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Effects of acute styrene and simultaneous noise exposure on auditory function in the guinea pig
Neurotoxicologyand Teratology,Vol. 15, pp. 151-155,1993
0892-0362/93$6.00 + .00 Copyright©1993PergamonPress Ltd.
Printedin the U.S.A. All rightsreserved.
Effects of Acute Styrene and Simultaneous Noise Exposure on Auditory Function in the Guinea Pig L A U R E N C E D. F E C H T E R
Departments o f Environmental Health Sciences and Otolaryngology, Head and Neck Surgery, The Johns Hopkins University, 615 North Wolfe Street, Baltimore M D 21205 Received 17 A u g u s t 1992; Accepted 17 N o v e m b e r 1992 FECHTER, L. D. Effects of acute styrene and simultaneous noise exposure on auditory function in the guinea pig. NEUROTOXICOL TERATOL 15(3) 151-155, 1993.-Although styrene has been demonstrated to disrupt vestibular function acutely, parallel studies have not been conducted in the auditory system. This article presents data on the effects of acute styrene administration by injection and inhalation on cochlear function. No deleterious effect of the maximally tolerated styrene dose on hearing was identified when cochlear function was assessed using a within-subjects design. When guinea pigs were administered styrene by inhalation during a single 7-h period, normal auditory function was observed both 1 and 7 days later as compared to chamber controls which did not receive styrene. In some instances, the interactive effects of noise and simultaneous styrene inhalation were studied to determine whether chemical exposure might enhance the disruptive effects of noise on hearing. While a persistent noise-induced hearing loss was observed 1 day following exposure, subjects administered styrene simultaneously did not show a greater hearing loss than those receiving noise alone. Finally, when a 7-day recovery period for noise-induced hearing loss was interposed before audiometric testing, the combined exposure to styrene and noise was not more potent than noise alone in elevating auditory thresholds. Although auditory dysfunction has been reported following subchronic styrene administration, the current results do not support an ototoxic effect of styrene at the level of the cochlea with short-term exposure. Styrene
Ototoxicity
Cochlea
Noise
Compound action potential
Cochlear microphonic
came more severe as test tone frequency increased up to 20 kHz (the highest frequency tested). This is consistent with the traditional preferential ototoxic injury to the basal turn of the cochlea observed with ototoxic drugs (7) and most ototoxic chemicals (5,6,11). Muijser et al. (2) studied high frequency hearing among workers exposed to styrene and those in the same plant whose jobs did not directly involve work with styrene. The latter might be considered a low dose group. Nonexposed controls selected from a different industry were also studied. They reported a small (approximately 8 dB) elevation in thresholds at 8 kHz among styrene workers as compared to the indirect exposure group. Threshold levels at five higher frequencies did not distinguish the two exposure groups although high variance levels, expected at such high tone frequencies, might obscure exposure effects. The present investigation was designed to study the effects of acute injection and a single 7-h styrene inhalation exposure on auditory function measured at the cochlea and on the potential for styrene to enhance noise-induced hearing loss. The protocol was specifically designed to allow a complete evaluation of auditory function across the guinea pig's auditory range, with emphasis on high frequency function. Based on the classic finding that ototoxicants produce their most pro-
STYRENE has been shown to disrupt both auditory and vestibular function in working populations and in laboratory animal models. The vestibular toxicity can develop after acute exposures and takes place in central vestibular pathways. Larsby et al. (1) and Tham et al. (8) demonstrated that IV infusion of styrene produced nystagmus during rotational challenge in rabbits and rats when arterial styrene levels reached 40-75 ~tg/g blood. 0dkvist et al. (4) subsequently reported enhanced saccade speed in human volunteers exposed to styrene by inhalation (87-139 ppm) producing blood styrene concentrations of approximately 3 ~tg/g. Accuracy of the saccade was not altered and nystagmus did not occur spontaneously or following rotation. Among workers chronically exposed to styrene, Tham et al. (9) reported evidence of disruption in smooth ocular pursuit of a moving fight stimulus and impaired visual suppression. It is not currently known whether acute exposures to styrene can alter auditory function, although auditory impairments have been reported following chronic exposure. Pryor et al. (5) reported a loss in auditory sensitivity for pure tones in weanling rats exposed to styrene at 800, 1000, or 1200 ppm for 14 h per day for 3 weeks using both behavioral and auditory brainstem evoked response measures. Impairments be151
152
FECHTER
nounced impairments at tone frequencies at the extreme high end of the subject's audible range (5,6,7,11), it would be predicted that the highest test tone frequencies would be more affected by styrene. Electrophysiological measurements were made from the cochlea to evaluate outer hair cell function and inner hair cell-spiral ganglion function in order to determine whether styrene has a cochlear site of action. METHOD
Subjects Forty-six male pigmented adult guinea pigs obtained from Murphy Labs served as experimental subjects. They were group housed in an AAALAC-approved animal facility with ambient sound levels of < 45 dB as measured on the A weighting scale (dBA). The A weighting scale maximizes sound energy in the human auditory range and thereby eliminates low frequency rumble below the guinea pigs' auditory range. Subjects had free access to food and water and were maintained on a 12L : 12D cycle. Audiometric testing and exposure to styrene occurred during the daylight portion of this cycle.
