Fit-for-purpose characterization of air-liquid-interface (ALI) in vitro exposure systems for e-vapor aerosol.

Air-liquid-interface (ALI) exposure systems deliver aerosol to the apical surface of cells which mimics the in vivo inhalation exposure conditions. It is necessary, however, to quantify the delivered amount of aerosol for ALI-based in vitro toxicity assessment. In this study, we evaluated two commercially available ALI exposure systems, a Vitrocell® Ames 48 (Ames 48) and a Vitrocell® 24/48 (VC 24/48), and the Vitrocell® VC1/7 smoking machine using a cig-a-like cartridge-based e-vapor device with a prototype formulation (containing 4% nicotine by weight). We characterized aerosol particle-size distribution, aerosol mass, and major chemical components (nicotine, propylene glycol, and glycerol) at the generation source and verified the repeatability of the aerosol generation. We determined aerosol delivery at the ALI by gravimetric analysis of mass collected on Cambridge filter pads and analytical quantitation of the buffer medium which showed that both aerosol mass and nicotine to an exposure insert linearly increased up to 400 puffs. The delivered aerosol mass covered a wide range of 0.8-3.4 mg per insert in the Ames 48 with variability (relative standard deviation, RSD) up to 12% and 1.1-6.4 mg per insert in the VC 24/48 with variability up to 15%. The delivered nicotine ranged approximately up to 200 µg per insert in both exposure systems. These results provided operation and aerosol delivery information of these ALI exposure systems for subsequent in vitro testing of e-vapor aerosols.

Variability of TSNA in U.S. Tobacco and Moist Smokeless Tobacco Products.

Tobacco-specific nitrosamines (TSNAs) have been of concern to the public health community for decades and their reduction through agricultural practices, plant breeding, and tobacco processing has also been a decades-long industry effort. Despite those efforts, TSNAs, though lower, continue to be constituents of concern in tobacco products. This paper examines the TSNA levels of dark air-cured, dark fire-cured, and burley tobaccos purchased in the United States by U.S. Smokeless Tobacco Company LLC (USSTC) and of nine finished USSTC moist smokeless tobacco products. TSNA values of the incoming purchased tobaccos and the finished products showed considerable variability. For the incoming tobaccos, the coefficient of variation was generally more than 100 % for each tobacco type and for each of the measured TSNAs. The relative TSNA variability of the finished tobacco products was also considerable, averaging approximately 25 %. It was also found that the measured values for the finished products averaged well above the proposed FDA NNN proposed product standard of 1.0 µg/g dry weight. Because of the large variability in NNN values, products would have to average well below FDA's proposed product standard to be consistently compliant.

Effect of cigarette smoking on nitric oxide, structural, and mechanical properties of mouse arteries.

Cigarette smoking (CS) is a major risk factor for vascular disease. The aim of this study was to quantitatively assess the influence of CS on mouse arteries. We studied the effect of short-term (6 wk) and long-term (16 wk) CS exposure on structural and mechanical properties of coronary arteries compared with that of control mice. We also examined the reversibility of the deleterious effects of CS on structural [e.g., wall thickness (WT)], mechanical (e.g., stiffness), and biochemical [e.g., nitric oxide (NO) by-products] properties with the cessation of CS. The left and right coronary arteries were cannulated in situ and mechanically distended. The stress, strain, elastic modulus, and WT of coronary arteries were determined. Western blot analysis was used to analyze endothelial NO synthase (eNOS) in the femoral and carotid arteries of the same mice, and NO by-products were determined by measuring the levels of nitrite. Our results show that the mean arterial pressure was increased by CS. Furthermore, CS significantly increased the elastic modulus, decreased stress and strain, and increased the WT and WT-to-radius ratio compared with those of control mice. The reduction of eNOS protein expression was found only after long-term CS exposure. Moreover, the NO metabolite was markedly decreased in CS mice after short- and long-term exposure of CS. These findings suggest that 16 wk of CS exposure can cause an irreversible deterioration of structural and elastic properties of mouse coronary arteries. The decrease in endothelium-derived NO in CS mice was seen to significantly correlate with the remodeling of arterial wall.

Dosimetry counts: molecular hypersensitivity may not drive pulmonary hyperresponsiveness.

