Analysis of rat testicular protein expression following 91-day exposure to JP-8 jet fuel vapor

Frank A. Witzmann1

Andrew Bobb2

G. Bruce Briggs3*Heather N. Coppage1

Rex A. Hess4

Junyu Li1

Nathan M. Pedrick1

Glenn D. Ritchie2

John Rossi III2

Kenneth R. Still2

1Department of Cellularand Integrative Physiology,Indiana University Schoolof Medicine,Indianapolis, IN, USA

2Naval Health ResearchCenter Detachment-Toxicology(NHRC-TD),Wright-Patterson Air Force Base,OH, USA

3Geo-Centers,Arlington, VA, USA

4Department ofVeterinary Biosciences,University of Illinois,Urbana, IL, USA

Analysis of rat testicular protein expressionfollowing 91-day exposure to JP-8 jet fuel vapor

We analyzed protein expression in preparations from whole testis in adult male Spra-gue-Dawley rats exposed for 6 h/d for 91 consecutive days to jet propulsion fuel-8(JP-8) in the vapor phase (0, 250, 500, or 1000 mg/m3 � 10%), simulating a range ofpossible human occupational exposures. Whole body inhalation exposures werecarefully controlled to eliminate aerosol phase, and subjects were sacrificed within48 h postexposure. Organ fractions were solubilized and separated via large-scale,high resolution two-dimensional electrophoresis, and gel patterns scanned, digitizedand processed for statistical analysis. Seventy-six different testis proteins were sig-nificantly increased or decreased in abundance in vapor-exposed groups, comparedto controls, and dose-response profiles were often nonlinear. A number of the pro-teins were identified by peptide mass fingerprinting and related to histopathologicalor physiological deficits shown in previously published studies to occur with repeatedexposure to hydrocarbon fuels or solvents. These results demonstrate a significanteffect of JP-8 exposure on protein expression, particularly in protein expression inthe rodent testis, and suggest that a 91 d exposure to jet fuel vapor induces changesof equal or greater magnitude to those reported previously for shorter duration JP-8aerosol exposures.

Keywords: Jet fuel / Rat / Testis / Toxicity / Two-dimensional gel electrophoresis PRO 0385

1 Introduction

Since the 1992–1996 US military transition from predom-inant use of JP-4 jet fuel (unleaded gasoline/keroseneblending), kerosene-based JP-8 formulations (JP-8 orJP-8�100) have been used to power virtually all US AirForce and US Army aircraft, vehicles and equipment.Additionally, Jet A and Jet A-1, jet fuels similar to JP-8except for the performance additive packages, havebeen used for over 20 years to power most domesticand international commercial flights [1]. Because almost60 billion gallons of kerosene-based jet fuels (JP-8, JP-8�100, JP-5, Jet A, Jet A-1) are consumed annually (ap-proximately 1.6 million barrels/day), there is extensiveopportunity for repeated direct exposure of numerouscommercial and military aircraft maintenance and opera-tions personnel, as well as those involved in fuel manufac-turing and transportation [2]. Furthermore, due to un-

avoidable fuel leakage, accidental spillage, and aircraftemergency fuel jettisoning into the atmosphere, there isample opportunity for nearly continual low level exposure(atmospheric, soil, ground water) of civilians working on,or living near airports and military flight lines [1].

Numerous animal studies of repeated dermal, respiratory,or oral exposure to kerosene based jet fuels or keroseneper se indicate significant and often persisting healtheffects in at least the pulmonary, immune, renal, hepatic,hematopoietic, and central nervous systems (CNS) [1].In several of these studies, very brief exposure (�1 h/dfor 5–7 d) of rats to JP-8 in aerosol phase, or repeateddermal exposure to neat JP-8 resulted in profound defi-cits in, respectively, the pulmonary and immune system[3–6] or immune system [7–9]. Additionally, oral gavageexposure of mice to JP-8 was shown to cause an increasein liver weight, a decrease in thymus weight, and a changein immunological function, as evidenced by a decreasein the plaque-forming response (PFC) [10]. In vitro JP-8exposure in human peripheral lymphocytes increasedDNA damage [11]. Finally, more prolonged exposure ofrats to JP-8 in the vapor phase (1000–1100 mg/m3, 6 h/d,for up to 60 d) resulted in persisting changes in the CNS,as well as in neurobehavioral capacity [12–13].

Correspondence: Frank A. Witzmann, Ph.D., Department ofCellular & Integrative Physiology, Indiana University School ofMedicine, Biotechnology Research and Training Center, Room308, 1345 W. 16th Street, Indianapolis, IN 46202, USAE-mail: [email protected]: +1-317-278-9739

Abbreviations: DiEGME, diethylene glycol monomethyl ether;EGME, ethylene glycol monomethyl ether; JP, jet propulsion fuel

1016 Proteomics 2003, 3, 1016–1027

* Deceased

2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 0173-0835/03/0606–1016 $17.50�.50/0

DOI 10.1002/pmic.200300385

Proteomics 2003, 3, 1016–1027 Testis protein expression in jet fuel exposed rats 1017

A number of published studies report human or animalreproductive toxicity in males from exposure to one ormore chemical constituents or additives of JP-8. Interest-ingly, concurrent exposure to more than one chemicalconstituent of JP-8 can, in some cases, result in antagon-ism of the expected male reproductive toxicity. This effectmay suggest possible nonlinearity in dose-responseeffects from increasing vapor concentrations of JP-8 thatmay contain different constituents or relative percentagesof those constituents.

Xiao et al. [14–15] examined the reproductive systems ofmale workers exposed to benzene, toluene and xylene.Each of these chemicals, when measured in the blood,was also measured in sperm. The sperm vitality andsperm motility was decreased in exposed workers, whilemean acrosin, gamma-GT, and lactate dehydrogenase-C4 (LDH-C4) relative activity were lower than in unex-posed controls. Ward et al. [16] reported testicular lesionsin mice exposed to 300 ppm benzene vapor, 6 h/d, 5 d/w,for 13 weeks. Yamada [17] measured reduced testes andaccessory reproductive organ weights, reduced acidphosphatase activity in the prostate, and reduced plasmatestosterone in male rats exposed briefly twice daily for7 d to xylene vapor, but not following similar exposuresto toluene or methanol. Chan et al. [18] detected in-creased incidence of testicular tumors in male rats ex-posed to ethylbenzene, another constituent of JP-8, byinhalation at 750 ppm 6 h/d, 5 d/w, for 2 years. De Martinoet al. [19] documented significant and progressive dam-age to the reproductive system of male rats following16–24 h/d inhalation exposure to 5000 ppm n-hexane.Lesions were first seen after 24 h of continuous exposureand involved both primary spermatocytes and sperma-tids at late stages of maturation. During more extendedexposures (16 h/d for 6 d/w) damage to the seminiferousepithelium increased for the first 7 days, with the epidi-dymis evidencing focal infiltration by inflammatory cells.Exposure for 6 weeks resulted in irreversible sterility.

