Association between polymorphism of interleukin-6 in the region -174G/C and tinnitus in the elderly with a history of occupational noise exposure.

UNLABELLED: Tinnitus is a symptom usually related to cochlear change that may arise from noise exposure and induces expression of proinflammatory cytokines including interleukin-6 (IL6). This study aimed to evaluate the association between the polymorphism of the IL6 gene in the region 174G/C and tinnitus in elderly with history of occupational noise exposure. SETTINGS AND
DESIGN: This was a cross-sectional study with a sample of 179 independent elderly individuals aged >60 years. Information on exposure to occupational noise was obtained by interviews. Audiological evaluation was performed using pure tone audiometry and genotyped through polymerase chain reaction by restriction fragment length polymorphism (PCR-RFLP). Data were analyzed using the chi-square test and the odds ratio (OR), with the significance level set at 5%. Among the study subjects, 24.6% were homozygous for the G allele, 39.7% were homozygous for the C allele, and 35.8% were heterozygous for IL6 (P > 0.05). Of these, 33.5% reported noise exposure history, with 42.5% having tinnitus. We found significant association between the genotype and allele frequencies of the IL6 -174 gene (rs1800795) and tinnitus among the elderly with history of exposure to occupational noise (P = 0.03). The elderly with the C allele were less likely to have tinnitus associated with history of exposure to occupational noise [OR = 0.167, confidence interval (CI) 95% 0.167-0.749; P = 0.004] when compared to those carrying the G allele. This study suggests that there is an association between polymorphisms in the IL6 gene at region - 174G/C and susceptibility to tinnitus.

Introduction

There has been an increase in the aging population in both developed countries and developing countries.[1] It is estimated that by 2040, developing countries will have 1 billion people aged 60 or older.[2] Given the high speed and extent of this growth, care for this particular group is essential so that they can age in good health with good quality of life.[3]

Tinnitus is a poorly understood auditory percept that occurs in the absence of an external stimulus and is typically associated with hearing loss.[4] It is a highly prevalent symptom worldwide and affects about 15% of Americans.[5] A study to determine the prevalence of tinnitus in elderly Nigerians and their impact on quality of life found that tinnitus was present in 14.1% of the population. The authors concluded that tinnitus is associated with treatable health conditions, such as otitis media, sinusitis, head injuries, noise, and hypertension.[6] In a study in Australia that evaluated 602 subjects, the buzz was present in 30.3%, being present for at least 6 years in 50% of cases. Despite tinnitus being described as mildly to extremely annoying by 67%, only 37% had sought professional help, and only 6% had received any treatment.[7]

Especially in the elderly, various metabolic and circulatory disorders that may be associated with exposure to occupational noise can cause various symptoms such as hearing loss, vertigo, and tinnitus.[38910]

The presence of inflammatory cells in the stationary state and its increase after lesions in the inner ear has been reported by several researchers.[11121314]

Noise exposure induces expression of proinflammatory cytokines, including tumor necrosis factor-α (TNF-α), interleukin-1b (IL1b), and interleukin-6 (IL6).[12] So et al. (2007) reported a transient upregulation of IL6 in animal models treated with cisplatin. Wakabayashi et al. (2010) investigated the effect of inhibiting IL6 using an anti-IL6 antibody (MR16-1) in mice. These authors found that MR16-1 showed a protective effect against noise-induced cochlear damage, mainly due to the suppression of neuronal loss and presumably through the relief of the inflammatory response. Similar data were found by Nakamoto et al. (2012). These authors suggested that the suppression of heat shock transcription factor 1 (HSF-1) proinflammatory cytokine in the cochlea by the administration of geranylgeranylacetone (GGA) can be an important way to protect the inner ear.

