, also has the potential to overwhelm local mitochondrial antioxidant systems, such as SOD2/MnSOD, that are used to quench superoxide and other ROS products of OXPHOS. This liability may be particularly significant in cells that are not normally expected or equipped to handle excessive OXPHOS, such as Schwann cells. Indeed, direct exposure of myelinating co-cultures of Schwann cells and sensory neurons to DCA resulted in reduced expression of myelin-associated proteins but not axonal structural proteins, emphasizing that Schwann cells may be the primary lesion site in DCA-induced neuropathy (Felitsyn, Stacpoole, & Notterpek, 2007). Schwann cell ATP production from glucose is predominately via glycolysis or the pentose phosphate pathway, rather than by OXPHOS. Unstressed adult myelinating Schwann cells do not require pyruvate-derived acetyl CoA to maintain myelination or axonal structure and function (Della-Flora Nunes et al., 2017), although it should also be noted that ablation of mitochondrial function in Schwann cells leads to axonopathy (Viader et al., 2011). Schwann cells in the PNS also express monocarboxylate transporters (Morrison et al., 2015; Domenech-Estevez

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et al., 2015) and are in a position to supply lactate to neurons as substrate for OXPHOS (Brown et al., 2012). Thus, while adult myelinating Schwann cells provide structural support to their axons and supply axons with glycolysis-derived nutrients, such as pyruvate and lactate, via the monocarboxylate transporter MCT-1 (Lee et al., 2012; Philips and Rothstein, 2017) and potentially neurotrophic factors, such as CNTF, they are metabolically relatively quiescent and exist in a Warburg-like state of reliance on aerobic glycolysis. The impact of DCA on Schwann cell mitochondrial function and ROS generation remains to be determined. As alluded to above, over-activation of OXPHOS by DCA for prolonged periods is but one suspected mechanism by which DCA induces peripheral neuropathy. Some studies have investigated the effect of DCA on mitochondrial bioenergetics, to better address this question in both cancer cells and mutant cell strains. Abildgaard and colleagues (Abildgaard, Dahl, Basse, et al., 2014) examined the drug’s action on the bioenergetics of mutated melanoma cell lines and primary melanocytes. DCA shifted metabolism towards mitochondrial respiration in mutant myeloma cells. Noteworthy was that mutant cells showed varied response to DCA in terms of ATP coupling and the respiratory control ratio (ratio between the maximal mitochondrial oxygen consumption rates (OCR) and the proton leak), suggesting that genetics may influence the degree to which mitochondrial bioenergetics is modified by DCA. In another example, oxygen consumption rates and extracellular acidification (metabolic flux) was measured in RPMI8226, JJN-3, and U266 human myeloma cells in the absence or presence of 5 mM DCA to determine maximal respiratory capacity and aerobic/ anaerobic ratio (Sanchez et al., 2013). DCA shifted metabolism in multiple myeloma cells away from lactate and toward OXPHOS, which was accompanied by increased superoxide production and apoptosis. While DCA at concentrations of 10 mM and 25 mM increased superoxide production in JJN-3 cells and RPMI8226-TGL cells, respectively, they did not generate superoxide in U266 cells, which displays no glucose to lactate conversion and is considered to be a “non-Warburg” acting multiple myeloma cell line. Thus, the effect of DCA on superoxide production in cell lines may depend on the specific metabolic profiles of the cells. In the context of neuropathies, superoxide production and oxidative damage is well documented to occur with many FDA-approved chemotherapeutic drugs in which peripheral neuropathy is often a dose-limiting toxicity. For example, cisplatin is widely used as a chemotherapeutic agent because it interferes with DNA replication and slows dividing cancerous

