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Antioxidant and pro-oxidant properties of boldine on hippocampal slices exposed to oxygen–glucose deprivation in vitro
NeuroToxicology 29 (2008) 1136–1140
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NeuroToxicology
Brief communication
Antioxidant and pro-oxidant properties of boldine on hippocampal slices exposed to oxygen–glucose deprivation in vitro Eduardo L. Konrath a,*, Katiane Santin b, Melissa Nassif b, Alexandra Latini b, Ame´lia Henriques a, Christianne Salbego b a b
Curso de Po´s-Graduac¸a˜o em Cieˆncias Farmaceˆuticas, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil Departamento de Bioquı´mica, Instituto de Cieˆncias Ba´sicas da Sau´de, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil
A R T I C L E I N F O
A B S T R A C T
Article history: Received 29 January 2008 Accepted 29 May 2008 Available online 11 June 2008
Boldine is one of the most potent natural antioxidants and displays some important pharmacological activities, such as cytoprotective and anti-inflammatory activities, which may arise from its free radical scavenging properties. Given that the pathogenesis of brain ischemia/reperfusion has been associated with an excessive generation of oxygen free radicals, the aim of this study was to evaluate the neuroproperties of boldine using hippocampal slices from Wistar rats exposed to oxygen and glucose deprivation (OGD), followed by reoxygenation, to mimic an ischemic condition. The OGD ischemic condition significantly impaired cellular viability, increased lactate dehydrogenase (LDH) leakage and increased free radical generation. In non-OGD slices, incubation with 100 mM boldine significantly increased LDH released into incubation media and decreased mitochondrial activity, suggesting an increase of tissue damage caused by boldine. However, slices incubated with 10 mM boldine during and after OGD exposure had significantly increased cellular viability with no effect on cell damage. Total reactive antioxidant potential (TRAP) levels measured for this alkaloid showed an antioxidant potential three times higher than Trolox, which acts as a peroxyl radical scavenger. Moreover, boldine prevented the increase in lipoperoxidation levels induced by ischemia, but higher concentrations potentiated this parameter. These results confirm the potent antioxidant properties of this alkaloid, and add evidence to support the need for further investigations in order to confirm the potential pro-oxidant effects of boldine at higher doses. ß 2008 Elsevier Inc. All rights reserved.
Keywords: Boldine Hippocampal slices Oxidative stress Pro-oxidant activity Antioxidant Neurotoxicity
1. Introduction Ischemia is defined as a severe reduction or complete blockage of blood flow, and this pathophysiological event results in cerebral damage, characterized as a prominent feature of stroke (Kirino and Sano, 1984; Price, 1999; Frantseva et al., 2001). It has been shown that a series of events, including depletion of cellular energy sources, release of excitatory amino acids, mitochondrial dysfunction, and excessive generation of free radicals has been involved with the pathogenesis of cerebral ischemia/reperfusion (White et al., 2000; Warner et al., 2004). In addition, an excessive formation of reactive oxygen species (ROS) has been implicated in cell damage to nervous tissue, which can lead to lipid, protein and
* Corresponding author at: Curso de Po´s-Graduac¸a˜o em Cieˆncias Farmaceˆuticas, Faculdade de Farma´cia, Universidade Federal do Rio Grande do Sul, Av Ipiranga 2752, 90610-000 Porto Alegre, RS, Brazil. Tel.: +55 51 3308 5258; fax: +55 51 3308 5243. E-mail address: [email protected] (E.L. Konrath). 0161-813X/$ – see front matter ß 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.neuro.2008.05.008
DNA oxidation, promoting chain reactions of membrane lipid peroxidation, and/or alterations in membrane fluidity. Several studies are being conducted to understand the actual antioxidant potential of boldine, a phenolic alkaloid found in the leaves and bark of the Chilean boldo tree (Peumus boldus Molina, Monimiaceae). Pure boldine has been shown to have cytoprotective, antitumoral, immunomodulator, anti-inflammatory, antipyretic and antiplatelet properties, which may arise from its well-established ability to scavenge reactive free radicals (O’Brien et al., 2005). In fact, its potent antioxidant activity has been demonstrated in a number of biological experimental models, and it was previously established that boldine acts as a very efficient scavenger for hydroxyl radicals, one of the most harmful reactive species found in vivo (Kringstein and Cederbaum, 1995; Youn et al., 2002). Considering that therapies available to decrease neuronal damage after stroke present minimal efficacy, the aim of the present study was to investigate the effects of this alkaloid on rat hippocampal slices submitted to ischemic conditions (OGD and reoxygenation). We used the OGD in vitro model of ischemia, a currently employed approach to investigate mechanisms of cell
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death and neuroprotection (Moro et al., 1998; Ca´rdenas et al., 2000), to evaluate the effect of boldine on the integrity of rat hippocampus after ischemia. Given that oxidative stress plays a major role in brain injury and that boldine is an important antioxidant molecule, we also investigated the thiobarbituric acid reactive substances (TBARS) test, a parameter of oxidative stress that represents an index of lipoperoxidation, as well as the antioxidant capacity for this alkaloid, assessed by the total reactive antioxidant potential (TRAP) level. Our prediction is that this phenolic compound might exert neuroprotection (Asencio et al., 1998; Youn et al., 2002; Loghin et al., 2003). 2. Materials and methods
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1987). After the recovery period, LDH activity was determined using a commercial kit (Doles Reagents, Goiaˆnia, Brazil). Each experiment was normalized by substracting the background levels of LDH produced from the ‘‘no-treatment’’ wells (Almli et al., 2001). The sample values were quantified using a standard curve. A mitochondrial viability assay was performed by the colorimetric 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) method. Briefly, after the recovery period, slices were incubated in medium containing 45 mg/ml MTT for 45 min at 37 8C. Only viable slices were able to reduce MTT into a purple formazan product that was soluble in dimethyl sulfoxide (DMSO) (Mosmann, 1983). The optical density was measured at 570 and 630 nm, and the net A570–A630 was taken as an index of cell viability (Siqueira et al., 2004).
