Developmental Immunotoxicity (DIT): Assays for Evaluating Effects of Exogenous Agents on Development of the Immune System

Jamie C. DeWitt1, Margie M. Peden‐Adams2, Deborah E. Keil3, Rodney R. Dietert4

1 Department of Pharmacology and Toxicology, Brody School of Medicine, East Carolina University, Greenville, 2 Harry Reid Center for Environmental Studies, University of Nevada Las Vegas, Las Vegas, 3 Medical Laboratory Sciences, Department of Pathology, University of Utah School of Medicine, Salt Lake City, 4 Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 18.15
DOI:  10.1002/0471140856.tx1815s51
Online Posting Date:  February, 2012
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Abstract

Developmental immunotoxicity (DIT) occurs when exposure to environmental risk factors prior to adulthood, including chemical, biological, physical, or physiological factors, alters immune system development. DIT may elicit suppression, hyperactivation, or misregulation of immune responses and may present clinically as decreased resistance to pathogens, allergic and autoimmune diseases, and inflammatory diseases. Immunotoxicity testing guidelines established by the Environmental Protection Agency for adult animals (OPPTS 8703.7800) require functional tests and immunophenotyping that are suitable for detecting immunomodulation, especially immunosuppression. However, evaluating immune function in offspring that are not fully immunocompetent yields results that are challenging to interpret. Therefore, this unit will describe an optimum exposure scenario, reference two assays (immunophenotyping and histopathology) appropriate for detecting immunomodulation in weaning‐age offspring, and reference four assays (immunophenotyping, histopathology, T cell‐dependent antibody responses, and delayed‐type hypersensitivity) appropriate for detecting immunomodulation in immunocompetent offspring. The protocol also will reference other assays (natural killer cell and cytotoxic T lymphocyte) with potential utility for assessing DIT. Curr. Protoc. Toxicol. 51:18.15.1‐18.15.14. © 2012 by John Wiley & Sons, Inc.

Keywords: Developmental immunotoxicity; immunophenotyping; histopathology; TDAR; DTH

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Exposure Scenario for Including All Critical Windows of Immune Development
  • Alternate Protocol 1: Exposure Scenario for Evaluating Limited Windows of Exposure
  • Support Protocol 1: Evaluation of DIT
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Exposure Scenario for Including All Critical Windows of Immune Development

  Materials
  • Rodents for testing: timed‐pregnant dams or dams and sires ordered as breeding pairs
  • Test agent in appropriate delivery vehicle, if necessary
  • Apparatus for delivering test agent (gavage needles, water bottles, instillation apparatus, etc.)
  • Apparatus for weighing dams and offspring
  • Data sheets for recording weights and other data
  • Additional reagents and equipment for evaluating DIT ( protocol 3)

Alternate Protocol 1: Exposure Scenario for Evaluating Limited Windows of Exposure

  Materials
  • Pregnant dams or weaning‐age offspring
  • Test agent in appropriate delivery vehicle, if necessary
  • Apparatus for delivering test agent (gavage needles, water bottles, instillation apparatus, etc.)
  • Apparatus for weighing dams and offspring
  • Data sheets for recording weights and other data
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Figures

