Br. J. exp. Path. (1977) 58, 663
INTESTINAL LESIONS INDUCED EXPERIMENTALLY BY METHOTREXATE A. BASKERVILLE AND D. BATTER-HATTON From the Microbiological Research Establishment, Porton, Salisbury, Wiltshire Received for publication July 4, 1977
Summary.-Mice were given up to 9 doses of methotrexate intermittently over a 3-week period. Inhibition of mitosis occurred in the duodenum, jejunum and ileum after the first injection, and after 2 doses the crypt epithelium showed megalocytosis and occasional abnormal mitotic figures. Further treatment produced degeneration of the epithelium of the villi, which became irregular and atrophic, and the amount of crypt tissue was greatly reduced. Focal ulceration and haemorrhage occurred in some animals. Changes in the caecum and colon developed later and were much milder. After withdrawal of methotrexate the intestinal mucosa rapidly recovered and was normal 1 week later.
IT is now well recognized that methotrexate, given orally or parenterally, can induce undesirable changes in the intestine, liver and other tissues (Taylor et al., 1950; Colsky, Greenspan and Warren, 1955; Trier, 1962; Coe and Bull, 1968). The drug is used to treat psoriasis and malignant diseases such as leukaemia, and its adverse side-effects are the result of its action as a folic acid antagonist. The present work was designed to study the development and time course of the changes in the different regions of the intestinal tract of mice after intermittent methotrexate therapy, and to examine the spontaneous recovery of the intestine after cessation of treatment. MATERIALS AND METHODS Male Porton strain specific-pathogen-free mice weighing 25-30 g were used in all the experiments. The drug preparation was the parenteral form of methotrexate sodium (Lederle, Gosport) reconstituted with sterile water for injection. The dose per day for each mouse was 1 mg in a volume of 041 ml, given by i.p. injection. In a pilot experiment to determine the tolerance of mice to this dose, a group of 60 animals received 1 mg methotrexate i.p. three times per week up to a maximum of 9 doses, a lapse of 48 or 72 h being allowed between doses. For the main sequential study mice were given
frorn 1 to 9 injections of methotrexate, and 5 animals were killed at each of these doses, 48 h after the last drug treatment. Five control mice inoculated with the same number of doses of 0.1 ml physiological saline were also killed at each stage. An additional recovery experiment was carried out in which 50 mice were given 4 doses each of 1 mg methotrexate over a period of 1 week, and 5 were then killed at 1, 2 and 5 days and 1, 2, 3 and 4 weeks after the final injection. An equal number of control animals given saline i.p. were also killed at each stage. All mice were killed with ether and necropsy was performed immediately after death. The gastrointestinal tract was removed from all animals and the following regions were fixed as separate tissues in 10% buffered neutral formalin: stomach, duodenum, jejunum, ileum, caecum, colon. From treated mice after 3, 6 and 9 injections and their controls, portions of ileum were also rapidly frozen as fresh tissue for histochemical studies. Cryostat sections of these unfixed blocks were cut at 8 ,im and stained to demonstrate acid and alkaline phosphatase activities at pH 6-5 and pH 9 0 respectively by the method of Gomori (1952), modified by Barka and Anderson (1965), using a-naphthyl phosphate as substrate. Sections were also stained to demonstrate lactate dehydrogenase activity by the polyvinyl alcohol technique of Chayen, Bitensky and Butcher (1973), and N-acetyl-flglucosaminidase by the diazo coupling method of Pugh and Walker (1961). After formalin fixation all other portions of the alimentary tract were processed by standard methods and embedded in paraffin wax. Sections cut at 5 ,um were stained by haematoxylin and
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eosin (H. & E.) and selected sections were also stained by the periodic acid-Schiff (PAS) and alcian blue (AB) methods, by Gordon and Sweet's method for reticulin, and by the Verhoeff-van Gieson technique.
RESULTS
Clinical and necropsy findings No clinical changes were observed after the first 3 injections of methotrexate, but after 4 doses many mice developed diarrhoea. Consistent necropsy findings were the presence of very fluid intestinal contents and a pale, translucent appearance of the wall of the small intestine. After 5 injections many mice were emaciated, had ruffled fur and were hunched and disinclined to move. After 6 doses a few mice showed paralysis of the hind limbs, and many had diarrhoea and appeared weak. The same pattern was evident until the termination of the experiment after the ninth injection. In the pilot mortality study 28/60 (46 6°%S) mice died. Deaths started to occur after the fourth treatment when 2 mice died, and the mortality continued to rise steadily until the ninth dose of the drug.
