Michael W. Conner

Time course of bleomycin-induced lung fibrosis.

Intratracheal instillation (IT) of bleomycin is a widely used experimental model for lung fibrosis. In this study we describe the time‐course of bleomycin‐induced lung fibrosis in mice using computer‐assisted morphometry. C57Bl/6J mice were treated with a single IT dose of bleomycin or control saline. Animals were killed 3, 6, 14 and 21 days post‐IT. Lung injury was evaluated by analysis of bronchoalveolar lavage (BAL) fluid, hydroxyproline concentration in the lung, routine light microscopic examination resulting in a semiquantitative morphological index (SMI) of lung injury, and quantitative morphological measurements (fibrosis fraction and alveolar wall area fraction) aided by optimas image analysis software. Changes in BAL fluid attributed to bleomycin treatment include increased total cell count (days 14 and 21), and increased percentage of neutrophils (days 3 and 6) followed by a sustained increase in lymphocytes (days 6, 14 and 21). Hydroxyproline levels increased in bleomycin‐treated mice on days 14 and 21. Median SMI grades were significantly elevated on days 3, 14 and 21. Computer‐assisted morphometry demonstrated a 3‐fold increase in fibrosis fraction and a 1.3‐fold increase in wall area fraction in bleomycin‐treated mice on day 14, with no further increase on day 21. These data also demonstrate that the most suitable time point for assessing lung fibrosis in this model is 14 days after IT instillation of bleomycin, based on the observation that at 14 days the animals developed extensive fibrosis, but had less variability in the fibrotic response and lower mortality than later at 21 days. Computer‐assisted morphometry provides objective and quantitative measurements that are a useful tool for the evaluation of bleomycin‐induced lung injury.

The Influence of Food Additives and Related Materials on Lower Bowel Structure and Function

Food additives, drugs, and other chemicals are known to influence the lower gastrointestinal tract under some defined conditions, resulting in morphological alterations in the mucosa and other tissues, deranged absorption and excretion of nutrients, and, in some cases, injury to other organs and tissues as a secondary phenomenon. Generally, in rats, hamsters, and dogs, there is increased absorption and urinary excretion of calcium, soft stools or diarrhea, and cecal enlargement. In the rat, hamster, and dog, renal lesions accompany the hypercalcemia and elevated excretion of calcium. These signs, symptoms, and lesions are typical of exposure to sugar alcohols (sorbitol, mannitol, xylitol, lactitol), lactose and caramel, some of the chemically modified food starches, and synthetic polydextrose. Soft stools and diarrhea, as well as cecal enlargement and variable hyperplasia of the colon mucosa, occur frequently when substances are absorbed incompletely in the small intestine and subjected to microbial metabolism in the cecum and colon. The remarkable cecal enlargement, mucosal hyperplasia and, when present, colonic mucosal hyperplasia are reversible, even when long-standing. Renal lesions are reversible only if exposure is of short duration, before significant mineralization and scarring has occurred. Morphological and functional anomalies associated with some of these substances are described and illustrated.

Coal combustion aerosols and sulfur dioxide: an interdisciplinary analysis.

Coal combustion produces both SO/sub 2/ and submicrometer metal oxides that can react to form irritant aerosols with potential effects on health. Experimental coal combustion studies defined conditions leading to the formation of submicrometer fume and characterized its composition. Effects of temperature and humidity during mixing on the reaction products of SO/sub 2/ and ZnO, which occur in the fume, were determined. These atmospheres produced altered pulmonary function and morphology in guinea pigs. Functional and morphologic changes were correlated. 42 references, 8 figures, 2 tables.

Lung injury in guinea pigs caused by multiple exposures to ultrafine zinc oxide: Changes in pulmonary lavage fluid

Metal oxide particles with diameters of less than 0.1 micron (ultrafine particles) are important products of fossil fuel combustion. Pulmonary lavage fluid was obtained from guinea pigs given 1, 2, or 3 consecutive, daily, 3-h, nose-only exposures to 0, 2.3, 5.9, or 12.1 mg/m3 of freshly generated zinc oxide (ZnO) particles with a projected area diameter of 0.05 micron. Exposure to ZnO at 5.9 or 12.1 mg/m3 was associated with increased protein, neutrophils, and activities of angiotensin-converting enzyme, alkaline phosphatase, acid phosphatase and lactate dehydrogenase in lavage fluid, and with histologic evidence of pulmonary damage characterized by centriacinar inflammation. The severity of inflammation, graded by the number of inflammatory foci per square centimeter of lung, correlated with the amount of protein and the activity of angiotensin-converting enzyme and other enzymes in lavage fluid. These results indicate that analysis of pulmonary lavage fluid is a useful and sensitive method for quantitative evaluation of pulmonary damage caused by inhalation of low levels of ultrafine ZnO.

