* National Institute for Occupational Safety and Health, Morgantown, West Virginia 26505;
Environmental Health Science, University of South Carolina-Beaufort, Beaufort, South Carolina 29902;
Department of Health Professions,
Marine Biomedicine and Environmental Science Center, and
¶ Department of Medicine/Pediatrics, Medical University of South Carolina, Charleston, South Carolina 29425.
Received May 21, 2003; accepted September 10, 2003
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ABSTRACT |
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Key Words: immunological parameters; host resistance; jet propulsion fuel-8; JP-8, thyroid hormones.
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INTRODUCTION |
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Similar to most petroleum distillates, JP-8 is a heterogeneous, kerosene-like mixture. It contains approximately 81% alkanes, primarily in the C8C17 range. The remaining portion consists of 1020% polycyclic aromatic hydrocarbons (PAHs) with relatively low levels of benzene, toluene, and xylenes (IARC, 1989; ATSDR, 1998
). The composition of JP-8 was 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.
Initial toxicological studies with JP-8 examined toxicity from 90-day exposures. For instance, F-344 rats and C57BL/6 mice that were exposed to JP-8 (01000 mg/m3) via inhalation did not exhibit tumor formation and displayed minimal treatment-related adverse effects (Mattie et al., 1991). Male rats did acquire
2-microglobulin nephropathy, but this was considered unique to this species and not extrapolatable to humans (Mattie et al., 1991
). This study was followed by another 90-day experiment examining the effects of oral exposure to neat JP-8 (03000 mg/kg/day) in Sprague-Dawley rats (Mattie et al., 1995
). Observations included perianal irritation, gastritis,
2-microglobulin nephropathy, and a dose-dependent decrease in body weight. No mortality or morbidity occurred even after exposure to high levels (3000 mg/kg) of JP-8 for 90 days, and no adverse consequences in liver pathology were noted. However, examination of hematological parameters identified significant increases in peripheral blood neutrophils and corresponding decreases in blood lymphocytes at all treatment levels (7503000 mg/kg/day of JP-8).
Subsequent investigations evaluated JP-8s effect on immune function and identified more profound and persistent effects. Immunotoxicity and pulmonary pathology were reported after 7-day exposures to aerosolized JP-8 jet fuel ranging from 1002500 mg/m3 (Harris et al., 1997a,b
,c
, 2000
; Hays et al., 1995
; Pfaff et al., 1996
; Robledo and Witten, 1999
). Immunological deficits in spleen and thymus organ weight and cellularity, lymphocytic subpopulations, and T-cell blastogenesis to the mitogen concanavalin A (Con A) were long lasting (Harris et al., 1997b
,c
). Dermal exposure to JP-8 (50 µl/day for 5 days or 250300 µl as a single exposure) also suppressed T-cell blastogenesis while impairing delayed and contact hypersensitivity in mice (Ullrich, 1999
). Suppression of T-cell proliferation was noted 34 days after a single dermal exposure to JP-8, and this effect persisted for approximately 3 weeks postexposure (Ullrich and Lyons, 2000
). Modulation of inflammatory cytokines has been suggested as a potential mechanism of JP-8-induced immunosuppression, as Ullrich (1999)
identified elevated interleukin-10 levels in the serum of dermally exposed mice. Moreover, our laboratory has also demonstrated immunotoxicity, including a decrease in thymus weight and cellularity and substantial deficits in humoral immune responses, after oral exposure to JP-8 for 7 days (Dudley et al., 2001
).
Few studies have evaluated the impact of JP-8 exposure during development. Cooper and Mattie (1996) established a no observed adverse effect level of 1000 mg/kg for a reduction in fetal rat body weight. Dermal exposure of pregant dams to kerosene, a primary component of JP-8, resulted in a live pup birth index of 97% with no significant changes in birth weight, mean number of live pups per litter, and weight gain in pups from treated and control groups (Schreiner et al., 1997
). Immunological function after gestational exposure to JP-8, however, has not been assessed in any study to date.
