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Review Article

Antioxidative Effects of Melatonin in Protection Against Cellular Damage Caused by Ionizing Radiation

Magorzata Karbownik^*, and Russel J. Reiter^*,1

^* Department of Cellular and Structural Biology, University of Texas Health Science Center, San Antonio, Texas 78229–3900; and
Department of Thyroidology, Institute of Endocrinology, Medical University of ód, 91–425 ód, Poland

	Abstract

Top Abstract Introduction Biological Effects of Ionizing... Antioxidative Properties of... Melatonin Counteracts... Concluding Remarks References

 
Ionizing radiation is classified as a potent carcinogen, and its injury to living cells is, to a large extent, due to oxidative stress. The molecule most often reported to be damaged by ionizing radiation is DNA. Hydroxyl radicals (^•OH), considered the most damaging of all free radicals generated in organisms, are often responsible for DNA damage caused by ionizing radiation. Melatonin, N-acetyl-5-methoxytryptamine, is a well-known antioxidant that protects DNA, lipids, and proteins from free-radical damage. The indoleamine manifests its antioxidative properties by stimulating the activities of antioxidant enzymes and scavenging free radicals directly or indirectly. Among known antioxidants, melatonin is a highly effective scavenger of ^•OH. Melatonin is distributed ubiquitously in organisms and, as far as is known, in all cellular compartments, and it quickly passes through all biological membranes. The protective effects of melatonin against oxidative stress caused by ionizing radiation have been documented in in vitro and in vivo studies in different species and in in vitro experiments that used human tissues, as well as when melatonin was given to humans and then tissues collected and subjected to ionizing radiation. The radioprotective effects of melatonin against cellular damage caused by oxidative stress and its low toxicity make this molecule a potential supplement in the treatment or co-treatment in situations where the effects of ionizing radiation are to be minimized.

	Introduction

Top Abstract Introduction Biological Effects of Ionizing... Antioxidative Properties of... Melatonin Counteracts... Concluding Remarks References

 
Ionizing radiation is classified as a potent carcinogen (1-4). When interacting with living cells, it causes a variety of changes depending on exposed and absorbed dose, duration of exposure and interval after exposure, and susceptibility of tissues to ionizing radiation. The sensitivity of cells to ionizing radiation depends especially on the rate of differentiation and accompanying factors of the tissue as well as on the efficiency of the intrinsic antioxidative defense systems (3, 5-7).

	Biological Effects of Ionizing Radiation Due to the Generation of Free Radicals

Top Abstract Introduction Biological Effects of Ionizing... Antioxidative Properties of... Melatonin Counteracts... Concluding Remarks References

 
Radiation injury to living cells is, to large extent, due to oxidative stress (1-9). Reactive oxygen species (ROS) and free radicals induced by partial reduction of oxygen (O₂) react with cellular macromolecules (i.e., nucleic acids, lipids, proteins, and carbohydrates) (10) and damage them. The interaction of ionizing radiation with living cells induces a variety of reaction products and a complex chain reaction in which many macromolecules and their degradation products participate. The assumption that destructive processes initiated by ionizing radiation begin exclusively in a single subcellular organelle is questionable.

Major biomarkers of oxidative damage to living cells are (i) lipid peroxidation (LPO) products, comprising volatile hydrocarbons measurable in exhaled air, such as ethane and penthane, and isoprostanes and aldehydic products measurable in tissues and body fluids; (ii) DNA-hydroxylation products (e.g., 8-hydroxy-2'-deoxyguanosine (8-OHdG)) and microscopic indices of damage such as chromosomal aberrations and micronuclei; and (iii) protein hydroxylation products such as oxidized amino acids (11, 12).

Oxidative Damage to DNA.
Ionizing radiation is a well-established carcinogen due to the resulting oxidative damage (1-3), and the molecule most often reported to be damaged by this physical agent is DNA. Interactions of ionizing radiation with DNA consist of the direct ionization of DNA (direct effect) (13) and its reaction with surrounding water molecules (the indirect effect), followed by DNA destruction by the induced radicals (^•OH, e^–• and, to much lesser extent, H^•). A wide variety of biochemical consequences of radiation-induced damage to DNA (5) occurs because of free-radical attack (14).

About 60%–70% of cellular DNA damage produced by ionizing radiation is estimated to be caused by ^•OH, formed from the radiolysis of water (8). ^•OH are considered the most damaging of all free radicals generated in organisms (15). All biological molecules are targets for ^•OH, and this radical reacts with them at a very high rate, exceeding 10⁹ M^-1 s^-1. Thus, the intracellular diffusion distance of ^•OH is on the order of Ångstroms (16), and the damage it produces is essentially at the site where it was generated. Of the DNA damage produced by ^•OH, oxidized bases, abasic sites, DNA-DNA intrastrand adducts, DNA single and double strand breaks, and DNA-protein cross-links are the most significant (17). These occur primarily by interaction of free radicals with DNA bases and, to a lesser extent, with DNA sugars (14).

