Oxidative stress in otosclerosis

Abstract

Objectives: Otosclerosis is a disease involving abnormal bone turnover in the human otic capsule that results in hearing loss. Several hypotheses have been suggested for the etiopathogenesis of otosclerosis; however, its etiology remains unclear.

Methods: This study evaluated the correlation between otosclerosis and levels of paraoxonase-1 (PON1), arylesterase, total antioxidant status, total oxidant status (TOS), oxidative stress index (OSI), total sulfhydryl (-SH) groups, lipid hydroperoxide, and ceruloplasmin in the serum of otosclerosis patients and healthy subjects with respect to oxidative stress.

Results: In our study, TOS and OSI levels were higher in the otosclerosis patients than in the controls. The PON1 levels showed that oxidative stress was severe, and as a result, antioxidants were consumed and depleted.

Discussion: When an imbalance between oxygen free radical production and antioxidative defense mechanisms occurs, reactive oxygen species levels may increase, which in turn may damage cells and tissues through the peroxidation of phospholipid membrane structures. The body initially responds with increased antioxidant production, but if the oxidative stress is severe, decreased antioxidant levels may result. This study reports expression levels of oxidative stress species in otosclerosis patients.

Keywords:

• Oxidative stress
• Otosclerosis

Introduction

Otosclerosis is a disease characterized by abnormal bone turnover in the human otic capsule and results in hearing loss.¹ The incidence of otosclerosis among Westerners is 0.5%, and 80% of cases are bilateral in nature. Otosclerosis occurs more frequently among women than men.^2,3 Several hypotheses have been proposed for the etiopathogenesis of otosclerosis; however, the exact etiology remains unclear. Oxidative stress and reactive oxygen species (ROS) have been associated with many types of hearing loss such as age-related, noise-induced, and drug ototoxicity-related hearing loss. Rudic et al. studied the involvement of Ang II and HNE (oxidative stress) in the pathophysiology of otosclerosis with a cultured human bone cell population, and their results support the hypothesis of the important effect of oxidative stress in bone growth regulation and remodeling.¹ In otosclerosis, ROS can be produced in otosclerotic foci and disperse into the inner ear through the cochlear endosteum, resulting in cochlear damage and sensorineural hearing loss. Therefore, the relationship between ROS and otosclerosis warrants further investigation.

Oxidation and reduction reactions in biological systems (redox reactions) have the potential to induce numerous metabolic changes. The generation of ROS is a normal result of any type of aerobic life. Under normal conditions, cells metabolize approximately 95% of the available oxygen without forming any toxic intermediates. Aerobic organisms need ground-state oxygen to survive. However, the use of oxygen during normal metabolism produces ROS, and some ROS are highly toxic and harmful to cells and tissues.⁴ The predominant ROS formed during the course of cellular metabolism is the superoxide radical (O•-2).⁵ Low-level ROS mediate many cellular activities, including differentiation, cell cycle progression or growth arrest, apoptosis and immunity. However, large amounts and/or the inadequate removal of ROS result in oxidative stress, as observed during inflammation, which may cause severe metabolic derangement and damage to biological macromolecules. Aerobic cells developed their own defenses through the antioxidant system, which controls ROS concentrations using enzymatic and non-enzymatic means.⁶ Paraoxonase-1 (PON1) is a high-density lipoprotein (HDL)-associated enzyme with three activities: paraoxonase (PON), arylesterase (ARYL), and dyazoxonase. PON protects low-density lipoproteins (LDLs) and HDLs from oxidation.⁷ Mackness et al. demonstrated the antioxidant activity of PON1 for the first time in 1991.⁸ Human serum PON and ARYL are enzymes of the esterase group that are encoded by the same gene and have similar active centers.⁹ ARYL is recognized as an antioxidant enzyme because it hydrolyzes lipid peroxides and oxidizes lipoproteins as PON. PON and ARYL have been reported to decrease in many patients with different systemic diseases.^10–14

