Direct Effect of Ceramide on the Mitochondrial Electron Transport Chain Leads to Generation of Reactive Oxygen Species

Ceramide is a sphingolipid that is generated in the signaling of inflammatory cytokines such as tumor necrosis factor (TNF), which exerts many functional roles depending on the cell type where it is produced. Since TNF cytotoxicity is mediated by overproduction of reactive oxygen species from mitochondria, we have examined the role of ceramide in generation of oxidative stress in isolated rat liver mitochondria. The present studies demonstrate that addition of N-acetylsphingosine (C2-ceramide) to mitochondria led to an increase of fluorescence of dihydrorhodamine 123 or dichlorofluorescein-stained mitochondria, indicating formation of hydrogen peroxide. Such effect was significant at 0.25 μM and maximal at 1-5 μM C2, decreasing at greater concentrations. This inductive effect of ceramide was mimicked by N-hexanoylsphingosine at the same concentration range, whereas the immediate precursor of C2, C2-dihydroceramide increased hydrogen peroxide at 1-5 μM. Sphingosine generated hydrogen peroxide at concentrations ≥10 μM, whereas diacylglycerol failed to increase hydrogen peroxide. The increase in hydrogen peroxide induced by C2 was not triggered by mitochondrial permeability transition as C2 did not induce mitochondrial swelling. Blocking electron transport chain at complex I and II prevented the increase in hydrogen peroxide induced by C2; however, interruption of electron flow at complex III by antimycin A potentiated the inductive effect of C2. Depletion of matrix GSH prior to exposure to ceramide resulted in a potentiated increase (2-fold) of hydrogen peroxide generation, leading to lipid peroxidation and loss of activity of respiratory chain complex IV compared with GSH-repleted mitochondria. Mitochondria isolated from TNF-treated cells showed an increase (2-3-fold) in the amount of ceramide compared with mitochondria from untreated cells. These results suggest that mitochondria are a target of ceramide produced in the signaling of TNF whose effect on mitochondrial electron transport chain leads to overproduction of hydrogen peroxide and consequently this phenomena may account for the generation of reactive oxygen species during TNF cytotoxicity. Ceramide is a sphingolipid that is generated in the signaling of inflammatory cytokines such as tumor necrosis factor (TNF), which exerts many functional roles depending on the cell type where it is produced. Since TNF cytotoxicity is mediated by overproduction of reactive oxygen species from mitochondria, we have examined the role of ceramide in generation of oxidative stress in isolated rat liver mitochondria. The present studies demonstrate that addition of N-acetylsphingosine (C2-ceramide) to mitochondria led to an increase of fluorescence of dihydrorhodamine 123 or dichlorofluorescein-stained mitochondria, indicating formation of hydrogen peroxide. Such effect was significant at 0.25 μM and maximal at 1-5 μM C2, decreasing at greater concentrations. This inductive effect of ceramide was mimicked by N-hexanoylsphingosine at the same concentration range, whereas the immediate precursor of C2, C2-dihydroceramide increased hydrogen peroxide at 1-5 μM. Sphingosine generated hydrogen peroxide at concentrations ≥10 μM, whereas diacylglycerol failed to increase hydrogen peroxide. The increase in hydrogen peroxide induced by C2 was not triggered by mitochondrial permeability transition as C2 did not induce mitochondrial swelling. Blocking electron transport chain at complex I and II prevented the increase in hydrogen peroxide induced by C2; however, interruption of electron flow at complex III by antimycin A potentiated the inductive effect of C2. Depletion of matrix GSH prior to exposure to ceramide resulted in a potentiated increase (2-fold) of hydrogen peroxide generation, leading to lipid