Physiology& Behavior,Vol. 50, pp. 397--401. ©Pergamon Press plc, 1991. Printed in the U.S.A.
0031-9384/91 $3.00 + .130
Hyperammonemia and Anorexia in Morris Hepatoma-Bearing Rats I W I L L I A M T. C H A N C E , * t
F U - S H E N G Z H A N G , * T E R I F O L E Y - N E L S O N * A N D J O S E F E. F I S C H E R *
*Department of Surgery, University of Cincinnati Medical Center ~Veterans Affairs Medical Center, 231 Bethesda Avenue, Cincinnati, OH 25267 R e c e i v e d 17 S e p t e m b e r 1990 CHANCE, W. T., F.-S. ZHANG, T. FOLEY-NELSON AND J. E. FISCHER. Hyperammonemia and anorexia in Morris hepatoma-bearing rats. PHYSIOL BEHAV 50(2) 397--401, 1991.--Inoculation of Buffalo rats with Morris hepatoma produced significant anorexia within four weeks and reduced body weight within two weeks. Blood ammonia concentration was increased by 113% when the rats were euthanized, five days after the development of anorexia. Infusing ammonium salts into normal Buffalo rats also induced anorexia at a blood ammonia concentration comparable to that observed in the tumor-beating rats. Although ammonia-infused rats exhibited expected increases in brain tyrosine, tryptophan, and metabolites of dopamine and serotonin, these alterations were attenuated in the tumor-bearing rats. These results indicate that hyperammonemia may be a general consequence of experimental cancer and that the increase in ammonia concentration may be of primary importance in the development of experimental cancer-induced anorexia. The rather small alterations in neurotransmitter metabolism in anoreetic tumor-bearing rats de,emphasize the role aberrations in DA and 5-HT systems in the development of experimental cancer anorexia. Cancer Anorexia Cachexia Tryptophan Nutrition Feeding
Ammonia
Neurotransmitters
THE development of anorexia and cachexia severely restricts therapeutic options for cancer patients (8,9). In addition, many neoplastic therapies may actually worsen the anorexia (14) leading to even greater depletion of lean body tissue in cancer patients. Although the prevalence of cancer-induced anorexia is widespread with approximately 75% of the cancer patients exhibiting disturbances in appetite (16), its etiology is not well understood. Studies employing animal models of cancer anorexia indicate that a humoraUy-elaborated factor appears to be of primary importance in maintaining the anorexia. Thus parabiotic rats that shared a portion of their blood supply with anorectic tumor-beating (TB) rats also exhibited a decrease in food intake (19). In addition, resection of the tumor mass was associated with the rapid return of normal feeding (6,19). Although several factors including toxic hormones (17), peptides (22) and tumor necrosis factor [TNF, (15)] have been suggested as mediators of the anorexia, increased blood concentrations of these substances have not been demonstrated consistently in anorectic TB organisms. In previous research (3) we reported that blood ammonia concentrations were doubled at the onset of anorexia in Fischer 344 rats beating methylcholanthrene-induced (MCA) sarcomas. We also observed (3) that infusing ammonium salts iv into normal rats induced anorexia at blood concentrations of ammonia that were equivalent to those observed in anorectic TB rats. In addition, we recently described rapid normalization of blood ammonia level following removal of the tumor mass in Fischer 344 rats (2). Therefore, hyperammonemia appears to be a major factor in the development of anorexia in Fischer 344 rats bearing MCA sarcomas.
Dopamine
Serotonin
Tyrosine
To test the generality of these observations, we conducted similar experiments in Buffalo rats bearing Morris hepatomas. Therefore, if hyperammonemia is a general feature of experimental cancer anorexia, one would hypothesize that this bepatoma would cause alterations similar to those observed previously with the MCA sarcoma. METHOD
Experiment 1 Frozen samples of the Morris hepatoma 5123 (M) were purchased from Bogden Laboratories (Biomeasure Inc., Hopkinton, MA) and established SC in Buffalo rats (200-250 g, Harlan Laboratories, Madison, WI). The tumor was transplanted SC two additional times at approximately monthly intervals before the initiation of the current experiment. Seventeen male Buffalo rats (250-325 g) served as subjects in this experiment. Ten of these rats were randomly selected to be anesthetized with ether and inoculated with fresh bepatoma tissue, provided by a donor from the colony. These inoculations were administered SC into the anterior-dorsal area using a 4 mm diameter trocar, which transferred approximately 50 mg of fresh tissue. The remaining seven rats were also anesthetized and subjected to the insertion of the empty trocar. All rats were housed individually and were maintained on Purina rat chow and water ad lib under a 12 h light/dark cycle. Food intake and body weight were assessed daily in the early part of the light phase of the light/dark cycle for the remainder of the experiment. Recorded values for food
1Supported by USPHS grant CA 48057 and a research grant from the Department of Veterans Affairs to W.T.C.
