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ORIGINAL ARTICLE
Year : 2020  |  Volume : 9  |  Issue : 3  |  Page : 183-192

Effects of Areca nut extracts on the neutrophil functions in blood and saliva samples of subjects with normal oral mucosa with and without Areca nut habit: A comparative study


1 Department of Oral Pathology and Microbiology, Maratha Mandal's NGH Institute of Dental Sciences, Belagavi, Karnataka, India
2 Department of Microbiology, Maratha Mandal's NGH Institute of Dental Sciences, Belagavi, Karnataka, India

Date of Submission29-Jul-2020
Date of Acceptance25-Aug-2020
Date of Web Publication30-Sep-2020

Correspondence Address:
Dr. Vijayalakshmi S Kotrashetti
Professor and Head, Maratha Mandal's NGH Institute of Dental Sciences and Research Centre, Belagavi - 590 010, Karnataka
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/JDRNTRUHS.JDRNTRUHS_124_20

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  Abstract 


Background: Areca nut, a major component in areca quid possesses genotoxic and carcinogenic properties. Studies suggest that areca nut chewing is associated with a higher prevalence of oral submucous fibrosis (OSMF) which could be also due to the detrimental effects of areca nut extract on the host defense system.
Objectives: To determine the effects of Areca nut extracts (ANE) of ripe (r-ANE) and tender (t-ANE) on the functions of neutrophils derived from peripheral blood and saliva samples in apparently healthy individuals with and without areca nut chewing habit.
Materials and Methods: Sixty subjects with clinically normal-appearing oral mucosa without any systemic disorders were divided into two groups of 30 each consisting of subjects without any habits grouped under the control group while subjects with areca nut chewing habit were grouped under the study group. Blood and saliva samples were collected from both groups. Neutrophil functions in both blood and saliva were assessed by Nitroblue Tetrazolium assay (NBT) and Phagocytic assay (PHG) using various concentration of t-ANE and r-ANE extracts.
Results: In both the groups, with increasing concentrations of t-ANE and r-ANE there was a drop in mean scores for both assays. A comparison between the groups revealed higher mean values in the control group than the study group which was statistically significant (P < 0.05).
Conclusion: Areca nut affects the neutrophil function significantly in ripe form than tender form. Saliva can prove to be an effective alternative material for analyzing the neutrophil function and hence the immune status in individuals. This may alter the defensive role of the oral cavity, and this may be one possible mechanism by which areca nut compromises the oral health of users of areca nut products increasing the susceptibility for various pathologies including oral submucous fibrosis (OSMF).

Keywords: Blood, NBT Assay, Neutrophils, PHG Assay, r-ANE, Saliva, t-ANE


How to cite this article:
Chandrarekha V, Kotrashetti VS, Bhat KG, Babji D. Effects of Areca nut extracts on the neutrophil functions in blood and saliva samples of subjects with normal oral mucosa with and without Areca nut habit: A comparative study. J NTR Univ Health Sci 2020;9:183-92

How to cite this URL:
Chandrarekha V, Kotrashetti VS, Bhat KG, Babji D. Effects of Areca nut extracts on the neutrophil functions in blood and saliva samples of subjects with normal oral mucosa with and without Areca nut habit: A comparative study. J NTR Univ Health Sci [serial online] 2020 [cited 2020 Oct 24];9:183-92. Available from: https://www.jdrntruhs.org/text.asp?2020/9/3/183/296826




