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Biomedical subjects

J A Last

Publications and source records attributed to J A Last.

At least 19 recordsLinked to original sources
PubMed1995

Rat lysyl hydroxylase: molecular cloning, mRNA distribution and expression in a baculovirus system.

A cDNA library from rat lung was screened with a chicken lysyl hydroxylase cDNA, and several overlapping rat lysyl hydroxylase cDNAs were isolated. The complete cDNA was 91 and 77% identical, respectively, to the human and chicken lysyl hydroxylase cDNAs at the protein level. By Northern blot, the rat lysyl hydroxylase cDNA recognized a single 3.2 kb mRNA that was present in a wide variety of rat tissues. In order to further confirm the identity of this cDNA, the cDNA was expressed in insect cells via a baculovirus vector. These cells produced an 85 kDa protein with lysyl hydroxylase activity. The recombinant lysyl hydroxylase had a specific activity and Km values for its substrates that were similar to those of the enzyme isolated from chick embryos. The fact that this single lysyl hydroxylase cDNA encodes a protein sufficient for lysyl hydroxylase activity is consistent with previous biochemical findings that lysyl hydroxylase only requires a single type of subunit for its activity.

Amino Acid Sequence↗
PubMed1995

Effects of exposure to environmental tobacco smoke on a human tracheobronchial epithelial cell line.

BEAS-2B cells, a human bronchial epithelial line immortalized by viral transformation, were exposed to sidestream tobacco smoke (STS) as a surrogate for environmental tobacco smoke (ETS) under biphasic culture conditions where the apical portion of the cells was in direct contact with the gas phase. Dose-dependent cytotoxicity was observed. In addition, induction of an as yet uncharacterized protein of molecular weight 45,000 was associated with exposure to STS. This protein might be part of a protective response of exposed cells, which do not show a classical heat shock response when exposed to STS. We conclude that STS and ETS can be directly cytotoxic to human airway epithelial cells in biphasic culture at concentrations not unreasonable for smoky indoor atmospheres. The model system described in this paper should be useful for studying the detailed mechanisms of cytotoxicity of, and protection from, ETS exposure in the human cells most directly exposed to ETS in vivo.

Bronchi↗
PubMed1995

Restrictive lung disease in rats exposed chronically to an urban profile of ozone.

The potential for irreversible lung impairment resulting from life-long ozone (O3) exposure remains uncertain. To address this question, young adult rats (male, F-344) were exposed to a simulated urban profile of O3 for 1, 3, 13, 52, or 78 wk, after which pulmonary function tests were performed. To assess reversibility of effects, cohorts from the 13-, 52-, and 78-wk groups were evaluated, respectively, after an additional 6, 27, and 17 wk of clean air. Static and dynamic lung properties were based on measurements of lung volume apportionment, respiratory system compliance (Crs), DLCO, multibreath N2 washout, and maximum expiratory flow-volume relationships. Electrocardiography was also performed in unanesthetized, restrained rats after 52 and 78 wk, as were determinations of wet and dry lung weights, lung collagen, and associated connective tissue crosslinks. Small (< 10%) but significant reductions in TLC and RV were noted after 13, 52, and 78 wk of O3 exposure. At 13 and 52 wk, N2 washout was enhanced, though at 78 wk it was similar to control. None of these changes appeared progressive with continued O3 exposure. Post exposure to clean air did not completely reverse the reduction in TLC. Additionally, Crs, though not affected during O3 exposure, decreased during the air recovery. No O3-related changes in collagen were apparent, however. Thus, near life-long exposure of F-344 rats to a worse-case, urban profile of O3 appears to have led to a functionally restrictive, i.e. "stiffened," lung without overt fibrosis. Furthermore, certain aspects of the O3-induced effect were not fully reversible.

Air Pollutants↗
PubMed1994

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↗
PubMed1994

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↗
PubMed1994

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↗
PubMed1994

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↗
PubMed1994

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↗
PubMed1992

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↗
PubMed1992

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↗
PubMed1992

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↗
PubMed1991

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↗
PubMed1991

Global atmospheric change: potential health effects of acid aerosol and oxidant gas mixtures.

Inhalation toxicology experiments in whole animals have demonstrated a remarkable lack of toxicity of sulfuric acid in the form of respirable aerosols, especially in rats and nonhuman primates. Thus, much of the current experimental emphasis has shifted to the evaluation of the potential health effects of acid aerosols as components of mixtures. Rats have been concurrently exposed to mixtures of ozone or nitrogen dioxide with respirable-sized aerosols of sulfuric acid, ammonium sulfate, or sodium chloride, or to each pollutant individually. Their responses to such exposures have been evaluated by various quantitative biochemical analysis of lung tissue or wash fluids ("lavage fluid") or by quantitative morphological methods ("morphometry"). Such studies have mainly been performed in the acute time frame due to the inherent limitations of the most sensitive assays available and have generally involved exposures for 1 to 9 days, depending on the assays used. Good correlations were found between the most sensitive biochemical indicators of lung damage (protein content of lung lavage fluid or whole lung tissue and lung collagen synthesis rate) and the exposure concentration of oxidant gas present alone or in mixtures with acidic aerosols showing interactive effects. Synergistic interaction between ozone and sulfuric acid aerosol was demonstrated to occur at environmentally relevant concentrations of both pollutants by several of the analytical methods used in this study. Such interactions were demonstrated at concentrations of ozone as low as 0.12 ppm and of sulfuric acid aerosol at concentrations as low as 5 to 20 micrograms/m3.(ABSTRACT TRUNCATED AT 250 WORDS)

Acids↗
PubMed1991

Synergistic effects of air pollutants: ozone plus a respirable aerosol.

Rats were concurrently exposed to mixtures of ozone or nitrogen dioxide and respirable-sized aerosols of sulfuric acid, ammonium sulfate, or sodium chloride, or to each pollutant individually. Their responses to such exposures were evaluated by various quantitative biochemical analyses of lung tissue or lavage fluids, or by morphometric analyses. Such studies were performed in the acute time frame, generally involving exposures of from one to nine days, depending on the assays used. Correlations between the biochemical and morphometric results were examined over a wide range of pollutant concentrations in the exposure chambers. Good correlations were found between the most sensitive biochemical indicators of lung damage--the protein content of lung lavage fluid or whole lung tissue and the rate of lung collagen synthesis--and the morphometric estimation of volume density or volume percent of the centriacinar lung lesion characteristically observed in animals exposed to ozone. Synergistic interaction between ozone and sulfuric acid aerosol was demonstrated to occur at environmentally relevant concentrations of both pollutants by several of the analytical methods used. Such interactions were demonstrated at concentrations of ozone as low as 0.12 parts per million (ppm)2 and of sulfuric acid aerosol at concentrations as low as 5 to 20 micrograms/m3. The acidity of the aerosol is a necessary (and apparently a sufficient) condition for such a synergistic interaction between an oxidant gas and a respirable aerosol to occur. A hitherto unexpected synergistic interaction between nitrogen dioxide and sodium chloride aerosol was found during these studies; it is hypothesized that this was due to formation of their acidic (anhydride) reaction product, nitrosyl chloride, in the chambers during exposure to the mixture. Preliminary experiments treating exposed animals in vivo with various free-radical scavengers suggested that dimethylthiourea, a hydroxyl-radical scavenger, might be protective against effects of ozone on rat lungs. This observation might have mechanistic implications, but further studies will be necessary to determine the significance of these findings.

Aerosols↗