Detection of Transgene Location in the CYP2A13/2B6/2F1-transgenic Mouse Model using Optical Genome Mapping Technology.

Most transgenic mouse models are generated through random integration of the transgene. The location of the transgene provides valuable information for assessing potential effects of the transgenesis on the host and for designing genotyping protocols that can amplify across the integration site, but it is challenging to identify. Here, we report the successful utility of optical genome mapping technology to identify the transgene insertion site in a CYP2A13/2B6/2F1-transgenic mouse model, which produces three human cytochrome P450 (P450) enzymes (CYP2A13, CYP2B6, and CYP2F1) that are encoded by neighboring genes on human chromosome 19. These enzymes metabolize many drugs, respiratory toxicants, and chemical carcinogens. Initial efforts to identify candidate insertion sites by whole genome sequencing was unsuccessful, apparently because the transgene is located in a region of the mouse genome that contains highly repetitive sequences. Subsequent utility of the optical genome mapping approach, which compares genome-wide marker distribution between the transgenic mouse genome and a reference mouse (GRCm38) or human (GRCh38) genome, localized the insertion site to mouse chromosome 14, between two marker positions at 4451324 base pair and 4485032 base pair. A transgene-mouse genome junction sequence was further identified through long-polymerase chain reaction amplification and DNA sequencing at GRCm38 Chr.14:4484726. The transgene insertion (∼2.4 megabase pair) contained 5-7 copies of the human transgenes, which replaced a 26.9-33.4 kilobase pair mouse genomic region, including exons 1-4 of Gm3182, a predicted and highly redundant gene. Finally, the sequencing results enabled the design of a new genotyping protocol that can distinguish between hemizygous and homozygous CYP2A13/2B6/2F1-transgenic mice. SIGNIFICANCE STATEMENT: This study characterizes the genomic structure of, and provides a new genotyping method for, a transgenic mouse model that expresses three human P450 enzymes, CYP2A13, CYP2B6, and CYP2F1, that are important in xenobiotic metabolism and toxicity. The demonstrated success in applying the optical genome mapping technology for identification of transgene insertion sites should encourage others to do the same for other transgenic models generated through random integration, including most of the currently available human P450 transgenic mouse models.

Effect of graphene-based nanomaterials on corneal wound healing in vitro.

Graphene-based nanomaterials (GBNs) are widely used due to their chemical and physical properties for multiple commercial and environmental applications. From an occupational health perspective, there is concern regarding the effects of inhalation on the respiratory system, and many studies have been conducted to study inhalation impacts on lung. Similar to the respiratory system, the eyes may also be exposed to GBNs and thus impacted. In this study, immortalized human corneal epithelial (hTCEpi) cells and rabbit corneal fibroblasts (RCFs) were used to investigate the toxicity of eight types of GBN: graphene oxide (GO; 400 nm), GO (1 µm), partially reduced graphene oxide (PRGO; 400 nm), reduced graphene oxide (RGO; 400 nm), RGO (2 µm), graphene (110 nm), graphene (140 nm), and graphene (1 µm). We next examined the effects of these GBNs on hTCEpi cell migration. We also determined whether the expression of α-smooth muscle actin (αSMA), a myofibroblast marker, is altered by the GBNs using RCFs. We found that RGO (400 nm) and RGO (2 µm) were highly toxic to hTCEPi cells and RCFs meanwhile, PRGO (400 nm) was toxic only to hTCEpi cells. In addition, PRGO (400 nm), RGO (400 nm), and RGO (2 µm) inhibited hTCEpi cell migration and significantly increased αSMA mRNA expression. Further study in vivo is required to determine if RGO nanomaterials delay corneal epithelial healing and induce scar formation.

Heterogeneous deposition of regular and mentholated little cigar smoke in the lungs of Sprague-Dawley rats.

