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Published ahead of print on December 23, 2004, doi:10.1165/rcmb.2004-0171OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 32, pp. 192-200, 2005
© 2005 American Thoracic Society
DOI: 10.1165/rcmb.2004-0171OC

Altered Expression of {gamma}-Synuclein and Detoxification-Related Genes in Lungs of Rats Exposed to JP-8

Luis A. Espinoza, Mohammad Valikhani, María J. Cossio, Theresa Carr, Mira Jung, Juanita Hyde, Mark L. Witten and Mark E. Smulson

Department of Biochemistry and Molecular Biology, and Department of Radiation Medicine, Georgetown University School of Medicine, Washington, DC; and Joan B. and Donald R. Diamond Lung Injury Laboratory, Department of Pediatrics, Arizona Health Sciences Center, Tucson, Arizona

Correspondence and requests for reprints should be addressed to Dr. Mark Smulson, Department of Biochemistry and Molecular Biology, Georgetown University School of Medicine, 3900 Reservoir Road NW, Washington, DC 20057. E-mail: smulson{at}georgetown.edu


   Abstract
Top
 Abstract
Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Many military personnel are at risk of lung damage or systemic toxicity as a result of exposure to the jet fuel JP-8. We have now used microarray analysis to characterize changes in the gene expression profile of lung tissue induced by exposure of rats to JP-8 at a concentration of 171 or 352 mg/m3 for 1 h/d for 7 d, with the higher dose estimated to mimic the level of occupational exposure in humans. The expression of 56 genes was significantly affected by a factor of <= 0.6 or >= 1.5 by JP-8 at the low dose. Eighty-six percent of these genes were downregulated by JP-8. The expression of 66 genes was similarly affected by JP-8 at the higher dose, with the expression of 42% of these genes being upregulated. Prominent among the latter genes was that for the centrosome-associated protein {gamma}-synuclein, whose expression was consistently increased. The expression of various genes related to antioxidant responses and detoxification, including those for glutathione S-transferases and cytochrome P450 proteins, were also upregulated. The microarray data were confirmed by quantitative RT-PCR analysis. Our extensive data set may thus provide important insight into the pulmonary response to occupational exposure to JP-8 in humans.

Key Words: JP-8 jet fuel • oligonucleotide microarrays • immunohistochemistry • {gamma}-synuclein • quantitative RT-PCR


   Introduction
Top
 Abstract
 Introduction
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Persistent exposure of the airway epithelium to environmental pollutants, such as aromatic hydrocarbons, may have deleterious effects on pulmonary function (1) and induce pathologic changes in lung tissue (2). JP-8 is a kerosene-based jet fuel that comprises a complex mixture of aliphatic and aromatic compounds and is extensively used by U.S. and NATO forces. Aircrew personnel routinely exposed to this fuel under normal working conditions possess chronically increased levels of fuel constituents in their breath (3). JP-8 contains several additives required by the military, including a corrosion inhibitor and lubricity enhancer, a fuel-system icing inhibitor, and a static dissipater, that are not present in the Jet A-1 fuel used by the commercial airline industry. The relatively low vapor pressure (1.8 mm Hg) and high flash point (38°C) of JP-8 are associated with a reduced potential for crash-related explosions, fires, and evaporative losses (4). The physical and chemical characteristics of JP-8 thus make it a desirable alternative to the JP-4 fuel used previously. However, the reduced vaporization of JP-8 increases its bioavailability, and the opportunities for human exposure are considerable.

Exposure to JP-8 has been associated with severe mental fatigue, headache, and skin irritation in pilots and other personnel who come into contact with this fuel (5). In addition, exposure to JP-8 at noncytotoxic levels induces loss of airway epithelial barrier function, an increase in respiratory permeability, dilation of intercellular spaces, and pulmonary edema (6, 7). Proteomics-based analyses of lung tissue of mice subjected to simulated occupational jet fuel exposure have revealed quantitative and qualitative differences in the expression of proteins that play roles in toxic or metabolic stress, detoxification, or cell structure, proliferation, or apoptosis (8, 9).

