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The effect of corticosteroid administration on bleomycin lung toxicity.

Bleomycin is well recognized as an active antineoplastic agent in the treatment of germ cell tumors. Pulmonary toxicity is the most significant complication of bleomycin administration. In this report, an attempt is made to modify both the incidence and severity of this side effect. One hundred eleven patients with advanced germ cell tumors were treated with a combination chemotherapy program that included the administration of 30 units (U) of bleomycin as a continuous infusion daily for 3 days every 3 weeks rather than a weekly bolus injection of a total of 360 U (mean dose received, 307 U). Also, 31 patients received high-dose steroids, which have been shown to modify bleomycin-induced pulmonary toxicity, for the treatment of chemotherapy-induced emesis. Changes in carbon monoxide diffusion capacity (DLCO) prompting cessation of bleomycin therapy occurred in 15 cases (bleomycin was stopped in one case due to dyspnea and lung infiltrates, and one patient suffered fatal respiratory failure probably due to bleomycin lung toxicity). Thus, probable bleomycin pulmonary toxicity changed the clinical treatment in 15.3% of the cases. On long-term follow-up, only two patients have demonstrated a residual decrease in DLCO. The incidence of a greater than 25% decrease in DLCO was 34% and was not significantly altered by the administration of steroids (P = 0.96). It is possible, however, that the low incidence of clinically significant and fatal pulmonary toxicity, as experienced in this group of patients, may be related to the infusion of bleomycin. It also is possible that the reversibility of the decrease in DLCO in 95% of the patients may be related to the duration and schedule of bleomycin administration. As bleomycin continues to be an important drug in the treatment of advanced germ cell tumors, further studies are warranted to evaluate the role of the continuous infusion of bleomycin as opposed to bolus therapy.

Administration, Oral↗

Analytical and biological inequivalence of two commercial formulations of the antitumor agent bleomycin.

Bleomycin is an antitumor agent which is a mixture of glycopeptides containing at least 55-75% bleomycin A2 and 25-32% bleomycin B2 fractional composition. Two bleomycin formulations, bleomycin sulfate, USP (Blenoxane, Bristol-Myers Squibb Oncology, Princeton, N.J.) and bleomycin HCI (Tianjin Hebei Pharmaceutical, Tianjin, China) were compared analytically and biologically. Reverse-phase high-performance liquid chromatography (HPLC) analyses using the USP methodology showed that Blenoxane contained primarily (69%) bleomycin A2 and 29.3% bleomycin B2. In contrast, Tianjin-supplied bleomycin HCI contained 97% bleomycin A5 fraction. In vitro tumor cell growth inhibition assays showed equivalent activity in human OVCAR-3 ovarian cancer cells and slightly greater potency in murine L-1210 leukemia cells for the Tianjin formulation. In C57/B1 mice bearing B-16 melanoma tumors, Tianjin-supplied bleomycin produced slightly greater tumor growth inhibition at the expense of greater drug-induced lethality at higher dose levels. These studies show there are significant differences in two international bleomycin formulations. These compositional differences lead to altered biologic effects.

Analysis of Variance↗

Bleomycin-induced pulmonary fibrosis in rat is associated with increased expression of collagen-binding heat shock protein (HSP) 47.

