Evaluation of OP acellular assays to predict PM-induced oxidative stress activity in the root system of a plant model organism

Over the last few decades, oxidative stress has been identified as one of the main mechanisms by which particulate matter (PM) exerts its adverse effects on living organisms (Li et al, 2015). During the last years, different acellular assays, such as dithiothreitol (DTT), ascorbic acid (AA) and 2′,7′-dichlorofluorescin (DCFH) assay, have been used for the evaluation of the oxidative potential (OP) of particular matter (PM) to predict PM capacity to generate reactive oxygen (ROS) and nitrogen (RNS) species in biological organisms (Kelly and Fussell, 2012). In a recent study, Simonetti et al (2018) applied the DTT, AA and DCFH assays to the soluble and insoluble fractions of seven types of widespread atmospheric dusts (brake dust - BD, pellet ash - PA, road dust - RD, certified urban dust NIST1648a - NIST, soil dust - S, coke dust - C and Saharan dust - SD) characterized by very different chemical compositions (Marcoccia et al, 2017), which can be associated to different adverse health effects. However, the assays have provided very different results for each PM-selected component and it was not possible to correctly predict PM-induced oxidative stress activity in living organisms. Relationships among PM oxidative potential and PM effects on the ROS and RNS generation pathways are still largely unknown. In this study, we applied the three OP assays (OPAA, OPDTT and OPDCFH) to the seven atmospheric dusts (BD, PA, RD, NIST, S, C and SD) and then we grew the seedlings of a plant model organism: Arabidopsis thaliana (L.) Heynh., in the presence of the PM-selected components to evaluate the bioaccumulation of element concentrations and their capacity to induce oxidative stress and root system alteration on the tested organism. A. thaliana root apparatus, which consists of primary root (PR) established during embryogenesis and post-embryonic lateral roots (LRs), is a common target organ of metal pollution whose morphology and development may be severely damaged by high levels of ROS and RNS. This study was aimed to assess the effects of the PM-selected components on the root system of the plant organism to find correlations among the OPs of the seven types of dust and their capacity to alter primary root length (PRL) and lateral root density (LRD) and to generate ROS and RNS (high levels of O2- and NO). Element concentrations of each dust accumulated in the tissues of A. thaliana were determined with the aim to identify the role of the single PM components in the alteration of the root system. Then, correlations among OPs of the dusts, element bioaccumulation and root morphology alteration were individuated by performing principal component analysis (PCA). Lastly, histochemical analyses of NO, O2- and lipid peroxidation content and distribution were performed to verify the induction of oxidative stress in the A. thaliana root apparatus. BD, PA and NIST showed the highest OPs and altered A. thaliana root morphology inhibiting primary root growth. High concentrations of Cs, Li, Nb, Pb, Sb, Sn, Tl, V, W and Zr were found to be accumulated in the tissues of the seedlings exposed to BD; Mn and W in the seedlings grown in the presence of PA; Al, Cd, Pb, Sn, Ti, V, W, Zr in the seedlings exposed to NIST. PCA revealed correlations among OPs of the dusts, element bioaccumulation and root morphology alteration, identifying the most responsible dust-associated elements affecting the model organism. OPDTT and OPDCFH were found to be well correlated with K and Mn concentrations accumulated in the A. thaliana seedlings exposed to PA. Since the OPs of the dusts were found to be positively correlated with primary root growth inhibition, BD, PA and NIST were identified as the PM-selected components with the highest potential to induced oxidative stress and root system alteration on A. thaliana. Moreover, the analysis of O2-, NO and lipid peroxidation content and distribution (Figure 1) in the A. thaliana roots confirmed the capacity of the PM-selected components to induce oxidative stress, leading to plant redox homeostasis alteration and root cellular damages, and verified the reliability of the OP assays for the prediction of the ROS and RNS generation pathways in biological organisms. Finally, our study validated A. thaliana as a suitable model for bio-indicator studies and encourage its employment for further investigations about the detection of PM toxicity on living organisms.