Mutation Research, 28 (1975) lO7-112
© Elsevier Scientific Publishing Company, Amsterdam--Printed in The Netherlands
107
C Y T O G E N E T I C E F F E C T OF BLEOMYCIN ON HUMAN LEUKOCYTES
IN VITRO
C. PROMCHAINANT
Department of Pathobiology, Faculty of Science, Mahidol University, Bangkok 4 (Thailand) (Received March isth, 1974) (Revision received September i7th, 1974)
SUMMARY
Human leukocytes treated with bleomycin (BLM) for clinical use, at concentrations of o.I, 0.5, I.O, IO and 50 ttg/ml were studied. Both chromosome- and chromatid-type aberrations were observed. The groups of larger chromosomes were more affected at every concentration. At dosages from o.I to IO #g/ml no significant difference of effects on chromosomes was observed. However, a dose-difference of about 500 times showed significant differences in effect both on the degree of chromosomal aberration and on mitotic indices.
INTRODUCTION BLM, a new antibiotic and antineoplastic drug, was discovered in 1962 by Dr. HAMAO UMEZAWAand his colleagues 1~. It is produced by Streptomyces verticillus, an actinomyces, which was isolated from soil in the neighborhood of a coal mine in Kahogun, Fukuoka prefecture, Japan. The drug is a complex which can be fractionated into sugar peptides of novel structures by means of chromatography. These peptides belong both to A groups (A1, As, Aa, A4, As, Ae, and A() and to B groups (B1, B~, B3, B4, Bs, and Be). BLM has biological effects on many types of organism ~. It inhibits DNA synthesis in cultured HeLa S3 cell 8; and an in vivo effect on an experimental tumor has been demonstrated in animals 11. An anti-cancer effect has also been demonstrated in the dog 7. BORNSTEIN investigated the effects of BLM of bone marrow in patients who were treated for up to one month, and chromosomal aberrations were found in those patients 1. This investigation aims to study the in vitro effect of BLM (clinically used form) on human chromosomes with the following objectives : (a) to ascertain whether or not BLM can cause any aberrations in chromosomes ; (b) to find out if those effects are dose-dependent ; (c) to determine the type of aberrations, if any, and (d) to study the effect on nfitotic activity. Abbreviations: BLM, bleomycin; PHA, phytohaemagglutinin.
I08
C. PROMCHAINANT
MATERIALS
AND
METHODS
Human leukocyte cultures used throughout this study were from 5 male donors, aged 22-27 years, selected at random. The male subjects were used to awfid bias due to hormonal variation during the menstrual cycle that has arisen with females. The leukocytes were stinmlated to mitosis by PHA-M (Wellcome). The method of culturing human leukocytes was slightly modified from that of MOORHEADCt al?. After the subjects have fasted for at least 3 h, 3o ml of the venous blood was drawn from each. The heparinized blood was allowed to stand at room temperature for 3o-6o rain before the leukocytes were separated from red blood cells. Then x ml of plasma-rich leukocytes was inoculated into 5 ml of warm (37 °) medium consisting of 8o(I{, TC I99 (Wellcome), zo% human AB serum and o.2 nfl of PHA-M (Wellcome). No antibiotics were added. Cultures were kept at 37 ° for 48 h before the treatment was started. BLM was diluted with three times-distilled water and then added to the cultures at the final concentrations of o.I, o.5, x, Io and 5o/~g/ml medium. The cultures were further incubated for 24 h before harvesting. At the end of incubation, o.o5 ml of o.o5% colchicine was added to arrest mitosis at metaphase. The cultures were then incubated for 3 h before being fixed in methanol :glacial acetic acid (3 : I) fixative. The preparation for metaphase karyotyping was done essentially according to the method described by MOORHEAD61 al.", but using the flame technique. The cells were stained with Giemsa. All slides were coded before scoring and only those cells carrying 40 chromosomes were studied. We attempted to score Ioo sufficiently well spread metaphase plates per dosage per individual bv a scanning method. The total number of cells showing chromosome aberration were carefully examined for identification of the type of aberration. The Denver classification system was used for standardization of karyotypet RESULTS
