Science Honors Research Proposal: Abstract Form Project title: The Effects of Buckminster Fullerenes on Mitochondrial Function Abstract (not to exceed 150 words): Since the discovery of Buckminster Fullerenes (C 60 ), hours have been devoted to the study of this carbon nanoparticle. C 60 has been investigated in such applications as photodynamic therapy of cancer, photovoltaic cells, semiconductors, and enzyme inhibition. Despite two decades of research, the biological effects of C 60 remain a mystery. The intention of this study is to examine the effects of Buckminster Fullerenes on mitochondrial function to determine the concentration of C 60 as well as the time frame of exposure that cause maximum dysfunction. Bovine heart mitochondria will be exposed to different doses of C 60 and tested for the ability to respire normally. To demonstrate that C 60 dosages result in varying effects on mitochondrial function, two mitochondrial enzymes will be measured. Hydrogen peroxide and lipid peroxidation will be measured as indicators of oxidative stress and membrane oxidation. The results will contribute to the growing field of nanotoxicology. 2
Science Honors Research Proposal: Project Description A. Title of Project. The Effects of Buckminster Fullerenes on Mitochondrial Function B. Goals and Objectives. Current research on the toxicity of C 60 and other carbon nanoparticles is at best inconsistent; this lack of consistency is due in part to researchers attempts to address the issue of C 60 solubility. However, recent studies indicate that photoexcited C 60 generates reactive oxygen species (ROS) (Yamakoshi, 1998; Lee, 2007) while other studies indicate DNA cleavage due to ROS generation. Mitochondria are major contributors to the oxidative stress that produces ROS in cells, and they are responsible for signaling cells to die. The size and hydrophobic behavior of C 60 lends itself to interactions with membranes as illustrated by Li in his 2007 article (Li, L. 2007). We propose to investigate the dosage of C 60 spheres necessary to affect mitochondrial function and to demonstrate that C 60 dosages result in varying effects on mitochondria. The hypothesis of this study is that mitochondrial membrane dysfunction and electron transport inhibition are caused by C 60 possibly by physical disruption of mitochondrial membranes leading to reactive oxygen species generation. The specific aim of this study is to investigate the dosages of C 60 that correlate to mitochondrial dysfunction, ROS generation and membrane disruption by lipid peroxidation. This study intends to eliminate some inconsistencies associated with solubility by using proteins in physiological buffers to minimize C 60 agglomeration and other changes in surface chemistry. In order to assess the dosage of C 60 spheres necessary to affect mitochondrial function and to demonstrate that C 60 dosages result in dysfunction, mitochondria will be exposed to varying concentrations of C 60 and tested for their ability to move electrons through the following enzymes: complex II (succinate dehydrogenase) and complexes II, III, and IV (succinate oxidase)of the electron transport chain. Activity of these enzyme systems are indicators of overall function. The C 60 exposed mitochondria will also be tested for hydrogen peroxide production and lipid peroxidation, which are indicators of oxidative stress and oxidative damage. C. Background and Significance. Man-made nanoparticles are gaining popularity among researchers in many fields. Some nanoparticles are carbon tubes and spheres while others are quantum dots made of gold, silver or 3
