Biochemical and morphological assessments of bleomycin pulmonary toxicity in rats

TOXICOLOGY

AND APPLIED

Biochemical

PHARMACOLOGY53,64-74(1980)

and Morphological Assessments Pulmonary Toxicity in Rats’ WAI-MINGTOM~

Pulmonary-Toxicology Minneapolis,

AND MARK R. MONTGOMERY

Laboratory and Pulmonary Disease Section, Minnesota 55417; and Department of Pharmacology, Minneapolis, Minnesota 55455

Received August

of Bleomycin

I, 1979; accepted

October

Veterans Administration Hospital, University of Minnesota,

24, 1979

Biochemical and Morphological Assessments of Bleomycin Pulmonary Toxicity in Rats. TOM, W.-M.,AND MoNTGoMERY,M.R.(~~~O). Toxicol.Appl.Pharmacol.53,64-74. Low doses (5 units) or high doses (15 units) of bleomycin were administered ip twice weekly to separate groups of male rats. A progressive dose-dependent reduction in body weight gain with a concomitant increase in wet lung weight to body weight ratio was observed over 6 weeks of treatments. Light microscopic confirmation of perivascular edema suggested that the increase in wet lung weight resulted from fluid accumulation. Although histological evidence of mild fibrosis was also present following low-dose treatment, only a slight increase in collagen content was observed biochemically. In vivo elevation of prolyl hydroxylase activity was observed in lungs of animals treated with low-dose bleomycin for 6 weeks. Highdose bleomycin regimen did not accelerate fibrosis, but rather inhibited the prolyl hydroxylase activity. In vitro, this enzyme activity was also stimulated at l- 10 pM concentration, but was inhibited at 0.1 mM. Lung angiotensin-converting enzyme activity was examined as an indication of damage to endothelial cells. This activity was unchanged after low-dose bleomycin treatments until 6 weeks, at which time a 5% decrease in activity was observed. High-dose bleomycin treatment produced a biphasic response in angiotensin-converting enzyme activity, with an initial 32% stimulation after 1.5 weeks of treatment followed by a 41% inhibition after 3 weeks. Kinetic analysis of the in vitro interaction of purified porcine plasma angiotensin-converting enzyme with bleomycin indicated that bleomycin acts as a competitive inhibitor with a Ki of 3.7 pM. Stimulation of angiotensin-converting enzyme activity following acute high-dose bleomycin administration could be due to the initial endothelial cell repair, while the inhibition at later stages may indicate extensive destruction of endothelial surface upon repeated bleomycin administration. Direct inhibition of the enzyme by the chelating effect of bleomycin may also be a possibility.

Bleomycin, an antitumor glycopeptide antibiotic isolated from the fermentation broth

of Streptomyces verticillus (Umezawa et al., 1966), is used in combination with other antineoplastic agents or radiotherapy for the treatment of various cancers (Crooke and Bradner, 1976; Friedman, 1978; Wasserman, 1978). The absence of myelotoxicity or immunosuppression (Andrews, 1972; Lehane and Lane, 1978) has been a principal advantage in the clinical use of bleomycin in cancer chemotherapy. Adverse side effects of bleomycin include anorexia, body

1 This work was supported in part by NIH Grant ESHL 01365. Portions of this work were presented at the joint meeting of the American Society of Pharmacology and Experimental Therapeutics and the Society of Toxicology, August 13- 17, 1978, at Houston, Texas; and the annual FASEB meeting, April l-10, 1979, at Dallas, Tex. * To whom corresoondence and requests for reprints should be addressed. 0041-008x/80/040064-1 1$02.00/0 Copyright 0 1980 by Academic Press, Inc. All rights of reproduction in any form reserved.

64

BLEOMYCIN

PULMONARY

weight reduction, fever, hypotension, and mucocutaneous and pulmonary toxicity (Blum er al., 1973). Among these, pulmonary toxicity is the most serious complication and commonly limits therapy with this drug (Comis, 1978; Willson, 1978). The pulmonary toxicity is dose-limiting and usually occurs between 4 and 10 weeks after initiation of bleomycin therapy. Pulmonary infiltration and fibrosis are common sequelae. Bleomycin-induced pulmonary toxicity has been studied in a variety of animal models (Adamson and Bowden, 1974; Aso et al., 1976; Bedrossian et al., 1977; McCullough et al., 1978; Ohwada et al., 1971; Schurig et al., 1979; Starcher et al., 1978; Sikic et al., 1978; Thrall et al., 1979; Tom and Montgomery, 1979). The characteristic fibrotic lesion appears to be the result of initial endothelial cell injury followed by epithelial cell damage, edema and intlammation, and epithelial cell hyperplasia. We have previously established an animal model in rats for the investigation of bleomycin-induced pulmonary toxicity by repeated ip administration of bleomycin for a period of 3 or 6 weeks (Tom and Montgomery, 1979). This communication reports the histological and biochemical confirmation of fibrosis in these animals. Lung collagen content and prolyl hydroxylase activity were determined as biochemical indicators of fibrosis. Lung angiotensinconverting enzyme was evaluated as a possible indication of damage to the capillary endothelium. METHODS Chemicals. Bleomycin was supplied by Bristol Laboratories, Syracuse, New York, as the copperfree sulfate, 15 biological units per ampoule (1.2- 1.7 units/m&. This preparation of bleomycin contained 55-70% bleomycin A, and 25-32% bleomycin B2 (Crooke and Bradner, 1976). L-[3,4-3H]Proline (specific activity, 55 Ci/mmol) was purchased from Amersham Radiochemical, Arlington Heights, Illinois, porcine angiotensin converting enzyme from Calbiochem, San Diego, California, Insta-gel from Packard Com-

