Journal of Applied Physiology
Vol. 83, No. 2, pp. 354-358, August 1997
ENVIRONMENT

Acutely administered melatonin reduces oxidative damage in lung and brain induced by hyperbaric oxygen

Marta I. Pablos, Russel J. Reiter, Jin-Ing Chuang, Genaro G. Ortiz, Juan M. Guerrero, Ewa Sewerynek, Maria T. Agapito, Daniela Melchiorri, Richard Lawrence, and Susan M. Deneke

Department of Cellular and Structural Biology and Department of Medicine, University of Texas Health Science Center at San Antonio, San Antonio, Texas 78284-7762

ABSTRACT

INTRODUCTION

MATERIALS AND METHODS

RESULTS

DISCUSSION

ACKNOWLEDGEMENTS

FOOTNOTES

REFERENCES

ABSTRACT

Pablos, Marta I., Russel J. Reiter, Jin-Ing Chuang, Genaro G. Ortiz, Juan M. Guerrero, Ewa Sewerynek, Maria T. Agapito, Daniela Melchiorri, Richard Lawrence, and Susan M. Deneke. Acutely administered melatonin reduces oxidative damage in lung and brain induced by hyperbaric oxygen. J. Appl. Physiol. 83(2): 354-358, 1997.Hyperbaric oxygen exposure rapidly induces lipid peroxidation and cellular damage in a variety of organs. In this study, we demonstrate that the exposure of rats to 4 atmospheres of 100% oxygen for 90 min is associated with increased levels of lipid peroxidation products [malonaldehyde (MDA) and 4-hydroxyalkenals (4-HDA)] and with changes in the activities of two antioxidative enzymes [glutathione peroxidase (GPX) and glutathione reductase (GR)], as well as in the glutathione status in the lungs and in the brain. Products of lipid peroxidation increased after hyperbaric hyperoxia, both GPX and GR activities were decreased, and levels of total glutathione (reduced+oxidized) and glutathione disulfide (oxidized glutathione) increased in both lung and brain areas (cerebral cortex, hippocampus, hypothalamus, striatum, and cerebellum) but not in liver. When animals were injected with melatonin (10 mg/kg) immediately before the 90-min hyperbaric oxygen exposure, all measurements of oxidative damage were prevented and were similar to those in untreated control animals. Melatonin's actions may be related to a variety of mechanisms, some of which remain to be identified, including its ability to directly scavenge free radicals and its induction of antioxidative enzymes via specific melatonin receptors.

free radicals; antioxidant enzymes; lipid peroxidation; pineal hormone

INTRODUCTION

THE EXPOSURE OF ANIMALS to hyperbaric oxygen (HBO) in excess of 1.5 atmospheres induces marked injury to the lungs and to the central nervous system (10). At 4 atmospheres, seizure activity begins in control animals between 100 and 200 min of exposure, followed by death of the animals with severe pulmonary edema between 200 and 400 min of exposure. The toxicity of HBO is thought to be mediated by the production of oxygen free radicals, which leads to lipid peroxidation and tissue damage (31). In the brain, hyperoxia also changes the concentration of several neurotransmitter amino acids such as -aminobutyric acid (GABA) (6) and induces oxidation of essential enzymes such as Na⁺-K⁺-dependent adenonsinetriphosphatase (12).

Free radical-induced damage is attenuated physiologically by enzymatic and nonenzymatic defense mechanisms. The enzymes involved in inactivating oxygen radicals or their reaction products include superoxide dismutase, glutathione peroxidase (GPX), glutathione reductase (GR), catalase, and glucose-6-phosphate dehydrogenase. Nonenzymatic antioxidative agents include the water-soluble antioxidants, e.g., glutathione (GSH), and the lipophilic radical quenchers -tocopherol and melatonin. Melatonin is an indole that is synthesized in and secreted from the pineal gland during the night (22). Its lipophilicity ensures that melatonin rapidly enters cells where it may accumulate in the nucleus (15). Recently, it was demonstrated that melatonin is a free radical scavenger (22) and antioxidant that protects cells against the damage induced by several oxidative agents including lipopolysaccharide (28), paraquat (14), and carbon tetrachloride (7). Melatonin's antioxidative effects may be due to its direct free radical-scavenging ability, to a stimulatory action on the detoxifying enzyme GPX, or to mechanisms that have not yet been identified (20).

