title: An approach for assessing estrogen receptor-mediated interactions in mixtures of three chemicals: a pilot study.
abstract: 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-alpha 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 17beta-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.
authors.affiliation: Toxicology and Environmental Research and Consulting, The Dow Chemical Company, 1803 Building, Midland, Michigan 48674, USA. gdcharles@dow.com
authors.forename: Grantley D
authors.initials: GD
authors.lastname: Charles
authors.forename: C
authors.initials: C
authors.lastname: Gennings
authors.forename: Timothy R
authors.initials: TR
authors.lastname: Zacharewski
authors.forename: B Bhaskar
authors.initials: BB
authors.lastname: Gollapudi
authors.forename: Edward W
authors.initials: EW
authors.lastname: Carney
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D001681:Biological Assay / Q000379:methods; D001943:Breast Neoplasms; D004054:Diethylstilbestrol / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004357:Drug Synergism; D004815:Epidermal Growth Factor / Q000494:pharmacology; D004958:Estradiol / Q000494:pharmacology; D004967:Estrogens / Q000494:pharmacology*; D004997:Ethinyl Estradiol / Q000494:pharmacology; D005260:Female; D017930:Genes, Reporter / Q000235:genetics; D006801:Humans; D007334:Insulin-Like Growth Factor I / Q000494:pharmacology; D051379:Mice; D008815:Mice, Inbred Strains; D009929:Organ Size / Q000187:drug effects; D011960:Receptors, Estrogen / Q000819:agonists* / Q000235:genetics; D014158:Transcription, Genetic / Q000187:drug effects; D014407:Tumor Cells, Cultured; D014599:Uterus / Q000187:drug effects / Q000473:pathology
chemical_list: D004967:Estrogens; D011960:Receptors, Estrogen; D004997:Ethinyl Estradiol; D004958:Estradiol; D004815:Epidermal Growth Factor; D007334:Insulin-Like Growth Factor I; D004054:Diethylstilbestrol