Chem-Biol.Intemctionu,24 (1979) 0 Elsevier/North-Holland Scientific
107-110 Publishers, Ltd.
107
Short Communications INHIBITIONOF CHEMICALLY INDUCED SISTER CHROMATID EXCHANGES BY ELASTATINAL *
KAZUO UMEZAWA, MUTSUKO SAWAMURA, TAIJIRO MATSUSHITA TAKASHI SUGIMURA
and
Department of Molecular Oncology, Institute of Medical Science, University of Tokyo, 4-6-l Shirokanedai, Minato-ku, Tokyo I08 (Japan) (Received July 21st, 1978) (Accepted August 2&h, 1978)
Introduction Several protease inhibitors of low molecular weight have been isolated from Streptomyces [l]. Among them antipain, an inhibitor of papain and trypsin, was reported to inhibit W-induced or tif-induced mutation and lambda prophage induction in Escherichia coli [Z]. Elastatinal (Fig. l), an elastase inhibitor [3,4], was found to inhibit chemically induced mutation in
Salmonella typhimurium [ 51. Recently, mutagens were shown to induce sister chromatid exchanges (SCEs) in Chinese hamster cells [6] and human lymphocytes [7 1. Measurement of SCE has been developed as a cytological method for detecting chemical mutagens [ 8,9]. This paper reports that elastatinal suppressed SCEs induced by N-methylN’-nitro-N-nitrosoganidine (MNNG) and by Nethyl-N’-nitro-N-nitrosoguanidine (ENNG) in human lymphocytes.
Mater-i& and methods Chemicals. Elastatinal was obtained from Research Resources Program for Cancer Research, Ministry of Education, Science and Culture. MNNG and ENNG were purchased from Aldrich Chemical Co., Ltd. Milwaukee, Wise. Cells. A lymphoblastoid cell line of human peripheral lymphocytes transformed by Epstein-Barr virus was kindly supplied by A. Oikawa [lo]. Methods. Differential staining of sister chromatids was done essentially as described by Perry and Wolff with slight modifications [ 111. Human lymphocytes were cultured in Roswell Park Memorial Institute Medium * This work was supported by Grants-in-Aid for Cancer Research from the Ministry of Education, Science and Culture and the Ministry of Health and Welfare (52-9) and a grant from the Princess Takamatsu Cancer Research Fund. Abbreviations: BrdU, 5-bromodeoxyuridine; ENNG, N-ethyl-N’-nitro-N-nitrosoguanidine; MNNG, N-methyl-N’-nitro-N-nitrosoguanidine; SCE, sister chromatid exchange.
H&,
CH3
NH HN+y’ ‘CH2
CH’
dH,
HIi kH2 'CW
60
A”, CH3
HOOC-;H-NH-CO-NH-;H-CO-N"-;"-CO-NH&$ Fig. 1.Structure
of elae&tinal.
1640 (Nissui Seiyaku Co., Tokyo) supplemented with 20% heat-inactivated foetal bovine serum (Grand Island Biological Co., Grand Island, N.Y.), 2 mM Lglutamine, and 50 pg/ml kanamycin. Exponentially growing 2 X lo6 cells were treated with 10 PM of 5-bromodeoxyuridine (BrdU) in 4 ml of medium for 48 h. Then, the medium with 10 PM BrdU was changed, and a mutagen, dissolved in a small amount of dimethylsulfoxide and elastatinal were added with 0.05 ml of water. After 42-h incubation, colcemide (final concentration, 2 lo-'M) was added for 3 h and the mitotic ceils were collected by centrifugation. The ceiis were treated for 15 min with 0.05 M KC1 and then fixed in methanol-acetic acid (3 : 1). Drops of cells in fixative were dried on glass, stained with Hoechst 33258 (0.5 &ml in l/15 M SSrensen buffer pH 7.0) for 12 min and rinsed briefly with redistilled water. The preparations were mounted in l/15 M Siirensen buffer pH 8.0, and the cover slips were ,inged with rubber cement. After exposing the slides to black light over-night, the cover slips were removed and the slides were incubated for 2 h at 60°C in water. The slides were then stained with 2% Giemsa for 15 min. l
Results and discussion Scoring 40 cells there were an average of 11 spontaneous
SCEs/cell in human lymphocytes. MNNG at concentrations of lo-‘M and 1P M induced 5.7 and 12.1 SCEs/cell, respectively, while 10e5 M MNNG inhibited mitosis. Addition of 10 &ml of elastatinal did not affect the spontaneous SCJ%, but reduced the SCEs induced by MNNG by about 45%, as shown in Table I. The differences at both concentrations of MNNG were significant by Student’s t-test (P < 0.001). Similarly, ENNG at concentrations of lo-’ M and 10m6 M induced 6.9 and 13.0 SCEs/cell, respectively, while 10Vs M ENNG inhibited mitosis. As shown in Table II, 10 pg/ml of elastatinal reduced the SCEs induced by ENNG by about 65%. The differences were also significant at both concentrations of ENNG (P < 0.001). In bacterial cells elastatinal inhibited chemically induced mutation [5] and lambda prophage induction (W. Troll, pers. comm.). Protease inhibitors have been suggested to inhibit error-prone DNA repair involved in
109 TABLE I INHIBITION BY ELASTATINAL
OF SCEs INDUCED BY MNNG
MNNG (M)
SC&/cell
S.D.
