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Effect of acetaminophen toxicity on erythrocyte osmotic fragility in the fisher rat
Camp. Biochem. Physrol. Vol. 79C, No. I, pp. 27.-30, 1984 Printed in Cireat Britain
0306-4492184$3.00 + 0.00 Pergamon Press Ltd
EFFECT OF ACETAMINOPHEN TOXICITY ON ERYTHROCYTE OSMOTIC FRAGILITY IN THE FISHER RAT KRISHAN
Departments
L.
RAHEJA,
STEPHEN A.
of Pharmacology,
LANDAW,
WILLEM
Medicine and Pathology, Medical Centers, Syracuse,
G.
LINSCHEER
and CHAIDONC CHO
Veterans Administration NY 13210, USA
and SUNY-Upstate
(Received 27 January 1984)
Abstract--l. The protective effect of propylthiouracil (PTU) pretreatment against acetaminophen-induced erythrocyte osmotic fragility was determined in the male Fisher rat. Hepatotoxicity was assessed for comparative purposes. 2. PTU (0.15%) was fed in chow for a period of 12 days. Acetaminophen (I g/kg body wt) was then administered orally by a stomach tube after an overnight fast. The rats were killed either 4 or 24 hr later. 3. Erythrocyte osmotic fragility was determined by the extent of hemolysis in various concentrations of NaCl solutions. Hepatotoxicity was assessed by a rise in serum transaminases and by histological examination of hepatic tissue. 4. PTU treatment when compared with control not only protected rats against acetaminophen-induced hepatotoxicity as reported before, but also protected against erythrocyte osmotic fragility. 5. The time course of acetaminophen toxicity seems to be similar for liver and erythrocyte since both showed damage after 24 hr but not after 4 hr of acetaminophen administration. 6. The data show that PTU pretreatment affords protection against acetaminophen-induced increased erythrocyte osmotic fragility even when their glutathione concentrations were not significantly different. suggesting that PTU per se has a protective effect.
INTRODUCTION
determine if the PTU protection was limited to liver or extends to other organs. The effect of a toxic dose of a~etaminophen was examined in control and PTU treated rats on changes in erythrocyte GSH concentrations and their susceptibility to osmotic fragility. For comparative purposes changes in hepatic GSH and hepatotoxicity were also determined. Moreover, it was hypothesized that if a relationship were to exist between acetaminophen-induced hepatic and erythrocyte damage, then blood could conveniently be used to determine possible hepatotoxicity.
Propylthiouracil (PTU) pretreatment of rats has been shown to protect them against acetaminopheninduced hepatotoxicity (Linscheer et al., 1980). The hepatic damage is associated with initial depletion of reduced hepatic glutathione (GSH) concentrations, followed by -covalent binding of the acetaminophen reactive metabolite(s) to hepatic macromolecules (Mitchell et al., 1973) to initiate the process which leads to tissue necrosis. The protective effect of PTU, however, was observed even in the presence of very low levels of hepatic GSH concentrations (Raheja et af., 1983) and was mostly independent of its hypothyroid effect (Raheja et af., 1982). These observations suggested that PTU per se probably has a protective effect and this is in agreement with the observations of Yamada ef al. (1980, 1982) that PTU can act as a substrate for g~utathione-~-transferase in vitro and can also decrease the covalent binding of acetaminophen metabolite(s) to hepatic macromolecules in vivo. Cell membrane defect is considered a terminal feature in most types of liver cell necrosis (Popper, 1975) and treatment modalities which prevent hepatic necrosis have also been reported to protect erythrocytes against osmotic damage (Stachura et al., 198 1). The membrane integrity of erythrocytes like hepatocytes is dependent upon optimal cellular GSH concentrations (Necheles et al., 1969; Spelberg et a/.; 1979). Literature review indicates that it has not been reported if a~etaminophen would also deplete erythrocyte glutathione as it does in hepatocytes (Linscheer et al., 1980; Raheja et al., 1983), and alter their osmotic fragility as an expression of cell damage. The study was, therefore, conducted to
MATERIALS
AND METHODS
Male Fisher rats which have been reported to be more susceptible to drug toxicity when compared to other strains of rats (Dent et al., 1980) were used in these studies. Rats were housed individually in raised wire screen bottom cages in a temperature-humidity controlled laboratory. A 12: 12 iight-dark cycle was maintained. Rats had always free access to drinking water and their respective diets. In the first experiment, 16 rats were used in four treatment groups. Two groups were fed plain chow while the other two groups were fed the same chow containing 0.15;; PTU for a period of 12 days. All rats were fasted overnight but had free access to drinking water. One of the two groups fed plain chow or chow with PTU were administered acetaminophen orally (1 g/kg body wt) by a stomach tube. Their respective controls were given an equivalent amount of saline, which was used as the vehicle for acetaminophen administration. After acetaminophen administration all rats had free access to their respective diet and water. Rats were weighed 24 hours later, they were bled by direct cardiac puncture under light ether anesthesia and then killed by exsanguination. Organs were examined for any gross pathology. Liver, kidney and thyroid gland were weighed. Part of the liver was preserved in 10% buffered formalin and 27
28
KRISHAN L. RAHEJA et al.
