Toxicology of Fluoroacetate: a Review, with Possible Directions for Therapy

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Toxicology of ﬂuoroacetate: A review, with possible directions for therapy research

Article in Journal of Applied Toxicology · March 2006 DOI: 10.1002/jat.1118 · Source: PubMed

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The user has requested enhancement of the downloaded file. 148JOURNALN. V. GONCHAROVOF APPLIED TOXICOLOGYET AL. J. Appl. Toxicol. 2006; 26: 148–161 Published online 26 October 2005 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jat.1118 Toxicology of ﬂuoroacetate: a review, with possible directions for therapy research

Nikolay V. Goncharov,1 Richard O. Jenkins2,* and Andrey S. Radilov1

1 Research Institute of Hygiene, Occupational Pathology and Human Ecology, Saint-Petersburg, Russia 2 School of Allied Health Sciences, De Montfort University, Leicester, UK Received 30 June 2005; Revised 16 August 2005; Accepted 5 September 2005

ABSTRACT: Fluoroacetate (FA; CH2FCOOR) is highly toxic towards humans and other mammals through inhibition of the enzyme aconitase in the tricarboxylic acid cycle, caused by ‘lethal synthesis’ of an isomer of fluorocitrate (FC). FA is found in a range of plant species and their ingestion can cause the death of ruminant animals. Some fluorinated compounds — used as anticancer agents, narcotic analgesics, pesticides or industrial chemicals — metabolize to FA as intermediate products. The chemical characteristics of FA and the clinical signs of intoxication warrant the re-evaluation of the toxic danger of FA and renewed efforts in the search for effective therapeutic means. Antidotal therapy for FA intoxication has been aimed at preventing fluorocitrate synthesis and aconitase blockade in mitochondria, and at providing citrate outflow from this organelle. Despite a greatly improved understanding of the biochemical mechanism of FA toxicity, ethanol, if taken immediately after the poisoning, has been the most acceptable antidote for the past six decades. This review deals with the clinical signs and physiological and biochemical mechanisms of FA intoxication to provide an explanation of why, even after decades of investigation, has no effective therapy to FA intoxication been elaborated. An apparent lack of integrated toxicological studies is viewed as a limiter of progress in this regard. Two principal ways of developing effective therapies for FA intoxication are considered. Firstly, competitive inhibition of FA interaction with CoA and of FC interaction with aconitase. Secondly, channeling the alternative metabolic pathways by orienting the fate of citrate via cytosolic aconitase, and by maintaining the flux of reducing equivalents into the TCA cycle via glutamate dehydrogenase. Copyright © 2005 John Wiley & Sons, Ltd.

KEY WORDS: fluoroacetate; compound 1080; fluorocitrate; monofluorides; aconitase; TCA; lethal synthesis; therapy; metabolic poison

Introduction The sodium salt of FA is known as ‘compound 1080’ and it is used in some countries for controlling the popu- The term fluoroacetate (FA) refers to a large series of lation of certain vertebrate species: in the USA and UK chemical compounds of the general formula CH2FCOOR. rodents are controlled in ships, sewers and warehouses; FA and other monofluorides are highly toxic compounds. also, coyotes are controlled by the use of FA-impregnated Their action characteristically involves a latent period, carcasses or collars on livestock; in Australia and New which for humans is from half to several hours even on Zealand rabbits, wallabies, goats, wild pigs, deer and exposure to lethal doses. opossums are controlled with the use of baits based on FA was first synthesized in 1896 and only much apple, carrot or grain; aerial sowing is used to control later found in Dichapetalum, Gastrolobium, Oxylobium, large or remote areas (Norris, 2001). To replace the Acacia and Palicourea plants prevailing in Australia, highly toxic FA, 1,3-difluoro-2-propanol, which is the southern Africa and South America (Oerlichs and major ingredient of the commercial product gliftor, was McEwan, 1961; McEwan, 1964; Vickery et al., 1973; proposed for pesticide applications. It is less toxic than De Oliveira, 1963; Aplin, 1971). The FA contents of FA (about 100 mg kg−1 in rats), but the mechanism of its Australian plants attain 5 g kg−1 dry weight (Hall, 1972), toxic action is similar to that of FA and involves its and their single or repeated ingestion cause the death of initial conversion into 1,3-difluoroacetone with the help ruminant animals, resulting in considerable economic of NAD-dependent alcohol dehydrogenase (Feldwick damage (Annison et al., 1960; McCosker, 1989; Minnaar et al., 1998). Fluoroacetamide (compound 1081) has been et al., 2000). used in Israel for field rodent control (Braverman, 1979). The conversion of fluoroacemide into FA in vivo occurs via hydrolysis by organophosphate-sensitive amidase * Correspondence to: Dr R. O. Jenkins, School of Allied Health Sciences, (Tecle and Casida, 1989). De Montfort University, The Gateway, Leicester LE1 9BH, UK. It was also found that a series of other compounds E-mail: [email protected] Contract/grant sponsor: BioIndustry Initiative Program, the US Department metabolize to FA as intermediate products: these are of State; Contract/grant number: ISTC BII-2629. the anticancer drugs 5-fluorouracil and fluoroethyl