The expression of cytokines may be influenced by genetic variation, resulting in pathogenic conditions,[15] and several studies have investigated single nucleotide polymorphisms (SNPs) as risk factors for inflammatory diseases.[16] These SNPs may affect the expression, secretion, and cellular transport of interleukins,[1718] and can also decrease the level of IL1 receptor antagonist (IL1Ra), which increases the production and activity of IL1β.[19]

−174 SNP in the region of the IL6 gene is the exchange of G to C, while the presence of the C allele is a protective function due to reduced production of IL6.[20]

While tinnitus is a symptom usually related to cochlear change that may arise from noise exposure, this study aimed to evaluate the association between the polymorphism in IL6 and the complaint of tinnitus with a history of occupational noise exposure in the environment by physically independent elderly Brazilians.

Methods

Study population

This cross-sectional study was approved by the Ethics Committee on Human Research of the University of North Paraná (0070/09). It is part of a wider research project, “Study on aging and longevity,” which the study has been conducted in Londrina in 2009. The city of Londrina (about 500,000 inhabitants) is located in the northern state of Paraná, Brazil.

From a population of 43,610 elderly enrolled in 38 basic health units in the urban area of the city, the sample size was set at 343 individuals, considering a 95% confidence interval (CI) and a margin of error of 5% 21. With the aim of sampling representativeness, random stratification of regions of the city and gender was done. The study included individuals aged 60 years or more, of both sexes, who were living independently, and at level 3 or 4 according to the classification proposed by Spirduso.[21] This rating assesses the level of independence of the elderly, with Level 1 indicating a lack of self-mobility and Level 5 indicating athletes. Elderly people who had an illness or limitation preventing the performance of the tests, such as a physical or mental disability, were excluded from the sample. All participants signed a free and informed consent form.

Audiological assessment

In order to accomplishment of research in relation to the audiologic data, routine audiological evaluation from Audiology Department of the Clinic of Speech, University of Northern Paraná (UNOPAR) based on the protocol for anamnesis by Miller, incluinding question about age, gender and tinnitus.[22]

Evaluation of occupational noise exposure

The assessment of occupational noise exposure was obtained through interviews with the elderly participants using a semistructured questionnaire. We collected information on the following: If work environment was noisy or not, how many years of work in a noisy environment, and whether they wore hearing aids. In addition, demographic characteristics were collected.

Genotyping for IL6 174G/C (rs1800795) gene polymorphism

Peripheral blood samples were collected in vacuum tubes containing 6% ethylenediaminetetraacetic acid (EDTA), and DNA was extracted from peripheral blood leukocytes, using the protocol described by Olerup and Zetterquist.[23]

Based on the original sample, the DNA was diluted to a concentration of 100 ng/μL. The concentration was determined by spectrophotometer at 260 nm and 280 nm (Biomate 3, Thermo Fisher Scientific, Madison, Wisconsin USA).

The G-to-C polymorphic site located at position −174 of the IL6 (rs1800795) gene was amplified by polymerase chain reaction (PCR), resulting in a 99-bp fragment. The PCR mixture contained a mixture of a buffer solution pH 8.0, 1× PCR buffer, 1.5 mM of MgCl2, 8 mM each of deoxyribonucleotide triphosphates, 1 μM of each primer, and 1 U Taq DNA polymerase (Invitrogen, Carlsbad, CA, USA). The primers used were F: 5’-TTGTCAAGACATGCCAAGTGCT-3’ and R: 5’ GCCTCAGAGACATCTCCAGTCC-3’ (Invitrogen, Carlsbad, CA, USA).[24]

PCR amplification was performed in a thermocycler (TC-512 Techne resistance, Burlington, NJ, USA) under the following conditions: initial denaturation at 95°C for 5 min followed by 30 cycles of 95°C for 1 min, 56°C for 1 min, and 72°C for 1 min, and final extension at 72°C for 5 min.

The PCR product was digested with 2 U of restriction enzyme NlaIII (Invitrogen, Carlsbad, CA, USA) overnight at 65°C. The digested fragments were separated on 2% agarose gel (Invitrogen Life Technologies, São Paulo, Brazil). A sample with known genotype was used as positive PCR control and ultrapure water was used as negative control.