JP-8 exposure also has adverse effects on the immune system, as revealed by decreases in the weight of the spleen and thymus, loss of viable immune cells, and impairment of immune function (10, 11). Mice subjected to controlled JP-8 inhalation thus exhibit an increased susceptibility to infection (11), immunosuppression (12), and altered immunologic memory function (13). Such immune impairment might be expected to promote tumorigenesis in the lungs of individuals who are continuously exposed to JP-8, given also that long-term dermal exposure of mice to petroleum middle distillates has been shown to result in an increase in skin tumor incidence (14). Exposure to JP-8 was recently shown to result in similar levels of benzene and higher levels of naphthalene in urine compared with those found in individuals exposed to cigarette smoke (15).

We have previously shown that exposure of several human and murine cell lines, including in a rat lung alveolar type II epithelial cells (RLE-6TN), to JP-8 in vitro induces biochemical and morphologic markers of apoptotic cell death such as activation of caspase-3, cleavage of poly(ADP-ribose) polymerase, chromatin condensation, membrane blebbing, release of cytochrome c from mitochondria, and cleavage of genomic DNA (16). Generation of reactive oxygen species (ROS) and depletion of intracellular reduced glutathione (GSH) were also shown to play important roles in the induction of programmed cell death by JP-8 (17). With the use of microarray analysis, we further demonstrated that human Jurkat T cells exposed to JP-8 manifested pronounced changes in the expression of genes related to the cellular response to oxidative stress or to apoptosis (18). In contrast, similar analysis with normal human epidermal keratinocytes revealed that exposure to JP-8 induced changes in the expression of genes whose products function in detoxification or regulation of cell growth (19). These findings might be relevant to the relative resistance of keratinocytes to JP-8 toxicity and support the notion that the induction of cell death by JP-8 may be cell type–specific (20).

Despite the detrimental effects of JP-8 on the pulmonary system in both humans and laboratory animals (11, 12, 21), the changes in gene expression in the lungs that underlie these effects have not previously been investigated systematically. We have therefore now applied the microarray approach to identify genes whose expression is substantially altered in the lungs of rats subjected to direct inhalation of JP-8 under controlled conditions.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 References
 
JP-8 Aerosol Generation and Animal Exposure
An aerosol of JP-8 (Wright-Patterson Air Force Base, Dayton, OH) was generated with an Ultra-Neb 99 nebulizer (DeVilbiss, Somerset, PA) and was allowed to mix with ambient air. The fuel–air mixture was drawn through a 12-port nose-only exposure chamber (IN-TOX, Albuquerque, NM) with the use of a constant vacuum. The JP-8 aerosol was characterized as described previously (21). Fischer 344 rats were exposed in quadruplicate for 1 h/d for 7 d to JP-8 at a mean concentration of 171 or 352 mg/m3, the latter treatment corresponding to a typical human exposure over a 7-d work period at an air base (10). The rationale for the first treatment was to assess whether the gene expression patterns, which are affected at the higher dose, are maintained or negatively altered at the much lower exposure dose. The animals were subjected to nose-only exposure to minimize oral ingestion of JP-8 during grooming and to simulate more closely occupational exposure in humans. A group of control rats was exposed in a similar manner to air only. At the end of the 7-d exposure period, the lungs of each rat were perfused with ice-cold PBS, removed, and rapidly placed in separate tubes containing RNAlater (Ambion, Austin, TX) at room temperature. All rats were housed in an American Association for Accreditation of Laboratory Animal Care–approved animal facility of the Department of Animal Resources at the University of Arizona Health Sciences Center.

Histology
Other animals in our study were subjected to analysis of lung pathology after exposure to JP-8. The lungs were fixed by intratracheal instillation of half-strength Karnovsky's fluid at a constant pressure of 20 cm H2O for 1 h. The trachea was then ligated and the lungs were incubated in fixative overnight. The fixed tissue was sliced, dehydrated, and embedded in paraffin for routine light microscopy. Adjacent slices were minced, exposed to osmium tetroxide, dehydrated, and embedded in Epon-Araldite (EM Science, Darmstadt, Germany) for examination with a Phillips CM12 electron microscope.

