Chromium distribution in an oropharyngeal aspiration model for hexavalent chromium in rats.

Hexavalent chromium [Cr(VI)] is a well-known and widespread environmental contaminant associated with a variety of adverse health effects, in particular lung cancer. The primary route of exposure in humans is through inhalation. Particulate forms of Cr(VI) are the most potent but in vivo studies are difficult. Intratracheal instillation requires highly trained surgical procedures which also limits the number of repeated exposures possible and thus requires high doses. Inhalation studies can deliver lower more chronic doses but are expensive and generate dangerous aerosols. We evaluated an oropharyngeal aspiration exposure route for zinc chromate particles in Wistar rats. Animals were treated once per week for 90 days. We found chromium accumulated in the lungs, blood, and reproductive tissues of all treated animals. Additionally, we found inflammatory indicators in the lung were elevated and circulating lymphocytes had increased chromosomal damage. These results show oropharyngeal aspiration provides a practicable exposure route for chronic and sub-chronic exposures of Cr(VI) particles.

Chronic + binge alcohol exposure promotes inflammation and alters airway mechanics in the lung.

INTRODUCTION: Alcohol use disorders are major risk factors for the development of and susceptibility to acute respiratory distress syndrome. Although these risks of alcohol consumption on the lung are well described, mechanisms by which alcohol abuse promotes acute lung injury are poorly understood. These gaps in our understanding are due, at least in part, to limitations of animal models to recapitulate human alcohol consumption. Recently, a new model of chronic plus binge alcohol exposure was developed that is hypothesized to better model drinking patterns of individuals with alcohol use disorders. Specifically, this paradigm models chronic consumption coupled with periodic bouts of heavy drinking. The impacts of this alcohol-exposure regimen on the lung are uncharacterized. Therefore, the goal of this study was to examine lung injury and inflammation in a well-characterized experimental model of chronic + binge alcohol exposure. METHODS: 10-week-old male C57Bl6/J mice were administered ethanol-containing (or isocaloric control) liquid diet for 10 days, followed by a single ethanol gavage (5 g/kg). Lung inflammation and pulmonary function were assessed. RESULTS: Ten days of ethanol-containing liquid diet alone (chronic) did not detectably affect any variables measured. However, ethanol diet plus gavage (chronic + binge) caused neutrophils to accumulate in the lung tissue and in the bronchoalveolar lavage fluid 24 h post-binge. This inflammatory cell recruitment was associated with airway hyper-responsiveness to inhaled methacholine, as indicated by elevated resistance, Newtonian resistance, and respiratory resistance. CONCLUSIONS: Taken together, the novel findings reveal that ethanol alone, absent of any secondary inflammatory insult, is sufficient to produce inflammation in the lung. Although these changes were relatively mild, they were associated with functional changes in the central airways. This animal model may be useful in the future for identifying mechanisms by which alcohol abuse sensitizes at-risk individuals to lung injury.

Inhibition of chlorine-induced airway fibrosis by budesonide.

Chlorine is a chemical threat agent that can be harmful to humans. Acute inhalation of high levels of chlorine results in the death of airway epithelial cells and can lead to persistent adverse effects on respiratory health, including airway remodeling and hyperreactivity. We previously developed a mouse chlorine exposure model in which animals developed inflammation and fibrosis in large airways. In the present study, examination by laser capture microdissection of developing fibroproliferative lesions in FVB/NJ mice exposed to 240 ppm-h chlorine revealed upregulation of genes related to macrophage function. Treatment of chlorine-exposed mice with the corticosteroid drug budesonide daily for 7 days (30-90 µg/mouse i.m.) starting 1 h after exposure prevented the influx of M2 macrophages and the development of airway fibrosis and hyperreactivity. In chlorine-exposed, budesonide-treated mice 7 days after exposure, large airways lacking fibrosis contained extensive denuded areas indicative of a poorly repaired epithelium. Damaged or poorly repaired epithelium has been considered a trigger for fibrogenesis, but the results of this study suggest that inflammation is the ultimate driver of fibrosis in our model. Examination at later times following 7-day budesonide treatment showed continued absence of fibrosis after cessation of treatment and regrowth of a poorly differentiated airway epithelium by 14 days after exposure. Delay in the start of budesonide treatment for up to 2 days still resulted in inhibition of airway fibrosis. Our results show the therapeutic potential of budesonide as a countermeasure for inhibiting persistent effects of chlorine inhalation and shed light on mechanisms underlying the initial development of fibrosis following airway injury.

