RAPID COMMUNICATION
Exacerbation of bleomycin-induced lung injury in mice by amifostine

Luis A. Ortiz1, Joseph A. Lasky1, Hana Safah2, Medel Reyes1, Alan Miller2, Giuseppe Lungarella3, and Mitchell Friedman1

1 Section of Pulmonary Diseases, Critical Care, and Environmental Medicine and 2 Tulane Cancer Center, Tulane University Medical Center, New Orleans, Louisiana 70112; and 3 Istituto di Patologia Generale, Universita di Siena, 53100 Siena, Italy


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Bleomycin (BLM) induces lung injury and fibrosis in the murine lung and enhances tumor necrosis factor (TNF)-alpha and collagen mRNA expression in the murine lung. Amifostine is a cytoprotective agent that protects normal tissues from the cytotoxic effects of chemo- and radiation therapy. We investigated the effect of amifostine in BLM-induced lung injury in mice. Mice received intraperitoneal amifostine (200 mg/kg) 30 min before and/or 1, 3, and 7 days after an intratracheal injection of saline or BLM (4 U/kg). The animals were killed 14 days after BLM exposure, and their lungs were studied for TNF-alpha and collagen mRNA expression, hydroxyproline content, and histopathology. Light microscopy demonstrated that amifostine exacerbated the BLM-induced lung injury in mice. Increased TNF-alpha mRNA expression as a result of BLM exposure was not modulated by amifostine treatment. In contrast, amifostine treatment enhanced the BLM-induced expression of alpha 1(I) procollagen mRNA in the lung. Similarly, mice treated with amifostine before BLM exposure accumulated significantly higher amounts of hydroxyproline (111 ± 5 µg/lung) than BLM-treated animals (90 ± 6 µg/lung). These data suggest that amifostine treatment exacerbates BLM-induced lung injury in mice.

tumor necrosis factor-alpha ; collagen


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

BLEOMYCIN (BLM) is an antineoplastic antibiotic used in the treatment of squamous cell carcinomas, lymphomas, and testicular carcinomas (3). The use of BLM as an antineoplastic drug is limited by the development of pulmonary toxicity (3). Induction of lung injury by BLM has been observed in all animal species, and animal models of BLM-induced lung injury have been used as representative models for the study of human pulmonary fibrosis (3). Although the mechanism(s) responsible for the BLM induction of lung injury is not completely understood, we know that BLM induces DNA strand scission and generates oxygen radicals and cytokine production in the lung (3). Tumor necrosis factor (TNF)-alpha has been shown to be critical in the pathogenesis of BLM-induced lung injury, and interventions that limit TNF-alpha expression in the lung of BLM-treated mice prevent the development of BLM-induced lung injury (15, 19).

Amifostine is an organic thiophosphate agent that has been shown to protect normal tissues from the cytotoxic damage induced by chemo- and radiation therapy (2). Pretreatment of cells and animals with amifostine provides protection against the cytotoxic effects of alkylating agents, organoplatinums, anthracyclines, and radiation (2, 5, 22). Cytoprotective levels of amifostine or its metabolites can be found in many organs, including the lungs, where amifostine has been shown to protect against the effects of radiation (2). The effects of amifostine on BLM-induced lung injury are not well known.

In the present study, we evaluated the possibility that amifostine would protect against BLM-induced lung injury in mice. Specifically, we evaluated the effects of amifostine on BLM induction of lung inflammation and fibrosis and on the expression of TNF-alpha and collagen mRNA.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Chemicals. Stock solutions (5 U/ml) of BLM (Blenoxane; kindly donated by Bristol-Myers, Princeton, NJ) and amifostine (kindly donated by US Bioscience) were prepared immediately before use with endotoxin-free water.

