1 Departments of Environmental Studies,
Medicine,
Medical Laboratory Science, and
§ Biochemistry and Molecular Biology, The Medical University of South Carolina, Charleston, South Carolina
Received August 14, 2000; accepted September 13, 2000
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ABSTRACT |
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Key Words: JP-8 jet fuel; immunotoxicity; aryl hydrocarbon receptor; CYP1A1/2; DBA/2 and B6C3F1 mice..
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INTRODUCTION |
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Furthermore, recent animal studies indicated that acute or chronic exposure to JP-8 might cause a range of effects including relatively minor toxicological effects in 90-day studies or impaired immune function after a single dermal exposure. For instance, a 90-day inhalation study using male and female rats indicated that JP-8 (01000 mg/m3) had limited toxicity and resulted in no tumor formation (Mattie et al., 1991). In male rats,
2-microglobulin protein droplet nephropathy was detected after JP-8 exposure; however, this observation appears to be unique in male rats exposed to hydrocarbons and not likely to extrapolate to human toxicity (Mattie et al., 1991
). A chronic 90-day, oral exposure to JP-8 (03000 mg/kg/day) in rats resulted in no mortality or morbidity with relatively minor or no adverse consequences in liver pathology, serum chemistries, and urological and hematological parameters. Primary effects noted in this chronic study included an increase in serum liver-enzyme levels (alanine aminotransferase and aspartate aminotransferase), perianal irritation, and a dose-dependent decrease in body weight (Mattie et al., 1995
). Additional rat studies indicated that JP-8 did not cause fetal malformation after oral JP-8 (02000 mg/kg/day) exposure in pregnant rats during gestation days 615. Thus, the no-observed-effect level of 500 mg/kg for pregnant dams and 1000 mg/kg for the fetal rat was established (Cooper and Mattie, 1996
). Reduced pulmonary function and induced lung pathology has also been reported in rats exposed to JP-8 via inhalation (Hays et al., 1995
; Pfaff et al., 1996
; Robledo and Witten, 1999
). More recently, studies have evaluated immune function in mice after administration of JP-8 by way of different exposure routes. Seven-day exposure to aerosolized JP-8 jet fuel ranging from 100-2500 mg/m3 caused persistent and substantial decreases in spleen and thymus organ weights and cellularity, changes in lymphocytic subpopulations, and compromised T-cell blastogenesis to the T-cell mitogen concanavalin A (Con A; Harris et al., 1997b,c). Similarly, dermal exposure to JP-8 (50 µl/day for 5 days or a 250300-µl single exposure) suppressed T-cell blastogenesis and impaired delayed and contact hypersensitivity (Ullrich, 1999
). Current studies in our laboratory have revealed that 7- or 14-day oral exposures to JP-8 are also immunotoxic (Dudley et al., 2000
; unpublished data). Thus, it is apparent from the current literature that profound changes have been noted in pulmonary and immune function by a variety of exposure routes, yet no mechanism has been suggested to account for these effects.
JP-8 is a kerosene-based complex mixture of aliphatic and aromatic hydrocarbons. The composition of JP-8 has been modified from previously used jet propulsion fuels (JP-4 and JP-5), in that it contains additives for static dissipation, anti-icing, and corrosion inhibition. Chemically, JP-8 is a complex mixture containing approximately 81% alkanes, primarily in the C8C17 range. The remaining portion includes 1020% polycyclic aromatic hydrocarbons (PAH) and low levels of benzene, toluene, and xylene (IARC, 1989; reviewed by Zeiger and Smith, 1998).
In mice, it is suggested that the manifestations of halogenated aromatic hydrocarbon (HAH) exposure, (i.e., polychlorinated biphenyls, dibenzo-p-dioxins, and dibenzofurans) including thymus involution, suppression in the splenic antibody response to TNP-LPS and SRBC, and liver hypertrophy are mediated through AhR (aryl hydrocarbon receptor) signal transduction (Gonzalez et al., 1998; Kerkvliet et al., 1990
; Staples et al., 1999
). Furthermore, studies have revealed that some strains of mice (C57BL/6 and B6C3F1) are more sensitive to the effects of aromatic hydrocarbons than other strains (DBA/2; Forkert, 1997; Poland et al., 1974; Vecchi et al., 1983). It is now understood that DBA/2 mice differ at the Ah locus (substitution of a valine for alanine at amino acid 375) resulting in a lower affinity cytosolic receptor (Poland et al., 1994
). Since this difference in sensitivity appears to reside solely at the Ah locus, any effects that are mediated by AhR signal transduction should occur at a lower dose in Ah-responsive mice and would only occur in Ah-nonresponsive mice at a higher, saturating dose. Understanding this difference has been advantageous in evaluating the role of the AhR in immunosuppression following exposure to a number of different PAHs (Lubet et al., 1984
; Silkworth et al., 1984
, 1995
; White et al., 1985
).
