Rethinking the dithiothreitol based PM oxidative potential: measuring DTT consumption versus ROS generation

Dithiothreitol (DTT);Reactive oxygen species (ROS)

Xiong, Qianshan ; Verma ; Vishal

关键词: Dithiothreitol (DTT);    Reactive oxygen species (ROS);   

Others  :  https://www.ideals.illinois.edu/bitstream/handle/2142/97630/XIONG-THESIS-2017.pdf?sequence=1&isAllowed=y

美国|英语

来源: The Illinois Digital Environment for Access to Learning and Scholarship

【 摘 要 】

Air pollution has been proved to be linked with adverse health effect through a large body of studies. This thesis focuses mainly on a major air pollutant, PM 2.5 (particulate matter with aerodynamic diameter less than 2.5µm), which has been associated with rising morbidity, increased possibility of obesity, respiratory diseases. The mechanism of how PM2.5 brings impact to human’s health has been studied intensively in recent years but the story behind it remains unclear. One hypothesis is that ROS (reactive oxygen species) catalyzed by ambient particulate matter will introduce excessive oxidative stress and further disrupt and destroy the cell. The thesis targeted to measure a specific ROS, hydroxyl radical (•OH) in dithiothreitol (DTT) assay. DTT assay has been a widely-used probe to measure the oxidative potential as it measures the consumption rate of DTT which presumably corresponds to the generation rate of a specific ROS - superoxide radicals (O2•-). However, this conventional assay fails to capture the most damaging ROS – •OH. Rather than taking an indirect measurement of oxidative potential introduced by ROS, this study directly measured the generation rate of •OH. Sodium terephthalate, which is able to oxidize •OH forming 2-hydroxyterephthalic acid (2-OHTA) was chosen as the probe to measure the concentration of generated •OH. 2-OHTA, a fluorescent compound, can be easily measured by a spectroflurometer at a wavelength of 425nm with the excitation wavelength at 310nm. Several environment-related organic compounds (9,10-phenanthrenequinone, 5-hydroxy-1,4-naphthoquinone, 1,2-naphthoquinone, 1,4-naphthoquinone) were tested to study the pattern of the generation of •OH. Metals such as copper(II), Manganese(II) and iron (II) which exist in the ambient particulate matter abundantly were also examined in this study. Surprisingly, different patterns were observed for both pure compounds and mixtures in DTT oxidation versus •OH generation. The efficiency order of quinones in •OH generation is 5H-1,4NQ>1,2-NQ>PQ>1,4-NQ, which is different from the efficiency order (PQ>5H-1,4NQ>1,2-NQ>1,4-NQ) in DTT oxidation. Cu(II), which is known to be a dominant metal in DTT oxidation at atmospherically relevant concentration (1µM) contributes almost negligibly to the •OH generation. Fe(II), which is mostly inactive in both DTT oxidation and •OH generation, with the presence of the other quinones it showed strong synergistic effect in •OH generation (mixture/sum = 1.6±0.2, 2.0±0.3, 1.6±0.3 and 2.2±0.3 for PQ, 1,2-NQ, 1,4-NQ and 5H-1,4NQ, respectively). Fenton reaction is responsible for the synergistic effect observed, as superoxide radicals (O2•-) formed by quinones are efficiently converted to •OH by Fe(II) through Fenton reaction. Ten ambient PM samples collected from an urban site were analyzed to investigate the correlation between DTT oxidation and •OH generation and no correlation was observed. The results show that DTT consumption and ROS-generation are two different aspects in DTT assay. Measuring both in the same assay is important to incorporate the synergistic contribution from different aerosol components and thus to provide a more comprehensive picture of the ROS activity of ambient aerosols.