cadmium. These simple particles are the platform on which researchers have built numerous novel inventions. C 60 in particular has been used in semiconductors, photovoltaic cells, inhibitors of HIV-1 protease (Friedman, S. 1993) and a human serum albumin complex (Belgorodsky, B. 2005). The photochemical properties of C 60 have yielded the most interesting biological application (Abrogas, 1991; Tokyama, 1993; Echegoyen, 1998; Williams, 1995); it can be used in photodynamic therapy of cancer as photoexcited C 60 generates reactive oxygen species which can cleave DNA and can cause cell death (Nakanishi, I, 2002). Studying the toxicity of C 60 is important as the increase in production and application of C 60 raises the potential of environmental and occupational exposure (Porter, A., 2006). An important aspect of studying its toxicity is assessing the dosage effects of short term exposures. This is especially important as the effects of C 60 remain to some extent enigmatic. Some research indicates that C 60, when combined with Vitamin E, will act as an antioxidant (Wang, I., 1999) while other studies dispute C 60 antioxidant effects by asserting that C 60 generates reactive oxygen species (Xia, 2006) and causes lipid peroxidation (Sayes, C., 2004). It has been reported that nanocrystals of C 60 caused a decrease in aerobic respiration rates (Fortner, J. 2005). It has been suggested by many (Buford, M. 2007) that the toxicity of C 60 could be dependent on degree of agglomeration, size of conglomerates, surface chemistry, surface charge, and production method. Furthermore, these traits can be affected by solvents ( Ruoff, 1993). D. Research Design and Methods In order to study the toxicity of C 60, electron transport inhibition, reactive oxygen species (ROS) production, and lipid peroxidation will be measured using submitochondrial particles and bovine heart mitochondria. The two tests that will be used in this study to analyze electron transport chain function are the Succinate Oxidase assay and the Succinate Dehydrogenase assay. Together these assays will provide insight into the chemically induced effects of C 60 (i.e. disruption of membrane integrity) (Knodeloch, 1990). The two other chemical tests that will be performed, in order to access the production of ROS and the resulting ROS damage, are the measurement of hydrogen peroxide production and the measurement of lipid peroxidation using the Thoibarbituric Acid assay (TBA assay). The general reaction components required for all chemical tests are a 25 mm potassium phosphate buffer, 1 mg/ml mitochondrial protein, 1 mm succinate and 2-50 mg/l C 60. All mitochondria will be incubated in a solution containing C 60 at 4
25 C and the mitochondrial reactions will be started with the addition of succinate. Aliquots will be removed from the reaction mixture at 0, 10, 20, and 30 minute intervals and used in the Succinate Oxidase, Succinate Dehydrogenase and TBA assays. The real time measurement of hydrogen peroxide production will be performed continuously. Two different control experiments will be run. One will contain all reaction components except C 60 and the other will contain all reaction components except mitochondria. In addition to containing no mitochondria, the reaction mixture of the second control will contain physiological reductants such as succinate and ascorbate. Preparation of C 60 solutions: The state of C 60 in solution is of great importance to toxicology studies as demonstrated by Buford and colleagues (2007). Prudent choices in solvents are essential for minimizing conglomerates of C 60 in solutions. For the purposes of this study, a 7.5% bovine serum albumin in phosphate buffer saline will be used to solvate C 60. Use of this solvent solution resulted in agglomerations with an area of 0.076 µm 2 and smaller (Buford, M. 2007). Also, solutions of C 60 will be sonicated for no more than one minute. This is done in order to incorporate C 60 into solution without developing conglomerates (Sager, 2007). For this same reason, the solutions will be stored in the absence of light and used before the development of conglomerates. UV-Visble spectra of the solutions will be collected in order to monitor the degree of agglomeration; an increase in agglomeration results in a broad peak at 400-500 nm range (Fortner, J. 2005). Isolation of Mitochondria: Mitochondria will be isolated from beef heart generously provided by A & B Foods in Toppenish, WA. The heart tissue is briefly homogenized in a buffer that is