Using Evidence Integration Techniques in the Development of Health-Based Occupational Exposure Levels.

Development of toxicology-based criteria such as occupational exposure levels (OELs) are rarely straightforward. This process requires a rigorous review of the literature, searching for patterns in toxicity, biological plausibility, coherence, and dose-response relationships. Despite the direct applicability, human data are rarely used primarily because of imprecise exposure estimates, unknown influence of assumptions, and confounding factors. As a result, high reliance is often placed on laboratory animal data. Often, data from a single study is typically used to represent an entire database to extrapolate an OEL, even for data-rich compounds. Here we present a holistic framework for evaluating epidemiological, controlled in vivo, mechanistic/in vitro, and computational evidence that can be useful in deriving OELs. It begins with describing a documented review process of the literature, followed by sorting of data into either controlled laboratory in vivo, in silico/read-across, mechanistic/in vitro, or epidemiological/field data categories. Studies are then evaluated and qualified based on rigor, risk of bias, and applicability for point of departure development. Other data (eg, in vitro, in silico estimates, read-across data and mechanistic information, and data that failed to meet the former criteria) are used alongside qualified epidemiological exposure estimates to help inform points of departure or human-equivalent concentrations that are based on toxic end points. Bayesian benchmark dose methods are used to estimate points of departure and for estimating uncertainty factors (UFs) to develop preliminary OELs. These are then compared with epidemiological data to support the OEL and the use and magnitude of UFs, when appropriate.

Immune modulation by chronic exposure to waterpipe smoke and immediate-early gene regulation in murine lungs.

OBJECTIVE: We investigated the effects of chronic waterpipe (WP) smoke on pulmonary function and immune response in a murine model using a research-grade WP and the effects of acute exposure on the regulation of immediate-early genes (IEGs). METHODS: WP smoke was generated using three WP smoke puffing regimens based on the Beirut regimen. WP smoke samples generated under these puffing regimens were quantified for nicotine concentration. Mice were chronically exposed for 6 months followed by assessment of pulmonary function and airway inflammation. Transcriptomic analysis using RNAseq was conducted after acute exposure to characterise the IEG response. These biomarkers were then compared with those generated after exposure to dry smoke (without water added to the WP bowl). RESULTS: We determined that nicotine composition in WP smoke ranged from 0.4 to 2.5 mg per puffing session. The lung immune response was sensitive to the incremental severity of chronic exposure, with modest decreases in airway inflammatory cells and chemokine levels compared with air-exposed controls. Pulmonary function was unmodified by chronic WP exposure. Acute WP exposure was found to activate the immune response and identified known and novel IEG as potential biomarkers of WP exposure. CONCLUSION: Chronic exposure to WP smoke leads to immune suppression without significant changes to pulmonary function. Transcriptomic analysis of the lung after acute exposure to WP smoke showed activation of the immune response and revealed IEGs that are common to WP and dry smoke, as well as pools of IEGs unique to each exposure, identifying potential biomarkers specific to WP exposure.

Gestational Exposure to Sidestream (Secondhand) Cigarette Smoke Promotes Transgenerational Epigenetic Transmission of Exacerbated Allergic Asthma and Bronchopulmonary Dysplasia.

Embryonic development is highly sensitive to xenobiotic toxicity and in utero exposure to environmental toxins affects physiological responses of the progeny. In the United States, the prevalence of allergic asthma (AA) is inexplicably rising and in utero exposure to cigarette smoke increases the risk of AA and bronchopulmonary dysplasia (BPD) in children and animal models. We reported that gestational exposure to sidestream cigarette smoke (SS), or secondhand smoke, promoted nicotinic acetylcholine receptor-dependent exacerbation of AA and BPD in mice. Recently, perinatal nicotine injections in rats were reported to induce peroxisome proliferator-activated receptor γ-dependent transgenerational transmission of asthma. Herein, we show that first generation and second generation progeny from gestationally SS-exposed mice exhibit exacerbated AA and BPD that is not dependent on the decrease in peroxisome proliferator-activated receptor γ levels. Lungs from these mice show strong eosinophilic infiltration, excessive Th2 polarization, marked airway hyperresponsiveness, alveolar simplification, decreased lung compliance, and decreased lung angiogenesis. At the molecular level, these changes are associated with increased RUNX3 expression, alveolar cell apoptosis, and the antiangiogenic factor GAX, and decreased expression of HIF-1α and proangiogenic factors NF-κB and VEGFR2 in the 7-d first generation and second generation lungs. Moreover, the lungs from these mice exhibit lower levels of microRNA (miR)-130a and increased levels of miR-16 and miR-221. These miRs regulate HIF-1α-regulated apoptotic, angiogenic, and immune pathways. Thus the intergenerational effects of gestational SS involve epigenetic regulation of HIF-1α through specific miRs contributing to increased incidence of AA and BPD in the progenies.