Immunohistochemistry
Formalin-fixed paraffin-embedded tissue sections were deparaffinized in xylene and dehydrated through a graded series of ethanol dilutions. Endogenous peroxide was blocked by incubation of samples in 0.3% hydrogen peroxidase. Tissues were then preblocked with normal rabbit serum at room temperature, followed by overnight incubation at 4°C with a polyclonal anti–{gamma}-synuclein goat antibody (dilution 1:50 in PBS; Santa Cruz, Santa Cruz, CA) or polyclonal anti-P450IIE1 rabbit antibody, kindly provided by Dr. Byoung J. Song (Laboratory of Membrane Biochemistry and Biophysics, National Institute of Alcohol Abuse and Alcoholism, NIH). Proteins were visualized using biotinylated antibodies in conjunction with the ABC system (Vector Laboratories, Burlingame, CA) followed by use of diaminobenzidine tetrahydrochloride as the chromogen. Subsequently, the sections were counterstained with hematoxylin, dehydrated, and mounted.

Isolation of RNA
Both lungs of each animal were collected, and total RNA was extracted with Trizol reagent (Invitrogen, Carlsbad, CA) and was purified with the use of an RNeasy Mini Kit (Qiagen, Valencia, CA). The concentration of the isolated RNA were determined by measurement of absorbance at 260 nm, and its high quality was verified by electrophoresis on a 1% agarose gel and ethidium bromide staining.

Microarray Analysis
Microarray analysis was performed using a GeneChip Rat Genome U34 Array A (Affymetrix, Santa Clara, CA), which contained > 8,000 probes including those targeted to ~ 7,000 full-length mRNAs and ~ 1,000 expressed sequence tags (ESTs). For each condition, four independent samples of 10 µg of total RNA were used to prepare the first strand of cDNA by incubation for 1 h at 42°C with purified T7-(dT) primer (Affymetrix) and SuperScript II reverse transcriptase (200 U/ml; Invitrogen). Second-strand synthesis was performed at 16°C for 2 h. The resulting double-stranded cDNA was then purified with a GeneChip Sample Cleanup Module (Qiagen) before use as a template for the synthesis of biotin-labeled cRNA with a BioArray RNA Transcript Labeling Kit (Enzo, New York, NY). The labeled cRNA was purified with the GeneChip Sample Cleanup Module, fragmented, and then subjected to hybridization with the microarray.

The microarray chips were scanned with a 428 Array Scanner (Affymetrix) and the data were analyzed with GeneSpring software (Silicon Genetics, Redwood City, CA). Data from each chip were normalized relative to the 50th percentile to eliminate interchip variation in hybridization intensity and to allow comparisons between chips. Data for each gene probe in the JP-8 samples were then normalized relative to the mean gene expression level in the control samples to yield an expression ratio for each gene. Genes with Affymetrix flag calls of "Absent" for more than 50% of the samples have unreliable signals due to nonspecific binding (high levels of Miss-Match signal) or low overall signal, and these were removed before further statistical analysis (Figure 1). Subsequent to flag filtering, in order for a gene's expression level to be considered significantly altered, two criteria were required to be satisfied: (1) differences in gene expression between JP-8 and control samples were assessed with the parametric Welch's t test resulting in a P value of <= 0.05, and (2) the mean expression ratios between JP-8 and control samples surpassed the threshold of <= 0.6 for downregulation or >= 1.5 for upregulation. Genes passing the flag filtering and overcoming the expression level criteria were considered to be biologically meaningful and are reported (Tables 1 and 2).



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  		Figure 1. Data analysis. (A and B) Scatter plots of genes that passed the initial flag-filtering restriction, which removed genes for which nonspecific binding accounted for most of the gene expression signal (signal was not reliable), for lung tissue derived from rats treated with JP-8 at 171 mg/m3 (A) or 352 mg/m3 (B). The plots show mean normalized gene expression for JP-8–treated tissue versus that for control tissue, and were constructed with the GeneSpring program. (C and D) Scatter plots of the 292 and 677 genes that passed the initial flag-filtering restriction and showed a statistically significant difference in expression level between samples of lung tissue of rats treated with JP-8 at 171 mg/m3 (C) or 352 mg/m3 (D) and control samples, respectively. The statistical significance (P <= 0.05) of differences in gene expression between the JP-8–treated and control samples was determined by Welch's t test. Colors represent the fold change in the expression of each gene (JP-8/control), with the green diagonal lines depicting expression ratio of 1.5, 1.0, and 0.6.

