Placental transfer of tritium-labelled prostaglandin A1 in the rat

Exp. Path. Bd. 10, S. 353-358 (1975) Teratology R~search Vtboratory, Department of Anatomy, University of Manitoba, Winnipeg, Canada

Placental transfer of tritium-Iabelled prostagIandin Al in the rat By T. V. N.

PERSAUD

and C. W.

JACKSON

With 4 figures (Received February 5, 1975) Key-words: pregnancy: metabolie etfect of prostagiandin Al; placental transfer; fetal development: 3H-Iabelling; kidney; lung; liver; gut; amniotic fluid; rat

Summary Pregnant Long Evans rat3 ware treated with 25,uCi 3H-prostaglandin Al on either gestational day 15, 18 or 21. The highest concentrations of radioactivity were found in maternal kidneys, lungs and liver. The lenis oi radioactivity in the gut and also in amniotic fluid markedly increased with time after isotope administration. Significant levels of radioactivity were present in fetal tissues and in the placenta.

The prostaglandins repre3ent a unique group of naturally occurring substances which have been used for the induction of labor, termination oI pregnancy and a3 antifertility agents. However, there is little information coneerning their metabolie effeets during pregnancy (PERSAUD 1974). An understanding of how the prostaglandins are rnetabolized and of their effeets on maternal and fetal rnetabolisrn is irnportant not only for deterrnining the safety of these substances in pregnant wornen, but also for the developrnent of new cornpounds with dt'sirable therapeutie propertie3 (WHO Chronicle 1973). The effeets of the long-acting prostagIandin Al on pregnaney and fetal developrnent in the rat were reported in a previous cornrnunication (JACK30N, PER3AUD and MOORE 1974). A further investigation was undertaken to deterrnine the placental transfer and tissue distribution of this substance.

Material and J.l1 ethods Tcitium·labliled prost'1gLvldin Al in eth'1nol: wlt~r (70: 30) solution, (New England Nuclear, specific activity: 110 Ci/mM), was diluted in sterile physiological saline and administered intravenously via the tail vein to pregnant Long-Evans rats (Mean weight 325 ± 15 grams) on various days of gestation (see table 1 for treatment schedule). Each animal received 25.0,uCi (0.076 mg PGA I /O.50 ml S~ vent) of the labelIed prostaglandin. The radiochemical purity of the substance, determined by thin layer chromatography using an ethyl acetate: methanol: water (80 : 20 : 50 V/V/V) solvent system, was found to be greater than 98%. Table 1. Day of gestation and interval of killing anima!s after injection with 3H-PGAI Gestational Day

Intervals after injection 5min 30min

15 18 21

* *

* *

60min

24 hrs

*

*

*

* Indicates anima I was killed at this time interval after injection of the labelIed prostaglandin. The animals were ki;led at various time periods with an overdose of ether after injection of the ahelled prostaglandin. Selected maternal and fetal tissues, whole embryos, and placentas were 24

Exp. Path. Bd. 10, H. 5/6

353

excised and frozen for subsequent determination of radioactivity levels. The tissues were thawed, weighed (10-20 mg; three sampIes of each tissue), dissolved in 0,6 ml NCS, and neutralized with 40 I 9 N acetic acid. 10 ml of a scintillation cocktail (5 g PPO; 0,5 g POPOP; 250 ml ethylene glycol monom ethyl ether; 750 ml toluene) were added and the sampIes counted three times for one minute in a Beckman LS 150 Liquid Scintillation Counter. The results were expressed as dpmjmg of wet tissue weight.

Results and discussion

The maternal and fetal tissue distribution of 3H-PGA1 andjor its metabolites in rats at days 15, 18 and 21 of gestation is shown in tables 2, 3 and 4. In the day 21 rats, the highest concentration of radioactivity in maternal tissues (descending order) was found in the kidney, liver and lung. The spleen, gut, uterus, heart, ovary and brain contained relatively little radioactivity 5 minutes after isotope administration. At 30 minutes the levels of radioactivity in the liver, kidney, lung, ovary, spleen and heart were significantly reduced; the concentrations in the uterus and brain showed uo marked change, but the gut level of radioactivity was significantly increased. 2400 W

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Fig. 1a. Distribution of 3H-PGA1 in matemal organs ~liver, kidney, Jung, gut, brain, uterus and heart) of day 21 pregnant rats. Fig. 1 b. Distribution of 3H-PGA1 in matemal organs (placenta, ovary and spleen) of day 21 pregnant rats.

354

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Time in Minutes

Time (Minutesl

Fig.2. Distribution of 3H-PGAI in the rat fetus on day 21 of gestation.