TOXICITY

65

pany, Downers Grove, Illinois. p-Dimethylaminobenzaldehyde was obtained from Fisher Scientific Company, Pittsburgh, Pennsylvania, and used without further purification, Other chemicals were best reagent grade. Treatment of animals. Male, CD strain SpragueDawley rats (180-200 g initial weight) were obtained from Charles River Breeding Laboratories, Wilmington, Massachusetts. Low doses (5 units) or high doses (15 units) of bleomycin (in 0.5 ml of 0.9% sodium chloride) were administered ip twice weekly to separate groups of rats for 1.5, 3, or 6 weeks. Controls received an equal volume of saline. Twenty hours following the last injection, animals were killed by cervical fracture and exsanguinated. The lungs were removed and dissected from the trachea. The wet weight of individual lobes was recorded. The left lung was perfused intrabronchially with a fixing solution (4% formaldehyde- 1% glutaraldehyde in 0.2 M phosphate buffer, pH 7.4) at a constant pressure of 30 cm water for 48 hr, embedded in paraffin, and sectioned at 6 pm for histological examination. Sections were stained with hematoxylin and eosin or Masson’s trichrome. The right upper lobe was used for the determination of hydroxyproline content and the right lower lobe for assay of prolyl hydroxylase and angiotensin-converting enzyme activities. Determination of hydroxyproline. Hydroxyproline was estimated colorimetricahy as described by Woessner (1961). The lobe was hydrolyzed in 6 N hydrochloric acid at 130°C for 16 hr in a sealed Pyrex tube. The hydrolysate was neutralized with 2 N NaOH and diluted with deionized water. Separate aliquots were used for hydroxyprohne assay and for the estimation of total protein content. Leucine was used as standard for the ninhydrin protein assay (Rosen, 1957). Preparation of labeled protocollagen. Tritium-labeled protocollagen was prepared from chick embryos with modification of the method of Peterkofsky and DiBlasio (1975). Frontal and thoracic bones (1-2 g) from 12- to lCday-old chick embryos were incubated with t-[3,4-3H]proline (400 &i) in modified Eagle’s minimum essential medium in the presence of 0.5 mM 2,2’-bipyridine at 37°C for 2.5 hr. The pulselabeled bones were washed and rinsed with cold 0.11 M NaCI-0.05 Tris-HCI, pH 7.4, which contained 1 mM L-prohne and homogenized in 0.5 M acetic acid with a Super-Dispax Tissuemizer Model 182 (Tekmar, Cincinnati, Ohio) at 12,000 rpm, twice at 15-set intervals. The suspension was stirred overnight at 4°C and then centrifuged at 20,OOOg for 30 min. The supernatant was dialyzed 16 hr against 0.4 M NaCI-0.1 M TrisHCI, pH 7.6, at 4°C and precipitated with ammonium sulfate (176 mg/ml supernatant). The pellet was resuspended and dialyzed against 0.2 M NaCl-0.05 M Tris-HCl, pH 7.6, for 16 hr at 4°C and subjected to fibril precipitation by incubation at 37°C for 1.5 hr.

