Biol Trace Elem Res (2014) 159:99–106 DOI 10.1007/s12011-014-9996-5
Mitigation of Lead-Induced Neurotoxicity by the Naringin: Erythrocytes as Neurons Substitute Markers Gamaleldin I. Harisa
Received: 21 March 2014 / Accepted: 24 April 2014 / Published online: 15 May 2014 # Springer Science+Business Media New York 2014
Abstract This study aimed to investigate the effect of lead (Pb) on neuronal nitric oxide synthase (nNOS) activity using erythrocytes as neurons surrogate markers. Moreover, the protective effect of naringin (NAR) against lead acetate (PbAc)-induced neurotoxicity was investigated. Human erythrocytes were incubated with L-arginine (L-Arg), Nω-nitro-L-Arginine methyl ester (L-NAME), NAR, PbAc, PbAc + L-Arg, PbAc + NAR, or PbAc + L-Arg +NAR. The present results revealed that incubation of erythrocytes with PbAc inhibited NOS activity and decreased nitrite levels as an index for nitric oxide (NO) production to values similar that of L-NAME as known NOS inhibitor. Likewise, PbAc induced a significant decrease in activities of ATPases and acetylcholinesterase compared to control cells. Furthermore, PbAc exposure significantly increased protein carbonyl content (PCC) and malondialdehyde (MDA) levels while significantly decrease the levels of reduced glutathione (GSH). On the contrary, incubation of erythrocytes with PbAc in the presence of L-Arg + NAR synergistically ameliorated the investigated parameters compared to erythrocytes incubated with PbAc alone. These data suggest that NAR can restore NO bioavailability in a situation of Pb-induced cellular damage. This attributed to antioxidant activity and restoration NOS activity.
Keywords Erythrocytes . Lead . Naringin . Nitric oxide synthase . Acetylcholinesterase . Oxidative stress G. I. Harisa (*) Department of Pharmaceutics, Kayyali Chair for Pharmaceutical Industry, College of Pharmacy, King Saud University, P.O. Box 2457, Riyadh 11451, Saudi Arabia e-mail: [email protected] G. I. Harisa Department of Biochemistry, College of Pharmacy, Al-Azhar University (Boys), Nasr City, Cairo, Egypt
Introduction Lead (Pb) is one of the health threat toxicants, its exposure can arise from contact with lead-based dyes, fertilizers, cosmetics, and motor vehicles exhaust. Pb exposure causes multiple organ dysfunctions, including nervous system [1]. Pbinduced damage to the cerebral cortex, hippocampus, and cerebellum. Pb-induced neurotoxicity by the inappropriate release of neurotransmitters, inhibition of ATPases and mimicking calcium effects [2]. Also, Pb alters N-methyL-aspartate (NMDA) receptor expression, disrupt redox homeostasis, and inhibit nitric oxide synthase (NOS) [2]. However, the mechanisms of Pb-induced neurotoxicity through NOS inhibition still not fully understood and require further studies. NOS plays an important role in the nervous system through the production of nitric oxide (NO) that regulate neuronal electrical activity [3]. There are three NOS isoforms: endothelial-NOS (eNOS), inducible NOS(iNOS), and neuronal NOS (nNOS) [4]. NOS uses L-Arginine (L-Arg) as substrate to produce NO [5]. NO is an important brain messenger released upon activation of the NMDA receptor and subsequent Ca2+-dependent activation of nNOS. NO is involved in neurological cascades that regulate learning and memory [2]. NO has been accredited with both pro-oxidant and antioxidant actions [6]. Abnormal NO production induces glutamate excitotoxicity and increases peroxynitrite formation in the neurons [6]. Extra-neuronal cells such as platelets, fibroblasts, and erythrocytes have been used to provide peripheral markers and a better understanding of the pathogenesis of the neuropathology [7]. In the last years, several studies have shown that NOS is present in the cytoplasm and plasma membrane of erythrocytes [8–10]. Also acetylcholine receptors are present on erythrocytes membrane acts as a NOS regulator [8]. Consequently, erythrocytes can act as non-neuronal regulators of neuronal NO signals [11]. Application of acetyl choline to
100
