ToxSci Advance Access originally published online on May 4, 2007
Toxicological Sciences 2007 98(2):510-525; doi:10.1093/toxsci/kfm101
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Published by Oxford University Press 2007.
JP-8 Jet Fuel Can Promote Auditory Impairment Resulting From Subsequent Noise Exposure in Rats
* Loma Linda VA Medical Center, Loma Linda, California 92357
The University of Georgia, Georgia 30602
CE-CERT, University of California, Riverside, California 92507
Loma Linda University School of Medicine, California 92350
1 To whom correspondence should be addressed at Research Service (151), Loma Linda VA Medical Center, 11201 Benton Street, Loma Linda, CA 92357. Fax: (909) 796-4508. E-mail: Larry.fechter{at}med.va.gov
Received February 28, 2007; accepted April 20, 2007
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODS AND MATERIALSRESULTSDISCUSSIONREFERENCES |
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We report on the transient and persistent effects of JP-8 jet fuel exposure on auditory function in rats. JP-8 has become the standard jet fuel utilized in the United States and North Atlantic Treaty Organization countries for military use and it is closely related to Jet A fuel, which is used in U.S. domestic aviation. Rats received JP-8 fuel (1000 mg/m3) by nose-only inhalation for 4 h and half of them were immediately subjected to an octave band of noise ranging between 97 and 105 dB in different experiments. The noise by itself produces a small, but permanent auditory impairment. The current permissible exposure level for JP-8 is 350 mg/m3. Additionally, a positive control group received only noise exposure, and a fourth group consisted of untreated control subjects. Exposures occurred either on 1 day or repeatedly on 5 successive days. Impairments in auditory function were assessed using distortion product otoacoustic emissions and compound action potential testing. In other rats, tissues were harvested following JP-8 exposure for assessment of hydrocarbon levels or glutathione (GSH) levels. A single JP-8 exposure by itself at 1000 mg/m3 did not disrupt auditory function. However, exposure to JP-8 and noise produced an additive disruption in outer hair cell function. Repeated 5-day JP-8 exposure at 1000 mg/m3 for 4 h produced impairment of outer hair cell function that was most evident at the first postexposure assessment time. Partial though not complete recovery was observed over a 4-week postexposure period. The adverse effects of repeated JP-8 exposures on auditory function were inconsistent, but combined treatment with JP-8 + noise yielded greater impairment of auditory function, and hair cell loss than did noise by itself. Qualitative comparison of outer hair cell loss suggests an increase in outer hair cell death among rats treated with JP-8 + noise for 5 days as compared to noise alone. In most instances, hydrocarbon constituents of the fuel were largely eliminated in all tissues by 1-h postexposure with the exception of fat. Finally, JP-8 exposure did result in a significant depletion of total GSH that was observable in liver with a nonsignificant trend toward depletion in the brain and lung raising the possibility that the promotion of noise-induced hearing loss by JP-8 might have resulted from oxidative stress.
Key Words: JP-8; jet fuel; auditory function; ototoxicity; inhalation exposure; rats.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODS AND MATERIALSRESULTSDISCUSSIONREFERENCES |
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A wealth of laboratory animal experiments along with industrial epidemiological studies have identified several chemical contaminants that can impair auditory function directly or potentiate the damaging effects of noise on the auditory system. Among such ototoxic agents are a variety of aromatic hydrocarbons including toluene (Crofton, 1994; Loquet, 1999; Pryor et al., 1983a
,b), styrene (Crofton et al., 1994; Lataye et al., 2001; Loquet et al., 1999; Pryor et al., 1987), ethyl benzene (Cappaert et al., 1999, 2000, 2001a, 2002), and mixed xylenes containing p-xylene (Cappaert et al., 2001b; Crofton et al., 1994; Pryor et al., 1987). Exposure to these agents over a period generally of 4–16 weeks yields a persistent auditory impairment that is particularly severe at mid-frequencies (c.f. Crofton et al., 1994). This loss is accompanied by damage to outer hair cells predominantly in regions of the cochlea corresponding to mid-frequency loss (Campo et al., 1997; Crofton et al., 1994; Loquet et al., 1999). Epidemiological research from workers often exposed to multiple solvents has associated auditory impairment with workplace exposures to toluene (e.g., Morata et al., 1997), styrene (Muijser et al., 1988; Sliwinska-Kowalska et al., 2003), and xylenes (Sliwinska-Kowalska et al., 2001).
A significant difficulty in assessing the risk of hearing loss from solvent exposure, however, is that aromatic hydrocarbons as a class do not uniformly disrupt hearing, but, rather, demonstrate agent-specific effects. For example, p-xylene is ototoxic and yields middle frequency hearing loss, while neither m-xylene or o-xylene disrupts auditory function or structure (Cappaert et al., 2001b; Gagnaire et al., 2001; Maguin et al., 2006). Similarly, ethyl benzene, toluene, and styrene are ototoxic while benzene does not share this ototoxic effect (Gagnaire and Langlais, 2005). In light of such disparate results among structurally similar chemicals, researchers have attempted to identify a specific mechanism of potential toxicity shared by the ototoxicants, but not by the nonototoxic agents. Disruption of intracellular calcium homeostasis (Liu and Fechter, 1997), disruption of membrane fluidity (Campo et al., 2001; Liu et al., 1997), free radical generation and oxidative stress (Fechter, 1999; Rao and Fechter, 2000), and disruption of efferent pathways synapsing at the cochlea (Lataye and Campo, personal communication) have been among the hypotheses advanced for how chemical contaminants can disrupt hearing. However, no such unifying concept has garnered convincing supporting evidence or is adequate to predict which hydrocarbons are ototoxicants and which are not. Consequently, it is important to test additional agents for potential ototoxicity in the hope of both enhancing risk assessment for solvent ototoxicity and, potentially, of finding a shared mechanism by adding compounds to the categories of ototoxic and nonototoxic compounds.
