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Influence of age on noise- and styrene-induced hearing loss in the Long–Evans rat
Environmental Toxicology and Pharmacology 19 (2005) 561–570
Influence of age on noise- and styrene-induced hearing loss in the Long–Evans rat Benoˆıt Pouyatos∗ , Pierre Campo1 , Robert Lataye1 Institut National de Recherche et de S´ecurit´e, Ave de Bourgogne, BP 27, 54501 Vandoeuvre, France Available online 30 January 2005
Abstract This paper reviews different investigations carried out with Long–Evans rats on the influence of age on the ototoxicity induced by styrene and on the vulnerability to noise. The first part of this article is focused on the differences in auditory susceptibility to noise (92 or 97 dB octave band noise centered at 8 kHz, 6 h/day, 5 days/week, 4 weeks) and styrene (700 ppm, 6 h/d, 5 d/w, 4 w) between young (three and half months) and old (24 months) Long–Evans rats. Auditory evoked potential measures revealed that the old rats tend to be more sensitive than young rats to higher noise levels (97 dB), but equally vulnerable to moderate levels (92 dB). By contrast, the aged rats were virtually insensitive to 700 ppm styrene compared to the young animals. Two additional studies were performed controlling and examining the influence of body weight and post-natal age on the sensitivity to styrene. Rats of the same age (21 weeks) and but having different body weight (∼310 g versus ∼410 g) did not show any difference of sensitivity to 700 ppm styrene, whereas 14-week-old rats with the same body weight as 21-week-old rats (∼350 g) revealed increased sensitivity to styrene. These results show that weight does not play a key role in the sensitivity to styrene, and suggest a long period of increased sensitivity to styrene during the first months of life. Published by Elsevier B.V. Keywords: Styrene; Noise; Hearing loss; Aging; Age; Weight
1. Introduction Aging and exposure to noise are undoubtedly the two variables responsible for most cases of hearing loss in humans. However, in the workplace, exposure to solvents can also be considered as an additional significant risk for hearing (M¨oller et al., 1990; Calebrese et al., 1996; Morata and Campo, 2001; Sliwinska-Kowalska et al., 2003). The effects of noise and solvents as well as the characteristics of age-related hearing loss (called presbycusis) have been extensively studied in the past 15 years, but surprisingly few studies have focused on the interactions among these different factors. The present article reviews the different ∗ Corresponding author. Present address: Jerry Pettis Memorial Veterans Medical Center, Research Service (151), 11201 Benton Street, Loma Linda, CA 92357, USA. Tel.: +1 909 825 7084x2816; fax: +1 909 796 4508. E-mail address: [email protected] (B. Pouyatos). 1 Fax: +33 3 83 50 20 96.
1382-6689/$ – see front matter. Published by Elsevier B.V. doi:10.1016/j.etap.2004.12.020
experiments performed in our laboratory to determine the influence of age (and aging) on the vulnerability to noise and styrene exposure. Some of the material included in this article was already published in Campo et al. (2003). The effects of aging on the peripheral and central auditory systems have been studied in different animals models (e.g. Miller et al., 1998; McFadden et al., 1997, 1998). Along with a loss of hearing sensitivity, aged subjects display several types of peripheral degenerations: vascular (Keithley et al., 1992; Gratton et al., 1996), neuronal (Keithley and Feldman, 1979), and sensory (Keithley and Feldman, 1982). Aging is also associated with a down-regulation in metabolism, which may underlie an increased sensitivity to stress agents and a decreased repair of tissues following stress. However, the question of relative vulnerability of aged subjects to noise and others environmental ototoxicants has been rarely addressed despite its obvious importance regarding the safety of aged workers. Miller et al. (1998) showed that aged mice were slightly more sensitive to high-level noise (0.5–40 kHz
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noise at 108 dB sound pressure level (SPL) for 45 min) than young ones; but he also stated that the influence of the genetic characteristic of the strain was predominant. Sun et al. (1994) demonstrated that older chinchilla ears were not more susceptible to noise damage than younger ears when exposed to 95 dB (0.5 kHz) for 36 days. McFadden et al. (1998) reported that old chinchillas are equally vulnerable to moderate-level noise (95 dB), but may be slightly more vulnerable to high-level noise (106 dB). Fraenkel et al. (2003) recently showed that, when exposed to 113 dB for 1 h or for 3 days (12 h noise/12 h quiet), 3- and 24-month-old Wistar rats exhibited similar recovery after noise exposure as well as similar permanent hearing loss. As far as the effect of aging on solvent ototoxicity is concerned, the literature is very poor. Only Li et al. (1992) demonstrated that toluene exposure could aggravate auditory deterioration in mice with a strong genetic predisposition to spontaneously precocious age-related hearing loss (C57Bl/6J), but not in a strain that show moderate signs of presbycusis (CBA/Ca). However, these authors did not determine whether aged animals were more sensitive to a solvent exposure regardless of the genetic background. In order to ameliorate the lack of data in the literature concerning influence of aging on the sensitivity to noise and on solvent ototoxicity, we performed an experiment in which young and old rats were exposed either to noise (experiment 1) or to styrene (experiment 2), a solvent with stronger ototoxicity potency than toluene (Loquet et al., 1999). Subsequently, the results obtained in this first series of experiments raised questions about the influence of body fat content on the sensitivity to styrene, as well as about the existence of a critical period of hypersensitivity to styrene during the first months of life in the rat. As a result, two investigations were added to discriminate the effect of weight (experiment 3) from the effect of age (experiment 4) on styrene ototoxicity. The rationale for these studies was that a difference in weight could constitute a stock reserve for the solvent prior to metabolizing it (Carlsson, 1981; Savolainen and Pf¨affli, 1978) and thus bias the results obtained in young and aged animals. In summary, the main goals of the present investigations were to establish the influence of aging on the auditory sensitivity to noise or styrene, and to compare styrene-induced hearing loss as a function of weight and post-natal age of the animals.
2. Methods 2.1. Animals and experimental design One hundred and fifty male Long–Evans rats were used in these investigations. All the animals were obtained from Janvier Laboratories (Le Genest Saint Isle, France). Except otherwise indicated, the animals received standard food (A04, UAR® , Villemoisson-sur-Orge, France) and tap water ad libitum. Lighting was on from 07:00 to 19:00 h, the temperature in the animal quarters was 22 ± 1 ◦ C and the relative humidity
Table 1 Experiments 1 and 2—treatment groups and experimental conditions Age at the begining of the exposure Experiment 1 14 w 14 w 14 w 24–26 m 24–26 m 24–26 m Experiment 2 14 w 14 w 24–26 m 24–26 m
Exposure
n
92 dB SPL (OBN/8 kHz), 6 h/d, 5 d/w, 4 w 97 dB SPL (OBN/8 kHz), 6 h/d, 5 d/w, 4 w Controls 92 dB SPL (OBN/8 kHz), 6 h/d, 5 d/w, 4 w 97 dB SPL (OBN/8 kHz), 6 h/d, 5 d/w, 4 w Controls
8 8 8 8 8 8
Styrene 700 ppm, 6 h/d, 5 d/w, 4 w Controls Styrene 700 ppm, 6 h/d, 5 d/w, 4 w Controls
13 13 14 14
OBN/8 kHz, octave band noise centered at 8 kHz; dB, decibel; SPL, sound pressure level; h, hour; d, day; w, week; m, month.