Correction to: electronic cigarette use behaviors and motivations among smokers and non-smokers.

After publication of the article [1], it has been brought to our attention that there is a funding acknowledgement missing. The authors would also like to include "Dr. Michael Joseph Blaha is funded by the American Heart Association Tobacco Regulatory Center, funding number: 1P50HL120163".

Airborne Particulate Matter Induces Nonallergic Eosinophilic Sinonasal Inflammation in Mice.

Exposure to airborne particulate matter (PM) has been linked to aggravation of respiratory symptoms, increased risk of cardiovascular disease, and all-cause mortality. Although the health effects of PM on the lower pulmonary airway have been extensively studied, little is known regarding the impact of chronic PM exposure on the upper sinonasal airway. We sought to test the impact of chronic airborne PM exposure on the upper respiratory system in vivo. Mice were subjected, by inhalation, to concentrated fine (2.5 µm) PM 6 h/d, 5 d/wk, for 16 weeks. Mean airborne fine PM concentration was 60.92 µm/m3, a concentration of fine PM lower than that reported in some major global cities. Mice were then killed and analyzed for evidence of inflammation and barrier breakdown compared with control mice. Evidence of the destructive effects of chronic airborne PM on sinonasal health in vivo, including proinflammatory cytokine release, and macrophage and neutrophil inflammatory cell accumulation was observed. A significant increase in epithelial barrier dysfunction was observed, as assessed by serum albumin accumulation in nasal airway lavage fluid, as well as decreased expression of adhesion molecules, including claudin-1 and epithelial cadherin. A significant increase in eosinophilic inflammation, including increased IL-13, eotaxin-1, and eosinophil accumulation, was also observed. Collectively, although largely observational, these studies demonstrate the destructive effects of chronic airborne PM exposure on the sinonasal airway barrier disruption and nonallergic eosinophilic inflammation in mice.

Impaired calcium signaling in muscle fibers from intercostal and foot skeletal muscle in a cigarette smoke-induced mouse model of COPD.

INTRODUCTION: Respiratory and locomotor skeletal muscle dysfunction are common findings in chronic obstructive pulmonary disease (COPD); however, the mechanisms that cause muscle impairment in COPD are unclear. Because Ca2+ signaling in excitation-contraction (E-C) coupling is important for muscle activity, we hypothesized that Ca2+ dysregulation could contribute to muscle dysfunction in COPD. METHODS: Intercostal and flexor digitorum brevis muscles from control and cigarette smoke-exposed mice were investigated. We used single cell Ca2+ imaging and Western blot assays to assess Ca2+ signals and E-C coupling proteins. RESULTS: We found impaired Ca2+ signals in muscle fibers from both muscle types, without significant changes in releasable Ca2+ or in the expression levels of E-C coupling proteins. CONCLUSIONS: Ca2+ dysregulation may contribute or accompany respiratory and locomotor muscle dysfunction in COPD. These findings are of significance to the understanding of the pathophysiological course of COPD in respiratory and locomotor muscles. Muscle Nerve 56: 282-291, 2017.

Electronic cigarette use behaviors and motivations among smokers and non-smokers.