Histopathological findings Changes were not found in the stomach of any methotrexate-treated mice. Lesions appeared synchronously in the duodenum, jejunum and ileum after only a single dose of the drug and were of similar severity in all regions. Changes in the caecum were minimal at this stage and were absent in the colon until 4 doses had been given. One dose.-There was inhibition of mitotic activity in the crypts of Lieberkuhn and a number of crypt cells were enlarged and intensely basophilic. The epithelium of the villi was normal. Two and three doses.-The nature of the lesion was the same in all regions of the intestine. It consisted of vacuolar degeneration and loss of staining affinity of the epithelial cells of the upper half of the villi, though the central core of lamina propria was normal (Fig. 1). The positive
PAS-staining reaction of the bruish border of affected epithelial cells had been lost. Cells in the crypts of Lieberkuhn were basophilic, many were enlarged and bizarre in shape and their vesicular nuclei were distorted and contained prominent nucleoli (Fig. 1). There were fewer mitotic figures in the crypt epithelium than in control sections and some of these were abnormal, having chromosomal bridges and clumped and degenerating chromosomes. Villi were more irregular than those of control animals but Brunner's glands in the duodenum appeared normal. Changes in the caecum were milder and confined to the surface epithelium, where the cells showed early degeneration. Four and five doses. There was now marked ballooning degeneration of epithelial cells throughout the small intestine and the epithelial layer frequently separated from the underlying lamina propria (Fig. 2). Goblet cells were greatly decreased in number and villi were misshapen and atrophic (Fig. 2). In some mice the lamnina propria and submucosa were oedematous and connective tissue cells were sparse, but more commonly there was hypercellularity of these regions, the majority of the cells being histiocytes and fibroblasts (Figs 2 and 3). The population of other inflammatory cells was similar to that seen in control animals. The amount of crypt tissue had diminished (Figs 2 and 3) and some of the remaining crypts in the small intestine were greatly dilated, showed megalocytosis and bizarre cell formation, and occasional abnormal mitoses (Fig. 4). Disintegration of some crypt cell nuclei was also found at this and later stages. Peyer's patches were undiminished in size when compared with those of control animals. After the fourth dose early degenerative changes were also found in the epithelium of the colon. Six doses.-Villi were short and irregular and much of their upper portions were necrotic. In some areas there was focal ulceration of the mucosa, which resulted in haemorrhage from the submucosa into the lumen. There was even less crypt tissue
INTESTINAL LESIONS INDUCED BY METHOTREXATE
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FIG. 1. Ileum of mouse after 3 injections of methotrexate, showing degeneration of epithelium. In the crypts there are large cells with few mitoses. An abnormal mitotic figure is in the centre of the field. H. & E. x220. FIG. 2.-Ileum, 4 injections of methotrexate. Atrophy of villi and separation of necrotic tips. There is little crypt tissue and the lamina propria and submucosa are filled by histiocytes and fibroblasts. H.&E. x8O. FIG. 3. Five doses of the drug. Ileum. Villi are irregular and crypt tissue is greatly reduced. H.&E. x80.
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than earlier and many crypt cells were distended by foamy, mucoid secretion which stained positively with the AB and PAS techniques (Fig. 5). Others were large and bizarre in shape and had abnormal nuclei (Figs 6 and 7). The lamina propria and submucosa in all sites were filled by histiocytes and fibroblasts. Changes in the colon were less severe than in the small intestine and its submucosal connective tissue remained normal. Seven to nine doses.-Villi were small, irregular and necrotic along much of their length. Crypt epithelium was scanty, and some of the glandular tissue was of the bizarre basophilic type seen at earlier stages. There was little evidence of mitotic activity. The remainder of the crypt epithelium was hypertrophic and cells and glands were distended by PAS-positive AB-positive mucus. The lumina of the crypts frequently contained refractile, eosinophilic, PAS-positive, AB-positive granules. The submucosa was filled by dense accumulations of histiocytes and fibroblasts, but there was no increase in other inflammatory cells. Changes in the duodenum affected only the villi and crypts, and Brunner's glands remained morphologically normal. In the caecum and colon lesions were less severe and not very extensive. They consisted of degeneration of some areas of the surface epithelium, accompanied by the formation of PAS-positive, AB-positive, mucus-filled cysts in the crypts and accumulation of mucus in the colonic lumen. There were isolated foci of ulceration and haemorrhage, which were not present in all treated mice. Peyer's patches throughout the intestinal tract appeared unaffected by the methotrexate treatment. Other organs.-Lesions were not present in the tongue or lungs at any stage of treatment. The changes produced in the liver, brain, kidneys and testes will be reported in another communication. Histochemical findings Enzymes demonstrated in the ileum were acid and' alkaline phosphatases,
