Fluoroacetate Toxicity Research Articles

50: A Zebrafish Model to Develop Mitochondrial Targeted Therapeutics for Sodium Fluoroacetate Toxicity

50: A Zebrafish Model to Develop Mitochondrial Targeted Therapeutics for Sodium Fluoroacetate Toxicity

“Lethal Synthesis” of Fluorocitrate by Citrate Synthase Explained through QM/MM Modeling

The term “lethal synthesis” was coined for enzymatic formation of fluorocitrate, but this classic problem of enzyme stereoselectivity remains poorly understood. Here, we show that high-level ab initio quantum mechanics/ molecular mechanics (QM/MM) modeling can accurately capture this enzymatic enantioselectivity and the results provide detailed insight into its origin. Citrate synthase (CS) performs the first reaction in the citric acid cycle: the formation of citrate from oxaloacetate and acetate in the form of acetyl-CoA. When fluoroacetyl-CoA (from fluoroacetate) is used as a substrate instead of acetyl-CoA, 2fluorocitrate is formed, which inhibits aconitase, the next enzyme in the citric acid cycle. This process is responsible for the lethal toxicity of fluoroacetate to humans and other mammals. The fluorocitrate enantiomer that is predominantly formed by CS, (2R,3R)-fluorocitrate (Figure 1), is the same enantiomer that specifically inhibits aconitase. The only (semi-)quantitative experimental study published to date indicates that the minor product of the enzymatic formation of fluorocitrate, (2S,3R)-fluorocitrate, amounts to 2–3% of the major product. Using transition-state theory, this translates to a difference in activation free energy (DDG ) of 2.06– 2.30 kcalmol . The causes of this selectivity remain uncertain. We have previously modeled the two initial reaction steps for the natural substrates, proton abstraction from acetylCoA and condensation with oxaloacetate (OAA), in CS with high-level QM/MM methods. For the reaction with fluoroacetyl-CoA (FaCoA), the distinction between enantiomers is made in the proton-abstraction step, where either an Eor a Z-enolate is formed. Here, we show that 1) the calculated relative energy of the enolates accurately predicts the experimentally observed enantiospecificity and 2) the enantiospecificity is mostly due to the inherent energy difference of the reacting species. A model of the enzyme with OAA and FaCoA bound was built for QM/MM simulation, with the Asp375 side chain from Cb, the methylthioester part of FaCoA, and OAA in the QM region. QM/MM molecular dynamics indicated that preE and pre-Z conformations in the CS active site can be sampled in the same trajectory. Five different approximate transition-state conformations for both E-enolate and Zenolate formation were generated by QM/MM umbrella sampling molecular dynamics (see the Supporting Information). These conformations were used to perform high-level QM/MM modeling of proton abstraction, using the established reaction coordinate r= d(OAsp375H) d(CFaCoAH). Geometries were optimized at the B3LYP/6-31+G(d)//MM level and energies calculated at the SCS-MP2/aug-cc-pVDZ// MM level. This describes the reaction of CS with acetyl-CoA accurately, in agreement with local coupled-cluster QM/MM results. The QM/MM energy profiles for enolate formation show correctly that formation of the E-enolate is preferred (Figure 2). Boltzmann-weighted energy differences between the E and Z profiles are 1.80 and 2.05 kcalmol 1 for the activation energy and reaction energy in this step, respectively. Entropy contributions for Eand Z-enolate formation are expected to be (nearly) identical (supported by AM1/MM free energy profiles, see the Supporting Information). The Figure 1. Conversion of fluoracetyl-CoA to fluorocitrate by CS.

Structural and biochemical studies of a fluoroacetyl-CoA-specific thioesterase reveal a molecular basis for fluorine selectivity.

