* Center for Clinical Research and Evidence-Based Medicine, University of Texas Health Science Center at Houston Medical School, 6431 Fannin, MSB 2.104, Houston, Texas 77030-1503;
Department of Veterinary Biosciences, University of Illinois at Urbana-Champaign, Urbana, Illinois 61802; and
Neurotoxicology Division, MD-74B, National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina 27711
Received December 24, 2001; accepted April 25, 2002
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ABSTRACT |
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Key Words: polychlorinated biphenyls; PCBs; Aroclor 1254; distortion product otoacoustic emissions; auditory evoked brainstem responses; Long-Evans rats.
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INTRODUCTION |
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Exposure to PCBs early in development causes a low-frequency (1 kHz) hearing loss in laboratory rats when tested using a modified reflex audiometry task (Crofton and Rice, 1999
; Crofton et al., 2000a
,b
; Goldey et al., 1995a
; Goldey and Crofton, 1998
). Rats exposed early in development to a commercial PCB mixture, Aroclor 1254 (A1254), also have significantly smaller amplitude auditory evoked brainstem responses (ABRs) at 1 and 4 kHz but not at 16 or 32 kHz (Herr et al., 1996
).
Goldey et al. (1995a) have hypothesized that PCB-induced auditory losses are caused by a reduction in thyroid hormones that are necessary for normal cochlear development (Uziel, 1986). There is empirical support for this hypothesis. Exposing pregnant and lactating rats to PCBs reduces circulating thyroid hormone concentrations in their offspring (Crofton et al., 2000b
; Goldey and Crofton, 1998
; Goldey et al., 1995a
), while postnatal thyroxine (T4)-replacement therapy partially ameliorates A1254-induced hearing loss (Goldey and Crofton, 1998
).
Crofton et al. (2000a) documented missing outer hair cells on the basilar membrane of the cochlea following perinatal exposure to A1254. Outer hair cell loss was confined to the apical and upper middle turns of the basilar membrane, consistent with the low-frequency hearing loss associated with A1254 exposure. Outer hair cell loss is the likely proximal cause of PCB-induced hearing loss. The effect of A1254 on outer hair cells was assessed in this study by measuring distortion product otoacoustic emissions (DPOAEs) in rats. ABRs were measured to determine whether A1254 exposure damages nerve cells in the auditory pathway as well as outer hair cells in the cochlea.
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MATERIALS AND METHODS |
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Apparatus and stimuli.
The instrumentation used to record the distortion product otoacoustic emissions (DPOAEs) consisted of a probe unit (2 Etymotic ER-2 insert earphones and an etymotic ER-10B ear canal microphone), an amplifier (etymotic 10B amplifier) providing 40 dB of gain, an Intel microprocessor-based computer, and an Ariel DSP-16+ signal processing board that generated the calibration stimuli, the stimuli evoking the DPOAEs, and the digitization of the analog signal from the ear canal. The sampling rates for both the analog-to-digital and digital-to-analog converters were 50 kHz. The software for generating the DPOAEs was CUBeDISPTM. The software was modified to add capabilities that included artifact rejection and response detection (Lasky et al., 1992). The distortion generated by the instrumentation was measured in 1-cc closed syringes to verify that the instrumentation did not distort the results.
The same hardware was used to generate the stimuli and digitize the responses for the auditory brainstem evoked response (ABR) recordings. A Grass P511K A.C. preamplifier amplified the responses 50,000 times and band pass filtered them with the 3 dB cut-offs at 100 Hz and 3 kHz. The roll-offs of the band pass filter were 12 dB/octave. The sampling rates to generate the click stimuli and digitize the responses were 10 kHz. Custom software controlled the recording of the response and generated the ABR stimuli, which were downloaded to the Ariel 16+ DSP board, where they were converted to analog signals.
