Comparative evaluation of Plasma and Salivary 8-isoprostane expression in Oral Leukoplakia and Oral Submucous Fibrosis patients

Abstract

Background and Objectives:

Among the various biofluids, 8-isoprostane is a relatively underexplored yet stable oxidative stress biomarker that can be detected in measurable quantities. It is a unique by-product of arachidonic acid lipid peroxidation, and in recent years, its expression has been investigated in various diseases, particularly in malignancies. Our study was performed to evaluate the level of 8-isoprostane and compare its expression in plasma and saliva samples of oral leukoplakia (OL), oral submucous fibrosis (OSMF) patients and healthy controls (HC).

Methodology:

Plasma and saliva samples were collected from clinically and histopathologically confirmed cases of OL, OSMF and HC, with 12 participants in each group. The 8-isoprostane levels in all samples were analysed using the enzyme-linked immunosorbent assay (ELISA) technique.

Results:

The expression of 8-isoprostane level was statistically significant (P < 0.05) in both plasma and saliva, denoting a steady elevation of 8-isoprostane level in oral potentially malignant disorder (OPMD) (OSMF and OL), when compared to HC. However, when compared to saliva, plasma 8-isoprostane levels were higher in the OL group, whereas the reverse trend was observed in the OSMF group, where salivary levels were higher.

Conclusion:

The study reveals a significant plasma and salivary 8-isoprostane level, where the concentration was higher in OPMD groups implying that saliva could be used as the diagnostic sample of choice for evaluating this oxidative stress marker.

INTRODUCTION

According to global statistics, cancer cases are expected to surge by 77% by 2050, reaching over 35 million new cases, compared to 20 million new cases and 10 million deaths due to cancer in 2022.[1] Projections indicate an upward trend in oral cancer metrics from 2022 to 2031, with the age-standardized incidence rate (ASIR) expected to reach 10.15 per 100,000 and the age-standardized prevalence rate (ASPR) estimated at 29.38 per 100,000 by 2031.[2] The current strategies aim at preventive measures, maintaining a healthy lifestyle and early identification of high-risk populations.[3] Tobacco and betelnut products contain carcinogens such as the hydrocarbons and strong nitrosamines, which are the main aetiological factors in the development of oral potentially malignant disorders (OPMD) and oral squamous cell carcinoma (OSCC).[4] The updated report and nomenclature of OPMD included the following lesions under OPMD, namely, oral leukoplakia (OL), erythroplakia, oral submucous fibrosis (OSMF), oral lichen planus (OLP), oral lichenoid reactions (OLL), actinic chelitis, palatal lesions in reverse smokers, oral lupus erythematosus (OLE), dyskeratosis congenita (DC) and oral graft vs host disease (OGVHD).[5]

The disease pathogenesis of OPMD and OSCC is centred around free radical damage and reactive oxygen species (ROS). ROS are exogenously and endogenously derived, produced in picomolar concentrations, which include hydrogen peroxide, superoxide, hydroxyl radical, peroxynitrite, etc.[6] A few oxidative stress markers, such as malondialdehyde (MDA), lipid hydroperoxides and volatile alkanes, have previously been evaluated in the saliva samples of patients diagnosed with OSMF.[7,8] An evolving stable biomarker of oxidative stress, increasingly recognized for its reliability as a gold standard, is 8-isoprostane (8-iso-PGF2α/15 F2t isoprostane). 8-isoprostane belongs to the group of prostaglandins and is involved in tissue fibrosis and malignancies.[9] Although biomarkers have been studied in various diagnostic samples, saliva has emerged as a preferred non-invasive biofluid for disease detection and prognostication in various disease conditions, including OPMD and OSCC.[10]

Our study aims to evaluate the expression of 8-isoprostane in the saliva and plasma of patients diagnosed with OSMF and OL, in comparison with healthy controls.

MATERIALS AND METHODS

The cross-sectional study was conducted at the Department of Oral Medicine and Radiology, with approval from the Institutional Ethical Committee (IEC No. CSP/21/JUL/96/383). Demographic details and biological samples (blood and saliva) were collected from all consecutive patients who reported to the dental outpatient department (OPD) and provided informed consent to participate in the study. The study was performed in adherence to the Helsinki Declaration of 1975, which was revised in October 2024. The study participants were categorized into the following groups:

• GROUP 1: Healthy controls (HC) (n = 12)

• GROUP 2: Oral leukoplakia (OL) (n = 12)

• GROUP 3: Oral submucous fibrosis (OSMF) (n = 12)

Groups 2 and 3 of our study included patients aged more than 18 years, both males and females, who were clinically and histopathologically diagnosed with OL and OSMF, respectively [Figures 1 and 2]. Healthy controls (Group 1) were selected as age- and gender-matched participants with no history of tobacco or betelnut use and no evidence of any systemic illness.