Nylen et al. [20] similarly reported severe damage tothe seminiferous tubules in rats exposed repeatedly ton-hexane, but curiously simultaneous administration of1000 ppm n-hexane � 1000 ppm toluene, or 1000 ppmn-hexane � 1000 ppm xylene significantly protected ani-mals from n-hexane induced testicular atrophy. Finally,ethylene monoethyl ether (EGME), a water miscibleorganic solvent, chemical industry emulsifier, and anti-icing agent used in jet fuels before 1996, has been shownto induce atrophy of the testes or loss of spermatocytesin rats and mice after as few as one exposure (Nagannoet al. [21]; Foster et al. [22]). EGME is a structural analogof diethylene monomethyl ether (DiEGME), the anti-icing agent used currently in JP-8 and JP-8�100. Afterrepeated exposure to an average of 9.9 mg/m3 EGME,

shipyard painters (also exposed to other paint-relatedtoxicants) exhibited increased prevalence of oligospermiaand azoospermia, and an increased odds ratio for a lowersperm count per ejaculate (Welch et al. [23]). More re-cently, Chung et al. [24] showed that daily co-exposureof rats to EGME (200 mg/kg) � toluene (250 mg/kg) �xylene (500 mg/kg) for 4 w induced less testicular atrophyin rats than did simple exposure to EGME. This study sug-gested that toluene and xylene reduced the metabolismof EGME to the toxic metabolite ethoxyacetic acid (EAA),thought to be responsible for reproductive toxicity effectsfrom EGME exposure.

Several previously published studies have used 2-D gelbased proteomic assay methods to evaluate JP-8 toxicityin mice or rats. Witzmann et al. [25] analyzed kidney tissuefrom male Swiss-Webster mice exposed (nose-only) to1000 mg/m3 JP-8 in aerosol phase for 1 h/d for 5 d, thensacrificed 48 h post-exposure. JP-8 exposure signifi-cantly altered the abundance of 56 proteins (6% of pro-teins tested). From these data, possible deficits in fourmajor functional categories were established: (1) cyto-skeletal organization; (2) protein synthesis or processingsystems; (3) metabolic enzymes; and (4) stress protein/detoxification system response. A similar approach wasused to identify possible markers in the pulmonary sys-tems of male Swiss-Webster mice exposed to JP-8 inthe aerosol phase (1000 or 2500 mg/m3) for 1 h/d for 7 d[26]. In that study, exposure to 2500 mg/m3 significantly(p � 0.001) increased the abundance of 34 proteins, whiledecreasing the abundance of 8. By comparison, an iden-tical duration exposure at 1000 mg/m3 resulted in signifi-cant modulation of the abundance of only one of these42 proteins (but 11 proteins, when p � 0.01). The identi-fied changes in lung protein abundance were related tofunctional deficits in: (1) protein synthesis or processing;(2) ultrastructure integrity; (3) toxic or metabolic stressand detoxification systems; or (4) control of carbon diox-ide handling, acid-base homeostasis, or pulmonary fluidsecretion.

With respect to reproductive toxicity, excluding dermalkerosene exposure in rats [27], few investigations havestudied the effects of jet fuel exposure on the male repro-ductive system. Only recently, Briggs et al. [28] reportedthat repeated exposure of male Sprague-Dawley rats toJP-8 in vapor phase, at levels approximating possibleoccupational exposures (0–1000 mg/m3), resulted in noobservable tissue histopathology or statistically signifi-cant changes in total sperm concentration or morphol-ogy. However, a linear and dose-related reduction inmean sperm motility was observed. It appears that re-peated exposure to at least some chemical componentsof JP-8 (i.e., those contained in JP-8 vapor) inducedchanges in spermatogenesis or sperm motility without

1018 F. A. Witzmann et al. Proteomics 2003, 3, 1016–1027

concomitant changes in testis histopathology. Based onthe results presented by Briggs et al. [28], the presentstudy attempted to identify JP-8 mediated changes atthe protein level that might both explain the motilitychanges and identify molecular level changes not obser-vable with traditional histopathology. Remaining frozentestis tissue from the same JP-8 exposed rats that wereexamined by Briggs et al. [28] was now subjected to pro-teomics analysis. Rats were exposed to room air controlconditions (0 mg/m3), to 250 mg/m3 (below the 8 h thresh-old limit value (TLV) for JP-8), 500 mg/m3 (between theTLV and 15 min short-term exposure level (STEL)), or1000 mg/m3 (at the interim STEL) JP-8 in vapor phase for6 h/d (typical work shift) for 91 consecutive days. It wasassumed that the vapor phase best modeled exposuresmost commonly encountered by military and commercialworkers with direct occupational exposure to JP-8, aswell those possibly encountered by the general public liv-ing near military bases, airports, or JP-8 manufacturing/storage facilities. Further, it was assumed that molecularmarkers of vapor exposure in rats that may occur in theabsence of easily observable health consequences mightprove useful in predicting human health effects fromcareer-long exposures.

2 Materials and methods

2.1 Materials

Acrylamide for the slab gels was purchased from Natio-nal Diagnostics (Atlanta, GA, USA) and for the IEF tubegels from Bio-Rad (Richmond, CA, USA). Other ultrapureelectrophoretic reagents were obtained from Bio-Rad,Sigma (St. Louis, MO, USA), or BDH (Poole, UK).Sequence grade trypsin was obtained from Promega(Madison, WI, USA). DTT was obtained from Calbiochem(La Jolla, CA, USA) and N-isopropyl iodoacetamide fromMolecular Probes (Eugene, OR, USA). All other chemi-cals used were reagent grade.

2.2 JP-8 jet fuel

A twenty-gallon sample of JP-8 was procured from the AirForce Research Laboratory (AFRL), Propulsion Directo-rate, Fuels Branch, Wright-Patterson Air Force Base OH.This sample consisted of a blending (designated POSF-3509) of JP-8 refinery streams procured from five differentdomestic and international manufacturers, minimizinglot effects. The blending occurred from newly procuredfuel lots into a clean and sealed container to minimizewater condensation, and possible microbial contamina-tion. The JP-8 sample contained (� 0.5% total v/v) threeperformance additives: a static dissipater (Stadis 450);a corrosion inhibitor/lubricity improver (DCI-4A); and an

icing inhibitor (DiEGME). A GC-MS analysis of the blendedfuel was conducted to ensure compliance with militaryJP-8 manufacturing specification MIL-T-83188D.