Pulmonary Function Testing in Animals.

Nanoparticles possess a number of useful properties that make them useful for a variety of industrial and commercial applications. The small size of nanoparticles means that they are respirable and can penetrate deep into the lung when inhaled. Because of this, there is interest in assessing possible toxic effects of nanoparticles on the respiratory system. Measurement of respiratory mechanics and pulmonary function represents a sensitive way of detecting pathological effects of inhaled substances on the lungs. Here we describe a procedure for conducting pulmonary function measurements in mice using the forced oscillation technique. Measurements of baseline lung mechanics are conducted in anesthetized, mechanically ventilated mice, followed by repeated measurements subsequent to inhalation challenge with aerosolized methacholine. General guidelines for data analysis are provided, and sample results are presented.

An Official American Thoracic Society Workshop Report: Chemical Inhalational Disasters. Biology of Lung Injury, Development of Novel Therapeutics, and Medical Preparedness.

This report is based on the proceedings from the Inhalational Lung Injury Workshop jointly sponsored by the American Thoracic Society (ATS) and the National Institutes of Health (NIH) Countermeasures Against Chemical Threats (CounterACT) program on May 21, 2013, in Philadelphia, Pennsylvania. The CounterACT program facilitates research leading to the development of new and improved medical countermeasures for chemical threat agents. The workshop was initiated by the Terrorism and Inhalational Disasters Section of the Environmental, Occupational, and Population Health Assembly of the ATS. Participants included both domestic and international experts in the field, as well as representatives from U.S. governmental funding agencies. The meeting objectives were to (1) provide a forum to review the evidence supporting current standard medical therapies, (2) present updates on our understanding of the epidemiology and underlying pathophysiology of inhalational lung injuries, (3) discuss innovative investigative approaches to further delineating mechanisms of lung injury and identifying new specific therapeutic targets, (4) present promising novel medical countermeasures, (5) facilitate collaborative research efforts, and (6) identify challenges and future directions in the ongoing development, manufacture, and distribution of effective and specific medical countermeasures. Specific inhalational toxins discussed included irritants/pulmonary toxicants (chlorine gas, bromine, and phosgene), vesicants (sulfur mustard), chemical asphyxiants (cyanide), particulates (World Trade Center dust), and respirable nerve agents.

Acute lung injury and persistent small airway disease in a rabbit model of chlorine inhalation.

Chlorine is a pulmonary toxicant to which humans can be exposed through accidents or intentional releases. Acute effects of chlorine inhalation in humans and animal models have been well characterized, but less is known about persistent effects of acute, high-level chlorine exposures. In particular, animal models that reproduce the long-term effects suggested to occur in humans are lacking. Here, we report the development of a rabbit model in which both acute and persistent effects of chlorine inhalation can be assessed. Male New Zealand White rabbits were exposed to chlorine while the lungs were mechanically ventilated. After chlorine exposure, the rabbits were extubated and were allowed to survive for up to 24h after exposure to 800ppm chlorine for 4min to study acute effects or up to 7days after exposure to 400ppm for 8min to study longer term effects. Acute effects observed 6 or 24h after inhalation of 800ppm chlorine for 4min included hypoxemia, pulmonary edema, airway epithelial injury, inflammation, altered baseline lung mechanics, and airway hyperreactivity to inhaled methacholine. Seven days after recovery from inhalation of 400ppm chlorine for 8min, rabbits exhibited mild hypoxemia, increased area of pressure-volume loops, and airway hyperreactivity. Lung histology 7days after chlorine exposure revealed abnormalities in the small airways, including inflammation and sporadic bronchiolitis obliterans lesions. Immunostaining showed a paucity of club and ciliated cells in the epithelium at these sites. These results suggest that small airway disease may be an important component of persistent respiratory abnormalities that occur following acute chlorine exposure. This non-rodent chlorine exposure model should prove useful for studying persistent effects of acute chlorine exposure and for assessing efficacy of countermeasures for chlorine-induced lung injury.

