TY - JOUR
AU - Carter, Dean E.
AB - Abstract The mechanism of arsine (AsH3) toxicity is not completely understood. The first cytotoxic effect of AsH3 is disruption of ion homeostasis, with a subsequent hemolytic action. The only accepted treatment for AsH3 toxicity is exchange transfusion of the blood. In this study the effect of sulfur, sulfur compounds, thiol-containing compounds, and thiol inhibitors on AsH3-induced disruption of membrane transport and hemolysis in human erythrocytes was investigated in vitro. Elemental sulfur, sodium thiosulfate, 5,5′-dithio-bis(2-nitrobenzoic acid), and meso-2,3-dimercaptosuccinic acid were successful in delaying hemolysis, but the most successful agent was the sulfhydryl inhibitor, N-ethylmaleimide (NEM). This indicated that sulfhydryl groups, possibly membrane sulfhydryls, are major factors in the hemolytic mechanism of AsH3. Measuring intracellular ion concentrations tested the effect of NEM on AsH3-induced disruption of membrane transport. AsH3 alone caused all ions tested to flow with their concentration gradients: Intracellular K+ and Mg++ decreased, whereas Na+, Cl–, and Ca++ increased. NEM was unable to prevent ion loss except for Ca++, whose increase was prevented for 1 h after AsH3 treatment. The influx of Ca++ in AsH3-treated erythrocytes is an irreversible event leading to hemolysis. Reduction of oxygenated hemoglobin to carboxyhemoglobin completely inhibited AsH3-induced hemolysis. In addition, AsH3 and NEM had no direct chemical interactions. We concluded that membrane sulfhydryl groups are likely targets of AsH3 toxicity, with NEM being able to prevent AsH3-induced hemolysis. arsine, thiols, chelator, human erythrocytes, intracellular ions, membrane disruption Arsine gas (AsH3) is the hydride of arsenic and its most acutely toxic form (threshold limit value = 50 ppb; ACGIH, 1982). Exposure to AsH3 was fatal in up to 25% of the reported human cases (Fowler and Weissberg, 1974). The erythrocyte is the main target of AsH3 since this cell undergoes hemolysis (loss of intracellular hemoglobin), due possibly to loss of membrane integrity. In human exposures to AsH3, clinical experience is consistent with intravascular hemolysis, and dark red urine (hematuria) is usually the first symptom. This is followed by abdominal pain, jaundice, and anemia, the triad of symptoms experienced following exposure to AsH3, which is possible from accidental release of the gas during some manufacturing processes. AsH3 is extensively used for epitaxial growth of gallium arsenide and as a dopant for silicon-based electronic devices in the semiconductor industry. Accidental exposure to AsH3 may also occur from any situation where arsenic-contaminated metals are treated with a strong acid, e.g., metal mining, paint, and herbicides (Buchanan, 1962). The mechanism of AsH3 toxicity is not clearly understood. Although AsH3 is a strong reducing agent, in this study it is proposed that its toxic mechanism occurs via oxidation of key erythrocyte components. Most of the oxidation hypotheses are in accord with published evidence reporting depletion of reduced gluthathione (GSH) and the oxidation of key sulfhydryl groups. Pernis and Magistretti (1960) reported reduction of GSH levels by AsH3 to less than 40% of control before hemolysis took place. Blair et al. (1990) also reported 60% reduction in erythrocyte GSH after a 4-h AsH3 exposure. Recent investigations have contradicted the importance of GSH (Hatlelid et al., 1995; Winski et al., 1997). Hatlelid et al. (1995) reported that thiol depletion only occurred after 4 h of AsH3 exposure and neither preceded nor coincided with hemolysis. Winski et al. (1997) reported no significant changes in GSH and glutathione disulfide (GSSG) after a 1-h incubation with 1 mM AsH3. Other authors have suggested that AsH3 interacts with important sulfhydryl groups located on the membrane Na+,K+-ATPase pump (the main mechanism for volume control), inducing cell swelling and lysis (Levinsky et al., 1970). In the erythrocyte, AsH3 first induces loss of intracellular potassium (Ayala-Fierro et al., 1999; Winski et al., 1997) and influx of extracellular sodium (Winski et al., 1997). Hemolysis follows AsH3-induced ion loss, for which the only accepted treatment is exchange transfusion of the blood (Klimecki and Carter, 1995). In this study, we investigated the effects of sulfur, a sulfur-containing compound, sulfhydryl-containing agents, and sulfhydryl-inhibitors on AsH3-induced ion loss and hemolysis. MATERIALS AND METHODS Chemicals Zinc arsenide, 2,3-butanedithiol, iron (II) sulfide, and pyridine, ACS grade, were obtained from Aldrich Chemical Co. (Milwaukee, WI). Mag-fura-2 and 6-methoxy-N-(3-sulfopropyl) quinolinium were purchased from Molecular Probes, Inc. (Eugene, OR). Sodium fluoride was purchased from Fluka-Gerantie (Switzerland). Sulfuric acid, urea, and all the blood collection supplies, including collection tubes containing heparin, were purchased from Fisher Scientific (Pittsburgh, PA). Carmustine (BCNU) was purchased from Bristol Lab. (Evansville, IN). Silver diethyldithiocarbamate, ACS grade, was purchased from J. T. Baker Chemical Co. (Phillipsbury, NJ). Meso-2,3-dimercaptosuccinic acid (DMSA), 1:1 Cysteine-DMSA, 2:1 Cysteine:DMSA, and 2,3-dimercapto-1-propanesulfonic acid (DMPS) were all generous gift from Dr. H. Vas Aposhian, Department of Pharmacology and Toxicology, The University of Arizona, Tucson, AZ. All other chemicals were ordered from Sigma Chemical Co. (St. Louis, MO). Blood Collection Blood was collected by venipuncture from healthy male and female volunteers (ages 22–40). The blood was sedimented by centrifugation (2000 rpm, 10 min) and rinsed