Life Sciences 78 (2005) 271 – 278 www.elsevier.com/locate/lifescie Aging affects oxidative state in hippocampus, hypothalamus and adrenal glands of Wistar rats Ionara Rodrigues Siqueira b,c,*, Cı́ntia Fochesatto a, Iraci Lucena da Silva Torres a, Carla Dalmaz a, Carlos Alexandre Netto a,b b a Departamento de Bioquı́mica Programa de Pós-Graduação em Ciências Biológicas-Fisiologia, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil c Centro Universitário UNIVATES, Lajeado, RS, Brazil Received 23 December 2004; accepted 20 April 2005 Abstract The aging process is associated with cognitive impairment and dysregulation of the hypothalamic – pituitary – adrenal (HPA) axis, as well as with oxidative stress. We determined some parameters of oxidative stress in homogenates of hippocampus, hypothalamus and adrenal glands from male 2-, 6- and 24-months-old Wistar rats. A significant age-dependent increase in the generation of free radicals was observed in hippocampus, hypothalamus and adrenal glands, as well as on lipid peroxidation in hippocampus and hypothalamus. The glutathione peroxidase (GPx) activity was significantly reduced in hypothalamus and hippocampus from 6-months-old rats; a decline on GPx and catalase activities in adrenal glands of 24-months-old animals was also present. Interestingly, a great decrease in total antioxidant capacity was found in all tissues tested. Reported findings support the idea that oxidative events participate on multiple neuroendocrine-metabolic impairments and suggest that the oxidative stress found in hippocampus, hypothalamus and adrenals might be associated with age-related physiological deficits. D 2005 Elsevier Inc. All rights reserved. Keywords: Oxidative stress; Free radicals; Antioxidant defenses; Hippocampus; Hypothalamus; Adrenal gland; Aging Introduction Hypothalamus and pituitary are closely related structures with the major role of integrating neural and endocrine systems. The hypothalamic – pituitary– adrenal (HPA) axis is responsible for the synthesis and release of steroid hormones, the most abundant being dehydroepiandrosterone (DHEA), DHEA sulfate (DHEAS), cortisol and aldosterone (Endoh et al., 1996). Aging is commonly associated with dysregulation of the HPA axis and with cognitive impairment. There is an increase in adrenal glucocorticoid secretion, as well as a decline in adrenal androgen synthesis and secretion in humans during the aging process (Yen and Laughlin, 1998). Several investigators have suggested that the hypothalamus may mediate the agerelated decline of physiological functions and changes of * Corresponding author. Centro III, Centro Universitário UNIVATES, Rua Barbedo 426, 03, 90110-260, Porto Alegre, RS, Brazil. Tel.: +55 51 37147000; fax: +55 51 37147001. E-mail address: ionara@ufrgs.br (I. Rodrigues Siqueira). 0024-3205/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2005.04.044 biological rhythms (Meites et al., 1987; Carlson and Sawada, 1996; Hofman, 1997); the hippocampus has also been implicated in this phenomenon (Jacobson and Sapolsky, 1991). Free radicals, such as superoxide radical anion, hydroxyl, alkoxyl and peroxyl radicals as well as hydrogen peroxide (H2O2), are continuously produced during oxidative metabolism and may provoke cell injury via direct oxidation to critical biological molecules, including the lipoperoxidation (LPO) and oxidative damage to proteins. The antioxidant defense system includes antioxidant enzymes (AOEs), such as superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx), as well as non-enzymatic antioxidants (glutathione, vitamins E and C). Previous work has shown that antioxidant function declines in almost all mammals during aging (Harman, 1992). Literature has generally reported that an altered endocrine function might be related to oxidative status. As for example, a moderate hypothyroidism extended mean lifespan (Ooka et al., 1983) and diminished lipid peroxidation (Pereira et al., 1994) in rats. Moreover, lifelong hyperthyroidism accelerated aging 272 I. Rodrigues Siqueira et al. / Life Sciences 78 (2005) 271 – 278 (Ooka and Shinkai, 1986) and enhanced lipid peroxidation (Pereira et al., 1994). Growth hormone (GH) administration increased both CAT and GPx activities, while transgenic mice with chronically high levels of GH showed a global decrease in AOE activities and decreased lifespan (Bartke, 1998; Youn et al., 1998; Brown-Borg et al., 1999). Considering endocrine changes and oxidative state reported during aging and that oxidative stress may possibly happen in regions modulating endocrine function, we decided to evaluate the oxidative status during aging in tissues that have been associated with the control of HPA axis, namely hypothalamus, adrenal gland and hippocampus. Age-related alterations on parameters of oxidative status, free radicals content, damage to macromolecules as well as the antioxidant capacity, were studied in hippocampus, hypothalamus and adrenal glands from 2 –3 months-, 6 months- and 20 – 21 months-old male Wistar rats. Free radical content Material and methods 2V–7V-dichlorofluorescein diacetate (DCFH-DA) was used as a probe for the assay of free radical content. This method does not determine the presence of specific free radicals, because DCFH may be oxidized by several reactive intermediates (Wang and Joseph, 1999). An aliquot of the sample was incubated with DCFH-DA (100 AM) at 37 -C for 30 min; the reaction was terminated by chilling the reaction mixture in ice. Formation of the oxidized fluorescent derivative (DCF) was monitored at excitation and emission wavelengths of 488 and 525 nm, respectively, using a fluorescence spectrophotometer (Hitachi F-2000). The free radical content was quantified using a DCF standard curve and results were expressed as pmol of DCF formed/mg protein. All procedures were performed in the dark and blanks containing only DCFHDA (no homogenate) were processed for measurement of autofluorescence (Sriram et al., 1997; Driver et al., 2000). Chemicals Assay of lipid peroxidation Thiobarbituric acid and Trolox were obtained from Merck, 2,2V-azobis (2-amidinopropane) dihydrochloride (ABAP) was obtained from Wako Chemicals USA, Inc., 2V–7V-dichlorofluorescein diacetate (DCFH-DA), 2V –7V-dichlorofluorescein (DCF), trichloroacetic acid (TCA), 2,4-dinitrophenylhydrazine (DNPH), guanidine hydrochloride, phenylmethylsulfonyl fluoride (PMSF), 5-amino-2,3-dihydro-1,4-phtalazinedione (luminol) and H2O2 stock solution were purchased from Sigma Chemical Co. The formation of thiobarbituric acid reactive substances (TBARS) was based on the methods described by Buege and Aust (1978). Aliquots of samples were incubated with 10% trichloroacetic acid (TCA) and 0.67% thiobarbituric acid (TBA). The mixture was heated (30 min) on a boiling water bath. Afterwards, n-butanol was added and the mixture was centrifuged. The organic phase was collected to measure fluorescence at excitation and emission wavelengths of 515 and 553 nm (Yagi, 1998), respectively; 1,1,3,3-tetramethoxypropane, which is converted to malondialdehyde (MDA), was used as standard. Animals and tissue preparation Male Wistar rats of distinct ages were used: 2 –3 monthsold, 6 months-old and 20– 21 months-old. It is well accepted that 2– 3 months-old rats are young adult animals, 6 monthsold are mature adult (Palomero et al., 2001) and that 20 –21month old ones are senescent (Gilad et al., 1990). Animals were maintained under standard conditions (12 h light / 12 h dark, temperature 22 T 2 -C); food (20% (w/w) protein commercial chow, Germani, Porto Alegre, RS, Brazil) and water were given ad libitum. All investigations and procedures were conducted in accordance with the Principles of Laboratory Animal Care (National Institute of Health) and the experimental protocol was approved by the University Animal Care Committee. Rats were killed by decapitation, hippocampi and hypothalamus were rapidly dissected out and adrenals were excised. They were instantaneously placed in liquid nitrogen and stored at 70 -C until biochemical measurements, when they were homogenized in 10 volumes of ice-cold phosphate buffer (0.1 M, pH 7.4) containing 140 mM KCl and 1 mM EDTA. In order to assay carbonyl levels and AOE activities, samples were homogenized in the presence of PMSF. The homogenate