RESPONSES OF LUNG CELLS TO METALS IN MANUFACTURED NANOPARTICLES
Impact/Purpose:
This proposal is based on the hypothesis that transition metals in particles induce pro-inflammatory signaling and cell damage through the production of reactive oxygen species. Established cell culture models and toxicology assays will be applied to the analysis of manufactured nanomaterials. Based on the literature and our own data, we expect that the small physical size and high surface area of nanoparticles ( d < 30 nm) will increase cellular uptake and increase induction of pro-inflammatory signaling compared to larger particles with the same elemental composition. In vitro studies with human and rat lung cells will evaluate the effects of manufactured nanoparticles in the as-sold condition, and the same materials after the particles have been subjected to surface modification simulating fire and wastewater treatment conditions. The emphasis will be on lower-cost nanomaterials that are sold in powder or liquid suspension form because these materials are expected to be produced and ultimately released in the largest amount.
Description:
In vitro assays with lung epithelial cells were used to compare pairs of micron-sized and nano-sized particles with the same nominal chemical composition for cytotoxicity and induction of the proinflammatory cytokine IL-6. Results suggested that nano-sized particles of metal oxides are not always more potent than micron sized particles. To provide toxicological perspective for risk assessment we compared the manufactured nanoparticles to micron-sized particles of soil-derived dust. While the pure metal oxides have proinflammatory effects on lung cells the potency (on a mass dose basis) is less than road dust. These results were published in Particle and Fibre Toxicology.
We had initial experimental indications that nanoparticles can cause artifacts in some established toxicology assays, presumably because the high surface area results in adsorption of proteins. We followed up on these indications, quantified some of the more important artifacts, and included these results in a publication.
Being able to detect and quantify nanoparticles in tissue is important for understanding the relative effects of solid metal oxide nanoparticles compared to soluble metal ions. We devoted efforts toward developing methods for quantifying solid nanoparticles in cultured cells and in lung tissue as a way to better understand the effect dose in particle toxicology experiments. We collaborated with researchers who have experience with particle detection by field flow-fractionation (FFF), and were able to demonstrate quantitative measurement of manufactured SiO2 particles in both lung cells and in rat lung tissue. These method development efforts involved adding known amounts of 70 nm particles to the biological sample followed by digestion and cleanup to remove the organic material that would interfere with the analysis. We were able to demonstrate detection of the 70 nm particles in rat lung tissue that contained a mixture of 70 and 250 nm particles. The results of this method demonstration were published in Particle and Fibre Toxicology, and a related publication appeared in Nano Letters.
The particle samples and particle characterization data from this study have been shared with another STAR grant (R833336, Dr. P. Moos, PI), which focuses on nanoparticles in the colon. This collaboration will result in publications that acknowledge EPA funding for both projects.
Record Details:
Record Type:PROJECT(
ABSTRACT
)
Start Date:10/01/2004
Completion Date:09/30/2007
Record ID:
89541
Keywords:
AIR, BIOAVAILABILITY, DOSE-RESPONSE, ULTRAFINE PARTICULATE MATTER, IN VITRO, MAMMALIAN, CELLULAR, METALS, TOXICOLOGY,
Related Organizations:
Role
:OWNER
Organization Name
:UNIVERSITY OF UTAH
Mailing Address
:200 S University St
Citation
:Salt Lake City
State
:UT
Zip Code
:84112
Project Information:
Approach
:A phased approach will be used to maximize useful results within the budget. In the first phase, low cost assays will be used to screen a wide range of samples with sufficient replicates for statistical power. This phase will emphasize measurement of cytotoxicity, induction of the proinflammatory cytokine IL-6, and dissolution rate in simulated lung fluid. Industrial collaborators will assist in prioritizing materials for testing and providing chemically similar materials in various sizes and grades. Materials selected in the screening phase will be used for more detailed, mechanistic studies. The second phase will test selected materials for particle uptake by the cells, for the induction of additional cytokines, and for the effect of antioxidants. Phase two physical characterization will include electron microscopy, BET surface area, zeta potential, and trace element analysis. In the third phase, the most inflammatory and most benign nanomaterials will be used in hypothesis-based toxicology experiments to evaluate plausible mechanisms by which the particles induce specific responses in cells. Cell culture toxicology studies with BEAS-2B cells, an immortalized human lung epithelial cell line, are emphasized and are consistent with the goal of refining, reducing, and replacing animal use. However, it is necessary to establish the relevance of cell culture data to whole animals and to human health. Experiments using normal macrophages and normal epithelial cells that are freshly harvested from rats will be conducted to test the ability of the cell culture assays to predict the induction of inflammation by specific nanomaterials.
Cost
:$332,958.00
Research Component
:Health Effects
Approach
:A phased approach will be used to maximize useful results within the budget. In the first phase, low cost assays will be used to screen a wide range of samples with sufficient replicates for statistical power. This phase will emphasize measurement of cytotoxicity, induction of the proinflammatory cytokine IL-6, and dissolution rate in simulated lung fluid. Industrial collaborators will assist in prioritizing materials for testing and providing chemically similar materials in various sizes and grades. Materials selected in the screening phase will be used for more detailed, mechanistic studies. The second phase will test selected materials for particle uptake by the cells, for the induction of additional cytokines, and for the effect of antioxidants. Phase two physical characterization will include electron microscopy, BET surface area, zeta potential, and trace element analysis. In the third phase, the most inflammatory and most benign nanomaterials will be used in hypothesis-based toxicology experiments to evaluate plausible mechanisms by which the particles induce specific responses in cells. Cell culture toxicology studies with BEAS-2B cells, an immortalized human lung epithelial cell line, are emphasized and are consistent with the goal of refining, reducing, and replacing animal use. However, it is necessary to establish the relevance of cell culture data to whole animals and to human health. Experiments using normal macrophages and normal epithelial cells that are freshly harvested from rats will be conducted to test the ability of the cell culture assays to predict the induction of inflammation by specific nanomaterials.
Cost
:$332,958.00
Research Component
:Nanotechnology
Project IDs:
ID Code
:R831723
Project type
:EPA Grant