- Email: [email protected]
Clastogenicity of lanthanides: induction of chromosomal aberration in bone marrow cells of mice in vivo
Genetic Toxicology
ELSEVIER
Mutation Research 341 (1995) 193-197
Clastogenicity of lanthanides: induction of chromosomal aberration in bone marrow cells of mice in vivo Anand M. Jha *, Akhilesh C. Singh Genetic Toxicology Laboratory, Department of Botany, Samastipur College, Samastipur-848134, India Received 18 April 1994; revision received 15 August 1994; accepted 30 August 1994
Abstract
Clastogenic properties of two lanthanide elements praseodymium (Pr) and neodymium (Nd) were evaluated by employing mouse in vivo chromosomal aberrations (CAs) assay. Praseodymium oxide (Pr6Oll) and neodymium oxide (Nd203) administered intraperitoneally to Swiss albino mice in vivo induced significant increase in the frequency of CAs in bone marrow cells, when compared to negative control. The number of CAs/cell and percent aberrant cells increased with an increase in the concentration and period of treatment. The effect was maximum when the cells were analysed 12 h after treatment, as compared to 6 and 24 h. This is the first report on the clastogenicity of these elements in mouse in vivo. Keywords: Lanthanides; Clastogenicity; Bone marrow cell; In vivo
I. Introduction
The rapid pace of industrialization during the last few decades has resulted in continuous dumping of hazardous chemicals, either in the original form or in combination with other chemicals. Metals and their compounds form a large proportion of chemical pollutants released by various industries. A large number of publications on the in vitro and in vivo mutagenicity, clastogenicity and carcinogenicity of metallic compounds are now available (Sharma and Talukder, 1987). The lanthanide elements, also known as rare earths, are f-block inner transition elements
* Corresponding author.
belonging to the group III B of the periodic table. They occur abundantly in the lithosphere and are used in various industries. The alloy of lanthanides, known as mish-metals, are used as an additive for making sunglasses, as an abrasive for polishing glasses and in the manufacture of lakes, dyes and paints for porcelain. Dimals which are salicylates of Pr and Nd are used as germicides. Therefore, the environmental and occupational exposure to these chemicals can not be ruled out. The impact of these chemicals on human health and the genetic damage caused by them are still largely unknown. The lanthanide elements that have been tested for their effect on mammalian system at molecular, cellular and histological levels, were positive (Bruce et al., 1963; Synder and Kyker, 1964; Giri et al., 1978, 1980). As a part of
0165-1218/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved
SSDI 0 1 6 5 - 1 2 1 8 ( 9 4 ) 0 0 0 6 2 - X
A.)I,[ Jha, A.C. Singh / Mutation Research 341 (1995) 193-197
194
the study assessing the cytotoxicity and clastogenicity of some lanthanide elements, we examined the capacity of the inorganic compound of praseodymium (Pr6Oll) and neodymium (Nd 203) to induce chromosomal aberrations (CAs) in vivo in bone marrow cells of mice.
were used as experimental material. Praseodymium oxide (Pr6Oll MW 1021.44) and neodymium oxide (Nd20 3 MW 336.48) were purchased from Loba Chemic Indoaustranal, P.O. Box 6136, Bombay, India. Primary stock solutions of both the compounds were prepared by dissoloving 1 g powder in 1 ml concentrated HC1. The final volume of the solutions (30 ml) was then made by adding 29 ml distilled water. Mice were injected i.p. with 5.13, 7.70, 15.40, and 38.50 mg/100 g b.w. of Pr6Oll and 5.30, 8.60, 17.20 and 43.0 mg/100 g b.w. of Nd20 3. The dosage se-
2. Materials and methods
Random bred Swiss albino mice (Mus musculus) of 8-10 weeks old and weighing 25-30 g
Table 1 C h r o m o s o m a l a b e r r a t i o n i n d u c e d by n e o d y m i u m oxide ( N D 2 0 3) in b o n e m a r r o w cells of Swiss albino mice Treatment
