Introduction

According to World Health Organization (WHO), Smokeless Tobacco (ST) is the single greatest cause of preventable death all over the world (WHO, 2008). The various forms of tobacco are smoked, chewed, sniffed, sucked and applied to teeth and gums (National Sample Survey Organization, 1999). Forms of chewing tobacco include crushed tobacco leaves mixed with slaked lime paste, khaini, gutkha, mishri (a roasted and powdered tobacco), mawa (a mixture of sliced areca nut, lime, kiwam and tobacco leaves), pan (piper betel leaf filled with sliced areca nut, lime and tobacco leaves) and dry snuff or tapkeer (use for inhalation) (Zakiullah et al., 2012). ST largely used in South Asia and mainly in India as compared to the Western world (WHO, 2009). In India, millions of people die every year due to tobacco use (Thomsen, Ahlbom, Bridges, & Rydzynski, 2008). The majority of cardiovascular diseases (CVD), cancers and lung diseases are directly credited to tobacco use (Thomsen et al., 2008).

ST use is documented in 120 nations of the world (Sinha, Suliankatchi, & Gupta, 2010). India has the largest number of ST users in the world. Out of the 346 million worldwide tobacco users, India alone has 152.4 million people and out of that, 80.8 million ST users (US NCI and WHO, 2016) and there has been a considerable rise in ST users among all age groups (Bhan et al., 2016). The financial burden due to tobacco-related diseases in India was US$ 22.4 billion in the year 2011- 2012 (MoHFW and WHO, 2014; Pednekar et al., 2016). The economic burden was more in females as compared to males due to ST use (US NCI and WHO, 2016).

In view of the various pharmacological actions of nicotine and its wide use in many regions of the world, chronic consumption of ST may affect the status of hematological parameters. However, there are limited studies available on the effect of ST use on hematological parameters in humans. The present study was undertaken to find out whether ‘smokeless tobacco’ causes any adverse effect on hematological parameters as well to evaluate the beneficial effects of vitamin C supplementation.

Materials and Methods

The present interventional study has an experimental approach and the case-control design was carried out in the Krishna Institute of Medical Sciences, Karad, during 2015–2019. The study protocol was approved by the Institutional Ethical Committee of KIMSDU, Karad (Ref. No: KIMSDU/IEC/01/2015). A total of 338 subjects aged between 31 to 60 years (168 ST chewers and 170 ST non-chewers) participated in the study. Experimental protocol and study objectives were explained to the participants and their written informed consent were obtained according to the Helsinki declaration (World Medical Association, 2013).

Inclusion Criteria

Healthy adults aged between 31 to 60 years with at least last one or more years of history of ST chewing ( dried tobacco leaves mixed with slaked lime) were selected as ST chewers. Age and sex-matched healthy adults with no history of tobacco use in any form were selected as controls.

Exclusion Criteria

Individuals with any type of CVD, autonomic, endocrine, metabolic dysfunction, liver disease, cancer, any type of long term and regular medication and alcoholic individuals were excluded from the present study.

EDTA anticoagulated blood was used for estimation of hematological parameters on SYSMEX XT- 1800 fully auto analyzer. Subjects of both groups were examined at the study baseline for hematological parameters. All study participants, ST chewers and controls were asked to consume 1gm of VC (Celin, Glaxo SmithKline) daily after a meal, respectively, for 45 days.

Regular follow up were taken twice in a week and subjects were advised to maintain their normal diet and continue their habitual physical activity. Subjects of both groups were examined at the end of the study (after supplementation of VC) for hematological parameters. Participants of the ST chewers group were further divided with respect to ST chewing duration in years. The data obtained from both study groups were analyzed and compared by using a suitable statistical test.

Results and Discussion

The present study showed that tobacco chewers had significantly deranged hematological parameters. In the present study, we observed that, WBC count (p=0.04), granulocytes % (p=0.0007), HCT (p=0.01) and MCV (p=0.04) were significantly increased whereas, monocytes % (p=0.002) and platelet count (p<0.0001) was significantly decreased in ST chewers as compared to controls (Table 1). Selected hematological parameters showed increased derangement with respect to increased ST chewing duration in years (Table 2). Lymphocytes % (p=0.02) and monocytes % (p=0.001) were significantly decreased with respect to increased ST chewing duration whereas granulocytes % (p=0.01) and platelets count (p<0.0001) were significantly decreased with respect to increased ST chewing duration.

In the present study, monocytes % (p= 0.008) shows a significant positive correlation with serum VC. WBC count shows a significant positive correlation with serum CTN (p=0.04) and a negative correlation with serum VC (p=0.05). However, platelet count (p=0.004) shows a significant negative correlation with serum CTN in ST chewers (Table 4).

In previous similar studies, it was concluded that Hb level and WBC count were increased whereas monocytes and basophils count were decreased in gutkha consumers as compared to controls (Dass, Jaganmohan, & Sravanakumar, 2013; Jaganmohan & Sarma, 2011; Rajsekhar, Ramgopal, Sridevi, & Narasimha, 2007).

