- Email: [email protected]
The effect of age on the activity of enzymes of peroxide metabolism in rat brain
I:~perimental Gerontology. Vol. 28, pp. 77-85, 1993 Printed in the USA. All rights reserved.
0531-5565/93 $6.00 + .00 Copyright © 1993 Pergamon Press Ltd.
THE EFFECT OF AGE ON THE ACTIVITY OF ENZYMES OF PEROXIDE METABOLISM IN RAT BRAIN
M A R I O VERTECHY, M I C H A E L B. COOPER, ~ ORLANDO G H I R A R D I and M . TERESA RAMACCI Institute for Research on Senescence, Sigma-Tau S.p.A., Via Pontina Km 30,400, Pomezia 00040, Rome, Italy and ~Department of Medicine, University College London, The Rayne Institute, University Street, London WC1E 6JJ, U.K.
Abstract - - The activity of some enzymes associated with peroxide metabolism and
cytochrome oxidase activity was measured in cortex, striatum, hypothalamus, and hippocampus from brains of rats aged either 4, 15, or 27 months. Cytochrome oxidase activity was greatest in the cortex, but no significant age-related changes in the activity of cytochrome oxidase, superoxide dismutase, or glutathione peroxidase were found in any of the brain areas. In contrast, glutathione reductase activity increased as a function of age in all regions. In general, the activity of catalase fell on maturation of the animal to adulthood and then showed a trend to increase with age. Key Words: aging, superoxide dismutase, glutathione peroxidase, glutathione reductase, catalase, brain
INTRODUCTION DURING NORMALaerobic respiration, the free radical of oxygen superoxide (02") is generated as a consequence of electron transfer by flavoproteins and quinones of the mitochondrial electron-transport chain and other similar redox reactions. 02" is rapidly converted to H202 by superoxide dismutase (EC 1.15. I. 1; SOD); this process being a major source of cellular H202. Although 0 7 itself may not be directly cytotoxic, the damaging effects of its accumulation within the cell could reside in its ability to react with H202, formed as a product of its own detoxification or from other enzymic reactions, giving rise to the highly reactive hydroxy (OH-) and singlet oxygen (~O2) radicals (Koppenol and Butler, 1977). From thermodynamic considerations, the spontaneous reaction is not favoured in the cell, but it can be catalysed by metals such as Fe 2÷ and Cu 2÷, the plasma content of the latter being known to increase with age (Harman, 1965). Cytochrome oxidase (EC 1.9.3.1; COX), the terminal component of the electron transport chain, contains both of these metals, and its reaction proceeds via bound hydroperoxide intermediates although
Correspondence to: M. Vertechy. (Received 16 August 1991 ; Accepted 21 January 1992) 77
78
M. V E R T E C H Y et aL
some free radicals are released from the active site (Fridovich and Handler, 1961 ; Chance et al., 1975). The 02" and OH. radicals have been implicated in the age-dependent nonspecific glycation of proteins, peroxidation of membrane lipids, and the cross-linking of proteins causing loss of organelle function and cell death (Cooper et al., 1981; Harris et aL, 1982; Slater, 1984; Hunt et al., 1988). In the brain, such reactions are thought to be involved in the synthesis and age-dependent accumulation of lipofuscin, the so-called aging pigment, and have been proposed as a general mechanism for the process of biological aging (Harman, 1982; Sohal and Brunk, 1989). In order to be protected against free radical production, a very low steady-state concentration of O2" is preserved in the cell at circa 10 -~j M by SOD (Chance et al., 1979). In mammalian tissues this enzyme exists in two forms: one containing Mn 2+ in the mitochondrion (SOD-Mito) and a cytosolic form containing Cu 2+ and Zn 2+ (SOD-Cyt). The reactions of catalase (EC i. 11.1.6; CT) or glutathione peroxidase (EC 1.11.1.9; GPX) are responsible for removal of H202, either acting together or singly to maintain a steady-state concentration at 10-8 M (Chance et aL, 1979). CT is active only against H202 as substrate, whereas GPX can also use lipid hydroperoxides, so providing a mechanism for the repair of membranes subjected to peroxidation, and is also a component enzyme of at least one pathway of lipid biosynthesis (Bhattacherjee and Eakins, 1984). Which of these two enzymes is most important in the removal of H202 is uncertain and probably depends on subcellular compartmentalization (Sies and Summer, 1975; Oshino and Chance, 1977). Neither of the reactions of CT and SOD require additional cofactors, but a supply of reduced glutathione (GSH) is necessary for glutathione peroxidase activity. Oxidized glutathione (GSSG) is then reduced by glutathione reductase (EC 1.6.4.2; GRD) at the expense of NADPH. Previous studies on the effects of age on the antioxidant enzyme content of brain have yielded conflicting results, and none of these studies have attempted to correlate the potential for oxidative metabolism with the enzyme content (Hothersall et al., 1981 ; Cao Dahn et al., 1983; Mizuno and Ohta, 1986; Scarpa et al., 1987). The aim of this project was to establish if changes occur in the capacity of brain tissue to utilize O~ and produce 02" and H202 in an age-related manner and to deal with their production by enzymic mechanism. We have measured the activity of COX both for its potential as a generator of free radicals and as a marker enzyme for the electron-transport chain in brain areas from young, mature, and old rats together with the activity of the enzyme systems responsible for the removal of O2- and H202 (SOD-Mito, SOD-Cyt, GPX, CT, and GRD). The areas chosen for study were cortex, hypothalamus, striatum, and hippocampus, the latter three regions containing aminergic terminals that may be particularly susceptible to free radical attack because catecholamines are readily oxidized by 02" (Graham, 1978). MATERIALS AND METHODS Male Sprague-Dawley rats, aged either 5, 15, or 27 months, were used in the study. Six animals of each age were studied and were permitted food and water ad libitum. The group of old rats were healthy, and at this age the mortality of a similar group of animals maintained on the same regime was 65%. On the same day, one animal from each group was guillotined and the brain immediately removed and chilled in ice cold 0.9% NaC1 (wt/vol). Cortex, hypothalamus, hippocampus, and striatum were dissected from the surrounding tissue on a chilled dissection board (Glowinski and Iversen, 1966), and placed into a hypo-
79
PEROXIDE METABOLISM IN RAT BRAIN
tonic solution containing KCI (25 mM), MgC12 (5 mM), dithiothreitol (100 ~M), EDTA ( 1 mM), and Tris (50 mM) pH. 7.2 in the proportion of 100 mg tissue per ml solution. The samples were homogenized at 0°C in a Potter type glass/teflon homogenizer, and COX activity present was measured using ferrocytochrome c as substrate (Smith, 1955). Under these conditions, the major part of the mitochondria are enclosed within synaptosomal membranes, and hence COX is inaccessible to exogenous substrate. Treatment with digitonin makes the membranes permeable to macromolecules. The optimal ratio of digitonin to homogenate protein required to give free access of the substrate to the enzyme was determined as follows. Samples of homogenate containing 200 ug protein were incubated with varying amounts of digitonin for 15 min at 0°C. Aliquots of the incubations were assayed for cytochrome oxidase acitivity (Vertechy et al., 1989). The results are shown in Fig. 1. It can be seen that at a ratio of 12.5:1 (mg digitonin:mg homogenate protein) COX activity expressed was maximal; this ratio was then used in all subsequent assays. The remainder of the homogenate was frozen and thawed once and then centrifuged at 6500 × g at 4"C for 10 min. The supernatant was decanted and analysed for enzyme activities. Protein was determined by a variation on the method of Lowry et al. ( 1951), modified for use with lipid containing samples (Markwell et al., 1978). GRD activity was measured by the method of Racker (1955), and GPX activity by the method ofTappel (1978). CT activity was measured by following the consumption of H202 (Aebi, 1984). Total SOD (SOD-Cyt + SOD-Mito) activity was determined by measuring inhibition of 02" induced reduction of cytochrome c (Floh6 and Otting, 1984). Following the inactivation of SOD-
250
_= oJ
200 o. c~
E
150
100
50
!
