HomeLarge Type Edition
HOME ARCHIVE SEARCH TABLE OF CONTENTS
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Services
Right arrow Download to citation manager
PubMed
Right arrow PubMed Citation
The Journals of Gerontology Series A: Biological Sciences and Medical Sciences 62:824-830 (2007)
© 2007 The Gerontological Society of America

Age-Associated Oxidative Macromolecular Damages in Rat Brain Regions: Role of Glutathione Monoester

Ganesan Murali and Chinnakkannu Panneerselvam

Department of Medical Biochemistry, Dr. ALM Postgraduate Institute of Basic Medical Sciences, University of Madras, Taramani Campus, Chennai, India.

Address correspondence to Chinnakkannu Panneerselvam, PhD, Department of Medical Biochemistry, Dr. ALM Postgraduate Institute of Basic Medical Sciences, University of Madras, Taramani Campus, Chennai – 600 113, India. E-mail: biomurali{at}gmail.com


   Abstract
Top
 Abstract
Methods
 Results
 Discussion
 References
 
The generation of reactive oxygen species (ROS) and resultant oxidative stress has been implicated in the mechanism of brain dysfunction due to age-related neurodegenerative diseases. We have evaluated the efficacy of glutathione monoester (GME) when administered intraperitoneally (12 mg/kg body weight) for 20 days on glutathione, ROS, superoxide anion production, lipid peroxidation (LPO), protein carbonyls, thiol status, oxidative DNA damage products such as 8-hydroxy deoxy guanosine and DNA protein cross-links in discrete brain regions of young and aged rats. An age associated increase in ROS, superoxide anion production, LPO, protein oxidation, and DNA damage products in cortex, striatum, and hippocampus was observed which was reversed by GME. Contradictorily, a decline in the levels of glutathione, total thiol, and nonprotein and protein thiols was observed which was also reversed upon GME administration. These findings suggest that GME administration inhibits free radical–induced oxidative macromolecular damage in aged rats and thereby protects the brain from ROS.

AN inescapable by-product of oxidative metabolism is the production of reactive oxygen species (ROS), which can damage lipids, nucleic acids, and proteins (1). The rate of generation of ROS in different species roughly correlates with life span, and is thought to represent a major determinant in defining the rate of aging and the development of numerous age-related diseases (2). Two tissues that are particularly vulnerable to oxidative stress are the central nervous system and muscle, both of which contain postmitotic cells, are liable to accumulate oxidative damage over time, and use a large amount of oxygen (3). Free radicals have previously been damaging many cellular components such as DNA (4), proteins (5), and lipids (6). Indeed, damage to DNA and proteins often may be more important than damage to lipids in oxidative stress situations in vivo (7).

Oxidative modifications of proteins in vivo may affect a variety of cellular functions involving proteins, which include receptors, signal transduction mechanisms, transport systems, and enzymes. They could also contribute to secondary damage to other biomolecules, for instance, inactivation of DNA repair enzymes and loss of fidelity of DNA polymerases in replicating DNA (8). Oxidative damage to proteins is reflected by an increase in the levels of protein carbonyls (PCO) (9). Reaction of free radicals, such as OH• or Formula with the side chains of lysine, arginine, proline, threonine, and glutamic acid residues of proteins leads to the formation of carbonyl derivatives (10). Furthermore, aldehydes such as 4-hydroxy-2-nonenal or MDA produced during lipid peroxidation (LPO) can be incorporated into proteins by reaction with either the {xi}-amino moiety of lysine or the sulfhydryl group of cysteine residues to form carbonyl derivatives (11). Carbonyl groups can also be introduced into proteins by glycation and glycoxidation reactions (12). Therefore, PCO groups provide a reasonable marker for free radical-induced protein oxidation. It has been shown that free radicals cause oxidation of protein thiol (PSH) groups (13). PSH may serve an antioxidant function by several mechanisms: They may preemptively scavenge oxidants, which initiate peroxidation, thus sparing vitamin E and/or lipids from attack (13,14).

A relationship between oxidative DNA damage and aging has been postulated based on studies that show inverse association between steady-state tissue levels of 8-hydroxy-2'-deoxyguanosine (8-OHdG) and life spans of different animal species (15). The damage to DNA is more variable, and attack by free radicals can produce, for example, structural damage (i.e., strand breaks), DNA protein cross-links, modification of the bases. One of the most common adducts formed from the reaction of oxyradicals with DNA is 8-OHdG (16) because, among all purine and pyrimidine bases, guanine is most prone to oxidation (17). DNA protein cross-links are formed by the addition of allyl radical of thymine to the C3 position of the tyrosine ring in a protein in the vicinity of DNA (18). These complexes generated by free radicals may be important DNA lesions creating genotoxicity because these complexes are in general persistent and not as readily repaired as that of other lesions (19).

