(The) effects of methotrexate upon the exocrine glands of pancreas in rats
Methotrexate, one of the folic acid antagonists, is widely used for chemotherapy of a variety of malignancy. However, clinical use of this is frequently limited due to its toxic effects on the hematopoietic and gastrointestinal systems.
The mechanism of anticancerous activity of folic acid antagonists is based on inhibition of mitoses by interference of synthesis specifically of desoxyribonucleic acid (DNA) and ribonucleic acid (RNA). This inhibitory effect of cellular proliferation is non-specific. It involves not only cancer cells, but also rapidly growing normal cells.
The matabolism of protein and the exocrine function of the pancreas are interrelated. Any significant changes in either one involves significant changes in the other. Pancreatic acinar cells, which serve the specific functions of synthesis, storage, and secretion of large amounts of protein in the form of digestive enzymes, seem to be particularly susceptible to damage by compounds affecting protein synthesis and secretion. Single amino acid deficiency and protein deprivation have also been shown to cause ultrastructural changes in the pancreatic acinar cells (Weisblum et al., 1962; Scott, 1966). Today we know that the pancreas assumes a key position in malnutrition but we still know very little about the relevant details of its action.
No experimental studies have been done as yet, concerning on the effects of repeated administration of Methotrexate on the pancreatic exocrine glands and the pancreatic enzyme system. Therefore, to study and characterize the histochemical and ultrastructural alterations in the exocrine glands of the pancreas and to compare the alterations in pancreatic enzymes, such as trypsin, lipase and amylase seems appropriate and important. concurrently, histochemical changes are precisely
studied at regular intervals, 1 day, 1 week and 2 weeks, after withdrawal of the Methotrexate treatment.
Materials and Methods
Healthy albino rats weighing around 200 grams were used. A total of 58 survived rats were subjected to the experiment. The animals were divided into 4 major groups and treated as follows.
Group Ⅰ : Normal untreated controls (18 rats)
Group Ⅱ : Methotrexate administration for 1 course (18 rats)
A. 1 day after cessation
B. 1 week after cessation
C. 2 weeks after cessation
Group Ⅲ : Methotrexate administration for 2 courses (30 rats)
A. 1 day after cessation
B. 1 week after cessation
C. 2 weeks after cessation
Group Ⅳ : Electron microscopy (12 rats)
The basic diet contained over 15% protein and 3% fat. Methotrexate was given intraperitoneally in a dosage of 0.3mg. per kg. of body weight daily for 5 consecutive days for the first course. A. second course of Methotrexate in a same dosage was given intraperitoneally beginning 2 weeks after the end of the first course.
During the experimental period, body weight was measured weekly. Surviving animals were sacrificed at the end of the experimental period. At autopsy, all the organs, particularly the pancreas and liver were examined grossly. Histochemical
examination of pancreas and liver, and ultrastructural examination of exocrine glands of pancreas were made.
Immediately after the autopay a part of the pancreas was removed for determination of the pancreatic enzymes. Preparation of the tissues was done by the method of Grossman (1943) and they were measured; trypsin by the methods of Anson (1938), lipase by Cherryand Crandall (1932), and amylase by Nelson (1944). For determination of erum amylase, at autopsy fresh blood was removed from the femoral artery and it was measured by the method of Nelson (1944).
For the light microscopic examination tissues were fixed in 10% formalin, and cut in 6 micron thickness after paraffin embedding, and stained with routine hematoxylin-eosin, Feulgen for desoxyribonucleic acid (DNA), methyl green pyronin for ribonucleic acid (RNA), chrome alum hematoxylin phroxine for zymogen granules, and periodic acid Schiff (PAS) for glycogen.
