The interaction of phenyldichloroarsine with erythrocytes

J BIOCHEM TOXICOLOGY Volume 4, Number 1, 1989

The Interaction of Phenyldichloroarsine with Erythrocytes Steven Chong, Kilian Dill, and Evelyn McGown Division of Biophysical Research, Letterman Army Institute of Research, Presidio of San Francisco, California 94129-6800, USA

ABSTRACT: The purpose of the study was to iden- tify binding sites of organic arsenic in the erythrocyte and to explain species differences in binding. Washed erythrocytes were exposed to graded concentrations of [U-14C]phenyldichloroarsine (PDA) in phosphate-buffered saline containing 0.1% glucose and 0.1% bovine serum albumin. At low PDA concentrations, all cells bound the arsenical rapidly (within 10 min) and quantitatively. Human, pig, hamster, guinea pig, and mouse erythrocytes approached saturation at 0.02-0.3 pmol PDA/109 cells, depending on the species. Satu- ration points correlated well with each respective species’ erythrocyte glutathione content. In contrast, rat erythrocytes showed no sign of saturation at PDA loads as high as 3.0 pmol/lOg cells. Hemolysates of PDA-treated erythrocytes were subjected to Sephadex G-75 gel filtration chromatography. I4C from rat hemolysate was distributed between the hemoglobin and small molecular weight (glutathione-containing) fractions. In all other species, the “C eluted almost exclusively with the glutathione-containing fractions. In equilibrium dialysis experiments, human hemoglobin did not bind PDA, whereas rat hemoglobin bound 2 PDAlmol with Kd = 5 pM. In conclusion, glutathione is the principal binding site of phenyldichloroarsine in erythrocytes. In most species, the arsenical does not bind to hemoglobin, even though it has free (titratable) sulfhydryls considerably in excess of the glutathione concentration. In rat erythrocytes, phenlydichloroarsine binds both to glutathione and to hemoglobin. Arsenical binding by rat hemoglobin is presumably due to the unique location of the extra titratable cysteine in that protein.

KEYWORDS: Arsenical, Sulfhydryl Reagent, Glu- tathione, Erythrocyte, Hemoglobin.

Received September 28, 1988. Address correspondence to Dr. Evelyn McGown, Division of

Biophysical Research, Letterman Army Institute of Research, Presidio of San Francisco, CA 94129-6800, USA.

INTRODUCTION

Organic arsenicals containing a single lipophilic alkyl or aryl group are among the most toxic arsenic compounds known (1,2). Examples include chlorovinyldichloroarsine (lewisite) and phenyldichloroarsine (PDA). Upon dermal contact, they cause severe local damage, readily penetrate tissue, and enter the blood stream to be distributed throughout the body (2,3). In the blood, lipophilic arsenicals are carried primarily by the erythrocytes, with little in the plasma, but they are readily transferred to other tissues.

Although it is well known that erythrocytes have a high capacity for arsenicals (1,2), the exact nature of the binding is not known. One would expect the binding site(s) to be sulfhydryl-containing molecules, the only known biological targets of trivalent arsenic. In the erythrocyte, therefore, the obvious candidates are hemoglobin and glutathione (GSH). Hemoglobin contributes the bulk (2 80%) of the exposed sulfhydryls, followed by glutathione with 20% or less (4). Erythrocyte membranes and ergothioneine account for less than 5% of the total free sulfhydryl content.

The purpose of this study was to characterize the binding of PDA by erythrocytes in vitro. Our first approach to the problem was to quantify the uptake of [U-14C]PDA. We then subjected the hemolysates to gel filtration chromatography to determine if the arsenical was protein bound. Upon finding that rat and human erythrocytes differed strikingly in their disposition of PDA, we expanded the study to survey other species.

MATERIALS AND METHODS

Reagents Phenyldichloroarsine was purchased from Re-

search OrganiclInorganic Chemical Corp., Sun Val-

0887-2082/89/$1.00 + .25 39

40 CHONG ET AL. Volume 4, Number 1, 1989

ley, CA. It was purified by distillation at 128°C under nitrogen at 15 torr, a procedure which removed any nonvolatile contaminants. The purity ( > 99%) was verified by infrared spectrophotometry. Stock solutions of PDA were prepared 50 mM in anhydrous ethanol. [U-14C]PDA (5.6 mCilmmo1) (Wizard Labo- ratories, Davis, CA) was diluted 1 to 10 with unlabeled PDA and distilled. Radiochemical purity was greater than 98% according to thin-layer chromatography on silica gel using benzenelacetic acidlisopro- panol(l7:1:2) as the elution solvent. Crystalline GSH and rat and human hemoglobin were purchased from Sigma Chemical Co., St. Louis, MO.

