Consequences of prolonged inhalation of ozone on F344/N rats: collaborative studies. Part XI: Integrative Summary.
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Administration, Inhalation↗
Biomedical subjects
J A Last
Publications and source records attributed to J A Last.
In vitro exposure of tracheobronchial epithelial cells and of tracheal explants to ozone.
An in vitro system for exposing respiratory epithelial cells or explant tissues to ozone has been developed and characterized. This system is designed to generate and monitor consistent, reproducible levels of ozone, over a range of concentrations, in a humidified atmosphere, and to allow an exposure time of 24 h or longer. Based on chemical analysis, highly reproducible concentrations of ozone are delivered throughout the chamber, with a coefficient of variation of < 5% between five replicate vials exposed to 0.5 ppm of ozone for 50 min. The viability of cultured human tracheobronchial epithelial cells, as measured by the ability to oxidize a vital dye, and of rat tracheal epithelium, as measured by total numbers of necrotic cells in tracheal explants, after ozone exposure was examined in this system. Responses of cultured cells to ozone exposure as measured by bioassay were consistent with the observed low level of variability of ozone concentration between replicate incubation dishes or vials. Responses of cultured cells to ozone were proportional to duration of exposure and inversely proportional to the volume of medium covering the cells. We conclude that this newly developed in vitro exposure system will allow relatively simple and convenient exposure of cultured cells or organs to ozone or other gaseous agents under highly controlled and reproducible conditions.
Air Pollution↗
Decreased fetal weights in rats exposed to sidestream cigarette smoke.
Pregnant Sprague-Dawley rats were exposed to sidestream cigarette smoke (SS) for 6 hr a day, at a concentration of 1 mg/m3 of respirable total suspended particulate material (TSP) on Days 3, 6-10, and 13-17 of pregnancy. Controls were kept in an identical chamber without smoke exposure. The animals were killed on Day 20 of gestation. No differences were found in maternal body weight gain or average daily food consumption between the smoke-exposed and control groups. The numbers of fetuses and of implantation sites per litter were comparable among the groups. None of the pups showed any gross malformations and no difference was found between controls and SS-exposed pups when examined for reduced skeletal ossifications. However, there was a small but significant reduction in mean pup weight. We conclude that intermittent exposure of rats to sidestream cigarette smoke at concentrations severalfold greater than those encountered in smokey public indoor environments causes intrauterine growth retardation.
Abnormalities, Drug-Induced↗
Ozone exposure of cultured cells and tissues.
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Animals↗
Effects of exposure to nicotine and to sidestream smoke on pregnancy outcome in rats.
Nicotine-delivering transdermal patches were applied to the back of timed-pregnant Sprague-Dawley rats. Pregnancy failure was 100% in animals exposed to 3.5 mg of nicotine per day during the entire pregnancy and 50% in animals exposed to the same amount during the first trimester. Application of 1.75 mg of nicotine per day resulted in a 50% pregnancy failure when exposure occurred during the entire pregnancy. In animals exposed for the first half of pregnancy to cigarette sidestream smoke, under conditions where plasma nicotine levels reached about 25% of those observed following exposure to 1.75 mg of nicotine per day, the average litter size was reduced by about 25%. It is concluded that continuous exposure to nicotine early during pregnancy may adversely affect pregnancy outcome in rats.
Administration, Cutaneous↗
Coordinated expression of a 45 kD protein and ozone toxicity in a human bronchial epithelial cell line.
The human bronchial epithelial cell line, BEAS-2B, which was immortalized by transformation with SV40 virus, when grown biphasically between 0.1 and 1.0 ppm of ozone and liquid medium showed increased release of Cr, decreased synthesis of various macromolecules, and decreased cell viability. Cell injury was a function of the concentration of ozone to which the cells were exposed. Furthermore, in proportion to the extent of cell injury, ozone exposure also induced and/or enhanced synthesis of a 45 kD protein but not any of the well-characterized heat shock proteins, e.g., HSP 70. Actinomycin D prevented enhanced synthesis of the 45 kD protein in cells exposed to ozone, suggesting transcriptional regulation of expression of the 45 kD protein. Enhanced synthesis of the 45 kD protein was not observed in cells treated with heat, cigarette smoke condensate, hydrogen peroxide, or bleomycin. High concentrations of glutathione added to the culture medium reduced ozone toxicity and ozone-enhanced synthesis of the 45 kD protein. These results suggest that ozone injury and enhanced expression of a gene encoding a 45 kD protein of as yet unknown function are coordinated in the SV40-immortalized bronchial epithelial cells.