Role of TRPA1 in acute cardiopulmonary toxicity of inhaled acrolein.

Acrolein is a highly toxic, volatile, unsaturated aldehyde generated during incomplete combustion as in tobacco smoke and indoor fires. Because the transient receptor potential ankyrin 1 (TRPA1) channel mediates tobacco smoke-induced lung injury, we assessed its role in high-level acrolein-induced toxicity in mice. Acrolein (100-275ppm, 10-30min) caused upper airway epithelial sloughing, bradypnea and oral gasping, hypothermia, cardiac depression and mortality. Male wild-type mice (WT, C57BL/6; 5-52weeks) were significantly more sensitive to high-level acrolein than age-matched, female WT mice. Both male and female TRPA1-null mice were more sensitive to acrolein-induced mortality than age- and sex-matched WT mice. Acrolein exposure increased lung weight:body weight ratios and lung albumin and decreased plasma albumin to a greater extent in TRPA1-null than in WT mice. Lung and plasma protein-acrolein adducts were not increased in acrolein-exposed TRPA1-null mice compared with WT mice. To assess TRPA1-dependent protective mechanisms, respiratory parameters were monitored by telemetry. TRPA1-null mice had a slower onset of breathing rate suppression ('respiratory braking') than WT mice suggesting TRPA1 mediates this protective response. Surprisingly, WT male mice treated either with a TRPA1 antagonist (HC030031; 200mg/kg) alone or with combined TRPA1 (100mg/kg) and TRPV1 (capsazepine, 10mg/kg) antagonists at 30min post-acrolein exposure (i.e., "real world" delay in treatment) were significantly protected from acrolein-induced mortality. These data show TRPA1 protects against high-level acrolein-induced toxicity in a sex-dependent manner. Post-exposure TRPA1 antagonism also protected against acrolein-induced mortality attesting to a complex role of TRPA1 in cardiopulmonary injury.

Diabetic Microvascular Disease and Pulmonary Fibrosis: The Contribution of Platelets and Systemic Inflammation.

Diabetes is strongly associated with systemic inflammation and oxidative stress, but its effect on pulmonary vascular disease and lung function has often been disregarded. Several studies identified restrictive lung disease and fibrotic changes in diabetic patients and in animal models of diabetes. While microvascular dysfunction is a well-known complication of diabetes, the mechanisms leading to diabetes-induced lung injury have largely been disregarded. We described the potential involvement of diabetes-induced platelet-endothelial interactions in perpetuating vascular inflammation and oxidative injury leading to fibrotic changes in the lung. Changes in nitric oxide synthase (NOS) activation and decreased NO bioavailability in the diabetic lung increase platelet activation and vascular injury and may account for platelet hyperreactivity reported in diabetic patients. Additionally, the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway has been reported to mediate pancreatic islet damage, and is implicated in the onset of diabetes, inflammation and vascular injury. Many growth factors and diabetes-induced agonists act via the JAK/STAT pathway. Other studies reported the contribution of the JAK/STAT pathway to the regulation of the pulmonary fibrotic process but the role of this pathway in the development of diabetic lung fibrosis has not been considered. These observations may open new therapeutic perspectives for modulating multiple pathways to mitigate diabetes onset or its pulmonary consequences.

Persistent effects of chlorine inhalation on respiratory health.

Chlorine gas is a toxic respiratory irritant that is considered a chemical threat agent because of the potential for release in industrial accidents or terrorist attacks. Chlorine inhalation damages the respiratory tract, including the airways and distal lung, and can result in acute lung injury. Some individuals exposed to chlorine experience a full recovery from acute injury, whereas others develop persistent adverse effects, such as respiratory symptoms, inflammation, and lung-function decrements. In animal models, chlorine can produce persistent inflammation, remodeling, and obstruction in large or small airways, depending on species. Airways with pseudostratified epithelia are repaired efficiently, with surviving basal epithelial cells serving as progenitor cells that repopulate the complement of differentiated cell types. Distal airways lacking basal cells are repaired less efficiently, leading to chronic inflammation and fibrosis at these sites. Persistent chlorine-induced airway disease in humans is treated with asthma medication to relieve symptoms. However, such treatment does not ameliorate the underlying disease pathogenesis, so treatments that are more effective at preventing initial development of airway disease after irritant gas exposure and at reversing established disease are needed.

