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Monosodium glutamate-induced damage in liver and kidney: a morphological and biochemical approach
Biomedicine & Pharmacotherapy 60 (2006) 86–91 http://france.elsevier.com/direct/BIOPHA/
Original article
Monosodium glutamate-induced damage in liver and kidney: a morphological and biochemical approach G.G. Ortiz a,*, O.K. Bitzer-Quintero b, C. Beas Zárate c, S. Rodríguez-Reynoso d, F. Larios-Arceo e, I.E. Velázquez-Brizuela a, F. Pacheco-Moisés f, S.A. Rosales-Corral a a Lab. de Desarrollo-Envejecimiento, Enfermedades Neurodegenerativas, División de Neurociencias, Centro de Investigación Biomédica de Occidente (CIBO), Instituto Mexicano del Seguro Social (IMSS), Sierra Mojada No. 800, cp 44340, Guadalajara, Jalisco, Mexico b Lab. de Neuroinmunomodulación, División de Neurociencias, CIBO-IMSS, Guadalajara, Jalisco, Mexico c Lab. de Neurobiología Celular y Molecular, División de Neurociencias, CIBO-IMSS, Guadalajara, Jalisco, Mexico d División de Investigación Quirúrgica, CIBO-IMSS, Guadalajara, Jalisco, Mexico e Departamento de Cirugía Pediátrica, UMAE-Pediatría, IMSS, Guadalajara, Jalisco, Mexico f Departamento de Química, CUCEI, Universidad de Guadalajara, Guadalajara, Jalisco, Mexico
Received 21 June 2005; accepted 11 July 2005 Available online 21 October 2005
Abstract It has been demonstrated that high concentrations of monosodium glutamate in the central nervous system induce neuronal necrosis and damage in retina and circumventricular organs. In this model, the monosodium glutamate is used to induce an epileptic state; one that requires highly concentrated doses. The purpose of this study was to evaluate the toxic effects of the monosodium glutamate in liver and kidney after an intra-peritoneal injection. For the experiment, we used 192 Wistar rats to carry out the following assessments: a) the quantification of the enzymes alanine aminotransferase and aspartate aminotransferase, b) the quantification of the lipid peroxidation products and c) the morphological evaluation of the liver and kidney. During the experiment, all of these assessments were carried out at 0, 15, 30 and 45 min after the intraperitoneal injection. In the rats that received monosodium glutamate, we observed increments in the concentration of alanine aminotransferase and aspartate aminotransferase at 30 and 45 min. Also, an increment of the lipid peroxidation products, in kidney, was exhibited at 15, 30 and 45 min while in liver it was observed at 30 and 45 min. Degenerative changes were observed (edema-degeneration-necrosis) at 15, 30 and 45 min. © 2006 Elsevier SAS. All rights reserved. Keywords: Monosodium glutamate; Epileptic state; Toxicity
1. Introduction L-glutamate is an excitatory neurotransmitter in the central nervous system (CNS) of mammals [1,2]. Glutamate is present in high levels in the brain and select groups of neurons. The endogenous L-glutamate, as the derived L-glutamate of exogenous precursors, is liberated in a Ca2+-dependent way after a depolarizing stimulus in the CNS [3,4]. Early studies in the 70’s, demonstrated that the administration of high concentrations of glutamate and other excitatory
*
Corresponding author. Tel.: +52 33 3668 3000 ext. 31951. E-mail address: [email protected] (G.G. Ortiz).
0753-3322/$ - see front matter © 2006 Elsevier SAS. All rights reserved. doi:10.1016/j.biopha.2005.07.012
amino acids to the nervous system, produced degeneration and neuronal death in certain cerebral regions and that these effects are related to the excitotoxicity or neuronal damage due to excessive neuronal excitation through a specific on-activation of their ionotropic receptors [2,5]. Two different groups of receptors, ionotropic and metabotropic, have been described for glutamate in the CNS. The ionotropic receptors are those that include; the N-methyl-D-aspartate (NMDA) type, the non-NMDA, the kainic receptor (KA) and the propionic alfa-amino-3-hydroxy-5-methyl-4-isoxasol (AMPA) [2,4–6]. The metabotropic receptors are present in the presynaptic membrane and do not form ion channels; they are associated with G proteins and respond to the stimulus of second intracellular messengers [6,7].
