Neurotoxicology and Teratology 44 (2014) 113–120
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Neuropharmacological and cochleotoxic effects of styrene. Consequences on noise exposures Pierre Campo ⁎, Thomas Venet, Aurélie Thomas, Chantal Cour, Céline Brochard, Frédéric Cosnier Institut National de Recherche et de Sécurité, Rue du Morvan, CS 60027, F-54519 Vandœuvre Cedex, France
a r t i c l e
i n f o
Article history: Received 28 February 2014 Received in revised form 21 May 2014 Accepted 28 May 2014 Available online 11 June 2014 Keywords: Noise Styrene Middle-ear reflex Combined exposure Risk assessment
a b s t r a c t Occupational noise exposure can damage workers' hearing, particularly when combined with exposure to cochleotoxic chemicals such as styrene. Although styrene-induced cochlear impairments only become apparent after a long incubation period, the pharmacological impact of styrene on the central nervous system (CNS) can be rapidly measured by determining the threshold of the middle-ear acoustic reflex (MER) trigger. The aim of the study was to evaluate the effects of a noise (both continuous and impulse), and a low concentration of styrene [300 ppm b (threshold limit value × 10) safety factor] on the peripheral auditory receptor, and on the CNS in rats. The impact of the different conditions on hearing loss was assessed using distortion product otoacoustic emissions, and histological analysis of cochleae. Although the LEX,8h (8-hour time-weighted average exposure) of the impulse noise was lower (80 dB SPL sound pressure level) than that of the continuous noise (85 dB SPL), it appeared more detrimental to the peripheral auditory receptors. A co-exposure to styrene and continuous noise was less damaging than exposure to continuous noise alone. In contrast, the traumatic effects of impulse noise on the organ of Corti were enhanced by co-exposure to styrene. The pharmacological effects of the solvent on the CNS were discussed to put forward a plausible explanation of these surprising results. We hypothesize that CNS effects of styrene may account for this apparent paradox. Based on the present results, the temporal structure of the noise should be reintroduced as a key parameter in hearing conservation regulations. © 2014 Elsevier Inc. All rights reserved.
1. Introduction Despite extensive preventive regulations, noise-induced hearing loss (NIHL) remains a major occupational health hazard. Exposure to loud noises causes cochlear damage, particularly to the hair cells located in the cochlea, which are particularly vulnerable to acoustic injury (Hamernik et al., 1989). To protect workers against NIHL, European and American frameworks and guidance documents for hearing conservation of workers require noise exposure to remain within limits: LEX,8h and the peak values (Directive 2003/10/EC, 2003; http://www. worksafebc.com). The strategy by which the danger related to noise is assessed relies on the equal energy principle over an 8-h workday (LEX,8h), which presumes that hearing damage is mainly a function of the acoustic energy received. Based on this assumption, the conservation of acoustic energy (the 3-dB exchange rate in the European Union
Abbreviations: CNS, central nervous system; dB SPL, Decibel sound pressure level; DPOAEs, distortion product oto-acoustic emissions; LEX,8h, equivalent continuous noise level calculated over 8 h; MER, middle-ear reflex; NIHL, noise-induced hearing loss; SDH, succinate dehydrogenase. ⁎ Corresponding author. Tel.: +33 3 83 50 21 55; fax: +33 3 83 50 20 96. E-mail address: [email protected] (P. Campo).
http://dx.doi.org/10.1016/j.ntt.2014.05.009 0892-0362/© 2014 Elsevier Inc. All rights reserved.
and the 5-dB exchange rate in the USA) would keep the hearing hazard constant. The permissible noise values are LEX,8h = 87 dB(A) in the European Union, and 90 dB(A) in the USA. Therefore, occupational exposures to noise must be maintained below these given limits. In addition to these limit values, peak values must not be raised. The peak values recommended in the USA and the European Union are quite similar: 140 dB and 137 dB SPL, respectively. While noise remains the predominant occupational hazard to hearing, there is growing evidence that a number of chemicals, such as organic solvents, used in industry may also affect hearing, or exacerbate the effects of occupational noise (Lataye et al., 2000; Sliwinska-Kowalska et al., 2003, 2004; Chen and Henderson, 2009; Morata et al., 2011; Sisto et al., 2013). The toxicological effects of co-exposures to noise and chemicals are complex, but highly relevant in the area of worker risk assessment. The literature on hearing conservation research has particularly focused on the effects of styrene (Morata et al., 2011; Morata and Campo, 2001; Campo et al., 2013b), which is often present in combination with noise in occupational settings. In occupational risk prevention however, although threshold limit values (TLVs) have been defined by the American Conference of Governmental Industrial Hygienist and the European agency for safety and health at work, no criterion to quantitatively assess the risk due to possible synergistic interactions between noise and chemicals has yet been proposed.
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In recent years, results have shown that the pharmacological effects induced by aromatic solvents on olivocochlear nuclei can, at least partly, explain the synergistic effects on hearing of combined exposure to noise and styrene (Campo et al., 2007; Venet et al., 2011). Indeed, many organic solvents such as styrene, ethylbenzene, xylene, or benzene, have been shown to affect ligand-gated ion channels in a ‘non-specific’ manner (Maguin et al., 2009; Cruz et al., 1998; Bale et al., 2002; Lopreato et al., 2003; Campo et al., 2013a). As a result, all these solvents may have similar pharmacological effects and modified the MER. In the current investigation, two types of noise, with similar spectra but different temporal structures (continuous vs. impulse), were tested to compare their impact on hearing. Through these experiments we wanted to compare the effects of two noise signals with the same spectrum but different temporal structures known to trigger the MER differently: the continuous noise was capable of triggering the MER, whereas the acoustic energy of the impulse noise penetrated the cochlea prior to triggering the MER. We also investigated a co-exposure “noise and styrene” to assess the neuropharmacological impact of a moderate styrene concentration on the traumatic effects of two noises with different temporal structures. The French TLV of styrene is 50 ppm, which is half of the value recommended by the USA (OSHA). Consequently, the styrene concentration tested in the current study was 6 times (safety factor) higher than the French TLV. Our results indicate that it is important to take the type of noise into account when assessing risks to human hearing, and that co-exposure to solvents should also be specifically considered in this context.
2. Materials and methods 2.1. Animals Adult male Brown-Norway (n = 114) rats weighing over 300 g were used in this experiment. The animals were purchased from Janvier breeders (Le Genest St Isle, St Berthevin, 53941, France). They were housed in individual cages (350 × 180 × 184 mm) with irradiated cellulose BCell8 bedding (ANIBED, Route de Lude, 72510 Pontvallain, France). All of the animals were 20 weeks old before starting experiments. Food and tap water were available ad libitum, except during the exposure period. The animals were maintained on a 12 h/12 h day/night cycle during experiments. Room temperature and relative humidity in the animal facility were 22 ± 2 °C and 55 ± 10%, respectively. The animal facilities have full accreditation (C54-547-10), and while conducting the research described in this article, investigators adhered to the Guide for Care and Use of Laboratory Animals promulgated by the European parliament and council (Directive 2010/63/EC, 2010/63/ EU, 22 September 2010). The present study was approved by the local ethics committee and the protocol was registered under reference CELMEA-2012-0030.
2.2. Protocol An overall picture of the experimental design is shown in Fig. 1. Individual steps in the experimental protocol are described below.
2.3. Anesthesia Hearing testing was performed in lightly anesthetized animals. DPOAEs were measured in lightly anesthetized animals. Anesthesia was induced by a single injection of a mixture of ketamine/xylazine (45/5 mg/kg). Body temperature was continuously monitored throughout the procedure using a rectal probe connected to a system maintaining a body temperature between 34 and 36 °C.
Fig. 1. Experimental protocol. Hearing of Brown Norway rats was tested using cubic distortion product oto-acoustic emissions (DPOAEs). Hearing loss was measured 28 days postexposure (HL = DPOAE 2 − DPOAE 1). Noise exposure involved exposure for 6 h either to continuous (LEX,8h = 85 dB SPL), or to an impulse noise (LEX,8h = 80 dB SPL). In some experiments, the animals were co-exposed to noise and styrene (300 ppm). The exposure conditions were: 6 hours (h) per day (d), 5 d/week (w) for 4 w. The animals were allowed to recover for 4 weeks in the animal facility before assessing the degree of hearing loss.