Development and assessment of countermeasure formulations for treatment of lung injury induced by chlorine inhalation.

Chlorine is a commonly used, reactive compound to which humans can be exposed via accidental or intentional release resulting in acute lung injury. Formulations of rolipram (a phosphodiesterase inhibitor), triptolide (a natural plant product with anti-inflammatory properties), and budesonide (a corticosteroid), either neat or in conjunction with poly(lactic:glycolic acid) (PLGA), were developed for treatment of chlorine-induced acute lung injury by intramuscular injection. Formulations were produced by spray-drying, which generated generally spherical microparticles that were suitable for intramuscular injection. Multiple parameters were varied to produce formulations with a wide range of in vitro release kinetics. Testing of selected formulations in chlorine-exposed mice demonstrated efficacy against key aspects of acute lung injury. The results show the feasibility of developing microencapsulated formulations that could be used to treat chlorine-induced acute lung injury by intramuscular injection, which represents a preferred route of administration in a mass casualty situation.

Crystalline silica-induced leukotriene B4-dependent inflammation promotes lung tumour growth.

Chronic exposure to crystalline silica (CS) causes silicosis, an irreversible lung inflammatory disease that may eventually lead to lung cancer. In this study, we demonstrate that in K-ras(LA1) mice, CS exposure markedly enhances the lung tumour burden and genetic deletion of leukotriene B4 receptor-1 (BLT1(-/-)) attenuates this increase. Pulmonary neutrophilic inflammation induced by CS is significantly reduced in BLT1(-/-)K-ras(LA1) mice. CS exposure induces LTB4 production by mast cells and macrophages independent of inflammasome activation. In an air-pouch model, CS-induced neutrophil recruitment is dependent on LTB4 production by mast cells and BLT1 expression on neutrophils. In an implantable lung tumour model, CS exposure results in rapid tumour growth and decreased survival that is attenuated in the absence of BLT1. These results suggest that the LTB4/BLT1 axis sets the pace of CS-induced sterile inflammation that promotes lung cancer progression. This knowledge may facilitate development of immunotherapeutic strategies to fight silicosis and lung cancer.

Abnormal epithelial structure and chronic lung inflammation after repair of chlorine-induced airway injury.

Chlorine is a toxic gas used in a variety of industrial processes and is considered a chemical threat agent. High-level chlorine exposure causes acute lung injury, but the long-term effects of acute chlorine exposure are unclear. Here we characterized chronic pulmonary changes following acute chlorine exposure in mice. A/J mice were exposed to 240 parts per million-hour chlorine or sham-exposed to air. Chlorine inhalation caused sloughing of bronchial epithelium 1 day after chlorine exposure, which was repaired with restoration of a pseudostratified epithelium by day 7. The repaired epithelium contained an abnormal distribution of epithelial cells containing clusters of club or ciliated cells rather than the uniformly interspersed pattern of these cells in unexposed mice. Although the damaged epithelium in A/J mice was repaired rapidly, and minimal airway fibrosis was observed, chlorine-exposed mice developed pneumonitis characterized by infiltration of alveoli with neutrophils and prominent, large, foamy macrophages. Levels of CXCL1/KC, CXCL5/LPS-induced CXC chemokine, granulocyte colony-stimulating factor, and VEGF in bronchoalveolar (BAL) fluid from chlorine-exposed mice showed steadily increasing trends over time. BAL protein levels were increased on day 4 and remained elevated out to day 28. The number of bacteria cultured from lungs of chlorine-exposed mice 4 wk after exposure was not increased compared with sham-exposed mice, indicating that the observed pneumonitis was not driven by bacterial infection of the lung. The results indicate that acute chlorine exposure may cause chronic abnormalities in the lungs despite rapid repair of injured epithelium.