BACKGROUND: The use of electronic cigarettes (EC) has risen exponentially over the past decade, including among never smokers, and ECs are now the most popular tobacco product among teenagers in the US. While, EC manufacturers utilize numerous marketing strategies to target both smokers and non-smokers, it is unclear how perceptions and behaviors differ between these two groups. METHODS: We conducted a survey of 320 adults either via online surveys or in Baltimore vape shops to determine demographics, behaviors, perceptions, and motivations underlying use of ECs. RESULTS: Our survey respondents were predominantly young, Caucasian males, 74% of whom identified themselves as former smokers, while 20% identified as current smokers and 6% were never smokers. Former smokers reported a longer history of EC use and higher nicotine concentrations than current smokers. For former and current smokers, the primary motivation for EC use was assistance to quit smoking, and nearly half indicated that they plan to reduce their nicotine concentration and eventually quit using ECs. Among former smokers, self-reports on use and measures of dependence were consistent with nicotine replacement as their primary motivation. The majority of former and current smokers also reported that their respiratory health had improved as a result of EC use, although this effect was stronger for former smokers. Never smokers reported less frequent EC use and dependence compared to former and current smokers. Their motivations for use were more commonly for enjoyment and popularity, and they displayed a reduced desire to eventually quit using ECs. CONCLUSIONS: These responses provide insight into the underlying thoughts and behaviors of smoking and non-smoking EC users and also suggest that never smoking EC users are an emerging demographic with different motivations and perceptions than those of current and former smokers.

Nrf2 reduces allergic asthma in mice through enhanced airway epithelial cytoprotective function.

Asthma development and pathogenesis are influenced by the interactions of airway epithelial cells and innate and adaptive immune cells in response to allergens. Oxidative stress is an important mediator of asthmatic phenotypes in these cell types. Nuclear erythroid 2-related factor 2 (Nrf2) is a redox-sensitive transcription factor that is the key regulator of the response to oxidative and environmental stress. We previously demonstrated that Nrf2-deficient mice have heightened susceptibility to asthma, including elevated oxidative stress, inflammation, mucus, and airway hyperresponsiveness (AHR) (Rangasamy T, Guo J, Mitzner WA, Roman J, Singh A, Fryer AD, Yamamoto M, Kensler TW, Tuder RM, Georas SN, Biswal S. J Exp Med 202: 47-59, 2005). Here we dissected the role of Nrf2 in lung epithelial cells and tested whether genetic or pharmacological activation of Nrf2 reduces allergic asthma in mice. Cell-specific activation of Nrf2 in club cells of the airway epithelium significantly reduced allergen-induced AHR, inflammation, mucus, Th2 cytokine secretion, oxidative stress, and airway leakiness and increased airway levels of tight junction proteins zonula occludens-1 and E-cadherin. In isolated airway epithelial cells, Nrf2 enhanced epithelial barrier function and increased localization of zonula occludens-1 to the cell surface. Pharmacological activation of Nrf2 by 2-trifluoromethyl-2'-methoxychalone during the allergen challenge was sufficient to reduce allergic inflammation and AHR. New therapeutic options are needed for asthma, and this study demonstrates that activation of Nrf2 in lung epithelial cells is a novel potential therapeutic target to reduce asthma susceptibility.

Source of biomass cooking fuel determines pulmonary response to household air pollution.

Approximately 3 billion people-half the worldwide population-are exposed to extremely high concentrations of household air pollution due to the burning of biomass fuels on inefficient cookstoves, accounting for 4 million annual deaths globally. Yet, our understanding of the pulmonary responses to household air pollution exposure and the underlying molecular and cellular events is limited. The two most prevalent biomass fuels in India are wood and cow dung, and typical 24-hour mean particulate matter (PM) concentrations in homes that use these fuels are 300 to 5,000 µg/m(3). We dissected the mechanisms of pulmonary responses in mice after acute or subchronic exposure to wood or cow dung PM collected from rural Indian homes during biomass cooking. Acute exposures resulted in robust proinflammatory cytokine production, neutrophilic inflammation, airway resistance, and hyperresponsiveness, all of which were significantly higher in mice exposed to PM from cow dung. On the contrary, subchronic exposures induced eosinophilic inflammation, PM-specific antibody responses, and alveolar destruction that was highest in wood PM-exposed mice. To understand the molecular pathways that trigger biomass PM-induced inflammation, we exposed Toll-like receptor (TLR)2-, TLR3-, TLR4-, TLR5-, and IL-1R-deficient mice to PM and found that IL-1R, TLR4, and TLR2 are the predominant receptors that elicit inflammatory responses via MyD88 in mice exposed to wood or cow dung PM. In conclusion, this study demonstrates that subchronic exposure to PM collected from households burning biomass fuel elicits a persistent pulmonary inflammation largely through activation of TLR and IL-1R pathways, which could increase the risk for chronic respiratory diseases.