2.4. Hearing testing Distortion-product otoacoustic emissions (DPOAEs) are low-level sounds generated by the cochlea through the middle-ear system. They can be measured by a sensitive microphone fitted into the outer-ear canal in anesthetized rats (Shaffer et al., 2003; Venet et al., 2011). The DPOAE procedure was performed inside a sound-attenuated booth. The custom-designed DPOAE probe consisted of two transducers generating f1 & f2 and a microphone measuring the acoustic pressure within the outer ear canal. Four couples of primary tones (f1–f2) were delivered to the left ear: (5–6), (8–9.6), (13.8–16.6), and (21.2–25.4) kHz. These four couples of frequencies were chosen to surround the hearing loss extend induced by an octave band noise centered at 8 kHz. At this intensity, the hearing loss was expected half an octave above the central frequency of the noise. The f1 to f2 ratio was always 1.2, as previously found suitable for rats (Henley et al., 1990). To simplify the presentation of results, hereafter each pair of primaries will be indicated only by the f2. The primary tones were produced by frequency synthesizers (Pulse, B&K 3110) and emitted by two miniature speakers (Microphone, B&K type 4191): the difference (L1–L2) was equal to 14 dB. This difference was chosen based on measurements carried out with five 6-month-old Brown Norway rats. L1–L2 was tested from 0 to 24 dB, in 2 dB steps; 14 dB gave the highest DPOAE level. Hearing was tested by acquiring DPOAEs through a series of input/output functions for L1 intensities ranging from 40 to 65 dB SPL (L2 from 26 to 51 dB SPL). Calibration, performed with a 1/8 inch microphone (B&K type 4138) placed in a specifically designed cavity of equivalent volume to the rat's outer ear canal, ensured that f1 and f2 were always emitted at the target intensities. DPOAEs were recorded with a microphone (Knowles FG 23329-C05) fitted into the calibration probe. The three transducers were enclosed in the same probe, the tip of which was pressed gently against the opening of the ear canal. The response was measured using an FFT analyzer (B&K PULSE 3110). DPOAE amplitude was determined from a linear spectrum averaging (N 382) over 4 s, each FFT epoch lasted 31.25 ms, with a 66.7% overlap. Baseline hearing was tested in 6-month-old animals just prior to the 4-week exposures. After exposure, the animals recovered for 4 weeks before re-testing hearing. The difference in DPOAE amplitudes (DPOAE2–DPOAE1) calculated at the end of the recovery period was considered as permanent hearing loss. 2.5. Styrene exposure For rats exposed to styrene, each animal was housed in an individual cage within an inhalation chamber designed to sustain dynamic and adjustable airflow (5–6 m3.h−1). The chambers were maintained at a negative pressure of no more than 3 mm H2O. Input air was filtered and conditioned to a temperature of 22 ± 1 °C and relative humidity of 55 ± 10%. Styrene was generated using a thermoregulated glass streamer. Delivered by the pump, the solvent was instantaneously vaporized upon contact with the heated surface and carried forward
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with an additional airflow through the streamer, into the main air inlet pipe of the exposure chambers. The rats were exposed to 300-ppm styrene, 6 h per day, 5 days per week, for four weeks. Controls (n = 8) were always ventilated with fresh air. 2.6. Noise exposures Two loudspeakers were placed in the ceiling of the chambers (12/16 cm) housing individual rats for the whole of the exposure period: 6 h/day, 5 days/week, 4 weeks. The animals were free to move about in the cage during noise exposure. 2.7. 6-h continuous noise The rats were exposed inside the inhalation chambers to an octave band noise centered at 8 kHz, which is located in the most sensitive frequency area in the rat (Fig. 2). The noise intensity was 86.2 dB SPL, over 6 h. This corresponds to a LEX,8h of 85 dB SPL, which is the upper exposure action value recommended in the European Directive 2003/10/EC (2003). 2.8. Impulse noise Each sound burst lasted 7.5 ms and was separated from the following one by 15 s quiet. The sound-absorbing acoustic material placed within the exposure chambers allowed a steady sound pressure level of 112 dB SPL to be maintained for 10 ms, followed by a steep decline. Thus, 90% of the acoustic energy was dissipated in 10 ms. In these conditions, the LEX,8h was calculated according to ISO 9612:2009 and was equal to 80 dB SPL. In rats, the mean latency required to trigger the middle-ear reflex (MER) was found to be 14 ms [range: 11 to 17 ms] (data not shown). Thus, a 15-s period between noise pulses was enough for the muscles to relax. As a result, each noise burst was fully absorbed by the cochlea, without attenuation due to the MER. The noise spectrum is shown in Fig. 2. 2.9. Recovery period
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windows were opened to allow cochleae to be perfused with a succinate dehydrogenase (SDH) incubation solution (0.05 M sodium succinate, 0.05 M phosphate buffer, and 0.05% tetranitro blue tetrazolium). Cochleae were then immersed in the solution for 50 min at 37 °C. Cochleae were then fixed with 2% glutaraldehyde for at least two days. Cochleae were dissected under a light microscope to remove remaining bone and the lateral wall, thus exposing the organ of Corti. The three turns of the organ of Corti were mounted in glycerin as a surface preparation to allow hair cells to be counted (cochleogram). The frequency-place map established by Müller (1991) was used to determine the positions of the frequency coordinates along the length of the organ of Corti. A description of the technique is detailed in Hu et al. (2008). SDH staining density was plotted as a function of location along the basilar membrane. Hair cell loss was quantified by counting the number of cells present (blue spots) on the organ of Corti. A cochleogram showing the percentage hair cell loss as a function of distance was plotted for each animal. The results were averaged across each group of animals for comparison between groups. 3. Statistical analysis Two-way (group, frequency) ANOVA was run to compare the variations of DPOAE amplitudes in exposed-subjects minus the variations of DPOAE amplitudes in controls. Each variation corresponded to [DPOAE2 − DPOAE1] exper − K. The constant K was the average of [DPOAE2 − DPOAE1] calculated in the associated control group. Multiple range tests were performed for each tested frequency using the Bonferroni method. The level of significance is indicated with * (p = 0.05) and *** (p = 0.001). Only significant differences between exposed animals and controls are presented in detail. The gray area reported in Figs. 3–7 corresponds to data excluded from statistical tests due to problems with signal noise ratio (SNR). Actually, only data obtained with the group “impulse plus styrene 300 ppm” had a bad SNR at the frequencies 17,520 and 9600 Hz at 40 and 45 dB. For Figs. 4 & 6, only two measurements carried out at 9600 Hz had a bad SNR at 40 dB SPL. To be homogeneous, all the data inferior to 50 dB SPL were excluded from the statistical analyses.
At the end of the noise/styrene exposures, the rats were returned to a quiet room in the animal facility. The conditions in this room were as follows: temperature, 20 °C; light period from 7.20 am to 6.30 pm.
4. Results
2.10. Cochleogram
All rats (n = 114) were in good health prior to anesthesia. Only one animal died during while under anesthesia. Before the start of exposure, the average body weights were almost identical for the control (308.8 ± 17.5 g), and exposed groups (308.1 ± 17.9 g). At the end of the exposure
After measuring the last DPOAEs, deep anesthesia was induced and cochleae were collected for examination. Both the round and oval
4.1. General health
Fig. 2. Spectrum of octave band noises centered at 8 kHz. Black area: continuous noise; gray area, impulse noise. The intensity of the impulse noise was measured for a 7-ms period, and for a 6-h exposure for the continuous noise.
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Fig. 3. Permanent hearing loss [(DPOAE2 − DPOAE1) exper − mean (DPOAE2 − DPOAE1) ctrl ] as a function of the primary intensity (L1). The rats were exposed to 300 ppm of styrene for 6 h/day, 5 d/week for 4 weeks. Each curve indicates the DPOAE amplitude obtained for the five pairs of frequencies tested. Hearing was measured 4 weeks after exposure. Values shown correspond to geometric mean ± confidence interval; exposed rats, n = 16; controls, n = 8.
period, the weights were slightly higher for the control animals (322.3 ± 19.1 g) than for the exposed animals (316.5 ± 21.4 g). In contrast, four weeks post-exposure, the exposed group had a higher mean weight (341.1.5 ± 19.0 g) than the control group (337.7 ± 20.8 g). However, these slightly differences were not significant.
Fig. 5. Permanent hearing loss [(DPOAE2 − DPOAE1) exper − mean (DPOAE2 − DPOAE1) ctrl ] as a function of the primary intensity. The rats were simultaneously exposed to a 6-h continuous noise with a LEX,8h of 85 dB SPL, and to 300 ppm of styrene. The exposure period lasted 6 h/day, 5 d/week for 4 w. Each curve indicates the DPOAE amplitude obtained for the five pairs of frequencies tested. Hearing was measured 4 weeks after exposure. Values shown correspond to geometric mean ± confidence interval; exposed rats, n = 15; controls, n = 8.
difference = −0.12 ± 1.10) the difference was not significant. In fact, no significant difference was found at 25,440 Hz for any of the experimental conditions tested in this study.
4.2.1. ppm styrene exposure Exposure to styrene (300 ppm) for 4 weeks caused small but significant changes to the DPOAE amplitudes measured in sedentary Brown Norway rats [Fgroupxfreq(3,1) = 4.11; p = 0.007] (Fig. 3). However, the difference was not significant for all the primaries tested. Multiplerange tests indicated that the differences between exposed (n = 10) and control animals (n = 8) were clearly significant at 95% at 9600 Hz (contrast difference = − 1.88 ± 0.94), at 17,500 Hz (contrast difference = − 1.76 ± 1.09), and just significant at 6000 Hz (contrast difference = − 0.73 ± 0.72). At 25,440 Hz (contrast
4.2.2. 6-h continuous noise exposure Exposure to continuous noise with a LEX,8h of 85 dB SPL for 6 h caused significant [Fgroupxfreq(3,1) = 15.9; p b 0.001] permanent hearing loss (Fig. 4). The maximum amplitude of hearing loss was approximately 5 dB at 17,500 Hz and 3 dB at 9600 Hz, with a primary intensity of 50 dB SPL. The observed deficits were in good agreement with the common finding of noise trauma placed half an octave above exposure frequency, since the animals were exposed to an octave band noise centered at 8 kHz (OBN,8kHz). Except at 25,440 Hz, multiple-range tests indicate that the differences between exposed (n = 16) and control animals (n = 8) were clearly significant (*) at 6000 Hz (contrast difference = − 1.28, with ± limits = 0.86), 9600 Hz (contrast difference = − 2.46, with ± limits = 1.25), and 17,500 Hz (contrast
Fig. 4. Permanent hearing loss [(DPOAE2 − DPOAE1)exper − mean (DPOAE2 −DPOAE1)ctrl] as a function of the primary intensity. The rats were exposed to a 6-h continuous noise with a LEX,8h of 85 dB SPL. Noise was emitted 6 h/day, 5 d/week for 4 weeks. Each curve indicates the DPOAE amplitude obtained for the five pairs of frequencies tested. Hearing was measured 4 weeks after exposure. Values shown correspond to geometric mean ± confidence interval; exposed rats, n = 16; controls, n = 8. The gray area corresponds to data excluded from statistical tests (as explained in Materials and methods).