Table 1. Effect of a toxrc dose of acetaminophen
administration
(1 g/kg body wt) on hepatotoxicity male Fisher rats
Control-acetaminophen Hepatic Hepatic
GSH (~mole,‘g) necrotic score
(k4) Hematocrit (‘Jo) Hemoglobin (gjdl) Erythrocyte membrane-SH concentration (nmole~mg membrane protein) Erythrocyte osmotic fraglhty ‘A hemolysis in: 0.4% NaCl solution 0.45% NaCl solution
PTU-acetaminophen
and erythrocyte
Control-saline
osmotic
fragility
in
PTU-saline
4.56 ? I .26b 3.8 k 0.2’
7.26 f 0.20” 1.5 + O.Sb
4.92 f0.18h 0
6.88 * 1.20” 0
40.8 14.5 38.3
f 3.3” * 1.1” + _ 7.3”
44.0 i 1.6” 14.7 * 2.2” 37.4 * 4.9”
43.3 k 0.4” 15.8 k 0.4” 34.0 k4.1”
43.4 f 0.6” 16.0 f 0.2’ 42.9 + 5.8”
78.2 i: 2.3” 45.6 i 4.6”
71.5 f 1.9b 32.2 f 2.1b
62.8 f 4.3b 39.5 + _ 3.0”b
57.8 + 7.6’ 32.2 + 4.1ab
Values are mean + SEM for four observations except for erythrocyte osmotic fagility which represents mean of eight observations. Values with a common superscript are not significantly different. Rats were killed 24 hr after acetaminophen administration. In another experiment where six control euthyroid and six PTU pretreated rats were given acetaminophen (1 g/kg body wt) and killed 24 hr later, erythrocyte osmotic fragility expressed as percentage hemolysis was significantly lower in PTU treated rats (61.4 f 2.7 and 19.3 f 1.9>, in 0.4 and 0.45% NaCl solutions respectively) compared to those for control euthyroid rats (78.1 + 3.0 and 34.3 f 6.4% in 0.4 and 0.45”, NaCl solutions respectively). The membrane-SH concentrations were not different being 49.7 f0.7 and 49.6 i 1.7 nmole~mg membrane protein for the PTU and control rats respectively.
the remainder was frozen in acetone-dry ice for later GSH determination. Blood was used for determination of hematocrit, hemoglobin, membrane sulfhydryl (SH) content and for erythrocyte osmotic fragility. Erythrocyte membrane protein profile was studied by using polyacrylamide gel electrophoresis in the presence and absence of dithiothreitol, a reducing agent. In the second experiment changes with time in hepatic and erythrocyte glutathione concentrations as well as hepatotoxicity and erythrocyte osmotic fragility were determined after a toxic dose of acetaminophen (1 g/kg body wt with 5 yCi [3H]acetaminophen/rat) in eight control and eight rats treated with PTU for 12 days. Four rats from each group were bled and killed 4 hr later and the remaining four 24 hr after acetaminophen administration as in the first experiment. Organs were examined and weighed, liver preserved in buffered formalin and in acetone-dry ice as in the first experiment for glutathione and glycogen determinations. The gastrointestinal (GI) tract with its contents was removed, homogenized in known volume of saline, and an determine the percentage of ahquot counted to [3H]acetaminophen administered remaining in the GI tract 4 and 24 hr later. Radioactivity remaining in blood and liver tissue was also determined at these time intervals. Blood was used for the determination of erythrocyte GSH concentrations, their osmotic fragility and for serum transaminases (SCOT, SGPT) activities. Serum glutamic oxalacetic transaminase (SGOT) and glutamic pyruvic transaminase (SGPT), hepatic GSH and glycogen determinations, tissue radioactivity and histopathology were assessed as reported before (Linscheer et al., 1980). Hematocrit, erythrocyte osmotic fragility, membrane suhhydryl (SH) contents, erythrocyte GSH concentrations, membrane protein contents and membrane protein SDS polyacrylamide gel electrophoretic pattern were determined by the method of Maile (1967), M&own et al. (1982) Beutler et al. (1963). Lowry et al. (1951) and Fairbanks et al. (1971) respectively. Since erythrocyte osmotic fragility values at 24 hr after acetaminophen administration were not statistically different in the two experiments, these were pooled for presentation and statistical analysis. Furthermore, another group of six control and six PTU treated rats were used for the erythrocyte osmotic fragility study, 24 hr after acetaminophen administration, to substantiate our findings. The data have been presented as mean + SEM. Statistical significance was determined by Student’s t-test and analysis of variance (Snedecor and Cochran, 1971) using the Tektronix 4051 Statistical Program Vol. 2. Comparisons between means with a P value of less than 0.05 were considered statistically significant.
