Mutation Research, 244 (1990) 179-183
179
Elsevier MUTLET 0357
Effects of aluminium sulphate on human leukocyte chromosomes in vitro Ajoy Kumar Roy, Geeta Talukder and Archana Sharma Human Genetics Unit, Centre for Advanced Study in Cell and Chromosome Research, Department of Botany, University of Calcutta, Calcutta - 700 019 (India)
(Accepted 5 January 1990)
Keywords: Micronuclei;Chromosomeaberrations; Sister-chromatidexchange;Aluminiumtoxicity
The progressive increase in the use of aluminium compounds has enhanced the possibility of human exposure. In spite of its low absorption rate, A1 accumulates in certain tissues, resulting in neurotoxic effects. An excess o f A1 has been related to the aetiology of various diseases such as renal dialysis dementia (Alfrey et al., 1976), senile dementia (Perl and Brody, 1983; Banks and Kastin, 1983) and Parkinson's dementia (Perl et al., 1982; Garruto et al., 1984). The accumulation of A1 in bone may also lead to dialysis-associated osteomalacia and osteitis fibrosa (Alfrey et al., 1976; Hodsman et al., 1982; Boyce et al., 1982; G o o d m a n et al., 1984). After absorption, A1 is distributed slowly and accumulates in bone and reticuloendothelial cells. The ionic form has a very high affinity for DNA, RNA and many mononucleotides (Ganrot, 1986) and complexes with DNA (Karlik et al., 1980; Dyrssen et al., 1987; Matsumoto, 1988). Information on the effects of A1 on chromosomes in vitro
Correspondence: Dr. A.K. Roy, Human GeneticsUnit, Centre for Advanced Study in Cell and Chromosome Research, Department of Botany, University of Calcutta, 35 Ballygunge Circular Road, Calcutta - 700019 (India).
in mammalian systems is relatively meagre (Roy, 1987; L6onard and Garber, 1988; Sharma and Talukder, 1987). The present work was undertaken to study the effect of A1 sulphate on human lymphocytes stimulated to proliferate in culture, as associated with factors such as the age and sex of the donor. Materials and methods Peripheral venous blood from normal healthy blood donors without addiction to tobacco and alcohol was collected in heparinised vials. Lymphocyte culture was carried out following standard techniques (Sharma and Sharma, 1980; Preston et al., 1987). Both male and female donors were used and belonged to 3 age groups: 0-10 years, 21-30 years and 41-50 years. Five donors from each group, of each sex, were used. RPMI 1640 (Gibco), supplemented with human A B + serum (20%) and phytohaemagglutinin (0.4 ml), was used as the culture medium. For each sample, one set of cultures was maintained as controls and one set was treated with aqueous aluminium sulphate solution in the dosage of 20 ttg/ml of medium. Each set consisted of 4 culture tubes, with 2 being for micronucleus analysis and
0165-7992/90/$ 03.50 © 1990Elsevier SciencePublishers B.V. (BiomedicalDivision)
180 p e r s o n (A.K.R.). F o r c a l c u l a t i o n o f breaks/cells, c h r o m a t i d breaks were t a k e n as a single break,
the other 2 being used for other purposes. The p a r a m e t e r s studied were mitotic index, proliferat i o n rate index, frequencies of c h r o m o s o m a l aber-
dicentrics a n d t r a n s l o c a t i o n s as 2 breaks. Gaps were n o t i n c l u d e d b u t recorded separately. The d a t a were analysed statistically with S t u d e n t ' s t test to c o m p a r e t r e a t m e n t a n d c o n t r o l sets. F u r t h e r analyses were c o n d u c t e d using a 3-way factorial
rations, m i c r o n u c l e i a n d sister-chromatid exchanges (SCEs). Cells were harvested after 72 h o f i n c u b a t i o n following the usual c o l c h i c i n e - h y p o t o n i c - a c e t i c : m e t h a n o l fixation a n d air-drying schedule. I n the study o f m i c r o n u c l e i , the hypotonic used was NaCI (0.9%) 9 parts a n d KC1 (0.56%) 1 part for 5 rain. F o r sister-chromatid ex-
A N O V A m o d e l to e x a m i n e i n t e r a c t i o n s o f different factors.