Animals and treatment regimen. All animal protocols were approved by the Tulane University (New Orleans, LA) Committee on the Use and Care of Animals. Specific pathogen-free female C57BL/6 mice (Charles River Laboratories, Kingston, NY) weighing 20-25 g (6-10 wk old) were housed in pathogen-free cabinets and provided with water ad libitum. Five groups of animal were assigned to receive treatment. The first two groups of animals received BLM (4 U/kg dissolved in 60 µl of normal saline) or saline (60 µl) as a control by intratracheal injection as previously described (15, 16). The third group of mice received a single dose of amifostine (200 mg/kg by intraperitoneal injection) 30 min before intratracheal exposure to BLM. The fourth group of mice received amifostine (200 mg/kg) 30 min before and 1, 3, and 7 days after intratracheal injection of BLM. The final treatment group of mice received amifostine alone (200 mg/kg) by intraperitoneal injection at 1, 3, and 7 days. There were 10 animals/treatment group.

BLM treatment. The animals were anesthetized with intraperitoneal tribromoethanol (Aldrich) and then exposed to BLM or saline as previously described (15, 16). Briefly, the trachea was exposed with the use of a sterile technique, and 4 U/kg of BLM in 0.06 ml of 0.9% NaCl were slowly instilled into the tracheal lumen. Control mice received the same volume of sterile saline. After exposure, the skin incision was closed and the animals were allowed to recover on a warming plate. The animals were anesthetized with tribromoethanol 14 days after BLM exposure, the descending aorta was severed, and the thorax was opened. The left lung was removed, frozen in liquid nitrogen, and stored at -80°C for subsequent analysis of hydroxyproline content or isolation of RNA.

cDNA probes. The following cDNA templates were used for the experiments. The 1,101-kb murine TNF-alpha plasmid pMuTNF was obtained from American Type Culture Collection (Manassas, VA) and has been described elsewhere (15, 16). The murine alpha 1(I) procollagen plasmid pMCollal-I, which contains a 321-bp insert from the EcoR I site near the translation stop codon to the Hind III site near the first stop codon, was graciously provided by Dr. Eero Vuorio (Turku University, Turku, Finland). This plasmid has also been designated pEVEHO.4. Murine 18S cDNA was obtained from American Type Culture Collection and was used as a loading control as previously described (15, 16).

Northern analysis. Tissues for RNA extraction from BLM-, amifostine-, and saline-exposed mice (5/treatment) were dissected and immediately snap-frozen in liquid nitrogen. Total RNA was extracted from the lung with a cesium chloride method (15, 16). RNA was separated by electrophoresis (20 µg/lane) on a 1.2% formaldehyde-agarose gel before transfer by capillary action to Immobilon-N transfer membranes (Millipore, Bedford, MA). The membranes were then dried for 2 h at 80°C. The membranes were hybridized overnight at 62°C with [32P]dCTP-labeled (ICN, Irvine, CA) random-primed cDNA probes. The murine TNF-alpha , alpha 1(I) procollagen, and 18S cDNAs described in cDNA probes were used as templates and were derived from restriction endonuclease digestion of their respective plasmids. The membranes were probed first for TNF-alpha , then stripped and reprobed for alpha 1(I) procollagen and, as a loading control, for 18S cDNA. Radiolabeled probes were generated with Ready-to-Go labeling kits (Pharmacia, Piscataway, NJ) and purified with TE Midi Select-D, G-50 spin columns (5' right-arrow 3'). Signal density for TNF-alpha was quantitated with a Bio-Rad Gs-670 scanner with Molecular Analyst software (Hercules, CA). For quantitation of alpha 1(I) procollagen expression membranes were exposed to a Fuji phosphorimager (Fujix BAS 1000) plate overnight and scanned. Quantitated analysis was determined with the use of McBAS 2.5 software (Fuji USA, Standford, CT). For each mRNA band, the results were normalized to the internal control (18S) and are expressed as a ratio of the bands being compared.