Fig. 6. Permanent hearing loss [(DPOAE2 − DPOAE1) exper − mean (DPOAE2 − DPOAE1)ctrl] as a function of the primary intensity. The rats were exposed to an impulse noise with a LEX,8h = 80 dB SPL. The exposure period lasted 6 h/day, 5 d/week for 4 weeks. Each curve indicates the DPOAE amplitude obtained for the five pairs of frequencies tested. Hearing was measured 4 weeks after exposure. Values shown correspond to geometric mean ± confidence interval; exposed rats, n = 16; controls, n = 8. The gray area corresponds to data excluded from statistical tests (as explained in Materials and methods).
4.2. Hearing tests
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difference = − 7.41 ± 2.76), at 9600 Hz (contrast difference = − 7.10 ± 1.66), and at 17,500 Hz (contrast difference = − 4.36 ± 0.71). The area affected was much more extensive than that obtained with the impulse noise alone. As above, the gray area on the figure corresponds to data excluded from statistical tests due to an inadequate signal-to-noise ratio.
Fig. 7. Permanent hearing loss [(DPOAE2 − DPOAE1) exper − mean (DPOAE2 − DPOAE1) ctrl ] as a function of the primary intensity. The rats were simultaneously exposed to an impulse noise with a LEX,8h of 80 dB SPL, and to 300 ppm of styrene. The exposure period lasted 6 h/day, 5 d/week for 4 weeks. Each curve indicates the DPOAE amplitude obtained for the five pairs of frequencies tested. Hearing was measured 4 weeks after exposure. Values shown correspond to geometric mean ± confidence interval; exposed rats, n = 16; controls, n = 8. The gray area corresponds to data excluded from statistical tests (as explained in Materials and methods).
difference = −4.66, with ± limits = 1.32). The gray area (primary intensities lower than 50 dB SPL) indicates data excluded from statistical tests due to a poor signal-to-noise ratio. Indeed, at these low intensities, the 2f1–f2 amplitudes were drowned in the background noise. 4.2.3. 6-h continuous noise plus 300-ppm styrene exposure Combining exposure to continuous noise (LEX,8h: 85 dB SPL) and 300-ppm styrene for 6 h also affected rats' hearing [Fgroupxfreq(3,1) = 5.98; p b 0.001] (Fig. 5). Surprisingly, a lower degree of hearing amplitude was lost than in experiments where the animals were exposed continuous noise (Fig. 4) or styrene (Fig. 3) alone. As previously, the differences between the exposed (n = 16) and control animals (n = 8) were not significant at 25,440 Hz, and nor was the difference calculated at 9600 Hz. However, multiple-range tests indicated significant (*) differences at 6000 Hz (contrast difference = − 1.71 ± 0.81), and 17,520 Hz (contrast difference = −1.37 ± 1.10). 4.2.4. Impulse noise exposure The impulse noise, with a LEX,8h of 80 dB, caused a significant difference in hearing between exposed (n = 15) and control rats (n = 8) (Fig. 6). The amplitudes of permanent hearing loss were 5-dB at 17,500 Hz, and 3-dB at 9600 Hz [Fgroupxfreq(3,1) = 14.42; p b 0.001]. In general, the amplitude of HL was quite similar to that obtained with the 6-h continuous noise alone, even though the LEX,8h was lower for the impulse noise: only 80 dB SPL compared to 85 dB SPL. In this series of experiments, the most severe damage was observed for 17,500 Hz (contrast difference = −3.44 ± 0.95) and 9600 Hz (contrast difference = − 3.73 ± 1.09). As with the continuous noise, the area most affected was positioned half an octave above OBN,8kHz. The gray area corresponds to data excluded from statistical tests for the same reasons as previously. 4.2.5. Impulse noise plus 300-ppm styrene exposure In contrast to the results obtained with the 6-h continuous noise, co-exposure to 300-ppm styrene and an impulse noise with a LEX,8h of 80 dB potentiated the traumatic impact of the noise [Fgroupxfreq(3,1) = 18.19; p b 0.001] (Fig. 7). Moreover, a 5-dB or greater amplitude of hearing loss was found at 6000, 9600 and 1750 Hz. Multiple-range tests indicate that the differences between exposed (n = 16) and control animals (n = 8) were significant (*), except at 25,440 Hz. The differences measured were: at 6000 Hz (contrast
4.2.6. Comparing hearing loss for the five experimental conditions The difference between the traumatic effects induced by the continuous noise and those induced by the impulse noise was not significant, but it must be remembered that the LEX,8h value for the continuous noise (85 dB SPL) was greater than for the impulse noise (80 dB SPL) (Fig. 8). There was a large and highly significant difference between the HL amplitudes obtained for the group exposed to “noise plus styrene” and all other groups [F(9,442) = 64.44; p b 0.0001]. The addition of styrene to the impulse noise significantly increased the HL amplitude obtained with the impulse noise alone (contrast difference = 10.84 ± 3.40), while co-exposure to styrene and continuous noise decreased the HL amplitude measured compared to the continuous noise alone (contrast difference = −5.26 ± 3.35). 4.3. Histological analyses 4.3.1. 6-h continuous noise plus 300-ppm styrene The average cochleogram based on histological observations from 5 rats exposed to both 6-h continuous noise and styrene is shown in Fig. 9. These cochleograms were similar to those obtained for control animals, with no major differences in terms of peaks present. The level of hair cell loss counted along the organ of Corti never exceeded 1% of all cells at any turn. Similar cochleograms were obtained with the control, 300-ppm styrene, 6-h continuous and “6-h continuous noise plus styrene” groups (data not shown). Although the permanent hearing loss induced by the 6-h continuous noise was significant, this was not visible on the compared cochleograms. 4.3.2. Impulse noise plus 300-ppm styrene Despite a LEX,8h lower than the European exposure action value (80 dB SPL), the impulse noise was as damaging as the 6-h continuous noise in terms of permanent loss of hearing amplitude. However, in histological terms, the impulse noise was more damaging than the continuous noise, with greater outer hair cell losses at the level of the first row (Fig. 10, gray bars). The highest cell losses due to the impulse noise appeared in the region corresponding to 16–20 kHz, with an approximately 17% hair cell loss.
Fig. 8. Comparison of hearing loss [(DPOAE2 − DPOAE1)exper − mean (DPOAE2 − DPOAE1) ctrl ] for the five experimental conditions. CN: continuous noise, IN: impulse noise, STYR: styrene, CTRL: controls. Y-axis: sum of hearing losses obtained at 6000, 9600, 17,500 and 25,400 Hz for primary intensities ranging from 50 to 65 dB.
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Fig. 9. Average cochleogram (n = 5/group) based on histological examination of cochlea from sedentary Brown Norway rats exposed either to 6-h continuous noise [LEX,8h = 85 dB(A)] or to 300-ppm styrene. Exposure schedule: 6 h/d, 5 d/w, 4 w. X-axis — upper trace: length (mm) of the spiral course of the organ of Corti. — lower trace: frequency-map. Y-axis: percent hair cell loss. IHC: inner hair cells; OHC1: first row of outer hair cells; OHC2: second row of outer hair cells; OHC3: third row of outer hair cells. The gray area indicates the frequency range explored using DPOAE-based hearing tests.
Combining exposure to impulse noise with styrene also affected the number of outer hair cells lost on the first row (Fig. 10, black bars). In these conditions, a larger proportion (27%) of cells was lost within the (16–20 kHz) frequency range. Thus, styrene potentiates the effect of impulse noise.
5. Discussion In this study we compared the effects of exposure to different types of noise (continuous and impulse), and co-exposure to styrene on hearing in Brown Norway rats.
Fig. 10. Average cochleogram (n = 5/group) from sedentary Brown Norway rats exposed either to impulse noise [LEX,8h = 80 dB(A)] (gray), or to impulse noise plus 300-ppm styrene (back). Exposure schedule: 6 h/d, 5 d/w, 4 w. X-axis — upper trace: length (mm) of the spiral course of the organ of Corti — lower trace: frequency-map. Y-axis: percent hair cell loss. IHC: inner hair cells; OHC1: first row of outer hair cells; OHC2: second row of outer hair cells; OHC3: third row of outer hair cells. The gray area indicates the frequency range explored using DPOAE-based hearing tests.
