Sulfite disrupts brain mitochondrial energy homeostasis and induces mitochondrial permeability transition pore opening via thiol group modification

Sulfite oxidase (SO) deficiency is biochemically characterized by the accumulation of sulfite, thiosulfate and S-sulfocysteine in tissues and biological fluids of the affected patients. The main clinical symptoms include severe neurological dysfunction and brain abnormalities, whose pathophysiology is still unknown. The present study investigated the in vitro effects of sulfite and thiosulfate on mitochondrial homeostasis in rat brain mitochondria. It was verified that sulfite per se, but not thiosulfate, decreased state 3, CCCP-stimulated state and respiratory control ratio in mitochondria respiring with glutamate plus malate. In line with this, we found that sulfite inhibited the activities of glutamate and malate (MDH) dehydrogenases. In addition, sulfite decreased the activity of a commercial solution of MDH, that was prevented by antioxidants and dithiothreitol. Sulfite also induced mitochondrial swelling and reduced mitochondrial membrane potential, Ca^2 + retention capacity, NAD(P)H pool and cytochrome c immunocontent when Ca^2 + was present in the medium. These alterations were prevented by ruthenium red, cyclosporine A (CsA) and ADP, supporting the involvement of mitochondrial permeability transition (MPT) in these effects. We further observed that N-ethylmaleimide prevented the sulfite-elicited swelling and that sulfite decreased free thiol group content in brain mitochondria. These findings indicate that sulfite acts directly on MPT pore containing thiol groups. Finally, we verified that sulfite reduced cell viability in cerebral cortex slices and that this effect was prevented by CsA. Therefore, it may be presumed that disturbance of mitochondrial energy homeostasis and MPT induced by sulfite could be involved in the neuronal damage characteristic of SO deficiency.     

Effect of sulfite on antioxidant enzymes and lipid peroxidation in normal and sulfite oxidase-deficient rat erythrocytes

Sulfite and related chemical such as sulfite salts and sulfur dioxide has been used as a preservative in food and drugs. This molecule has also been generated from the catabolism of sulfur-containing amino acids. Sulfite is a very reactive and potentially toxic molecule and has to be detoxified by the enzyme sulfite oxidase (SOX). The aim of this study was to investigate the effects of ingested sulfite on erythrocyte antioxidant status by measuring glucose-6-phosphate dehydrogenase (G-6-PD), superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) activities and oxidant status by measuring thiobarbituric acid reactive substances (TBARS) in normal and SOX-deficient rats. Rats were assigned to four groups (n = 10 rats/group) as follows; control (C), sulfite (CS), deficient (D), and deficient + sulfite (DS). SOX deficiency was established by feeding rats a low molybdenum diet and adding to their drinking water 200 ppm tungsten (W). Sulfite (25 mg/kg) was administered to the animals via their drinking water. At the end of 6 weeks, Erythrocyte G-6-PD, SOD, and GPx but not CAT activities were found to be significantly increased with and without sulfite treatment in SOX-deficient groups. Sulfite treatment alone was also significantly increased erythrocytes’ SOD activity in CS group compared to control. TBARS levels were found to be significantly increased in CS and DS groups and decreased in D group. When SOX-deficient rats treated with sulfite, TBARS level was still higher than other groups. In conclusion, these results suggested that erythrocyte antioxidant capacity, a defense mechanism against the oxidative challenge, increased by endogenous and exogenous sulfite due to its oxidant nature. This increase was also observed in CS and DS groups but it was insufficient to prevent lipid peroxidation.     

Sulfite-oxido-reductase is involved in the oxidation of sulfite in Desulfocapsa sulfoexigens during disproportionation of thiosulfate and elemental sulfur