Figure 1.

(a and b) Clinical image shows raised, well-demarcated, white plaque with a cracked mud appearance and hyperpigmentation; (c-e) Clinical image shows blanching of buccal mucosa, bud-shaped uvula and restricted mouth opening

Figure 2.

(a) - Histopathological image shows atrophic stratified squamous epithelium, with underlying connective tissue showing hyalinization, fibrosis, with few inflammatory infiltrates, suggestive of oral submucous fibrosis (Hematoxylin & Eosin stain (H&E) stain, 10 × magnification); (b) - Histopathological image shows hyperparakeratotic stratified squamous epithelium, with few collagen fibres and chronic inflammatory cell infiltrate in underlying connective tissue, suggestive of oral leukoplakia (H&E stain, 10 × magnification)

Patients who had been diagnosed with any other OPMD, patients under treatment for OL and OSMF, patients with other medically compromised conditions, long-term medications, pregnant and lactating mothers and patients with oral manifestations of chronic infection or inflammatory disorders were excluded from the study.

About 2.5 ml of peripheral blood was collected using a standardized phlebotomy technique from patients and was stored in an EDTA (ethylene diamine tetra-acetic acid) vacutainer. About 3 ml of an unstimulated salivary sample was collected in a 15 ml centrifuge tube from the study participants by the drooling method. Resultant supernatant of the centrifuged (3500 rpm for 10 min at 4°C) samples was aliquoted and stored for further analysis in -80°C. 8-epi-PGF2α (8‐Epi-Prostagladin F2 Alpha) Competitive-ELISA Kit (Product Number: E-El-0041), manufactured by Elabscience, Houston, TX, USA, was used for performing enzyme-linked immunosorbent assay (ELISA) using the standard protocol and was further analysed using the Semi-Automatic ELISA plate Analyser, (MULTISKAN-EX, manufactured by Thermo Scientific).

RESULTS

A total of 72 samples, including both saliva and plasma, were taken for the study, 12 from each group. IBM SPSS Statistics for Windows, Version 23.0 (Armonk, NY: IBM Corp) software was used for the evaluation of 8-isoprostane levels and Mann–Whitney test, chi-square test and Kruskal–Wallis test were used for statistical analysis.

The data showed a wide range (20–70 years) of sample distribution and also showed a higher male (n = 26) than female (n = 10) participants, in all three groups [Table 1].

Table 1.

Age and Gender data showing sample distribution among the three groups

Variable		Control	Leukoplakia	OSMF
Age	20-29	5	1	0
	31-39	4	3	2
	40-49	3	3	5
	50 and above	0	5	5
Sex	Male	6	9	11
	Female	6	3	1

OSMF: Oral submucous fibrosis

The comparison of the differences in the mean between plasma and salivary 8-isoprostane levels of OSMF, OL and HC denoted increased levels of 8-isoprostane in OPMDs than in HC, which was statistically significant [Figure 3 and Table 2].

Figure 3.

Correlation of plasma and salivary 8-isoprostane levels of the OSMF, OL and HC OSMF: Oral submucous fibrosis; OL: Oral leukoplakia; HC: Healthy controls

Table 2.

Comparison of mean plasma and salivary 8-isoprostane levels of the OSMF, OL and HC

Group		Mean	SD	P
Plasma	OSMF	579.48	164.16	0.005
	OL	706.46	74.27	0.012
	HC	481.12	179.85	0.008
Saliva	OSMF	122.30	94.40	0.005
	OL	99.76	68.28	0.012
	HC	38.30	24.53	0.008

OSMF: Oral submucous fibrosis; OL: Oral leukoplakia; HC: Healthy controls; SD: Standard deviation

The plasma levels of 8-isoprostane were shown to be higher in OPMD than HC, and on comparison between the different OPMDs, an increased expression was observed in OL when compared to the OSMF group. The levels were found to be statistically significant [Figure 3 and Table 2]. The mean levels of salivary 8-isoprostane were 122.3 ng/ml, 99.76 ng/ml and 38.31 ng/ml, respectively, for OSMF, OL and HC. The levels were statistically significant [Figure 3 and Table 2].