Increased accumulation of collagens in extracellular matrix (ECM) is mainly responsible for bleomycin-induced pulmonary fibrosis in rats. This study was designed to assess whether increased collagen accumulation in bleomycin-induced pulmonary fibrosis is associated with heat shock protein (HSP) 47, a molecular chaperone for collagen biosynthesis. We investigated the expression of type I and type III collagens and HSP47 in bleomycin-induced pulmonary fibrosis. Fifteen male Wistar rats were divided into two groups; group I: bleomycin-induced pulmonary fibrosis; group II: PBS-treated age-matched control rats. Pulmonary fibrosis was induced by injecting a single dose of bleomycin sulphate (5 U/kg body weight) intratracheally. Three bleomycin-treated rats and two age-matched control rats were sacrificed at the end of each of the 1st, 2nd and 4th weeks of the experiment. In bleomycin-treated rats, histological examination revealed pulmonary fibrosis, which increased with time. Increased type I and type III collagen desposition was observed in the lungs of all the bleomycin-treated rats. Weak immunostaining of HSP47 was noted in the control lungs. In contrast, strong immunostaining for HSP47 was seen in all the bleomycin-treated fibrotic lungs. In addition, increased numbers of phenotypically altered myofibroblasts (alpha-smooth muscle actin immunopositive) and fibroblast (vimentin immunopositive) were seen in bleomycin-treated lungs and found to express HSP47. Parallel increase of collagens and their molecular chaperone HSP47 expression was found in the bleomycin-treated lungs, and their co-localization could be detected by double immunostaining. Overexpression of HSP47 may play a significant part in the excessive assembly of collagens and could contribute in this way to the fibrosis found in bleomycin-treated rat lungs.

Animals↗

Antitumor and toxic effects of combination chemotherapy with bleomycin and a phenothiazine anticalmodulin agent.

Phenothiazines and structurally related calmodulin antagonists acted synergistically with bleomycin in killing malignant cells in culture. For exploration of the potential clinical importance of this observation, the effects of administering the combination of chlorpromazine and bleomycin in vitro and in vivo against the transplantable B16 melanoma were studied. The toxicity of the combination to bone marrow and lungs was also determined. Combined chlorpromazine and bleomycin therapy reduced the concentration of chlorpromazine required to inhibit cellular growth of B16 melanoma cells by greater than half (25 vs. 60 microM). Against the transplanted tumor, after 3 weeks of treatment the tumor sizes (in cubic millimeters) were 540 +/- 100 (vehicle), 520 +/- 120 (chlorpromazine), 170 +/- 10 (bleomycin), and 80 +/- 20 (bleomycin + chlorpromazine, P less than .01 vs. bleomycin alone). The median time required to reach a tumor size of 100 mm3 was 15 days for vehicle and chlorpromazine, 17 days for bleomycin, and 26 days for chlorpromazine and bleomycin together. The doubling time of the tumor was also increased 3.5-fold above control with the two-drug combination. At doses of bleomycin and chlorpromazine that increased antitumor activity, no pulmonary fibrosis, measured histologically and biochemically, was detected. At higher doses of bleomycin, the addition of chlorpromazine diminished lung fibrosis. The combination of drugs was not more toxic to hematopoietic precursors than chlorpromazine alone. These studies suggested that the addition of a phenothiazine to bleomycin can augment the therapeutic efficacy of bleomycin in vivo without substantially increasing its toxicity.

Animals↗

Effects of the 21-aminosteroid, U74389F, on bleomycin-induced pulmonary fibrosis in rats.

OBJECTIVE: To determine if a new class of agents, the 21-aminosteroids, which are reportedly potent inhibitors of iron-dependent lipid peroxidation, could protect rats from bleomycin-induced pulmonary fibrosis. SUBJECTS: Fifty-five adult male Sprague-Dawley rats. DESIGN: Prospective, randomized, blinded, controlled trial. INTERVENTIONS: The rats were subjected to intratracheal bleomycin (or saline vehicle), and were then treated with the 21-aminosteroid, U74389F (20 mg/kg/day), or vehicle, for the next 7 days. MEASUREMENTS AND MAIN RESULTS: At 21 days after bleomycin administration, pulmonary fibrosis was assessed histologically as percent of lung fields with evidence of fibrosis. Pulmonary fibrosis was assessed biochemically by measuring pulmonary elastin and hydroxyproline content. To determine if a protective effect of U74389F was linked to the 21-aminosteroid's ability to suppress lipid peroxidation, two products of lipid peroxidation were assayed in the lungs at 7 and 14 days after bleomycin exposure. By histologic assessment, the 21-aminosteroid-treated, bleomycin-exposed animals were found to have significantly decreased the extent of pulmonary fibrosis when compared with the bleomycin control group (mean 48.6 +/- 20.0 [SD] % [n = 9] vs. 68.4 +/- 19.6% [n = 11]; p < .05). In addition, lung elastin was decreased by approximately 75% (p < .05) and hydroxyproline was decreased by approximately 50% (NS) in the 21-aminosteroid-treated group when compared with the bleomycin control group. At 7 and 14 days after bleomycin exposure, all bleomycin-exposed animals had evidence of increased lipid peroxidation (conjugated dienes and thiobarbituric acid-reactive substances), but the 21-aminosteroid-treated, bleomycin-exposed animals had significantly decreased evidence of lipid peroxidation when compared with bleomycin controls. CONCLUSIONS: The 21-aminosteroid can substantially protect animals from bleomycin-induced pulmonary fibrosis and may prove useful in other lung diseases where iron-dependent, free-radical reactions and/or lipid peroxidation are presumed mechanisms of toxicity.

