Teratogenicity of Cytochalasin D in the Mouse 1 THOMAS H. SHEPARD AND J E A N C. GREENAWAY Central Laboratory for Human Embryology, Department of Pediatrics, School of Medicine, University of Washington,Seattle 98195
ABSTRACT Cytochalasin D, a mold metabolite identified in food, was injected intraperitoneally in doses of 0.4 to 0.9 mg per kilogram on gestational days 7 through 11 and produced evidence of teratogenicity in two out of three strains of mice. Exencephaly, hypognathia and axial skeletal defects were found in strains C57BLI6J and BALB/c while no increase in defects was observed in the Swiss Webster strain. In all three strains, a significantly increased resorption rate was found. Oral doses of approximately 7.0 mg per kg on days 7 through 11 in the BALB/c produced exencephaly in the offspring.Autoclaved cytochalasin D retained its teratogenic potential. Cytochalasins are a class of metabolites produced by mold species (Aldridge et al., '67; Aldridge and Turner, '69) identified as contaminants of potatoes (Scott et al., '75), tomatoes (Pribela, '75), pecans (Wells, '76), rice Glinsukon e t al., '73) and millet (Patwardhan e t al., '74). The reports of their effects on cells (Carter, '72; Wessels e t al., '71), especially with contractility, led us to speculate upon their possible teratogenic effect on neurulation which is in part produced by intrinsic cell forces (Schroeder, '70). Inhibition of neural tube closure by cytochalasin B has been demonstrated in explanted chick embryos (Linville and Shepard, '72; Karfunkel, '72). As a n extension of the teratogenicity studies in the chick explant system, we have found that cytochalasin D is teratogenic at concentrations one-hundredth of the teratogenic dose for cytochalasin B (Greenaway et al., '77). In this communication, teratogenicity test results of cytochalasin D in the pregnant mouse are presented along with a discussion of their possible significance as an environmental agent causally related to the human defects anencephaly and meningomyelocele. METHODS
The three strains of mice used were BALB/c (Flow Laboratories, Dublin, Virginia); C57BL/6J (Jackson Laboratories, Bar Harbor, Maine); and Swiss Webster (Simonson Laboratories, Gilroy, California). The females were caged overnight with males and if plugs were detected the next morning, that morning was TERATOLOGY, 16:
131-136.
considered the start of day 0 of gestation. The mice were fed Purina Lab Chow and water ad lib. The cytochalasin D was obtained from Aldrich Chemical Company. When its purity was checked by running in two separate silica gel G thin layer chromatographic systems, a single migrating spot was observed. The solvent systems were chloroform-acetone (9:1, v/v) and toluene-ethylacetate-88%formic acid (5:4:1, v/v) (Scott et al., '75). For the heat inactivation studies the cytochalasin was autoclaved at 12OOC for 15 minutes. The LD-50 for each strain was studied after a single intraperitoneal dose given in the morning to animals on a 10-hour night dark cycle. Six female animals were used a t each of three effective levels. The calculations were made by the method of Litchfield and Wilcoxon ('49). Single daily intraperitoneal injections of cytochalasin D in 0.05 ml of dimethyl sulfoxide (DMSO) were made on days 7-11 of gestation. The dose in mg/kg/day ranged from 0.61.0 for BALB/c, 0.8-1.0 for C57BL/6J and 0.30.76 for the Swiss Webster dams. Controls received 0.05 ml of DMSO intraperitoneally on the same gestational days. Two additional groups of BALB/c dams were treated, one with autoclaved intraperitoneal doses and the other with oral doses. The autoclaved cytochalasin was injected daily on days 7-11 of Received Nov. 19, '76. Accepted June 6, '77. I Supported by the National Institute of Child Health and Human Development HD08554. Presented in part to the Teratology Society on June 23, 1976, Camel. California. Dr. Shepad is an affiliateof the Child Development and Mental Retardation Center
131
132
THOMAS H. SHEPARD AND JEAN C. GREENAWAY
pregnancy. A single daily gavage feeding of 250 p g of cytochalasin D in 0.5 ml of corn oil was given via passage of a paraplast-tipped polyethylene tube through a small plastic tube on which the animal was biting. These treatments were given on days 7-11 of gestation. The dose ranged from 7.1-7.6 mg per kg which was approximately ten times greater than the intraperitoneal dose. The dams were sacrificed and autopsy was performed on day 17 or 18. The sites, resorptions and corpora lutea of pregnancy were counted and the fetuses were examined, weighed and crown-rump length was measured. After fixation, approximately one-half of the fetuses were razor-sectioned by the method of Wilson ('65); the other half were cleared in potassium hydroxide and stained with Alizarin Red S (Dawson, '26). In order to study early genesis of the malformations, some embryos were examined on days 9
through 12 after intraperitoneal treatment of the mothers from day 7. These embryos were removed from the uterus under Hanks solution and examined with a dissecting microscope. Statistical analyses (chi-square with correction for continuity and Student's t-test) were done by the method of Edwards ('55). RESULTS
The LD-50 from a single intraperitoneal dose was very similar among the strains studied, being 2.2 mg/kg for the BALB/c and the C57BL/6J and 2.1 mg/kg for the Swiss Webster (table 1).A t daily doses of 1 mg/kg the Swiss Webster animals died on the second or third day necessitating a reduction in the administered dose in subsequently treated animals of this strain. In the other two strains, there was no maternal mortality a t doses near 1mg per kg per day.