The mechanisms of PAH-induced immunotoxicity are not clear; however, several possibilities have been postulated, which include interaction with the Ah-receptor, membrane perturbation, altered interleukin production, modulation of intracellular Ca+2 mobilization, and metabolic activation to reactive metabolites (reviewed by White et al., 1994). Many PAHs are carcinogenic and their ability to cause cancer has been linked to the Ah-receptor (Kerkvliet et al., 1990; Okey et al., 1984a
, b
; Piskorska-Pliszczynska et al., 1986
) and the immunotoxicity of PAHs is thought to be related to their carcinogenic potency (White and Holsapple 1984
; White et al., 1985
, 1994
). Generally, the bay-region diols (diol epoxides) of the parent are the most toxic metabolites and are considered responsible for the carcinogenic effects, and it has been suggested that these metabolites may mediate the observed immunosuppression as shown by the diol epoxides of benzo[a]pyrene (BaP) and 7, 12-dimethylbenz[a]anthracene (DMBA; White et al., 1994). However, metabolic activation of parent PAHs is not always required for immunosuppression. BaP was shown to suppress in vitro Ab production directly (White and Holsapple, 1984
) and it has been suggested that immunosuppression by chrysene is not related to metabolites (Silkworth et al., 1995
). Regardless, current thought centers around the correlation between PAH carcinogenicity and immunotoxicity and the role of the AhR and bioactivated metabolites in PAH-induced immunosuppression.
The immunotoxicity of HAHs like 2,3,7,8-tetrachlordibenzo-p-dioxin (TCDD), which are resistant to enzymatic metabolism, is most likely due to effects or products resulting from activation of the Ah gene complex. However, the immunotoxicity of PAHs that bind the AhR is probably due to an increased formation of bioactivated metabolites. Quantitative studies to evaluate protein expression of the AhR and CYP1A1 following in vivo and in vitro TCDD exposure reveal a rapid reduction in the AhR and increased levels of CYP1A1 in total cell and tissue lysates (Davarinos and Pollenz, 1999; Pollenz, 1996
; Pollenz et al., 1998
). For example, Sprague-Dawley rats given a single oral dose of TCDD demonstrated a reduction in AhR protein levels in spleen, liver, lung and thymus 8 h after exposure (Pollenz et al., 1998
). This is consistent with reduced AhR protein in the palate of developing mice (Abbott et al., 1994
) and in reproductive organs from rats treated with TCDD (Roman et al., 1998
). It is possible that other HAHs and PAHs that are strong AhR ligands may result in similar AhR reduction. Studies in our laboratory have indicated that oral exposure to JP-8 causes a reduction in thymic mass, an increase in liver mass, and suppression of antibody responses that are considered in HAH exposures to be mediated via AhR signal transduction. Moreover, many PAHs are known to bind to the AhR (Piskorska-Pliszczynska et al., 1986
). Currently, there are few reports that evaluate a complex mixture of aliphatic and aromatic hydrocarbons like JP-8 and its role as a possible AhR ligand. To address this issue, experiments were undertaken to evaluate the effects of JP-8 on thymus weight and cellularity, liver weight, specific IgM antibody responses, and the expression of the AhR and CYP1A1 in AhR-responsive and AhR-nonresponsive mice. Additionally, a murine hepatic cell line was used to evaluate the expression of AhR, CYP1A1, and a TCDD-inducible reporter gene following in vitro JP-8 administration.
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MATERIALS AND METHODS |
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Treatment.
