Different setups of ascorbic acid a-cellular assay for measuring oxidative stress potential of airborne particulate

As there is increasing evidence that the mechanism of adverse effects caused by inhaled ambient particulate matter (PM) is mediated by the generation of reactive oxygen species (ROS), the oxidative potential (OP) has been proposed as a biologically relevant metric for assessing PM toxicity. Among the different cell-free assays developed for measuring OP, in this work we characterize the ascorbic acid assay, as an inexpensive and user-friendly method based on spectrophotometric measurements of depletion rate of ascorbate oxidized by redox-active species (OPAA). Given the important role of lung lining fluid antioxidants in ROS formation, the purpose of our study is to investigate the effects of different compositions of antioxidants to be more representative surrogate lung fluid (SLF). In addition to ascorbate (AA),we include typical lung concentrations of reduced glutathione (GSH) and urate (UA), which are naturally occurring in the lung fluid, and citrate (Cit), that is a good proxy for proteins that mobilize iron in the lung fluid. To address this, we quantified OPAA from standard solutions of two transition metals − Cu2+ and Fe2+ − and three quinones − 1,2-naphthoquinone (1,2-NPQ), 1,4-naphthoquinone (1,4-NPQ) and 9,10-phenantrenequinone (9,10-PNQ) − which are commonly found in atmospheric PM. We find that the antioxidant composition of our SLF significantly affects the AA depletion rate from all the investigated species, but with different dependence on the mixture complexity. Both citrate and glutathione decrease OPAA from Cu, if singly added to AA, and from Fe, if present in combination. This behaviour may be ascribed to formation of metal complexes, altering the reactivity of the metals. GSH suppress AA oxidation also for the investigated quinones, mainly if combined with Cit, independently of the additional presence of urate. These effects are likely due to the increase on the reductant properties of the composite SFL, due to the concomitant presence of several antioxidants. Although it is impossible to in vitro reproduce the complexity of particle−lung interactions, the composite surrogate lung lining fluid investigated in this work may represent an useful experimental set up for a screening assay for the oxidative potential of ambient PM.