Inhibition of chlorine-induced pulmonary inflammation and edema by mometasone and budesonide.

Chlorine gas is a widely used industrial compound that is highly toxic by inhalation and is considered a chemical threat agent. Inhalation of high levels of chlorine results in acute lung injury characterized by pneumonitis, pulmonary edema, and decrements in lung function. Because inflammatory processes can promote damage in the injured lung, anti-inflammatory therapy may be of potential benefit for treating chemical-induced acute lung injury. We previously developed a chlorine inhalation model in which mice develop epithelial injury, neutrophilic inflammation, pulmonary edema, and impaired pulmonary function. This model was used to evaluate nine corticosteroids for the ability to inhibit chlorine-induced neutrophilic inflammation. Two of the most potent corticosteroids in this assay, mometasone and budesonide, were investigated further. Mometasone or budesonide administered intraperitoneally 1h after chlorine inhalation caused a dose-dependent inhibition of neutrophil influx in lung tissue sections and in the number of neutrophils in lung lavage fluid. Budesonide, but not mometasone, reduced the levels of the neutrophil attractant CXCL1 in lavage fluid 6h after exposure. Mometasone or budesonide also significantly inhibited pulmonary edema assessed 1 day after chlorine exposure. Chlorine inhalation resulted in airway hyperreactivity to inhaled methacholine, but neither mometasone nor budesonide significantly affected this parameter. The results suggest that mometasone and budesonide may represent potential treatments for chemical-induced lung injury.

Differential susceptibility of inbred mouse strains to chlorine-induced airway fibrosis.

Chlorine is a reactive gas that is considered a chemical threat agent. Humans who develop acute lung injury from chlorine inhalation typically recover normal lung function; however, a subset can experience chronic airway disease. To examine pathological changes following chlorine-induced lung injury, mice were exposed to a single high dose of chlorine, and repair of the lung was analyzed at multiple times after exposure. In FVB/NJ mice, chlorine inhalation caused pronounced fibrosis of larger airways that developed by day 7 after exposure and was associated with airway hyperreactivity. In contrast, A/J mice had little or no airway fibrosis and had normal lung function at day 7. Unexposed FVB/NJ mice had less keratin 5 staining (basal cell marker) than A/J mice in large intrapulmonary airways where epithelial repair was poor and fibrosis developed after chlorine exposure. FVB/NJ mice had large areas devoid of epithelium on day 1 after exposure leading to fibroproliferative lesions on days 4 and 7. A/J mice had airways covered by squamous keratin 5-stained cells on day 1 that transitioned to a highly proliferative reparative epithelium by day 4 followed by the reappearance of ciliated and Clara cells by day 7. The data suggest that lack of basal cells in the large intrapulmonary airways and failure to effect epithelial repair at these sites are factors contributing to the development of airway fibrosis in FVB/NJ mice. The observed differences in susceptibility to chlorine-induced airway disease provide a model in which mechanisms and treatment of airway fibrosis can be investigated.

Repair of tracheal epithelium by basal cells after chlorine-induced injury.

BACKGROUND: Chlorine is a widely used toxic compound that is considered a chemical threat agent. Chlorine inhalation injures airway epithelial cells, leading to pulmonary abnormalities. Efficient repair of injured epithelium is necessary to restore normal lung structure and function. The objective of the current study was to characterize repair of the tracheal epithelium after acute chlorine injury. METHODS: C57BL/6 mice were exposed to chlorine and injected with 5-ethynyl-2'-deoxyuridine (EdU) to label proliferating cells prior to sacrifice and collection of tracheas on days 2, 4, 7, and 10 after exposure. Airway repair and restoration of a differentiated epithelium were examined by co-localization of EdU labeling with markers for the three major tracheal epithelial cell types [keratin 5 (K5) and keratin 14 (K14) for basal cells, Clara cell secretory protein (CCSP) for Clara cells, and acetylated tubulin (AcTub) for ciliated cells]. Morphometric analysis was used to measure proliferation and restoration of a pseudostratified epithelium. RESULTS: Epithelial repair was fastest and most extensive in proximal trachea compared with middle and distal trachea. In unexposed mice, cell proliferation was minimal, all basal cells expressed K5, and K14-expressing basal cells were absent from most sections. Chlorine exposure resulted in the sloughing of Clara and ciliated cells from the tracheal epithelium. Two to four days after chlorine exposure, cell proliferation occurred in K5- and K14-expressing basal cells, and the number of K14 cells was dramatically increased. In the period of peak cell proliferation, few if any ciliated or Clara cells were detected in repairing trachea. Expression of ciliated and Clara cell markers was detected at later times (days 7-10), but cell proliferation was not detected in areas in which these differentiated markers were re-expressed. Fibrotic lesions were observed at days 7-10 primarily in distal trachea. CONCLUSION: The data are consistent with a model where surviving basal cells function as progenitor cells to repopulate the tracheal epithelium after chlorine injury. In areas with few remaining basal cells, repair is inefficient, leading to airway fibrosis. These studies establish a model for understanding regenerative processes in the respiratory epithelium useful for testing therapies for airway injury.