Animals↗

Oxidative cell wall damage mediated by bleomycin-Fe(II) in Saccharomyces cerevisiae.

Bleomycin mediates cell wall damage in the yeast Saccharomyces cerevisiae. Bleomycin treatments in the presence of Fe(II) increased the rate of spheroplast formation by lytic enzymes by 5- to 40-fold. Neither Fe(III) nor other tested ions caused significant cell wall damage in the presence of bleomycin. The effect of bleomycin-Fe(II) on the cell wall mimicked the characteristics of bleomycin-Fe(II)-mediated DNA damage in dependence on aeration, inhibition by ascorbate, and potentiation by submillimolar concentrations of sodium phosphate. Bleomycin-mediated cell wall damage was time and dose dependent, with incubations as short as 20 min and drug concentrations as low as 3.3 x 10(-7)M causing measurable cell wall damage in strain CM1069-40. These times and concentrations are within the range of effectiveness for bleomycin-mediated DNA damage and for the cytotoxicity of the drug. Although Fe(III) was inactive with bleomycin and O2, the bleomycin-Fe(III) complex damaged walls and lysed cells in the presence of H2O2. H2O2 causes similar activation of bleomycin-Fe(III) in assays of DNA scission. These results suggest that an activated bleomycin-Fe-O2 complex disrupts essential cell wall polymers in a manner analogous to bleomycin-mediated cleavage of DNA.

Bleomycin↗

Bleomycin induces alveolar epithelial cell death through JNK-dependent activation of the mitochondrial death pathway.

Exposure to bleomycin in rodents induces lung injury and fibrosis. Alveolar epithelial cell death has been hypothesized as an initiating mechanism underlying bleomycin-induced lung injury and fibrosis. In the present study we evaluated the contribution of mitochondrial and receptor-meditated death pathways in bleomycin-induced death of mouse alveolar epithelial cells (MLE-12 cells) and primary rat alveolar type II cells. Control MLE-12 cells and primary rat alveolar type II cells died after 48 h of exposure to bleomycin. Both MLE-12 cells and rat alveolar type II cells overexpressing Bcl-X(L) did not undergo cell death in response to bleomycin. Dominant negative Fas-associating protein with a death domain failed to prevent bleomycin-induced cell death in MLE-12 cells. Caspase-8 inhibitor CrmA did not prevent bleomycin-induced cell death in primary rat alveolar type II cells. Furthermore, fibroblast cells deficient in Bax and Bak, but not Bid, were resistant to bleomycin-induced cell death. To determine whether the stress kinase JNK was an upstream regulator of Bax activation, MLE-12 cells were exposed to bleomycin in the presence of an adenovirus encoding a dominant negative JNK. Bleomycin-induced Bax activation was prevented by the expression of a dominant negative JNK in MLE-12 cells. Dominant negative JNK prevented cell death in MLE-12 cells and in primary rat alveolar type II cells exposed to bleomycin. These data indicate that bleomycin induces cell death through a JNK-dependent mitochondrial death pathway in alveolar epithelial cells.

Adaptor Proteins, Signal Transducing↗

Potentiation of bleomycin-induced lung injury by exposure to 70% oxygen. Morphologic assessment.

