- Email: [email protected]
Effect of thiols oxidation on lipid peroxidation in rat liver mitochondria
Chem.-Biol. Interactions, 19 (1977) 383--386 © Elsevier/North-Holland Scientific Publishers Ltd.
383
EFFECT OF THIOLS OXIDATION ON LIPID PEROXIDATION IN RAT LIVER MITOCHONDRIA
A. BINDOLI, L. C A V A L L I N I and N. SILIPRANDI Institute of Biological Chemistry, University of Padova, and Centro Studio Fisiologia Mitocondriale C.N.R., Padova (Italy) (Received July 22nd, 1977) (Accepted Sept. 17th, 1977)
Introduction
The role of ascorbic acid in accelerating the rate of lipid peroxide formation was firstpointed out by Deutsch [1] and confirmed by Elliotand Libet [2]. Ottolenghi found that mitochondrial suspensions undergo a rapid peroxide formation when relativelysmall amounts of ascorbic acid are added to the incubation medium [3]. A non~nzymatic mechanism consisting of a cooxidation of ascorbic acid and unsaturated fats was assumed. Interesting correlations between mitochondrial swelling and lipid peroxidation induced by Fe 2+, ascorbate and G S S G + G S H have been reported by Schneider et al. [4]. O n the other hand Utley et al. [5] found that incubation of livermicrosomes with sulfhydryl reagents, such as HgCI2, N E M or p C M B resulted in the formation of lipid peroxides. These results are consistent with the possibility that thiol-reacting agents produce changes in protein conformation thereby rendering the protein bound iron availablefor catalysisof endogenous lipid peroxidation. The present study describes the influence of diamide (diazenedicarboxylic acid bis (N,N
Results and Discussion
Fig. 1, relative to the time course of Malondialdehyde (MDA) formation, shows that, as previously described by Ottolenghi [3], rat liver mitochondrial suspensions did not form peroxide upon incubation in the usual medium (trace 4), but peroxide formation took place when 0.2 m M ascorbic acid was added (trace 2). Ascorbate-supported peroxide formation started after a period of 30 min at 25°C and then proceeded with an increasing rate. Dj-mide, a thiol oxidizing agent, did not induce per se any peroxide Abbreviations: MDA, malondialdehyde; NEM, N~thylmaleimide.
384
15
(OIAMIASCORBATE
/o(~
_z i,i
E O
w _J O IE c
30
I
I
60
90
MINUTES Fig. 1. M D A f o r m a t i o n in rat liver m i t o e h o n d r i a . The assay system consisting o f 125 mM KC1 and 25 mM Tris-buffer, p H 7.4 c o n t a i n e d 1 m g / m l o f m i t o e h o n d r i a l protein. M D A formed was d e t e r m i n e d by the thiobarbituri¢ acid m e t h o d as described by Ottolenghi [ 3 l. F o r further i n f o r m a t i o n see text.
formation (trace 3). However, when mitochondria were pretreated for 20 min with 0.3 mM diamide and then repeatedly washed to avoid any interference of diamide with ascorbate, the rate and the extent of peroxide formation were strongly enhanced by 0.2 mM ascorbate (trace 1). It has been consequently assumed t h a t in mitochondrial membranes the non-enzymatic process of peroxide formation is prevented until membrane thiols are preserved. When a critical a m o u n t of thiols has been oxidized by diamide, peroxide formation is no longer prevented and proceeded autocatalitically. In Fig. 2 the e x t e n t o f peroxidation initiated by ascorbate 0.2 mM and the c o n c o m i t a n t decrease of thiol groups as a function of diamide concentration
385 o
o DIAMIDE PRETREATED
-',
.~ NEM
PRETREATED
~e
MITOCHONDRIA
MITOCHONDRIA
,¢.,"
,,3
.~
n." ¢.9
i
nr C)
g~00 t~ O
ININ;
r.,,.
1
i
!