2.3 Animals

Male Sprague-Dawley rats (Crl:CD (BR)), 50 d of age uponreceipt, were procured from Charles River Laboratories(Raleigh, NC, USA). Upon arrival, rats were singly housedin hanging polycarbonate shoebox cages with cellulosefiber contact bedding (Cell-Sorb Plus; A. W. Products,New Philadelphia, OH, USA). Rats were held in quarantinefor 14 d, and were inspected on a daily basis by the veter-inary services staff throughout the study. During quaran-tine and subsequent whole body inhalation exposures,fresh pelleted food (Purina Formulab �5002; Purina Mills,St. Louis, MO, USA) and fresh conditioned (reverse osmo-sis) water were available ad libitum in the home cages.Food and water were withheld during the 6 h/d inhalationexposures to minimize oral ingestion of JP-8. During the91 d period of inhalation exposure, home cage rackshousing control and vapor-exposed rats were locatedwithin individual Bio-Clean air mass displacement unitsthat provided a constant supply of high efficiency particu-late air, eliminating off-gassing exposure of control ani-mals. A 12 h light:dark cycle was maintained with fullspectrum fluorescent lights on from 6:30 am – 6:30 pm;rats were exposed between the hours of 8:00 am and2:00 pm. Throughout the experiment, animal room airtemperature and humidity was maintained at 23 � 2�Cand 55� 15%, respectively.

2.4 Exposures and vapor concentrations

Whole body inhalation exposures occurred 6 h/d for 91consecutive d (546 h) in four identical 690-LTHRU cham-bers. For the daily exposures, rats were placed in individ-ual housing spaces within one of two 8-rat wire meshcages, within one of four consistently assigned exposurechambers. A semirandomization procedure was used todetermine daily animal placement, such that individualrats experienced vapor exposure an approximately equalnumber of times in each of the 16 housing locations withinthe assigned exposure chamber. For each vapor expo-sure, neat JP-8 fuel was directed through a heatedJ-tube, temperature controlled to maximize vaporizationwith minimal formation of aerosol. The vaporized fuelwas introduced into a (counter-current) room air sourceflowing into each 670 L chamber at 55� 3 L/min. For con-trol exposures, rats were identically exposed to filteredroom air inflowing into an identical 670 L chamber at arate of 55 � 3 L/min. Tubing directing the JP-8 vaporflow was externally heated to minimize condensation,


and contained a J-tube trap to contain and measure (ap-proximately 2%) condensed vapor. Gelman (Ann Arbor,MI, USA) 25 mm, extra thick glass fiber filters were usedto sample chamber atmospheres for aerosol. Chamberatmospheres were quantified by infrared (IR) spectrome-try at 20 min intervals using the IR absorbance band be-tween 3.4 and 3.5 microns, calibrated with known massconcentrations of hexane. Chamber atmospheres were,then, adjusted by controlling raw fuel flow and/or J-tubetemperature to maintain target concentrations �10%.The four exposure group (n = 5) conditions are summa-rized in Table 1. Following daily exposures, rats wereexamined by the veterinary services staff and returned toindividual home cages for 18 h.

Table 1. Summary of JP-8 vapor exposure and controlconditions

JP-8exposuregroup

Vaporexposure(mg/m3 � 10%)

Days ofexposure(6 h/d)

n

Control 0 91 5Low 250 91 5Moderate 500 91 5High 1000 91 5

2.5 Tissue preparation

Within 24–48 h postexposure, fuel vapor exposed and aircontrol rats were sacrificed by CO2 overdose. Testes wererapidly dissected and flash frozen for subsequent proteom-ics assay. Each frozen testis was bisected manually into su-perior and inferior halves. The inferior half was returned tothe �80�C freezer, and the superior half was placed in a 50mL beaker along with 8 vol of a solution containing 9 M urea,4% Igepal CA-630 ([octylphenoxy] polyethoxyethanol;Sigma, St. Louis, MO, USA), 1% DTT and 2% carrierampholytes (pH 8–10.5) and thoroughly minced with surgi-cal scissors. The minced samples were then placed in 5 mLDuall (Kimble/Kontes, Vineland, NJ, USA) ground-glasstissue grinders and manually homogenized. After completesolubilization at room temperature for 120 min, sampleswere centrifuged at 100 000�g for 30 min using a Beck-man (Beckman-Coulter, Fullerton, CA, USA) TL-100 ultra-centrifuge to remove nucleic acid and insoluble materials,and the supernates stored at �45�C.

2.6 2-DE and image analysis

Two-dimensional electrophoresis was performed essen-tially as described previously [29]. Twenty �L of each ofthe twenty solubilized testis samples were placed on

each of twenty IEF tube gels (23�0.15 cm) containing3.3% acrylamide (diacrylyl-piperazine as a cross-linker),9 M urea, 2% Igepal CA-630, 2% carrier ampholyte(BDH pH 4–8). The IEF gels were run simultaneously for27 000 Vh using progressively increasing voltage (500 Vfor 1 h, 750 V for 1 h, 1000 V for 1 h, and 1400 V for thebalance of the run) for a total of 28 500 Vh. A computer-controlled gradient casting system was used to prepare20 second dimension SDS gradient slab gels in whichthe acrylamide concentration varied linearly from 9% to19%T. First dimension IEF tube gels were loaded directlyonto the slab gels without equilibration. Second dimen-sion slab gels were run in parallel, in a 20-gel slab electro-phoresis tank at 8�C for 18 h at 160 V. Slab gels werestained using a colloidal Coomassie Blue G-250 proce-dure [30] in covered plastic boxes, ten gels per box. Gelswere fixed in 1.5 L of 50% ethanol/2% phosphoric acidovernight followed by three 30 min washes in 2 L of deion-ized water. Gels were transferred to 1.5 L of 30% meth-anol/17% ammonium sulfate/3% phosphoric acid for 1 hfollowed by addition of 1 g of powdered Coomassie BlueG-250 stain. After 96 h, the 20 gels were washed severaltimes with water and scanned at 95.3 �m/pixel resolutionusing a GS-800 Calibrated Imaging Densitometer (Bio-Rad). The resulting 12-bit images were analyzed usingPDQuest software (Bio-Rad, V6.2) running under Win-dows 2000 on a PC workstation. Background was sub-tracted and peaks for the protein spots located andcounted. Because total spot counts and the total opticaldensity are directly related to the total protein concentra-tion, individual protein quantities were thus expressedas parts-per-million (ppm) of the total integrated opticaldensity. A reference pattern was constructed and eachof the 20 gels in the matchset was matched to the refer-ence gel. Numerous proteins that were uniformly ex-pressed in all patterns were used as landmarks to facili-tate rapid gel matching. Statistical comparisons betweenindividual protein abundances were conducted by calcu-lation of Student’s t-test within the PDQuest analysis andafter data export to Excel.

