A Mechanism of Sulfite Neurotoxicity

Abstract

Exposure of Neuro-2a and PC12 cells to micromolar concentrations of sulfite caused an increase in reactive oxygen species and a decrease in ATP. Likewise, the biosynthesis of ATP in intact rat brain mitochondria from the oxidation of glutamate was inhibited by micromolar sulfite. Glutamate-driven respiration increased the mitochondrial membrane potential (MMP), and this was abolished by sulfite but the MMP generated by oxidation of malate and succinate was not affected. The increased rate of production of NADH from exogenous NAD+ and glutamate added to rat brain mitochondrial extracts was inhibited by sulfite, and mitochondria preincubated with sulfite failed to reduce NAD+. Glutamate dehydrogenase (GDH) in rat brain mitochondrial extract was inhibited dose-dependently by sulfite as was the activity of a purified enzyme. An increase in the Km (glutamate) and a decrease in Vmax resulting in an attenuation in Vmax/Km (glutamate) at 100 μm sulfite suggest a mixed type of inhibition. However, uncompetitive inhibition was noted with decreases in both Km (NAD+) and Vmax, whereas Vmax/Km (NAD+) remained relatively constant. We propose that GDH is one target of action of sulfite, leading to a decrease in α-ketoglutarate and a diminished flux through the tricarboxylic acid cycle accompanied by a decrease in NADH through the mitochondrial electron transport chain, a decreased MMP, and a decrease in ATP synthesis. Because glutamate is a major metabolite in the brain, inhibition of GDH by sulfite could contribute to the severe phenotype of sulfite oxidase deficiency in human infants. Exposure of Neuro-2a and PC12 cells to micromolar concentrations of sulfite caused an increase in reactive oxygen species and a decrease in ATP. Likewise, the biosynthesis of ATP in intact rat brain mitochondria from the oxidation of glutamate was inhibited by micromolar sulfite. Glutamate-driven respiration increased the mitochondrial membrane potential (MMP), and this was abolished by sulfite but the MMP generated by oxidation of malate and succinate was not affected. The increased rate of production of NADH from exogenous NAD+ and glutamate added to rat brain mitochondrial extracts was inhibited by sulfite, and mitochondria preincubated with sulfite failed to reduce NAD+. Glutamate dehydrogenase (GDH) in rat brain mitochondrial extract was inhibited dose-dependently by sulfite as was the activity of a purified enzyme. An increase in the Km (glutamate) and a decrease in Vmax resulting in an attenuation in Vmax/Km (glutamate) at 100 μm sulfite suggest a mixed type of inhibition. However, uncompetitive inhibition was noted with decreases in both Km (NAD+) and Vmax, whereas Vmax/Km (NAD+) remained relatively constant. We propose that GDH is one target of action of sulfite, leading to a decrease in α-ketoglutarate and a diminished flux through the tricarboxylic acid cycle accompanied by a decrease in NADH through the mitochondrial electron transport chain, a decreased MMP, and a decrease in ATP synthesis. Because glutamate is a major metabolite in the brain, inhibition of GDH by sulfite could contribute to the severe phenotype of sulfite oxidase deficiency in human infants. Sulfite is formed from sulfur dioxide, an environmental pollutant. It is also generated endogenously by the metabolism of sulfur-containing amino acids such as methionine and cysteine and from sulfate in response to bacterial lipopolysaccharide (1Mitsuhashi H. Nojima Y. Tanaka T. Ueki K. Maezawa A. Yano S. Naruse T. J. Leukoc. Biol. 1998; 64: 595-599Crossref PubMed Scopus (47) Google Scholar). The most common exogenous source is sulfiting agents used as preservatives in dried fruits and vegetables (2Lester M.R. J. Am. Coll. Nutr. 1995; 14: 229-232Crossref PubMed Scopus (138) Google Scholar) and in wine where millimolar concentrations have been reported (3Casella I.G. Contursi M. Desimoni E. Analyst. 2002; 127: 647-652Crossref PubMed Scopus (35) Google Scholar). Sulfite is also used as a stabilizer in many drugs administered to patients (4Altland K. Winter P. Neurogenetics. 