RESULTS
except for the thyroid gland, were not affected either by PTU or acetaminophen treatment. The thyroid gland in the 12 days PTU treated rats was significantly enlarged (25.6 k 1.1 mg/lOO g body wt) when compared with euthyroid controls (7.4 + 1.1 mg). The effect of acetaminophen administration to control and PTU treated rats on hepatic GSH concentrations and necrotic score, on blood hematocrit, hemoglobin, on erythrocyte membrane-SH contents as well as on erythrocyte osmotic fragility 24 hr after its administration is shown in Table 1. The PTU pretreated rats given acetaminophen had significantly higher hepatic GSH concentrations when compared to similarly treated control rats and these values were not different from the respective PTU treated and control rats given saline instead of acetaminophen. The necrotic score of 3.8 k 0.2 for the control given acetaminophen was significantly higher compared to 1.5 _t 0.8 for the PTU treated rats. Neither PTU treatment nor acetaminophen administration had any effect on hematocrit or hemoglobin. Although membrane SH-contents were not erythrocyte different, erythrocyte osmotic fragility expressed as a percentage of hemolysis was significantly greater in the control rats compared to PTU treated rats given acetaminophen, in 0.40 and 0.45’j/, sodium chloride solutions (Table 1). Also shown in Table I are results of the other six control and six PTU treated rats given acetaminophen and killed 24 hr later. Densitometric scans of the erythrocyte membrane sodium dodecyl polyacrylamide gel electrophoresis for the PTU treated and control rats with or without acetaminophen, 24 hr after its administration (Fig. 1) shows that the membrane protein pattern was similar in various treatment groups. The results of the second experiment on the effect of acetaminophen 4 and 24 hr after its administration on serum transaminases, hepatic GSH and glycogen concentrations, on erythrocyte glutathione and their osmotic fragility in the PTU treated and control rats are shown in Table 2. Acetaminophen administration did not affect serum transaminases (SGOT, SGPT) in either treatment group after 4 hr but after 24 hr both The
organ
weights,
Acetaminophen
PTU-Acet
PTU-Saline
and RBC damage
Control-Saline
Control-Acet
Fig. I. Densitometric scans for various protein bands of the polyacrylamide gels of erythrocyte membranes prepared from PTU pretreated hypothyroid and control euthyroid rats 24 hr after oral acetaminophen or saline administration.
SGOT and SGPT were significantly elevated (7968 f 2709 and 4428 + 1348 respectively) in the control rats compared to 101 f 19 and 51 k 20 sigma units/ml respectively for the PTU treated rats. Although hepatic GSH concentrations were significantly higher for the PTU treated rats (3.48 f 0.18 p mol/g) compared to euthyroid controls (2.02 + 0.18) at 4 hr, necrosis was not observed in either group. After 24 hr the GSH values for both groups had increased and it was significantly higher for the PTU group. Hepatic glycogen was also very low at 4 hr in both groups and stayed low at 24 hr in the control rats (4.3 k 2.0mg/g) compared to 30.6 f 6.3 in the PTU treated rats. Like hepatic GSH, erythrocyte GSH concentrations were lower at 4 hr compared to 24 hr values but there was no difference between control and PTU treated rats at either time interval. Osmotic fragility like hepatic necrosis was not different at 4 hr after acetaminophen administration in eurthyroid control and PTU treated rats (76.4 + 2.0 and 72.8 f 3.1%) but it was significantly higher for the control rats compared to PTU treated rats after 24 hours (80.6 k 1.7 and 72.5 k 2.3% respectively). The percentage of the administered acetaminophen radioactivity recovered from whole
gastro-intestinal tract, per ml of serum and per g of liver tissue 4 hr after acetaminophen administration in the PTU treated rats was 34.3 f 2.9, 0.54 k 0.01 and 0.64 f 0.03 and at 24 hr was 5.9 + 0.5, 0.07 f 0.005 and 0.07 f 0.008 respectively. S&ilar values for the euthyroid controls at 4 hr were 71.3 f 3.6, 0.32 + 0.02 and 0.55 k 0.06 and at 24 hr 11 .O & 1.6, 0.11 f 0.02 and 0.10 k 0.01% respectively. DISCUSSION
Serum transaminases, elevation of which is considered an index of hepatic damage in response to various xenobiotics, suggest that acetaminophen did not cause hepatic damage after 4 hr but did so after 24 hr of its administration. Cellular damage to the erythrocyte membrane as expressed in terms of its susceptibility to osmotic lysis, also showed no change after 4 hr but a significant increase after 24 hr of acetaminophen administration. Erythrocyte and hepatic glutathione concentrations were similarly affected as both were lower at 4 hr compared to 24 hr determinations. Although there was no difference in erythrocyte glutathione or erythrocyte membrane-SH contents in the control and PTU treated rats, eryth-
Table 2. Effect of a toxic dose of acetaminophen (1 g/kg body wt) administration on serum transaminases and hepatic and ervthrocvte zlutathione concentrations in control and PTU oretreated male Fisher rat Control-acetaminophen Hours after acetaminophen 24 4 Serum glutamic oxalacetic acid transaminase (sigma units/ml) Serum glutamic pyruvic transaminase (sigma umts/ml) Hepatic glutathione (pmoleig) Hepatic glycogen (mg/g) Erythrocyte glutathione (me/100 ml oacked RBC)
PTU-acetaminophen administration 74 4
87 * 14b
7968 k 2709”
74*
IIh
101 f 19t
31i4b
4428 * 13484
35*
IIb
51 f 20h
2.02 It 0.18’ 7.8 & l.Ob 53.4 * I .8h
4.28 i 1.46b 4.3 * 2.0b 79.8 * 1 I .8db
3.48 f 0.1 Sb 6.0 f O.gh 59.3 * 3.7b
7.68 + 0.60” 30.6 f 6.3” 89.4 * 5.4”
Values are mean f SEM for four observations. Values with a common superscript are not significantly different. The erythrocyte osmotic fragility was not different after 4 hr with values of 76.4 k 2.0 and 72.8 f 3.1 for the control and PTU treated rats but was significantly different after 24 hr with values of 80.6 i 1.7 and 72.5 + 2.3 for the two respective groups. The hepatic glycogen concentrations for the control and PTU treated rats not given acetaminophen and killed at 24 hr were 66.0 + 3.8 and 59.5 i 2.3 mgfg respectively.
