High-precision genetic mapping of behavioral traits in the diversity outbred mouse population.

Historically our ability to identify genetic variants underlying complex behavioral traits in mice has been limited by low mapping resolution of conventional mouse crosses. The newly developed Diversity Outbred (DO) population promises to deliver improved resolution that will circumvent costly fine-mapping studies. The DO is derived from the same founder strains as the Collaborative Cross (CC), including three wild-derived strains. Thus the DO provides more allelic diversity and greater potential for discovery compared to crosses involving standard mouse strains. We have characterized 283 male and female DO mice using open-field, light-dark box, tail-suspension and visual-cliff avoidance tests to generate 38 behavioral measures. We identified several quantitative trait loci (QTL) for these traits with support intervals ranging from 1 to 3 Mb in size. These intervals contain relatively few genes (ranging from 5 to 96). For a majority of QTL, using the founder allelic effects together with whole genome sequence data, we could further narrow the positional candidates. Several QTL replicate previously published loci. Novel loci were also identified for anxiety- and activity-related traits. Half of the QTLs are associated with wild-derived alleles, confirming the value to behavioral genetics of added genetic diversity in the DO. In the presence of wild-alleles we sometimes observe behaviors that are qualitatively different from the expected response. Our results demonstrate that high-precision mapping of behavioral traits can be achieved with moderate numbers of DO animals, representing a significant advance in our ability to leverage the mouse as a tool for behavioral genetics.

Short-term pulmonary response to inhaled JP-8 jet fuel aerosol in mice.

B6.A.D. (Ahr(d)/Nat(s)) mice were utilized to investigate the short-term pulmonary response to JP-8 jet fuel (JP-8) aerosol inhalation. Mice were nose-only exposed to atmospheres of 0 to 118 mg/m3 for 1 h/d over a period of 7 days to further test the hypothesis that JP-8 concentrations below the permissible exposure level (PEL) of 350 mg/m3 will induce lung injury. At 24 to 30 hours after the final exposure, pulmonary function and respiratory permeability were measured on anesthetized mice and then randomly assigned for bronchoalveolar lavage or histopathology. Bronchoalveolar lavage fluid (BALF) was analyzed for total protein, lactic dehydrogenase (LDH), N-acetyl-beta-D-glucosaminidase (NAG), and cytology. Respiratory permeability increases were observed following doses of 48 and 118 mg/m3 and were supported by concomitant BALF increases in total protein and LDH. Conversely, NAG and alveolar macrophage levels decreased following the same exposure concentrations. Morphological lung injury was characterized by the targeting of bronchiolar epithelium and consisted of perivascular edema, Clara cell vacuolization, and necrosis. Alveolar injury included sporadic pulmonary edema, intra-alveolar hemorrhage, and alterations in type II epithelial cells. These results indicate that repeated inhalation of aerosolized JP-8 induces physiological, biochemical, cellular, and morphological lung injury. This study also provides evidence for the reevaluation of the 350 mg/m3 PEL for more volatile petroleum distillates with regard to respirable aerosols.

Effects of short-term JP-8 jet fuel exposure on cell-mediated immunity.

The U.S. Air Force has implemented the widespread use of JP-8 jet fuel in its operations, although a thorough understanding of its potential effects upon exposed personnel is unclear. Exposure to environmental toxicants such as JP-8 may have significant effects on host physiology. Jet fuel exposure has been shown to cause human liver dysfunction, abnormal electroencephalograms, shortened attention spans, and decreased sensorimotor speed. Previous studies have shown that short-term, low-concentration JP-8 exposure had significant effects on the immune system; e.g., decreased viable immune cell numbers, decreased immune organ weights, and loss of immune function that persisted for extended periods of time (i.e., up to 4 weeks post-exposure). In the current study, an in-depth analysis of the effects of JP-8 exposure on cellular immunity was performed. Short-term (7 days, 1 h/day), low-concentration (1000 mg/m3) exposures were conducted in mice, and T cell and natural killer (NK) cell functions were analyzed 24 h after the last exposure. The exposure regimen was found to almost completely ablate NK cell function, as well as significantly suppress the generation of lymphokine-activated killer (LAK) cell activity. Furthermore, JP-8 exposure suppressed the generation of cytotoxic T lymphocyte (CTL) cells from precursor T cells, and inhibited helper T cell activity. These findings demonstrate that JP-8 jet fuel exposure has significant detrimental effects on immune functions of exposed individuals. JP-8 jet fuel should be considered a potential and significant immunotoxicant. Chronic exposure to JP-8 may have serious implications to the long-term health of exposed individuals.

