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The Journals of Gerontology Series A: Biological Sciences and Medical Sciences 62:687-695 (2007)
© 2007 The Gerontological Society of America

Melatonin Is Able to Reduce the Apoptotic Liver Changes Induced by Aging Via Inhibition of the Intrinsic Pathway of Apoptosis

Virginia Molpeceres, José L. Mauriz, María V. García-Mediavilla, Paquita González, Juan P. Barrio and Javier González-Gallego

Ciberehd and Institute of Biomedicine, University of León, Spain.

Address correspondence to Javier González-Gallego, MD, PhD, Department of Biomedical Sciences, University of León, 24071-León, Spain. E-mail: jgonga{at}unileon.es


   Abstract
Top
 Abstract
Methods
 Results
 Discussion
 References
 
We examined the effect of daily melatonin supplementation on liver apoptosis induced by aging in rats. Young (3-month-old) and aged (24-month-old) Wistar rats were supplemented daily with melatonin in their drinking water (20 mg/L) for 4 weeks. Aged rats showed increases in the liver concentration of thiobarbituric acid-reactive substances and in the oxidized/reduced glutathione ratio. These increases were accompanied by apoptotic ultrastructural alterations and increases in cytochrome c mitochondrial release, Bax to Bcl-2 relative expression, and activity of caspase-3. No significant changes were observed in Fas-ligand (Fas-L) expression and caspase-8 activity. Melatonin administration was able to abrogate changes detected in aged rats. Data suggest that liver apoptotic cell death is induced by reactive oxygen species, via the intrinsic signalling pathway, and that the antiapoptotic action provided by melatonin is related to its antioxidant effect, with reduction of cytochrome c release by the modulation of Bcl-2 and Bax genes.

AGING is characterized by a general decline in physiological function that leads to morbidity and mortality (1). Although aging is an extremely complex, multifactorial process that has been the subject of considerable speculation (2), accumulated evidence identifies oxidative stress with age as a source of damage to cellular structure and function. The redox state of the cell is a consequence of the precise balance between the levels of oxidizing and reducing equivalents, such as reactive oxygen species (ROS) and endogenous antioxidants. Oxidative stress is a condition in which the redox balance between oxidant and antioxidant is disrupted, tilting the equilibrium toward an oxidized state (3). The free radical theory of aging proposes that aging and some related diseases are, at least in part, a consequence of oxidative stress (4).

Apoptosis (or programmed cell death) is an active process critical for the development and maintenance of cellular homeostasis in adults (5). Two major pathways have been described regulating apoptosis: the extrinsic pathway, in which cell plasma membrane receptors act as the starting point of the apoptotic process, and the intrinsic pathway, in which mitochondria play a central role. In both intrinsic and extrinsic pathways, activation of caspases plays an indispensable role. Caspases (cysteine-dependent aspartate-specific proteases) are a family of proteases with the characteristic of cleaving to aspartate residues in their substrates (6). Numerous reports suggest that aging is accompanied by alterations in the apoptotic behavior of a variety of cell types and tissues, with modification on caspase activities (5). It remains, however, to be established whether enhanced levels of apoptosis serve as a self-protective mechanism to remove increased numbers of dysfunctional cells as a result of aging, or whether they play a destructive role, causing excessive cell death and the decline of organ function attendant with aging (7).

Several observations suggest a possible implication of ROS as signaling molecules in apoptosis. The increase of ROS generation or the depletion of endogenous antioxidants can promote apoptosis (8), and apoptosis can be delayed or inhibited by antioxidants in various types of cells (9). Although a direct connection between apoptosis and aging has not been established, some data suggest that oxidative stress may elicit its effects on aging via regulation of apoptosis (10,11).

Melatonin (N-acetyl-5-methoxytryptamine) is the major hormone of the pineal gland, but it has been detected in many other tissues. It is a highly lipophilic molecule that crosses cell membranes to easily reach subcellular compartments including mitochondria, where it seems to accumulate in high concentrations (12). Melatonin is able to prevent oxidative stress both through its free radical scavenging effect and by directly increasing antioxidant activity (13), and different studies have demonstrated its protective role against oxidative damage induced by drugs, toxins, and different diseases (14–16). It is known that endogenous melatonin production diminishes in elderly persons (17) and that the total antioxidative capacity of serum correlates well with its melatonin levels in humans (18). Moreover, melatonin shows beneficial effects against aging in rats, preventing lipid peroxidation and other mechanisms related to oxidative stress (19,20). Thus, the reduction in melatonin with age may be a factor in the elevated oxidative damage observed in the elderly population (21).