  Introduction Top


Areca-nut (AN) or betel quid/tobacco leaf chewing habits are widely prevalent in many parts of Asia and migrant communities. Many betel-quid products in different parts of the world are not actually chewed; rather, they are placed in the mouth or applied to the oral cavity and remain in contact with the oral mucosa. Nevertheless, it is recommended that they all be considered as part of the betel quid chewing habit. Betel quid (BQ) chewing is a popular habit in India and Southeast Asia which is highly associated with oral cancer and oral submucous fibrosis.[1] Areca nut is a seed of the fruit of Areca catechu an oriental palm. It has a characteristic astringent and slightly bitter taste. It is consumed at different stages of maturity according to preference.[2] Areca nut may be used fresh, dried, or cured before use, by sun-drying, baking, or roasting. The major constituents of the nut are carbohydrates, fats, proteins, crude fiber, polyphenols (flavonols and tannins), alkaloids, and mineral matter.[1],[2],[3] Variations in the concentrations of the various constituents may occur in nuts from different geographical locations and also according to the degree of maturity of the nut. Of the chemical ingredients, tannins, alkaloids, and some minerals have adverse effects on tissues that have been subjected to detailed study. Studies have revealed the genotoxic, carcinogenic, embryotoxic, and immunotoxic potentials of AN. In addition, ANE generates reactive oxygen species (ROS) and induce DNA damage in vitro.[1],[2],[4] The ROS generated from chewing BQ may cause oxidative damage. The ripe and fully grown AN without husk is used in BQ chewing in many countries, whereas fresh and tender areca nut with husk is consumed in Taiwan. Liu et al. reported that ripe ANE (r-ANE) is more cytotoxic than tender ANE (t-ANE) to Chinese hampster ovary (CHO-K1).[5] In addition, r-ANE induces higher oxidative DNA damage levels when compared with t-ANE.[1]

Studies have shown that there is an impairment of neutrophil functions in conditions like aggressive periodontitis and certain oral mucosal diseases.[2],[6],[7] Recent concept that has emerged during the last decade is that of a hyperactive or primed neutrophil that highlights the destructive aspect of neutrophil-mediated tissue damage concept. We are already aware of the immune role in the pathogenesis of certain pre-malignancies. This has opened up new avenues of research for the diagnosis of pre-malignant lesions and conditions or that of the possible changes that take place in the areca nut/tobacco habituates prior to the clinical presentation of the same. As neutrophils represent the first line of the host defense mechanism against microbial infection or foreign source.[8] Approximately one million neutrophils enter the oral cavity via the gingival crevicular fluid every minute which protects the oral mucosa against all insults through mechanisms such as secreting hydrolytic enzymes and microbial killing.[9],[10] Little is known about the influence of areca quid on the immune system.[11] The aim of the present study was to determine the effect of different concentrations of areca nut extract both t-ANE and r-ANE on the functions of human neutrophils derived from saliva and peripheral blood in apparently healthy individuals with and without areca nut chewing habits.


  Materials and Methods Top


The subjects for the study were recruited from Outpatient Department, of the Institute. The samples were analyzed at Department of Molecular Biology and Immunology of the Institute. The study was commenced after obtaining institutional ethical committee clearance. Ethical approval was obtained from the ethics committee and the date of approval 15/06/2013.

Healthy individuals who visited the outpatient department for routine oral examination and dental treatment were enrolled. The subjects were equally divided into two groups as follows:

Group 1: Healthy individuals who possessed good physical and mental health with the absence of any generalized disease/illness and who did not have any form of areca nut and tobacco-associated habits and presented with clinically appearing normal oral mucosa (NOM).

Group 2: Individuals without a history of any systemic/oral disease and absence of any detectable lesion in the oral mucosa and with any form of areca nut chewing habit (any variety of areca nut alone, areca nut coupled with slaked lime, areca nut with tobacco leaves) and duration of habit for minimum period of 2 yrs and above were considered.

In both the groups, individuals between the age group of 20–45 yrs were considered.

The medical records of these patients were reviewed and information concerning gender, age, and their areca nut habit were noted.

In both groups 1 and 2 individuals showing any abnormal changes in the oral mucosa such as white, red, or red and white oral lesions in the form of patch, plaque, and reticular areas, or if any atrophic or erosive areas were not considered.

Information on patient's habits (both groups) was sought by direct interviews of the subjects which included a history of areca nut chewing habit. In non-habituates group, all subjects were selected based on having no prior or current use of any form of areca nut.