 

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  		TABLE 1. Differential expression of genes (classified according to function) in the lungs of rats exposed to JP-8 at a dose of 171 mg/m3

 

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  		TABLE 2. Differential expression of genes (classified according to function) in the lungs of rats exposed to JP-8 at a dose of 352 mg/m3

 
Quantitative RT-PCR Analysis
To confirm the differential expression of genes identified by microarray analysis, we used reverse transcription (RT) and real-time polymerase chain reaction (PCR) analysis. Total RNA (5 µg) was incubated for 50 min at 42°C with SuperScript II reverse transcriptase in a final volume of 20 µl, and the resulting cDNA was subjected to PCR with specific primers designed with the use of Primer Express software (Applied Biosystems, Foster City, CA). The amplification reaction was performed in 96-well optical trays and in a final volume of 25 µl containing 2.5 µl SYBR Green PCR buffer, 1.25 U of AmpliTaq Gold, 0.5 U of AmpErase, 0.96 mM deoxynucleoside triphosphates, 6 mM MgCl2 (SYBR Green PCR Core Reagents Kit; PE Biosystems, Warrington, UK), 0.6 µM forward and reverse primers, and 3 µl of cDNA. Primers specific for glyceraldehyde-3-phosphate dehydrogenase cDNA were used as a control. Real-time monitoring of the reaction was performed with an ABI PRISM 7700 Sequence Detection System (Applied Biosystems). PCR was performed in triplicate and the mean value of the triplicates was used for quantitation.


   RESULTS
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 References
 
Effect of JP-8 on Lung Histology
Light microscopic examination of the lungs of rats exposed to an aerosol of JP-8 at a concentration of 171 or 352 mg/m3 for 1 h/d for 7 d showed deterioration of the alveolar–capillary barrier and sporadic areas of red blood cell accumulation within alveolar spaces (Figure 2A). Occasional formation of perivascular edema was also apparent. Changes in the airway epithelium were minimal. Ultrastructural evaluation of lung tissue focused on alveolar type II epithelial cells and the terminal bronchial airway epithelium (Figure 2B). The number and size of surfactant-producing lamellar bodies appeared to be increased in alveolar type II epithelial cells of rats exposed to JP-8. Osmiophilic inclusion-like bodies were also apparent within the lamellar bodies or vacuoles of these cells in the JP-8–treated animals. There were no apparent differences at the light or electron microscopy level between lung tissues exposed to JP-8 at the low or higher dose (data not shown).



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  		Figure 2. Histology of lung tissue derived from rats exposed to JP-8 at a concentration of 352 mg/m3. (A) Light microscopy. Staining with hematoxylin-eosin revealed alveolar infiltration of red blood cells and pulmonary edema with areas of marked alveolar collapse and moderate perivascular edema. Original magnification: x100. (B) Transmission electron microscopy. Alveolar type II epithelial cells (E2) manifested an increase in the size and number of lamellar bodies (L) as well as vacuolization of the endoplasmic reticulum (ER) (1-µm thickness x 100).

 
Transcriptional Profiling of the Rat Lung Response to JP-8
We compared the gene expression profiles of lung tissue isolated from rats exposed to JP-8 at 171 or 352 mg/m3 with that of lung tissue derived from control animals exposed to air alone. The jet fuel significantly affected the expression of 56 and 66 genes at the low (Table 1) and higher (Table 2) doses, respectively. The expression of most (48/56, 86%) of the genes affected by JP-8 at 171 mg/m3 was downregulated, whereas that of 42% (28/66) of the genes affected by JP-8 at 352 mg/m3 was upregulated. The affected genes were classified according to their biological function, being divided into eight or nine groups for the low and high doses of JP-8, respectively (Tables 1 and 2). The diverse biological functions of the affected genes likely reflect the chemical complexity of the fuel.

