Klinische Wochenschrift
Klin Wochenschr (1991) 69:1012-1017
9 Springer-Verlag 1991
The Role of Oxygen-Derived Free Radicals in Two Models of Experimental Acute Pancreatitis: Effects of Catalase, Superoxide Dismutase, Dimethylsulfoxide, and Allopurinol M.L. Steer, P.L. Rutledge, R.E. Powers, M. Saluja, and A.K. Saluja Department of Surgery, Beth Israel Hospital, Harvard Digestive Diseases Center, Harvard Medical School, Boston
Summary. The role of oxygen-derived free radicals was evaluated in two models of experimental acute pancreatitis by testing the effects of agents which either reduce oxygen-derived free radical generation or scavenge those free radicals. Those agents (catalase, superoxide dismutase, polyethylene glycol-superoxide dismutase, dimethylsulfoxide, and allopurinol) were evaluated using the choline-deficient ethionine-supplemented diet-induced model of acute hemorrhagic pancreatic necrosis and the supramaximal caerulein stimulation model of acute interstitial edematous pancreatitis. In both models, the only effect associated with administration of the test agents was a reduction in the degree of pancreatic edema. These results suggest that oxygen-derived free radicals may play an important role in the development of pancreatic edema during pancreatitis but that those free radicals do not play an important role in the development of acinar cell injury. Key words" Ethionine - Digestive enzymes - Mice - Cats - Secretogogues
Studies employing several different experimental models of acute pancreatitis have suggested that oxygen-derived free radicals may play an important role in the evolution of that disease. Evidence supporting that suggestion can be divided into four groups as follows. First, increased pancreatic tissue levels of oxygen-derived free radicals during experimental acute pancreatitis have been measured using electron spin resonance spectroscopy and cheAbbreviations: CDE = choline-deficient ethionine supplemented; PEG=polyethylene glycol; DMSO=dimethyl sulfoxide
miluminescence (Nonaka etal. 1989a; Gough et al. 1990). Second, altered tissue levels of enzymes which either generate or scavenge oxygenderived free radicals have been demonstrated during experimental pancreatitis (Dabrowski et al. 1988; Nonaka et al. 1989b). Third, products of lipid peroxidation, presumably resulting from interaction of oxygen derived free radicals with membrane lipids, have been noted during experimental pancreatitis (Dabrowski et al. 1988; Schoenberg et al. 1989; Schoenberg et al. 1990). Fourth, drugs which either scavenge or inhibit the generation of oxygen-derived free radicals have been reported to have a beneficial effect on experimental models of acute pancreatitis (Sanfey et al. 1984; Guice et al. 1986; Sarr et al. 1987; Wisner et al. 1988; Koiwai etal. 1989; Schoenberg etal. 1989; Schoenberg et al. 1990). In spite of this evidence, the actual importance of oxygen-derived free radicals to the development of pancreatitis and the mechanism by which those radicals might act in the disease remain uncertain. In this communication, we report the results of our studies which have evaluated the role of oxygen-derived free radicals in two dissimilar models of experimental acute pancreatitis. Those were the model of acute hemorrhagic necrotizing pancreatitis which follows feeding young female mice a choline-deficient ethionine-supplemented (CDE) diet (Lombardi et al. 1977) and the model of acute interstitial edematous pancreatitis which follows infusion of rats with a supramaximally stimulating dose of the CCK analog caerulein (Lampel and Kern 1977). We have tested the effects of several agents which either reduce the generation of oxygen-derived free radicals or scavenge those radicals. In both models, a reduction in edema formation was the major effect noted.
