Glutathione Reductase Inhibition and Methylated Arsenic Distribution in Cd1 Mice Brain and Liver
http://www.100md.com
《毒物学科学杂志》
Instituto de Neurobiología, Instituto de Investigaciones Biomédicas, UNAM, HP 70-228, Civdad Universitaria, 04510 DF, Mexico
Sección de Toxicología, CINVESTAV-IPN, AP 14-740, 07000 DF, México
ABSTRACT
Inorganic arsenic exposure via drinking water has been associated with cancer and serious injury in various internal organs, as well as with peripheral neuropathy and diverse effects in the nervous system. Alterations in memory and attention processes have been reported in exposed children, whereas adults acutely exposed to high amounts of inorganic arsenic showed impairments in learning, memory, and concentration. Glutathione (GSH) is extensively involved in the metabolism of inorganic arsenic, and both arsenite and its methylated metabolites have been shown to be potent inhibitors of glutathione reductase (GR) in vitro. Brain would be more susceptible to GR inhibition because of the decreased activities of superoxide dismutase (SOD) and catalase reported in this tissue. To investigate whether GR inhibition could be documented in vivo, we determined the activity and levels of GR in brain as well as in liver, the main organ of arsenic metabolism in mice exposed to 2.5, 5, or 10 mg/kg/day of sodium arsenite over a period of 9 days. In contrast to what has been observed in vitro, significant inhibition of the expression and activity of GR was observed only at the highest concentration used (10 mg/kg/day) in both organs. Although the disposition of arsenicals was higher in liver, significant amounts of inorganic and methylated arsenic forms were determined in the brain of exposed animals. The formation of monomethylarsenic (MMA) and dimethylarsenic (DMA) metabolites in the brain was confirmed by incubating brain slices for 24, 48, and 72 h with sodium arsenite.
Key Words: arsenite; biotransformation; glutathione reductase inhibition; brain.
INTRODUCTION
Inorganic arsenic exposure via drinking water has been associated with cancer and serious injury in various internal organs, as well as with peripheral neuropathy and diverse effects in the circulatory and nervous system. Acute intoxication that produces initial gastrointestinal or cardiovascular symptoms can be followed by the delayed onset of central or peripheral nervous system involvement. During subacute or chronic exposure, inorganic arsenic can occasionally result in subclinical or overt peripheral neuropathy (Yip et al., 2002). In children exposed to inorganic arsenic throughout their whole lives, hearing impairment (Bencko and Symon, 1977) and lower scores in verbal intelligence quotient (IQ) have been reported (Calderon et al., 2001; Wasserman et al., 2004). While alterations in memory and attention processes have been reported in adolescents exposed to high levels of inorganic arsenic in well water (Tsai et al., 2003), adults acutely exposed showed impairments in learning, memory, and concentration (Bolla-Wilson and Bleecker, 1987; Franzblau and Lilis, 1989; Morton and Caron, 1989).
Similarly, inorganic arsenic exposure has been related to behavioral alterations in rodents, such as disruption in operant learning (Nagaraja and Desiraju, 1994), alterations in locomotor activity (Chattopadhyay et al., 2002a; Chattopadhyay et al., 2002b; Itoh et al., 1990; Pryor et al., 1983; Rodriguez et al., 2001, 2002), and increases in errors in the delayed alternation task tested in a T maze (Rodriguez et al., 2001, 2002). Some of these alterations could be related to changes in cholinergic, monoaminergic, GABAergic, and glutamatergic systems in several brain regions such as striatum, midbrain, hypothalamus, and hippocampus of rodents exposed to arsenicals (Delgado et al., 2000; Kannan et al., 2001; Mejia et al., 1997; Nagaraja and Desiraju, 1993, 1994; Rodriguez et al., 2001, 2003; Tripathi et al., 1997; Valkonen et al., 1983).
In humans and in many mammalian species exposed to inorganic arsenic, at least four metabolites that can exert toxic effects have been identified in tissues and fluids. They originate in a metabolic process that is carried out in two steps: (1) the reduction of the pentavalent species to trivalency in the presence of glutathione (GSH) or thioredoxin and (2) oxidative methylation, whereby inorganic arsenic is converted to its monomethyl and dimethyl arsenic forms (MMA, DMA, respectively). Thus, both pentavalent and trivalent methylarsenic forms are intermediates or products of this pathway (Lin et al., 2002; Thomas et al., 2004).
Few studies have attempted to unravel the mechanism of toxicity of inorganic arsenic exposure in the central nervous system (CNS). In vitro studies demonstrated that As exposure caused apoptosis in cortical and cerebellar neurons, whereas dimethylarsinic acid exposure induced apoptosis in cerebellar neurons only. In both types of cell cultures, apoptosis was induced by caspase activation (Namgung and Xia, 2000, 2001). A report of an in vivo study demonstrated the formation of hydroxyl radicals in the rat striatum, which were induced by direct infusion of sodium arsenite via a microdialysis probe (García-Chavez et al., 2003). However, there are no previous in vivo studies relating the adverse effects of arsenical exposure to the presence of methylated arsenic species in brain, which could be in to some extent responsible for the neurotoxic alterations reported.
