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ABSTRACT |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Many possible treatments for pulmonary fibrosis have been investigated, but except for some current clinical trials, none have succeeded in clinical trials. On the basis of the antioxidant action of
bilirubin (BIL), we examined the effects of hyperbilirubinemia on
the development of bleomycin (BLM)-induced pulmonary fibrosis in rats. The animals' plasma BIL level was kept within 3 and 10 mg/dl
by repeated intravenous infusion of a high dose of BIL. We studied
the inhibitory effects of hyperbilirubinemia on BLM-induced pulmonary fibrosis through histopathologic and biochemical analyses. Mortality of rats with BLM-induced pulmonary fibrosis was
significantly lower in the three groups with hyperbilirubinemia.
The ameliorating effect of hyperbilirubinemia on pulmonary fibrosis was shown by lung histology, as well as by a decreased lung
content of hydroxyproline and reduced bronchoalveolar lavage
fluid (BALF) concentration of transforming growth factor (TGF)-
1.
The number of polymorphonuclear leukocytes and lymphocytes in
BALF was also decreased in the groups with hyperbilirubinemia.
Furthermore, oxidative metabolites of BIL in urine were present at
significantly higher levels in BLM-treated rats with hyperbilirubinemia than in those without hyperbilirubinemia. These data suggest that the antioxidative action of BIL can attenuate BLM-induced
pulmonary fibrosis, partly by inhibiting lung inflammation and
production of TGF-1.
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Keywords: pulmonary fibrosis; bleomycin; bilirubin; antioxidant
Interstitial pulmonary fibrosis is a consequence of many types of severe or sustained lung inflammation. Bleomycin (BLM), a mixture of glycopeptides derived from Streptomyces verticillus, is a potent chemotherapeutic agent and is known to produce pulmonary fibrosis in humans as well as in experimental animals. The mechanisms by which BLM causes pulmonary fibrosis are not yet clearly understood, but it is generally believed that superoxide radicals generated by BLM itself cause direct injury to epithelial or endothelial cells in the lung (1). Such an initial lung injury may subsequently increase the influx of activated inflammatory cells into lung parenchyma (2, 3). The inflammatory cells (e.g., alveolar macrophages [AM] or polymorphonuclear cells) produce reactive oxygen species (ROS), and these ROS may be important contributors to the pathogenesis of BLM-induced pulmonary fibrosis (1).
Many possible treatments for pulmonary fibrosis have been investigated, but none have succeeded in clinical trials (4), except for some limited therapeutic effects in the treatment of idiopathic pulmonary fibrosis (IPF) (7). Orally administered N-acetylcysteine has been shown to ameliorate BLM-induced pulmonary fibrosis, and clinical trials of antioxidants are currently underway in patients with IPF (8, 9). Bilirubin (BIL), a product of heme degradation by heme oxygenase (10), is regarded as a powerful antioxidant substance in vitro (11) and as a very effective physiologic antioxidant in vivo (12). In addition, Nakagami and colleagues reported that conjugated BIL directly inhibited inflammatory responses at the complement-reacting step (13). However, the effects of BIL on pulmonary fibrosis have not been studied. We therefore examined whether a high level of circulating BIL in rats inhibits the pulmonary fibrosis induced by a single intratracheal instillation of BLM (14).
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METHODS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Animals and Surgery
A high level of circulating BIL was achieved by repeated intravenous infusion of a high dose of BIL into specific pathogen free male Wistar rats, weighing from 260 to 280 g (Funabashi Farm, Shizuoka, Japan) as described by Roger and colleagues (15). All surgical procedures were done on animals under anesthesia achieved with an intraperitoneal injection of sodium pentobarbital (50 mg/kg). A jugular vein was catheterized with a polyethylene catheter (O.D. = 1.0 mm; Atom Medical Co., Tokyo, Japan). The catheter was threaded under the animal's skin and up the back through a small opening in the skin, and it was then threaded through a small hole (diameter = 3 mm) in a stainless steel chest band (width = 2 cm) and into and through the lumen of a wire spring (I.D. = 3 mm; length = 30 cm). The chest band was fixed to the skin with sutures. At one end, the wire spring was attached to the chest band. At the other end, the wire spring was threaded through a hole (diameter = 3 cm) in the cage roof, and this end of the wire ring was free to move. The end of the catheter was closed, and the lumen of the catheter was filled with saline containing heparin sulfate (10 U/ml) until the catheter was used for BIL injection. Only one rat was transferred to each cage (Toyo Riko, Tokyo, Japan), in order to avoid having the catheter bitten by other rats. BIL (4.8 mg/ml; Sigma Chemical, St. Louis, MO) was dissolved in a mixture of 0.5 M NaOH, 0.055 M phosphate buffer, and 5% bovine serum albumin at pH 7.4 (10:20:70, vol/vol/vol). BIL was infused twice a day through the catheter with a syringe pump (Terumo, Tokyo, Japan), in the morning (9 A.M.) and in the evening (6 P.M.), with the recipient animal in an awake state. A loading dose of 115.2 mg/kg of BIL was administered over 180 min. To instill BLM into the lung, we dissolved 0.2 ml of bleomycin hydrochloride (Nippon Kayaku, Tokyo, Japan) in sterile saline (7.5 U/kg body weight) and injected the resulting solution through a needle inserted between the cartilaginous rings of the trachea. The experiments described in this section were performed on all rats according to the guidelines for the care and use of laboratory animals of the Tohoku University Animal Experiment Ethics Committee. Blood was taken from the rat tail vein, and plasma BIL content was measured by SRL Co. (Tokyo, Japan).
