Effect of TRPV4 Antagonist GSK2798745 on Chlorine Gas-Induced Acute Lung Injury in a Swine Model.

As a regulator of alveolo-capillary barrier integrity, Transient Receptor Potential Vanilloid 4 (TRPV4) antagonism represents a promising strategy for reducing pulmonary edema secondary to chemical inhalation. In an experimental model of acute lung injury induced by exposure of anesthetized swine to chlorine gas by mechanical ventilation, the dose-dependent effects of TRPV4 inhibitor GSK2798745 were evaluated. Pulmonary function and oxygenation were measured hourly; airway responsiveness, wet-to-dry lung weight ratios, airway inflammation, and histopathology were assessed 24 h post-exposure. Exposure to 240 parts per million (ppm) chlorine gas for ≥50 min resulted in acute lung injury characterized by sustained changes in the ratio of partial pressure of oxygen in arterial blood to the fraction of inspiratory oxygen concentration (PaO2/FiO2), oxygenation index, peak inspiratory pressure, dynamic lung compliance, and respiratory system resistance over 24 h. Chlorine exposure also heightened airway response to methacholine and increased wet-to-dry lung weight ratios at 24 h. Following 55-min chlorine gas exposure, GSK2798745 marginally improved PaO2/FiO2, but did not impact lung function, airway responsiveness, wet-to-dry lung weight ratios, airway inflammation, or histopathology. In summary, in this swine model of chlorine gas-induced acute lung injury, GSK2798745 did not demonstrate a clinically relevant improvement of key disease endpoints.

Prenatal airborne polycyclic aromatic hydrocarbon exposure, altered regulation of peroxisome proliferator-activated receptor gamma (Ppar)γ, and links with mammary cancer.

Environmental exposure to polycyclic aromatic hydrocarbons (PAH) has been shown to be associated with chronic disease outcomes through multiple mechanisms including altered regulation of the transcription factor peroxisome proliferator-activated receptor gamma (Ppar) γ. Because PAH exposure and Pparγ each have been associated with mammary cancer, we asked whether PAH would induce altered regulation of Pparγ in mammary tissue, and whether this association may underlie the association between PAH and mammary cancer. Pregnant mice were exposed to aerosolized PAH at proportions that mimic equivalent human exposures in New York City air. We hypothesized that prenatal PAH exposure would alter Pparγ DNA methylation and gene expression and induce the epithelial to mesenchymal transition (EMT) in mammary tissue of offspring (F1) and grandoffspring (F2) mice. We also hypothesized that altered regulation of Pparγ in mammary tissue would associate with biomarkers of EMT, and examined associations with whole body weight. We found that prenatal PAH exposure lowered Pparγ mammary tissue methylation among grandoffspring mice at postnatal day (PND) 28. However, PAH exposure did not associate with altered Pparγ gene expression or consistently with biomarkers of EMT. Finally, lower Pparγ methylation, but not gene expression, was associated with higher body weight among offspring and grandoffspring mice at PND28 and PND60. Findings suggest additional evidence of multi-generational adverse epigenetic effects of prenatal PAH exposure among grandoffspring mice.

Characterization of aerosols from hemp-derived pre-roll joints.

OBJECTIVE: Availability and consumer use of hemp products is rapidly increasing, but little work has been done to assess aerosol emissions of hemp pre-rolls. The objective of this research was to characterize the aerosol of pre-rolled joints from hemp material enriched for production of cannabigerol (CBG) that were smoked on a test system mimicking human use patterns. MATERIALS AND METHODS: Aerosol emissions were collected and analyzed using glass microfiber filters and charcoal cartridges. The aerosol was screened for nine phytocannabinoids and 19 terpenes. RESULTS: Three phytocannabinoids (CBG, cannabichromene (CBC), and delta-9-tetrahydrocannabinol (THC)) were detected and quantified at a mean (SD) concentration of 19.4 (4.7), 0.48 (0.01), and 0.40 (0.04) mg per pre-roll, respectively. Five terpenes ((-)-α-bisabolol, (-)-guaiol, ß-caryophyllene, nerolidol, and α-humulene) were detected and quantified at an average concentration of 352.7 (112.0), 194.3 (66.4), 106.0 (50.4), 28.3 (9.3), and 27.7 (11.2) µg per pre-roll, respectively. Particle size distribution testing via aerodynamic particle sizer and inertial impactor showed that average size of emitted aerosols was 0.77 (0.0) and 0.54 (0.1) µm, respectively. CONCLUSIONS: This study describes methodology for characterization of cannabinoid and terpene dose in emitted aerosols and aerosolization efficiency from hemp pre-rolls. It also presents these data for one of the marketed products.

Composition of aerosols from thermal degradation of flavors used in ENDS and tobacco products.

