Effect of Chronic Consumption of Smokeless Tobacco (Snuff) on Liver Enzymes of Males in Maiduguri, Northeast Nigeria 1 -

and ALP respectively Smokeless tobacco snuff consumption is as dangerous as cigarette smoking. It is now considered a significant source of morbidity and mortality owing to the effect of the numerous chemical constituents. Thus, this study was designed to investigate the effect of chronic consumption of Smokeless Tobacco (Snuff) on Liver Enzymes of males in Maiduguri and its environs, Northeast Nigeria. In this study we recruited 109 individuals who were smokeless tobacco snuffers and 97 healthy controls. It was a prospective case control study performed in adult males (30-50 years and 23 – 48 years for case and control respectively) with mean age of the case and control subjects of 37.12 ± 10.21and From the 33.64 ± 3.29 respectively. Serum Alanine amino results of the current study and the literature transferase (ALT), Serum Aspartate amino reviewed it is evident that tobacco snuff may transferase (AST) and serum Alkaline likely be one of the causes of several liver phosphatase (ALP) were estimated using kinetic diseases. Therefore, our study might be helpful methods by Cobas C311(Roche/Hitachi) in creating awareness on the hazards of using chemistry auto analyzer. The estimated Serum smokeless tobacco products (Snuff), among our AST, ALT, and ALP, levels were compared using population who are using smokeless tobacco. un-paired student's t test between the two groups. Also, the liver enzyme levels were evaluated on the duration of the smokeless tobacco consumption and correlation was determined.

