Mutation Research, 242 (1990) 1-7
1
Elsevier MUTGEN 01567
Studies on the genotoxicity of endosulfan, an organochlorine insecticide, in mammalian germ cells Nina Pandey, Freny Gundevia, A.S. Prem and P.K. Ray Environmental Microbiology Section, Industrial Toxicology Research Centre, Mahatma Gandhi Marg, Lucknow - 226 001 (India)
(Received 3 November 1989) (Revision received13 February 1990) (Accepted 19 February 1990)
Keywords: Endosulfan; Dominant lethal mutation; Sperm abnormality; Mouse germ cells
Summary The genotoxic potential of endosulfan was assessed in mouse germ cells by 2 in vivo tests: the dominant lethal and the sperm shape abnormality test. At higher doses, endosulfan induced dominant lethal mutations in one mating interval (36-42 days) post treatment. A statistically significant dose-dependent increase in sperm abnormalities was observed with endosulfan treatment. At high doses the sperm count decreased up to 39%. No change in sperm motility was observed at any dose level. On the basis of the present in vivo results, it appears that endosulfan has a damaging effect on spermatogonial cells as well as on sperm morphology.
Endosulfan, an organochlorine insecticide, is extensively used for the control of pests in India. It has several biological effects on animals and human beings. Extensive studies have been made on the biophysical and biochemical aspects of endosulfan toxicity (Dikshith et al., 1984). However, very limited studies on its mutagenic effect have been reported. Its genotoxic effect on somatic cells demonstrated the induction of chromosome aberrations in human lymphocytes (Yoder et al., 1973), and micronuclei in mouse bone marrow (Usha Rani et al., 1980). Velazquez et al. (1984) reported it to be mutagenic in germ cells of
in germ cells of male mice. The dominant lethal test has been employed to assess the genetic damage induced in animals exposed to the mutagenic agent (Green et al., 1985). It measures the frequency of embryonic deaths due to induced chromosomal abnormalities in the germ cells of the treated animals. The sperm abnormality test also has the potential to identify chemicals that induced spermatogenic dysfunction and perhaps heritable mutations (Wyrobek et al., 1983). Most of the agents that induce sperm abnormalities are mutagenic in other germ-cell assays (Wyrobek and Bruce, 1975; Heddle and Bruce, 1977).
Drosophila melanogaster.
In the present study, an attempt has been made to evaluate the genotoxic properties of endosulfan
Materials and methods Animals
Correspondence: Dr. P.K. Ray, Director, Industrial Toxicology Research Centre, Post Box No. 80, Mahatma Gandhi Marg, Lucknow-226001 (India).
Swiss albino mice, maintained at the Industrial Toxicology Research Centre, were employed in the study. All animals were given standard diet and water ad libitum.
0165-1218/90/$03.50 © 1990 ElsevierSciencePublishers B.V. (BiomedicalDivision)
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~3
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Test compound Endosulfan (Thiodan, technical purity 97.03%) was purchased from Hoechst Pharmaceuticals Ltd., Bombay and cyclophosphamide was purchased from Sigma Chemicals (U.S.A.).
Experimental protocol The dominant lethal assay was performed following the described protocol of Bateman (1984). The LDs0 of endosulfan administered i.p. to male mice was determined using Weil's method (Weil, 1952). Three groups of animals (20 animals per group) were treated with endosulfan dissolved in dimethyl sulphoxide for 5 consecutive days at dose levels of 9.8, 12.7 and 16.6 mg/kg b.w. (i.p.). The total doses administered were 49.0, 63.5 and 83.0 mg/kg b.w. After the treatment, each male was caged with a virgin female. A 7-day sequential mating procedure was used over a total of 8 mating intervals. The uterine contents were determined 11 days after the day of separation from the males. The frequency of induced dominant lethal mutations was calculated as: Dominantlethal mutations: Liveimplantsper femaleof the test group 1 Liveimplantsper femaleof the controlgroup xlO0 The caudae epididymides were excised, weighed, minced in 2 ml of physiological saline, dispersed and filtered to exclude large tissue fragments. Smears were prepared on clean slides after staining the sperms with Eosin Y (aqueous) and coded. 200 sperm per animal were examined for morphological abnormalities by light microscopy at 100 × 10 magnification with a green filter, and counts were determined using a haemocytometer. Percentage motility was assessed. The weight of each testis was recorded.
