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Cadmium sulfate does not induce sister chromatid exchanges in human lymphocytes in vitro
173
Toxicology Letters, 37 (1987) 173-175 Elsevier
TXL 01801
CADMIUM SULFATE DOES NOT INDUCE SISTER CHROMATID EXCHANGES IN HUMAN LYMPHOCYTES IN VITRO (Cadmium; mutagenicity;
sister chromatid
EWA BASSENDOWSKA-KARSKA
exchanges)
and MARIA ZAWADZKA-KOS
Department of Hygiene, MedicaI Academy, Jaracza, 63, 90-2.51 tddi
(Poland)
(Received 5 January 1987) (Revision received 23 March 1987) (Accepted 25 March 1987)
SUMMARY Mutagenic properties of cadmium sulfate were analyzed by the method of sister chromatid exchanges (SCEs) in lymphocytes of human peripheral blood taken from 4 donors. No significant increase was found in the mean frequency of SCEs in lymphocytes exposed to 1.6 x lO-3 mM, 3.1 x 10e3 mM and 6.2 x 10e3 mM CdS04 in culture.
INTRODUCTION
Cadmium is a metal widely spread in the environment. Both the general population and the people occupationally exposed are at risk of cadmium absorption. In the areas of insignificant contamination the daily dietary cadmium intake was estimated to range between 15 and 60 fig per person. Cadmium resorption in lungs does not exceed 0.1 pg per day [ 1,2]. The quantities quoted do not cause intoxications or symptoms of disease. Nevertheless some authors claim cadmium compounds to lead to chromosome and chromatid aberrations in vitro [2,3]. This was the reason why the present research on mutagenic properties of cadmium sulfate was undertaken. MATERIALS AND METHODS
Analysis was carried out in human lymphocytes of peripheral blood taken from 4 healthy donors (3 women and 1 man). Cultures were prepared according to Wolff 0378-4274/87/$ 03.50 0 1987 Elsevier Science Publishers B.V. (Biomedical Division)
174
and Perry [4]. Heparinized venous blood of 0.5 ml collected with a syringe was added to a culture medium consisting of medium F-10 (Flow Lab.), L-glutamine (Koch-Light), penicillin, streptomycin and bovine serum (produced in Poland). Each culture contained 5 bg/ml bromodeoxyuridine (Koch-Light) and 0.15 ml phytohemagglutinin (Wellcome). Finally an aqueous solution of cadmium sulfate was added to obtain concentrations of 1.6 x 10m3 mM, 3.1 x lo- 3 mM and 6.2 x lo- 3 mM. Positive control cultures were those where mitomycin C (Kyowa) was added in concentrations of 1.5 x 10e4 mM. A pilot study was the basis for selecting CdS04 concentrations: Concentrations lower than 1.6 x lo- 3 mM did not result in any differences in SCE frequency as compared with controls. However, concentrations higher than 6.2 x low3 mM decreased the mitotic activity of the culture by 80% as related to controls. The cells remained in darkness at 37°C and within 72 h most showed 2 cycles of DNA replication. Mitosis was stopped after 70 h when colcemid was added (0.5 pg/ml). At 72 h, the cultures were centrifuged for 10 min at 3000 rpm. After the supernatant had been removed with a pipette, the cells were rinsed with 75 mM KC1 and then centrifuged again. The supernatant was removed after rinsing the cells 3 times with methanol and acetic acid (3: 1). At the end of centrifugation about 0.5 ml of suspended cells were left in the test tubes. The cells were then speckled with a pipette onto a wet and chilled glass and were stained by the method of Antoshina [5]. After dipping the glasses in SSC solution (0.3 M sodium chloride and 0.03 M trisodium citrate in aqu. dist.) they were heated to 60°C in a water bath. During incubation the cultures were irradiated with a UV lamp placed 15 cm over the bath. After 25 min the glasses were rinsed in distilled water and dried. For staining a mixture of 0.1% azur II solution and 0.1% eosin solution in phosphate buffer (pH 6.9) was used. The pigments contained the following: 1 ml of eosin solution and 2.5 ml azur II solution were added to 15 ml of phosphate buffer. Staining time was 2 min. The cultures were rinsed in distilled water, air-dried and eluted with xylene. On the average, 50 cells at metaphase were analyzed for each cadmium sulfate concentration. No exogenous metabolizing system was used. Statistical evaluation of the results was done with the use of variance analysis and Student’s t-test. RESULTS
AND
DlSCUSSlON
The results of the experiments imply that the concentrations of cadmium sulfate under study did not significantly increase the mean frequency of exchanges in lymphocytes in human peripheral blood as compared with the control (Table I). Baker [6] quoting data of others pointed out the discrepancy in the results referring to a mutagenic potential of cadmium compounds. Some authors described mutagenic properties of CdS04 and CdS when studying mammalian chromosomes in vitro. Others denied such an impact of CdC12 and Cd(NO& on mammalian cells in vitro. The present investigation confirms the results of the latter authors.
