Effects of ozone on evaporative water loss and thermoregulatory behavior of marine toads (Bufo marinus).

Ozone (O(3)) is a strong pulmonary irritant and causes a suite of respiratory tract inflammatory responses in humans and other mammals. In addition to lung injury, rodents exposed to O(3) exhibit a pronounced decrease in core body temperature at rest, which may offer a protective effect against O(3) damage. The effects of O(3) on other vertebrates have not been studied. Compared to individuals exposed to air (N=34), Bufo marinus toads exposed to O(3) (N=32) for 4 h lost 3.78 g body mass (adjusted mean from analysis of covariance, body mass mean+/-SD, 90.1+/-21.90 g). We tested the thermoregulatory responses of 22 toads in a thermal gradient 1, 24, and 48 h after 4-h exposure to air (N=11) or 0.8 ppm O(3) (N=11). Individual toad thermal preferences were also significantly repeatable across all trials (intraclass correlation=0.66, P <0.001). We did not observe a direct effect of O(3) exposure on the preferred body temperatures (PBT) of toads. However, O(3) exposure did have an indirect effect on selected temperatures. Ozone-exposed toads with higher evaporative water loss rates, in turn, also selected lower PBT, voluntary minimum, and voluntary maximum temperatures 24 h post-exposure. Ozone exposure may thus alter both water balance and thermal preferences in anuran amphibians.

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.

Xantusiid lizards have low energy, water, and food requirements.

Lizards in the family Xantusiidae (the night lizards) are known to have resting metabolic rates that are only half those of other lizards of comparable size. We evaluated whether xantusiids also have low field metabolic rates (FMR) and food requirements by measuring FMR and water flux rates with doubly labeled water in three xantusiid species in their natural habitats. Free-living Xantusia vigilis, Xantusia henshawi, and Xantusia riversiana processed energy and water very slowly, about one-third as fast as do other reptiles of similar size. Xantusiid lizards have a distinctive life history that is characterized by very slow growth and low reproductive rates, and they are intensely reclusive. This general lifestyle is also found in some species that live in environments with scarce food resources, such as in caves and in arid habitats, and these species may also have relatively low energy requirements.

Toxicity of chemical components of ambient fine particulate matter (PM 2.5) inhaled by aged rats.

The toxicity of two important chemical components of fine ambient particulate matter (PM 2.5)-ammonium bisulfate (ABS) and elemental carbon (C)-was studied using aged (senescent) rats. The study tested the hypotheses that fine particle exposure can damage lungs and impair host defenses in aged rats and that ozone would potentiate the toxicity of these particles. Ammonium bisulfate aerosols were generated by nebulization of dilute aqueous solutions. Elemental carbon was generated from an aqueous suspension of carbon black. Carbon and ABS mixtures were generated by nebulization of a suspension of carbon black in a dilute aqueous solution of ABS. Rats were exposed, nose-only, for 4 h a day, three consecutive days a week, for 4 weeks. The rats were exposed to one of six atmospheres: (1) purified air; (2) C, 50 microg m(-3), 0.3 microm mass median aerodynamic diameter (MMAD); (3) ABS, 70 microg m(-3), 0.3 microm MMAD; (4) O3, 0.2 ppm; (5) ABS + C, 0.46 microm MMAD; and (6) ABS + C + O3, 0.45 microm MMAD. Data were analyzed using ANOVA and Tukey multiple comparison tests; a two-tailed significance level of 0.05 was used. The nuclei of lung epithelial and interstitial cells were examined to determine the labeling of the DNA of dividing cells by 5-bromo-2-deoxyuridine and to identify the location of injury-repair-related cell replication. Increased labeling of both epithelial and interstitial lung cells occurred following all pollutant exposures. Although epithelial cells are most likely impacted by inhaled particles first, the adjacent interstitial cells were the cells that showed the greatest degree of response. Exposure to the ABS + C + O3 mixture resulted in losses of lung collagen and increases in macrophage respiratory burst and phagocytic activities that were statistically significant. Our results demonstrate that ozone can increase the toxicity of inhaled particles (or vice versa), and suggest that detailed study of mixtures could provide a more comprehensive understanding of the mechanisms by which inhaled pollutants adversely affect human health.