66

TOM AND MONTGOMERY

The supematant which contained the labeled protocollagen was stored at -20°C in small aliquots. Linear formation of hydroxylated collagen as a function of time and enzyme concentration was established for each substrate preparation. Preparation of 9000g supernatant. The minced right lower lobe was washed and homogenized twice at 15-set intervals in 3 vol of ice-cold 0.15 M KCl-0.02 M Tris-HCl, pH 7.4, with a Super-Dispax Tissuemizer. The resulting homogenate was centrifuged at 9000g for 15 min. The supematant protein concentration was measured as described by Sutherland et al. (1949) with bovine serum albumin as standard. Preparation of microsomal fraction. The 9000g supernatant was centrifuged at 165,OOOg for 45 min at 5°C. The pellet was suspended in 0.02 M Tris-HCl0.15 M KC1 buffer, pH 7.4 (Tris-HCI buffer), and resedimented at 165,OOOgfor an additional 45 min. The resulting pellet was resuspended in Tris-HCl buffer to 10 mg/ml concentration. Protein concentration was determined as described by Sutherland et al. (1949). Prolyl hydroxylase assay. Prolyl hydroxylase activity was estimated by the release of tritiated water from the hydroxylation of labeled protocollagen (Peterkofsky and DiBlasio, 1975). The standard assay mixture had a pH of 7.6 and contained 40 mmol Tris-HCl, 1.0 mmol cw-ketoglutarate, 1.0 mmol sodium ascorbate, 0.2 mm01 ferrous ammonium sulfate, 0.5 mm01 dithiothreitol, 0.4 mg/ml catalase, 2 mg/ml bovine serum albumin, 50,000 cpm of labeled protocollagen, and 0.1 mg of either the 9000s or microsomal fraction protein in a final volume of 100 ~1. Incubation performed was at 37°C for 15 min and was terminated with 0.4 ml of 6.25% trichloroacetic acid, followed by the addition of 15 ~1 of bovine serum albumin (25 mg/ml) as coprecipitant. After centrifugation at 1SOOgfor 5 min. the supernatant was eluted through a 0.5 x 2-cm column of washed Bio-Rad AC SOW-X8 ion-exchange resin. The precipitate was resuspended in 5% trichloroacetic acid and resedimented. The supematant was then applied again to the column followed by washing with 0.5 ml of deionized water. The effluent (1.5 ml) was dissolved in 10 ml of Insta-Gel and counted in a Beckman LS-1OOC scintillation counter. Angiotensin-converting enzyme assay. This enzyme activity was determined from hydrolysis of an artificial substrate, hippuryl-histidyl-leucine (HHL) as described by Yang and Neff (1972). The reaction mixture contained 2.5 mM HHL, 0.3 mM sodium chloride and either 0.1 mg 9000g supematant or 250 pg of porcine serum angiotensin-converting enzyme (EC 3.4.15.1) (activity: 2.5 nmol/mg-hr-’ of angiotensin I converted to angiotensin II) in a final volume of 100 pl of 0.5 M potassium phosphate buffer, pH 8.3. Incubation was at 37°C for 30 min. Following the addition of 1% (w/v) p-phthaldehyde in methanol, the liberated histidy]-leucine (HL) was quantitated with an Aminco-

Bowman Fluorometer 495 nm).

(excitation

365 nm, emission

Statistical analysis. Data obtained from bleomycintreated groups were compared to control groups with the unpaired Student’s r test (two-tailed). p < 0.05 was selected as statistical significance. Kinetic constants were determined by Wilkinson regression analysis (Wilkinson, l%l).

RESULTS A progressive increase in body weight of 30,75, and 127% was observed in control groups at the end of the 1.5, 3, and 6 weeks of treatments, respectively. As seen in Table 1, a slight weight loss occurred in both treated groups at 1.5 weeks. In the 3-week treatment, the weight of the animals in the low-dose group was 70% that of the controls. After 6-week treatment of low-dose bleomycin, the body weight was about 50% of control. The mortality was 10% for the 6week low-dose group. Some animals showed signs of labored respiration, nail and hair loss, with occasional skin hyperpigmentation. The weight of the animals in the highdose group was only half that of control after 3 weeks. Due to the high mortality (>80%) in 6 weeks in the high-dose group, this treatment group was not available for investigation. The lungs of bleomycin-treated animals were edematous although their wet weights were not different from control. However, when expressed as the ratio of wet lung weight to body weight, a dose-dependent increase in this index of fluid accumulation was apparent in the treated animals (Table 1). Significant increases of 28, 44, and 81% in wet lung weight/body weight occurred in the low-dose group at the end of the 1.5, 3-, and 6-week treatments, respectively. High doses of bleomycin treatment resulted in a 45% increase in the 1.5-week group and a 100% increase at 3 weeks. Morphological confirmation of perivascular edema was evident in lung sections from all bleomycin-treated animals (Fig. 1). For comparison, no significant difference in wet liver weight/body weight was observed

BLEOMYCIN

PULMONARY TABLE

EFFECT

OF BLEOMYCIN

ON BODY

67

TOXICITY

1

WEIGHT

AND

WET

LUNG

WEIGHT

IN RATS

Weeks of treatment 1.5

3

6

Body weight (g) Control” Low-dose bleomycin’ High-dose bleomycin’

241 2 9 (lo)* 201 -r- 8 (lO)d 177 * 7 (10)d

317 -c 7 (11) 226 rt 5 (ll)d 153 2 10 (8)d

387 k 11 (12) 183 + 13 (lDd -

Wet lung weight (g) Control Low-dose bleomycin High-dose bleomycin

1.28 c 0.04 (10) 1.36 + 0.05 (10) 1.38 ? 0.06 (10)

