Estimation of salivary nitric oxide in oral precancer patients R Metgud1, C Anandani2, K Singh3 1Department

of Oral and Maxillofacial Pathology, Pacific Dental College and Hospital, Paher University, Udaipur, of Oral and Maxillofacial Pathology, College of Dental Sciences and Research Centre, Bopal, Ahmedabad, Gujarat, and 3Department of Public Health Dentistry, Siddhpur Dental College, Siddhpur, Gujarat, India 2Department

Accepted December 10, 2014

Abstract The role of nitric oxide (NO) in the initiation, promotion and progression of cancer has been the subject of speculation and conflicting reports in the literature. The high incidence of oral cancer and precancer has been linked to tobacco chewing and smoking habits; NO is considered an indicator of tobacco-related diseases. We compared salivary NO levels in oral precancer and normal patients. Unstimulated whole saliva was collected from 15 patients with oral precancer (group 1) and 15 healthy age and sex matched subjects (group 2). Salivary nitrite levels were estimated using a colorimetric method and a spectrophotometer. The salivary nitrite concentration of group 2 (median  4.21 μg/ml) was significantly less than for group 1 (median  12.91 μg/ ml). We have added evidence concerning involvement of NO in the pathogenesis of oral cancer, but whether it is a potentially carcinogenic agent at the concentration at which it is present in oral precancer patients requires further evaluation. Key words: cancer, nitric oxide, nitrite, oral precancer, saliva Nitric oxide (NO) has been regarded as simply a toxic pollutant in cigarette smoke and smog. NO now has become the subject of intense research in many fields of medicine and science (Brennan and Moncada 2002, Korde Choudhari et al. 2012). NO is a short-lived endogenously produced gas that is synthesized by a complex family of NO synthase (NOS) enzymes that use L-arginine as a substrate. Three isoforms of NOS have been characterized. NOS1, also known as nNOS, is the isoform first purified and cloned from neuronal tissue. NOS3, or eNOS, is the isoform first found in endothelial cells; these two isoforms also are termed constitutive, because they are expressed continuously in neurons and endothelial cells, respectively. They also depend on a rise in tissue calcium concentration to be active and to produce low, transient concentrations

Correspondence: Dr. Chitra Anandani, Department of Oral and Maxillofacial Pathology, College of Dental Sciences and Research Centre, Bopal, Ahmedabad, Gujarat, India. Email: [email protected] © 2015 The Biological Stain Commission Biotechnic & Histochemistry 2015, 90(4): 302–308.

DOI: 10.3109/10520295.2014.998282

of NO. By contrast, NOS2, the calcium-independent isoform, which also is called iNOS, can be induced by macrophages, natural killer cells, T cells and various cytokines including interleukin-1, interferon gamma and tumor necrosis factor α. Its induction results in continuous production of NO for many hours or even days (Choudhari et al. 2013, Jenkins et al. 1995). When produced in appropriate amounts and at appropriate times, NO is a key signaling molecule for vasodilation, respiration, cell migration, immune response and apoptosis. On the other hand, excess and unregulated NO synthesis has been implicated in various inflammatory and pathophysiological conditions including cancer (Korde Choudhari et al. 2012). The molecular structure of NO makes available an unpaired electron, which makes it a highly reactive free radical (Brennan and Moncada 2002). Because it is unstable and has a short half-life, it must be estimated by its end products, nitrites (NO2) and nitrates (NO3) (Beevi et al. 2004). Nitrates are taken up by leafy vegetables such as lettuce and spinach and they are dissolved in drinking water. When 302