lactate dehydrogenase and n-acetyl-3glucosaminidase. After 3 doses of methotrexate there was some loss of activity of all the enzymes from the epithelium of the villi and upper crypts of the ileum when compared with the abundant staining detected in tissue from control mice. Six and 9 injections of the drug produced a complete loss of activity of the enzymes from the villous epithelium and crypts. Recovery after withdrawal of drug The first sign of healing of the intestinal lesions was at 5 days, when mitoses in most crypts were normal in number and appearance, and crypt tissue was again abundant. Villi were normal in length and shape, and villous epithelium was restored, except at the tip where the cells still showed ballooning degeneration. In a few areas of the intestine crypts were distended by mucus and lined by large mucus-filled cells. Seven days after receiving the fourth dose of methotrexate all regions of the intestine had returned to morphological normality, and this was maintained up to the end of the period studied at 4 weeks. There were comparable numbers of mitoses to those found in control tissues, and abnormal mitotic figures were no longer a feature. Evidence that the intestine was also functionally normal was provided by a dramatic improvement in the clinical condition of the mice after 1 week, and by the return to the mucosa of the activities of acid and alkaline phosphatase, lactate dehydrogenase and N-acetyl-f-glucoseaminidase. DISCUSSION
Folate deficiency and consequent damage to the intestinal mucosa of mice was induced by methotrexate after only a single injection of the drug and the damage rapidly progressed with further methotrexate therapy. The jejunum and ileum proved to be the most susceptible regions of the alimentary tract. The
INTESTINAL LESIONS INDUCED BY METHOTREXATE
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FIa. 4.-Abnormal cells and nuclei in ileal crypts after 5 injections of methotrexate. H. & E. x 240. FIG. 5.-Mucoid metaplasia, ileum, 6 doses. Villi are very short. H. & E. x 175. FIG. 6.-Jejunal crypts after 6 injections. The dilated crypt is lined by flattened cells, one of which shows abnormal mitosis. H. & E. x 245. FIG. 7.-Ileum, 6 injections of methotrexate. Crypts contain large and bizarre cells. H. & E. x 175.
stomach remained normal throughout the experiment and the caecum and colon were only mildly affected. Lesions were of the same type at different stages of
methotrexate therapy, and consisted of degeneration of epithelial cells, distortion of villi, and inhibition of crypt cell replication.
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The underlying lesion in the intestine was of greatly reduced crypt cell proliferation, and this resulted in shortening of villi and a decrease in the mucosal surface area available for absorption. The vacuolar degeneration of the epithelial cells over most of the surface of the villi was also reported in the early experimental work on methotrexate (Phillips and Thiersch, 1949). This cannot be attributed to a normal desquamation process, and may be the direct result of a local reduction of folate in the intestinal epithelial cells, which methotrexate has been shown to produce (Selhub, Brin and Grossowicz, 1973). Changes in the crypts included megalocytosis and greatly reduced mitotic activity, together with some abnormal mitoses. Methotrexate ac-ts on crypt cells by inhibiting the enzyme dihydrofolate reductase, and this in turn reduces the supply to the cells of pyrimidine deoxythymidine triphosphate (Bertino and Johns, 1972). This impairs DNA synthesis and is responsible for the reduced crypt cell replication. The effect on DNA synthesis also causes breaking and despirallization of chromosomes (Hoffbrand et al., 1976), which may be the explanation for the abnormal mitotic figures observed in the jejunal and ileal crypts. The lesion is thus similar to that described in the intestine of patients with pernicious anaemia (Foroozan and Trier, 1967) and after repeated exposure to X-irradiation (Trier and Browning, 1966), except that in the present study much of each villus actually became necrotic. Trier (1962) recorded the histological lesions in the human jejunum produced by a single large dose of methotrexate. The inhibition of mitosis in crypt epithelium was the same as described here, but the single dose did not produce degeneration of the epithelial cells of the villi. The clinical signs seen in prolonged methotrexate therapy of man, however, indicate that changes in the intestinal tract similar to those seen in mice do occur. The small intestine itself acts as the main site of folate adsorption from food,
the jejunum and upper ileum being particularly important (Rosenberg, 1976). It is therefore probable that once the epithelium of the villi is damaged by the initial doses of methotrexate and its absorptive capacity reduced, then further deficiency of folate rapidly occurs until the drug is discontinued and the crypt cells can replicate again. In the later stages of methotrexate intoxication the intestinal crypts which remained exhibited marked mucoid metaplasia, many of them being distended by huge mucus-filled cells. The reason for this metaplasia is obscure, though the composition of the mucus was a similar mixture of acidic and neutral glycoproteins to that found in the normal animals. Although ulceration of the mouth and tongue, and occasionally also lung lesions, have been reported in patients undergoing prolonged methotrexate therapy (Condit, 1960; Clarysse et al., 1969) such changes were not observed, even histologically, in the experimental mice. This may be due to different susceptibility of the two species, or it may require much longer exposure of mice to the drug. Recovery after a course of 4 injections of methotrexate was surprisingly rapid, and at 1 week the mucosa of all regions of the intestine had returned from a state of severe damage to normal. This reflects not only the very short crypt cell generation time of about 19 h and an epithelial cell renewal time of 2 to 3 days in this region in the mouse (Leblond and Messier, 1958; Quastler and Sherman, 1959) but also indicates that remaining crypt cells are capable of rapid recovery from the specific injury produced by methotrexate. This is in contrast to the slow regeneration which occurs in the jejunum of patients with corrected pernicious anaemia (Foroozan and Trier, 1967). In that condition, even though the mitotic rate has been shown to return to normal within a few days of treatment, the disappearance of megalocytosis and the restoration of normal villous architecture require many weeks.
