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Effect of β-mercaptoethylamine on reproduction of the rat
TOXICOLOGY
Effect
AND
APPLIED
PHAHMACOLOGY
11,
523-528
of P-Mercaptoethylamine R. P.
RELILES
(1967)
on Reproduction W. J. SCOTT,
AND
of the
Rat
JR.
Woodard Research Corporation, Herndon, Virginia 22070 Receiced
August
28,
1967
p-Mercaptoethylamine (MEA) or cysteamine was first reported by Bacq et al. in 1951 as an effective protective agent against ionizing radiation. Since then it has been widely studied and often used as a reference standard protective agent. The pharmacology and toxicology of MEA have been reviewed by Bacq ( 1965). Although its protective action on fetuses of irradiated pregnant mammals has been well documented (Rugh and Clugston, 1956; Rugh, 1957; Woollam and Millen, 1958; Rugh and Grupp, 1960; Ershoff et al., 1962), the data related to the teratologic effects of MEA are meager. Reyss-Brion (1962) reported that when given alone MEA reduced the percent survival and increased the frequency of malformed embryos, although it did protect the chick embryo against the teratogenic effects of radiation as shown by a reduction in severity of malformations when compared to irradiated controls. We, have studied the effects of MEA on the reproductive capacity of the rat. METHOD
Male and female Charles River CD weanling rats1 acclimated to laboratory conditions for 1 week, were housed in temperature-controlled quarters and allowed free access to food’ and water. The rats were randomly placed into five groups as shown in Table 1. MEA, expressed in terms of free base was incorporated into the diet in appropriate concentrations to provide the desired dosage levels and fed to the rats the entire duration of the study. The male rats were weighed at 2-week intervals. The females were similarly weighed, except during gestation and lactation. Weighing was discontinued during these periods in order to minimize the occurrence of cannibalism or neglect of litters. After 70 days of drug administration the rats were mated. The females were allowed to deliver naturally. At birth of each litter the number of live and stillborn were counted and their condition and weights were noted. Immediately after birth of the pups, litters from 10 producing females of group IV (375 mg/kg, females only) were placed with 10 producing females of group I (control); and the Iitters from those females of group I (control) 1 The Charles River Breeding Massachusetts. ’ Purina Laboratory Chow meal,
Laboratories, Ralston
Inc.,
Purina 523
351
Company,
Ballardvale St. Louis,
Street, Missouri.
Wilmington,
524
n.
Group
1'.
BELILES
*4m
Number
I
III IV V
J.
scam-.
JR.
Dosage level (mg/kg/dag)
per group
40 50 40 40 PO 20 20 20 20 20
II
w.
males females males females males females males females males females
0 (control) IJ (control) 375 375 375 0 0 375 75 75
were placed with the 10 females of group IV (375 mg/kg, females only) until weaning. Survivors at weaning were carefully examined at gross necropsy. After weaning of the first litters, the rats were again mated and the identical procedure was followed, except that females were paired with different males. Gonads from rats in group I (control) and group II (375 mg/kg, both sexes) were preserved in 10% formalin. After sectioning they were stained with hematoxylin and eosin and then examined histopathologically. RESULTS
Mortality of treated parent rats was limited to one low level female (75 mg/kg) and thus is of no significance. Mean body weights at week 10 are shown in Table 2. Body weight gain TABLE THE
EFFECT
OF @-MERCAPTOETHYLAMINE
Dose Groups Untreated males (groups I and IV) Treated males (groups II and III) Treated males (group Untreated (groups Treated (groups Treated (grow
2
ON MEAN BY DOS\GE
(mg/k/day)
BODY
WEIGHT
Number animals
of
OF RATS
Mean body weight (g)
-
60
436
375
60
370
20
419
-
70
251
375
60
217
75
00
217
7.5
VI females I and III) females II and IV) females VI
COMBINED
MEA
REPRODUCTIVE
525
EFFECTS
for control and male rats fed 75 mg/kg/day of MEA was comparable throughThe rats treated with 375 mg/kg/day MEA revealed out the investigation. a significant reduction in body weight gain throughout the study. Findings at the time of birth are summarized in Table 3. Statistical analysis (chi-square) of the pregnancy ratios indicated a significant decrease in the number of litters produced at the first mating in all groups treated with 375
mg/kg/day. Results of the second mating revealed a significant reduction ber of litters produced in group II (375 mg/kg, both sexes).
