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Further studies on the trichosanthin-induced termination of pregnancy
CONTRACEPTION
FURTHER STUDIES ON THE TRICHOSANTHIN-INDUCED
TERMINATION
OF PREGNANCY
I.F. Lau, Ph.D., S.K. Saksena, Ph.D. and M.C. Chang, Ph.D., Sc.D* Worcester
Foundation
Shrewsbury,
for Experimental
Massachusetts
Biology
01545
ABSTRACT Trichosanthin (TCS), a protein from the root extract of Trichosanthis kirilowii, terminated pregnancy when injected once in 15-day pregnant'm2 mg/doe) but failed to interrupt pregnancy in l2-day pregnant rabbits even at higher doses. -In vitro release of progesterone (A4P) from the maternal or fetal placental tissue into the incubation medium was not affected by TCS. When the distribution bf '25I-TCS was raced in 12-day pregnant mice, persistently higher concentration of r 251-radioactivity was detected in the kidney. By contrast, in other organs including the reproductive organs, blood serum and amniotic fluid f251-radioactivity declined between 12-48 h after treatment. The low 1251-radioactivity in both the reproductive organs and amniotic fluid suggests a possible barrier beween the embryo and maternal blood. It is suggested that TCS might be acting directly on the placental unit, causing fetal death and dislodging of the placenta. Administration of TCS to PD-19 mice or PD-28 rabbits resulted in premature delivery. This effect of TCS in pregnant mice was comparable to the administration of PGF2,.
Accepted
for publication
* Research
JANUARY
Career Awardee
December
26, 1979
(HD-18,334)
1980 VOL. 21 NO. 1
of NICHD
77
CONTRACEPTION INTRODUCTION The abortifacient effects of Trichosanthin, a protein purified from the root of Trichosanthis kirilowii, in several-mammalian species have been well documented (l-5). Althouqh a sinqle iniection of 2 ma Trichosanthin given either on 17- or 22-day of pregnancy induced abortion in 100% of the rabbits within 48-72 h, the same dose failed to terminate pregnancy in 6- or lo-day pregnant does (4). Numerous modes of action of Trichosanthin have been put forward (see 4). In women and monkey, Trichosanthin-induced abortion has been attributed to its direct action on the syncytiotrophoblasts where necrosis of placental villi and obstruction of blood vessels occurred (3,6). We postulated that Trichosanthin terminates pregnancy in rabbits and mice, not just due to an incurrence of luteolysis but also a consequence of its toxic effects on fetal or maternal placenta or both (4). However, exogenous progesterone, orolactin alone or in combination with human chorionic qonadotrooin given twice daily failed to prevent termination of pregnancy induced by Trichosanthin in the rabbit (5). To substantiate this statement and to further investigate other pharmacological effects of Trichosanthin on pregnancy, the present study aimed at a) to study the effects of 2 mg Trichosanthin on pregnancy in day 12 or 15 pregnant rabbits; b) to study the in vitro accumulation of progesterone in the incubation medium containxgx1 or maternal placenta in the presence of Trichosanthin in rabbits; c) to study the labor induction potential of Trichosanthin in rabbits and mice and d to study the distribution of 125Iodine after a single injection of '2 & I-Trichosanthin in 12-day pregnant mice. MATERIALS
AND METHODS
Animals Fourteen-day timed pregnant mice purchased from Charles River Breeding Laboratories were housed (3/cage) in quarantine until the 19th day of pregnancy. Estrous rabbits of mixed breeds were mated to fertile bucks and the day of mating was assigned as day 0 of pregnancy (PD-0). The pregnant rabbits were kept in individual cages under standard conditions. Experiment
1:
Effect of a single injection of Trichosanthin 12-day or 15-day pregnant rabbits.
(TCS) on
Twelve-day or 15-day pregnant rabbits were intraperitoneally injected with 2-3 or 2 mg of TCS, respectively. The does were observed every 8 h for vaginal bleeding and/or expulsion of uterine contents. The treated females were then sacrificed on day 18 or 19 of pregnancy and the number of corpora lutea, live and dead embryos were counted. Experiment
2:
In vitro accumulation
of progesterone
(A4P)
in the incuba-
tion medium containing rabbit fetal or maternal in the presence of TCS.
placenta
Twelve-day (day when administration of TCS is ineffective in terminating pregnancy) or 15-day (day when TCS terminates pregnancy)
78
JANUARY
1980 VOL. 21 NO. 1
CONTRACEPTION pregnant rabbits were killed by overdose of Diabutal. The fetal and maternal placentae were cleaned on precooled (4°C) glass plate, minced and weighed. Approximately 350-400 mg of the minced maternal or fetal placentae tissue was incubated in 4 or 3 ml, respectively, of KRBG (Kreb-Ringer Buffer supplemented with 1% glucose) containing O-25 ug TCS /ml for 3 h. Incubation was carried out in a Dubnoff Metabolic Shaker under an atmosphere of 02:CO2 (95:5) at 37°C. The samples were spinned at 2,500 x g in a refrigerated centrifuge and 1 ml medium was used for the estimation of A4P concentration. Experiment
3:
Induction of labor by TCS or Prostaglandin-F2,
(PGF2,).
