TY - JOUR
AU - Fulton,, Sherry
AB - Abstract This study was undertaken to identify the ototoxic potential of two jet fuels presented alone and in combination with noise. Rats were exposed via a subacute inhalation paradigm to JP-8 jet fuel, a kerosene-based fuel refined from petroleum, and a synthetic fuel produced by the Fischer-Tropsch (FT) process. Although JP-8 contains small (∼5%) concentrations of aromatic hydrocarbons some of which known to be ototoxic, the synthetic fuel does not. The objectives of this study were to identify a lowest observed adverse effect level and a no observed adverse effect level for each jet fuel and to provide some preliminary, but admittedly, indirect evidence concerning the possible role of the aromatic hydrocarbon component of petroleum-based jet fuel on hearing. Rats (n = 5–19) received inhalation exposure to JP-8 or to FT fuel for 4 h/day on five consecutive days at doses of 500, 1000, and 2000 mg/m3. Additional groups were exposed to various fuel concentrations followed by 1 h of an octave band of noise, noise alone, or no exposure to fuel or noise. Significant dose-related impairment in the distortion product otoacoustic emissions (DPOAE) was seen in subjects exposed to combined JP-8 plus noise exposure when JP-8 levels of at least 1000 mg/m3 were presented. No noticeable impairment was observed at JP-8 levels of 500 mg/m3 + noise. In contrast to the effects of JP-8 on noise-induced hearing loss, FT exposure had no effect by itself or in combination with noise exposure even at the highest exposure level tested. Despite an observed loss in DPOAE amplitude seen only when JP-8 and noise were combined, there was no loss in auditory threshold or increase in hair cell loss in any exposure group. JP-8 jet fuel, Fischer-Tropsch fuel, hearing loss, noise-induced hearing loss This investigation was undertaken to determine the ototoxic potential of two types of jet fuels both by themselves and in the presence of noise. The study was predicated upon limited epidemiological and laboratory data suggesting that traditional petroleum-based jet fuels may be ototoxic, the high probability for combined exposure to jet fuel and to noise in a wide range of occupations related to airplane operations, and the known relationship between exposure to specific aromatic hydrocarbons contained in jet fuels at low concentrations and permanent hearing loss. Both occupational epidemiological studies (Abbate et al., 1993; Fuente et al., 2009; Morata et al., 1997; Sliwinska-Kowalska et al., 2001, 2003; Vrca et al., 1996, 1997) and controlled laboratory studies (Campo et al., 1997, 2001; Cappaert et al., 1999, 2000, 2001a,b; Crofton et al., 1994; Gagnaire and Langlais, 2005; Lataye et al., 2003; Loquet et al., 1999; McWilliams et al., 2000; Pryor et al., 1983, 1987) have documented hearing loss resulting from several aromatic hydrocarbons, including toluene, ethylbenzene, and p-xylene. Each of these aromatic hydrocarbons is found in JP-8 jet fuel. The current investigation was designed to evaluate the ototoxic potential of two types of jet fuels: JP-8, a traditional petroleum-derived fuel, and a synthetic fuel produced by the Fischer-Tropsch (FT) process. Although JP-8 (designated as MIL-DTL-83133) contains aromatic hydrocarbons (25% max), the synthetic fuel does not. This study was undertaken based on an investigation of U.S. Air Force employees with occupational exposure to noise and jet fuels (JP-4 and JP-8) containing aromatic hydrocarbons (Kaufman et al., 2005). The Kaufman study, though small in size, suggests that jet fuel may increase hearing loss in a chronic exposure model (a larger odds ratio for hearing loss with 12 years of exposure than for 3 years of exposure). Moreover, the odds ratio associated with duration of fuel exposure exceeded that obtained for age. However, because all subjects did have a history of noise exposure, it is not clear whether the fuel by itself might have produced some ototoxicity. In addition, it is not clear whether or not the aromatic hydrocarbon component of the fuel is responsible for the hearing loss. The Kaufman study also focuses attention on the likelihood that individuals exposed to jet fuel will also have significant risk for substantial noise exposure. The current study was based on an initial laboratory investigation in which the effects of controlled inhalation exposure to JP-8 jet fuel were studied on auditory function in rats (Fechter et al., 2007). Those data demonstrated that fuel exposure by itself had no effect on auditory function assessed using either distortion product otoacoustic emissions (DPOAE) or on pure-tone auditory thresholds assessed by measuring the occurrence of a compound action potential (CAP) of ∼1 μV. However, exposure to JP-8 concentrations at levels substantially higher than the Department of Defense permissible exposure level (PEL) of 350 mg/m3 did enhance the adverse effects of moderate noise exposure on DPOAE amplitude. Specifically, successive exposure first to JP-8 jet fuel (1000 mg/m3 for 4 h/day × 5 days) followed by a 1-h exposure to 100dBlin octave band noise (OBN) centered at a frequency of 8 kHz yielded a persistent reduction in the DPOAE. The noise exposure alone produced minimal impairments in this measure of outer hair cell (OHC) function. The purposes of this investigation were twofold: (1) to compare JP-8 and FT fuels for their ototoxic potential when presented by themselves and in combination with noise exposure and (2) to determine whether the effects of fuel exposure