Refinement of the acute inhalation limit test for inert, nano-sized dusts by an in silico dosimetry-based evaluation: case study for the dissolution of a regulatory dilemma.

This case study aims to describe the dilemma faced when exposing rats to very high concentrations of fine, pulverulent materials for acute inhalation studies and to address the regulatory question of whether the effects seen here are relevant to humans and the subject of classification according to the Globally Harmonized System of Classification and Labeling of Chemicals (GHS). Many powders match the definition of nanomaterials in the EU; therefore, information on acute inhalation testing of powders up to the GHS cutoff of 5 mg/L is required. However, testing rats at such a high aerosol concentration can cause physical obstruction of the airways and even mortality by suffocation. Therefore, to evaluate whether the physical effects on airway obstruction in rats exposed to 5 mg/L for 4 hours and alternative exposures to 1 and 2 mg/L are relevant for humans, an in silico evaluation of aerosol deposition was conducted using the multiple-path particle dosimetry (MPPD) model. For this evaluation, actual exposure conditions for an organic, nano-sized pigment which produced 100% lethality in rats at 5 mg/L, but not at 1 mg/L, were used to assess the potential for airway obstruction in rats and accordingly in humans. As an indicator of the potential for airway obstruction, the ratio of the diameter of the deposited, aggregated aerosol to airway diameter was calculated for each exposure condition. For rats exposed to 5 mg/L for 4 h, approximately 75% of tracheobronchial and 22% of pulmonary/alveolar airways were considered vulnerable to significant or complete obstruction (ratios >0.5). In humans, an equivalent exposure resulted in just over 96% of human tracheobronchial airways that received deposited mass to airway diameter ratios between 0.3 and 0.4 (nasal) or 0.4 and 0.5 (oral), with no airways with ratios >0.5. For the pulmonary/alveolar region, ∼88% of the airways following nasal or oral breathing were predicted to have deposited aerosol diameter to airway diameter ratios <0.1, with no airways with ratios >0.5. Thus, the in silico results obtained for rats are in line with the pathological findings of the animal test. The predicted results in humans, however, affirm the hypothesis of a rat-specific high dose effect which does not justify a classification according to GHS.

In Silico Quantification of Intersubject Variability on Aerosol Deposition in the Oral Airway.

The extrathoracic oral airway is not only a major mechanical barrier for pharmaceutical aerosols to reach the lung but also a major source of variability in lung deposition. Using computational fluid dynamics, deposition of 1−30 µm particles was predicted in 11 CT-based models of the oral airways of adults. Simulations were performed for mouth breathing during both inspiration and expiration at two steady-state flow rates representative of resting/nebulizer use (18 L/min) and of dry powder inhaler (DPI) use (45 L/min). Consistent with previous in vitro studies, there was a large intersubject variability in oral deposition. For an optimal size distribution of 1−5 µm for pharmaceutical aerosols, our data suggest that >75% of the inhaled aerosol is delivered to the intrathoracic lungs in most subjects when using a nebulizer but only in about half the subjects when using a DPI. There was no significant difference in oral deposition efficiency between inspiration and expiration, unlike subregional deposition, which shows significantly different patterns between the two breathing phases. These results highlight the need for incorporating a morphological variation of the upper airway in predictive models of aerosol deposition for accurate predictions of particle dosimetry in the intrathoracic region of the lung.

Advancing New Approach Methodologies (NAMs) for Tobacco Harm Reduction: Synopsis from the 2021 CORESTA SSPT-NAMs Symposium.

New approach methodologies (NAMs) are emerging chemical safety assessment tools consisting of in vitro and in silico (computational) methodologies intended to reduce, refine, or replace (3R) various in vivo animal testing methods traditionally used for risk assessment. Significant progress has been made toward the adoption of NAMs for human health and environmental toxicity assessment. However, additional efforts are needed to expand their development and their use in regulatory decision making. A virtual symposium was held during the 2021 Cooperation Centre for Scientific Research Relative to Tobacco (CORESTA) Smoke Science and Product Technology (SSPT) conference (titled "Advancing New Alternative Methods for Tobacco Harm Reduction"), with the goals of introducing the concepts and potential application of NAMs in the evaluation of potentially reduced-risk (PRR) tobacco products. At the symposium, experts from regulatory agencies, research organizations, and NGOs shared insights on the status of available tools, strengths, limitations, and opportunities in the application of NAMs using case examples from safety assessments of chemicals and tobacco products. Following seven presentations providing background and application of NAMs, a discussion was held where the presenters and audience discussed the outlook for extending the NAMs toxicological applications for tobacco products. The symposium, endorsed by the CORESTA In Vitro Tox Subgroup, Biomarker Subgroup, and NextG Tox Task Force, illustrated common ground and interest in science-based engagement across the scientific community and stakeholders in support of tobacco regulatory science. Highlights of the symposium are summarized in this paper.

