Among 150 chemicals of gasoline small amounts of benzene, toluene, xylene, and sometimes lead contains were included (Ekpenyong and Asuquo, 2017). Thus, many of the gasoline exposure harmful effects are due to its individual chemicals (Rodamilans et al., 1996). Its fumes have been considered as a major route of air pollution. The latter affect immunity as well as the functions of liver, kidneys, lungs, etc. (Asefaw et al., 2020). Therefore, its fume inhalation is one of the most important routes of absorption during occupational periods. The concentration of inhaled fume and length of exposure determine its toxicity (Ekpenyong and Asuquo, 2017).
Macrophages play a crucial role in immune defense, immune monitoring as well as immunomodulation which may be assessed using different immunological parameters. These include assessment of TNF-α, and interleukins (IL); including IL-10 and IL-12. These soluble components are essential part of human defense mechanisms against invaders (Hernández-Urzúa and Alvarado-Navarro, 2001); including gasoline exposure (Jabbar and Ali, 2020). These cytokines are produced by activated monocytes (Esche et al., 2000). As an effector molecule in innate immunity, Toll-like receptors are believed to play a critical role in initiating the subsequent inflammatory responses against endogenous and/or exogenous stimuli, a mechanism which is characterized by production of pro-inflammatory mediators such as TNF- α (Garantziotis et al., 2010). In this study, the level of TNF-α was increased in sera of attendants when compared to that of the control group. In 2003, Han et al. reported that, activated macrophages can defend against pathogen invasion by increasing the secretion of pro-inflammatory cytokines, nitric oxide or other interleukins. This is already the case in the present study in which NO serum level was increased in sera of gasoline stations male attendants; especially after lengthening of exposure time. To confirm, the occupational exposure to low-level of benzene and the joint action of toluene–xylene- as individual’s components of gasoline (Rodamilans et al., 1996) showed to stimulate circulating monocytes immune response with subsequent elevation of serum TNF-α. The elevation of serum TNF-α level after gasoline exposure, in turn, may lead one to expect the involvement of an M1 classically activated macrophage in the immune-mediated toxicity of gasoline exposure. These results confirm those of Haro-García et al. (2012) who reported that the occupational exposure to benzene–toluene–xylene mixture (BTX) can stimulate TNF-α, IL-10 and IL-12 production by peripheral blood mononuclear cells )AL-Rrubaei et al., 2020).
The role of AMCase in pulmonary disease is somewhat controversial. Elias and coworkers, (2005) and others reported that, AMCase has an important role in the pathology of asthma by inducing neutrophil as well as eosinophil infiltration into lung tissues causing airway hyper-responsiveness (Donnelly and Barnes, 2004 and Shuhui et al., 2009). Therefore, highly expression of AMCase in OVA—sensitized mice in lung tissue of asthmatic patients was reported by Zhu et al. (2004). Further, the decrease in eosinophil and neutrophil recruitment to the lungs of OVA-sensitized mice which was ameliorated by chemical inhibition of AMCase allergic inflammation induced by Donnelly and Barnes (2004) confirm the role of AMCase in lung disorders. In contrast, Fitz and colleagues (2012) reported that, despite the absence of chitinase activity in models of pulmonary inflammation and allergic airway disease, the absence of AMCase had no significant effect on cellularity or airway responses (Fitz et al., 2012). The latter may be the case in the present study. Taken together, M1- but not M2 macrophage activation was mediated during occupational exposure to gasoline. Also, Wynn et al. (2016) found no role for AMCase in airway disease induced by house dust mite allergens, although AMCase was essential for developing a protective type II immune response to intestinal nematodes (Vannella et al., 2016). Locksley and coworkers (2017) showed that elder mice spontaneously develop pulmonary fibrosis and die earlier than (WT( wild type controls. This may be at least in part due to failure to digest environmental chitin, related fragments or any accumulated fragments in the lungs (Van Dyken et al., 2017); including gasoline. These investigators concluded that AMCase production by specific lung epithelial cells is important for chitin degradation in the airways. As evidenced from our results, gasoline exposure may lake these activities; confirming an M1 induced polarization of activated macrophage during gasoline exposure. To our knowledge, most of the previous investigators identified macrophages as the primary source of AMCase, whereas others identified murine and human neutrophils as a major source of this enzyme (Chang et al., 2001). Also, there is only one other paper which reported significant elevation of AMCase by neutrophils from patients with type-2 diabetes compared with that of the healthy individuals (Carrion et al., 2019).