On PD-19. preqnant mice were caqed individually. Groups of 4-12 mice were treated between 0700-0730 h with a single-intraperitoneal iniection of either 0.2 ml saline. TCS (25. 75 or 150 uoj in 0.2 ml saline or PGF2, (25, 50 or 75 pg) in 0;2 ml saline (Table III-j.The treated mice were observed every hour. Live or dead pups were removed from the cage whenever they were found. The time when the first pup(s) was found and the duration of parturition were recorded. Pregnant rabbits received intraperitoneally 2.0 ml saline or 2 mg TCS in 2.0 ml saline between 0830-0900 h on PD-28. They were then observed periodically. Live or dead fetuses were removed from the cage and the time of the appearance of the first pup(s) and the duration of parturition were recorded. Experiment
4:
Distribution
of TCS in 12-day pregnant mouse.
Trichosanthin (TCS) was labelled with 125Iodine (7). The reaction time of TCS (450 ug) with chloramine-T was 60 seconds. Purified TCS fraction (No. 4, from Sephadex G-75 column, Sp. act. 2.2 uCi/ug) was diluted with phosphate buffer saline (pH 7.4). Each 12-day pregnant mouse received intraperitoneally 0.2 ml aliquot containing 9 uCi of 125ITCS. Groups of 4-5 mice were anesthetized at 12, 24, or 48 h after '25ITCS and blood was collected from the abdominal aorta. Amniotic fluid was also collected. Aliquots of blood serum (0.1 ml) or amniotic fluid (0.1 -0.2 ml) were used for the estimation of 1251-radioactivity. Maternal placenta, fetal placenta, uterus, kidney, liver, spleen, a piece of thigh muscle (each %lOO mg) and ovary were procured after the whole animal was perfused with cool saline. The minced tissues, blood serum and AF were.then digested with 0.5 ml 1N NaOH at 60“ overnight. After neutralizinq with an equal amount of 1N HCl. 10 ml Aouasol (Packard) was added to each vial and the amount of radioactivity was determined using a liquid scintillation counter (Beckman, LS-8000) which had an efficiency of 95% for 125Iodine. Progesterone
determination:
The concentration of progesterone (A4P) in incubation medium (1.0 ml) was carried out after extracting with 10 ml anhydrous ether. Tritiated A4P (~1000 cpm) was added for the determination of procedural losses. The dried extract was dissolved in 1.0 ml of iso-octane and A4P was eluted on micro-celite columns (8). The concentration of A4P was determined by radioimmunoassay (8). Antisera to A4P was supplied by Dr. G.E. Abraham.
JANUARY
1980 VOL. 21 NO. 1
79
CONTRACEPTION
RESULTS Intraperitoneal injection of 2.0 ml phosphate buffer saline (PBS) on PD-12 and PD-15 did not affect pregnancy in the rabbit. When autopsied on PD-31 (Table I), the embryos were viable and the pups appeared to be normal. A differential response towards TCS treatment was observed when does were treated on PD-12 or PD-15. Dose as high as 3 mg /rabbit of TCS given on PD-12 was ineffective, whereas 2 mg TCS/rabbit given intraperitoneally on PD-15 induced termination of pregnancy in all treated females. Different concentrations of TCS did not interfere with the release of ASP from either the m ternal or fetal placental tissue (Table II). However, a much higher A$P concentration was recorded in placental tissue from PD-12 canpared with the one obtained from 15-day pregnant does (6-fold for fetal and P-fold for maternal placenta).
In the mouse, normal parturition started from late PD-19 to PD-21 The incidence of dead pups was while the majority gave birth on PD-21. 1.7% of the total pups born (Table III). Adminstration of TCS at 25 pg /mouse on PD-19 did not alter the length of gestation. On the other hand, TCS administered at doses of 75 or 150 pg/mouse advanced the day of parturition and all treated mice delivered between late PD-19 and A higher rate of fetal death (28.6%)was encountered in noon of PD-20. pregnant mice treated with 150 ug/mice of TCS. A dose of 25 pg/mouse resulted in a shortened gestation length accompanied by an inof PGF crease 3" proportion of stillborns (62.3 and 56.7%, respectively). It can
Table I.
Effect of a single intraperitoneal (TCS) on pregnancy in the rabbit. Number/doe
Treatmenttt
'$$ii:
Control
12 and 15
TCS
injection of Trichosanthin
at autopsy+
Percent of pregnancy termination
Corpus luteum
Live Embryo
8,10,14,7,10, 12,ll
8,9,12,7,10, 10,ll
2-3 mg
12
11,9,12,14,13, 14,ll
8,7,9,11,10, 14,7
2 mg*
15
10,8,13,14,12
O,O,O,O,O
PD = Day of pregnancy,
0 100
and day of mating was considered
as PD 0
t Control animals were examined on PD-31 while those treated with TCS were autopsied on PD-18 or PD-19, respectively. tt Controls were treated with 2.0 ml phosphate buffer saline (PBS). Trichosanthin was administered in 2 ml PBS. * All does aborted within 2-4 days while vaginal bleeding occurred within 36-48 hr after treatment.
