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Gender difference in glutathione metabolism during aging in mice
Experimental Gerontology 38 (2003) 507–517 www.elsevier.com/locate/expgero
Gender difference in glutathione metabolism during aging in mice Hong Wanga, Honglei Liub, Rui-Ming Liub,* a
Department of Immunology, School of Medicine, University of Alabama at Birmingham (UAB) 1665 University Blvd, Birmingham, AL 35294-0022 USA b Department of Environmental Health Sciences, School of Public Health, University of Alabama at Birmingham (UAB) 1665 University Blvd, Birmingham, AL 35294-0022 USA Received 23 September 2002; received in revised form 20 January 2003; accepted 13 February 2003
Abstract Oxidative damage of the macromolecules increases with age and has been suggested to contribute importantly to the aging process and the pathogenesis of many age-related diseases. However, what causes such an increase in the oxidative damage of the macromolecules and whether male and female have the same susceptibility are not clear. In this study, we demonstrated for the first time that although the concentrations of GSH were similar between young male and female mice in most tissues examined and GSH content declined with age in both genders, male mice seemed to experience more dramatic age-associated change in GSH content than did female mice in many tissues. The age-related decline in the GSH content in both male and female mice was also associated with a decrease in the amounts of glutamate cysteine ligase (GCL) mRNAs and proteins as we have reported previously in male rats, further suggesting an important role of GCL in maintaining GSH homeostasis during the aging process. The results from this study may reveal an important basis underlying the genderassociated differences in the longevity and the susceptibility to certain age-related diseases, and also further suggest that the decreased synthesis, which is mainly due to the down regulation of GCL gene expression, may be responsible for the age-associated decline in GSH content. q 2003 Elsevier Science Inc. All rights reserved. Keywords: Glutathione; Aging; Gender; Glutamate cysteine ligase; g-glutamylcysteine synthetase
1. Introduction The mechanism underlying aging, an inevitable biological process that affects most living organisms, is still an area of significant controversy as are the mechanisms of agerelated diseases such as cancer, neurodegenerative diseases, diabetes, cataract, and cardiovascular diseases. Reactive oxygen species (ROS), generated endogenously or exogenously, cause damages to DNA, RNA, lipids, and proteins. Accumulated evidence indicates that oxidative damage of these macromolecules increases with age and in various age-related diseases. Therefore, it has been suggested that oxidative stress contributes importantly to the aging process and the pathogeneses of age-associated diseases (Jenner, Abbreviations: GCL, glutamate cysteine ligase; GCLC, glutamate cysteine ligase catalytic subunit; GCLM, glutamate cysteine ligase modifier subunit; GSH, glutathione; GSSG, glutathione disulfide; BALF, bronchoalveolar lavage fluid; GST, glutathione-S-transferase. * Corresponding author. Tel.: þ 205-934-7028; fax: þ 205-975-6341. E-mail address: [email protected] (R.-M. Liu).
1996; Mezzetti et al., 1996; Multhaup et al., 1997; Stadtman and Berlett, 1997; Behl, 1999; Lu et al., 1999; Markesbery, 1999; Hamilton et al., 2001). Although intensive studies have been done, the mechanism underlying such increased oxidative damage of the macromolecules in aged animals, however, has not been completely elucidated. It has also been well documented that man has an average lifespan short than woman (Ortmeyer, 1979; Dhar, 2000; Dhar, 2001; Alexiou et al., 2002), and that some of agerelated diseases show gender preferences. The mechanisms underlying such gender-related differences in the lifespan and the susceptibility to certain diseases are largely unknown. As oxidative stress has been implicated in the aging process and the pathogeneses of many age-related diseases, it will be interesting to know whether there is any difference between male and female in terms of their abilities to maintain oxidant and antioxidant balance during the aging process. Glutathione (GSH), the most abundant intracellular nonprotein thiol, participates in many important biological
0531-5565/03/$ - see front matter q 2003 Elsevier Science Inc. All rights reserved. doi:10.1016/S0531-5565(03)00036-6
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processes such as the synthesis of cellular compounds, the cell cycle, and the regulation of the gene expression. The most important function of this tri-peptide, perhaps, is to detoxify oxidants and electrophiles by acting as a cofactor in glutathione peroxidase or glutathione-S-transferase catalyzed reactions. A large body of evidence has shown that increasing GSH concentration can protect cells against oxidative damage while decreasing GSH can promote such damages. Although many studies, including those from this laboratory, have shown that GSH content decreases with age in many tissues from different animal species (Hazelton and Lang, 1980; Ravindranath et al., 1989; De and Darad, 1991; Iantomasi et al., 1993; Favilli et al., 1994; Teramoto et al., 1994; Christon et al., 1995; Yang et al., 1995; Nakata et al., 1996; Sanz et al., 1997; Samiec et al., 1998; Liu and Choi, 2000; Sasaki et al., 2001; Liu 2002), some other studies indicated that there was no significant change or even increase in GSH content during the aging process (Stio, 1994 #4491; Hussain, 1995 #3135; Leeuwenburgh, 1994 #4378; Nakata et al., 1996). Whether such discrepancies between the results from different laboratories reflect species difference or other factors remain to be determined. Furthermore, as most of the studies to examine ageassociated change in GSH metabolism were conducted in one gender or another, it is not clear whether there is any difference between genders in terms of GSH metabolism during the aging process. A systematical comparison of ageassociated change in GSH content between male and female will certainly help us to better understand the mechanisms underlying the gender differences in the longevity and the susceptibility to certain diseases. There are several mechanisms by which cells maintain their intracellular GSH homeostasis, including GSH redox cycling, direct up take, and de novo synthesis. De novo GSH synthesis is a two-step process. The first step, the synthesis of g-glutamylcysteine catalyzed by glutamate cysteine lagase (GCL, EC 6.3.2.2), is the rate-limiting step in this process. Increased evidence suggests that GCL plays a critical role in maintaining GSH homeostasis not only for the resting cells but also for oxidant challenged cells (Shi et al., 1994a,b; Liu et al., 1996a,b; Choi et al., 1997; Choi et al., 2000; Liu et al., 2001). Whether this enzyme also plays an important role in maintaining GSH homeostasis during aging process needs to be studied further. In this study, we systematically compared the ageassociated changes in GSH content and GCL gene expression in 10 tissues/organs between male and female mice. Our results indicated that although there was no significant difference in the GSH content between young male and female mice in most tissues examined and GSH content decreased with age in both male and female mice, male mice seemed to be more vulnerable to such ageassociated decline than female mice did. The age-dependent decline in GSH content in both male and female mice was also associated with a down regulation of GCL gene expression, as we have previously shown in the rat tissues.
The results from this study may reveal an important basis underlying the gender-associated differences in the longevity and the susceptibility to certain age-related diseases, and also further suggest that the decreased synthesis, which is mainly due to the down regulation of GCL gene expression, may be responsible for the age-associated decline in GSH content.
2. Materials and methods 2.1. Animals Young (3-month), adult (12-month), and aged (24month) C57BL/6 male and female mice were purchased from Harlan Sprague Darley/National Institute of Aging and were acclimated for one week in our animal facilities prior to the initiation of our experiments. The animals were maintained on a 12 h light/dark cycle at 22 8C with free access to water and food. The mice were euthanatized with 100 mg/kg sodium pentobarbital and placed in a supine position. Trachea was cannulated, and bronchoalveolar lavage was performed using room temperature saline. After lavage, heart, lung, liver, kidney, spleen as well as brain were removed; cerebral cortex, cerebellum, and brain stem were dissected immediately. The samples for GSH measurement were washed twice in cold PBS and sonicated immediately in 10% perchloric acid (PCA)-2 mM EDTA solution. The remaining tissue samples were frozen in liquid nitrogen immediately for further analyses. All procedures involving animals were approved by the Institutional Animal Care and Use Committees at the University of Alabama at Birmingham. 2.2. Reagents TRIzol Reagent, an RNA isolation solution, was from Life Science Technologies (Grand Island, N.Y.). QuickHyb solution and salmon sperm DNA were from Stratagene (La Jolla, CA). Polyvinylidene fluoride membranes were purchased from Millipore Company. Enhanced chemiluminescence reagent and Hyperfilm ECL film were from Amersham. All high performance liquid chromatography solvents were Baker Analyzed HPLC-grade reagents from VWR scientific (San Diego, CA). All chemicals used were at least analytical grade. 2.3. Measurement of GSH, GSSG, and cysteine content The concentrations of tissue GSH, GSSG, and cysteine were determined by HPLC as described previously (Liu et al., 1998). Briefly, the tissues were cut and washed with cold PBS twice, then sonicated in 10% PCA containing 2 mM EDTA and 16.67 mM of g-glutamyl-glutamic acid, the latter of which served as an internal standard. After centrifugation, 10 mM iodoacetic acid in 0.2 mM cresol
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purple was added to the supernatant, and the pH was adjusted to 8 – 9. The mixture was incubated in the dark at room temperature for 15 min. Then 1% dinitrobenzene (DNB) was added and the reaction mixture, incubated at 4 8C overnight. The amounts of GSH, GSSG, and cysteine were measured by HPLC with standards being run under the same conditions. GSH, GSSG, and cysteine contents were calculated based on the internal standard and standard curves. The total GSH content was calculated as 1GSH þ 2 GSSG.
