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.     

Ceruloplasmin, Copper, Selenium, Iron, Zinc, and Manganese Levels in Normal and Sulfite Oxidase Deficient Rat Plasma: Effects of Sulfite Exposure

A noticeable effect of sulfite treatment was observed on the plasma ceruloplasmin ferroxidase activity of rats with normal sulfite oxidase activity when compared to normal controls. The plasma levels of selenium, iron, and zinc were unaffected by sulfite in normal and sulfite oxidase (SOX)-deficient rats. While plasma level of Mn was decreasing, plasma Cu level increased in SOX-deficient rats. Treating SOX-deficient groups with sulfite did not alter plasma level of Mn but made plasma level of Cu back to its normal level. This is the first evidence that Cu and Mn status were affected in experimental sulfite oxidase deficiency induced by low molybdenum diet with tungsten addition deserving further research to determine the underlying mechanisms of these observations in experimental sulfite oxidase deficiency.     

Sulfite leads to neuron loss in the hippocampus of both normal and SOX-deficient rats

Sulfites are compounds commonly used as preservatives in foods, beverages and pharmaceuticals. Sulfite is also endogenously generated during the metabolism of sulfur-containing amino acids and drugs. It has been shown that sulfite is a highly toxic molecule. Many studies have examined the effects of sulfite toxicity, but the effect of ingested sulfite on the number of neurons in the hippocampus has not yet been reported. The present study was undertaken to investigate the effect of ingested sulfite on pyramidal neurons by counting cells in CA1 and CA3-2 subdivisions of the rat hippocampus. For this purpose, rats were assigned to one of four groups (6 rats per group): control (C), sulfite (S), deficient (D) and deficient+sulfite (DS). Sulfite oxidase deficiency was established by feeding rats a low molybdenum diet and adding 200ppm tungsten (W) to their drinking water. Sulfite (70mg/kg) was also administered to the animals via their drinking water. At the end of the experimental period, the rats were sacrificed by exsanguination under anesthesia, and their brains and livers quickly removed. The livers were used for a SOX activity assay, and the brains were used for neuronal counts in a known fraction of the CA1 and CA3-2 subdivisions of the left hippocampus using the optical fractionator method, which is a stereological method. The results showed that sulfite treatment caused a significant decrease in the total number of pyramidal neurons in three subdivisions of the hippocampus (CA1 and CA3-2) in the S, D and DS groups compared with the control group. It is concluded that exogenous administration of sulfite causes loss of pyramidal neurons in CA1 and CA3-2 subdivisions in both normal and SOX deficient rat hippocampus. This finding provides supporting evidence that sulfite is a neurotoxic molecule.     

Effect of sulfite exposure on zinc, iron, and copper levels in rat liver and kidney tissues

Sulfite is a potentially toxic molecule that might enter the body via ingestion, inhalation, or injection. For cellular detoxification, mammalians rely on sulfite oxidase to convert sulfite to sulfate. The purpose of this research was to determine the effect of sulfite on zinc, iron, and copper levels in rat liver and kidney tissues. Forty normal and sulfite oxidase-deficient male albino rats were divided into four groups that included untreated controls (group C), a sulfite-supplemented group that received 70 mg sodium metabisulfite per kilogram per day (group S), a sulfite oxidase-deficient group (group D), and a sulfite oxidase-deficient group that was also given 70 mg sodium metabisulfite per kilogram per day (group DS). The iron and zinc levels in the liver and kidney in groups S and DS were not affected by sulfite treatment compared to their respective controls (groups C and D). Sulfite exposure led to an increase of kidney copper content in the S group when compared to untreated controls. The kidney copper levels were significantly increased in the unexposed deficient rats, but it was not different than that of the deficient rats that were given oral sulfite treatment. These results suggest that kidney copper levels might be affected by exogenous or endogenous sulfite. An erratum to this article is available at .     

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.     