twice with glucose-containing phosphate-buffered saline (Glucose-PBS, pH 7.4) in order to remove plasma and buffy coat. The packed cells were either used immediately or stored overnight at 4°C in the presence of glucose-PBS. One-day-old cells were the oldest cells used in all experiments. Arsine Generation AsH3 was generated by the method of Hatlelid et al. (1995). Briefly, zinc arsenide was reacted with 50% sulfuric acid to generate the AsH3 gas that was bubbled into 0.02-M phosphate buffer or PBS to the desired concentration, using argon as the carrier gas. AsH3 concentration in the phosphate buffer of PBS was determined by reaction with 0.55% diethyldithiocarbamate in pyridine, followed by spectrophotometric determination of this product at 510 nm.3 Studies on Arsine-Induced Hemolysis For these studies, fresh human blood was obtained from healthy volunteers as described previously. All incubations were performed at 37°C in a Lab-Line® Orbit Environ-Shaker Incubator (Lab-Line Instruments, Inc., Melrose Park, IL). Hemoglobin leakage into the extracellular fluid was considered a direct measure of total erythrocyte death or hemolysis. It was determined as described by Winski et al., 1997. The compounds studied as possible hemolytic inhibitors were divided into 3 groups. The first group consisted of elemental sulfur (15.6 mM); sodium thiosulfate (15.6 mM); sodium fluoride (5.8 mM); 2,3-butanedithiol (5.8 mM); meso-2,3-dimercaptosuccinic acid (DMSA, 15.6 mM); 1:1 cysteine:DMSA (15.6 mM); 2:1 cysteine:DMSA (15.6 mM); N-acetylcysteine (15.6 mM); and 2,3-dimercapto-1-propanesulfonic acid (DMPS, 15.6 mM). The erythrocytes (0.25% hematocrit) were incubated with these compounds 2 h prior to AsH3 addition, added simultaneously to AsH3, or added 5 min after AsH3. The treatment solutions consisted of 0.25 ml packed erythrocytes, 0.25 ml of the inhibitor (4× their final concentration), 0.25 ml PBS, and 0.25 ml of 2 mM AsH3 solution prepared in PBS (final AsH3 concentration was 0.5 mM). The negative control consisted of 25% hematocrit in PBS (AsH3 and inhibitor excluded) while the positive control (AsH3-treated cells) consisted of 25% hematocrit, 0.5 ml of PBS, and 0.25 ml 2mM AsH3 (only inhibitor excluded). The second group consisted of N-ethylmaleimide (NEM, 0.5 mM); 5,5′-dithio-bis(2-nitrobenzoic acid) (DTNB, 0.5 mM); and a cocktail containing Carmustine (BCNU, 0.5 mM), L-buthionine-[S,R]-sulfoximine (BSO, 0.5 mM), and maleic acid diethyl ester (DEM, 0.5 mM). For this group, the erythrocytes were incubated with these compounds 30 min before AsH3 treatment. The treatment solutions and controls were prepared as described above. In this experiment, another incubation containing 0.25 ml of the inhibitor and 0.25 ml of PBS in place of AsH3, was prepared. The third group consisted of erythrocytes pretreated with 100% carbon monoxide for 20 min before AsH3 treatment. The formation of carboxyhemoglobin was verified spectrophotometrically via hemoglobin binding. The negative control consisted of 0.25 ml of carbon monoxide-treated erythrocytes and 0.75 ml of PBS (25% hematocrit). The positive control consisted of 0.25 ml of oxygenated erythrocytes, 0.5 ml of PBS, and 0.25 ml of 2 mM AsH3 (final AsH3 concentration was 0.5 mM). The treatment group consisted of carbon monoxide-treated erythrocytes, 0.5 ml of PBS, and 0.25 ml of 2 mM AsH3. The reaction was started after the addition of AsH3. At 30, 60, and 120 min aliquots were taken, transferred to clean microcentrifuge tubes, spun at 16,000 rpm for seven s, and 50-μl supernatant analyzed for hemoglobin (Winski et al., 1997). Incubations from all treatment groups had a final volume of 1 ml with a 25% hematocrit (0.25ml whole erythrocytes). AsH3 -NEM Interactions The results from the hemolysis studies indicated that NEM was the most potent inhibitor of AsH3-induced hemolysis. In order to determine whether NEM has its effect by direct interaction with AsH3 or by an indirect action, the disappearance of AsH3 was measured over time in the presence of NEM. Controls consisted of 1 mM AsH3 or NEM prepared in PBS by adding 0.5 ml of 2 mM AsH3 or NEM to 0.5 ml PBS. The interaction of AsH3 with NEM was investigated in a solution containing 1 mM AsH3 and NEM prepared by adding 0.5 ml 2 mM AsH3 to 0.5 ml 2 mM NEM. The samples were incubated at 37°C and aliquots were removed at 5, 15, 30, and 60 min to determine AsH3 concentrations as described earlier. Effect of NEM on AsH3-induced Disruption of Ion Homeostasis Human erythrocytes were treated with control (PBS), AsH3, NEM, or AsH3/NEM at the same concentrations described earlier. At 5, 15, 30, and 60 min, a 125-μl aliquot was removed from the incubation reaction, the cells washed twice with a 310-mOsm-tris base solution (pH 7.4), and centrifuged at 16,000 rpm for 7 s, discarding the supernatant each time. The washed cell pellet was added to 1 ml water in order to lyse the cells. In order to normalize ion leakage data to total hemoglobin, total hemoglobin was measured by adding 50 μl cell lysate to 3.5 ml Drabkin's reagent (Winski et al., 1997). The intracellular ions were determined as follows: Potassium (K+). Intracellular K+ was determined by flame emission photometry (Bacharach Coleman model 51 Ca flame photometer) based on the method of Winski et al. (1997). Magnesium (Mg++). Intracellular Mg++ was measured based on the fluorescence procedure of Günther et al. (1994) with few modifications. Briefly, after the cell washing and lysing, 50 μl of the lysate was added to 0.7 ml of H2O and 2.25 ml of 3.41-μM mag-fura-2 in a fluorometric cuvette. Fluorescent excitation scans (300–400 nm) were performed on a Hitachi F-2000 Fluorescence Spectrophotometer with an emission λ of 505 nm using an 11% filter. Scan traces were done at 335 nm and 378 nm, and the ratios of the fluorescence intensities at these 2 wavelengths were calculated with the appropriate standards and dilutions. Chloride (Cl.