Journal of Neuroscience Research 26: 120-128 (1990)

Influence of Oxidative Stress on the Age-Linked Alterations of the Cerebral Glutathione System G. Benzi, F. Marzatico, 0. Pastoris, and R.F. Villa Institute of Pharmacology, University of Pavia, Pavia, Italy

The glutathione system (reduced and oxidized glutathione; redox index) was studied in the forebrain of male Wistar rats of 5 , 15, and 25 months of age following the administration for 2 months in drinking water of chemicals that induce oxidative stress: paraquat and diethyldithiocarbamate (DDCj to increase superoxide radical formation, aminotriazole and hydrogen peroxide to increase hydroxyl radical generation, as well as diamide and ferrous chloride to decrease the glutathione cycle activity. Chronic oral administration of phosphatidylcholine for 2 months was evaluated in 25-month-old rats. Aging accentuated the changes produced by chemicals that induce oxidative stress; i.e., the changes in the glutathione redox index were most pronounced in the forebrains of the older paraquat-, DDC-, H,O,-, and diamidetreated rats. Markedly different adaptative changes occurred within the various drug groups. The reduced glutathione was increased (by paraquat, DDC and aminotrazole), decreased (by H,O,j or unchanged (by iron and diamide). Furthermore, in older rats, paraquat and DDC increased the glutathione redox index, whereas H,O, and diarnide decreased the glutathione redox index or were ineffective (i.e., aminotriazole, iron). The glutathione redox index alterted by chronic drug administration was modified by the concomitant administration of phosphatidylcholine. Key words: aging, brain aging, forebrain, prooxidants

INTRODUCTION The wear-and-tear theory of aging statcs that damage randomly accumulates with time in biopolymer molecules, decreasing the ability of the brain to preserve and maintain homeostasis. In this view, aging is suggested to result from the random deleterious effects of free radicals that are produced in the transfer of electrons and that are responsible for a significant part of the damage that oc0 1990 Wiley-Liss, Inc.

curs (Harman, 1956, 195 1984; Pryor, 1977, 1982, 1984, 1986). However, I ) the oxyradicals suspected of causing cellular damage are so short-lived as to be difficult to detect directly in vivo; 2) somc typcs of oxyradicals (e.g., superoxide radicals) themselves are not damaging to most biopolymer molecules; 3 ) the biochemical process by which some oxyradicals (e.g. superoxide radicals) are converted to more damaging species (e.g., superoxide radicals) is not unequivocally established; 4) adding antioxidants (e.g., carotenoids and vitamin Ej to the diet has littlc or no effect on increasing mammalian maximum life span; 5 ) defective absorption of antioxidants (e.g., vitamin E) induces pathologies but not acceleratcd aging; and 6) enhanced metabolic rate increases the intracellular concentration of free radicals. Moreover, mammals on calorie-restricted diets do not necessarily exhibit lowcr metabolic rate although they show longer life spans. It is at present difficult to establish a causeieffect relationship between free radicals present in low, steadystate concentrations in neurons and brain aging, which occurs very slowly and has poorly specific markers. Of these, the most suitable is the glutathione system because of its primary role in protecting cells against the toxic action of both partial reduccd oxygen intermediates and lipid hydroperoxides that can be produced, e.g., from ccrebral unsaturated fatty acid (Aebi and Sutcr, 1974; Christophersen, 1968; Flohe, 1971: Orlowski and Karkowsky, 1976). Thus a rational and productive approach may be to magnify the age-rclated modifications of the glutathione system by oxidative stress, initiating damage by reactions that involve radicals and shorten life span (Pryor, 1981; Pryoret al., 1982; Sies, 1985). To the extent that various types of oxidative threats mimic accelerated aging, they should result in the accelerated al~

Received Junc 12, 1989; revised October 5 , 1989; accepted November I . 1989. Address reprint requests to Prof. Gianni Benr.i, Istituto di Farmacologia. Facolti di Scienze, Univcrsita di Pavia, Piazza Botta I I , 27 100 Pavia. Italy.