consumed, nitrates are absorbed from the upper gastrointestinal tract. Nitrates in plasma are concentrated in saliva by the salivary glands through an active transport mechanism. Salivary nitrates are reduced to nitrites in the oral cavity, depending on the activity of salivary nitrate reductase, which produces concentrations of nitrites in the saliva 1,000 times greater than in plasma (Lundberg et al. 2009). Nikov et al. (2003) reported that oral microflora also play a role in nitrate reduction and several nitrite producing organisms in human saliva have been identified including Veillonella species, Staphylococcus aureus, Staphylococcus epidermidis, Nocardia species, Corynebacterium psuedodiphtheriticum and Fusobacterium nucleatum. Seventy percent of ingested nitrites are formed by oral flora. Nitrites are important promoters of carcinogenesis, because they react with amines and amides to form carcinogenic nitrosamines (Bahar et al. 2007). Bodis and Haregewoin (1993) reported that freshly secreted human saliva contains measurable and sometimes high levels of NO. The possible cellular sources of salivary NO include nerve endings, salivary gland tissue, endothelial cells and macrophages that respond to oral bacterial products (Sunitha and Shanmugam 2006). NO and NOS have been studied in human malignant tumors including breast, gynecological, lung, brain, gastric, colorectal, renal cell, pancreatic, prostrate, bladder, stomach, and head and neck cancers. There are few reports in the literature, however, concerning salivary NO levels in oral precancer patients (Brennan and Moncada 2002, Choudhari et al. 2013). Therefore, we estimated salivary NO levels by measuring its nitrite product in oral precancer and normal patients.

Collection of saliva All subjects were given a thorough oral prophylaxis a day before sample collection and were asked to avoid a diet that alters NO levels, e.g.. spinach, lentils, garlic, egg yolk, brown bread, nuts, and other foods rich in arginine. After fasting overnight, subjects were asked to rinse their mouths with betadine mouth rinse for 2 min early the next morning to reduce the number of bacteria. After waiting for 1 min, approximately 5 ml freshly secreted, unstimulated whole saliva was collected in a sterile container containing 0.5 ml 1 N sodium hydroxide. Sodium hydroxide served as a stabilizer, because nitrite is unstable in acid solution. Two tenths milliliter 0.5 M zinc sulfate was added to an aliquot of 3 ml of saliva and mixed. Treatment with zinc sulfate removes proteins and other substances that could inhibit chromogen formation by nitrite. The mixture then was centrifuged for 10 min at 885.45  g and the supernatant was decanted and stored at 20° C until use. Nitrite quantification Nitrite was quantified using the method described by Diaconu et al. (2001). Briefly, a reddish purple azo dye is produced at pH 2.0–2.5 by coupling diazotized compound with 1-naphthylamine. Deproteinized samples (2 ml) were placed in glass tubes and diluted to 10 ml with distilled water. Four tenths milliliter 4-aminobenzene sulfonic acid solution (1.6 g% in 30% glacial acetic acid) was added, which forms a diazotized compound. The tubes were placed in ice for 15 min, 0.4 ml 1-naphthylamine solution (0.5 g% in 30% glacial acetic acid) was added and 20 min later the absorbance at 520 nm was measured by a spectrophotometer at room temperature.

Material and methods Our study protocol was approved by the Ethical Committee of Pacific Dental College and Hospital and informed consent was obtained from all participants. Subjects were divided into two groups: group 1 comprised 15 patients with clinically diagnosed oral precancer and group 2 comprised 15 healthy age and sex matched controls, who were free of health-compromising habits. To avoid interference with NO detection, individuals with immune disorders, asthmatic conditions, inflammatory or infectious diseases of the mouth, periodontitis or other systemic diseases, or undergoing glucocorticoid treatment were excluded. The clinical and socio-demographic data for the patients in our study group are given in Table 1.

Table 1. Clinical and socio-demographic details Characteristics male female clinical type leukoplakia oral submucous fibrosis lichen planus tobacco habits chewers smokers chewers  smokers

No. of patients 11 4 5 8 2 3 2 7

Subjects were 22–48 years old.