Among bases and, generally among nucleic acid components, guanine is the most susceptible DNA target for oxidative reactions mediated by ^•OH and other free radicals, and it exhibits the lowest ionization potential (17). Thus, one of the most mutagenic lesions, and the most abundant lesion formed in irradiated chromatin is 8-hydroxyguanine (18-21). Once formed, this product can be repaired by several mechanisms (22). Under laboratory conditions, one of the most common and abundant measurable oxidative DNA base adducts is 8-OHdG. This is reported to be a key biomarker related to carcinogenesis (23). The increased oxidation of guanine bases after exposure to ionizing radiation has been observed in numerous studies as well as in different tissues and species (18, 23-25).

The interaction of free radicals with sugar moieties leads to the cleavage of the sugar-phosphate backbone of DNA followed by single-strand breaks that undergo repair processes relatively easily (1). On the other hand, double-strand breaks have more serious consequences. Double-strand breaks are well correlated with the cytotoxic effects of ionizing radiation and are considered the primary lesion involved in cellular death (26).

If DNA repair mechanisms, which are induced after exposure to ionizing radiation, are inefficient, the damaged DNA strands that are copied during replication lead to mutagenesis and carcinogenesis (3). The damaging effects of ionizing radiation lead to cell death and are associated with an increased risk for numerous genetically determined diseases (27).

Among other indices of DNA damage caused by ionizing radiation are chromosome aberrations and micronuclei formation; these are apparent when irradiated cells are observed microscopically (28-33).

Oxidative Damage to Biological Membranes.
Besides DNA, lipids and proteins are also attacked by free radicals induced by ionizing radiation (9, 34, 35). The initiated chain reaction caused by ionizing radiation leads to the formation of a variety of degradation products in biological membranes including products of lipid breakdown. These degradation products induce changes in membrane structure and function that are exaggerated by the accumulation of free radical–mutilated proteins. These degradation products make membranes more rigid (less fluid) and furthermore, after they migrate out of the membrane they react with proteins and nucleic acids, thereby contributing to DNA damage and mutagenesis. Significant changes in structure and function of membranes result in cell death via apoptosis (36).

In a lipid-rich environment, as in membranes, both the phospholipid acyl chain and phospholipid backbone as well as cholesterol are subject to radiation damage. However, polyunsaturated hydrocarbon moieties of the phospholipids are particularly sensitive to attack by reactive oxygen species. Exposure of liposomes to -radiation confirms the particular susceptibility of polyunsaturated acyl chains to oxidative damage (37). Thus, the radical chain reactions induced by ionizing radiation result in the production of hydroperoxides at the unsaturated sites of lipid acyl chains; hydroperoxide residues change the hydrophobic interactions between adjacent chains of phospholipids allowing easier penetration of water molecules to the most external portion of the bilayer (38), thereby altering the electric constant across the bilayer (39). The altered gradient of water concentrations throughout the membrane has relevant biological consequences since it changes membrane substructure directly and indirectly leads to the degradation of lipids and proteins. Membrane damage caused by ionizing radiation is of particular importance because these altered molecules are not repaired after exposure either to low fractionated doses or to a single acute dose of ionizing radiation (40).

Another commonly measured parameter of lipid damage after ionizing radiation exposure is thiobarbituric acid reactive substances (TBARS). These products, which include malondialdehyde (MDA) and 4-hydroxyalkenals (4-HDA), result from the interaction of free radicals with polyunsaturated fatty acids (PUFA). The method for measuring TBARS, however, has some limitations (41, 42), and the quantity of TBARS measured is highly dependent on the interval after ionizing radiation (41, 43-46). The formation of TBARS as an index of lipid breakdown is a complex process which is due to the high susceptibility of PUFA to free radicals, the production of lipid peroxyl radicals (LOO^•), endoperoxides and hydroperoxides (47), and, thereafter, the propagation of a chain reaction leading to the extensive lipid damage (48). During this process the fine structure of the biological membranes is modified gradually in different ways and to different extents by end products of lipid peroxidation. Thus, the lack of detection of TBARS in membranes shortly after oxidative stress (e.g., ionizing radiation), and their increasing levels at later intervals is the result of a chain reaction in membranes and the induction of a variety of indirect mechanisms related to the decrease in antioxidant capacity of the membranes.

Another biochemical parameter used as an index of radiation-induced damage to membranes is the level of isoprostanes. In humans, ethane exhaled from the respiratory system is known to be increased after total body irradiation (49). The level of conjugated dienes and ketone dienes, other indices of LPO, also increase in rat liver after exposure to ionizing radiation (45).