The total antioxidant status (TAS), total oxidant status (TOS), and oxidative stress index (OSI) are used to determine the oxidant/antioxidant balance within an individual. Ceruloplasmin (Cp) is considered a preventive plasma antioxidant because it sequesters transition metals, thereby preventing them from participating in free radical reactions. Another antioxidant group is the total sulfhydryl (-SH) group, which plays a crucial role in protecting cells from oxidative damage by interacting with the electrophilic group of ROS as the first and major member of the physiological antioxidant defense system. Enzymes and proteins with free SH groups have antioxidant characteristics. Lipid hydroperoxide (LOOH) is a well-known marker of oxidative stress formed from unsaturated phospholipids, glycolipids, and cholesterol.^4,15

To the best of our knowledge, no experimental study has investigated PON and ARYL activities and TOS, TAS, OSI, LOOH, SH, and Cp levels in the serum of patients with otosclerosis. Therefore, the aim of the present study was to investigate these oxidants (LOOH, TOS, and OSI) and antioxidants (TAS, PON, ARYL, SH, and Cp) in the serum of patients with otosclerosis.

Materials and methods

The Local Ethics Committee approved this study. Informed consent was obtained from all patients, and all procedures were performed in accordance with the Declaration of Helsinki.

Patients with chronic systemic diseases (cardiovascular, pulmonary, liver, renal, or hematologic diseases), malignancies, or who chronically used medication were excluded from the study. Patients were diagnosed via history and routine otoscopic, otomicroscopic and audiologic evaluations. The participants were also clinically evaluated during surgery by the otoneurologist. The patients who were surgically diagnosed with otosclerosis were enrolled in the study. Serum was collected from each patient before any medical or surgical intervention and after fasting overnight in the Gaziantep University Otolaryngology Department. Blood serum was collected from 32 Caucasian patients with otosclerosis and 50 Caucasian healthy individuals for this study.

Measurement of PON and ARYL activity

The PON activity measurements were performed in the absence (basal activity) and presence of NaCl (salt-stimulated activity). The rate of paraoxon hydrolysis (diethyl-p-nitrophenylphosphate) was measured by monitoring the increase in absorbance at 412 nm at 25°C. The amount of generated p-nitrophenol was calculated from the molar absorptivity coefficient at pH 10.5, which was 18.290 M⁻¹ cm⁻¹.¹³ PON activity was expressed as U/l serum. Phenylacetate was used as a substrate to measure the ARYL enzyme activity. Enzymatic activity was calculated from the molar absorptivity coefficient of the produced phenol (1310 mol/cm). One unit of ARYL was defined as 1 μmol phenol generated per minute under the above conditions and expressed as U/L serum.^9,16

TAS measurements

The TAS level was measured using a colorimetric method that was introduced by Erel (Rel Assay# Diagnostics kit, Mega Tip, Gaziantep, Turkey).¹⁷ In this method, hydroxyl radicals, the most potent type of radical, were created via a Fenton reaction. In this reaction, hydroxyl radicals are produced by mixing a hydrogen peroxide solution with a ferrous ion. In the assay, the ferrous ion solution present in Reagent 1 is mixed with hydrogen peroxide, which is present in Reagent 2. The resultant radicals, including the brown-colored dianisidinyl radical cation, which is produced by the hydroxyl radical, are also potent radicals. In this assay, the antioxidative effect of the sample was measured against the potent free radical reactions initiated by the produced hydroxyl radical. The assay produced excellent precision values, which were lower than 3% (the coefficient of variation). The results are expressed as mmol Trolox equiv./L.¹⁷

TOS measurements

The serum TOS levels were determined using a novel automated measurement method of Erel.¹⁶ The oxidant present in the sample oxidized the ferrous ion-o-dianisidine complex to form ferric ions. The oxidation reaction was enhanced by the glycerol molecules present in the reaction medium. In an acidic medium, ferric ions formed a colorful complex of xylenol orange. The intensity of the color, which was measured with a spectrophotometer, was related to the total amount of oxidant molecules present in the sample. The assay was calibrated with hydrogen peroxide. The results are expressed as μmol H2O2 equiv./L.