Disturbance of brain energy and redox homeostasis provoked by sulfite and thiosulfate: Potential pathomechanisms involved in the neuropathology of sulfite oxidase deficiency

Sulfite oxidase (SO) deficiency is biochemically characterized by tissue accumulation and high urinary excretion of sulfite, thiosulfate and S-sulfocysteine. Affected patients present severe neurological symptoms and cortical atrophy, whose pathophysiology is still poorly established. Therefore, in the present work we investigated the in vitro effects of sulfite and thiosulfate on important parameters of energy metabolism in the brain of young rats. We verified that sulfite moderately inhibited the activity of complex IV, whereas thiosulfate did not alter any of the activities of the respiratory chain complexes. It was also found that sulfite and thiosulfate markedly reduced the activity of total creatine kinase (CK) and its mitochondrial and cytosolic isoforms, suggesting that these metabolites impair brain cellular energy buffering and transfer. In contrast, the activity of synaptic Na⁺,K⁺-ATPase was not altered by sulfite or thiosulfate. We also observed that the inhibitory effect of sulfite and thiosulfate on CK activity was prevented by melatonin, reduced glutathione and the combination of both antioxidants, as well as by the nitric oxide synthase N^ω-nitro-l-arginine methyl ester, indicating the involvement of reactive oxygen and nitrogen species in these effects. Sulfite and thiosulfate also increased 2′,7′-dichlorofluorescin oxidation and hydrogen peroxide production and decreased the activity of the redox sensor aconitase enzyme, reinforcing a role for oxidative damage in the effects elicited by these metabolites. It may be presumed that the disturbance of cellular energy and redox homeostasis provoked by sulfite and thiosulfate contributes to the neurological symptoms and abnormalities found in patients affected by SO deficiency.     

Thiols are main determinants of total antioxidant capacity of cellular homogenates

While the total antioxidant capacity (TAC) of blood plasma is mainly accounted for by urate, TAC of cell interior can be expected to depend more on other antioxidants, especially glutathione and protein -SH groups. We studied TAC of homogenates of several lines of cultured cells subjected to the action of thiol-modifying agents. Comparison of changes of TAC of the homogenates and of the level of total thiols (determined with a biradical spin label) demonstrates that alterations in cellular thiol content is the main determinant of changes of TAC of cell homogenates. These results show that estimation of TAC of cell extracts may be a useful parameter of assessment of oxidative stress, primarily of oxidation of thiol groups, yielding information different than TAC of body fluids.     

Serum prolidase activity and oxidative–antioxidative status in patients with developmental dysplasia of the hip and its relationship with radiographic severity

Background: We aimed to investigate serum prolidase activity and to investigate its association with oxidative–antioxidative status in patients with developmental dysplasia of the hip (DDH).

Methods: Oxidative status parameters, including lipid hydroperoxide (LOOH), total oxidant status (TOS), and the oxidative stress index (OSI), and antioxidative status parameters, free sulfhydryl groups (Total –SH), and total antioxidative capacity (TAC), as well as serum prolidase activity were assessed in patients with DDH (n?=?93), and in healthy controls (n?=?82). The severity of dysplasia was evaluated according to the Tonnis grading system.

Results: Serum prolidase activity and the oxidant parameters (LOOH, TOS, and OSI) were significantly higher and the antioxidant parameters (Total –SH and TAC) were significantly lower in patients with DDH compared to the controls (P?P?P?Conclusion: Increased levels of serum prolidase activity, LOOH, TOS, and OSI, and decreased levels of total –SH and TAC, may be associated with DDH, and these parameters may be useful adjunctive tools to assess the severity of DDH.     

Sulfite Inhibition of Photosystem II in Illuminated Spinach Leaves

PS II activity (dichlorophenolindophenol photoreduction) inchloroplasts isolated from sulfite-treated spinach leaves inlight was inhibited but not in darkness. Sulfite treatment decreasedthe variable part of fluorescence induction and the fluorescenceintensities of emissions at 685 and 694 nm at 77K, but it hadno effect when sulfite was administered together with DCMU.These results indicate that sulfite inactivates the PS II reactioncenter when electron transport takes place. (Received August 5, 1983; Accepted November 25, 1983)     

Molecular basis of the biological function of molybdenum. Developmental patterns of sulfite oxidase and xanthine oxidase in the rat

The developmental patterns of the molybdenum-containing enzymes sulfite oxidase and xanthine oxidase and of the mitochondrial enzymes adenylate kinase and succinate-cytochrome c reductase in rat liver are reported. Adenylate kinase and succinate-cytochrome c reductase develop in parallel with total liver protein and are maximal 5 days after birth. Sulfite oxidase, which is also a mitochondrial protein, shows its largest increase in activity between 5 and 11 days after birth. The appearance of sulfite oxidase and xanthine oxidase proteins parallels very closely the development of their respective activities. Xanthine oxidase activity is extremely low in rats prior to weaning at 21 days. Development of activity of this enzyme may be related to the protein nutritional status of the young animal. The development of both sulfite oxidase and xanthine oxidase activities is very much impaired by administration of tungsten to the pregnant rats for 20 days before birth of the litters. Apparently normal development of sulfite oxidase protein, however, leads to the accumulation of inactive molecules in the livers of offspring of tungsten-fed rats. Development of adenylate kinase and succinate-cytochrome c reductase activities is not affected by tungsten treatment.     