on auditory function were dose related. Our expectation was that JP-8 would be a more potent ototoxicant owing to the aromatic components that it contains, although we could not rule out the potential that other components of this complex hydrocarbon mixture might contribute to the ototoxicity. METHODS AND MATERIALS A total of 133 pigmented male Long-Evans rats (250–300 g) obtained from Harlan (Livermore, CA) were used in these studies. All the rats were used in auditory function studies (n = 5–19) both with repeated noninvasive assessment of OHC function and a single assessment of auditory thresholds conducted 4 weeks following the end of exposures (Fig. 1). Group numbers are larger in the control and noise-alone conditions because these subjects were always run in parallel with each of the fuels at each exposure concentration. The subjects were housed in plastic cages with free access to food and water. Temperature was maintained at 21°C ± 1°C, and fluorescent lights were on from 6:30 A.M. to 6:30 P.M. The Loma Linda VA Medical Center Institutional Animal Care and Use Committee approved all the experimental protocols. All exposures and testing were performed during the daytime. All subjects received an initial screen of auditory function using DPOAE testing to ensure that they had normal hearing. Group assignments were made based on these initial tests and were designed to ensure equivalent baseline responses across all groups. FIG. 1. Open in new tabDownload slide Schematic representation of experimental design detailing repeated measurements of DPOAE prior to and following exposure, the temporal relationship between fuel and noise exposure, and the time of the final assessment of auditory threshold using the CAP. FIG. 1. Open in new tabDownload slide Schematic representation of experimental design detailing repeated measurements of DPOAE prior to and following exposure, the temporal relationship between fuel and noise exposure, and the time of the final assessment of auditory threshold using the CAP. Exposure Procedures Jet fuels. Three different doses of each jet fuel were used. The median dose of 1000 mg/m3 for 4 h was chosen based on earlier reports (Fechter et al., 2007) that this dosage was effective in potentiating noise-induced hearing loss (NIHL). This dose is higher than the permissible concentration for the workplace (namely an 8 h time weighted average (TWA) of 350 mg/m3). Two other doses of JP-8 were used: 500 mg/m3 dose and 2000 mg/m3. FT fuel exposures were also conducted at levels of 500, 1000, and 2000 mg/m3. Doses are expressed in terms of total hydrocarbon levels. The fuel was supplied from a stock maintained by the fuels branch at the Wright-Patterson Air Force Base (Dayton, OH). It consisted of a blend of jet fuel obtained from various refineries to which was added the JP-8 fuel additive package consisting of diethylene glycol monomethyl ester to inhibit ice formation and both static and corrosion inhibitors. The FT fuel was also supplied by the Fuels Branch at Wright-Patterson Air Force Base. A single lot of each fuel type was used to complete all the studies described here. The fuels were stored in sealed 1-l opaque bottles at 4°C upon arrival to minimize evaporation. The fuel generation system consisted of a stainless steel constant rate atomizer based on the original design of Liu and Lee (1975). Large drops generated in the system were impacted onto a stainless steel plate in the system and removed. Fuel stock that was not volatilized was collected and eliminated such that fresh stock fuel was always used. The fuel was maintained at room temperature during the exposure. The aerosolized fuel was immediately quenched with dry clean air to reduce particle coagulation and achieve desired total hydrocarbon concentrations. The diluted air was then fed to the nose-only exposure system. Fuel concentration was monitored in real time during the exposure using a Thermo Electron (Franklin, MA) model 51C-HT total hydrocarbon analyzer. The profiles for the vapor generated for the exposure is shown in Figure 2. FIG. 2. Open in new tabDownload slide Comparison of 19 constituents (C4–C13) of aerosolized JP-8 and FT vapors used for inhalation exposures. For both fuels, the nominal total hydrocarbon level was 1000 mg/m3. The levels that were achieved were 932.7 mg/m3 for JP-8 and 1041 mg/m3 for FT fuel. TMB refers to trimethylbenzene. Approximately 25% of the inhalation material is accounted for by these 19 constituents. FIG. 2. Open in new tabDownload slide Comparison of 19 constituents (C4–C13) of aerosolized JP-8 and FT vapors used for inhalation exposures. For both fuels, the nominal total hydrocarbon level was 1000 mg/m3. The levels that were achieved were 932.7 mg/m3 for JP-8 and 1041 mg/m3 for FT fuel. TMB refers to trimethylbenzene. Approximately 25% of the inhalation material is accounted for by these 19 constituents. The rats were exposed to JP-8 or FT using a nose-only exposure system (CH Technologies, Westwood, NJ) that was designed to minimize the potential for fuel deposition on the subjects’ fur and subsequent ingestion during grooming. The rats were habituated for 3 days to the clear Plexiglas restraint cylinders that held the rats during the exposure. The habituation occurred in the rat’s home cage and was designed to minimize stress associated with the restraint procedure. Noise exposure. Within each experiment, 6–19 rats were assigned to receive noise exposure alone or noise exposure that followed the jet fuel inhalation. The noise