2.7 Peptide mass fingerprinting

Protein spots whose abundance differed betweenexposure groups along with other spots from thestained and image-analyzed 2-D gels were cut fromthe gel robotically using the Protean 2D Spot Cutter(Bio-Rad), placed in each of 94 wells of a 96-well plate,along with one gel blank and serum albumin in acryl-amide, and processed using the MassPREP Station(Waters, Milford, MA, USA). In this automated system,the excised protein spots were destained with 100 mM

ammonium bicarbonate 50% acetonitrile followed by


100% acetonitrile, reduced with 10 mM DTT in 100 mM

ammonium bicarbonate, alkylated with 55 mM iodoace-tamide in 100 mM ammonium bicarbonate, and trypti-cally digested using Promega sequence grade, modifiedtrypsin at a final concentration of 13 ng/�L in 100 mM

ammonium bicarbonate for 14 h (overnight). The result-ing peptides were extracted by addition of 10 �L trypsinsolution to the wells and refrigerating the plate for30 min. Four �L of peptide extract was then placedonto each corresponding position (A1-H12) on theMALDI target plate, air dried, and the applicationrepeated until all extract buffer was used up. When thepeptide sample targets were dry, each was overlayedwith 1 �L of matrix (10 mg/mL �-cyano-4-hydroxycin-namic acid 0.05% TFA) and analyzed by MALDI-TOFMS using a MicroMass M@LDI system (MicroMass).Prior to data collection, the instrument was calibrated

using peptide standards and internal standards basedon tryptic autolysis peaks (842.5099 and 2211.1045 Da)were used for calibration.

Proteins were identified by manual ProFound (GenomicSolutions, Ann Arbor, MI, USA) database searches usingthe mass lists obtained from MALDI spectra of 188 spotscut from the gels. A Z-score of 1.30, corresponding to the90th percentile, was the threshold for what was consid-ered a positive identification. This means that for a givenpeptide mass search with a Z-score of at least 1.30, nomore than 10% of random matches could yield higherZ-scores. In reality, over 80% of the identified proteinslisted in Table 3 scored above the 95th percentile andnearly half scored at the 99th. The coordinate location inthe 2-DE pattern of each of the proteins listed in Table 3appears in Fig. 1.

Figure 1. 2-DE pattern of rat testis proteins, solubilized and separated on 23.5 cm IEF tube gelsand 20�25 cm slab gels, and stained with colloidal Coomassie blue as described in Section 2.6.The horizontal dimension represents a working pH gradient of approx. 4–7.5 and the vertical anacrylamide gradient of 9–19%. The protein spots were analyzed by peptide mass fingerprintingusing MALDI-MS and those successfully identified are designated by their spot number (SSP) inthe PDQuest reference pattern. The corresponding protein names appear in Table 3.


3 Results and discussion

3.1 General health of rats

Despite exposure for 546 h to JP-8 vapor, there were nodeaths, no obvious evidence of carcinogenicity, or othervisible signs of health, behavioral or irritancy disordersamong any of the rats. During the 6 h daily exposures,the majority of the vapor exposed and control animalsexhibited sleep or quiescence, minimizing the possibilitythat the exposure scenario induced excessive stress.There were no significant differences in either mean bodyweights or rates of growth between any JP-8 exposuregroup and the control group for the duration of the 91 dexposures.

3.2 JP-8 vapor exposure effects on proteinexpression

Figure 1 illustrates the result of 2-DE separation of rattestis proteins. The specific gel pattern shown was usedas the reference pattern for matchset construction withinthe imaging analysis (PDQuest) and thus serves as asuitable example of the typical testis whole homogenateprotein pattern. Image analysis of all 20 gel patterns indi-cated that a number of testis proteins (77/1320, or 5.8%)were significantly (p � 0.05) increased or decreased inabundance following exposure to JP-8 vapor. As shownin Table 2, repeated exposure to the lowest JP-8 vapordose (250 mg/m3) resulted in a five-fold higher number ofproteins with increased abundance than did exposure tothe highest dose (1000 mg/m3). In contrast, exposure tothe highest dose level resulted in significantly reducedabundance of twice as many proteins as the lowest doseexposure. Across all 77 proteins altered by the various ex-posures, proteomic analyses of individual protein abun-dance, as a function of JP-8 vapor dose, seldom reflecteda strict linear dose relationship. As is shown in Table 3, ofthe 19 identified proteins that were significantly changedin abundance at any JP-8 concentration, only four in-creased linearly in abundance as a function of dose:

Table 2. Number of testis proteins significantly (p�0.05)increased or decreased in abundance versuscontrols in rats exposed repeatedly to JP-8vapor

Effect Low Moderate High

Increased abundance 41 15 8Decreased abundance 9 9 18Total 50 24 26

Hsp86, nicotinic acetylcholine receptor alpha subunit,serum albumin, and T-complex protein 1. By the sameanalysis, no identified protein decreased linearly in abun-dance as a function of JP-8 dose exposure. In the remain-ing 15 cases of significantly altered expression acrossall doses, there was no clear linear relationship betweenJP-8 dose (250–1000 mg/m3) and change from baselinein protein abundance. This suggests that human riskassessment of complex mixtures like JP-8 should: (1)consider data from both low and high dose exposures;(2) not assume linear extrapolation among dose responseeffects; and (3) thus should not exclude nonlinear doseresponse data. While the expression of many proteinsdisplayed interesting trends relative to JP-8 dose (someof which appear in Table 3 listed as NC for no change),internal variation rendered these changes statisticallyinsignificant and therefore no speculative discussion ofthese will be attempted.

