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Genetic effects of microwave radiation in mice
Mutation Research, 103 (1982) 39-42
39
Elsevier Biomedical Press
GENETIC EFFECTS OF MICROWAVE RADIATION IN MICE
S.N. G O U D a, M.V. USHA RANI b, P.P. REDDY b, O.S. REDDI b, M.S. RAO a and V.K. SAXENA c
aDepartment of Genetics, Osmania University, Hyderabad-500 007; blnstitute of Genetics, Begumpet, Hyderabad-500 016; and cElectronics Corporation o f India Limited, Hyderabad (India) (Accepted 7 May 1981)
Microwave radiations are being increasingly used in radar, space research, telecommunication, medicine and in ovens. Microwaves have been found to induce chromatid and chromosomal aberrations in human lymphocyte cultures [1], mice spermatogonia [8], Chinese hamster corneal epithelial cells [131, root tips of garlic [7] and Vicia faba [41. In the present study we have investigated the mutagenic potential of 2450 MHz microwave radiation in mice using the dominant lethal test and sperm abnormality assay. MATERIALS AND M E T H O D S
Dominant lethal test 25 male mice of Swiss albino strain (8 weeks old) were taken in plastic porous tubes and exposed to whole body microwave radiation from a microwave diathermy unit (ECIL) at 2450 MHz, CW and at a power density of 170 mW/cm 2 for 70 sec. An equal number of unexposed males served as controls. Immediately after treatment each male was caged with 3 virgin females, which were replaced at weekly intervals for 3 consecutive weeks. Females were autopsied 13 days after the midweek of their caging and scored for total implantations, dead implantation (early and late deaths) and live embryos. Dominant lethals were calculated in terms of pre-, post-implantation loss and total loss. Post-implantation loss was evaluated by the dead implants/female or dead implants/total implants × 100. Pre-implantation loss was calculated by comparing the total implantation per female in control and treated series. The total loss was made on the basis of live implants per female [5]. The results were analysed for their statistical significance by chi square, t-test and analysis of variance. Sperm abnormality assay The treated males were killed by cervical dislocation on the 35th day after treatment. Cauda epididymis was dissected out and minced finely in 0.9% 0165 - 7992/82/0000 - 0000/$02.75 © Elsevier Biomedical Press
40
physiological saline. It was stained with 0.1 °7o eosin Y and slides were prepared [11]. The coded slides were analysed for sperm head morphology and tested by t-test. RESULTS A N D DISCUSSION
Dominant lethal mutations indicate major genetic damage and have been used in mammals for testing the mutagenic potential of physical and chemical agents. The genetic basis of dominant lethality is chiefly the induction of structural and numerical chromosomal aberrations. In the present study microwave exposure decreased significantly (P < 0.05) the fertility index (Table 1) in all 3 weeks of matings. The post-implantation loss was enhanced significantly (P < 0.01) in all the post-meiotic stages and it was highest in the spermatid stage. The pre-implantation and total losses were also increased significantly (P < 0.01) in the 2nd and 3rd weeks of mating. Sperm abnormality assay is one of the newly introduced tests in the protocols of mutagenicity screening. The relevance of this test gains support from the following three findings. The mutagenic agents were found to induce a high proportion of sperm shape abnormalities. Sperm morphology is determined by a number of autosomal and sex-linked genes. Sperm abnormalities were found to be heritable in simple Mendelian fashion [11]. As is evident from Table 2 there is a significant (P < 0.01) increase in the frequency of abnormal sperms in the microwave exposed male mice when compared to controls.
