Grantee Research Project Results
2005 Progress Report: Iron Oxide Nanoparticle-Induced Oxidative Stress and Inflammation
EPA Grant Number: R831722Title: Iron Oxide Nanoparticle-Induced Oxidative Stress and Inflammation
Investigators: Elder, Alison C.P. , Oberdörster, Günter , Yang, Hong , Finkelstein, Jack
Institution: University of Rochester
EPA Project Officer: Hahn, Intaek
Project Period: September 1, 2004 through February 28, 2008
Project Period Covered by this Report: September 1, 2004 through August 31,2005
Project Amount: $335,000
RFA: Exploratory Research to Anticipate Future Environmental Issues: Impacts of Manufactured Nanomaterials on Human Health and the Environment (2003) RFA Text | Recipients Lists
Research Category: Nanotechnology , Safer Chemicals , Human Health
Objective:
The manufacture of nanoparticles (< 100 nm diameter) of varying shapes and compositions has exploded over the last several years with applications ranging from diagnostic imaging to nanoscale molecular construction, thus making intentional and unintentional human exposures likely. Although much has been learned recently about their synthesis, very little is known about cellular or organ responses upon contact with nanoparticles. A defining feature of nanoparticles is their large specific surface area; thus, it is possible that current concepts of dose expressed as mass concentration, which is very low for nanoparticles, may fail in predicting exposure outcomes if this feature is not taken into account. We hypothesize that the small size of nanoparticles contributes to their evasion of normal particle clearance mechanisms; increases the likelihood of contact with cells of many types, particularly epithelial cells; and allows their translocation to sites distant from the original exposure. We hypothesize further that this contact results in inflammation and oxidant stress and that the large surface area of the nanoparticles potentiates their effects.
The objective of this research project is to determine if nanoparticles: (1) induce oxidative stress and toxicity in cultured epithelial and endothelial cells; (2) cause lung inflammation or extrapulmonary effects after in vivo exposure; and (3) are translocated to extrapulmonary sites. Nanoparticles may gain access to tissues in humans through inhalation or via the blood (i.e., oral, dermal, or inhalation exposure). Thus, for in vitro studies, we will use human alveolar epithelial and umbilical vein endothelial cells and monitor the uptake of nanoparticles as well as inflammation- and oxidative stress-related responses using a range of doses. For in vivo studies, we will use F-344 rats for intratracheal instillation and intravenous injection exposures. Endpoints related to lung inflammation, inflammatory cell activation, and oxidative stress as well as those indicating vascular endothelial injury and acute phase responses will be assessed. Comparisons will be made with ultrafine (20 nm) titanium oxide (TiO2) particles. Tissues from exposed rats (lung, liver, and olfactory bulb) will also be examined for the presence of the particles following intratracheal and intranasal instillations and intravenous injection exposures. We anticipate that this research project will provide dose-, size-, and composition-related toxicological information about nanoparticles. The insight gained regarding mechanisms of response to nanoparticles and their disposition after exposure can be used to predict outcomes of human exposures in environmental, occupational, and therapeutic settings.
Progress Summary:
We have been using Fe2O3 nanoparticles as a model system for better understanding the cellular and tissue responses following exposure to nano-sized materials. In a series of several in vitro tests, however, we found that dipalmitoylphosphatidylcholine (DPPC)-coated 20 nm Fe2O3 nanoparticles were not stable over long periods of time in culture medium. Agglomeration occurred within 1 to 2 hours of incubation in culture medium, presumably because of salt destabilization of the fatty acid coating. Incidentally, the agglomerated Fe2O3 nanoparticles did not induce adverse effects in cultured lung epithelial cells (A549luc) up to 800 μg/well (38 μg/cm2). No changes in lactate dehydrogenase (LDH) release or luciferase reporter activity were detected. Ultrafine TiO2 (20 nm) was used as a comparison and similar results were obtained for the same mass dose range.
Until a coating can be devised that confers greater stability to the Fe2O3 nanoparticles in cell culture systems, we have decided to move to other model systems. One such system is the liquid phase-synthesis of several nanoparticle shapes made of platinum (Teng, et al., 2005). This system allows us to test the hypothesis that shape and surface area (SA) can predict response. Another similar system that can be used for our studies is based on gold. A central hypothesis of the current research is that nanoparticles cause oxidative stress and this can be tested on several levels, including the acellular, cellular, and tissue. The platinum shapes demonstrated a range of reactive oxygen species (ROS)-generating capacity, with the highest activity being found for the flowers and the spheres made from flowers. In addition, although the spheres made from the multipods are actually smaller by almost half as compared to the flower spheres, their ROS activity is lower. We hypothesize that nanoparticle SA plays a role in the observed differences in ROS generating capacity (i.e. platinum flowers and spheres made from flowers have greater SA as compared to multipods and spheres made from multipods) and currently are obtaining SA measurements for the platinum shapes. The platinum shapes have, by mass, lower ROS-generating capacity than the gold nanoparticles. Gold nanorods (50 x 15 nm) have the lowest activity; gas phase-generated (10, 18 nm) and colloidal (5 nm) gold nanoparticles have similar activity by mass. Interestingly, coating the colloidal gold nanoparticles with bovine serum albumin (BSA) dramatically reduced ROS activity (by approximately 8 times).