2.1. Animals and reagents 2.4. Total radical-trapping antioxidant potential: TRAP assay Adult male Wistar rats (180–200 g; about 8 weeks of age) were used in the present study. The animals were kept under standard conditions (12 h light/dark, 22 2 8C) with food and water ad libitum. The procedures were designed to minimize suffering and limit the number of animals used. Animal care was approved by the Ethics Committee of the Federal University of Rio Grande do Sul, Brazil. Boldine and all other chemicals were purchased from Sigma Chemical Co., St. Louis, MO, USA. The purity of the alkaloid was checked by HPLC (high-performance liquid chromatography) analysis (99%). 2.2. Oxygen and glucose deprivation (OGD) followed by reoxygenation Animals were killed by decapitation, the hippocampi were quickly dissected out and transverse sections (400 mm) were prepared using a McIlwain tissue chopper. Hippocampal slices were divided into two equal sets (control and OGD), transferred to separate 24-well culture plates and pre-incubated for 15 min in HEPES-buffered saline solution (mM): 120 NaCl, 2 KCl, 0.5 CaCl2, 10 MgSO4, 26 NaHCO3, 1.18 KH2PO4 and 11 glucose (pH 7.2) in a tissue culture incubator at 37 8C with 95% O2/5% CO2 (Siqueira et al., 2004; Ca´rdenas et al., 2000). High magnesium and low calcium concentrations were used in order to prevent N-methyl-Daspartate (NMDA) receptor-mediated damage (Aitken et al., 1995). After pre-incubation, the medium in the control plate was replaced with another HEPES-buffered saline solution (mM): 120 NaCl, 2 KCl, 2 CaCl2, 1.19 MgSO4, 2.6 NaHCO3, 1.18 KH2PO4 and 11 glucose (pH 7.2) (Cimarosti et al., 2001; Tavares et al., 2001). Control slices were incubated for 45 min in a tissue culture incubator at 37 8C with 95% O2/5% CO2 in the presence or absence of boldine (concentrations of 1–100 mM). After 45 min, the control medium was replaced by a fresh one and slices incubated for 180 min under the same conditions. To model ischemic conditions, OGD slices were washed twice after pre-incubation with a medium without glucose, and incubated for 45 min (OGD period) in the presence or absence of boldine (1–100 mM) at 37 8C in an anaerobic chamber saturated with nitrogen, as described elsewhere (Siqueira et al., 2004; Porciu´ncula et al., 2003). After 45 min (OGD period), media from both control and OGD slices were removed and the two groups received medium with glucose. Slices were then incubated for 180 min (recovery period) in an incubator in the presence or absence of boldine (1–100 mM), as indicated. Control and OGD experiments were run concomitantly using four slices of the same animal in each plate. 2.3. Assessment of cellular damage and viability: LDH and MTT assays Cellular injury was quantified by measuring lactate dehydrogenase (LDH) released into the incubation medium (Koh and Choi,
This assay is based on luminol-enhanced chemiluminescence (CL) measurement, induced by an azo initiator (Lissi et al., 1992, 1995). TRAP was determined by measuring the CL intensity of a reaction mixture containing 10 mM 2,20 -azobis (2-amidinopropane) (ABAP), a source of peroxyl radicals, and 4 mM luminol in glycine buffer (0.1 M, pH 8.6). The CL generated was measured in a scintillation counter (Beckman) working in the out of coincidence mode. The addition of Trolox (antioxidant standard, 300 mM) or boldine solutions (10–200 mM) decreases CL to basal levels for a period (induction time) proportional to the concentration of antioxidants (TRAP), until luminol radicals are regenerated, when the antioxidants are totally consumed. A comparison of the induction time, after addition of known concentrations of Trolox and boldine, allows the expression of TRAP values as equivalents of Trolox concentration. 2.5. Assessment of lipoperoxidation levels: TBARS assay TBARS was determined, as previously described (Yagi, 1998). Hippocampal slices homogenates were incubated with 10% (w/v) trichloroacetic acid and 0.67% (w/v) thiobarbituric acid in 7.1% (w/ v) sodium sulfate. The mixture was heated for 60 min in a boiling water bath. Afterwards, n-butanol was added and the resulting pink stained TBA-RS was extracted and centrifuged. The organic phase was collected and quantified by measuring its fluorescence at excitation and emission wavelengths of 515 and 553 nm, respectively. The results were normalized by the protein content (Lowry et al., 1951). The calibration curve was performed using 1,1,3,3-tetramethoxypropane (TMP), and each curve point was subjected to the same treatment as that of the homogenates. 2.6. Statistical analysis Data were analyzed by one-way ANOVA followed by the Duncan’s multiple range test when the F test was significant. All analyses were performed using the Statistical Package for the Social Sciences (SPSS) software. Differences were considered statistically significant, if P < 0.05. 3. Results 3.1. Effects of boldine on neural injury and cellular viability in rat hippocampal slices submitted to OGD As shown in Fig. 1A and B, boldine per se potentiated the ischemic effect, given that hippocampal non-OGD slices incubated with 100 mM boldine showed a significant increase (50%) in cytosolic LDH released into the incubation media (F5,11 = 15.76,
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Fig. 1. Effect of different concentrations of boldine on cellular integrity and viability. Hippocampal slices from rats were exposed to oxygen and glucose deprivation (OGD) for 45 min and reoxygenation for 180 min—the ischemic groups. After treatments, cellular integrity was accessed by LDH released to the medium (A), and mitochondrial viability was measured by MTT assay (B). Results are expressed as percentages of the control non-OGD groups. Columns represent mean S.E.M. for five independent experiments performed in triplicate. Open bars represent non-OGD groups and filled bars represent the OGD-reoxygenation groups. *: values significantly different from those of non-OGD group; +: values significantly different between control group; #: values significantly different between OGD group, as determined by one-way ANOVA followed by Duncan’s test (P < 0.05).
