Effect of intratracheally administered anticancer drugs on lung hydroxyproline content

Toxicology Letters, 30 (1986) 63-70 Elsevier

63

TOXLett. 1527

EFFECT OF INTRATRACHEALLY ADMINISTERED ANTICANCER DRUGS ON LUNG HYDROXYPROLINE CONTENT (Lung; pulmonary; cyclophosphamide;

fibrosis; hydroxyproline; collagen; BCNU; bleomycin; doxorubicin; dactinomycin; mitomycin c; vincristine)

JAMES P. KEHRER, YU-CHEN C. LEE and ROBIN D. SMITH Division of ~harmaco~~~ and Toxicology, College of Pharmacy, The U~iversiiy of Texas at Austin, Austin, TX 78712-1074 fU.S.A.j (Received October 8th, 1985) (Revision received November lOth, 1985) (Accepted November 24th, 1985)

SUMMARY The systemic administration of the anticancer drug bleomycin is associated with the devefopment of lung damage and fibrosis. intratracheal (it.) instillation of bleomycin has often been employed in animal models of this lesion to more rapidly elicit lung damage. However, various drugs and chemicals are known to induce lung damage and fibrosis when given i.t. The results presented here demonstrate that, in addition to bfeomycin, the i.t. instillation of dactinomycin, mitomycin c, doxorubicin, and cyclophosphamide resulted in increased lung levels of hydroxyproline, an indicator of fibrosis. In contrast, increases were not seen following i.t. doses of 5-fluorouracil or vincristine, and 1,3-bis(2-chloroethyl)-1-nitrosourea(BCNU) actually decreased lung hydroxyproline content. These findings suggest that i.t. treatments with bleomycin may produce lung damage more representative of anticancer drugs in general than the specific lesion which is produced when bleomycin is administered systemically. The mechanisms underlying i.t. drug-induced increases in lung hydroxyproline are not known, but may be related to the ability of cell-cycle-nonspecific anticancer drugs to directly damage alveolar epithelial cells.

INTRODUCTION

Numerous anticancer drugs including BCNU (carmustine), bleomycin, cyclophosphamide, methotrexate, and mitomycin c induce lung damage and pulmonary fibrosis following their systemic administration to man undergoing cancer chemotherapy [I ,2]. Similar lung lesions have been produced in various Abbreviations:

BCNU, 1,3-bis(2-chloroethyI)-I-nitrosourea;

it., intratracheal.

0378-4274/86/~ 03.50 0 Elsevier Science Publishers B.V. (Biomedical Division)

64

animal species receiving these drugs by the parenteral route, although this damage is often difficult to induce and may be delayed and variable. The rapid induction of lung damage in animals has been particularly difficult to achieve with bleomycin. This drug has attracted considerable interest because, although it lacks hematopoietic effects, it produces lung damage and fibrosis in about 5% of treated patients [2]. The lung damage produced following the it. instillation of bleomycin appears to produce a disease model which closely resembles the histological and physiological picture of bleomycin toxicity and idiopathic pulmonary fibrosis in humans [3]. The it. route of administration, in contrast to the parenteral route, rapidly induces lung damage and fibrosis in most species and has therefore achieved widespread use by investigators studying the mechanisms and pathology of bleomycin-induced lung disease. The pulmonary fibrosis which develops following the i.t. administration of bleomycin could be a specific response to this drug or a general response which would be seen with other cytotoxins. The it. instillation of numerous agents, including dusts such as silica [4] and chemicals such as cadmium chloride [5] or trisodium citrate and acid-citrate-dextrose [6], among others, induces lung damage and fibrosis. The mechanisms underlying these effects have not been conclusively established, but several investigators have hypothesized that necrosis of type I alveolar epithelial cells is an important factor [4,6,7]. Since anticancer drugs are toxic to normal as well as neoplastic cells, they would be expected to damage type I cells following it. administration and may induce lung damage and fibrosis when given by this route. The purpose of the present study was to examine the pulmonary toxicity of a series of widely used anticancer drugs administered by the i.t. route to determine whether agents, in addition to bleomycin, were capable of inducing fibrosis. Increases in total lung hydroxyproline were monitored as an index of collagen content 181. The results indicate that other anticancer drugs will increase pulmonary hydroxyproline content when given it. There did not appear to be any correlation between drugs known to elicit lung damage when administered parenterally, and those producing lung damage when administered i.t. The anticancer drugs most likely to produce increased hydroxyproline levels when given i.t. were cell-cycle-nonspecific agents such as doxorubicin, dactinomycin, mitomycin c and cyclophosphamide. MATERIALS AND METHODS