We hypothesize that one important target for nanoparticle effects is the epithelium lining the gas exchange region of the lung (alveoli). Stably transfected luciferase-interleukin (IL)-8 A549 type II alveolar epithelial cells were exposed to aggregates of DPPC-coated Fe2O3 nanoparticles to measure oxidative stress; no significant increases in response were observed (as was also observed for ultrafine TiO2). We have also tested some of the platinum shapes using this cell culture system; dose-related responses to platinum flowers and multipods were assessed. There was a small dose-dependent (0-500 μg/well; 0-24 μg/cm2) increase in reporter activity with the flowers, but not with the multipods, that was not related to increased LDH release.
In addition to contact with the epithelium, we further suggest that nanoparticles can move from the epithelial surface in the alveoli to gain access to the blood, thus interacting with endothelial cells. We have, therefore, initiated studies using cultured primary human umbilical vein endothelial cells (HUVEC) to investigate the effects of nanoparticle exposure on endothelial target cells. We found that LDH and baseline IL-6 responses were stable through passage 10. We also observed a dose-related increase in IL-6 release in response to colloidal gold (12-188 μg/well; 0.6-8.9 μg/cm2) from passage 4 through 10 without accompanying changes in LDH; this increase was manifest by more than a doubling of IL-6 release from low to high-dose nano-gold (20 nm). Interestingly, these changes occurred in the presence of BSA coating and despite the fact that the coated gold nanoparticles appeared to agglomerate within the first hour of culture at 37°C. The responses to the gold colloids were slightly higher than those to ultrafine TiO2, which was used at a higher dose range (and caused increases in LDH release).
A key issue that we are addressing in our research project is whether and under what conditions nanoparticles are taken up by various cell types. We have preliminary data from in vivo exposures using rats in which colloidal gold nanoparticles were intratracheally instilled (50 μg). Lung tissue was removed from one rat 24 hours following instillation and the alveolar regions were examined using electron microscopy (EM). The preliminary results suggest that RSA-coated gold nanoparticles are taken up by alveolar macrophages and found within epithelial cells. A nanoparticle of a size that appeared to indicate a singlet was found in a cytoplasmic bleb of a type II alveolar epithelial cell; no nanoparticles could be found within epithelial cells from the rat treated with uncoated colloidal gold. Some cells appeared to be heavily loaded with gold and the nanoparticles often were found inside lysosomes. In addition, the nanoparticles were found both as singlets and agglomerates, or clusters, within the cells. It is not yet known, however, how the particles were taken up. Samples from an earlier time point (30 mins) are currently being examined. In addition to these in vivo uptake data, we are also currently working with human umbilical vein endothelial cells to study uptake after in vitro exposures, in this case using exposed cells that have been embedded in gelatin and then sectioned for EM analyses. Singlets and agglomerates of colloidal gold can also be found within these cells.
Future Activities:
We will focus our efforts over the next several months on in vitro analyses of nanoparticle effects and intend to move to in vivo evaluations by the middle of the calendar year. The specific activities that are planned for the immediate future include continuing the work to define the conditions under which agglomeration occurs and studies on cellular uptake of various nanoparticles using chemical detection and microscopic methods to determine if uptake is required to produce oxidative stress.
References:
Teng XW, Liang XY, Maksimuk S, Yang H. Synthesis of porous platinum nanoparticles. Small 2006;2(2):249-253.
Journal Articles:
No journal articles submitted with this report: View all 9 publications for this projectSupplemental Keywords:
nanoparticles, oxidative stress, inflammation, endothelial cells, epithelial cells,, Health, Scientific Discipline, PHYSICAL ASPECTS, ENVIRONMENTAL MANAGEMENT, Health Risk Assessment, Risk Assessments, Physical Processes, Biology, Risk Assessment, lung injury, lung epithelial cells, toxicology, exposure, nanotechnology, human exposure, lung inflamation, iron oxide nanoparticles, cellular response to nanoparticles, exposure assessment, human health riskProgress 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.