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Melatonin but not vitamins C and E maintains glutathione homeostasis in t-butyl hydroperoxide-induced mitochondrial oxidative stress

Departamento de Fisiología, Instituto de Biotecnología, Universidad de Granada, E-18012 Granada, Spain

¹Correspondence: Departamento de Fisiología, Facultad de Medicina, Avda. de Madrid 11, E-18012 Granada, Spain. E-mail: dacuna{at}goliat.ugr.es

SPECIFIC AIM

To assess whether the antioxidant properties of melatonin are suitable to maintain GSH homeostasis counteracting mitochondrial oxidative stress.

PRINCIPAL FINDINGS

1. Effect of melatonin, NAC, ascorbate, and Trolox on mitochondrial GSH and GSSG levels
In basal conditions, i.e., mitochondria incubated in the absence of t-BHP, melatonin (100 nM) significantly (P<0.001) increased the content of GSH and decreased that of GSSG in brain (GSH: 6.82±0.78 to 10.13±0.42 nmol/mg prot) and liver (GSH: 11.54±0.89 to 19.43±0.80 nmol/mg prot; GSSG: 1.44±0.22 to 0.06±0.007 nmol/mg prot) mitochondria. The other antioxidants tested were unable to exert a similar effect of melatonin, and only the dose of 1000 µM Trolox increased GSH levels (11.50 ± 0.43 nmol/mg prot) in brain mitochondria.

After incubation with 100 µM t-BHP, practically all GSH was oxidized to GSSG (Fig. 1) . Melatonin (100 nM) counteracted these toxic effects, recovering the basal levels of GSH and GSSG. In this situation, i.e., t-BHP-induced oxidative stress, the other antioxidants were unable to recover the GSH-GSSG balance. In other experiments, we found that t-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) was enough to neutralize the effects of t-BHP. Thus, melatonin not only prevents but also counteracts GSH oxidation by t-BHP. NAC, ascorbate, and Trolox were without effect in these experiments.

View larger version (24K): [in this window] [in a new window]   Figure 1. 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, followed by the addition of melatonin (100 nM), NAC (1000 µM), ascorbate (1000 µM), or Trolox (1000 µM). : control; •: melatonin 100 nM; : NAC, 1000 µM; : ascorbate 1000 µM; : Trolox 1000 M. Mean of six experiments per group, each assayed in duplicate. *P< 0.001 vs. 10 min.

2. Effect of melatonin, NAC, ascorbate, and Trolox on mitochondrial GPx and GRx activities
Melatonin (100 nM) significantly (P<0.001) increased the activity of the GPx in brain (1.01 0.10 to 4.61±0.32 µmol/min · mg prot) and liver (0.47±0.04 to 4.02±0.31 µmol/min · mg prot) mitochondria by four- and eightfold, respectively, compared with the basal levels of these enzymes. The activity of GRx in both brain (GRx: 0.11±0.008 to 0.16±0.008 µmol/min · mg prot) and liver (GRx: 0.007±0.02 to 0.16±0.006 µmol/min · mg prot) was also significantly (P<0.001) increased by melatonin. Neither NAC, ascorbate, nor Trolox were able to modify the activity of the glutathione-related enzymes.

The effect of incubation with t-BHP drastically reduced to undetectable values the activity of both GPx and GRx. These effects were significantly (P<0.001) counteracted by melatonin (100 nM), but not by NAC, ascorbate, or Trolox.

3. Effect of melatonin, NAC, ascorbate, and Trolox on mitochondrial hydroperoxides
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 (P<0.001) reduced both basal (1.21±0.03 to 0.82±0.05 nmol/mg prot) and t-BHP-induced (2.38±0.17 to 1.42±0.14 nmol/mg prot) mitochondrial hydroperoxide production. Regarding the other antioxidants tested, only the dose of 1000 µM Trolox reduced the levels of t-BHP-induced hydroperoxides (P<0.05), but was without effect on their basal production.