–). Intracellular Cl– was measured based on the fluorescence procedure of Calafut and Dix (1995) with few modifications. Briefly, after the cell washing and lysing, 50 μl of the lysate was added to 0.7 ml of H2O and 2.25 ml of 20 μM 6-methoxy-N-(3-sulfopropyl)-quinolinium (SPQ) in a fluorometric cuvette. Fluorescent excitation scans (300–400 nm) were performed as described for Mg++ with an emission λ of 450 nm using an 11% filter. A scan trace was done at 352 nm in order to calculate intracellular chloride concentrations with the appropriate standards and dilutions. Calcium (Ca++). Intracellular Ca++ was measured by atomic absorption spectroscopy based on the method of Harrison and Long (1968) with few changes. Briefly, after the cell washing and lysing, the proteins were precipitated. The supernatant was measured for Ca++ content using a Perkin-Elmer Atomic Absorption Spectrophotometer with a Perkin-Elmer hollow cathode, calcium/magnesium lamp set at a wavelength of 422 nm with the appropriate standards and dilutions. Effect of Ouabain and Bumetanide on K+ Transport in the Presence of AsH3 The effect of blocking K+ transport on AsH3-induced potassium efflux was studied based on the method described by Dwight and Hendry (1996). Briefly, 6 separate incubations were used, each containing 0.25 ml of packed erythrocytes, 0.25 ml of PBS, and 0.25 ml of either PBS, 0.1 mM ouabain in PBS, or 0.1 mM ouabian/0.1 mM bumetanide. The samples were pre-incubated at 37°C for 5 min. Either 0.25 ml of 2 mM AsH3 or PBS was added to the samples. Aliquots (125 μl) were removed at 0, 2, 5, 10, and 15 min and the cells were rinsed twice, lysed, and the proteins precipitated. The resulting supernatant was assayed for potassium using the flame photometry method described earlier. Data Analysis Individual experiments were performed at least in duplicate, and sample number (n) refers to the number of at least 3 separate experiments. All data are expressed as the mean ± standard deviation. Values are denoted with an asterisk (*) if statistically different from controls at p < 0.05, using one-way analysis of variance (ANOVA) with Bonferroni's multiple comparison tests (GraphPad Prism, GraphPad Software, Inc., San Diego, CA). RESULTS Effects of Sulfur, Sulfhydryl Agents, and Sulfhydryl Inhibitors on AsH3-induced Hemolysis The effect of certain compounds on hemolysis was studied by first pre-incubating erythrocyte suspensions with the chosen compound for various time points at 37°C prior to AsH3 treatment. For sulfur and sodium thiosulfate, erythrocytes were pre-incubated with these compounds for 2 h, added simultaneously with AsH3, or added 5 min after AsH3 addition. For DMSA, erythrocytes were allowed to pre-incubate for 2 h. For carbon monoxide-treated erythrocytes, solutions were bubbled for 20 min with pure carbon monoxide gas, and the formation of carboxyhemoglobin was verified spectrally. For N-ethylmaleimide (NEM), erythrocytes were pre-incubated with this compound for 30 min before AsH3 treatment. Effect of sulfur-containing compounds on AsH3-induced hemolysis. Human erythrocytes were pre-incubated with 15.6-mM sulfur for 2 h, or sulfur was added simultaneously with AsH3, or sulfur was added 5 min after the AsH3 addition. All 3 sulfur-treatment groups (2 h previous to, added simultaneously, or 5 min after) were statistically different from the AsH3-treated erythrocytes at 1- and 2-h time points. The best protection was found at 1-h incubation in which all the sulfur treatments decreased AsH3-induced hemolysis about 30–70% (Fig. 1, top). At 2-h incubation, all sulfur treatments except that one when sulfur was added 5 min after AsH3, also protected, but to a lesser extent. Pre-incubation of human erythrocytes with sulfur for 2 h offered the better protection against AsH3-induced hemolysis at both time points, decreasing hemolysis 33 and 59%, respectively. Sulfur added simultaneously with AsH3 offered protection, decreasing AsH3-induced hemolysis about 11% at 2 h. Finally, sulfur added 5 min after AsH3 addition offered the least protection but decreased AsH3induced hemolysis about 3% by 2 h (Fig. 1, top). When erythrocytes were treated with sodium thiosulfate at the same conditions as sulfur, it was not as effective as sulfur in decreasing total hemolysis. At 60 min, 2 out of 3 thiosulfate treatment groups produced about 15% hemolysis (Fig. 1, bottom). The only treatment group that inhibited hemolysis by 60% at 60 min was the sample pre-incubated with thiosulfate 2 h prior to AsH3 addition. At the 2-h incubation, the samples containing thiosulfate added simultaneously or 5 min after AsH3 addition showed about 35% loss of intracellular hemoglobin (Fig. 1, bottom). This is still a considerably large amount of the total intracellular hemoglobin. At this time point, thiosulfate added 2 h prior to AsH3 addition was the most effective at inhibiting hemolysis, similarly to sulfur-treated erythrocytes. Effect of the sulfhydryl-agent (DMSA) and the sulfhydryl-inhibitor (NEM) on AsH3-induced hemolysis. The ability of the heavy metal chelator sulfhydryl-agent, DMSA, to inhibit hemolysis was also investigated. Only a 2-h DMSA pre-incubation was used for this particular compound. This compound exhibited a similar pattern when compared to sulfur- and thiosulfate-treated erythrocytes. At 60 and 120 min, hemolysis was inhibited significantly by DMSA as compared to AsH3-treated erythrocytes. The best inhibition effect was