UTILITY OF THE MURINE ERYTHROLEUKEMIC CELL (MELC) IN ASSESSING MECHANISMS OF ACTION OF DNA-ACTIVE DEVELOPMENTAL TOXICANTS - APPLICATION TO 5-FLUOROURACIL

Murine erythroleukemic cells (MELC) exposed to 2'-deoxy-5-azacytidine (D-AZA) or to the active cyclophosphamide (CP) metabolites phosphoramide mustard (PAM) and 4-hydroxycyclophosphamide (OHCP) exhibit cell-cycle perturbations similar to those seen in limb bud nuclei of gestational day (GD) 10 CD-1 mouse embryos exposed in utero to D-AZA or CP, respectively. The similarities in response suggest MELC may be a useful model for determining mechanisms of action of DNA-active developmental toxicants. As such, we used the MELC model to investigate the mechanism of action of 5-fluorouracil (5-FU), an antimetabolite that induced in GD 14 rat fetuses an apparent S-phase accumulation in limb cells 8 hr after in utero exposure, but S-phase depletion in liver cells 24 hr postexposure. MELC timed-recovery and synchronization studies suggest that in proliferative tissues, 5-FU induces an early S-phase accumulation, followed by a synchronous, concentration-dependent delay in progression through the cell cycle. Consequently, it is the tissue-specific rate of delay, rather than different mechanisms of action, that results in apparent tissue-specific perturbations. Moreover, growth and cell-cycle data suggest that cells entering S phase (when TS activity is greatest) are the most sensitive to 5-FU toxicity. Assays of the TS activity of recovering MELC reveal that although the initial extent of TS inhibition does not appear to be concentration-dependent, the time to recovery is, suggesting that the rate of S-phase progression is closely associated with TS activity. Together, the induction of similar cell-cycle perturbations in embryonic/fetal tissues and MELC following exposure to CP (or CP metabolites), D-AZA, or 5-FU, as well as the adaptability of MELC to a variety of kinetic assays suggests that, for those developmental toxicants suspected of inducing cell-cycle perturbations in embryonic/fetal tissues, MELC may prove useful for elucidating mechanisms of action.