Aim: The cardiovascular toxicity of unheated and heated flavorants and their products as commonly present in electronic cigarette liquids (e-liquids) was evaluated previously in vitro. Based on the results of in vitro assays, cinnamaldehyde, eugenol, menthol, and vanillin were selected to conduct a detailed chemical analysis of the aerosol generated following heating of each compound both at 250 and 750 °C. Materials and Methods: Each flavoring was heated in a drop-tube furnace within a quartz tube. The combustion atmosphere was captured using different methods to enable analysis of 308 formed compounds. Volatile organic compounds (VOCs) were captured with an evacuated Summa canister and assayed via gas chromatography interfaced with mass spectrometry (GC-MS). Carbonyls (aldehydes and ketones) were captured using a 2,4-dinitrophenylhydrazine (DNPH) cartridge and assayed via a high-performance liquid chromatography-ultra-violet (HPLC-UV) assay. Polyaromatic hydrocarbons (PAHs) were captured using an XAD cartridge and filter, and extracts were assayed using GC-MS/MS. Polar compounds were assayed after derivatization of the XAD/filter extracts and analyzed via GC-MS. Conclusion: At higher temperature, both cinnamaldehyde and menthol combustion significantly increased formaldehyde and acetaldehyde levels. At higher temperature, cinnamaldehyde, eugenol, and menthol resulted in increased benzene concentrations. At low temperature, all four compounds led to higher levels of benzoic acid. These data show that products of thermal degradation of common flavorant compounds vary by flavorant and by temperature and include a wide variety of harmful and potentially harmful constituents (HPHCs).

Prenatal polycyclic aromatic hydrocarbons, altered ERα pathway-related methylation and expression, and mammary epithelial cell proliferation in offspring and grandoffspring adult mice.

BACKGROUND: Airborne polycyclic aromatic hydrocarbons (PAH) possess carcinogenic and endocrine disrupting properties linked to mammary tumorigenesis. These effects may be initiated during a prenatal period of susceptibility to PAH activation of the aryl hydrocarbon receptor (Ahr) and through downstream effects on estrogen receptor (Er) α. PURPOSE: We hypothesized prenatal airborne PAH exposure induces sustained effects in female adult wild type BALB/cByj mice detected in the offspring (F1) and grandoffspring (F2) generation. We hypothesized these effects would include altered expression and epigenetic regulation of Erα and altered expression of aryl hydrocarbon receptor repressor (Ahrr, Ahrr/aryl hydrocarbon receptor nuclear translocator (Arnt), and breast cancer type 1 susceptibility (Brca1). Further, we hypothesized that PAH would induce precancerous outcomes such as epithelial cell proliferation and epithelial cell hyperplasia in mammary glands of adult female offspring and grandoffspring. RESULTS: Prenatal ambient PAH exposure lowered Erα mRNA expression (F1 and F2: p<0.001 for each) and induced methylation in the Erα promoter in mammary tissue in offspring and grandoffspring mice on postnatal day (PND) 60. Prenatal PAH lowered Brca1 mRNA (F1: p=0.002, F2: p=0.02); Erα mRNA was correlated with Brca1 (F1: r=0.42, p=0.02; F2: r=0.53, p=0.005). Prenatal PAH lowered Ahrr (F1: p=0.03, F2: p=0.009) and raised Arnt mRNA expression (F1: p=0.01, F2: p=0.03). Alterations in Erα mRNA (F2: p<0.0001) and Ahrr (F2: p=0.02) in the grandoffspring mice also occured by PND 28, and similarly occurred in the dam on postpartum day (PPD) 28. Finally, prenatal PAH was associated with higher mammary epithelial cell proliferation in the offspring (p=0.02), but not grandoffspring mice, without differences in the frequency of mammary cell hyperplasia. These results did not differ after adjustment by each candidate gene expression level. CONCLUSIONS: Prenatal PAH exposure induces DNA methylation and alters gene expression in the Erα-mediated pathway across generations, and suggests that functional outcomes such as mammary cell proliferation also may occur in offspring as a result.

Disposition and metabolism of ethylene glycol 2-ethylhexyl ether in Sprague Dawley rats, B6C3F1/N mice, and in vitro in rat hepatocytes.

Ethylene glycol 2-ethylhexyl ether (EGEHE) is a solvent used in a variety of applications.We report disposition and metabolism of EGEHE following a single gavage or dermal administration of 50, 150 or 500 mg/kg [14C]EGEHE in rats and mice and in vitro in rat hepatocytes.EGEHE was cleared rapidly in rat hepatocytes (half-life ∼4 min) with no sex difference.EGEHE was well- and moderately absorbed following oral administration (rats: 80-96%, mice: 91-95%) and dermal application (rats: 25-37%, mice: 22-24%), respectively, and rapidly excreted in urine.[14C]EGEHE-derived radioactivity was distributed to tissues (oral: 2.3-7.2%, dermal: 0.7-2.2%) with liver and kidney containing the highest levels in both species.EGEHE was extensively metabolised with little to no parent detected in urine. The alkoxyacetic acid metabolite, which has previously been shown to mediate toxicities of other shorter-chain ethylene glycol ethers, was not detected.There were no apparent dose, species or sex differences in disposition and metabolism of EGEHE, except that the exhaled volatile compounds were greater in mice (19-20%) compared with rats (<2%).These studies address a critical gap in the scientific literature and provide data that will inform future studies designed to evaluate toxicity of EGEHE.