I
!
I
5 10 15 20 RATIO OF DIGITONIN: HOMOGENATEPROTEIN (g/g)
I 25
FIG. 1. The effect ofdigitonin on the expression ofcytochrome o×idase activity. Brain homogenates and digitonin were incubated together as described in the Materials and Methods section. Each point represents the mean values of triplicate determinations,
80
M. VERTECHYetal.
Mito (Geller and Winge, 1984), SOD-Cyt activity was determined as previously and SODMito activity calculated as the difference between total and SOD-Cyt activities. Assays were performed in triplicate with a Beckmann DU40 spectrophotometer.
Statistical analysis of data Data from each group were tested for the normal distribution by the Wilk-Shapiro test, and in no cases were the data skewed. The effect of the age of the animals on the enzymic composition of the tissues was assessed by analysis of variance (ANOVA). The level of significance to reject the hypothesis of equal variance among the populations of rats of different ages was set at 0.05. Regional differences in enzyme activity were compared by t test and where appropriate, correlation (r) was obtained by regression analysis. RESULTS The distribution of COX between the brain regions from young, adult, and old rats are shown in Fig. 2. At each age studied, hippocampus, hypothalamus, and striatum had comparable levels of COX activity. However, the activity present in cortex at all ages was greater than in the other regions (p < 0.05, paired t-test; cortex vs. striatum, hippocampus, or hypothalamus at either 5, 15, or 27 months old), but in common with hippocampus, hypothalamus, and striatum, no significant age-dependent changes in activity were 400 -A c
.[ 300
iI
t
--
1
I
J 200 --
100 -x 0
I
I 0n -
t~
I
coJ Hi .st !'Hy 5 MONTHS
C0 Hi St H~
C01 Hi St Hy
15 MONTHS
27 MONTHS
FIG. 2. Cytochrome oxidase activity in brain regions. COX was assayed as described in Materials and Methods. The bars show the mean _+ SD. Co: cortex, Hi: hippocampus, St: striatum, Hy: hypothalamus.* p < 0.05 when compared to any other region (t test).
81
PEROXIDEMETABOLISMIN RAT BRAIN
observed. Table 1 shows the activity of SOD-Mito, SOD-Cyt, GPX, GRD, and CT in the brain regions. The cortex does not contain increased SOD-Mito activity to compensate for the greater COX activity. In consequence, the ratio of SOD-Mito to COX activity is lower in this region than the others, so providing some support for the hypothesis that cortex is more predisposed to oxidative damage than other brain regions. On the basis of protein content, GPX and both forms of SOD were found to be evenly distributed throughout the brain areas and showed no statistically significant age-related changes in activity. In contrast, whilst not differing significantly in distribution between the brain areas, both CT and GRD did show age-related changes in activity (p < 0.05, ANOVA). In the case of GRD, there was a progressive and statistically significant increase in activity with age in all of the brain areas studied. The rise in activity correlated linearly as a function of age (hippocampus: r = 0.96; striatum: r = 0.99; hypothalamus: r = 0.99; cortex: r = 0.97). The activity of CT showed a different response to age. A trend for the activity to fall was observed between the ages of 5 and 15 months, whilst aging from 15 to 27 months reversed this to some extent, although these effects were only statistically significant in the striatum (p < 0.05, ANOVA). DISCUSSION Of the brain areas studied, cortex was found to contain the greatest COX activity, demonstrating a greater requirement for mitochondrial oxidation in this tissue. No significant changes in activity were found to occur with aging, indicating that a decrease in mitochondrial content of the tissues is not primarily responsible for age-related degeneration of the brain areas. Despite having a higher COX content, there was no compensatory TABLE 1. PEROXIDIC ENZYME ACTIVITIESIN BRAIN REGIONS OF RATS OF DIFFERENT AGES
Brain region
Age Glutathione (months) peroxidase
Glutathione reductase Catalase
SOD-Mito
SOD-Mito/('OX SOD-Cvt
Hippocampus 5 15 27
111 _+ 8 96 ± 8 111 _+ 12
20.0_+ 1.0" 21.0±0.9" 24.5 ± 1.1"
58_+ 6 41 _+ 2 49 ± 5
12_+ 3 10_+ 3 10 ± 1
0.07_+ 0.02 0.06_+0.02 0.06 ± 0 . 0 2
18_+ 3 17_+ 3 17 + 3
5 15 27
120 + 7 123_+ 10 129 + 5
22.9 +_ 1.5" 24.9 + 1.6" 28.7 + 0.8*
49 + 2*t 35 +_ 2* 43 +_ 2 " I
14 + 4 18 _+ 2 15 + 2
0.07 + 0.02 0.10_+ 0.02 0.09 + 0.02
14_+ 1 13 + 3 15 _+ 1
5 15 27
128_+ 10 108 + 10 104 ± 7
18.7 + 1.5" 20.6_+ 1.4" 23.2 + 1.0"
68 + 8 56 + 5 50 + 4
13 + 2 10 + 2 10 ± 2
0.08 + 0.02 0.05 + 0 . 0 1 0.06 ± 0.02
18_+ 2 20 + 3 20_+ 3
5 15 27
107 ± 6 111 + 8 116 +_ 5
2 0 . 0 + 0.8* 23.5 + 1.0" 25.1 + 1.3"
47_+ 4 39 ± 4 42 + 3
10 + 3 9_+ 2 11 + 2
0.04_+ 0.01 0.04_+ 0.01 0.04_+0.01
13 _+ 1 11 _+ 2 14 + 1
Striatum
Hypothalamus
Cortex
The data show the mean _+ SE of the values obtained from each group of rats. Enzyme activities are expressed as follows--glutathione peroxidase and glutathione reductase: ~mol N A D P H oxidized/min/g protein; catalase: umol H202 consumed/min/g protein; superoxide dismustase: units/mg protein. SOD-Mito: superoxide dismutase mitochondrion, COX = cytochrome oxidase, SOD-Cyt = cytosolic form ofsuperoxide dismutase. *p < 0.05, A N O V A of activity in each region with respect to age. tP < 0.05, unpaired t-test, animals aged 5 or 27 months vs. 15 months.