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Although the LEx,8h for the 6-h continuous noise was higher (85 dB SPL) than that (80 dB SPL) for the impulse noise, the latter was more harmful to the cochlea. Thus, the impulse noise led to significant outer hair cell losses along the first row (up to 20% between 16 and 20 kHz). This was unexpected given that the hearing loss measured based on DPOAEs was quite similar for the two types of noise. Based on these results, the use of the LEX,8h, and more generally the equal energy principle, is inaccurate and therefore does not effectively prevent damage/risk to workers' hearing due to impulse noises. Thus, the temporal structure (steady vs. impulse) of noises should be taken into consideration in a hearing conservation program, as was the case in the past. Indeed, several previous studies suggested that the equal energy principle was only valid for low-intensity and not high-intensities noises (Roberto et al., 1985; Hamernik et al., 1993; Lataye and Campo, 1996). For this reason, the European and North American hearing conservation programs recommend LEx,8h values while also limiting the permissible duration of noise exposure. Thus, regardless of the duration, workers should not be exposed to threshold noise levels without wearing individual hearing protectors. The limit levels defined were 140 dB SPL for the USA, and 137 dB SPL for the European hearing conservation programs. In the experiments described here, the maximum intensity (peak level) reached with the impulse noise exposure was 128 dB SPL, which is quite a lot lower than either of these noise exposure limits. Given the traumatic effects observed with the impulse noise tested, it seems clear that the noise limits recommended on both sides of the Atlantic are not low enough. Since it appears difficult to reduce these values in different settings, it would be more realistic to consider the temporal structure of noises as a major parameter in hearing conservation programs worldwide by weighting impulse noises. Beyond the intensity, the nature of the noise can affect the efficiency of natural protective mechanisms such as the middle-ear acoustic reflex (MER). While a 6-h continuous noise can be attenuated by the MER, the impact of an impulse noise cannot (Dunn et al., 1991; Lataye and Campo, 1996). Indeed, the acoustic energy of this type of noise is dissipated into the cochlea before the MER is triggered (Borg et al., 1984; Stevin, 1986). The protective role of the MER therefore becomes insignificant for impulse noises, resulting in a difference between noises even when the LEX,8h are similar, and close to the permissible values. In the present investigation, the noise spectrum was an octave band noise centered at 8 kHz, which means that the spectral width of the noise ranged from 5.6 up to 11.2 kHz, with an intensity of 86.2 dB SPL over the 6-h exposure (Fig. 2). According to Murata et al. (1986), the acoustic MER is readily elicited by an 80-dB noise at 6 kHz. As a result, the traumatic impact of a continuous moderate-intensity noise, such as that used in here, can be reduced by the MER. The combination of continuous noise with 300-ppm styrene also gave unexpected results, with lower levels of permanent HL than those obtained for the continuous noise alone. In contrast, a synergistic effect was obtained in the “impulse noise plus 300-ppm styrene” condition. Once again, the main difference between these two experimental conditions was their capacity to trigger the MER. In Campo et al. (2007), toluene and styrene had the same action on the middle-ear acoustic reflex. Recently, the same team has shown that a low concentration of toluene modified the amplitude of the MER triggered in rats (Venet et al., 2011; Campo et al., 2013a). Moreover, the aforementioned authors showed that styrene, like ethylbenzene replicates the toluene effects, when administrated by inhalation (data not published yet). Thus, styrene like toluene and other aromatic solvents could have two distinct effects: a cochleotoxic effect which is observed in the cochlea after a long incubation period, and a rapid pharmacological impact on the central nervous system (CNS), decreasing the threshold beyond which the MER is triggered (Campo et al., 2001). This effect appears to be due to hyperpolarization of the neuronal membrane involved in the MER. In an in vitro investigation with toluene, Magnusson et al. (1998) estimated this hyperpolarization to be in the 2–5 mV range. But whatever the value, modification of membrane polarization could
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explain why the MER threshold decreases in the presence of aromatic solvents to explain why co-exposure to noise and solvents can modify the impact of continuous noises on subjects' hearing. Given the duration of exposure and the moderate intensity of the continuous noise used in the current investigation, styrene would decrease the MER trigger threshold (and would increase the MER amplitude) by neuropharmacological mechanisms. This effect would counterbalance the cochleotoxic effect of styrene, which requires a longer period of exposure, or a higher concentration that those used in this experiment. In stark contrast, co-exposure to impulse noise and styrene increased the vulnerability of the organ of Corti, probably due to inefficient triggering of the MER. This clearly illustrates the risk that NIHL could be potentiated by concomitant exposure to styrene. In summary, our results show that a moderate concentration of styrene potentiates the cochlear damage caused by impulse noise, whereas it reduces it when caused by continuous noise. The explanation for this paradox would be that styrene has a slow cochleotoxic effect, but a rapid pharmacological impact on the CNS which would modify the efficiency of the middle-ear acoustic reflex. This specificity could explain the apparent discrepancies in the literature with short exposure periods (Sass-Kortsak et al., 1995). In any case, it must be remembered that impulse noises are more damaging to hearing than continuous noises, and that their traumatic effects can be potentiated by styrene. From a preventive point of view, application of the equal energy principle over an 8-h workday (LEX,8h), combined with noise exposure limits are not enough to prevent NIHL. The temporal structure of the noise should be reintroduced as a key parameter in hearing conservation programs. It would also be worthwhile to include a test to measure the threshold of the MER trigger to determine the pharmacological impact of solvents on the auditory CNS. Conflict of interest The authors declare that they do not have any actual or potential conflict of interest including any financial, personal or other relationships with other people or organizations that could inappropriately influence, or be perceived to influence, this work. Acknowledgments The authors would like to thank Hervé Nunge and Sylvie Michaux for the chemical analyses performed in this study, Marie-Joseph Décret, and Lionel Dussoul for their help in rat handling and husbandry. References Bale A, Smothers CT, Woodward JJ. Inhibition of neuronal nicotinic acetylcholine receptors by the abused solvent, toluene. Br J Pharmacol 2002;137:375–83. Borg E, Counter S, Röster G. Theories of middle-ear muscle functions. In: Silman Shlomo, editor. The acoustic reflex: Basic principles and clinical and applications; 1984. p. 77–9. Campo P, Lataye R, Loquet G, Bonnet P. Styrene-induced hearing loss: a membrane insult. Hear Res 2001;154:170–80. Campo P, Maguin K, Lataye R. Effects of aromatic solvents on acoustic reflexes mediated by central auditory pathways. Toxicol Sci 2007;99:582–90. Campo P, Venet T, Thomas A, Cour C, Castel B, Nunge H, et al. Inhaled toluene can modulate the effects of anesthetics on the middle-ear acoustic reflex. Neurotoxicol Teratol 2013a;35:1–6. Campo P, Morata T, Hong O. Chemical exposure and hearing loss. Dis Mon 2013b;59(4): 119–38. Chen GD, Henderson D. Cochlear injuries induced by the combined exposure to noise and styrene. Hear Res 2009;254:25–33. Cruz SL, Mirshahi T, Thomas B, Balster RL, Woodward JJ. Effects of the abused solvent toluene on recombinant N-methyl-D-aspartate and non-N-methyl-D-aspartate receptors expressed in Xenopus oocytes. J Pharmacol Exp Ther 1998;286:334–40. Directive 2003/10/EC. On the minimum health and safety requirements regarding the exposure of workers to the risks arising from physical agents (noise). Off J Eur Communities 2003;L042:38–44. Directive 2010/63/EC. Protection of animals used for scientific purposes. Off J Eur Communities 2010;L 276:33.
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Dunn D, Davis R, Merry C, Franks R. Hearing loss in chinchilla from impact and continuous noise exposure. J Acoust Soc Am 1991;90:1979–85. Hamernik R, Patterson J, Turrentine G, Ahroon WA. The quantitative relation between sensory cell loss and hearing thresholds. Hear Res 1989;38:199–212. Hamernik R, Ahroon W, Hsueh K, Lei SF, Davis RI. Audiometric and histological differences between the effects of continuous and impulse noise exposures. J Acoust Soc Am 1993;93:2088–95. Henley CM, Owings MH, Stagner BB, Martin GK, Lonsbury-Martin BL. Postnatal development of 2f1–f2 otoacoustic emissions in pigmented rat. Hear Res 1990;43:141–8. Hu BH, Henderson D, Yang WP. The impact of mitochondrial energetic dysfunction on apoptosis in outer hair cells of the cochlea following exposure to intense noise. Hear Res 2008;236:11–21. ISO 9612:2009. Acoustics — Determination of occupational noise exposure — Engineering method; 2009 (ISO 9612:2009). Lataye R, Campo P. Applicability of the Leq as a damage-risk criterion: an animal experiment. J Acoust Soc Am 1996;99:1621–31. Lataye R, Campo P, Loquet G. Combined effects of noise and styrene exposure on hearing function in the rat. Hear Res 2000;139:86–96. Lopreato G, Phelan R, Borghese C, Beckstead MJ, Mihic SJ. Inhaled drugs of abuse enhance serotonin-3 receptor function. Drug Alcohol Depend 2003;70:11–5. Magnusson A, Sulaiman M, Dutia M, Tham R. Effects of toluene on tonic firing and membrane properties of rat medial vestibular nucleus neurones in vitro. Brain Res 1998; 779:334–7. Maguin K, Campo P, Parietti-Winkler C. Toluene can perturb the neuronal voltagedependent Ca2+ channels involved in the middle-ear reflex. Toxicol Sci 2009;107: 473–81. Morata T, Campo P. Auditory function after single or combined exposure to styrene: a review. In: Prasher D, Henderson D, Kopke R, Salvi R, Hamernik R, editors. Noise-
induced hearing loss/Basic mechanisms, prevention and control. nRn London publications; 2001. p. 293–304. Morata T, Sliwinska-Kowalska M, Johnson AC, Starck J, Pawlas K, et al. A multicentre study on the audiometric findings of styrene-exposed workers. Int J Audiol 2011;50: 652–60. Müller M. Frequency representation in the rat cochlea. Hear Res 1991;51:247–54. Murata K, Ito S, Horikawa J, Minami S. The acoustic middle ear muscle reflex in albino rats. Hear Res 1986;23:169–83. Roberto M, Hamernik R, Salvi R, Henderson D, Milone R. Impact noise and the equal energy hypothesis. J Acoust Soc Am 1985;77:1514–20. Sass-Kortsak A, Corey P, Robertson J. An investigation of the association between exposure to styrene and hearing loss. Ann Epidemiol 1995;5:15–24. Shaffer LA, Withnell RH, Dhar S, Lilly DJ, Goodman SS, Harmon KM. Sources and mechanisms of DPOAE generation: implications for the prediction of auditory sensitivity. Ear Hear 2003;24(5):367–79. Sisto R, Cerini L, Gatto M, Gherardi M, Gordiani A, Sanjust F. Otoacoustic emission sensitivity to exposure to styrene and noise. J Acoust Soc Am 2013;134:3739–48. Sliwinska-Kowalska M, Zamyslowska-Szmytke E, Szymczak W, Kotylo P, Fiszer M, Wesolowski W, et al. Ototoxic effects of occupational Exposure to styrene and coexposure to styrene and noise. J Occup Environ Med 2003;45:15–24. Sliwinska-Kowalska M, Sliwinska-Kowalska M, Zamyslowska-Szmytke E, Szymczak W, Kotylo P, Fiszer M, et al. Effects of coexposure to noise and mixture of organic solvents on hearing in dockyard workers. J Occup Environ Med 2004;46:30–8. Stevin G. Simulation of the middle ear acoustic reflex applied to damage-risk for hearing produced by burst fire. Basic and Applied Aspects of Noise-Induced Hearing Loss, vol. 111. NATO ASI Series; 1986. p. 271–80. Venet T, Rumeau C, Campo P, Thomas A, Chantal C. Neuronal circuits involved in the middle-ear acoustic reflex. Toxicol Sci 2011;119:146–55.
