Inhalation exposure of animals.

Relative advantages and disadvantages and important design criteria for various exposure methods are presented. Five types of exposures are discussed: whole-body chambers, head-only exposures, nose or mouth-only methods, lung-only exposures, and partial-lung exposures. Design considerations covered include: air cleaning and conditioning; construction materials; losses of exposure materials; evenness of exposure; sampling biases; animal observation and care; noise and vibration control, safe exhausts, chamber loading, reliability, pressure fluctuations; neck seals, masks, animal restraint methods; and animal comfort. Ethical considerations in use of animals in inhalation experiments are also discussed.

Factors influencing the deposition of inhaled particles.

Because the initial deposition pattern of inhaled particles of various toxic agents determines their future clearance and insult to tissue, respiratory tract deposition is important in assessing the potential toxicity of inhaled aerosols. Factors influencing the deposition of inhaled particles can be classified into three main areas: (1) the physics of aerosols, (2) the anatomy of the respiratory tract and (3) the airflow patterns in the lung airways. In the physics of aerosols, the forces acting on a particle and its physical and chemical properties, such as particle size or size distribution, density, shape, hygroscopic or hydrophobic character, and chemical reactions of the particle will affect the deposition. With respect to the anatomy of the respiratory tract, important parameters are the diameters, the lengths, and the branching angles of airway segments, which determine the deposition. Physiological factors include airflow and breathing patterns, which influence particle deposition. Various lung models used in predicting particle deposition are reviewed and discussed. The air-way structures of various animal species are compared, showing the unique structure of the human lung compared to the animal species under study. Regional deposition data in man and dog are reviewed. Recent deposition data for small rodents are presented, showing regional difference in deposition with the right apical lobe having the highest relative deposition.

Inhalation exposure methodology.

Modern man is being confronted with an ever-increasing inventory of potentially toxic airborne substances. Exposures to these atmospheric contaminants occur in residential and commercial settings, as well as in the workplace. In order to study the toxicity of such materials, a special technology relating to inhalation exposure systems has evolved. The purpose of this paper is to provide a description of the techniques which are used in exposing laboratory subjects to airborne particles and gases. The various modes of inhalation exposure (whole body, head only, nose or mouth only, etc.) are described at length, including the advantages and disadvantages inherent to each mode. Numerous literature citations are included for further reading. Among the topics briefly discussed are the selection of appropriate animal species for toxicological testing, and the types of inhalation studies performed (acute, chronic, etc.).

Health effects of acid aerosols formed by atmospheric mixtures.

Under ambient conditions, sulfur and nitrogen oxides can react with photochemical products and airborne particles to form acidic vapors and aerosols. Inhalation toxicological studies were conducted, exposing laboratory animals, at rest and during exercise, to multicomponent atmospheric mixtures under conditions favorable to the formation of acidic reaction products. Effects of acid and ozone mixtures on early and late clearance of insoluble radioactive particles in the lungs of rats appeared to be dominated by the oxidant component (i.e., the mixture did cause effects that were significantly different from those of ozone alone). Histopathological evaluations showed that sulfuric acid particles alone did not cause inflammatory responses in centriacinar units of rat lung parenchyma (expressed in terms of percent lesion area) but did cause significant damage (cell killing followed by a wave of cell replication) in nasal respiratory epithelium, as measured by uptake of tritiated thymidine in the DNA of replicating cells. Mixtures of ozone and nitrogen dioxide, which form nitric acid, caused significant inflammatory responses in lung parenchyma (in excess of effects seen in rats exposed to ozone alone), but did not damage nasal epithelium. Mixtures containing acidic sulfate particles, ozone, and nitrogen dioxide damaged both lung parenchyma and nasal epithelia. In rats exposed at rest, the response of the lung appeared to be dominated by the oxidant gas-phase components, while responses in the nose were dominated by the acidic particles. In rats exposed at exercise, however, mixtures of ozone and sulfuric acid particles significantly (2.5-fold) elevated the degree of lung lesion formation over that seen in rats exposed to ozone alone under an identical exercise protocol.

A stereoradiographic technique and its application to the evaluation of lung casts.