The enzymatic pathways of elemental sulfur and thiosulfate disproportionation were investigated using cell-free extract of Desulfocapsa sulfoexigens. Sulfite was observed to be an intermediate in the metabolism of both compounds. Two distinct pathways for the oxidation of sulfite have been identified. One pathway involves APS reductase and ATP sulfurylase and can be described as the reversion of the initial steps of the dissimilatory sulfate reduction pathway. The second pathway is the direct oxidation of sulfite to sulfate by sulfite oxidoreductase. This enzyme has not been reported from sulfate reducers before. Thiosulfate reductase, which cleaves thiosulfate into sulfite and sulfide, was only present in cell-free extract from thiosulfate disproportionating cultures. We propose that this enzyme catalyzes the first step in thiosulfate disproportionation. The initial step in sulfur disproportionation was not identified. Dissimilatory sulfite reductase was present in sulfur and thiosulfate disproportionating cultures. The metabolic function of this enzyme in relation to elemental sulfur or thiosulfate disproportionation was not identified. The presence of the uncouplers HQNO and CCCP in growing cultures had negative effects on both thiosulfate and sulfur disproportionation. CCCP totally inhibited sulfur disproportionation and reduced thiosulfate disproportionation by 80% compared to an unamended control. HQNO reduced thiosulfate disproportionation by 80% and sulfur disproportionation by 90%.     

Significance of plant sulfite oxidase

Sulfite oxidizing activities are known since years in animals, microorganisms, and also plants. Among plants, the only enzyme well characterized on molecular and biochemical level is the molybdoenzyme sulfite oxidase (SO). It oxidizes sulfite using molecular oxygen as electron acceptor, leading to the production of sulfate and hydrogen peroxide. The latter reaction product seems to be the reason why plant SO is localized in peroxisomes, because peroxisomal catalase is able to decompose hydrogen peroxide. On the other hand, we have indications for an additional reaction taking place in peroxisomes: sulfite can be nonenzymatically oxidized by hydrogen peroxide. This will promote the detoxification of hydrogen peroxide especially in the case of high amounts of sulfite. Hence we assume that SO could possibly serve as "safety valve" for detoxifying excess amounts of sulfite and protecting the cell from sulfitolysis. Supportive evidence for this assumption comes from experiments where we fumigated transgenic poplar plants overexpressing ARABIDOPSIS SO with SO(2) gas. In this paper, we try to explain sulfite oxidation in its co-regulation with sulfate assimilation and summarize other sulfite oxidizing activities described in plants. Finally we discuss the importance of sulfite detoxification in plants.     

Mechanism of sulfite cytotoxicity in isolated rat hepatocytes

Sulfite (SO(3)(2-)) has been widely used as preservative and antimicrobial in preventing browning of foods and beverages. SO(2), a common air pollutant, also is capable of producing sulfite and bisulfite depending on the pH of solutions. A molybdenum-dependent mitochondrial enzyme, sulfite oxidase, oxidizes sulfite to inorganic sulfate and prevents its toxic effects. In the present study, sulfite toxicity towards isolated rat hepatocytes was markedly increased by partial inhibition of cytochrome a/a(3) by cyanide or by putting rats on a high-tungsten/low-molybdenum diet, which result in inactivation of sulfite oxidase. Sulfite cytotoxicity was accompanied by a rapid disappearance of GSSG followed by a slow depletion of reduced glutathione (GSH). Depleting hepatocyte GSH beforehand increased cytotoxicity of sulfite. On the other hand, dithiothreitol (DTT), a thiol reductant, added even 1h after the addition of sulfite to hepatocytes, prevented cell death and restored hepatocyte GSH levels. Sulfite cytotoxicity was also accompanied by an increase of oxygen uptake, reactive oxygen species (ROS) formation and lipid peroxidation. Cytochrome P450 inhibitors, metyrapone and piperonyl butoxide also prevented sulfite-induced cytotoxicity and lipid peroxidation. Desferroxamine and antioxidants also protected the cells against sulfite toxicity. These findings suggest that cytotoxicity of sulfite is mediated by free radicals as ROS formation increases by sulfite and antioxidants prevent its toxicity. Reaction of sulfite or its free radical metabolite with disulfide bonds of GSSG and GSH results in the compromise of GSH/GSSG antioxidant system leaving the cell susceptible to oxidative stress. Restoring GSH content of the cell or protein-SH groups by DTT can prevent sulfite cytotoxicity.     