Copyright © 2005 John Wiley & Sons, Ltd. J. Appl. Toxicol. 2006; 26: 148–161 TOXICOLOGY OF FLUOROACETATE 149 nitrosourea, N-(2-fluoroethyl) derivatives of the narcotic substrate coordination to a specific iron atom in this analgesics normeperidin and normetazocin, the industrial cluster, Fea (Lauble et al., 1992). All four 2-fluorocitrate chemicals fluoroethanol and 1-(di)halo-2-fluoroethanes isomers were synthesized and purified to show that (Yamashita et al., 2004; Arellano et al., 1998; Yeh (−)-erythro-2-flurocitrate (2R, 3R) is the single inhibitory and Cheng, 1994; Reifenrath et al., 1980; Tisdale and isomer (Carrell et al., 1970). The reversible character of Brennan, 1985; Tecle and Casida, 1989; Keller et al., competitive inhibition of the enzyme was established 1996; Feldwick et al., 1998). Finally, industrial ‘achieve- (Villafranca and Platus, 1973; Brand et al., 1973; Eanes ments’ have led to the appearance of FA in fog and rain and Kun, 1974). Furthermore, it was shown that aconitase (Rompp et al., 2001). removes fluoride ion from (−)-erythro-2-fluorocitrate Formulators and pest control workers are the largest (2R, 3R isomer) with a stoichiometry of 1 F− per enzyme occupational risk group (Norris, 2001). Exposure to a molecule (Kent et al., 1985; Tecle and Casida, 1989), stock solution during formulation and dermal or respira- whereas from (+)-erythro-2-fluorocitrate (2S, 3S) with tory exposure during application of baits, as well as a stoichiometry of about 1 F− per substrate (Lauble accidental or intentional acute intoxications are the main et al., 1996). These findings imply that (−)-erythro-2- human health concerns. The chemical characteristics of fluorocitrate is responsible for aconitase inhibition. Kent FA, such as high solubility in water and lack of specific et al. (1985) suggested that the defluorination generates taste, and the clinical signs of intoxication warrant re- an actual aconitase inhibitor, 4-hydroxy-trans-aconitate evaluation of the toxic danger of FA and renewed efforts (HTA), which was later confirmed by Lauble et al. in the search for effective therapeutic means. This review (1996). (−)-Erythro-2-flurocitrate is a ‘passive’ aconitase deals with the clinical signs and physiological and bio- inhibitor of a sort, whose activity is associated with a chemical mechanisms of FA intoxication to provide certain sequence of conversions induced by aconitase a clearer understanding of why, even after decades of (mechanism-based inhibitor). Initially it converts to investigation, no effective therapy has been elaborated. fluoro-cis-aconitate, the latter then takes up hydroxyl and The review also addresses the question: is there now a loses fluoride to form HTA that binds very tightly, but gleam of hope for developing one? not covalently, to the enzyme. The bond strength can be judged by the slow displacement of HTA from its complex with aconitase by the natural aconitase substrate Aconitase and Molecular Mechanism isocitrate; HTA is detectable only at a 106-fold excess of of the Toxic Action of FA isocitrate (Lauble et al., 1994; Lauble et al., 1996). The HTA-aconitase complex involves four hydrogen bonds no The mechanism of the inhibitory effect of FA on less than 2.7 Å long, which hold together HTA, a water aconitase [citrate (isocitrate) hydro-lyase, EC 4.2.1.3] is molecule bound to the [4Fe-4S] cluster, Asp165 and one of the most interesting and long-standing problems His167. In comparison, isocitrate has only one such bond. in biochemistry. In the organism, FA undergoes a series of metabolic conversions resulting in the synthesis of an extremely toxic compound, fluorocitrate (FC); this Toxicokinetics and Mechanism of FA process was named ‘lethal synthesis’ (Peters, 1952). FC Detoxication is formed by the enzymatic condensation of fluoroacetyl CoA with oxaloacetate, catalysed by citrate (si)-synthase Defluorination is carried out mainly by anionic proteins (EC 4.1.3.7). Initially, FC was considered to be a com- with glutathione transferase activity. In addition, there petitive aconitase inhibitor. However, in the early 1990s are about 10% of proteins in the anionic fraction that do it was speculated that FC acts as a ‘suicide substrate’ in not have the glutathione transferase activity but do carry the sense that it has a high affinity for aconitase at any out defluorination; also, there are cationic enzymes with concentration of the competitive citrate (Clarke, 1991). glutathione transferase activity, which are responsible Competitive reaction would have been indicated by re- for approximately 20% of cytosolic defluorination of placement of FC by citrate at increased concentration of FA (Wang et al., 1986). The major detoxication pathway the latter, but this is not the case. of FA involves glutathione-dependent defluorination By the mid-1990s the toxic action of FA was fairly via nucleophilic attack at the β-carbon atom, result- well understood, and all its stages had been estab- ing in the formation of fluoride ion and S- lished. Aconitase effects conversion of citrate to isocitrate carboxymethylglutathione, with subsequent cleavage of through an obligatory intermediate, cis-aconitate. The the latter into constituent amino acids and excretion stereochemical features of the dehydration/rehydration within urine as an S-(carboxymethyl) conjugate com- reactions are such that cis-aconitate should bind with plex (Mead et al., 1979; Tecle and Casida, 1989). aconitase in two different ways swung 180° to the Cα-Bβ The activity of enzymes responsible for defluorination bond (Gawron and Mahajan, 1966). Aconitase includes a depends on the concentration of glutathione (GSH), [4Fe-4S] cluster and the catalytic conversion involves with maximum activity at 5 mmol l−1. At a saturating

GSH concentration, the apparent enzymatic affinity con- (apathy, flabbiness) for 30–120 min; (2) excitement for −1 stant (Km) for FA defluorination is 7 mmol l . The up to 30 min; and (3) convulsions for 2–4 min. Animals main GSH-dependent enzyme that defluorinates FA is not and humans usually die after 2–4 such cycles. The death identical to liver GSH-dependent S-transferases (Soiefer results from the following reasons (separately or in com- and Kostyniak, 1983). This is the so-called FA-specific bination): (1) asphyxia during convulsions; (2) cardiac defluorinase, an enzyme that is distinct in its biochemical arrest or ventricular fibrillation; (3) CNS depression and immunological characteristics from multiple cationic associated with respiratory or cardiovascular disorders and anionic glutathione S-transferase isoenzymes. The (Pattison, 1959). defluorinase has an acidic isoelectric point (pH = 6.4) and There are considerable species-specific variations in a molecular weight of 41 kD, with a predominant subunit clinical signs of exposure to FA and in sensitivity to of 27 kD (Soiefer and Kostyniak, 1984). Increased blood the poison (Chenoweth, 1949). Thus, the mean lethal concentrations of fluoride ions can serve as a criterion for dose varies from 0.05 mg kg−1 in dogs to 150 mg kg−1 in intoxication with FA. The highest defluorinating activity opossum. The most general criterion of tolerance or, vice is characteristic of liver, with kidney, lungs, heart and versa, animal sensitivity to FA is the rate of metabolism. testicles ranked below in order of decreasing activity. Thus, the metabolism of FA in skink lizards Tiliqua Defluorinating activity was not found in the brain rugosa is an order of magnitude slower than in rats (Soiefer and Kostyniak, 1983). Rattus norvegicus, and the lethal dose for lizards is two The elimination half-life of FA is generally no less orders of magnitude higher than for rats (Twigg et al., than 2 days, which causes long-term toxicity in tissues of 1986). The low rate of metabolic processes suggests that poisoned animals and sets up a risk of secondary poison- FA slowly converts into fluorocitrate, and this makes ing (Aulerich et al., 1987). After administration of possible more effective excretion and detoxication of FA. sodium FA at a dose of 0.25 mg kg−1, its concentration in There is also a correlation between nutrition mode and rat plasma was 0.26 µgml−1 at 1 h and 0.076 µgml−1 at toxic effect of FA; the cardiovascular system is mainly 12 h (Eason and Turck, 2002). The average percentage affected in herbivores, while the CNS is mainly affected distribution of FA (organic fluoride) in rat plasma, liver in carnivores. Consequently, four groups were recognized and kidney were 7.05, 5.07 and 1.68, respectively in terms of clinical signs of intoxication (Chenoweth and (Egekeze and Oehme, 1979a). After FA was administered Gilman, 1946; see Table 1). The first comprised herbiv- orally to sheep and goats at a dose of 0.1 mg kg−1, ores, such as rabbits, goats, sheep, cattle and horses, the elimination half-life was found to be 10.8 h and in which exposure to FA caused ventricular fibrillation 5.4 h respectively. The highest concentration of FA without notable CNS disorder (Marais, 1944; Chenoweth, after 2.5 h was 0.098 µgml−1 in blood plasma, and next 1949; Egyed, 1973). The second group comprised carni- were kidney (0.057 µgg−1), muscles (0.042 µgg−1) and vores, such as dogs, with guinea pigs being an exception, liver (0.021 µgg−1). After 96 h, only traces of FA in which the CNS was primarily affected (Chenoweth (<0.008 µgg−1) were found in all tissues (Eason et al., and Gilman, 1946; Egyed and Shupe, 1971). In dogs, 1994). However, according to Gooneratne et al. (1995), a species most sensitive to FA, intoxication symptoms the plasma elimination half-life in rabbits subjected to a appear after a latent period of 1–10 h (Chenoweth and sublethal dose of sodium FA was 1.1 h. The concentra- Gilman, 1946; Egyed and Shupe, 1971). Interestingly, tions of FA in rabbit muscles, kidney, and liver were histological changes in dogs are similar to those in much higher than in plasma. sheep; mainly hemorrhagic gastroenteritis and coagulating necrosis in certain organs (Egyed and Shupe, 1971). The third group comprised animals in which the clinical Toxicodynamics and Clinical Signs of FA pattern of intoxication was similar to that characteristic of Intoxication the second group animals, but slightly less pronounced. This group comprised rats and hamsters relatively The clinical pattern of FA intoxication at any administra- tolerant to FA. After exposure to high FA doses, death tion mode characteristically involves a latent period from usually occurred within 4–6 h as a result of respiratory 0.5 to 6 h (Chenoweth, 1949). The length of the latent depression, ventricular fibrillation occasionally developing period depends on the time required for: (1) hydrolysis of in rats (Chenoweth and Gilman, 1946; Pattison, 1959). fluoroacetates to monofluoroacetic acid; (2) synthesis of The fourth group comprised cats, pigs and rhesus mon- an effective quantity of fluorocitrate; and (3) disturbance keys for which combined reaction on exposure to FA of intracellular processes, that manifest themselves in including both CNS and cardiovascular affects are char- clinical signs of the exposure (Pattison, 1959; Atzert, acteristic. On acute poisoning, independent of administra- 1971; Egekeze and Ochme, 1979b). Acute poisoning tion mode, the following symptoms are observed in these mainly affects the central nervous and cardiovascular animals: adynamia, salivation, vomiting, frequent defeca- systems. The visual symptoms of intoxication appear tion, pupil dilatation, nystagmus, accelerated respiration, as cycles, each involving three phases: (1) depression enhanced excitability, tremor and clonic-tonic convulsions

