Complexation of Arsenic Species in Rabbit Erythrocytes - Chemical

Chem. Res. Toxicol. 1994, 7, 621-627

621

Complexation of Arsenic Species in Rabbit Erythrocytes Marielle Delnomdedieu,*>tytMufeed M. Basti,$ Miroslav Styblo,$J' James D. Otvos,§ and David J. Thomas* Center for Environmental Medicine, University of North Carolina, Chapel Hill, North Carolina 27599, Pharmacokinetics Branch, Environmental Toxicology Division, Health Effects Research Laboratory, US.Environmental Protection Agency, Research Triangle Park, North Carolina 27711, Department of Biochemistry, North Carolina State University, Raleigh, North Carolina 27695, and Curriculum in Toxicology, University of North Carolina, Chapel Hill, North Carolina 27599 Received March 24, 1994@ The binding of arsenite, As(III), and arsenate, As(V), by molecules in the intracellular compartment of rabbit erythrocytes has been studied by lH- and 31P-NMRspectroscopy, uptake of 7 3 A ~and , ultrafiltration experiments. For intact erythrocytes t o which 0.1-0.4 mM arsenite was added, direct evidence was obtained for entry of 76% within V 2 h and subsequent binding of As(I1I)by intracellular glutathione and induced changes in the hemoglobin structure (NMR), likely due t o binding of As(II1). These results were compared with the effect of addition of As(V) on intact erythrocytes and revealed that a smaller amount of As(V) (-25%) enters the cells; the main fraction of As(V) enters the phosphate pathway, depletes ATP, and increases Pi. In contrast, As(II1) did not affect the ATP level. Both lH- and 31P-NMR data indicated striking differences between As(II1) and As(V) behavior when incubated with rabbit erythrocytes. These differences were confirmed by 73Asuptake and binding experiments. meso-2,3Dimercaptosuccinic acid (DMSA), a dithiol ligand, released glutathione from its arsenite complexes in erythrocytes.

Introduction Sulfhydryl-containing molecules are critical in both the reductive metabolism and biomethylation of arsenic (As) (1-6). Because glutathione (GSH) is the most abundant sulfhydryl-containingmolecule in the cellular milieu (71, much emphasis has focused recently on examining the interaction of As with this molecule. Studies utilizing NMR techniques have shown that GSH can reduce As(V) to As(II1) and subsequently form a (GS)sAs(III)complex (8, 9). This mode of complexation is consistent with earlier results that GSH forms a 1:2 adduct with phenyldichloroarsine (IO). The transfer of As(II1) from the (GS)&(III) complex to a dithiol-containing molecule, meso-2,3-dimercaptosuccinicacid (DMSA),' has also been demonstrated using NMR techniques (11). The complexation of As(II1) by three closely spaced cysteines (residues 640,656, and 661) on the rat glucocorticoid receptor (12, 13) and the complexation of As(II1) by lipoic acid (6,8thioctic acid) are examples of the critical role of sulfhydryl-containing molecules in mediating As metabolism in the cell. Considerable evidence suggests that As(V) is reduced to As(II1) in tissues of rats, rabbits, mice, and humans (14-1 7). However, the molecular basis of the reduction of As(V) in vivo has not been well characterized. Given the ability of GSH to reduce AsW) in a chemically-defined aqueous system (9,111, it was considered likely to serve

* Correspondence should be addressed to this author at the Center for Environmental Medicine, U S . EPA-HERL MD 74, Research Triangle Park, NC 27711; Ph: (919) 541-1847; Fax: (919) 541-5394. + Center for Environmental Medicine, University of North Carolina. U S . EPA. 9 North Carolina State University. 'I Curriculum in Toxicology, University of North Carolina. Abstract published in Advance ACS Abstracts, August 1, 1994. Abbreviations: DMSA, meso-2,3-dimercaptosuccinicacid; SEFT, spin-echo Fourier transform; FID, free induction decay; NPSH, nonprotein sulfhydryl groups.