Trisomy represses Apc(Min)-mediated tumours in mouse models of Down's syndrome.

Epidemiological studies spanning more than 50 yr reach conflicting conclusions as to whether there is a lower incidence of solid tumours in people with trisomy 21 (Down's syndrome). We used mouse models of Down's syndrome and of cancer in a biological approach to investigate the relationship between trisomy and the incidence of intestinal tumours. Apc(Min)-mediated tumour number was determined in aneuploid mouse models Ts65Dn, Ts1Rhr and Ms1Rhr. Trisomy for orthologues of about half of the genes on chromosome 21 (Hsa21) in Ts65Dn mice or just 33 of these genes in Ts1Rhr mice resulted in a significant reduction in the number of intestinal tumours. In Ms1Rhr, segmental monosomy for the same 33 genes that are triplicated in Ts1Rhr resulted in an increased number of tumours. Further studies demonstrated that the Ets2 gene contributed most of the dosage-sensitive effect on intestinal tumour number. The action of Ets2 as a repressor when it is overexpressed differs from tumour suppression, which requires normal gene function to prevent cellular transformation. Upregulation of Ets2 and, potentially, other genes involved in this kind of protective effect may provide a prophylactic effect in all individuals, regardless of ploidy.

Chronic intermittent hypoxia induces atherosclerosis via activation of adipose angiopoietin-like 4.

RATIONALE: Obstructive sleep apnea is a risk factor for dyslipidemia and atherosclerosis, which have been attributed to chronic intermittent hypoxia (CIH). Intermittent hypoxia inhibits a key enzyme of lipoprotein clearance, lipoprotein lipase, and up-regulates a lipoprotein lipase inhibitor, angiopoietin-like 4 (Angptl4), in adipose tissue. The effects and mechanisms of Angptl4 up-regulation in sleep apnea are unknown. OBJECTIVES: To examine whether CIH induces dyslipidemia and atherosclerosis by increasing adipose Angptl4 via hypoxia-inducible factor-1 (HIF-1). METHODS: ApoE(-/-) mice were exposed to intermittent hypoxia or air for 4 weeks while being treated with Angptl4-neutralizing antibody or vehicle. MEASUREMENTS AND MAIN RESULTS: In vehicle-treated mice, hypoxia increased adipose Angptl4 levels, inhibited adipose lipoprotein lipase, increased fasting levels of plasma triglycerides and very low density lipoprotein cholesterol, and increased the size of atherosclerotic plaques. The effects of CIH were abolished by the antibody. Hypoxia-induced increases in plasma fasting triglycerides and adipose Angptl4 were not observed in mice with germline heterozygosity for a HIF-1α knockout allele. Transgenic overexpression of HIF-1α in adipose tissue led to dyslipidemia and increased levels of adipose Angptl4. In cultured adipocytes, constitutive expression of HIF-1α increased Angptl4 levels, which was abolished by siRNA. Finally, in obese patients undergoing bariatric surgery, the severity of nocturnal hypoxemia predicted Angptl4 levels in subcutaneous adipose tissue. CONCLUSIONS: HIF-1-mediated increase in adipose Angptl4 and the ensuing lipoprotein lipase inactivation may contribute to atherosclerosis in patients with sleep apnea.

Oxidized phospholipids impair pulmonary antibacterial defenses: evidence in mice exposed to cigarette smoke.