P < 0.001) and a significant decrease in MTT reduction of slices (18.5%), both compared to control non-OGD groups (F5,12 = 7.90, P < 0.001). As expected, oxygen and glucose deprivation, followed by reoxygenation, resulted in marked changes in both cellular damage assessed by LDH release (118% increase in LDH efflux; F5,13 = 5.13, P < 0.001) and in cellular viability (38% decreased MTT reduction; F5,11 = 4.84, P < 0.001) in hippocampal slices. Moreover, during the ischemic process, both boldine concentrations, of 50 and 100 mM, significantly increased the LDH leakage (30% and 40%, respectively), as compared to the OGD control group, and 100 mM boldine also significantly decreased MTT reduction of slices exposed (40%) to OGD. These results indicate that the presence of high boldine concentrations causes cellular damage, affecting cell integrity as well as the mitochondrial viability. Interestingly, at the concentration of 10 mM, this alkaloid seems to significantly increase MTT reduction of slices exposed to OGD (56%), as compared to the OGD non-treated group, with no significant effect on the LDH efflux from OGD treated slices, suggesting a neuroprotective effect. 3.2. Effects of boldine on lipoperoxidation levels and the total antioxidant capacity Boldine exhibited intense free radical scavenging properties, as manifested in the reduction of luminescence intensity assessed by the TRAP measurement (Fig. 2). Observing the curves for both compounds, the trapping effect for boldine is to be considered higher than that observed for Trolox, a reference scavenger molecule. Next, the effects of boldine on lipoperoxidation levels of hippocampal slices of rats submitted to OGD are shown in Fig. 3. A significant increase in this parameter was observed in hippocampal slices submitted to OGD (60%; F5,18 = 2.98, P < 0.001), in respect to non-OGD slices. The presence of 10 and 50 mM boldine
Fig. 2. Effect of different concentrations of boldine on the TRAP measurement, as compared to Trolox, a reference scavenger molecule. Data are representative of three independent experiments.
significantly prevented this effect (approximately 45% and 17.5%, respectively), indicating in this case a possible neuroprotective effect for this alkaloid. Although TBARS levels within non-OGD slices do not significantly differ for any concentration tested, compared to non-treated group, OGD-slices in the presence of 100 mM boldine potentiated the lipoperoxidation induced by ischemia (F5,12 = 27.15, P < 0.001), indicating a pro-oxidant effect. 4. Discussion Free radicals appear to play an important role in pathogenesis in neurodegenerative processes, such as Alzheimer and Parkinson diseases and brain injury secondary to hypoxia and/or ischemia events (Aitken et al., 1995; White et al., 2000). In this context,
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Fig. 3. Effect of different concentrations of boldine on lipoperoxidation by TBARS. Hippocampal slices from rats were exposed to oxygen and glucose deprivation (OGD) for 45 min and reoxygenation for 180 min—the ischemic groups. Results are expressed as nmol TBARS per mg protein. Columns represent mean S.E.M. for four independent experiments performed in triplicate. Open bars represent non-OGD groups and filled bars represent the OGD-reoxygenation groups. *: values significantly different from those of non-OGD group; #: values significantly different from those of OGD group, as determined by one-way ANOVA followed by Duncan’s test (P < 0.05).
experimental in vitro ischemia in hippocampal slices is a widely used model in the literature and offers many advantages, allowing the study of the mechanisms of protection and neuronal damage (Moro et al., 1998; De Alba et al., 1999). Recently, the development of effective therapies for brain ischemia-induced damage has also considered free radicals and mitochondria as new targets (Clemens, 2000; Morin et al., 2001). It has been widely reported that boldine possesses potent free radical abilities (O’Brien et al., 2005), and we confirmed here, for the first time to our knowledge, such an effect through the investigation of the total reactive antioxidant potential assessed by TRAP measurement. This method is most likely due to trapping peroxyl radicals, and antioxidants can inhibit this chemiluminescence proportionally to the total antioxidant potential (Lissi et al., 1995; Desmarchelier et al., 1998). Although Trolox was added at higher concentrations than boldine, it can be seen that its curves are situated between those for all concentrations tested for the alkaloid, indicating that this compound may have an antioxidant potential of about three times higher than that of the standard peroxyl radical scavenger, Trolox, supporting all other previous findings for boldine. Consistent with previous studies, the exposure of hippocampal slices to OGD followed by reoxygenation, resulted in increased LDH in the medium, which is a consequence of cell damage or death (Koh and Choi, 1987), given that hippocampus is one of the brain regions most vulnerable to oxidative stress (Candelario-Jalil et al., 2001). Mitochondrial viability was also significantly affected, when compared to control (non-OGD) groups. Incubation of rat hippocampal slices with boldine (50 and 100 mM) during the OGD and reoxygenation periods led to an increase on the amount of cytosolic lactate dehydrogenase leaked from the cells into the culture media, as compared to ischemic control (OGD non-treated) slices. In addition, 100 mM boldine produced a significant efflux of LDH from non-OGD treated slices, which confirms a potential toxicity. A decrease in MTT reduction (100 mM) in slices treated with this alkaloid, as compared to control groups (OGD and nonOGD) was also verified. Taken together, these results indicate a neurotoxic effect for this substance, observed significantly at the highest concentrations, probably due to a pro-oxidant effect of boldine, as seen in Fig. 1. Furthermore, OGD followed by reoxygenation led to an increase in free radicals production, expressed indirectly by the increase in TBARS levels found in OGD slices. Oxidative stress induces cellular damage and lipoperoxidation, which may lead to alterations in membranes, producing significant changes in their biophysical properties (Joseph et al., 2000). Our findings showed that 10 and 50 mM boldine prevented the malondihaldeyde liberation by