175
TABLE
I
SISTER
CHROMATID
EXPOSED CdS04 concentration
IN LYMPHOCYTES
EXCHANGES
TO CADMIUM Donor
Analyzed
Exchange
cells (n)
Range
3.1 x lo--’
1O-3
Mean
1
50
2-9
4.66
2
50
3-10
5.60 f
3
44
2-8
4.55
4
50
l-9
5.01 f
1.93 2.38
+I 1.61 1.86
1
50
l-12
4.92
2
50
6.86 k 2.38
3
50
3-13 l-11
4
50
2-12
5.00 -t 1.99 5.42 + 2.26
t
5.84 _t 2.08
1
50
1-12
2
50
2-15
6.20 f
2.78
3
50
2-11
6.02
1.91
4
50
2-13
5.86 1 2.56
f
50
2-9 I-10
4.06 + 1.73
50
3
50
1-13
5.08 + 2.25
4
50
1-8
4.12
+ 1.61
con-
1
22
15-39
24.45
f 6.09
1.5 X
2
24
20-48
31.88
+ 8.01
10e4 mM
3
50
22-44
30.70
f
mitomycin
C 4
32
17-40
26.25
+ 5.96
Positive trol:
4.97
* 1.66
1
control
of
difference
+ SD
in group
2
Negative
BLOOD
Significance
+ SD
in cell
1.6x 1O-3
PERIPHERAL
frequency Mean
(mM)
6.2x
OF HUMAN
SULFATE
5.08 ? 2.01
f
5.66 k 2.32
P> 0.05
5.88 + 2.54
P> 0.05
4.59
f
28.73
5.63
P>O.O5
1.88
1.96
P
* 6.86
REFERENCES K.J. B.C.
Yost, Cadmium, Lakkad,
chloride Toxicol.,
S.K.
the environment Tigam,
on cell division
A.B.
S.H.
K.N.
and chromosomes
health:
Thakore
in Chinese
an overview, and
hamster
Experientia,
B.B. Chatterjee, ovary
40 (1984) 157.
Effect
of cadmium
cells, Bull. Environ.
Contam.
36 (1986) 342.
T. Ochi, M. Mogi, M. Watenabe Chinese
and human
Karnik,
hamster
Wolff
and M. Ohsawa,
cells by short-term
and P. Perry,
Insights
and the lack of both isolabelling
treatment
Induction
with cadmium
on chromosome
and heterolabelling
structure
of chromosomal chloride, from
as determined
aberrations
Mutat.
Res.,
sister chromatid
in cultured
137 (1984) 103. exchange
by the FPG technique,
ratios
Exp. Cell
Res., 93 (1975) 23. M.M. Antoshina and N.A. without using fluorochromes,
Porjadkowa, A technique for differential CitoL Cienet., 12 (1978) 349.
R.S.U. Baker, Evaluation of metals in in vitro assays. of action, Toxicol. Environ. Chem., 7 (1984) 191.