TABLE EFFECT
OF TREATMENTS
WITH
in the num-
3
,%MERCAPTOETHYLAMINE
ON VARIOUS
PARAMETERY
MEASURED
AT PARTURITION
No. litters _____ Mating
Mean
No. hred
number live pups per litter
of
Number of dead pups
Number of litter mean birth wt. < 5 g
18
4
33
1
Control
First Second
48/50
First Second
30/40
9.2
15/40
8.2
First Second
15/20
12.0
14
6
17/20
9.7
16
“z
First Second
16/20
3.1
14/20
9.4
10.3 9.9
40/50
MEA
ME.4
N EA
MBA First Second
19/20 16/20
(375
(375
(575
mglkg-both
mg/kg~males
mglkg-females
(75 mg/kg-both 9.6 10.8
sexes) 10 62
1 2
only)
only) 2 1
1 4
seres) 11 3
0 0
Histopathologic examination of the gonads from group I (control) and group II (375 mg/kg, both sexes) was performed in an attempt to postulate a mechanism for the reduced number of litters. No difference was evident between tissues from control and treated animals. The number of pups per litter in each group was compared with the appropriate control data, and in first litters of groups II (375 mg/kg, both sexes) and IV (375 mg/kg, females only), a significant reduction was evident. Results from the second mating indicated a decrease in the number of pups per litter in group II rats (375 mg/kg, both sexes). Examination of the fetuses at birth indicated that all were within normal limits except those indicated below:
526
H.
I’.
Dosage level (mg/kg/dag)
BELILES
AND
1%‘. J. S(:OTl-,
Number affected
Control
Sutnher examined
1 4
375, both sexes 375, males only
JR.
Type of nbnormalitj
891
Brnchygnathia Arthrogr~posis
39x 334
I
Arthragryposis
The frequency of occurrence does not appear to be significantly different in treated and control groups. Data obtained at the time of weaning are summarized in Table 4. These data indicate that treatment of the lactating female with 375 mg/kg/day of MEA increases the mortality and reduces growth in surviving pups while nursing regardless of whether or not the pups were from treated females or were fostered pups from control females. Conversely, when pups from treated females were fostered to control females, weanling survival was unaffected. TABLE ON THE Prws
EFFECT
4
OF TREATMENT WITH @-MERCAPTOETHYLAMINE DURING LACTATION Number
group
L).im
of litters
mean Treatment
TO THE
weaning
Percent
< 30 g
weight
alive
pups
at weaning
('O?h-01
Group I pups First and second
litters
7/w
90.4
Group IV pups First and second
litters
2p0
90.4
First
and second
litters
First
and second
litters
MEA
(575
mglkg-both
se.wry)
“L9/30 MEA
(875
mglkg-,rnules
52.3 only)
1 /so XE.4
(375
mg/kg-females
93.4 only)
Group I pups First and second
litters
14/13
46.1
GTOU~ IV pups First and second
litters
7/s
50.7
First
litters
MEA and second
(75 my/kg-both 7125
sews) 89.8
DISCUSSION
Bacq (1965) reported growth inhibition and death after 10-30 days in young rats fed cysteamine at 0.35-0.45% in the diet. In our study the highlevel rats received 0.46% cysteamine free base, in the diet. Reduction of body weight gain was evident throughout all treated groups excepting males re-
MEA
REPRODUCTIVE
,527
EFFECTS
ceiving 7.5 mg/kg/day (0.09% in the diet), but no drug mortality was observed. Cysteamine has reportedly produced lesions identical to those of lathyrism due to aminopropionitrile (the active principle of sweet pea) (Dasler, 1955). In this study no gross evidence of lathyrism was observed in the parent rats. The additional reduction in the number of litters produced by group II females (375 mg/kg/day, both sexes) at the second litter is thought to be related to the continued administration of MEA. Of notable interest is the necessity of feeding