397
398
CHANCE, ZHANG, FOLEY-NELSON AND FISCHER
intake were corrected for spillage, which was minimal. Individual TB rats were decapitated in the early part of the light phase when their food intake per 100 g body weight was at least one gram lower than the mean daily intake by control rats for three consecutive days. At sacrifice blood was collected for the deterruination of plasma ammonia concentration by the glutamate dehydrogenase method (10). The gastrocnemius muscles were taken, lyophylized and subjected to protein determination (11) as an index of cachexia. Brains were removed and dissected rapidly over ice into hypothalamus and corpus striatum according to our previously reported methods (3). The concentrations of norepinephrine (NE), dopamine (DA) and serotonin (5-HT), the amino acid precursors, tyrosine (TYR) and tryptophan (TRP), and the metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA) and 5-hydroxyindoleacetic acid (5-HIAA), were determined in brain regions following homogenization in 1 N formic acid/acetone (15/85) by HPLC-EC according to our previously published methods (3). Following homogenization and deproteinization in 5% sulfosalicylic acid, concentrations of free glutamine were determined in the cerebral cortex using a Beckman (Palo Alto, CA) 121MB automated amino acid analyzer as reported previously (3).
Experiment 2 This experiment was conducted to determine whether the infusion of ammonium salts into Buffalo rats would induce anorexia and cause alterations in neurochemistry similar to those observed in Fischer 344 rats. Twenty-seven male Buffalo rats (275-350 g) were purchased from Harlan Laboratories. Following two weeks of acclimation to the laboratory environment, these rats were anesthetized with pentobarbital (45 mg/kg) and silastic catheters (#602-155, Dow Coming, Midland, MI) were implanted into the external jugular veins of 16 rats as published previously (3). The eleven remaining rats were also anesthetized and underwent sham operations. Immediately following surgery, the catheter of each rat was connected to a feed-through swivel (Harvard Biosciences, Cambridge, MA) and a peristaltic pump (#502, Harvard Biosciences) infused normal saline at a rate of 2 ml/h. Saline continued to be infused at this rate for next seven days to permit the return of normal feeding in these groups. At the end of this period, eight rats were begun on infusion (2 ml/h) of ammonium salts at a concentration of 0.2 M (equal parts of ammonium acetate and ammonium bicarbonate, pH=7.6) for three days, which was increased to 0.3 M for two additional days to mimic the gradual increase in blood ammonia observed in TB rats. The remaining eight carmulated rats continued to be infused with sterile saline. All rats were maintained on rat chow and water ad lib during this experiment. Intake of rat chow and body weight were recorded daily. At the conclusion of these infusion schedules all rats were decapitated and subjected to the analyses outlined for experiment 1. The data produced by these experiments were evaluated statisticaUy by analysis of variance techniques, with individual means being compared post hoe by Scheffe's multiple range test or t tests as appropriate. All procedures in this report conform to NIH guidelines and were approved by the University of Cincinnati Animal Care Committee. RF.SULTS
Experiment 1 Rats inoculated with the Morris bepatoma first exhibited consistently reduced food intake (1 g/100 g b.wt. less than control mean) between days 21 and 40 following tumor inoculation. The
Bm ot o o
0~0
6-
0---~0~0------0-~--~0 ,
0- t O
4.
p ( 0.01 vu CONTROL 1 ~
O - - O C O N T R O L (n = 7) • - - • TUMOR (n = 10)
2. Z
1
Z
DAYS PRIOR TO SACRIFICE
FIG. 1. Mean-SEM daily intake of rat chow by control and tumorbearing rats. The data are grouped according to the days prior to sacririce.