We have initiated a broad-based program aimed at understanding the molecular basis of fluorine specificity in enzymatic systems, and in this context, we report crystallographic and biochemical studies on a fluoroacetyl-coenzyme A (CoA) specific thioesterase (FlK) from Streptomyces cattleya. Our data establish that FlK is competent to protect its host from fluoroacetate toxicity in vivo and demonstrate a 10(6)-fold discrimination between fluoroacetyl-CoA (k(cat)/K(M) = 5 × 10⁷ M⁻¹ s⁻¹) and acetyl-CoA (k(cat)/K(M) = 30 M⁻¹ s⁻¹) based on a single fluorine substitution that originates from differences in both substrate reactivity and binding. We show that Thr 42, Glu 50, and His 76 are key catalytic residues and identify several factors that influence substrate selectivity. We propose that FlK minimizes interaction with the thioester carbonyl, leading to selection against acetyl-CoA binding that can be recovered in part by new C═O interactions in the T42S and T42C mutants. We hypothesize that the loss of these interactions is compensated by the entropic driving force for fluorinated substrate binding in a hydrophobic binding pocket created by a lid structure, containing Val 23, Leu 26, Phe 33, and Phe 36, that is not found in other structurally characterized members of this superfamily. We further suggest that water plays a critical role in fluorine specificity based on biochemical and structural studies focused on the unique Phe 36 "gate" residue, which functions to exclude water from the active site. Taken together, the findings from these studies offer molecular insights into organofluorine recognition and design of fluorine-specific enzymes.

Sodium Fluoroacetate Poisoning

Sodium fluoroacetate was introduced as a rodenticide in the US in 1946. However, its considerable efficacy against target species is offset by comparable toxicity to other mammals and, to a lesser extent, birds and its use as a general rodenticide was therefore severely curtailed by 1990. Currently, sodium fluoroacetate is licensed in the US for use against coyotes, which prey on sheep and goats, and in Australia and New Zealand to kill unwanted introduced species. The extreme toxicity of fluoroacetate to mammals and insects stems from its similarity to acetate, which has a pivotal role in cellular metabolism. Fluoroacetate combines with coenzyme A (CoA-SH) to form fluoroacetyl CoA, which can substitute for acetyl CoA in the tricarboxylic acid cycle and reacts with citrate synthase to produce fluorocitrate, a metabolite of which then binds very tightly to aconitase, thereby halting the cycle. Many of the features of fluoroacetate poisoning are, therefore, largely direct and indirect consequences of impaired oxidative metabolism. Energy production is reduced and intermediates of the tricarboxylic acid cycle subsequent to citrate are depleted. Among these is oxoglutarate, a precursor of glutamate, which is not only an excitatory neurotransmitter in the CNS but is also required for efficient removal of ammonia via the urea cycle. Increased ammonia concentrations may contribute to the incidence of seizures. Glutamate is also required for glutamine synthesis and glutamine depletion has been observed in the brain of fluoroacetate-poisoned rodents. Reduced cellular oxidative metabolism contributes to a lactic acidosis. Inability to oxidise fatty acids via the tricarboxylic acid cycle leads to ketone body accumulation and worsening acidosis. Adenosine triphosphate (ATP) depletion results in inhibition of high energy-consuming reactions such as gluconeogenesis. Fluoroacetate poisoning is associated with citrate accumulation in several tissues, including the brain. Fluoride liberated from fluoroacetate, citrate and fluorocitrate are calcium chelators and there are both animal and clinical data to support hypocalcaemia as a mechanism of fluoroacetate toxicity. However, the available evidence suggests the fluoride component does not contribute. Acute poisoning with sodium fluoroacetate is uncommon. Ingestion is the major route by which poisoning occurs. Nausea, vomiting and abdominal pain are common within 1 hour of ingestion. Sweating, apprehension, confusion and agitation follow. Both supraventricular and ventricular arrhythmias have been reported and nonspecific ST- and T-wave changes are common, the QTc may be prolonged and hypotension may develop. Seizures are the main neurological feature. Coma may persist for several days. Although several possible antidotes have been investigated, they are of unproven value in humans. The immediate, and probably only, management of fluoroacetate poisoning is therefore supportive, including the correction of hypocalcaemia.