Table 1. Lethal doses and main clinical signs for some animals of the four groups, after the classiﬁcations of Chenoweth and Gilman (1946), and for humans

Group Animal Lethal dose Clinical signs References (mg kg−1)

1 Rabbits 0.5 Tachycardia and ventricular fibrillation without notable Marais, 1944; Quin and Clark, 1947; Chenoweth, Goats 0.6 CNS disorder; partial or complete heart block; edema, 1949; Egyed, 1973; Nwude et al., 1977 Sheep 2 congestion, acute vasculitis; dyspnoea and anoxic convulsions 2 Dogs 0.05 The CNS is primarily affected; foaming at the mouth and Chenoweth, Gilman, 1946; Chenoweth, 1949; nostrils, accelerated respiration; enhanced motive activity Egyed and Shupe, 1971; Buch et al., 1976 and barking; vomiting, frequent urination and defecation; recurrent clonic-tonic convulsions, disorganization of respiratory rhythm and respiratory center depression 3 Rats 0.1–5 Respiratory depression and ventricular fibrillation; tremor Chenoweth and Gilman, 1946; Chenoweth, 1949; Mice 0.5–19 and enhanced excitability; clonic-tonic convulsions; Pattison, 1959; Hayes, 1963 asthenia, ataxia, bradycardia; focal hemorrhage in lungs 4 Cats 0.2 Both CNS and cardiovascular affection; adynamia, Chenoweth and Gilman, 1946; Chenoweth, 1949; Pigs 0.4–1 sialorrhea, vomiting, frequent defecation, pupil dilatation, Pattison, 1959; Gammie, 1980 Rhesus 4–15 nystagmus, accelerated respiration, enhanced excitability, monkeys tremor, and clonic-tonic convulsions; cardiac arrhythmia and ventricular fibrillation; respiratory failure ?? Humans 2–10 Clonic-tonic convulsions; depression; loss of consciousness; Egekeze and Oehme, 1979b; Harrison et al., metabolic acidosis; hypotension; cardiac rhythm 1952; Chi et al., 1996, 1999; Reigart et al., disturbances (tachycardia, bradycardia, asystolia, ventricular 1975; Montoya and Lopez, 1983; Chung, 1984; fibrillations); respiratory failure; hypocalcemia and Gajdusek and Lutheer, 1950; McTaggart, 1970; hypopotassiemia; renal failure Gosselin et al., 1976; Robinson et al., 2002

(Chenoweth and Gilman, 1946; Gammie, 1980). After the most characteristic intoxication signs involve generalized spasmodic attack monkeys were deeply depressed ment- recurrent clonic-tonic convulsions alternating with deep ally and physically. One more attack of the same strength depression (Pattison, 1959; Robinson et al., 2002). A is very rare. Simultaneously, signs of cardiac arrhythmia sudden loss of consciousness (Gajdusek and Lutheer, up to ventricular fibrillation were observed, that were 1950; Gosselin et al., 1976) and coma (Gajdusek and responsible for the death of monkeys (Chenoweth, 1949; Lutheer, 1950; Trabes et al., 1983) may occur. These Pattison, 1959). Similar cardiovascular disorders devel- symptoms were associated with metabolic acidosis oped in cats and pigs, but unlike monkeys, cats generally (Taitelman et al., 1983a; Chi et al., 1996), hypotension died of respiratory depression resulting from direct (Harrison et al., 1952; Chi et al., 1996, 1999), as well CNS affection, whereas pigs died both of ventricular as cardiac rhythm disturbances, such as tachycardia fibrillation and of respiratory depression. (Reigart et al., 1975; Trabes et al., 1983), bradycardia The method of classification into four groups was re- (Gajdusek and Lutheer, 1950), asystolia (Gajdusek vised recently, following the conclusion that the general and Lutheer, 1950; Pridmore, 1978) and sustained acceptance of it obscured the fact that the principle signs ventricular fibrillations (Reigart et al., 1975; Montoya and of FA poisoning were common to most vertebrate species Lopez, 1983). Death occurs in 3 h to 5 days of heart (Sherley, 2004). The division of animals into cardiac and block, arrhythmia, or respiratory failure (Reigart et al., neurological symptomatic groups is considered to be 1975; Montoya and Lopez, 1983). Important diagnostic unsatisfactory as it ignores common neurological signs in symptoms are arrhythmia, a strongly changed shape of all the groups (e.g. ataxia, tremor, myotonic convulsions, the T wave, as well as prolongation of the QT interval muscular weakness, hypersensitivity and partial para- (Pattison, 1959; Chi et al., 1996). The shock resulting lysis). It also fails to recognize the neurological basis of from diminished systemic vascular resistance and signs/symptoms such as retching and vomiting, agitation, increased cardiac output was also observed (Chi et al., epigastric pain, incontinence and excessive salivation, 1999). Hypocalcemia (42%) and hypopotassiemia (65%) all of which are consistent with over-stimulation of the were the common electrolyte abnormalities. Kidneys are autonomic nervous system. The cardiac response in a among the most sensitive organs: acute FA poisoning pure form was determined just for a limited number resulted in acute renal failure associated with uremia of animals, but central nervous system involvement is (Chung, 1984). The most severe and irreversible sequelae clearly widespread. of acute FA poisoning are encephalopathia and in-life The clinical pattern of the acute poisoning in humans brain damage. Long after an acute poisoning (from 1.5 is similar to that observed in rhesus monkeys (Chenoweth to 9 years) psychic disorders, ataxia, tendency for and Gilman, 1946; Chenoweth, 1949; Hayes, 1963). The epileptoid seizures, extremity muscular hypertension,

