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.

Early-life ozone exposure modulates region-specific gene expression in the developing rat lung.

Early-life ozone exposure disrupts normal patterns of lung development, but the molecular determinants underlying these changes are not well understood. This study aimed to elucidate changes in gene expression following episodic ozone exposure to identify potential mechanisms of ozone-mediated impairments in lung development. Rat pups were exposed to either filtered air or ozone (0.5 ppm, 6 hr./day, 5 days/week) from postnatal day (PND) 7-28 (16 dams total with 8 pups each, 4 M & 4 F) and sacrificed at either PND 30-31 or PND 80-84. Lung microdissection isolated major regions for RNA-Seq analysis. Ozone modified inherent differences in gene expression between lung regions in both male and female rat pups, whereas statistically significant changes in gene expression directly attributed to ozone were only identified in females. The greatest number of differentially expressed genes was observed between the distal airways and the parenchyma of ozone-exposed juvenile female rats, with 355 genes being differentially expressed. Genes modulating epithelial-to-mesenchymal transition, cell growth, and adhesion were differentially expressed in the parenchyma of ozone exposed juvenile females, suggesting that episodic ozone exposure may affect branching morphogenesis and lung cell growth. Importantly, our study provides novel targets for future experiments investigating the impact of ozone on lung development.

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.

Alteration of glycosphingolipid metabolism by ozone is associated with exacerbation of allergic asthma characteristics in mice.

Asthma is a common chronic respiratory disease exacerbated by multiple environmental factors. Acute ozone exposure has previously been implicated in airway inflammation, airway hyperreactivity, and other characteristics of asthma, which may be attributable to altered sphingolipid metabolism. This study tested the hypothesis that acute ozone exposure alters sphingolipid metabolism within the lung, which contributes to exacerbations in characteristics of asthma in allergen-sensitized mice. Adult male and female BALB/c mice were sensitized intranasally to house dust mite (HDM) allergen on days 1, 3, and 5 and challenged on days 12-14. Mice were exposed to ozone following each HDM challenge for 6 h/day. Bronchoalveolar lavage, lung lobes, and microdissected lung airways were collected for metabolomics analysis (N = 8/sex/group). Another subset of mice underwent methacholine challenge using a forced oscillation technique to measure airway resistance (N = 6/sex/group). Combined HDM and ozone exposure in male mice synergistically increased airway hyperreactivity that was not observed in females and was accompanied by increased airway inflammation and eosinophilia relative to control mice. Importantly, glycosphingolipids were significantly increased following combined HDM and ozone exposure relative to controls in both male and female airways, which was also associated with both airway resistance and eosinophilia. However, 15 glycosphingolipid species were increased in females compared with only 6 in males, which was concomitant with significant associations between glycosphingolipids and airway resistance that ranged from R2 = 0.33-0.51 for females and R2 = 0.20-0.34 in male mice. These observed sex differences demonstrate that glycosphingolipids potentially serve to mitigate exacerbations in characteristics of allergic asthma.

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.

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.

Metallic Engineered Nanomaterials and Ocular Toxicity: A Current Perspective.

The ocular surface, comprised of the transparent cornea, conjunctiva, and protective tear film, forms a protective barrier defending deeper structures of the eye from particulate matter and mechanical trauma. This barrier is routinely exposed to a multitude of naturally occurring and engineered nanomaterials (ENM). Metallic ENMs are particularly ubiquitous in commercial products with a high risk of ocular exposure, such as cosmetics and sunscreens. Additionally, there are several therapeutic uses for metallic ENMs owing to their attractive magnetic, antimicrobial, and functionalization properties. The increasing commercial and therapeutic applications of metallic ENMs come with a high risk of ocular exposure with poorly understood consequences to the health of the eye. While the toxicity of metallic ENMs exposure has been rigorously studied in other tissues and organs, further studies are necessary to understand the potential for adverse effects and inform product usage for individuals whose ocular health may be compromised by injury, disease, or surgical intervention. This review provides an update of current literature on the ocular toxicity of metallic ENMs in vitro and in vivo, as well as the risks and benefits of therapeutic applications of metallic ENMs in ophthalmology.

Cytotoxicity of 2D engineered nanomaterials in pulmonary and corneal epithelium.