Lung hydroxyproline content. Lung collagen content was quantitated by measuring the total hydroxyproline content of the lung. Lung hydroxyproline concentration was determined spectrophotometrically as previously described (15, 16). Briefly, stored left lungs were homogenized in 5% trichloroacetic acid (1:9 wt /vol) and centrifuged for 10 min at 4,000 g. The pellet was then washed twice with distilled water and hydrolyzed for 16 h at 100°C in 6 N HCl. Hydroxyproline in the hydrolysate was assessed colorimetrically at 561 nm with p-dimethylaminobenzaldehyde. The results are expressed as micrograms of hydroxyproline per left lung.

Morphology. The heart was perfused with 0.9% NaCl to remove residual blood, and the right lung was fixed in situ for 2 h by the intratracheal instillation of 10% neutral Formalin (Sigma, St. Louis, MO) at a constant pressure of 20 cmH2O and preserved in fixative for 24 h. Lung tissues were then sectioned sagittally and embedded in paraffin. Four-micrometer-thick sections were generated. The slides were stained with hematoxylin and eosin or Masson trichrome staining for light-microscopic examination. The sections were examined by two pathologists blinded to the exposure protocol. The degree of lung injury and fibrosis in 40 fields/lung at a magnification of ×100 was determined as previously described (15, 16).

Statistics. All values are expressed as means ± SE. Differences between the treatment groups for hydroxyproline data were analyzed with ANOVA with Fisher's protected least significant difference test for pairwise comparison (Statview 4, Abacus Concepts, Berkeley, CA). A P value < 0.05 was considered significant. Two-tailed Student's t-test was used for comparison of densitometric values from Northern blots.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Effect of amifostine on BLM-induced lung injury in mice. The intratracheal exposure of mice to BLM, but not to saline or intraperitoneal amifostine alone (Fig. 1A), resulted in the development of subpleural areas of inflammation (accumulations of macrophages, lymphocytes, and fibroblasts) that extended into the lung parenchyma and involved the bronchi and vasculature (Fig. 1B). Within the areas of BLM-induced inflammation, the lung parenchyma was consolidated, with the loss of normal alveolar architecture. Microscopy analysis demonstrated an involvement of 25% of the lung parenchyma in animals treated with BLM alone. The amount of BLM-induced lung inflammation was increased in the lungs of those animals that had received amifostine, and the inflammatory reaction involved 100% of the subpleural area of the lung. The amifostine-induced increase in lung inflammation was observed regardless of whether the mice received amifostine before (Fig. 1C) or after (Fig. 1D) the instillation of BLM.


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Fig. 1.   Effect of amifostine on bleomycin (BLM)-induced lung injury in mice. Shown are representative low-power magnification (×200) photomicrographs of lung obtained from C57BL /6 mice (n = 10/treatment) 14 days after endotracheal injection of saline (control; A), BLM alone (B), amifostine followed by BLM (C), and BLM followed by amifostine (D) as described in MATERIALS AND METHODS. BLM treatment induced inflammation and fibrosis, with predominance in subpleural regions of lung. Amifostine treatment exacerbated amount of lung inflammation and produced honeycomb cysts (arrows). BR, bronchiole; TB, terminal bronchiole; PL, pleural surface; V, vein; AD, alveolar duct. Bar, 20 µm.

Effect of amifostine on TNF-alpha and collagen mRNA expression in the lung of BLM-treated mice. Because BLM challenge of mice is associated with enhanced TNF-alpha and alpha 1(I) procollagen gene upregulation in the lung, we evaluated the possibility that any potential protective effect of amifostine could be mediated by modulating the lung expression of these genes. BLM exposure resulted in enhanced TNF-alpha and alpha 1(I) procollagen mRNA expression in the murine lung 14 days after BLM exposure as assessed by Northern analysis. Compared with control mice, all groups of BLM-treated mice demonstrated an obvious increase in TNF-alpha mRNA expression in their lungs (Fig. 2). Densitometric study of the Northern analysis did not reveal significant differences in TNF-alpha mRNA expression between mice that received BLM plus amifostine (optical density = 0.54 ± 0.14 and 0.63 ± 0.11 arbitrary units before and after, respectively, BLM exposure) and mice that received BLM treatment alone (optical density = 0.66 ± 0.18 arbitrary units). Compared with control animals, BLM treatment resulted in a significant increase (3.4 ± 0.32-fold increase) in alpha 1(I) procollagen mRNA expression in the lung (P < 0.05; Fig. 2). This BLM-induced upregulation in alpha 1(I) procollagen mRNA expression was marginally enhanced in animals pretreated with amifostine (4.4 ± 1.2-fold increase) and was significantly increased in animals that received amifostine after BLM exposure (6.3 ± 0.93-fold increase; P < 0.05 compared with animals that received BLM treatment alone).


