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Correlation of the activities of antioxidant enzymes superoxide dismutase and glutathione reductase with micronuclei counts among areca nut chewers of Manipuri population using exfoliative cytology: A preliminary study


1 Department of Oral Pathology and Microbiology, Dental College, RIMS, Imphal, Manipur, India
2 Department of Biochemistry, Manipur University, Imphal, Manipur, India
3 Department of Conservative Dentistry and Endodontics, Dental College, RIMS, Imphal, Manipur, India

Date of Submission23-Aug-2020
Date of Decision03-Oct-2020
Date of Acceptance12-Jan-2021
Date of Web Publication17-Jul-2021

Correspondence Address:
Ngairangbam Sanjeeta,
Department of Oral Pathology & Microbiology, Dental College, RIMS, Imphal - 795004, Manipur
India
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/jcrt.JCRT_1227_20

 > Abstract 


Context: Areca nut (AN) is a potent cytotoxic and genotoxic agent. Oxidative stress-induced by chewing of AN can cause DNA damage leading to nuclear anomalies such as micronuclei (MN) and also alters antioxidant defense mechanisms, leading to genomic instabilities and the development of oral cancer.
Aims: The aim of this study is to study the correlation between the levels of glutathione reductase (GR) and superoxide dismutase (SOD) in exfoliated buccal mucosal cells and the genotoxicity levels (MN count) in chronic AN chewers.
Materials and Methods: The present study was conducted with the approval of the Research Ethics Board in 60 individuals; 40 cases (Group I–20 raw AN chewers, Group II–20 dried areca with tobacco chewers), and 20 controls as Group III in the age group of 18–68 years who attended the outpatient department of our college. Estimation of SOD and GR and MN assessment was done using buccal exfoliated cells.
Statistical Analysis Used: Statistical analysis was performed using the Student's t-test and one-way ANOVA.
Results: Antioxidant levels were found to be significantly reduced in both Group I and Group II in comparison to the control group. Group II showed significantly reduced level of GR in comparison to Group I. The MN count was significantly increased in Group II in comparison to Group I. The MN counts showed an inverse correlation to the activities of the antioxidant enzymes. Greater activities of antioxidant enzymes correlated with decreased MN counts.
Conclusions: Detection of MN in AN chewers with or without tobacco can be a useful biomarker for clinical screening procedures that may be used as a risk marker for oral cancer. It is important to increase the awareness programs to educate the public about the deleterious effects of AN chewing, emphasize on early intervention of AN chewing habit and thus prevent the development of oral cancer.

Keywords: Antioxidant, areca nut, exfoliative cytology, micronuclei, tobacco



How to cite this URL:
Sanjeeta N, Banerjee S, Mukherjee S, Devi T P, Nandini D B, Aparnadevi P. Correlation of the activities of antioxidant enzymes superoxide dismutase and glutathione reductase with micronuclei counts among areca nut chewers of Manipuri population using exfoliative cytology: A preliminary study. J Can Res Ther [Epub ahead of print] [cited 2021 Jul 29]. Available from: https://www.cancerjournal.net/preprintarticle.asp?id=321711




 > Introduction Top


Areca nut (AN) is chewed by about 10% of the world's population.[1],[2] India has the largest betel-quid consuming population in the world.[3] Chewing of AN is deeply rooted in the tradition and age-old cultural practices of the people of Manipur. AN is a potent cytotoxic and genotoxic agent that has also been classified as carcinogenic to humans by the International Agency for Research on Cancer.[4] Reactive-oxygen species (ROS) such as superoxide anion, hydroxyl radical and hydrogen peroxide, highly toxic to cells, are formed in the body when the body is exposed to cytotoxic and genotoxic agents such as carcinogens. Substantial amounts of ROS are generated in the oral cavity during the chewing of AN. Thereby, oxidative damage to DNA of buccal mucosal cells is observed in AN chewers which indicates genetic damage in oral mucosal cells.[5] ROS that are generated in the body are efficiently taken care of by the highly powerful antioxidant system of the cell without any untoward effect. The antioxidant system includes the cellular antioxidant enzymes and the free radical scavengers that normally protect a cell from the toxic effects of the ROS.[6] Antioxidant enzymes such as superoxide dismutase (SOD) and glutathione reductase (GR) can directly counterbalance the oxidant attack and protect the cells against DNA damage.[7] SOD is a decisive antioxidant enzyme in aerobic cells, which is responsible for the elimination of superoxide radicals. SOD converts two toxic species: Superoxide O2 and hydrogen peroxide into water. This diminishes the toxic effects of superoxide radical and other radicals formed by secondary reactions. Glutathione prevents oxidative stress in humans by scavenging singlet oxygen, hydroxy radicals, and electrophiles. The ratio of oxidized glutathione (GSSG) and reduced glutathione (GSH) in a cell helps in maintaining oxidative balance. This oxidative balance is mediated by the enzyme GR which catalyzes the reduction of GSSG to GSH.