Two-dimensional (2D) engineered nanomaterials are widely used in consumer and industrial goods due to their unique chemical and physical characteristics. Engineered nanomaterials are incredibly small and capable of being aerosolized during manufacturing, with the potential for biological interaction at first-contact sites such as the eye and lung. The unique properties of 2D nanomaterials that make them of interest to many industries may also cause toxicity towards epithelial cells. Using murine and human respiratory epithelial cell culture models, we tested the cytotoxicity of eight 2D engineered nanomaterials: graphene (110 nm), graphene oxide (2 um), graphene oxide (400 nm), reduced graphene oxide (2 um), reduced graphene oxide (400 nm), partially reduced graphene oxide (400 nm), molybdenum disulfide (400 nm), and hexagonal boron nitride (150 nm). Non-graphene nanomaterials were also tested in human corneal epithelial cells for ocular epithelial cytotoxicity. Hexagonal boron nitride was found to be cytotoxic in mouse tracheal, human alveolar, and human corneal epithelial cells. Hexagonal boron nitride was also tested for inhibition of wound healing in alveolar epithelial cells; no inhibition was seen at sub-cytotoxic doses. Nanomaterials should be considered with care before use, due to specific regional cytotoxicity that also varies by cell type. Supported by U01ES027288 and T32HL007013 and T32ES007059.

Role of Lung P450 Oxidoreductase in Paraquat-Induced Collagen Deposition in the Lung.

Paraquat (PQ) is an agrochemical known to cause pulmonary fibrosis. PQ-induced collagen deposition in the lung is thought to require enzymatic formation of PQ radicals, but the specific enzymes responsible for this bioactivation event in vivo have not been identified. We tested the hypothesis that lung P450 oxidoreductase (POR or CPR) is important in PQ-induced lung fibrosis in mice. A lung-Cpr-null mouse model was utilized, which undergoes doxycycline-induced, Cre recombinase-mediated deletion of the Por gene specifically in airway Club cells and alveolar type 2 cells in the lung. The lungs of lung-Cpr-null mice and their wild-type littermates were collected on day 15 after a single intraperitoneal injection of saline (control) or PQ (20 mg/kg). Lung tissue sections were stained with picrosirius red for detection of collagen fibrils. Fibrotic lung areas were found to be significantly smaller (1.6-fold for males and 1.4-fold for females) in PQ-treated lung-Cpr-null mice than in sex- and treatment-matched wild-type mice. The levels of collagen in lung tissue homogenate were also lower (1.4-2.3-fold; p < 0.05) in PQ-treated lung-Cpr-null mice compared to PQ-treated wild-type mice. In contrast, plasma PQ toxicokinetic profiles were not different between sex-matched wild-type and lung-Cpr-null mice. Taken together, these results indicate that lung POR plays an important role in PQ-induced pulmonary fibrosis.

Metal Oxide Engineered Nanomaterials Modulate Rabbit Corneal Fibroblast to Myofibroblast Transformation.

Purpose: Corneal keratocyte-fibroblast-myofibroblast (KFM) transformation plays a critical role in corneal stromal wound healing. However, the impact of engineered nanomaterials (ENMs), found in an increasing number of commercial products, on this process is poorly studied. This study investigates the effects of metal oxide ENMs on KFM transformation in vitro and in vivo. Methods: Cell viability of rabbit corneal fibroblasts (RCFs) was tested following treatment with 11 metal oxide ENMs at concentrations of 0.5 to 250 µg/ml for 24 hours. Messenger RNA (mRNA) and protein expression of αSMA, a marker of myofibroblast transformation, were measured using RCFs after exposure to 11 metal oxide ENMs at a concentration that did not affect cell viability, in media containing either 0 or 10 ng/ml of TGF-ß1. Additionally, the effect of topical Fe2O3 nanoparticles (NPs) (50 ng/ml) on corneal stromal wound healing following phototherapeutic keratectomy (PTK) was determined. Results: V2O5, Fe2O3, CuO, and ZnO ENMs were found to significantly reduce cell viability as compared to vehicle control and the other seven metal oxide ENMs tested. V2O5 nanoflakes significantly reduced mRNA and protein αSMA concentrations in the presence of TGF-ß1. Fe2O3 NPs significantly increased αSMA mRNA expression in the presence of TGF-ß1 but did not alter αSMA protein expression. Topically applied Fe2O3 NPs in an in vivo rabbit corneal stromal wound healing model did not delay healing. Conclusions: Fe2O3 NPs promote corneal myofibroblast induction in vitro but do not impair corneal stromal wound healing in vivo. Translational Relevance: These experimental results can apply to human nanomedical research.

Metabolomics of Lung Microdissections Reveals Region- and Sex-Specific Metabolic Effects of Acute Naphthalene Exposure in Mice.