An analysis of the functional significance of the JP-8-mediated protein expression alterations (and absenceof effect) listed in Table 3 is complex, as the exact roleof many of these proteins in testis function is conjec-tural. Additionally, many (49) of the altered proteinsremain to be identified. Nonetheless, the following isan attempt to combine the observed alterations withthe putative role assigned to the identified proteinsaltered by JP-8 vapor exposure in the literature, so thatpossible functional consequences in the testis can behypothesized.

In rats exposed to 250 mg/m3 JP-8 vapor, there was asignificant two-fold increase (p � 0.03) in mitochondrialaldehyde dehydrogenase (ALDH) abundance. How-ever, ALDH expression was unaffected in both the mod-erate and high exposure groups. ALDH is commonlyexpressed in a number of organ systems, including tes-tis [30] where it is located principally in the Leydig cell[31] and is known to increase in response to the pres-ence of alcohols or glycols [32]. Furthermore, ethyleneglycol monomethylether (EGME), a structural analog ofDiEGME (diethylene glycol monomethyl ether), the anti-icing additive in JP-8, is metabolized by ALDH and possi-bly lactate dehydrogenase (LDH) to the major metabolitemethoxyacetic acid (MAA). EGME or MAA have beenwidely implicated in male reproductive system toxicity,including disruption of spermatogenesis [33]. While therelationship between exposure to DiEGME and expres-sion of ALDH has not been explored, Hobson et al. [24]have indicated that dermal exposure (1 g/kg/d) of guineapigs to DiEGME 5 d/w for 13 weeks resulted in increasedactivity of LDH, whose abundance in the present studywas increased (p � 0.03) 16% by the 250 mg/m3 expo-sure.


Table 3. Identified proteins from whole testis and the effect of prolonged JP-8 jet fuel exposure on mean proteinabundance

SSP z-Score

% Mr pI Name Change Con-trol

250mg/m3

p 500mg/m3

p 1000mg/m3

p

5304 1.58 22 38.7 5.7 aryl-hydrocarbon interacting protein-like 1

NC 2784 3210 0.2 2976 0.7 2789 1.0

2818 2.22 24 111.5 5.1 150 kDa oxygen-regulated proteinprecursor

� 2405 2225 0.2 1966 0.05 2158 0.6

2409 1.86 45 57.5 5.1 26S protease regulatory subunit 6B(TAT-binding protein-7) (TBP-7)

NC 947 989 0.6 1073 0.3 927 0.8

2726 2.11 40 72.1 5.1 78 kDa glucose-regulated proteinprecursor (grp 78)

NC 3863 3922 0.9 3631 0.6 4190 0.6

2706 2.19 36 72.5 5.1 78 kDa glucose-regulated proteinprecursor (grp 78)

� 3193 4777 0.06 3949 0.2 3620 0.5

2415 2.43 42 42.3 5.2 actin alpha NC 3619 2063 0.3 4113 0.8 4154 0.83407 2.43 50 42.1 5.3 actin beta NC 7527 8175 0.6 8407 0.6 7884 0.82909 1.30 4 256.9 5.0 A-kinase anchor protein �/� 1772 2547 0.05 1784 1.0 1184 0.55416 2.43 46 48.7 6.1 Aldehyde dehydrogenase, mito-

chondrial (ALDH1)�/NC 1580 3330 0.03 1771 0.8 1743 0.8

5507 2.43 31 48.7 6.1 Aldehyde dehydrogenase, mitochondrial(ALDH CLASS 2) (ALDH1)

NC 338 448 0.4 524 0.5 398 0.5

7314 2.39 34 36.2 6.3 aldehyde reductase 1 (low Km aldosereductase)

NC 6982 6926 0.9 7067 0.9 6611 0.3

8308 2.43 28 39.6 6.8 aldolase C (AA 1-362) NC 1326 990 0.6 2378 0.3 1742 0.5806 1.39 10 184.6 4.9 alpha 1 type V collagen NC 5738 4686 0.1 5899 0.8 5623 0.91618 2.43 42 73.2 5.5 aminopeptidase B NC 152 236 0.3 193 0.5 183 0.58708 2.27 9 99.6 6.3 androgen receptor NC 357 550 0.6 609 0.5 541 0.79107 2.15 43 24.6 7.0 androgen receptor-associated

protein 24� 2609 2501 0.7 2366 0.6 3640 0.01

310 1.64 22 35.4 5.0 annexin V NC 1777 1994 0.3 1831 0.8 1762 0.94603 2.00 9 68 5.6 apoptosis inhibitor 2 NC 12921 13577 0.8 8483 0.2 18068 0.1203 1.71 45 34 5.3 arylamine N-acetyltransferase NC 1839 1983 0.3 1978 0.4 1816 0.97416 2.43 27 46.7 6.7 aspartate transaminase (EC 2.6.1.1),

cytosolic – ratNC 330 240 0.5 199 0.2 275 0.61

615 1.54 16 48.2 4.3 calreticulin � 5077 5787 0.02 6003 0.08 5707 0.056509 2.43 42 57.8 6.0 chaperonin containing TCP-1 beta

subunit (Mus musculus)NC 3157 3612 0.1 2852 0.7 3151 1.0

2504 2.43 38 50.1 4.8 class I beta-tubulin NC 1216 1528 0.2 1099 0.6 930 0.42505 2.43 39 50.1 4.8 class I beta-tubulin NC 2020 1635 0.4 1764 0.5 1716 0.46002 2.43 32 15.7 5.9 Cu-Zn superoxide dismutase NC 6232 6160 0.8 6565 0.3 5871 0.31805 2.43 33 92.7 4.7 Endoplasmin (94 kDa glucose-regulated

protein) (GRP94)NC 4481 3840 0.5 3357 0.2 3969 0.6

7402 1.72 18 47.4 6.2 Enolase, 1 alpha NC 5680 6084 0.5 5695 0.9 5462 0.54406 1.35 27 49.4 5.4 eukaryotic initiation factor 5 (eIF-5) NC 1135 1243 0.3 1031 0.6 1128 1.03422 2.43 22 51.1 5.0 GDP-dissociation inhibitor 1 NC 169 162 0.9 184 0.7 159 0.85711 2.43 19 88.5 6.1 glucocorticoid receptor NC 133 160 0.7 137 0.9 147 0.93507 1.65 12 52.6 5.1 glucokinase NC 135 136 0.9 83 0.3 154 0.67101 1.31 27 25.9 6.1 glutathione S-transferase Yb4 gene NC 4724 4464 0.2 4337 0.3 3715 0.32806 2.43 16 91.2 5.1 GRIP-associated protein 1 short form NC 613 459 0.4 520 0.6 367 0.32506 2.43 43 50.8 4.9 H�-transporting ATP synthase; beta