The present study is novel because it is an attempt to estimate the levels of the antioxidant enzymes SOD and GR in exfoliated buccal mucosal cells of chronic raw AN chewers and dried AN with tobacco chewers, whereas majority of the studies till date has been carried out on blood and other body fluids and tissues.

Our study aimed to (i) Estimate biochemically the levels of antioxidants such as GR and SOD in chronic chewers of AN with or without tobacco, (ii) Estimate AN genotoxicity by assessing the number of micronuclei (MN) in users of AN with or without tobacco, and (iii) Examine if there is any correlation between dietary antioxidants and MN count.


 > Materials and Methods Top


Before commencing the study, an ethical clearance was obtained from the Research Ethics Board of our Institute. A total of 60 individuals reporting to the outpatient department of our institute participated in the study. They belonged to the same ethnic background residing in Manipur state in the north-eastern region of India. All the participants were selected from those attending the department of oral medicine and radiology of our institute after subjecting them to a selection process that included a questionnaire-based interview about the details of AN consumption, diet and dietary supplements, medical history, etc. Informed consents were obtained from the participants. The participants were aged between 18 and 68 years. They were divided into three groups of 20 each: Group I – Indigenous individuals who chewed raw AN, lime, betel leaf without tobacco for not <2 years, Group II – Indigenous individuals who chewed dried AN, lime, betel leaf with tobacco for not <2 years, and Group III – control group comprising of healthy indigenous individuals who did not have any AN chewing habit. Individuals who are on medication or habits that may alter the level of antioxidant enzymes and MN count, with visible oral mucosal lesions, those suffering from systemic diseases such as diabetes, hypertension, cardiovascular diseases, renal dysfunction, or liver disorders or with history of treatment for the same that may alter the antioxidant enzyme levels and MN count were excluded from the study.

Chemicals/materials

All the chemicals used in this study were purchased from Sigma Aldrich (Sigma-Aldrich, Kolkata, West Bengal, India.) and Qualigens (ThermoFisher Scientific), Kolkata, West Bengal, India.

Collection of oral mucosal cells

The exfoliated buccal mucosal cells were collected by gently scraping the buccal mucosa at the site of AN placement by using a moistened wooden spatula, and the cells were collected in sterile vials containing 0.9% normal saline for the biochemical analysis. For the assessment of MN count, the exfoliated cells were evenly spread on clean glass slides and air dried.

Giemsa staining

The slides were fixed in 100% ethanol, stained with working Giemsa solution (Himedia) for 10 min and observed under the light microscope under ×40 magnification. For scoring of MN, 1000 cells were counted using ×100 magnification. For identification and scoring of MN, the proposed method of Tolbert was followed.[8] As per Tolbert's description, MN should have smooth perimeter which suggests the presence of the membrane. MN should have same intensity and texture as the nucleus and should be located in the cytoplasm in the same focal plane as the nucleus. The diameter of MN should be 1/3–1/6th of nucleus. The buccal mucosal squames among the controls showed no MN [Figure 1]a while buccal mucosal smears from the cases revealed the presence of MN around the nucleus [Figure 1]b.
Figure 1: (a) Image showing buccal mucosal squames taken from controls with no micronuclei. (b) Image showing buccal mucosal squames from the cases that revealed presence of micronuclei around the nucleus

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Preparation of cell lysate

Cells were washed with 0.9% saline thrice to maintain the isotonicity. After each wash, the cells were centrifuged at 3000 rpm for 10 min. The supernatant was discarded, and pellet was incubated with ice cold distilled water at 4°C for 1 h to enable cold osmolysis of cells. The cell lysate was subjected to biochemical activity assays.

