Studies on the Metabolism of Monofluroacetate in the Liver of a Frog

Many workers found that the poisoning animals with sodium fluoroacetate led quickly to a marked increase of citrate in the various tissues, but it failed to find such result in the liver in vivo as well as in vitro. In the liver of a frog, however, an accumulation of a large amount of citric acid was found 6 hours after the poison was orally administered to the animal. To decide whether the accumulation of citric acid in the liver of a frog is due to the formation of fluorocitrate in the liver itself or to the transport of fluorocitrate formed in other organs into the liver, we carried out the experiments using the mitochondrial particles prepared from a frog liver. And it was found that the oxygen uptake of mitochondria was inhibited and that the citrate synthesis of the particle increased by the addition of fluoroacetate with pyruvate and malate as substrates.     

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.     

Metabolism of fluoroacetate in the skink (Tiliqua rugosa) and the rat (Rattus norvegicus)

Administration of 100 mg sodium fluoroacetate (compound 1080) per kilogram body weight to T. rugosa resulted in a 3.4-fold increase in plasma citrate levels 48 h after dosing while administration of 3 mg sodium fluoroacetate per kilogram body weight to R. norvegicus produced a fivefold increase in plasma citrate levels within 4 h. Administration of 300 mg sodium fluoroacetate per kilogram body weight reduced the oxygen consumption of the skink by between 2.5 and 11% while in the rat, 2 mg sodium fluoroacetate per kilogram body weight reduced oxygen consumption by between 28 and 57%. Aconitate hydratase activity in extracts of liver acetone powders from T. rugosa was less inhibited by (-)erythrofluorocitrate (Ki: 0.065 mM) than that in extracts derived from R. norvegicus (Ki: 0.026 mM). The rate of defluorination of fluoroacetate in erythrocytes and in extracts of liver acetone powders of T. rugosa was 8- and 4.5-fold greater, respectively, than that found in similar preparations from R. norvegicus. A rapid rate of defluorination together with a low reliance on aerobic respiration favoured detoxification of fluoroacetate in T. rugosa rather than its conversion into fluorocitrate. Though defluorination in this species helped to minimize the immediate effects of fluoroacetate on aerobic respiration, it resulted in rapid depletion of liver glutathione levels.     

Experiments on the fluoroacetate metabolism of Dichapetalum cymosum (Gifblaar)

It was shown that application of fluoroacetate to leaf disks of Dichapetalum cymosum (Gifblaar) did not lead to an inhibition of oxygen uptake or accumulation of citrate, in contrast to the 'control plant' Parimarium capense which lacks fluoroacetate. The addition of fluorocitrate did, however, inhibit the oxygen uptake of both plants and caused an accumulation of citrate. From the results it was deduced that either citrate synthetase or acetate thiokinase from D. cymosum had different affinities for the fluorinated derivative and the 'normal' metabolite. The addition of fluoropyruvate to leaf disks caused a decrease in oxygen uptake and no change in the citrate concentration. From this it was deduced that fluoropyruvate inhibited pyruvate oxidase in both plants. It was concluded that the tolerance of D. cymosum to such high concentrations of fluoroacetate may be ascribed to the fact that the 'lethal synthesis' of fluorocitrate does not take place in the plant most probably because citrate synthetase has different affinities for fluoroacetylcoenzyme A and acetylcoenzyme A.     

Studies on the Abnormal Metabolism in Animals Poisoned with Sodium Fluoroacetate

The changes of metabolisms in the calf, dog and rat following administeration of fluoroacetate were studied. It was found that citrate is accumulated in the tissues, and increased amounts of acetone bodies are excreted in the urine of the animals which received fluoroacetate, and their urinary nitrogen also increases. All these abnormal accumulations and excretions of the rat after injection of fluoroacetate diminish within 24hrs., suggesting the decrease of the toxity or the excretion of the poison substance.

Significant amounts of organic fluorine compounds were detected in various tissues of fluoroacetate-treated calves.

The lethal dose limit of fluoroacetate for the calf could not be determined. As for the dog, 0.02 mg/kilo of fluoroacetate seemed to be fatal.     

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 -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.     

