Circulating factors that modify lung cell DNA synthesis following exposure to inhaled oxidants. II. Effect of serum and lavage on lung pneumocytes following exposure of adult rats to 1 ppm ozone

Adult rats were exposed to 1 ppm (1.96 mg/m3) ozone or air for 2 wk. Animals were sacrificed at 3, 5, 7, or 14 d after the onset of exposure, and samples of plasma and lung lavage were obtained. Heat-inactivated plasma and lavage from animals exposed to ozone for 5 or 7 d significantly increased DNA synthesis by lung pneumocytes compared with plasma or lavage from air-exposed animals. Fractionation of plasma and lavage samples indicated that the factor responsible had an isoelectric point of 6.45-6.75, and a molecular weight of 38 +/- 3 kDa. This factor has a dose-dependent effect on lung pneumocyte DNA synthesis in culture. It has no effect on cultured fibroblast DNA synthesis, and is distinct from a previously described factor in the plasma of these ozone-exposed animals that enhances fibroblast DNA synthesis. The factor is detectable within 5 d of exposure, and may hold some promise as a marker of early oxidant lung injury.     

Circulating factors that modify lung cell DNA synthesis following exposure to inhaled oxidants. I. Effect of serum and lavage on lung fibroblasts following exposure of adult rats to 1 ppm ozone

Adult rats were exposed to 1 ppm (1.96 mg/m3) ozone or air for 2 wk. Animals were sacrificed at 3, 5, 7, or 14 d after the onset of exposure, and samples of plasma and lung lavage were obtained. Heat-inactivated plasma, from animals exposed to ozone for 7 or 14 d, significantly increased DNA synthesis by lung fibroblasts compared with plasma from air-exposed animals. Fractionation of plasma and lavage samples indicated that the factor responsible had an isoelectric point of 6.45-6.75 and a molecular weight of 32 +/- 2 kDa. This factor has a dose-dependent effect on lung fibroblast DNA synthesis in culture, but no significant effect on cultured pneumocyte DNA synthesis. The factor is detectable within 72 h of exposure, and may hold some promise as a marker of early oxidant lung injury.     

Time study on development and repair of lung injury following ozone exposure in rats

The aim of this study was to investigate the time course of lung injury in rats during acute and subchronic ozone exposure and during postexposure recovery. Rats were continuously exposed to 0.4 ppm ozone ( approximately 0.8 mg O(3)/m(3)) for 1, 3, 7, 28, or 56 days. Recovery from 3 days of exposure was studied at day 7, 14, and 28; recovery from 7 days of exposure was studied at day 14, 28, and 56, recovery from 28 days of exposure was studied at day 35 and 56, and recovery from 56 days of exposure was studied at day 136. The study included a correlated biochemical and morphological analysis of inflammatory responses, structural changes, and collagen content. The acute inflammatory response, as measured by an increase of polymorphonuclear cells and plasma protein in bronchoalveolar lavage (BAL) fluid, reached a maximum at day 1 and resolved largely within 6 days during ongoing exposure. Numbers of macrophages in BAL fluid increased progressively up to day 56, and slowly returned to near control levels when exposure was followed by postexposure recovery. Histological examination and morphometry of the lungs revealed centriacinar inflammatory responses throughout ozone exposure. Centriacinar thickening of septa was observed at day 7. Ductular septa, thickened progressively at days 7, 28, and 56 of exposure, showed increased collagen upon exposure at day 28, which was further enhanced at exposure at day 56. Increased collagen content in lungs, as measured biochemically by hydroxyproline concentration, was observed at exposure day 56. Collagen content was not different from control at day 56 when 7 or 28 days of exposure was followed by postexposure recovery. After continuous ozone exposure, respiratory bronchioles were present in an increasing degree, and remained present after a recovery period. The results of this study clearly show that after continuous exposure to O(3) some acute effects, such as protein and albumin content, and neutrophil influx in BAL fluid, returned to control levels within a few days. However, other parameters, such as the alveolar macrophage response and structural changes such as the presence of terminal bronchioles, thickening of ductular septa by enhanced cellularity, and collagen formation, persisted or progressively increased during continued exposure. Postexposure recovery seems to partly resolve these subchronic responses (macrophages response, septal cellularity), whereas other effects (collagen increase and respiratory bronchioles formation) do not disappear.     