*

@

this role in the cell. Previous studies have shown that the complexation of various metals [e.g., Zn2+,(CH&Pb+, CH3Hg+,Hg2+lwith GSH and possibly hemoglobin (Hb) can be examined by lH-NMR spectroscopy (18-21 using spin-echo Fourier transform (SEFT) techniques to detect interactions. This noninvasive method has the advantage of examining metal-sulfhydryl interaction in intact cells. This approach should avoid the redistribution of metal among binding sites that may occur during the separation and purification of cellular components. The present work examines the reduction of As(V) and the binding of As(II1) in intact rabbit erythrocytes and in hemolysate by lH SEFT-NMR spectroscopy. Initially, spectra were measured for the more abundant small molecules of the intracellular compartment of erythrocytes (e.g., GSH) by eliminating interfering resonances from Hb protons. A shorter spin-echo delay period, ZZ, was then used to observe aromatic Hb resonances. The results for intact erythrocytes and hemolysates to which As(II1) was added indicated that As(II1) was complexed by intracellular GSH and that this complex modifies the structure of Hb. Parallel studies with AdV) showed no significant binding to GSH or Hb, even with 25%uptake. In contrast, 31P-NMRindicated that As(V) perturbs the cellular metabolism of phosphorus by depleting highenergy nucleotides (e.g., ATP) whereas As(II1) did not affect ATP levels at a similar or higher intracellular concentration. Thus, these findings indicated that primary targets of As(V) differ from those of As(II1). The chemical reversibility of the As(111)binding to GSH was established by using DMSA, a vicinal dithiol, which releases GSH from complexes with As(II1) in aqueous solution (11). Hence, differences in the intracellular fates of As(V) and As(II1) were corroborated by NMR data and biochemical studies of the distribution and binding of this metalloid.

0893-228x/94/2707-0621$04.50/0 0 1994 American Chemical Society

Delnomdedieu et al.