80
JANUARY
1980 VOL. 21 NO. 1
CONTRACEPTION
be seen that both TCS and PGF2, when administered at doses sufficient to initiate premature delivery tended to prolong the duration of parturition (4.5 - 6.5 h and 4.9 - 6.0 h vs 3.0 h for the control). The normal gestation lenqth in the New Zealand mixed breed rabbits varied between 31-32 days (Table IV). However, a single intraperitoneal injection of 2 mg TCS at 0830 h of PD-28 induced premature labor within 14-23 h. All treated does delivered between PD-28 and PO-29. Seventyfive percent of the premature fetuses were dead before they were All TCS-treated rabbits had excessive bleeding and prolonged expelled. duration of labor (4.9 vs 3.5 in the control). Of all th internal organs studied, kidney showed the highest concentration of ?2% radioactivity p' ich did not decline with time until 48 h after a single injection of 251-TCS (Table V). By contrast, other or ans, blood serum and amniotic fluid showed substantial decrease in 122 I radioactivity bet een 12-48 h post-1251-TCS injection. The uterus exhibited the highest r25 I-radioactivity among the reproductive organs and the blood serum had at least lo-fold more l25I radioactivity than was found in amniotic fluid.
Table II. Effect of different concentrations of Tricgosanthin (TCS) on the -in vitro release of progesterone (A P) from the fetal and maternal placenta of the rabbit at different stages of pregnancy.
Day of Pregnancy
Mean h4P Treatment (us/ml)
Maternal
Control
12
TCS
TCS
placenta
Fetal placenta
2141 + 208
14240 f 414
1
2160 f 73
11569 + 734
5
2387 2 86
14560 -c686
25
2098 f 96
14279 + 1863
1260 + 84
2338 f 86
1
1312 f 147
2036 f 92
5
1316 f. 121
2077 f 125
25
1292 f 120
2319 f 138
Control
15
(pg/g, tissue) f SEM t
t Each value represents the mean of 4-5 determinations.
JANUARY
1980 VOL. 21 NO. 1
81
CONTRACEPTION
Table III.
Effects of trichosanthin in the mouse.
1 All treatments
82
were given
or prostaglandin
FEa on parturition
on PD 19 at 0700hour.
JANUARY 1980VOL.21NO.1
CONTRACEPTION
Table IV.
*
Induction of parturition
TRICHOSANTHIN
PBS
PHOSPHATE
WAS GIVEN BUFFER
by trichosanthin
INTRAPERITONEALLY
BETWEEN
in the rabbit.
0830-0900 HOUR
ON
PD28.
SALINE
DISCUSSION The observation that 2 or 3 mg TCS given on PO-12 did not affect pregnancy, but a dose of 2 mg TCS injected on PD-15 terminated pregnancy in all rabbits suggests that the detrimental effects of TCS on pregnancy are not obvious until the 15th day of pregnancy. These results also support our hypothesis that TCS is less effective in terminating early pregnancy (4); however, as pregnancy progresses, administration of TCS becomes more effective in the termination of pregnancy. Since does treated on PD-12 with TCS did not show any change in circulatory progesterone (A4P) while significant drop in A4P was encountered in does receiving TCS on PD-15 (unpublished data), it appears that suppression of h4P production by corpus luteum is a prerequisite for the interruption of pregnancy by TCS. It is likely that corpora lutea younger than 15-day are resistant to TCS. During midterm and later stages of pregnancy,TCS is able to interfere with normal luteal function, leading to an impaired feto-maternal relationship. Also, physical separation of fetal placenta from maternal placenta or the maternal placenta from the uterine wall seems to be more difficult during early part of pregnancy as compared with midterm or later part of pregnancy (unpublished data). This close approximation between fetal and maternal placenta might serve as a protective measure against the abortifacient action of TCS.
JANUARY
1980 VOL. 21 NO. 1
83
CONTRACEPTION
Table V.
Distribution
of Iodine-la5
in different
organs and body fluids
at various times after a single intraperitoneal I 125-Trichosanthin
injection of
in 12-day pregnant mice. t Hours after I'25 Trichosanthin
Samples
injection tt
12
24
48
19.0 f 3.0
35.0 f 6.1
14.0 f 2.7
144.0 it 16.9
117.2 f 7.8
59.5 f 3.6
Fetal placenta
44.3 f 5.0
26.7 + 0.8
14.0 * 1.2
Ovary
60.8 f 7.4
41.5 * 3.1
21.0 f 1.8
165.0 + 20.0
105.3 f 3.8
89.0 f 13.0
65.8 + 2.9
Organs
(Mean cpm/mg f SEM)
Muscle Maternal
placenta
Uterus Hammary gland Kidney
6350.0 f 901 232.3 f 36.6
Liver Body Fluids
45.8 + 2.3
8449.0 + 1430 7294.0 + 1012 232.0 _* 11.4
168.8 -+ 18.3
(Mean cpm /ml + SEM)
Blood serum Amniotic
61.3 f 10.8
64353.0 + 8275 39908.0 i 626 10493.0 -+ 17.7
fluid
t Each reading
represents
4842.0 * 615
3228.0 f 519
1038.0 4 89
the mean of at least 4 determinations.
tt cpm injected = 19.0 x 106 cpm/mouse.