TABLE 1 EFFECT OF M I C R O W A V E R A D I A T I O N ON D O M I N A N T L E T H A L MUTATIONS IN MALE MICE Observation
Total females Fertility index (070) Total implants per female Live implants per female Dead implants per female Post-implantation loss (070)
2nd week
I st week
3rd week
C
T
C
T
C
T
75
75
75
75
75
75
84.0
60.0
78.6
62.6
80.0
58.6
8.73 + 0.22
8.53 ± 0.24
8.34 + 0.32
6.39 + 0.35
9.02 + 0.26
7.05 ± 0.28
8.30 ± 0.30
7.53 ± 0.21
7.89 + 0.23
5.31 _+ 0.18
8.53 :t: 0.42
5.61 _+0.38
0.43 ± 0.19
1.00 _+ 0.22
0.44 ± 0.17
1.06 _+ 0.23
0.48 _+ 0.21
1.43 _+ 0.29
4.90
11.72
5.28
16.66
5.36
C, control; T, treated. Fertility index (070) = (number of fertile females/number of total females) x 100.
20.32
41
TABLE 2 RESULTS ON SPERM-SHAPE ABNORMALITIES IN MICE EXPOSED TO MICROWAVE R A D I A T I O N S (2450 MHz) Group
Number of animals
Total number of sperms counted
Abnormal sperm (%) (~ _+ S.E.)
Control Treated
8 8
8000 8000
1.42 _+ 0.26 5.67* _ 0.39
From each animal 1000 sperms were analysed. *P 0.01.
Our results are in agreement with those of Manikowska et al. [8] who reported a significant increase in the frequency of chromosomal rearrangements (translocations) and autosomal univalents in the microwave exposed mice of Balb/c strain. They exposed the animals to a very high frequency (9.4 GHz) and for a chronic period of 2 weeks, 1 h/day. Varma and Traboulay [9] in their histological study on the testes of microwave-exposed (1.7 GHz, 10-50 mW/cm 2, 30-40 min) Swiss albino mice found the lumen of the seminiferous tubules to be empty with complete disintegration of spermatids and sertoli cells. However, when tested with 3.0 GHz the injuries were minimal. In their later investigations they reported that microwave alters the hyperchromicity and melting temperature of the mouse testicular DNA, suggesting that microwave causes DNA strand separation [10]. Cieciura and Minecki [2, 3], when they exposed rats to microwaves of 2860 MHz at a power density of 60-90 mW/cm 2 for 1-2 min, found hemorrhages, degeneration of testicular vesicles and abnormal maturation of spermatozoa. They also observed a decrease in the activities of certain testicular enzymes such as 5-nucleotidase, ATPase, alkaline phosphatase and succinic dehydrogenase. A higher frequency of chromosomal breaks was reported in the corneal epithelial cells of Chinese hamsters exposed to 2450 MHz microwaves at 100 mW/cm 2 for 30 min [13]. Microwaves were also found to induce a high frequency of chromosomal and chromatid aberrations in choroid and bone-marrow cells of rat kangaroo irradiated in vitro [12]. Hamnerius et al. [6] reported no significant mutagenic activity in Drosophila. They exposed Drosophila larvae to 2450 MHz for 6 h and scored for eye pigmentation mutants. This is a new test system which measures somatic mutations. Its relationship to other conventional mutagenicity test systems has not been well established. Whereas we have employed standard protocols which are routinely employed in assessing the mutagenic potential of environmental agents in the germ cells of mice. In summary, the present study reveals that microwaves induce dominant lethal mutations in post-meiotic stages and sperm-shape abnormalities in mice. Further studies are needed involving acute and chronic treatments in all the stages of spermatogenesis.