622 Chem. Res. Toxicol., Vol. 7, No. 5, 1994

Experimental Procedures Chemicals. GSH, DMSA, D-glucose, sodium chloride, disodium arsenate (NazHAs04),tris(hydroxymethy1)aminomethane (Tris), 2-propanol, and acetic acid were obtained from Sigma (St. Louis, MO) and used as received. Arsenic trichloride (hC13) was purchased from Strem (Newburyport, MA). Radiolabeled carrier-free [73As]sodi~m arsenate was obtained from Los Alamos Meson Production Facility (Los Alamos, NM), and [73Aslarsenite was prepared by the method of Reay and Asher (22). Caution: Arsenic compounds are hazardous chemicals and should be handled carefully, using appropriate protection for carcinogens. Sample Preparation. Blood was collected from ear veins of New Zealand White rabbits (Hazelton, Denver, PA) using heparin-containing vacutainers (Vacutainer System, Rutherford, NJ). Erythrocytes were separated from plasma by centrifugation a t 3000g for 5 min a t 4 "C. Packed erythrocytes were washed three times with 2 volumes of modified Krebs-Ringer buffer (23) and collected by centrifugation at 3000g for 5 min a t 4 "C; packed cells for lH-NMR were washed twice with 1 volume of 154 mM NaCl and 0.1% D-glucose solution prepared in DzO (Cambridge Isotope Laboratories, Woburn, MA). The washes replaced most of the intracellular HzO with DzO. Packed erythrocyte samples for 31P-NMR were processed without glucose; glucose was added directly to the samples a t the beginning of the exposure to As. Tubes were incubated in a bath at 35 "C. Hemolysate was prepared by adding an equal volume of DzO to washed packed erythrocytes. After 1 h a t 0 "C membranes were removed by centrifugation a t 17000g for 20 min at 4 "C. The pH of erythrocytes and hemolysates, with or without addition of arsenic, ranged between 7.1and 7.3. Blood samples for 'H-NMR were kept at 4 "C prior to acquisition and analyzed within 24 h. lH-NMR spectra were recorded immediately aRer addition of As(II1) and A s 0 . For ultrafiltration experiments, samples containing As(II1) or A s 0 were incubated 30 min a t 25 "C to match the history of NMR samples. Stock solutions of 50 mM As(II1) and As(V) solutions were prepared in DzO and stored under argon until used. NMR Spectroscopy. SEFT 'H-NMR spectra were acquired a t 25 "C on a GE-Omega 500-MHz spectrometer using a spinecho pulse sequence (90"-t~-180"-t~-acquisition) (18-21,2427). A t~ of 0.015s was used when Hb resonances were being observed and a 0.06 s value was used when resonances from small molecules were observed (28). Spectra were measured on 0.5 mL packed or hemolyzed cells in 5 mm tubes. The free induction decay (FID) was collected with 8K data points using a spectral width of 4300 Hz (t2 = 0.06 s) or 6000 Hz ( t = ~ 0.015 s), and 192 transients were collected for each spectrum. Chemical shifts are reported relative to the methyl resonance of DSS (HDO 4.76 ppm). Before Fourier transformation, FID's were multiplied by an exponential function which induced a 0.2 Hz line broadening. 31P-NMR spectra were acquired at 35 "C with a broad-band probe on 2 mL packed cells in 10 mm tubes. FID was collected with 8K data points, a spectral width of 6400 Hz, 600 transients, and p90 of 70 ps. A n exponential multiplication of 5 Hz was applied to process the FIDs. Spectra were collected after incubation times from 0 to 5 h. Chemical shifis are reported relative to H3P04. Nonprotein Sulfhydryl Groups (NPSH) and H b Determination. NPSH and Hb contents were determined on freshly prepared packed rabbit erythrocytes and hemolysates with the same pool of cells used for NMR measurements. NPSH (mainly GSH) contents were determined in supernates prepared from erythrocytes and hemolysates adjusted to a final 5-sulfosalicylic acid concentration of 5%. Ellman's reagent [40 mg of 53'dithiobis(2-nitrobenzoic acid) in 100 mL of 1% sodium citrate1 and GSH was used to was used to detect NPSH at 412 nm (29), prepare a standard calibration curve. Total Hb was measured by spectrophotometry using Drabkin's reagent (Sigma total Hb determination kit, St. Louis, MO). Human Hb was used to prepare a calibration curve.

hl

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Figure 1. (A) lH spin-echo NMR spectrum of 0.5 mL of packed rabbit erythrocytes in HDO/saline/glucose solution. A tz value of 0.06s was used in the spin-echo pulse sequence. Resonances of interest are identified according to refs 20 and 21. (B-E) Effect of increasing concentrations of As(II1) on erythrocytes. As(II1)concentrations were (B) 0.1 mM, (C) 0.2 mM, (D) 0.3 mM, and (E) 0.4 mM. The arrows indicate the resonances of Cysa and Cysp protons of GSH. Biochemical Separation Methods. (A) Uptake by Packed Erythrocytes. Intact erythrocytes (100 pL) were incubated 30 min a t 25 "C with 0.4 mM [73Aslarseniteor [73Aslarsenate (4 replicates). Cells were then washed once with 154 mM NaCl and 0.1% D-glUCOSe solution in DzO and separated by centrifugation (5 min, SOOOg, 25 "C), and the cell pellet was radioassayed for 73As, (B)Ultrafiltration. Microconcentrators with a 10 000-Da cutoff (Microcon, Amicon) were used to separate proteins from hemolysates (100pL) containing radiolabeled 73As(111 or V). Ultrafiltrates and retentates were radioassayed to determine the percentage of total 73Aspresent in low and high molecular weight fractions.

