Despite there are several clinical associations, the direct or indirect toxicity of the lung by ST is certainly not at the level caused by smoked tobacco. Inflammatory changes leads to lung cancer, cardiovascular, non-cancer respiratory diseases, chronic obstructive pulmonary disease (COPD) and digestive system-related diseases . It is known that cigarette smoke causes the exacerbation of asthma as determined by functional airway hyperreactivity and increased levels of blood eosinophilia in Ova-sensitized mice . However, studies of the relationship between ST and asthma are very rare in the literature. In fact, in our previous study, we showed that shot-term administration of ST in a rat model of allergic asthma clearly contributed to the worsening of lung inflammation, intensifies the oxidative stress state induced by Ova-challenge in rats, which was obviously affirmed by the lung histopathological changes observed in this study . To the best of our knowledge, there is no study available showing ST consumption effects in asthmatic patients.
The findings of Ukoha et al. (2012)  indicated that chronic tobacco consumption might put the body at some risk of adverse hematological and homeostatic conditions. Mukherjee reported that the adverse effects of ST on hematological parameters are not less than smoking .
Nicotine as an element present in all tobacco products might alter the suprarenal glands to produce more catecholamine , which may influence leukocytosis causing damage and inflammation to tissues .
In the present study, WBC count was significantly increased in ST users as compared to controls. A similar significant increase in WBC count was also seen in previous studies [12, 31–34].
The granulocytes, mainly neutrophils, significantly increased in the present study in ST users compared to control, which may be linked with continuous inflammation of tissues. Neutrophils are well known to produce cytotoxic material which harmful to lung functions .
In our study, monocytes number were significantly increased in ST users than controls. Few studies confirm the association of ST with higher levels of monocytes [35, 36], which indicates the presence of infection in the individuals who consume ST.
However, lymphocytes did not show any significant change in ST uses in the present study. According to previous studies, inadequate pulmonary function in ST users might be responsible for stimulating erythropoiesis for fulfilling the demands of oxygen to the tissues .
The increased hemoglobin (Hb) levels in ST users in contrast to the control group in the present study is possibly secondary to hypoxic stimuli exerted by ST. In fact, high levels of carbon Monoxide (CO) are present in smokeless tobacco as well as in cigarette smoke. Furthermore, smokers may possess continuously rise levels of carboxy – Hb in the blood . CO lowers the affinity of hemoglobin for oxygen by binding with Hb to form carboxy – Hb. This affects the capacity of Hb to carry and deliver oxygen to different tissues of the body . Changes in arterial oxygen tension are known to affect erythropoietin production. The production of erythropoietin is known to be affected by changes in arterial oxygen tension.
In hypoxic conditions erythropoietin production is enhanced as a result more erythrocytes are produced by erythropoiesis . Thus, the increase in Hb levels is quite obvious and expected.
In the present study, hematocrit (HCT) levels were significantly decreased as compared to control. ST is also well known for its effects on physiological processes. In a recent study, ST users revealed an alteration in RBC morphology. Electron microscopy scanning demonstrated modifications in the RBC membranes with fine “bubble-like” protrusions lacking their discoid shape. ST ingredients disrupt individuals’ cellular metabolism that contributes to shape changes and scale which have enormous consequences in terms of health maintenance .
This RBC morphology alteration can explain the significantly increased RDW levels in ST users as compared to the control in the present study.
Mean platelet volume (MPV) is an essential indicator of platelet activation. The size of the platelet, activity and the function of the platelet are correlated. Larger platelets are more active than smaller ones . There are very few studies relating to the effect of ST on platelets. In the present study, MPV levels are significantly decreased in ST users and the asthmatic group as compared to the control. This is in agreement with a previous study .
C-reactive protein (CRP) is an acute-phase reactant secreted by hepatocytes in response to circulating inflammatory cytokines. It has long been used clinically to evaluate the presence and degree of inflammation because CRP blood levels increase as much as 1.000-fold within 24 hours after the onset of inflammation .
In our present study, CRP levels are significantly increased in asthmatic patients as compared to the control group. Our results are consistent with previous studies which confirmed that CRP is increased in asthmatic patients than in healthy control, and may be a useful biomarker of airway inflammation in non-smoking asthmatic patients without complications, such as heart disease, hypertension, hyperlipidemia, chronic obstructive pulmonary disease, or infection [42, 43].
Smokeless tobacco causes a significant rise in CRP levels and inflammatory cells. The significant rise of CRP among ST users can be attributed to the inflammatory response in the body . Thus, our results showed that CRP levels are significantly increased in ST users and asthmatic ST users groups as compared to the control group. Our findings are similar to those of Costello et al. (2013) and Furie et al. (2000), in which authors reported that both smokeless and addictive tobacco use leads to higher levels of CRP [44, 45].
IgE is an immunoglobulin that plays a significant role in chronic inflammatory allergic diseases and acute allergic reactions . It is a central mediator in atopic asthma, which is produced by sensitized allergen-specific B cells . Our study showed a highly significant increase of total IgE levels in patients with allergic asthma in comparison to the control. These findings are in accordance with previous studies [48–50]. This is due to the stimulated Th2 cells that are known to produce higher levels of s IL-4 and IL-13 which mediate the development of eosinophils and stimulate B-cells to secrete the specific immunoglobulin E . The eosinophilic phenotype is associated with an intense production of IL-5 and IL-13 .