Mechanism of dissimilatory sulfite reduction by Desulfovibrio desulfuricans: purification of a membrane-bound sulfite reductase and coupling iwth cytochrome c 3 and hydrogenase

The localization of the dissimilatory sulfite reductase in Desulfovibrio desulfuricans strain Essex 6 was investigated. After treatment of the cells with lysozyme, 90% of the sulfite reductase activity was found in the membrane fraction, compared to 30% after cell rupture with the French press. Sulfite reductase was purified from the membrane (mSiR) and the soluble (sSiR) fractiion. On SDS-PAGE, both mSiR and sSiR exhibited three bands at 50, 45 and 11 kDa, respectively. From their UV/VIS properties (distinct absorption maxima at 391, 410, 583, 630 nm, enzymes as isolated) and the characteristic red fluorescence in alkaline solution, mSiR and sSiR were identified as desulfoviridin. Sulfite reductase (HSO₃ ^-H₂S) activity was reconstituted by coupling of mSiR to hydrogenase and cytochrome c ₃ from D. desulfuricans. The specific activity of mSiR was 103 nmol H₂ min^-1 mg^-1, and sulfide was the major product (72% of theoretical yield). No coupling was found with sSiR under these conditions. Furthermore, carbon monoxide was used to diferentiate between the membrane-bound and the soluble sulfite reductase. In a colorimetric assay, with photochemically reduced methyl viologen as redox mediator, CO stimulated the activity of sSiR significantly. CO had no effect in the case of mSiR. These studies documented that, as isolated, both forms of sulfite reductase behaved differently in vitro. Clearly, in D. desulfuricans, the six electron conversion HSO₃ ^-H₂S was achieved by a membranebound desulfoviridin without the assistance of artificial redox mediators, such as methyl viologen.Abbreviations SiR sulfite reductase - mSiR sulfite reductase purified from membranes - sSiR sulfite reductase purified from the soluble fraction Enzymes Sulfite reductase, EC 1.8.99.1 Cytochrome c ₃ hydrogenase, EC 1.12.2.1     

Validation of a novel in vitro assay using ultra performance liquid chromatography–mass spectrometry (UPLC/MS) to detect and quantify hydroxylated metabolites of BDE-99 in rat liver microsomes

The purpose of this study was to develop and validate an ultra performance liquid chromatography–mass spectrometry (UPLC/MS) method to investigate the hepatic oxidative metabolism of 2,2′,4,4′,5-pentabromodiphenyl ether (BDE-99), a widely used flame retardant and ubiquitous environmental contaminant. Hydroxylated metabolites were extracted using liquid-to-liquid extraction, resolved on a C₁₈ column with gradient elution and detected by mass spectrometry in single ion recording mode using electrospray negative ionization. The assay was validated for linearity, accuracy, precision, limit of quantification, range and recovery. Calibration curves were linear (R² ≥ 0.98) over a concentration range of 0.010–1.0 μM for 4-OH-2,2′,3,4′,5-pentabromodiphenyl ether (4-OH-BDE-90), 5′-OH-2,2′,4,4′,5-pentabromodiphenyl ether (5′-OH-BDE-99) and 6′-OH-2,2′,4,4′,5-pentabromodiphenyl ether (6′-OH-BDE-99), and a concentration range of 0.0625–12.5 μM for 2,4,5-tribromophenol (2,4,5-TBP). Inter- and intra-day accuracy values ranged from −2.0% to 6.0% and from −7.7% to 7.3%, respectively, and inter- and intra-day precision values ranged from 2.0% to 8.5% and from 2.2% to 8.6% (n = 6), respectively. The limits of quantification were 0.010 μM for 4-OH-BDE-90, 5′-OH-BDE-99 and 6′-OH-BDE-99, and 0.0625 μM for 2,4,5-TBP. Recovery values ranged between 85 and 100% for the four analytes. The validated analytical method was applied to identify and quantify hydroxy BDE-99 metabolites formed in vitro. Incubation of BDE-99 with rat liver microsomes yielded 4-OH-BDE-90 and 6′-OH-BDE-99 as major metabolites and 5′-OH-BDE-99 and 2,4,5-TBP as minor metabolites. To our knowledge, this is the first validated UPLC/MS method to quantify hydroxylated metabolites of PBDEs without the need of derivatization.     