The tripeptide GSH (L--glutamyl-cysteinyl-glycine) is the most important intracellular nonprotein thiol in mammalian cells. It is found at concentrations of 1–3 mM in human and monkey brain cells (Slivka et al., 1987), and it possesses reducing and nucleophilic properties. It can exist in either a reduced (GSH) or oxidized (GSSG) form. Glutathione reductase (GR, EC 1.6.4.2) is an ubiquitous flavoenzyme that catalyzes the NAD(P)H-dependent reduction of GSSG to GSH. This enzyme maintains adequate levels of the cellular GSH pool, which is critical not only for maintaining the cellular redox status by keeping sulfhydryl groups of cytosolic proteins in their reduced form (Dringen et al., 2000) but also because numerous toxic or potentially toxic compounds, including some metals, are either taken up by or removed from the cells by GSH-mediated pathways (Chouchane and Snow, 2001). In comparison to other organs, brain is more susceptible to oxidative damage because of the high oxygen utilization (20% of oxygen consumed by the body), high iron content, presence of unsaturated fatty acids, and decreased activities of detoxifying enzymes such as superoxide dismutase (SOD), catalase, and GR, as compared with kidney and liver (Bharath et al., 2002; Dringen et al., 2000). GSH serves as an antioxidant, reacting with free radicals and organic peroxides; it is also a substrate of glutathione peroxidase (Maher and Schubert, 2000) and a coenzyme of glutathione-S-transferase and thioltransferases among other enzymes (Arrigo, 1999).
Furthermore, GSH is extensively involved in the metabolism of inorganic arsenic, specifically in the reduction of pentavalent arsenicals (arsenate AsV, methylarsonic acid MMAV, and dimethylarsinic acid DMAsV) to their trivalent form (arsenite AsIII, methylarsonic acid MMAIII, and dimethylarsinous acid DMAIII, respectively), and the addition of GSH to rat liver extracts promoted arsenite methylation (Thomas et al., 2001, 2004; Waters et al., 2004). Both arsenite and its methylated metabolites have been shown to be potent inhibitors of GR in vitro (Styblo et al., 1995, 1997; Chouchane and Snow, 2001). Thus it appears that exposure to arsenicals would compromise the antioxidant mechanisms by consuming GSH and inhibiting the enzyme responsible for its recycling in brain, an organ highly susceptible to oxidative damage. To investigate this hypothesis, we exposed mice to 2.5, 5, or 10 mg/kg/day of sodium arsenite for 9 days and determined the activity and levels of GR, as well as the formation of methylated arsenic metabolites in brain. Several studies have demonstrated that, compared with other organs such as lung and kidney, liver is the primary arsenic-methylating organ (Hughes et al., 2003; Kitchin et al., 1999), and therefore the liver could also be affected by GR inhibition. Thus, we performed similar determinations in liver of GR levels and activity, measuring the disposition of arsenicals, which served as a reference for the metabolic activity observed in brain. The formation of methylated metabolites in brain was further confirmed by organotypic cultures of brain slices.
MATERIALS AND METHODS
Exposure protocol. CD-1 male mice obtained from the animal care facility of the Institute for Neurobiology and the Biomedical Research Institute of the National Autonomous University of Mexico (UNAM) were acclimated for one week to the vivarium conditions. Subsequently, they
One day after the last dose of inorganic arsenic, mice were sacrificed by cervical dislocation, followed by decapitation. Brain and liver were extracted and washed in ice-cold isotonic saline solution to remove debris and blood. For immunoblotting determination, tissues were homogenized in ice-cold 0.1 M phosphate saline buffer (PBS) pH 7.4, aliquoted in the presence of a proteinase inhibitor cocktail (Sigma-Aldrich), and frozen by immersion in liquid nitrogen. For inorganic arsenic and its methylated metabolites, determination tissue was frozen at –80°C until processing.
Organotypic culture. The procedure used was adapted from Stoppini et al. (1991). Briefly, after cervical dislocation, adult mice were plunged into a 70% alcohol solution and decapitated, and the brain was rapidly removed. The brain was placed in a vibratome chamber filled with sterile artificial cerebrospinal fluid (ACSF: 126 mM NaCl; 3 mM KCl; 1 mM MgCl2; 26 mM NaHCO3; 2 mM CaCl2; 10 mM glucose; ascorbic acid 200 μM and thiourea 200 μM; oxygenated at least 1 h before use). Eight 400-μm longitudinal slices were obtained from each brain and stabilized for 1 h in ACSF before culturing. Two slices were then placed on pre-wetted membranes (30-mm diameter; 0.4 μm pore size; Millicell-CM, Millipore, Billerica, MA) in 6-well cluster plates containing 1.1 ml of culture media (50% DMEM + Hepes; 25% horse serum and 25% Hank's solution from GIBCO [Invitrogen, Carlsbad, CA]; 6.5 mg/ml glucose final concentration (Sigma-Aldrich); penicillin and streptomycin (GIBCO); pH 7.2. Sodium arsenite was dissolved in the culture medium to obtain a non-cytotoxic (Styblo et al., 2002) final concentration of 0.1 μM. Petri dishes were placed in an incubator at 36°C with a 5% CO2-enriched atmosphere for 24, 48, and 72 h. Non-treated slices were used as controls. At the end of the experiment, the tissue and culture medium were pooled and assessed for methylated forms of arsenic, as described below.
Determination of methylated arsenic species. Liver and brain homogenates and culture media were used to measure the levels of arsenic species by hydride generation–atomic absorption spectroscopy after column chromatographic separation of inorganic arsenic and its metabolites: monomethylarsenic (MMA), dimethylarsenic (DMA), and trimethylarsenic (TMAO), as described by Hughes et al. (2003).
Glutathione reductase activity assay.To measure GR activity, homogenates were thawed at room temperature and centrifuged at 700 x g for 10 min, after which 20 μl of supernatant was added to quartz cuvettes containing a fresh solution of 0.44 mM GSSG, 0.30 M EDTA, in 0.1 M phosphate buffer—pH 7.0—and 0.036 M NADPH was added just before the enzymatic determination as the starting reagent. The assay was run at 340 nm for 4 min with absorbance readings taken every 30 s. GR activity was estimated using NADPH extinction coefficient of 6.2 mM–1 · cm–1 and expressed as mU/mg of protein. Protein content was determined using the Bradford assay (BioRad Laboratories, Hercules, CA) (Bradford, 1976).