Effects of ozone on lung and somatic growth. Pair fed rats after ozone exposure and recovery periods

Minor differences in lung growth and development during childhood have been considered as a potential cause of rapid decline in pulmonary function in adulthood. Inhalation of ozone commonly causes changes in both body weight and lung volumes, which complicates interpretation of any changes in lung growth. The effects of ozone on lung growth were studied in rats which were pair fed. This technique permitted comparison of ozone-exposed and filtered-air control rats of the same body weight and body size as well as age and sex. Exposure was to filtered air or to 0.64 or 0.96 ppm ozone (UV standard) 8 h/night for 42 nights. A second control group was fed ad libitum and exposed to only filtered air. Half the rats were studied at the end of the 42-night exposures, the rest after a 42-day post-exposure period during which all rats were fed ab libitum and breathed filtered air. Rats examined at the end of the exposure period had larger saline and fixed lung volumes. These larger lungs had greater volumes of parenchyma, alveoli and respiratory bronchioles. Some of these changes persisted throughout a 42-day post-exposure period. Ozone inhalation by young rats alters lung growth and development in ways likely to be detrimental and those changes persist after ozone inhalation stops.     

Rat lung metallothionein and heme oxygenase gene expression following ozone and zinc oxide exposure.

We have conducted exposures in rats to determine pulmonary responses following inhalation of two common components of welding fumes, zinc oxide and ozone. To examine their effects on target-inducible gene expression, we measured mRNA levels of two metal-responsive genes, metallothionein (MT) and heme oxygenase (HO), in lung tissue by RNA slot-blot analysis. A 3-hr exposure to ZnO fume via a combustion furnace caused a substantial elevation in lung MT mRNA at all concentrations tested. Exposures to 5 and 2.5 mg/m3 ZnO resulted in peak 8-fold increases in MT mRNA levels (compared to air-exposed control animal values) immediately after exposure, while 1 mg/m3 ZnO exposure caused a 3.5-fold elevation in MT mRNA. These levels returned to approximate control gene expression values 24 hr after exposure. In addition, ZnO exposure caused an immediate elevation in lung HO gene expression levels, with 8-, 11-, and 5-fold increases observed after the same ZnO exposure levels (p < 0.05). Like MT gene induction, HO mRNA values returned to approximate control levels 24 hr after exposure. In striking contrast to the induction of MT and HO gene expression after ZnO exposures, there was no elevation in gene expression following a 6-hr exposure to 0.5 and 1 ppm ozone, even when lungs were examined as late as 72 hr after exposure. Our results demonstrate the induction of target gene expression following the inhalation of ZnO at concentrations equal to, and below, the current recommended threshold limit value of 5 mg/m3 ZnO. Furthermore, the lack of effect of ozone exposure on MT and HO gene expression suggests no involvement of these genes in the acute respiratory response to this oxidant compound.     

Responsiveness to cocaine challenge in adult rats following prenatal exposure to cocaine

Adult rats that were gestationally exposed to cocaine and control offspring were examined for their sensitivity to challenge doses of cocaine. Offspring were derived from Sprague-Dawley dams that had received subcutaneous injections of 40 mg/kg per 3 cc cocaine hydrochloride daily on gestational days 8–20, pair-fed dams that were injected with saline, and nontreated control dams. In order to investigate the sensitivity to challenge doses of cocaine, offspring were assessed in adulthood for locomotor activity, cocaine drug discrimination, and the time course of cocaine in brain tissue following acute cocaine challenge. Adult offspring prenatally exposed to cocaine were observed to exhibit a reduced sensitivity to the discriminative stimulus effects of cocaine as evidenced by a significant shift to the right in the dose-response curve of cocaine discrimination. No prenatal treatment effects were observed in terms of the temporal patterns of cocaine discrimination or with regard to brain levels of cocaine. In addition, baseline locomotor activity and locomotor responses to challenge doses of cocaine were comparable across the prenatal treatment groups. Thus, prenatal cocaine exposure reduced sensitivity of offspring to the discriminative stimulus properties of cocaine without altering either the distribution of cocaine to the brain or the sensitivity of the offspring to the locomotor stimulant effects of cocaine.     

