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.

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.

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.

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.

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.

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.

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.

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.

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.

Global long-range transport and lung cancer risk from polycyclic aromatic hydrocarbons shielded by coatings of organic aerosol.

Polycyclic aromatic hydrocarbons (PAHs) have toxic impacts on humans and ecosystems. One of the most carcinogenic PAHs, benzo(a)pyrene (BaP), is efficiently bound to and transported with atmospheric particles. Laboratory measurements show that particle-bound BaP degrades in a few hours by heterogeneous reaction with ozone, yet field observations indicate BaP persists much longer in the atmosphere, and some previous chemical transport modeling studies have ignored heterogeneous oxidation of BaP to bring model predictions into better agreement with field observations. We attribute this unexplained discrepancy to the shielding of BaP from oxidation by coatings of viscous organic aerosol (OA). Accounting for this OA viscosity-dependent shielding, which varies with temperature and humidity, in a global climate/chemistry model brings model predictions into much better agreement with BaP measurements, and demonstrates stronger long-range transport, greater deposition fluxes, and substantially elevated lung cancer risk from PAHs. Model results indicate that the OA coating is more effective in shielding BaP in the middle/high latitudes compared with the tropics because of differences in OA properties (semisolid when cool/dry vs. liquid-like when warm/humid). Faster chemical degradation of BaP in the tropics leads to higher concentrations of BaP oxidation products over the tropics compared with higher latitudes. This study has profound implications demonstrating that OA strongly modulates the atmospheric persistence of PAHs and their cancer risks.

In vitro metabolism of benzo[a]pyrene-7,8-dihydrodiol and dibenzo[def,p]chrysene-11,12 diol in rodent and human hepatic microsomes.

Polycyclic aromatic hydrocarbons (PAHs) are contaminants that are ubiquitously found in the environment, produced through combustion of organic matter or petrochemicals, and many of which are procarcinogens. The prototypic PAH, benzo[a]pyrene (B[a]P) and the highly carcinogenic dibenzo[def,p]chrysene (DBC) are metabolically activated by isoforms of the P450 enzyme superfamily producing benzo[a]pyrene-7,8-dihydrodiol (B[a]P diol), dibenzo[def,p]chrysene-11,12 diol (DBC diol). Each of these diols can be further metabolized by cytochrome P450 enzymes to highly reactive diol-epoxide metabolites that readily react with DNA or by phase II conjugation facilitating excretion. To complement prior in vitro metabolism studies with parent B[a]P and DBC, both phase I metabolism and phase II glucuronidation of B[a]P diol and DBC diol were measured in hepatic microsomes from female B6129SF1/J mice, male Sprague-Dawley rats, and female humans. Metabolic parameters, including intrinsic clearance and Michaelis-Menten kinetics were calculated from substrate depletion data. Mice and rats demonstrated similar B[a]P diol phase I metabolic rates. Compared to rodents, human phase I metabolism of B[a]P diol demonstrated lower overall metabolic capacity, lower intrinsic clearance at higher substrate concentrations (>0.14µM), and higher intrinsic clearance at lower substrate concentrations (<0.07µM). Rates of DBC diol metabolism did not saturate in mice or humans and were highest overall in mice. Higher affinity constants and lower capacities were observed for DBC diol glucuronidation compared to B[a]P diol glucuronidation; however, intrinsic clearance values for these compounds were consistent within each species. Kinetic parameters reported here will be used to extend physiologically based pharmacokinetic (PBPK) models to include the disposition of B[a]P and DBC metabolites in animal models and humans to support future human health risk assessments.

Spatially-Resolved Proteomics: Rapid Quantitative Analysis of Laser Capture Microdissected Alveolar Tissue Samples.