Salivary nitric oxide in oral precancer 303

1.2

0.8

A significant linear relation was found between the standard sodium nitrite concentrations and their absorbance values (correlation coefficient  0.991, p  0.001), which enabled prediction of an unknown parameter (nitrite concentration) using the linear regression equation with an accuracy of 99.1% (Fig. 1, Table 2). Using this equation, the nitrite concentrations (x) for both groups were obtained using the known values for absorbance (y) (Figs. 2,3). The maximum absorbance value for group 1 was 1.896, the minimum value was 0.791 and the median value was 1.263. For group 2, the maximum absorbance value was 0.85, the minimum value was 0.689 and the median value was 0.781. Comparison of groups 1 and 2 using Mann-Whitney U test revealed a statistically significant difference (p  0.003) between the absorbance values for the two groups (Table 3). The maximum nitrite concentration for group 1 was 25.32; the minimum was 4.47 and the median was 12.91. For group 2, the maximum nitrite concentration was 5.59, the minimum was 2.56 and the median was 4.21. Comparison of groups 1 and 2 using Mann-Whitney U test showed a statistically significant difference (p  0.001) between the nitrite concentrationsfor the two groups (Table 4). 304

0.8 0.812

y = 0.0532x + 0.5543 R2 = 0.9914

0.4 0.2

Statistical analysis

Results

0.678

0.716

0.6

0

Data were analyzed using SPSS version 15.0 (Chicago, IL). Descriptive statistics were obtained, and means and standard deviation were calculated. Mann-Whitney U test was used to determine the significance of difference of the median of absorbance values and nitrite concentrations of both groups; a p value  0.05 was considered significant. Karl Pearson’s correlation was used to assess the relation of nitrite concentration and the absorbance values of standard solutions.

1.071 0.977

1 Absorbance A520

A series of standards was prepared using sodium nitrite solutions of varying concentrations from 0.25–10 μg/ml. A graph of absorbance vs. concentration was constructed for these standards to create a standard curve. A plot of absorbance vs. distilled water measured at 520 nm served as the blank value. The optical densities of the saliva samples then were measured and compared to the standard curve to determine the corresponding concentrations of nitrite.

0

2

4

6

8

10

12

NO2Concentration µg/ml

Fig. 1. Calibration curve of standard sodium nitrite solution.

Discussion Oral cancer is the third most common cancer in India (Patel et al. 2009); the age standardized incidence rate is reported to be 12.6/100,000 people (Khan 2012). Oral cancer usually is preceded by premalignant states including leukoplakia, erythroplakia, lichen planus and oral submucous fibrosis with a transformation rate ranging from 0 to 20% in 1–30 years depending on the type of lesion (Shah et al. 2011). The increased prevalence of oral cancer in the Indian subcontinent appears to be due to genetic predisposition, smoking and other tobacco habits, alcohol, spicy food, and neglect of oral and overall health (Khan 2012). Oral squamous cell carcinoma (OSCC) ultimately is the result of DNA damage. Free radicals, such as reactive oxygen species (ROS) and reactive nitrogen species (RNS), are important agents of DNA damage (Beevi et al. 2004). Interaction of NO with O2 or O2 causes formation of reactive nitrogen species, such as dinitrogen trioxide and peroxynitrite, that can induce two types of chemical stress, nitrosative and oxidative. Dinitrogen trioxide is a potent nitrosating agent that can N- and S-nitrosate a variety of biological targets to produce potentially carcinogenic nitrosamines and nitrosothiol derivatives. Peroxynitrite is a powerful oxidant that oxidizes thiols or thioethers,

Table 2. Correlation of absorbance values and sodium nitrite concentrations of the standard solutions

Parameter

Mean ⴞ SD

NaNO2 absorbance

5.417  2.8534 0.842  0.1523

Biotechnic & Histochemistry 2015, 90(4): 302–308

Correlation coefficient

p

0.991

0.001

GROUP I

NITRITE CONCENTRATION

30 25 20 15 10 5 0

0.791 0.964 1.01 1.069 1.116 1.198 1.202 1.263 1.302 1.406 1.5 1.578 1.605 1.702 1.896 ABSORBANCE VALUE

Fig. 2. Nitrite concentration for group 1.

