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KMID : 0378019670100070033
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New Medical Journal
1967 Volume.10 No. 7 p.33 ~ p.47
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Effect of Methotrexate and 5-Fluorouracil on Fetuses and Placentas of Rat
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Abstract
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Methotrexate and 5-fluorouracil are the most commonly used anticancerous drugs at present and their effectiveness is due to antagonistic action with folic acid and pyrimidine, respectively, which are essential in the synthesis of DNA and RNA through which cell reproduction is achieved (Heiderberger et aI. 1957, Rich et al. 1953, Werkheiser 1963, Bertino et al. 1954).
The ideal anticancer drug should affect selectively on neoplastic tissue without adverse effect to normal one and non-neoplastic tissue proliferation. However, methotrexate and 5-fluorouracil is non-selective metabolic inhibitor affecting any type of cells which are in the process of active proliferation (Paschkiss et al. 1959). Nevertheless, clinical experience with these drugs showed some preferential action on certain types of tumors than others (Ansfield et al. 1962, Schneiderman, 1962, Hertz. 1963).
The knowledge of the action of methotrexate and 5-fluorouracil on neoplastic and non-neoplastic tissue is being accumulated rapidly. Yet it is still far from complete. The present study is aimed to investigate the effect of methotrexate and 5-fluorouracil on different types of tissue,. including placenta, using fetuses in which all tissue elements are rapidly proliferating.
Materials and Methods
Seventy female albino rats, weighing from 200 to 255 gms, were used in the experiment. They were kept for 3 weeks and proved to be not pregnant. Then, animals were divided into 7 groups and treated as follows.
Group I : normal pregnancy control
Group 11 : Methotrexate from the 10 th day of pregnancy
Group lil : Methotrexate from the 14 th day of pregnancy
Group 1V : Methotrexate from the 17 th day of pregnancy
Group V : 5-fluorouracil from the 10 th day of pregnancy
Group VI : 5-fluorouracil from the 14 th day of pregnancy
Group VII: 5-fluorouracil from the 17 th day of pregnancy
Artificial mating was achieved by the housing with a :vale rat at pre-estrus cycle. Copulation was confirmed by the presence of spermatozoa on vaginal smear, one day after the mating and then it was regarded as the first day of pregnancy. Methotrexate (Lederle Co. product) was given orally in a dose of 0.3 mg per kg of the body weight once a day for five consecutive days starting from the Io th, 14 th and 17 th day of pregnancy in respective groups. And the 5-fluorouracil (Roche Co. product) were given by intraperitoneal iniection in a dose of 20 mg per kg of body weight once a day for 5 consecutive days starting from the 10 th, 14 th and 17 th day of pregnancy.
All animals were killed at the 21 st day of pregnancy which is the normal delivery day proved by previous experiment (Chung 1966). Then, entire uterus was removed to examine grossly on gravida segments to count absorbed and degenerating gravida segments as well as normal ones.
After the external examination the uterus was opened and the fetuses and placentas were separated each other and from the uterus. Fetuses were classified into four groups; 1) normal alive, 2) dead, 3) deformed, and 4) absorbed. Normal alived and dead fetuses were weighed. The placenta was measured in three dimensions; length, width, and thickness, to compare the size.
For the miscroscopical examinations; fetuses were bisected sagitally through median line and one half was blocked. The placentas were sectioned and both were blocked. A piece of the liver and bone marrow of the maternal rats were also blocked to check the effect of drugs on maternal tissue. All tissue were fixed in 4% neutral formalin and embeded in paraffin. IN7icrosections were made in 5 to 6 te. thickness and stained routinely with hemaxoylin and eosin.
Results and Discussion
A. Macroscopic Observations
The numbers of conceptus ranged from 7 to 10 per rat with average of 8.30.37 in normal pregnancy control group, and all of the fetuses were alive without deformity. Treatment with methotrexate or 5-fluorouracil brought a large numbers of early fetal death followed by absorption, some deformed and dead fetuses, depending on the time of administration of the drugs during the pregnancy course. When the drug was given on the 10 th day of pregnancy
the majority of the fetuses were dead and absorbed, while if it is given on the 14 th day deformed 3 fetuses as wcli as absorbed and dead fetuses were observed, and when the drugs were given from the 17th day, fetuses grew relatively normal but some found dead.