The lungs of rats exposed to JP-8 at the occupationally relevant dose of 352 mg/m3 manifested a 5.5-fold increase in expression of the gene for {gamma}-synuclein, a centrosomal protein that plays an important role in the regulation of cell growth and that is present at a relatively low level in normal lung tissue. The expression of genes whose products contribute to the cellular response to oxidative stress or to toxicants, including glutathione S-transferases (Gsta1, GstaYc2) and cytochromes P450 (CYP2C13, Cyp2e1), was also prominently increased in lung tissue from rats exposed to JP-8 at the higher dose. The expression of these genes has been shown to be induced by Nrf2, a transcription factor that protects against oxidative or chemically induced cellular damage in various organs, including the lungs (22). In contrast, the expression of none of the apoptosis-related genes represented on the microarray was affected by JP-8 at either dose, consistent with the results of the histologic analysis showing minimal cell damage in the lungs of the JP-8–treated rats. The abundance of mRNAs for various structural proteins, including myosin heavy chain 7 (Myh7), {alpha}-actin, and ß-actin, was increased by exposure to JP-8 at 352 mg/m3, whereas that of the mRNA for high molecular weight microtubule-associated protein 2 (HMW-MAP2) was decreased. Expression of the gene for the inositol 1,4,5-trisphosphate receptor (InsP3R1), a ligand-gated Ca2+ channel that mediates the release of Ca2+ from intracellular stores (23), was reduced by a factor of 10 in the lungs of rats exposed to JP-8 at 352 mg/m3. In this regard, we have previously shown that JP-8–induced fragmentation of DNA during apoptosis in RLE-6TN cells is Ca2+-dependent (16, 17). Expression of the genes for aquaporin 1 (Aqp1) and aquaporin 4 (Aqp4), proteins that mediate water transport in various tissues including the lungs, was increased and decreased, respectively, in the lungs of rats exposed to JP-8 at 352 mg/m3.

Validation of JP-8–Induced Changes in Gene Expression by Quantitative RT-PCR
To confirm the validity of the JP-8–induced changes in gene expression detected by microarray analysis, we examined the abundance of selected gene transcripts by RT and real-time PCR with gene-specific primers. Quantitative analysis of the RT-PCR data yielded differential expression ratios highly similar to those obtained by microarray analysis (Figure 3). The results of these experiments demonstrated that expression of all these genes were significantly altered in lung tissues exposed to JP-8 relative to the control samples.



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  		Figure 3. Confirmation of microarray data by RT and real-time PCR analysis. The abundance of transcripts of selected genes identified by microarray analysis was examined in lung tissue isolated from rats exposed to JP-8 at 171 mg/m3 (A) or 352 mg/m3 (B) by quantitative RT-PCR analysis. The amount of each mRNA in each JP-8 sample was normalized by that of glyceraldehyde-3-phosphate dehydrogenase mRNA and was then expressed relative to the corresponding value for control tissue (solid bars). Data are from an experiment that was repeated a total of three times with samples from three different animals per condition. The differential gene expression ratios (JP-8/control) obtained by microarray analysis are shown for comparison (open bars).

 
Validating JP-8–Induced Changes in Lung Tissue at the Protein Level by Immunohistochemistry
To demonstrate that the protein level validate the expression ratios from our microarray data we evaluated {gamma}-synuclein and P450IIE1 levels by immunohistochemistry (Figure 4). At a JP-8 jet fuel concentration of 352 mg/m3 we found the first signs of pathologic injury at the terminal brochioles, which consisted of minor changes in the airway epithelium. This finding is consistent with the present studies of immunohistochemical measurements of {gamma}-synuclein (Figures 4B–4E) and P450IIE1 (Figures 4G and 4H) at the same site. The immunoreactivity for both proteins was higher in lung tissue samples from animals treated with the JP-8 concentration of 352 mg/m3 than in control samples. Our results confirmed increased levels of these proteins in agreement with the microarray and RT-PCR results (Table 2 and Figure 3).



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  		Figure 4. Representative immunostaining of {gamma}-synuclein and P450IIE1 distribution in Fischer 344 lung tissues exposed to 352 mg/m3 JP-8 jet fuel by immunoalkaline phosphatase assay. Lung tissues samples of Fischer 344 rat exposed to room temperature air that were stained with {gamma}-synuclein (A) and P450IIE1 (F) antibodies, respectively. Immunohistochemical staining of {gamma}-synuclein (B–E) and P450IIE1 (G and H) in lung tissue samples of Fischer 344 rat exposed to 352 mg/m3 JP-8 jet fuel. Magnification: A–C, F–H, x10; D and E, x40.

