The antioxidative property of green tea against iron-induced oxidative stress in rat brain

The antioxidative property of green tea against iron-induced oxidative stress was investigated in the rat brain both in vivo and in vivo. Incubation of brain homogenates at 37 degrees C for 4 hours in vitro increased the formation of Schiff base fluorescent products of malonaldehyde, an indicator of lipid peroxidation. Auto-oxidation (without exogenous iron) of brain homogenates was inhibited by green tea extract in a concentration-dependent manner. Moreover, incubation with iron (1 microM) elevated lipid peroxidation of brain homogenates after 4-hour incubation at 37 degrees C. Co-incubation with green tea extract dose-dependently inhibited the iron-induced elevation in lipid peroxidation. For the in vivo studies: ferrous citrate (iron, 4.2 nmoles) was infused intranigrally and induced degeneration of the nigrostriatal dopaminergic system of rat brain. An increase in lipid peroxidation in substantia nigra as well as a decrease in dopamine content in striatum was observed seven days after the iron infusion. Intranigral infusion of green tea extract alone did not increase, and in some cases, even decreased lipid peroxidation in substantia nigra. Co-infusion of green tea extract prevented oxidative injury induced by iron. Both iron-induced elevation in lipid peroxidation in substantia nigra and iron-induced decrease in dopamine content in striatum were suppressed. Oral administration of green tea extract for two weeks did not prevent the iron-induced oxidative injury in nigrostriatal dopaminergic system. Our results suggest that intranigral infusion of green tea extract appears to be nontoxic to the nigrostriatal dopaminergic system. Furthermore, the potent antioxidative action of green tea extract protects the nigrostriatal dopaminergic system from the iron-induced oxidative injury.     

Carboxyfullerene prevents iron-induced oxidative stress in rat brain

Carboxyfullerene, a water-soluble carboxylic acid derivative of a fullerene, was investigated as a protective agent against iron-induced oxidative stress in the nigrostriatal dopaminergic system of anesthetized rats. Intranigral infusion of exclusive carboxyfullerene did not increase lipid peroxidation in substantia nigra or deplete dopamine content in striatum. Infusion of ferrous citrate (iron II) induced degeneration of the nigrostriatal dopaminergic system. An increase in lipid peroxidation in substantia nigra as well as decreases in K+-evoked dopamine overflow and dopamine content in striatum were observed 7 days after the infusion. Co-infusion of carboxyfullerene prevented iron-induced oxidative injury. Furthermore, tyrosine hydroxylase-immunoreactive staining showed that carboxyfullerene inhibited the iron-induced loss of the dopaminergic nerve terminals in striatum. The antioxidative action of carboxyfullerene was verified by in vitro studies. Incubation of brain homogenates increased the formation of the Schiff base fluorescent products of malonaldehyde, an indicator of lipid peroxidation. Both autooxidation (without exogenous iron) and iron-induced elevation of lipid peroxidation of brain homogenates were suppressed by carboxyfullerene in a dose-dependent manner. Our results suggest that intranigral infusion of carboxyfullerene appears to be nontoxic to the nigrostriatal dopaminergic system. Furthermore, the potent antioxidative action of carboxyfullerene protects the nigrostriatal dopaminergic system from iron-induced oxidative injury.     

Coexistence of zinc and iron augmented oxidative injuries in the nigrostriatal dopaminergic system of SD rats

Clinical studies have demonstrated an excess of transition metals, including zinc and iron, in the substantia nigra (SN) of Parkinson's patients. In the present study, the neurotoxic effect of zinc was investigated using iron as a positive control. Addition of zinc or iron to brain homogenates increased lipid peroxidation. Zinc was less potent than iron in inducing lipid peroxidation. Coincubation with desferrioxamine prevented zinc- and iron-induced lipid peroxidation. Furthermore, glutathione (GSH), S-nitroso-N-acetylpenicillamine, or melatonin inhibited zinc-induced lipid peroxidation. The oxidative effect of zinc was further investigated in anesthetized rats. Seven days after intranigral infusion of zinc, lipid peroxidation was elevated in the infused SN, and dopamine content and tyrosine hydroxylase-positive axons were decreased in the ipsilateral striatum. Zinc was less potent than iron in inducing neurodegeneration in vivo. L-Buthionine-[S,R]-sulfoximine pretreatment (i.c.v.), which depletes cellular GSH levels, enhanced zinc-induced oxidative injuries in the nigrostriatal dopaminergic system. Moreover, simultaneous infusion of zinc and iron appeared to augment oxidative injuries in rat brain. Taken together, our results demonstrate that intranigral infusion of zinc caused degeneration of the nigrostriatal dopaminergic system in rat brain. Furthermore, coexistence of zinc and iron augmented oxidative injuries in rat brain. These findings indicate that both zinc and iron contribute to the etiology of Parkinsonism.     