Animal and Erythrocyte Experiments Sprague-Dawley rates (Ruttus nomegicus), Swiss

Webster mice (Mus musculus), and Syrian golden hamsters (Mesocricetus auratus) were purchased from Charles River, Wilmington, MA. Hartley guinea pigs (Cuviu porceZZus) were purchased from Simonsen Labs, Gilroy, CA, and York duroc pigs from Bos- well's Farm, Corcoran, CA. Small animals were sacrificed with carbon dioxide gas and exsanguinated by cardiac puncture. Blood was obtained from pigs through indwelling venous catheters. Human blood was obtained from volunteers by venipuncture. All blood was collected into ethylenediaminetetraacetic acid (EDTA)-containing syringes, and sample ma- nipulations were done at room temperature, unless specified otherwise.

Blood samples were centrifuged for 10 min at 400 g , the plasma and buffy coat were removed, and the erythrocytes were resuspended to the original volume in phosphate-buffered saline (pH 7.4) containing 0.1% glucose and 0.1% bovine serum albumin (PBS-G-A). The centrifuge step was repeated, and the cells were resuspended to 2 x lo9 cellslmL in PBS-G-A. They were then mixed with an equal volume of PBS-G-A containing 20 pM [14C]PDA (1.4 x lo6 DPMlpmol). Unlabeled PDA (serially diluted) was added in a 10 pL aliquot to each tube to achieve a final PDA concentration of 0.01 to 3.76 mM and a final cell concentration of lo9 cellslml. After a 10-min incubation, the tubes were centrifuged for 1 min at 15,000 G, and 14C was determined in 0.5 mL of the supernatant with a Packard Tricarb 4530 liquid scintil- lation counter.

Gel Filtration Chromatography Washed erythrocytes were incubated for 10 min

at room temperature with 0.24 pmol [14C]PDA1109 cells (enough to saturate the estimated primary binding sites). Excess PDA was removed by washing three times in PBS-G-A, and the final pellets were hemolyzed in water. The hemolysates were centri-

fuged at 15,000 g for 15 min to pack the membranes as a pellet, and 1 mL of each supernatant was applied to a Sephadex G-75 (Pharmacia) column (2 x 25 cm) that had been preequilibrated with PBS, pH 7.0. The flow rate was adjusted to 0.2-0.3 mLlmin, and l-mL fractions were collected for measurement of 14C, hemoglobin, and GSH. Total recovery of 14C from the columns ranged from 82 to 88%.

Hemoglobin-PDA Dialysis Experiments Rat and human hemoglobin were purchased from

Sigma or prepared freshly. In the latter case, washed erythrocytes were hemolyzed in water and centrifuged to remove stroma. GSH with other small molecular weight material was removed by passing the hemolysate through a mixed-bed ion-exchange column (26 x 100 cm, BioRad AG501-X8), which yielded a solution with conductivity less than 10 pmhos. The hemoglobin preparations were diluted to 0.1 mM in PBS, pH 7.0, graded concentrations of [14C]PDA were added, and the solutions were placed in dialysis chambers. They were dialyzed with gentle agitation against equal volumes of PBS, pH 7.0, at 5°C through a membrane with a cutoff of 3,500 Da. Samples were removed from both chambers at timed intervals for 14C measurements until equilibrium was reached (24-48 hr). The data were evaluated by standard ligand binding techniques (primarily Scat- chard plots) (5).