Styrene Exposures Injection. Styrene was administered by IP injection in some subjects to allow within subject comparisons of auditory function prior to and then immediately following styrene administration. The dosage selected, two injections of 0.75 ml each spaced 30 min apart, was selected based on pilot experiments which showed that a dose of 2 ml styrene was lethal when administered to two subjects. Inhalation. Styrene vapor was generated by evaporating styrene monomer at 40°C within a heated glass vessel. Styrene was injected into the vessel at a controlled rate (approximately 70/zl/min to achieve chamber concentratins of 500 ppm and 150 #l/min to attain 1200 ppm) designed to maintain constant chamber styrene concentrations. A Miran infrared detector was used to monitor styrene levels in the exposure chamber in real time over the 7 h of exposure. Manual adjustments in styrene injection rates were made when chamber concentrations deviated by more than 10 ppm from the desired exposure level. Ambient sound levels in the chamber were < 50 dBA.
Simultaneous Noise and Styrene Exposures In some experiments, subjects received simultaneous exposure to styrene by inhalation and broad-band noise for 7 h. This noise was generated by a white noise generator and presented to subjects at an intensity of 95 dB using the Aweighting scale. This sound level was chosen because it produced a mild, temporary auditory threshold shift observable one day following exposure but no permanent disruption of auditory function. A 90 dBA time weighted average (TWA) is considered the permissible exposure level for workers although an 85 dB T W A is recommended (3). Subjects in the control group which received neither styrene nor noise were also placed in the inhalation chamber for 7 h during which they were exposed to a stream of air equivalent in volume to that given styrene-exposed subjects but not to additional noise. Experimental design. Three experiments were performed. Experiment 1 was designed to evaluate the effects of acute, IP styrene administration on auditory function using a withinsubjects design. Auditory function was assessed in all subjects (n = 3) prior to injection of styrene monomer (0.75 ml/sub-
ject) and 30 min following exposure. Because this dose did not alter auditory potentials, all subjects received a second administration of 0.75 ml/subject styrene immediately following the 30 min evaluation of auditory function and auditory potentials were assessed 30 rain following this second dose. Untreated control subjects (n = 3) were also tested according to the same time schedule. For Experiment 2, styrene exposures were effected by inhalation and auditory thresholds were assessed 18-22 h later. Four groups of five subjects were exposed as follows: styrene only group = 500 ppm styrene for 7 h at chamber noise levels; styrene + noise group = the same level of styrene plus 95 dBA white noise; noise only group = 95 dBA white noise alone; chamber control group = exposure to the chamber with no noise or styrene. Experiment 3 entailed inhalation exposure to styrene at either 500 (n = 5) or 1200 (n = 5) ppm for 7 h and simultaneous noise exposure at 95 dBA, but audiometric testing was delayed until 1 week following exposure to provide time for recovery from the noiseinduced threshold shift observed in Experiment 2. Control groups were treated with 95 dBA noise (n = 5) or were placed in a chamber with no added noise or styrene (n = 5).