c h a n g e counts, c h r o m o s o m a l a b e r r a t i o n s , pro-
Results
liferation rate index a n d mitotic index, 5 - b r o m o d e o x y u r i d i n e (Sigma, 6 # g / m l ) was a d d e d to the culture m e d i a a n d the p r e p a r a t i o n s were stained
Mitotic index A l u m i n i u m sulphate i n d u c e d a slight increase in
following the fluorescence plus G i e m s a ( F P G ) t e c h n i q u e (Perry a n d W o l f f , 1974). 2000 ceils were s c a n n e d per i n d i v i d u a l sample for mitotic index a n d m i c r o n u c l e i , 250-300 metaphases for replica-
the mitotic index in l y m p h o c y t e cultures prepared u s i n g b l o o d o b t a i n e d f r o m male d o n o r s in the first 2 age groups. In the oldest age g r o u p , however, the mitotic index was decreased significantly (P < 0.05). I n female subjects, the change in mitotic index
t i o n index, 80-100 first-cycle m e t a p h a s e s for c h r o m o s o m a l a b e r r a t i o n s a n d 100 second-cycle
following t r e a t m e n t was not statistically significant (Table 1).
m e t a p h a s e s for sister-chromatid exchanges. Observ a t i o n s for all p a r a m e t e r s were m a d e using the same slide. All cultures were d o n e in replicate and, for each p a r a m e t e r , o b s e r v a t i o n s were t a k e n f r o m
Replication index A l t h o u g h n o t statistically significant, the cell cycle for mitogenic stimulated lymphocytes was delayed in all o f the treated cultures as a f u n c t i o n
b o t h culture tubes a n d pooled. Slides were coded a n d scored b l i n d by a single TABLE 1
FREQUENCY OF ABERRATIONS IN HUMAN LYMPHOCYTE CULTURE AFTER TREATMENT WITH ALUMINIUM SULPHATE (20 #g/ml OF MEDIUM) FOR 72 h IN VITRO Sex
Age
Mitotic index
Micronuclei/cell (%)
Breaks/cell (%)
group
Control
Treated
Control
Treated
Control
Treated
Male
I II III Total
2.66 2.01 1.55 2.07
+ 1.31 + 0.84 ___0.48 + 0.99
3.51 2.03 0.95 2.16
_+ 1.18 _+ 0.92 _+ 0.31" _+ 1.36
0.60 1.12 0.97 0.90
_+ 0.26 + 0.32 + 0.48 +_ 0.41
0.81 1.70 1.46 1.33
_+ 0.24 _+ 0.37* + 0.65 _+ 0.57
7.3 7.2 9.2 7.90
_+ 2.54 + 5.07 _+ 2.40 _+ 3.43
6.9 11.0 10.1 9.3
+ 2.71 +_ 6.18 _+ 3.82 _+ 4.53
Female
I II III Total
2.47 1.97 2.15 2.20
+ 0.63 _+ 1.04 + 1.80 + 1.18
1.87 1.59 1.81 1.76
_+ 0.99 _+ 0.71 _ 1.30 _+ 0.96
1.00 1.22 0.70 0.98
_+ 0.94 + 0.42 _-!-0.34 + 0.62
1.55 1.62 1.60 1.59
_+ 0.26 _+ 0.39 ___0.67* _+ 0.44**
6.0 8.6 5.0 6.6
+_ 4.24 + 3.03 _+ 1.28 + 3.27
9.5 13.3 11.7 11.5
_+ 3.70 _+ 5.03 + 4.26* _+ 4.35**
Total (male and female)
2.13 _+ 0.07
Data are expressed as means _+ SD. *P
179
Elsevier MUTLET 0357
Effects of aluminium sulphate on human leukocyte chromosomes in vitro Ajoy Kumar Roy, Geeta Talukder and Archana Sharma Human Genetics Unit, Centre for Advanced Study in Cell and Chromosome Research, Department of Botany, University of Calcutta, Calcutta - 700 019 (India)
(Accepted 5 January 1990)
Keywords: Micronuclei;Chromosomeaberrations; Sister-chromatidexchange;Aluminiumtoxicity