Antioxidants that accumulate in brain and neuronal tissue are potential candidates for prevention or treatment of disorders involving oxidative damage. In particular, thiol antioxidants may be good candidates for use in brain disorders. Glutathione monoester (GME) contains an ester group, esterified to the glycine of glutathione (GSH). GME is lipophilic, and enters cells more readily than GSH (20); thus, GME provides direct intracellular availability of GSH (21). The traditional role of GSH is that of a free radical scavenger; the ability of GSH to nonenzymatically scavenge both singlet oxygen and OH• radical (22) provides the first line of antioxidant defense. Thus the overall objective of the present study was to investigate the efficacy of GME against the accumulation of protein oxidation and DNA damage in brain regions like cortex, striatum, and hippocampus of young and aged rats.


   METHODS
Top
 Abstract
 Methods
Results
 Discussion
 References
 
Source of Chemicals
GME, bovine serum albumin (BSA), 2',7'-dichlorodihydrofluorescein-diacetate (DCFH-DA), dinitrophenylhydrazine (DNPH), dithiodinitrobenzoic acid (DTNB), 8-OHdG, alkaline phosphatase, and nuclease P1 were purchased from Sigma (St. Louis, MO). All other chemicals used were of analytical grade that were procured from Sisco Research Laboratories, Mumbai, India.

Animals
Male albino rats of Wistar strain used in this study were obtained from King's Institute of Preventive Medicine (Chennai, India) and were maintained in a clean rodent room. Animals were housed 2–3 per cage, and cages were fitted with stainless steel wire mesh bottoms and maintained at a temperature of 28 ± 1°C with a 12-hour light/dark cycle. The animals were fed a pellet diet (Hindustan Lever Limited, Mumbai, India) and tap water ad libitum. The commercial rat feed contained 5% fat, 21% protein, 55% nitrogen-free extract, and 4% fiber (wt/wt), with adequate mineral and vitamin contents. The laboratory animal protocol used for this study was approved by the Committee for the Purpose of Control and Supervision on Experimental Animals (CPCSEA) at the University of Madras, Chennai, India.

Experimental Protocol
The animals were divided into four groups consisting of six animals each:

GME (12 mg/kg body weight/day) was dissolved in 0.89% physiological saline and administered intraperitoneally for 20 days. Control animals received physiological saline alone. On completion of experimental period, animals were killed by decapitation. Brains were excised immediately; regions were separated according to the method of Glowinski and Iversen (23) and immersed in ice-cold physiological saline. A 10% homogenate of the brain regions was prepared and used for biochemical analysis.

Assay of LPO
Lipid peroxides were estimated in the brain regions (the cortex, striatum, and hippocampus) using a modified thiobarbituric acid (TBA) test described by Ohkawa and colleagues (24) using malondialdehyde (MDA) as the standard. Briefly, 0.1 mL of homogenate of brain regions was added to the test tube containing 0.2 mL of 8.1% sodium dodecyl sulfate (SDS), 1.5 mL of 20% acetic acid solution (pH 3.5), and 1.5 mL of 0.8% TBA solution. The mixture was diluted to 4 mL with distilled water and heated at 95°C for 60 minutes. After cooling on ice, the samples were extracted with 4.0 mL of a mixture of n-butanol and pyridine (15:1, vol/vol). After centrifugation at 800 x g for 10 minutes, the organic phase was collected and the absorbance measured at a wavelength of 532 nm. The concentration of TBA was determined using the extinction coefficient of 1.56 x 105 M–1 cm–1.

Reduced GSH Level
The level of reduced GSH was measured by the method of Moron and colleagues (25). One-milliliter of homogenate was precipitated with 1 mL of 10% TCA, and the precipitate was removed by centrifugation. To 0.5 mL of the supernatant, 2 mL of 0.6 mM DTNB in 0.2 M sodium phosphate was added, and the total volume was made up to 3 mL with phosphate buffer. The absorbance was read at 412 nm.

Determination of ROS Levels
Generation of ROS was determined using DCFH as the probe with 10% homogenate, according to Lebel and colleagues (26). In brief, the assay buffer contained 20 mM Tris–HCl, 130 mM KCl, 5 mM MgCl2, 20 mM NaH2PO4, 30 mM glucose, and 5 mM 2,7-dichlorofluorescein (DCF). The assay medium was incubated at 37°C for 15 minutes and 1 µmoL of H2O2 was added into the mixture at the end of assay. DCF formation was measured using excitation at 488 nm and emission at 525 nm for 30 minutes using a Shimadzu fluorescence spectrometer (model UV-1700; Shimadzu Pvt Ltd, Singapore). Protein was estimated by the method of Lowry and colleagues (27).

Cytochrome c Reduction Assay
Superoxide anion (O2•–) production was measured by the cytochrome c reduction assay of Babior and colleagues (28). One milliliter of the reaction mixture contained 1 mg of protein and 0.05 mM cytochrome c in the incubation buffer. Placing the reaction mixture on ice terminated the reactions. The mixtures were centrifuged for 10 minutes, and the supernatant was transferred to clean tubes for spectrophotometric measurement at 550 nm. Absorbance values were converted to nanomoles of cytochrome c reduced/15 min/mg of protein using the extinction coefficient 2.1 x 10–4 M–1 cm–1.