Metabolic differences betweenEscherichia coli cultures growing aerobically and anaerobically in the presence of fluoroacetate

The amount of citrate and pyruvate increased during the stationary phase of anEscherichia coli B culture growing in a synthetic medium, aerobically in the presence of glucose. In an anaerobic culture the amount of citrate was minute and did not rise during the stationary phase; the level of pyruvate in the stationary phase rose only slightly. Fluoroacetate blocked the growth of cells both aerobically and anaerobically. Cultures growing aerobically in the presence of fluoroaeetate displayed an increased accumulation of citrate as compared with the control. In anaerobic cultures citrate did not accumulate in the presence of inhibitory concentrations of fluoroacetate. In the presence of 2mm fluoroacetate cells grew aerobically somewhat more slowly at first, then inhibition ceased and finally the growth yield was greater than in the control. The obtained data indicate that citrate accumulated at first was partly utilized for growth under these conditions. The results are discussed from the point of view of differences in the metabolism of aerobically and anaerobically grown cells.     

Tissue slices for the evaluation of metabolism-based toxicity with the example of diclofenac

Drug-induced organ injury is a multifaceted process, involving numerous cell types and mediators, and remains a significant safety issue in pharmaceutical development and clinical therapy. Organotypic in vitro models, including precision-cut tissue slices, possess the multi-cellular, structural and functional features of in vivo tissue to facilitate the elucidation of mechanisms of drug-induced organ injury and to characterize species susceptibilities. This study reviews diclofenac-induced hepatotoxicity and presents data comparing the metabolism, specific binding of diclofenac products to cellular proteins and the effects on liver function in rat, monkey and human liver slices. Concentration- and time-dependent increases in specific protein binding demonstrate the progressive nature of the toxicity. Altered liver function correlated with the species differences in the extent of diclofenac metabolism (rat > monkey or human). Liver injury was not detectable within 24 h, unlike specific protein binding, yet it developed by 48 h and lower concentrations of diclofenac exhibited effects by 72 h, demonstrating that continued metabolism and the accumulation of specific protein binding could lead to altered cell function. The decline of liver slice ATP levels at concentrations not affecting GSH levels implicates mitochondrial dysfunction as a primary indicator of hepatotoxicity, of which oxidative stress may be a contributing cause. Diclofenac affected monkey liver slices function at similar concentrations as rat liver slices, while human liver slices exhibited less extensive specific protein binding and required higher diclofenac concentrations to alter cell function.     

Oxalate accumulation from citrate by Aspergillus niger. II. Involvement of the tricarboxylic acid cyclase.

Carbon-14 was incorporated into oxalate and CO2 from either citrate-1,5-14C, succinate-1,4-14C, or fumarate-1,4-14C by cultures of Aspergillus niger pregrown on a medium which contained glucose as the sole carbon source and which did not allow citrate accumulation. In cell-free extracts of mycelium forming oxalate and CO2 from added citrate the following enzymes of the tricarboxylic acid (TCA) cycle were identified: citrate synthase CE 4.1.3.7), aconitate hydratase (EC4.2.1.3), NAD and NADP-dependent isocitrate dehydrogenase (EC 1.1.1.41, 1.1.1.42), (alpha-oxoglutarate dehydrogenase (EC 1.2.4.2), succinate dehydrogenase (EC 1.3.99.1), fumarate hydratase (EC 4.2.1.2), and malate dehydrogenase (EC 1.1.1.37). The in vitro activity of aconitate hydratase and of NADP-dependent isocitrate dehydrogenase was shown to be almost identical to the rate of in vivo degradation of citrate or to exceed this rate. The degradation of citrate to oxalate was inhibited completely by 9 mM fluoroacetate. It is concluded that the TCA cycle is involved in the formation of oxalate from citrate.     

Assessment of some critical factors in the freezing technique for the cryopreservation of precision-cut rat liver slices

A number of studies on the cryopreservation of precision-cut liver slices using various techniques have been reported. However, the identification of important factors that determine cell viability following cryopreservation is difficult because of large differences between the various methods published. The aim of this study was to evaluate some important factors in the freezing process in an effort to find an optimized approach to the cryopreservation of precision-cut liver slices. A comparative study of a slow and a fast freezing technique was carried out to establish any differences in tissue viability for a number of endpoints. Both freezing techniques aim at the prevention of intracellular ice formation, which is thought to be the main cause of cell death after cryopreservation. Subsequently, critical variables in the freezing process were studied more closely in order to explain the differences in viability found in the two methods in the first study. For this purpose, a full factorial experimental design was used with 16 experimental groups, allowing a number of variables to be studied at different levels in one single experiment. It is demonstrated that ATP and K(+) content and histomorphology are sensitive parameters for evaluating slice viability after cryopreservation. Subsequently, it is shown that freezing rate and the cryopreservation medium largely determine the residual viability of liver slices after cryopreservation and subsequent culturing. It is concluded that a cryopreservation protocol with a fast freezing step and using William's Medium E as cryopreservation medium was the most promising approach to successful freezing of rat liver slices of those tested in this study.     

