The Effect of Deferoxamine on Bleomycin-induced Lung
Fibrosis in the Hamster1- 3
DAVID B. CHANDLER and JACK D. FULMER
Introduction
Bleomycin, an antineoplastic com-
pound consisting of a mixture of cyto-
toxic glycopeptides, is used therapeuti-
cally against a variety of squamous cell
carcinomas and lymphomas and may
produce pulmonary toxicity (1, 2). Ini-
tially, lung disease manifests itself as a
pneumonitis, which in some patients
progresses to interstitial fibrosis (2-4).
Intratracheally administered bleomycin
is widely used to produce an animal
model for the development of pulmo-
nary fibrosis (5, 6). The cytotoxicity of
bleomycin has been found to be related
to its ability to induce intracellular
chain breakage of DNA by way of a fer-
rous ion-molecular oxygen mechanism
(7). Furthermore, studies that analyzed
the reaction products of bleomycin and
DNA in the presence of iron and O2
demonstrated the production of su-
peroxide radicals, hydroxyl radicals, and
the presence of iron-catalyzed lipid
peroxidation (8-11). Although the pul-
monary fibrosis caused by bleomycin
has been investigated biochemically and
morphologically (5, 12-14), its mecha-
nism has not been established. It is as-
sumed that lipid peroxidation might be
related to the pulmonary fibrosis, but
involvement of the immune and inflam-
matory systems have also been sug-
gested (14-16). Therefore, it might be
possible that iron could participate in
bleomycin-induced lung injury by cata-
lyzing oxygen radical formation and
lipid peroxidation.
In order to evaluate the possible role
of ferric ion in bleomycin-induced pul-
monary fibrosis, we studied the effects
of deferoxamine, a ferric ion chelator,
on the severity of bleomycin-induced
lung injury in hamsters. Our results
showed that deferoxamine treatment
significantly decreases the morphologic
and biochemical evidence of bleomycin-
induced pulmonary fibrosis.
Methods
Forty male Golden Syrian hamsters weigh-
596
SUMMARY Bleomycin Is a commonly used antineoplastic compound that can produce a dose-
and time-dependent pneumonitis and fibrosis In humans. The mechanism of bleomycin-induced
fibrosis is not known. However, current data suggest that the production of oxygen radicals by way
of a terrous ion-molecular oxygen mechanism might be related to the pulmonary fibrosis. Therefore,
we evaluated the possibility that parenterally admlnlnstered deteroxamlne, an Iron chelatlng com-
pound, could modulate the morphologic and biochemical estimates of bleomycin-induced lung fibro-
sis In hamsters. Deteroxamine pretreatment and dally injection for 21 days after intratracheally ad-
ministered bleomycin resulted in a 33% reduction In lung collagen accumulation compared with
that after bleomycin treatment alone. However, fibrosis was stili present In the bleomycin-deteroxamlne
group; the animals showed a 142 and 150% Increase in lung collagen compared with that in saline-
and deteroxamine-treated control animals, respectively. Morphologic estimates of the severity of
fibrosis In the bleomycln-deferoxamlne treatment group were reduced when compared with the
bleomycin treatment group alone, but was Increased compared with saline- and saline-deteroxamine-
treated control animals. These data indicate that deteroxamine treatment reduces the severity of
bleomycin-Induced pulmonary fibrosis In hamsters. The mechanism might be by the prevention
of iron-catalyzed, free-radical formation. AM REV RESPIR DIS 1985; 131:000-000
ing 90 to 100 g were used (Engle Laborato- intraperitoneally) via tracheostomies accord-
ries, Farmersburg, IN). They were housed 5 ing to established techniques (5, 6). After in-
per cage and fed laboratory chow and water tratracheal treatment, saline or deferoxamine
ad libitum. Bleomycin sulfate (Blenoxanel!!l) administration was continued twice daily until
was donated by Bristol Laboratory (Division the animals were killed.
of Bristol-Myers Co., Syracuse, NY). Defer- Twenty-one days after the intratracheal
oxamine mesylate (Desferall!!l) was purchased treatment, hamsters were weighed, anesthe-
from CIBA Pharmaceutical Co. (Division of tized with sodium pentobarbital (60 to 70
CIBA-Geigy Corp., Summit, NJ). mg/kg given intraperitoneally), and killed
by exsanguination. The thoracic cavity was
Experimental Protocol opened, and the lungs were perfused free of
Four groups of animals were compared in this blood in situ with saline (4° C), as described
study: (1) a control group (saline group) (n=5) previously (6). The trachea was then cannu-
in which animals were administered a single lated, and lung and heart were removed en
injection intratracheally of 0.33 ml sterile sa- bloc. The right main-stem bronchus was iso-
line and received 0.05 ml of sterile saline in- lated and ligated, and the right lung was re-
tramuscularly twice daily, (2) a deferoxamine moved for collagen determination. The left
(DF) group (n = 5) in which animals were ad- lung was then reexpanded with 10070 neutral
ministered a single injection intratracheally buffered formalin and fixed overnight at 25.0
of 0.33 ml sterile saline, and received 5.0 mg cm H 2 0 pressure. The lung was sectioned at
of deferoxamine in 0.05 ml sterile saline
intramuscularly, twice daily (3) a bleomycin-
deferoxamine (Bleo-DF) group (n=15) in (Received in originalform July 24, 1984 and in re-
which animals received a single injection vised form October 30, 1984)
intratracheally of 1 U bleomycin sulfate in 0.33 I From the Division of Pulmonary and Critical
ml sterile saline and received 5.0 mg of Care Medicine, Department of Medicine, Univer-
deferoxamine in 0.05 mg sterile saline in- sity of Alabama in Birmingham, and the Veterans
tramuscularly twice daily, and (4) a bleomy- Administration Medical Center, Birmingham, Al-
cin (Bleo) group (n = 15) in which animals re- abama.
