Aerosolised 5-azacytidine suppresses tumour growth and reprogrammes the epigenome in an orthotopic lung cancer model.

BACKGROUND: Epigenetic silencing by promoter methylation and chromatin remodelling affects hundreds of genes and is a causal event for lung cancer. Treatment of patients with low doses of the demethylating agent 5-azacytidine in combination with the histone deacetylase inhibitor entinostat has yielded clinical responses. The subcutaneous dosing route for consecutive days and reduced bioavailability of 5-azacytidine because of inactivation by cytidine deaminase may limit the expansion of epigenetic therapy into Phase III trials. To mitigate these barriers, an aerosol of 5-azacytidine was generated and characterised. METHODS: The effect of aerosol vs systemic delivery of 5-azacytidine on tumour burden and molecular response of engrafted lung tumours in the nude rat was compared. RESULTS: Pharmacokinetics revealed major improvement in the half-life of 5-azacytidine in lung tissue with aerosol delivery. Aerosolised 5-azacytidine significantly reduced lung tumour burden and induced global demethylation of the epigenome at one-third of the comparable effective systemic dose. High commonality for demethylation of genes was seen in tumours sampled throughout lung lobes and across treated animals receiving the aerosolised drug. CONCLUSION: Collectively, these findings show that aerosolised 5-azacytidine targets the lung, effectively reprogrammes the epigenome of tumours, and is a promising approach to combine with other drugs for treating lung cancer.

Cigarette smoke-induced hypercapnic emphysema in C3H mice is associated with increases of macrophage metalloelastase and substance P in the lungs.

The authors tested whether macrophage metalloelastase (MMP-12) and substance P (SP) were increased in the cigarette smoke (CS)-exposed female C3H/HeN mice with hypercapnic emphysema. The authors found that as compared to control (filtered air), 16 weeks of CS exposure significantly up-regulated mRNA and protein levels of MMP-12, the ratio of MMP-12/tissue inhibitor of matrix metalloproteinase-1, and SP/preprotachykinin-A (a precursor to SP) in the lungs. Importantly, a significant correlation was found between MMP-12 and SP, and between MMP-12/SP and the degrees of hypoxemia/hypercapnia denoted in CS-exposed mice. These data suggest a possible involvement of SP and MMP-12 in the pathogenesis of severe COPD.

Blunted ventilatory response to hypoxia/hypercapnia in mice with cigarette smoke-induced emphysema.

It has been reported that the degree of emphysema induced by chronic cigarette smoke (CS) is greater in female C3H/HeN mice as compared to other mouse strains. We hypothesized that these mice would develop the similar major characteristics seen in hypercapnic patients with chronic obstructive pulmonary disease (COPD), including emphysema, pulmonary inflammation, hypercapnia/hypoxemia, rapid breathing, and attenuated ventilatory response (AVR). Mice were exposed either to CS or filtered air (FA) for 16 weeks. After exposure, arterial blood gases and minute ventilation were measured before and during chemical challenges in anesthetized and spontaneously breathing mice. We found that as compared to FA, CS exposure caused emphysema and pulmonary inflammation associated with: (1) hypercapnia and hypoxemia, (2) rapid breathing, and (3) AVR to 25 breaths of pure N(2), 5% CO(2) alone, and 5% CO(2) coupled with 10% O(2). The similarity of these pathophysiological characteristics between our mouse model and COPD patients suggests that this model could be effectively applied to study COPD pathophysiology, especially central mechanisms of the AVR genesis.

Effects of concurrent ozone exposure on the pathogenesis of cigarette smoke-induced emphysema in B6C3F1 mice.