Statistical analysis The mean percentages, standard errors and deviations were calculated. The level of significance between the treated and control groups was determined with Student's t-test. Results
The dominant lethal results are summarised in Table 1. All females that had implants were clas-
sifted as fertile. The results do not indicate any adverse effect of the compound on fertility in the dose range tested except during 1 post-treatment mating interval. The post-implantation loss observed in the sixth week with the highest dose (83.0 mg/kg b.w.) differed significantly from the control value (Table 1). With the lower dose levels (49.0 and 63.5 mg/kg b.w.) no induction of dominant lethals could be observed. A dose of 83 mg/kg b.w. significantly induced dominant lethal mutations during the mating interval of 36-42 days. In accordance with the experience of Brewen et al. (1975), it was found that the evidence of broken chromosomes at the first meiotic division correlated with dominant lethality. It is expected that the broken chromosomes are eventually lost at anaphase resulting in a monosomic embryo that subsequently dies in utero. Endosulfan also induced various types of abnormalities in sperm shape. Many aberrant sperms displayed double heads, big heads, amorphous types, coiled tail etc. (Fig. 1). The percentage of different abnormalities is listed in Table 2. A statistically significant increase in sperm abnormalities was observed, the maximum being 23.0% at the highest dose level. Data in Table 3 present the effect of endosulfan on testis weight, sperm count and motility. Endosulfan exposure (108 mg/kg b.w.) caused a significant decrease in testis weight. A statistically significant dose-dependent decrease (34-39%) in sperm count was observed with higher doses. Sperm motility remained unaffected at all dose levels. Discussion
Utilising the dominant lethal test different stages in gametogenesis may be scored for induced mutation, depending upon the interval between treatment and fertilisation. If spermatogenesis is not affected by the treatment the various gametogenic stages are sampled in the mouse during the different post-treatment mating intervals: spermatozoa (1-7 days), spermatids (8-21 days), spermatocytes (22-35 days) followed by differentiating spermatogonia and gonial stem cells (Oakberg, 1975). In the present study the dominant lethality
resulting from exposure to endosulfan is significant only in the sixth week and with the highest dose level (83 m g / k g b.w.). The mating interval (sixth week) indicates that damage that can result
in dominant mutation is induced specifically in spermatogonia. A marked reduction in total implants was observed with the same dose. The results indicate that the induction of mutation is
TABLE 1 I N C I D E N C E OF D O M I N A N T LETHALITY IN MALE MICE I N D I F F E R E N T POST-TREATMENT WEEKS A F T E R 5 DAYS OF REPEATED T R E A T M E N T W I T H E N D O S U L F A N Dose (mg/kg/day)
Mating interval (days)
Females with implants
Live i m p l a n t s / pregnant female Mean _+SE
Dead i m p l a n t s / pregnant female Mean _+SE
Total i m p l a n t s / pregnant female Mean _+SE
Corpora l u t e a / pregnant female Mean _+SE
Induced dominant lethal mutation (%)
Control (DMSO)
1- 7 8-14 15-21 22-28 29-35 36-42 43-49 50-56
18 16 18 18 17 18 16 16
8.0_+1.00 7.5-+0.75 7.1-+0.30 6.1_+0.79 7.1+1.05 9.0+0.40 7.0+2.08 6.6-+0.60
1.0 0.4 0.3 0.1 0.7 0.0 0.2 0.4
+0.57 +0.29 -+0.21 _+0.16 _+0.18 +0.00 +0.25 _+0.24
9.0+0.57 8.0+0.72 7.5+0.42 6.3_+0.84 7.8_+1.18 9.0-+0.40 7.2_+2.05 7.2_+0.73
1- 7 8-14 15-21 22-28 29-35 36-42 43-49 50-56
16 17 18 18 18 18 16 16
7.3+0.66 7.0+0.0 7.5+0.0 6.7+0.46 7.0+0.0 9.3+0.66 6.5-t-0.95 7.0+1.09
0.0 0.0 0.4 0.6 0.5 0.0 0.2 0.8