Fluoroacetate-Mediated Toxicity of Fluorinated Ethanes

A series of 1-(di)halo-2-fluoroethanes reported in the literature to be nontoxic or of low toxicity were found to be highly toxic by the inhalation route. Experiments were performed that showed the compounds, 1,2-difluoroethane, 1-chloro-2-fluoroethane, 1-chloro-1,2-difluoroethane, and 1-bromo-2-fluoroethane to be highly toxic to rats upon inhalation for 4 hr. All four compounds had 4-hr approximate lethal concentrations of ≤100 ppm in rats. In contrast, 1,1-difluoroethane (commonly referred to as HFC-152a) has very low acute toxicity with a 4-hr LC50 of >400,000 ppm in rats. Rats exposed to the selected toxic fluoroethanes showed clinical signs of fluoroacetate toxicity (lethargy, hunched posture, convulsions). 1,2-Difluoroethane, 1-chloro-2-fluoroethane, 1-chloro-1,2-difluoroethane, and 1-bromo-2-fluoroethane were shown to increase concentrations of citrate in serum and heart tissue, a hallmark of fluoroacetate intoxication.19F NMR analysis confirmed that fluoroacetate was present in the urine of rats exposed to each toxic compound. Fluorocitrate, a condensation product of fluoroacetate and oxaloacetate, was identified in the kidney of rats exposed to 1,2-difluoroethane. There was a concentration-related elevation of serum and heart citrate in rats exposed to 0–1000 ppm 1,2-difluoroethane. Serum citrate was increased up to 5-fold and heart citrate was increased up to 11-fold over control citrate levels. Metabolism of 1,2-difluoroethane by cytochrome P450 (most likely CYP2E1) is suspected because pretreatment of rats or mice with SKF-525A, disulfiram, or dimethyl sulfoxide prevented or delayed the toxicity observed in rats not pretreated. Experimental evidence indicates that the metabolism of the toxic fluoroethanes is initiated at the carbon–hydrogen bond, with metabolism to fluoroacetate via an aldehyde or an acyl fluoride. The results of these studies show that 1-(di)halo-2-fluoroethanes are highly toxic to rats and should be considered a hazard to humans unless demonstrated otherwise.

Commentary on ‘The toxicity of fluoroacetate and the tricarboxylic acid cycle’ by C. Liébecq and R.A. Peters Biochim. Biophys. Acta 3 (1949) 215–230

Commentary on ‘The toxicity of fluoroacetate and the tricarboxylic acid cycle’ by C. Liébecq and R.A. Peters Biochim. Biophys. Acta 3 (1949) 215–230

Significance of Sulfhydryl Compounds in the Manifestation of Fluoroacetate Toxicity to the Rat, Brush-tailed Possum, Woylie and Western Grey Kangaroo

Levels of citrate in kidneys and livers of rats with normal glutathione levels increased 6.8 and 1.7-fold respectively 2 h after dosing with 1.5 mg of compound 1080 (= 95% sodium fluoroacetate) per kilogram body weight. In animals with liver glutathione levels 15% of normal, increases in plasma and liver citrate levels after dosing with fluoroacetate were significantly greater than those of control animals. Cysteamine and N-acetylcysteine, like glutathione, partially protected aconitate hydratase from fluorocitrate inhibition in rat liver preparations but were unable to replace glutathione as a substrate for the defluorination of fluoroacetate in vitro. N-Acetylcysteine did not diminish plasma citrate levels of glutathione-deficient rats dosed with fluoroacetate, while cysteamine inhibited the rate of in vivo defluorination in glutathione-deficient brush-tailed possums. It is suggested that non-physiological sulfhydryl compounds are ineffective antidotes to fluoroacetate intoxication in vivo. The in vivo defluorination patterns of four mammal species with differing sensitivities to fluoroacetate did not indicate a direct relationship between tolerance and rate of defluorination and it is also suggested that a high level of activity of the glutathione-S-transferase responsible for the defluorination of fluoroacetate is not the major mechanism for circumventing fluoroacetate toxicity in resistant mammals.