The purpose of the present study was to determine the possible protective role of melatonin against the damage induced by HBO in several tissues of rats. Liver was selected because of its high rate of free radical generation. Lung was chosen because of its primary exposure to the high PO₂ and because significant lung damage occurs during HBO exposure. The brain was investigated because of its high content of polyunsaturated fatty acids and its increased susceptibility to free radical generation and damage and because seizures are an early indicator of physiological damage in HBO-exposed animals.

MATERIALS AND METHODS

Before the study, male Sprague-Dawley rats (300 ± 25 g) were maintained in a light- and temperature-controlled room with water and food being available ad libitum. At 0900 on the day of the experiment, the rats were injected intraperitoneally with 10 mg/kg of melatonin or with diluent immediately before exposure to 100% oxygen at 4 atmospheres. Controls (non-HBO-exposed animals) were injected with diluent at the same time the melatonin-treated rats received their injections.

Animals were exposed to HBO for 90 min in individual Plexiglas chambers with continuous oxygen flow. The animals were observed continuously during this period. Rats were killed immediately after exposure. Liver and lungs were collected and frozen on solid CO₂. The brain was also collected and dissected into five parts (cerebral cortex, hippocampus, hypothalamus, striatum, and cerebellum). All tissues were kept frozen at 80°C until analyses were performed. Because only eight individual chambers were available for HBO exposure, the experiment was repeated three times to provide 12 animals/group. Control animals were kept in a cage in normal room air in the vicinity of the exposure chambers. After biochemical analyses were made, the data from the experiments were combined and subjected to an analysis of variance followed by Scheffé's test for significant differences.

The products of lipid peroxidation, i.e., malonaldehyde (MDA) and 4-hydroxyalkenals (4-HDA), were measured in all tissues. Tissues were homogenized in ice-cold 20 mM tris(hydroxymethyl)aminomethane buffer (pH = 7.4) with a Polytron-like stirrer to produce 1:10 homogenates. Homogenates were centrifuged at 3,000 g for 30 min at 4°C. The supernatant was collected and immediately assayed for products of lipid peroxidation (i.e., MDA and 4-HDA). A Bioxytech LPO-586 kit was used for these measurements as previously described (14).

GPX activity was measured by a coupled reaction with GR by using cumene hydroperoxide as substrate and measurement of the decrease in NADPH absorbance at 340 nm as reported previously (16). GR was assayed after the decrease in the NADPH absorbance at 340 nm in presence of glutathione disulfide (oxidized glutathione; GSSG) (9).

Total GSH (reduced GSH plus GSSG) and GSSG levels were assayed in tissue 1:5 homogenates (wt/vol). The homogenates were centrifuged in a Beckman desktop centrifuge at 15,500 revolutions/min for 15 min at 4°C. The supernatants were used to measure total GSH and GSSG.

Proteins were measured by using Bio-Rad Protein Assay kit II (Bio-Rad Laboratories, Hercules, CA) and used albumin as standard.

RESULTS

The changes in the levels of MDA+4-HDA as a consequence of HBO are shown in Figs. 1 and 2. Although no statistically significant changes were detected in the degree of lipid peroxidation in liver, the lungs showed a large increase in lipid peroxidation products (~72%) after HBO exposure for 90 min (Fig. 1). In lung, pretreatment of the animals with melatonin not only inhibited the stimulatory effect of HBO on lipid peroxidation but also depressed levels below those measured in the control animals not exposed to HBO. In each of the five neural tissues studied, HBO exposure significantly increased levels of MDA+4-HDA; in each case, the increase was prevented when the animals had been pretreated with melatonin (Fig. 2).

No changes in either GPX or GR activity in the liver occurred as a consequence of either HBO exposure or melatonin treatment (Fig. 3). However, in lung tissue both GPX and GR activities were significantly reduced by HBO exposure. Melatonin pretreatment reversed the inhibitory effects of HBO on the activity of both enzymes. The same effect was observed in neural tissues. Again, HBO reduced enzyme activities, with the reductions being prevented by melatonin pretreatment (Fig. 4).