Dose (mg/100g
Gap a
CtB
b.w.) 6 h harvest N e g a t i v e control Mitomycin C ( + v e control) N e o d y m i u m oxide (Nd203)
CAs/cell c
Total CA b ChB
CF
6
5
-
58 10 12 14 18
28 6 8 13 15
27 4 4 10 10
6
7
7
0.15 5.30
65 10
8.60 17.20 43.00
0.15 5.30 8.60 17.20 43.00
RC
Ex
%Abn a cells
St + PI
-
-
-
0.010
1.00
18 -
16 -
-
0.190 0.020
16.20 1.20
2
2 4
3
-
0.024 0.050 0.068
1.80 2.80 4.60
-
-
-
-
-
0.014
1.40
40 8
55 12
8 -
15 16
9 10
14 19
-
22
12
18
2
22 4 5
22 4
4
0.294 0.040 0.046 0.066 0.082
21.40 2.00 3.00 4.00 5.60
7
10
0.020
1.80
49 6 9 10 14
22 6 8 7 5
0.164 0.028 0.032 0.032 0.038
14.60 2.00 2.40 2.60 3.60
T r e n d test p < 0.001 12 h h a r v e s t N e g a t i v e control Mitomycin C ( + ve control) N e o d y m i u m oxide ( N d 2 0 3)
-
T r e n d test p < 0.001 24 h h a r v e s t N e g a t i v e control Mitomycin C ( + v e control) N e o d y m i u m oxide ( N d 2 0 3)
0.15 5.30 8.60 17.20 43.00
28 8 6 7 10
2 -
19 2 2 3
11 1
5
T r e n d test p < 0.001 a I n c l u d e s b o t h c h r o m a t i d a n d i s o c h r o m a t i d gap. b 500 m e t a p h a s e s analysed p e r dose a n d 100 p e r a n i m a l . c N u m b e r of C A s / c e l l (excluding g a p a n d stickiness + pulverization). d P e r c e n t a g e a b n o r m a l cells include stickiness + p u l v e r i z a t i o n w h e r e e v e r r e c o r d e d . CtB, C h r o m a t i d b r e a k ; ChB, c h r o m o s o m e b r e a k ; CF, c e n t r i c fusion; RC, ring c h r o m o s o m e . Ex, E x c h a n g e ; St + P1, stickiness + pulverization.
A.M. Jha, A.C. Singh /Mutation Research 341 (1995) 193-197
195
for each dose and for each sampling time. 2 h before killing each animal received a freshly prepared colchicine solution at the rate of 4 m g / k g b.w. through i.p. injection. The animals were killed by cervical dislocation at 6, 12 and 24 h after treatment. Bone marrow from both the femurs was collected by flushing with 0.075 M potassium chloride (pre-warmed at 37°C), incubated at 37°C for 25 min, centrifuged and fixed in cold fixative (3 : 1 ethanol-glacial acetic acid). The samples were washed twice in fixative, dropped on clean chilled slides, flame dried and stained in
lected for administration were determined according to the guidelines suggested for in vivo cytogenetic assay (Preston et al., 1987). The selection of maximum dose was based on depression in mitotic index (not more than 50%)in the bone marrow cells, measured at the same sampling times as those used for chromosomal aberration assay. 0.15 m g / 1 0 0 g b.w. of mitomycin C was used as positive control. Negative control animals received 0.1 ml of solvent (1 ml concentrated HC1 in 29 ml distilled water) through i.p. injection. Five animals (3 males and 2 females) were used
Table 2 C h r o m o s o m a l a b e r r a t i o n i n d u c e d by p r a s e o d y m i u m oxide ( P r 6 O l l ) in b o n e m a r r o w cells of Swiss albino mice Treatment
Dose (mg/100g
Gap a
CtB
b.w.) 6 h harvest N e g a t i v e control Mitomycin C ( + ve control) P r a s e o d y m i u m oxide (Pr6Oll)
-
0.15 5.13 7.70 15.40 38.50
CAs/cell c
Total CA b ChB
6
5
58 7 8 11 9
28 8 13 11 15
7
7
65 7 11 14 10
40 8 9 13 14
55 10 14 15 19
7
10
.
49 4 5 13 7
22 5 8 9 9
28 5 9 11 13
CF
27 2 7 9 12
RC
6 2 3
Ex
18 1 2 3 5
St + PI
%Abn d cells
-
-
0.010
1.00
16 2
-
0.190 0.022 0.044 0.050 0.074
16.20 2.00 3.20 3.60 5.20
0.014
1.40
0.294 0.036 0.054 0.078 0.088
21.40 2.40 3.20 5.60 6.40
0.020
1.80
0.064 0.024 0.040 0.054 0.056
14.60 1.80 3.00 4.40 5.00
T r e n d test p < 0.001 12 h harvest N e g a t i v e control Mitomycin C ( + ve control) P r a s e o d y m i u m oxide (Pr 6 O l l )
-
0.15 5.13 7.70 15.40 38.50
8 . 3 3
22 . 4 6 6
.
22
-
2 2
2 4
.
T r e n d test p < 0.001 24 h harvest N e g a t i v e control Mitomycin C (+ve control) P r a s e o d y m i u m oxide (Pr6Oll)
0.15 5.13 7.70 15.40 38.50
.
. 2 2 1
.