Uptake of sulfate,sulfite and thiosulfate by proton-anion symport in Desulfovibrio desulfuricans

pH changes and sulfide production upon addition of sulfate, sulfite or thiosulfate to non-buffered H₂-saturated cell suspensions of Desulfovibrio desulfuricans were studied by means of electrodes. The addition of these electron acceptors resulted in a rapid alkalinization of the suspension which was accompanied by sulfide production. At-2° C, alkalinization without immediate sulfide production could be obtained. After addition of ³⁵S-labelled sulfate at-2° C, the label was found to be concentrated 7,500-fold in the cells, while 2 protons per sulfate molecule had disappeared from the outer bulk phase. Alkalinization and sulfide production from micromolar electron acceptor additions depended on the transmembraneous proton gradient ( pH), and were reversibly inhibited in alkaline solution (pH>8.0) or by the protonophore carbonylcyanide m-chlorophenylhydrazone (CCCP). Protonophore-inhibited sulfide production from sulfite or thiosulfate could be restored if the cell membranes were permeabilized by the detergent cetyltrimethylammonium bromide (CTAB), or if downhill transport was made possible by the addition of electron acceptors at millimolar concentrations. Sulfate was not reduced under these conditions, presumably because the cells did not contain ATP for its activation. K⁺-and Na⁺-ionophores such as nigericin, valinomycin or monensin appeared to be of limited efficiency in D. desulfuricans. In most experiments, sulfate reduction was inhibited by the K⁺–H⁺ antiporter nigericin in the presence of K⁺, but not by the thiocyanate anion or the K⁺-transporter valinomycin. The results indicate that sulfate, sulfite and thiosulfate are taken up by proton-anion symport, presumably as undissociated acids with an electroneutral mechanism, driven by the transmembraneous pH gradient ( pH) or by a solute gradient. Kinetics of alkalinization and sulfide production in cells grown with different electron acceptors revealed that D. desulfuricans has different specific uptake systems for sulfate and thiosulfate, and obviously also for sulfite. It is proposed that the electron acceptor transport finally will not consume net energy during growth in buffered medium: The protons taken up during active electron acceptor transport leave the cell with the reduced end-product by simple passive diffusion of H₂S.Abbreviations CCCP carbonyl cyanide m-chlorophenylhydrazone - FCCP carbonyl cyanide p-trifluoromethoxy phenylhydrazone - CTAB cethyltrimethylammonium bromide     

Evidence That Antioxidants Prevent the Inhibition of Na+,K+-ATPase Activity Induced by Octanoic Acid in Rat Cerebral Cortex in Vitro

The objective of the present study was to investigate the in vitro effects of octanoic acid, which accumulates in medium-chain acyl-CoA dehydrogenase (MCAD) deficiency and in Reye syndrome, on key enzyme activities of energy metabolism in the cerebral cortex of young rats. The activities of the respiratory chain complexes I–IV, creatine kinase, and Na⁺, K⁺-ATPase were evaluated. Octanoic acid did not alter the electron transport chain and creatine kinase activities, but, in contrast, significantly inhibited Na⁺, K⁺-ATPase activity both in synaptic plasma membranes and in homogenates prepared from cerebral cortex. Furthermore, decanoic acid, which is also increased in MCAD deficiency, and oleic acid strongly reduced Na⁺, K⁺-ATPase activity, whereas palmitic acid had no effect. We also examined the effects of incubating glutathione and trolox (-tocopherol) alone or with octanoic acid on Na⁺, K⁺-ATPase activity. Tested compounds did not affect Na⁺, K⁺-ATPase activity by itself, but prevented the inhibitory effect of octanoic acid. These results suggest that inhibition of Na⁺, K⁺-ATPase activity by octanoic acid is possibly mediated by oxidation of essential groups of the enzyme. Considering that Na⁺, K⁺-ATPase is critical for normal brain function, it is feasible that the significant inhibition of this enzyme activity by octanoate and also by decanoate may be related to the neurological dysfunction found in patients affected by MCAD deficiency and Reye syndrome.     