42 REFERENCES 1 Baranski, S., P. Czerski and S. Szmigielski, The influence of microwaves on mitosis in vitro and in vivo, Postepy Fiz. Medycznej., 6 (1971) 93-94. 2 Cieciura, L., and L. Minecki, Distribution and activity of certain hydrolytic enzymes in testicles of rats subjected to microwaves, Med. Pracy, 15 (1964) 159-161. 3 Cieciura, L., and L. Minecki, Histopathologic changes in testicles of hypothermic microwave irradiated rats, Med. Pracy, 27 (1966) 507-508. 4 Cieciura, L., H. Hiber, L. Minecki and G. Andry Szak, The influence of microwave radiation on the mitosis in root tips of the bean Viciafaba, Med. Pracy, 22 (1971) 211-212. 5 Epstein, S.S., and G. Rohrborn, Recommended procedures for testing genetic hazards from chemicals based on the induction of dominant lethal mutations in mammals, Nature (London), 230 (1971) 459-460. 6 Hamnerius, Y., H. Olofsson and B. Rasmuson, A negative test for mutagenic action of microwave radiation in Drosophila melanogaster, Mutation Res., 68 (1979) 217-223. 7 Heller, J.H., in: S.F. Cleary (Ed.), Biological Effects and Health Implications of Microwave Radiation, Symp. Proc. US Dept. of Health, Education and Welfare, Report BRH/DBE 70-2 (PB 193858) Rockville, MD, 1970, p. ll6. 8 Manikowska, E., J.M. Luciani, B. Servantie, P. Czerski, J. Obrenovitch and A. Stahl, Effects of 9.4 GHz microwave exposure on meiosis, Experientia, 35 (1979) 388-390. 9 Varma, M.M., and E.A. Traboulay, Biological effects of microwave radiation on the testes of Swiss mice, Experientia, 31 (1975) 301-303. l0 Varma, M.M., and E.A. Traboulay, Comparison of native and microwave irradiated DNA, Experientia, 33 (1977) 1649-1650. 11 Wyrobek, A.J., and W.R. Bruce, Induction of sperm shape abnormalities, in: A. Hollaender (Ed.), Chemical Mutagens, Voi. 5, Plenum, New York, 1978, p. 257. 12 Yao, K.T.S., and M.M. Jiles, in: S.F. Cleary (Ed.), Biological Effects and Health Implications of Microwave Radiation, Symp. Proc. US Dept. of Health, Education and Welfare, Report BRH/DBE 70-2 (PB 193858) Rockville, MD, 1970, p. 123. 13 Yao, K.T.S., Microwave induced chromosomal aberrations, in corneal epithelium of Chinese hamsters, J. Hered., 69 0978) 409-412.
39
Elsevier Biomedical Press
GENETIC EFFECTS OF MICROWAVE RADIATION IN MICE
S.N. G O U D a, M.V. USHA RANI b, P.P. REDDY b, O.S. REDDI b, M.S. RAO a and V.K. SAXENA c
aDepartment of Genetics, Osmania University, Hyderabad-500 007; blnstitute of Genetics, Begumpet, Hyderabad-500 016; and cElectronics Corporation o f India Limited, Hyderabad (India) (Accepted 7 May 1981)
Microwave radiations are being increasingly used in radar, space research, telecommunication, medicine and in ovens. Microwaves have been found to induce chromatid and chromosomal aberrations in human lymphocyte cultures [1], mice spermatogonia [8], Chinese hamster corneal epithelial cells [131, root tips of garlic [7] and Vicia faba [41. In the present study we have investigated the mutagenic potential of 2450 MHz microwave radiation in mice using the dominant lethal test and sperm abnormality assay. MATERIALS AND M E T H O D S