Assessments of lung toxicity to Acrawax C following acute inhalation exposure

Acrawax is a trademark for a series of synthetic waxes which are used as flatteners in paint, and lubricants in plastics, and these materials have been routinely regarded as nuisance dusts. Due to a paucity of information regarding the pulmonary toxicity of this material, we investigated the effects of acute inhalation of Acrawax C in rats. CD rats were exposed to aerosols of Acrawax C for 6 hours at 112 mg/m3. Fluids and cells from sham and exposed animals were recovered by bronchoalveolar lavage (BAL) and measured for cellular and biochemical parameters at 0, 24, 48, 172 hrs (8 days), and 1 month postexposure. Pulmonary macrophages (PM) were cultured and studied for in vitro and in vivo phagocytosis, as well as surface morphology. The lungs of additional animals exposed to Acrawax were fixed for assessment by histopathology, and transmission electron microscopy. Our results showed that Acrawax C exposure produced a mild inflammatory response at 24 hours postexposure, but cell differentials were not significantly different from controls at 48 hrs after exposure. BAL levels of lactate dehydrogenase, alkaline phosphatase and protein were slightly different from controls only at 8 days postexposure, and had returned to control values by 1 month of recovery. Acrawax exposure had no adverse effects on either morphology or the phagocytic capacity of pulmonary macrophages recovered from exposed animals. Histopathologic analysis of lung tissue from Acrawax C-exposed rats revealed normal lung architecture. Based on acute studies, our results suggest that the response to inhaled Acrawax C is not substantially different from the response to other nuisance dusts such as carbonyl iron and titanium dioxide.     

Attenuation and recovery of pulmonary injury in rats following short-term,repeated daily exposure to ozone

Controlled human and epidemiology studies have demonstrated that during repeated exposure to ozone (O(3)) attenuation of lung function responses may occur. It is yet unknown whether inflammatory and biochemical effects in lower airways of humans, as observed upon single O(3) exposure, also show a diminutive response following repeated exposure to O(3). The aim of this study was to investigate inflammatory, permeability, and histopathological responses in lungs of rats following repeated daily O(3) exposure and to study the time course of attenuation and recovery of these effects using single O(3) challenges at various postexposure times. To aid in animal-to-human extrapolation, this study and a previously reported human study (Devlin et al., 1997) were designed with similar protocols. Wistar rats were exposed for 5 consecutive days to 0.4 ppm O(3) for 12 h/night. Subsequently, the time course of postexposure recovery was determined by a single challenge of 12 h to 0.4 ppm O(3) after a 5-, 10-, 15-, or 20-day recovery period. Bronchoalveolar lavage (BAL) examination and histopathology were performed 12 h after this O(3) challenge. To quantify the magnitude of the O(3) response, results were compared with a group exposed only once for 12 h to 0.4 ppm O(3) and sacrificed simultaneously. The results demonstrate that a single exposure of 0.4 ppm O(3) causes marked permeability and inflammatory responses in lower airways of rats, as evidenced by enhanced BAL fluid levels of proteins, fibronectin, interleukin (IL)-6, and inflammatory cells. However, 5 days of exposure to 0.4 ppm O(3) for 12 h/night resulted in a complete disappearance of these responses, resulting in BAL fluid values that were not different from those observed in unexposed controls. Postexposure analyses of pulmonary response to O(3) challenges demonstrated that these attenuated responses show a gradual recovery. The data indicate that with respect to BAL fluid levels of albumin, IL-6, and number of macrophages and neutrophils, the period for lung tissue to regain its full susceptibility and responsiveness to O(3) following a 5-day preexposure period is approximately 15-20 days. Remarkably, the total protein and fibronectin responses in BAL fluid still exhibited an attenuated response to an O(3) challenge at 20 days postexposure. Morphometry (number of BrdU-labeled cells in terminal bronchiolar epithelium, and number of alveolar macrophages) showed that after a recovery of 5-10 days following a 5-day preexposure the response to a challenge was identical to that after a single exposure. These results suggest that complete repair from lower airway inflammation caused by short-term, repeated exposure to O(3) may take longer than previously assumed.     