Manuscripts published in this issue were the matter of a Symposium held at the University of Ulm, April 24-27, 1991
M.L. Steer et al. : Oxygen-Derived Free Radicals and Pancreatitis
Materials and Methods
The experiments utilizing the diet-induced model of hemorrhagic pancreatitis were performed using female CD-1 mice (12-14gm) purchased from Charles River Laboratories, Inc., Wilmington, MA. The CDE diet was prepared as previously described (Lombardi et al. 1975). Experiments utilizing the supramaximal secretagogue-induced model of interstitial edematous pancreatitis were performed using male Wistar rats (180-220 gin) also purchased from Charles River Laboratories. Catalase (44200 ~t/mg), dimethylsulfoxide (DMSO, 99%), allopurinol, and caerulein were purchased from Sigma Chemical Co., St. Louis, MO. Superoxide dismutase, coupled to polyethylene glycol (PEG-superoxide dismutase) was purchased from Enzon Inc., South Plainfield, NJ. D1ethionine was purchased from U.S. Biochemical Corp., Cleveland, Ohio. Denatured catalase was prepared by extensive boiling and polytron cavitation of active catalase, and contained no detectable catalase activity. Experimental Protocols
For studies involving diet-induced pancreatitis (Rutledge et al. 1987), mice were fasted for 24 h, fed 3 gins/mouse of the CDE diet over the next 36 h, fasted for the subsequent 24 h and then placed back on regular laboratory chow or sacrificed. The drugs tested were catalase or denatured catalase (1 x 107 ~t/kg given intraperitoneally q6H beginning 24 h before the CDE diet), allopurinol (40 mg/kg/day given in the drinking water beginning 2 4 h before the CDE diet), and DMSO (10 mg/kg/day given in the drinking water beginning 24 h before the CDE diet). For studies involving secretagogue-induced pancreatitis (Saluja et al. 1986), unanesthetized rats were infused with 5 ~tg/ kg/h of caerulein via a jugular vein catheter for 3.5 h. The drugs tested with this model were catalase (5 x 105 ~t/kg/h given IV), allopurinol (8 rag/ kg/h given IV), superoxide dismutase (1.3 x 105 ~t/ kg/h given IV), and PEG-superoxide dismutase (5 x 104 ~t/kg/h given IV). Animals were infused with the test drugs or vehicle for 1 h before and during the entire 3.5 h of caerulein infusion. Measuremen ts
Immediately after sacrifice of the animals, samples of pancreas were removed, trimmed of fat, and used for measurement of pancreatic amylase activity, quantitation of pancreatic water content, and light microscopic examination as previously de-
1013
scribed (Rutledge et al. 1987; Saluja et al. 1985). Serum amylase activity and the subcellular distribution of cathepsin B were also evaluated using techniques previously described (Rutledge et al. 1987; Saluja et al. 1985). Mortality data were analyzed using the technique of Kaplan and Meier (1958) and evaluated using the X 2 method with Yates' correction. Results
Diet-Induced Pancreatitis
Administration of the CDE diet resulted in an increase in serum and pancreatic amylase activity, an increase in pancreatic water content (i.e., edema), and mortality rate of approximately 80%. Catalase. Administration of active catalase reduced the magnitude of hyperamylasemia (CDE diet alone=l15___16~t/ml, CDE diet plus catalase=21_+4 ~t/ml) and the degree of pancreatic edema (dry/wet weight ratio of CDE diet alone = 17.6 ___0.9% ; CDE diet plus catalase = 20.9 +_0.8%, P
Klin Wochenschr (1991) 69:1012-1017
9 Springer-Verlag 1991
The Role of Oxygen-Derived Free Radicals in Two Models of Experimental Acute Pancreatitis: Effects of Catalase, Superoxide Dismutase, Dimethylsulfoxide, and Allopurinol M.L. Steer, P.L. Rutledge, R.E. Powers, M. Saluja, and A.K. Saluja Department of Surgery, Beth Israel Hospital, Harvard Digestive Diseases Center, Harvard Medical School, Boston
Summary. The role of oxygen-derived free radicals was evaluated in two models of experimental acute pancreatitis by testing the effects of agents which either reduce oxygen-derived free radical generation or scavenge those free radicals. Those agents (catalase, superoxide dismutase, polyethylene glycol-superoxide dismutase, dimethylsulfoxide, and allopurinol) were evaluated using the choline-deficient ethionine-supplemented diet-induced model of acute hemorrhagic pancreatic necrosis and the supramaximal caerulein stimulation model of acute interstitial edematous pancreatitis. In both models, the only effect associated with administration of the test agents was a reduction in the degree of pancreatic edema. These results suggest that oxygen-derived free radicals may play an important role in the development of pancreatic edema during pancreatitis but that those free radicals do not play an important role in the development of acinar cell injury. Key words" Ethionine - Digestive enzymes - Mice - Cats - Secretogogues
Studies employing several different experimental models of acute pancreatitis have suggested that oxygen-derived free radicals may play an important role in the evolution of that disease. Evidence supporting that suggestion can be divided into four groups as follows. First, increased pancreatic tissue levels of oxygen-derived free radicals during experimental acute pancreatitis have been measured using electron spin resonance spectroscopy and cheAbbreviations: CDE = choline-deficient ethionine supplemented; PEG=polyethylene glycol; DMSO=dimethyl sulfoxide
miluminescence (Nonaka etal. 1989a; Gough et al. 1990). Second, altered tissue levels of enzymes which either generate or scavenge oxygenderived free radicals have been demonstrated during experimental pancreatitis (Dabrowski et al. 1988; Nonaka et al. 1989b). Third, products of lipid peroxidation, presumably resulting from interaction of oxygen derived free radicals with membrane lipids, have been noted during experimental pancreatitis (Dabrowski et al. 1988; Schoenberg et al. 1989; Schoenberg et al. 1990). Fourth, drugs which either scavenge or inhibit the generation of oxygen-derived free radicals have been reported to have a beneficial effect on experimental models of acute pancreatitis (Sanfey et al. 1984; Guice et al. 1986; Sarr et al. 1987; Wisner et al. 1988; Koiwai etal. 1989; Schoenberg etal. 1989; Schoenberg et al. 1990). In spite of this evidence, the actual importance of oxygen-derived free radicals to the development of pancreatitis and the mechanism by which those radicals might act in the disease remain uncertain. In this communication, we report the results of our studies which have evaluated the role of oxygen-derived free radicals in two dissimilar models of experimental acute pancreatitis. Those were the model of acute hemorrhagic necrotizing pancreatitis which follows feeding young female mice a choline-deficient ethionine-supplemented (CDE) diet (Lombardi et al. 1977) and the model of acute interstitial edematous pancreatitis which follows infusion of rats with a supramaximally stimulating dose of the CCK analog caerulein (Lampel and Kern 1977). We have tested the effects of several agents which either reduce the generation of oxygen-derived free radicals or scavenge those radicals. In both models, a reduction in edema formation was the major effect noted.
