Spectrophotometric cell-free assays for measurement of the oxidative potential of atmospheric aerosol.

Oxidative stress has been suggested as an important underlying mechanism of action by which exposure to ambient particulate matter (PM) may lead to adverse health effects in humans. Oxidative stress results when the generations of reactive oxygen species (ROS), or free radicals, exceed the available antioxidant defenses. For assessment of the capacity of a PM sample to catalyze ROS generation, the oxidative potential (OP) has been proposed, as a measure of the ability of PM to oxidize target molecules, i.e. by generating ROS in environments without living cells. Among the various assays developed for measuring OP, in this study two common methods are investigated and compared in terms of different sensitivity to the ROS generating compounds. The dithiothreitol (DTT) assay measures the presence of reactive oxygen species via consumption of DTT to form the DTT-disulfide due to transfer electrons from DTT to ROS by recycling chemicals such as quinones. The reaction was stopped at designated time points by addition of trichloroacetic acid. The subsequent loss of DTT is followed by its reaction with 5,5’-dithiobis-2-nitrobenzoic acid (DTNB) to form 2-nitro-5-mercaptobenzoic acid, which is monitored spectrophotometrically at 412nm. The linear rate of DTT loss is measured (expressed as μmol DTT min-1). The ascorbate (AA) depletion assay measures the ability of PM to deplete ascorbic acid: the reaction kinetic is followed by measuring AA absorption at 265 nm. The results are expressed as μmol min-1of AA depletion. The performances of the two assays were investigated with standard solutions of individual redox-active species that are common in ambient PM, such as quinones and transition metals. Quinones –mainly phenanthrenequinone and 1,2-naphthoquinone–were found more efficient at oxidizing DTT compared to the less reactive transition metals, i.e., copper, manganese, nickel, chromium, iron. On the contrary, the AA assay is most sensitive to transition metals – mainly copper, chromium, iron and nickel – in comparison to quinones. The obtained information is very relevant to understand the relative importance of metals and organics towards ROS generation from ambient particles. Both methods were applied to real ambient PM2.5samples collected at urban sites in winter. The preliminary results show that the two OP assays can provide complementary data to be used as a good quantitative chemical assay for oxidant generation and toxicity measurement of PM. Future studies will focus on relationship with chemical characterization of PM samples to assess the role of organic and inorganic species in generating redox activity.