Effect of Diethyl Maleate and Glutathione on. Linoleate Peroxidation NANCY F E R N A N D E Z a, ALFONSO VALENZUELAa . *, V I R G I N I A FERNANDEZ b and LUIS A. VIDELAb, aUnidad de Bioqufmica, Instituto de Nutricidn Y Tecnologfa de los Alimento$, Universidad de Chile, Casilla 15138, Santiago 11; and bUnidad de Bioqutlwica, Divisidn de Ciencias Mddica$ Occidente, Facultad de Medicina, Universidad de Chile, Ca$illa 10455, Correo Central, Santiago, Chile

393

ABSTRACT

The antioxidant effect of diethyl maleate and reduced glutathione was studied in an in vitro peroxidizing system. Linoleate peroxidation, measured as conjugated diene formation, was not altered by diethyl maleate (2 or 4 raM), whereas the addition of reduced glutathione (1, 2 or 4 mM) elicited a marked and progressive reduction. This effect of glutathione is not modified by diethyl maieate. ]'he inhibition of linoleate peroxidation by glutathione was found concomitantly with a decrease in the concentration of its reduced form and a corresponding increase in glutathione disulfide levels, so that the total equivalents of reduced glutathione in the system remained constant. It is concluded that diethyl maleate does not have antioxidant properties in a peroxidizing system, as found for reduced glutathione. Lipids 17:393-395, 1982.

Recently, a metabolic interrelationship be- tween reduction in the concentration of re- duced glutathione (GSH) and the stimulation of lipoperoxidative processes in the liver cell was observed following acute ethanol intoxication in rats (1). The lipoperoxidative pressure in- duced by ethanol was suggested to be a conse- quence of both an increased generation of oxygen-related free radicals (2) and the low GSH levels (1), thus impairing the protective systems of the hepatocyte against peroxide toxicity.

Diethyl maleate is known to be a powerful GSH-depleting agent in the cell (3). Its mecha- nism of action has been proposed to be exerted by a conjugation with the tripeptide, forming a complex that is released to the extracellular medium (3). Studies carried out in rats have revealed that the administration of diethyl maleate in vivo produces a faster and greater effect of liver GSH levels than ethanol ingestion (4) without a concomitant enhancement of lipid peroxidation (4-6). It is known that unsaturated compounds such as polyunsatu- rated fatty acids are susceptible to hydrogen abstration by free radicals, leading to the production of fatty aeyl radicals and to an autoxidation reaction in the presence of oxygen (7). Thus, it is conceivable that the lack of effect of diethyl maleate on hepatic lipid peroxidation (4-6) may be a consequence, in part, of an antioxidant action related to its unsaturated structure that could scavenge the free radicals involved in the stimulation of lipid peroxidation. In view of these observa- tions, the possible antioxidant effects of diethyl maleate were evaluated in an in vitro peroxidiz- ing system formed by linoleic acid and Fe ~+ (8), and were compared to those elicited by GSH.

M A T E R I A L S A N D METHODS

The peroxidizing system was prepared by emulsifying 1 ml of linoleic acid with 10 ml of distilled water containing 0.5 ml of Tween-60 (90% w/v) with agitation. The emulsion ob- tained was neutralized with 1 N KOH, resus- pended in 100 ml of 0.05 M potassium phos- phate buffer, pH 7.0, and made up to a final volume of 150 ml with dis-tilled water. This system was incubated at 37 C and peroxidation was initiated in all experiments by adding FeSO4 to a final concentration of 500 /~M Fe 2+ (8). In the nonperoxidizing system, lino- leic acid was replaced by water. Diethyl maleate and/or GSH were added at time zero as indi- cated in Figure 1.

Linoleate peroxidation was assessed by conjugated diene formation. Aliquots of 0.2 ml of the incubation medium were taken every 30 min and were added to 7.8 ml of 50% v/v ethanol at room temperature. Conjugated dienes were measured at 233 nm according to Hasse and Dunkley (8). Results were expressed as mmol of peroxide/ml of incubation medium by using t h e e = 2.52 x I04 M -1 x cm -1 (9).

Glutathione measurements were done enzy- maritally in aliquots of 40/zl of the incubation medium taken every hour. GSH was measured using methylglyoxal and glyoxalase I at 240 nm and glutathione disulfide (GSSG) was deter- mined with NADPH and glutathione reductase at 340 nm in the same aliquots (10).

All the reagents used were obtained from Sigma (St. Louis) except for diethyl maleate (Aldrich, Milwaukee, WI).

RESULTS A N D DISCUSSION

Data presented in Figure 1 show that the

LIPIDS, VOL. 17, NO. 5 (1982)

394 COMMUNICATIONS

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addition of Fe 2§ to a final concentration of 500 pM induces the peroxidation of linoleate as evidenced by the enhancement of conjugated diene formation during the incubation period of 4 hr. A similar lipoperoxidative response is obtained when diethyl maleate is added to the peroxidizing system at a final concentration of 2 or 4 mM (Fig. 1A). This indicates that the GSH-depleting agent diethyl maleate does not

FIG. 1. (A) Effect of diethyl maleate (DEM) on linoleate peroxidation induced in vitro by Fe 2+. Control (e) peroxidizing system with no addition. Addition of (o) 2 mM or (zx) 4 mM DEM. Each point represents the mean +- SEM for values obtained in duplicate from 5 separate experiments. (B) Effect of reduced glutathione (GSH) on linoleate peroxidation induced in vitro by Fe ~2. Control (o) peroxidizing system with no addition. Addition of (o) 1 raM; (z~) 2 mM or (A) 4 mM GSH. Each point represents the mean • SEM for values obtained in duplicate from 5 separate experiments. (C) Effect of diethyl maleate (DEM) and reduced glutathione (GSH) on linoleate peroxidation induced by Fe +2. Control (e) peroxidizing system with no additions. Addition of (o) 2 mM DEM; (A) 2 mM GSH and (zx) 2 mM DEM + 2 mM GSH. Each point represents the mean -+ SEM for values obtained in duplicate from 5 separate experiments.

have any protective effect on linoleate peroxi- dation induced by Fe 2+ in the in vitro condi- tions used. These results suggest that the lack of stimulation of hepatic lipid peroxidation by diethyl maleate when given in vivo (4,6) or added to isolated rat liver cells ( 5 ) d o e s not seem to be related to an antioxidant property of the agent due to its unsaturated structure.