BACKGROUND: Quantifying the dose and distribution of tobacco smoke in the respiratory system is critical for understanding its toxicity, addiction potential, and health impacts. Epidemiologic studies indicate that the incidence of lung tumors varies across different lung regions, suggesting there may be a heterogeneous deposition of smoke particles leading to greater health risks in specific regions. Despite this, few studies have examined the lobar spatial distribution of inhaled particles from tobacco smoke. This gap in knowledge, coupled with the growing popularity of little cigars among youth, underscores the need for additional research with little cigars. RESULTS: In our study, we analyzed the lobar deposition in rat lungs of smoke particles from combusted regular and mentholated Swisher Sweets little cigars. Twelve-week-old male and female Sprague-Dawley rats were exposed to smoke particles at a concentration of 84 ± 5 mg/m3 for 2 h, after which individual lung lobes were examined. We utilized Inductively Coupled Plasma Mass Spectrometry to quantify lobar chromium concentrations, serving as a smoke particle tracer. Our findings demonstrated an overall higher particle deposition from regular little cigars than from the mentholated ones. Higher particle deposition fraction was observed in the left and caudal lobes than other lobes. We also observed sex-based differences in the normalized deposition fractions among lobes. Animal study results were compared with the multi-path particle dosimetry (MPPD) model predictions, which showed that the model overestimated particle deposition in certain lung regions. CONCLUSIONS: Our findings revealed that the particle deposition varied between different little cigar products. The results demonstrated a heterogenous deposition pattern, with higher particle deposition observed in the left and caudal lobes, especially with the mentholated little cigars. Additionally, we identified disparities between our measurements and the MPPD model. This discrepancy highlights the need to enhance the accuracy of models before extrapolating animal study results to human lung deposition. Overall, our study provides valuable insights for estimating the dose of little cigars during smoking for toxicity research.

In vitro airway models from mice, rhesus macaques, and humans maintain species differences in xenobiotic metabolism and cellular responses to naphthalene.

The translational value of high-throughput toxicity testing will depend on pharmacokinetic validation. Yet, popular in vitro airway epithelia models were optimized for structure and mucociliary function without considering the bioactivation or detoxification capabilities of lung-specific enzymes. This study evaluated xenobiotic metabolism maintenance within differentiated air-liquid interface (ALI) airway epithelial cell cultures (human bronchial; human, rhesus, and mouse tracheal), isolated airway epithelial cells (human, rhesus, and mouse tracheal; rhesus bronchial), and ex vivo microdissected airways (rhesus and mouse) by measuring gene expression, glutathione content, and naphthalene metabolism. Glutathione levels and detoxification gene transcripts were measured after 1-h exposure to 80 µM naphthalene (a bioactivated toxicant) or reactive naphthoquinone metabolites. Glutathione and glutathione-related enzyme transcript levels were maintained in ALI cultures from all species relative to source tissues, while cytochrome P450 monooxygenase gene expression declined. Notable species differences among the models included a 40-fold lower total glutathione content for mouse ALI trachea cells relative to human and rhesus; a higher rate of naphthalene metabolism in mouse ALI cultures for naphthalene-glutathione formation (100-fold over rhesus) and naphthalene-dihydrodiol production (10-fold over human); and opposite effects of 1,2-naphthoquinone exposure in some models-glutathione was depleted in rhesus tissue but rose in mouse ALI samples. The responses of an immortalized bronchial cell line to naphthalene and naphthoquinones were inconsistent with those of human ALI cultures. These findings of preserved species differences and the altered balance of phase I and phase II xenobiotic metabolism among the characterized in vitro models should be considered for future pulmonary toxicity testing.

Inhalation of Silver Silicate Nanoparticles Leads to Transient and Differential Microglial Activation in the Rodent Olfactory Bulb.