30
KRISHAN L. RAHEJA et d.
rocyte osmotic fragility was consistently greater in controls, suggesting that PTU per se or its induced hypothyroidism has a protective effect. Such an effect of PTU per se independent of its effect on hepatic GSH or its hypothyroid effect has been previously reported by us against acetaminophen-induced hepatotoxicity (Raheja et ul., 1982, 1983). It is not likely that increased erythrocyte osmotic fragility in the control rats compared to PTU treated rats given acetaminophen is related to changes in their membrane protein composition because the SDS gel electrophoretic pattern with or without dithiothreitol was similar in the two treatment groups given acetaminophen. Moreover, it was not different from their respective controls given only isotonic saline instead of acetaminophen (Fig. 1). The protective effect of PTU treatment could not be due to decreased intestinal absorption of the drug, because the absorption of acetaminophen was actually greater in the PTU treated rats 4 hr after its gavage. The greater rise in hepatic glycogen and giutathione levels in the PTU treated rats compared to control rats, from 4 to 24 hr after acetaminophen administration is possibly a reflection of less damage in the PTU treated rats and, therefore, their better capacity to recuperate from the toxic effect. Although our results show that changes in erythrocyte osmotic fragility do not precede hepatic necrosis and thus could not predict pending necrosis, as we had hypothesized, determination of erythrocyte osmotic fragility could prove to be a convenient in vitro method to test individual susceptibility to toxicity of various xenobiotics which are metabolized like acetaminophen by the cytochrome P-450 dependent mixed function oxidase enzyme system. Our data show that the time course of increase in erythrocyte osmotic fragility and hepatotoxicity subsequent to a toxic dose of acetaminophen is similar, suggesting that acetaminophen toxicity probably affects all organs whose integrity normally depends upon optimal glutathione concentrations. Furthermore, PTU pretreatment protects both liver and erythrocytes against acetaminophen toxicity in even the more susceptible Fisher rat than the Wistar rat which we previously used
in all our
studies.
Ackno~~ledgements~The authors are grateful to Robert Ciuancial, Katherine Larsson and Carol Sterling for their excellent technical assistance and to Sally Michalski for her secretarial help. We also thank Dr. D. G. Patel, Barbara Kopp Geriatric Research Center, for kindly supplying us with the experimental animals. This work was supported in part by the Veterans Administration, US Public Health Service Grant GM 27801, and National Aeronautics and Space Administration Contract No. A62829B. REFERENCES Beutler E., Duron 0. and Kelley B. M. (1963) Improved method for the determination of blood glutathione. .I. Lab. c/in. Med. 61, 882-888.
Dent J. G., Graichen M. E., Schnell S. and Lasker J. (1980) Constitute and induced hepatic microsomal cytochrome P-450 monoxygenase activities in male Fisher-344 and CD rats. Tax. appl. Pharmac. 52, 45-53. Fairbanks G., Steck T. L. and Wallach D. J. H. (1971) Electrophoretic analysis of the major polypeptides of the human erythrocyte membrane. Biochemistry 10, 26062617. Linscheer W. G., Raheja K. L., Cho C. and Smith N. J. (1980) Mechanism of the protective effect of propylthiouracil against acetaminophen (Tylenol) toxicity in the rat. Gustroenterology 78, 100-107. Lowry O., Rosenbrough N. J., Farr A. L. and Randall R. J. (1951) Protein measurement with Folin uhenol reaeent. J. hiol. Chem. 193, 265-275. Maile J. B. (1967)/ Lahoratorv Medicine-Hemutolow. ,>_ 3rd edn, p. 586. Mosby, St. Louis. McGown E. L., O’Connor R. J. and Neher J. W. (1982) Erythrocyte filterability, fragility and membrane proteins in Folic acid deficient guinea Dies. J. Nutr. 112. 