In addition, our results showed that total IgE levels are significantly increased in ST users and asthmatic ST users groups in comparison to the control group. Our results are in agreement with previous studies [51, 52]. Furthermore, our data showed a significant increase of total IgE in the asthmatic ST users group as compared to the asthmatic group. This elevation among ST users can be explained as nicotine the main component in tobacco products increases mucosal permeability allowing easier and greater access of allergens to sub epithelial lymphoid tissue and this rise in IgE levels indicates an increased probability of type 1 hypersensitivity reaction (allergy), which also explains the allergy symptoms experienced ST users .
IL-5 is the most important Th2 cytokine associated with eosinophils, and it can regulate most aspects of eosinophil behavior including eosinophil growth, maturation, differentiation, survival, and activation . Therefore, this cytokine exerts key functions in the pathogenesis of eosinophilic asthma, which is often therapeutically responsive to corticosteroids because of its effective ability to induce eosinophil apoptosis . Interleukin-5 acts as a homodimer, and is essential for the maturation of eosinophils in the bone marrow and their release into the blood .
In the present study, IL-5 levels were significantly increased in asthmatic patients in comparison to the control. Our results are in agreement with previous studies, which demonstrated that higher serum IL-5 concentrations were detected in subjects with severe disease in comparison to healthy control subjects [54, 55].
Allergen inhalation increases the production of IL-5 in the airways as measured in bronchoalveolar lavage cells  and induced sputum . Interestingly, eosinophils are one of the cell types responsible for this rise. Moreover, allergen inhalation rises the number of peripheral blood eosinophils and lymphocytes containing intracellular IL-5 . The relatively high numbers of lymphocytes positive for IL-5 suggest that IL-5 is contained not only in CD4+ TH2 cells but also in other lymphocytes, including CD8+ and CD4− CD8− cells . In addition, it has been shown that IL-5 is the predominant eosinophil active cytokine present in BAL fluids during allergen-induced late-phase inflammation and may play a key role in the pathophysiology of allergen-induced, eosinophil-predominant airway inflammation .
Our results also showed a significant increase of IL-5 levels in asthmatic ST users in comparison with the control group and also with the asthmatic patients. Previous studies have demonstrated that tobacco smoking is associated with the increase of the bronchoalveolar levels of IL-5 in: acute eosinophilic pneumonia , in a model of house dust mite asthma , in allergic rhinitis mice . Furthermore, Cozen et al., showed that when genotype, age, and gender are accounted for, smoking appears to be associated with increased capacity to secrete IL-5 . Their study supports earlier observations [65, 66] and suggests that tobacco smoke may exacerbate or even lead to asthma through “priming” of immune cells toward a Th2 phenotype, possibly through an IgE independent pathway.
Nitric oxide (NO) is now well recognized for its involvement in diverse biological processes, including vasodilation, bronchodilation, and regulation of inflammatory-immune processes . Thus, it is not surprising that the role of NO in asthma has been under investigation. Accumulating evidence indicates that NO plays a role in the regulation of airway function both in health and disease. Indeed, exhaled NO has been detected in normal subjects and asthmatics [68–70].
In the present study, NO levels were significantly increased in asthmatic patient compared to the control group. These findings are in concordance with many previous studies which have shown increased NO levels in asthmatic patients [71–73]. The exact pathophysiological role of NO in the airways and lungs is complex. On the one hand, it may act as a proinflammatory mediator predisposing to the development of airway hyperresponsiveness (AHR) [74, 75]. On the other, under physiological conditions NO acts as a weak mediator of smooth muscle relaxation, and protects against AHR . In exhaled air, NO appears to originate in the airway epithelium, as a result of NOS2 up-regulation which occurs with inflammation [73, 77]. Thus, exhaled NO may be regarded as an indirect marker for up-regulation of airway inflammation .
Furthermore, our results showed a significant increase in NO levels in ST users compared to the control group. As well as, in asthmatic ST users compared to asthmatic patients. Many previous studies have demonstrated that ST consumption led to elevated NO levels compared to control [78–80].
Smokeless tobacco includes specific chemicals like polycyclic aromatic hydrocarbons, N‑Nitrosamines aromatic amines, ethylene oxide, 1,3‑butadine, and other tobacco‑specific nitrosamines. Tobacco causes increased generation of free radicals and reactive oxygen species, such as NO, superoxide anions, hydroxyl radicals, etc. . In contrast, the major constituent of tobacco, namely nicotine and its active metabolite cotinine, have been shown to stimulate NO production neurologically and also shown to stimulate angiogenesis and promotes tumor growth thought to be mediated by the production of NO and other factors [82–84].
According to Chan et al. (2009) and Aldakheel et al. (2016) in patients suffering from asthma, the imbalance between reactive oxygen species (ROS) and antioxidants leads to in oxidative stress due to the development of airway inflammation secondary to the actions of inflammatory mediators [85, 86]. Oxidative stress is believed to play a crucial role in the pathophysiology of asthma because several of the characteristic changes in the airways can be produced by the actions of ROS .
