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| Year : 2004
| Volume
: 7 | Issue : 25 | Page
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| Critical period for styrene ototoxicity in the rat |
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R Lataye, B Pouyatos, P Campo, AM Lambert, G Morel
Institut National de Recherche et de Sécurité‚ Vandoeuvre, France
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The current experiments were undertaken to determine whether or not styrene-induced hearing loss in the rat depends more on the existence of a critical period between 14 and 21 weeks of age than on body weight. For these purposes, two experiments were carried out with mature Long-Evans rats. In the first experiment, two groups of 5-month old rats, but having different body weight (slim: 314 g vs. fat: 415 g) were exposed to 700 ppm styrene for 4 consecutive weeks, 5 days per week, 6 hours per day. In the second experiment, two groups of rats having the same weight: 345 g, but different ages (14- vs. 21- week old) were exposed to styrene in strictly identical experimental conditions. Auditory sensitivity was tested by recording evoked potentials from the inferior colliculus. Surface preparations of the organ of Corti were also performed to complete the investigation. At the end of the six week recovery period following the styrene exposure, a 7 dB permanent threshold shift (PTS) was obtained with the same age animals regardless of the body weight. Consequently, weight was not a major factor in styrene-induced hearing loss. Age was a more critical factor in determining higher sensitivity to styrene. Indeed, the three months old group had 23.5 dB PTS, whereas the five months old group had only a 7.7 dB PTS at 16 kHz. Thus, a 15 dB difference of PTS was obtained between the rats having the same weight but different age. While the weight does not play a major role in styrene ototoxicity, there is a critical period whose duration lasts more than three months and for which the susceptibility to styrene is enhanced. Keywords: Critical period, Ototoxicity, Styrene, Solvent, Bodyweight
How to cite this article:
Lataye R, Pouyatos B, Campo P, Lambert A, Morel G. Critical period for styrene ototoxicity in the rat. Noise Health 2004;7:1-10 |
| Introduction | |  |
Styrene monomer is the principal precursor in the industrial production of artificial polymer materials. Consequently, styrene is widely used in industry by workers (Miller et al., 1994; Nylander-French et al., 1999). Although exposure levels have been decreased in most industrialized countries during the last decade, the number of workers with regular exposure to solvents is still large (Johnson and Nylen, 1995). Human studies have shown that chronic exposures to styrene can cause auditory deficits in workers (Moller et al., 1990; Calabrese et al., 1996; Morata and Campo, 2001; SliwinskaKowalska et al., 2003). Concurrently, numerous animal experiments have proved that styrene can severely disrupt the auditory function, specifically in the mature rat (Yano et al., 1992; Crofton et al., 1994; Campo et al., 2001, Pouyatos et al., 2002).
Hearing capacity in mammals is known to decline as a result of ageing. Age-related hearing loss is called presbycusis. While several authors have studied the age-related changes in rats (Borg, 1982; Keithley et al., 1992; Palombi et al., 1996), to the best of our knowledge, only Campo et al. (2003) have studied the cochlear effects of styrene in young and aged Long-Evans rats. The results were both clear and unexpected : the auditory function of aged rats were less sensitive to styrene than that of young rats. However, these findings were opened to a number of criticisms because of the large difference of body weight between the young (300 g) and the aged rats (500 g) used in the experiments. It is true that such a difference in weight: roughly 200 g, could constitute a storage compartment for the solvent prior to its metabolism and thus explain the discrepancy of vulnerability obtained between young and aged animals. Because of the lack of data in the literature regarding the influence of body weight on styrene ototoxicity, the authors have studied this particular parameter by carrying out two successive experiments.
In the first experiment, two groups of rats being the same age: 5 months old, but having different body weight (slim: 314 g vs. fat: 415 g) were exposed to 700 ppm styrene, 6 hours per day, 5 days per week for 4 consecutive weeks.
In the second experiment, two groups of rats having the same weight: 345 g, but being of different ages (3 months old vs. 5 months old ) were exposed in the same experimental conditions than previously. The choice of the dose of styrene and the choice of the age of the rats (3 months old) were determined in order to compare the results of the current study with those obtained in the prior study (Campo et al., 2003).
In summary, the main goal of the present investigation was to compare styrene-induced hearing loss as a function of (1) the weight, and (2) the postnatal age of the animals.
