Quantifying the modulation effect of imidazoles on reactive oxygen species generation by quinones and atmospheric humic-like substances

Abstract

Ambient particulate matter (PM) can cause adverse health effects via their ability to produce reactive oxygen species (ROS). Humic-like substances (HULIS), a complex mixture of amphiphilic organic compounds, have been demonstrated to contain the majority of redox activity in the water-extractable organic fraction of PM. Apart from redox-active quinones, reduced organic nitrogen compounds, such as alkaloids resulting from biomass burning emissions, are also found among HULIS constituents. In this study, we examined the redox activities of pyridine, imidazole and their alkyl derivatives using a cell-free dithiothreitol (DTT) assay under simulated physiological conditions (37 °C, pH = 7.40). These compounds were found to have little redox activity on their own as measured by the DTT assay, but they enhanced ROS generation catalyzed by 1,4-naphthoquinone (1,4-NQ), 1,2-naphthoquinone (1,2-NQ), 9,10-phenanthrenequinone (9,10-PQ), and HULIS isolated from multiple aerosol samples. The enhancement effect by the individual nitrogen-containing bases was determined to be proportional to their amount in the assay solutions. It is postulated that the underlying mechanism involves the unprotonated N atom acting as a H-bonding acceptor to facilitate hydrogen-atom transfer in the ROS generation cycle. The enhancement capability was found to increase with their basicity (i.e., pK_a of their conjugated acids, BH⁺), consistent with the proposed mechanism for enhancement. Among the imidazole homologues, a linear relationship was observed between the enhancement factors (in log scale) of the unprotonated form of the imidazole compounds (B) and the pKa of their conjugated acids (BH⁺). This relationship predicts that the range of alkyl imidazole homologues (C₆–C₁₃) observed in atmospheric HULIS would be 1.5–4.4 times more effective than imidazole in facilitating HULIS-mediated ROS generation. As for the redox activity of HULIS, it was found that the DTT consumption rate per μg of HULIS increased with HULIS dose. A quadratic curve of zero intercept yields excellent fitting for the relationship between DTT consumption rate and the amount of HULIS, which is consistent with the characteristics of HULIS containing both ROS-active constituents and components enhancing ROS activities. During these tests, the protocol of DTT assay was also investigated. Chelex 100 resin seems to be a better choice for the treatment of phosphate buffer, as metal chelators (e.g., DTPA and EDTA) added may cause unexpected effects on the test. But it should be noted that Chelex 100 resin was absent in the buffer during the DTT assay, thus cannot eliminate the DTT response from residual metals in samples. Resin treatment is not recommended on HULIS samples as it would remove HULIS significantly. Our work reveals that the ability of atmospheric PM organics to catalyze generation of ROS in cells could be affected by co-existing redox inactive organic constituents, which need to be considered when studying the dose–response relationship of atmospheric PM, and suggests further work optimizing DTT assay protocol and deploying multiple assays be conducted to better quantify redox capabilities and enhancement effects of the HULIS components.

Date of Award	2015
Original language	English
Awarding Institution	The Hong Kong University of Science and Technology