Przysucha et al. (2020) reported that, mammals are mainly express chitinases and chitinase-like proteins which are secreted by phagocytes (mainly neutrophils and macrophages). These cells are induced at the sites of inflammation, infection and tissue remodeling. All of these lead one to suggest that, these proteins play active roles in the anti-infective defense and repair responses. However, major evidence for involvement of chitinase-like proteins in lung diseases was reported by Tabata et al. (2018) and by Patel and Goyal (2017).
M2 macrophage activation is a major and a common feature of COPD with increased YKL-40 protein levels which can reflect M2 macrophage polarization (Da Silva et al., 2008). In general, polarization occurs during the macrophage activation process and is controlled by three variables: microenvironment, cytokines and epigenetics (Mills et al., 2000).
Given together, TNF-α could be used as a marker for M1 and AMCase as a marker for M2. Therefore and in our opinion, the assay of TNF-α/AMCase ratio can be used to estimate the dominance of M1 over that of M2. In this regard, this ratio was found to be very significantly elevated (p < 0.01) in Gasoline Station male attendants when compared with the corresponding ratio of the healthy control. Such increment may lead one to suggest that M1 polarization in macrophages was the major under these research conditions. Thus, gasoline exposure may act as a stimulus for inflammation as infection, debris, or apoptotic bodies do. Therefore, the activated macrophages switch their activation to be a classical one. As before, the outcome of the classical activation will result in the involvement of pro-inflammatory mode of organ damage following gasoline exposure. These represent that such exposure may trigger other pro-inflammatory including TNF-α which is actually the case in the present.
The paradigm of the M1/M2 subtypes was suggested to a parallel that of Th1/ Th2 cells (Martinez and Gordon, 2014). Since Th1 provide immunity against intracellular bacteria and virus (Bystrom et al., 2020). Gasoline may be an alternative of them. This is because it is freely diffuse into the exposed cell, causing pro-inflammatory. The latter mediate other inflammation processing which eventually cause organ damage, including fibrosis. In addition to the production of TNF-α by the activated macrophage, (Murray et al., 2014) inducible nitric oxide synthase (iNOS) is produced with a resultant increase in NO serum level (Sica and Mantovani, 2012). Furthermore, M1 cells produce iNOS, which stimulates the production of nitric oxide (NO) (Funes et al., 2018). On other hand, the M2 subtype produces an NO inhibitor. Additionally, M1 polarization uses STAT-1 as a transcription factor. In general, the complete mechanism of macrophage polarization is not yet fully understood. Since NO mean level in sera of the exposed attendants was increased, one can suggest that M2 polarization was inhibited; thus, favoring M1 polarization. Also, in vitro studies on human macrophages have shown that serotonin may have an important role in this activation process (Dominguez-Soto et al., 2017). Whether, gasoline exposure can affect serotonin level or not was not yet be evaluated in this study. Also, the increment in NO/AMCase ratio, especially the blood of attendants who were long exposed, confirms M1 polarization of macrophage (Gershon and Tack, 2007 and Wu et al., 2019). The M1 and M2 pathways are antithetic: while one destroys, the other repairs and an imbalance between these pathways could lead to the appearance of autoimmune diseases, metabolic instability and even cancer (Orecchioni et al., 2019).