The effects of a single intratracheal instillation of bleomycin followed by exposure to 70% oxygen for 72 h were studied in hamsters. Mortality increased markedly among hamsters exposed to 70% oxygen for 72 h after bleomycin instillation, compared with animals receiving bleomycin and breathing room air. The lethal dose required to kill 50% of the hamsters at 30 days (LD50, 30 day) for bleomycin alone was 0.73 U/100 g body weight, whereas the LD50, 30 day for bleomycin followed by 70% oxygen fell to 0.23 U/100 g body weight. Using morphometry and light microscopy, we found that the amount of diseased lung increased in hamsters given bleomycin with hyperoxia compared with that in those treated with bleomycin alone. After 0.20 U bleomycin and air, 2.8 +/- 1.6% of the lung was abnormal, but with 0.20 U bleomycin followed by 70% oxygen, 42.7 +/- 17.9% of the lung was abnormal. At bleomycin doses that produced no apparent lesions, the addition of 70% oxygen for 72 h produced focal interstitial fibrosis at 30 days. Neither mortality nor significant histologic changes were seen in hamsters treated with saline followed by exposure to 70% oxygen for 72 h. This study demonstrates that hyperoxia potentiates bleomycin damage and suggests that the use of elevated oxygen concentrations in patients being treated with bleomycin should be minimized.

Air↗

Fourteen-membered ring macrolides inhibit vascular cell adhesion molecule 1 messenger RNA induction and leukocyte migration: role in preventing lung injury and fibrosis in bleomycin-challenged mice.

BACKGROUND AND OBJECTIVE: Although the pathogenesis of interstitial pneumonia and pulmonary fibrosis are not well understood, it has been reported that inflammatory cells, especially neutrophils, and the injurious substances produced by them play important roles in the progression of interstitial pneumonia and subsequent fibrosis. Erythromycin and other 14-membered ring macrolides (14-MRMLs) have been reported to improve the survival of patients with diffuse panbronchiolitis by antineutrophil and several other anti-inflammatory mechanisms. The present study was undertaken to investigate the effects of 14-MRMLs on an experimental model of bleomycin-induced acute lung injury and subsequent fibrosis in mice. METHODS: Bleomycin was administered IV to ICR mice. At 28 days after bleomycin injection, fibrotic foci were histologically observed in left lung tissues, and hydroxyproline content in right lung tissues was chemically analyzed. The inhibitory effects of 14-MRMLs were assessed by overall comparison between control (normal saline solution [NS] alone), untreated (bleomycin alone), and treated (bleomycin plus 14-MRMLs) groups. For evaluation of early-phase inflammation, cell populations in BAL fluid and induction of messenger RNA (mRNA) of adhesion molecules (E-selectin, P-selectin, intercellular adhesion molecule 1 [ICAM-1], and vascular cell adhesion molecule 1 [VCAM-1]) in lung tissues were examined at 0 to 13 days after bleomycin treatment. These parameters were also compared with those for the control (NS alone), 14-MRML untreated (bleomycin alone), and 14-MRML pretreated (bleomycin plus 14-MRML pretreated) groups. RESULTS: Bleomycin-induced pulmonary fibrosis was inhibited by erythromycin and other 14-MRMLs on day 28 after bleomycin injection in ICR mice, especially those pretreated with 14-MRMLs. Hydroxyproline content in lung tissues was also decreased in the 14-MRML-pretreated groups. The number of neutrophils in BAL fluid significantly increased, with two peaks at 1 day and 9 days (from 6 to 11 days) after bleomycin administration. 14-MRMLs significantly inhibited both peaks of neutrophil infiltration into the airspace. Changes in mRNA expression of adhesion molecules (E-selectin, P-selectin, ICAM-1, VCAM-1) were associated with leukocyte migration into the airspace. 14-MRMLs clearly inhibited the induction of VCAM-1 mRNA, and tended to attenuate that of ICAM-1 mRNA, but inhibited the induction of neither E-selectin mRNA nor P-selectin mRNA. CONCLUSION: These findings indicate that attenuation of inflammatory cell migration into the airspace by 14-MRMLs, especially of neutrophils and macrophages, resulted in inhibition of lung injury and subsequent fibrosis. 14-MRMLs clearly attenuated the expression of VCAM-1 mRNA during the early phase of bleomycin-induced lung injury, and this might be one mechanism of inhibition of neutrophil and macrophage migration into the airspace by 14-MRMLs. This may be one mechanism of the anti-inflammatory and antifibrotic effects of 14-MRMLs. These findings suggest that prophylactic administration of 14-MRMLs may be clinically efficacious in preventing acute exacerbation of interstitial pneumonia and acute lung injury.

