Grantee Research Project Results
2009 Progress Report: Innate Immune Response of an Aquatic Vertebrate Model to Manufactured Nanoparticles Assessed Using Genomic Markers
EPA Grant Number: R833319Title: Innate Immune Response of an Aquatic Vertebrate Model to Manufactured Nanoparticles Assessed Using Genomic Markers
Investigators: Klaper, Rebecca , Goetz, Frederick , Chen, Jian
Institution: University of Wisconsin - Madison
EPA Project Officer: Hahn, Intaek
Project Period: April 1, 2008 through April 15, 2011
Project Period Covered by this Report: April 1, 2009 through March 31,2010
Project Amount: $398,810
RFA: Exploratory Research: Nanotechnology Research Grants Investigating Environmental and Human Health Effects of Manufactured Nanomaterials: a Joint Research Solicitation-EPA, NSF, NIOSH, NIEHS (2006) RFA Text | Recipients Lists
Research Category: Nanotechnology , Safer Chemicals
Objective:
The overall objective of this project is to assess the innate immune reaction of an aquatic model, the rainbow trout, to manufactured nanomaterials of varying chemistries at levels not inducing cellular toxicity. This research will create a mechanism with which to test other nanomaterials, provide data to support ecological risk assessments, and ultimately inform decisions as to which materials will be the safest to industrialize and use with respect to aquatic environments.
Progress Summary:
Our investigations have focused on testing a wide concentration range (0.1-100 µg/ml) for each nanomaterial to determine toxicity to macrophage cells using two tests for cytotoxicity: 1) the metabolic reduction of resazurin and 2) lactate dehydrogenase activity (LDH). We have found that cell viability is not impacted by nanomaterial exposures up to 10 µg/ml concentrations. In addition, surfactants alone as well as chemicals used for functionalization also are benign at these concentrations when it comes to cell viability.
Based on cytotoxicity data, we have chosen concentrations of nanomaterials below cytotoxic levels to test for effects on targeted gene expression. We have chosen TNFα (tumor necrosis factor) and COX2 (cyclooxygenase) as representative of proinflammatory genes; IFNα (interferon) and IP-10 (interferon inducible protein) as genes associated with the antiviral response; and IL l0 (interleukin) and TGFβ-1 (transforming growth factor) as representative of the anti-inflammatory (alternative/Th2) pathway. Each gene has been assayed for expression in several tissue concentrations using RT-PCR (QPCR) to investigate any dose-dependent effects on expression. Our results indicate that fullerenes as well as single wall nanotubes and multiwall nanotubes are stimulatory to primary immune cells. All cause an increase in candidate proinflammatory genes that is equivalent to stimulation of positive controls at our highest concentrations however nanomaterials begin to differ in their stimulation of the immune system at lower doses (below 1 µg/ml). We are now elucidating this response further with the final stage of our project using directed microarray experiments. Our data indicate that chemicals used for functionalization may also stimulate the immune response and that this response is equivalent to the nanoparticle alone.
Our goals for this project have been completely aligned with what we have accomplished so far. The main findings from our preliminary data include:
- Trout macrophages are proving to be a sensitive tool to investigate the effects of nanoparticles (NP) on gene expression.
- Side-chains used on NPs may have significant effects on their own. For example, cyclodextrin is a stimulator of inflammatory gene expression in trout macrophages.
- Surfactants used to solubilize NPs may have significant effects on gene expression. Deoxycholate and tetrahydrofuran are both stimulatory of inflammatory gene expression in trout macrophages.
- C60 fullerenes stimulate inflammatory gene expression in trout macrophages and functionalization of these nanomaterials may decrease this effect.
- There is a dose-response effect of nanomaterials on the immune response of trout macrophages with differences seen in the inflammatory responses at levels at or below 1 µg/ml.
Because we have found a significant impact of surfactants on stimulation of our cell culture system, we have moved completely away from using these suspensions and instead we are "stirring" our particles into suspension without the surfactant present with the understanding that this creates a slight alteration of the external aggregation. Of course, the functionalized nanoparticles were created to negate the need for the surfactant in the first place, so these treatments have not been a problem.
Journal Articles on this Report : 1 Displayed | Download in RIS Format
| Other project views: | All 18 publications | 1 publications in selected types | All 1 journal articles |
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Klaper R, Arndt D, Setyowati K, Chen J, Goetz F. Functionalization impacts the effects of carbon nanotubes on the immune system of rainbow trout, Oncorhynchus mykiss. Aquatic Toxicology 2010;100(2):211-217. |
R833319 (2009) R833319 (Final) |
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Supplemental Keywords:
risk assessment, dose response, exposure, immunology, ecological effects, nanotoxicology, immunotoxicology, genomics, biomarkers, Health, Scientific Discipline, Health Risk Assessment, Risk Assessments, Environmental Chemistry, bioavailability, nanomaterials, manufactured nanomaterials, environmental risks, aquatic ecosystem, biogeochemistry, biological pathways, bioaccumulation, nanotechnology, nanochemistry, nanoparticle toxicity, cellular response to nanoparticles, toxicologic assessmentRelevant Websites:
Progress and Final Reports:
Original AbstractThe perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.