Glutathione reductase immunoblots. Aliquots of homogenates containing equal concentration of proteins (previously determinated by Bradford method) were electrophoresed as described by Laemmli (1970), in 4% stacking gels and 10% running gels in reducing conditions [25 mM b-mercaptoethanol (BioRad) or 10 mM dithiotreitol (BioRad)]. The proteins were visualized by Western blotting. Primary rabbit polyclonal antibodies against yeast and bovine GR developed in our laboratory were used to determine the amount of the enzyme present in brain and liver homogenates. A secondary peroxidase-coupled antibody against rabbit IgG (Santa Cruz Biotechnology, Santa Cruz, CA) was used to reveal the presence of the protein in the membranes. Immunocomplexes were detected with 3',3-diaminobenzidine and hydrogen peroxide. The analysis of the immunoblots was performed using a densitometer AMBIS Optical Image System; Scanalytics. The software used was Quantity One (Bio Rad) version 4.0.
Statistical analysis. The arsenic distribution data of each tissue and in brain slices were evaluated by one-way analysis of variance (ANOVA) using GraphPad Instat, version 3.06 (GraphPad, San Diego, CA). In cases of significant effects (P < 0.05), a Tukey-Kramer Multiple Comparison test was performed. Dose–response effects for the distribution of arsenicals were explored using Spearman's nonparametric correlation with a significance level of P < 0.05. Data of GR activities and GR Western blot bands intensity, analyzed using Quantity One (BioRad) software, were also evaluated by one-way ANOVA followed by a Tukey-Kramer Multiple Comparison test when significant (P < 0.05) effects were observed.
Ethics. The experiments reported in this article were carried out following the guidelines stated in ‘Principles of Laboratory Animal Care’ (NIH publication #85–23, revised 1985) and the ‘Norma Oficial Mexicana de la Secretaría de Agricultura, Ganadería, Desarrollo Rural, Pesca y Alimentación’ (SAGARPA) titled ‘Especificaciones técnicas para la producción, cuidado y uso de los animales de laboratorio’ (Clave NOM-062-ZOO-1999) (published in August 2001).
RESULTS
There were no gross changes in the general appearance among groups exposed to 2.5, 5, or 10 mg/kg/day of sodium arsenite and the control group. Similarly, no differences in body weight were observed among control and treated animals (data not shown). Arsenic species were determined in brain homogenates from five animals per dose [F,F(3,19) = 12.859 – 583.73, P < 0.0001] and in four liver homogenates from four animals per dose [F,F(3,15) = 77.831 – 165.34, P < 0.0001] (Figs. 1A and 1B). Total arsenic (TAs) concentration in tissue samples reported in this article is the sum of the concentrations of inorganic arsenic (As), MMA, DMA, and TMAO. TAs increased linearly with dose in both brain and liver (Fig. 2A). Control mice showed low levels of arsenic, because there are small amounts of As (0.087–0.097 μg/g of inorganic arsenic and no traces of MMA and DMA) in the Harlan 7004 Diet (Harlan, Indianapolis, IN).
Methylated Arsenic Species in Brain and Liver
The distribution of arsenicals was tissue specific: In brain, the concentration of DMA increased linearly with dose (Fig. 1A). Brains from the group exposed to 10 mg of arsenite distinctively showed the highest concentration of DMA. A significant accumulation of MMA was also observed (Fig. 1A), although it was not as evidently related with dose as the dimethylated species was. Inorganic arsenic increased linearly with dose in liver, but a different pattern was observed in brain, because the amount of inorganic arsenic decreased linearly with dose (Fig. 2B). In addition TMAO was detected only in the livers from mice treated with the highest dose of arsenite, but it was not present in brain at all (Fig. 1A).
To confirm that methylation of arsenic was occurring in brain, brain slices were incubated in the presence of 0.1 μM of sodium arsenite. The time course for formation of methylated metabolites is consistent with our observations in vivo (Fig. 3); DMA concentration increased with incubation time, while inorganic arsenic and MMA decreased.
Glutathione Reductase Activity and Western Blot
Arsenite exposure caused significant inhibition of GR activity in brain [F(3, 34) = 3.27, P = 0.03] and liver [F(3, 35) = 3.96, P = 0.015] from mice exposed to 10 mg/kg/day of arsenite compared with controls or to groups exposed to 2.5 and 5 mg/kg/day of arsenite. This inhibition was not associated with a significant reduction in the amount of the enzyme in the homogenates observed in the densitometric analysis of immunoblots (Fig. 4A and 4B).
DISCUSSION
Subchronic oral exposure to sodium arsenite showed a dose-related distribution of As and of its methylated metabolites in brain, as well as in liver, of mice.
Some reports suggest that inorganic arsenic is able to cross the blood–brain barrier (Vahter and Marafante, 1985; Klaassen, 1996; Rodriguez et al., 2001). There is no information available about arsenical species distribution in brain after acute or sustained arsenical exposure. The results of the present study corroborate the entrance of As in brain and demonstrate the presence of the methylated metabolites MMA and DMA in this organ. Also, the proportion of methylated metabolites increased with dose, and this was clearly evident in the DMA species. Thus, at the highest dose of arsenite administered (10 mg/kg/day), the main arsenic methylated species found in brain was DMA (Fig. 1A).
Total As concentration in liver was higher than in brain, although the metabolite DMA was an exception for brain. The group exposed to 10 mg/kg/day of arsenite showed three times the amount of DMA in brain as the amount determined in liver. Selective accumulation of DMA in other organs such as lung and bladder have been also observed in mice in a repeated-dose experiment with sodium arsenate (Hughes et al., 2003). Although inorganic arsenic increased linearly with dose in liver, the opposite pattern was observed in brain (Fig. 2), and a similar dose response effect was observed in rat brain (Rodríguez et al., 2001), suggesting that transport of inorganic arsenic to the brain may be limited by the blood–brain barrier. The presence of MMA and DMA in brain could be the result of blood distribution and/or brain methylation. The formation of MMA and DMA in brain slices showed that brain is capable of methylation of inorganic arsenic. In this respect, a recent study has reported that brain tissue expresses arsenic methyltransferase (Waters et al., 2003) and that enzymatic activity of MMAV reductase was higher in brain than in bladder, spleen, or liver of male golden Syrian hamsters (Sampayo-Reyes et al., 2000). In human cell lines, levels of arsenic methyltransferase mRNA have been correlated with the capacity to convert arsenite into methylated metabolites (Lin et al., 2002; Styblo et al., 2000).