Bleomycin↗
Collagen mRNA content and distribution in the lungs of rats exposed to ozone.
cDNAs, synthesized by the polymerase chain reaction (PCR) technique, were used to quantify mRNA concentrations for the alpha 1 (I) and alpha 1 (III) chains of collagen types I and III and for beta-actin in the lungs of rats exposed to either filtered air or to 1.2 ppm of ozone. The alpha 1 (I) procollagen mRNA concentration was increased by about 30% in the lungs of rats exposed to ozone, while the concentrations of the mRNAs for alpha 1 (III) procollagen and for beta-actin were the same in the lungs of control and ozone-exposed animals. The lungs from the rats exposed to 1.2 ppm of ozone preferentially synthesized type I collagen as compared with controls. Sites of increased expression of the alpha 1 (I) procollagen mRNA were detected by in situ hybridization in lung sections embedded in paraffin prepared from rats exposed either to filtered air or to ozone. The lungs from rats treated with ozone focally expressed increased amounts of alpha 1 (I) procollagen mRNA in the lung parenchyma at the septal tips and the bronchiole-alveolar duct junctions. They also showed an apparent diffuse increase in alpha 1 (III) procollagen mRNA expression. We conclude that exposure of rats to high concentrations of ozone causes a specific increase in the lung content of mRNA for the major chain of type I collagen.
Actins↗
Ozone, NO, and NO2: oxidant air pollutants and more.
This article reviews the acute and chronic toxicity of the three oxidant air pollutants ozone, nitric oxide (NO), and nitrogen dioxide (NO2). The toxicity of binary mixtures of NO2 with other inhaled agents is also discussed. Newer studies are emphasized, especially those published in the last 5 years or still in press. Very recent data from our laboratory that suggest a new cellular mechanism of importance in lung injury in animals exposed to mixtures of ozone and NO2 that may have general relevance with regard to the effects of oxidant air pollutants on the lung are also presented.
Air Pollutants↗
Consequences of prolonged inhalation of ozone on Fischer-344/N rats: collaborative studies. Part I: Content and cross-linking of lung collagen.
Male and female Fischer-344 rats were exposed either to filtered air (controls) or to 0.12, 0.5, or 1.0 parts per million (ppm)* ozone for six hours per day, five days per week, for 20 months. We examined collagen deposition in lung tissue from these animals to determine whether or not chronic exposure of rats to ozone causes pulmonary fibrosis, as defined biochemically. Several techniques were used to study collagen deposition in the lungs of the animals. These methods included biochemical quantification by analysis of 4-hydroxyproline in lung tissue hydrolysates. The hydroxylysine-derived cross-links in mature collagen were quantified to estimate biochemically the excess of fibrotic collagen in the lung tissue. Biochemical analysis indicated excess collagen in the female rats exposed to 0.5 or 1.0 ppm ozone. Collagen in the lungs of the females also contained relatively more hydroxylysine-derived cross-links than did the lung collagen from age-matched control animals that had breathed only filtered air. Exposure of Fischer-344 rats for 20 months to 0.5 or 1.0 ppm ozone was associated with excess fibrotic lung collagen deposition as defined histologically. In female rats, exposure was also associated with excess deposition as determined biochemically. There was no indication of any significant changes in the lungs of any of the rats exposed to 0.12 ppm ozone, but the number of animals in this group was far too small to conclude whether this was a true no-observable-effect level. We conclude that chronic exposure of rats for 20 months to ozone at concentrations of 0.5 ppm or above for six hours per day, five days per week, causes mild to moderate lung fibrosis, as defined histologically and, in female rats, biochemically. The significance of these observations with regard to health risks to humans chronically inhaling ozone at ambient levels in polluted air remains to be determined.
Air Pollutants↗
Tendon hypertrophy is associated with increased hydroxylation of nonhelical lysine residues at two specific cross-linking sites in type I collagen.
