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Polyamines reduces lipid peroxidation induced by different pro-oxidant agents
Brain Research 1008 (2004) 245 – 251 www.elsevier.com/locate/brainres
Research report
Polyamines reduces lipid peroxidation induced by different pro-oxidant agents Na´dia Ale´ssio Velloso Belle´, Gerusa Duarte Dalmolin, Graciela Fonini, Maribel Antonello Rubin, Joa˜o Batista Teixeira Rocha * Departamento de Quı´mica, Centro de Cieˆncias Naturais e Exatas, Universidade Federal de Santa Maria, Campus UFSM, Santa Maria, RS 97105-900, Brazil Accepted 11 February 2004 Available online 15 April 2004
Abstract Polyamines, among other functions, are considered to act as a free radical scavenger and antioxidant. The quinolinic acid (QA), sodium nitroprusside (SNP) and iron (Fe+ 2) stimulate production of free radicals and lipid peroxidation. In the present study, we investigated the free radical and/or aldehyde scavenger effects of polyamines spermine and spermidine on thiobarbituric acid reactive species (TBARS) production induced by QA, SNP, Fe+ 2/EDTA system and free Fe2 + in rat brain. Spermine and spermidine inhibited QA-induced TBARS production; however spermine was a better antioxidant than spermidine. Spermine also inhibited SNP-, Fe+ 2/EDTA- and free Fe2 +-induced TBARS production, but had a modest effect. Spermidine, in turn, also discreetly inhibited SNP-, Fe+ 2/EDTA- and free Fe2 +-induced TBARS production. In the presence of MK-801, QA-induced TBARS production was considerably more inhibited by polyamines. In addition, arcaine does not affect the reducer effect of polyamines. The present findings suggest that the observed effects of polyamines are not related to the activation of NMDA receptor but with their antioxidant and free radical scavenger properties. D 2004 Elsevier B.V. All rights reserved. Theme: Disorders of the nervous system Topic: Neurotoxicity Keywords: Polyamine; Scavenger; Free radical; Antioxidant; Lipid peroxidation
1. Introduction Free radicals are now accepted as important mediators of tissue injury in several neurodegenerative models [6,22,28,45,49]. The brain is particularly susceptible to free radical damage because of its high utilization of oxygen and its relatively low concentration of antioxidants enzymes and free radicals scavengers. These free radicals may attack membrane lipids, proteins and nucleic acids to cause cell damage or death [24,25,36,37]. During oxidative stress, cell injury can be amplified by reactive compounds that are by-products of oxidative damage including malondialdehyde, 4-hydroxyalkenals and numerous 2-alkenals [17]. Based on the observation of elevated levels of the * Corresponding author. Tel.: +55-55-220-8140; fax: +55-55-2208031. E-mail address: [email protected] (J.B.T. Rocha). 0006-8993/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2004.02.036
aldehydes and/or their respective macromolecular adducts, these lipid peroxidation products appear to contribute to the etiology of a number of chronic diseases including neurodegenerative conditions, chronic inflammatory diseases, alcoholic liver disease, cardiovascular disorders and diabetic complications [3,10,13,15,54]. Pharmacological efforts to attenuate oxidative injury in degenerative diseases have typically focused on drugs with antioxidant properties [48]. Such approaches provide a ‘first line of defense’ against free radicals, but do not target secondary products of oxidative stress, including numerous reactive a,h-unsaturated aldehydes. A complementary strategy involves identification of low-molecular weight drugs possessing nucleophilic centers (e.g. primary amine groups) that exhibit high reactivity toward endogenous aldehydes. In biological systems such drugs might act as ‘aldehyde scavengers’, sparing cellular constituents and slowing disease progression [46].
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The polyamines (spermine, spermidine and putrescine) are ornitine metabolic products, which are found at high concentrations in the brain [2,57]. The primary and secondary amine moieties of polyamines always carry a charge at physiological pH, resulting in low molecular weight ‘organic cations’, the most important characteristic required for ‘aldehyde scavengers’. This polycationic quality has attracted the attention of researchers and led to the hypothesis that polyamines could affect physiological systems by binding to anionic sites, such as those associated with nucleic acids and membrane phospholipids [27]. These amines are involved with numerous cellular functions, including free radical scavenger, antioxidant and antiinflammatory properties [1,14,16,19,23,32]. In addition, recent experimental evidence supports a role for polyamines as transcriptional regulators in the nucleus of neural eukaryotic cells [30]. However, the precise mechanisms by which polyamines exert their regulatory effects are unresolved. It have been reported that spermine inhibits both NADPH- and ascorbic acid-dependent lipid peroxidation in liver microsomes [29]. Quinolinic acid (QA) is a neuroactive metabolite of the tryptophan-kinurenine pathway which can be produced by macrophages and microglia [11,51]. It is present in both the human and rat brain [58] and it has been implicated in the pathogenesis of a variety of human neurological diseases [7,31]. QA is recognized pharmacologically as an endogenous glutamate agonist with a relative selectivity for the NMDA receptor in the brain [20]. Since it is not readily metabolized in the synaptic cleft, it stimulates the NMDA receptor for prolonged periods. This sustained stimulation results in opening of calcium channels causing Ca+ 2 influx followed by Ca+ 2-dependent enhancement of free radical production leading to molecular damage and often to cell death [9,52]. In addition, it has been reported that QA was able to stimulate lipid peroxidation in rat brain homogenates [39,43]. These findings have suggested the involvement of free radicals and oxidative stress in the pattern of toxicity elicited by QA in the nervous system [41]. Sodium nitroprusside (SNP) has been suggested to cause cytotoxicity through the release of cyanide and/or nitric oxide (NO) [4,5]. There is a growing number of recent studies concerning the role of NO, a molecule that is regarded as universal neuronal messenger in the central nervous system, in the pathophysiology of such disorders as Alzheimer’s and Parkinson’s diseases, stroke, trauma, seizure disorders, etc. [8,12,34,55]. NO is a free radical with short half-life ( < 30 s). Although NO acts independently, it also may cause neuronal damage in cooperation with other reactive oxygen species (ROS) [26,35]. Therefore, the aim of this study was to verify the potential protective effects of spermine and spermidine against thiobarbituric reactive species (TBARS) production induced by different pro-oxidant agents and provide an hypothesis of the mechanisms by which polyamines exert their regulatory effects.
