Exponentially growing Chinese hamster V79 cells were exposed to Adriamycin either in phosphate buffered saline (PBS) or fresh growth medium (F-med) supplemented with various amounts of NaCl in the range between 50–1000 mm and survival was measured by the colony forming assay. Compared to the survival obtained after exposure of cells to isotonic (140 mm NaCl) PBS (D0 = 0.16 µg/ml, Dq = 0.49 µg/ml) a potentiation in cell killing was observed after treatment in hypotonic (50 mm NaCl) PBS (D0 = 0.08 µg/ml, Dq = 0.19 µg/ml) and a reduction in cell killing after treatment in hypertonic (500 mm NaCl) PBS (D0 = 0.36 µg/ml, Dq = 0.55 µg/ml). Cells exposed to Adriamycin in F-med were more sensitive to Adriamycin (D0 = 0.1 µg/ml, Dq = 0.27 µg/ml) than cells exposed to Adriamycin in PBS, but cell killing was reduced when the medium was made hypertonic by the addition of NaCl (500 mm NaCl) (D0 = 0.23 µg/ml, Dq = 0.45 µg/ml). The amount of Adriamycin accumulated in the cells during treatment was measured in a spectrophotofluorometer and was found to vary as a function of the treatment medium and NaCl concentration. Cells exposed to Adriamycin in PBS (isotonic) were accumulating three to four times less drug than cells exposed to Adriamycin in F-med. Less Adriamycin (two to three times) was also accumulated in cells treated in hypertonic (500 mm NaCl) F-med. Compared to the Adriamycin accumulation observed after exposure to cells in isotonic PBS, an increase was observed after exposure in hypotonic PBS (2.3 times) but no change after exposure in hypertonic PBS (500 mm NaCl). Adriamycin-induced DNA damage was assayed with the alkaline filter elution technique and it was found to increase after treatment in hypotonic PBS and to decrease after treatment in hypertonic PBS. The modification in the survival curve slope and DNA damage induction observed after exposure in hypotonic PBS was quantitatively similar to the modification in intracellular drug concentration (factor of 2.3, comparison based on the results obtained in isotonic PBS). However, after exposure of cells to Adriamycin in hypertonic PBS, a reduction by a factor of 20 was observed in the induction of DNA damage but a reduction only by a factor of 2.3 was observed in cell killing with no modification of intracellular Adriamycin concentration. Exposure of cells to the Adriamycin analogue N-trifluoroacetyladriamycin-14-valerate (AD32) resulted in a dose- and time-dependent cell killing which was enhanced after incubation in hypotonic or hypertonic PBS. The importance of Adriamycin binding to DNA for cytotoxicity and its modification by the ionic strength of the medium used for the treatment are considered for the interpretation of the results obtained.
Supported in part by USPHS Grant 1-R01-CA42026 awarded by the National Cancer Institute, Department of Health and Human Services.