Electrophysiological Procedures Compound action potential and cochlear microphonic measurement from the round window. The propogated dectrophysiological response of the cochlea measured at the round window to tone pips consists of a complex waveform. The initial negative potential (N1) corresponds to the compound action potential generated by the Type 1 spiral ganglion cells which are the initial post-synaptic elements of the cochlea. This potential is dependent not only on the integrity of the presynaptic (inner hair cells) and postsynaptic (Type 1 spiral ganglion) elements of the cochlea but also on outer hair cells which influence inner hair cell sensitivity. This potential occurs approximately 2 msec following onset o f the tone stimulus. The thresholds for evoking an N1 potential at each of 11 test tone frequencies were measured to evaluate cochlear function using a round window electrode. The stimulation of the cochlea with constant tone stimuli also elicits an ac waveform which is phase locked to the tone frequency and reflects the mechanical movement of outer hair cells in response to sound. This waveform, the cochlear microphonic, can be measured by filtering the signal from the round window and amplifying only that frequency which matches that used as the sound stimulus delivered to the ear canal. The cochlear microphonic was employed as a measure of outer hair cell function. Specifically, the sound intensity for the same 11 tone frequencies used to measure N1 above were adjusted upward until a criterion voltage of 1 #V RMS was obtained. These values were used to plot a cochlear microphonic isopotential curve relating sound level in dB sound pressure level (SPL) at each test tone frequency to the criterion response. The outer hair cell is the primary generator of the cochlear microphonic and the sound intensity necessary to produce a cochlear microphonic signal of 1 #V is increased when outer hair cells are damaged. The N1 component of the compound action potential response represents the action potential generated at the Type 1 spiral ganglion cell in response to potentials generated at the inner hair cell. A selective elevation in the sound intensity necessary to elicit the N1 response when the amplitude of the cochlear microphonic is normal is interpreted as either a pre- or post-synaptic impairment. Subjects were anaesthetized with urethane (1.5 g/kg IP);
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were determined by the presentation of tone pips of 10 msec duration with 1 msec onset-offset ramps presented at a rate of 9/sec. Eleven pure tones at approximately 1/2 octave intervals between 2 and 40 kHz were presented to the subjects and thresholds are expressed in dB SPL referred to 20/zPa. The cochlear microphonic was elicited by constant pure tone stimuli of the same frequencies as described above for the compound action potential. The signal was high pass filtered above 300 Hz, preamplified x 1000, as above and filtered using a Stanford Research Systems (Palo Alto, CA) Model SR530 lock-in amplifier to select ac signals in the electrophysiological signal corresponding to the frequency o f the stimulating tone. Tone intensity was increased until a 1 #V RMS signal was achieved. Subsequent to the last determination of the compound action potential and the cochlear microphonic, the sound intensity within approximately l mm of the tympanic membrane was measured directly for each subject using a I / 2 " Bruel and Kjaer 4134 microphone (Naerum, Denmark) coupled to a
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FIG. 1. Compound action potential thresholds in animals injected with styrene (A) and in uninjected controls tested at the same time intervals (B).
supplemental injections o f 0• 15 g / k g IP were used as necessary to maintain an adequate level of anaesthesia. Lidocaine (2°70) was injected at all incision loci to block pain. The surgery was conducted in an IAC 122A audiometric booth (Industrial Acoustics Company, New York, NY). A d c heating pad was used to maintain body temperature at 39 + 1 °C as measured by a rectal probe• The animals were tracheotomized, the right pinna was removed, and the right auditory bulla was then exposed using a ventral approach. A low voltage lamp was directed at the cochlea to prevent cooling. The active electrode was a teflon insulated 40 ga. silver wire ( A - M Systems, Inc., Everett, WA). A silver/silver chloride reference electrode was placed in a neck muscle. A plexiglas speculum was inserted into the external auditory canal to accommodate an ACO Pacific (Belmont, CA) Model 7013 1/2" pressure microphone used for stimulus delivery. The compound action potentials were bandpass filtered between 300 and 1000 Hz and preamplified × 10 by a Grass (Quincy, MA) dc preamplifier (Model P16) and then amplified x 100 by a Grass ac preamplifier (Model P15). Compound action potential thresholds (detection o f a NI wave of approximately 1 #V amplitude observed on a digital oscilloscope)
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probe tube inserted into the speculum. The subject was then sacrificed by a lethal injection of sodium pentobarbital. Separate split plot factorial analyses of variance (ANOVA) (Statpak, Northwest Analytical Inc., Portland, OR) were used to analyze the compound action potential thresholds and the 1 ~V cochlear microphonic isopotential curves for each experiment. Group condition was assessed between subjects and auditory function across tone frequency was analyzed within subjects. RESULTS Acute styrene injection failed to increase the auditory threshold as detected by tone intensity necessary to elicit the N1 component of the compound action potential (see Fig. 1A) and the cochlear microphonic response (see Fig. 2A). Although the number of subjects studied was small, the use of a within-subjects design, the small, and homogeneous variability seen in each condition, and the similarity between the data obtained in the uninjected controls (see Figs. IB and 2B) and