Range
Cell scored
0 lo-’ lO+j 10 Erg/ml elastatinal 0 lo-’ 1o-6
11.0 16.7 23.1
2.5 3.4 6.0
6-16 10-24 14-40
40 40 40
2.8 3.7 4.7
8-18 7-20 8-32
40 40 40
+ 10.7 13.5 17.8
TABLE II INHIBITION BY ELASTATINAL ENNG (M)
SCEs/cell
0 10.3 10 -I 17.2 1o-6 23.3 10 pg/ml elastatinai + 0 10.4 lo-’ 12.5 1O-6 15.6
OF SCEs INDUCED BY ENNG S.D.
Range
CelI scored
2.5 2.6 4.1
6-16 13-22 16-38
40 40 40
2.8 2.4 2.6
6-22 9-18 10-22
40 40 40
mutagenesis and phage induction [ 21. SCE is a phenomenon which may be related to the repair of damaged DNA, but its aetiology is uncertain [12]. Elastatinal inhibits porcine pancreatic elastase, its Ki value being 2.4 lo-’ M, with acetyl-L-alanyl-L-alanyl-L-a.lanine-p-nitroanilide as substrate (T. Aoyagi, pers. comm.). Induction of SCEs may involve elastase like activity in human lymphocytes. The authors wish to thank Drs. A. Oikawa and H. Tohda, Department of Pharmacology, Research Institute for Tuberculosis and Cancer, Tohoku University, Sendai for valuable suggestions and for supplying cells. T. Aoyagi and H. Umezawa, Structures and activities of protease inhibitors of microbial origin, in E. Reich, D.B. Rifkin and E. Shaw (Eds.), Proteases and Biological Control, Cold Spring Harbor Laboratory, Cold Spring Harbor, 1975, pp. 429-454. MS. Meyn, T. Rossman and W. Troll, A protease inhibitor blocks SOS functions in Escherichia coli: antipain prevents A repressor inactivation, ultraviolet mutagenesis, and filamentous growth, Proc. Natl. Acad. Sci. USA, 74 (1977) 1152. H. Umezawa, T. Aoyagi, A. Okura, H. Morishima, T. Takeuchi and Y. Okami, Elastatinal, a new elastase inhibitor produced by Actinomycetes, J. Antibiot., 26 (1973) 787. A. Okura, H. Morishima, T. Takita, T. Aoyagi, T. Takeuchi and H. Umezawa, The structure of elastatinal, an elestase inhibitor of microbial origin, J. Antibiot., 28 (1975) 337.
110 5 K. Umexawa, T. Matauahima and T. Sugimura, Antimutagenic effect of elastatinal, a protease inhibitor from Actinomycetes. Proc. Acad. Japan, Ser. B, 53 (1977) 30. 6 P. Perry and H-J. Evans, Cytological detection of mutagen-carcinogen exposure by sister chromatid exchange. Nature (Lond.), 258 (1975) 121. 7 S-A. Latt, Sister chromatid exchanges, indices of human chromosome damage and repair: detection by fluorescence and induction by mitomycin C, Proc. Natl. Acad. Sci. USA, 71(1974) 3162. 8 Hd. Evans, Cytological methods for detecting chemical mutagens, in A. Hollaender (Ed.), Chemical Mutagens, Vol. 4, Plenum Press, New York, 1976, pp. l-29. 9 A. Kin&la, S. Mousset, C. Szpirer and M. Radman, Fixation and expression of recessive mutations as a model to study carcinogenesis, in P.C. Hanawalt and EC. Friedberg (Eds.), DNA Repair Mechanisms, Academic Press, New York, in press. 10 H. Tohda, A. Oikawa, T. Katsuki, Y. Hinuma and M. Seiji, A convenient method of establishing permanent lines of xeroderma pigmentoeum cells, Cancer Res., 38 (1978) 253. 11 P. Perry and 6. Wolff, New Qiemsa method for the differential staining of sister chromaticls, Nature (Bond.), 251 (1974) 156. 12 C-J. Bostock and A.T. Sumner, The eucaryotic chromosome. North Holland, Amsterdam 1978, pp. 466-467.