The antioxidant status of patients subjected to total body irradiation.

BACKGROUND AND PURPOSE: Total body irradiation (TBI) is a routine preconditioning procedure for the treatment of leukemia and aplastic anemia, prior to bone marrow transplantation (BMT). Ionizing radiation generates reactive oxygen derived species (ROS) that can be removed by antioxidants. Our purpose is to determine the antioxidant status of patients undergoing TBI by evaluating the oxidant stress and their antioxidant capacity. MATERIAL AND METHODS: We evaluated by cyclic voltammetry (CV) the total antioxidant capacity (TAC) in plasma of 14 patients undergoing TBI prior to BMT. The levels of the antioxidants, ascorbic acid (AA) and uric acid (UA) were determined by HPLC-ECD. The oxidant stress level was calculated by the ratio [dehydro ascorbic acid]/total ascorbic acid]. RESULTS: TAC was reduced by 36% (p < 0.02) but after 4 months recovered to a level 22% higher than before the treatment (p < 0.05). Both, AA and UA, decreased following irradiation by 84% (p < 0.02) and 24% (p < 0.05) respectively, but returned to a level of 21% and 320% after 4 months compared to baseline values. The changes in [UA] were affected by Allopurinol (xanthine oxidase inhibitor), given as a routine pretransplant therapy until day -1. The [dehydroascorcbic acid]/[total ascorbic acid] (%) was 45% (range of normal controls = 13.2 +/- 1.5%) and increased by 69% following TBI. In order to obtain a decrease in the TAC of plasma in vitro, comparable to that in vivo, a 1000 fold higher dose of irradiation was required. CONCLUSIONS: TBI caused a pronounced decrease in antioxidant capacity and an excessive increase in oxidant stress. We assume that TBI alters antioxidant homeostasis greatly enhancing the stress damage. CV measurements may lead to a better understanding of the balance between oxidant stress and antioxidant utilization, and to a reconsideration of the routine use of Allopurinol as pretreatment for TBI, and antioxidant support before and/or after TBI.     

Molecular mechanism of intramolecular electron transfer in dimeric sulfite oxidase

Sulfite oxidase (SOX) is a homodimeric molybdoheme enzyme that oxidizes sulfite to sulfate at the molybdenum center. Following substrate oxidation, molybdenum is reduced and subsequently regenerated by two sequential electron transfers (ETs) via heme to cytochrome c. SOX harbors both metals in spatially separated domains within each subunit, suggesting that domain movement is necessary to allow intramolecular ET. To address whether one subunit in a SOX dimer is sufficient for catalysis, we produced heterodimeric SOX variants with abolished sulfite oxidation by replacing the molybdenum-coordinating and essential cysteine in the active site. To further elucidate whether electrons can bifurcate between subunits, we truncated one or both subunits by deleting the heme domain. We generated three SOX heterodimers: (i) SOX/Mo with two active molybdenum centers but one deleted heme domain, (ii) SOX/Mo_C264S with one unmodified and one inactive subunit, and (iii) SOX_C264S/Mo harboring a functional molybdenum center on one subunit and a heme domain on the other subunit. Steady-state kinetics showed 50% SOX activity for the SOX/Mo and SOX/Mo_C264S heterodimers, whereas SOX_C264S/Mo activity was reduced by two orders of magnitude. Rapid reaction kinetics monitoring revealed comparable ET rates in SOX/Mo, SOX/Mo_C264S, and SOX/SOX, whereas in SOX_C264S/Mo, ET was strongly compromised. We also combined a functional SOX Mo domain with an inactive full-length SOX R217W variant and demonstrated interdimer ET that resembled SOX_C264S/Mo activity. Collectively, our results indicate that one functional subunit in SOX is sufficient for catalysis and that electrons derived from either Mo^(IV) or Mo^(V) follow this path.     

Purification and some properties of sulfite:ferric ion oxidoreductase from Thiobacillus ferrooxidans.