Although the action of TCS was suggested to be at the placental level (l), we observed no significant alterations in the amount of ASP released from the PD-12 or PD-15 fetal or maternal tissue into the incubation medium in the presence of graded concentrations of TCS (Table II). It can be argued that the incubation period of 3 h was not enough to initiate any appreciable changes in release of a4P produced by the placenta. In another study, except for a decrease of ASP content in the fetal placenta from rabbits killed on PD-13 (24 h after 2 mg TCS),there was no significant change in A4P concentration in the blood serum or amniotic fluid or in the maternal placenta from PD-13 or PD-16 rabbits exposed 24 h earlier to 2 mg TCS. All these evidences suggest that TCS probably acts directly on the placenta.
84
JANUARY
1980 VOL. 21 NO. 1
CONTRACEPTION
In order to further explore the site of action of TCS, 125Ilabelled purified TCS was injecteQ intraperitoneally in 12-day pregnant A large proportion of the 25 I-radioactivity was recovered in the mice. blood serum while the amniotic fluid contained very little (Table V). Since TCS is a protein, it is not unlikely that there exists a selective barrier (the placenta and embryonic membranes) between the blood and amniotic fluid which preven ed a free transport of TCS. It is understandable that most of the t251-radioactivity was detected in the blood serum which serves as a carrier. However, even after extensive removal of blood by perfusion, 125 I-radioactivity could still be det cted in different organs. In fact, a differential affinity between T~~I-TCS and issues was evident. The ovary, uterus and mammary glands showed low t25 I, whereas, the kidney exhibited a high 1251-radioactivity at all times,and followed by the liver and spleen. In this context, immunofluorescent studies also revealed specific fluorescence first in trophoblastic and decidual cells in the vicinitv of the blood vessels in the placental spongy zone, extending into the-trophoblasts of labyrinth (2). The intensity of fluorescence increased with time but showed a tendency of decline 24 h later. No fluorescence was observed in fetal tissue. Our observations and those of Wang et al. (3) and Xiongsa. (6) were that TCS was not detected in fetal ns=e which could be attributed to the poor uptake o '251,TCS by the uterine and placental tissue. A persistent and high 125 I-radioactivity in the kidney indicate that it is the major site of elimination of TCS. The results of the present study also demonstrate that TCS induces premature delivery in the mouse and rabbit, a pharmacological effect similar to a well-established labor inducing agent, PGF2, (9). In both species, premature delivery was accomplished by excessive bleeding. Nevertheless, the animals appeared to be normal after delivery and the uterus was clean (without residual tissue) at autopsy. Occurrence of a high prooortion oi stillborn whether premature lab&-was induced by TCS or PGF,. (Tables III and 1V)mioht be a result of severe uterine contractionLsOL . The data presented here suggest that TCS lends itself to be a promising mid-term to late pregnancy intercepting and labor inducing Trichosanthin probably acts directly on the placenta and its agent. inhibitory effect on progesterone secretion might alter the feto-maternal placental relationship. ACKNOWLEDGEMENTS This investigation was supported by a grant (HD-03472) from NICHD and a small contract (78076) fran World Health Organization, Geneva. Trichosanthin was a gift from Dr. Y.H. Wang, Shanghai Institute of Cell Biology, PRC. Thanks are due to Ms. Lois E. Hager for typing the manuscript.
JANUARY 1980 VOL. 21 NO. 1
85
CONTRACEPTION
REFERENCES
86
1.
The Second Laboratory, Shanghai Institute of Experimental Biology: Studies of the mechanicsm of abortion induced by Trichosanthin. (In English) Scientia Sinica, 19: 811-827 (1976)
2.
Hsu, K.J., Jiang, W.S., Chen, Y.F. and Huang, M.B. Studies on the site of action of induced abortion by Trichosanthin. (In Chinese) Acta Zoologica Sinica, 22: 149-155 (1976)
3.
Wang, Y.T., Ju, R.M., Huang, J.H., and Hsu, K.C. Investigations on the injurious effects of the abortifacient Trichosanthin to monkey placental villi. (In Chinese) Acta Zoologica Sinica, 22: 156-165 (1976)
4.
Chang, M.C., Saksena, S.K., Lau, I.F., and Wang, Y.H. Induction of midterm abortion by Trichosanthin in laboratory animals. Contraception, 19: 175-184 (1979)
5.
Saksena, S-K., Chang, M.C. and Lau, I.F. Termination in rabbit and mouse by Trichosanthin. Contraception,
6.
Xiong, Y.Z., Pan, P., Xu, S.Y., Zhang, J., Wang, Y.H., Zuo, J.K., and Gu, Z. Study on the effects of Trichosanthin on human trophoblast cells in vitro. (In Chinese). Acta Zoologica Sinica, 22: 172-179 (197iq
7.
Greenwood, F.G., Hunter, W.M. and Glover, J.S. The preparation of 1311-labelled human growth hormone of high specific radioactivity. Biochem. J., 89: 114-123 (1963).
8.
Saksena, S.K., Lau, I.F., and Chang, M.C. Inhibition of the conversion of testosterone into 5a-dihydrotestosterone in the reproductive organs of the male rat . Steroids, 27: 751-757 (1976)
9.