Naphthalene is a ubiquitous environmental contaminant produced by combustion of fossil fuels and is a primary constituent of both mainstream and side stream tobacco smoke. Naphthalene elicits region-specific toxicity in airway club cells through cytochrome P450 (P450)-mediated bioactivation, resulting in depletion of glutathione and subsequent cytotoxicity. Although effects of naphthalene in mice have been extensively studied, few experiments have characterized global metabolomic changes in the lung. In individual lung regions, we found metabolomic changes in microdissected mouse lung conducting airways and parenchyma obtained from animals sacrificed at 3 timepoints following naphthalene treatment. Data on 577 unique identified metabolites were acquired by accurate mass spectrometry-based assays focusing on lipidomics and nontargeted metabolomics of hydrophilic compounds. Statistical analyses revealed distinct metabolite profiles between the 2 lung regions. Additionally, the number and magnitude of statistically significant exposure-induced changes in metabolite abundance were different between airways and parenchyma for unsaturated lysophosphatidylcholines, dipeptides, purines, pyrimidines, and amino acids. Importantly, temporal changes were found to be highly distinct for male and female mice with males exhibiting predominant treatment-specific changes only at 2 h postexposure. In females, metabolomic changes persisted until 6 h postnaphthalene treatment, which may explain the previously characterized higher susceptibility of female mice to naphthalene toxicity. In both males and females, treatment-specific changes corresponding to lung remodeling, oxidative stress response, and DNA damage were observed. Overall, this study provides insights into potential mechanisms contributing to naphthalene toxicity and presents a novel approach for lung metabolomic analysis that distinguishes responses of major lung regions.

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.

The aryl hydrocarbon receptor as a target of environmental stressors - Implications for pollution mediated stress and inflammatory responses.

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor regulating the expression of genes, for instance encoding the monooxygenases cytochrome P450 (CYP) 1A1 and CYP1A2, which are important enzymes in metabolism of xenobiotics. The AHR is activated upon binding of polycyclic aromatic hydrocarbons (PAHs), persistent organic pollutants (POPs), and related ubiquitous environmental chemicals, to mediate their biological and toxic effects. In addition, several endogenous and natural compounds can bind to AHR, thereby modulating a variety of physiological processes. In recent years, ambient particulate matter (PM) associated with traffic related air pollution (TRAP) has been found to contain significant amounts of PAHs. PM containing PAHs are of increasing concern as a class of agonists, which can activate the AHR. Several reports show that PM and AHR-mediated induction of CYP1A1 results in excessive generation of reactive oxygen species (ROS), causing oxidative stress. Furthermore, exposure to PM and PAHs induce inflammatory responses and may lead to chronic inflammatory diseases, including asthma, cardiovascular diseases, and increased cancer risk. In this review, we summarize findings showing the critical role that the AHR plays in mediating effects of environmental pollutants and stressors, which pose a risk of impacting the environment and human health.

Outdoor Air Pollution and New-Onset Airway Disease. An Official American Thoracic Society Workshop Report.

Although it is well accepted that air pollution exposure exacerbates preexisting airway disease, it has not been firmly established that long-term pollution exposure increases the risk of new-onset asthma or chronic obstruction pulmonary disease (COPD). This Workshop brought together experts on mechanistic, epidemiological, and clinical aspects of airway disease to review current knowledge regarding whether air pollution is a causal factor in the development of asthma and/or COPD. Speakers presented recent evidence in their respective areas of expertise related to air pollution and new airway disease incidence, followed by interactive discussions. A writing committee summarized their collective findings. The Epidemiology Group found that long-term exposure to air pollution, especially metrics of traffic-related air pollution such as nitrogen dioxide and black carbon, is associated with onset of childhood asthma. However, the evidence for a causal role in adult-onset asthma or COPD remains insufficient. The Mechanistic Group concluded that air pollution exposure can cause airway remodeling, which can lead to asthma or COPD, as well as asthma-like phenotypes that worsen with long-term exposure to air pollution, especially fine particulate matter and ozone. The Clinical Group concluded that air pollution is a plausible contributor to the onset of both asthma and COPD. Available evidence indicates that long-term exposure to air pollution is a cause of childhood asthma, but the evidence for a similar determination for adult asthma or COPD remains insufficient. Further research is needed to elucidate the exact biological mechanism underlying incident childhood asthma, and the specific air pollutant that causes it.

Age-Dependent Translocation of Gold Nanoparticles across the Air-Blood Barrier.

Do immature lungs have air-blood barriers that are more permeable to inhaled nanoparticles than those of fully developed mature lungs? Data supporting this notion and explaining the underlying mechanisms do not exist as far as we know. Using a rat model of postnatal lung development, here the data exactly supporting this notion, that is, significantly more gold nanoparticles (NPs) cross from the air space of the lungs to the rest of the body in neonates than in adults, are presented. Moreover, in neonates the translocation of gold NPs is not size dependent, whereas in adult animals smaller NPs cross the air-blood lung barrier much more efficiently than larger NPs. This difference in air-blood permeability in neonate versus adult animals suggests that NP translocation in the immature lungs may follow different rules than in mature lungs. Supporting this notion, we propose that the paracellular transport route may play a more significant role in NP translocation in immature animals, as suggested by protein expression studies. Findings from this study are critical to design optimal ways of inhalation drug delivery using NP nanocarriers for this age group, as well as for better understanding of the potential adverse health effects of nanoparticle exposures in infants and young children.