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experimental subjects (see Figs. 1A and 2A) provide strong evidence that styrene administered by IP injection does not alter auditory thresholds acutely. Neither the main effect of treatment nor the treatment x frequency interaction was statistically significant (F < 1.0). Figures 3 and 4 present the effects of styrene inhalation in the presence and absence of additional noise on the compound action potential threshold and cochlear microphonic isopotential curve one day following exposure. Figure 3 clearly shows an elevation of approximately 20 dB in the compound action potential thresholds at mid-frequencies (8, 12, and 16 kHz) in both the noise only and styrene plus noise groups. Styrene by itself did not elevate compound action potential thresholds above control levels. However, ANOVA identified differences between-treatment groups which approached statistical significance, F(3, 16) = 3.0726, p = 0.0577. Auditory thresholds varied significantly over tone frequency in a fashion consistent with published (10) frequency-sensitivity functions, F(10, 160) = 36.5017, p < 0.0001. Of more importance here, a significant group by frequency interaction was found, F(30, 160) =
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LACK OF STYRENE OTOTOXICITY
155
2.3543, p < 0.001, reflecting the elevation in auditory thresholds at mid-frequencies shown by the noise-only and noiseplus styrene groups. A very limited elevation in the 1 ;tV cochlear microphonic isopotential curve is suggested at the lowest test frequencies among all treated subjects. However, an ANOVA did not confirm that group differences met the accepted criterion for statistical significance, F(3, 1 6 ) = 2.683, p > 0.08. While statistically significant differences were found among tone frequencies in the isopotential curve, F(10, 160) = 183.3799, p < 0.0001, a significant treatment group by frequency interaction was not found, F(30, 160) = 1.4265,p > 0.08. The effects of noise and styrene on cochlear function were examined further in Experiment 3 by interposing a I week recovery period between experimental exposures and audiometric testing and by including an additional dose of styrene. One week following exposure there appeared to be a small elevation in auditory thresholds among subjects in the 500 ppm styrene plus noise group in relation to untreated controls (see Fig. 5). Similar findings were not evident in the group receiving the higher dose of 1200 ppm styrene plus noise; in fact, thresholds for this latter group were equivalent to the subjects which had been exposed to noise alone. Reliable differences between treatment groups were not found, F(3, 16) = 2.1840, p 0.1, nor was a significant interaction seen between group conditions and tone frequency, F(30, 160) = 1.0538,p > 0.1. Figure 6 depicts the effect of styrene plus noise exposure on the cochlear microphonic recorded 1 week following exposure. There is a suggestion in the data of a limited elevation in the sound level necessary to elicit the criterion cochlear microphonic value among all treatment groups relative to untreated controls. An ANOVA showed that the effect of treatment did meet statistical significance, F(3, 16) = 3.279, p < 0.05. Subjects receiving combined exposure to styrene plus noise did not show an enhanced loss in cochlear microphonic sensitivity relative to the subjects which received noise alone.
at levels well above the threshold limit value (TLV). Neither IP administration approximating the maximally tolerated dose nor inhalation exposure at doses as high as 500 ppm disrupted auditory thresholds or outer hair cell function indexed by the cochlear microphonic response. When noise was combined with styrene exposure in an attempt to evaluate ototoxicity under conditions of cochlear stress, the results were negative; styrene did not enhance the loss of auditory function due to noise alone. However, all subjects receiving noise, whether or not styrene was present, did show a loss in threshold sensitivity at appropriate sound frequencies based on the noise power spectrum delivered. The results obtained one week following combined exposure to 500 ppm styrene plus noise are suggestive of a limited loss in auditory sensitivity, but the effect was not sufficiently robust to reach statistical reliability nor was it dose-related. The extent of loss observed, 15 dB at the maximally affected frequency, is a modest one. The source of the loss in cochlear microphonic sensitivity observed 1 week following combined noise and styrene exposure in all treatment groups is not clear. It seems unlikely that it is a result of noise-induced injury to the cochlea because auditory thresholds reflected by the N1 response were unaffected. Damage to outer hair cells would also be expected to impair cochlear tuning such that loss in the threshold to elicit an action potential would be observed. The fact that the cochlear microphonic was most affected at low frequencies is consistent with the presence of excess fluid around the round window due to the surgical preparation. Whereas the data do not demonstrate an acute cochlear effect of styrene, it is still possible that acute styrene exposure may disrupt central auditory processes. The techniques used here are not appropriate for detecting such effects. In addition, there are data from both humans (2) and laboratory animals (5) which demonstrate styrene ototoxicity following relatively long-term exposure. ACKNOWLEDGEMENTS
DISCUSSION The results of these experiments provide little evidence that acute styrene administration can alter cochlear function even
This work was supported in part by PHS Grant Nos. ES02852 and ES03819. I thank Dr. Lei Yao for his technical support and Dr. David Bassctt for setting up the inhalation exposure system.
REFERENCES 1. Larsby, B.; Tham, R.; (3dkvist, L. M.; Hydtn, D.; Bunnfors, I.; Aschan, (3. Exposure of rabbits to styrene: Electronystagmographic findings correlated to the styrene level in blood and cerebrospinal fluid. Scand. J. Work Environ. Hlth. 4:60-65; 1978. 2. Muijser, H.; Hoogendijk, E. M. (3.; Hooisma, J. The effects of occupational exposure to styrene on high-frequency hearing thresholds. Toxicology 49:331-340; 1988. 3. National Institute for Occupational Safety and Health: Publications on noise and hearing. Cincinnati, OH: U.S. Department of Health and Human Services; 1991. 4. (3dkvist, L. M.; Larsby, B.; Tham, R.; .~hlfeldt, H.; Anderson, B.; Eriksson, B.; Liedgren, S. R. C. Vestibulo-oculomotor disturbances in humans exposed to styrene. Acta Otolaryngol. 94: 487-493; 1982. 5. Pryor, (3. T.; Rebert, C. S.; Howd, R. A. Hearing loss in rats caused by inhalation of mixed xylenes and styrene. J. Appl. Toxicoi. 7:55-61; 1987.