Results 'H-NMR Spectroscopy. SEFT 'H-NMR spectra of rabbit erythrocytes were measured using a spin-echo delay period, t2, of 0.06 s. Most of the Hb resonances are selectively eliminated because their spin-spin relaxation times are short relative to the delay period. Resonances in the 4.8-1.0 ppm region (Figure 1A) arise from small molecules in the erythrocyte cytosol such as glycine, lactic acid, ergothioneine (el), creatine, and glutathione (18-21, 24-27). The phase differences of these signals were due to differing degrees of phase modulation accumulated during the spin-echo delays arising from proton-proton spin coupling. Resonances

NMR Studies of Arsenic in Rabbit Erythrocytes

c Chem. Res. Toxicol., Vol. 7, No. 5, 1994 623

His143-CZ

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Figure 3. Effect of increasing concentrations of h ( V ) on packed rabbit erythrocytes @EFT NMR spectra with 72 = 0.06 8). Adv) concentrations were (A) zero, (B)0.4mM, and (C) 3.2 mM. The arrows indicate the resonances of Cysa and Cysp protons of GSH.

0.4mM

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Figure 2. Portions of the IH spin-echo NMR spectra obtained for the same samples as in Figure 1,using a 9 value of 0.015 s in the spin-echo pulse sequence.Resonances indicated by arrows correspond to C-4 and C-2 hydrogens of some of the Hb imidazole side chains.

in the 6.85-8.3 ppm region are due to imidazole groups of some of the Hb histidine residues (28,30,31f and can be observed using a t 2 value of 0.015 s (see Figure 2A). Figure 1A is an expanded portion of the SEFT spectrum of rabbit erythrocytes (tz = 0.06 s). Figure 1B shows the spectrum for the same cells immediately after addition of As(II1) to a final concentration of 0.1 mM. Significant changes were observed for the a-CH and j3-CH2 protons of the cyteinyl residue of GSH (indicated by arrows), indicating the complexation of As(II1) by intracellular GSH. With higher concentrations of As(II1) (Figure lC,D), the resonances of Cysa and Cysj3 of GSH were significantly broader and decreased in intensity. Addition of more As(1II) to a final concentration of 0.4 mM led to the complete disappearance of the resonance of the j3-CH2 protons of the cyteinyl residue (Figure 1E). Further addition of As(II1) did not induce any changes of the spectrum. Modifications in resonances of cysteinyl protons were correlated with a stepwise decrease in intensity and broadening of the Glya proton signal of GSH. There were no apparent changes in the resonances of intracellular glycine, creatine, lactate, or ergothioneine, indicating no detectable complexation of As(II1) by these potential ligands. Results presented in Figure 2 indicate that As(II1) also induced small but significant structural changes in Hb. As As(II1) was added, resonances in the 6.85-7.5 and 7.5-8.5 ppm regions were due to the C-4 and C-2 hydrogens, respectively, of the imidazole side chains.

Small but significant stepwise upfield shifts (0.1 ppm) and a small decrease in intensity of the resonances a t 6.98 and 7.87 ppm were observed. Especially notable were the broadening and decrease in intensity of the resonance at 8.47 ppm, which was previously assigned to His97, located close to Cys93 (32). The SEW spectra of a sample containing purified rabbit Hb and As(II1) were collected; a similar decrease and broadening of the His97 resonance could be observed (data not shown). To ensure that His resonance modifications were not arising from a direct interaction of As(II1) with His, As(II1) was added to a buffered solution of histidine (pH 7.3); no spectral modification was observed when compared to the control (data not shown). These results support the hypothesis that As(II1)binds to Cys93 and not to other neighboring residues of Hb. Figure 3 shows SEFT spectra (72 = 0.06 s) of packed erythrocytes obtained in the presence of increasing concentrations of As(V). Trace A shows the spectrum obtained for packed cells without added As(V). No significant changes were observed h after addition of 0.4 and 3.2 mM As(V), respectively (traces B and C). The arrows in Figure 3 indicate resonances of Cysa and Cysp protons of GSH. At 9.3 mM As(V) (data not shown), a concentration 50 times higher than that of As(II1)which induced maximal effects on packed cells, spectral features were greatly broadened. However, the chemical shifts of GSH protons or of other small molecules were not significantly altered. The resonance a t about 1.29 ppm was assigned to the methyl protons of lactate, a product of red cell metabolism of glucose (24). Its increase indicates that the cells continued to metabolize glucose during the time between the recording of spectra A, B, and C. Using the spin-echo sequence with ~2 = 0.015 s to observe Hb, no significant changes of Hb resonances could be detected (data not shown).