The deformity observed was rather minor sort and principally limited to the tail and extremities. The weight of normal and dead fetuses also showed considerable variations among the experimental groups. The average weight of fetus in normal pregnancy control group was 4.0{). 10 gm, while it was less in the groups treated with methotrexate or 5-fluoruracil, and the earlier was the date of drug given the fetal weight decreased more.
The size of the placenta was also markedly influenced by the administration of the methotrexate or 5-fluorouracil. The placenta in the animals treated with drugs showed smaller size, and the smallest -as observed in the group treated at the earlier date of pregnancy.
These changes on fetus and placenta were qualitatively similar between the groups treated either with methotrexate or 5-fluorouracil and some of quantitative difference was probably due to the dose of each drug rather than different mechanisms Mvovled.
B. Microscopic Observation.
Fetuses in the animals treated with methotrexate or 5-fluorouracil from the 10 th day of pregnancy were mostly necrotized and absorbed, but a few fetuses survived in this groups, showed no notable alterations. However, fetuses from the 14 th day of pregnancy showed pre-mature o_ immature histologic pictures of internal organs as well as the elements derived from ectoderm. But the fetuses from the animals treated with either drugs from the 17 th day of pregnancy showed ncTmal development and maturation of all tissue elements, and in occasional cases early degenerative changes of the liver and the mucosa of gastro-intestinal tract was noted.
The placenta showed marked degenerative changes with necrosis of yolk sac and labyrinth
{ in the groups treated with methotrexate or 5-fluorouracil-from the 10th day of pregnancy. These changes appeared to be largely secondary to the fetal death in addition to toxic effects of the drugs. The placentas of the animals treated with drugs from the 14 th and 17 th day of
I pregnancy showed much less degree of degenerative changes, and the changes were marked congestion in the labyrinth with fibrin deposition and early degenerative changes. The trophoblasts in the junctional zone were most resistant to both drugs, and the decidua was more
n
vulnerable to the 5-fluorouracil.
Besides the fetus and placenta, the liver and bone marrow from the maternal rat were also examined histologically. The liver from the animals treated with methotrexate showed enlarged and hyperchromatic nuclei and mild degree of extramedullary hematopoiesis when the drug was given on the 10th day, while in the animals treated after the 14th day showed cloudy swelling and mild fatty degeneration in the liver cells_ The liver of the animals treated with 5-fluorouracil showed more marked degree of hepatic changes than that of methotrexate treated animals. The liver from the animals treated with 5-fluoouracil from the 10 th day showed moderate degree of cloudy and fatty degeneration with band like atrophy of hepatic cells at periportal areas, but no extramedullary hematopoiesis was noted. When the drug was given after the 14th day of pregnancy, the liver showed increased degree of cloudy swelling and fatty changes, including multiple patchy parenchymal necrosis in one of the animals treated from -the 17 th day..
Degeneration of the bone marrow was more marked in the groups treated with 5-fluorouracil than methetrexate, and the most marked degenerative change was observed in the anima´s treated with 5-fluorouracil from the 34th day of pregnancy.
In summary, the results obtained by present experiment showed that the effect of methotrexate and 5-fluorouracil on rapidly growing fetal tissues and placenta is not selective to any special tissue element or organ but general one as evidenced by retarded weight gain, pre-maturity and immaturity without major deform ity..And if any selectivity is present, it is dependent on the rate of cell multiplication as evidenced by the fact that when the dreg was administered´ from the earlier date of pregnancy the, mire death and deformed fetuse-s were observed.
Some of the quantitative difference between the effect of methotrexate and 5-fluoreuracil is likely due to the dose effect rather than any specific mechanism is involved.
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KEYWORD
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