Airway hyperresponsiveness is one measure of allergic asthma. One such test, the methacholine challenge, uses an inhaled aerosol to induce changes in resistance to breathing. The test is also used to test hyperresponsiveness in rodent models of asthma. For two varieties of mice, the B6C3F1 and the Balb/c, exposure to aerosolized methacholine demonstrates that the Balb/c is 12x more responsive based on the concentration of methacholine in the solution used to produce the inhaled aerosol (the normally accepted dose-metric). Here we show that the 12x difference in exposure disappears when measurements of airway dimensions of generations 1-6 are used first to calculate deposited mass of methacholine; and second to account for the physiology of airway constriction and pressure drop. These observations in mice provide one explanation of how some hyperresponsive subjects can have no underlying molecular sensitivity; and how constriction in the upper airways can have greater impact on breathing than constriction of airway generations 6-16.

Methods for modeling particle deposition as a function of age.

The purpose of this paper is to review the application of mathematical models of inhaled particle deposition to people of various ages. The basic considerations of aerosol physics, biological characteristics and model structure are presented along with limitations inherent in modern modeling techniques. Application of the models to children and senescent adults has been largely based on extrapolating anatomical and physiological data from young adults to match the changes observed during growth and aging. Sample results are included for total particle deposition and deposition in the bronchial and pulmonary regions. The models proposed provide particle deposition predictions that are consistent with the scant measurements available. The models discussed appear to be on firm theoretical grounds, but they are largely limited in application to simple aerosols and average individuals. Also, additional validation of the computational predictions is needed.

Effects of breathing parameters on sidestream cigarette smoke deposition in a hollow tracheobronchial model.

The effects of variations in cyclic breathing parameters (i.e., tidal volume and breath frequency) have been the subject of few studies devoted to the deposition of submicrometer aerosols in the human respiratory tract. Therefore, a series of experiments was performed to investigate whether the deposition efficiency (DE) of sidestream cigarette smoke is altered by varying tidal volume and breath frequency in a child-size hollow tracheobronchial (TB) model while maintaining a fixed minute ventilation rate of 5 L/min. Under cyclic flow conditions with tidal volumes of 100 mL (50 breaths/min), 250 mL (20 breaths/min), 500 mL (10 breaths/min) and 750 mL (6.7 breaths/min), sidestream cigarette smoke was passed through replicas of an idealized hollow TB model. The smoke deposits were extracted and then quantitated spectrophotometrically. The experiments revealed a significant difference in DE between the 100-mL tidal volume (DE = 6.0%) and the 750-mL tidal volume (DE = 11.1%). Under equivalent steady flow conditions, the mean DE was 21.5%. A trend was evident in the data--DE increased as tidal volume increased (and breathing frequency decreased)--suggesting that the influence of diffusion and secondary flows on DE becomes greater as the air residence time increases and the degree of air turbulence decreases. The results provide evidence of the importance of breathing parameters when attempting to model in vivo deposition of environmental tobacco smoke and other similar-size respirable aerosols.

Deposition of monodisperse particles in hollow models representing adult and child-size tracheobronchial airways.

A series of experiments was performed to determine deposition efficiencies of four sizes of radiolabeled monodisperse particles in custom-made hollow tracheobronchial models. The particles had geometric diameters of about 1, 5, 10, and 15 microm. The tracheobronchial models, consisting of a trachea and two or more additional generations, had dimensions representative of a typical adult, a 7-y-old child, and a 4-y-old child; the child-size models were appropriately scaled-down replicas of the adult-size model. Each deposition experiment was conducted using a steady inspiratory airflow representative of low physical activity for the appropriate age of individual: 20 L min(-1) for the adult; 9 L min(-1) for the 7-y-old; 5.5 L min(-1) for the 4-y-old. The results indicate that deposition efficiency of the particles increased substantially (up to 35 times) in all three models as particle diameter increased from 1-15 microm, undoubtedly as a result of particle impaction and sedimentation-related phenomena. An analysis of variance demonstrated the occurrence of statistically-significant (p < 0.05) main effects of hollow model size and particle size; the interaction between the two parameters was also significant. In general, deposition efficiencies of the various sizes of particles were greater in the child-size models than in the adult-size model; this effect may have risk assessment implications. In addition, the results obtained experimentally agreed more closely with those predicted using a radiation-protection mathematical particle deposition formulation as the particle size increased for each of the sizes of models.

Effects of nitrogen dioxide on respiratory tract clearance in the ferret.

