Statistical analysis of interactive cytotoxicity in human epidermal keratinocytes following exposure to a mixture of four metals

  • Chris Gennings
  • Walter H. Carter
  • Julie A. Campain
  • Dong-soon Bae
  • Raymond S. H. Yang


Exposure to mixtures of chemicals is an important and relevant environmental issue. Of particular interest is the detection and characterization of departure of biological effects from additivity. Methodology based on the assumption of additivity is used in fitting single-chemicaldata. Interactionsare determined and characterized by making comparisons between the observed and predicted responses at mixtures along a fixed ratio ray of the component substances. Two simultaneous tests are developed for testing for any departure from additivity. Multiple comparisons procedures are used to compare observed responses to that predicted under additivity. A simultaneous confidence band on the predicted responses along the mixture ray is also developed. The methods are illustrated with cytotoxicity data that arise when human epidermal keratinocytes are exposed to a mixture of arsenic, chromium, cadmium, and lead. Synergistic, antagonistic, and additive cytotoxicities were observed at different dose levels of the four-metal mixture.

Key Words

Simultaneous inference Synergism 


  1. ATSDR. (1997), 1997 CERCLA Priority List of Hazardous Substances That Will Be the Subjects of Toxicological Profiles and Support Document, Washington, DC: Agency for Toxic Substances and Disease Registry, U.S. Department of Health and Human Services.Google Scholar
  2. Berenbaum, M. C. (1985), “The Expected Effect of a Combination of Agents: The General Solution,” Journal of Theoretical Biology, 114, 413–431.CrossRefGoogle Scholar
  3. Berenbaum, M. C. (1989), “What Is Synergy?,” Pharmacological Reviews, 41, 93–141.Google Scholar
  4. Calabrese, E. J. (1997), “Hormesis Revisited: New Insights Concerning the Biological Effects of Low-Dose Exposures to Toxins,” Environmental Law Reporter, 27, 10526–19532.Google Scholar
  5. Calabrese, E. J., and Baldwin, L. A. (1997a), “The Dose Determines the Stimulation (and Poison): Development of a Chemical Hormesis Database.” International Journal of Toxicology, 16, 545–559.CrossRefGoogle Scholar
  6. — (1997b), “A Quantitatively-Based Methodology for the Evaluation of Chemical Hormesis,” Human Ecological Risk Assessment, 3, 545–554.Google Scholar
  7. Choi, E. J., Toscano, D. G., Ryan J. A., Riedel, N., and Toscano, W. A. (1991), “Dioxin Induces Transforming Growth Factor-α in Human Keratinocytes,” Journal of Biological Chemistry, 266, 9591–9597.Google Scholar
  8. Cohen, M. D., Kargacin, C. B., Klein, C. B., and Costa, M. (1993), “Mechanisms of Chronium Carcinogenicity and Toxicity,” Critical Reviews in Toxicology, 23, 255–281.CrossRefGoogle Scholar
  9. De Rosa, C. T., Johnson, B. L., Fay, M., Hansen, H., and Mumtaz, M. M. (1996), “Public Health Implications of Hazardous Waste Sites: Findings, Assessment and Research,” Food and Chemical Toxicology, 34, 1131–1138.CrossRefGoogle Scholar
  10. Fay, R. M., and Mumtaz, M. M. (1996), “Development of a Priority List of Chemical Mixtures Occurring at 1188 Hazardous Waste Sites, Using the Haz Dat Database,” Food and Chemical Toxicology, 34, 1163–1165.CrossRefGoogle Scholar
  11. Gaido, K. W., Maness, S. C., Leonard, L. S., and Greenlee, W. F. (1992), “2,3,7,8-Tetrachlorodibenzo-p-Dioxin-Dependent Regulation of Transforming Growth Factors-α and β2 Expression in a Human Keratinocyte Cell Line Involves Both Transcriptional and Posttranscriptional Control,” Journal of Biological Chemistry, 267, 245591–24595.Google Scholar
  12. Gennings, C., and Carter, W. H., Jr., (1995), “Utilizing Concentration—Response Data From Individual Components to Detect Statistically Significant Departures From Additivity in Chemical Mixtures,” Biometrics, 51, 1264–1277.MATHCrossRefGoogle Scholar
  13. Gennings, C., Schwartz, P., Carter, W. H., Jr., and Simmons, J. E. (1997), “Detection of Departures From Additivity in Mixtures of Many Chemicals With a Threshold Model,” Journal of Agricultural, Biological, and Environmental Statistics, 2, 198–211.CrossRefMathSciNetGoogle Scholar