Labhsetwar, A.P. Prostaglandins and studies related to reproduction in laboratory animals. In: Prostaglandins and Reproduction. Ed. S.M.M. Karim, University Park Press, Baltimore. pp. 241-270 (1975)
JANUARY
of pregnancy In Press.
1980 VOL. 21 NO. 1
FURTHER STUDIES ON THE TRICHOSANTHIN-INDUCED
TERMINATION
OF PREGNANCY
I.F. Lau, Ph.D., S.K. Saksena, Ph.D. and M.C. Chang, Ph.D., Sc.D* Worcester
Foundation
Shrewsbury,
for Experimental
Massachusetts
Biology
01545
ABSTRACT Trichosanthin (TCS), a protein from the root extract of Trichosanthis kirilowii, terminated pregnancy when injected once in 15-day pregnant'm2 mg/doe) but failed to interrupt pregnancy in l2-day pregnant rabbits even at higher doses. -In vitro release of progesterone (A4P) from the maternal or fetal placental tissue into the incubation medium was not affected by TCS. When the distribution bf '25I-TCS was raced in 12-day pregnant mice, persistently higher concentration of r 251-radioactivity was detected in the kidney. By contrast, in other organs including the reproductive organs, blood serum and amniotic fluid f251-radioactivity declined between 12-48 h after treatment. The low 1251-radioactivity in both the reproductive organs and amniotic fluid suggests a possible barrier beween the embryo and maternal blood. It is suggested that TCS might be acting directly on the placental unit, causing fetal death and dislodging of the placenta. Administration of TCS to PD-19 mice or PD-28 rabbits resulted in premature delivery. This effect of TCS in pregnant mice was comparable to the administration of PGF2,.
Accepted
for publication
* Research
JANUARY
Career Awardee
December
26, 1979
(HD-18,334)
1980 VOL. 21 NO. 1
of NICHD
77
CONTRACEPTION INTRODUCTION The abortifacient effects of Trichosanthin, a protein purified from the root of Trichosanthis kirilowii, in several-mammalian species have been well documented (l-5). Althouqh a sinqle iniection of 2 ma Trichosanthin given either on 17- or 22-day of pregnancy induced abortion in 100% of the rabbits within 48-72 h, the same dose failed to terminate pregnancy in 6- or lo-day pregnant does (4). Numerous modes of action of Trichosanthin have been put forward (see 4). In women and monkey, Trichosanthin-induced abortion has been attributed to its direct action on the syncytiotrophoblasts where necrosis of placental villi and obstruction of blood vessels occurred (3,6). We postulated that Trichosanthin terminates pregnancy in rabbits and mice, not just due to an incurrence of luteolysis but also a consequence of its toxic effects on fetal or maternal placenta or both (4). However, exogenous progesterone, orolactin alone or in combination with human chorionic qonadotrooin given twice daily failed to prevent termination of pregnancy induced by Trichosanthin in the rabbit (5). To substantiate this statement and to further investigate other pharmacological effects of Trichosanthin on pregnancy, the present study aimed at a) to study the effects of 2 mg Trichosanthin on pregnancy in day 12 or 15 pregnant rabbits; b) to study the in vitro accumulation of progesterone in the incubation medium containxgx1 or maternal placenta in the presence of Trichosanthin in rabbits; c) to study the labor induction potential of Trichosanthin in rabbits and mice and d to study the distribution of 125Iodine after a single injection of '2 & I-Trichosanthin in 12-day pregnant mice. MATERIALS
AND METHODS
Animals Fourteen-day timed pregnant mice purchased from Charles River Breeding Laboratories were housed (3/cage) in quarantine until the 19th day of pregnancy. Estrous rabbits of mixed breeds were mated to fertile bucks and the day of mating was assigned as day 0 of pregnancy (PD-0). The pregnant rabbits were kept in individual cages under standard conditions. Experiment
1:
Effect of a single injection of Trichosanthin 12-day or 15-day pregnant rabbits.
(TCS) on
Twelve-day or 15-day pregnant rabbits were intraperitoneally injected with 2-3 or 2 mg of TCS, respectively. The does were observed every 8 h for vaginal bleeding and/or expulsion of uterine contents. The treated females were then sacrificed on day 18 or 19 of pregnancy and the number of corpora lutea, live and dead embryos were counted. Experiment
2:
In vitro accumulation
of progesterone
(A4P)
in the incuba-
tion medium containing rabbit fetal or maternal in the presence of TCS.
placenta
Twelve-day (day when administration of TCS is ineffective in terminating pregnancy) or 15-day (day when TCS terminates pregnancy)
78
JANUARY
1980 VOL. 21 NO. 1
CONTRACEPTION pregnant rabbits were killed by overdose of Diabutal. The fetal and maternal placentae were cleaned on precooled (4°C) glass plate, minced and weighed. Approximately 350-400 mg of the minced maternal or fetal placentae tissue was incubated in 4 or 3 ml, respectively, of KRBG (Kreb-Ringer Buffer supplemented with 1% glucose) containing O-25 ug TCS /ml for 3 h. Incubation was carried out in a Dubnoff Metabolic Shaker under an atmosphere of 02:CO2 (95:5) at 37°C. The samples were spinned at 2,500 x g in a refrigerated centrifuge and 1 ml medium was used for the estimation of A4P concentration. Experiment
3:
Induction of labor by TCS or Prostaglandin-F2,
(PGF2,).
