**3. Oxidative stress and genotoxic damage related with coal exposure**

One important aspect to consider about the coal exposure is the amount of products generated during the coal combustion. The burning of coal, in order to generate electricity, produces flue gasses and particulate materials like coal fly ashes and residues as scoria and bottom ash. The finer particles (coal fly ash) are obtained by mechanical or electrostatic precipitation of the dust in suspension in the gases produced by combustion, while the coarser particles fall to the bottom by gravity and are removed at the bottom of the boiler [43, 44].

The combustion temperature is an important factor that determines the physical properties of the particles. In the combustion of conventional high temperature (>1400°C), the main aluminosilicate melts and condenses to form spherical particles. The coal fly ash particles produced are mostly irregularly shaped and contain a complex mixture consisting of unburned carbon; oxides; quartz; elements such as aluminum, silicon, calcium, iron, nickel, arsenic, chromium, copper, lead, cadmium, zinc [45, 46], and PAHs [47].

The coal fly ash has a relatively low toxicity as compared with coal or quartz [45]. Studies have determined the role of coal fly ash particle size and the release of iron, which leads to generation of radicals and oxidative stress. In this context, it was demonstrated the ability of coal fly ash release of bioavailable iron, which triggers processes and redox oxidant production [48]. In addition, it was shown that interleukin 8 (IL-8) levels in human lung epithelial cells are increased in response to coal fly ash and vary with the bioavailability of iron, as a function of source of coal and particle size [49]. The smaller size fraction had more stimulatory activity, which may be related to the fact that iron is more concentrated in this fraction. Particle size is a critical factor because a larger surface area allows more significant transport of metal and other adsorbed components, increasing the pulmonary toxicity of particulate matter (PM) [50].

The particles are classified according to their aerodynamic diameter (in micrometer) in coarse (PM 10), fine (PM 2.5), ultrafine (PM 0.1) [51]. The smaller particles are more harmful with respect to health effects because of their very high alveolar deposition fraction, large surface area, chemical composition, ability to induce inflammation, and potential to translocate to the circulation to extrapulmonary organs [52–54]. These particles could trigger persistent lung inflammation compared to the coarse particles in addition to the exposure to genotoxic compounds, which are contained in the particles [26, 55].

Depending on the toxicity, the chemical properties, and the concentration in air, coal and coal fly ash particles can constitute a risk to exposed workers. When these particles are inhaled and deposited in the lungs, they can lead to health risks due to the leaching of genotoxic compounds and altered immunological mechanisms affecting the lung parenchyma causing diseases [56]. These nanometric particles are very small, which allows them to penetrate the biological organs and affect its normal function. More specifically, as the particle load in the lung increases the alveolar macrophages and epithelial cells are activated, leading to the release of inflammatory mediators, ROS, enzymes (elastases, proteases, collagenases), cytokines [tumor necrosis factor alpha (TNF-α), interleukins], and growth factors (TGF-β) that control and stimulates the fibrosis, genotoxic events, and cell death [45, 57, 58].

Persistent inflammatory processes have been accepted as a crucial factor in the pathogenesis. In Zhai et al. [59], was investigated whether systemic TNF-α, soluble TNF-α receptors (p55, p75), IL-6, and soluble IL-6 receptor could be markers of biological activities of Chinese CWP. Interestingly, those results suggest that serum levels of TNF receptors and IL-6 are associated with the fibrotic process of CWP and serum cytokine levels may be correlated with the severity of CWP. In the pathogenesis of these respiratory diseases related with coal exposure, oxidative damage plays a key role. Either acting in association or independently, the chemical and physical characteristics can lead to the generation of ROS and oxidative stress [60, 61].

These particles are chemically heterogeneous and can be a source of oxidants by themselves ("acellular" mechanisms), due to their composition, such as oxides, metals, and PAHs [26]. Soluble metals (transition) associated to the particle can increase the generation of ROS by Haber-Weiss reactions. PAHs may be metabolically activated and induce ROS and oxidative stress, also forming bulky adducts or strand breaks on DNA [50, 62, 63].

Another way of generating oxidants is via cellular. Once in the lungs, alveolar macrophages are activated and generate large amounts of ROS, and chemoattractant factors of other inflammatory cells such as monocytes and neutrophils are released, which amplify this response generating more oxidants [64]. The particle size is a critical factor, because very large particles are difficult to phagocytose, leading to the process of incomplete or "frustrated" phagocytosis aggravating the response [65, 66].

