One of the fundamental principles of chemical mixture toxicity that remains to be clarified is whether there is a relationship between the type of combined effect obtained and the molecular site or sites of action of the agents. The work proposed in this application is designed to help determine whether such a relationship exists, thereby providing a more sound fundamental basis from which to perform more accurate risk assessments. The approach involves chemical mixture toxicity testing using the previously developed and evaluated frog embryo mixture toxicity assay. The chemicals evaluated in combination are osteolathyrogens, chemicals which inhibit lysyl oxidase (LO) catalyzed cross-linking reactions for developing connective tissue fibers. Osteolathyrogens have several potential sites of action: the LO active site, the organic cofactor for LO, chelation of copper from LO, and blockage of sites on the developing fibers where the normal cross-linking reactions take place. Through the use of UV-VIS spectrophotometry and spectrofluorimetry, each of twelve to fifteen osteolathyrogens will be examined for potential reactivity with the organic cofactor for LO and for their potential to chelate copper from the enzyme. The results of these studies will be compared with results from the mixture toxicity assay. Preliminary data suggests that mixture toxicity resembles the dose-addition combined effect, generally, but with results for some combinations being significantly different, statistically, from dose-addition - the combined effect expected for agents working at the same molecular site. By testing chemicals in two-, three- and four-chemical combinations that have been demonstrated to work at the same or at different molecular sites, it should be possible to evaluate the current theoretical framework for combined effect/site of actions models. Upon completion, the project should provide an answer as to whether there is a relationship between the type of combined effect obtained and the molecular site(s) of action of the agents.
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