+0.00 +0.00 +0.20 +0.16 +0.50 +0.0 +0.2 +0.20
7.3+0.66 7.05-0.0 8.0+0.37 7.4+0.44 7.5+0.50 9.3+0.66 6.7+0.75 7.8+__0.80
8.6+0.33 7.5+0.18 8.5+0.42 7.6+0.37 9.0+1.00 10.0___ 0.0 10.0+0.40 9.2+0.56
8.75 6.66 -5.63 -9.83 1.40 -3.33 7.14 6.06
1- 7 8-14 15-21 22-28 29-35 36-42 43-49 50-56
18 18 18 17 17 16 17 17
7.5+0.56 7.0+0.00 7.4+0.74 6.7+1.10 7.0+0.0 8.6 + 1.20 7.8+0.48 7.8+0.37
0.0 0.0 0.4 0.2 0.5 0.0 0.0 0.8
+0.00 +0.00 +0.24 +0.23 +0.5 + 0.0 +0.0 +0.22
7.5+0.56 7.0+0.00 7.8+0.86 7.0+0.91 7.5+0.50 8.6 -+ 1.20 7.8+0.48 8.6-t-0.60
9.1___0.69 7.0-1-0.00 8.6+0.81 7.2+0.85 9.0+1.00 10.9 + 1.20 9.4+0.50 9.0+0.70
6.25 6.66 4.22 9.83 1.44 4.44 11.42 -18.10
1- 7 8-14 15-21 22-28 29-35 36-42 43-49 50-56
16 17 16 16 18 16 18 18
8.3+0.61 6.8-+1.31 6.8+0.48 6.6-+0.75 6.6+0.75 2.2+1.25 7.0_+1.15 7.05:0.70
0.6 +0.40 0.2 -+0.20 0.6 -+0.30 0.8 -t-0.29 0.8 _ + 0 . 2 9 2.25-+1.25 0.0 -t- 0.00 0.4 -+0.24
8.8_+0.52 7.0-+1.41 7.4-+0.40 7.3-+0.67 7.3+0.67 4.5+1.19 7.0+1.15 7.4+0.50
9.3+0.54 9.2-+0.86 10.6+0.50 8.5_+0.73 8.5-+0.73 6.7-+0.62 8.3-+0.88 8.6_+0.67
-3.75 9.33 4.22 -7.57 7.04 75.55 * * *
1- 7 8-14 15-21
17 16 16
4.6+0.67 4.0+0.70 3.2+0.86
1.6 +0.50 3.0 +0.31 3.6 +0.72
6.2+0.48 7.0+0.63 6.8+0.66
7.2+0.58 7.8+0.58 8.4+0.24
42.63 * * 46.66 * * 54.92 * * *
9.3-+0.33 7.4-+0.92 8.3_+0.49 7.0_+0.68 9.0_+0.61 9.7_+0.85 9.0_+0.57 9.4_+0.50
Endosulfan 9.8
12.7
16.6
-
-
-
- 6.06
Cyclophosphamide 120
** P < 0 . 0 1 ;
***P
1
Elsevier MUTGEN 01567
Studies on the genotoxicity of endosulfan, an organochlorine insecticide, in mammalian germ cells Nina Pandey, Freny Gundevia, A.S. Prem and P.K. Ray Environmental Microbiology Section, Industrial Toxicology Research Centre, Mahatma Gandhi Marg, Lucknow - 226 001 (India)
(Received 3 November 1989) (Revision received13 February 1990) (Accepted 19 February 1990)
Keywords: Endosulfan; Dominant lethal mutation; Sperm abnormality; Mouse germ cells
Summary The genotoxic potential of endosulfan was assessed in mouse germ cells by 2 in vivo tests: the dominant lethal and the sperm shape abnormality test. At higher doses, endosulfan induced dominant lethal mutations in one mating interval (36-42 days) post treatment. A statistically significant dose-dependent increase in sperm abnormalities was observed with endosulfan treatment. At high doses the sperm count decreased up to 39%. No change in sperm motility was observed at any dose level. On the basis of the present in vivo results, it appears that endosulfan has a damaging effect on spermatogonial cells as well as on sperm morphology.
Endosulfan, an organochlorine insecticide, is extensively used for the control of pests in India. It has several biological effects on animals and human beings. Extensive studies have been made on the biophysical and biochemical aspects of endosulfan toxicity (Dikshith et al., 1984). However, very limited studies on its mutagenic effect have been reported. Its genotoxic effect on somatic cells demonstrated the induction of chromosome aberrations in human lymphocytes (Yoder et al., 1973), and micronuclei in mouse bone marrow (Usha Rani et al., 1980). Velazquez et al. (1984) reported it to be mutagenic in germ cells of
in germ cells of male mice. The dominant lethal test has been employed to assess the genetic damage induced in animals exposed to the mutagenic agent (Green et al., 1985). It measures the frequency of embryonic deaths due to induced chromosomal abnormalities in the germ cells of the treated animals. The sperm abnormality test also has the potential to identify chemicals that induced spermatogenic dysfunction and perhaps heritable mutations (Wyrobek et al., 1983). Most of the agents that induce sperm abnormalities are mutagenic in other germ-cell assays (Wyrobek and Bruce, 1975; Heddle and Bruce, 1977).
Drosophila melanogaster.