Videos

Literature Cited

   Ayadi, A., Ferrand, G., Goncalves da Cruz, I., and Warot, X. 2011. Mouse breeding and colony management. Curr. Protoc. Mouse Biol. 1:239‐264.
   Brown, R.C., Barone, S. Jr., and Kimmel, C.A. 2008. Children's health risk assessment: Incorporating a lifestage approach into the risk assessment process. Birth Defects Res. B Dev. Reprod. Toxicol. 83:511‐521.
   Burleson, G.R. and Burleson, F.G. 2008. Testing human biological in animal host resistance models. J. Immunotoxicol. 5:23‐31.
   Burleson, G.R., Burleson, F.G., and Dietert, R.R. 2010. The cytotoxic T lymphocyte assay for evaluating cell‐mediated immune function. Methods Mol. Biol. 598:195‐205.
   Burns‐Naas, L.A., Hastings, K.L., Ladics, G.S., Makris, S.L., Parker, G.A., and Holsapple, M.P. 2008. What's so special about the developing immune system? Int. J. Toxicol. 27:223‐254.
   DeWitt, J.C., Peden‐Adams, M.M., Keil, D.E., and Dietert, R.R. 2011. Current status of developmental immunotoxicity: Early‐life patterns and testing. Toxicol. Pathol. 2011 Nov 22. [Epub ahead of print].
   Dietert, R.R. and Holsapple, M.P. 2007. Methodologies for developmental immunotoxicity (DIT) testing. Methods 41:123‐131.
   Dietert, R.R. and DeWitt, J. 2010. Developmental immunotoxicity (DIT): The why, when, and how of DIT testing. Methods Mol. Biol. 598:17‐25.
   Dietert, R.R., Etzel, R.A., Chen, D., Halonen, M., Holladay, S.D., Jarabek, A.M., Landreth, K., Peden, D.B., Pinkerton, K., Smialowicz, R.J., and Zoetis, T. 2000. Workshop to identify critical windows of exposure for children's health: Immune and respiratory systems work group summary. Environ. Health Perspect. 108:483‐490.
   Heyser, C.J. 2003. Assessment of developmental milestones in rodents. Curr. Protoc. Neurosci. 25:8.18.1‐8.18.15.
   Holsapple, M.P., Burns‐Naas, L.A., Hastings, K.L., Ladics, G.S., Lavin, A.L., Makris, S.L., Yang, Y., and Luster, M.I. 2005. A proposed testing framework for developmental immunotoxicology (DIT). Toxicol. Sci. 83:18‐24.
   Luebke, R.W., Chen, D.H., Dietert, R., Yang, Y., King, M., and Luster, M.I. 2006. The comparative immunotoxicity of five selected compounds following developmental or adult exposure. J. Toxicol. Environ. Health B Crit. Rev. 9:1‐26.
   Luo, Y. and Dorf, M.E. 1993. Delayed‐type hypersensitivity. Curr. Protoc. Immunol. 55:4.5.1‐4.5.5.
   Luster, M.I., Dietert, R.R., Germolec, D.R., Luebke, R.W., and Makris, S.L. 2008. Developmental immunotoxicology. In Encyclopedia of Environmental Health ( B. Sonawane and R. Brown, eds.). Elsevier, Oxford, UK.
   Moser, V.C., Walls, I., and Zoetis, T. 2005. Direct dosing of preweaning rodents in toxicity testing and research: Deliberations of an ILSI RSI expert working group. Int. J. Toxicol. 24:87‐94.
   Smialowicz, R.J., Brundage, K.M., and Barnett, J.B. 2007. Immune system ontogeny and developmental immunotoxicology. In Immunotoxicology and Immunopharmacology, 3rd ed. ( R. Luebke, R. House, and I. Kimber, eds.). pp. 327‐345. CRC Press, Boca Raton, Florida.
   Tonk, E.C., Verhoef, A., de la Fonteyne, L.J., Waalkens‐Berendsen, I.D., Wolterbeek, A.P., van Loveren, H., and Piersma, A.H. 2011. Developmental immunotoxicity in male rats after juvenile exposure to di‐n‐octyltin dichloride (DOTC). Reprod. Toxicol. 32:341‐348.
   Zeller, R. 1989. Fixation, embedding, and sectioning of tissues, embryos, and single cells. Curr. Protoc. Mol. Biol. 7:14.1.1‐14.1.8.
Key References
   UNIT 13.5 (Current Protocols in Toxicology)
  Details methods for examining apparently nongravid uteri.
   UNIT 16.2 (Current Protocols in Toxicology)
  Details methods for mating rodents and managing offspring prior to weaning.
   UNIT 18.6 (Current Protocols in Toxicology)
  Details methods for evaluating the natural killer cell cytotoxicity and cytotoxic T lymphocyte activity.
   UNIT 18.8 (Current Protocols in Toxicology)
  Details methods for determining immune cell phenotypes using flow cytometry.
   UNIT 18.11 (Current Protocols in Toxicology)
  Details methods for measuring the T cell‐dependent antibody response.
   Ayadi et al., 2011. See above.
  This Current Protocols in Mouse Biology unit details methods for establishing a mouse breeding colony and management of the colony after successful breeding.
   Heyser, 2003. See above.
  This Current Protocols in Neuroscience unit provides a detailed overview of what developmental endpoints to assess in rodents and how to assess them.
   Luo and Dorf, 1993. See above.
  This Current Protocols in Immunology unit details methods for eliciting and measuring the delayed‐type hypersensitivity response.
   Zeller, 1989. See above.
  This Current Protocols in Molecular Biology unit details methods for collecting tissues and preparing them for histological analysis.
Internet Resources
   https://www.aalaslearninglibrary.org.
  The Learning Library of the American Association for Laboratory Animal Science (AALAS) provides materials for training researchers in appropriate methods for handling, caring for, and using animals in research.
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