400
Chem. Res. Toxicol. 2008, 21, 400–407
Reversal of Arsenic-Induced Hepatic Apoptosis with Combined Administration of DMSA and Its Analogues in Guinea Pigs: Role of Glutathione and Linked Enzymes Deepshikha Mishra, Ashish Mehta, and Swaran J. S. Flora* DiVision of Pharmacology and Toxicology, Defence Research and DeVelopment Establishment, Jhansi Road, Gwalior-474 002, India ReceiVed September 5, 2007
Arsenicosis, due to contaminated drinking water in the Indo-Bangladesh region, is a serious health hazard in terms of morbidity and mortality. Reactive oxygen species (ROS) generated due to arsenic toxicity have been attributed as one of the initial signals that impart cellular toxicity, which is controlled by the internal antioxidant glutathione (GSH). In the present study, we investigated (i) the role of GSH and its linked enzymes, glutathione peroxidase and glutathione reductase, in reversing chronic arsenic toxicity using a thiol chelating agent, meso-2,3-dimercaptosuccinic acid (DMSA), or one of its analogues individually or in combination; (ii) if alterations in the carbon side chain of DMSA increased efficacy; and (iii) whether the combination therapy enhance arsenic removal from hepatic tissue and prevent hepatic apoptosis. Results indicated that chronic arsenic exposure led to a ROS-mediated, mitochondrial-driven, caspase-dependent apoptosis in hepatic cells with a significant increase in glutathione disulfide (GSSG) levels and decreased glutathione reductase levels. Monotherapy with DMSA and its analogues did show minimal recovery postchelation. However, the combination of DMSA with long carbon chain analogues like monoisoamyl DMSA (MiADMSA) or monocyclohexyl DMSA (MchDMSA) showed a better efficacy in terms of reducing the arsenic burden as well as reversing altered biochemical variables indicative of oxidative stress and apoptosis. We also observed that GSH and its linked enzymes, especially glutathione reductase, play a vital role in scavenging ROS, maintaining GSH pools, and providing clinical recoveries. On the basis of the above observations, we recommend that combinational therapy of DMSA and its long carbon chain analogues MiADMSA or MchDMSA would be more effective in arsenic toxicity. Introduction Arsenic is a metalloid that is ubiquitously present in water, soil, and air from natural and anthropogenic sources. Arsenic, which is found in several different chemical and oxidation states (-3, 0, +3, and +5), causes acute and chronic adverse health effects (1). Over 200 million people worldwide are at risk, out of which 112 million are residing in West Bengal, India (2), and Bangladesh, areas where groundwater arsenic concentrations exceed the World Health Organization maximum permissible level of 50 µg/L (3). Arsenic is known to interfere with a number of bodily functions of the central nervous system, hematopoietic system, liver, and kidneys (4–6) and has also been associated with cancer (7). The tripeptide GSH1 (L-γ-glutamyl-cysteinyl-glycine) is the most important intracellular nonprotein thiol in mammalian cells (8). GSH possesses reducing and nucleophilic properties and can exist in either reduced (GSH) or oxidized (GSSG) form (9). The maintenance of an adequate cellular GSH pool is not only critical for normal cellular redox status (10) but also for the removal of numerous potentially toxic compounds, including * To whom correspondence should be addressed. Tel: +91 751 2344301. Fax: +91 751 2341148. E-mail:
[email protected] or sjsflora@ drde.drdo.in. 1 Abbreviations: DMSA, meso-2,3-dimercaptosuccinic acid; MiADMSA, monoisoamyl DMSA; MchDMSA, monocyclohexyl DMSA; MmDMSA, monomethyl DMSA; ROS, reactive oxygen species; MMP, mitochondria membrane potential; SOD, superoxide dismutase; GSH, glutathione; GSSG, glutathione disulfide; GPx, glutathione peroxidase; GR, glutathione reductase; TBARS, thiobarbituric acid reactive substances; ALT, alanine aminotransaminase; AST, aspartate aminotransaminase; CAT, catalase.