Engineered silver nanoparticles (AgNPs), including silver silicate nanoparticles (Ag-SiO2 NPs), are used in a wide variety of medical and consumer applications. Inhaled AgNPs have been found to translocate to the olfactory bulb (OB) after inhalation and intranasal instillation. However, the biological effects of Ag-SiO2 NPs and their potential nose-to-brain transport have not been evaluated. The present study assessed whether inhaled Ag-SiO2 NPs can elicit microglial activation in the OB. Adult Sprague-Dawley rats inhaled aerosolized Ag-SiO2 NPs at a concentration of 1 mg/ml for 6 hours. On day 0, 1, 7, and 21 post-exposure, rats were necropsied and OB were harvested. Immunohistochemistry on OB tissues were performed with anti-ionized calcium-binding adapter molecule 1 and heme oxygenase-1 as markers of microglial activation and oxidative stress, respectively. Aerosol characterization indicated Ag-SiO2 NPs were sufficiently aerosolized with moderate agglomeration and high-efficiency deposition in the nasal cavity and olfactory epithelium. Findings suggested that acute inhalation of Ag-SiO2 NPs elicited transient and differential microglial activation in the OB without significant microglial recruitment or oxidative stress. The delayed and differential pattern of microglial activation in the OB implied that inhaled Ag-SiO2 may have translocated to the central nervous system via intra-neuronal pathways.

Comparison of acute respiratory epithelial toxicity for 4-Methylimidazole and naphthalene administered by oral gavage in B6C3F1 mice.

4-Methylimidazole (4MEI) is a contaminant in food and consumer products. Pulmonary toxicity and carcinogenicity following chronic dietary exposures to 4MEI is a regulatory concern based on previous rodent studies. This study examined acute pulmonary toxicity in B6C3F1 mice from 6 h to 5 days after oral gavage with a single dose of 150 mg/kg 4MEI, a double dose delivered 6 h apart, or vehicle controls. Oral gavage of 150 mg/kg naphthalene, a prototypical Club cell toxicant, was used as a positive control. Intrapulmonary conducting airway cytotoxicity was assessed in fixed-pressure inflated lungs using qualitative histopathology scoring, quantitative morphometric measurement of vacuolated and exfoliating epithelial cells, and immunohistochemistry. 4MEI treatment did not change markers of cytotoxicity including the mass of vacuolated epithelium, the thickness of the epithelium, or the distributions of epithelial proteins: secretoglobin 1A1, proliferating cell nuclear antigen, calcitonin gene-related peptide, and myeloperoxidase. 4MEI and vehicle controls caused slight cytotoxicity with rare vacuolization of the epithelium relative to the severe bronchiolar epithelial cell toxicity found in the naphthalene exposed mice at terminal bronchioles, intrapulmonary airways, or airway bifurcations. In summary, 4MEI caused minimal airway epithelial toxicity without characteristic Club Cell toxicity when compared to naphthalene, a canonical Club Cell toxicant.

Toxicokinetic Interaction between Hepatic Disposition and Pulmonary Bioactivation of Inhaled Naphthalene Studied Using Cyp2abfgs-Null and CYP2A13/2F1-Humanized Mice with Deficient Hepatic Cytochrome P450 Activity.