G.G. Ortiz et al. / Biomedicine & Pharmacotherapy 60 (2006) 86–91
The neurotoxicity that is induced by an on-activation of these glutamatergic receptors has been associated with diverse neurodegenerative diseases [8], as well as the excitotoxicity by nutritious ingestion of glutamate in the form of monosodic salts when consumed in high concentrations [3,9]. It has also been demonstrated that the administration of monosodium glutamate (MSG) to immature animals induces destruction in certain regions of the brain that lack a blood–brain barrier, such as the arcuatus nucleus of the hypothalamus that is involved in the regulation of neuroendocrine functions [2,3,8]. However, these demonstrations have ignored the effects of the systemic administration of MSG that can develop high concentrations in organs such as liver and kidney; even when the presence of glutamatergic receptors has been demonstrated outside the CNS [2,6,8,10]. These sub-types of receptors have been observed as the NMDA-R1, GluR 2/3 and mGluR 2/3 in liver, kidney, lungs, spleen and testicles [2,6,8,10]. Therefore, with the objective to know if a dose of 4 mg MSG per gram of body weight is toxic when administered systemically to rats; we studied the biochemical and morphological changes in the liver and kidney; activity of alanine aminotransferase (ALAT) and aspartate aminotransferase (ASAT), enzymes used as markers of liver function [11], as well as the lipid peroxidation marker of membranal damage for free radicals [9,12]. 2. Materials and methods One hundred and ninety-two Wistar male rats (225–250 g each) were obtained from CIBO-IMSS and housed in Plexiglas cages, three animals per cage. The animal rooms were windowless with automatic temperature (22 ± 1 °C) and light control (light on at 07 h:00 min and off at 21 h:00 min; 14 h light/10 h dark). The rats received standard laboratory chow (Purina) and water ad libitum. The animals were then divided into two groups: I) 96 animals for the quantification of the ALAT and ASAT enzymes and II) 96 animals to quantify the lipid peroxidation products (MDA and 4-HDA) and the morphological evaluation. These groups in turn were subdivided into: 1) control group (C) that received physiologic saline solution. 2) Sodium chloride solution (1.38 mg/g) equimolar to MSG (SSE) and 3) Experimental group that received MSG a dose of 4 mg/g of body weight (23.6 mMol/kg) by way of intra-peritoneal injection (ip). The animals were sacrificed at the following times: 0, 15, 30 and 45 min after the ip. injection of the solutions (N = 8 each group). The rats were anesthetized and blood samples were taken by cardiac puncture. The samples were placed in dry tubes without anticoagulant to obtain serum for enzymatic assays. ALAT and ASAT concentrations were measured in a Metrolab (1600 plus) spectro-photometer using kits from Beckman. The products of lipid peroxidation, malonaldehyde (MDA) and 4-hydroxyalkenals (4-HDA), were measured in liver and kidney. Tissues were homogenized in ice-cold 20 mM tris (hydroxymethyl) aminomethane buffer (pH 7.4) with a polytron-
87
like stirrer to produce 1:10 homogenates. Homogenates were centrifuged at 3000 × g for 30 min at 4 °C. The supernatant was collected and immediately assayed for products of lipid peroxidation (MDA and 4-HDA). An LPO kit was used for these measurements as previously described (Oxford Biomedical Res. Inc.) [13]. Immediately after the blood had been taken, the rats were perfused with a phosphate buffered saline solution (1000 UI/l) and procaine (1 g/l) was added at a pH 7.4 for 2 min, followed by perfusion of 4% phosphate buffered paraformaldehyde solution, for 10 min [14]. Both solutions were perfused at 140 cm of water: the liver and kidney were postfixed by immersion for 90 min, dehydrated in an ethanol series and embedded in paraplast-plus. Sections measuring 6 μm were stained with hematoxylin and eosin for light microscopy. Data were analyzed by one-way analysis of variance (ANOVA). If the F values were significant, Student–Newman– Keul’s test was used to compare groups. The level of significance was accepted at P < 0.05. 3. Results 3.1. Enzymes At 0 min, increased activity of the ASAT enzyme in the SSE group was observed; however, incremented ASAT enzymatic activity in the MSG group was present at 15, 30 and 45 min (P < 0.001) (Fig. 1a). Incremented ALAT activity was present from 15 min similarly for both SSE and MSG groups (P < 0.001) (Fig. 1b). 