Examination of the Adverse Effects of Exposure to Gaseous and Particulate Oxidant Air Pollutants in Human Airway Epithelial Cells

Human exposure to ambient air pollution is a pervasive global public health problem. Ambient levels of air pollutants, such as particulate matter and ozone, are associated with multiple adverse health effects, including increases in the incidence of morbidity and mortality. The underlying mechanism(s) responsible for the adverse effects of most air pollutants is not well understood. However, oxidative stress has been implicated as being a major contributor to the mechanism of toxic action of numerous gaseous and particulate air pollutants. The lungs serve as the primary route of exposure for air pollutants, making cells of the respiratory epithelia principal targets for many of the toxicological outcomes of air pollution exposure. The concentrations of gaseous and particulate matter (PM) air pollutants are primary determinants of the pulmonary toxicity resultant from air pollutant exposure. The study of oxidative responses to air pollutant exposure requires that a number of methodological challenges be overcome. The studies of this dissertation purposely address these challenges in the following manner: 1) Development and implementation of imaging methodologies for the investigation of effects resulting from particulate and gaseous air pollutant exposure to Human Airway Epithelial Cells (HAEC); 2) Examination of the cellular mechanisms that underlie oxidative stress responses to air pollution exposures in HAEC using live cell imaging methodologies; and 3) Examination of factors that mediate air pollution-induced changes in intracellular redox status. The major features of this body of work were able to validate and establish significant methodologies for examining the interaction of nano-scaled particulates with cellular environments, and observe oxidative alterations in the intracellular redox environment of oxidant-exposed cells in real-time. Moreover, these findings reveal that exposure to oxidative air pollutants, such as ozone, induces a profound increase in the intracellular glutathione redox potential of human airway epithelial cells that is indicative of an oxidant-dependent impairment of redox homeostasis in the cell. Cumulatively, this work advances current toxicological knowledge regarding the spatiotemporal interaction of gaseous and particulate air pollutants with cellular environments, while producing effective methodologies for the assessment of implications resulting from air pollutant exposure. Furthermore, the methodologies described herein can be used in broader toxicological applications assessing similar endpoints from other types of xenobiotic exposures.Doctor of Philosoph

Live-cell imaging approaches for the investigation of xenobiotic-induced oxidant stress

Oxidant stress is arguably a universal feature in toxicology. Research studies on the role of oxidant stress induced by xenobiotic exposures have typically relied on the identification of damaged biomolecules using a variety of conventional biochemical and molecular techniques. However, there is increasing evidence that low-level exposure to a variety of toxicants dysregulates cellular physiology by interfering with redox-dependent processes

Darkfield-Confocal Microscopy detection of nanoscale particle internalization by human lung cells

<p>Abstract</p> <p>Background</p> <p>Concerns over the health effects of nanomaterials in the environment have created a need for microscopy methods capable of examining the biological interactions of nanoparticles (NP). Unfortunately, NP are beyond the diffraction limit of resolution for conventional light microscopy (~200 nm). Fluorescence and electron microscopy techniques commonly used to examine NP interactions with biological substrates have drawbacks that limit their usefulness in toxicological investigation of NP. EM is labor intensive and slow, while fluorescence carries the risk of photobleaching the sample and has size resolution limits. In addition, many relevant particles lack intrinsic fluorescence and therefore can not be detected in this manner. To surmount these limitations, we evaluated the potential of a novel combination of darkfield and confocal laser scanning microscopy (DF-CLSM) for the efficient 3D detection of NP in human lung cells. The DF-CLSM approach utilizes the contrast enhancements of darkfield microscopy to detect objects below the diffraction limit of 200 nm based on their light scattering properties and interfaces it with the power of confocal microscopy to resolve objects in the z-plane.</p> <p>Results</p> <p>Validation of the DF-CLSM method using fluorescent polystyrene beads demonstrated spatial colocalization of particle fluorescence (Confocal) and scattered transmitted light (Darkfield) along the X, Y, and Z axes. DF-CLSM imaging was able to detect and provide reasonable spatial locations of 27 nm TiO₂particles in relation to the stained nuclei of exposed BEAS 2B cells. Statistical analysis of particle proximity to cellular nuclei determined a significant difference between 5 min and 2 hr particle exposures suggesting a time-dependant internalization process.</p> <p>Conclusions</p> <p>DF-CLSM microscopy is an alternative to current conventional light and electron microscopy methods that does not rely on particle fluorescence or contrast in electron density. DF-CLSM is especially well suited to the task of establishing the spatial localization of nanoparticles within cells, a critical topic in nanotoxicology. This technique has advantages to 2D darkfield microscopy as it visualizes nanoparticles in 3D using confocal microscopy. Use of this technique should aid toxicological studies related to observation of NP interactions with biological endpoints at cellular and subcellular levels.</p

Monitoring Intracellular Redox Changes in Ozone-Exposed Airway Epithelial Cells

Background: The toxicity of many xenobiotic compounds is believed to involve oxidative injury to cells. Direct assessment of mechanistic events involved in xenobiotic-induced oxidative stress is not easily achievable. Development of genetically encoded probes designed for monitoring intracellular redox changes represents a methodological advance with potential applications in toxicological studies

Effective intra-S checkpoint responses to UVC in primary human melanocytes and melanoma cell lines