Animals and chemicals Male BALB/c mice, 9-14 weeks of age, bred and maintained in the Animal Resources Center of the University of Texas at Austin, were used for these studies. Dactinomycin (actinomycin D) was obtained as Cosmegen (40:1 mannitol) from Merck, Sharp and Dohme (West Point, PA), cyclophosphamide as Cytoxan from

65

Mead Johnson (Evansville, IN), vincristine as Oncovin from Eli Lilly and Co. (Indianapolis, IN), 5fluorouracil from Roche (Nutley, NJ) and BCNU as BiCNU and mitomycin c as Mutamycin from Bristol (Syracuse, NY). Doxorubicin, as adriamycin (5:l lactose), was obtained as a gift from Adria Laboratories (Columbus, OH) and bleomycin, as outdated vials of Blenoxane, was a gift from Bristol. Previous work has demonstrated that outdated bleomycin retains its pulmonary toxicity ]9]. Treatments Intratracheal injections were performed transorally with the animals under halothane anesthesia. A small incision was made in the neck of each animal to verify placement of the catheter before instillation of the drug. Drug concentrations were adjusted with isotonic saline (or 10% ethanol for BCNU) so that the injection volume was 75 ~1 in all cases. The incision was then closed with a wound clip and the animals were maintained for 21 days with food and water ad lib. Control mice received equal volumes of isotonic saline or 10% ethanol in isotonic saline. Bleomycin, at an it. dose of 0.1 units [lo] or following a single intraperitoneal dose of 100 mg/kg [l I], damages lung tissue in BALB/c mice. Assuming 1.5 mg bleomycin/unit and a parenteral therapeutic dose of about 0.5 units/kg, a factor of 0.2 times this dose was used to select initial i.t. doses of the other anticancer drugs. Doses were increased or decreased as necessary until lung damage or significant mortality was observed. The dose of BCNU was limited to 300 pglmouse by its solubility in the 10% ethanol vehicle used for this drug. Hydroxyproline assay Mice were killed by cervical dislocation 21 days after the i.t. instillation of each drug. Lung tissue was removed, frozen and lyophilized. The dried tissue was weighed and hydrolyzed in 4 ml 6 N HCl at 107°C for I8 h. The hydrolysate was neutralized with 10 N NaOH and adjusted to 8 ml with water. A 0. l-ml aliquot of this solution was assayed for hydroxyproline by oxidizing it to pyrrole with chloramine T and measuring the absorbance at 560 nm of the chromophore formed by the reaction of pyrrole with p-dimethylaminobenzaldehyde [ 121. Statistics All data are expressed as means k S.E. The data from B~NU-treated mice were compared to 10% ethanol-treated controls with Student’s t-test. All other treatment groups were compared to saline-treated controls with Dunnett’s test [ 131. A P value of co.05 was considered significant. RESULTS

Table I shows the various drugs and dosages administered

i-t. to mice. Deaths

66

TABLE

1

MORTALITY

FOLLOWING

INTRATRACHEAL

INSTILLATION

OF VARIOUS

ANTICANCER

DRUGS Dosea Drug

&g/mouse)

Dactinomycin BCNU Bleomycin Cyclophosphamide

Number

Mortality

treated

(Q)

(mglkg)

10

0.4

7

0

25

1

5

100

300

0

12

10

0.10 units

4

10

0

0.35 units

14

5

100

1000

40

10

0

5000

200

10

30b

10

10

Doxorubicin

60

Fluorouracil

700

28

10

0

5000

200

5

20

100

4

10

60

1.2

10

10

Mitomycin

c

Vincristine

30

a Mice were dosed per animal. mice weighed

Dosages

expressed

as mg/kg

were calculated

with the assumption

that the

25 g.

b This dose approached 12 h after

2.4

dosing

the solubility

limit of the cyclophosphamide

and may have been unrelated

to the cytotoxicity

in water. The deaths occurred

within

of this drug.

were observed at the maximal dose of all drugs tested except BCNU. The greatest mortality was seen following the highest doses of bleomycin, dactinomycin and mitomycin c. Mice treated with these agents rapidly lost weight and deaths occurred from 2-5 days after the initial treatment. Dosages of these drugs were therefore decreased, as needed, to achieve the survival of enough animals for analysis of lung hydroxyproline content. The total hydroxyproline content of lung tissue from mice, 21 days after i.t. treatment with various anticancer drugs, is shown in Fig. 1. Compared to results from saline-treated controls, there was a significant increase in lung hydroxyproline following administration of bleomycin, doxorubicin, dactinomycin, mitomycin c, and the highest dose of cyclophosphamide. In contrast, there was a significant decrease in lung hydroxyproline with BCNU, while fluorouracil and vincristine did not produce measurable changes at the doses tested. Lung tissue from mice treated with fluorouracil, vincristine or the lowest dose of cyclophosphamide appeared normal in terms of gross morphology. Treatments with the other anticancer drugs resulted in hemorrhagic areas throughout the lung. In addition, lungs from mitomycin c-treated mice failed to float when placed in water indicating severe consolidation.