The multitude of functions assigned to the mammalian liver and its central role in human physiology suggest that it may be particularly susceptible to the insults of aging, and one consequence of aging appears to be an increase in the incidence of liver pathologies, which may compromise hepatic function in elderly persons (22). Hepatic aging involves alterations in redox status resulting in enhanced oxidant production and an increased inflammatory response (23), which may be partially prevented by antioxidants (24). Although protection against liver oxidative stress by melatonin has been reported in senescence-accelerated mice (25), and the effects of melatonin on liver apoptosis have been studied in experimental cholestasis (26), no information exists on whether melatonin may influence the mechanism of programmed cell death in the aging liver. Thus, in the present study we compared the effects of 4-week melatonin supplementation on markers of oxidative stress and on various mediators of apoptosis in the liver of young (3-month-old) and aged (24-month-old) rats.


   METHODS
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 Abstract
 Methods
Results
 Discussion
 References
 
Animals and Experimental Procedures
All study protocols were reviewed and approved by the University of León Animal Care Committee, and experiments were performed in accordance with the Guiding Principles for Research Involving Animals (U.S. National Academy of Sciences [NAS]). Male Wistar rats were caged at a temperature of 22°C, with a 12-hour light/dark cycle and free access to food and water until the time of experiments. Animals were divided into four groups (n = 8): young rats (3 months old), aged rats (24 months old), young melatonin-treated rats, and aged melatonin-treated rats. Melatonin (Sigma Chemical Company, St. Louis, MO) was prepared three times a week by dissolving the drug (16 mg) in ethanol (1 mL, 100%, vol/vol). This solution was then diluted with drinking water to a final concentration of 20 mg/L. Water bottles were covered with aluminium foil, and melatonin was administered daily at the onset of the scotophase for 4 weeks. Rats drank about 25 mL/day, and the average daily intake of melatonin was estimated to be 1 mg/kg/day, which was expected to rise 20–30 times normal plasma melatonin levels (27,28). Appropriate dilutions were made to the drinking melatonin solution to match individual variations in water consumption on the previous day. After the experimental period, the animals were decapitated and exsanguinated and the livers were immediately removed.

Biochemical Markers of Oxidative Stress
Oxidized glutathione (GSSG) and reduced glutathione (GSH) analysis was performed fluorimetrically. Briefly, 250 mg of tissue was homogenized in 0.1 M sodium phosphate 5 mM EDTA buffer (pH 8.0) with 25% phosphoric acid at a proportion of 1:20. The mixture was centrifuged at 100,000 g for 30 minutes at 4°C, the supernatant was collected, and 500 µL was diluted with 4.5 mL of buffer. Two spectrophotometry cuvettes per sample were prepared with 1.8 mL of phosphate-EDTA buffer, 100 µL of supernatant, and 100 µL of O-phthalaldehyde. After incubating for 15 minutes at 4°C, a spectrofluorometric reading was obtained at an excitation wavelength of 350 nm and an emission wavelength of 420 nm. To find the percentage of glutathione corresponding to oxidized and reduced forms, 500 µL of the sample supernatant was incubated with 20 µL of 4-vinylpyridine for 30 minutes; to this mixture 4.5 mL of 0.1 N NaOH was added. A 100-µL portion of this mixture was then processed as described above to determine GSSG. GSH was obtained by subtracting GSSG from total glutathione.

The amount of aldehydic products generated by lipid peroxidation was quantified by measuring the concentration of thiobarbituric acid–reactive substances (TBARS). For this purpose a final amount of 3 mg of protein per sample was assayed. The samples were incubated at 90°C for 30 minutes after adding 500 µL of 0.37% thiobarbituric acid in 15% trichloroacetic acid, then were centrifuged at 4°C at 2000 g for 15 minutes. Spectrophotometric absorbance was determined in the supernatant at 535 nm.