erythrocytes increases the concentration of nitrite as an index for NO production [8]. However, no enough data is available about this issue. Several studies have indicated that neurotransmission signals are present in body fluids and peripheral blood cells [12]. Therefore, erythrocytes can be utilized as peripheral alternate biomarkers for neurons [13] and mercury-induced vascular cell damage [14]. Erythrocyte acetylcholinesterase (AChE) was investigated as surrogate biomarkers of Pb exposure [12]. Naringin(NAR) is a flavonoid found in grapefruit citrus species that possesses diverse biological and pharmacological properties including anti-inflammatory, anticarcinogenic, lipid-lowering, and antioxidant activities [15]. Moreover, flavonoids decrease accumulation of heavy metal in blood and tissue [3]. Several studies showed that NAR possesses neuroprotective activity against neuron damage [16]. Moreover, they exert protection against oxidative stress, modulate calcium homeostasis, and modulated NOS pathways [16]. Despite the beneficial effects of flavonoids, mechanisms by which NAR elicits neuroprotective effects need more investigations. The available data about Pb-induced neurotoxicity through inhibition of NOS is contradictory and incompletely understood. Therefore, this study sought to investigate the effect of Pb on NOS activity and NO production using erythrocytesNOS as neuron surrogate markers. However, no enough data are available about the protective effect of NAR against Pbinduced neurotoxicity. Herein, the possible neuroprotective effect of NAR through modulation of NOS activity was investigated.
Materials and Methods Materials Lead acetate (PbAc), L-Arg, N -nitro-L-Arginine methyl ester(L-NAME), and naringin (NAR) were purchased from Sigma-Aldrich (St. Louis, MO, USA). All other reagents were of the highest commercial grade unless indicated were obtained from Sigma Chemical Co. (St. Louis, MO, USA). A Spectro UV–vis Split Beam PC spectrophotometer (Model UVS-2800, Labomed, Inc., USA) and CT5 centrifuge were used in this investigation.
Harisa
phosphate-buffered saline (PBS, pH 7.4) with repeated centrifugation for 5 min at 1,500 rpm. The protocol for this study conformed to the guidelines of the Institutional Ethical Committee. The erythrocyte suspension was prepared at a haematocrit of 45 % in incubation buffer. The erythrocyte samples were divided into eight groups of six samples each: Group 1:
Erythrocyte suspension without any treatment, (control group). Group 2: Erythrocyte suspension was incubated with 1 mM of L-NAME [17], (L-NAME group). Group 3: Erythrocyte suspension was incubated with 1 mM of L-Arg [17], (L-Arg group). Group 4: Erythrocyte suspension was incubated with 1 mM of NAR [18], (NAR group). Group 5: Erythrocyte suspension was incubated with 100 μM of PbAc [19], (PbAc group). Group 6: Erythrocyte suspension treated with L-Arg + PbAc, ( L-Arg + PbAc group). Group 7: Erythrocyte suspension treated with NAR + PbAc, (NAR + PbAc group). Group 8: Erythrocyte suspension treated with L-Arg + NAR, + PbAc. (L-Arg +NAR + PbAc group). All samples in the erythrocyte suspension groups were incubated at 37 °C for 24 h. All drugs were dissolved in PBS to achieve the required drug concentration. The PBS contained 150 mM NaCl, 1.9 mM NaH2PO4, and 8.1 mM KH2PO4. The incubation buffer contained 8.1 % NaCl, 0.94 % KCl, 0.143 % MgCl2, 1.8 % glucose, 2.3 % N-(2-hydroxyethyl) piperazine-N-2-ethanesulfonic acid buffer (HEPES), and 40 mg/L gentamicin. After 24 h of incubation at 37 °C with moderate agitation, erythrocytes were hemolyzed by the addition of an equal volume of distilled water and mixed well. The hemolysate was then diluted with distilled water and used for the determination of glutathione reductase (GR), glutathione peroxidase (GPx), catalase (CAT) and superoxide dismutase (SOD), malondialdehyde (MDA) level, protein carbonyl content (PCC), and reduced glutathione (GSH). Erythrocytes membrane preparation was used for determination of membrane-bound enzymes Na+/K+-ATPase, Ca2+-ATPase, Mg2+-ATPase, NOS, and AChE.