Histopathologic Evaluation
Rats were killed under sodium pentobarbital anesthesia (100 mg/kg, intraperitoneally) at 21 d after intratracheal instillation of either BLM or saline. Each lung was fixed with 10% formaldehyde neutral buffer solution for a period of at least 48 h and was then embedded in paraffin. Sequential 3-µm sections of the lung were stained with hematoxylin and eosin (H & E). Severity of fibrosis was assessed semiquantitatively according to the method described by Ashcroft and coworkers (16). The grade of pulmonary fibrosis was scored in a blinded fashion on a scale of from 0 to 8 by examining 30 randomly chosen regions per sample at a magnification of ×100. Criteria for grading pulmonary fibrosis were as follows: Grade 0 = normal lung; Grade 1 = minimal fibrous thickening of alveolar or bronchiolar walls; Grade 3 = moderate thickening of the walls without obvious damage to the lung architecture; Grade 5 = increased fibrosis with definite damage to the lung structure and the formation of fibrous bands or small fibrous masses; Grade 7 = severe distortion of the lung structure and large fibrous areas; Grade 8 = total fibrous obliteration of the field. If there was any difficulty in deciding between two odd-numbered categories, the field was given the intervening even-numbered grade. The pulmonary fibrosis score was expressed as a mean grade of fibrosis for each sample.
Leukocyte Analysis in Bronchoalveolar Lavage Fluid
Sampling of bronchoalveolar lavage fluid (BALF) was also done at 21 d
as described previously (17, 18). An aliquot of the fluid was portioned
for total differential cell counts. The remainder was centrifuged at
270 × g for 5 min at 4° C. The supernatant was stored at 
80° C for
the assay of transforming growth factor (TGF)-1. Total leukocyte
counts were done on the BALF as described previously (17, 18).
Assay of Hydroxyproline and TGF-1
Hydroxyproline in the rat lung was assayed at Day 21 according to the
commonly used procedure of colorimetric measurement by SRL Co.
(19, 20). We also measured the concentration of TGF-1 in BALF at
Day 21, using an enzyme-linked immunosorbent assay (ELISA) kit
(Amersham Ltd, Amersham, Buckinghamshire, UK), which detects active TGF-1.
Measurement of BIL Oxidative Metabolites in Rat Urine
To measure BIL oxidative metabolites (BOM) in urine, we kept rats in a special cage designed to collect urine (Toyo Riko). One week after intratracheal instillation of BLM, rat urine was collected for 24 h and the urine BOM content was measured with an ELISA as described by Yamaguchi and associates (21).
Experimental Protocols
To study the effects of hyperbilirubinemia on the survival rate after intratracheal instillation of BLM, we randomized rats into four groups: a BLM group, which was injected with the vehicle for BIL and received intratracheal instillation of BLM; and three BIL+BLM groups that received intratracheal instillation of BLM plus repeated intravenous infusion of BIL from 7 d before intratracheal instillation of BLM, or concurrently with intratracheal instillation of BLM, or 7 d after intratracheal instillation of BLM until the end of the experimental phase of the study.