2. Materials and methods 2.1. Chemicals Tris(hydroximethyl)aminomethane, spermine, spermidine, arcaine, quinolinic acid, thiobarbituric acid and malonaldehyde bis-(dimethyl acetal) (MDA) were obtained from Sigma (St. Louis, MO, USA). MK-801 was obtained from Research Biochemicals International (Natick, MA, USA). Sodium nitroprusside was obtained from Merck (Darmstadt, Germany). Ethylenediaminetetraacetic (EDTA), HCl and acetic acid were obtained from Merck (Rio de Janeiro, RJ, Brazil). Ammonium iron(II) sulfate was obtained from Reagen (Rio de Janeiro, RJ, Brazil). 2.2. Animals All animal procedures were in strict accordance with the NIH Guide for the Care and Use of Laboratory Animals. In these experiments, every effort was made to reduce the number and the suffering of the animals used. Two- to three-month old Wistar rats (200 – 250 g) from our own breeding colony were used. They were maintained at 25 jC, on a 12-h light/12-h dark cycle, with free access to food and water. 2.3. Production of TBARS Rats were decapitated under mild ether anesthesia and the cerebral tissue (whole brain) was rapidly dissected, placed on ice and weighed. Tissues were immediately homogenized in cold 10 mM Tris –HCl, pH 7.5 (1/10, w/v) with 10 up-and-down strokes at approximately 1200 rev/min in a Teflon-glass homogenizer. The homogenate was centrifuged for 10 min at 3000 g to yield a pellet that was discarded and a low-speed supernatant (S1). Just after the end of centrifugation, an aliquot of 200 Al or 100 Al of S1 was incubated for 1 h at 37 jC and then used for lipid peroxidation determination. One rat brain was used per experiment. A summary of all tested substances, their concentrations and combinations is depicted in Table 1. Production of TBARS were determined as described by Ohkawa et al. [33] excepting that the buffer of color reaction have a pH of 3.4. The color reaction was developed by adding 300 Al 8.1% SDS to S1, followed by sequential addition of 600 Al acetic acid/HCl (pH 3.4) and 600 Al 0.8% TBA. This mixture was incubated at 95 jC for 1 h. TBARS produced were measured at 532 nm and the absorbance was compared to that of a standard curve obtained using MDA. 2.4. Statistical analysis Data were analyzed statistically by two- or three-way ANOVA, followed by Duncan’s multiple range test when
N.A.V. Belle´ et al. / Brain Research 1008 (2004) 245–251 Table 1 Concentrations of spermine (SPM), spermidine (SPD), quinolinic acid (QA), sodium nitroprusside (SNP), MK-801, arcaine, Fe2 +/EDTA complex and Fe2 + SPM
AQ
SNP
MK801
ARCAINE
Fe2 +/ EDTA
Fe2 +
0.000033 – 2 mM 2500 AM 0.00033 – 5 AM 2000 AM 30 – 1000 AM 2 mM 1 mM 1000 AM 2 mM 10 – 1000 AM 10 – 2500 AM 100 AM 1000 – 1 – 100 2500 AM AM SPD
QA
SNP
MK801
ARCAINE
Fe2 +/ EDTA
Fe2 +
0.000033 – 2 mM 2000 AM 330 – 5 AM 20,000 AM 100 – 2 mM 1 mM 2000 AM 1000 AM 2 mM 10 – 1000 AM 10 – 2500 AM 100 AM 1000 and 1 – 100 2500 AM AM
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induced TBARS production in a concentration-dependent manner, but have negligible effect in the basal TBARS production. These effects were confirmed by the significant spermidine vs. QA interaction ( F(5,36) = 5.38, P < 0.05) and IC50 for QA-induced TBARS production by spermidine was 1300 AM (Fig. 1B). 3.2. Quinolinic acid vs. MK-801 vs. Polyamines The influence of MK-801, a classical antagonist of NMDA receptor, on the pro-oxidant effect of QA and antioxidant effect of polyamines is depicted in Fig 2. Three-way ANOVA revealed the following second order significant interactions: spermine vs. QA ( F(4,60) = 19.80, P < 0.001), spermine vs. MK-801 interaction ( F(4,60) = 4.82, P < 0.01) and MK-801 vs. QA ( F(1,60) = 80.47, P < 0.001). As observed above (Fig. 1A), spermine reduced considerably the TBARS production induced by
The concentrations presented in the same row of SPM or SPD indicates that the substances were tested in combination with them.
appropriate. Significant main effects will be presented only when two- or three-way interactions were not significant. Differences between groups were considered to be significant when P < 0.05.