Adaptive and non-adaptive responses in rats exposed to ozone, alone and in mixtures, with acidic aerosols.

Healthy young adult (300 g) Sprague-Dawley rats were exposed for 1-day or 5-day periods, nose only, to purified air (CA) or four different pollutant atmospheres. Pollutant atmospheres included (a) 0.2 ppm ozone; (b) 0.4 ppm O3; (c) a low concentration mixture of ozone and sulfuric acid-coated carbon particles (0.2 ppm, 100 microg/m(3) and 50 microg/m(3), respectively); and (d) a high-concentration O3 and sulfuric acid-coated carbon particle mixture (0.4 ppm, 500 microg/m(3) and 250 microg/m(3), respectively). Following 1-day exposures to the high O3 concentration, significant (p< or =.05) decreases were observed in respiratory tidal volumes and significant increases were observed in lung inflammatory response. Following 5-day exposures to 0.4 ppm ozone, tidal volumes and lung inflammation were not significantly different from those seen in CA controls. In contrast, following 5-day exposures to the high-concentration O3-particle mixture, lung inflammation was increased significantly relative to that seen after 1-day high concentration mixture exposure or after CA exposure. Macrophage Fc-receptor binding, an important immunological function of macrophages, was significantly depressed after 5-day exposures to either the high- or low-concentration O3-particle mixtures compared to 1-day exposures or to CA. Thus, at the concentrations tested, repeated exposures to O3 produced diminished responses in breathing pattern changes and lung parenchymal injuries compared to acute, single exposures. This diminution was not observed after exposures to mixtures of acidic particles plus ozone. We conclude that mixtures of ozone and acidic particles may alter adaptive mechanisms that have been reported by us and others after repeated exposures to ozone alone.

Chronic exposure to ozone and nitric acid vapor results in increased levels of rat pulmonary putrescine.

In the past decade, there has been growing public concern for the human health effects of exposure to environmental pollutants. Ozone (O3) is one of the most reactive components of photochemical air pollution. Despite extensive investigations by many laboratories on the functional, biochemical, and cellular effects of O3 exposure in humans, animals, and in vitro systems, questions remain concerning the potential adverse effects to human health represented by chronic near-ambient exposure to this environmental pollutant. In the present investigation, the influence of inhalation of O3 and nitric acid (HNO3) vapor on polyamine levels was examined in rat lungs. Male F344/N rats were exposed nose-only to 0.15 ppm O3 and 50 microg/m3 HNO3 vapor alone and in combination for 4 hours/day. 3 days/week for a total of 40 weeks. At this time the animals were sacrificed and their lungs were examined for polyamine contents. Exposure to O3 and O3 plus HNO3 vapor caused a significant increase in the putrescine content of the lung compared to the air-exposed controls (P < 0.05). The concentrations of pulmonary spermidine and spermine were not significantly increased by exposure to either O3 or HNO3 vapor alone or in combination compared to the air-exposed controls. The role of polyamines in repair and anti-inflammatory processes has been discussed.

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.

Chronic inhalation exposure to ozone and nitric acid elevates stress-inducible heat shock protein 70 in the rat lung.

