These data suggest a role of melatonin on mitochondrial homeostasis. The objective of this work was to study the influence of melatonin in the redox balance of mitochondrial GSH‐GSSG, in both liver and brain mitochondria incubated with tert‐butyl hydroperoxide ( t‐ BHP). This drug induces oxidative damage with important consequences for the GSH redox status ( 15 , 17 , 21 ). t‐BHP oxidizes pyridine nucleotides depleting mitochondrial GSH pool ( 8 ), and the drug also inhibits the activity of GPx and GRx. To evaluate the relevance of melatonin effect, we compared it with other classical antioxidants including N‐acetylcysteine (NAC), ascorbate (vitamin C) and Trolox (a soluble form of vitamin E).

The neurohormone melatonin displays notable antioxidant properties either in vivo and in vitro ( 13 ‐ 15 ). Melatonin behaves as a more efficient scavenger of free radicals than GSH and vitamin E ( 16 ‐ 19 ); it counteracts lipid peroxidation and avoids the oxygen radical attack to cytosolic proteins and DNA ( 20 ‐ 22 ). The efficiency of melatonin as endogenous antioxidant is partially due to its lipophilicity, which means that melatonin is ubiquitously located into the cell ( 23 ). Furthermore, melatonin maintains GSH homeostasis in different models of excitotoxicity ( 21 , 24 ‐ 26 ); it stimulates the activity of GPx ( 27 , 28 ) and increases gene expression for antioxidant enzymes such as GPx and manganese and cooper‐zinc superoxide dismutases ( 29 , 30 ). Recent data have shown that melatonin increases the activity of the oxidative complexes I and IV in both liver and brain mitochondria and it counteracts the ruthenium red‐induced inhibition of these enzymes ( 31 ). Melatonin also avoids the damage caused by bacterial lipopolysaccharide on liver and lung ( 32 ).

Glutathione (GSH) and its related enzymes glutathione peroxidase (GPx) and reductase (GRx) are the main mitochondrial antioxidant system (6‐8). Mitochondria do not contain catalase and are therefore largely, if not entirely, dependent on GSH and its recycling enzymes ( 6 ). Mitochondria do not synthesize GSH but obtain it by from cytosol through a multicomponent transport system, which explains the remarkable ability of mitochondria to take up and retain GSH ( 6 , 9 ). Studies in liver and kidney preparations have concluded that in chemical‐induced oxidative injury involving GSH depletion, it was the depletion of the mitochondrial GSH pool rather that of the cytosolic GSH pool critical for development of irreversible cellular damage ( 10 , 11 ). The GSH‐GSSG status is decisive to maintenance of numerous aspects of mitochondrial function, including membrane structure and integrity, intramitochondrial redox status, calcium homeostasis, and the activity of numerous mitochondrial enzymes containing essential sulfhydryl groups ( 9 , 12 ). When GSH levels are greatly decreased, H2O2 accumulates, and this leads to extensive mitochondrial damage. Other antioxidants may be involved in the protection of mitochondria, but mitochondrial GSH homeostasis is essential to ensure the protection against the oxidative damage of mitochondria and thus of the whole cell ( 6 ).

Mitochondria is the major intracellular source of superoxide anion (O 2 .‐ ) and hydrogen peroxide (H 2 O 2 ) because of the fixation of molecular oxygen in the respiratory chain ( 1 , 2 ). Small inefficiencies in the mitochondrial electron transport chain produce background levels of radical oxygen species (ROS) that can lead to severe mitochondrial dysfunction and cell death (2‐5).

Data are expressed as mean Å SE. A two‐way analysis of variance (ANOVA), followed by a Dunnet's post hoc test when appropriate, was used to compare means between groups. A P value of less than 0.05 was considered to be statistically significant.