To address the impact of JP-8 on the developing immune system, this study examined a battery of immunological endpoints in mice following in utero exposure to JP-8. Assessment of immune function was comprehensive and included evaluation of immunological parameters at weaning (age 3 weeks) and at adulthood (age 8 weeks). This study also evaluated thyroid hormone levels and host susceptibility to bacterial or tumor cell challenge at adulthood.
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MATERIALS AND METHODS |
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Mice.
Adult, timed-pregnant female C57BL/6N mice mated with C3H/HeJ male mice were purchased from Harlan-Teklab, Madison, WI. Pups generated from this mating were B6C3F1, a common strain used in immunotoxicology testing. The time-pregnant mice were transported via air-conditioned truck on day 3 or 4 of gestation to an AAALAC approved animal facility at the Medical University of South Carolina. Mouse chow (Tek Lab Sterilizable Rodent Diet, formula no. 8656; Harlan Tek Lab, Madison WI) and water were provided ad libidum. Pregnant dams were housed 4 per cage and then housed individually 1 day prior to birthing. Bedding, food, and water were changed twice a week, and mice were observed daily.
Exposure and experimental design.
Pregnant dams were gavaged on gestation days 615 daily with 1000 or 2000 mg/kg JP-8 (100 µl gavage volume) in a carrier of olive oil. Control animals received olive oil only (100 µl gavage). At least ten dams were randomly assigned to each treatment group. Typical litter size ranged from 4 to 7, and pups remained with dams until age 21 days, when they were weaned, separated by gender, and housed 4 per cage. In the litters born, gender distribution was approximately 49% male and 51% female in control and treatment groups. A male and female from each litter of each treatment group were selected for testing for various assays at either 3 or 8 weeks of age. Select immunological, hematological, or thyroid parameters were assessed in B6C3F1 pups either at weaning (age 21 days; 3 weeks old) or at maturity (age 56 days; 8 weeks old). Typically, each experiment involved 46 mice per treatment group, depending on the distribution of male and female mice/litter available for the assays. Experiments were repeated at least twice unless otherwise stated.
Body weight.
Body weight was measured weekly in pups from birth to 8 weeks of age. Known standard weights were used to verify balance calibration prior to use (DI-800, Denver Instruments, Arvado, CO).
Organ weight and total cellularity.
Kidney, liver, thymus, and spleen weights were measured at weaning (3 weeks) or at adulthood (8 weeks). Bone marrow cells were aseptically collected from the medullary cavity of one femur from each mouse. Peritoneal macrophages were obtained via sterile peritoneal lavage. Total cellularity was determined for spleen, thymus, bone marrow, and from peritoneal lavage using a Coulter Model ZM particle counter (Coulter Electronics, Hileah, FL). The Coulter counter was calibrated with cell suspensions that were verified using a hemacytometer.
Flow cytometric evaluation of thymic and splenic lymphocytic subpopulations.
Spleen or thymus cells were labeled with fluorescent (phycoerythrin or fluorescein isothiocyanate) mouse IgG2 monoclonal antibodies specific for murine CD4 or CD8. In addition, splenocytes were separately labeled with rat anti-mouse B220 to semiquantitate the percentage of B-cells in the spleen. Details of this labeling procedure are described previously (Peden-Adams et al., 2001). Splenic and thymic CD4/8 lymphocytic populations were assessed at weaning (3 weeks) and at adulthood (8 weeks), while anti-B220 populations in the spleen were determined at adulthood only. Data are represented as absolute number of cells as determined by multiplying the percent gated cells by the total number of nucleated cells obtained by the Coulter Counter.
Hematology.
Whole blood in EDTA collection tubes was sent to Antech Diagnostics (Farmington, NY) for complete blood count (CBC) analysis with white blood cell (WBC) differential. CBC analysis included erythrocyte and leukocyte number, hemoglobin, and hematocrit. Hematology was assessed at weaning (3 weeks) and at adulthood (8 weeks).