3. Results 3.1. Quinolinic acid vs. Polyamines Spermine (0.000033– 3.33 AM) did not change the basal production of TBARS by brain homogenates. QA elicited a marked increase in TBARS production and this effect was not modified by spermine at the same concentrations range (data not shown). Similarly, spermidine (0.00003 –0.002 AM) did not change the basal production of TBARS. QA caused a marked increase in TBARS production which was not counteracted by these concentrations of spermidine (data not shown). At higher concentrations tested, spermine caused a pronounced reduction in a concentration-dependent manner in TBARS production induced by QA, whereas it had a modest effect on the basal TBARS production. These effects were confirmed by the significant spermine vs. QA interaction ( F(8,37) = 10.28, P < 0.001). The calculated IC50 for TBARS production induced by QA was about 230 AM of spermine (Fig. 1A). Similarly, spermidine also reduced QA-
Fig. 1. Effect of different concentrations of spermine (A) and spermidine (B) on basal and QA (2 mM)-induced TBARS production in whole rat brain homogenates. Data shown mean F S.E.M. values averaged from three independent experiments performed in duplicate and are presented as percentage of control values obtained in absence of polyamines and QA. Control value was 144.38 nmol MDA/g tissue/h for spermine and 176.63 nmol MDA/g tissue/h for spermidine.
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3.3. Quinolinic acid vs. Arcaine vs. Polyamines Arcaine, an antagonist of binding sites of polyamines on NMDA receptor [38], did not alter the basal or QA-induced TBARS production in the presence or absence of polyamines (data not shown). 3.4. Sodium nitroprusside vs. Polyamines Two-way ANOVA indicated that spermine caused a reduction in basal and SNP-induced TBARS production, when tested at 2 mM (Fig. 3A). In fact, ANOVA indicated a significant main effect of spermine ( F(9,40) = 3.87, P < 0.01) and of SNP ( F(1,40) = 262.88, P < 0.001). In parallel to spermine, spermidine also reduce the basal and SNP-triggered TBARS production, when tested at 20 mM. Two-way ANOVA yield a significant main effect of spermidine ( F(5,24) = 5.15, P < 0.01) and of SNP ( F(1,24) = 622.62, P < 0.001; Fig. 3B).
Fig. 2. Effect of different concentrations of spermine (A) and spermidine (B) on basal, MK-801 (1 mM), QA (2 mM) and QA (2 mM) plus MK-801 (1 mM) TBARS production in whole rat brain homogenates. Data shown mean F S.E.M. values averaged from four independent experiments performed in duplicate and are presented as percentage of control values obtained in absence of polyamines, QA and MK-801. Control value was 180.17 nmol MDA/g tissue/h for spermine and 185.83 nmol MDA/g tissue/ h for spermidine.
QA. Addition of MK-801 (1 mM) caused a marked reduction on the TBARS production stimulated by QA. Furthermore, in the presence of QA and MK-801, increasing concentrations of spermine caused an additional decrease in TBARS production to levels lower than that observed for basal condition or only in the presence of MK-801 (Fig. 2A). Three-way ANOVA revealed the following significant second order interactions: spermidine vs. QA ( F(4,60) = 11.25, P < 0.001) and QA vs. MK-801 ( F(1,60) = 83.70, P < 0.001). As observed above (Fig. 1B), spermidine reduced considerably the TBARS production induced by QA. Inclusion of 1 mM of MK-801 caused a marked reduction on the TBARS production stimulated by QA. Furthermore, in the presence of QA and MK-801, increasing concentrations of spermidine caused an additional decrease in TBARS production to levels lower than that observed for basal condition or only in the presence of MK-801 (Fig. 2B).
Fig. 3. Effect of different concentrations of spermine (A) and spermidine (B) on basal and SNP (5 AM)-induced TBARS production in whole rat brain homogenates. Data shown mean F S.E.M. values averaged from three independent experiments performed in duplicate and are presented as percentage of control values obtained in absence of polyamines and SNP. Control value was 155.83 nmol MDA/g tissue/h for spermine and 159.33 nmol MDA/g tissue/h for spermidine.