3.2. Lipid peroxidation In the second experiment (LPO/morphology) MSG (4 mg/g body weight) increased products of lipid peroxidation (MDA and 4-HAD). In the liver, the MSG group presented increments in the levels of MDA and 4-HDA at 30 and 45 min and the SSE group only at 45 min (Fig. 2a, b). 3.3. Morphological analysis Macroscopically, the livers and kidneys of MSG-injected rats had an external appearance that was pale in color, clear signs of congestion and edema with loss of hepatic borders. When the liver and kidney were cut, edema and congestion had increased at 45 min after the injection of MSG. Microscopic examination exhibited degenerative changes in liver and kidney at all the times studied as compared to controls (Figs. 3 and 4). In liver, a turbid swelling was seen at 30 min as a pale, fine cytoplasmic granulation; there were also small vesicles, the nuclei were contracted, rounded and hyperchromatic. The nucleoli and chromatin could not been seen. The hepatic cords were disrupted at 45 min and the cytoplasm had swollen and more vesicles were visible. The nuclei were intensely stained and excentric in some cells (Fig. 3a–e). The kidney showed degenerative changes in all the experimental groups studied. Glomeruli were denser in the MSG-injected
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G.G. Ortiz et al. / Biomedicine & Pharmacotherapy 60 (2006) 86–91
Fig. 1. a) Serum alanine aminotransferase (ALAT), and b) aspartic aminotransferase (ASAT) activity. Incremented ASAT enzymatic activity in the MSG group was present at 15, 30 and 45 min. On the other hand, incremented ALAT activity was present from 15 min.
animals. Glomerular cells exhibited hyper-chromatic nuclei. Cloudy swelling and hydropic degeneration were seen in the convoluted tubules, especially at 30–45 min. In some instances perinuclear halos and disruption of the cells, and necrosis were observed (Fig. 4a–e). 4. Discussion MSG, a dose of 4 mg/g of body weight administered by intra-peritoneal injection to rats, is toxic for the liver and the kidney. In this study, starting from 15 min post injection, we observed high levels of ALAT and ASAT (Fig. 1a, b), which indicate that the serum concentration of these enzymes fluctuates with the hepatic damage. The localization of ALAT and ASAT in the hepatocyte is cytoplasmatic; the MSG cytotoxic
Fig. 2. a) Lipid peroxidation (MDA and 4-HAD) in liver, and b) lipid peroxidation (MDA and 4-HAD) in kidney. In liver and kidney, the MSG group exhibited increments in the levels of MDA and 4-HDA at 15, 30 and 45 min. The SSE group only at 45 min.
effect induced tissue damage and enzyme release increasing their serum levels. The circulating MSG was dissociated in sodium (Na+) and L-glutamate. The L-glutamate crosses the mesothelial peritoneal cells and arrives at the bloodstream by means of a transport system using ATP. A part of the L-glutamate in the cell conjugates, in order to be eliminated, and another part is transformed into glutamine [15]. When this occurs, the cells try to repair some of the damages by using enzymes that are present in the smooth endoplasmic reticulum but the cell is not able to completely remove the excess glutamine. Probably, for this reason, the liver (15 min) presented cloudy swelling (turbid swelling) at 30 min. It is possible to observe vesicular degeneration and necrosis at 45 min. When the L-glutamate arrives in high concentrations through the renal artery, the kidney tries to excrete it. The renal corpuscle receives the L-glutamate through the afferent arter-
G.G. Ortiz et al. / Biomedicine & Pharmacotherapy 60 (2006) 86–91
89
Fig. 3. a) and b): CTL, and SSE groups: hepatocyte (arrow), Kupffer cell (small arrow), sinusoid space (asterisk), both groups exhibited normal structures; c) MSG group at 15 min: hyperchromatic nuclei (large arrow), turbid swelling (medium arrow), Kupffer cell (small arrow); d) MSG at 30 min: contracted and hyperchromatic nuclei with perinuclear halos (large and medium arrow), lipid degeneration (pick arrow), hyperchromatic and hypertrophic Kupffer cell (small arrow), sinusoidal space (asterisk); e) MSG at 45 min: excentric, hyperchromatic and contracted nuclei with halos (large arrow), cytoplasm swollen and disrupted (middle arrow), hyperchromatic, and hypertrophic Kupffer cell (small arrow), sinusoidal space (asterisk) H&E × 260.
