An efficient multiple-exposure analysis of the toxicity of crisnatol, a DNA intercalator in phase II clinical trials

Abstract

To investigate the toxicity and mechanism of action of crisnatol (CRS), a new DNA intercalator currently in phase II clinical trials, we analyzed cellular and nuclear flow cytometric (FCM) parameters of murine erythroleukemic cells (MELC) exposed to a range of CRS concentrations over three exposure conditions: short-term (4 h), long-term (24 h), and short-term with recovery (4 h+/19 h-). At 0.5-1.0 microM CRS, 4 h exposure results in a reversible G2-phase block, while 24 h exposure results in greater than G2 polyploidy. At 5-10 microM CRS concentrations, cells exhibit persistent retardation of S-phase progression or irreversible G2 and/or greater than G2 blocks, depending on duration of exposure. Cells terminally blocked in G2 exhibit increased nuclear/cellular volumes and increased nuclear fluorescein isothiocyanate (protein) staining, suggestive of unbalanced growth. At 25-50 microM CRS concentrations, MELC exhibit severe membrane perturbation (loss of viability) regardless of exposure. In contrast, following similar exposures to an inactive isomer of CRS, MELC exhibit minimal cell cycle effects, suggesting that cell cycle kinetics may be a useful criterion for assessing potential efficacy. Similar analyses with different classes of chemotherapeutic agents reveal that the range of induced cellular/nuclear perturbations varies with the class of compound used. Taken together, these results suggest that drug toxicity can vary with both concentration and duration of exposure and, as such, a selective multiple-exposure FCM analysis may better represent the spectrum of drug action for drug development and pharmacodynamic studies.