ranged from 50 to 55%. The animals were housed in individual cages (35 cm × 18 cm × 18.4 cm). While conducting the research described in this article, the investigators adhered to the Guide for Care and Use of Laboratory Animals, as mandated by the French Conseil d’Etat in D´ecret no. 87-848 published in the French Journal Officiel on 20 October 1987, and the principles of the declaration of Helsinki. Because, the relationship between aging and noiseinduced hearing loss was not clear, we carried out the experiment 1 in which young (14-week-old) and old (26-month-old) Long–Evans rats were exposed to an octave band noise centered at 8 kHz (OBN/8 kHz) presented at 92 or 97 dB, 6 h/day, 5 days/week for 4 weeks (6 h/d, 5 d/w for 4 w). These parameters were chosen to induce a permanent threshold shift (PTS) of 10–30 dB in the vicinity of the frequency range injured by styrene. In the experiment 2, young and old rats (same ages as in experiment 1) were exposed to styrene by inhalation at a concentration of 700 ppm, 6 h/d, 5 d/w for 4 w. Table 1 summarizes the experimental groups and exposure conditions used in experiments 1 and 2. The weight parameter was studied in experiment 3 by exposing two groups of rats of the same age (21 weeks old), but having different body weight (slim: ∼310 g versus fat: ∼410 g) to 700 ppm styrene (6 h/d, 5 d/w, 4 w). Twenty-four rats of 10 weeks of age on their arrival in the animal facility were used. Twelve of them received standard animal food in controlled quantities (UAR® A04: 16% proteins, 4% fibers, 5% minerals, 3% lipids, 12% moisture, 60% nitrogen free extract) to reach an average body weight of 312 g, whereas 12 others received an enriched food (UAR® A03: 21.4% proteins, 3.9% fibers, 5.7% minerals, 5.1% lipids, 51.7% nitrogen free extract) to reach an average weight of 411 g at the beginning of the exposure; 11 weeks after their arrival. Due to the diet, a 100-g difference was obtained between the two groups of animals at the age of 21 weeks. In the experiment 4, two groups of rats having the same weight (∼345 g), but being of different ages (14 and 21 weeks old) were exposed to 700 ppm styrene (6 h/d, 5 d/w, 4 w) to
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Table 2 Experiment 3 and 4—treatment groups and experimental conditions Parameter tested
Age at the beginning of the exposure (w)
Weight (g)
Exposure
n
21 21 21 21
314 ± 3 310 ± 3 g 408 ± 3 415 ± 3
Styrene 700 ppm, 6 h/d, 5 d/w, 4 w Controls Styrene 700 ppm, 6 h/d, 5 d/w, 4 w Controls
6 6 6 6
14 14 21 21
342 ± 16 335 ± 11 353 ± 6 351 ± 16
Styrene 700 ppm, 6 h/d, 5 d/w, 4 w Controls Styrene 700 ppm, 6 h/d, 5 d/w, 4 w Controls
6 6 6 6
Experiment 3 Weight Experiment 4 Postnatal age
h, hour; d, day; w, week; g, gram; ppm, parts per million.
test the effect of post-natal age on the sensitivity to styrene. Twelve 7-week-old and 12 14-week-old rats were used. The youngest rats received the enriched food (UAR® A03) to reach a target weight of 345 g, whereas the oldest were fed with a standard breeding food (UAR® A04) to keep their weight close to the target weight. At the beginning of the exposure, the youngest rats were 14 weeks old, whereas the oldest were 21 weeks old. The experimental groups and exposure parameters used in experiments 3 and 4 are summarized in Table 2. All the experiments reported in the present paper allowed comparing the noise effects with those induced by styrene on hearing in young adult and aged populations of Long–Evans rats. Auditory function was tested “within subject” by recording the near field evoked potentials from the inferior colliculus before and 6 weeks after exposure in order to calculate PTS. After the last physiological measurement, the histological damage was assessed by counting hair cells of the organ of Corti.
by the center of the animal’s head. The targeted intensity is obtained by adjusting the level of the continuous pure tone. The electrical signal from the implanted electrode was amplified (×2000) and filtered between 30 and 3000 Hz. Averaged auditory evoked potentials were obtained from 260 presentations. An amplitude trough-to-peak (N1-P1) of 15 V of the response was considered as the threshold value. For each animal, an audiogram was obtained prior to styrene exposure (T1) and 6 weeks after exposure (T2). Permanent threshold shifts were defined as PTS = T2 − T1. 2.3. Noise exposure The rats were exposed either to 92 ± 1 dB SPL or to 97 ± 1 dB SPL OBN/8 kHz for 6 h/d, 5 d/w for 4 consecutive weeks. They were housed alone in individual cages with a speaker above the cages. Controls were maintained in the same conditions without noise. 2.4. Styrene exposure
2.2. Hearing testing Rats were deeply anesthetized by the i.p. administration of ketamine (45 mg/kg) and xylazine (6 mg/kg). A tungsten electrode was implanted into the right inferior colliculus, and a second electrode was placed in the rostral cranium just below the dura mater to serve as the ground electrode. These two electrodes were then fastened to a transistor socket and fixed with dental cement to the skull. One month after surgery, audiometric testing was performed in a soundproof booth on awake rats placed in a restraining device. The generation and the signal treatment were performed with a Tucker–Davis Technologies apparatus. The acoustic stimuli (two cycles for the rise/fall ramp, four cycles for the plateau) were gated sinusoidal stimuli at 2, 3, 4, 5, 6, 8, 10, 12, 16, 20, 24 and 32 kHz, presented at a rate of 20/s, with an analysis window of 30 ms. The stimuli were transduced by a speaker (JBL, 2405) positioned 15 cm from the left pinna. For each frequency, the acoustic calibration was carried out by measuring the sound pressure level emitted by the speaker, when it was driven by a continuous pure tone. The sound field was calibrated by positioning a microphone at a point normally occupied
The animals were housed in individual cages placed in inhalation chambers and exposed to 700 ppm styrene vapors (Sigma–Aldrich, 99%) for 6 h/d, 5 d/w, 4 w. The controls were housed in similar chambers ventilated with fresh air. The chambers (200 l) were maintained at a negative pressure of no more than 3 mm H2 O in order to sustain a dynamic and adjustable airflow (10–20 m3 /h). The input air was filtered and conditioned to a temperature of 22–24 ◦ C and a relative humidity of 50–55%. The styrene was vaporized by bubbling an additional airflow through a flask containing the test compound. The solvent concentration in the chambers was measured by collecting atmosphere samples through glass tubes packed with activated charcoal. Styrene samples were desorbed with carbon disulfide and analyzed by a gas chromatograph (GC: Intersmat, 120FB I.G.C. model, France) using oxylene as the internal standard. These analyses allowed daily calibration of another GC used for the continuous monitoring of exposure level; this GC was equipped with a flame ionization detector and an automatic gas-sampling valve. Concentration measurements were performed at regular intervals (0.5 min).
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2.5. Histology At 6 weeks post-exposure, the animals were anesthetized with a heavy dose of ketamine (75 mg/kg), and then fixed by transcardiac perfusion with 300 ml of a tri-aldehyde fixative (3% glutaraldehyde, 2% formaldehyde, 1% acrolein and 2.5% DMSO in buffer pH 7.4). The cochleae were harvested and fixed again by perilymphatic perfusion. Following the primary 24-h fixation, the cochleae were post-fixed with 1% OsO4 in buffer pH 7.4 for 1 h and finally washed again with buffer. The organ of Corti was dissected in 70% ethanol and mounted in glycerin for counting of hair cells. The frequencyplace map established by M¨uller (1991) was used to superimpose the frequency coordinates on the length coordinates of the organ of Corti. 2.6. Statistical analysis The statistical analyses were made with NCSS software (Kaysville, UT). The electrophysiological data obtained in the experiments in which the effect of age was investigated were analyzed with repeated measure analyses of variances (ANOVAs) using the “treatment” and “age” as the between subject factors, and “frequency” as the within factor. For the experiment in which the effect of weight was investigated, the “weight” was used as a between subject factor instead of the “age”. Planned post hoc comparisons were performed between treatment groups using the Tukey–Kramer test. p = 0.05 was considered as the significance threshold.
3. Results 3.1. Influence of aging on the auditory sensitivity to noise or styrene 3.1.1. Experiment 1: noise Fig. 1 presents the PTS calculated in young and old animals exposed to 92 and 97 dB noises, compared to the
Fig. 1. Permanent threshold shifts (PTS) vs. frequencies, ranging from 2 to 32 kHz, obtained from young (14-week-old) and old (24–26-month-old) noise-exposed rats and age-matched controls (n = 8 per group). The noise was an octave band noise centered at 8 kHz at (a) 92 dB or (b) 97 dB sound pressure level (SPL), 6 h/d, 5 d/w, 4 w. Error bars: 95% half-confidence interval.