carefully maintained at ph 7.8. The heart tissue is then centrifuged using differential centrifugation at 1600 g and then 26,000 g in order to separate other organelles from the desired mitochondria. The mitochondria is washed in buffer to remove contaminates, hemoglobin and lipids and then stored at -20 C until use. A stock solution of 10mg/mL of mitochondrial protein per milliliter buffer is made using a ratio. Incubations will be prepared from 1.0 ml of the 10mg/ml stock solution, 7.4 ph potassium phosphate buffer, and the C 60 stock solutions. Succinate Dehydrogenase Assay: Succinate Dehydrogenase activity will be measured spectrophotometrically. In this assay, the reduction of 2,6-dichloroindophenol (DCIP) will be 5
monitored in the presence of the artificial electron acceptor phenazine methosulfate (PMS) at 600 nm. An aliquot of 0.65 ml will be used in this reaction. The reaction solution will contain 25 nm potassium phosphate buffer ph 7.4, 10 mm succinate, and 200 microliters of 200 micrograms mitochondrial protein (Trounce, 1996). Succinate Oxidase Activity: In order to assess electron transport function, mitochondria that are exposed to C 60 will be tested for their ability to move electrons through complexes II, III, and IV in the electron transport chain. This is done by measuring the disappearance of oxygen from solution as oxygen is converted to water at complex IV of the mitochondrial electron transport chain and thus is consumed in the reaction. This can be done because oxygen is the final electron acceptor in the electron transport chain. A Clarke electrode will be used for this measurement. The reaction flask used in combination with the Clarke electrode is a 1.6 ml water jacketed glass chamber. The Clarke electrode will be calibrated between zero and 100% air in a solution of buffer. This is accomplished by measuring the percent of air in the buffer after shaking the reaction buffer at ambient atmospheric pressure (100%) and then purging the oxygen from this solution with nitrogen gas (0%). The Succinate Oxidase reaction mixture will contain 25 mm potassium phosphate buffer ph 7.4, 5 mm succinate as the electron donor, 1 mg protein/ml mitochondria and 2-50 mg/l C 60. Two control reactions will contain either: 1) all components but C 60, or 2) all components but mitochondria. This second control, conducted in the absence of mitochondria, will indicate whether C 60 is reduced by physiological substrates (e.g., succinate) to produce ROS which are not detected by the Clarke electrode and are measured as oxygen consumption. Determining ROS generation and ROS damage. The measurement of ROS generation is critical in the assessment of the toxicity of a substance. ROS can be generated when a chemical takes electrons from the electron transport chain. Once the chemical has acquired the electron, it is termed a free radical and can initiate a radical process in which oxygen is reduced to form superoxide anion radical, hydrogen peroxide and hydroxyl radical. These chemical species can have devastating effects on living cells. Cells have enzymes known as dismutases that convert dangerous ROS into less reactive compounds. However, when an ROS attacks the lipid molecules of membranes, another radical reaction occurs that produces lipid peroxides. This reaction is known as lipid peroxidation and the 6
resulting polar lipid peroxides will move toward the surrounding aqueous solution ultimately causing membrane disruption. Measuring Hydrogen peroxide: The Apollo 4000 hydrogen peroxide probe will be used to measure the production of hydrogen peroxide in real time over a 30 minute period (Boveris, 2002). The Apollo 4000 probe is capable of detecting a hydrogen peroxide concentration range of 10 nm to 1 mm. The Apollo probe will be calibrated via a standard curve produced from hydrogen peroxide standards. From the incubations of protein and C 60, aliquots will be removed and used in the measurement of oxygen consumption, lipid peroxidation, and Succinate Dehydrogenase activity. Aliquots will be taken at 0, 10, 20 and 