Developmental Immunotoxicity (DIT): Assays for Evaluating Effects of Exogenous Agents on Development of the Immune System
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- Abstract
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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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- 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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Basic Protocol 1: Exposure Scenario for Including All Critical Windows of Immune Development
Materials
Alternate Protocol 1: Exposure Scenario for Evaluating Limited Windows of Exposure
Materials
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Figure 18.15.1 Schematic of protocol for developmental immunotoxicity as outlined by Dietert and Holsapple (), with inclusion of rat/mouse and human time frames for immunological development (Dietert et al., ). In this protocol, DIT is assessed at 6 weeks of age in the offspring. Exposure can occur during gestation only, during lactation only, or during gestation and lactation (stopping at weaning of offspring), or can continue past weaning at postnatal day (PND) 21 and through adulthood to encompass earlier measurements in the offspring. GD = gestation day, GW = gestation week. It is advised, except in specific cases where exposure to the agent of concern occurs only during certain developmental time points, that exposure occur from gestation (beginning at conception when using breeding pairs by starting exposure to the pairs when they are placed together or no later than GD6 when using time‐pregnant animals) through PND42. View Image -
Figure 18.15.2 Potential experimental breakdown of pregnant dams and offspring for DIT assessment. View Image -
Figure 18.15.3 Example form for recording birth from breeding pairs or time pregnant mice (in the case of time pregnant mice change breeding pair ID to the animal ID number for the time pregnant dam). The comments section can include information on survivability of the offspring. Not all offspring may survive to weaning age, due to various reasons related to the compound, or at early stages, to the dam's actions. Dead offspring and the date can be recorded here or may be recorded on a different form. View Image -
Figure 18.15.4 (A ) Example form for monitoring individual dam weights and dosing over the course of the study. (B ) Example litter/individual offspring weight form. For ID marking, the correct information (i.e., ear or tail or litter box number) can be circled at the top of the column to indicate the ID information below in the column. Thus, these form examples can be used for litter weights, and then for individual weights once offspring are weaned, by simply changing which ID marker type is circled once the offspring are weaned. Additionally, during this time the dam ID can be circled as such while collecting litter weights, and the individual offspring ID can then be added and circled as such when individual offspring weights are collected. View Image
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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