Evaluation of dose-response curve analysis in delineating shared or different molecular sites of action for...

Single-chemical and mixture concentration-response curves generated using a frog embryo model were examined for value in assessing whether chemicals exert toxic effects at the same or at different molecular sites of action. Toxicity tests were conducted on a series of osteolathyrogens, i.e. chemicals that inhibit cross-linking of developing connective tissue fibers. Induction of osteolathyrism, which manifests as lesions in the notochord of exposed tadpoles, has several possible molecular sites of action, including agent-cofactor reactivity during the enzyme-mediated cross-linking process. UV-VIS spectrophotometry of osteolathyrogen-cofactor reactivity (i.e. in vitro analysis) was coupled with the 96-h frog embryo mixture toxicity assay (i.e. in vivo toxicity) to compare molecular sites of action for several osteolathyrogens with the combined osteolathyritic effects of the agents. Single-chemical concentration-response curves were used to calculate theoretical curves for the dose-addition model of combined effect. Slope and EC(50) values for both theoretical and experimental mixture curves were then generated to statistically examine the hypothesis that agents with shared sites of action have dose-response curve (DRC) slopes that are similar when given alone and in combination, and slope and EC(50) values that, when administered together, are consistent with those calculated for dose-addition. For combinations of cofactor-binding agents (semicarbazide, thiosemicarbazide, aminoacetonitrile), slope values were generally similar with additivity quotients near 1.0 (1.0=dose-additive) and combined osteolathyritic effects that were consistent with dose-addition. None of these were true for combinations that included agents that did not show rapid cofactor binding (β-aminopropionitrile, methyleneaminoacetonitrile). The results suggest that DRC analysis could be a useful tool for delineating common or different molecular sites of toxic action and that the approaches used warrant further study for evaluating the mechanistic basis for combined effects of toxicants.