Particulate matter (PM) pollution is a serious global problem, especially in urban and industrialized areas [1
], threatening both human and environmental health [2
]. A growing number of scientific studies have demonstrated links between exposure to ambient PM and adverse health outcomes in human, especially related to cardio-pulmonary diseases [3
] and neurological disorders [4
]. Over the last few decades, oxidative stress has been identified as one of the key mechanisms by which PM exerts its negative impacts on cellular systems and, thus, on living organisms [5
]. Oxidative stress is due to the imbalance between the generation of reactive oxygen species (ROS) alongside reactive nitrogen species (RNS) and the antioxidant defenses [3
]. Particulate matter’s capacity to elicit damaging oxidative reactions and inflammations is defined as oxidative potential (OP) [7
]. Measurements of particles’ OP are considered a promising and integrative method for assessing health impacts induced by PM [8
]. In fact, it is increasingly recognized that this PM property is more closely associated with adverse health impacts than ordinarily used PM mass concentrations [9
]. In this context, many acellular assays were developed to measure oxidative potential in order to gain a proxy of this PM capability [10
]. There is still no agreement regarding the most representative assay to determine oxidative potential [11
], but most commonly used acellular tests include the ascorbic acid (AA) [12
] and dithiothreitol (DTT) [13
] assays which consist of mimicking the consumption of a physiological and a surrogate antioxidant, respectively, and 2′,7′-dichlorofluorescin (DCFH) by which particle-bound ROS are determined [14
]. Oxidative potential acellular assays present several advantages such as ease of application, a clear and wide description in the literature that make them replicable, and applicability to a high number of samples during monitoring campaigns by generating large data sets with the aim of studying different redox components for detailed investigation [11
]. However, several studies showed that each assay is sensitive to different pathways of ROS/RNS formation [17
], and results are also responsive to different ROS/RNS generated by PM components and sources, meaning that each OP assay is seemingly linked to different health endpoints [15
]. Therefore, the combined application of different acellular methods on the same samples is strongly suggested. Although some recent studies were focused on the in vivo evaluation of PM’s negative impacts on living organisms [18
], knowledge about the relationships between OP and PM’s toxicological effects still presents some important gaps [6
Other still unresolved issues are related to the assay’s design and the influence of the operative conditions by which OP tests are performed. In fact, over recent years, many researchers underlined how test conditions could influence collected data. For example, recent studies analyzed the effect derived by the choices of extraction solvent (methanol and/or water) and sampling filter type (quartz or Teflon filter) on different OP assays [17
]. Other researchers studied the impact of sonication-derived free radicals on OP results and suggested alternative extraction methods such as vortexing, magnetic stirrer or orbital shaking to avoid ultrasound-induced radicals [23
]. The influence of filter-storage conditions also merits further investigation. Moreover, OP assays are commonly performed on PM water-soluble fractions, and another still debated issue concerns the lack of an appropriate standard protocol for measuring the water-insoluble oxidative potential [15
]. An additional important point is related to the stability of the species: short-life oxidant species can react and redox equilibria, and among PM native species, this could occur during the sample storage and extraction phase. In this regard, it is possible that, within the conditions that influence OP results, there is also the reaction and/or competition between oxidant and reducing species naturally occurring in PM. In fact, PM is a heterogeneous and complex mixture of particles [25
] that varies in composition and it is very difficult to identify the exact chemical constituents [26
]. Some studies proved the presence of species with likely antioxidant and reducing characteristics such as phenols from wood burning [27
], phenolic compounds from different sources [28
], pollen [29
], airborne bioaerosols that are biological in origin [30
], and biological and vegetable components [31
This research was aimed at clarifying the latter aspect by considering the application of an acellular procedure to evaluate the presence of reducing species in PM samples. The 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay is a rapid, simple, and widely used method to evaluate the antioxidant potential of a compound or an extract [32
], and it is commonly used for vegetable juice [33
], olive oils, and wines [34
]. The assay is known to be sensitive towards some classes of reducing species such as phenolic and polyphenolic compounds [35
]. 2,2-Diphenyl-1-picrylhydrazyl is a stable free radical by virtue of the delocalization of the spare electron over the molecule [36
], and it accepts electrons or hydrogen radicals from donor compounds [37
The assay is based on the quantitative measurement of the scavenging capacity of antioxidants towards DPPH free radicals [32
] by the decrease in absorbance [38
]. This test provides information on the antioxidants’ capacity to donate hydrogen atoms or electrons [37
]. To date, no DPPH standard experimental procedures exist; various researchers have used widely different protocols [40
] based on their treated samples which differ in DPPH concentration, reaction time, and reaction solvent. To our knowledge, this assay has never been applied to PM before.
The aim of this work was to evaluate the applicability of the DPPH assay to PM for measuring reducing potential (RP) due to the presence of antioxidant species which could, in the future, integrate the information about redox properties of PM obtained by OP assays. In this work, the DPPH assay was applied to seven types of widespread components of PM produced by specific emission sources and characterized by very different chemical compositions [19
] (i.e., urban dust certified for its elemental content, UD; brake dust, BD; Saharan dust, SD; coke dust C; calcitic soil dust, CSD; incinerator dust, ID; and certified diesel particulate matter, D). Moreover, the DPPH assay was applied to PM2.5
field samples collected during a short monitoring campaign.
In this study, the DPPH radical scavenging assay was adapted and preliminarily applied to PM with the aim of verifying its possible use as an acellular method for estimating the presence of reducing species. The results showed that the assay was sufficiently sensitive to be applied to 24 h PM samples collected by samplers working at 2.3 m3/h with a good repeatability and linearity.
The described preliminary application of the DPPH assay revealed the presence of reducing species in several components of atmospheric PM derived from various emission sources and, thus, with very different chemical composition; the highest values were measured in urban dust, brake dust, and diesel dust. These outcomes seem to be mainly due to the reactions involving DPPH and organic fraction, but further studies are required to identify the species responsible for DPPH scavenging.
The RPDPPH test is a cost-effective, rapid, and simple acellular method thus offering the same advantages of the OP assays. In the future, the combined application of all these tests to wider sample sets will permit to better understand redox equilibria among PM native species that might occur during sample storage, extraction phase, and/or application of oxidative potential assays. Furthermore, it is worth considering that reducing species could reasonably react with oxidizing ones, also when PM gets in contact with biological systems, thus exerting a possible opposition to oxidative stress.
The availability of a suitable assay for routinely estimate the amount of reducing species in PM samples could constitute a new and potentially useful tool for acquiring new insight in the field of its redox behavior and health effect.