In vitro hypoxic cytotoxicity of nitroimidazoles: uptake and cell cycle phase specificity.

The hypoxic cytotoxicity of four different 2-nitroimidazoles of similar electron affinities but different lipophilicities was compared using EMT6/Ro mouse mammary tumor cells in exponential growth phase in severely (less than 20 ppm) hypoxic conditions. The relative cytotoxicities were misonidazole (MISO) = desmethylmisonidazole (9963) greater than SR-2508 much greater than SR-2555 indicating that the compounds with the lowest lipophilicity were less cytotoxic. The rates of uptake of these compounds were MISO greater than 9963 greater than SR-2508 = SR-2555. These data together with comparisons of the amounts of cell-associated compounds indicate that the similarity in toxicity of MISO and 9963 can be related to a general similarity in their pharmacokinetics, but that other unknown factors must be considered to explain the relative toxicity of SR-2508 and SR-2555. In other experiments, EMT6/Ro cells synchronized using centrifugal elutriation were most sensitive in hypoxia to MISO at the late G1--early S phase of the cell cycle. These data indicate the importance of considering cellular and subcellular distribution of these nitroimidazoles as well as possible cell cycle specificity for cytotoxicity in interpreting relative effectiveness of different compounds in responses of mixed populations of cells in cultures or tumors.

Cytotoxicity of hypoxic cell sensitizers in multicell spheroids.

Multicell tumor spheroids of EMT6/Ro cells were used to evaluate the cytotoxicity of several hypoxic cell sensitizers. The number of clonogenic cells per spheroid was determined after different exposure periods and concentrations of misonidazole, Ro-05-9963, and SR 2508 which have similar electron affinities. The kinetics of cytotoxicity were similar for each drug and the clonogenic fraction was reduced by about 0.5 to a plateau level after 24 hours at 3.0 mM for spheroids grown in 20% O2. Extended exposure periods caused additional cytotoxicity for both Ro-05-9963 and SR 2508. Lower concentrations (0.5 mM) of these sensitizers were not cytotoxic even in spheroids grown in low concentrations of oxygen (2.5% O2) to increase the hypoxic fraction. However, at cytotoxic concentrations (3.0 mM) spheroids grown in this low oxygen concentration exhibited almost twice as much cytotoxicity. No cytotoxicity was produced at 3.0 mM misonidazole in small spheroids without necrotic centers and hypoxic cells. In addition to being cytotoxic, continuous exposure of spheroids to high concentrations (3.0 mM) of misonidazole were cytostatic. The spheroid experiments were predictive of the relative effectiveness of the different sensitizers for EMT6/Ro tumors in vivo but the rate and extent of cytotoxicity was greater in tumors especially at low concentrations. In both spheroids and tumors there was little change in growth rate after 24 hours or single intraperitoneal exposures respectively, even with concentrations which reduced the clonogenic fraction by 0.9. This was related to a rapid repopulation of cells and increase in growth fraction after misonidazole cytotoxicity. Addition of adriamycin immediately after misonidazole cytotoxicity resulted in an apparent supra-additive overall response. The significance of these results for interpretation of tumor properties and responses in vivo and for tumor therapy were discussed.

Evidence for the presence of secondary calcium phosphate in bone and its stabilization by acid production.

Calvaria from three 4-day-old chicks were incubated in a variety of buffers to study the parameters controlling the equilibration of bone with its supporting fluid environment. Strong inferential chemical evidence was obtained for the presence in viable bone of some calcium phosphate phase of high solubility apparently governed by the Ksp of CaHPO4-2H2O. In dead bone, this phase underwent spontaneous conversion to a higher Ca/P ratio. In viable bone the soluble phase appeared to be stabilized by the metabolic production of acid (lactate) as revealed by the effects of selective inhibitors.

Further studies on the nature of fluid compartmentalization in chick calvaria.

Labeled mannitol and polyethylene glycol (mol. wt. 4000), PEG, were used as space markers to estimate the fluid compartmentalization of calvaria taken from 3-5-day-old chicks. The mannitol results indicated that about 80% of the water in these bones is extracellular. Only a small fraction of this extracellular space was available to the diffusion of polyehtylene glycol. Adsorption studies in vitro and prior incubation of the tissues with hydrolytic enzymes indicated that the phenomenon of exclusion of PEG was primarily physical in nature. The polyethylene polymer is excluded from the water of hydration of the mineral phase. Apparently much of the extracellular water (two-thirds) of bone is in the form of crystal hydration.