Ethylmalonic Acid Inhibits Mitochondrial Creatine Kinase Activity from Cerebral Cortex of Young Rats in Vitro

Short-chain acyl-CoA dehydrogenase (SCAD) deficiency is an inherited metabolic disorder biochemically characterized by tissue accumulation of predominantly ethylmalonic acid (EMA) and clinically by neurological dysfunction. In the present study we investigated the in vitro effects of EMA on the activity of the mitochondrial (Mi-CK) and cytosolic (Cy-CK) creatine kinase isoforms from cerebral cortex, skeletal muscle, and cardiac muscle of young rats. CK activities were measured in the mitochondrial and cytosolic fractions prepared from whole-tissue homogenates of 30-day-old Wistar rats. The acid was added to the incubation medium at concentrations ranging from 0.5 to 2.5 mM. EMA had no effect on Cy-CK activity, but significantly inhibited the activity of Mi-CK at 1.0 mM and higher concentrations in the brain. In contrast, both Mi-CK and Cy-CK from skeletal muscle and cardiac muscle were not affected by the metabolite. We also evaluated the effect of the antioxidants glutathione (GSH), ascorbic acid, and a-tocopherol and the nitric oxide synthase inhibitor L-NAME on the inhibitory action of EMA on cerebral cortex Mi-CK activity. We observed that the drugs did not modify Mi-CK activity per se, but GSH and ascorbic acid prevented the inhibitory effect of EMA when co-incubated with the acid. In contrast, L-NAME and -tocopherol could not revert the inhibition provoked by EMA on Mi-CK activity. Considering the importance of CK for brain energy homeostasis, it is proposed that the inhibition of Mi-CK activity may be associated to the neurological symptoms characteristic of SCAD deficiency.     

Assimilatory reduction of sulfate and sulfite by methanogenic bacteria

A variety of sulfur-containing compounds were investigated for use as medium reductants and sulfur sources for growth of four methanogenic bacteria. Sulfide (1 to 2 mM) served all methanogens investigated well. Methanococcus thermolithotrophicus and Methanobacterium thermoautotrophicum Marburg and delta H grew well with S0, SO3(2-), or thiosulfate as the sole sulfur source. Only Methanococcus thermolithotrophicus was able to grow with SO4(2-) as the sole sulfur source. 2-Mercaptoethanol at 20 mM was greatly inhibitory to growth of Methanococcus thermolithotrophicus on SO4(2-) or SO2(2-) and Methanobacterium thermoautotrophicum Marburg on SO3(2-) but not to growth of strain delta H on SO3(2-). Sulfite was metabolized during growth by Methanococcus thermolithotrophicus. Sulfide was produced in cultures of Methanococcus thermolithotrophicus growing on SO4(2-), SO3(2-), thiosulfate, and S0. Methanobacterium thermoautotrophicum Marburg was successfully grown in a 10-liter fermentor with S0, SO3(2-), or thiosulfate as the sole sulfur source.     

Effect of sulfite treatment on total antioxidant capacity, total oxidant status, lipid hydroperoxide, and total free sulfydryl groups contents in normal and sulfite oxidase-deficient rat plasma

Sulfites, which are commonly used as preservatives, are continuously formed in the body during the metabolism of sulfur-containing amino acids. Sulfite oxidase (SOX) is an essential enzyme in the pathway of the oxidative degradation of sulfite to sulfate protecting cells from sulfite toxicity. This article investigated the effect of sulfite on total antioxidant capacity (TAC), total oxidant status, lipid hydroperoxide (LOOH), and total free sulfydryl groups (-SH) levels in normal and SOX-deficient male albino rat plasma. For this purpose, rats were divided into four groups: control, sulfite-treated, SOX-deficient, and sulfite-treated SOX-deficient groups. SOX deficiency was established by feeding rats a low molybdenum diet and adding to their drinking water 200 ppm tungsten. Sulfite (70 mg/kg) was administered to the animals via their drinking water. SOX deficiency together with sulfite treatment caused a significant increase in the plasma LOOH and total oxidant status levels. -SH content of rat plasma significantly decreased by both sulfite treatment and SOX deficiency compared to the control. There was also a significant decrease in plasma TAC level by sulfite treatment. In conclusion, sulfite treatment affects the antioxidant/oxidant balance of the plasma cells of the rats toward oxidants in SOX-deficient groups.     