. 19 2 3 3 2
11 2 3
3 3
T r e n d test p < 0.001 a I n c l u d e s b o t h c h r o m a t i d a n d i s o c h r o m a t i d gap. t, 500 m e t a p h a s e a n a l y s e d p e r d o s e a n d 100 p e r animal. c N u m b e r of C A s / c e l l (excluding g a p and stickiness + pulverization). d P e r c e n t a g e a b n o r m a l cells i n c l u d e s stickiness + p u l v e r i z a t i o n w h e r e e v e r recorded. CtB, C h r o m a t i d b r e a k ; C h B c h r o m o s o m e b r e a k ; CF, c e n t r i c fusion; RC, ring c h r o m o s o m e . Ex, E x c h a n g e ; St + P1, stickiness + pulverization.
A.M. Jha, A.C. Singh / Mutation Research 341 (1995) 193-197
196
buffered Giemsa. The slides were coded. 100 metaphases/animal (500 metaphases/dose) were screened for the presence of CAs. The number and type of CAs were scored. Data recorded were evaluated as the number of CAs/cell ( C A s / c e l l excluding gaps and stickiness + pulverizations)and percent aberrant cells (excluding gaps). Chromatid and isochromatid gaps, chromatid and chromosome breaks, chromatid and chromosome exchanges, ring chromosomes, centric fusions, stickiness and pulverization were recorded as chromosomal abnormalities. For statistical analysis one tailed trend test (Margolin et al., 1986) and two-way A N O V A (Harter, 1960) were employed.
Two-way A N O V A (Table 3) showed significant differences between the treatments as regards percent aberrant ceils and CAs/cell. CAs recorded were chromatid and chromosome breaks, gaps, exchanges, centric fusion, ring chromosomes, stickiness and pulverizations. Chromatid and chromosome breaks were present in higher frequencies than the other types and were induced by all the doses used. The frequencies of chromatid breaks were higher at 6 h sampling time whereas chromosome breaks were present in higher frequencies at 12 h sampling time. All other types were recorded only after treatment with the higher doses of Pr6Otl and N d 2 0 3.
3. Results
4. Discussion
Tables 1 and 2 record data on the induction of CAs in bone marrow ceils of mice following in vivo exposure to different doses of Pr6011 and N d 2 0 3 for 6, 12 and 24 h. A single exposure to mice of different doses of Pr6011 and N d 2 0 3 significantly enhanced the frequency of CAs at metaphase. The number of CAs/cell and percent aberrant cells increased, when compared to negative control, with an increase in dose. The frequencies also increased with the lapse of time attaining a maximal at 12 h sampling time. The number of CAs/cell and percent aberrant cells, as shown by a one-tailed trend test, showed a positive trend with the dose of Pr6011 and N d 2 0 3.
Mouse in vivo chromosomal aberration assays have been recognised as one of the sensitive endpoints to test the genotoxicity of industrial pollutants. Much is known about the ability of metallic compounds to induce CAs in bone marrow cells of laboratory animals (Agarwal et al., 1990; Ghosh, 1991; Ghosh and Talukder, 1990; Sharma and Talukder, 1987). The lanthanide elements, although are being utilized in many industries, have not come in for much genotoxic testing. Only a few of them have gone under shortterm in vivo genotoxicity assays. Among the lanthanides, lanthanum chloride has been reported to induce dose- and duration-related increase in the frequency of chromosomal aberratins in vivo in mice (Das, 1991; De and Sharma, 1981). The clastogenic effect has been attributed to the property of lanthanum to form complex with DNA (Rosoff and Spencer, 1979). Cerium as a nitrate induced breaks and reduced the mitotic index in rat bone marrow in vivo. This element is known to affect the nucleotides and to accumulate in bone through its affinity with phosphates, leading to its cytotoxic actifvity (Giri et al., 1979; Sharma and Talukder, 1987). Cerium has also been reported to affect the lipid metabolism in liver (Synder and Kyker, 1969) and as nitrate, DNA, R N A and protein synthesis in liver, kidney, lung and brain of rat (Giri et al., 1978).
Table 3 Two-way A N O V A of percent aberrant cells and CAs/cell showing significant difference among different doses and durations following in vivo treatment Sources of
df
Mean squares
F-ratio
variation
Pr6Oll
N d 2 0 3 Pr6Oll
Nd20 3
% Aberrant cells Duration 2 Concentration 4 Error 8
0.87 8.74 0.24
1.17 4.87 0.28
3.62 36.41"*
4.17 19.39 **
CAs/cell Duration Concentration Error
0.0004 0.0017 0.00005
0.0055 0.0102 0.0012
8.00 * 34.00 **
4.58 * 8.50 *
2 4 8
*'** Significant at 5% and 1% level respectively.