Defluorination: A possible mechanism of detoxification in rats exposed to fluoroacetate

An earlier study described a defluorinating system in the 105 000 × g supernatant fraction of rat liver which is stimulated in vitro by glutathione and inhibited by maleate. This work suggested the possible role of this defluorinating system in the detoxification of fluoroacetate in vivo. The current study reports the enhanced toxicity of fluoroacetate in animals in which liver glutathione has been depleted. Diethylmaleate pretreatment was found to reduce liver glutathione levels to 10% of control values 30 min after dosing. In animals pretreated with diethylmaleate the LD 50 of fluoroacetate was reduced by 35%. Increased toxicity of fluoroacetate with depletion of liver glutathione concentrations is consistent with a protective role for the glutathione requiring fluoroacetate defluorinating system in rat liver.

Fluoroacetate toxicity.

Fluoroacetate toxicity.

Studies on the biochemical basis of susceptibility and resistance of the housefly to fluoroacetate

1. Administration of fluoroacetate to sensitive houseflies in amounts close to the L.D. 50 range (0.25–0.3 μg/fly) brought about a prompt elevation of their citrate content. With about 10-fold higher doses of fluoroacetate a concurrent increase of both citrate and pyruvate levels took place in the fly tissues. 2. Incubation of sarcosomes of the sensitive housefly strain in the presence of oxidizable substrates and fluoroacetate resulted in accumulation of citrate, inhibition of respiration and uncoupling of oxidative phosphorylation. The magnitude of the effects varied considerably with the different substrates used, being particularly pronounced with pyruvate and malate and inappreciable with succinate and α-glycerophosphate. 3. The respiratory inhibition induced by a brief exposure in the cold of housefly sarcosomes to fluoroacetate, persisted after the sarcosomes had been washed free from fluoroacetate. The toxic effect of fluoroacetate on the respiratory chain could be prevented by an excess of simultaneously added acetate. 4. The susceptibility of the respiratory function of the sarcosomes to fluoroacetate inhibition was abolished by sonication. The unresponsiveness of the sonicated sarcosomes to fluoroacetate was attended by a loss of their respiratory chain phosphorylation activity. 5. Sarcosomes derived from a partially resistant housefly strain, when incubated in the presence of fluoroacetate, failed to accumulate citrate, but displayed the characteristic respiratory-inhibition response. Sarcosomes from a highly resistant strain showed no impairment of their functional capacity by fluoroacetate. However, all the different housefly strains tested proved to be equally sensitive to the deleterious effect of fluorocitrate on sarcosomal respiration. 6. The possible biochemical mechanisms underlying the toxicity of fluoroacetate in the housefly are considered with particular reference to the altered response of the target systems exhibited by the fluoroacetate-resistant strains.

Fluoroacetate toxicity in Thiobacillus neapolitanus and its relevance to the problem of obligate chemoautotrophy.

Fluoroacetate was extremely toxic toThiobacillus neapolitanus strainC, retarding growth even at 10-9m. Inhibition could be relieved by relatively high concentrations of acetate or propionate. Inhibited cultures eventually recovered from inhibition and grew in the presence of fluoroacetate over the concentration range 10-9 to 10-5m. The recovery represented a recommencement of division of the total population, as it was shown that 60–100% of the organisms inoculated on to agar containing as much as 10-3m fluoroacetate formed colonies after lags as long as 37 days. Even longer lags occurred with more fluoroacetate, but fewer organisms survived. Fluoroacetate appeared specifically to inhibit the Krebs' cycle through fluorocitrate synthesis; this confirmed that the cycle is essential to the autotrophic metabolism.

Differential toxicity of fluoroacetate to heart, kidney and brain mitochondria of the living rat

Mitochondria were prepared from the heart, kidney and brain of rats intoxicated with fluoroacetate. Incubation with several substrates showed depression of respiration of heart mitochondria with pyruvate and succinate, and depression of respiration of kidney mitcohondria with pyruvate, succinate, citrate, β-oxobutyrate and l-glutamate; no inhibition of respiration was detected in brain mitochondria. Phosphorylative capacity, as expressed by the P:O ratio, was not affected in any of the tissues. However, a reduction of the respiratory control was observed in all cases.

