Bleomycin Lung Injury Research Articles

Angiotensin-Converting Enzyme N-Terminal Inactivation Alleviates Bleomycin-Induced Lung Injury

Bleomycin has potent anti-oncogenic properties for several neoplasms, but drug administration is limited by bleomycin-induced lung fibrosis. Inhibition of the renin-angiotensin system has been suggested to decrease bleomycin toxicity, but the efficacy of such strategies remains uncertain and somewhat contradictory. Our hypothesis is that, besides angiotensin II, other substrates of angiotensin-converting enzyme (ACE), such as the tetrapeptide N-acetyl-seryl-aspartyl-lysyl-proline (AcSDKP), play a significant role in controlling fibrosis. We studied bleomycin-induced lung injury in normotensive mice, termed N-KO and C-KO, which have point mutations inactivating either the N- or C-terminal catalytic sites of ACE, respectively. N-KO, but not C-KO mice, have a marked resistance to bleomycin lung injury as assessed by lung histology and hydroxyproline content. To determine the importance of the ACE N-terminal peptide substrate AcSDKP in the resistance to bleomycin injury, N-KO mice were treated with S-17092, a prolyl-oligopeptidase inhibitor that inhibits the formation of AcSDKP. In response to bleomycin injection, S-17092-treated N-KO mice developed lung fibrosis similar to wild-type mice. In contrast, the administration of AcSDKP to wild-type mice reduced lung fibrosis due to bleomycin administration. This study shows that the inactivation of the N-terminal catalytic site of ACE significantly reduced bleomycin-induced lung fibrosis and implicates AcSDKP in the mechanism of protection. These data suggest a possible means to increase tolerance to bleomycin and to treat fibrosing lung diseases.

An Essential Role for Resident Fibroblasts in Experimental Lung Fibrosis Is Defined by Lineage-Specific Deletion of High-Affinity Type II Transforming Growth Factor β Receptor

Fibrotic response to lung injury depends on development of a fibrogenic population of myofibroblasts. The importance of resident interstitial fibroblasts and role of transforming growth factor β (TGFβ) in this process is unclear. To define the importance of TGFβ signaling in resident lung fibroblasts in the development of experimental pulmonary fibrosis. A compound genetic strategy in which mice homozygous for a floxed high-affinity type II TGFβ receptor (TβRII) allele were crossed with a transgenic strain harboring a fibroblast-specific transgene encoding ligand-dependent Cre-recombinase was used. TβRII was deleted by postnatal administration of tamoxifen over 5 days to compound mutant mice with appropriate littermate controls. Illumina microarray gene profiling and quantitative reverse transcriptase-polymerase chain reaction were used to confirm anergy to TGFβ in explanted lung fibroblasts. Bleomycin lung injury was used to induce lung fibrosis, which was analyzed by histology and biochemical methods. Immunofluorescence was used to define cell populations after lung injury. There was significant attenuation of fibrosis in mice after deletion of TβRII in resident fibroblasts. At 7 days after injury the number of fibrocytes and myofibroblasts was substantially reduced. Potential regulators of fibrosis were suggested by gene expression profiles that identified key candidate profibrotic genes, including connective tissue growth factor and endothelin-1 expressed by wild-type but not mutant lung fibroblasts. Intact TGFβ signaling in resident pulmonary fibroblasts is essential for pulmonary fibrosis to develop. Our data support a key regulatory role of these cells in determining fibrocyte recruitment and myofibroblast differentiation.