chain, mitochondrialNC 3507 3753 0.5 3559 0.8 3503 1.0


Table 3. Continued

SSP z-Score


250mg/m3

p 500mg/m3

p 1000mg/m3

p

5001 2.43 56 14.8 5.9 heart fatty acid binding protein NC 4956 5034 0.8 5604 0.2 5314 0.23717 2.43 43 71.1 5.4 heat shock 70 kDa protein 8; Heat

shock cognate protein 70NC 4532 5719 0.2 4038 0.7 5072 0.5

3505 2.43 19 57.8 4.8 heat shock factor 2 � 741 936 0.01 852 0.3 895 0.23704 2.43 24 85.2 4.9 heat shock protein 86 �/� 2278 1034 0.3 2987 0.05 2657 0.72711 2.07 29 85.2 4.9 heat shock protein 86 � 3103 4460 0.2 4794 0.2 5342 0.059201 1.56 21 29.2 6.5 hydroxyacyl glutathione hydrolase;

round spermatid protein RSP29NC 2957 2839 0.7 3030 0.9 3421 0.2

9203 2.28 32 30.9 7.8 inducible carbonyl reductase NC 1198 1241 0.8 1509 0.21 1535 0.23722 1.39 15 71.7 5.4 integrin beta-7 subunit � 279 604 0.02 632 0.01 472 0.2107 2.11 28 22.5 4.9 interleukin-18 � 467 556 0.04 595 0.007 496 0.53816 2.15 4 145.4 5.3 JNK/SAPK-associated protein-1

(Mus musculus)NC 238 209 0.7 434 0.4 337 0.4

4218 1.90 45 36.9 5.7 lactate dehydrogenase B � 4467 5298 0.03 4660 0.9 5033 0.43609 2.43 17 66.8 5.2 lamin B1 � 574 816 0.002 597 0.8 846 0.21110 2.43 57 27.9 4.7 mitochondrial import stimulation factor

S1 chainNC 466 955 0.3 838 0.3 602 0.6

4303 2.43 21 39.6 5.3 NDRG1 related protein NDRG2a2 NC 481 546 0.7 474 1 530 0.58609 2.43 14 71.3 6.7 nicotinic acetylcholine receptor

alpha 4 subunit� 264 658 0.4 971 0.1 2020 0.001

4409 2.43 40 49.6 5.4 nuclear RNA helicase, DECD variant ofDEAD box family

NC 315 318 1 406 0.2 323 0.9

4819 2.43 37 95.2 5.5 Osmotic stress protein 94 (Heat shock70-related protein APG-1)

NC 2908 3001 0.7 2765 0.6 3217 0.3

4822 2.43 33 95.2 5.5 Osmotic stress protein 94 (Heatshock 70-related protein APG-1)

� 2403 2767 0.06 2501 0.7 2680 0.4

3406 1.40 27 53.6 5.4 peripherin NC 1133 1353 0.2 1324 0.3 1290 0.35103 2.43 44 24.9 6.0 peroxiredoxin 5; anti-oxidant protein 2 NC 492 500 0.9 692 0.4 654 0.63104 2.28 48 20.9 5.5 phosphatidylethanolamine binding

proteinNC 13526 14393 0.5 14026 0.5 13957 0.6

3103 1.64 26 19.9 5.1 proline-rich protein NC 3893 5581 0.3 5464 0.3 5433 0.35415 2.43 44 49.8 5.0 proteasome (prosome, macropain)

26S subunit, ATPase 3NC 565 797 0.2 559 0.9 776 0.1

8209 1.67 17 23.1 7.0 proteasome (prosome, macropain)subunit, beta type, 2

NC 1199 1410 0.5 1170 0.9 1480 0.4

6501 2.43 26 57.1 5.9 Protein disulfide isomerase (Disulfideisomerase ER-60) (ERp60)

NC 2008 3674 0.3 1664 0.8 3344 0.4

2411 1.32 26 47.6 4.9 Protein disulfide isomerase (Proteindisulfide isomerase P5)

NC 1651 1549 0.7 1672 0.9 1983 0.1

3601 2.43 23 73.1 5.0 protein disulfide isomerase relatedprotein

NC 355 1534 0.2 859 0.3 872 0.3

509 1.68 19 45.5 4.3 protein kinase C-binding proteinZeta1

� 171 256 0.01 255 0.002 155 0.8

4307 2.43 37 35.9 5.1 Recombinant Rat Annexin V, TripleMutant (T72k, S144k, S228k)

NC 1739 1776 0.8 1418 0.3 1688 0.8

7006 2.43 50 14.8 6.8 ribosomal protein S12, cytosolic NC 3678 3762 0.8 3084 0.3 3045 0.34609 1.43 29 59.4 5.4 RP58 protein NC 2398 2790 0.2 2178 0.5 2544 0.78312 2.43 31 38.8 7.9 serine dehydratase NC 5737 5970 0.8 6236 0.7 7425 0.3


Table 3. Continued

SSP z-Score


250mg/m3

p 500mg/m3

p 1000mg/m3

p

4807 1.70 11 183.1 5.5 serine proteinase (EC 3.4.21.-)PC6B – mouse

NC 682 875 0.3 607 0.8 804 0.8

6615 1.39 28 68.7 6.4 Serum albumin � 21471 18429 0.5 29682 0.2 41175 0.055611 1.34 31 53 5.5 serum albumin – mouse (fragment) NC 741 1090 0.2 1454 0.3 2424 0.37607 1.87 7 65.4 6.0 SNERG-1 protein, Sertoli cells NC 3927 3838 0.7 3389 0.1 2743 0.38315 2.43 26 38.8 6.8 sorbitol dehydrogenase NC 1600 1687 0.8 1497 0.8 1505 0.83503 1.52 17 59.4 5.2 sorting nexin I NC 1623 1947 0.08 1756 0.4 1821 0.32416 1.50 19 49.8 5 spermatogenic cell/sperm-associated