Considering three different scenarios with respect to exposure to particles, the generation of oxidative stress, inflammation, and oxidative DNA damage, several authors questioned whether the lung inflammation may be related to secondary genotoxic effects. They also questioned if phenomena of oxidative stress, inflammation and DNA damage are independent or interrelated, whether oxidative stress stimulates inflammatory processes, or inflammation mediated by particles cause oxidative stress, or even if it is possible that particles may cause both phenomena of oxidative stress and inflammation but for different mechanisms of action [26, 61].

In normal physiological conditions, there is a balance between ROS generation and antioxidant defenses. However, the continuous inhalation of particles may interfere in this equilibrium leading to oxidative stress process in the lung. Consequently, a high loading of particles alters the oxidant-antioxidant balance, leading to oxidative damage and the beginning of patholog‐ ical processes [67]. The most important effects of ROS in the lung include damage to cell membranes by lipid peroxidation process, protein oxidation, and DNA damage in target cells [27].

As seen in **Figure 1**, oxidative DNA damage can have many consequences, from cell death and tissue destruction to cell proliferation. Furthermore, ROS can also act as regulators in signaling pathways intracellularly and transcription factors of a variety of genes including those of proinflammatory cytokines, adhesion molecules, and proto-oncogenes [68].

*In vitro* effects induced by coal exposure have been described in different cells such as murine alveolar type II epithelial cells (C10) [69] and in 7TD1 cells [70]. ROS generation and oxidative damage by coal fly ash particles have been described in different cell lines, in human peripheral blood mononuclear cells [71], in rat alveolar macrophages (NR8383) [72], in BEAS-2B human lung epithelial cells [73], and in rat lung epithelial (RLE) cells [74].

**Figure 1.** Main pathways associated with the generation of oxidative damage and the development of diseases induced by coal and coal fly ash particles.

ROS induce point mutations and CAs in cells. Many inhaled toxic substances contained in the particles contribute to oxidative modification that has as target of attack specific components of the cytoplasm and the nucleus. Such changes include DNA breakage, DNA oxidative modification, base modifications, alterations in the DNA sequence, poly-ADP ribosylation, activation of kinases, activation of proto-oncogenes, and inactivation of tumor suppressor genes. Persistent generation of ROS generated by mineral particles indestructible or engulfed incompletely leads to damage to organelles keys [59, 61, 75]. The oxidation of C8 deoxygua‐ nosine (dG), resulting in 7-dihydro-8-oxo-2′-deoxyguanosine (8-oxodG), is the most common oxidative lesion generated by ROS. The proportion of 8-oxodG/dG has been considered as a biomarker of oxidative stress and has been studied in relation to exposure to mineral particles *in vitro* and *in vivo* [76].

Human biomonitoring studies about the effects of exposure to coal and residues using different biomarkers have been conducted around the world. In this context, our group has obtained interesting findings in workers exposed to coal mining in Colombia and Brazil. In Rohr et al. [77], was found that Brazilian workers with occupational exposure to coal had significantly increased genetic damage in peripheral blood lymphocytes compared with unexposed individuals. Exposed workers presented lower average levels of thiobarbituric acid reactive substances (TBARS) and catalase activity (CAT). In addition, DNA damage evaluated by human buccal micronucleus cytome (BMCyt) assay was observed in mine workers, which could be a consequence of oxidative damage resulting from exposure to coal residue mixtures [78].

In Colombia, DNA damage in lymphocytes of coal open-cast mining workers using the cytokinesis-blocked micronucleus test and the comet assay were observed [79]. Also, in buccal mucosa samples, the micronucleus frequencies and nuclear buds were significantly higher in the exposed group than in non-exposed control group. Interestingly, blood samples of Colombian mining workers analyzed showed higher values of silicon and aluminum charac‐ teristic elements of coal particles, compared with the control group [80]. All these studies converge to a point: the compounds contained in the particles may be related to ROS genera‐ tion, DNA damage, and formation of pro-mutagenic adducts.

These are important findings if we consider that oxidative DNA damage can lead to long-term risk of cancer and other diseases caused by air pollution by these particles. In **Table 1**, can be observed an overview of key studies on the genotoxicity in human population exposed to coal and coal combustion products. These studies demonstrated DNA damage using different methods, related with inorganic elements and oxidative stress.





**Table 1.** Overview of key studies on the genotoxicity in human population exposed to coal and coal combustion products.