Glutathione reductase activity assay

GR activity assay was performed as described by Smith et al., in 1988[9] with modifications. Herein, 0.5 ml cell lysate was mixed with 0.5 ml of 5% Trichloroacetic acid (TCA) to precipitate the protein. After precipitation of proteins, the solution was centrifuged at 3000 rpm for 10 min. The supernatant was collected, and the pellet was discarded. Then, 0.5 ml of supernatant was mixed with 2 ml of 0.2 M Phosphate buffer (pH 8.0). The mix was incubated with 0.5 ml of 0.6 mM 5-5'-dithiobis (2-nitro benzoic acid) (DTNB, Ellman's reagent) in 0.2 M phosphate buffer at the room temperature for 5 min. For blank measurements, DTNB solution was incubated in the similar condition with 5% TCA instead of cell lysate. A deep yellow-colored product developed in the study samples due to the formation of 5-thionitrobenzoic acid. The absorbance was recorded at 412 nm in a ultraviolet-visible (UV-VIS) spectrophotometer, and the amount of reduced glutathione in cell lysate was deduced by Beer Lambert law using L-Glutathione as standard.

Superoxide dismutase activity assay

SOD activity assay was performed as described by Marklund et al,[10] with some modifications. Briefly, 0.5 ml cell lysate of each study groups was mixed with 0.25 ml of absolute ethanol and 0.15 ml of chloroform for membrane denaturation and protein extraction. After 15 min of shaking in an orbital shaker at 200 rpm, the suspension was centrifuged for 10 min at 3000 rpm. The supernatant containing the protein fraction including SOD was collected, and the pellet was discarded. For study samples, 0.5 ml supernatant was mixed with 2 ml of 0.05 M Tris-(pH 7.4), 0.5 ml pyrogallol (2 mM), and 1 ml of water. To monitor pyrogallol autoxidation, control sets were prepared. 0.5 ml of 2 mM pyrogallol was added to 2 ml of 0.05 M and 0.1 M Tris HCl (pH 7.4 and 8.2, respectively) and 1.5 ml distilled water. Autooxidation rate of pyrogallol increases with increase in alkalinity of the solution and is indicated by the linear increase in A420 nm per min as the absorbance was recorded for 3 min in a UV VIS spectrophotometer in both sets of control tubes. In the presence of SOD in sample tubes at pH 7.4, pyrogallol autooxidation is prevented as the change in absorbance is recorded for 3 min. 1 unit of SOD enzyme activity is defined as the amount of enzyme that inhibited autooxidation of pyrogallol by 50%. Activity of SOD is estimated as units/ml of cell lysate.

Statistical analysis

Descriptive statistics were calculated by using the SPSS software version 20. One-way ANOVA test was applied to compare the mean values among the three study groups for selected variables. P < 0.05 was taken as statistically significant.


 > Results Top


The demographic details of the study participants are shown in [Table 1]. The mean duration of the chewing habit for those who chew raw AN without tobacco was 14.5 ± 2.4 years for Group I and 7.4 ± 0.8 years for Group II.
Table 1: Characteristics of the study population

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Antioxidant levels were significantly reduced, and MN counts were increased in both Group I and Group II individuals in comparison to the control group. Group II (those who chew dried AN with lime and tobacco showed significantly reduced level of GR in comparison to Group I (those who chew raw AN with lime without tobacco). The MN count was significantly increased in Group II in comparison to Group I [Table 2], [Table 3], [Table 4], [Table 5].
Table 2: Comparison of superoxide dismutase activity, glutathione reductase activity, and micronuclei counts between Groups I and III

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Table 3: Comparison of superoxide dismutase activity, glutathione reductase activity, and micronuclei counts between Groups II and III

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Table 4: Comparison of superoxide dismutase activity, glutathione reductase activity, and micronuclei counts between Groups I and II

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Table 5: One-way ANOVA test comparing superoxide dismutase assay, glutathione reductase assay, and micronuclei counts among the study groups

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Both antioxidants showed decreased activity in Group I and Group II compared to Group III individuals. Mean activity of SOD was 0.0163 ± 0.00059, 0.0141 ± 0.00119, and 0.0263 ± 0.001139 (Units/ml) in Group I, Group II, and Group III, respectively [Table 1]. For GR, the mean enzyme activities were 0.01745 ± 0.000749, 0.01035 ± 0.000629, and 0.02165 ± 0.001067 (Units/ml) in Group I, Group II, and Group III, respectively [Table 1].