At 60 minutes after administration of the isotope significant decreases in radioactivity were found in the liver, kidney, lung, gut, and uterus while the ovary, spleen, heart and brain remained relatively constant. A notable level of radioactivity was found in the placenta at 5 minutes and significant decreases occurred from 5 to 30, and 30 to 60 mi nut es (figs. 1 a and 1 b). Low levels of radioactivity (descending order) were found in fetal liver, lung, kidney and brain (fig. 2). At 30 minutes the levels significantly increased in fetal liver, kidney and gut, but decreased in the lungs; no change was detected in fetal brain. At 60 minutes the level of radioactivity was significantly increased in fetal lung, but decreased in fetal liver. Fetal brain, gut and kidney showed no significant changes. On gestational day 15, 30 minutes after administration of the isotope, the highest concentration of radioactivity in maternal tissues (descending order) was found in the gut, liver, kidney and spleen (table 2). The ovary, lung, heart, brain and uterus contained relatively little radioactivity. Notable levels of radioactivity were found in the placenta, whole fetus, amniotic fluid, and in the remains of one conceptus that was partially resorbed. Table 2. Distribution of 3H-prostaglandin Al in day 15 pregnant rat Tissues Maternal Liver Kidney Lung Gut Ovay SpIeren Heart Uterus Brain PIaeenta Whole Fetus Amniotic Fluid Resorption

Time (minutes) after isotope administration: 30 mins 347 ± 344 ± 140 ± 1,620 ± 145 ± 221 ± 134 ± 56.7± 67.1 ± 71.7 ± 24.0 ± 21,300 ± 47.0 ±

57*) 25 55 230 36 7 9 4.2 10.0 11.6 2.1 6,800**) 2.3

*) Mean dpm per minute/mg wet tissue ± standard error.

**) l\fean dpm per minute/mI ± standard error. 24*

355

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Fig.3a. Distribution of 3H·PGA1 in matern al organs (lung, liver, kidney, ovary, uterus and heart) of day 18 pregnant rats. Fig.3b. Distribution of 3H·PGA1 in maternaiorgans (spleen, gut and brain) of day 18 pregnant rats.

At day 18 of gestation, the highest concentration of radioactivity in maternal tisssue (descending order) was found in the kidney, liver and lung. The gut, ovary, uterus, heart, spleen and brain (five minutes after administration of the isotope) contained relatively little radioactivity. At 60 minutes, the levels in liver, kidney, lung and ovary were significantly decreased. The gut, spleen, heart and uterus showed no marked changes, but the brain concentration of radioactivity was significantly increased (figs. 3 a and 3 b). At 24 hours, al! maternal tissues studied showed a significant decrease in the concentration of radioactivity, except for the spleen which showed no marked difference. Radioactivity was detected in the placenta five minutes after maternal administration. This was significantly increased at 60 minutes, but was reduced at 24 hours (fig. 4). Low levels of radioactivity (descending order) were found in fetal liver and in fetal brain. At 60 minutes, the levels of radioactivity in all fetal l.issues were significantly increased; no marked changes were detected at 24 houro (fig. 4). A notable cuncentration of radioactivity was found in the amniotic fluid at 5 minutes which was significantly increased at 24 hours (table 3). The maternal tissue distribution of tritium-Iabelled prostagiandin Al and/or its metabolites showed initial high levels in the liver, kidney and lung. Relatively low levels of

356

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Fig.4. Distribution of H-PGAI in the rat fetus on day 18 of gestation. Table 3. Distribution of 3H-prostaglandin Al in day 18 pregnant rats Tissues Maternal Liver Kidney Lung Gut Ovary Spleen Heart Uterus Brain Placenta Fetal Whole Fetus Liver Brain Amniotic Fluid

Time after isotope administration 5 min 60 min p***)

24 hrs

659 ± 70 207 ± 8 1,422 ± 64 252 ± 13 191 ± 13 71.8 ± 8.0 89.0 ± 1.6 89.7 ± 7.5 58.2 ± 3.8 42.4 ± 2.5 50.8 ± 11.7 71.7 ± 9.7 51.6 ± 5.5 51.6 ± 8.5 57.0 ± 7.2 77.5 ± 18.6 26.3 ± 0.7 37.9 ± 3.1 37.2 ± 6.0 52.1 ± 5.9

40.3 ± 35.3 ± 33.1 ± 27.1 ± 22.0 ± 67.5 ± 34.9 ± 26.4 ± 22.9 ± 29.6 ±

15.5 ± 0.8 27.3 ± 4.9 12.4 ± 0.5 8,520 820**)

24.4 ± 2.7 42.1 ± 5.9 19.7 ± 3.1

< 0.001 < 0.001 < 0.001 NS

< 0.005 NS NS NS < 0.005 < 0.05

< 0.01 < 0.05 < 0.02

20.2 ± 33.3 ± 24.2 ± 58,600 ±

p****) 0.8 2.2 5.0 2.1 2.7 11.0 1.0 0.7 2.7 2.2 0.7 3.5 3.0 2,100

< < < < <

NS

0.001 0.001 0.005 0.001 0.001

< 0.05 < 0.01

< 0.005 < 0.005 NS NS NS

*) Mean dpm per minute/mg wet tissue ± standard error. **) Mean dpm per minute/mI ± standard error. ***) Significance of difference between 5 and 60 minutes was determined by STUDENT'S "t"-test. NS = not significant ****) Significance of difference between 60 minutes and 24 hrs was determined by STUDENT'S "t"-test.

