Study of Glutathione S Transferases and Malondialdehyde Levels in Male Smokers from Vidharbha Region, India

Introduction: Free radicals are compounds having two unpaired electrons. Free radicals oxidize macromolecule in the body, including proteins, nucleic acids, and lipids. The body uses antioxidants to fight free radicals. Antioxidants may come from the diet or be generated internally (endogenous). Endogenous antioxidants include superoxide dismutase, glutathione (GSH), catalase, and glutathione peroxidase. Aim: To study of glutathione s transferases and malondialdehyde (MDA) levels in male smokers from Vidharbha region attending Datta Meghe Medical College Materials and Methods: A minimum sample size of 80 was used with 40 smoking males and 40 non-smoking males. This was a cross-sectional analytical observational analysis conducted in the Original Research Article Ambad et al.; JPRI, 33(36B): 167-172, 2021; Article no.JPRI.71077 168 medical study. Result: The mean GSH concentrations of the smoking and non-smoking research participants were 1.69 ± 0.97 mol/L and 2.20 ± 1.40 mol/L, respectively, when plasma GSH concentrations were measured. Smokers had lower plasma GSH concentrations than non-smokers, but the disparity was not statistically significant (p=0.1885) suggests that smoking has no discernible effect on GSH levels. Conclusion: Smoking tends to reduce plasma GSH while rising plasma MDA levels in men. Owing to a loss of antioxidant potential and a higher oxidative load, smokers are more susceptible to oxidant stress. Young smokers, in particular, should quit smoking as soon as possible before developing health complications in order to reap the full benefits of quitting.


INTRODUCTION
Free radicals are compounds having two unpaired electrons form. Free radicals oxidize any macromolecule in the body, including proteins, nucleic acids, and lipids. Malondialdehyde (MDA) and deoxyguanosine P are carbonyl compounds formed when proteins, nucleic acids, and lipids are oxidized by free radicals. [1,2] Our body uses antioxidants to remove free radicals. Antioxidants may be endogenous. Endogenous antioxidants include superoxide dismutase, glutathione, catalase, and glutathione peroxidase. GSH is one antioxidant that is often examined to see how a rise in free radicals affects the body. Glutathione has the molecular formula C10 H17 N3 O6 S and its molecular weight is 307.3235 g/mol. Glutathione is an antioxidant that is made up of the amino acids glutamate, cysteine, and glycine. Glutathione can be found in any cell in the body, but the liver has the highest concentration. The cysteine thiol (SH) group is responsible for glutathione's biological activity and acts as a proton donor. Toxic agents are introduced into the body by smoking. Inhaled cigarette smoke contains toxic chemicals that are harmful to one's skin. Smoking remains a public health concern worldwide, especially in developed countries like Indonesia. Toxic compounds in tobacco smoke, such as tar, nicotine, lead, and carbon monoxide, are considered to be harmful to one's health. The use of smoking raises blood lead levels. [5] Furthermore, smoking can impair lung capacity and increase the risk of chronic obstructive pulmonary disease and skeletal muscle weakness over time. A research on laboratory animals found that tobacco smoke intensified the death of pulmonary alveolar cells. Free radicals may be induced by the gaseous and particulate components of tobacco smoke. Free radicals were found in smokers' periodontal tissues, according to the findings of a report. Another study found that exposure to nicotine, whether electronic or traditional, would disrupt rat spermatogenesis by increasing free radical levels. Since free radicals are highly reactive molecules, they can readily react with other substances. A rise in oxidants (free radicals) and/or a reduction in antioxidants in the body is referred to as oxidative stress. The MDA concentration is one of the most commonly used markers to assess oxidative stress. MDA is the product of free radicals peroxiding lipids. A rise in MDA levels in the body means an increase in free radicals. Aside from MDA, glutathione is another marker that is commonly used to assess oxidative stress (GSH). GSH is an endogenous antioxidant that aids in the cleavage of free radicals. The level of GSH reduces under oxidative stress. In addition to MDA and GSH, oxidative stress markers include superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase, and carbonyl compounds. Smoking has been related to increased aging, cell death, and disease via the process of oxidative stress. There is a propensity for promoter of free radicals to rise and antioxidants to decrease in smokers. However, these experiments included people of all ages, from infants to the elderly. In a study by Koubaa et al.
[6] MDA concentrations were shown to be lower in smokers than in nonsmokers. In a study of thirty healthy adult subjects, no significant variations in GSH and MDA concentrations were seen in smokers (15 smokers and 15 non-smokers). Latest study on the relationship between smoking and MDA and GSH concentrations in smokers was agreed to be conducted due to the large number of smokers in Indonesia, especially among university students, and the conflicting findings of previous studies. [7] Since disturbances in glutathione synthesis and metabolism cause impaired glutathione activity as an antioxidant, glutathione levels in the body become a significant factor to consider. 8 The amount of malondialdehyde released as a result of lipid peroxidation is used to determine antioxidant activity. Malondialdehyde has the chemical formula C3H4O2 and is a volatile molecule. According to Suparno et al. 9 high levels of plasma MDA, an oxidative stress marker, may trigger DNA disorders. Thus the study aims to evaluate glutathione s transferases and malondialdehyde levels in male smokers from vidharbha region