82
M. VERTECHY
et a l
increase in SOD-Mito activity in cortex, so giving support to the hypothesis that cortex is more susceptible to damage caused by oxidative stress than the other regions (Loomis et al., 1976; Mizuno and Ohta, 1986). The effect of aging on COX and other mitochondrial activities has been studied in a variety of animal and human tissues (Miquel et al., 1980). Apart from a recent study performed on human liver samples that demonstrated fall in mitochondrial function with age (Yen et al., 1990), other studies show little change in muscle mitochondrial activities with age, although mitochondria from the myocardium from old rats are reported to produce more OZ than young animals (Nohl and Hegner, 1978; Vertechy et al., 1989; Muscari et al., 1990). We found no significant age-related changes in activity of either SOD-Mito or SOD-Cyt in any of the brain regions studied. Small changes in the total SOD activity in brain and other tissues have been reported (Reiss and Gershon, 1976; Mavelli et al., 1978; Mizuno and Ohta, 1986), but since the turnover of substrate by SOD is very rapid (Chance et al., 1979), it seems unlikely that the enzyme is ever saturated with 02 ~, and that under normal conditions the capacity of the brain areas to dismutate 02 ~ to H202 is essentially unimpaired by aging. Instead of conferring additional protection to the organism, overproduction of SOD is reported to lead to brain damage and does not extend life span (Kellog and Fridovich, 1976; Ono and Okada, 1984; Elroy-Stein et al., 1986; Epstein et al., 1987; Seto et al., 1990). This suggests that it is the enzymes of H202 metabolism that are unable to respond to increased production of their substrate, with the result that direct or indirect reactions of H202 are allowed to initiate cellular damage. One explanation for the fall in CT activity observed between the ages of 5 and 15 months could be that, in growing animals, enhanced H202 generation is due to the growth of brain tissue and necessitates greater CT activity than in adults. Although statistically significant only in the striatum, there was a trend for CT activity to increase between adult and aged animals, suggesting that some kind of age-related stress is put on H202 metabolism. Increased mitochondrial H202 formation as a function of age has been demonstrated in insects (Sohal, 1991). This could be caused by an age-related increase in activity of other H202 producing enzymes such as monamine oxidase as well as a greater extent of dismutation ofO2 ~formed by the respiratory electron-transport chain (Robinson, 1975; StrollinBenedetti and Keane, 1980). In contrast to our results and those of other reports (Reiss and Gershon, 1976; Mavelli et al., 1978; Mizuno and Ohta, 1986; Scarpa et al., 1987), a recent study performed on homogenates of whole brain have reported lowered activity of both total SOD and catalase during aging, coupled with a decreased content of the respective mRNA species (Semesei et al., 1991). The reason for this disparity between this finding and the results presented here and in other studies is not clear, but may depend on assay conditions employed and environmental conditions in which the animals were maintained. In line with other findings obtained using homogenates of whole brain (Hothersall et al., 1981 ), we found no significant changes in GPX activity in the specific brain regions during aging, and in consequence, a failure in this pathway alone is an unlikely mechanism for age-induced damage. Small increases in activity during development of young animals to 18 months of age have been reported (Mizuno and Ohta, 1986; Scarpa et al., 1987), but at variance with these reports, the effect of age on Se2+-dependent and non-Se 2+-dependent GPX in whole brain has been reported to cause a decrease in activity; the reason for the differing results is uncertain (Zhang et al., 1989). Perhaps the most significant finding of this project is the progressive increase in G R D
PEROXIDE METABOLISM IN RAT BRAIN
83
activity throughout life in all of the brain areas studied. Glutathione participates in a variety of important biochemical processes apart from peroxide metabolism, and the maintenance of an adequate supply of GSH is essential for their normal function. A glutathione-CoA mixed disulphide is formed in vivo during hydroperoxide metabolism and is also a substrate for G R D (Carlsberg and Mannervick, 1977; Crane et al., 1982; Dyer and Wilken, 1972). The total glutathione content of the brain decreases with age (Hazelton and Lang, 1980), and given that oxidative stress and aging cause an increase in the level of GSSG in the brain (Zhang et al., 1990), increased G R D activity could be induced in an attempt by the cell to maintain the availability of both CoA and GSH. It has been proposed that, instead of being a continuous process, the accumulation of free radical damage is dependent on episodes of disease (Pryor, 1988; Benzi et al., 1990). Superimposed on an underlying and progressively increasing stress placed on the maintenance of a supply of GSH, periods of oxidative stress in old age could cause more free radical-induced damage than a similar stress in young animals. The rate of accumulation of free radical damage is dependent on the frequency and severity of episodes of disease or anorexia as well as the age of the animal. Acknowledgments - - We would like to thank N. Finocchio and A. Cernilli for expert technical assistance.
REFERENCES AEBI, H. Catalase in vitro. Methods Enzymol. 105, 121-126, 1984. BHATTACHERJEE, P. and EAKINS, K.E. Lipoxygenase products: Mediation of inflammatory responses and inhibition of their formation. In: Leucotrienes--Chemistry and Biology. Chakrin, W.C. and Bailey, D.M. (Editors), pp. 195-214, Academic Press, London, 1984. BENZI, G., MARZATICO, F., PASTORIS, O., and VILLA, R.F. Influence of oxidative stress on the age-linked alterations of the cerebral glutathione system. J. Neurosci. Res. 26, 120-128, 1990. CARLBERG, I. and MANNERVICK, B. Purification by affinity chromatography of yeast glutathione reductase, the enzyme responsible for NADPH-dependant reduction of the mixed disulfide of co-enzyme A and glutathione. Biochim. Biophys. Acta 484, 268-274, 1977. CHANCE, B., SARONIO, C., and LEIGH, J.S. Functional intermediates in reaction ofcytochrome oxidase with oxygen. Proc. Natl. Acad. Sci. U. S. A. 72, 1635-1640, 1975. CHANCE, B., SIES, H., and BOVERIS, A. Hydroperoxide metabolism in mammalian organs. Physiol. Rev. 59, 527-605, 1979. COOPER, M.B., ESTALL, M.R., and RABIN, B.R. The use ofproteinases to determine the topological location ofcytochrome P450 in vesicles derived from smooth endoplasmic reticulum of rat liver. Biochem J. 196, 585589, 1981. CRANE, D,, HAUSSINGER, D., and SIES, H. Rise of co-enzyme A-glutathione mixed disulphide during hydroperoxide metabolism in perfused rat liver. Eur. J. Biochem. 127, 575-578, 1982. DYAR, R.E. and WILKEN, D.R. Rat liver levels of co-enzyme A-glutathione disulphide and of enzymes of its metabolism. Arch. Biochem. Biophys. 153, 619-626, 1972. ELROY-STEIN, O., BERNSTEIN, Y., and GRONER, Y. Overproduction of human Cu/Zn-superoxide dismutase in transfected cells: Extenuation of paraquat-mediated cytotoxicity and enhancement of lipid peroxidation. EMBO ,L 5, 615-622, 1986. EPSTEIN, C.J., AVRAHAM, K.B., LOVETT, M., SMITH, S., ELROY-STEIN, O., ROTMAN, G., BRY, C., and GRONER, Y. Transgenic mice with increased Cu/Zn-superoxide dismutase activity: Animal model of dosage effects in Down Syndrome. Proc. Natl. Acad. Sci. U. S. A. 84, 8044-8048, 1987. FLOHI~, L. and (~TTING, F. Superoxide dismutase assays. Methods Enzymol. 105, 101-104, 1984. FRIDOVICH, I. and HANDLER, P. Detection of free radicals generated during enzymic oxidation by the initiation of sulphite oxidation. J. Biol. Chem. 236, 1836-1840, 1961. GELLER, B.L. and WlNGE, D.R. Subcellular distribution of superoxide dismutases. Methods Enzymol. 105, 113-114, 1984.