Copyright © 2005 John Wiley & Sons, Ltd. J. Appl. Toxicol. 2006; 26: 148–161 152 N. V. GONCHAROV ET AL. spastic tetraplegia, blindness of central (cortical) origin Tissue citrate seems to be the only biochemical and diffuse brain atrophy were observed (Pridmore, 1978; parameter whose qualitative (but not quantitative) trends Trabes et al., 1983). In adults, the lowest effect level of are not a matter of contention. In cases where respiration FA, producing reversible intoxication symptoms, was is maintained mostly by acetate oxidation, the conversion found to be 0.1 mg kg−1 (Temple and Edwards, 1985). In of FA into FC is accompanied by an especially fast farm workers who were chronically exposed to low doses accumulation of citrate (Buffa et al., 1973). The citrate of FA for 10 years, neurologic and mild hepatic dys- concentration in tissues starts to increase 30 min after functions as well as renal tubular lesions were observed administration, attains a maximum within 4–6 h, and falls (Hayes and Laws, 1991). to the control level within 40 h (Pattison, 1959). On acute FA intoxication, the rat heart and serum citrate levels increase 8–15- and 5–10-fold, respectively. However, in Pathogenesis: Physiological and dogs, the heart and serum citrate levels increase as little Biochemical Features of FA Action as 2–3-fold, and the heart ATP level decreases only slightly. An increase in citrate level is directly related to The pathogenesis of FA poisoning is intricate and not yet the respiratory activity of the tissue: metabolically active clear. Published reports of physiological and biochemical tissues, such as heart, kidney and spleen, accumulate responses to FA are rather controversial. The toxicolog- citrate to the greatest extent. However, liver, whose ical pattern is better understood — provided the animal respiratory and metabolic activities are also high, tends to species, metabolic features of one or another organ or accumulate little citrate (Cole et al., 1955; Twigg et al., tissue, intoxication degree and dynamics of parameters 1986). are taken into account. The series of pathological The increase in cellular citrate induces lesions in glu- phenomena is triggered in the tricarboxylic acid (TCA) cose metabolism through inhibition of the key regulatory cycle, which predetermines the entire complex of anoma- enzyme of glycolysis, phosphofructokinase (Bowman, lous processes and the degree of affection. The effect 1964; Peters, 1972). Moreover, succinate dehydrogenase of FA on the physiological biochemical and functional activity is also inhibited (Fanshier et al., 1964), though status of cells, organs and tissues is directly related to this requires very high concentrations of FC (Kun, 1969). the level of cellular oxidative metabolism. For instance, Pyruvate dehydrogenase activity may increase two-fold neither sodium fluoroacetate nor sodium malonate in the presence of FA, and this effect is associated (succinate dehydrogenase inhibitor) are capable of inhib- with FA-induced inhibition of pyruvate dehydrogenase iting phagocytosis, since TCA cycle activity is poorly kinase, which is responsible for inactivating the pyruvate developed in macrophages (Cifarelli et al., 1979). Prob- dehydrogenase complex via phosphorylation (Taylor ably of importance are the relative activities of different et al., 1977). The kinase inhibition cannot be explained metabolic pathways: glycolysis and pentosophosphate by the absence of ATP, since FA had no effect on the shunt, pyruvate dehydrogenase and thiokinase ways of tissue ATP level in the experiment. At the same time, the carbon input to the TCA cycle, and transamination. accumulation of citrate suggested aconitase inhibition. The biochemical and physiological effects of FA Citrate inhibited guanylate cyclase in rat hepatocytes include: excessive citrate accumulation (Buffa and Peters, (Dohi and Murad, 1981), whereas FA in adipose tissue 1950; Egyed and Brisk, 1965; Buffa et al., 1973); dis- reduced the basal and hormone-stimulated cyclic AMP turbed citrate transport in mitochondria (Eanes et al., levels by inhibition of adenylate cyclase and had no 1972; Kirsten et al., 1978); increased lactate level (Engel effect on the activity of cAMP phosphodiesterase (Taylor et al., 1954; Schultz et al., 1982; Taitelman et al., et al., 1977). 1983b); deviations in carbohydrate metabolism regulation One of the adverse effects of excess citrate is disturbed (Elliott and Phillips, 1954; Bobyleva-Guarriero et al., electrolyte and acid-base balance in the organism. Thus 1984); decreased level of free fatty acids (Liang, 1977); systemically administered FA reduced arterial pH (Szerb decreased tissue level of macroergotic compounds and Redondo, 1993). Further development of acidosis (Williamson, 1967; Stewart et al., 1970); increased is associated with lactate accumulation, which retards adenosine level (Liang, 1977); decreased oxygen glycolysis as well by the feedback mechanism. Lactic consumption (Saito, 1990); disturbed balance of acid accumulation in cerebrospinal fluid, along with bivalent cations, specifically Ca2+ (Buffa and Peters, citrate accumulation, is considered to be one of the 1950; Bosakowski and Levin, 1986); acid-base imbalance reasons for a coma with convulsions (Stewart et al., (Taitelman et al., 1983b; Szerb and Redondo, 1993); 1970). increased ammonium level (Stewart et al., 1970); Experiments with a biologically active isomer of changed γ-aminobutyric acid level (Maytnert and Kaji, FC, (−)-erythro-fluorocitrate, showed that 50 pmol l−1 1962; Stewart et al., 1970); increased plasma levels of of this compound selectively and irreversibly inhibited phosphorus and various enzymes (Bgin et al., 1972); mitochondrial citrate metabolism; not only ATP synthe- hemodynamic disorders (Liang, 1977). sis, but also cytoplasmic fatty acid synthesis associated