iole, it is absorbed, filtrated, and crosses the membrane damaging the cell. The convoluted proximal tubules were more susceptible to damage in comparison to the distal convoluted tubules. The kidney at 15, and 30 min exhibited edema; hydropic degeneration and necrosis were observed at 45 min. Glutamate, a major excitatory amino acid neurotransmitter is also an endogenous excitotoxin. The effects of the glutamate excitotoxicity in different brain regions, and lipid peroxidation
are well documented [16]. In recent studies, the daily administration of MSG significantly increased levels of MDA and 4HDA in the hepatic microsomes [9]. In this study, the increase of lipid peroxidation products (MDA and 4-OH alkenals) was the response of the liver and kidney damage. At the same times, there were observed increases in malonaldehyde and 4-OH alkenals as well as ALAT and ASAT enzymes. The morphology of the damage shows a
90
G.G. Ortiz et al. / Biomedicine & Pharmacotherapy 60 (2006) 86–91
Fig. 4. a) and b): CTL and SSE groups: Glomeruli (G), pick arrows showed nuclei of the tubular cell, both groups exhibited normal structures; c) MSG at 15 min: dense glomeruli with hyperchromatic nuclei (G) and arterial spaces (large arrow), dense material in the Bowman’s capsule (middle arrows), small arrows exhibited cloudy swelling and hydropic degeneration, tubular space (asterisk); d) MSG at 30 min: Glomeruli (G), arterial spaces (arterial spaces), we observed a height concentration of dense material in the Bowman’s capsule (middle arrow); e) MSG at 45 min: condensed and hyperchromatic glomeruli (G), hyperchromatic nuclei (arrow), hydropic degeneration and necrosis were observed H&E × 260.
correlation between the progressive damage and the lipid peroxidation products, especially during the 30 and 45 min after glutamate administration. In liver the steatosis and necrosis were observed with high levels of malonaldehyde and 4-OH alkenals. In kidney, very similar responses were exhibited with hydropic degeneration and necrosis. All of these data could be explained by the excitotoxic role of the glutamate.
Finally, relatively little attention has been paid to functional expression of glutamate signaling molecules in peripheral tissues. Evidence is emerging for a role of glutamate as an extra-cellular signal mediator in several organs and systems, in addition to an excitatory amino acid neurotransmitter role in the CNS [2,17,18]. Recent molecular biological analysis gives support to the expression of particular glutamate signaling molecules in a
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variety of different neuronal and non-neuronal peripheral locations, including liver and kidney [2]. The results of this study provide strong motivation to investigate the systemic effects of the MSG. It is clear that this model induced neuroexcitotoxic damage, as well as exhibiting a strong and diffuse peripheral damage, probably in all the organs and systems.
[7] Schwendt M, Jezova D. Glutamate receptors and transporters in the brain and peripheral tissues. Cesk Fysiol 2001;50(2):43–56.
5. Conclusions
[10] Carlton SM. Peripheral excitatory amino acids. Curr Opin Pharmacol 2001;1(1):52–6.
In this study, monosodium glutamate-induced damage in liver and kidney, as demonstrated by biochemical and morphological methods.
[11] Wissing H, Khun I. The effect of desfluorane in liver function markers in infants and children. Report to study and pertinent marries report. Records Anesthesiol Scand 2000;44(9):1149–53.
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Takamori S, Rhee JS, Rosenmund C, Jahn R. Identification of to vesicular glutamate transporter that defines to glutamatergic phenotype in neurons. Nature 2000;407(6801):189–94. Hinoi E, Takeda T, Ueshima T, Tsuchihashi Y, Yoneda Y. Glutamate signaling in peripheral tissues. Eur J Biochem 2004;271:1–13. Olney JW. Excitotoxic amino acids and neuropsychiatric disorders. Annu Rev Pharmacol Toxicol 1990;30:47–71. Cotman CW, Kahle JS. Ulas. Excitatory amino acid neurotransmitters 2000; File:///E/ens/articles/00000750/tx1.htm. Sinor JD, Shen D, Venneti S, Blitzblau RC, Leszkiewicz DN, Rosenberg PA, et al. NMDA and glutamate evoke excitotoxicity at distinct cellular locations in cortical rat neurons in vitro. J Neurosci 2000;20 (23):8831–7. Gill SS, Pulido OM. Glutamate receptors in peripheral tissues: current knowledge, future research, and implications for toxicology. Toxicol Pathol 2001;29(2):208–23.