The evaluation of oxidative damage to membranes by fluorescence spectroscopic methods is often used because the techniques are both sensitive and reliable (40, 42, 50-52). The motion of spin labels and fluorescence probes intercalated into membranes results from their interaction with both lipids and proteins (53). Two common specific lipophilic probes (i.e., 1,6-diphenyl-1,3,5-hexatriene (DPH) and 1-[4-(trimethylammonium)phenyl]-6-phenyl-1,3,5-hexatriene p-toluene sulfonate (TMA-DPH)), are used to measure membrane fluidity (the inverse of membrane rigidity). Of these two probes, TMA-DPH, anchored close to the polar heads of the phospholipid bilayer (the more rigid part of the membrane), seems to be superior to DPH in estimating membrane fluidity. Anisotropy values recorded for TMA-DPH are usually higher, and they usually appear earlier than those for DPH after exposure to ionizing radiation (52). The reliability of membrane fluidity rather than the measurement of TBARS in biological membranes, under certain experimental conditions, results for different reasons. The techniques that use ethidium bromide to reveal changes in membrane permeability show that these alterations begin 6–8 hr after exposure to irradiation (54). The changes in membrane fluidity depend on both the lipid and protein composition of the specific membrane being investigated (55).

Changes in membrane fluidity after exposure to ionizing radiation provides information not only about the lipid bilayer environment but also about changes in the amount of protein damage and the accumulation of water. The latter alteration can change intra- and intermolecular forces that determine the structure of the multilayered membrane, thus influencing the phospholipid heads within the depth of the membrane. As noted above, exposure of biological membranes to ionizing radiation also seriously damages intrinsic proteins. Detectable graded changes in the structure and function of proteins due to the action of reactive oxygen species (56) are reported after increasing doses of ionizing radiation (35, 57, 58). Thus, alterations in the properties of biological membranes caused by ionizing radiation are due to complex processes involving both lipids and proteins. Therefore, changes in membrane fluidity reliably represent the alterations in membrane structure, whereas changes in LPO result only from lipid damage.

Protective Action of Ionizing Radiation Applied in Low Doses.
The majority of studies using ionizing radiation are devoted to its negative effects, related especially to genetic damage seen after high radiation doses (4). However, a substantial body of data provides evidence for a protective action of ionizing radiation when applied in low doses (59, 60). Ionizing radiation at low levels brings about what are referred to as adaptive responses and the stimulation of protective physiological mechanisms. These adaptive responses are specific for doses typically below 0.5 Gy (61). The antioxidant properties of ionizing radiation at low doses as well as the protective effects of pretreatment with low-dose ionizing radiation against the damaging effects of high doses have been observed in a variety of studies using different parameters of oxidative damage (e.g., chromosome aberrations and micronuclei formation in human blood lymphocytes (12, 28) and the level of LPO and the activities of the antioxidant enzymes (62-64)). Thus, the effects of low-dose ionizing radiation are similar to those brought about by antioxidants. Interestingly, in a comparative study of ionizing radiation at a 50 cGy dose and the antioxidant melatonin, these factors reveal a similar protective action against chemically induced LPO in mouse brain (64).

Physiological Defense Against Oxidative Stress.
Efficient defense and repair mechanisms exist in living cells to protect against oxidant species (65, 66). The antioxidative defense system is composed of methods to (i) transfer sensitive material to compartments better protected from the action of reactive species, (ii) complex transition metals, a potential source of electrons, thereby rendering them unreactive, (iii) inhibit vulnerable processes such as DNA replication, (iv) repair damaged molecules, (v) initiate apoptosis, and, possibly the most important considering the lability to internal and external modifying factors, (vi) activate antioxidant enzymes and finally (vii) use a variety of direct free radical scavengers (65, 66). Of the enzymes involved in antioxidative defense, particularly well documented are the antioxidative properties of the superoxide dismutases (SOD), glutathione peroxidases (GSH-Px), and catalase (CAT) (11).

Since the amount of DNA damage caused by ionizing radiation is correlated with the intensity of oxidative stress (1-3), with the efficacy of defense mechanisms that metabolize toxic intermediates (67) and with intrinsic repair mechanisms (22), there are several potential means to reduce macromolecular damage due to ionizing radiation. Since radiation-induced cellular DNA damage is primarily attributed to the damaging effects of free radicals, molecules with direct free radical scavenging properties are particularly promising as radioprotectors. The involvement of free radical scavengers in protecting against radiation damage is emphasized by the observation that whole body irradiation decreases the total antioxidant capacity of the organism and depletes the levels of known antioxidants such as ascorbic acid and uric acid as they are used in the scavenging of free radicals (68).

A number of different substances have been examined as to their radioprotective effects against cellular damage caused by ionizing radiation. Several antioxidants have been proven to be efficient protectors against ionizing radiation (e.g., 5-aminosalicylic acid and ascorbate against 8-OHdG formation in calf thymus DNA (24), flavonoids, especially luteolin, against lipid peroxidation, and micronuclei formation in mouse peripheral blood erythrocytes (69) and panthotenol against lipid peroxidation in rat liver (45). These free radical scavengers presumably have protective effects via similar actions.