hippocampal slices during ischemia, and the obtained values in OGD groups are significantly different as compared to the control OGD group. Interestingly, at 100 mM, boldine significantly increased (20%) the lipoperoxidation induced by OGD, which confirms a putative toxicity for boldine at higher doses for the neural cells. On the other hand, it is noteworthy that only 10 mM boldine could display beneficial actions at all levels tested, since it was able to significantly increase MTT reduction (as compared to OGD group), as well as the best lipoperoxidation inhibition against ischemia (45%), comparing all OGD groups. In addition, this concentration promoted no significant imbalance in LDH release, with regard to both OGD and non-OGD groups. Thus, the anti- and pro-oxidant effects observed for boldine on hippocampal slices were clear, depending on the concentration tested. Accordingly, many other data suggest that, depending upon its concentration and the cell type, polyphenols such boldine and other antioxidants may have opposing effects on some biological parameters, presenting both anti- and pro-oxidant activities (Cao et al., 1997; Quincozes-Santos et al., 2007). It is known that polyphenols, ascorbic acid and carotenoids may act as antioxidants against free radicals but they can also exert pro-oxidant activities under certain experimental conditions, such as the presence of transitional metals (Cao et al., 1997; Rietjens et al., 2002). Similarly, the oxidation of these molecules produces superoxide anions, H2O2 and a mixture of some quinones and semi-quinones, all of which are potentially cytotoxic (Halliwell, 2008). Previous studies demonstrated such effects for some boldine-related alkaloids such as apomorphine, which can act as a peroxyl radicals scavenger and, at the same time, increase the deoxyribose degradation by iron– EDTA in the presence of H2O2 (Ubeda et al., 1993), in a dosedependent manner. Research conducted over the years has demonstrated the ability of boldine to exert cytoprotective effects in models of oxidative stress-induced damage. Furthermore, the effectiveness of boldine in preventing various oxidative-stress-related pharmacological effects includes anti-inflammatory, antipyretic, antitumour promoting, antiplatelet and anti-atherogenic effects (O’Brien et al., 2005; Jime´nez and Speisky, 2000). As such, we tried to corroborate the fact that antioxidants may be potential neuroprotective substances in in vitro ischemic events. However, the presence of higher boldine concentrations in hippocampal slices, inclusive during ischemia, induced toxic effects, including cellular death, as characterized by the increase in LDH efflux and a decrease in mitochondrial viability, when compared to control groups (ischemic and non-ischemic). To our knowledge, this is the first report demonstrating such neuroproperties for boldine, an alkaloid extensively studied for its antioxidant properties.
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Although the understanding of the molecular mechanisms involved in the proposed neurotoxic/pro-oxidant actions of boldine is incomplete, we suggest that these effects could be related to our concentration range of 10–100 mM, based on other in vitro assays, given that the cerebral biodisponibility of this alkaloid is many times lower (Jime´nez and Speisky, 2000). These authors demonstrated that the highest boldine concentrations found in brain tissue (18 and 24 mM) were found after 30 min of an oral administration of 50 and 75 mg boldine/kg, respectively, indicating that the neuroprotective effects found in vitro herein could be achieved in vivo, since this alkaloid crosses the blood–brain barrier. Secondly, additional work is required before a comprehensive knowledge of boldine’s anti- and pro-oxidant effects, as well as other in vitro and in vivo ischemia models, in order to characterize its potential neuroproperties. Our results support the idea that boldine, at lower concentrations, has a potential role in neuroprotection against OGD, followed by reoxygenation in vitro, and could be beneficial in brain disorders involving oxidative stress, such as ischemic disorders. Higher concentrations become neurotoxic for hippocampal slices, which could be due to a pro-oxidant effect. In conclusion, the present study adds evidence of new pharmacological effects for the alkaloid boldine, considered to be an experimentally confirmed potent free radical scavenger. This in vitro study provides new pharmacological and toxicological data to this alkaloid, but further research is necessary and recommendations are suggested to define the actual potential of boldine for its use in humans, such as other in vivo ischemia models. Acknowledgements This work was supported by the Brazilian funding agents Coordenac¸a˜o de Aperfeic¸oamento de pessoal de Nı´vel Superior (CAPES), Conselho Nacional de Desenvolvimento Cientı´fico (CNPq) and Pro´-Reitoria de Pesquisa e Po´s-Graduac¸a˜o da Universidade Federal do Rio Grande do Sul (PROPESQ). We are also grateful to Prof. Dr. Carmem Gottfried for her excellent review of the manuscript. References Aitken P, Breese G, Dedek F, Edwards F, Espanol M, Larkman P, et al. Preparative methods for brain slices: a discussion. J Neurosci Methods 1995;59:139–49. Almli L, Hamrick S, Koshy A, Tauber M, Ferriero D. Multiple pathways of neuroprotection against oxidative stress and excitotoxic injury in immature primary hippocampal neurons. Dev Brain Res 2001;132:121–9. Asencio M, Delaquerrie`re B, Cassels B, Speisky H, Comoy E, Protais P. Biochemical and behavioral effects of boldine and glaucine on dopamine systems. Biochem Behav 1998;62:7–13. Candelario-Jalil E, Mhadu N, Al-Dalain S, Martinez G, Leo´n O. Time course of oxidative damage in different brain regions following transient cerebral ischemia in gerbils. Neurosci Res 2001;41:233–41. Cao G, Sofic E, Prior R. Antioxidant and prooxidant behavior of flavonoids: structure– activity relationships. Free Radical Biol Med 1997;22:749–60. Ca´rdenas A, Moro M, Hurtado O, Leza J, Lorenzo P, Castrillo A, et al. Implication of glutamate in the expression of inducible nitric oxide synthase after oxygen and glucose deprivation in rat forebrain slices. J Neurochem 2000;74:2041–8. Cimarosti H, Rodnight R, Tavares A, Paiva R, Valentim L, Rocha E, et al. An investigation of the neuroprotective effect of lithium in organotypic slice cultures of rat hippocampus exposed to oxygen and glucose deprivation. Neurosci Lett 2001;315:33–6.