Increased phosphorylated extracellular signal-regulated kinase immunoreactivity associated with proliferative and morphologic lung alterations after chrysotile asbestos inhalation in mice.

Activation of extracellular signal-regulated kinases (ERK) has been associated with the advent of asbestos-associated apoptosis and proliferation in mesothelial and alveolar epithelial cells and may be linked to the development of pulmonary fibrosis. The objective of studies here was to characterize the development of inflammation, cellular proliferation, and fibrosis in asbestos-exposed C57Bl/6 mice in relationship to patterns of ERK phosphorylation. Inflammation occurred after 10 and 20 days of asbestos exposure as evidenced by increases in total protein and neutrophils in bronchoalveolar lavage fluid. Increases in cell proliferation were observed at 30 days in bronchiolar epithelia and at 4, 14, and 30 days in the alveolar compartment of the lung. Trichrome-positive focal lesions of pulmonary fibrosis developed at 30 days in the absence of elevations in lung hydroxyproline or procollagen mRNA levels. Striking increases in ERK phosphorylation were observed within pulmonary epithelial cells at sites of developing fibrotic lesions after 14 and 30 days of inhalation. In addition to characterizing a murine inhalation model of asbestosis, we provide the first evidence showing activation of ERK signaling within lung epithelium in vivo, following inhalation of asbestos fibers.

Substance P as prophylaxis for JP-8 jet fuel-induced immunotoxicity.

Previous studies have shown that short-term, low-concentration JP-8 exposure had significant effects on the immune system that persisted for extended periods of time. It was found that administration of aerosolized substance P (SP) was able to protect exposed animals from JP-8-induced immune changes, whereas administration of SP antagonists compounded the deleterious effects ofjet fuel exposure. Thus, SP administration appears to be a relatively simple and efficient means to reverse the immunotoxicity due to hydrocarbon exposure. In the current study, aerosolized SP was analyzed for its potential prophylactic ability to counteract JP-8-induced immunotoxicity. It was observed that concentrations as low as 1 nM were effective in ameliorating the effects of JP-8 exposure on the immune system. SP administered before JP-8 exposure could prophylactically protect both the spleen and thymus from significant organ weight loss, but could not completely restore immune cell numbers to normal, baseline levels. Furthermore, SP treatment could be delayed as long as 1 h postexposure and reverse the effects of jet fuel exposure on immune organ weight loss and immune cell recovery. Significantly, SP could be given 15 min pre-JP-8 exposure but neither 1 nor 6 h pre-JP-8 exposure, and prevent immune dysfunction as measured in mitogenesis assays. However, SP could be delayed up to 6 h post-JP-8 exposure and still almost completely restore immune function. Thus, SP appears able to both prevent and reverse the immunotoxicological effects associated with JP-8 exposure. These results also provide insight into the manner in which JP-8 jet fuel mediates its effects on the immune system.

Jet fuel-induced immunotoxicity.