1999; 2: 183-188Crossref PubMed Scopus (41) Google Scholar). Interestingly, its presence in a dexamethasone preparation increased the neurotoxicity of excitotoxic agents (5Baud O. Laudenbach V. Evrard P. Gressens P. Pediatr. Res. 2001; 50: 706-711Crossref PubMed Scopus (77) Google Scholar) suggesting that it could have an effect on neuronal cells. Humans can oxidize sulfite ( SO32−) to sulfate ( SO42−) by sulfite oxidase (SO), 1The abbreviations used are: SO, sulfite oxidase; ROS, reactive oxygen species; Ap5A, P1,P5-di(adenosine-5′)pentaphosphate; CCCP, carbonylcyanide-m-chlorophenylhydrazone; DEPMPO, 5-diethoxyphosphoryl-5-methyl-1-pyrroline-n-oxide; GDH, glutamate dehydrogenase; JC-1, 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide; MDH, malate dehydrogenase; MDCK, Madin-Darby canine kidney cells; MMP, mitochondrial membrane potential; MTT, thiazolyl blue or 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide; OK, opossum kidney cells. 1The abbreviations used are: SO, sulfite oxidase; ROS, reactive oxygen species; Ap5A, P1,P5-di(adenosine-5′)pentaphosphate; CCCP, carbonylcyanide-m-chlorophenylhydrazone; DEPMPO, 5-diethoxyphosphoryl-5-methyl-1-pyrroline-n-oxide; GDH, glutamate dehydrogenase; JC-1, 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide; MDH, malate dehydrogenase; MDCK, Madin-Darby canine kidney cells; MMP, mitochondrial membrane potential; MTT, thiazolyl blue or 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide; OK, opossum kidney cells. a mitochondrial enzyme. However, the SO activity in human liver was reported to be only 10% that of rat liver (6Johnson J.L. Rajagopalan K.V. J. Clin. Invest. 1976; 58: 543-550Crossref PubMed Scopus (87) Google Scholar). Although SO is expressed in human lung (7Millard J. Parsons R.B. Waring R.H. Williams A.C. Ramsden D.B. Mol. Pathol. 2003; 56: 270-274Crossref PubMed Scopus (9) Google Scholar), its activity was reported to be low (8Beck-Speier I. Hinze H. Holzer H. Biochim. Biophys. Acta. 1985; 841: 81-89Crossref PubMed Scopus (39) Google Scholar), which may be why some asthmatic subjects react adversely to sulfite in food or atmospheric sulfur dioxide (9Bush R.K. Taylor S.L. Holden K. Nordlee J.A. Busse W.W. Am. J. Med. 1986; 81: 816-820Abstract Full Text PDF PubMed Scopus (97) Google Scholar). The comprehensive literature over a 33-year period also shows a relationship between sulfite as food additives and asthma (10Reus K.E. Houben G.F. Stam M. Dubois A.E. Ned. Tijdschr. Geneeskd. 2000; 144: 1836-1839PubMed Google Scholar). Sulfite oxidase is a dimeric metallohemoprotein with molybdenum and protoheme as prosthetic groups (11Cohen H.J. Fridovich I. Rajagopalan K.V. J. Biol. Chem. 1971; 246: 374-382Abstract Full Text PDF PubMed Google Scholar, 12Johnson J.L. Rajagopalan K.V. Cohen H.J. J. Biol. Chem. 1974; 249: 859-866Abstract Full Text PDF PubMed Google Scholar). The catalytic molybdenum centers of SO from various species of animals appear to be identical as examined by electron paramagnetic resonance (13Kessler D.L. Rajagopalan K.V. J. Biol. Chem. 1972; 247: 6566-6573Abstract Full Text PDF PubMed Google Scholar, 14Astaskin A.V. Railsimring A.M. Feng C. Johnson J.L. Rajagopalan K.V. Enemark J.H. J. Am. Chem. Soc. 2002; 124: 6109-6118Crossref PubMed Scopus (41) Google Scholar). However, SO activity from human liver was almost 10 to 20 times lower than levels found in rat and chicken liver, and it was suggested that the decreased reactivity of the human enzyme could be due to nonfunctional molybdenum centers (6Johnson J.L. Rajagopalan K.V. J. Clin. Invest. 1976; 58: 543-550Crossref PubMed Scopus (87) Google Scholar). A deficiency of SO in humans could be due to a mutation in the SO gene or in any of the several genes encoding the synthesis of molybdopterins (15Mudd S.H. Irrevere F. Laster L. Science. 1967; 156: 1599-1602Crossref PubMed Scopus (121) Google Scholar, 16Irreverre F. Mudd S.H. Heizer W.D. Laster L. Biochem. Med. 1967; 1: 187-199Crossref Scopus (113) Google Scholar, 17Johnson J.L. Rajagopalan K.V. J. Clin. Invest. 