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2.6. Statistics Data were expressed as mean ^ SEM and evaluated by one-way ANOVA. Statistical significance was determined by Fisher LSD test. P , 0.05 was considered significant.
3. Results 3.1. GSH content was decreased more in male mouse tissues than in female mouse tissues during the aging process
2.4. Western blotting analysis of GCL protein The amounts of GCL catalytic (GCLC) and modifier (GCLM) subunit proteins were determined by Western analysis as described before (Liu et al., 1998). Briefly, the tissues were cut and washed in cold PBS buffer and homogenized in 0.25 M sucrose buffer containing 2 mg/ml leupeptin and aprotinin, and 50 mg/ml PMSF. The homogenates were then centrifuged at 3,000 £ g for 10 min at 4 8C and the supernatant was centrifuged at 10,000 £ g for 20 min and then, at 105,000 £ g for 30 min at 4 8C. After ultracentrifugation, the supernatant was used for determining the amounts of GCL proteins by Western blotting using specific anti GCLC or anti GCLM subunit antibodies. The positions of GCLC and GCLM bands were identified according to molecular weight markers and rat kidney, a positive control, which contains high concentrations of GCL proteins. The X-ray films were scanned using ScanJet 4C/T deskscan and the signal was semi-quantitated, using Sigmascan program. 2.5. Northern hybridization analysis of GCL mRNAs A GCLC cDNA (804 bp) and a GCLM cDNA (1001 bp), which were reversely transcribed from rat kidney RNA and from L2 cell RNA, respectively, and amplified by PCR (Shi et al., 1994a,b; Tian et al., 1997), were labelled with [a-32P] dCTP using a random-primer DNA labelling kit from Life Technologies (Gaithersburg, MD). The GCL mRNA content was determined by Northern hybridization analysis as described previously (Liu et al., 1998). Briefly, total RNA was extracted with TRIzol Reagent (Life Technologies, Grand Island, N.Y.) according to the protocol provided by the manufacturer. Twenty mg RNA from each sample was resolved on a 1.2% agarose gel and transferred onto a Nylon membrane. Hybridization was carried out with GCLM cDNA, GCLC cDNA, and 18S cDNA probes subsequently at 60 8C using Quikhyb (Stratagene) solution. After hybridization, the membranes were washed twice with 2 £ sodium chloride-sodium citrate (SSC) buffer/0.1% sodium dodecyl sulfate (SDS) for 15 min at room temperature, then twice with 0.1 £ SSC/0.1% SDS for 15 min each at 50 8C. The membranes were scanned and radioactivity was quantitated with an InstantImager (Packard Instrument Company, Meriden, CT).
In order to see whether there was any gender difference in GSH metabolism during the aging process, we compared age-associated changes in GSH content in various tissues between male and female mice. The results showed that there was no significant difference in GSH content between young male and female mice in the most tissues examined except the lung, where a significantly higher GSH content was found in female versus male (5.87 ^ 0.74 in male versus 9.97 ^ 0.92 in female). The mechanism underlying such a difference in GSH content in the lung tissue between young male and female mice is not clear. Most interestingly, the results also indicated that although GSH content decreased with increasing age in both male and female mice, the male mice experienced more dramatic decrease than female mice in many tissues. In male mice, 7 out of 10 tissues, cells, or body fluids examined showed an age-associated decline in GSH content while in female mice, only 4 showed the same phenomenon (Figs. 1 and 2, and Table 1). Specifically, GSH content decreased with age in the BALF, lung, and cerebellum in male mice but not in female mice. Of 4 tissues/organs (liver, spleen lymphocytes, brainstem, and cerebral cortex), where GSH content was decreased with age in both male and female mice, 2 (spleen lymphocytes and brainstem) showed a bigger decline in male than in female mice. The only organ that experienced a bigger age-associated decline in GSH content in female than in male mice was the liver. GSH content decreased by 21% and 34% in the liver of 12- and 24-month old male mice, respectively, but by 32% and 55% in the liver of 12- and 24-month old female mice, respectively, as compared with 3-month old mice (Table 1). No age-associated change was found in GSH content in red blood cells (RBC), kidney, or heart from either male or female mice. There was also no significant age or gender difference in either cysteine or GSSG content in any of the tissues or cells that were examined (data not shown). 3.2. The amounts of GCL proteins decreased with age in male and female mouse tissues that showed an agedependent decline in GSH content GCL is the rate-limiting enzyme in de novo GSH synthesis and plays an important role in maintaining GSH homeostasis. In order to explore the mechanism underlying
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Fig. 1. Age-associated decline in GSH content in male mouse tissues. GSH content was measured using a HPLC assay as described in Section 2. Values represent means ^ SEM of 5–10 mice. (a) Significantly different from 3-month old mice (p , 0.05); (b) Significantly different from 12 month-old mice (p , 0.05).
Fig. 2. Age-associated decline in GSH content in female mouse tissues. GSH content was measured using a HPLC assay as described in Section 2. Values represent means ^ SEM of 5–10 mice. (a) Significantly different from 3-month old mice (p , 0.05); (b) Significantly different from 12 month-old mice (p , 0.05).
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Table 1 Comparison of age-associated change in GSH content between male and female mice Tissues
Liver BALF SL Lung Cortex Cerebellum Brainstem
a b c d
gender
M F M F M F M F M F M F M F
GSH (%)a 3 mo.
12 mo.
24 mo.
100.0 ^ 6.7 (10) 100.0 ^ 4.6 (8) 100.0 ^ 15.1 (6) 100.0 ^ 33.9 (8) 100.0 ^ 5.5 (10) 100.0 ^ 5.5 (8) 100.0 ^ 9.6 (6) 100.0 ^ 9.2 (8) 100.0 ^ 4.9 (5) 100.0 ^ 1.9 (8) 100.0 ^ 5.8 (5) 100.0 ^ 2.8 (8) 100.0 ^ 5.8 (5) 100.0 ^ 3.9 (8)
78.7 ^ 11.3 (10) 68.0 ^ 5.6 (8)b 47.2 ^ 13.5 (9)b 119.1 ^ 35.2 (8) 79.0 ^ 20.5 (7) 91.7 ^ 4.5 (8) 119.4 ^ 9.1 (5) 126.0 ^ 9.3 (8) 86.2 ^ 1.7 (5)b 97.8 ^ 4.0 (8) 73.8 ^ 7.1 (5)b 95.6 ^ 3.8 (8)d 88.1 ^ 3.7 (5)b 87.6 ^ 2.5 (8)b
66.4 ^ 8.0 (10)b 44.4 ^ 2.6 (8)b,c,d 52.2 ^ 7.9 (5)b 54.5 ^ 11.8 (8) 63.7 ^ 4.5 (8)b 79.9 ^ 5.8 (8)b,d 73.7 ^ 4.4 (6)b 114.8 ^ 5.0 (8)d 79.3 ^ 2.2 (5)b 84.5 ^ 2.3 (8)b,c 75.5 ^ 1.4 (5)b 89.9 ^ 3.0 (8)d 70.1 ^ 9.6 (5)b 88.5 ^ 2.7 (8)b,d
BALF: bronchoalveolar lavage fluid; SL, spleenic lymphocytes. GSH was reported as % of change from the 3 month old group, which was arbitrarily set as 100%. Significantly different from 3 month old group. Significantly different from 12 month old group. Significantly different from male mice with the same age.