Collection of samples

A total of 2 ml of Un-stimulated whole saliva and 5 ml of peripheral blood were collected under sterile conditions.

Method of extract preparation of r-ANE and t-ANE

Both Ripe and Tender Areca nut seeds were obtained. They were further processed to obtain the respective extracts using Soxhlet apparatus (Borosil Company Ltd). The extract solutions were further processed using rotary evaporation apparatus and were evaporated in the china dish in order to obtain dried or crude extracts. Later they were packed in air-tight containers and were stored at 4°C for further use. The aqueous extract solutions were prepared as and when required freshly and various dilutions of both r-ANE and t-ANE were prepared following the serial dilution method. The various dilutions prepared for t-ANE were 100, 200, 400, 800, 1600 μg/ml and for r-ANE were 12.5, 25, 50, 100, 200 μg/ml. The prepared various dilutions of aqueous extracts of r-ANE and t-ANE, a major alkaloid (Arecoline) and phenolic component (catechin) of areca nut were examined for their effects on the defensive functions of human neutrophils. Exposure of neutrophils derived from saliva and peripheral blood to r-ANE and t-ANE was done in a dose-dependent manner.

Isolation of neutrophils from blood and saliva

The blood sample was divided into two parts, one part of the whole blood was kept aside and the other part of whole blood was used to isolate the neutrophils. We preferred to isolate a relatively pure population of white blood cells with minimum manipulation of blood. The blood samples were collected in tubes containing EDTA as an anticoagulant. For separation of WBCs, the blood sample was diluted with saline and an equal amount of 3% gelatine was added to it. The tubes were made to stand upright for about 45 min till all the RBCs settle down. The supernatant was collected in a fresh tube, centrifuged and washed with saline and the WBCs were counted in Neubauer's chamber and then diluted with Hank's balanced salt solution (HBSS) to get a specific concentration. Similar procedure was done for the freshly collected saliva samples. The cell viability of isolated neutrophils from both blood and saliva samples was assessed by Trypan blue exclusion assay.[12],[13]

The defensive functions of the neutrophils were examined under the following two tests:

(a) NitroblueTetrazolium Test (NBT Assay) (b) Phagocytic assay (PHG assay)

For NBT assay whole blood was used and for PHG assay neutrophil isolate was used

PROCEDURE OF EACH ASSAY:

NBT Assay:

To 100 μl of anti-coagulated whole blood, 50 μl of 0.34% NBT was added and to the above mixture, 150 μl of HBSS was added. Twelve such tubes were prepared. Of them 10 tubes were considered as test where the different concentrations of t-ANE (100, 200, 400, 800, 1600 μg/ml) and r-ANE (12.5, 25, 50, 100, 200 μg/ml) were added. Of the remaining 2 tubes, 50 μl of endotoxin C prepared from E. coli was added to 1 tube which served as a positive control. Another tube was left untreated which served as the negative control. Then all the tubes were incubated at 37°C for 20 min and later kept at room temperature for 20 min. Later all tubes were mixed well and a thin film of smear on the glass slide was prepared, dried, and fixed with methanol that was subsequently stained with Giemsa stain and were studied under oil immersion using a light microscope.[12],[13] Similar procedure was followed for saliva using isolate neutrophils

Interpretation: Number of neutrophils stimulated (with formazan crystals) was calculated. For the tube containing endotoxin, more than 80% of phagocytic cells had reduced NBT and demonstrated as bluish-black granules in the cytoplasm. [Figure 1] and [Figure 2] The percentage of NBT reduced cells was calculated for all the samples collected with respect to individual concentration and type of ANE respectively.
Figure 1: Photomicrograph showing Stimulated neutrophils in the BLOOD smears under NBT Assay (Giemsa Stain, ×100)

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Figure 2: Photomicrograph showing Stimulated neutrophils in the SALIVA smears under NBT Assay (Giemsa Stain, ×100)

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PHG Assay:

In PHG assay, twelve tubes were prepared to which 100 μl of HBSS, 100 μl of leukocyte suspension, and 100 μl of heat-killed C. albicans were added. As a positive control, 100 μl of patient's serum was added to one tube. Another tube considered as negative control was left untreated. Remaining 10 tubes were all treated with 100 μl of different concentrations of both r-ANE and t-ANE, respectively. Later contents of all the tubes were mixed thoroughly and were incubated at 37°C for 30 min. Following which they were centrifuged and the smears were prepared from the deposit obtained. The smears prepared from the deposit were fixed with methanol, stained with Giemsa stain, and were subsequently observed under a light microscope.[12],[13] The number of candida ingested per cell were counted and expressed as mean particle number (MPN) [Figure 3] and [Figure 4]. Similar procedure was followed for salivary neutrophil isolates.
Figure 3: Photomicrograph showing ingested candida (6) by the neutrophils in the BLOOD smears under PHG Assay (Giemsa Stain, ×100)

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Figure 4: Photomicrograph showing ingested candida (6) by the neutrophils in the SALIVA smears under PHG Assay (Giemsa Stain, ×100)

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Statistical analysis

The obtained results of both NBT assay and PHG assay of blood and saliva samples were Compared and statistically analyzed using Student's t-test.


  Results Top


Out of 30 subjects in the control group, there were 22 males (73.3%) and 08 females (26.7%) while in the study group out of 30 subjects, there were 24 males (80%) and 06 females (20%), respectively. The average age of all the subjects in our study was 30 years.

NBT assay of blood sample

In the control group, the mean percentages of the NBT reduced cells of samples treated with various concentrations of t-ANE range from 32.10 ± 14.50 at a concentration of 100 mcg/ml to 17.07 ± 7.56 at concentration of 1600 mcg/ml and the mean percentages of NBT reduced cells of samples treated with various concentrations of r-ANE range from 20.70 ± 10.19 at concentration of 12.5 mcg/ml to 12.13 ± 6.79 at concentration of 200 mcg/ml.

In the study group, the mean percentages of the NBT reduced cells of samples treated with various concentrations of t-ANE range from 41.10 ± 16.46 at a concentration of 100 mcg/ml to 22.53 ± 11.61 at a concentration of 1600 mcg/ml and the mean percentages of NBT reduced cells of samples treated with various concentrations of r-ANE range from 28.80 ± 14.64 at a concentration of 12.5 mcg/ml to 16.40 ± 10.83 at concentration of 200 mcg/ml.

There was a reduction in the mean values with increasing concentrations of t-ANE and r-ANE in both control and study groups.

A comparison of the data made between the groups showed higher mean values in the study group than the control group. These differences between both the groups were statistically significant at all concentrations of t-ANE and r-ANE except at concentration of 200 mcg/ml of r-ANE (P < 0.005) [Graph 1].



NBT assay of saliva sample

In the control group, the mean percentages of the NBT reduced cells of samples treated with various concentrations of t-ANE range from 49.20 ± 12.45 at concentration of 100 mcg/ml to 27.19 ± 14.49 at concentration of 1600 mcg/ml and the mean percentages of NBT reduced cells of samples treated with various concentrations of r-ANE range from 39.40 ± 15.93 at concentration of 12.5 mcg/ml to 29.19 ± 10.75 at concentration of 200 mcg/ml.

In the study group the mean percentages of the NBT reduced cells of samples treated with various concentrations of t-ANE range from 44.88 ± 11.03 at concentration of 100 mcg/ml to 27.12 ± 8.53 at concentration of 1600 mcg/ml and the mean percentages of NBT reduced cells of samples treated with various concentrations of r-ANE range from 38.42 ± 11.88 at concentration of 12.5 mcg/ml to 23.31 ± 10.47 at concentration of 200 mcg/ml.