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Contents lists available at ScienceDirect
NeuroToxicology
Brief communication
Antioxidant and pro-oxidant properties of boldine on hippocampal slices exposed to oxygen–glucose deprivation in vitro Eduardo L. Konrath a,*, Katiane Santin b, Melissa Nassif b, Alexandra Latini b, Ame´lia Henriques a, Christianne Salbego b a b
Curso de Po´s-Graduac¸a˜o em Cieˆncias Farmaceˆuticas, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil Departamento de Bioquı´mica, Instituto de Cieˆncias Ba´sicas da Sau´de, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil
A R T I C L E I N F O
A B S T R A C T
Article history: Received 29 January 2008 Accepted 29 May 2008 Available online 11 June 2008
Boldine is one of the most potent natural antioxidants and displays some important pharmacological activities, such as cytoprotective and anti-inflammatory activities, which may arise from its free radical scavenging properties. Given that the pathogenesis of brain ischemia/reperfusion has been associated with an excessive generation of oxygen free radicals, the aim of this study was to evaluate the neuroproperties of boldine using hippocampal slices from Wistar rats exposed to oxygen and glucose deprivation (OGD), followed by reoxygenation, to mimic an ischemic condition. The OGD ischemic condition significantly impaired cellular viability, increased lactate dehydrogenase (LDH) leakage and increased free radical generation. In non-OGD slices, incubation with 100 mM boldine significantly increased LDH released into incubation media and decreased mitochondrial activity, suggesting an increase of tissue damage caused by boldine. However, slices incubated with 10 mM boldine during and after OGD exposure had significantly increased cellular viability with no effect on cell damage. Total reactive antioxidant potential (TRAP) levels measured for this alkaloid showed an antioxidant potential three times higher than Trolox, which acts as a peroxyl radical scavenger. Moreover, boldine prevented the increase in lipoperoxidation levels induced by ischemia, but higher concentrations potentiated this parameter. These results confirm the potent antioxidant properties of this alkaloid, and add evidence to support the need for further investigations in order to confirm the potential pro-oxidant effects of boldine at higher doses. ß 2008 Elsevier Inc. All rights reserved.
Keywords: Boldine Hippocampal slices Oxidative stress Pro-oxidant activity Antioxidant Neurotoxicity
1. Introduction Ischemia is defined as a severe reduction or complete blockage of blood flow, and this pathophysiological event results in cerebral damage, characterized as a prominent feature of stroke (Kirino and Sano, 1984; Price, 1999; Frantseva et al., 2001). It has been shown that a series of events, including depletion of cellular energy sources, release of excitatory amino acids, mitochondrial dysfunction, and excessive generation of free radicals has been involved with the pathogenesis of cerebral ischemia/reperfusion (White et al., 2000; Warner et al., 2004). In addition, an excessive formation of reactive oxygen species (ROS) has been implicated in cell damage to nervous tissue, which can lead to lipid, protein and
* Corresponding author at: Curso de Po´s-Graduac¸a˜o em Cieˆncias Farmaceˆuticas, Faculdade de Farma´cia, Universidade Federal do Rio Grande do Sul, Av Ipiranga 2752, 90610-000 Porto Alegre, RS, Brazil. Tel.: +55 51 3308 5258; fax: +55 51 3308 5243. E-mail address: [email protected] (E.L. Konrath). 0161-813X/$ – see front matter ß 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.neuro.2008.05.008
DNA oxidation, promoting chain reactions of membrane lipid peroxidation, and/or alterations in membrane fluidity. Several studies are being conducted to understand the actual antioxidant potential of boldine, a phenolic alkaloid found in the leaves and bark of the Chilean boldo tree (Peumus boldus Molina, Monimiaceae). Pure boldine has been shown to have cytoprotective, antitumoral, immunomodulator, anti-inflammatory, antipyretic and antiplatelet properties, which may arise from its well-established ability to scavenge reactive free radicals (O’Brien et al., 2005). In fact, its potent antioxidant activity has been demonstrated in a number of biological experimental models, and it was previously established that boldine acts as a very efficient scavenger for hydroxyl radicals, one of the most harmful reactive species found in vivo (Kringstein and Cederbaum, 1995; Youn et al., 2002). Considering that therapies available to decrease neuronal damage after stroke present minimal efficacy, the aim of the present study was to investigate the effects of this alkaloid on rat hippocampal slices submitted to ischemic conditions (OGD and reoxygenation). We used the OGD in vitro model of ischemia, a currently employed approach to investigate mechanisms of cell
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death and neuroprotection (Moro et al., 1998; Ca´rdenas et al., 2000), to evaluate the effect of boldine on the integrity of rat hippocampus after ischemia. Given that oxidative stress plays a major role in brain injury and that boldine is an important antioxidant molecule, we also investigated the thiobarbituric acid reactive substances (TBARS) test, a parameter of oxidative stress that represents an index of lipoperoxidation, as well as the antioxidant capacity for this alkaloid, assessed by the total reactive antioxidant potential (TRAP) level. Our prediction is that this phenolic compound might exert neuroprotection (Asencio et al., 1998; Youn et al., 2002; Loghin et al., 2003). 2. Materials and methods
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1987). After the recovery period, LDH activity was determined using a commercial kit (Doles Reagents, Goiaˆnia, Brazil). Each experiment was normalized by substracting the background levels of LDH produced from the ‘‘no-treatment’’ wells (Almli et al., 2001). The sample values were quantified using a standard curve. A mitochondrial viability assay was performed by the colorimetric 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) method. Briefly, after the recovery period, slices were incubated in medium containing 45 mg/ml MTT for 45 min at 37 8C. Only viable slices were able to reduce MTT into a purple formazan product that was soluble in dimethyl sulfoxide (DMSO) (Mosmann, 1983). The optical density was measured at 570 and 630 nm, and the net A570–A630 was taken as an index of cell viability (Siqueira et al., 2004).