mean-+SEM day of initial anorexia in these rats was day 2 7 - 3 . As illustrated in Fig. 1, TB rats as a group exhibited significantly reduced (p
0031-9384/91 $3.00 + .130
Hyperammonemia and Anorexia in Morris Hepatoma-Bearing Rats I W I L L I A M T. C H A N C E , * t
F U - S H E N G Z H A N G , * T E R I F O L E Y - N E L S O N * A N D J O S E F E. F I S C H E R *
*Department of Surgery, University of Cincinnati Medical Center ~Veterans Affairs Medical Center, 231 Bethesda Avenue, Cincinnati, OH 25267 R e c e i v e d 17 S e p t e m b e r 1990 CHANCE, W. T., F.-S. ZHANG, T. FOLEY-NELSON AND J. E. FISCHER. Hyperammonemia and anorexia in Morris hepatoma-bearing rats. PHYSIOL BEHAV 50(2) 397--401, 1991.--Inoculation of Buffalo rats with Morris hepatoma produced significant anorexia within four weeks and reduced body weight within two weeks. Blood ammonia concentration was increased by 113% when the rats were euthanized, five days after the development of anorexia. Infusing ammonium salts into normal Buffalo rats also induced anorexia at a blood ammonia concentration comparable to that observed in the tumor-beating rats. Although ammonia-infused rats exhibited expected increases in brain tyrosine, tryptophan, and metabolites of dopamine and serotonin, these alterations were attenuated in the tumor-bearing rats. These results indicate that hyperammonemia may be a general consequence of experimental cancer and that the increase in ammonia concentration may be of primary importance in the development of experimental cancer-induced anorexia. The rather small alterations in neurotransmitter metabolism in anoreetic tumor-bearing rats de,emphasize the role aberrations in DA and 5-HT systems in the development of experimental cancer anorexia. Cancer Anorexia Cachexia Tryptophan Nutrition Feeding
Ammonia
Neurotransmitters
THE development of anorexia and cachexia severely restricts therapeutic options for cancer patients (8,9). In addition, many neoplastic therapies may actually worsen the anorexia (14) leading to even greater depletion of lean body tissue in cancer patients. Although the prevalence of cancer-induced anorexia is widespread with approximately 75% of the cancer patients exhibiting disturbances in appetite (16), its etiology is not well understood. Studies employing animal models of cancer anorexia indicate that a humoraUy-elaborated factor appears to be of primary importance in maintaining the anorexia. Thus parabiotic rats that shared a portion of their blood supply with anorectic tumor-beating (TB) rats also exhibited a decrease in food intake (19). In addition, resection of the tumor mass was associated with the rapid return of normal feeding (6,19). Although several factors including toxic hormones (17), peptides (22) and tumor necrosis factor [TNF, (15)] have been suggested as mediators of the anorexia, increased blood concentrations of these substances have not been demonstrated consistently in anorectic TB organisms. In previous research (3) we reported that blood ammonia concentrations were doubled at the onset of anorexia in Fischer 344 rats beating methylcholanthrene-induced (MCA) sarcomas. We also observed (3) that infusing ammonium salts iv into normal rats induced anorexia at blood concentrations of ammonia that were equivalent to those observed in anorectic TB rats. In addition, we recently described rapid normalization of blood ammonia level following removal of the tumor mass in Fischer 344 rats (2). Therefore, hyperammonemia appears to be a major factor in the development of anorexia in Fischer 344 rats bearing MCA sarcomas.
Dopamine
Serotonin
Tyrosine
To test the generality of these observations, we conducted similar experiments in Buffalo rats bearing Morris hepatomas. Therefore, if hyperammonemia is a general feature of experimental cancer anorexia, one would hypothesize that this bepatoma would cause alterations similar to those observed previously with the MCA sarcoma. METHOD
Experiment 1 Frozen samples of the Morris hepatoma 5123 (M) were purchased from Bogden Laboratories (Biomeasure Inc., Hopkinton, MA) and established SC in Buffalo rats (200-250 g, Harlan Laboratories, Madison, WI). The tumor was transplanted SC two additional times at approximately monthly intervals before the initiation of the current experiment. Seventeen male Buffalo rats (250-325 g) served as subjects in this experiment. Ten of these rats were randomly selected to be anesthetized with ether and inoculated with fresh bepatoma tissue, provided by a donor from the colony. These inoculations were administered SC into the anterior-dorsal area using a 4 mm diameter trocar, which transferred approximately 50 mg of fresh tissue. The remaining seven rats were also anesthetized and subjected to the insertion of the empty trocar. All rats were housed individually and were maintained on Purina rat chow and water ad lib under a 12 h light/dark cycle. Food intake and body weight were assessed daily in the early part of the light phase of the light/dark cycle for the remainder of the experiment. Recorded values for food
1Supported by USPHS grant CA 48057 and a research grant from the Department of Veterans Affairs to W.T.C.