OSI calculation

The OSI is the ratio of the TOS to the TAS. To calculate the OSI, the TAS was multiplied by 10 and normalized to the TOS. The results are reported as arbitrary units (AU).

Lipidperoxide (LOOH) measurements

The amount of LOOH was measured by an automated method using xylenol orange. In this method, LOOH oxidizes ferrous ions to ferric ions. The produced ferric ions form a colored mega complex with xylenol orange. The absorbance was measured at 570 nm 12.^18–20

Cp measurements

Cp enzymatic activity was assayed according to Erel's Cp measurement method.²⁰ Using this assay, ferrous ions are oxidized to ferric ions via Cp ferroxidase activity.⁹ The results are expressed as U/L.

Measurement of SH groups in the serum samples

The free -SH groups in the serum samples were measured according to the method described by Ellman and modified by Hu et al.^21,22

Statistical analyses

A Mann–Whitney U test was used to compare the variables between the two groups. SPSS 22.0 for Windows (Chicago, IL, USA) was used for all statistical analyses, and P values <0.05 were considered significant.

Results

The patient group included 32 patients (13 males, 19 females) with a mean age of 34.45 years (max: 47, min: 25), and the control group included 50 age-related healthy subjects. The mean duration of complaints was 44 months (12–120 months), and the mean hearing loss of the involved ear was 52 dB (38–75 dB, standard deviation 7.9 dB). The mean LDL levels were 143 mg/dl (min: 125, max: 176), and the mean HDL levels were 41.61 mg/dl (min: 35, max: 55). The mean ALT levels were 30.3 mg/dl (24–36), and the mean AST levels were 36.21 mg/dl (27–47). Seven patients had unilateral otosclerosis, whereas 25 patients had bilateral otosclerosis. The data obtained from the measurements are given in table (Table 1).

Table 1 Parameters of otosclerosis patients and control individuals. OS: Otosclerosis patients

	Groups	N	Mean ± Standard deviation	P
Arylesterase	Control	50	147.02 ± 34.82	0.401
	OS	32	140.2410 ± 36.6192
LOOH	Control	50	5.12 ± 0.96	0.000
	OS	32	15.94 ± 4.78
TOS	Control	50	13.53 ± 3.30	0.000
	OS	32	26.15 ± 8.14
PON	Control	50	91.31 ± 22.32	0.013
	OS	32	79.86 ± 15.19
SH	Control	50	0.48 ± 0.06	0.003
	OS	32	0.43 ± 0.05
Cp	Control	50	795.08 ± 102.87	0.003
	OS	32	886.01 ± 162.63
TAS	Control	50	1.10 ± 0.19	0.001
	OS	32	0.94 ± 0.21
OSI	Control	50	1.24 ± 0.32	0.000
	OS	32	2.92 ± 1.26

Serum TOS, OSI, Cp, and LOOH levels were significantly higher in the otosclerosis patients (TOS: 26.15± 8.14 μmol H2O2 equiv./L, OSI: 2.92 ± 1.26 AU, Cp: 886.01±162.63 U/L, LOOH: 15.94±4.78 μmol/L) than in the controls (TOS: 13.53±3.30 μmol H2O2 equiv./L, OSI: 1.24±0.32 AU, Cp:795.08±102.87 U/L, and LOOH: 5.12±0.96 μmol/L) P < 0.005. Serum TAS and SH levels and PON1 activity were significantly lower in the otosclerosis patients (TAS: 0.94±0.21 mmol Trolox equiv./L, SH: 0.43±0.05 mmol/L, and PON:79.86 ±15.19 U/l serum) than in the controls (TAS:1.10±0.19 mmol Trolox equiv./L SH: 0.48±0.06 mmol/L and PON: 91.31±22.32 U/l serum) P < 0.005. Serum ARYL activity was not significantly different between the Otosclerosis (ARYL: 140.24±36.61 U/l serum) and control groups (ARYL:147.02±34.82 U/l serum) P = 0.401. The measured data showed no statistically significant differences between male and female otosclerosis patients (Table 2).