Following a one-week acclimation period after shipment, mice were randomly assigned to control or treatment groups (45 mice/group). The control group was administered a vehicle of olive oil while the treatment groups were administered 1000 or 2000 mg/kg/day of JP-8 (kindly provided by Wright-Patterson Air Force Base) prepared in the vehicle. The exposure period was 7 days and the route of exposure was via gavage. Mice used for the positive control were treated with TCDD (Radian International, Austin, TX). TCDD was dissolved in olive oil and mice were gavaged with 15 µg/kg for 3 consecutive days. The IACUC of the Medical University of SC approved all animal procedures.
Organ weights and cellularities.
After mice were euthanized by C02 asphyxiation, thymus, lung and liver were carefully dissected and weighed using a Mettler Toledo balance. The liver and lung were placed immediately on ice and then frozen at 80°C. The thymus was transferred to 3 ml complete media and a single cell suspension was made by grinding the organ between two sterile glass slides. Thymic cell counts were measured using a Coulter Counter (Model ZM) and are reported as total cellularity for each respective organ. Liver and thymus weights were reported as a somatic index (organ weight/body weight x 100).
Jerne plaque-forming assay.
Four days prior to euthanization, mice were injected intraperitoneally with 0.1 ml of a 20% sheep red-blood-cell suspension (SRBC, Biowhittaker) in PBS. All sheep red blood cells for the experiments were drawn from a single, donor animal. As described above, mouse spleens were homogenized, cell densities counted, and suspensions diluted to 2 x 106 cells/ml in complete media. The number of plaque-forming cells was determined using the Cunningham modification of the Jerne plaque assay (Cunningham and Szenberg, 1968). Data are reported as plaque-forming cells (PFC)/million cells.
Cell culture.
Wt Hepa1c1c7 cells were added in 12-well plates at a density of 5 x 106 cells/ml in DMEM and allowed to grow to confluence. Plates were placed in a 37° C incubator at 5% CO2. JP-8 was dissolved in dimethyl sulfoxide (DMSO) (Fisher, Fair Lawn, NJ) and delivered in a volume of 10 µl to achieve a final DMSO concentration of 0.05%. The JP-8 concentrations used were 500, 100, or 10 ppm. 3-methylcholanthrene (3MC, Lot 88H3402, Sigma) served as the positive control and was prepared in DMSO and added at a concentration of 5 µM/well. Cells only and DMSO control wells were included in all experiments.
Preparation of tissue and whole-cell lysates.
Tissue and whole cell lysates were prepared as previously described by Pollenz (1996). Briefly, a portion of the frozen lung was thawed, separated, and weighed. Approximately 0.1 g of tissue was homogenized in 1 ml of 1X lysis buffer. Four hundred and fifty µl were removed and a total volume of 500 µl was achieved by adding 10% Nonidet P-40. Samples were sonicated on ice 3 times for 10 s each, and 50 µl was removed for protein determination, using the Bio-Rad Bradford Reagent (Bio-Rad, Hercules, CA) with bovine serum albumin as the standard. The remainder was combined with an equal volume of 2X sample buffer, heated at 95°C for 5 min, and stored at 80°C.
For whole cell lysates, cell monolayers were washed once with cold phosphate-buffered saline (PBS) (Mediatech, Cellgro), trypsinized, and centrifuged at 340g for 5 min. After decanting the supernatant, cell pellets were redissolved in 0.5 ml PBS and centrifuged at 1000 x g for 2 min. Pellets were then suspended in 0.1 ml of 1X lysis buffer and sonicated for 10 s. A small portion of the homogenate was removed for protein determination and the remainder was brought to equal volume with 2X sample buffer. Samples were heated at 95°C for 5 min and then stored at 80°C.
Microsomal fraction preparation.
The livers were weighed and placed in pre-chilled tubes. Homogenization buffer was added at 3 times the tissue weight. Next, samples were completely homogenized, transferred to pre-chilled centrifuge tubes, and spun at 20,000 x g (Beckman JA-20) for 10 min (4°C). The supernatant was transferred to another set of pre-chilled centrifuge tubes and spun at 10,000 x g for an additional 20 min. This supernatant was again transferred to pre-chilled tubes and spun at 105,000 x g (Beckman L5-50 Ultra) for 1 h. Finally, the supernatant (cytosolic fraction) was discarded and the remaining pellet (microsomal fraction) was washed with ice-cold wash buffer and resuspended in 0.5 ml resuspension buffer. The pellet was then homogenized using a drill-driven pestle at low speed. A small amount was removed for protein determination and the remainder was brought to equal volume with 2X sample buffer, heated at 95°C for 5 min, and stored at 80°C until needed.