Samples were always homogenized using a glass Teflon Potter-Elvehjem homogenizer (B. Braun, Melsungen, Germany). The entire procedure was performed using a Beckman centrifuge (model J2-21 M/E; Beckman Instruments, Palo Alto, CA).

Ultrastructural Analysis
For transmission electron microscopy analysis, tissues were randomly collected from different regions of the surface of the different liver lobes. The samples were immediately fixed with a modified Karnovsky fixative (2% glutaraldehyde + 4% buffered formalin [0.1 mol/L phosphate buffer]) for 2 hours, followed by osmication (2% OsO4 for 2 hours). Tissue samples were then dehydrated and embedded in epoxy resin. Ultrathin sections were stained with uranyl acetate and lead nitrate and were observed under a JEM-1010 transmission electron microscope (JEOL, Tokyo, Japan).

Caspase-3 and Caspase-8 Activities
Lysates were prepared by homogenizing liver tissue in 0.25 mM sucrose, 1 mM EDTA, 10 mM Tris, and a protease inhibitor cocktail (Roche, Mannheim, Germany). The lysates were then centrifuged at 14,000 g for 10 minutes at 4°C, and supernatants (50 µg of protein) were incubated for 1 hour at 37°C in HEPES buffer containing 100 µM concentrations of the specific fluorogenic substrates (7-amino-4-methylcoumarin N-acetyl-L-aspartyl-L-glutamyl-L-valyl-L-aspartic acid amide [Ac-DEVD-AMC] and N-acetyl-Ile-Glu-Thr-Asp-7-amino-4-trifluoromethylcoumarin [Ac-IEDT-AFC] for caspase-3 and 8, respectively). Cleavage of the caspase substrates was monitored using a spectrofluorimeter (Hitachi F-2000 fluorimeter; Hitachi LTD, Tokyo, Japan) at excitation/emission wavelengths of 380/460 nm for caspase-3 and 400/505 nm for caspase-8, respectively. Activity was expressed as fluorescence units per milligram of protein per minute of incubation.

Western Blot Analysis
Western blot analyses were performed on cytosolic or mitochondrial extracts (29). Cytosolic extracts were prepared by liver tissue homogenization in 0.25 mM sucrose, 1 mM EDTA, 10 mM Tris, and 1% protease inhibitor cocktail (Roche). The homogenate was centrifuged at 4°C for 30 minutes at 13,000 g. Mitochondrial extracts were prepared by homogenization of liver tissue in 250 mM mannitol, 70 mM sucrose, 2 mM EDTA, 20 mM HEPES (pH 7.4), 0.45% bovine serum albumin (BSA), and 1% protease inhibitor cocktail (Roche). The homogenate was centrifuged at 4°C for 10 minutes at 750 g, and the supernatant obtained was centrifuged at 12,000 g for 10 minutes. Pellet was resuspended in 250 mM mannitol, 70 mM sucrose, 3 mM HEPES buffer (pH 7.4), and 0.1% BSA with protease inhibitor cocktail, and was centrifuged at 12,000 g for 10 minutes. Finally, the mitochondrial pellet was resuspended in 250 mM sucrose, 2 mM EDTA, 3 mM HEPES (pH 7.4), 0.1% BSA, and 1% protease inhibitor cocktail.

For Western blot analysis of Bcl-2, Bax, cytochrome c, and Fas-ligand (Fas-L), protein extracts (50–100 µg) were separated by 10%–15% sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis for 1.5 hours at 100 V and then blotted on polyvinylidene difluoride (PVDF) membranes (Amersham Pharmacia, Little Chalfont, U.K.). The membranes were then blocked with 5% nonfat dry milk in phosphate-buffered saline (PBS) buffer containing 0.05% Tween 20 (PBST) for 30 minutes at 37°C and probed overnight at 4°C with polyclonal anti-Bcl-2, anti-Bax, anticytochrome c, and anti-Fas-L (Santa Cruz Biotechnology, Santa Cruz, CA) at 1:200–1:400 dilution with PBST containing 3% nonfat dry milk. Equal loading of protein was demonstrated by probing the membranes with a rabbit anti-ß-actin polyclonal antibody (1:1000–1:4000 dilution; Sigma). After washing with TBST, bound primary antibody was detected with horseradish peroxidase (HRP)-conjugated antigoat antibody (DAKO, Glostrup, Denmark), and blots were developed using an enhanced chemiluminescence detection system (ECL kit; Amersham Pharmacia). Densitometry analysis of specific bands was performed by Scion Image software (Scion Corporation, Frederick, MD).