Stereoradiographs have been used on occasion for three-dimensional reconstruction and measurement of objects in radiology and radiotherapy. The lack of a good stereoradiographic technique has limited the uses of steroradiographic exposures. In this paper, the principle of the double-image is outlined and a method of applying this principle to stereoradiographic exposures is developed. A computer program has been developed from geometrical considerations to analyze the stereobronchogram and to calculate the dimensions of the objects in question. The applications of this technique are discussed and its use in evaluating lung casts is described.

A rodent treadmill for inhalation toxicological studies and respirometry.

A 10-runway treadmill was enclosed for inhalation toxicological studies of rodents under exercise exposure to environmental pollutants. The exposure system was lined with sheet stainless steel to minimize scrubbing of charged particles and reactive gases. Average metabolic gas exchange of exercising animals was derived from measurements of inlet or outlet airflow and data from an O2 analyzer in conjunction with either a CO2 or N2 analyzer. An airflow rate of 400 l X min-1 ensured a response time of 1 min to reach 95% of a step change in metabolic rate and held scrubbing losses of an O3 test atmosphere to less than 2% of treadmill inlet concentration. Gas exchange averaged for 10 rats during incremental exercise up to their highest collective performance was similar to published data for rats tested individually.

Surfactant dysfunction after inhalation of nitric oxide.

To study whether nitric oxide (NO) affects surfactant function, 36 young rats inhaled one of the following humidified environments for 24 h: 1) air; 2) 95% O2; 3) air and 100 parts/million (ppm) NO; and 4) 95% O2 and 100 ppm NO. The treatments did not change the recovery of phospholipid from bronchoalveolar lavage (BAL). Exposure to NO of animals that breathed either air or 95% O2 increased the minimum surface tension of surfactant from BAL at low (1.5 mumol/ml), but not at high (4 mumol/ml), phosphatidylcholine concentration. After inhaled NO, the nonsedimentable protein of BAL decreased the surface activity of surfactant (1 mumol phosphatidylcholine/ml) more than the protein from the controls. NO treatment of animals that breathed either air or 95% O2 affected neither the quantity nor the molecular weight distribution of nonsedimentable protein. Hyperoxia increased the amount of the nonsedimentable protein, whereas NO increased the iron saturation of transferrin. The surfactant fraction and the nonsedimentable protein from BAL were separately exposed to 80 ppm NO in vitro. NO exposure had no effect on the surface activity of surfactant fraction. NO exposure of nonsedimentable protein from the control animals (no NO) increased the inhibition of the surface activity and changed the adsorption spectrum of the protein, suggesting conversion of hemoglobin to methemoglobin. Nonsedimentable protein from NO-exposed animals contained methemoglobin. We propose that surfactant dysfunction caused by inhaled NO is in part due to alteration of protein(s) in epithelial lining fluid that in turn inactivates surfactant.

Respiratory tract responses to repeated inhalation of an oxidant and acid gas-particle air pollutant mixture.

The purpose of this study was to examine a broad range of toxicologic responses in rats exposed to a multi-component pollutant atmosphere. Cumulative and adaptive respiratory tract responses to 3 concentrations of an inhaled particle-oxidant mixture were examined in Fisher 344 N rats exposed 4 h/day, 3 days/week for 4 weeks. The mixtures contained O3, NO2, NH4HSO4, carbon particles, and HNO3 vapor. Irritant-induced, rapid-shallow breathing responses were present during the first 4-h exposure to medium and high concentrations. Successive exposures showed diminished responses in medium concentrations and exacerbated responses in high concentrations. At the end of 4 weeks, rats exposed to high concentrations exhibited lung lesions. Lavaged pulmonary macrophages showed dose-dependent depressions of Fc-receptor binding and phagocytosis. Lung tissue macrophages showed dose-dependent increases in acid phosphatase staining density and carbon particles. Respiratory tract clearance of tracer particles was not significantly affected by the exposures. Broncho-alveolar epithelial permeability was increased by the high concentration. Epithelial cell-proliferation labeling showed a dose-dependent increase at all levels of the respiratory tract. Progressively exacerbated breathing-pattern responses at high concentrations were associated with lung lesions and high cell-proliferation labeling in the nose transitional epithelium and terminal bronchioles. Attenuating or adaptive breathing-pattern responses occurred in the presence of smaller, but in many cases still significant, compromise of respiratory functions. Either attenuating or exacerbated breathing-pattern responses can occur in the presence of a significant dose-dependent compromise of other respiratory functions and lung tissue injury.