30 minute intervals. Determining Lipid peroxidation: The amount of lipid peroxidation that occurs will be measured using the Thiobarbituric Acid Assay. This Assay measures the aldehydes formed as a result of lipid peroxidation. An aliquot of the solution used in the measurement of hydrogen peroxide production will be removed and added to a tube containing 10% trichloracetic acid, 2% butylated hydroxytoluene, and 0.67% Thiobarbituric acid. This solution will be heated for 20 min at 90 C and will then be centrifuged. The remaining supernatant will be measured spectrophotometrically at 530 nm. The samples will be compared to standard curves created using malondialdehyde in the same reaction. Data Analysis: The data that will be gathered from the Clarke Electrode will be analyzed using Microsoft Excel. The linear portion of the curve formed after the introduction of the electron donor succinate will be used to calculate initial rates of oxygen consumption. The slope of the line represents the rate of O 2 consumed per minute per mg mitochondrial protein. The average activity for each time of exposure will be calculated and graphed alongside the average activity of the control. The data will be presented as percent of control. The same calculations will be performed on the data collected from the spectrophotometric assays. All data will be reported as means and standard deviations of at least triplicate determinations. E. Dissemination Plan. The results of this study will be presented at SOURCE 2009, national or regional ACS meetings in 2009, and at the Murdock College Science Research Symposium. If the results of this study are significant, they will be submitted to a peer-reviewed journal such as Nanotoxicology for publication. 7
F. Literature Cited. Abrogas,J.; Drmanyan, C.; Foote, C.; Rubin, Y.; Diederich, M.; Alvarez, M.; Anz, S.; Whetten, R.; Photophysical Properties of C 60. Journal of Physical Chemistry: 95; 11-12; 1991. Belgorodsky, B; Fedeev, L; Ittah, V; Benyamini, H; Zelner, S; Huppert, D; Kotlyar, A; Gozin, M; Formation and Characterization of Stable Human Serum Albumin Tris-Malonic Acid [C 60 ]Fullerene Complex. Bioconjugate Chemistry: 16; 1058-1062; 2005. Boveris, A.; Alvarez,S.; Bustamante, J.; Valdez.; Measurement of superoxide radical and hydrogen peroxide production in isolated cells and subcellular organelles. Methods in Enzymology: 349; 280-287; 2002. Buford, M; Hamilton, R; Holian, A; A Comparison of dispersion media for various engineered carbon nanoparticles. Particle and Fiber Toxicology; 4; 1-9; 2007 Echegoyen, L.; Echegoyen, L.; Electrochemistry of Fullerenes and their Derivatives. Accounts of Chemical Research: 31; 593-601; 1998. Fortner, J.; Lyon, D.; Sayes, C.; Falkner, L.; Hotze, M.; Alemany, L.; Tao, Y.; Ausman, K.; Colvin, V.; Hughes, J; C 60 in Water: Nanocrystal Formation and Microbial Response. Environmantal Science and Technology: 39; 4307-4316; 2005. Friedmen, S.; DeCamp, D.; Sijbesma, R.; Srdanov, G.; Wudl, F.; Keyon, G.; Inhibition of the HIV-1 Protease by fullerene Derivatives Model Building studies and Expermental Verfication. Journal of the American Chemical Society; 115; 6506-6506; 1993. Knodeloch, L.; Blondin, G.; Read, H.; Harkin, J.; Assessment of Chemical Toxicity Using Mammalian Mitochondrial Electron Transport Particles; Archives of Environmental Contamination and Toxicology: 19; 828-835; 1990. Lee, J; Fortner, J; Hughes, J; Kim, J; Photochemical production of reactive Oxygen species By C 60 in the Aqueous Phase During UV Irradiation. Environmental Science & Technology: 41; 2529-2535; 2007. Li, L; Davande, H; Bedrov, D; Smith, G; A Molecular Dynamics Stimulation Study of C 60 Fullerenes Inside a Dimyristoylphosphatidylcholine Lipid Bilayer. Journal of Physical Chemistry B: 111; 4067-4072; 2007. Nakanishu, I; Fukuzumi, S; Konishi, T; Ohkubo, K; Fujitsuka, M; Ito,O; Miyata, N; DNA Cleavage via Superoxide Anion Formed in Photoindinduced Electron Transfer from NADH to γ-cyclodextrin-bicapped C 60 in an Oxygen-Saturated Aqueous Solution. Journal of Physical Chemistry: 106; 2372-2380; 2002. Porter, A.; Muller, K.; Skepper, J.; Midgley, P.; Welland, M.; Uptake of C60 by human monocyte macrophages, its localization and implications for toxicity: Studied by high resolution electron microscopy and electro tomography. Acta Biomaterialia: 2; 409-419; 2006. 8