92-97. Mitchell J. R., Jollow D. i., Potter-W. Z., Gillette J. R. and Brodie B. B. (1973) Acetaminophen-induced hepatic necrosis. IV. Protective role of glutathione. J. Pharmac. exp. Ther. 187, 211-217. Necheles T. F., Maldonado N., Barguet-Chediak A. and Allen D. M. (1969) Homozygous erythrocyte glutathioneperoxidase deficiency: Clinical and biochemical studies. Blood 33, 164-169. Popper H. (1975) Morphologic features of hepatocellular necrosis in human disease. In Puthogenesis and Mechanism of Liver Cell Necrosis (Edited by Keppler D.), pp. 15-24. MTP Press Ltd, Lancaster. Potter W. Z., Thorgeirsson S. S., Jollow D. J. and Mitchell J. R. (1974) Acetaminophen-induced hepatic necrosis. V. Correlation of hepatic necrosis, covalent binding and glutathione depletion in hamsters. Pharmacology 12, 129-143. Raheja K. L., Linscheer W. G., Cho C. and Mahany D. (1982) Protective effect of propylthiouracil independent of its hypothyroid effect on acetaminophen toxicity in the rat. J. Pharmac. exp. Ther. 220, 427432. Raheja K. L., Linscheer W. G. and Cho C. (1983a) Prevention of acetaminophen hepatotoxicity by propylthiouracil in the glutathione depleted rat. Camp. Biothem. Physiol. 76C, 9-14. Raheja K. L., Linscheer W. G. and Cho C. (1983b) Hepatotoxicity and metabolism of acetaminophen in male and female rats. J. toxic. Enuir. Health 12, 143-158. Snedecor G. M. and Cochran W. G. (1967) Statistical Methods, 6 edn, p. 59. Iowa State University Press, Ames. Soeifbere S. P.. Boxer L. A.. Corash L. M. and Schulman I J. D. 6979) improved erythrocyte survival with high-dose vitamin E in chronic hemolyzing G-6-PD and glutathione synthetase deficiencies. Ann. Infer. Med. 90, I
53-54. Stachura J., Tarnawski A., Ivey K. J., Mach T., Bogdal J., Szczudrawa J. and Klimczyk B. (1981) Prostaglandin protection of carbon tetrachloride-induced liver cell necrosis in the rat. Gastroenterology 81, 211-217. Yamada T. and Kaolowitz N. (1980) Propvlthiouracil-A substrate for the glutathione S‘-transferases that competes with glutathione. J. biol. Chem. 255, 3508-3513. Yamada T., Ludwig S., Kuhlenkamp J. and Kaplowitz N. (1982) Direct protection against acetaminophen hepatotoxicity by propylthiouracil. J. clin. Invest. 67, 688-695.
0306-4492184$3.00 + 0.00 Pergamon Press Ltd
EFFECT OF ACETAMINOPHEN TOXICITY ON ERYTHROCYTE OSMOTIC FRAGILITY IN THE FISHER RAT KRISHAN
Departments
L.
RAHEJA,
STEPHEN A.
of Pharmacology,
LANDAW,
WILLEM
Medicine and Pathology, Medical Centers, Syracuse,
G.
LINSCHEER
and CHAIDONC CHO
Veterans Administration NY 13210, USA
and SUNY-Upstate
(Received 27 January 1984)
Abstract--l. The protective effect of propylthiouracil (PTU) pretreatment against acetaminophen-induced erythrocyte osmotic fragility was determined in the male Fisher rat. Hepatotoxicity was assessed for comparative purposes. 2. PTU (0.15%) was fed in chow for a period of 12 days. Acetaminophen (I g/kg body wt) was then administered orally by a stomach tube after an overnight fast. The rats were killed either 4 or 24 hr later. 3. Erythrocyte osmotic fragility was determined by the extent of hemolysis in various concentrations of NaCl solutions. Hepatotoxicity was assessed by a rise in serum transaminases and by histological examination of hepatic tissue. 4. PTU treatment when compared with control not only protected rats against acetaminophen-induced hepatotoxicity as reported before, but also protected against erythrocyte osmotic fragility. 5. The time course of acetaminophen toxicity seems to be similar for liver and erythrocyte since both showed damage after 24 hr but not after 4 hr of acetaminophen administration. 6. The data show that PTU pretreatment affords protection against acetaminophen-induced increased erythrocyte osmotic fragility even when their glutathione concentrations were not significantly different. suggesting that PTU per se has a protective effect.
INTRODUCTION
determine if the PTU protection was limited to liver or extends to other organs. The effect of a toxic dose of a~etaminophen was examined in control and PTU treated rats on changes in erythrocyte GSH concentrations and their susceptibility to osmotic fragility. For comparative purposes changes in hepatic GSH and hepatotoxicity were also determined. Moreover, it was hypothesized that if a relationship were to exist between acetaminophen-induced hepatic and erythrocyte damage, then blood could conveniently be used to determine possible hepatotoxicity.
