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Induction of dominant lethal mutations in male mice by potassium dichromate
Mutation Research, 103(1982)345-347
345
Elsevier BiomedicalPress
Induction of dominant lethal mutations in male mice by potassium dichromate Y.V. Paschin, T . A . Zacepilova and V.I. K o z a c h e n k o Institute of General Genetics, Academy of Sciences of the U.S.S.R., Moscow 117809 (U. S. S.R.)
(Accepted 3 September 1981)
In recent years heavy metals are considered as environmentally important pollutants. By different tests mutagenic activity of the hexavalent chromium ion has been shown in bacteria, fungi, mammalian somatic cells etc. [1-4, 8, 9]. But no studies on mammalian germ cells are available for estimation of genetic activity of low-dose exposure to this agent. The special danger of chromium compounds is due to their bioaccumulation [5, 7]. The purpose of the present study is to investigate the frequency of dominant lethal mutations in mouse male germ cells at the different stages of gametogenesis after treatment with hexavalent chromium ion.
Materials and methods Test agent. Potassium dichromate (PD), analytical grade, composition: 99.9% water-soluble active substance, the rest includes by-products as follows: insoluble substances, 0.002%; chlorides (CI), 0.002%; sulphates (SO4), 0.01%; substances sedimented by ammonia (A1, Fe, etc.), 0.002°7o; calcium (Ca), 0.02%; sodium (Na), 0.02%. A n i m a l s . Mature (2-3 months) hybrid mice (CBA × C57B1/6J) approximately 27-30 g b.w. D o m i n a n t lethal test. After treatment with 0.5, 1.0, 1.5, 2.0, 10.0 and 20.0 mg. kg -~ PD, single, i.p. or with 1.0 and 2.0 mg. kg -~ PD daily, i.p. for 21 days each male was mated with 2 untreated virgin females for 7 days. At the end of a week, female mice were removed and replaced by 2 new females. The replacement of females was continued for 4 consecutive weeks. Pregnant females were killed by cervical dislocation 12-14 days after conception. Number of implants, live and dead embryos were scored. The determination of the frequency of dominant lethal mutations in male mice was based on the postimplantation loss. xZ-test was used for statistical evaluation of results obtained in dominant lethal experiments.
0165-7992/82/0000-0000/$02.75 © ElsevierBiomedicalPress
346 TABLE 1 F R E Q U E N C Y OF D O M I N A N T L E T H A L M U T A T I O N S I N D U C E D BY P D A F T E R SINGLE A N D CHRONIC INJECTIONS Stage o f
Dose o f P D
N u m b e r o f females
Effective
N u m b e r of
spermato-
(mg • k g - l
Mated
matings
Alive
Dead
genesis
b.w.)
(%)
embryos
embryos
Pregnant
Postimplantation
(L)
(D)
lethality D
L+D
Single injection Sperm
2.0 10.0 20.0 Control
30 30 30 27
24 24 24 24
80.0 80.0 80.0 88.8
211 247 219 202
8 7 14 6
3.6 2.7 6.0 2.9
Late spermatids
2.0 10.0 20.0 Control
30 30 30 27
27 24 27 24
90.0 80.0 90.0 88.8
246 214 206 203
7 5 16 7
2.8 2.3 7.2 3.3
Early spermatids
2.0 10.0 20.0 Control
30 30 30 27
24 24 24 25
80.0 80.0 80.0 92.5
221 197 168 220
4 12 33 7
1.9 5.7 16.4"** 3.1
Late spermatocytes
2.0 10.0 20.0 Control
30 30 30 27
24 28 27 25
80.0 93.0 90.0 92.5
112 190 195 228
7 11 16 7
5.9 5.5 7.6** 3.0
Chronic injections Sperm
1.0 2.0 Control
30 30 27
24 22 25
80.0 73.3 92.5
233 143 228
5 14 7
2.1 8.2** 3.0
Late spermatids
1.0 2.0 Control
30 30 27
21 26 24
70.0 86.6 88.8
178 198 200
10 23 5
5.3 10.6"* 2.5
Early spermatids
1.0 2.0 Control
30 30 27
21 22 25
70.0 73.3 92.5
192 173 227
5 13 7
2.5 6.3* 2.9
