Melatonin reduces phenylhydrazine-induced oxidative damage to cellular membranes: evidence for the involvement of iron

The International Journal of Biochemistry & Cell Biology 32 (2000) 1045–1054 www.elsevier.com/locate/ijbcb

Melatonin reduces phenylhydrazine-induced oxidative damage to cellular membranes: evidence for the involvement of iron Ma*gorzata l Karbownik a,b, Russel J. Reiter a,*, Joaquin J. Garcia a, Dun-Xian Tan a a

Department of Cellular and Structural Biology, Uni6ersity of Texas Health Science Center, Mail Code 7762, 7703 Floyd Curl Dri6e, San Antonio, TX 78229 -3900, USA b Department of Thyroidology, Institute of Endocrinology, Medical Uni6ersity of L * o´dz´, 5 Dr Sterling St., 91 -425 Lodz, Poland Received 28 June 2000; received in revised form 16 August 2000; accepted 17 August 2000

Abstract Phenylhydrazine and iron overload result in augmented oxidative damage and an increased likelihood of cancer. Melatonin is a well known antioxidant and free radical scavenger. The aim of this study was to determine whether melatonin would protect against phenylhydrazine-induced oxidative damage to cellular membranes and to evaluate the possible role of iron in this process. Changes in lipid peroxidation and microsomal membrane fluidity were estimated after the treatment of rats with phenylhydrazine (15 mg/kg body weight, daily, 7 days) alone and melatonin or ascorbic acid (15 mg/kg body weight, two times daily, 8 days), or their combination. Additionally, lipid peroxidation was measured in liver homogenates from untreated and melatonin or ascorbic acid-treated rats in vivo and exposed to iron in vitro. Melatonin, but not ascorbic acid, reduced phenylhydrazine-induced lipid peroxidation in vivo in spleen (3.1690.06 vs. 3.8390.12 nmol/mg protein, PB 0.05) and plasma (7.739 0.52 vs. 9.969 0.71 nmol/ml, P B0.05) and attenuated the decrease in hepatic microsomal membrane fluidity (1/polarization, 3.068 9 0.007 vs. 3.027 90.008, PB0.05). In vitro exposure to iron significantly enhanced the lipid peroxidation in liver homogenates from untreated (3.34 90.75 vs. 1.259 0.28, P B 0.05) or ascorbic acid-treated rats (2.729 0.39 vs. 0.88 90.06, PB0.05) but not from melatonin-treated rats (1.49 9 0.55 vs. 0.68 9 0.20, NS). It is concluded that free radical mechanisms are involved in the toxicity of phenylhydrazine and that the antioxidant melatonin, but not ascorbic acid, reduces the toxic affects of phenylhydrazine in vivo and of iron in vitro in cell membranes. Therefore, melatonin co-treatment in conditions of iron overload may prove beneficial. © 2000 Elsevier Science Ltd. All rights reserved. Abbre6iations: CBC, complete blood count; EDTA, ethylenediamine tetraacetic acid; GSH, reduced glutathione; HEPES, N-[2-hydroxyethyl]piperazine-N%-[2-ethanesulphonic acid]; H2O2, hydrogen peroxide; LOO’, peroxyl radical; MDA +4-HDA, ’ malondialdehyde+ 4-hydroxyalkenals; NO’, nitric oxide; ONOO−, peroxynitrite anion; 1O2, singlet oxygen; O− 2 , superoxide anion radical; OH, hydroxyl radical; PHZ, phenylhydrazine; THF, tetrahydrofuran; TMA-DPH, 1-[4-(trimethylammonium)phenyl]-6phenyl-1,3,5-hexatriene p-toluene sulphonate. * Corresponding author. Tel.: +1-210-5673859; fax: + 1-210-5676948. E-mail address: [email protected] (R.J. Reiter). 1357-2725/00/$ - see front matter © 2000 Elsevier Science Ltd. All rights reserved. PII: S 1 3 5 7 - 2 7 2 5 ( 0 0 ) 0 0 0 5 6 - X

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Keywords: Melatonin; Phenylhydrazine; Iron; Membrane oxidative damage; Cancer