exposure selected was designed to produce a permanent impairment in auditory function, but one small enough such that additive or potentiating effects of chemical exposure could also be detected (e.g., Pouyatos et al., 2005; Rao and Fechter, 2000). The noise level used was an octave band (OBN) centered at 8 kHz. The noise level measured within this octave was set between 100 and 102 dBlin and measured between 97 and 99 dB on the A weighting scale that reflects the human audiogram and which is used for setting permissible workplace exposures for humans. Current OSHA standards have established a PEL for noise of 90 dB using the A weighting scale for an 8 h TWA. A 5-dB exchange rate is utilized for intermittent noise and for noise that does not persist for 8 h. Based on this rule, the equivalent human PEL for a 4-h time period would be 95 dB (A) and 105 dB (A) for 1 h. Thus, the noise used in these studies would fall just under the exposure limit permitted by OSHA for workplace exposures. However, noise exposure at the level used in this study would certainly be rated by a human observer as “extremely annoying” (Ouis, 2003). According to Dobie (1993), sound levels of 100 dB are similar to those experienced in using a blow dryer or snowmobile, whereas sound levels of 90 dB might be experienced using a power lawnmower. Noise levels of 130 dB might be experienced at 30 m from a jet engine. Table 1 summarizes the exposure treatments used in each experiment, and Figure 1 provides a summary of the experimental design. TABLE 1 Summary of Exposure Groups and Group Subject Number Jet fuel (4 h/day × 5 days) Without noise Noise 100–102 dB (1 h × 5 days) 0 mg/m3 fuel 19 (control) 13 JP-8 (mg/m3)     500 5 7     1000 11 11     2000 6 12 Fischer-Tropsch (mg/m3)     500 6 8     1000 13 8     2000 7 8 Jet fuel (4 h/day × 5 days) Without noise Noise 100–102 dB (1 h × 5 days) 0 mg/m3 fuel 19 (control) 13 JP-8 (mg/m3)     500 5 7     1000 11 11     2000 6 12 Fischer-Tropsch (mg/m3)     500 6 8     1000 13 8     2000 7 8 Open in new tab TABLE 1 Summary of Exposure Groups and Group Subject Number Jet fuel (4 h/day × 5 days) Without noise Noise 100–102 dB (1 h × 5 days) 0 mg/m3 fuel 19 (control) 13 JP-8 (mg/m3)     500 5 7     1000 11 11     2000 6 12 Fischer-Tropsch (mg/m3)     500 6 8     1000 13 8     2000 7 8 Jet fuel (4 h/day × 5 days) Without noise Noise 100–102 dB (1 h × 5 days) 0 mg/m3 fuel 19 (control) 13 JP-8 (mg/m3)     500 5 7     1000 11 11     2000 6 12 Fischer-Tropsch (mg/m3)     500 6 8     1000 13 8     2000 7 8 Open in new tab Exposures to noise were conducted immediately following the fuel exposure in a reverberant 40-l chamber. This successive exposure was necessitated by the very limited size of the restraint tubes within which the animals were housed during fuel exposure and was designed to capture a time interval during which tissue hydrocarbon levels remain maximal (Fechter et al., 2007). Air exchange rate within the noise exposure chamber was 12.5 lpm (providing ∼17 changes per hour) with airflow being monitored by a Dwyer Instruments (Michigan City, IN) flow gauge. Broadband noise was generated by a function-generator (Model DS335; Stanford Research System, Menlo Park, CA) and bandpass filtered (Frequency Devices, 9002, Haverhill, MA) to provide an OBN with center frequency of 8 kHz. The roll-off for the filter system was 48 dB/octave. This signal was amplified by a HCA1000A Parasound Amplifier (Parasound Products, Inc., San Francisco, CA) and fed to speakers (Vifa D25AG-06, Videbaek, Denmark) located ∼5 cm above the subjects’ wire-cloth enclosure. The sound intensity was measured at the level of the rats’ pinnae. Sound pressure measurements were made using a Quest Type 1 sound pressure meter with 1/3 octave filter set (models 1700 and OB300, Oconomowoc, WI). The noise spectrum has previously been published (Pouyatos et al., 2007). Sound levels in the exposure chamber were maximal and essentially flat between 6.3 and 10 kHz. The levels were ∼7 dB lower at 5 and 12.5 kHz. The acoustic intensity was about 20 dB below maximum at 4 and 16 kHz. Noise levels varied less than 2 dB within the exposure chamber. The subjects were placed within small wire-cloth enclosures (15 × 13 × 11 cm) within the chamber. The rats were conscious and free to move within the enclosures. The noise level in the fuel exposure restraint tubes during fuel exposure was below 60 dBlin at all 1/3 octave bands between 1.25 and 12.5 kHz. Auditory Assessment Hair cell functional assessment: DPOAE. Hair cell function was assessed repeatedly within subjects by means of a noninvasive method known as DPOAE testing. Because this method permits repeated testing within subjects, it can track impairment that may occur between a pre-exposure baseline and various time points following an exposure. In this way, both transient and permanent impairments in auditory function as well as the recovery rate can be estimated in each subject. The DPOAE test relies upon the finding that the intact cochlea is able to generate measurable sound energy when stimulated with two simultaneous tones known as “primary tones” and designated as frequencies “f1” and “f2.” Because the sound energy generated by the cochlea consists of different frequencies than the primary tones they are spoken of as “distortion products.” A particularly robust distortion product is the cubic distortion product which is defined algebraically as 2f1 − f2. If the ratio of f1/f2 is kept constant as the frequency of f2 is swept along the subject’s audiometric