Zinc-induced apoptosis in substantia nigra of rat brain: neuroprotection by vitamin D3

Accumulation of transition metals has been suggested to be responsible for the deteriorated nigrostriatal dopaminergic system in Parkinson's patients. In the present study, the mechanism underlying the zinc-induced neurotoxicity was investigated in the nigrostriatal dopaminergic system in vivo. Our 6-methoxy-8-paratoluene sulfonamide quinoline fluorescence study showed zinc translocation in the infused nigral cells after intranigral infusion of zinc. Furthermore, lipid peroxidation in the zinc-infused substantia nigra was consistently elevated 4 h to 7 d after the infusion. At the same time, an abrupt increase in cytosolic cytochrome c content in the infused substantia nigra was observed 4 h after zinc infusion and gradually decreased to basal levels 7 d after infusion. Both TUNEL-positive neurons and DNA fragmentation, indicatives of apoptosis, were detected in the zinc-infused substantia nigra. Furthermore, striatal dopamine content was reduced 7 d after the infusion. In attempt to prevent zinc-induced neurotoxicity, vitamin D3 was systemically administered. Zinc-induced increases in lipid peroxidation and cytosolic cytochrome c in the infused substantia nigra were prevented by this treatment. Moreover, zinc-induced reduction in striatal dopamine content was attenuated after vitamin D3 treatment. Our in vivo data suggest that zinc-induced oxidative stress may result in apoptosis followed by reduced dopaminergic function in the nigrostriatal dopaminergic system. Furthermore, vitamin D3 prevented zinc-induced oxidative injuries in the rat brain.     

Melatonin reduces Fenton reaction-induced lipid peroxidation in porcine thyroid tissue

Free radicals and reactive oxygen species (ROS) participate in physiological and pathological processes in the thyroid gland. Bivalent iron cation (ferrous, Fe(2+)), which initiates the Fenton reaction (Fe(2+) + H2O2 --> Fe(3+) + *OH + OH(-)) is frequently used to experimentally induce oxidative damage, including that caused by lipid peroxidation. Lipid peroxidation is involved in DNA damage, thus indirectly participating in the early steps of carcinogenesis. In turn, melatonin is a well-known antioxidant and free radical scavenger. The aim of the study was to estimate the effect of melatonin on basal and iron-induced lipid peroxidation in homogenates of the porcine thyroid gland. In order to determine the effect of melatonin on the auto-oxidation of lipids, thyroid homogenates were incubated in the presence of that indoleamine in concentrations of 0.0, 0.00001, 0.0001, 0.001, 0.01, 0.1, 0.25, 0.5, 1.0, 2.5, or 5.0 mM. To study melatonin effects on iron-induced lipid peroxidation, the homogenates were incubated in the presence of FeSO(4) (40 microM) plus H2O2 (0.5 mM), and, additionally, in the presence of melatonin in the same concentrations as above. The degree of lipid peroxidation was expressed as the concentration of malondialdehyde + 4-hydroxyalkenals (MDA + 4-HDA) per mg protein. Melatonin, in a concentration-dependent manner, decreased lipid peroxidation induced by Fenton reaction, without affecting the basal MDA + 4-HDA levels. In conclusion, melatonin protects against iron + H2O2-induced peroxidation of lipids in the porcine thyroid. Thus, the indoleamine would be expected to prevent pathological processes related to oxidative damage in the thyroid, cancer initiation included.     