A.M. Jha, A.C. Singh / Mutation Research 341 (1995) 193-197 The results of the present investigation demonstrate that Praseodymium and Neodymium a r e c l a s t o g e n i c in v i v o in m o u s e . T h e c h r o m o s o m a l d a m a g i n g activity o f t h e s e e l e m e n t s in b o n e m a r r o w cells o f m i c e is in a g r e e m e n t w i t h t h e i r c l a s t o g e n i c p r o p e r t i e s in Vicia faba r o o t tip cells ( J h a a n d S i n g h , 1994). B o t h t h e e l e m e n t s i n d u c e d d o s e r e l a t e d i n c r e a s e in t h e n u m b e r o f C A s / c e l l a n d p e r c e n t a g e o f a b e r r a n t cells at all s a m p l i n g p e r i o d s , w h e n c o m p a r e d to n e g a t i v e c o n t r o l . C e l l s s a m p l e d 12 h a f t e r e x p o s u r e s h o w e d h i g h e r n u m b e r o f C A s / c e l l as c o m p a r e d to 6 a n d 24 h. D e c r e a s e in t h e p e r c e n t a g e o f a b e r r a n t cells a f t e r 24 h i.p. i n j e c t i o n s u g g e s t r e d u c t i o n in t h e survival r a t e o f t h e a f f e c t e d cells a n d t h e i r s u b s e quent elimination. T h e p r e s e n t s t u d y t h u s r e p o r t s t h e first in v i v o clastogenicity of two lanthanide elements p r a s e o d y m i u m (Pr) a n d n e o d y m i u m ( N d ) as m e a s u r e d by c h r o m o s o m a l a b e r r a t i o n assays. F u r t h e r s t u d i e s , s u c h as in v i v o m i c r o n u c l e u s a n d S C E t e s t a n d t e s t i c u l a r toxicity o f t h e e l e m e n t s , a r e in p r o g r e s s to v a l i d a t e o u r f i n d i n g s .
Acknowledgement T h e a u t h o r s a r e t h a n k f u l to P r o f e s s o r P . N . Sinha, Head, Department of Botany, Samastipur College, Samastipur, India for facilities provided d u r i n g this study.
References Agarwal, K., A. Sharma and G. Talukder (1990) Clastogenic effects of copper sulphate on the bone marrow chromosomes of mice in vivo, Mutation Res., 243, 1-6. Bruce, D.W., B.E. Heitbrink and K.P. DuBois (1963) The
197
acute mammalian toxicity of rare earth nitratesand oxides, Toxicol. Appl. Pharmacol., 5, 750-759. Das, T. (1991) Clastogenic effects of lanthanum chloride on bone marrow chromosomes of mice in vivo, Proc. Symp. Environment and Genome, EMSI, pp. 268-271. De, S.K. and A. Sharma (1981) Mutachromosomal effects of lanthanum chloride on mammalian bone marrow, Curr. Sci., 50, 189-190. Giri, A.K., A. Sharma and G. Talkuder (1978) Cytochemical effects of cobalt sulphate and ceric sulphate on mammalian system, Bull. Inst. P.G. Med. Edu. Res., 20, 145147. Girl, A.K., R. Sanyal, G. Talukder and A. Sharma (1979) Cytological and cytochemical changes induced through certain heavy metals on mammalian system, Natl. Acad. Sci. Lett., 2, 391-394. Giri, A.K., R. Banerjee, G. Talukder and A. Sharma(1980) Mutagenic effects of certain common metal toxicants on mammalian system, Proc. Indian Acad. Sci. (Anim. Sci.), 89, 311-331. Ghosh S. (1991) Clastogenic effects of ziroconium oxychloride in ralation to dose and duration of exposure in bone marrow cells of mice in vivo, Proc. Symp. Environment and Genome, EMSI, pp. 222-225. Ghosh, A., A. Sharma and G. Talukder (1990) Clastogenic effects of cesium chloride in bone marrow cells of mice in vivo, Mutation Res., 244, 295-298. Hatter, H.L. (1960) Biometrics, 16, 671. Jha, A.M. and A.C. Singh (1994) Clastogenicity of lanthanides induction of micronuclei in root tips of V.iciafaba, Mutation Res., 322, 169-172. Margolin, B.H., M.A. Resnik, J.Y. Rimpo, P. Archer, S.M. Galloway, A.D. Bloom and E. Zeiger (1986) Statistical analysis for in vitro cytogenetic assays using Chinese hamster ovary cells, Environ. Mol. Mutagen., 8, 183-204. Preston, R.J., B.D. Dean, S. Galloway, H Holden, A.F. McFee and M. Shelby (1987) Mammalian in vivo cytogenetic assay: analysis of chromosome aberrations in bone marrow cells, Mutation Res., 189, 157-165. Rosoff, B. and H. Spencer (1979) Binding of rare earths to serum proteins and DNA, Clin. Chim. Acta, 93, 311-319. Sharma, A. and G. Talukder (1987) Effects of metals on chromosomes of higher organism, Environ. Mutagen., 9, 191-226. Synder, F., E.A. Cress and G.C. Kyker (1959) Lipid responses to intravenous rare earths in rats, J. Lipid Res., 1,125-131.