In another previous study (Kumar, Binawara, Beniwal, & Sharma, 2017) observed that Hb, RBC and WBC count were significantly increased, whereas; lymphocytes, monocytes and platelet count were significantly decreased in tobacco chewers as compared to controls. The findings of Ukoha indicated that chronic tobacco consumption might put the body at some risk of adverse hematological and haemostatic conditions (Ukoha, Dimkpa, & Maduka, 2012). Roan Mukherjee reported that the adverse effects of gutkha on hematological parameters are not less than smoking (Mukherjee & Chatterjee, 2013).

Nicotine present in tobacco might affect the suprarenal glands to release more catecholamine, which may influence leukocytosis causing damage and inflammation to tissues (Ewing & Clarke, 1982). In the present study, WBC count was significantly increased in tobacco chewers as compared to controls. A similar significant increase in WBC count was also seen in previous studies (Jaganmohan et al., 2011; Saeed et al., 2005).

The granulocytes, mainly neutrophils %, significantly increased in the present study, may be linked with continuous inflammation of tissues. Neutrophils are well known to produce cytotoxic material which harmful to lung functions (Carel, Tockman, & Baser, 1988; Gillum, 1991).

In our study, monocytes % was significantly decreased in tobacco chewers than controls; this variation of monocytes % might be due to placid adverse effects on lungs (Jensen, Pedersen, Frederiksen, & Dahl, 1998).

However, lymphocytes % did not show any significant change in tobacco chewers in the present study. According to previous studies, inadequate pulmonary function in tobacco and gutkha chewers might be responsible for stimulating erythropoiesis for fulfilling the demands of oxygen to the tissues (Shahla, Tahmina, Stuti, Rastogi, & Joyita, 2007).

Blood indices such as MCV, MCHC, and MCH relate to individual RBCs as well as indicators of anemia. A lowered level of MCV related to iron deficiency anemia and increased levels are related to vitamin deficiency anemia (Kalburgi, Koregol, Muley, Warad, & Patil, 2013).

In the present study, the MCV level (p=0.04) was significantly increased in tobacco chewers as compared to controls. A similar increase in the MCV level was found in the previous study (Biswas, Manna, & Das, 2015).

However, after VC supplementation, the MCV level was decreased. It suggested that tobacco chewers had vitamin deficiency due to nicotine-induced oxidative stress. Whereas another study reported a significant decrease in the level of MCV in tobacco users (Dass et al., 2013; Shukla, Khaitan, Gupta, & Naik, 2019).

In the present study, MCH and MCHC level did not show a significant change in tobacco chewers as compared to controls. However, some previous studies reported that the MCH level was found to be significantly increased in their studies (Biswas et al., 2015; Dass et al., 2013; Shukla et al., 2019).

We studied the effect of VC supplementation in controls as well as in tobacco chewers independently for hematological parameters (Table 3).

In the control group, WBC count (p=0.01) significantly decreased and Hb content (p<0.003) was significantly increased after supplementation of VC as compared to their baseline values.

Whereas, in tobacco chewers group WBC count (p<0.001) and granulocytes % (p<0.0001) were significantly decreased and lymphocytes % (p=0.008), monocytes % (p<0.0001), RBC count (p=0.01) and Hb content (p=0.006) were significantly increased after supplementation of VC as compared to baseline values.

Vitamin C enhances the absorption of iron which is helpful to improve hemoglobin level in girls (Tutu & Rohimah, 2014). Vitamin C involves a hydrogen transfer rather than electron transfer and neutralizes harmful free radicals and reverted hemotoxicity (Ashraf, Abdel-Razik, Farrag, & Hany, 2017).

In the previous study, Hounkpatin reported the protective effect of VC supplementation on deranged hematological parameters in Wistar rats (Hounkpatin et al., 2012).

Ibitoroko reported that hemotoxicity caused by gasoline was reversed by supplementation VC in Albino rats (George & Adegoke, 2011), the similar result reported in rabbits (Ovuru & Ekweozor, 2004), in Herring gulls and Atlantic puffins (Leighton et al., 1985), in African catfish (Sunmonu & Oloyede, 2008) and in Wistar rats (Akaninwor & Okeke, 2007).

Soronnadi reported that platelet count was significantly decreased after supplementation of VC (Soronnadi et al., 2013). This suggested that oral VC supplementation has beneficial effects on haemostatic dysfunction in chronic tobacco users.

Conclusion

ST has adverse effects on hematological parameters such as an increase in WBC count, granulocytes %, HCT and MCV and decreases monocytes % and platelets count whereas, insignificant changes were observed in lymphocytes %, RBC count, Hb, MCH and MCHC. Increased tobacco chewing duration produce increased adverse effects on health. In addition, it was concluded that long term supplementation with 1 g/day of vitamin C may help in improving deranged hematological parameters in ST chewers.

Funding Support

The authors would be like to thank Krishna Institute of Medical Sciences, “Deemed to be Uiversity," for funding the research.

Conflict of Interest

The authors declare that they have no conflict of interest.