GSH-PDA Interaction Experiments The interaction of PDA and GSH could not be

evaluated with the same methods as was the hemoglobin-PDA interaction, for two reasons: (1) ordinary dialysis membranes are permeable to GSH and (2) the Scatchard model does not apply to reactions in which the 1igand:substrate stoichiometry is less than 1:l (6). To get around the first problem, we devised an experiment in which PDA-loaded erythrocytes served as "minidialysis bags" in which the substrate (GSH) was entrapped but the ligand (PDA) was free to traverse the membrane. Total GSH was held constant by using equal numbers of erythrocytes in a series of tubes. The fraction of bound PDA versus free PDA was controlled, not by varying the amount of PDA added, but instead by varying the total (extracellular) volume. Human erythrocytes were exposed to sufficient PDA to obtain at least 90% saturation of GSH. They were then washed three times in PBS and resuspended. Equal aliquots were placed into tubes and diluted with PBS such that the final erythrocyte concentration ranged from 6 x lo5 to 2 x l o9 cellslml. In this way, the total GSH concentration was held constant because of its intracellular location, but the fraction of PDA bound

Volume 4, Number 1, 1989 ORGANIC ARSENIC AND ERYTHROCYTES 41

decreased with increasing extracellular volume. The erythrocyte suspensions were centrifuged after 1 hr, the supernatants carefully removed, and the I4C was determined in the supernatant (free PDA) and the pellet.

The second problem was due to the stoichiometry of the GSH-PDA reaction (given below) and to the fact that PDA (the measured variable) is considered to be the “ligand” and GSH the ”substrate.”

- 2HC1 4 - AsC12 + 2GSH 4 - As(GS)2

The 1igand:substrate ratio is 1:2, in contrast to a ratio of 2:l for the Hb-PDA interaction. The Scatchard model assumes the ligandlsubstrate is greater than or equal to 1, in which case the fraction of occupied substrate sites is a function of one variable only, the free ligand concentration. When the 1igand:substrate stoichiometry is less than 1:1, however, the fraction of substrate sites occupied is a function of two variables, the free ligand and substrate concentrations (6) . Because Scatchard analysis was not appro- priate, we estimated the overall dissociation constant (the product of the stepwise dissociation constants) by calculating the apparent & at each point in the dilution series and taking the average of those data points spanning 2040% saturation of GSH. The calculations required several assumptions which could not be rigorously proven. Initial GSH concentration [GSHini,] was calculated by dividing the total GSH in the erythrocyte suspension by the hematocrit of that suspension. The free PDA concentration was calculated from supernatant 14C. PDA boundlGSH was taken as the PDA in the pelletlthe total GSH in the pellet. All PDA in the pellet was assumed to be 4- AS(GS)2. [GSH], [+-As(GS)& and Kd were calculated according to the following equations:

[PDA] [ GSH] Kd=

[$-As(GS)21 [4- As(GS)~] = [PDA boundlGSH][GSHwt]

[GSH] = [GSHhit] - 2 x [4 - As(GS),]

For these experiments, we made three crucial assumptions that could not be rigorously proven, but that we believe are valid. Firstly, we assumed that greater than 90% of cell-associated PDA was bound to GSH. Second, we assumed that the erythrocyte membrane is freely permeable. This latter assumption is based on our observations that PDA binding to erythrocytes reaches equilibrium within minutes and that, upon washing the cells repeatedly, small amounts continue to leach out at each resuspension step and reestablish a low extracellular PDA concentration. Third, we have disregarded the first step in the association process that would produce the PDA-GSH monoadduct species. High-performance liquid chromatography (HPLC) and nuclear magnetic resonance (NMR) evidence indicates that this species exists only as an exceedingly minor component. Addition of GSH to PDA produces +-AS(GS)~ preferentially.

GSH and Hemoglobin Analysis Erythrocyte GSH (before PDA treatment) was

measured with an assay based on the catalytic action of GSH on the reduction of 5,5’-dithiobis-(2-nitro- benzoic acid) in the presence of glutathione reductase and NADPH (7). In gel chromatography fractions, GSH was assayed spectrophotometrically with 4,4‘- dithiodipyridine, which reacts irreversibly with sulfhydryls (8). The latter reagent was not inhibited by the presence of PDA and, thus, could be used on PDA-containing samples. Hemoglobin was assayed by the standard cyanornethemoglobin method (9).

RESULTS

PDA Uptake by Erythrocytes Uptake of PDA by erythrocytes reached equilib-

rium in less than 5 min. Rat erythrocytes clearly had a higher capacity for the arsenical than did the other species. Even at PDA loads as high as 3 pmol/109 cells, over 80% was bound (Figure 1) (higher PDA loads caused hemolysis). In contrast to the rat, other species’ erythrocytes showed signs of saturation (Figure 1). In order to focus on the uptake at low PDA concentrations, the data were plotted as extracellular versus total PDA (rat and mouse erythrocytes are illustrated in Figure 2). At the lowest PDA levels, all species’ erythrocytes took up PDA quantitatively. There was essentially no extracellular I4C until the

4,001

/

0.00 1.00 2.00 3.00 4.00

TOTAL PDA (ILm0~/109 cells)

FIGURE 1. Uptake of FDA by erythrocytes of several species. Erythrocyte suspensions (lo9 cellsiml) were exposed for 10 min to graded concentrations of [“C] PDA and centrifuged, and the radioactivity of the supernatant was measured.