produced at 1 h (Fig. 2, top). The most potent inhibitor of AsH3-induced hemolysis was NEM. At a final concentration of only 0.5 mM, NEM was able to cause a significant, 2-fold decrease in the total amount of hemoglobin released from the erythrocyte (Fig. 2, bottom). The decrease in AsH3-induced hemolysis was similar at both 1 and 2 h. At a final NEM concentration of 1 mM, the total amount of hemoglobin released upon AsH3 addition was insignificant as compared to negative controls (data not shown). Other sulfhydryl-containing compounds such as 2,3-butanedithiol (5.8 mM), DMPS (15.6 mM), DTNB (0.5 mM), 1:1 cysteine:DMSA (15.6 mM), and 2:1 cysteine:DMSA (15.6 mM), were effective at significantly decreasing the amount of hemoglobin released from erythrocytes treated with AsH3 (data not shown). These compounds were not as potent as NEM, therefore they were not chosen as hemolysis inhibitors for future membrane studies. Other sulfhydryl-inhibitors such as N-acetylcysteine (15.6 mM), sodium fluoride (5.8 mM), and the cocktail BCNU/BSO/DEM (0.5 mM) were ineffective at blocking hemolysis (data not shown). Effect of Carbon Monoxide on AsH3-induced Hemolysis The importance of structural changes in hemoglobin on AsH3-induced hemolysis was studied by converting all reduced, oxygenated hemoglobin to reduced carboxyhemoglobin. The formation of carboxyhemoglobin was verified spectrally by lysing an aliquot of the erythrocyte suspension in a disposable cuvette containing H2O. The hemoglobin in the erythrocytes was assumed to be 100% carboxyhemoglobin if the spectra was identical to the spectra of carboxyhemoglobin found in the literature. Carbon monoxide completely inhibited AsH3-induced hemolysis for the entire time course of 2 h, compared to the effect of AsH3 on oxygenated erythrocytes. At 30, 60, and 120 min, the percent of hemolysis remained at control levels (Fig. 3). Interactions between AsH3 and NEM NEM was the most potent agent at preventing AsH3-induced hemolysis. In order to determine if NEM has a direct interaction with AsH3, a test was performed that measured the disappearance of AsH3 in PBS in the presence or absence of NEM. Based on the method developed by Hatlelid et al. (1995), the disappearance of AsH3 was measured at 5, 15, 30, and 60 min. A 1-mM AsH3 solution by itself undergoes spontaneous release into the headspace of the incubation tube. By 15 min, half of the AsH3 has been released from the solution into the headspace (Fig. 4). The incubation containing 1 mM AsH3 with 1 mM NEM demonstrated the same trend as seen with AsH3 alone (Fig. 4). Therefore, it seemed that NEM and AsH3 have no direct interactions, since the disappearance rate of AsH3 was not increased by the presence of NEM. Effect of NEM on Arsine-Induced Disruption of Ionic Gradients The AsH3 effect on the intracellular ion concentrations was determined in order to assess the erythrocyte health status at various times after AsH3 exposure. The electrolytes potassium (K+), sodium (Na+), magnesium (Mg++), chloride (Cl–), and calcium (Ca++) were all measured intracellularly to determine whether these ions followed their concentration gradients upon AsH3 exposure. Also, we examined the effect of NEM on preventing any electrolyte leakage that AsH3 might cause. Finally, the possibility of the combination of ouabain and bumetanide to prevent AsH3 from reaching reactive sites on the potassium pumps was studied. This was assayed as a function of potassium leakage. Effect of NEM on the arsine-induced loss of intracellular potassium and magnesium. Human erythrocytes were exposed to AsH3, and intracellular potassium (K+) in intact erythrocytes was measured by flame photometry. All points were normalized to total intracellular hemoglobin of intact red cells. Treatment with AsH3 produced a 55% loss of intracellular K+ levels in erythrocytes at 5 min., and by 30 min this K+ loss reached 70% (Fig. 5, top). NEM was only partially effective at preventing K+ loss at 15 and 30 min compared to AsH3-treated cells. Meanwhile, NEM by itself caused about a 35% loss of intracellular K+, although by 60 min K+ levels were back to 85% of controls (Fig. 5, top). Intracellular sodium levels (Na+) increased in proportion to K+ loss, indicating a significant loss of membrane function (data not shown). Intracellular magnesium levels (Mg++) were measured in intact erythrocytes using a fluorometric Mg++ chelator (mag-fura-2). Mg++ concentrations were also normalized to total hemoglobin in intact erythrocytes. Treatment with AsH3 caused a 15% drop in intracellular Mg++ levels at 5 min and this decrease continued for the next 25 min, with Mg++ concentrations rising slightly (Fig. 5, bottom). Treatment with NEM alone also caused a significant 15% loss in intracellular Mg++ levels over the entire 30-min time course (Fig. 5, bottom). Therefore, it was found that NEM was ineffective at protecting the erythrocyte from intracellular Mg++ loss caused by AsH3. Effect of NEM on the arsine-induced increase of intracellular chloride and calcium. Chloride levels (Cl–) inside intact erythrocytes were measured using a fluorometric assay with the chloride chelator, SPQ. In the presence of AsH3, intracellular Cl– concentration was increased by 70% at 5 min, reaching a maximum at 15 min (100% increase, Fig. 6, top). Given alone or in combination with AsH3, NEM was again ineffective at preventing chloride ions from moving with its concentration gradient, which showed at least a 100% increase in Cl– levels by 5 min (Fig. 6, top). All points were normalized to total intracellular hemoglobin of intact cells. One of the most important mediators of cell death is intracellular calcium (Ca++) concentration. Ca++ was