In the absence of a clear understanding of the mechanisms of ototoxicity, there remain several rational bases for choosing test compounds. First, given that many hydrocarbons can promote noise-induced hearing loss (NIHL) yielding additive effects, it is appropriate to test compounds that workers commonly experience in noisy environments. The existence of a human population exposed to such compounds with and without noise exposure provides an appropriate group to which research findings may be applied and for which epidemiological data can be collected. Secondly, it appears rational to select agents that share with noise a potential common pathway leading to injury. Third, given that the exposure of workers to hydrocarbons is frequently to complex mixtures, it is appropriate to determine whether or not there is an additivity of effect among mixture constituents even though the concentrations of individual known ototoxicant components are small. Therefore, in this manuscript, we report on the transient and persistent effects of JP-8 jet fuel exposure with and without subsequent noise exposure on auditory function in rats.
JP-8 has become the common jet fuel utilized in the United States and North Atlantic Treaty Organization (NATO) countries for military use and it is closely related to Jet A fuel which is used in U.S. domestic aviation. Thus, there is a large population potentially exposed to this agent. With an estimated 5 billion gallons of JP-8 jet fuel used by the U.S. military and NATO each year, JP-8 jet fuel is the chemical agent having the broadest range of exposure among service personnel (National Research Council of the National Academies, 2003). JP-8 is used, not only in aircraft, but also as a heat source, as an obscurant for military vehicles, as a degreaser, and even to reduce blowing sand in desert settings (National Research Council of the National Academies, 2003). According to a recent National Research Council of the National Academies (2003) report, the pattern and extent of usage make JP-8 "...probably responsible for the most common and abundant potential chemical exposure of DoD and NATO personnel." Clearly, jet fuel exposure is commonly associated with potentially significant noise exposure.
There is, in fact, limited epidemiological evidence that jet fuel exposure among airforce personnel may increase the odds ratio for hearing loss in workers exposed for 3–12 years. Kaufman et al. (2005) reported on pure-tone threshold impairment among a relatively small sample of aircraft maintenance and similar personnel with mixed exposure to both JP-4 and JP-8 + noise relative to workers exposed to noise alone using a cross-sectional retrospective design. They found an increased relative risk of hearing loss among workers exposed to jet fuel + noise for 3–12 years as compared to individuals exposed to comparable noise without fuel exposure. Duration of JP-4 exposure rather than estimated concentration appeared to be a significant predictor of effect. However, the population tested was small and the jet fuel exposure was predominantly to JP-4 as JP-8 was introduced rather late relative to the exposure duration considered in this research. Thus, there is a need for systematic study of JP-8 alone under controlled exposure conditions.
Beyond the suggestive epidemiological data, the potential for significant public health relevance, and the known ototoxic constituents of JP-8, there is yet another reason for studying whether this fuel might interact with noise in producing hearing loss. Boulares et al. (2002) suggest that JP-8 might exert at least some of its toxicity by interfering with antioxidant potential at the cellular level. These authors showed that JP-8 (80 µg/ml) produced apoptotic changes in RLE-6TN cells obtained from rat lung epithelium after 12 h of exposure. This effect was enhanced by pretreatment with buthionine sulfoximine and hydrogen peroxide. Both of these treatments would be expected to enhance the risk of oxidative stress. They also demonstrated that JP-8 actually reduced cellular glutathione (GSH) levels by approximately 40% after a 1-h exposure. The damaging effects of JP-8 on epithelial cells could be reduced or inhibited by treatment with exogenous GSH, and by treatment with the antioxidant drug, L-N-acetylcysteine. The tripeptide GSH serves a critical protective role against oxidative stress and its importance in protecting the cochlea both from ototoxic drugs and from noise has received significant attention in recent years. Given that NIHL has been associated with oxidative stress (Henderson et al., 1999; Pourbakht and Yamasoba, 2003; Ohinata et al., 2000; Ohlemiller et al., 1999, 2000; Seidman et al., 1993; Yamane et al., 1995; Yamasoba et al., 1999), a depletion in GSH, if it were to occur in vivo, would offer a mechanism by which this jet fuel might potentiate NIHL.
Finally, it might be predicted that JP-8 would have considerable ototoxic potential since it contains several ototoxic components of JP-8, among which are toluene, ethyl benzene, and p-xylene. However, each of these aromatic hydrocarbons makes up only a small proportion of this fuel and the anticipated concentration of any of these agents would be quite small relative to the known ototoxicity of each of these constituents individually.
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METHODS AND MATERIALS |
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TOPABSTRACTINTRODUCTIONMETHODS AND MATERIALSRESULTSDISCUSSIONREFERENCES |
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Subjects
A total of 98 pigmented male Long–Evans rats (250–300 g) obtained from Harlan (Indianapolis, IN) were employed in these studies. Of these, 84 rats were used for auditory function studies (n = 6–9) and 14 were used to generate toxicokinetic and biochemical data. The subjects were housed with free access to food and water in their home cages. Temperature was maintained at 21 ± 1°C and 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.
Exposure Procedures
Three separate experiments were conducted to investigate the effects of JP-8 exposure with and without subsequent noise exposure on auditory function in rats. Additional studies focused entirely on JP-8 kinetics and on the effects of JP-8 exposure on GSH levels. A diagram of experimental exposure protocols is provided in Figure 1. Of the three auditory experiments, the first one entailed a single 4-h exposure to 1000 mg/m3 JP-8 followed, in some subjects, by a 4-h octave band of noise (OBN) exposure (105 dB). The second experiment utilized a repeated exposure design in which the rats were treated daily for 5 days with 1000 mg/m3 JP-8 for 4 h per day followed by a 4-h octave band noise exposure centered at 8 kHz at an intensity of 97 dB sound pressure level (SPL). The third experiment also used repeated exposure to JP-8 using the same parameters as in study 2. However, the noise exposure duration was reduced to 1 h and the noise exposure was increased to 102 dB. The reduction in noise duration was designed to match the noise exposure more closely to the residence time of the JP-8 components in biological tissue. The increase in noise intensity was designed to yield a limited auditory impairment among the rats receiving only noise for such a short duration.