Changes in lipid synthesis in rat liver during development

1. Lipogenesis, as measured by the incorporation of ¹⁴C-labelled glucose or acetate into fatty acids in liver slices, is high in foetal and adult rat liver but is low in the liver of the suckling rat, especially with glucose as substrate. 2. The rate of synthesis of non-saponifiable lipids from glucose is about 15 times as great in the liver of the 18-day foetus as in adult liver. Activity in the newborn is negligible. 3. Glucose incorporation into fat is strongly concentration-dependent in liver slices from the adult and 2-week-old rat, but less markedly so in liver slices from the foetus. 4. Changes in the activity of hepatic citrate-cleavage enzyme (ATP–citrate lyase) occur in parallel with the changes in the extent of fatty acid formation, supporting the participation of this enzyme in lipogenesis. However, NADP–malate dehydrogenase, a potential source of reduced nucleotide coenzyme for lipogenesis in the adult, could not be detected in foetal rat liver.     

The biochemical toxicology of 1,3-difluoro-2-propanol,the major ingredient of the pesticide Gliftor: The potential of 4-methylpyrazole as an antidote

Administration to rats of 1,3-difluoro-2-propanol (100 mg kg⁻¹ body weight), the major ingredient of the pesticide gliftor, resulted in accumulation of citrate in the kidney after a 3 hour lag phase. 1,3-Difluro-2-propanol was found to be metabolized to 1,3-difluoroacetone and ultimately to the aconitate hydratase inhibitor (-) erythrofluorocitrate and free fluoride. The conversion of 1,3-difluoro-2-propanol to 1,3-difluoroacetone was found to be catalyzed by an NAD⁺-dependent alcohol dehydrogenase, while the defluorination was attributed to microsomal monooxygenase activity induced by phenobarbitone and inhibited by piperonyl butoxide. 4-Methylpyrazole was found to inhibit both of these processes in vitro and when administered (90 mg kg⁻¹ body weight) to rats, 2 hours prior to 1,3-difluoro-2-propanol, eliminated signs of poisoning, prevented (-) erythrofluorocitrate synthesis, and markedly decreased citrate and fluoride accumulation in vivo. 4-Methylpyrazole also appeared to diminish (-) erythrofluorocitrate synthesis from fluoroacetate in vivo, and this was attributed to its capacity to inhibit malate dehydrogenase activity. The antidotal potential of 4-methylpyrazole and the potential for 1,3-difluoro-2-propanol to replace fluoroacetate (compound 1080) as a vertebrate pesticide is discussed. © 1997 John Wiley & Sons, Inc. J Biochem Toxicol 12: 41–52, 1998     

Effects of pyruvate and other metabolites on cyclic GMP levels in incubations of rat hepatocytes and kidney cortex

Pyruvate increased cyclic GMP levels in rat hepatocytes. The effects were observed without or with 1-methyl-3-isobutylxanthine. Lactate, acetate, oxaloacetate, alpha-ketoglutarate, succinate, acetoacetate and beta-hydroxybutyrate also increased cyclic GMP levels. Some compounds increased cyclic GMP in kidney cortex slices. The effects were dependent upon Ca2+ in the medium. Cyclic AMP was increased 30-50% by some of these substances with 2.6 mM Ca2+. Rotenone, oligomycin, antimycin, dinitrophenol, KCN, and arsenate decreased GTP and ATP, basal cyclic GMP and the pyruvate effect, but did not alter cyclic AMP. Although fluoroacetate alone had no effect on cyclic nucleotides, GTP, or ATP, it potentiated the pyruvate effect on cyclic GMP. Adenosine and guanosine increased cyclic GMP and GTP to a similar extent of 30-50%. Aminooxyacetate, cycloserine, pentenoic acid and mepacrine decreased the pyruvate effect while cycloserine or mepacrine alone increased cyclic GMP. Citrate and mepacrine inhibited soluble and particulate guanylate cyclase from rat liver while cycloserine and acetoacetate increased guanylate cyclase activity. None of the other compounds altered guanylate cyclase activity. These results indicate that various metabolites and inhibitors can alter cyclic GMP accumulation in hepatocytes and renal cortex slices. Several mechanisms may be involved in these effects.     