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cells; however, adverse side effects include nephrotoxicity and neurotoxicity due, in part, to oxidative stress (Yu, Chen, Dubrulle, et al., 2018). Cisplatinrelated toxicity also involves decreased flux from pyruvate to lactate, which may also explain cisplatin sensitivity in some individuals (Yu et al., 2018). The oxidative damage caused by ROS generated from therapeutic doses of DCA may lead to DNA damage, oxidized proteins, and lipid peroxidation in neuronal cells, responses observed in other tissues such as the liver (Austin, Okita, Okita, et al., 1995; Parrish, Austin, Stevens, et al., 1996). In the case of glia, it is plausible that a sustained metabolic shift in peripheral cells from a predominately glycolytic to an oxidative phenotype may create an ROS-enriched environment deleterious to peripheral glia function and survival. Oxidative damage to proteins and lipids in the mitochondria can also lead to mitophagy, or the selective degradation of mitochondria by autophagy; this mechanism has been investigated in vitro with DCA. Using the SH-SY5Y neuroblastoma cell line, Pajuelo-Reguera, Ala´n, Oleja´r, et al. (2015) reported that cells treated with DCA for 16 h decreased the number of viable cells at doses >5 mM. Moreover, there was a dose-dependent decrease in membrane proteins such as Tim23, PINK1, Parkin, further suggesting that autophagy is involved in the degradation of mitochondrial membrane components. The shape of the mitochondria also changed with DCA exposure. The control group tended to have a mitochondrial structure that was elongate, with interconnected filaments. However, as the DCA concentration increased, the mitochondrial network contained shorter and fragmented filaments, and there were clusters of mitochondria filaments within the cell cytosol. Thus, DCA acted to impair mitochondrial integrity in the neuroblastoma cells at higher doses (30–60 mM). Other studies have investigated DCA with a focus on autophagic processes within cancerous cells, suggesting that it has both protective and initiating effects in various cell types ( Jia, Wang, Xia, et al., 2017; Lin, Hill, Andrejeva, et al., 2014; Lu, Zhou, Hou, et al., 2018). When inquiries into the potential role for mitochondrial fusion/fission in DCA-induced neuropathy are made, it becomes clear that there is little evidence to refute or support such a case. This knowledge gap must be addressed; the dynamin proteins regulate this process and little is known about how DCA may affect the expression or function of this important class of mitochondrial structure and function. In a study by Sun, Li, and Xie (2016), the protective effects of DCA following brain injury were investigated in the neonatal mouse brain. Transcripts related to fission/fusion (e.g., mitofusion, optic atrophy 1) were measured,

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and there was evidence that DCA modulated the expression of transcripts controlling this process in the CNS. Noteworthy is that major enzymes that facilitate mitochondrial fission, such as dynamin-related protein 1, have a critical role in hyperalgesia and neuropathic pain (Ferrari, Chum, Bogen, Reichling, & Levine, 2011) and this mechanism may be one that explains DCA-induced neuropathy. Indeed, diabetic neuropathies have a component of altered balance of mitochondrial fission and function (Vincent et al., 2010). Small molecule inhibitors of fission/fusion may prove useful treating DCA-induced neuropathy, given that more traditional antioxidants have been less effective at mitigating neurotoxicity. One aspect of DCA neurotoxicity may also be at the level of axonal transport, a process that requires sufficient ATP. Energy deficits within the cell are associated with loss of protein transport systems within the neuron and axonopathy may ensue. Impaired axonal transport has been reported in both inherited/acquired peripheral neuropathy (Prior, Van Helleputte, Benoy, et al., 2017) as well as in diabetic neuropathy models (Pesaresi, Giatti, Spezzano, et al., 2018). Genetic defects or chemically-induced damage to cytoskeletal proteins and regulators of transport systems, such as deacetylase (HDAC6) in the neuron can lead to axonal damage. While recent studies report on the relationship between the axonal transport system and chemotherapy-induced peripheral neuropathy (Fukuda, Li, & Segal, 2017), such data are lacking for a direct role for DCA in this process. Thus, studies are required to better understand DCA-associated peripheral neuropathy in relation to axonal injury. As peripheral neuropathy related to DCA is reversible, it will become important to identify the events leading up to the injury as well as the repair pathways called upon within the cell to mitigate the damage.