Dominant lethal test 25 male mice of Swiss albino strain (8 weeks old) were taken in plastic porous tubes and exposed to whole body microwave radiation from a microwave diathermy unit (ECIL) at 2450 MHz, CW and at a power density of 170 mW/cm 2 for 70 sec. An equal number of unexposed males served as controls. Immediately after treatment each male was caged with 3 virgin females, which were replaced at weekly intervals for 3 consecutive weeks. Females were autopsied 13 days after the midweek of their caging and scored for total implantations, dead implantation (early and late deaths) and live embryos. Dominant lethals were calculated in terms of pre-, post-implantation loss and total loss. Post-implantation loss was evaluated by the dead implants/female or dead implants/total implants × 100. Pre-implantation loss was calculated by comparing the total implantation per female in control and treated series. The total loss was made on the basis of live implants per female [5]. The results were analysed for their statistical significance by chi square, t-test and analysis of variance. Sperm abnormality assay The treated males were killed by cervical dislocation on the 35th day after treatment. Cauda epididymis was dissected out and minced finely in 0.9% 0165 - 7992/82/0000 - 0000/$02.75 © Elsevier Biomedical Press
40
physiological saline. It was stained with 0.1 °7o eosin Y and slides were prepared [11]. The coded slides were analysed for sperm head morphology and tested by t-test. RESULTS A N D DISCUSSION
Dominant lethal mutations indicate major genetic damage and have been used in mammals for testing the mutagenic potential of physical and chemical agents. The genetic basis of dominant lethality is chiefly the induction of structural and numerical chromosomal aberrations. In the present study microwave exposure decreased significantly (P < 0.05) the fertility index (Table 1) in all 3 weeks of matings. The post-implantation loss was enhanced significantly (P < 0.01) in all the post-meiotic stages and it was highest in the spermatid stage. The pre-implantation and total losses were also increased significantly (P < 0.01) in the 2nd and 3rd weeks of mating. Sperm abnormality assay is one of the newly introduced tests in the protocols of mutagenicity screening. The relevance of this test gains support from the following three findings. The mutagenic agents were found to induce a high proportion of sperm shape abnormalities. Sperm morphology is determined by a number of autosomal and sex-linked genes. Sperm abnormalities were found to be heritable in simple Mendelian fashion [11]. As is evident from Table 2 there is a significant (P < 0.01) increase in the frequency of abnormal sperms in the microwave exposed male mice when compared to controls.
TABLE 1 EFFECT OF M I C R O W A V E R A D I A T I O N ON D O M I N A N T L E T H A L MUTATIONS IN MALE MICE Observation
Total females Fertility index (070) Total implants per female Live implants per female Dead implants per female Post-implantation loss (070)
2nd week
I st week
3rd week
C
T
C
T
C
T
75
75
75
75
75
75
84.0
60.0
78.6
62.6
80.0
58.6
8.73 + 0.22
8.53 ± 0.24
8.34 + 0.32
6.39 + 0.35
9.02 + 0.26
7.05 ± 0.28
8.30 ± 0.30
7.53 ± 0.21
7.89 + 0.23
5.31 _+ 0.18
8.53 :t: 0.42
5.61 _+0.38
0.43 ± 0.19
1.00 _+ 0.22
0.44 ± 0.17
1.06 _+ 0.23
0.48 _+ 0.21
1.43 _+ 0.29
4.90
11.72
5.28
16.66
5.36
C, control; T, treated. Fertility index (070) = (number of fertile females/number of total females) x 100.
20.32
41
TABLE 2 RESULTS ON SPERM-SHAPE ABNORMALITIES IN MICE EXPOSED TO MICROWAVE R A D I A T I O N S (2450 MHz) Group
Number of animals
Total number of sperms counted
Abnormal sperm (%) (~ _+ S.E.)
Control Treated
8 8
8000 8000
1.42 _+ 0.26 5.67* _ 0.39
From each animal 1000 sperms were analysed. *P 0.01.