Animals↗

Dose-related effects of Ampligen (poly(I).poly(C12U)), a mismatched double-stranded RNA, in a bleomycin-mouse model of pulmonary fibrosis.

The antifibrotic effect of the mismatched double-stranded RNA, Ampligen (poly(I).poly(C12U)), was evaluated in a bleomycin-mouse model of pulmonary fibrosis. Mice received a single intratracheal dose of bleomycin (0.125 U/mouse) or saline (50 microL) at the beginning of the experiment, followed by 5 or 6 intraperitoneal injections of Ampligen (1.0, 5.0, 10.0, 15.0, or 25.0 mg/kg) or saline at regular intervals for 2 weeks. Ampligen did not produce increased mortality or weight loss by itself. However, it produced varying degrees of mortality in combination with bleomycin. Five injections of 10 mg/kg Ampligen or three injections of 25 mg/kg Ampligen plus three injections of 10 mg/kg Ampligen in combination with bleomycin .produced significant reductions in lung collagen accumulation as indicated by lung hydroxyproline content compared to the bleomycin control group. Animals receiving bleomycin plus Ampligen at all dosages had significantly reduced prolyl hydroxylase activity compared to the bleomycin control group. Lipid peroxidation and bronchoalveolar lavage fluid (BALF)-supernatant protein content for the groups receiving bleomycin plus Ampligen were not reduced compared to the bleomycin control group. In the BALF-supernatant, the activity of acid phosphatase, a lysosomal enzyme produced by neutrophils, monocytes, and macrophages, was significantly decreased in the group receiving bleomycin plus 10 mg/kg Ampligen. Also, selected BALF differential immune cell counts were reduced in some of the groups receiving bleomycin plus Ampligen, but not in a consistent or dose-dependent manner. The results of this study indicate that Ampligen can significantly reduce the bleomycin-induced increased collagen accumulation and may be therapeutically useful in the management of lung fibrosis in humans.

Acid Phosphatase↗

Biochemical and cellular determinants of bleomycin cytotoxicity.

Bleomycin is a mixture of cytotoxic glycopeptides which function as mininucleases, binding to DNA and producing single and double strand breaks by the formation of an activated oxygen complex. Bleomycin is an effective agent against a few human cancers, notably lymphomas, testicular and ovarian germ cell cancers and certain squamous carcinomas. Most human cancers are resistant to bleomycin a priori, however, and those which are initially sensitive frequently develop resistance to the drug during therapy. Several potential modes of resistance to bleomycin have been identified in cell culture and animal tumour models, although their relative importance in determining the responsiveness of human cancers to the drug is not well understood. Bleomycin is selectively toxic to cells in the M and G2 phases of the cell cycle, and generally more effective against actively dividing rather than resting cells. Thus, the cytokinetic state of the tumour cell population is an important determinant of drug activity. Oxygen is an essential substrate for bleomycin's action, with the degree of cytotoxicity directly related to ambient oxygen. Both acutely and chronically hypoxic cells form a substantial fraction of the cell population of many tumours, and may serve as a reservoir of cells resistant to bleomycin on this epigenetic basis. Metabolic inactivation of bleomycin is a mechanism of resistance to the drug in some cells and may influence toxicity in normal tissues. Bleomycin hydrolase activity is low in lungs and skin, the two major sites of normal tissue toxicities, and levels of this enzyme have been elevated in some but not all tumour cell lines selected for resistance to bleomycin. The capacity to repair or withstand single and double strand DNA breaks may also be an important determinant of resistance to the drug. Most yeast and mammalian cell mutants, which are hypersensitive to ionizing radiation because of defects in DNA repair, are also more sensitive to bleomycin than wild-type cells. A number of agents which interact with membranes or inhibit DNA repair, such as ethanol, lidocaine, verapamil and caffeine, have been reported to sensitize cells to bleomycin in vitro.