This study was designed to investigate whether the changes in lysine hydroxylation known to occur in hypertrophic tendon occur randomly or at specific lysine residues in the type I collagen molecule. Peptides corresponding to the two known major cross-linking sites of type I collagen (a lysine (or hydroxylysine) at position 9N cross-linked to a hydroxylysine at 930 and a lysine (or hydroxylysine) at position 16C cross-linked to a hydroxylysine at position 87) were prepared by collagenase digestion, size fractionation, and separation by high performance liquid chromatography from normal chicken tendon and from chicken tendon subjected to increased tensile load as a result of muscle hypertrophy. The ratio of the difunctional cross-links dihydroxylysinonorleucine to hydroxylysinonorleucine in normal tendon is 0.75:1; this ratio is increased to 6:1 in hypertrophic tendon. The dihydroxylysinonorleucine to hydroxylysinonorleucine ratio is increased to the same extent in samples of the purified cross-linked peptides derived from both the N-terminal and C-terminal lysine aldehyde residues. On the other hand, the relative hydroxylysine content of preparations of the pooled larger helical peptides obtained by cyanogen bromide digestion of normal and hypertrophic tendons was essentially identical. These results demonstrate that there is a specific increase in hydroxylation of only the N- and C-terminal non-helical lysine residues that participate in the formation of the reducible difunctional cross-links of type I collagen in hypertrophic tendon, while the extent of hydroxylation of lysine residues in the helical regions is not affected. The specific mechanism by which the enzyme lysyl hydroxylase acting on its substrate can distinguish between lysine residues destined to be in non-helical versus helical regions in a nascent collagenous peptide that has not yet attained its final secondary structure remains to be defined.
Amino Acid Sequence↗
Effects of 20 months of ozone exposure on lung collagen in Fischer 344 rats.
Fischer 344 rats were exposed to filtered air (controls) or to 0.12, 0.5, or 1.0 ppm of ozone for 6 h/day, 5 days/week, for 20 months. We examined lung collagen deposition and metabolism in tissue from these animals to determine whether chronic exposure of rats to ozone causes pulmonary fibrosis. We observed excess stainable collagen in the centriacinar region of lungs from the rats exposed to 0.5 or 1.0 ppm of ozone. Biochemical analysis indicated a slight, yet significant, excess collagen deposition in the female rats exposed to 0.5 or 1.0 ppm of ozone. Collagen in the lungs of the females also contained relatively more hydroxylysine-derived crosslinks than did lung collagen from age-matched control animals. No excess of type I procollagen mRNA could be appreciated by in situ hybridization in lungs of the rats exposed to 1.0 ppm of ozone for 20 months, although this mRNA was detected in occasional alveolar interstitial cells at 2 months of exposure to ozone under the same protocol. These findings indicate that chronic exposure of rats to ozone causes mild, persistent fibrosis. The significance of these observations with regard to human health risks of chronically inhaling ozone at ambient levels in polluted air remains to be determined.
Animals↗
Airway epithelial labeling index as an indicator of ozone induced lung injury.
Rats were implanted subcutaneously with a bromodeoxyuridine-filled minipump and then were exposed to ozone delivered at a low dose rate (0.4 ppm during 12 h per night) or at a high dose rate (0.8 ppm during 6 h per night). Three and 7 days after pump implantation the cumulative labeling indices were measured in the alveolar zone and in the airways. Greater alveolar labeling indices were observed 7 days after implantation of the minipumps than after 3 days in all groups, but no ozone-related changes were found in the alveoli of rats in either experimental group at either time. After 3 or 7 days, the labeling index in the large intrapulmonary airways and in the terminal bronchioli of the rats exposed to the higher dose rate (0.8 ppm) was increased. In rats exposed to the lower dose rate (0.4 ppm) the labeling index was significantly elevated in the terminal bronchioli after 3 days and in both the terminal bronchioli and large intrapulmonary airways after 7 days. In the terminal bronchioli the extent of cell proliferation appeared to be defined by dose rate rather than by cumulative exposure. It is concluded that measurement of the airway labeling index is a sensitive indicator of the response of the rat lung to acute exposure to ozone.
Animals↗
Alveolar and airway cell kinetics in the lungs of rats exposed to nitrogen dioxide, ozone, and a combination of the two gases.