age-matched controls. The 92-dB noise induced threshold shifts at frequencies ranging from 8 to 16 kHz, with the peak occurring near 10–12 kHz, which corresponds to approximately one-half octave above 8 kHz, the center frequency of the exposure. At this noise intensity, there was no obvious difference between the young and aged groups in terms of PTS. At 97 dB SPL, the PTS were large from 8 up to 32 kHz regardless of the age of the animals. However, the average amplitude of the PTS measured in the old rats was 10 dB greater than in the young animals in that frequency range. The electrophysiological measures were analyzed using two different repeated-measure ANOVAs, one for each noise level. The ANOVAs showed significant effect of both 92 dB noise [F(1,28) = 36.08; p < 0.0001] and 97 dB noise [F(1,28) = 197.36; p < 0.0001] on PTS. The age effect was significant for the 97 dB noise [F(1,28) = 21.27; p < 0.0002], but not for the 92 dB noise [F(1,28) = 1.01; p = 0.32]. Post hoc comparisons using Tukey–Kramer multiple-comparison tests showed that the PTS measured in the old rats exposed to 97 dB were significantly (p < 0.05) different from the PTS measured in young animals. PTS measured in old and young rats exposed to 92 dB were not significantly different. The hair cell loss obtained in the different experimental groups is presented in Fig. 2. The average cochleogram obtained with the young controls revealed very small amounts of hair cell loss (<1%) scattered along the organ of Corti. Consequently, it is not shown. By contrast, the cochleae from old controls (Fig. 2a) displayed significant OHC loss particularly at the extreme apex (OHC3 : 50%; OHC2 : 28.5%; OHC1 : 22% at 0.5 kHz). Despite this damage, the auditory thresholds measured in old animals were just slightly higher (by an average of 2 dB between 2 and 32 kHz) than those measured in young rats (Campo et al., 2003). The 92 dB noise exposure did not induce any hair cell loss in any animals (not shown), but the 97 dB intensity caused significant OHC loss in both young (Fig. 2a) and old (Fig. 2b) rats. The cochleogram obtained from young rats shows that the cell losses of the first row (9.3%) were greater than those of the second (3.8%) and third (2%) rows between 12 and 34 kHz. The averaged OHC loss induced by the 97 dB exposure was quantitatively not greater in the aged rats than in the young rats, but the losses in the aged group were surprisingly located in a higher frequency range: from 23 to 37 kHz. 3.1.2. Experiment 2: styrene Fig. 3 shows the styrene-induced hair cell loss observed in young and old animals after a 4-week exposure to a concentration of 700 ppm. Surprisingly, only the young rats showed significant hearing loss, up to 15 dB at 20 kHz. No difference was obtained between controls and aged styrene-treated rats. The ANOVA showed significant effect of 700 ppm styrene [F(1,50) = 10.95; p < 0.002] on PTS. The age effect was also significant [F(1,50) = 19.67; p < 0.0001]. Post hoc comparisons using Tukey–Kramer multiple-comparison tests showed that the PTS measured in the old rats exposed to
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Fig. 3. Permanent threshold shift (PTS) vs. frequencies, ranging from 2 to 32 kHz, obtained from young (14-week-old; n = 13) and old (24–26-monthold; n = 14) styrene exposed animals, and age-matched controls (young: n = 13; old: n = 14). Styrene exposure: 700 ppm, 6 h/d, 5 d/w, 4 w. Error bars represent the 95% half-confidence intervals.
700 ppm styrene were significantly (p < 0.05) different from the PTS measured in young animals. Consistent with physiological results, the styrene-induced cochlear damage was much larger in young than in old subjects. In the young cochleae (Fig. 4a), the styrene exposure caused up to 61% of OHC loss on the third row between 1 and 28 kHz, with a gradual decrease for OHC2 and OHC1 . No IHC was observed in any styrene-exposed cochlea. The OHC loss observed in the old animals exposed to styrene (Fig. 4b) in the same conditions was strikingly smaller, when the aged-related hair cell loss (observed in aged controls): the percentages were ∼23% at the level of the OHC1 , ∼3% at OHC2 and ∼2% at OHC3 . Similarly, the IHC were not injured after styrene in the old cochleae.
3.2. Influence of weight and post-natal age on styrene ototoxicity
Fig. 2. Average cochleogram obtained from (a) old control rats (n = 9), (b) young (n = 5) and (c) old (n = 5) 97 dB sound pressure level (SPL) exposed rats. Noise schedule: 6 h/d, 5 d/w, 4 w. Abscissa, upper trace: length (mm) of the entire spiral course of the organ of Corti from the bottom of the hook; lower trace: frequency-map according to M¨uller (1991). Ordinate: hair cell loss in percent. IHC: inner hair cells; OHC1 : first row of outer hair cells; OHC2 : second row; OHC3 : third row. Error bars represent the S.E.M.
3.2.1. Experiment 3: weight Fig. 5 presents the hearing loss and the cochleogram obtained in 310 and 410 g animals exposed to 700 ppm styrene. Styrene induced very similar threshold shift in both groups (Fig. 5a), approximately 8 dB at the vicinity of 16 kHz. The ANOVA showed significant effect of 700 ppm styrene [F(1,20) = 6.88; p < 0.02] on PTS, but no effect of weight [F(1,20) = 0.00; p = 0.98]. Post hoc comparisons using Tukey–Kramer multiple-comparison tests confirmed that the PTS measured in the heavier rats exposed to 700 ppm styrene were not significantly (p < 0.05) different from the PTS measured in the lighter animals. As expected, the cochleograms obtained from 310 and 410 g animals were similar (Fig. 5b and c). Hair cell loss was massive in the third row for which 58% of the cells were missing from 2 to 27 kHz, peaking around 5 and 20 kHz. The second row was less damaged (13%) than the third, but more than the first row (5%). The high-frequency region (above 30 kHz) and the IHCs were typically well preserved.
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Fig. 4. Average (n = 5 per group) cochleograms obtained from (a) young and (b) old 700 ppm styrene-exposed rats. Styrene exposure: 6 h/d, 5 d/w, 4 w. Abscissa, upper trace: length (mm) of the entire spiral course of the organ of Corti from the bottom of the hook; lower trace: frequency-map according to M¨uller (1991). Ordinate: hair cell loss in percent. IHC: inner hair cells; OHC1 : first row of outer hair cells; OHC2 : second row; OHC3 : third row. Error bars represent the S.E.M.
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Fig. 5. (a) Permanent threshold shifts (PTS) vs. frequency obtained from styrene-exposed rats (n = 6 per group) having the same age (21-week-old) but having different weight (310 g vs. 410 g). Styrene exposure: 700 ppm, 6 h/d, 5 d/w, 4 w. Each point represents the mean and the bars represent the 95% half-confidence intervals. Average (n = 5 per group) cochleograms obtained from the styrene-exposed (b) 310 g and (c) 410 g groups. The animals were 31 weeks old when the cochleae were harvested (21 weeks old at the beginning of the exposure). Abscissa, upper trace: length (mm) of the entire spiral course of the organ of Corti from the bottom of the hook; lower trace: frequency-map according to M¨uller (1991). Ordinate: hair cell loss in percent. IHC: inner hair cells; OHC1 : first row of outer hair cells; OHC2 : second row; OHC3 . The bars represent the S.E.M.
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Fig. 6. (a) Permanent threshold shift (PTS) vs. frequencies, ranging from 2 to 32 kHz, obtained from styrene-exposed rats (n = 6 per group). The animals have the same weight (345 g) but different age: 14 vs. 21 weeks old (at the beginning of the exposure). Styrene exposure: 700 ppm, 6 h/d, 5 d/w, 4 w. Each point represents the mean and the bars represent the 95% half-confidence intervals. Average cochleogram obtained from (b) the 14-week-old rats, (c) the 21-week-old rats (age at the beginning of the exposure). Abscissa, upper trace: length (mm) of the entire spiral course of the organ of Corti from the bottom of the hook; lower trace: frequency-map according to M¨uller (1991). Ordinate: hair cell loss in percent. IHC: inner hair cells; OHC1 : first row of outer hair cells; OHC2 : second row; OHC3 .
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3.2.2. Experiment 4: post-natal age Fig. 6 shows the hearing loss and the cochlear damage obtained in the 14- and 21-week-old rats exposed to 700 ppm styrene. The younger animals were obviously more sensitive to styrene, as shown by physiological and histological data. Respectively, 23.5 and 8 dB styrene-induced hearing loss were obtained at the vicinity of 16 kHz in 14- and 21week-old animals (Fig. 6a). The ANOVA confirmed the significant effect of 700 ppm styrene [F(1,20) = 31.63; p < 0.0001] on PTS. The effect of age was also significant [F(1,20) = 5.54; p < 0.03]. Post hoc comparisons using Tukey–Kramer multiple-comparison tests confirmed that the PTS measured in the 14-week-old rats exposed to 700 ppm styrene were greater (p < 0.05) than PTS measured in the older animals. The OHC loss obtained in the 14- and 21-week-old rats exposed to 700 ppm styrene differed mainly at the level of the first two row of OHC: in the 14-week group, the styrene exposure caused 21.5% of OHC loss in the first row and 39.8% in the second row in the 16–25-kHz region, while only 9.2% of OHC were missing from the first row, and 16% from the second row in the 21-week-old group in the same region.