42 CHONGETAL. Volume 4, Number 1, 1989

FIGURE 2. Low-level PDA binding by mouse and rat .32-

erythrocytes ( lo9 celldml). The number of primary binding sites was taken to be the threshold where the plot rose above the x axis. The threshold was estimated by visual inspection of the data points.

.28-

5 .24- > E a .20- - d n. ~ .16- 5 3 yJ .12-

d 5 .on-

4

r

.04-

0- 0 .10 .20 .30 .40 S O .60 .70 .80 .90 1.00 1.10

TOTAL PDA (pmol/ml)

PDA level exceeded a threshold region. Above that threshold, most PDA remained extracellular (except for rat), indicating little additional uptake. The rat erythrocyte also exhibited quantitative uptake at low PDA concentrations and a distinct threshold region. Above the threshold some of the PDA remained extracellular, but binding still continued at greater than 80% of total. We defined the threshold values (normalized to cell number) as "primary binding sites" and estimated their numbers by visually ex- trapolating the plots back to the x axis. The number of primary binding sites varied considerably between species and ranged from a low of 0.02 pmol/109 cells in pig erythrocytes to a high of 0.28 pmol/109 cells in guinea pig erythrocytes (Table 1). These values were all an order of magnitude lower than the free sulfhydryl content of hemoglobin (1.8-3.6 pmol/109 cells), but they were similar to the GSH contents of the respective erythrocytes (Table 1 and Figure 3). Primary PDA binding sites and GSH both were lowest in the pig and highest in the guinea pig erythrocyte. The estimated number of primary binding sites was approximately one-half of the erythrocyte GSH content.

TABLE 1. Primary PDA Binding Sites and GSH Content of Erythrocytes

PDA Binding Sites GSH Content Species (n) ( p ~ ~ l l l o ~ cells)" (pmol/109 cells)"

pig (2) 0.02 0.045 (0.04-0.05) Hamster (2) 0.05 0.15 (0.14-0.17) Human (3) 0.075 (0.07-0.08) 0.20 (0.13-0.24) Rat (4) 0.10 (0.08-0.14) 0.15 (0.13-0.17) Mouse (3) 0.10 (0.09-0.12) 0.16 (0.13-0.20) Guinea Pig (2) 0.26 (0.24-0.28) 0.43 (0.40-0.46)

Intracellular PDA Location When [I4C]PDA-tagged erythrocytes were lysed

and centrifuged to remove the membranes, almost all of the radioactivity remained in the supernatant (all species examined). This suggested that any membrane-bound PDA was minor compared to that bound by the cytoplasm. Gel filtration chromatography was then used to examine the intracellular distribution of PDA between small and large molecular weight (protein) material. In the mouse hemolysate, the bulk of the [I4C]PDA eluted with small molecular weight fractions and was clearly separated from hemoglobin (Figure 4A). In contrast, the ra- diolabel in rat hemolysate was distributed between the large and small molecular weight fractions (Figure

7 G

G

H H E 0 4 j P P

00 ! 1 I I I , ! , I I I 00 0 5 10 15 20 25 30 35 4 0 4 5 50

GLUTATHIONE (pmol/109 4 1 s )

FIGURE 3. Scatter plot of estimated primary PDA binding sites versus erythrocyte GSH content. Each letter represents results from one experiment with pig (P), hamster (H), mouse (M), rat (R), human (H), and guinea pig (G). Mean (range).


w

l- 0 5 10 I5 2 0 2 5 30 35 4 0 4 5 50 5 5 60

301 h ~ ~ ~ 1 , 10

5

0

FRACTION NUMBER

A

0-0 DMP lo3 A---A Hb m g / m l x 0 1 0. ..... -0 GSH nmol /ml . 0 5

0 5 10 I5 2 0 25 30 35 4 0 45 50 5 5 60

FRACTION NUMBER B

FIGURE 4. (A) Gel filtration chromatogram of hemolysate from mouse erythrocytes that had been exposed to 0.24 pmol ['*C]PDA/ lo9 cells (B) Gel filtration chromatogram of hemolysate from rat erythrocytes that had been exposed to 0.24 pmol [14C]PDA/109 cells.