range, it is possible to detect impairment of the hair cells as a drop in DPOAE amplitude. In these experiments, the ratio of f1/f2 was maintained at 1.25 and the f2 frequency was swept from 3.1 to 63 kHz in 0.1-octave increments. Tone intensities were set at 55 dB for f1 and 35 dB for f2. This difference in tone intensity was selected to maximize the amplitude of the DPOAE (Whitehead et al., 1995). The f1 and f2 primaries were presented through two separate realistic dual radial horn tweeters (Radio Shack, Tandy Corp., Ft Worth, TX). The tones were delivered to the outer ear canal through a probe that also contained an emissions microphone assembly (Etymotic Research, ER-10B+, Elk Grove Village, IL). The tones were sampled, synchronously averaged, and Fourier analyzed for geometric mean frequencies. Delivery of the primary test tones and computation of the 2f1 − f2 distortion product amplitude were accomplished by a DSP board (model PCI-4461; National Instruments, Austin, TX) controlled by a specially formulated program written using LabVIEW version 7.1 (National Instruments). The related noise floors were estimated by averaging the levels of the ear canal sound pressure for the two fast Fourier transform frequency bins below the DPOAE frequency (i.e., for 3.75 Hz below the DPOAE). A hard-walled cavity that approximated the size of the rat outer ear canal was used to calibrate the tonal stimuli. For both stimulus protocols, DPOAEs were considered to be present when they were at least 3 dB above the noise floor. DPOAE testing was accomplished in a single-walled audiometric booth while rats were lightly anaesthetized with ketamine (44 mg/kg) and xylazine (7 mg/kg) injected im. Normal body temperature was maintained using a dc heating unit built into the table supporting the rat. Each subject was first tested at least 3 days after arrival in the laboratory and prior to any experimental treatment. The subjects were retested at 4 days after the end of the experimental treatment, and, again, 4 weeks after exposure. Each DPOAE test required ∼3 min to perform. Audiometric threshold assessment: CAP. In contrast to the repeated assessment of OHC function by the DPOAE method, assessment of auditory threshold, a marker of neural activity in the auditory branch of the eighth cranial nerve, requires nonsurvival surgery. Threshold assessment was performed 4 weeks following the end of all experimental exposures by recording the CAPs from the round window for pure tones between 2 and 40 kHz in ∼½ octave steps. The CAP is a marker of synchronous auditory nerve action potentials elicited by pure-tone stimuli. Auditory thresholds were assessed in a double-walled audiometric booth. Preparation of subjects for CAP assessment required nonsurvival surgical procedures performed under anesthesia (87 mg/kg ketamine and 13 mg/kg xylazine). The auditory bulla was opened via a ventrolateral approach to allow the placement of a fine (outer diameter 0.1 mm) Teflon-coated silver wire electrode (A-M Systems, Inc., Carlsborg, WA) onto the round window. A silver chloride reference electrode was inserted into neck musculature. The cochlea was warmed using a low-voltage high-intensity lamp. Tonal stimuli were generated and shaped using a SoundMax Integrated Digital Audio board. A custom program running within LabVIEW 7.1 (National Instruments) was used to control stimulus intensity, frequency, and timing. Each pure-tone stimulus consisted of a 10 ms burst with 1 ms onset and offset ramps. Tones were presented at a frequency of 9.7/s. The computer program allowed tones to be augmented in 1 dB intensity steps until a discernable CAP was identified on a digital oscilloscope by the experimenter. The CAP signals evoked by pure tones were amplified ×1000 between 0.1 and 1.0 kHz with a Grass A.C. preamplifier (Model P15, W. Warwick, RI). The sound level necessary to generate a visually detectable CAP response averaged over four sweeps on a digital oscilloscope (approximate response amplitude of 1 mV measured as the output of the preamplifier) was identified. Identification of the N1 response was based on shape of the response as well as its temporal relationship to the onset of the tonal stimulus. The CAP threshold was defined as the highest stimulus intensity at which the N1 response was no longer observed against the noise background. Statistical testing. Initial ANOVA tests were utilized to assess potential group differences in DPOAE amplitude prior to any experimental treatment. Group assignment was used as a between-subjects variable, whereas frequency served as a within-subjects variable. Subsequently, the shift in DPOAE amplitude from the subjects’ pre-exposure level was calculated, and these difference scores were subjected to a repeated measures ANOVA test where treatment served as the between-subjects variable and frequency was assessed within subjects. The range of frequencies analyzed ranged from 5.2 to 16.9 kHz as this corresponds to the frequency range that can be predicted to be susceptible to NIHL from an octave band of noise centered at 8 kHz. It also eliminates the frequency range of ∼20–25 kHz where instabilities occur in the DPOAE response due to outer ear canal resonance. Separate analyses were conducted for each type of fuel. A Greenhouse–Geiser correction was applied in all instances. Post hoc analyses were conducted using Bonferroni pairwise multiple comparisons. Subsequently, direct comparisons were made between DPOAE amplitude shifts between the JP-8 + noise and the FT + noise