the age-associated decline in GSH content in mouse tissues, we further compared the amounts of the GCL catalytic and modifier subunit proteins between three age groups in both male and female mice. Western analyses were performed with the samples from liver, spleenic lymphocytes, and brainstem tissues as GSH content was decreased with aging in these tissues in both male and female mice. The results
showed that there was an age-associated decrease in the amounts of GCL catalytic and/or modifier subunit proteins in all three tissues in both male and female mice (Figs. 3 and 4). The patterns of changes in GCL proteins with age matched the patterns of the changes in the GSH content, e.g. that GCL proteins decreased more in the spleen lymphocytes and the brainstem but less in the liver of male mice
Fig. 3. Age-associated decline in GCL protein content in male mouse tissues. The amounts of GCLC and GCLM proteins were determined by Western analysis as described in Section 2. The X-ray films were scanned with a Desk-scanner and semi-quantitated using Sigmascan. The results were expressed as percentages of the 3-month group. Values represent means ^ SEM of 4 –5 samples. (a) Significantly different from 3-month old mice (p , 0.05); (b) Significantly different from 12 month-old mice (p , 0.05).
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Fig. 4. Age-associated decline in GCL protein content in female mouse tissues. The amounts of GCLC and GCLM proteins were determined by Western analysis as described in Section 2. The X-ray films were scanned with a Desk-scanner and semi-quantitated using Sigmascan. The results were expressed as percentages of the 3-month group. Values represent means ^ SEM of 4–8 samples. (a) Significantly different from 3-month old mice (p , 0.05); (b) Significantly different from 12 month-old mice (p , 0.05).
during the aging process as compared to that in female mice (Table 2). 3.3. Decreased amounts of GCL proteins were associated with a decline in the GCL mRNA content GCL mRNA content in the liver and brainstem of both male and female mice was further analyzed to see whether the age-associated decrease in GCL protein content was due to a decreased GCL mRNA expression. The results showed
that, as compared to the 3 month-old groups, only GCLC mRNA was decreased (by 39%) in the 24 month-old male mouse liver while both GCLC and GCLM mRNAs were decreased (by 37% and 20%, respectively) in the 24 monthold female mouse liver. In the brainstem, the amounts of both GCLC and GCLM mRNAs were decreased (by 38% and 66%, respectively) in the 24 month-old male mice while only the GCLC was decreased (by 16%) in the 24 month-old female mice, as compared to the 3 month-old mouse groups (Fig. 5(a) and (b)). The results indicated that decreased gene
Table 2 Comparison of age-associated changes in GCL protein content between male and female mice Tissues
Liver
Gene
GCLC GCLM
SL
GCLC GCLM
Brainstem
GCLC GCLM
a b c d
Gender
M F M F M F M F M F M F
GCL proteins (%)a 3 mo.
12 mo.
24 mo.
100.0 ^ 6.9 (4) 100.0 ^ 10.1 (8) 100.0 ^ 19.0 (4) 100.0 ^ 10.0 (8) 100.0 ^ 4.7 (4) 100.0 ^ 4.6 (4) 100.0 ^ 19.2 (4) 100.0 ^ 12.7 (4) 100.0 ^ 12.0 (5) 100.0 ^ 21.2 (4) 100.0 ^ 24.0 (5) 100.0 ^ 14.3 (4)
59.2 ^ 3.8 (4)b 69.8 ^ 15.4 (8) 196.0 ^ 30.5 (4) 57.2 ^ 11.9 (8)c 67.1 ^ 4.3 (4)b 83.6 ^ 9.3 (4) 94.9 ^ 11.6 (4) 92.2 ^ 12.8 (4) 59.1 ^ 6.2 (4)b 96.2 ^ 14.6 (4)c 83.5 ^ 20.7 (4) 116.8 ^ 19.6 (4)
71.0 ^ 4.0 (4)b 54.4 ^ 2.4 (8)b,c 118 ^ 39.0 (4) 36.6 ^ 2.8 (8)b,d,c 38.8 ^ 1.4 (4)b,d 72.6 ^ 5.5 (4)b,c 61.6 ^ 15.8 (4) 75.4 ^ 7.4 (4) 49.2 ^ 7.4 (4)b 85.9 ^ 15.7 (4)c 54.9 ^ 10.9 (4)b 107.2 ^ 21.0 (4)c
SL, Spleenic lymphocytes GCL protein content was reported as % of change from the 3 month old group, which was arbitrarily set as 100%. Significantly different from 3 month old group. Significantly different from male mice with the same age. Significantly different from 12 month old group.
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Fig. 5. GCL mRNA content was decreased with age in the liver and brainstem of both male and female mice. The amounts of both GCLC and GCLM mRNAs were determined by Northern analyses as described in Section 2. 18S was used to normalize GCLC as well as GCLM signals. The results were expressed as percentages of the 3-month group after normalized by 18S. (A) age-associated changes in GCL mRNAs in male mouse liver and brainstem. (B) Age-associated changes in GCL mRNAs in female mouse liver and brainstem. The values represent means ^ SEM of 3– 5 samples. (a) significantly different from 3 month mice (p , 0.05); (b) significantly different from 12 month mice (p , 0.05).
expression might be responsible for the age-associated decline in the GCL protein content.
4. Discussion Although numerous studies have shown that GSH content decreases with age in various animal species, whether male and female animals experience the same change during the aging process is not clear. In this study, we systematically compared age-associated changes in GSH metabolism between male and female mice. Our results showed for the first time that, although GSH content
decreased with age in both male and female mice, male mice seemed to experience more dramatic age-associated decline than did female mice in most tissues examined. Of 10 tissues or cells or body fluid examined, 7 showed an agedependent decrease in GSH content in male mice while only 4 showed the same phenomenon in female mice. The extent of age-associated decrease in GSH content was also greater in 2 of the 4 tissues that showed an age-associated decline in GSH content in both genders. The only exception to this trend was the liver. GSH content in the liver decreased more in female mice than in male mice during the aging process. The mechanism underlying such a difference in the ageassociated change in GSH content between male and female
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mice is unclear. One possible explanation may be that the amount of some hormones, which are involved in the regulation of GSH homeostasis and to which different tissues/organs have different sensitivities, decreases with different rates in male and female. More work is needed to answer this question. Although it is a well-known phenomenon that GSH content decreases with age, the underlying mechanism is also not completely understood. GSH depletion may occur when cells are exposed to massive oxidants, when GSSG reductase activity decreases, which limits the redox cycle of GSSG, and/or when the rate of GSH synthesis is decreased. A long-term low-level oxidative stress, which is most likely the case during the aging process, however, usually increases rather than decreases intracellular GSH content as it can induce the expression of various enzymes involved in GSH synthesis (Liu et al., 1996a,b; Liu et al., 1998; Moellering et al., 1998; Moellering et al., 1999; Liu et al., 2001). An age-associated decrease in GCL activity accompanied by a decline in GSH content has been reported in different tissues of various animal species (Sethna et al., 1982; Rathbun, 1984; Rathbun, 1986; Iantomasi et al., 1993; Favilli et al., 1994; Stio et al., 1994). In the previous studies, we have shown that GSH content decreased with age in several organs/tissues in male Fisher 344 rats, which was due, at least in part, to a down regulation of GCL gene expression (Liu and Choi, 2000; Liu, 2002). In this study, we showed that the age-associated decline in GSH content in both male and female mice was also associated with a decreased GCL gene expression. These data further indicated an important role of GCL in maintaining GSH homeostasis for different animal species during the aging process. Although we did not measure GSSG reductase activity in this study, we did not observe any age-associated increase in GSSG content, an indicator of massive oxidant production or a decrease in GSSG reductase activity, in any of the tissues examined. These results suggest that the ageassociated decline in GSH content in mouse tissues does not result from either an increased oxidant production or a decreased GSSG reductase activity. This conclusion is supported by the results form several other studies (Hazelton and Lang, 1980; Chen et al., 1989; Richie, 1992; Yang et al., 1995; Nakata et al., 1996; Liu, 2002). Another important observation from this study was that there was no significant difference in GSH content between young male and female mice in most tissues, cells, and body fluid examined. Although the regulation of GSH homeostasis has been well studied at the cellular level, the factors regulating GSH homeostasis in whole organisms have not been completely revealed. Estrogen is believed to be one of the hormones that may regulate GSH homeostasis under physiological situation as estrogen replacement therapy has been reported to increase total thiol level in the plasma of postmenopausal women (Konukoglu et al., 2000) and estradiol treatment increased GSH content in canine myocardial tissue (Kim et al., 1998). As female has much
