Succinate prodrugs as treatment for acute metabolic crisis during fluoroacetate intoxication in the rat.

Sodium fluoroacetate (FA) is a metabolic poison that systemically inhibits the tricarboxylic acid (TCA) cycle, causing energy deficiency and ultimately multi-organ failure. It poses a significant threat to society because of its high toxicity, potential use as a chemical weapon and lack of effective antidotal therapy. In this study, we investigated cell-permeable succinate prodrugs as potential treatment for acute FA intoxication. We hypothesized that succinate prodrugs would bypass FA-induced mitochondrial dysfunction, provide metabolic support, and prevent metabolic crisis during acute FA intoxication. To test this hypothesis, rats were exposed to FA (0.75 mg/kg) and treated with the succinate prodrug candidate NV354. Treatment efficacy was evaluated based on cardiac and cerebral mitochondrial respiration, mitochondrial content, metabolic profiles and tissue pathology. In the heart, FA increased concentrations of the TCA metabolite citrate (+ 4.2-fold, p < 0.01) and lowered ATP levels (- 1.9-fold, p < 0.001), confirming the inhibition of the TCA cycle by FA. High-resolution respirometry of cardiac mitochondria further revealed an impairment of mitochondrial complex V (CV)-linked metabolism, as evident by a reduced phosphorylation system control ratio (- 41%, p < 0.05). The inhibition of CV-linked metabolism is a novel mechanism of FA cardiac toxicity, which has implications for drug development and which NV354 was unable to counteract at the given dose. In the brain, FA induced the accumulation of ß-hydroxybutyrate (+ 1.4-fold, p < 0.05) and the reduction of mitochondrial complex I (CI)-linked oxidative phosphorylation (OXPHOSCI) (- 20%, p < 0.01), the latter of which was successfully alleviated by NV354. This promising effect of NV354 warrants further investigations to determine its potential neuroprotective effects.

Ethanol and caffeine age-dependently alter brain and retinal neurochemical levels without affecting morphology of juvenile and adult zebrafish (Danio rerio).

Adolescent alcohol exposure in humans is predictive of adult development of alcoholism. In rodents, caffeine pre-exposure enhances adult responsiveness to ethanol via a pathway targeted by both compounds. Embryonic exposure to either compound adversely affects development, and both compounds can alter zebrafish behaviors. Here, we evaluate whether co-exposure to caffeine and/or alcohol in adolescence exerts neurochemical changes in retina and brain. Zebrafish (Danio rerio) were given daily 20 min treatments to ethanol (1.5% v/v), caffeine (25-100 mg/L), or caffeine + ethanol for 1 week during mid-late adolescence (53-92 days post fertilization (dpf)) or early adulthood (93-142 dpf). Immediately after exposure, anatomical measurements were taken, including weight, heart rate, pigment density, length, girth, gill width, inner and outer eye distance. Brain and retinal tissue were subsequently collected either (1) immediately, (2) after a short interval (2-4d) following exposure, or (3) after a longer interval that included an acute 1.5% ethanol challenge. Chronic ethanol and/or caffeine exposure did not alter anatomical parameters. However, retinal and brain levels of tyrosine hydroxylase were elevated in fish sacrificed after the long interval following exposure. Protein levels of glutamic acid decarboxylase were also increased, with the highest levels observed in 70-79 dpf fish exposed to caffeine. The influence of ethanol and caffeine exposure on neurochemistry demonstrates specificity of their effects during postembryonic development. Using the zebrafish model to assess neurochemistry relevant to reward and anxiety may inform understanding of the mechanisms that reinforce co-addiction to alcohol and stimulants.

Neurochemical and Behavioral Consequences of Ethanol and/or Caffeine Exposure: Effects in Zebrafish and Rodents.

Zebrafish are increasingly being utilized to model the behavioral and neurochemical effects of pharmaceuticals and, more recently, pharmaceutical interactions. Zebrafish models of stress establish that both caffeine and ethanol influence anxiety, though few studies have implemented coadministration to assess the interaction of anxiety and reward-seeking. Caffeine exposure in zebrafish is teratogenic, causing developmental abnormalities in the cardiovascular, neuromuscular, and nervous systems of embryos and larvae. Ethanol is also a teratogen and, as an anxiolytic substance, may be able to offset the anxiogenic effects of caffeine. Co-exposure to caffeine and alcohol impacts neuroanatomy and behavior in adolescent animal models, suggesting stimulant substances may moderate the impact of alcohol on neural circuit development. Here, we review the literature describing neuropharmacological and behavioral consequences of caffeine and/or alcohol exposure in the zebrafish model, focusing on neurochemistry, locomotor effects, and behavioral assessments of stress/anxiety as reported in adolescent/juvenile and adult animals. The purpose of this review is twofold: (1) describe the work in zebrafish documenting the effects of ethanol and/or caffeine exposure and (2) compare these zebrafish studies with comparable experiments in rodents. We focus on specific neurochemical pathways (dopamine, serotonin, adenosine, GABA), anxiety-type behaviors (assessed with a novel tank, thigmotaxis, shoaling), and locomotor changes resulting from both individual and co-exposure. We compare findings in zebrafish with those in rodent models, revealing similarities across species and identifying conservation of mechanisms that potentially reinforce coaddiction.

Succinate prodrugs in combination with atropine and pralidoxime protect cerebral mitochondrial function in a rodent model of acute organophosphate poisoning.

Pesticides account for hundreds of millions of cases of acute poisoning worldwide each year, with organophosphates (OPs) being responsible for the majority of all pesticide-related deaths. OPs inhibit the enzyme acetylcholinesterase (AChE), which leads to impairment of the central- and peripheral nervous system. Current standard of care (SOC) alleviates acute neurologic-, cardiovascular- and respiratory symptoms and reduces short term mortality. However, survivors often demonstrate significant neurologic sequelae. This highlights the critical need for further development of adjunctive therapies with novel targets. While the inhibition of AChE is thought to be the main mechanism of injury, mitochondrial dysfunction and resulting metabolic crisis may contribute to the overall toxicity of these agents. We hypothesized that the mitochondrially targeted succinate prodrug NV354 would support mitochondrial function and reduce brain injury during acute intoxication with the OP diisopropylfluorophosphate (DFP). To this end, we developed a rat model of acute DFP intoxication and evaluated the efficacy of NV354 as adjunctive therapy to SOC treatment with atropine and pralidoxime. We demonstrate that NV354, in combination with atropine and pralidoxime therapy, significantly improved cerebral mitochondrial complex IV-linked respiration and reduced signs of brain injury in a rodent model of acute DFP exposure.

Differential behavioral effects of ethanol pre-exposure in male and female zebrafish (Danio rerio).

Alcohol exposure in adolescence is a contributing factor toward reward-seeking behavior in adulthood. This reward-seeking behavior is assessed in animal models using the conditioned place preference (CPP) paradigm. In this study, ethanol-induced change in time spent by zebrafish on the initially non-preferred tank side was studied by conditioning adult zebrafish to ethanol dissolved in water (0.00% 1.00%; 1.25%; 1.50%; 1.60%; 1.75% vol/vol) paired with an initially non-preferred environment. Following a single conditioning cycle, fish swam unrestricted in the CPP chamber to assess changes in preference. Daily 20-min pre-exposure to ethanol for 1 week during the juvenile stage starting at either 20days post fertilization (dpf) or 40 dpf altered percent time spent on the ethanol-paired side in adulthood in a dose-dependent and sex-dependent manner. The results suggest that male and female zebrafish are an effective model in which to investigate behavioral correlates of ethanol-induced changes in neural circuits implicated in reward and anxiety.