was centrifuged at 960 g for 10 min and the supernatant, a suspension of mixed and preserved organelles, was used. Determination of protein carbonyl levels Protein carbonyl content was determined according to Levine et al. (1990). Aliquots of samples were incubated with streptomycin sulfate solution (1%) for 15 min. The mixture was centrifuged at 3600 g; the supernatant was divided and incubated for 1 h, at room temperature, with 10 mM 2,4dinitrophenylhydrazine (DNPH) in 2 M HCl, or only with 2 M HCl (blank). The protein was precipitated by adding an equal volume of 20% TCA. After centrifugation (8600 g), the pellet was washed three times with ethanol: ethyl acetate (1 : 1) to remove excess DNPH. The precipitated protein was redissolved in 6 M guanidine HCl and the absorbance of solutions was measured at 370 nm. Carbonyl content was calculated taking the extinction coefficient of 22 mM 1 cm 1 / mg of protein. Total reactive antioxidant potential (TRAP) assay TRAP and TAR are based on luminol-enhanced chemiluminescence measurement, induced by an azo initiator (Lissi et al., 1992, 1995; Evelson et al., 2001). The reaction mixture contained ABAP 10 mM, source of peroxyl radicals, and luminol (4 mM) in glycine buffer (0.1 M, pH 8.6). The chemiluminescence (CL) generated was measured in a scintil- 273 I. Rodrigues Siqueira et al. / Life Sciences 78 (2005) 271 – 278 lation counter (Beckman) working in the out of coincidence mode. The addition of Trolox (antioxidant standard, 200 nM) or samples (5.0 or 1.0 Al of sample) decreases CL to basal levels for a period (induction time) proportional to the concentration of antioxidants. The TRAP values were calculated as equivalents of Trolox concentration per milligram of protein. assessing the initial decrease of luminescence calculated as the ratio ‘‘I o / I’’, where ‘‘I o’’ is the CL in the absence of additives, and ‘‘I’’ is the CL after addition of the 20 nM Trolox, or the samples (1 Al). TAR values were expressed as equivalents of Trolox concentration per milligram of protein (Lissi et al., 1992, 1995). Superoxide dismutase activity Total antioxidant reactivity (TAR) assay The reaction mixture contained 2 mM ABAP and 6 mM luminol in glycine buffer. TAR values were determined by SOD activity was determined using a RANSOD kit (Randox labs., USA) which is based on the procedure described by Delmas-Beauvieux (1995). This method employs pmol DCF formed/mg protein (a) # 8 6 * * * 4 2 0 HC HT young adult pmoles MDA/mg ptotein mature adult (b) 0,4 AD old # 0,3 * 0,2 0,1 0 HC HT young adult nmol DNPH/mg protein 180 mature adult AD old (c) 120 60 0 HC AD HT young adult mature adult old Fig. 1. Effects of aging on free radical content (a), on lipoperoxidation levels (b), and protein damage (c), in hippocampus (HC), hypothalamus (HT) and adrenal glands (AD) in 2 – 3 months (young adult), 6 months (mature adult) and 20 – 21 months (old) male Wistar rats. Results are expressed as mean T SEM from six to eight samples per group. * Denotes values significantly different from those of young group, # same, when compared to those of young and mature adult rats, as determined by ANOVA followed by Duncan’s test ( P < 0.05). 274 I. Rodrigues Siqueira et al. / Life Sciences 78 (2005) 271 – 278 xanthine and xanthine oxidase to generate superoxide radicals that react with 2-(4-iodophenyl)-3-(4-nitrophenol)-5-phenyltetrazolium chloride to form a red formazan dye which is assayed spectrophotometrically at 505 nm at 37 -C. The inhibition on production of the chromogen is proportional to the activity of SOD present in the sample. Catalase activity The homogenate was incubated with ethanol (10%) and Triton (10%). The activity was assayed at 25 -C by determining the rate of degradation of H2O2 at 240 nm in 10 mM potassium phosphate buffer (pH 7.0). The extinction coefficient of 43.6 mM 1 cm 1 was used for calculation. One unit is defined as 1 pmol of H2O2 consumed per minute and the specific activity is reported as units per milligram protein (Aebi, 1984). Glutathione peroxidase activity GPx activity was determined according to Wendel (1981). The reaction was carried out at 25 -C in 600 