The presence of small amounts of TMAO in liver, the major site of arsenic methylation (Sakurai et al., 2004), suggests that DMA was methylated to TMAO in this tissue, whereas this mechanism apparently was not available to brain DMA, which therefore accumulated. The smaller proportion of MMA present in both tissues in all arsenite-exposed groups suggests that the methylation rate is higher for the first and second methylation steps, whereas the third step might require certain accumulation of DMA or inorganic arsenic to occur. Trivalent methylated arsenicals are considered more cytotoxic than either pentavalent or trivalent inorganic arsenic (Styblo et al., 2002). Although the methodology employed here to determine methylated metabolites cannot ascertain if they are in their trivalent or pentavalent form, trivalent MMA and DMA have been found in humans chronically exposed to arsenic in drinking water (Del Razo et al., 2001). Because of the high affinity of arsenic for sulfhydryl groups, it has been proposed that methylathed arsenicals are in the trivalent state when they become protein bound (Del Razo et al., 2001; Lin et al., 1999). Thus, it can be assumed that methylated trivalent species can be found in liver and brain where they might inhibit important antioxidant enzymes such as GR and GPx, among others. The interaction of As with GSH and related enzymes seems to be a very important factor for the mechanism of arsenical toxicity. GR is an integral component of the antioxidant defense mechanism, and its inhibition by arsenicals, especially by trivalent methylated forms, has been documented in vitro in short-term treatment studies (Styblo and Thomas, 1995; Styblo et al., 1997; Chouchane and Snow, 2001). Our results showed that a significant inhibition of GR in liver and brain occurred in vivo only when the animals
The decrease in GR activity could lead to imbalance in redox status due to decrements in GSH levels in brain. Some studies have tried to correlate alterations in GSH with neurodegenerative disorders such as Alzheimer's disease, amyotrophic lateral sclerosis, schizophrenia, and Parkinson's disease (Bharath et al., 2002; Wullner et al., 1996).
Loss of glutathione and consequent oxidative damage have been suggested to be early signaling events in apoptotic cell death. A decrease in GSH triggers the activation of neuronal 12-lipoxygenase, which leads to the production of peroxides, an influx of calcium, and, ultimately, cell death (Schulz et al., 2000). Thus arsenite exposure caused apoptosis by caspase activation in cortical cell cultures, whereas in cerebellar cell cultures, apoptosis was induced by arsenite or DMA, the former being more toxic (Namgung and Xia, 2000, 2001).
Choline, folate, vitamin B6, and vitamin B12 metabolism interact at the point where homocysteine is converted to methionine, the substrate of methionine adenosyltranferase, to produce S-adenosyl-L-methionine (SAM; Niculescu and Zeisel, 2002), the most important methyl donor in the arsenic biomethylation process (Fig. 5). Biomethylation of arsenic is considered in most mammal species the major route of arsenic biotransformation and elimination (Tice et al., 1997). Studies of animal models of methionine, choline, and folate deficiency reported altered methylation and elimination of arsenic (Marafante and Vather, 1986; Vather and Marafante, 1987; Tice et al., 1997; Spiegelstein et al., 2003). Our results showed, on the one hand, that important arsenic biomethylation activity occurs in mice brain, and that such activity might be impaired by nutritional deficiencies. On the other hand, the toxic effects of arsenic in the nervous system could be attributable not only to the direct action of the more toxic trivalent arsenicals on enzymes (Styblo and Thomas, 1995; Styblo et al., 1997; Chouchane and Snow, 2001), to oxidative stress (García-Chavez et al., 2003), and to a decrease in GR activity (present study), but also to biotransformation of arsenic. Arsenical biotransformation might alter the homeostasis of SAM and its intermediate metabolites, including methionine, choline, folate, and vitamins B6 and B12, some of which are important substrates or cofactors in neurotransmitter metabolism (acetylcholine, monoamines, amino acids) (Zeisel and Blusztajn, 1994; Bottiglieri et al., 2000; Meck and Williams, 2003) or in the development of the CNS (Rodriguez et al., 2002), especially in genetically susceptible organisms (Wlodarczyk et al., 2001). In this respect, one clinical case report described a 16-year-old girl with mental deterioration, paraparesis areflexia, and bilateral Babinski signs caused by exposure to copper acetate arsenite. Laboratory blood tests revealed hyperhomocysteinemia caused by deficiency of 5,10-methylene-tetrahydrofolate reductase (MTHFR), the enzyme that converts 5,10 methylene tetrahydrofolate to 5-methyltetrahydrofolate (Brouwer et al., 1992), a cofactor necessary to for the conversion of homocysteine to methionine (Fig. 5). It is well known that the plasma homocysteine concentration is modulated by nutritional status for folate, vitamin B6, and vitamin B12 (Cuskelly et al., 2001).
Findings from the present study support the notion that arsenic is an environmental toxin that can affect the CNS through several pathways. Arsenic enters the CNS, accumulates, and undergoes biomethylation in the brain. The neurotoxicity of arsenicals could be due to the adverse effects of the parent compound or to the effects of the methylated metabolites on antioxidant enzymes and neurotransmitter metabolism, which could account for the behavioral alterations reported in animal models. More studies are needed to understand the relationship of inorganic arsenic and the methylated arsenic metabolites and their toxic effects in different organs.