exposure groups relative to their baseline DPOAE levels. Results obtained with a p value < 0.05 are reported as statistically significant. The CAP data were evaluated by separate repeated measures ANOVA in which treatment served as a between-subjects variable and test frequency was assessed within subjects. RESULTS Baseline DPOAE profiles for the cubic distortion product (2f1 − f2) obtained prior to any experimental treatment are presented as Supplementary Data accompanying this manuscript for rats used in the JP-8 and FT fuel exposures, respectively. In general, the groups had quite similar DPOAE amplitudes particularly within the 1.5 octave range that was analyzed statistically. Subjects in the JP-8 1000 mg/m3 only exposure group tended to have slightly higher DPOAE amplitudes than the other treatment groups, whereas those in the JP-8 500 mg/m3 + noise group had somewhat lower initial DPOAE levels. Among rats used in the FT exposures, baseline DPOAE amplitudes were slightly elevated in rats that were in the FT 1000 mg/m3 + noise exposure group and somewhat depressed at the very highest frequencies for rats in the FT 1000 mg/m3 group. Nevertheless, repeated measures ANOVAs conducted with “treatment assignment” as the between-subjects variable failed to detect any significant differences among the treatment groups at baseline (Fs < 1.0). Moreover, because DPOAE loss was based on the shift in DPOAE amplitude within subjects, these minor deviations in DPOAE amplitude seen in rats prior to experimental treatment were not considered to seriously impede evaluation of subsequent DPOAE loss. Figure 3A compares the shift in DPOAE amplitude 4 days and 4 weeks following treatment with noise alone with that seen in the subjects exposed to no experimental treatment (Fig. 3B). It is apparent in these figures that while untreated control subjects demonstrate a very stable DPOAE response over the 4 weeks of DPOAE testing (Fig. 3B), that the noise-treated rats show a broad loss in DPOAE amplitude 4 days following treatment followed by substantial recovery of function at the 4-week test period (Fig. 3A). The loss of DPOAE amplitude in the noise-exposed rats initially approaches 20 dB of loss over a range of frequencies that exceeds the OBN (identified by shading) that constitutes their noise exposure. Four weeks after noise treatment, the extent of DPOAE impairment has shrunk to no more than 10 dB and this is clearly limited to the frequencies within the OBN. FIG. 3. Open in new tabDownload slide Comparison between shifts in DPOAE amplitude from baselines among rats exposed to noise treatment alone (A) and control subjects (B). The shaded area denotes the range of frequencies contained in the noise exposure. Following a statistically significant effect of treatment (F7/80 = 14.27, p < 0.001), Bonferroni’s multiple comparisons determined a reliable difference between the control and noise-treated rats 4 days after exposure while no significant difference was observed between groups 4 weeks after exposure. FIG. 3. Open in new tabDownload slide Comparison between shifts in DPOAE amplitude from baselines among rats exposed to noise treatment alone (A) and control subjects (B). The shaded area denotes the range of frequencies contained in the noise exposure. Following a statistically significant effect of treatment (F7/80 = 14.27, p < 0.001), Bonferroni’s multiple comparisons determined a reliable difference between the control and noise-treated rats 4 days after exposure while no significant difference was observed between groups 4 weeks after exposure. Figure 3 presents DPOAE profiles for rats that received the three doses of JP-8 fuel alone, whereas Figure 4 presents comparable data for the three doses of FT fuel alone. For the JP-8 fuel (Fig. 4), DPOAE amplitudes were remarkably stable from baseline levels for fuel exposure levels of 500 and 1000 mg/m3 remaining well within a 5-dB range of baseline. For rats exposed to 2000 mg/m3 JP-8, however, there was an initial loss in DPOAE amplitude observed at the 4-day test period for test frequencies above the frequency range that would have been predicted if these subjects had received noise exposure. This loss was essentially gone at the 4-week test period. FIG. 4. Open in new tabDownload slide Dose-response data demonstrating the effects of exposure to JP-8 at (A) 500, (B) 1000, and (C) 2000 mg/m3 for 4 h/day for 5 days on DPOAE amplitude. There were no statistically significant effects of JP-8 exposure on DPOAE relative to control subjects. FIG. 4. Open in new tabDownload slide Dose-response data demonstrating the effects of exposure to JP-8 at (A) 500, (B) 1000, and (C) 2000 mg/m3 for 4 h/day for 5 days on DPOAE amplitude. There were no statistically significant effects of JP-8 exposure on DPOAE relative to control subjects. Comparable data collected for rats exposed to FT fuel exposure (Fig. 5) are considerably more variable. Here, it appears that rats exposed to 500 mg/m3 FT exposure show a loss in DPOAE amplitude at the 4-day postexposure test with recovery at the 4-week time point. However, there is no indication of a dose-response relationship. DPOAE amplitudes were very stable for rats exposed to FT at 1000 mg/m3 and were somewhat depressed for the rats at the 2000 mg/m3 exposure level. The loss of DPOAE amplitude in the range of 20 kHz is difficult to evaluate because these DPOAE responses generated in this frequency region are distorted by the normal resonance characteristics of the ear canal such that a noticeable trough is present