107-110 Publishers, Ltd.
107
Short Communications INHIBITIONOF CHEMICALLY INDUCED SISTER CHROMATID EXCHANGES BY ELASTATINAL *
KAZUO UMEZAWA, MUTSUKO SAWAMURA, TAIJIRO MATSUSHITA TAKASHI SUGIMURA
and
Department of Molecular Oncology, Institute of Medical Science, University of Tokyo, 4-6-l Shirokanedai, Minato-ku, Tokyo I08 (Japan) (Received July 21st, 1978) (Accepted August 2&h, 1978)
Introduction Several protease inhibitors of low molecular weight have been isolated from Streptomyces [l]. Among them antipain, an inhibitor of papain and trypsin, was reported to inhibit W-induced or tif-induced mutation and lambda prophage induction in Escherichia coli [Z]. Elastatinal (Fig. l), an elastase inhibitor [3,4], was found to inhibit chemically induced mutation in
Salmonella typhimurium [ 51. Recently, mutagens were shown to induce sister chromatid exchanges (SCEs) in Chinese hamster cells [6] and human lymphocytes [7 1. Measurement of SCE has been developed as a cytological method for detecting chemical mutagens [ 8,9]. This paper reports that elastatinal suppressed SCEs induced by N-methylN’-nitro-N-nitrosoganidine (MNNG) and by Nethyl-N’-nitro-N-nitrosoguanidine (ENNG) in human lymphocytes.
Mater-i& and methods Chemicals. Elastatinal was obtained from Research Resources Program for Cancer Research, Ministry of Education, Science and Culture. MNNG and ENNG were purchased from Aldrich Chemical Co., Ltd. Milwaukee, Wise. Cells. A lymphoblastoid cell line of human peripheral lymphocytes transformed by Epstein-Barr virus was kindly supplied by A. Oikawa [lo]. Methods. Differential staining of sister chromatids was done essentially as described by Perry and Wolff with slight modifications [ 111. Human lymphocytes were cultured in Roswell Park Memorial Institute Medium * This work was supported by Grants-in-Aid for Cancer Research from the Ministry of Education, Science and Culture and the Ministry of Health and Welfare (52-9) and a grant from the Princess Takamatsu Cancer Research Fund. Abbreviations: BrdU, 5-bromodeoxyuridine; ENNG, N-ethyl-N’-nitro-N-nitrosoguanidine; MNNG, N-methyl-N’-nitro-N-nitrosoguanidine; SCE, sister chromatid exchange.
H&,
CH3
NH HN+y’ ‘CH2
CH’
dH,
HIi kH2 'CW
60
A”, CH3
HOOC-;H-NH-CO-NH-;H-CO-N"-;"-CO-NH&$ Fig. 1.Structure
of elae&tinal.
1640 (Nissui Seiyaku Co., Tokyo) supplemented with 20% heat-inactivated foetal bovine serum (Grand Island Biological Co., Grand Island, N.Y.), 2 mM Lglutamine, and 50 pg/ml kanamycin. Exponentially growing 2 X lo6 cells were treated with 10 PM of 5-bromodeoxyuridine (BrdU) in 4 ml of medium for 48 h. Then, the medium with 10 PM BrdU was changed, and a mutagen, dissolved in a small amount of dimethylsulfoxide and elastatinal were added with 0.05 ml of water. After 42-h incubation, colcemide (final concentration, 2 lo-'M) was added for 3 h and the mitotic ceils were collected by centrifugation. The ceiis were treated for 15 min with 0.05 M KC1 and then fixed in methanol-acetic acid (3 : 1). Drops of cells in fixative were dried on glass, stained with Hoechst 33258 (0.5 &ml in l/15 M SSrensen buffer pH 7.0) for 12 min and rinsed briefly with redistilled water. The preparations were mounted in l/15 M Siirensen buffer pH 8.0, and the cover slips were ,inged with rubber cement. After exposing the slides to black light over-night, the cover slips were removed and the slides were incubated for 2 h at 60°C in water. The slides were then stained with 2% Giemsa for 15 min. l
Results and discussion Scoring 40 cells there were an average of 11 spontaneous
SCEs/cell in human lymphocytes. MNNG at concentrations of lo-‘M and 1P M induced 5.7 and 12.1 SCEs/cell, respectively, while 10e5 M MNNG inhibited mitosis. Addition of 10 &ml of elastatinal did not affect the spontaneous SCJ%, but reduced the SCEs induced by MNNG by about 45%, as shown in Table I. The differences at both concentrations of MNNG were significant by Student’s t-test (P < 0.001). Similarly, ENNG at concentrations of lo-’ M and 10m6 M induced 6.9 and 13.0 SCEs/cell, respectively, while 10Vs M ENNG inhibited mitosis. As shown in Table II, 10 pg/ml of elastatinal reduced the SCEs induced by ENNG by about 65%. The differences were also significant at both concentrations of ENNG (P < 0.001). In bacterial cells elastatinal inhibited chemically induced mutation [5] and lambda prophage induction (W. Troll, pers. comm.). Protease inhibitors have been suggested to inhibit error-prone DNA repair involved in
109 TABLE I INHIBITION BY ELASTATINAL
OF SCEs INDUCED BY MNNG
MNNG (M)
SC&/cell
S.D.