Copyright © 2005 John Wiley & Sons, Ltd. J. Appl. Toxicol. 2006; 26: 148–161 TOXICOLOGY OF FLUOROACETATE 153 with intramitochondrial generation of citric acid, were intoxication can even change into hypoglycemia (Boquist inhibited to an equal degree (Eanes et al., 1972; Brand et al., 1988). For this reason, the effect of FA was even et al., 1973; Eanes and Kun, 1974). Since aconitase is not considered to be similar to that of insulin (Zieve et al., involved directly in triglyceride metabolism, the existence 1983). However, a fundamental difference was that FA of a (−)-erythro-fluorocitrate inhibitory site hindering intoxication produced glycogen depletion in organs and citrate transport through the inner mitochondrial mem- tissues (Godoy et al., 1968; Boquist et al., 1988). There brane was suggested (Eanes et al., 1972; Kirsten et al., is evidence that 1 and 2 h after exposure to FA the 1978). glycogen level in animals decreased by 75% and 90%, It was proposed that changes in carbohydrate meta- respectively (Buffa et al., 1973; Zhou et al., 1984). Such bolism result not only from the direct effect of FA, FC decreases may result from an indirect effect dependent on and excess of citrate on the enzymatic systems of peri- adrenaline secretion or sympathetic regulation (Buffa pheral organs and tissues, but may also be associated et al., 1973), although extensive studies failed to provide with imbalance in sympathetic and hormonal regulation a clear explanation for the glycogen depletion (Elliott (Cole et al., 1955; Buffa et al., 1973). Thus some authors and Phillips, 1954; Williamson, 1967; Buffa et al., 1973; assigned the increase of blood glucose to reduced insulin Wiedemann et al., 1983). De novo glycogen synthesis secretion by pancreatic β-cells affected by FA (Cole was also retarded, by 85%–90% on average (Zhou et al., et al., 1955; Karam and Grodsky, 1962). Actually, insulin 1984). A negative modulation of gluconeogenesis in secretion is controlled by glucose and neurohumoral hepatocytes of poisoned animals was reported (Dickson factors, such as acetylcholine (ACh) that activates the and Langslow, 1977; Baunister and O’Neil, 1981). This phospholipase C and Ca2+ signaling pathway. Glucose is effect was associated with a number of factors, such as: inherently incapable of inducing insulin secretion without reduced activity of the TCA cycle; inactivation of lactate increasing the concentration of intracellular Ca2+, which dehydrogenase; blockade of the malate shuttle; accumu- in turn depends on ATP regeneration by the metabolism lation of NADH in the cytoplasm; and decrease of of glucose per se and other energy substrates. TCA mitochondrial oxaloacetic acid for phosphoenolpyruvate cycle blockage by FA almost completely suppresses carboxylase. ACh-induced mobilization of intracellular calcium. Under Hormonal regulation probably still has a role in FA these conditions, ACh can increase the concentration of intoxication and serves to decrease the concentration of Ca2+ in β-cells only if an alternative energy substrate is plasma calcium. This is assigned to a disturbance in ionic present, such as glutamine (Schofl et al., 2000). re-absorption in the kidneys, resulting from a decrease Along with hyperglycemia, a serious hyperketonemia in the concentration of parathyroid hormones, which is observed, which is also characteristic of diabetes normally favor calcium re-absorption in the distal seg- (Williamson, 1967; Taitelman et al., 1983b). This is ments of convoluted tubules (Buffa and Peters, 1950; associated with depletion of tissue oxalic acid, resulting Perez and Frindt, 1977). Along with the decrease in from TCA cycle blockade (Engel et al., 1954; Buffa plasma calcium, enhanced urinary excretion of Ca2+, up et al., 1973; Taitelman et al., 1983b). The attendant to 0.173 mg min−1 (normally about 0.06 mg min−1), was formation of two-carbon fragments reaches its maximum observed (Perez and Frindt, 1977). Therefore, the primary 20–25 h after intoxication (Cole et al., 1955). Condensa- effect of citrate on both calcium and parathyroid hormone tion of the two-carbon fragments affords acetoacetate levels is not excluded. Impaired Ca2+ re-absorption aggra- and other ketone bodies, enhancing acidosis, attenuating vates hypocalcemia and its consequences (Buffa and glucose utilization and increasing the citrate level (Miller Peters, 1950; Peters et al., 1972). FA decreases the et al., 1982). Evidence for the analogy with diabetes calcium level in animals by 27% within 40 min (Peters was given by the fact that in adipose tissue FA inhibited et al., 1972). This decrease may be responsible for the hormone-stimulated lipolysis, simultaneously decreasing so-called ‘hypocalcemic tetanus’ (Roy et al., 1980) that cyclic AMP levels by inhibition of adenylate cyclase manifests itself in typical convulsions, blood clotting (Taylor et al., 1977). Moreover, FA accelerated glucose lesions and hypotension leading to vascular collapse and conversion into fatty acids in their experiments. Such death (Arena, 1970). It was also found that the degree of a combination of the antilipolytic and lipogenic effects hypocalcemia correlates with prolongation of the Q-T suggested that FA and insulin act by a common mech- interval in ECG, which is a consequence of a broad range anism. Subsequently, however, it was reported that of cardiac arrhythmias, from scarcely detected ventricular insulin administration had no effect on the degree of FA fibrillations to various atrioventricular blocks (Buffa and intoxication in general and no symptomatic influence on Peters, 1950). the ‘fluoroacetate diabetes’ in particular (Reichelt, 1979). Both in vivo and in vitro experiments showed that The actual reason for FA-induced hyperglycemia was the acute FA poisoning decreases the ATP level in rat heart blockade of glucose utilization, resulting from phospho- by 40% within 6 h and by 55% within 18 h. The concen- fructokinase inhibition (Bowman, 1964; Peters, 1972). trations of ADP and AMP increase within a few hours Moreover, hyperglycemia initially developed upon FA and then decrease. The decrease in heart ATP level is

Copyright © 2005 John Wiley & Sons, Ltd. J. Appl. Toxicol. 2006; 26: 148–161 154 N. V. GONCHAROV ET AL. accompanied by a decrease of ATP in other organs and suppression of glia in the retrotrapezoid nucleus causes tissues (Bowman, 1964; Koenig and Patel, 1970; Stewart temporary and reversible acidiﬁcation of the extracellular et al., 1970; Cremer-Lacuara et al., 1980). The changes medium (Erlichman et al., 1998). Together with the in ATP concentration are not associated with uncoupling natural hypercapnic carbonate acidiﬁcation of the extra- of respiration and phosphorylation (Fairhurst et al., 1958; cellular medium (normocapnia) this activates the dia- Stewart et al., 1970), as evidenced by the normal ADP/ phragmal nerve by 43% and increases the expired minute O (or P/O) indices. Moreover, evidence is available that ventilation by 38% (Erlichman et al., 1998; Holleran FA may have no effect on the ATP levels in certain et al., 2001). During hypercapnia, breathing frequency organs and tissues. Thus FA had no effect on ATP and is also enhanced. Exposure to FA results in attaining

