Manganese in toenails is associated with hearing loss at high frequencies in humans

<p><b>Purpose:</b> Elevated hearing thresholds from high frequencies are known to be one of the hallmarks of age-related hearing loss. Our recent study showed accumulation of manganese (Mn) in inner ears resulting in acceleration of age-related hearing loss in mice orally exposed to Mn. However, there is no evidence showing an association between Mn in non-invasive biological samples and hearing loss in humans evaluated by pure tone audiometry (PTA). In this study, we evaluated Mn in non-invasive biological samples as a possible biomarker for hearing loss in humans.</p> <p><b>Materials and methods:</b> We determined hearing levels by PTA and Mn levels in toenails, hair and urine with an inductively coupled plasma mass spectrometer (ICP-MS) in 145 healthy subjects in Bangladesh.</p> <p><b>Results:</b> Multivariable analyses showed that Mn levels in toenails, but not in hair and urine samples, were significantly associated with hearing loss at 8 kHz and 12 kHz. Moreover, our experimental study showed a significant correlation between Mn levels in inner ears and nails, but not hair, in mice orally exposed to Mn.</p> <p><b>Conclusions:</b> The results provide novel evidence that Mn in toenails is a possible biomarker for hearing loss at high frequencies in humans.</p

Cigarette Smoking Causes Hearing Impairment among Bangladeshi Population

<div><p>Lifestyle including smoking, noise exposure with MP3 player and drinking alcohol are considered as risk factors for affecting hearing synergistically. However, little is known about the association of cigarette smoking with hearing impairment among subjects who carry a lifestyle without using MP3 player and drinking alcohol. We showed here the influence of smoking on hearing among Bangladeshi subjects who maintain a lifestyle devoid of using MP3 player and drinking alcohol. A total of 184 subjects (smokers: 90; non-smokers: 94) were included considering their duration and frequency of smoking for conducting this study. The mean hearing thresholds of non-smoker subjects at 1, 4, 8 and 12 kHz frequencies were 5.63±2.10, 8.56±5.75, 21.06±11.06, 40.79±20.36 decibel (dB), respectively and that of the smokers were 7±3.8, 13.27±8.4, 30.66±12.50 and 56.88±21.58 dB, respectively. The hearing thresholds of the smokers at 4, 8 and 12 kHz frequencies were significantly (<i>p</i><0.05) higher than those of the non-smokers, while no significant differences were observed at 1 kHz frequency. We also observed no significant difference in auditory thresholds among smoker subgroups based on smoking frequency. In contrast, subjects smoked for longer duration (>5 years) showed higher level of auditory threshold (62.16±19.87 dB) at 12 kHz frequency compared with that (41.52±19.21 dB) of the subjects smoked for 1-5 years and the difference in auditory thresholds was statistically significant (<i>p</i><0.0002). In this study, the Brinkman Index (BI) of smokers was from 6 to 440 and the adjusted odds ratio showed a positive correlation between hearing loss and smoking when adjusted for age and body mass index (BMI). In addition, age, but not BMI, also played positive role on hearing impairment at all frequencies. Thus, these findings suggested that cigarette smoking affects hearing level at all the frequencies tested but most significantly at extra higher frequencies.</p></div

Characteristics of the participants according to smoking status.

<p>^¶The subjects were categorized underweight, normal weight and overweight when the BMI was found <18.5, 18.5–25 and >25 kg/m², respectively.</p><p>Characteristics of the participants according to smoking status.</p

Effect of smoking on hearing level.

<p>Auditory thresholds (mean ± S.D) from 1 to 12 kHz frequencies in non-smoker ‘control’ (Brinkman index [BI = 0]; <i>n</i> = 94) and smokers (6 ≤ BI ≤ 440; <i>n</i> = 90) are shown. Smokers showed significantly (<i>p</i> = 0.0001) higher auditory thresholds than non-smokers at 4, 8 and 12 kHz frequencies.</p

Beam crossing test for LFN-exposed mice.