On PD-19. preqnant mice were caqed individually. Groups of 4-12 mice were treated between 0700-0730 h with a single-intraperitoneal iniection of either 0.2 ml saline. TCS (25. 75 or 150 uoj in 0.2 ml saline or PGF2, (25, 50 or 75 pg) in 0;2 ml saline (Table III-j.The treated mice were observed every hour. Live or dead pups were removed from the cage whenever they were found. The time when the first pup(s) was found and the duration of parturition were recorded. Pregnant rabbits received intraperitoneally 2.0 ml saline or 2 mg TCS in 2.0 ml saline between 0830-0900 h on PD-28. They were then observed periodically. Live or dead fetuses were removed from the cage and the time of the appearance of the first pup(s) and the duration of parturition were recorded. Experiment
4:
Distribution
of TCS in 12-day pregnant mouse.
Trichosanthin (TCS) was labelled with 125Iodine (7). The reaction time of TCS (450 ug) with chloramine-T was 60 seconds. Purified TCS fraction (No. 4, from Sephadex G-75 column, Sp. act. 2.2 uCi/ug) was diluted with phosphate buffer saline (pH 7.4). Each 12-day pregnant mouse received intraperitoneally 0.2 ml aliquot containing 9 uCi of 125ITCS. Groups of 4-5 mice were anesthetized at 12, 24, or 48 h after '25ITCS and blood was collected from the abdominal aorta. Amniotic fluid was also collected. Aliquots of blood serum (0.1 ml) or amniotic fluid (0.1 -0.2 ml) were used for the estimation of 1251-radioactivity. Maternal placenta, fetal placenta, uterus, kidney, liver, spleen, a piece of thigh muscle (each %lOO mg) and ovary were procured after the whole animal was perfused with cool saline. The minced tissues, blood serum and AF were.then digested with 0.5 ml 1N NaOH at 60“ overnight. After neutralizinq with an equal amount of 1N HCl. 10 ml Aouasol (Packard) was added to each vial and the amount of radioactivity was determined using a liquid scintillation counter (Beckman, LS-8000) which had an efficiency of 95% for 125Iodine. Progesterone
determination:
The concentration of progesterone (A4P) in incubation medium (1.0 ml) was carried out after extracting with 10 ml anhydrous ether. Tritiated A4P (~1000 cpm) was added for the determination of procedural losses. The dried extract was dissolved in 1.0 ml of iso-octane and A4P was eluted on micro-celite columns (8). The concentration of A4P was determined by radioimmunoassay (8). Antisera to A4P was supplied by Dr. G.E. Abraham.
JANUARY
1980 VOL. 21 NO. 1
79
CONTRACEPTION
RESULTS Intraperitoneal injection of 2.0 ml phosphate buffer saline (PBS) on PD-12 and PD-15 did not affect pregnancy in the rabbit. When autopsied on PD-31 (Table I), the embryos were viable and the pups appeared to be normal. A differential response towards TCS treatment was observed when does were treated on PD-12 or PD-15. Dose as high as 3 mg /rabbit of TCS given on PD-12 was ineffective, whereas 2 mg TCS/rabbit given intraperitoneally on PD-15 induced termination of pregnancy in all treated females. Different concentrations of TCS did not interfere with the release of ASP from either the m ternal or fetal placental tissue (Table II). However, a much higher A$P concentration was recorded in placental tissue from PD-12 canpared with the one obtained from 15-day pregnant does (6-fold for fetal and P-fold for maternal placenta).
In the mouse, normal parturition started from late PD-19 to PD-21 The incidence of dead pups was while the majority gave birth on PD-21. 1.7% of the total pups born (Table III). Adminstration of TCS at 25 pg /mouse on PD-19 did not alter the length of gestation. On the other hand, TCS administered at doses of 75 or 150 pg/mouse advanced the day of parturition and all treated mice delivered between late PD-19 and A higher rate of fetal death (28.6%)was encountered in noon of PD-20. pregnant mice treated with 150 ug/mice of TCS. A dose of 25 pg/mouse resulted in a shortened gestation length accompanied by an inof PGF crease 3" proportion of stillborns (62.3 and 56.7%, respectively). It can
Table I.
Effect of a single intraperitoneal (TCS) on pregnancy in the rabbit. Number/doe
Treatmenttt
'$$ii:
Control
12 and 15
TCS
injection of Trichosanthin
at autopsy+
Percent of pregnancy termination
Corpus luteum
Live Embryo
8,10,14,7,10, 12,ll
8,9,12,7,10, 10,ll
2-3 mg
12
11,9,12,14,13, 14,ll
8,7,9,11,10, 14,7
2 mg*
15
10,8,13,14,12
O,O,O,O,O
PD = Day of pregnancy,
0 100
and day of mating was considered
as PD 0
t Control animals were examined on PD-31 while those treated with TCS were autopsied on PD-18 or PD-19, respectively. tt Controls were treated with 2.0 ml phosphate buffer saline (PBS). Trichosanthin was administered in 2 ml PBS. * All does aborted within 2-4 days while vaginal bleeding occurred within 36-48 hr after treatment.