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The dose of JP-8 used in all three experiments (1000 mg/m3 for 4 h corresponding to a time-weighted average [TWA] of 500 mg/m3) was selected because it represents a dose just slightly above the current human permissible exposure level (PEL) (an 8 h TWA of 350 mg/m3) and because this exposure level has been used in other published experimental research on health effects of JP-8 (e.g., Drake et al., 2003; Witzmann et al., 1999, 2000). 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). Current OSHA standards have established a PEL for noise of 90 dB using the A weighting scale for an 8 h TWA. The A weighting scale provides an average noise exposure level that reflects the sensitivity of the human audiogram to different sound frequencies. Sound levels that are measured using this scale are expressed as dB (A). The equivalent exposure for a 4-h time period would be 95 dB (A) and for 1 h 105 dB (A). The current Occupational Safety and Health Administration (OSHA) action level for noise, however, is 85 dB (A). Additional rats were used to provide tissues for monitoring biomarkers of exposure (n = 11) and for determining total GSH levels (n = 3). These rats received 4 h exposure to 1000 mg/m3 JP-8, but were not noise exposed. The rats in this study were euthanized either immediately following exposure or after a 1- or 3-h recovery period. These studies are described more fully below.
JP-8 Exposure
The rats were exposed to JP-8 using a nose-only exposure system (CH Technologies, Westwood, NJ), which was designed to minimize the potential for fuel deposition on the subjects' fur and subsequent ingestion during grooming. The rats were habituated for at least 1 day 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. The fuel was kindly supplied from a stock maintained by the fuels branch at the Wright-Patterson Airforce 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. Once the standard fuel arrived in this laboratory, it was stored in sealed 1-l opaque bottles at 4°C to minimize evaporation. The JP-8 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 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 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 constantly during the exposure using a Ratfisch (Poing, Germany) Model RS-55CA total hydrocarbon analyzer.
Particle size distributions were obtained with a MSP Corporation micro-orifice uniform deposit impactor (MOUDI, Model 100, Shoreview, MN) and a scanning electrical mobility spectrometer (SEMS) (Wang and Flagan, 1989). The SEMS was comprised of a TSI 3077 85Kr neutralizer (St Paul, MN), a TSI 3081 long column cylindrical differential mobility analyzer (DMA), and a TSI 3760A condensation particle counter. It was operated at sheath and excess flow rates of 2.5 l/min, aerosol and monodisperse flow rate of 0.25 l/min. Voltage was exponentially ramped from –40 to –7000 V with a 60-s scan time to obtain electrical mobility diameter measurements ranging from 28 to 700 nm. Size distributions were obtained using the data inversion algorithm of Collins et al. (2002). The MOUDI was operated at 30 l/min with aerosols collected in 10 size fractions between 0.056 and 18 µm onto pretared and conditioned aluminum substrates
Detailed chemical composition of the aerosolized fuel was also conducted. Gaseous hydrocarbons with 12 carbon or less were also analyzed qualitatively and quantitatively using a Hewlett-Packard (Palo Alto, CA) 5890 Series II Plus gas chromatograph (GC) utilizing a DB-5 60-m column (J&W Scientific, Davis, CA) and a flame ionization detector following the SAE 930142HP Method-1 and Method-2 specifications for C1–C4 and C4–C12 analyses, respectively. Larger hydrocarbons including polyaromatic hydrocarbons were captured on quartz and polyurethane foam substrates and subsequently analyzed following a modified EPA-TO13A protocol on an Agilent (Palo Alto, CA) 6890 Gas Chrmoatograph/5973 Mass Spectrometer in electrical ionization mode following a modified EPA-TO13A method described in Shah et al. (2005).
Characterization of Exposure
Specific JP-8 components were quantified in tissues from a separate group of subjects not used for auditory testing. They were euthanized at one of several survival times following a single 4 h nose-only exposure to JP-8 (1000 mg/m3). Subjects were euthanized immediately after the exposure (0 h), 1- and 3-h postexposure. The tissues chosen, lung, blood, liver, fat, and brain, were selected in order to estimate exposure in the tissue representing the portal of entry (lung), the route of systemic exposure (blood), the predominant organ of metabolism (liver), a primary reservoir for the hydrocarbons (fat), and an organ of interest in terms of target of toxicity (brain). While the cochlea would have been a more relevant organ of potential toxicity, early pilot studies showed that the hydrocarbon levels obtained in this small tissue sample were below the level of detection. Data obtained with respect to time during which appreciable fuel could be found in the subjects' organs were subsequently used to establish the duration of noise exposure used in the last experiment reported in this manuscript. The rats were exposed to 1000 mg/m3 JP-8 fuel for 4 h and were euthanized immediately, 1- and 3-h postexposure under ketamine (87 mg/kg) and xylazine (13 mg/kg) anesthesia. First a blood sample was obtained via a cardiac puncture. The rats were then decapitated and samples of brain, lung, liver, and perirenal fat were harvested. The tissues were immediately placed into 4-ml glass autosampler vials with Teflon caps (National Scientific, Rockwood, TN) which were tightly sealed and stored at –20°C. The samples were shipped on ice to the University of Georgia where the analytical work was performed. Preliminary studies of sample stability have been carried out using tissues spiked with standards at the time of euthanasia and with variable delays that would occur if a sample remained on the autosampler. The recovery of hydrocarbons in these studies demonstrates that such samples were stable over a 40-h time period. The procedure used for assessing tissue JP-8 components is fully described in Campbell and Fisher (2007). Briefly, a headspace solid phase microextraction–GC/MS (SPME-GC/MS) method was developed to measure low levels (e.g., low ng/ml) of individual hydrocarbons in biological tissues that were not detectable by liquid extraction based on preliminary experiments. A weighed amount of tissue (1.0 ml of heparinized blood or 0.5 g tissue) was placed in a 10-ml screw cap headspace vial (Supelco, Inc., Bellefone, PA). After the addition of 3 ml of 33% salt water (0.33 g/ml NaCl in deionized water), the tissue was quickly chopped with iris scissors, 1 µl of internal standard solution (150 µg/ml d26-dodecane) added, the vials were then capped, vortexed, and placed on the autosampler for extraction and analysis. Spiked tissue served as standards for creating calibrations. Tissue standards were prepared to eliminate differences in partitioning between tissue, salt water, and air during analysis. Unexposed tissue was prepared in the same manner as described for exposed samples and subsequently spiked with 1 µl of a surrogate mixture containing the 23 components listed in Tables 2–3 including the internal standard. The SPME-GC/MS system consisted of a CombiPAL autosampler with SPME adaptor (CTC Analytics, Zwingen, Switzerland) and a CP-3800 GC (Varian, Inc., Walnut Creek, CA) with a Saturn 2200 Ion Trap MS operated in scan mode. The ion trap parameters were set to the optimized values for hydrocarbons previously determined by Joos et al. (2003). Quantitative analysis was performed with the Saturn GC/MS Workstation (Version 6.40, Varian, Inc.). The standard curves ranged from 3.91 to 500 ng/ml for blood and 7.8 to 1000 ng/g for liver. Standard curves were prepared each time samples were analyzed.
