1 Section of Pulmonary
Diseases, Bleomycin (BLM) induces lung injury and
fibrosis in the murine lung and enhances tumor necrosis factor
(TNF)-
tumor necrosis factor- 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)- 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- 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 cDNA probes. The following cDNA
templates were used for the experiments. The 1,101-kb murine TNF- 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- 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.
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.
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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-
and collagen mRNA expression,
hydroxyproline content, and histopathology. Light microscopy
demonstrated that amifostine exacerbated the BLM-induced lung injury in
mice. Increased TNF-
mRNA expression as a result of BLM exposure was
not modulated by amifostine treatment. In contrast, amifostine
treatment enhanced the BLM-induced expression of
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.
; collagen
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
has been shown to be critical in the pathogenesis of
BLM-induced lung injury, and interventions that limit TNF-
expression in the lung of BLM-treated mice prevent the development of
BLM-induced lung injury (15, 19).
and collagen mRNA.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
80°C for subsequent analysis of hydroxyproline content or isolation of RNA.
plasmid pMuTNF was obtained from American Type Culture Collection
(Manassas, VA) and has been described elsewhere (15, 16). The murine
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).
,
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-
, then stripped and reprobed for
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'
3').
Signal density for TNF-
was quantitated with a Bio-Rad Gs-670
scanner with Molecular Analyst software (Hercules, CA). For
quantitation of
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.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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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- and
collagen mRNA expression in the lung of BLM-treated
mice. Because BLM challenge of mice is associated with
enhanced TNF-
and
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-
and
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-
mRNA expression in their lungs (Fig. 2). Densitometric study of the Northern
analysis did not reveal significant differences in TNF-
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
1(I) procollagen mRNA
expression in the lung (P < 0.05;
Fig. 2). This BLM-induced upregulation in
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).
|
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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DISCUSSION |
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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- 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-
gene in the lung tissue (15, 16,
19). This upregulated TNF-
expression, which occurs mostly in
alveolar macrophages, results in increased secretion of biologically
active TNF-
protein that precedes the accumulation of collagen in
the lung (15, 16, 19). Moreover, measures that limit TNF-
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- 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- in the lungs of BLM-treated mice. Upregulation of TNF-
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-
expression and develop
BLM-induced lung injury and fibrosis (15). Piguet and Vesin
(20) demonstrated that neutralizing TNF-
in the lung by the administration of neutralizing anti-TNF-
antibodies or soluble TNF-
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-
receptor-deficient mice
(which are developed in a C57BL /6 background) demonstrate a
decreased expression of profibrotic cytokines (such as transforming
growth factor-
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-
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- 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.
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ACKNOWLEDGEMENTS |
---|
We thank Mary Chelles and Boioang Tonthat for technical assistance in the preparation of the lung tissues and Northern analysis, respectively.
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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.
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