Effects of inhaled air pollution on markers of integrity, inflammation, and microbiota profiles of the intestines in Apolipoprotein E knockout mice.

Air pollution exposure is known to contribute to the progression of cardiovascular disease (CVD) and there is increasing evidence that dysbiosis of the gut microbiome may also play a role in the pathogenesis of CVD, including atherosclerosis. To date, the effects of inhaled air pollution mixtures on the intestinal epithelial barrier (IEB), and microbiota profiles are not well characterized, especially in susceptible individuals with comorbidity. Thus, we investigated the effects of inhaled ubiquitous air-pollutants, wood-smoke (WS) and mixed diesel and gasoline vehicle exhaust (MVE) on alterations in the expression of markers of integrity, inflammation, and microbiota profiles in the intestine of atherosclerotic Apolipoprotein E knockout (ApoE-/-) mice. To do this, male 8 wk-old ApoE-/- mice, on a high-fat diet, were exposed to either MVE (300 µg/m3 PM), WS; (∼450 µg/m3 PM), or filtered air (FA) for 6 h/d, 7 d/wk, for 50 d. Immunofluorescence and RT-PCR were used to quantify the expression of IEB components and inflammatory factors, including mucin (Muc)-2, tight junction (TJ) proteins, matrix metalloproteinase (MMP)-9, tumor necrosis factor (TNF)-α, and interleukin (IL)-1ß, as well as Toll-like receptor (TLR)-4. Microbial profiling of the intestine was done using Illumina 16S sequencing of V4 16S rRNA PCR amplicons. We observed a decrease in intestinal Muc2 and TJ proteins in both MVE and WS exposures, compared to FA controls, associated with a significant increase in MMP-9, TLR-4, and inflammatory marker expression. Both WS and MVE-exposure resulted in decreased intestinal bacterial diversity, as well as alterations in microbiota profiles, including the Firmicutes: Bacteroidetes ratio at the phylum level. Our findings suggest inhalation exposure to either MVE or WS result in alterations in components involved in mucosal integrity, and also microbiota profiles and diversity, which are associated with increased markers of an inflammatory response.

Metabolism and disposition of 2-hydroxy-4-methoxybenzophenone, a sunscreen ingredient, in Harlan Sprague Dawley rats and B6C3F1/N mice; a species and route comparison.

2-Hydroxy-4-methoxybenzophenone (HMB) is a common ingredient in personal care products and used as an UV stabilizer. In these studies, disposition and metabolism of [14C]HMB in rats and mice was assessed following single gavage administration (10, 100, or 500 mg/kg), single IV administration (10 mg/kg), or dermal application (0.1, 1, 10, or 15 mg/kg).Following gavage administration, [14C]HMB was well absorbed and excreted mainly in urine (39-57%) and feces (24-42%) with no apparent difference between doses, species or sexes. Distribution of HMB in tissues was minimal in rats (0.36%) and mice (<0.55%).Distribution of HMB following dermal application was comparable to that following gavage administration; no differences between doses, sexes, or species were observed but absorption varied between dose vehicles. Light paraffin oil had the highest absorption and excretion (98% of the HMB dose absorbed).In rats, HMB slowly appeared in the systemic circulation (Tmax ∼2-6 h) and had poor bioavailability (F%<1).Urine metabolites for both species and all routes included HMB, HMB-glucuronide, 2,4-dihydroxybenzophenone (DHB), DHB-glucuronide, and DHB-sulfates, and novel minor dihydroxy metabolites including 2,5-dihydroxy-4-methoxybenzophenone.In vitro hepatic metabolism in mice differed from human and in vivo metabolism especially for phase II conjugates.

Disposition and metabolism of 2,2'-dimorpholinodiethyl ether in sprague dawley rats and B6C3F1/N mice after oral, intravenous administration, and dermal application.