Episodic elevation of air pollutants may exacerbate respiratory distress associated with chronic obstructive pulmonary disease (COPD), yet few experiments have been performed to determine how continuously polluted atmospheres may contribute to the etiology of COPD, in general and pulmonary emphysema in particular. This study describes the effects of concurrent exposure to ozone (O(3)) in the pathogenesis of cigarette smoke (CS)-induced emphysema in the mouse. Female B6C3F1 mice were whole-body exposed either to filtered air (FA) or to mainstream CS at a concentration of 250 mg total particulate material/m(3) for 6 h/day, 5 days/wk for 15 or 32 wk. Concurrently, mice were exposed either to FA or to O(3) at 0.3 ppm for 8 h/night, 5 nights/wk for the same time periods. At necropsy, mouse lungs were lavaged, and bronchoalveolar lavage fluid (BALF) was analyzed for inflammatory cell numbers, total protein, lactate dehydrogenase (LDH) and alkaline phosphatase (AP) activities, superoxide production by isolated alveolar macrophages, glutathione content, inflammatory cytokines, and proteolytic activity. Other lungs were inflated at constant pressure for 6 h with formalin for fixation, routine histopathology, and stereology. After 32 wk of exposure, CS with or without concurrent O(3) exposure produced stereologic evidence of emphysema as previously described. Concurrent O(3) exposure did not worsen any of these parameters, nor did O(3) by itself cause stereologic changes that were consistent with emphysema. The O(3) exposure caused only slight elevations of BALF macrophages, while CS exposure caused marked increases in the numbers of both BALF macrophages and neutrophils. Neutrophils in the BALF in response to CS exposure were also more numerous at 32 wk than at 15 wk. Exposure to CS caused an increase in BALF total protein, LDH, AP, and interleukin (IL)-1beta. After 32 wk, CS exposure was associated with decreased superoxide production from isolated alveolar macrophages. The CS exposure elevated BALF total glutathione primarily at 15 wk. Overall, O(3) had little effect on endpoints that were significantly affected by CS exposure. We conclude that concurrent O(3) exposure has no effect on the induction of emphysema by CS in this animal model.

Effects of subchronic inhalation exposure of rats to emissions from a diesel engine burning soybean oil-derived biodiesel fuel.

There is increasing interest in diesel fuels derived from plant oils or animal fats ("biodiesel"), but little information on the toxicity of biodiesel emissions other than bacterial mutagenicity. F344 rats were exposed by inhalation 6 h/day, 5 days/wk for 13 wk to 1 of 3 dilutions of emissions from a diesel engine burning 100% soybean oil-derived fuel, or to clean air as controls. Whole emissions were diluted to nominal NO(x) concentrations of 5, 25, or 50 ppm, corresponding to approximately 0.04, 0.2, and 0.5 mg particles/m(3), respectively. Biologically significant, exposure-related effects were limited to the lung, were greater in females than in males, and were observed primarily at the highest exposure level. There was a dose-related increase in the numbers of alveolar macrophages and the numbers of particles in the macrophages, as expected from repeated exposure, but no neutrophil response even at the highest exposure level. The macrophage response was reduced 28 days after cessation of the exposure. Among the high-level females, the group mean lung weight/body weight ratio was increased, and minimal, multifocal bronchiolar metaplasia of alveolar ducts was observed in 4 of 30 rats. Lung weights were not significantly increased, and metaplasia of the alveolar ducts was not observed in males. An increase in particle-laden macrophages was the only exposure-related finding in lungs at the intermediate and low levels, with fewer macrophages and fewer particles per macrophage at the low level. Alveolar histiocytosis was observed in a few rats in both exposed and control groups. There were statistically significant, but minor and not consistently exposure-related, differences in body weight, nonpulmonary organ weights, serum chemistry, and glial fibrillary acidic protein in the brain. There were no significant exposure-related effects on survival, clinical signs, feed consumption, ocular toxicity, hematology, neurohistology, micronuclei in bone marrow, sister chromatid exchanges in peripheral blood lymphocytes, fertility, reproductive toxicity, or teratology. This study demonstrated modest adverse effects at the highest exposure level, and none other than the expected physiological macrophage response to repeated particle exposure at the intermediate level.

Refinement of an orthotopic lung cancer model in the nude rat.