Introduction 30 and 50 years and the mean age of the Case and Tobacco which is botanically known as control subjects were 37.12 ± 10.21and 33.64 ± Nicotiana tabacum is a perennial herbaceous 3.29 respectively. The mean(average) of the plant which is the most commonly grown of all duration of tobacco snuff intake was 10.52 ± 7.02 plants in the genius Nicotiana (Ugbor et al., years. 2013). More than 65 species of tobacco plant are known to exist. Tobacco is a general name for 15.45 ± 3.32, 22.00 ± 5.10 and 22.00 ± 5.10 any product prepared from the leaves of these respectively) as 10.42 ± plants. The plant is commercially grown in many 2.36, 8.88 ± 3.14 and 31.14 ± 4.60 for AST, ALT countries where the leaves are used to process the ) and the differences were statistically significant at p< 0.05. Also, the serum levels of the liver enzymes (AST, ALT, and ALP) were evaluated according to duration of snuff intake and the liver enzymes were found to be higher in people who uses the tobacco snuff for 11-20 years. However, the duration of the snuff intake was correlated with the serum levels of the liver enzymes. There was strong correlation between duration of snuff intake and serum levels of AST and ALT (r=0.648 and r=0.741 respectively) and the relationship were statistically significant at p<0.05. But the serum level of ALP was weekly correlated with the duration of snuff intake and the relationship was not statistically significant at p>0.05.
The mean Serum level of AST, ALT, and ALP of tobacco snuffers were found to be higher ( compared to controls ( production of tobacco products. The products are butanone), 4-methyl-nitrosamino)-4-(3prepared in different forms for different use such p y r i d y l ) -b u t a n a l ( N N A ) , a n d Nas for smoking as in cigarettes, cigars and pipes Nitrosonornicotine, heavy metals (Cadmium, and can also be consumed as in snuff, and Mercury etc.) and 23 polycyclic aromatic chewing tobacco (Rudgley and Richard, 2017).
hydrocarbons which has been implicated with Globally, non-communicable diseases such as tobacco associated cancers and diseases (Ugbor chronic respiratory disease, ischemic heart et al., 2013heart et al., , Jasmine et al., 2019. The major diseases, cancers, and diabetes are the leading ingredient in tobacco, alkaloid nicotine is causes of death and these diseases are found to be metabolized and detoxified in the liver. Nicotine associated with tobacco use (WHO, 2008 and inhibits antigen mediated signaling in T-cells and GATS, 2014). In middle-and low-income this block the proliferation and differentiation of countries, about 38 million people die each year lymphocytes and suppression of antibody from this disease (WHO, 2010). In India, about forming cells. This leads to increased production 274.9 million people use tobacco out of which of pro-inflammatory cytokines (IL-1, IL-6 and 163.7 million use only smokeless tobacco, 68.9 TNF-a) which are involved in liver cell injury million only smokers and 42.3 million users of (El-zayadi, 2006). Studies have shown that there both smoking and smokeless as per Global Adult is a decrease in antioxidants enzymes [ hepatic Tobacco Survey India (GATS) (GATS, 2014).
Glutathione (GSH), Glutathione peroxidase (GPx), Super oxide dismutase (SOD) and In Nigeria, tobacco snuff is prepared and catalase (CAT)] and increase lipid peroxidase produced locally from the tobacco leaves mixed (LPx) (Pramod, 2006). These factors lead to with other substances such as potash, black inflammation of the liver (Mitchell et al., 2013;pepper, alligator pepper, salt, ayaro, cloves and Velayutharaj et al., 2013). However, the effect in most products flavors and fragrances are depends on the amount and duration of added. The products are commercially available consumption of the smokeless snuff and differ according to the nature and (Velayutharaj et al., 2013). c o m p o n e n t s m i x t u r e s ( S h a a y a u a n d Mohammed, 2019). The consumption is on the However, there is paucity of data on the effect of increase in most developing countries including chronic consumption of smokeless tobacco Nigeria. The increase in the consumption and the (snuff) on liver enzymes in male consumers in availability of this product (Snuff) may be this area. Hence, it is imperative to assess the related to lack of legislation against its usage and liver enzymes of people who consume these therefore promoting its widespread marketing products. We believe that this study will help to making it available and affordable for the users.
increase awareness about harmful effect of the The increase in the consumption may also be toxic chemicals present in tobacco snuff related to some belief that these products have products of this area. some medicinal benefits such as cure of catarrh, headache, pile, and also energy booster and a Materials and Methods sexual enhancer thus causing potential addiction This was a prospective case control study. A total (Ugbor et al., 2013). Again, others use it as of 206 adult males were recruited for the study tradition in social gathering in unspecified out of which 109 were cases (Tobacco Snuff quantities without the knowledge of its potential Users) between the ages 30 and 50 years and 97 dangers to health (Musa et al., 2019). It is this were control between the age of 30 and 48 years. addiction and irrational use of these products that Controls were healthy subjects who have the may expose the users to numerous health risks. same dietary feeding habits and who had no Tobacco products contains more than 490 toxic previous history of using tobacco in any form. Test Principle informed to the paticipants in a language they AST understand and written informed written consent AST in the sample catalyzes the transfer of an w a s o b t a i n e d f r o m t h e p a r t i c i p a n t s .
amino group between L-aspartate and 2-Sociodemographic data was obtained using a oxoglutarate to form oxaloacetate and Lquestionnaire.
glutamate. The oxaloacetate then reacts with NADH, in the presence of malate dehydrogenase Blood sample collection (MDH), to form NAD+. Five milliliters (5 ml) of blood specimen was AST collected from each subject for biochemical L-Aspartate + 2-oxoglutarate oxaloacetate + analysis. The blood was collected aseptically L-glutamate from the antecubital vein using sterile disposable 5 ml syringe and transferred into an MDH + + appropriately labeled plain container and Oxaloacetate + NADH + H L-malate + NAD allowed to clot at room temperature. The samples were centrifuged at 4000 revolutions per minute The rate of the NADH oxidation is directly (rpm) for 5 minutes and serum separated into proportional to the catalytic AST activity. It is another appropriately labeled sample determined by measuring the decrease in containers(cryovial) and stored frozen until the absorbance. time of analysis.
ALT Anthropometry: ALT in the sample catalyzes the reaction Anthropometric measurements were also taken between transfer L-alanine and 2-oxoglutarate. for each subject. The rate of the NADH oxidation is directly alcohol in any form and those who are on proportional to the catalytic ALT activity. It is traditional medicine.
determined by measuring the decrease in absorbance.

Estimation of biochemical parameters
All biochemical analysis was conducted at ALP university of Maiduguri teaching hospital, Colorimetric assay in accordance with a standard Maiduguri, Borno state Nigeria.
method. In the presence of magnesium and Zinc ions, p-nitrophenyl phosphate is cleaved by The following biochemical parameters were phosphatases into phosphate and p-nitrophenol. e s t i m a t e d b a s e d o n e s t a b l i s h e d ALP spectrophotometric and automated procedures, p-nitrophenyl phosphate + H2O Phosphate + approved by the International Federation of p-nitrophenol Clinical Chemistry and Laboratory medicine (IFCC). Serum Alanine amino transferase The p-nitrophenol released is directly (ALT), Serum Aspartate amino transferase proportional to the catalytic ALP activity. It is (AST) and serum Alkaline phosphatase (ALP) determined by measuring the increase in were estimated using kinetic methods by Cobas absorbance. C311(Roche/Hitachi) chemistry auto analyzer.

Statistical analysis:
± 3.32, 22.00 ± 5.10 and 22.00 ± 5.10 All the data were expressed as Mean ± SD; respectively) as 10.42 ± unpaired Students t-test was used to compare the 2.36, 8.88 ± 3.14 and 31.14 ± 4.60 for AST, ALT data between the two groups of smokeless and ALP respectively tobacco Snuffers and healthy control. A p value <0.05 was statistically considered significant for all statistical tests. SPSS 20.0 package was used for all statistical analysis.