Concentration of hydroperoxides (nmol/mg prot) into the mitochondria membranes was determined according to the principle of rapid Fe 2 + to Fe 3 + peroxide‐mediated oxidation under acid conditions ( 41 ). Hydroperoxide production was induced with i‐BPH, a potent inductor of peroxidation and, after incubation at 37°C for 30 min, xylenol orange was added. The mixture was incubated at room temperature for 70 min and the absorbance at 560 nm was noted. A standard curve was constructed with known concentrations of t ‐BPH.

Mitochondrial fractions were sonicated 30 s at 38 watts in 50 mM PBS, pH 7.4 and centrifuged 30 min at 1,000 g at 4°C. GPx activity (µmol/min‐ mg prot) was spectrophotometrically measured in the supernatant (25 μl) by a coupled reaction with GRx using cumene hydroperoxide as substrate ( 40 ). To measure GRx, the samples were treated as for the GPx assay. The supernatant obtained (20 μI) was added to a working solution containing EDTA‐Na 2 and GSSG. After incubation for 4 min at 37°C, the spectrophotometric determination of the GRx activity (µmol/min· mg prot) was started by addition of 5 μl of NADPH, and the oxidation of the NADPH was followed each 30 s for 4 min at 340 nm. Nonenzymatic NADPH oxidation was subtracted from the overall rate.

Both oxidized (GSSG) and reduced (GSH) forms (nmol/mg prot) were measured using a spectrophotometric assay ( 39 ). Mitochondrial fractions were sonicated in picric acid, centrifuged at 1,000 g for 15 min at 4°C, and the resultant supernatant was used immediately. To measure GSSG form, the GSH content in the sample (if any) was masked with 2 μl of 2‐vinylpyridine and the sample centrifuged at 1,000 g for 1 min at 4°C. For each assay, six GSH standard concentrations ranged from 0.0625 to 4.0 nmol/ml were prepared. Two hundred microliters of standards plus 200 μl of the mitochondrial supernatant (diluted 1:40 and 1:2 for GSH and GSSG assay, respectively) were incubated for 3 min at 30°C in the presence of 700 μl of 0.3 mM NADPH and 100 μl of 6 mM DTNB. After incubation, 5 μl of glutathione reductase (200 IU/ml) was added, and changes in absorbance were determined each 30 s for 2 min at 412 nm.

On the day of the experiment, pellets containing submitochondrial fractions were thawed, suspended in 1 ml of the incubation medium corresponding to the complex to be measured, and homogenized in a Polytron (position 5, 1 stroke x 7 s). To determine the complex I activity, submitochondrial fractions (0.2 to 0.4 mg protein/ml) were incubated in 4 ml (2 min at 25°C) of a medium comprising 120 mM KCl, 25 mM potassium‐MOPS 5 mM MgCl 2 , 5 mM potassium‐phosphate, 0.5 mM EDTA‐Tris, pH 7.2, at 25°C. The activity of the NADH‐CoQ oxidoreductase (complex I, in nmol reduced ferricyanide/min‐ mg prot) was measured following the rate of reduction of ferricyanide (1.5 mM) from the wavelength difference 440‐490 nm in a Spectronic 710 (Bausch and Lomb, Rochester, N.Y.) spectrophotometer in the presence of 0.8 μM antimycin and 5 mM azide ( 36 ). The reaction was started by the addition of 1 mM NADH. To measure the activity of the succinate cytochrome c oxidoreductase (complex II+III), submitochondrial fractions (0.2‐0.4 mg protein/ml) were incubated in 1 ml (2 min at 37 °C) of a medium containing a final reaction mixture of 7.8 mM sodium succinate, 0.78 mM sodium azide, 0.16 mM EDTA, 0.05 ml 10% BSA, 7.8 mM sodium‐phosphate buffer, pH 7.4. The reaction was started by the addition of 0.1 ml of 1% oxidized cytochrome c , and the complex II‐III activity was determined by the optical density changes at 550 nm ( 37 ). The activity of the cytochrome c oxidase (complex IV, in nmol oxidized cytochrome c /min‐ mg prot) was measured in a 0.8 ml medium containing submitochondrial fractions (0.2‐0.4 mg protein/ml) and 75 mM potassium‐phosphate pH 7.5 at 25 °C. The reaction was started by adding 0.2 ml of 0.5 % cytochrome c previously reduced with sodium borohydride, and measuring the absorbance at 550 nm ( 38 ).