67

350

300

250 t zK? gs

(10) 2oo

P-’ 150

100

SC

. mg) Treatment

0 saline

BCNU

Fig. 1. Total lung hydroxyproline was analyzed in parentheses MITO, ferent

VIN

= N. Abbreviations: c; DACT,

saline-instilled

5-FU,

dactinomycin;

control

CP

in mice treated

21 days after the i.t. instillation

mitomycin from

5-w

content

of each drug. 5fluorouracil; BLEO,

MIT0

i.t. with various

DACT

Data are presented VIN,

bleomycin;

vincristine;

DOX,

BLEO

anticancer

00X

agents.

as mean CP,

doxorubicin.

Lung tissue

+ S.E. Values

cyclophosphamide; *Significantly

dif-

mice (P
DISCUSSION

Although BALB/c mice have been found to be relatively resistant to bleomycininduced lung damage [ 141, this strain is generally an excellent model for the production of pulmonary fibrosis following treatment with a variety of toxic agents. In addition, BALB/c mice maintain a relatively constant size after reaching maturity and survive despite severe lung damage (personal observation). The results presented here demonstrate that bleomycin and cyclophosphamide,

68

which readily damage mouse lung when administered systematically [11,15], also elicit damage when given it. to BALB/c mice. The i.t. doses of bleomycin and cyclophosphamide employed have been reported previously to induce lung damage and fibrosis in mice [10,16]. The 32% increase in hydroxyproline after cyclophosphamide and the 49% increase after bleomycin were similar to the results shown in these previous reports. In addition to bleomycin and cyclophosphamide, the it. instillation of doxorubicin, dactinomycin, and mitomycin c increased lung collagen content, an indicator of fibrosis [8,12]. Although systemic mitomycin has been associated with pulmonary fibrosis in human patients [17], there is no evidence this occurs in mice and the other anticancer drugs tested have not been linked to lung damage [ 1,2]. Increases in lung hydroxyproline content after the i.t. instillation of anticancer drugs appears, therefore, to be unrelated to the ability of systemic treatments to damage lung tissue. Increased lung hydroxyproline was not seen in mice treated with fluorouracil, vincristine, or BNCU. 3 weeks have been found to be sufficient for fibrosis to develop in mice treated with a variety of pneumotoxins [7,10,11], but it is possible that effects would have appeared if the animals were analyzed after a longer period of time. The reasons why some, but not all anticancer drugs were able in produce fibrosis when instilled i.t. are not known. Those agents which increased lung collagen could be broadly classified as cell-cycle-nonspecific while those which did not alter lung collagen were cell-cycle-specific. The classification of anticancer drugs by their action on the cell cycle is, however, imprecise at best. Bleomycin, the classic lung-toxic anticancer drug, might be an exception to this grouping since it has been classified as cell-cycle-specific. However, the cytotoxic mechanisms of bleomycin remain rather obscure and may involve other factors [18]. BCNU, which slightly decreased lung collagen, has been classified as cell-cycle-nonspecific [19] providing another potential exception to such a grouping, although some workers have indicated this drug has cell-cycle-specific characteristics [ 181. The decreased lung collagen measured following the i.t. instillation of BCNU may be related to its general debilitating effects on mice or to its lipid solubility which also prevented the testing of higher doses. Although histopathological analyses were not performed as part of this study, the gross pathology and changes in lung hydroxyproline content measured demonstrated that i.t. treatments with several different anticancer drugs induce lung damage and fibrosis. The basis for this toxic effect is unclear, but appears to be at least moderately related to whether the drug is cell-cycle-nonspecific. The lung damage produced when anticancer drugs are administered i.t. may, therefore, be more representative of their overall cytotoxicity rather than of a specific lung toxicity as is seen following parenteral treatments with bleomycin or cyclophosphamide.

69

ACKNOWLEDGEMENTS

This work was supported by American Cancer Society grants CH-263 and CH-263A. We thank Mr. Emmanuel Enemkpali for his excellent technical assistance. J.P.K. is the recipient of Research Career Development Award number HL 01435 from the National Heart, Lung and Blood Institute and holds the Gustavus Pfeiffer Centennial Fellowship in Pharmacology.

REFERENCES 1 J.P. Kehrer

and S. Kacew,

Systemically

applied

chemicals

that damage

lung tissue, Toxicology,

toxicity

of antineoplastic

35

(1985) 251-293. 2 S.J. Ginsberg

and R.L.

