Toxicokinetic parameters of toluene in the rat and guinea pig: a comparative study.

Toluene is the most widely used industrial solvent. It has been shown to cause cochlear disruptions in rats but markedly less ototoxic effects in guinea pigs. Susceptibility to the ototoxic properties of toluene is, therefore, species specific. In recent publications, an important difference in the solvent concentration in blood has been identified when rats and guinea pigs were exposed in strictly identical experimental conditions. Solvent concentrations in blood were greater in rats than in guinea pigs. The present studies were designed to compare blood affinity and toxicokinetic parameters of toluene in an attempt to understand the susceptibility differences in both species. The in vitro experiment, in which the headspace concentration of toluene was measured within a sealed vial containing blood, highlighted the greater toluene partition coefficient in rat than in guinea pig blood. The in vivo experiment showed that 10min after a single intravenous administration of 28µL of toluene, the solvent concentration is approximately two-fold lower in guinea pig than in rat blood. Based on the toxicokinetic parameters of toluene and on the relative partition coefficient of toluene in blood, it seems plausible that guinea pigs are not susceptible to organic solvents because the solvent concentration in blood does not reach the concentration required to induce permanent damage. Attempts to explain differences of vulnerability between the rat and guinea pig are addressed in the present paper.

Combined effects of noise and gentamicin on hearing in the guinea pig.

The last ten years, the use of gentamicin has increased due to antibiotic resistance among bacterial pathogens. One of the side effects of gentamicin is its toxicity on hearing. Several authors had even pointed out synergistic effects of gentamicin and noise on hearing. It was therefore reasonable to think that the damaging effects of noise could be emphasized by a gentamicin treatment of the subjects. In order to test the applicability of the Leq8h for estimating the hazard of noise on animals treated with a non-ototoxic dose of gentamicin (40 mg/kg for 8 days), two experiments were carried out with guinea pigs. The animals were exposed to octave band noises centred at 8 kHz and treated with gentamicin either simultaneously or sequentially with regard to the noise exposure. Two noise exposures having different acoustic energy, respectively Leq8h = 85 dB and 98.8 dB SPL, were tested. The auditory function of the guinea pigs was tested by recording auditory-evoked potentials. The electrophysiological findings were completed by histological data. The gentamicin treatment tested in the current studies did not cause any auditory permanent threshold shift neither cochlear disruptions, although the treatment could be considered as approximately ten times the therapeutic dose used in human. The auditory deficit induced by the mixed exposures to noise and gentamicin did not worsen the noise effect alone in our experimental conditions. As a result, the European value recommended for noise exposure (Leq8h=85 dB) seems to be robust enough to protect gentamicin-treated workers.

Combined effects of noise and styrene on hearing: comparison between active and sedentary rats.

In this study, two investigations were carried out with adult Long-Evans rats exposed to increasing concentrations of styrene. In the first experiment, the hearing of rats, which were forced to walk in a special wheel during the exposure, was compared to that of rats which were sleepy in their cage. The active rats were exposed to styrene concentrations ranging from 300 to 600 ppm, whereas the sedentary rats were exposed from 500 to 1000 ppm for 4 weeks, 5 days per week, 6 hours per day. In the second experiment, designed to evaluate the hearing risks at threshold limit values, active rats were exposed either to a noise having a Leq8h of 85 dB (equivalent level of a continuous noise for a typical 8-h workday), or to 400-ppm styrene or to a simultaneous exposure to noise and styrene. In both experiments, auditory function was tested by auditory-evoked potentials from the inferior colliculus and completed by morphological analyses of the organ of Corti. The results of the first experiment showed that the same amount of styrene-induced hearing loss can be obtained by using concentrations approximately 200 ppm lower in active rats than in sedentary rats. The second investigation showed that, in spite of the low-intensity noise and the low-concentration of styrene, there is a clear risk of potentiation of styrene-induced hearing loss by noise. These findings and exposure conditions were discussed and extrapolated with regard to the risk assessment for human beings. The authors propose to decrease the French threshold limit value of styrene for ensuring a high level of protection for human hearing.

Combined effects of noise and styrene exposure on hearing function in the rat.