Brain Aging and Oxidative Stress

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Fig. I . Production of oxygen-derived radicals involvcd in the antioxidant activity of the glutathione system. Mechanisms of action of chcmicals charged with producing oxidative stress.

teration of the glutathione redox state. Hence, the primary purpose of the present study was to evaluate thc age-related putative changes in the glutathione system induced in the rat forebrain by a variety of chcmicals charged with producing oxidative stress (Fig. 1) by 1) increasing the superoxide radical concentration, i.e., the herbicide paraquat or thc supcroxide dismutase inhibitor

diethyldithiocarbamate (DDC); 2) increasing the hydrogen peroxide concentration, i.e., either the catalase inhibitor aminotriazole or H,O, administration; 3) increasing hydroxyl radical production, i.e., ferrous chloride administration; 4) decreasing the glutathione cycle function, i.e., the SH oxidant diamide. A significant age-related decrease in the rate of

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synthesis of both choline and ethanolamine glycerophospholipids takes place in the brain during aging (Gaiti et al., 1982a,b). The resulting increase in the cholesterol to phospholipid molar ratio induces an age-dependent increase in cerebral membrane viscosity (Sun and Sun, 1979), and these changes are associated with behavioral dysfunctions (Lippa et al., 1980; Strong et al., 1980). Phosphatidylcholine 1) is one of the major constituents of cell membranes and also appears in lipoproteins and 2) is one of the major sources of arachidonic acid, which serves as a precursor for the formation of prostaglandins, leukotrienes, and other substances linked to the abovementioned peroxidation processes. Furthermore, phosphatidylcholine and/or its metabolite(s) interferes with both the cytosolic form of superoxide dismutase and the glutathione reductase in the brains of 25-month-old rats (Benzi et al., 1989h). To date, there is little information on the age-related influence of phosphatidylcholine on the cerebral glutathione antioxidant system. This prompted our studies on phosphatidylcholine administration during induced oxidative stress in aging.

MATERIALS AND METHODS Animals The evaluations were carried out on male Wistar rats aged 5 , 15, or 25 months. The animals were kept under constant environmental conditions (temperature 22°C + 5%; circadian rhythm 12 hr light and 12 hr dark) and fed normal laboratory diet as pellets. Experimental Plan Twenty-one groups of five rats aged 3, 13, or 23 months were analyzed after 2 months of maintenance under standard environmental conditions with water ad libitum or replacement of drinking water by 1 :20 diluted stock solution of 2 mM paraquat, 10 mM DDC, 2 mM aminotriazole, 100 mM hydrogen peroxide, 2 mM ferrous chloride, or 2 mM diamide. Biochemical evaluations were performed at 5 , 15, or 25 months of age. Fourteen lots of five “treated” rats aged 23 months 1) were watered either with water ad libitum or with diluted stock solution of paraquat, DDC, aminotriazole, H,O,, FeCl,, or diamide for 2 months and at the same time 2) were administered orally for 2 months vehicle or phosphatidylcholine (EPL + polyunsaturated soybean phospholipid material) in a dose of 100 mg . kg -‘/day.

oxidized (GSSG) glutathione be curtailed during the processing of tissues for analyses and 2) no air oxidation of reduced glutathione occurs. Thus the frozen forebrain was immediately powdercd under liquid nitrogen (Microdismembrator, Braun) and stored at - 80°C. The assay of glutathione by 5,5’-dithio-bis-(2-nitrobenzoic) acid in frozen forebrain powder was carried out within 3 hr,utilizing perchloric acid and N-ethyl-maleimide in the presence of ethylenediamidetetraacetic acid for the extraction procedure (Cooper et al., 1980; Lowry ct al., 1964; Slivka e t a ] . , 1987; Tietze, 1969). The glutathione redox index was calculated as: ([GSH] + 2[GSSG])/ (2rGSSGI x 100).

Statistical Analysis Two statistical tests (ANOVA and Dunnett’s tests) were applied to the results (P < 0.01) after we checked the homogeneity of variance by Bartlett‘s test.

RESULTS Age-Related Changes in Forebrain Glutathione System An age-related increase in reduced glutathione concentration was observed (Fig. 2) in forebrains of rats from 5 to 15 months of life. Subsequently, the rcduced glutathione concentration declined in 25-month-old rats and was approximately 90% of the level of the 5-monthold rats. The forebrain concentration of oxidized glutathione slightly increased during aging. The glutathione redox index increased in rats from 5 to 15 months of life, with a subsequent decline in 25-month-old rats.