	Antioxidative Properties of Melatonin and its Potential Protection Against Carcinogenesis

Top Abstract Introduction Biological Effects of Ionizing... Antioxidative Properties of... Melatonin Counteracts... Concluding Remarks References

 
Melatonin (N-acetyl-5-methoxytryptamine), which was discovered about 40 years ago, is a ubiquitously acting molecule with several functions. It was initially recognized as a molecule related to neuroendocrine physiology, especially reproductive physiology (70, 71). Thereafter, melatonin was found to be involved in the control of circadian rhythms and possibly sleep processes in diurnal species (72, 73). In more recent years, melatonin's ability to influence immune function (74) and cancer growth (75), and its antioxidative properties have been uncovered (76).

Distribution and Mechanisms of Action.
Important features of a satisfactory antioxidant are its wide distribution within tissues and cells as well as in subcellular compartments, its ability to cross morphophysiological barriers, and its rapid transport into cells. The apparent ability of melatonin to enter all cells in the organism and all cellular compartments relates to its physical and chemical properties. Melatonin is highly lipid (77) and somewhat aqueous (78) soluble. Binding proteins have been described for melatonin, which may account for the ability of cells to concentrate it against a gradient. Within cell membranes, melatonin may align itself in a position to scavenge free radicals in both the lipid and aqueous environments of the membrane (79, 80).

Within a single cell, melatonin has been found in different compartments (e.g., in the cytosol (81), nucleus (81-83), and cellular membranes (79)); however, highest concentrations of the indole may be presented in the nucleus (82, 83). Thus, melatonin may be in highest concentrations in the portion of the cell (the nucleus) that contains the most sensitive target molecule to ionizing radiation, namely, DNA.

The widespread subcellular distribution of melatonin may allow it to interact with all molecules, thereby reducing oxidative damage to molecules in both the lipid and aqueous environments of the cell. Certainly, this view is supported by the experimental observations showing that melatonin protects lipids in membranes, proteins in the cytosol, and DNA in the nucleus from free radical damage (84).

Clearly, some effects of melatonin are mediated via membrane (85-87) and possibly by nuclear receptors (88-91). Its direct free radical scavenging effects, however, are receptor-independent (92, 93). Some of melatonin's indirect antioxidative actions (e.g., stimulation of antioxidative enzymes) may well involve specific receptors for the indole (84).

Both membrane and nuclear receptors for melatonin have been identified. Membrane receptors seem to be widespread but have been most thoroughly investigated in the brain (85) and immune system (86). These receptors are members of the G-protein-linked receptor superfamily (87). Melatonin also influences a number of intracellular events that rely on calcium via binding to calmodulin (94). Of recent interest is the identification of nuclear binding sites for melatonin. Melatonin nuclear binding sites were initially described in the liver (88, 89) and subsequently found essentially in all cells (90, 91).

At least in part, the marked protective effects of melatonin against oxidative stress are aided by its ability to cross all biological membranes and the fact that it does not require a receptor for this action. The fact that melatonin diffuses easily and quickly passes through all biological membranes to enter cells and their subcellular compartments is documented (84, 95). The organs of origin of melatonin are still the subject of scientific investigation; however, the pineal gland is generally agreed to be a primary source of this and other indoleamines (84). The highest production of pineal melatonin occurs at night, resulting in high nocturnal concentrations in blood, and consequently, in other bodily fluids as well. In bodily fluids such as the saliva, the nighttime increase in the concentration of melatonin is less than that measured in the blood (84). Whether similar 24-hr fluctuations of melatonin occur within cells remains essentially uninvestigated.

There is some evidence that any external or internal insult that initiates free radical generation in an organism may lead to a reduction in circulating melatonin levels (96-99). This feature of melatonin is especially valuable in case of the exposure to ionizing radiation. Ionizing radiation causes oxidative damage to radiosensitive tissues within an extremely short period, and possible protection against it would require the rapid transfer of an antioxidant to the sensitive cells. At this point, it is not known whether there is a differential uptake of melatonin by various tissues. The presence of melatonin or even its production by radiosensitive tissues such as the retina (100), gastrointestinal tract (101), ovaries (102), testes (103), and bone marrow (104) suggests that tissues easily damaged by ionizing radiation may either produce or quickly take up melatonin from the blood.

Melatonin as a Free Radical Scavenger.
The ability of melatonin to scavenge free radicals is undoubtedly an important property in its protection against oxidative stress. The discovery that melatonin is effective in antioxidative defense is related to the finding that under both in vitro and in vivo conditions this molecule directly scavenges the highly toxic ^•OH to form cyclic 3-hydroxymelatonin (3-OHM), a stable metabolite of melatonin (105-112). Melatonin scavenges ^•OH with a very high rate constant, roughly on the order of 2.7 x 10¹⁰ M^-1 s^-1 (107). Of particular significance is the fact that the exposure of rats to whole body irradiation increases the level of urinary 3-OHM (113). These levels correlate well with the number ^•OH generated in vivo (110). Moreover, melatonin given prior to whole body irradiation caused a further increase in 3-OHM formation when compared with the effect of the treatment with melatonin only (110). Thus, the metabolite of melatonin, 3-OHM, may be a valuable biomarker of in vivo ^•OH generation. The finding of 3-OHM in human and rat urine under normal physiological conditions (110) provides direct evidence that endogenously produced melatonin scavenges ^•OH to form its metabolite, 3-OHM. The fact that oxidative damage caused by ionizing radiation is known to be related to the action of ^•OH (8) makes the finding of Tan et al. (105, 110) and other investigators (106-109, 111, 112) particularly important. Interestingly, some methoxylated and hydroxylated indoles, particularly 5-methoxytryptamine (105, 107), 6-chloromelatonin, 5-hydroxytryptamine, 6-hydroxymelatonin (107), and pinoline (6-methoxy-tetrahydro-beta-carboline) (112) also have some ability to scavenge ^•OH.