both sexes MEA to produce this effect in respect to the first litters. The mechanism by which MEA produces this effect remains to be explained. Reduction in the number of pups per litter in groups II and IV at the first mating and Group II at the second mating is most likely attributable to death of the conceptus followed by resorption sometime between fertilization and parturition. This effect may possibly be due to a direct drug effect or an indirect effect resulting from lowered food intake reflected by decreased body weight gain. A reduction in growth and an increase in mortality of the pups nursing dams treated with 375 mg/kg/day indicates either a detrimental effect on lactation or a decreased palatability of the milk. MEA should be concentrated in the milk due to its pK, (8.35) and the difference of pH be.tween blood (7.4) and milk (6.6). SUMMARY The effects of p-mercaptoethylamine administration on reproduction in two successive matings of rats were studied. MEA reduced the body weight gain of parent rats when administered at a level of 375 mg/kg/day. MEA reduced the number of first litters produced in all groups receiving 375 mgl’kg/day. The number of litters produced when both sexes were fed 375 mg/kg/day was reduced at the second mating. The number of first litter pups was reduced in those litters in which both parents or the dam alone was fed MEA at a level of 375 mg/kg/day. However, at the second mating a reduction in the nmnber of pups per litter was induced only when both parents were fed 375 mg/kg/day of MEA. The frequency of occurrence of congenital malformations was not significant in litters from parents treated with MEA. The growth was reduced and the mortality increased in litters which suckled dams treated with 375 mg/kg of MEA regardless of whether the pups during fetal life developed in a treated or control dam. ACKNOWLEDGMENT This General. WRAIR,
study was supported by contract number DA-49-193%MD-2458, The @-mercaptoethylamine was furnished by the Division WRAMC, Washington, D.C.
Office of r\ledicinal
of the Surgeon Chemistry of
REFERENCES BACQ, Z. M. (1965). “Chemical Protection against Ionizing Radiation” (I. N. Kugelmass, p. 60. Thomas, Springfield, Illinois. BACQ, Z. M., HERVE, A., LECONTE, J., FISHER, P., BLAVIEH, J., &cHAhiPs, G., LEBIHAN, and RAYET, P. (1951). Protection contre le rayonnement S par la @-mercaptoethylamine. Arch. Intern. Physiol. 59, 442. DASLEH, W. (1955). Production of experimental lathyrism in the rat by two different substitrrted ethylamines. Proc. Sm. Exptl. Biol. Med. 88, 196-199.
ed.), H.,
beta-
528
R.
I’.
BELILES
AND
W.
J. SCOTT,
JR.
ERSHOFF, B. H., STEERS, C. IV., JR., and KRUGER, L. (1962). Elfects of rndioprot~ctivt~ agtmt on foot deformities and gait defects in prenatally x-irradiated rat. Proc. Sot. L.rrrll. Riol. Med. 111, 391-394. REYSS-BRION, M. ( 1962). Protection par la cyst&amine de jeunes embryons de porrlct soumis ultkrieurement a une irradiation aux rayons x. Arch. Anat. Pistol. Emhryol. Xorm. Exptl. 44, Suppl., 197. RUGH, R. (1957). Relative value of cysteamine and cystamine as radioprotectivc agents for fetal and adult mice. Am. 1. Physiol. 189, 31-35. RUGH, R., and CLUGSTON, H. (1956). Protection of mouse fetus against x-irradiation death. Science 123, 28-29. RUGH, R., and GRUPP, E. (1960). Protection of the embryo against the congenital and lethal effects of x-irradiation. I. Atompwzxis 6, 143-148. WOOLLAM, D. H. M., and MILLEN, J. W. (1958). The influence of cysteamine on the teratogenic action of x-irradiation. Nature 182, 1801.