[8] Gill SS, Mueller RW, McGuire PF, Refined OM. Potential target sites in peripheral tissues for excitatory neurotransmission and excitotoxicity. Toxicol Pathol 2000;28(2):277–85. [9] Choudhary P, Malik VB, Puri S, Ahluwalia P. Studies on the effects of monosodium glutamate on hepatic microsomal lipid peroxidation, calcium, ascorbic acid and glutathione and its dependent enzymes in adult male mice. Toxicol Lett 1996;89(1):71–6.
[12] Montal M. Mitochondria, glutamate neurotoxicity and the death cascade. Biochem Bioph Rec 1998;1366:113–26. [13] García JJ, Reiter RJ, Karbownik M, Calvo JR, Ortiz GG, Tan DX, et al. N-acetylserotonin suppresses hepatic microsomal membrane rigidity associated with lipid peroxidation. Eur J Pharmacol 2001;428(2):169–75. [14] Feria-Velasco A, Karnovsky Y. Optima preservación ultraestructural del sistema nervioso central por perfusión intravascular con glutaraldehido. Arch Inv Méd 1970;1:201–20. [15] Walker R, Lupien JR. The safety evaluation of monosodium glutamate. J Nutr 2000;130(45 suppl):1049S–1052S. [16] Singh P, Mann KA, Mangat HK, Kaur G. Prolonged glutamate excitotoxicity: effects of mitochondrial antioxidants and antioxidant enzymes. Mol Cell Biochem 2003;243(1–2):139–45. [17] Watkins JC. L-Glutamate ace to central neurotransmitter: looking back. Biochem Soc Trans 2000;1:297–309 [28 (4)]. [18] R., Medina-brow walls L, Rock F. 1999. On the relationship between extracellular glutamate, hyperexcitation and neurodegeneration, in alive. Neurochem Int 1999;34:23–31.
Original article
Monosodium glutamate-induced damage in liver and kidney: a morphological and biochemical approach G.G. Ortiz a,*, O.K. Bitzer-Quintero b, C. Beas Zárate c, S. Rodríguez-Reynoso d, F. Larios-Arceo e, I.E. Velázquez-Brizuela a, F. Pacheco-Moisés f, S.A. Rosales-Corral a a Lab. de Desarrollo-Envejecimiento, Enfermedades Neurodegenerativas, División de Neurociencias, Centro de Investigación Biomédica de Occidente (CIBO), Instituto Mexicano del Seguro Social (IMSS), Sierra Mojada No. 800, cp 44340, Guadalajara, Jalisco, Mexico b Lab. de Neuroinmunomodulación, División de Neurociencias, CIBO-IMSS, Guadalajara, Jalisco, Mexico c Lab. de Neurobiología Celular y Molecular, División de Neurociencias, CIBO-IMSS, Guadalajara, Jalisco, Mexico d División de Investigación Quirúrgica, CIBO-IMSS, Guadalajara, Jalisco, Mexico e Departamento de Cirugía Pediátrica, UMAE-Pediatría, IMSS, Guadalajara, Jalisco, Mexico f Departamento de Química, CUCEI, Universidad de Guadalajara, Guadalajara, Jalisco, Mexico
Received 21 June 2005; accepted 11 July 2005 Available online 21 October 2005
Abstract It has been demonstrated that high concentrations of monosodium glutamate in the central nervous system induce neuronal necrosis and damage in retina and circumventricular organs. In this model, the monosodium glutamate is used to induce an epileptic state; one that requires highly concentrated doses. The purpose of this study was to evaluate the toxic effects of the monosodium glutamate in liver and kidney after an intra-peritoneal injection. For the experiment, we used 192 Wistar rats to carry out the following assessments: a) the quantification of the enzymes alanine aminotransferase and aspartate aminotransferase, b) the quantification of the lipid peroxidation products and c) the morphological evaluation of the liver and kidney. During the experiment, all of these assessments were carried out at 0, 15, 30 and 45 min after the intraperitoneal injection. In the rats that received monosodium glutamate, we observed increments in the concentration of alanine aminotransferase and aspartate aminotransferase at 30 and 45 min. Also, an increment of the lipid peroxidation products, in kidney, was exhibited at 15, 30 and 45 min while in liver it was observed at 30 and 45 min. Degenerative changes were observed (edema-degeneration-necrosis) at 15, 30 and 45 min. © 2006 Elsevier SAS. All rights reserved. Keywords: Monosodium glutamate; Epileptic state; Toxicity
1. Introduction L-glutamate is an excitatory neurotransmitter in the central nervous system (CNS) of mammals [1,2]. Glutamate is present in high levels in the brain and select groups of neurons. The endogenous L-glutamate, as the derived L-glutamate of exogenous precursors, is liberated in a Ca2+-dependent way after a depolarizing stimulus in the CNS [3,4]. Early studies in the 70’s, demonstrated that the administration of high concentrations of glutamate and other excitatory
*
Corresponding author. Tel.: +52 33 3668 3000 ext. 31951. E-mail address: [email protected] (G.G. Ortiz).