 

   DISCUSSION
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
References
 
Recent studies performed both in vitro and in vivo have shown that JP-8 exposure induces apoptotic cell death as well as pathologic injury in both lung cell lines and tissue (15, 17). Furthermore, personnel exposed to this fuel exhibit levels of JP-8 components in their breath that are 40 times greater than ambient levels (3). In the present study, with the use of microarray analysis, we have determined the gene expression profiles of lung tissue of rats subjected to short-term intermittent exposure to JP-8 at two different concentrations in a simulated occupational exposure model. Among the > 8,000 transcripts analyzed, we identified 122 mRNAs whose abundance was significantly increased (>= 1.5-fold) or decreased (<= 0.6-fold) by JP-8, suggesting that JP-8 toxicity is mediated by several signaling pathways. We will focus our discussion on the results obtained with the JP-8 concentration of 352 mg/m3, because this dose more closely mimics that experienced by air force personnel exposed to this fuel.

The gene whose expression was upregulated to the greatest extent by JP-8 at 352 mg/m3 was that for {gamma}-synuclein. This protein is abundant in the peripheral nervous system, but is also expressed at a relatively low level in normal breast, ovary, and lung tissue (24, 25). Synuclein-{gamma} is a centrosome-associated protein that has been implicated in the regulation of intracellular vesicular trafficking (26) as well as in signal transduction in neuronal and non-neuronal cells (25). Microarray analysis recently showed that expression of the {gamma}-synuclein gene was upregulated in the brain of rats treated with cocaine (27). We feel it is quite novel to have found a marked expression in lung tissues exposed to inflammatory conditions. Furthermore, {gamma}-synuclein was found to promote cancer cell survival and to inhibit apoptosis induced by cellular stress or chemotherapeutic drugs through modulation of intracellular signaling by mitogen-activated protein kinase (MAPK) pathways (28). In the present study the increases in lung permeability (21) as well as the morphologic changes associated with impaired barrier function (perivascular edema) and the early signs of pulmonary fibrosis (apparent from an increase in the number and size of surfactant-secreting lamellar bodies) (2, 29) induced by inhalation of JP-8 might be related to modification of the cytoskeleton by overexpression of {gamma}-synuclein in lung cells.

Glutathione S-transferases (GSTs) mediate detoxification by catalyzing the conjugation of xenobiotics and their electrophilic metabolites with GSH, and they play an important protective role against toxicants in the lung (30). Consistent with such a role, we have now shown that the expression of the genes for Gsta1 and GstaYc2 was increased in the lungs of rats exposed to the higher dose of JP-8. Similarly, the abundance of transcripts for the cytochrome P450 isoforms CYP2C13 and Cyp2e1 was increased by JP-8 exposure. The cytochrome P450 family of proteins efficiently metabolizes certain aromatic and hydrophobic components of JP-8, such as naphthalene. Therefore its presence in the lung was not unexpected, because it undoubtedly plays a role in bioactivation of JP8 components and thus contributes to reduction in oxidative stress in this organ when exposed to the fuel. Indeed, increased levels of 1- and 2-naphthol have been detected in the urine of individuals exposed to JP-8 (15). Previous proteomics analyses have also shown that the expression of GSTs is markedly increased in lung tissue from mice and in brain tissue from rats exposed to JP-8 (9, 31). Repeated inhalation of toxicants might be expected to result in the generation of high levels of ROS by inflammatory cells in the airways and alveolar spaces and in the consequent depletion of endogenous antioxidants. Such an excess of free radicals can result in deficits in pulmonary cell function that may lead to various pulmonary diseases. We previously showed that exposure of human Jurkat T cells to JP-8 induced the expression of various genes related to the cellular stress response or apoptosis (18). We have also previously shown that JP-8 induces the generation of ROS, collapse of the mitochondrial membrane potential, and depletion of GSH in a rat lung epithelial (RLE-6TN) cell line, and that the JP-8–induced activation of caspase-3 in these cells was inhibited by thiol antioxidants (17). Together, these results suggest that antioxidant-related proteins may be critical for protection of lung tissue against the toxic effects of JP-8.

Exposure to JP-8 has been associated with deleterious effects on pulmonary function, including an increase in pulmonary resistance and permeability and the accumulation of inflammatory cells (7, 32). Repetitive exposure to this fuel also induces long-term effects on the immune system, as evidenced by reduced numbers of viable immune cells, decreased weight of immune organs, and loss of immune function (11, 33). Indeed, we have shown that JP-8 induces a series of events in lymphocytes that lead to the activation of genes that trigger irreversible cell death by apoptosis (18). In this regard, the expression of immunoglobulin genes in lung tissue was downregulated by JP-8 in the present study. Such effects on the immune system may increase the susceptibility of personnel exposed to JP-8 to infectious agents as well as confer a predisposition to the development of autoimmune disease as a result of immune suppression (11).