The specialty amine catalyst 2,2'-dimorpholinodiethyl ether (DMDEE) is a high-production volume chemical used in the production of flexible foam, high-resilient molded foam, and in coatings and adhesives. The disposition and metabolism of [14C]DMDEE (20 or 200 mg/kg) were determined in male ane female rats and mice after oral and intravenous administration and dermal application. In male and female rats, following a single oral administration, [14C]DMDEE was well-absorbed and excreted rapidly and extensively via urine (75-93%) and some in feces (∼4-8%). The total radioactivity in tissues at 24 h and 72 h (males only) following oral administration was 8-10% and ∼4%, respectively, suggesting considerable tissue distribution. A moderate amount of the total tissue radioactivity in kidney and liver were unextractable suggesting covalent binding of [14C]DMDEE-derived products in tissue macromolecules. Absorption following a single dermal application in rats was significant (∼64%) with a similar disposition pattern to oral. The oral and dermal disposition of [14C]DMDEE in male and female mice was similar to rats. Urinary products of DMDEE identified were oxidative metabolism of the morpholine ring. Coadministration of DMDEE with nitrite in rats didn't produce the rodent carcinogen, N-nitrosomorpholine.

Pulmonary and Regional Deposition of Nebulized and Dry Powder Aerosols in Ferrets.

The utilization of ferrets as a non-clinical model for disease is rapidly increasing within drug development. Many of these models include respiratory diseases that involve targeted drug delivery via nose-only inhalation. While the deposition patterns within other non-clinical models (mice, rats, canines, and non-human primates) have been well studied, the local and regional deposition of aerosols in ferrets has not been well characterized. Therefore, inhalation aerosols were developed, radiolabeled and the radiolabeling methods validated to support SPECT-CT imaging and quantification of regional deposition within ferrets. The studies were conducted with one liquid formulation and one dry powder formulation (two concentrations of dry powder). Additionally, both aerosols were polydisperse and therefore reflect the majority of pharmaceutical aerosols. Overall, the studies showed lung deposition fractions between 5 and 10% with median aerodynamic particle sizes of 2.5 and 2.8 µm. The lung deposition fraction of the liquid aerosol was ~ 9%, nearly double observed in rats with a similarly sized aerosol. Analysis of respiratory tract (oropharynx, laryngopharynx, trachea, bifurcation area, and lung) deposition indicates increased deposition of the liquid aerosol compared to the dry powder aerosol, however, when this analysis was refined to the pulmonary region (trachea, bifurcation, and lung) the deposition was similar between formulations. These data provide the first description of the regional deposition of inhalation aerosols in ferrets with standard nose-only inhalation procedures. These data can be used for calculations of both total and regional doses within ferret inhalation drug delivery.

Disposition of ß-N-methylamino-l-alanine (L-BMAA), a neurotoxin, in rodents following a single or repeated oral exposure.

ß-N-methylamino-l-alanine (L-BMAA) is produced by cyanobacteria (blue-green algae). Human exposure to L-BMAA occurs via consumption of L-BMAA-contaminated water and food. It is speculated that exposure to L-BMAA, and subsequent brain accumulation, may contribute to an increased incidence of neurodegenerative diseases indicating the need to evaluate risk of L-BMAA exposure to humans. As an initial step in this process, we have evaluated disposition following a single or repeated gavage administration of 1, 10 or 100mg/kg [14C]L-BMAA in rats and mice. L-BMAA was well absorbed following a single gavage administration with minimal dose, species, or sex-related effect. In both species, the main excretion route was as exhaled CO2 (46-61%) with 7-13% and 1.4-8% of the administered dose excreted in the urine and feces, respectively. L-BMAA was distributed to all tissues examined; the total radioactivity in tissues increased with the dose and was significant in both species (8-20%). In male rats, L-BMAA was slowly eliminated from blood and tissues (half-lives ≥48h). Following 1, 5 and 10days of dosing in male rats, levels in tissues increased with the number of doses demonstrating potential for accumulation of BMAA-derived equivalents. There was no greater affinity for accumulation in the brain compared to other organs and tissues. Following repeated exposure in rats, amino acid mass shifts associated with L-BMAA were detected in brain peptides. However, the low frequency of occurrence suggests that the substitution of an amino acid with L-BMAA is not significant relative to substitutions and/or modifications by other L-BMAA-derived equivalents.

Exposure to traffic-generated air pollutants mediates alterations in brain microvascular integrity in wildtype mice on a high-fat diet.