[Table 2] shows the comparison of activities of enzymes SOD assay, GR assay, and MN counts between Group I and III that were analyzed by the t-test. Antioxidant activities of the enzymes SOD (0.0263 ± 0.001139) and GR (0.0263 ± 0.001139) were higher in Group III (Control group) compared to the enzyme activities in Group I (raw AN without tobacco chewers) with reduced activity of the antioxidants SOD (0.01745 ± 0.000749) and GR (0.01745 ± 0.000749). This indicates that the activity of antioxidants is reduced in Group I individuals in comparison to Group III individuals. However, MN counts were increased in Group I individuals (1.6 ± 0.169) compared to control Group III (0.95 ± 0.088) indicating genotoxicity in the individuals who chew raw AN without tobacco (Group I individuals).

[Table 3] shows the comparison of activities of enzymes SOD Assay, GR Assay and MN counts between Group II and III as revealed by the t-test. Antioxidant activities of the enzymes SOD (0.0141 ± 0.00119) and GR (0.01035 ± 0.000629) were reduced in Group II (dried AN with tobacco chewers) compared to the enzyme activities in Group III (control group) showing increased activity of the antioxidants SOD (0.0263 ± 0.001139) and GR (0.02165 ± 0.001067). This indicates the activity of antioxidants is reduced in Group II individuals who have the habit of chewing dried AN with tobacco in comparison to Group III control individuals who do not have such chewing habits. However, MN counts were significantly increased in Group II individuals (2.2 ± 0.258) compared to control Group III (0.95 ± 0.088) indicating genotoxicity in individuals who chew dried AN with tobacco (Group II individuals).

[Table 4] compares the activities of enzymes SOD, GR, and MN counts between Group I (raw AN without tobacco chewers) and Group II (dried AN with tobacco chewers). Antioxidant activities of the enzymes SOD (0.0163 ± 0.00059) and GR (0.01745 ± 0.000749) were higher in Group I compared to the enzyme activities of the antioxidants SOD (0.0141 ± 0.00119) and GR (0.01035 ± 0.000629) in Group II individuals. The activity of antioxidant enzyme GR is significantly reduced (P = 0.000) in Group II individuals who have the habit of chewing dried AN with tobacco in comparison to Group I individuals who chew raw AN without tobacco. MN counts were significantly increased in Group II individuals (2.2 ± 0.258) compared to Group I (1.6 ± 0.169) indicating greater genotoxicity in individuals who chew dried AN with tobacco (Group II individuals). [Table 2], [Table 3], [Table 4] shows increase in MN count. [Table 5] shows the comparison between SOD assay, GR assay, and MN count among the study groups which was found to be statistically significant (P = 0.000).


 > Discussion Top


ROS are formed in the oral cavity due to chewing of AN. Antioxidants that are formed in the body counteract the oxidative stress due to these ROS. The activities of first-line defense antioxidant enzymes SOD and GR have been estimated in the serum and saliva of smokers and tobacco chewers and in various pathological states to study the effects on the antioxidant enzymes as a result of the oxidative stress.

Endogenous antioxidant enzyme level measurement is of great importance as it may indicate future health.[11] In the present study, we have used exfoliative cytology, a noninvasive procedure to study the antioxidant status of buccal exfoliated cells in AN chewers. To the best of our knowledge, no similar studies have been carried out in AN chewer, although Gururaj et al., in 2004, have estimated intracellular antioxidant levels in smokers and nonsmokers using exfoliative cytology.[11] Our study showed reduction in the activity of both the antioxidants enzymes SOD and GR in the habit groups when compared with the control group.

Gurudath et al., in 2012, also found significantly reduced antioxidant enzymes in smokers when compared to nonsmokers.[7] Oxidative stress could be induced due to tobacco-induced free radical formation. Similar results were also reported by Hemalatha et al., Khanna et al., and Patel et al., where it is clearly shown that the use of tobacco suppressed the activity of antioxidant enzymes.[12],[13],[14] Other studies also reported reduced antioxidant enzyme levels in various pathological conditions. Uikey et al., in 2012, reported decreased serum levels of antioxidant enzymes in oral submucous fibrosis (OSMF) patients which was associated with carcinoma development in OSMF.[15]

Gurudath et al. observed decreased erythrocyte SOD levels in OSF, oral leukoplakia, and oral cancer patients when compared to control patients.[7] There could be a possible association between reduction in the levels of antioxidant enzymes with neoplastic progression as a gradual reduction of antioxidant enzymes from normal mucosa to precancerous lesion to carcinoma is observed. In our study, the activity of SOD between the groups and controls is statistically significant. SOD activity in both AN chewing groups is lower than control non-AN chewing group because its activity could be suppressed by the AN-specific nitrosamines (ASNAs) and ROS that is formed during the chewing of AN thereby disbalancing the antioxidant activity in the cell.