INTESTINAL LESIONS INDUCED BY METHOTREXATE
We are grateful for the assistance of Miss M. Orman, Mrs I. Francis and Miss D. Harwood. REFERENCES BARKA, T. & ANDERSON, P. J. (1965) Histochemistry; Theory, Practice and Bibliography, 2nd ed. Hoeber Medical Division. New York: Harper & Row. BERTINO, J. R. & JOHNS, D. G. (1972) Folate Antagonists. In Cancer Chemotherapy. II. Ed. I. Bradsky and S. B. Kahn. New York: Grune and Stratton, p. 9. CHAYEN, J., BITENSKY, L. & BUTCHER, R. G. (1973) Practical Histochemistry. London and New York: John Wiley and Sons. CLARYSSE, A. M., CATHEY, W. J., CARTWRIGHT, G. E. & WINTROBE, M. M. (1969) Pulmonary Disease Complicating Intermittent Therapy with Methotrexate. J. Amer. Med. Ass., 209, 1861. COE, R. 0. & BULL, F. E. (1968) Cirrhosis Associated with Methotrexate Treatment of Psoriasis. J. Amer. Med. Ass., 206, 1515. COLSKY, J., GREENSPAN, E. M. & WARREN, T. N. (1955) Hepatic Fibrosis in Children with Acute Leukaemia after Therapy with Folic Acid Antagonists. Archs Path., 59, 198. CONDIT, P. T. (1960) Studies on the Folic Acid Vitamins. II. The Acute Toxicity of Amethopterin in Man. Cancer, 13, 222. FOROOZAN, P. & TRIER, J. S. (1]967) Mucosa of the Small Intestine in Pernicious Anaemia. New Engl. J. Med., 277, 553. GOMORI, G. (1952) Microscopic Histochemistry; Principles and Practice. Chicago: University of
Chicago.
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HOFFBRAND, A. V., GANESHAGURU, K., HOOTON, J. W. L. & TRIPP, E. (1976) Megaloblastic Anaemia; Initiation of DNA Synthesis in Excess of DNA Chain Elongation as the Underlying Mechanism. Clinics in Haermatol. 5, 727. LEBLOND, C. P. & MESSIER, B. (1958) Renewal of Chief Cells and Goblet Cells in the Small Intestine as Shown by Radioa.utography after Injection of Thymidine-H3 into Mice. Anat. Rec., 132, 247. PHILLIPS, F. S. & THIFRSCH, J. B. (1949) Studies of the Actions of 4-Amino-pteroylglutamic acid in Rats and Mice. J. Pharmacol. exp. Ther., 95, 303. PUGH, D. & WALKER, P. G. (1961) The Localization of N-acetyl-B-glucos-aminidase in Tissues. J. Histochem. Cytochem., 9, 242. QUASTLER, H. & SHERMAN, F. M. (1959) Cell Population Kinetics in the Intestinal Epithelium of the Mouse. Exp. Cell Res., 17, 420. ROSENBERG, I. H. (1976) Absorption and Malabsorption of Folates. Clinics in Haematol., 5, 589. SELHUB, J., BRIN, H. & GROSSOWICZ, N. (1973) Uptake and Reduction of Radioactive Folate by Everted Sacs of Rat Small Intestine. Eur. J. Biochem., 33, 433. TAYLOR, S. G., HASS, G. M., CRUMRINE, J. L. & SLAUGHTER, D. P. (1950) Toxic Reactions of 4Amino-pteroylglutamic acid (Aminopterin) in Patients with Far Advanced Neoplastic Disease. Cancer, 3, 493. TRIER, J. S. (1962) Morphologic Alterations Induced by Methotrexate in Mucosa of Human Proximal Intestine. I. Serial Observations by Light Microscopy. Gastroenterology, 42, 295. TRIER, J. S. & BROWNING, T. H. (1966) Morphologic Response of Mucosa of Human Small Intestine to X-ray Exposure. J. clin. Invest., 45, 194.