Propylthiouracil (PTU) pretreatment of rats has been shown to protect them against acetaminopheninduced hepatotoxicity (Linscheer et al., 1980). The hepatic damage is associated with initial depletion of reduced hepatic glutathione (GSH) concentrations, followed by -covalent binding of the acetaminophen reactive metabolite(s) to hepatic macromolecules (Mitchell et al., 1973) to initiate the process which leads to tissue necrosis. The protective effect of PTU, however, was observed even in the presence of very low levels of hepatic GSH concentrations (Raheja et af., 1983) and was mostly independent of its hypothyroid effect (Raheja et af., 1982). These observations suggested that PTU per se probably has a protective effect and this is in agreement with the observations of Yamada ef al. (1980, 1982) that PTU can act as a substrate for g~utathione-~-transferase in vitro and can also decrease the covalent binding of acetaminophen metabolite(s) to hepatic macromolecules in vivo. Cell membrane defect is considered a terminal feature in most types of liver cell necrosis (Popper, 1975) and treatment modalities which prevent hepatic necrosis have also been reported to protect erythrocytes against osmotic damage (Stachura et al., 198 1). The membrane integrity of erythrocytes like hepatocytes is dependent upon optimal cellular GSH concentrations (Necheles et al., 1969; Spelberg et a/.; 1979). Literature review indicates that it has not been reported if a~etaminophen would also deplete erythrocyte glutathione as it does in hepatocytes (Linscheer et al., 1980; Raheja et al., 1983), and alter their osmotic fragility as an expression of cell damage. The study was, therefore, conducted to
MATERIALS
AND METHODS
Male Fisher rats which have been reported to be more susceptible to drug toxicity when compared to other strains of rats (Dent et al., 1980) were used in these studies. Rats were housed individually in raised wire screen bottom cages in a temperature-humidity controlled laboratory. A 12: 12 iight-dark cycle was maintained. Rats had always free access to drinking water and their respective diets. In the first experiment, 16 rats were used in four treatment groups. Two groups were fed plain chow while the other two groups were fed the same chow containing 0.15;; PTU for a period of 12 days. All rats were fasted overnight but had free access to drinking water. One of the two groups fed plain chow or chow with PTU were administered acetaminophen orally (1 g/kg body wt) by a stomach tube. Their respective controls were given an equivalent amount of saline, which was used as the vehicle for acetaminophen administration. After acetaminophen administration all rats had free access to their respective diet and water. Rats were weighed 24 hours later, they were bled by direct cardiac puncture under light ether anesthesia and then killed by exsanguination. Organs were examined for any gross pathology. Liver, kidney and thyroid gland were weighed. Part of the liver was preserved in 10% buffered formalin and 27
28
KRISHAN L. RAHEJA et al.
Table 1. Effect of a toxrc dose of acetaminophen
administration
(1 g/kg body wt) on hepatotoxicity male Fisher rats
Control-acetaminophen Hepatic Hepatic
GSH (~mole,‘g) necrotic score
(k4) Hematocrit (‘Jo) Hemoglobin (gjdl) Erythrocyte membrane-SH concentration (nmole~mg membrane protein) Erythrocyte osmotic fraglhty ‘A hemolysis in: 0.4% NaCl solution 0.45% NaCl solution
PTU-acetaminophen
and erythrocyte
Control-saline
osmotic
fragility
in
PTU-saline
4.56 ? I .26b 3.8 k 0.2’
7.26 f 0.20” 1.5 + O.Sb
4.92 f0.18h 0
6.88 * 1.20” 0
40.8 14.5 38.3
f 3.3” * 1.1” + _ 7.3”
44.0 i 1.6” 14.7 * 2.2” 37.4 * 4.9”
43.3 k 0.4” 15.8 k 0.4” 34.0 k4.1”
43.4 f 0.6” 16.0 f 0.2’ 42.9 + 5.8”
78.2 i: 2.3” 45.6 i 4.6”
71.5 f 1.9b 32.2 f 2.1b
62.8 f 4.3b 39.5 + _ 3.0”b
57.8 + 7.6’ 32.2 + 4.1ab
Values are mean + SEM for four observations except for erythrocyte osmotic fagility which represents mean of eight observations. Values with a common superscript are not significantly different. Rats were killed 24 hr after acetaminophen administration. In another experiment where six control euthyroid and six PTU pretreated rats were given acetaminophen (1 g/kg body wt) and killed 24 hr later, erythrocyte osmotic fragility expressed as percentage hemolysis was significantly lower in PTU treated rats (61.4 f 2.7 and 19.3 f 1.9>, in 0.4 and 0.45% NaCl solutions respectively) compared to those for control euthyroid rats (78.1 + 3.0 and 34.3 f 6.4% in 0.4 and 0.45”, NaCl solutions respectively). The membrane-SH concentrations were not different being 49.7 f0.7 and 49.6 i 1.7 nmole~mg membrane protein for the PTU and control rats respectively.