Stem cell assay.
This procedure was based on previous studies assessing granulocyte-macrophage progenitors and erythrocyte progenitors (Bradley et al., 1966; Phillips et al., 1997
). Briefly, bone marrow cells were aseptically washed and collected from the medullary cavity of one femur from each mouse. Cell counts were determined using Coulter counter and 4.5 x 104 total cells were added to MethocultTM medium (GF M3434, Stem Cell Technologies, Inc., Vancouver, BC) for the colony assay. Cultures were performed in duplicate and incubated at 37°C and 5% CO2 for 10 days. Total numbers of colonies were counted using an inverted microscope. A colony was defined as 50 cells or more. Total colony forming units (CFU) per 105 bone marrow cells were reported. This parameter was assessed in F1 adults only.
Natural killer cell (NK) activity.
An in vitro cytotoxicity assay using 51Cr-labeled Yac-1 cells (TIB 160, ATCC, Manassas, VA) was used as based on a previous study by Duke et al.(1985) and modified slightly as described in detail by Peden-Adams et al.(2001)
. The results were expressed in lytic units per 107 splenocytes, using 10% lysis as the reference point (Bryant et al., 1992
). NK activity was assessed in F1 adults only.
Nitric oxide production by peritoneal macrophages.
Peritoneal macrophages were aseptically isolated by peritoneal lavage and incubated for 24 hours with 10 µg/ml of lipopolysaccharide (LPS) and 500 Units/ml of interferon-gamma (IFN-). These methods are described previously (Keil et al., 1995
). Nitric oxide was indirectly measured by spectrophotometrically quantitating nitrite, a stable endproduct of nitric oxide generation. A standard nitrite curve was established for each experiment, and data were expressed in µM of nitrite production. Control cells contained macrophages only without any stimulant and background was always less than 0.5 µM. This endpoint was assessed at 8 weeks only.
Phagocytosis.
Peritoneal macrophages were aseptically isolated by peritoneal lavage. The cell density was determined and adjusted to 2 x 105/well. Macrophages were added to sterile Lab-Tek tissue culture chamber/slides (NalgeNunc International, Naperville, IL.) and incubated for 1 hour in a humidified incubator set at 37°C and 5% CO2. Cells were washed with prewarmed medium to remove nonadhered leukocytes. Washed Listeria monocytogenes (serotype 4 of strain 1930 3) was added at an approximate ratio of 10 bacterial cells per 1 macrophage. Sterile normal mouse serum (10%) was added to permit opsonization of bacteria. After 4 hours of incubation, the macrophages were aseptically washed 10 times to remove nonphagocytized bacteria. The remaining cells were fixed, stained with Wrights stain, and then a coverslip was applied. The number of bacteria associated per macrophage was determined using a light microscope. To reduce subjectivity in the reading process, at least 2 independent reviewers enumerated bacteria per macrophage. Phagocytosis was assessed at 8 weeks only.
Mitogen-induced lymphocyte proliferation.
Splenic lymphocyte proliferation was measured by the standard 3H-thymidine uptake assay as described in detail by Peden-Adams et al.(2001) with specific modifications. The final concentration of concanavalin A was 5 µg/ml, and the cells were incubated for 48 hours before a 12-hour tritiated thymidine pulse. Results are reported as the net cpm (cpm stimulated wells - cpm unstimulated wells).
Plaque forming cell response to T-dependent antigen, sRBC.
The plaque-forming cell (PFC) response was assessed in F1 adult mice only by using the Cunningham modification of the Jerne assay (Cunningham and Szenberg, 1968; Jerne and Nordin, 1963
). This method is described in detail by Peden-Adams et al.(2001)
. Results are reported as PFCs/million splenocytes.
B16F10 melanoma tumor challenge model.