higher plasma estrogen level than male, one would expect that the GSH content is higher in female than in male, at least in the tissues that are estrogen sensitive. Interestingly, our results showed that there was no significant difference in the GSH content between young male and female mice in most tissues examined except the lung, in which a significant higher level of GSH was found in female mice vs. male mice. Although no such systematic comparison has ever been previously reported, a few sporadic studies using different animal species have shown similar results. Igarashi reported that GSH content in the liver of young rats was not significantly different between the two genders (Igarashi et al., 1983). Ferris (Ferris et al., 1995) and Kume-Kick (Kume-Kick et al., 1996) demonstrated that there was no difference in the GSH content in the brains of young male and female rats. Furthermore, Erden-Inal showed that the GSH content in the blood from men and women was same (Erden-Inal et al., 2002). These results, including ours, suggest that sex hormones may not be involved in the regulation of GSH homeostasis under physiological conditions, at least in these tissues/organs. We would like to mention that although most studies indicated that GSH content decreased with age, a few reports showed a different trend (Stio, 1994 #4491; Hussain, 1995 #3135; Leeuwenburgh, 1994 #4378; Nakata et al., 1996). The reason for such discrepancies between the results from different laboratories may be due to the differences in the experimental conditions including animal species and ages, etc. For example, Hazelton reported that GSH content decreased with age in the kidney and heart of male C57BL mice (Hazelton and Lang, 1980) while we did not observe any age-associated change in GSH content in these two organs form the same animal species. The difference may result from the age of the animals, which was used to represent an old group (31 month-old in their experiment verse 24 month-old in our experiment). On the other hand, Hussain reported (Hussain et al., 1995) that GSH content was significantly increased in the cerebellum and brainstem of old male mice (C57BL) as compared with the young male mice while we observed an opposite phenomenon in this same animal species. The difference between our experiments and theirs was that the animals were fasting for 16 h before sacrificed in our experiments (in order to minimize the effect of food and circadian rhythm of hormones on the GSH content) but not in their experiments. The biological significance of such difference between male and female in GSH metabolism during the aging process needs to be further explored. Interestingly, it has been well documented that man has an average lifespan shorter than woman (Ortmeyer, 1979; Dhar, 2000; Dhar, 2001; Alexiou et al., 2002). Although the mechanism trigging the aging process has not been completely elucidated yet, oxidative damage of the macromolecules has been proposed to be one of the important contributors. Therefore, a lower capacity to maintain GSH homeostasis in male animals, probably in man too, as compared with
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female animals during the aging process may contribute to their shorter lifespan. Consistent with this notion, Honda and Richie reported that increasing GSH level extended not only the life of cultured human diploid fibroblasts (Honda and Matsuo, 1988) but also the life span of mosquito (Richie et al., 1987). Dietary calorie restriction, which has been shown to be able to extend the life span of various animal species, also increases GSH content (Richie et al., 1994; Taylor et al., 1995; Armeni et al., 1998). Nevertheless, whether GSH content also decreases more in man than in women during the aging process and if it does, whether a lower capacity of maintaining GSH homeostasis in man as compared with woman contributes to their shorter lifespan are very important questions to be answered. It is also well documented that the susceptibility to certain age-related diseases such as cancers, cardiovascular diseases, and neurodegenerative diseases differs between two genders (Berko, 1989; Jarvik et al., 1995; Corrao et al., 1997; Lapane et al., 2001; Bella et al., 2002; Chen et al., 2002; Radzikowska et al., 2002). Although the mechanism underlying such a gender difference is unknown in the most cases, the difference in GSH metabolism has been suggested to play a role in some situations. For example, lung cancer is a leading cause of death in men in the United States. The incidence of the lung cancer increases with age and is higher in man than in woman (Alexiou et al., 2002; Koyi et al., 2002). Although the environmental and behavioural factors contribute importantly to such a gender difference, the difference in the genetic background related to GSH metabolism has also been shown to be responsible. Polymorphism of glutathione-S-transferase (GST), an enzyme that catalyzes GSH conjugate formation, has been shown to be associated with a high risk of the lung cancer in different populations (Hou et al., 2001; Matsuzoe et al., 2001; Reszka and Wasowicz, 2001; Miller et al., 2002). Furthermore, it has also been reported recently that polymorphisms of GCL, the rate-limiting enzyme in de novo GSH synthesis, may determine lung cancer survival (Yang et al., 2002). In this study, we found that GSH content was significantly lower in the lung of young male mice as compared with young female mice, and decreased with age in male mice but not in female mice. Whether such a difference in GSH metabolism in the lung between male and female also exists in human and if it does, whether such a gender difference contributes to the different susceptibility of man and women to lung cancer remain to be explored further. Summary. The present study demonstrated for the first time that although GSH content decreased with age in both male and female mice, male mice experienced more dramatic age-associated change in GSH content than did female mice. There was no significant difference in the GSH content between young male and female mice in most tissues examined except the lung, in which a significantly
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lower level was found in male compared with female mice. Age-associated decline in GSH content in both male and female mice was also associated with a down regulation of GCL gene expression as we have reported previously in rats, further suggesting an important role of GCL in maintaining GSH homeostasis during the aging process. The results from this study may reveal an important basis for the genderassociated differences in the longevity and the susceptibility to certain age-related diseases. Acknowledgements This project was funded by National Institute Health grant 1 R01 ES11831-01 and American lung Association research grant RG-057-N. The authors would like to acknowledge Dr Henry Jay Forman for his advice, Dr Jinah Choi, Dr Dale Dickinson and Dr Karen Iles for their critical review of the manuscript, as well as Xu Jia for her technical assistance.