INTESTINAL LESIONS INDUCED EXPERIMENTALLY BY METHOTREXATE A. BASKERVILLE AND D. BATTER-HATTON From the Microbiological Research Establishment, Porton, Salisbury, Wiltshire Received for publication July 4, 1977
Summary.-Mice were given up to 9 doses of methotrexate intermittently over a 3-week period. Inhibition of mitosis occurred in the duodenum, jejunum and ileum after the first injection, and after 2 doses the crypt epithelium showed megalocytosis and occasional abnormal mitotic figures. Further treatment produced degeneration of the epithelium of the villi, which became irregular and atrophic, and the amount of crypt tissue was greatly reduced. Focal ulceration and haemorrhage occurred in some animals. Changes in the caecum and colon developed later and were much milder. After withdrawal of methotrexate the intestinal mucosa rapidly recovered and was normal 1 week later.
IT is now well recognized that methotrexate, given orally or parenterally, can induce undesirable changes in the intestine, liver and other tissues (Taylor et al., 1950; Colsky, Greenspan and Warren, 1955; Trier, 1962; Coe and Bull, 1968). The drug is used to treat psoriasis and malignant diseases such as leukaemia, and its adverse side-effects are the result of its action as a folic acid antagonist. The present work was designed to study the development and time course of the changes in the different regions of the intestinal tract of mice after intermittent methotrexate therapy, and to examine the spontaneous recovery of the intestine after cessation of treatment. MATERIALS AND METHODS Male Porton strain specific-pathogen-free mice weighing 25-30 g were used in all the experiments. The drug preparation was the parenteral form of methotrexate sodium (Lederle, Gosport) reconstituted with sterile water for injection. The dose per day for each mouse was 1 mg in a volume of 041 ml, given by i.p. injection. In a pilot experiment to determine the tolerance of mice to this dose, a group of 60 animals received 1 mg methotrexate i.p. three times per week up to a maximum of 9 doses, a lapse of 48 or 72 h being allowed between doses. For the main sequential study mice were given
frorn 1 to 9 injections of methotrexate, and 5 animals were killed at each of these doses, 48 h after the last drug treatment. Five control mice inoculated with the same number of doses of 0.1 ml physiological saline were also killed at each stage. An additional recovery experiment was carried out in which 50 mice were given 4 doses each of 1 mg methotrexate over a period of 1 week, and 5 were then killed at 1, 2 and 5 days and 1, 2, 3 and 4 weeks after the final injection. An equal number of control animals given saline i.p. were also killed at each stage. All mice were killed with ether and necropsy was performed immediately after death. The gastrointestinal tract was removed from all animals and the following regions were fixed as separate tissues in 10% buffered neutral formalin: stomach, duodenum, jejunum, ileum, caecum, colon. From treated mice after 3, 6 and 9 injections and their controls, portions of ileum were also rapidly frozen as fresh tissue for histochemical studies. Cryostat sections of these unfixed blocks were cut at 8 ,im and stained to demonstrate acid and alkaline phosphatase activities at pH 6-5 and pH 9 0 respectively by the method of Gomori (1952), modified by Barka and Anderson (1965), using a-naphthyl phosphate as substrate. Sections were also stained to demonstrate lactate dehydrogenase activity by the polyvinyl alcohol technique of Chayen, Bitensky and Butcher (1973), and N-acetyl-flglucosaminidase by the diazo coupling method of Pugh and Walker (1961). After formalin fixation all other portions of the alimentary tract were processed by standard methods and embedded in paraffin wax. Sections cut at 5 ,um were stained by haematoxylin and
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eosin (H. & E.) and selected sections were also stained by the periodic acid-Schiff (PAS) and alcian blue (AB) methods, by Gordon and Sweet's method for reticulin, and by the Verhoeff-van Gieson technique.
RESULTS
Clinical and necropsy findings No clinical changes were observed after the first 3 injections of methotrexate, but after 4 doses many mice developed diarrhoea. Consistent necropsy findings were the presence of very fluid intestinal contents and a pale, translucent appearance of the wall of the small intestine. After 5 injections many mice were emaciated, had ruffled fur and were hunched and disinclined to move. After 6 doses a few mice showed paralysis of the hind limbs, and many had diarrhoea and appeared weak. The same pattern was evident until the termination of the experiment after the ninth injection. In the pilot mortality study 28/60 (46 6°%S) mice died. Deaths started to occur after the fourth treatment when 2 mice died, and the mortality continued to rise steadily until the ninth dose of the drug.
