TRANSPORT OF AMINOTHIOL RADIOPROTECTORS INTO MAMMALIAN-CELLS - PASSIVE DIFFUSION VERSUS MEDIATED UPTAKE

Water:n-octanol partition coefficients (K-D) were determined for a ser ies of radioprotective thiols to ascertain whether these could be used to estimate reliably their rates of uptake into mammalian cells by pa ssive diffusion. Values of K-D, determined for thiols in 0.1 M potassi um phosphate, pH 7.4, at 22 degrees C were: N-(2-mercaptoethyl)-1,3-di aminopropane (WR-1065, WRSH), 2.0 x 10(3); dithiothreitol, 1.4; 2-merc aptoethanol, 1.7; cysteamine, 180; 3-mercaptopropanoic acid, 450; merc aptosuccinic acid, 5 x 10(6) (extrapolated value). Predictions of upta ke rates by passive diffusion into mammalian cells using these values and values for the membrane diffusion rate derived from empirical eval uation of appropriate values from the literature for erythrocyte perme ability paralleled the experimental rates for WR-1065 and dithiothreit ol but were about threefold lower. Although the utility of K-D, values for quantitative prediction of uptake rates is limited, the analysis clearly indicated that uptake of aminothiols having three or more ioni zed amino groups will not occur at useful rates by passive diffusion. Studies of WR-1065 import by Chinese hamster V79-171 cells at micromol ar levels of WR-1065 revealed an uptake that could not be explained by passive diffusion. This uptake was not inhibited by substrates for co mmon amino acid transport systems but was inhibited by polyamines and by 1 mill DTT, which suggested that WR-33278 (WRSSWR) formed by oxidat ion of WRSH was being. transported by a polyamine transport system. Th is was confirmed by shelving that WRSSWR is imported efficiently by V7 9-171 cells treated with D,L-2-difluoromethylornithine to deplete intr acellular polyamines and hence enhance their transport. Spermine inhib ited uptake of WRSSWR and WRSSWR inhibited uptake of [C-14]spermine, c onfirming that a common system is involved in the uptake of these simi lar molecules, both having +4 charge. Tt was shown that after import W RSSWR is reduced to WRSH and that uptake at low micromolar concentrati ons of WRSSWR results in marked cellular concentration of the drug. Th ese results indicate that the spermidine/spermine transport system map also provide a feasible route for import of radioprotective aminothio ls bearing net charges of +3 or +4 into mammalian cells. (C) 1996 by R adiation Research Society.