397
398
CHANCE, ZHANG, FOLEY-NELSON AND FISCHER
intake were corrected for spillage, which was minimal. Individual TB rats were decapitated in the early part of the light phase when their food intake per 100 g body weight was at least one gram lower than the mean daily intake by control rats for three consecutive days. At sacrifice blood was collected for the deterruination of plasma ammonia concentration by the glutamate dehydrogenase method (10). The gastrocnemius muscles were taken, lyophylized and subjected to protein determination (11) as an index of cachexia. Brains were removed and dissected rapidly over ice into hypothalamus and corpus striatum according to our previously reported methods (3). The concentrations of norepinephrine (NE), dopamine (DA) and serotonin (5-HT), the amino acid precursors, tyrosine (TYR) and tryptophan (TRP), and the metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA) and 5-hydroxyindoleacetic acid (5-HIAA), were determined in brain regions following homogenization in 1 N formic acid/acetone (15/85) by HPLC-EC according to our previously published methods (3). Following homogenization and deproteinization in 5% sulfosalicylic acid, concentrations of free glutamine were determined in the cerebral cortex using a Beckman (Palo Alto, CA) 121MB automated amino acid analyzer as reported previously (3).
Experiment 2 This experiment was conducted to determine whether the infusion of ammonium salts into Buffalo rats would induce anorexia and cause alterations in neurochemistry similar to those observed in Fischer 344 rats. Twenty-seven male Buffalo rats (275-350 g) were purchased from Harlan Laboratories. Following two weeks of acclimation to the laboratory environment, these rats were anesthetized with pentobarbital (45 mg/kg) and silastic catheters (#602-155, Dow Coming, Midland, MI) were implanted into the external jugular veins of 16 rats as published previously (3). The eleven remaining rats were also anesthetized and underwent sham operations. Immediately following surgery, the catheter of each rat was connected to a feed-through swivel (Harvard Biosciences, Cambridge, MA) and a peristaltic pump (#502, Harvard Biosciences) infused normal saline at a rate of 2 ml/h. Saline continued to be infused at this rate for next seven days to permit the return of normal feeding in these groups. At the end of this period, eight rats were begun on infusion (2 ml/h) of ammonium salts at a concentration of 0.2 M (equal parts of ammonium acetate and ammonium bicarbonate, pH=7.6) for three days, which was increased to 0.3 M for two additional days to mimic the gradual increase in blood ammonia observed in TB rats. The remaining eight carmulated rats continued to be infused with sterile saline. All rats were maintained on rat chow and water ad lib during this experiment. Intake of rat chow and body weight were recorded daily. At the conclusion of these infusion schedules all rats were decapitated and subjected to the analyses outlined for experiment 1. The data produced by these experiments were evaluated statisticaUy by analysis of variance techniques, with individual means being compared post hoe by Scheffe's multiple range test or t tests as appropriate. All procedures in this report conform to NIH guidelines and were approved by the University of Cincinnati Animal Care Committee. RF.SULTS
Experiment 1 Rats inoculated with the Morris bepatoma first exhibited consistently reduced food intake (1 g/100 g b.wt. less than control mean) between days 21 and 40 following tumor inoculation. The
Bm ot o o
0~0
6-
0---~0~0------0-~--~0 ,
0- t O
4.
p ( 0.01 vu CONTROL 1 ~
O - - O C O N T R O L (n = 7) • - - • TUMOR (n = 10)
2. Z
1
Z
DAYS PRIOR TO SACRIFICE
FIG. 1. Mean-SEM daily intake of rat chow by control and tumorbearing rats. The data are grouped according to the days prior to sacririce.
mean-+SEM day of initial anorexia in these rats was day 2 7 - 3 . As illustrated in Fig. 1, TB rats as a group exhibited significantly reduced (p