peroxidation and loss of activity of respiratory chain complex IV compared with GSH-repleted mitochondria. Mitochondria isolated from TNF-treated cells showed an increase (2-3-fold) in the amount of ceramide compared with mitochondria from untreated cells. These results suggest that mitochondria are a target of ceramide produced in the signaling of TNF whose effect on mitochondrial electron transport chain leads to overproduction of hydrogen peroxide and consequently this phenomena may account for the generation of reactive oxygen species during TNF cytotoxicity. INTRODUCTIONTumor necrosis factor (TNF) 1The abbreviations used are: TNFtumor necrosis factorAAantimycin ABSObuthionine-L-sulfoximineC2N-acetylsphingosineC6N-hexanoylsphingosineC2DHdihydro-C2DAG1,2-diacylglycerolDEMdiethylmaleateDHRdihydrorhodamine 123DCF2′-7′-dichlorofluoresceinDCFDA2′-7′-dichlorofluorescin diacetateMOPS4-morpholinepropanesulfonic acidNF-κBnuclear factor κBPC-PLCphosphatidylcholine-dependent phospholipase CQubiquinoneROSreactive oxygen speciesSMasesphingomyelinaseTTFAthenoyltrifluoroacetone. is a cytokine produced by a wide variety of cell types whose production is up-regulated in a number of stressful and pathological conditions (1Beutler B. Cerami A. Nature. 1986; 320: 584-588Google Scholar, 2Tracey K.J. Wei H Manogue K.R. Fong Y. Hesse D.G. Nguyen H.T. Kuo G.C. Beutler B. Cotran R.S. Cerami A. J. Exp. Med. 1988; 167: 1211-1227Google Scholar, 3Fiers W. FEBS Lett. 1991; 285: 199-212Google Scholar). TNF exerts a pleiotropic mode of action on multiple cell functions including regulation of immune responses, host defense reactions, and gene regulation. In addition, its role as a mediator of cytotoxicity on certain susceptible transformed cell lines has been well documented (4Brach M.A. Gruss H.J. Asano Y. DeVos S. Ludwig W.D. Mertelsmann R. Herrmann F. Cancer Res. 1992; 52: 2197-2201Google Scholar, 5Elbaz O. Budel L.M. Hoogerbrugge H. Touw I.P. Delwel R. Mahmoud L.A. Lowenberg B. J. Clin. Invest. 1991; 87: 838-841Google Scholar, 6Brach M.A. Gruss H.J. Scott C. Herrmann F. Mol. Cell. Biol. 1993; 13: 4824-4830Google Scholar, 7Belka C. Wiegmann K. Adam D. Holland R. Neuloh M. Herrmann F. Kronke M. Brach M.A. EMBO J. 1995; 14: 1156-1165Google Scholar). Upon binding to its receptor subtypes, TNF evokes a complicated array of intracellular signals, including G-coupled activation of phospholipase A2, release of arachidonic acid, DAG production, and activation of protein kinase C, some of which may participate in the chain of reactions that result in cell killing (5Elbaz O. Budel L.M. Hoogerbrugge H. Touw I.P. Delwel R. Mahmoud L.A. Lowenberg B. J. Clin. Invest. 1991; 87: 838-841Google Scholar, 6Brach M.A. Gruss H.J. Scott C. Herrmann F. Mol. Cell. Biol. 1993; 13: 4824-4830Google Scholar, 7Belka C. Wiegmann K. Adam D. Holland R. Neuloh M. Herrmann F. Kronke M. Brach M.A. EMBO J. 1995; 14: 1156-1165Google Scholar). An overproduction of ROS has been proposed as an important mechanism to mediate the cytotoxic and gene regulating effects that TNF exerts on tumor cells (8Jones A.L. Selby P. Cancer Surv. 1989; 8: 817-836Google Scholar,9Schulze-Ostholl K. Beyaert R. Vandevoorde V. Haegeman G. Fiers W. J. Biol. Chem. 1992; 267: 5317-5323Google Scholar, 10Schulze-Oshoff K. Bakker A.C. Vanhaesebroeck B. Beyaert R. Jacob W.A. Fiers W. EMBO J. 1993; 12: 3095-3104Google Scholar, 11Goosens V. Grooten J. De Vos K. Fiers W. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8115-8119Google Scholar, 12Adamson G.M. Billings R.E. Arch. Biochem. Biophys. 1992; 294: 223-229Google Scholar).Ceramide has attracted considerable attention due to its role as an intracellular effector molecule that mimics some of the biological effects exerted by inflammatory cytokines such as TNF (13Kolesnick R. Golde D.W. Cell. 1994; 77: 325-328Google Scholar, 14Kolesnick R. Fuks Z. J. Exp. Med. 1995; 181: 1949-1952Google Scholar, 15Hannun Y. J. Biol. Chem. 1994; 269: 3125-3128Google Scholar, 16Zhang Y. Kolesnick R. Endocrinology. 