The dependence on dicarboxylic acids and energy of citrate accumulation in depleted rat liver mitochondria

The accumulation of some organic anions in the space inaccessible to sucrose of rat liver mitochondria was measured. In untreated mitochondria anions were apparently concentrated from 1mm applied concentration by between five- and 22-fold, depending on their charge. After depletion of endogenous reserves either with uncoupling agent or with oligomycin uptakes were decreased. The accumulation of citrate was restored by combinations of a dicarboxylic acid (malate, succinate, maleate or meso-tartrate) and energy. The energy could either be provided by oxidation of a suitable dicarboxylic acid or from ascorbate in the presence of tetramethylphenylenediamine, or from ATP. The restoration of citrate uptake is not necessarily accompanied by a gain of K(+), but a cation- and energy-linked citrate uptake can be induced with valinomycin. When citrate is added to mitochondria in the presence of malate the latter is competitively displaced. The anion accumulation could arise from an internal energy-linked positive potential.     

Acetyl transport mechanisms in the nervous system. The oxoglutarate shunt and fatty acid synthesis in the developing rat brain

Abstract Radioactive acetyl groups and lipids are produced from dl -[5-¹⁴C]glutamate. Degradation studies indicate that approximately 90 per cent of the radioactivity is localized in the original carboxyl groups of the two carbon unit. Since these results are shown not to be due to a ¹⁴CO₂ fixation, it is concluded that the oxoglutarate shunt as an acetyl group transport system is functional in brain. The highest ratio of fatty’acid activity/CO₂ activity in this pathway is found in the newborn rat brain and steadily decreases with development. This pattern is observed with incubations of brain slices with labelled glutamate or citrate and is similar to the changes observed in the activity of the citrate cleavage enzyme with brain maturation. In contrast to the previous studies with liver preparations, the conversion of [2-¹⁴C]- and [5-¹⁴C]glutamate to fatty acids is relatively small. This is particularly true during the period of maximal lipid synthesis.     

Histochemical studies on the uptake of horseradish peroxidase by rat kidney slices

When rat kidney slices were incubated in the presence of horseradish peroxidase, there was an energy-dependent uptake of the protein by the cells of the kidney tubules. The uptake was greatest in the proximal convoluted tubules and in the thick ascending limbs of the loops of Henle; it was abolished by cold, anoxia, 2,4-dinitrophenol, and fluoroacetate, and was more readily depressed by unfavorable metabolic conditions in the proximal convoluted tubules than in the thick ascending limbs. Protein uptake was inhibited when the kidney slices were incubated in electrolyte-free media. In sodium chloride solutions, uptake was reduced as sodium was progressively replaced by choline, and ouabain inhibited uptake in the proximal convoluted tubules, but not in the thick ascending limbs. To a limited extent, lithium could replace sodium in the incubation medium with no depression of peroxidase uptake. These results suggest that a sodium-stimulated, ouabain-sensitive ATPase may be involved in the uptake of protein by cells of the kidney tubule. The intracellular transport of peroxidase in cells of the proximal convoluted tubules was abolished by cold, anoxia, and 2,4-dinitrophenol, but it was not affected by concentrations of ouabain which inhibited the uptake of the protein.     

Locations of amino acids in brain slices from the rat. Tetrodotoxin-sensitive release of amino acids

1. Amino acids, particularly glutamate, gamma-aminobutyrate, aspartate and glycine, were released from rat brain slices on incubation with protoveratrine (especially in a Ca(2+)-deficient medium) or with ouabain or in the absence of glucose. Release was partially or wholly suppressed by tetrodotoxin. 2. Tetrodotoxin did not affect the release of glutamine under various incubation conditions, nor did protoveratrine accelerate it. 3. Protoveratrine caused an increased rate of formation of glutamine in incubated brain slices. 4. Increased K(+) in the incubation medium caused release of gamma-aminobutyrate, the process being partly suppressed by tetrodotoxin. 5. Incubation of brain slices in a glucose-free medium led to increased production of aspartate and to diminished tissue contents of glutamates, glutamine and glycine. 6. Use of tetrodotoxin to suppress the release of amino acids from neurons in slices caused by the joint action of protoveratrine and ouabain (the latter being added to diminish reuptake of amino acids), it was shown that the major pools of glutamate, aspartate, glycine, serine and probably gamma-aminobutyrate are in the neurons. 7. The major pool of glutamine lies not in the neurons but in the glia. 8. The tricarboxylic cycle inhibitors, fluoroacetate and malonate, exerted different effects on amino acid contents in, and on amino acid release from, brain slices incubated in the presence of protoveratrine. Fluoroacetate (3mm) diminished the content of glutamine, increased that of glutamate and gamma-aminobutyrate and did not affect respiration. Malonate (2mm) diminished aspartate and gamma-aminobutyrate content, suppressed respiration and did not affect glutamine content. It is suggested that malonate acts mainly on the neurons, and that fluoroacetate acts mainly on the glia, at the concentrations quoted. 9. Glutamine was more effective than glutamate as a precursor of gamma-aminobutyrate. 10. It is suggested that glutamate released from neurons is partly taken up by glia and converted there into glutamine. This is returned to the neurons where it is hydrolysed and converted into glutamate and gamma-aminobutyrate.     