Late spermatocytes
1.0 2.0 Control
30 30 27
23 26 24
76.0 86.6 88.8
202 217 201
5 4 5
2.4 1.8 2.5
*P _< 0.05. **P _< 0,02. ***P _< 0,001,
x 100(%)
347
Results and discussion A l l females with i m p l a n t a t i o n s were classified as fertile. T a b l e 1 presents d a t a on t h e live a n d d e a d e m b r y o s , p r o g e n y o f males t h a t experience the influence o f P D after single a n d c h r o n i c a d m i n i s t r a t i o n . The single injections o f doses r a n g i n g f r o m 0.5 to 2.0 m g - k g - ~ s h o w e d n o effect, a n d hence the effect o f 2.0 m g - k g - 1 o n l y is p r e s e n t e d in T a b l e 1. As follows f r o m a n analysis o f the results for single a n d c h r o nic t r e a t m e n t s , P D i n d u c e d a statistically significant decrease in the survival o f e m b r y o s f r o m cells t r e a t e d as early s p e r m a t i d s a n d late s p e r m a t o c y t e s (Table 1). T h e r e a r e a few d a t a on c h r o m a t e b i o t r a n s f o r m a t i o n in the h u m a n b o d y a n d i n c o r p o r a t i o n into the cell. A d e c r e a s e o f m u t a g e n i c activity o f Cr 6÷ has been o b s e r v e d a f t e r in v i t r o t r e a t m e n t with a rat-liver m i c r o s o m e f r a c t i o n a n d h u m a n e r y t h r o c y t e lysates. These d a t a m a y be e x p l a i n e d by t r a n s f o r m a t i o n o f the Cr 6+ ion t o less m u t a g e n i c C r 3÷ [10, 6]. It is still u n k n o w n in which v a l e n t f o r m the c h r o m i u m ion m a y be a c c u m u l a t e d in m a m m a l i a n tissues. T h e p r e s e n t s t u d y shows t h a t P D o v e r c o m e s the m a m m a l i a n b l o o d - t e s t i c u l a r b a r r i e r a n d so m a y induce m u t a t i o n s which m a y be t r a n s m i t t e d to s u b s e q u e n t generations.
References 1 Bigaliev, A.B., R.K. Bigalieva, M.S. Elemesova, N. Schpak, G. Pynhasov and A. Pisman, Influence of chromates on rat chromosomes, in: Problems of Experimental and Clinical Medicine, Alma-Ata, 1973, pp. 30-32 [in Russian]. 2 Bigaliev, A.B., M.S. Elemesova and R.K. Bigalieva, Chromosomal aberrations at the somatic mammalian cells induced by chromates, Cytol. Genet., 10 (1976) No. 3,222-224 [in Russian]. 3 Bigaliev, A.B., M.N. Turebaev, M.S. Elemesova and R.K. Bigalieva, Tissue culture as test system for the evaluation of the mutagenic potentials of industrial pollutants, in: Genetic Consequences of Environmental Pollution, Vol. 2, Institute for General Genetics, Acad. Sci. U.S.S.R., Moscow, 1977, pp. 78-84 [in Russian]. 4 Bonatti, S., M. Meini and A. Abbondandolo, Genetic effects of potassium dichromate, Mutation Res., 38 (1976) 147-150. 5 Lbonard, A., G.N. Deknudt and M. Debackere, Cytogenetic investigations on leukocytes of cattle intoxicated with heavy metals, Toxicology, 2 (1974) 269-276. 6 LOfroth, G., The mutagenicity of hexavalent chromium is decreased by microsomal metabolism, Naturwiss., 65 (1978) 5207-5208. 7 Miheev, M.I., Hazardous Industrial Substances, Vol. 3, Chemistry, Leningrad, pp. 486-498 [in Russian]. 8 Newbold, R.F., J. Ames and J.R. Connel, The cytotoxic, mutagenic and clastogenic effects of chromium-containing compounds on mammalian cells in culture, Mutation Res., 67 (1979) 55-62. 9 Nishioka, H., Mutagenic activities of metal compounds in bacteria, Mutation Res., 31 (1975) 185-193. 10 Petrilli, F.L., and S. de Flora, Metabolic deactivation of hexavalent chromium mutagenicity, Mutation Res., 54 (1978) 139-147.