the remainder was frozen in acetone-dry ice for later GSH determination. Blood was used for determination of hematocrit, hemoglobin, membrane sulfhydryl (SH) content and for erythrocyte osmotic fragility. Erythrocyte membrane protein profile was studied by using polyacrylamide gel electrophoresis in the presence and absence of dithiothreitol, a reducing agent. In the second experiment changes with time in hepatic and erythrocyte glutathione concentrations as well as hepatotoxicity and erythrocyte osmotic fragility were determined after a toxic dose of acetaminophen (1 g/kg body wt with 5 yCi [3H]acetaminophen/rat) in eight control and eight rats treated with PTU for 12 days. Four rats from each group were bled and killed 4 hr later and the remaining four 24 hr after acetaminophen administration as in the first experiment. Organs were examined and weighed, liver preserved in buffered formalin and in acetone-dry ice as in the first experiment for glutathione and glycogen determinations. The gastrointestinal (GI) tract with its contents was removed, homogenized in known volume of saline, and an determine the percentage of ahquot counted to [3H]acetaminophen administered remaining in the GI tract 4 and 24 hr later. Radioactivity remaining in blood and liver tissue was also determined at these time intervals. Blood was used for the determination of erythrocyte GSH concentrations, their osmotic fragility and for serum transaminases (SCOT, SGPT) activities. Serum glutamic oxalacetic transaminase (SGOT) and glutamic pyruvic transaminase (SGPT), hepatic GSH and glycogen determinations, tissue radioactivity and histopathology were assessed as reported before (Linscheer et al., 1980). Hematocrit, erythrocyte osmotic fragility, membrane suhhydryl (SH) contents, erythrocyte GSH concentrations, membrane protein contents and membrane protein SDS polyacrylamide gel electrophoretic pattern were determined by the method of Maile (1967), M&own et al. (1982) Beutler et al. (1963). Lowry et al. (1951) and Fairbanks et al. (1971) respectively. Since erythrocyte osmotic fragility values at 24 hr after acetaminophen administration were not statistically different in the two experiments, these were pooled for presentation and statistical analysis. Furthermore, another group of six control and six PTU treated rats were used for the erythrocyte osmotic fragility study, 24 hr after acetaminophen administration, to substantiate our findings. The data have been presented as mean + SEM. Statistical significance was determined by Student’s t-test and analysis of variance (Snedecor and Cochran, 1971) using the Tektronix 4051 Statistical Program Vol. 2. Comparisons between means with a P value of less than 0.05 were considered statistically significant.
RESULTS
except for the thyroid gland, were not affected either by PTU or acetaminophen treatment. The thyroid gland in the 12 days PTU treated rats was significantly enlarged (25.6 k 1.1 mg/lOO g body wt) when compared with euthyroid controls (7.4 + 1.1 mg). The effect of acetaminophen administration to control and PTU treated rats on hepatic GSH concentrations and necrotic score, on blood hematocrit, hemoglobin, on erythrocyte membrane-SH contents as well as on erythrocyte osmotic fragility 24 hr after its administration is shown in Table 1. The PTU pretreated rats given acetaminophen had significantly higher hepatic GSH concentrations when compared to similarly treated control rats and these values were not different from the respective PTU treated and control rats given saline instead of acetaminophen. The necrotic score of 3.8 k 0.2 for the control given acetaminophen was significantly higher compared to 1.5 _t 0.8 for the PTU treated rats. Neither PTU treatment nor acetaminophen administration had any effect on hematocrit or hemoglobin. Although membrane SH-contents were not erythrocyte different, erythrocyte osmotic fragility expressed as a percentage of hemolysis was significantly greater in the control rats compared to PTU treated rats given acetaminophen, in 0.40 and 0.45’j/, sodium chloride solutions (Table 1). Also shown in Table I are results of the other six control and six PTU treated rats given acetaminophen and killed 24 hr later. Densitometric scans of the erythrocyte membrane sodium dodecyl polyacrylamide gel electrophoresis for the PTU treated and control rats with or without acetaminophen, 24 hr after its administration (Fig. 1) shows that the membrane protein pattern was similar in various treatment groups. The results of the second experiment on the effect of acetaminophen 4 and 24 hr after its administration on serum transaminases, hepatic GSH and glycogen concentrations, on erythrocyte glutathione and their osmotic fragility in the PTU treated and control rats are shown in Table 2. Acetaminophen administration did not affect serum transaminases (SGOT, SGPT) in either treatment group after 4 hr but after 24 hr both The
organ
weights,
Acetaminophen
PTU-Acet
PTU-Saline
and RBC damage
Control-Saline
Control-Acet
Fig. I. Densitometric scans for various protein bands of the polyacrylamide gels of erythrocyte membranes prepared from PTU pretreated hypothyroid and control euthyroid rats 24 hr after oral acetaminophen or saline administration.