Results shown in Table IA indicate that the numbers of cells with chromosome aberrations in control groups and groups treated with a BLM dose of o.I/~g/ml was significantly different (Po.o5). This is evident from the TABLE
IA
AVERAGE YIELD NUMBER
(PERCENT)
OF C H R O M O S O M E A B E R R A T I O N S
IN K A R Y O T Y P I C
G R O U P S AN1) A V E R A G E
OF C E L L S \VITIt A L L T Y P E S OF C H R O M O S O M E A B E R R A T I O N
BLM (ttg/ml)
Scoring metaplates
Percentage of aberrations in chromosome groups A B C D E F
Control O,1 0. 5 i,o 10.O
ioo IOO too too IOO
o.i6 O.51 0.38 o.99 I,O 5
0.0 5 0.55 0.55 o.6o 0.90
0.25 o.49 0.65
O.I6 o-I9 o.22 O.I6
5o.o
zoo
t.0o
* .25
o.88
o.44
0.0 4 O,I1
--o.o 3 O.O 3
-
G
-
A w'rage number O/aberrant cells (all types of aberration) 1.0o
-
7.0°
-
,~.,~o
-
-
i4.~o i6.6o
_
._
24.40
-
In vitro EFFECTS OF BLEOMYCIN TABLE
109
IB
RESULTS OF t-TEST IN COMPARING THE NUMBER OF CELLS CARRYING CHROMOSOME ABERRATIONS FOR VARIOUS DOSES OF B L M
BLM
dosage
Control-o. i O.l-O. 5 o.I--I.O O.I--IO O.1--5o o.5--1 .o o.5--1o 0.5--50 I.O--IO I.O--50 lO--5o
te 6.65a o.83b 1.8I b 1.88 i° 7.IIa 1.44 b 1.53 b 6.29 a 1.88 b 2.56b 2.32b
a p < 0.05 . l0 p > 0.05 . e C a l c u l a t i o n of t v a l u e : t h e n u m b e r of cells w i t h c h r o m o s o m a l (cf. T a b l e I A , f i n a l c o l u m n ) .
aberrations for each dose was used
results of Student t-tests applying between dosages as shown in Table IB. There was a significant difference of effect between o.I and 5o #g/ml and 0. 5 and 5o #g/ml doses (P
© Elsevier Scientific Publishing Company, Amsterdam--Printed in The Netherlands
107
C Y T O G E N E T I C E F F E C T OF BLEOMYCIN ON HUMAN LEUKOCYTES
IN VITRO
C. PROMCHAINANT
Department of Pathobiology, Faculty of Science, Mahidol University, Bangkok 4 (Thailand) (Received March isth, 1974) (Revision received September i7th, 1974)
SUMMARY
Human leukocytes treated with bleomycin (BLM) for clinical use, at concentrations of o.I, 0.5, I.O, IO and 50 ttg/ml were studied. Both chromosome- and chromatid-type aberrations were observed. The groups of larger chromosomes were more affected at every concentration. At dosages from o.I to IO #g/ml no significant difference of effects on chromosomes was observed. However, a dose-difference of about 500 times showed significant differences in effect both on the degree of chromosomal aberration and on mitotic indices.
INTRODUCTION BLM, a new antibiotic and antineoplastic drug, was discovered in 1962 by Dr. HAMAO UMEZAWAand his colleagues 1~. It is produced by Streptomyces verticillus, an actinomyces, which was isolated from soil in the neighborhood of a coal mine in Kahogun, Fukuoka prefecture, Japan. The drug is a complex which can be fractionated into sugar peptides of novel structures by means of chromatography. These peptides belong both to A groups (A1, As, Aa, A4, As, Ae, and A() and to B groups (B1, B~, B3, B4, Bs, and Be). BLM has biological effects on many types of organism ~. It inhibits DNA synthesis in cultured HeLa S3 cell 8; and an in vivo effect on an experimental tumor has been demonstrated in animals 11. An anti-cancer effect has also been demonstrated in the dog 7. BORNSTEIN investigated the effects of BLM of bone marrow in patients who were treated for up to one month, and chromosomal aberrations were found in those patients 1. This investigation aims to study the in vitro effect of BLM (clinically used form) on human chromosomes with the following objectives : (a) to ascertain whether or not BLM can cause any aberrations in chromosomes ; (b) to find out if those effects are dose-dependent ; (c) to determine the type of aberrations, if any, and (d) to study the effect on nfitotic activity. Abbreviations: BLM, bleomycin; PHA, phytohaemagglutinin.