Fluoroacetate poisoning: comparison of synthetic fluorocitric acid with the enzymically synthesized fluorotricarboxylic acid.

THE hypothesis advanced earlier to account for the toxicity of fluoroacetate (FCH2.COONa) has been proved recently by the isolation of an enzymically synthesized mono-fluorotricarboxylic acid in crystalline form1, prepared from the products of interaction of kidney tissue with fumarate and fluoroacetate. Though there was much to suggest that it was mono-fluorocitric acid, this had not been rigorously proved by a comparison of the infra-red spectrum with a synthetic specimen. The recent synthesis by one of us (D. E. A. R., in the press) of fluorocitric acid has made it possible to compare the infra-red spectra of the barium salts of this with the barium salt from 1.5 mgm. of the enzymically synthesized (natural) crystalline compound isolated in Oxford (by R. A. P. and R. W. W.).

Biochemistry of fluoroacetate poisoning; the isolation and some properties of the fluorotricarboxylic acid inhibitor of citrate metabolism.

It has been suggested that the toxicity of fluoroacetate is due to the enzymic synthesis of a fluorotricarboxylic acid, which ‘jams’ the tricarboxylic acid cycle at the citrate stage. This communication presents the proof of this hypothesis. The inhibitory substance for citrate metabolism synthesized by enzymic action from fluoroacetate has been isolated as a compound in crystalline form of great potency. Under the conditions of test it inhibits the disappearance of approximately 300 times its weight of citric acid in 30 min. The final isolation involved a separation from citric acid by the use of ion-exchange resin, and fractional extraction with ether. It is a monofluorotricarboxylic acid, as shown by its migration on a paper chromatogram, by its fluorine content (estimated spectrochemically), and by its titration curve. It does not give the colour reaction with sodium sulphide for pentabromacetone produced from citric acid by the usual methods. It gives an infra-red band which may be expected from a C-F bond. By a process of exclusion, it is considered to be a fluorocitric acid; a final decision must await synthesis.

The toxicity of fluoroacetate and the tricarboxylic acid cycle

1. 1. The hypothesis that fluoroacetate is a competitive inhibitor for acetate in tissue metabolism ( Bartlett and Barron) has been examined and found incapable of explaining some of the enzymatic effects of the poison. 2. 2. Using a homogenate from guinea pig kidney, free from residual oxidizable substrates, and reinforced with Mg ++ and adenine nucleotides, it was found that this readily oxidizes fumarate and citrate. In presence of fluoroacetate, citrate accumulates during the oxidation of fumarate, without an accompanying accumulation of acetate. 3. 3. With the same homogenate, fumarate and pyruvate together give 20–50% increased oxygen uptake as compared with fumarate alone, and there is even formation of citrate in absence of poison. Fluoroacetate produces inhibition without accumulation of acetate, but this is less than with fumarate alone. 4. 4. Fluoroacetate has no action upon the pyruvate dehydrogenase component of the brain pyruvate oxidase system. It also has no effect upon the activity of the enzymes aconitase, oxalosuccinate decarboxylase, fumarase and oxaloacetate decarboxylase, as tested by a pigeon liver preparation. 5. 5. The fact that no single enzymatic reaction has been found to be inhibited by fluoroacetate 6. and yet that the reactions of the tricarboxylic cycle are stopped with accumulation of citrate requires a hypothesis in addition to that proposed by Barron and Bartlett. 7. 6. It is suggested that, in the kidney preparation, fluoroacetate is not the inhibitor but that it is transformed into another substance which is inhibitory. 8. 7. The accumulation of citrate can be observed with concentrations of 0.05 mM (15 μg Nafluoroacetate per bottle) which approximates to the amounts causing toxic effects in vivo. 9. 8. It was confirmed that malonate inhibits the enzymatic decarboxylation of oxaloacetate. 10. 9. Some experiments upon kidney brei are described.