Negative impact of Gutkha on certain blood parameters of Swiss mice.
Children are using chewable tobacco products as their first step towards tobacco use (Zickler 2006). Gutkha forms a major category of smokeless tobacco in India and South-East Asia. It is a flavoured and sweetened dry mixture of areca nut, catechu, slaked lime, cardamom, menthol, fennel seeds and tobacco (Gupta et al 1992); Nigam et al 2001).
Unfortunately, both gutkha and pan masala have been strongly implicated in the incidence of Oral Sub-Mucous Fibrosis, a precancerous lesion (Gupta and Ray 2003, Nair et al 2004; WHO, 2006; Sami et. al. 2006). Long term harmful effects of gutkha have been fairly well established (Nigam et al 2001; Gupta 2001). We investigated the short term effects of Gutkha on the haematological parameters and liver function of Swiss mice. Based upon the study and a survey of the oral cancer patients in Patna, a community service on the topic 'Tobacco and Health Hazards' was organized for streetchildren to make them aware of the harmful effects of gutkha on health
MATERIALS AND METHODS
Gutkha packets were purchased from the market and ground to form powder. Small doses of 2% of the powder were prepared following Kumar et al. (2003). Eight pure bred swiss male mice of the weight 25 grams were selected from the college's animal house. They were kept in polypropylene cages with wire mesh and a water bottle. Bedding comprised of rice husk. The mice were housed in an ambient temperature of 25[degrees] C[+ or -] 2[degrees] C with 12 hours light dark cycle. Out of eight mice four were kept in 'control' category. The other four were fed with the 2% gutkha mixed with chapattis (made of whole grain wheat flour) once every morning for one month. Each of the treated mice were kept in separate cage to ensure that each of them consumed the full dose of the gutkha. All the eight mice were supplied with equal ration of food and it was seen that the entire food was consumed. The animals had free access to water. After 30 days all the mice were sacrificed one by one. They were anaesthesized with chloroform, dissected and their blood was collected by puncturing the heart in plastic vials containing EDTA. The blood samples were immediately sent to the laboratory for analysis.
The data were analyzed using 't' test for matched pairs.
RESULTS AND DISCUSSION
No mortality occurred during the study period. The mice did not show any abnormal behaviour. They consumed the complete ration of food supplied to them on daily basis for the experimental period (30 days). None of the organs of the treated mice upon dissection showed any abnormal growth.
The treated mice had significantly higher level of total leucocytes (t =3.394, d.f.=3, P<0.05), neutrophil (t =4.285, d.f.=3, P<0.05), monocyte (t =7.142, d.f.=3, P<0.01) and eosinophil (t =4.318, d.f.=3, P<0.05) as compared to the control ones (Table 1).
Leucocytes have a wide range of biological effects and increased leucocyte count shows signs of inflammation which may be responsible for carcinogenic effects or tissue damage. Nigam et al. (2001) have shown that long term exposure to pan masala with tobacco induces the formation of tumour in mice.
While the erythrocyte count (t =1.747, NS) and haemoglobin (t =1.737, NS) content did not vary significantly between the control and treated mice, there was significant rise in Packed Cell Volume (PCV) (t =3.564, d.f.=3, P<0.05), Mean Corpuscular Haemoglobin (MCH) (t =6.123, d.f.=3, P<0.01), Mean Corpuscular Haemoglobin Concentration (MCHC) (t =4.214, d.f.=3, P<0.05), Mean Corpuscular Volume (MCV) (t =4.621, d.f.=3, P<0.02) and platelet count (t =28.5, d.f.=3, P<0.01) in the treated mice as compared to the control ones (Table 2).
Increased value of PCV reflects problem in the bone marrow which starts manufacturing more RBCs in order to carry enough oxygen throughout the body. This is commonly associated with inadequate lung function. The results showed a fall in Haemoglobin level, but it was not statistically significant. However, there was a rise in MCV and MCH level suggesting occurrence of anaemia which may be associated with liver damage. Kilinc et al.(2004) also reported higher counts of PCV and MCV in humans using Maras powder, a form of smokeless tobacco. A study by Choudhary et al (1994) showed that 35% of tobacco chewers were affected with mouth ulcers and 46 % with anaemia.