DISCUSSION

The current study was done to compare the expression of oxidative stress marker, namely, 8-isoprostane, in OL and OSMF patients in plasma and saliva. Over the past two decades, oxidative stress and ROS have been recognized as major aetiological factors that pose a substantial risk of malignant transformation in OPMD. Significant research has been conducted regarding the enzymes MDA (malondialdehyde) and SOD (superoxide dismutase), evaluating their potential role in the disease pathogenesis of OPMD and OSCC.[11] The potential role of oxidative stress markers, specifically MDA and 8-hydroxy-2’-deoxyguanosine (8-OHdG), has been established by the meta-analysis on OSMF, which revealed their increased expression.[12] In the present study, both plasma and salivary 8-isoprostane levels showed significant differences. The mean concentration of 8-isoprostane in patients with OPMD was higher than that in healthy controls. Among the OPMD subgroups, plasma 8-isoprostane levels were notably elevated in the OL group, while salivary levels were higher in the OSMF group.

Increased ROS levels along with reactive nitrogen species (RNS) and decreased concentrations of enzymatic and non-enzymatic antioxidants have been demonstrated in OPMD and OSCC. Overexpression of NADPH oxidase (NOX), Cyclooxygenase (COX) and similar enzymes, which are produced from inflammatory cells, fibroblasts and epithelial cells, is a major source of ROS.[13] ROS in turn activates the proteins, which leads to the transcription of genes associated with cell proliferation, regulation and apoptosis, influencing cancer progression.[14] The imbalance between the ROS and antioxidant defence mechanisms is a major stimulant for oxidative stress.[15] When ROS reacts with specific polyunsaturated fatty acids like arachidonic acid, lipid peroxidation occurs, forming a stable derivative named 8-isoprostane.[16]

Products of betelnut and tobacco trigger increased oxidant levels, leading to ROS formation, which acts crucially in the malignant transformation of OSMF and OL, making these disease conditions have the highest malignant transformation rate (MTR) among OPMDs.[10,17,18,19,20] Oxidative stress biomarkers are one of the reliable soluble biological fluid mediators measured in saliva.[21] However, ROS and free radicals are used by polymorphonuclear neutrophils (PMNs) to maintain the normal commensals of the oral stoma, thereby justifying higher plasma 8-isoprostane levels in OL patients.[17,20] Longer duration of chewing and stalking the quid of betelnut or pan leads to a sustained and steady increase in ROS levels, causing oxidative damage.[22] This theory justifies the resultant higher salivary 8-isoprostane levels in OSMF patients observed in the present study.

As per the literature, 8-isoprostane was studied in smokers,[23] smokers compared with non-smokers,[24] oral leukoplakia[6] and oral lichen planus.[25] Plasma was widely used as a sample when compared to saliva. Only one study has been carried out comparing both the plasma and saliva, but it has compared the 8-isoprostane levels in OSMF and OSCC, in which OSCC values were higher.[7] In our study, the estimated levels of 8-isoprostane were significant and higher in OPMD groups than in HC in both saliva and plasma. Since the collection of saliva is non-invasive, non-technique sensitive, cost-effective and has no risk of vessel injury or infection, it may be considered as a promising diagnostic tool for the detection of 8-isoprostane. However, replication of results in different settings with larger samples would be required for translational use of salivary 8-isoprostane from benchside to chairside.

The limitation of the present study is the smaller sample size and the non-inclusion of OSCC as one of the study groups to assess its expression. This would have enabled us to quantify the expression with respect to the underlying malignant potential. The current study adds yet another evidence to the literature regarding the potential role of 8-isoprostane as a potent diagnostic marker, with an indication of its utility in saliva as the diagnostic sample.

CONCLUSION

The current study revealed demonstrable measures of 8-isoprostane in plasma and saliva samples of OSMF and OL patients. Our analysis reported significantly higher levels of salivary 8-isoprostane in OSMF and increased expression of plasma 8-isoprostane in OL, when compared to HC. Hence, the potential role of saliva as an alternative diagnostic tool can be considered, especially for disease monitoring in OPMD.

Institutional review board statement

The study was conducted in accordance with the Declaration of Helsinki and approved by the Institutional Review Board of Sri Ramachandra Institute of Higher Education and Research (IEC No. CSP/21/JUL/96/383) for studies involving humans.

Informed consent statement

Informed consent was obtained from all patients involved in the study.

Data availability statement

The study did not report any additional data.

Conflicts of interest

There are no conflicts of interest.

Funding Statement

Nil.