TABLE 1
Acute toxicity of cytochalasin D in three strains of mice Sex
Average weight
Route
Vehicle
No.of animals
LD-50' (mg/kg)
Swiss Webster
F
31.5
ip
DMSO
24
BALB/c
F
21.5
ip
DMSO
18
C57BL16J
F
29.1
ip
DMSO
18
2.1 (2.44-1.81) 2.2 (2.55-1.9 2.2 (2.55-1.9)
Strain
Slopefunction '
1.24 1.23 1.23
' Calculated by method of Litchfield and Wilcoxon ('49); values in parentheses are 95% confidence limits TABLE 2
Cytochalasin D teratogenicity studies in three strains of mice Strain
Dose mgikge SD
Route
Litters
Surviving fetuses
%
resorption8
No. of fetuses malformed
Litters with malformed
Type of defects
0.82*0.15 DMSO only Autoclaved 0.7450.02
fP' 'P
12 6
70
60 4
12 3 0
6 EX, 7 SR
55
ip
9
96
21
14
Gavaged Gavaged
5 5
44 57
10 9
13 0
C57BLhJ
7.4k0.07 Corn oil 0.5 ml 0.9450.09
8 EX, 1OMPH, 1SR with CP and 4 SR 13 EX
iP
10
37
37'
12
DMSO only 0.4450.16 DMSO only
iP
Swiss Webster
7 10 5
38 104 62
8 24 2
2 2 0
BALB/c
!P 1P
2 EX, 5 SR, 3 Hgn, 1CP + Anoph, RA 2 Anoph partial 1SR, 1Polydacty
EX. exencephaly; SR. skeletal reduction; Hgn, hypognathia; CP, cleft palate; Polydact. Polydactyly; RA. renal anomaly; Omph, omphalocele; Anoph, anophthalmia. ' Intraperitoneal. P
ABSTRACT Cytochalasin D, a mold metabolite identified in food, was injected intraperitoneally in doses of 0.4 to 0.9 mg per kilogram on gestational days 7 through 11 and produced evidence of teratogenicity in two out of three strains of mice. Exencephaly, hypognathia and axial skeletal defects were found in strains C57BLI6J and BALB/c while no increase in defects was observed in the Swiss Webster strain. In all three strains, a significantly increased resorption rate was found. Oral doses of approximately 7.0 mg per kg on days 7 through 11 in the BALB/c produced exencephaly in the offspring.Autoclaved cytochalasin D retained its teratogenic potential. Cytochalasins are a class of metabolites produced by mold species (Aldridge et al., '67; Aldridge and Turner, '69) identified as contaminants of potatoes (Scott et al., '75), tomatoes (Pribela, '75), pecans (Wells, '76), rice Glinsukon e t al., '73) and millet (Patwardhan e t al., '74). The reports of their effects on cells (Carter, '72; Wessels e t al., '71), especially with contractility, led us to speculate upon their possible teratogenic effect on neurulation which is in part produced by intrinsic cell forces (Schroeder, '70). Inhibition of neural tube closure by cytochalasin B has been demonstrated in explanted chick embryos (Linville and Shepard, '72; Karfunkel, '72). As a n extension of the teratogenicity studies in the chick explant system, we have found that cytochalasin D is teratogenic at concentrations one-hundredth of the teratogenic dose for cytochalasin B (Greenaway et al., '77). In this communication, teratogenicity test results of cytochalasin D in the pregnant mouse are presented along with a discussion of their possible significance as an environmental agent causally related to the human defects anencephaly and meningomyelocele. METHODS
The three strains of mice used were BALB/c (Flow Laboratories, Dublin, Virginia); C57BL/6J (Jackson Laboratories, Bar Harbor, Maine); and Swiss Webster (Simonson Laboratories, Gilroy, California). The females were caged overnight with males and if plugs were detected the next morning, that morning was TERATOLOGY, 16:
131-136.