Laser capture microdissection (LCM)-enabled region-specific tissue analyses are critical to better understand complex multicellular processes. However, current proteomics workflows entail several manual sample preparation steps and are challenged by the microscopic mass-limited samples generated by LCM, impacting measurement robustness, quantification and throughput. Here, we coupled LCM with a proteomics workflow that provides fully automated analysis of proteomes from microdissected tissues. Benchmarking against the current state-of-the-art in ultrasensitive global proteomics (FASP workflow), our approach demonstrated significant improvements in quantification (~2-fold lower variance) and throughput (>5 times faster). Using our approach we for the first time characterized, to a depth of >3,400 proteins, the ontogeny of protein changes during normal lung development in microdissected alveolar tissue containing only 4,000 cells. Our analysis revealed seven defined modules of coordinated transcription factor-signaling molecule expression patterns, suggesting a complex network of temporal regulatory control directs normal lung development with epigenetic regulation fine-tuning pre-natal developmental processes.

Human Microdosing with Carcinogenic Polycyclic Aromatic Hydrocarbons: In Vivo Pharmacokinetics of Dibenzo[def,p]chrysene and Metabolites by UPLC Accelerator Mass Spectrometry.

Metabolism is a key health risk factor following exposures to pro-carcinogenic polycyclic aromatic hydrocarbons (PAHs) such as dibenzo[def,p]chrysene (DBC), an IARC classified 2A probable human carcinogen. Human exposure to PAHs occurs primarily from the diet in nonsmokers. However, little data is available on the metabolism and pharmacokinetics in humans of high molecular weight PAHs (≥4 aromatic rings), including DBC. We previously determined the pharmacokinetics of DBC in human volunteers orally administered a microdose (29 ng; 5 nCi) of [14C]-DBC by accelerator mass spectrometry (AMS) analysis of total [14C] in plasma and urine. In the current study, we utilized a novel "moving wire" interface between ultraperformance liquid chromatography (UPLC) and AMS to detect and quantify parent DBC and its major metabolites. The major [14C] product identified in plasma was unmetabolized [14C]-DBC itself (Cmax = 18.5 ±15.9 fg/mL, Tmax= 2.1 ± 1.0 h), whereas the major metabolite was identified as [14C]-(+/-)-DBC-11,12-diol (Cmax= 2.5 ±1.3 fg/mL, Tmax= 1.8 h). Several minor species of [14C]-DBC metabolites were also detected for which no reference standards were available. Free and conjugated metabolites were detected in urine with [14C]-(+/-)-DBC-11,12,13,14-tetraol isomers identified as the major metabolites, 56.3% of which were conjugated (Cmax= 35.8 ± 23.0 pg/pool, Tmax = 6-12 h pool). [14C]-DBC-11,12-diol, of which 97.5% was conjugated, was also identified in urine (Cmax = 29.4 ± 11.6 pg/pool, Tmax = 6-12 h pool). Parent [14C]-DBC was not detected in urine. This is the first data set to assess metabolite profiles and associated pharmacokinetics of a carcinogenic PAH in human volunteers at an environmentally relevant dose, providing the data necessary for translation of high dose animal models to humans for translation of environmental health risk assessment.

Total and regional deposition of inhaled aerosols in supine healthy subjects and subjects with mild-to-moderate COPD.