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Neurotoxicology and Teratology journal homepage: www.elsevier.com/locate/neutera
Neuropharmacological and cochleotoxic effects of styrene. Consequences on noise exposures Pierre Campo ⁎, Thomas Venet, Aurélie Thomas, Chantal Cour, Céline Brochard, Frédéric Cosnier Institut National de Recherche et de Sécurité, Rue du Morvan, CS 60027, F-54519 Vandœuvre Cedex, France
a r t i c l e
i n f o
Article history: Received 28 February 2014 Received in revised form 21 May 2014 Accepted 28 May 2014 Available online 11 June 2014 Keywords: Noise Styrene Middle-ear reflex Combined exposure Risk assessment
a b s t r a c t Occupational noise exposure can damage workers' hearing, particularly when combined with exposure to cochleotoxic chemicals such as styrene. Although styrene-induced cochlear impairments only become apparent after a long incubation period, the pharmacological impact of styrene on the central nervous system (CNS) can be rapidly measured by determining the threshold of the middle-ear acoustic reflex (MER) trigger. The aim of the study was to evaluate the effects of a noise (both continuous and impulse), and a low concentration of styrene [300 ppm b (threshold limit value × 10) safety factor] on the peripheral auditory receptor, and on the CNS in rats. The impact of the different conditions on hearing loss was assessed using distortion product otoacoustic emissions, and histological analysis of cochleae. Although the LEX,8h (8-hour time-weighted average exposure) of the impulse noise was lower (80 dB SPL sound pressure level) than that of the continuous noise (85 dB SPL), it appeared more detrimental to the peripheral auditory receptors. A co-exposure to styrene and continuous noise was less damaging than exposure to continuous noise alone. In contrast, the traumatic effects of impulse noise on the organ of Corti were enhanced by co-exposure to styrene. The pharmacological effects of the solvent on the CNS were discussed to put forward a plausible explanation of these surprising results. We hypothesize that CNS effects of styrene may account for this apparent paradox. Based on the present results, the temporal structure of the noise should be reintroduced as a key parameter in hearing conservation regulations. © 2014 Elsevier Inc. All rights reserved.
1. Introduction Despite extensive preventive regulations, noise-induced hearing loss (NIHL) remains a major occupational health hazard. Exposure to loud noises causes cochlear damage, particularly to the hair cells located in the cochlea, which are particularly vulnerable to acoustic injury (Hamernik et al., 1989). To protect workers against NIHL, European and American frameworks and guidance documents for hearing conservation of workers require noise exposure to remain within limits: LEX,8h and the peak values (Directive 2003/10/EC, 2003; http://www. worksafebc.com). The strategy by which the danger related to noise is assessed relies on the equal energy principle over an 8-h workday (LEX,8h), which presumes that hearing damage is mainly a function of the acoustic energy received. Based on this assumption, the conservation of acoustic energy (the 3-dB exchange rate in the European Union
Abbreviations: CNS, central nervous system; dB SPL, Decibel sound pressure level; DPOAEs, distortion product oto-acoustic emissions; LEX,8h, equivalent continuous noise level calculated over 8 h; MER, middle-ear reflex; NIHL, noise-induced hearing loss; SDH, succinate dehydrogenase. ⁎ Corresponding author. Tel.: +33 3 83 50 21 55; fax: +33 3 83 50 20 96. E-mail address: [email protected] (P. Campo).
http://dx.doi.org/10.1016/j.ntt.2014.05.009 0892-0362/© 2014 Elsevier Inc. All rights reserved.
and the 5-dB exchange rate in the USA) would keep the hearing hazard constant. The permissible noise values are LEX,8h = 87 dB(A) in the European Union, and 90 dB(A) in the USA. Therefore, occupational exposures to noise must be maintained below these given limits. In addition to these limit values, peak values must not be raised. The peak values recommended in the USA and the European Union are quite similar: 140 dB and 137 dB SPL, respectively. While noise remains the predominant occupational hazard to hearing, there is growing evidence that a number of chemicals, such as organic solvents, used in industry may also affect hearing, or exacerbate the effects of occupational noise (Lataye et al., 2000; Sliwinska-Kowalska et al., 2003, 2004; Chen and Henderson, 2009; Morata et al., 2011; Sisto et al., 2013). The toxicological effects of co-exposures to noise and chemicals are complex, but highly relevant in the area of worker risk assessment. The literature on hearing conservation research has particularly focused on the effects of styrene (Morata et al., 2011; Morata and Campo, 2001; Campo et al., 2013b), which is often present in combination with noise in occupational settings. In occupational risk prevention however, although threshold limit values (TLVs) have been defined by the American Conference of Governmental Industrial Hygienist and the European agency for safety and health at work, no criterion to quantitatively assess the risk due to possible synergistic interactions between noise and chemicals has yet been proposed.
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In recent years, results have shown that the pharmacological effects induced by aromatic solvents on olivocochlear nuclei can, at least partly, explain the synergistic effects on hearing of combined exposure to noise and styrene (Campo et al., 2007; Venet et al., 2011). Indeed, many organic solvents such as styrene, ethylbenzene, xylene, or benzene, have been shown to affect ligand-gated ion channels in a ‘non-specific’ manner (Maguin et al., 2009; Cruz et al., 1998; Bale et al., 2002; Lopreato et al., 2003; Campo et al., 2013a). As a result, all these solvents may have similar pharmacological effects and modified the MER. In the current investigation, two types of noise, with similar spectra but different temporal structures (continuous vs. impulse), were tested to compare their impact on hearing. Through these experiments we wanted to compare the effects of two noise signals with the same spectrum but different temporal structures known to trigger the MER differently: the continuous noise was capable of triggering the MER, whereas the acoustic energy of the impulse noise penetrated the cochlea prior to triggering the MER. We also investigated a co-exposure “noise and styrene” to assess the neuropharmacological impact of a moderate styrene concentration on the traumatic effects of two noises with different temporal structures. The French TLV of styrene is 50 ppm, which is half of the value recommended by the USA (OSHA). Consequently, the styrene concentration tested in the current study was 6 times (safety factor) higher than the French TLV. Our results indicate that it is important to take the type of noise into account when assessing risks to human hearing, and that co-exposure to solvents should also be specifically considered in this context.
2. Materials and methods 2.1. Animals Adult male Brown-Norway (n = 114) rats weighing over 300 g were used in this experiment. The animals were purchased from Janvier breeders (Le Genest St Isle, St Berthevin, 53941, France). They were housed in individual cages (350 × 180 × 184 mm) with irradiated cellulose BCell8 bedding (ANIBED, Route de Lude, 72510 Pontvallain, France). All of the animals were 20 weeks old before starting experiments. Food and tap water were available ad libitum, except during the exposure period. The animals were maintained on a 12 h/12 h day/night cycle during experiments. Room temperature and relative humidity in the animal facility were 22 ± 2 °C and 55 ± 10%, respectively. The animal facilities have full accreditation (C54-547-10), and while conducting the research described in this article, investigators adhered to the Guide for Care and Use of Laboratory Animals promulgated by the European parliament and council (Directive 2010/63/EC, 2010/63/ EU, 22 September 2010). The present study was approved by the local ethics committee and the protocol was registered under reference CELMEA-2012-0030.
