Methotrexate (MTX) Effect on Pancreatic Enzymes in Leukemic Mice DAVID BRANSKI, MD, E M A N U E L L E B E N T H A L , MD, ARNOLD I. FREEMAN, MD, J O H N E. FISHER, MD, TERRY F. HATCH, MD, and JOSEPH KRASNER, PhD
We evaluated the effects of M T X on normal and leukemic mice (L-1210) in order to determine the effects of M T X on the pancreas in both leukemic and normal control mice. The mice were divided into two experiments according to their treatment schedule. The first group consisted of mice with L-1210 leukemia and normal controls treated with escalating doses of M T X or with placebo at 10, 15, 20, 25, 30, or 45 mg/kg. These mice were all arbitrarily sacrificed at 48 hr. The second group consisted of mice with L-1210 leukemia and normal controls treated with an arbitrary dose of M T X (20 mg/kg) or with placebo and sacrificed at spaced time intervals of l2, 24, 48, and 96 hr after M T X administration. Both L-1210 and normal nonleukemic mice were examined for histological changes in the pancreas as well as pancreatic zymogen enzyme levels. In general, the maximal effect on the pancreas occurred at a dose of 20 mg/kg o f M T X and at 48 hr following M T X administration. Histologically, in the L-1210 leukemic mice, there was leukemic infiltration, diminished acini, and fatty changes in the pancreas; these findings were less pronounced following treatment with M T X compared to those treated with placebo. All pancreatic enzymes in untreated leukemic mice were significantly diminished (P < 0.001) compared t O normal mice. Amylase and lipase were decreased more than trypsin and chymotrypsin. The zymogen enzymes in MTX-treated normal mice responded in a hyperbolar fashion, falling to a nadir at 20 mg/kg of MTX. In contrast, in the MTX-treated leukemic mice, the zymogen enzymes increased significantly reaching a peak at 20 mg/kg of M T X and then falling off with increasing M T X doses.
In human acute leukemia in relapse, it is common to see infiltrates of these neoplastic cells throughout the body, including those of the gastrointestinal tract. Normal cells within the small intestine (1-3), the large intestine (2-4), the liver (5), and the pancreas (6, 7) may be damaged as the result of direct compression by the leukemic cells as well as by obstruction of their normal blood supply. Leukemic Manuscript received November 1, 1978; revised manuscript received July 10, 1979; accepted July 13, 1979. From the Division of Gastroenterology and Nutrition, Department of Pediatrics, School of Medicine, State University of New York; The Children's Hospital of Buffalo; and Roswell Park Memorial Institute. Dr. David Branski is a Postdoctoral Research Fellow on leave from the Bikur Cholim General Hospital, Jerusalem, Israel. Address for reprint requests: Dr. Emanuel Lebenthal, Division of Gastroenterology and Nutrition, Children's Hospital of Buffalo, 219 Bryant Street, Buffalo, New York 14222.
infiltration may be so extensive as to obliterate the normal histological architecture. Consequently, cell function may be altered, resulting in impairment of organ function. Exocrine pancreatic disturbances (8) and depressed levels of pancreatic exocrine enzymes in duodenal fluids (9) have been described both in patients with leukemia and pancreatic tumors. Methotrexate (MTX, amethopterin) is a folic acid antagonist used extensively in the treatment of acute lymphocytic leukemia, which binds to dihydrofolic acid reductase resulting in the inhibition of nucleic acid synthesis (10). However, MTX may affect normal as well as leukemic cells. The purpose of the study was to elucidate the effects of MTX and of acute leukemia on the enzymes and histopathology of the pancreas. A leukemic
Digestive Diseases and Sciences, Vol. 24, No. ll (November 1979)
0163-2116/79/1100-0865503.00/1 9 1979DigestiveDiseaseSystems,Inc.
865
BRANSKI ET AL
Fig 1. Photomicrograph shows pancreas heavily infiltrated with leukemic cells. Pancreatic tissue is identified at left of picture. It and interstitial tissue are infiltrated by abundant leukemic cells.
mouse model (L-1210) was e m p l o y e d since it has been the major screening tool of the National Cancer Institute Drug Development P r o g r a m (11).