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Liu, R.M., Gao, L., et al., 1998. gamma-glutamylcysteine synthetase, mRNA stabilization and independent subunit transcription by 4hydroxy-2-nonenal. Am. J. Physiol. 275 (5.1), L861–L869. Lu, C.Y., Lee, H.C., et al., 1999. Oxidative damage elicited by imbalance of free radical scavenging enzymes is associated with large-scale mtDNA deletions in aging human skin. Mutat. Res. 423 (1/2), 11–21. Markesbery, W.R., 1999. The role of oxidative stress in Alzheimer disease. Arch. Neurol. 56 (12), 1449–1452. Matsuzoe, D., Hideshima, T., et al., 2001. Glutathione S-transferase mu1 null genotype is associated with K-ras gene mutation in lung adenocarcinoma among smokers. Carcinogenesis 22 (8), 1327–1330. Mezzetti, A., Lapenna, D., et al., 1996. Systemic oxidative stress and its relationship with age and illness. Associazione Medica Sabin (see comments). J. Am. Geriatr. Soc. 44 (7), 823–827. Miller, D.P., Liu, G., et al., 2002. Combinations of the variant genotypes of GSTP1, GSTM1, and p53 are associated with an increased lung cancer risk. Cancer Res. 62 (10), 2819–2823. Moellering, D., McAndrew, J., et al., 1998. Nitric oxide-dependent induction of glutathione synthesis through increased expression of gamma-glutamylcysteine synthetase. Arch. Biochem. Biophys. 358 (1), 74 –82. Moellering, D., McAndrew, J., et al., 1999. The induction of GSH synthesis by nanomolar concentrations of NO in endothelial cells, a role for gamma-glutamylcysteine synthetase and gamma-glutamyl transpeptidase. FEBS Lett. 448 (2/3), 292–296. Multhaup, G., Ruppert, T., et al., 1997. Reactive oxygen species and Alzheimer’s disease. Biochem. Pharmacol. 54 (5), 533– 539. Nakata, K., Kawase, M., et al., 1996. Effects of age on levels of cysteine, glutathione and related enzyme activities in livers of mice and rats and an attempt to replenish hepatic glutathione level of mouse with cysteine derivatives. Mech. Aging Dev. 90 (3), 195–207. Ortmeyer, L.E., 1979. Female’s natural advantage? Or, the unhealthy environment of males? The status of sex mortality differentials. Women Health 4 (2), 121–133. Radzikowska, E., Glaz, P., et al., 2002. Lung cancer in women, age, smoking, histology, performance status, stage, initial treatment and survival. Population-based study of 20 561 cases. Ann. Oncol. 13 (7), 1087–1093. Rathbun, W.B., 1984. Lenticular glutathione synthesis, rate-limiting factors in its regulation and decline. Curr. Eye Res. 3 (1), 101–108. Rathbun, W.B., 1986. Activity of glutathione synthesis enzymes in the rhesus monkey lens related to age, a model for the human lens. Curr. Eye Res. 5 (2), 161– 166. Ravindranath, V., Shivakumar, B.R., et al., 1989. Low glutathione levels in brain regions of aged rats. Neurosci. Lett. 101 (2), 187 –190. Reszka, E., Wasowicz, W., 2001. Significance of genetic polymorphisms in glutathione S-transferase multigene family and lung cancer risk. Int. J. Occup. Med. Environ. Health 14 (2), 99–113. Richie, J.P. Jr., 1992. The role of glutathione in aging and cancer. Exp. Gerontol. 27 (5/6), 615–626. Richie, J.P. Jr., Mills, B.J., et al., 1987. Correction of a glutathione deficiency in the aging mosquito increases its longevity. Proc. Soc. Exp. Biol. Med. 184 (1), 113 –117. Richie, J.P. Jr., Leutzinger, Y., et al., 1994. Methionine restriction increases blood glutathione and longevity in F344 rats. Faseb J. 8 (15), 1302–1307. Samiec, P.S., Drews-Botsch, C., et al., 1998. Glutathione in human plasma, decline in association with aging, age- related macular degeneration, and diabetes. Free Radical Biol. Med. 24 (5), 699–704. Sanz, N., Diez-Fernandez, C., et al., 1997. Age-dependent modifications in rat hepatocyte antioxidant defense systems. J. Hepatol. 27 (3), 525 –534. Sasaki, T., Senda, M., et al., 2001. Age-related changes of glutathione content, glucose transport and metabolism, and mitochondrial electron transfer function in mouse brain. Nucl. Med. Biol. 28 (1), 25–31.
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Gender difference in glutathione metabolism during aging in mice Hong Wanga, Honglei Liub, Rui-Ming Liub,* a
Department of Immunology, School of Medicine, University of Alabama at Birmingham (UAB) 1665 University Blvd, Birmingham, AL 35294-0022 USA b Department of Environmental Health Sciences, School of Public Health, University of Alabama at Birmingham (UAB) 1665 University Blvd, Birmingham, AL 35294-0022 USA Received 23 September 2002; received in revised form 20 January 2003; accepted 13 February 2003
Abstract Oxidative damage of the macromolecules increases with age and has been suggested to contribute importantly to the aging process and the pathogenesis of many age-related diseases. However, what causes such an increase in the oxidative damage of the macromolecules and whether male and female have the same susceptibility are not clear. In this study, we demonstrated for the first time that although the concentrations of GSH were similar between young male and female mice in most tissues examined and GSH content declined with age in both genders, male mice seemed to experience more dramatic age-associated change in GSH content than did female mice in many tissues. The age-related decline in the GSH content in both male and female mice was also associated with a decrease in the amounts of glutamate cysteine ligase (GCL) mRNAs and proteins as we have reported previously in male rats, further suggesting an important role of GCL in maintaining GSH homeostasis during the aging process. The results from this study may reveal an important basis underlying the genderassociated differences in the longevity and the susceptibility to certain age-related diseases, and also further suggest that the decreased synthesis, which is mainly due to the down regulation of GCL gene expression, may be responsible for the age-associated decline in GSH content. q 2003 Elsevier Science Inc. All rights reserved. Keywords: Glutathione; Aging; Gender; Glutamate cysteine ligase; g-glutamylcysteine synthetase
1. Introduction The mechanism underlying aging, an inevitable biological process that affects most living organisms, is still an area of significant controversy as are the mechanisms of agerelated diseases such as cancer, neurodegenerative diseases, diabetes, cataract, and cardiovascular diseases. Reactive oxygen species (ROS), generated endogenously or exogenously, cause damages to DNA, RNA, lipids, and proteins. Accumulated evidence indicates that oxidative damage of these macromolecules increases with age and in various age-related diseases. Therefore, it has been suggested that oxidative stress contributes importantly to the aging process and the pathogeneses of age-associated diseases (Jenner, Abbreviations: GCL, glutamate cysteine ligase; GCLC, glutamate cysteine ligase catalytic subunit; GCLM, glutamate cysteine ligase modifier subunit; GSH, glutathione; GSSG, glutathione disulfide; BALF, bronchoalveolar lavage fluid; GST, glutathione-S-transferase. * Corresponding author. Tel.: þ 205-934-7028; fax: þ 205-975-6341. E-mail address: [email protected] (R.-M. Liu).
1996; Mezzetti et al., 1996; Multhaup et al., 1997; Stadtman and Berlett, 1997; Behl, 1999; Lu et al., 1999; Markesbery, 1999; Hamilton et al., 2001). Although intensive studies have been done, the mechanism underlying such increased oxidative damage of the macromolecules in aged animals, however, has not been completely elucidated. It has also been well documented that man has an average lifespan short than woman (Ortmeyer, 1979; Dhar, 2000; Dhar, 2001; Alexiou et al., 2002), and that some of agerelated diseases show gender preferences. The mechanisms underlying such gender-related differences in the lifespan and the susceptibility to certain diseases are largely unknown. As oxidative stress has been implicated in the aging process and the pathogeneses of many age-related diseases, it will be interesting to know whether there is any difference between male and female in terms of their abilities to maintain oxidant and antioxidant balance during the aging process. Glutathione (GSH), the most abundant intracellular nonprotein thiol, participates in many important biological
0531-5565/03/$ - see front matter q 2003 Elsevier Science Inc. All rights reserved. doi:10.1016/S0531-5565(03)00036-6
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processes such as the synthesis of cellular compounds, the cell cycle, and the regulation of the gene expression. The most important function of this tri-peptide, perhaps, is to detoxify oxidants and electrophiles by acting as a cofactor in glutathione peroxidase or glutathione-S-transferase catalyzed reactions. A large body of evidence has shown that increasing GSH concentration can protect cells against oxidative damage while decreasing GSH can promote such damages. Although many studies, including those from this laboratory, have shown that GSH content decreases with age in many tissues from different animal species (Hazelton and Lang, 1980; Ravindranath et al., 1989; De and Darad, 1991; Iantomasi et al., 1993; Favilli et al., 1994; Teramoto et al., 1994; Christon et al., 1995; Yang et al., 1995; Nakata et al., 1996; Sanz et al., 1997; Samiec et al., 1998; Liu and Choi, 2000; Sasaki et al., 2001; Liu 2002), some other studies indicated that there was no significant change or even increase in GSH content during the aging process (Stio, 1994 #4491; Hussain, 1995 #3135; Leeuwenburgh, 1994 #4378; Nakata et al., 1996). Whether such discrepancies between the results from different laboratories reflect species difference or other factors remain to be determined. Furthermore, as most of the studies to examine ageassociated change in GSH metabolism were conducted in one gender or another, it is not clear whether there is any difference between genders in terms of GSH metabolism during the aging process. A systematical comparison of ageassociated change in GSH content between male and female will certainly help us to better understand the mechanisms underlying the gender differences in the longevity and the susceptibility to certain diseases. There are several mechanisms by which cells maintain their intracellular GSH homeostasis, including GSH redox cycling, direct up take, and de novo synthesis. De novo GSH synthesis is a two-step process. The first step, the synthesis of g-glutamylcysteine catalyzed by glutamate cysteine lagase (GCL, EC 6.3.2.2), is the rate-limiting step in this process. Increased evidence suggests that GCL plays a critical role in maintaining GSH homeostasis not only for the resting cells but also for oxidant challenged cells (Shi et al., 1994a,b; Liu et al., 1996a,b; Choi et al., 1997; Choi et al., 2000; Liu et al., 2001). Whether this enzyme also plays an important role in maintaining GSH homeostasis during aging process needs to be studied further. In this study, we systematically compared the ageassociated changes in GSH content and GCL gene expression in 10 tissues/organs between male and female mice. Our results indicated that although there was no significant difference in the GSH content between young male and female mice in most tissues examined and GSH content decreased with age in both male and female mice, male mice seemed to be more vulnerable to such ageassociated decline than female mice did. The age-dependent decline in GSH content in both male and female mice was also associated with a down regulation of GCL gene expression, as we have previously shown in the rat tissues.