2.2. Protocol An overall picture of the experimental design is shown in Fig. 1. Individual steps in the experimental protocol are described below.
2.3. Anesthesia Hearing testing was performed in lightly anesthetized animals. DPOAEs were measured in lightly anesthetized animals. Anesthesia was induced by a single injection of a mixture of ketamine/xylazine (45/5 mg/kg). Body temperature was continuously monitored throughout the procedure using a rectal probe connected to a system maintaining a body temperature between 34 and 36 °C.
Fig. 1. Experimental protocol. Hearing of Brown Norway rats was tested using cubic distortion product oto-acoustic emissions (DPOAEs). Hearing loss was measured 28 days postexposure (HL = DPOAE 2 − DPOAE 1). Noise exposure involved exposure for 6 h either to continuous (LEX,8h = 85 dB SPL), or to an impulse noise (LEX,8h = 80 dB SPL). In some experiments, the animals were co-exposed to noise and styrene (300 ppm). The exposure conditions were: 6 hours (h) per day (d), 5 d/week (w) for 4 w. The animals were allowed to recover for 4 weeks in the animal facility before assessing the degree of hearing loss.
2.4. Hearing testing Distortion-product otoacoustic emissions (DPOAEs) are low-level sounds generated by the cochlea through the middle-ear system. They can be measured by a sensitive microphone fitted into the outer-ear canal in anesthetized rats (Shaffer et al., 2003; Venet et al., 2011). The DPOAE procedure was performed inside a sound-attenuated booth. The custom-designed DPOAE probe consisted of two transducers generating f1 & f2 and a microphone measuring the acoustic pressure within the outer ear canal. Four couples of primary tones (f1–f2) were delivered to the left ear: (5–6), (8–9.6), (13.8–16.6), and (21.2–25.4) kHz. These four couples of frequencies were chosen to surround the hearing loss extend induced by an octave band noise centered at 8 kHz. At this intensity, the hearing loss was expected half an octave above the central frequency of the noise. The f1 to f2 ratio was always 1.2, as previously found suitable for rats (Henley et al., 1990). To simplify the presentation of results, hereafter each pair of primaries will be indicated only by the f2. The primary tones were produced by frequency synthesizers (Pulse, B&K 3110) and emitted by two miniature speakers (Microphone, B&K type 4191): the difference (L1–L2) was equal to 14 dB. This difference was chosen based on measurements carried out with five 6-month-old Brown Norway rats. L1–L2 was tested from 0 to 24 dB, in 2 dB steps; 14 dB gave the highest DPOAE level. Hearing was tested by acquiring DPOAEs through a series of input/output functions for L1 intensities ranging from 40 to 65 dB SPL (L2 from 26 to 51 dB SPL). Calibration, performed with a 1/8 inch microphone (B&K type 4138) placed in a specifically designed cavity of equivalent volume to the rat's outer ear canal, ensured that f1 and f2 were always emitted at the target intensities. DPOAEs were recorded with a microphone (Knowles FG 23329-C05) fitted into the calibration probe. The three transducers were enclosed in the same probe, the tip of which was pressed gently against the opening of the ear canal. The response was measured using an FFT analyzer (B&K PULSE 3110). DPOAE amplitude was determined from a linear spectrum averaging (N 382) over 4 s, each FFT epoch lasted 31.25 ms, with a 66.7% overlap. Baseline hearing was tested in 6-month-old animals just prior to the 4-week exposures. After exposure, the animals recovered for 4 weeks before re-testing hearing. The difference in DPOAE amplitudes (DPOAE2–DPOAE1) calculated at the end of the recovery period was considered as permanent hearing loss. 2.5. Styrene exposure For rats exposed to styrene, each animal was housed in an individual cage within an inhalation chamber designed to sustain dynamic and adjustable airflow (5–6 m3.h−1). The chambers were maintained at a negative pressure of no more than 3 mm H2O. Input air was filtered and conditioned to a temperature of 22 ± 1 °C and relative humidity of 55 ± 10%. Styrene was generated using a thermoregulated glass streamer. Delivered by the pump, the solvent was instantaneously vaporized upon contact with the heated surface and carried forward
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with an additional airflow through the streamer, into the main air inlet pipe of the exposure chambers. The rats were exposed to 300-ppm styrene, 6 h per day, 5 days per week, for four weeks. Controls (n = 8) were always ventilated with fresh air. 2.6. Noise exposures Two loudspeakers were placed in the ceiling of the chambers (12/16 cm) housing individual rats for the whole of the exposure period: 6 h/day, 5 days/week, 4 weeks. The animals were free to move about in the cage during noise exposure. 2.7. 6-h continuous noise The rats were exposed inside the inhalation chambers to an octave band noise centered at 8 kHz, which is located in the most sensitive frequency area in the rat (Fig. 2). The noise intensity was 86.2 dB SPL, over 6 h. This corresponds to a LEX,8h of 85 dB SPL, which is the upper exposure action value recommended in the European Directive 2003/10/EC (2003). 2.8. Impulse noise Each sound burst lasted 7.5 ms and was separated from the following one by 15 s quiet. The sound-absorbing acoustic material placed within the exposure chambers allowed a steady sound pressure level of 112 dB SPL to be maintained for 10 ms, followed by a steep decline. Thus, 90% of the acoustic energy was dissipated in 10 ms. In these conditions, the LEX,8h was calculated according to ISO 9612:2009 and was equal to 80 dB SPL. In rats, the mean latency required to trigger the middle-ear reflex (MER) was found to be 14 ms [range: 11 to 17 ms] (data not shown). Thus, a 15-s period between noise pulses was enough for the muscles to relax. As a result, each noise burst was fully absorbed by the cochlea, without attenuation due to the MER. The noise spectrum is shown in Fig. 2. 2.9. Recovery period
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windows were opened to allow cochleae to be perfused with a succinate dehydrogenase (SDH) incubation solution (0.05 M sodium succinate, 0.05 M phosphate buffer, and 0.05% tetranitro blue tetrazolium). Cochleae were then immersed in the solution for 50 min at 37 °C. Cochleae were then fixed with 2% glutaraldehyde for at least two days. Cochleae were dissected under a light microscope to remove remaining bone and the lateral wall, thus exposing the organ of Corti. The three turns of the organ of Corti were mounted in glycerin as a surface preparation to allow hair cells to be counted (cochleogram). The frequency-place map established by Müller (1991) was used to determine the positions of the frequency coordinates along the length of the organ of Corti. A description of the technique is detailed in Hu et al. (2008). SDH staining density was plotted as a function of location along the basilar membrane. Hair cell loss was quantified by counting the number of cells present (blue spots) on the organ of Corti. A cochleogram showing the percentage hair cell loss as a function of distance was plotted for each animal. The results were averaged across each group of animals for comparison between groups. 3. Statistical analysis Two-way (group, frequency) ANOVA was run to compare the variations of DPOAE amplitudes in exposed-subjects minus the variations of DPOAE amplitudes in controls. Each variation corresponded to [DPOAE2 − DPOAE1] exper − K. The constant K was the average of [DPOAE2 − DPOAE1] calculated in the associated control group. Multiple range tests were performed for each tested frequency using the Bonferroni method. The level of significance is indicated with * (p = 0.05) and *** (p = 0.001). Only significant differences between exposed animals and controls are presented in detail. The gray area reported in Figs. 3–7 corresponds to data excluded from statistical tests due to problems with signal noise ratio (SNR). Actually, only data obtained with the group “impulse plus styrene 300 ppm” had a bad SNR at the frequencies 17,520 and 9600 Hz at 40 and 45 dB. For Figs. 4 & 6, only two measurements carried out at 9600 Hz had a bad SNR at 40 dB SPL. To be homogeneous, all the data inferior to 50 dB SPL were excluded from the statistical analyses.
At the end of the noise/styrene exposures, the rats were returned to a quiet room in the animal facility. The conditions in this room were as follows: temperature, 20 °C; light period from 7.20 am to 6.30 pm.
4. Results
2.10. Cochleogram
All rats (n = 114) were in good health prior to anesthesia. Only one animal died during while under anesthesia. Before the start of exposure, the average body weights were almost identical for the control (308.8 ± 17.5 g), and exposed groups (308.1 ± 17.9 g). At the end of the exposure
After measuring the last DPOAEs, deep anesthesia was induced and cochleae were collected for examination. Both the round and oval
4.1. General health
Fig. 2. Spectrum of octave band noises centered at 8 kHz. Black area: continuous noise; gray area, impulse noise. The intensity of the impulse noise was measured for a 7-ms period, and for a 6-h exposure for the continuous noise.
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Fig. 3. Permanent hearing loss [(DPOAE2 − DPOAE1) exper − mean (DPOAE2 − DPOAE1) ctrl ] as a function of the primary intensity (L1). The rats were exposed to 300 ppm of styrene for 6 h/day, 5 d/week for 4 weeks. Each curve indicates the DPOAE amplitude obtained for the five pairs of frequencies tested. Hearing was measured 4 weeks after exposure. Values shown correspond to geometric mean ± confidence interval; exposed rats, n = 16; controls, n = 8.
period, the weights were slightly higher for the control animals (322.3 ± 19.1 g) than for the exposed animals (316.5 ± 21.4 g). In contrast, four weeks post-exposure, the exposed group had a higher mean weight (341.1.5 ± 19.0 g) than the control group (337.7 ± 20.8 g). However, these slightly differences were not significant.