Inhibition of chlorine-induced lung injury by the type 4 phosphodiesterase inhibitor rolipram.

Chlorine is a highly toxic respiratory irritant that when inhaled causes epithelial cell injury, alveolar-capillary barrier disruption, airway hyperreactivity, inflammation, and pulmonary edema. Chlorine is considered a chemical threat agent, and its release through accidental or intentional means has the potential to result in mass casualties from acute lung injury. The type 4 phosphodiesterase inhibitor rolipram was investigated as a rescue treatment for chlorine-induced lung injury. Rolipram inhibits degradation of the intracellular signaling molecule cyclic AMP. Potential beneficial effects of increased cyclic AMP levels include inhibition of pulmonary edema, inflammation, and airway hyperreactivity. Mice were exposed to chlorine (whole body exposure, 228-270 ppm for 1 h) and were treated with rolipram by intraperitoneal, intranasal, or intramuscular (either aqueous or nanoemulsion formulation) delivery starting 1h after exposure. Rolipram administered intraperitoneally or intranasally inhibited chlorine-induced pulmonary edema. Minor or no effects were observed on lavage fluid IgM (indicative of plasma protein leakage), KC (Cxcl1, neutrophil chemoattractant), and neutrophils. All routes of administration inhibited chlorine-induced airway hyperreactivity assessed 1 day after exposure. The results of the study suggest that rolipram may be an effective rescue treatment for chlorine-induced lung injury and that both systemic and targeted administration to the respiratory tract were effective routes of delivery.

Tumor necrosis factor-α mediates interactions between macrophages and epithelial cells underlying proinflammatory gene expression induced by particulate matter.

Ambient particulate matter (PM) exposure is known to have adverse effects on respiratory health, but the underlying mechanisms remain obscure. We tested the hypothesis that macrophages and epithelial cells synergize to produce maximal cytokine release in response to PM exposure, thereby promoting inflammatory responses. We developed a co-culture model using MLE-12 (mouse lung epithelial) cells and RAW 264.7 (mouse monocyte/macrophage) cells. MLE-12 cells produced KC (Cxcl1) but not tumor necrosis factor-α (TNF), and KC was upregulated only at high levels of urban particulate matter (UPM; NIST 1648a). RAW 264.7 cells produced TNF but not KC, and TNF production was increased by treatment with UPM. In contrast, KC production was upregulated by co-culture of MLE-12 and RAW 264.7 cells, and it was further increased by treatment with a concentration of UPM that had no effect on MLE-12 cells alone. Multiplex cytokine assay revealed a similar pattern of synergistic production of MIG (Cxcl9) and IP-10 (Cxcl10) in co-cultures in response to UPM. TNF was implicated as mediating the synergistic increase in KC production because TNF upregulated KC production in MLE-12 cells, and UPM-induced KC production in co-cultures could be inhibited by a TNF blocking antibody. Intratracheal instillation of UPM into both wild-type and TNF receptor knockout mice resulted in increased TNF production in lavage fluid and increased TNF mRNA expression in cells recovered from lavage fluid. Additionally, UPM instillation into wild-type mice resulted in increased neutrophils and KC in lavage fluid, and these were inhibited in UPM-exposed TNF receptor knockout mice. These results are consistent with a model in which PM activates TNF production in macrophages which in turn stimulates epithelial cells to produce proinflammatory cytokines such as KC. The findings suggest a potential mechanism by which inhaled PM induces inflammation in the lung.