Superoxide dismutase inhibits radiation-induced lung injury in hamsters.

An animal model of pulmonary radiation-induced lung injury was established in the hamster and the effects of pretreatment with recombinant human CuZn superoxide dismutase (SOD) on the development of the lesion were evaluated. Hamsters exposed to a single irradiation dose of 2000 cGy delivered to the thorax were treated with 150 mg/kg body weight of SOD or an equivalent volume of saline intraperitoneally 75 min and subcutaneously 5 min before receiving irradiation. At 4, 8, and 16 weeks following irradiation, pulmonary injury was evaluated by the grading of morphologic changes semiquantitatively, measurement of lung hydroxyproline content, and analysis of bronchoalveolar lavage fluid for total and differential cell counts and total protein concentration. Radiation-induced lung injury in saline-pretreated animals was documented at 16 weeks by histologic morphology and increased protein in bronchoalveolar lavage fluid. SOD protected against radiation-induced pulmonary injury as indicated by the absence of severe histopathologic changes and prevention of elevation in bronchoalveolar lavage protein levels. The beneficial effects of SOD in preventing radiation-induced pulmonary toxicity suggests that this recombinant enzyme may play a role in protection against radiation-induced pulmonary injury in humans.

Amelioration of bleomycin-induced pulmonary injury by cyclosporin A

This study evaluated the effect of cyclosporin-A (CyA), a potent immunosuppressive drug, on Bleomycin (Bleo)-induced pulmonary inflammation in hamsters. Pulmonary injury was induced by a single intratracheal (i.t.) instillation of Bleo. Four groups of 10 male Syrian hamsters each received one of four treatments: (1) i.t. Bleo and daily intraperitoneal (i.p.) injections of CyA starting 1 day before i.t. instillation of Bleo (Bleo-CyA); (2) i.t. Bleo and i.p. injections of saline (Bleo-Sal); (3) i.t. saline and i.p. CyA (Sal-CyA); (4) i.t. saline and i.p. saline (Sal-Sal). Animals were sacrificed 14 days after i.t. treatment. Lung injury was evaluated histologically, biochemically, and by analysis of bronchoalveolar lavage (BAL) fluid. Treatment of hamsters with CyA significantly ameliorated the Bleo-induced lung injury, as determined by a semiquantitative morphological index that assesses the severity and extent of the injury on a scale of 0-3. Lung hydroxyproline measurements were lower in Bleo-CyA compared to Bleo-Sal, comparable to Sal-Sal and Sal-CyA controls. The percentage of neutrophils, eosinophils, and lymphocytes in BAL fluid was higher in Bleo-Sal and Bleo-CyA animals when compared with control Sal-CyA or Sal-Sal animals. A further increase in percentage of eosinophils was observed in Bleo-CyA compared with Bleo-Sal animals (13.3 +/- 6.6% [mean +/- SE] and 3.7 +/- 2.1%, respectively, p = .0007). BAL fluid protein content was higher in Bleo-Sal compared to Sal-Sal animals, but BAL fluid protein content from Bleo-CyA was not significantly different from that of Bleo-Sal animals. These results indicate that CyA ameliorates the Bleo-induced inflammation but does not prevent leakage of plasma protein or cells into the airspaces. The increased eosinophil numbers in Bleo-CyA-treated hamsters suggests enhanced production of interleukin-4 and -5.

Mapping of β-Adrenergic Receptors in Rat Lung: Effect of Isoproterenol

β-Adrenergc receptors were mapped in the rat lung using a light microscopic technique that identifies binding sites for H-labeled dihydroalprenolol in intact slide-mounted tissue sections, which were also used for biochemical analysis of ligand binding. Grain density measurements were combined with morphometric data. These results indicate that, although over 97% of the specific binding is to cells of alveolar walls, ^-adrenergic receptors are present and can be quantified in tissues that represent a small fraction of total lung mass, such as bronchial epithelium and smooth muscle of bronchial walls and pulmonary vessels. Repeated administration of isoproterenol decreased the receptor number, as determined biochemically and from grain density measurements, in all anatomic regions studied, but did not alter the distribution of binding.

