This study was conducted at the outpatient Departments of Oral Pathology and Microbiology and Oral Medicine and Radiology, Meenakshi Ammal Dental College and Hospital, Chennai, Tamil Nadu, India. The subjects comprised of four groups; Group I comprised of 30 subjects as healthy controls, Group II comprised of 30 subjects with smoking habit, Group III comprised of 30 subjects with tobacco chewing habit, Group IV comprised of 30 subjects with oral lichenoid reactions. The patients were in the age group of 20-50 years, without any medically compromised conditions or systemic illness. Group I had 12 males and 18 females, group II had 30 males with only smoking habit, group III with 26 males and 4 females with only tobacco chewing habit and group IV had 22 males and 8 females. Ten patients in group IV had the habit of smoking (group IVa), 16 patients with the habit of chewing tobacco (group IVb), and the rest 4 with both smoking and tobacco chewing habits (group IVc).

Written consent of the patient was obtained and a biopsy was performed to confirm the clinical diagnosis of lichenoid reaction with histopathological findings. The subjects in the experimental groups were asked to rinse their mouth with povidone iodine mouth rinse for 2 minutes. After waiting for a minute, freshly secreted unstimulated saliva, about 1ml was collected in a sterile container. The sample was then diluted with 10ml of phosphate buffered saline to neutralise the pH. The mixture was then centrifuged for 5 minutes at 3000 rpm and the supernatant fluid was stored at 20°C until use. The substrate to be determined in our study was nitrite and hence, known concentrations of sodium nitrite solutions were prepared for evaluation. Sodium nitrite solutions in varying concentrations from 10μg-50μg were prepared and test tubes were labelled as S1 – S5 respectively. Aliquots of these solutions (whose concentration were known) were reacted with Griess reagent which was prepared by using 1% Sulfanilamide, 0.1% Napthylethylene Diamine Dihydrochloride, 2.5% Phosphoric acid and incubated at room temperature for 10 mins to ensure complete reaction takes place. The purplish pink colour thus obtained was read in spectrophotometer and the absorbance optical density (O.D) was recorded. This was taken as standard optical density (SOD).

In the same way, 1ml of the saliva sample were taken from each patient in our study group and was incubated with an equal volume of Griess reagent in a test tube for 10 minutes at room temperature. The colour change in the test tube was read in a spectrophotometer at 545nm and absorbance was recorded. This represented the test optical density (TOD). The concentration of nitrite in the saliva was calculated using the formula

TOD/SOD x concentration of the standard solution.

The levels of nitric oxide were recorded for controls and the study groups.

Ethical Approval: All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. The written consent form was approved by the ethics committee.

Multiple comparisons between groups using One-Way ANOVA and using Post-HOC Tukey HSD analysis were done using version 16.0 of SPSS software. Values were found to be statistically significant (p-value <0.05) for all the parameters taken into consideration. In group I, the salivary nitric oxide levels were in the range from 3.04μg/dl to 5.186μg/dl with a mean of 3.928μg/dl and in group II, the salivary nitric oxide levels were in the range from 6.08μg/dl to 7.154μg/dl with a mean of 6.648μg/dl. Group III had salivary nitric oxide levels in the range from 5.768μg/dl to 6.841μg/dl with a mean of 5.929μg/dl and patients in group IV had the salivary nitric oxide levels were in the range from 8.808μg/dl to 16.006μg/dl with a mean of 11.880μg/dl [Table/Fig-1,2 and 3].

There was a significant increase in the nitric oxide levels among the four groups with maximum values seen in group IV consisting of patients with oral lichenoid reactions. However, the increase was not significant among group II and group III. Comparisons between group II (smokers) and group IVa (smoking associated with lichenoid reactions) showed a definite increase in values. Moreoever, Group III (tobacco chewers) were compared with group IVb (tobacco chewing associated with lichenoid reactions) and nitric oxide levels were increased in Group IVb. Intra comparisons between Group IVa and IVb were done and the value was increased in the latter and was stastically significant.

Lichenoid reaction, similar to oral lichen planus is an inflammatory lesion with inflammatory cells such as lymphocytes and plasma cells distributed in the stromal tissue. Since, the usage of tobacco products and oral lichenoid reactions produce an inflammatory reaction, cells like T lymphocytes and macrophages serve as a source of nitric oxide, thereby causing cell injury and damage. Nitric oxide can react with other radicals to form cytotoxic compounds, such as peroxynitrite, which can cause DNA damage and protein modifications by formation of carcinogenic nitrosamines or by inhibiting DNA repair mechanism [7,8]. It can also react directly with a variety of enzymes and other proteins to either activate or inhibit their functions. Inducible nitric oxide synthase (iNOS) is capable of producing sustained concentrations of nitric oxide in the micromolar range and hence, this molecule is principally involved in inflammatory processes and cancer formation [9].

In the present study, the levels of nitric oxide in the saliva of patients with smoking, tobacco nut chewing habits and patients with oral lichenoid reactions was compared with healthy controls. Nitric oxide levels can be estimated from blood, exhaled air, saliva, urine and also from polymerase chain reaction (PCR) and immunohistochemical analysis. In this study, saliva was used for the estimation of nitric oxide level as it was readily available, easier to collect and the procedure was non-invasive. The nitric oxide levels in saliva were measured by a calorimetric assay based on Griess reagent, described by Ohashi et al., [10].

The results obtained in the present study showed elevated salivary nitric oxide levels in smokers, tobacco chewers and patients with oral lichenoid reactions when compared to normal controls. An increase in nitric oxide levels were also seen in patients with lichenoid reactions when compared with smokers and tobacco chewers. The results even showed elevated levels of nitric oxide in patients with tobacco chewing associated oral lichenoid reactions with oral lichenoid reaction patients associated with smoking and this was also found to be statistically significant (p ≤ 0.05).