2.1. Animals and reagents 2.4. Total radical-trapping antioxidant potential: TRAP assay Adult male Wistar rats (180–200 g; about 8 weeks of age) were used in the present study. The animals were kept under standard conditions (12 h light/dark, 22 2 8C) with food and water ad libitum. The procedures were designed to minimize suffering and limit the number of animals used. Animal care was approved by the Ethics Committee of the Federal University of Rio Grande do Sul, Brazil. Boldine and all other chemicals were purchased from Sigma Chemical Co., St. Louis, MO, USA. The purity of the alkaloid was checked by HPLC (high-performance liquid chromatography) analysis (99%). 2.2. Oxygen and glucose deprivation (OGD) followed by reoxygenation Animals were killed by decapitation, the hippocampi were quickly dissected out and transverse sections (400 mm) were prepared using a McIlwain tissue chopper. Hippocampal slices were divided into two equal sets (control and OGD), transferred to separate 24-well culture plates and pre-incubated for 15 min in HEPES-buffered saline solution (mM): 120 NaCl, 2 KCl, 0.5 CaCl2, 10 MgSO4, 26 NaHCO3, 1.18 KH2PO4 and 11 glucose (pH 7.2) in a tissue culture incubator at 37 8C with 95% O2/5% CO2 (Siqueira et al., 2004; Ca´rdenas et al., 2000). High magnesium and low calcium concentrations were used in order to prevent N-methyl-Daspartate (NMDA) receptor-mediated damage (Aitken et al., 1995). After pre-incubation, the medium in the control plate was replaced with another HEPES-buffered saline solution (mM): 120 NaCl, 2 KCl, 2 CaCl2, 1.19 MgSO4, 2.6 NaHCO3, 1.18 KH2PO4 and 11 glucose (pH 7.2) (Cimarosti et al., 2001; Tavares et al., 2001). Control slices were incubated for 45 min in a tissue culture incubator at 37 8C with 95% O2/5% CO2 in the presence or absence of boldine (concentrations of 1–100 mM). After 45 min, the control medium was replaced by a fresh one and slices incubated for 180 min under the same conditions. To model ischemic conditions, OGD slices were washed twice after pre-incubation with a medium without glucose, and incubated for 45 min (OGD period) in the presence or absence of boldine (1–100 mM) at 37 8C in an anaerobic chamber saturated with nitrogen, as described elsewhere (Siqueira et al., 2004; Porciu´ncula et al., 2003). After 45 min (OGD period), media from both control and OGD slices were removed and the two groups received medium with glucose. Slices were then incubated for 180 min (recovery period) in an incubator in the presence or absence of boldine (1–100 mM), as indicated. Control and OGD experiments were run concomitantly using four slices of the same animal in each plate. 2.3. Assessment of cellular damage and viability: LDH and MTT assays Cellular injury was quantified by measuring lactate dehydrogenase (LDH) released into the incubation medium (Koh and Choi,
This assay is based on luminol-enhanced chemiluminescence (CL) measurement, induced by an azo initiator (Lissi et al., 1992, 1995). TRAP was determined by measuring the CL intensity of a reaction mixture containing 10 mM 2,20 -azobis (2-amidinopropane) (ABAP), a source of peroxyl radicals, and 4 mM luminol in glycine buffer (0.1 M, pH 8.6). The CL generated was measured in a scintillation counter (Beckman) working in the out of coincidence mode. The addition of Trolox (antioxidant standard, 300 mM) or boldine solutions (10–200 mM) decreases CL to basal levels for a period (induction time) proportional to the concentration of antioxidants (TRAP), until luminol radicals are regenerated, when the antioxidants are totally consumed. A comparison of the induction time, after addition of known concentrations of Trolox and boldine, allows the expression of TRAP values as equivalents of Trolox concentration. 2.5. Assessment of lipoperoxidation levels: TBARS assay TBARS was determined, as previously described (Yagi, 1998). Hippocampal slices homogenates were incubated with 10% (w/v) trichloroacetic acid and 0.67% (w/v) thiobarbituric acid in 7.1% (w/ v) sodium sulfate. The mixture was heated for 60 min in a boiling water bath. Afterwards, n-butanol was added and the resulting pink stained TBA-RS was extracted and centrifuged. The organic phase was collected and quantified by measuring its fluorescence at excitation and emission wavelengths of 515 and 553 nm, respectively. The results were normalized by the protein content (Lowry et al., 1951). The calibration curve was performed using 1,1,3,3-tetramethoxypropane (TMP), and each curve point was subjected to the same treatment as that of the homogenates. 2.6. Statistical analysis Data were analyzed by one-way ANOVA followed by the Duncan’s multiple range test when the F test was significant. All analyses were performed using the Statistical Package for the Social Sciences (SPSS) software. Differences were considered statistically significant, if P < 0.05. 3. Results 3.1. Effects of boldine on neural injury and cellular viability in rat hippocampal slices submitted to OGD As shown in Fig. 1A and B, boldine per se potentiated the ischemic effect, given that hippocampal non-OGD slices incubated with 100 mM boldine showed a significant increase (50%) in cytosolic LDH released into the incubation media (F5,11 = 15.76,
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Fig. 1. Effect of different concentrations of boldine on cellular integrity and viability. Hippocampal slices from rats were exposed to oxygen and glucose deprivation (OGD) for 45 min and reoxygenation for 180 min—the ischemic groups. After treatments, cellular integrity was accessed by LDH released to the medium (A), and mitochondrial viability was measured by MTT assay (B). Results are expressed as percentages of the control non-OGD groups. Columns represent mean S.E.M. for five independent experiments performed in triplicate. Open bars represent non-OGD groups and filled bars represent the OGD-reoxygenation groups. *: values significantly different from those of non-OGD group; +: values significantly different between control group; #: values significantly different between OGD group, as determined by one-way ANOVA followed by Duncan’s test (P < 0.05).