Application of a new approach method (NAM) for inhalation risk assessment.

The US Environmental Protection Agency (USEPA) and other regulatory authorities have been working to utilize in vitro studies with human cells and in silico modelling to reduce the use of vertebrate animals for evaluating chemical risk. Using the Source-to-Outcome framework, a novel mathematical procedure was developed to estimate the human equivalent concentration (HEC) for inhalation risk assessment based upon the relevant aerosol characterization, respiratory dosimetry modelling, and endpoints derived from an in vitro assay using human respiratory epithelial tissue. The procedure used the retained doses at the various areas of the inhalation tract estimated from a computational fluid-particle dynamics (CFPD) model coupled with a simple clearance model. The effect of exposure was derived from an in vitro assay. The magnitude of exposure and the particle size distributions (PSDs) of the external aerosol droplets were obtained from Unit Exposure values published by the USEPA and published monitoring studies, respectively. The Source-to-Outcome approach incorporates external and internal exposure metrics with the toxicity pathway. The information was then integrated to conduct a risk assessment for agricultural operators exposed to products containing chlorothalonil (CTN), a broad-spectrum fungicide. The HECs for three different PSDs considered in this work ranged from 0.043 to 0.112 mg-CTN/L for nasal and oral breathing. These were compared with the estimated average daily exposure concentration for six representative application scenarios. The resulting margins of exposure (MOEs) ranged from 230 to 70,000 depending on the application scenario. This New Assessment Method (NAM) that combined human in silico and human in vitro methods, eliminated the typical uncertainties associated with extrapolation from rodent studies, with their associated interspecies toxicokinetics and toxicodynamics differences. The intraspecies toxicodynamics and toxicokinetics, are still relevant and may need to be used in an inhalation risk assessment. The NAM presented in this work is not chemical-specific and may be applied to conduct an inhalation risk assessment for workers as well as bystanders who could be exposed to aerosol particles of any cytotoxic respiratory irritant.

Translating dosimetry of Dibenzo[def,p]chrysene (DBC) and metabolites across dose and species using physiologically based pharmacokinetic (PBPK) modeling.

Dibenzo[def,p]chrysene (DBC) is an environmental polycyclic aromatic hydrocarbon (PAH) that causes tumors in mice and has been classified as a probable human carcinogen by the International Agency for Research on Cancer. Animal toxicity studies often utilize higher doses than are found in relevant human exposures. Additionally, like many PAHs, DBC requires metabolic bioactivation to form the ultimate toxicant, and species differences in DBC and DBC metabolite metabolism have been observed. To understand the implications of dose and species differences, a physiologically based pharmacokinetic model (PBPK) for DBC and major metabolites was developed in mice and humans. Metabolism parameters used in the model were obtained from experimental in vitro metabolism assays using mice and human hepatic microsomes. PBPK model simulations were evaluated against mice dosed with 15 mg/kg DBC by oral gavage and human volunteers orally microdosed with 29 ng of DBC. DBC and its primary metabolite DBC-11,12-diol were measured in blood of mice and humans, while in urine, the majority of DBC metabolites were obeserved as conjugated DBC-11,12-diol, conjugated DBC tetrols, and unconjugated DBC tetrols. The PBPK model was able to predict the time course concentrations of DBC, DBC-11,12-diol, and other DBC metabolites in blood and urine of human volunteers and mice with reasonable accuracy. Agreement between model simulations and measured pharmacokinetic data in mice and human studies demonstrate the success and versatility of our model for interspecies extrapolation and applicability for different doses. Furthermore, our simulations show that internal dose metrics used for risk assessment do not necessarily scale allometrically, and that PBPK modeling provides a reliable approach to appropriately account for interspecies differences in metabolism and physiology.