Animals↗

Effects of iron, copper, cobalt, and their chelators on the cytotoxicity of bleomycin.

Bleomycin is widely used for treating several types of human tumors as well as a variety of experimental tumors. The ability of this antibiotic to bind and to damage DNA has been proposed to be responsible for its antitumor effect. Bleomycin is also a good chelator for several metals, e.g., iron, copper, and others. Bleomycin:metal complexes have been investigated in detail particularly for their action on isolated DNA. The conclusions from these studies indicate that metal-chelated bleomycin either is ineffective or more effective in damaging DNA. In this paper, we tested the effect of iron, copper, cobalt, and their chelators on bleomycin cytotoxicity. Our results suggest that chelating bleomycin with copper or adding an iron chelator (deferoxamine), diethylenetriamine pentaacetic acid, and a copper chelator (penicillamine) shows no effect on bleomycin cytotoxicity. On the other hand, iron dextran and a metal chelator, diethyldithiocarbamate (DDC), with bleomycin show enhanced cytotoxicity. Cobalt-chelated bleomycin is not cytotoxic but is cytotoxic when combined with DDC. We suggest that different mechanisms are contributing to the enhanced toxicity of bleomycin with iron dextran and DDC. Bleomycin acts as a ferrous oxidase which promotes the iron toxicity. In the case of DDC, it can act as a reducing agent or it can help to maintain the bleomycin:metal complex in the reduced form which can generate radicals.

Animals↗

Inhibition of bleomycin-induced pulmonary fibrosis by lipopolysaccharide.

Current evidence suggests that bleomycin toxicity may be attributable to its DNA degradative activity possibly via generation of free radicals and O2 metabolites as mediators. Since lipopolysaccharide (LPS) has been known to provide protection against O2 toxicity, which is correlated with increased activity of O2 metabolite-detoxifying enzymes, the effect of this agent on bleomycin-induced pulmonary fibrosis was examined. Endotracheal bleomycin administration caused increased lung collagen synthesis. A single intraperitoneal injection of LPS (500 micrograms/kg) at day zero significantly decreased these increases. Total bleomycin-induced lung collagen increase was also significantly reduced. LPS alone had no significant effect on total lung catalase activity. Glutathiione peroxidase activity, however, was significantly decreased by 15.8% compared to untreated animals at 2 days after LPS treatment and remained unchanged at other time points. In addition, superoxide dismutase activity was significantly elevated by 30% above untreated animals only at 14 days after LPS administration and remained unchanged at other time points. Endotracheal bleomycin administration alone caused significant reductions in catalase activity at 2 days and 2 weeks after treatment, whereas glutathione peroxidase activity increased above control untreated animals at 2 and 4 weeks, respectively. Superoxide dismutase activity was unaffected by bleomycin treatment. Pretreatment with LPS before bleomycin prevented these reductions or caused increases in the activities of these enzymes at 2 days. Glutathione peroxidase was increased and was significantly greater than those animals treated with bleomycin alone. Catalase also was higher in the LPS plus bleomycin group (by 22.2%, p less than 0.05) than the bleomycin group alone. Compared to the effects on lung collagen synthesis and content, LPS treatment resulted in much less dramatic changes in total lung antioxidant enzyme activities. This discrepancy between the intensity of LPS effects on lung O2 metabolite-detoxifying enzymes and that on pulmonary fibrosis implies that the LPS-ameliorating effect on pulmonary fibrosis could not be totally explained by increased ability to detoxify O2 metabolites. Rather, the data would favor the possibility that LPS inhibits bleomycin-induced pulmonary fibrosis either by its known immunosuppressive effects or some other unknown mechanism. The former would be in agreement with previous data which suggest that an intact immune response is necessary for complete expression of the fibrogenic response to bleomycin.

