Cellular transport and accumulation of thiotepa in murine, human, and avian cells

Because the transport and accumulation of thiotepa by cells has not been characterized, these process were investigated with [14C]thiotepa and cultured L1210 or freshly obtained human or avian RBC. The octanol:phosphate buffered saline partition coefficient of thiotepa was 2.4 +/- 0.1 (n = 8). With this value, the permeability coefficient (P) for thiotepa was estimated to be between 2.8 X 10(-4) and 1.81 X 10(-3) cm/s and the half-life of accumulation of thiotepa by L1210 cells was estimated to be 0.063-0.40 s. Thiotepa accumulation by cells was measured after incubation of cell with [14C]thiotepa and subsequent harvesting of cells by centrifugation through silicone fluid. Thiotepa accumulation by L1210 cells was biphasic. The initial phase was rapid essentially complete by 10 s. The amount of cell-associated 14C increased linearly with increasing extracellular concentrations of thiotepa or with increasing size of the cell pellet. The absolute amount of cell-associated 14C was consistent with that expected if the [14C]thiotepa had been evenly distributed in the incubation medium and a volume equal to that of the cell pellet had been sampled and counted. This rapid phase of thiotepa accumulation was not slowed when cells were incubated on ice. The second phase of [14C]thiotepa accumulation occurred at a rate much slower than that of the initial phase. This slower phase of drug accumulation was linear for at least 5 h. The rate of 14C accumulation increased progressively over a range of extracellular thiotepa concentrations between 5 and 100 nmol/ml and could not be saturated under acceptable tissue culture conditions. The slower rate of 14C accumulation was ablated by incubation cells on ice and was reduced by 30-50% in the presence of 1 mM sodium azide or 2,4-dinitrophenol. The slow rate of accumulation of 14C reflected summation of a relatively stable or constant amount of exchangeable 14C an an amount of nonexchangeable 14C which increased linearly from almost undetectable levels at the start of the experiment to amounts approximately equal to those of exchangeable radioactivity after 5 h. The initial association of [14C]thiotepa with both human and avian RBCs was also very rapid. Avian RBCs also exhibited a slow rate of 14C accumulation which was linear for at least 5 h which was 15-20% that of L1210 cells. Human RBCs did not exhibit a slower rate of 14C accumulation and essentially all of the 14C associated with human RBCs was exchangeable for the 5 h duration of the experiment.(ABSTRACT TRUNCATED AT 400 WORDS)