During growth and development, young children are periodically exposed to relatively high concentrations of various air contaminants, including tobacco smoke and environmental pollutants generated by fossil fuel use. The effects of these exposures on respiratory function and lung development are difficult to determine because of interindividual variation and lack of accurate dosimetry. To provide information on the effects of chronic exposure to a common indoor and outdoor pollutant during lung development, a study was performed to assess the effects of exposure to two concentrations of nitrogen dioxide (NO2; 0.5 or 10 ppm) on tracer particle clearance from the airways of ferrets exposed during postnatal respiratory tract development. Separate groups of ferrets were exposed nose-only to the test atmospheres or clean air 4 h/d, 5 d/wk, for either 8 or 15 wk. Those animals exposed for 8 wk were subsequently housed in a filtered air environment until the particle clearance measurements commenced at 3 wk prior to the end of the 15-wk exposure protocol. Radiolabeled (51Cr) tracer particles were deposited in the respiratory tract of all animals by inhalation, and the clearance rates from the head and thoracic regions were separately monitored for 18 d. No significant effects of the NO2 exposure on head airways clearance were seen. In contrast, the rates of particle clearance from the thorax of both the 8- and 15-wk groups exposed to 10 ppm NO2 were significantly reduced, and did not differ from each other. Thoracic clearance was also reduced in animals exposed to 0.5 ppm, but the rate was not significantly different from that of the clean air exposed controls. These results show that NO2 at moderate concentrations caused highly significant changes in the deep lung of the juvenile ferret, and suggest that impairment of the clearance function may be only slowly recovered after chronic exposure.

Low-cost wind tunnel for aerosol inhalation studies.

A low-cost wind tunnel for aerosol studies has been designed, constructed, and evaluated for aerosol uniformity with 2- and 0.46-micron particles. A commercial nebulizer was used to produce the suspended test particles, and a custom-made, four-hole injector was used to introduce the aerosol into the wind tunnel. A commercially available optical particle counter measured the particle concentration. Performance tests of the velocity profile and particle concentration distribution at two flow rates showed that the system performs well for small particles.

The effect of dead space on inhaled particle deposition.

Mathematical models which have been developed to predict the deposition of particles in the conducting airways of the lung require simplified anatomical models of the dimensions and geometry of the bronchial airways. In order to produce valid deposition predictions, the computed volumes of the conducting airways must be realistic in comparison to anatomical dead space. This requirement must be met even as the developing lung grows to maturity and then undergoes aging. The effect of these age-related changes on predicted particle deposition efficiencies has not been well studied. Numerous authors have suggested that differences in lung volumes (total lung capacity, functional residual capacity, dead space and tidal volume) may account for significant variations between predicted or observed particle deposition but no general age-specific relationship has been proposed. New models are proposed to describe changes in dead space as functions of age and body size, and methods to adjust existing anatomical models to various dead space predictions are given. Also, the effect of these modifications to anatomical models on particle deposition efficiencies are simulated for a variety of breathing patterns for models scaled to represent young children, adults, and aged persons.

Inflated, dried whole lung specimens.

A method is described for preparing fully-inflated whole lung specimens that are suitable for instruction or research purposes. Undamaged lungs are removed from the body and then tracheally cannulated and lavaged with tap water more than 250 times. The treatment also includes rinsing blood from vessels with water. A final filling of the lung with alcohol is optional. The multiply rinsed lung is drained and inflated to 30 cm of H2O pressure with dehumidified air and held at that pressure until the tissue is completely dry. The resulting specimens are light in color and appear to be permanent if stored properly.

Growth of the ferret tracheobronchial tree.

Because the ferret is being used increasingly in inhalation toxicology and lung physiology studies, it is necessary to better understand the airway structure of its tracheobronchial tree. Previously published information does not include dimensions of bronchi and bronchioles in either adult or growing ferrets. The airway structure of interest for calculating inhaled particle deposition patterns includes airway lengths, diameters and branching angles in each generation. Measurements of these dimensions were obtained for several selected airway paths on replica casts. Casts were made in-situ in four male litter mates age 14 hours, 9.5 days, 16.5 days and 56 days. These data demonstrate, that as with human lung growth, body length at a given age is a good predictor of airway lengths and diameters. Airway branch angles do not appear to change significantly during growth. Sufficient measurements were made to provide dimensions of a typical tracheobronchial pathway for this species. This pathway begins with the trachea and ends at the terminal bronchiole. The morphometric data were not sufficient to determine whether or not the number of tracheobronchial generations increase or decrease postnatally.

