Lester W. Schwartz

Biochemical and structural alterations of hamster lungs in response to intratracheal administration of bleomycin

Abstract One unit of bleomycin was administered intratracheally to hamsters and an equivalent volume of saline to controls. Morphological changes within lungs of bleomycin-treated hamsters consisted of a diffuse hemorrhagic interstitial pneumonia at 7 days post-treatment. Lungs contained less hemorrhage and edema but increased numbers of mononuclear inflammatory cells and thickened interalveolar walls at 14 days post-treatment. A diffuse mononuclear cell infiltrate with multifocal areas of fibrosis later predominated. The protein, RNA, and DNA levels in bleomycin-treated hamsters were consistently and significantly elevated at 4 (except DNA), 7, 14, 21, and 28 days after treatment. The Ca 2+ levels in lungs of these animals at the corresponding times were increased by 158, 194, 36, 22, and 8% without any change in plasma Ca 2+ . Lung soluble collagen was increased by 124, 207, 121, and 30% at 7, 14, 21, and 28 days, respectively, after bleomycin treatment. The increases in insoluble collagen were 65, 108, 132, and 91% at the corresponding times. The lung cAMP levels at 4, 7, 14, 21, and 28 days were 176, 164, 132, 158, and 96% of control, respectively, and cGMP at the corresponding times were 50, 81, 222, 198, and 137% of control. These data suggest that shifts in the intracellular levels of cAMP, cGMP, and Ca 2+ are associated with early lung changes induced by bleomycin insult and may serve as indicators to gauge the severity and progression of lung damage.

Stimulation by Cigarette Smoke of Glutathione Peroxidase System Enzyme Activities in Rat Lung

This study was undertaken to evaluate the effect of in vivo cigarette smoke exposure on glutathione peroxidase--related enzyme systems of the rat lung. These enzymes, acting in concert, are thought to be responsible for disposing of toxic lipid peroxides in pulmonary tissue. Thirty-day-old rats were exposed to thirteen cigarettes per day for 21 days with a Walton reverse-smoking exposure apparatus. After 21 days of smoke exposure, the activities of glutathione peroxidase, glutathione reductase, and glucose-6-phosphate dehydrogenase were increased 34%, 24%, and 38%, respectively, over control values. This level of cigarette smoke exposure did not cause detectable histological lesions. We present the hypothesis that short-term, low-level cigarette smoke exposure is capable of initiating metabolic alterations in lung cells at exposures at which histological changes are not detectable by light microscopy.

Morphological effects of prolonged exposure to ozone and sulfuric acid aerosol on the rat lung

Abstract The purpose of this study was to determine the pulmonary effects of a combination of ozone (0.5 ppm) and sulfuric acid aerosol (1 mg/m 3 ) and to assess the possibility of interactive effects. Groups of Sprague-Dawley rats were continuously exposed to the pollutants, either individually or combined, for 3, 50, 90, or 180 days. After 180 days of exposure, additional groups breathed clean air for a further 62 days. Morphological evaluation included light microscopy, autoradiography, scanning electron microscopy (SEM), and transmission electron microscopy. Quantification of pulmonary centriacinar inflammatory cell response was performed by SEM. The results clearly demonstrated that exposure to 0.5 ppm ozone for 180 days resulted in a persistent inflammatory response in the pulmonary centriacinar region together with a structural modification of the terminal bronchiole—proximal alveolar duct junction. Sulfuric acid aerosol did not induce pulmonary morphological changes, nor did it potentiate lesions produced by simultaneous ozone exposure. After termination of the 62-day postexposure period, ozone and ozone plus sulfuric acid postexposure rats demonstrated a marked diminution in the intensity of the pulmonary centriacinar inflammatory response and a partial restoration of normal centriacinar structure.

cAMP-specific phosphodiesterase inhibitor, rolipram, reduces eosinophil infiltration evoked by leukotrienes or by histamine in guinea pig conjunctiva

The effect of rolipram, an isozyme IV-selective inhibitor of cAMP-specific phosphodiesterase, was evaluated in a guinea pig eye model of tissue eosinophilia. (R)-rolipram was administered by gavage to guinea pigs 1 h prior to topical ocular challenge with a mixture of leukotrienes (LTs) (10 ng LTB4 + 1000 ng LTD4/eye) or with histamine dihydrochloride (1 mg/eye). Conjunctivae were evaluated histologically 6 h after challenge. Eosinophil counts per millimeter of conjunctival epithelium in LT-challenged animals that received (R)-rolipram at dosages of 0.1, 0.3, 1, 3, or 10 mg/kg were reduced by 63, 63, 84, 81 and 90% respectively, compared to LT-challenged controls. Reduction was statistically significant (P<0.05) at all dosages. Eosinophil counts per millimeter of epithelium in histamine-challenged animals that received 10 mg/kg (R)-rolipram were reduced by 79% compared to histamine-challenged controls (P<0.01). The results indicate that (R)-rolipram inhibits the response to two distinct classes of mediator in this model of eosinophil infiltration, adding support to the contention that isozyme IV-selective cAMP phosphodiesterase inhibitors offer therapeutic potential for human asthma.