6. Pryor, G. T.; Rebert, C. S.; Dickinson, J.; Feeney, E. M. Factors affecting toulene-induced ototoxicity in rats. Neurobehav. Toxicol. Teratol. 6:223; 1984. 7. Rybak, L. P. Drug ototoxicity. Ann. Rev. Pharmacol. Toxicol. 26:79; 1986. 8. Tham, R.; Larsby, B.; Eriksson, B.; Bunnfors, I.; ~dkvist, L.; Liedgren, C. Electronystagmographic findings in rats exposed to styrene or toluene. Acta Otolaryngol. 93:107-112; 1982. 9. Tham, R.; Larsby, B.; M611er.; Niklasson, M.; ~3dkvist, L. M. Vestibulotoxicity of organic solvents. Proceedings of the 4rth International Conference on the Combined Effects of Environmental Factors 101-105; 1991. 10. Young, J. S.; Fechter, L. D. Reflex inhibition procedures for animal audiometry: A technique for assessing ototoxicity. J. Acous. Soc. Amer. 73:1686; 1983. 11. Young, J. S.; Upchurch, M. B.; Kaufman, M. J.; Fechter, L. D. Carbon monoxide exposure potentiates high-frequency auditory threshold shifts induced by noise. Hear. Res. 26:37; 1987.
0892-0362/93$6.00 + .00 Copyright©1993PergamonPress Ltd.
Printedin the U.S.A. All rightsreserved.
Effects of Acute Styrene and Simultaneous Noise Exposure on Auditory Function in the Guinea Pig L A U R E N C E D. F E C H T E R
Departments o f Environmental Health Sciences and Otolaryngology, Head and Neck Surgery, The Johns Hopkins University, 615 North Wolfe Street, Baltimore M D 21205 Received 17 A u g u s t 1992; Accepted 17 N o v e m b e r 1992 FECHTER, L. D. Effects of acute styrene and simultaneous noise exposure on auditory function in the guinea pig. NEUROTOXICOL TERATOL 15(3) 151-155, 1993.-Although styrene has been demonstrated to disrupt vestibular function acutely, parallel studies have not been conducted in the auditory system. This article presents data on the effects of acute styrene administration by injection and inhalation on cochlear function. No deleterious effect of the maximally tolerated styrene dose on hearing was identified when cochlear function was assessed using a within-subjects design. When guinea pigs were administered styrene by inhalation during a single 7-h period, normal auditory function was observed both 1 and 7 days later as compared to chamber controls which did not receive styrene. In some instances, the interactive effects of noise and simultaneous styrene inhalation were studied to determine whether chemical exposure might enhance the disruptive effects of noise on hearing. While a persistent noise-induced hearing loss was observed 1 day following exposure, subjects administered styrene simultaneously did not show a greater hearing loss than those receiving noise alone. Finally, when a 7-day recovery period for noise-induced hearing loss was interposed before audiometric testing, the combined exposure to styrene and noise was not more potent than noise alone in elevating auditory thresholds. Although auditory dysfunction has been reported following subchronic styrene administration, the current results do not support an ototoxic effect of styrene at the level of the cochlea with short-term exposure. Styrene
Ototoxicity
Cochlea
Noise
Compound action potential
Cochlear microphonic
came more severe as test tone frequency increased up to 20 kHz (the highest frequency tested). This is consistent with the traditional preferential ototoxic injury to the basal turn of the cochlea observed with ototoxic drugs (7) and most ototoxic chemicals (5,6,11). Muijser et al. (2) studied high frequency hearing among workers exposed to styrene and those in the same plant whose jobs did not directly involve work with styrene. The latter might be considered a low dose group. Nonexposed controls selected from a different industry were also studied. They reported a small (approximately 8 dB) elevation in thresholds at 8 kHz among styrene workers as compared to the indirect exposure group. Threshold levels at five higher frequencies did not distinguish the two exposure groups although high variance levels, expected at such high tone frequencies, might obscure exposure effects. The present investigation was designed to study the effects of acute injection and a single 7-h styrene inhalation exposure on auditory function measured at the cochlea and on the potential for styrene to enhance noise-induced hearing loss. The protocol was specifically designed to allow a complete evaluation of auditory function across the guinea pig's auditory range, with emphasis on high frequency function. Based on the classic finding that ototoxicants produce their most pro-