1. Introduction The hydrazines — which are extensively used in laboratory, industrial and therapeutical settings — are toxic and can cause irreversible cellular damage [1]. A variety of toxic effects of the hydrazines have been described, including autoimmune disturbances in humans [2,3], human leukemogenesis [4], alterations in the liver, kidney, central nervous system [1], hemolytic anemia [5,6], and cancer [7 – 9]. Phenylhydrazine (PHZ) intoxication leads to hemolysis resulting in severe hemolytic anemia and reticulocytosis [5,6], and to hepatic and splenic iron overload [10] causing a number of pathophysiological changes, e.g. fatty liver [11] and hepatocyte necrosis [12]. PHZ-induced free iron release [10], followed by free radical generation, is a likely mechanism of its toxicity. It is known, for example, that PHZ induces oxidative damage to hemoglobin [13], to membrane phospholipids and proteins in human erythrocytes [14,15] and it generates free radicals and reactive oxygen species [16,17]. PHZ is one of the most potent carcinogens belonging to the hydrazine family of molecules [7]. Iron often plays a central role in oxidative stress since, once released from its binding molecules, it becomes free iron which acts locally [18], as well as diffusing to adjacent cells [19], where it generates free radicals that destroy essential macromolecules. In conditions of iron overload an increased risk of cancer, e.g. hepatocellular carcinoma [20], has been noted. Similarly, human and mouse colon cancer related to dietary iron overload [21,22] have been reported. Melatonin and vitamin C (ascorbic acid) are commonly used to reduce experimentally-induced oxidative damage, and both are recommended as supplements in the course of human diseases related to oxidative stress. Melatonin, the chief indoleamine produced by the pineal gland, an antioxidant and free radical scavenger [23 – 29], effectively protects cellular compartments against oxidative damage caused by a variety of carcino-

gens [30–33]. A wide range of mechanisms are involved in melatonin’s antioxidative effects [25]. While ascorbic acid also is an antioxidant under most conditions, in the presence of transition metals it can be strongly prooxidant [34]. The aim of the present study was to compare the effects of melatonin or ascorbic acid against experimentally induced oxidative damage to cellular membranes induced by PHZ where free radicals are believed to be involved.

2. Materials and methods

2.1. Chemicals The LPO-586 kit for lipid peroxidation was obtained from Calbiochem (La Jolla, CA), 1-[4(trimethylammonium)phenyl]-6-phenyl-1,3,5-hexatriene p-toluene sulphonate (TMA-DPH) from Molecular Probes (Eugene, OR), and phenylhydrazine hydrochloride, L-ascorbic acid and ferrous sulfate from Sigma (St. Louis, MO). Pure melatonin was a gift from Helsinn Chemicals SA (Biasca, Switzerland). Other chemicals used were of analytical grade and came from commercial sources. TMA-DPH was diluted in tetrahydrofuran (THF) (the final concentration of THF in the incubation volume was 0.4% and of 66.7 nM TMA-DPH).

2.2. Animals The procedures used in the study were approved by the Institutional Animal Care and Use Committee. Fifty-four male Sprague–Dawley rats (weighing  250 g) were housed in plexiglas cages (three animals per cage) in a windowless room with automatically regulated temperature (229 2°C) and lighting (14-h light/10-h dark, with lights on from 06.00 to 20.00 h). The animals received standard chow and water ad libitum. After 1 week

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of acclimatization, the rats were randomly divided into seven groups with seven (groups I and II) or eight (groups III–VII) animals per group. The animals in these groups were injected with the following substances; groups III, IV, VI and VII — PHZ (15 mg/kg body weight) in freshly prepared 0.9% NaCl, once daily (at 08.00 h) for 7 days; groups II, IV and VII-melatonin (15 mg/kg body weight) in freshly prepared 0.9% NaCl/ethanol (v/v, 20/1), twice daily (at 08.00 h and 16.00 h) for 8 days; groups V, VI and VII-ascorbic acid (15 mg/kg body weight) in freshly prepared 0.9% NaCl, twice daily (at 08.00 h and 16.00 h) for 8 days. The control rats, which did not receive either PHZ, melatonin or ascorbic acid, were injected with their solvents, i.e. 0.9% NaCl or 0.9% NaCl per ethanol (v/v, 20/1) at the time points mentioned above. Injections with melatonin or ascorbic acid lasted 1 day longer than the injections with PHZ. All the substances were administered intraperitoneally in the volume of 0.5 ml per injection. Rats were killed by decapitation on the day following the last injections. Peripheral blood was collected into sterile vacutainer tubes containing K3 EDTA. The blood was used for measurement of complete blood count (CBC) and for obtaining plasma (2500× g, 10 min, 4°C). The liver and spleen were collected, frozen in solid CO2 and stored at − 80°C until assay.

