Abstract
Screening methods for detecting single chemicals and chemical mixtures that have endocrine effects are of increasing importance. One such assay used by many laboratories to screen for potential estrogenicity is the estrogen receptor (ER) transcriptional activation assay, also known as an ER “reporter gene assay.” When applied in screening situations or to mechanistic studies, the assay appears to be quite reliable in giving a qualitative indication of a compound’s potential to activate ER-regulated target genes. Reporter gene assays also are advantageous for more complex applications, such as the analysis of chemical mixtures, but such applications present some challenges for statistical analysis. In particular, the need to transiently transfect cells with DNA constructs each time a reporter gene experiment is run may be a major factor contributing to a significant amount of interexperiment variability in strictly reproducing concentration-effect curves. This article reports on the use of nonlinear mixed models to account for the different sources of variability in analyzing such data. A population-averaged model is selected for use in the analysis of mixtures of chemicals. The models are illustrated with data from an ER reporter gene assay used to analyze tertiary mixtures of chemicals expected to exhibit additivity or synergy based on prior scientific reports. The models yielded the expected conclusions, and thus, validated the statistical approach.
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Arcaro, K. F., and Grethery, J. F. (1997), “Estrogenic Responses of Human Breast Cancer Cells to Combinations of Hydroxylated PCBs, Pesticides and 17-b Estradiol,” Toxicologist, 36, Supplement 1, 295.
Arnold, S. F., Bergeron, J. M., Gran, D. Q., Collings, B. M., Vonier, P. M., Crews, D., Toscano, Jr., W. A., and BcLachlan, J. A. (1997a), “Synergistic Responses of Steroidal Estrogens in Vitro (yeast) and in Vivo (turtles),” Biochemical and Biophysical Research Communications, 235, 336–342.
Arnold, S. F., Vonier, B. M., Collins, B. M., Klotz, D. M., Guillette, Jr. L. J., and McLachlan, J. A. (1997b), “In Vitro Synergistic Interaction of Alligator and Human Estrogen Receptors with Combinations of Environmental Chemicals,” Environmental Health Perspectives, 105, Suppl. 3, 615–618.
Arnold, S. F., Klotz, D. M., Collins, B. M., Vonier, P. M., Guillette Jr., L. J., and McLachlan, J. A. (1996), “Synergistic Activation of Estrogen Receptor with Combinations of Environmental Chemicals,” Science, 272, 1489–1492.
Ashby, J., Lefevre, P. A., Odum, J., Harris, C. A., Routledge, E. J., and Sumpter, J. P. (1997), “Synergy Between Synthetic Oestrogens?” Nature, 385, 494.
Berenbaum, M. C. (1985), “The Expected Effect of a Combination of Agents: The General Solution,” Journal of Theoretical Biology, 114, 413–431.
Carter, W. H., Jr., Gennings, C., Staniswalis, J. G., Campbell, E. D., and White, K. L. Jr. (1988), A Statistical Approach to the Construction and Analysis of Isobolograms,” Journal of American College Toxicology, 7, 963–973.
Charles, D. G., Gennings, C., Zacharewski, T. R., Gollapudi, B. B., and Carney, E. W. (2001a), “An Approach for Assessing Estrogen Receptor-Mediated Interactions in Mixtures of Three Chemicals: Proof of Principle,” Toxicological Sciences, under review.
Charles, D. G., (2001b), “Assessment of Interactions of Diverse Ternary Mixtures in an Estrogen Receptor-α Reporter Assay,” Toxicology and Applied Pharmacology, in press.
Cho, H., and Katzenellenbogen, B. S. (1993), “Synergistic Activation of Estrogen Receptor-Mediated Transcription by Estradiol and Protein Kinase Activators,” Molecular Endocrinology, 7, 441–452.
Crews, D., Bergeron, J. M., and McLachlan, J. A. (1995), “The Role of Estrogen in Turtle Sex Determination and the Effects of PCBs,” Environmental Health Perspectives, 103, Suppl. 7, 73–77.