Previous studies using Cyp2abfgs-null (lacking all genes of the Cyp2a, 2b, 2f, 2g, and 2s subfamilies), CYP2A13/2F1-humanized, and liver-Cpr-null (LCN) mice showed that although hepatic cytochrome P450 (P450) enzymes are essential for systemic clearance of inhaled naphthalene (a possible human carcinogen), both hepatic and extrahepatic P450 enzymes may contribute to naphthalene-induced lung toxicity via bioactivation. Herein, we aimed to further understand the toxicokinetics of inhaled naphthalene in order to provide a basis for predicting the effects of variations in rates of xenobiotic disposition on the extent of target tissue bioactivation. We assessed the impact of a hepatic deficit in naphthalene metabolism on the toxicokinetics of inhaled naphthalene using newly generated Cyp2abfgs-null-and-LCN and CYP2A13/2F1-humanized-and-LCN mice. We determined plasma, lung, and liver levels of naphthalene and naphthalene-glutathione conjugate, a biomarker of naphthalene bioactivation, over time after naphthalene inhalation. We found that the loss of hepatic naphthalene metabolism severely decreased naphthalene systemic clearance and caused naphthalene to accumulate in the liver and other tissues. Naphthalene release from tissue, as evidenced by the continued increase in plasma naphthalene levels after termination of active inhalation exposure, was accompanied by prolonged bioactivation of naphthalene in the lung. In addition, transgenic expression of human CYP2A13/2F1 in the respiratory tract caused a reduction in plasma naphthalene levels (by 40%, relative to Cyp2abfgs-null-and-LCN mice) and corresponding decreases in naphthalene-glutathione levels in the lung in mice with hepatic P450 deficiency, despite the increase in local naphthalene-bioactivating P450 activity. Thus, the bioavailability of naphthalene in the target tissue has a significant effect on the extent of naphthalene bioactivation in the lung. SIGNIFICANCE STATEMENT: In this study, we report several novel findings related to the toxicokinetics of inhaled naphthalene, the ability of which to cause lung carcinogenesis in humans is a current topic for risk assessment. We show the accumulation of naphthalene in the liver and lung in mice with compromised hepatic cytochrome P450 (P450) activity; the ability of tissue-stored naphthalene to redistribute to the circulation after termination of active inhalation exposure, prolonging exposure of target tissues to naphthalene; and the ability of non-CYP2ABFGS enzymes of the lung to bioactivate naphthalene. These results suggest potentially large effects of deficiencies in hepatic P450 activity on naphthalene tissue burden and bioactivation in human lungs.

Naphthalene DNA Adduct Formation and Tolerance in the Lung.

Naphthalene (NA) is a respiratory toxicant and possible human carcinogen. NA is a ubiquitous combustion product and significant component of jet fuel. The National Toxicology Program found that NA forms tumors in two species, in rats (nose) and mice (lung). However, it has been argued that NA does not pose a cancer risk to humans because NA is bioactivated by cytochrome P450 monooxygenase enzymes that have very high efficiency in the lung tissue of rodents but low efficiency in the lung tissue of humans. It is thought that NA carcinogenesis in rodents is related to repeated cycles of lung epithelial injury and repair, an indirect mechanism. Repeated in vivo exposure to NA leads to development of tolerance, with the emergence of cells more resistant to NA insult. We tested the hypothesis that tolerance involves reduced susceptibility to the formation of NA-DNA adducts. NA-DNA adduct formation in tolerant mice was examined in individual, metabolically-active mouse airways exposed ex vivo to 250 µΜ 14C-NA. Ex vivo dosing was used since it had been done previously and the act of creating a radioactive aerosol of a potential carcinogen posed too many safety and regulatory obstacles. Following extensive rinsing to remove unbound 14C-NA, DNA was extracted and 14C-NA-DNA adducts were quantified by AMS. The tolerant mice appeared to have slightly lower NA-DNA adduct levels than non-tolerant controls, but intra-group variations were large and the difference was statistically insignificant. It appears the tolerance may be more related to other mechanisms, such as NA-protein interactions in the airway, than DNA-adduct formation.

Impact of hepatic P450-mediated biotransformation on the disposition and respiratory tract toxicity of inhaled naphthalene.