These investigations aimed to answer the question raised in a prior experiment, so the same experimental tools were kept to compare the data obtained in both studies. Therefore, auditory function was tested by recording the near field auditory evoked potentials from the inferior colliculus which is the major brainstem auditory structure receiving inputs from most other auditory nuclei, and the same histological analysis was performed on the organ of Corti.
| Methods | | |
Subjects
To avoid sex related changes in several hepatic enzymes in the rat (Chengelis, 1988), only male Long-Evans rats were used in this investigation. The animals were obtained from Janvier Laboratories in France.
Experiment 1
Twenty four rats being 10 weeks old at their arrival in the animal facility were used for this experiment. Twelve of them were put on a caloric restrained diet (UAR-A0410: 16% proteins, 4% fibres, 5% minerals, 3% lipids, 12% moisture, 60% nitrogen free extract) to reach an average body weight of 312g, whereas twelve others received a rich food (UAR-A03:21.4% proteins, 3.9% fibres, 5.7% minerals, 5.1% lipid, 51.7 % nitrogen free extract) to reach an average weight of 411g at the beginning of the exposure, so 11 weeks after their arrival. Due to the diet, a 100g difference was obtained between the two groups of animals at 21 weeks of age.
Experiment 2
Twelve rats being 7 weeks old and twelve others being 14 weeks old on their arrival in the animal facility were used for this experiment. The youngest rats received a rich food to reach a target weight of 345g, whereas the oldest were fed with a standard breeding food (UAR-A0410) 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. [Table - 1] summaries the age and the weight of the animals used in both experiments.
Regardless of the experiments, tap water was available ad libitum. Lighting was on from 7am to 7pm. The temperature in the animal quarters was 22±1°C and the relative humidity ranged from 50 to 55%. 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 Decret n°87-848 published in the French Journal Officiel on October, 20th, 1987, and the principles of the declaration of Helsinki.
Audiometry
Because the method has been already detailed in a companion publication (Campo et al., 1997), only a cursory description of it is presented hereafter. Rats were deeply anesthetized by the i.p. administration of a mixture of ketamine and xylazine at a dosage of 45 and 6 mg/kg respectively. Animals were then placed on a stereotaxic table while a tungsten electrode was implanted into the right inferior colliculus. A second electrode was implanted 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 30ms. 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 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 (x2000) and filtered between 30 and 3000 Hz. Averaged auditory evoked potentials were obtained from 260 presentations. An amplitude trough-to-peak (N1P1) of 15 µv of the response was considered as the threshold value. For each animal, an audiogram was obtained prior to styrene exposure (T1), at the end of exposure (T2), and six weeks after exposure (T3). Compound and permanent threshold shifts were respectively defined as : CTS = T2 - T1 and PTS = T3 - T1.
Styrene exposure
The animals (n=24) were exposed to 700 ppm styrene vapors (Sigma-Aldrich, 99 %), for 6 h/d, 5 d/w for 4 consecutive weeks in inhalation chambers. The chambers contained 8 animals housed in individual cages. Neither food nor water was given to the animals during exposure. Simultaneously, the controls were housed in similar chambers ventilated with fresh air. Designed to sustain a dynamic and adjustable airflow (4-8 m 3 /h), the chambers (200 L) were maintained at a negative pressure of no more than 3 mm H 2 O. The input air was filtered and conditioned to a temperature of 22-24 C and a relative humidity of 50-65 %. 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 o-xylene 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).
Histology
At six weeks postexposure, the animals were deeply anesthetized with a heavy dose of ketamine (75 mg/kg), and then fixed by trans cardiac perfusion with 300 ml of a tri-aldehyde fixative: 3% glutaraldehyde, 2% formaldehyde, 1% acrolein and 2.5% DMSO in 0.08 M sodium cacodylate buffer, pH = 7.4 after flushing out red blood cells with buffer. The temporal bones were then removed, the tympanic bullae opened, and the cochleae fixed again by perilymphatic perfusion. Following the primary 24 h-fixation, the cochleae were postfixed with 1% OsO4 in 0.08 M cacodylate buffer (pH = 7.4) for 1 h and finally washed in a trihydrate solution of sodium cacodylate. The cochleae were dissected in 70% ethanol at room temperature. The organ of Corti was dissected and mounted in glycerin. Hair cells were counted as present when either the stereocilia, the cuticular plate or the cell nucleus could be visualized. No attempt was made to assess the degree of possible cellular damage to surviving cells. The frequency-place map established by Muller (1991) was used to superimpose the frequency coordinates on the length coordinates of the organ of Corti. A cochleogram showing the percentage of hair cell loss as a function of distance from the base of the cochlea was plotted for each animal. The results were averaged across each group of animals for comparison between groups.