84
M. V E R T E C H Y et al.
GLOWINSKI, J. and IVERSEN, L.L. Regional studies on catecholamines in the rat brain. J. Neurochem. 13, 655-669, 1966. GRAHAM, D.G. Oxidation pathways for catechol amines in the genesis of neuromelanin and cytotoxic quinones. Mol. Pharamcol. 14, 633-643, 1978. HARMAN, D. The free radical theory of aging. In: Free Radicals in Bioh~g),. Vol. 5, Pryor, W.A. (Editor), pp. 255-271, Academic Press, New York, NY, 1982. HARMAN, D.J. The free radical theory of aging: Effect of age on serum copper levels. J. Geronto/. 20, 151-153, 1965. HARRIS, E.J., BOOTH, R., and COOPER, M.B. The effect of superoxide generation on the ability of mitochondria to take up and retain Ca 2~ . FEBS Lett. 146, 267-272, 1982. HAZELTON, G.A. and LANG, C.A. Glutathone content of tissues in the aging mouse. Biochem. J 188, 25-30, 1980. HOTHERSALL, J.S., EL-HASSAN, A., McCLEAN, P., and GREENBAUM, A.L. Age related changes in enzymes of rat brain 2,- redox systems linked to NADPH and glutathione. Enzyme 26, 271-276, 1981. HUNT, J.V., DEAN, R.T., and WOLFF, S.P. Hydroxyl radical production and autoxidative glycosylation. Biochem. J. 256, 205-212, 1988. KELLOG, E.W., Ill and FRIDOVICH, I. Superoxide dismutase in the rat and mouse as a function of age and longevity. J. Gerontol. 31,405-408, 1976. KOPPENOL, W.H. and BUTLER, J. Mechanism of reactions involving singlet oxygen and the superoxide anion. FEBS Left, 83, 1-6, 1977. LOOMIS, T.C., YEE, G., and STAHL, W.L. Regional and subcellular distribution of superoxide dismutase in brain. ExT~erientia 32, 1374-1376, 1976. LOWRY, O.H., ROSEBROUGH, N.J., FARR, A.L., and RANDALL, R.J. Protein measurement with the Folin-henol reagent. J. Biol. (Ttem. 193, 265-275, 1951. MARKWELL, M.A.K., HAAS, S.M., BIEBER, L.L., and TOLBERT, N.E. A modification of the Lowry procedure to simplify protein determinations in membrane and lipoprotein samples. Anal. Biochem. 87, 206210, 1978. MAVELLI, l., MONDOVI, B., FEDERICO, R., and ROTILIO, G. Superoxide dismutase activity in developing rat brain. J. Neurochem. 31,363-364, 1978. MIQUEL, J., ECONOMOS, A.C., FLEMING, J., and JOHNSON, J.E., JR. Mitochondrial role in cell aging. L~¥p. Geronto/. 15, 575-591, 1980. MIZUNO. Y. and OHTA, K. Regional distributions of thiobarbituric acid-reactive products, activities of enzymes regulating the metabolism of oxygen free radicals and some related enzymes in adult and aged rat brain. J Neurochem. 46, 1344- 1352, 1986. MUSCARI, C.. FRASCARO, M., GUARNIERI, C., and CALDARERA, C.M. Mitochondrial function and superoxide generation from submitochondrial particles of aged rat hearts. Biochim. Biophys. Acta 1015, 200204, 1990. NOHL, tt. and HEGNER, D. Do mitochondria produce oxygen radicals in vivo? Eur. J. Biochem. 82, 563-567, 1978. ONO, T. and OKADA, S. Unique increase in superoxide dismutase level in brains of long-living animals. Exp. Geronto/. 19, 349-354, 1984. OSHINO, N. and CHANCE, B. Properties of glutathione release observed during reduction of organic hydroperoxide, demethylation of amino pyrine and oxidation of some substances in perfused rat liver, and their implications for the physiological function ofcatalase. Biochemisto, 162, 509-525, 1977. PRYOR, W.A. The free-radical theory of aging revisited: A critique and a suggested disease specific theory. In: Modern Biological Theories ~[Aging. Vol. 31 (Aging), Warner, H.R., Butler, R.N., Sprott, R.L., and Schneider, M.B. (Editors), pp. 89-105, Raven Press, New York, NY, 1988. RACKER, E. Glutathione reductase (liver and yeast). Methods Enzymol. 2, 722-729, 1955. REISS, U. and GERSHON, D. Comparison of cytoplasmic superoxide dismutase in liver heart and brain of aging rats and mice. Biochem. Biophys. Res. Commun. 73, 255-262, 1976. ROBINSON, D.S. Changes in monoamine oxidase and monoamines in human development and aging. Fed Proc. 34, 103-107, 1975. SCARPA, M., RIGO, A., VIGLINO, P., STEVANTO, R., BRACCO, F., and BATTESTIN, L. Age dependence on the level of the enzymes involved in the protection against active oxygen species in the rat brain. Proc. Soc. E~p. Biol. MecL 185, 129-133, 1987.
PEROXIDE METABOLISM IN RAT BRAIN
85
SEMESEI, 1., RAO, G., and RICHARDSON, A. Expression ofsuperoxide dismutase and catalase in rat brain as a function of age. Mech. AgeingDev. 58, 13-19, 1991. SETO, N.O.L., HAYASHI, S., and TENER, G. Overexpression of Cu-Zn superoxide dismutase in drosophila does not affect life span. Proc. Nat. Acad. Sci. U. S. A. 87, 4270-4274, 1990. SIES, H. and SUMMER, K.-H. Hydroperoxide-metabolizing systems in rat liver. Eur. J. Biochern. 57, 503-512, 1975. SLATER, T.F. Free radical mechanisms in tissue injury. Biochem. J. 222, 1-15, 1984. SMITH, L. Spectrophotometric assay ofcytochrome c oxidase. Methods Biochern. Anal, 2, 427-434, 1955. SOHAL, R.S. Hydrogen peroxide production by mitochondria may be a biomarker of aging. Mech. Ageing Dev. 60, 189-198, 1991. SOHAL, R. S. and BRUNK, U.T. Lipofuscin as an indicator of oxidative stress and aging. Adv. Exp. Med. Biol. 266, 17-30, 1989. STROLLIN-BENEDETTI, M. and KEANE, P.E. Differential changes in monoamine oxidase A and B activities in the aging rat brain. Z Neurochem. 35, 1026-1032, 1980. TAPPEL, A.L. Glutathione peroxidase and hydroperoxides. In: Methods Enzymol. 52, 506-513, 1978. VERTECHY, M., COOPER, M.B., GHIRARD1, O., and RAMACC1, M.T. Antioxidant enzym~ activities in heart and skeletal muscle of rats of different ages. Exp. Gerontol. 24, 211-218, 1989. YEN, T.-C., CHEN, Y.-S., KING, K.-L., YEH, S.-H., and WEI, Y.-H. Liver mitochondrial respiratory functions decline with age. Biochem. Biophys. Res. Commun. 165, 944-1003, 1990. ZHANG, L., MAIORINO, M., ROVERI, A., and URSINI, F. Phospholipid hydroperoxide glutathione peroxidase specific activity in tissues of rats of different ages and comparison with other glutathione peroxidases. Biochirn. Biophys. Acta 1006, 140-143, 1989.