Combined exposure to both noise and aromatic solvents such as styrene is common in many industries. In order to study the combined effects of simultaneous exposure to both noise and styrene on hearing, male adult Long-Evans rats were exposed either to 750 ppm styrene alone, to a 97 dB SPL octave band of noise centered at 8 kHz, or to a combination of noise and styrene. The exposure duration was 6 h/day, 5 days/week, for 4 consecutive weeks. Auditory function was tested over a frequency range from 2 to 32 kHz by recording near field potentials from the inferior colliculus, whereas histopathological analyses of the cochleae were performed with conventional morphometric approaches. Whereas both noise and styrene each caused permanent threshold shifts, the mechanisms of cochlear damage were different. Noise-induced hearing loss was mainly related to injuries of the stereocilia, whereas styrene-induced hearing loss was related to outer hair cell losses. Following the combined exposure, the threshold elevations as well as the cell losses exceeded the summed loss caused by noise and by styrene alone in the range of 8-16 kHz. Therefore, these results suggest that the two ototoxicants can cause a permanent synergistic loss of auditory sensitivity.

Styrene-induced hearing loss: a membrane insult.

Styrene is an aromatic solvent widely used as a precursor for polystyrene plastics in many factories which produce glass-reinforced plastic. This solvent has been shown to disrupt the auditory system in both humans and animals. In order to study the sequence of events which could explain the cochlear impairments, a time course experiment was carried out with 6-month-old rats. Male Long Evans rats were exposed to 1000 ppm styrene for 6 h/day, 5 days/week, for either 1, 2, 3, or 4 consecutive weeks. Auditory function was tested by recording the near field evoked potentials from the inferior colliculus, and histological analyses of the cochleae were performed with light and transmission electron microscopy. The electrophysiological results support a toxic mid-frequency process which keeps worsening even after the end of the exposure. The histological findings demonstrate that supporting cells are the first targets of the solvent. Then, the outer hair cells of the third row (OHC3) are disrupted, followed successively by OHC2 and OHC1 from the basal (20 kHz) to the upper turn (4 kHz) of the cochlea. Basically, the disorganization of the membranous structures could be the starting point for the cochlear injury induced by styrene. This paper presents a hypothesis that the accumulation of K+ in the spaces of Nuel underlies the toxic effects of styrene.

Combined effects of exposure to styrene and ethanol on the auditory function in the rat.

In order to study the auditory effects of a metabolic interaction between ethanol and styrene, a first group of rats was gavaged once a day with ethanol (4 g/kg), a second group was exposed to 750 ppm styrene by inhalation, and a third group was exposed to both ethanol and styrene (5 days/week, 4 weeks). Auditory function was tested by recording brainstem (inferior colliculus) auditory evoked potentials, and cochlear hair cell loss was estimated by light microscopy. Cytochrome P450 2E1 and the main urinary styrene metabolites, namely mandelic, phenylglyoxylic and hippuric acids, were measured by high-performance liquid chromatography to check the effects of ethanol on styrene metabolism. In our experimental conditions, ethanol alone did not have any effect on auditory sensitivity, whereas styrene alone caused permanent threshold shifts and outer hair cell damage. Hearing and outer hair cell losses were larger after the exposure to both ethanol and styrene than those induced by styrene alone, indicating a clear potentiation of styrene ototoxicity by ethanol. As expected, metabolic data showed that ethanol alters styrene metabolism and can therefore be considered a modifying factor of styrene toxicokinetics.

Combined effects of a simultaneous exposure to noise and toluene on hearing function.

To study the combined effects of noise and toluene on auditory function, three experimental groups of Long-Evans adult rats were used. The first group was exposed to toluene (2000 ppm, 6 h/day, 5 days/week, 4 weeks), the second group to an octave band of noise centered at 8 kHz (92 dB SPL), and the last group to a simultaneous exposure to toluene and noise. Auditory function was tested by recording brainstem (inferior colliculus) auditory-evoked potentials. The auditory deficit induced by the combined exposure exceeded the summated losses caused by toluene alone and by noise alone within the range (2-32 kHz) of test frequencies. The nature of the cochlear damage induced by noise alone (injured stereocilia) or by toluene alone (outer hair cells loss) is different.