Exposure to an Environmental Mixture of Polycyclic Aromatic Hydrocarbons Induces Hepatic Cytochrome P450 Enzymes in Mice.

Cytochrome P450 enzymes (CYPs) play an important role in bioactivating or detoxifying polycyclic aromatic hydrocarbons (PAHs), common environmental contaminants. While it is widely accepted that exposure to PAHs induces CYPs, effectively increasing rates of xenobiotic metabolism, dose- and time-response patterns of CYP induction are not well-known. In order to better understand dose- and time-response relationships of individual CYPs following induction, we exposed B6129SF1/J mice to single or repeated doses (2-180 µmol/kg/d) of benzo[a]pyrene (BaP) or Supermix-10, a mixture of the top 10 most abundant PAHs found at the Portland Harbor Superfund Site. In hepatic microsomes from exposed mice, we measured amounts of active CYPs using activity-based protein profiling and total CYP expression using global proteomics. We observed rapid Cyp1a1 induction after 6 h at the lowest PAH exposures and broad induction of many CYPs after 3 daily PAH doses at 72 h following the first dose. Using samples displaying Cyp1a1 induction, we observed significantly higher metabolic affinity for BaP metabolism (Km reduced 3-fold), 3-fold higher intrinsic clearance, but no changes to the Vmax. Mice dosed with the highest PAH exposures exhibited 1.7-5-fold higher intrinsic clearance rates for BaP compared to controls and higher Vmax values indicating greater amounts of enzymes capable of metabolizing BaP. This study demonstrates exposure to PAHs found at superfund sites induces enzymes in dose- and time-dependent patterns in mice. Accounting for specific changes in enzyme profiles, relative rates of PAH bioactivation and detoxification, and resulting risk will help translate internal dosimetry of animal models to humans and improve risk assessments of PAHs at superfund sites.

New Approach Methodology for Assessing Inhalation Risks of a Contact Respiratory Cytotoxicant: Computational Fluid Dynamics-Based Aerosol Dosimetry Modeling for Cross-Species and In Vitro Comparisons.

Regulatory agencies are considering alternative approaches to assessing inhalation toxicity that utilizes in vitro studies with human cells and in silico modeling in lieu of additional animal studies. In support of this goal, computational fluid-particle dynamics models were developed to estimate site-specific deposition of inhaled aerosols containing the fungicide, chlorothalonil, in the rat and human for comparisons to prior rat inhalation studies and new human in vitro studies. Under bioassay conditions, the deposition was predicted to be greatest at the front of the rat nose followed by the anterior transitional epithelium and larynx corresponding to regions most sensitive to local contact irritation and cytotoxicity. For humans, simulations of aerosol deposition covering potential occupational or residential exposures (1-50 µm diameter) were conducted using nasal and oral breathing. Aerosols in the 1-5 µm range readily penetrated the deep region of the human lung following both oral and nasal breathing. Under actual use conditions (aerosol formulations >10 µm), the majority of deposited doses were in the upper conducting airways. Beyond the nose or mouth, the greatest deposition in the pharynx, larynx, trachea, and bronchi was predicted for aerosols in the 10-20 µm size range. Only small amounts of aerosols >20 µm penetrated past the pharyngeal region. Using the ICRP clearance model, local retained tissue dose metrics including maximal concentrations and areas under the curve were calculated for each airway region following repeated occupational exposures. These results are directly comparable with benchmark doses from in vitro toxicity studies in human cells leading to estimated human equivalent concentrations that reduce the reliance on animals for risk assessments.

Ventilation Modulation and Nanoparticle Deposition in Respiratory and Olfactory Regions of Rabbit Nose.