Experimental bleomycin lung injury and preventive effects of urokinase

Experimental bleomycin lung injury and preventive effects of urokinase

Protection of bleomycin-induced fibrosis and inflammation by taurine

Taurine has been shown to protect against lung injury induced by various oxidants including ozone, nitrogen dioxide, amiodarone, and paraquat and to protect against bleomycin-induced lung injury in combination with niacin. In this study, Spraque-Dawley rats were treated with 5% taurine in the drinking water for 10 days prior to bleomycin instillation. Fibrosis in the rats pretreated with taurine (BT) was absent, along with fewer inflammatory infiltrates compared to the untreated rats (BW). A significant decrease in the number of PMNs and a decrease in hydroxyproline levels were found in the bronchoalveolar lavage fluid in the BT group compared to the BW group. By immunohistochemical staining, inducible nitric oxide synthase was evident in the lungs of bleomycin-treated rats, and minimal when rats were treated with taurine. Tumor necrosis factor-alpha (TNF-α) as measured by immunohistochemical staining, was present in lungs of both taurine-treated and untreated rats, but was more abundant in the BW group compared to the BT group. In addition, decreased ICAM presentation was detected by EM immunogold staining in the BT group compared to the BW group. These data demonstrate that rats pretreated with 5% taurine in their drinking water prior to bleomycin instillation are protected from fibrosis, inflammatory infiltrates, as well as nitric oxide and TNF-α production, which are hallmarks of bleomycin lung injury.

Bleomycin-induced pulmonary fibrosis is attenuated by a monoclonal antibody targeting HER2

The importance of HER2/HER3 signaling in decreasing the effects of lung injury was recently demonstrated. Transgenic mice unable to signal through HER2/HER3 had significantly less bleomycin-induced pulmonary fibrosis and showed a survival benefit. Based on these data, we hypothesized that pharmacological blockade of HER2/HER3 in vivo in wild-type mice would have the same beneficial effects. We tested this hypothesis in a bleomycin lung injury model using 2C4, a monoclonal antibody directed against HER2 that blocks HER2/HER3 signaling. The administration of 2C4 before injury decreased the effects of bleomycin at days 15 and 21 after injury. HER2/HER3 blockade resulted in less collagen deposition (362.8 +/- 37.9 compared with 610.5 +/- 27.1 microg/mg; P = 0.03) and less lung morphological changes (injury score of 1.99 +/- 1.55 vs. 3.90 +/- 0.76; P < 0.04). In addition, HER2/HER3 blockade resulted in a significant survival advantage with 50% vs. 25% survival at 30 days (P = 0.04). These results confirm that HER2 signaling can be pharmacologically targeted to reduce lung fibrosis and remodeling after injury.

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-PE66(4Glu), 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-PE66(4Glu). 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.

Expression of mutant human epidermal receptor 3 attenuates lung fibrosis and improves survival in mice

Neuregulin-1 (NRG-1), binding to the human epidermal growth factor receptor HER2/HER3, plays a role in pulmonary epithelial cell proliferation and recovery from injury in vitro. We hypothesized that activation of HER2/HER3 by NRG-1 would also play a role in recovery from in vivo lung injury. We tested this hypothesis using bleomycin lung injury of transgenic mice incapable of signaling through HER2/HER3 due to lung-specific dominant-negative HER3 (DNHER3) expression. In animals expressing DNHER3, protein leak, cell infiltration, and NRG-1 levels in bronchoalveolar lavage fluid increased after injury, similar to that in nontransgenic littermate control animals. However, HER2/HER3 was not activated, and DNHER3 animals displayed fewer lung morphological changes at 10 and 21 days after injury (P = 0.01). In addition, they contained 51% less collagen in injured lungs (P = 0.04). Transforming growth factor-beta1 did not increase in bronchoalveolar lavage fluid from DNHER3 mice compared with nontransgenic littermate mice (P = 0.001), suggesting that a mechanism for the decreased fibrosis was lack of transforming growth factor-beta1 induction in DNHER3 mice. Severe lung injury (0.08 units bleomycin) resulted in 80% mortality of nontransgenic mice, but only 35% mortality of DNHER3 transgenic mice (P = 0.04). Thus inhibition of HER2/HER3 signaling protects against pulmonary fibrosis and improves survival.