Previous In vitro studies indicated that phagocyte-dependent generation of NO at concentrations greater than 400–500 nM triggers apoptosis in nearby cells (Uehara et al., 2015). This effect may act in an in a way which is similar to specialized pro-resolving mediators to dampen and/or to reverse inflammatory responses by neutralization and speeding the clearance rate of pro-inflammatory cells from the inflamed tissues (Li et al., 2006). Thus, the over production of NO in sera of attendants may lead one to propose its involvement in cell debris clearance or damaged cell apoptosis.
Variation of both neutrophil and lymphocytes, and their ratio (NLR) comprehensively indicated immune status change (Chen and Yang, 2020). Thus, the correlations of the reduction of this ratio with length of gasoline exposure in male attendants confirm a state of immune disturbance after exposure. Thus, the elevation of leucocytic count after such exposure was the case in the present study. In this regard, Hu et al., (2020) explored the diagnostic value of PLR; as a hematological parameter, and they concluded that such parameter is a classic indicator of inflammation. These confirm other inflammatory mechanisms included herein.
Oxidative stress occurs as a consequence of the imbalance between pro-oxidants and antioxidants. This imbalance is due to excessive accumulation of reactive oxygen species (ROS) or antioxidant depletion or both with a resultant increase in cellular damages (Poljsak et al., 2013). In this regard, the levels of superoxide anion (O2−), hydrogen peroxide, hydroxyl radical (.OH) which may be increased as a consequence of disturbance in the antioxidant enzymes listed herein enhance a state of oxidative. Thus, inhalation of the gasoline fumes is a quite common cause of oxidant/antioxidant status disturbance with a possible involvement of lung related disease; a prerequisite for inflammation persistence. In vitro study by Domej (2014) confirms these inter-relationships. To confirm, Uzma et al. (2010) argue such imbalance to be due to enhanced benzene metabolism and formation of hydroquinone and 1, 2, 4 benzene triol. The latter help to generate ROS as well as to impair antioxidant defense system. These together induce oxidative stress as well as immune suppression (Uzma et al., 2010). Jabir et al., (2016) added that ROS are generated by the dust particles of the inhaled gasoline and by the oxidative burst of macrophages and neutrophils activated during their phagocytosis. All together mediate the persistence of inflammation among attendants.
Total antioxidant capacity (TAC) is a dynamic equilibrium affected by interactions between each serum antioxidative constituent, where antioxidants collaboration supplies human body with greater protection against free radicals than any antioxidant lonely. Thus, TAC considers the cumulative effect of all antioxidants present in blood and body fluids (Stocks and Donnandy, 1971). Thus, TAC cause free radicals like OH- radical, H2O2, HOCl or O2− radical attempts to destroy any of the cellular integrity by acting on the lipid bi layer of the cellular plasma membrane this may be the case in the present study (Wu et al., 2013) with subsequent formation of MDA (Wright and Welbourne, 2002) revealed that MDA increases from the reaction between (OH.) radicals and cellular poly unsaturated fatty acids, resulting in loss of cell. Increased oxidative stress, as well as lipid peroxidation represented by elevated levels of MDA gives an indication of cellular damage that is usually accompanied by reduction of TAC, SOD, CAT activities and GSH content (Mohammed et al., 2016). GSH helps in detoxification and protecting cells from ROS, while both SOD and CAT enzymes are supplementary in function. They are the primary antioxidant defense components that catalyze superoxide dismutation radicals (O2−) to H2O2 which is then converted to H2O by CAT. The present data showed depletion of TAC, SOD, CAT and GSH but accumulation of MDA, in the lysed RBCs which mediate increased risk of RBCs oxidative damage on prolonged gasoline exposure. Further, ROS can induce direct damage in RBCs membranes due to continuous oxidative degenerations as well as biochemical, physical and structural changes during their life span with subsequent removal from circulation by reticule-endothelial cells (Pac, 2017). All together distort the oxygen transport and carrying capacity of RBCs. This mechanism may be one of the causative factors of the significant reduction in RBCs count in long lived exposed attendants. Another prospect for RBCs damage may occur indirectly through benzene, as it is one of the most important constituents of gasoline (Ali et al., 2019).