Buffers and antibodies.
Unless otherwise specified, all reagents used in these experiments were purchased from Sigma (St. Louis, MO). The following buffers were used. Gel loading buffer (2X) (125 mM Tris, pH 6.8, 4% SDS, 25% glycerol, 4mM EDTA, 0.5ml ß mercaptoethanol, 0.005% bromophenol blue), TBS (50 mM Tris, 150 mM NaCl, pH 7.6), TTBS (50 mM Tris, 0.2% Tween, 150 mM NaCl, pH 7.5), TTBS+ (50 mM Tris, 0.5% Tween 20, 300 mM NaCl, pH 7.5), Blotto (5% dry milk in TTBS), 2X lysis buffer (50 mM HEPES, 40 mM Na molybdate, 10 mM EGTA, 6 mM MgCl2, 20% Glycerol, pH 7.4), 1X trypsin-EDTA, homogenization buffer (250 mM sucrose, 20 mM Tris, pH 7.4), wash buffer (250 mM sucrose, 80 mM Tris, 25 mM KCl, pH 7.4), and resuspension buffer (250 mM sucrose, 80 mM Tris, 25 mM KCl, 20% glycerol, pH 7.4). Tissue culture media used were complete media (RPMI-1640 with 10% fetal calf serum and 50,000 IU/l pen/50 µg/l strep) and Dulbecco's modification of Eagle's Medium (DMEM) (with 5% fetal calf serum and 50,000 IU/l pen/50 µg/l strep) (Cellgro, Mediatech). The antibodies used for the Western blots were affinity-purified anti-mouse AhR raised against a bacterial expressed portion of the mouse AhR (amino acids 1416), anti-rat polyclonal CYP1A1/2 or monoclonal CYP1A1 (Xenotech, Kansas City, KS), and anti-ß actin. The secondary antibody was goat anti-rabbit or anti-mouse IgG conjugated to horseradish peroxidase (GAR/GAM-HRP) (Santa Cruz Biotech).
Gel electrophoresis and Western blotting.
Tissue, cell lysates, or microsomes were electrophoresed on a 412% Tris/glycine gel (Novex, San Diego, CA) and transferred to nitrocellulose membranes. Membranes were blocked in 2 changes of BLOTTO for 30 min and stained overnight at 4°C with primary antibodies (anti-mouse AhR (A-1A), anti-rat CYP1A1/2, or anti-ß-actin). The next day, membranes were washed in 2 changes of BLOTTO for 15 min and then incubated for 1 h with GAR/GAM-HRP at room temperature. Next, membranes were washed in 2 changes of BLOTTO and 2 changes TTBS+ for 15 min each. Following a final 5-min wash in TBS, membranes were subjected to electrochemiluminesence (ECL) according to the manufacturer's specifications (NEN, Boston, MA).
Eukaryotic transfection and reporter gene assays.
Approximately 5 x 106 Hepa-1c1c7 cells were placed into 60-mm culture dishes and incubated at 37°C for 1624 h. PgudLuc1.1 (200 ng) (Garrison et al., 1996) and 500 ng pSV-ß galactisidase (Promega, Madison WI) were than transfected into cells with LipofectAMINE reagent, as detailed by the manufacturer (Gibco, Gaithersburg, MD). PGudLuc1.1 contains a 484-bp fragment from the mouse CYP1A1 promoter that contains 4 XRE sequences upstream of a luciferase reporter gene. Following a 24-h recovery period, cells were incubated in the presence of TCDD (2 nm), DMSO (0.02%), or JP-8 for an additional 8 h. Cells were then scraped from plates in reporter lysis buffer (Promega) and ß-Gal luciferase activities were determined as specified by the manufacturer. Normalized luciferase activity is reported as a percentage of control and the SEM for 3 replicates.
Statistics.
All experiments were repeated at least twice. The data were evaluated for normality and homogeneity prior to performing a one-way analysis of variance followed by Dunnett's statistical test (p < 0.05). Statistical analysis was performed using Minitab statistical software (Version 12.1). Statistics were not calculated for mice exposed to TCDD, due to the small sample size (n = 2).