ACKNOWLEDGMENTS
Authors wish to thank the technical help of Patricia Guzman and Roxana Navarrete.
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Sección de Toxicología, CINVESTAV-IPN, AP 14-740, 07000 DF, México
ABSTRACT
Inorganic arsenic exposure via drinking water has been associated with cancer and serious injury in various internal organs, as well as with peripheral neuropathy and diverse effects in the nervous system. Alterations in memory and attention processes have been reported in exposed children, whereas adults acutely exposed to high amounts of inorganic arsenic showed impairments in learning, memory, and concentration. Glutathione (GSH) is extensively involved in the metabolism of inorganic arsenic, and both arsenite and its methylated metabolites have been shown to be potent inhibitors of glutathione reductase (GR) in vitro. Brain would be more susceptible to GR inhibition because of the decreased activities of superoxide dismutase (SOD) and catalase reported in this tissue. To investigate whether GR inhibition could be documented in vivo, we determined the activity and levels of GR in brain as well as in liver, the main organ of arsenic metabolism in mice exposed to 2.5, 5, or 10 mg/kg/day of sodium arsenite over a period of 9 days. In contrast to what has been observed in vitro, significant inhibition of the expression and activity of GR was observed only at the highest concentration used (10 mg/kg/day) in both organs. Although the disposition of arsenicals was higher in liver, significant amounts of inorganic and methylated arsenic forms were determined in the brain of exposed animals. The formation of monomethylarsenic (MMA) and dimethylarsenic (DMA) metabolites in the brain was confirmed by incubating brain slices for 24, 48, and 72 h with sodium arsenite.
Key Words: arsenite; biotransformation; glutathione reductase inhibition; brain.
INTRODUCTION
Inorganic arsenic exposure via drinking water has been associated with cancer and serious injury in various internal organs, as well as with peripheral neuropathy and diverse effects in the circulatory and nervous system. Acute intoxication that produces initial gastrointestinal or cardiovascular symptoms can be followed by the delayed onset of central or peripheral nervous system involvement. During subacute or chronic exposure, inorganic arsenic can occasionally result in subclinical or overt peripheral neuropathy (Yip et al., 2002). In children exposed to inorganic arsenic throughout their whole lives, hearing impairment (Bencko and Symon, 1977) and lower scores in verbal intelligence quotient (IQ) have been reported (Calderon et al., 2001; Wasserman et al., 2004). While alterations in memory and attention processes have been reported in adolescents exposed to high levels of inorganic arsenic in well water (Tsai et al., 2003), adults acutely exposed showed impairments in learning, memory, and concentration (Bolla-Wilson and Bleecker, 1987; Franzblau and Lilis, 1989; Morton and Caron, 1989).
Similarly, inorganic arsenic exposure has been related to behavioral alterations in rodents, such as disruption in operant learning (Nagaraja and Desiraju, 1994), alterations in locomotor activity (Chattopadhyay et al., 2002a; Chattopadhyay et al., 2002b; Itoh et al., 1990; Pryor et al., 1983; Rodriguez et al., 2001, 2002), and increases in errors in the delayed alternation task tested in a T maze (Rodriguez et al., 2001, 2002). Some of these alterations could be related to changes in cholinergic, monoaminergic, GABAergic, and glutamatergic systems in several brain regions such as striatum, midbrain, hypothalamus, and hippocampus of rodents exposed to arsenicals (Delgado et al., 2000; Kannan et al., 2001; Mejia et al., 1997; Nagaraja and Desiraju, 1993, 1994; Rodriguez et al., 2001, 2003; Tripathi et al., 1997; Valkonen et al., 1983).
In humans and in many mammalian species exposed to inorganic arsenic, at least four metabolites that can exert toxic effects have been identified in tissues and fluids. They originate in a metabolic process that is carried out in two steps: (1) the reduction of the pentavalent species to trivalency in the presence of glutathione (GSH) or thioredoxin and (2) oxidative methylation, whereby inorganic arsenic is converted to its monomethyl and dimethyl arsenic forms (MMA, DMA, respectively). Thus, both pentavalent and trivalent methylarsenic forms are intermediates or products of this pathway (Lin et al., 2002; Thomas et al., 2004).
Few studies have attempted to unravel the mechanism of toxicity of inorganic arsenic exposure in the central nervous system (CNS). In vitro studies demonstrated that As exposure caused apoptosis in cortical and cerebellar neurons, whereas dimethylarsinic acid exposure induced apoptosis in cerebellar neurons only. In both types of cell cultures, apoptosis was induced by caspase activation (Namgung and Xia, 2000, 2001). A report of an in vivo study demonstrated the formation of hydroxyl radicals in the rat striatum, which were induced by direct infusion of sodium arsenite via a microdialysis probe (García-Chavez et al., 2003). However, there are no previous in vivo studies relating the adverse effects of arsenical exposure to the presence of methylated arsenic species in brain, which could be in to some extent responsible for the neurotoxic alterations reported.
The tripeptide GSH (L--glutamyl-cysteinyl-glycine) is the most important intracellular nonprotein thiol in mammalian cells. It is found at concentrations of 1–3 mM in human and monkey brain cells (Slivka et al., 1987), and it possesses reducing and nucleophilic properties. It can exist in either a reduced (GSH) or oxidized (GSSG) form. Glutathione reductase (GR, EC 1.6.4.2) is an ubiquitous flavoenzyme that catalyzes the NAD(P)H-dependent reduction of GSSG to GSH. This enzyme maintains adequate levels of the cellular GSH pool, which is critical not only for maintaining the cellular redox status by keeping sulfhydryl groups of cytosolic proteins in their reduced form (Dringen et al., 2000) but also because numerous toxic or potentially toxic compounds, including some metals, are either taken up by or removed from the cells by GSH-mediated pathways (Chouchane and Snow, 2001). In comparison to other organs, brain is more susceptible to oxidative damage because of the high oxygen utilization (20% of oxygen consumed by the body), high iron content, presence of unsaturated fatty acids, and decreased activities of detoxifying enzymes such as superoxide dismutase (SOD), catalase, and GR, as compared with kidney and liver (Bharath et al., 2002; Dringen et al., 2000). GSH serves as an antioxidant, reacting with free radicals and organic peroxides; it is also a substrate of glutathione peroxidase (Maher and Schubert, 2000) and a coenzyme of glutathione-S-transferase and thioltransferases among other enzymes (Arrigo, 1999).
