Chem.-Biol. Interactions, 19 (1977) 383--386 383 © Elsevier/North-Holland Scientific Publishers Ltd.

EFFECT OF THIOLS OXIDATION ON LIPID PEROXIDATION IN RAT LIVER MITOCHONDRIA

A. BINDOLI, L. CAVALLINI 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 forma-

tion was first pointed out by Deutsch [1] and confirmed by Elliot and Libet [2]. Ottolenghi found that mitochondrial suspensions undergo a rapid per- oxide formation when relatively small 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 GSSG + GSH have been reported by Schneider et al. [4]. On the other hand Utley et al. [5] found that incubation of liver micro- somes with sulfhydryl reagents, such as HgCI2, NEM or pCMB resulted in the formation of lipid peroxides. These results are consistent with the possibility that thiol-reacting agents produce changes in protein conforma- tion thereby rendering the protein bound iron available for catalysis of endo- genous lipid peroxidation.

The present study describes the influence of diamide (diazenedicarboxylic acid bis (N,N<limethylamide)) and N~thylmaleimide (NEM), both of which affect thiols by different mechanisms, on the non~nzymatic formation of lipid peroxide in rat liver mitochondria upon addition of ascorbate.

Results and Discussion Fig. 1, relative to the time course of Malondialdehyde (MDA) formation,

shows that, as previously described by Ottolenghi [3], rat liver mitochon- drial suspensions did not form peroxide upon incubation in the usual medium (trace 4), but peroxide formation took place when 0.2 mM 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

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Fig. 1. MDA format ion in rat liver mi toehondr ia . The assay system consisting o f 125 mM KC1 and 25 mM Tris-buffer, pH 7.4 conta ined 1 mg/ml o f mi toehondr ia l protein . MDA formed was de te rmined by the thiobarbi tur i¢ acid me thod as described by Ot to lenghi [ 3 l . Fo r further in format ion 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 interfer- ence 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 that in mitochondrial membranes the non-enzymatic process of peroxide formation is prevented until membrane thiols are preserved. When a critical amount of thiols has been oxidized by diamide, peroxide formation is no longer prevented and proceeded autoca- talitically.

In Fig. 2 the extent of peroxidation initiated by ascorbate 0.2 mM and the concomitant decrease of thiol groups as a function of diamide concentration

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Fig. 2. MDA formation and SH groups remaining in rat liver mitochondria after pretreat- ment with increasing amounts of 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. Mitochon- dria 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 took place only with concentrations of diamide above 0.2 mM which oxidized 15--20% of thiols.

Parallel experiments carried out with NEM also showed that lipid peroxid- ation 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, what- ever 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 protect ion of DTE against spontaneous ageing of mitochondria [8] can be interpreted as a preservation by this re- ducing agent of vicinal thiols from oxidation to disulfide bonds and conse- quent ly an induced prevention from peroxide formation.

It has been reported that the content of titrable --SH in rat liver mitochon- dria decreases [9] while mitochondrial potential peroxidation increases with the rat age [10] . Hence a correlation between --SH disappearance and in- creased 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 peroxida- tion 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 mitochon-

dria 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.