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Fig. 2.   Effect of amifostine on tumor necrosis factor-alpha (TNF) and collagen mRNA expression in lungs of BLM-treated mice. Shown is Northern blot analysis of TNF, alpha 1(I) collagen, and 18S (loading control) mRNA expression in mouse lung 14 days after intratracheal injection of saline, BLM alone, amifostine followed by BLM (A /BLM), and BLM followed by amifostine (BLM/A) as described in MATERIALS AND METHODS. BLM treatment induced expression of TNF and alpha 1(I) collagen mRNA in mouse lungs. Amifostine treatment (either before or after BLM exposure) did not alter BLM-enhanced TNF mRNA expression in mouse lungs. In contrast, amifostine treatment enhanced alpha 1(I) collagen mRNA expression in lungs of BLM/A-treated mice. Blots illustrate results obtained from 1 set of mice exposed to control, BLM, A /BLM, or BLM/A. The same results were obtained with 2 additional sets of animals.

Effect of amifostine on lung collagen content after BLM exposure. The end point of BLM-induced lung fibrosis in mice is increased lung collagen deposition. Lung collagen content was measured as lung hydroxyproline content 14 days after BLM exposure. The results are shown in Fig. 3. The administration of amifostine alone did not result in a difference in lung collagen content (62 ± 2 µg/lung) compared with that in saline-treated mice (71.5 ± 7 µg/lung). Compared with control mice, there was a significant increase in hydroxyproline content in the lungs of BLM-treated mice (90 ± 6 µg/lung; P < 0.05). There was a further significant increase in lung collagen content in the animals that were pretreated with amifostine before BLM exposure compared with that in the animals that received BLM alone (111 ± 5 vs. 90 ± 6 µg/lung; P < 0.05). The lung collagen content of the mice that received multiple doses of amifostine after BLM exposure (94 ± 6 µg/lung) was also slightly greater than that observed in the group of animals that received BLM alone, but this difference did not achieve significant levels (P > 0.05).


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Fig. 3.   Effect of amifostine on lung collagen content after BLM exposure. Lung hydroxyproline (HP) levels were measured in murine lungs 14 days after a single endotracheal injection of saline (control), BLM alone, A /BLM, or BLM/A as described in MATERIALS AND METHODS. Data are means ± SE obtained from 5 animals/group. * BLM treatment resulted in significant increase in HP compared with that in saline-exposed mice. dagger  Amifostine pretreatment significantly increased HP content compared with that in mice receiving BLM treatment alone.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Lung injury that leads to pulmonary fibrosis is an important undesirable effect for many chemotheraphy protocols currently used. Recently, the use of amifostine as a cytoprotective agent has been successfully established (2). Amifostine is an organic thiophosphate agent that protects normal tissues from the cytotoxic damage induced by chemo- and radiation therapy (2). Amifostine is dephosphorylated by cell membrane-bound alkaline phosphatase and incorporated into normal tissues where it exerts its cytoprotective role (2). Pretreatment of cells and animals with amifostine provides protection against the cytotoxic effects of alkylating agents, organoplatinums, anthracyclines, and radiation (1, 2, 5, 22). Cytoprotective levels of amifostine or its metabolites can be found in many organs including the lungs (2).