Furthermore, GSH is extensively involved in the metabolism of inorganic arsenic, specifically in the reduction of pentavalent arsenicals (arsenate AsV, methylarsonic acid MMAV, and dimethylarsinic acid DMAsV) to their trivalent form (arsenite AsIII, methylarsonic acid MMAIII, and dimethylarsinous acid DMAIII, respectively), and the addition of GSH to rat liver extracts promoted arsenite methylation (Thomas et al., 2001, 2004; Waters et al., 2004). Both arsenite and its methylated metabolites have been shown to be potent inhibitors of GR in vitro (Styblo et al., 1995, 1997; Chouchane and Snow, 2001). Thus it appears that exposure to arsenicals would compromise the antioxidant mechanisms by consuming GSH and inhibiting the enzyme responsible for its recycling in brain, an organ highly susceptible to oxidative damage. To investigate this hypothesis, we exposed mice to 2.5, 5, or 10 mg/kg/day of sodium arsenite for 9 days and determined the activity and levels of GR, as well as the formation of methylated arsenic metabolites in brain. Several studies have demonstrated that, compared with other organs such as lung and kidney, liver is the primary arsenic-methylating organ (Hughes et al., 2003; Kitchin et al., 1999), and therefore the liver could also be affected by GR inhibition. Thus, we performed similar determinations in liver of GR levels and activity, measuring the disposition of arsenicals, which served as a reference for the metabolic activity observed in brain. The formation of methylated metabolites in brain was further confirmed by organotypic cultures of brain slices.
MATERIALS AND METHODS
Exposure protocol. CD-1 male mice obtained from the animal care facility of the Institute for Neurobiology and the Biomedical Research Institute of the National Autonomous University of Mexico (UNAM) were acclimated for one week to the vivarium conditions. Subsequently, they
One day after the last dose of inorganic arsenic, mice were sacrificed by cervical dislocation, followed by decapitation. Brain and liver were extracted and washed in ice-cold isotonic saline solution to remove debris and blood. For immunoblotting determination, tissues were homogenized in ice-cold 0.1 M phosphate saline buffer (PBS) pH 7.4, aliquoted in the presence of a proteinase inhibitor cocktail (Sigma-Aldrich), and frozen by immersion in liquid nitrogen. For inorganic arsenic and its methylated metabolites, determination tissue was frozen at –80°C until processing.
Organotypic culture. The procedure used was adapted from Stoppini et al. (1991). Briefly, after cervical dislocation, adult mice were plunged into a 70% alcohol solution and decapitated, and the brain was rapidly removed. The brain was placed in a vibratome chamber filled with sterile artificial cerebrospinal fluid (ACSF: 126 mM NaCl; 3 mM KCl; 1 mM MgCl2; 26 mM NaHCO3; 2 mM CaCl2; 10 mM glucose; ascorbic acid 200 μM and thiourea 200 μM; oxygenated at least 1 h before use). Eight 400-μm longitudinal slices were obtained from each brain and stabilized for 1 h in ACSF before culturing. Two slices were then placed on pre-wetted membranes (30-mm diameter; 0.4 μm pore size; Millicell-CM, Millipore, Billerica, MA) in 6-well cluster plates containing 1.1 ml of culture media (50% DMEM + Hepes; 25% horse serum and 25% Hank's solution from GIBCO [Invitrogen, Carlsbad, CA]; 6.5 mg/ml glucose final concentration (Sigma-Aldrich); penicillin and streptomycin (GIBCO); pH 7.2. Sodium arsenite was dissolved in the culture medium to obtain a non-cytotoxic (Styblo et al., 2002) final concentration of 0.1 μM. Petri dishes were placed in an incubator at 36°C with a 5% CO2-enriched atmosphere for 24, 48, and 72 h. Non-treated slices were used as controls. At the end of the experiment, the tissue and culture medium were pooled and assessed for methylated forms of arsenic, as described below.
Determination of methylated arsenic species. Liver and brain homogenates and culture media were used to measure the levels of arsenic species by hydride generation–atomic absorption spectroscopy after column chromatographic separation of inorganic arsenic and its metabolites: monomethylarsenic (MMA), dimethylarsenic (DMA), and trimethylarsenic (TMAO), as described by Hughes et al. (2003).
Glutathione reductase activity assay.To measure GR activity, homogenates were thawed at room temperature and centrifuged at 700 x g for 10 min, after which 20 μl of supernatant was added to quartz cuvettes containing a fresh solution of 0.44 mM GSSG, 0.30 M EDTA, in 0.1 M phosphate buffer—pH 7.0—and 0.036 M NADPH was added just before the enzymatic determination as the starting reagent. The assay was run at 340 nm for 4 min with absorbance readings taken every 30 s. GR activity was estimated using NADPH extinction coefficient of 6.2 mM–1 · cm–1 and expressed as mU/mg of protein. Protein content was determined using the Bradford assay (BioRad Laboratories, Hercules, CA) (Bradford, 1976).