Determination of PCO Content
PCO content was determined by the most common and reliable method based on the reaction of carbonyl groups with DNPH to form a 2,4-dinitrophenylhydrazone as suggested by Levine and colleagues (29). Briefly, 100 µL of the brain homogenate was incubated with 0.5 mL of DNPH for 60 minutes. Subsequently, protein was precipitated from the solution using 20% TCA. The pellet was washed after centrifugation (3400 x g) with an ethyl acetate/ethanol (1:1, vol/vol) mixture three times to remove excess DNPH. The final protein pellet was dissolved in 1.5 mL of 6 M guanidine hydrochloride. The carbonyl content was evaluated in a spectrophotometer at a wavelength of 370 nm. A standard curve of BSA was included in each assay to determine the linearity and to measure the extent of derivatization. The results were presented in nanomoles per milligram of protein.

Determination of PSH Levels
Aliquots of 250 µL of tissue homogenate were mixed in 5-mL test tubes with 750 µL of 0.2 M Tris buffer (pH 8.2) and 50 µL of 0.01 M DTNB. The mixture was made up to 5 mL with 3950 µL of absolute methanol. A reagent blank and a sample blank were prepared in a similar manner. The test tubes were stoppered with rubber caps and color developed after 15 minutes. The reaction mixture was then centrifuged at approximately 3000 x g at room temperature for 15 minutes. The absorbances of the supernatants were read in a spectrometer at 412 nm. The molar extinction coefficient at 412 nm was 13,100 in both total thiol and non-PSH procedures. Aliquots of 250 µL of the homogenates were mixed in 5-mL test tubes with 200 µL of distilled H2O and 50 µL of 50% TCA. The test tubes were shaken intermittently for 10 minutes and centrifuged for 15 minutes at approximately 3000 x g. Two hundred microliters of the supernatant was mixed with 400 µL of 0.4 M Tris buffer (pH 8.9) followed by 10 mL of DTNB, and the sample was shaken on a shaker. The absorbance was read within 5 minutes of the addition of DTNB at 412 nm against a reagent blank with no homogenate. The PSH groups were calculated by subtracting the non-PSH from total thiol (30).

Measurement of 8-OHdG by HPLC
DNA was extracted by the method of Gross-Bellard and colleagues (31). The measurement of 8-OHdG by HPLC was determined by the method of Ito and colleagues (32). Isolated DNA was dissolved in 20 mM acetate buffer (pH 5.0) and digested to deoxynucleosides with 8 units of nuclease P1 at 37°C for 30 minutes and then with 1.3 units of alkaline phosphatase at 37°C for 1 hour in 0.1 M Tris–HCl buffer (pH 7.5). The resulting deoxynucleosides were injected into a C18 column equipped with both an ultraviolet (UV) detector and an electrochemical detector. The eluent/mobile phase used was 10% aqueous methanol containing 12.5 mM citric acid, 25 mM sodium acetate, 30 mM NaOH, and 10 mM acetic acid at a flow rate of 1 mL/min. The levels of 8-OHdG in the samples were measured based on the peak height of authentic 8-OHdG with electrochemical detector and the UV absorbance at 254 nm.

Estimation of DNA Protein Cross-Links
The extent of DNA protein cross-links was assayed by the method of Zhitkovich and Costa (33). Five hundred microliters of tissue homogenate was lysed with 500 µL of SDS, and the DNA was sheared by passing the cell lysates through a micropipette. From this, 500 µL was taken and used to estimate DNA. To the remaining 500 µL of sample, 500 µL of 100 mM KCl and 20 mM Tris (pH 7.5) were added. The contents were mixed by vortexing and then heated for 10 minutes at 65°C in a water bath. Samples were then placed in ice for 5 minutes to form potassium-SDS (K-SDS). The precipitate was collected by centrifugation at 6000 x g for 5 minutes. The supernatant was collected, and the pellet was resuspended in KCl–Tris by brief vortexing. The samples were again heated at 65°C for 10 minutes and the washing, as well as the heating steps described above, was repeated twice. Protein-linked DNA was released from the final K-SDS precipitate by treatment with proteinase K at 200 mg/mL in 500 µL of solution containing 100 mM KCl, 20 mM Tris (pH 7.5), and 10 mM EDTA. Then, the samples were centrifuged at 12,000 x g for 10 minutes at 4°C, and the supernatant was taken to determine the quantity of DNA. DNA was quantified both as free and K-SDS-precipitable protein-bound DNA by the addition of Hoechst 33258 and by fluorescence detection. Results were reported as a percentage of protein-bound DNA of total DNA. Percentage of DNA precipitated by K-SDS is an indicator of the amount of DNA associated with protein cross-links.

Statistical Analysis
The results are expressed as mean ± standard deviation (SD). Differences between groups were assessed by analysis of variance (ANOVA) using the SPSS software package for Windows (version 7.5; SPSS, Chicago, IL). Post hoc testing was performed for inter-group comparisons using the least significance difference (LSD) test; statistical significance at p value <.001 has been given respective symbols in the tables.


   RESULTS
Top
 Abstract
 Methods
 Results
Discussion
 References
 
LPO
To evaluate the possible consequences of oxidative stress in the brain, LPO was measured in different regions of brain (Table 1). There were significant increases in LPO in cortex (1.7-fold), striatum (1.5-fold), and hippocampus (1.3-fold) of aged rats as compared with the respective regions of young rats. GME administration lowered brain regional LPO approximately 34% in cortex, 31% in striatum, and 23% in hippocampus. No marked change was observed in GME-treated young rats.