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1980 VOL. 21 NO. 1
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be seen that both TCS and PGF2, when administered at doses sufficient to initiate premature delivery tended to prolong the duration of parturition (4.5 - 6.5 h and 4.9 - 6.0 h vs 3.0 h for the control). The normal gestation lenqth in the New Zealand mixed breed rabbits varied between 31-32 days (Table IV). However, a single intraperitoneal injection of 2 mg TCS at 0830 h of PD-28 induced premature labor within 14-23 h. All treated does delivered between PD-28 and PO-29. Seventyfive percent of the premature fetuses were dead before they were All TCS-treated rabbits had excessive bleeding and prolonged expelled. duration of labor (4.9 vs 3.5 in the control). Of all th internal organs studied, kidney showed the highest concentration of ?2% radioactivity p' ich did not decline with time until 48 h after a single injection of 251-TCS (Table V). By contrast, other or ans, blood serum and amniotic fluid showed substantial decrease in 122 I radioactivity bet een 12-48 h post-1251-TCS injection. The uterus exhibited the highest r25 I-radioactivity among the reproductive organs and the blood serum had at least lo-fold more l25I radioactivity than was found in amniotic fluid.
Table II. Effect of different concentrations of Tricgosanthin (TCS) on the -in vitro release of progesterone (A P) from the fetal and maternal placenta of the rabbit at different stages of pregnancy.
Day of Pregnancy
Mean h4P Treatment (us/ml)
Maternal
Control
12
TCS
TCS
placenta
Fetal placenta
2141 + 208
14240 f 414
1
2160 f 73
11569 + 734
5
2387 2 86
14560 -c686
25
2098 f 96
14279 + 1863
1260 + 84
2338 f 86
1
1312 f 147
2036 f 92
5
1316 f. 121
2077 f 125
25
1292 f 120
2319 f 138
Control
15
(pg/g, tissue) f SEM t
t Each value represents the mean of 4-5 determinations.
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Table III.
Effects of trichosanthin in the mouse.
1 All treatments
82
were given
or prostaglandin
FEa on parturition
on PD 19 at 0700hour.
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CONTRACEPTION
Table IV.
*
Induction of parturition
TRICHOSANTHIN
PBS
PHOSPHATE
WAS GIVEN BUFFER
by trichosanthin
INTRAPERITONEALLY
BETWEEN
in the rabbit.
0830-0900 HOUR
ON
PD28.
SALINE
DISCUSSION The observation that 2 or 3 mg TCS given on PO-12 did not affect pregnancy, but a dose of 2 mg TCS injected on PD-15 terminated pregnancy in all rabbits suggests that the detrimental effects of TCS on pregnancy are not obvious until the 15th day of pregnancy. These results also support our hypothesis that TCS is less effective in terminating early pregnancy (4); however, as pregnancy progresses, administration of TCS becomes more effective in the termination of pregnancy. Since does treated on PD-12 with TCS did not show any change in circulatory progesterone (A4P) while significant drop in A4P was encountered in does receiving TCS on PD-15 (unpublished data), it appears that suppression of h4P production by corpus luteum is a prerequisite for the interruption of pregnancy by TCS. It is likely that corpora lutea younger than 15-day are resistant to TCS. During midterm and later stages of pregnancy,TCS is able to interfere with normal luteal function, leading to an impaired feto-maternal relationship. Also, physical separation of fetal placenta from maternal placenta or the maternal placenta from the uterine wall seems to be more difficult during early part of pregnancy as compared with midterm or later part of pregnancy (unpublished data). This close approximation between fetal and maternal placenta might serve as a protective measure against the abortifacient action of TCS.
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Table V.
Distribution
of Iodine-la5
in different
organs and body fluids
at various times after a single intraperitoneal I 125-Trichosanthin
injection of
in 12-day pregnant mice. t Hours after I'25 Trichosanthin
Samples
injection tt
12
24
48
19.0 f 3.0
35.0 f 6.1
14.0 f 2.7
144.0 it 16.9
117.2 f 7.8
59.5 f 3.6
Fetal placenta
44.3 f 5.0
26.7 + 0.8
14.0 * 1.2
Ovary
60.8 f 7.4
41.5 * 3.1
21.0 f 1.8
165.0 + 20.0
105.3 f 3.8
89.0 f 13.0
65.8 + 2.9
Organs
(Mean cpm/mg f SEM)
Muscle Maternal
placenta
Uterus Hammary gland Kidney
6350.0 f 901 232.3 f 36.6
Liver Body Fluids
45.8 + 2.3
8449.0 + 1430 7294.0 + 1012 232.0 _* 11.4
168.8 -+ 18.3
(Mean cpm /ml + SEM)
Blood serum Amniotic
61.3 f 10.8
64353.0 + 8275 39908.0 i 626 10493.0 -+ 17.7
fluid
t Each reading
represents
4842.0 * 615
3228.0 f 519
1038.0 4 89
the mean of at least 4 determinations.
tt cpm injected = 19.0 x 106 cpm/mouse.