4. Discussion The findings of the first experiments showed conclusively that 24–26-month-old rats are, by and large, equally vulnerable to moderate-level noise (92 dB SPL) as 14-week-old rats, but may be more vulnerable to moderate–high-level noise (97 dB SPL), specifically in the high-frequency region. By contrast, older rats were very resistant to styrene ototoxic effect compared to the younger animals. In the second part of this paper, we show that this surprising greater sensitivity to styrene of the younger animals could be explained by the existence of a long period of hypersensitivity to solvents during the first months of life. We also demonstrate that the sensitivity to styrene of 21-week-old rats does not seem to be influenced by their body weight. More detailed information on these experiments is available in Campo et al. (2003). The fact that old animals are more sensitive to higher noise exposure and equally sensitive to lower noise exposures than young animals has already been reported in previous works with chinchillas (McFadden et al., 1998), and mice (Miller et al., 1998). Conversely, our results are inconsistent with those obtained by Fraenkel et al. (2003) in the Wistar rat. They did not obtain any difference in susceptibility between young and aged animals, which were approximately the same age as our subjects. This discrepancy may be due to either the difference of strain (Wistar versus Long–Evans), noise intensity (113 dB versus <97 dB), or noise duration (1 h or 3 days versus 4 weeks). Clearly, some more work has to be done to determine whether aging should be considered as a significant risk factor. Regarding the histological findings, the hair cell loss obtained in the young and aged rats exposed to 97 dB SPL was
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low relative to the electrophysiological data. However, it is well known that the noise effects cause predominant stereocilia damage but moderate hair cell loss (Hamernik and Qiu, 2000). Therefore, the strict quantitative hair cell counts performed in these studies probably underestimate the damage. If the amount of hair cell loss was similar in young and aged animals, the location was different: it was centered at 20–22 kHz region in the young subjects, and near the 30 kHz place in the aged animals. The reason for such a difference in tonotopicity of the trauma is unknown. One hypothesis is that the hair cell losses already present in the organ of Corti of the aged rats could modify the mechanical properties of the basilar membrane, and thereby the location of the trauma. Styrene-induced hearing loss has already been reported in rats (Yano et al., 1992; Crofton et al., 1994; Campo et al., 2001; Pouyatos et al., 2002), but to the best of our knowledge, no data have been published on styrene effects as a function of age of the exposed subjects. Contrary to all expectations, old rats were very resistant (PTS = ∼0 dB) to the ototoxicity of styrene compared to young animals (PTS = ∼15 dB). Aged rats showed only fairly small hair cell loss in the third row of OHCs. Such a loss of OHCs was modest with regard to that observed in the young rats, and apparently insufficient to modify the functional results. According to Miller et al. (1998), aging is associated with a decrease in the metabolic activity that could enhance the vulnerability of the old subject to environmental insults. If this concept can explain the difference of sensitivity to noise between young and aged animals, it cannot explain the styrene effects since old ears showed better resistance to styrene. The difference in sensitivity to styrene between age groups could be due to a weight difference between animals since the aged rats weighted more than the young rats (500 g versus 300 g) at the beginning of exposure. By controlling the weight difference between two different groups of the same age, we expected to determine the influence of the body weight on the effects of styrene intoxication. The results revealed that a 100-g difference in the body weight does not play a major role in the ototoxic process, at least in our experimental conditions. So the hypothetical capacity of fat compartment to capture the solvents (Carlsson, 1981; Savolainen and Pf¨affli, 1978) and therefore to decrease their ototoxic potency was not confirmed. By contrast, our results demonstrate that age is an important factor in the susceptibility to styrene since the styrene exposure produced larger PTS in older than in younger animals. Therefore, a 7-week difference of age at the beginning of the rat life can have a dramatic influence on the ototoxic potency of styrene. This finding validates the hypothesis of a period of hypersensitivity to styrene. This long “critical period” is atypical in comparison to the critical periods already described for noise or aminoglycosides. Indeed, Rybalko and Syka (2001) showed that the vulnerability to noise is higher during the 5 first post-natal weeks, and Pujol (1986) demonstrated that the ear is more sensitive to aminoglycosides during the 3 first post-natal weeks. It is
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hard to think that this critical period of sensitivity to solvents can be correlated with maturational events in the cochlea. Indeed, cochlear structures and functions are fully developed and mature at the age of 3 months, the rat cochlea reaching its adult-like properties at the end of the third week (Roth and Bruns, 1992). It is necessary to find other explanations for our results. The metabolic changes in the liver could be the clue of the problem. The enzymatic reactions involved in the hepatic metabolism of styrene are an initial oxidation to styrene oxide, catalyzed by cytochrome P-450. Then styrene oxide is a substrate for epoxide hydrolase and glutathioneS-transferase to be metabolized in phenylethylene glycol and mercapturic acids, respectively (Bond, 1989). Hence, the age-related changes in liver enzymes, particularly the cytochrome P-450, the epoxide hydrolase and the glutathione-S-transferase might explain the difference of susceptibility stated between young and adult rats. Indeed, Chengelis (1988) showed that (1) total cytochrome P-450 increases substantially during the first weeks of life (from 12 to 34 nmol/g) reaching its peak at week 26 in males and; (2) the amount of epoxide hydrolase increases significantly between week 16 and 24 to reach 160 nmol/g; (3) in the mean time, glutathione-S-transferase doubles between week 16 and 24 tending to plateau at 63 nmol/g. Presumably, these agerelated changes in metabolism have the potential to change the sensitivity to solvent. Hence, the young rats would have a reduced metabolic activity that might explain the difference of vulnerability between the 14- and the 21-week-old groups. Acknowledgments The authors would like to thank the organizers of the 9th International Neurotoxicology Association for offering the opportunity to present our results during the student symposium. The authors are also grateful to Christian Barth´el´emy for his technical assistance, and Laurence D. Fechter for his comments on the manuscript. This study was supported by European grant QLK4-2000-00293 and by Institut National de Recherche et de S´ecurit´e. References Bond, J., 1989. Review of the toxicology of styrene. Crit. Rev. Toxicol. 19 (3), 227–249. Calebrese, G., Martini, A., Sessa, G., Cellini, M., Bartolucci, G.B., Marcuzzo, G., De Rosa, E., 1996. Otoneurological study in workers exposed to styrene in the fiberglass industry. Int. Arch. Occup. Environ. Health 68, 219–223. Campo, P., Lataye, R., Loquet, G., Bonnet, P., 2001. Styrene-induced hearing loss: a membrane insult. Hear. Res. 154 (1/2), 170–180. Campo, P., Pouyatos, B., Lataye, R., Morel, G., 2003. Is the aged rat ear more susceptible to noise or styrene damage than the young ear? Noise Health 5, 1–18. Carlsson, A., 1981. Distribution and elimination of 14 C-styrene in rat. Scand. J. Work Environ. Health 7, 45–50. Chengelis, C., 1988. Age- and sex-related changes in epoxide hydrolase, UDP-glucuronosyl transferase, glutathione S-transferase, and
PAPS sulphotransferase in Sprague–Dawley rats. Xenobiotica 18, 1225–1237. Crofton, K.M., Lassiter, T., Rebert, C., 1994. Solvent-induced ototoxicity in rats: an atypical selective mid-frequency hearing deficit. Hear. Res. 80, 25–30. Fraenkel, R., Freeman, S., Sohmer, H., 2003. Susceptibility of young adult and old rats to noise-induced hearing loss. Audiol. Neurootol. 8, 129–139. Gratton, M.A., Schmiedt, R.A., Schulte, B.A., 1996. Age-related decreases in endocochlear potential are associated with vascular abnormalities in the stria vascularis. Hear. Res. 102, 181–190. Hamernik, R.P., Qiu, W., 2000. Correlations among evoked potential thresholds, distortion product otoacoustic emissions and hair cell loss following various noise exposures in the chinchilla. Hear. Res. 150, 245–257. Keithley, E.M., Feldman, M.L., 1982. Hair cell counts in an age-graded series of rat cochleas. Hear. Res. 8, 249–262. Keithley, E.M., Feldman, M.L., 1979. Spiral ganglion cell counts in an age-graded series of rat cochleas. J. Comp. Neurol. 188 (3), 429–442. Keithley, E.M., Ryan, A.F., Feldman, M.L., 1992. Cochlear degeneration in aged rats of four strains. Hear. Res. 59, 171–178. Li, H.S., Johnson, A.C., Borg, E., Hoglund, G., 1992. Auditory degeneration after exposure to toluene in two genotypes of mice. Arch. Toxicol. 66, 382–386. Loquet, G., Campo, P., Lataye, R., 1999. Comparison of tolueneinduced and styrene-induced hearing losses. Neurotoxicol. Teratol. 21, 689–697. McFadden, S., Campo, P., Quaranta, N., Henderson, D., 1997. Agerelated decline of auditory function in the chinchilla. Hear. Res. 111, 114–126. McFadden, S., Campo, P., Ding, D., Quaranta, N., 1998. Effects of noise on inferior colliculus evoked potentials and cochlear anatomy in young and aged chinchillas. Hear. Res. 117, 81–96. Miller, J., Dolan, D., Raphael, Y., Altschuler, R., 1998. Interactive effects of aging with noise induced hearing loss. Scand. Audiol. 27, 53–61. M¨oller, C., Odkvist, L., Larsby, B., Tham, R., Ledin, T., Bergholtz, L., 1990. Otoneurological findings in workers exposed to styrene. Scand. J. Work Environ. Health 16, 189–194. Morata, T.C., Campo, P., 2001. Auditory function after single or combined exposure to styrene: a review. In: Prasher, D., Henderson, D., Kopke, R., Salvi, R., Hamernik, R. (Eds.), Noise-induced Hearing Loss: Basic Mechanisms, Prevention and Control. nRn publications, London, pp. 293–304. M¨uller, M., 1991. Frequency representation in the rat cochlea. Hear. Res. 51, 247–254. Pouyatos, B., Campo, P., Lataye, R., 2002. Use of DPOAEs for assessing hearing loss caused by styrene in the rat. Hear. Res. 265, 156–164. Pujol, R., 1986. Periods of sensitivity to antibiotic treatment. Acta Otolaryngol. 429, 29–33. Roth, B., Bruns, V., 1992. Postnatal development of the rat organ of Corti. I and II Anat. Embryology 185, 559–581. Rybalko, N., Syka, J., 2001. Susceptibility to noise exposure during postnatal development in rats. Hear. Res. 155, 32–40. Savolainen, H., Pf¨affli, P., 1978. Accumulation of styrene monomer and neurochemical effects of long-term inhalation exposure in rats. Scand. J. Work Environ. Health 4 (2), 78–83. Sliwinska-Kowalska, M., Zamyslowska-Szmytke, E., Szymczak, W., Kotylo, P., Fiszer, M., Wesolowski, W., Pawlaczyk-Luszczynska, M., 2003. Ototoxic effects of occupational exposure to styrene and coexposure to styrene and noise. J. Occup. Environ. Med. 45 (1), 15–24. Sun, J.C., Bohne, B.A., Harding, G.W., 1994. Is the older ear more susceptible to noise damage? Laryngoscope 104 (10), 1251–1258. Yano, B.L., Dittenber, D.A., Albee, R.R., Mattsson, J.L., 1992. Abnormal auditory brain stem responses and cochlear pathology in rats induced by an exaggerated styrene exposure regimen. Toxicol. Pathol. 20 (1), 1–6.
