Transport of aminothiol radioprotectors into mammalian cells: passive diffusion versus mediated uptake.

Water:n-octanol partition coefficients (KD) were determined for a series of radioprotective thiols to ascertain whether these could be used to estimate reliably their rates of uptake into mammalian cells by passive diffusion. Values of KD determined for thiols in 0.1 M potassium phosphate, pH 7.4, at 22 degrees C were: N-(2-mercaptoethyl)-1,3-diaminopropane (WR-1065, WRSH), 2.0 x 10(3); dithiothreitol, 1.4; 2-mercaptoethanol, 1.7; cysteamine, 180; 3-mercaptopropanoic acid, 450; mercaptosuccinic acid, 5 x 10(6) (extrapolated value). Predictions of uptake rates by passive diffusion into mammalian cells using these values and values for the membrane diffusion rate derived from empirical evaluation of appropriate values from the literature for erythrocyte permeability paralleled the experimental rates for WR-1065 and dithiothreitol but were about threefold lower. Although the utility of KD values for quantitative prediction of uptake rates is limited, the analysis clearly indicated that uptake of aminothiols having three or more ionized amino groups will not occur at useful rates by passive diffusion. Studies of WR-1065 import by Chinese hamster V79-171 cells at micromolar levels of WR-1065 revealed an uptake that could not be explained by passive diffusion. This uptake was not inhibited by substrates for common amino acid transport systems but was inhibited by polyamines and by 1 mM DTT, which suggested that WR-33278 (WRSSWR) formed by oxidation of WRSH was being transported by a polyamine transport system. This was confirmed by showing that WRSSWR is imported efficiently by V79-171 cells treated with D,L-2-difluoromethylornithine to deplete intracellular polyamines and hence enhance their transport. Spermine inhibited uptake of WRSSWR and WRSSWR inhibited uptake of [14C]spermine, confirming that a common system is involved in the uptake of these similar molecules, both having +4 charge. It was shown that after import WRSSWR is reduced to WRSH and that uptake at low micro-molar concentrations of WRSSWR results in marked cellular concentration of the drug. These results indicate that the spermidine/spermine transport system may also provide a feasible route for import of radioprotective aminothiols bearing net charges of +3 or +4 into mammalian cells.