metals, from the cells via GSH-mediated pathways (11, 12). This GSH pool is mainly controlled by the conversion of GSSG to GSH by an ubiquitous flavoenzyme, glutathione reductase (GR). Glutathione is extensively involved in the metabolism of inorganic arsenic, specifically in the reduction of pentavalent arsenicals to their trivalent form, and experiments have shown that the addition of GSH to rat liver extracts promoted arsenite methylation (13, 14). Both arsenite and its methylated metabolites have been known to be potent inhibitors of GR in vitro (15–17); thus, exposure to arsenicals would compromise the antioxidant mechanisms by consuming GSH and inhibiting the enzyme responsible for its recycling and may eventually lead to cell death. Few studies have attempted to unravel the mechanism of arsenic toxicity in vitro studies and have demonstrated that arsenic exposure causes apoptosis via caspase activations (18, 19). Recently, Santra et al. (20) also demonstrated mitochondrial-dependent apoptosis in mouse liver after arsenic treatment. Chelation therapy with 2,3-dimercaprol (BAL) has been the basis for the medical treatment of arsenic poisoning, but the serious side effects of BAL have opened new avenues in this area. meso-2,3-dimercaptosuccinic acid (DMSA) has been tried successfully in animals (21, 22) as well as in a few cases of human arsenic poisoning (23). In an experimental study, we provided in vivo evidence of oxidative stress in a number of major organs of arsenic-exposed rats and showed that these effects can be mitigated by pharmacological intervention that encompasses combined treatment with N-acetylcysteine and DMSA (24). However, a double blind, randomized, controlled
10.1021/tx700315a CCC: $40.75 2008 American Chemical Society Published on Web 12/29/2007
Therapy for Arsenic-Induced Hepatic Apoptosis
Figure 1. Structure of chelating agents (A) DMSA, (B) MiADMSA, (C) MChDMSA, and (D) MmDMSA.
trial study conducted on patients from arsenic-affected West Bengal (India) regions with oral administration of DMSA suggested that it was not effective in producing any clinical or biochemical benefits or any histopathological improvements of skin lesions (25). To achieve optimal chelation as compared to DMSA, a large number of esters of DMSA have been synthesized. These esters are mainly the mono- and dimethyl esters of DMSA that have been studied experimentally and have shown better potential in mobilizing arsenic from tissues in mice (22, 26). The present study was planned (i) to address the role of glutathione and linked enzymes in inducing oxidative stress and its recovery by chelation therapy; (ii) to determine if alternations in the carbon side chain of DMSA could help in achieving better clinical recoveries; (iii) to determine if combined chelation of succimer (DMSA) (Figure 1A), which is a hydrophilic chelator, and its analogue [monoisoamyl DMSA (MiADMSA), monocyclohexyl DMSA (MchDMSA), and monomethyl DMSA (MmDMSA)] (Figure 1B,C,D), which are lipophilic in nature and presently have a minimal clinical role, could be better options for the treatment of chronic arsenic poisoning; (iv) to determine whether combination therapy mobilizes arsenic from the target organ; and (v) to determine whether biochemical variables indicative of oxidative stress and cell death are recovered.
Experimental Procedures Chemicals and Reagents. DMSA was procured from Sigma (St. Louis, MO), while sodium arsenite was obtained from Merck
Chem. Res. Toxicol., Vol. 21, No. 2, 2008 401 (Germany). All other analytical laboratory chemicals and reagents were purchased from Merck (Germany), Sigma, or BDH Chemicals. Ultrapure water prepared by Millipore (New Delhi, India) was used throughout the experiment to avoid metal contamination and for the preparation of reagents/buffers used for various biochemical assays in our study. MiADMSA, MchDMSA, and MmDMSA were synthesized in our synthetic chemistry division, by the controlled esterification of DMSA, with their corresponding alcohol in acidic medium (27). The product was purified (purity 99.9%) and characterized using spectral and analytical methods before experimentation. The chemicals were stored in desiccators at 4 °C to avoid oxidation and thermal decomposition. DMSA and MiADMSA were dissolved in 5% sodium bicarbonate solutions, respectively. All of the antidote solutions were prepared immediately before use. The dosing volume amounted to 4 mL/kg body weight. Animal and Treatments. All of the experiments were performed on male guinea pigs weighing 400 ( 50 g. Animals were obtained from the animal house facility of the Defense Research and Development Establishment (DRDE) (Gwalior). The animal ethical committee of DRDE (Gwalior, India) approved the protocols for the experiments. Prior to dosing, they were acclimatized for 7 days to light from 0600 to 1800 h alternating with 12 h of darkness. The animals were housed in stainless steel cages in an airconditioned