Over 85% of people with lung cancer eventually succumb to this disease, largely because current chemotherapies are ineffective. The testing and validation of promising new approaches generally rely on achieving responses with cell lines in vitro or in tumor xenografts in nude mice. However, quite often the results seen with these models are not recapitulated in the clinic, thus necessitating the need for better animal models of lung cancer for preclinical testing of new therapies. One promising model is that of orthotopic lung cancer, where xenografts of human lung cancer are established in lungs of immunodeficient rodents. The problems associated with this model include poor rates of engraftment, limited tumor multiplicity, and a heightened risk for surgical trauma. The purpose of our study was to develop an efficient approach to engraftment of orthotopic tumors throughout the lungs of the Rowett nude rat. Initially, we augmented immunosuppression in the rats with whole-body X-irradiation and then used orotracheal cannulas to intratracheally instill human cancer cells from the Calu-6 cell line. This protocol produced a low rate of engraftment and low tumor multiplicity. The hypothesis that slight disruption of the pulmonary epithelium or the surfactant layer would allow better tumor engraftment was tested by coadministration of either pancreatic elastase or ethylenediaminetetraacetic acid (EDTA) along with the cell instillations. Lung tumor engraftment was evaluated 8 weeks after instillation. The inclusion of elastase or EDTA with the Calu-6 cells resulted in an 80-100% engraftment rate, respectively. Coadministration of EDTA resulted in significantly larger and greater numbers of tumors/lung than those in elastase-treated animals. Temporal studies demonstrated that small nodules were scattered throughout the lung parenchyma 5 weeks after instilling Calu-6 cells and EDTA. These nodules grew to coalesce and form large masses that effaced >75% of the parenchyma at 9 weeks postinstillation. The refinements made through our studies have led to the development of an orthotopic lung cancer model that should facilitate the evaluation of novel therapies designed to treat or impede lung cancer development.

Response of F344 rats to inhalation of subclinical levels of sarin: exploring potential causes of Gulf War illness.

Subclinical, repeated exposures of F344 rats to sarin resulted in brain alterations in densities of chlonergic receptor subtypes that may be associated with memory loss and cognitive dysfunction. The exposures also depressed the immune system. The rat appears to be a good model for studying the effects of subclinical exposure to a nerve gas.

Particle clearance and histopathology in lungs of C3H/HeJ mice administered beryllium/copper alloy by intratracheal instillation.

Beryllium/copper (BeCu) alloys are commonly used in the electronics, automotive, consumer, defense, and aerospace industries. Some individuals exposed occupationally to BeCu alloys have developed chronic beryllium disease. However, little is known of the toxicity and fate of BeCu alloys in the respiratory tract. To begin to address this question, we investigated the pulmonary toxicity and clearance of BeCu alloy (2% Be; 98% Cu) in mice. Groups of 40 female C3H/HeJ mice were administered 12.5, 25, and 100 microg BeCu alloy or 2 and 8 microg Be metal by intratracheal instillation. Mice were sacrificed at 1 h and 1, 7, 14, and 28 days postinstillation. Left lungs were evaluated for histopathological change. Right lungs were analyzed for Be and Cu content. Twenty-five percent of the high-dose BeCu mice and 7.5% of the mid-dose BeCu mice died within 24 h of dosing. Acute pulmonary lesions included acute alveolitis and interstitial inflammation. Type II epithelial cell hyperplasia and centriacinar fibrosis were present by 7 days after dosing. Lesions persisted through 28 days after instillation. No lesions attributable to alloy exposure were present in liver or kidney. Be metal instillation caused no deaths and minimal pulmonary changes over the time studied, indicating that the pulmonary lesions were due to Cu rather than Be. Cu cleared the lung with a half-time of 0. 5-2 days. Be cleared with a half-time of several weeks or longer. Results of this study suggest that exposure to BeCu alloy is more acutely toxic to lung than Be metal. The results of tissue analyses also indicate that, while the Cu component of the alloy clears the lung rapidly, Be is retained and may accumulate upon repeated exposure.

Cyclooxygenase-2 expression is abundant in alveolar type II cells in lung cancer-sensitive mouse strains and in premalignant lesions.