1995; 136: 4157-4160Google Scholar, 17Hannun Y.A. Bell R.M. Science. 1989; 243: 500-507Google Scholar, 18Spiegel S. Merrill A.H. FASEB J. 1996; 10: 188-1397Google Scholar). In addition to its de novo biosynthesis, which is initiated by the condensation of serine and palmitoyl-CoA, ceramide can also be generated by sphingomyelin hydrolysis. Thus, enzymes that hydrolyze sphingomyelin such as sphingomyelinases stand as regulators of intracellular ceramide levels and consequently ceramide-mediated functions. These enzymes are key components of the so-called sphingomyelin pathway, an ubiquitous system that functions in transducing the signals of cytokines to the cell interior (13Kolesnick R. Golde D.W. Cell. 1994; 77: 325-328Google Scholar, 14Kolesnick R. Fuks Z. J. Exp. Med. 1995; 181: 1949-1952Google Scholar, 15Hannun Y. J. Biol. Chem. 1994; 269: 3125-3128Google Scholar).Sphingomyelinase is known to exist in two forms depending on their intracellular localization and pH optima (13Kolesnick R. Golde D.W. Cell. 1994; 77: 325-328Google Scholar, 14Kolesnick R. Fuks Z. J. Exp. Med. 1995; 181: 1949-1952Google Scholar, 15Hannun Y. J. Biol. Chem. 1994; 269: 3125-3128Google Scholar, 16Zhang Y. Kolesnick R. Endocrinology. 1995; 136: 4157-4160Google Scholar). A Mg2+-dependent membrane-bound with a neutral pH optima initiates signaling by generating ceramide at or near the vicinity of the plasma membrane. In addition to the membrane-associated enzyme, another cytosolic neutral SMase independent of Mg2+ has been identified and partially purified, which appears to hydrolyze intracellular sphingomyelin stores to initiate signaling (19Okazaki T. Bielawska A. Domal N. Bell R.M. Hannun Y.A. J. Biol. Chem. 1994; 269: 4070-4077Google Scholar). The signal initiated by these enzymes is then transmitted further down in the signaling cascade by activation of ceramide-activated protein phosphatases and ceramide-dependent protein kinases (13Kolesnick R. Golde D.W. Cell. 1994; 77: 325-328Google Scholar, 14Kolesnick R. Fuks Z. J. Exp. Med. 1995; 181: 1949-1952Google Scholar, 15Hannun Y. J. Biol. Chem. 1994; 269: 3125-3128Google Scholar). In addition to the neutral SMase forms, an acidic SMase form has also been identified, displaying an pH optima around 5, the bulk of which seems to be located at the lysosomes/endosomal compartment. Although it appears that acidic SMase plays a role in signaling, the molecular mechanism of its activation and recruitment during signaling is unclear. Indirect evidence have suggested that DAG generated by PC-PLC activates the acidic enzyme at or near the plasma membrane since inhibitors of PC-PLC prevent activation of the acidic SMase. This hypothesis, which implies a redistribution of the enzyme from the lysosomal compartment to or near the plasma membrane, requires further verification (20Schütze S. Potthoff K. Machleidt T. Berkovic D. Wiegmann K. Kronke M. Cell. 1992; 71: 756-776Google Scholar,21Wiegmann K. Schütze S. Machleidt T. Witte D. Kronke M. Cell. 1994; 78: 1005-1015Google Scholar). Despite the existence of the neutral and acidic SMases, an alkaline form of the enzyme has been described recently, although its role in signaling remains to be defined (22Nyberg L. Duan R.D. Axelson J. Nilsson A. Biochim. Biophys. Acta. 1996; 1300: 42-48Google Scholar).There is compelling evidence to propose ceramide as a second messenger in the sphingomyelin pathway similar to DAG in the glycerophospholipid pathway. The role that ceramide fulfills within the cell are numerous and of varied nature (13Kolesnick R. Golde D.W. Cell. 1994; 77: 325-328Google Scholar, 14Kolesnick R. Fuks Z. J. Exp. Med. 1995; 181: 1949-1952Google Scholar, 15Hannun Y. J. Biol. Chem. 1994; 269: 3125-3128Google Scholar, 16Zhang Y. Kolesnick R. Endocrinology. 