Statistical analysis
The Statgraphics® plus (version 5) software was used to run all the statistical analyses. A multifactor ANOVA, with "styrene", "weight" and "age" as between-subject factors, and tone "frequency" as within subjects, was run. This ANOVA allowed evaluation of either the "styrene" "weight" or "age" effect, or interactions such as "styrene x age", "styrene x weight". When needed, the ANOVA test was completed by a post-hoc Bonferroni's test which allows comparison of the means at each frequency. Alpha levels of 0.05 (significant at 95%) were used for the significance of the tests.
| Results | | |
Hearing testing
The mean auditory [brainstem (inferior colliculus)-evoked potentials] thresholds obtained from 14-week (n = 8) and 21-week groups (n = 8) rats are plotted in [Figure - 1]. The best frequency sensitivity (10.8 dB SPL) is located at 24 kHz. The threshold differences between the two groups were not significant.
Experiment 1
Styrene induced hearing loss
[Figure - 2] shows significant CTS and PTS following the styrene exposure [F styrene (1, 20) = 5.99, p = 0.002] for both the slim and fat groups of rats. A 8dB styrene-induced hearing loss was located at the vicinity of 16 kHz [F styrene*frequency (11, 362) = 2.05, p = 0.005], regardless of the groups of rats. Consequently, no significant difference was obtained between slim and fat groups [F weight (1, 20) = 0.09, p = 0.7681]. No significant recovery could be measured from the CTS induced by styrene at the end of the six week post-exposure period.
Cochleogram
The cochleogram obtained with the control group revealed that small amounts of hair cell loss can be observed along the organ of Corti, but they do not exceed 1% of all cells for any turn. [Figure - 3]a,b illustrates the mean (n=5) cochleogram obtained from the styrene-exposed slim and fat groups. There is not a large difference between these groups ; the fat group shows a slight trend to have more losses than the slim group. Hair cell losses are massive for both styrene groups in the third row for which 58 %
of the cells are missing from 2 to 27 kHz, peaking around 5 and 20 kHz. As expected, the second row is less damaged (13 %) than the third, but more than the first row (5 %). OHCs for frequencies above 30 kHz and IHC seem to be well preserved.
Experiment 2
Styrene-induced hearing loss
[Figure - 4] shows the CTS and PTS values as a result of styrene exposure for the 14week and 21week groups. Respectively, 23.5 and 8dB styrene-induced hearing loss were obtained at the vicinity of 16 kHz. Consequently, the 14week rats were more susceptible to styrene than the 21week rats [F age (1, 10) = 21.22, p = 0.0002].
Cochleogram
[Figure - 5]a corresponds to the mean (n=5) cochleogram obtained with the 14week group, and [Figure - 5]b to that obtained with the 24week group exposed to styrene. The losses in the third row were massive (80.3% between 2 and 30 kHz) for both groups. The most striking difference appeared at the vicinity of 20 kHz in the second and first rows. For the audiometric frequencies ranging from 16 kHz to 25 kHz, the styrene exposure caused in the 14week group: 21.5% of OHC loss in the first row and 39.8% in the second row. As far as the 21week group was concerned, only 9.2% of OHC were missing in the first row, and 16% in the second row.
| Discussion | | |
Despite the fact that a significant fraction of styrene and its metabolites accumulates in the fat of the rat (Carlsson, 1981), the results obtained in the first investigation demonstrate that a 100g difference in the body weight did not play a major role in the ototoxic process.
The fat of the animal which could constitute a storage compartment for the solvent (Savolainen and Pfaffli, 1978) and therefore decrease the ototoxic potency of solvent was not confirmed in our experimental conditions. In fact, the rapidity with which the tissues or the target organs are reached by the solvent might be determinant in terms of ototoxic potency. Since brain and cochlea are two richly perfused tissues, they could be reached by the solvent before being stocked by the fat compartment of the animals.