Sulfite:ferric ion oxidoreductase in the plasma membrane of Thiobacillus ferrooxidans AP19-3 was purified to an electrophoretically homogeneous state. The enzyme had an apparent molecular weight of 650,000 and was composed of two subunits (M(rs), 61,000 and 59,000) as estimated by sodium sulfate-polyacrylamide gel electrophoresis. The Michaelis constants of sulfite:ferric ion oxidoreductase for Fe3+ and sulfite ions were 1.0 and 0.071 mM, respectively. Sulfite:ferric ion oxidoreductase suffered from end product inhibition by 1 mM Fe2+.     

Insights into a key sulfite scavenger enzyme sulfite oxidase (<Emphasis Type="Italic">SOX</Emphasis>) gene in plants

Sulfite oxidase (SOX) is a crucial molybdenum cofactor-containing enzyme in plants that re-oxidizes the sulfite back to sulfate in sulfite assimilation pathway. However, studies of this crucial enzyme are quite limited hence this work was attempted to understand the SOXs in four plant species namely, Arabidopsis thaliana, Solanum lycopersicum, Populus trichocarpa and Brachypodium distachyon. Herein studied SOX enzyme was characterized with both oxidoreductase molybdopterin binding and Mo-co oxidoreductase dimerization domains. The alignment and motif analyses revealed the highly conserved primary structure of SOXs. The phylogeny constructed with additional species demonstrated a clear divergence of monocots, dicots and lower plants. In addition, to further understand the phylogenetic relationship and make a functional inference, a structure-based phylogeny was constructed using normalized RMSD values in five superposed models from four modelled plant SOXs herein and one previously characterized chicken SOX structure. The plant and animal SOXs showed a clear divergence and also implicated their functional divergences. Based on tree topology, monocot B. distachyon appeared to be diverged from other dicots, pointing out a possible monocot–dicot split. The expression patterns of sulfite scavengers including SOX were differentially modulated under cold, heat, salt and high light stresses. Particularly, they tend to be up-regulated under high light and heat while being down-regulated under cold and salt stresses. The presence of cis-regulatory motifs associated with different stresses in upstream regions of SOX genes was thus justified. The protein–protein interaction network of AtSOX and network enrichment with gene ontology (GO) terms showed that most predicted proteins, including sulfite reductase, ATP sulfurylases and APS reductases were among prime enzymes involved in sulfite pathway. Finally, SOX–sulfite docked structures indicated that arginine residues particularly Arg374 is crucial for SOX–sulfite binding and additional two other residues such as Arg51 and Arg103 may be important for SOX–sulfite bindings in plants.     

Thiols are Main Determinants of Total Antioxidant Capacity of Cellular Homogenates

While the total antioxidant capacity (TAC) of blood plasma is mainly accounted for by urate, TAC of cell interior can be expected to depend more on other antioxidants, especially glutathione and protein -SH groups. We studied TAC of homogenates of several lines of cultured cells subjected to the action of thiol-modifying agents. Comparison of changes of TAC of the homogenates and of the level of total thiols (determined with a biradical spin label) demonstrates that alterations in cellular thiol content is the main determinant of changes of TAC of cell homogenates. These results show that estimation of TAC of cell extracts may be a useful parameter of assessment of oxidative stress, primarily of oxidation of thiol groups, yielding information different than TAC of body fluids.     

Structural insights into sulfite oxidase deficiency

Sulfite oxidase deficiency is a lethal genetic disease that results from defects either in the genes encoding proteins involved in molybdenum cofactor biosynthesis or in the sulfite oxidase gene itself. Several point mutations in the sulfite oxidase gene have been identified from patients suffering from this disease worldwide. Although detailed biochemical analyses have been carried out on these mutations, no structural data could be obtained because of problems in crystallizing recombinant human and rat sulfite oxidases and the failure to clone the chicken sulfite oxidase gene. We synthesized the gene for chicken sulfite oxidase de novo, working backward from the amino acid sequence of the native chicken liver enzyme by PCR amplification of a series of 72 overlapping primers. The recombinant protein displayed the characteristic absorption spectrum of sulfite oxidase and exhibited steady state and rapid kinetic parameters comparable with those of the tissue-derived enzyme. We solved the crystal structures of the wild type and the sulfite oxidase deficiency-causing R138Q (R160Q in humans) variant of recombinant chicken sulfite oxidase in the resting and sulfate-bound forms. Significant alterations in the substrate-binding pocket were detected in the structure of the mutant, and a comparison between the wild type and mutant protein revealed that the active site residue Arg-450 adopts different conformations in the presence and absence of bound sulfate. The size of the binding pocket is thereby considerably reduced, and its position relative to the cofactor is shifted, causing an increase in the distance of the sulfur atom of the bound sulfate to the molybdenum.     