Toluene ototoxicity in rats: assessment of the frequency of hearing deficit by electrocochleography.

To identify the frequency range most sensitive to toluene-induced auditory damage, the auditory function of adult Long-Evans rats exposed to 1750 ppm of toluene (6 h/day, 5 days/week, 4 weeks), was tested by recording auditory-evoked potentials directly from the round window of the cochlea. The present electrocochleographic findings do not support a specific mid- to high-frequency loss of auditory sensitivity. On the contrary, the electrophysiologic data, obtained for audiometric frequencies ranging from 2 to 32 kHz, showed a hearing deficit not only in the mid-frequency region (12-16 kHz), but also in the mid-low-frequency region (3-4 kHz). Actually, the effect of toluene was independent of the frequency in our experimental conditions. Histological analysis was consistent with electrophysiologic data because a broad loss of outer hair cells occurred in both mid- and mid-apical coil of the organ of Corti.

Comparison of toluene-induced and styrene-induced hearing losses.

Toluene and styrene are industrial solvents that can severely damage the auditory function in adult rats. In the present study, toluene (1000 to 2000 ppm) and styrene doses (500 to 1500 ppm) were investigated according to the same schedule: 6 hours per day, 5 days per week, for 4 consecutive weeks. The auditory function of the animals was tested by recording evoked potentials from the inferior colliculus over a frequency range from 2 to 32 kHz, whereas pathological data were evaluated by conventional histologic techniques. The permanent threshold shifts (PTS) were obtained with a styrene dose 2.4 times lower than that of the toluene. The slope of the regression line (PTS/doses) was 2.1 steeper with styrene than that obtained with toluene in the same experimental conditions. The sequence of histopathological events along the organ of Corti, especially the orderliness and the location of the traumas, was similar for paired concentrations of styrene and toluene, which were respectively 650 ppm, 1500 ppm for the first match, and 850 ppm, 1750 ppm for the second one. Both electrophysiological and histological findings point out the higher ototoxic potency of the styrene compared to that of the toluene. Assumptions concerning the ototoxic mechanism are addressed in the present paper.

Toluene-induced hearing loss: a mid-frequency location of the cochlear lesions.

Inhaled toluene (from 1000 to 2000 ppm, 6 h/day, 5 days/week, 4 weeks) is anototoxic solvent that severely damaged the cochlea in adult Long-Evans rats. Auditory function was tested by recording near field potentials from the inferior colliculus. Surprisingly, the electrophysiologic results did not reflect all the cochlear damage observed by histology. Loss of outer hair cells of the organ of Corti occurred in all toluene-treated rats in middle and mid-apical turns, whereas the basal turn of the cochlea was fairly well preserved. The third row of outer hair cells was more injured than the second row, which itself was more injured than the first row. The locations of the cochlear lesions are reported in the present study with regard to the toluene dose.

Cochlear pathology induced by styrene.

Hair cells, spiral fibers and spiral ganglion cells (SGCs) coming from cochleae of styrene-treated Long-Evans rats were counted in order to assess the extent and location of the cochlear injury after the solvent inhalation. If the hair cells, and more specifically the outer hair cells (OHCs), were undoubtedly the first targets of inhaled styrene, the histological results of the present study would seem to indicate that neurons of the spiral ganglion were also injured with increasing styrene doses. The degenerative process of SGCs and spiral fibers within the osseous lamina was predominant in the middle and mid-basal turn. The electrophysiological data, obtained by recording near-field potentials from the inferior colliculus, reflected the damages of the SGCs and fibers but were not consistent with the histopathological data of the organ of Corti. Because of the weak correlation between the styrene-induced injury at the level of the organ of Corti and that induced at the level of the spiral ganglion, it is likely that two different intoxication routes exist within the cochlea. Such an assumption is discussed in the present paper.

Combined effects of simultaneous exposure to toluene and ethanol on auditory function in rats.