ELSEVIER
Mutation Research 341 (1995) 193-197
Clastogenicity of lanthanides: induction of chromosomal aberration in bone marrow cells of mice in vivo Anand M. Jha *, Akhilesh C. Singh Genetic Toxicology Laboratory, Department of Botany, Samastipur College, Samastipur-848134, India Received 18 April 1994; revision received 15 August 1994; accepted 30 August 1994
Abstract
Clastogenic properties of two lanthanide elements praseodymium (Pr) and neodymium (Nd) were evaluated by employing mouse in vivo chromosomal aberrations (CAs) assay. Praseodymium oxide (Pr6Oll) and neodymium oxide (Nd203) administered intraperitoneally to Swiss albino mice in vivo induced significant increase in the frequency of CAs in bone marrow cells, when compared to negative control. The number of CAs/cell and percent aberrant cells increased with an increase in the concentration and period of treatment. The effect was maximum when the cells were analysed 12 h after treatment, as compared to 6 and 24 h. This is the first report on the clastogenicity of these elements in mouse in vivo. Keywords: Lanthanides; Clastogenicity; Bone marrow cell; In vivo
I. Introduction
The rapid pace of industrialization during the last few decades has resulted in continuous dumping of hazardous chemicals, either in the original form or in combination with other chemicals. Metals and their compounds form a large proportion of chemical pollutants released by various industries. A large number of publications on the in vitro and in vivo mutagenicity, clastogenicity and carcinogenicity of metallic compounds are now available (Sharma and Talukder, 1987). The lanthanide elements, also known as rare earths, are f-block inner transition elements
* Corresponding author.
belonging to the group III B of the periodic table. They occur abundantly in the lithosphere and are used in various industries. The alloy of lanthanides, known as mish-metals, are used as an additive for making sunglasses, as an abrasive for polishing glasses and in the manufacture of lakes, dyes and paints for porcelain. Dimals which are salicylates of Pr and Nd are used as germicides. Therefore, the environmental and occupational exposure to these chemicals can not be ruled out. The impact of these chemicals on human health and the genetic damage caused by them are still largely unknown. The lanthanide elements that have been tested for their effect on mammalian system at molecular, cellular and histological levels, were positive (Bruce et al., 1963; Synder and Kyker, 1964; Giri et al., 1978, 1980). As a part of
0165-1218/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved
SSDI 0 1 6 5 - 1 2 1 8 ( 9 4 ) 0 0 0 6 2 - X
A.)I,[ Jha, A.C. Singh / Mutation Research 341 (1995) 193-197
194
the study assessing the cytotoxicity and clastogenicity of some lanthanide elements, we examined the capacity of the inorganic compound of praseodymium (Pr6Oll) and neodymium (Nd 203) to induce chromosomal aberrations (CAs) in vivo in bone marrow cells of mice.
were used as experimental material. Praseodymium oxide (Pr6Oll MW 1021.44) and neodymium oxide (Nd20 3 MW 336.48) were purchased from Loba Chemic Indoaustranal, P.O. Box 6136, Bombay, India. Primary stock solutions of both the compounds were prepared by dissoloving 1 g powder in 1 ml concentrated HC1. The final volume of the solutions (30 ml) was then made by adding 29 ml distilled water. Mice were injected i.p. with 5.13, 7.70, 15.40, and 38.50 mg/100 g b.w. of Pr6Oll and 5.30, 8.60, 17.20 and 43.0 mg/100 g b.w. of Nd20 3. The dosage se-
2. Materials and methods
Random bred Swiss albino mice (Mus musculus) of 8-10 weeks old and weighing 25-30 g
Table 1 C h r o m o s o m a l a b e r r a t i o n i n d u c e d by n e o d y m i u m oxide ( N D 2 0 3) in b o n e m a r r o w cells of Swiss albino mice Treatment
Dose (mg/100g
Gap a
CtB
b.w.) 6 h harvest N e g a t i v e control Mitomycin C ( + v e control) N e o d y m i u m oxide (Nd203)
CAs/cell c
Total CA b ChB
CF
6
5
-
58 10 12 14 18
28 6 8 13 15
27 4 4 10 10
6
7
7
0.15 5.30
65 10
8.60 17.20 43.00
0.15 5.30 8.60 17.20 43.00
RC
Ex
%Abn a cells
St + PI
-
-
-
0.010
1.00
18 -
16 -
-
0.190 0.020
16.20 1.20
2
2 4
3
-
0.024 0.050 0.068
1.80 2.80 4.60
-
-
-
-
-
0.014
1.40
40 8
55 12
8 -
15 16
9 10
14 19
-
22
12
18
2
22 4 5
22 4
4
0.294 0.040 0.046 0.066 0.082
21.40 2.00 3.00 4.00 5.60
7
10
0.020
1.80
49 6 9 10 14
22 6 8 7 5
0.164 0.028 0.032 0.032 0.038
14.60 2.00 2.40 2.60 3.60
T r e n d test p < 0.001 12 h h a r v e s t N e g a t i v e control Mitomycin C ( + ve control) N e o d y m i u m oxide ( N d 2 0 3)
-
T r e n d test p < 0.001 24 h h a r v e s t N e g a t i v e control Mitomycin C ( + v e control) N e o d y m i u m oxide ( N d 2 0 3)
0.15 5.30 8.60 17.20 43.00
28 8 6 7 10
2 -
19 2 2 3
11 1
5
T r e n d test p < 0.001 a I n c l u d e s b o t h c h r o m a t i d a n d i s o c h r o m a t i d gap. b 500 m e t a p h a s e s analysed p e r dose a n d 100 p e r a n i m a l . c N u m b e r of C A s / c e l l (excluding g a p a n d stickiness + pulverization). d P e r c e n t a g e a b n o r m a l cells include stickiness + p u l v e r i z a t i o n w h e r e e v e r r e c o r d e d . CtB, C h r o m a t i d b r e a k ; ChB, c h r o m o s o m e b r e a k ; CF, c e n t r i c fusion; RC, ring c h r o m o s o m e . Ex, E x c h a n g e ; St + P1, stickiness + pulverization.