Changes in Forebrain Glutathione system by chronic exposure to oxidative Stress Putative changes in the state of the forebrain glutathione system (Fig. 3) were induced by the intake for 2 months of chemicals charged with producing oxidative stress (Fig. 1): paraquat, DDC, aminotriazole, hydrogen peroxide, ferrous chloride, or diamide. Compared with controls, reduced glutathione concentrations increased by about 13%, 15541, and 22% in paraquat-fed 5 - , 15-, and 25-month-old rats, respectively, the oxidized glutathione concentrations being practically unchanged. Compared with controls, the glutathione redox index increased by about 19%’in paraquat-fed 25-month-old rats. Chronic administration for 2 months of DDC increased by 1996, 17%, and 22% the forebrain reduced Analytical Techniques glutathione in 5 , 1 5 , and 25-month-old rats, respecAnimals were sacrificed between 9:OO and 10:30 tively. The oxidized glutathione concentrations remained unaffected in all age groups. The glutathione redox index AM (24 hr after the last day of administration), and brains increased by 17% in DDC-fed 25-month-old rats comwere frozen in liquid nitrogen prior to dissection. The proper assessment of cerebral glutathione con- pared with controls. Reduced glutathione concentrations increased by centration requires that I ) oxidation of reduced (GSB) to

Brain Aging and Oxidative Stress

123

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5 months

15months

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GSH

GSSG

25months

Redox Index

Fig. 2. Concentrations of reduced glutathione (GSH; mM) and oxidized glutathione (GSSG; pM) evaluated in forebrain from untrcated rats aged 5 , 15, and 25 month, (abscissa). The glu-

tathione redox index (arbitrary units) is calculated as: ([GSH] + 2[GSSG]):(2[GSSG] x 100). Each column represents the mean * S.E.M. (n = 5). S, differs from 5-month-old rats

20%, 1296, and 18% in aminotriazole-fed rats at 5 , 15, and 25 months, respectively. Aminotriazole increased oxidized glutathione by about 15% in all age groups, and the glutathione redox index remained unchanged. Compared with the controls, reduced glutathione concentrations decreased to 86%, 78%, and 70% in H,O,-fed 5 , IS-, and 25-month-old rats, respectively. The oxidized glutathione concentrations increased by about 36% in H,O,-fed 5- and 15-month-old rats and by about 62% in 25-month-old rats. H,O, decreased the glutathione redox index to 67570, 59%, and 44% in 5 - , 1 5 , and 25-mohth-old rats, respectively. There were no significant differences in forebrain reduced glutathione between controls and FeC1,-administered rats of the different ages tested. Oxidized glutathione concentration increased by 20-22% in both 15and 25-month-old rats. The glutathione redox index declined to 78% in 15-month-old rats administered FeC1,. Oxidized glutathione concentrations increased by about 14%, 17%, and 25% in diamide-fed 5-, 15-, and 25-month-old rats, respectively, compared with controls. There were no differences in forebrain reduced glutathione between controls and diamide-administered rats of the different ages tested. The glutathione redox index declined to about 73% in 25-month-old rats administered diamide. In the forebrains of 25-month-old rats, chronic oral administration for 2 months of phosphatidylcholine induced (Fig. 4) 1) a decrease in the oxidized glutathione concentration and an increase in the glutathione redox index in both DDC- and diamide-treated animals and 2) a decrease in the oxidized glutathione concentration, consistent with an increase in both reduced glutathione concentration and glutathione redox index in H,O,-ad-

ministered animals. Phosphatidylcholine did not alter the effects of paraquat, aminotriazole. or iron administration.

DISCUSSION The free radical theory of aging postulates that in mammalian aging oxygen is the main source of damaging irreversible reactions characterized by the transient presence of highly reactive intermediates bearing unpaired electrons. The glutathione system is currently thought to play a predominant role in the defense of the central nervous system (CNS) against random free radical release. It may be easier to detect glutathione system alterations if the time course of the aging process is shortened by a variety of oxidative stress (Allen et al., 1983, 1984a,b, 1985; Matkovics and Novak, 1977; Sohal et al., 1984, 1985). With few exceptions (Renzi et al., I989a), this prediction has never been systematically tested in mammalian brain. Thus, in the present research, the effects of chronic administration for 2 months in drinking water of chemicals that induce oxidative stress were evaluated in the forebrain of 5 , 15- and 25-month-old rats. Paraquat increases the production of superoxide radical by NADPH-diaphorase interference, giving rise to an autoxidizable form that reacts with dioxygen to generate 0; (Bus et al., 1975, 1976; Hassan and Fridovich, 1979). However, paraquat toxicity may be due to biochemical mechanisms other than 0; production (Misra and Gorsky, 1981; Shu et al., 1979). DDC is a copper chelator that exerts multiple effects on cellular functions, causing an increase in superoxide radical concentration by marked inactivation of cyanide-qcnsitive as