The progressive increase in the use of aluminium compounds has enhanced the possibility of human exposure. In spite of its low absorption rate, A1 accumulates in certain tissues, resulting in neurotoxic effects. An excess o f A1 has been related to the aetiology of various diseases such as renal dialysis dementia (Alfrey et al., 1976), senile dementia (Perl and Brody, 1983; Banks and Kastin, 1983) and Parkinson's dementia (Perl et al., 1982; Garruto et al., 1984). The accumulation of A1 in bone may also lead to dialysis-associated osteomalacia and osteitis fibrosa (Alfrey et al., 1976; Hodsman et al., 1982; Boyce et al., 1982; G o o d m a n et al., 1984). After absorption, A1 is distributed slowly and accumulates in bone and reticuloendothelial cells. The ionic form has a very high affinity for DNA, RNA and many mononucleotides (Ganrot, 1986) and complexes with DNA (Karlik et al., 1980; Dyrssen et al., 1987; Matsumoto, 1988). Information on the effects of A1 on chromosomes in vitro
Correspondence: Dr. A.K. Roy, Human GeneticsUnit, Centre for Advanced Study in Cell and Chromosome Research, Department of Botany, University of Calcutta, 35 Ballygunge Circular Road, Calcutta - 700019 (India).
in mammalian systems is relatively meagre (Roy, 1987; L6onard and Garber, 1988; Sharma and Talukder, 1987). The present work was undertaken to study the effect of A1 sulphate on human lymphocytes stimulated to proliferate in culture, as associated with factors such as the age and sex of the donor. Materials and methods Peripheral venous blood from normal healthy blood donors without addiction to tobacco and alcohol was collected in heparinised vials. Lymphocyte culture was carried out following standard techniques (Sharma and Sharma, 1980; Preston et al., 1987). Both male and female donors were used and belonged to 3 age groups: 0-10 years, 21-30 years and 41-50 years. Five donors from each group, of each sex, were used. RPMI 1640 (Gibco), supplemented with human A B + serum (20%) and phytohaemagglutinin (0.4 ml), was used as the culture medium. For each sample, one set of cultures was maintained as controls and one set was treated with aqueous aluminium sulphate solution in the dosage of 20 ttg/ml of medium. Each set consisted of 4 culture tubes, with 2 being for micronucleus analysis and
0165-7992/90/$ 03.50 © 1990Elsevier SciencePublishers B.V. (BiomedicalDivision)
180 p e r s o n (A.K.R.). F o r c a l c u l a t i o n o f breaks/cells, c h r o m a t i d breaks were t a k e n as a single break,
the other 2 being used for other purposes. The p a r a m e t e r s studied were mitotic index, proliferat i o n rate index, frequencies of c h r o m o s o m a l aber-
dicentrics a n d t r a n s l o c a t i o n s as 2 breaks. Gaps were n o t i n c l u d e d b u t recorded separately. The d a t a were analysed statistically with S t u d e n t ' s t test to c o m p a r e t r e a t m e n t a n d c o n t r o l sets. F u r t h e r analyses were c o n d u c t e d using a 3-way factorial
rations, m i c r o n u c l e i a n d sister-chromatid exchanges (SCEs). Cells were harvested after 72 h o f i n c u b a t i o n following the usual c o l c h i c i n e - h y p o t o n i c - a c e t i c : m e t h a n o l fixation a n d air-drying schedule. I n the study o f m i c r o n u c l e i , the hypotonic used was NaCI (0.9%) 9 parts a n d KC1 (0.56%) 1 part for 5 rain. F o r sister-chromatid ex-
A N O V A m o d e l to e x a m i n e i n t e r a c t i o n s o f different factors.