Fig. 5. Permanent hearing loss [(DPOAE2 − DPOAE1) exper − mean (DPOAE2 − DPOAE1) ctrl ] as a function of the primary intensity. The rats were simultaneously exposed to a 6-h continuous noise with a LEX,8h of 85 dB SPL, and to 300 ppm of styrene. The exposure period lasted 6 h/day, 5 d/week for 4 w. Each curve indicates the DPOAE amplitude obtained for the five pairs of frequencies tested. Hearing was measured 4 weeks after exposure. Values shown correspond to geometric mean ± confidence interval; exposed rats, n = 15; controls, n = 8.
difference = −0.12 ± 1.10) the difference was not significant. In fact, no significant difference was found at 25,440 Hz for any of the experimental conditions tested in this study.
4.2.1. ppm styrene exposure Exposure to styrene (300 ppm) for 4 weeks caused small but significant changes to the DPOAE amplitudes measured in sedentary Brown Norway rats [Fgroupxfreq(3,1) = 4.11; p = 0.007] (Fig. 3). However, the difference was not significant for all the primaries tested. Multiplerange tests indicated that the differences between exposed (n = 10) and control animals (n = 8) were clearly significant at 95% at 9600 Hz (contrast difference = − 1.88 ± 0.94), at 17,500 Hz (contrast difference = − 1.76 ± 1.09), and just significant at 6000 Hz (contrast difference = − 0.73 ± 0.72). At 25,440 Hz (contrast
4.2.2. 6-h continuous noise exposure Exposure to continuous noise with a LEX,8h of 85 dB SPL for 6 h caused significant [Fgroupxfreq(3,1) = 15.9; p b 0.001] permanent hearing loss (Fig. 4). The maximum amplitude of hearing loss was approximately 5 dB at 17,500 Hz and 3 dB at 9600 Hz, with a primary intensity of 50 dB SPL. The observed deficits were in good agreement with the common finding of noise trauma placed half an octave above exposure frequency, since the animals were exposed to an octave band noise centered at 8 kHz (OBN,8kHz). Except at 25,440 Hz, multiple-range tests indicate that the differences between exposed (n = 16) and control animals (n = 8) were clearly significant (*) at 6000 Hz (contrast difference = − 1.28, with ± limits = 0.86), 9600 Hz (contrast difference = − 2.46, with ± limits = 1.25), and 17,500 Hz (contrast
Fig. 4. Permanent hearing loss [(DPOAE2 − DPOAE1)exper − mean (DPOAE2 −DPOAE1)ctrl] as a function of the primary intensity. The rats were exposed to a 6-h continuous noise with a LEX,8h of 85 dB SPL. Noise was emitted 6 h/day, 5 d/week for 4 weeks. Each curve indicates the DPOAE amplitude obtained for the five pairs of frequencies tested. Hearing was measured 4 weeks after exposure. Values shown correspond to geometric mean ± confidence interval; exposed rats, n = 16; controls, n = 8. The gray area corresponds to data excluded from statistical tests (as explained in Materials and methods).
Fig. 6. Permanent hearing loss [(DPOAE2 − DPOAE1) exper − mean (DPOAE2 − DPOAE1)ctrl] as a function of the primary intensity. The rats were exposed to an impulse noise with a LEX,8h = 80 dB SPL. The exposure period lasted 6 h/day, 5 d/week for 4 weeks. Each curve indicates the DPOAE amplitude obtained for the five pairs of frequencies tested. Hearing was measured 4 weeks after exposure. Values shown correspond to geometric mean ± confidence interval; exposed rats, n = 16; controls, n = 8. The gray area corresponds to data excluded from statistical tests (as explained in Materials and methods).
4.2. Hearing tests
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difference = − 7.41 ± 2.76), at 9600 Hz (contrast difference = − 7.10 ± 1.66), and at 17,500 Hz (contrast difference = − 4.36 ± 0.71). The area affected was much more extensive than that obtained with the impulse noise alone. As above, the gray area on the figure corresponds to data excluded from statistical tests due to an inadequate signal-to-noise ratio.
Fig. 7. Permanent hearing loss [(DPOAE2 − DPOAE1) exper − mean (DPOAE2 − DPOAE1) ctrl ] as a function of the primary intensity. The rats were simultaneously exposed to an impulse noise with a LEX,8h of 80 dB SPL, and to 300 ppm of styrene. The exposure period lasted 6 h/day, 5 d/week for 4 weeks. Each curve indicates the DPOAE amplitude obtained for the five pairs of frequencies tested. Hearing was measured 4 weeks after exposure. Values shown correspond to geometric mean ± confidence interval; exposed rats, n = 16; controls, n = 8. The gray area corresponds to data excluded from statistical tests (as explained in Materials and methods).
difference = −4.66, with ± limits = 1.32). The gray area (primary intensities lower than 50 dB SPL) indicates data excluded from statistical tests due to a poor signal-to-noise ratio. Indeed, at these low intensities, the 2f1–f2 amplitudes were drowned in the background noise. 4.2.3. 6-h continuous noise plus 300-ppm styrene exposure Combining exposure to continuous noise (LEX,8h: 85 dB SPL) and 300-ppm styrene for 6 h also affected rats' hearing [Fgroupxfreq(3,1) = 5.98; p b 0.001] (Fig. 5). Surprisingly, a lower degree of hearing amplitude was lost than in experiments where the animals were exposed continuous noise (Fig. 4) or styrene (Fig. 3) alone. As previously, the differences between the exposed (n = 16) and control animals (n = 8) were not significant at 25,440 Hz, and nor was the difference calculated at 9600 Hz. However, multiple-range tests indicated significant (*) differences at 6000 Hz (contrast difference = − 1.71 ± 0.81), and 17,520 Hz (contrast difference = −1.37 ± 1.10). 4.2.4. Impulse noise exposure The impulse noise, with a LEX,8h of 80 dB, caused a significant difference in hearing between exposed (n = 15) and control rats (n = 8) (Fig. 6). The amplitudes of permanent hearing loss were 5-dB at 17,500 Hz, and 3-dB at 9600 Hz [Fgroupxfreq(3,1) = 14.42; p b 0.001]. In general, the amplitude of HL was quite similar to that obtained with the 6-h continuous noise alone, even though the LEX,8h was lower for the impulse noise: only 80 dB SPL compared to 85 dB SPL. In this series of experiments, the most severe damage was observed for 17,500 Hz (contrast difference = −3.44 ± 0.95) and 9600 Hz (contrast difference = − 3.73 ± 1.09). As with the continuous noise, the area most affected was positioned half an octave above OBN,8kHz. The gray area corresponds to data excluded from statistical tests for the same reasons as previously. 4.2.5. Impulse noise plus 300-ppm styrene exposure In contrast to the results obtained with the 6-h continuous noise, co-exposure to 300-ppm styrene and an impulse noise with a LEX,8h of 80 dB potentiated the traumatic impact of the noise [Fgroupxfreq(3,1) = 18.19; p b 0.001] (Fig. 7). Moreover, a 5-dB or greater amplitude of hearing loss was found at 6000, 9600 and 1750 Hz. Multiple-range tests indicate that the differences between exposed (n = 16) and control animals (n = 8) were significant (*), except at 25,440 Hz. The differences measured were: at 6000 Hz (contrast
4.2.6. Comparing hearing loss for the five experimental conditions The difference between the traumatic effects induced by the continuous noise and those induced by the impulse noise was not significant, but it must be remembered that the LEX,8h value for the continuous noise (85 dB SPL) was greater than for the impulse noise (80 dB SPL) (Fig. 8). There was a large and highly significant difference between the HL amplitudes obtained for the group exposed to “noise plus styrene” and all other groups [F(9,442) = 64.44; p b 0.0001]. The addition of styrene to the impulse noise significantly increased the HL amplitude obtained with the impulse noise alone (contrast difference = 10.84 ± 3.40), while co-exposure to styrene and continuous noise decreased the HL amplitude measured compared to the continuous noise alone (contrast difference = −5.26 ± 3.35). 4.3. Histological analyses 4.3.1. 6-h continuous noise plus 300-ppm styrene The average cochleogram based on histological observations from 5 rats exposed to both 6-h continuous noise and styrene is shown in Fig. 9. These cochleograms were similar to those obtained for control animals, with no major differences in terms of peaks present. The level of hair cell loss counted along the organ of Corti never exceeded 1% of all cells at any turn. Similar cochleograms were obtained with the control, 300-ppm styrene, 6-h continuous and “6-h continuous noise plus styrene” groups (data not shown). Although the permanent hearing loss induced by the 6-h continuous noise was significant, this was not visible on the compared cochleograms. 4.3.2. Impulse noise plus 300-ppm styrene Despite a LEX,8h lower than the European exposure action value (80 dB SPL), the impulse noise was as damaging as the 6-h continuous noise in terms of permanent loss of hearing amplitude. However, in histological terms, the impulse noise was more damaging than the continuous noise, with greater outer hair cell losses at the level of the first row (Fig. 10, gray bars). The highest cell losses due to the impulse noise appeared in the region corresponding to 16–20 kHz, with an approximately 17% hair cell loss.
Fig. 8. Comparison of hearing loss [(DPOAE2 − DPOAE1)exper − mean (DPOAE2 − DPOAE1) ctrl ] for the five experimental conditions. CN: continuous noise, IN: impulse noise, STYR: styrene, CTRL: controls. Y-axis: sum of hearing losses obtained at 6000, 9600, 17,500 and 25,400 Hz for primary intensities ranging from 50 to 65 dB.