The extent of lipoperoxidative processes in the cell depends on the balance between peroxi- dant and antioxidant systems (11,12). Among the antioxidant systems, GSH has been postu- lated as the most important one (13). The antioxidant actions of GSH can be visualized in its participation with either the catabolism of cellular peroxides formed by free-radical- induced lipid peroxidation (11,12) or with the direct interception of free radical species (14). In both mechanisms, GSSG is formed (11,12, 14).

The addition of GSII to the peroxidizing system elicited a drastic and progressive inhibi- tion of linoleate peroxidation when added to a final concentration of 1, 2 or 4 raM, respective- ly (Fig. 1B). This antioxidant effect of GSH was observed concomitantly with a decrease in its concentration in the peroxidizing system as a function of time (Fig. 2A), suggesting an interaction between GSH and the free radicals generated in this condition that could form glutathionyl radicals (GS ~ and GSSG by homologous binding (14). This view is further supported by the fact that the decrease in GSH concentration is accompanied by a correspond- ing increase in GSSG levels (Fig. 2A), so that the concentration of total GSH equivalents (GSH + 2GSSG) of the peroxidizing system remains constant throughout the incubation period (Fig. 2A). The possibility of a sponta- neous oxidation of GSH in the in vitro condi-

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FIG. 2. (A) Time course of the changes in the concentration of reduced glutathione (GSH), gluta- thione disulfide (GSSG) and total GSH equivalents (GSH + 2GSSG) in the peroxidizing system. (B) Time course of the changes in GSH concentration in the nonperoxidizing system. Each point represents the mean -+ SEM for values obtained in duplicate from 5 separate experiments.

t.ions used can be discarded as its concen t r a t i on is no t al tered when added to a nonperox id iz ing sys tem (Fig. 2B).

Diethyl maleate was unable to mod i fy the an t iox idan t ef fect of GSH in the in vitro peroxidiz ing sys tem (Fig. 1C), indicat ing that an in terac t ion be tween die thyl maleate and GSH does no t seem to occur in this situa- t ion. In the cell, however , this in te rac t ion s e e m s to occur med ia ted by an enzymat i c conjugat ion catalyzed by the g luta th ione-S-alkenetransfer- ase sys tem (15).

ACKNOWLEDGMENTS

This work was supported in part by Grant B-1162-

811-3 from the Servicio de Desarrollo Cientilieo, Arti[tico y de Cooperacidn [nternacionai, Universidad de Chile, and by the Research Assoeiateship Program (L.A. Videla) from the Natural Sciences and Engineer- ing Research Council of Canada. The technical assist- ance of R. Guerra and C. Almeyda is acknowledged. The secretarial assistance of F. Rodrikuez is appre- ciated.

REFERENCES

1. Videla, L.A., Ferndndez, V., Ugarte, G., Valen- zuela, A., and Villanueva, A. (1980) FEBS Lett. 111,6-10.

2. Valenzuela, A., Ferngndez, N, Ferndndez, V., Ugarte, G., Videla, L.A., Guerra, R., and Villa- nueva, A. (1980) FEBS Lett. I l l , 11-13.

3. Chasseaud, L.F. (1976) in Glutathione: Metabo- lism and Function (Arias, I.M., and Jakoby, W.B., eds.) pp. 77-114, Raven Press, New York, NY.

4. Juanet, A., Tabak, M., Videla, L.A., and Valen- zuela, A. (1981) IRCS Med. Sci. 9, 388.

5. Hogberg, J., and Kristoferson, A. (1977) Eur. J. Biochem. 74, 77-82.

6. Wendel, A., Feuerstein, S., and Kong, K.H. (1979) Biochem. Pharmacol. 28, 2051-2055.

7. Mead, J.F. (1976) in Free Radicals in Biology (Pryor, W.A., ed.) Vol. 1, pp. 51-68, Academic Press, New York, NY.

8. Hasse, G., and Dunkley, W. (1969) J. Lipid Res. 10, 55-58.

9. Buege, J.A., and Aust, S.D. (1978) Methods Enzymol. 52,302-310.

10. Bernt, F., and Bergmeyer, H.U. (1974) in Meth- ods of Enzymatic Analysis (Bergmeyer, H.U., ed.) Vol. 4, pp. 1643-1647, Academic Press, New York, NY.

11. Chance, B., Sies, H., and Boveris, A. (1979) Physiol. Rev. 59, 527-605.

12. Sies, H., Wahllander, A., Waydhas, C., Soboll, S., and Haberle, D. (1980) Adv. Enz. Reg. 18, 303-320.

13. Meister, A. (1975) in Metabolic Pathways (Green- berg, D.M., ed.) Vol. 7, pp. 101-188, Academic Press, New York, NY.

14. Kosower, N.S., and Kosower, E.M. (1979) Int. Rev. Cytol. 54, 109-160.

15. Chaaseaud, L.F. (1973) Drug Metab. Rev. 2, 185- 220.

[Received Oc tobe r 1, 1981 ]

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