The total GSH and GSSG levels were measured in both the nonneural and neural tissues collected (Table 1). In general, total GSH and GSSG levels increased to a relatively small degree (10-20%) in all tissues, although the observed increases were not statistically significant in all cases (e.g., in liver) because of the variation among the samples. The relative percentage of GSSG in the total level was also increased in most cases. In no case was there any elevation of total GSH or GSSG in the melatonin+HBO group compared with the unexposed controls.

Table  1.   Total glutathione, glutathione disulfide, and glutathione disulfide-to-total glutathione ratio in all tested tissues Tissue Total GSH, µmol/g tissue GSSG, µmol/g tissue %GSSG/ Total GSH Liver   Control 4.03 ± 0.39  0.358 ± 0.10  8.87 ± 0.77    HBO 4.79 ± 0.14  0.451 ± 0.03  9.54 ± 0.25    HBO + Melatonin 4.41 ± 0.92  0.405 ± 0.12  8.97 ± 0.13  Lung   Control 1.83 ± 0.033  0.081 ± 0.003  4.41 ± 0.11    HBO 1.89 ± 0.091  0.089 ± 0.005 4.68 ± 0.12    HBO + Melatonin 1.83 ± 0.015  0.080 ± 0.008  4.29 ± 0.08  Cerebral cortex   Control 1.68 ± 0.052  0.038 ± 0.002  2.28 ± 0.04    HBO 1.84 ± 0.162 0.046 ± 0.001* 2.52 ± 0.12   HBO + Melatonin 1.71 ± 0.058  0.040 ± 0.003  2.35 ± 0.05  Hippocampus   Control 1.44 ± 0.041  0.091 ± 0.002  6.32 ± 0.05    HBO 1.54 ± 0.050* 0.106 ± 0.003* 6.81 ± 0.06*   HBO + Melatonin 1.46 ± 0.013  0.095 ± 0.005  6.69 ± 0.26  Hypothalamus   Control 1.14 ± 0.06  0.051 ± 0.011  4.38 ± 0.35    HBO 1.35 ± 0.09* 0.085 ± 0.006* 6.27 ± 0.07*   HBO + Melatonin 1.09 ± 0.05  0.031 ± 0.003  2.82 ± 0.30  Striatum   Control 1.48 ± 0.02  0.065 ± 0.015  4.28 ± 0.75    HBO 1.57 ± 0.06 0.079 ± 0.003 5.30 ± 0.32   HBO + Melatonin 1.36 ± 0.10  0.063 ± 0.006  4.58 ± 0.05  Cerebellum   Control 1.24 ± 0.03  0.028 ± 0.006  2.25 ± 0.25    HBO 1.72 ± 0.19* 0.039 ± 0.002* 2.29 ± 0.01    HBO + Melatonin 1.21 ± 0.11  0.027 ± 0.005  2.19 ± 0.05 Values are means ± SE; n, 12 animals/group. GSH, glutathione; GSSG, oxidized glutathione (GSH disulfide); HBO, hyperbaric O2. See MATERIALS AND METHODS for details. Significantly greater than control levels: * P < 0.01; P < 0.05.

DISCUSSION

Melatonin was recently discovered to be a free radical scavenger that protects cells from damage induced by a variety of oxidants (7, 14, 20, 28). In this study, we demonstrate for the first time that melatonin protects against HBO-induced lipid peroxidation in several tissues of rats. HBO exposure induced damage in lung and brain, whereas the changes observed in the liver were not statistically significant. In addition to increased levels of products of lipid peroxidation, reductions in antioxidative enzymes and alterations in both total and GSSG concentrations were observed. In all tissues tested except in liver, HBO induced a significant increase in levels of MDA+4-HDA. An increase in both lipid peroxides and reactive oxygen species may lead to an inactivation of detoxifying enzymes such as GPX by splitting of the peptide chain (18). These findings are consistent with previous studies that demonstrate that GPX activity is depressed after the treatment of animals with oxidants such as lead (25) and vanadate (24). In the present study, pretreatment of the HBO-exposed rats with melatonin prevented the drop in GPX activity associated with HBO; this was not unexpected because it was shown earlier that, pharmacologically, melatonin stimulates GPX activity (22). GR activity was also found to be inhibited after HBO exposure. This inhibition may be a consequence of oxidative stress-induced decrease in cellular-reducing equivalents (11). Oxidant stress may also be involved in increases in GSH synthesis as well as the increased accumulation of GSSG as seen in Table 1 (8).