We determined whether a decrease in hepatic microsomal cytochrome P450 activity would impact lung toxicity induced by inhalation exposure to naphthalene (NA), a ubiquitous environmental pollutant. The liver-Cpr-null (LCN) mouse showed decreases in microsomal metabolism of NA in liver, but not lung, compared to wild-type (WT) mouse. Plasma levels of NA and NA-glutathione conjugates (NA-GSH) were both higher in LCN than in WT mice after a 4-h nose-only NA inhalation exposure at 10ppm. Levels of NA were also higher in lung and liver of LCN, compared to WT, mice, following exposure to NA at 5 or 10ppm. Despite the large increase in circulating and lung tissue NA levels, the level of NA-GSH, a biomarker of NA bioactivation, was either not different, or only slightly higher, in lung and liver tissues of LCN mice, relative to that in WT mice. Furthermore, the extent of NA-induced acute airway injury, judging from high-resolution lung histopathology and morphometry at 20h following NA exposure, was not higher, but lower, in LCN than in WT mice. These results, while confirming the ability of extrahepatic organ to bioactivate inhaled NA and mediate NA's lung toxicity, suggest that liver P450-generated NA metabolites also have a significant, although relatively small, contribution to airway toxicity of inhaled NA. This hepatic contribution to the airway toxicity of inhaled NA may be an important risk factor for individuals with diminished bioactivation activity in the lung.

Aerosolized Silver Nanoparticles in the Rat Lung and Pulmonary Responses over Time.

Silver nanoparticle (Ag NP) production methods are being developed and refined to produce more uniform Ag NPs through chemical reactions involving silver salt solutions, solvents, and capping agents to control particle formation. These chemical reactants are often present as contaminants and/or coatings on the Ag NPs, which could alter their interactions in vivo. To determine pulmonary effects of citrate-coated Ag NPs, Sprague-Dawley rats were exposed once nose-only to aerosolized Ag NPs (20 nm [C20] or 110 nm [C110] Ag NPs) for 6 hr. Bronchoalveolar lavage fluid (BALF) and lung tissues were obtained at 1, 7, 21, and 56 days postexposure for analyses. Inhalation of Ag NPs, versus citrate buffer control, produced significant inflammatory and cytotoxic responses that were measured in BALF cells and supernatant. At day 7, total cells, protein, and lactate dehydrogenase were significantly elevated in BALF, and peak histopathology was noted after C20 or C110 exposure versus control. At day 21, BALF polymorphonuclear cells and tissue inflammation were significantly greater after C20 versus C110 exposure. By day 56, inflammation was resolved in Ag NP-exposed animals. Overall, results suggest delayed, short-lived inflammatory and cytotoxic effects following C20 or C110 inhalation and potential for greater responses following C20 exposure.

Women and Lung Disease. Sex Differences and Global Health Disparities.

There is growing evidence that a number of pulmonary diseases affect women differently and with a greater degree of severity than men. The causes for such sex disparity is the focus of this Blue Conference Perspective review, which explores basic cellular and molecular mechanisms, life stages, and clinical outcomes based on environmental, sociocultural, occupational, and infectious scenarios, as well as medical health beliefs. Owing to the breadth of issues related to women and lung disease, we present examples of both basic and clinical concepts that may be the cause for pulmonary disease disparity in women. These examples include those diseases that predominantly affect women, as well as the rising incidence among women for diseases traditionally occurring in men, such as chronic obstructive pulmonary disease. Sociocultural implications of pulmonary disease attributable to biomass burning and infectious diseases among women in low- to middle-income countries are reviewed, as are disparities in respiratory health among sexual minority women in high-income countries. The implications of the use of complementary and alternative medicine by women to influence respiratory disease are examined, and future directions for research on women and respiratory health are provided.

Alterations in the proteome of the respiratory tract in response to single and multiple exposures to naphthalene.