Effect
AND
APPLIED
PHAHMACOLOGY
11,
523-528
of P-Mercaptoethylamine R. P.
RELILES
(1967)
on Reproduction W. J. SCOTT,
AND
of the
Rat
JR.
Woodard Research Corporation, Herndon, Virginia 22070 Receiced
August
28,
1967
p-Mercaptoethylamine (MEA) or cysteamine was first reported by Bacq et al. in 1951 as an effective protective agent against ionizing radiation. Since then it has been widely studied and often used as a reference standard protective agent. The pharmacology and toxicology of MEA have been reviewed by Bacq ( 1965). Although its protective action on fetuses of irradiated pregnant mammals has been well documented (Rugh and Clugston, 1956; Rugh, 1957; Woollam and Millen, 1958; Rugh and Grupp, 1960; Ershoff et al., 1962), the data related to the teratologic effects of MEA are meager. Reyss-Brion (1962) reported that when given alone MEA reduced the percent survival and increased the frequency of malformed embryos, although it did protect the chick embryo against the teratogenic effects of radiation as shown by a reduction in severity of malformations when compared to irradiated controls. We, have studied the effects of MEA on the reproductive capacity of the rat. METHOD
Male and female Charles River CD weanling rats1 acclimated to laboratory conditions for 1 week, were housed in temperature-controlled quarters and allowed free access to food’ and water. The rats were randomly placed into five groups as shown in Table 1. MEA, expressed in terms of free base was incorporated into the diet in appropriate concentrations to provide the desired dosage levels and fed to the rats the entire duration of the study. The male rats were weighed at 2-week intervals. The females were similarly weighed, except during gestation and lactation. Weighing was discontinued during these periods in order to minimize the occurrence of cannibalism or neglect of litters. After 70 days of drug administration the rats were mated. The females were allowed to deliver naturally. At birth of each litter the number of live and stillborn were counted and their condition and weights were noted. Immediately after birth of the pups, litters from 10 producing females of group IV (375 mg/kg, females only) were placed with 10 producing females of group I (control); and the Iitters from those females of group I (control) 1 The Charles River Breeding Massachusetts. ’ Purina Laboratory Chow meal,
Laboratories, Ralston
Inc.,
Purina 523
351
Company,
Ballardvale St. Louis,
Street, Missouri.
Wilmington,
524
n.
Group
1'.
BELILES
*4m
Number
I
III IV V
J.
scam-.
JR.
Dosage level (mg/kg/dag)
per group
40 50 40 40 PO 20 20 20 20 20
II
w.
males females males females males females males females males females
0 (control) IJ (control) 375 375 375 0 0 375 75 75
were placed with the 10 females of group IV (375 mg/kg, females only) until weaning. Survivors at weaning were carefully examined at gross necropsy. After weaning of the first litters, the rats were again mated and the identical procedure was followed, except that females were paired with different males. Gonads from rats in group I (control) and group II (375 mg/kg, both sexes) were preserved in 10% formalin. After sectioning they were stained with hematoxylin and eosin and then examined histopathologically. RESULTS
Mortality of treated parent rats was limited to one low level female (75 mg/kg) and thus is of no significance. Mean body weights at week 10 are shown in Table 2. Body weight gain TABLE THE
EFFECT
OF @-MERCAPTOETHYLAMINE
Dose Groups Untreated males (groups I and IV) Treated males (groups II and III) Treated males (group Untreated (groups Treated (groups Treated (grow
2
ON MEAN BY DOS\GE
(mg/k/day)
BODY
WEIGHT
Number animals
of
OF RATS
Mean body weight (g)
-
60
436
375
60
370
20
419
-
70
251
375
60
217
75
00
217
7.5
VI females I and III) females II and IV) females VI
COMBINED
MEA
REPRODUCTIVE
525
EFFECTS
for control and male rats fed 75 mg/kg/day of MEA was comparable throughThe rats treated with 375 mg/kg/day MEA revealed out the investigation. a significant reduction in body weight gain throughout the study. Findings at the time of birth are summarized in Table 3. Statistical analysis (chi-square) of the pregnancy ratios indicated a significant decrease in the number of litters produced at the first mating in all groups treated with 375
mg/kg/day. Results of the second mating revealed a significant reduction ber of litters produced in group II (375 mg/kg, both sexes).