Impaired mouse mammary gland growth and development is mediated by melatonin and its MT1G protein-coupled receptor via repression of ERα, Akt1, and Stat5.

To determine whether melatonin, via its MT(1) G protein-coupled receptor, impacts mouse mammary gland development, we generated a mouse mammary tumor virus (MMTV)-MT1-Flag-mammary gland over-expressing (MT1-mOE) transgenic mouse. Increased expression of the MT(1) -Flag transgene was observed in the mammary glands of pubescent MT1-mOE transgenic female mice, with further significant increases during pregnancy and lactation. Mammary gland whole mounts from MT1-mOE mice showed significant reductions in ductal growth, ductal branching, and terminal end bud formation. Elevated MT(1) receptor expression in pregnant and lactating female MT1-mOE mice was associated with reduced lobulo-alveolar development, inhibition of mammary epithelial cell proliferation, and significant reductions in body weights of suckling pups. Elevated MT(1) expression in pregnant and lactating MT1-mOE mice correlated with reduced mammary gland expression of Akt1, phospho-Stat5, Wnt4, estrogen receptor alpha, progesterone receptors A and B, and milk proteins ß-casein and whey acidic protein. Estrogen- and progesterone-stimulated mammary gland development was repressed by elevated MT(1) receptor expression and exogenous melatonin administration. These studies demonstrate that the MT(1) melatonin receptor and its ligand melatonin play an important regulatory role in mammary gland development and lactation in mice through both growth suppression and alteration of developmental paradigms.

Deviations from Haber's Law for multiple measures of acute lung injury in chlorine-exposed mice.

Chlorine gas is considered a chemical threat agent that can cause acute lung injury. Studies in the early 20th century on war gases led Haber to postulate that the dose of an inhaled chemical expressed as the product of gas concentration and exposure time leads to a constant toxicological effect (Haber's Law). In the present work, mice were exposed to a constant dose of chlorine (100 ppm-h) delivered using different combinations of concentration and time (800 ppm/7.5 min, 400 ppm/15 min, 200 ppm/30 min, and 100 ppm/60 min). Significant effects of exposure protocol on survival evaluated 6 h after exposure were observed, ranging from 0% for the 7.5-min exposure to 100% for the 30- and 60-min exposures. Multiple parameters indicative of lung injury were examined to determine if any aspects of lung injury were differentially affected by the exposure protocols. Most parameters (pulmonary edema, neutrophil influx, and levels of protein, immunoglobulin M, and the chemokine KC [Cxcl1] in lavage fluid) indicated that lung injury was most pronounced for the 15-min exposure and least for the 60-min exposure. In contrast, changes in pulmonary function at baseline and in response to inhaled methacholine were similar following the three exposure regimens. The results indicate that the extent of lung injury following chlorine inhalation depends not only on total dose but also on the specifics of exposure concentration and time, and they suggest that evaluation of countermeasures against chlorine-induced lung injury should be performed using multiple types of exposure scenarios.

Mitigation of chlorine lung injury by increasing cyclic AMP levels.

Chlorine is considered a chemical threat agent to which humans may be exposed as a result of accidental or intentional release. Chlorine is highly reactive, and inhalation of the gas causes cellular damage to the respiratory tract, inflammation, pulmonary edema, and airway hyperreactivity. Drugs that increase intracellular levels of the signaling molecule cyclic AMP (cAMP) may be useful for treatment of acute lung injury through effects on alveolar fluid clearance, inflammation, and airway reactivity. This article describes mechanisms by which cAMP regulates cellular processes affecting lung injury and discusses the basis for investigating drugs that increase cAMP levels as potential treatments for chlorine-induced lung injury. The effects of beta(2)-adrenergic agonists, which stimulate cAMP synthesis, and phosphodiesterase inhibitors, which inhibit cAMP degradation, on acute lung injury are reviewed, and the relative advantages of these approaches are compared.