Galactosamine-Induced Cell Death in Primary Cultures ... - Europe PMC

Galactosamine-Induced Cell Death in Primary Cultures of Rat Hepatocytes Francis A. X. Schanne, BS, Richard G. Pfau, BS, and John L. Farber, MD

Primary cultures of rat hepatocytes were exposed to 0.5 mM D-galactosamine. After 36 hours, only 10-20% of the original cells were viable, as assessed by trypan blue exclusion. In the absence of galactosamine, there was no loss of viability over this same period. The addition of 3 mM uridine to the culture medium completely prevented the cell death produced by galactosamine. Glucosamine had no effect on the viability of the hepatocytes. The extent of galactosamine-induced cell death was dependent upon the concentration of Ca++ ions in the culture medium. With the only source of Ca++ that added with the fetal calf serum, galactosamine had only a very slight effect on viability. With higher Ca++ than with the fetal calf serum, galactosamine had only a very slight effect on viability. With higher Ca++ concentrations, from 0.9 to 3.6 mM, the viability ranged from 75% to 31% 18 hours after treatment with galactosamine. The addition of 1.4 pM chlorpromazine to culture medium containing 1.8 mM Ca"+ decreased the extent of the galactosamine-induced cell death. This protective effect was progressively reduced by raising the Ca++ concentration to 3.6 and 5.4 mM. Chlorpromazine given to intact rats 2 hours after treatment with 400 mg/kg galactosamine prevented the appearance of liver cell necrosis. At the same time, chlorpromazine prevented the increases in liver cell Ca"+ content. These results indicate that many of the features of the effect of galactosamine on intact rat liver cells can be reproduced in primary cultures of these same cells. The data also support the hypothesis that a disturbance in intracellular Ca" homeostasis leading to accumulations of these ions is causally related to the cell death produced by galactosamine. (Am J Pathol 1980, 100:25-38)

PRIMARY CULTURES of adult rat hepatocytes are increasingly being used in the study of the biochemical mechanisms underlying toxic cell injury. These cells are easily obtained in high yields with good viability, and they can be maintained in culture for several days. Such cultures allow more control over and manipulation of the extracellular environment than is possible in vivo. Recent studies in our laboratory have employed primary cultures of rat hepatocytes to analyze the role of extracellular calcium ions in toxic cell death."2 The killing of these cells by 10 different toxins that act directly on cellular membranes without prior metabolic activation was dependent upon extracellular Ca". In the absence of Ca++ ions in the culture medium, none of the toxins produced cell death. When exposed to the same From the Department of Pathology and the Fels Research Institute, Temple University School of Medicine, Philadelphia, Pennsylvania. Supported by Grants AM-19154 and CA-12073 from the National Institutes of Health. Accepted for publication January 29, 1980. Address reprint requests to John L. Farber, MD, Department of Pathology, Temple University Health Sciences Center, 3400 N. Broad Street, Philadelphia, PA 19140. 0002-9440/80/0709-0025$01.00 25 i American Association of Pathologists

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American Journal of Pathology

agents in the presence of Ca", at least 50-90% of the cells were dead. A two-step mechanism was proposed to explain the killing of the hepatocytes. A disruption of the integrity of the plasma membrane by any one of a number of different mechanisms is followed by a common functional consequence in the influx of extracellular Ca++ across the injured membrane and down the steep electrochemical gradient that separates the outside from the inside of all cells in the body. These same steps may occur in the sequence of events that leads to the toxic death of liver cells in the intact animal. Accumulation of Ca++ ions accompanies the death of liver cells produced by a wide variety of hepatotoxins including carbon tetrachloride,37 thioacetamide,8'9 galactosamine,'0 dimethylnitrosamine,"i and aflatoxin Bl." In order to provide more direct evidence for the active role of such Ca++ accumulations in the liver cell death produced by these agents, it seemed necessary to reproduce their toxicity in vitro. We chose to concentrate initially on galactosamine for several reasons. Previous studies demonstrated the presence of plasma membrane injury that is accompanied by alterations in intracellular Ca++ homeostasis.'0 Both the plasma membrane alterations and the increased Ca++ content are prevented and reversed by uridine.1' This fact suggested that a similar action of uridine could be used to insure the specificity of any lethal cell injury in vitro. Finally, the metabolism of galactosamine is not dependent upon the microsomal mixed-function oxidase system, which is known to decline very rapidly in primary cultures of rat hepatocytes. The present report describes the initial results of the study of galactosamine-induced death of primary cultures of rat hepatocytes. The essential features of the in vivo system have been reproduced in vitro, and data are presented implicating Ca++ ions as mediators of cell death. Materials and Methods Hepatocytes were prepared from nonfasted female Wistar rats (Charles River Farms), weighing 150-200 g, by collagenase perfusion, as described previously.' Yields of 2-4 x 108 cells/liver with 85-90% viability (trypan blue exclusion) were obtained. The hepatocytes were plated in plastic 25-sq cm cultures flasks (Coming) at a density of 5-7 x 104 cells/sq cm in Williams E (Flow Laboratories) containing 100 mM Hepes buffer, pH 7.4, 10% inactivated (56 C for 10 minutes) fetal calf serum (Flow), 50 ,g/ml garamycin, and 0.02 U/ml insulin (Complete Williams Medium). After incubation in a humidified atmosphere of 5% CO2-95% air for 90 minutes to allow attachment of the cells, the cultures were rinsed three times with prewarmed Hanks' balanced salt solution (HBSS) (Microbiological Associates) to remove all unattached and dead cells. The hepatocytes were then incubated in Complete Williams Medium with the additions indicated in text. In one experiment (Text-figure 3) the rinsed hepatocytes were incubated in Williams E that had been prepared without any added CaCl2 (Flow) but with the other additions listed above. CaCl2 was added to the concentrations indicated in the text. D-Galactosamine, uridine,