2.3. Measurement of protein Protein was measured using the method of Bradford [35], with bovine serum albumin as the standard.

2.4. In 6itro-induced lipid peroxidation Approximately, 100 mg of tissue from untreated animals (group I) and from rats given either melatonin (group II) or ascorbic acid (group V) were homogenized (Euro Turrax T20B homogenizer) in ice cold 50 mM Tris buffer (pH 7.4) (10%, w/v). Two aliquots of homogenates from each animal were incubated in a water bath at 37°C, one in the presence of ferrous sulfate (15 mM) and hydrogen peroxide (H2O2, 0.1 mM) to

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generate free radicals, and the other in the absence of these compounds.

2.5. Measurement of products of lipid peroxidation The concentrations of malondialdehyde+ 4-hydroxyalkenals (MDA+4-HDA), as products of lipid peroxidation, were measured in liver homogenates after in vitro-induced lipid peroxidation, and in homogenates of liver and spleen, and in blood plasma, obtained after treatment of the rats in vivo. Homogenates were centrifuged at 10 000× g for 10 min at 4°C. The obtained supernatant or plasma (200 ml) were mixed with 650 ml of methanol:acetonitrile (1:3, v:v) solution containing N-methyl-2-phenylindole and vortexed. After adding 150 ml 15.4 M methanesulfonic acid, incubation was carried out at 45°C for 40 min. The concentration of MDA+ 4-HDA was measured spectrophotometrically at the absorbance at 586 nm using a solution of 10 mM 4-hydroxynonenal as standard. The level of lipid peroxidation is expressed as the amount of MDA+4-HDA (nmol) per mg protein.

2.6. Microsomal membrane isolation and measurement of membrane fluidity Liver microsomes were isolated according to a method used in our laboratory [36,37]. Liver was homogenized in 140 mM KCl per 20 mM HEPES buffer (pH 7.4) (1:10 w/v) and the resulting suspension was centrifuged at 1000× g for 10 min at 4°C. The pellets containing nuclei were removed, and the supernatant was centrifuged at 105 000× g for 60 min at 4°C. The pellets were resuspended in buffer and centrifuged at 10 000× g for 15 min at 4°C. This centrifugation resulted in the separation of microsomes (supernatant) from mitochondria (pellets). The supernatant was centrifuged at 105 000× g for 60 min at 4°C. The obtained microsomal pellets were resuspended in 140 mM KCl per 20 mM HEPES buffer (1:1, w/v) and kept at − 80°C until assay. Membrane fluidity was measured in triplicates. Microsomes (0.5 mg/ml microsomal protein) were resuspended in 50 mM Tris–HCl buffer (pH 7.4)

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(3 ml of final volume), vortexed for 1 min in the presence TMA-DPH and then incubated with shaking at 37°C for 30 min to ensure the uniform incorporation of the probe into the membranes. Fluorescence measurements were performed using a Perkin–Elmer LS-50 Luminescence Spectrometer equipped with a circulating water bath to maintain the temperature of 22 9 0.1°C during the assay. Excitation and emission wavelength of 360 and 430 nm were used, respectively. The emission intensity of vertically polarized light was detected by an analyzer oriented parallel (IVV), or perpendicular (IVH) to the excitation plane. A correction factor for the optical system (G) was used. Polarization (P) was calculated as follows: P =



IVV − GIVH IVV − GIVH



Since an inverse relationship exists between membrane fluidity and polarization, the membrane fluidity is expressed as the inverse of P (1/P).

Fig. 1. Concentrations of malondialdehyde + 4-hydroxyalkenals (MDA +4-HDA) in spleen homogenates collected from rats treated with phenylhydrazine (PHZ, 15 mg/kg body weight) for 6 days, melatonin (Mel, 15 mg/kg body weight) two times daily for 8 days or ascorbic acid (AA, 15 mg/kg body weight) two times daily for 8 days. Data (mean 9 S.E.) are concentrations of MDA + 4-HDA (nmol per mg of protein). Seven or eight rats per group. *PB 0.05 vs. control, vs. Mel, vs. PHZ+ Mel; vs. AA and vs. PHZ+ AA + Mel.