Besides ^•OH, several other reactive species are also scavenged by melatonin. Compared with ^•OH, these agents are considered to be less toxic and less involved in the damage caused by ionizing radiation. However, several are involved in radical chain reactions that directly or indirectly influence cellular damage. Since ionizing radiation causes lipid damage as well (34, 35, 37-39) and because LOO^• propagates the radical chain reaction in the lipid environment, the reported ability of melatonin to scavenge LOO^• (114-118) is of understandable interest. One report even claims that melatonin is more effective in scavenging LOO^• than is vitamin E (114), which is regarded as a premier lipid antioxidant. Although the superiority of melatonin to vitamin E in scavenging LOO^• has not been confirmed (117), the efficacy of melatonin as a lipid antioxidant, regardless of the mechanisms involved, is not questioned (50, 51, 119, 120).

Melatonin has also been shown to quench singlet oxygen (¹O₂) (121), a high energy form of O₂ that exhibits high toxicity at the molecular level. Also the peroxynitrite anion (ONOO^-), the highly destructive product of the interaction between the superoxide anion radical (O₂^-•) and nitric oxide (NO^•), is scavenged by melatonin (122, 123). There is also evidence that melatonin directly neutralizes NO^• (124). Melatonin, as it donates an electron in the process of detoxifying electron-deficient reactive oxygen species (92, 93, 106, 107, 125), itself becomes a radical, the melatonyl cation radical; this radical has very low reactivity (92). The melatonyl cation radical may be recycled back to melatonin (126) and theoretically reacts with the O₂^-• to produce N¹-acetyl-N²-formyl-5-methoxykynuramine (92). If melatonin reacts indirectly with O₂^-•, it would reduce the formation of the hydrogen peroxide (H₂O₂) and, consequently, the formation of ^•OH. Finally, evidence has also accumulated to show that melatonin directly detoxifies H₂O₂ (Tan et al., unpublished data). Since H₂O₂ is the precursor of the ^•OH, its removal reduces formation of the ^•OH.

Effects of Melatonin on the Activities of Enzymes Involved in Antioxidative Defense.
The main antioxidant enzymes are SOD, CAT, GSH-Px, glutathione reductase (GSH-Rd), and glucose-6-phosphate dehydrogenase (G6PD). The influence of melatonin on the activities of each of these enzymes showed that the indole stimulated the activity of each of them (84, 119, 127, 128). By increasing the activities of antioxidant enzymes, melatonin reduces the number of free radicals or ROS generated and increases the production of molecules protecting against oxidative stress.

SOD dismutases O₂^-• to H₂O₂, decreasing the amount of O₂^-• and the formation of ONOO^-. Melatonin increases tissue mRNA levels of two isoforms (manganese/copper SOD) of this enzyme (129, 130). Thus, melatonin may decrease the quantity of O₂^-• in two ways, directly by stimulating SOD and indirectly when the melatonyl cation radical scavenges it.

Extensive studies have been conducted on melatonin's influence on glutathione (GSH) metabolism. GSH is a well-known antioxidant. Melatonin stimulates the activity of GSH-Px, which transforms H₂O₂ to O₂ (131-135). In this process GSH is oxidized, thus forming oxidized glutathione (GSSG). The reduced form, GSH, is replenished by the action of the enzyme GSH-Rd, the activity of which is also stimulated by melatonin (133, 134). The latter process requires the co-factor NADPH generated from NADP in the reaction catalyzed by G6PD; melatonin also reportedly increases the activity of this enzyme (136). Recently melatonin was also shown to stimulate the activity of -glutamylcysteine synthetase (-GCS) (137), the rate-limiting enzyme in GSH synthesis. The stimulation of -GCS is mediated by activator protein-1 (AP-1), an oxidative stress-responsive transcription factor. The process in question was accompanied by the increased number of cells in the G₀/G₁ phase, which suggests that melatonin, while stimulating GSH synthesis, suppresses cell proliferation (137). In general, the ability of melatonin to increase the metabolism of GSH, an effective antioxidant, is well documented. Finally, melatonin was recently found to stimulate CAT (138), thereby further reducing H₂O₂ levels and ^•OH generation.