!
I
0.1
0.2
0.3
0.4
0.5
DIAMIDE
OR
NEbl
CONCENTRATION (mM)
Fig. 2. MDA formation and SH groups remaining in rat liver mitochondria after pretreatment with increasing amounts o f diamide and NEM. Rat liver mitochondria (2.5 mg/ml) were preincubated at 25°C for 20 rain in 0.125 M KCI, 20 mM Tris-buffer pH 7.4 in the presence of various amounts of diamide and NEM. At the end of incubation, mitochondria were rapidly centrifuged, washed twice and resuspended in the same medium. Mitochondria pretreated with sulfhydryl reagents were partly incubated with 0.2 mM ascorbate for 90 rain for MDA development (determined as described in Fig. 1) and partly utilized for SH groups determination. The unreacted SH groups were titrated with DTNB [6] in a mixture containing 0.2 M Tris--HCl buffer pH 8.1, 5 mM EDTA, 0.3% Sodium dodecyl sulphate, 1 mM DTNB and about 1 mg protein. The values are expressed as percentage of the control.
are reported. MDA formation t o o k place only with concentrations of diamide above 0.2 mM which oxidized 15--20% of thiols. Parallel experiments carried o u t with NEM also showed that lipid peroxidation did not occur until approximately the same amount of thiols had been affected. It appears that a critical decrease of about 15% of membrane thiols, whatever the experimental condition imposed, constitute a prerequisite for lipid peroxide formation in mitochondria. Therefore it is conceivable that any condition, for instance spontaneous ageing [7] involving a decrease of thiol groups potentially promoted lipid peroxidation.
386 On the light of present results the protection of DTE against spontaneous ageing o f mitochondria [8] can be interpreted as a preservation by this reducing agent of vicinal thiols from oxidation to disulfide bonds and consequently an induced prevention from peroxide formation. It has been reported that the content of titrable --SH in rat liver mitochondria decreases [9] while mitochondrial potential peroxidation increases with the rat age [ 1 0 ] . Hence a correlation between --SH disappearance and increased peroxidation either in vivo or in vitro can be assumed.
1 H.F. Deutsch, B.E. Kline and H.P. Rusch, The oxidation of phospholipids in the presence of ascorbic acid and carcinogenic chemicals, J. Biol. Chem., 141 (1941) 529. 2 K.A. Elliott and B. Libet, Oxidation of phospholipid catalyzed by iron compounds with ascorbic acid, J. Biol. Chem., 152 (1944) 617. 3 A. Ottolenghi, Interaction of ascorbic acid and mitochondriai lipides, Arch. Biochem. Biophys., 79 (1959) 355. 4 A.K. Schneider, E.E. Smith and F.E. Hunter Jr., Correlation of oxygen consumption with swelling and lipid peroxide formation when mitochondria are treated with the swelling-inducing agents Fe 2÷, glutathione, ascorbate or phosphate, Biochemistry, 3 (1964) 1470. 5 H.G. Utley, F. Bernheim and P. Hochstein, Effect of sulfhydryl reagents on peroxidation in microsomes, Arch. Biochem. Biophys., 118 (1967) 29. 6 G.L. Ellman, Tissue sulfhydryl groups, Arch. Biochem. Biophys., 82 (1959) 70. 7 M.V. Riley and A.L. Lehninger, Changes in sulfhydryl groups of rat liver mitochondria during swelling and contraction, J. Biol. Chem., 239 (1964) 2083. 8 D. Siliprandi, G. Scutari, F. Zoccarato and N. Siliprandi, Action of diamide on some energy linked processes of rat liver mitochondria, FEBS Lett., 42 (1974) 197. 9 T.G. Lastovskaya, Content of sulfhydryl groups in some tissues and mitochondria of rats of various ages, Chem. Abstr., 74 (1971) 29895 j. 10 M.C. Barret and A.A. Horton, Age-related changes in lipid peroxidation in rat liver mitochondria, Biochem. Soc. Trans., 3 (1975) 124.