nitrates tyrosine residues, nitrates and oxidizes guanosine, degrades carbohydrates, initiates lipid oxidation and cleaves DNA (Choudhari et al. 2013). In its initial stages, OSCC often is asymptomatic and not diagnosed or treated until it reaches an advanced stage. Although biopsy is considered the gold standard for diagnosis of oral cancers, the reliability of an appropriate site to biopsy is problematic. To the contrary, biochemical markers could provide evidence of changes in the tissues that eventually develop into malignancies. Detection of alterations of fluids during early stages of oral cancer could make possible early diagnosis prior to the development of visible tumors (Nayyar et al. 2012, Ravindran and Deepa 2012). Saliva is a multiconstituent oral fluid and is a promising diagnostic tool, because it is easier to collect than blood and it is inexpensive, noninvasive and easy to use for screening methods (Deepa and Thirrunavukkarasu 2010, Kaufman and Lamster 2002). ROS and RNS induce OSCC. These compounds are produced mainly as a result of smoking, alcohol,

NITRITE CONCENTRATION

6

food, drink or volatile sources that enter the oral cavity freely (Bahar et al. 2007). Evidence for the role of NO in carcinogenesis includes the fact that both iNOS expression and NO products have been found in various human cancers (Joshi et al. 1996, Zeillinger et al. 1996, Siegert et al. 2002, Wolf et al. 2000, Sun et al. 2005, Roy et al. 2007). Rosbe et al. (1995) identified iNOS in head and neck squamous cell carcinoma, which suggests that it plays a role in tumor growth. It has been reported also that expression of iNOS or plasma/serum or salivary levels of NO or its products is significantly higher in OSCC, which links it to the pathogenesis of oral cancer (Korde Choudhari et al. 2012). Gallo et al. (2003) analyzed the correlation between iNOS activity and p53 gene status and found that p53 gene mutations may be responsible for the iNOS up-regulation that frequently is seen in OSCC. This hypothesis is supported by the fact that a negative feedback loop appears to exist between NO production and the p53 tumor suppressor gene (Ambs et al. 1998). The

GROUP II

5 4 3 2 1 0

0.689 0.698 0.722 0.723 0.758 0.769 0.772 0.781 0.787 0.791 0.798 0.809 0.821 0.83 0.85 ABSORBANCE VALUE

Fig. 3. Nitrite concentration for group 2.

Salivary nitric oxide in oral precancer 305

Table 3. Comparison of absorbance values Group

Median

p

group 1 group 2

1.263 0.781

0.003

n  15.

genotoxic and cytotoxic effects of NO that are produced in excess at local sites due to up-regulation of iNOS would be expected to be cancelled in part by activation of the wild type p53 gene. For cancer cells that have a mutant p53 gene or lack the gene completely, however, dysregulation or up-regulation of NO subsequently occurs and potentially leads to a cancerous state (Rajendran and Varkey 2007). We found a significant (p  0.001) increase in salivary nitrite concentration in patients with oral precancer (median  12.91 μg/ml) compared to healthy individuals (median  4.21 μg/ml). Also, 80% of oral precancer patients had a history of either smoking or smokeless tobacco consumption. Rasheed et al. (2007) reported that tobacco components initiate an inflammatory response that could cause generation of ROS and RNS, which causes lipid oxidation, enhanced NO production and a deranged antioxidant defense system in tobacco users. The damage to genes sustained by elevated reactive species could be a mechanism by which cancer develops from long term tobacco abuse. Carcinogenesis, including oral cancer, can be induced by chronic inflammation (Choi et al. 2008). The inflammatory cells and cytokines found in the stroma near the tumor are more likely to contribute to tumor development, progression and metastasis than to mount an effective anti-tumor response by the host. Genetic damage initiates the development of cancer and some types of inflammation may foster its development (Balkwill et al. 2001). The cytokine-releasing inflammatory cells activate iNOS, which ultimately results in increased production of NO (Brennan et al. 2002). Taken together, these observations suggest that NO is a biomarker for inflammation and can be used to estimate the risk of cancer in precancer patients or in healthy tobacco users (Choudhari et al. 2013). Table 4. Comparison of estimated nitrite concentrations Group

Median

p

group 1 group 2

12.91 4.21

0.001

n  15.