The ability of urban oxidant and acid air pollutants to induce heat shock proteins (HSPs) in the mammalian lung is not known. Such proteins are known to be correlated with environmental stress and pathophysiological conditions. In this study, stress-inducible HSP 70 was assessed by slot-blotting in rat lungs (N=10 per group) following inhalation exposures for 4 h per day, 3 days per week for 40 weeks to the following pollutants: (a) purified air;(b) 0.15 ppm ozone (O3);(c)50 micrograms/m3 nitric acid (HNO3); or(d) a combination of both 0.15 ppm O3 and 50 micrograms/m3 HNO3. At 24 h following the last exposure, samples from the right apical lobe of the lung were obtained for either slot-blotting or gel electrophoretic separation, subsequent protein immunoblotting, and chemiluminescence detection of HSP 70 levels. Experiments demonstrate that stress-inducible HSP 70 was present constitutively in the control lungs and was separable from the constitutive form of HSP 70. Slot-blotting analysis demonstrate that the O3 and HNO3 exposures alone produced significant elevations of HSP70. Specifically, either O3 or HNO3 alone significantly elevated lung stress-inducible HSP 70 levels by 277% and 221% respectively, above control levels. The group exposed to combined O3 and HNO3 showed a 177% elevation in lung stress-inducible HSP 70 that was significantly greater that the group inhaling purified air, but this effect was less than the effects of either pollutant component alone. Moreover, all exposure groups were significantly different from one another. These results indicate that stress-inducible HSP 70 in the rat lung is highly elevated after chronic inhalation exposures to both O3 and HNO3 when administered either alone or in combination within the range of urban ambient concentrations.

Calibration of respiratory gas exchange measurements in inhalation toxicology studies.

The use of simplifying assumptions for determining respiratory gas exchange is associated with substantial errors in the estimates of oxygen consumption (VO2) and carbon dioxide production (VCO2). Studies were done to estimate the magnitude of these errors under common exposure conditions, and a simple procedure that reveals these errors is described for calibrating an open flow respirometer. The errors associated with various simplifying assumptions ranged from -1 to -21% for VO2, 0.1 to 15% for VCO2, and 4 to 45% for the respiratory exchange ratio (R). The calibration was performed with a standard calibration gas elevated in CO2 and depressed in O2 relative to air and bled into the respirometer at a measured flow rate. Dilution of the gas into the respirometer airstream simulates the effect of respiratory gas exchange, and expected values of VO2, VCO2, and R compared to measured values provide a check of accuracy of flow and gas fraction measurements, a test for system leaks, and a test for the effects of any simplifying assumptions on the calculation of respiratory gas exchange.

The effects of exercise on dose and dose distribution of inhaled automotive pollutants.

The purpose of this study was to determine how changes in ventilation rate and in the entry route of air pollutants into the respiratory tract (nose versus mouth breathing) affected the respiratory tract uptake and penetration of inhaled gaseous and particulate pollutants associated with automobile emissions. Experiments were performed with female beagle dogs exposed while standing at rest or while exercising on a treadmill at 5 km/hour and a 7.5 percent grade. Dogs were exposed to nitrogen dioxide at concentrations of 1 and 5 parts per million (ppm), to formaldehyde at 2 and 10 ppm, and to an aerosol of ammonium nitrate particles (0.3 micron mass median aerodynamic diameter) at 1 mg/m3. Total respiratory system uptake and effects on breath time, expired tidal volume, fractional expiration time, minute ventilation, respiratory gas exchange, ventilation equivalents for oxygen and carbon dioxide, and dynamic pulmonary resistance and compliance were measured in exercising and resting dogs exposed for two hours to 5 ppm nitrogen dioxide and 10 ppm formaldehyde in combination with 1 mg/m3 of ammonium nitrate particles. Regional penetration of pollutants through oral and nasal airways and pollutant uptake in the lung were measured in a separate group of six tracheostomized dogs standing at rest while being exposed to nitrogen dioxide, formaldehyde, and ammonium nitrate particles. Hypercapnic stimulation was used to modify ventilation rates in the tracheostomized dogs while pollutant penetration and uptake were measured. Dogs exposed to 5 ppm of nitrogen dioxide at rest tended to breathe more rapidly (p less than 0.05) and more shallowly (a nonsignificant trend) than dogs exposed to purified air. The changes observed were similar in direction, but of smaller magnitude, to changes observed when the same dogs were exposed during exercise to ozone at 0.6 ppm in a separate study. Rapid-shallow breathing was not observed when the dogs were exposed during exercise to 5 ppm nitrogen dioxide. Dogs exposed to a mixture of 10 ppm formaldehyde and 1 mg/m3 ammonium nitrate particles during exercise showed a shift to larger tidal volume breathing, but the response was much less pronounced than the slow-deep breathing pattern response observed in a separate study of dogs exposed to 10 ppm formaldehyde alone. The total respiratory system uptake of formaldehyde from the formaldehyde and ammonium nitrate mixture was larger than that measured for 10 ppm of formaldehyde alone in another exercise and exposure study.(ABSTRACT TRUNCATED AT 400 WORDS)

Breathing pattern and metabolic rate responses of rats exposed to ozone.