Results
Two hundred and seven subjects were recruited for this study. One hundred and nine (109) 52.7% were case (Tobacco snuffers) and ninety-seven (97) 46.9% were control. The mean age of the Case and control subjects were 37.12 ± 10.21and 33.64 ± 3.29 respectively. Other anthropometric variables are as indicated in table1.The mean(average) of the duration of tobacco snuff intake was 10.52 ± 7.02.

15.45
compared to controls ( ) and the differences were statistically significant at p< 0.05 as shown in table 2. Also, the serum levels of the liver enzymes (AST, ALT, and ALP) were evaluated according to duration of snuff intake as indicated in table3. The liver enzymes were found to be higher in people who uses the tobacco snuff for 11-20 years. However, the duration of the snuff intake was correlated with the serum levels of the liver enzymes. There was strong correlation between duration of snuff intake and serum levels of AST and ALT (r=0.648 and r=0.741 respectively) and the relationship were statistically significant at p<0.05. But the serum level of ALP was weakly correlated with the duration of snuff intake and the relationship was not statistically significant at p<0.05 as shown in The mean Serum level of AST, ALT, and ALP of table4. This relationship is evident from the tobacco snuffers were found to be higher ( scatterplots in figures 1, 2 and 3.     (Ugbor et al ,2013). This study butanone), 4-methyl-nitrosamino)-4-(3-also evaluated the liver enzymes according to the p y r i d y l ) -b u t a n a l ( N N A ) , a n d N -duration of the tobacco snuff intake. The enzyme Nitrosonornicotine, heavy metals (Cadmium, levels were found to be higher in people who Mercury etc.) and 23 polycyclic aromatic uses tobacco snuff for a period of 11-20years of hydrocarbons which has been implicated with intake. However, there was strong correlation tobacco associated cancers and diseases (Ugbor between duration of tobacco snuff intake and et al., 2013). serum levels of AST and ALT (r=0.648 and r=0.741 respectively) and the relationship were In this study we recruited 109 individuals who statistically significant at p<0.05. The serum were smokeless tobacco snuffers and 97 healthy level of ALP was weakly correlated (r=0.126) controls. It was a prospective case control study with the duration of tobacco snuff intake and performed in adult males between the ages 30 -there was no statistically significant relationship 50 years.
(p=0.072). This study therefore indicates that In this study we tried to assess the effect of tobacco snuff on the liver. We evaluated the mean serum liver enzyme levels of AST, ALT and ALP as a marker of liver damage in individuals who use tobacco snuff and compared with those of control individuals. The result showed higher levels of the liver enzymes in tobacco snuffers as Conclusions compared to the control subjects and there were From the results of the current study and the literature significant statistical differences at p<0.05. This reviewed it is evident that tobacco snuff may likely be finding was in agreement with the work of one of the causes of several liver diseases since its Velayutharaj Alwar et. al (2013) who reported production and consumption is growing rapidly and is similar finding in India. In a similar research alarmingly becoming prevalent in most part our conducted by Ugbor et.al (2013) in Enugu country, Nigeria. This may be due to lack of legislation Nigeria, the authors reported similar findings on against its usage and therefore making its widespread the liver enzymes AST, ALT and ALP. However, marketing, availability, affordability and perhaps may they reported that ALP shows no statistically also be due to the belief that it has some medicinal significant increase which is contrary to our effects with consequence of potential addiction and finding. In another research conducted by effect on health. Shaayau and Mohammed (2019) in Sokoto Nigeria, the researchers reported their findings in Therefore, our study might be helpful in creating Wister albino Rats similar to our findings. Our awareness on the hazards of using smokeless finding is also in agreement with the report of tobacco products (Snuff), among our population ) who reported that liver who are using smokeless tobacco Snuff. enzymes increase significantly at p<0.05 The high level of serum enzymes observed in our study may be due to the effect of Nicotine and other phytochemical constituents present in Tobacco snuff and which has hepatocellular toxicity. These chemicals are implicated in the Legislation against the use of this smokeless inhibition of antigen mediated signaling in T-tobacco snuff consumption similar to that of Cells blocking the proliferation and tobacco smoking should be established Al-Mukhaini et al (2017 tobacco snuff toxicity on liver and the observed changes were duration dependent. This is in agreement with report of Ugbor et al (2013) that potash-tobacco dust (local tobacco snuff) is toxic to the liver and the observed changes were dose and duration dependent.

Recommendations
Further Cohort study is suggested to evaluate the incidence rate of the effect of tobacco snuff consumption.