Influence of age on noise- and styrene-induced hearing loss in the Long–Evans rat Benoˆıt Pouyatos∗ , Pierre Campo1 , Robert Lataye1 Institut National de Recherche et de S´ecurit´e, Ave de Bourgogne, BP 27, 54501 Vandoeuvre, France Available online 30 January 2005
Abstract This paper reviews different investigations carried out with Long–Evans rats on the influence of age on the ototoxicity induced by styrene and on the vulnerability to noise. The first part of this article is focused on the differences in auditory susceptibility to noise (92 or 97 dB octave band noise centered at 8 kHz, 6 h/day, 5 days/week, 4 weeks) and styrene (700 ppm, 6 h/d, 5 d/w, 4 w) between young (three and half months) and old (24 months) Long–Evans rats. Auditory evoked potential measures revealed that the old rats tend to be more sensitive than young rats to higher noise levels (97 dB), but equally vulnerable to moderate levels (92 dB). By contrast, the aged rats were virtually insensitive to 700 ppm styrene compared to the young animals. Two additional studies were performed controlling and examining the influence of body weight and post-natal age on the sensitivity to styrene. Rats of the same age (21 weeks) and but having different body weight (∼310 g versus ∼410 g) did not show any difference of sensitivity to 700 ppm styrene, whereas 14-week-old rats with the same body weight as 21-week-old rats (∼350 g) revealed increased sensitivity to styrene. These results show that weight does not play a key role in the sensitivity to styrene, and suggest a long period of increased sensitivity to styrene during the first months of life. Published by Elsevier B.V. Keywords: Styrene; Noise; Hearing loss; Aging; Age; Weight
1. Introduction Aging and exposure to noise are undoubtedly the two variables responsible for most cases of hearing loss in humans. However, in the workplace, exposure to solvents can also be considered as an additional significant risk for hearing (M¨oller et al., 1990; Calebrese et al., 1996; Morata and Campo, 2001; Sliwinska-Kowalska et al., 2003). The effects of noise and solvents as well as the characteristics of age-related hearing loss (called presbycusis) have been extensively studied in the past 15 years, but surprisingly few studies have focused on the interactions among these different factors. The present article reviews the different ∗ Corresponding author. Present address: Jerry Pettis Memorial Veterans Medical Center, Research Service (151), 11201 Benton Street, Loma Linda, CA 92357, USA. Tel.: +1 909 825 7084x2816; fax: +1 909 796 4508. E-mail address: [email protected] (B. Pouyatos). 1 Fax: +33 3 83 50 20 96.
1382-6689/$ – see front matter. Published by Elsevier B.V. doi:10.1016/j.etap.2004.12.020
experiments performed in our laboratory to determine the influence of age (and aging) on the vulnerability to noise and styrene exposure. Some of the material included in this article was already published in Campo et al. (2003). The effects of aging on the peripheral and central auditory systems have been studied in different animals models (e.g. Miller et al., 1998; McFadden et al., 1997, 1998). Along with a loss of hearing sensitivity, aged subjects display several types of peripheral degenerations: vascular (Keithley et al., 1992; Gratton et al., 1996), neuronal (Keithley and Feldman, 1979), and sensory (Keithley and Feldman, 1982). Aging is also associated with a down-regulation in metabolism, which may underlie an increased sensitivity to stress agents and a decreased repair of tissues following stress. However, the question of relative vulnerability of aged subjects to noise and others environmental ototoxicants has been rarely addressed despite its obvious importance regarding the safety of aged workers. Miller et al. (1998) showed that aged mice were slightly more sensitive to high-level noise (0.5–40 kHz
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noise at 108 dB sound pressure level (SPL) for 45 min) than young ones; but he also stated that the influence of the genetic characteristic of the strain was predominant. Sun et al. (1994) demonstrated that older chinchilla ears were not more susceptible to noise damage than younger ears when exposed to 95 dB (0.5 kHz) for 36 days. McFadden et al. (1998) reported that old chinchillas are equally vulnerable to moderate-level noise (95 dB), but may be slightly more vulnerable to high-level noise (106 dB). Fraenkel et al. (2003) recently showed that, when exposed to 113 dB for 1 h or for 3 days (12 h noise/12 h quiet), 3- and 24-month-old Wistar rats exhibited similar recovery after noise exposure as well as similar permanent hearing loss. As far as the effect of aging on solvent ototoxicity is concerned, the literature is very poor. Only Li et al. (1992) demonstrated that toluene exposure could aggravate auditory deterioration in mice with a strong genetic predisposition to spontaneously precocious age-related hearing loss (C57Bl/6J), but not in a strain that show moderate signs of presbycusis (CBA/Ca). However, these authors did not determine whether aged animals were more sensitive to a solvent exposure regardless of the genetic background. In order to ameliorate the lack of data in the literature concerning influence of aging on the sensitivity to noise and on solvent ototoxicity, we performed an experiment in which young and old rats were exposed either to noise (experiment 1) or to styrene (experiment 2), a solvent with stronger ototoxicity potency than toluene (Loquet et al., 1999). Subsequently, the results obtained in this first series of experiments raised questions about the influence of body fat content on the sensitivity to styrene, as well as about the existence of a critical period of hypersensitivity to styrene during the first months of life in the rat. As a result, two investigations were added to discriminate the effect of weight (experiment 3) from the effect of age (experiment 4) on styrene ototoxicity. The rationale for these studies was that a difference in weight could constitute a stock reserve for the solvent prior to metabolizing it (Carlsson, 1981; Savolainen and Pf¨affli, 1978) and thus bias the results obtained in young and aged animals. In summary, the main goals of the present investigations were to establish the influence of aging on the auditory sensitivity to noise or styrene, and to compare styrene-induced hearing loss as a function of weight and post-natal age of the animals.
