(DMAIII) by Red Blood Cells - American Chemical Society

Yamato Shiobara, Yasumitsu Ogra, and Kazuo T. Suzuki*. Graduate School of Pharmaceutical Sciences, Chiba University, Chiba 263-8522, Japan. Received ...
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Animal Species Difference in the Uptake of Dimethylarsinous Acid (DMAIII) by Red Blood Cells Yamato Shiobara, Yasumitsu Ogra, and Kazuo T. Suzuki* Graduate School of Pharmaceutical Sciences, Chiba University, Chiba 263-8522, Japan Received July 5, 2001

The animal species difference in the metabolism of arsenic was studied from the viewpoint of the mechanism underlying its distribution in the form of dimethylated arsenic in red blood cells (RBCs). Dimethylarsinic (DMAV) and dimethylarsinous (DMAIII) acids were incubated with rat, hamster, mouse, and human RBCs, and the uptake rates and chemical forms of arsenic were determined. Although DMAV was practically not or taken up slowly by RBCs of all the present animal species, DMAIII was taken efficiently in the order of rat > hamster > human, RBCs of mice taking it up less efficiently and with a different pattern from the former three animals. Further, although DMAIII taken up by rat RBCs was retained, that by hamster ones was effluxed in the form of DMAV. The uptake of DMAIII and efflux of DMAV took place much more slowly in human RBCs than rat and hamster ones. The uptake of DMAIII by RBCs was inhibited on the oxidation of glutathione with diamide. Incubation of DMAIII, but not of DMAV, with a hemolysate produced a high molecular weight complex, which increases in the presence of glutathione, suggesting that DMAIII taken up by RBCs is retained through the formation of a complex with protein(s) specific to animal species, and effluxed from RBCs after being oxidized to DMAV. These results indicate that DMA is taken up by RBCs in the form of DMAIII, and that the uptake and efflux rates are dependent on the animal species, the effluxed arsenic being DMAV. The present results suggest that the uptake of DMA by RBCs is an additional contributing factor to the animal species difference in the metabolism of arsenic in addition to the reduction and methylation capacity in the liver.

Introduction Arsenic has long been known to cause the toxicity in humans (1), and has been correlated convincingly with cancers of the skin, lungs, liver, kidneys, and bladder (25). It has been generally believed that reduction and methylation of inorganic arsenic comprise detoxification mechanisms (3, 6-9). The toxicity of arsenic is known to be manifested differently depending on the animal species, owing to differences in the metabolic pathways (3, 10-13). The rat is one of the most tolerant species among animal species (10), and arsenite (iAsIII) is effectively transformed to dimethylated arsenic (DMA), and then the arsenic (possibly in the form of DMA) is mostly distributed to red blood cells (RBCs) (14, 15), which we also confirmed in a separate experiment (data not shown). On the other hand, the hamster is one of the animal species most sensitive to arsenic, and arsenite is methylated less efficiently than in rats and distributed to RBCs in only small quantities (16-18). Arsenite is known to be taken up by and methylated in the liver, and then DMA is distributed possibly in the form of DMA to RBCs in rats, but not in hamsters (1618). These observations suggest that the reduction and methylation capacity of the liver and/or the process of * To whom correspondence should be addressed. Fax/Phone: 8143-290-2891. E-mail: [email protected]. 1 Abbreviations: iAsIII, arsenite; iAsV, arsenate; DMA, dimethylated arsenic; DMAV, dimethylarsinic acid; DMAIII, dimethylarsinous acid; RBCs, red blood cells; diamide, azodicarboxylic acid bis(dimethylamide); DIDS, 4,4′-diisothiocyano-2,2′-stilbene disulfonic acid disodium salt; GSH, glutathione.

Scheme 1. Reduction and Methylation Pathways for Arsenic

uptake of DMA by RBCs may differ among animal species and be related with the difference in manifestation of the toxicity of arsenic among animal species. Inorganic arsenic has been proposed to be metabolized in the body through successive reduction and methylation reactions leading to the major urinary metabolite DMA, as illustrated in Scheme 1. Arsenite injected intravenously into rats was not taken up or at least not effectively taken up by RBCs, but was taken up by the liver, and then the arsenic accumulated in the RBCs in our preliminary experiment (data not shown). Further, the methylation reaction seems to be not or less possible in RBCs than in liver. Therefore, arsenic is assumed to

10.1021/tx015537k CCC: $20.00 © 2001 American Chemical Society Published on Web 09/01/2001