2.7. Measurement of whole blood constituents CBC was performed on a Cell-Dyn 4000 Hematology analyzer. Automated reticulocyte counts were measured using a modified New Methylene blue procedure also performed on a Cell-Dyn 4000 Hematology analyzer.

2.8. Statistical analyses Results are expressed as mean9S.E. The data were statistically analyzed using a one-way analysis of variance (ANOVA) followed by a StudentNewman-Keuls test. Statistical significance was determined at a level of B 0.05.

3. Results Treatment of rats with PHZ increased lipid peroxidation, measured by the level of MDA+ 4HDA, in both liver and spleen homogenates, as well as in blood plasma, when compared with the control, melatonin- and ascorbic acid-treated rats. Increased membrane MDA+4-HDA levels were also seen in the homogenates of rats treated with both PHZ and ascorbic acid; these increases were of similar magnitude as those in the PHZ-treated animals. When melatonin was given to animals injected with PHZ or treated with both PHZ and ascorbic acid, the increases in MDA+ 4-HDA levels in spleen (Fig. 1) and plasma (Fig. 2) were prevented. The levels of lipid peroxidation products in the liver of melatonin-treated rats were also reduced but the difference did not reach statistical significance (data not shown). The injections of PHZ caused a significant decrease in hepatic microsomal membrane fluidity (an increase in membrane rigidity) when compared with the control, melatonin- or ascorbic acid injected rats (Fig. 3). Melatonin treatment in animals given PHZ or PHZ plus ascorbic acid significantly prevented changes in microsomal membrane fluidity. Conversely, membrane fluidity in hepatic microsomes from rats treated with PHZ plus ascorbic acid was even lower than that in animals given PHZ only.

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Fig. 2. Concentrations of malondialdehyde + 4-hydroxyalkenals (MDA +4-HDA) in blood plasma collected from rats treated with phenylhydrazine (PHZ, 15 mg/kg body weight) for 6 days, melatonin (Mel, 15 mg/kg body weight) two times daily for 8 days or ascorbic acid (AA, 15 mg/kg body weight) two times daily for 8 days. Data (mean 9S.E.) are concentrations of MDA +4-HDA (nmol per ml of plasma). Seven or eight rats per group. *P B0.05 vs. control, vs. Mel, vs. PHZ + Mel; vs. AA and vs. PHZ+AA +Mel.

Changes in the concentrations of MDA+ 4HDA in the homogenates of liver collected from control animals or from rats given either melatonin or ascorbic acid and then incubated with ferrous sulfate are presented in Fig. 4. In vitro exposure to iron significantly increased lipid peroxidation in liver from untreated or ascorbic acidtreated animals. Conversely, in vivo treatment with melatonin effectively protected against the increased lipid peroxidation in liver induced by incubation in the presence of iron. The results of CBC revealed marked decreases in the concentration of hemoglobin and the number of erythrocytes in animals treated with PHZ (ranging 7.5–12.0 g/dl and 2.28 – 4.20 × 106/mm3, respectively) when compared with the control or melatonin-treated animals (14.4 – 17.10 g/dl and 6.82 –9.38×106/mm3, respectively). Concurrent treatment of PHZ-injected rats with either melatonin or ascorbic acid did not prevent these PHZinduced effects (data not shown).

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Fig. 3. Membrane fluidity (the inverse of membrane rigidity) of hepatic microsomal membranes collected from rats treated with phenylhydrazine (PHZ, 15 mg/kg body weight) for 6 days, melatonin (Mel, 15 mg/kg body weight) two times daily for 8 days or ascorbic acid (AA, 15 mg/kg body weight) two times daily for 8 days. Data (mean9 S.E.) are expressed as an inverse of polarization (1/polarization). Seven or eight rats per group. *PB0.05 vs. control, vs. Mel and vs. AA. **PB0.05 vs. PHZ and vs. PHZ+ AA; ***PB0.05 vs. PHZ.