Aerosol deposition in the nose as a function of body size.

The effect of body size on nasal doses from inhaled aerosols has not been measured directly in people. Two basic types of computational models are used to calculate inhaled particle deposition in adults. One type uses an impaction parameter that incorporates particle aerodynamic diameter and the average airflow rate. The second type uses the nasal pressure drop and particle aerodynamic diameter. Although both types of models have been adjusted to give reasonably accurate deposition efficiencies for adults, they predict very different deposition efficiencies when they are applied to young children. This is not surprising because the airflow-type model has no body-size-dependent parameters, unlike the pressure-drop-type model. The objective of our studies was to test these two types of computational models using idealized hollow nasal models of two sizes, representing the adult and young child. The results indicate that a pressure-drop relationship fits the aerosol deposition data very well. When the properly scaled physiological air flows and minute ventilations are used in a nasal dose calculation, the young child is seen to have potentially larger nasal doses than those of an adult.

Postnatal enlargement of human tracheobronchial airways and implications for particle deposition.

In support of predictions for inhaled particle deposition, morphometric measurements were taken on 20 replica airway casts of people aged 11 days to 21 years. Measurements of right upper lobe airway lengths, diameters, and branching angles were made such that a growth model suitable as input to predictive equations for particle deposition efficiency was obtained. The tracheobronchial airways growth was describable by linear regressions on body length. The length-to-diameter ratio of growing airways did not change in any simple way as a function of airway generation. Airflow rates for a given state of physical activity for various ages were found from previously published data to be describable by linear regressions on body mass. Three states of physical exertion-low activity, light exertion, and heavy exertion-were used for modeling purposes. The computed particle deposition efficiencies indicate that under most circumstances smaller (younger) people will have greater tracheobronchial deposition efficiencies than larger (older) people. For example, tracheobronchial dose on a per kilogram body mass basis for 5-micron-diameter particles may be more than 6 times higher in the resting newborn than in the resting adult assuming equivalent deposition efficiencies above the larynx.

Tracheobronchial airway structure as revealed by casting techniques.

Research quality tracheobronchial airway casts, prepared in the intact thorax, have proved to be useful in comparative mammalian anatomic studies. Grossly, casts trimmed free of alveoli are quite different in appearance for different species. Overall organ shape, tracheal length/diameter ratio, presence or absence of a tracheal bronchus, and degree of branching symmetry constitute the major gross characteristics. Detailed morphometric measurements performed on such casts reveal important species differences in branch shape, number of divisions in the tree, and variations in such structure as a function of airway generation number. Of the mammalian tracheobronchial casts examined to date, those of humans have several distinctive characteristics. Their overall shape is the most nearly spherical; most other mammals have lungs that are significantly longer along the tracheal axis in relation to their width or thickness. Human branches are typically relatively symmetric with respect to both daughter tube diameter ratio and daughter branch angle ratio. In short, of all of the studied mammalian lungs those of humans appear to be the least heterogeneous.

Effects of exercise on coronary collateralization--angiographic studies of six patients in a supervised exercise program.

The existence of coronary collaterals in man and a positive correlation of their extent with the degree of coronary artery obstruction has been shown. The theory that functional collateralization associated with coronary occlusive disease is a response to local hypoxia is widely advocated. It was theorized here that coronary heart disease patients might enhance coronary collateralization through exercise-induced myocardial hypoxia. This pilot study was carried out to test the specificity of coronary arteriography for measuring changes in collateralization after a period of exercise training. Reports of follow-up examinations 7 years after program termination are also included. Six volunteer, male subjects--3 months to 3 years post-myocardial infarction--completed 10-12 months of medically-supervised exercise. All had an intensive cardiovascular work-up, serial treadmill exercise tests and coronary arteriography before and after training. All demonstrated the expected physical, physiological, metabolic, and psychological benefits. Two showed some definite, increased collateralization, however, in both of these the changes may have been a response to some extension of the occlusive disease and not exclusively an exercise effect. It was evident that minute changes in coronary collaterals are detectable by selective coronary arteriography, but that the specific effect of exercise on the development of collaterals could only be determined by a large-scale, controlled program with randomization of the multiple, uncontrollable variables among exercise and non-exercise populations.