Delnomdedieu et al.

624 Chem. Res. Toxicol., Vol. 7, No. 5, 1994 Cysa

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Figure 4. Effect of increasing concentrations of As(II1) on hemolyzed rabbit erythrocytes (SEFT NMR spectra with tz = 0.06 s). As(II1) concentrations were (A) zero, (B) 0.4 mM, and (C) 0.8 mM. "he arrows indicate the resonances of Cysa and Cysp protons of GSH.

In order to determine if plasma was required to reduce AdV) to As(II1)before its entry into erythrocytes, packed cells (0.5 mL) were mixed with 0.5 mL of fresh plasma and As(V) was added to a final concentration of 0.4 mM. After 2 h of incubation, the cells were separated from the plasma by centrifugation (5 min, 3000g,4 "C) and SEFT spectra of the cell pellet were collected. Spectra obtained from cells exposed to As(V) in the presence of plasma did not differ from those obtained from cells exposed to As(V) in the absence of plasma (data not shown). To determine whether the lack of effect of As(V) on GSH and Hb was only due to its inability to cross the erythrocyte membranes, or rather to its interaction with different intracellular binding sites, 0.4 mM As(V) was added to hemolysate. SEFT spectra of this sample showed no changes, proving that the membrane was not the limiting step for the interaction between As(V) and GSH or Hb (data not shown). To verify that the hemolysate system was similar to the packed erythrocyte system, the effect of a range of As(II1) concentrations on the SEFT spectra of hemolysate was examined (Figure 4). In the presence of 0.4 mM As(II1) (trace B), the intensity of the Cysa and Cysp protons of GSH decreased as described previously for intact erythrocytes. With addition of 0.8 mM As(III), the signals of those protons disappeared. The Glya protons of GSH also exhibited a stepwise decrease in intensity as the signals of the Cys protons disappear, as already observed with intact erythrocytes.

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Figure 5. Effect of increasing concentrations of DMSA on packed rabbit erythrocytes containing 0.2 mM As(II1) (SEFT NMR spectra with zz = 0.06 s). DMSA concentrations were (A) zero, (B) 0.4 mM, (C) 0.8 mM, and (D) 1.2 mM. The arrows indicate the resonances of Cysa and Cysp protons of GSH. Determination of NPSH and Hb. Determination of NPSH and Hb contents in packed and hemolyzed erythrocyte samples was as follows: packed erythrocytes contained 0.70 mM NPSH and 11g of hemoglobiddl of blood. For hemolysates, NPSH was 0.38 mM and Hb was 6-7 g/dL of blood. Reversibility. It was recently shown in aqueous solution that a dithiol molecule could displace As(1II) from its GSH complex (11). To determine whether the binding of As(II1) to intracellular sites (GSH, Hb) was reversible, a range of DMSA from 0.2 to 1.2 mM was investigated in 0.5-mL samples of packed erythrocytes containing 0.2 mM As(II1). SEFT spectra are presented in Figure 5. Trace A displays the SEFT spectrum of packed cells incubated with 0.2 mM As(II1) as described previously (Figure 10. The small differences that can be observed between Figures 1C and 5A are attributable to two different blood donors. Trace B was obtained after addition of 0.4 mM DMSA. The resonances of Cy@ reappeared as well as the one of Cysa from free GSH, proving that the presence of a dithiol species released GSH from its As(II1) complex. With a concentration of 0.8 mM DMSA (trace C) this phenomenon was more pronounced; the signals of Cysa and Cysp were comparable to those from a sample containing packed erythrocytes without As(II1). No further spectral changes were observed with addition of 1.2 mM DMSA, indicating saturation a t 0.8 mM DMSA. It is important to notice that even at 1.2 mM the DMSA signal could not be observed: a SEFT spectrum of a solution of DMSA a t