Except when indicated, mitochondrial membranes (100 μg protein) were incubated with or without ί‐BHP at 37°C for 30 min and in the presence and absence of the other antioxidants used. Melatonin (2.5% ethanol:water) was added at a final concentration of 100 nM. In previous dose‐response (1 nM‐100 μM) experiments, we found that melatonin doses of 1‐10 nM were enough to influence mitochondrial homeostasis significantly. We chose 100 nM for the present experiments because at this dose the antioxidant effects of melatonin and its action on mitochondrial respiratory complexes activity were clearer. NAC, ascorbate (Asc), and Trolox (Tx) were added at a final concentrations of 10, 100 and 1,000 μM each. Control experiments were done by adding the vehicle in lieu of the antioxidant.

Mitochondria from rat brain and liver were purified as described elsewhere ( 33 ) with some modifications ( 31 ). Rats were killed by neck dislocation; the brain and liver were quickly removed, placed in dry cold ice, and processed immediately. All procedures were carried out at 4°C using fresh tissue samples. Forebrain and liver were weighed, diced, diluted in 8 vol of 0.25 mM sucrose, and homogenized with a Teflon pestle (Stuart Scientific, mod. SS2) (3 strokes in position 500 plus 10 strokes in position 300). After twice centrifugation at 800 g for 10 min, the supernatant obtained was subsequently centrifuged at 8,500 g for 10 min. The pellet was resuspended in 4 vol 0.25 mM sucrose, homogenized with a Polytron (3 strokes x 7 s in position 3) and centrifuged at 8,500 g for 10 min. This step was repeated twice to obtain the mitochondrial fraction pellet that was frozen to ‐80°C. To measure the specific activity of the mitochondrial oxidative complexes, submitochondrial particles were prepared by freezing and thawing (twice) the mitochondrial pellets suspended at 10 mg protein/ml in 140 mM KCl, 20 mM Tris‐HCl (pH 7.4), and homogenized in a Polytron with 1 stroke x 10 s in position 3 ( 34 ). Protein concentration in mitochondria and in submitochondrial particles was determined using bovine serum albumin as standard ( 35 ). The procedure permits the separation of particulate subfractions of mitochondria containing the bulk of the respiratory chain components. Electron microscopy studies were done to assess the breaking of the outer mitochondrial membrane (data not shown).

Activity of the respiratory chain complex I (A) and IV (B) in brain (cross‐hatched columns) and liver (hatched columns) mitochondria Rat brain and liver mitochondrial fractions were incubated with melatonin, NAC, ascorbate or Trolox, and the specific activity of each complex was determined as stated in Materials and Methods. Mean of six experiments per group, each assayed in duplicate. * P < 0.01 and ** P <0.001 vs. control.

To study any effect of these compounds on the electron transport chain, we measured the respiratory complex I, II‐III and IV activity in submitochondrial fractions incubated with melatonin (100 nM), NAC (100 μM), ascorbate (100 μM) and Trolox (100 μM). The results show that only melatonin significantly increased the activity of the complex I and IV in both brain and liver mitochondria (Fig. 3 ). The activity of complex II‐III was not affected by these antioxidants (data not shown).

Basal (open columns) and t ‐BHP‐induced (hatched columns) hydroperoxides in mitochondria Mitochondrial fractions were incubated with or without 100‐ μM t‐BHP at 37 °C for 30 min and in the presence of melatonin, NAC, Asc or Tx. Mean of six experiments per group, each assayed in duplicate. # P <0.001 vs. basal control; * P <0.05 and ** P < 0.001 vs. t‐BHP control.