Paraoxonase and arylesterase activities in fibromyalgia

We aimed to evaluate the association of serum paraoxonase and arylesterase activities and oxidative/antioxidative status in patients with fibromyalgia. Forty-two patients with fibromyalgia and 53 healthy controls were included in the study. Serum paraoxonase and arylesterase activities were measured spectrophotometrically. Oxidative and antioxidative status were evaluated by measuring serum lipid hydroperoxide (LOOH) levels, total antioxidant status (TAS) and free sulfhydryl groups (-SH = total thiol). Lipid parameters were determined by routine laboratory methods. Serum paraoxonase and arylesterase activities, and TAS were lower in patients with fibromyalgia than in controls (P < 0.001, for all), and the -SH level was also lower in the patient group (P = 0.03). LOOH levels were higher in the patient group than in controls (P = 0.01). Our results suggest that patients with fibromyalgia were exposed to oxidative stress, and paraoxonase and arylesterase activities were decreased in these patients. Patients with fibromyalgia might be prone to development of atherosclerosis with reduced paraoxonase and arylesterase activities.     

Boron ameliorates arsenic‐induced DNA damage,proinflammatory cytokine gene expressions,oxidant/antioxidant status,and biochemical parameters in rats

Arsenic, an element found in nature, causes hazardous effects on living organisms. Meanwhile, natural compounds exhibit protective effects against hazardous substances. This study evaluated the effects of boron against arsenic‐induced genotoxicity and altered biochemical parameters in rats. Thirty‐five male Wistar albino rats were equally divided into five groups, and the experimental period lasted 30 days. One group was used as the control, and another group was treated with 100 mg/L arsenic in drinking water. The other groups were orally treated with 5, 10, and 20 mg/kg boron plus arsenic (100 mg/L via drinking water). Arsenic caused changes in biochemical parameters, total oxidant/antioxidant status, and DNA damage in mononuclear leukocytes. Moreover, it increased IFN‐γ, IL‐1β, TNF‐α, and NFκB mRNA expression levels in rat tissue. However, boron treatment improved arsenic‐induced alterations in biochemical parameters and increases in DNA damage and proinflammatory cytokine gene expressions.     

Somatostatin analogue,Octreotide, improves restraint stress-induced liver injury by ameliorating oxidative stress,inflammatory response,and activation of hepatic stellate cells

The aim of this study is to investigate the effect of somatostatin (SST) analogue, Octreotide, on some features of liver injury induced by immobilization stress (IS) in adult male albino rats. Eighteen adult male albino rats were randomly divided into three equal groups: control, IS, and Octreotide-treated stressed groups. Octreotide (40 μg/kg body weight, subcutaneously) was administrated twice daily for 8 days during the exposure to IS. Octreotide was found to reduce the IS significantly and induce elevations in the plasma level of corticosterone, liver transaminases, and tumor necrosis factor α (TNF-α) as compared with IS group. Furthermore, Octreotide administration has significantly elevated the decline in the total antioxidant capacities (TAC) and lowered the elevated malondialdehyde (MDA) levels observed with IS in the hepatic tissue. Additionally, Octreotide treatment provided protection against the histopathological changes in the stressed liver in the form of significant reduction in the mean number of degenerated hepatocytes, the area % of collagen fibers, and glial fibrillary acid protein (GFAP) immunostaining with a significant increase in the mean number of normal hepatocytes. In conclusion, stressed rats showed disturbed liver functions and its oxidant–antioxidant status with highly expression hepatic stellate cells (HSCs), which were all improved by Octreotide administration, SST analogue.     

Evidence for sulfite as an essential metabolite for human peripheral lymphocytes

Sulfite has been identified as an essential metabolite by means of growth studies using a chemically-defined, protein-free medium for culture of human peripheral lymphocytes. Sulfite reduced the amount of cysteine required for optimum growth by at least four-fold. In some subjects, sulfite stimulated growth even in the presence of optimal amounts of cysteine indicating that lymphocytes of some individuals are unable to convert cysteine to sulfite in adequate amounts.