In the present study, an attempt has been made to evaluate the genotoxic properties of endosulfan
Materials and methods Animals
Correspondence: Dr. P.K. Ray, Director, Industrial Toxicology Research Centre, Post Box No. 80, Mahatma Gandhi Marg, Lucknow-226001 (India).
Swiss albino mice, maintained at the Industrial Toxicology Research Centre, were employed in the study. All animals were given standard diet and water ad libitum.
0165-1218/90/$03.50 © 1990 ElsevierSciencePublishers B.V. (BiomedicalDivision)
al
~3
io
,
,1 ~
Test compound Endosulfan (Thiodan, technical purity 97.03%) was purchased from Hoechst Pharmaceuticals Ltd., Bombay and cyclophosphamide was purchased from Sigma Chemicals (U.S.A.).
Experimental protocol The dominant lethal assay was performed following the described protocol of Bateman (1984). The LDs0 of endosulfan administered i.p. to male mice was determined using Weil's method (Weil, 1952). Three groups of animals (20 animals per group) were treated with endosulfan dissolved in dimethyl sulphoxide for 5 consecutive days at dose levels of 9.8, 12.7 and 16.6 mg/kg b.w. (i.p.). The total doses administered were 49.0, 63.5 and 83.0 mg/kg b.w. After the treatment, each male was caged with a virgin female. A 7-day sequential mating procedure was used over a total of 8 mating intervals. The uterine contents were determined 11 days after the day of separation from the males. The frequency of induced dominant lethal mutations was calculated as: Dominantlethal mutations: Liveimplantsper femaleof the test group 1 Liveimplantsper femaleof the controlgroup xlO0 The caudae epididymides were excised, weighed, minced in 2 ml of physiological saline, dispersed and filtered to exclude large tissue fragments. Smears were prepared on clean slides after staining the sperms with Eosin Y (aqueous) and coded. 200 sperm per animal were examined for morphological abnormalities by light microscopy at 100 × 10 magnification with a green filter, and counts were determined using a haemocytometer. Percentage motility was assessed. The weight of each testis was recorded.
Statistical analysis The mean percentages, standard errors and deviations were calculated. The level of significance between the treated and control groups was determined with Student's t-test. Results
The dominant lethal results are summarised in Table 1. All females that had implants were clas-
sifted as fertile. The results do not indicate any adverse effect of the compound on fertility in the dose range tested except during 1 post-treatment mating interval. The post-implantation loss observed in the sixth week with the highest dose (83.0 mg/kg b.w.) differed significantly from the control value (Table 1). With the lower dose levels (49.0 and 63.5 mg/kg b.w.) no induction of dominant lethals could be observed. A dose of 83 mg/kg b.w. significantly induced dominant lethal mutations during the mating interval of 36-42 days. In accordance with the experience of Brewen et al. (1975), it was found that the evidence of broken chromosomes at the first meiotic division correlated with dominant lethality. It is expected that the broken chromosomes are eventually lost at anaphase resulting in a monosomic embryo that subsequently dies in utero. Endosulfan also induced various types of abnormalities in sperm shape. Many aberrant sperms displayed double heads, big heads, amorphous types, coiled tail etc. (Fig. 1). The percentage of different abnormalities is listed in Table 2. A statistically significant increase in sperm abnormalities was observed, the maximum being 23.0% at the highest dose level. Data in Table 3 present the effect of endosulfan on testis weight, sperm count and motility. Endosulfan exposure (108 mg/kg b.w.) caused a significant decrease in testis weight. A statistically significant dose-dependent decrease (34-39%) in sperm count was observed with higher doses. Sperm motility remained unaffected at all dose levels. Discussion