With increasing concentrations of t-ANE and r-ANE there was a drop in mean percentages of NBT reduced cells in both study and control groups. A comparison between both groups revealed overall higher mean values in the control group than study group with statistical significant difference at a concentration of 200 mcg/ml (P < 0.01). [Graph 2]



Comparison of t-ANE and r-ANE values from blood sample

In both the study and control groups, the mean values scored higher across all concentrations of t-ANE compared to r-ANE which were statistically significant (P < 0.05) [Bar diagram 1].



Comparison of t-ANE and r-ANE values from saliva sample

In both the study and control groups, the mean values scored higher across all concentrations of t-ANE compared to r-ANE. However, the differences were statistically significant in two concentrations only 100/12.5 mcg/ml and 200/25 mcg/ml of control group (P < 0.05) [Bar diagram 2].



PHG assay of blood sample

In the control group the mean scores of mean particle numbers of samples treated with various concentrations of t-ANE range from 3.17 ± 0.65 at concentration of 100 mcg/ml to 1.07 ± 0.37 at concentration of 1600 mcg/ml and the mean of mean particle numbers of samples treated with various concentrations of r-ANE range from 2.37+/-0.56 at concentration of 12.5 mcg/ml to 1.00 ± 0.26 at concentration of 200 mcg/ml.

In the study group the mean of mean particle numbers of samples treated with various concentrations of t-ANE range from 2.73 ± 0.83 at concentration of 100 mcg/ml to 1.20 ± 0.41 at concentration of 1600 mcg/ml and the mean of mean particle numbers of samples treated with various concentrations of r-ANE range from 2.17 ± 0.65 at concentration of 12.5 mcg/ml to 1.03 ± 0.32 at concentration of 200 mcg/ml.

With increasing concentrations of t-ANE and r-ANE the mean particle numbers have shown a reduction in both the groups. A comparison drawn between both the groups revealed an overall higher mean scores with control group but with statistically significant difference only with 100 mcg/ml of t-ANE and 25 mcg/ml of r-ANE (P < 0.05) [Graph 3].



PHG assay of saliva sample

In the control group the mean scores of mean particle numbers of samples treated with various concentrations of t-ANE range from 2.89 ± 0.70 at concentration of 100 mcg/ml to 1.00 ± 0.27 at concentration of 1600 mcg/ml and the mean of mean particle numbers of samples treated with various concentrations of r-ANE range from 2.00 ± 0.48 at concentration of 12.5 mcg/ml to 0.93 ± 0.26 at concentration of 200 mcg/ml.

In the study group the mean scores of mean particle numbers of samples treated with various concentrations of t-ANE range from 2.41 ± 0.63 at concentration of 100 mcg/ml to 1.07 ± 0.25 at concentration of 1600 mcg/ml and the mean of mean particle numbers of samples treated with various concentrations of r-ANE range from 1.79 ± 0.49 at concentration of 12.5 mcg/ml to 0.90 ± 0.40 at concentration of 200 mcg/ml.

With increasing concentrations of t-ANE and r-ANE the mean particle numbers have shown a reduction in both the groups. A comparison drawn between both the groups revealed an overall higher mean scores with the control group but with statistically significant difference only with 100 mcg/ml and 400 mcg/ml of t-ANE and 50 mcg/ml of r-ANE (P < 0.05) [Graph 4].



Comparison of t-ANE and r-ANE values from blood sample

In both the study and control groups the mean values scored higher across all concentrations of t-ANE compared to r-ANE except in 1600/200 mcg/ml in the control group and 400/50 mcg/ml, 800/100 mcg/ml, and 1600/200 mcg/ml of the study group which was statistically insignificant (P > 0.05) [Bar diagram 3].



Comparison of t-ANE and r-ANE values from saliva sample

In both the study and control groups, the mean values scored higher across all concentrations of t-ANE compared to r-ANE. However, the differences were statistically insignificant across 1600/200 mcg/ml concentration of the control group and 800/100 mcg/ml and 1600/200 mcg/ml concentration of study group (P > 0.05) [Bar diagram 4].