On the contrary, the results of the second experiment demonstrated that age is an important factor to take into consideration in the sensitivity to styrene since the styrene exposure produced larger and broader PTS in older than in younger animals. The age effect had been already identified in a prior publication in which the hearing of 26month old rats was much less damaged than 3month old rats exposed to 700ppm styrene in the same experimental conditions (14 versus 21 weeks of age). The age difference in the current study is somewhat smaller (two months) than in the previous study. Therefore, a 2month period at the beginning of the rat life can have a dramatic influence on the ototoxic potency of a 700ppm styrene exposure for 4 consecutive weeks, 5 days per week for 6 hours per day. Based on these findings, the variation of sensitivity stated in Campo et al. (2003) between the young and the aged groups was not really the result of a lower sensitivity to solvent of the aged rats, but rather the result of a greater sensitivity of the 3month old rats compared with that of mature rats. Since the same styrene exposure (700ppm, 6h/d, 5d/w for 4w) is more ototoxic in young rats (3month old) than in young adult (5month old) or aged (26month old) rats, a period of increased sensitivity to solvent ototoxicity appears to exist. This "critical period" for solvents which extends during a period lasting between 3 and 5 months (in conditions of exposure used in our experiments), is considerably longer than that to acoustic trauma: the 5 first postnatal weeks according to Rybalko and Syka, (2001), or to aminoglycosides: the 3 first postnatal weeks according to Pujol (1986) and Lenoir et al. (1986).It is hard to think that this critical period of sensitivity to solvents can be closely correlated with specific maturational events in the cochlea. Indeed, cochlear structures and functions are fully developed and mature at 3 postnatal months, the rat cochlea reaching its adult-like properties at the end of the third week (Roth and Bruns, 1992). Definitive maturation of the auditory system exceeds the period of cochlea development according to morphological criteria. However, the difference in solvent susceptibility may be connected with metabolic changes in the cochlea. Zelck et al. (1993), and Whitlon et al. (1999) suggested that the biochemical immaturity of detoxification enzymes of the cochlea in juvenile animals continues to be present 5 weeks after birth and can account for the higher susceptibility to ototoxic agents. For instance, the gluthathioneS-transferase composition in the cochlea reaches its standard value by one month of age (Whitlon et al., 1999).
Taking into account the metabolic changes in the cochlea, we are still far from the 3 months corresponding to the youngest rats tested in the second experiment. It is necessary to find other arguments to explain 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 of styrene to styrene oxide, catalyzed by cytochrome P450, 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 and in their induction, particularly the cytochrome P450, the epoxide hydrolase and the glutathione-Transferase might explain the difference of susceptibility stated between young and adult rats. Indeed, Chengelis (1988) showed that (1) total cytochrome P450 increases a lot during the first weeks [from 12 to 34 nmol/g] to peak at week 26 in males and; (2) the amount of epoxide hydrolase increases significantly between week 16 to 24 to reach 160 nmol/g; (3) in the mean time, glutathione-S-transferase doubles between week 16 to week 24 tending to plateau at 63 nmol/g. These enzymes are of interest because of their potential involvement in detoxifying styrene and their reactive intermediates. It is therefore reasonable to think that the age-related changes in metabolism have the potential to change the sensitivity to solvent. These changes can influence study results and their interpretation. A certain amount of enzyme is needed to assume an efficient detoxication. The young rats would have a reduced metabolic activity which might explain the difference of vulnerability between the 14week old and the 21week old groups.
| Acknowledgment | | |
The authors wish to thank Christian Barthelemy for his technical assistance and Onofrio Bevilacqua for taking care of the animals. This study was supported by European grant QLK42000-00293 and by Institut National de Recherche et de Securite[27].
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Correspondence Address:
P Campo
Institut National de Recherche et de Sécurité‚ Avenue de Bourgogne, BP 27, 54501, Vandoeuvre
France
 Source of Support: None, Conflict of Interest: None  | Check |
PMID: 15703144 
[Figure - 1], [Figure - 2], [Figure - 3], [Figure - 4], [Figure - 5]
[Table - 1] |
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