2. Methods 2.1. Animals and experimental design One hundred and fifty male Long–Evans rats were used in these investigations. All the animals were obtained from Janvier Laboratories (Le Genest Saint Isle, France). Except otherwise indicated, the animals received standard food (A04, UAR® , Villemoisson-sur-Orge, France) and tap water ad libitum. Lighting was on from 07:00 to 19:00 h, the temperature in the animal quarters was 22 ± 1 ◦ C and the relative humidity
Table 1 Experiments 1 and 2—treatment groups and experimental conditions Age at the begining of the exposure Experiment 1 14 w 14 w 14 w 24–26 m 24–26 m 24–26 m Experiment 2 14 w 14 w 24–26 m 24–26 m
Exposure
n
92 dB SPL (OBN/8 kHz), 6 h/d, 5 d/w, 4 w 97 dB SPL (OBN/8 kHz), 6 h/d, 5 d/w, 4 w Controls 92 dB SPL (OBN/8 kHz), 6 h/d, 5 d/w, 4 w 97 dB SPL (OBN/8 kHz), 6 h/d, 5 d/w, 4 w Controls
8 8 8 8 8 8
Styrene 700 ppm, 6 h/d, 5 d/w, 4 w Controls Styrene 700 ppm, 6 h/d, 5 d/w, 4 w Controls
13 13 14 14
OBN/8 kHz, octave band noise centered at 8 kHz; dB, decibel; SPL, sound pressure level; h, hour; d, day; w, week; m, month.
ranged from 50 to 55%. The animals were housed in individual cages (35 cm × 18 cm × 18.4 cm). While conducting the research described in this article, the investigators adhered to the Guide for Care and Use of Laboratory Animals, as mandated by the French Conseil d’Etat in D´ecret no. 87-848 published in the French Journal Officiel on 20 October 1987, and the principles of the declaration of Helsinki. Because, the relationship between aging and noiseinduced hearing loss was not clear, we carried out the experiment 1 in which young (14-week-old) and old (26-month-old) Long–Evans rats were exposed to an octave band noise centered at 8 kHz (OBN/8 kHz) presented at 92 or 97 dB, 6 h/day, 5 days/week for 4 weeks (6 h/d, 5 d/w for 4 w). These parameters were chosen to induce a permanent threshold shift (PTS) of 10–30 dB in the vicinity of the frequency range injured by styrene. In the experiment 2, young and old rats (same ages as in experiment 1) were exposed to styrene by inhalation at a concentration of 700 ppm, 6 h/d, 5 d/w for 4 w. Table 1 summarizes the experimental groups and exposure conditions used in experiments 1 and 2. The weight parameter was studied in experiment 3 by exposing two groups of rats of the same age (21 weeks old), but having different body weight (slim: ∼310 g versus fat: ∼410 g) to 700 ppm styrene (6 h/d, 5 d/w, 4 w). Twenty-four rats of 10 weeks of age on their arrival in the animal facility were used. Twelve of them received standard animal food in controlled quantities (UAR® A04: 16% proteins, 4% fibers, 5% minerals, 3% lipids, 12% moisture, 60% nitrogen free extract) to reach an average body weight of 312 g, whereas 12 others received an enriched food (UAR® A03: 21.4% proteins, 3.9% fibers, 5.7% minerals, 5.1% lipids, 51.7% nitrogen free extract) to reach an average weight of 411 g at the beginning of the exposure; 11 weeks after their arrival. Due to the diet, a 100-g difference was obtained between the two groups of animals at the age of 21 weeks. In the experiment 4, two groups of rats having the same weight (∼345 g), but being of different ages (14 and 21 weeks old) were exposed to 700 ppm styrene (6 h/d, 5 d/w, 4 w) to
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Table 2 Experiment 3 and 4—treatment groups and experimental conditions Parameter tested
Age at the beginning of the exposure (w)
Weight (g)
Exposure
n
21 21 21 21
314 ± 3 310 ± 3 g 408 ± 3 415 ± 3
Styrene 700 ppm, 6 h/d, 5 d/w, 4 w Controls Styrene 700 ppm, 6 h/d, 5 d/w, 4 w Controls
6 6 6 6
14 14 21 21
342 ± 16 335 ± 11 353 ± 6 351 ± 16
Styrene 700 ppm, 6 h/d, 5 d/w, 4 w Controls Styrene 700 ppm, 6 h/d, 5 d/w, 4 w Controls
6 6 6 6
Experiment 3 Weight Experiment 4 Postnatal age
h, hour; d, day; w, week; g, gram; ppm, parts per million.
test the effect of post-natal age on the sensitivity to styrene. Twelve 7-week-old and 12 14-week-old rats were used. The youngest rats received the enriched food (UAR® A03) to reach a target weight of 345 g, whereas the oldest were fed with a standard breeding food (UAR® A04) to keep their weight close to the target weight. At the beginning of the exposure, the youngest rats were 14 weeks old, whereas the oldest were 21 weeks old. The experimental groups and exposure parameters used in experiments 3 and 4 are summarized in Table 2. All the experiments reported in the present paper allowed comparing the noise effects with those induced by styrene on hearing in young adult and aged populations of Long–Evans rats. Auditory function was tested “within subject” by recording the near field evoked potentials from the inferior colliculus before and 6 weeks after exposure in order to calculate PTS. After the last physiological measurement, the histological damage was assessed by counting hair cells of the organ of Corti.
by the center of the animal’s head. The targeted intensity is obtained by adjusting the level of the continuous pure tone. The electrical signal from the implanted electrode was amplified (×2000) and filtered between 30 and 3000 Hz. Averaged auditory evoked potentials were obtained from 260 presentations. An amplitude trough-to-peak (N1-P1) of 15 V of the response was considered as the threshold value. For each animal, an audiogram was obtained prior to styrene exposure (T1) and 6 weeks after exposure (T2). Permanent threshold shifts were defined as PTS = T2 − T1. 2.3. Noise exposure The rats were exposed either to 92 ± 1 dB SPL or to 97 ± 1 dB SPL OBN/8 kHz for 6 h/d, 5 d/w for 4 consecutive weeks. They were housed alone in individual cages with a speaker above the cages. Controls were maintained in the same conditions without noise. 2.4. Styrene exposure
2.2. Hearing testing Rats were deeply anesthetized by the i.p. administration of ketamine (45 mg/kg) and xylazine (6 mg/kg). A tungsten electrode was implanted into the right inferior colliculus, and a second electrode was placed in the rostral cranium just below the dura mater to serve as the ground electrode. These two electrodes were then fastened to a transistor socket and fixed with dental cement to the skull. One month after surgery, audiometric testing was performed in a soundproof booth on awake rats placed in a restraining device. The generation and the signal treatment were performed with a Tucker–Davis Technologies apparatus. The acoustic stimuli (two cycles for the rise/fall ramp, four cycles for the plateau) were gated sinusoidal stimuli at 2, 3, 4, 5, 6, 8, 10, 12, 16, 20, 24 and 32 kHz, presented at a rate of 20/s, with an analysis window of 30 ms. The stimuli were transduced by a speaker (JBL, 2405) positioned 15 cm from the left pinna. For each frequency, the acoustic calibration was carried out by measuring the sound pressure level emitted by the speaker, when it was driven by a continuous pure tone. The sound field was calibrated by positioning a microphone at a point normally occupied
The animals were housed in individual cages placed in inhalation chambers and exposed to 700 ppm styrene vapors (Sigma–Aldrich, 99%) for 6 h/d, 5 d/w, 4 w. The controls were housed in similar chambers ventilated with fresh air. The chambers (200 l) were maintained at a negative pressure of no more than 3 mm H2 O in order to sustain a dynamic and adjustable airflow (10–20 m3 /h). The input air was filtered and conditioned to a temperature of 22–24 ◦ C and a relative humidity of 50–55%. The styrene was vaporized by bubbling an additional airflow through a flask containing the test compound. The solvent concentration in the chambers was measured by collecting atmosphere samples through glass tubes packed with activated charcoal. Styrene samples were desorbed with carbon disulfide and analyzed by a gas chromatograph (GC: Intersmat, 120FB I.G.C. model, France) using oxylene as the internal standard. These analyses allowed daily calibration of another GC used for the continuous monitoring of exposure level; this GC was equipped with a flame ionization detector and an automatic gas-sampling valve. Concentration measurements were performed at regular intervals (0.5 min).
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2.5. Histology At 6 weeks post-exposure, the animals were anesthetized with a heavy dose of ketamine (75 mg/kg), and then fixed by transcardiac perfusion with 300 ml of a tri-aldehyde fixative (3% glutaraldehyde, 2% formaldehyde, 1% acrolein and 2.5% DMSO in buffer pH 7.4). The cochleae were harvested and fixed again by perilymphatic perfusion. Following the primary 24-h fixation, the cochleae were post-fixed with 1% OsO4 in buffer pH 7.4 for 1 h and finally washed again with buffer. The organ of Corti was dissected in 70% ethanol and mounted in glycerin for counting of hair cells. The frequencyplace map established by M¨uller (1991) was used to superimpose the frequency coordinates on the length coordinates of the organ of Corti. 2.6. Statistical analysis The statistical analyses were made with NCSS software (Kaysville, UT). The electrophysiological data obtained in the experiments in which the effect of age was investigated were analyzed with repeated measure analyses of variances (ANOVAs) using the “treatment” and “age” as the between subject factors, and “frequency” as the within factor. For the experiment in which the effect of weight was investigated, the “weight” was used as a between subject factor instead of the “age”. Planned post hoc comparisons were performed between treatment groups using the Tukey–Kramer test. p = 0.05 was considered as the significance threshold.