ceived a single injection intratracheally of 1 2 Supported by Training Grant No. HL-07553-
U bleomycin sulfate in 0.33 ml sterile saline 01 from the National Institutes of Health, the Re-
search Service of the Veterans Administration, and
and received 0.05 ml of sterile saline intramus-
by a grant from the American Lung Association.
cularly twice daily. Each group was pretreated
with either saline or deferoxamine for 5 days.
3 Requests for reprints should be addressed to
David B. Chandler, Ph.D., Division of Pulmonary
Intratracheally administered bleomycin or sa- and Critical Care Medicine, Department of Medi-
line was then administered under sodium pen- cine, 323 Lyons-Harrison Research, University of
tobarbital anesthesia (60 to 70 mg/kg given Alabama in Birmingham, Birmingham, AL 35294.
DEFEROXAMINE AND BLEOMYCIN-INDUCED WNG FIBROSIS IN THE HAMSTER
5-/Am intervals and stained with hematoxylin-
eosin. The severity of fibrosis in these sec-
tions was graded using a modification of pub-
lished criteria (17): none (0), no evidence of
fibrosis; mild (1 + ), focal regions of fibrosis
involving less than 100/0 of lung; moderate
(2+), more extensive fibrosis involving 20 to
40% of lung; severe (3 + ), widespread fibro-
sis involving 40 to 60% of lung; extensive
(4+), fibrosis involving greater than 60% of
lung. All morphologic studies were made
blindly by one of us (JDF) without knowl-
edge of the treatment.
Collagen Determination
Collagen determination was performed on the
right lung using a colormetric assay for
hydroxyproline as described previously (18).
Briefly, Type I skin collagen was extracted,
purified, and used as a control. An aliquot
of right lung from each animal was hydro-
lyzed in 6 N HCI for 16 h at 120 0 C and as-
sayed in triplicate for hydroxyproline. Hy-
droxyproline constituted approximately
12.5% of the purified Type I skin collagen;
therefore, an estimate of lung collagen was
made by multiplying mean hydroxyproline
weight by 8 (18). From these measurements,
total lung collagen was calculated.
Statistical Analysis
The data are expressed as the mean ± stan-
dard error of the mean (SEM). Differences
between the treatment groups were tested
using a one-way analysis of variance
(ANOVA). The Scheffe test was used to de-
termine the p value for multiple comparisons.
The level of significance was taken to be p
< 0.05.
Results
The effects of intratracheal administra-
tion of bleomycin on percent of initial
body weight showed significant differ-
ences between treatment groups (p <
0.001). The body weight of the Bleo
group decreased significantly to 95.0%
of their initial body weight when com-
pared with either the saline group
(120.8%) or the OF group (120.60/0). No
other comparisons showed any differ-
ences.
Collagen accumulation per lung after
bleomycin treatment with and without
deferoxamine treatment is shown in fig-
ure 1. The lung collagen after treatments
showed significant differences between
groups (p < 0.001). Furthermore, the
Bleo group without deferoxamine re-
sulted in a significant increase in lung
collagen (col) (9.54 mg col/lung) when
compared with either the saline group
(4.48 mg col/lung), the OF group (4.22
mg col/lung), or the Bleo-OF group (6.35
mg col/lung). No difference between
the saline and OF, saline and Bleo-OF,
or OF and Bleo-OF groups were seen.
c
~--
uo §'"
c,
"'"
g'E
~-
u established and is described by the Fen-
Fig. 1. The effects of deferoxamine or saline treat-
ment on total lung collagen in hamsters given bleomy-
cin. Each value represents the mean ± SEM of 5
hamsters for the saline and deferoxamine groups and
6 hamsters for the Bleo-DF and Bleo groups. ANOVA
between experimental groups: p < 0.001; p < 0.05 be-
tween saline and Bleo; OF and Blea; and Bleo and
Bleo-DF groups.