even in control subjects. FIG. 5. Open in new tabDownload slide Dose-response data demonstrating the effects of exposure to FT fuel at (A) 500, (B) 1000, and (C) 2000 mg/m3 for 4 h/day for 5 days on DPOAE amplitude. There were no statistically significant effects of FT fuel exposure on DPOAE relative to control subjects. FIG. 5. Open in new tabDownload slide Dose-response data demonstrating the effects of exposure to FT fuel at (A) 500, (B) 1000, and (C) 2000 mg/m3 for 4 h/day for 5 days on DPOAE amplitude. There were no statistically significant effects of FT fuel exposure on DPOAE relative to control subjects. Finally, Figures 6 and 7 portray the effects of combined exposure to JP-8 + noise (Fig. 6) and of FT fuel + noise (Fig. 7) on DPOAE amplitudes between baseline values and 4 days and 4 weeks following exposure for all dosage levels. FIG. 6. Open in new tabDownload slide Dose-response data demonstrating the effects of combined exposure to JP-8 at (A) 500, (B) 1000, and (C) 2000 mg/m3 for 4 h/day and to noise for 1 h/day for 5 days on DPOAE amplitude. There were no statistically significant differences among groups. Following a statistically significant effect of treatment (F7/80 = 14.27, p < 0.001), Bonferroni’s multiple comparisons determined a reliable difference between the control group and groups treated with noise alone, JP-8 1000 mg/m3 + noise, and JP-8 2000 mg/m3 + noise. Moreover, the JP-8 2000 mg/m3 + noise group showed significantly lower DPOAE amplitudes than did noise subjects (Fig. 3). At 4 weeks after exposure, only the JP-8 1000 mg/m3 + noise and JP-8 2000 mg/m3 + noise groups differed significantly from control levels. The shaded area denotes the range of frequencies contained in the noise exposure. FIG. 6. Open in new tabDownload slide Dose-response data demonstrating the effects of combined exposure to JP-8 at (A) 500, (B) 1000, and (C) 2000 mg/m3 for 4 h/day and to noise for 1 h/day for 5 days on DPOAE amplitude. There were no statistically significant differences among groups. Following a statistically significant effect of treatment (F7/80 = 14.27, p < 0.001), Bonferroni’s multiple comparisons determined a reliable difference between the control group and groups treated with noise alone, JP-8 1000 mg/m3 + noise, and JP-8 2000 mg/m3 + noise. Moreover, the JP-8 2000 mg/m3 + noise group showed significantly lower DPOAE amplitudes than did noise subjects (Fig. 3). At 4 weeks after exposure, only the JP-8 1000 mg/m3 + noise and JP-8 2000 mg/m3 + noise groups differed significantly from control levels. The shaded area denotes the range of frequencies contained in the noise exposure. FIG. 7. Open in new tabDownload slide Dose-response data demonstrating the effects of combined exposure to FT fuel at (A) 500, (B) 1000, and (C) 2000 mg/m3 for 4 h/day and to noise for 1 h/day for 5 days on DPOAE amplitude. Following a statistically significant effect of treatment (F7/79 = 7.15, p < 0.001), Bonferroni’s multiple comparisons determined a reliable difference between the control group and groups treated with noise and with FT 500 mg/m3 + noise. Four days after exposure with the noise-only group showing the larger shift. Four weeks after exposure, there were no differences between noise-treated and control rats and only a significant effect of FT 1000 mg/m3 alone relative to noise-only subjects. The shaded area denotes the range of frequencies contained in the noise exposure. FIG. 7. Open in new tabDownload slide Dose-response data demonstrating the effects of combined exposure to FT fuel at (A) 500, (B) 1000, and (C) 2000 mg/m3 for 4 h/day and to noise for 1 h/day for 5 days on DPOAE amplitude. Following a statistically significant effect of treatment (F7/79 = 7.15, p < 0.001), Bonferroni’s multiple comparisons determined a reliable difference between the control group and groups treated with noise and with FT 500 mg/m3 + noise. Four days after exposure with the noise-only group showing the larger shift. Four weeks after exposure, there were no differences between noise-treated and control rats and only a significant effect of FT 1000 mg/m3 alone relative to noise-only subjects. The shaded area denotes the range of frequencies contained in the noise exposure. Figure 6 portrays a very clear dose-related impairment of the DPOAE response as JP-8 levels increase from 500 to 1000 and 2000 mg/m3. Notably, all these subjects received the same noise exposure. For rats exposed to JP-8 at 500 mg/m3 + noise, a small loss in DPOAE amplitude can be observed. However, this loss is actually smaller than that produced by noise exposure alone (Fig. 3A). At both JP-8 levels of 1000 and 2000 mg/m3, however, a statistically significant reduction (see below) in DPOAE amplitude is observed 4 days after treatment than was seen with noise alone (Fig. 3A) and the recovery of function that occurs 4 weeks following treatment is also more limited than that seen in the rats exposed to noise alone. DPOAE loss approaches 30 dB for rats exposed either to 1000 or to 2000 mg/m3 JP-8 exposure + noise 4 days after exposure. DPOAE values for the rats exposed to 1000 and 2000 mg/m3 JP-8 + noise are still depressed 20–25 dB at 4 weeks after exposure. At the latter time point, noise-exposed rats had only a limited 10-dB loss of DPOAE amplitude within a single noise octave. For rats exposed to FT fuel + noise (Fig. 7), a different picture emerges. For rats exposed to FT 500 mg/m3 + noise, DPOAE levels were depressed at the initial postexposure measurement to levels no different from those