The results from this study may reveal an important basis underlying the gender-associated differences in the longevity and the susceptibility to certain age-related diseases, and also further suggest that the decreased synthesis, which is mainly due to the down regulation of GCL gene expression, may be responsible for the age-associated decline in GSH content.
2. Materials and methods 2.1. Animals Young (3-month), adult (12-month), and aged (24month) C57BL/6 male and female mice were purchased from Harlan Sprague Darley/National Institute of Aging and were acclimated for one week in our animal facilities prior to the initiation of our experiments. The animals were maintained on a 12 h light/dark cycle at 22 8C with free access to water and food. The mice were euthanatized with 100 mg/kg sodium pentobarbital and placed in a supine position. Trachea was cannulated, and bronchoalveolar lavage was performed using room temperature saline. After lavage, heart, lung, liver, kidney, spleen as well as brain were removed; cerebral cortex, cerebellum, and brain stem were dissected immediately. The samples for GSH measurement were washed twice in cold PBS and sonicated immediately in 10% perchloric acid (PCA)-2 mM EDTA solution. The remaining tissue samples were frozen in liquid nitrogen immediately for further analyses. All procedures involving animals were approved by the Institutional Animal Care and Use Committees at the University of Alabama at Birmingham. 2.2. Reagents TRIzol Reagent, an RNA isolation solution, was from Life Science Technologies (Grand Island, N.Y.). QuickHyb solution and salmon sperm DNA were from Stratagene (La Jolla, CA). Polyvinylidene fluoride membranes were purchased from Millipore Company. Enhanced chemiluminescence reagent and Hyperfilm ECL film were from Amersham. All high performance liquid chromatography solvents were Baker Analyzed HPLC-grade reagents from VWR scientific (San Diego, CA). All chemicals used were at least analytical grade. 2.3. Measurement of GSH, GSSG, and cysteine content The concentrations of tissue GSH, GSSG, and cysteine were determined by HPLC as described previously (Liu et al., 1998). Briefly, the tissues were cut and washed with cold PBS twice, then sonicated in 10% PCA containing 2 mM EDTA and 16.67 mM of g-glutamyl-glutamic acid, the latter of which served as an internal standard. After centrifugation, 10 mM iodoacetic acid in 0.2 mM cresol
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purple was added to the supernatant, and the pH was adjusted to 8 – 9. The mixture was incubated in the dark at room temperature for 15 min. Then 1% dinitrobenzene (DNB) was added and the reaction mixture, incubated at 4 8C overnight. The amounts of GSH, GSSG, and cysteine were measured by HPLC with standards being run under the same conditions. GSH, GSSG, and cysteine contents were calculated based on the internal standard and standard curves. The total GSH content was calculated as 1GSH þ 2 GSSG.
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2.6. Statistics Data were expressed as mean ^ SEM and evaluated by one-way ANOVA. Statistical significance was determined by Fisher LSD test. P , 0.05 was considered significant.
3. Results 3.1. GSH content was decreased more in male mouse tissues than in female mouse tissues during the aging process
2.4. Western blotting analysis of GCL protein The amounts of GCL catalytic (GCLC) and modifier (GCLM) subunit proteins were determined by Western analysis as described before (Liu et al., 1998). Briefly, the tissues were cut and washed in cold PBS buffer and homogenized in 0.25 M sucrose buffer containing 2 mg/ml leupeptin and aprotinin, and 50 mg/ml PMSF. The homogenates were then centrifuged at 3,000 £ g for 10 min at 4 8C and the supernatant was centrifuged at 10,000 £ g for 20 min and then, at 105,000 £ g for 30 min at 4 8C. After ultracentrifugation, the supernatant was used for determining the amounts of GCL proteins by Western blotting using specific anti GCLC or anti GCLM subunit antibodies. The positions of GCLC and GCLM bands were identified according to molecular weight markers and rat kidney, a positive control, which contains high concentrations of GCL proteins. The X-ray films were scanned using ScanJet 4C/T deskscan and the signal was semi-quantitated, using Sigmascan program. 2.5. Northern hybridization analysis of GCL mRNAs A GCLC cDNA (804 bp) and a GCLM cDNA (1001 bp), which were reversely transcribed from rat kidney RNA and from L2 cell RNA, respectively, and amplified by PCR (Shi et al., 1994a,b; Tian et al., 1997), were labelled with [a-32P] dCTP using a random-primer DNA labelling kit from Life Technologies (Gaithersburg, MD). The GCL mRNA content was determined by Northern hybridization analysis as described previously (Liu et al., 1998). Briefly, total RNA was extracted with TRIzol Reagent (Life Technologies, Grand Island, N.Y.) according to the protocol provided by the manufacturer. Twenty mg RNA from each sample was resolved on a 1.2% agarose gel and transferred onto a Nylon membrane. Hybridization was carried out with GCLM cDNA, GCLC cDNA, and 18S cDNA probes subsequently at 60 8C using Quikhyb (Stratagene) solution. After hybridization, the membranes were washed twice with 2 £ sodium chloride-sodium citrate (SSC) buffer/0.1% sodium dodecyl sulfate (SDS) for 15 min at room temperature, then twice with 0.1 £ SSC/0.1% SDS for 15 min each at 50 8C. The membranes were scanned and radioactivity was quantitated with an InstantImager (Packard Instrument Company, Meriden, CT).
In order to see whether there was any gender difference in GSH metabolism during the aging process, we compared age-associated changes in GSH content in various tissues between male and female mice. The results showed that there was no significant difference in GSH content between young male and female mice in the most tissues examined except the lung, where a significantly higher GSH content was found in female versus male (5.87 ^ 0.74 in male versus 9.97 ^ 0.92 in female). The mechanism underlying such a difference in GSH content in the lung tissue between young male and female mice is not clear. Most interestingly, the results also indicated that although GSH content decreased with increasing age in both male and female mice, the male mice experienced more dramatic decrease than female mice in many tissues. In male mice, 7 out of 10 tissues, cells, or body fluids examined showed an age-associated decline in GSH content while in female mice, only 4 showed the same phenomenon (Figs. 1 and 2, and Table 1). Specifically, GSH content decreased with age in the BALF, lung, and cerebellum in male mice but not in female mice. Of 4 tissues/organs (liver, spleen lymphocytes, brainstem, and cerebral cortex), where GSH content was decreased with age in both male and female mice, 2 (spleen lymphocytes and brainstem) showed a bigger decline in male than in female mice. The only organ that experienced a bigger age-associated decline in GSH content in female than in male mice was the liver. GSH content decreased by 21% and 34% in the liver of 12- and 24-month old male mice, respectively, but by 32% and 55% in the liver of 12- and 24-month old female mice, respectively, as compared with 3-month old mice (Table 1). No age-associated change was found in GSH content in red blood cells (RBC), kidney, or heart from either male or female mice. There was also no significant age or gender difference in either cysteine or GSSG content in any of the tissues or cells that were examined (data not shown). 3.2. The amounts of GCL proteins decreased with age in male and female mouse tissues that showed an agedependent decline in GSH content GCL is the rate-limiting enzyme in de novo GSH synthesis and plays an important role in maintaining GSH homeostasis. In order to explore the mechanism underlying
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Fig. 1. Age-associated decline in GSH content in male mouse tissues. GSH content was measured using a HPLC assay as described in Section 2. Values represent means ^ SEM of 5–10 mice. (a) Significantly different from 3-month old mice (p , 0.05); (b) Significantly different from 12 month-old mice (p , 0.05).
Fig. 2. Age-associated decline in GSH content in female mouse tissues. GSH content was measured using a HPLC assay as described in Section 2. Values represent means ^ SEM of 5–10 mice. (a) Significantly different from 3-month old mice (p , 0.05); (b) Significantly different from 12 month-old mice (p , 0.05).