Experimental Procedures Determination of Antioxidant Enzymes Blood samples were collected from apparently healthy adult men (aged 45–52 years), in heparinized tubes and centrifuged at 1,500 rpm for 5 min. The volunteers were advised not to take any drugs or dietary supplements four weeks prior to the experiment. The plasma and buffy coat were removed by aspiration to eliminate leukocytes and platelets. The erythrocytes were then washed three times with cold isotonic
CAT activity was determined using H2O2 as substrate according to method of Aebi [20]. SOD activity was estimated according to Beauchamp and Fridovich [21]. GR activity was measured according to method of Pinto and Bartley [22]. GPx activity was estimated using the method of Rotruck et al. [23].
Naringin Inhibits Lead-Induced Neurotoxicity
101
Determination of MDA, PCC, GSH, and Nitrite
Table 1 Erythrocytes ratio of GPx/GR, SOD/GPx and SOD/CAT activities as well as ratios GSSG/GSH after exposure to different studied agents
Lipid peroxidation was demonstrated by spectrophotometric measurement of MDA [24]. PCC was quantified on the basis of reaction with 2,4 dinitrophenylhydrazine [25]. GSH content was estimated according to method of Ellman [26]. Nitrite determination was based on the reduction of nitrate to nitrite by nitrate reductase and was performed as described by Green et al. [27].
Groups
Determination of Membrane-Bound Enzymes Erythrocyte membrane was prepared according the method of Dodge et al. [28] Activities of Na+/K+-ATPase, Ca2+-ATPase, and Mg2+-ATPase were determined in erythrocyte membrane preparation using ATP as the substrate in the presence of Na+, K+, Mg2+, and Ca2+ ions [7, 29, 30], respectively. After incubation, the liberated phosphorus content was estimated by method of Fiske and Subbarow [31]. NOS activity was measured using L-Arg substrate as described by Mckee et al. [32]. AChE activity was measured using acetylthiocholine iodide as substrate [33]. Total protein content was assayed according to method of Lowry et al. [34] using bovine serum albumin as standard. Hemoglobin was measured as cyanmethemoglobin [35]. Statistical Analysis Data are expressed as the mean ± SD of each group. The data were evaluated by one-way ANOVA followed by the Tukey– Kramer test for multiple comparisons. A 0.05 level of probability was used as the criterion for significance.
Results The present results showed that the ratios of SOD /CAT, SOD/ GPx, GPx/GR, and oxidized glutathione/GSH (GSSG/GSH) were significantly higher in PbAc-treated erythrocytes compared to either control, L-Arg, L-NAME, or NAR-treated groups. However, incubation of erythrocytes NAR or L-Arg plus NAR simultaneously with PbAc ameliorate these ratios compared to PbAc incubated erythrocytes. L-Arg treatment alone did not prevent lead-induced increase in these ratios. Table 1 displays these results. As shown in Table 2, incubation of erythrocytes with L-NAME, L-Arg, or NAR alone had no significant effect on the MDA, PCC, and nitrite levels compared to control cells. On the other side, PbAc exposure significantly increased levels of both MDA and PCC. The effect of PbAc was alleviated by simultaneous incubation with NAR or L-Arg + NAR. However, nitrite levels were significantly decreased in erythrocytes exposed to PbAc at value similar to that of
GPx/GR
Control
2.54±0.52 2.72±0.49 L-NAME 2.77±0.4 NAR 2.37±0.4 PbAc 4.24±0.7a PbAc + L-Arg 3.14±0.5a PbAc + NAR 2.91±0.5b PbAc + L-Arg + NAR 2.80±0.4b L-Arg
SOD/GPx SOD/ CAT
GSSG/ GSH
29.9±3.8 26.0±4.5 26.3±2.7 24.3±2.7 53.5±6.9a 44.7±5.9a 36.4±4.2b 32.8±3.8b
0.05±0.02 0.03±0.01 0.04±0.01 0.02±0.01 0.31±0.09a 0.24±0.06a 0.07±0.03b 0.06±0.02b
10.2±1.4 9.22±1.3 9.92±1.4 8.94±1.1 19.8±2.1a 14.4±1.7a 12.7±1.6b 10.9±1.5b
Data are presented as mean ± S.D., n=6. Multiple comparisons were achieved using one-way ANOVA followed by Tukey–Kramer as postANOVA test a
Indicates a significant increase from control
b
Indicates a significant decreased from lead-exposed aliquots respectively at p
Mitigation of Lead-Induced Neurotoxicity by the Naringin: Erythrocytes as Neurons Substitute Markers Gamaleldin I. Harisa
Received: 21 March 2014 / Accepted: 24 April 2014 / Published online: 15 May 2014 # Springer Science+Business Media New York 2014