The objective of this study was to assess potential functional attenuation or inactivation of the intra-S checkpoint during melanoma development. Proliferating cultures of skin melanocytes, fibroblasts and melanoma cell lines were exposed to increasing fluences of UVC and intra-S checkpoint responses were quantified. Melanocytes displayed stereotypic intra-S checkpoint responses to UVC qualitatively and quantitatively equivalent to those previously demonstrated in skin fibroblasts. In comparison to fibroblasts, primary melanocytes displayed reduced UVC-induced inhibition of DNA strand growth and enhanced degradation of p21Waf1 after UVC, suggestive of enhanced bypass of UVC-induced DNA photoproducts. All nine melanoma cell lines examined, including those with activating mutations in BRAF or and NRAS oncogenes, also displayed proficiency in activation of the intra-S checkpoint in response to UVC irradiation. The results indicate that bypass of oncogene-induced senescence during melanoma development was not associated with inactivation of the intra-S checkpoint response to UVC-induced DNA replication stress

Histopathologic changes in the uterus, cervix and vagina of immature CD-1 mice exposed to low doses of perfluorooctanoic acid (PFOA) in a uterotrophic assay

The estrogenic and antiestrogenic potential of perfluorooctanoic acid (PFOA) was assessed using an immature mouse uterotrophic assay and by histologic evaluation of the uterus, cervix and vagina following treatment. Female offspring of CD-1 dams were weaned at 18 days old and assigned to groups of equal weight, and received 0, 0.01, 0.1, or 1 mg PFOA/kg BW/d by gavage with or without 17-β estradiol (E2, 500 μg/kg/d) from PND18-20 (n=8/treatment/block). At 24 hr after the third dose (PND 21), uteri were removed and weighed. Absolute and relative uterine weights were significantly increased in the 0.01 mg/kg PFOA only group. Characteristic estrogenic changes were present in all E2-treated mice; however, they were minimally visible in the 0.01 PFOA only mice. These data suggest that at a low dose PFOA produces minimal histopathologic changes in the reproductive tract of immature female mice, and does not antagonize the cellular effects of E2

Comparative Effects of Various Metals on the Generation of Oxidative Damage Using 8-Hydroxy-2’-Deoxyguanosine as a Biomarker

In the recent years of scientific investigation, the role of DNA in disease pathology has become increasingly important. In attempts to understand the effects of foreign materials and energies on DNA structures and interactions, several types of genetic damage have been characterized. One type of genetic malfunction that is of interest to this laboratory is oxidative damage to DNA. Oxidative damage can result when cells are exposed to stimuli such as metallic ions, creating free radicals from metabolic processes that can attack and damage cellular components such as DNA that lead to elevated levels of oxidative stress. The objective of this investigation was to examine the effects of various metallic members of the periodic table in relation to their ability to induce oxidative damage of a particular DNA constituent. Furthermore, any possible effect of time and temperature was examined via changes made to a standard reaction setup in attempts to understand the impact of these variables on the generation of the analyte of interest. The investigation into the effects of various metals on oxidative damage was monitored and assessed by means of HPLC analysis of 8-Hydroxy-2- 1 2 deoxyguanosine, a specific biomarker of oxidative stress. The metals of interest include iron, manganese, cobalt, copper, lead, chromium, nickel, cadmium, mercury, and zinc. The results of this study found iron to be by far the most potent inducer of oxidative damage followed by manganese, cobalt, chromium, copper, nickel, lead, and cadmium respectively. In relation to the time and temperature study, two general trends were -revcalcd that relate to an increase in temperature over time. Firstly, certain metals tend to exude an innate ability to increase or decrease the concentration of 8-0HdG detected :,:\u270/ over time. Secondly, the effects of increased temperature on the outcomes of the aforementioned trends seem to only push these reactions further in the directions in which they were already compelled, causing either a noticeable nse or reduction tn concentration of 8-0HdG produce

Monitoring Intracellular Redox Changes in Ozone-Exposed Airway Epithelial Cells

Background: The toxicity of many xenobiotic compounds is believed to involve oxidative injury to cells. Direct assessment of mechanistic events involved in xenobiotic-induced oxidative stress is not easily achievable. Development of genetically encoded probes designed for monitoring intracellular redox changes represents a methodological advance with potential applications in toxicological studies. Objective: We tested the utility of redox-sensitive green fluorescent protein (roGFP)–based redox sensors for monitoring real-time intracellular redox changes induced by xenobiotics in toxicological studies. Methods: roGFP2, a reporter of the glutathione redox potential (E(GSH)), was used to monitor E(GSH) in cultured human airway epithelial cells (BEAS-2B cells) undergoing exposure to 0.15–1.0 ppm ozone (O(3)). Cells were imaged in real time using a custom-built O(3) exposure system coupled to a confocal microscope. Results: O(3) exposure induced a dose- and time-dependent increase of the cytosolic E(GSH). Additional experiments confirmed that roGFP2 is not directly oxidized, but properly equilibrates with the glutathione redox couple: Inhibition of endogenous glutaredoxin 1 (Grx1) disrupted roGFP2 responses to O(3), and a Grx1-roGFP2 fusion protein responded more rapidly to O(3) exposure. Selenite-induced up-regulation of GPx (glutathione peroxidase) expression–enhanced roGFP2 responsiveness to O(3), suggesting that (hydro)peroxides are intermediates linking O(3) exposure to glutathione oxidation. Conclusion: Exposure to O(3) induces a profound increase in the cytosolic E(GSH) of airway epithelial cells that is indicative of an oxidant-dependent impairment of glutathione redox homeostasis. These studies demonstrate the utility of using genetically encoded redox reporters in making reliable assessments of cells undergoing exposure to xenobiotics with strong oxidizing properties