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Paraquat DDC Amino Trlazole Hydrogen Perox

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Fig. 3. Reduced and oxidized glutathione and glutathione redox index evaluated in forebrain from rats aged 5 , 15, and 25 months (abscissa) fed normal laboratory diet (as pellets) with distilled water, or administered for 2 months in their drinking

water paraquat, diethyldithiocarbamatc (DDC) , aminotriazole, hydrogen peroxide, ferrous chloride (iron), or diamide. Each column represents the means 2 S.E.M. (n = 5 ) . S, differs from distilled water-fed rats.

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PI Untreated Legend Fig. 4. Reduced and oxidized glutathione and glutathione redox index evaluated in forebrain from 25-month-old rats fed normal laboratory diet (as pellets) with distilled watcr, or chronically administered for 2 months in their drinking water paraquat, diethyldithiocarbamate (DCC), aminotriazolc. hy-

Ph.chol-treated drogen peroxide (H-perox), ferrous chloride (iron), or diamide; at the samc time, the rats werc orally treated for 2 months with vehiclc or phosphatidylcholine ( 100 mgikgiday). Each column S . E . M . (n 5 ) . S, differs from verepresents the means hicle-treated rats.

*

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Benzi et al.

TABLE I. Effects of Chemicals That Produce Oxidative Stress’ Oxidized glutathione

Reduced glutathione Chemicals Paraquat Diethyldithiocarbainatc Aminotriazole Hydrogen peroxide Ferrous chloride Diamide

Glutathione redox index

Mechanism of action

Adult

Senescent

Adult

Sene\cent

Adult

Senejcent

Increase in superoxide radical production lnhibiton of supcroxidc dismutase activity Inhihition o f catalase activity Substrate availability for hydroxyl radical formation Catalyiation or hydmxyl radical-generating reactions Oxidation of reduced glutathione

t 7 t &

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-

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-

t t

-

~

t

1‘

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-

t t

-

1

11 1

t t

-

“Alterations of the forebrain glutathione system of 5-month-old rats (adult rats) or 25-month-old rats (senescent rats)

well as cyanide-insensitive superoxide dismutase both in vitro and in vivo (Heikkila and Cohen, 1977; Heikkila et al., 1976; Sohal ct al., 1984). As is summarized in Table I. the chronic intake for 2 months of either paraquat or DDC 1) enhances the concentrations of reduced glutathione in the forebrains of the rats uf the different ages tested and 2) increases the glutathione redox index in the older rats, the oxidized glutathione always being unchanged. Aminotriazole inhibits the catalase activity because of its irreversible binding with the protein moiety of the enzyme (Matgoliash et al., 1960). The concentrations of both reduced and oxidized glutathione (Table 1) are greater in the forebrain of the aminotriazole-administered rats than in the controls at all ages tested, the glutathione redox index always being unchanged. The action of aminotriazole could be related to the increase in H,O, concentrations by catalase inhibition. However, the chronic H,O, ingestion for 2 months induces a marked decrease in the glutathione redox index, particularly in the forebrains of older rats (Table I). Unfortunately, it cannot be determined on the basis of the present model whether the decrease in the glutathione redox state is directly linked to the high intracellular concentration of H,O, or is triggered by the intermediate or molecular products of the breakdown of H,02 that can react directly with reduced glutathione (Ross et al., 1985). The discordance between the effect induced by H,O, administration and the effect induced by arninotriazole administration could be tentatively explained by the different H,O, concentrations (Imre and Juhasz. 1987; Sohal. 1988). The markedly poor effects induced by ferrous chloride ingestion for 2 months may be related to the active control of in vivo iron uptake. According to current opinion, iron should play an important role in the aging process because 1) it plays in vitro a catalytic role in many free radical reactions, most significantly in the formation of the hydroxyl, alkoxy, and lipid-peroxy radicals (Halliwell and Guteridge, 1984); 2) it magnifies the power of the thiols to promote the formation of superoxide anions, hyroxyl radicals and epoxides (Misra, 1974; Rowley and