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Noise Exposure
Exposures to noise were conducted immediately following the JP-8 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. Air exchange rate within the chamber was 12.5 lpm (providing approximately 17 changes per hour) with airflow being monitored by a Dwyer Instruments (Michigan City, IN) flow gauge. The subjects were placed within small wire-cloth enclosures (15 x 13 x 11 cm) within the chamber. They were conscious and free to move within the enclosures. Broadband noise was generated by a function-generator (Stanford Research System, Model DS335, 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 SAE 2200 Power Amplifier (Scientific Audio Electronics Inc., Los Angeles, CA) and fed to speakers (Vifa D25AG-05, Videbaek, Denmark) located approximately 5 cm above the subjects' wire-cloth enclosure. The sound intensity measured at the level of the rats' pinnae was 97, 102, or 105 dBlin SPL in the octave band centered at 8 kHz depending upon the specific experiment. 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 the same between 6.3 and 10 kHz. The levels were approximately 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 noise level in the fuel exposure chamber was assessed when no additional sound was applied. Noise levels during JP-8 exposure were below 60 dB SPL at all sound frequencies.
Auditory Assessment
Hair cell functional assessment: distortion product otoacoustic emissions.
Hair cell function was assessed by means of a noninvasive test method known as the distortion product otoacoustic emissions (DPOAE) test. This method permits repeated testing within subjects and, thereby, can trace impairment that may occur between a preexposure baseline and various time points following an exposure. In this way, both transient and permanent impairments 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 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 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, as a function of length along the basilar membrane. In these experiments, the ratio of f1/f2 was maintained at 1.25 in order to yield maximal distortion products. The f2 frequency was swept from 3.2 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, where they acoustically mixed to avoid artifactual distortion. Ear-canal sound pressure levels, measured by an emissions microphone assembly (Etymotic Research, ER-10B+, Elk Grove Village, IL) embedded in the probe, were sampled, synchronously averaged, and Fourier analyzed for geometric mean frequencies [(f1 x f2)0.5] ranging from 5.6 to 19.7 kHz (i.e., f2 = 6.3–22.5 kHz) by a computer-based DSP board. Corresponding noise floors were computed by averaging the levels of the ear-canal sound pressure for five frequency bins above and below the DPOAE frequency bin (± 54 Hz). For test frequencies above 20.1 kHz, a computer-controlled dynamic-signal analyzer (Hewlett-Packard Model 3561A) was used. 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. No artifact DPOAEs were ever measured in this test cavity. 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 while rats were lightly anaesthetized with ketamine (44 mg/kg) and xylazine (7 mg/kg). Normal body temperature was maintained using a dc heating unit built into the surgical table. 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 3 days and/or 1 week after the end of the experimental treatment, and again 4 weeks postexposure. Each DPOAE test required approximately 6 min to perform.
Audiometric threshold assessment: compound action potential.
Threshold assessment was performed 4 weeks following the end of experimental exposures by recording compound action potentials (CAPs) from the round window for pure tones between 2 and 40 kHz in approximately 
octave steps. Auditory thresholds were assessed in a double walled audiometric booth. The subjects were anaesthetized with xylazine (13 mg/kg, im) and ketamine (87 mg/kg, im) and normal body temperature was maintained using a dc heating unit built into the surgical table. The auditory bulla was opened via a ventrolateral approach to allow the placement of a fine (od 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. The CAP signals evoked by pure tones were amplified x1000 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.
Histological assessment.
Immediately after CAP measurements, rats were decapitated and the cochleae harvested. Within 2 min, the cochleae were fixed by perilymphatic perfusion with 1 ml of a trialdehyde fixative (3% glutaraldehyde, 2% formaldehyde, 1% acrolein, and 2.5% dimethyl sulfoxide in phosphate buffered saline pH 7.4). Following the primary 24-h fixation, the tissue was first washed with 0.1M phosphate buffered saline, postfixed with 2% OsO4 in water for 2 h, and finally washed again with 0.1M phosphate buffered saline. The organ of Corti was dissected in 70% ethanol and mounted in glycerin for counting of hair cells. Cells were counted as present either when the stereocilia, the cuticular plate or the cell nucleus could be visualized. No attempt was made to assess the degree of possible cellular damage to surviving cells. The frequency-place map established by Muller (1991) was used to superimpose the frequency coordinates on the length coordinates of the organ of Corti. This "map" reflects the fact that the cochlea is organized tonotopically with high frequency sound producing maximum stimulation of cells in the base, and low frequency sound in the apex. A cochleogram showing the percentage of hair cell loss as a function of distance from the base of the cochlea was plotted for each animal. The results were averaged across each group of subjects for comparison between groups. The software used for counting cochlear hair cells was developed by R. Lataye and Dr. P. Campo from the "Institut National de Recherche et Sécurité" (Nancy, France).
Statistical testing.