Overexpression of cyclooxygenase-2 (COX-2) is seen in a high percentage of human colon tumors, lung adenocarcinomas and other cancers. Inhibition of this enzyme represses human colon tumorigenesis and decreases lung tumor multiplicity in 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone-exposed A/J mice. The purpose of this investigation was to characterize the expression of cyclooxygenase-2 (COX-2) during tumor progression in the A/J mouse lung and to compare the results with expression in other cancer-susceptible and several cancer-resistant mouse strains. Analysis of normal A/J mouse lung showed that type II alveolar epithelial cells express high levels of COX-2 protein and mRNA, indicating that COX-2 is present constitutively in this tumor progenitor cell prior to any carcinogen exposure. Examination of lung-cancer-resistant (C3H/HeJ, C57BL/6J, DBA/2J) and other lung-cancer-susceptible (A/WySnJ, SWR/J) strains showed similar levels of COX-2 mRNA expression in the three susceptible strains and lower levels of expression in two of the resistant strains, indicating a possible correlation between COX-2 expression in type II cells and lung cancer susceptibility. COX-2 protein expression was observed in A/J lung tumors at all stages of development. Variation and occasional absence of protein expression were also observed in A/J lung tumors, particularly in adenomas and adenocarcinomas, suggesting that COX-2 is not obligatory for maintenance of the malignant phenotype. In support of this conclusion, treatment of xenografted cell lines derived from malignant murine pulmonary tumors with COX-2 inhibitors produced only a slight repression of growth. However, the frequent expression of COX-2 in early lesions in the A/J mouse lung combined with the known reduction in tumor number in animals treated with COX-2 inhibitors prior to carcinogen exposure indicate that COX-2 could be a promising target for lung cancer chemoprevention. In addition, high levels of COX-2 expression in the normal tumor-progenitor cells of lung-cancer-sensitive mice indicate that COX-2 may play a role in lung cancer susceptibility.

Animal models of emphysema and their relevance to studies of particle-induced disease.

Emphysema is a pulmonary disease that may be exacerbated by inhaled particles. Over the years, many animal models of emphysema have been developed that may be useful in studying the effects of inhaled particles on humans with emphysema. Models have been described in many species, and many approaches have been described for inducing emphysema. Emphysema in humans is a parenchymal component of chronic obstructive pulmonary disease and frequently coexists in a complex with disease of the airways such as bronchitis. Animal models of emphysema usually recapitulate only one or a few aspects of this complex disease. Thus, the emphysema model must be selected carefully in order to answer specific questions about the interactive effects of particles and emphysema.

Enhanced pulmonary epithelial replication and axial airway mucosubstance changes in F344 rats exposed short-term to mainstream cigarette smoke.

Cigarette smoking is associated with respiratory diseases that may be caused by injury to specific pulmonary cells. The injury may manifest itself as site-specific enhanced cellular replication. In this study, rats were exposed either to mainstream cigarette smoke (CS; 250 mg total particulate matter/m(3)) or to filtered air (FA) for 6 h/day, 5 days/week, for 2 weeks. In one group, cells in S-phase were labeled over 7 days by bromodeoxyuridine (BrdU) released from implanted osmotic pumps (pump labeled), while another group received BrdU by injection 2 h prior to necropsy (pulse labeled). Morphometry showed that the type II epithelial BrdU labeling index (LI) was significantly elevated in the CS-exposed animals of both labeling groups. The axial airway and terminal bronchiolar LIs were enhanced by CS only in the pump-labeled group. In a third group (pulse labeled), 2 weeks of recovery following exposure to CS allowed a normalization in the type II LI. In the pump-labeled rats, the CS-induced elevation of the type II LI was greater than the LI elevation in conducting airways, suggesting that the parenchyma may have been injured more than the conducting airways. The terminal bronchiolar LI in the pump-labeled group, regardless of exposure, was significantly greater than the axial airway LI. Pump labeling, in contrast to pulse labeling, could therefore discern differences among replication rates of conducting airway epithelium in different regions of the lung. Mucosubstance (MS) within the axial airway epithelium was quantified by morphometry. The CS exposure did not increase the total number of MS-containing cells or the total number of axial airway epithelial cells, but there was a phenotype change in the MS cells. Neutral MS cells (periodic acid-Schiff-positive) were significantly decreased, while acid MS cells (alcian blue-positive) were slightly increased by CS exposure. Either cell replication and differentiation or differentiation alone may have changed the phenotype in the MS cell population.