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3.5. Fe2+/EDTA vs. Polyamines Two-way ANOVA yielded a significant spermine vs. Fe2 +/EDTA interaction ( F(4,40) = 4.16, P < 0.01). Fe2 +/ EDTA caused a marked increase in TBARS production that was partially reduced by high concentrations of spermine (Fig. 4A). The calculated IC50 for TBARS production induced Fe2 +/EDTA was about 2630 AM of spermine. Similarly, two-way ANOVA showed a significant spermidine vs. Fe2 +/EDTA interaction ( F(4,40) = 9.70, P < 0.01). High concentrations of spermidine partially decrease Fe2 +/ EDTA-induced TBARS production. The IC50 for Fe2 +/ EDTA-induced TBARS production by spermidine was not possible to calculate due to the weak effect of this polyamine (Fig. 4B). 3.6. Free Fe2+ vs. Polyamines Increasing concentrations of Fe2 + stimulated significantly lipid peroxidation in brain homogenates and spermine counteracted this stimulation. The inhibitory effect of sper-
Fig. 5. Effect of different concentrations of iron on basal and 1 or 2.5 mM spermine (A) and 1 or 2.5 mM spermidine (B) TBARS production in whole rat brain homogenates. Data shown mean F S.E.M. values averaged from four independent experiments performed in duplicate and are presented as percentage of control values obtained in absence of iron and polyamines. Control value was 185.75 nmol MDA/g tissue/h for spermine and 187.46 nmol MDA/g tissue/h for spermidine.
mine was more evident at the higher concentration of Fe2 + tested (Fig. 5A). Two-way ANOVA revealed, in addition to the main effects of Fe2 +and spermine, a significant spermine vs. Fe2 + interaction ( F(6,36) = 5.58, P < 0.001). Spermidine also reduced Fe 2 + -induced lipid peroxidation ( F(2,24) = 32.7, P < 0.001; Fig. 5B).
4. Discussion
Fig. 4. Effect of different concentrations of spermine (A) and spermidine (B) on basal and Fe2 +/EDTA (100 AM)-induced TBARS production in whole rat brain homogenates. Data shown mean F S.E.M. values averaged from five independent experiments performed in duplicate and are presented as percentage of control values obtained in absence of polyamines and Fe2 +/EDTA. Control value was 158.10 nmol MDA/g tissue/h for spermine and 176.00 nmol MDA/g tissue/h for spermidine.
The main findings of the present study can be summarized in the following topics: (1) spermine and spermidine reduced QA-induced lipid peroxidation; (2) spermine and spermidine, at relatively high concentrations, have a modest inhibitory effect against sodium nitroprusside-induced lipid peroxidation; and (3) at relatively high concentrations, both amines have a modest effect as inhibitors of Fe2 +- or Fe2 +/ EDTA-induced TBARS production. Taken together, these results can indicate that the antioxidant effect of these polyamines is more specific for certain
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toxins or pro-oxidant situations. In fact, at physiological concentrations, spermine has a considerable inhibitory effect against QA-induced TBARS production, whereas spermidine has a modest inhibitory effect. In the presence of sodium nitroprusside or iron, the antioxidant effects of these polyamines were observed only at supraphysiological concentrations of the amines [47]. The modest effect of polyamines against nitroprusside-induced TBARS formation indicates some specificity of action of these amines. In all cases, spermine was a more potent reducer of TBARS production caused by these three pro-oxidant agents. These results can be related to the presence of more amine groups in the structure of spermine, when compared with spermidine. In the present study, we realized that the pro-oxidant effects of QA are attribute to sustained stimulation of NMDA receptors, which induces free radical production and posterior lipid peroxidation [18,44]. This assumption is supported by the fact that MK-801, a classical antagonist of NMDA receptors [42,59,60], suppresses TBARS production induced by QA. Neurophysiological data indicates that the effect of spermine and spermidine on NMDA receptor activation is biphasic. At low concentrations, polyamines enhance NMDA-evoked whole-cell currents, whereas higher concentrations of polyamines produced less or even inhibited NMDA receptor mediated currents [40,50,56]. So, at low micromolar concentrations (which are reported to facilitate the activation of NMDA receptor), spermine and spermidine did not modify TBARS production. These results indicate that the observed effects of polyamines are not related to the activation of NMDA receptor. Accordingly, the observed effects probably are related with free radical and/or aldehyde scavenger activity of polyamines. Probably these amines functions like free radical scavengers since for ‘aldehyde scavenger’ activity its necessary mainly low molecular weight and primary amino groups, founded in both polyamines [46]. It well knows that iron and iron complexes stimulate lipid peroxidation in cells [21]. The mechanism(s) that underlies the antioxidant activity of polyamines measured in the presence of Fe2 + have been credited to the formation of a ternary complex with the phospholipid polar head and the Fe3 +, which in turn retards Fe2 + oxidation, reduces generation of reactive species [53]. Formation of such kind of ternary complexes in our assay system is possible and can, at least in part, explain the antioxidant activity of spermine and spermidine. However, formation of such complexes are probably not involved in the protection caused by polyamines, because spermine and spermidine had only a weak protection effect against Fe2 +-induced lipid peroxidation. In conclusion, the results of the present investigation support a scavenger and antioxidant role for polyamines in brain. However, the antioxidant activity of these compounds varied considerably depending on the agent used to induce TBARS formation and, to a less extent, on the
polyamine considered. In fact, spermine was superior to spermidine as scavenger and/or antioxidant in all situations tested. Of particular importance, at physiological concentrations, polyamines reduced the TBARS production induced by QA, an endogenous neurotoxin that activates NMDA receptors and is supposed be increased in neurodegenerative diseases such as Huntington [7,31], raise the possibility of their use as therapeutic or preventive agents against these pathologic situations.