The etymotic ER-10B ear canal microphone measured the sound pressure levels of the DPOAE stimuli. With the probe in the ear canal, the CUBeDISPTM software initially presented chirps to determine the frequency response of the canal with the probe in place. The frequency response was used to adjust the output of the etymotic ER-2 insert earphones to the specified levels. The 100-µs rarefaction click stimuli for the ABRs was calibrated as peak equivalent, sound-pressure level (peSPL on a linear scale) in a 2-cc coupler with a Bruel and Kjaer sound-level meter (Type 2230) and 4144 microphone (frequency range 2.68,000 Hz).
Procedures.
Testing was conducted in an isolated room in a laboratory at the University of Illinois. Background noise level in the testing room was 58 dB, C-weighted scale, 54 dB, A-weighted scale (Radio Shack Realistic Sound Level Meter, catalog # 332050) SPL. Rats were sedated with 0.4 ml/kg ketamine/xylazine (87:13 mg/ml) ip. Otoscopy preceded testing. No external or middle ear pathology or obstructions were identified by otoscopy in any of the rats. Subsequent to otoscopy, the rats were placed on their right sides (left ear up) and the probe was positioned in the ear canal. Electrodes were also attached. The ABR testing immediately followed the DPOAE testing.
Total test time was approximately 3045 min per rat. The room temperature was 20°C. The rats were placed on a heating pad (medium temperature setting) to maintain body temperature, which was not measured during testing. All testing was conducted without prior knowledge of a rats experimental condition, to avoid potential bias.
DPOAE testing.
DPOAEs were generated by simultaneously presenting 2 sinusoids differing in frequency (the lower frequency primary is f1 and the higher frequency primary is f2) and recording the sound pressure in the sealed ear canal. Because the cochlear mechanical response to sound is nonlinear, nonlinear distortions can be used to characterize cochlear function. The 2f1f2 distortion product is the most robust of the distortion products recorded.
Initially, 25 primary stimulus pairs were presented sequentially. These 25 stimuli differed in frequency (f2/f1 = 1.2; f2s = 20.0, 17.4, 15.1, 13.2, 11.4, 9.9, 8.6, 7.5, 6.4, 5.6, 4.9, 4.2, 3.7, 3.2, 2.7, 2.4, 2.1, 1.8, 1.5, 1.3, 1.1, 0.9, 0.8, 0.6, and 0.5 kHz). They were presented from the highest to the lowest frequency pairs. Each stimulus pair was presented for a total of 4096 artifact-free ms. The level of the lower frequency primary (L1) was 59 dB SPL and the level of the higher frequency primary (L2) was 49 dB SPL. L1 was presented 10 dB greater than L2 because larger DPOAEs are associated with those primary stimulus level differences. A fast Fourier transform (FFT) was calculated on the time-averaged response and the amplitudes of the distortion product and noise (estimated by the mean amplitude of the 3 lower- and higher-frequency bins adjacent to the distortion product) were measured. After recording the DPOAEs to the 25 primary stimulus-pairs, a second replicate 25 stimulus-pair sequence was presented. DPOAEs to stimulus pairs differing in frequency are referred to as Dpgrams, which are used to assess cochlear function loss across the range of frequencies presented.
For these data, a DPOAE was defined as a reliable response (and not noise) by a maximum-likelihood ratio test (Lasky et al., 1992). That test calculated the probability that the measured DPOAE came from the distribution of background noise near the DPOAE frequency recorded in the ear canal. DPOAEs unlikely (p < 0.05) to be noise were identified as responses. Both replicate DPOAEs to the same stimulus pairs had to be significant (p < 0.05) in order to be considered a response. Suprathreshold responses were determined by the averaged amplitude of the significant replicate DPOAEs to the L1 = 59 and L2 = 49 dB SPL stimuli. All rats had significant DPOAEs for f2s from 11,400 to 3200 Hz. For f2s > 11,400 Hz, the instrumentation could not generate the stimuli at the specified levels for all rats, given the resonance characteristics of their ear canals. Significant responses declined rapidly with decreasing frequency below f2 = 2051 Hz (50% at f2 = 1758 Hz, 0% by f2 = 1074 Hz). The noise levels in the rat ear canal increased with decreasing frequency, contributing to the difficulty recording DPOAEs to low-frequency stimuli. In addition, the input impedances to low frequencies were high because of small outer and middle ears.