Interpretation
staining
of sister chromatid
of data and possible
mechanisms
Toxicology Letters, 37 (1987) 173-175 Elsevier
TXL 01801
CADMIUM SULFATE DOES NOT INDUCE SISTER CHROMATID EXCHANGES IN HUMAN LYMPHOCYTES IN VITRO (Cadmium; mutagenicity;
sister chromatid
EWA BASSENDOWSKA-KARSKA
exchanges)
and MARIA ZAWADZKA-KOS
Department of Hygiene, MedicaI Academy, Jaracza, 63, 90-2.51 tddi
(Poland)
(Received 5 January 1987) (Revision received 23 March 1987) (Accepted 25 March 1987)
SUMMARY Mutagenic properties of cadmium sulfate were analyzed by the method of sister chromatid exchanges (SCEs) in lymphocytes of human peripheral blood taken from 4 donors. No significant increase was found in the mean frequency of SCEs in lymphocytes exposed to 1.6 x lO-3 mM, 3.1 x 10e3 mM and 6.2 x 10e3 mM CdS04 in culture.
INTRODUCTION
Cadmium is a metal widely spread in the environment. Both the general population and the people occupationally exposed are at risk of cadmium absorption. In the areas of insignificant contamination the daily dietary cadmium intake was estimated to range between 15 and 60 fig per person. Cadmium resorption in lungs does not exceed 0.1 pg per day [ 1,2]. The quantities quoted do not cause intoxications or symptoms of disease. Nevertheless some authors claim cadmium compounds to lead to chromosome and chromatid aberrations in vitro [2,3]. This was the reason why the present research on mutagenic properties of cadmium sulfate was undertaken. MATERIALS AND METHODS
Analysis was carried out in human lymphocytes of peripheral blood taken from 4 healthy donors (3 women and 1 man). Cultures were prepared according to Wolff 0378-4274/87/$ 03.50 0 1987 Elsevier Science Publishers B.V. (Biomedical Division)
174
and Perry [4]. Heparinized venous blood of 0.5 ml collected with a syringe was added to a culture medium consisting of medium F-10 (Flow Lab.), L-glutamine (Koch-Light), penicillin, streptomycin and bovine serum (produced in Poland). Each culture contained 5 bg/ml bromodeoxyuridine (Koch-Light) and 0.15 ml phytohemagglutinin (Wellcome). Finally an aqueous solution of cadmium sulfate was added to obtain concentrations of 1.6 x 10m3 mM, 3.1 x lo- 3 mM and 6.2 x lo- 3 mM. Positive control cultures were those where mitomycin C (Kyowa) was added in concentrations of 1.5 x 10e4 mM. A pilot study was the basis for selecting CdS04 concentrations: Concentrations lower than 1.6 x lo- 3 mM did not result in any differences in SCE frequency as compared with controls. However, concentrations higher than 6.2 x low3 mM decreased the mitotic activity of the culture by 80% as related to controls. The cells remained in darkness at 37°C and within 72 h most showed 2 cycles of DNA replication. Mitosis was stopped after 70 h when colcemid was added (0.5 pg/ml). At 72 h, the cultures were centrifuged for 10 min at 3000 rpm. After the supernatant had been removed with a pipette, the cells were rinsed with 75 mM KC1 and then centrifuged again. The supernatant was removed after rinsing the cells 3 times with methanol and acetic acid (3: 1). At the end of centrifugation about 0.5 ml of suspended cells were left in the test tubes. The cells were then speckled with a pipette onto a wet and chilled glass and were stained by the method of Antoshina [5]. After dipping the glasses in SSC solution (0.3 M sodium chloride and 0.03 M trisodium citrate in aqu. dist.) they were heated to 60°C in a water bath. During incubation the cultures were irradiated with a UV lamp placed 15 cm over the bath. After 25 min the glasses were rinsed in distilled water and dried. For staining a mixture of 0.1% azur II solution and 0.1% eosin solution in phosphate buffer (pH 6.9) was used. The pigments contained the following: 1 ml of eosin solution and 2.5 ml azur II solution were added to 15 ml of phosphate buffer. Staining time was 2 min. The cultures were rinsed in distilled water, air-dried and eluted with xylene. On the average, 50 cells at metaphase were analyzed for each cadmium sulfate concentration. No exogenous metabolizing system was used. Statistical evaluation of the results was done with the use of variance analysis and Student’s t-test. RESULTS
AND
DlSCUSSlON
The results of the experiments imply that the concentrations of cadmium sulfate under study did not significantly increase the mean frequency of exchanges in lymphocytes in human peripheral blood as compared with the control (Table I). Baker [6] quoting data of others pointed out the discrepancy in the results referring to a mutagenic potential of cadmium compounds. Some authors described mutagenic properties of CdS04 and CdS when studying mammalian chromosomes in vitro. Others denied such an impact of CdC12 and Cd(NO& on mammalian cells in vitro. The present investigation confirms the results of the latter authors.