Despite substantial development of sophisticated subject-specific computational models of aerosol transport and deposition in human lungs, experimental validation of predictions from these new models is sparse. We collected aerosol retention and exhalation profiles in seven healthy volunteers and six subjects with mild-to-moderate COPD (FEV1 = 50-80%predicted) in the supine posture. Total deposition was measured during continuous breathing of 1 and 2.9 µm-diameter particles (tidal volume of 1 L, flow rate of 0.3 L/s and 0.75 L/s). Bolus inhalations of 1 µm particles were performed to penetration volumes of 200, 500 and 800 mL (flow rate of 0.5 L/s). Aerosol bolus dispersion (H), deposition, and mode shift (MS) were calculated from these data. There was no significant difference in total deposition between healthy subjects and those with COPD. Total deposition increased with increasing particle size and also with increasing flow rate. Similarly, there was no significant difference in aerosol bolus deposition between subject groups. Yet, the rate of increase in dispersion and of decrease in MS with increasing penetration volume was higher in subjects with COPD than in healthy volunteers (H: 0.798 ± 0.205 vs. 0.527 ± 0.122 mL/mL, p=0.01; MS: -0.271±0.129 vs. -0.145 ± 0.076 mL/mL, p=0.05) indicating larger ventilation inhomogeneities (based on H) and increased flow sequencing (based on MS) in the COPD than in the healthy group. In conclusion, in the supine posture, deposition appears to lack sensitivity for assessing the effect of lung morphology and/or ventilation distribution alteration induced by mild-to-moderate lung disease on the fate of inhaled aerosols. However, other parameters such as aerosol bolus dispersion and mode shift may be more sensitive parameters for evaluating models of lungs with moderate disease.

Anatomical Details of the Rabbit Nasal Passages and Their Implications in Breathing, Air Conditioning, and Olfaction.

The rabbit is commonly used as a laboratory animal for inhalation toxicology tests and detail knowledge of the rabbit airway morphometry is needed for outcome analysis or theoretical modeling. The objective of this study is to quantify the morphometric dimension of the nasal airway of a New Zealand white rabbit and to relate the morphology and functions through analytical and computational methods. Images of high-resolution MRI scans of the rabbit were processed to measure the axial distribution of the cross-sectional areas, perimeter, and complexity level. The lateral recess, which has functions other than respiration or olfaction, was isolated from the nasal airway and its dimension was quantified separately. A low Reynolds number turbulence model was implemented to simulate the airflow, heat transfer, vapor transport, and wall shear stress. Results of this study provide detailed morphological information of the rabbit that can be used in the studies of olfaction, inhalation toxicology, drug delivery, and physiology-based pharmacokinetics modeling. For the first time, we reported a spiral nasal vestibule that splits into three paths leading to the dorsal meatus, maxilloturbinate, and ventral meatus, respectively. Both non-dimensional functional analysis and CFD simulations suggested that the airflow in the rabbit nose is laminar and the unsteady effect is only significantly during sniffing. Due to the large surface-to-volume ratio, the maxilloturbinate is highly effective in warming and moistening the inhaled air to body conditions. The unique anatomical structure and respiratory airflow pattern may have important implications for designing new odorant detectors or electronic noses. Anat Rec, 299:853-868, 2016. © 2016 Wiley Periodicals, Inc.

Hepatic Cytochrome P450 Activity, Abundance, and Expression Throughout Human Development.

Cytochrome P450s are oxidative metabolic enzymes that play critical roles in the biotransformation of endogenous compounds and xenobiotics. The expression and activity of P450 enzymes varies considerably throughout human development; the deficit in our understanding of these dynamics limits our ability to predict environmental and pharmaceutical exposure effects. In an effort to develop a more comprehensive understanding of the ontogeny of P450 enzymes, we employed a multi-omic characterization of P450 transcript expression, protein abundance, and functional activity. Modified mechanism-based inhibitors of P450s were used as chemical probes for isolating active P450 proteoforms in human hepatic microsomes with developmental stages ranging from early gestation to late adult. High-resolution liquid chromatography-mass spectrometry was used to identify and quantify probe-labeled P450s, allowing for a functional profile of P450 ontogeny. Total protein abundance profiles and P450 rRNA was also measured, and our results reveal life-stage-dependent variability in P450 expression, abundance, and activity throughout human development and frequent discordant relationships between expression and activity. We have significantly expanded the knowledge of P450 ontogeny, particularly at the level of individual P450 activity. We anticipate that these results will be useful for enabling predictive therapeutic dosing, and for avoiding potentially adverse and harmful reactions during maturation from both therapeutic drugs and environmental xenobiotics.