345
Elsevier BiomedicalPress
Induction of dominant lethal mutations in male mice by potassium dichromate Y.V. Paschin, T . A . Zacepilova and V.I. K o z a c h e n k o Institute of General Genetics, Academy of Sciences of the U.S.S.R., Moscow 117809 (U. S. S.R.)
(Accepted 3 September 1981)
In recent years heavy metals are considered as environmentally important pollutants. By different tests mutagenic activity of the hexavalent chromium ion has been shown in bacteria, fungi, mammalian somatic cells etc. [1-4, 8, 9]. But no studies on mammalian germ cells are available for estimation of genetic activity of low-dose exposure to this agent. The special danger of chromium compounds is due to their bioaccumulation [5, 7]. The purpose of the present study is to investigate the frequency of dominant lethal mutations in mouse male germ cells at the different stages of gametogenesis after treatment with hexavalent chromium ion.
Materials and methods Test agent. Potassium dichromate (PD), analytical grade, composition: 99.9% water-soluble active substance, the rest includes by-products as follows: insoluble substances, 0.002%; chlorides (CI), 0.002%; sulphates (SO4), 0.01%; substances sedimented by ammonia (A1, Fe, etc.), 0.002°7o; calcium (Ca), 0.02%; sodium (Na), 0.02%. A n i m a l s . Mature (2-3 months) hybrid mice (CBA × C57B1/6J) approximately 27-30 g b.w. D o m i n a n t lethal test. After treatment with 0.5, 1.0, 1.5, 2.0, 10.0 and 20.0 mg. kg -~ PD, single, i.p. or with 1.0 and 2.0 mg. kg -~ PD daily, i.p. for 21 days each male was mated with 2 untreated virgin females for 7 days. At the end of a week, female mice were removed and replaced by 2 new females. The replacement of females was continued for 4 consecutive weeks. Pregnant females were killed by cervical dislocation 12-14 days after conception. Number of implants, live and dead embryos were scored. The determination of the frequency of dominant lethal mutations in male mice was based on the postimplantation loss. xZ-test was used for statistical evaluation of results obtained in dominant lethal experiments.
0165-7992/82/0000-0000/$02.75 © ElsevierBiomedicalPress
346 TABLE 1 F R E Q U E N C Y OF D O M I N A N T L E T H A L M U T A T I O N S I N D U C E D BY P D A F T E R SINGLE A N D CHRONIC INJECTIONS Stage o f
Dose o f P D
N u m b e r o f females
Effective
N u m b e r of
spermato-
(mg • k g - l
Mated
matings
Alive
Dead
genesis
b.w.)
(%)
embryos
embryos
Pregnant
Postimplantation
(L)
(D)
lethality D
L+D
Single injection Sperm
2.0 10.0 20.0 Control
30 30 30 27
24 24 24 24
80.0 80.0 80.0 88.8
211 247 219 202
8 7 14 6
3.6 2.7 6.0 2.9
Late spermatids
2.0 10.0 20.0 Control
30 30 30 27
27 24 27 24
90.0 80.0 90.0 88.8
246 214 206 203
7 5 16 7
2.8 2.3 7.2 3.3
Early spermatids
2.0 10.0 20.0 Control
30 30 30 27
24 24 24 25
80.0 80.0 80.0 92.5
221 197 168 220
4 12 33 7
1.9 5.7 16.4"** 3.1
Late spermatocytes
2.0 10.0 20.0 Control
30 30 30 27
24 28 27 25
80.0 93.0 90.0 92.5
112 190 195 228
7 11 16 7
5.9 5.5 7.6** 3.0
Chronic injections Sperm
1.0 2.0 Control
30 30 27
24 22 25
80.0 73.3 92.5
233 143 228
5 14 7
2.1 8.2** 3.0
Late spermatids
1.0 2.0 Control
30 30 27
21 26 24
70.0 86.6 88.8
178 198 200
10 23 5
5.3 10.6"* 2.5
Early spermatids
1.0 2.0 Control
30 30 27
21 22 25
70.0 73.3 92.5
192 173 227
5 13 7
2.5 6.3* 2.9
Late spermatocytes
1.0 2.0 Control
30 30 27
23 26 24
76.0 86.6 88.8
202 217 201
5 4 5