Animals↗

TNF and IL-6 mediate MIP-1alpha expression in bleomycin-induced lung injury.

Previously, macrophage inflammatory protein-1alpha (MIP-1alpha), a member of the C-C chemokine family, has been implicated in bleomycin-induced pulmonary fibrosis, a model of the human disease idiopathic pulmonary fibrosis. Neutralization of MIP-1alpha protein with anti-MIP-1alpha antibodies significantly attenuated both mononuclear phagocyte recruitment and pulmonary fibrosis in bleomycin-challenged CBA/J mice. However, the specific stimuli for MIP-1alpha expression in the bleomycin-induced lesion have not been characterized. In this report, two mediators of the inflammatory response to bleomycin, tumor necrosis factor (TNF) and interleukin-6 (IL-6), were evaluated as putative stimuli for MIP-1alpha expression after bleomycin challenge in CBA/J mice. Elevated levels of bioactive TNF and IL-6 were detected in bronchoalveolar lavage (BAL) fluid and lung homogenates from bleomycin-treated CBA/J mice at time points post-bleomycin challenge, which precede MIP-1alpha protein expression. Treatment of bleomycin-challenged mice with soluble TNF receptor (sTNFr) or anti-IL-6 antibodies significantly decreased MIP-1alpha protein expression in the lungs. Furthermore, normal alveolar macrophages secreted elevated levels of MIP-1alpha protein in response to treatment with TNF plus IL-6 or bleomycin plus IL-6, but not TNF, bleomycin, or IL-6 alone. Finally, leukocytes recovered from the BAL fluid of bleomycin-challenged mice secreted higher levels of MIP-1alpha protein, compared to controls, when treated with TNF alone. Based on the data presented here, we propose that TNF and IL-6 are part of a cytokine network that modulates MIP-1alpha protein expression in the profibrotic inflammatory lesion during the response to intratracheal bleomycin challenge.

Animals↗

Isolation and characterization of Saccharomyces cerevisiae mutants with enhanced resistance to the anticancer drug bleomycin.

Bleomycin is an antitumor agent believed to act by damaging DNA. It is currently used for treating testicular carcinomas, but other types of cancers such as ovarian and colon are resistant to the drug from the outset. The mechanism involved in allowing cells to confer resistant to bleomycin is not known. We exploited the power of yeast genetics to isolate for the first time several bleomycin-resistant mutants derived from a strain deleted for the IMP2 gene encoding a transcriptional co-activator. imp2Delta mutants are known to be hypersensitive to bleomycin, monovalent and divalent cations, and high pH. The suppressors of imp2Delta showed extreme resistance to bleomycin and also either fully or partially rescued the phenotypes associated with the imp2Delta mutant, suggesting that bleomycin resistance is linked to other phenotypes. Using fluorescently labeled bleomycin, we demonstrated that two bleomycin-resistant variants, MAY1 and MAY2, were compromised for uptake of the drug, as compared with the parent. In contrast, the imp2Delta mutant showed a substantial increase in the uptake of fluorescently labeled bleomycin. We further showed that strains MAY1 and MAY2 contain a reduced amount of a plasma membrane protein, which binds to (57)Co-labeled bleomycin and is believed to mediate drug entry into the cell. We propose that the bleomycin-resistant mutants are likely defective in a process responsible for transporting the drug into the cell.

Antimetabolites, Antineoplastic↗

Genetic changes and bioassays in bleomycin- and phleomycin-treated cells, and their relationship to chromosomal breaks.