Range
Cell scored
0 lo-’ lO+j 10 Erg/ml elastatinal 0 lo-’ 1o-6
11.0 16.7 23.1
2.5 3.4 6.0
6-16 10-24 14-40
40 40 40
2.8 3.7 4.7
8-18 7-20 8-32
40 40 40
+ 10.7 13.5 17.8
TABLE II INHIBITION BY ELASTATINAL ENNG (M)
SCEs/cell
0 10.3 10 -I 17.2 1o-6 23.3 10 pg/ml elastatinai + 0 10.4 lo-’ 12.5 1O-6 15.6
OF SCEs INDUCED BY ENNG S.D.
Range
CelI scored
2.5 2.6 4.1
6-16 13-22 16-38
40 40 40
2.8 2.4 2.6
6-22 9-18 10-22
40 40 40
mutagenesis and phage induction [ 21. SCE is a phenomenon which may be related to the repair of damaged DNA, but its aetiology is uncertain [12]. Elastatinal inhibits porcine pancreatic elastase, its Ki value being 2.4 lo-’ M, with acetyl-L-alanyl-L-alanyl-L-a.lanine-p-nitroanilide as substrate (T. Aoyagi, pers. comm.). Induction of SCEs may involve elastase like activity in human lymphocytes. The authors wish to thank Drs. A. Oikawa and H. Tohda, Department of Pharmacology, Research Institute for Tuberculosis and Cancer, Tohoku University, Sendai for valuable suggestions and for supplying cells. T. Aoyagi and H. Umezawa, Structures and activities of protease inhibitors of microbial origin, in E. Reich, D.B. Rifkin and E. Shaw (Eds.), Proteases and Biological Control, Cold Spring Harbor Laboratory, Cold Spring Harbor, 1975, pp. 429-454. MS. Meyn, T. Rossman and W. Troll, A protease inhibitor blocks SOS functions in Escherichia coli: antipain prevents A repressor inactivation, ultraviolet mutagenesis, and filamentous growth, Proc. Natl. Acad. Sci. USA, 74 (1977) 1152. H. Umezawa, T. Aoyagi, A. Okura, H. Morishima, T. Takeuchi and Y. Okami, Elastatinal, a new elastase inhibitor produced by Actinomycetes, J. Antibiot., 26 (1973) 787. A. Okura, H. Morishima, T. Takita, T. Aoyagi, T. Takeuchi and H. Umezawa, The structure of elastatinal, an elestase inhibitor of microbial origin, J. Antibiot., 28 (1975) 337.
110 5 K. Umexawa, T. Matauahima and T. Sugimura, Antimutagenic effect of elastatinal, a protease inhibitor from Actinomycetes. Proc. Acad. Japan, Ser. B, 53 (1977) 30. 6 P. Perry and H-J. Evans, Cytological detection of mutagen-carcinogen exposure by sister chromatid exchange. Nature (Lond.), 258 (1975) 121. 7 S-A. Latt, Sister chromatid exchanges, indices of human chromosome damage and repair: detection by fluorescence and induction by mitomycin C, Proc. Natl. Acad. Sci. USA, 71(1974) 3162. 8 Hd. Evans, Cytological methods for detecting chemical mutagens, in A. Hollaender (Ed.), Chemical Mutagens, Vol. 4, Plenum Press, New York, 1976, pp. l-29. 9 A. Kin&la, S. Mousset, C. Szpirer and M. Radman, Fixation and expression of recessive mutations as a model to study carcinogenesis, in P.C. Hanawalt and EC. Friedberg (Eds.), DNA Repair Mechanisms, Academic Press, New York, in press. 10 H. Tohda, A. Oikawa, T. Katsuki, Y. Hinuma and M. Seiji, A convenient method of establishing permanent lines of xeroderma pigmentoeum cells, Cancer Res., 38 (1978) 253. 11 P. Perry and 6. Wolff, New Qiemsa method for the differential staining of sister chromaticls, Nature (Bond.), 251 (1974) 156. 12 C-J. Bostock and A.T. Sumner, The eucaryotic chromosome. North Holland, Amsterdam 1978, pp. 466-467.