0531-5565/93 $6.00 + .00 Copyright © 1993 Pergamon Press Ltd.
THE EFFECT OF AGE ON THE ACTIVITY OF ENZYMES OF PEROXIDE METABOLISM IN RAT BRAIN
M A R I O VERTECHY, M I C H A E L B. COOPER, ~ ORLANDO G H I R A R D I and M . TERESA RAMACCI Institute for Research on Senescence, Sigma-Tau S.p.A., Via Pontina Km 30,400, Pomezia 00040, Rome, Italy and ~Department of Medicine, University College London, The Rayne Institute, University Street, London WC1E 6JJ, U.K.
Abstract - - The activity of some enzymes associated with peroxide metabolism and
cytochrome oxidase activity was measured in cortex, striatum, hypothalamus, and hippocampus from brains of rats aged either 4, 15, or 27 months. Cytochrome oxidase activity was greatest in the cortex, but no significant age-related changes in the activity of cytochrome oxidase, superoxide dismutase, or glutathione peroxidase were found in any of the brain areas. In contrast, glutathione reductase activity increased as a function of age in all regions. In general, the activity of catalase fell on maturation of the animal to adulthood and then showed a trend to increase with age. Key Words: aging, superoxide dismutase, glutathione peroxidase, glutathione reductase, catalase, brain
INTRODUCTION DURING NORMALaerobic respiration, the free radical of oxygen superoxide (02") is generated as a consequence of electron transfer by flavoproteins and quinones of the mitochondrial electron-transport chain and other similar redox reactions. 02" is rapidly converted to H202 by superoxide dismutase (EC 1.15. I. 1; SOD); this process being a major source of cellular H202. Although 0 7 itself may not be directly cytotoxic, the damaging effects of its accumulation within the cell could reside in its ability to react with H202, formed as a product of its own detoxification or from other enzymic reactions, giving rise to the highly reactive hydroxy (OH-) and singlet oxygen (~O2) radicals (Koppenol and Butler, 1977). From thermodynamic considerations, the spontaneous reaction is not favoured in the cell, but it can be catalysed by metals such as Fe 2÷ and Cu 2÷, the plasma content of the latter being known to increase with age (Harman, 1965). Cytochrome oxidase (EC 1.9.3.1; COX), the terminal component of the electron transport chain, contains both of these metals, and its reaction proceeds via bound hydroperoxide intermediates although
Correspondence to: M. Vertechy. (Received 16 August 1991 ; Accepted 21 January 1992) 77
78
M. V E R T E C H Y et aL
some free radicals are released from the active site (Fridovich and Handler, 1961 ; Chance et al., 1975). The 02" and OH. radicals have been implicated in the age-dependent nonspecific glycation of proteins, peroxidation of membrane lipids, and the cross-linking of proteins causing loss of organelle function and cell death (Cooper et al., 1981; Harris et aL, 1982; Slater, 1984; Hunt et al., 1988). In the brain, such reactions are thought to be involved in the synthesis and age-dependent accumulation of lipofuscin, the so-called aging pigment, and have been proposed as a general mechanism for the process of biological aging (Harman, 1982; Sohal and Brunk, 1989). In order to be protected against free radical production, a very low steady-state concentration of O2" is preserved in the cell at circa 10 -~j M by SOD (Chance et al., 1979). In mammalian tissues this enzyme exists in two forms: one containing Mn 2+ in the mitochondrion (SOD-Mito) and a cytosolic form containing Cu 2+ and Zn 2+ (SOD-Cyt). The reactions of catalase (EC i. 11.1.6; CT) or glutathione peroxidase (EC 1.11.1.9; GPX) are responsible for removal of H202, either acting together or singly to maintain a steady-state concentration at 10-8 M (Chance et aL, 1979). CT is active only against H202 as substrate, whereas GPX can also use lipid hydroperoxides, so providing a mechanism for the repair of membranes subjected to peroxidation, and is also a component enzyme of at least one pathway of lipid biosynthesis (Bhattacherjee and Eakins, 1984). Which of these two enzymes is most important in the removal of H202 is uncertain and probably depends on subcellular compartmentalization (Sies and Summer, 1975; Oshino and Chance, 1977). Neither of the reactions of CT and SOD require additional cofactors, but a supply of reduced glutathione (GSH) is necessary for glutathione peroxidase activity. Oxidized glutathione (GSSG) is then reduced by glutathione reductase (EC 1.6.4.2; GRD) at the expense of NADPH. Previous studies on the effects of age on the antioxidant enzyme content of brain have yielded conflicting results, and none of these studies have attempted to correlate the potential for oxidative metabolism with the enzyme content (Hothersall et al., 1981 ; Cao Dahn et al., 1983; Mizuno and Ohta, 1986; Scarpa et al., 1987). The aim of this project was to establish if changes occur in the capacity of brain tissue to utilize O~ and produce 02" and H202 in an age-related manner and to deal with their production by enzymic mechanism. We have measured the activity of COX both for its potential as a generator of free radicals and as a marker enzyme for the electron-transport chain in brain areas from young, mature, and old rats together with the activity of the enzyme systems responsible for the removal of O2- and H202 (SOD-Mito, SOD-Cyt, GPX, CT, and GRD). The areas chosen for study were cortex, hypothalamus, striatum, and hippocampus, the latter three regions containing aminergic terminals that may be particularly susceptible to free radical attack because catecholamines are readily oxidized by 02" (Graham, 1978). MATERIALS AND METHODS Male Sprague-Dawley rats, aged either 5, 15, or 27 months, were used in the study. Six animals of each age were studied and were permitted food and water ad libitum. The group of old rats were healthy, and at this age the mortality of a similar group of animals maintained on the same regime was 65%. On the same day, one animal from each group was guillotined and the brain immediately removed and chilled in ice cold 0.9% NaC1 (wt/vol). Cortex, hypothalamus, hippocampus, and striatum were dissected from the surrounding tissue on a chilled dissection board (Glowinski and Iversen, 1966), and placed into a hypo-
79
PEROXIDE METABOLISM IN RAT BRAIN
tonic solution containing KCI (25 mM), MgC12 (5 mM), dithiothreitol (100 ~M), EDTA ( 1 mM), and Tris (50 mM) pH. 7.2 in the proportion of 100 mg tissue per ml solution. The samples were homogenized at 0°C in a Potter type glass/teflon homogenizer, and COX activity present was measured using ferrocytochrome c as substrate (Smith, 1955). Under these conditions, the major part of the mitochondria are enclosed within synaptosomal membranes, and hence COX is inaccessible to exogenous substrate. Treatment with digitonin makes the membranes permeable to macromolecules. The optimal ratio of digitonin to homogenate protein required to give free access of the substrate to the enzyme was determined as follows. Samples of homogenate containing 200 ug protein were incubated with varying amounts of digitonin for 15 min at 0°C. Aliquots of the incubations were assayed for cytochrome oxidase acitivity (Vertechy et al., 1989). The results are shown in Fig. 1. It can be seen that at a ratio of 12.5:1 (mg digitonin:mg homogenate protein) COX activity expressed was maximal; this ratio was then used in all subsequent assays. The remainder of the homogenate was frozen and thawed once and then centrifuged at 6500 × g at 4"C for 10 min. The supernatant was decanted and analysed for enzyme activities. Protein was determined by a variation on the method of Lowry et al. ( 1951), modified for use with lipid containing samples (Markwell et al., 1978). GRD activity was measured by the method of Racker (1955), and GPX activity by the method ofTappel (1978). CT activity was measured by following the consumption of H202 (Aebi, 1984). Total SOD (SOD-Cyt + SOD-Mito) activity was determined by measuring inhibition of 02" induced reduction of cytochrome c (Floh6 and Otting, 1984). Following the inactivation of SOD-
250
_= oJ
200 o. c~
E
150
100
50
!