The physiological data were evaluated by separate repeated measures analysis of variance (ANOVA) tests that were run on the DPOAE data and on the CAP data in each experiment using a univariate approach. A Greenhouse–Geiser correction was applied in all instances. Post hoc analyses were conducted using Bonferroni pair-wise multiple comparisons. Results obtained with a p value < 0.05 are reported as statistically significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODS AND MATERIALSRESULTSDISCUSSIONREFERENCES |
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Hydrocarbon Concentrations in Aerosolized JP-8 Fuel
Characterization of the JP-8 exposure showed that the overwhelming proportion of hydrocarbons consisted of a vapor phase with only 1–5% reflecting aerosol particles. Those particles that were delivered had a mass median aerodynamic range that was in the rats' respirable range. MOUDI analysis showed that all of the measurable mass from the fuel aerosol was in the size range of 1 µm and below. Approximately 40% of the fuel aerosol by mass was in the size range of 0.56–1 µm, 40% was between 0.32 and 0.56 µm and 20% was in the range of 0.18–0.32 µm. For the chemical analysis of aerosolized JP-8 fuel, on average 72% was identified and quantified. The remaining (28%) were unidentified. Twenty-five hydrocarbons are presented in Table 1 based on their abundance and toxicity. In the JP-8 analysis, decane, nonane, undecane, and 1,2,4-trimethylbenzene constitute the largest identified components of the fuel in gas phase (accounting for 56% in the total gas phase quantified). In the aerosol phase, fluorene is the most abundant hydrocarbon. It was found that the aerosolized JP-8 fuel consists predominantly of gas-phase hydrocarbons, which account for 99.5% of the total. C2–C5 hydrocarbons account for only 1% of the total hydrocarbon (gas + aerosol phase). The most abundant unidentified hydrocarbons were C9, followed by C8, and C10. These three hydrocarbons account for 96% of the total unspeciated hydrocarbons.
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Tissue Hydrocarbon Concentrations
Table 2 portrays the concentration of JP-8 components recovered from blood, liver, and lung immediately following exposure (0 h), 1 h following the end of exposure and 3 h following exposure. Fat and brain hydrocarbon levels are shown in Table 3. As shown in Table 2, hydrocarbon concentrations in lung while generally high immediately following exposure show a marked decline by 1-h postexposure and, with the exception of two trimethylbenzenes and straight chain hydrocarbons of dodecane and above are undetectable 3 h following exposure. Blood hydrocarbon levels generally follow those seen in the lung except that the initial levels are substantially lower in blood than in lung. And with the exceptions of dodecane, tridecane, tetradecane, and pentadecane the hydrocarbons have been rapidly removed from the blood and stored in fat.
Fat hydrocarbon levels (see Table 3) far exceed those in either lung or blood for all but these four long chain hydrocarbons which appeared to preferentially partition into the lung tissue reflecting the portal of entry for aerosol droplets. That fat does serve as a storage depot for the hydrocarbon components studied is further demonstrated by the persistence of these compounds over a 3-h postexposure period. While fat hydrocarbon levels reached a peak level immediately following the exposure, these values remained fairly constant over the time period studied with a few notable exceptions. Among the hydrocarbons showing a 30–40% decline in the first 3 h following JP-8 exposure were the ototoxic agents toluene (44% decline), p-xylene (31% decline), and ethylbenzene (30% decline). By contrast, in lung and blood, hydrocarbon levels fell rapidly between 0- and 1-h postexposure and, aside from the four long chain hydrocarbons (dodecane–pentadecane) and two trimethylbenzenes, the hydrocarbons were undetectable at 3-h postexposure.
Brain hydrocarbon concentrations were quantifiable only at the time point immediately after exposure (see Table 3). At the immediate postexposure time point the levels of toluene, ethylbenzene, m-xylene, o-xylene, and nonane in brain approached those levels found in the blood. Also, decane and 1,2,3-trimethylbenzenene, and naphthalene showed concentrations in brain that approached the blood levels at the immediate time point after exposure.
Auditory Effects of a Single Exposure to JP-8 with and without Noise
Figures 2A–D present the results of Experiment I. This study was designed to assess the effects of a single exposure to JP-8 (1000 mg/m3) with (Fig. 2A) and without (Fig. 2C) subsequent 105 dB noise exposure (OBN centered at 8 kHz, for 4 h) on auditory function. DPOAE amplitudes are also provided for rats that received noise alone (Fig. 2B) and for control rats that did not receive experimental treatment (Fig. 2D). The frequency range of the noise band is shown by the shaded area in each graph. As anticipated, exposure to noise alone (Fig. 2B) produces a persistent loss in DPOAE amplitude of approximately 10 dB that begins within the noise band and extends to frequencies slightly more than one octave band above the center frequency of the noise. This noise-induced impairment is stable between the first DPOAE assessment made 1 week following exposure and the 4-week postexposure test. Combined treatment with JP-8 fuel followed by noise (Fig. 2A) produces a greater depression in the DPOAE amplitudes as compared to the noise only treatment (Fig. 2B) at both the 1- and 4-week postexposure time points. Combined exposure to JP-8 + noise yields a depression in DPOAE values as great as 10 dB below those observed in rats receiving only noise, or 20 dB below those measured in control rats. Interestingly, there appears to be little evidence that combined exposure expands the frequency range over which DPOAEs are depressed relative to subjects treated with noise alone. By contrast, DPOAE amplitudes do not shift over time from the baseline measure among the rats that received JP-8 alone (Fig. 2C) and the untreated control rats (Fig. 2D).
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The effects of JP-8 and noise on CAP auditory thresholds are shown in Figure 3. These data provide a second method for evaluating auditory function following experimental treatment and provide direct assessment of the output of the cochlea to the brain. While the differences among treatment groups are small, control subjects do have the most sensitive pure-tone auditory thresholds of all groups. After the control subjects, rats receiving JP-8 alone and noise alone had slightly elevated auditory thresholds, followed by the rats exposed to JP-8 + noise, which display the highest (least sensitive) thresholds, especially at 12 kHz. Notably, this frequency is
octave higher than the center of the noise band; a frequency that would be expected to show maximal disruption due to noise alone.