A.M. Jha, A.C. Singh /Mutation Research 341 (1995) 193-197
195
for each dose and for each sampling time. 2 h before killing each animal received a freshly prepared colchicine solution at the rate of 4 m g / k g b.w. through i.p. injection. The animals were killed by cervical dislocation at 6, 12 and 24 h after treatment. Bone marrow from both the femurs was collected by flushing with 0.075 M potassium chloride (pre-warmed at 37°C), incubated at 37°C for 25 min, centrifuged and fixed in cold fixative (3 : 1 ethanol-glacial acetic acid). The samples were washed twice in fixative, dropped on clean chilled slides, flame dried and stained in
lected for administration were determined according to the guidelines suggested for in vivo cytogenetic assay (Preston et al., 1987). The selection of maximum dose was based on depression in mitotic index (not more than 50%)in the bone marrow cells, measured at the same sampling times as those used for chromosomal aberration assay. 0.15 m g / 1 0 0 g b.w. of mitomycin C was used as positive control. Negative control animals received 0.1 ml of solvent (1 ml concentrated HC1 in 29 ml distilled water) through i.p. injection. Five animals (3 males and 2 females) were used
Table 2 C h r o m o s o m a l a b e r r a t i o n i n d u c e d by p r a s e o d y m i u m oxide ( P r 6 O l l ) in b o n e m a r r o w cells of Swiss albino mice Treatment
Dose (mg/100g
Gap a
CtB
b.w.) 6 h harvest N e g a t i v e control Mitomycin C ( + ve control) P r a s e o d y m i u m oxide (Pr6Oll)
-
0.15 5.13 7.70 15.40 38.50
CAs/cell c
Total CA b ChB
6
5
58 7 8 11 9
28 8 13 11 15
7
7
65 7 11 14 10
40 8 9 13 14
55 10 14 15 19
7
10
.
49 4 5 13 7
22 5 8 9 9
28 5 9 11 13
CF
27 2 7 9 12
RC
6 2 3
Ex
18 1 2 3 5
St + PI
%Abn d cells
-
-
0.010
1.00
16 2
-
0.190 0.022 0.044 0.050 0.074
16.20 2.00 3.20 3.60 5.20
0.014
1.40
0.294 0.036 0.054 0.078 0.088
21.40 2.40 3.20 5.60 6.40
0.020
1.80
0.064 0.024 0.040 0.054 0.056
14.60 1.80 3.00 4.40 5.00
T r e n d test p < 0.001 12 h harvest N e g a t i v e control Mitomycin C ( + ve control) P r a s e o d y m i u m oxide (Pr 6 O l l )
-
0.15 5.13 7.70 15.40 38.50
8 . 3 3
22 . 4 6 6
.
22
-
2 2
2 4
.
T r e n d test p < 0.001 24 h harvest N e g a t i v e control Mitomycin C (+ve control) P r a s e o d y m i u m oxide (Pr6Oll)
0.15 5.13 7.70 15.40 38.50
.
. 2 2 1
.
