BIOLOGICALLY-BASED DOSE-RESPONSE MODELING IN DEVELOPMENTAL TOXICOLOGY- BIOCHEMICAL AND CELLULAR SEQUELAE OF 5-FLUOROURACIL EXPOSURE IN THEDEVELOPING RAT

Mechanistically based dose-response models for developmental toxicity require elucidation of critical biological events that intervene betwe en maternal exposure and adverse developmental outcome. We have examin ed some of the major events in the rat embryo/fetus following a subcut aneous injection of 5-fluorouracil (5-FU; 0-40 mg/kg) to the dam on Da y 14 of gestation. This treatment resulted in reduced fetal weight tha t was significant at doses of 20 mg/kg and higher, generalized reduced ossification at doses above 25 mg/kg, and wavy ribs at doses of 30 mg /kg and higher. Numerous malformations including cleft palate and hind limb defects were substantially increased at doses of 35 and 40 mg/kg. 5-FU inhibits thymidylate synthetase (TS), resulting in inhibited gro wth of rapidly proliferating tissues. To identify early events in the pathogenesis of hindlimb defects, we examined the effects of 5-FU on T S activity, cell cycle, growth, and morphology in the developing hindl imb as a function of dose and time. The rate of decline of TS activity following 5-FU exposure was dose related, although maximal inhibition and recovery were similar at doses within (20 and 40 mg/kg) and below (10 mg/kg) the range of detectable developmental toxicity. Flow cytom etric analysis of nuclei from embryonic hindlimbs revealed a transient increase in the percentage of cells in S phase and decrease in G(0)/G (1) phase 8 hr after maternal injection of 20-40 mg 5-FU/kg, but not a t lower doses. Reduction in growth and morphometric changes of hindlim bs were observed only after maternal exposure to 40 mg/kg. The tissue specificity of these effects was examined by comparing the hindlimb wi th other embryonic tissues. There was also a dose-related decline of T S activity in the embryonic liver. However, the pattern of recovery of TS activity and cell cycle alterations were different in the liver th an in the hindlimb, probably reflecting the higher cell proliferative rate in the liver at this stage. We have derived a quantitative, empir ical model for induction of hindlimb defects based on TS inhibition an d subsequent cellular events following 5-FU exposure. The model predic ted a dose response similar to that of the observed data although the predicted curve was shifted toward lower doses. These results suggest that while this model may not capture all of the critical events invol ved in the induction of hindlimb defects following maternal exposure t o 5-FU, it does reflect a central mechanism of its developmental toxic ity. Biologically based dose-response modeling provides a framework fo r testing mechanistic hypotheses, and developing such models should ul timately improve our ability to perform risk assessments. (C) 1994 Aca demic Press, Inc.