room with the temperature maintained at 25 ( 2 °C. The guinea pigs were allowed standard animal’s chow diet (Lipton, India; metal content of diet, in ppm dry weight: Zn, 45; Cu, 10; Mn, 55; Fe, 70; and Co, 5) throughout the experiment. Forty-five animals were randomized into two groups and were treated as indicated for a period of 4 months: group I, no treatment (drinking water) (n ) 5); and group II, 25 ppm sodium arsenite (in drinking water) (n ) 40). After 4 months, arsenic-exposed animals were randomly divided into eight groups and given the following treatments consecutively for 5 days: group IIA, saline (n ) 5); group IIB, DMSA (0.3 mM/kg, orally, once daily; n ) 5); group IIC, MiADMSA (0.3 mM/kg, orally, once daily; n ) 5); group IID, MchDMSA (0.3 mM/kg, orally, once daily; n ) 5); group IIE, MmDMSA (0.3 mM/kg, orally, once daily; n ) 5); group IIF, DMSA (0.15 mM/kg, orally, once daily) + MiADMSA (0.15 mM/kg, ip, once daily; n ) 5); group IIG, DMSA (0.15 mM/kg, orally, once daily) + MchDMSA (0.15 mM/kg, ip, once daily; n ) 5); and group IIH, DMSA (0.15 mM/kg, orally, once daily) + MmDMSA (0.15 mM/kg, ip, once daily; n ) 5). Arsenic exposure was stopped during chelation therapy. The selection of the doses of each of the chelators used in the study was based on our previous studies. These doses were at physiologically attainable concentrations. At the end of treatment, all animals were sacrificed under light ether anesthesia and blood was collected through intracardiac puncture for serum separation. Liver tissue samples were removed and washed with normal saline, and all the extraneous materials were removed before weighing. Tissues were kept at ice-cooled conditions at all times. Reactive Oxygen Species (ROS). The amount of ROS in the liver was measured using 2′,7′-dichlrofluorescin diacetate (DCFDA), which gets converted into highly fluorescent DCF by cellular peroxides (including hydrogen peroxide). The assay was performed as described by Socci et al. (28). Briefly, 1% liver homogenate was prepared in ice-cold 40 mM Tris-HCl buffer (pH 7.4), and this was further diluted to 0.25% with the same buffer (40 mM Tris-HCl, pH 7.4) and placed on ice. The samples were divided into two equal fractions (2 mL each). In one fraction, 40 µL of 1.25 mM DCF-DA prepared in methanol was added for ROS estimation, whereas only 40 µL of methanol was added to the other fraction that served as a control for tissue autofluorescence. All samples were incubated for 15 min in a 37 °C water bath. The fluorescence was determined at 488 nm excitation and 525 nm emission using a fluorescence reader (Perkin-Elmer, LS-55, United Kingdom). Liver ROS readings were expressed as arbitrary fluorescence intensity units (FIU at 530 nm). Mitochondrial Membrane Potential (MMP) (∆Ψm). The MMP was measured using a JC-1 probe as described previously
402 Chem. Res. Toxicol., Vol. 21, No. 2, 2008 with minor modifications (29). Briefly, liver homogenate was centrifuged at 800g for 5 min, and the supernatant was collected, which was again recentrifuged at 1000g for 10 min. An equal number of cells from each group was taken after counting and incubated for 10 min with 10 µM JC-1 at 37 °C. The cells were washed and resuspended in phosphate-buffered saline, and the fluorescence was measured. Subsequently, the changes in fluorescence were monitored at two different wavelengths with excitation at 485 nm and with emission at 530 nm and the other excitation at 535 nm and emission at 590 nm. The ratio of the reading at 590 nm to the reading at 530 nm (590:530 ratio) was considered as the relative ∆Ψm value. RNA Extraction and RT-PCR. Liver tissue RNA was extracted from different treated groups using the TRIzol method. One microgram of RNA was converted to cDNA using superscript reverse transcriptase. PCR was performed with the initial denaturation cycle at 94 °C for 5 min, followed by 35 cycles of 94 °C for 30 s, 60 °C for 30 s, 72 °C for 60 s, and followed by final extension at 72 °C for 5 min. The PCR-amplified products were then subjected to electrophoresis on 1.5% agarose gels. The primers used for the study were bcl2 (sense, CTT TGT GGA ACT GTA CGG CCC CAG CAT GCG; antisense, ACA GCC TGC AGC TTT GTT TCA TGG TAC ATC; 231 bps; accession #M16506), bax (sense, GGG AAT TCT GGA GCT GCA GAG GAT GAT T; antisense, GCG GAT CCA AGT TGC CAT CAG CAA ACA T; 96 bps; accession #L22472), and β-actin (sense, TAT GGA GAA GAT TTG GCA CC; antisense, GTC CAG ACG CAG GAT GGC AT; 300 bps; accession #J00691). The band intensities were further analyzed using Image J software, and values were then normalized using β-actin as an internal control to plot the graphs. Caspase 3. The caspase 3 assay was performed using the Caspase 3 Colorimetric Assay Kit per the manufacturer’s instructions (Sigma, United States). Briefly, the single cell suspension was prepared as described above, and an equal number of cells was taken after counting. The cells were centrifuged, and