Our results are in agreement with those of Manikowska et al. [8] who reported a significant increase in the frequency of chromosomal rearrangements (translocations) and autosomal univalents in the microwave exposed mice of Balb/c strain. They exposed the animals to a very high frequency (9.4 GHz) and for a chronic period of 2 weeks, 1 h/day. Varma and Traboulay [9] in their histological study on the testes of microwave-exposed (1.7 GHz, 10-50 mW/cm 2, 30-40 min) Swiss albino mice found the lumen of the seminiferous tubules to be empty with complete disintegration of spermatids and sertoli cells. However, when tested with 3.0 GHz the injuries were minimal. In their later investigations they reported that microwave alters the hyperchromicity and melting temperature of the mouse testicular DNA, suggesting that microwave causes DNA strand separation [10]. Cieciura and Minecki [2, 3], when they exposed rats to microwaves of 2860 MHz at a power density of 60-90 mW/cm 2 for 1-2 min, found hemorrhages, degeneration of testicular vesicles and abnormal maturation of spermatozoa. They also observed a decrease in the activities of certain testicular enzymes such as 5-nucleotidase, ATPase, alkaline phosphatase and succinic dehydrogenase. A higher frequency of chromosomal breaks was reported in the corneal epithelial cells of Chinese hamsters exposed to 2450 MHz microwaves at 100 mW/cm 2 for 30 min [13]. Microwaves were also found to induce a high frequency of chromosomal and chromatid aberrations in choroid and bone-marrow cells of rat kangaroo irradiated in vitro [12]. Hamnerius et al. [6] reported no significant mutagenic activity in Drosophila. They exposed Drosophila larvae to 2450 MHz for 6 h and scored for eye pigmentation mutants. This is a new test system which measures somatic mutations. Its relationship to other conventional mutagenicity test systems has not been well established. Whereas we have employed standard protocols which are routinely employed in assessing the mutagenic potential of environmental agents in the germ cells of mice. In summary, the present study reveals that microwaves induce dominant lethal mutations in post-meiotic stages and sperm-shape abnormalities in mice. Further studies are needed involving acute and chronic treatments in all the stages of spermatogenesis.
42 REFERENCES 1 Baranski, S., P. Czerski and S. Szmigielski, The influence of microwaves on mitosis in vitro and in vivo, Postepy Fiz. Medycznej., 6 (1971) 93-94. 2 Cieciura, L., and L. Minecki, Distribution and activity of certain hydrolytic enzymes in testicles of rats subjected to microwaves, Med. Pracy, 15 (1964) 159-161. 3 Cieciura, L., and L. Minecki, Histopathologic changes in testicles of hypothermic microwave irradiated rats, Med. Pracy, 27 (1966) 507-508. 4 Cieciura, L., H. Hiber, L. Minecki and G. Andry Szak, The influence of microwave radiation on the mitosis in root tips of the bean Viciafaba, Med. Pracy, 22 (1971) 211-212. 5 Epstein, S.S., and G. Rohrborn, Recommended procedures for testing genetic hazards from chemicals based on the induction of dominant lethal mutations in mammals, Nature (London), 230 (1971) 459-460. 6 Hamnerius, Y., H. Olofsson and B. Rasmuson, A negative test for mutagenic action of microwave radiation in Drosophila melanogaster, Mutation Res., 68 (1979) 217-223. 7 Heller, J.H., in: S.F. Cleary (Ed.), Biological Effects and Health Implications of Microwave Radiation, Symp. Proc. US Dept. of Health, Education and Welfare, Report BRH/DBE 70-2 (PB 193858) Rockville, MD, 1970, p. ll6. 8 Manikowska, E., J.M. Luciani, B. Servantie, P. Czerski, J. Obrenovitch and A. Stahl, Effects of 9.4 GHz microwave exposure on meiosis, Experientia, 35 (1979) 388-390. 9 Varma, M.M., and E.A. Traboulay, Biological effects of microwave radiation on the testes of Swiss mice, Experientia, 31 (1975) 301-303. l0 Varma, M.M., and E.A. Traboulay, Comparison of native and microwave irradiated DNA, Experientia, 33 (1977) 1649-1650. 11 Wyrobek, A.J., and W.R. Bruce, Induction of sperm shape abnormalities, in: A. Hollaender (Ed.), Chemical Mutagens, Voi. 5, Plenum, New York, 1978, p. 257. 12 Yao, K.T.S., and M.M. Jiles, in: S.F. Cleary (Ed.), Biological Effects and Health Implications of Microwave Radiation, Symp. Proc. US Dept. of Health, Education and Welfare, Report BRH/DBE 70-2 (PB 193858) Rockville, MD, 1970, p. 123. 13 Yao, K.T.S., Microwave induced chromosomal aberrations, in corneal epithelium of Chinese hamsters, J. Hered., 69 0978) 409-412.