I
!
I
5 10 15 20 RATIO OF DIGITONIN: HOMOGENATEPROTEIN (g/g)
I 25
FIG. 1. The effect ofdigitonin on the expression ofcytochrome o×idase activity. Brain homogenates and digitonin were incubated together as described in the Materials and Methods section. Each point represents the mean values of triplicate determinations,
80
M. VERTECHYetal.
Mito (Geller and Winge, 1984), SOD-Cyt activity was determined as previously and SODMito activity calculated as the difference between total and SOD-Cyt activities. Assays were performed in triplicate with a Beckmann DU40 spectrophotometer.
Statistical analysis of data Data from each group were tested for the normal distribution by the Wilk-Shapiro test, and in no cases were the data skewed. The effect of the age of the animals on the enzymic composition of the tissues was assessed by analysis of variance (ANOVA). The level of significance to reject the hypothesis of equal variance among the populations of rats of different ages was set at 0.05. Regional differences in enzyme activity were compared by t test and where appropriate, correlation (r) was obtained by regression analysis. RESULTS The distribution of COX between the brain regions from young, adult, and old rats are shown in Fig. 2. At each age studied, hippocampus, hypothalamus, and striatum had comparable levels of COX activity. However, the activity present in cortex at all ages was greater than in the other regions (p < 0.05, paired t-test; cortex vs. striatum, hippocampus, or hypothalamus at either 5, 15, or 27 months old), but in common with hippocampus, hypothalamus, and striatum, no significant age-dependent changes in activity were 400 -A c
.[ 300
iI
t
--
1
I
J 200 --
100 -x 0
I
I 0n -
t~
I
coJ Hi .st !'Hy 5 MONTHS
C0 Hi St H~
C01 Hi St Hy
15 MONTHS
27 MONTHS
FIG. 2. Cytochrome oxidase activity in brain regions. COX was assayed as described in Materials and Methods. The bars show the mean _+ SD. Co: cortex, Hi: hippocampus, St: striatum, Hy: hypothalamus.* p < 0.05 when compared to any other region (t test).
81
PEROXIDEMETABOLISMIN RAT BRAIN
observed. Table 1 shows the activity of SOD-Mito, SOD-Cyt, GPX, GRD, and CT in the brain regions. The cortex does not contain increased SOD-Mito activity to compensate for the greater COX activity. In consequence, the ratio of SOD-Mito to COX activity is lower in this region than the others, so providing some support for the hypothesis that cortex is more predisposed to oxidative damage than other brain regions. On the basis of protein content, GPX and both forms of SOD were found to be evenly distributed throughout the brain areas and showed no statistically significant age-related changes in activity. In contrast, whilst not differing significantly in distribution between the brain areas, both CT and GRD did show age-related changes in activity (p < 0.05, ANOVA). In the case of GRD, there was a progressive and statistically significant increase in activity with age in all of the brain areas studied. The rise in activity correlated linearly as a function of age (hippocampus: r = 0.96; striatum: r = 0.99; hypothalamus: r = 0.99; cortex: r = 0.97). The activity of CT showed a different response to age. A trend for the activity to fall was observed between the ages of 5 and 15 months, whilst aging from 15 to 27 months reversed this to some extent, although these effects were only statistically significant in the striatum (p < 0.05, ANOVA). DISCUSSION Of the brain areas studied, cortex was found to contain the greatest COX activity, demonstrating a greater requirement for mitochondrial oxidation in this tissue. No significant changes in activity were found to occur with aging, indicating that a decrease in mitochondrial content of the tissues is not primarily responsible for age-related degeneration of the brain areas. Despite having a higher COX content, there was no compensatory TABLE 1. PEROXIDIC ENZYME ACTIVITIESIN BRAIN REGIONS OF RATS OF DIFFERENT AGES
Brain region
Age Glutathione (months) peroxidase
Glutathione reductase Catalase
SOD-Mito
SOD-Mito/('OX SOD-Cvt
Hippocampus 5 15 27
111 _+ 8 96 ± 8 111 _+ 12
20.0_+ 1.0" 21.0±0.9" 24.5 ± 1.1"
58_+ 6 41 _+ 2 49 ± 5
12_+ 3 10_+ 3 10 ± 1
0.07_+ 0.02 0.06_+0.02 0.06 ± 0 . 0 2
18_+ 3 17_+ 3 17 + 3
5 15 27
120 + 7 123_+ 10 129 + 5
22.9 +_ 1.5" 24.9 + 1.6" 28.7 + 0.8*
49 + 2*t 35 +_ 2* 43 +_ 2 " I
14 + 4 18 _+ 2 15 + 2
0.07 + 0.02 0.10_+ 0.02 0.09 + 0.02
14_+ 1 13 + 3 15 _+ 1
5 15 27
128_+ 10 108 + 10 104 ± 7
18.7 + 1.5" 20.6_+ 1.4" 23.2 + 1.0"
68 + 8 56 + 5 50 + 4
13 + 2 10 + 2 10 ± 2
0.08 + 0.02 0.05 + 0 . 0 1 0.06 ± 0.02
18_+ 2 20 + 3 20_+ 3
5 15 27
107 ± 6 111 + 8 116 +_ 5
2 0 . 0 + 0.8* 23.5 + 1.0" 25.1 + 1.3"
47_+ 4 39 ± 4 42 + 3
10 + 3 9_+ 2 11 + 2
0.04_+ 0.01 0.04_+ 0.01 0.04_+0.01
13 _+ 1 11 _+ 2 14 + 1
Striatum
Hypothalamus
Cortex
The data show the mean _+ SE of the values obtained from each group of rats. Enzyme activities are expressed as follows--glutathione peroxidase and glutathione reductase: ~mol N A D P H oxidized/min/g protein; catalase: umol H202 consumed/min/g protein; superoxide dismustase: units/mg protein. SOD-Mito: superoxide dismutase mitochondrion, COX = cytochrome oxidase, SOD-Cyt = cytosolic form ofsuperoxide dismutase. *p < 0.05, A N O V A of activity in each region with respect to age. tP < 0.05, unpaired t-test, animals aged 5 or 27 months vs. 15 months.