Difference in Arsenic (DMAIII) Uptake by RBCs

be effluxed in the form of DMA from the liver into the bloodstream and then taken up by RBCs. Although it has been documented that the reduction and methylation capacity is different among animal species (3, 6-9), it is not known whether there is any difference in the uptake of DMA among RBCs of different animal species. It is also not known why arsenic accumulates preferentially in RBCs in rats (16-18). Therefore, in the present study, we examined in vitro the difference in the uptake of DMA by RBCs. DMA can be in two forms, dimethylarsinous (DMAIII) and dimethylarsinic (DMAV) acids, the latter form being proposed to be the major urinary metabolite. However, the former form was shown to be present as a urinary metabolite in humans together with the latter form (1922), suggesting that DMA may be effluxed from liver to bloodstream in both forms. It should be noted that DMAIII and MMAIII have been detected in the liver of hamsters (23). In the present study, we examined whether there is any difference in the uptake of DMA among RBCs of different animal species and also difference in the chemical species of DMA between DMAIII and DMAV. RBCs were prepared from three animal species, i.e., rats, mice, and hamsters of different arsenic metabolisms (10), and also from humans. DMAIII was prepared from DMAV in our laboratory (22, 24). The present study revealed that only DMAIII, i.e., not DMAV, is taken up efficiently by RBCs, and further the uptake is dependent on the animal species, the uptake by rat RBCs being most effective. These results suggest that the species difference in the metabolism of arsenic, and hence its toxicity, can be attributed not only to the reduction and methylation capacity leading to DMA in the liver but also to the reduction capacity of DMAV to DMAIII in the liver followed by the uptake/retention capacity for DMAIII of RBCs. Further, the mechanism underlying the uptake/retention of DMA by RBCs was examined.

Experimental Procedures Caution. Inorganic arsenic compounds have been established as human carcinogens (3). Ingestion of inorganic arsenic may cause cancer of the skin, urinary bladder, kidney, lung, and liver as well as with disorders of the circulatory and nervous system. Reagents. Sodium dimethlyarsinite (DMAV), sodium thiosulfate, sodium disulfite, sulfuric acid, and nitric acid of reagent grade were purchased from Wako Pure Chemicals Industries Ltd., Osaka, Japan. Trizma Hydrochloride, Trizma Base and azodicarboxylic acid bis(dimethylamide) (diamide) were purchased from Sigma, St. Louis, MO. 4,4′-Diisothiocyano-2,2′stilbene disulfonic acid disodium salt (DIDS) was purchased from ICN Pharmaceuticals, Inc., Costa Mesa, CA. The arsenic standard solution (1000 ppm) used for ICP MS was purchased from SPEX CentiPrep, Metuchen, NJ. DMAIII was prepared from DMAV according to the method of Reay and Asher (24). Briefly, 0.28 g of sodium disulfite was dissolved in 15 mL of 50 mM Tris-HCl buffered saline (TBS, pH 7.4), and then 2 mL of 1% sodium thiosulfate was added to the solution. After the addition of 0.1 mL of concentrated sulfuric acid to the mixture, 1 vol of it was mixed with one volume of a DMAV solution (2 mM), followed by incubation at room temperature for 2 h. This reaction mixture was subsequently diluted with the mobile phase for HPLC to the final concentration of 5-20 nM and then analyzed by the HPLC-ICP MS (mass spectrometry with ionization by inductively coupled argon plasma) method.