measured in intact erythrocytes using atomic absorption spectroscopy with units reported in nmol Ca++/g Hb. Treatment with AsH3 caused a large, significant increase in intracellular Ca++ levels at 15 min, and this continued increasing up to 1 h (Fig. 6, bottom). AsH3 caused about a 4-fold increase in intracellular Ca++ concentrations. At 30 min, the time point where AsH3 produces significant hemolysis in human erythrocytes, there was observed about a 4.5-fold increase in Ca++ levels. NEM was able to prevent AsH3-induced increase in intracellular Ca++ levels. Intracellular Ca++ levels in NEM-treated erythrocytes remained at control levels for the duration (60 min) of the study (Fig. 6, bottom). Effects of Ouabain and Humetanide on AsH3-induced Hemolysis The effects of ouabain and bumetanide on AsH3-induced hemolysis were studied in order to test whether AsH3 specifically targeted the various potassium pumps located around the erythrocyte cell membrane. Ouabain and bumentanide are known potassium-pump inhibitors and therefore could potentially block an AsH3-binding site. Ouabain and bumetanide had no effect on blocking or even delaying hemolysis caused by AsH3 (data not shown). The two agents were also unsuccessful at depleting the cell of intracellular potassium via passive diffusion (efflux). This result may be due to the fact that the 2 agents only block 2 potassium pumps on the erythrocyte membrane, which has a total of 5 potassium transporters. DISCUSSION In this study, the effects of sulfur, sulfur compounds, and thiol-containing compounds, as well as thiol inhibitors on AsH3-induced hemolysis were investigated. It has been shown in vitro that the aging and denaturation of heme proteins involves the oxidation of globin, including its sulfhydryl groups, and that such oxidation is inhibited by reduced glutathione (Allen and Jandl, 1961; Harley and Mauer, 1961). In rat erythrocytes, Winski and Carter (1995) demonstrated that intracellular GSH was depleted when sodium arsenate [As(V)] was added in the incubation reaction. In that study, GSH was oxidized, but not to GSSG; instead, the formation of mixed disulfides with proteins (ProS-SG) was postulated. Kosower and co-workers (1977) postulated that exposing rat blood to sulfhydryl oxidizing agents caused the formation of mixed disulfides between GSH and hemoglobin. It is clear that sulfhydryls play a major role in the mechanism of AsH3-induced hemolysis. Therefore, it was hypothesized that using a sulfhydryl-blocking agent could prevent an AsH3-induced change in the hemoglobin protein or other proteins and should decrease the degree of hemolysis. Kensler et al. (1946) showed that certain monothiols and dithiols were successful in preventing hemolysis caused by AsH3. In our study, sulfur-containing compounds and compounds that are known to interact with sulfhydryl groups were tested for their ability to block hemolysis observed in erythrocytes incubated with AsH3. Compounds containing sulfur included an elemental sulfur suspension and sodium thiosulfate. Others included the well-documented heavy-metal chelator, DMSA (Mückter et al., 1993), and the common alkylating sulfhydryl agent, NEM (Jacob and Jandl, 1962; Winski and Carter, 1995). In each incubation, the compounds were allowed to equilibrate with the surrounding erythrocytes for at least 30 min before AsH3 addition. The most successful and potent compound in inhibiting AsH3-induced hemolysis was the sulfhydryl blocker, NEM. At equimolar concentrations with AsH3, NEM was able to decrease hemolysis significantly 2 h after the addition of AsH3. NEM is a very effective agent at blocking sulfhydryl groups. In these studies, its mechanism of action is thought to be a blockade of these sulfhydryl sites from reacting with AsH3 or a reactive metabolite. Although NEM does enter the cell, other agents such as p-mercuribenzoate (Jacob and Jandl, 1962), sulfur, and Ellman's reagent do not enter the erythrocyte, although they are effective at sulfhydryl and/or hemolysis blockade. Based on these results and others (Jacob and Jandl, 1962; Reglinski et al., 1988), it can be concluded that the shape and integrity of the erythrocyte exposed to sulfhydryl inhibitors prior to AsH3 treatment in vitro depend ultimately upon membrane sulfhydryl status and not intracellular sulfhydryl blockade. The erythrocyte membrane is rich in sulfhydryl-group functions. Sulfhydryl groups are crucial to the maintenance of the shape and motility of the erythrocyte membrane. The meshwork of the cytoskeleton is based on a variety of sulfhydryl interactions between such membrane proteins as actin, ankyrin, spectrin, band 3, and band 4.1 (Bennett, 1985). Sulfhydryl blocking agents such as NEM are known to alter the spectrin dimer-tetramer equilibrium (Smith and Palek, 1983). Although NEM protects against AsH3-induced hemolysis, it still causes a decrease in membrane stability (Chasis and Mohandas, 1986) and eventually hemolysis (Jacob and Jandl, 1962). The time course for hemolysis is different for AsH3 and NEM. At 2 h, the process of hemolysis is over with 0.5 mM AsH3, while it is just beginning with 0.5 mM NEM. A rapid increase in hemolysis is also observed after 30 min pre-incubation with AsH3, while hemolysis caused by NEM is a slow, steady rise in released hemoglobin. Therefore, the two compounds likely have similar mechanisms of hemolysis (disruption of membrane framework), but NEM is able to block sulfhydryl groups which prevents the rapid hemolysis observed with AsH3. Elemental sulfur and sodium thiosulfate were somewhat effective at decreasing the extent of AsH3-induced hemolysis. Sulfur is practically insoluble in phosphate-buffered saline (PBS) and