GTP, or on cyclic nucleotide levels in hepatocytes maximum ventilation at 4% CO2 against 8%–10% in in vitro, whereas rotenone, oligomycin, antimycin, control hypercapnic trials (Holleran et al., 2001). The dinitrophenol, potassium cyanide and arsenate decreased physiologic effects of FC, at least its ventilatory effects, GTP, ATP and cyclic GMP levels (Dohi and Murad, can be reversed even on repeated exposure, implying a 1981). Brain mitochondria were completely unaffected high restorative potential of astrocytes (Holleran et al., by citrate accumulation, whereas kidney mitochondria 2001). Also, FA selectively affects potassium conduct- reduced their oxidative activity (Corsi and Granata, ance in astrocytes, thus affecting neuronal signal pro- 1967). pagation. The blockade of astrocyte metabolism impairs fast synaptic transmission and induces a delayed excitation, probably resulting from a combination of reduced Pathogenesis: Action of FA on the Cells repolarization of neurons and persistent depolarization of of Nervous System astrocytes (Hulsmann et al., 2003). It was established in the 1960s that FA only partially Research into the effect of FA on the nervous system is (by 35%–55%) blocked the TCA cycle in nerve tissues closely related to the discovery of the role of glia in the (Patel and Koenig, 1968). Both FA and FC were found functional activity of brain. At least four mechanisms of to affect the composition of brain amino acids (Clarke astrocyte effect on neuron function are presently known: and Nicklas, 1970; Paulsen et al., 1987). Thus, FC (1) lactate metabolism (Schurr et al., 1997; Kitano et al., administered to the brain decreased the level of glutamine 2003); (2) glutamate uptake from the synaptic gap (Aoki and aspartate but increased that of alanine. Glutamic acid et al., 1987; Szerb and O’Regan, 1988); (3) ionic regu- mostly metabolizes in glia, unlike glucose whose meta- lation in the extracellular medium (Orkand and Opava, bolism occurs primarily in neurons (Muir et al., 1986). 1994; Erlichman et al., 1998); and (4) reciprocal calcium- Regardless of the fact that glutamine synthesis is ATP- dependent interaction providing synaptic plasticity dependent, FA suppressed glutamine synthesis in glia (Vernadakis, 1996). while having no effect on ATP synthesis and oxygen The rate of acetate metabolism in astrocytes, measured consumption (Benjamin and Verjee, 1980). Similarly, by the yield of CO2, is about 18 times as high as that in an experiment on primary astrocyte cultures showed cortical synaptosomes. At the same time, the activity of that FA and FC only slightly decrease the astrocyte acetyl-CoA synthase, the ﬁrst enzymatic step in acetate ATP level, but decrease by almost 30% the energy- utilization, was greater in synaptosomes than in astrocytes dependent glutamate uptake and by 60%–65% the (5.0 and 2.9 nmol min−1 per milligram of protein, respect- astrocyte glutamine production in the absence of ively). The principal difference in the acetate metabolism exogenous glutamate and aspartate (a measure of carbon rates is explained by the different transport functions; ﬂux through the TCA cycle). When 50 µm of glutamate acetate uptake by astrocytes, unlike synaptosomes, was present in the incubation medium, glutamine produc- rapidly increases and follows saturation kinetics (Vmax = tion remained at the control level (Swanson and Graham, −1 −1 −1 498 nmol mg protein min , Km = 9.3 mmol l ). Acetate 1994). However, in another experiment on astrocyte transport is mediated by a monocarboxylate carrier cultures, FC (100 µM) not only inhibited glutamine and inhibited by FA. Moreover, the transport is synthesis from acetate and glucose, but also reduced the inhibited by L-lactate, pyruvate, propionate, α-cyano-4- ATP level by 40% (Hassel et al., 1994). Other evidence hydroxycynnamate (Waniewski and Martin, 1998). is available to show that FC does reduce the ATP level Having penetrated into astrocytes at one site, FA can (Hertz et al., 1996; Lian and Stringer, 2004). This contro- diffuse into other cells through gap junctions. Evidence versy can be understood in view of the fact that no FC- for this is provided by fast diffusion of the dye Lucifer induced ATP reduction was observed in the presence Yellow, which was introduced into one astrocyte and of 2.5 mmol l−1 of glutamine (Hassel et al., 1994). With then detected in approx 100 cells (Ransom, 1995). Citrate increasing concentration of FC, glutamine utilization also accumulates largely in astrocytes; it is readily increased and glucose utilization decreased. The experi- released and then effectively penetrates other astro- ment revealed the possibility for switching the normal cytes (Westergaard et al., 1994). Selective FA-induced natural metabolic pathway: 2-oxoglutarate, rather than