I08
C. PROMCHAINANT
MATERIALS
AND
METHODS
Human leukocyte cultures used throughout this study were from 5 male donors, aged 22-27 years, selected at random. The male subjects were used to awfid bias due to hormonal variation during the menstrual cycle that has arisen with females. The leukocytes were stinmlated to mitosis by PHA-M (Wellcome). The method of culturing human leukocytes was slightly modified from that of MOORHEADCt al?. After the subjects have fasted for at least 3 h, 3o ml of the venous blood was drawn from each. The heparinized blood was allowed to stand at room temperature for 3o-6o rain before the leukocytes were separated from red blood cells. Then x ml of plasma-rich leukocytes was inoculated into 5 ml of warm (37 °) medium consisting of 8o(I{, TC I99 (Wellcome), zo% human AB serum and o.2 nfl of PHA-M (Wellcome). No antibiotics were added. Cultures were kept at 37 ° for 48 h before the treatment was started. BLM was diluted with three times-distilled water and then added to the cultures at the final concentrations of o.I, o.5, x, Io and 5o/~g/ml medium. The cultures were further incubated for 24 h before harvesting. At the end of incubation, o.o5 ml of o.o5% colchicine was added to arrest mitosis at metaphase. The cultures were then incubated for 3 h before being fixed in methanol :glacial acetic acid (3 : I) fixative. The preparation for metaphase karyotyping was done essentially according to the method described by MOORHEAD61 al.", but using the flame technique. The cells were stained with Giemsa. All slides were coded before scoring and only those cells carrying 40 chromosomes were studied. We attempted to score Ioo sufficiently well spread metaphase plates per dosage per individual bv a scanning method. The total number of cells showing chromosome aberration were carefully examined for identification of the type of aberration. The Denver classification system was used for standardization of karyotypet RESULTS
Results shown in Table IA indicate that the numbers of cells with chromosome aberrations in control groups and groups treated with a BLM dose of o.I/~g/ml was significantly different (Po.o5). This is evident from the TABLE
IA
AVERAGE YIELD NUMBER
(PERCENT)
OF C H R O M O S O M E A B E R R A T I O N S
IN K A R Y O T Y P I C
G R O U P S AN1) A V E R A G E
OF C E L L S \VITIt A L L T Y P E S OF C H R O M O S O M E A B E R R A T I O N
BLM (ttg/ml)
Scoring metaplates
Percentage of aberrations in chromosome groups A B C D E F
Control O,1 0. 5 i,o 10.O
ioo IOO too too IOO
o.i6 O.51 0.38 o.99 I,O 5
0.0 5 0.55 0.55 o.6o 0.90
0.25 o.49 0.65
O.I6 o-I9 o.22 O.I6
5o.o
zoo
t.0o
* .25
o.88
o.44
0.0 4 O,I1
--o.o 3 O.O 3
-
G
-
A w'rage number O/aberrant cells (all types of aberration) 1.0o
-
7.0°
-
,~.,~o
-
-
i4.~o i6.6o
_
._
24.40
-
In vitro EFFECTS OF BLEOMYCIN TABLE
109
IB
RESULTS OF t-TEST IN COMPARING THE NUMBER OF CELLS CARRYING CHROMOSOME ABERRATIONS FOR VARIOUS DOSES OF B L M
BLM
dosage
Control-o. i O.l-O. 5 o.I--I.O O.I--IO O.1--5o o.5--1 .o o.5--1o 0.5--50 I.O--IO I.O--50 lO--5o
te 6.65a o.83b 1.8I b 1.88 i° 7.IIa 1.44 b 1.53 b 6.29 a 1.88 b 2.56b 2.32b
a p < 0.05 . l0 p > 0.05 . e C a l c u l a t i o n of t v a l u e : t h e n u m b e r of cells w i t h c h r o m o s o m a l (cf. T a b l e I A , f i n a l c o l u m n ) .
aberrations for each dose was used
results of Student t-tests applying between dosages as shown in Table IB. There was a significant difference of effect between o.I and 5o #g/ml and 0. 5 and 5o #g/ml doses (P