Al of solution containing 100 mM pH 7.7 potassium phosphate buffer, 1 mM EDTA, 0.4 mM sodium azide, 2 mM GSH, 0.1 mM NADPH, 0.62 U of GSH reductase. The activity of selenium-dependent GPx was measured taking tert-butyl-hydroperoxide as the substrate at 340 nm. The contribution of spontaneous NADPH oxidation was always subtracted from the overall reaction rate. GPx activity was expressed as nanomole NADPH oxidized per minute per milligram protein. (a) U/ mg protein 60 40 20 0 HC nmol NADPH oxidized/min/mg protein young adult HT mature adult AD old (b) 15 12 9 * * 6 3 0 * * * HC HT young adult mature adult AD old (c) mU/ mg protein 3000 2500 * 2000 1500 1000 500 0 HC HT young adult mature adult AD old Fig. 2. Effects of aging on activities of antioxidant enzymes, superoxide dismutase (a), glutathione peroxidase (b), catalase (c) in hippocampus (HC), hypothalamus (HT) and adrenal glands (AD) at different ages. Results are expressed as mean T SEM from six to eight samples per group. * Denotes values significantly different from those of young group, as determined by ANOVA followed by Duncan’s test ( P < 0.05). I. Rodrigues Siqueira et al. / Life Sciences 78 (2005) 271 – 278 Protein assay The total protein concentrations were determined using the method described by Lowry et al. (1951) with bovine serum albumin as the standard. Statistical analysis Data were analyzed using analysis of variance (ANOVA) with post hoc analysis performed by the Duncan’s multiple range test. A difference was considered significant when P < 0.05 level. Results are expressed as mean T standard error mean (SEM). Results The brain areas examined, hippocampus and hypothalamus, showed a similar pattern of results with regard to oxidative stress. A significant age-dependent increase in free radical levels was observed in both regions, since old, 20– 21 monthsold, rats showed DCF levels higher than those of young. Interestingly, we found higher DCF levels in hippocampus already in mature animals, as shown in Fig. 1 (Panel A). % vs. young group 120 90 * * * * # 60 30 0 HC HT young adult mature adult AD old (b) % vs. young group 120 * * * # 80 40 0 HT HC young adult We measured TBARS and protein carbonyl levels to investigate the effect of aging on oxidatively modified lipids and proteins. Panel B (Fig. 1) shows that TBARS levels were increased in hippocampus and hypothalamus in aged rats; on the other hand, no significant differences on the protein carbonyl content were found (Panel C). The effects of aging on the activities of SOD, CAT, and GPx are depicted in Fig. 2. There were no changes in SOD activity (Panel A); however the GPx activity was significantly lower in mature adult and aged rats as compared to young ones in both hypothalamus and hippocampus. GPx and CAT activities were significantly reduced in adrenals from old rats (Panels B and C). The effect of aging on tissue antioxidant capacity was investigated using TAR and TRAP assays, as seen in Fig. 3. It is worth noting the significant age-dependent decrease in TRAP and TAR levels revealed in the hippocampus from mature (about 30%) and old rats (about 40% and 50%, respectively), indicating an important reduction on the total antioxidant capacity. A decrease in TAR levels (about 40%) was observed in hypothalamus from senescent rats; decreases in TAR and TRAP levels (about 25%) in adrenals from mature and old rats were also shown. Discussion (a) 150 275 mature adult AD old Fig. 3. Effects of aging on total antioxidant capacity, measured by the total antioxidant reactivity (TAR, panel a) and the total antioxidant potential (TRAP, panel b) assays in hippocampus (HC), hypothalamus (HT) and adrenal glands (AD). Results are expressed as percentage of the young adult group. Columns represent mean T SEM for six to eight determinations. The absolute mean of TAR values for young groups were 88 T 14 (hippocampus), 42 T 7 (hypothalamus) and 260 T 68 (adrenals); the absolute mean of TRAP values were 41 T 5 (hippocampus), 22 T 3 (hypothalamus) and 116 T 25 (adrenals) equivalents pmol Trolox / mg protein. * Denotes values significantly different from those of young group, # same, when