Search

Clonogenic survival and drug content for Chinese hamster V79-171 cells incubated in suspension with WR-1065 prior to gamma irradiation have been determined. Factors that might influence the radioprotection by WR-1065 were investigated in control studies. Intracellular drug levels studied ranged between 0-36 nmol per 10(6) cells. In control studies, it was established that extracellular drug toxicity was not significant for cells in suspension at 10(6) per milliliter over short periods but was important when residual drug was present above 2 microM in the final plating of cells. Accumulation of intracellular drug above 30 nmol per 10(6) cells produced significant cytotoxicity in unirradiated cells. Irradiation with doses as high as 150 Gy produced no significant change in the total drug level or the thiol/disulfide ratio, either for the drug in the cells or for the drug in the medium. Preirradiation with 8 Gy did not change the ability of cells to import the drug but did appear to increase the cytotoxicity of the intracellular drug at levels above 25 nmol per 10(6) cells. There was no qualitative difference in the ability of WR-1065 to protect viable cells preirradiated with 8 Gy compared with protection of unirradiated cells. For a given gamma-ray dose from 2 to 40 Gy, there is a limiting value for surviving fraction which cannot be increased by further elevation of the intracellular drug level in V79-171 cells. Such limiting radioprotection was demonstrated for HT-29/SP-ld, HeLa, Me-180-VCII and OV-2008-VI human tumor cells.

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.

The radioprotector WR-1065 (N-(2-mercaptoethyl)-1,3-diaminopropane) has been shown to be the active moiety involved in protecting mammalian cells from the cytotoxic and mutagenic effects of ionizing radiation after administration of WR-1065 or the phosphorylated form, WR-2721. Initial experiments demonstrated that, in our hands, WR-1065 protects Chinese hamster AA8 cells from killing by (a) mechanism(s) other than induction of hypoxia. AA8 cells were then incubated in the presence of [14C]WR-1065 to determine whether association of WR-1065 in vivo was random or targeted to the nucleus or the nuclear matrix. The kinetics of incorporation of labeled material showed rapid incorporation for the first 30 min and little, if any, additional incorporation over the next 2.5 h. Examination of nuclei and nucleoids generated from the AA8 cells indicated that approximately 10% of the drug was localized in the nucleus and the drug that remained was not dislodged with repeated washes of the filters. Association kinetics of the drug with nuclei and nucleoids indicated that there was little increase in drug association with time, suggesting that there may be a limited number of strong association sites in the nucleus, but these sites are either with DNA or with matrix proteins. Exposure of the AA8 cells to 6 Gy of 60Co gamma rays did not significantly alter the association of the drug with AA8 cells. Incubating AA8 cells in [14C]WR-1065 for 30 min and then incubating in drug-free medium indicated that nearly all of the drug was lost from cells within the first 5 min of incubation in drug-free medium. The low level of tightly bound matrix-associated label may be important in generating alterations in matrix organization that have been observed previously in this laboratory.