Besides the antioxidative enzymes mentioned above, the activity of one pro-oxidative enzyme (i.e., nitric oxide synthase (NOS)), is also altered by melatonin. Melatonin has been found to inhibit the activity of NOS (139-141), the enzyme catalyzing the formation of NO^•. Melatonin, by inhibiting NOS activity, decreases the formation of NO^• and the product of its interaction with O₂^-•, ONOO^-. The reported effects of melatonin on the activity of anti- and pro-oxidative enzymes are summarized in Table I.

In aging studies in rats, Reiter et al. (146) observed much higher 8-OHdG and MDA+4-HDA levels and increased microsomal membrane rigidity (indices of oxidative damage, which are also caused by ionizing radiation) in a variety of organs collected from 25-month-old rats when compared with 2-month-old controls; when the rats were pinealectomized at the age of 2 months, which caused a relative melatonin deficiency, this brought about a further increase in these parameters of oxidative damage when measured in rats at the age of 25 months. Since both aging and pinealectomy (147-149) led to a reduction in pineal-derived melatonin, it is suggested that a reduction in physiological levels of this indoleamine may contribute to the increase in oxidative damage in advanced age.

That endogenously produced melatonin contributes to the total antioxidant status of the organism has been documented. In both rats and humans, the total antioxidative capacity of the serum correlated with the levels of melatonin (150, 151). Thus, they exhibit a synchronous circadian rhythm with nocturnal peaks and day-time troughs (150, 151). Nocturnal exposure to light or treatment with antibodies against melatonin brought about a reduction of nocturnal peak of the total antioxidant status (151). Furthermore, the total antioxidant status, like melatonin, exhibited a more pronounced circadian rhythm in young individuals, and an aging-dependent decreasing night-day amplitude (151). Similarly, a correlation exists between the circadian rhythm of melatonin and phagocytosis in ring dove heterophils; at the same time a higher level of O₂^-• production by phagocytes occurred during the light period and a lower level during the day (152).

The implication of these studies is that melatonin at physiological levels contributes to the total antioxidant status of organisms. Likewise, it is well documented that supplementing animals with melatonin increases the capacity of the organism to resist oxidative damage.

Other Properties of Melatonin Confirming its Potential Antioxidant Capacity in Protection Against Damage Caused by Ionizing Radiation.
The contribution of the immune system to defense against oxidative stress is probably not a direct one. However, the efficiency of the immune system improves the function of other systems and mechanisms in living organisms. The connection between melatonin and the neuroimmunoendocrine system is well known (74) and, additionally, a connection between the immune system and antioxidant status was recently suggested (152).

Melatonin has been reported to protect against apoptosis in rat neurons in vitro (121), in rat thymocytes in vivo and in vitro (153), and mouse bone marrow cells in vivo and in vitro (154). In view of the protective action of melatonin against apoptosis and radiation-induced diurnal changes in apoptosis (155), a preventive effect of melatonin against apoptosis caused by ionizing radiation seems probable, but requires experimental proof.

Melatonin as an Antioxidant and Free Radical Scavenger in Defense Against Different Carcinogens—Examples.
The literature related to the protective role of melatonin against oxidative damage caused by carcinogens, other than ionizing radiation, is abundant. The indoleamine highly efficiently protects against damage of all macromolecules (i.e., DNA (108, 143, 156-161), lipids (127, 128, 162-164) and proteins (165). The protection of nuclear DNA from reactants that generate free radicals and duplicate the effects of ionizing radiation (i.e., from ionizing radiation mimics) are especially noteworthy (166).

	Melatonin Counteracts Carcinogenic Action of Ionizing Radiation by Antioxidative Mechanisms—Experimental Evidence

Top Abstract Introduction Biological Effects of Ionizing... Antioxidative Properties of... Melatonin Counteracts... Concluding Remarks References

 
Studies on the protective effects of melatonin against damage caused by ionizing radiation began soon after the discovery of its antioxidative properties. Actually, the first study that revealed the antioxidative effects of melatonin to a physical agent was conducted using ultraviolet light (UV), which shares many features with ionizing radiation. In 1993, Tan et al. (105) reported that melatonin scavenges ^•OH generated in vitro when H₂O₂ was exposed to ultraviolet light. In vivo as well, UV light damage to the skin is known to be prevented by melatonin. Using a similar model, the potency of melatonin in protecting against ^•OH generated by ionizing radiation was confirmed under in vivo conditions (110). In the meantime, a number of studies have tested melatonin's efficacy in protecting against ionizing radiation both in vivo and in vitro.

In Vivo Studies in Animals.
Already in 1994, Blickenstaff et al. (167) carried out studies illustrating the radioprotective actions of melatonin in Swiss ND4 mice. They noted that their exposure to 950 cGy of whole body radiation caused the death of all animals 12 days later, whereas when mice were pretreated with melatonin (1.076 mmol/kg body wt), 43% of the irradiated mice survived at least 30 days after treatment. Interestingly, homologs of melatonin obtained by acylation of 5-methoxytryptamine revealed protective effects against ionizing radiation as well; maximal survival rate was achieved with homologs having acyl chain lengths of six or eight carbons.