1.70 * 0.12 (11) 1.75 * 0.08 (11) 1.75 + 0.08 (8)

1.84 * 0.09 (12) 1.54 lr 0.10 (11)’ -

Lung/body ratio (%) Control Low-dose bleomycin High-dose bleomycin

0.53 + 0.02 (10) 0.68 + 0.03 (10) 0.77 + 0.04 (10)

0.54 k 0.03 (11) 0.78 k 0.04 (11)” 1.07 2 0.06 (8)d

0.48 2 0.03 (12) 0.87 k 0.06 (ll)d -

” 0.9% saline, 0.5 ml ip, twice weekly. b Data are expressed as means ? SE. Number in parentheses indicate numbers of animals. c Bleomycin, 5 units in 0.5 ml saline ip, twice weekly. d p < 0.01, compared to controls, Student’s t test. r Bleomycin, 15 units in 0.5 ml saline ip, twice weekly. ‘p < 0.05, compared to controls, Student’s t test.

among the three groups at any time (data not shown). To investigate the development of pulmonary fibrosis, lung collagen content was estimated by the quantitation of total lung hydroxyproline and is also expressed as the ratio of hydroxyproline to total ninhydrin reactive materials (in terms of leucine equivalents). There were no differences in collagen content among the control, lowdose, and high-dose groups in the 1.5 and 3-week studies (Table 2). A 23% elevation in lung collagen content (p < 0.05) was detected in the 6-week low-dose group. However, no significant increase in collagen/total protein was observed (p = 0.08). This result may agree with the observation of scattered focal areas of mild pulmonary fibrosis (Fig. 2) which was apparent in several but not all Masson’s trichrome sections prepared from this group. Disruption of the alveolar structure and collagen deposition at the periphery were also

observed in sections from several of these animals. The high-dose treatment failed to produce any detectable fibrosis in 3 weeks. Prolyl hydroxylase, the rate-limiting enzyme in collagen biosynthesis (Prockop et al., 1976), catalyzes the hydroxylation of prolyl residues of protocollagen prior to glycosylation and crosslinking. The enzyme requires a-ketoglutarate, ascorbate, and ferrous ion. This enzyme activity was unchanged at the end of 1.5 or 3 weeks of the low-dose treatment (Fig. 3). However, the enzyme activity was elevated 87% (p < 0.05) when measured at the 6-week period. The increase in lung prolyl hydroxylase activity agrees with the slight increase in lung collagen content at 6 weeks (Table 2) and the presence of focal fibrotic lesions (Fig. 2). High doses of bleomycin did not alter the prolyl hydroxylase activity at the end of 1.5 weeks. A slight inhibition of the enzyme activity (17%) (p < 0.05) was observed after 3-week treatment.

68

TOM

AND

MONTGOMERY

BLEOMYCIN

PULMONARY TABLE

69

TOXICITY

2

EFFECTOF BLEOMYCIN ON RAT LUNGCOLLAGEN

CONTENT

Weeks of treatment 1.5

3

6

Lung hydroxyproline” ControP Low-dose bleomycind High-dose bleomycid

11.1 5 0.4 (5) 10.4 +- 0.6 (5) 10.1 + 1.0 (5)

15.9 _c 0.9 (5) 13.8 ” 1.6 (5) 13.0 rf- 1.5 (4)

16.5 _c 0.9 (6) 20.3 + 0.8 (5) -

Leucine equivalents of lung protein0 Control Low-dose bleomycin High-dose bleomycin

1.03 + 0.02 (5) 0.98 it 0.07 (5) 0.92 -+ 0.06 (5)

1.36 + 0.07 (5) 1.27 t 0.08 (5) 0.99 r 0.10 (4)

1.20 + 0.07 (6) 1.20 rt 0.14 (5) -

HydroxyprolineAeucine ratio (X lOa) Control Low-dose bleomycin High-dose bleomycin

10.7 r 0.3 (5) 10.7 r 0.4 (5) 10.9 % 0.5 (5)

11.8 ” 0.5 (5) 10.8 f 0.7 (5) 13.0 k 0.1 (4)

13.9 f 0.7 (6) 17.6 r 1.8 (5)” -

n Lung collagen content of the right upper lobes is expressed as pmol hydroxyproline per g wet lung tissue. h 0.9% saline, 0.5 ml ip, twice weekly. r Data are expressed as mean r SE. Number in parentheses indicate numbers of animals. ‘r Bleomycin, 5 units in 0.5 ml saline ip, twice weekly. (’ p < 0.05, compared to controls, Student’s t test. f Bleomycin, 15 units in 0.5 ml saline ip, twice weekly. y Lung protein content of the right upper lobes is expressed as mmol leucine equivalent of ninhydrin reactive substances per g wet lung tissue. h p = 0.08, compared to control, Student’s t test.