Chem. Res. Toxicol., Vol. 14, No. 10, 2001 1447 Uptake of Arsenic Compounds by Red Blood Cells. Male Wistar rats and ICR mice, and Syrian hamsters were purchased at 8 weeks of age from Clea Japan Co., Tokyo, Japan and Japan SLC Inc., Hamamatsu, Japan, respectively. The animals were fed a commercial diet (CE-2; Clea Japan Co.) and tap water ad libitum. RBCs of each animal species were prepared from heparinized blood, and were separated from plasma and a buffy coat by centrifuging at 1600g for 15 min. The separated RBCs were resuspended in TBS, and then centrifuged to remove residual plasma, only erythrocytes being detected under microscopic examination. DMAIII or DMAV was added to heparinized whole blood of Wistar rats or to suspensions (10% in TBS) of rat, hamster, mouse, and human RBCs at the concentration of 15 µg/mL incubation solution, followed by incubation at 37 °C for up to 4 h. RBCs were removed by centrifugation at 8000g for 10 s, and then after ashing with HNO3 and H2O2, the concentration of arsenic in the medium was determined with an ICP MS (HP 4500; Yokogawa Analytical Systems Co., Musashino, Japan). The chemical form of arsenic in the medium was determined with a cation exchange column (RSpak NN 614, 6.0 × 150 mm; Showa Denko, Tokyo) by the HPLC-ICP MS method (22). To confirm the uptake but not the adsorption of DMAIII on the outer membrane of RBCs, DMAIII was incubated in a 10% suspension of rat RBCs or in a 10% suspension of the membrane fraction separated from rat RBCs for 1 h under the same conditions as mentioned above. The concentrations of arsenic in the incubation media and the membrane fractions were determined with an ICP MS. Arsenic was recovered in the RBCs at 80.6% of the dose, i.e., 78.8% in the lysate, 0.97% in the membrane fraction, and 0.82% in the precipitate. On the other hand, when DMAV was incubated in a 10% suspension of the membrane fraction, arsenic was recovered mostly (95.8% of the dose) in the incubation medium without being precipitated with the membrane fraction. Suspensions (10% RBCs in TBS) of rat, hamster, mouse, and human RBCs were incubated with different concentrations of diamide for 20 min at 37 °C, and then incubated with DMAIII and DMAV for 1 h at 37 °C. Rat RBCs were lysed by freezing and thawing, and the resulting lysate was centrifuged at 15000g for 20 min to obtain a supernatant fraction. DMAIII and DMAV were incubated in the supernatant fraction in the presence or absence of 1 mM GSH at 37 °C for 1 h. The distribution of arsenic was determined on a gel filtration column (Asahipak GS 220 HQ, 7.6 × 250 mm; Showa Denko) by the HPLC-ICP MS method. Determination of Arsenic Concentrations and Their Chemical Forms. The concentrations of arsenic in whole blood, plasma and medium were determined after wet-ashing with HNO3 and H2O2, at m/z 75, with an ICP-MS. A 0.02 mL aliquot of plasma or medium containing an arsenic compound was applied to a cation exchange column (NN-614), and then the column was eluted with 36 mM formic acid/2 mM ammonium formate buffer, pH 2.81, at 25 °C, with an HPLC (PU 610; GL Sciences Co., Tokyo) at the flow rate of 0.6 mL/ min. The eluent was introduced directly into the nebulizer tube of the ICP-MS to detect arsenic (m/z 75).

Results Arsenic administered orally to rats in the form of arsenite (AsIII) was efficiently taken up by and accumulated in RBCs (data not shown), in the form of dimethylated As (DMA) as also reported in the literature (14, 15). Contrary to the accumulation of DMA in RBCs in vivo, commercially available DMA was taken up only slowly and marginally in vitro by the RBCs in heparinized whole blood of rats, as shown in Figure 1. With higher and lower concentrations of arsenic than the

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Figure 1. Uptake of dimethylarsinic acid (DMAV) by red blood cells (RBCs) of rats and the effect of medium glucose on the uptake. DMAV was incubated in heparinized blood of Wistar rats at a concentration of 15 µg/mL blood at 37 °C for up to 8 h in the presence of glucose at doses of 0, 5, 20, and 40 g/L. The dotted horizontal line shows the concentration of arsenic distributed evenly in the plasma and RBCs.

dotted line, arsenic was not or less efficiently taken up and effectively taken up by RBCs, respectively. Although the concentration of arsenic in the plasma decreased marginally and very slowly in vitro in whole blood, the decrease was enhanced in the presence of glucose depending on its concentration, as shown in Figure 1. Nevertheless, the uptake of arsenic was limited and slow compared with that in vivo (arsenic accumulates in RBCs within several hours after an intravenous injection of arsenite into rats, unpublished observation) and it seems to be too slow for uptake by RBCs, being rather filtered out by the glomerulus. DMA is present in two forms, DMAV and DMAIII, the former being a commercially available form. The DMA used in the experiment in Figure 1 was DMAV, which may explain the slow and limited uptake. Therefore, the uptake of DMA in the reduced form, DMAIII, was examined, as shown in Figure 2. Commercially available DMAV was reduced to DMAIII according to the reported procedure (22, 24), as shown in Figure 2A. The two forms of DMA were incubated in heparinized blood, as shown in Figure 2B. The reduced DMA (DMAIII) was taken up by RBCs within 10 min, which suggests that DMA is secreted from the liver in the form of DMAIII and then taken up efficiently by RBCs. This also suggests that DMAV is taken up much less and slowly by RBCs than DMAIII and hence is filtered out by the glomerulus rather than being taken up by RBCs in vivo. Although DMAV was added to heparinized whole blood at the concentration of 15 µg of As/mL whole blood, the arsenic concentration was already lowered than the calculated level of 21.2 µg/mL plasma (hematocrit value, 40.7%) immediately after the incubation and then increased to the calculated level 10 min after the incubation (Figure 2). This rapid change suggests the homogeneous distribution of arsenic between RBCs and plasma, followed by the relocalization in the plasma. However, it was not detected in a suspension of rat RBCs, as shown in Figure 3A. The results in Figure 2 indicate that the effective uptake of DMA by RBCs in vivo can be attributed either to the effective reduction of DMAV to DMAIII in the liver or the effective uptake of DMAIII by RBCs. Then, the latter possibility was examined in vitro by comparing the