1976; 58: 551-556Crossref PubMed Scopus (44) Google Scholar, 18Schindelin H. Kisker C. Rajagopalan K.V. Adv. Prot. Chem. 2001; 58: 47-94Crossref PubMed Scopus (66) Google Scholar). The associated severe neurological dysfunction characterized by dislocation of ocular lenses, mental retardation, and attenuated growth of the brain suggests that neuronal cells are highly susceptible to sulfite toxicity. Indeed, SO activity measured in whole brain of some laboratory animals was consistently low compared with other tissues (19Tejnorova I. Toxicol. Appl. Pharmacol. 1978; 44: 251-256Crossref PubMed Scopus (33) Google Scholar, 20Cabre F. Marin C. Cascante M. Canela E.I. Biochem. Med. Metab. Biol. 1990; 43: 159-162Crossref PubMed Scopus (33) Google Scholar). Measurement of the expression of SO in human tissues in our laboratory concurred with this observation (21Woo W.H. Yang H.Y. Wong K.P. Halliwell B. Biochem. Biophys. Res. Commun. 2003; 305: 619-623Crossref PubMed Scopus (34) Google Scholar). Four missense mutations were characterized in cell lines from patients with isolated sulfite oxidase deficiency (22Kisker C. Schindelin H. Pacheco A. Wehbi W.A. Garett R.M. Rajagopalan K.V. Enemark J.H. Rees D.C. Cell. 1997; 91: 973-983Abstract Full Text Full Text PDF PubMed Scopus (436) Google Scholar). A substitution of G to A of the cDNA of liver SO resulted in an Arg-to-Gln substitution at amino acid residue 190 (23Garrett R.M. Johnson J.L. Graf T.N. Feigenbaum A. Rajagopalan K.V. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6394-6398Crossref PubMed Scopus (123) Google Scholar). Another arginine residue (Arg-160) was recently reported to be essential for the binding of sulfite near the molybdenum cofactors in human SO (24Feng C. Wilson H.L. Hurley K.V. Enemark J.H. 2003; PubMed Scopus Google Scholar), whereas the residue was to the and catalytic activity of the H.L. Rajagopalan K.V. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). the in the of SO, is on the by which of sulfite neuronal was in rat neuronal cell to sulfite M. P. Halliwell B. J. 1998; PubMed Scopus Google Scholar). 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H. of the of of the of cells were from was from and human liver cells were in 100 of G and and of with 10% in a with at Measurement of and ATP in and were H. J.A. Biol. Med. 1999; PubMed Scopus Google Scholar). The of ATP was on the have been reported in our J. M. Halliwell B. Wong K.P. Full Text Full Text PDF PubMed Scopus Google Scholar). of as reported A. C. 2003; 44: PubMed Scopus Google Scholar) was a of C. F. Sci. 1997; PubMed Scopus Google Scholar) and by J. 1990; PubMed Scopus Google Scholar). of were by by The were and with in 20 and at a with and The was at for to cell and The was at for The mitochondrial was in of was on of of and The was by at for The near the of and was and in of the was added to the at for to a of as measured by the Biochem. 1976; PubMed Scopus Google Scholar). of was used to isolated mitochondria K. M. Res. 2001; PubMed Scopus Google Scholar). A of was in and at was with to 10 μm for brain mitochondria were in and for at The was in of this was added of the and at for by as The mitochondrial was in and a for by was by an at at and the was through a is a mitochondria is by the potential M. C. 1995; PubMed Scopus Google Scholar). A of in was to 20 μm for an were added 10 of rat brain 100 of 10 and of 20 μm An of the was a for by with by an at The was through a and the of the through of a succinate was to the and one of the was examined as for both and Measurement of mitochondrial membrane potential was measured on a The was at by an of was a by 10 of isolated brain mitochondrial extract between and of and 10 μm The in the were with a of were the for and the for the was for a by the of the and the of the from to were at the other the a for times or and the were for the and for the of an period of or succinate was to the mitochondria by the of sulfite or The concentrations