Patients with COPD are associated with poor pulmonary anti-bacterial innate defenses, which increase the risk for frequent acute exacerbations caused by bacterial infection. Despite elevated numbers of phagocytes (macrophages and neutrophils), airways of patients with COPD show stable bacterial colonization. A defect in the phagocytic ability of alveolar macrophages (AMs) is one of the primary reasons for failure to clear the invading bacteria in airways of smokers and COPD patients and also in mice exposed to cigarette smoke (CS). Oxidative stress, as a result of CS exposure is implicated; however, the factors or mediators that inhibit phagocytic activity of AMs in lungs of smokers remain unclear. In the current study, we provide evidence that accumulation of oxidized phospholipids (Ox-PLs) mediate inhibition of phagocytic function of AMs in CS-exposed mice. Mice exposed to 6months of CS showed impaired bacterial phagocytosis and clearance by AMs and elevated levels of Ox-PLs in bronchoalveolar lavage fluid (BALF), compared to mice exposed to room air. Intratracheal instillation of oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (OX-PAPC) inhibited phagocytic activity of AMs and impaired pulmonary bacterial clearance in mice. In vitro studies demonstrated that exposure of J774 macrophages to OX-PAPC inhibited bacterial phagocytosis and clearance. However, pre-treatment of OX-PAPC with the monoclonal antibody EO6, which specifically binds to oxidized phospholipid but not native phospholipid, abolished OX-PAPC induced inhibition of bacterial phagocytosis and clearance. Incubation of BALF retrieved from CS-exposed mice impaired bacterial phagocytosis by J774 macrophages, which was abolished by pre-treatment of BALF with the EO6 antibody. In conclusion, our study shows that Ox-PLs generated following chronic CS exposure could play a crucial role in inhibiting phagocytic function of AMs and thus impair pulmonary anti-bacterial innate defenses in CS-exposed mice. Therapeutic approaches that augment pulmonary antioxidant defenses could be beneficial in reducing oxidative stress-driven impairment of phagocytosis by AMs in smokers and COPD patients.

Telomere length is a determinant of emphysema susceptibility.

RATIONALE: Germline mutations in the enzyme telomerase cause telomere shortening, and have their most common clinical manifestation in age-related lung disease that manifests as idiopathic pulmonary fibrosis. Short telomeres are also a unique heritable trait that is acquired with age. OBJECTIVES: We sought to understand the mechanisms by which telomerase deficiency contributes to lung disease. METHODS: We studied telomerase null mice with short telomeres. MEASUREMENTS AND MAIN RESULTS: Although they have no baseline histologic defects, when mice with short telomeres are exposed to chronic cigarette smoke, in contrast with controls, they develop emphysematous air space enlargement. The emphysema susceptibility did not depend on circulating cell genotype, because mice with short telomeres developed emphysema even when transplanted with wild-type bone marrow. In lung epithelium, cigarette smoke exposure caused additive DNA damage to telomere dysfunction, which limited their proliferative recovery, and coincided with a failure to down-regulate p21, a mediator of cellular senescence, and we show here, a determinant of alveolar epithelial cell cycle progression. We also report early onset of emphysema, in addition to pulmonary fibrosis, in a family with a germline deletion in the Box H domain of the RNA component of telomerase. CONCLUSIONS: Our data indicate that short telomeres lower the threshold of cigarette smoke-induced damage, and implicate telomere length as a genetic susceptibility factor in emphysema, potentially contributing to its age-related onset in humans.

Targeting Nrf2 with the triterpenoid CDDO-imidazolide attenuates cigarette smoke-induced emphysema and cardiac dysfunction in mice.

Chronic obstructive pulmonary disease (COPD), which comprises emphysema and chronic bronchitis resulting from prolonged exposure to cigarette smoke (CS), is a major public health burden with no effective treatment. Emphysema is also associated with pulmonary hypertension, which can progress to right ventricular failure, an important cause of morbidity and mortality among patients with COPD. Nuclear erythroid 2 p45 related factor-2 (Nrf2) is a redox-sensitive transcription factor that up-regulates a battery of antioxidative genes and cytoprotective enzymes that constitute the defense against oxidative stress. Recently, it has been shown that patients with advanced COPD have a decline in expression of the Nrf2 pathway in lungs, suggesting that loss of this antioxidative protective response is a key factor in the pathophysiological progression of emphysema. Furthermore, genetic disruption of Nrf2 in mice causes early-onset and severe emphysema. The present study evaluated whether the strategy of activation of Nrf2 and its downstream network of cytoprotective genes with a small molecule would attenuate CS-induced oxidative stress and emphysema. Nrf2(+/+) and Nrf2(-/-) mice were fed a diet containing the potent Nrf2 activator, 1-[2-cyano-3-,12-dioxooleana-1,9(11)-dien-28-oyl]imidazole (CDDO-Im), while being exposed to CS for 6 months. CDDO-Im significantly reduced lung oxidative stress, alveolar cell apoptosis, alveolar destruction, and pulmonary hypertension in Nrf2(+/+) mice caused by chronic exposure to CS. This protection from CS-induced emphysema depended on Nrf2, as Nrf2(-/-) mice failed to show significant reduction in alveolar cell apoptosis and alveolar destruction after treatment with CDDO-Im. These results suggest that targeting the Nrf2 pathway during the etiopathogenesis of emphysema may represent an important approach for prophylaxis against COPD.