1995; 136: 4157-4160Google Scholar). It has been shown that ceramide plays a critical role in apoptosis, proliferation, cellular senescence, and gene regulation through activation of transcription factors such as NF-κB (20Schütze S. Potthoff K. Machleidt T. Berkovic D. Wiegmann K. Kronke M. Cell. 1992; 71: 756-776Google Scholar,21Wiegmann K. Schütze S. Machleidt T. Witte D. Kronke M. Cell. 1994; 78: 1005-1015Google Scholar). However, the possibility that ceramide may interact with mitochondria leading to production of ROS has not been documented to our knowledge, and constitutes the basis of the present report.Mitochondria are one of the most important cellular sources of ROS due to its quantitative consumption of molecular oxygen. Since ceramide appears as an important mediator of the effects elicited by TNF and due to the participation of mitochondria in the TNF-induced ROS production (9Schulze-Ostholl K. Beyaert R. Vandevoorde V. Haegeman G. Fiers W. J. Biol. Chem. 1992; 267: 5317-5323Google Scholar, 10Schulze-Oshoff K. Bakker A.C. Vanhaesebroeck B. Beyaert R. Jacob W.A. Fiers W. EMBO J. 1993; 12: 3095-3104Google Scholar, 11Goosens V. Grooten J. De Vos K. Fiers W. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8115-8119Google Scholar, 12Adamson G.M. Billings R.E. Arch. Biochem. Biophys. 1992; 294: 223-229Google Scholar), the purpose of the present work was to analyze the effects of ceramide and other sphingolipids, on the production of hydrogen peroxide in isolated mitochondria from rat liver. Furthermore, since reduced GSH is the only defense provided to metabolize peroxides generated from the electron transport chain through GSH redox cycle (23Kaplowitz N. Aw T.Y. Ookhtens M. Annu. Rev. Pharmacol. Toxicol. 1985; 25: 715-744Google Scholar), we determined the role of mitochondrial GSH in modulating the production of hydrogen peroxide and its consequences upon incubation of mitochondria with ceramide. Our studies demonstrate for the first time that addition of ceramide to mitochondria results in a dose-dependent increase in hydrogen peroxide, which is prevented when complex I and II of respiration are inhibited. Furthermore, mitochondria from TNF-treated hepatocytes displayed an increased level of ceramide supporting the role of ceramide as an intermediate in the TNF-induced ROS generation from mitochondria. Depletion of matrix GSH prior to exposure to ceramide results in an additional increase of hydrogen peroxide, which peroxidizes lipids from mitochondria resulting in loss of mitochondrial function. These results suggest that ceramide produced in the signaling of TNF is responsible, at in for some of the TNF-induced cytotoxic acid, SMase ceramide and from and from N-acetylsphingosine N-hexanoylsphingosine and C2-dihydroceramide from and from was from diacylglycerol kinase was from activity of was from and of isolated as described on rat and in C. Ookhtens M. N. J. Clin. Invest. 1991; 87: C. A. A. J. N. J. Clin. Invest. 1994; Scholar). determined a II and by was determined by and by the in the of hepatocytes in the or of TNF or ceramide with fluorescence or for by to the generation of peroxides by fluorescence a with a was was a with an on a and with a with II The of the in the of by a the fluorescence of the was in the fluorescence for of fluorescence are the result of fluorescence in in the of from the in to a of Mitochondria and with liver mitochondria isolated by J. Biol. Scholar). of mitochondria was by the activity of in mitochondria to that of was determined by the as oxygen consumption in and of respiration a oxygen with or as for respiratory for complex I or II as described C. A. A. J. N. J. Clin. Invest. 1994; C. A. A. N. Mol. Pharmacol. 