Abstract This study aimed to investigate the effect of lead (Pb) on neuronal nitric oxide synthase (nNOS) activity using erythrocytes as neurons surrogate markers. Moreover, the protective effect of naringin (NAR) against lead acetate (PbAc)-induced neurotoxicity was investigated. Human erythrocytes were incubated with L-arginine (L-Arg), Nω-nitro-L-Arginine methyl ester (L-NAME), NAR, PbAc, PbAc + L-Arg, PbAc + NAR, or PbAc + L-Arg +NAR. The present results revealed that incubation of erythrocytes with PbAc inhibited NOS activity and decreased nitrite levels as an index for nitric oxide (NO) production to values similar that of L-NAME as known NOS inhibitor. Likewise, PbAc induced a significant decrease in activities of ATPases and acetylcholinesterase compared to control cells. Furthermore, PbAc exposure significantly increased protein carbonyl content (PCC) and malondialdehyde (MDA) levels while significantly decrease the levels of reduced glutathione (GSH). On the contrary, incubation of erythrocytes with PbAc in the presence of L-Arg + NAR synergistically ameliorated the investigated parameters compared to erythrocytes incubated with PbAc alone. These data suggest that NAR can restore NO bioavailability in a situation of Pb-induced cellular damage. This attributed to antioxidant activity and restoration NOS activity.
Keywords Erythrocytes . Lead . Naringin . Nitric oxide synthase . Acetylcholinesterase . Oxidative stress G. I. Harisa (*) Department of Pharmaceutics, Kayyali Chair for Pharmaceutical Industry, College of Pharmacy, King Saud University, P.O. Box 2457, Riyadh 11451, Saudi Arabia e-mail: [email protected] G. I. Harisa Department of Biochemistry, College of Pharmacy, Al-Azhar University (Boys), Nasr City, Cairo, Egypt
Introduction Lead (Pb) is one of the health threat toxicants, its exposure can arise from contact with lead-based dyes, fertilizers, cosmetics, and motor vehicles exhaust. Pb exposure causes multiple organ dysfunctions, including nervous system [1]. Pbinduced damage to the cerebral cortex, hippocampus, and cerebellum. Pb-induced neurotoxicity by the inappropriate release of neurotransmitters, inhibition of ATPases and mimicking calcium effects [2]. Also, Pb alters N-methyL-aspartate (NMDA) receptor expression, disrupt redox homeostasis, and inhibit nitric oxide synthase (NOS) [2]. However, the mechanisms of Pb-induced neurotoxicity through NOS inhibition still not fully understood and require further studies. NOS plays an important role in the nervous system through the production of nitric oxide (NO) that regulate neuronal electrical activity [3]. There are three NOS isoforms: endothelial-NOS (eNOS), inducible NOS(iNOS), and neuronal NOS (nNOS) [4]. NOS uses L-Arginine (L-Arg) as substrate to produce NO [5]. NO is an important brain messenger released upon activation of the NMDA receptor and subsequent Ca2+-dependent activation of nNOS. NO is involved in neurological cascades that regulate learning and memory [2]. NO has been accredited with both pro-oxidant and antioxidant actions [6]. Abnormal NO production induces glutamate excitotoxicity and increases peroxynitrite formation in the neurons [6]. Extra-neuronal cells such as platelets, fibroblasts, and erythrocytes have been used to provide peripheral markers and a better understanding of the pathogenesis of the neuropathology [7]. In the last years, several studies have shown that NOS is present in the cytoplasm and plasma membrane of erythrocytes [8–10]. Also acetylcholine receptors are present on erythrocytes membrane acts as a NOS regulator [8]. Consequently, erythrocytes can act as non-neuronal regulators of neuronal NO signals [11]. Application of acetyl choline to
100
erythrocytes increases the concentration of nitrite as an index for NO production [8]. However, no enough data is available about this issue. Several studies have indicated that neurotransmission signals are present in body fluids and peripheral blood cells [12]. Therefore, erythrocytes can be utilized as peripheral alternate biomarkers for neurons [13] and mercury-induced vascular cell damage [14]. Erythrocyte acetylcholinesterase (AChE) was investigated as surrogate biomarkers of Pb exposure [12]. Naringin(NAR) is a flavonoid found in grapefruit citrus species that possesses diverse biological and pharmacological properties including anti-inflammatory, anticarcinogenic, lipid-lowering, and antioxidant activities [15]. Moreover, flavonoids decrease accumulation of heavy metal in blood and tissue [3]. Several studies showed that NAR possesses neuroprotective activity against neuron damage [16]. Moreover, they exert protection against oxidative stress, modulate calcium homeostasis, and modulated NOS pathways [16]. Despite the beneficial effects of flavonoids, mechanisms by which NAR elicits neuroprotective effects need more investigations. The available data about Pb-induced neurotoxicity through inhibition of NOS is contradictory and incompletely understood. Therefore, this study sought to investigate the effect of Pb on NOS activity and NO production using erythrocytesNOS as neuron surrogate markers. However, no enough data are available about the protective effect of NAR against Pbinduced neurotoxicity. Herein, the possible neuroprotective effect of NAR through modulation of NOS activity was investigated.