5. Future avenues of potential research Prior studies have demonstrated that DCA causes oxidative damage to lipid membranes in a variety of organs (Larson and Bull, 1992) and that chronic pre-treatment with DCA exaggerates the effect of an acute dose on liver cell membrane lipid peroxidation (Austin et al., 1995). Induction of oxidative damage has also been proposed to contribute to the capacity of DCA to kill cancer cells (Kankotia & Stacpoole, 2014; McCarty, Barroso-Aranda, & Contreras, 2010). We previously demonstrated that systemic DCA treatment causes oxidative damage in rat peripheral nerve that is coincident with functional and structural indices of peripheral neuropathy

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(Calcutt et al., 2009). To test the hypothesis that oxidative stress contributes to DCA-induced neuropathy, we investigated the efficacy of the antioxidant ellagic acid (75 mg/kg/day) on indices of large and small fiber neuropathy in mice treated with 1000 mg/kg/day DCA for 4 weeks. Ellagic acid is a polyphenol that occurs naturally in berries, pomegranates and other fruits and nuts. It has a number of actions on the nervous system, including antioxidant effects via both iron chelation and free radical scavenging (Ahmed et al., 2016). Ellagic acid treatment completely prevented both large fiber MNCV slowing and paw thermal hypoalgesia indicative of small sensory fiber neuropathy (Fig. 5). While we cannot yet ascribe the efficacy of ellagic acid solely to its antioxidant properties, these data encourage further studies investigating the role of DCA-induced oxidative damage in its capacity to cause peripheral neuropathy. They also offer a potential adjuvant therapy to mitigate this unwanted side effect, thereby allowing optimal dosing of DCA when treating primary mitochondrial diseases, cancer and other disorders of metabolic integration. Transcriptomic-based approaches have been utilized to uncover downstream cellular targets and pathways associated with DCA-modulation of OXPHOS in both cancerous and healthy cells. This approach acts to broaden perspectives on the global molecular responses to DCA, independent of whether it is acting in a beneficial or toxic manner. To date, studies on global expression patterns associated with DCA are limited and further work is needed to identify the molecular pathways associated with DCAinduced neuropathy. Bonnet, Archer, Allalunis-Turner, et al. (2007) investigated the effects of DCA on human cultured A549 lung carcinoma and 60 50

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Fig. 5 Ellagic acid prevents DCA-induced neuropathy: MNCV (left panel) and paw thermal response latency (right panel) in adult female Swiss Webster mice treated with vehicle (control) or 1000 mg/kg/day DCA  75 mg/kg/day ellagic acid (EA) for 4 weeks. Data are mean  SEM of N ¼ 9–11/group. Statistical comparisons by one-way ANOVA with Dunnett’s post-hoc test. *** ¼ P < 0.001 vs DCA (Lopez and Calcutt, unpublished data).