Measurements of cardiopulmonary response in awake rats during acute exposure to near-ambient concentrations of ozone

Cardiopulmonary responses during acute exposure to near-ambient (less than or equal to 1.0 ppm) concentrations of ozone (O3) have not been reported for the unanesthetized rat. Such data on species sensitivity are crucial for the extrapolation of animal data to man. Therefore, this study was conducted to obtain functional measurements on awake rats using head-out plethysmographs and intrapleural or carotid artery catheters during a 135-min exposure to 0.0, 0.12, 0.25, 0.5 or 1.0 ppm O3. Carbon dioxide was added during alternate 15-min periods of the exposure to increase ventilation, much like the use of exercise in human O3 exposure studies. The results established that frequency of breathing was increased and tidal volume was decreased as a function of both the concentration and the duration of exposure. Breathing mechanics and cardiopulmonary measures were only marginally affected. Differences in the response of individual rats revealed that as O3 concentration increased, the proportion of rats responding and the magnitude of the response was increased. These data indicate that, for similar functional responses, the rat's sensitivity to O3 is comparable to that observed in man.     

Initial and residual toxicity following acute exposure of developing male rats to dibromochloropropane

Male Fischer 344 rats were given a single, sc injection of 1,2-dibromo-3-chloropropane (DBCP) at 6 or 25 days of age. One group of treated animals was killed 1 to 3 days afterward to compare the dose and time relationships of the acute toxic response of neonatal and weanling male rats to DBCP and another group at approximate sexual maturity (approximately 120 days of age) to detect residual toxic effects resulting from acute exposure. The 6-day-old rats were more susceptible than the 25-day-old rats to the acute toxic effects of DBCP, as characterized by reduced 48-hr survival, renal dysfunction, and renal and hepatic necrosis over the dose range of 80 to 320 mg/kg. The lowest dose tested, 20 mg/kg, and all higher doses reduced subsequent body and gonadal weight gains, and caused hypospermatogenesis or seminiferous tubular atrophy in animals exposed at 6 days of age and killed at sexual maturity. Similar effects were observed in animals exposed at 25 days of age, except that doses of 160 mg/kg or greater were required to produce residual toxic effects. These data indicate enhanced susceptibility of neonatal male rats to the gonadotoxic effects of dibromochloropropane, including the possibility of apparent irreversible injury caused by acute exposure.     

Effects of maternal high-fat diet and sedentary lifestyle on susceptibility of adult offspring to ozone exposure in rats

Epidemiological and experimental data suggest that obesity exacerbates the health effects of air pollutants such as ozone (O₃). Maternal inactivity and calorically rich diets lead to offspring that show signs of obesity. Exacerbated O₃ susceptibility of offspring could thus be manifested by maternal obesity. Thirty-day-old female Long-Evans rats were fed a control (CD) or high-fat (HF) (60% calories) diet for 6 wks and then bred. GD1 rats were then housed with a running wheel (RW) or without a wheel (SED) until parturition, creating four groups of offspring: CD-SED, CD-RW, HF-SED and HF-RW. HF diet was terminated at PND 35 and all offspring were placed on CD. Body weight and %fat of dams were greatest in order; HF-SED?>?HF-RW?>?CD-SED?>?CD-RW. Adult offspring were exposed to O₃ for two consecutive days (0.8?ppm, 4?h/day). Glucose tolerance tests (GTT), ventilatory parameters (plethysmography), and bronchoalveolar fluid (BALF) cell counts and protein biomarkers were performed to assess response to O₃. Exercise and diet altered body weight and %fat of young offspring. GTT, ventilation and BALF cell counts were exacerbated by O₃ with responses markedly exacerbated in males. HF diet and O₃ led to significant exacerbation of several BALF parameters: total cell count, neutrophils and lymphocytes were increased in male HF-SED versus CD-SED. Males were hyperglycemic after O₃ exposure and exhibited exacerbated GTT responses. Ventilatory dysfunction was also exacerbated in males. Maternal exercise had minimal effects on O₃ response. The results of this exploratory study suggest a link between maternal obesity and susceptibility to O₃ in their adult offspring in a sex-specific manner.     