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RESULTS |
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DISCUSSION |
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The use of both responsive and nonresponsive mouse strains and cell lines deficient in the ARNT protein or the AhR have been useful in evaluating the role of the AhR in PAH toxicity. Since nonresponsive strains (DBA/2, AKR, SJL) differ in both the quantity and affinity binding of the cytosolic AhR, comparing the reaction of these strains to a responsive strain following PAH exposure may elucidate the role of the AhR in the toxic response. In particular, two descriptive endpoints have proven reliable and consistent: onset of thymus involution and hepatomegaly (Fernandez-Salguero et al., 1995; Kerkvliet et al, 1990; Staples et al., 1999
). DBA/2 (nonresponsive) mice typically require 10 times the amount of TCDD to produce an equivalent degree of thymus atrophy or hepatomegaly (Kerkvliet et al., 1990
; Poland and Knunston, 1982; Silkworth and Vecchi, 1985
; White et al., 1985
), and the AhR knockout mouse is resistant to hepatomegaly and liver hypertrophy caused by TCDD (Fernandez-Salguero et al., 1996
). Therefore, an agent that operates through AhR-signal transduction pathways typically produces a response in B6C3F1 mice while having no effect in DBA/2 mice. The results of the present study suggest that both B6C3F1 and DBA/2 mice were equally sensitive when comparing either decreases in thymus weight and cellularity or increases in liver weight following JP-8 exposure. Based on these observations, JP-8 may not mediate its toxicological effects via an AhR mechanism.
However, TCDD caused an equivalent increase in liver weight in both B6C3F1 and DBA/2 mice. This is a limitation in the study, because the level of TCDD used (15 µg/kg repeated exposure for 3 days) was high. These results are consistent with a previous report demonstrating that a high dose of TCDD (20 µg/kg) resulted in an increase in liver weight in both B6C3F1 and DBA/2 mice (Kerkvliet et al., 1990) indicating that this level of exposure is above the dose-response curve. It is expected that the 10-fold difference in sensitivity to TCDD between B6C3F1 and DBA/2 mice would only be apparent at lower doses of TCDD. Nevertheless, TCDD did serve as a positive control in other aspects in that it caused a significant decrease in thymus weight and cellularity in B6C3F1 mice but not in DBA/2 mice, and down-regulated AhR while increasing CYP1A1 protein levels.
Another reliable biomarker of AhR activation is CYP1A1 expression. Induction of CYP1A1 is regulated primarily by the AhR, although there are reports that phenobarbital (Sadar et al., 1996) and some insecticides (Delescluse et al., 1998
) are able to induce CYP1A1 without binding to the AhR. In the present study, the results from the in vivo and in vitro Western blots identifying the expression pattern of CYP1A1 after JP-8 exposures were not comparable with 3MC or TCDD (known AhR ligands). This would suggest that JP-8 does not exert its toxicity via an AhR-mediated mechanism. However, it could not be precluded that JP-8 may bind minimally to the AhR with enough affinity to cause a small increase in CYP1A1 activity without remarkable increases in CYP1A1 protein expression. Therefore, in the present study, a luciferase reporter gene assay was utilized using Hepa-1 cells integrated with an XRE-driven reporter vector to directly measure CYP1A1 activity via AhR-ligand binding. It was demonstrated that JP-8 (250 ppm) did induce small, yet significant increases in reporter gene activity above control but this activity was well below that induced by TCDD. This may suggest that JP-8 is a weak AhR ligand that binds with enough affinity to cause small increases in CYP1A1 activity without appreciable increases in protein expression. However, without directly assessing the binding affinity of JP-8, this is speculation, because the increase in activity could be due to undefined non-AhR-mediated pathways.
Down-regulation of the AhR has been demonstrated in vivo and in different cell culture systems in vitro following exposure to TCDD (Pollenz et al., 1998). Additionally, a recent report has shown that both B6C3F1 and DBA/2 mice demonstrated reductions in AhR protein and upregulation of CYP1A1 following exposure to 3MC (Forkert, 1997
). Thus, it was hypothesized that if JP-8 were immunotoxic through AhR signal-transduction pathways, a down-regulation of the AhR would be expected in JP-8-exposed animals and in murine Hepa-1 cells. Despite the fact that JP-8 was able to increase luciferase reporter-gene activity, the present study indicates that JP-8 did not down-regulate the AhR at a wide range of exposures. In vitro and in vivo exposures overlap, in that the highest in vitro exposure of 500 ppm may be considered similar to the 500 mg/kg in vivo exposure that has been established as the lowest observable immunological effect level in range-finding studies performed in our laboratory. For example, an exposure of 500 mg/kg for 14 days suppressed the PFC response in ranges of 47 to 72% of control (unpublished data). It is unclear if the mechanisms of TCDD and PAH immunotoxicity are related specifically to down-regulation of the AhR, as the low affinity AhR ligand 2,7-dichlorodibenzo-p-dioxin produces similar effects to TCDD (Holsapple et al., 1986a
, b
).