of sulfite, and are as in of ATP by reported J. M. Halliwell B. Wong K.P. Full Text Full Text PDF PubMed Scopus Google Scholar) was with one an of T. Y. J. Biochem. PubMed Scopus Google Scholar), was to for its to ATP of was added μm and μm of the and succinate was added at in the presence or of 100 μm sulfite. a where a of is the were also at concentrations that a to concentrations reported in the whole The for and succinate in the brain expressed in were and of were added at and the μm was to to the of the oxidation of which is a of succinate The was in with 10 of isolated mitochondria of at for It was by for and the were or at for ATP. or were at for 10 in a at The was and of 10 or times were on the of ATP that could be from a of of a that a of the were used for ATP the in the cell The was times in and 100 of this was added The by the was measured the of in a J. M. Halliwell B. Wong K.P. Full Text Full Text PDF PubMed Scopus Google Scholar, Biochem. 1997; 246: PubMed Scopus (33) Google Scholar). Measurement of of GDH and in formed by GDH and was measured on a with of The mitochondrial extract was from of intact mitochondria that been at It was in a and of mitochondrial extract of were used in a of of for GDH and of for were at with glutamate and NAD+ in the presence or of μm sulfite. of glutamate and NAD+ was with sulfite at the or the of the were to that the and sulfite not with the of the of of Sulfite on of a preparation of GDH from liver with activity of of and NAD+ in a of of at as NADH in the presence or of μm sulfite was at on a for 10 of the GDH of GDH was in a of of as NADH was measured for at a of were in the and presence of and 100 μm sulfite glutamate and NAD+ and NAD+ and The Km and Vmax were generated the in and The production of ROS, measured by the oxidation of was of Neuro-2a and PC12 cells to micromolar concentrations of sulfite for ATP measured was by 100 μm sulfite, with decrease in both cell lines However, cell by the was not not were in and human liver cells with to and the decrease in ATP was in compared with neuronal of ATP. cells in were to sulfite of to μm for A decrease in ATP measured by the as reported J. M. Halliwell B. Wong K.P. Full Text Full Text PDF PubMed Scopus Google Scholar) was in and human liver cells. for and decreases in ATP as of that mitochondria from rat brain by and by intact mitochondria for of of intact mitochondria was by the of the JC-1, mitochondrial its as was added exogenous with of the of succinate oxidation the of with an increase in MMP was by the increased of of the rat brain mitochondria also and respiration on of succinate not of intact mitochondria by rat brain mitochondria 100 of in 100 of was with μm at for of in the of exogenous the of with some of the by exogenous succinate also of and of of of ATP from of Glutamate by or ATP was in the of exogenous and with μm mitochondria and this respiration was inhibited by Ap5A, a of C. F. Sci. 1997; PubMed Scopus Google Scholar). of exogenous of the resulting ATP to be of of such as or α-ketoglutarate electron transport to ATP. biosynthesis of ATP was not by A is that in the presence of a highly electron transport by the added most of the added was by However, was added in The biosynthesis of ATP from glutamate and in the presence of Ap5A, was inhibited by μm sulfite in a with almost inhibition at 100 μm inhibition was not with or succinate a where glutamate was with the other at concentrations that concentrations in the whole brain of Scholar), the inhibition by sulfite on ATP biosynthesis was not The decrease in ATP biosynthesis from glutamate oxidation by μm sulfite was not by DEPMPO, a of sulfite M. T. H. Biol. Med. 1999; PubMed Scopus Google Scholar), as in of ATP in isolated rat brain mitochondria rat brain mitochondria of were in the and presence of μm and μm by for at ATP was by the are expressed in μm of μm μm μm μm μm A of in a of sulfite on mitochondrial GDH activity rat brain mitochondria were with in the or presence of 10 to 100 μm sulfite for at by for μm was also and this is