1995; in a a in the of of TNF or inhibitors of respiratory for to as in the fluorescence incubation was in the of in and at when to mitochondria, the concentration of the did not mitochondria only whose did not the fluorescence of of of mitochondria fluorescence and for at a flow on mitochondrial fluorescence and a at and of from of was through a in of the a The of the fluorescence by mitochondria with in the or of ceramide was determined the and as fluorescence from to the of was in by of mitochondria with or for by of the by mitochondria of GSH from rat liver by in with for or in for as described C. A. A. J. N. J. Clin. Invest. 1994; Ookhtens M. N. J. Clin. Invest. 1989; Scholar). These mitochondrial GSH to of not and GSH determined by as described C. Ookhtens M. N. J. Clin. Invest. 1991; 87: C. A. A. J. N. J. Clin. Invest. 1994; of and peroxide was determined Mitochondria with the μM, in the or of ceramide or other electron transport inhibitors was determined at for and for with of and C. A. A. N. Mol. Pharmacol. 1995; R. Biochem. Scholar). of was with concentrations of hydrogen peroxide of hydrogen peroxide as described C. A. A. N. Mol. Pharmacol. 1995; peroxidation was determined by of fluorescence of as described S. 1992; 13: Scholar). Mitochondria with C2 with and fluorescence determined at for and for of was by at An increase in mitochondrial results in a in rat liver mitochondria in a of μM μM pH at or to mitochondrial at 1-5 μM, and at was determined at of the was induced by the or by incubation of mitochondria with and and prevented by with A and of from TNF-treated hepatocytes isolated by as described C. A. A. J. N. J. Clin. Invest. 1994; Scholar). Ceramide was by the diacylglycerol kinase as described Kolesnick J. Biol. Chem. Bell R.M. Biochem. 1989; Scholar). from mitochondria and and in of of μM acid, of and diacylglycerol kinase at pH at the was by of lipids with of and of lipid of the and ceramide by on as and by was by The level of ceramide was by with a generated known of ceramide type for of for multiple mitochondrial by of by INTRODUCTIONTumor necrosis factor (TNF) 1The abbreviations used are: TNFtumor necrosis factorAAantimycin ABSObuthionine-L-sulfoximineC2N-acetylsphingosineC6N-hexanoylsphingosineC2DHdihydro-C2DAG1,2-diacylglycerolDEMdiethylmaleateDHRdihydrorhodamine 123DCF2′-7′-dichlorofluoresceinDCFDA2′-7′-dichlorofluorescin diacetateMOPS4-morpholinepropanesulfonic acidNF-κBnuclear factor κBPC-PLCphosphatidylcholine-dependent phospholipase CQubiquinoneROSreactive oxygen speciesSMasesphingomyelinaseTTFAthenoyltrifluoroacetone. is a cytokine produced by a wide variety of cell types whose production is up-regulated in a number of stressful and pathological conditions (1Beutler B. Cerami A. Nature. 1986; 320: 584-588Google Scholar, 2Tracey K.J. Wei H Manogue K.R. Fong Y. Hesse D.G. Nguyen H.T. Kuo G.C. Beutler B. Cotran R.S. Cerami A. J. Exp. Med. 1988; 167: 1211-1227Google Scholar, 3Fiers W. FEBS Lett. 1991; 285: 199-212Google Scholar). TNF exerts a pleiotropic mode of action on multiple cell functions including regulation of immune responses, host defense reactions, and gene regulation. In addition, its role as a mediator of cytotoxicity on certain susceptible transformed cell lines has been well documented (4Brach M.A. Gruss H.J. Asano Y. DeVos S. Ludwig W.D. Mertelsmann R. Herrmann F. Cancer Res. 1992; 52: 2197-2201Google Scholar, 5Elbaz O. Budel L.M. Hoogerbrugge H. Touw I.P. Delwel R. Mahmoud L.A. Lowenberg B. J. Clin. Invest. 1991; 87: 838-841Google Scholar, 6Brach M.A. Gruss H.J. Scott C. Herrmann F. Mol. Cell. Biol. 1993; 13: 4824-4830Google Scholar, 7Belka C. Wiegmann K. Adam D. Holland R. Neuloh M. Herrmann F. Kronke M. Brach M.A. EMBO J. 1995; 14: 1156-1165Google Scholar). Upon binding to its receptor subtypes, TNF evokes a complicated array of intracellular signals, including G-coupled activation of phospholipase A2, release of arachidonic acid, DAG production, and activation of protein kinase C, some of which may participate in the chain of reactions that result in cell killing (5Elbaz O. Budel L.M. Hoogerbrugge H. Touw I.P. Delwel R. Mahmoud L.A. Lowenberg B. J. Clin. Invest. 