Angeli's salt induces neurotoxicity in dopaminergic neurons in vivo and in vitro

In this study, we investigated the hypothesis that the pro-oxidative properties of Angeli's salt (AS), a nitroxyl anion (HNO/NO -) releasing compound, cause neurotoxicity in dopaminergic neurons. The pro-oxidative properties were demonstrated in vitro by measuring hydroxylation products of salicylate and peroxidation of lipids under various redox conditions. AS (0-1000 μM) released high amounts of hydroxylating species in a concentration dependent manner. AS also increased lipid peroxidation in brain homogenates at concentrations below 100 μM, while inhibiting it at 1000 μM concentration. The AS induced pro-oxidative effects were completely suppressed by copper (II), which converts nitroxyl anion to nitric oxide, as well as by a potent nitroxyl anion scavenger glutathione. Neurotoxicity towards dopaminergic neurons was tested in rat nigrostriatal dopaminergic system in vivo and by using primary mesencephalic dopaminergic neuronal cultures in vitro . Intranigral infusion of AS (0-400 nmol) caused neurotoxicity reflected as a dose dependent decrease of striatal dopamine seven days after treatment. The effect of the 100 nmol dose was more pronounced when measured 50 days after the infusion. Neurotoxicity was also confirmed as a decrease of tyrosine hydroxylase positive neurons in the substantia nigra. Neither sulphononoate, a close structural analog of AS, nor sodiumnitrite caused changes in striatal dopamine, thus reflecting lack of neurotoxicity. In primary dopaminergic neuronal cultures AS reduced [ 3 H] dopamine uptake with concentrations over 200 μM confirming neurotoxicity. In line with the quite low efficacy to increase lipid peroxidation in vitro , infusion of AS into substantia nigra did not cause increased formation of fluorescent products of lipid peroxidation. These results support the hypothesis that AS derived species oxidize critical thiol groups, rather than membrane lipids, potentially leading to protein oxidation/dysfunction and demonstrated neurotoxicity. These findings may have pathophysiological relevance in case of excess formation of nitroxyl anion.     

Relative efficacies of indole antioxidants in reducing autoxidation and iron-induced lipid peroxidation in hamster testes

Increased iron stores are associated with free radical generation and carcinogenesis. Lipid peroxidation is involved in DNA damage, thus indirectly participating in the early steps of tumor initiation. Melatonin and structurally related indoles are effective in protecting against oxidative stress. The aim of the study was to compare the relative efficacies of melatonin, N-acetylserotonin (NAS), indole-3-propionic acid (IPA), and 5-hydroxy-indole-3-acetic acid (5HIAA) in altering basal and iron-induced lipid peroxidation in homogenates of hamster testes. To determine the effect of the indoles on the autoxidation of lipids, homogenates were incubated in the presence of each agent in concentrations of 0.0, 0.01, 0.05, 0.1, 0.25, 0.5, 0.75, 1.0, 2.0, 2.5, or 5.0 mM. To study their effects on induced lipid peroxidation, homogenates were incubated with FeSO(4) (30 microM + H(2)O(2) (0.1 mM) + each of the indoles in the same concentrations as above. The degree of lipid peroxidation was expressed as concentrations of malondialdehyde + 4-hydroxyalkenals (MDA + 4-HDA) per mg protein. The indoles decreased both basal and iron-related lipid peroxidation in a concentration-dependent manner. Melatonin reduced basal MDA + 4-HDA levels when used at the concentrations of 0.25 mM or higher, and prevented iron-induced lipid peroxidation at concentrations of 1.0, 2.0, 2.5, or 5.0 mM. The lowest effective concentrations of NAS required to lower basal and iron-related lipid peroxidation were 0.05 mM and 0.25 mM, respectively. IPA, only when used in the highest concentrations of 2.5 mM or 5 mM inhibited basal lipid peroxidation levels and it was ineffective on the levels of MDA + 4-HDA due to iron damage. 5HIAA reduced basal lipid peroxidation when used at concentrations of 0.25 mM or higher, and it prevented iron-induced lipid peroxidation only at the highest applied concentration (5 mM). In conclusion, melatonin and related indoles at pharmacological concentrations protect against both the autoxidation of lipids as well as induced peroxidation of lipids in testes. In doing so, these agents would be expected to reduce testicular cancer that is initiated by products of lipid peroxidation.     