0753-3322/$ - see front matter © 2006 Elsevier SAS. All rights reserved. doi:10.1016/j.biopha.2005.07.012
amino acids to the nervous system, produced degeneration and neuronal death in certain cerebral regions and that these effects are related to the excitotoxicity or neuronal damage due to excessive neuronal excitation through a specific on-activation of their ionotropic receptors [2,5]. Two different groups of receptors, ionotropic and metabotropic, have been described for glutamate in the CNS. The ionotropic receptors are those that include; the N-methyl-D-aspartate (NMDA) type, the non-NMDA, the kainic receptor (KA) and the propionic alfa-amino-3-hydroxy-5-methyl-4-isoxasol (AMPA) [2,4–6]. The metabotropic receptors are present in the presynaptic membrane and do not form ion channels; they are associated with G proteins and respond to the stimulus of second intracellular messengers [6,7].
G.G. Ortiz et al. / Biomedicine & Pharmacotherapy 60 (2006) 86–91
The neurotoxicity that is induced by an on-activation of these glutamatergic receptors has been associated with diverse neurodegenerative diseases [8], as well as the excitotoxicity by nutritious ingestion of glutamate in the form of monosodic salts when consumed in high concentrations [3,9]. It has also been demonstrated that the administration of monosodium glutamate (MSG) to immature animals induces destruction in certain regions of the brain that lack a blood–brain barrier, such as the arcuatus nucleus of the hypothalamus that is involved in the regulation of neuroendocrine functions [2,3,8]. However, these demonstrations have ignored the effects of the systemic administration of MSG that can develop high concentrations in organs such as liver and kidney; even when the presence of glutamatergic receptors has been demonstrated outside the CNS [2,6,8,10]. These sub-types of receptors have been observed as the NMDA-R1, GluR 2/3 and mGluR 2/3 in liver, kidney, lungs, spleen and testicles [2,6,8,10]. Therefore, with the objective to know if a dose of 4 mg MSG per gram of body weight is toxic when administered systemically to rats; we studied the biochemical and morphological changes in the liver and kidney; activity of alanine aminotransferase (ALAT) and aspartate aminotransferase (ASAT), enzymes used as markers of liver function [11], as well as the lipid peroxidation marker of membranal damage for free radicals [9,12]. 2. Materials and methods One hundred and ninety-two Wistar male rats (225–250 g each) were obtained from CIBO-IMSS and housed in Plexiglas cages, three animals per cage. The animal rooms were windowless with automatic temperature (22 ± 1 °C) and light control (light on at 07 h:00 min and off at 21 h:00 min; 14 h light/10 h dark). The rats received standard laboratory chow (Purina) and water ad libitum. The animals were then divided into two groups: I) 96 animals for the quantification of the ALAT and ASAT enzymes and II) 96 animals to quantify the lipid peroxidation products (MDA and 4-HDA) and the morphological evaluation. These groups in turn were subdivided into: 1) control group (C) that received physiologic saline solution. 2) Sodium chloride solution (1.38 mg/g) equimolar to MSG (SSE) and 3) Experimental group that received MSG a dose of 4 mg/g of body weight (23.6 mMol/kg) by way of intra-peritoneal injection (ip). The animals were sacrificed at the following times: 0, 15, 30 and 45 min after the ip. injection of the solutions (N = 8 each group). The rats were anesthetized and blood samples were taken by cardiac puncture. The samples were placed in dry tubes without anticoagulant to obtain serum for enzymatic assays. ALAT and ASAT concentrations were measured in a Metrolab (1600 plus) spectro-photometer using kits from Beckman. The products of lipid peroxidation, malonaldehyde (MDA) and 4-hydroxyalkenals (4-HDA), were measured in liver and kidney. Tissues were homogenized in ice-cold 20 mM tris (hydroxymethyl) aminomethane buffer (pH 7.4) with a polytron-