Effect of IL-2-Bax, a novel interleukin-2-receptor-targeted chimeric protein, on bleomycin lung injury.

The role of lymphocytes in the pathogenesis of lung fibrosis is not clear, but the weight of the evidence supports a pro‐fibrotic effect for lymphocytes. The high‐affinity interleukin‐2 receptor (haIL‐2R) is expressed on activated, but not quiescent, T lymphocytes. This selective expression of haIL‐2R provides the basis for therapeutic strategies that target IL‐2R‐expressing cells. We hypothesized that elimination of activated lymphocytes by IL‐2R‐targeted chimeric proteins might ameliorate lung fibrosis. We investigated the effects of IL‐2‐Bax, a novel apoptosis‐inducing IL‐2R‐targeted chimeric protein, on bleomycin‐induced lung injury in mice. Treatment groups included (i) a single intratracheal instillation of bleomycin and twice‐daily intraperitoneal injections of IL‐2‐Bax; (ii) intratracheal bleomycin and intraperitoneal IL‐2‐PE664Glu, an older‐generation chimeric protein; (iii) intratracheal bleomycin/intraperitoneal PBS; (iv) intratracheal saline/intraperitoneal PBS. Lung injury was evaluated 14 days after intratracheal instillation by cell count in bronchoalveolar lavage (BAL) fluid, semi‐quantitative and quantitative histomorphological measurements and by biochemical analysis of lung hydroxyproline. Bleomycin induced a BAL lymphocytosis that was significantly attenuated by IL‐2‐Bax and IL‐2‐PE664Glu. However, morphometric parameters and lung hydroxyproline were unaffected by the chimeric proteins. These results show that IL‐2‐Bax reduces the lymphocytic infiltration of the lungs in response to bleomycin, but this effect is not accompanied by a decrease in lung fibrosis.

Anguidine-induced testicular injury in Lewis rats☆

Anguidine (diacetoxyscirpenol, DAS) and other trichothecene mycotoxins are potent inhibitors of protein synthesis and injure organs with rapidly dividing cell populations, including the testis. Testicular structure and function were studied in male Lewis rats 1, 3, 7, 30, 60, and 90 days after exposure at age 12 weeks to anguidine at 1.7 mg/kg body weight given by ip injection. The dose was equivalent to 75% of the ip LD50. Anguidine caused a gradual decline in testicular weight beginning 30 days after treatment. Sperm production was also reduced by 30 days, and the frequency of hypocellular seminiferous tubules increased by day 60. There was no evidence of recovery by 90 days. These changes are consistent with injury to proliferating cells early in the maturation sequence. Epididymal sperm reserves were reduced by 3 days after anguidine administration, prior to the reduction in sperm production, suggesting premature release of spermatozoa from the epididymis.

Changes in pulmonary lavage fluid of guinea pigs exposed to ultrafine zinc oxide with adsorbed sulfuric acid

Ultrafine metal oxide particles (diameters less than 0.1 microns) and sulfur dioxide are important products of coal combustion. Interaction of these products in the effluent stream results in formation of ultrafine particles with adsorbed sulfur compounds, including sulfuric acid. The toxicity of ultrafine zinc oxide particles with adsorbed sulfuric acid was evaluated by comparing pulmonary lavage fluid from guinea pigs exposed for 1, 2, 3, 4, or 5 consecutive daily 3-h periods to ultrafine zinc oxide generated in the presence of sulfur dioxide (ZnO + SO2) to pulmonary lavage fluid from guinea pigs exposed to an equivalent concentration of ultrafine ZnO. Two groups of guinea pigs exposed either to SO2 or to particle-free furnace gas served as additional controls. Cells, protein, and activities of lactate dehydrogenase, acid phosphatase, and alkaline phosphatase were increased in lavage fluid obtained from guinea pigs exposed to ZnO + SO2 as compared to guinea pigs exposed to ZnO. These results demonstrate the potential importance of ultrafine metal oxides as carries of sulfuric acid derived from fossil fuel combustion.