Air pollution-exposure is associated with detrimental outcomes in the central nervous system (CNS) such as cerebrovascular disorders, including stroke, and neurodegenerative diseases. While the mechanisms of these CNS-related outcomes involved have not been fully elucidated, exposure to traffic-generated air pollutants has been associated with altered blood brain barrier (BBB) integrity and permeability. The current study investigated whether inhalation exposure to mixed vehicle emissions (MVE) alters cerebral microvascular integrity in healthy 3 mo old C57BL/6 mice, as well as whether exposure-mediated effects were exacerbated by a high-fat (HF) vs. low-fat (LF) diet. Mice on each diet were randomly assigned to be exposed to either filtered air (FA) or MVE [100PM/m3 vehicle emissions mixture: 30µg PM/m3 gasoline engine + 70µg PM/m3 diesel engine emissions; median size ~ 60nm; particle mass size distribution median of ~ 1µm (range: < 0.5-20µm)] for 6h/d, 7d/wk, for 30d. Using sodium fluorescein as a tracer, we observed a significant increase in BBB permeability in both HF + MVE exposed and HF + FA animals, compared to LF + FA controls. Exposure to HF + MVE also led to a significant increase plasma ox-LDL and ox-LDL scavenger receptors (LOX-1 and CD-36) expression in the cerebral vasculature. Histological analysis revealed decreased expression of TJ protein, claudin-5, associated with increased matrix metalloproteinase (MMP)-9 activity and oxidative stress in the cerebral vasculature of HF + MVE mice, compared to LF + MVE. Such findings indicate that inhalation exposure to traffic-generated pollutants, coupled with a HF diet, results in altered BBB integrity and increased ox-LDL signaling in the cerebral vasculature in a wildtype animal model.

Microglial priming through the lung-brain axis: the role of air pollution-induced circulating factors.

Air pollution is implicated in neurodegenerative disease risk and progression and in microglial activation, but the mechanisms are unknown. In this study, microglia remained activated 24 h after ozone (O3) exposure in rats, suggesting a persistent signal from lung to brain. Ex vivo analysis of serum from O3-treated rats revealed an augmented microglial proinflammatory response and ß-amyloid 42 (Aß42) neurotoxicity independent of traditional circulating cytokines, where macrophage-1 antigen-mediated microglia proinflammatory priming. Aged mice exhibited reduced pulmonary immune profiles and the most pronounced neuroinflammation and microglial activation in response to mixed vehicle emissions. Consistent with this premise, cluster of differentiation 36 (CD36)(-/-) mice exhibited impaired pulmonary immune responses concurrent with augmented neuroinflammation and microglial activation in response to O3 Further, aging glia were more sensitive to the proinflammatory effects of O3 serum. Together, these findings outline the lung-brain axis, where air pollutant exposures result in circulating, cytokine-independent signals present in serum that elevate the brain proinflammatory milieu, which is linked to the pulmonary response and is further augmented with age.-Mumaw, C. L., Levesque, S., McGraw, C., Robertson, S., Lucas, S., Stafflinger, J. E., Campen, M. J., Hall, P., Norenberg, J. P., Anderson, T., Lund, A. K., McDonald, J. D., Ottens, A. K., Block, M. L. Microglial priming through the lung-brain axis: the role of air pollution-induced circulating factors.

The role of the lectin-like oxLDL receptor (LOX-1) in traffic-generated air pollution exposure-mediated alteration of the brain microvasculature in Apolipoprotein (Apo) E knockout mice.

Recent studies have shown a strong correlation between air pollution-exposure and detrimental outcomes in the central nervous system, including alterations in blood brain barrier (BBB) integrity, neuroinflammation, and neurodegeneration. However, the mechanisms mediating these pathologies have not yet been fully elucidated. We have previously reported that exposure to traffic-generated air pollution results in increased circulating oxidized low-density lipoprotein (oxLDL), associated with alterations in BBB integrity, in atherosclerotic Apolipoprotein E null (ApoE-/-) mice. Thus, we investigated the role of the lectin-like oxLDL receptor (LOX)-1 in mediating these deleterious effects in ApoE-/- mice exposed to a mixture of gasoline and diesel engine exhaust (MVE: 100 PM µg/m3) for 6 h/d, 7d/week, for 30 d by inhalation. Concurrent with exposures, a subset of mice were treated with neutralizing antibodies to LOX-1 (LOX-1 Ab) i.p., or IgG (control) i.p., every other day during exposures. Resulting brain microvascular integrity, tight junction (TJ) protein expression, matrix metalloproteinase (MMP)-9/-2 activity, ROS, and markers of cellular adhesion and monocyte/macrophage sequestration were assessed. MVE-exposure resulted in decreased BBB integrity and alterations in microvascular TJ protein expression, associated with increased LOX-1 expression, MMP-9/-2 activities, and lipid peroxidation, each of which was attenuated with LOX-1 Ab treatment. Furthermore, MVE-exposure induced cerebral microvascular ROS and adhesion molecules, expression of which was not normalized through LOX-1 Ab-treatment. Such findings suggest that alterations in brain microvascular structure and integrity observed with MVE-exposure may be mediated, at least in part, via LOX-1 signaling.

Surface area-dependence of gas-particle interactions influences pulmonary and neuroinflammatory outcomes.