c h a n g e counts, c h r o m o s o m a l a b e r r a t i o n s , pro-
Results
liferation rate index a n d mitotic index, 5 - b r o m o d e o x y u r i d i n e (Sigma, 6 # g / m l ) was a d d e d to the culture m e d i a a n d the p r e p a r a t i o n s were stained
Mitotic index A l u m i n i u m sulphate i n d u c e d a slight increase in
following the fluorescence plus G i e m s a ( F P G ) t e c h n i q u e (Perry a n d W o l f f , 1974). 2000 ceils were s c a n n e d per i n d i v i d u a l sample for mitotic index a n d m i c r o n u c l e i , 250-300 metaphases for replica-
the mitotic index in l y m p h o c y t e cultures prepared u s i n g b l o o d o b t a i n e d f r o m male d o n o r s in the first 2 age groups. In the oldest age g r o u p , however, the mitotic index was decreased significantly (P < 0.05). I n female subjects, the change in mitotic index
t i o n index, 80-100 first-cycle m e t a p h a s e s for c h r o m o s o m a l a b e r r a t i o n s a n d 100 second-cycle
following t r e a t m e n t was not statistically significant (Table 1).
m e t a p h a s e s for sister-chromatid exchanges. Observ a t i o n s for all p a r a m e t e r s were m a d e using the same slide. All cultures were d o n e in replicate and, for each p a r a m e t e r , o b s e r v a t i o n s were t a k e n f r o m
Replication index A l t h o u g h n o t statistically significant, the cell cycle for mitogenic stimulated lymphocytes was delayed in all o f the treated cultures as a f u n c t i o n
b o t h culture tubes a n d pooled. Slides were coded a n d scored b l i n d by a single TABLE 1
FREQUENCY OF ABERRATIONS IN HUMAN LYMPHOCYTE CULTURE AFTER TREATMENT WITH ALUMINIUM SULPHATE (20 #g/ml OF MEDIUM) FOR 72 h IN VITRO Sex
Age
Mitotic index
Micronuclei/cell (%)
Breaks/cell (%)
group
Control
Treated
Control
Treated
Control
Treated
Male
I II III Total
2.66 2.01 1.55 2.07
+ 1.31 + 0.84 ___0.48 + 0.99
3.51 2.03 0.95 2.16
_+ 1.18 _+ 0.92 _+ 0.31" _+ 1.36
0.60 1.12 0.97 0.90
_+ 0.26 + 0.32 + 0.48 +_ 0.41
0.81 1.70 1.46 1.33
_+ 0.24 _+ 0.37* + 0.65 _+ 0.57
7.3 7.2 9.2 7.90
_+ 2.54 + 5.07 _+ 2.40 _+ 3.43
6.9 11.0 10.1 9.3
+ 2.71 +_ 6.18 _+ 3.82 _+ 4.53
Female
I II III Total
2.47 1.97 2.15 2.20
+ 0.63 _+ 1.04 + 1.80 + 1.18
1.87 1.59 1.81 1.76
_+ 0.99 _+ 0.71 _ 1.30 _+ 0.96
1.00 1.22 0.70 0.98
_+ 0.94 + 0.42 _-!-0.34 + 0.62
1.55 1.62 1.60 1.59
_+ 0.26 _+ 0.39 ___0.67* _+ 0.44**
6.0 8.6 5.0 6.6
+_ 4.24 + 3.03 _+ 1.28 + 3.27
9.5 13.3 11.7 11.5
_+ 3.70 _+ 5.03 + 4.26* _+ 4.35**
Total (male and female)
2.13 _+ 0.07
Data are expressed as means _+ SD. *P