1991; 87: 838-841Google Scholar, 6Brach M.A. Gruss H.J. Scott C. Herrmann F. Mol. Cell. Biol. 1993; 13: 4824-4830Google Scholar, 7Belka C. Wiegmann K. Adam D. Holland R. Neuloh M. Herrmann F. Kronke M. Brach M.A. EMBO J. 1995; 14: 1156-1165Google Scholar). An overproduction of ROS has been proposed as an important mechanism to mediate the cytotoxic and gene regulating effects that TNF exerts on tumor cells (8Jones A.L. Selby P. Cancer Surv. 1989; 8: 817-836Google Scholar,9Schulze-Ostholl K. Beyaert R. Vandevoorde V. Haegeman G. Fiers W. J. Biol. Chem. 1992; 267: 5317-5323Google Scholar, 10Schulze-Oshoff K. Bakker A.C. Vanhaesebroeck B. Beyaert R. Jacob W.A. Fiers W. EMBO J. 1993; 12: 3095-3104Google Scholar, 11Goosens V. Grooten J. De Vos K. Fiers W. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8115-8119Google Scholar, 12Adamson G.M. Billings R.E. Arch. Biochem. Biophys. 1992; 294: 223-229Google Scholar).Ceramide has attracted considerable attention due to its role as an intracellular effector molecule that mimics some of the biological effects exerted by inflammatory cytokines such as TNF (13Kolesnick R. Golde D.W. Cell. 1994; 77: 325-328Google Scholar, 14Kolesnick R. Fuks Z. J. Exp. Med. 1995; 181: 1949-1952Google Scholar, 15Hannun Y. J. Biol. Chem. 1994; 269: 3125-3128Google Scholar, 16Zhang Y. Kolesnick R. Endocrinology. 1995; 136: 4157-4160Google Scholar, 17Hannun Y.A. Bell R.M. Science. 1989; 243: 500-507Google Scholar, 18Spiegel S. Merrill A.H. FASEB J. 1996; 10: 188-1397Google Scholar). In addition to its de novo biosynthesis, which is initiated by the condensation of serine and palmitoyl-CoA, ceramide can also be generated by sphingomyelin hydrolysis. Thus, enzymes that hydrolyze sphingomyelin such as sphingomyelinases stand as regulators of intracellular ceramide levels and consequently ceramide-mediated functions. These enzymes are key components of the so-called sphingomyelin pathway, an ubiquitous system that functions in transducing the signals of cytokines to the cell interior (13Kolesnick R. Golde D.W. Cell. 1994; 77: 325-328Google Scholar, 14Kolesnick R. Fuks Z. J. Exp. Med. 1995; 181: 1949-1952Google Scholar, 15Hannun Y. J. Biol. Chem. 1994; 269: 3125-3128Google Scholar).Sphingomyelinase is known to exist in two forms depending on their intracellular localization and pH optima (13Kolesnick R. Golde D.W. Cell. 1994; 77: 325-328Google Scholar, 14Kolesnick R. Fuks Z. J. Exp. Med. 1995; 181: 1949-1952Google Scholar, 15Hannun Y. J. Biol. Chem. 1994; 269: 3125-3128Google Scholar, 16Zhang Y. Kolesnick R. Endocrinology. 1995; 136: 4157-4160Google Scholar). A Mg2+-dependent membrane-bound with a neutral pH optima initiates signaling by generating ceramide at or near the vicinity of the plasma membrane. In addition to the membrane-associated enzyme, another cytosolic neutral SMase independent of Mg2+ has been identified and partially purified, which appears to hydrolyze intracellular sphingomyelin stores to initiate signaling (19Okazaki T. Bielawska A. Domal N. Bell R.M. Hannun Y.A. J. Biol. Chem. 1994; 269: 4070-4077Google Scholar). The signal initiated by these enzymes is then transmitted further down in the signaling cascade by activation of ceramide-activated protein phosphatases and ceramide-dependent protein kinases (13Kolesnick R. Golde D.W. Cell. 1994; 77: 325-328Google Scholar, 14Kolesnick R. Fuks Z. J. Exp. Med. 1995; 181: 1949-1952Google Scholar, 15Hannun Y. J. Biol. Chem. 1994; 269: 3125-3128Google Scholar). In addition to the neutral SMase forms, an acidic SMase form has also been identified, displaying an pH optima around 5, the bulk of which seems to be located at the lysosomes/endosomal compartment. Although it appears that acidic SMase plays a role in signaling, the molecular mechanism of its activation and recruitment during signaling is unclear. Indirect evidence have suggested that DAG generated