therefore unlikely to cross the erythrocyte membrane. It is possible that sulfur forms mixed disulfides with extracellular membrane sulfhydryl groups causing the formation of mixed glutathione-intracellular membrane protein disulfides. This mechanism of membrane blockade is postulated for the membrane-impermeant and sulfhydryl specific compound, 5,5′-dithiobis(2-nitrobenzoic acid) or Ellman's reagent (Reglinski et al., 1988). Indeed, the Ellman's reagent was somewhat effective at inhibiting the amount of hemolysis caused by AsH3 (data not shown). Therefore, sulfur could be acting by oxidizing extracellular sulfhydryl groups, leading in turn to the blockade of intracellular membrane sulfhydryl groups by glutathione. It is known that the only successful treatment of arsine poisoning is exchange transfusion (Klimecki and Carter, 1995). Chelators of arsenic such as dimercaprol (BAL) offer no protection against erythrocyte destruction caused by arsine although it might exert a protective effect against the long-term consequences of arsine poisoning (Fowler and Weissberg, 1974; Pinto et al., 1950). Other arsenic chelators that have not been examined for their ability to stop AsH3-induced hemolysis showed some ability to delay hemolysis. DMSA is unable to cross the cell membrane (Zheng et al., 1990), and therefore, its mechanism of action is probably identical to that of sulfur and thiosulfate. As mentioned before, the mechanism might be that proposed for Ellman's reagent, which is blockading of intracellular membrane sulfhydryl groups by GSH. This may indicate that sulfhydryl groups located on the endofacial side of the erythrocyte membrane are important to the mechanism of hemolysis. The compounds DMSA, DMPS, and 2,3-butanedithiol were all successful at preventing hemolysis while glutathione, cysteine, and N-acetylcysteine were incapable of preventing hemolysis. Structurally, DMSA, DMPS, and 2,3-butanedithiol are dithiol compounds in that they each contain two sulfhydryl groups per molecule. Meanwhile, glutathione, cysteine, and N-acetylcysteine contain only one sulfhydryl unit. Based on this finding, it is suggested that dithiols are more successful agents than monothiols at protecting against AsH3-induced hemolysis. Indeed, dithiols have been shown to offer greater protection from AsH3 than monothiols (Kensler et al., 1946). The possibility that there is a direct, chelation-type reaction between NEM and AsH3 was eliminated, based on the observation that NEM had no effect on the rate of disappearance of AsH3 in an aqueous solution. Therefore, NEM was chosen for subsequent studies involving the effect of AsH3 on erythrocyte transport and function. It has been recognized for 38 years that agents that interact with membrane sulfhydryl groups alter the ion permeability of erythrocytes (Jacob and Jandl, 1962). More recently, it was discovered that these agents, specifically NEM, affect specific transport pathways. NEM has been shown to stimulate a latent K+,Cl− cotransport, which appears kinetically identical to that stimulated by swelling of these cells (Ihrig et al., 1992; Lauf et al., 1984). NEM has also been reported to inhibit Na+,K+,Cl− cotransport in human erythrocytes (Lauf et al., 1984). Therefore, the available literature tends to conclude that membrane sulfhydryl groups may play a role in the regulation of certain ion transport processes by changes in cell volume, the effect of which could be mimicked in cells of normal volume by directly modifying these sulfhydryls with NEM (Haas and Harrison, 1989). Since AsH3 seemed to modify sulfhydryl groups, it seems likely that this hemolytic agent causes disturbances in volume control of the human erythrocyte. It appears that AsH3 causes a general, nonspecific disruption of all ion gradients. In the case of the high intracellular electrolytes, K+ and Mg++, there was a significant time-dependent efflux of these ions in erythrocytes treated with AsH3. Arsine-induced loss of intracellular K+ has been reported in human (Winski et al., 1997) and rat (Ayala-Fierro et al., 1999) erythrocytes. The intracellular concentrations of these electrolytes decreased based on their concentration gradients favoring their efflux. AsH3 also caused an influx of the electrolytes Na+, Cl–, and Ca++. Winski et al. (1997) also reported an influx of Na+ into erythrocytes exposed to AsH3. This agrees with the fact that these ions are higher in extracellular concentration; this causes them to enter the erythrocyte when the membrane function is disrupted. NEM is known to alter membrane transport and may compete with AsH3 for specific transporter sulfhydryl sites. Treatment of erythrocytes with NEM caused a decrease in intracellular K+ levels, and therefore was only somewhat effective at preventing AsH3-induced K+ loss. This K+ loss can be explained due to the fact that NEM inhibit the Na+,K+,Cl− cotransport (Lauf et al., 1984) while stimulating a usually latent K+,Cl− contransporter (Lauf et al., 1984; Ihrig et al., 1992). This stimulation of the latent K+,Cl− cotransporter was observed in these experiments with a steady rebound in intracellular K+ levels over 1.5 h-incubation with NEM. The effect of NEM on Mg++ transport seems to be inhibitory although there is very limited data to support this finding. There is some evidence that NEM inhibits Ca++,Mg++-ATPase (Cameron and Smariga, 1981; Cha and Lee, 1976) supporting the finding that NEM decreases intracellular Mg++ concentrations. NEM was ineffective in preventing AsH3-induced increase in intracellular Cl– concentration. NEM alone caused Cl– influx into the erythrocyte in agreement with its concentration gradient, then there was a gradual decrease