BACKGROUND: Deleterious consequences of exposure to traffic emissions may derive from interactions between carbonaceous particulate matter (PM) and gaseous components in a manner that is dependent on the surface area or complexity of the particles. To determine the validity of this hypothesis, we examined pulmonary and neurological inflammatory outcomes in C57BL/6 and apolipoprotein E knockout (ApoE-/-) male mice after acute and chronic exposure to vehicle engine-derived particulate matter, generated as ultrafine (UFP) and fine (FP) sizes, with additional exposures using UFP or FP combined with gaseous copollutants derived from fresh gasoline and diesel emissions, labeled as UFP + G and FP + G. RESULTS: The UFP and UFP + G exposure groups resulted in the most profound pulmonary and neuroinflammatory effects. Phagocytosis of UFP + G particles via resident alveolar macrophages was substantial in both mouse strains, particularly after chronic exposure, with concurrent increased proinflammatory cytokine expression of CXCL1 and TNFα in the bronchial lavage fluid. In the acute exposure paradigm, only UFP and UFP + G induced significant changes in pulmonary inflammation and only in the ApoE-/- animals. Similarly, acute exposure to UFP and UFP + G increased the expression of several cytokines in the hippocampus of ApoE-/- mice including Il-1ß, IL-6, Tgf-ß and Tnf-α and in the hippocampus of C57BL/6 mice including Ccl5, Cxcl1, Il-1ß, and Tnf-α. Interestingly, Il-6 and Tgf-ß expression were decreased in the C57BL/6 hippocampus after acute exposure. Chronic exposure to UFP + G increased expression of Ccl5, Cxcl1, Il-6, and Tgf-ß in the ApoE-/- hippocampus, but this effect was minimal in the C57BL/6 mice, suggesting compensatory mechanisms to manage neuroinflammation in this strain. CONCLUSIONS: Inflammatory responses the lung and brain were most substantial in ApoE-/- animals exposed to UFP + G, suggesting that the surface area-dependent interaction of gases and particles is an important determinant of toxic responses. As such, freshly generated UFP, in the presence of combustion-derived gas phase pollutants, may be a greater health hazard than would be predicted from PM concentration, alone, lending support for epidemiological findings of adverse neurological outcomes associated with roadway proximity.

Metabolomic changes in murine serum following inhalation exposure to gasoline and diesel engine emissions.

The adverse health effects of environmental exposure to gaseous and particulate components of vehicular emissions are a major concern among urban populations. A link has been established between respiratory exposure to vehicular emissions and the development of cardiovascular disease (CVD), but the mechanisms driving this interaction remain unknown. Chronic inhalation exposure to mixed vehicle emissions has been linked to CVD in animal models. This study evaluated the temporal effects of acute exposure to mixed vehicle emissions (MVE; mixed gasoline and diesel emissions) on potentially active metabolites in the serum of exposed mice. C57Bl/6 mice were exposed to a single 6-hour exposure to filtered air (FA) or MVE (100 or 300 µg/m(3)) by whole body inhalation. Immediately after and 18 hours after the end of the exposure period, animals were sacrificed for serum and tissue collection. Serum was analyzed for metabolites that were differentially present between treatment groups and time points. Changes in metabolite levels suggestive of increased oxidative stress (oxidized glutathione, cysteine disulfide, taurine), lipid peroxidation (13-HODE, 9-HODE), energy metabolism (lactate, glycerate, branched chain amino acid catabolites, butrylcarnitine, fatty acids), and inflammation (DiHOME, palmitoyl ethanolamide) were observed immediately after the end of exposure in the serum of animals exposed to MVE relative to those exposed to FA. By 18 hours post exposure, serum metabolite differences between animals exposed to MVE versus those exposed to FA were less pronounced. These findings highlight complex metabolomics alterations in the circulation following inhalation exposure to a common source of combustion emissions.

Changes in HPBMC markers of immmune function following controlled short-term inhalation exposures of humans to hardwood smoke.

Previous studies have shown that complex mixtures containing particulate matter (PM) and polycyclic aromatic hydrocarbons (PAHs) produce systemic immunotoxicity in animal models following inhalation exposures. While we and others have shown that emissions associated with hardwood smoke (HWS), cigarette smoke and diesel exhaust can suppress the immune systems of animals in vitro and in vivo, there have been few immune function studies on human peripheral blood mononuclear cells (HPBMC) following exposure of humans to HWS. Our work shows that T cells are an important targets of PM and PAH immunotoxicity. These studies were conducted on HPBMC from 14 human volunteers receiving four 2 h nightly exposures to clean air or HWS at a concentration of 500 ug/m(3). We measured anti-CD3/anti-CD28 stimulated T-cell proliferation and HPBMC cytokine production in cell supernatants, including interleukin 1ß (IL-1ß), tumor necrosis factor α (TNF-α), interleukin 6 (IL-6), interleukin 8 (IL-8), TH1 cytokines γIFN and IL-2, TH2 cytokine IL-4, Th17 cytokine interleukin 17A (IL-17A) and interleukin 10 (IL-10). We analyzed results using analysis of variance (ANOVA), t-tests and Pearson correlation. Results showed that there was significant variation in the amount of T-cell proliferation observed following polyclonal activation with anti-CD3/anti-CD28 antibodies in both the air and HWS-exposed groups. There was not a significant effect of HWS on T-cell proliferation. However, we did find a strong relationship between the presence of proinflammatory cytokines (IL-1ß, TNF-α, IL-6, but not IL-8) and the amount of T-cell proliferation seen in individual donors, demonstrating that brief exposures of humans to HWS can produce changes in systemic immunity that is associated with proinflammatory cytokines.