MATERIALS AND METHODS The study group, consisting of 80 female mice nine weeks old (strain DBA/2Ha), was given intraperitoneal (ip) inoculations of 1 x 106 leukemic cells (L-1210). An additional 72 female mice, nine weeks old, were injected with normal saline to serve as nonleukemic controls. The mice were maintained on a standard Purina Chow diet and tap water, and the two groups were fed comparable diets. The mice were watched daily for fecal consistency. Two separate experiments were performed. In the first experiment five days after inoculation both leukemic and nonleukemic mice were given either MTX or saline ip. Five days after inoculation of leukemic cells, MTX was administered to 42 mice in escalating doses of 5, 10, 15, 20, 25, 30, or 45 mg/kg body weight. Thus, each group of 6 mice received a different dose of MTX. Due to mortality from handling the mice, the size of groups varied from 4
866
to 6 animals. Mice were sacrificed 48 hr after the administration of MTX or saline placebo. In the second experiment following a single ip injection of MTX of 20 mg/kg body weight to 30 mice, the mice were sacrificed in groups of 4 to 6 at 12, 24, 48, 72, and 96 hr after drug injection. The mice were sacrificed by cervical dislocation. The pancreas was immediately resected and divided into two portions. The first portio n was immediately analyzed for zymogen enzymes or stored at - 2 0 ~ C for subsequent enzymatic assays. The second portion from the tail of the pancreas was placed into 10% formalin solution for histopathologic analysis. Because of obvious gross findings in the leukemic mice, a blind histological analysis was not performed. The f r o z e n pancreatic tissue was subsequently thawed, weighed, and diluted 1:10 with cold distilled water and homogenized in a Potter-Elvehjem homogenizer, since this technique of preservation for enzyme analysis has been shown not to result in the loss of enzyme activity. Trypsinogen and" chymotrypsinogen were activated by the addition of a small amount of enterokinase. The following enzyme determinations were performed Digestive Diseases and Sciences, Vol. 24, No. 11 (November 1979)
PANCREAS IN LEUKEMIA
Lipase
Amylose
Trypsin 14
i40 -
E]Normal(fO) r
_[
120
i2
Ct cmolrypsin 3
ioo
~
60 40
i
20
p
We evaluated the effects of M T X on normal and leukemic mice (L-1210) in order to determine the effects of M T X on the pancreas in both leukemic and normal control mice. The mice were divided into two experiments according to their treatment schedule. The first group consisted of mice with L-1210 leukemia and normal controls treated with escalating doses of M T X or with placebo at 10, 15, 20, 25, 30, or 45 mg/kg. These mice were all arbitrarily sacrificed at 48 hr. The second group consisted of mice with L-1210 leukemia and normal controls treated with an arbitrary dose of M T X (20 mg/kg) or with placebo and sacrificed at spaced time intervals of l2, 24, 48, and 96 hr after M T X administration. Both L-1210 and normal nonleukemic mice were examined for histological changes in the pancreas as well as pancreatic zymogen enzyme levels. In general, the maximal effect on the pancreas occurred at a dose of 20 mg/kg o f M T X and at 48 hr following M T X administration. Histologically, in the L-1210 leukemic mice, there was leukemic infiltration, diminished acini, and fatty changes in the pancreas; these findings were less pronounced following treatment with M T X compared to those treated with placebo. All pancreatic enzymes in untreated leukemic mice were significantly diminished (P < 0.001) compared t O normal mice. Amylase and lipase were decreased more than trypsin and chymotrypsin. The zymogen enzymes in MTX-treated normal mice responded in a hyperbolar fashion, falling to a nadir at 20 mg/kg of MTX. In contrast, in the MTX-treated leukemic mice, the zymogen enzymes increased significantly reaching a peak at 20 mg/kg of M T X and then falling off with increasing M T X doses.