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Separate repeated measures ANOVA tests were run on the DPOAE data and on the CAP threshold data. In both cases, "treatment" served as a between-subject factor and "frequency" served as a within-subject factor. For the DPOAE data only, "time" of measurement with respect to experimental treatment was also included as a second within-subject factor. For the DPOAE analysis, both treatment (F(3,30) = 5.49; p < 0.005) and time (F(2,60) = 11.74; p < 0.0001) main effects were significant. The interaction of these two treatments was also statistically significant (F(6,60) = 7.44; p <0.0001), indicating that the different treatment groups showed significantly different effects at the various postexposure time points. Bonferroni pair-wise multiple post hoc comparisons indicated that both the combined treatment of JP-8 + noise and the effect of noise alone were reliably different from the control treatment. No other significant group differences were observed.
The CAP data were also subjected to a two-way ANOVA with "treatment" considered as a between-subject variable and test "frequency" as a within-subject variable. This analysis did show a significant effect of treatment (F(3,26) = 3.09; p < 0.05) and of frequency (F(10,260) = 26.04, p = 0.0001). However, the interaction between treatment and test frequency was not statistically significant (F < 1.0). Thus, the basic shape of the rat audiometric curve was not selectively shifted at specific frequencies among the treated rats. Post hoc comparisons using Bonferroni multiple comparisons test between group means disclosed that rats treated with JP-8 + noise differed in their auditory threshold sensitivity from the control group. No other between group differences reached statistical significance.
Counts of missing outer hair cells across the length of the cochlea (cytocochleagrams) are presented in Figures 4A–D. Both rats receiving noise alone (Fig. 4B) and those receiving combined treatment with JP-8 + noise (Fig. 4A) show a slight elevation in the number of missing hair cells relative to control (Fig. 4D) and JP-8 alone subjects (Fig. 4C). However, there is no clear evidence that combined treatment under the exposure conditions used in this study increases the loss of hair cells relative to rats receiving only noise exposure.
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Auditory Effects of Repeated JP-8 Exposure with and without Noise
Experiments II and III were completed to evaluate the effects of repeated JP-8 exposure + noise. Since key groups differed in terms of the intensity and duration of noise exposure, the results are described individually for each study. Given that the rats in the JP-8 alone condition were identical in both studies, comparison of the outcomes from JP-8 alone can be viewed as a replication.
Figures 5A–D portray the effects of repeated experimental treatments to JP-8 and 4 h of noise at 97 dB on DPOAE amplitudes recorded over an interval of 4 weeks postexposure. For comparative purposes, the DPOAE amplitudes for control subjects are shown in Figure 5D and rats receiving 97-dB noise treatment alone are shown in Figure 5B. All groups that received an experimental treatment show a decrease in the DPOAE response, while untreated DPOAE levels are stable in the control subjects (Fig. 5D).
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Rats treated with this mild noise treatment alone (Fig. 5B) show a decrease in DPOAE amplitudes of approximately 10 dB in a limited frequency range at the initial postexposure measurement performed 3 days following the noise treatment. This group shows limited recovery of DPOAE amplitude at the 4-week postexposure test. The loss in DPOAE amplitude observed in the noise-treated rats is centered approximately
octave band above the center of the noise band to which they were exposed. Repeated exposure to JP-8 alone for 5 days (Fig. 5C) produces a distinct decrease in DPOAE amplitude between approximately 8–12 kHz. The loss in DPOAE amplitude approaches 20 dB at a maximum although there is some evidence for a slight recovery of function observed 4 weeks after exposure. Nevertheless, the persistent loss of DPOAE amplitude still approximates 15 dB even at the 4-week time period.
Rats receiving successive daily exposures for 5 days to JP-8 followed by 97 dB noise exposure (Fig. 5A) show a rather broad loss in DPOAE amplitude that extends initially from the lower range of the noise band to a point approximately two octaves above the center frequency of the noise. This loss shrinks considerably at the 4-week postexposure time point although permanent loss of about 10 dB can be observed over approximately one octave band starting toward the upper half of the noise band.
Auditory threshold determinations conducted 4 weeks following the last experimental exposure are presented in Figure 6. As in the prior study, group differences are quite small, but there appears to be a slight loss in threshold sensitivity among experimental groups in the rats' area of greatest hearing sensitivity, 12–16 kHz.
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Separate repeated measures ANOVA tests were run on the DPOAE data and on the CAP threshold data. In both cases, treatment served as a between-subjects measure and frequency served as a within-subject measures. For the DPOAE data, a second within-subjects measure, time of measurement with respect to experimental treatment, was also included. For the DPOAE analysis, treatment (F(3,20) = 7.46, p < 0.0025) and time relative to experimental exposure (F(2,1720) = 7.55, p < 0.0025) were statistically significant. The interaction of treatment with time failed to meet statistical significance (F(6,1720) = 2.12, p = 0.0726). The main effects of frequency (F(43,1720) = 138.83, p < 0.0001) and the interaction of treatment with frequency (F(129,860) = 2.67, p < 0.0001) were significant. Bonferroni pair-wise multiple post hoc comparisons indicated that the combined treatment of JP-8 + noise was reliably different from noise alone and from the control treatment. No other significant group differences were observed.
The CAP data were also subjected to a two-way ANOVA with treatment considered as a between-subjects variable and test frequency as a within-subject variable. This analysis did not show a significant effect of treatment (F < 1.0); only frequency (F(10,200) = 85.71, p < 0.0001) was significant. Thus, the basic shape of the rat audiometric curve was not selectively shifted at specific frequencies among the treated rats.