observed for rats exposed to noise alone. These same FT + noise subjects showed full recovery of the DPOAE response 4 weeks after treatment. Similarly, the rats treated with FT 1000 mg/m3 + noise showed no loss in DPOAE amplitude at either postexposure test period and thereby appeared to show better acoustic function than did noise only–exposed rats. Finally, rats exposed to FT 2000 mg/m3 + noise showed a loss in DPOAE amplitude at both the 4-day and 4-week test periods that were equivalent to the loss produced by noise alone. In contrast to the effects of fuel and noise exposure on the functional ability of OHCs reflected in the DPOAE assessment, the measure of auditory threshold sensitivity did not differentiate among the treatment groups for either fuel at any exposure level. This is exemplified for the highest fuel exposure levels in Figures 8A and 8B. Although control subjects generally showed the most sensitive pure-tone audiograms and although noise exposure alone tended to increase the auditory thresholds, there was no evidence that exposure to JP-8 jet fuel (Fig. 8A) or to FT jet fuel (Fig. 8B) produced additional impairment of this measure. FIG. 8. Open in new tabDownload slide Auditory thresholds assessed using the compound action potential in rats exposed to (A) JP-8 fuel + noise, noise alone, or untreated controls or (B) FT fuel + noise, noise alone, or untreated controls There were no statistically significant differences among groups. FIG. 8. Open in new tabDownload slide Auditory thresholds assessed using the compound action potential in rats exposed to (A) JP-8 fuel + noise, noise alone, or untreated controls or (B) FT fuel + noise, noise alone, or untreated controls There were no statistically significant differences among groups. Statistical conclusions JP-8 fuel. Based on significant overall ANOVAs, the effects of JP-8 and noise were evaluated both at 4 days after exposure and at 4 weeks after exposure using two-way ANOVAs comparing the shift in DPOAE amplitudes postexposure by treatment group (between subjects) and frequency (within subjects). Four days following JP-8 exposure, a repeated measures ANOVA showed a significant effect of treatment (F7/17 = 14.27, p < 0.0001) and of frequency (F17/1360 = 18.79, p < 0.0001). Bonferroni’s multiple comparisons test demonstrated that the noise alone, JP-8 1000 mg/m3 + noise, and JP-8 2000 mg/m3 + noise groups all differed significantly from the control group. In addition, rats exposed to JP-8 2000 mg/m3 + noise showed significantly greater loss in DPOAE amplitude than did rats exposed to noise alone. Direct comparisons between the shifts in DPOAE amplitude resulting from JP-8 + noise exposure and FT + noise exposure were also undertaken. Post hoc analyses using Bonferroni multiple comparisons test showed that the loss in DPOAE was significantly greater in the JP-8 1000 + noise and the JP-8 2000+ noise groups than it was in any of the FT + noise groups. No significant difference in DPOAE disruption was observed between the JP-8 500 + noise group and any of the FT + noise groups. Four weeks after exposure, the JP-8 1000 mg/m3 + noise and JP-8 2000 mg/m3 + noise groups still showed significantly greater loss of DPOAE amplitude than did the control group, whereas the noise-exposed subjects were no longer significantly different from controls. However, the difference between noise alone–treated subjects and those exposed to either JP-8 1000 mg/m3 + noise or JP-8 mg/m3 + noise was no longer sufficiently robust to reach statistical significance. FT fuel. At 4 days following the end of exposure, significant effects of treatment (F7/79 =7.15, p < 0.0001) and of frequency (F17/1343 = 17.43, p < 0.0001) were found. Among treatment groups, both noise-alone and FT 500 mg/m3 + noise groups showed significantly greater declines in DPOAE amplitude from baseline than did control subjects. And subjects exposed only to noise showed the greatest loss in DPOAE amplitude. There were no differences observed between rats exposed to noise alone and those exposed to FT fuel + noise. Four weeks following exposure, the main effects of treatment (F7/78 = 2.69, p < 0.05) and frequency (F17/1326 = 10.47, p < 0.0001) were still significant. However, at this time point, Bonferroni’s comparisons showed that noise alone was no longer significantly different from control subjects and that the only treatment groups that did differ from each other were the noise alone and FT 1000 mg/m3. As was the case at the 4-day postexposure test, direct comparisons between the shifts in DPOAE amplitude resulting from JP-8 + noise exposure and FT + noise exposure were also undertaken. Post hoc analyses using Bonferroni multiple comparisons test showed that the loss in DPOAE was significantly greater in the JP-8 1000 + noise and the JP-8 2000+ noise groups than it was in any of the FT + noise groups. No significant difference in DPOAE disruption was observed between the JP-8 500 + noise group and any of the FT + noise groups. DISCUSSION This experiment has focused on the vulnerability of auditory function to exposure from two different jet fuels with and without subsequent noise exposure. The results demonstrate that neither JP-8 nor FT fuel by itself is able to permanently disrupt OHC function as reflected in the DPOAE even at doses roughly three times the permissible human exposure level on a TWA basis. The data do suggest that 2000 mg/m3 JP-8 alone under the exposure conditions used here might produce a transient impairment of OHC function observed at least 