Rats carrying minipumps filled with 5-bromo-2'deoxyuridine were exposed to ozone, NO2, or a mixture of the two gases using four different protocols: (A) ozone 0.2 ppm, NO2 3.6 ppm, or their mixture for 24 hr a day; (B) ozone 0.2 ppm, NO2 7.2 ppm, or their mixture for 12 hr per night; (C) ozone 0.6 ppm, NO2 10.8 ppm, or their mixture for 8 hr per night; and (D) ozone 0.8 ppm, NO2 14.4 ppm, or their mixture for 6 hr per night. After three consecutive daily exposures, the animals were returned to filtered air and killed 7 days after implantation of the minipump. Alveolar labeling indices were comparable to control values except in the group of animals exposed for 6 hr nightly to a combination of 0.8 ppm of ozone and 14.4 ppm of NO2. Labeling indices in the peripheral airways were the most sensitive exposure index since they were significantly increased over control values in all animals exposed to ozone, NO2, or a mixture of the two gases, regardless of concentration or exposure duration. Labeling indices increased with elevated dose rate, i.e., concentration of the gases in the inspired air. The response to the combined gases was greater than the calculated sum of the responses to the two individual gases for the three higher dose rates in the large airways and for the highest dose rate in the peripheral airways. The results led to the following conclusions: (1) By the criterion of analysis of cell kinetics in rat large and peripheral airways, neither ozone, NO2, nor their mixture follows Haber's law (c x t = k) over the concentration ranges studied; and (2) at the higher dose rates studied, there is a more than additive (synergistic) airway response to the combination of ozone and NO2.
Animals↗
A new model of progressive pulmonary fibrosis in rats.
Sprague-Dawley rats were exposed for 6 h daily to 0.8 ppm of ozone and 14.4 ppm of nitrogen dioxide. Approximately 7 to 10 wk after the initiation of exposure, animals began to demonstrate respiratory insufficiency and severe weight loss. About half of the rats died between Days 55 and 78 of exposure; no overt ill effects were observed in animals exposed to filtered air, to ozone alone, or to nitrogen dioxide. Biochemical findings in animals exposed to ozone and nitrogen dioxide included increased lung content of DNA, protein, collagen, and elastin, which was about 300% higher than the control values. The collagen-specific crosslink hydroxy-pyridinium, a biomarker for mature collagen in the lung, was decreased by about 40%. These results are consistent with extensive breakdown and remodeling of the lung parenchyma and its associated vasculature. Histopathologic evaluation showed severe fibrosis, alveolar collapse, honeycombing, macrophage and mast cell accumulation, vascular smooth muscle hypertrophy, and other indications of severe progressive interstitial pulmonary fibrosis and end-stage lung disease. This unique animal model of progressive pulmonary fibrosis resembles the final stages of human idiopathic pulmonary fibrosis and should facilitate studying underlying mechanisms and potential therapy of progressive pulmonary fibrosis.
Administration, Inhalation↗
Nonenzymatic glycation of type I collagen. The effects of aging on preferential glycation sites.
The present study was designed to investigate the effects of aging on preferential sites of glucose adduct formation on type I collagen chains. Two CNBr peptides, one from each type of chain in the type I tropocollagen molecule, were investigated in detail: alpha 1(I)CB3 and alpha 2CB3-5. Together these peptides comprise approximately 25% of the total tropocollagen molecule. The CNBr peptides were purified from rat tail tendon, obtained from animals aged 6, 18, and 36 months, by ion exchange chromatography, gel filtration, and high-performance liquid chromatography (HPLC). Sugar adducts were radiolabeled by reduction with NaB3H4. Glycated tryptic peptides were prepared from tryptic digests of alpha 2CB3-5 and alpha 1(I)CB3 by boronate affinity chromatography and HPLC. Peptides were identified by sequencing and by compositional analysis. Preferential sites of glycation were observed in both CB3 and alpha 2CB3-5. Of the 5 lysine residues in CB3, Lys-434 was the favored glycation site. Of the 18 lysine residues and 1 hydroxylysine residue in alpha 2CB3-5, 3 residues (Lys-453, Lys-479, and Lys-924) contained more than 80% of the glucose adducts on the peptide. Preferential glycation sites were highly conserved with aging. In collagen that had been glycated in vitro, the relative distribution of glucose adducts in old animals differed from that of young animals. In vitro experiments suggest that primary structure is the major determinant of preferential glycation sites but that higher order structure may influence the relative distribution of glucose adducts among these preferred sites.
Aging↗
Synergistic interaction of nitrogen dioxide and ozone on rat lungs: acute responses.