Food Quality Protection Act of 1996, Public Law 104-170, 104th Congress, August 3, 1996.
Gennings, C., and Carter, Jr., W. H. (1995), “Utilizing Concentration-Response Data from Individual Components to Detect Statistically Significant Departures from Additivity in Chemical Mixtures,” Biometrics, 51, 1264–1277.
Gennings, C., Carter, W. H., Jr., Campain, J. A., Bae, D., and Yang, R. S. H. (2002), “Statistical Analysis of Interactive Cytotoxicity in Human Epidermal Keratinocytes Following Exposure to a Mixture of Four Metals,” Journal of Agricultural, Biological, and Environmental Statistics, 7, 58–73.
Kelly, C., and Rice, J. (1990), “Monotone Smoothing with Application to Dose-Response Curves and the Assessment of Synergism,” Biometrics, 46, 1071–1085.
Loewe, S. (1953), “The Problem of Synergism and Antagonism of Combined Drugs,” Arzneimittle forshung, 3, 285–290.
Loewe, S., and Muischnek, H. (1926), “Uber kombinationswirkunger. 1. Mitteilung: Hiltsmittel der fragstellung,” Naunyn-Schmiedebergs, Arch. Pharmacol, 114, 313–326.
Ramamoorthy, K., Wang, F., Chen, I.-C., Norris, J. D., McDonnell, D. P., Leonard, L. S., Gaido, K. W., Bocchinfuso, W. P., Korach, K. S., and Safe, S. (1997), “Estrogenic Activity of a Dieldrin/Toxaphene Mixture in the Mouse Uterus, MCF-7 Human Breast Cancer Cells, and Yeast-Based Estrogen Receptor Assays: No Apparent Synergism,” Endocrinology, 138, 1520–1527.
Safe, S., Ramamoorthy, K., Wang, F., Chen, I.-C., Norris, J. D., McDonnel, D. P., Gaido, K. W., Bocchinfuso, W. P., and Dorach, K. S. (1997), “Estrogenic Activity of a Dieldrin/Toxaphene Mixture in the Mouse Uterus, MCF-7 Human Breast Cancer Cells and Yeast-Based Estrogen Receptor Assays: No Apparent Synergism,” Toxicologist, 36, Supplement 1, 294.
Soto, A. M., Fernandez, M. F., Juizzi, M. F., Oles Karasko, A. S., and Sonnenschein, C. (1997), “Developing a Marker of Exposure to Zenoestrogen Mixtures in Human Serum,” Environmental Health Perspective 105, Supplement 3, 647–654.
Vonesh, E. F., and Chinchilli, V. M. (1997), Linear and Nonlinear Models for the Analysis of Repeated Measurements, New York: Marcel Dekker, Inc.
Wiese, T. E., Lambright, C. R., and Kelce, W. R. (1997), “Lack of Synergistic Estrogen Effects of Dieldrin and Endosulfan Mixtures on MCF-7 and MVLN Cells,” Toxicologist 36, Supplement 1, 294.
Zacharewski, T., Berhane, K., Gillesby, B., and Burnison, K. (1995), “Detection of Estrogen- and Dioxin-like Activity in Pulp and Paper Mill Black Liquor Ef uent Using in vitro Recombinant Receptor/Reporter Gene Assays,” Environmental Science Technology, 29, 2140–2146.
Zeger, S. L., Liang, K. L., and Albert, P. S. (1988), “Models for Longitudinal Data: A Generalized Estimating Equation Approach,” Biometrics, 44, 1049–1060.
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Gennings, C., Charles, G., Gollapudi, B. et al. Analysis of resulting data from estrogen receptor reporter gene assays. JABES 8, 84–104 (2003). https://doi.org/10.1198/1085711031030
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DOI: https://doi.org/10.1198/1085711031030