Statistical Analysis
Results are expressed as mean values ± standard error of the mean (SEM). The data were compared by analysis of variance (ANOVA); when the analysis indicated the presence of a significant difference, the means were compared with the Newman–Keuls test. Significance was accepted at p <.05. Values were analyzed using the statistical package SPSS 13.0 (SPSS Inc., Chicago, IL).


   RESULTS
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 Abstract
 Methods
 Results
Discussion
 References
 
To measure oxidative stress induced by aging, both TBARS concentration and the GSSG/GSH ratio were analyzed in the different experimental groups. Liver concentration of TBARS significantly increased in the aged rats (+58%), whereas values did not significantly differ from the young in aged animals receiving melatonin (Figure 1A). Aging also induced a significant increase in the GSSG/GSH ratio (+20%), which was prevented by melatonin (Figure 1B).


Figure 01
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  		Figure 1. Effect of aging and melatonin (MEL) treatment on liver oxidative stress. A, Thiobarbituric acid–reactive substances (TBARS) concentration. B, Oxidized glutathione/reduced glutathione (GSSG/GSH) ratio. *p <.05, compared with young group. #p <.05, compared with aged group

 
Transmission electron microscopy analysis of liver tissue confirmed a normal ultrastructural aspect of hepatocytes in young rats, whereas ultrastructural lesions occurred in aged animals. These lesions were detected in hepatocytes from the different lobes, and included nuclear condensation, perinuclear clustering of mitochondria, and peripheral displacement of the rough endoplasmic reticulum (Figure 2, top). All ultrastructural modifications were markedly alleviated by melatonin (Figure 2, bottom).


Figure 02
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  		Figure 2. Ultrastructural changes induced in the liver by aging and melatonin (MEL). Top, Aged group. Aging induced nuclear condensation, perinuclear clustering of mitochondria, and peripheral displacement of the rough endoplasmic reticulum. Original magnification 5000x. Bottom, Aged+MEL group. Melatonin was able to reduce all ultrastructural changes induced by aging. Original magnification 5000x

 
Caspase-8 is a key initiator caspase mediating Fas-L-induced apoptosis by the death receptor pathway of apoptosis, in which the binding of Fas to Fas-L induces receptor clustering and formation of death-induced signaling complexes. To determinate whether this pathway was activated in aged livers, we examined the activity of caspase-8 by measuring cleavage of the specific substrate Ac-IEDT-AFC at excitation/emission wavelengths of 400/505, and the protein content of Fas-L by blotting its cytosolic protein with anti-Fas-L antibody. There was no significant difference in caspase-8 activity and Fas-L protein among aged and young rats (Figure 3A and B, respectively). Moreover, no changes were induced by melatonin treatment.


Figure 03
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  		Figure 3. Effect of aging and melatonin treatment on caspase-8 activity and Fas ligand (Fas-L) protein content in the liver. A, Caspase-8 activity. Activity was determined using specific substrate N-acetyl-Ile-Glu-Thr-Asp-7-amino-4-trifluoromethylcoumarin (Ac-IEDT-AFC). B, Fas-L protein content. Top: Western blot analysis. Equal loading of proteins is illustrated by ß-actin bands. Cytosolic protein was separated on 15% sodium dodecyl sulfate (SDS)–polyacrylamide gels and blotted with anti-Fas-L antibody. Bottom: Densitometry of Fas-L Western blot. Densitometric analysis was performed, and the arithmetic mean ± standard error of the mean (SEM) is represented. The y axis indicates arbitrary units. UAF, Arbitrary units of fluorescence

 
In contrast, we found modifications of the intrinsic pathway of apoptosis. Cytochrome c is known as a mitochondrial intermembrane protein, released by changes in mitochondrial membrane permeability, that finally activates caspase-3, leading to apoptosis. Increases in the cytosolic cytochrome c protein content (+117%) and reductions in the mitochondrial cytochrome c protein content (–47%) were detected in aged rats (Figure 4A and B, respectively). Densitometric analysis of Western blots revealed that melatonin was able to prevent these changes; values for both cytosolic and mitochondrial cytochrome c protein levels did not significantly differ from those in young animals (Figure 4).