IL-10 inhibits inflammation but does not affect fibrosis in the pulmonary response to bleomycin

Bleomycin yields pulmonary injury characterized by inflammation that proceeds to fibrosis. The production of IL-10 by pulmonary macrophages is increased in the inflammation that accompanies bleomycin lung injury. In the present study, IL-10 deficient and wildtype mice received 0.075 units of bleomycin intratracheally at day 0 and were sacrificed at day 7 or day 14. At day 7, pulmonary inflammation was increased in IL-10-deficient mice as reflected by increased representation of CD3+ and CD4+ lymphocytes and GR-1+ pulmonary granulocytes in the bronchoalveolar lavage (BAL) fluid. Pulmonary interstitial CD80+ and CD86+ mononuclear cells were increased in situ. At day 14, mononuclear cell inflammation was comparable between groups but pulmonary eosinophils were increased in the wildtype. There was no difference in the degree of pulmonary fibrosis, as judged by histology or lung hydroxyproline content. Lung chemokine expression of MIP-1α/β, MIP-2, and eotaxin was increased at days 7 and 14 with a trend towards increased MCP-1 expression at day 14. The findings suggest an immunomodulatory role for IL-10 in the inflammatory response but not in the pulmonary fibrosis yielded by bleomycin.

Role of interferon-gamma in the evolution of murine bleomycin lung fibrosis.

IFN-gamma production is upregulated in lung cells (LC) of bleomycin-treated C57BL/6 mice. The present study characterizes the time course, cellular source, and regulation of IFN-gamma expression in bleomycin-induced lung injury. IFN-gamma mRNA in LC from bleomycin-treated mice peaked 3 days after intratracheal instillation. IFN-gamma protein levels were increased at 6 days, as was the percentage of LC expressing IFN-gamma. CD4+, CD8+, and natural killer cells each contributed significantly to IFN-gamma production. IL-12 mRNA levels were increased at 1 day in LC of bleomycin-treated mice. Anti-IL-12 and anti-IL-18 antibodies decreased IFN-gamma production by these cells. To define the role of endogenous IFN-gamma in the evolution of bleomycin lung injury, we compared the effect of bleomycin in mice with a targeted knockout mutation of the IFN-gamma gene (IFN-gamma knockout) and wild-type mice. At 14 days after intratracheal bleomycin, total bronchoalveolar lavage cell counts and lung hydroxyproline were decreased in IFN-gamma knockouts compared with wild-type animals. There was no difference in morphometric parameters of fibrosis. Our data show that enhanced IFN-gamma production in the lungs of bleomycin-treated mice is at least partly IL-12 and IL-18 dependent. Absence of IFN-gamma in IFN-gamma knockout mice does not increase pulmonary fibrosis. Endogenous IFN-gamma may play a proinflammatory or profibrotic role in bleomycin-induced lung fibrosis.

IL-12p40−/− Mice Treated with Intratracheal Bleomycin Exhibit Decreased Pulmonary Inflammation and Increased Fibrosis

Pulmonary lymphohistiocytic inflammation and fibrosis characterize bleomycin (BLM) lung injury. IL-12, a p70 cytokine produced primarily by macrophages and dendritic cells, promotes T-helper-1-mediated inflammation. IL-12 production by blood monocytes and bronchoalveolar large mononuclear cells (BAMC) was investigated at Days 1–14 following intratracheal administration of BLM. In the lung, BAMC showed a large peak of IL-12 expression at Day 5 that returned rapidly toward baseline. IL-12p40−/− mice treated with BLM intratracheally showed less pulmonary mononuclear cell inflammation at Day 7 than wild-type controls, whereas pulmonary fibrosis and hydroxyproline content were increased in IL-12p40−/− mice at Day 14. The expression of IP-10, RANTES, and eotaxin were decreased in IL-12p40−/− mice and lung IL-6 expression was increased, all compared to controls. We conclude that IL-12 promotes the lymphohistiocytic response to BLM and may inhibit the late development of pulmonary fibrosis.