Materials and Methods Materials Lead acetate (PbAc), L-Arg, N -nitro-L-Arginine methyl ester(L-NAME), and naringin (NAR) were purchased from Sigma-Aldrich (St. Louis, MO, USA). All other reagents were of the highest commercial grade unless indicated were obtained from Sigma Chemical Co. (St. Louis, MO, USA). A Spectro UV–vis Split Beam PC spectrophotometer (Model UVS-2800, Labomed, Inc., USA) and CT5 centrifuge were used in this investigation.
Harisa
phosphate-buffered saline (PBS, pH 7.4) with repeated centrifugation for 5 min at 1,500 rpm. The protocol for this study conformed to the guidelines of the Institutional Ethical Committee. The erythrocyte suspension was prepared at a haematocrit of 45 % in incubation buffer. The erythrocyte samples were divided into eight groups of six samples each: Group 1:
Erythrocyte suspension without any treatment, (control group). Group 2: Erythrocyte suspension was incubated with 1 mM of L-NAME [17], (L-NAME group). Group 3: Erythrocyte suspension was incubated with 1 mM of L-Arg [17], (L-Arg group). Group 4: Erythrocyte suspension was incubated with 1 mM of NAR [18], (NAR group). Group 5: Erythrocyte suspension was incubated with 100 μM of PbAc [19], (PbAc group). Group 6: Erythrocyte suspension treated with L-Arg + PbAc, ( L-Arg + PbAc group). Group 7: Erythrocyte suspension treated with NAR + PbAc, (NAR + PbAc group). Group 8: Erythrocyte suspension treated with L-Arg + NAR, + PbAc. (L-Arg +NAR + PbAc group). All samples in the erythrocyte suspension groups were incubated at 37 °C for 24 h. All drugs were dissolved in PBS to achieve the required drug concentration. The PBS contained 150 mM NaCl, 1.9 mM NaH2PO4, and 8.1 mM KH2PO4. The incubation buffer contained 8.1 % NaCl, 0.94 % KCl, 0.143 % MgCl2, 1.8 % glucose, 2.3 % N-(2-hydroxyethyl) piperazine-N-2-ethanesulfonic acid buffer (HEPES), and 40 mg/L gentamicin. After 24 h of incubation at 37 °C with moderate agitation, erythrocytes were hemolyzed by the addition of an equal volume of distilled water and mixed well. The hemolysate was then diluted with distilled water and used for the determination of glutathione reductase (GR), glutathione peroxidase (GPx), catalase (CAT) and superoxide dismutase (SOD), malondialdehyde (MDA) level, protein carbonyl content (PCC), and reduced glutathione (GSH). Erythrocytes membrane preparation was used for determination of membrane-bound enzymes Na+/K+-ATPase, Ca2+-ATPase, Mg2+-ATPase, NOS, and AChE.