NMR Studies of Arsenic in Rabbit Erythrocytes the same pH would display a sharp peak at 3.3 ppm. Also, a SEFT spectrum of a n aqueous solution containing As(II1)-DMSA (pH 7.3) will display three sets of resonances: a broad peak at 4.0 ppm and two small peaks a t 3.6 and 1.4 ppm. These are also absent from the spectra presented on Figure 5B-D. Notably, high concentrations of DMSA broadened the spectra. A similar broadening was observed after adding 0.8 mM DMSA to erythrocytes, without As (data not shown). Because spectral broadening occurred in samples containing DMSA a t a high concentration, the complete reversibility of complex formation could not be demonstrated. 73AsUptake Experiments. As uptake experiments were carried out with erythrocytes as described in the Experimental Procedures. With 0.4 mM 73As(III),76% of 73Aswas found in the cells after '12 h (0.304 mM). A similar experiment with 7 3 A s 0found only 25%(0.1 mM) of 73As(V)taken up by intact erythrocytes. Ultrafiltration Experiments. Hemolysate from 73As(III)- or 73As(V)-loadedcells was ultrafiltered to determine the extent of protein binding of As. For As(II1)-containing lysate, 39% of 73Aswas bound to the protein fraction. About 28%of the protein-bound 73As(III) was not removed by exhaustive washing of the retentate with DzO. For As(V)-containing lysate, 36% was associated with the protein fraction. 31P-NMRSpectroscopy. 31P-NMR spectra were obtained for intact erythrocytes without As, with 0.8 mM As(III1, or with 0.8 mM As(V) after incubation at 35 "C for up to 5 h (Figure 6). The control (trace A) and the As(II1) sample did not show any significant variation of ATP resonance intensities over a 5 h incubation period. Resonances were identified according to ref 33. In contrast, when As(V) was added to packed erythrocytes, a significant decrease in ATP resonance intensities was observed at 3.5 h, without changes in the chemical shifts and with the appearance of Pi signal. ATP signals were no longer observable after 5 h incubation, and Pi resonance increased correlatively. No changes of the phosphate chemical shifts occurred, proving that the modifications observed were not due to variation of internal pH (34).

Discussion As(II1) forms complexes with a variety of different donor groups; however, sulfhydryls are generally considered to be the most important binding site for As(II1)in biological systems. The complex of As(II1) with GSH has been recently characterized as (GS)&(III) (8, 9). In rabbit erythrocytes, the most abundant sulfhydrylcontaining molecules are Hb (-5 mM), with eight SH groups, GSH (-2.8 mM), ergothioneine (-0.2 mM), and membrane proteins which contain about 6% of the total sulfhydryls of erythrocytes (35). On the basis of our previous studies of As(II1) complexation with monothiol (GSH) and dithiol molecules (DMSA)in aqueous solution (9,11), the aim of the present study was to determine if As(II1)-GSH complexes would be formed in rabbit erythrocytes and to compare the efficacy of the two As species, As(II1) and As(V), in formation of the complex. The results presented in Figures 1, 2, and 4 indicate that As(II1) is rapidly complexed by GSH and most likely by Hb in both erythrocytes and hemolysates. Thus, As(II1) binds to the sulfhydryl group of GSH in intact rabbit erythrocytes a t physiological pH; this observation confirms and extends previous work performed in aque-

Chem. Res. Toxicol., Vol. 7, No. 5, 1994 626

0.8mM As(V) after 5h Incubation

0.8mM As(V) after 3h1/2 incubation

0.8mM As(iIi) from 0 to 5h Incubation

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Figure 6. Effect of As(II1) or A s 0 on packed rabbit erythrocytes after incubation at 35 "C for up to 5 h (31P-NMRspectra).