Characterization of bleomycin lung injury by nuclear magnetic resonance: correlation between NMR relaxation times and lung water and collagen content.

The response of the NMR relaxation times (T(1), CPMG T(2), and Hahn T(2)) to bleomycin-induced lung injury was studied in excised, unperfused rat lungs. NMR, histologic, and biochemical (collagen content measurement) analyses were performed 1, 2, 4, and 8 weeks after intratracheal instillation of saline (control lungs) or 10 U/kg bleomycin sulfate. The control lungs showed no important NMR, water content, histologic, or collagen content changes. The spin-spin relaxation times for the fast and intermediate components of the CPMG decay (T(2f) and T(2i), respectively) increased 1 week after bleomycin injury (acute inflammatory stage) and then progressively decreased during the following 2-8 weeks (i.e., with the development of the chronic, fibrotic stage of the injury). The slow component (T(2s)) showed no significant changes. The response of T(1) and the slow component of the Hahn T(2) was, on the whole, similar to that of CPMG T(2f) and T(2i). T(1) changes were very small. Lung water content increased 1 week after injury. Histologic and biochemical assessment of collagen showed that collagen content was close to control at 1 week, but markedly increased at 2, 4, and 8 weeks. T(1) and T(2) data were directly correlated with lung water content and inversely correlated with collagen content. Our results indicate that NMR relaxation time measurements (particularly T(2)) reflect the structural changes associated with bleomycin injury. The prolonged T(2) relaxation times observed in the acute stage are related to the presence of edema, whereas the subsequent decrease in these values marks the stage of the collagen deposition (fibrotic stage). CPMG-T(2) and Hahn-T(2) measurements can be valuable as a potentially noninvasive method for characterizing bleomycin-induced lung injury and pathologically related lung disorders.

Modulation of bleomycin‐induced pulmonary toxicity in the hamster by the antioxidant amifostine

BACKGROUND Bleomycin produces lung fibrosis in a wide variety of species. In humans, it can cause significant morbidity and mortality when used to treat malignancies such as lymphoma and testicular carcinoma. In rodents, it has been extensively used to study key mechanisms of lung injury and repair. Bleomycin pulmonary toxicity is mediated, at least in part, by the generation of active oxygen species. Amifostine, an aminothiol compound, is a cytoprotectant that is used with many antitumor agents and can act as a potent scavenger of free radicals. The authors hypothesized that amifostine could ameliorate bleomycin lung injury. METHODS Hamsters weighing 120 g were given an intraperitoneal (IP) injection of amifostine (200 mg/kg, 1180 mg/m2) or saline with intratracheal (IT) bleomycin (1 unit) or saline, followed by daily IP amifostine or saline for 6 days. Lungs were assessed on Day 2 for acute lung injury, which was determined by wet-to-dry lung weight ratios. On Day 21, histologic assessment of fibrosis and biochemical analysis of lung hydroxyproline content were performed. RESULTS No significant differences in morbidity or mortality were observed among the groups. Animals who received IT bleomycin, when compared with controls, had increased lung water measurements on Day 2 that were consistent with acute inflammation; on Day 21, they had pulmonary fibrosis, as measured by morphometric analysis, as well as increased hydroxyproline content. For animals treated with amifostine and bleomycin, significant decreases in wet-to-dry lung weight ratios were observed (mean ± standard deviation, 4.5 ± 1.2 vs. 10.2 ± 2.7), as well as significant decreases in the percentage of fibrosis per lung (15.03% ± 3.27 vs. 37.26% ± 5.76) and hydroxyproline content (1.132 ± 0.30 vs. 1.831 ± 0.243). CONCLUSIONS Amifostine significantly decreased the amount of acute lung injury and subsequent fibrosis in the hamster model of bleomycin-induced lung injury. Cancer 1998;83:2008-2014. © 1998 American Cancer Society.