We also detected increased expression of genes related to the extracellular matrix or cytoskeleton in lung tissue of rats exposed to JP-8 at 352 mg/m3. Among these genes was that for myosin heavy chain polypeptide 7 (Myh7), the altered expression of which has been associated with chronic lung hyperinflation in humans (34) as well as with severe emphysema in rats (35). Proteomics analyses have also shown that repeated inhalation of JP-8 alters the expression of several proteins related to the maintenance of cell structure in addition to those related to detoxification and cell proliferation (8, 9). We also found that JP-8 downregulated the expression of the gene for high molecular weight microtubule-associated protein 2 (HMW-MAP2), a protein important in microtubule assembly. This protein has been proposed as a specific marker for pulmonary carcinoid tumor and small cell carcinoma (36). Interestingly, a reduced expression of this gene has been recently detected in whole brain tissue of rats exposed to JP-8 (37). It is conceivable that persistent altered expression of extracellular matrix or cytoskeleton genes on cells exposed to this jet fuel may induce morphologic and functional changes, thus increasing the risk of development of diseases such as pulmonary fibrosis (2, 21, 29).

The abundance of transcripts for the inositol 1,4,5-trisphosphate receptor was greatly reduced in the lung tissue of rats exposed to JP-8 at the higher dose. We have previously shown that JP-8 induces the release of Ca2+ from intracellular stores in RLE-6TN cells, and that this effect is related to the depletion of GSH and to mitochondrial and downstream events that lead to apoptosis in these cells (17). Treatment with exogenous GSH or the thiol-containing antioxidant N-acetyl cysteine protected against the toxic effects of JP-8 and other toxicants in several cell lines (17, 38). Oxidized glutathione, a product of oxidative stress, has been shown to reduce the Ca2+ content of inositol 1,4,5-trisphosphate–sensitive stores in pulmonary endothelial cells (23). Furthermore, high levels of intracellular Ca2+ are implicated in airway hyperresponsiveness and allergic pulmonary inflammation (39).

Although previous proteomic analysis was performed in lung tissue from mice (8) and our experiments were designed in lungs derived from rats, the integration of those protein data sets with the present gene expression profile may provide important clues to predict the cellular perturbations that are regulated by the JP-8 toxicant in simulated occupational models. The comparison of these experiments albeit in different rodents using approximately the same dose of JP-8 revealed a substantial agreement on genes associated with extracellular matrix such as actin and myosin genes which were upregulated as consequence of JP-8 treatment. Increased expression of these contractile filament genes may imply a protective response of cells to the oxidative stress induced by this jet fuel. Furthermore, proteomic data obtained from lungs of animals exposed at higher doses of JP-8 (1,000 and 2,500 mg/m3) (9) to that used in the present study still demonstrated a remarkable correspondence in the upregulation of certain detoxification genes such as GSTs, which are known to play an important role in the defense mechanisms against the toxic effects of JP-8 components.

In conclusion, we have shown that short-term exposure of rats to JP-8 under conditions that mimic the occupational exposure of Air Force personnel to this jet fuel induced marked changes in the expression of various genes with diverse functions. Prominent among these genes, however, were those whose functions are related to defense against oxidative and toxicant-induced stress. The expression data provided by our study may help to identify genes whose products are largely responsible for the protection of lung cells against the toxic effects of JP-8 in vivo.


   Acknowledgments
 
L.A.E. has no declared conflicts of interest; M.V. has no declared conflicts of interest; M.J.C. has no declared conflicts of interest; T.C. has no declared conflicts of interest; M.J. has no declared conflicts of interest; J.H. has no declared conflicts of interest; M.L.W. has no declared conflicts of interest; and M.E.S. has no declared conflicts of interest.

The authors are grateful to Dr. Edmund A. Gehan from the Department of Biomathematics and Biostatistics of Georgetown University for many helpful discussions.


   Footnotes
 
This work was supported by grants: F49620-94-1-0297 to M.W. and F49620-01-1-0263 and FA9550-04-1-0395 to M.E.S., from the US Air Force Office of Scientific Research.

Received in original form May 20, 2004

Received in final form November 23, 2004


   References
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 

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