Figure 2. Chemical reduction of dimethylarsinic (DMAV) to dimethylarsinous (DMAIII) acids, and uptake of DMAIII by rat RBCs. (A) The distributions of arsenic on a cation exchange column with the HPLC-ICP MS method were determined for commercially available DMAV and its reduced form, DMAIII. (B) DMAIII or DMAV was incubated in heparinized blood of rat origin, and the concentration of arsenic was plotted against the incubation period. The dotted horizontal line shows the concentration of arsenic distributed evenly in the plasma and RBCs.

Figure 3. Species differences in the uptake of dimethylarsinous acid (DMAIII) by rat, hamster, mouse and human RBCs in vitro. DMAIII or DMAV was incubated with suspended RBCs (10% RBCs in Tris-HCl buffered saline) of rat (A), hamster (B), mouse (C), and human (D) origin, and the concentration of arsenic was plotted against the incubation period. The dotted horizontal line shows the concentration of arsenic distributed evenly in the medium and RBCs.

uptake of DMAIII by rat, hamster, mouse, and human RBCs, as shown in Figure 3. As DMAIII was effectively taken up by RBCs without the presence of plasma constituents, DMAIII and DMAV were incubated with RBC suspensions in Tris-HCl buffered saline instead of heparinized whole blood in the present experiment. It was also confirmed that the decrease of arsenic in the suspension medium of RBCs is due to the uptake of DMA by RBCs but not due to the adsorption on the outer

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Figure 4. Changes in the chemical form of arsenic in the medium on incubation of DMAIII with rat, hamster, and human RBCs. DMAIII was incubated with suspended RBCs (10% RBCs in Tris-HCl buffered saline) of rat (A), hamster (B), and human (C) origin, or without RBCs in Tris-HCl buffered saline (D), at 37 °C for up to 4 h, and then the distribution of arsenic in the medium was determined on a cation exchange column by the HPLC-ICP MS method. The vertical bar indicates the detection level for arsenic.

membrane of RBCs, as described in the Experimental Procedures. DMAIII was taken up by RBCs at different rates depending on the animal species, the uptake by the rat RBCs being the most efficient, followed by hamster, mouse and human ones. On the other hand, DMAV was taken up less efficiently than DMAIII by the RBCs of all animal species, being distributed homogeneously between the RBCs and medium. DMAIII was shown to be taken up differently by RBCs depending on the animal species, as shown in Figure 3. However, there is an alternative explanation; DMAIII may be taken up and retained in rat RBCs, while it may be taken up but not retained in hamster, mouse, and human RBCs. Therefore, the chemical form of arsenic in the medium was determined by the HPLC-ICP MS method, as shown in Figure 4. DMAIII was effectively taken up by rat RBCs, while the intensity of DMAV peak did not change with incubation time, as shown in Figure 4A. On the other hand, in the case of hamster RBCs, DMAIII decreased with time (though less efficiently than that of rat ones), as shown in Figure 4B, at the same time, the intensity of the DMAV peak increased with the incubation time and became greater than that of the DMAIII peak after 1 h. The results in Figure 4, panels A and B, suggest that, in rats, DMA is secreted from the liver into the bloodstream in the form of DMAIII and then taken up by and retained in the RBCs. However, in hamsters, DMAIII is taken up by RBCs and oxidized to DMAV and then effluxed from the RBCs without retaining them. The chemical forms of arsenic in the medium were also determined after incubation of DMAIII with human RBCs, as shown in Figure 4C. The DMAIII peak decreased less slowly for human RBCs than for hamster ones, and the DMAV peak increased much more slowly than in the case of hamsters, suggesting that the oxidation and efflux are less efficient in human than in rat RBCs. Although