considered the start of day 0 of gestation. The mice were fed Purina Lab Chow and water ad lib. The cytochalasin D was obtained from Aldrich Chemical Company. When its purity was checked by running in two separate silica gel G thin layer chromatographic systems, a single migrating spot was observed. The solvent systems were chloroform-acetone (9:1, v/v) and toluene-ethylacetate-88%formic acid (5:4:1, v/v) (Scott et al., '75). For the heat inactivation studies the cytochalasin was autoclaved at 12OOC for 15 minutes. The LD-50 for each strain was studied after a single intraperitoneal dose given in the morning to animals on a 10-hour night dark cycle. Six female animals were used a t each of three effective levels. The calculations were made by the method of Litchfield and Wilcoxon ('49). Single daily intraperitoneal injections of cytochalasin D in 0.05 ml of dimethyl sulfoxide (DMSO) were made on days 7-11 of gestation. The dose in mg/kg/day ranged from 0.61.0 for BALB/c, 0.8-1.0 for C57BL/6J and 0.30.76 for the Swiss Webster dams. Controls received 0.05 ml of DMSO intraperitoneally on the same gestational days. Two additional groups of BALB/c dams were treated, one with autoclaved intraperitoneal doses and the other with oral doses. The autoclaved cytochalasin was injected daily on days 7-11 of Received Nov. 19, '76. Accepted June 6, '77. I Supported by the National Institute of Child Health and Human Development HD08554. Presented in part to the Teratology Society on June 23, 1976, Camel. California. Dr. Shepad is an affiliateof the Child Development and Mental Retardation Center
131
132
THOMAS H. SHEPARD AND JEAN C. GREENAWAY
pregnancy. A single daily gavage feeding of 250 p g of cytochalasin D in 0.5 ml of corn oil was given via passage of a paraplast-tipped polyethylene tube through a small plastic tube on which the animal was biting. These treatments were given on days 7-11 of gestation. The dose ranged from 7.1-7.6 mg per kg which was approximately ten times greater than the intraperitoneal dose. The dams were sacrificed and autopsy was performed on day 17 or 18. The sites, resorptions and corpora lutea of pregnancy were counted and the fetuses were examined, weighed and crown-rump length was measured. After fixation, approximately one-half of the fetuses were razor-sectioned by the method of Wilson ('65); the other half were cleared in potassium hydroxide and stained with Alizarin Red S (Dawson, '26). In order to study early genesis of the malformations, some embryos were examined on days 9
through 12 after intraperitoneal treatment of the mothers from day 7. These embryos were removed from the uterus under Hanks solution and examined with a dissecting microscope. Statistical analyses (chi-square with correction for continuity and Student's t-test) were done by the method of Edwards ('55). RESULTS
The LD-50 from a single intraperitoneal dose was very similar among the strains studied, being 2.2 mg/kg for the BALB/c and the C57BL/6J and 2.1 mg/kg for the Swiss Webster (table 1).A t daily doses of 1 mg/kg the Swiss Webster animals died on the second or third day necessitating a reduction in the administered dose in subsequently treated animals of this strain. In the other two strains, there was no maternal mortality a t doses near 1mg per kg per day.
TABLE 1
Acute toxicity of cytochalasin D in three strains of mice Sex
Average weight
Route
Vehicle
No.of animals
LD-50' (mg/kg)
Swiss Webster
F
31.5
ip
DMSO
24
BALB/c
F
21.5
ip
DMSO
18
C57BL16J
F
29.1
ip
DMSO
18
2.1 (2.44-1.81) 2.2 (2.55-1.9 2.2 (2.55-1.9)
Strain
Slopefunction '
1.24 1.23 1.23
' Calculated by method of Litchfield and Wilcoxon ('49); values in parentheses are 95% confidence limits TABLE 2
Cytochalasin D teratogenicity studies in three strains of mice Strain
Dose mgikge SD
Route
Litters
Surviving fetuses
%
resorption8
No. of fetuses malformed
Litters with malformed
Type of defects
0.82*0.15 DMSO only Autoclaved 0.7450.02
fP' 'P
12 6
70
60 4
12 3 0
6 EX, 7 SR
55
ip
9
96
21
14
Gavaged Gavaged
5 5
44 57
10 9
13 0
C57BLhJ
7.4k0.07 Corn oil 0.5 ml 0.9450.09
8 EX, 1OMPH, 1SR with CP and 4 SR 13 EX
iP
10
37
37'
12
DMSO only 0.4450.16 DMSO only
iP
Swiss Webster
7 10 5
38 104 62
8 24 2
2 2 0
BALB/c
!P 1P
2 EX, 5 SR, 3 Hgn, 1CP + Anoph, RA 2 Anoph partial 1SR, 1Polydacty
EX. exencephaly; SR. skeletal reduction; Hgn, hypognathia; CP, cleft palate; Polydact. Polydactyly; RA. renal anomaly; Omph, omphalocele; Anoph, anophthalmia. ' Intraperitoneal. P
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