Histopathological findings Changes were not found in the stomach of any methotrexate-treated mice. Lesions appeared synchronously in the duodenum, jejunum and ileum after only a single dose of the drug and were of similar severity in all regions. Changes in the caecum were minimal at this stage and were absent in the colon until 4 doses had been given. One dose.-There was inhibition of mitotic activity in the crypts of Lieberkuhn and a number of crypt cells were enlarged and intensely basophilic. The epithelium of the villi was normal. Two and three doses.-The nature of the lesion was the same in all regions of the intestine. It consisted of vacuolar degeneration and loss of staining affinity of the epithelial cells of the upper half of the villi, though the central core of lamina propria was normal (Fig. 1). The positive
PAS-staining reaction of the bruish border of affected epithelial cells had been lost. Cells in the crypts of Lieberkuhn were basophilic, many were enlarged and bizarre in shape and their vesicular nuclei were distorted and contained prominent nucleoli (Fig. 1). There were fewer mitotic figures in the crypt epithelium than in control sections and some of these were abnormal, having chromosomal bridges and clumped and degenerating chromosomes. Villi were more irregular than those of control animals but Brunner's glands in the duodenum appeared normal. Changes in the caecum were milder and confined to the surface epithelium, where the cells showed early degeneration. Four and five doses. There was now marked ballooning degeneration of epithelial cells throughout the small intestine and the epithelial layer frequently separated from the underlying lamina propria (Fig. 2). Goblet cells were greatly decreased in number and villi were misshapen and atrophic (Fig. 2). In some mice the lamnina propria and submucosa were oedematous and connective tissue cells were sparse, but more commonly there was hypercellularity of these regions, the majority of the cells being histiocytes and fibroblasts (Figs 2 and 3). The population of other inflammatory cells was similar to that seen in control animals. The amount of crypt tissue had diminished (Figs 2 and 3) and some of the remaining crypts in the small intestine were greatly dilated, showed megalocytosis and bizarre cell formation, and occasional abnormal mitoses (Fig. 4). Disintegration of some crypt cell nuclei was also found at this and later stages. Peyer's patches were undiminished in size when compared with those of control animals. After the fourth dose early degenerative changes were also found in the epithelium of the colon. Six doses.-Villi were short and irregular and much of their upper portions were necrotic. In some areas there was focal ulceration of the mucosa, which resulted in haemorrhage from the submucosa into the lumen. There was even less crypt tissue
INTESTINAL LESIONS INDUCED BY METHOTREXATE
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FIG. 1. Ileum of mouse after 3 injections of methotrexate, showing degeneration of epithelium. In the crypts there are large cells with few mitoses. An abnormal mitotic figure is in the centre of the field. H. & E. x220. FIG. 2.-Ileum, 4 injections of methotrexate. Atrophy of villi and separation of necrotic tips. There is little crypt tissue and the lamina propria and submucosa are filled by histiocytes and fibroblasts. H.&E. x8O. FIG. 3. Five doses of the drug. Ileum. Villi are irregular and crypt tissue is greatly reduced. H.&E. x80.
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than earlier and many crypt cells were distended by foamy, mucoid secretion which stained positively with the AB and PAS techniques (Fig. 5). Others were large and bizarre in shape and had abnormal nuclei (Figs 6 and 7). The lamina propria and submucosa in all sites were filled by histiocytes and fibroblasts. Changes in the colon were less severe than in the small intestine and its submucosal connective tissue remained normal. Seven to nine doses.-Villi were small, irregular and necrotic along much of their length. Crypt epithelium was scanty, and some of the glandular tissue was of the bizarre basophilic type seen at earlier stages. There was little evidence of mitotic activity. The remainder of the crypt epithelium was hypertrophic and cells and glands were distended by PAS-positive AB-positive mucus. The lumina of the crypts frequently contained refractile, eosinophilic, PAS-positive, AB-positive granules. The submucosa was filled by dense accumulations of histiocytes and fibroblasts, but there was no increase in other inflammatory cells. Changes in the duodenum affected only the villi and crypts, and Brunner's glands remained morphologically normal. In the caecum and colon lesions were less severe and not very extensive. They consisted of degeneration of some areas of the surface epithelium, accompanied by the formation of PAS-positive, AB-positive, mucus-filled cysts in the crypts and accumulation of mucus in the colonic lumen. There were isolated foci of ulceration and haemorrhage, which were not present in all treated mice. Peyer's patches throughout the intestinal tract appeared unaffected by the methotrexate treatment. Other organs.-Lesions were not present in the tongue or lungs at any stage of treatment. The changes produced in the liver, brain, kidneys and testes will be reported in another communication. Histochemical findings Enzymes demonstrated in the ileum were acid and' alkaline phosphatases,