  Discussion Top


Areca nut is commonly referred to as betel nut, as it is often chewed wrapped in betel leaves (paan). The term areca originated from a South Asian word root during the 16th century. The habit has many harmful effects on health.[14] The International Agency for Research on Cancer concluded after reviewing the published medical research that chewing areca nut is carcinogenic to humans. Various compounds present in the nut, most importantly arecoline (the primary psychoactive ingredient), contribute to histological changes in the oral mucosa. As with chewing tobacco, its use is discouraged by preventive efforts. The Areca nut contains flavonoids like tannins, areca tannin, gallic acid, and four main alkaloids like arecoline, arecaidine, guvacine, and guvacoline which due to persistent contact, serves as a constant source of chemical irritation to the oral mucosa which provokes chronic inflammatory reaction characterized by the presence of activated T cells, macrophages, and various inflammatory mediators. One of those important cells that act in the host defense mechanism is neutrophils. These cells play a major role in pathogenesis of OSMF.[3],[14],[15]

These cells are recruited to sites of infection by sensing, and migrating toward, a gradient of chemotactic substances and they are capable of phagocytosis and killing a wide range of bacteria. Under normal circumstances, in the absence of infection, neutrophils roll along the vascular endothelium, transiently attaching to and detaching from their surface. This is mediated by lectin binding glycoproteins, L-selectin on the surface of neutrophils, and E-selectins on the endothelial cell. As the rolling neutrophils near the focus of infection, they respond to chemo-attractants such as complement components, IL-8, platelet-activating factor, and microbial peptides generated at the infection site leading to activation of integrins present on the surface of neutrophils. These integrins subsequently induce tighter adherence of neutrophils to vascular endothelial cells and cessation of rolling. Neutrophils then detach from endothelium and migrate through a gap in the endothelial lining created by contraction of juxtaposed vascular endothelial cells. After their migration into the extravascular space, neutrophils migrate along chemotactic gradients to the focus of infection. In the tissues, neutrophils proceed with the ingestion and killing of microbes and it happens through adhesion, opsonization, ingestion, and by intracellular killing.[12],[13],[16]

The release of reactive oxygen intermediates (ROI) such as superoxide radicals and hydrogen peroxide is an important component of neutrophil's bactericidal machinery.[16] These reactions are mediated by NADPH oxidase and the neutrophils defective in this enzyme cannot generate ROI leading to defective microbial killing. The production of superoxide can be tested by the reduction of cytochrome c and the extracellular release of H2O2 can be measured by horseradish peroxidase induced oxidation of phenol red. The principle of the NBT assay performed in our study is based upon the above reactions where the cells that were exposed to the yellow dye NBT. This test gives us the information about phagocytic function since the dye is not taken into cells except by phagocytosis and also about metabolic function since the intracellular reduction depends upon the production of ROIs.[17] Most methods for measuring phagocytosis rely on the uptake of particles by phagocytes over a brief time period. The number of candida ingested per cell was counted and expressed as mean particle number (MPN). Under normal circumstances, each neutrophil should have MPN of ≥4.[16],[17]

Very few studies exist in literature which have evaluated the neutrophil functions in the saliva samples. No comparative studies exist in NOM between healthy individuals as well as on areca nut and tobacco habituates. In our study, the overall obtained results of NBT assay in blood, there was a significant difference between control and study groups and the mean values were comparatively higher in the study group than controls which could be due to the primed state or hyper-stimulated state of the neutrophils.[13],[16] This could be due to overall already posed insults or stress from the areca nut and tobacco ingredients. Similar comparison made in saliva samples resulted in a significant difference with respect to only t-ANE at concentration of 200 μg/ml which could be due to scanty material obtained from saliva samples. However, the mean values of the study group were comparatively less than that of controls which implies the affected function of the neutrophils.

The comparison of different concentrations of t-ANE and r-ANE in control and study groups, where all the results obtained were significant in blood while in saliva only a few concentrations showed significant difference. The mean values of r-ANE are less than t-ANE in both blood and saliva samples suggesting r-ANE variety hampered neutrophil functions more than t-ANE. Thus, our results suggest that ANE may alter the functions of neutrophils compromising the defense system which might hamper the immune mechanism in the individuals. Through hampered neutrophil functions, this could be one of the possible role of immune mechanism in the pathogenesis of OSMF in individuals with areca nut chewing habit.