Acknowledgements This study was supported by CNPq (474241/2003-3, 523761/95-3 and 500096/2003-1), CAPES and FAPERGS (01/1338.9).
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Research report
Polyamines reduces lipid peroxidation induced by different pro-oxidant agents Na´dia Ale´ssio Velloso Belle´, Gerusa Duarte Dalmolin, Graciela Fonini, Maribel Antonello Rubin, Joa˜o Batista Teixeira Rocha * Departamento de Quı´mica, Centro de Cieˆncias Naturais e Exatas, Universidade Federal de Santa Maria, Campus UFSM, Santa Maria, RS 97105-900, Brazil Accepted 11 February 2004 Available online 15 April 2004
Abstract Polyamines, among other functions, are considered to act as a free radical scavenger and antioxidant. The quinolinic acid (QA), sodium nitroprusside (SNP) and iron (Fe+ 2) stimulate production of free radicals and lipid peroxidation. In the present study, we investigated the free radical and/or aldehyde scavenger effects of polyamines spermine and spermidine on thiobarbituric acid reactive species (TBARS) production induced by QA, SNP, Fe+ 2/EDTA system and free Fe2 + in rat brain. Spermine and spermidine inhibited QA-induced TBARS production; however spermine was a better antioxidant than spermidine. Spermine also inhibited SNP-, Fe+ 2/EDTA- and free Fe2 +-induced TBARS production, but had a modest effect. Spermidine, in turn, also discreetly inhibited SNP-, Fe+ 2/EDTA- and free Fe2 +-induced TBARS production. In the presence of MK-801, QA-induced TBARS production was considerably more inhibited by polyamines. In addition, arcaine does not affect the reducer effect of polyamines. The present findings suggest that the observed effects of polyamines are not related to the activation of NMDA receptor but with their antioxidant and free radical scavenger properties. D 2004 Elsevier B.V. All rights reserved. Theme: Disorders of the nervous system Topic: Neurotoxicity Keywords: Polyamine; Scavenger; Free radical; Antioxidant; Lipid peroxidation
1. Introduction Free radicals are now accepted as important mediators of tissue injury in several neurodegenerative models [6,22,28,45,49]. The brain is particularly susceptible to free radical damage because of its high utilization of oxygen and its relatively low concentration of antioxidants enzymes and free radicals scavengers. These free radicals may attack membrane lipids, proteins and nucleic acids to cause cell damage or death [24,25,36,37]. During oxidative stress, cell injury can be amplified by reactive compounds that are by-products of oxidative damage including malondialdehyde, 4-hydroxyalkenals and numerous 2-alkenals [17]. Based on the observation of elevated levels of the * Corresponding author. Tel.: +55-55-220-8140; fax: +55-55-2208031. E-mail address: [email protected] (J.B.T. Rocha). 0006-8993/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2004.02.036
aldehydes and/or their respective macromolecular adducts, these lipid peroxidation products appear to contribute to the etiology of a number of chronic diseases including neurodegenerative conditions, chronic inflammatory diseases, alcoholic liver disease, cardiovascular disorders and diabetic complications [3,10,13,15,54]. Pharmacological efforts to attenuate oxidative injury in degenerative diseases have typically focused on drugs with antioxidant properties [48]. Such approaches provide a ‘first line of defense’ against free radicals, but do not target secondary products of oxidative stress, including numerous reactive a,h-unsaturated aldehydes. A complementary strategy involves identification of low-molecular weight drugs possessing nucleophilic centers (e.g. primary amine groups) that exhibit high reactivity toward endogenous aldehydes. In biological systems such drugs might act as ‘aldehyde scavengers’, sparing cellular constituents and slowing disease progression [46].
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The polyamines (spermine, spermidine and putrescine) are ornitine metabolic products, which are found at high concentrations in the brain [2,57]. The primary and secondary amine moieties of polyamines always carry a charge at physiological pH, resulting in low molecular weight ‘organic cations’, the most important characteristic required for ‘aldehyde scavengers’. This polycationic quality has attracted the attention of researchers and led to the hypothesis that polyamines could affect physiological systems by binding to anionic sites, such as those associated with nucleic acids and membrane phospholipids [27]. These amines are involved with numerous cellular functions, including free radical scavenger, antioxidant and antiinflammatory properties [1,14,16,19,23,32]. In addition, recent experimental evidence supports a role for polyamines as transcriptional regulators in the nucleus of neural eukaryotic cells [30]. However, the precise mechanisms by which polyamines exert their regulatory effects are unresolved. It have been reported that spermine inhibits both NADPH- and ascorbic acid-dependent lipid peroxidation in liver microsomes [29]. Quinolinic acid (QA) is a neuroactive metabolite of the tryptophan-kinurenine pathway which can be produced by macrophages and microglia [11,51]. It is present in both the human and rat brain [58] and it has been implicated in the pathogenesis of a variety of human neurological diseases [7,31]. QA is recognized pharmacologically as an endogenous glutamate agonist with a relative selectivity for the NMDA receptor in the brain [20]. Since it is not readily metabolized in the synaptic cleft, it stimulates the NMDA receptor for prolonged periods. This sustained stimulation results in opening of calcium channels causing Ca+ 2 influx followed by Ca+ 2-dependent enhancement of free radical production leading to molecular damage and often to cell death [9,52]. In addition, it has been reported that QA was able to stimulate lipid peroxidation in rat brain homogenates [39,43]. These findings have suggested the involvement of free radicals and oxidative stress in the pattern of toxicity elicited by QA in the nervous system [41]. Sodium nitroprusside (SNP) has been suggested to cause cytotoxicity through the release of cyanide and/or nitric oxide (NO) [4,5]. There is a growing number of recent studies concerning the role of NO, a molecule that is regarded as universal neuronal messenger in the central nervous system, in the pathophysiology of such disorders as Alzheimer’s and Parkinson’s diseases, stroke, trauma, seizure disorders, etc. [8,12,34,55]. NO is a free radical with short half-life ( < 30 s). Although NO acts independently, it also may cause neuronal damage in cooperation with other reactive oxygen species (ROS) [26,35]. Therefore, the aim of this study was to verify the potential protective effects of spermine and spermidine against thiobarbituric reactive species (TBARS) production induced by different pro-oxidant agents and provide an hypothesis of the mechanisms by which polyamines exert their regulatory effects.