P < 0.001) and a significant decrease in MTT reduction of slices (18.5%), both compared to control non-OGD groups (F5,12 = 7.90, P < 0.001). As expected, oxygen and glucose deprivation, followed by reoxygenation, resulted in marked changes in both cellular damage assessed by LDH release (118% increase in LDH efflux; F5,13 = 5.13, P < 0.001) and in cellular viability (38% decreased MTT reduction; F5,11 = 4.84, P < 0.001) in hippocampal slices. Moreover, during the ischemic process, both boldine concentrations, of 50 and 100 mM, significantly increased the LDH leakage (30% and 40%, respectively), as compared to the OGD control group, and 100 mM boldine also significantly decreased MTT reduction of slices exposed (40%) to OGD. These results indicate that the presence of high boldine concentrations causes cellular damage, affecting cell integrity as well as the mitochondrial viability. Interestingly, at the concentration of 10 mM, this alkaloid seems to significantly increase MTT reduction of slices exposed to OGD (56%), as compared to the OGD non-treated group, with no significant effect on the LDH efflux from OGD treated slices, suggesting a neuroprotective effect. 3.2. Effects of boldine on lipoperoxidation levels and the total antioxidant capacity Boldine exhibited intense free radical scavenging properties, as manifested in the reduction of luminescence intensity assessed by the TRAP measurement (Fig. 2). Observing the curves for both compounds, the trapping effect for boldine is to be considered higher than that observed for Trolox, a reference scavenger molecule. Next, the effects of boldine on lipoperoxidation levels of hippocampal slices of rats submitted to OGD are shown in Fig. 3. A significant increase in this parameter was observed in hippocampal slices submitted to OGD (60%; F5,18 = 2.98, P < 0.001), in respect to non-OGD slices. The presence of 10 and 50 mM boldine
Fig. 2. Effect of different concentrations of boldine on the TRAP measurement, as compared to Trolox, a reference scavenger molecule. Data are representative of three independent experiments.
significantly prevented this effect (approximately 45% and 17.5%, respectively), indicating in this case a possible neuroprotective effect for this alkaloid. Although TBARS levels within non-OGD slices do not significantly differ for any concentration tested, compared to non-treated group, OGD-slices in the presence of 100 mM boldine potentiated the lipoperoxidation induced by ischemia (F5,12 = 27.15, P < 0.001), indicating a pro-oxidant effect. 4. Discussion Free radicals appear to play an important role in pathogenesis in neurodegenerative processes, such as Alzheimer and Parkinson diseases and brain injury secondary to hypoxia and/or ischemia events (Aitken et al., 1995; White et al., 2000). In this context,
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Fig. 3. Effect of different concentrations of boldine on lipoperoxidation by TBARS. Hippocampal slices from rats were exposed to oxygen and glucose deprivation (OGD) for 45 min and reoxygenation for 180 min—the ischemic groups. Results are expressed as nmol TBARS per mg protein. Columns represent mean S.E.M. for four independent experiments performed in triplicate. Open bars represent non-OGD groups and filled bars represent the OGD-reoxygenation groups. *: values significantly different from those of non-OGD group; #: values significantly different from those of OGD group, as determined by one-way ANOVA followed by Duncan’s test (P < 0.05).
experimental in vitro ischemia in hippocampal slices is a widely used model in the literature and offers many advantages, allowing the study of the mechanisms of protection and neuronal damage (Moro et al., 1998; De Alba et al., 1999). Recently, the development of effective therapies for brain ischemia-induced damage has also considered free radicals and mitochondria as new targets (Clemens, 2000; Morin et al., 2001). It has been widely reported that boldine possesses potent free radical abilities (O’Brien et al., 2005), and we confirmed here, for the first time to our knowledge, such an effect through the investigation of the total reactive antioxidant potential assessed by TRAP measurement. This method is most likely due to trapping peroxyl radicals, and antioxidants can inhibit this chemiluminescence proportionally to the total antioxidant potential (Lissi et al., 1995; Desmarchelier et al., 1998). Although Trolox was added at higher concentrations than boldine, it can be seen that its curves are situated between those for all concentrations tested for the alkaloid, indicating that this compound may have an antioxidant potential of about three times higher than that of the standard peroxyl radical scavenger, Trolox, supporting all other previous findings for boldine. Consistent with previous studies, the exposure of hippocampal slices to OGD followed by reoxygenation, resulted in increased LDH in the medium, which is a consequence of cell damage or death (Koh and Choi, 1987), given that hippocampus is one of the brain regions most vulnerable to oxidative stress (Candelario-Jalil et al., 2001). Mitochondrial viability was also significantly affected, when compared to control (non-OGD) groups. Incubation of rat hippocampal slices with boldine (50 and 100 mM) during the OGD and reoxygenation periods led to an increase on the amount of cytosolic lactate dehydrogenase leaked from the cells into the culture media, as compared to ischemic control (OGD non-treated) slices. In addition, 100 mM boldine produced a significant efflux of LDH from non-OGD treated slices, which confirms a potential toxicity. A decrease in MTT reduction (100 mM) in slices treated with this alkaloid, as compared to control groups (OGD and nonOGD) was also verified. Taken together, these results indicate a neurotoxic effect for this substance, observed significantly at the highest concentrations, probably due to a pro-oxidant effect of boldine, as seen in Fig. 1. Furthermore, OGD followed by reoxygenation led to an increase in free radicals production, expressed indirectly by the increase in TBARS levels found in OGD slices. Oxidative stress induces cellular damage and lipoperoxidation, which may lead to alterations in membranes, producing significant changes in their biophysical properties (Joseph et al., 2000). Our findings showed that 10 and 50 mM boldine prevented the malondihaldeyde liberation by