Fig. 4. Concentrations of malondialdehyde +4-hydroxyalkenals (MDA+4-HDA) in liver homogenates collected from control rats, animals treated with melatonin (Mel, 15 mg/kg body weight) two times daily for 8 days or animals treated with ascorbic acid (AA, 15 mg/kg body weight) two times daily for 8 days and incubated in the presence of ferrous sulfate (15 mM) and hydrogen peroxide (H2O2, 0.1 mM). Data (mean9 S.E.) represents concentrations of MDA +4-HDA (nmol per mg of protein). Sample size equals 7 or 8. *PB0.05 vs. control, **PB 0.05 vs. Control +Fe2 + and vs. AA + Fe2 + ; ***PB0.05 vs. AA.

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4. Discussion PHZ-induced damage to cellular membranes observed in the present study is in agreement with previous findings [10,14,18]. The increased level of lipid peroxidation products in liver and spleen, following PHZ treatment, was anticipated since the liver is the main storage site for iron and PHZ induces striking increases in the free iron concentration in both liver and spleen [10]. Iron-induced lipid peroxidation is a well-studied phenomenon [36 – 38]. Additionally, the reduction in membrane fluidity in hepatic microsomes due to PHZ, as seen in the present studies, are in agreement with PHZ-induced damage not only to lipids [14] but also to proteins [39] and with iron-induced lipid [10,18] and protein [40] damage. Indeed, alterations in membrane structure due to both PHZ [14] and iron [36,37] treatment have been previously observed. An increased amount of lipid peroxidation products after PHZ treatment was also found in blood plasma. These products conceivably derived from solid tissues, when cellular membranes were exposed to oxidative stress caused by PHZ and/or iron. Thus, we feel that the rise in the level of lipid peroxidation products in the plasma represents, to some extent, the general systemic oxidative damage to membrane lipids caused by PHZ treatment. Melatonin effectively protects against the increases in lipid peroxidation in spleen and plasma and against the changes in microsomal membrane fluidity in liver brought about by PHZ treatment. The mechanisms of action of melatonin as an antioxidant are complex. Melatonin, due to its small size and high lipophilicity, crosses biological membranes easily and reaches all subcellular compartments [41]. The indoleamine exhibits both lipophilic [42] and hydrophilic [43] properties, and in membranes it localizes in a superficial position of the lipid bilayers near the polar heads of membrane phospholipids [44]. Melatonin protects both the membrane lipids [45,46] and proteins [47] from oxidative damage. The indoleamine effectively protects liver and kidney [48], as well as lung and spleen [32] against lipid peroxidation due to d-aminolevulinic acid, the mechanism of which also involves iron. Melatonin directly or indirectly

neutralizes a variety of free radicals and reactive species. The indoleamine directly detoxifies hydrogen peroxide (H2O2) [29,49] and secondarily the superoxide anion radical (O2 − ’) [24], both of which are generated by PHZ [16,17]. Furthermore, efficient protection of melatonin against the highly toxic hydroxyl radical (’OH) has been shown in both in vivo and in vitro studies [27,29]. Also, melatonin has been shown to scavenge directly nitric oxide (NO’) [50], peroxynitrite anion (ONOO−) [51] and singlet oxygen (1O2) [52]. The finding that melatonin scavenges the peroxyl radical (LOO’) [53,54] is controversial; the fact that melatonin limits lipid peroxidation probably relates to its ability to scavenge the initiating agents, e.g. ’OH, ONOO−, etc. Melatonin is also known to stimulate the activity of several antioxidant enzymes, i.e. glutathione peroxidase, which metabolizes H2O2 to H2O, glutathione reductase which converts glutathione disulfide back to reduced glutathione (GSH), glucose-6-phosphate dehydrogenase which enzymatically promotes the formation of NADPH, an important cofactor for glutathione reductase [25,55] and g-glutamylcysteine synthetase [56], the rate limiting enzyme in the synthesis of GSH, an important intracellular antioxidant. Furthermore, melatonin stimulates mRNA levels for the antioxidative enzyme, superoxide dismutase, while it is believed to inhibit the activity of at least one enzyme which promotes free radical generation, i.e. nitric oxide synthase [23–25,55]. Since, PHZ decreases the content of GSH, an important intracellular antioxidant, in rat liver [57] and in erythrocytes [18], the actions of melatonin which lead to increased production of GSH [25] may be of particular importance. Specifically, which of the many antioxidative actions of melatonin provide protection against oxidative stress caused by PHZ and iron remain to be defined. PHZ-induced iron release, followed by the production of reactive species and free radicals results in the increased lipid peroxidation [18]. Lipid peroxidation products are known to interact with DNA and thereby contribute to carcinogenesis [58]. Chronic treatment with PHZ results not only in membrane and DNA damage but also in an increase in the hepatic g-glutamyl