To examine the effects of hyperbilirubinemia and BLM on BLM-induced pulmonary fibrosis, on BLM-induced changes in the hydroxyproline content of the lung, on TGF-1 content, on leukocyte number
in BALF, and on the urine concentration of BOM, we randomized
rats into four groups. The control group was injected with the vehicle for BIL and received intratracheal saline instillation; the BIL group
was injected with BIL and received intratracheal saline instillation;
the BLM group was injected with the vehicle for BIL and received intratracheal BLM instillation; and the BIL+BLM group was injected
with BIL and received intratracheal BLM instillation. Rats received
repeated intravenous infusions of BIL or of the saline that was the vehicle for BIL from 7 d before the intratracheal instillation of either
BLM or saline until the end of the experimental phase of the study.
Statistical Analysis
Survival rates were evaluated with a log-rank test over a period of 21 d. All values are displayed as mean ± SD. Statistical analysis was done with Student's unpaired t test. Significance was accepted at a value of p < 0.05.
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RESULTS |
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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First, we confirmed the plasma BIL concentration after the start of repeated intravenous infusion of a high dose of BIL. To determine the changes in plasma BIL concentration from before to after BIL infusion and to examine whether the plasma BIL concentration was kept at a high level for 4 wk, we measured the plasma BIL concentration before and after BIL infusion in the morning (9 A.M.) and in the evening (6 P.M.) on Day 2 and again 4 wk after the start of the repeated intravenous infusion of BIL. The plasma BIL concentration was between 3 mg/dl and 10 mg/dl for at least 4 wk after the start of repeated intravenous infusion of BIL. The plasma BIL concentration measured 10 min before the BIL infusion in the morning (9 A.M.) on Day 2 after the start of the repeated intravenous infusion of BIL (3.2 ± 0.1 [mean ± SD] mg/dl, n = 7) was no different from that measured 4 wk after the start of the repeated intravenous infusion of BIL (3.3 ± 0.1 mg/dl, n = 7; p > 0.02). Likewise, the plasma BIL concentration measured 10 min after the 180 min of BIL infusion in the morning (9 A.M.) on Day 2 after the start of the repeated intravenous infusion of BIL (7.8 ± 0.4 mg/dl, n = 7) was no different from that measured 4 wk after the start of the repeated intravenous infusion of BIL (7.9 ± 0.4 mg/dl, n = 7; p > 0.02). The plasma BIL concentration measured 10 min before the BIL infusion in the evening (6 P.M.) on Day 2 after the start of the repeated intravenous infusion of BIL (4.4 ± 0.2 mg/dl, n = 7) was no different from that measured 4 wk after the start of the repeated intravenous infusion of BIL (4.3 ± 0.3 mg/dl, n = 7; p > 0.02). Furthermore, the plasma BIL concentration measured 10 min after the 180 min of BIL infusion in the evening (6 P.M.) on Day 2 after the start of the repeated intravenous infusion of BIL (9.1 ± 0.3 mg/dl, n = 7) was no different from that measured 4 wk after the start of the repeated intravenous infusion of BIL (9.2 ± 0.3 mg/dl, n = 7; p > 0.02).
On the basis of these results, we decided to begin the investigational phase of the study 1 wk after the start of repeated intravenous infusion of a high dose of BIL. The survival rate of each group is shown in Figure 1. In the BLM group (n = 25), the rats died consistently every week. In contrast, although some rats in the BIL+BLM group died in the first 10 d, no rats died on the following days. On Day 21 after intratracheal instillation of BLM, the survival rate of the BIL+BLM group that received infusion of BIL from 7 d before intratracheal instillation of BLM to the end of the experimental phase of the study was significantly higher than that of the BLM group (p < 0.05; n = 25) (Figure 1). Likewise, when the repeated intravenous infusion of BIL was started at the same time as intratracheal instillation of BLM, the survival rate was significantly higher than that of the BLM group (p < 0.05; n = 25) (Figure 1). Furthermore, when the repeated intravenous infusion of BIL was started 7 d after intratracheal instillation of BLM, the survival rate was significantly higher than that of the BLM group (p < 0.05; n = 15) (Figure 1). There were no significant differences among the survival rates for the three BIL+BLM groups. No death occurred in either the BIL group or in the control group during the 3-wk experimental phase of the study.