MATERIALS AND METHODS
Study comprising randomly selected smokers (≤10 cigarette per day aged 25 to 49 yrs) The participants' willingness to participate in the study was recorded by signing informed consent that included their name, age, gender, smoking frequency, and daily cigarette consumption. This was a cross-sectional analytical observational analysis conducted in Dept of Biochemistry, Datta Meghe Medical College and Shalinitai Meghe Hospital and Research Center.
Study Period: September 2020 to January 2021.

Sample Collection
A 3-mL syringe was used to extract a 2 mL blood sample from each (non-fasting) study participant. The blood samples were centrifuged at 5000 rpm for 2 minutes to extract plasma, which was then collected at -20°C before being used to measure GSH and MDA levels. The Biochemistry and Molecular Biology Laboratories of Datta Meghe Medical College's Faculty of Medicine and Shalinitai Meghe Hospital and Research Centre. assessed plasma GSH and MDA concentrations. Using the Sigma GSH and MDA assay kits, the levels of plasma GSH and MDA is calculated biochemically. The Sigma GSH assay kit was used to biochemically test plasma GSH concentrations. GSH and MDA levels were assayed by readymade quality control assayed kits manufactured by M/S-chemicals, USA. Below are the criteria for the sampling.

 Inclusion criteria:
a. The participants in the survey were all smokers between the ages of 25 to 49. b. The only condition for participation was that the men were in good health.

 Exclusion Criteria:
a. Males who were sick or with a diagnosis of a metabolic disease were number one. b. Such as diabetes mellitus, heart disease, and kidney disease.

Results
Smoking survey participants had an average age of 20.0 ±2.03 years, a mean smoking length of 6.0 ± 1.0 years, and an average daily cigarette consumption of 10.0 ± 3.34 cigarettes. Table 1 shows the features of the subjects.
The mean GSH concentrations of the smoking and non-smoking study participants were 1.69 ± 0.97 mol/L and 2.20 ± 1.40 mol/L, respectively, when plasma GSH concentrations were measured (Table 2). Smokers had lower plasma GSH concentrations than non-smokers, but the disparity was statistically non-significant (p=0.1885). This suggests that smoking has no discernible effect on GSH levels. The study group had a mean MDA concentration of 2.80 ± 2.11 nmol/L, while the non-smoking study subjects had a mean MDA concentration of 1.46±1.28 nmol/L, according to the results. As a result, smokers had higher plasma MDA concentrations than non-smokers, but the disparity was not statistically important (P = 0.0200) ( Table 2).