At 8 weeks of age, mice were injected intravenously with B16F10 melanoma cells (1 x 105 total cells/mouse; ATCC, Manassas, VA) (Holsapple et al., 1988). Fourteen days following tumor challenge, mice were euthanized, and the lungs were removed and placed in labeled vials containing Bouins fixative. The number of nodules was enumerated using a dissecting microscope by at least two independent readers.
Listeria monocytogenes challenge model.
A stock of Listeria monocytogenes (serotype 4 of strain 19303) was used to challenge mice. Bacterial colony counts were performed on the liver and spleen of mice 4 days postintravenous challenge with Listeria. The liver and spleen are major sites for Listeria replication, and bacteria can be detected as soon as 2472 hours after challenge. In previous studies with mice, an LD8 was reported at 2000 colony forming units (CFU) (Bleavins et al., 1995). Therefore, a target challenge range of 2000 CFU/mouse was determined using the information from previous studies in addition to range-finding studies performed in this laboratory. This challenge level was also consistent with levels of septicemia in range-finding studies 4 days postchallenge. To determine levels of septicemia, liver and spleen were aseptically removed and weighed. Premeasured portions of each organ were diluted in sterile saline, 1:10, 1:102, 1:103, 1:104, and 1:105. One ml of each dilution was added to prewarmed brain heart infusion agar and poured into a sterile petri dish. These plates were incubated overnight at 37°C and enumerated after 24-hour incubation. F1 adult mice were challenged with Listeria at 8 weeks of age and euthanized 4 days post challenge.
Thyroid hormones.
Serum thyroid hormone levels were measured in 8-week-old, adult mice exposed in utero to JP-8. Total serum T3 and T4 were measured by means of RIA commercial kits for canine T3 and T4 measurement (Diagnostic Products Incorporated, Los Angeles, CA) and were conducted in accordance with the kit/manufacturers instructions. These kits are recommended by the manufacturer for murine testing, as sufficient cross reactivity between murine and canine thyroid hormones has been reported. Other published studies have utilized these kits when evaluating murine thyroid hormone levels (Goya et al., 1995; Hotz et al., 1997
; Sagartz et al., 1997
; Wagle et al., 1994
).
Statistical analysis.
Using JMP 4.0.2 (SAS Inst. Inc., 2000), data were tested for normality (Shapiro-Wilks) and homogeneity (Bartletts), and if needed, appropriate transformations were made. Because differences between sexes could be expected and could skew mean values, differences were tested for sex by treatment and sex within treatment interaction. If no sex-dependent effect was observed, then male and female data were combined. If significant sex by treatment (sex x treatment) or sex within treatment (sex[treatment]) differences were observed (p 0.05), these data were separated by gender and analyzed for treatment effects via ANOVA. Statistical significance was determined using a one-way ANOVA (p
0.05). When significant differences were detected by ANOVA, Dunnetts Comparison was used to compare treatment groups and controls.
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RESULTS |
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Thymic and Splenic Lymphocytic Subpopulations
A significant increase was noted in the absolute number of thymic CD4-/CD8+ lymphocytes of 3-week-old male mice exposed in utero to 2000 mg/kg JP-8 (Table 2). There were also several differences noted between male and female mice in the thymic CD4-/CD8+ subpopulation at both 3 and 8 weeks of age. No significant alterations in splenic CD4/8 lymphocytic subpopulations were detected at weaning or adulthood in F1 mice (data not shown). Additionally, the absolute number of splenic B-cells (B220+) was not altered in adult F1 mice (data not shown).
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IgM Antibody Plaque-Forming Cell (PFC) Assay
In F1 adult mice, decreases of 46% and 81% in the IgM plaque-forming cell response were observed in the 1000 and 2000 mg/kg treatment groups, respectively. Both male and female mice were uniformly affected (Fig. 3). This parameter was not assessed at 3 weeks of age.
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Splenic Natural Killer (NK) Cell Function
No significant change was observed in splenic natural killer cell activity in either male or female F1 adult mice following exposure to 1000 or 2000 mg/kg JP-8 in utero (data not shown).