compared to those of young and mature adult rats, as determined by ANOVA, followed by Duncan’s test ( P < 0.05). We reported clear age-associated changes on free radical levels and antioxidant defenses in the critical HPA axis tissues studied, the hippocampus and hypothalamus, as well as in adrenal glands, what strongly suggests a relationship between oxidative status and neuroendocrine dysfunction; that could probably affect longevity. The hippocampus and hypothalamus from 21-months-old rats presented alterations in various parameters that implicate the existence of a strong oxidative stress (elevated free radicals and lipid peroxidation levels, along with impaired antioxidant activity). It has been shown that the hippocampus might be involved in the age-related HPA dysregulation, since this structure modulates axis activation and contains a high concentration of glucocorticoid receptors (Sapolsky, 1992). In addition, the hippocampus is known to be functionally impaired in aged rat populations, the main change observed so far being synaptic loss (Geinisman, 1999). Notably, mature rats (6-months-old) exhibited an increase of free radical content in hippocampus, paralleled by reduced antioxidant capacity, including decreased GPx activity in both hippocampus and hypothalamus, along with decreased TRAP and TAR levels in hippocampus and adrenals. Although both assays do evaluate enzymatic and non-enzymatic defenses (Evelson et al., 2001), TRAP levels indicate the antioxidant concentration in the sample, while the TAR test indicates antioxidant reactivity (Lissi et al., 1995; Desmarchelier et al., 1997). The overall tissue antioxidant status (TRAP and TAR indexes) is decreased with aging in all areas studied, which may imply their inability to modulate oxidative changes. It follows that even a small enhancement on free radical 276 I. Rodrigues Siqueira et al. / Life Sciences 78 (2005) 271 – 278 production would exceed the ability of cells to defend themselves against these substances. Interestingly, these data indicate that oxidative events in the hippocampus — HPA axis occur already in early stages of life, before apparent senescence, and are in agreement with those of Herman and colleagues (2001), who found HPA up-regulation and hyperresponsiveness to hypoxia in middle-aged Fischer 344 rats. Hauck and Bartke (2000), investigating the effect of differences in GH status, showed consistent age-related alterations in CAT and SOD activities using mice with GH deficiency and transgenic mice overexpressing human GH. The GH deficient mice exhibited prolonged lifespan, as well as higher hypothalamic SOD and CAT activities than controls, while the overexpression in human GH lead to reduced lifespan and enhanced hypothalamic SOD activity (Hauck and Bartke, 2000). The distinct lifespan presented by these mice could be related to high SOD activity in the absence of parallel changes in GPx and/or CAT activities, so allowing the accumulation of H2O2 that, in the presence of metal ions, leads to formation of the highly reactive hydroxyl radical. Similarly, our results demonstrated an age-related significant difference on GPx activity in hypothalamus, suggesting also an accumulation of H2O2 that could also be related to differences in life expectancy. Increased free radical content here reported could explain, at least in part, the altered AOEs activities presented on Fig. 2; superoxide radical itself could contribute to the observed diminished activity of CAT and GPx, since it is reported to interact and inactivate both enzymes (Kono and Fridovich, 1982; Blum and Fridovich, 1985). It is important to point out that the differences in AOE activities and total antioxidant capacity do not necessarily imply a causal mechanism in the aging process, however the decrease in free radical detoxification is one possible explanation for the increased free radical production and lipid peroxidation. The high LPO basal levels in adrenals could be tentatively explained by elevated amounts of polyunsaturated fatty acids (PUFA) (Staats et al., 1988). It was proposed that extensive antioxidant protection is needed by the adrenal cortex