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M059K glioblastoma cells. At the gene level, DCA treatment altered cell processes associated with mitochondrial redox status, ion channel function, proliferation and apoptosis. In another study, Galgamuwa, Hardy, Dahlstrom, et al. (2016) investigated whether DCA co-administered with cisplatin would ameliorate or exacerbate cisplatin-induced toxicity in the kidney. To determine if there was evidence for DCA-mediated nephroprotection, whole transcriptome sequencing was conducted in the kidneys of mice treated with saline, DCA, cisplatin or both compounds. Co-treatment with DCA and cisplatin revealed that DCA reversed cisplatin-induced expression of many genes involved in apoptosis, DNA damage response, p53 expression and MAPK signaling pathways. As expected, several of the differentially expressed transcripts were also related to aerobic respiration, ATP synthesis and fatty acid oxidation pathways. Interestingly, DCA’s effect on the transcriptome was greater when co-administered with cisplatin, whereas few transcripts were altered in cells exposed to DCA alone. Overall, the study provides mechanistic evidence that DCA prevented cisplatin-induced ATP depletion by maintaining glucose and fatty acid oxidation pathways, in addition to identifying novel transcript responses to DCA. Based on these limited studies, there is evidence that DCA affects molecular pathways associated with metabolism, OXPHOS and apoptosis in multiple tissues. However, a significant knowledge gap is how DCA is specifically affecting the nervous system at the molecular level. DCA is a PDK inhibitor that shifts metabolic capacity of cells; what is less clear is how cells of the PNS respond to such changes in metabolic activity. For example, do specific cell types evoke a transcriptional response to compensate for energy deficits, up-regulate anti-oxidant responses or activate DNA repair mechanisms to repair DNA from ROS damage? While in vitro studies point to processes that encompass oxidative damage and cellular repair, there are likely additional mechanisms associated with both DCA’s therapeutic and neurotoxic effects. The lack of molecular data on a global scale is a significant obstacle for therapeutic intervention to mitigate or prevent DCA-induced neuropathy. Transcriptome-based studies have determined some of the primary signaling pathways associated with other forms of peripheral neuropathy. For example, Hinder et al. (2017) identified molecular responses in the sciatic nerve and dorsal root ganglion of a diabetic mouse strain to reveal molecular pathways associated with neuropathy. RNAseq and functional enrichment analysis identified mitochondrial dysfunction, OXPHOS, glycolysis, valine degradation, fatty acid beta-oxidation and the pentose phosphate pathway

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as canonical pathways enriched following treatment with the anti-diabetic medication pioglitazone. The study reported that both inflammation and mitochondrial dysfunction appear to underlie nerve dysfunction. Transcripts that were identified as playing a role in small and/or large fiber dysfunction included collagen, type I, alpha 1, SRC kinase signaling inhibitor 1, spondin 2, bone morphogenetic protein 3, and laminin, gamma 2. Such an approach can be taken with DCA to reveal gene regulatory networks and functional pathways associated with DCA-induced PN. Baseline studies on how the peripheral nerves respond to physical injury may offer a strong comparative dataset for chemical-induced injury. Transcriptomics has been conducted in both Schwann cells and dorsal root ganglia and also in response to peripheral nerve injury (Chang, Viader, Varghese, et al., 2013; Hu, Huang, Hu, et al., 2016). While these studies provide insight into the mechanisms underlying nerve damage and recovery, data are scant for chemical-induced injury to the peripheral nerves and a deeper understanding of the mechanisms (e.g., lipid dysregulation, inflammation, demyelination, changes in membrane potential) are needed. Recent studies are integrating Big Data into regulatory frameworks using computational approaches. For example, de Anda-Ja´uregui, McGregor, Guo, et al. (2019) compiled 234 drugs using a text-mining approach to identify a list of agents associated with drug-induced peripheral neuropathy from the Drugs@FDA database. There were 98 drugs identified as those that can induce neuropathy and 12,438 genes were extracted. The computational network approach produced a short list of 27 genes that are commonly associated with neuropathy and these included RELB proto-oncogene, NF-kB subunit 14 (transcription factor), phosphoinositide-3-kinase regulatory subunit 2 (signal transduction), asteroid homolog 1 (uncharacterized), histone deacetylase 7 (histone modification), ciliary neurotrophic factor receptor (neurite outgrowth), URB2 ribosome biogenesis 2 homolog (S. cerevisiae) (uncharacterized), radial spoke head 14 homolog (microtubule organization), elongin A2 (transcription elongation), ganglioside induced differentiation associated protein 1 (mitochondrial metabolism), coagulation factor XII (coagulation), and potassium calcium-activated channel subfamily N member 4 (ion channel), among others. Rich data sets such as these can be used to identify putative co-treatments for DCA-induced peripheral neuropathy. What of other emerging approaches that can be used to better understand DCA-induced neurotoxicity and its association to peripheral neuropathy? Experimental data have demonstrated that a range of pharmaceuticals have

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epigenetic mechanisms of action, modifying the genome in such a way as to increase detrimental effects in individuals. These mechanisms include methylation of DNA, histone modification, and regulation of non-coding RNA. There is recent evidence that peripheral neuropathy may have an epigenetic component in diseases such as diabetes (Fachrul, Utomo, & Parikesit, 2018). DCA may also act via these mechanisms to induce reversible peripheral neuropathy. Chronic treatments using DCA may impact these mechanisms, which can limit tolerance or alter efficacy of the treatment in patients treated with DCA long term. Fig. 6 summarizes some of the putative molecular mechanisms associated with DCA-induced peripheral neuropathy.