In human acute leukemia in relapse, it is common to see infiltrates of these neoplastic cells throughout the body, including those of the gastrointestinal tract. Normal cells within the small intestine (1-3), the large intestine (2-4), the liver (5), and the pancreas (6, 7) may be damaged as the result of direct compression by the leukemic cells as well as by obstruction of their normal blood supply. Leukemic Manuscript received November 1, 1978; revised manuscript received July 10, 1979; accepted July 13, 1979. From the Division of Gastroenterology and Nutrition, Department of Pediatrics, School of Medicine, State University of New York; The Children's Hospital of Buffalo; and Roswell Park Memorial Institute. Dr. David Branski is a Postdoctoral Research Fellow on leave from the Bikur Cholim General Hospital, Jerusalem, Israel. Address for reprint requests: Dr. Emanuel Lebenthal, Division of Gastroenterology and Nutrition, Children's Hospital of Buffalo, 219 Bryant Street, Buffalo, New York 14222.
infiltration may be so extensive as to obliterate the normal histological architecture. Consequently, cell function may be altered, resulting in impairment of organ function. Exocrine pancreatic disturbances (8) and depressed levels of pancreatic exocrine enzymes in duodenal fluids (9) have been described both in patients with leukemia and pancreatic tumors. Methotrexate (MTX, amethopterin) is a folic acid antagonist used extensively in the treatment of acute lymphocytic leukemia, which binds to dihydrofolic acid reductase resulting in the inhibition of nucleic acid synthesis (10). However, MTX may affect normal as well as leukemic cells. The purpose of the study was to elucidate the effects of MTX and of acute leukemia on the enzymes and histopathology of the pancreas. A leukemic
Digestive Diseases and Sciences, Vol. 24, No. ll (November 1979)
0163-2116/79/1100-0865503.00/1 9 1979DigestiveDiseaseSystems,Inc.
865
BRANSKI ET AL
Fig 1. Photomicrograph shows pancreas heavily infiltrated with leukemic cells. Pancreatic tissue is identified at left of picture. It and interstitial tissue are infiltrated by abundant leukemic cells.
mouse model (L-1210) was e m p l o y e d since it has been the major screening tool of the National Cancer Institute Drug Development P r o g r a m (11).
MATERIALS AND METHODS The study group, consisting of 80 female mice nine weeks old (strain DBA/2Ha), was given intraperitoneal (ip) inoculations of 1 x 106 leukemic cells (L-1210). An additional 72 female mice, nine weeks old, were injected with normal saline to serve as nonleukemic controls. The mice were maintained on a standard Purina Chow diet and tap water, and the two groups were fed comparable diets. The mice were watched daily for fecal consistency. Two separate experiments were performed. In the first experiment five days after inoculation both leukemic and nonleukemic mice were given either MTX or saline ip. Five days after inoculation of leukemic cells, MTX was administered to 42 mice in escalating doses of 5, 10, 15, 20, 25, 30, or 45 mg/kg body weight. Thus, each group of 6 mice received a different dose of MTX. Due to mortality from handling the mice, the size of groups varied from 4
866
to 6 animals. Mice were sacrificed 48 hr after the administration of MTX or saline placebo. In the second experiment following a single ip injection of MTX of 20 mg/kg body weight to 30 mice, the mice were sacrificed in groups of 4 to 6 at 12, 24, 48, 72, and 96 hr after drug injection. The mice were sacrificed by cervical dislocation. The pancreas was immediately resected and divided into two portions. The first portio n was immediately analyzed for zymogen enzymes or stored at - 2 0 ~ C for subsequent enzymatic assays. The second portion from the tail of the pancreas was placed into 10% formalin solution for histopathologic analysis. Because of obvious gross findings in the leukemic mice, a blind histological analysis was not performed. The f r o z e n pancreatic tissue was subsequently thawed, weighed, and diluted 1:10 with cold distilled water and homogenized in a Potter-Elvehjem homogenizer, since this technique of preservation for enzyme analysis has been shown not to result in the loss of enzyme activity. Trypsinogen and" chymotrypsinogen were activated by the addition of a small amount of enterokinase. The following enzyme determinations were performed Digestive Diseases and Sciences, Vol. 24, No. 11 (November 1979)
PANCREAS IN LEUKEMIA
Lipase
Amylose
Trypsin 14
i40 -
E]Normal(fO) r
_[
120
i2
Ct cmolrypsin 3
ioo
~
60 40
i
20
p