DISCUSSION
The age group in study group were 25to 49 years old and had been smoking for an average of 5 years. In terms of average smoking length, it can be calculated that the research participants began smoking when they were 13to 17 years old, i.e. when they were teenagers (in junior or senior high school). The causes that cause these teenagers to start smoking at such a young age include social influence, self-actualization, a labile temperament, and a high level of curiosity. The results of this study matched those of Reimondos et al.
[10] and Subandrate et al. [11] reported that the average age of smoking initiation was 13to 15 years. Our smokers were habitual or routine smokers who smoked an average of nine cigarettes a day. This is in line with previous studies, which showed that smokers consumed an average of 10to 11 cigarettes a day, or one pack.
Toxic agents are introduced into the body by smoking. Over 2000 chemicals enter the lungs as gaseous or particulate matter as you smoke. Toxic chemicals in cigarette smoke, such as tar, nicotine, lead, carbon monoxide, nitrogen monoxide, benzopyrene, and nitrosamines, are metabolized in the liver and produce oxidants, or free radicals [12] Long-term and continuous smoking causes toxic compounds to accumulate in the bloodstream, resulting in an uptick in free radicals including hydroxyl and superoxide radicals. When free radicals increase, oxidant markers like MDA rise, whereas antioxidant markers, level of GSH, glutathione peroxidase, catalase, and superoxide dismutase diminished. [13] In this analysis, smokers had lower mean GSH concentrations than non-smokers, but the disparity was not statistically important.
According to the findings of Abdul-Rasheed et al.
[14] smokers' glutathione concentrations are not significantly lower than non-smokers' glutathione concentrations. In another analysis, however, GSH concentrations were observed to be lower in smokers as compare to non-smokers. Free radicals such as hydroxyl radicals, hydrogen peroxide, and superoxide anions may have increased as a result of the accumulation of cigarette smoke oxidants. Endogenous antioxidants like GSH become more competitive when free radicals increase, lowering their plasma concentrations. GSH is a tripeptide with a sulfhydryl group that can cleave free radicals. GSH may specifically quench free radicals or cleave hydroxyl radicals and superoxide anions by acting as a cofactor of glutathione peroxidase, glutathione transhydrogenase, and superoxide dismutase. Present study reported that smokers had higher mean MDA levels than non-smokers, but the difference was not statistically significant. In an analysis performed by Erguder et al. [15] similar non-significant findings were found, who reported no significant variation in serum MDA concentration between smokers and nonsmokers. In contrast, a survey of 22-25 year-old Indian students documented a significant increase in serum MDA concentration in smokers relative to non-smokers. This may have been possible due to oxidative stress induced by the build-up of toxic compounds and superoxide radicals caused by long-term and frequent tobacco use. MDA is the end product of a series of free radical-mediated lipid peroxidation reactions. MDA concentrations that rise mean that the cells are under oxidative stress. [16][17][18][19][20]

CONCLUSION
The smoking tends to reduce plasma GSH while rising plasma MDA levels in smokers. Owing to a loss of antioxidant potential and a higher oxidative load, smokers are more susceptible to oxidant stress. Young smokers, in particular, should quit smoking as soon as possible before developing health complications in order to reap the full benefits of quitting. Present study supports hypothesis that any form of tobacco is dangerous to health.

DISCLAIMER
The products used for this research are commonly and predominantly use products in our area of research and country. There is absolutely no conflict of interest between the authors and producers of the products because we do not intend to use these products as an avenue for any litigation but for the advancement of knowledge. Also, the research was not funded by the producing company rather it was funded by personal efforts of the authors.

CONSENT AND ETHICAL APPROVAL
As per international standard or university standard guideline participant consent and ethical approval has been collected and preserved by the authors.

ACKNOWLEDGEMENT
Author acknowledges the immense help received from the scholars whose articles are cited and included in references of this manuscript. The author is also grateful to authors / editors / publishers of all those articles, journals, and books from where the literature for this article has been reviewed and discussed. Author also acknowledges Department of Medicine, Datta Meghe Medical College, Nagpur.