Evaluation of plasma von Willebrand factor as a biomarker for acute arterial damage in rats

Plasma von Willebrand factor (vWF) was evaluated as a potential biomarker of acute arterial damage in rats after a vasotoxic dose of the dopaminergic vasodilator, fenoldopam (FP). Male Sprague-Dawley rats were given FP or isotonic saline by subcutaneous injection, and plasma vWF was measured at 2, 6, and 24 hours after challenge. Mean plasma vWF values increased in FP-treated rats compared to controls at 2 hours (167 vs 122%; p < 0.05) and 6 hours postdose (172 vs 130%; p < 0.01) but were comparable to control values after 24 hours. Mesenteric arterial lesions were observed microscopically in all FP-treated rats 24 hours postdose but were not present in rats at 1, 2, 4, 6, or 8 hours after FP challenge. Further, plasma vWF concentrations increased in saline-treated rats after only the minimal perturbation of repeated venipuncture. These results indicate an early, minimal, and transient release of vWF that precedes the onset of morphologically evident vascular damage. The minimal increases in plasma vWF concentrations were of limited predictive value, may be more reflective of an acute-phase reactant response, and were not considered a reliable biomarker of acute FP-induced arterial damage in the rat.

Transcriptional Profiling of Laser Capture Microdissected Rat Arterial Elements: Fenoldopam-induced Vascular Toxicity as a Model System:

Transcriptional profiling of specific elements of vasculature from animal models of vascular toxicity is an approach to gain insight into molecular mechanisms of vascular injury. Feasibility of using laser capture microdissection (LCM) to evaluate differential gene expression in selected elements of mesenteric arteries (MA) from untreated rats and rats given a single vasotoxic dose of 100 mg/kg Fenoldopam and euthanized 1 or 4 hours postdose was assessed. Regions of MA (endothelial cells [EC] and vascular smooth muscle cells [VSMC]) were selectively microdissected from optimal-cutting-temperature (O.C.T.)-embedded-frozen tissue sections. RNA was isolated, linearly amplified (LA), and hybridized to Affymetrix GeneChips®. Enrichment for specific vascular elements was evident by unique gene-expression profiles. Statistical analysis indicated that Fenoldopam treatment resulted in differential expression of 333 versus 458 genes in EC and 371 versus 618 genes in VSMC at the 1-hour or 4-hour time point, respectively. Analysis of regulated EC and VSMC genes common to both time points identified several gene functions or pathways affected by treatment. Several genes were identified in EC and/or VSMC that have not been previously linked to vascular structure or function. These data indicate that tissue–element-enrichment by LCM in conjunction with LA and GeneChip analysis offers a refined approach for assessment of injury-mediated transcriptome changes in distinct elements of the vasculature.

An ontogenic study of histamine and mast cells in the fetal rhesus monkey

This study was designed to determine the presence of cell-bound histamine and mast cells, if any, in the lung and skin of nonhuman primate fetuses and neonates. The results indicate that cell-bound histamine can be detected in both tissues of the rhesus monkey fetus by 60 days of gestation, the earliest age examined, at levels that are 2% to 3% of levels present in yearling animals. Levels increase markedly during the least 1 1/2 mo of gestation; however, at birth, levels are only 12% to 46% of those observed in the yearling and are lower than levels observed at 150 days of gestation. Most cell numbers in lung and skin of the fetus closely parallel histamine content.

Characteristics and effect of antiinflammatory drugs on adriamycin-induced inflammation in the mouse paw

A subplantar injection of 5–100 μg adriamycin in the mouse hind paw produced a biphasic inflammatory response. The first phase peaked at 2h while the second, more severe phase peaked at four to five days. The magnitude of inflammation was dose related. Administration of [3H]adriamycin revealed that 78% of the drug was lost from the paw within one day. The loss of the remaining drug followed a biphasic decay curve. The first-phase half-life was 1.2 days, and the second-phase half-life was 16.0 days. Vascular permeability, as measured by the leakage of intravenously administered [125I]albumin, was increased between day 4 and day 8. Pathologically, the paw had mild edema and hemorrhage by 4 h after adriamycin injection. The most severe pathological response was seen at 5 days with diffuse inflammation characterized by edema of the dermis, cellular debris, and mononuclear inflammatory cells. By 10 days the inflammatory response was still present but the edema was milder. The antihistamine diphenhydramine, an H1-blocker, inhibited the first phase of inflammation at the highest dose tested but had no effect on the second phase of inflammation. The antihistamine metiamide, an H2-blocker; the antiserotonin drug, ρ-chlorophenylalanine; and the antiinflammatory drugs, aspirin, hydrocortisone, and ibuprofen failed to antagonize adriamycin-induced inflammation at 2 h or 5 days after adriamycin injection. Indomethacin reduced the inflammation after 5 days but only at toxic dose levels.

Effects of p38 MAP Kinase Inhibitors on the Differentiation and Maturation of Erythroid Progenitors

In rodents, p38 MAP kinase inhibitors (p38is) induce bone marrow hypocellularity and reduce reticulocyte and erythrocyte counts. To identify target cell populations affected, a differentiating primary liquid erythroid culture system using sca-1+cells from mouse bone marrow was developed and challenged with p38is SB-203580, SB-226882, and SB-267030. Drug-related alterations in genes involved at different stages of erythropoiesis, cell-surface antigen expression (CSAE), burst-forming unit erythroid (BFU-E) colony formation, and cellular morphology (CM), growth (CG), and viability were evaluated. CSAE, CM, and decreases in BFU-E formation indicated delayed maturation, while CG and viability were unaffected. Terminal differentiation was delayed until day 14 versus day 7 in controls. CSAE demonstrated higher percentages of sca-1+cells after day 2 and reduced percentages of ter119+ cells after day 7 in all treated cultures. Real-time reverse transcriptase polymerase chain reaction revealed a transient delay in expression of genes involved at early, intermediate, and late stages of erythropoiesis, followed by rebound expression at later time points. Results demonstrate p38is do not irreversibly inhibit erythrogenesis but induce a potency-dependent, transient delay in erythropoietic activity. The delay in activity is suggestive of effects on sca-1+bone marrow cells caused by alterations in expression of genes related to erythroid commitment and differentiation resulting in delayed maturation.