  14. Germolec, D. R., Spalding, J., Boorman, G. A., Wilmer, J. L., Yoshida, T., Simeonova, P. P., Bruccoleri, A., Kayama, F., Gaido, K., Tennant, R., Burleson, F., Dong, W., Lang, R. W., and Luster, M. I. (1997), “Arsenic Can Mediate Skin Neoplasia by Chronic Stimulation of Keratinocyte-Derived Growth Factors,” Mutation Research, 386, 209–218.CrossRefGoogle Scholar
  15. Germolec, D. R., Yoshida, T., Gaido, K., Wilmer, J. L., Simeonova, P. P., Kayama, F., Burleson, F., Dong, W., Lange, R. W., and Luster, M. I. (1996), “Arsenic Induces Overexpression of Growth Factors in Human Keratinocytes,” Toxicology Applied Pharmacology, 141, 308–318.Google Scholar
  16. Glick, A. B., Lee, M. M., Darwiche, N., Kulkarni, A. B., Karlsson, S., and Yuspa, S. H. (1994), “Targeted Deletion of the TGFβ1 Gene Causes Rapid Progression to Squamous Cell Carcinoma,” Genes Development, 8, 2429–2440.CrossRefGoogle Scholar
  17. Glick, A. B., Sporn, M. B., and Yuspa, S. H. (1991), “TGF-β1 and TGF-α in Primary Keratinocytes and Papillomas Expressin v-Ha-ras,” Molecular Carcinogenicity, 4, 210–219.CrossRefGoogle Scholar
  18. Hochberg, Y. (1990), “A Sharper Bonferroni Procedure for Multiple Tests of Significance,” Biometrika, 75, 800–802.CrossRefMathSciNetGoogle Scholar
  19. Kachinskas, D. J., Qin, Q., Phillips, M. A., and Rice, R. H. (1997), “Arsenic Suppression of Human Keratinocyte Programming,” Mutation Research, 386, 253–261.CrossRefGoogle Scholar
  20. Kelly C., and Rice, J. (1990), “Monotone Smoothing With Application to Dose—Response Curves and the Assessment of Synergism,” Biometrics, 46, 1071–1085.CrossRefGoogle Scholar
  21. Loewe, S. (1953), “The Problem of Synergism and Antagonism of Combined Drugs,” Arzneimittle Forshung, 3, 285–290.Google Scholar
  22. Loewe, S., and Muischnek, H. (1926), “Uber Kombinationswirkunger. I. Mitteilung: Hiltsmittel der gragstellung. Naunyn-Schmiedebergs,” Archives of Pharmacology, 114, 313–326.CrossRefGoogle Scholar
  23. Miller, R. J., Jr. (1981), Simultaneous Statistical Inference (2nd ed.), New York: McGraw Hill.MATHGoogle Scholar
  24. Mosmann, T. (1983), “Rapid Colorimetric Assay for Cellular Growth and Survivals: Application to Proliferation and Cytotoxicity Assays,” Journal of Immunological Methods, 65, 55–63.CrossRefGoogle Scholar
  25. Pietenpol, J. A., Holt, J. T., Stein, R. W., and Moses, H. L. (1990), “Transforming Growth Factor β1-Suppression of c-myc Gene Transcription: Role in Inhibition of Keratinocyte Proliferation,” Proceedings of the National Academy of Sciences, USA, 87, 53–65.CrossRefGoogle Scholar
  26. Punnonen, K., Denning, M. F., Rhee, S. G., and Yuspa, S. H. (1994), “Differences in the Regulation of Phosphatidylinositol-Specific Phospholipase C in Normal and Neoplastic Keratinocytes,” Molecular Carcinogenicity, 10, 216–225.CrossRefGoogle Scholar
  27. Seber, G. A., and Wild, C. J. (1989), Nonlinear Regression, New York: Wiley.MATHCrossRefGoogle Scholar
  28. Stebbing, A. R. D. (1982), “Hormesis—The Stimulation of Growth by Low Levels of Inhibitors.” Science of the Total Environment. 22, 213–234.CrossRefGoogle Scholar
  29. — (1997), “A Theory for Growth Hormesis,” BELLE Newsletter, 6, 1–11.Google Scholar
  30. Ye, J., Zhang, X., Young, H. A., Mao, Y., and Shi, X. (1995), “Chromium(V1)-Induced Nuclear Factor-κB Activation in Intact Cells Via Free Radical Reactions,” Carcinogenesis, 16, 2401–2405.CrossRefGoogle Scholar
  31. Yen, H.-T., Chiang, L.-C., Wen, K.-H., Chang, S.-F., Tsai, C.-C., Yu, C.-C., Yu, C.-L., and Yu, H.-S. (1996), “Arsenic Induces Interleuk in-8 Expression in Cultured Keratinocytes,” Archives Dermatological Research, 288, 716–717.CrossRefGoogle Scholar

Copyright information

© International Biometric Society 2002

Authors and Affiliations

  • Chris Gennings
    • 1
  • Walter H. Carter
    • 1
  • Julie A. Campain
    • 2
  • Dong-soon Bae
    • 2
  • Raymond S. H. Yang
    • 2
    • 3
  1. 1.Department of BiostatisticsVirginia Commonwealth UniversityRichmond
  2. 2.Center for Environmental Toxicology and TechnologyColorado State UniversityFt. Collins
  3. 3.Department of Environmental HealthColorado State UniversityFt. Collins

Personalised recommendations