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KMID : 0378019710140070063
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New Medical Journal
1971 Volume.14 No. 7 p.63 ~ p.82
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The Effects of Anticancerous Agents (Methotrexate and 5-fluorouracil) on the Placenta of the Rat
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Abstract
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In recent years many studies have been made about the mechanism of action of antimetabolites, and the effects on the cancerous and non-cancerous tissues or organs. Most of antimetabolites act as in￢hibitors of nucleic acid synthesis, although the site of inhibition or block is different according to the type of antimetabolites (Heiderberger et al., 1957, Rich et al., 1958; Werkheiser 1963; Bertino.. et al., 1964).
Methotrexate and 5-fluorouracil are the most commonly used anticancerous agents in clinical practice. The Methotrexate is an antimetabolite which interferes with the participation of folinic acid in nucleic acid synthesis, and thus disrupts the mitotic process, and 5-fluorouracil is antagonistic in action with pyrimidine which is essential in the synthesis of DNA and RNA.
Methotrexate and 5-fluorouracil inhibit the growth of any rapidly growing types of nori-cancerous cells. (Heiderberger et al.,, 1956; Clarkson and Lawrence, 1961; Heiderberger and Anspfield, 1963; Lipsett, 1963; Shin, 1967; Choo, 1968).
Present investigation is designed to study the morphologic changes of the rat placenta, especially ultrastructural alteration in trophoblasts of the labyrinth in the rat placenta.
Materials and Methods
Female albino rats weighing around 200 gms were used in this experiment. They were kept from
male albino rats for 4 weeks and proved to be not pregnant. The animals were divided into groups and
treated as following.
Group I : Normal untreated pregnant groups(10 animals) a. the 12th day of pregnancy (2 animals) b. the 15th day of pregnancy (2 animals) c. the 17th day of pregnancy (2 animals) d. the 19th day of pregnancy (2 animals) e. the 21st day of pregnancy (2 animals)
Group II : Methotrexate injected group (20 animals) a. the 12th day of pregnancy (4 animals) b. the 15th day of pregnancy (4 animals) c. the 17th day of pregnancy (4 animals) d. the 19th day of pregnancy (4 animals) e. the 21st day of pregnancy (4 animals)
Group II[ : 5-fluorouracil injected group (20 animals) a. the 12th day of pregnancy (4 animals) b. the 15th day of pregnancy (4 animals) c. the 17th day of pregnancy (4 animals)
d. the 19th day of pregnancy (4 animals)
e. the 21st day of pregnancy (4 animals)
Extrus cycle was determined by the vaginal smear methods of Long and Evans(1922) and mating was achieved by housing a male and female rat together, in the pre-estrus cycle. Copulation was confirmed by the presence of spermatozoa on vaginal smear, one day after mating and this was regarded as the first day of pregnancy.
Methotrexate (Lederle Lab. Div. product) was given by intramuscular injection in a dose of 0.3mg per kg of body weight once a day for five consecutive days starting from the 7th, 10th, 12th, 14th, and 17th day of pregnancy. The 5-fluorouracil (SAF. Hoffmann-La Roche and Co. Ltd.) was given by intraperitoneal injection in a dose of 20mg per kg of body weight once a day for five consecutive days starting from the 7th, 10th, 12th, 14th and 17th day of pregnancy.
The normal untreated animals were killed at the 12th, 15th, 17th, 19th and 21st day of pregnancy. The anticarcinogen treated animals were killed also at the 12th, 15th, 19th, and 21st day of pregnancy just one day after 5 consecutive days of treatment starting from the 7th, 10th, 12th, 14th and 16th day of pregnancy in respective groups.
The entire uterus was removed and then cross sections from pregnant segments were taken and embedded in paraffin after fixation in 10% neutral formalin.
The microsections were made in 5 to 6 p thickness, and hematoxylin-eosin, periodic acid Schiff (PAS), methyl-green pyronin, and Feulgen staining were applied to each section for histologic and histochemical studies of the placenta.