Table 2 Differences according to gender (male versus female)

Gender difference among the levels of	@tas	@looh	@arylest	@sh	@osi	@serulop	@pon	@tos
Asymp. Sig. (2-tailed)	0.762	0.897	0.387	0.187	0.226	0.489	0.226	0.489

Discussion

This study evaluated the levels of PON, ARYL, TAS, TOS, OSI, SH, LOOH, and Cp in the serum of otosclerosis patients and healthy subjects with respect to oxidative stress. When the levels of ROS production and antioxidative defense mechanisms are unbalanced, ROS levels may increase, which in turn may damage cells and tissues through the peroxidation of phospholipid membrane structures.⁵ PON1 is one of the most important antioxidant enzymes in the human body and has PON, ARYL, and dyazoxonase activity. Human serum PON1 and ARYL protect against oxidative stress. Human serum PON catalyzes the hydrolysis of the organophosphate paraoxon into nontoxic products. In some studies, PON inhibits the ROS-induced oxidation of cell membrane lipids that leads to acute and chronic inflammation.^7–9 The role of PON has been previously reported for some diseases.^{7,11–14,18–30} In this study, the antioxidant capacity and oxidative stress marker levels differed between the otosclerosis patients and controls. An association between ROS and myringosclerosis was previously reported. Shigemi et al. reported that ROS may be related to the chronicity of OME.³¹ To our knowledge, this report is the first published study of serum PON and ARYL enzyme activity, oxidative status and antioxidant enzymes in patients with otosclerosis. Inflammation of the middle ear increases ROS production in the blood.³² When oxidative stress increases and the levels of antioxidant defense enzymes decrease, the subsequent inflammation (free radicals) may contribute to otic capsule inflammation. The pathological activation of osteoclasts in otosclerosis is associated with chronic inflammation.³³ This process is mediated by pro-inflammatory cytokines.^34,35

In our study, TOS and OSI levels were higher in the otosclerosis patients than in the controls. The PON levels indicated that oxidative stress was severe resulting in antioxidant consumption and depletion. The body initially responds by increasing antioxidant production. However, if the oxidative stress is severe, decreased antioxidant levels may result. Although this study quantified the expression levels of oxidative stress species in otosclerosis patients, it had several limitations. One limitation is the lack of stapes footplate and middle ear mucosa biopsies due to ethical issues. These biopsies may have helped determine the oxidative and antioxidative status and enzyme activity histopathologically. Second, we studied a limited number of cases. Future studies with more cases and histopathological examinations that determine cytokine expression in individuals with a genetic predisposition should be conducted. Increased oxidative stress results in increased antioxidant production. However, severely increased oxidative stress will consume and decrease the levels of antioxidants. We suggest that oxidative stress is increased in otosclerosis patients. According to our results, oxidative stress and antioxidant enzyme imbalances are more severe in otosclerosis patients than in normal subjects. This finding must be investigated in future studies.

In conclusion, otosclerosis is a complex bone remodeling disease of the human otic capsule that most likely results from an imbalance between cell survival and apoptosis. This imbalance may be triggered by impaired oxidative metabolism. Additionally, certain gene mutations, viral infections, autoimmune-inflammatory mechanisms, and hormonal and metabolic factors that disturb bone metabolism may contribute to the etiopathogenesis of otosclerosis. Because the objective diagnosis of otosclerosis is based on the histopathological examination of the stapes footplate, prospective clinical studies based on comprehensive histopathological and molecular biological and biochemical analyses are necessary to evaluate the background of the disease and determine the optimal medical treatment for otosclerosis.

Disclaimer statements

Contributors All authors contributed equally.

Funding None.

Conflicts of interest There is no conflicts of interest.

Ethics approval Ethical committee has approved this study.