3. Results 3.1. Influence of aging on the auditory sensitivity to noise or styrene 3.1.1. Experiment 1: noise Fig. 1 presents the PTS calculated in young and old animals exposed to 92 and 97 dB noises, compared to the
Fig. 1. Permanent threshold shifts (PTS) vs. frequencies, ranging from 2 to 32 kHz, obtained from young (14-week-old) and old (24–26-month-old) noise-exposed rats and age-matched controls (n = 8 per group). The noise was an octave band noise centered at 8 kHz at (a) 92 dB or (b) 97 dB sound pressure level (SPL), 6 h/d, 5 d/w, 4 w. Error bars: 95% half-confidence interval.
age-matched controls. The 92-dB noise induced threshold shifts at frequencies ranging from 8 to 16 kHz, with the peak occurring near 10–12 kHz, which corresponds to approximately one-half octave above 8 kHz, the center frequency of the exposure. At this noise intensity, there was no obvious difference between the young and aged groups in terms of PTS. At 97 dB SPL, the PTS were large from 8 up to 32 kHz regardless of the age of the animals. However, the average amplitude of the PTS measured in the old rats was 10 dB greater than in the young animals in that frequency range. The electrophysiological measures were analyzed using two different repeated-measure ANOVAs, one for each noise level. The ANOVAs showed significant effect of both 92 dB noise [F(1,28) = 36.08; p < 0.0001] and 97 dB noise [F(1,28) = 197.36; p < 0.0001] on PTS. The age effect was significant for the 97 dB noise [F(1,28) = 21.27; p < 0.0002], but not for the 92 dB noise [F(1,28) = 1.01; p = 0.32]. Post hoc comparisons using Tukey–Kramer multiple-comparison tests showed that the PTS measured in the old rats exposed to 97 dB were significantly (p < 0.05) different from the PTS measured in young animals. PTS measured in old and young rats exposed to 92 dB were not significantly different. The hair cell loss obtained in the different experimental groups is presented in Fig. 2. The average cochleogram obtained with the young controls revealed very small amounts of hair cell loss (<1%) scattered along the organ of Corti. Consequently, it is not shown. By contrast, the cochleae from old controls (Fig. 2a) displayed significant OHC loss particularly at the extreme apex (OHC3 : 50%; OHC2 : 28.5%; OHC1 : 22% at 0.5 kHz). Despite this damage, the auditory thresholds measured in old animals were just slightly higher (by an average of 2 dB between 2 and 32 kHz) than those measured in young rats (Campo et al., 2003). The 92 dB noise exposure did not induce any hair cell loss in any animals (not shown), but the 97 dB intensity caused significant OHC loss in both young (Fig. 2a) and old (Fig. 2b) rats. The cochleogram obtained from young rats shows that the cell losses of the first row (9.3%) were greater than those of the second (3.8%) and third (2%) rows between 12 and 34 kHz. The averaged OHC loss induced by the 97 dB exposure was quantitatively not greater in the aged rats than in the young rats, but the losses in the aged group were surprisingly located in a higher frequency range: from 23 to 37 kHz. 3.1.2. Experiment 2: styrene Fig. 3 shows the styrene-induced hair cell loss observed in young and old animals after a 4-week exposure to a concentration of 700 ppm. Surprisingly, only the young rats showed significant hearing loss, up to 15 dB at 20 kHz. No difference was obtained between controls and aged styrene-treated rats. The ANOVA showed significant effect of 700 ppm styrene [F(1,50) = 10.95; p < 0.002] on PTS. The age effect was also significant [F(1,50) = 19.67; p < 0.0001]. Post hoc comparisons using Tukey–Kramer multiple-comparison tests showed that the PTS measured in the old rats exposed to
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Fig. 3. Permanent threshold shift (PTS) vs. frequencies, ranging from 2 to 32 kHz, obtained from young (14-week-old; n = 13) and old (24–26-monthold; n = 14) styrene exposed animals, and age-matched controls (young: n = 13; old: n = 14). Styrene exposure: 700 ppm, 6 h/d, 5 d/w, 4 w. Error bars represent the 95% half-confidence intervals.
700 ppm styrene were significantly (p < 0.05) different from the PTS measured in young animals. Consistent with physiological results, the styrene-induced cochlear damage was much larger in young than in old subjects. In the young cochleae (Fig. 4a), the styrene exposure caused up to 61% of OHC loss on the third row between 1 and 28 kHz, with a gradual decrease for OHC2 and OHC1 . No IHC was observed in any styrene-exposed cochlea. The OHC loss observed in the old animals exposed to styrene (Fig. 4b) in the same conditions was strikingly smaller, when the aged-related hair cell loss (observed in aged controls): the percentages were ∼23% at the level of the OHC1 , ∼3% at OHC2 and ∼2% at OHC3 . Similarly, the IHC were not injured after styrene in the old cochleae.
3.2. Influence of weight and post-natal age on styrene ototoxicity
Fig. 2. Average cochleogram obtained from (a) old control rats (n = 9), (b) young (n = 5) and (c) old (n = 5) 97 dB sound pressure level (SPL) exposed rats. Noise schedule: 6 h/d, 5 d/w, 4 w. Abscissa, upper trace: length (mm) of the entire spiral course of the organ of Corti from the bottom of the hook; lower trace: frequency-map according to M¨uller (1991). Ordinate: hair cell loss in percent. IHC: inner hair cells; OHC1 : first row of outer hair cells; OHC2 : second row; OHC3 : third row. Error bars represent the S.E.M.
3.2.1. Experiment 3: weight Fig. 5 presents the hearing loss and the cochleogram obtained in 310 and 410 g animals exposed to 700 ppm styrene. Styrene induced very similar threshold shift in both groups (Fig. 5a), approximately 8 dB at the vicinity of 16 kHz. The ANOVA showed significant effect of 700 ppm styrene [F(1,20) = 6.88; p < 0.02] on PTS, but no effect of weight [F(1,20) = 0.00; p = 0.98]. Post hoc comparisons using Tukey–Kramer multiple-comparison tests confirmed that the PTS measured in the heavier rats exposed to 700 ppm styrene were not significantly (p < 0.05) different from the PTS measured in the lighter animals. As expected, the cochleograms obtained from 310 and 410 g animals were similar (Fig. 5b and c). Hair cell loss was massive in the third row for which 58% of the cells were missing from 2 to 27 kHz, peaking around 5 and 20 kHz. The second row was less damaged (13%) than the third, but more than the first row (5%). The high-frequency region (above 30 kHz) and the IHCs were typically well preserved.
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Fig. 4. Average (n = 5 per group) cochleograms obtained from (a) young and (b) old 700 ppm styrene-exposed rats. Styrene exposure: 6 h/d, 5 d/w, 4 w. Abscissa, upper trace: length (mm) of the entire spiral course of the organ of Corti from the bottom of the hook; lower trace: frequency-map according to M¨uller (1991). Ordinate: hair cell loss in percent. IHC: inner hair cells; OHC1 : first row of outer hair cells; OHC2 : second row; OHC3 : third row. Error bars represent the S.E.M.
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Fig. 5. (a) Permanent threshold shifts (PTS) vs. frequency obtained from styrene-exposed rats (n = 6 per group) having the same age (21-week-old) but having different weight (310 g vs. 410 g). Styrene exposure: 700 ppm, 6 h/d, 5 d/w, 4 w. Each point represents the mean and the bars represent the 95% half-confidence intervals. Average (n = 5 per group) cochleograms obtained from the styrene-exposed (b) 310 g and (c) 410 g groups. The animals were 31 weeks old when the cochleae were harvested (21 weeks old at the beginning of the exposure). Abscissa, upper trace: length (mm) of the entire spiral course of the organ of Corti from the bottom of the hook; lower trace: frequency-map according to M¨uller (1991). Ordinate: hair cell loss in percent. IHC: inner hair cells; OHC1 : first row of outer hair cells; OHC2 : second row; OHC3 . The bars represent the S.E.M.