STYRENE has been shown to disrupt both auditory and vestibular function in working populations and in laboratory animal models. The vestibular toxicity can develop after acute exposures and takes place in central vestibular pathways. Larsby et al. (1) and Tham et al. (8) demonstrated that IV infusion of styrene produced nystagmus during rotational challenge in rabbits and rats when arterial styrene levels reached 40-75 ~tg/g blood. 0dkvist et al. (4) subsequently reported enhanced saccade speed in human volunteers exposed to styrene by inhalation (87-139 ppm) producing blood styrene concentrations of approximately 3 ~tg/g. Accuracy of the saccade was not altered and nystagmus did not occur spontaneously or following rotation. Among workers chronically exposed to styrene, Tham et al. (9) reported evidence of disruption in smooth ocular pursuit of a moving fight stimulus and impaired visual suppression. It is not currently known whether acute exposures to styrene can alter auditory function, although auditory impairments have been reported following chronic exposure. Pryor et al. (5) reported a loss in auditory sensitivity for pure tones in weanling rats exposed to styrene at 800, 1000, or 1200 ppm for 14 h per day for 3 weeks using both behavioral and auditory brainstem evoked response measures. Impairments be151
152
FECHTER
nounced impairments at tone frequencies at the extreme high end of the subject's audible range (5,6,7,11), it would be predicted that the highest test tone frequencies would be more affected by styrene. Electrophysiological measurements were made from the cochlea to evaluate outer hair cell function and inner hair cell-spiral ganglion function in order to determine whether styrene has a cochlear site of action. METHOD
Subjects Forty-six male pigmented adult guinea pigs obtained from Murphy Labs served as experimental subjects. They were group housed in an AAALAC-approved animal facility with ambient sound levels of < 45 dB as measured on the A weighting scale (dBA). The A weighting scale maximizes sound energy in the human auditory range and thereby eliminates low frequency rumble below the guinea pigs' auditory range. Subjects had free access to food and water and were maintained on a 12L : 12D cycle. Audiometric testing and exposure to styrene occurred during the daylight portion of this cycle.
Styrene Exposures Injection. Styrene was administered by IP injection in some subjects to allow within subject comparisons of auditory function prior to and then immediately following styrene administration. The dosage selected, two injections of 0.75 ml each spaced 30 min apart, was selected based on pilot experiments which showed that a dose of 2 ml styrene was lethal when administered to two subjects. Inhalation. Styrene vapor was generated by evaporating styrene monomer at 40°C within a heated glass vessel. Styrene was injected into the vessel at a controlled rate (approximately 70/zl/min to achieve chamber concentratins of 500 ppm and 150 #l/min to attain 1200 ppm) designed to maintain constant chamber styrene concentrations. A Miran infrared detector was used to monitor styrene levels in the exposure chamber in real time over the 7 h of exposure. Manual adjustments in styrene injection rates were made when chamber concentrations deviated by more than 10 ppm from the desired exposure level. Ambient sound levels in the chamber were < 50 dBA.
Simultaneous Noise and Styrene Exposures In some experiments, subjects received simultaneous exposure to styrene by inhalation and broad-band noise for 7 h. This noise was generated by a white noise generator and presented to subjects at an intensity of 95 dB using the Aweighting scale. This sound level was chosen because it produced a mild, temporary auditory threshold shift observable one day following exposure but no permanent disruption of auditory function. A 90 dBA time weighted average (TWA) is considered the permissible exposure level for workers although an 85 dB T W A is recommended (3). Subjects in the control group which received neither styrene nor noise were also placed in the inhalation chamber for 7 h during which they were exposed to a stream of air equivalent in volume to that given styrene-exposed subjects but not to additional noise. Experimental design. Three experiments were performed. Experiment 1 was designed to evaluate the effects of acute, IP styrene administration on auditory function using a withinsubjects design. Auditory function was assessed in all subjects (n = 3) prior to injection of styrene monomer (0.75 ml/sub-
ject) and 30 min following exposure. Because this dose did not alter auditory potentials, all subjects received a second administration of 0.75 ml/subject styrene immediately following the 30 min evaluation of auditory function and auditory potentials were assessed 30 rain following this second dose. Untreated control subjects (n = 3) were also tested according to the same time schedule. For Experiment 2, styrene exposures were effected by inhalation and auditory thresholds were assessed 18-22 h later. Four groups of five subjects were exposed as follows: styrene only group = 500 ppm styrene for 7 h at chamber noise levels; styrene + noise group = the same level of styrene plus 95 dBA white noise; noise only group = 95 dBA white noise alone; chamber control group = exposure to the chamber with no noise or styrene. Experiment 3 entailed inhalation exposure to styrene at either 500 (n = 5) or 1200 (n = 5) ppm for 7 h and simultaneous noise exposure at 95 dBA, but audiometric testing was delayed until 1 week following exposure to provide time for recovery from the noiseinduced threshold shift observed in Experiment 2. Control groups were treated with 95 dBA noise (n = 5) or were placed in a chamber with no added noise or styrene (n = 5).
