Year of Publication

2012

Degree Name

Doctor of Philosophy (PhD)

Document Type

Doctoral Dissertation

College

Arts and Sciences

Department

Chemistry

First Advisor

Dr. D. Allan Butterfield

Abstract

Advancing applications of engineered nanomaterials (ENM) in various fields create the opportunity for intended (e.g. drug and gene delivery) or unintended (e.g. occupational and environmental) exposure to ENM. However, the knowledge of ENM-toxicity is lagging behind their application development. Understanding the ENM hazard can help us to avoid potential human health problems associated with ENM applications as well as to increase their public acceptance. Ceria (cerium [Ce] oxide) ENM have many current and potential commercial applications. Beyond the traditional use of ceria as an abrasive, the scope of ceria ENM applications now extends into fuel cell manufacturing, diesel fuel additives and for therapeutic intervention as a putative antioxidant. However, the biological effects of ceria ENM exposure have yet to be fully defined. Both pro-and anti-oxidative effects of ceria ENM exposure are repeatedly reported in literature. EPA, NIEHS and OECD organizations have nominated ceria for its toxicological evaluation. All these together gave us the impetus to examine the oxidative stress effects of ceria ENM after systemic administration.

Induction of oxidative stress is one of the primary mechanisms of ENM toxicity. Oxidative stress plays an important role in maintaining the redox homeostasis in the biological system. Increased oxidative stress, due to depletion of antioxidant enzymes or molecules and / or due to increased production of reactive oxygen (ROS) or nitrogen (RNS) species may lead to protein oxidation, lipid peroxidation and/or DNA damage. Increased protein oxidation or lipid peroxidation together with antioxidant protein levels and activity can serve as markers of oxidative stress.

To investigate the oxidative stress effects and the mechanisms of ceria-ENM toxicity, fully characterized ceria ENM of different sizes (~ 5nm, 15nm, 30nm, 55nm and nanorods) were systematically injected into rats intravenously in separate experiments. Three brain regions (hippocampus, cortex and cerebellum) were harvested from control and ceria treated rats after various exposure periods for oxidative stress assessment. The levels of oxidative stress markers viz. protein carbonyl (PC), 3-nitrotyrosine (3NT), and protein bound 4-hydroxy-2-trans-nonenal (HNE) were evaluated for each treatment in each control and treated rat organ. Further, the levels and activities of antioxidant proteins, such as catalase, glutathione peroxidase (GPx), glutathione reductase (GR), super oxide dismutase (SOD), were measured together with levels of heat shock proteins heme oxygenase -1 and 70 (HO-1 and Hsp-70). In addition, the levels of pro-inflammatory cytokines IL-1β, TNF-α, pro-caspase-3, and autophagy marker LC-3A/B were measured by Western blot technique. In agreement with the literature-proposed model of oxidative stress hierarchy mechanism of ENM-toxicity, the statistical analysis of all the results revealed that the ceria ENM-induced oxidative stress mediated biological response strongly depends on the exposure period and to some extent on the size of ceria ENM. More specifically, a single intravenous injection of ceria ENM induced tier-1 (phase-II antioxidant) response after shorter exposure periods (1 h and 20 h) in rat brain. Upon failure of tier-1 response after longer exposure periods (1 d to 30 d), escalated oxidative stress consequently induced tier-2 and tier-3 oxidative stress responses. Based on our observations made at chronic exposure period (90 d) after the single i.v. injection of ceria ENM, we could extend the model of oxidative stress hierarchy mechanisms for ceria-ENM-induced toxicity. Considering the evaluation of all the oxidative stress indices measured in 3-brain regions, oxidative stress effects were more prominent in hippocampus and the least in cerebellum, but no specific pattern or any significant difference was deduced.

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