by ATP from μm in the presence μm was by the The were from μm μm μm in a of ATP from oxidation of a to in and were to a The were added or at concentrations in glutamate succinate malate and α-ketoglutarate which concentrations in whole brain of Scholar). are of compared to the sulfite. the other of of compared to the sulfite. the other of in a of MMP by of as by intact rat brain mitochondria added was and of also The of glutamate the mitochondria with an increase in the and a decrease in the at a increase of the with a decrease of the an was with 100 μm sulfite was A of the MMP was and the decrease of the for several Likewise, isolated mitochondria were by malate and succinate but sulfite failed to the MMP in both and suggesting that sulfite is to on the mitochondrial a sulfite action on the MMP but of mitochondria was by a of μm CCCP, a The decrease in MMP almost and MMP to as in to the but by the isolated rat brain mitochondria the as and formed for 10 The relationship between the species was consistently in of the by an increase in the was of of glutamate malate and succinate of mitochondria with sulfite abolished the action of glutamate the MMP in but that with malate as and it effect succinate was the of Sulfite on of Glutamate and oxidation of glutamate and malate by in rat brain mitochondrial extract was by the of the presence of NAD+ and glutamate or NADH was and this was inhibited in the GDH by 100 μm sulfite but with malate as A inhibition was from μm sulfite in the GDH of the mitochondrial extract with sulfite abolished the GDH but inhibited the activity with and sulfite added to a of NADH that not in the of of NADH not of GDH by activity of GDH was inhibited by sulfite in a from μm this was measured by the of NADH are in with Km and Vmax in the presence and of of sulfite action on glutamate dehydrogenase of GDH was in the and presence of and 100 μm sulfite at in a of of NADH was at in a for the of Vmax/Km generated from a mixed type of inhibition was measured with glutamate and whereas uncompetitive inhibition was with NAD+ and and μm μm μm and μm μm in a have to an in of human sulfite oxidase deficiency J. Johnson J.L. Garett R.M. Rajagopalan K.V. J.H. PubMed Scopus Google Scholar). other of molybdenum which SO from resonance also suggested M. T. I. J. 2001; Google Scholar). It was that neuronal was caused by mitochondrial by sulfite, which mitochondrial membrane leading to decreased ATP or C. C. L. J. Sci. 2002; PubMed Scopus Google Scholar). it was that the was a dysfunction of brain that sulfite a and decrease in ATP synthesis in rat brain mitochondria on a major in brain of ATP was also in and human liver cells have been reported with rat kidney mitochondria and and cells J. M. Halliwell B. Wong K.P. Full Text Full Text PDF PubMed Scopus Google Scholar). However, other and were added at concentrations that concentrations in the whole brain, the effect of sulfite on ATP production was not It appear ATP generated from other could for the inhibition on However, not the levels of various in brain mitochondria or in human brain the human brain is on glutamate than the other is to be the effect of sulfite may in It also be that of sulfite is to in human brain than in rat brain SO activity was reported to be 10 times (6Johnson J.L. Rajagopalan K.V. J. Clin. Invest. 1976; 58: 543-550Crossref PubMed Scopus (87) Google Scholar). a decrease in an was of and human liver cells to sulfite in are a of B. 2003; PubMed Scopus Google Scholar), and this was by an increase in the of in the sulfite. However, sulfite increased the production Although a decrease in ATP and an increase in to exogenous sulfite were in both neuronal and in of sulfite is to in neuronal cells compared with and cells SO activity (19Tejnorova I. Toxicol. Appl. Pharmacol. 