87
like stirrer to produce 1:10 homogenates. Homogenates were centrifuged at 3000 × g for 30 min at 4 °C. The supernatant was collected and immediately assayed for products of lipid peroxidation (MDA and 4-HDA). An LPO kit was used for these measurements as previously described (Oxford Biomedical Res. Inc.) [13]. Immediately after the blood had been taken, the rats were perfused with a phosphate buffered saline solution (1000 UI/l) and procaine (1 g/l) was added at a pH 7.4 for 2 min, followed by perfusion of 4% phosphate buffered paraformaldehyde solution, for 10 min [14]. Both solutions were perfused at 140 cm of water: the liver and kidney were postfixed by immersion for 90 min, dehydrated in an ethanol series and embedded in paraplast-plus. Sections measuring 6 μm were stained with hematoxylin and eosin for light microscopy. Data were analyzed by one-way analysis of variance (ANOVA). If the F values were significant, Student–Newman– Keul’s test was used to compare groups. The level of significance was accepted at P < 0.05. 3. Results 3.1. Enzymes At 0 min, increased activity of the ASAT enzyme in the SSE group was observed; however, incremented ASAT enzymatic activity in the MSG group was present at 15, 30 and 45 min (P < 0.001) (Fig. 1a). Incremented ALAT activity was present from 15 min similarly for both SSE and MSG groups (P < 0.001) (Fig. 1b). 3.2. Lipid peroxidation In the second experiment (LPO/morphology) MSG (4 mg/g body weight) increased products of lipid peroxidation (MDA and 4-HAD). In the liver, the MSG group presented increments in the levels of MDA and 4-HDA at 30 and 45 min and the SSE group only at 45 min (Fig. 2a, b). 3.3. Morphological analysis Macroscopically, the livers and kidneys of MSG-injected rats had an external appearance that was pale in color, clear signs of congestion and edema with loss of hepatic borders. When the liver and kidney were cut, edema and congestion had increased at 45 min after the injection of MSG. Microscopic examination exhibited degenerative changes in liver and kidney at all the times studied as compared to controls (Figs. 3 and 4). In liver, a turbid swelling was seen at 30 min as a pale, fine cytoplasmic granulation; there were also small vesicles, the nuclei were contracted, rounded and hyperchromatic. The nucleoli and chromatin could not been seen. The hepatic cords were disrupted at 45 min and the cytoplasm had swollen and more vesicles were visible. The nuclei were intensely stained and excentric in some cells (Fig. 3a–e). The kidney showed degenerative changes in all the experimental groups studied. Glomeruli were denser in the MSG-injected
88
G.G. Ortiz et al. / Biomedicine & Pharmacotherapy 60 (2006) 86–91
Fig. 1. a) Serum alanine aminotransferase (ALAT), and b) aspartic aminotransferase (ASAT) activity. Incremented ASAT enzymatic activity in the MSG group was present at 15, 30 and 45 min. On the other hand, incremented ALAT activity was present from 15 min.
animals. Glomerular cells exhibited hyper-chromatic nuclei. Cloudy swelling and hydropic degeneration were seen in the convoluted tubules, especially at 30–45 min. In some instances perinuclear halos and disruption of the cells, and necrosis were observed (Fig. 4a–e). 4. Discussion MSG, a dose of 4 mg/g of body weight administered by intra-peritoneal injection to rats, is toxic for the liver and the kidney. In this study, starting from 15 min post injection, we observed high levels of ALAT and ASAT (Fig. 1a, b), which indicate that the serum concentration of these enzymes fluctuates with the hepatic damage. The localization of ALAT and ASAT in the hepatocyte is cytoplasmatic; the MSG cytotoxic
Fig. 2. a) Lipid peroxidation (MDA and 4-HAD) in liver, and b) lipid peroxidation (MDA and 4-HAD) in kidney. In liver and kidney, the MSG group exhibited increments in the levels of MDA and 4-HDA at 15, 30 and 45 min. The SSE group only at 45 min.