Functional and inflammatory alterations in the lung following exposure of rats to nitrogen mustard

Nitrogen mustard is a vesicant that causes damage to the respiratory tract. In these studies, we characterized the acute effects of nitrogen mustard on lung structure, inflammatory mediator expression, and pulmonary function, with the goal of identifying mediators potentially involved in toxicity. Treatment of rats (male Wistar, 200-225 g) with nitrogen mustard (mechlorethamine hydrochloride, i.t., 0.25 mg/kg) resulted in marked histological changes in the respiratory tract, including necrotizing bronchiolitis, thickening of alveolar septa, and inflammation which was evident within 24 h. This was associated with increases in bronchoalveolar lavage protein and cells, confirming injury to alveolar epithelial regions of the lung. Nitrogen mustard administration also resulted in increased expression of inducible nitric oxide synthase and cyclooxygenase-2, pro-inflammatory proteins implicated in lung injury, in alveolar macrophages and alveolar and bronchial epithelial cells. Expression of connective tissue growth factor and matrix metalloproteinase-9, mediators regulating extracellular matrix turnover was also increased, suggesting that pathways leading to chronic lung disease are initiated early in the pathogenic process. Following nitrogen mustard exposure, alterations in lung mechanics and function were also observed. These included decreases in baseline static compliance, end-tidal volume and airway resistance, and a pronounced loss of methacholine responsiveness in resistance, tissue damping and elastance. Taken together, these data demonstrate that nitrogen mustard induces rapid structural and inflammatory changes in the lung which are associated with altered lung functioning. Understanding the nature of the injury induced by nitrogen mustard and related analogs may aid in the development of efficacious therapies for treatment of pulmonary injury resulting from exposure to vesicants.     

Splenomegaly in mice following exposure to ambient levels of ozone

Female strain A/J mice were exposed to 0.31 ppm (608 micrograms/m3) ozone continuously for 103 h every other week for 6 mth. Following an additional period of 5 mth in a filtered air environment, animals were killed and examined for evidence of altered spleen weight. It was observed that animals exposed to ozone had a greater spleen weight, and spleen to body weight ratio than air-breathing controls. In some of the ozone-exposed mice, pronounced splenomegaly was noted. Increased spleen weight appears to be another example of an extrapulmonary effect of ozone inhalation.     

Influence of hyperthyroidism on rat lung cytokine production and nuclear factor-kappaB activation following ozone exposure

Results from previous studies indicate that hyperthyroidism increases the risk of ozone-induced lung toxicity. To better understand the processes that might contribute to the increased pulmonary inflammatory response to ozone in hyperthyroidism, we evaluated bronchoalveolar lavage fluid levels of selected cytokines in control and hyperthyroid rats after exposure to air or ozone. In addition, we assessed whether there is a relative increase in nuclear factor-kappa B (NF-kappaB) binding activity in cells harvested by bronchoalveolar lavage from hyperthyroid rats following the inhalation of ozone. A hyperthyroid condition was induced by the administration of thyroxine (0.5 mg/kg body weight) for 7 days. Control rats received vehicle injections. The animals were then exposed by inhalation to air or ozone (2 ppm for 3 h) and studied 18 h following the exposure. Bronchoalveolar lavage levels of MIP-2 and MCP-1 were increased in both control and hyperthyroid rats by ozone exposure. However, the increases in hyperthyroid rats were much greater, MIP-2 1.5-fold and MCP-1 11-fold, when compared to levels in controls following ozone. These changes appeared to be relatively specific; bronchoalveolar lavage fluid levels of interleukin (IL)-6, IL-4, and IL-10 were generally low or nondetectable across all of the studied groups at the 18-h postexposure time point. We also found that NF-kappaB binding activity was increased at both 4 and 18 h following ozone exposure in bronchoalveolar lavage cell extracts from hyperthyroid rats relative to the activity in control samples. Collectively, these results suggest that mechanisms contributing to the enhanced pulmonary inflammatory response to ozone in a hyperthyroid state include an increase in NF-kappaB activation and an upregulation of chemokine production.     