1978; 44: 251-256Crossref PubMed Scopus (33) Google Scholar, 20Cabre F. Marin C. Cascante M. Canela E.I. Biochem. Med. Metab. Biol. 1990; 43: 159-162Crossref PubMed Scopus (33) Google Scholar) its oxidation to sulfate could contribute to the decrease in ATP in neuronal compared with cells The increase in and decreased ATP suggested that the target of sulfite action could be the isolated rat brain oxidation of glutamate with of was found to be inhibited by sulfite in a with as as decrease in the presence of 100 μm sulfite, mitochondria are a decrease in ATP was not by the presence of which to suggest that reactive sulfite are not is a to with sulfite M. T. H. Biol. Med. 1999; PubMed Scopus Google Scholar). The biosynthesis of ATP from the oxidation of or succinate not by sulfite. our of of mitochondrial ATP MMP, and dehydrogenase GDH was found to be to the action of sulfite. an inhibition of GDH and the associated of the MMP and ATP production from the oxidation of glutamate was decreased and Although activity was also inhibited by sulfite, the effect was only a preparation of mitochondria was only inhibition was and The biosynthesis of ATP from the oxidation of succinate or α-ketoglutarate was not suggesting that are It was that sulfite on GDH a decrease in Vmax and an increase in Km for glutamate in the presence of μm sulfite resulting in a decrease in Vmax/Km of a mixed type of inhibition. the other the concentrations of NAD+ were uncompetitive inhibition with decreases in Km for NAD+ and Vmax were and the Vmax/Km remained relatively sulfite could to the or to the the source of for brain is Scholar) of which of its oxidation in to the cycle D.L. F. 2003; PubMed Scopus Google Scholar). is an amino acid in the brain, and it can be to which is in concentrations than the cycle it to mitochondrial of GDH by an of sulfite in the brain of subjects with human SO deficiency an as in J. Johnson J.L. Garett R.M. Rajagopalan K.V. J.H. PubMed Scopus Google Scholar, M. T. I. J. 2001; Google Scholar, C. C. L. J. Sci. 2002; PubMed Scopus Google Scholar). inhibition of GDH by sulfite, the of α-ketoglutarate be in the brain the GDH in the of to α-ketoglutarate A Res. 2001; PubMed Scopus Google Scholar). tricarboxylic acid cycle such as malate and succinate be decreased of a diminished flux through the tricarboxylic acid the to for GDH inhibition in of ATP production in not the of the enzyme of the tricarboxylic acid α-ketoglutarate been reported to be inhibited in isolated by C. L. V. J. 1999; PubMed Scopus Google Scholar, L. V. J. 2000; PubMed Google Scholar). enzyme is to a that to and 2000; PubMed Scopus Google Scholar). generated by sulfite in Neuro-2a and PC12 cells could the inhibition of GDH and α-ketoglutarate dehydrogenase to an of glutamate to the tricarboxylic acid The of a of glutamate with brain dysfunction is glutamate is Sulfite in a dexamethasone preparation was to increase the neurotoxicity of excitotoxic agents (5Baud O. Laudenbach V. Evrard P. Gressens P. Pediatr. Res. 2001; 50: 706-711Crossref PubMed Scopus (77) Google Scholar). Glutamate is also the of an Interestingly, a of GDH activity was reported in a where GDH activity was decreased to a than other mitochondrial S. S. C. PubMed Scopus Google Scholar), suggesting that GDH is to that it not inhibited by from μm A. S. B. and K. P. GDH been found to be in of the human brain A. V. A. J. 1986; PubMed Scopus Google Scholar), and it is to that its inhibition by sulfite is for the in of a with SO deficiency S.L. Am. J. 2002; Google Scholar). We propose that the deficiency is the of an inhibition of GDH with decrease in α-ketoglutarate and other tricarboxylic acid cycle an decrease in NADH flux through the mitochondrial electron transport chain, a MMP, and a decrease in ATP were in this The of mitochondrial could be or it could the brain to by other leading to of with this (15Mudd S.H. Irrevere F. Laster L. Science. 1967; 156: 1599-1602Crossref PubMed Scopus (121) Google Scholar, 16Irreverre F. Mudd S.H. Heizer W.D. Laster L. Biochem. Med. 1967; 1: 187-199Crossref Scopus (113) Google Scholar, 17Johnson J.L. Rajagopalan K.V. J. Clin. Invest. 1976; 58: 551-556Crossref PubMed Scopus (44) Google Scholar). ATP is also for the synthesis of or a decrease in sulfate and ATP synthesis of and other in the