Assimilatory reduction of sulfate and sulfite by methanogenic bacteria.

A variety of sulfur-containing compounds were investigated for use as medium reductants and sulfur sources for growth of four methanogenic bacteria. Sulfide (1 to 2 mM) served all methanogens investigated well. Methanococcus thermolithotrophicus and Methanobacterium thermoautotrophicum Marburg and delta H grew well with S0, SO3(2-), or thiosulfate as the sole sulfur source. Only Methanococcus thermolithotrophicus was able to grow with SO4(2-) as the sole sulfur source. 2-Mercaptoethanol at 20 mM was greatly inhibitory to growth of Methanococcus thermolithotrophicus on SO4(2-) or SO2(2-) and Methanobacterium thermoautotrophicum Marburg on SO3(2-) but not to growth of strain delta H on SO3(2-). Sulfite was metabolized during growth by Methanococcus thermolithotrophicus. Sulfide was produced in cultures of Methanococcus thermolithotrophicus growing on SO4(2-), SO3(2-), thiosulfate, and S0. Methanobacterium thermoautotrophicum Marburg was successfully grown in a 10-liter fermentor with S0, SO3(2-), or thiosulfate as the sole sulfur source.     

Formation of thiosulfate and trithionate during sulfite reduction by washed cells of Desulfovibrio desulfuricans

Deenergized cells of Desulfovibrio desulfuricans strain Essex 6 formed trithionate and thiosulfate during reduction of sulfite with H₂ or formate. The required conditions were pretreatment with the uncoupler carbonylcyanide m-chlorophenylhydrazone (CCCP), low concentration of the electron donor H₂ or formate (25–200 M) and the presence of sulfite in excess (>250 M). The cells formed up to 20 M thiosulfate, and variable amounts of trithionate (0–9 M) and sulfide (0–62 M). Tetrathionate was not produced. Sulfate could not replace sulfite in these experiments, as deenergized cells cannot activate sulfate. However, up to 5 M thiosulfate was produced by cells growing with H₂ and excess sulfate in a chemostat. Micromolar concentrations of trithionate were incompletely reduced to thiosulfate and sulfide by washed cells in the presence of CCCP. Millimolar trithionate concentrations blocked the formation of sulfide, even in the absence of CCCP, and caused thiosulfate accumulation; sulfide formation from sulfate, sulfite or thiosulfate was stopped, too. Trithionate reduction with H₂ in the presence of thiocyanate was coupled to respiration-driven proton translocation (extrapolated H⁺/H₂ ratios of 1.5±0.6). Up to 150 M trithionate was formed by washed cells during oxidation of sulfite plus thiosulfate with ferricyanide as electron acceptor (reversed trithionate reductase activity). Cell breakage resulted in drastic decrease of sulfide formation. Cell-free extract reduced sulfite incompletely to trithionate, thiosulfate, and sulfide. Thiosulfate was reduced stoichiometrically to sulfite and sulfide (thiosulfate reductase activity). The formation of sulfide from sulfite, thiosulfate or trithionate by cell-free extract was blocked by methyl viologen, leading to increased production of thiosulfate plus trithionate from sulfite, or increased thiosulfate formation from trithionate. Our study demonstrates for the first time the formation of intermediates during sulfite reduction with whole cells of a sulfate-reducing bacterium oxidizing physiological electron donors. All results are in accordance with the trithionate pathway of sulfite reduction.With gratitude dedicated to Prof. Dr. Norbert Pfennig on occasion of his 65th birthday     

Hydrogen sulfide-producing kinetics of Shewanella oneidensis in sulfite and thiosulfate respiration