In this study, the catalase and GSH activities were very significantly decreased when compared to that of the healthy control. The MDA and NO levels were significantly increased depending on duration of exposure. The activity of SOD in RBCS was significantly changes depend on age. TAC was non-significant decreased when compared with that of the control. This study agrees with Odewabi et al. (2014) who observed elevation of MDA level and reductions in the activities of SOD and CAT among gasoline filling station attendants, and disagree with (Malini and Maithily, 2017) who observed no significant difference in SOD, TAC and MDA levels.
Cerna et al., (2007) added that, benzene affects blood production by affecting the bone marrow. Further, in Korean industries, an excessive risk of hematopoietic diseases because of relatively high past exposure to benzene. CBC is the main assessment of gasoline toxicity in human (Edokpolo et al., 2015). In addition, gasoline hematotoxicity extended also to leucocytes, the primary protective line from infectious agents (John and Hole, 1992). The decrease in RBCS could also be attributed to the cytotoxic effects of the various gasoline constituents. As a result, the reduction in RBCS cause hemoglobin reduction as well, which was reported herein (Festus and Fan-Osuala, 2013 and Riaz et al., 2014). The toxic components, especially those in gasoline fumes, have been reported to change blood chemistry and induce anemia by causing bone marrow hypoplasia in experimental animals (Dacie and Lewis, 2001).
In this study, Hb and PLT were significantly decreased when compared with the control values depending on age. These agree with Al-jadaan and Alkinany (2017) and Okonkwo et al. (2016) but disagree with Mahmood (2012). The last outher observed that most of the hematological markers are not significantly affected via exposure to petroleum products; only the number of platelets showed significant decrease (13%) compared to that of the controls. The induced inflammatory process due to petrol vapor exposure could significantly reduce the hematological parameters and stimulate some immune processes. It has been established that chronic exposure to benzene may damage the precursors; namely the stem cells, and stromal cells resulting in bone marrow suppression due to the presence of toxic compounds that turn to oxidative compounds and thus mediate oxidative stress (Agrawal et al., 2018).
In this study, the increase in leucocytic count, neutrophil and lymphocytes of attendants disagree with that of Jabbar and Ali, (2020) who found a significant decline in total leucocytic count. These immune cells have protective functions against the invading infectious agents as well as the invading dusts or fume that can include gasoline. Their finding suggests gasoline containing aromatic hydrocarbons to be a contributor to such significant decrease in leucocytic count and their differential types. Thus, the suppression in bone marrow function after gasoline exposure and the failure in the migration of phagocytic cells may be expected and could also reduce the number of neutrophils and monocytes in a way that could affect the immune processes of the workers. These changes may make the attendants to be more vulnerable and susceptible to various infective agents (Ray and Kolls, 2017). The exposure may also be attributed to changes in numbers of lymphocytes, peripheral blood mononuclear cells (PBMCs) and macrophages, impaired responses to mitogens, and depression of neutrophil functions. Most of these changes are reported herein in the present study. Different studies have found that exposure to benzene results in a reduction in both circulating B and T lymphocytes in vivo and reduction in mitogen-stimulated lymphocytes proliferation (Fayed et al., 2017). Unfortunately, differentiations between B- and T-lymphocytes were not included in this but the sums of them were included; i.e. total lymphocytes. Benzene may damage the lymphocytes producing system, which regulates and inhibit both hematopoiesis and immunity. These increase sensitivity to autoimmune development, hypersensitivity, cancer, and infectious diseases (Abou-El-wafa et al., 2015). The exposure also affects the proliferation of Th1 and Th2-mediated responses. These impair humoral immunity that produces IL-6. This stimulates the differentiation and interaction of strong antibody responses but inhibits many of the phagocytic function of the cells. However, additional and further studies are required to confirm this hypothesis (Ali et al., 2019).
Studies on experimental animals and humans have shown that many chemicals suppress the immune response, leading to increased incidence of influenza and common cold (Dacie and Lewis, 2001 and Azari, 2012).