Protein adduction is considered to be critical to the loss of cellular homeostasis associated with environmental chemicals undergoing metabolic activation. Despite considerable effort, our understanding of the key proteins mediating the pathologic consequences from protein modification by electrophiles is incomplete. This work focused on naphthalene (NA) induced acute injury of respiratory epithelial cells and tolerance which arises after multiple toxicant doses to define the initial cellular proteomic response and later protective actions related to tolerance. Airways and nasal olfactory epithelium from mice exposed to 15 ppm NA either for 4 h (acute) or for 4 h/day × 7 days (tolerant) were used for label-free protein quantitation by LC/MS/MS. Cytochrome P450 2F2 and secretoglobin 1A1 are decreased dramatically in airways of mice exposed for 4 h, a finding consistent with the fact that CYPs are localized primarily in Clara cells. A number of heat shock proteins and protein disulfide isomerases, which had previously been identified as adduct targets for reactive metabolites from several lung toxicants, were upregulated in airways but not olfactory epithelium of tolerant mice. Protein targets that are upregulated in tolerance may be key players in the pathophysiology associated with reactive metabolite protein adduction. All MS data have been deposited in the ProteomeXchange with identifier PXD000846 (http://proteomecentral.proteomexchange.org/dataset/PXD000846).

Evolution of silver nanoparticles in the rat lung investigated by X-ray absorption spectroscopy.

Following a 6-h inhalation exposure to aerosolized 20 and 110 nm diameter silver nanoparticles, lung tissues from rats were investigated with X-ray absorption spectroscopy, which can identify the chemical state of silver species. Lung tissues were processed immediately after sacrifice of the animals at 0, 1, 3, and 7 days post exposure and the samples were stored in an inert and low-temperature environment until measured. We found that it is critical to follow a proper processing, storage and measurement protocol; otherwise only silver oxides are detected after inhalation even for the larger nanoparticles. The results of X-ray absorption spectroscopy measurements taken in air at 85 K suggest that the dominating silver species in all the postexposure lung tissues were metallic silver, not silver oxide, or solvated silver cations. The results further indicate that the silver nanoparticles in the tissues were transformed from the original nanoparticles to other forms of metallic silver nanomaterials and the rate of this transformation depended on the size of the original nanoparticles. We found that 20 nm diameter silver nanoparticles were significantly modified after aerosolization and 6-h inhalation/deposition, whereas larger, 110 nm diameter nanoparticles were largely unchanged. Over the seven-day postexposure period the smaller 20 nm silver nanoparticles underwent less change in the lung tissue than the larger 110 nm silver nanoparticles. In contrast, silica-coated gold nanoparticles did not undergo any modification processes and remained as the initial nanoparticles throughout the 7-day study period.

Ozone-induced airway epithelial cell death, the neurokinin-1 receptor pathway, and the postnatal developing lung.

Children are uniquely susceptible to ozone because airway and lung growth continue for an extensive period after birth. Early-life exposure of the rhesus monkey to repeated ozone cycles results in region-specific disrupted airway/lung growth, but the mediators and mechanisms are poorly understood. Substance P (SP), neurokinin-1 receptor (NK-1R); and nuclear receptor Nur77 (NR4A1) are signaling pathway components involved in ozone-induced cell death. We hypothesize that acute ozone (AO) exposure during postnatal airway development disrupts SP/NK-1R/Nur77 pathway expression and that these changes correlate with increased ozone-induced cell death. Our objectives were to 1) spatially define the normal development of the SP/NK-1R/Nur77 pathway in conducting airways; 2) compare how postnatal age modulates responses to AO exposure; and 3) determine how concomitant, episodic ozone exposure modifies age-specific acute responses. Male infant rhesus monkeys were assigned at age 1 mo to two age groups, 2 or 6 mo, and then to one of three exposure subgroups: filtered air (FA), FA+AO (AO: 8 h/day × 2 days), or episodic biweekly ozone exposure cycles (EAO: 8 h/day × 5 days/14-day cycle+AO). O3 = 0.5 ppm. We found that 1) ozone increases SP/NK-1R/Nur77 pathway expression in conducting airways, 2) an ozone exposure cycle (5 days/cycle) delivered early at age 2 mo resulted in an airway that was hypersensitive to AO exposure at the end of 2 mo, and 3) continued episodic exposure (11 cycles) resulted in an airway that was hyposensitive to AO exposure at 6 mo. These observations collectively associate with greater overall inflammation and epithelial cell death, particularly in early postnatal (2 mo), distal airways.