Ruoff, R.; Tse; D.; Malhotra, R.; Lorents; D.; Solubility of C 60 in a Variety of solvents; Journal of Physical Chemistry: 97; 3379-3383; 1993. Sager, T.; Porter, D.; Robinson, V.; Lindsley, W.; Schwegler-Berry, D.; Castranova, V.; Improved method to disperse nanoparticles for in vitro and in vivo investigation of toxicity. Nanotoxicology: 2; 118-129; 2007. Sayes, C.; Fortner, J.; Guo, W.; Lyon, D.; Boyd, A.; Ausman, K.; Tao, Y.; Sitharaman, B.; Wilson, L.; Hughes, J.; West, J.; Colvin, V. The Differential Cytotoxicity of Water- Soluble Fullerenes. Nano Letters: 4; 1881-1887; 2004. Sayes, C.; Gobin, A.; Ausman, K.; Mendez, J.; West, J.; Colvin, V. Nano- C60 cytotoxicity is due to lipid peroxidation. Biomaterials: 26; 7587-7595; 2005. Tokyama, H.; Yamage, S. Nakamura, E.; Photoinduced Biochemical Activity of Fullerene Carboxylic Acid. Journal of the American Chemical Society: 115; 7918-7919; 1993. Trounce, I.; Kim, Y.; Jun, A.; Wallace, D.; Assessment of Mitochondrial Oxidative Phosphorylation in Patient Muscle Biopsies, lymphoblasts, and Transmitochondrial Cell Lines. Methods in Enzymology: 264; 484-509; 1996. Wang, I.; Tai, L.; Lee, D.; Kanakamma, P.; Shen, C.; Luh, T.; Cheng, C.; Hwang, K. C60 and Water-Soluble fullerene derivatives as Antioxidants Against Radical-Initiated lipid peroxidation. Journal of the American Chemical Society: 42; 4614-4620; 1999. Williams, R.; Zwier, M.; Verhoeven, J.; Photoinduced Intramolecular Electron Transfer in a Briged C 60 (Accecptor) Aniline (Donor) System. Photophysical Properties of the First Fullerene Diad; Journal of the American Chemical Society: 117; 4093-4099; 1995. Xia, T.; Kovochich, M.; Brant, J.; Hotze, M.; Sempf, J.; Oberley, T.; Sioutas, C.; Yeh, J. Wiesner, M.; Nel, A.; Comparison of the Ablilities of Ambient and Manufactured Nanoparticle To Induce Cellular Toxicity according to a Oxidative Stress Paradigm. Nano Letters: 6; 1794-1807; 2006. Yamakoshi, Y.; Umezawa, N.; Ryu, A.; Arakane, K.; Miyata, N.; Goda, Y.; Masumizu, T.;Nagano, T.; Active Oxygen Species Generated from Photoexcited Fullerene (C 60 ) as potential Medicines: O 2 - versus 1 O 2 ;. Journal of the American Chemical Society: 125; 12803-12809; 2003. 9
4) Science Honors Research Proposal - Budget Cover Sheet Attach a detailed list of the equipment, supplies, travel, or other items that are required for your research project (do not include travel to conferences). List each item separately, specify its cost, and briefly describe why it is necessary for your project. Also describe other sources of funding for which you have applied in support of this project. Summarize your expenses in the table below: Pending Requested from Supplies and Materials cost /received Science Honors Supplies H 2 O 2 probes $900.00 - $700.00 Equipment Other C 60 & Bovine Serum Albumin $318.50 - $300.00 Totals: $1218.50 $400 $1000.00 List other sources of support: 1. Chemistry Research Foundation account, 2. Existing supplies and chemicals in lab. Project title: The Effects of Buckminster Fullerenes on Mitochondrial Function. 10
Science Honors Research Proposal - Budget Justification A. Supplies ($900) a. Funds are requested for the following supplies i. H 2 O 2 probes 3 per package ($900) the probes are necessary for the measurement of hydrogen peroxide. B. Equipment ($0) C. Project-related travel ($0) D. Other ($318.50) a. Chemicals and solvents Some of the chemicals and solvents needed for the proposed experiments are very expensive. Funds are requested for the following: i. One gram of C 60 ($150.00) This compound is essential for all the proposed experiments. ii. Other chemicals and solvents ($168.50) Fifty grams of Bovine Serum Albumin are essential for the proposed solutions of C 60. iii. All other necessary chemicals and solvents exist in lab. 11