2. Materials and methods 2.1. Chemicals Tris(hydroximethyl)aminomethane, spermine, spermidine, arcaine, quinolinic acid, thiobarbituric acid and malonaldehyde bis-(dimethyl acetal) (MDA) were obtained from Sigma (St. Louis, MO, USA). MK-801 was obtained from Research Biochemicals International (Natick, MA, USA). Sodium nitroprusside was obtained from Merck (Darmstadt, Germany). Ethylenediaminetetraacetic (EDTA), HCl and acetic acid were obtained from Merck (Rio de Janeiro, RJ, Brazil). Ammonium iron(II) sulfate was obtained from Reagen (Rio de Janeiro, RJ, Brazil). 2.2. Animals All animal procedures were in strict accordance with the NIH Guide for the Care and Use of Laboratory Animals. In these experiments, every effort was made to reduce the number and the suffering of the animals used. Two- to three-month old Wistar rats (200 – 250 g) from our own breeding colony were used. They were maintained at 25 jC, on a 12-h light/12-h dark cycle, with free access to food and water. 2.3. Production of TBARS Rats were decapitated under mild ether anesthesia and the cerebral tissue (whole brain) was rapidly dissected, placed on ice and weighed. Tissues were immediately homogenized in cold 10 mM Tris –HCl, pH 7.5 (1/10, w/v) with 10 up-and-down strokes at approximately 1200 rev/min in a Teflon-glass homogenizer. The homogenate was centrifuged for 10 min at 3000 g to yield a pellet that was discarded and a low-speed supernatant (S1). Just after the end of centrifugation, an aliquot of 200 Al or 100 Al of S1 was incubated for 1 h at 37 jC and then used for lipid peroxidation determination. One rat brain was used per experiment. A summary of all tested substances, their concentrations and combinations is depicted in Table 1. Production of TBARS were determined as described by Ohkawa et al. [33] excepting that the buffer of color reaction have a pH of 3.4. The color reaction was developed by adding 300 Al 8.1% SDS to S1, followed by sequential addition of 600 Al acetic acid/HCl (pH 3.4) and 600 Al 0.8% TBA. This mixture was incubated at 95 jC for 1 h. TBARS produced were measured at 532 nm and the absorbance was compared to that of a standard curve obtained using MDA. 2.4. Statistical analysis Data were analyzed statistically by two- or three-way ANOVA, followed by Duncan’s multiple range test when
N.A.V. Belle´ et al. / Brain Research 1008 (2004) 245–251 Table 1 Concentrations of spermine (SPM), spermidine (SPD), quinolinic acid (QA), sodium nitroprusside (SNP), MK-801, arcaine, Fe2 +/EDTA complex and Fe2 + SPM
AQ
SNP
MK801
ARCAINE
Fe2 +/ EDTA
Fe2 +
0.000033 – 2 mM 2500 AM 0.00033 – 5 AM 2000 AM 30 – 1000 AM 2 mM 1 mM 1000 AM 2 mM 10 – 1000 AM 10 – 2500 AM 100 AM 1000 – 1 – 100 2500 AM AM SPD
QA
SNP
MK801
ARCAINE
Fe2 +/ EDTA
Fe2 +
0.000033 – 2 mM 2000 AM 330 – 5 AM 20,000 AM 100 – 2 mM 1 mM 2000 AM 1000 AM 2 mM 10 – 1000 AM 10 – 2500 AM 100 AM 1000 and 1 – 100 2500 AM AM
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induced TBARS production in a concentration-dependent manner, but have negligible effect in the basal TBARS production. These effects were confirmed by the significant spermidine vs. QA interaction ( F(5,36) = 5.38, P < 0.05) and IC50 for QA-induced TBARS production by spermidine was 1300 AM (Fig. 1B). 3.2. Quinolinic acid vs. MK-801 vs. Polyamines The influence of MK-801, a classical antagonist of NMDA receptor, on the pro-oxidant effect of QA and antioxidant effect of polyamines is depicted in Fig 2. Three-way ANOVA revealed the following second order significant interactions: spermine vs. QA ( F(4,60) = 19.80, P < 0.001), spermine vs. MK-801 interaction ( F(4,60) = 4.82, P < 0.01) and MK-801 vs. QA ( F(1,60) = 80.47, P < 0.001). As observed above (Fig. 1A), spermine reduced considerably the TBARS production induced by
The concentrations presented in the same row of SPM or SPD indicates that the substances were tested in combination with them.
appropriate. Significant main effects will be presented only when two- or three-way interactions were not significant. Differences between groups were considered to be significant when P < 0.05.