Chronic exposure to jet fuel has been shown to cause human liver dysfunction, emotional dysfunction, abnormal electroencephalograms, shortened attention spans, and to decrease sensorimotor speed (3-5). Exposure to potential environmental toxicants such as jet fuel may have significant effects on host systems beyond those readily visible (e.g., physiology, cardiology, respiratory, etc.), e.g., the immune system. Significant changes in immune function, even if short-lived, may have serious consequences for the exposed host that may impinge affect susceptibility to infectious agents. Major alterations in immune function that are long lasting may result in an increased likelihood of development and/or progression of cancer, as well as autoimmune diseases. In the current study mice were exposed 1 h/day for 7 days to a 1000-mg/m3 concentration of aerosolized jet fuel obtained from various sources (JP-8, JP-8+100 and Jet A1) and of differing compositions to simulate occupational exposures. Twenty-four hours after the last exposure the mice were analyzed for effects on the immune system. It was observed that exposure to all jet fuel sources examined had detrimental effects on the immune system. Decreases in viable immune cell numbers and immune organ weights were found. Jet fuel exposure resulted in differential losses of immune cell populations in the thymus. Further, jet fuel exposure resulted in significantly decreased immune function, as analyzed by mitogenesis assays. Suppressed immune function could not be overcome by the addition of exogenous growth factors known to stimulate immune function. Thus, short-term, low-concentration exposure of mice to aerosolized jet fuel, regardless of source or composition, caused significant deleterious effects on the immune system.

Modulation of bronchial epithelial cell barrier function by in vitro jet propulsion fuel 8 exposure.

The loss of epithelial barrier integrity in bronchial and bronchiolar airways may be an initiating factor in the observed onset of toxicant-induced lung injuries. Acute 1-h inhalation exposures to aerosolized jet propulsion fuel 8 (JP-8) have been shown to induce cellular and morphological indications of pulmonary toxicity that was associated with increased respiratory permeability to 99mTc-DTPA. To address the hypothesis that JP-8 jet fuel-induced lung injury is initiated through a disruption in the airway epithelial barrier function, paracellular mannitol flux of BEAS-2B human bronchial epithelial cells was measured. Incubation of confluent cell cultures with non-cytotoxic concentrations of JP-8 or n-tetradecane (C14), a primary constituent of JP-8, for a 1-h exposure period resulted in dose-dependent increases of paracellular flux. Following exposures of 0.17, 0.33, 0.50, or 0.67 mg/ml, mannitol flux increased above vehicle controls by 10, 14, 29, and 52%, respectively, during a 2-h incubation period immediately after each JP-8 exposure. C14 caused greater mannitol flux increases of 37, 42, 63, and 78%, respectively, following identical exposure conditions. The effect on transepithelial mannitol flux reached a maximum at 12 h and spontaneously reversed to control values over a 48-h recovery period, for both JP-8 and C14 exposure. These data indicate that non-cytotoxic exposures to JP-8 or C14 exert a noxious effect on bronchial epithelial barrier function that may preclude pathological lung injury.

Functional alterations of alveolar macrophages subjected to smoke exposure and antioxidant lazaroids.

Acute inhalation of diesel fuel-polycarbonate plastic (DFPP) smoke causes severe lung injury, leading to acute respiratory distress syndrome (ARDS) and death. It has been reported that the initiation of acute lung injury is associated with the activation of pulmonary alveolar macrophages (PAM). To further explore the pathogenesis, alveolar macrophages (AM) of New Zealand rabbits ventilated and exposed to a 60 tidal volume of DFPP smoke in vivo were recovered at 1 h post-smoke. Smoke exposure induced significant increases in both mRNA and protein levels for PAM tumor necrosis factor-alpha (TNF-alpha), when compared to smoke control. Smoke also induced a biphasic response (inhibited at 2 h, enhanced at 24 h after cell isolation) in the production of superoxide (O2-) by PAM. However, aerosolized lazaroid, U75412E (1.6 mg/kg body weight), significantly attenuated smoke-induced expression in AM TNF-alpha at the protein level but not at the mRNA level, and smoke-induced changes in AM production of O2-. This study suggests that highly expressing AM TNF-alpha following smoke may be a key contributor to the cascade that establishes an acute injury process and exacerbates oxidant-derived cell injury. Whereas, the lazaroid may ameliorate smoke-induced lung injury by attenuating AM TNF-alpha release, in addition to its primary antioxidative mechanism.