View this table:
[in this window]
[in a new window]

  		Table 1. Effect of Glutathione Monoester on Level of Glutathione and Lipid Peroxidation in Various Brain Regions of Control and Experimental Rats.

 
GSH
There was a marked decline in reduced GSH levels in striatum (33%), cortex (29%), and hippocampus (26%) in aged rats as compared to young rats (Table 1). GME supplementation significantly increased the GSH level in aged rats (p <.001). There were no significant changes in GME-treated young rats.

ROS
Examination of differences between young and aged brains revealed higher levels of ROS as well as increased levels of oxidative stress markers in aged animals as compared to young animals. Table 2 depicts the levels of ROS in control and GME-treated young and aged rats. Discrete brain regions of aged rats showed a significant increase (p <.001) in ROS levels. Supplementation of GME appeared to markedly reduce the ROS levels in brain regions of aged rats when compared to aged control rats. Administration of GME did not show any significant change in the levels of ROS in young rats.


View this table:
[in this window]
[in a new window]

  		Table 2. Effect of Glutathione Monoester on Levels of Oxidant Production and Protein Carbonyl in Brain Regions of Control and Experimental Rats.

 
Superoxide Anion
Superoxide anion radical (O2•–) can be formed by one-electron reduction of molecular oxygen or by one-electron oxidation of hydrogen peroxide. Table 3 shows the levels of superoxide anion in brain regions of control and experimental rats. There was a significant increase (p <.001) in superoxide anion levels in brain regions of aged rats when compared to those in young rats. Administration of GME notably reduced the superoxide anion levels in aged rats. There were no significant alterations in GME-administered young rats in all the three brain regions.


View this table:
[in this window]
[in a new window]

  		Table 3. Effect of Glutathione Monoester on Levels of Superoxide Anion in Brain Regions of Control and Experimental Rats.

 
PCO Content
Protein oxidative damage was evident in different regions of the brain of young and aged rats, and an age-linked increase in PCO content was observed (Table 2). There were notable differences in the degree of increase among the brain regions of aged rats; the greatest increase was evident for cortex (63%), then hippocampus (61%), followed by striatum (57%). Simultaneous GME administration proved to be more effective (p <.001) in suppressing elevated PCO levels in aged rats when compared to aged control rats. GME administration did not bring any marked change in the levels of PCO in young rats.

PSH
Table 4 represents the levels of total thiol, non-PSH, and protein-bound thiol in brain regions of control and experimental rats. The levels of total thiol were found to be significantly decreased in cortex, striatum, and hippocampus of aged rats (35%, 38%, and 34%, respectively) when compared to young rats. A notable (43%, 46%, and 45%, respectively) increase in total thiol levels was observed in GME-administered aged rats. Similarly, the levels of non-PSH were also found to be significantly decreased (39%, 42%, and 37%, respectively) in the cortex, striatum, and hippocampus regions of aged rats. Administration of GME for 20 days significantly (47%, 35%, and 32%, respectively) enhanced the non-PSH levels in aged rats when compared to aged control rats. There was a significant (32%, 40%, and 34%, respectively) decrease in the levels of protein-bound thiol in the cortex, striatum, and the hippocampus of aged rats when compared to young rats. Aged rats supplemented with GME showed a marked elevation (40%, 47%, and 42%, respectively) in the level of protein-bound thiol when compared with control aged rats. Supplementation of GME to young rats did not bring about any marked alterations in thiol status.


View this table:
[in this window]
[in a new window]

  		Table 4. Effect of Glutathione Monoester on Level of Thiols in Brain Regions of Control and Experimental Rats.

 
DNA Protein Cross-Links
Table 5 demonstrates the levels of DNA protein cross-links in brain regions of control and experimental rats before and after supplementation of GME. DNA protein complexes were increased by 1.7-fold in cortex, 1.8-fold in striatum, and 1.6-fold in hippocampus of aged rats when compared to young rats. Administration of GME for 20 days significantly reduced DNA protein complexes in aged rats by 1.6-fold in cortex, 1.7-fold in striatum, and 1.6-fold in hippocampus when compared to aged control rats.


View this table:
[in this window]
[in a new window]

  		Table 5. Effect of Glutathione Monoester on Levels of DNA-Protein Cross-Links and 8-Hydroxy-2'-Deoxyguanosine (8-OHdG) in Brain Regions of Control and Experimental Rats.

 
8-OHdG
Table 5 represents the level of 8-OHdG in nuclear DNA of brain regions in control and GME-treated young and aged rats. A significant increase of 8-OHdG levels was detected in all the three brain regions of aged rats when compared to young controls (p <.001). Administration of GME for 20 days to aged rats apparently reduced the levels of 8-OHdG in all the brain regions (decrease of 34% in cortex, 29% in striatum, and 32% in hippocampus). There were no significant alterations in GME-administered young rats in all the three brain regions.