the pellet was suspended in 1× lysis buffer and incubated on ice for 15 min, followed by centrifugation at 20000g for 15 min at 4 °C. The supernatant was collected. Samples were mixed with assay buffer with or without caspase 3 inhibitor (Ac-DEVD-CHO) for control. Finally, caspase 3 substrate (Ac-DEVD-pNA) was added to all of the samples and incubated at 37 °C for 60 min. The absorbance was recorded at 405 nm. The data are a representation of three experiments. Alanine Aminotransferase (ALT) and Aspartate Aminotransferase (AST). Serum ALT and AST were measured following the method of Flora et al. (30). Briefly, the assay system contained 1 mL of buffer/substrate solution and 0.2 mL of serum and was incubated for exactly 60 min for ALT and 30 min for AST at 37 °C in a water bath. One milliliter of chromogen solution was added, mixed, and allowed to stand for 20 min at room temperature, and 10 mL of a 0.4 N NaOH was added subsequently. The extinction was read at 505 nm against a blank. The controls were run in parallel, and the substrate was added after deproteinization. Glutathione Peroxidase (GPx) and GR. The GPx activity was measured as described in detail in our earlier publication (31). The reaction mixture contained 0.3 mL of phosphate buffer (0.1 M, pH 7.4), 0.2 mL of GSH (2 mM), 0.1 mL of sodium azide (10 mM), 0.1 mL of H2O2 (1 mM), and 0.3 mL of supernatant [as prepared for measuring the superoxide dismutase (SOD) activity]. The complete mixture was incubated at 37 °C for 15 min, and the reaction was terminated by adding 0.5 mL of 5% TCA. Tubes were centrifuged at 1500g for 5 min, and the supernatant was collected. Two hundred microliters of phosphate buffer (0.1 M, pH 7.4) and 0.7 mL of DTNB (0.4 mg/mL) were added to 0.1 mL of reaction supernatant. After they were mixed, the absorbance was recorded at 420 nm. To measure the GR activity (31), homogenates were thawed at room temperature and centrifuged at 700g for 10 min. Twelve microliters of supernatant was added to quartz cuvettes containing a fresh solution of 0.44 mM GSSG and 0.30 M EDTA in 0.1 M phosphate buffer (pH 7.0), and 0.036 M NADPH was added just
Mishra et al. 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. Hepatic Reduced and Oxidized Glutathione Level. Liver GSH and GSSG estimation were performed as described in our previous paper (32). Briefly, 0.25 g of tissue sample was homogenized on ice with 3.75 mL of 0.1 M phosphate-0.005 M EDTA buffer (pH 8.0) and 1 mL of 25% HPO3, which was used as a protein precipitant. The homogenate (4.7 mL) was centrifuged at 100000g for 30 min at 4 °C. For the GSH assay, 0.5 mL of supernatant and 4.5 mL of phosphate buffer (pH 8.0) were mixed. The final assay mixture (2.0 mL) contained 100 µL of supernatant, 1.8 mL of phosphate-EDTA buffer, and 100 µL of O-phthaldehyde (OPT; 1000 µL/mL in absolute methanol, prepared fresh). After they were mixed, the fluorescence was determined at 420 nm with an excitation wavelength of 350 nm using a spectrofluorometer (PerkinElmer, LS-55, United Kingdom). For the GSSG assay, 0.5 mL of supernatant was incubated at room temperature with 200 µL of 0.04 mol/L N-ethylmaleimide solution for 30 min. To this mixture, 4.3 mL of 0.1 mol/L NaOH was added. A 100 µL sample of this mixture was taken for the measurement of GSSG using the procedure described above for the GSH assay, except that 0.1 mol/L NaOH was used as the diluents instead of phosphate buffer (32). Catalase (CAT) Activity. The CAT activity was assayed following the procedure of Pande and Flora (33) at room temperature. A 100 µL amount of tissue extract was placed on an ice bath for 30 min, and then, for another 30 min at room temperature, 10 µL of Triton-X100 was added to each tube. In a cuvette containing 200 µL of phosphate buffer and 50 µL of tissue extract was added 250 µL of 0.066 M H2O2 (in phosphate buffer); a decrease in the optical density was measured at 240 nm for 60 s. The molar extinction coefficient of 43.6 M cm-1 was used to determine the CAT activity. One unit of activity was equal to the mol of H2O2 degraded/min/mg protein. Thiobarbituric Acid Reactive Substances (TBARS) and SOD Activity. Hepatic TBARS and SOD were estimated by the methods described in detail in our earlier publication (31). Elemental Analysis. The arsenic concentrations in liver and urine were measured after wet acid digestion using a microwave digestion system (Anton Paar Multiwave 3000, Austria, Europe). Samples were brought to a constant volume, and determination of tissue arsenic was performed using an autosampler (AS-72) and graphite furnace (MHS) fitted with an atomic absorption spectrophotometer (AAS, Perkin-Elmer model AAnalyst 100). Statistical Analysis. Data were expressed as means ( SEM. Data comparisons were carried out using one-way analysis of variance followed by Tukey’s post-test to compare means between the different treatment groups. The difference (with or without chelation) with a p value