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In the histologic studies, lungs from the BLM group showed a diffuse and marked infiltration of inflammatory cells and increased alveolar wall thickness with typical fibrotic changes (Figure 2a). In contrast, lungs from the BIL+BLM group receiving repeated intravenous infusion of BIL from 7 d before intratracheal instillation of BLM to the end of the experimental phase of the study showed fewer fibrotic lesions and local infiltrations of inflammatory cells (Figure 2b). Lungs of rats from the BIL group did not show the identifiable lesions observed in either the BLM group or the BIL+BLM group (Figure 2c), demonstrating that hyperbilirubinemia itself does not cause any change to the lung. No infectious pneumonia was seen in any lung from any group. The grades of fibrosis in the four groups are presented in Table 1. The BLM group had a significantly higher pulmonary fibrosis score than any of the other groups (p < 0.001). The pulmonary fibrosis scores for the BIL+BLM group that received repeated intravenous infusion of BIL from 7 d before the intratracheal instillation of BLM to the end of the experimental phase of the study was significantly lower than that for the BLM group (p < 0.001).
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The lung hydroxyproline content of the study groups, an index of collagen accumulation, is shown in Table 1. The lung hydroxyproline content in the BLM group was significantly higher than in the control group. On the other hand, the lung hydroxyproline content in the BIL+BLM group treated with repeated intravenous infusion of BIL from 7 d before the intratracheal instillation of BLM to the end of the experimental phase of the study remained at the same level as in the control group, and it was significantly lower than the level in the BLM group (p < 0.001).
All BALF were obtained at 21 d after intratracheal instillation of BLM. The leukocyte profiles in the BALF from the four study groups of animals are shown in Figure 3. Although the number of alveolar macrophages (the predominant cell type in BALF) did not differ significantly among the four groups, the numbers of lymphocytes (p < 0.05) and neutrophils (p < 0.05) in the BLM group were significantly higher than in the control group. The numbers of lymphocytes and neutrophils were significantly reduced in the BIL+BLM group receiving repeated intravenous infusion of BIL from 7 d before the intratracheal instillation of BLM to the end of the experimental phase of the study, as compared with the numbers of these cells in the BLM group.
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, Control; 
, BIL+Saline; 
, BLM; 
, BIL+BLM.
The TGF-1 concentration in the BALF of the BIL group
was almost the same as that in the control group. The TGF-1
concentration in the BALF of the BLM group was significantly higher than that of the control group (Table 1). In contrast, the TGF-1 concentration in the BALF of the BIL+
BLM group receiving repeated intravenous infusion of BIL
from 7 d before the intratracheal instillation of BLM to the
end of the experimental phase of the study was significantly lower than that in the BLM group (Table 1).
The urine content of BOM in the BIL group was almost the same as that in the control group (p > 0.4). In contrast, the urine BOM content in the BLM group was slightly higher than that in the control group (p < 0.001). Furthermore, the urine BOM content in the BIL+BLM group receiving repeated intravenous infusion of BIL from 7 d before the intratracheal instillation of BLM to the end of the experimental phase of the study was higher than in the BLM group (Table 1).
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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The hallmark of interstitial pulmonary fibrosis is the deposition
of collagen in the alveolar walls, which causes derangement of
the gas-exchanging units of the lung. In the present study, we
demonstrated the inhibitory effects of hyperbilirubinemia on pulmonary fibrosis caused by BLM through histopathologic
and biochemical analysis of the lung. Hyperbiliruminemia inhibited BLM-induced increases in the hydroxyproline content
of the lung and the TGF-1 content in BALF, two biologic
markers of pulmonary fibrosis (19, 20, 22).
BOM were isolated by Yamaguchi and associates (25). They also demonstrated increases in urine BOM in a patient undergoing laparotomy, and they suggested that BOM are markers of oxidation of BIL and that BIL may have a protective effect against oxidative stress in vivo (25). Therefore, the increase in BOM in rat urine that we observed in the present study might provide evidence of BIL oxidation in vivo after the injection of BLM. An increase in BOM in the urine in the BIL+BLM group suggests that BIL might be used as an antioxidant against the oxidative action of BLM and that the consumption of BIL resulting from this might partly relate to inhibition of lung inflammation and reduced collagen deposition in the lung. These findings suggest that BIL may play a crucial role in protection against BLM-induced oxidation in the lung.
Cigarette smoking also causes oxidative stress to the lung and is an obvious single risk factor for pulmonary emphysema. Serum BIL concentrations of smokers are significantly lower than those of nonsmokers (26), suggesting that in vivo, BIL may act as an antioxidant via oxidative degradation per se.