Modulation of bleomycin-induced pulmonary toxicity in the hamster by the antioxidant amifostine

Bleomycin produces lung fibrosis in a wide variety of species. In humans, it can cause significant morbidity and mortality when used to treat malignancies such as lymphoma and testicular carcinoma. In rodents, it has been extensively used to study key mechanisms of lung injury and repair. Bleomycin pulmonary toxicity is mediated, at least in part, by the generation of active oxygen species. Amifostine, an aminothiol compound, is a cytoprotectant that is used with many antitumor agents and can act as a potent scavenger of free radicals. The authors hypothesized that amifostine could ameliorate bleomycin lung injury. Hamsters weighing 120 g were given an intraperitoneal (IP) injection of amifostine (200 mg/kg, 1180 mg/m2) or saline with intratracheal (IT) bleomycin (1 unit) or saline, followed by daily IP amifostine or saline for 6 days. Lungs were assessed on Day 2 for acute lung injury, which was determined by wet-to-dry lung weight ratios. On Day 21, histologic assessment of fibrosis and biochemical analysis of lung hydroxyproline content were performed. No significant differences in morbidity or mortality were observed among the groups. Animals who received IT bleomycin, when compared with controls, had increased lung water measurements on Day 2 that were consistent with acute inflammation; on Day 21, they had pulmonary fibrosis, as measured by morphometric analysis, as well as increased hydroxyproline content. For animals treated with amifostine and bleomycin, significant decreases in wet-to-dry lung weight ratios were observed (mean +/- standard deviation, 4.5+/-1.2 vs. 10.2+/-2.7), as well as significant decreases in the percentage of fibrosis per lung (15.03%+/-3.27 vs. 37.26%+/-5.76) and hydroxyproline content (1.132+/-0.30 vs. 1.831+/-0.243). Amifostine significantly decreased the amount of acute lung injury and subsequent fibrosis in the hamster model of bleomycin-induced lung injury.

Polymerization of fibrinogen in murine bleomycin-induced lung injury.

Bleomycin lung injury in mice leads to an acute alveolitis followed by a fibroproliferative response characterized by the accumulation of extracellular matrix. Because distinct regions of the fibrin(ogen) molecule have unique in vitro biological effects on cells, we quantified, localized, and biochemically characterized the molecular form of extravascular fibrin(ogen) in methoxyflurane anesthetized, bleomycin-injured mice. Bleomycin or saline (controls) was administered intratracheally, and lung tissue was harvested and analyzed at several times thereafter. Immunoreactive fibrin tissue content increased to a maximal 50-fold over controls in a temporal and spatial pattern paralleling that of alveolitis and maximal fibroproliferation. The generation of gamma-gamma-chain dimers and alpha-chain polymers, together with the loss of free alpha- and gamma-chains, indicates that the fibrin is predominantly covalently cross-linked. In fibroproliferative-phase lungs, the fibrin fibrils are branched and colocalize with those of collagen at the electron microscopic level. These observations strongly suggest that fibrin is a significant molecular effector of the in vivo fibroproliferative response after lung injury.

