The Role of Particulate Matter-Associated Zinc in Cardiac Injury in Rats

Background: Exposure to particulate matter (PM) has been associated with increased cardiovascular morbidity; however, causative components are unknown. Zinc is a major element detected at high levels in urban air.Objective We investigated the role of PM-associated zinc in cardiac injury. Methods: We repeatedly exposed 12- to 14-week-old male Wistar Kyoto rats intratracheally (1×/week for 8 or16 weeks) to a) saline (control); b) PM having no soluble zinc (Mount St. Helens ash, MSH); or c) whole-combustion PM suspension containing 14.5 μg/mg of water-soluble zinc at high dose (PM-HD) and d ) low dose (PM-LD), e) the aqueous fraction of this suspension (14.5 μg/mg of soluble zinc) (PM-L), or f ) zinc sulfate (rats exposed for 8 weeks received double the concentration of all PM components of rats exposed for 16 weeks). Results: Pulmonary inflammation was apparent in all exposure groups when compared with saline (8 weeks greater than 16 weeks). PM with or without zinc, or with zinc alone caused small increases in focal subepicardial inflammation, degeneration, and fibrosis. Lesions were not detected in controls at 8 weeks but were noted at 16 weeks. We analyzed mitochondrial DNA damage using quantitative polymerase chain reaction and found that all groups except MSH caused varying degrees of damage relative to control. Total cardiac aconitase activity was inhibited in rats receiving soluble zinc. Expression array analysis of heart tissue revealed modest changes in mRNA for genes involved in signaling, ion channels function, oxidative stress, mitochondrial fatty acid metabolism, and cell cycle regulation in zinc but not in MSH-exposed rats. Conclusion: These results suggest that water-soluble PM-associated zinc may be one of the causal components involved in PM cardiac effects

Abstract 3007: Intracellular detection of hypoxia in live cells

Abstract Hypoxia is an important phenomenon in many physiological processes and involved in many human diseases including cancer. Inflammation can lead to significant hypoxia in tissues. The study of hypoxia has been complicated with the lack of proper instrumentation to induce hypoxia in cells and image cells under hypoxic conditions. Here, we describe a live cell-based method to conveniently measure hypoxia using a new Image-iT® Hypoxia Probe and a specialized microscope incubator which can control oxygen concentrations down to 1%, The Image-iT® Hypoxia Probe is an oxygen sensing fluorescent probe, is quenched with increasing oxygen concentrations, and has excitation and emission peaks of 483 and 616 nm respectively. The probe is sensitive to varying concentrations of oxygen and can detect as low as 1% O2 concentrations in cells. Imaging of cells with the Image-iT® Hypoxia Probe in the incubator prevents re-oxygenation of cells and gives more precise measurement of hypoxia in cells, allowing for reversible and dynamic measurements of hypoxia in cells. Using this system, we measured hypoxia in several cell lines including A549, HeLa and U-2 OS. The Image-iT® Hypoxia Probe is also very good at detecting hypoxia in 3D tumor spheroids generated using different methods. The new hypoxia probe gives good signal to noise with more than 3-fold changes at 5% O2 levels with robust statistics. The Image-iT® Hypoxia Probe provides a good system for precise, robust and reproducible measurements of hypoxia in cells. Citation Format: Bhaskar S. Mandavilli, Michael O'Grady. Intracellular detection of hypoxia in live cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3007. doi:10.1158/1538-7445.AM2015-3007</jats:p

Abstract 512: Cell-based analysis of oxidative stress, lipid peroxidation and lipid peroxidation-derived protein modifications using fluorescence microscopy

Abstract Oxidative stress plays an important role in the progression of several diseases including inflammation, atherosclerosis, aging and age-related degenerative disorders. Reactive oxygen species damage membrane bound lipids resulting in lipid peroxidation-derived protein modifications. Cell-based measurements of oxidative stress, lipid peroxidation and protein carbonylation by traditional fluorescence microscopy provide a powerful platform to quantitate oxidative stress and lipid peroxidation. Here, we used three different approaches to measure oxidative stress and lipid peroxidation in cells by fluorescence microscopy. 1) Two new fluorogenic probes, CellROX™ Orange and CellROX ™ Green Reagents to measure oxidative stress in cells, 2) Image-iT® Lipid Peroxidation Kit for a ratiometric determination of lipid peroxidation in live cells 3) Click-iT® Lipid Peroxidation Imaging Kit, a click chemistry-based approach which utilizes incorporation of an alkyne-modified unsaturated fatty acid analog, linoleamide, into the cellular membranes. The resulting oxidation products, like 9, 12-dioxo-10(E) dodecenoic acid (DODE) can readily modify proteins and these modifications were readily detected in fixed cells by the copper-catalyzed click reaction using fluorescent azides. Using these approaches, we measured oxidative stress and lipid peroxidation caused by several oxidants in cells. Increases in oxidative stress, lipid peroxidation, and protein modifications were assessed by high content imaging and analysis as well as traditional fluorescence microscopy. In the models tested, at least 2-3 fold increases were observed compared to controls was and responses were successfully inhibited by antioxidants. The three strategies described here provide powerful new tools for the assessment of oxidative stress in cells and convey distinct advantages over existing cell-based methods. Citation Format: Bhaskar S. Mandavilli, Robert Aggeler. Cell-based analysis of oxidative stress, lipid peroxidation and lipid peroxidation-derived protein modifications using fluorescence microscopy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 512. doi:10.1158/1538-7445.AM2014-512</jats:p