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Table 1 Comparison of age-associated change in GSH content between male and female mice Tissues
Liver BALF SL Lung Cortex Cerebellum Brainstem
a b c d
gender
M F M F M F M F M F M F M F
GSH (%)a 3 mo.
12 mo.
24 mo.
100.0 ^ 6.7 (10) 100.0 ^ 4.6 (8) 100.0 ^ 15.1 (6) 100.0 ^ 33.9 (8) 100.0 ^ 5.5 (10) 100.0 ^ 5.5 (8) 100.0 ^ 9.6 (6) 100.0 ^ 9.2 (8) 100.0 ^ 4.9 (5) 100.0 ^ 1.9 (8) 100.0 ^ 5.8 (5) 100.0 ^ 2.8 (8) 100.0 ^ 5.8 (5) 100.0 ^ 3.9 (8)
78.7 ^ 11.3 (10) 68.0 ^ 5.6 (8)b 47.2 ^ 13.5 (9)b 119.1 ^ 35.2 (8) 79.0 ^ 20.5 (7) 91.7 ^ 4.5 (8) 119.4 ^ 9.1 (5) 126.0 ^ 9.3 (8) 86.2 ^ 1.7 (5)b 97.8 ^ 4.0 (8) 73.8 ^ 7.1 (5)b 95.6 ^ 3.8 (8)d 88.1 ^ 3.7 (5)b 87.6 ^ 2.5 (8)b
66.4 ^ 8.0 (10)b 44.4 ^ 2.6 (8)b,c,d 52.2 ^ 7.9 (5)b 54.5 ^ 11.8 (8) 63.7 ^ 4.5 (8)b 79.9 ^ 5.8 (8)b,d 73.7 ^ 4.4 (6)b 114.8 ^ 5.0 (8)d 79.3 ^ 2.2 (5)b 84.5 ^ 2.3 (8)b,c 75.5 ^ 1.4 (5)b 89.9 ^ 3.0 (8)d 70.1 ^ 9.6 (5)b 88.5 ^ 2.7 (8)b,d
BALF: bronchoalveolar lavage fluid; SL, spleenic lymphocytes. GSH was reported as % of change from the 3 month old group, which was arbitrarily set as 100%. Significantly different from 3 month old group. Significantly different from 12 month old group. Significantly different from male mice with the same age.
the age-associated decline in GSH content in mouse tissues, we further compared the amounts of the GCL catalytic and modifier subunit proteins between three age groups in both male and female mice. Western analyses were performed with the samples from liver, spleenic lymphocytes, and brainstem tissues as GSH content was decreased with aging in these tissues in both male and female mice. The results
showed that there was an age-associated decrease in the amounts of GCL catalytic and/or modifier subunit proteins in all three tissues in both male and female mice (Figs. 3 and 4). The patterns of changes in GCL proteins with age matched the patterns of the changes in the GSH content, e.g. that GCL proteins decreased more in the spleen lymphocytes and the brainstem but less in the liver of male mice
Fig. 3. Age-associated decline in GCL protein content in male mouse tissues. The amounts of GCLC and GCLM proteins were determined by Western analysis as described in Section 2. The X-ray films were scanned with a Desk-scanner and semi-quantitated using Sigmascan. The results were expressed as percentages of the 3-month group. Values represent means ^ SEM of 4 –5 samples. (a) Significantly different from 3-month old mice (p , 0.05); (b) Significantly different from 12 month-old mice (p , 0.05).
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Fig. 4. Age-associated decline in GCL protein content in female mouse tissues. The amounts of GCLC and GCLM proteins were determined by Western analysis as described in Section 2. The X-ray films were scanned with a Desk-scanner and semi-quantitated using Sigmascan. The results were expressed as percentages of the 3-month group. Values represent means ^ SEM of 4–8 samples. (a) Significantly different from 3-month old mice (p , 0.05); (b) Significantly different from 12 month-old mice (p , 0.05).
during the aging process as compared to that in female mice (Table 2). 3.3. Decreased amounts of GCL proteins were associated with a decline in the GCL mRNA content GCL mRNA content in the liver and brainstem of both male and female mice was further analyzed to see whether the age-associated decrease in GCL protein content was due to a decreased GCL mRNA expression. The results showed
that, as compared to the 3 month-old groups, only GCLC mRNA was decreased (by 39%) in the 24 month-old male mouse liver while both GCLC and GCLM mRNAs were decreased (by 37% and 20%, respectively) in the 24 monthold female mouse liver. In the brainstem, the amounts of both GCLC and GCLM mRNAs were decreased (by 38% and 66%, respectively) in the 24 month-old male mice while only the GCLC was decreased (by 16%) in the 24 month-old female mice, as compared to the 3 month-old mouse groups (Fig. 5(a) and (b)). The results indicated that decreased gene
Table 2 Comparison of age-associated changes in GCL protein content between male and female mice Tissues
Liver
Gene
GCLC GCLM
SL
GCLC GCLM
Brainstem
GCLC GCLM
a b c d
Gender
M F M F M F M F M F M F
GCL proteins (%)a 3 mo.
12 mo.
24 mo.
100.0 ^ 6.9 (4) 100.0 ^ 10.1 (8) 100.0 ^ 19.0 (4) 100.0 ^ 10.0 (8) 100.0 ^ 4.7 (4) 100.0 ^ 4.6 (4) 100.0 ^ 19.2 (4) 100.0 ^ 12.7 (4) 100.0 ^ 12.0 (5) 100.0 ^ 21.2 (4) 100.0 ^ 24.0 (5) 100.0 ^ 14.3 (4)
59.2 ^ 3.8 (4)b 69.8 ^ 15.4 (8) 196.0 ^ 30.5 (4) 57.2 ^ 11.9 (8)c 67.1 ^ 4.3 (4)b 83.6 ^ 9.3 (4) 94.9 ^ 11.6 (4) 92.2 ^ 12.8 (4) 59.1 ^ 6.2 (4)b 96.2 ^ 14.6 (4)c 83.5 ^ 20.7 (4) 116.8 ^ 19.6 (4)
71.0 ^ 4.0 (4)b 54.4 ^ 2.4 (8)b,c 118 ^ 39.0 (4) 36.6 ^ 2.8 (8)b,d,c 38.8 ^ 1.4 (4)b,d 72.6 ^ 5.5 (4)b,c 61.6 ^ 15.8 (4) 75.4 ^ 7.4 (4) 49.2 ^ 7.4 (4)b 85.9 ^ 15.7 (4)c 54.9 ^ 10.9 (4)b 107.2 ^ 21.0 (4)c
SL, Spleenic lymphocytes GCL protein content was reported as % of change from the 3 month old group, which was arbitrarily set as 100%. Significantly different from 3 month old group. Significantly different from male mice with the same age. Significantly different from 12 month old group.
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Fig. 5. GCL mRNA content was decreased with age in the liver and brainstem of both male and female mice. The amounts of both GCLC and GCLM mRNAs were determined by Northern analyses as described in Section 2. 18S was used to normalize GCLC as well as GCLM signals. The results were expressed as percentages of the 3-month group after normalized by 18S. (A) age-associated changes in GCL mRNAs in male mouse liver and brainstem. (B) Age-associated changes in GCL mRNAs in female mouse liver and brainstem. The values represent means ^ SEM of 3– 5 samples. (a) significantly different from 3 month mice (p , 0.05); (b) significantly different from 12 month mice (p , 0.05).
expression might be responsible for the age-associated decline in the GCL protein content.