SGOT and SGPT were significantly elevated (7968 f 2709 and 4428 + 1348 respectively) in the control rats compared to 101 f 19 and 51 k 20 sigma units/ml respectively for the PTU treated rats. Although hepatic GSH concentrations were significantly higher for the PTU treated rats (3.48 f 0.18 p mol/g) compared to euthyroid controls (2.02 + 0.18) at 4 hr, necrosis was not observed in either group. After 24 hr the GSH values for both groups had increased and it was significantly higher for the PTU group. Hepatic glycogen was also very low at 4 hr in both groups and stayed low at 24 hr in the control rats (4.3 k 2.0mg/g) compared to 30.6 f 6.3 in the PTU treated rats. Like hepatic GSH, erythrocyte GSH concentrations were lower at 4 hr compared to 24 hr values but there was no difference between control and PTU treated rats at either time interval. Osmotic fragility like hepatic necrosis was not different at 4 hr after acetaminophen administration in eurthyroid control and PTU treated rats (76.4 + 2.0 and 72.8 f 3.1%) but it was significantly higher for the control rats compared to PTU treated rats after 24 hours (80.6 k 1.7 and 72.5 k 2.3% respectively). The percentage of the administered acetaminophen radioactivity recovered from whole
gastro-intestinal tract, per ml of serum and per g of liver tissue 4 hr after acetaminophen administration in the PTU treated rats was 34.3 f 2.9, 0.54 k 0.01 and 0.64 f 0.03 and at 24 hr was 5.9 + 0.5, 0.07 f 0.005 and 0.07 f 0.008 respectively. S&ilar values for the euthyroid controls at 4 hr were 71.3 f 3.6, 0.32 + 0.02 and 0.55 k 0.06 and at 24 hr 11 .O & 1.6, 0.11 f 0.02 and 0.10 k 0.01% respectively. DISCUSSION
Serum transaminases, elevation of which is considered an index of hepatic damage in response to various xenobiotics, suggest that acetaminophen did not cause hepatic damage after 4 hr but did so after 24 hr of its administration. Cellular damage to the erythrocyte membrane as expressed in terms of its susceptibility to osmotic lysis, also showed no change after 4 hr but a significant increase after 24 hr of acetaminophen administration. Erythrocyte and hepatic glutathione concentrations were similarly affected as both were lower at 4 hr compared to 24 hr determinations. Although there was no difference in erythrocyte glutathione or erythrocyte membrane-SH contents in the control and PTU treated rats, eryth-
Table 2. Effect of a toxic dose of acetaminophen (1 g/kg body wt) administration on serum transaminases and hepatic and ervthrocvte zlutathione concentrations in control and PTU oretreated male Fisher rat Control-acetaminophen Hours after acetaminophen 24 4 Serum glutamic oxalacetic acid transaminase (sigma units/ml) Serum glutamic pyruvic transaminase (sigma umts/ml) Hepatic glutathione (pmoleig) Hepatic glycogen (mg/g) Erythrocyte glutathione (me/100 ml oacked RBC)
PTU-acetaminophen administration 74 4
87 * 14b
7968 k 2709”
74*
IIh
101 f 19t
31i4b
4428 * 13484
35*
IIb
51 f 20h
2.02 It 0.18’ 7.8 & l.Ob 53.4 * I .8h
4.28 i 1.46b 4.3 * 2.0b 79.8 * 1 I .8db
3.48 f 0.1 Sb 6.0 f O.gh 59.3 * 3.7b
7.68 + 0.60” 30.6 f 6.3” 89.4 * 5.4”
Values are mean f SEM for four observations. Values with a common superscript are not significantly different. The erythrocyte osmotic fragility was not different after 4 hr with values of 76.4 k 2.0 and 72.8 f 3.1 for the control and PTU treated rats but was significantly different after 24 hr with values of 80.6 i 1.7 and 72.5 + 2.3 for the two respective groups. The hepatic glycogen concentrations for the control and PTU treated rats not given acetaminophen and killed at 24 hr were 66.0 + 3.8 and 59.5 i 2.3 mgfg respectively.
30
KRISHAN L. RAHEJA et d.
rocyte osmotic fragility was consistently greater in controls, suggesting that PTU per se or its induced hypothyroidism has a protective effect. Such an effect of PTU per se independent of its effect on hepatic GSH or its hypothyroid effect has been previously reported by us against acetaminophen-induced hepatotoxicity (Raheja et ul., 1982, 1983). It is not likely that increased erythrocyte osmotic fragility in the control rats compared to PTU treated rats given acetaminophen is related to changes in their membrane protein composition because the SDS gel electrophoretic pattern with or without dithiothreitol was similar in the two treatment groups given acetaminophen. Moreover, it was not different from their respective controls given only isotonic saline instead of acetaminophen (Fig. 1). The protective effect of PTU treatment could not be due to decreased intestinal absorption of the drug, because the absorption of acetaminophen was actually greater in the PTU treated rats 4 hr after its gavage. The greater rise in hepatic glycogen and giutathione levels in the PTU treated rats compared to control rats, from 4 to 24 hr after acetaminophen administration is possibly a reflection of less damage in the PTU treated rats and, therefore, their better capacity to recuperate from the toxic effect. Although our results show that changes in erythrocyte osmotic fragility do not precede hepatic necrosis and thus could not predict pending necrosis, as we had hypothesized, determination of erythrocyte osmotic fragility could prove to be a convenient in vitro method to test individual susceptibility to toxicity of various xenobiotics which are metabolized like acetaminophen by the cytochrome P-450 dependent mixed function oxidase enzyme system. Our data show that the time course of increase in erythrocyte osmotic fragility and hepatotoxicity subsequent to a toxic dose of acetaminophen is similar, suggesting that acetaminophen toxicity probably affects all organs whose integrity normally depends upon optimal glutathione concentrations. Furthermore, PTU pretreatment protects both liver and erythrocytes against acetaminophen toxicity in even the more susceptible Fisher rat than the Wistar rat which we previously used
in all our
studies.
Ackno~~ledgements~The authors are grateful to Robert Ciuancial, Katherine Larsson and Carol Sterling for their excellent technical assistance and to Sally Michalski for her secretarial help. We also thank Dr. D. G. Patel, Barbara Kopp Geriatric Research Center, for kindly supplying us with the experimental animals. This work was supported in part by the Veterans Administration, US Public Health Service Grant GM 27801, and National Aeronautics and Space Administration Contract No. A62829B. REFERENCES Beutler E., Duron 0. and Kelley B. M. (1963) Improved method for the determination of blood glutathione. .I. Lab. c/in. Med. 61, 882-888.
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