Toxicological Sciences 68, 349-360 (2002)
Copyright © 2002 by the Society of Toxicology
ENDOCRINE TOXICOLOGY |
An Approach for Assessing Estrogen Receptor-Mediated Interactions in Mixtures of Three Chemicals: A Pilot Study
* Toxicology and Environmental Research and Consulting, The Dow Chemical Company, 1803 Building, Midland, Michigan 48674;
Department of Biostatistics, Virginia Commonwealth University, Richmond, Virginia 23298; and
Department of Biochemistry and Molecular Biology and National Food Safety and Toxicology Center, Michigan State University, East Lansing, Michigan 48824
Most studies investigating interactions among endocrine-active chemicals have been limited to binary mixtures. This study reports on the preliminary evaluation an in vitro MCF-7 cell ER-
reporter gene system, coupled with a statistical methodology adapted for assessing interactions within ternary (3-chemical) mixtures. Two mixtures were initially chosen for assessment of the in vitro system’s ability to detect additivity (mixture A) as well as greater-than-additive (mixture B) responses. Mixture A was composed of 17ß-estradiol (E2), ethinyl estradiol, and diethylstilbestrol and served as a control for additivity, whereas mixture B (E2, epidermal growth factor, insulin-like growth factor-I) was selected to model greater-than-additive interactions based on previous in vitro studies. After generating complete dose–response curves for each chemical, ternary mixtures were then tested in a full factorial design (4 concentrations per chemical, 64 treatment groups). A response surface was estimated using a nonlinear mixed model, and the observed responses were statistically analyzed for departures from the responses expected under the assumption of additivity. Mixture A exhibited additivity in vitro when the chemicals were present at concentrations in the linear range of their individual dose-response curves. For mixture B, in vitro analysis resulted in the additivity hypothesis being rejected (p < 0.001) because of a greater-than-additive interaction, as expected. A limited in vivo evaluation of mixture A was performed in the immature mouse uterotrophic assay (27 treatment groups), which agreed with the in vitro assessment of no significant departure from additivity ( p = 0.903). These findings demonstrate the ability of this in vitro methodology to detect additive, greater-than-additive, and less-than-additive interactions within ternary mixtures, which now allows for the assessment of environmentally relevant mixtures.
Key Words: mixture; endocrine; estrogen receptor; additivity; diethylstilbestrol; 17ß-estradiol; response surface; synergy.
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