TABLE EFFECT
OF TREATMENTS
WITH
in the num-
3
,%MERCAPTOETHYLAMINE
ON VARIOUS
PARAMETERY
MEASURED
AT PARTURITION
No. litters _____ Mating
Mean
No. hred
number live pups per litter
of
Number of dead pups
Number of litter mean birth wt. < 5 g
18
4
33
1
Control
First Second
48/50
First Second
30/40
9.2
15/40
8.2
First Second
15/20
12.0
14
6
17/20
9.7
16
“z
First Second
16/20
3.1
14/20
9.4
10.3 9.9
40/50
MEA
ME.4
N EA
MBA First Second
19/20 16/20
(375
(375
(575
mglkg-both
mg/kg~males
mglkg-females
(75 mg/kg-both 9.6 10.8
sexes) 10 62
1 2
only)
only) 2 1
1 4
seres) 11 3
0 0
Histopathologic examination of the gonads from group I (control) and group II (375 mg/kg, both sexes) was performed in an attempt to postulate a mechanism for the reduced number of litters. No difference was evident between tissues from control and treated animals. The number of pups per litter in each group was compared with the appropriate control data, and in first litters of groups II (375 mg/kg, both sexes) and IV (375 mg/kg, females only), a significant reduction was evident. Results from the second mating indicated a decrease in the number of pups per litter in group II rats (375 mg/kg, both sexes). Examination of the fetuses at birth indicated that all were within normal limits except those indicated below:
526
H.
I’.
Dosage level (mg/kg/dag)
BELILES
AND
1%‘. J. S(:OTl-,
Number affected
Control
Sutnher examined
1 4
375, both sexes 375, males only
JR.
Type of nbnormalitj
891
Brnchygnathia Arthrogr~posis
39x 334
I
Arthragryposis
The frequency of occurrence does not appear to be significantly different in treated and control groups. Data obtained at the time of weaning are summarized in Table 4. These data indicate that treatment of the lactating female with 375 mg/kg/day of MEA increases the mortality and reduces growth in surviving pups while nursing regardless of whether or not the pups were from treated females or were fostered pups from control females. Conversely, when pups from treated females were fostered to control females, weanling survival was unaffected. TABLE ON THE Prws
EFFECT
4
OF TREATMENT WITH @-MERCAPTOETHYLAMINE DURING LACTATION Number
group
L).im
of litters
mean Treatment
TO THE
weaning
Percent
< 30 g
weight
alive
pups
at weaning
('O?h-01
Group I pups First and second
litters
7/w
90.4
Group IV pups First and second
litters
2p0
90.4
First
and second
litters
First
and second
litters
MEA
(575
mglkg-both
se.wry)
“L9/30 MEA
(875
mglkg-,rnules
52.3 only)
1 /so XE.4
(375
mg/kg-females
93.4 only)
Group I pups First and second
litters
14/13
46.1
GTOU~ IV pups First and second
litters
7/s
50.7
First
litters
MEA and second
(75 my/kg-both 7125
sews) 89.8
DISCUSSION
Bacq (1965) reported growth inhibition and death after 10-30 days in young rats fed cysteamine at 0.35-0.45% in the diet. In our study the highlevel rats received 0.46% cysteamine free base, in the diet. Reduction of body weight gain was evident throughout all treated groups excepting males re-
MEA
REPRODUCTIVE
,527
EFFECTS
ceiving 7.5 mg/kg/day (0.09% in the diet), but no drug mortality was observed. Cysteamine has reportedly produced lesions identical to those of lathyrism due to aminopropionitrile (the active principle of sweet pea) (Dasler, 1955). In this study no gross evidence of lathyrism was observed in the parent rats. The additional reduction in the number of litters produced by group II females (375 mg/kg/day, both sexes) at the second litter is thought to be related to the continued administration of MEA. Of notable interest is the necessity of feeding