Listeria Monocytogenes Challenge
At 8 weeks of age, F1 mice were challenged with Listeria monocytogenes. No significant change in susceptibility to this infection was observed in mice exposed to 1000 or 2000 mg/kg JP-8 in utero as determined by bacterial counts in the spleen and liver on day 4 of infection (Fig. 4). However, male mice had significantly lower splenic infection levels than females.
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DISCUSSION |
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JP-8 exposure levels and administration route were determined using data from human exposure guidelines (NAVOSH Standards Update Board, 1992), published studies in rodents (Cooper and Mattie, 1996
; Mattie et al., 1991
, 1995
), and range-finding experiments performed in this laboratory. Although oral administration is not the most relevant route of exposure in humans, it alone offers delivery of JP-8 in its entirety without prior fractionation of components (reviewed in National Research Council, 2003a
). This stems from the fact that JP-8 is a complex mixture of components having extreme variability in chemical and physical properties (e.g., it contains both light and heavy fractions differing in volatility), rendering it difficult to qualitatively and quantitatively assess exposure and dose via inhalation and dermal routes. For example, direct skin application of JP-8 generally results in greater exposure to the higher-molecular-weight fraction of JP-8, while low-molecular-weight, volatile fractions evaporate preferentially. When generating JP-8 atmospheres for inhalation studies, the process used can enrich specific components of JP-8 (reviewed in National Research Council, 2003a
). Although much effort has been applied to model inhalation and dermal penetration, technical limitations in measurement technology for these routes make the accurate and comprehensive assessment of actual exposure difficult. As this was the first project to assess the role of JP-8 during immunological development, oral exposure was utilized to consider JP-8 in its entirety.
Of the immunological effects noted following oral exposure in the pregnant dams, B-cell proliferative responses were transiently suppressed in the developing offspring and fully reestablished by 8 weeks of age. In contrast, previous studies with mature adult mice indicated that JP-8 did not alter B-cell proliferative responses after oral administration of either 500, 1000, or 2000 mg/kg JP-8 for 7 or 14 days (Peden-Adams et al., 2001; Scharstein et al., 2002
). Although not a permanent effect, this does suggest that B-cells may be more susceptible to the effects of JP-8 during in utero development.
The converse can be described for T-cells. Proliferative responses with T-cells were unaffected after in utero exposure to JP-8, yet there are several reports indicating that T-lymphocytic proliferative responses were suppressed in adult mice after dermal or inhalation exposure to JP-8 (Harris et al., 1997a,b
; Ullrich, 1999
; Ullrich and Lyons, 2000
), and this suppression was long-term, as recovery was not evident until 28 days postexposure (Harris et al., 1997c
). In contrast to these publications, we have previously reported that oral exposure to JP-8 does not affect T-proliferative responses (Peden-Adams et al., 2001
; Scharstein et al., 2002
). Therefore, it might be more reasonable to expect that alterations in this function appear to be contingent on the route of exposure to JP-8.
The PFC response is an immunological parameter that evaluates the effective interaction of antigen-presenting cells, T-cells, and antibody-producing B-cells to mount a primary IgM response. It is considered highly predictive of immunotoxicity, thereby suggesting increased susceptibility to disease (Luster et al., 1992). We have previously reported that primary immunological responses to sRBCs were dose-responsively suppressed when adult mice were exposed to JP-8 (Dudley et al., 2001
; Peden-Adams et al., 2001
), but this alteration was temporary, because function was fully restored by 7 days postexposure (Scharstein et al., 2002
). However, the fact that there was a significant deficit in humoral responses in F1 mice at 8 weeks is disturbing, demonstrating that this alteration is persistent and the developing immune system appears to be at greater risk from JP-8 exposure.