Rats were exposed for 6 hr per day to either ozone alone (0.2-0.8 ppm), nitrogen dioxide (NO2) alone (3.6-14.4 ppm), or to combinations of these two oxidant air pollutants. Their response was quantified by changes in the total protein content of lung lavage supernatants or by changes in the content of specific cell types in the lung lavage pellets. A concentration-dependent synergistic response was observed when rats were exposed to the combination of ozone and NO2. Apparent threshold concentrations for the observation of synergistic interaction between ozone and NO2 were assay specific, with epithelial cell content of lung lavage fluid being the most sensitive parameter evaluated, showing positive interaction (greater than additive response) at the lowest concentrations tested. Concurrent exposure to ozone and NO2 was necessary to elicit greater than additive responses; no such interactions were seen upon sequential exposure to ozone or NO2 in either order of presentation. Based upon apparent disappearance rates of ozone in the chambers during exposure of rats to ozone and NO2, we modelled the predicted outcomes based upon the assumption that the two oxidant gases were reacting to form nitrogen pentoxide (N2O5) in the chambers. Agreement between predicted concentrations of ozone and NO2 and those actually observed was excellent. Based upon such modelling estimates and our acute toxicological data, we conclude that synergistic toxicologic interactions between ozone and NO2 are found only at concentrations very much higher than would be encountered in environmental or occupational settings. It remains to be determined whether there are any chronic toxicological responses to exposure to combinations of ozone and NO2 at concentrations below the thresholds for observing acute responses.
Administration, Inhalation↗
Concentration-response relationships of rat lungs to exposure to oxidant air pollutants: a critical test of Haber's Law for ozone and nitrogen dioxide.
Exposure protocols were designed to ask whether lung damage in rats exposed to either ozone or nitrogen dioxide is proportional to dose rate or to cumulative dose. Thus, the response of rats to a constant product of concentration of oxidant air pollutant and time of exposure (C x T) was evaluated for 3-day exposures over a fourfold range of concentrations of ozone (0.2-0.8 ppm) or of nitrogen dioxide (3.6-14.4 ppm) for exposure durations of 6-24 hr per day. The response of rat lungs was quantified by changes in total protein content of lung lavage supernatants or by changes in content of specific cell types in lung lavage pellets. The results of these experiments clearly demonstrate that acute lung damage is a function of cumulative dose (that is, C x T product) for the three highest dose rates tested. However, when exposure duration is extended to include the entire 24-hr period (the lowest dose rate tested), there is a marked attenuation of pulmonary response. Rats were also exposed to mixtures of ozone and nitrogen dioxide with the C x T product held constant. Our results clearly demonstrate that when rats are exposed to combinations of ozone and nitrogen dioxide, lung damage is a function of peak concentration rather than a function of cumulative dose. This deviation from Haber's Law is attributed to a concentration-dependent, synergistic (greater than additive) response to this specific mixture of oxidant air pollutants.
Administration, Inhalation↗
Silica increases cytosolic free calcium ion concentration of alveolar macrophages in vitro.
Rat alveolar macrophages were exposed to silica dust (quartz) suspended in culture medium (SiO2, dry particle size less than 5 microns in diameter) and fluctuation in their cytosolic free calcium content ([Ca2+]i) was detected in cell monolayers with a fluorescent calcium probe (Indo-1AM). Cytosolic free calcium content was correlated with lactate dehydrogenase (LDH) release, an index of cell damage. SiO2 induced a concentration- and time-dependent increase of cytosolic free Ca2+ ion concentration and LDH release. [Ca2+]i was increased about fivefold when cells were exposed to 200 micrograms of SiO2 per milliliter (3 ml per dish) for 2 hr. [Ca2+]i changed within 15 min of SiO2 treatment, whereas LDH release was measurably increased only after 30 min. Chelation of extracellular Ca2+ by 2 mM ethylene glycol bis(beta-aminoethyl ether) N,N'-tetraacetate did not prevent SiO2-induced fluctuation of macrophage [Ca2+]i, but did partially prevent the SiO2-induced increase in LDH release (p less than 0.01). We conclude that a very early event in SiO2-induced damage of alveolar macrophages involves mobilization of intracellular calcium pools to increase [Ca2+]i. These results suggest that SiO2-induced macrophage damage, a key event in the development of silicosis, may involve perturbation of intracellular calcium homeostasis.
Animals↗