DMAIII can be oxidized spontaneously to DMAV, this oxidation was not observed under the present conditions without RBCs, as shown in Figure 4D. The results shown in Figure 4 suggest that the species difference in the uptake of DMA by RBCs is not due to the difference in the channel responsible to the transport through the membrane. Nevertheless, the responsible channel seems to be important as to understanding the mechanism underlying the species difference in the metabolism of arsenic. The anion exchange carrier (band 3 protein) is the major integral protein of RBCs and catalyses the passage of anions across the membrane (25, 26). The possibility of the transport of DMAIII through the common channel for phosphate, carbonate, and sulfate was examined as to the uptake of DMAIII by adding phosphate to suspensions of rat and hamster RBCs. Phosphate did not affect the uptake of DMAIII up to 5 mM (data not shown). Further, a specific inhibitor of band 3 protein, DIDS (27), was not effective under the conditions under which the transport of selenite into RBCs is effectively inhibited (28), as shown in Figure 5; the uptake and oxidation of DMAIII by rat RBCs and also by hamster RBCs did not change in the presence of 50 µM DIDS, as shown in Figure 5, panels A, B and C, D, respectively. The animal species difference in the uptake of DMAIII by RBCs is, thus, assumed to be dependent on the difference in the oxidation/retention of arsenic in RBCs. Since trivalent arsenic compounds are known to conjugate with glutathione (GSH) (29, 30), the effect of GSH depletion on the uptake/retention of arsenic in RBCs was examined in the presence of GSH-depleter diamide in the RBC suspension, as shown in Figure 6. DMAIII and DMAV were incubated with rat RBCs in the presence of different concentrations of the oxidizing agent for GSH, diamide (31), for 1 h. The uptake of DMAIII decreased depending on the dose of diamide, while the concentration of DMAV in the medium did not show

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Figure 5. Effect of 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS) on the uptake of dimethylarsinous acid (DMAIII) on incubation with rat and hamster RBCs. DMAIII was incubated with suspended RBCs (10% RBCs in Tris-HCl buffered saline) of rat (A, B) and hamster (C, D) origin in the presence (B, D) and absence (A, C) of 50 µM DIDS at 37 °C for up to 4 h, and then the distribution of arsenic in the medium was determined on a cation exchange column by the HPLC-ICP MS method. The vertical bar indicates the detection level for arsenic.

Figure 6. Effect of glutathione oxidation with diamide on the uptake of dimethylarsinous acid (DMAIII) by rat RBCs. Rat RBCs in Tris-HCl buffered saline (10%) were incubated with different concentrations of diamide for 20 min at 37 °C, and then with DMAIII (A) and DMAV (B) for 1 h at 37 °C. The distribution of arsenic in the medium was determined on a cation exchange column by the HPLC-ICP MS method. The vertical bar indicates the detection level for arsenic.

observable changes, as shown in Figure 6A. DMAV was not taken up by rat RBCs under the present conditions, as shown in Figure 6B. These results suggest that DMAIII is not retained in RBCs in the absence of GSH. However, the intensity of the DMAV peak did not change with the presence of diamide, suggesting that DMAIII was not oxidized even under GSH-depleted conditions in rat RBCs. DMAIII incubated with hamster RBCs changed to DMAV, as shown in Figure 4B. This observation can be explained by that DMAIII is oxidized to DMAV in RBCs and then effluxed into the medium because DMAIII is retained less effectively under less reductive conditions (Figure 6A). However, at the same time, the intensity of

the DMAV peak did not increase with rat RBCs even under more oxidized conditions with depletion of GSH by means of diamide, as shown in Figure 6A. Therefore, it is of importance to identify the chemical forms of arsenic in RBCs. Arsenic accumulates in control rat RBCs due to contaminating inorganic arsenic in food and the arsenic in the hemolysate of control rats was detected mostly in the void volume of a gel filtration column, as shown in Figure 7A. The major sulfur peak (m/z 34) was also detected at the void volume and GSH was detected as a small peak at 13.3 min. The DMAIII and DMAV peaks did not change on incubation with GSH (Figure 7B, parts a and b). However, the DMAIII but not the DMAV peak decreased on incubation with a hemolysate, together with an increase in the arsenic peak at the void volume (compare Figure 7, part c in panel B with part b in panels B and A). GSH further enhanced the decrease in the DMAIII peak and the increase in the arsenic peak at the void volume (compare Figure 7B, part d with part c). On the other hand, DMAV was not affected by incubation with GSH and the hemolysate, as shown in Figure 7C. These results suggest that DMAIII, but not DMAV, forms a complex with constituent(s) in RBCs in the presence of GSH, and that arsenic is retained in RBCs in the form of a high molecular weight complex.