Manuscripts published in this issue were the matter of a Symposium held at the University of Ulm, April 24-27, 1991
M.L. Steer et al. : Oxygen-Derived Free Radicals and Pancreatitis
Materials and Methods
The experiments utilizing the diet-induced model of hemorrhagic pancreatitis were performed using female CD-1 mice (12-14gm) purchased from Charles River Laboratories, Inc., Wilmington, MA. The CDE diet was prepared as previously described (Lombardi et al. 1975). Experiments utilizing the supramaximal secretagogue-induced model of interstitial edematous pancreatitis were performed using male Wistar rats (180-220 gin) also purchased from Charles River Laboratories. Catalase (44200 ~t/mg), dimethylsulfoxide (DMSO, 99%), allopurinol, and caerulein were purchased from Sigma Chemical Co., St. Louis, MO. Superoxide dismutase, coupled to polyethylene glycol (PEG-superoxide dismutase) was purchased from Enzon Inc., South Plainfield, NJ. D1ethionine was purchased from U.S. Biochemical Corp., Cleveland, Ohio. Denatured catalase was prepared by extensive boiling and polytron cavitation of active catalase, and contained no detectable catalase activity. Experimental Protocols
For studies involving diet-induced pancreatitis (Rutledge et al. 1987), mice were fasted for 24 h, fed 3 gins/mouse of the CDE diet over the next 36 h, fasted for the subsequent 24 h and then placed back on regular laboratory chow or sacrificed. The drugs tested were catalase or denatured catalase (1 x 107 ~t/kg given intraperitoneally q6H beginning 24 h before the CDE diet), allopurinol (40 mg/kg/day given in the drinking water beginning 2 4 h before the CDE diet), and DMSO (10 mg/kg/day given in the drinking water beginning 24 h before the CDE diet). For studies involving secretagogue-induced pancreatitis (Saluja et al. 1986), unanesthetized rats were infused with 5 ~tg/ kg/h of caerulein via a jugular vein catheter for 3.5 h. The drugs tested with this model were catalase (5 x 105 ~t/kg/h given IV), allopurinol (8 rag/ kg/h given IV), superoxide dismutase (1.3 x 105 ~t/ kg/h given IV), and PEG-superoxide dismutase (5 x 104 ~t/kg/h given IV). Animals were infused with the test drugs or vehicle for 1 h before and during the entire 3.5 h of caerulein infusion. Measuremen ts
Immediately after sacrifice of the animals, samples of pancreas were removed, trimmed of fat, and used for measurement of pancreatic amylase activity, quantitation of pancreatic water content, and light microscopic examination as previously de-
1013
scribed (Rutledge et al. 1987; Saluja et al. 1985). Serum amylase activity and the subcellular distribution of cathepsin B were also evaluated using techniques previously described (Rutledge et al. 1987; Saluja et al. 1985). Mortality data were analyzed using the technique of Kaplan and Meier (1958) and evaluated using the X 2 method with Yates' correction. Results
Diet-Induced Pancreatitis
Administration of the CDE diet resulted in an increase in serum and pancreatic amylase activity, an increase in pancreatic water content (i.e., edema), and mortality rate of approximately 80%. Catalase. Administration of active catalase reduced the magnitude of hyperamylasemia (CDE diet alone=l15___16~t/ml, CDE diet plus catalase=21_+4 ~t/ml) and the degree of pancreatic edema (dry/wet weight ratio of CDE diet alone = 17.6 ___0.9% ; CDE diet plus catalase = 20.9 +_0.8%, P