The in vitro interaction of bleomycin with lung prolyl hydroxylase was also examined (Fig. 4). With 1 to 10 FM bleomycin, a stimulation of prolyl hydroxylase activity was observed in both microsomal and 9000g supematant fractions. However, at 100 ,UM concentration, there was a complete inhibition of prolyl hydroxylase activity in both preparations. The inhibition could not be reversed by addition of either exogenous ferrous iron or additional uketoglutarate. In the presence of Fe (II) and molecular oxygen, bleomycin has been shown to generate reactive superoxide anion radical (Sausville et al., 1978; Oberley and Buettner, 1979; Sugiura and Kikuchi, 1978). If the observed bleomycin inhibition of prolyl hydroxylase activity is mediated by superoxide, the addition of superoxide dismutase should alleviate the effect of

bleomycin. However, superoxide dismutase was ineffective to reverse such inhibition. Angiotensin-converting enzyme is located on the luminal membrane of capillary endothelial cells. Since these cells may be one of the initial sites of pulmonary damage by bleomycin (Adamson and Bowden, 1974), the activity of lung angiotensinconverting enzyme was also examined (Fig. 5). Treatment with the low dose had no effect on angiotensin-converting enzyme activity at IS- and 3-week periods, but a 27 + 6% (p < 0.01) decrease was observed in the 6-week treatment group. On the other hand, high-dose treatment initially (1.5 weeks) stimulated the angiotensin-converting enzyme to 32 2 10% (p < 0.01) above control activity, followed by a 41 -+ 7% (p < 0.01) inhibition at 3 weeks. The in vitro effect of bleomycin on puri-

TOMANDMONTGOMERY

BLEOMYCIN

PULMONARY

FIG. 3. Effect of in vivo bleomycin on lung prolyl hydroxylase activity. Data are expressed as percent of control enzyme activity. Separate groups of rats (N = 4-6) were injected ip twice weekly with saline (C); low-dose bleomycin, 5 units (L) or high-dose bleomytin, 15 units (H) for 1.5 weeks (spotted bars), 3 (solid bars) or 6 weeks (crosshatched bars). Data are represented as ,? 2 SE. *p < 0.05: **p < 0.01.

fied porcine plasma angiotensin-converting enzyme (EC 3.4.15.1) was investigated. A concentration-dependent inhibition of this enzyme activity was observed at concentration greater than 10 PM. Kinetic analysis suggests that bleomycin may inhibit the enzyme in a competitive manner with a Ki Of 3.7 PM. DISCUSSION Our schedule of low-dose bleomycin treatment in rats requires 12 separate

1000 2 e

z :: 2% B :: +

Microsomol

71

TOXICITY

injections spaced over 6 weeks to produce mild fibrosis. Increasing the bleomycin dose threefold failed to accelerate the fibrosis. Thus it appears that time rather than drug quantity may be important in the development of fibrotic lesions. As reported recently, a minimum duration of 4 to 5 weeks was also necessary to produce pulmonary fibrosis in rats after SC administration of 24 units/kg of bleomycin three times per week (Schurig et al., 1979). The dose-dependent reduction in total body weight and in weight gain in all bleomycin-treated animals may be due to the direct cytotoxic action on cell growth. Bleomycin has been demonstrated to inhibit DNA and protein synthesis in a variety of cell systems (Mtiller et al., 1976). Anorexia, as experienced by most bleomycin-treated patients, could also be another explanation for reduction in body weight gain (Blum et al., 1973). Wet lung weight/body weight increased during bleomycin treatment. Histological confirmation of perivascular edema suggests that the increase in this ratio could be the consequence of accumulation of excessive extracellular fluid. However, mononuclear cell infiltration, hyperplasia of type II alveolar cells and collagen deposition

Fraction

800 600 400

0

Conml No E"Z

NO Slrn

0.1 1.0 IO Km 8lm (@I)

NO Blrn

a.1 10 ICI too 8lm

(JJM)

FIG. 4. Effect of bleomycin on lung prolyl hydroxylase activity. Enzyme activity is expressed as amount of tritiated water released from tritiated protocollagen per 0.1 mg protein per 15 min. Data represent mean f SD of duplicate samples.