effect induced tissue damage and enzyme release increasing their serum levels. The circulating MSG was dissociated in sodium (Na+) and L-glutamate. The L-glutamate crosses the mesothelial peritoneal cells and arrives at the bloodstream by means of a transport system using ATP. A part of the L-glutamate in the cell conjugates, in order to be eliminated, and another part is transformed into glutamine [15]. When this occurs, the cells try to repair some of the damages by using enzymes that are present in the smooth endoplasmic reticulum but the cell is not able to completely remove the excess glutamine. Probably, for this reason, the liver (15 min) presented cloudy swelling (turbid swelling) at 30 min. It is possible to observe vesicular degeneration and necrosis at 45 min. When the L-glutamate arrives in high concentrations through the renal artery, the kidney tries to excrete it. The renal corpuscle receives the L-glutamate through the afferent arter-
G.G. Ortiz et al. / Biomedicine & Pharmacotherapy 60 (2006) 86–91
89
Fig. 3. a) and b): CTL, and SSE groups: hepatocyte (arrow), Kupffer cell (small arrow), sinusoid space (asterisk), both groups exhibited normal structures; c) MSG group at 15 min: hyperchromatic nuclei (large arrow), turbid swelling (medium arrow), Kupffer cell (small arrow); d) MSG at 30 min: contracted and hyperchromatic nuclei with perinuclear halos (large and medium arrow), lipid degeneration (pick arrow), hyperchromatic and hypertrophic Kupffer cell (small arrow), sinusoidal space (asterisk); e) MSG at 45 min: excentric, hyperchromatic and contracted nuclei with halos (large arrow), cytoplasm swollen and disrupted (middle arrow), hyperchromatic, and hypertrophic Kupffer cell (small arrow), sinusoidal space (asterisk) H&E × 260.
iole, it is absorbed, filtrated, and crosses the membrane damaging the cell. The convoluted proximal tubules were more susceptible to damage in comparison to the distal convoluted tubules. The kidney at 15, and 30 min exhibited edema; hydropic degeneration and necrosis were observed at 45 min. Glutamate, a major excitatory amino acid neurotransmitter is also an endogenous excitotoxin. The effects of the glutamate excitotoxicity in different brain regions, and lipid peroxidation
are well documented [16]. In recent studies, the daily administration of MSG significantly increased levels of MDA and 4HDA in the hepatic microsomes [9]. In this study, the increase of lipid peroxidation products (MDA and 4-OH alkenals) was the response of the liver and kidney damage. At the same times, there were observed increases in malonaldehyde and 4-OH alkenals as well as ALAT and ASAT enzymes. The morphology of the damage shows a
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G.G. Ortiz et al. / Biomedicine & Pharmacotherapy 60 (2006) 86–91
Fig. 4. a) and b): CTL and SSE groups: Glomeruli (G), pick arrows showed nuclei of the tubular cell, both groups exhibited normal structures; c) MSG at 15 min: dense glomeruli with hyperchromatic nuclei (G) and arterial spaces (large arrow), dense material in the Bowman’s capsule (middle arrows), small arrows exhibited cloudy swelling and hydropic degeneration, tubular space (asterisk); d) MSG at 30 min: Glomeruli (G), arterial spaces (arterial spaces), we observed a height concentration of dense material in the Bowman’s capsule (middle arrow); e) MSG at 45 min: condensed and hyperchromatic glomeruli (G), hyperchromatic nuclei (arrow), hydropic degeneration and necrosis were observed H&E × 260.
correlation between the progressive damage and the lipid peroxidation products, especially during the 30 and 45 min after glutamate administration. In liver the steatosis and necrosis were observed with high levels of malonaldehyde and 4-OH alkenals. In kidney, very similar responses were exhibited with hydropic degeneration and necrosis. All of these data could be explained by the excitotoxic role of the glutamate.
Finally, relatively little attention has been paid to functional expression of glutamate signaling molecules in peripheral tissues. Evidence is emerging for a role of glutamate as an extra-cellular signal mediator in several organs and systems, in addition to an excitatory amino acid neurotransmitter role in the CNS [2,17,18]. Recent molecular biological analysis gives support to the expression of particular glutamate signaling molecules in a
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variety of different neuronal and non-neuronal peripheral locations, including liver and kidney [2]. The results of this study provide strong motivation to investigate the systemic effects of the MSG. It is clear that this model induced neuroexcitotoxic damage, as well as exhibiting a strong and diffuse peripheral damage, probably in all the organs and systems.
[7] Schwendt M, Jezova D. Glutamate receptors and transporters in the brain and peripheral tissues. Cesk Fysiol 2001;50(2):43–56.
5. Conclusions
[10] Carlton SM. Peripheral excitatory amino acids. Curr Opin Pharmacol 2001;1(1):52–6.
In this study, monosodium glutamate-induced damage in liver and kidney, as demonstrated by biochemical and morphological methods.
[11] Wissing H, Khun I. The effect of desfluorane in liver function markers in infants and children. Report to study and pertinent marries report. Records Anesthesiol Scand 2000;44(9):1149–53.
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