Histologically, changes observed with
bleomycin injury in this study were simi-
lar to those reported previously with this
agent (14). The severity of fibrotic le-
sions in the lung after treatments ex-
hibited significant differences between
groups (p < 0.001). As seen in table 1,
the grade of histologic lesion was signif-
icantly (p < 0.05) increased in the Bleo
(+ 3.1) and Bleo-OF groups (+ 1.2) when
compared with the saline group. In ad-
dition, the Bleo group exhibited signifi-
cant elevation in lesion grade compared
with that in the OF group. Furthermore,
the grade of histologic lesions in the
Bleo group was significantly elevated
above that in the Bleo-OF group.
Discussion
The use of bleomycin in the study of
pulmonary fibrosis has been well
documented (19-22). The exact mecha-
nism by which bleomycin induces lung
fibrosis is not understood. It is known
that bleomycin can generate reactive ox-
ygen species such as superoxide radicals
and hydroxyl radicals (8-10). Bleomycin
was also found to increase lipid peroxi-
dation in liver and lung microsomal
fractions (23). Whether the generation
of superoxide or hydroxyl radicals and
lipid peroxidation contributes to the
pathogenesis of bleomycin-induced pul-
monary fibrosis is not known.
In the present study, we examined the
effect of deferoxamine, a ferric ion che-
lator, on bleomycin-induced collagen
accumulation. It was found that defer-
oxamine treatment reduced the amount
of collagen accumulation per lung and
lesion severity after bleomycin injury.
The data from this study do not allow
the determination of the specific mech-
597
anism by which deferoxamine is func-
tioning. However, the ability of iron to
serve as a redox reagent in the metabo-
lism of oxygen (24, 25), and the mecha-
nism of iron-catalyzed hydroxyl radical
formation by the reaction of ferric ion
(Fe III) and superoxide ion has been well
ton reaction (24-27). Recently, deferox-
amine has been shown to effectively pre-
vent the formation of iron-catalyzed
hydroxyl radicals from superoxide an-
ion radical (28) and associated lipid
peroxidation (29), wherein ferric ion is
believed to be required as an initiation
factor (30).
The possible reduction in hydroxyl
radical formation by deferoxamine as
proposed previously might have an ad-
ditional effect other than prevention of
lipid peroxidation. Recent evidence has
also shown that hydroxyl radical scav-
engers inhibit lymphocyte mitogenesis
(31). This mechanism could be impor-
tant because the lymphocyte appears to
modulate the proliferation of fibro-
blasts, and collagen synthesis and secre-
tion by fibroblasts (15, 32, 33). In the
present study the lesions in the Bleo-OF
group appeared more diffuse and to have
a decreased amount of mononuclear
cells than did those in the Bleo group.
Therefore, the ability of deferoxamine
to decrease hydroxyl radical formation
might decrease lymphocyte proliferation
and, secondarily, reduce collagen accu-
mulation after bleomycin treatment.
In addition to its effects on hydroxyl
radical generation, deferoxamine has
been reported to modulate fibroblast
proliferation and collagen synthesis.
Oeferoxamine has been shown to de-
crease the in vitro proliferation of lung
fibroblasts and to inhibit prolyl hydrox-
ylase activity in fibroblasts cultured for
24 days (34). However, in an earlier study,
TABLE 1
THE EFFECT OF DEFEROXAMINE OR
SALINE TREATMENT ON THE MORPHOLOGIC
SEVERITY OF BLEOMYCIN-INDUCED FIBROSIS
Treatment Grade
Saline 0.0 ± 0.0·
Bleo 3.1 ± 0.4t:t:§
Bleo-DF 1.2 ± 0.2t
OF 0.4 ± 0.2
• Mean ± SEM of 3 hamsters for the saline group, 6 ham-
sters in the bleomycin group, 6 in the bleomycin-deferoxamine
group, and 4 in the deferoxamine group. ANOVA between ex-
perimental groups: p < 0.001.
t p < 0.05 when compared with saline group.
* p < 0.05 when compared with deferoxamine group.
§ p < 0.05 when compared with bleomycin-deferoxamine
group.
598
deferoxamine was found to stimulate
prolyl hydroxylase activity in vivo (35).
The effect of deferoxamine on prolyl hy-
droxylase activity appears controversial.
In summary, deferoxamine does de-
crease but does not eliminate collagen ac-
cumulation and lesion severity in
bleomycin-induced lung fibrosis. The
mechanism by which deferoxamine is act-
ing to reduce lung injury is unknown.
However, its ability to decrease hydroxyl
radical formation and subsequent hy-
droxyl-radical-induced lipid peroxidation
and possible secondary effects on lym-
phocyte proliferation might play sig-
nificant roles in the sequelae of events
leading to bleomycin-induced collagen
accumulation.
Acknowledgment
We wish to thank Mrs. Wynola Fuller for ex-
pert technical assistance and Mrs. Victoria
Jones for expert editorial assistance.
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