Effect of Buthionine Sulfoximine, a Synthesis Inhibitor of the Antioxidant Glutathione, on the Murine Nigrostriatal Neurons

Abstract: This study analyzed the effects of acute systemic treatment with buthionine sulfoximine (BSO), a synthesis inhibitor of the antioxidant reduced glutathione (GSH), on dopaminergic neurons of the murine nigrostriatal pathway. Part 1 of the study established a dose-response curve and the temporal pattern of GSH loss and recovery in the substantia nigra and striatum following acute BSO treatment. Part 2 of the study determined the effect of acute BSO treatment on the morphology and biochemistry of nigrostriatal neurons. We found that decreases in GSH levels had profound morphological effects, including decreased catecholamine fluorescence per cell, increased levels of lipid peroxidation and lipofuscin accumulation, and increased numbers of dystrophic axons in dopaminergic neurons of the nigrostriatal pathway. However, no measurable effects were observed in biochemical levels of either dopamine or its metabolites. These changes mimic those that have been reported to occur in the nigrostriatal system of rodents with advancing age. Our data suggest that reduction of GSH via BSO treatment results in the same types of nigrostriatal degenerative effects that occur during the aging process and consequently is a good model system for examining the role of GSH in protecting this area of the brain against the harmful effects of age-related oxidative stress.     

Intranigral iron infusion in the rat

Iron is known to induce lipid perocidation and recent evidence indicates that both iron and lipid peroxidation are elevated in the substantia nigra in Parkinson's disease (PD). To test whether excess intranigral iron induces lipid peroxidation, we infused an iron citrate solution (0.63 nmol in 0.25 μL) into the rat substantia nigra and measured nigral thiobarbituric acid reactive products at 1-h, 1-d, 1-wk, and 1-mo postinfusion. In a separate group of iron-infused animals, histologic analysis within the substantia nigra through 1-mo postinfusion was accomplished by thionine- and iron-staining, with concurrent assessment of striatal neurochemical markers. Concentrations of nigral thiobarbituric acid reactive products were significantly elevated at 1 h and 1 d in iron-infused animals compared to vehicle-infused and unoperated animals, with a return to control values by 1 wk. Similarly, striatal dopamine turnover was acutely elevated, suggesting damage to dopaminergic neurons, which was confirmed histologically. Although iron-staining within the iron diffusionary area was increased through the postinfusion month, there was an apparent progression of the cellular character of staining from predominantly neuronal to reactive glial and finally to oligodendroglial by 1 mo postinfusion. this progression of cellular iron-staining may indicate a shifting of infused iron to a more bound unreactive form, thus explaining only an acute elevation in lipid peroxidation through 1 d following intranigral iron infusion. The data indicate that damage to nigral neurons induced by iron infusion is transciently associated with a marker of oxidative damage and supports the possibility that iron-induced oxidative stress contributes to the pathogenesis of PD.     

Role of oxidative stress in paraquat-induced dopaminergic cell degeneration

Systemic treatment of mice with the herbicide paraquat causes the selective loss of nigrostriatal dopaminergic neurons, reproducing the primary neurodegenerative feature of Parkinson's disease. To elucidate the role of oxidative damage in paraquat neurotoxicity, the time-course of neurodegeneration was correlated to changes in 4-hydroxy-2-nonenal (4-HNE), a lipid peroxidation marker. When mice were exposed to three weekly injections of paraquat, no nigral dopaminergic cell loss was observed after the first administration, whereas a significant reduction of neurons followed the second exposure. Changes in the number of nigral 4-HNE-positive neurons suggest a relationship between lipid peroxidation and neuronal death, since a dramatic increase in this number coincided with the onset and development of neurodegeneration after the second toxicant injection. Interestingly, the third paraquat administration did not cause any increase in 4-HNE-immunoreactive cells, nor did it produce any additional dopaminergic cell loss. Further evidence of paraquat-induced oxidative injury derives from the observation of nitrotyrosine immunoreactivity in the substantia nigra of paraquat-treated animals and from experiments with ferritin transgenic mice. These mice, which are characterized by a decreased susceptibility to oxidative stress, were completely resistant to the increase in 4-HNE-positive neurons and the cell death caused by paraquat. Thus, paraquat exposure yields a model that emphasizes the susceptibility of dopaminergic neurons to oxidative damage.     