   DISCUSSION
Top
 Abstract
 Methods
 Results
 Discussion
References
 
Neuronal tissue is particularly susceptible to oxidative damage due to high oxygen consumption coupled with modest antioxidant defense strategies and high concentrations of iron and polyunsaturated fatty acids, which renders the tissue susceptible to oxygen radicals (34). A decrease in the level of GSH with advancing age has been observed in the present study, which is consistent with earlier reports (35). Decline in GSH level may affect the redox state, thereby affecting the total GSH pool of the cell. GSH and GSH-related enzymes thus play a key role in protecting the cell against the effects of ROS (36). GSH protects cellular constituents from oxidative damage by reacting directly with oxidants or by acting as the substrate for GSH peroxidase to scavenge peroxides (37). GSH also promotes the antioxidant properties of vitamin C and vitamin E by maintaining these nutrients in a reduced state (38). After GME supplementation, aged rats showed a significant increase in GSH levels; this protective measure might be due to the ability of GME to improve GSH status.

In the present investigation, ROS production was markedly increased in cortex, striatum, and hippocampus of aged rats when compared to young rats, and our results are in agreement with those of Schreiber and colleagues (39). Though an aged brain as a whole would be susceptible to injury by free radicals, the hippocampus and striatum are more prone to oxidative damage due to a higher oxygen consumption rate in those regions (40) and the presence of nonheme iron, which is catalytically involved in the production of ROS (41). The difference in ROS burden in various brain regions is due to well known heterogeneity and metabolic compartmentalization of brain mitochondria (42). The protective effect of GME against an age-related increase in ROS production is connected with its ability to directly scavenge ROS. Evidence is growing that GSH plays an important role in the detoxification of ROS in brain (43), and it has been reported that GSH protects cells from ROS, electrophiles, and xenobiotics (44).

In the present study, superoxide anion levels were significantly increased in aged rats when compared to young rats. During oxidative stress in the neuronal cells, there occurs an increased intracellular calcium ion concentration (Ca2+) in the rat brain (45). This increased Ca2+ ion concentration can induce the irreversible conversion of xanthine dehydrogenase to xanthine oxidase, which in turn catalyzes the oxidation of xanthine to provide a source of O2•–. In addition, auto-oxidation of dopamine in brain could also serve as a source of superoxide anion (46). These above-mentioned mechanisms could be the chief possibilities for the production of O2•–. GME supplementation markedly reduced the Formula levels in aged rats, and this protective measure might be due to the superoxide-scavenging ability of GSH (22).

The present findings display an increase in the steady-state level of protein oxidative damage, as indicated by the increase in the concentration of PCO in aged rats. Protein oxidation, an exothermic event during which peptides react with free radicals, causes the modification of several amino acids, protein aggregation, and protein fragmentation. Carbonyl level is probably the most common method used for assessing oxidative modification of proteins (47). An age-associated decline in GSH level as observed in our present study could have led to protein denaturation and aggregation subsequent to PSH oxidation (48). GME administration decreased the levels of PCO in discrete brain regions of aged rats, suggesting its ability to provide most direct availability of GSH and thereby reduce the concentration of PCO. GME is cell permeable and is effectively transported into the cerebrospinal fluid; it therefore provides the most direct and convenient means available to increase intracellular GSH concentration (21,49,50).

In the present study, aged rats showed a significant increase in LPO as evidenced by the increase in MDA levels in discrete regions (cortex, striatum, and hippocampus) of brain. The observed differences in the levels of LPO products in various regions may be due to the differences in their iron content and also in their oxygen consumption rate, which influence the production of ROS. MDA could be further cross-linked with proteins to form PCO derivatives. The reduction in MDA levels on GME administration suggests the free radical scavenging ability of GME. Santoscoy and colleagues (51) suggested that GME therapy at a dosage of 12 mg/body kg weight significantly diminished the spinal cord LPO.

Thiol compounds are regarded as the natural reservoir of reductive capacity of a cell. The most important role played by thiols in vivo is their function as components of the intracellular and extracellular redox buffer. The present study demonstrated an age-dependent decline in total thiol, non-PSH, and protein-bound thiol concentration in the discrete brain regions. This finding indicates that the efficiency of S-thiolation as a mechanism of antioxidant defense decreases with age, which creates an increased risk of irreversible oxidation of –SH groups of proteins (52). The reduction in the thiol status suggests depleted antioxidant capacity of the brain, and thus a disturbance in the balance between different redox forms of thiols that in turn lead to impaired protection of protein –SH groups upon irreversible oxidation. GME supplementation significantly increased the thiol levels in aged rats because GSH is considered to be the most prevalent and important intracellular non-PSH/sulfhydryl compound in mammalian cells (53,54). Rajasekaran and colleagues (55) also reported the prevention of LPO and loss of thiol groups in buthionine sulfoximine (BSO) administered rats on GME therapy.