383
EFFECT OF THIOLS OXIDATION ON LIPID PEROXIDATION IN RAT LIVER MITOCHONDRIA
A. BINDOLI, L. C A V A L L I N I and N. SILIPRANDI Institute of Biological Chemistry, University of Padova, and Centro Studio Fisiologia Mitocondriale C.N.R., Padova (Italy) (Received July 22nd, 1977) (Accepted Sept. 17th, 1977)
Introduction
The role of ascorbic acid in accelerating the rate of lipid peroxide formation was firstpointed out by Deutsch [1] and confirmed by Elliotand Libet [2]. Ottolenghi found that mitochondrial suspensions undergo a rapid peroxide formation when relativelysmall amounts of ascorbic acid are added to the incubation medium [3]. A non~nzymatic mechanism consisting of a cooxidation of ascorbic acid and unsaturated fats was assumed. Interesting correlations between mitochondrial swelling and lipid peroxidation induced by Fe 2+, ascorbate and G S S G + G S H have been reported by Schneider et al. [4]. O n the other hand Utley et al. [5] found that incubation of livermicrosomes with sulfhydryl reagents, such as HgCI2, N E M or p C M B resulted in the formation of lipid peroxides. These results are consistent with the possibility that thiol-reacting agents produce changes in protein conformation thereby rendering the protein bound iron availablefor catalysisof endogenous lipid peroxidation. The present study describes the influence of diamide (diazenedicarboxylic acid bis (N,N
Results and Discussion
Fig. 1, relative to the time course of Malondialdehyde (MDA) formation, shows that, as previously described by Ottolenghi [3], rat liver mitochondrial suspensions did not form peroxide upon incubation in the usual medium (trace 4), but peroxide formation took place when 0.2 m M ascorbic acid was added (trace 2). Ascorbate-supported peroxide formation started after a period of 30 min at 25°C and then proceeded with an increasing rate. Dj-mide, a thiol oxidizing agent, did not induce per se any peroxide Abbreviations: MDA, malondialdehyde; NEM, N~thylmaleimide.
384
15
(OIAMIASCORBATE
/o(~
_z i,i
E O
w _J O IE c
30
I
I
60
90
MINUTES Fig. 1. M D A f o r m a t i o n in rat liver m i t o e h o n d r i a . The assay system consisting o f 125 mM KC1 and 25 mM Tris-buffer, p H 7.4 c o n t a i n e d 1 m g / m l o f m i t o e h o n d r i a l protein. M D A formed was d e t e r m i n e d by the thiobarbituri¢ acid m e t h o d as described by Ottolenghi [ 3 l. F o r further i n f o r m a t i o n see text.
formation (trace 3). However, when mitochondria were pretreated for 20 min with 0.3 mM diamide and then repeatedly washed to avoid any interference of diamide with ascorbate, the rate and the extent of peroxide formation were strongly enhanced by 0.2 mM ascorbate (trace 1). It has been consequently assumed t h a t in mitochondrial membranes the non-enzymatic process of peroxide formation is prevented until membrane thiols are preserved. When a critical a m o u n t of thiols has been oxidized by diamide, peroxide formation is no longer prevented and proceeded autocatalitically. In Fig. 2 the e x t e n t o f peroxidation initiated by ascorbate 0.2 mM and the c o n c o m i t a n t decrease of thiol groups as a function of diamide concentration
385 o
o DIAMIDE PRETREATED
-',
.~ NEM
PRETREATED
~e
MITOCHONDRIA
MITOCHONDRIA
,¢.,"
,,3
.~
n." ¢.9
i
nr C)
g~00 t~ O
ININ;
r.,,.
1
i
!
!