Comparison of melatonin, vitamin E and L-carnitine in the treatment of neuro- and hepatotoxicity induced by thioacetamide

This study was designed to evaluate and compare the effect of melatonin, vitamin E and L-carnitine on brain and liver oxidative stress and liver damage. Oxidative stress and hepatic failure were produced by a single dose of thioacetamide (TAA) (150 mg kg(-1)) in Wistar rats. A dose of either melatonin (3 mg kg(-1)) vitamin E (20 mg kg(-1) ) or L-carnitine (100 mg kg(-1)) was used. Blood samples were taken from the neck vasculature in order to determine ammonium, blood urea nitrogen (BUN) and liver enzymes. Lipid peroxidation products, glutathione (GSH) content and antioxidative enzymes were determined in cerebral and hepatic homogenates. The results showed a decrease in BUN and in the antioxidant enzymes activities and GSH in the brain and liver. Likewise, TAA induced significant enhancement of lipid peroxidation products levels in both liver and brain, as well as in ammonia values. Melatonin, vitamin E and L-carnitine, although melatonin more significantly, decreased the intensity of the changes produced by the administration of TAA alone. Furthermore melatonin combined with TAA, decreased the ammonia levels and increased the BUN values compared with TAA animals. Also it was more effective than vitamin E or L-carnitine in these actions. These data show the protective effect of these agents, especially melatonin, against oxidative stress and hepatic damage present in fulminant hepatic failure.     

Effects of melatonin on oxidative stress and spatial memory impairment induced by acute ethanol treatment in rats

Melatonin has recently been suggested as an antioxidant that may protect neurons from oxidative stress. Acute ethanol administration produces both lipid peroxidation as an indicator of oxidative stress in the brain and impairs water-maze performance in spatial learning and memory tasks. The present study investigated the effect of melatonin against ethanol-induced oxidative stress and spatial memory impairment. The Morris water maze was used to evaluate the cognitive functions of rats. Thiobarbituric acid reactive substances (TBARS), which are the indicators of lipid peroxidation, and the activities of antioxidative enzymes (glutathione peroxidase and superoxide dismutase) were measured in the rat hippocampus and prefrontal cortex which form interconnected neural circuits for spatial memory. Acute administration of ethanol significantly increased TBARS levels in the hippocampus. Combined melatonin-ethanol treatment caused a significant increase in glutathione peroxidase activities and a significant decrease of TBARS in the rat hippocampus. In the prefrontal cortex, there was only a significant decrease of TBARS levels in the combined melatonin-ethanol receiving group as compared to the ethanol-treated group. Melatonin did not affect the impairment of spatial memory due to acute ethanol exposure, but melatonin alone had a positive effect on water maze performances. Our study demonstrated that melatonin decreased ethanol-induced lipid peroxidation and increased glutathione peroxidase activity in the rat hippocampus.     

Protective effects of melatonin on myocardial ischemia/reperfusion induced infarct size and oxidative changes

Free radicals, calcium overloading and loss of membrane phospholipids play an important role in the development of ischemia/reperfusion (I/R) injury. Melatonin is a well-known antioxidant and free radical scavenger. Melatonin may also reduce the intracellular calcium overloading and inhibit lipid peroxidation. This study was designed to investigate the effects of melatonin on the I/R-induced cardiac infarct size in an in vivo rat model. We also investigated glutathione (GSH) levels, an antioxidant the levels of which are influenced by oxidative stress, and malondialdehyde (MDA) levels, which is an index of lipid peroxidation. To produce cardiac damage, the left main coronary artery was occluded for 30 min, followed by 120 min reperfusion, in anesthetized rats. Melatonin (10 mg/kg) or vehicle was given 10 min before ischemia via the jugular vein. Infarct size, expressed as the percentage of the risk zone, was found significantly greater in I/R group than in the melatonin-treated I/R group. MDA levels were significantly higher, but GSH levels were lower in the I/R group than in the control group. Melatonin significantly reduced the MDA values and increased the GSH levels. These results suggest that oxidative stress contributes to myocardial I/R injury and melatonin administration exerts a mitigating effect on infarct size. Furthermore, the results indicated that melatonin improves the antioxidant capacity of the heart and attenuates the degree of lipid peroxidation after I/R.     