by PC-PLC activates the acidic enzyme at or near the plasma membrane since inhibitors of PC-PLC prevent activation of the acidic SMase. This hypothesis, which implies a redistribution of the enzyme from the lysosomal compartment to or near the plasma membrane, requires further verification (20Schütze S. Potthoff K. Machleidt T. Berkovic D. Wiegmann K. Kronke M. Cell. 1992; 71: 756-776Google Scholar,21Wiegmann K. Schütze S. Machleidt T. Witte D. Kronke M. Cell. 1994; 78: 1005-1015Google Scholar). Despite the existence of the neutral and acidic SMases, an alkaline form of the enzyme has been described recently, although its role in signaling remains to be defined (22Nyberg L. Duan R.D. Axelson J. Nilsson A. Biochim. Biophys. Acta. 1996; 1300: 42-48Google Scholar).There is compelling evidence to propose ceramide as a second messenger in the sphingomyelin pathway similar to DAG in the glycerophospholipid pathway. The role that ceramide fulfills within the cell are numerous and of varied nature (13Kolesnick R. Golde D.W. Cell. 1994; 77: 325-328Google Scholar, 14Kolesnick R. Fuks Z. J. Exp. Med. 1995; 181: 1949-1952Google Scholar, 15Hannun Y. J. Biol. Chem. 1994; 269: 3125-3128Google Scholar, 16Zhang Y. Kolesnick R. Endocrinology. 1995; 136: 4157-4160Google Scholar). It has been shown that ceramide plays a critical role in apoptosis, proliferation, cellular senescence, and gene regulation through activation of transcription factors such as NF-κB (20Schütze S. Potthoff K. Machleidt T. Berkovic D. Wiegmann K. Kronke M. Cell. 1992; 71: 756-776Google Scholar,21Wiegmann K. Schütze S. Machleidt T. Witte D. Kronke M. Cell. 1994; 78: 1005-1015Google Scholar). However, the possibility that ceramide may interact with mitochondria leading to production of ROS has not been documented to our knowledge, and constitutes the basis of the present report.Mitochondria are one of the most important cellular sources of ROS due to its quantitative consumption of molecular oxygen. Since ceramide appears as an important mediator of the effects elicited by TNF and due to the participation of mitochondria in the TNF-induced ROS production (9Schulze-Ostholl K. Beyaert R. Vandevoorde V. Haegeman G. Fiers W. J. Biol. Chem. 1992; 267: 5317-5323Google Scholar, 10Schulze-Oshoff K. Bakker A.C. Vanhaesebroeck B. Beyaert R. Jacob W.A. Fiers W. EMBO J. 1993; 12: 3095-3104Google Scholar, 11Goosens V. Grooten J. De Vos K. Fiers W. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8115-8119Google Scholar, 12Adamson G.M. Billings R.E. Arch. Biochem. Biophys. 1992; 294: 223-229Google Scholar), the purpose of the present work was to analyze the effects of ceramide and other sphingolipids, on the production of hydrogen peroxide in isolated mitochondria from rat liver. Furthermore, since reduced GSH is the only defense provided to metabolize peroxides generated from the electron transport chain through GSH redox cycle (23Kaplowitz N. Aw T.Y. Ookhtens M. Annu. Rev. Pharmacol. Toxicol. 1985; 25: 715-744Google Scholar), we determined the role of mitochondrial GSH in modulating the production of hydrogen peroxide and its consequences upon incubation of mitochondria with ceramide. Our studies demonstrate for the first time that addition of ceramide to mitochondria results in a dose-dependent increase in hydrogen peroxide, which is prevented when complex I and II of respiration are inhibited. Furthermore, mitochondria from TNF-treated hepatocytes displayed an increased level of ceramide supporting the role of ceramide as an intermediate in the TNF-induced ROS generation from mitochondria. Depletion of matrix GSH prior to exposure to ceramide results in an additional increase of hydrogen peroxide, which peroxidizes lipids from mitochondria resulting in loss of mitochondrial function. These results suggest that ceramide produced in the signaling of TNF is responsible, at in for some of the TNF-induced cytotoxic