Early alterations of lung injury following acute smoke exposure and 21-aminosteroid treatment.

In a simulated fire-related smoke exposure protocol, New Zealand white rabbits were utilized to investigate the potential effects of the 21-aminosteroid (lazaroid) analog U75412E on the early events of acute lung injury. Inhalation of a total of 1.6 mg/kg U75412E aerosolized at a rate of 1.53 mg/min at 0.5 hr after smoke exposure significantly attenuated the extent of lung injury at 1 hr, as evidenced by decreased bronchoalveolar lavage (BAL) concentration of total protein, 6-keto-prostaglandin F1-alpha, and blood gas defect. Histopathologic examination demonstrated that the lazaroid significantly attenuated smoke-induced lung injury as evidenced by a decrease in wet lung/body weight ratio, necrosis, and sloughing of airway epithelial cells. Electron microscopy showed that the lazaroid decreased smoke-induced interstitial edema and the vacuolization of alveolar type II epithelium (21.6 +/- 9.7 vs 8.5 +/- 3.6 vacuoled blebs/cell, smoke only vs smoke + lazaroid). However, U75412E did not attenuate smoke-induced changes in BAL concentration of tumor necrosis factor-alpha, total cell count, and granulocyte percentage. These observations suggest that U75412E may exert its action through cooperative mechanisms, such as the modulation of arachidonic acid metabolism, in addition to its characterized antioxidative effects.

NK1-receptor activation prevents hydrocarbon-induced lung injury in mice.

Recent evidence suggests that neurokinin (NK)-receptor activation may have a protective role in maintaining lung integrity when challenged by airborne toxicants such as sulfur dioxide, ozone, acrolein, or hydrocarbons. To investigate the effect of NK1-receptor activation on hydrocarbon-induced lung injury, B6.A.D. (Ahr d/Nats) mice received subchronic exposures to JP-8 jet fuel (JP-8). Lung injury was assessed by the analysis of pulmonary physiology, bronchoalveolar lavage fluid, and morphology. Hydrocarbon exposure to target JP-8 concentrations of 50 mg/m3, with saline treatment, was characterized by enhanced respiratory permeability to 99mTc-labeled diethylenetriaminepentaacetic acid, alveolar macrophage toxicity, and bronchiolar epithelial damage. Mice administered [Sar9,Met(O2)11]substance P, an NK1-receptor agonist, after each JP-8 exposure had the appearance of normal pulmonary values and tissue morphology. In contrast, endogenous NK1-receptor antagonism by CP-96345 administration exacerbated JP-8-enhanced permeability, alveolar macrophage toxicity, and bronchiolar epithelial injury. These data indicate that NK1-receptor activation may have a protective role in preventing the development of hydrocarbon-induced lung injury, possibly through the modulation of bronchiolar epithelial function.

Short-term exposure to JP-8 jet fuel results in long-term immunotoxicity.

Chronic exposure to jet fuel has been shown to have adverse effects on human liver function, to cause emotional dysfunction, to cause abnormal electroencephalograms, to cause shortened attention spans, and to decrease sensorimotor speed. Due to the decision by the United States Air Force to implement the widespread use of JP-8 jet fuel in its operations, a thorough understanding of its potential effects upon exposed personnel is both critical and necessary. Exposure to potential environmental toxicants such as JP-8 may have significant effects on host systems beyond those readily visible (i.e., physiology, cardiology, respiratory, etc.); e.g., the immune system. Previous studies have shown that short-term, low concentration JP-8 exposure had significant effects on the immune system, which should have serious consequences for the exposed host in terms of susceptibility to infectious agents. If these alterations in immune function were long-lasting, it might also result in an increased likelihood of development and/or progression of cancer, as well as autoimmune disease. In the current study, mice were exposed for 1 h/day for 7 days to a moderate (1000 mg/m3) and a high (2500 mg/m3) concentration of aerosolized JP-8 jet fuel to stimulate occupational exposures. One to 28 days after the last exposure the mice were analyzed for effects of the exposure on their immune systems. It was observed that decrease in viable immune cell numbers and immune organ weights found at 24 h after exposure persisted for extended periods of time. Further, JP-8 exposure resulted in significantly decreased immune infection, as analyzed by mitogenesis assays, which persisted for up to 4 weeks post-exposure. Thus, short-term exposure of mice to JP-8 jet fuel caused significant toxicological effects on the immune system, which were long-lasting and persistent. It appears that the immune system may be the most sensitive indicator of toxicological damage due to JP-8 exposure. Such long-term changes in immune status may have significant effects on the health of the exposed individual.