Tat-binding protein� 350 427 0.04 461 0.007 387 0.3

5609 2.43 26 60.9 5.9 T-complex polypeptide 1 � 545 632 0.2 583 0.5 789 0.05622 2.43 43 69.8 5.4 testis-specific heat shock protein-

related gene hst70NC 274 196 0.5 290 0.9 175 0.2

2604 2.43 46 69.8 5.4 testis-specific heat shock protein-related gene hst70

NC 178 69 0.1 529 0.4 506 0.5


NC 12921 13577 0.8 8483 0.2 18068 0.1


NC 631 834 0.3 1098 0.1 840 0.3


NC 1223 1170 0.8 799 0.2 912 0.3

5107 2.43 68 24.9 5.6 thiol-specific antioxidant protein;1-Cys peroxiredoxin

NC 2612 3990 0.2 3633 0.3 3249 0.6

3014 1.74 42 11.3 5.1 translocase of inner mitochondrialmembrane 8

NC 530 430 0.5 402 0.5 776 0.5

2411 1.31 29 50.8 4.9 Tubulin alpha NC 1651 1549 0.7 1672 0.9 1983 0.12401 2.43 31 50.4 4.8 Tubulin beta chain 15 NC 497 759 0.1 569 0.6 359 0.45110 1.82 23 20.2 5.6 ubiquinone biosynthesis protein coq7

homologNC 2209 2509 0.3 2359 0.7 2046 0.7

8612 2.43 15 67.9 6.6 vacuolar protein sorting protein 33a NC 2197 2306 0.9 2432 0.7 2932 0.39603 2.43 12 67.9 6.6 vacuolar protein sorting protein 33a NC 1474 1376 0.7 1820 0.3 1185 0.26810 2.36 37 117.3 5.8 Vinculin (Mus musculus) NC 1652 1759 0.7 1873 0.4 2027 0.2

SSP = spot number in PDQuest; Z-score from ProFound; %= sequence coverage; (�) indicates abundance increase and(�) decrease relative to control

In view of the temperature sensitivity of spermatogenesis,it is not surprising that the major stress proteins (hsp andgrp) are commonly expressed in the testis of rodent.These include 70 kDa hsp-1, hsp-2, and hsp-3; 70 kDatestis-specific hsp (hsp70t); 70 kDa heat shock cognateprotein (hsc70); 75 kDa glucose-regulated protein (grp75);78 kDa glucose-regulated protein (grp78) [35]; and heatshock 70-related APG-1 (osmotic stress protein 94) [36]as well as regulators of the stress response such as heatshock factors 1 and 2 [37]. Repeated exposure of malerats to JP-8 resulted in differential expression of severalproteins related to this stress protein family. Increased

expression of heat shock factor 2 (low dose), totalhsp70t (e.g., sum of SSP 622, 2604, 4603, 4608, 4611charge isoforms, see Table 3) (high dose), Grp78 (lowdose), and Hsp86 (moderate and high doses) suggests ageneralized cellular stress response at those individualexposure levels. In addition, T-complex polypeptide 1(TCP-1, also known as chaperonin containing TCP-1(CCT)) expression rose at high-dose exposure. TCP-1 isclassified as one of the Type II molecular chaperones,the variety that has a specific role in the folding of newlysynthesized tubulins and actins [38] and in the mouse isencoded by testis-specific gene (Cctz-2) [39] identified


here. Previously, in in vitro test systems, TCP-1 levelswere up-regulated under continuous chemical stresswith sodium arsenite as well as during recovery fromchemical stress caused by sodium arsenite or a prolineanalogue, L-azetidine-2-carboxylic acid [40]. These re-sults suggest that the stress proteins and chaperonesmentioned above all may play a role in the recovery ofcells from possible protein damage inflicted by JP-8,assisting in the folding of proteins that are actively synthe-sized and/or renatured as a consequence of prolongedexposure to the volatile components of JP-8. The slight(22%) but significant increase in Tat binding protein-1(TBP-1) may be related to the response described above.TBP-1 is abundant in the testis and shows particularexpression in spermatogonia, spermatocytes, sperma-tids and epididymal sperm [41]. TBP-1 function is linkedto the 26S proteasome and appears to be involved innumerous germ cell activities including ATP-dependentdegradation of ubiquinized proteins [42]. Surprisingly,the abundance of another chaperone, the endoplasmicreticular 150 kDa oxygen-regulated protein (ORP150),was decreased, particularly at the moderate JP-8 ex-posure. Though the mechanism is unclear, in neuronsORP150 is involved in cytosolic free calcium regulation[43], is induced by hypoxia/ischemia, and is absent innormoxia. The reason for ORP150’s decreased expres-sion in response to JP-8 is likewise unclear.

The interleukin-1 system is present in testis [44] and isspeculated to be involved in autocrine, paracrine andendocrine regulation of testicular cells and indirectlythe process of spermatogenesis. However, interleukin-18(identified as SSP 107, Table 3) is associated with re-sponses in tissue injury and induces apoptosis [45] inresponse to JP-8 exposure. Although an increase inapoptosis was not observed in the present study, itshould be noted that both interleukin-18 and lamin B(SSP 3609, Table 3) are associated with the induction ofapoptosis [46]. This raises the possibility that JP-8 expo-sure, which has already been shown to induce apoptosisin the lung [47, 48], may also have induced the beginningstages of apoptosis in the exposed testis. Any directeffect of interleukins on Leydig cell function followingJP-8 exposure was not evident, as there did not appearto be morphological changes in the Leydig cell population(unpublished observation). Lamin B is found along thenuclear envelop of all cell types in the seminiferous tubule,including Sertoli cells, primitive type A spermatogonia,preleptotene, leptotene, zygotene and pachytene sper-matocytes, and round spermatids [48].

Lamin B1 is a protein kinase c (PKC) binding protein [49],but nothing is known regarding this activity in the testis.However, it is noteworthy that JP-8 caused changes in

the expression of another PKC binding protein in the tes-tis, Zeta1 (SSP 509), a 50% increase at low and moderateexposure. PKC binding proteins are abundant in the testisstarting with the onset of spermiogenesis with highexpression of specific mRNA in the elongating spermatids[50]. Changes in these proteins following JP-8 exposureraise the possibility that subtle effects were taking placein the Sertoli cell cytoplasm or in the developing germcells both of which depend on stable microtubule forma-tions for normal spermatogenesis. Another up-regulated(50%) cytoskeleton-related protein, A-kinase anchor pro-tein (AKAP) (SSP2909, Table 3), is expressed in high levelsin the testis and is closely associated with the microtubuleelements of the sperm flagellum [51]. Interestingly, it hasalso been suggested that AKAP 220 may play a role intargeting type II PKA for cAMP-responsive peroxisomalevents [52]. The nature of its altered expression here iselusive, but certainly warrants further investigation ofpotential long-term effects of JP-8 on spermatozoa for-mation and maturation.