72

TOM AND MONTGOMERY

may also contribute to increased lung mass (Aiolfi et al., 1978). Only a few of our treated rats developed scattered focal areas of pulmonary fibrosis. Our observation of a significant elevation in total lung collagen (p < 0.05) with no change in protein content at the end of 6-week low-dose bleomycin treatment indicates that the rate of collagen synthesis may have slightly exceeded that of noncollagenous protein synthesis. Increased collagen content has been reported to occur from 4 to 10 weeks after bleomycin administration in other animal models (McCullough et al., 1978: Sikic et al., 1978; Starcher et ul., 1978). However, pulmonary fibrosis may occur without large changes in total lung collagen content. Fibrosis may involve destruction of type I collagen with simultaneous deposition of type III collagen, resulting in no net increase in collagen content (Seyer et al., 1976). The newly formed, loosely packed connective tissues may give a misleading histological appearance of enhanced collagen deposition (Orthoefer et al., 1976). The observation of an increase in cutaneous insoluble collagen content in mice with simultaneous decrease in collagenase activity after bleomycin treatment supports this hypothesis (Ichihashi et nl., 1973). The prolyl hydroxylase activity in lung tissue generally indicates its capacity for collagen synthesis (Prockop et al., 1976). The elevated enzyme activity observed in the 6-week low-dose group strongly suggests that fibrotic process had been initiated by this time. Interestingly, high doses of bleomycin treatment for 3 weeks caused a slight inhibition of this enzyme activity (83 t 5% of control). We have previously reported the stimulation of lung prolyl hydroxylase activity by 1 to 10 PM bleomycin in vitro (Tom and Montgomery, 1978). Stimulation of fetal rat prolyl hydroxylase by 10 PM bleomycin also has been reported by Takeda et al. (1978). This group concluded subsequently

FIG. 5. Effect of in ~ivo bleomycin treatments on lung angiotensin-converting enzyme activity. Data are expressed as percentage of control rate of enzyme activity. Code as in Fig. 3. Control rate = 286 nmol histidyl-leucine releasedimg-hr-‘. N = 4-6. **p < 0.01.

that the formation of a bleomycin-iron (II) complex was necessary for the stimulation of prolyl hydroxylase activity (Takeda et al., 1979). It has also been demonstrated that l,lO-phenanthroline at IO PM concentration markedly stimulated prolyl hydroxylase activity in confluent fibroblast cultures. This stimulation may be due to the removal of metal ions or metal ion-containing substances, which are inhibitory toward the enzyme, by complex formation (Chvapil et al., 1977). Recently, kinetic analysis suggested that both positive and negative cooperativity of intrinsic binding of ferrous ion to the allosteric sites of the enzyme may exist (Cumming et al., 1978). Thus, it is possible that bleomycin may exert its biphasic, concentration dependent effect on prolyl hydroxylase activity by influencing the binding of ferrous ion to the enzyme. Other than prolyl hydroxylase, the synthesis of extracellular ground substances of connective tissues such as acidic glycosaminoglycans and hyaluronic acid synthetase are also stimulated by bleomycin in cultured fibroblasts at about 1 FM concentration (Otsuka et a/. , 1976, 1978). If an initial site of bleomycin-induced lung damage is the capillary endothelial cells, the activity of the angiotensin converting enzyme might reflect cellular damage. Lung and serum activities of this enzyme have been utilized in the detection of acute bleomycin toxicity (Vals et al., 1979). In

BLEOMYCIN

PULMONARY

this study, no change in lung angiotensin converting enzyme activity was observed until 6 weeks of low-dose bleomycin treatment although appreciable histological evidence of pulmonary edema was obvious as early as 1.5 weeks. However, edema formation may merely reflect alterations in capillary permeability and not necessary direct damage to endothelial cells. Interestingly, a stimulation of angiotensin-converting enzyme activity was observed after 1.5 weeks of high-dose bleomytin treatment, followed by an inhibition at 3 weeks. A similar pattern has been reported in mice (Vals et al., 1979). The stimulation of the angiotensin-converting enzyme activity following acute high-dose bleomycin could be due to initial endothelial cell repair. It has been demonstrated in an acute lung injury study with oxygen that the endothelial cells are the first to begin to divide before epithelial and interstitial cells (Hogg et al., 1979). However, the subsequent inhibition of this enzyme activity might reflect destruction of the endothelial surface upon repeated challenges with high doses of bleomycin. This assumption awaits detailed morphological examination at the electron microscopic level. Direct inhibition of this metalloenzyme by the potent chelating effect of bleomycin may also be a possibility. Kinetic analysis indicated that bleomycin is a competitive inhibitor of the enzyme with an apparent Ki much smaller than those of other chelating agents such as 8-hydroxyquinoline, dimercaprol, 2-mercaptoethanol and dithiothreitol (Cushman and Cheung, 1971). REFERENCES ADAMSON, I. Y. R., AND BOWDEN, D. H. (1974). The pathogenesis of bleomycin-induced pulmonary fibrosis in mice. Amer. J. Pathol. 77, 185-198. AIOLFI, S., BIANCHI, A., GANDOLA, L., SERVIDA, E., SOCCINI, F., AND TANSINI. G. (1978). Bleomycin: pulmonary toxicity in rats. In Current Chemotherapy: Proc. Chemotherapy