I
0.1
0.2
0.3
0.4
0.5
DIAMIDE
OR
NEbl
CONCENTRATION (mM)
Fig. 2. MDA formation and SH groups remaining in rat liver mitochondria after pretreatment with increasing amounts o f diamide and NEM. Rat liver mitochondria (2.5 mg/ml) were preincubated at 25°C for 20 rain in 0.125 M KCI, 20 mM Tris-buffer pH 7.4 in the presence of various amounts of diamide and NEM. At the end of incubation, mitochondria were rapidly centrifuged, washed twice and resuspended in the same medium. Mitochondria pretreated with sulfhydryl reagents were partly incubated with 0.2 mM ascorbate for 90 rain for MDA development (determined as described in Fig. 1) and partly utilized for SH groups determination. The unreacted SH groups were titrated with DTNB [6] in a mixture containing 0.2 M Tris--HCl buffer pH 8.1, 5 mM EDTA, 0.3% Sodium dodecyl sulphate, 1 mM DTNB and about 1 mg protein. The values are expressed as percentage of the control.
are reported. MDA formation t o o k place only with concentrations of diamide above 0.2 mM which oxidized 15--20% of thiols. Parallel experiments carried o u t with NEM also showed that lipid peroxidation did not occur until approximately the same amount of thiols had been affected. It appears that a critical decrease of about 15% of membrane thiols, whatever the experimental condition imposed, constitute a prerequisite for lipid peroxide formation in mitochondria. Therefore it is conceivable that any condition, for instance spontaneous ageing [7] involving a decrease of thiol groups potentially promoted lipid peroxidation.
386 On the light of present results the protection of DTE against spontaneous ageing o f mitochondria [8] can be interpreted as a preservation by this reducing agent of vicinal thiols from oxidation to disulfide bonds and consequently an induced prevention from peroxide formation. It has been reported that the content of titrable --SH in rat liver mitochondria decreases [9] while mitochondrial potential peroxidation increases with the rat age [ 1 0 ] . Hence a correlation between --SH disappearance and increased peroxidation either in vivo or in vitro can be assumed.
1 H.F. Deutsch, B.E. Kline and H.P. Rusch, The oxidation of phospholipids in the presence of ascorbic acid and carcinogenic chemicals, J. Biol. Chem., 141 (1941) 529. 2 K.A. Elliott and B. Libet, Oxidation of phospholipid catalyzed by iron compounds with ascorbic acid, J. Biol. Chem., 152 (1944) 617. 3 A. Ottolenghi, Interaction of ascorbic acid and mitochondriai lipides, Arch. Biochem. Biophys., 79 (1959) 355. 4 A.K. Schneider, E.E. Smith and F.E. Hunter Jr., Correlation of oxygen consumption with swelling and lipid peroxide formation when mitochondria are treated with the swelling-inducing agents Fe 2÷, glutathione, ascorbate or phosphate, Biochemistry, 3 (1964) 1470. 5 H.G. Utley, F. Bernheim and P. Hochstein, Effect of sulfhydryl reagents on peroxidation in microsomes, Arch. Biochem. Biophys., 118 (1967) 29. 6 G.L. Ellman, Tissue sulfhydryl groups, Arch. Biochem. Biophys., 82 (1959) 70. 7 M.V. Riley and A.L. Lehninger, Changes in sulfhydryl groups of rat liver mitochondria during swelling and contraction, J. Biol. Chem., 239 (1964) 2083. 8 D. Siliprandi, G. Scutari, F. Zoccarato and N. Siliprandi, Action of diamide on some energy linked processes of rat liver mitochondria, FEBS Lett., 42 (1974) 197. 9 T.G. Lastovskaya, Content of sulfhydryl groups in some tissues and mitochondria of rats of various ages, Chem. Abstr., 74 (1971) 29895 j. 10 M.C. Barret and A.A. Horton, Age-related changes in lipid peroxidation in rat liver mitochondria, Biochem. Soc. Trans., 3 (1975) 124.