The 2f1f2 DPOAE thresholds were determined by reducing the levels of the primaries in 10 dB steps until no significant responses (p < 0.05 by a maximum-likelihood ratio test) could be recorded at any frequency. Replicates were recorded at each stimulus level. All other stimulus and recording parameters were kept constant. The DPOAE thresholds were defined by the lowest level f2 that elicited a significant DPOAE for both replicates.
ABRs.
The ABR stimuli were presented immediately after the DPOAEs without repositioning the probe. The ABR stimulus was a 65-dB peSPL 100-µs click (broadband stimulus) presented at a rate of 26.8/s. Replicate responses (each representing the averaged responses to 500 clicks) were recorded to all stimuli. The responses were differential voltage recordings from needle electrodes placed in the scalp at the vertex (noninverting electrode) and ipsilateral mastoid (inverting electrode). The ground was placed in the back of the neck.
Figure 1 presents an example of replicate waveforms recorded to the 65-dB peSPL broadband-stimulus click. Four positive peaks (local amplitude maxima) were recorded in all but 2 of the rats in the first 8 ms following stimulus onset. Peak 3 was absent in the 2 rats (one control and one A1254) with missing peaks. Peak 3 and not peak 4 was judged missing by the latencies of the recorded peaks. The 4 peaks were labeled according to the nomenclature adopted by Herr et al. (1996). The average latency and amplitude (peak to following trough) of the 2 replicates were used in analyzing the data. ABR click thresholds were recorded by attenuating the click stimulus in 10-dB steps until no reproducible response could be recorded. Reproducible and nonreproducible responses were subjectively judged. A response was defined if any of the 4 peaks (invariably peak 2) was scored in both replicates. The stimulus level was raised in 5-dB increments until a response was reestablished, thus determining the ABR threshold within 5 dB. The ABR waveforms and thresholds were recorded with no knowledge of each rats experimental condition.
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RESULTS |
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ANOVAs, 2 (group) x 2 (gender), were calculated for frequencies that had adequate sample sizes (more than half of the subjects in each group with responses) but were not included in the three-way ANOVA reported above because of missing data (see Table 1). At 17.4, 15.1, and 13.2 kHz, A1254 rats had smaller DPOAEs than control rats although the differences were significant only at 15.1 kHz (F(1,14) = 5.10; p = 0.040). No other main effects or interactions were significant at these frequencies. There were no data at 20.0 kHz, because our instrumentation could not produce the specified sound levels at that frequency. There were significant group main effects at each of the 2 lowest frequencies (2.7 kHz: F(1,17) = 5.82, p = 0.027; and 2.4 kHz: F(1,19) = 21.75, p < 0.001) with adequate sample sizes. However, neither the gender main effect nor the group x gender interaction was significant at these frequencies, suggesting that both sexes were equally affected by A1254 at the lowest frequencies tested.3
The group differences at the 2 lowest frequencies were a little over 8 dB. Three of the 11 A1254-treated rats at each of these frequencies were excluded because they failed to elicit a reliable DPOAE by a maximum-likelihood ratio test. In contrast, none of the control rats for the 2.4 kHz stimulus and 2 of the 15 control rats were excluded for the 2.7 kHz stimulus. Because more A1254-treated rats failed to elicit reliable DPOAEs, the 8-dB loss may be an underestimate of the actual size of the A1254-induced loss.
The DPOAE threshold data for males and females are presented in Figure 3. DPOAE thresholds in the A1254-exposed rats were elevated relative to controls, particularly at the lowest frequencies tested. The gender differences were not as large as they were for DPOAE suprathreshold amplitudes.
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Again, at the lowest frequencies, the group differences may have been underestimates, because reliable DPOAEs could not be recorded at the highest stimulus levels presented. For rats that failed to produce a reliable DPOAE at the highest stimulus levels, we assigned a threshold of 54-dB SPL, 5 dB higher than the most intense L2 presented to assess thresholds. Thus, these rats were assigned a conservative estimate of their threshold, which happened more often for the A1254-exposed rats than controls for the 2.1 (1 of 15 controls and 6 of 11 A1254-exposed rats) and the 2.4 and 2.7 kHz stimuli, as described above. Therefore, the thresholds for the A1254-exposed rats at these frequencies may have been underestimated.