A similar survival study was conducted by Vijayalaxmi et al. (168) using CD2-F1 mice. They observed that the exposure of mice to 815 cGy of ionizing radiation resulted in only a 45%–50% survival rate after 30 days; pretreatment with melatonin at a dose of 125 mg/kg body wt slightly increased the survival rate to 60%, whereas melatonin at a dose of 250 mg/kg body wt significantly increased survival to 85%.

Other studies have examined lipid damage after ionizing radiation and the protective effects of melatonin. Exposure of Wistar female rats to ionizing radiation at two doses of 360 cGy radiation given in an interval of 12 hr, brought about, expectedly, significant increases in the levels of MDA in ovaries and plasma 6 hr after radiation exposure (169). Pretreatment of the rats with melatonin at a dose of 100 mg/kg body wt decreased the level of MDA in both tissues when compared with that in irradiated animals. A study performed on Wistar:Han SPF rats has found that melatonin administered in drinking water (dose not specified) for 15 days prevented the increase in hepatic lipids and thymic triacylglycerol levels caused by continuous irradiation at a daily dose of 96 mGy and/or dimethylbenz(a)anthracene treatment, also given daily for 15 days (170).

Experiments have also been performed to examine the early effect of ionizing radiation on both in vivo DNA and membrane damage in rats. Whole body irradiation at a dose of 800 cGy to Sprague-Dawley male rats increased oxidation of guanine bases in DNA isolated from liver collected 12 hr later (Karbownik et al., unpublished data). Similarly, radiation exposure decreased membrane fluidity of hepatic microsomes, although it did not influence lipid breakdown as evaluated by the level of MDA+4-HDA in liver and other tissues. These early effects of ionizing radiation on DNA damage and membrane fluidity were, expectedly, prevented by melatonin injected at a dose of 50 mg/kg body wt at 120, 90, 60, and 30 min prior to radiation exposure.

Another study performed by Vijayalaxmi et al. (171), using CD2-F1 male mice, revealed a protective effect against ionizing radiation-induced damage to the genome of hematopoietic cells. The exposure of mice to whole body irradiation at a dose of 150 cGy resulted in a reduced percentage of polychromatic erythrocytes in the peripheral blood and bone marrow cells and an increased incidence of micronuclei in the same cells measured 24 hr later. However, melatonin, when injected 1 hr prior to irradiation, in doses of 5 mg/kg body wt or 10 mg/kg body wt, partially reversed the damaging effect of radiation with the higher dose being more effective.

A similar study to one described above was performed using mice. Whole body irradiation at a dose of 1.5 Gy brought about a marked increase in the frequency of micronuclei in polychromatic erythrocytes and of chromosomal aberrations in spermatogonia and spermatocytes 24 hr later (172). Melatonin given at a dose of 10 mg/kg body wt, 1 hr prior to irradiation, significantly decreased the effects of this exposure. The authors of the present survey underline that they do not entirely agree with the interpretation of the results in the report by Badr et al. (172). Their conclusion related to "a cytotoxic effect of melatonin at a relatively high dose (10 mg/kg body wt)" (172) is not supported, in the opinion of the authors of the present survey, by either their findings (172) or other literature. The very small increase in micronuclei in polychromatic erythrocytes after a 10 mg/kg body wt dose of melatonin, when compared with the effect of ionizing radiation, would seem irrelevant. Worth underlining is the fact that melatonin did not influence any other examined parameter of chromosomal damage and, when applied before exposure to radiation, decreased the damaging effect of ionizing radiation in all measured parameters, micronuclei included (172). These in vivo studies are summarized in Table II.

Using similar experimental conditions, an additional study by Vijayalaxmi et al. (30) revealed a significant increase in micronuclei formation after exposure of cells to -irradiation at a dose of 1.5 Gy. In this study there was both an increased number of cytokinesis-blocked binucleate cells with micronuclei and an increased total number of micronuclei. Preincubation (20 min) with melatonin at concentrations of either 0.5, 1.0, or 2.0 mM reduced, in a concentration-dependent manner, the number of binucleate cells by 32.5%, 45.4%, and 61.6% and the total number of micronuclei by 36.0%, 50.0%, and 63.7%, respectively. These in vitro studies are summarized in Table III.

Additionally, on the same population of blood lymphocytes under similar experimental conditions but using a lower dose of ionizing radiation (100 cGy), Vijayalaxmi et al. (32) examined the extent of primary DNA damage determined by the length of DNA migration and the fluorescence intensity of the nuclear tail using the alkaline Comet assay. Ingestion of melatonin reduced -radiation-induced increases in lengths of DNA migration by 15.6% and 20.9% after 1 and 2 hr, respectively. Also, the fluorescence intensity measured in the Comet tail decreased by 12.9%–20.5% at 1 hr and by 18.5%–24.0% at 2 hr as the result of melatonin treatment. In this study, an inverse correlation between serum melatonin concentrations and the number of DNA-damaged cells was apparent. These results are summarized in Table IV.