4B). In both cases, the first I4C peak eluted slightly ahead of the hemoglobin peak, which suggested that some PDA was bound to protein(s) larger than hemoglobin. The second I4C peak was consistently one to two fractions behind the GSH peak. This was probably due to a partial separation of free GSH from the GSH-PDA adduct. Chromatograms of human and guinea pig hemolysates were essentially identical to that of the mouse. These results indicated that, in the rat erythrocyte, PDA was bound both to protein and to small molecular weight material, presumably GSH. In the other species examined, PDA was bound almost exclusively to GSH.

Equilibrium Dialysis Analysis of PDA Binding to Hemoglobin and GSH

When PDA was put into dialysis chambers with human hemoglobin, the arsenical readily dialyzed

away. This observation supported the conclusion drawn from the gel chromatography experiments that human hemoglobin does not bind PDA. Under the same conditions, rat hemoglobin retained the arsenical. The Scatchard plots for PDA-rat hemoglobin interaction were linear, whether the data were obtained with hemoglobin freshly prepared from hemolysate (Figure 5) or with commercial (met)hemoglobin. The slope reciprocals indicated at Kd value of 5 pM (4.4-5.2 pM). The calculated number of binding sites per hemoglobin tetramer was 2.3 for the fresh preparations and 1.6 for commercial (met)hemoglobin (averages of two experiments each).

The apparent overall Kd for the PDA-GSH interaction in intact human erythrocytes was 2.7 & 0.9 x 10-lo M2 (mean plus or minus the standard deviation of five separate experiments, 8-11 data points per experiment).

DISCUSSION

GSH is the primary target of PDA in the erythrocyte. This conclusion is based on several lines of evidence. First, the number of primary binding sites in each species is approximately half the respective erythrocyte GSH content, which is consistent with the stoichiometry of the PDA-GSH reaction. Thus, the 10-fold difference in primary PDA binding sites between the pig and guinea pig erythrocyte is explained by similar species differences in erythrocyte GSH content. Second, most of the PDA taken up by the erythrocyte is not protein bound, but is associated with small molecular weight material (with the exception of the rat, which is discussed shortly). GSH, being the major nonprotein sulfhydryl in the erythrocyte, is the obvious candidate. Third, we

\ n 2 0.2- 3 0 m

0.1 -

0.0 0.5 1.0 1.5 2.0 2.5

BOUND

FIGURE 5. Scatchard plot of PDA interaction with rat hemoglobin (freshly prepared). Bound, PDA boundlGSH; Free, free PDA, nmollml. For this set of data, Kd = llslope = 5.2 x M and n = 2.3.

44 CHONGETAL. Volume 4, Number 1, 1989

previously used *H spin-echo (NMR) spectroscopic techniques and obtained evidence that PDA interacts with GSH in the intact guinea pig erythrocyte (10). We have also reacted PDA with GSH in vitro, purified the reaction product, and characterized it as a 1:2 adduct of PDA and glutathione (11).

In the rat, PDA binds to hemoglobin as well as GSH, which explains why rat erythrocytes have a higher capacity for arsenicals. The equilibrium dialysis experiments were complicated by precipitation problems. Rat hemoglobin is notoriously heteroge- neous and readily forms a precipitate below pH 8.6 (12). Thus, we were forced to raise the pH to 8.6 or to deal with partially precipitated samples. Surpris- ingly, the equilibrium dialysis results were virtually identical whether the experiment was done at pH 7.0 (in which case we took care to get representative aliquots of the suspensions) or at pH 8.6. The data indicated that rat hemoglobin binds 2 mol PDA per mole hemoglobin (tetramer) with Kd of 5 pM.