Discussion The most significant difference in the metabolism of arsenic among diverse animals has been recognized to be that in the distribution of arsenic in RBCs of tolerant rats and sensitive hamsters, arsenic being distributed and retained mostly in rat RBCs (14, 15), but being distributed slightly in RBCs and excreted into the urine in the case of hamsters (16-18). However, the mechanism underlying the difference in the metabolism has not been elucidated. Therefore, the present study was focused on the process of uptake of arsenic by RBCs based on the speciation of arsenic at each metabolic step. The speciation of arsenic by HPLC-ICP MS, HPLCHG-AFS, or other methods has been carried out for

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Chem. Res. Toxicol., Vol. 14, No. 10, 2001 1451 Scheme 2. Proposed Mechanisms Underlying the Uptake and Retention of Dimethylated Arsenic (DMAs) by Red Blood Cells (RBCs)

Figure 7. Formation of a high molecular weight complex between arsenic of dimethylarsinous acid (DMAIII) and protein(s) in rat RBCs. Rat RBCs were lysed by freezing and thawing, and the resulting lysate was centrifuged at 15000g for 20 min to give a supernatant fraction (A). DMAIII (B) and DMAV (C) were incubated in the supernatant fraction in the presence or absence of glutathione (GSH) at 37 °C for 1 h. The distributions of arsenic and sulfur (S) were determined on a gel filtration column by the HPLC-ICP MS method at m/z 75 and 34, respectively. The vertical bar indicates the detection level for arsenic.

understanding the mechanism underlying the metabolism of arsenic in the body and in the ecosystem (19-22, 32, 33). The separation, into DMAIII and DMAV, and detection of DMA seem to shed new light on the mechanism underlying the toxicity of arsenic because of the proposed metabolic pathway for arsenic in Scheme 1. In fact, DMAIII has been demonstrated in the urine of people inhabiting in an arsenic-contaminated area (19-22). The present study revealed for the first time that DMAIII, but not DMAV, is the chemical form of arsenic taken up by RBCs, and further that the uptake of DMAIII by RBCs is dependent on the animal species. Uptake by RBCs or excretion at the glomerulus seems to determine the metabolic destination and distribution of small molecular weight DMA, and slower uptake by RBCs in vitro suggests lower uptake by RBCs and higher excretion into the urine in vivo. Further, the lower uptake by RBCs may cause the higher incidence of the distribution of DMAIII to other organs/tissues, which may explain the higher incidence of the toxicity of arsenic in animals with less arsenic distributed in RBCs. Although more data for diverse animal species are required, the present observations as to the order of the rates of uptake of DMAIII by RBCs and its oxidation/efflux to DMAV seem to be related with the animal species difference in the metabolism and, hence, the toxicity of arsenic. Styblo et al. (34) and Styblo and Thomas (35) have reported that MMAIII and DMAIII, in a manner similar to arsenite, but unlike MMAV and DMAV, exhibit high affinity for specific cellular proteins. In the present study,

the retention of DMAIII by RBCs decreased with depletion of GSH, and DMAIII formed a complex with protein(s) in RBCs, especially in the presence of GSH. These observations can be explained as follows: DMAIII forms a complex with sulfur-rich specific protein(s) that can be reduced by GSH, and then the complex is retained in RBCs. However, when the concentration of GSH is not sufficient to reduce the specific protein(s), DMAIII is oxidized to DMAV through reduction of the specific protein(s), DMAV being effluxed, as proposed in Scheme 2. Then, the animal species difference in the retention/ efflux of arsenic by RBCs can be attributed to the difference in the specific protein(s) and/or GSH concentration in RBCs. The uptake of DMAIII by RBCs in Figures 4 and 5 can be divided into two distinct processes, i.e., the uptake of DMAIII and the oxidation of DMAIII to DMAV, followed by the efflux of DMAV. The former process took place faster in the order of rat > hamster > human, but the latter process in the order of hamster > human > rat. The animal species differences in these two processes lead to variation in the metabolism and toxicity of arsenic in addition to the differences in the reduction and methylation processes in the liver. Therefore, it should be noted that the uptake of DMA by RBCs is an additional contributing factor to the reduction and methylation capacity for the animal species difference in the metabolism and toxicity of arsenic.

Acknowledgment. This study was supported by Grants-in-Aid of Ministry of Education, Science, Technology, Sports and Culture (no. 12470509).

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