In Experiment III, the noise duration was limited to a 1-h time period immediately after JP-8 exposure and the noise intensity was increased to 102 dB. The effects of the different experimental treatments on DPOAEs are presented in Figures 7A–D. Rats treated with JP-8 + noise (Fig. 7A) show a loss in DPOAE amplitude of 20–25 dB over a broad range of frequencies beginning just below the midpoint of the noise band and extending to 50–55 kHz. Limited recovery of DPOAE amplitude is apparent 4 weeks postexposure. A similar though somewhat less extensive pattern of loss and partial recovery also occurs among rats treated only with the noise exposure (Fig. 7B). The effect of JP-8 alone (Fig. 7C) is quite small among this group of experimental animals particularly in relationship to the effect seen in the previous study (see Fig. 5C). DPOAE amplitudes are relatively stable among control rats although some apparent loss is observable at the last DPOAE assessment. The basis for this loss is not clear. However, if it reflects true differences in equipment sensitivity or some undetected health issue that also exists among the treated rats, it would imply that somewhat more recovery of function might have been observed in these treated groups were DPOAE levels truly stable across test periods.
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As in the previous two studies, the groups are rather similar in their auditory thresholds with both the noise alone and control subjects appearing to be most sensitive to pure tones and with the combined exposure group having somewhat poorer thresholds than the other group (see Fig. 8). However, these effects are quite limited both in degree and in the extent over frequency. Comparison among cytocochleagrams (Figs. 9A–D) obtained from the four groups shows very limited and scattered loss of outer hair cells in both the control subjects (Fig. 9D) and those treated with JP-8 alone (Fig. 9C). These two groups are indistinguishable from each other. Somewhat greater loss of outer hair cells is observable in rats exposed only to noise (Fig. 9B) compared to controls (Fig. 9D). The loss of outer hair cells appears to be even greater in the combined exposure group than it is in the noise alone group with particular increases in outer hair cell loss in the basal turn of the cochlea where high frequency auditory encoding occurs (Fig. 9A). And it is consistent with evidence of high frequency loss in DPOAE amplitude.
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Statistical analyses of the DPOAE data confirm significant main effects of frequency (F(43, 98) = 151.3, p < 0.0001) and of time relative to the exposures (F(3,69) = 6.41, p < 0.001). The treatment main effect failed to meet significance. However, a significant interaction between treatment and time of test (F(9,69) = 3.01, p < 0.005) was seen along with a significant interaction of treatment and frequency (F(129, 989) = 2.67, p < 0.0001).
Likewise, in the analysis of the CAP data, treatment failed to meet statistical significance (F < 1.0) although both frequency (F(10, 190) = 32.98, p < 0.0001) and the treatment by frequency interaction (F(30, 190) = 1.82, p < 0.01) were significant.
JP-8 Exposure and its Effect on Tissue GSH Levels
The effect of JP-8 exposure on liver, lung, and brain total GSH levels measured immediately following exposure (0 h), 1-h postexposure, and 3-h postexposure is portrayed in Figure 10. It is apparent that JP-8 produces a marked depletion in GSH levels in liver tissue at all time points relative to the unexposed controls and that there appears to be some recovery of GSH at the 3-h time point. The data from lung and brain are less dramatic. GSH levels appear to fall following JP-8 exposure, but the extent of this depletion is very limited. A global ANOVA showed significant effects of JP-8 treatment (F(3,24) = 11.60, p < 0.0001) and both significant differences among tissue types (F(2,24) = 134.02, p < 0.0001) and, more importantly, a significant interaction between tissue type and time of GSH assay following JP-8 (F(6,24) = 6.91, p < 0.0005). Step-down analyses showed that GSH depletion was significant in liver where the control GSH levels were significantly different from both the immediate and 1-h post–JP-8-treated rats. GSH levels for the 3-h recovery group were not significantly different from controls pointing toward a recovery at this time point. Neither brain nor lung GSH levels were significantly depleted in JP-8 treated rats compared to controls (p values > 0.1).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODS AND MATERIALSRESULTSDISCUSSIONREFERENCES |
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The current study identifies a limited, yet consistent alteration in cochlear function and in hair cell survival following exposure to JP-8 jet fuel when it is given in combination with noise exposure. Both after a single inhalation exposure as well as repeated exposures over the course of 5 days, JP-8 exposure increases the cochlea's susceptibility to noise exposure. This is most readily seen in the DPOAE amplitude assessment. In all three experiments, rats treated with both JP-8 and noise show a more profound depression in the DPOAE than do rats exposed to noise alone. When the effects of treatment on auditory function were assessed by the CAP, a measure of auditory threshold, the combined treatment group showed a tendency toward less sensitive pure-tone auditory thresholds, but the effect reached statistical significance only in the initial experiment when rats received a single JP-8 exposure with and without noise exposure. That the effect is more apparent in the DPOAE test than on the CAP measurement may reflect several different factors; the exposure may preferentially disrupt outer hair cells leaving other portions of the cochlea functioning relatively normally. In the current research, the extent of outer hair cell loss is not noticeably greater when combined JP-8 and noise exposure were given on only 1 day (see Figs. 4A and 4B). However, with repeated exposure there does appear to be somewhat greater outer hair cell death with the combined exposure than noise alone (see Figs. 9A and 9B). In addition, the DPOAE test because it permits multiple assessments of auditory function in the same subject can make more obvious a loss in the cochlea than the single assessment of the CAP.
The effects of JP-8 alone on the cochlea are somewhat more difficult to discern. While in the initial repeated exposure experiment a loss in the DPOAE response was observed in the JP-8 only treatment group, the effect was not replicated in the subsequent study which used a comparable exposure protocol with respect to JP-8 exposure. In both the 1 day and repeated exposure models where cytocochleograms were prepared, there was no discernable increase in hair cell loss between JP-8 exposed rats and unexposed control rats. However, it should be noted that even the repeated exposures used in this study were relatively short compared to the exposure schedules that have been used by others in evaluating the ototoxicity of aromatic hydrocarbons.