(A and B)Unchanged spectra over 5 h incubation for a control and an erythrocyte sample with 0.8 mM As(III), respectively. (C and D) 0.8 mM AsW) after 3.5 and 5 h incubation, respectively. DPG, 2,3-diphosphoglycerate; Pi, inorganic phosphate.

ous solution (8,9).However, the effect of As(II1)binding on the NMR properties of GSH was somewhat different for GSH in erythrocytes compared to aqueous solution. Titration of GSH with As(II1) in aqueous solution caused changes in the chemical shifts of Cysa and Cysp resonances of GSH (data not shown). However, these resonances did not disappear in aqueous solution as they did in erythrocytes. The disappearance of the GSH resonances in As(II1)-exposederythrocytes, mostly for Cysa, Cysa protons and the broadness of Glya protons, suggested a significant decrease in the GSH spin-spin relaxation time (T2)complexed by As(II1). It can be proposed that a ternary complex involving GSH, As(III), and a macromolecule, possibly Hb, was formed. In this ternary complex GSH would have motional characteristics (and thus T2 values of its protons) similar to those of Hb protons. Experiments with As(II1) in hemolysate yielded the same results as found with erythrocytes. The slight difference in the saturation concentration for As(II1) was probably due to GSH released in the cytosolic fraction during cell lysis. The small but significant stepwise upfield shift (0.1 ppm), decrease in intensity, and broadening of the resonances at 6.98, 7.87, and 8.47 ppm as erythrocytes were titrated with As(II1) suggested that As(II1) bound to the /Ichain of Hb. This interaction of As(II1) with Hb was particularly indicated by the perturbation in the resonance of His97 (8.47 ppm) which is located near

626 Chem. Res. Toxicol., Vol. 7, No. 5, 1994 Cys93 (321, a probable site of As(II1) binding to Hb. The broadness of the peak indicated a decrease in local mobility of the His97 imidazole side chain upon As(II1) addition. These histidine resonances have been found to be affected by Zn2+ (181, Cd2+ (251, and Hg2+ (21), suggesting that these metals and As(II1) induce similar changes (possibly due to binding) in Hb. The large decrease in the intensity of the Glya protons of GSH (Figure 1)was unexpected as our previous 13CNMR results ( 9 )indicated that the predominant As(II1) binding site was the sulfhydryl group of GSH. The absence of changes in the Glya chemical shift was also consistent with no complexation of As(II1) by the glycine carboxylate group. Rabenstein and co-workers (19)have also observed a similar decrease in the Glya signal which was not due to complexation. They showed that the intensity of a n AB pattern was phase modulated in the SEFT spectrum, which caused the considerable decrease in its intensity even though complexation was a t the relatively distant sulfhydryl group. The fate of As(V) in rabbit erythrocytes was quite different from that of As(II1). Following ' / 2 h incubation with 0.4 mM As(V), erythrocytes attained a final concentration of only 100 pM. In contrast, erythrocytes incubated '/2 h with 0.4 mM As(II1) attained a final concentration of 304 pM. NMR data display no significant perturbation of GSH or Hb signals in As(V)-exposed cells, indicating that neither molecule binds As(V) to a significant degree. For example, even at a concentration about 50 times greater than that of As(II1)which consumed all of cellular GSH by the formation of As(II1) complex, very little As was associated with GSH in As(V)-exposedcells; furthermore, a t this high As(V) concentration, there was no evidence of an interaction of AsW) with other observable intracellular ligands. These findings underline the fact that, in erythrocytes, unlike what was shown in aqueous solution containing GSH and As(V) (9),As(V) was not reduced to As(II1) during the time course of the experiment (30 min). As shown by 31P-NMR,As(V) accumulated in rabbit erythrocytes enters the phosphate pool of the cell and depleted high-energy nucleotides (ATP). Although 31P-NMRdata presented here required slightly different experimental conditions (35 "C, 0.8 mM As), similar results could be obtained at 25 "C and 0.4 mM As;however, the depetion of high-energy metabolites took longer and the control erythrocyte preparations showed signs of metabolic slowdown. These results are supported by an earlier report that As(V) incubated with rat liver mitochondria generated a n arsenate ester (36). Its formation depended upon the electron transport chain and oxidative phosphorylation. In rat kidney slices, phosphate also inhibited the uptake and metabolism of As(V) ( 171, and exposure of mice and rat to high doses of arsenate produced damage of hepatic mitochondrial function related to a significant accumulation of As in these organelles (37). In contrast, rabbits injected with arsenite did not show any retention in hepatic, renal, or pulmonary mitochondrial fractions (38). The antidote DMSA, a vicinal dithiol molecule (39), readily released GSH from the (GS)&(III) complex, forming a (DMSA)3(As(III))2complex (211. To evaluate the physiological relevance of this result, increasing concentrations of DMSA were added to intact erythrocytes containing 0.2 mM As(II1). As presented in Figure 5 , 0.8 mM DMSA restored GSH resonances, reflecting a total release of reduced GSH, as was previously observed