4 days following exposure. However, this conclusion would be strengthened by additional studies using exposure concentrations of JP-8 greater than 2000 mg/m3. By contrast, when noise exposure was combined with the JP-8 jet fuel using a successive exposure schedule, rats that received either 1000 or 2000 mg/m3 of that fuel showed greater impairment of the DPOAE response 4 days after exposure than did the rats exposed only to noise. And even 4 weeks after exposure, these two highest JP-8 + noise groups were significantly inferior to the control subjects, whereas the noise-only subjects had substantially recovered to their baseline DPOAE amplitude. There was no evidence that exposure to 500 mg/m3 JP-8 could increase sensitivity to noise exposure under the conditions used here, thus establishing a no-effect level. This finding that JP-8 + noise exposure significantly reduced the DPOAE response relative to the subjects exposed to noise alone replicates the results of a similar study conducted in this laboratory using 1000 mg/m3 JP-8 and a higher noise exposure level (105 dB) (Fechter et al., 2007). Not withstanding the impairment in DPOAE amplitude among the rats exposed to JP-8 fuel followed by noise exposure, there was no shift in pure-tone auditory thresholds among these subjects as measured by the tone intensity necessary to elicit a CAP response. This finding extends the results of an earlier study in which combined exposure to JP-8 + noise produced more robust impairment of DPOAE levels than it did on the auditory threshold response measured by impairment of CAP threshold (Fechter et al., 2007). That JP-8 + noise exposure yielded impairment of the DPOAE response, but not auditory threshold requires further discussion. These two measures of auditory function are measuring somewhat different physiological processes. The DPOAE response arises primarily from the motile behavior of the OHCs in response to sound and is highly dependent on intact stereocilia on these accessory sensory cells. By contrast, the CAP represents the final output from the cochlea to central auditory pathways and, as such, is dependent on multiple cell types, including inner hair cells, the actual sense receptors for sound, and the spiral ganglion cells on which they synapse, as well as the OHCs. The finding of differential outcomes on the DPOAE and CAP measures is not at all unusual. Verpy et al. (2008) recently demonstrated in mice deficient in sterocilin, a key protein in the stereocilia, that the DPOAE response could be eliminated while a normal CAP could be recorded. And Guthrie (2008) reviews data demonstrating loss of DPOAE input/output functions in children treated with aminoglycoside antibiotics when auditory thresholds are still reported to be normal. That the DPOAE response alone was impaired suggests a less serious cochlear impairment than one that also disrupted the CAP. Nevertheless, the DPOAE disruption observed following the higher two doses of JP-8 + noise does appear to be a permanent effect in as much as it was still apparent 4 weeks following the end of experimental exposure. In contrast to the enhanced sensitivity to noise produced by JP-8, the rats that received the FT fuel did not show a dose-dependent enhancement in NIHL. Indeed, in isolated cases, it appears as if the loss of DPOAE amplitude is actually smaller among rats receiving the low and middle exposure levels of FT fuel followed by noise than it is in the noise-treated group alone. It is uncertain whether these differences represent a true protection against NIHL (hormesis) or a chance difference among individual subjects. It is difficult to speculate upon a biological mechanism by which FT fuel might convey a protective effect relative to noise. This issue should be elucidated by future studies which might be able to replicate the effect. The basis for the difference between the effect of the two jet fuels on NIHL was not tested directly, but a leading candidate is the absence of ototoxic aromatic hydrocarbons from the FT fuel. In other respects, the JP-8 and FT fuels used in these studies show rather similar chemical profiles. OHCs are also known to be selectively or preferentially sensitive to a number of aromatic hydrocarbons including specific aromatic hydrocarbons, such as toluene, ethylbenzene, p-xylene and also to styrene. Gagnaire and Langlais (2005) have attempted to identify chemical structures that can account for this ototoxicity. The finding that JP-8 but not the FT fuel was able to enhance the disruptive effects of noise on hearing is also an important finding that might relate to the absence of aromatic hydrocarbons in the FT fuel. Whatever its cause, however, it does suggest that this synthetic fuel has less ototoxic potential than JP-8. FUNDING This material is based on work supported in part by the Office of Research and Development Rehabilitation Research and Development Service Department of Veterans Affairs by Merit Review (C3575R and 6006) and by a Senior Career Research Scientist award (C4613L). Additional support was received from the American Petroleum Institute. 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TI - Ototoxic Potential of JP-8 and a Fischer-Tropsch Synthetic Jet Fuel following Subacute Inhalation Exposure in Rats
JF - Toxicological Sciences
DO - 10.1093/toxsci/kfq110
DA - 2010-07-01
UR - https://www.deepdyve.com/lp/oxford-university-press/ototoxic-potential-of-jp-8-and-a-fischer-tropsch-synthetic-jet-fuel-RJCQs8V0GM
SP - 239
EP - 248
VL - 116
IS - 1
DP - DeepDyve
ER -