82
M. VERTECHY
et a l
increase in SOD-Mito activity in cortex, so giving support to the hypothesis that cortex is more susceptible to damage caused by oxidative stress than the other regions (Loomis et al., 1976; Mizuno and Ohta, 1986). The effect of aging on COX and other mitochondrial activities has been studied in a variety of animal and human tissues (Miquel et al., 1980). Apart from a recent study performed on human liver samples that demonstrated fall in mitochondrial function with age (Yen et al., 1990), other studies show little change in muscle mitochondrial activities with age, although mitochondria from the myocardium from old rats are reported to produce more OZ than young animals (Nohl and Hegner, 1978; Vertechy et al., 1989; Muscari et al., 1990). We found no significant age-related changes in activity of either SOD-Mito or SOD-Cyt in any of the brain regions studied. Small changes in the total SOD activity in brain and other tissues have been reported (Reiss and Gershon, 1976; Mavelli et al., 1978; Mizuno and Ohta, 1986), but since the turnover of substrate by SOD is very rapid (Chance et al., 1979), it seems unlikely that the enzyme is ever saturated with 02 ~, and that under normal conditions the capacity of the brain areas to dismutate 02 ~ to H202 is essentially unimpaired by aging. Instead of conferring additional protection to the organism, overproduction of SOD is reported to lead to brain damage and does not extend life span (Kellog and Fridovich, 1976; Ono and Okada, 1984; Elroy-Stein et al., 1986; Epstein et al., 1987; Seto et al., 1990). This suggests that it is the enzymes of H202 metabolism that are unable to respond to increased production of their substrate, with the result that direct or indirect reactions of H202 are allowed to initiate cellular damage. One explanation for the fall in CT activity observed between the ages of 5 and 15 months could be that, in growing animals, enhanced H202 generation is due to the growth of brain tissue and necessitates greater CT activity than in adults. Although statistically significant only in the striatum, there was a trend for CT activity to increase between adult and aged animals, suggesting that some kind of age-related stress is put on H202 metabolism. Increased mitochondrial H202 formation as a function of age has been demonstrated in insects (Sohal, 1991). This could be caused by an age-related increase in activity of other H202 producing enzymes such as monamine oxidase as well as a greater extent of dismutation ofO2 ~formed by the respiratory electron-transport chain (Robinson, 1975; StrollinBenedetti and Keane, 1980). In contrast to our results and those of other reports (Reiss and Gershon, 1976; Mavelli et al., 1978; Mizuno and Ohta, 1986; Scarpa et al., 1987), a recent study performed on homogenates of whole brain have reported lowered activity of both total SOD and catalase during aging, coupled with a decreased content of the respective mRNA species (Semesei et al., 1991). The reason for this disparity between this finding and the results presented here and in other studies is not clear, but may depend on assay conditions employed and environmental conditions in which the animals were maintained. In line with other findings obtained using homogenates of whole brain (Hothersall et al., 1981 ), we found no significant changes in GPX activity in the specific brain regions during aging, and in consequence, a failure in this pathway alone is an unlikely mechanism for age-induced damage. Small increases in activity during development of young animals to 18 months of age have been reported (Mizuno and Ohta, 1986; Scarpa et al., 1987), but at variance with these reports, the effect of age on Se2+-dependent and non-Se 2+-dependent GPX in whole brain has been reported to cause a decrease in activity; the reason for the differing results is uncertain (Zhang et al., 1989). Perhaps the most significant finding of this project is the progressive increase in G R D
PEROXIDE METABOLISM IN RAT BRAIN
83
activity throughout life in all of the brain areas studied. Glutathione participates in a variety of important biochemical processes apart from peroxide metabolism, and the maintenance of an adequate supply of GSH is essential for their normal function. A glutathione-CoA mixed disulphide is formed in vivo during hydroperoxide metabolism and is also a substrate for G R D (Carlsberg and Mannervick, 1977; Crane et al., 1982; Dyer and Wilken, 1972). The total glutathione content of the brain decreases with age (Hazelton and Lang, 1980), and given that oxidative stress and aging cause an increase in the level of GSSG in the brain (Zhang et al., 1990), increased G R D activity could be induced in an attempt by the cell to maintain the availability of both CoA and GSH. It has been proposed that, instead of being a continuous process, the accumulation of free radical damage is dependent on episodes of disease (Pryor, 1988; Benzi et al., 1990). Superimposed on an underlying and progressively increasing stress placed on the maintenance of a supply of GSH, periods of oxidative stress in old age could cause more free radical-induced damage than a similar stress in young animals. The rate of accumulation of free radical damage is dependent on the frequency and severity of episodes of disease or anorexia as well as the age of the animal. Acknowledgments - - We would like to thank N. Finocchio and A. Cernilli for expert technical assistance.
REFERENCES AEBI, H. Catalase in vitro. Methods Enzymol. 105, 121-126, 1984. BHATTACHERJEE, P. and EAKINS, K.E. Lipoxygenase products: Mediation of inflammatory responses and inhibition of their formation. In: Leucotrienes--Chemistry and Biology. Chakrin, W.C. and Bailey, D.M. (Editors), pp. 195-214, Academic Press, London, 1984. BENZI, G., MARZATICO, F., PASTORIS, O., and VILLA, R.F. Influence of oxidative stress on the age-linked alterations of the cerebral glutathione system. J. Neurosci. Res. 26, 120-128, 1990. CARLBERG, I. and MANNERVICK, B. Purification by affinity chromatography of yeast glutathione reductase, the enzyme responsible for NADPH-dependant reduction of the mixed disulfide of co-enzyme A and glutathione. Biochim. Biophys. Acta 484, 268-274, 1977. CHANCE, B., SARONIO, C., and LEIGH, J.S. Functional intermediates in reaction ofcytochrome oxidase with oxygen. Proc. Natl. Acad. Sci. U. S. A. 72, 1635-1640, 1975. CHANCE, B., SIES, H., and BOVERIS, A. Hydroperoxide metabolism in mammalian organs. Physiol. Rev. 59, 527-605, 1979. COOPER, M.B., ESTALL, M.R., and RABIN, B.R. The use ofproteinases to determine the topological location ofcytochrome P450 in vesicles derived from smooth endoplasmic reticulum of rat liver. Biochem J. 196, 585589, 1981. CRANE, D,, HAUSSINGER, D., and SIES, H. Rise of co-enzyme A-glutathione mixed disulphide during hydroperoxide metabolism in perfused rat liver. Eur. J. Biochem. 127, 575-578, 1982. DYAR, R.E. and WILKEN, D.R. Rat liver levels of co-enzyme A-glutathione disulphide and of enzymes of its metabolism. Arch. Biochem. Biophys. 153, 619-626, 1972. ELROY-STEIN, O., BERNSTEIN, Y., and GRONER, Y. Overproduction of human Cu/Zn-superoxide dismutase in transfected cells: Extenuation of paraquat-mediated cytotoxicity and enhancement of lipid peroxidation. EMBO ,L 5, 615-622, 1986. EPSTEIN, C.J., AVRAHAM, K.B., LOVETT, M., SMITH, S., ELROY-STEIN, O., ROTMAN, G., BRY, C., and GRONER, Y. Transgenic mice with increased Cu/Zn-superoxide dismutase activity: Animal model of dosage effects in Down Syndrome. Proc. Natl. Acad. Sci. U. S. A. 84, 8044-8048, 1987. FLOHI~, L. and (~TTING, F. Superoxide dismutase assays. Methods Enzymol. 105, 101-104, 1984. FRIDOVICH, I. and HANDLER, P. Detection of free radicals generated during enzymic oxidation by the initiation of sulphite oxidation. J. Biol. Chem. 236, 1836-1840, 1961. GELLER, B.L. and WlNGE, D.R. Subcellular distribution of superoxide dismutases. Methods Enzymol. 105, 113-114, 1984.