Auditory Evoked Brainstem Responses
Figure 4 presents the latencies and amplitudes for the 4 peaks to the 65-dB peSPL stimulus. Data were not presented separately by gender, because there were no gender-related differences associated with A1254 exposure on these measures. The differences in peak latencies and amplitudes between the A1254-exposed rats and the controls were small and did not consistently favor either group. Separate 2 (group) x 2 (gender) x 3 (peak: 1A, 2, and 4) repeated-measures ANOVAs were calculated for the ABR latency and amplitude data. The only significant effect was the peak main effect (F(2,40) = 2358.41, p < 0.001 for latency and F(2,40) = 157.35, p < 0.001 for amplitude). Peak 3 could not be reliably scored in all rats and was not included in these analyses. Instead, separate 2 (group) x 2 (gender) ANOVAs were calculated for peak 3 latency and amplitude. Neither the main effects nor the interaction were significant for those analyses.
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DISCUSSION |
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Frequency Effects
Our results confirm that A1254-induced hearing loss is greater at low frequencies. However, we also reported losses at frequencies for which Goldey, Crofton, and colleagues (Goldey and Crofton, 1995a; Crofton et al., 2000b) have reported no significant loss. The threshold elevations recorded at frequencies greater than 1 kHz in this study were smaller than those reported, but yet, were statistically significant because of the smaller variability in DPOAE measurements. The standard errors of the mean for DPOAE amplitudes and DPOAE thresholds in this study were about half those for behavioral thresholds in the reflex audiometry paradigm used by Goldey, Crofton, and colleagues (Goldey and Crofton, 1995a; Crofton et al., 2000b
).
Gender Effects
While gender-specific effects on neurobehavioral function have been reported following perinatal PCB exposure (Davenport, et al., 1976; Geller et al., 2001
; Widholm et al., 2001
), previous studies have shown that males and females are equally affected with respect to thyroid hormone reductions and hearing loss (Crofton and Rice, 1999
; Crofton et al., 2000b
; Goldey et al., 1995a
; Rice and Hayward, 1999
). Similarly, in this study there was no gender effect on DPOAE amplitudes at the lowest frequencies reliably assessed (2.7 and 2.4 kHz), consistent with the results of prior studies. Gender differences were only recorded at higher frequencies. Why there would be a gender effect at higher but not lower frequencies is not obvious but could be explained if females were adversely affected by A1254 earlier in development. Alternatively, this result could be explained by chance (a type I error). Additional research is needed to interpret the recorded gender effects.
Effects on the ABR
The discrepancy between the ABR results in this study (no effect) and those of Herr et al. (1996, reduced amplitudes and prolonged latencies) may be explained, in part, by the frequencies and levels of the stimuli presented. Herr et al. used equal energy pure tone pips. We used a 100-µs click, a broadband stimulus with a null at 10,000 Hz. However, the acoustic waveform is shaped by the auditory transducer, and therefore, the spectrum of the click presented in this study rolls off at both the low- and high-frequency ends. The effective stimulus is also determined by the anatomy and physiology of the rat. ABRs to 100-µs clicks correlate best with hearing thresholds from 2000 to 4000 Hz (Stapells et al., 1994) in humans. Rat outer and middle ears have higher resonance frequencies than humans (Hemila et al., 1995
). Rats also have more sensitive higher-frequency hearing. Thus, it is likely that rat ABRs to clicks are generated by higher frequencies than in the human. At the stimulus level (65-dB peSPL) and effective frequencies (probably higher than 24 kHz) presented in this study, Herr et al. (1996) reported no effect of A1254 exposure on the ABR, consistent with the results of this study.
