2007 Progress Report: Effects of Ingested Nanoparticles on Gene Regulation in the ColonEPA Grant Number: R833336
Title: Effects of Ingested Nanoparticles on Gene Regulation in the Colon
Investigators: Moos, Philip J. , Cutler, Ned S. , Veranth, John
Institution: University of Utah
EPA Project Officer: Hahn, Intaek
Project Period: April 1, 2007 through April 30, 2009
Project Period Covered by this Report: April 1, 2007 through April 30, 2008
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 , Health Effects , Health , Safer Chemicals
This study will focus on ingestion exposure to lower-cost, high-production-volume manufactured nanoparticles (5 - 100 nm diameter) of carbon black, SiO2, Al2O3, TiO2, ZnO and Fe2O3. These materials are produced in large quantity and are being marketed as specialty chemicals for use in consumer and industrial products. We will focus on the effects of nanoparticles on colon cells because colon cancer is a major cause of death that is related to environmental and lifestyle factors, and chronic inflammation promotes cancer. In vitro evidence that environmental or occupational exposure to nanoparticles leads to increased colon tissue inflammation or altered regulation of cell cycle genes would suggest a need for comprehensive in vivo toxicology studies. A negative result in this study, that is, evidence that nanoparticles are benign to colon cells, would be reassuring to industry and the public. Consumer web sites are expressing concern regarding nanoparticles in cosmetics and topical skin products, but few peer-reviewed studies are available that deal with the toxicology of these materials. Carbon black from hydrocarbon sources is not approved for foods or cosmetics, but carbon black is widely used in pigments, inks, and photocopy toners, and as a filler in rubber products. Specialty chemical suppliers are actively marketing new grades of these materials with smaller primary particle size as they pursue the nanomaterials market.
The central hypothesis to be tested was that ingested manufactured nanoparticles are taken up by inflamed colon cells, translocate to the nucleus, and alter gene transcription thereby further increasing inflammation and leading ultimately to development of pathological conditions, including cancer.
We set out to determine the effect the metal oxide particulate matter (PM) would have on the viability of the RKO and CaCo-2 cells in the presence or absence of TNFα pretreatment. We chose to focus first on the nano-sized PM and if we found a result indicating toxicity, we could then evaluate the effect of the size of the PM by using micron-sized particles of the same material. We evaluated concentrations ranging from 1 to 100 μg/cm2 using a formazan-style viability assay that could rapidly screen for cellular toxicity. These experiments demonstrated that most of the metal oxide nanoparticles (Al2O3, TiO2, SiO2, and Fe2O3) were not toxic, carbon black showed modest toxicity, primarily at the highest concentrations, and ZnO displayed the most toxicity. The TNFα pretreatment did not dramatically alter the sensitivity of the RKO and CaCo-2 cells to any of the PM. Since ZnO was clearly the most toxic in both cell lines, we evaluated the effect of the size of the particles by using micron-sized PM and found that even the micronZnO tended to show dose-dependent toxicity.
In separate experiments, we prepared samples from all nanoPM and ran representative microarray experiments and we are currently following up with selective QPCR as a validation method. For the expression analysis, we used high doses (50 μg/cm2) of most of the PM except for ZnO (5 μg/cm2 to minimize toxicity) and Fe2O3 (10 μg/cm2 due to difficulties in RNA collection at 50 μg/cm2). All of the experiments were two color hybridizations and the largest set was comparing untreated RKO or CaCo-2 cells compared to these cells treated with TNFα, nanoPM, or TNFα plus nanoPM. With this initial dataset, we first set out to determine if TNFα had an effect on these cell lines and determine if there was a consistent or non-specific effect of the nanoPM. Using multiclass Significance Analysis, we compared the groups of cells treated with TNFα, nanoPM, or TNFα plus nanoPM and found that the TNFα treatment expression profile dominated the output suggesting: 1) that TNFα did activate a signaling pathway (though not IL-6), and 2) that the nanoPM does not have a non-specific or consistent effect on these cells. The second evaluation of the data was another multiclass Significance Analysis, but this time each nanoPM treatment, regardless of TNFα treatment was compared to each other nanoPM treatment to determine if specific PM had distinctive effects on transcription. This analysis indicated that TiO2 and ZnO displayed transcriptional effects with ZnO having the most pronounced effect. Since ZnO gave the most robust response, we tried to evaluate micron-sized ZnO as well, and we found similar changes in expression. Therefore, the data suggests that multiple pathways are activated by the ZnO including; stress response pathways, Zn metabolism and transport genes, and genes that suggest alterations in redox pathways. Using Principle Components Analysis, we determined that the ZnO treatment resulted in the largest portion of the variation of the dataset, suggesting that this was the most pronounced effect. This pronounced ZnO effect was masked in the first analysis since we were asking for a common effect among the nanoPM and, as already indicated, most of the PM did not demonstrate a transcriptional response.
We plan to pursue the effects of the ZnO PM on the colon cell lines. First, we will selectively follow up the microarray results with QPCR by evaluating genes that are characteristic of select pathways to validate the exciting results. The other major goals will be to determine the origin of the Zn that appears to be driving the transcriptional response, and determine if the mechanism of action of the ZnO mediated cell death.