Cigarette smoke exposure produces more evidence of emphysema in B6C3F1 mice than in F344 rats.

Cigarette smoke (CS) causes pulmonary emphysema in humans, but results of previous studies on CS-exposed laboratory animals have been equivocal and have not clearly demonstrated progression of the disease. In this study, morphometry and histopathology were used to assess emphysema in the lungs of B6C3F1 mice and Fischer-344 rats. The animals were exposed, whole-body, to CS at a concentration of 250 mg total particulate matter/m3 for 6 h/day, 5 days/week, for either 7 or 13 months. Morphometry included measurements of parenchymal air space enlargement (alveolar septa mean linear intercept [Lm], volume density of alveolar air space [VVair]), and tissue loss (volume density of alveolar septa [VVspt]). In addition, centriacinar intra-alveolar inflammatory cells were counted to assess species differences in the type of inflammatory response associated with CS exposure. In mice, many of the morphometric parameters indicating emphysema differed significantly between CS-exposed and control animals. In CS-exposed rats, only some of the parameters differed significantly from control values. The Lm in both CS-exposed mice and rats was increased at 7 and 13 months, indicating an enlargement of parenchymal air spaces, but the VVair was increased significantly only in CS-exposed mice. The VVspt was decreased at both time points in mice, but not in rats, indicating damage to the structural integrity of parenchyma. Morphologic evidence of tissue destruction in the mice included alveoli that were irregular in size and shape and alveoli with multiple foci of septal discontinuities and isolated septal fragments. Morphometric differences in the mice at 13 months were greater than at 7 months, suggesting a progression of the disease. Inflammatory lesions within the lungs of mice contained significantly more neutrophils than those lesions in rats. These results suggest that B6C3F1 mice are more susceptible than F344-rats to the induction of emphysema by this CS exposure regimen and that in mice the emphysema may be progressive. Furthermore, the type of inflammatory response may be a determining factor for species differences in susceptibility to emphysema induction by CS exposure.

Characterization of rat pancreatic glutathione S-transferases by chromatofocusing, reverse-phase high-performance liquid chromatography, and immunohistochemistry.

The cytosolic glutathione S-transferases (GSTs) are a family of phase II detoxifying isoenzymes that catalyze the interaction of the tripeptide thiol glutathione (GSH) with a wide variety of reactive and often toxic or carcinogenic electrophilic substrates. Pancreatic GSTs, however, have only been partially characterized. In this study, pancreatic cytosolic GSTs from male Fisher 344 rats were semipurified by affinity chromatography and then analyzed for isoenzyme content by chromatofocusing (fast protein liquid chromatography) and for subunit content by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and reverse-phase high-performance liquid chromatography. In addition, polyclonal rabbit antisera were produced against homodimeric isoenzymes purified from rat liver and kidney, including the alpha class isoenzymes 1-1 and 2-2, the mu class isoenzyme 4-4, and the pi class isoenzyme 7-7. These antisera were used in immunohistochemical (IHC) studies of the distribution of the pancreatic GSTs. A range of 0.5-1.6% of the total protein in rat pancreatic cytosol was found to be GST protein. The most abundant subunits present were the pi subunit 7 and mu subunits 3 and 4. Using modified methodology, smaller amounts of the alpha subunit 2 and the mu subunit 6 were detected, whereas very small amounts of the alpha subunits 1 and 8 were present. The IHC demonstrated that the GSTs were in large part limited to the duct system of the exocrine pancreas, with positive staining of endothelial cells and stroma observed for the alpha and mu subunits. Isoenzymes containing the alpha subunit 2 were preferentially expressed in centroacinar cells and small ductules, whereas those containing the mu subunit 4 and the pi subunit 7 were more prevalent within larger ductules and ducts. The lumens of the largest ducts also contained the two subunits 4 and 7. It is concluded that the acinar cells of the exocrine pancreas may lack the protection against electrophilic toxic and carcinogenic agents provided by the ductular system by GSTs.