Our results showed a significant increase in malondialdehyde (MDA) levels in asthmatic patients compared to the control. MDA has been widely studied as a major reactive aldehyde resulting from the peroxidation of biological membrane polyunsaturated fatty acids, It is used as an indicator to identify damaged tissues by a series of chain reactions . Analyses confirmed that in patients with asthma, the MDA concentrations is high in several biological fluids [89–92]. These results indicate that increased production of the ROS may lead to increasing oxidative injury, which has been implicated in the pathogenesis of asthma .
Moreover, MDA levels in the present study showed a significant increase in ST users compared to the control as well as in asthmatic ST users compared to asthmatic patients. Several earlier studies have demonstrated that ST use increases lipid peroxidation in comparison with non-users [11, 94–96]. Furthermore, in our recent study, we have shown that ST administration to Ova-sensitized rats causes a significant increase of MDA levels compared to Ova-sensitized rats. In fact, nicotine and tobacco-specific nitrosamines could reinforce the increased ROS production, and decreased antioxidant defense leads to lipid peroxidation and protein oxidation. Likewise, several studies can be found that the various ingredients of smokeless tobacco extract were more toxic than pure nicotine alone in the induction of ROS formation and disparity of redox state .
In contrast, the lung and blood are endowed with several antioxidants, including GSH, superoxide dismutase (SOD), catalase, vitamin E, and vitamin C, to opposite the oxidant-mediated response [98, 99]. Therefore, the increase of the oxidant burden rate associated with such inflammatory disease (asthma) may lead to physiological changes in serum levels of the antioxidant . Glutathione (GSH) is considered a key molecule in the antioxidant pathways. This tripeptide is found in the cytosol and extracellular spaces, such as the lining fluid of the lung and plasma [100, 101].
The obtained results in the present study show a significant decrease in GSH levels in asthmatic patients compared to normal subjects. These data are in agreement with previous studies that reported that the levels of GSH in asthmatic patients decrease compared to the control subjects [93, 102–105]. This significant decrease of GSH levels in asthmatic patients may correlate with the asthma exacerbation and the low antioxidant defense. Moreover, the decrease in GSH levels in asthmatic patients may be due to increased consumption of GSH  or a lack of the amino acids found in the structure of glutathione . Sackesen et al. (2008) observed lower levels in glycine and glutamic acid in children with asthma, and they suggested that this might result from excessive use of these amino acids for increased glutathione production to cope with free radicals . Likewise, several studies have shown that oxidized glutathione (GSSG) and total glutathione (GSH and GSSG) levels are higher in the erythrocyte hemolysate, plasma, bronchial washing and BAL fluid of asthmatic patients [108–110]. Since it is already understood that increased levels of GSSG correspond to increased glutathione oxidation , and GSH level reduction, only GSH was considered in this study.
Furthermore, GSH levels in the present study show a significant decrease in ST users compared to the control group. These findings are in agreement with many previous studies which demonstrate that ST consumption reduces GSH levels in human and in rats [10, 111–113]. Therefore, the toxicity of ST in numerous organs in particular the lung might be linked to the formation of the radical species. The diverse elements of ST such as: tobacco, betel quid, areca nut, and catechu, among others, have also been reported to be toxic in experimental animals [114–116]. Moreover, during the metabolism of smoked tobacco many electrophiles are generated which are detoxified by the use of GSH . Consequently, the decreased GSH levels increase the free radical burden due to ineffective removal of ROS from the tissues, which results in increased lipid peroxidation. In addition, enhanced lipid peroxidation with a concomitant decrease in reduced GSH is indicative of oxidative stress, which provides evidence to show the relationship between lipid peroxidation, tissue damage, and inflammation .
ALT (alanine transaminase) and AST (aspartate transaminase) are the liver enzymes, which play an important role in protein metabolism and are markers of liver function [119, 120]. Compared to the control group, the level of ALT and AST in asthmatic patients is elevated. This finding agrees with many previous studies . The main causes of high ALT and AST values have been identified as hepatocellular damage and inflammation. However, levels of both enzymes are sometimes falsely lowered . Similarly, increased levels of LAP and GGT showed in asthmatic patients in the present study is an indicator of liver injury. Furthermore, the obtained results of the present study showed a significant increase of ALT, AST and GGT levels in ST users compared to the control group. Many previous studies showed elevated levels of these hepatic enzymes due to ST consumption [10, 96]. Elevated levels of ALT, AST and GGT in ST users might be related to damage and destruction of the liver tissue as ST contains ingredients of hepatotoxic agent, which induces microsomal enzyme of liver cells .
Uric acid the final product of purine degradation, acts as an antioxidant by virtue of its ability to tightly bound iron and copper . Thus, the decreased levels of urea in asthmatic patients could be explained by its conjugation with ROS, the temporal order of antioxidant consumption in human blood plasma exposed to a constant flux of aqueous peroxy radicals is vitamin C, bilirubin, uric acid and vitamin E .