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Fig. 9. Average cochleogram (n = 5/group) based on histological examination of cochlea from sedentary Brown Norway rats exposed either to 6-h continuous noise [LEX,8h = 85 dB(A)] or to 300-ppm styrene. Exposure schedule: 6 h/d, 5 d/w, 4 w. X-axis — upper trace: length (mm) of the spiral course of the organ of Corti. — lower trace: frequency-map. Y-axis: percent hair cell loss. IHC: inner hair cells; OHC1: first row of outer hair cells; OHC2: second row of outer hair cells; OHC3: third row of outer hair cells. The gray area indicates the frequency range explored using DPOAE-based hearing tests.
Combining exposure to impulse noise with styrene also affected the number of outer hair cells lost on the first row (Fig. 10, black bars). In these conditions, a larger proportion (27%) of cells was lost within the (16–20 kHz) frequency range. Thus, styrene potentiates the effect of impulse noise.
5. Discussion In this study we compared the effects of exposure to different types of noise (continuous and impulse), and co-exposure to styrene on hearing in Brown Norway rats.
Fig. 10. Average cochleogram (n = 5/group) from sedentary Brown Norway rats exposed either to impulse noise [LEX,8h = 80 dB(A)] (gray), or to impulse noise plus 300-ppm styrene (back). Exposure schedule: 6 h/d, 5 d/w, 4 w. X-axis — upper trace: length (mm) of the spiral course of the organ of Corti — lower trace: frequency-map. Y-axis: percent hair cell loss. IHC: inner hair cells; OHC1: first row of outer hair cells; OHC2: second row of outer hair cells; OHC3: third row of outer hair cells. The gray area indicates the frequency range explored using DPOAE-based hearing tests.
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Although the LEx,8h for the 6-h continuous noise was higher (85 dB SPL) than that (80 dB SPL) for the impulse noise, the latter was more harmful to the cochlea. Thus, the impulse noise led to significant outer hair cell losses along the first row (up to 20% between 16 and 20 kHz). This was unexpected given that the hearing loss measured based on DPOAEs was quite similar for the two types of noise. Based on these results, the use of the LEX,8h, and more generally the equal energy principle, is inaccurate and therefore does not effectively prevent damage/risk to workers' hearing due to impulse noises. Thus, the temporal structure (steady vs. impulse) of noises should be taken into consideration in a hearing conservation program, as was the case in the past. Indeed, several previous studies suggested that the equal energy principle was only valid for low-intensity and not high-intensities noises (Roberto et al., 1985; Hamernik et al., 1993; Lataye and Campo, 1996). For this reason, the European and North American hearing conservation programs recommend LEx,8h values while also limiting the permissible duration of noise exposure. Thus, regardless of the duration, workers should not be exposed to threshold noise levels without wearing individual hearing protectors. The limit levels defined were 140 dB SPL for the USA, and 137 dB SPL for the European hearing conservation programs. In the experiments described here, the maximum intensity (peak level) reached with the impulse noise exposure was 128 dB SPL, which is quite a lot lower than either of these noise exposure limits. Given the traumatic effects observed with the impulse noise tested, it seems clear that the noise limits recommended on both sides of the Atlantic are not low enough. Since it appears difficult to reduce these values in different settings, it would be more realistic to consider the temporal structure of noises as a major parameter in hearing conservation programs worldwide by weighting impulse noises. Beyond the intensity, the nature of the noise can affect the efficiency of natural protective mechanisms such as the middle-ear acoustic reflex (MER). While a 6-h continuous noise can be attenuated by the MER, the impact of an impulse noise cannot (Dunn et al., 1991; Lataye and Campo, 1996). Indeed, the acoustic energy of this type of noise is dissipated into the cochlea before the MER is triggered (Borg et al., 1984; Stevin, 1986). The protective role of the MER therefore becomes insignificant for impulse noises, resulting in a difference between noises even when the LEX,8h are similar, and close to the permissible values. In the present investigation, the noise spectrum was an octave band noise centered at 8 kHz, which means that the spectral width of the noise ranged from 5.6 up to 11.2 kHz, with an intensity of 86.2 dB SPL over the 6-h exposure (Fig. 2). According to Murata et al. (1986), the acoustic MER is readily elicited by an 80-dB noise at 6 kHz. As a result, the traumatic impact of a continuous moderate-intensity noise, such as that used in here, can be reduced by the MER. The combination of continuous noise with 300-ppm styrene also gave unexpected results, with lower levels of permanent HL than those obtained for the continuous noise alone. In contrast, a synergistic effect was obtained in the “impulse noise plus 300-ppm styrene” condition. Once again, the main difference between these two experimental conditions was their capacity to trigger the MER. In Campo et al. (2007), toluene and styrene had the same action on the middle-ear acoustic reflex. Recently, the same team has shown that a low concentration of toluene modified the amplitude of the MER triggered in rats (Venet et al., 2011; Campo et al., 2013a). Moreover, the aforementioned authors showed that styrene, like ethylbenzene replicates the toluene effects, when administrated by inhalation (data not published yet). Thus, styrene like toluene and other aromatic solvents could have two distinct effects: a cochleotoxic effect which is observed in the cochlea after a long incubation period, and a rapid pharmacological impact on the central nervous system (CNS), decreasing the threshold beyond which the MER is triggered (Campo et al., 2001). This effect appears to be due to hyperpolarization of the neuronal membrane involved in the MER. In an in vitro investigation with toluene, Magnusson et al. (1998) estimated this hyperpolarization to be in the 2–5 mV range. But whatever the value, modification of membrane polarization could
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explain why the MER threshold decreases in the presence of aromatic solvents to explain why co-exposure to noise and solvents can modify the impact of continuous noises on subjects' hearing. Given the duration of exposure and the moderate intensity of the continuous noise used in the current investigation, styrene would decrease the MER trigger threshold (and would increase the MER amplitude) by neuropharmacological mechanisms. This effect would counterbalance the cochleotoxic effect of styrene, which requires a longer period of exposure, or a higher concentration that those used in this experiment. In stark contrast, co-exposure to impulse noise and styrene increased the vulnerability of the organ of Corti, probably due to inefficient triggering of the MER. This clearly illustrates the risk that NIHL could be potentiated by concomitant exposure to styrene. In summary, our results show that a moderate concentration of styrene potentiates the cochlear damage caused by impulse noise, whereas it reduces it when caused by continuous noise. The explanation for this paradox would be that styrene has a slow cochleotoxic effect, but a rapid pharmacological impact on the CNS which would modify the efficiency of the middle-ear acoustic reflex. This specificity could explain the apparent discrepancies in the literature with short exposure periods (Sass-Kortsak et al., 1995). In any case, it must be remembered that impulse noises are more damaging to hearing than continuous noises, and that their traumatic effects can be potentiated by styrene. From a preventive point of view, application of the equal energy principle over an 8-h workday (LEX,8h), combined with noise exposure limits are not enough to prevent NIHL. The temporal structure of the noise should be reintroduced as a key parameter in hearing conservation programs. It would also be worthwhile to include a test to measure the threshold of the MER trigger to determine the pharmacological impact of solvents on the auditory CNS. Conflict of interest The authors declare that they do not have any actual or potential conflict of interest including any financial, personal or other relationships with other people or organizations that could inappropriately influence, or be perceived to influence, this work. Acknowledgments The authors would like to thank Hervé Nunge and Sylvie Michaux for the chemical analyses performed in this study, Marie-Joseph Décret, and Lionel Dussoul for their help in rat handling and husbandry. References Bale A, Smothers CT, Woodward JJ. Inhibition of neuronal nicotinic acetylcholine receptors by the abused solvent, toluene. Br J Pharmacol 2002;137:375–83. Borg E, Counter S, Röster G. Theories of middle-ear muscle functions. In: Silman Shlomo, editor. The acoustic reflex: Basic principles and clinical and applications; 1984. p. 77–9. Campo P, Lataye R, Loquet G, Bonnet P. Styrene-induced hearing loss: a membrane insult. Hear Res 2001;154:170–80. Campo P, Maguin K, Lataye R. Effects of aromatic solvents on acoustic reflexes mediated by central auditory pathways. Toxicol Sci 2007;99:582–90. Campo P, Venet T, Thomas A, Cour C, Castel B, Nunge H, et al. Inhaled toluene can modulate the effects of anesthetics on the middle-ear acoustic reflex. Neurotoxicol Teratol 2013a;35:1–6. Campo P, Morata T, Hong O. Chemical exposure and hearing loss. Dis Mon 2013b;59(4): 119–38. Chen GD, Henderson D. Cochlear injuries induced by the combined exposure to noise and styrene. Hear Res 2009;254:25–33. Cruz SL, Mirshahi T, Thomas B, Balster RL, Woodward JJ. Effects of the abused solvent toluene on recombinant N-methyl-D-aspartate and non-N-methyl-D-aspartate receptors expressed in Xenopus oocytes. J Pharmacol Exp Ther 1998;286:334–40. Directive 2003/10/EC. On the minimum health and safety requirements regarding the exposure of workers to the risks arising from physical agents (noise). Off J Eur Communities 2003;L042:38–44. Directive 2010/63/EC. Protection of animals used for scientific purposes. Off J Eur Communities 2010;L 276:33.
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