4. Discussion Although numerous studies have shown that GSH content decreases with age in various animal species, whether male and female animals experience the same change during the aging process is not clear. In this study, we systematically compared age-associated changes in GSH metabolism between male and female mice. Our results showed for the first time that, although GSH content
decreased with age in both male and female mice, male mice seemed to experience more dramatic age-associated decline than did female mice in most tissues examined. Of 10 tissues or cells or body fluid examined, 7 showed an agedependent decrease in GSH content in male mice while only 4 showed the same phenomenon in female mice. The extent of age-associated decrease in GSH content was also greater in 2 of the 4 tissues that showed an age-associated decline in GSH content in both genders. The only exception to this trend was the liver. GSH content in the liver decreased more in female mice than in male mice during the aging process. The mechanism underlying such a difference in the ageassociated change in GSH content between male and female
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mice is unclear. One possible explanation may be that the amount of some hormones, which are involved in the regulation of GSH homeostasis and to which different tissues/organs have different sensitivities, decreases with different rates in male and female. More work is needed to answer this question. Although it is a well-known phenomenon that GSH content decreases with age, the underlying mechanism is also not completely understood. GSH depletion may occur when cells are exposed to massive oxidants, when GSSG reductase activity decreases, which limits the redox cycle of GSSG, and/or when the rate of GSH synthesis is decreased. A long-term low-level oxidative stress, which is most likely the case during the aging process, however, usually increases rather than decreases intracellular GSH content as it can induce the expression of various enzymes involved in GSH synthesis (Liu et al., 1996a,b; Liu et al., 1998; Moellering et al., 1998; Moellering et al., 1999; Liu et al., 2001). An age-associated decrease in GCL activity accompanied by a decline in GSH content has been reported in different tissues of various animal species (Sethna et al., 1982; Rathbun, 1984; Rathbun, 1986; Iantomasi et al., 1993; Favilli et al., 1994; Stio et al., 1994). In the previous studies, we have shown that GSH content decreased with age in several organs/tissues in male Fisher 344 rats, which was due, at least in part, to a down regulation of GCL gene expression (Liu and Choi, 2000; Liu, 2002). In this study, we showed that the age-associated decline in GSH content in both male and female mice was also associated with a decreased GCL gene expression. These data further indicated an important role of GCL in maintaining GSH homeostasis for different animal species during the aging process. Although we did not measure GSSG reductase activity in this study, we did not observe any age-associated increase in GSSG content, an indicator of massive oxidant production or a decrease in GSSG reductase activity, in any of the tissues examined. These results suggest that the ageassociated decline in GSH content in mouse tissues does not result from either an increased oxidant production or a decreased GSSG reductase activity. This conclusion is supported by the results form several other studies (Hazelton and Lang, 1980; Chen et al., 1989; Richie, 1992; Yang et al., 1995; Nakata et al., 1996; Liu, 2002). Another important observation from this study was that there was no significant difference in GSH content between young male and female mice in most tissues, cells, and body fluid examined. Although the regulation of GSH homeostasis has been well studied at the cellular level, the factors regulating GSH homeostasis in whole organisms have not been completely revealed. Estrogen is believed to be one of the hormones that may regulate GSH homeostasis under physiological situation as estrogen replacement therapy has been reported to increase total thiol level in the plasma of postmenopausal women (Konukoglu et al., 2000) and estradiol treatment increased GSH content in canine myocardial tissue (Kim et al., 1998). As female has much
higher plasma estrogen level than male, one would expect that the GSH content is higher in female than in male, at least in the tissues that are estrogen sensitive. Interestingly, our results showed that there was no significant difference in the GSH content between young male and female mice in most tissues examined except the lung, in which a significant higher level of GSH was found in female mice vs. male mice. Although no such systematic comparison has ever been previously reported, a few sporadic studies using different animal species have shown similar results. Igarashi reported that GSH content in the liver of young rats was not significantly different between the two genders (Igarashi et al., 1983). Ferris (Ferris et al., 1995) and Kume-Kick (Kume-Kick et al., 1996) demonstrated that there was no difference in the GSH content in the brains of young male and female rats. Furthermore, Erden-Inal showed that the GSH content in the blood from men and women was same (Erden-Inal et al., 2002). These results, including ours, suggest that sex hormones may not be involved in the regulation of GSH homeostasis under physiological conditions, at least in these tissues/organs. We would like to mention that although most studies indicated that GSH content decreased with age, a few reports showed a different trend (Stio, 1994 #4491; Hussain, 1995 #3135; Leeuwenburgh, 1994 #4378; Nakata et al., 1996). The reason for such discrepancies between the results from different laboratories may be due to the differences in the experimental conditions including animal species and ages, etc. For example, Hazelton reported that GSH content decreased with age in the kidney and heart of male C57BL mice (Hazelton and Lang, 1980) while we did not observe any age-associated change in GSH content in these two organs form the same animal species. The difference may result from the age of the animals, which was used to represent an old group (31 month-old in their experiment verse 24 month-old in our experiment). On the other hand, Hussain reported (Hussain et al., 1995) that GSH content was significantly increased in the cerebellum and brainstem of old male mice (C57BL) as compared with the young male mice while we observed an opposite phenomenon in this same animal species. The difference between our experiments and theirs was that the animals were fasting for 16 h before sacrificed in our experiments (in order to minimize the effect of food and circadian rhythm of hormones on the GSH content) but not in their experiments. The biological significance of such difference between male and female in GSH metabolism during the aging process needs to be further explored. Interestingly, it has been well documented that man has an average lifespan shorter than woman (Ortmeyer, 1979; Dhar, 2000; Dhar, 2001; Alexiou et al., 2002). Although the mechanism trigging the aging process has not been completely elucidated yet, oxidative damage of the macromolecules has been proposed to be one of the important contributors. Therefore, a lower capacity to maintain GSH homeostasis in male animals, probably in man too, as compared with
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female animals during the aging process may contribute to their shorter lifespan. Consistent with this notion, Honda and Richie reported that increasing GSH level extended not only the life of cultured human diploid fibroblasts (Honda and Matsuo, 1988) but also the life span of mosquito (Richie et al., 1987). Dietary calorie restriction, which has been shown to be able to extend the life span of various animal species, also increases GSH content (Richie et al., 1994; Taylor et al., 1995; Armeni et al., 1998). Nevertheless, whether GSH content also decreases more in man than in women during the aging process and if it does, whether a lower capacity of maintaining GSH homeostasis in man as compared with woman contributes to their shorter lifespan are very important questions to be answered. It is also well documented that the susceptibility to certain age-related diseases such as cancers, cardiovascular diseases, and neurodegenerative diseases differs between two genders (Berko, 1989; Jarvik et al., 1995; Corrao et al., 1997; Lapane et al., 2001; Bella et al., 2002; Chen et al., 2002; Radzikowska et al., 2002). Although the mechanism underlying such a gender difference is unknown in the most cases, the difference in GSH metabolism has been suggested to play a role in some situations. For example, lung cancer is a leading cause of death in men in the United States. The incidence of the lung cancer increases with age and is higher in man than in woman (Alexiou et al., 2002; Koyi et al., 2002). Although the environmental and behavioural factors contribute importantly to such a gender difference, the difference in the genetic background related to GSH metabolism has also been shown to be responsible. Polymorphism of glutathione-S-transferase (GST), an enzyme that catalyzes GSH conjugate formation, has been shown to be associated with a high risk of the lung cancer in different populations (Hou et al., 2001; Matsuzoe et al., 2001; Reszka and Wasowicz, 2001; Miller et al., 2002). Furthermore, it has also been reported recently that polymorphisms of GCL, the rate-limiting enzyme in de novo GSH synthesis, may determine lung cancer survival (Yang et al., 2002). In this study, we found that GSH content was significantly lower in the lung of young male mice as compared with young female mice, and decreased with age in male mice but not in female mice. Whether such a difference in GSH metabolism in the lung between male and female also exists in human and if it does, whether such a gender difference contributes to the different susceptibility of man and women to lung cancer remain to be explored further. Summary. The present study demonstrated for the first time that although GSH content decreased with age in both male and female mice, male mice experienced more dramatic age-associated change in GSH content than did female mice. There was no significant difference in the GSH content between young male and female mice in most tissues examined except the lung, in which a significantly
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lower level was found in male compared with female mice. Age-associated decline in GSH content in both male and female mice was also associated with a down regulation of GCL gene expression as we have reported previously in rats, further suggesting an important role of GCL in maintaining GSH homeostasis during the aging process. The results from this study may reveal an important basis for the genderassociated differences in the longevity and the susceptibility to certain age-related diseases. Acknowledgements This project was funded by National Institute Health grant 1 R01 ES11831-01 and American lung Association research grant RG-057-N. The authors would like to acknowledge Dr Henry Jay Forman for his advice, Dr Jinah Choi, Dr Dale Dickinson and Dr Karen Iles for their critical review of the manuscript, as well as Xu Jia for her technical assistance.
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