Protection from JP-8 jet fuel induced immunotoxicity by administration of aerosolized substance P.

Chronic exposure to jet fuel has been shown to cause human liver dysfunction, emotional dysfunction, abnormal electroencephalograms, shortened attention spans, and decreased sensorimotor speed. The United States Air Force has decided to implement the widespread use of JP-8 jet fuel in its operations, although a thorough understanding of its potential effects upon exposed personnel is unclear. Exposure to potential environment toxicants such as JP-8 may have significant effects on host physiology. Previous studies in mice have shown that short-term, low concentration JP-8 exposure had significant effects on the immune system; e.g., decreased viable immune cell numbers, decreased immune organ weights, and loss on immune function that persisted for extended periods of time (i.e., up to 4 weeks post-exposure). Previous studies have shown that JP-8 induced pulmonary dysfunction was associated with a decrease in levels of the neuropeptide substance P (SP) in lung lavage fluids. It was found that administration of aerosolized SP was able to protect exposed animals from such JP-8 induced pulmonary changes. In the current study, aerosolized SP was analyzed for its effects on JP-i induced immunotoxicity in exposed mice. It was observed that SP administration could protect JP-8 exposed animals from losses of viable immune cell numbers, but not losses in immune organ weights. Further, exposure of animals to SP inhibitors generally increased the immunotoxicity of JP-8 exposure. SP appeared to act on all immune cell populations equally as analyzed by flow cytometry, as no one immune cell population appeared to be preferentially protected by SP. Also, SP administration was capable of protecting JP-8 exposed animals from loss of immune function at all concentrations of JP-8 utilized (250-2500 mg/m3). Significantly, SP only needed to be administered for 15 minutes after JP-8 exposure, and was active at both 1 microM and 1 nM concentrations. Thus, SP administration appears to be a relatively simple and efficient means to reverse the immunotoxicity due to hydrocarbon exposure.

Fractal analysis of lung alveoli during the acute phase vs. repair phase of an adenoviral infection in canines.

Acute viral respiratory infections are commonly associated with alterations in lung growth. Recently, fractal techniques have been demonstrated to show changes in alveolar perimeter after canine adenovirus type 2 (CAV2) infection in a beagle puppy model. In the present study, we investigated whether the fractal dimension (Df) of the alveolar perimeter was changed in the acute phase (2-3 weeks after inoculation, 131d CAV2 group) or during the recovery phase (approximately 22 weeks after inoculation, 235d CAV2 group) after a single bout of CAV2. There were sham CAV2 groups, 130d and 238d controls, corresponding to the CAV2 groups. The Df of alveolar perimeter length was significantly increased in the 235d CAV2 puppies compared to all of the other beagle puppy groups. On the other hand, the fractal dimensions for alveolar perimeter length for the other beagle puppy groups were very similar. In a related human study of patients (age range of 25 h to 19 y, N = 11), who died of non-respiratory causes, showed no consistent change in Df of alveolar perimeter length with normal lung growth and development. We conclude that fractal analysis of alveolar perimeter length can be used as an index of permanent lung injury after insult to the growing lungs.

Immunotoxicological effects of JP-8 jet fuel exposure.

