Gl. Newton et al., TRANSPORT OF AMINOTHIOL RADIOPROTECTORS INTO MAMMALIAN-CELLS - PASSIVE DIFFUSION VERSUS MEDIATED UPTAKE, Radiation research, 146(2), 1996, pp. 206-215
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.