Figure 04
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  		Figure 4. Effect of aging and melatonin (MEL) treatment on liver cytochrome c. A, Cytosolic cytochrome c protein content. B, Mitochondrial cytochrome c protein content. Top: Western blot analysis. Equal loading of proteins is illustrated by ß-actin bands. Cytosolic or mitochondrial protein was separated on 10% sodium dodecyl sulfate (SDS)–polyacrylamide gels and blotted with cytochrome c antibody. Bottom: Densitometry of cytochrome c Western blot. Densitometric analysis was performed, and the arithmetic mean ± standard error of the mean (SEM) is represented. The y axis indicates arbitrary units. *p <.05, compared with young group. #p <.05, compared with aged group

 
Different Bcl-2 family members have been shown to exert a regulatory function on the intrinsic pathway. Members of this family can be divided into an antiapoptotic family, which includes Bcl-2, and an apoptotic family, which includes Bax. The ratio of antiapoptotic Bcl-2 to proapoptotic Bax contributes to the susceptibility of a given cell to apoptosis. We found a significant increase in Bax protein level (+20%) and a parallel reduction of Bcl-2 protein level (–20%) in the mitochondria of aged rats (Figure 5A and B), and the Bcl-2/Bax ratio was significantly lower in comparison to young animals (.44 ±.02 vs.83 ±.10, p <.05). These effects were blocked by melatonin administration (Figure 5A and B, respectively), resulting in a Bcl-2/Bax ratio (.87 ±.03) that did not significantly differ from that in untreated young rats.


Figure 05
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  		Figure 5. Effect of aging and melatonin (MEL) treatment on liver Bax and Bcl-2. A, Mitochondrial Bax protein content. B, Mitochondrial Bcl-2 protein content. Top: Western blot analysis. Equal loading of proteins is illustrated by ß-actin bands. Mitochondrial protein was separated on 12% sodium dodecyl sulfate (SDS)–polyacrylamide gels and blotted with Bax or Bcl-2 antibodies, respectively. Bottom: Densitometry of Bax or Bcl-2 Western blot. Densitometric analysis was performed, and the arithmetic mean ± standard error of the mean (SEM) is represented. The y axis indicates arbitrary units. *p <.05, compared with young group. #p <.05, compared with aged group

 
Cytochrome c is known as a mitochondrial substrate that finally activates the downstream effector caspase-3, leading to apoptosis. To determine whether caspase-3 was activated by aging, supernatants from liver lysates were incubated with the specific substrate Ac-DEVD-AMC, and cleavage of the substrate was monitored at an excitation wavelength of 380 nm and an emission wavelength of 460 nm. Our results show that aging induced an important increase in caspase-3 activity (+86%), which was prevented by melatonin (Figure 6).


Figure 06
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  		Figure 6. Effect of aging and melatonin (MEL) treatment on caspase-3 activity. Activity was determined using specific substrate N-acetyl-L-aspartyl-L-glutamyl-L-valyl-L-aspartic acid amide (Ac-DEVD-AMC). *p <.05, compared with young group. #p <.05, compared with aged group. UAF, Arbitrary units of fluorescence

 

   DISCUSSION
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 Methods
 Results
 Discussion
References
 
The senescent liver exhibits a number of characteristics consistent with oxidative injury, and many studies have examined the effect of aging on the oxidative status in this mammalian tissue. In rat liver, aging has been associated with enhanced ROS generation and oxidative stress (30,31). Liver concentrations of GSH are inversely proportional to those of TBARS, showing a peak of GSH concentration in young animals that is followed by its gradual decrease and an increase in TBARS with aging (32). Lower concentrations of GSH in senescent tissues can be explained by the increased rate of oxidation in old rats and the increase in GSH utilization in the removal of lipid peroxides and other peroxides. TBARS measurement has some limitations related mainly to its nonspecificity (33), and the GSSG/GSH ratio is a useful alternative index in determining oxidative stress in tissues (34). The parallel increase in both TBARS concentration and the GSSG/GSH ratio observed by us in the livers of aged rats is consistent with previous reports (23,30) and supports the presence of enhanced oxidative stress in the liver of senescent rats.