6. Summary Dichloroacetate (DCA) is unique in that it is both an environmental contaminant, present in treated drinking water, and an investigational drug for the treatment of various metabolic diseases and cancer. While mechanisms underlying DCA action are many (e.g., inhibition of GSTZ1, noncompetitive inhibitor of HMG-CoA-reductase), the primary effect is direct regulation of mitochondrial PDC/PDK axis. PDC is a critical homeostat for sustaining adequate energy stores (OXPHOS). Studies have described the pathobiology of many acquired diseases involving pathological perturbation of the PDC/PDK axis, leading to impairment of mitochondrial OXPHOS and oxidative damage. However, while DCA is considered an investigational drug, emerging translational and clinical research suggests a wide therapeutic window exists for DCA, provided it can be administered with adequate tolerability and safety. The single limiting factor for its use

Oxidave stress OXPHOS ROS

DNA Methylaon

Mitophagy

ER stress

Peripheral Neuropathy

Fig. 6 Proposed mechanisms of action for DCA-induced neurotoxicity. These cell processes are unlikely to work in isolation, each playing some role in the peripheral neuropathy. DCA may interact directly with the epigenome to affect transcriptional activity.

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clinically is peripheral neuropathy. Thus, investigations into the pathogenesis of DCA-induced neuropathy may identify sites for intervention. Therapeutic uses of DCA remain broad and are actively pursued in ongoing clinical trials; defining the balance between toxicity, such as peripheral neuropathy, and therapy remains paramount and could lead to a breakthrough in treatments for a number of debilitating diseases.

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Further reading Fuhrmann, D., & Els€asser, H. P. (2018). Schwann cell Myc-interacting zinc-finger protein 1 without pox virus and zinc finger: Epigenetic implications in a peripheral neuropathy. Neural Regeneration Research, 13(9), 1534–1537. Lindner, R., Puttagunta, R., Nguyen, T., et al. (2014). DNA methylation temporal profiling following peripheral versus central nervous system axotomy. Scientific Data, 1, 140038. Liu, W., & Zhou, C. (2012). Corticosterone reduces brain mitochondrial function and expression of mitofusin, BDNF in depression-like rodents regardless of exercise preconditioning. Psychoneuroendocrinology, 37(7), 1057–1070. Perez-Siles, G., Ly, C., Grant, A., et al. (2016). Pathogenic mechanisms underlying X-linked Charcot-Marie-Tooth neuropathy (CMTX6) in patients with a pyruvate dehydrogenase kinase 3 mutation. Neurobiology of Disease, 94, 237–244. Tao, L., Kramer, P. M., Ge, R., et al. (1998). Effect of dichloroacetic acid and trichloroacetic acid on DNA methylation in liver and tumors of female B6C3F1 mice. Toxicological Sciences, 43(2), 139–144. Velpula, K. K., Guda, M. R., Sahu, K., et al. (2017). Metabolic targeting of EGFRvIII/ PDK1 axis in temozolomide resistant glioblastoma. Oncotarget, 8(22), 35639–35655. Wehmas, L. C., DeAngelo, A. B., Hester, S. D., et al. (2017). Metabolic disruption early in life is associated with latent carcinogenic activity of dichloroacetic acid in mice. Toxicological Sciences, 159(2), 354–365. Zhang, Y., Brasher, A. L., Park, N. R., et al. (2018). High activity before breeding improves reproductive performance by enhancing mitochondrial function and biogenesis. The Journal of Experimental Biology, 221. Pt 7.