Delnomdedieu et al. in aqueous solution (111. Because DMSA does not enter the erythrocytes (40), it is likely that mass action dissociates the intracellular As(III)(GS)3 complex and yields As(III), which is transported to the extracellular compartment of the assay system. A significant broadening was also observed in Hb spectra upon addition of DMSA, with or without As(II1). Ciriolo and co-workers (41 reported that addition of GSH (molecule that does not cross membranes) to intact erythrocytes was undetectable by spin-echo techniques. They attributed this phenomenon to noncovalent interactions of GSH with the membrane surface in a very fast exchange dynamic that average out the relaxation values characteristic of the membrane-bound coumpounds. They also observed that extracellular GSH induced a transduction of reducing power through a thiol-rich membrane protein via a thiol/ disulfide interchange mechanism. By analogy, it is possible that DMSA, which releases As(II1) from erythrocytes, will also be undetected by NMR. However, transduction of the reducing power of DMSA via a thiol/ disulfide exchange will result in spectral broadening. The work presented here establishes by three different physical methods (NMR, radioisotope uptake, and ultrafiltration) striking differences between the behavior of As(II1) and As(V) in rabbit erythrocytes after V2-h incubation. As(II1) readily enters the cells and binds to GSH and possibly Hb. The interaction of As(II1) with Hb may involve direct complexation and the formation of ternary complexes of As(III)(GS)and Hb. In contrast, a smaller amount of AdV) enters the cells, inducing a depletion in high-energy nucleotides (ATP) and a rise in Pi level, without binding to GSH or Hb. Differences in the penetrance of As(II1) and As(V) into mouse erythrocytes have been reported (42). A possible explanation for the differences in uptake is the ionization a t physiologic pH for the two forms: As(II1) is un-ionized [As(III)(OH)3, lowest pKa = 9.231 whereas As(V) bears two negative charges [As(V)(O~H)~-, lowest pKa = 2.201 (22). The differences between As(II1) and As(V) may explain the more rapid excretion and lower toxicity of As(V) (43-46). Binding of As(II1) to GSH and other target molecules (e.g., Hb) may yield species which are more persistent than As(V). Concentration-dependent differences in capacity for As(V) reduction in cells have not been explored, but whole-body retention of As(V) and As(II1) differed in mice exposed to high doses of these agents (42). Because inorganic As is rapidly converted into mono- and dimethylated forms in most species (47), additional studies of the fate of these organic arsenicals in erythrocytes are underway.

Acknowledgment. M.D. was supported by funds provided by the U.S.Environmental Protection Agency through the Center for Environmental Medicine (Cooperative Agreement CR8176431, and M.S. was supported by the Curriculum in Toxicology (Training Grant T901915), University of North Carolina, Chapel Hill, NC. We thank Karen Herbin-Davis, Mantech Environmental Technology, for her outstanding technical assistance. The research described in this paper has been reviewed by the Health Effects Research Laboratory of the U.S. Environmental Protection Agency and approved for publication. Approval does not signify that the contents necessarily reflect the views and policy of the Agency, nor does the mention of trade names or commercial products constitute endorsement, or recommendation for use.

NMR Studies of Arsenic in Rabbit Erythrocytes

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