in intracellular Cl– indicating the activation of the latent K+,Cl− cotransporter. This gradual decrease in intracellular Cl– was also observed for the AsH3-treated erythrocytes suggesting the possibility of AsH3-induced activation of the normally latent K+,Cl− cotransporter. Determining intracellular Na+ levels is important in testing the hypothesis proposed by Levinsky and co-workers (1970) that the target site for AsH3 is the Na+,K+-ATPase pump on the membrane, which is the main mechanism for volume control. This pump is dependent on a sulfhydryl group, which senses changes in cell volume. Recently, this hypothesis has been disproved, based on the observation that ATPase activity was not inhibited by AsH3 while ATP levels remained the same as controls (Winski et al., 1997). In this study the Na+ and K+ gradients were not selectively disrupted by AsH3 suggesting that this pump is not the AsH3 target. Treatment of erythrocytes with NEM caused intracellular Na+ levels to increase. This finding is in agreement with other observations that NEM enhances Na+ influx due to nonspecific interaction with membrane-bound sulfhydryl groups (Sha'afi and Naccache, 1974). The most important finding about the NEM effect on protecting the erythrocyte from AsH3-induced disruption of membrane transport processes was that observed with Ca++. NEM was very successful at preventing the rapid Ca++ influx induced by AsH3. This agreed with the finding that a sulfhydryl inhibitor structurally similar to NEM, diamide, locks Na+/Ca++ exchange in dog erythrocytes in an “on” conformation (Parker, 1986). NEM also blocks dephosphorylation of Ca++-ATPase, indicating that the pump is unable to turn off (Cha and Lee, 1976). Therefore, when AsH3 is present, NEM offers its hemolytic protection to erythrocytes by facilitating Ca++ efflux. This Ca++ influx seems to be the irreversible, final event of AsH3-induced hemolysis. The influx of Ca++ has been shown to induce morphological changes within the human erythrocyte. The normal discocyte shape of the erythrocyte can be modified by a series of distinct transitions, due to an increase in intracellular Ca++ concentrations. Whatmore and co-workers (1992) proved that prolonged exposure of erythrocytes to Ca++ caused significant proteolysis of key cytoskeletal elements such as band 4.1 and ankyrin. Loss of these elements is considered to be an irreversible event with respect to the shape and membrane integrity of the erythrocyte. Morphological studies have been performed on AsH3-treated erythrocytes using formaldehyde/glutaraldehyde fixation (Winski et al., 1997). AsH3 also caused morphological changes strikingly similar to increased intracellular calcium levels evident by the increase in echinocytes, which are pre-lytic cells. Since K+ efflux is the first sign of AsH3 toxicity, it was necessary to determine if sulfhydryl sites on potassium transporters are the primary targets of AsH3. Ihrig and co-workers (1992) report that there are 5 potassium transporters located on the erythrocyte membrane with Na+,K+-ATPase (ouabain sensitive) and Na+,K+,Cl− cotransport (bumetanide sensitive), accounting for 86% of the total K+ transport. By blocking these transporters with ouabain and bumetanide, it was hypothesized that these transporters could be protected from AsH3 toxicity. This was not the case since hemolysis occurred in the inhibited erythrocytes at the same rate as that with AsH3-treated erythrocytes. Therefore, it seemed that these transporters are not the main targets of AsH3. AsH3-induced K+ efflux was also unaffected by the presence of these inhibitors. When AsH3 was absent, the inhibitors had no effect on intracellular potassium levels compared to negative controls. This is in agreement with the fact that there are 3 other potassium pumps located on the erythrocyte membrane, and the finding that a net potassium uptake still occurs even in the presence of ouabain and bumetanide (Dørup and Clausen, 1994). In conclusion, membrane-sulfhydryl groups are likely targets of AsH3 toxicity since the sulfhydryl inhibitor, N-ethylmaleimide is able to prevent AsH3-induced hemolysis. AsH3 causes the destruction of ion gradients across the cell membrane as evidenced by changes in the intracellular concentrations of the electrolytes K+, Mg++, Na+, Cl–, and Ca++. NEM was found to prevent the loss of the Ca++ gradient during AsH3 toxicity. The Ca++ influx is the toxic, irreversible endpoint of hemolysis caused by AsH3. FIG. 1. View largeDownload slide Effect of sulfur-containing compounds on arsine-induced hemolysis. Elemental sulfur: human erythrocytes were exposed simultaneously to control (PBS) (square), 0.5 mM AsH3 (circle), or 15.6 mM sulfur (diamond). Human erythrocytes were also pre-incubated with 15.6 mM sulfur for 2 h and then treated with 0.5 mM AsH3 (up triangle), or sulfur was added 5 min after AsH3 (down triangle). Sodium thiosulfate: human erythrocytes were exposed simultaneously to control (PBS) (square), 0.5 mM AsH3 (circle), or 15.6 mM sodium thiosulfate (diamond). Human erythrocytes were also pre-incubated with 15.6 mM sodium thiosulfate for 2 h and then treated with 0.5 mM AsH3 (up triangle) or sodium thiosulfate was added 5 min after AsH3 (down triangle). Values are mean ± SD; n = 3, *p < 0.05. FIG. 1. View largeDownload slide Effect of sulfur-containing compounds on arsine-induced hemolysis. Elemental sulfur: human erythrocytes were exposed simultaneously to control (PBS) (square), 0.5 mM AsH3 (circle), or 15.6 mM sulfur (diamond). Human erythrocytes were also pre-incubated with 15.6 mM sulfur for 2 h and then treated with 0.5 mM AsH3 (up triangle), or sulfur was added 5 min after AsH3 (down triangle). Sodium