Chronic exposure to jet fuel has been shown to have adverse effects on human liver function, to cause emotional dysfunction, to cause abnormal electroencephalograms, to cause shortened attention spans, and to decrease sensorimotor speed (3-5). Due to the decision by the United States Air Force to implement the widespread use of JP-8 jet fuel in its operations, a thorough understanding of its potential effects upon exposed personnel is both critical and necessary. Exposure to potential environmental toxicants such as JP-8 may have significant effects on host systems beyond those readily visible (e.g., physiology, cardiology, respiratory, etc.); e.g., the immune system. Significant changes in immune consequences, even if short-lived, may have serious consequences for the exposed host that may impinge affect susceptibility to infectious agents. Major alterations in immune function that are long-lasting may result in an increased likelihood of development and/or progression of cancer, as well as autoimmune diseases. In the current study mice were exposed for 1h/day for 7 days to varying concentrations of aerosolized JP-8 jet fuel to simulate occupational exposures. Twenty-four hours after the last exposure the mice were analyzed for effects on their immune systems. It was observed that even at exposure concentrations as low as 100 mg/m3 detrimental effects on the immune system occurred. Decreases in viable immune cell numbers and immune organ weights were found. Jet fuel exposure resulted in losses of different immune cell subpopulations depending upon the immune organ being examined. Further, JP-8 exposure resulted in significantly decreased immune function, as analyzed by mitogenesis assays. Suppressed immune function could not be overcome by the addition of exogenous growth factors known to stimulate immune function. Thus, short-term, low concentration exposure of mice to JP-8 jet fuel caused significant toxicological effects on the immune system. It appears that the immune system may be the most sensitive indicator of toxicological damage due to JP-8 exposure, as effects were seen at concentrations of jet fuel that did not evidence change in other biological systems. Such changes may have significant effects on the health of the exposed individual.

A free radical scavenger (Lazaroid U75412E) attenuates tumor necrosis factor-alpha generation in a rabbit model of smoke-induced lung injury.

The lazaroid (21-aminosteroid) analogue U75412E was evaluated in rabbits exposed to diesel fuel-polycarbonate plastic smoke. Inhalation of total of 4.6 mg U75412E aerosolized at a rate of 1.53 mg/min for 3 min before or after smoke significantly prevented or limited the extent of alveolar hypoventilation, interstitial edema, and tumor necrosis factor-alpha (TNF-alpha) by pulmonary alveolar macrophages (PAM) ex vivo observed at 2 h. The smoke-induced changes in wet lung/body weight ratios and the production of superoxide (O2-) by PAM ex vivo were also attenuated by the drug treatment after smoke exposure (p < 0.05). This study suggests that lazaroids may ameliorate the oxygen-radical-initiated cytokine processes and inflammation cascade as a result of the smoke insult.

The prophylactic effects of U75412E pretreatment in a smoke-induced lung injury rabbit model.

The effects of the lazaroid analogue U75412E (21-[4-(3-ethylamino-2-pyridinyl)-1-piperazinyl]-16 alpha-methylpregna-1,4,9]-(11)-triene-3,20-dione) were examined in an acute lung injury rabbit model. Standard doses of 0, 8 and 16 mM U75412E were aerosolized and ventilated into the lungs for 3 min. via an endotracheal tube. A 60 tidal volume dose of diesel fuel-polycarbonate plastic smoke was then instilled, followed by mechanical ventilation for one hour. Pretreatment with 16 mM U75412E significantly increased blood PaO2 and pH values, and decreased blood PaCO2 as compared to smoke only exposures. It also significantly decreased the total cell counts and granulocytes in bronchoalveolar lavage fluid, and the ability of pulmonary alveolar macrophages to produce tumour necrosis factor-alpha in vitro after cell isolation and culture. Histopathology indicated that 16 mM U75412E pretreatment attenuated increases in wet lung/body weight ratios, inflammatory focus, and interstitial oedema associated with smoke insult. In summary, U75412E pretreatment may possess the potential to improve acute smoke-induced lung injury, in part, through modulation of tumour necrosis factor-alpha production from pulmonary alveolar macrophages.