Alleviation of iron induced oxidative stress by the grape fruit flavanone naringin in vitro

Iron is an essential element that participates in several metabolic activities of cells; however, excess iron is a major cause of iron-induced oxidative stress and several human diseases. The protective effect of naringin, a grape fruit flavanone, was studied in iron overloaded isolated mouse liver mitochondria, where the isolated mitochondrial fraction was incubated with various concentrations of naringin before ferric ion loading. Iron overloading of mitochondrial fraction resulted in an increase in lipid peroxidation, protein oxidation, and DNA damage, whereas iron overload reduced the glutathione (GSH) concentration, glutathione-S-transferase (GST), glutathione peroxidase (GSHPx), catalase and superoxide dismutase (SOD) activities. Pretreatment of mitochondrial fraction with naringin inhibited iron-induced lipid peroxidation, protein oxidation, and DNA damage. Conversely, naringin supplementation arrested iron-induced depletion in the GSH contents, GSHPx, GST, SOD and catalase activities significantly. Ferric iron reduction assay revealed that naringin could not reduce ferric iron into ferrous iron indicating that it did not exhibit prooxidant activity. Iron free coordination site assay indicated that naringin was unable to occupy all the active sites of iron indicating that naringin did not completely chelate iron. Our study demonstrates that naringin was able to share the burden of endogenous oxidants by inhibiting the iron-induced depletion of all important antioxidant enzymes as well as GSH and may act as a good antioxidant.     

Prophylactic action of melatonin against cyclophosphamide-induced oxidative stress in mice

The present study investigated the prophylactic influence of melatonin against cyclophosphamide-induced oxidative stress in mouse tissues. Lipid peroxidation, reduced glutathione (GSH), glutathione disulphide (GSSG), glutathione peroxidase (GSH-Px) and serum phosphatase levels were analyzed in brain, spleen liver, lungs, kidney and testes. Fifteen days oral administration with melatonin (0.1 mg/kg bw per day) before treatment checked the augmentation of the level of lipid peroxidation, blood GSSG and acid phosphatase caused by an acute treatment with a radiomimetic drug, cyclophosphamide (75 mg/kg bw). Cyclophosphamide-induced depletion in the level of GSH, GSH-Px and alkaline phosphatase was made up statistically significant by chronic melatonin administration given orally. The results indicate the antioxidative properties of melatonin resulting into its prophylactic property against the cyclophosphamide-induced biochemical alterations. The finding support the idea that melatonin is a potent free-radical scavenger and antioxidant.     

Semichronic Inhibition of Glutathione Reductase Promotes Oxidative Damage to Proteins and Induces both Transcription and Translation of Tyrosine Hydroxylase in the Nigrostriatal System

We have evaluated the effect of N,N-bis (2-chloroethyl)-N-nitrosourea (BCNU), an inhibitor of glutathione reductase (GR), on the oxidative status along with the integrity of the nigrostriatal dopaminergic system of the rat. The oxidative status was studied by the quantification of carbonyl groups coupled to protein homogenates. Moreover, the specific oxidations in glial fibrillary acidic protein (GFAP) and neurofilament-200 (NF-200) were also measured. The results show that oxidative damage in proteins in the nigrostriatal system is confined to the striatum. Specific carbonyl groups coupled to native NF-200 and GFAP were also increased. These changes were accompanied by reactive astrocytosis in striatum but not in substantia nigra. In substantia nigra, decreased levels of dopamine (DA) and 3,4-dihydroxyphenylacetic acid (DOPAC) were observed following BCNU treatment. In contrast, DA levels were increased in the striatum along with an overall decrease in the ratios of DA metabolites to DA. We also studied the mRNA levels for tyrosine hydroxylase (TH) and the dopamine transporter (DAT) by in situ hybridization. TH mRNA but not DAT mRNA was significantly induced in substantia nigra following BCNU treatment, which was consistent with significant elevations in TH enzyme amount and activity and unchanged DA uptake in striatum. All these results support the DA free radical hypothesis and the key role of the striatal glutathione system in protecting the striatal system against oxidative stress.     

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

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 (3.16+/-0.06 vs. 3.83+/-0.12 nmol/mg protein, P<0.05) and plasma (7. 73+/-0.52 vs. 9.96+/-0.71 nmol/ml, P<0.05) and attenuated the decrease in hepatic microsomal membrane fluidity (1/polarization, 3. 068+/-0.007 vs. 3.027+/-0.008, P<0.05). In vitro exposure to iron significantly enhanced the lipid peroxidation in liver homogenates from untreated (3.34+/-0.75 vs. 1.25+/-0.28, P<0.05) or ascorbic acid-treated rats (2.72+/-0.39 vs. 0.88+/-0.06, P<0.05) but not from melatonin-treated rats (1.49+/-0.55 vs. 0.68+/-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.     

