Grantee Research Project Results
2008 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: September 1, 2007 through August 31,2008
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
Approach:
A range of water-soluble C60 and carbon nanotubes with different chemical compositions and surface chemistries will be synthesized and tested for their effects on trout macrophages. A trout primary macrophage cell culture system will be used to determine the 1) dose versus cell viability for each synthesized nanomaterial type, 2) level of expression (by quantitative PCR) of marker genes associated with inflammatory, antiviral, and anti-inflammatory responses with respect to nanomaterial dose at levels that have no deleterious effect on cell viability, and 3) global patterns of gene expression for those materials that cause significant changes in marker genes using custom trout immune microarrays.
Progress Summary:
First year investigations have focused on testing a wide concentration range (0.1-100 ug/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 ug/ml concentrations. In addition, surfactants alone as well as chemicals used for functionlization are also 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 IL10 (interleukin) and TGFβ-1 (transforming growth factor) as representative of the antiinflammatory (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. Our data indicate that chemicals used for functionalization may also stimulate the immune response and that this response is equivalent to the nanoparticle alone.
Future Activities:
While the results of QPCR will provide important information concerning the expression of several macrophage genes, we realize that candidate gene expression analysis may be limited in determining the overall pathway(s) that are being stimulated in trout macrophages by different nanomaterials. Therefore, we will use a more global gene expression analysis to assess gene expression profiles with selected nanomaterials using a directed gene chip. For a subset of nanoparticles as described above we will perform microarray analysis. Nanomaterials and concentrations with the greatest capacity to stimulate or suppress the marker genes from quantitative PCR will be chosen for microarray analysis.
Journal Articles:
No journal articles submitted with this report: View all 18 publications for this projectSupplemental 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, biological pathways, biogeochemistry, bioaccumulation, nanochemistry, nanotechnology, nanoparticle toxicity, cellular response to nanoparticles, toxicologic assessmentRelevant Websites:
Laboratory of Dr. Rebecca Klaper Exit
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.