The rabbit nose's ability to filter out inhaled agents is directly related to its defense to infectious diseases. The knowledge of the rabbit nose anatomy is essential to appreciate its functions in ventilation regulation, aerosol filtration and olfaction. The objective of this study is to numerically simulate the inhalation and deposition of nanoparticles in a New Zealand white (NZW) rabbit nose model with an emphasis on the structure-function relation under normal and sniffing conditions. To simulate the sniffing scenario, the original nose model was modified to generate new models with enlarged nostrils or vestibules based on video images of a rabbit sniffing. Ventilations into the maxilloturbinate and olfactory region were quantified with varying nostril openings, and deposition rates of inhaled aerosols ranging from 0.5 nm to 1000 nm were characterized on the total, sub-regional and local basis. Results showed that particles which deposited in the olfactory region came from a specific area in the nostril. The spiral vestibule played an essential role in regulating flow resistance and flow partition into different parts of the nose. Increased olfactory doses were persistently predicted in models with expanded nostrils or vestibule. Particles in the range of 5-50 nm are more sensitive to the geometry variation than other nanoparticles. It was also observed that exhaled aerosols occupy only the central region of the nostril, which minimized the mixing with the aerosols close to the nostril wall, and potentially allowed the undisruptive sampling of odorants. The results of this study shed new light on the ventilation regulation and inhalation dosimetry in the rabbit nose, which can be further implemented to studies of infectious diseases and immunology in rabbits.

Lipid Coverage in Nanospray Desorption Electrospray Ionization Mass Spectrometry Imaging of Mouse Lung Tissues.

Lipids are a naturally occurring group of molecules that not only contribute to the structural integrity of the lung preventing alveolar collapse but also play important roles in the anti-inflammatory responses and antiviral protection. Alteration in the type and spatial localization of lipids in the lung plays a crucial role in various diseases, such as respiratory distress syndrome (RDS) in preterm infants and oxidative stress-influenced diseases, such as pneumonia, emphysema, and lung cancer following exposure to environmental stressors. The ability to accurately measure spatial distributions of lipids and metabolites in lung tissues provides important molecular insights related to lung function, development, and disease states. Nanospray desorption electrospray ionization (nano-DESI) and other ambient ionization mass spectrometry techniques enable label-free imaging of complex samples in their native state with minimal to absolutely no sample preparation. However, lipid coverage obtained in nano-DESI mass spectrometry imaging (MSI) experiments has not been previously characterized. In this work, the depth of lipid coverage in nano-DESI MSI of mouse lung tissues was compared to liquid chromatography tandem mass spectrometry (LC-MS/MS) lipidomics analysis of tissue extracts prepared using two different procedures: standard Folch extraction method of the whole lung samples and extraction into a 90% methanol/10% water mixture used in nano-DESI MSI experiments. A combination of positive and negative ionization mode nano-DESI MSI identified 265 unique lipids across 20 lipids subclasses and 19 metabolites (284 in total) in mouse lung tissues. Except for triacylglycerols (TG) species, nano-DESI MSI provided comparable coverage to LC-MS/MS experiments performed using methanol/water tissue extracts and up to 50% coverage in comparison with the Folch extraction-based whole lung lipidomics analysis. These results demonstrate the utility of nano-DESI MSI for comprehensive spatially resolved analysis of lipids in tissue sections. A combination of nano-DESI MSI and LC-MS/MS lipidomics is particularly useful for exploring changes in lipid distributions during lung development, as well as resulting from disease or exposure to environmental toxicants.

An integrated experimental-computational approach for predicting virulence in New Zealand white rabbits and humans following inhalation exposure to Bacillus anthracis spores.

Inhalation of Bacillus anthracis spores can lead to an anthrax infection that can be fatal. Previously published mathematical models have extrapolated kinetic rates associated with bacterial growth in New Zealand White (NZW) rabbits to humans, but to date, actual measurements of the underlying processes associated with anthrax virulence between species have not been conducted. To address this knowledge gap, we have quantified species-specific rate constants associated with germination, proliferation, and immune cell inactivation of B. anthracis Sterne using an in vitro test platform that includes primary lung epithelial and immune cells. The generated data was then used to develop a physiologically based biokinetic model (PBBK) which quantitatively compares bacterial growth and mean time to death under lethal conditions in rabbits and humans. Simulations based upon our in vitro data and previously published in vivo data from rabbits indicate that disease progression is likely to be faster in humans than in NZW rabbits under comparable total deposited dose conditions. With the computational framework established, PBBK parameters can now be refined using experimental data for lethal B. anthracis strains (e.g. Ames) under identical conditions in future studies. The PBBK model can also be linked to existing aerosol dosimetry models that account for species-specific differences in aerosol deposition patterns to further improve the human health risk assessment of inhalation anthrax.

Benzo[ a]pyrene Induction of Glutathione S-Transferases: An Activity-Based Protein Profiling Investigation.