The 100-bp DNA molecular weight marker (Ladder, Invitrogen, Waltham, Massachusetts, USA) was included on each gel stained with SYBR Safe (Invitrogen Life Technologies, São Paulo, Brazil) and visualized under ultraviolet (UV) illumination. The reading and interpretation of agarose gels were done with the LabImage L-PIX (HE) 1D-L340 (Loccus Biotecnologia, São Paulo, Brazil) program. Fragments of 13 bp, 227 bp, and 59 bp (allele G) and fragments of 13 bp, 118 bp, 109 bp, and 59 bp (allele C) were observed.

Statistical analysis

Data analysis was performed with IBM SPSS Statistics 20.0 software (IBM Corporation Software Group, New York, USA). To verify the association between genotype frequency and tinnitus related to a history of occupational noise exposure, the chi-square test was performed. The Hardy–Weinberg equilibrium was tested in each group using the chi-square test. The odds ratio (OR) with 95% CI was calculated. The significance level was set at 5%.

Results

Of the total of 343 subjects, molecular genetics procedures were conducted in 179 elderly. Of these, 33.5% reported noise exposure history, with 42.5% having tinnitus. Of the 66.5% without noise exposure history, 28.5% had tinnitus. The mean age was 68.9 ± 5.25 years, with a higher proportion (65.4%) of female elderly [Table 1].

Table 1

General characteristics and allele/genotypes frequencies among elderly (N = 179)

Characteristics	N	(%)
Gender
Male	62	34.6
Female	117	65.4
Age (years)
60-64	56	31.2
65-74	101	56.5
75+	22	12.3
Mean age=68.9 (SD=5.25)
Groups
Tinnitus with history of occupational noise-exposure	60	33.5
Tinnitus without history of occupational noise-exposure	119	66.5
Genotypic frequency
GG	44	24.6
CC	71	39.7
GC	64	35.8
Allelic frequency
G	152	42.4
C	206	57.6

SD = Standard deviation, GG = Wild-type homozygotes, CC = Mutant homozygotes, GC = Heterozygotes

Of the patients included, 24.6% were homozygous for the G allele, 39.7% were homozygous for the C allele, and 35.8% were heterozygous for IL6 [Table 1]. The genotype distribution for the IL6 gene was in agreement with Hardy–Weinberg equilibrium (P > 0.05).

We found significant association between the genotype and allele frequencies of the IL6 −174 gene (rs1800795) and tinnitus among the elderly with history of exposure to occupational noise (P = 0.03) [Table 2]. The elderly with the C allele were less likely to have tinnitus associated with history of exposure to occupational noise (OR = 0.167, CI 95% 0.167-0.749; P = 0.004) when compared to those carrying the G allele.

Table 2

Association between genotype frequency for the IL6 174G/C gene polymorphism and tinnitus related to history of occupational noise exposure (N = 179)

Occupational noise exposure	Genotyping	No tinnitus N (%)	With tinnitus N (%)	P value and chi-square test
No history	Genotypic frequency			P=0.477
	GG	07 (8.0)	2 (06.5)	χ2=1.48
	CC	52 (59.1)	15 (48.4)
	GC	29 (33.0)	14 (45.2)
	Allelic frequency			P=0.475
	G	43 (24.4)	18 (29.0)	χ2=0.509
	C	133 (75.6)	44 (71.0)
With history	Genotypic frequency			P=0.031
	GG	22 (48.9)	13 (86.7)	χ2=6.771
	CC	4 (08.9)	00 (0.0)
	GC	19 (42.2)	02 (13.3)
	Allelic frequency			P=0.011
	G	63 (70.0)	28 (93.3)	χ2=6.68
	C	27 (30.0)	02 (06.7)

However, we did not find any significant association between the genotypic frequency of the IL6 -174 gene (rs1800795) and tinnitus among the elderly without considering history of exposure to occupational noise [Table 3].

