br Conclusions The present protocol for localizing or spotti

Conclusions The present protocol for localizing or “spotting” the site of action of an antioxidant in a micro-heterogeneous medium is based on the differences in its reactivity vis-à-vis the series of amphiphobic TEMPO derivatives 1a–f. Plots of the relative antioxidant effectiveness of a given AO towards probes 1a–f yield cut-off curves, which can be convex, if the AO is in a more BS-181 microenvironment, or concave, if it is in a more hydrophobic microenvironment than the probe. The degree of convexity of these curves is also a measure of the selectivity by the probe series. The more hydrophilic (or more hydrophobic) the AO microenvironment, the more pronounced is the convexity (concavity) of the resulting cut-off curve. LogP values are not reliable, when comparing different antioxidant activities as a result of different sites of reaction. They do not take into account the relative orientations of probe and antioxidant, and the resulting proximity of their reacting groups, a decisive factor for measuring antioxidant activities. By contrast, cut-off curves like those depicted in Fig. 4, yield precious information regarding the accessibility of the AO by the probe. The paradoxical behaviour of the series of TEMPO probes 1a–f may thus be used to identify the site of action of a known antioxidant in micro-heterogeneous media. Very often, food extracts consist of complex mixtures of antioxidants in emulsified media, with unknown structures and partitioning behaviour. In such cases, global parameters are used to characterize the antioxidant activity of the food sample, often with misleading values, since they do not take into consideration the actual site of action of the antioxidant. The result of such a simplification has been recognized before: “unless we take these factors into consideration, we cannot correctly evaluate antioxidant activity” (Niki, 2002). This becomes a very difficult task, if the nature and composition of all antioxidants present in a food sample and their site of action are not known. The simple protocol described here may be used as a first step to tackle this problem, and to characterize or “spot” the major site of action of a complex mixture of antioxidants, even when they are not known, and no information about their partitioning in the heterogeneous system is available.
Acknowledgements This work was financed by CONICYT/FONDECYT project 1160486. CA thanks Financiamiento Basal para Centros Científicos y Tecnológicos de Excelencia, under project FB0807.
The impact of air pollution on human health remains a critical worldwide concern. Epidemiologic studies have consistently identified air pollution to be associated with increased morbidity and mortality (Lodovici and Bigagli, 2011, Seaton et al., 1995). Various air pollutants, including fine and ultrafine particles, ozone, nitrogen oxides, polycyclic aromatic hydrocarbons and transition metals, can act as free radical initiators generating reactive oxygen species (ROS), which directly attack cellular DNA (Lodovici and Bigagli, 2011). Oxidative stress, which results from the imbalance of reactive oxygen species (ROS) generation and antioxidant enzymes, may induce damage of tissue, lipids, proteins, and nucleic acids, and therefore, plays a critical role in environment related diseases in humans including cancer, asthma, respiratory diseases, and arteriosclerosis (Lodovici and Bigagli, 2011). When a cell sustains oxidative stress, antioxidants present in the cell respond to quench the reactive oxygen species (ROS). Phase I and II metabolic enzymes interact with foreign and toxic compounds in the body (Delfino et al., 2011, Sies, 1997), and therefore, are important for regulating the balance between the overproduction and destruction of ROS within the cell. Glutathione peroxidase (GPx), a key front line defense phase II enzyme, is responsible for breaking down hydrogen peroxide and additional peroxides into less toxic compounds, and reduces the formation of free hydroxyl radicals (Ceballos-Picot et al., 1996). Glutathione peroxidase (GPx) requires glutathione (GSH) as a co-factor producing glutathione disulfide (GSSG) as a product. Glutathione reductase (GR) is responsible for the reduction of GSSG to glutathione (GSH), which is critical for maintaining glutathione levels and minimizing oxidative stress in cells (Carlberg and Mannervik, 1985). Glutathione S-transferases (GST), a large family of Phase II enzymes, use glutathione to detoxify xenobiotic substances to less toxic products that can be removed from the body (Sies, 1997). Among all antioxidant enzymes, GPx has been considered as the most important given its higher affinity to hydrogen peroxide compared to catalase, which also catalyzes hydrogen peroxide into water and oxygen (Baud et al., 2004, Davis and Uthus, 2003, Valko et al., 2006). Moreover, the treatment of using GPx-1 as a therapeutic target might have potential benefits for COPD patients against hydrogen peroxide and the peroxynitrite molecules from cigarette smoke-induced inflammation and emphysema (József and Filep, 2003, Vlahos and Bozinovski, 2013). In addition, it has been shown that deletion of the genes regulating glutathione enzyme activity influences the level of DNA adducts and development of cancer, specifically lung cancer (Nielsen et al., 1996).