Breathing pattern (frequency and tidal volume), minute ventilation (VE), oxygen consumption (MO2), and rectal temperature (TR) were measured from rats exposed to 0.8, 0.6, 0.4, and 0.2 ppm O3 to determine the relation between breathing pattern responses to O3 and metabolic rate. In 0.8 ppm O3, rapid-shallow breathing began at 60 min exposure while VE and MO2 declined beginning at 40 min. In comparison to clean air exposed animals, rats (n = 8) during the third hour of 0.8 ppm O3 exposure had a 27% increase in frequency, 35% decline in tidal volume, 20% decrease in VE, 24% decrease in MO2, and 1.3 degrees C decrease in TR. At lower O3 concentrations, responses were diminished in magnitude, and in rats exposed to 0.2 ppm O3, only MO2 was significantly decreased, irritant-induced depression of VE did not imply a state of hypoventilation or hypoxia because ventilation equivalent for O2 (VE/MO2) did not decline during O3 exposures. Body temperature and metabolic rate depression have not been observed during the development of rapid-shallow breathing in dogs or humans exposed to these low levels of O3, and the present observations in rats may reflect a more labile thermoregulatory physiology among rodents. Ventilatory and metabolic rate depression in response to irritant inhalation can be an effective pulmonary defense in rodents and other heterothermic mammals.

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.

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.

Effects of exercise exposure on toxic interactions between inhaled oxidant and aldehyde air pollutants.

Respiratory tract injury resulting from inhalation of mixtures of ozone (O3) and nitrogen dioxide (NO2) and of O3 and formaldehyde (HCHO) was studied in Sprague-Dawley rats under exposure conditions of rest and exercise. Focal inflammatory injury induced in lung parenchyma by O3 exposure was measured morphometrically and HCHO injury to the nasal respiratory epithelium was measured by cell turnover using tritium-labeled thymidine. Mixtures of O3 (0.35 or 0.6 ppm) with NO2 (respectively 0.6 or 2.5 ppm) doubled the level of lung injury produced by O3 alone in resting exposures to the higher concentrations and in exercising exposures to the lower concentrations. Formaldehyde (10 ppm) mixed with O3 (0.6 ppm) resulted in reduced lung injury compared to O3 alone in resting exposures, but exercise exposure to the mixture did not show an antagonistic interaction. Nasal epithelial injury from HCHO exposure was enhanced when O3 was present in a mixture. Mixtures of O3 and NO2 at high and low concentrations formed respectively 0.73 and 0.02 ppm nitric acid (HNO3) vapor. Chemical interactions among the oxidants, HNO3, and other reaction products (N2O5 and nitrate radical) and lung tissue may be the basis for the O3-NO2 synergism. Increased dose and dose rate associated with exercise exposure may explain the presence of synergistic interaction at lower concentrations than observed in resting exposure. No oxidation products were detected in O3-HCHO mixtures, and the antagonistic interaction observed in lung tissue during resting exposure may result from irritant breathing pattern interactions.

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.

Enhancement of ozone-induced lung injury by exercise.

Rats were exposed for up to 3.75 h to 0.20-0.80 ppm O3 under conditions of rest and treadmill exercise up to 30 m/min, 20% grade, to assess the importance of exposure duration, O3 concentration, and exercise on lung tissue injury. Focal lung parenchymal lesions increased in abundance and severity in response to the three variables; however, exercise was the most important. Lesion response to exercise was greater than that predicted by a simple proportion to estimated effective dose of O3. The results emphasize the importance of including exercise in assessment of possible adverse health effects of exposure to airborne pollutants.