Electrophysiological Procedures Compound action potential and cochlear microphonic measurement from the round window. The propogated dectrophysiological response of the cochlea measured at the round window to tone pips consists of a complex waveform. The initial negative potential (N1) corresponds to the compound action potential generated by the Type 1 spiral ganglion cells which are the initial post-synaptic elements of the cochlea. This potential is dependent not only on the integrity of the presynaptic (inner hair cells) and postsynaptic (Type 1 spiral ganglion) elements of the cochlea but also on outer hair cells which influence inner hair cell sensitivity. This potential occurs approximately 2 msec following onset o f the tone stimulus. The thresholds for evoking an N1 potential at each of 11 test tone frequencies were measured to evaluate cochlear function using a round window electrode. The stimulation of the cochlea with constant tone stimuli also elicits an ac waveform which is phase locked to the tone frequency and reflects the mechanical movement of outer hair cells in response to sound. This waveform, the cochlear microphonic, can be measured by filtering the signal from the round window and amplifying only that frequency which matches that used as the sound stimulus delivered to the ear canal. The cochlear microphonic was employed as a measure of outer hair cell function. Specifically, the sound intensity for the same 11 tone frequencies used to measure N1 above were adjusted upward until a criterion voltage of 1 #V RMS was obtained. These values were used to plot a cochlear microphonic isopotential curve relating sound level in dB sound pressure level (SPL) at each test tone frequency to the criterion response. The outer hair cell is the primary generator of the cochlear microphonic and the sound intensity necessary to produce a cochlear microphonic signal of 1 #V is increased when outer hair cells are damaged. The N1 component of the compound action potential response represents the action potential generated at the Type 1 spiral ganglion cell in response to potentials generated at the inner hair cell. A selective elevation in the sound intensity necessary to elicit the N1 response when the amplitude of the cochlear microphonic is normal is interpreted as either a pre- or post-synaptic impairment. Subjects were anaesthetized with urethane (1.5 g/kg IP);
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supplemental injections o f 0• 15 g / k g IP were used as necessary to maintain an adequate level of anaesthesia. Lidocaine (2°70) was injected at all incision loci to block pain. The surgery was conducted in an IAC 122A audiometric booth (Industrial Acoustics Company, New York, NY). A d c heating pad was used to maintain body temperature at 39 + 1 °C as measured by a rectal probe• The animals were tracheotomized, the right pinna was removed, and the right auditory bulla was then exposed using a ventral approach. A low voltage lamp was directed at the cochlea to prevent cooling. The active electrode was a teflon insulated 40 ga. silver wire ( A - M Systems, Inc., Everett, WA). A silver/silver chloride reference electrode was placed in a neck muscle. A plexiglas speculum was inserted into the external auditory canal to accommodate an ACO Pacific (Belmont, CA) Model 7013 1/2" pressure microphone used for stimulus delivery. The compound action potentials were bandpass filtered between 300 and 1000 Hz and preamplified × 10 by a Grass (Quincy, MA) dc preamplifier (Model P16) and then amplified x 100 by a Grass ac preamplifier (Model P15). Compound action potential thresholds (detection o f a NI wave of approximately 1 #V amplitude observed on a digital oscilloscope)
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2.3543, p < 0.001, reflecting the elevation in auditory thresholds at mid-frequencies shown by the noise-only and noiseplus styrene groups. A very limited elevation in the 1 ;tV cochlear microphonic isopotential curve is suggested at the lowest test frequencies among all treated subjects. However, an ANOVA did not confirm that group differences met the accepted criterion for statistical significance, F(3, 1 6 ) = 2.683, p > 0.08. While statistically significant differences were found among tone frequencies in the isopotential curve, F(10, 160) = 183.3799, p < 0.0001, a significant treatment group by frequency interaction was not found, F(30, 160) = 1.4265,p > 0.08. The effects of noise and styrene on cochlear function were examined further in Experiment 3 by interposing a I week recovery period between experimental exposures and audiometric testing and by including an additional dose of styrene. One week following exposure there appeared to be a small elevation in auditory thresholds among subjects in the 500 ppm styrene plus noise group in relation to untreated controls (see Fig. 5). Similar findings were not evident in the group receiving the higher dose of 1200 ppm styrene plus noise; in fact, thresholds for this latter group were equivalent to the subjects which had been exposed to noise alone. Reliable differences between treatment groups were not found, F(3, 16) = 2.1840, p 0.1, nor was a significant interaction seen between group conditions and tone frequency, F(30, 160) = 1.0538,p > 0.1. Figure 6 depicts the effect of styrene plus noise exposure on the cochlear microphonic recorded 1 week following exposure. There is a suggestion in the data of a limited elevation in the sound level necessary to elicit the criterion cochlear microphonic value among all treatment groups relative to untreated controls. An ANOVA showed that the effect of treatment did meet statistical significance, F(3, 16) = 3.279, p < 0.05. Subjects receiving combined exposure to styrene plus noise did not show an enhanced loss in cochlear microphonic sensitivity relative to the subjects which received noise alone.