Oxidative damage to DNA is a major cause of the aging process. It causes depurination, depyrimidination, single strand breaks, double strand breaks, and apoptosis (56). These damages accumulate particularly in nonreplicating cells and lead to a decline in the production of messenger RNA (mRNA) with age and thereby the function of postmitotic cells (57). In our present study, the level of 8-OHdG was increased in cortex, striatum, and hippocampus of aged rats under investigation. The possible reason might be the inefficient endogenous antioxidant system, which is unable to completely prevent oxidative DNA damage even under physiological conditions (58). The improvement in the antioxidant status by GME in aged rats could play a central role in preventing the accumulation of 8-OHdG. Superoxide (O2•–), a by-product of aerobic metabolism, has been implicated in the production of oxidative DNA damage. An increase in superoxide production during aging as observed in our present study could result in increased DNA damage. It has been reported that antioxidant vitamins like vitamin E and C have protective effects against the accumulation of 8-OHdG in guinea pigs (59). GME, by its GSH-promoting activity, in turn promotes vitamin C and E, as these antioxidants are tightly linked (55). The restoration of normal levels of vitamins E and C after GME supplementation thus helps the brain in reducing accumulation of 8-OHdG by free radicals. In addition, GSH also plays a vital role in the synthesis of the deoxyribonucleotide precursors of DNA (60).

DNA protein complexes represent bulky molecular lesions that are hardly repaired, thereby interfering with the transcriptional functions and causing cell death whenever these lesions fall in an essential region (61). Our present observations exhibit that the level of DNA protein cross-links increased in discrete brain regions of aged rats. Cross-linking between DNA and histones and nonhistone proteins would inhibit DNA repair by the inhibition of poly(ADP-ribosyl)ation of histones and by the inactivation of the repair enzyme. Depletion of GSH during aging increases superoxide formation resulting in the production of hydroxy radicals, which in turn leads to DNA damage and neuronal apoptosis (62), and these hydroxyl radicals attack both sugar and base moieties, leading to sugar fragmentation, strand scission, and base adducts (63). Administration of GME to aged rats significantly decreased the DNA protein cross-link levels. It has been reported that GME provides direct intracellular availability of GSH (21), which can scavenge superoxide ions, thereby reducing the formation of hydroxy radicals and consequent DNA protein cross-links. Apart from this, GSH is particularly effective against the highly toxic hydroxy radicals for which there are no enzymatic defenses (64).

Brain GSH can be increased safely using different treatment strategies, and an increase of brain GSH will result in clinical benefits and neuroprotection in animal models or in human diseases (43). The present results, which represent the direct evidence of increased oxidative macromolecular damage in aging brain regions are of particular significance because analysis of macromolecular damage may represent a novel tool for the study of initial stages of neuronal degeneration. This study also divulges that administration of GME alleviates age-associated macromolecular damages.