3. Results 3.1. Quinolinic acid vs. Polyamines Spermine (0.000033– 3.33 AM) did not change the basal production of TBARS by brain homogenates. QA elicited a marked increase in TBARS production and this effect was not modified by spermine at the same concentrations range (data not shown). Similarly, spermidine (0.00003 –0.002 AM) did not change the basal production of TBARS. QA caused a marked increase in TBARS production which was not counteracted by these concentrations of spermidine (data not shown). At higher concentrations tested, spermine caused a pronounced reduction in a concentration-dependent manner in TBARS production induced by QA, whereas it had a modest effect on the basal TBARS production. These effects were confirmed by the significant spermine vs. QA interaction ( F(8,37) = 10.28, P < 0.001). The calculated IC50 for TBARS production induced by QA was about 230 AM of spermine (Fig. 1A). Similarly, spermidine also reduced QA-
Fig. 1. Effect of different concentrations of spermine (A) and spermidine (B) on basal and QA (2 mM)-induced TBARS production in whole rat brain homogenates. Data shown mean F S.E.M. values averaged from three independent experiments performed in duplicate and are presented as percentage of control values obtained in absence of polyamines and QA. Control value was 144.38 nmol MDA/g tissue/h for spermine and 176.63 nmol MDA/g tissue/h for spermidine.
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3.3. Quinolinic acid vs. Arcaine vs. Polyamines Arcaine, an antagonist of binding sites of polyamines on NMDA receptor [38], did not alter the basal or QA-induced TBARS production in the presence or absence of polyamines (data not shown). 3.4. Sodium nitroprusside vs. Polyamines Two-way ANOVA indicated that spermine caused a reduction in basal and SNP-induced TBARS production, when tested at 2 mM (Fig. 3A). In fact, ANOVA indicated a significant main effect of spermine ( F(9,40) = 3.87, P < 0.01) and of SNP ( F(1,40) = 262.88, P < 0.001). In parallel to spermine, spermidine also reduce the basal and SNP-triggered TBARS production, when tested at 20 mM. Two-way ANOVA yield a significant main effect of spermidine ( F(5,24) = 5.15, P < 0.01) and of SNP ( F(1,24) = 622.62, P < 0.001; Fig. 3B).
Fig. 2. Effect of different concentrations of spermine (A) and spermidine (B) on basal, MK-801 (1 mM), QA (2 mM) and QA (2 mM) plus MK-801 (1 mM) TBARS production in whole rat brain homogenates. Data shown mean F S.E.M. values averaged from four independent experiments performed in duplicate and are presented as percentage of control values obtained in absence of polyamines, QA and MK-801. Control value was 180.17 nmol MDA/g tissue/h for spermine and 185.83 nmol MDA/g tissue/ h for spermidine.
QA. Addition of MK-801 (1 mM) caused a marked reduction on the TBARS production stimulated by QA. Furthermore, in the presence of QA and MK-801, increasing concentrations of spermine caused an additional decrease in TBARS production to levels lower than that observed for basal condition or only in the presence of MK-801 (Fig. 2A). Three-way ANOVA revealed the following significant second order interactions: spermidine vs. QA ( F(4,60) = 11.25, P < 0.001) and QA vs. MK-801 ( F(1,60) = 83.70, P < 0.001). As observed above (Fig. 1B), spermidine reduced considerably the TBARS production induced by QA. Inclusion of 1 mM of MK-801 caused a marked reduction on the TBARS production stimulated by QA. Furthermore, in the presence of QA and MK-801, increasing concentrations of spermidine caused an additional decrease in TBARS production to levels lower than that observed for basal condition or only in the presence of MK-801 (Fig. 2B).
Fig. 3. Effect of different concentrations of spermine (A) and spermidine (B) on basal and SNP (5 AM)-induced TBARS production in whole rat brain homogenates. Data shown mean F S.E.M. values averaged from three independent experiments performed in duplicate and are presented as percentage of control values obtained in absence of polyamines and SNP. Control value was 155.83 nmol MDA/g tissue/h for spermine and 159.33 nmol MDA/g tissue/h for spermidine.