Gluconeogenesis in the kidney and liver slices of a lizard, Uromastix hardwickii

1. The liver and kidney of the lizard Uromastix hardwickii have much higher contents of carbohydrate than have been reported for the corresponding rat tissues. Most of this carbohydrate still remains in the tissue even after a long preincubation. 2. Kidney slices of this lizard release both glucose and other carbohydrates into the medium. Hence glucose release alone, as demonstrated for rats, cannot be used as a good criterion of gluconeogenesis in this lizard. Moreover, the results obtained by glucose release did not agree with those in which the total carbohydrate was estimated in the slice and medium. 3. l-Glutamate, l-aspartate, dl-valine, l-proline, l-cysteine, l-lactate and succinate stimulated gluconeogenesis in the kidney slices, whereas citrate, l-alanine, l-serine, glycine, l-arginine and l-leucine did not. In liver slices only l-glutamate increased gluconeogenesis. 4. New carbohydrate formation in the kidney and liver slices after incubation with various substrates indicated that gluconeogenesis as well as the amino acid metabolism in this animal may be somewhat different from that of mammals.     

Is fluoroacetate-specific defluorinase a glutathione S-transferase?

Fluoroacetate-specific defluorinase (FSD) is a critical enzyme in the detoxication of fluoroacetate. This study investigated whether FSD can be classed as a glutathione S-transferase (GST) isoenzyme with a high specificity for fluoroacetate detoxication metabolism. The majority of FSD and GST activity, using 1-chloro-2,4-dinitrobenzene (CDNB) and 1,2-epoxy-3-(p-nitrophenoxy)propane (EPNP) as GST substrates, in rat liver was cytosolic. GSTT1 specific substrate, EPNP caused a slight non-competitive inhibition of FSD activity. CDNB, a general substrate of GST isoenzyme, was a more potent non-competitive inhibitor of FSD activity. The fluoroacetate defluorination activity by GST isoenzymes was determined in this study. The results showed that the GSTZ1C had the highest fluoroacetate defluorination activity of the various GST isoenzymes studied, while GSTA2 had a limited activity toward fluoroacetate. The human GSTZ1C recombinant protein then was purified from a human GSTZ1C cDNA clone. Our experiments showed that GSTZ1C catalysed fluoroacetate defluorination. GSTZ1 shares many of the characteristics of FSD; however, it accounts only for 3% of the total cytosolic FSD activity. GSTZ1C based enzyme kinetic studies has low affinity for fluoroacetate. The evidence suggests that GSTZ1 may not be the major enzyme defluorinating fluoroacetate, but it does detoxify the fluoroacetate. To clarify the identity of enzymes responsible for fluoroacetate detoxication, further studies of the overall FSD activity are needed.     

Fatty acid oxidation in human and rat heart. Comparison of cell-free and cellular systems

Oxidation rates of palmitate and activities of the mitochondrial marker enzymes cytochrome c oxidase and citrate synthase have been determined in homogenates, isolated mitochondria and slices of human and rat heart and in calcium-tolerant rat cardiac myocytes. Homogenates and mitochondria from rat heart showed a 6- and 2.5-fold higher palmitate oxidation rate than the corresponding preparations from human heart. From the palmitate oxidation rates and cytochrome c oxidase and citrate synthase activities as parameters, the mitochondrial protein contents of human and rat heart were calculated to be about 18 and 45 mg/g wet weight, respectively. Based on citrate synthase activities, the fatty acid oxidation rates were about the same in homogenates and isolated mitochondria, much lower in myocytes and lowest in slices. In the cellular systems the palmitate molecule was more completely oxidized than in homogenates or isolated mitochondria. Fatty acid oxidation rates were concentration-dependent in slices, but not with myocytes. With the cellular systems, palmitate oxidation was synergistically stimulated by the addition of carnitine, coenzyme A and ATP to the incubation medium. This stimulation could be attributed only partly to an increased oxidation in damaged cells.