Glutathione reductase immunoblots. Aliquots of homogenates containing equal concentration of proteins (previously determinated by Bradford method) were electrophoresed as described by Laemmli (1970), in 4% stacking gels and 10% running gels in reducing conditions [25 mM b-mercaptoethanol (BioRad) or 10 mM dithiotreitol (BioRad)]. The proteins were visualized by Western blotting. Primary rabbit polyclonal antibodies against yeast and bovine GR developed in our laboratory were used to determine the amount of the enzyme present in brain and liver homogenates. A secondary peroxidase-coupled antibody against rabbit IgG (Santa Cruz Biotechnology, Santa Cruz, CA) was used to reveal the presence of the protein in the membranes. Immunocomplexes were detected with 3',3-diaminobenzidine and hydrogen peroxide. The analysis of the immunoblots was performed using a densitometer AMBIS Optical Image System; Scanalytics. The software used was Quantity One (Bio Rad) version 4.0.
Statistical analysis. The arsenic distribution data of each tissue and in brain slices were evaluated by one-way analysis of variance (ANOVA) using GraphPad Instat, version 3.06 (GraphPad, San Diego, CA). In cases of significant effects (P < 0.05), a Tukey-Kramer Multiple Comparison test was performed. Dose–response effects for the distribution of arsenicals were explored using Spearman's nonparametric correlation with a significance level of P < 0.05. Data of GR activities and GR Western blot bands intensity, analyzed using Quantity One (BioRad) software, were also evaluated by one-way ANOVA followed by a Tukey-Kramer Multiple Comparison test when significant (P < 0.05) effects were observed.
Ethics. The experiments reported in this article were carried out following the guidelines stated in ‘Principles of Laboratory Animal Care’ (NIH publication #85–23, revised 1985) and the ‘Norma Oficial Mexicana de la Secretaría de Agricultura, Ganadería, Desarrollo Rural, Pesca y Alimentación’ (SAGARPA) titled ‘Especificaciones técnicas para la producción, cuidado y uso de los animales de laboratorio’ (Clave NOM-062-ZOO-1999) (published in August 2001).
RESULTS
There were no gross changes in the general appearance among groups exposed to 2.5, 5, or 10 mg/kg/day of sodium arsenite and the control group. Similarly, no differences in body weight were observed among control and treated animals (data not shown). Arsenic species were determined in brain homogenates from five animals per dose [F,F(3,19) = 12.859 – 583.73, P < 0.0001] and in four liver homogenates from four animals per dose [F,F(3,15) = 77.831 – 165.34, P < 0.0001] (Figs. 1A and 1B). Total arsenic (TAs) concentration in tissue samples reported in this article is the sum of the concentrations of inorganic arsenic (As), MMA, DMA, and TMAO. TAs increased linearly with dose in both brain and liver (Fig. 2A). Control mice showed low levels of arsenic, because there are small amounts of As (0.087–0.097 μg/g of inorganic arsenic and no traces of MMA and DMA) in the Harlan 7004 Diet (Harlan, Indianapolis, IN).
Methylated Arsenic Species in Brain and Liver
The distribution of arsenicals was tissue specific: In brain, the concentration of DMA increased linearly with dose (Fig. 1A). Brains from the group exposed to 10 mg of arsenite distinctively showed the highest concentration of DMA. A significant accumulation of MMA was also observed (Fig. 1A), although it was not as evidently related with dose as the dimethylated species was. Inorganic arsenic increased linearly with dose in liver, but a different pattern was observed in brain, because the amount of inorganic arsenic decreased linearly with dose (Fig. 2B). In addition TMAO was detected only in the livers from mice treated with the highest dose of arsenite, but it was not present in brain at all (Fig. 1A).
To confirm that methylation of arsenic was occurring in brain, brain slices were incubated in the presence of 0.1 μM of sodium arsenite. The time course for formation of methylated metabolites is consistent with our observations in vivo (Fig. 3); DMA concentration increased with incubation time, while inorganic arsenic and MMA decreased.
Glutathione Reductase Activity and Western Blot
Arsenite exposure caused significant inhibition of GR activity in brain [F(3, 34) = 3.27, P = 0.03] and liver [F(3, 35) = 3.96, P = 0.015] from mice exposed to 10 mg/kg/day of arsenite compared with controls or to groups exposed to 2.5 and 5 mg/kg/day of arsenite. This inhibition was not associated with a significant reduction in the amount of the enzyme in the homogenates observed in the densitometric analysis of immunoblots (Fig. 4A and 4B).
DISCUSSION
Subchronic oral exposure to sodium arsenite showed a dose-related distribution of As and of its methylated metabolites in brain, as well as in liver, of mice.
Some reports suggest that inorganic arsenic is able to cross the blood–brain barrier (Vahter and Marafante, 1985; Klaassen, 1996; Rodriguez et al., 2001). There is no information available about arsenical species distribution in brain after acute or sustained arsenical exposure. The results of the present study corroborate the entrance of As in brain and demonstrate the presence of the methylated metabolites MMA and DMA in this organ. Also, the proportion of methylated metabolites increased with dose, and this was clearly evident in the DMA species. Thus, at the highest dose of arsenite administered (10 mg/kg/day), the main arsenic methylated species found in brain was DMA (Fig. 1A).
Total As concentration in liver was higher than in brain, although the metabolite DMA was an exception for brain. The group exposed to 10 mg/kg/day of arsenite showed three times the amount of DMA in brain as the amount determined in liver. Selective accumulation of DMA in other organs such as lung and bladder have been also observed in mice in a repeated-dose experiment with sodium arsenate (Hughes et al., 2003). Although inorganic arsenic increased linearly with dose in liver, the opposite pattern was observed in brain (Fig. 2), and a similar dose response effect was observed in rat brain (Rodríguez et al., 2001), suggesting that transport of inorganic arsenic to the brain may be limited by the blood–brain barrier. The presence of MMA and DMA in brain could be the result of blood distribution and/or brain methylation. The formation of MMA and DMA in brain slices showed that brain is capable of methylation of inorganic arsenic. In this respect, a recent study has reported that brain tissue expresses arsenic methyltransferase (Waters et al., 2003) and that enzymatic activity of MMAV reductase was higher in brain than in bladder, spleen, or liver of male golden Syrian hamsters (Sampayo-Reyes et al., 2000). In human cell lines, levels of arsenic methyltransferase mRNA have been correlated with the capacity to convert arsenite into methylated metabolites (Lin et al., 2002; Styblo et al., 2000).