<p>ICR mice that had been exposed to LFN (n = 12) and to HFN (n = 10) and non-exposed mice (n = 12) were examined by a beam crossing test. The number of mice that fell from the beam (i.e., imbalance behavior) is shown as “Failure” in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039807#pone-0039807-t002" target="_blank">Table 2</a>. The number of mice crossing the beam without falling is shown as “Success” in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039807#pone-0039807-t002" target="_blank">Table 2</a>. P values were obtained by chi square analysis. **<i>p</i><0.01.</p

Decreased number of vestibular hair cells with increased levels of oxidative stress in LFN-exposed mice.

<p>(A, B) Immunohistochemical analysis with anti-calbindin D28k for vestibules in LFN-exposed (B) and non-exposed mice (A). (C-F) Enhanced oxidative stress levels in vestibules of LFN-exposed mice. Vestibules of LFN-exposed (D, F) and non-exposed mice (C, E) were immunohistochemically stained by an anti-Ox-PC antibody (DLH3) (C, D) and anti-D-βeta-Asp antibody (E, F). Vestibules of LFN-exposed mice showed stronger signals (D, F, arrows) than those of non-exposed mice (C, E). Scale bars: 20 µm. (G-I) Percentage (means ± SD) of calbindin-positive hair cells (G) and positive areas of anti-Ox-PC antibody (H) and anti-D-βeta-Asp antibody (I) in vestibules from LFN-exposed mice (LFN, black bar, n = 7) and non-exposed mice (Cont, gray bar, n = 7). Significant difference (*, <i>p</i><0.05) from non-exposed mice was analyzed by the Mann-Whitney <i>U</i> test.</p

Exposure to LFN affects gait pattern of ICR mice.

<p>(A) After exposure to LFN (center panel) and HFN (right panel), front and back paws of mice were dipped in red or green paint, and mice walked across a box lined with paper. Non-exposed mice (left panel) are also shown. LFN-exposed mice display shorter stride length and winding gait patterns (center panel, arrows). (B) Quantification of stride length. Strides (mean ± SD) for seven mice (each group) were assessed. A total of 40–50 steps for each group were determined. Significant difference (**, <i>p</i><0.01) from the non-exposure group was analyzed by the Mann-Whitney <i>U</i> test.</p

Typical low frequency noise levels of electric devices in experimental rooms.

<p>Noise levels (means ± SD) were measured by a noise level meter and calculated as an average of five repeated measurements. Noise levels were measured at a distance of approximately 20 cm from the devices shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039807#pone-0039807-t001" target="_blank">Table 1</a>. Background level (mean ± SD) of low frequency noise at 100 Hz was 35.7±2.7 dB SPL in 5 experimental rooms without noise-generating devices shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039807#pone-0039807-t001" target="_blank">Table 1</a>.</p

Frequency distributions of noise used in this study.

<p>Frequency distributions (means ± SD) of (A) low frequency noise (LFN; 0.1 kHz) and (B) high frequency noise (HFN; 16 kHz) are presented. Noise levels from a speaker at a distance of 10 cm in a soundproof room were measured by a noise level meter and calculated as average of five repeated measurements. Background levels measured in a soundproof room without noise-generating devices were subtracted from noise levels from the speaker. Whole noise levels of (A) LFN and (B) HFN measured by the noise level meter without FFT analyzing software were almost the same (70 dB SPL).</p

Effects of duration of smoking on hearing level.

<p>Auditory thresholds (mean± S.D) from 1 kHz to 12 kHz frequencies in non-smoker ‘control’ (n = 94) and smokers are shown. Based on duration of smoking, smokers were divided into 2 subgroups as smoked for 1–5 years (n = 23), and for more than 5 years (n = 67). The difference in auditory threshold between smokers smoked for 1–5 years and >5 years was statistically significant (<i>p</i> = 0.005) at 12 kHz frequency, however, the values were not found significant at all other frequencies (<i>p</i>>0.05).</p