84
M. V E R T E C H Y et al.
GLOWINSKI, J. and IVERSEN, L.L. Regional studies on catecholamines in the rat brain. J. Neurochem. 13, 655-669, 1966. GRAHAM, D.G. Oxidation pathways for catechol amines in the genesis of neuromelanin and cytotoxic quinones. Mol. Pharamcol. 14, 633-643, 1978. HARMAN, D. The free radical theory of aging. In: Free Radicals in Bioh~g),. Vol. 5, Pryor, W.A. (Editor), pp. 255-271, Academic Press, New York, NY, 1982. HARMAN, D.J. The free radical theory of aging: Effect of age on serum copper levels. J. Geronto/. 20, 151-153, 1965. HARRIS, E.J., BOOTH, R., and COOPER, M.B. The effect of superoxide generation on the ability of mitochondria to take up and retain Ca 2~ . FEBS Lett. 146, 267-272, 1982. HAZELTON, G.A. and LANG, C.A. Glutathone content of tissues in the aging mouse. Biochem. J 188, 25-30, 1980. HOTHERSALL, J.S., EL-HASSAN, A., McCLEAN, P., and GREENBAUM, A.L. Age related changes in enzymes of rat brain 2,- redox systems linked to NADPH and glutathione. Enzyme 26, 271-276, 1981. HUNT, J.V., DEAN, R.T., and WOLFF, S.P. Hydroxyl radical production and autoxidative glycosylation. Biochem. J. 256, 205-212, 1988. KELLOG, E.W., Ill and FRIDOVICH, I. Superoxide dismutase in the rat and mouse as a function of age and longevity. J. Gerontol. 31,405-408, 1976. KOPPENOL, W.H. and BUTLER, J. Mechanism of reactions involving singlet oxygen and the superoxide anion. FEBS Left, 83, 1-6, 1977. LOOMIS, T.C., YEE, G., and STAHL, W.L. Regional and subcellular distribution of superoxide dismutase in brain. ExT~erientia 32, 1374-1376, 1976. LOWRY, O.H., ROSEBROUGH, N.J., FARR, A.L., and RANDALL, R.J. Protein measurement with the Folin-henol reagent. J. Biol. (Ttem. 193, 265-275, 1951. MARKWELL, M.A.K., HAAS, S.M., BIEBER, L.L., and TOLBERT, N.E. A modification of the Lowry procedure to simplify protein determinations in membrane and lipoprotein samples. Anal. Biochem. 87, 206210, 1978. MAVELLI, l., MONDOVI, B., FEDERICO, R., and ROTILIO, G. Superoxide dismutase activity in developing rat brain. J. Neurochem. 31,363-364, 1978. MIQUEL, J., ECONOMOS, A.C., FLEMING, J., and JOHNSON, J.E., JR. Mitochondrial role in cell aging. L~¥p. Geronto/. 15, 575-591, 1980. MIZUNO. Y. and OHTA, K. Regional distributions of thiobarbituric acid-reactive products, activities of enzymes regulating the metabolism of oxygen free radicals and some related enzymes in adult and aged rat brain. J Neurochem. 46, 1344- 1352, 1986. MUSCARI, C.. FRASCARO, M., GUARNIERI, C., and CALDARERA, C.M. Mitochondrial function and superoxide generation from submitochondrial particles of aged rat hearts. Biochim. Biophys. Acta 1015, 200204, 1990. NOHL, tt. and HEGNER, D. Do mitochondria produce oxygen radicals in vivo? Eur. J. Biochem. 82, 563-567, 1978. ONO, T. and OKADA, S. Unique increase in superoxide dismutase level in brains of long-living animals. Exp. Geronto/. 19, 349-354, 1984. OSHINO, N. and CHANCE, B. Properties of glutathione release observed during reduction of organic hydroperoxide, demethylation of amino pyrine and oxidation of some substances in perfused rat liver, and their implications for the physiological function ofcatalase. Biochemisto, 162, 509-525, 1977. PRYOR, W.A. The free-radical theory of aging revisited: A critique and a suggested disease specific theory. In: Modern Biological Theories ~[Aging. Vol. 31 (Aging), Warner, H.R., Butler, R.N., Sprott, R.L., and Schneider, M.B. (Editors), pp. 89-105, Raven Press, New York, NY, 1988. RACKER, E. Glutathione reductase (liver and yeast). Methods Enzymol. 2, 722-729, 1955. REISS, U. and GERSHON, D. Comparison of cytoplasmic superoxide dismutase in liver heart and brain of aging rats and mice. Biochem. Biophys. Res. Commun. 73, 255-262, 1976. ROBINSON, D.S. Changes in monoamine oxidase and monoamines in human development and aging. Fed Proc. 34, 103-107, 1975. SCARPA, M., RIGO, A., VIGLINO, P., STEVANTO, R., BRACCO, F., and BATTESTIN, L. Age dependence on the level of the enzymes involved in the protection against active oxygen species in the rat brain. Proc. Soc. E~p. Biol. MecL 185, 129-133, 1987.
PEROXIDE METABOLISM IN RAT BRAIN
85
SEMESEI, 1., RAO, G., and RICHARDSON, A. Expression ofsuperoxide dismutase and catalase in rat brain as a function of age. Mech. AgeingDev. 58, 13-19, 1991. SETO, N.O.L., HAYASHI, S., and TENER, G. Overexpression of Cu-Zn superoxide dismutase in drosophila does not affect life span. Proc. Nat. Acad. Sci. U. S. A. 87, 4270-4274, 1990. SIES, H. and SUMMER, K.-H. Hydroperoxide-metabolizing systems in rat liver. Eur. J. Biochern. 57, 503-512, 1975. SLATER, T.F. Free radical mechanisms in tissue injury. Biochem. J. 222, 1-15, 1984. SMITH, L. Spectrophotometric assay ofcytochrome c oxidase. Methods Biochern. Anal, 2, 427-434, 1955. SOHAL, R.S. Hydrogen peroxide production by mitochondria may be a biomarker of aging. Mech. Ageing Dev. 60, 189-198, 1991. SOHAL, R. S. and BRUNK, U.T. Lipofuscin as an indicator of oxidative stress and aging. Adv. Exp. Med. Biol. 266, 17-30, 1989. STROLLIN-BENEDETTI, M. and KEANE, P.E. Differential changes in monoamine oxidase A and B activities in the aging rat brain. Z Neurochem. 35, 1026-1032, 1980. TAPPEL, A.L. Glutathione peroxidase and hydroperoxides. In: Methods Enzymol. 52, 506-513, 1978. VERTECHY, M., COOPER, M.B., GHIRARD1, O., and RAMACC1, M.T. Antioxidant enzym~ activities in heart and skeletal muscle of rats of different ages. Exp. Gerontol. 24, 211-218, 1989. YEN, T.-C., CHEN, Y.-S., KING, K.-L., YEH, S.-H., and WEI, Y.-H. Liver mitochondrial respiratory functions decline with age. Biochem. Biophys. Res. Commun. 165, 944-1003, 1990. ZHANG, L., MAIORINO, M., ROVERI, A., and URSINI, F. Phospholipid hydroperoxide glutathione peroxidase specific activity in tissues of rats of different ages and comparison with other glutathione peroxidases. Biochirn. Biophys. Acta 1006, 140-143, 1989.