A number of in vitro and in vivo studies have documented the ability of both physiological and pharmacological concentrations of melatonin to protect against free radical damage (12). When melatonin has been compared to the antioxidant capacity of either vitamin E or C in vivo, it has frequently been far superior (35). In addition, melatonin is known to regulate and/or maintain the intracellular concentration of GSH (36,37). If there is validity to the widely accepted free radical theory of aging (4), then it is justifiable to consider a potential role for melatonin in these processes. It is known that endogenous melatonin production diminishes in elderly persons (17), and this decrease closely correlated with a decrease in the total antioxidant capacity of human serum with age (18). In our study, melatonin was able to reduce both the GSSG/GSH ratio and the TBARS concentration in the liver, preventing completely the oxidative stress induced by aging. This finding confirms previous studies indicating that antioxidant molecules are able to preserve hepatic function in aged animals (24,38).

It is generally accepted that oxidative stress is the underlying inducer of apoptosis in hepatocytes (39). It has been shown that hepatocytes isolated from livers of old rats are more sensitive to apoptosis by oxidants such as t-butyl-hydroperoxide (7), and aging appears to increase the number of the TUNEL (terminal deoxynucleotidyl-transferase-mediated dUTP nick end labeling)-positive cells in rats. Whether TUNEL-positive staining alone indicates definitively that apoptosis has occurred has become increasingly controversial, as DNA fragmentation may also occur in the late phases of necrosis, and this fragmentation can be detected by the TUNEL assay (40). For this reason, specific mediators were measured in our study to confirm whether oxidative stress was accompanied by alterations in the different pathways of apoptosis and whether these effects could be prevented by melatonin administration. Ultrastructural studies were also carried out to recognize the presence of cells fulfilling morphological criteria of apoptosis, including ultrastructural mitochondrial changes.

We observed a normal ultrastructural aspect of hepatocytes in young rats, whereas ultrastructurally intact hepatocytes coexisted in old rats with cells exhibiting lesions that included nuclear condensation, perinuclear clustering of mitochondria, and peripheral displacement of the rough endoplasmic reticulum. Moreover, an outer-membrane rupture was observed in some mitochondria. Similar findings in different models of liver apoptosis have been reported by other authors (41,42), and a number of studies have demonstrated that mitochondrial integrity also declines as a function of age (43). In our study these ultrastructural modifications were markedly alleviated by melatonin. Previous research has shown that melatonin can suppress the subcellular apoptotic changes in models of liver apoptosis induced by aflatoxin B1 (44), doxorubicin-induced cardiotoxicity (45), or apoptosis by ultraviolet B (UV-B) treatment in promonocytic leukemia cells (46).

Caspase-3 is an executioner caspase that represents a point of convergence of intrinsic and extrinsic apoptotic pathways (5). When activated, it is responsible for the proteolytic cleavage of a broad range of cellular proteins that contribute to the characteristic apoptotic morphological changes described above. Our data indicated an important increase in caspase-3 activity in the liver of aged rats, which was prevented by melatonin. Although the possibility exists that the increase of liver apoptosis could be due to a major sensitivity to death stimuli during killing, Zhang and colleagues (7) have demonstrated that hepatocytes isolated from old animals are more sensitive to cell death by oxidative stress than are hepatocytes isolated from young animals. Therefore, in the liver of aged rats, most probably there are cells undergoing death at any time.

Two major pathways that regulate apoptosis have been described: the extrinsic and the intrinsic pathways. The two apoptotic pathways are not mutually exclusive in hepatocytes, but are closely interrelated (5). In contrast, in the extrinsic pathway, Fas and other death receptors are activated by binding to their cognate ligand, leading to recruitment and cleavage of procaspase-8 to its active form, which subsequently cleaves and activates downstream caspases, such as caspase-3 (47). Effects of aging in the extrinsic apoptosis pathway are still controversial. It has been described that the level of Fas messenger RNA (mRNA) increases with age in rat liver (48), and this result is consistent with the finding that Fas is a major regulator of liver homeostasis (49). Moreover, it has been reported that methyl methanesulfonate (MMS)-induced apoptosis, which signals through the Fas pathway, is compromised in aged liver (50). However, other authors have found that caspase-8 is not activated by aging in the hepatic tissue of rats (7). In our study, no changes in Fas-L protein content or in caspase-8 activity were observed in aged animals, whether supplemented or not with melatonin.