radioactivity were found in all other maternal tissues studied with the lowest level in the brain. All tissues studied showed significant decreases at 60 minutes and 24 hours following administration of the labelIed prostaglandin. In the brain, however, the concentration of radioactivity remained relatively constant at all time periods studied. The level of radioactivity in the gut increased from 5 to 30 minutes, then decreased by 60 minutes and 24 hours. This suggests that one route of rapid excretion of prostagIandin Al and/or its metabolites may be via the biliary tract. Placental concentrations of radioactivity were comparable to maternal uterus, spleen and heart levels. The levels of radioactivity followed a similar relationship of decreasing concentration with time.

357

The significant levels of radioactivity detected in fetal tissues within five minutes of administration of the labelIed prostagIandin indicate that the intaet moleeule readily passes to the fetus. Further, thc finding of either relatively constant or increasing levels of radioactivity in fetal tissues at subsequent time intervals suggests that the fetus may not be in a position to metabolize prostaglandins as readily as maternal tissues (see also KIRTON and FORBES 1971). This suggestion is supported by the finding of marked increases in the concentration of radioactivity in thc amniotic fluid. It would therefore appear from these findings that the prostagIandin may act directIy on the developing coneeptus and so produee its effeets. Table 4. Distribution oE 3H-pr03t:tglitndin Al in day 21 pregnant rats Tissues Maternal Liver Kidney Lung Gut Ovary Spleen Heart Uterus Brain Placenta Fetal Liver Kidney Lung Gut Brain

Time (minntes) after isotope administration p**) 5 30 1,690 ± 2,010 ± 336 ± 139 ± 113 ± 145 ± 12fi ± 128 ± 36.4 ± 146 ±

110*) 230 23 19 10 5 5 29 0.6 16

39.5 ± 20.8 ± 30.0 ± 33.3 ± 19.6 ±

3.0 2.4 2.6 2.7 2.9

53fi ± 474 ± 185 + 776± 50.8 ± 62.6 ± GO.8 ± 93.1 ± 38.8± 91.6 ±

28 2 10 36 2.4 1.1 1.6 18.6 3.1 6.3

56.2 ± 26.8 ± 24.3 ± 41.1 ± 21.0 ±

1.3 1.3 1.4 3.6 2.3

< 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001

NS NS

< 0.005

< < < <

NS

0.001 0.02 0.05 0.05

p***)

60 319 ± 187 ± 80.3 ± 84.1 ± 43.4± 74.7 ± 60.9 ± 45.9 ± 39.1 ± 80.2 ±

28 13 4.G 0.0 6.6 11.1 3.3 3.2 3.2 4.5

34.6 ± 29.4 ± 39.1 ± 44.7 ± 23.0±

2.6 1.5 7.0 4.4 2.3

< < < <

0.001 0.001 0.001 0.001

NS NS NS < 0.02 NS < 0.05

< 0.001

~S

< 0.01

NS NS

*) Mean dpm per minute/mg wet tissue ± standard error **) Significance of difference between 5 and 30 minutes was determined by STUDENT'S "t"-test. NS = not significant. ***) Significance of difference between 30 and 60 minutes was determined by STUDENT'S "t"-test

Acknowledgements This investigation was supported by a grant from the Medical Research Council of Canada and by Ciba-Geigy Canada Ltd. We wish to thank Miss MARLENE WrEBE and Mr. GrLBERT SAM for valuable technical assistance and Miss ROBERTA BrEDRoN for typing the manuscript.

Literature JACKSON, C. W., T. V. N. PERSAUD and K. L. MOORE, ProstagIandin Al effects on pregnancy and fetal development in rats following intra-uterine administration. Ar.at. Hec. 178, 516 (1974). KIRTON, K. T., and A. A. FORBEs, Abortificient efficiency of prostagIandin F z'" administered intraamniotically to Rhesus monkeys. Contraception 4,31-35 (1971). PERSAUD, T. V. N., Embryonie and fetal development. In: RAMWELL, P. W., The Prostaglandins. Volume II. Plenum Press, New York 1974. WHO Chronicle 27, 356-362 (1973). Author's address: T. V. N. PERSAUD, M. D., Ph. D., D. Sc., M. R. C. Path., Teratolcgy REsearch Laboratory, Department of Anatomy, University of Manitoba, Bannatyne & Emily, Winnipeg, Manitoba, Canada R 3E OW3.

358