Further a significant fall in the blood sugar level (t =4.389, d.f.=3, P<0.05) was noted in the treated mice as compared to the control one, while there was significant rise in the level of liver enzymes, Serum Glutamic Oxaloacetic Transaminase (SGOT) (t =12.247, d.f.=3, P<0.01) and Serum Glutamic Pyruvic Transaminase (SGPT) (t =4.074, d.f.=3, P<0.05) in the treated mice (Table 3). These enzymes are normally present in the liver cells and are released into the blood when the liver is damaged. Low blood sugar level may also result due to liver damage. Studies by Bagchi et al (1995); Nigam et al (2001) showed negative effects of tobacco on histopathology of lung, liver, kidney, testis, ovary, oesophagus and stomach. Avti et al (2006) found inflammatory effects of smokeless tobacco on liver and lungs.
The study indicates that gutkha may cause adverse effect on blood parameters and may have direct toxic effects on liver and other organs, even when it is consumed for a short period of time.
We are highly obliged to our college Principal, Dr. Sister Doris D' Souza for providing the necessary facilities to conduct the study.
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Shahla Yasmin *, Neha, Tahmina, Mamta, S. Stuti, Nidhi Rastogi and Joyita Das
Deptt. of Zoology, Patna Women's College, Bailey Road, Patna- 800 001, Bihar, India.
* Head, E-mail- email@example.com
Table 1 : Comparison of leucocyte counts in mice treated ones (duration of experiment = 30 days) Mice Groups Control (n = 4) Treated (n = 4) Total Leucocyte 10475 [+ or -] 143.61 11575 [+ or -] 268.87 * (per cu. mm) Neutrophil (%) 34 [+ or -] 1.68 42 [+ or -] 2.71 * Lymphocyte (%) 60.5 [+ or -] 0.5 58.75 [+ or -] 5.15 Monocyte (%) 2 [+ or -] 0 7 [+ or -] 0.71 *** Eosinophil (%) 2 [+ or -] 0.41 6.75 [+ or -] 0.75 * Basophil (%) 0 0 "t" test for matched pairs, * = P< 0.05, *** = P< 0.01 Table 2 : Comparison of erythrocyte parameters in mice treated with 2% gutkha versus control ones (duration of experiment = 30 days) Mice Groups Control (n = 4) Treated (n = 4) Total R.B.C. 3.23 [+ or -] 0.09 3.65 [+ or -] 2.23 (million per cu. mm) PCV (%) 49.5 [+ or -] 1.19 55.5 [+ or -] 1.19 * MCH (pg) 34.25 [+ or -] 0.85 39.25 [+ or -] 0.85 *** MCHC (%) 31.75 [+ or -] 0.85 38.9 [+ or -] 0.92 * MCV (cu. microns) 95.5 [+ or -] 0.5 101 [+ or -] 1.47 ** Haemoglobin (g) 13.25 [+ or -] 0.10 13.15 [+ or -] 1.14 Platelets (lacs/cu.mm.) 4.23 [+ or -] 0.13 5.0 [+ or -] 1.11 *** "t" test for matched pairs, * = P< 0.05, ** = P< 0.02, *** = P< 0.01 Table 3 : Comparison of blood sugar level and level of liver enzymes in mice treated with 2% gutkha versus control ones (duration of experiment = 30 days Mice Groups Control (n = 4) Blood Sugar (mg/dl) 69.5 [+ or -] 2.63 63 [+ or -] 1.22 * SGPT or ALT 50.0 [+ or -] 0.82 59.75 [+ or -] 1.65 * (Units/ml) SGOT or AST 49.0 [+ or -] 1.29 59.0 [+ or -] 0.58 *** (Units/ml) "t" test for matched pairs, * = P< 0.05, *** = P< 0.01