BIL is an efficient scavenger of ROS (27, 28) and interacts chemically with the superoxide anion (29). Its activity as a chain-breaking antioxidant has been shown (11, 30). Therefore, BIL might act as a scavenger of oxidants in lung inflammation after treatment with BLM (1). The increase in BOM that we observed in rat urine could be a partial indicator of the total oxidative stress occurring under such conditions. In mice, however, the activities of antioxidants including superoxide dismutase, catalase, and glutathione peroxidase were observed to decrease at 2 d after treatment with BLM and then to increase at 4 d after treatment with BLM (31), suggesting that BLM might affect antioxidant content and these antioxidants might inhibit the oxidative action of BLM. The concentration of oxidized extracellular glutathione increases in the BALF of patients with lung fibrosis (32), although we did not measure it in the present study. Further studies are needed to clarify the mechanisms involved in these findings.
Besides the direct oxidative action of BLM in injuring lung
tissue, the simultaneous activation of inflammatory cells is an essential process in the development of pulmonary fibrosis
(1). Of the cytokines associated with fibrosis in experimental animal models and patients with pulmonary fibrosis, TGF-1
is the most potent regulator of inflammation (22) and induces connective tissue synthesis (33, 34). Giri and colleagues
reported that antibodies to TGF-1 reduced lung collagen accumulation in BLM-treated mice (35). Our finding that the
content of TGF-1 in BALF obtained from the BIL+BLM
group of rats in our study was significantly lower than that obtained from the BLM group was consistent with these reports.
Alveolar macrophages are a major source of TGF-1 in
interstitial pulmonary inflammation (17). However, in the
present study, the numbers of macrophages in BALF did not
differ among the four study groups of animals. According to
other reports, total cells in BALF increase in the early weeks
after BLM treatment (18). This early-stage alteration of cell
populations after BLM treatment is mainly due to an increase
in polymorphonuclear cells and lymphocytes (4, 18), whereas
the number of alveolar macrophages does not increase (4).
The significant increase in polymorphonuclear cells as well as
lymphocytes in BALF that we observed in the present study
are consistent with these reports (4, 18). Thus, the time course
of changes in cell numbers after administration of BLM may
differ among cell types. Because the increased concentration of TGF-1 in BALF from fibrosing lung was not found to always be accompanied by an increased number of alveolar
macrophages, we speculate that activation of alveolar macrophages is necessary to maintain the high concentration of TGF-1
in BALF (36).
BIL is highly reactive by itself (15, 37, 38), although it has an antioxidant effect (27, 37). Its toxic effects are dose-dependent. More than 10 mg/dl of BIL and a ratio of BIL to albumin that exceeds 1 are toxic for neonatal erythrocytes (37) and reduce local cerebral metabolic rates for glucose in immature rats (15). We did not examine organ injury other than in the lung in the BIL rat group. However, we observed no inflammatory changes in the rat lung, including the hemorrhage observed in patients with kernicterus (38). Therefore, less than 10 mg/dl of BIL given by intravenous injection does not appear to have a significant toxic effect on rats. In fact, all of the rats in the BIL group survived throughout the experiments.
In summary, we have described a beneficial effect of hyperbilirubinemia on pulmonary fibrosis induced by the administration of BLM in rats, as found through morphologic and biochemical analyses. The increased BOM in urine from the BIL+ BLM group of animals in our study suggests that the antioxidant action of BIL may partly explain the inhibition of pulmonary fibrosis in this model. The data described here support the idea that BIL is not only an important antioxidant in serum (39, 40), but can also play an important antioxidant role in pulmonary fibrosis. Further studies are required to establish the safe and appropriate conditions for clinical use of bilirubin.
Many possible treatments for pulmonary fibrosis have been investigated, but none have succeeded in clinical trials (5, 6). Orally administered N-acetylcysteine has been shown to ameliorate BLM-induced pulmonary fibrosis, and clinical trials of antioxidants are currently underway in patients with IPF (8, 9). Recently, treatment with interferon gamma-1b and prednisolone has also been proposed for the treatment of IPF (7). Because BIL exists naturally in human serum, the data in the present study might support it too as a possible candidate for the treatment of pulmonary fibrosis.
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Footnotes |
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Correspondence and requests for reprints should be addressed to Hidetada Sasaki M.D., Ph. D., Professor and Chairman, Department of Geriatric and Respiratory Medicine, Tohoku University School of Medicine, Sendai, Japan 980-8574.
(Received in original form March 27, 2001 and accepted in revised form November 5, 2001).
Acknowledgments: The authors thank Mr. Grant Crittenden for correcting the English in this article.
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References |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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