PGI synthase overexpression protects against bleomycin-induced mortality and is associated with increased Nqo 1 expression.

The mortality rate for acute lung injury (ALI) is reported to be between 35-40%, and there are very few treatment strategies that improve the death rate from this condition. Previous studies have suggested that signaling through the prostaglandin (PG) I(2) receptor may protect against bleomycin-induced ALI in mice. We found that mice that overexpress PGI synthase (PGIS) in the airway epithelium were significantly protected against bleomycin-induced mortality and had reduced parenchymal consolidation, apoptosis of lung tissue, and generation of F(2)-isoprostanes compared with littermate wild-type controls. In addition, we show for the first time in both in vivo and in vitro experiments that PGI(2) induced the expression of NADP (H): quinoneoxidoreductase 1 (Nqo 1), an enzyme that prevents the generation of reactive oxygen species. PGI(2) induction of Nqo 1 provides a possible novel mechanism by which this prostanoid protects against bleomycin-induced mortality and identifies a potential therapeutic target for human ALI.

Heightened endoplasmic reticulum stress in the lungs of patients with chronic obstructive pulmonary disease: the role of Nrf2-regulated proteasomal activity.

RATIONALE: Nuclear factor erythroid 2-related factor 2 (Nrf2), an important regulator of lung antioxidant defenses, declines in chronic obstructive pulmonary disease (COPD). However, Nrf2 also regulates the proteasome system that degrades damaged and misfolded proteins. Because accumulation of misfolded proteins in the endoplasmic reticulum (ER) causes ER stress and ER stress-induced apoptosis, Nrf2 may potentially prevent ER stress-mediated apoptosis in COPD. OBJECTIVES: To determine whether Nrf2-regulated proteasome function affects ER stress-mediated apoptosis in COPD. METHODS: We assessed the expression of Nrf2, Nrf2-dependent proteasomal subunits, proteasomal activity, markers of ER stress, and apoptosis in emphysematous lungs of mice exposed to cigarette smoke (CS) as well as peripheral lung tissues from normal control subjects and patients with COPD. MEASUREMENTS AND MAIN RESULTS: Compared with wild-type mice, emphysematous lungs of CS-exposed Nrf2-deficient mice exhibited markedly lower proteasomal activity and elevated markers of ER stress and apoptosis. Furthermore, compared with normal control subjects, lungs of patients with mild and advanced COPD showed a marked decrease in the expression of Nrf2-regulated proteasomal subunits and total proteasomal activity. However, they were associated with greater levels of ER stress and apoptosis markers. In vitro studies have demonstrated that enhancing proteasomal activity in Beas2B cells either by sulforaphane, an activator of Nrf2, or overexpression of Nrf2-regulated proteasomal subunit PSMB6, significantly inhibited cigarette smoke condensate (CSC)-induced ER stress and cell death. CONCLUSIONS: Impaired Nrf2 signaling causes significant decline in proteasomal activity and heightens ER stress response in lungs of patients with COPD and CS-exposed mice. Accordingly, pharmacological approaches that augment Nrf2 activity may protect against COPD progression by both up-regulating antioxidant defenses and relieving ER stress.

Sildenafil inhibits superoxide formation and prevents endothelial dysfunction in a mouse model of secondhand smoke induced erectile dysfunction.