For electron microscopic examinations, immediately after killing the rats, the three uteri were opened and placentas were separated from the uterine wall, and then the placental tissues were cut in 1 cu mm size and fixed in 1% osmic acid in veronal buffer of pH 7.4, followed by dehydration in graded alcohol. They were embedded in Epon 812 and cut at 400 to 500A thickness wih a glass knife After staining with uranyl acetate and lead hydroxide, observation was made with Hitachi´ 11-E model electron microscope.
Results and Summary
The placentas of the normal rats on the 12th, 15th, 17th, 19th and 21st day of pregnancy showed no abnormal developmental or histologic findings.
The placentas on the 10th day of pregnancy, treated with methotrexate from the 7th day of pre￢gnancy, showed marked necroses and degenerative changes in the decidua, and degenerative changes of yolk sac and mild inflammatory cell infiltration in the decidua. Labyrinth and junctional zones were not identified in this group.
The placentas on the 15th day, treated with methotrexate from the 10th day of pregnancy, showed moderate degenerative changes of the trophoblasts in the labyrinth and junctional zone, and focal necroses in the decidua were noted.
The placenta on the 17th day of pregnancy, treated with methotrexate from the 12th day of pregn￢ancy, showed also moderate degenerative changes of trophoblasts in the labyrinth and junctional zone, and also mild degenerative changes and mild focal necroses in the decidua.
The placentas on the 19th and 21st day of pregnancy, treated with methotrexate from the 14th and 16th day of pregnancy, showed mild degenerative changes of trophoblasts, but the maturation of placental elements was almost normal.
The placenta on the 10th day of pregnancy, treated with 5-fluorouracil from the 7th day of preg￢nancy, showed marked degenerative changes and necroses were noted in the decidua and early stages of the labyrinth and junctional zone were identified.
The placenta on the 15th day of pregnancy, treated with 5-fluorouracil from the 10th day of
pregnancy, showed moderate degenerative changes of trophoblasts in the labyrinth and junctional zone, and mild focal necroses were noted. The placenta on the 17th day of pregnancy, treated with 5-flu￢orouracil from the 12th day of pregnancy showed, moderate degenerative changes of torphoblasts in the labyrinth and junctional zone. The placenta on the 1901 and 21st day of pregnancy showed no abnormal histologic alteration and the maturation of placental elements were the same as normal control groups. -
Methyl-green pyronin and Feulgen stains revealed mild to moderately decreased amounts of pyr￢onophilic granules in the cytoplasm and Feulgen positive material in nuclei from the early stage of pregnancy treated with methotrexate and 5-fluorouracil, but only mildly decreased pyronin and Feulgen positive materials in the later stage of pregnancy, that is, those treated with anticancerous agents when the maturation of placentas were completed.
Ultrastructural examination of labyrinth trophoblast cells in the rat placentas was performed.
There are three layers of trophoblast throughout the labyrinth, an outer, middle and inner layer, separating the maternal blood spaces from fetal blood vessels and other extraembryonic connective tissue elements.
The structural changes showed enlargement of nuclei and nucleoli and mild segregation of nucleoli. These were more marked in the methotrexate treated groups than the 5-fluorouracil treated groups.
Cytoplasmic alteration showed dilatation of rough endoplasmic reticulum (RER) with detached ribosome, hyperplasia of smooth endoplasmic reticulum and swelling of mitochondria.
These changes were more marked in 5-fluorouracil treated groups than methotrexate groups. In addition, increased numbers of lipid,, multivesicular bodies and myeline bodies were seen in both groups.
In summary, the results obtained by this experiment showed that methotrexate brought more morphological changes than 5-fluorouracil in the early stage- .of development, that is, the rapidly developing stage of the placenta.
The later stage of development treated with these anticancerous agents brought mild morphological changes. This experiment indicates that these anticancerous agents are more effective in the early stage of development especially the premitotic stage.
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KEYWORD
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