Carcinogenic responses of transgenic heterozygous p53 knockout mice to inhaled 239PuO2 or metallic beryllium.

The transgenic heterozygous p53+/- knockout mouse has been a model for assessing the tumorigenicity of selected carcinogens administered by noninhalation routes of exposure. The sensitivity of the model for predicting cancer by inhaled chemicals has not been examined. This study addresses this issue by acutely exposing p53+/- mice of both sexes by nose-only inhalation to either air (controls), or to 1 of 2 levels of 239PuO2 (500 or 100 Bq 239Pu) or beryllium (Be) metal (60 or 15 micrograms). Additional wild-type p53+/+ mice were exposed by inhalation to either 500 Bq of 239PuO2 or 60 micrograms of Be metal. These carcinogens were selected because they operate by differing mechanisms and because of their use in other pulmonary carcinogenesis studies in our laboratory. Four or 5 of the 15 mice per sex from each group were sacrificed 6 mo after exposure, and only 2 pulmonary neoplasms were observed. The remainder of the mice were held for life-span observation and euthanasia as they became moribund. Survival of the p53+/- knockout mice was reduced compared to the p53+/+ wild-type mice. No lung neoplasms were observed in p53+/- mice exposed to air alone. Eleven of the p53+/- mice inhaling 239PuO2 developed pulmonary neoplasms. Seven p53+/+ mice exposed to 239PuO2 also developed pulmonary neoplasms, but the latency period for pulmonary neoplasia was significantly shorter in the p53+/ mice. Four pulmonary neoplasms were observed in p53+/- mice exposed to the higher dose of Be, whereas none were observed in the wild-type mice or in the heterozygous mice exposed to the lower dose of Be. Thus, both p53+/- and p53+/+ mice were susceptible to 239Pu-induced carcinogenesis, whereas the p53+/- but not the p53+/+ mice were susceptible to Be-induced carcinogenesis. However, only 2 pulmonary neoplasms (1 in each of the 239PuO2 exposure groups) were observed in the 59 p53+/ mice that were sacrificed or euthanatized within 9 mo after exposure, indicating that the p53+/- knockout mouse might not be appropriate for a 6-mo model of carcinogenesis for these inhaled carcinogens.

The cruciferous nitrile, crambene, induces rat hepatic and pancreatic glutathione S-transferases.

Indoles and isothiocyanates found in cruciferous vegetables have been implicated as chemopreventive agents against carcinogenesis. The bioactivities of chemically related cruciferous nitriles, including 1-cyano-2-hydroxy-3-butene (crambene), however, have not been thoroughly evaluated. Crambene causes a prolonged elevation of rat hepatic and pancreatic glutathione and induces the GSH S-transferases (GSTs). Because elevated GST activity against the model substrate chlorodinitrobenzene does not reflect individual isoenzyme induction, quantitative HPLC evaluation of specific GST subunits is necessary to fully assess the range of GST isoenzymes induced by crambene. Accordingly, male Fischer 344 rats were given, via esophageal intubation, either 100 (Experiment 1) or 50 mg crambene/kg body wt (Experiment 2) once daily for 7 days. GSTs were extracted from hepatic cytosol by affinity chromatography, and the individual subunits that comprise the various isoenzymes were quantified by reverse-phase HPLC to gain an estimate of induction. In addition, pancreatic GST subunits were assessed in the low-dose experiment. In parallel with increased GST activity, crambene caused a generalized induction of GST subunits in both liver and pancreas, but the pattern of subunit induction was tissue dependent. In the liver, alpha subunits 1 and 2 and the mu subunit 3 were induced approximately 2-fold, while the mu subunit 4 was induced only 1.5-fold. In the pancreas, the alpha subunit 2 was induced to a much larger extent (2.6-fold) than the other subunits (from no induction to 1.6 fold). These results suggests that crambene-mediated GST induction mechanisms vary from tissue to tissue. Potential chemoprevention provided by crambene against GST-metabolized carcinogens or toxins may differ between liver and pancreas because of differences in the degree and pattern of induction.