lactate dehydrogenase and n-acetyl-3glucosaminidase. After 3 doses of methotrexate there was some loss of activity of all the enzymes from the epithelium of the villi and upper crypts of the ileum when compared with the abundant staining detected in tissue from control mice. Six and 9 injections of the drug produced a complete loss of activity of the enzymes from the villous epithelium and crypts. Recovery after withdrawal of drug The first sign of healing of the intestinal lesions was at 5 days, when mitoses in most crypts were normal in number and appearance, and crypt tissue was again abundant. Villi were normal in length and shape, and villous epithelium was restored, except at the tip where the cells still showed ballooning degeneration. In a few areas of the intestine crypts were distended by mucus and lined by large mucus-filled cells. Seven days after receiving the fourth dose of methotrexate all regions of the intestine had returned to morphological normality, and this was maintained up to the end of the period studied at 4 weeks. There were comparable numbers of mitoses to those found in control tissues, and abnormal mitotic figures were no longer a feature. Evidence that the intestine was also functionally normal was provided by a dramatic improvement in the clinical condition of the mice after 1 week, and by the return to the mucosa of the activities of acid and alkaline phosphatase, lactate dehydrogenase and N-acetyl-f-glucoseaminidase. DISCUSSION
Folate deficiency and consequent damage to the intestinal mucosa of mice was induced by methotrexate after only a single injection of the drug and the damage rapidly progressed with further methotrexate therapy. The jejunum and ileum proved to be the most susceptible regions of the alimentary tract. The
INTESTINAL LESIONS INDUCED BY METHOTREXATE
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FIa. 4.-Abnormal cells and nuclei in ileal crypts after 5 injections of methotrexate. H. & E. x 240. FIG. 5.-Mucoid metaplasia, ileum, 6 doses. Villi are very short. H. & E. x 175. FIG. 6.-Jejunal crypts after 6 injections. The dilated crypt is lined by flattened cells, one of which shows abnormal mitosis. H. & E. x 245. FIG. 7.-Ileum, 6 injections of methotrexate. Crypts contain large and bizarre cells. H. & E. x 175.
stomach remained normal throughout the experiment and the caecum and colon were only mildly affected. Lesions were of the same type at different stages of
methotrexate therapy, and consisted of degeneration of epithelial cells, distortion of villi, and inhibition of crypt cell replication.
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The underlying lesion in the intestine was of greatly reduced crypt cell proliferation, and this resulted in shortening of villi and a decrease in the mucosal surface area available for absorption. The vacuolar degeneration of the epithelial cells over most of the surface of the villi was also reported in the early experimental work on methotrexate (Phillips and Thiersch, 1949). This cannot be attributed to a normal desquamation process, and may be the direct result of a local reduction of folate in the intestinal epithelial cells, which methotrexate has been shown to produce (Selhub, Brin and Grossowicz, 1973). Changes in the crypts included megalocytosis and greatly reduced mitotic activity, together with some abnormal mitoses. Methotrexate ac-ts on crypt cells by inhibiting the enzyme dihydrofolate reductase, and this in turn reduces the supply to the cells of pyrimidine deoxythymidine triphosphate (Bertino and Johns, 1972). This impairs DNA synthesis and is responsible for the reduced crypt cell replication. The effect on DNA synthesis also causes breaking and despirallization of chromosomes (Hoffbrand et al., 1976), which may be the explanation for the abnormal mitotic figures observed in the jejunal and ileal crypts. The lesion is thus similar to that described in the intestine of patients with pernicious anaemia (Foroozan and Trier, 1967) and after repeated exposure to X-irradiation (Trier and Browning, 1966), except that in the present study much of each villus actually became necrotic. Trier (1962) recorded the histological lesions in the human jejunum produced by a single large dose of methotrexate. The inhibition of mitosis in crypt epithelium was the same as described here, but the single dose did not produce degeneration of the epithelial cells of the villi. The clinical signs seen in prolonged methotrexate therapy of man, however, indicate that changes in the intestinal tract similar to those seen in mice do occur. The small intestine itself acts as the main site of folate adsorption from food,
the jejunum and upper ileum being particularly important (Rosenberg, 1976). It is therefore probable that once the epithelium of the villi is damaged by the initial doses of methotrexate and its absorptive capacity reduced, then further deficiency of folate rapidly occurs until the drug is discontinued and the crypt cells can replicate again. In the later stages of methotrexate intoxication the intestinal crypts which remained exhibited marked mucoid metaplasia, many of them being distended by huge mucus-filled cells. The reason for this metaplasia is obscure, though the composition of the mucus was a similar mixture of acidic and neutral glycoproteins to that found in the normal animals. Although ulceration of the mouth and tongue, and occasionally also lung lesions, have been reported in patients undergoing prolonged methotrexate therapy (Condit, 1960; Clarysse et al., 1969) such changes were not observed, even histologically, in the experimental mice. This may be due to different susceptibility of the two species, or it may require much longer exposure of mice to the drug. Recovery after a course of 4 injections of methotrexate was surprisingly rapid, and at 1 week the mucosa of all regions of the intestine had returned from a state of severe damage to normal. This reflects not only the very short crypt cell generation time of about 19 h and an epithelial cell renewal time of 2 to 3 days in this region in the mouse (Leblond and Messier, 1958; Quastler and Sherman, 1959) but also indicates that remaining crypt cells are capable of rapid recovery from the specific injury produced by methotrexate. This is in contrast to the slow regeneration which occurs in the jejunum of patients with corrected pernicious anaemia (Foroozan and Trier, 1967). In that condition, even though the mitotic rate has been shown to return to normal within a few days of treatment, the disappearance of megalocytosis and the restoration of normal villous architecture require many weeks.