. 19 2 3 3 2
11 2 3
3 3
T r e n d test p < 0.001 a I n c l u d e s b o t h c h r o m a t i d a n d i s o c h r o m a t i d gap. t, 500 m e t a p h a s e a n a l y s e d p e r d o s e a n d 100 p e r animal. c N u m b e r of C A s / c e l l (excluding g a p and stickiness + pulverization). d P e r c e n t a g e a b n o r m a l cells i n c l u d e s stickiness + p u l v e r i z a t i o n w h e r e e v e r recorded. CtB, C h r o m a t i d b r e a k ; C h B c h r o m o s o m e b r e a k ; CF, c e n t r i c fusion; RC, ring c h r o m o s o m e . Ex, E x c h a n g e ; St + P1, stickiness + pulverization.
A.M. Jha, A.C. Singh / Mutation Research 341 (1995) 193-197
196
buffered Giemsa. The slides were coded. 100 metaphases/animal (500 metaphases/dose) were screened for the presence of CAs. The number and type of CAs were scored. Data recorded were evaluated as the number of CAs/cell ( C A s / c e l l excluding gaps and stickiness + pulverizations)and percent aberrant cells (excluding gaps). Chromatid and isochromatid gaps, chromatid and chromosome breaks, chromatid and chromosome exchanges, ring chromosomes, centric fusions, stickiness and pulverization were recorded as chromosomal abnormalities. For statistical analysis one tailed trend test (Margolin et al., 1986) and two-way A N O V A (Harter, 1960) were employed.
Two-way A N O V A (Table 3) showed significant differences between the treatments as regards percent aberrant ceils and CAs/cell. CAs recorded were chromatid and chromosome breaks, gaps, exchanges, centric fusion, ring chromosomes, stickiness and pulverizations. Chromatid and chromosome breaks were present in higher frequencies than the other types and were induced by all the doses used. The frequencies of chromatid breaks were higher at 6 h sampling time whereas chromosome breaks were present in higher frequencies at 12 h sampling time. All other types were recorded only after treatment with the higher doses of Pr6Otl and N d 2 0 3.
3. Results
4. Discussion
Tables 1 and 2 record data on the induction of CAs in bone marrow ceils of mice following in vivo exposure to different doses of Pr6011 and N d 2 0 3 for 6, 12 and 24 h. A single exposure to mice of different doses of Pr6011 and N d 2 0 3 significantly enhanced the frequency of CAs at metaphase. The number of CAs/cell and percent aberrant cells increased, when compared to negative control, with an increase in dose. The frequencies also increased with the lapse of time attaining a maximal at 12 h sampling time. The number of CAs/cell and percent aberrant cells, as shown by a one-tailed trend test, showed a positive trend with the dose of Pr6011 and N d 2 0 3.
Mouse in vivo chromosomal aberration assays have been recognised as one of the sensitive endpoints to test the genotoxicity of industrial pollutants. Much is known about the ability of metallic compounds to induce CAs in bone marrow cells of laboratory animals (Agarwal et al., 1990; Ghosh, 1991; Ghosh and Talukder, 1990; Sharma and Talukder, 1987). The lanthanide elements, although are being utilized in many industries, have not come in for much genotoxic testing. Only a few of them have gone under shortterm in vivo genotoxicity assays. Among the lanthanides, lanthanum chloride has been reported to induce dose- and duration-related increase in the frequency of chromosomal aberratins in vivo in mice (Das, 1991; De and Sharma, 1981). The clastogenic effect has been attributed to the property of lanthanum to form complex with DNA (Rosoff and Spencer, 1979). Cerium as a nitrate induced breaks and reduced the mitotic index in rat bone marrow in vivo. This element is known to affect the nucleotides and to accumulate in bone through its affinity with phosphates, leading to its cytotoxic actifvity (Giri et al., 1979; Sharma and Talukder, 1987). Cerium has also been reported to affect the lipid metabolism in liver (Synder and Kyker, 1969) and as nitrate, DNA, R N A and protein synthesis in liver, kidney, lung and brain of rat (Giri et al., 1978).
Table 3 Two-way A N O V A of percent aberrant cells and CAs/cell showing significant difference among different doses and durations following in vivo treatment Sources of
df
Mean squares
F-ratio
variation
Pr6Oll
N d 2 0 3 Pr6Oll
Nd20 3
% Aberrant cells Duration 2 Concentration 4 Error 8
0.87 8.74 0.24
1.17 4.87 0.28
3.62 36.41"*
4.17 19.39 **
CAs/cell Duration Concentration Error
0.0004 0.0017 0.00005
0.0055 0.0102 0.0012
8.00 * 34.00 **
4.58 * 8.50 *
2 4 8
*'** Significant at 5% and 1% level respectively.