at levels well above the threshold limit value (TLV). Neither IP administration approximating the maximally tolerated dose nor inhalation exposure at doses as high as 500 ppm disrupted auditory thresholds or outer hair cell function indexed by the cochlear microphonic response. When noise was combined with styrene exposure in an attempt to evaluate ototoxicity under conditions of cochlear stress, the results were negative; styrene did not enhance the loss of auditory function due to noise alone. However, all subjects receiving noise, whether or not styrene was present, did show a loss in threshold sensitivity at appropriate sound frequencies based on the noise power spectrum delivered. The results obtained one week following combined exposure to 500 ppm styrene plus noise are suggestive of a limited loss in auditory sensitivity, but the effect was not sufficiently robust to reach statistical reliability nor was it dose-related. The extent of loss observed, 15 dB at the maximally affected frequency, is a modest one. The source of the loss in cochlear microphonic sensitivity observed 1 week following combined noise and styrene exposure in all treatment groups is not clear. It seems unlikely that it is a result of noise-induced injury to the cochlea because auditory thresholds reflected by the N1 response were unaffected. Damage to outer hair cells would also be expected to impair cochlear tuning such that loss in the threshold to elicit an action potential would be observed. The fact that the cochlear microphonic was most affected at low frequencies is consistent with the presence of excess fluid around the round window due to the surgical preparation. Whereas the data do not demonstrate an acute cochlear effect of styrene, it is still possible that acute styrene exposure may disrupt central auditory processes. The techniques used here are not appropriate for detecting such effects. In addition, there are data from both humans (2) and laboratory animals (5) which demonstrate styrene ototoxicity following relatively long-term exposure. ACKNOWLEDGEMENTS
DISCUSSION The results of these experiments provide little evidence that acute styrene administration can alter cochlear function even
This work was supported in part by PHS Grant Nos. ES02852 and ES03819. I thank Dr. Lei Yao for his technical support and Dr. David Bassctt for setting up the inhalation exposure system.
REFERENCES 1. Larsby, B.; Tham, R.; (3dkvist, L. M.; Hydtn, D.; Bunnfors, I.; Aschan, (3. Exposure of rabbits to styrene: Electronystagmographic findings correlated to the styrene level in blood and cerebrospinal fluid. Scand. J. Work Environ. Hlth. 4:60-65; 1978. 2. Muijser, H.; Hoogendijk, E. M. (3.; Hooisma, J. The effects of occupational exposure to styrene on high-frequency hearing thresholds. Toxicology 49:331-340; 1988. 3. National Institute for Occupational Safety and Health: Publications on noise and hearing. Cincinnati, OH: U.S. Department of Health and Human Services; 1991. 4. (3dkvist, L. M.; Larsby, B.; Tham, R.; .~hlfeldt, H.; Anderson, B.; Eriksson, B.; Liedgren, S. R. C. Vestibulo-oculomotor disturbances in humans exposed to styrene. Acta Otolaryngol. 94: 487-493; 1982. 5. Pryor, (3. T.; Rebert, C. S.; Howd, R. A. Hearing loss in rats caused by inhalation of mixed xylenes and styrene. J. Appl. Toxicoi. 7:55-61; 1987.
6. Pryor, G. T.; Rebert, C. S.; Dickinson, J.; Feeney, E. M. Factors affecting toulene-induced ototoxicity in rats. Neurobehav. Toxicol. Teratol. 6:223; 1984. 7. Rybak, L. P. Drug ototoxicity. Ann. Rev. Pharmacol. Toxicol. 26:79; 1986. 8. Tham, R.; Larsby, B.; Eriksson, B.; Bunnfors, I.; ~dkvist, L.; Liedgren, C. Electronystagmographic findings in rats exposed to styrene or toluene. Acta Otolaryngol. 93:107-112; 1982. 9. Tham, R.; Larsby, B.; M611er.; Niklasson, M.; ~3dkvist, L. M. Vestibulotoxicity of organic solvents. Proceedings of the 4rth International Conference on the Combined Effects of Environmental Factors 101-105; 1991. 10. Young, J. S.; Fechter, L. D. Reflex inhibition procedures for animal audiometry: A technique for assessing ototoxicity. J. Acous. Soc. Amer. 73:1686; 1983. 11. Young, J. S.; Upchurch, M. B.; Kaufman, M. J.; Fechter, L. D. Carbon monoxide exposure potentiates high-frequency auditory threshold shifts induced by noise. Hear. Res. 26:37; 1987.