   Footnotes
Top
 Abstract
 Methods
 Results
 Discussion
 References
 
Decision Editor: Huber R. Warner, PhD

Received December 15, 2006

Accepted April 16, 2007


   References
Top
 Abstract
 Methods
 Results
 Discussion
 References
 

  1. Berlett BS, Stadtman ER. Protein oxidation in aging, disease, and oxidative stress. J Biol Chem. 1997;272:20313-20316.[Free Full Text]
  2. Finkel T, Holbrook NJ. Oxidants, oxidative stress and the biology of ageing. Nature. 2000;408:239-247.[Medline]
  3. Pansarasa O, Castagna L, Colombi B, Vecchiet J, Felzani G, Marzatico F. Age and sex differences in skeletal muscle: role of reactive oxygen species. Free Radic Res. 2000;33:287-293.[Medline]
  4. Sohal RS, Agarwal S, Sohal BH. Oxidative stress and aging in the Mongolian gerbil (Meriones unguiculatus). Mech Ageing Dev. 1995;81:15-25.[Medline]
  5. Stadtman ER, Levine RL. Protein oxidation. Ann N Y Acad Sci. 2000;899:191-208.[Medline]
  6. Cai Q, Tian L, Wei H. Age-dependent increase of indigenous DNA adducts in rat brain is associated with a lipid peroxidation product. Exp Gerontol. 1996;31:373-385.[Medline]
  7. Stadtman ER, Oliver CN. Metal-catalyzed oxidation of proteins. Physiological consequences. J Biol Chem. 1991;266:2005-2008.[Free Full Text]
  8. Evans P, Lyras L, Halliwell B. Measurement of protein carbonyls in human brain tissue. Methods Enzymol. 1999;300:145-156.[Medline]
  9. Mecocci P, Fano G, Fulle S, et al. Age-dependent increases in oxidative damage to DNA, lipids, and proteins in human skeletal muscle. Free Radic Biol Med. 1999;26:303-308.[Medline]
  10. Stadtman ER, Berlett BS. Reactive oxygen-mediated protein oxidation in aging and disease. Chem Res Toxicol. 1997;10:485-494.[Medline]
  11. Uchida K, Stadtman ER. Covalent attachment of 4-hydroxynonenal to glyceraldehyde-3-phosphate dehydrogenase. A possible involvement of intra- and intermolecular cross-linking reaction. J Biol Chem. 1993;268:6388-6393.[Abstract/Free Full Text]
  12. Baynes JW. Role of oxidative stress in development of complications in diabetes. Diabetes. 1991;40:405-412.[Abstract]
  13. Dubey A, Forster MJ, Lal H, Sohal RS. Effect of age and caloric intake on protein oxidation in different brain regions and on behavioral functions of the mouse. Arch Biochem Biophys. 1996;333:189-197.[Medline]
  14. Takenaka Y, Miki M, Yasuda H, Mino M. The effect of alpha-tocopherol as an antioxidant on the oxidation of membrane protein thiols induced by free radicals generated in different sites. Arch Biochem Biophys. 1991;285:344-350.[Medline]
  15. Adelman R, Saul RL, Ames BN. Oxidative damage to DNA: relation to species metabolic rate and life span. Proc Natl Acad Sci U S A. 1988;85:2706-2708.[Abstract/Free Full Text]
  16. Dizdaroglu M. Chemical determination of free radical-induced damage to DNA. Free Radic Biol Med. 1991;10:225-242.[Medline]
  17. Wu LL, Chiou CC, Chang PY, Wu JT. Urinary 8-OHdG: a marker of oxidative stress to DNA and a risk factor for cancer, atherosclerosis and diabetics. Clin Chim Acta. 2004;339:1-9.[Medline]
  18. Margolis SA, Coxon B, Gajewski E, Dizdaroglu M. Structure of a hydroxyl radical induced cross-link of thymine and tyrosine. Biochemistry. 1988;27:6353-6359.[Medline]
  19. Costa M. Analysis of DNA-protein complexes induced by chemical carcinogens. J Cell Biochem. 1990;44:127-135.[Medline]
  20. Anderson ME, Powrie F, Puri RN, Meister A. Glutathione monoethyl ester: preparation, uptake by tissues, and conversion to glutathione. Arch Biochem Biophys. 1985;239:538-548.[Medline]
  21. Anderson ME, Meister A. Glutathione monoesters. Anal Biochem. 1989;183:16-20.[Medline]
  22. Coyle JT, Puttfarcken P. Oxidative stress, glutamate, and neurodegenerative disorders. Science. 1993;262:689-695.[Abstract/Free Full Text]
  23. Glowinski J, Iversen LL. Regional studies of catecholamines in rat brain. I. The disposition in [3H] NE, [3H] dopamine and [3H] DOPA in various regions of the brain. J Neurochem. 1966;13:655-669.[Medline]
  24. Ohkawa H, Ohishi N, Yagi K. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal Biochem. 1979;95:351-358.[Medline]
  25. Moron MS, Depierre JW, Mannervik B. Levels of glutathione, glutathione reductase and glutathione S-transferase activities in rat lung and liver. Biochim Biophys Acta. 1979;582:67-78.[Medline]
  26. Lebel CP, Ischiropoulos H, Bondy SC. Evaluation of the probe 2',7'-dichlorofluorescin as an indicator of reactive oxygen species formation and oxidative stress. Chem Res Toxicol. 1992;5:227-231.[Medline]
  27. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951;193:265-275.[Free Full Text]
  28. Babior BM, Kipnes RS, Curnutte JT. Biological defense mechanisms. The production by leukocytes of superoxide, a potential bactericidal agent. J Clin Invest. 1973;52:741-744.[Medline]
  29. Levine RL, Garland D, Oliver CN, et al. Determination of carbonyl content in oxidatively modified proteins. Methods Enzymol. 1990;186:464-478.[Medline]
  30. Sedlak J, Lindsay RH. Estimation of total, protein bound, and nonprotein sulfhydryl groups in tissue with Ellman's reagent. Anal Biochem. 1968;25:192-205.[Medline]
  31. Gross-Bellard M, Oudet P, Chambon P. Isolation of high molecular weight DNA from mammalian cells. Eur J Biochem. 1973;36:32-38.[Medline]
  32. Ito K, Inoue S, Yamamoto K, Kawanishi S. 8-Hydroxydeoxyguanosine formation at the 5' site of 5'-GG-3' sequences in double-stranded DNA by UV radiation with riboflavin. J Biol Chem. 1993;268:13221-13227.[Abstract/Free Full Text]