Although the action of TCS was suggested to be at the placental level (l), we observed no significant alterations in the amount of ASP released from the PD-12 or PD-15 fetal or maternal tissue into the incubation medium in the presence of graded concentrations of TCS (Table II). It can be argued that the incubation period of 3 h was not enough to initiate any appreciable changes in release of a4P produced by the placenta. In another study, except for a decrease of ASP content in the fetal placenta from rabbits killed on PD-13 (24 h after 2 mg TCS),there was no significant change in A4P concentration in the blood serum or amniotic fluid or in the maternal placenta from PD-13 or PD-16 rabbits exposed 24 h earlier to 2 mg TCS. All these evidences suggest that TCS probably acts directly on the placenta.
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In order to further explore the site of action of TCS, 125Ilabelled purified TCS was injecteQ intraperitoneally in 12-day pregnant A large proportion of the 25 I-radioactivity was recovered in the mice. blood serum while the amniotic fluid contained very little (Table V). Since TCS is a protein, it is not unlikely that there exists a selective barrier (the placenta and embryonic membranes) between the blood and amniotic fluid which preven ed a free transport of TCS. It is understandable that most of the t251-radioactivity was detected in the blood serum which serves as a carrier. However, even after extensive removal of blood by perfusion, 125 I-radioactivity could still be det cted in different organs. In fact, a differential affinity between T~~I-TCS and issues was evident. The ovary, uterus and mammary glands showed low t25 I, whereas, the kidney exhibited a high 1251-radioactivity at all times,and followed by the liver and spleen. In this context, immunofluorescent studies also revealed specific fluorescence first in trophoblastic and decidual cells in the vicinitv of the blood vessels in the placental spongy zone, extending into the-trophoblasts of labyrinth (2). The intensity of fluorescence increased with time but showed a tendency of decline 24 h later. No fluorescence was observed in fetal tissue. Our observations and those of Wang et al. (3) and Xiongsa. (6) were that TCS was not detected in fetal ns=e which could be attributed to the poor uptake o '251,TCS by the uterine and placental tissue. A persistent and high 125 I-radioactivity in the kidney indicate that it is the major site of elimination of TCS. The results of the present study also demonstrate that TCS induces premature delivery in the mouse and rabbit, a pharmacological effect similar to a well-established labor inducing agent, PGF2, (9). In both species, premature delivery was accomplished by excessive bleeding. Nevertheless, the animals appeared to be normal after delivery and the uterus was clean (without residual tissue) at autopsy. Occurrence of a high prooortion oi stillborn whether premature lab&-was induced by TCS or PGF,. (Tables III and 1V)mioht be a result of severe uterine contractionLsOL . The data presented here suggest that TCS lends itself to be a promising mid-term to late pregnancy intercepting and labor inducing Trichosanthin probably acts directly on the placenta and its agent. inhibitory effect on progesterone secretion might alter the feto-maternal placental relationship. ACKNOWLEDGEMENTS This investigation was supported by a grant (HD-03472) from NICHD and a small contract (78076) fran World Health Organization, Geneva. Trichosanthin was a gift from Dr. Y.H. Wang, Shanghai Institute of Cell Biology, PRC. Thanks are due to Ms. Lois E. Hager for typing the manuscript.
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REFERENCES
86
1.
The Second Laboratory, Shanghai Institute of Experimental Biology: Studies of the mechanicsm of abortion induced by Trichosanthin. (In English) Scientia Sinica, 19: 811-827 (1976)
2.
Hsu, K.J., Jiang, W.S., Chen, Y.F. and Huang, M.B. Studies on the site of action of induced abortion by Trichosanthin. (In Chinese) Acta Zoologica Sinica, 22: 149-155 (1976)
3.
Wang, Y.T., Ju, R.M., Huang, J.H., and Hsu, K.C. Investigations on the injurious effects of the abortifacient Trichosanthin to monkey placental villi. (In Chinese) Acta Zoologica Sinica, 22: 156-165 (1976)
4.
Chang, M.C., Saksena, S.K., Lau, I.F., and Wang, Y.H. Induction of midterm abortion by Trichosanthin in laboratory animals. Contraception, 19: 175-184 (1979)
5.
Saksena, S-K., Chang, M.C. and Lau, I.F. Termination in rabbit and mouse by Trichosanthin. Contraception,
6.
Xiong, Y.Z., Pan, P., Xu, S.Y., Zhang, J., Wang, Y.H., Zuo, J.K., and Gu, Z. Study on the effects of Trichosanthin on human trophoblast cells in vitro. (In Chinese). Acta Zoologica Sinica, 22: 172-179 (197iq
7.
Greenwood, F.G., Hunter, W.M. and Glover, J.S. The preparation of 1311-labelled human growth hormone of high specific radioactivity. Biochem. J., 89: 114-123 (1963).
8.
Saksena, S.K., Lau, I.F., and Chang, M.C. Inhibition of the conversion of testosterone into 5a-dihydrotestosterone in the reproductive organs of the male rat . Steroids, 27: 751-757 (1976)
9.
Labhsetwar, A.P. Prostaglandins and studies related to reproduction in laboratory animals. In: Prostaglandins and Reproduction. Ed. S.M.M. Karim, University Park Press, Baltimore. pp. 241-270 (1975)
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of pregnancy In Press.
1980 VOL. 21 NO. 1