Comis,

The pulmonary

agents,

Sem. Oncol.,

9

(1982) 34-5 1. 3 G.L.

Snider,

hamsters

J.A.

Hayes

and A.L.

by endotracheal

1099-I 108. 4 D.H. Bowden and I.Y.R. in the generation 5 C.E. Cross, tratracheal

ST.

Omaye,

6 T. Mitsuhashi, pulmonary

D.C.

M. Shimazaki,

fibrosis

induced

(1985) 261-270. 7 W.M. Haschek and H.P. macol.,

Rifas,

fibrosis, G.K.

J. Pathol.,

Hasegawa

chloride

pulmonary

fibrosis

Am. Rev. Respir.

Pulmonary

Reddy,

Biochem.

H. Kuwahara, citrate

in

117 (1978)

inflammatory

Biochemical

Pharmacol.,

response

effects

- a possible

of in-

28 (1979) 381-388.

T. Sakai and H. Masuda,

and acid-citrate-dextrose,

fibrosis

produced

Dis.,

144 (1984) 149-161.

and K.A.

on rat lung,

Y. Chanoki, by trisodium

Witschi,

interstitial

and stereology,

The role of cell injury and the continuing

pulmonary

of cadmium

Chronic

Pathology

Adamson,

of silicotic

instillation

Korthy,

bleomycin.

Exp.

mechanism,

Experimental

Mol. Pathol.,

Toxicol.

42

Appl. Phar-

51 (1979) 475-487.

8 S.I. Rennard,

V.J. Ferrans,

and P. Nettesheim

(Eds.),

K.H.

Bradley

Mechanisms

and R.G. Crystal,

in Respiratory

Lung connective

Toxicology,

tissue, in H. Witschi

Vol. II, CRC Press,

Boca Raton

FL, 1982, pp. 115-153. 9 A. Schlein,

J.E. Schurig,

bleomycin

tested before

10 P.J.

Hakkinen,

tiating

C.C. Morse,

effects

of oxygen

mium chloride, 11 P.J.

C.D. Baca, W.T. Bradner and after its expiration

Hakkinen,

J.W.

Whiteley

Appl.

drugs,

and H.R.

W.L.

15 V.E. Gould

fibrosis,

Hightower

Am.

Hyperoxia,

22 (1979) 265-268. 17 S. Martino, L.H.

Baker,

and H.R.

manganese

Pharmacol.,

studies of

Witschi,

Poten-

tricarbonyl,

cad-

67 (1983) 55-69.

but not thoracic

lung damage

and R.V. Smith,

Rev. Respir.

and J. Miller,

(1975) 513-522. Pulmonary 16 D. Popenoe,

Appl.

toxicity

65 (1981) 727-728.

x-irradiation,

in mice, Am. Rev. Respir.

The many faces of an increase

potenDis.,

126

in lung collagen,

5 (1985) 240-250.

Lab., 11 (1979) 32-45. 14 D.J. Schrier, R.G. Kunkel and S.H. Phan, pulmonary

Witschi,

Pulmonary Rep.,

W.M. Haschek

Toxicol.

and R.C. Lindenschmidt,

Toxicol.,

Treat.

by methylcyclopentadienyl

and cyclophosphamide-induced

(1982) 281-285. 12 H.P. Witschi, A.F. Tryka 13 R.E. Wilcox,

W.E. Dalbey,

in lungs damaged

oleic acid, and antitumor

tiates bleomycin-

Fundam.

F.M. Martin,

and S.T. Crooke,

date, Cancer

Sclerosing

effects R.J.

data analysis

The role of strain variation

Dis.,

alveolitis

J.J.

in biomedical in murine

research,

Am.

bleomycin-induced

127 (1983) 63-66.

of intratracheal Pollard,

Post-hoc

induced

by cyclophosphamide,

cyclophosphamide, Correa

and

M.D.

Proc. DeMattia,

Am. J. Pathol., West. Pharmacol. Pulmonary

81 Sot.,

toxicity

of

70

mitomycin,

in S.K.

Developments,

Carter

Academic

18 R.L. Schilsky and J.W. Yarbro, bro (Eds.), 19 H.C.

Toxicity

Hoegland,

bro (Eds.),

S.T.

Cooke

New York,

of Chemotherapy,

Mitomycin

of antineoplastic

Grune

complications

(Eds.),

c: Current

Status

and

New

1979, pp. 231-242.

Pharmacology

of Chemotherapy,

Hematologic

Toxicity

and

Press,

and Stratton,

of cancer

Grune

drugs,

chemotherapy,

and Stratton,

in M.C. Perry and J.W. Yar-

New York,

1984, pp. 21-59.

in M.C. Perry and J.W. Yar-

New York,

1984, pp. 433-448.