Cytotoxic drug‐induced pulmonary disease in infants and children

The increased survival rate of malignant diseases due to more aggressive treatments contributes to the occurrence of drug-induced pulmonary diseases (DIPD). We reviewed, retrospectively over a 10-year period, 15 children (8 girls) who presented a DIPD. Their mean age was 9 years (range, 1 to 17 years), with an underlying malignant disease in 14 (9 leukemias). Three typical patterns have emerged from this analysis: (1) acute hypersensitivity lung disease caused by methotrexate (in 6 patients) or azathioprine (in 1 patient). This acute syndrome consisted of alveolar-interstitial infiltrate with a hypercellularity on bronchoalveolar lavage (BAL) (mean, 714,286 cells/mL; range, 180,000-2,940,000 cells/mL) and an increase of lymphocyte counts (mean, 39%; range 11-64%) with predominantly CD8-suppressor/cytotoxic lymphocytes. Inhibition of leukocyte migration or leukocyte aggregation in the presence of low drug concentrations was positive in the 5 cases tested. Lung function tests showed a restrictive pattern and the outcome of DIPD was always favorable. (2) Chronic pneumonitis/fibrosis was seen in 6 patients who received a variable association of cyclophosphamide (3 patients), bleomycin (2 patients), BCNU (2 patients), and melphalan (1 patient). Symptoms of an alveolar-interstitial pneumonitis developed progressively. BAL showed a moderate increase of total cell numbers (mean, 495,000 cells/mL; range, 150,000-900,000 cells/mL). Lung function tests showed a restrictive pattern. Despite corticosteroid treatment in 4 children, one died after bleomycin lung injury and 2 had functional lung impairment. (3) Noncardiogenic pulmonary edema occurred in 2 patients with leukemia treated with recombinant interleukin II. BAL showed hypercellularity and outcome was rapidly favorable.(ABSTRACT TRUNCATED AT 250 WORDS)

Bleomycin injury of the lung in a mast-cell-deficient model.

Lung mast cell hyperplasia and fibrosis is induced by bleomycin lung injury. The role of the mast cell in this process of injury and resultant fibrosis is unclear. Mutant mi/mi mice, profoundly mast-cell-deficient, were treated with intraperitoneal bleomycin and demonstrated minimal acute inflammatory and chronic fibrotic responses. Lung histamine values determined at 14 and 42 days after bleomycin injury in mi/mi mice were not increased compared to untreated mi/mi animals. However, lung histamine levels in normal mice demonstrated a 300% increase over controls on Day 14 after bleomycin injury, and then returned to baseline by Day 42. The mi/mi BAL cell recovery at 2 weeks after injury and lung hydroxyproline levels at 4 weeks after injury were not altered from baseline. The normal litter mates, in contrast, demonstrated significant increases compared to controls in both of these parameters (p < 0.01, p < 0.04). Although the mi/mi mouse is also deficient in basophils, natural killer cells and functional osteoclasts, there is no evidence of lowered pulmonary defense mechanism and neutrophils and alveolar macrophages are present in normal numbers. This investigation supports the hypothesis that the mast cell contributes to bleomycin-induced lung injury and fibrosis.

Alveolar Epithelial Cell Keratin Expression during Lung Development

Defining the expression and organization of keratins has provided insight into epithelial cell differentiation during tissue development and remodeling. We have used monoclonal antibodies to examine keratin distribution in lung epithelial cells in the rat from the preglandular phase of gestation to the adult. Of particular interest were the distributions of keratin No. 18 and keratin No. 19, since previous results have suggested these keratins may be important in alveolar epithelial cell transitions occurring in adult remodeling lung and in cultured type II cells. The epithelial tubes at 15 days of gestation do not react with 24A3 monoclonal antibody to keratin No. 18, nor is this antigen apparent by gel or immunoblot analysis. Staining is apparent at day 16, however, showing a light punctate pattern at the basal edge of the cells, and becomes prominent by day 17, with intensity greatest in the larger airway tubes. The intensity and number of cells in the parenchyma staining with 24A3 peaks at postnatal days 5 to 10, when proliferation and cytodifferentiation of type I and type II cells is most active. In the adult, staining of type II cells is present mainly at the cell periphery, and occasional reactive attenuated type I-like cells can be observed. Keratin No. 19 immunoreactivity is not present in the primitive epithelial tube until 19 days' gestation but predominantly stains type II pneumocytes in the adult rat lung throughout the entire cell. AE3 antibody to basic keratins stains similarly to keratin No. 19. We conclude that keratin No. 18 is expressed at high levels in type II cells during development in periods of intense proliferation and alveolarization. This correlates with our previous observations on keratin expression following bleomycin lung injury.

