Dissertation/Thesis Abstract

Mechanistic studies of water soluble fullerenes as free radical scavengers, biological antioxidants and NF-kappaB inhibitors
by Rawashdeh, Rabeah Yousef, Ph.D., The University of North Carolina at Greensboro, 2014, 168; 3624223
Abstract (Summary)

Since their discovery, fullerenes and fullerene derivatives have been the subject of intense research because of their unique properties and potential applications. The combination of a network of conjugated double bonds with high electron affinity makes them good electron acceptors, so that, despite being described commonly as antioxidants in the biological literature, they may act mechanistically as oxidants. Because of their ability to accept electrons, fullerenes can quench reactive oxygen species (ROS), particularly free radicals, by accepting their unpaired electron. Overproduction of ROS interferes with cell signaling and apoptosis, as well as causing generalized damage to biomolecules, through their interaction with lipids, DNA and proteins, thereby contributing to chronic diseases. The pro-inflammatory transcription factor NF-κB is known to be activated by ROS; modulation of NF-κB signaling is well established as a way to control inflammatory immune responses.

Although there are numerous studies on the biological roles of fullerenes, their mechanism of action is poorly understood. The main focus of this research is to clarify redox properties and bioactivity of fullerene derivatives, including their fundamental redox properties, their interaction with NF-κB and their biocompatibility involving the possibility of ROS generation vs. elimination by free radical scavenging. Different C60 and C70 fullerene derivatives used in these studies include hydroxylated fullerenes (C 60OHx and C70OHx), carboxyfullerene (C3), amphiphilic liposomal malonylfullerene (ALM), fullerene-tetraglycolate TGA and TTA, which were studied in comparison to common antioxidants such as ascorbic acid and N-acetyl cysteine.

The results from the ferric reducing antioxidant power (FRAP) assay demonstrate that fullerenes are very poor reducing agents, whereas the results of electro-paramagnetic resonance spectroscopy (EPR) confirmed their radical scavenging properties vs. superoxide ion. Taken together, the results of both studies (FRAP and EPR) emphasize the need to distinguish between antioxidant and antiradical properties of fullerenes, as the latter can occur via oxidation of free radical species.

Regarding their potential future use in clinical applications, an important finding is that hydroxyl and carboxy fullerene derivatives exposed to ambient conditions in living cells neither produce ROS nor cause any cytotoxicity at nanomolar to micromolar concentrations, as shown using proliferation assays and redox sensitive fluorescent dyes. On the other hand, TTA and C60 OHx fullerenes were found to have a distinct effect on mitochondrial function of cells, implying their interference with proton transfer process of mitochondrial membrane. However, this effect is apparently not sufficient to decrease cell viability.

The activity of fullerenes as potential anti-inflammatory drugs was investigated using several cell-based assays involving an NF-κB reporter gene assay, coupled with visualization of nuclear translocation of exogenous (transfected) NF-κB, achieved via a construct with the NF-κB p65 gene fused to green fluorescent protein. The fullerene derivatives C3 and C60OHx were found to be effective inhibitors of NF-κB driven gene expression at micromolar concentrations. However, unlike some antioxidants such as α-lipoic acid, they did not inhibit PMA-induced nuclear translocation of exogenous NF-κB even at high concentration (40 μM). Along with western blot analysis, this result strongly suggests that fullerene derivatives block NF-κB signaling downstream of the cytosolic activation pathway (the site of action of many antioxidants), possibly inhibiting NF-κB activation at a step penultimate to DNA binding in the nucleus (e.g., by interfering with the mandatory reduction of NF-κB by thioredoxin).

These studies suggest that the molecular basis of action of water soluble fullerenes has both similarities and differences to typical antioxidants. Like many antioxidants, they are active as NF-κB inhibitors, suggesting a potential role as novel anti-inflammatory agents, as they have low overall toxicity. However, they have a distinct mechanism of action, such as scavenging free radicals by oxidation, and lack activity as direct reducing agents.

Indexing (document details)
Advisor: Taylor, Ethan Will
Commitee: Haddy, Alice E., Kepley, Christopher L., LaJeunesse, Dennis R., Sandros, Marinella G.
School: The University of North Carolina at Greensboro
Department: Chemistry and Biochemistry
School Location: United States -- North Carolina
Source: DAI-B 75/10(E), Dissertation Abstracts International
Source Type: DISSERTATION
Subjects: Molecular biology, Toxicology, Surgery, Nanoscience
Keywords: Anti-inflammatory agents, Biological antioxidants, Free radical scavengers, Fullerenes
Publication Number: 3624223
ISBN: 978-1-303-97369-7
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