306

The literature contains few reports concerning the role of NO in oral precancer. Brennan et al. (2000) showed that iNOS expression was correlated with the severity of oral dysplasia and that it was increased also in cases that showed positive staining for p53. These investigators also suggested that the correlation between iNOS and vascular endothelial growth factor expression in oral dysplasia might provide an understanding of the complex transformation of oral epithelial dysplasia to invasive carcinoma and the role of angiogenesis in the process (Brennan et al. 2002). Chen et al. (2002) suggested that an iNOS-dependent mechanism may be involved in malignant transformation of oral submucous fibrosis, verrucous hyperplasia and verrucous carcinoma. Rajendran and Varkey (2007) investigated iNOS expression in oral submucous fibrosis and correlated it with different grades of epithelial dysplasia that are associated with the disease. Because NO is a vasodilator, these investigators also suggested that iNOS expression might cause the vasodilation of sinusoids that characterizes the disease. Sunitha and Shanmugam (2006) and Panjwani et al. (2013) found increased salivary NO levels in oral lichen planus and recurrent aphthous stomatitis patients, and suggested that psychological stress causes NO release and increases neural NOS activity. Because both of these diseases are associated with stress, NO may play a role in their pathogenesis. Some investigators (Beevi et al. 2004, Bahar et al. 2007, Patel et al. 2009, Korde et al. 2011, Hegde et al. 2012) have evaluated the serum, plasma and salivary levels of NO together with lipid oxidation products and antioxidants in oral precancer and cancer patients, and found elevated levels of NO products (nitrite, nitrate and total NO) and lipid oxidation products together with a decreased total antioxidant level. All of these investigators suggested that the oxidative DNA damage that occurs due to interplay between RNS and ROS and a deranged antioxidant defense system plays an important role in oral carcinogenesis. The significantly higher concentration of NO end products could result from either a generalized increase in NO synthesis throughout the body of the cancer patient or increased NO degradation promoted by oxidative stress. By contrast to its ability to promote tumor growth, NO also has been reported to have tumoricidal effects. Shang et al. (2002), Harada et al. (2004), Zhao et al. (2005) and Avci et al. (2009) reported that NO derived from macrophages, Kupffer cells, natural killer cells and endothelial cells participates in tumoricidal activity against many types of tumors. These investigators suggested that NO has a cyto-

Biotechnic & Histochemistry 2015, 90(4): 302–308

static or cytotoxic effect on tumor cells at concentrations greater than the concentration at which it promotes tumor activity and may be a useful therapeutic agent for cancer management in the future. Although tumoricidal roles have been proposed for NO, most experiments have been performed in vitro and similar findings have not been reported for cancer patients. Instead, it has been suggested that NO concentrations in OSCC and other solid tumors are at least one to two orders of magnitude less than those required to produce the effects. At its usual concentration, NO is thought to facilitate tumor growth and dissemination (Choudhari et al. 2013, Brennan and Moncada 2002, Korde Choudhari et al. 2012). We have investigated the potential involvement of NO in the pathogenesis of oral cancer. Whether it acts as a tumor promoting agent at its usual concentration in oral precancer requires further evaluation. A long term clinical study of oral precancerous states with a larger sample size must be undertaken to ascertain the role of NO in the initiation and promotion of oral carcinogenesis. Particularly in Asian countries, including India, where oral cancer is related to smoking habits and where diet is not rich in antioxidants due to the low socio-economic status of the population, the role of NO requires better understanding.

Acknowledgments The authors thank Mr. Sanjay Bais and other staff members from Pacific College of Pharmacy for their expertise and help with the spectrophotometric method. The authors also are grateful to the faculty members in the Department of Oral and Maxillofacial Pathology for their support. Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this paper.

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Biotechnic & Histochemistry 2015, 90(4): 302–308

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Estimation of salivary nitric oxide in oral precancer patients.

The role of nitric oxide (NO) in the initiation, promotion and progression of cancer has been the subject of speculation and conflicting reports in th...
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