Rat hemoglobin differs from other species in that it has a higher titratable sulfhydryl (SH) content (4 versus 2 SHlmol) and higher total sulfhydryl content (10 versus 6 SHlmol) (4). The additional titratable cysteine, located at position 13 of the alpha chain (12), is presumably the PDA binding location in rat hemoglobin because the other titratable cysteine (0- 93) is in the same location as that of species whose hemoglobin did not bind PDA (13). Assuming rat hemoglobin binds 2 PDAlmol, the theoretical binding capacity of the rat erythrocyte (Hb + GSH) should be no more than 1.0 pmoll109 cells. But the actual capacity appears to be at least 3 pmol/109 cells (Figure 1). A likely explanation is that, at the higher PDA loads, Hb is partially denatured and additional sulfhydryls are exposed.

Even in the rat erythrocyte, GSH appears to be the preferred target at low PDA concentrations. This was inferred from the uptake experiments, where the threshold region was similar to the GSH content and considerably lower than that predicted by the hemoglobin content. It can also be calculated from the Kd values and the intracellular GSH and Hb concentrations that PDA binds preferentially to GSH in the rat erythrocyte. By rearranging the binding equations to isolate [PDA]boundl[PDA]l~ree on the left side and taking the initial free GSH to be 2 mM and hemoglobin-sulfhydryl concentrations to be 5 mM x 2 SHI mol, one obtains the following:

[+ - AS-Hb] [Hb-SH] 1 x lo-’ [PDAlfree Kd 5 ~ 1 0 - ~

- 2 ~ 1 0 3 - - -

Thus, at low PDA concentrations, the GSH adduct is favored over the Hb adduct by a factor of 7. As PDA

is increased, GSH should become nearly saturated before significant binding to Hb occurs. There was less 14C in the rat GSH chromatographic fractions than in the mouse GSH fractions, however, even though the erythrocytes had been exposed to equal amounts of PDA (Figures 4A, 4B). The explanation is obvious in the equations above. Because [GSH], but not [Hb-SH], is squared, the GSH-PDA equilibrium is more concentration dependent than the Hb-PDA equilibrium. One can easily calculate that, when the cells are hemolyzed (and the reactants are diluted by approximately 17-fold), Hb becomes favored over GSH. Thus, the water hemolysis step causes partial redistribution from GSH to hemoglobin.

None of the other species’ hemoglobin bound PDA appreciably. In the gel chromatograms, a small amount of protein-associated I4C in those hemolysates eluted distinctly ahead of the hemoglobin, sug- gesting that some minor protein(s) bind PDA; these could be cytoplasmic proteins or membrane frag- ments remaining after the centrifugation step. Even in the rat hemolysate, some 14C eluted slightly ahead of hemoglobin. The 14C and GSH in the gel chromatograms were also not strictly superimposable. The most likely explanation is that some free GSH is generated and partially separated during gel chromatography as a result of dilution and the rapid associationldissociation kinetics. When a standard solution containing [14C]PDA and GSH standards in a 1:2 ratio was chromatographed, the GSH peak was one fraction ahead of the 14C peak.

Mercurials readily react with hemoglobin, and, in fact, they have long been used to titrate the accessible SH groups (14, 15). Human hemoglobin binds 2 mol organic mercury (R-Hg-) at the 0-93 cysteine, accompanied by lowered heme-heme interaction and altered oxygen affinity (15). In the intact human red cell, part of methyl mercury is bound to hemoglobin and the remainder to GSH, with the binding affinity to GSH approximately 6 times greater (16, 17). It is not obvious why PDA does not bind to most hemoglobins when its (larger) mercuric counterpart, phenylmercuric chloride, does so readily. Trivalent organic arsenicals such as PDA are noted for reacting with 2 sulfhydryls per mole. Although a 1:l adduct with GSH can be detected by high-performance liquid chromatography (HPLC) (V. Boyd and E. L. McGown, unpublished results), the 1:2 adduct appears by far to be the most stable (11). Thus, one might presume that PDA does not bind to most species’ hemoglobins because of the lack of a second sulfhydryl ligand. The puzzling question then becomes why does rat hemoglobin bind PDA when the additional cysteine (a-13) is not located near another sulfhydryl? One possibility is that a carboxyl-containing amino acid (e.g., glutamic acid-116) is situated near the a-cysteine-13 such that PDA can chelate to a


sulfur and an oxygen atom. Precedence for such an unusual complex is found in the literature in the form of phenylarsine oxide-mercaptoacetic acid adduct (18).

ACKNOWLEDGMENT

The authors thank R. J . O’Connor for his assist- ance with the radiolabeled PDA.

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Documents

The interaction of phenyldichloroarsine with erythrocytes