PURPOSE: We determined the effect of passive secondhand cigarette smoke on 1) erectile function in vivo, 2) molecular mechanisms involved in penile vascular function, and 3) erectile function and penile molecular signaling in the presence of phosphodiesterase type 5 inhibitor therapy. MATERIALS AND METHODS: Four groups of mice were used, including group 1--controls, group 2--mice exposed to 3 weeks of secondhand smoke (5 hours per day for 5 days per week), group 3--control plus sildenafil (100 mg/kg per day) and group 4--smoke exposed plus sildenafil (100 mg/kg per day). Cavernous nerve electrical stimulation and intracavernous injection of acetylcholine were done to assess erectile function. Constitutive and inducible nitric oxide synthase activity, reactive oxygen species generation, nitrotyrosine formation and superoxide anion levels were assessed. RESULTS: Decreased erectile responses to cavernous nerve electrical stimulation and impaired endothelium dependent erectile responses to ACh in mice exposed to secondhand smoke were observed. Superoxide anion was increased in endothelial and corporeal smooth muscle cells of smoking mouse penises. In mice exposed to secondhand smoke constitutive nitric oxide synthase activity was decreased, and inducible nitric oxide synthase activity, reactive oxygen species generation and nitrotyrosine formation increased. Sildenafil therapy restored constitutive nitric oxide synthase activity in the penis of smoking mice, decreased inducible nitric oxide synthase activity, reactive oxygen species generation and nitrotyrosine formation, and improved erectile responses to cavernous nerve electrical stimulation and acetylcholine. CONCLUSIONS: Short-term exposure to secondhand smoke impairs erectile function through excessive penile reactive oxygen species signaling and inducible nitric oxide synthase activity. Decreased penile constitutive nitric oxide synthase activity may be attributable to the decreased endothelial nitric oxide synthase activity resulting from increased oxidative stress. Sildenafil therapy restored nitric oxide synthase activity and decreased reactive oxygen species signaling, resulting in improved erectile function.

Complex contributions of Ets2 to craniofacial and thymus phenotypes of trisomic "Down syndrome" mice.

Ts65Dn mice have segmental trisomy for orthologs of about half of the genes on human chromosome 21, including Ets2. These mice develop anomalies of the cranial skeleton and thymus that parallel those in Down syndrome. Overexpression of the Ets2 transcription factor gene was posited to be sufficient to produce these craniofacial and thymus deficits in transgenic mice that constitutively overexpress a processed Ets2 transcript under a promiscuous promoter [Sumarsono et al. (1996); Nature 379:534-537; Wolvetang et al. (2003); Hum Mol Genet 12:247-255]. Evaluation of trisomic mice with varying copy numbers of a properly regulated Ets2 gene indicated increased dosage of Ets2 was not sufficient to produce effects on thymus and most of the cranial anomalies seen in Ts65Dn mice. However, mesoderm-derived cranial skeletal elements are significantly more affected in Ts65Dn, Ets2(+/-) mice compared to Ts65Dn littermates suggesting a differential interaction of Ets2-related processes with mesoderm-derived and neural crest-derived formative tissues. Our results support the growing evidence for interactions among multiple genes contributing to developmental perturbations resulting in variation in complex Down syndrome phenotypes.

Deletion of Nrf2 enhances susceptibility to eosinophilic sinonasal inflammation in a murine model of rhinosinusitis.

BACKGROUND: Oxidative stress exacerbates lower airway diseases including asthma and chronic obstructive pulmonary disease (COPD); however, its role in upper airway (sinonasal) chronic inflammatory disorders is less clear. Nuclear erythroid 2 p45-related factor (Nrf2) is an endogenous mechanism that upon activation invokes an antioxidant response pathway via nuclear translocation and upregulation of cytoprotective genes. We sought to determine whether deletion of Nrf2 enhances susceptibility to allergic sinonasal inflammation in vivo. METHODS: Nrf2-/- mice were subjected to the ovalbumin (Ova)-induced murine model of rhinosinusitis and indices of sinonasal inflammation and epithelial barrier dysfunction were assessed. RESULTS: We show that deletion of Nrf2 results in enhances indices of allergen-induced sinonasal inflammation including aggravated eosinophil accumulation and goblet cell hyperplasia. An exaggerated increase in epithelial derived inflammatory cytokines including interleukin 33 (IL-33) and thymic stromal lymphopoietin (TSLP) was observed in the nasal lavage fluid and sinonasal mucosal tissue of Nrf2-/- mice. Furthermore, Nrf2-/- mice showed heightened Ova-induced barrier dysfunction as measured by serum albumin accumulation in nasal lavage fluid of mice. CONCLUSION: These data show that the endogenous Nrf2 pathway limits Ova-induced sinonasal inflammation, epithelial derived inflammatory cytokine production, and epithelial barrier dysfunction in vivo and identify a potential therapeutic target in the management of allergic sinonasal inflammatory disorders. This is the first study to our knowledge which shows that Nrf2 regulates allergic inflammation in the sinonasal cavity in vivo.