The rational for recording the ABR was to determine whether there were A1254-induced neural deficits indicated by a greater effect on later peaks than on the earlier peaks. Neural insult is unlikely to be frequency-specific, because there is little reason to expect that A1254 exposure would only affect neurons relaying information from restricted regions of the cochlea. Thus, frequency-specific stimuli were not presented. There is little evidence from this study or the Herr et al. (1996) study to indicate that A1254 exposure affects neural transmission in the auditory nerve and brainstem.
Implications for Humans
Species differences in the timing of cochlear development are significant enough to warrant caution in generalizing results from rodent models to humans. Cochlear functional development occurs postnatally in rats. In contrast, the functional maturation of the human cochlea begins about the 20th week postconception and is completed by the 35th week (Pujol and Uziel, 1988; Sulik, 1995
). Many of the anatomical changes that occur during that time interval concern the outer hair cells (the targets of PCB ototoxicity in rats and the likely generators of DPOAEs) and their support cells. Assuming that the critical period for PCB-induced auditory insult corresponds to the period of functional maturation of the cochlea, the human would be most vulnerable to PCB ototoxicity during the last half of pregnancy when the only route of PCB exposure is via placental transfer. A recent epidemiological study suggests that prenatal PCB exposure may be associated with subtle auditory deficits in children (Grandjean et al., 2001
). Based on these findings, further epidemiological studies of auditory function in children exposed to PCBs prenatally are warranted. Recording otoacoustic emissions could contribute significantly to that research. Otoacoustic emissions reflect the hypothesized site of PCB damage, and they can be recorded reliably and efficiently in humans of all ages (Robinette and Glattke, 1997
).
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ACKNOWLEDGMENTS |
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NOTES |
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1 To whom correspondence should be addressed. Fax: (713) 500-0519. E-mail: robert.e.lasky{at}uth.tmc.edu.
2 A 2 (group) x 2 (gender) x 10 (f2 frequency) repeated-measures ANOVA was calculated for the 8 litters, each contributing a male and a female rat. Group was a between-subjects (litters) factor and gender and frequency were within-subjects (litters) factors. The group (F(1,6) = 6.69, p = 0.041) and the frequency (F(9,54) = 55.40, p < 0.001) main effects were significant. The group x gender interaction approached significance (F(1,6) = 3.70, p = 0.103). The controls had larger amplitude DPOAEs than A1254-exposed rats. That difference was more apparent for females and at lower frequencies. The trends in the data were the same as those observed in the total sample.
3 ANOVAs, 2 (group) x 2 (gender), with gender as a within-subjects (litters) factor, were calculated at 17.4, 15.1, 13.2, 2.7, 2.4, and 2.1 kHz for the 8 litters, each litter contributing a male and a female rat. Only 2 effects were significant: the group main effect for the 2.7 (F(1,3) = 18.09, p = 0.024) and 2.4 (F(1,5) = 7.32, p = 0.042) kHz stimuli. Group x gender interactions were not significant at the lowest frequencies analyzed (F(1,3) = 0.14, p = 0.737 for f2 = 2.7 kHz; F(1,5) = 0.13, p = 0.734 for f2 = 2.4 kHz; F(1,3) = 0.47, p = 0.544 for f2 = 2.1 kHz).
4 A 2 (group) x 2 (gender) x 17 (f2 frequency) repeated-measures ANOVA was calculated for the 8 litters, each contributing a male and a female rat. Group was a between-subjects (litters) factor and gender and frequency were within-subjects (litters) factors. The frequency (F(16,96) = 44.23, p < 0.001) main effect and the group x frequency interaction (F(16,96) = 1.81, p = 0.040) were significant. Elevated thresholds for the A1254-treated rats were more apparent at low than high frequencies, as they were for the total sample.
5 The complementary analyses on the sub-sample of rats from the 8 litters, contributing both a male and a female each, were 2 (group) x 2 (gender) ANOVAs. Gender was a within-subjects (litters) factor. Only the main effect for group (F(1,6) = 11.94, p = 0.014) at 3.2 kHz was significant, although the group main effect at 2.7 kHz approached significance (F(1,6) = 4.77, p = 0.072).
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