Halliwell, 1981); 3) it induces a stimulation of soluble fluorescent materials (Sohal et al., 198.5; Tappel, 1980) identical to lipofuscin, which is increased in vivo by iron administration in the housel‘ly (Sohal and Lamb, 1977); and 4) it accumulates with age in poikilotherms (Massie et al., 198.5). In the present research, chronic dietary iron intake failed to modify the reduced glutathione concentrations and the glutathione redox index in forebrains from the rats of different ages tested. However, I ) the iron uptake is regulated by well-established machanism; 2) the rcsults from in vitro studies must not be compared with the present situation in vivo; and 3 ) it cannot be ruled out that the “turnover” of glutathione is influenced by ferrous chloride, since reduced glutathione can be directly utilized to react with iron itself to produce thyol radicals, which can react with molecular oxygen to form superoxide anion (Misra, 1974). Many chemicals capable of oxidizing reduced glutathione are usually utilized in the experimental field. In this case, the chemicals react with any -SH group, so critical membrane -SH groups on the outside of cells may react even before reduced glutathione inside the cells does. However, diamide seems to be a stable and rather specific oxidant of glutathione (Kosower and Kosower, 1969; Kosower et al., 1972). As is indicated in Table I, chronic diamide intake increases the oxidized glutathione concentrations in forebrains from the rats at the different ages tested, whereas the reduced glutathione remains unchanged. The glutathione redox index decreases in the forebrains of diamide-administered older rats. Chronic oral treatment for 2 months with phosphatidylcholine modifies both the oxidized glutathione concentration and the glutathione redox index in rats, whereas the glutathione redox index is markedly altered by some chemicals (H,O, DDC, diamide). In contrast, chronic oral treatment with phosphatidylcholine is ineffective in rats, whereas the glutathione redox index is unmodified by other chemicals (paraquat, arninotriazolc, iron). These two biochemical trends taken together sug-

Brain Aging and Oxidative Stress

gest that the altered glutathione system can be modified by exogenous interventions. The interference of the chronic administration of phosphatidylcholine and/or its metabolite(s) with the cerebral glutathione system could be related to the influence on some cerebral enzyme activities, i.e., cytosolic superoxide dismutase and glutathione reductase (Benzi et al., 1989b). In conclusion, the results of the present study indicate that 1 ) aging magnifies the modifications induced by chemicals charged with producing oxidativc stress; i.e., the changes in the glutathione redox index are greater in the forebrains of paraquat-. DDC-. HZOZ-,and diarnide-administered older rats than in youngcr ones; 2) markedly different adaptative changes occur in mammalian brain in response to the various chemicals charged of producing oxidative stress; i .e., the reduced glutathione may be increased (by paraquat, DDC, and aminotriazole), decreased (by H,O,), or unchanged (by iron and diamide); and 3) the altered glutathione redox index could be modified by exogenous interventions (e.g., phosphatidylcholine administration). These observations taken together suggest that, although there is considerable support for the free radical theory of aging, our data are not completely in accordance with its prediction. Of these data, perhaps the most disappointing is that in older brains some cheniicals regarded as prooxidants (i, .e., paraquat, DDC) increase the glutathione redox index, whereas other chemicals (i.e., H,O,, diamide) decrease the glutathione redox index or are ineffective (i.e., aminotriazole, iron). The glutathione redox state is the result of the complex balance that exists between prooxidants and antioxidant within neurons. Thus the markedly different response of the glutathione redox index to the various chemical tested cannot be adequately explained by the hypothesis that augmentation or depression of one antioxidant defense causes a compensatory change in another related or overlapping antioxidant system. It is more likely that during aging the quality and the intensity of the single stress to which the brain is subjected decide both the type and the extent of the radical-mediated reactions. These considerations suggest that specific oxidative events influence the glutathione redox state of the neuron.

ACKNOWLEDGMENTS We thank Gemma Garlaschi, Gianfranca Corbellini, and Domenica Minisci for their secretarial work. The technical assistance of Luigi Maggi, Giovanni Arioli, and Giorgio Coscia is gratefully acknowledged.

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