High-dose corticosteroids during bleomycin-induced alveolitis in the rat do not suppress the accumulation of hyaluronan (hyaluronic acid) in lung tissue

A single intratracheal injection of bleomycin in rats induced, 4 days later, a considerable accumulation of hyaluronan (hyaluronate, hyaluronic acid) in the lung tissue. This connective tissue reaction was quantified biochemically by analysing hyaluronan (HA) in bronchoalveolar lavage fluid (BAL) and lung tissue extracts. The molecular weight of the HA recovered during lavage was 0.2-0.3 X 10(6) daltons. The HA accumulation was related to an increase in lung water content and associated with an increased influx of eosinophils, neutrophils and lymphocytes into BAL fluid. High-dose corticosteroid treatment (prednisolone 15 mg.kg-1 bw per day) to bleomycin injured rats had no effect on the lung tissue content of HA, the recovery of HA during BAL or the molecular weight of HA accumulated in the alveolar space. Furthermore, steroids did not influence the increased lung water content, or the appearance of inflammatory cells in lavage fluid. These findings indicate that the early connective tissue response to the bleomycin lung injury is mediated by a mechanism which is unaffected by systemic high-dose corticosteroids.

ICRF 187 and polyhydroxyphenyl derivatives fail to protect against bleomycin induced lung injury

The potential protective effects of ICRF 187, Didox, Amidox and VF 165 were investigated in models of bleomycin, or bleomycin and hyperoxia induced lung injury. ICRF 187, a bispiperazinedione compound, is a strong chelating agent which blocks a number of free radical mediated processes. The polyhydroxyphenyl derivatives, Didox, Amidox and VF 165, demonstrate degrees of Fe chelating activities and free radical scavenging abilities. Hamsters treated with 5.0 U/kg bleomycin followed by treatment with ICRF 187 or Didox exhibited similar mortality to the bleomycin alone treated group. In a second study, a low dose of bleomycin (1.2 U/kg) was used followed by exposure to 70% oxygen. Treatment with ICRF 187, Didox, Amidox, or VF 165 failed to protect against lung injury; with the ICRF 187 and Amidox groups exhibiting significantly increased rates of mortality over that seen in animals treated only with bleomycin and hyperoxia. No animals treated with the agents alone died. Histopathology documented that all bleomycin-treated hamsters died of severe pneumonitis. Additionally, in the agent-treated groups there was a prominent proliferation of type II pneumocytes, which demonstrated marked anaplasia, a feature not typical of early bleomycin and hyperoxia lung injury. In conclusion, ICRF 187 and the polyhydroxyphenyl derivative, Amidox, paradoxically increase bleomycin- and hyperoxia-induced lung injury. The possible mechanisms of these interaction include: (1) increased availability of Fe to bleomycin; (2) interference with the healing process; or (3) inhibition of endogenous protective effects of SOD.

Lack of enhancement of bleomycin lung injury in vitamin E-deficient rats

DNA breakage caused by bleomycin may be due to the formation of bleomycin-ferrous complexes, which activate molecular oxygen to form activated oxygen species or free radicals. If bleomycin administration results in intracellular free radical formation, then peroxidation of cellular membranes may be a prominent mechanism in producing bleomycin lung toxicity. In order to explore this hypothesis, we performed experiments instilling bleomycin intratracheally into vitamin E-deficient and control rats. Exhaled ethane production and formation of lung tissue malonaldehyde were measured as indices of lipid peroxidation. Mortality and serial changes in lung collagen content were also measured to assess increases in lung injury in the vitamin E-deficient rats. No increases in ethane exhalation occurred in the groups receiving bleomycin as compared to the matched controls receiving saline. Lung malonaldehyde was found to increase at 96 hours following bleomycin instillation. No increases in mortality or the formation of lung collagen occurred in vitamin E-deficient rats treated with bleomycin as compared to control rats given bleomycin. These results suggest that vitamin E-deficient rats do not have enhanced lung toxicity following bleomycin instillation and that although free radical activation may be occurring, lipid peroxidation does not appear to be a prominent mechanism in producing bleomycininduced pulmonary toxicity.