2.4 1.8 2.5
*P _< 0.05. **P _< 0,02. ***P _< 0,001,
x 100(%)
347
Results and discussion A l l females with i m p l a n t a t i o n s were classified as fertile. T a b l e 1 presents d a t a on t h e live a n d d e a d e m b r y o s , p r o g e n y o f males t h a t experience the influence o f P D after single a n d c h r o n i c a d m i n i s t r a t i o n . The single injections o f doses r a n g i n g f r o m 0.5 to 2.0 m g - k g - ~ s h o w e d n o effect, a n d hence the effect o f 2.0 m g - k g - 1 o n l y is p r e s e n t e d in T a b l e 1. As follows f r o m a n analysis o f the results for single a n d c h r o nic t r e a t m e n t s , P D i n d u c e d a statistically significant decrease in the survival o f e m b r y o s f r o m cells t r e a t e d as early s p e r m a t i d s a n d late s p e r m a t o c y t e s (Table 1). T h e r e a r e a few d a t a on c h r o m a t e b i o t r a n s f o r m a t i o n in the h u m a n b o d y a n d i n c o r p o r a t i o n into the cell. A d e c r e a s e o f m u t a g e n i c activity o f Cr 6÷ has been o b s e r v e d a f t e r in v i t r o t r e a t m e n t with a rat-liver m i c r o s o m e f r a c t i o n a n d h u m a n e r y t h r o c y t e lysates. These d a t a m a y be e x p l a i n e d by t r a n s f o r m a t i o n o f the Cr 6+ ion t o less m u t a g e n i c C r 3÷ [10, 6]. It is still u n k n o w n in which v a l e n t f o r m the c h r o m i u m ion m a y be a c c u m u l a t e d in m a m m a l i a n tissues. T h e p r e s e n t s t u d y shows t h a t P D o v e r c o m e s the m a m m a l i a n b l o o d - t e s t i c u l a r b a r r i e r a n d so m a y induce m u t a t i o n s which m a y be t r a n s m i t t e d to s u b s e q u e n t generations.
References 1 Bigaliev, A.B., R.K. Bigalieva, M.S. Elemesova, N. Schpak, G. Pynhasov and A. Pisman, Influence of chromates on rat chromosomes, in: Problems of Experimental and Clinical Medicine, Alma-Ata, 1973, pp. 30-32 [in Russian]. 2 Bigaliev, A.B., M.S. Elemesova and R.K. Bigalieva, Chromosomal aberrations at the somatic mammalian cells induced by chromates, Cytol. Genet., 10 (1976) No. 3,222-224 [in Russian]. 3 Bigaliev, A.B., M.N. Turebaev, M.S. Elemesova and R.K. Bigalieva, Tissue culture as test system for the evaluation of the mutagenic potentials of industrial pollutants, in: Genetic Consequences of Environmental Pollution, Vol. 2, Institute for General Genetics, Acad. Sci. U.S.S.R., Moscow, 1977, pp. 78-84 [in Russian]. 4 Bonatti, S., M. Meini and A. Abbondandolo, Genetic effects of potassium dichromate, Mutation Res., 38 (1976) 147-150. 5 Lbonard, A., G.N. Deknudt and M. Debackere, Cytogenetic investigations on leukocytes of cattle intoxicated with heavy metals, Toxicology, 2 (1974) 269-276. 6 LOfroth, G., The mutagenicity of hexavalent chromium is decreased by microsomal metabolism, Naturwiss., 65 (1978) 5207-5208. 7 Miheev, M.I., Hazardous Industrial Substances, Vol. 3, Chemistry, Leningrad, pp. 486-498 [in Russian]. 8 Newbold, R.F., J. Ames and J.R. Connel, The cytotoxic, mutagenic and clastogenic effects of chromium-containing compounds on mammalian cells in culture, Mutation Res., 67 (1979) 55-62. 9 Nishioka, H., Mutagenic activities of metal compounds in bacteria, Mutation Res., 31 (1975) 185-193. 10 Petrilli, F.L., and S. de Flora, Metabolic deactivation of hexavalent chromium mutagenicity, Mutation Res., 54 (1978) 139-147.