There is some evidence for the protective role of melatonin against radiation-induced damage in the endocrine system. It was observed that pretreatment with melatonin partially reversed histoenzymological changes in rat thyroid gland caused by the exposure to 8 Gy of -radiation (174). In another study that used morphometric parameters, melatonin was shown to decrease the height of the thyroid follicular cells and nuclear volume of these cells obtained from 8 Gy-irradiated rats (175). Melatonin treatment also inhibited the effect of -irradiation at a dose of 8 Gy on several morphometric characteristics of the seminiferous tubules in rats (176). Similarly, the treatment of rats for 2 weeks with melatonin at a daily dose of 0.2 mg significantly decreased what the authors referred to as the nuclear and cytoplasm surface of Leydig cells in rats exposed to 8 Gy radiation (177).

Toxicology of Melatonin.
Melatonin has been tested over a wide range of doses from physiological to high pharmacological concentrations in different species as to its potential toxicology. There is little evidence at this point to suggest major unfavorable effects of melatonin. The usual doses given in vivo are as follows: 10 mg/kg body wt in mice (171), 100 mg/kg body wt in rats (169), 200 mg/kg body wt in rats (Karbownik et al., unpublished data), 250 mg/kg body wt in mice (167, 168), 250 mg/kg in pregnant rats (178), or even 800 mg/kg body wt in mice, rabbits, cats, and dogs (179). Recently, no toxicity of melatonin to mouse embryos was observed when it was given during pregnancy at doses up to 200 mg/kg body wt (180). Particularly worth underlining are studies on human volunteers, in which no adverse side effects have been observed after oral administration of melatonin in doses of 1–300 mg (31, 32, 181-187) and 1 gram of melatonin daily for 30 days (188).

Prooxidative effects of melatonin used in suprapharmacological doses have been described only in a few situations, namely in isolated red blood cells (189) and isolated retinal photoreceptors (190). It is important to note these findings, but their significance to the in vivo situation remains unknown.

	Concluding Remarks

Top Abstract Introduction Biological Effects of Ionizing... Antioxidative Properties of... Melatonin Counteracts... Concluding Remarks References

 
The mechanisms of the radioprotective effects of melatonin seem to consist particularly of its scavenging ^•OH and other free radicals and reactive oxygen intermediates, and its ability to modify the activity of enzymes participating in oxidative stress. Besides the obvious antioxidative mechanisms by which melatonin functions in the protection of DNA, it also may influence DNA repair enzymes directly and stimulate intracellular signals indirectly for the activation of genes responsible for protein synthesis related to DNA repair. These latter processes deserve more extensive investigation in relation to melatonin.

A decreased total antioxidant capacity of plasma of patients exposed to whole body irradiation for the purpose of reducing tumor growth or because of its immunosuppressive effect has been reported (68). Since the total antioxidant capacity of human serum positively correlates with its melatonin content (150), supplementing patients, who are exposed therapeutically or diagnostically to ionizing radiation, with melatonin may have some benefit.

On the basis of the evidence described in the present survey, the following features make melatonin a potentially useful radioprotector: (i) direct and indirect scavenging of free radicals (105-112, 114-124); (ii) the ability to stimulate the activity of antioxidant enzymes (109, 131-137) and to inhibit the activity of a pro-oxidative enzyme (139-141); (iii) distribution in all tissues, cells, and cellular compartments throughout the organism (84, 95); (iv) quick diffusion through all biological membranes (84, 95); (v) ability to provide radioprotection without receptor interaction (92, 93); (vi) presence in radiosensitive tissues (100-104); (vii) tolerance to high doses of melatonin by living organisms and little toxicity in the species tested (31, 32, 167-171, 178-188); (viii) antioxidant efficacy in physiological concentrations (143-146, 150-152); (ix) correlation of melatonin concentration with total antioxidant activity (150, 151); (x) antioxidative protection against all macromolecules (i.e., DNA (29-33, 108, 143, 156-161, 171), lipids (127, 128, 162-164) and proteins (169); (xi) ease of oral administration; and (xii) high bioavailability when administered by virtually any route.

The evidence summarized herein, related to radioprotective effects of melatonin against cellular damage caused by oxidative stress, make this molecule a potential supplement that should be considered in the treatment or co-treatment in situations where the effects of ionizing radiation are to be minimized (e.g., during routine therapies, in occupational settings where accidental exposure may occur, during space travel, or after nuclear accidents, and where high radiation persists).

	Footnotes

 
One author (M.K.) was supported by an American Cancer Society International Fellowship for Beginning Investigators. Research by the authors was supported in part by Amoun Pharmaceutical Company.

¹ To whom requests for reprints should be addressed at the Department of Cellular and Structural Biology, University of Texas Health Science Center, 7703 Floyd Curl Dr., Mail Code 7762, San Antonio, TX 78229–3900. E-mail: Reiter{at}uthscsa.edu

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