To put the current data into a broader context, many previous investigations have determined that aromatic hydrocarbons and some other isolated compounds are able to impair auditory function and enhance sensitivity of the cochlea to noise. Most of those investigations have entailed protracted experimental treatments and the effects of exposure have most commonly been reported following 4–16 weeks of exposure 5 days per week. Pryor et al. (1983a,b, 1991) demonstrated that toluene exposure induces marked sensorineural hearing loss in adult and weanling rats exposed to toluene vapors between 1200 and 2000 ppm for a duration of 1 or 2 weeks. Subsequent research determined that the functional threshold of toluene-induced hearing loss is close to 1300 ppm for a 4-week long exposure (Loquet, 1999). Shorter exposure times, to toluene, for example, have tended to produce only transient disruption in auditory function with full recovery occurring subsequently (e.g., McWilliams et al., 2000). Low-level occupational exposure to an average of 97 ppm toluene for 12–14 years had an apparent effect on hearing in 40 rotogravure workers when auditory brainstem response (ABR) results were compared to a group of 40 nonexposed workers (Abbate et al., 1993). In two other studies by Vrca et al. (1996, 1997), ABRs in workers exposed to average toluene concentrations of about 50 ppm for an average of 21.4 years were found to be affected, with a significant decrease in all wave amplitudes. Additionally, a cross-sectional study of 124 Brazilian workers exposed to various levels of noise and a variety of organic solvents, including toluene at concentrations ranging from 0.037 to 244 ppm (midpoint = 122 ppm), showed hearing loss in nearly half of the exposed workers (Morata, et al, 1997).
Laboratory animal data on the auditory effect of ethyl benzene come from a group of studies from a single laboratory conducted by Cappaert et al. (1999, 2002a) in rats. These results suggest that ethyl benzene is the most potent ototoxic organic solvent known today. The functional and histologic patterns of the hearing loss induced by ethyl benzene are very similar to the effects of toluene or styrene (i.e., mid-frequency hearing loss; selective outer cell damage; potentiation of NIHL; rats are sensitive but not guinea pigs), suggesting a common ototoxic mechanism (Cappaert et al., 1999, 2001a, 2002), but the threshold ototoxic dose of ethylbenzene (300–400 ppm for 5 days, Cappaert et al., 2000) is comparatively low relative to toluene.
In the current study, evidence of mild auditory impairment was observed following a complex chemical exposure followed by noise. That components other than the widely recognized ototoxicants toluene, ethylbenzene, and p-xylene may be partly responsible for this effect is suggested by the fact that the concentration of known ototoxic agents in the JP-8 exposure we employed was approximately 8 ppm for toluene, 10 ppm for ethylbenzene, and 15 ppm for xylenes. These concentrations are far lower than those that have been reported previously as ototoxic. Subsequent studies now in progress are determining whether or not longer-term JP-8 exposures yield a more consistent impairment of cochlear function.
The current report shows that JP-8 also can enhance vulnerability to noise with effects apparent even after 1 week of exposure. The effects we have observed are not large, but they do show a consistent pattern of impairment and some evidence of increased hair cell death. This finding may be important both given the number of workers who are exposed to JP-8 jet fuel and in light of the fact that the concentration of individual hydrocarbon constituents of JP-8 employed are rather small. While the current experimental design is not able to identify the ototoxic components of JP-8, the current report gives additional credence to the preliminary findings of Kaufman et al. (2005) showing that occupational exposure to jet fuel may enhance auditory dysfunction in workers also exposed to noise. It must be noted that the PEL for JP-8 in the workplace is 350 mg/m3 while our model employed a concentration of 1000 mg/m3 for 4 h.
The mechanism by which JP-8 exposure potentiates NIHL is not clear at this point. The current work does verify some in vitro studies suggesting that JP-8 might deplete GSH levels (Boulares et al., 2002) thereby reducing the antioxidant potential of tissue and establishing a potential oxidative stress when noise exposure is involved. In the current study, the decrement in GSH is most apparent in the liver, but fails to reach statistical reliability either in lung or brain tissue. Future studies will be essential to determine whether or not JP-8 exposure can depress cochlear GSH levels and whether evidence of oxidative stress can be observed when the fuel exposure is combined with noise.
Several different noise exposure conditions were used in this study; the objective in each case being to produce a very limited impairment in cochlear function given the duration or repetition of noise exposure in each individual experiment. Noise levels varied from a single 105 dB OBN for 4 h to five repeated 1-h noise exposures at 97 dB. Currently, OSHA has established a permissible workplace noise exposure level of 90 dB (A) averaged over an 8-h time interval. OSHA also permits a trade-off of 5 dB for intermittent noise. That is, 105 dB (A) noise exposure for 1 h, 100 dB (A) exposure for 2 h, 95 dB (A) noise exposure for 4 h are all considered to be equivalent to the exposure standard of 90 dB (A) exposure for 8 h. Using this rubric, the noise exposures employed in the two repeated exposure studies were within OSHA guidelines while the exposure level in the initial study exceeded the OSHA guideline by 10 dB.
The toxicokinetic data obtained in this study are useful in conducting future studies because they indicate how short-lived the constituents of JP-8 fuel are in biological tissue using an inhalation exposure model. In considering more extensive repeated measures designs, it would appear that any increase in JP-8 toxicity will not be explicable in terms of a build-up of xenobiotic in tissue providing that the fat does not become a longer-term store for this material.
The current report provides initial data supporting an interaction between JP-8 and noise exposure on hearing. While it does not establish a mechanism of action or the constituents of the fuel that are ototoxic, it does begin to provide some dosimetry that may be useful in evaluating workers for adverse effects of exposure and testable hypotheses with respect to mechanism.
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ACKNOWLEDGMENTS |
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This research was funded by a Merit review grant from the VA Rehabilitation Research and Development Service (grant # C3575R) and was dependent upon the research facilities at the Jerry Pettis Memorial VA Medical Center, Loma Linda, CA. Additional support was obtained from the Air Force Office of Scientific Research and from the American Petroleum Institute. JP-8 jet fuel was kindly provided by Dr Tim Edwards of the Air Force Research Laboratory's Propulsion Directorate/Turbine Engine Division/Fuels Branch, Wright-Patterson Airforce Base, Dayton, OH.
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