thiosulfate: human erythrocytes were exposed simultaneously to control (PBS) (square), 0.5 mM AsH3 (circle), or 15.6 mM sodium thiosulfate (diamond). Human erythrocytes were also pre-incubated with 15.6 mM sodium thiosulfate for 2 h and then treated with 0.5 mM AsH3 (up triangle) or sodium thiosulfate was added 5 min after AsH3 (down triangle). Values are mean ± SD; n = 3, *p < 0.05. FIG. 2. View largeDownload slide Effect of the heavy metal chelator, DMSA, and the alkylating sulfhydryl agent, NEM, on arsine-induced hemolysis. DMSA: human erythrocytes were exposed to control (PBS) (square), 0.5 mM AsH3 (circle), or were preincubated with 15.6 mM DMSA for 2 h prior to AsH3 addition (triangle). NEM: human erythrocytes were exposed to control (PBS) (square), 0.5mM AsH3 (circle), or were preincubated with 0.5 mM NEM for 30 min prior to AsH3 addition (triangle). One sample was only incubated with 0.5mM NEM (diamond) (These data overlap with control). Values are mean ± S.D; n = 3, *p < 0.05. FIG. 2. View largeDownload slide Effect of the heavy metal chelator, DMSA, and the alkylating sulfhydryl agent, NEM, on arsine-induced hemolysis. DMSA: human erythrocytes were exposed to control (PBS) (square), 0.5 mM AsH3 (circle), or were preincubated with 15.6 mM DMSA for 2 h prior to AsH3 addition (triangle). NEM: human erythrocytes were exposed to control (PBS) (square), 0.5mM AsH3 (circle), or were preincubated with 0.5 mM NEM for 30 min prior to AsH3 addition (triangle). One sample was only incubated with 0.5mM NEM (diamond) (These data overlap with control). Values are mean ± S.D; n = 3, *p < 0.05. FIG. 3. View largeDownload slide Effect of carbon monoxide (CO) on arsine-induced hemolysis. Human erythrocytes were bubbled with CO gas or were oxygenated for 20 min. The formation of carboxyhemoglobin was verified spectrally in the CO-treated erythrocytes. CO-treated erythrocytes were incubated with PBS (control) (square), or with 0.5mM AsH3 (triangle). Oxygenated erythrocytes were incubated with 0.5 mM AsH3 (circle). Values are mean ± SD; n = 3, *p < 0.05. Note: Data from control (CO-treated erythrocytes) and CO-treated erythrocytes + 0.5 mM AsH3 overlap. FIG. 3. View largeDownload slide Effect of carbon monoxide (CO) on arsine-induced hemolysis. Human erythrocytes were bubbled with CO gas or were oxygenated for 20 min. The formation of carboxyhemoglobin was verified spectrally in the CO-treated erythrocytes. CO-treated erythrocytes were incubated with PBS (control) (square), or with 0.5mM AsH3 (triangle). Oxygenated erythrocytes were incubated with 0.5 mM AsH3 (circle). Values are mean ± SD; n = 3, *p < 0.05. Note: Data from control (CO-treated erythrocytes) and CO-treated erythrocytes + 0.5 mM AsH3 overlap. FIG. 4. View largeDownload slide Effect of N-Ethylmaleimide (NEM) on arsine concentration. Human erythrocytes were incubated with 1 mM AsH3 (circle), 1 mM AsH3 + 1 mM NEM (square), or with 1 mM NEM (triangle) for 1 h. 150-μl aliquots were removed at the indicated time points and assessed for AsH3 remaining at incubation. Values are mean ± SD; n = 3, *p < 0.05. FIG. 4. View largeDownload slide Effect of N-Ethylmaleimide (NEM) on arsine concentration. Human erythrocytes were incubated with 1 mM AsH3 (circle), 1 mM AsH3 + 1 mM NEM (square), or with 1 mM NEM (triangle) for 1 h. 150-μl aliquots were removed at the indicated time points and assessed for AsH3 remaining at incubation. Values are mean ± SD; n = 3, *p < 0.05. FIG. 5. View largeDownload slide Effect of NEM on the arsine-induced loss of intracellular potassium (K+) and magnesium (Mg++) levels. Human erythrocytes were exposed to 0.5mM AsH3 (circle), 0.5 mM NEM (square), or AsH3 + NEM (triangle). Values are mean ± SD; n = 3 for K+, n = 4 for Mg++, *p < 0.05. FIG. 5. View largeDownload slide Effect of NEM on the arsine-induced loss of intracellular potassium (K+) and magnesium (Mg++) levels. Human erythrocytes were exposed to 0.5mM AsH3 (circle), 0.5 mM NEM (square), or AsH3 + NEM (triangle). Values are mean ± SD; n = 3 for K+, n = 4 for Mg++, *p < 0.05. FIG. 6. View largeDownload slide Effect of NEM on the arsine-induced increase of intracellular chloride (Cl–) and calcium (Ca++) levels. Human erythrocytes were exposed to control (diamond) (lower panel), 0.5 mM AsH3 (circle), 0.5 mM NEM (square), or AsH3 + NEM (triangle). Values are mean ± SD; n = 5, *p < 0.05. FIG. 6. View largeDownload slide Effect of NEM on the arsine-induced increase of intracellular chloride (Cl–) and calcium (Ca++) levels. Human erythrocytes were exposed to control (diamond) (lower panel), 0.5 mM AsH3 (circle), 0.5 mM NEM (square), or AsH3 + NEM (triangle). Values are mean ± SD; n = 5, *p < 0.05. 1 Present Address: Diagnostic Markers, Inc., 3601 S. Clarkson St. Suite 420, Englewood, CO 80110. 2 To whom correspondence should be addressed at the Department of Pharmacology and Toxicology, College of Pharmacy, Rm. 228, Tucson, AZ 85721. Fax: (520) 626-2466. E-mail: carter@pharmacy.arizona.edu. 3 AsH3 is a toxic gas and appropriate precautions should be taken. All procedures should be performed in an approved fume hood. A saturated potassium-permanganate solution trap, in-line after the aqueous trap, should be used to prevent the release of AsH3 during its generation. 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TI - The Effects of Sulfur, Thiol, and Thiol Inhibitor Compounds on Arsine-Induced Toxicity in the Human Erythrocyte Membrane
JF - Toxicological Sciences
DO - 10.1093/toxsci/55.2.468
DA - 2000-06-01
UR - https://www.deepdyve.com/lp/oxford-university-press/the-effects-of-sulfur-thiol-and-thiol-inhibitor-compounds-on-arsine-eZ1FKbcO1Q
SP - 468
EP - 477
VL - 55
IS - 2
DP - DeepDyve
ER -