175
TABLE
I
SISTER
CHROMATID
EXPOSED CdS04 concentration
IN LYMPHOCYTES
EXCHANGES
TO CADMIUM Donor
Analyzed
Exchange
cells (n)
Range
3.1 x lo--’
1O-3
Mean
1
50
2-9
4.66
2
50
3-10
5.60 f
3
44
2-8
4.55
4
50
l-9
5.01 f
1.93 2.38
+I 1.61 1.86
1
50
l-12
4.92
2
50
6.86 k 2.38
3
50
3-13 l-11
4
50
2-12
5.00 -t 1.99 5.42 + 2.26
t
5.84 _t 2.08
1
50
1-12
2
50
2-15
6.20 f
2.78
3
50
2-11
6.02
1.91
4
50
2-13
5.86 1 2.56
f
50
2-9 I-10
4.06 + 1.73
50
3
50
1-13
5.08 + 2.25
4
50
1-8
4.12
+ 1.61
con-
1
22
15-39
24.45
f 6.09
1.5 X
2
24
20-48
31.88
+ 8.01
10e4 mM
3
50
22-44
30.70
f
mitomycin
C 4
32
17-40
26.25
+ 5.96
Positive trol:
4.97
* 1.66
1
control
of
difference
+ SD
in group
2
Negative
BLOOD
Significance
+ SD
in cell
1.6x 1O-3
PERIPHERAL
frequency Mean
(mM)
6.2x
OF HUMAN
SULFATE
5.08 ? 2.01
f
5.66 k 2.32
P> 0.05
5.88 + 2.54
P> 0.05
4.59
f
28.73
5.63
P>O.O5
1.88
1.96
P
* 6.86
REFERENCES K.J. B.C.
Yost, Cadmium, Lakkad,
chloride Toxicol.,
S.K.
the environment Tigam,
on cell division
A.B.
S.H.
K.N.
and chromosomes
health:
Thakore
in Chinese
an overview, and
hamster
Experientia,
B.B. Chatterjee, ovary
40 (1984) 157.
Effect
of cadmium
cells, Bull. Environ.
Contam.
36 (1986) 342.
T. Ochi, M. Mogi, M. Watenabe Chinese
and human
Karnik,
hamster
Wolff
and M. Ohsawa,
cells by short-term
and P. Perry,
Insights
and the lack of both isolabelling
treatment
Induction
with cadmium
on chromosome
and heterolabelling
structure
of chromosomal chloride, from
as determined
aberrations
Mutat.
Res.,
sister chromatid
in cultured
137 (1984) 103. exchange
by the FPG technique,
ratios
Exp. Cell
Res., 93 (1975) 23. M.M. Antoshina and N.A. without using fluorochromes,
Porjadkowa, A technique for differential CitoL Cienet., 12 (1978) 349.
R.S.U. Baker, Evaluation of metals in in vitro assays. of action, Toxicol. Environ. Chem., 7 (1984) 191.
Interpretation
staining
of sister chromatid
of data and possible
mechanisms