Inhaled PYY(3-36) dry-powder formulation for appetite suppression.

OBJECTIVE: Peptide YY3-36 [PYY(3-36)] has shown efficacy in appetite suppression when dosed by injection modalities (intraperitoneal (IP)/subcutaneous). Transitioning to needle-free delivery, towards inhalation, often utilizes systemic pharmacokinetics as a key endpoint to compare different delivery methods and doses. Systemic pharmacokinetics were evaluated for PYY3-36 when delivered by IP, subcutaneous, and inhalation, the systemic pharmacokinetics were then used to select doses in an appetite suppression pharmacodynamic study. METHODS: Dry-powder formulations were manufactured by spray drying and delivered to mice via nose only inhalation. The systemic plasma, lung tissue, and bronchoalveolar lavage fluid pharmacokinetics of different inhalation doses of PYY(3-36) were compared to IP and subcutaneous efficacious doses. Based on these pharmacokinetic data, inhalation doses of 70:30 PYY(3-36):Dextran T10 were evaluated in a mouse model of appetite suppression and compared to IP and subcutaneous data. RESULTS: Inhalation pharmacokinetic studies showed that plasma exposure was similar for a 2 × higher inhalation dose when compared to subcutaneous and IP delivery. Inhalation doses of 0.22 and 0.65 mg/kg were for efficacy studies. The results showed a dose-dependent (not dose proportional) decrease in food consumption over 4 h, which is similar to IP and subcutaneous delivery routes. CONCLUSIONS: The pharmacokinetic and pharmacodynamics results substantiate the ability of pharmacokinetic data to inform pharmacodynamics dose selection for inhalation delivery of the peptide PYY(3-36). Additionally, engineered PYY(3-36):Dextran T10 particles delivered to the respiratory tract show promise as a non-invasive therapeutic for appetite suppression.

Part 1. Assessment of carcinogenicity and biologic responses in rats after lifetime inhalation of new-technology diesel exhaust in the ACES bioassay.