In the present study, the level of SOD in dried AN with tobacco chewers is slightly lower than raw AN without tobacco chewers which could be because they may be more prone to oxidative stress due to the increasing load of free radicals in the body due to the chemical carcinogens, Tobacco-specific nitrosamines (TSNAs) present in tobacco in addition to ASNAs. Level of GR in raw AN without tobacco chewers was found to be greater than dried AN with tobacco chewers though the level in both cases group was lesser than the control group.

Decreased activity of GR in dried AN with tobacco chewers could be due to reduced cellular antioxidant glutathione. Chewing of AN with tobacco results in high exposure to carcinogenic TSNAs and carcinogens as well as the generation of ROS that could cause increased oxidative stress with reduced detoxification that can cause DNA damage.[4] Due to the added tobacco in addition to the AN, there is the formation of more ROS that may overtake or overwhelm the antioxidant protective effects thus producing more oxidative damages and stress. Greater levels of GR in raw AN chewers could be explained by the fact that cellular antioxidant production is not suppressed in these chewers resulting in increased detoxification of ROS that are formed due to chewing of AN. The activity of the enzyme GR is dependent on the constant availability of reduced glutathione. Increasing dietary intake of GSH-rich foods or dietary supplements of GSH may have chemopreventive potential to reduce AN-associated oral lesions.[16] In addition, raw AN chewers are also not exposed to the harmful effects of tobacco as these chewers do not chew tobacco. Increased activity of GR in raw AN chewers could be due to the consumption of glutathione/anti-oxidant-rich food that the local indigenous people take in their daily diet. All the participants comprising the study group did not give any history of intake of antioxidants as dietary supplements. Whether the increased activity of GR that we found in raw AN chewers can be attributed to the natural/traditional diet of the indigenous population needs to be investigated. This traditional diet includes raw leaves, young inflorescences, tender stalks, and other plant parts which can act as medicine/nutrient supplement in the diet since time immemorial.[17] The local diet is also rich in phyto-chemicals besides being a rich source of Vitamin C, Vitamin K, Vitamin E, carotenoids, folate, magnesium, selenium, potassium, zinc, fiber, etc.,[17] GSH (at a concentration of 1.95 and 2.6 mmol/L) and cysteine (at a concentration of 4 and 8 mmol/L) have been reported to prevent arecoline-induced cytotoxicity.[16]

In our study, MN count was higher in Groups I and II in comparison to the control group (Group III). The MN count in exfoliated oral mucosal cells of chewers of raw AN without tobacco was 1.6 ± 0.169, that of dried AN with tobacco chewers, 2.2 ± 0.258 and the control group showed 0.95 ± 0.088. Dried AN with tobacco chewers showed significantly increased MN count in comparison to chewers of raw AN without tobacco which was similarly reported by Stich et al. in 1982.[18] Our results are supported by several other studies where increased MN count were also seen in the exfoliated buccal mucosal cells of AN chewers as compared to healthy individuals.[19],[20],[21],[22],[23],[24],[25],[26] The reduction in activities in antioxidant enzymes SOD and GR in Group I and Group II in our study correlates with the genotoxicity levels found in these habit groups as we observed increased MN count in the AN chewers compared to the control group. Similarly, increased activities of both SOD and GR were observed in the nonchewers groups which also showed lower MN count.


 > Conclusions Top


The activity of antioxidant enzymes SOD and GR was significantly reduced in cases group in comparison to the control group. AN with tobacco chewers showed significantly reduced level of GR in comparison to raw AN without tobacco chewers. The MN count was found to be significantly increased in individuals chewing AN with tobacco compared to individuals chewing raw AN without tobacco. The MN count showed significant correlation with the antioxidant activities as observed in the different groups. The MN counts showed an inverse correlation to the activities of the antioxidant enzymes. Greater activities of antioxidant enzymes correlated with decreased MN counts. Detection of MN in AN chewers with or without tobacco can be a useful biomarker for clinical screening procedures that may prove to be an effective risk marker for oral cancer. In addition, the benefits of an antioxidant rich diet can be highlighted for early prevention of oral cancer. It is important to increase awareness programs to educate the public about the deleterious effects of AN chewing, emphasize on early intervention of AN chewing habit and thus prevent the development of oral cancer. The results of this preliminary study are to be validated with a larger sample size.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
 > References Top

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[PUBMED]  [Full text]  


    Figures

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  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5]



 

 
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