Semichronic inhibition of glutathione reductase promotes oxidative damage to proteins and induces both transcription and translation of tyrosine hydroxylase in the nigrostriatal system

We have evaluated the effect of N,N-bis (2-chloroethyl)-N-nitrosourea (BCNU), an inhibitor of glutathione reductase (GR), on the oxidative status along with the integrity of the nigrostriatal dopaminergic system of the rat. The oxidative status was studied by the quantification of carbonyl groups coupled to protein homogenates. Moreover, the specific oxidations in glial fibrillary acidic protein (GFAP) and neurofilament-200 (NF-200) were also measured. The results show that oxidative damage in proteins in the nigrostriatal system is confined to the striatum. Specific carbonyl groups coupled to native NF-200 and GFAP were also increased. These changes were accompanied by reactive astrocytosis in striatum but not in substantia nigra. In substantia nigra, decreased levels of dopamine (DA) and 3,4-dihydroxyphenylacetic acid (DOPAC) were observed following BCNU treatment. In contrast, DA levels were increased in the striatum along with an overall decrease in the ratios of DA metabolites to DA. We also studied the mRNA levels for tyrosine hydroxylase (TH) and the dopamine transporter (DAT) by in situ hybridization. TH mRNA but not DAT mRNA was significantly induced in substantia nigra following BCNU treatment, which was consistent with significant elevations in TH enzyme amount and activity and unchanged DA uptake in striatum. All these results support the DA free radical hypothesis and the key role of the striatal glutathione system in protecting the striatal system against oxidative stress.     

Iron-Melanin Interaction and Lipid Peroxidation: Implications for Parkinson''s Disease

The vulnerability of substantia nigral (SN) melaninized dopamine neurons to neurodegeneration in Parkinson's disease and the selective increases of iron and basal lipid peroxidation in SN indicate that iron-melanin interaction could be crucial to the pathogenesis of this disease. The present study describes, for the first time, the identification and characterization of a high-affinity (KD = 13 nM) and a lower affinity (KD = 200 nM) binding site for iron on dopamine melanin. The binding of iron to melanin is dependent on pH and the concentration of melanin. Iron chelators, U74500A, desferrioxamine, and to less extent 1,10-phenanthroline and chlorpromazine, but not the Parkinson-inducing neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, can inhibit the binding of iron to melanin and iron-induced lipid peroxidation. Although melanin alone diminishes basal lipid peroxidation in rat cortical homogenates, it can also potentiate that initiated by iron, a reaction inhibited by desferrioxamine. In the absence of an identifiable exogenous or endogenous neurotoxin in idiopathic Parkinson's disease, iron-melanin interaction in pars compacta of SN may be a strong candidate for the cytotoxic component of oxygen radical-induced neurodegeneration of melaninized dopamine neurons.     

Protective effects of melatonin and indole-3-propionic acid against lipid peroxidation, caused by potassium bromate in the rat kidney

Potassium bromate (KBrO(3)) is classified as a carcinogenic agent. KBrO(3) induces tumors and pro-oxidative effects in kidneys. Melatonin is a well known antioxidant and free radical scavenger. Indole-3-propionic acid (IPA), an indole substance, also reveals antioxidative properties. Recently, some antioxidative effects of propylthiouracil (PTU)-an antithyroid drug-have been found. The aim of the study was to compare protective effects of melatonin, IPA, and PTU against lipid peroxidation in the kidneys and blood serum and, additionally, in the livers and the lungs, collected from rats, pretreated with KBrO(3). Male Wistar rats were administered KBrO(3) (110 mg/kg b.w., i.p., on the 10th day of the experiment) and/or melatonin, or IPA (0.0645 mmol/kg b.w., i.p., twice daily, for 10 days), or PTU (0.025% solution in drinking water, for 10 days). The level of lipid peroxidation products-malondialdehyde + 4-hydroxyalkenals (MDA + 4-HDA)-was measured spectrophotometrically in thyroid homogenates. KBrO(3), when injected to rats, significantly increased lipid peroxidation in the kidney homogenates and blood serum, but not in the liver and the lung homogenates. Co-treatment with either melatonin or with IPA, but not with PTU, decreased KBrO(3)-induced oxidative damage to lipids in the rat kidneys and serum. In conclusion, melatonin and IPA, which prevent KBrO(3)-induced lipid peroxidation in rat kidneys, may be of great value as protective agents under conditions of exposure to KBrO(3).