Utilising the dominant lethal test different stages in gametogenesis may be scored for induced mutation, depending upon the interval between treatment and fertilisation. If spermatogenesis is not affected by the treatment the various gametogenic stages are sampled in the mouse during the different post-treatment mating intervals: spermatozoa (1-7 days), spermatids (8-21 days), spermatocytes (22-35 days) followed by differentiating spermatogonia and gonial stem cells (Oakberg, 1975). In the present study the dominant lethality
resulting from exposure to endosulfan is significant only in the sixth week and with the highest dose level (83 m g / k g b.w.). The mating interval (sixth week) indicates that damage that can result
in dominant mutation is induced specifically in spermatogonia. A marked reduction in total implants was observed with the same dose. The results indicate that the induction of mutation is
TABLE 1 I N C I D E N C E OF D O M I N A N T LETHALITY IN MALE MICE I N D I F F E R E N T POST-TREATMENT WEEKS A F T E R 5 DAYS OF REPEATED T R E A T M E N T W I T H E N D O S U L F A N Dose (mg/kg/day)
Mating interval (days)
Females with implants
Live i m p l a n t s / pregnant female Mean _+SE
Dead i m p l a n t s / pregnant female Mean _+SE
Total i m p l a n t s / pregnant female Mean _+SE
Corpora l u t e a / pregnant female Mean _+SE
Induced dominant lethal mutation (%)
Control (DMSO)
1- 7 8-14 15-21 22-28 29-35 36-42 43-49 50-56
18 16 18 18 17 18 16 16
8.0_+1.00 7.5-+0.75 7.1-+0.30 6.1_+0.79 7.1+1.05 9.0+0.40 7.0+2.08 6.6-+0.60
1.0 0.4 0.3 0.1 0.7 0.0 0.2 0.4
+0.57 +0.29 -+0.21 _+0.16 _+0.18 +0.00 +0.25 _+0.24
9.0+0.57 8.0+0.72 7.5+0.42 6.3_+0.84 7.8_+1.18 9.0-+0.40 7.2_+2.05 7.2_+0.73
1- 7 8-14 15-21 22-28 29-35 36-42 43-49 50-56
16 17 18 18 18 18 16 16
7.3+0.66 7.0+0.0 7.5+0.0 6.7+0.46 7.0+0.0 9.3+0.66 6.5-t-0.95 7.0+1.09
0.0 0.0 0.4 0.6 0.5 0.0 0.2 0.8
+0.00 +0.00 +0.20 +0.16 +0.50 +0.0 +0.2 +0.20
7.3+0.66 7.05-0.0 8.0+0.37 7.4+0.44 7.5+0.50 9.3+0.66 6.7+0.75 7.8+__0.80
8.6+0.33 7.5+0.18 8.5+0.42 7.6+0.37 9.0+1.00 10.0___ 0.0 10.0+0.40 9.2+0.56
8.75 6.66 -5.63 -9.83 1.40 -3.33 7.14 6.06
1- 7 8-14 15-21 22-28 29-35 36-42 43-49 50-56
18 18 18 17 17 16 17 17
7.5+0.56 7.0+0.00 7.4+0.74 6.7+1.10 7.0+0.0 8.6 + 1.20 7.8+0.48 7.8+0.37
0.0 0.0 0.4 0.2 0.5 0.0 0.0 0.8
+0.00 +0.00 +0.24 +0.23 +0.5 + 0.0 +0.0 +0.22
7.5+0.56 7.0+0.00 7.8+0.86 7.0+0.91 7.5+0.50 8.6 -+ 1.20 7.8+0.48 8.6-t-0.60
9.1___0.69 7.0-1-0.00 8.6+0.81 7.2+0.85 9.0+1.00 10.9 + 1.20 9.4+0.50 9.0+0.70
6.25 6.66 4.22 9.83 1.44 4.44 11.42 -18.10
1- 7 8-14 15-21 22-28 29-35 36-42 43-49 50-56
16 17 16 16 18 16 18 18
8.3+0.61 6.8-+1.31 6.8+0.48 6.6-+0.75 6.6+0.75 2.2+1.25 7.0_+1.15 7.05:0.70
0.6 +0.40 0.2 -+0.20 0.6 -+0.30 0.8 -t-0.29 0.8 _ + 0 . 2 9 2.25-+1.25 0.0 -t- 0.00 0.4 -+0.24
8.8_+0.52 7.0-+1.41 7.4-+0.40 7.3-+0.67 7.3+0.67 4.5+1.19 7.0+1.15 7.4+0.50
9.3+0.54 9.2-+0.86 10.6+0.50 8.5_+0.73 8.5-+0.73 6.7-+0.62 8.3-+0.88 8.6_+0.67
-3.75 9.33 4.22 -7.57 7.04 75.55 * * *
1- 7 8-14 15-21
17 16 16
4.6+0.67 4.0+0.70 3.2+0.86
1.6 +0.50 3.0 +0.31 3.6 +0.72
6.2+0.48 7.0+0.63 6.8+0.66
7.2+0.58 7.8+0.58 8.4+0.24
42.63 * * 46.66 * * 54.92 * * *
9.3-+0.33 7.4-+0.92 8.3_+0.49 7.0_+0.68 9.0_+0.61 9.7_+0.85 9.0_+0.57 9.4_+0.50
Endosulfan 9.8
12.7
16.6
-
-
-
- 6.06
Cyclophosphamide 120
** P < 0 . 0 1 ;
***P