The Health Effects Institute and its partners conceived and funded a program to characterize the emissions from heavy-duty diesel engines compliant with the 2007 and 2010 on-road emissions standards in the United States and to evaluate indicators of lung toxicity in rats and mice exposed repeatedly to 2007-compliant new-technology diesel exhaust (NTDE*). The a priori hypothesis of this Advanced Collaborative Emissions Study (ACES) was that 2007-compliant on-road diesel emissions "... will not cause an increase in tumor formation or substantial toxic effects in rats and mice at the highest concentration of exhaust that can be used ... although some biological effects may occur." This hypothesis was tested at the Lovelace Respiratory Research Institute (LRRI) by exposing rats by chronic inhalation as a carcinogenicity bioassay. Indicators of pulmonary toxicity in rats were measured after 1, 3, 12, 24, and 28-30 months of exposure. Similar indicators of pulmonary toxicity were measured in mice, as an interspecies comparison of the effects of subchronic exposure, after 1 and 3 months of exposure. A previous HEI report (Mauderly and McDonald 2012) described the operation of the engine and exposure systems and the characteristics of the exposure atmospheres during system commissioning. Another HEI report described the biologic responses in mice and rats after subchronic exposure to NTDE (McDonald et al. 2012). The primary motivation for the present chronic study was to evaluate the effects of NTDE in rats in the context of previous studies that had shown neoplastic lung lesions in rats exposed chronically to traditional technology diesel exhaust (TDE) (i.e., exhaust from diesel engines built before the 2007 U.S. requirements went into effect). The hypothesis was largely based on the marked reduction of diesel particulate matter (DPM) in NTDE compared with emissions from older diesel engine and fuel technologies, although other emissions were also reduced. The DPM component of TDE was considered the primary driver of lung tumorigenesis in rats exposed chronically to historical diesel emissions. Emissions from a 2007-compliant, 500-horsepower-class engine and after treatment system operated on a variable-duty cycle were used to generate the animal inhalation test atmospheres. Four groups were exposed to one of three concentrations (dilutions) of exhaust combined with crankcase emissions, or to clean air as a negative control. Dilutions of exhaust were set to yield average integrated concentrations of 4.2, 0.8, and 0.1 ppm nitrogen dioxide (NO2). Exposure atmospheres were analyzed by daily measurements of key effects of NTDE in the present study were generally consistent with those observed previously in rats exposed chronically to NO2 alone. This suggests that NO2 may have been the primary driver of the biologic responses to NTDE in the present study. There was little evidence of effects characteristic of rats exposed chronically to high concentrations of DPM in TDE, such as an extensive accumulation of DPM within alveolar macrophages and inflammation leading to neoplastic transformation of epithelia and lung tumors. components and periodic detailed physical-chemical characterizations. Exposures were conducted 16 hours/day (overnight, during the rats' most active period), 5 days/week. Responses to exposure were evaluated via hematology, serum chemistry, bronchoalveolar lavage (BAL), lung cell proliferation, histopathology, and pulmonary function. The exposures were accomplished as planned, with average integrated exposure concentrations within 20% of the target dilutions. The major components from exhaust were the gaseous inorganic compounds, nitrogen monoxide (NO), NO2, and carbon monoxide (CO). Minor components included low concentrations of DPM and volatile and semi-volatile organic compounds (VOCs and SVOCs). Among the more than 100 biologic response variables evaluated, the majority showed no significant difference from control as a result of exposure to NTDE. The major outcome of this study was the absence of pre-neoplastic lung lesions, primary lung neoplasia, or neoplasia of any type attributable to NTDE exposure. The lung lesions that did occur were minimal to mild, occurred only at the highest exposure level, and were characterized by an increased number and prominence of basophilic epithelial cells (considered reactive or regenerative) lining distal terminal bronchioles, alveolar ducts, and adjacent alveoli (termed in this report "Hyperplasia; Epithelial; Periacinar"), which often had a minimal increase in subjacent fibrous stroma (termed "Fibrosis; Interstitial; Periacinar"). Slight epithelial metaplastic change to a cuboidal morphology, often demonstrating cilia, was also noted in some animals (termed "Bronchiolization"). In addition to the epithelial proliferation, there was occasionally a subtle accumulation of pulmonary alveolar macrophages (termed "Accumulation; Macrophage") in affected areas. The findings in the lung progressed slightly from 3 to 12 months, without further progression between 12 months and the final sacrifice at 28 or 30 months. In addition to the histologic findings, there were biochemical changes in the lung tissue and lavage fluid that indicated mild inflammation and oxidative stress. Generally, these findings were observed only at the highest exposure level. There was also a mild progressive decrease in pulmonary function, which was more consistent in females than males. Limited nasal epithelial changes resulted from NTDE exposure, including increases in minor olfactory epithelial degeneration, hyperplasia, and/or metaplasia. Increases in these findings were present primarily at the highest exposure level, and their minor and variable nature renders their biologic significance uncertain. Overall, the findings of this study demonstrated markedly less severe biologic responses to NTDE than observed previously in rats exposed similarly to TDE. Further, the effects of NTDE in the present study were generally consistent with those observed previously in rats exposed chronically to NO2 alone. This suggests that NO2 may have been the primary driver of the biologic responses to NTDE in the present study. There was little evidence of effects characteristic of rats exposed chronically to high concentrations of DPM in TDE, such as an extensive accumulation of DPM within alveolar macrophages and inflammation leading to neoplastic transformation of epithelia and lung tumors.

Carbon capture and sequestration: an exploratory inhalation toxicity assessment of amine-trapping solvents and their degradation products.

Carbon dioxide (CO2) absorption with aqueous amine solvents is a method of carbon capture and sequestration (CCS) from flue gases. One concern is the possible release of amine solvents and degradation products into the atmosphere, warranting evaluation of potential pulmonary effects from inhalation. The CCS amines monoethanolamine (MEA), methyldiethanolamine (MDEA), and piperazine (PIP) underwent oxidative and CO2-mediated degradation for 75 days. C57bl/6N mice were exposed for 7 days by inhalation of 25 ppm neat amine or equivalant concentration in the degraded mixture. The aqueous solutions were nebulized to create the inhalation atmospheres. Pulmonary response was measured by changes in inflammatory cells in bronchoalveolar lavage fluid and cytokine expression in lung tissue. Ames mutagenicity and CHO-K1 micronucleus assays were applied to assess genotoxicity. Chemical analysis of the test atmosphere and liquid revealed complex mixtures, including acids, aldehydes, and other compounds. Exposure to oxidatively degraded MEA increased (p < 0.05) total cells, neutrophils, and lymphocytes compared to control mice and caused inflammatory cytokine expression (statistical increase at p < 0.05). MEA and CO2-degraded MEA were the only atmospheres to show statistical (p < 0.05) increase in oxidative stress. CO2 degradation resulted in a different composition, less degradation, and lower observed toxicity (less magnitude and number of effects) with no genotoxicity. Overall, oxidative degradation of the amines studied resulted in enhanced toxicity (increased magnitude and number of effects) compared to the neat chemicals.