AAAR 35th Annual Conference October 17 - October 21, 2016 Oregon Convention Center Portland, Oregon, USA
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Rethinking Dithiothreitol based PM Oxidative Potential – Measuring Antioxidant Consumption versus ROS Generation
Qianshan Xiong, Runran Wang, Haoran Yu, VISHAL VERMA, University of Illinois Urbana-Champaign
Abstract Number: 649 Working Group: Health Related Aerosols
Abstract The capability of ambient particles (PM) to generate reactive oxygen species (ROS) is proposed as a biologically relevant property. Among the various methods developed to measure the oxidative potential of ambient PM, dithiothreitol (DTT) assay has been most widely used. In this assay, the rate of DTT oxidation catalyzed by the ambient PM is measured, which is assumed to be proportional to the rate of superoxide radical generation. These superoxide radicals can further react with the metals (e.g. Fe and Cu) to generate hydroxyl radicals. However, in the conventional protocol, where we measure the rate of oxidation of DTT, this important step of hydroxyl radical generation is not captured. This is probably a great caveat of DTT assay causing an underestimation of the role of transition metals, which are otherwise known to generate ROS in biological system. To overcome this limitation, for the first time, we measured the hydroxyl radical generation catalyzed by ambient PM in the DTT assay. This is accomplished by adding sodium terephthalate in the reaction vial, which gets oxidized by hydroxyl radicals to 2- hydroxyterephthalic acid (2-OHTA). 2-OHTA is a highly fluorescent compound and the fluorescence intensity is measured at 425 nm (excitation wavelength 310 nm). Preliminary tests conducted with pure compounds revealed new insights on the pattern of ROS generation in DTT assay. For example, four quinones known to oxidize DTT in the efficiency order of phenanthrenequinone (PQN)>5-hydroxy-1,4-naphthoquinone (5-hydroxy-1,4-NQN)>1,2-naphthoquinone (1,2-NQN)>1,4-naphthoquinone (1,4-NQN), showed a different efficiency order (5-hydroxy-NQN> 1,2-NQN> PQN> 1,4-NQN) in the hydroxyl radicals generation. Cu, which is a dominant metal in the DTT assay, has a long lag period (up to 2-5 hours depending on the concentration of Cu) in the hydroxyl radicals generation. Most importantly, ROS generation by both quinones and Cu was enhanced by 2-3 folds in presence of Fe, which was supposed to be inactive in the DTT assay. Our results show that DTT-oxidation and ROS-generation are two different aspects, and measuring both in the same assay is important to incorporate the contribution from all major aerosol components and to provide a comprehensive picture of the total ROS activity of ambient aerosols.