Table 3

Association between genotype frequency for the IL6 174G/C gene polymorphism and tinnitus (N = 179)

Tinnitus	Genotyping IL6			P
	GG N (%)	GC N (%)	CC N (%)
No tinnitus	25 (56.8)	35 (54.7)	43 (60.6)	0.783
With tinnitus	19 (43.2)	29 (45.3)	28 (39.4)

(χ2 = 0.488)

Discussion

Tinnitus is more challenging than many other health conditions because there is no objective measurement of the condition and no consensus regarding diagnostic assessment.[25] Although the mechanism and the role of proinflammatory cytokines in noise-induced hearing loss (NIHL) and tinnitus are not yet well understood, the SNP present results in obvious pathological consequences (Satoh et al. 2003). The functionality of SNPs with respect to gene expression is an important subject for disease association studies (Dinarello 2011) and may be useful regarding diagnostic approach in tinnitus.

An association was found between the IL6 allele and tinnitus with history of exposure to occupational noise (χ2 = 6.771; P = 0,031). The elderly with the C allele are less likely to have tinnitus as a result of occupational exposure to noise when compared to those carrying the G allele (OR = 0.167, CI 95%, 0.037-0.749; P = 0.019). However, it is stated that the presence of the C allele results in a lower expression of IL6 after an inflammatory stimulus compared with the G allele, based on the belief that the CC genotype confers a protective effect against the development of comorbidities.[26]

Fujioka et al.[12] indicated the possibility of inflammatory changes in the cochlea after noise stimulus, and demonstrated for the first time the induction of proinflammatory cytokines in the cochlea exposed to noise and observed the increased expression of TNF-α, IL1b and IL6, 3 h after noise exposure. These authors pointed out that this mechanism is self-protection against exposure to large amounts of noise, and the consequent excessive expression of interleukins for long periods should worsen the cochlear function.

So et al.[13] showed that IL6 acts as an inducer of acute cochlear damage, as after exposure the animals with cisplatin IL6, there was a transient upregulation. Some studies have shown that inhibition of IL6 production presents a cochlear protective effect. Wakabayashi et al.[14] used anti-IL6 antibody (MR10-1) to inhibit IL6 production in mice by finding a protective effect for noise-induced cochlear injury. Along the same line of research, Nakamoto et al.[27] found that GGA stimulates expression of the HSF gene, which, in turn, inhibits the inflammation of the inner ear protecting the cochlea.

It should be noted that these strong evidences of the association of IL6 with cochlear changes related to noise have been observed through research in animal models.[12131427]

Evidence in humans related to proinflammatory interleukins and NIHL was recently reported by our group, were we investigated the association between the polymorphism of the interleukin (IL)-1β gene with complaints of hearing loss due to occupational noise exposure. The polymorphism in this gene has not been shown to be associated with NIHL in the evaluated elderly.[28] But subsequent findings showed that polymorphism in the IL6 gene should contribute to the risk of NIHL in elderly Brazilians.[29]

A recent study cites that inflammatory responses occur in the inner ear under various adverse conditions, including overstimulation with noise, although an association between the proinflammatory cytokines is rarely reported.[12] In addition, a study of a relaxing training program indicated that in individuals with chronic tinnitus symptoms, it can lead to significant reduction of stress, depression, anger, and perception of tinnitus, in parallel with a reduction of TNF-α.[30]

In this study, it can be seen that the frequency of G allele was higher in the group of elderly with history of occupational noise exposure and tinnitus (n = 28) than in the group with no history of occupational noise exposure and tinnitus (n = 18).

Conclusion

An association between the polymorphism of the IL6 gene in the region 174G/C and tinnitus in elderly with history of occupational noise exposure was found. The elderly with the C allele were less likely to have tinnitus associated with history of exposure to occupational noise (OR = 0.167, CI 95% 0.167-0.749; P = 0.004) when compared to those carrying the G allele.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.