The plaque-forming cell response is a sensitive indicator of immunological disruption and has been widely used to demonstrate immunosuppression following exposure to PAHs and HAHs. Exposure to TCDD has been shown to inhibit IgM secretion through AhR pathways in vitro (Sulentic et al., 1998) and via non-AhR-mediated pathways (Davis and Safe, 1991
). It has also been demonstrated that from a battery of compounds, only those which were ligands for the AhR caused a decrease in the humoral immune response to sheep red blood cells in B6C3F1 mice (Silkworth et al., 1984
). However, AhR-independent pathways have also been described for suppression in the PFC response (Davis and Safe, 1991
; Kerkvliet et al., 1990
; Lubet et al., 1984
). To our knowledge, this is the first study that describes decreases in the PFC response in B6C3F1 and DBA/2 mice following exposure to JP-8, and this further supports observations in other studies that JP-8 is immunotoxic (Harris et al., 1997a
, b
, c
; Ullrich, 1999
).
Jet fuel, like most petroleum distillates, is a heterogeneous mixture of kerosene, benzene, toluene, xylenes, and other aliphatics and PAHs. Some of JP-8's components have been previously studied in single-exposure studies. However, identifying the mechanism of toxicity caused by a mixture such as JP-8 is considerably more challenging. Kerosene and benzene are substantial components of JP-8 and are both known to cause hepatic, hematological, and immune toxicity. Specifically, benzene is known to lead to progressive bone marrow degeneration, leukopenia, and disruptions in the drug metabolic system (Abraham, 1996). Similarly, rats exposed to kerosene demonstrated increases in liver weights, decreases in the relative weights of the spleen and thymus, and decreased activity of enzymes (benzo[a]pyrene hydroxylase) involved in the metabolism of environmental chemicals including PAHs (Rao et al., 1984
; Upreti et al., 1989
). Thus, it is important to consider that kerosene and/or benzene may be important contributors to JP-8's induced immunotoxicity, not only by a potential direct effect, but perhaps by an indirect effect via an alteration in metabolic enzymes leading to the formation of more reactive and immunotoxic metabolites.
The recent, large-scale conversion of jet-fuel use from JP-4 to JP-8 in the military environment has increased the incidence of reported human health effects. Thus, our understanding of the molecular mechanisms leading to respiratory or immune dysfunction caused by JP-8 will facilitate how human exposure limits should be assessed. For example, recent work by Witzmann et al. (1999) has utilized electrophoretic techniques to examine the expression of cytosolic proteins and their responses to JP-8 jet-fuel exposure. In these studies, it was shown that inhalation of jet-fuel vapors caused increases of GST in liver and kidney homogenates (Witzmann et al., 2000). More studies are needed to determine the metabolic enzymatic profile of this complex mixture of JP-8. This approach would be more feasible in evaluating health risks of JP-8 as compared to the tedious job of isolating individual components of JP-8 that may or may not cause toxicity.
In conclusion, our most compelling evidence to indicate a non AhR-mediated mechanism of JP-8 immunotoxicity are the results from the Western blots in vitro and in vivo, demonstrating neither induction of CYP1A1 protein or down-regulation of the AhR. There were minimal increases in XRE-driven luciferase activity following JP-8 exposure of Hepa-1 cells. In further support of these findings, B6C3F1 and DBA/2 mice exposed to moderate to high levels of JP-8 displayed increases in liver weight, suppression of the PFC response, and reduced thymus weight and cellularity. Thus, the immunotoxic effects were similar between the Ah-responsive and -nonresponsive mouse strains, indicating that the AhR is not likely to have played a direct role in JP-8 immunotoxicity following a 7-day oral exposure.
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ACKNOWLEDGMENTS |
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NOTES |
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