Short versus long silver nanowires: a comparison of in vivo pulmonary effects post instillation.

BACKGROUND: Silver nanowires (Ag NWs) are increasingly being used to produce touchscreens for smart phones and computers. When applied in a thin film over a plastic substrate, Ag NWs create a transparent, highly-conductive network of fibers enabling the touch interface between consumers and their electronics. Large-scale application methods utilize techniques whereby Ag NW suspensions are deposited onto substrates via droplets. Aerosolized droplets increase risk of occupational Ag NW exposure. Currently, there are few published studies on Ag NW exposure-related health effects. Concerns have risen about the potential for greater toxicity from exposure to high-aspect ratio nanomaterials compared to their non-fibrous counterparts. This study examines whether Ag NWs of varying lengths affect biological responses and silver distribution within the lungs at different time-points. METHODS: Two different sizes of Ag NWs (2 µm [S-Ag NWs] and 20 µm [L-Ag NWs]) were tested. Male, Sprague-Dawley rats were intratracheally instilled with Ag NWs (0, 0.1, 0.5, or 1.0 mg/kg). Broncho-alveolar lavage fluid (BALF) and lung tissues were obtained at 1, 7, and 21 days post exposure for analysis of BAL total cells, cell differentials, and total protein as well as tissue pathology and silver distribution. RESULTS AND CONCLUSIONS: The two highest doses produced significant increases in BAL endpoints. At Day 1, Ag NWs increased total cells, inflammatory polymorphonuclear cells (PMNs), and total protein. PMNs persisted for both Ag NW types at Day 7, though not significantly so, and by Day 21, PMNs appeared in line with sham control values. Striking histopathological features associated with Ag NWs included 1) a strong influx of eosinophils at Days 1 and 7; and 2) formation of Langhans and foreign body giant cells at Days 7 and 21. Epithelial sloughing in the terminal bronchioles (TB) and cellular exudate in alveolar regions were also common. By Day 21, Ag NWs were primarily enclosed in granulomas or surrounded by numerous macrophages in the TB-alveolar duct junction. These findings suggest short and long Ag NWs produce pulmonary toxicity; thus, further research into exposure-related health effects and possible exposure scenarios are necessary to ensure human safety as Ag NW demand increases.

Resolving multi-image spatial lipidomic responses to inhaled toxicants by machine learning.

Regional responses to inhaled toxicants are essential to understand the pathogenesis of lung disease under exposure to air pollution. We evaluated the effect of combined allergen sensitization and ozone exposure on eliciting spatial differences in lipid distribution in the mouse lung that may contribute to ozone-induced exacerbations in asthma. Lung lobes from male and female BALB/c mice were cryosectioned and acquired by high resolution mass spectrometry imaging (MSI). Processed MSI peak annotations were validated by LC-MS/MS data from scraped tissue slides and microdissected lung tissue. Images were normalized and segmented into clusters. Interestingly, segmented clusters overlapped with stained serial tissue sections, enabling statistical analysis across biological replicates for morphologically relevant lung regions. Spatially distinct lipids had higher overall degree of unsaturated fatty acids in distal lung regions compared to proximal regions. Furthermore, the airway and alveolar epithelium exhibited significantly decreased sphingolipid and glycerophospholipid abundance in females, but not in males. We demonstrate the potential role of lipid saturation in healthy lung function and highlight sex differences in regional lung lipid distribution following ozone exposure. Our study provides a framework for future MSI experiments capable of relative quantification across biological replicates and expansion to multiple sample types, including human tissue.

Impact of aging and ergothioneine pre-treatment on naphthalene toxicity in lung.