Influence of steroid aerosol treatments on the clearance of inhaled gold particles.

Female Long Evans rats were used to test the hypothesis that in haled triamcinolone acetonide accelerates the rate of clearance of particles from the lung. Three groups of animals inhaled a radioactive gold aerosol, which functioned as a tracer of respiratory tract clearance, and then were subjected to various inhalation treatments. The group treated with triamcinolone acetonide aerosol showed a significant acceleration in the rate of early clearance, but the total amount of tracer particles cleared in the first day was not significantly increased. Inhaled triamcinolone acetonide appears to accelerate the translocation of foriegn particles from small airways to larger ones, but stimulation of clearance does not appear to be a large effect.

Uncertainties relating to the health effects of particulate air pollution: the US EPA's particle standard.

Although the epidemiologic associations between urban particulate air pollution and human mortality and morbidity have been accumulating for several years, the causal agents (a specific chemical component, a specific particle size range, one or more pollutant combinations, etc.), and the physiological mechanisms behind the associations have yet to be identified. Significant questions regarding confounding effects due to weather, indoor air pollutant exposures and co-pollutants (that accompany particulate matter) stubbornly remain. The events in the United States began with recent epidemiological associations, followed by a lawsuit forcing the US EPA to accelerate the standard-setting process, and finally controversy over the scientific basis of the new standard. In contrast to the potential risks posed by particulate air pollution, many of the sources of such particles are positive contributors to human health; control measures to meet the proposed standard may therefore produce offsetting enhanced mortality and morbidity. In order to establish the information required for well-informed public health policies, a substantial research program is needed because of uncertainties relating to, the affected individuals, the potential causal agents, and the consequences of particle-control activities. Not only are the remaining scientific questions significant, but the particle exposure/health effects associations also call into question some of the current scientific assumptions relating to the nature of effects of population exposures to low concentrations of pollutants.

Physical characterization of incense aerosols.

Experiments were performed to study the physical characteristics of smoke aerosols generated by burning three types of stick incense in a 4 m3 clean room. Sidestream cigarette smoke was also examined under the same conditions to provide a comparison. Among the parameters measured were (a) masses of aerosol, carbon monoxide and nitrogen oxides generated by burning the incense or cigarettes, (b) rates of decay of the particles from the air, and (c) estimates of count median particle size during a 7 h period post-burning. There was variability among the types of incense studied with respect to many of the parameters. Also, as a general trend, the greater the initial particulate mass concentration, the more rapid the rate of decay of the smoke. In relation to the quantity of particulate generated, cigarette smoke was found to produce proportionally larger quantities of carbon monoxide and nitrogen oxides than did incense. Due to the fact that burning incense was found to generate large quantities of particulate (an average of greater than 45 mg/g burned, as opposed to about 10 mg/g burned for the cigarettes), it is likely, in cases in which incense is habitually burned in indoor settings, that such a practice would produce substantial airborne particulate concentrations.

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.

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.

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.

Adaptations to ozone in reference to mucociliary clearance.

Adaptation to ozone in respiratory tract mucociliary clearance was investigated in this study. Eighty rats inhaled insoluble radioactively labeled particles in order to permit monitoring of clearance. The respiratory tract and the feces were counted for radioactivity at fixed intervals to determine clearance rates. A brief challenge to 1.2 ppm of ozone following particle deposition caused a substantial delay in rapid (mucociliary) clearance. This delay (or "ozone effect") however, was eliminated by brief pre-exposure to 0.8 ppm of ozone 3 days prior to deposition of particles. When a 13-day period intervened between the ozone pre-exposure and challenge, a substantial delay (or "ozone effect") was again seen. Thus, the pre-exposure to ozone appeared to afford essentially complete protection at 3 days, and no protection by 13 days.

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.

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.

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.