Influence of cell crowding on toxicity of nitroheterocycles

Cell density (no. of cells per unit area or volume) during drug treatment may play a role of considerable importance in the interpretation of drug toxicity experiments performed in vitro. Chinese hamster V-79 and mouse L-929 cells exposed to nitroheterocycles under aerobic conditions are considerably more sensitive to the cytotoxic effects of these drugs when incubated at low cell density (10² cells/cm² or 10⁴ cells/ml) than at higher cell density (10⁴ cells/cm² or 10⁶ cells/ml). This may be related to diffusion limitations when cells are in contact and to the ability of dense cell suspensions to inactivate drugs. In contrast, under anaerobic conditions, more toxicity is observed at high cell density than at low cell density, perhaps due to local effects of toxic metabolites. Toxicity appears to correlate with intracellular drug levels under both aerobic and hypoxic conditions.The chemical nature of the fluorescent species has not yet been determined. However, it is likely that loss of the nitro group reduces but does not abolish fluorescence. When L-cells were used to metabolize AF-2 under hypoxia so that only 20% of the parent compound remained (with nitro group intact), 50% of the fluorescence was still present (unpublished results). Cells incubated with AF-2 under air or nitrogen show little decrease in fluorescence intensity for several hours after drug removal suggesting that the fluorescent compound being observed was bound intracellularly. As yet, we have no reason to suspect that the fluorescent products bound under hypoxia differ from those bound under air. Therefore, cell density dependent toxicity of nitroheterocycles under aerobic conditions may be related to diffusion limitations when cells are in close contact, as well as drug inactivation by cells at high cell densities.