The presence of small amounts of TMAO in liver, the major site of arsenic methylation (Sakurai et al., 2004), suggests that DMA was methylated to TMAO in this tissue, whereas this mechanism apparently was not available to brain DMA, which therefore accumulated. The smaller proportion of MMA present in both tissues in all arsenite-exposed groups suggests that the methylation rate is higher for the first and second methylation steps, whereas the third step might require certain accumulation of DMA or inorganic arsenic to occur. Trivalent methylated arsenicals are considered more cytotoxic than either pentavalent or trivalent inorganic arsenic (Styblo et al., 2002). Although the methodology employed here to determine methylated metabolites cannot ascertain if they are in their trivalent or pentavalent form, trivalent MMA and DMA have been found in humans chronically exposed to arsenic in drinking water (Del Razo et al., 2001). Because of the high affinity of arsenic for sulfhydryl groups, it has been proposed that methylathed arsenicals are in the trivalent state when they become protein bound (Del Razo et al., 2001; Lin et al., 1999). Thus, it can be assumed that methylated trivalent species can be found in liver and brain where they might inhibit important antioxidant enzymes such as GR and GPx, among others. The interaction of As with GSH and related enzymes seems to be a very important factor for the mechanism of arsenical toxicity. GR is an integral component of the antioxidant defense mechanism, and its inhibition by arsenicals, especially by trivalent methylated forms, has been documented in vitro in short-term treatment studies (Styblo and Thomas, 1995; Styblo et al., 1997; Chouchane and Snow, 2001). Our results showed that a significant inhibition of GR in liver and brain occurred in vivo only when the animals
The decrease in GR activity could lead to imbalance in redox status due to decrements in GSH levels in brain. Some studies have tried to correlate alterations in GSH with neurodegenerative disorders such as Alzheimer's disease, amyotrophic lateral sclerosis, schizophrenia, and Parkinson's disease (Bharath et al., 2002; Wullner et al., 1996).
Loss of glutathione and consequent oxidative damage have been suggested to be early signaling events in apoptotic cell death. A decrease in GSH triggers the activation of neuronal 12-lipoxygenase, which leads to the production of peroxides, an influx of calcium, and, ultimately, cell death (Schulz et al., 2000). Thus arsenite exposure caused apoptosis by caspase activation in cortical cell cultures, whereas in cerebellar cell cultures, apoptosis was induced by arsenite or DMA, the former being more toxic (Namgung and Xia, 2000, 2001).
Choline, folate, vitamin B6, and vitamin B12 metabolism interact at the point where homocysteine is converted to methionine, the substrate of methionine adenosyltranferase, to produce S-adenosyl-L-methionine (SAM; Niculescu and Zeisel, 2002), the most important methyl donor in the arsenic biomethylation process (Fig. 5). Biomethylation of arsenic is considered in most mammal species the major route of arsenic biotransformation and elimination (Tice et al., 1997). Studies of animal models of methionine, choline, and folate deficiency reported altered methylation and elimination of arsenic (Marafante and Vather, 1986; Vather and Marafante, 1987; Tice et al., 1997; Spiegelstein et al., 2003). Our results showed, on the one hand, that important arsenic biomethylation activity occurs in mice brain, and that such activity might be impaired by nutritional deficiencies. On the other hand, the toxic effects of arsenic in the nervous system could be attributable not only to the direct action of the more toxic trivalent arsenicals on enzymes (Styblo and Thomas, 1995; Styblo et al., 1997; Chouchane and Snow, 2001), to oxidative stress (García-Chavez et al., 2003), and to a decrease in GR activity (present study), but also to biotransformation of arsenic. Arsenical biotransformation might alter the homeostasis of SAM and its intermediate metabolites, including methionine, choline, folate, and vitamins B6 and B12, some of which are important substrates or cofactors in neurotransmitter metabolism (acetylcholine, monoamines, amino acids) (Zeisel and Blusztajn, 1994; Bottiglieri et al., 2000; Meck and Williams, 2003) or in the development of the CNS (Rodriguez et al., 2002), especially in genetically susceptible organisms (Wlodarczyk et al., 2001). In this respect, one clinical case report described a 16-year-old girl with mental deterioration, paraparesis areflexia, and bilateral Babinski signs caused by exposure to copper acetate arsenite. Laboratory blood tests revealed hyperhomocysteinemia caused by deficiency of 5,10-methylene-tetrahydrofolate reductase (MTHFR), the enzyme that converts 5,10 methylene tetrahydrofolate to 5-methyltetrahydrofolate (Brouwer et al., 1992), a cofactor necessary to for the conversion of homocysteine to methionine (Fig. 5). It is well known that the plasma homocysteine concentration is modulated by nutritional status for folate, vitamin B6, and vitamin B12 (Cuskelly et al., 2001).
Findings from the present study support the notion that arsenic is an environmental toxin that can affect the CNS through several pathways. Arsenic enters the CNS, accumulates, and undergoes biomethylation in the brain. The neurotoxicity of arsenicals could be due to the adverse effects of the parent compound or to the effects of the methylated metabolites on antioxidant enzymes and neurotransmitter metabolism, which could account for the behavioral alterations reported in animal models. More studies are needed to understand the relationship of inorganic arsenic and the methylated arsenic metabolites and their toxic effects in different organs.
ACKNOWLEDGMENTS
Authors wish to thank the technical help of Patricia Guzman and Roxana Navarrete.
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