Aging increases susceptibility to lung disease, but the topic is understudied, especially in relation to environmental exposures with the bulk of rodent studies using young adults. This study aims to define the pulmonary toxicity of naphthalene (NA) and the impacts of a dietary antioxidant, ergothioneine (ET), in the liver and lungs of middle-aged mice. NA causes a well-characterized pattern of conducting airway epithelial injury in the lung in young adult mice, but NA's toxicity has not been characterized in middle-aged mice, aged 1-1.5 years. ET is a dietary antioxidant that is synthesized by bacteria and fungi. The ET transporter (ETT), SLC22A4, is upregulated in tissues that experience high levels of oxidative stress. In this study, middle-aged male and female C57BL/6 J mice, maintained on an ET-free synthetic diet from conception, were gavaged with 70 mg/kg of ET for five consecutive days. On day 8, the mice were exposed to a single intraperitoneal NA dose of 50, 100, 150, or 200 mg/kg. At 24 hours post NA injection samples were collected and analyzed for ET concentration and reduced (GSH) and oxidized glutathione (GSSG) concentrations. Histopathology, morphometry, and gene expression were examined. Histopathology of mice exposed to 100 mg/kg of NA suggests reduction in toxicity in the terminal airways of both male (p ≤ 0.001) and female (p ≤ 0.05) middle-aged mice by the ET pretreatment. Our findings in this study are the first to document the toxicity of NA in middle-aged mice and show some efficacy of ET in reducing NA toxicity.

Age-specific effects on rat lung glutathione and antioxidant enzymes after inhaling ultrafine soot.

Vehicle exhaust is rich in polycyclic aromatic hydrocarbons (PAHs) and is a dominant contributor to urban particulate pollution (PM). Exposure to PM is linked to respiratory and cardiovascular morbidity and mortality in susceptible populations, such as children. PM can contribute to the development and exacerbation of asthma, and this is thought to occur because of the presence of electrophiles in PM or through electrophile generation via the metabolism of PAHs. Glutathione (GSH), an abundant intracellular antioxidant, confers cytoprotection through conjugation of electrophiles and reduction of reactive oxygen species. GSH-dependent phase II detoxifying enzymes glutathione peroxidase and glutathione S-transferase facilitate metabolism and conjugation, respectively. Ambient particulates are highly variable in composition, which complicates systematic study. In response, we have developed a replicable ultrafine premixed flame particle (PFP)-generating system for in vivo studies. To determine particle effects in the developing lung, 7-day-old neonatal and adult rats inhaled 22 µg/m(3) PFP during a single 6-hour exposure. Pulmonary GSH and related phase II detoxifying gene and protein expression were evaluated 2, 24, and 48 hours after exposure. Neonates exhibited significant depletion of GSH despite higher initial baseline levels of GSH. Furthermore, we observed attenuated induction of phase II enzymes (glutamate cysteine ligase, glutathione reductase, glutathione S-transferase, and glutathione peroxidase) in neonates compared with adult rats. We conclude that developing neonates have a limited ability to deviate from their normal developmental pattern that precludes adequate adaptation to environmental pollutants, which results in enhanced cytotoxicity from inhaled PM.

Matrix metalloproteinase-7 coordinates airway epithelial injury response and differentiation of ciliated cells.

Matrix metalloproteinase-7 (MMP7) expression is quickly up-regulated after injury, and functions to regulate wound repair and various mucosal immune processes. We evaluated the global transcriptional response of airway epithelial cells from wild-type and Mmp7-null mice cultured at an air-liquid interface. The analysis of differentially expressed genes between genotypes after injury revealed an enrichment of functional categories associated with inflammation, cilia, and differentiation. Because these analyses suggested that MMP7 regulated ciliated cell formation, we evaluated the recovery of the airway epithelium in wild-type and Mmp7-null mice in vivo after naphthalene injury, which revealed augmented ciliated cell formation in the absence of MMP7. Moreover, in vitro studies evaluating cell differentiation in air-liquid interface cultures also showed faster ciliated cell production under Mmp7-null conditions compared with wild-type conditions. These studies identified a new role for MMP7 in attenuating ciliated cell differentiation during wound repair.