For electron microscopic examination of the changes in acute stage (1 hour, 6 hours and 18 hours), small pieces of pancreatic tissue were fixed in 1% osmium tetraoxide in Veronal buffer at pH of 7.4. The tissues were dehydrated at 4℃ for 2 hours in a graded series of ethanol solutions and embedded in Epon 812 and 815. Ultrathin sections were cut with glass knife in 400 to 500 Å and were stained with uranyl acetate and with lead hydroxide. Sections were examinetd in the Hitachii Ⅱ E electron microscope.
Results and Summary
The body weight in untreated normal controls gradually increased, but remained stationary in the experimental groups.
Pancreatic lipase and especially trypsin decreased one day after cessation in Group Ⅱ, and showed a simultaneous increment one week after cessation in Group Ⅲ. The serum amylase and also pancreatic amylase were not significantly altered in one course of Methotrexate (Group Ⅱ), but did concomitantly increased significantly two weeks after cessation in Group Ⅲ. The decrease of these enzymes appeared first in lipase and then trypsin, and serum and pancreatic amylase were unchanged. It appeared that the enzyme activity seems to be returned normal before complete recovery of the altered morphology.
The light microscopic examination of the exocrine glands of pancreas included routine check of hematoxylin eosin stain, and study of histochemical changes using special stains, such as feulgen for DNA, methyl green pyronin for RNA, chrome alum
hematoxylin phloxine for zymogen granules, and peridic acid Schiff's reaction for glycogen.
The tissue changes in the acinar cells of pancreas in hematoxylin eosin stain showed dissociation of acinar cells, degeneration, atrophy, vacuolation, and focal necrosis. These changes were more marked in subgroup A of Group Ⅱ than those of
subgroup A of Group Ⅲ. Subgroup B and C of Group Ⅱ and Group Ⅲ showed milder changes than those of subgroups A, but patchy scattered focal changes were still noted.
The distribution of DNA was generally slight in alterations which appeared slighly decreased in subgroups A of Group Ⅱ and Group Ⅲ. The distribution of RNA was abundant in normal controls particularly around the nucleus. It was greatly decreased in subgroups A of Group Ⅱ and Group Ⅲ, but only mildly decreased in Group Ⅲ. Subgroups B and C of Group Ⅱ and Group Ⅲ gradually returned to normal. Zymogen granules were mainly located in the apical area of the acinar cytoplasm in normal controls (Ⅰ), but were greatly decreased in subgroups A of Group Ⅱ and Group Ⅲ, and mildly decreased in Group Ⅲ. The reduction in zymogen granules gradually returned to normal in subgroups B and C of Group Ⅲ.
Histopathologic changes of the liver included cloudy swelling, fatty metamorphosis, focal atrophy or necrosis, and extramedullary erythropoiesis. PAS positive materials were disappeared particularly around the central veins.
Ultrastructural changes of the acinar cells of the pancreas (Group Ⅳ) were those of progressive irregular dilatation of endoplasmic reticulum, increased number of free ribosomes in the cytoplasmic matrix, dilated Golgi complex surrounded by small
smooth vesicles and containing small vesicles and distended large vacuoles, and immature zymogen granules. These changes were gradually altered by the time lapsed. Zymogen granules were located at the mid portion of the acinar cytoplasm early, but later mostly located at apical area of the acinar cytoplasm.
In summary, the administration of Methotrexate induced a variety of morphological changes in the exocrine glands of the pancreas, such as dissociation of acinar cells, atrophy of cytoplasm, reduction in the amount of zymogen granules and RNA,
which were most marked in the Groups of one day after cessation and there were minimal focal changes or almost complete recovery in the Groups of 2 weeks after cessation. The changes in Group Ⅲ were milder than those in Group Ⅱ, which seemed to be due to refractory tolerance after the repeated administration of
Methotrexate. Alterations in pancreatic enzymes were generally parallel to the morphological changes, but appeared to be more rapidly recovered than the morphological alterations. Ultrastructural changes included dilatation of endoplasmic reticulum, increased free ribosomes, dilated Golgi complex, immature zymogen granules and decreased number of prozymogen and zymogen granules.