In contrast, in the intrinsic pathway, various apoptosis-inducing signals (particularly oxidative stress) directly or indirectly change mitochondrial membrane permeability and cause release of mitochondrial intermembrane proteins, including cytochrome c (6), which lead to the activation of caspase-9 and downstream caspases, such as caspase-3 (6). The mechanisms of cytochrome c release can be multiple and remain controversial, but both promotion of the formation of cytochrome c channels in the mitochondrial outer membrane by proapoptotic Bcl-2 family members, and opening of mitochondrial permeability transition (MPT) pores leading to the release of cytochrome c by the outer membrane ruptures, could be involved (51). Oxidative stress has been reported to damage components of the MPT pores, such as mitochondrial adenine nucleotide translocase (52), and it is also known that p53, which in turn affects the expression of both Bax and Bcl-2, can be turned on by oxidative stress (53,54). We detected modifications in the cytochrome c protein level, with a release from the mitochondria to the cytosol induced by aging. Melatonin was able to inhibit these changes and normalize both the cytosolic and mitochondrial cytochrome c content. Similar results have been described recently in a model of neural apoptosis induced by homocysteine in hippocampus, with inhibition on cytochrome c release by melatonin administration (55). The inhibitory effects of melatonin on cytochrome c release could be related to its function as an antioxidant and free radical scavenger (56,57).

The intrinsic pathway involves signals originating from various stimuli which are potently inhibited by Bcl-2 (58). Diverse investigations have shown that cellular adenosine triphosphate (ATP) depletion initiates the translocation of Bax, a proapoptotic Bcl-2 family member protein, from the cytosol to the outer mitochondrial membrane by mechanisms that remain unclear. The translocation of Bax causes mitochondrial dysfunction and swelling, and can induce the efflux of cytochrome c to the cytosol (51). Bcl-2 functions to prevent cell death, whereas Bax appears to accelerate the cell death signal (58). Our results confirm a significant decrease in Bcl-2 content accompanied by the increase of Bax on mitochondria. Furthermore, melatonin treatment increased the levels of antiapoptotic Bcl-2 and reduced the proapoptotic protein Bax. Although there is a report that melatonin induces cell death in human B-lymphoma cells by downregulation of Bcl-2 and cytochrome c release (59), most studies support an antiapoptotic role. Thus, the ability of melatonin to enhance the level of Bcl-2 has been demonstrated in rat brain (55), and it has been shown that melatonin treatment is able to prevent H2O2-induced apoptosis by regulating Bax expression in a model of cultured rat astrocytes (60). Recent research indicates that early melatonin supplementation significantly reduces upregulated expression of Bax and caspase-3 in a transgenic mouse model of Alzheimer's disease (61). Protective effects of melatonin appear to be related to its antioxidant capacity, which limits loss of intramitochondrial glutathione and lowers mitochondrial protein damage (62), and to the improvement in the electron transport chain activity (63). A major consequence is the prevention of the harmful reduction in the mitochondrial membrane potential that may trigger MPT pores opening and the apoptotic cascade (64).

The present data support that, in our model of aging, caspase-3 activation and apoptotic cell death is induced by ROS, via the intrinsic signaling pathway, and that the antiapoptotic action provided by melatonin is related to its antioxidant effect, with reduction of cytochrome c release by the modulation of Bcl-2 and Bax genes. Nevertheless, mammalian tissues are differentiated to exert specific functions, and whereas apoptosis may contribute during aging to the loss of irreplaceable cells and the development of degenerative diseases in differentiated tissues, defective apoptosis, by avoiding the removal of damaged cells, may lead to cancer in regenerative tissues (5,10). Further studies are, thus, necessary to clarify the potential therapeutic use of the inhibition of oxidative stress and the intrinsic apoptotic pathway induced by melatonin in elderly persons.


   Acknowledgments
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 Abstract
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This work has been partially supported by Junta de Castilla y León (Spain).


   Footnotes
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 Discussion
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Decision Editor: Huber R. Warner, PhD

Received June 2, 2006

Accepted January 31, 2007


   References
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 Discussion
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