10th

International

Congress

on

(W. Siegenthaler and R. Ltithy, eds.),

TOXICITY

73

Vol. II, pp. 1225-1228. American Society for Microbiology, Washington, DC. ANDREWS, E. J. (1972). An in vivo evaluation of the immunosuppressive action of bleomycin. Cancer Res. 32, 1993-1994. Aso, Y., YONEDA, K., AND KIKKAWA, Y. (1976). Morphologic and biochemical study of pulmonary changes induced by bleomycin in mice. Lab. Invest. 35, 558-568. BEDROSSIAN, C. W. M., GREENBERG, S. D., YAWN, D. H., AND O’NEAL, R. M. (1977). Experimentally induced bleomycin sulfate pulmonary toxicity: histopathology and ultrastructural study in the pheasant. Arch. Pathol. Lab. Med. 101, 248-254. BLUM, R. H., CARTER, S. K., AND ACRE, K. A. (1973). A clinical review of bleomycin-A new antineoplastic agent. Cancer 31, 903-914. CHVAPIL, M., MISIOROWSKI, R., TILLEMA, L., AND HERRING, C. (1977). Stimulation of the activity of prolyl hydroxylase in 3T3 fibroblasts by l,lOphenanthroline. Biochim. Biophys. Acta 497, 488498. COMIS, R. L. (1978). Bleomycin pulmonary toxicity. In Bleomycin: Current Status And New Developments (S. K. Carter, S. T. Crooke, and H. Umezawa, eds.), pp. 279-291. Academic Press, New York. CROOKE, S. T., AND BRADNER, W. T. (1976). Bleomytin, a review. J. Med. 7, 333-428. CUMMING, R. W., THOMPSON, J., AND KOPPEL, J. L. (1978). Iron: A possible heterotropic effector of prolyl hydroxylase. Biochim. Biophys. Acta 523, 533-537. CUSHMAN, D. W., AND CHEUNG, H. S. (1971). Spectrophotometric assay and properties of the angiotensin-converting enzyme of rabbit lung. Biothem. Pharmacol. 20, 1637-1648. FRIEDMAN, M. A. (1978). A review of the bleomycin experience in the United States. Rec. Results Cancer Res. 63, 152- 168. HOGG, J. C., STAUB, N. C., BERGOFSKY, E. H., AND VREIM, C. E. (1979). Workshop on the pulmonary endothelial cell. Amer. Rev. Resp. Dis. 119, 165170. ICHIHASHI, M., SHINKAI, H., TAKEI, M., AND SANO, S. (1973). Analysis of the mechanism of bleomycininduced cutaneous fibrosis in mice. J. Antibiot. 26, 238-242. LEHANE, D. E., AND LANE, M. (1978). The immunopharmacology of bleomycin in man. In Bleomycin: Current Status And New Developments (S. K. Carter, S. T. Crooke, and H. Umezawa, eds.), pp. 143-150. Academic Press, New York. MCCULLOUGH, B. M., COLLINS, J. F., JOHANSON, W. G., JR., AND GROVER, F. L. (1978). Bleomycininduced diffuse interstitial pulmonary fibrosis in baboons. J. Clin. Invest. 61, 79-88. MULLER, W. E. G.. ROHDE, H. J., AND ZAHN, R. K.