The lung injury observed in response to chemotherapeutic agents is characterized by the development of an exaggerated cell proliferation within the lung, expansion of the extracellular matrix with an increased deposition of collagen, and pulmonary fibrosis (21). The exposure of mice to BLM is associated with the development of lung injury and fibrosis, with identical histological characteristics to that observed in humans (7, 10).

The mechanism(s) responsible for the BLM induction of lung injury is not completely understood. The acute toxicity of BLM in the lung has been attributed to DNA strand scission and oxygen radical production (3, 6). BLM complexes with ferrous ions and molecular oxygen between pairs of DNA and induces single- and double-strand DNA breakage (3, 6). However, because the half-life of BLM in the lung is short (<32 min after an intratracheal instillation in mice) (9), it has been suggested that the fibrotic response to BLM is mediated by activation of immune cells within the lung and the release of inflammatory mediators such as cytokines (3, 8, 15, 19). Among these cytokines, TNF-alpha has been shown to be critical in the pathogenesis of BLM-induced lung injury (6, 15, 16, 19). The challenge of mice with BLM is associated with the upregulated expression of the TNF-alpha gene in the lung tissue (15, 16, 19). This upregulated TNF-alpha expression, which occurs mostly in alveolar macrophages, results in increased secretion of biologically active TNF-alpha protein that precedes the accumulation of collagen in the lung (15, 16, 19). Moreover, measures that limit TNF-alpha expression in the lungs of BLM-treated mice prevent the development of BLM-induced lung inflammation and fibrosis (15, 16, 19).

Limited information is available regarding the potential use of amifostine to prevent the BLM induction of lung injury. Preclinical data from in vitro systems demonstrated protection of amifostine against BLM toxicity of intestinal crypt cells (18). In addition, the active free thiol metabolite of amifostine, WR-1065, attenuated the formation of single-strand DNA breakages and reduced the induction of mutagenesis in Chinese hamster cells exposed to BLM (13). Recently, in preliminary studies, DeBruijn et al. (4) evaluated the effect of amifostine on BLM-induced mortality in mice. They compared the mortality in groups of mice that were pretreated with amifostine (200 mg/kg) or saline followed by BLM (20 mg/kg) injected subcutaneously twice a week and reported a 50% reduction in mortality in animals that received amifostine plus BLM compared with animals that received BLM alone. They also reported a decrease in the amount of infiltrating alveolar macrophages in the lungs of amifostine and BLM-treated mice compared with animals that were treated with BLM alone. Nici et al. (14) have recently reported a protective effect of amifostine in BLM-induced pulmonary toxicity in hamsters. These authors demonstrated that amifostine was able to decrease the amount of lung injury and subsequent hydroxyproline accumulation in the lung of hamsters treated with an endotracheal injection of BLM. The authors proposed the possibility that amifostine may successfully modulate the altered redox state, which may contribute to the proliferative response observed in BLM-induced lung injury.

In contrast to the data suggesting a protective effect of amifostine on BLM-induced lung injury, we have found in the present study that the treatment of mice with amifostine, either as a single dose before or as multiple doses after a single intratracheal injection of BLM, exacerbated the BLM induction of lung inflammation and fibrosis in mice. We also found that the addition of amifostine to BLM treatment resulted in increased expression of the collagen gene and increased lung collagen accumulation compared with that in animals that received BLM alone. Furthermore, amifostine treatment did not diminish the increase in TNF-alpha mRNA expression observed in the lung of BLM-treated mice. These data demonstrate that amifostine exacerbates BLM-induced lung injury in mice.

The differences between our results and those from DeBruijn et al. (4) and Nici et al. (14) are difficult to reconcile and may represent differences in the metabolism of BLM or differences in the species and strain used in the experimental design (8). Supporting our observation that amifostine may potentiate BLM toxicity are data demonstrating that pretreatment of human lymphocytes with amifostine before BLM exposure increases chromosomal damage and enhances BLM clastogenic activity (11). The mechanisms by which amifostine enhances the clastogenic activity of BLM are unknown but may involve the potentiation of BLM activity by enhancing redox mechanisms (11). Amifostine is a sulfhydryl compound that is able to donate electrons that promote iron reduction inside the BLM molecule, thereby permitting BLM activation (11). Furthermore, amifostine is able to bind to DNA, generating changes in the conformation of the major groove of DNA that may promote BLM binding to the 4' position of deoxyribose in the minor groove (11).