because of its vulnerability to peroxidative injury, considering the amount of PUFA and the various P450 isozymes that are involved in steroid hormone synthesis, potential sources of oxygen radicals (Hornsby and Crivello, 1983). Additional evidence was obtained by Azhar and colleagues (1995), who found a reduction in the efficiency of antioxidant defenses in adrenals from aged animals. Together with a reduction in antioxidant defenses, they showed a decline in steroidogenesis and proposed that these changes may lead to oxidative damage of membrane or cytosolic factors important to cholesterol transport; as a consequence, cholesterol cannot reach the appropriate mitochondrial sites for side chain cleavage. In the present work, we did not find changes on lipoperoxidation levels on crude homogenates. However, considering the importance of the antioxidant system in steroidogenesis, the great reduction on antioxidant system in adrenal glands could contribute to age-related impairments in this metabolic pathway. Important endocrine changes on peripheral and pituitary hormones are associated to aging process. The adrenosenescence is accompanied by low serum levels of androstenedione, DHEA, DHEAS, progesterone and aldosterone, and by high levels of glucocorticoids (Guazzo et al., 1996), although the gonado-senescence is marked by low serum levels of 17h-estradiol in females and of testosterone in males. Therefore, both an increase in adrenocorticotropic hormone and a decrease of other pituitary hormones, such as GH, thyroid-stimulating, luteinizing and follicle-stimulating hormones, have been found in aging (Straub et al., 2001). Some hormones are physiological modulators of antioxidant enzyme activities (Bolzan et al., 1995), such as the glucocorticoids that reduce GPx activity in rat macrophages (Pereira et al., 1995). Recently, the involvement of the pineal gland in the aging processes has been proposed (Reiter, 1995), since melatonin content changes both in liquor and plasma with aging (Liu et al., 1999; Skene et al., 1990). Conversely, melatonin has been widely reported as a potent antioxidant (Marshall et al., 1996; Reiter et al., 1997; 2001) that protects against LPO (Carneiro and Reiter, 1998) and increases both activity and expression of antioxidant enzymes (Rodriguez et al., 2004). Therefore, the age-related decrease in melatonin levels may contribute to aging process. Conclusion Present data show strong and consistent evidence for ageassociated alterations on oxidative status in hippocampus, hypothalamus and adrenal glands, providing support to the hypothesis that oxidative damage in these areas may have a significant role in aging of Wistar rats. We suggest that the increase on free radical content, paralleled by a decrease of antioxidant defenses, are important factors contributing to the aging process. To what extent antioxidant dysfunction results in macromolecules’ oxidative damage in these tissues is a question that merits further investigation. It is conceivable that both enhancement of free radicals’ generation and impairment of antioxidant defenses here reported might be involved in agerelated metabolic changes. Acknowledgments We gratefully acknowledge financial support by Programa dos Núcleos de Excelência (PRONEX), Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul (FAPERGS), Coordenação de Aperfeiçoamento de Pessoal de Nı́vel Superior (CAPES), Conselho Nacional de Desenvolvimento Cientı́fico e Tecnológico (CNPq) and Pró-Reitoria de Pesquisa da Universidade Federal do Rio Grande do Sul (PROPESQ-UFRGS). References Aebi, H., 1984. Catalase in vitro. Methods in Enzymology 105, 121 – 126. Azhar, S., Cao, L., Reaven, E., 1995. Alteration of the adrenal antioxidant defense system during aging in rats. The Journal of Clinical Investigation 96, 1414 – 1424. I. Rodrigues Siqueira et al. / Life Sciences 78 (2005) 271 – 278 Bartke, A., 1998. Growth hormone and aging. Endocrine 8, 103 – 108. Blum, J., Fridovich, I., 1985. Inactivation of glutathione peroxidase by superoxide radical. Archives of Biochemistry and Biophysics 240, 500 – 508. 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