Time dependence of accumulation and binding of inorganic and organic arsenic species in rabbit erythrocytes

The uptake by rabbit erythrocytes of 0.4 mM arsenite, As(III), arsenate, As(V), monomethylarsinate, MMA(V) and dimethylarsonate, DMA(V) were compared over 24 h. In membrane-free hemolysate, the distribution of As between proteins (10 kDa) and ultrafiltrate was determined by ultrafiltration and arsenic species in the ultrafiltrate were identified by thin layer chromatography methods. 1H spin-echo Fourier transform NMR was used to follow the binding of these arsenic species to glutathione (GSH). 31P-NMR was used to observe their effects on high-energy adenine nucleotide levels (ATP, ADP). These results demonstrate that As(III) readily accumulates in cells, reaches a quasi-plateau at 78% of the total As in the incubation system after 1 h and 88% of the total As after 24 h. On average, 20% of the total erythrocyte As(III) burden is associated with the protein fraction, particularly with hemoglobin (Hb). About 68% of the erythrocyte As(III) burden is bound to GSH. As(III) has no effect on ATP levels during a 5-h incubation. By comparison, As(V) enters erythrocytes more slowly (53% of the total As after 5 h). Erythrocytes take up 81% of the As(V) in the reaction system after a 24 h incubation. Of the total As burden in As(V)-exposed erythrocytes, 22% was associated with the proteins (10 kDa) and possibly reduced to As(III) and 59% was in the ultrafiltrate (8% as As(III) and 51% as As(V)). This finding indicates that, over a 24 h incubation period, the reduction of As(V) to As(III) may account for 30% of the total As in rabbit erythrocytes. As(V) present in the erythrocytes enters the phosphate pool and depletes ATP. In comparison, about 65% of the total MMA(V) or about 44% of the total DMA(V) in the incubation system is taken up by rabbit erythrocytes during a 24 h incubation. Neither organoAs species perturbed the Hb signals observed by spin-echo Fourier transform NMR and the binding to GSH was minimal. Unlike As(V), MMA(V) and DMA(V) do not perturb phosphate metabolism, showing that, despite their pentavalent oxidation state, these arsenic species are not analogs for phosphate.