both sexes MEA to produce this effect in respect to the first litters. The mechanism by which MEA produces this effect remains to be explained. Reduction in the number of pups per litter in groups II and IV at the first mating and Group II at the second mating is most likely attributable to death of the conceptus followed by resorption sometime between fertilization and parturition. This effect may possibly be due to a direct drug effect or an indirect effect resulting from lowered food intake reflected by decreased body weight gain. A reduction in growth and an increase in mortality of the pups nursing dams treated with 375 mg/kg/day indicates either a detrimental effect on lactation or a decreased palatability of the milk. MEA should be concentrated in the milk due to its pK, (8.35) and the difference of pH be.tween blood (7.4) and milk (6.6). SUMMARY The effects of p-mercaptoethylamine administration on reproduction in two successive matings of rats were studied. MEA reduced the body weight gain of parent rats when administered at a level of 375 mg/kg/day. MEA reduced the number of first litters produced in all groups receiving 375 mgl’kg/day. The number of litters produced when both sexes were fed 375 mg/kg/day was reduced at the second mating. The number of first litter pups was reduced in those litters in which both parents or the dam alone was fed MEA at a level of 375 mg/kg/day. However, at the second mating a reduction in the nmnber of pups per litter was induced only when both parents were fed 375 mg/kg/day of MEA. The frequency of occurrence of congenital malformations was not significant in litters from parents treated with MEA. The growth was reduced and the mortality increased in litters which suckled dams treated with 375 mg/kg of MEA regardless of whether the pups during fetal life developed in a treated or control dam. ACKNOWLEDGMENT This General. WRAIR,
study was supported by contract number DA-49-193%MD-2458, The @-mercaptoethylamine was furnished by the Division WRAMC, Washington, D.C.
Office of r\ledicinal
of the Surgeon Chemistry of
REFERENCES BACQ, Z. M. (1965). “Chemical Protection against Ionizing Radiation” (I. N. Kugelmass, p. 60. Thomas, Springfield, Illinois. BACQ, Z. M., HERVE, A., LECONTE, J., FISHER, P., BLAVIEH, J., &cHAhiPs, G., LEBIHAN, and RAYET, P. (1951). Protection contre le rayonnement S par la @-mercaptoethylamine. Arch. Intern. Physiol. 59, 442. DASLEH, W. (1955). Production of experimental lathyrism in the rat by two different substitrrted ethylamines. Proc. Sm. Exptl. Biol. Med. 88, 196-199.
ed.), H.,
beta-
528
R.
I’.
BELILES
AND
W.
J. SCOTT,
JR.
ERSHOFF, B. H., STEERS, C. IV., JR., and KRUGER, L. (1962). Elfects of rndioprot~ctivt~ agtmt on foot deformities and gait defects in prenatally x-irradiated rat. Proc. Sot. L.rrrll. Riol. Med. 111, 391-394. REYSS-BRION, M. ( 1962). Protection par la cyst&amine de jeunes embryons de porrlct soumis ultkrieurement a une irradiation aux rayons x. Arch. Anat. Pistol. Emhryol. Xorm. Exptl. 44, Suppl., 197. RUGH, R. (1957). Relative value of cysteamine and cystamine as radioprotectivc agents for fetal and adult mice. Am. 1. Physiol. 189, 31-35. RUGH, R., and CLUGSTON, H. (1956). Protection of mouse fetus against x-irradiation death. Science 123, 28-29. RUGH, R., and GRUPP, E. (1960). Protection of the embryo against the congenital and lethal effects of x-irradiation. I. Atompwzxis 6, 143-148. WOOLLAM, D. H. M., and MILLEN, J. W. (1958). The influence of cysteamine on the teratogenic action of x-irradiation. Nature 182, 1801.