Copyright © 2005 John Wiley & Sons, Ltd. J. Appl. Toxicol. 2006; 26: 148–161 TOXICOLOGY OF FLUOROACETATE 155 being used for glutamine formation is used in the TCA Therapy for FA Intoxication cycle, whereas if an exogenous glutamine is present it is used in the TCA cycle via conversion to 2-oxoglutarate, Antidotal therapy for FA intoxication is aimed at i.e. glutamine serves as an energy substrate. Similar re- developing a means of preventing FC synthesis and action pathways were found in the renal cortex (Yu et al., aconitase blockade in mitochondria, and to provide citrate 1976). Glutamate dehydrogenase (GDH) is an exclusively outflow from mitochondria. In view of the fact that mitochondrial enzyme. The concentration of GDH in acetate groups prevent FA from metabolism into FC, neurons is low, unlike that in astroglia, where the pres- much emphasis has been placed on identifying com- ence of GDH depends not only on the proximity of pounds capable of donating acetate groups. Monoacetin glutamatergic fibers and terminals, but also on the act- (glycerol monoacetate) (Chenoweth, 1949; Egyed, 1971), ivity of proximate neurons, irrespective of their function; acetamide (Giller, 1956; Egyed and Shlosberg, 1977), deficiencies of glial GDH may provoke the cytotoxic cortisol acetate (Cole et al., 1955) and ethanol (Hutchens effects of excessive glutamate and aspartate levels (Aoki et al., 1949; Chenoweth, 1949) have been tried as anti- et al., 1987). dotes. Ethanol was the first and, probably, is the most Inhibition of glial glutamate uptake may be respon- effective FA antidote known; its oxidation increases sible for the convulsions caused by FA treatment in vivo blood acetate level and inhibits FC production (Hutchens (Szerb and Issekutz, 1987), whereas the reasons for con- et al., 1949; Chenoweth, 1949). On FA poisoning in vulsions observed in some animals are not established rabbits at a dose of 0.5 mg kg−1 and administering ethanol conclusively. There is an opinion that the toxic effect of (800 mg kg−1) 5 min after poisoning, 6 of 10 animals died FA is largely associated with citrate chelation of calcium (100% mortality in control), and on FA poisoning at a ions (Fonnum et al., 1997). Intrathecal injection of dose of 0.25 mg kg−1, only one animal died (6 of 10 in fluorocitrate in mice results in convulsions after an aver- control). Apart from inhibition of FC synthesis, ethanol age latency of 15 s, while intracerebroventricular injection decreases hyperglycemia (Prasanna and Ramakrishnan, produced convulsions after 36.5 min, and required higher 1984) and increases GABA levels in the cerebral hemi- doses to achieve this effect. Intrathecal injection of citrate spheres and cerebellum (Sytinskii et al., 1986). On acute produces the same effects. These findings imply that the FA intoxication in humans, immediate oral administration main target of FC and citrate, and the site where convul- of 40–60 ml of 96% ethanol has been advised, followed sions are generated, is the spinal cord. Glutamate, lactate, by 5%–10% ethanol intravenously at 1.0–1.5 g kg−1 EDTA and EGTA produced convulsions similar to FA. during the first hour and 0.1 g kg−1 each hour during the These compounds are structurally unrelated to FA, but following 6–8 h (Hutchens et al., 1949). A therapeutic have one common feature with it: the ability to chelate effect was also observed on simultaneous administration calcium ions, as judged from the fact that their neuro- of ethanol and acetate (Hutchens et al., 1949; Tourtelotte toxicity is considerably reduced by coadministration and Coon, 1949). However, these regimens with ethanol of calcium (Hornfeldt and Larson, 1990). Along with are not commonly applied at resuscitation units because increased citrate level, activation of anaerobic neuronal of delayed admission, the usual lack of exact information glucose metabolism and accumulation of lactic acid in about the poisoning and the similarity of the clinical the cerebrospinal fluid may also be responsible for a signs to those of a number of natural diseases. So treat- coma with convulsions (Stewart et al., 1970). Disorders ment of the patients is essentially symptomatic and sup- in γ-aminobutyric acid (GABA) metabolism associated portive, with special attention to be focused on stabilizing with inhibition of the TCA cycle have been reported. cardiac and CNS functions (Dorman, 1990; Chi et al., It is well known that GABA is synthesized in neurons 1996, 1999; Norris, 2001). exclusively (Szerb and Issekutz, 1987). After FA injec- Monoacetin and acetamide also increase the acetate tion, the GABA concentration in various brain regions level in the organism (Buck et al., 1976; Egekeze and first increases and then decreases concurrently with Oehme, 1979b). It was established that for successful the initiation of clonic-tonic convulsions (Peters, 1952; therapy more than 100 mg kg−1 of monoacetin is required Maytnert and Kaji, 1962; Stewart et al., 1970). Citrate (Engel et al., 1954; Rammel et al., 1985). Monoacetin chelation of zinc and other bivalent ions in the nerve functions to decrease the level of ketone bodies. Intrave- tissue results in an enhancement of the signal activity nous injection at a dose of 0.5 mg kg−1 immediately after of N-methyl-D-aspartate (NMDA) receptors (Westergaard intoxication and every 30 min over the course of the et al., 1995). Convulsions are aggravated by an increase following 5 h is recommended (Engel et al., 1954). in ammonium concentration by 84% on average (Patel Decreased citrate concentrations in the heart, brain and and Koenig, 1968) and up to 225% in brain, 80–100 min kidney of experimental animals were also noted. It was after fluoroacetate injection (Turner and Whittle, 1983). established that monoacetin not only prevents but also Hyperammoniemia was assumed to arise from a disorder reverses the neurotoxic and cardiotoxic effects of FA in the urea cycle responsible for ammonium assimilation in monkey even if administered 30 min after the poison- (Iles and Jack, 1980). ing, when convulsions have only started to develop;

Copyright © 2005 John Wiley & Sons, Ltd. J. Appl. Toxicol. 2006; 26: 148–161 156 N. V. GONCHAROV ET AL. it normalized cardiac rhythm and smoothed cerebral sodium succinate (240 mg kg−1) (Omara and Sisodia, bioelectrical activity (Egyed, 1971). The therapeutic 1990), but it may hardly be considered to be more effec- effect of acetamide was studied on various endotherms tive in comparison with ethanol, taking into consideration (Giller et al., 1953; Giller, 1956; Egyed and Schultz, their experimental approach. 1986). It was found that this compound provides a The tranquilizer diazepam was used for prevention or protective effect in sheep if administered at a dose of cessation of generalized convulsions (McTaggart, 1970). 2gkg−1 before or concurrently with FA. Protection of Barbiturates, such as sodium thiopental (Taitelman et al., rats or guinea pigs on lethal FA poisonings is attained at 1983b; Trabes et al., 1983), were also used to reduce the a single oral administration at 0.5 and 5 g kg−1, respect- risk of local ischemia and brain edema in cases where ively (Egyed and Schultz, 1986). Acetamide exerts no tranquilizers were insufficiently effective against convul- therapeutic effect when applied 30 min after FA poison- sions (Arena, 1970). To compensate hypocalcemia, intra- ing (Giller et al., 1953; Giller, 1956). The negative venous calcium gluconate, thiosulfate or chloride were effects of monoacetin and acetamide comprise aggrava- applied (Gajdusek and Lutheer, 1950; Brockmann et al., tion of hyperglycemia and metabolic acidosis, damage to 1955; Taitelman et al., 1983b). Calcium treatment has capillaries and hemolysis of erythrocytes (Engel et al., been shown to reduce muscular agitation, normalizes 1954). Moreover, these compounds can increase citrate myocardial retraction and increases survival (Roy et al., concentration in various organs (Egyed and Shlosberg, 1980, 1982). 1973). For these reasons, monoacetin and acetamide Cardiac arrhythmias are frequently treated with β- cannot be recommended for humans (Rammel et al., blockers (Anaprilin®, Obsidan®). It was shown that 1985; Egyed and Schultz, 1986; Spoerke et al., 1986). sodium FA has no effect on adrenergic receptors; for this Cortisol acetate offers certain advantages, since, apart reason both α- and β-blockers were ineffective (Burande from the primary effect (inhibition of FC synthesis), it et al., 1983). However, evidence is available for the pro- prevents ketosis. At the same time, hyperglycemia is tective effect of obsidan in poisoned laboratory animals enhanced (Cole et al., 1955). (Huang et al., 1980). The cardiac arrhythmias associated Another group of antidotes was proposed whose effect with FA poisoning were effectively treated with novo- was caused by the ability to activate the transport of TCA cainamide (Harrison et al., 1952; Brockmann et al., 1955; cycle intermediates through mitochondrial membranes. Arena, 1970). The acute cardiovascular failure associated To this end, fluoromaleic acid was proposed (Peters, with the disturbance of cardiac rhythm and transmittance 1972). However, the mechanism of its action was not is abolished by reserpin. This effect is enhanced by established, and the positive effect was negligible. At the simultaneous application of oxytocin, a hormone that is same time, Buffa et al. (1972) proposed malate as an also used for the treatment of cardiac arrhythmias (Huang activator of citrate transport. It was assumed that malate et al., 1980; Beaulnes et al., 1964). competes with FA for binding to the synthase-hydrolase carrier system. A positive effect was obtained in vitro, whereas the results in vivo were not so encouraging. Are there Alternative Strategies to Glutathione and a series of SH-containing compounds Development of Effective Therapies? (cysteamine and N-acetylcysteine) were tried in vitro and Discussion proved rather effective remedies, protecting transferases from the inhibiting effect of FC and preventing aconitase Paradoxical as it may seem, decades of FA studies have blockade in mitochondria (Mead et al., 1985). However, led to the opinion that therapeutic success in FA and these preparations were incapable of replacing glutathione similar poisonings is more dependent upon symptomatic in enzymatic FA defluorination reactions and have not and supportive care rather than the use of antidotal found practical application. therapy (Dorman, 1990; Norris, 2001). In spite of the fact In view of the fact that FA intoxication disturbs the that great progress has been made in understanding the TCA cycle, succinate, malate and glutamate were tested, mechanism of action of FA, it is still ‘good old ethanol’ but none exhibited a protective effect (Hutchens et al., that has been the most acceptable therapeutic agent for 1949). In experiments on isolated rat kidney tubules the past 60 years, and only if administered immediately incubated with FA, however, positive results with 2- after the poisoning. Why have more effective therapies oxoglutarate were obtained (Wiedemann et al., 1983). not been found? We argue that it is largely a conse- Increased ATP levels and delayed citrate accumulation quence of specialization within the scientific endeavors were noted. Calcium gluconate (neutralization of hypo- undertaken, with an apparent lack of integral toxicolog- calcemia), sodium 2-oxoglutarate and sodium succinate ical studies. There is already a great deal of scientific were also tested. All three individually, as well as a com- knowledge upon which new approaches to the develop- bination of calcium gluconate and sodium 2-oxoglutarate, ment of an effective therapy can be based. For example, proved ineffective. A therapeutic effect was obtained with Gardner’s discovery of high sensitivity of aconitase a combination of calcium gluconate (130 mg kg−1) and to inhibition by superoxide generated during cellular