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental contaminants generated from combustion of carbon-based matter. Upon ingestion, these molecules can be bioactivated by cytochrome P450 monooxygenases to oxidized toxic metabolites. Some of these metabolites are potent carcinogens that can form irreversible adducts with DNA and other biological macromolecules. Conjugative enzymes, such as glutathione S-transferases or UDP-glucuronosyltransferases, are responsible for the detoxification and/or facilitate the elimination of these carcinogens. While responses to PAH exposures have been extensively studied for the bioactivating cytochrome P450 enzymes, much less is known regarding the response of glutathione S-transferases in mammalian systems. In this study, we investigated the expression and activity responses of murine hepatic glutathione S-transferases to benzo[ a]pyrene exposure using global proteomics and activity-based protein profiling for chemoproteomics, respectively. Using this approach, we identified several enzymes exhibiting increased activity including GSTA2, M1, M2, M4, M6, and P1. The activity of one GST enzyme, GSTA4, was found to be downregulated with increasing B[ a]P dose. Activity responses of several of these enzymes were identified as being expression-independent when comparing global and activity-based data sets, possibly alluding to as of yet unknown regulatory post-translational mechanisms.

Spatial distribution of marker gene activity in the mouse lung during alveolarization.

This data is a curated collection of visual images of gene expression patterns from the pre- and post-natal mouse lung, accompanied by associated mRNA probe sequences and RNA-Seq expression profiles. Mammalian lungs undergo significant growth and cellular differentiation before and after the transition to breathing air. Documenting normal lung development is an important step in understanding abnormal lung development, as well as the challenges faced during a preterm birth. Images in this dataset indicate the spatial distribution of mRNA transcripts for over 500 different genes that are active during lung development, as initially determined via RNA-Seq. Images were systematically acquired using high-throughput in situ hybridization with non-radioactive digoxigenin-labeled mRNA probes across mouse lungs from developmental time points E16.5, E18.5, P7, and P28. The dataset was produced as part of The Molecular Atlas of Lung Development Program (LungMAP) and is hosted at https://lungmap.net. This manuscript describes the nature of the data and the protocols for generating the dataset.

Toxicokinetics of benzo[a]pyrene in humans: Extensive metabolism as determined by UPLC-accelerator mass spectrometry following oral micro-dosing.

Benzo[a]pyrene (BaP), is a known human carcinogen (International Agency for Research on Cancer (IARC) class 1). The remarkable sensitivity (zepto-attomole 14C in biological samples) of accelerator mass spectrometry (AMS) makes possible, with de minimus risk, pharmacokinetic (PK) analysis following [14C]-BaP micro-dosing of humans. A 46 ng (5 nCi) dose was given thrice to 5 volunteers with minimum 2 weeks between dosing and plasma collected over 72 h. [14C]-BaPeq PK analysis gave plasma Tmax and Cmax values of 1.25 h and 29-82 fg/mL, respectively. PK parameters were assessed by non- compartment and compartment models. Intervals between dosing ranged from 20 to 420 days and had little impact on intra-individual variation. DNA, extracted from peripheral blood mononuclear cells (PBMCs) of 4 volunteers, showed measurable levels (LOD ~ 0.5 adducts/1011 nucleotides) in two individuals 2-3 h post-dose, approximately three orders of magnitude lower than smokers or occupationally-exposed individuals. Little or no DNA binding was detectable at 48-72 h. In volunteers the allelic variants CYP1B1*1/*⁎1, *1/*3 or *3/*3 and GSTM1*0/0 or *1 had no impact on [14C]-BaPeq PK or DNA adduction with this very limited sample. Plasma metabolites over 72 h from two individuals (one CYP1B1*1/*1 and one CYP1B1*3/*3) were analyzed by UPLC-AMS. In both individuals, parent [14C]-BaP was a minor constituent even at the earliest time points and metabolite profiles markedly distinct. AMS, coupled with UPLC, could be used in humans to enhance the accuracy of pharmacokinetics, toxicokinetics and risk assessment of environmental carcinogens.

High-Fat Diets Alter the Modulatory Effects of Xenobiotics on Cytochrome P450 Activities.