Except in the liver, where no statistically significant alterations were detected, melatonin pretreatment inhibited the rise in MDA+4-HDA levels induced by HBO, restored normal enzyme activities, and maintained total GSH and GSSG levels at those seen in control animals. These effects of melatonin are consistent with an antioxidative role of this indole. The antioxidative effects of melatonin are probably mediated by at least two different mechanisms: one mechanism is receptor independent and involves the direct scavenging of toxic radicals by melatonin (13, 17, 20, 23, 26, 30). Both the hydroxyl radical (30) and the peroxyl radical (17, 26) are reported to be directly scavenged by melatonin. Furthermore, the indolyl cation radical, produced when melatonin contributes an electron to the hydroxyl radical, is believed to scavenge the superoxide anion radical (23), further reducing the number of toxic hydroxyl radicals that can be generated. A second means may involve melatonin's receptor-mediated effect on the genome, with the subsequent stimulation of radical detoxifying enzymes. Cellular membranes in a number of organs have been shown to possess melatonin receptors (29), and nuclear binding sites for melatonin have also been identified (1, 2). An action of melatonin via a nuclear binding site is consistent with previous studies that show the nuclear localization of melatonin (11, 24), which has been reported to be linked to gene expression (4).

Another potential mechanism whereby melatonin may protect against oxidative damage is due to its inhibition of nitric oxide synthase (NOS). NOS generates nitric oxide, which can degrade into the highly toxic hydroxyl radical in the presence of superoxide anion radicals. Previous studies by Pozo et al. (19) and Bettahi and colleagues (3) have shown that melatonin inhibits NOS activity in the cerebellum and hypothalamus, respectively. Future studies with both NOS and specific receptor agonists and antagonists will help to clarify the multiple mechanisms whereby melatonin acts directly and indirectly as an antioxidant.

There is always concern in studies of this type that cellular changes in certain constituents, e.g., GSSG, may have occurred in the interval between the time the animal was killed and the time the tissue was frozen rather than their being a result of the treatment. In the present study the tissues were collected and frozen on solid CO₂ within 30 s of death of the animal. Even this interval may be sufficient to account for a percentage of the change measured. However, because the measurements are consistent with one another in terms of the direction of change and so on, it is felt that the data accurately reflect the levels of the constituents at the time the animals were killed.

Melatonin given to otherwise untreated animals, i.e., not oxidatively challenged, usually has no effect on measured parameters of oxidative stress. In one study, however, melatonin was shown in vitro to reduce even basal levels of noninduced MDA+4-HDA in brain, lung, and liver (27). A similar observation was made herein, where the MDA+4-HDA concentrations in the lungs and cerebellum of the HBO exposed melatonin-treated rats were lower than those measured in the controls (Figs. 1 and 2, respectively). Also, melatonin has not been shown to exhibit prooxidant activity (5, 13).

Blood melatonin levels in the rat are normally derived primarily from the pineal gland, which produces only small amounts of the indole during the day and larger amounts at night (21); this circadian rhythm in melatonin production is reflected in a similar cycle in the blood, with nighttime blood melatonin levels being 10-15 times higher than those measured during the day. The dosage of melatonin (10 mg/kg) used in the present study created much higher levels in the blood than normally exist at night; thus the dosage used was pharmacological rather than physiological. Also, with regard to the 40-min half-life of melatonin in the blood, pharmacological levels of melatonin persisted in the blood, and presumably in the tissues, during the 90-min HBO-exposure period.

ACKNOWLEDGEMENTS

This work was supported by North Atlantic Treaty Organization Grant (M. I. Pablos).

FOOTNOTES

Address for reprint requests: R. J. Reiter, Dept. of Cellular and Structural Biology, The Univ. of Texas Health Science Center, 7703 Floyd Curl Drive, San Antonio, TX 78284-7762.

Received 25 November 1996; accepted in final form 25 March 1997.

REFERENCES