Final Report: Using Flathead Minnow Microarrays to Test Toxicity of Nanoparticles

EPA Contract Number: EPD08026
Title: Using Flathead Minnow Microarrays to Test Toxicity of Nanoparticles
Investigators: Carter, Barbara J.
Small Business: EcoArray Inc.
EPA Contact: Manager, SBIR Program
Phase: I
Project Period: March 1, 2008 through August 31, 2008
Project Amount: $69,500
RFA: Small Business Innovation Research (SBIR) - Phase I (2008) RFA Text |  Recipients Lists
Research Category: Small Business Innovation Research (SBIR) , SBIR - Nanotechnology

Description:

The overall goals of this Phase 1 grant were to employ microarrays to identify differentially expressed genes in fathead minnows after acute exposure to nanotubes. This Phase 1 study of nanotubes should help validate the expediency and affordability of the high-density fathead minnow microarrays for compound screening and use in environmental toxicology. The data were analyzed to determine what pathways in the fathead minnow are affected by exposure to nanotubes.  This information should enable us to identify “genetic fingerprints” and to use the database we developed as a tool for identifying contaminants in unknown situations (class prediction), which may lead to useful interpretation of human health issues. 

Summary/Accomplishments (Outputs/Outcomes):

We showed that, for the multi-walled carbon nanotubes (MWCNT) that we studied, microarrays clearly identify biological impact through genotypic change, even when there are no obvious changes in phenotype.  The MWCNT caused no major changes in phenotype, but more than 400 genes were differentially expressed.  We also noted that MWCNT are not easily taken up in water, so we had to use a dispersant, sodium dodecylbenzene sulfonate (NaDDBS) to get enough of the material into water to develop a relevant range of concentrations.  We found the following:

  • Toxicity.  None noted up to the level of 1.0 mg/L (nominal).
  • Histopathologies.  There were no statistically significant abnormalities detected in the liver, ovary, heart or digestive tract for any of the treatments in either size of MWCNT.  The gills of fish exposed to the smaller MWCNT (< 8 nm) showed a statistically significant (p = 0.014) difference in the number of fused lamellae in the 0.1 and 0.3 mg/L doses, and none present in the controls.
  • H2O control vs. NaDDBS control.  Using the acceptance criteria (p < 0.05 and fold change > 2), there are NO differences in gene expression between the water controls and the NaDDBS controls (2.2. mg/L) for gill or ovary after 48 h exposure to > 50 nm MWCNTs.  
  • Differential Gene Expression (p < 0.005 and fold change > 2).  From the microarray analysis, we observed a substantial transcriptional response to exposure in each of the tissues, with more than 400 genes exhibiting altered expression in each of the tissues.  However, there was very little commonality in the transcriptional response of these three tissues to a given MWCNT.  In addition, the response of a single tissue (gill) to both sizes of nanotubes showed as many differences as similarities.  Overall, the effect of the MWCNTs on FHMs varies with the parameters of dose, nanotube size, and tissue.  The majority of the differentially expressed genes from each tissue are involved in the biological process category 0006xxx, regulating transport, transcription and protein functions. 

Conclusions:

The purpose of the study was to determine whether microarrays are a feasible technology for identifying the biological impact of carbon nanotubes.  We showed that, for MWCNT, microarrays clearly identify biological impact through genotypic change, even when there are no obvious changes in phenotype.  This is an important development, and we believe microarrays and associated data analysis can be developed into the testing method for nanomaterials that the EPA seeks.  (The Draft Nanomaterial Research Strategy (NRS) of January 2008, notes that “(t)he complexities faced by analysis for nanomaterials in environmental matrices may prove intractable to conventional instrumented approaches of analysis. The eventual solution may well evolve from the development of new analytical approaches using arrays of standardized assays based on biological/biochemical endpoints.” (NRS, pp.28-29)).
 
The products we can commercialize as a result of this research consist of our existing high-density fathead minnow gene microarrays, which have been made more valuable by this demonstration of efficacy, and the associated Gene Expression Database we are developing.  The “product” is a package of services for:  (1) testing and monitoring of nanomaterials that are discharged into the environment, (2) screening of new compounds before they are produced by industry, and (3) basic toxicology research.  The arrays can be used as a screening tool for nanomaterials before they are released into the environment as well as to assess the presence and distribution of various contaminants in the field (i.e., for class prediction) and help with site remediation.  To develop the database to broadly apply to other nanomaterials will require more exposures, although we have started this work in related research involving nano iron (RNIP) and titanium dioxide.
 
The EPA has written several white papers on the use of genomics for Regulatory and Risk Assessment Applications in the last several years (https://www.epa.gov/osa/genomics.htm).  As a result of these white papers, we expect microarrays to be incorporated into standard assays for water testing by the EPA and other agencies in the future.  EcoArray is developing microarrays in fathead minnows, which is a standard species used by the EPA for water testing, as well as other species (e.g., Sheepshead minnow for salt water, daphnia magna, and bass) that are useful models for environmental toxicology.
 
Our products will be used initially in three areas of toxicology, all of which relate to human health, which is the ultimate target of testing.  These are:  (1) testing and monitoring of compounds that are discharged into the environment, (2) screening of compounds produced by industry, and (3) basic toxicology research.  We estimate full market size for these three market segments at $450 million by 2010.  The market for use of microarrays has grown to $1 billion in less than 10 years, primarily in human health research.  The applications in the markets EcoArray addresses, primarily environmental toxicology and compound screening, are just as compelling and have not yet been developed.  We estimate the market potential for EcoArray’s products to approach $450 million over the next 5-8 years from virtually zero today. 

Journal Articles:

No journal articles submitted with this report: View all 1 publications for this project

Supplemental Keywords:

small business, SBIR, EPA, nanoparticles, manufacturing products, industrial products, commercial products, nanomaterials, nanotechnology, aquatic environment, exposure, toxicity, human health, nanotubes, fathead minnow, microstructure, surface properties, industrial waste, aerosols, nanoscale products, by-products, nanotechnology industries, aquatic biota, ingestion, epithelial boundaries, phagocytosis, endocytosis, cellular immunity, intracellular digestion, toxicological testing, environmental contaminants, reproduction, gene microarray, in vitro assay, in vivo assay, in vivo exposure, mechanistic outcome, acute exposure, genetic fingerprints, compound screening, environmental toxicology, scientific discipline, health, toxicology, risk assessments, health risk assessment, genetics, altered gene expression, nanotechnology, toxicologic assessment, genetic analysis, nanochemistry, DNA array, bioinformatics, microarray processing, hybridization,, Health, Scientific Discipline, Toxicology, Genetics, Health Risk Assessment, Risk Assessments, altered gene expression, nanochemistry, genetic analysis, nanotechnology, microarray studies