hippocampal slices during ischemia, and the obtained values in OGD groups are significantly different as compared to the control OGD group. Interestingly, at 100 mM, boldine significantly increased (20%) the lipoperoxidation induced by OGD, which confirms a putative toxicity for boldine at higher doses for the neural cells. On the other hand, it is noteworthy that only 10 mM boldine could display beneficial actions at all levels tested, since it was able to significantly increase MTT reduction (as compared to OGD group), as well as the best lipoperoxidation inhibition against ischemia (45%), comparing all OGD groups. In addition, this concentration promoted no significant imbalance in LDH release, with regard to both OGD and non-OGD groups. Thus, the anti- and pro-oxidant effects observed for boldine on hippocampal slices were clear, depending on the concentration tested. Accordingly, many other data suggest that, depending upon its concentration and the cell type, polyphenols such boldine and other antioxidants may have opposing effects on some biological parameters, presenting both anti- and pro-oxidant activities (Cao et al., 1997; Quincozes-Santos et al., 2007). It is known that polyphenols, ascorbic acid and carotenoids may act as antioxidants against free radicals but they can also exert pro-oxidant activities under certain experimental conditions, such as the presence of transitional metals (Cao et al., 1997; Rietjens et al., 2002). Similarly, the oxidation of these molecules produces superoxide anions, H2O2 and a mixture of some quinones and semi-quinones, all of which are potentially cytotoxic (Halliwell, 2008). Previous studies demonstrated such effects for some boldine-related alkaloids such as apomorphine, which can act as a peroxyl radicals scavenger and, at the same time, increase the deoxyribose degradation by iron– EDTA in the presence of H2O2 (Ubeda et al., 1993), in a dosedependent manner. Research conducted over the years has demonstrated the ability of boldine to exert cytoprotective effects in models of oxidative stress-induced damage. Furthermore, the effectiveness of boldine in preventing various oxidative-stress-related pharmacological effects includes anti-inflammatory, antipyretic, antitumour promoting, antiplatelet and anti-atherogenic effects (O’Brien et al., 2005; Jime´nez and Speisky, 2000). As such, we tried to corroborate the fact that antioxidants may be potential neuroprotective substances in in vitro ischemic events. However, the presence of higher boldine concentrations in hippocampal slices, inclusive during ischemia, induced toxic effects, including cellular death, as characterized by the increase in LDH efflux and a decrease in mitochondrial viability, when compared to control groups (ischemic and non-ischemic). To our knowledge, this is the first report demonstrating such neuroproperties for boldine, an alkaloid extensively studied for its antioxidant properties.
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Although the understanding of the molecular mechanisms involved in the proposed neurotoxic/pro-oxidant actions of boldine is incomplete, we suggest that these effects could be related to our concentration range of 10–100 mM, based on other in vitro assays, given that the cerebral biodisponibility of this alkaloid is many times lower (Jime´nez and Speisky, 2000). These authors demonstrated that the highest boldine concentrations found in brain tissue (18 and 24 mM) were found after 30 min of an oral administration of 50 and 75 mg boldine/kg, respectively, indicating that the neuroprotective effects found in vitro herein could be achieved in vivo, since this alkaloid crosses the blood–brain barrier. Secondly, additional work is required before a comprehensive knowledge of boldine’s anti- and pro-oxidant effects, as well as other in vitro and in vivo ischemia models, in order to characterize its potential neuroproperties. Our results support the idea that boldine, at lower concentrations, has a potential role in neuroprotection against OGD, followed by reoxygenation in vitro, and could be beneficial in brain disorders involving oxidative stress, such as ischemic disorders. Higher concentrations become neurotoxic for hippocampal slices, which could be due to a pro-oxidant effect. In conclusion, the present study adds evidence of new pharmacological effects for the alkaloid boldine, considered to be an experimentally confirmed potent free radical scavenger. This in vitro study provides new pharmacological and toxicological data to this alkaloid, but further research is necessary and recommendations are suggested to define the actual potential of boldine for its use in humans, such as other in vivo ischemia models. Acknowledgements This work was supported by the Brazilian funding agents Coordenac¸a˜o de Aperfeic¸oamento de pessoal de Nı´vel Superior (CAPES), Conselho Nacional de Desenvolvimento Cientı´fico (CNPq) and Pro´-Reitoria de Pesquisa e Po´s-Graduac¸a˜o da Universidade Federal do Rio Grande do Sul (PROPESQ). We are also grateful to Prof. Dr. Carmem Gottfried for her excellent review of the manuscript. References Aitken P, Breese G, Dedek F, Edwards F, Espanol M, Larkman P, et al. Preparative methods for brain slices: a discussion. J Neurosci Methods 1995;59:139–49. Almli L, Hamrick S, Koshy A, Tauber M, Ferriero D. Multiple pathways of neuroprotection against oxidative stress and excitotoxic injury in immature primary hippocampal neurons. Dev Brain Res 2001;132:121–9. Asencio M, Delaquerrie`re B, Cassels B, Speisky H, Comoy E, Protais P. Biochemical and behavioral effects of boldine and glaucine on dopamine systems. Biochem Behav 1998;62:7–13. Candelario-Jalil E, Mhadu N, Al-Dalain S, Martinez G, Leo´n O. Time course of oxidative damage in different brain regions following transient cerebral ischemia in gerbils. Neurosci Res 2001;41:233–41. Cao G, Sofic E, Prior R. Antioxidant and prooxidant behavior of flavonoids: structure– activity relationships. Free Radical Biol Med 1997;22:749–60. Ca´rdenas A, Moro M, Hurtado O, Leza J, Lorenzo P, Castrillo A, et al. Implication of glutamate in the expression of inducible nitric oxide synthase after oxygen and glucose deprivation in rat forebrain slices. J Neurochem 2000;74:2041–8. Cimarosti H, Rodnight R, Tavares A, Paiva R, Valentim L, Rocha E, et al. An investigation of the neuroprotective effect of lithium in organotypic slice cultures of rat hippocampus exposed to oxygen and glucose deprivation. Neurosci Lett 2001;315:33–6.
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