4. Effect of melatonin, NAC, ascorbate, and Trolox on mitochondrial oxidative complexes
To study any effect of these compounds on the electron transport chain, we measured the activity of respiratory complexes I, II-III, and IV 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 complexes I and IV in both brain and liver mitochondria (Fig. 2) . The activity of complex II-III was not affected by these antioxidants.

View larger version (46K): [in this window] [in a new window]   Figure 2. 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 were determined as stated in the Principal Findings. Mean of six experiments per group, each assayed in duplicate. *P < 0.01 and **P < 0.001 vs. control.

CONCLUSIONS AND SIGNIFICANCE

The main conclusion of this study is the demonstration, for the first time, that melatonin regulates glutathione redox status in brain and liver mitochondria, correcting it when it is disrupted by oxidative stress. An additional significant conclusion of this work is that melatonin improves respiratory chain activity increasing the activity of the complex I and IV. Although the concentration of melatonin used in the study was above its maximum plasma concentration (1 nM), we found intramitochondrial melatonin levels 100-fold higher than those of plasma. Altogether, these results support a physiological role of melatonin on mitochondrial homeostasis, a role not sustained by the other endogenous antioxidants tested, i.e., vitamins C and E and NAC.

In basal conditions, melatonin increased the 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 and on the activity and gene expression for some antioxidant enzymes, including glutathione-related enzymes. The increase in GSH content and the decrease in hydroperoxide level are related phenomena. If melatonin diminish mitochondrial hydroperoxides, the mitochondria do not consume GSH to remove them. Thus, melatonin action has two main consequences for these organelle: 1) causing a cyclic stimulation of the activity of GPx and GRx that regenerates GSH to be used in other antioxidant processes by the mitochondria, and 2) improving mitochondrial function by detoxifying hydroperoxides.

Another key finding of our study is that melatonin not only maintains a good redox status in basal conditions, but also in t-BHP-induced oxidative stress. It is well known that t-BHP dramatically increases the level of mitochondrial hydroperoxides and produces large amounts of ROS responsible for oxidative damage, partially due to the depletion of GSH. Micromolar concentration of t-BHP causes 80–90% oxidation of the mitochondrial GSH, and the high level of GSSG inhibits GPx and saturates GRx. Besides, t-BHP-induced ROS affects GRx, impairing the structure of this SH-dependent enzyme and causing oxidation of pyridine nucleotides such as NADPH, a limiting cofactor for GRx. Thus, melatonin recovers mitochondria from an irreversible oxidative damage due to 100 µM t-BHP. 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. Figure 3 summarizes the sites at which melatonin acts as antioxidant into the mitochondria in both basal and t-BHP-induced oxidative stress conditions.

View larger version (35K): [in this window] [in a new window]   Figure 3. Drawing of mitochondrial inner membrane and mitochondrial matrix showing the sites of melatonin action. Melatonin increases both the GPx and GRx activities, increasing GSH available to mitochondria. Melatonin also scavenges reactive oxygen species (R.O.S.) produced either by t-BHP or by electron transport chain itself. In both cases, melatonin undergoes an oxidation donating an electron (e-), yielding N-acetyl-5-methoxykynuramine.

From the findings of this study, two main questions arise: 1) are the antioxidant properties of melatonin the cause of the observed increase of complex I and IV activities? and 2) are these increases in complex I and IV activities followed by an increment in mitochondrial ATP synthesis? 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. The ability of melatonin to function as a direct free radical scavenger is related to its electron donating ability. Since the redox potential for melatonin is close to -700 mV (Prof. R. J. Reiter, personal communication), the data suggest that melatonin can improve respiratory chain activity by electron donation, an effect lacking in the other antioxidants (Fig. 3) . If the electron transport chain is coupled to oxidative phosphorylation, it is reasonable to presume that an increase in ATP synthesis should follow the melatonin effect. Thus, a direct effect of melatonin on respiratory chain complexes and its role on ATPase activity and/or ATP synthesis should be studied further.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.99-0865fje

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