Copyright © 2005 John Wiley & Sons, Ltd. J. Appl. Toxicol. 2006; 26: 148–161 TOXICOLOGY OF FLUOROACETATE 157 hyperoxia. This effect is comparable to the effects of FA Table 2. Toxicological assessment of METIS effective- or fluorocitrate in vitro (Gardner et al., 1994). Further- ness upon acute poisoning of female rats with sodium more, aconitase was shown to be equally highly sensitive fluoroacetate (SFA) to nitric oxide (Andersson et al., 1998). Reactive SFA dose Lethal Probit analysis oxygen species (ROS) and nitric oxide (NO) inhibit the (mg kg−1) frequency after Litchfield- mitochondrial and cytoplasmic aconitase via reaction with and (therapy) Wilcoxon the [4Fe-4S]2+ cluster, the mitochondrial aconitase being more sensitive to NO (Castro et al., 1998). This means 1.17 (none) 1/6 LD16 = 1.179 1.4 (none) 3/6 LD50 = 1.368 that ROS and NO could serve as competitive antagonists 1.75 (none) 6/6 (1.215 ÷ 1.540) of FC to prevent its inhibitory action on aconitase. It was LD84 = 1.586 also shown that peroxynitrite inhibits aconitase activity in 3.0 (METIS) 0/6 LD16′ = 3.199 3.5 (METIS) 9/18 LD50′ = 3.476 rat heart ventricular homogenates (Cheung et al., 1998). 4.0 (METIS) 6/6 (3.292 ÷ 3.670) Since it reacts with sulfhydryl groups, conditions should LD84′ = 3.778 ′ be developed to avoid the formation of peroxynitrite at KLD16 = LD16 /LD16 = 2.7 ′ KLD50 = LD50 /LD50 = 2.5 effective concentrations. K = LD84′/LD84 = 2.3 The monoenzymic mechanism of FA action was LD84 once disputed, based on the following reasoning: if METIS therapy involved administration of an antidote at 10 min and at 2 h fluoroacetyl CoA is a structural analog of acetyl CoA, after poisoning. their competition for the active site of an enzyme should formation, which would maintain the flux of reducing affect metabolic reactions involving acetyl CoA (Fanshier equivalents into the TCA cycle and ATP synthesis (Yu et al., 1964). Today, this reasoning indicates that not only et al., 1976). Evidence in favor of the compensatory would it be a good idea to look for a new biochemical glutamate utilization was also provided by an experiment signs of the intoxication (for example, N-acetylation), but on the physiology of dogs exposed to FA; the fact that also that substances other than acetate that competitively exposure to sublethal doses of FA has no effect on bind with CoA may provide an effective therapy (for oxygen consumption and ATP level was explained by the example, the well-known CoA binding metabolites such utilization of glutamate and aspartate, that enter the Krebs as malonate, methylmalonate, propionate and succinate). cycle after aconitase, as well as by the compensatory Citrate transport blockade by FA does not necessarily occur in all the organs and tissues. It was shown that effect of citrate (Liang, 1977). about 32% of the citrate synthesized in mitochondria In summary, two principal ways of developing effec- passes into the extramitochondrial space (Buffa et al., tive therapies for FA intoxication have been highlighted. 1972). Evidence is available for citrate transport and/or Firstly, competitive inhibition of FA interaction with diffusion from mitochondria to cytosol in liver and CoA and FC interaction with aconitase, and secondly, heart, with subsequent utilization by extramitochondrial channeling the alternative metabolic pathways. Both of aconitase (which is almost unaffected by FA intoxication) these approaches have been developed by our scientific and NADP-dependent isocitrate dehydrogenase (Max team and very encouraging results have been obtained. and Purvis, 1965). These processes should be considered There is now the matter of patenting, but the resulting adaptive and positive, since they serve for reducing data of one of the approaches may be presented oxygen uptake: the NADPH formed needs not to be (Table 2). The first experimental therapeutic means called K oxidized in the respiratory chain and can take part in METIS showed very good coefficient of efficiency ( ) at glutathione-dependent defluorinating reactions. On the the level of 2.5. other hand, due to the liberation of citrate from mito- Acknowledgements—This work is supported by the BioIndustry Initia- chondria, the cytoplasm is also provided with carbon for tive Program (BII, Director — Dr Jason Rao) of the US Department of fatty acid and steroid synthesis, as well as with NAD+ by State, ISTC grant BII-2629. ATP-dependent citrate lyase and malate dehydrogenase (Denton and Halestrap, 1979). 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