Cytochrome P450 monooxygenase (P450) enzymes metabolize critical endogenous chemicals and oxidize nearly all xenobiotics. Dysregulated P450 activities lead to altered capacity for drug metabolism and cellular stress. The effects of mixed exposures on P450 expression and activity are variable and elusive. A high-fat diet (HFD) is a common exposure that results in obesity and associated pathologies including hepatotoxicity. Herein, we report the effects of cigarette smoke on P450 activities of normal weight and HFD induced obese mice. Activity-based protein profiling results indicate that HFD mice had significantly decreased P450 activity, likely instigated by proinflammatory chemicals, and that P450 enzymes involved in detoxification, xenobiotic metabolism, and bile acid synthesis were effected by HFD and smoke interaction. Smoking increased activity of all lung P450 and coexposure to diet effected P450 2s1. We need to expand our understanding of common exposures coupled to altered P450 metabolism to enhance the safety and efficacy of therapeutic drug dosing.

Extracellular matrix in lung development, homeostasis and disease.

The lung's unique extracellular matrix (ECM), while providing structural support for cells, is critical in the regulation of developmental organogenesis, homeostasis and injury-repair responses. The ECM, via biochemical or biomechanical cues, regulates diverse cell functions, fate and phenotype. The composition and function of lung ECM become markedly deranged in pathological tissue remodeling. ECM-based therapeutics and bioengineering approaches represent promising novel strategies for regeneration/repair of the lung and treatment of chronic lung diseases. In this review, we assess the current state of lung ECM biology, including fundamental advances in ECM composition, dynamics, topography, and biomechanics; the role of the ECM in normal and aberrant lung development, adult lung diseases and autoimmunity; and ECM in the regulation of the stem cell niche. We identify opportunities to advance the field of lung ECM biology and provide a set recommendations for research priorities to advance knowledge that would inform novel approaches to the pathogenesis, diagnosis, and treatment of chronic lung diseases.

Time-resolved proteome profiling of normal lung development.

Biochemical networks mediating normal lung morphogenesis and function have important implications for ameliorating morbidity and mortality in premature infants. Although several transcript-level studies have examined normal lung development, corresponding protein-level analyses are lacking. Here we performed proteomics analysis of murine lungs from embryonic to early adult ages to identify the molecular networks mediating normal lung development. We identified 8,932 proteins, providing a deep and comprehensive view of the lung proteome. Analysis of the proteomics data revealed discrete modules and the underlying regulatory and signaling network modulating their expression during development. Our data support the cell proliferation that characterizes early lung development and highlight responses of the lung to exposure to a nonsterile oxygen-rich ambient environment and the important role of lipid (surfactant) metabolism in lung development. Comparison of dynamic regulation of proteomic and recent transcriptomic analyses identified biological processes under posttranscriptional control. Our study provides a unique proteomic resource for understanding normal lung formation and function and can be freely accessed at Lungmap.net.

Towards High-Resolution Tissue Imaging Using Nanospray Desorption Electrospray Ionization Mass Spectrometry Coupled to Shear Force Microscopy.

Constant mode ambient mass spectrometry imaging (MSI) of tissue sections with high lateral resolution of better than 10 µm was performed by combining shear force microscopy with nanospray desorption electrospray ionization (nano-DESI). Shear force microscopy enabled precise control of the distance between the sample and nano-DESI probe during MSI experiments and provided information on sample topography. Proof-of-concept experiments were performed using lung and brain tissue sections representing spongy and dense tissues, respectively. Topography images obtained using shear force microscopy were comparable to the results obtained using contact profilometry over the same region of the tissue section. Variations in tissue height were found to be dependent on the tissue type and were in the range of 0-5 µm for lung tissue and 0-3 µm for brain tissue sections. Ion images of phospholipids obtained in this study are in good agreement with literature data. Normalization of nano-DESI MSI images to the signal of the internal standard added to the extraction solvent allowed us to construct high-resolution ion images free of matrix effects. Graphical Abstract ᅟ.

LungMAP: The Molecular Atlas of Lung Development Program.