A.M. Jha, A.C. Singh / Mutation Research 341 (1995) 193-197 The results of the present investigation demonstrate that Praseodymium and Neodymium a r e c l a s t o g e n i c in v i v o in m o u s e . T h e c h r o m o s o m a l d a m a g i n g activity o f t h e s e e l e m e n t s in b o n e m a r r o w cells o f m i c e is in a g r e e m e n t w i t h t h e i r c l a s t o g e n i c p r o p e r t i e s in Vicia faba r o o t tip cells ( J h a a n d S i n g h , 1994). B o t h t h e e l e m e n t s i n d u c e d d o s e r e l a t e d i n c r e a s e in t h e n u m b e r o f C A s / c e l l a n d p e r c e n t a g e o f a b e r r a n t cells at all s a m p l i n g p e r i o d s , w h e n c o m p a r e d to n e g a t i v e c o n t r o l . C e l l s s a m p l e d 12 h a f t e r e x p o s u r e s h o w e d h i g h e r n u m b e r o f C A s / c e l l as c o m p a r e d to 6 a n d 24 h. D e c r e a s e in t h e p e r c e n t a g e o f a b e r r a n t cells a f t e r 24 h i.p. i n j e c t i o n s u g g e s t r e d u c t i o n in t h e survival r a t e o f t h e a f f e c t e d cells a n d t h e i r s u b s e quent elimination. T h e p r e s e n t s t u d y t h u s r e p o r t s t h e first in v i v o clastogenicity of two lanthanide elements p r a s e o d y m i u m (Pr) a n d n e o d y m i u m ( N d ) as m e a s u r e d by c h r o m o s o m a l a b e r r a t i o n assays. F u r t h e r s t u d i e s , s u c h as in v i v o m i c r o n u c l e u s a n d S C E t e s t a n d t e s t i c u l a r toxicity o f t h e e l e m e n t s , a r e in p r o g r e s s to v a l i d a t e o u r f i n d i n g s .
Acknowledgement T h e a u t h o r s a r e t h a n k f u l to P r o f e s s o r P . N . Sinha, Head, Department of Botany, Samastipur College, Samastipur, India for facilities provided d u r i n g this study.
References Agarwal, K., A. Sharma and G. Talukder (1990) Clastogenic effects of copper sulphate on the bone marrow chromosomes of mice in vivo, Mutation Res., 243, 1-6. Bruce, D.W., B.E. Heitbrink and K.P. DuBois (1963) The
197
acute mammalian toxicity of rare earth nitratesand oxides, Toxicol. Appl. Pharmacol., 5, 750-759. Das, T. (1991) Clastogenic effects of lanthanum chloride on bone marrow chromosomes of mice in vivo, Proc. Symp. Environment and Genome, EMSI, pp. 268-271. De, S.K. and A. Sharma (1981) Mutachromosomal effects of lanthanum chloride on mammalian bone marrow, Curr. Sci., 50, 189-190. Giri, A.K., A. Sharma and G. Talkuder (1978) Cytochemical effects of cobalt sulphate and ceric sulphate on mammalian system, Bull. Inst. P.G. Med. Edu. Res., 20, 145147. Girl, A.K., R. Sanyal, G. Talukder and A. Sharma (1979) Cytological and cytochemical changes induced through certain heavy metals on mammalian system, Natl. Acad. Sci. Lett., 2, 391-394. Giri, A.K., R. Banerjee, G. Talukder and A. Sharma(1980) Mutagenic effects of certain common metal toxicants on mammalian system, Proc. Indian Acad. Sci. (Anim. Sci.), 89, 311-331. Ghosh S. (1991) Clastogenic effects of ziroconium oxychloride in ralation to dose and duration of exposure in bone marrow cells of mice in vivo, Proc. Symp. Environment and Genome, EMSI, pp. 222-225. Ghosh, A., A. Sharma and G. Talukder (1990) Clastogenic effects of cesium chloride in bone marrow cells of mice in vivo, Mutation Res., 244, 295-298. Hatter, H.L. (1960) Biometrics, 16, 671. Jha, A.M. and A.C. Singh (1994) Clastogenicity of lanthanides induction of micronuclei in root tips of V.iciafaba, Mutation Res., 322, 169-172. Margolin, B.H., M.A. Resnik, J.Y. Rimpo, P. Archer, S.M. Galloway, A.D. Bloom and E. Zeiger (1986) Statistical analysis for in vitro cytogenetic assays using Chinese hamster ovary cells, Environ. Mol. Mutagen., 8, 183-204. Preston, R.J., B.D. Dean, S. Galloway, H Holden, A.F. McFee and M. Shelby (1987) Mammalian in vivo cytogenetic assay: analysis of chromosome aberrations in bone marrow cells, Mutation Res., 189, 157-165. Rosoff, B. and H. Spencer (1979) Binding of rare earths to serum proteins and DNA, Clin. Chim. Acta, 93, 311-319. Sharma, A. and G. Talukder (1987) Effects of metals on chromosomes of higher organism, Environ. Mutagen., 9, 191-226. Synder, F., E.A. Cress and G.C. Kyker (1959) Lipid responses to intravenous rare earths in rats, J. Lipid Res., 1,125-131.