Metabolism and Lung Toxicity of Inhaled Naphthalene: Effects of Postnatal Age and Sex.

Human exposure to naphthalene (NA), an acute lung toxicant and possible human carcinogen, is primarily through inhalation. Acute lung toxicity and carcinogenesis are thought to be related because the target sites for both are similar. To understand susceptibility of the developing lung to cytotoxicity of inhaled NA, we exposed neonatal (7 days), juvenile (3 weeks), and adult mice to 5 or 10 ppm NA vapor for 4 h. We measured vacuolated airway epithelium morphometrically, quantified NA and NA-glutathione levels in plasma and lung, and quantified gene expression in microdissected airways. NA inhalation caused airway epithelial cytotoxicity at all ages, in both sexes. Contrary to a previous study that showed the greatest airway epithelial cytotoxicity in neonatal mice following intraperitoneal NA injection, we observed the most extensive airway epithelial toxicity in older, juvenile, animals exposed to NA by inhalation. Juvenile female animals were the most susceptible. Furthermore, NA inhalation in juvenile animals resulted in damage to conducting airway Club cells that was greater in proximal versus distal airways. We also found NA tissue burden and metabolism differed by age. Gene expression pathway analysis was consistent with the premise that female juvenile mice are more predisposed to damage; DNA damage and cancer pathways were upregulated. Our data demonstrate special susceptibility of young, juvenile mice to NA inhalation-induced cytotoxicity, highlight the importance of route of exposure and airway location in toxicity of chemicals in the developing lung, and provide metabolic and molecular insights for further identification of mechanisms underlying age and sex differences in NA toxicity.

Naphthalene genotoxicity: DNA adducts in primate and mouse airway explants.

Naphthalene (NA) is a ubiquitous environmental pollutant and possible human carcinogen that forms tumors in rodents with tissue/regional and species selectivity. This study seeks to determine whether NA is able to directly adduct DNA in an ex vivo culture system. Metabolically active lung tissue was isolated and incubated in explant culture with carbon-14 labeled NA (0, 25, 250 µM) or 1,2-naphthoquinone (NQ), followed by AMS analyses of metabolite binding to DNA. Despite relatively low metabolic bioactivation in the primate airway, dose-dependent NA-DNA adduct formation was detected. More airway adducts were detected in female mice (4.7-fold) and primates (2.1-fold) than in males of the same species. Few adducts were detected in rat airway or nasal epithelium. NQ, which is a metabolic product of NA, proved to be even more potent, with levels of adduct formation 70-80-fold higher than seen when tissues were incubated with the parent compound NA. This is the first study to demonstrate NA-DNA adduct formation at a site of carcinogenesis, the mouse lung. Adducts were also detected in non-human primate lung and with a NQ metabolite of NA. Taken together, this suggests that NA may contribute to in vivo carcinogenesis through a genotoxic mechanism.

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.

Prenatal Bisphenol A Exposure Alters Epithelial Cell Composition in the Rhesus Macaque Fetal Oviduct.

Bisphenol A (BPA) is an endocrine disrupting compound that is a pervasive environmental contaminant. Although it has been reported to affect the development of a variety of fetal reproductive tissues, data on the effect of fetal BPA exposure on oviducts were extremely limited and were only available in mice. To determine if there are adverse effects of gestational BPA exposure on fetal oviduct, we exposed pregnant rhesus macaques with female fetuses to oral or nonoral BPA during the last trimester of gestation (day 100 to term). After the treatment, fetal oviducts were collected for morphology evaluation. BPA exposure altered the percentages of different cell types (ciliated, nonciliated, and secretory) in the fetal oviduct and resulted in a significant high ciliated cell population in the BPA-exposed fetal oviduct. The distribution of ciliated cells on the epithelium in the BPA-exposed fetal oviduct was also altered. Gestational BPA exposure reduced the expression of mucosubstance and uteroglobin in secretory cells in the fetal oviduct. A comparison of the outcome of the fetal oviduct studies with similar outcomes previously reported in the lung from the same fetuses demonstrates that BPA exhibits opposite effects in these two organs. In conclusion, the BPA-associated alterations in the fetal oviduct could potentially affect the oviduct morphology and function later in life with a negative impact on fertility. The mechanisms of action of the differential response in the oviduct and the lung to BPA exposure require further investigation.