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transpeptidase expression [10], a phenomenon associated with multistage carcinogenesis. Additionally, a positive correlation between in vivo DNA damage and the likelihood of cancer due to PHZ treatment has been found [7]. Therefore, the fact that melatonin reversed early changes related to oxidative stress, namely changes in lipid peroxidation and membrane fluidity, caused by PHZ, may be important in reducing the incidence of cancer due to PHZ action. Considering the multiple actions of melatonin, the indoleamine may be important in reducing carcinogenesis in all phases where oxidative damage resulting from PHZ treatment is involved [26,59]. In fact, melatonin is a well known oncostatic agent [60]. Some comments are required regarding the apparent greater efficacy of melatonin in the spleen than in the liver relative to melatonin’s protection against lipid peroxidation. This may relate to the higher free iron storage in liver after PHZ treatment [10], and, therefore, more free radical generation and oxidative damage. In liver, PHZ-induced changes in microsomal membrane fluidity were effectively prevented by melatonin. This is probably due to the fact that MDA+ 4HDA, measured in the current study, represent only a portion of the lipid peroxidation products; thus, they are not equivalent to the total damaged lipid in the membranes. Changes in membrane fluidity depend on both lipid and protein damage and, therefore, fluidity constitutes a better index of structural and functional changes in cellular membranes. It is known that changes in membrane fluidity and in the concentration of MDA+ 4-HDA do not always run in parallel [33]. Of particular interest is the finding that in vivo treatment with melatonin protected against ironinduced lipid peroxidation in liver homogenates in vitro, whereas another antioxidant, ascorbic acid, did not. This observation shows that administering melatonin to organisms decreases organ susceptibility to oxidative stress even after the tissues are oxidatively challenged in vitro. Clearly, tissues from melatonin-treated rats are capable of resisting iron-induced oxidative damage more effectively than are tissues of animals not given melatonin. This is the first study to show that

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melatonin given in vivo has a carry-over effect to the in vitro situation. A variety of studies have shown that iron-overloaded hepatocytes exhibit an increased susceptibility to oxidative stress [61]. Based on the current findings, treatment with an antioxidant such as melatonin may reduce some of the toxic consequencies of excessive free iron in cells. Chronic administration of PHZ in a high dose was applied in our study and, as in other reports [6], this was followed by marked anemia. Neither melatonin nor ascorbic acid were able to reduce the severity of PHZ-induced anemia, i.e. the antioxidants did not protect against the PHZ-induced decrease in the concentration of hemoglobin and the number of erythrocytes. Hemolysis and associated anemia are complex processes, not exclusively related to oxidative stress, but possibly involving mechanisms which are not influenced by antioxidants. For example, in another study sulfhydryl oxidation and protein aggregation in hemoglobin-free human erythrocyte membranes were not prevented by any of the antioxidants tested or by antioxidative enzymes [15]. As shown in this study, ascorbic acid was clearly less protective than melatonin in terms of the toxicity of PHZ. This is likely due to the fact that ascorbic acid converts Fe3 + to Fe2 + which then reduces H2O2 to the highly toxic ’OH. It is well known that ascorbic acid in combination with iron induces oxidative DNA damage [62] and illustrates the ambiguity of ascorbic acid concerning its anti- and prooxidative effects. Worth stressing, is the finding, that the combined treatment with PHZ and ascorbic acid applied in the present study did not result in typical oxidative damage when rats were simultaneously injected with melatonin. Thus, melatonin protects against the action of PHZ even when it is combined with ascorbic acid. In summary, it is concluded that exogenously administered melatonin reduces at least some of the toxic effects of in vivo PHZ administration. The results also show that giving melatonin in vivo protects the tissues from subsequent in vitro oxidative challenges.

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Acknowledgements Ma*gorzata l Karbownik has been supported by an American Cancer Society International Fellowship for Beginning Investigators. Research was supported in part by a grant from Amoun Pharmaceutical Company.

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