The National Heart, Lung, and Blood Institute is funding an effort to create a molecular atlas of the developing lung (LungMAP) to serve as a research resource and public education tool. The lung is a complex organ with lengthy development time driven by interactive gene networks and dynamic cross talk among multiple cell types to control and coordinate lineage specification, cell proliferation, differentiation, migration, morphogenesis, and injury repair. A better understanding of the processes that regulate lung development, particularly alveologenesis, will have a significant impact on survival rates for premature infants born with incomplete lung development and will facilitate lung injury repair and regeneration in adults. A consortium of four research centers, a data coordinating center, and a human tissue repository provides high-quality molecular data of developing human and mouse lungs. LungMAP includes mouse and human data for cross correlation of developmental processes across species. LungMAP is generating foundational data and analysis, creating a web portal for presentation of results and public sharing of data sets, establishing a repository of young human lung tissues obtained through organ donor organizations, and developing a comprehensive lung ontology that incorporates the latest findings of the consortium. The LungMAP website (www.lungmap.net) currently contains more than 6,000 high-resolution lung images and transcriptomic, proteomic, and lipidomic human and mouse data and provides scientific information to stimulate interest in research careers for young audiences. This paper presents a brief description of research conducted by the consortium, database, and portal development and upcoming features that will enhance the LungMAP experience for a community of users.

In vitro exposure systems and dosimetry assessment tools for inhaled tobacco products: Workshop proceedings, conclusions and paths forward for in vitro model use.

In 2009, the passing of the Family Smoking Prevention and Tobacco Control Act facilitated the establishment of the FDA Center for Tobacco Products (CTP), and gave it regulatory authority over the marketing, manufacture and distribution of tobacco products, including those termed 'modified risk'. On 4-6 April 2016, the Institute for In Vitro Sciences, Inc. (IIVS) convened a workshop conference entitled, In Vitro Exposure Systems and Dosimetry Assessment Tools for Inhaled Tobacco Products, to bring together stakeholders representing regulatory agencies, academia and industry to address the research priorities articulated by the FDA CTP. Specific topics were covered to assess the status of current in vitro smoke and aerosol/vapour exposure systems, as well as the various approaches and challenges to quantifying the complex exposures in in vitro pulmonary models developed for evaluating adverse pulmonary events resulting from tobacco product exposures. The four core topics covered were: a) Tobacco Smoke and E-Cigarette Aerosols; b) Air-Liquid Interface-In Vitro Exposure Systems; c) Dosimetry Approaches for Particles and Vapours/In Vitro Dosimetry Determinations; and d) Exposure Microenvironment/Physiology of Cells. The 2.5-day workshop included presentations from 20 expert speakers, poster sessions, networking discussions, and breakout sessions which identified key findings and provided recommendations to advance these technologies. Here, we will report on the proceedings, recommendations, and outcome of the April 2016 technical workshop, including paths forward for developing and validating non-animal test methods for tobacco product smoke and next generation tobacco product aerosol/vapour exposures. With the recent FDA publication of the final deeming rule for the governance of tobacco products, there is an unprecedented necessity to evaluate a very large number of tobacco-based products and ingredients. The questionable relevance, high cost, and ethical considerations for the use of in vivo testing methods highlight the necessity of robust in vitro approaches to elucidate tobacco-based exposures and how they may lead to pulmonary diseases that contribute to lung exposure-induced mortality worldwide.

Lipidomics reveals dramatic lipid compositional changes in the maturing postnatal lung.

Lung immaturity is a major cause of morbidity and mortality in premature infants. Understanding the molecular mechanisms driving normal lung development could provide insights on how to ameliorate disrupted development. While transcriptomic and proteomic analyses of normal lung development have been previously reported, characterization of changes in the lipidome is lacking. Lipids play significant roles in the lung, such as dipalmitoylphosphatidylcholine in pulmonary surfactant; however, many of the roles of specific lipid species in normal lung development, as well as in disease states, are not well defined. In this study, we used liquid chromatography-mass spectrometry (LC-MS/MS) to investigate the murine lipidome during normal postnatal lung development. Lipidomics analysis of lungs from post-natal day 7, day 14 and 6-8 week mice (adult) identified 924 unique lipids across 21 lipid subclasses, with dramatic alterations in the lipidome across developmental stages. Our data confirmed previously recognized aspects of post-natal lung development and revealed several insights, including in sphingolipid-mediated apoptosis, inflammation and energy storage/usage. Complementary proteomics, metabolomics and chemical imaging corroborated these observations. This multi-omic view provides a unique resource and deeper insight into normal pulmonary development.