Experimental Procedures Determination of Antioxidant Enzymes Blood samples were collected from apparently healthy adult men (aged 45–52 years), in heparinized tubes and centrifuged at 1,500 rpm for 5 min. The volunteers were advised not to take any drugs or dietary supplements four weeks prior to the experiment. The plasma and buffy coat were removed by aspiration to eliminate leukocytes and platelets. The erythrocytes were then washed three times with cold isotonic
CAT activity was determined using H2O2 as substrate according to method of Aebi [20]. SOD activity was estimated according to Beauchamp and Fridovich [21]. GR activity was measured according to method of Pinto and Bartley [22]. GPx activity was estimated using the method of Rotruck et al. [23].
Naringin Inhibits Lead-Induced Neurotoxicity
101
Determination of MDA, PCC, GSH, and Nitrite
Table 1 Erythrocytes ratio of GPx/GR, SOD/GPx and SOD/CAT activities as well as ratios GSSG/GSH after exposure to different studied agents
Lipid peroxidation was demonstrated by spectrophotometric measurement of MDA [24]. PCC was quantified on the basis of reaction with 2,4 dinitrophenylhydrazine [25]. GSH content was estimated according to method of Ellman [26]. Nitrite determination was based on the reduction of nitrate to nitrite by nitrate reductase and was performed as described by Green et al. [27].
Groups
Determination of Membrane-Bound Enzymes Erythrocyte membrane was prepared according the method of Dodge et al. [28] Activities of Na+/K+-ATPase, Ca2+-ATPase, and Mg2+-ATPase were determined in erythrocyte membrane preparation using ATP as the substrate in the presence of Na+, K+, Mg2+, and Ca2+ ions [7, 29, 30], respectively. After incubation, the liberated phosphorus content was estimated by method of Fiske and Subbarow [31]. NOS activity was measured using L-Arg substrate as described by Mckee et al. [32]. AChE activity was measured using acetylthiocholine iodide as substrate [33]. Total protein content was assayed according to method of Lowry et al. [34] using bovine serum albumin as standard. Hemoglobin was measured as cyanmethemoglobin [35]. Statistical Analysis Data are expressed as the mean ± SD of each group. The data were evaluated by one-way ANOVA followed by the Tukey– Kramer test for multiple comparisons. A 0.05 level of probability was used as the criterion for significance.
Results The present results showed that the ratios of SOD /CAT, SOD/ GPx, GPx/GR, and oxidized glutathione/GSH (GSSG/GSH) were significantly higher in PbAc-treated erythrocytes compared to either control, L-Arg, L-NAME, or NAR-treated groups. However, incubation of erythrocytes NAR or L-Arg plus NAR simultaneously with PbAc ameliorate these ratios compared to PbAc incubated erythrocytes. L-Arg treatment alone did not prevent lead-induced increase in these ratios. Table 1 displays these results. As shown in Table 2, incubation of erythrocytes with L-NAME, L-Arg, or NAR alone had no significant effect on the MDA, PCC, and nitrite levels compared to control cells. On the other side, PbAc exposure significantly increased levels of both MDA and PCC. The effect of PbAc was alleviated by simultaneous incubation with NAR or L-Arg + NAR. However, nitrite levels were significantly decreased in erythrocytes exposed to PbAc at value similar to that of
GPx/GR
Control
2.54±0.52 2.72±0.49 L-NAME 2.77±0.4 NAR 2.37±0.4 PbAc 4.24±0.7a PbAc + L-Arg 3.14±0.5a PbAc + NAR 2.91±0.5b PbAc + L-Arg + NAR 2.80±0.4b L-Arg
SOD/GPx SOD/ CAT
GSSG/ GSH
29.9±3.8 26.0±4.5 26.3±2.7 24.3±2.7 53.5±6.9a 44.7±5.9a 36.4±4.2b 32.8±3.8b
0.05±0.02 0.03±0.01 0.04±0.01 0.02±0.01 0.31±0.09a 0.24±0.06a 0.07±0.03b 0.06±0.02b
10.2±1.4 9.22±1.3 9.92±1.4 8.94±1.1 19.8±2.1a 14.4±1.7a 12.7±1.6b 10.9±1.5b
Data are presented as mean ± S.D., n=6. Multiple comparisons were achieved using one-way ANOVA followed by Tukey–Kramer as postANOVA test a
Indicates a significant increase from control
b
Indicates a significant decreased from lead-exposed aliquots respectively at p