Three experimental groups and one control group of Long-Evans rats were used to study the combined effects of toluene and ethanol on auditory function. The first experimental group was exposed to toluene vapors (1750 ppm, 6 h/day, 5 days/week, 4 weeks), the second one was daily gavaged with a saline solution of ethanol (4 g/kg, 4 weeks), and the last group was simultaneously exposed to both toluene and ethanol. Auditory function was tested by recording brain stem (inferior colliculus) auditory-evoked potentials for audiometric frequencies ranging from 2 to 32 kHz. Urinary hippuric acid was dosed to check the toluene metabolism during the experiments. Ethanol clearly modified the toluene metabolism in the present experimental conditions. As a result, the hearing loss induced by a simultaneous exposure to both ethanol and toluene was larger than that induced by exposure to toluene alone.

Noise and solvent, alcohol and solvent: two dangerous interactions on auditory function.

While noise exposure is the most significant contributor to occupational hearing loss, recent evidence points to solvents and their interactions as additional contributors to occupational deafness. Furthermore, due to the metabolic competitive inhibition between aromatic solvents and ethanol, the solvent toxicity can be even enhanced in certain circumstances. So, two dangerous interactions: noise and solvents, solvents and ethanol deserve to be taken into consideration in an exhaustive preventive policy. Based on the investigations reviewed in the present study, it appears that the combined effects of an exposure to noise and solvent exceed the summation of the damage produced by each agent alone. Such a statement can be also made for a combined exposure to solvent and ethanol. It is therefore important to bear in mind that noise effects can be exacerbated by non-acoustic agents. Thus, if our noise regulations have to be more effective, it is necessary to take into consideration the ototoxic effects of noise in a multifactorial environment.

Is the aged rat ear more susceptible to noise or styrene damage than the young ear?

Noise- and styrene-induced hearing and hair cell loss were studied in young (3 months) and aged (24-26 months) Long-Evans rats. The animals were exposed 6 h/d, 5 d/w for 4 weeks to (a) broadband noise centered at 8 kHz (92 or 97dB SPL), or b) styrene (700 ppm). Auditory sensitivity was tested by recording evoked potentials from the inferior colliculus. Histological analyses of the organ of Corti, stria vascularis, and the spiral ganglions were also performed. Aged controls showed outer hair cell (OHC) loss at the basal and apical regions of the organ of Corti, and an increase in pigmentation concomitant to a decrease in vascularization of the stria vascularis, along with elevated thresholds relative to young controls. The 92-dB noise caused similar threshold shifts in both age groups, whereas the 97-dB noise caused more threshold shifts in the aged group compared to the young group. Recovery of the hearing thresholds depended both on the intensity of the noise and on the age of the animals. Aged rats had minimal hair cell loss as a result of styrene exposure, whereas young animals showed significant OHC loss, particularly in third row. Despite significant loss of OHCs, the young subjects showed styrene-induced threshold shifts only at high frequencies. In summary, the data show that : (a) there is an influence of age on both noise-induced and styrene-induced threshold shift and hair cell loss in rats and (b) the cochlea appear to have a redundancy in the number of OHCs, thus threshold shift does not necessarily occur with significant OHC loss.

Critical period for styrene ototoxicity in the rat.

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.

Applicability of the L(eq) as a damage-risk criterion: an animal experiment.

Within the European communities, the L(eq8h) is used as a damage-risk criterion (DRC) for hearing protection. The use of such a DRC supposes that sound exposures with equal energy imply equal risk for noise-induced hearing loss (NIHL). The aim of the present study, carried out with guinea pigs, was to test the applicability of the L(eq8h) for estimating the hazard of different noises having the same spectrum and acoustic energy. Therefore, only the temporal structure of noises was a variable (continuous, intermittent, or impulsive) and each noise had a L(eq8h) of 92 dB. The results indicate the L(eq8h) is not an accurate DRC. It overestimates the NIHL for intermittent and short-continuous noises, whereas it seems to be useful for moderate-intensity noises (95 dB) and for the impulse noise (101 dB). Furthermore, the data point out the notion of ¿critical intensity¿ which seems to play a determining role in the transition between metabolic and mechanical mechanisms involved in the acoustic trauma. Since the critical intensity and the existence of two mechanisms are parameters that the L(eq8h) cannot take into consideration, recommendations on the safety program of hearing loss prevention are discussed.