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Fig. 6. (a) Permanent threshold shift (PTS) vs. frequencies, ranging from 2 to 32 kHz, obtained from styrene-exposed rats (n = 6 per group). The animals have the same weight (345 g) but different age: 14 vs. 21 weeks old (at the beginning of the exposure). Styrene exposure: 700 ppm, 6 h/d, 5 d/w, 4 w. Each point represents the mean and the bars represent the 95% half-confidence intervals. Average cochleogram obtained from (b) the 14-week-old rats, (c) the 21-week-old rats (age at the beginning of the exposure). Abscissa, upper trace: length (mm) of the entire spiral course of the organ of Corti from the bottom of the hook; lower trace: frequency-map according to M¨uller (1991). Ordinate: hair cell loss in percent. IHC: inner hair cells; OHC1 : first row of outer hair cells; OHC2 : second row; OHC3 .
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3.2.2. Experiment 4: post-natal age Fig. 6 shows the hearing loss and the cochlear damage obtained in the 14- and 21-week-old rats exposed to 700 ppm styrene. The younger animals were obviously more sensitive to styrene, as shown by physiological and histological data. Respectively, 23.5 and 8 dB styrene-induced hearing loss were obtained at the vicinity of 16 kHz in 14- and 21week-old animals (Fig. 6a). The ANOVA confirmed the significant effect of 700 ppm styrene [F(1,20) = 31.63; p < 0.0001] on PTS. The effect of age was also significant [F(1,20) = 5.54; p < 0.03]. Post hoc comparisons using Tukey–Kramer multiple-comparison tests confirmed that the PTS measured in the 14-week-old rats exposed to 700 ppm styrene were greater (p < 0.05) than PTS measured in the older animals. The OHC loss obtained in the 14- and 21-week-old rats exposed to 700 ppm styrene differed mainly at the level of the first two row of OHC: in the 14-week group, the styrene exposure caused 21.5% of OHC loss in the first row and 39.8% in the second row in the 16–25-kHz region, while only 9.2% of OHC were missing from the first row, and 16% from the second row in the 21-week-old group in the same region.
4. Discussion The findings of the first experiments showed conclusively that 24–26-month-old rats are, by and large, equally vulnerable to moderate-level noise (92 dB SPL) as 14-week-old rats, but may be more vulnerable to moderate–high-level noise (97 dB SPL), specifically in the high-frequency region. By contrast, older rats were very resistant to styrene ototoxic effect compared to the younger animals. In the second part of this paper, we show that this surprising greater sensitivity to styrene of the younger animals could be explained by the existence of a long period of hypersensitivity to solvents during the first months of life. We also demonstrate that the sensitivity to styrene of 21-week-old rats does not seem to be influenced by their body weight. More detailed information on these experiments is available in Campo et al. (2003). The fact that old animals are more sensitive to higher noise exposure and equally sensitive to lower noise exposures than young animals has already been reported in previous works with chinchillas (McFadden et al., 1998), and mice (Miller et al., 1998). Conversely, our results are inconsistent with those obtained by Fraenkel et al. (2003) in the Wistar rat. They did not obtain any difference in susceptibility between young and aged animals, which were approximately the same age as our subjects. This discrepancy may be due to either the difference of strain (Wistar versus Long–Evans), noise intensity (113 dB versus <97 dB), or noise duration (1 h or 3 days versus 4 weeks). Clearly, some more work has to be done to determine whether aging should be considered as a significant risk factor. Regarding the histological findings, the hair cell loss obtained in the young and aged rats exposed to 97 dB SPL was
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low relative to the electrophysiological data. However, it is well known that the noise effects cause predominant stereocilia damage but moderate hair cell loss (Hamernik and Qiu, 2000). Therefore, the strict quantitative hair cell counts performed in these studies probably underestimate the damage. If the amount of hair cell loss was similar in young and aged animals, the location was different: it was centered at 20–22 kHz region in the young subjects, and near the 30 kHz place in the aged animals. The reason for such a difference in tonotopicity of the trauma is unknown. One hypothesis is that the hair cell losses already present in the organ of Corti of the aged rats could modify the mechanical properties of the basilar membrane, and thereby the location of the trauma. Styrene-induced hearing loss has already been reported in rats (Yano et al., 1992; Crofton et al., 1994; Campo et al., 2001; Pouyatos et al., 2002), but to the best of our knowledge, no data have been published on styrene effects as a function of age of the exposed subjects. Contrary to all expectations, old rats were very resistant (PTS = ∼0 dB) to the ototoxicity of styrene compared to young animals (PTS = ∼15 dB). Aged rats showed only fairly small hair cell loss in the third row of OHCs. Such a loss of OHCs was modest with regard to that observed in the young rats, and apparently insufficient to modify the functional results. According to Miller et al. (1998), aging is associated with a decrease in the metabolic activity that could enhance the vulnerability of the old subject to environmental insults. If this concept can explain the difference of sensitivity to noise between young and aged animals, it cannot explain the styrene effects since old ears showed better resistance to styrene. The difference in sensitivity to styrene between age groups could be due to a weight difference between animals since the aged rats weighted more than the young rats (500 g versus 300 g) at the beginning of exposure. By controlling the weight difference between two different groups of the same age, we expected to determine the influence of the body weight on the effects of styrene intoxication. The results revealed that a 100-g difference in the body weight does not play a major role in the ototoxic process, at least in our experimental conditions. So the hypothetical capacity of fat compartment to capture the solvents (Carlsson, 1981; Savolainen and Pf¨affli, 1978) and therefore to decrease their ototoxic potency was not confirmed. By contrast, our results demonstrate that age is an important factor in the susceptibility to styrene since the styrene exposure produced larger PTS in older than in younger animals. Therefore, a 7-week difference of age at the beginning of the rat life can have a dramatic influence on the ototoxic potency of styrene. This finding validates the hypothesis of a period of hypersensitivity to styrene. This long “critical period” is atypical in comparison to the critical periods already described for noise or aminoglycosides. Indeed, Rybalko and Syka (2001) showed that the vulnerability to noise is higher during the 5 first post-natal weeks, and Pujol (1986) demonstrated that the ear is more sensitive to aminoglycosides during the 3 first post-natal weeks. It is
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hard to think that this critical period of sensitivity to solvents can be correlated with maturational events in the cochlea. Indeed, cochlear structures and functions are fully developed and mature at the age of 3 months, the rat cochlea reaching its adult-like properties at the end of the third week (Roth and Bruns, 1992). It is necessary to find other explanations for our results. The metabolic changes in the liver could be the clue of the problem. The enzymatic reactions involved in the hepatic metabolism of styrene are an initial oxidation to styrene oxide, catalyzed by cytochrome P-450. Then styrene oxide is a substrate for epoxide hydrolase and glutathioneS-transferase to be metabolized in phenylethylene glycol and mercapturic acids, respectively (Bond, 1989). Hence, the age-related changes in liver enzymes, particularly the cytochrome P-450, the epoxide hydrolase and the glutathione-S-transferase might explain the difference of susceptibility stated between young and adult rats. Indeed, Chengelis (1988) showed that (1) total cytochrome P-450 increases substantially during the first weeks of life (from 12 to 34 nmol/g) reaching its peak at week 26 in males and; (2) the amount of epoxide hydrolase increases significantly between week 16 and 24 to reach 160 nmol/g; (3) in the mean time, glutathione-S-transferase doubles between week 16 and 24 tending to plateau at 63 nmol/g. Presumably, these agerelated changes in metabolism have the potential to change the sensitivity to solvent. Hence, the young rats would have a reduced metabolic activity that might explain the difference of vulnerability between the 14- and the 21-week-old groups. Acknowledgments The authors would like to thank the organizers of the 9th International Neurotoxicology Association for offering the opportunity to present our results during the student symposium. The authors are also grateful to Christian Barth´el´emy for his technical assistance, and Laurence D. Fechter for his comments on the manuscript. This study was supported by European grant QLK4-2000-00293 and by Institut National de Recherche et de S´ecurit´e. References Bond, J., 1989. Review of the toxicology of styrene. Crit. Rev. Toxicol. 19 (3), 227–249. Calebrese, G., Martini, A., Sessa, G., Cellini, M., Bartolucci, G.B., Marcuzzo, G., De Rosa, E., 1996. Otoneurological study in workers exposed to styrene in the fiberglass industry. Int. Arch. Occup. Environ. Health 68, 219–223. Campo, P., Lataye, R., Loquet, G., Bonnet, P., 2001. Styrene-induced hearing loss: a membrane insult. Hear. Res. 154 (1/2), 170–180. Campo, P., Pouyatos, B., Lataye, R., Morel, G., 2003. Is the aged rat ear more susceptible to noise or styrene damage than the young ear? Noise Health 5, 1–18. Carlsson, A., 1981. Distribution and elimination of 14 C-styrene in rat. Scand. J. Work Environ. Health 7, 45–50. Chengelis, C., 1988. Age- and sex-related changes in epoxide hydrolase, UDP-glucuronosyl transferase, glutathione S-transferase, and
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