INTESTINAL LESIONS INDUCED BY METHOTREXATE
We are grateful for the assistance of Miss M. Orman, Mrs I. Francis and Miss D. Harwood. REFERENCES BARKA, T. & ANDERSON, P. J. (1965) Histochemistry; Theory, Practice and Bibliography, 2nd ed. Hoeber Medical Division. New York: Harper & Row. BERTINO, J. R. & JOHNS, D. G. (1972) Folate Antagonists. In Cancer Chemotherapy. II. Ed. I. Bradsky and S. B. Kahn. New York: Grune and Stratton, p. 9. CHAYEN, J., BITENSKY, L. & BUTCHER, R. G. (1973) Practical Histochemistry. London and New York: John Wiley and Sons. CLARYSSE, A. M., CATHEY, W. J., CARTWRIGHT, G. E. & WINTROBE, M. M. (1969) Pulmonary Disease Complicating Intermittent Therapy with Methotrexate. J. Amer. Med. Ass., 209, 1861. COE, R. 0. & BULL, F. E. (1968) Cirrhosis Associated with Methotrexate Treatment of Psoriasis. J. Amer. Med. Ass., 206, 1515. COLSKY, J., GREENSPAN, E. M. & WARREN, T. N. (1955) Hepatic Fibrosis in Children with Acute Leukaemia after Therapy with Folic Acid Antagonists. Archs Path., 59, 198. CONDIT, P. T. (1960) Studies on the Folic Acid Vitamins. II. The Acute Toxicity of Amethopterin in Man. Cancer, 13, 222. FOROOZAN, P. & TRIER, J. S. (1]967) Mucosa of the Small Intestine in Pernicious Anaemia. New Engl. J. Med., 277, 553. GOMORI, G. (1952) Microscopic Histochemistry; Principles and Practice. Chicago: University of
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HOFFBRAND, A. V., GANESHAGURU, K., HOOTON, J. W. L. & TRIPP, E. (1976) Megaloblastic Anaemia; Initiation of DNA Synthesis in Excess of DNA Chain Elongation as the Underlying Mechanism. Clinics in Haermatol. 5, 727. LEBLOND, C. P. & MESSIER, B. (1958) Renewal of Chief Cells and Goblet Cells in the Small Intestine as Shown by Radioa.utography after Injection of Thymidine-H3 into Mice. Anat. Rec., 132, 247. PHILLIPS, F. S. & THIFRSCH, J. B. (1949) Studies of the Actions of 4-Amino-pteroylglutamic acid in Rats and Mice. J. Pharmacol. exp. Ther., 95, 303. PUGH, D. & WALKER, P. G. (1961) The Localization of N-acetyl-B-glucos-aminidase in Tissues. J. Histochem. Cytochem., 9, 242. QUASTLER, H. & SHERMAN, F. M. (1959) Cell Population Kinetics in the Intestinal Epithelium of the Mouse. Exp. Cell Res., 17, 420. ROSENBERG, I. H. (1976) Absorption and Malabsorption of Folates. Clinics in Haematol., 5, 589. SELHUB, J., BRIN, H. & GROSSOWICZ, N. (1973) Uptake and Reduction of Radioactive Folate by Everted Sacs of Rat Small Intestine. Eur. J. Biochem., 33, 433. TAYLOR, S. G., HASS, G. M., CRUMRINE, J. L. & SLAUGHTER, D. P. (1950) Toxic Reactions of 4Amino-pteroylglutamic acid (Aminopterin) in Patients with Far Advanced Neoplastic Disease. Cancer, 3, 493. TRIER, J. S. (1962) Morphologic Alterations Induced by Methotrexate in Mucosa of Human Proximal Intestine. I. Serial Observations by Light Microscopy. Gastroenterology, 42, 295. TRIER, J. S. & BROWNING, T. H. (1966) Morphologic Response of Mucosa of Human Small Intestine to X-ray Exposure. J. clin. Invest., 45, 194.