The nearly eight-fold increase in alpha 4 subunit of thenicotinic acetylcholine (Ach) receptor in the high-doseJP-8 exposure group is difficult to explain. The testisdoes not appear to contain cholinergic nerve fibers [53],but does contain nACh receptors [54]. These receptorscan be found in blood vessels, mature germ cells and fla-gella of the sperm but not in the interstitial cells. Thoughnicotine has been shown to exert a negative effect ontesticular function [55], and alpha nACh is up-regulatedby interferon-gamma in the thymus [56], the underlyingreason for an apparent JP-8 related up-regulation of thenACh subunit in the testis remains unclear.

Integrins are ubiquitously expressed on the surface ofcells and enable cell-to-cell communication. The identifi-cation of SSP 3722 as the �7 integrin is somewhat sur-prising as this integrin type is localized in the lymphocytewhere it is involved in homing [57]. Numerous subtypesare found in the testis [58], but integrin �7 has not beendescribed previously. A low abundance protein presentin control testis, its increased abundance at low and mod-erate JP-8 exposures may be related to an injury-relatedincrease in the local lymphocyte population. However, it isalso possible that, despite a reasonably high Z-score of1.39 (92%), �7 may have been misidentified and is actu-ally one of the resident integrins. A potential explanationfor changes in integrin expression is found in one of themore subtle observations that the lumen of certain semi-niferous tubules was dilated after JP-8 exposure. It is wellestablished that integrins are involved in mechanicalstress to cells and tissues and thus, stretching of theseminiferous epithelium may have altered the expressionof the integrin family of genes in the testis [59].


The effects of JP-8 on the male reproductive systemdescribed above are subtle. We are in the process ofanalyzing the testis in more detail but we currently havepreliminary data suggesting that JP-8 may have inducedan effect not only in the testis (proteomic response), butalso an effect on the excurrent ducts of the testis. Thiseffect would be related to fluid reabsorption in the headof the epididymis, as evidenced by the accumulation offluid in the rete testis and seminiferous tubule lumens(Hess, unpublished results). The basic pathophysiologicalmechanism involves the inhibition of fluid reabsorption bythe efferent ductule epithelium. This epithelium is respon-sible for reabsorbing nearly 96% of the luminal fluidsarriving from the rete testis, or the seminiferous tubuleeffluent [60]. Disruption of this physiology results in eitherthe accumulation of fluid or total occlusion, both of whichcreates back pressure on the rete testis and seminiferoustubules, which ultimately will lead to atrophy of the testis[61]. Occlusion of the efferent ductules is not likely to haveoccurred in this study because testicular swelling or evendoubling of testicular weight would have been a rapidresponse, which was not observed. Therefore, the mostlikely event was an inhibition of fluid reabsorption withsubsequent accumulation of luminal fluid, which wouldexplain the dilation of rete testis and seminiferous tubules.This is supported by the observation that serum albumin(SSP 6615, Table 3) was increased (nearly two-fold) inthe high-dose JP-8 treated testis. Such a response couldindicate that fluid transport or even blood flow was dis-rupted, particularly in view of the fact that serum albuminis abundant in the male reproductive tract, with con-centrations reaching 17% of the rete testis fluid in theram [62].

The observation that repeated low-dose JP-8 vapor ex-posure resulted in far more proteins with increased abun-dance than did exposure to the highest dose, and thatexposure to the highest dose level resulted in significantlyreduced abundance of twice as many proteins as thelow dose exposure (Table 2) is difficult to explain but notunusual. This phenomenon has been observed consis-tently with JP-8 vapor and aerosol exposures in otherorgans (unpublished results) and its explanation requiresadditional study. Whether the alterations are related toinjury versus adaptive or repair mechanisms, or perhapsto a more complex hormetic [63] response remains to bedetermined.

4 Concluding remarks

Previously published studies have reported male repro-ductive system toxicity in humans or animals exposed tohydrocarbon solvents. JP-8 jet fuel is a complex mixturecontaining over 228 hydrocarbon constituents, three per-

formance additives, and at least four of the solvents pre-viously identified as toxic to the male reproductive sys-tem. We used a 2-D gel based proteomic approach toinvestigate the potential reproductive toxicity in male ratsof 91 d (6 h/d) exposure to JP-8 in vapor phase. The goalof this animal research was identification of changes inprotein abundance that may, in humans, predict subtleacute changes, or more complex chronic changes inreproductive system integrity or function. In the rat testis,it was clearly demonstrated that JP-8 vapor exposure,mimicking human occupational exposure scenarios, issufficient to induce significant changes in the abundanceof a number of different proteins with diverse or, in somecases, unknown functional significance. In the majority ofcases, significant changes in the abundance of a specificprotein occurred to one or two of the three JP-8 expo-sures concentrations, and in many cases there was anonlinear dose response. A simple explanation for thisobservation is that JP-8 vapor is itself a complex mixturethat varies in content as a function of the concentrationgenerated. It has been demonstrated, for example, thatsignificant antagonism in toxic effects on the reproductivesystem can occur among hydrocarbon solvents duringcertain concurrent exposures. This suggests that evensubtle changes in the formulation of widely used jet fuels,including changes in performance additive packages(e.g., ongoing conversion from JP-8 to the JP-8�100 for-mulation) and batch variations among and within jet fuelmanufacturing operations (i.e., manufacturing to meetfuel performance and not fuel content requirements),may require toxicological evaluation prior to widespreadimplementation.

While there is minimal evidence of reproductive toxicityin humans exposed occupationally to JP-8, there have,however, been no published efforts to scientifically evalu-ate this possibility. Thus, the protein abundance modula-tions observed in the present 91 d animal study are some-what disquieting, raising the possibility that, by extra-polation, JP-8 may have significant effects in humans,especially when exposed to the fuel during careers thatmay exceed 30 years. Admittedly, the approach takenhere is limited in its sensitivity to very low abundance pro-teins, and the nature and functional significance of identi-fied changes in protein expression remains to be fullyexplained by further research, such as field testing ofcareer fuel workers for subtle changes in reproductivesystem function.

This effort was supported by grants or general fundingfrom the Office of Naval Research (ONR), the Navy Envi-ronmental Health Center (NEHC), and the US Air ForceOffice of Scientific Research (AFOSR �F49620-96-1-0156). The opinions expressed in this paper are strictlythose of the authors and do not necessarily represent the


views of the Indiana University School of Medicine, theUniversity of Illinois, the US Navy, Department of Defense,or the military at large.

Received October 15, 2002

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Analysis of rat testicular protein expression following 91-day exposure to JP-8 jet fuel vapor

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