Because the method to detect hydroperoxides is more sensitive in liver mitochondria than in brain mitochondria ( 41 ), hydroperoxides were measured in the former. Melatonin significantly reduced both basal and t ‐BHP‐induced mitochondrial hydroperoxide production (Fig. 2 ). Regarding the other antioxidants tested, only the dose of 1,000 μM Trolox reduced the levels of t ‐BHP‐induced hydroperoxides, but it was without effect on their basal production.

Melatonin increase the activity of the GPx in brain and liver mitochondria by 4 and 8 times, respectively, compared with the basal levels of these enzymes (Table 3 ). Neither NAC, ascorbate nor Trolox was able to modify the activity of the glutathione‐related enzymes.

Percentage of total glutathione oxidized by t ‐BHP treatment Brain (A) and liver (B) mitochondria (2.5 mg prot/ml) were incubated with 100 μM of t‐BHP for 10 min and followed by the addition of melatonin (100 nM), NAC (1,000 μM), ascorbate (1,000 μM) or Trolox (1,000 μM). ■: Control; ●: melatonin 100 nM; ○: NAC, 1,000 μM; ▼: ascorbate 1,000 μM; □: Trolox 1,000 M. Mean of six experiments per group, each assayed in duplicate. * P <0.001 vs. 10 min.

After incubation with 100 μM f‐BHP, practically all GSH was oxidized to GSSG (Table 2 ). Melatonin (100 nM) counteracted these toxic effects, recovering the basal levels of GSH and GSSG. In this situation, i.e., i‐BHP‐induced oxidative stress, the other antioxidants were unable to recovery GSH‐GSSG balance. In other experiments, we found that i‐BHP induced an oxidation of the 90% of total glutathione after 10 min of incubation (Fig. 1 ). Another 10 min of incubation in the presence of melatonin (100 nM) were enough to neutralize the effects of i‐BHP. Thus, melatonin not only prevents but also counteracts GSH oxidation by i‐BHP. NAC, ascorbate and Trolox were without effect in these experiments.

In basal conditions, i.e., mitochondria incubated in absence of i‐BHP, melatonin (100 nM) significantly increased the content of GSH and decreased that of GSSG in brain and liver mitochondria (Table 1 ). The other antioxidants tested were unable to exert a similar effect of melatonin, and only the dose of 1,000 μM Trolox increased GSH levels in brain mitochondria.

DISCUSSION

This study shows by the first time that melatonin, but not other endogenous antioxidants such as vitamins C and E, regulates glutathione redox status in brain and liver mitochondria, correcting it when it was disrupted by oxidative stress. In basal conditions, melatonin increased mitochondrial GSH pool, decreasing GSSG content. Moreover, melatonin reduced the mitochondrial hydroperoxide level and stimulated the activity of the two enzymes involved in the GSH‐GSSG balance: GPx and GRx. These results on mitochondria resemble data published elsewhere showing the effects of melatonin on GSH homeostasis in brain tissue (24) and on the activity and gene expression for some antioxidant enzymes, including glutathione‐related enzymes (27‐30). The increase in GSH content and the decrease in hydroperoxide level are related phenomena. If melatonin diminishes mitochondrial hydroperoxides, the mitochondria do not consume GSH to remove them. Thus, melatonin action has two main consequences for these organelle: a) causing a cyclic stimulation of the activity of GPx and GRx regenerates GSH to be used in other antioxidant processes by the mitochondria, and b) improves mitochondrial function detoxifying hydroperoxides.

Mitochondria are the most important source for free radicals into the cell (1‐3). Mitochondria produces O 2 •‐ and H 2 O 2 , and they should be detoxified (through GSH) to avoid the production of the highly toxic ‐ OH. Melatonin is a potent free radical scavenger (13‐16), and one molecule of melatonin scavenges two molecules of free radicals (one molecule of O 2 •‐ and one of ‐ OH, or two molecules of ‐ OH) (13, 42). Taken together, these data and the results of the present study support an important role for melatonin to maintain mitochondrial GSH homeostasis, simultaneously removing oxygen free radicals produced during oxidative stress.

