Comparison of adriamycin uptake in chick embryo heart and liver cells an murine L5178Y lymphoblasts in vitro: role of drug uptake in cardiotoxicity
Johnson, B.A.; Cheang, M.S.; Goldenberg, G.J.
Cancer Research 46(1): 218-223
1986
ISSN/ISBN: 0008-5472 PMID: 3940192 Document Number: 273249
The mechanism of uptake of Adriamycin was investigated in chick embryo heart and liver cells and in murine L5178Y lymphoblasts in vitro. Drug uptake at 4.degree. C for 15 s due to rapid association accounted for a cell:medium distribution ratio of 104 .+-. 14 (SE) in heart cells, 10.2 .+-. 1.3 in liver cells, and 10.3 .+-. 1.5 in L5178Y lymphoblasts. On thin-layer chromatographic analysis, 98% of the radioactivity migrated with the mobility of intact drug, suggesting that drug metabolism was negligible for at least 30 min in both heart and tumor cells. A time course of drug uptake was somewhat different in heart cells compared to that noted for liver of L15178Y cells. The steady state for drug uptake was reached more promptly in heart cells; apparent equilibrium was observed at 6 min in heart cells, at approximately 20 min in L5178Y lymphoblasts, but was not attained by 25 min in liver cells. Temperature dependence of drug uptake also differed in the three cell types; drug uptake was most temperature sensitive in L5178Y cells, intermediate in liver cells, and least temperature dependent in heart cells. Separation of heart, liver, and leukemic cells into membrane and cytosol fractions demonstrated that, at 1 and 30 min, more than 75% of the drug was associated with the membrane fraction. Trichloroacetic acid extraction of cell constituents revealed that, at 1 min, the acid-soluble fraction amounted to 32 .+-. 2% of radioactivity in heart cells and 37 .+-. 2% in L5178Y cells. Ethanol extraction of these cells demonstrated that, at 1 min, ethanol-soluble components accounted for 49 .+-. 2% of radioactivity in heart cells and 27 .+-. 2% in leukemic cells. The finding of a large component of rapid association together with evidence of prompt drug binding to cellular constituents made evaluation of unidirectional drug influx impractical. Accordingly, an investigation was undertaken of Adriamycin efflux from chick embryo heart and liver cells and L5178Y lymphoblasts, after the cells had been loaded with drug for various time intervals. In all three cell types, efflux was rapid down to a plateau level, representing nonexchangeable drug. As the period of time for loading cells was increased, there was a progressive rise in the level of nonexchangeable drug. Equilibration of the nonexchangeable pool occurred more rapidly in heart cells than in either liver or leukemic cells. In heart cells, the nonexchangeable pool of drug reached an apparent steady state by 12 min, whereas nonexchangeable drug continued to increase for at least 30 min in liver cells and 60 min in leukemic cells. After 1 min, intracellular Adriamycin concentration was 1.98 .+-. 0.12 mM in heart cells, 0.18 .+-. 0.01 mM in liver cells, and 0.18 .+-. 0.02 mM in L5178Y cells; after 30 min, the drug levels were 2.22 .+-. 0.17 mM in heart cells, 0.50 .+-. 0.80 mM in liver cells, and 0.55 .+-. 0.06 mM in leukemic cells. The higher cellular concentration of Adriamycin in heart cells, relative to those found in liver and tumor cells, and perhaps the more rapid rate of equilibration of both total and nonexchangeable drug in heart cells may explain, at least in part, the marked sensitivity of cardiac tissue to drug toxicity.