The recombinogenicity of damaged chromosomes in diploid Saccharomyces cerevisiae cells treated with bleomycin and structurally related phleomycin was measured, along with aneuploidy and mutation events. Phleomycin was substantially (up to 26-fold) more effective than bleomycin in producing genetic changes at all concentrations, even when colony-forming abilities of cells growing in the presence of bleomycin or phleomycin were similar. These results suggest that the DNA lesions produced by the two structurally related analogs could differ in their nature or frequency, or could be processed differently by the cells. Bioassays were developed and used to compare the cytotoxicities of freshly dissolved bleomycin and phleomycin with the cytotoxicities of lysates prepared from bleomycin- and phleomycin-treated cells. Unexpectedly, lysates prepared from bleomycin-treated cells were 1.5-3.5 times more cytotoxic than freshly dissolved bleomycin after 45-min treatments (3-33 x 10(-6) M). In contrast, lysates prepared from phleomycin-treated cells were 3-38 times less cytotoxic than freshly dissolved phleomycin (0.5-6.4 x 10(-6) M). Cytotoxicities of all lysates were higher after 36-h treatments than after 45-min treatments. At 3.3 x 10(-6) M, this increase was eightfold for bleomycin and 15-fold for phleomycin. Nevertheless, lysates from phleomycin-treated cells were considerably more cytotoxic than lysates from bleomycin-treated cells or freshly prepared bleomycin, consistent with the higher effectiveness of phleomycin than bleomycin in producing chromosomal breaks, genetic changes, and cell killing.

Aneuploidy↗

Effects of aminoguanidine and antioxidant erdosteine on bleomycin-induced lung fibrosis in rats.

Reactive oxygen and nitrogen species have been implicated in the pathogenesis of bleomycin-induced lung fibrosis. The effects of aminoguanidine and erdosteine on the bleomycin-induced lung fibrosis were evaluated in rats. The animals were placed into five groups: Vehicle + vehicle, vehicle + bleomycin (2.5 U/kg), bleomycin + aminoguanidine (200 mg/kg), bleomycin + erdosteine (10 mg/kg), and bleomycin + erdosteine + aminoguanidine. Bleomycin administration resulted in prominent lung fibrosis as measured by lung hydroxyproline content and lung histology, which is completely prevented by erdosteine and aminoguanidine. A strong staining for nitro tyrosine antibody in lung tissue and increased levels of lung NO were found in bleomycin group, that were significantly reduced by aminoguanidine and erdosteine. Aminoguanidine and erdosteine significantly prevented depletion of superoxide dismutase and glutathione peroxidase and elevated myeloperoxidase activities, malondialdehyde level in lung tissue produced by bleomycin. Data presented here indicate that aminoguanidine and erdosteine prevented bleomycin-induced lung fibrosis and that nitric oxide mediated tyrosine nitration of proteins plays a significant role in the pathogenesis of bleomycin-induced lung fibrosis. Also our data suggest that antifibrotic affect of antioxidants may be due to their inhibitory effect on nitric oxide generation in this model.

Animals↗

Spontaneous cleavage of bleomycin-induced abasic sites in chromatin and their mutagenicity in mammalian shuttle vectors.

The stability of oxidized abasic sites induced by bleomycin and neocarzinostatin was examined in chromatin reconstituted from a supercoiled plasmid and core histones. Most of the drug-induced abasic sites were found to undergo spontaneous cleavage in chromatin, probably by reaction with histone amine groups. However, there was considerable heterogeneity in the rate of spontaneous cleavage, with some sites being cleaved almost immediately and some remaining intact even after 7 h. Bleomycin-induced abasic sites with closely opposed strand breaks were more unstable than lone abasic sites. Neocarzinostatin-induced abasic sites, which have a different chemical structure, were cleaved somewhat more slowly than those induced by bleomycin. To assess the mutagenic potential of bleomycin-induced abasic sites, bleomycin-treated shuttle vectors were transfected into mammalian cells, and mutations in progeny plasmids were sequenced. Bleomycin treatment resulted primarily in deletions of various sizes in the shuttle vectors, including a number of one-base deletions occurring at potential bleomycin damage sites. However, under certain conditions, substitutions occurring at expected sites of bleomycin attack were also observed. The results suggest that bleomycin-induced abasic sites have only a slight potential to produce base substitutions in mammalian cells and that a substantial fraction of the double-strand breaks induced by bleomycin and most of the double-strand breaks induced by neocarzinostatin are the result of spontaneous cleavage of abasic sites with closely opposed strand breaks. Inaccurate repair of these double-strand breaks may account for the large deletions, and perhaps the one-base deletions, induced by bleomycin.

Apurinic Acid↗