  33. Zhitkovich A, Costa M. A simple, sensitive assay to detect DNA protein crosslinks in intact cells and in vivo. Carcinogenesis. 1992;13:1485-1489.[Abstract/Free Full Text]
  34. O'Donnell E, Lynch MA. Dietary antioxidant supplementation reverses age-related neuronal changes. Neurobiol Ageing. 1998;19:461-467.
  35. Subathra M, Shila S, Devi MA, Panneerselvam C. Emerging role of Centella asiatica in improving age-related neurological antioxidant status. Exp Gerontol. 2005;40:707-715.[Medline]
  36. Nordberg J, Arner ES. Reactive oxygen species, antioxidants, and the mammalian thioredoxin system. Free Radic Biol Med. 2001;31:1287-1312.[Medline]
  37. Beutler E. Nutritional and metabolic aspects of glutathione. Annu Rev Nutr. 1989;9:287-302.[Medline]
  38. May JM, Qu ZC, Whitesell RR, Cobb CE. Ascorbate recycling in human erythrocytes: role of GSH in reducing dehydroascorbate. Free Radic Biol Med. 1996;20:543-551.[Medline]
  39. Schreiber SJ, Megow D, Raupach A, Victorov IV, Dirnagl U. Age-related changes of oxygen free radical production in the rat brain slice after hypoxia: on-line measurement using enhanced chemiluminescence. Brain Res. 1995;703:227-230.[Medline]
  40. Floyd RA, Carney JM. Age influence on oxidative events during brain ischemia/reperfusion. Arch Gerontol Geriatr. 1991;12:155-177.[Medline]
  41. Hill JM, Switzer RC. The regional distribution and cellular localization of iron in the rat brain. Neuroscience. 1984;11:595-603.[Medline]
  42. Villa RF, Gorini A, Zanada F, Benzi G. Action of L-acetylcarnitine on different cerebral mitochondrial populations from hippocampus. Arch Int Pharmacodyn Ther. 1986;279:195-211.[Medline]
  43. Dringen R. Metabolism and functions of glutathione in brain. Prog Neurobiol. 2000;62:649-671.[Medline]
  44. Sies H. Glutathione and its role in cellular functions. Free Radic Biol Med. 1999;27:916-921.[Medline]
  45. Annunziato L, Amoroso S, Pannaccione A, et al. Apoptosis induced in neuronal cells by oxidative stress: role played by caspases and intracellular calcium ions. Toxicol Lett. 2003;139:125-133.[Medline]
  46. Olanow CW. A radical hypothesis for neurodegeneration. Trends Neurosci. 1993;16:439-444.[Medline]
  47. Butterfield DA, Koppal T, Howard B, et al. Structural and functional changes in proteins induced by free radical-mediated oxidative stress and protective action of the antioxidants N-tert-butyl-alpha-phenylnitrone and vitamin E. Ann N Y Acad Sci. 1998;854:448-462.[Medline]
  48. Freeman ML, Huntley SA, Meredith MJ, Senisterra GA, Lepock J. Destabilization and denaturation of cellular protein by glutathione depletion. Cell Stress Chaperones. 1997;2:191-198.[Medline]
  49. Robinson MK, Ahn MS, Rounds JD, Cook JA, Jacobs DO, Wilmore DW. Parenteral glutathione monoester enhances tissue antioxidant stores. JPEN J Parenter Enteral Nutr. 1992;16:413-418.[Abstract/Free Full Text]
  50. Wernerman J, Hammarqvist F. Modulation of endogenous glutathione availability. Curr Opin Clin Nutr Metab Care. 1999;2:487-492.[Medline]
  51. Santoscoy C, Rios C, Franco-Bourland RE, et al. Lipid peroxidation by nitric oxide supplements after spinal cord injury: effect of antioxidants in rats. Neurosci Lett. 2002;330:94-98.[Medline]
  52. Malgorzata I, Grazyna C, Elzbieta L, Edward B, Lidia W. Plasma levels of total, free and protein bound thiols as well as sulfane sulfur in different age groups of rats. Acta biochimica Polonica. 2004;51:815-824.[Medline]
  53. Meister A. Glutathione metabolism and its selective modification. J Biol Chem. 1988;263:17205-17208.[Free Full Text]
  54. Wullner U, Loschmann PA, Schulz JB, et al. Glutathione depletion potentiates MPTP and MPP+ toxicity in nigral dopaminergic neurones. Neuroreport. 1996;7:921-923.[Medline]
  55. Rajasekaran NS, Devaraj H, Devaraj SN. The effect of glutathione monoester (GME) on glutathione (GSH) depleted rat liver. J Nutr Biochem. 2002;13:302-306.[Medline]
  56. Rao KS. Non-apoptotic DNA fragmentation: a molecular pointer of ageing. Curr Sci. 1998;74:894-901.
  57. Holmes GE, Bernstein C, Bernstein H. Oxidative and other DNA damages as the basis of ageing: a review. Mutat Res. 1992;275:305-315.[Medline]
  58. Halliwell B. Reactive oxygen species and the central nervous system. J Neurochem. 1992;59:1609-1623.[Medline]
  59. Cadenas S, Barja G, Poulsen HE, Loft S. Oxidative DNA damage estimated by oxo8dG in the liver of guinea-pigs supplemented with graded dietary doses of ascorbic acid and alpha-tocopherol. Carcinogenesis. 1997;18:2373-2377.[Abstract/Free Full Text]
  60. Meister A, Anderson ME. Glutathione. Annu Rev Biochem. 1983;52:711-760.[Medline]
  61. Oleinick NL, Chiu SM, Ramakrishnan N, Xue LY. The formation, identification, and significance of DNA-protein cross-links in mammalian cells. Br J Cancer Suppl. 1987;8:135-140.[Medline]
  62. Ratan RR, Murphy TH, Baraban JM. Oxidative stress induces apoptosis in embryonic cortical neurons. J Neurochem. 1994;62:376-379.[Medline]
  63. Carlsson J, Granberg GP, Nyberg GK, Edlund MB. Bactericidal effect of cysteine exposed to atmospheric oxygen. Appl Environ Microbiol. 1979;37:383-390.[Abstract/Free Full Text]
  64. Bains JS, Shaw CA. Neurodegenerative disorders in humans: the role of glutathione in oxidative stress-mediated neuronal death. Brain Res Rev. 1997;25:335-358.[Medline]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Services
Right arrow Download to citation manager
PubMed
Right arrow PubMed Citation

HOME ARCHIVE SEARCH TABLE OF CONTENTS