Hung SL et al. studied the effects of areca nut extracts on the functions of human neutrophils derived from bloodin vitro and their results demonstrated that ripe and tender ANEs reduced the antibacterial activity and the superoxide anion production of neutrophils. This effect may contribute to a less efficient elimination of bacteria from the periodontal environment. They concluded that the inhibition of the antimicrobial functions of neutrophils may alter the microbial ecology of the oral cavity affecting periodontal health.[1] In another study by Hung SL et al. stated that the components of areca nut reduced the human neutrophil uptake of A. actinomycetemcomitans ATCC 33384 which may compromise the periodontal health of areca quid chewers.[11],[18] A Study conducted by Patil KG and Metgud SC on the effects of areca nut extracts on phagocytosis of A. actinomycetemcomitans ATCC 33384 by neutrophils in patients with chronic periondontitis revealed both ripe and tender areca nut extract affected the neutrophil function in healthy and patients with chronic periodontitis. Ripe areca nut extract significantly altered the neutrophil functions more than tender areca nut extract which was similar to our findings.[2] Similarly, Ho WH et al. studied the effects of areca nut extract on the apoptosis pathways in human neutrophils and they observed that ANE reduced early apoptosis, but increased the primary necrosis of neutrophils. They suggested that areca nut may regulate death pathways in neutrophils stating it as one of the mechanisms in compromising the periodontal health.[4]

Katayama K et al. assessed the effect of age, lifestyle, and health behavior on neutrophil function by NBT method and stated that all the factors like age, lifestyle factors like subjective stress, exercise habits, smoking habits, alcohol drinking habit, self-perceived health status are correlated with the neutrophil functions. An age-related decrease in the neutrophil phagocytosis and bactericidal ability was stated. So, we considered middle-aged healthy individuals where neutrophil functions would not have been affected.[19],[20]

In our study, the Phagocytosis assay yielded mean scores of mean particle numbers which were found to be significantly lower in the study group at certain concentrations which indicates the affected phagocytotic function of the neutrophils.

From the data, it is evident that the phagocytotic function is less affected in t-ANE than r-ANE which suggests that r-ANE is more harmful than t-ANE. The various concentrations of both t-ANE and r-ANE that were employed in the study helped us to conclude that an increase in the concentration affected neutrophil functions proportionally. The results obtained using saliva samples were almost similar to the results of the blood samples. Saliva can be used as an alternative to the blood sample in studying the neutrophil functions. r-ANE is more harmful than t-ANE and r-ANE might have a more detrimental effect on oral mucosa in long term duration.


  Conclusion Top


In conclusion, areca nut causes reduction in the neutrophilic function. Certain subjective errors with saliva samples could be possible due to the presence of epithelial cells and microbial flora masking up the neutrophils and also scanty yield of cells. Nevertheless, saliva can prove to be an effective alternative material for analyzing the neutrophil function as it is non-invasive and least uncomfortable to the patient. Improvisation in the neutrophil isolation techniques may prove to be of value which may assess neutrophil function as a valuable method to analyze the immune status of an individual. Further the subjects who showed altered neutrophil functions significantly have to be recalled and kept on regular follow-up in order to evaluate any possible changes in the oral mucosa and also possible association with the occurrence of OSMF. Further longitudinal studies will add up a value in this regard in order to better understand the role of affected neutrophils in areca nut chewers for any pathological changes in the oral mucosa.

Acknowledgements

We would like to thank Dr. Ravi Shirahatti, Head and Professor, Department of Public Health Dentistry, Maratha Mandal's NGH Institute of Dental Sciences and Research Centre, Belagavi, Karnataka for helping with the statistical analysis.

Financial support and sponsorship

Self-funded.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

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