The schedule of amifostine treatment used in the present study was selected based on our knowledge that any neutralizing activity of the BLM effect in the mouse lung must take place shortly after the endotracheal injection of this drug. A recent study (12) demonstrated that amifostine and its principal metabolite (WR-1065) concentrate in the lung tissue very rapidly (5-10 min) after peritoneal injection. That study with a single intraperitoneal injection of amifostine (200 mg/kg) into different murine strains demonstrated that >3% of the total injected dose can be found in the lung 30 min after injection. In a similar manner, the same intraperitoneal dose of amifostine given to Fischer 344 rats generated lung concentrations in excess of 3,000 µmol/l and reduced 30-day mortality in cisplatinum-treated mice. These experimental data predict cytoprotective levels of amifostine in the lung at the time of BLM exposure and suggest that amifostine may be potentiating the cytotoxic effects of BLM as judged by the enhanced inflammatory and fibrotic response observed in the lung of amifostine plus BLM-treated mice.

An important observation of the present work is the fact that amifostine treatment did not alter the upregulated expression of TNF-alpha in the lungs of BLM-treated mice. Upregulation of TNF-alpha gene expression in the lung is a fundamental aspect of the pathogenesis of BLM-induced lung injury in mice (4). Murine strains have been characterized as BLM sensitive (C57BL /6) or BLM resistant (BALB/c) according to their ability to upregulate TNF-alpha expression and develop BLM-induced lung injury and fibrosis (15). Piguet and Vesin (20) demonstrated that neutralizing TNF-alpha in the lung by the administration of neutralizing anti-TNF-alpha antibodies or soluble TNF-alpha receptors prevents the development of lung injury and the subsequent accumulation of collagen in the lungs of BLM-treated mice. More recently, Liu et al. (12) and Ortiz et al. (17) have shown that TNF-alpha receptor-deficient mice (which are developed in a C57BL /6 background) demonstrate a decreased expression of profibrotic cytokines (such as transforming growth factor-beta and platelet-derived growth factor) and are protected from the fibroproliferative effects of BLM, silica, and asbestos. Therefore, because amifostine did not diminish the TNF-alpha mRNA expression, these data argue against an amifostine-mediated protective effect on BLM-induced lung injury.

In summary, the present study suggests that amifostine exacerbates the BLM induction of lung inflammation and collagen accumulation in the murine lung. These data also suggest that amifostine does not alter TNF-alpha mRNA expression and may increase collagen mRNA expression in the lungs of BLM-treated mice. Further studies analyzing the mechanisms by which amifostine exacerbates BLM toxicity in the lung are necessary to clarify the potential interactions in the use of BLM and amifostine in clinical practice.


    ACKNOWLEDGEMENTS

We thank Mary Chelles and Boioang Tonthat for technical assistance in the preparation of the lung tissues and Northern analysis, respectively.


    FOOTNOTES

This work was supported in part by National Heart, Lung, and Blood Institute Grants HL-03569 (to L. A. Ortiz) and HL-03374 (to J. A. Lasky) and a grant from US Biosience (Palo Alto, CA).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: L. A. Ortiz, Section of Pulmonary Diseases, Critical Care, and Environmental Medicine, Dept. of Medicine SL9, Tulane Univ. Medical Center, New Orleans, LA 70112-2699 (E-mail: lortiz{at}mailhost.tcs.tulane.edu).

Received 14 April 1999; accepted in final form 2 September 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Lung Cell Mol Physiol 277(6):L1239-L1244
0002-9513/99 $5.00 Copyright © 1999 the American Physiological Society




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