74

TOM

AND

(1976). Effect of bleomycin on DNA, RNA, enzymes, chromatin and on local sarcoma. J. Mol. Med. 1, 173-186. OBERLEY, L. W., AND BUETTNER, G. R. (1979). The production of hydroxyl radical by bleomycin and iron (II). FEBS Left. 97, 47-49. OHWADA, H., KATSUKI, H., AND HAYASAKI, Y. (197I). Histopathological and histochemical studies of experimental pulmonary fibrosis in bleomycingiven rats. In Blenoxane New Drug Application. Summary available by the Freedom of Information Act. FDA, Rockville, Md. ORTHOEFER, .I. G., BHATNAGER, R. S., RAHMAN, A., YANG, Y. Y., LEE, S. D., AND STARA, J. F. (1976). Collagen and prolyl hydroxylase levels in lungs of beagles exposed to air pollutants. Environ. Res. 12, 299-305. OTSUKA, K., MUROTA, S., AND MORI, Y. (1976). Stimulatory effect of bleomycin on the synthesis of acidic glycosaminoglycans in cultured fibroblasts derived from rat carrageenin granuloma. Biochim. Biophys. Acta 444, 359-368. OTSUKA, K., MUROTA, S., AND MORI, Y. (1978). Stimulatory effect of bleomycin on the hyaluronic acid synthetase in cultured fibroblasts. B&hem. Pharmacol. 27, 155 I- 1554. PETERKOFSKY, B., AND DIBLASIO, R. (1975). Modification of the tritium-release assays for prolyl and lysyl hydroxylases using Dowex-50 columns. Anal. Biothem. 66, 279-286. PROCKOP, D. J., BERG, R. A., KIVIRIKKO, K. I., AND UITTO, J. (1976). Intracellular steps in the biosynthesis of collagen. In Biochemistry of Collagen (G. N. Ramachandran and A. H. Reddi, eds.), pp. 163-273. Plenum, New York. ROSEN, H. (1957). A modified ninhydrin calorimetric analysis for amino acids. Arch. Biochem. Biophys. 67, 10-15. SAUSVILLE, E. A., STEIN, R. W., PEISACH, J., AND HORWITZ, S. B. (1978). Properties and products of the degradation of DNA by bleomycin and iron (II). Biochemistry I7, 2746-2754. SCHURIG, J. E., FLORCZYK, A. P., HIRTH, R. S., BRADNER, W. T., AND BVYNISKI, J. P. (1979). A rat model of bleomycin-induced pulmonary fibrosis. Fed. Proc. Fed. Amer. Sot. Exp. Biol. 38, 522. SEYER, J. M., HUTCHESON, E. H., AND KANG, A. H. (1976). Collagen polymorphism in idiopathic chronic pulmonary fibrosis. J. Clin. Invest. 57, 1498-1507. SIKIC, B. I., YOUNG, D. M., MIMNAUGH, E. D., AND GRAM, T. E. (1978). Quantification of bleomycin pulmonary toxicity in mice by changes in lung hydroxyproline content pathology. Cancer Res. &ARCHER, B. C., KUHN, (1978). Increased elastin

and morphometric 38, 787-792. C., AND OVERTON, and collagen content

histoJ. E. in the

MONTGOMERY lungs of hamsters receiving an intratracheal injection of bleomycin. Amer. Rev. Resp. Dis. 117, 299-305. SUGIURA, Y., AND KIKUCHI. T. (1978). Formation of superoxide and hydroxy radicals in iron (II)bleomycin-oxygen system: electron spin resonance detection by spin trapping. J. Antibiot. 31, 13101312. SUTHERLAND, E. W., CORI. C. F., HAYNES, R.. AND OLSEN, N. S. (1949). Purification of the hyperglycemic-glycogenolytic factor from insulin and from gastric mucosa. J. Biol. Chem. 180, 825-837. TAKEDA, K., KAWAI, S., KATO, F., TETSUKA. T., AND KONNO, K. (1978). Stimulation of prolyl hydroxylase activity by bleomycin. J. Antihiot. 31,701-704. TAKEDA, K., KATO, F., KAWAI, S., AND KONNO, K. (1979). The effect of bleomycin on prolyl hydroxylase and DNA chain breakage: Structure-activity relationship. J. Ant&or. 32, 43-47. THRALL, R. S., MCCORMICK, J. R., JACK, R. M., MCREYNOLDS, R. A.. AND WARD, P. A. (1979). Bleomycin-induced pulmonary fibrosis in the rat. Inhibition by indomethacin. Amer. J. Pathol. 95, 117-130. TOM, W.-M., AND MONTGOMERY, M. R. (1978). Interaction of bleomycin with iron-dependent enzyme systems of the lung. Pharmacologist 20, 238. TOM, W.-M., AND MONTGOMERY, M. R. (1979). Bleomycin-induced pulmonary toxicity: morphological and biochemical changes. Fed. Proc. Fed. Amer. Sot. Exp. Biol. 38, 582. UMEZAWA, H., MAEDA, K., TAKEVCHI, T.. AND AKAMI (1966). New antibiotics: bleomycin A and B. J. Anti&or. 19, 200-209. VALS, T. S., MOLTENI, A., MATTIOLI, L., SOBONYA, D., AND BARTH, R. (1979). Effects of chronic bleomycin administration on serum and lung angiotensin I converting enzyme in mice. Fed. Pror. Fed. Amer. Sot. Exp. Biol. 38, 964. WASSERMAN, T. H. (1978). Bleomycin and radiotherapy: an overview of clinical studies in the United States. In Bleomycin: Current Status And New Derlelopments (S. K. Carter, S. T. Crooke. and H. Umezawa, eds.), pp. 253-265. Academic Press, New York. WILKINSON, G. N. (1961). Statistical estimations in enzyme kinetics. Biochem. J. 80, 324-332. WILLSON, J. K. V. (1978). Pulmonary toxicity of antineoplastic drugs. Cancer Treat. Rep. 62, 20032008. WOESSNER, J. F., JR. (1961). The determination of hydroxyproline in tissue and protein samples containing small proportions of this amino acid. Arch. Biochem. Biophys. 93, 440-447. YANG, H.-Y. T., AND NEFF, N. H. (1972). Distribution and properties of angiotensin converting enzyme of rat brain. J. Neurochem. 19, 2443-2450.