Non-Invasive Probing Cellular Oxidative Stress and Antioxidants Therapeutic Effectiveness

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PROJECT SUMMARY. Particulate matter (PMs) that induces oxidative stress have prominent impacts and adverse health effects including increased cardiovascular and pulmonary diseases. The World Health Organization recognizes diesel engine particles (DEPs) as mutagenic and carcinogenic to humans and the International Agency for Research on Cancer (IARC) classified diesel engine exhaust as a human group I carcinogen. It has been established that generation of reactive oxygen species (ROS) and oxidative stress is an important toxicological mechanism of particulate air pollution-induced lung cancer and disorders. However, the pathophysiological mechanism of air pollutant mediated pulmonary toxicity still remains unclear. This is primarily due to the lack of efficient test systems, mimicking human air pollutant inhalation exposure scenarios. Furthermore, it is unclear, but important, to understand whether epithelial cells from different respiratory tract regions respond to PMs similarly or differently and how to develop measurement technology to assess responses in both in vitro and in vivo. Dietary interventions with antioxidants (e.g., resveratrol), polyunsaturated fatty acids (PUFA, such as docosahexaenoic acid or DHA) are an attractive therapeutic approach to ameliorate air pollution-induced respiratory toxicity. Current methods to detect cellular ROS are fluorescence-based, suffering several drawbacks: each fluorescent dye is limited to a couple of ROS species, not detecting the overall ROS production; single-cell resolution is not achievable; lack of both molecule-level details and temporal specificity; and the photobleaching problem. The primary goal of this proposal is to test the application of a non-invasive, non-destructive, label-free Raman spectroscopy technique in combination with machine learning analysis to assess cellular oxidative stress and cytoprotection by antioxidants. Our central hypothesis is that subcellular and biochemical composition alterations induced by oxidative stress and intracellular ROS production can be quantitatively detected in the cell by Raman spectroscopy using the combinational analysis of respective spectral markers of the fingerprint and the silent regions. To accomplish this goal, we propose the following specific aims: 1) In vitro toxicity assessment of epithelial cells exposed to DEP and the effects of antioxidants; 2) Identification of oxidative stress-associated spectral markers in the fingerprint region and development of intracellular ROS sensing nanoprobes in the silent region; and 3) Pre-validation of Raman-derived spectral markers and ROS quantitation on the lung tissues isolated from DEP exposed mice. We expect that successful completion of these aims will yield a wealth of new knowledge regarding the mechanistic link(s) between cellular oxidative stress biomarkers and non-physiological spectral markers. Further, we expect that these experiments will establish a new technical platform for screening antioxidant activities that can benefit the discovery of new therapeutic agents to battle PM-induced lung disease and other oxidative stress induced inflammatory diseases.