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3.5. Fe2+/EDTA vs. Polyamines Two-way ANOVA yielded a significant spermine vs. Fe2 +/EDTA interaction ( F(4,40) = 4.16, P < 0.01). Fe2 +/ EDTA caused a marked increase in TBARS production that was partially reduced by high concentrations of spermine (Fig. 4A). The calculated IC50 for TBARS production induced Fe2 +/EDTA was about 2630 AM of spermine. Similarly, two-way ANOVA showed a significant spermidine vs. Fe2 +/EDTA interaction ( F(4,40) = 9.70, P < 0.01). High concentrations of spermidine partially decrease Fe2 +/ EDTA-induced TBARS production. The IC50 for Fe2 +/ EDTA-induced TBARS production by spermidine was not possible to calculate due to the weak effect of this polyamine (Fig. 4B). 3.6. Free Fe2+ vs. Polyamines Increasing concentrations of Fe2 + stimulated significantly lipid peroxidation in brain homogenates and spermine counteracted this stimulation. The inhibitory effect of sper-
Fig. 5. Effect of different concentrations of iron on basal and 1 or 2.5 mM spermine (A) and 1 or 2.5 mM spermidine (B) TBARS production in whole rat brain homogenates. Data shown mean F S.E.M. values averaged from four independent experiments performed in duplicate and are presented as percentage of control values obtained in absence of iron and polyamines. Control value was 185.75 nmol MDA/g tissue/h for spermine and 187.46 nmol MDA/g tissue/h for spermidine.
mine was more evident at the higher concentration of Fe2 + tested (Fig. 5A). Two-way ANOVA revealed, in addition to the main effects of Fe2 +and spermine, a significant spermine vs. Fe2 + interaction ( F(6,36) = 5.58, P < 0.001). Spermidine also reduced Fe 2 + -induced lipid peroxidation ( F(2,24) = 32.7, P < 0.001; Fig. 5B).
4. Discussion
Fig. 4. Effect of different concentrations of spermine (A) and spermidine (B) on basal and Fe2 +/EDTA (100 AM)-induced TBARS production in whole rat brain homogenates. Data shown mean F S.E.M. values averaged from five independent experiments performed in duplicate and are presented as percentage of control values obtained in absence of polyamines and Fe2 +/EDTA. Control value was 158.10 nmol MDA/g tissue/h for spermine and 176.00 nmol MDA/g tissue/h for spermidine.
The main findings of the present study can be summarized in the following topics: (1) spermine and spermidine reduced QA-induced lipid peroxidation; (2) spermine and spermidine, at relatively high concentrations, have a modest inhibitory effect against sodium nitroprusside-induced lipid peroxidation; and (3) at relatively high concentrations, both amines have a modest effect as inhibitors of Fe2 +- or Fe2 +/ EDTA-induced TBARS production. Taken together, these results can indicate that the antioxidant effect of these polyamines is more specific for certain
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toxins or pro-oxidant situations. In fact, at physiological concentrations, spermine has a considerable inhibitory effect against QA-induced TBARS production, whereas spermidine has a modest inhibitory effect. In the presence of sodium nitroprusside or iron, the antioxidant effects of these polyamines were observed only at supraphysiological concentrations of the amines [47]. The modest effect of polyamines against nitroprusside-induced TBARS formation indicates some specificity of action of these amines. In all cases, spermine was a more potent reducer of TBARS production caused by these three pro-oxidant agents. These results can be related to the presence of more amine groups in the structure of spermine, when compared with spermidine. In the present study, we realized that the pro-oxidant effects of QA are attribute to sustained stimulation of NMDA receptors, which induces free radical production and posterior lipid peroxidation [18,44]. This assumption is supported by the fact that MK-801, a classical antagonist of NMDA receptors [42,59,60], suppresses TBARS production induced by QA. Neurophysiological data indicates that the effect of spermine and spermidine on NMDA receptor activation is biphasic. At low concentrations, polyamines enhance NMDA-evoked whole-cell currents, whereas higher concentrations of polyamines produced less or even inhibited NMDA receptor mediated currents [40,50,56]. So, at low micromolar concentrations (which are reported to facilitate the activation of NMDA receptor), spermine and spermidine did not modify TBARS production. These results indicate that the observed effects of polyamines are not related to the activation of NMDA receptor. Accordingly, the observed effects probably are related with free radical and/or aldehyde scavenger activity of polyamines. Probably these amines functions like free radical scavengers since for ‘aldehyde scavenger’ activity its necessary mainly low molecular weight and primary amino groups, founded in both polyamines [46]. It well knows that iron and iron complexes stimulate lipid peroxidation in cells [21]. The mechanism(s) that underlies the antioxidant activity of polyamines measured in the presence of Fe2 + have been credited to the formation of a ternary complex with the phospholipid polar head and the Fe3 +, which in turn retards Fe2 + oxidation, reduces generation of reactive species [53]. Formation of such kind of ternary complexes in our assay system is possible and can, at least in part, explain the antioxidant activity of spermine and spermidine. However, formation of such complexes are probably not involved in the protection caused by polyamines, because spermine and spermidine had only a weak protection effect against Fe2 +-induced lipid peroxidation. In conclusion, the results of the present investigation support a scavenger and antioxidant role for polyamines in brain. However, the antioxidant activity of these compounds varied considerably depending on the agent used to induce TBARS formation and, to a less extent, on the
polyamine considered. In fact, spermine was superior to spermidine as scavenger and/or antioxidant in all situations tested. Of particular importance, at physiological concentrations, polyamines reduced the TBARS production induced by QA, an endogenous neurotoxin that activates NMDA receptors and is supposed be increased in neurodegenerative diseases such as Huntington [7,31], raise the possibility of their use as therapeutic or preventive agents against these pathologic situations.
Acknowledgements This study was supported by CNPq (474241/2003-3, 523761/95-3 and 500096/2003-1), CAPES and FAPERGS (01/1338.9).
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