Despite the substantial decrease in humoral immunity, no increase in susceptibility to Listeria monocytogenes was observed after in utero exposure to JP-8. This is not surprising, as humoral immunity does not play a large role during the early stages of this infection. During this early window of active infection, immunological defenses depend largely on neutrophils, macrophages, NK cells, and T-cells (Czuprynski et al., 1994; reviewed by Edelson and Unanue, 2000
). Furthermore, T-cell, NK, and macrophage cell function were not impaired in the F1 mice and could effectively contribute to immunological protection. Considering the substantial deficits in humoral immunity caused by JP-8, future studies might assess additional host challenge models that depend largely on humoral immunity for protection, such as the group B Streptococcus bacterial challenge model (Barnes et al., 1992
).
Immunological resistance to tumors was impaired, and this effect was evident in adult animals. The tumor cell line utilized in this study is syngeneic to one of the parent strains (C57BL/6), and clearance requires the function of CD4(+) T-cells, CD8(+) T-cells, and natural killer (NK) cells (reviewed by Wu and Fleischmann, 2001). This deficit may not be unique to the in utero exposure, bacause there are preliminary observations suggesting that B16F10 melanoma tumor growth was less regulated in adult mice exposed to JP-8 via inhalation (National Research Council, 2003b
). However, the fact that tumor susceptibility was significantly enhanced when the F1 offspring were fully mature is striking, indicating long-lasting vulnerability of the developing immune system to the effects of JP-8. Also noteworthy is that, while susceptibility to B16F10 melanoma cells was significantly impaired, NK function exhibited normal levels of cytolytic activity. This study also demonstrates an important role for the incorporation of host challenge models in developmental immunotoxicology evaluations, as resistance to melanoma tumor challenge was impaired with no warning by any of the other immunological endpoints evaluated in this study.
In addition to the immunotoxicological assessment, a cursory evaluation of thyroid function was performed. Not only is proper thyroid function necessary for healthy development, several reports indicate that thyroid hormones can modulate immunological function (Blalock et al., 1984; Coutelier et al., 1990
; Kanazawa et al., 1992
; Marazuela et al., 1995
; Montecino-Rodriguez et al., 1997
). JP-8 exposure during fetal development affected thyroid function, leading to deficits of circulating T4 levels in serum from adult progeny. While such a deficiency in T4 may suggest a hypothyroid state, examination of thyroid stimulating hormone (TSH) and thyroid histopathology would be of value in corroborating dysfunction. Within the scope of this study, it is unknown if the alteration in T4 is linked to any of the changes observed in immunological function, although this would appear to be a worthwhile line of inquiry.
The potential for exposure to JP-8 during pregnancy appears to be substantial, because nearly 200,000 women are on active duty in the Armed Forces (Institute of Medicine, 1995). Of the women on active duty, approximately 94% are of childbearing age (Institute of Medicine, 1995
). Potential exposure also extends to the aviation industry, as commercial jet fuel is essentially JP-8 devoid of certain additives that impart properties of military significance. Aside from the fact that women are an ever-increasing population in the military and aviation industry, many civilians reside near environmentally contaminated sites where jet fuel has been reportedly spilled or leaked from supply lines or storage tanks. A list of national priority sites provided by the U.S. Environmental Protection Agency (http://www.epa.gov/superfund/sites/index.htm) describe thousands of gallons of jet fuel contaminating soil and ground water, associated with U.S. Air Force bases bordering residential areas. Thus, it was appropriate to investigate the possible implications of JP-8 exposure during developmental periods in offspring. As this is the first report to demonstrate that the developing immune system is vulnerable to the effects of JP-8, it is difficult to extrapolate the results of this study to those that might occur in humans exposed in utero via the dermal or inhalation routes.
Despite the fact that several immunological and hematological parameters assessed in this study were unaffected, in utero exposure to JP-8 caused long-lasting and possibly permanent deficits in humoral immunity, T4 levels, and tumor resistance. While much work remains to be done, these results suggest that the developing immune system is sensitive to the effects of JP-8, resulting in effects persisting into adulthood.
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ACKNOWLEDGMENTS |
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NOTES |
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