Considering the other antioxidant evaluated, only Trolox showed some effect, but at a dose 104 times higher that that of melatonin. Therefore, melatonin is a much more efficient antioxidant than Trolox, conceivably because melatonin, but not Trolox or the other antioxidants, stimulates the activity of the glutathione‐related enzymes. Although ascorbate and vitamin E have important antioxidant properties, our results confirm previous data supporting melatonin as a better endogenous antioxidant than vitamins C and E and glutathione (13,16, 18, 19, 26, 43). The use of NAC in this study was considered for a negative control. NAC displays antioxidant properties because it is used for glutathione synthesis by the cell. Since glutathione synthesis takes place in the cytosol where mitochondria obtain it by transport, in our experimental paradigm (i.e., isolated mitochondria) NACs do not should show any effect.

Another key finding of our study is that melatonin not only maintains a good redox status in basal conditions, but is able to counteract the oxidative damage induced by t‐BHP on mitochondria, recovering GSH levels and scavenging hydroperoxides. Melatonin also recovers the activity GPx and GRx inhibited by i‐BHP. It is well known that t‐BHP dramatically increases the level of mitochondrial hydroperoxides and produces large amounts of ROS responsible for oxidative damage (6, 8, 12), partially due to the depletion of GSH. At a low dose of i‐BHP, GPx and GRx may recover GSH from GSSG. Micromolar concentrations of i‐BHP cause 80‐90% oxidation of the mitochondrial GSH, and the high level of GSSG inhibits GPx and saturates GRx (8). GRx will be also blocked because t‐BHP‐induced ROS directly impair the structure of this SH‐dependent enzyme (44) and t‐BHP itself causes oxidation of pyridine nucleotides such as NADPH, a limiting cofactor for GRx (8, 12, 44). Our results suggest that melatonin, but not other antioxidants, recovers mitochondria from an irreversible oxidative damage due to 100 μM t‐BHP.

Some authors described that compounds behaving either as electron donors to the mitochondrial electron transport chain or as mitochondrial respiring substrates support the reduction of GSSG formed during oxidative stress (8, 45). The ability of melatonin to function as a direct free radical scavenger may relate to its electron donating ability (15, 42). The data suggest that melatonin can perhaps improve respiratory chain activity by electron donation, an effect lacking in the other antioxidants. Supporting this hypothesis is the increased activity of complex I and IV found after mitochondrial incubation with melatonin but not ascorbate or Trolox.

The dose of melatonin used in this study (100 nM) is above its normal plasma concentration measured during its nocturnal peak (1 nM), suggesting a pharmacological effect of the neurohormone. However, we recently reported a significant effect of 1‐10 nM melatonin on mitochondrial respiratory chain (31). On the other hand, it is suggested that melatonin may accumulate in some cellular structures such as cell membrane, cytosol and nuclei (23, 46), and we found an intramitochondrial melatonin levels 100 times higher than those of plasma (unpublished data). Altogether, these data support a physiological role of melatonin in mitochondrial homeostasis.

Many reports describe protective effects of melatonin in degenerative disorders and in different models of oxidative stress such as MPTP‐induced Parkinson's disease, kainate‐induced neurotoxicity, lipopolysaccharide‐induced sepsis and aging (22, 24, 25, 32, 47‐50). A final common pathway of these diseases is the oxidative damage of the cell, including mitochondria, an effect mainly due to a severe alteration of glutathione redox status (51, 52). It has been suggested that the ability of a cell to synthesize glutathione rapidly in response to a stress may be as important or perhaps more important than the initial cellular level of glutathione (6). From our results it can be assumed that melatonin maintains GSH homeostasis and counteracts oxidative damage in mitochondria without requiring GSH synthesis de novo. Thus, melatonin behaves as an endogenous mitochondrial protector that may be used in clinical treatments against oxidative damage‐dependent diseases.