The results imply that tobacco smoking, tobacco chewing habit and lichenoid reactions can induce inflammatory changes in the oral mucosa. Chronic inflammation would play an important role in causing genetic damage and inducing tissue proliferation by oxidative damage of the DNA products derived from inflammation which induces enzymes, such as nitric oxide synthase, which in turn produces nitric oxide [11].

The values of salivary nitric oxide levels among the healthy controls correlated with the values of the control group in the study conducted by Sunitha M and Shanmugam [6]. Bodis and Haregewoin, found reduced salivary nitric oxide levels in smokers compared to control subjects [12]. This was in contrast with the present study which showed an elevated nitric oxide levels in smokers compared to controls. This could probably be due to different products of nitric oxide that were analysed. Nitrogen dioxide (NO₂) was evaluated in the former study compared to nitrite evaluated in our present study. Fresh cigarette smoke contains nitric oxide primarily with little or no nitrogen dioxide [13]. Also, hypoxia is a synergistic inducer of iNOS expression and hence, favours the reaction between nitric oxide and oxygen resulting in nitrosative modifications [14]. This could explain the reason for increased nitric oxide levels in smokers in the present study compared to the former.

Ohashi et al., have reported that increase in salivary nitric oxide levels in oral lichen planus and recurrent apthous ulcerations is a consequence of cell damage [10]. Sunitha M and Shanmugam S also found increased levels of nitric oxide in lichen planus compared to controls and concluded that free radicals represent one route of pathogenesis and excess of salivary nitric oxide may have a pathophysiological implication for erosive and ulcerative lesions in oral lichen planus and recurrent aphthous ulceration [6]. The present study show elevated nitric oxide levels in patients with tobacco usage and a more elevated status in patients with oral lichenoid reactions.

Inducible nitric oxide synthase expression was upregulated in oral submucous fibrosis in a study conducted by Rajendran R and Shirley Varkey and their study group consisted of patients with the habit of areca nut chewing [15]. Nair UJ et al., first demonstrated that aqueous extracts of areca-nut and catechu were capable of generating free radicals at specific pH that are produced during auto oxidation of polyphenols in saliva of the tobacco users [16]. In accordance, there was an increased nitric oxide levels in patients in group III and increased more in patients with oral lichenoid reactions associated with tobacco chewing habit as seen in group IV.

Lichenoid reactions are a form of type IV contact hypersensitivity reaction [17] and according to Ross et al., nitric oxide production has been shown to be increased by tenfold in hypersensitivity reaction [18] and the present study showed increased nitric oxide levels in group 4.

Continuous local irritation and trauma caused by betel quid and pan masala can generate chronic inflammation, oxidative stress and cytokine production. Invitro studies have demonstrated the adhesive nature of areca-nut particles to cultured oral mucosal cells, leading to morphological changes and membrane damage. Oxidative stress and the generation of reactive oxygen species can drive affected cells to proliferation, senescence or cell death. Components of areca nut stimulate the release of inflammatory mediators such as prostaglandin E2 (PGE2), interleukin-6 (IL-6) and tumour necrosis factor α (TNF-α) from primary cultured human oral keratinocytes. Activation of complement was demonstrated invitro using aqueous extracts of loose leaf chewing tobacco, dry snuff and moist snuff [19]. Earlier studies have shown that pan masala and gutkha have clastogenic and carcinogenic effects [20].

In any lichenoid lesion, whether reactive or dysplastic, there is the presence of localized mononuclear cell infiltration within the superficial lamina propria [21]. These T lymphocytes in the lamina propria produces increased levels of interleukin-6 (IL-6) and granulocyte macrophage colony stimulating factors (GM-CSF) and can be stimulated to produce more tumour necrosis factor-α (TNF-α) by IL-1β, IL-6 and GM-CSF. These cytokines, interleukins and TNF are capable of activating T cells, which in turn produces more iNOS and thus contribute to the release of nitric oxide. It is seen that lichenoid reactions being inflammatory, show increased nitric oxide levels due to the release of various inflammatory mediators, and these levels can be further enhanced by tobacco and its products, which explains the reason why there is an increased nitric oxide levels in patients with oral lichenoid reactions associated with the tobacco chewing habit. Increased nitric oxide levels can in turn cause DNA modifications and damage, progressing the cell to malignancy.

Studies have been done on the level of nitric oxide in smokers, smokeless tobacco users, lichen planus and ulcerative lesions and mostly on tumours, but very few studies have been conducted on oral lichenoid lesions and its aetiopathogenesis [22–24]. This study was an attempt to analyse the role of nitric oxide in oral lichenoid reactions. The results in our study were analysed using a spectrophotometer which requires a minimum saliva sample of 1ml for estimation. If the quantity is less than 1ml, it has to be diluted so as to make the volume up to 1ml to be analysed in the spectrophotometer. By diluting this sample, chances of error may occur which can interfere with the accuracy of the result. This was overcome in the present study by collecting adequate saliva sample from patients.

Although, at present we cannot completely confirm whether the over expression of iNOS in smokers, tobacco chewers and lichenoid reactions is mediated via hypoxia or due to the release of cytokines by inflammatory mediators as a result of tobacco or betel nut. Usage of tobacco products has produced lichenoid reactions in the buccal mucosa and the reasoning, at the molecular level is still debatable.

Either increased levels of nitric oxide contributed to the development of these oral lichenoid lesions or whether these oral lesions contributed to elevated nitric oxide levels or if they have a synergistic effect on each other is still unknown. Although, it appears reasonable to suspect that the increased activity of the enzyme may, at least in part, indicate that nitric oxide has a pathophysiological role in these conditions.