Shewanella oneidensis is a model species for aquatic ecosystems and plays an important role in bioremediation, biofuel cell manufacturing and biogeochemical cycling. S. oneidensis MR-1 is able to generate hydrogen sulfide from various sulfur species; however, its catalytic kinetics have not been determined. In this study, five in-frame deletion mutants of S. oneidensis were constructed and their H₂S-producing activities were analyzed. SirA and PsrA were the two major contributors to H₂S generation under anoxic cultivation, and the optimum SO₃²⁻ concentration for sulfite respiration was approximately 0.8 mM, while the optimum S₂O₃²⁻ concentration for thiosulfate respiration was approximately 0.4 mM. Sulfite and thiosulfate were observed to interfere with each other during respiration, and a high concentration of sulfite or thiosulfate chelated extracellular free-iron but did not repress the expression of sirA or psrA. Nitrite and nitrate were two preferred electron acceptors during anaerobic respiration; however, under energy-insufficient conditions, S. oneidensis could utilize multiple electron acceptors simultaneously. Elucidiating the stoichiometry of H₂S production in S. oneidensis would be helpful for the application of this species in bioremediation and biofuel cell manufacturing, and would help to characterize the ecophysiology of sulfur cycling.     

Brain Na+,K(+)-ATPase inhibition induced by arginine administration is prevented by vitamins E and C

Hyperargininemia is a metabolic disorder caused by deficiency of arginase activity resulting in tissue accumulation of arginine and neurological dysfunction. We have previously demonstrated that arginine induces oxidative stress and decreases Na⁺,K⁺-ATPase in rat midbrain. In the present study we investigated the action of vitamins E and C on the inhibition of Na⁺,K⁺-ATPase provoked by arginine in the midbrain of 60-day-old rats. Animals were pretreated for 1 week with daily IP administration of saline (control) or vitamins E (40 mg/kg) and C (100 mg/kg). Twelve h after the last injection, animals received one injection of arginine (0.8 mol/g of body weight) or saline. Chemiluminescence was significantly increased, whereas total antioxidant capacity and Na⁺,K⁺-ATPase activity were significantly decreased. Furthermore, treatment with vitamins E and C prevented these effects. If these effects also occur in the human condition, it is possible that antioxidant administration might slow the progression of neurodegeneration in this disorder.     

Inhibition of Rat Brain Na+, K+-ATPase Activity Induced by Homocysteine Is Probably Mediated by Oxidative Stress

The objective of the present study was to investigate the effects of preincubation of hippocampus homogenates in the presence of homocysteine or methionine on Na⁺, K⁺-ATPase and Mg²⁺-ATPase activities in synaptic membranes of rats. Homocysteine significantly inhibited Na⁺, K⁺-ATPase activity, whereas methionine had no effect. Mg²⁺-ATPase activity was not altered by the metabolites. We also evaluated the effect of incubating glutathione, cysteine, dithiothreitol, trolox, superoxide dismutase and GM1 ganglioside alone or incubation with homocysteine on Na⁺, K⁺-ATPase activity. Tested compounds did not alter Na⁺, K⁺-ATPase and Mg²⁺-ATPase activities, but except for trolox, prevented the inhibitory effect of homocysteine on Na⁺, K⁺-ATPase activity. These results suggest that inhibition of this enzyme activity by homocysteine is possibly mediated by free radicals and may contribute to the neurological dysfunction found in homocystinuric patients.     

Inhibition of thyroid secretion by sodium fluoride in vitro

NaF mimicked the activation by thyrotropin of iodide binding to proteins and of glucose C-I oxidation but not the accumulation of intracellular colloid droplets or the stimulation of secretion in dog thyroid slices in vitro. On the contrary, NaF inhibited the two latter thyrotropin effects. The inhibitory action of F⁻ was partially relieved by the addition of glucose to the medium; it was mimicked by sodium oxamate. These data suggest that NaF depresses the endocytosis of colloid and thyroid secretion by inhibiting aerobic glycolysis in the follicular cell. NaF inhibited the activation of colloid droplet accumulation and secretion by N⁶,O^2′-dibutyryl-adenosine 3′,5′-monophosphate (dibutyryl cyclic AMP) and the accumulation of cyclic AMP in thyrotropin-stimulated slices. This suggests an inhibition at the level of both cyclic AMP accumulation and cyclic AMP action. The inhibition by NaF and sodium oxamate of colloid droplet formation and thyroid secretion but not of glucose C-I oxidation in stimulated slices further confirms our conclusion that the latter effect is not merely a consequence of the activation by thyrotropin of colloid endocytosis.