Neurobehavioral assessment following e-cigarette refill liquid exposure in adult rats

The present study was conducted to assess the toxic effect of e-cigarette refill liquid on cognitive and motor functions in adult rats. Animals were administered 28?μl/kg of body weight of e-liquid with/without a dose of 0.5?mg of nicotine/kg of body weight, using the intraperitoneally route for a period of 4 weeks. They were then evaluated by novel object recognition test (NORT) and spontaneous alternation T-maze test for cognitive functions. Results indicated that e-liquid without nicotine induced, in the NORT, a decrease in time exploring the novel object during the test session and lower discrimination and recognition indexes compared to control and e-liquid with nicotine treated rats. Furthermore, short-term spatial memory was affected after e-liquid treatment in the spontaneous alternation T-maze test, identifying recognition memory impairments. However, none of the treatments altered motor functions assessed by inclined plane test, Kondziela’s inverted screen test and weights test. Cell cytotoxicity assessment following e-liquid exposure showed a significant decrease in hippocampal cell viability, but no change in cortical cell viability. Thereby, e-liquid without nicotine causes cognitive impairments, especially on the hippocampus. Based on these results, more extensive assessments on e-cigarettes must be carried out.     

Pulmonary responses of acute exposure to ultrafine iron particles in healthy adult rats

As critical constituents of ambient particulate matter, transition metals such as iron may play an important role in health outcomes associated with air pollution. The purpose of this study was to determine the respiratory effects of inhaled ultrafine iron particles in rats. Sprague Dawley rats 10-12 weeks of age were exposed by inhalation to iron particles (57 and 90 microg/m(3), respectively) or filtered air (FA) for 6 h/day for 3 days. The median diameter of particles generated was 72 nm. Exposure to iron particles at a concentration of 90 microg/m(3) resulted in a significant decrease in total antioxidant power along with a significant induction in ferritin expression, GST activity, and IL-1beta levels in lungs compared with lungs of the FA control or of animals exposed to iron particles at 57 microg/m(3). NFkappaB-DNA binding activity was elevated 1.3-fold compared with that of control animals following exposure to 90 microg/m(3) of iron, but this change was not statistically significant. We concluded that inhalation of iron particles leads to oxidative stress associated with a proinflammatory response in a dose-dependent manner. The activation of NFkappaB may be involved in iron-induced respiratory responses, but further studies are merited.     

Time course of response to ozone exposure in healthy adult females

Ozone exposure causes acute decrements in pulmonary function, increases airway responsiveness, and changes the breathing pattern. We examined these responses in 19 ozone-responsive (DeltaFEV(1) > 5%) young females exposed to both air and 0.35 ppm ozone. The randomized 75-min exposures included two 30-min exercise periods at V(E) approximately 40 L/min. Responses were measured before, during, and after exposure and at 18 and 42 h postexposure. FVC, FEV(1), and FIV(0.5) decreased (p <.01) immediately postexposure by 13.2%, 19.9%, and 20.8%, respectively, and the airway responsiveness was significantly increased. Raw increased (p <.05), while TGV remained essentially unchanged. At 18 h postexposure, the airways were still hyperresponsive and FEV(1) and FIV(0.5) were still 5% below the preexposure levels. There were no residual effects in any of the variables at 42 h postexposure. During exercise in ozone the tidal volume was decreased (-14%) and respiratory frequency increased (+15%). The changes in airway responsiveness were not related to changes in spirometric measurements. We found no significant differences between postair and postozone mouth occlusion pressure (Pm(0.1)) and the hypercapnic response to CO(2) rebreathing. We conclude that ozone induced typical acute changes in airway responsiveness and that ventilatory (exercise), spirometric (inspiratory and expiratory), and plethysmographic pulmonary function may show some residual effects for up to 18 h after exposure. The ozone-induced alteration in breathing pattern during exercise does not appear to be related to a change in ventilatory drive.     

Individual variability in human lung function responses to ozone exposure

Ozone is a common photochemical air pollutant which is present in the ambient air of many urban areas at concentrations sufficient to produce acute respiratory effects in humans. Because individuals vary considerably in the magnitude of their responses to ozone exposure, it is difficult to estimate the number of individuals in a given population who are experiencing adverse effects. Consequently risk and benefits analysis for various regulatory scenarios cannot be carried out with precision. As an aid to risk assessment this paper presents a method of predicting the proportion of individuals in the population who experience a particular health effect. Risk equations predicting the proportion of individuals experiencing lung function decrements as a function of ozone concentration, duration of exposure, and age are presented.