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GALACTOSAMINE-INDUCED CELL DEATH

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and glucosamine were obtained from Sigma Chemical Company and chlorpromazine-HCl from Smith, Kline and French. Cell viability was assayed by trypan blue exclusion. Trypan blue (0.4% in 0.15% NaCl, Grand Island Biological Co.) was added directly to the cultures at a final concentration of 0.01%. Within 10 minutes the number of attached cells that excluded the dye was counted using a 10-sq mm eyepiece grid in an' inverted microscope at 200X magnification. Viability is expressed as the percentage of the number of unstained cells counted immediately after the initial 90-minute incubation. We made all measurements by counting 5 fields each of triplicate cultures. The results are the mean ± SD of the triplicate cultures. For the in vivo studies, female Wistar rats weighing 150-175 g were fasted overnight prior to use. All compounds were administered by intraperitoneal injection: D-galactosamine at a dose of 400 mg/kg as a 20 mg/ml solution and chlorpromazine at a dose of 20 mg/kg as a 10 mg/ml solution. Control animals received an equal volume of 0.9% NaCl. We determined total liver calcium content by atomic absorption spectroscopy as previously described,") employing a Perkin Elmer 460 Spectrometer with a hollow cathode calcium lamp. We obtained histologic sections by fixing sections of the livers in 10%N buffered formalin, embedding them in paraffin, sectioning them, and staining them with he-

matoxylin-eosin.

Results

Freshly prepared rat hepatocytes readily attach to plastic tissue culture dishes. After an initial 90-minute incubation, the culture medium can be replaced with fresh medium, and all of the viable cells seeded will then be firmly adherent. The small number of nonviable cells present in the original preparation of hepatocytes will not attach and should be discarded with the medium change. Text-figure 1 indicates that there is no loss of viability of the attached hepatocytes maintained in culture for at least the next 36 hours. In this case and in all subsequent studies, viability was measured by trypan blue exclusion. We have shown previously that viability measured by this technique yields the same results as that given by two independent assays, the hydrolysis of fluorescein diacetate and the plating

efficiency.' In the presence of 0.5 mM D-galactosamine in the tissue culture medium, there is a progressive loss of viable hepatocytes. Text-figure 1 indicates that by 36 hours 80-90% of the hepatocytes are dead. These data are based on the measurement of viable, attached cells. The effect of galactosamine cannot be explained as simply a release of viable cells into the medium. At each of time points indicated by the data in Text-figure 1, many cells that do not exclude trypan blue are still attached to the culture dishes. In addition, at each of the time points indicated in Text-figure 1, the medium was collected and centrifuged. The resulting pellet was stained with trypan blue, and the cells were counted. At no point were any viable cells observed in the medium. However, the total number of cells represented by the sum of attached viable cells plus attached and floating nonviable cells did decrease progressively with time. This finding implies that


SCHANNE ET AL

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TEXT-FIGURE 1-Effect D-galactosamine on the viability of primary cultures of rat hepatocytes. Isolated rat hepatocytes were prepared as described in Materials and Methods. After an initial 90-minute incubation to allow attachment of all viable cells, fresh medium was added either with (broken line) or without (solid line) 0.5 mM D-galactosamine. Viability was assessed by trypan blue exclusion at the times indicated. The effect of galactosamine at 12, 24, and 36 hours is significantly different from the control, with P < 0.001 on the basis of the Student t distribution for the comparison of independent samples.27

of the dead cells were being lysed, either before or after release into the medium. The death of rat liver cells in the intact animal produced by galactosamine can be prevented by the simultaneous administration of uridine. Similarly, uridine will prevent the death of cultured hepatocytes produced by galactosamine. Text-figure 2 shows that the addition of 3 mM uridine to the culture medium along with 0.5 mM galactosamine preserves the viability of the cells. As in Text-figure 1 and in the absence of uridine, 80-90% of the cells were killed by the addition of galactosamine. Uridine alone had no effect on the viability of the cells. In the intact animal, glucosamine at the same dosage as galactosamine does not produce liver cell necrosis.'2 Text-figure 2 also indicates that glucosamine has no effect on the viability of hepatocytes in culture. The data, then, in Textfigure 2 indicate that the toxicity of galactosamine to cultured rat hepatocytes shares two important features of the effect of this agent on liver cells in the intact animal, prevention by uridine and specificity for the amino sugar of galactose, as opposed to that of glucose. some

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A major objective of the development of an in vitro system to study galactosamine toxicity was to assess the role of Ca++ ions in the cell death produced by this compound. In vivo, galactosamine produces a very early disturbance in liver cell calcium homeostasis.'0 The increased liver cell Ca++ content is prevented by uridine and correlates closely with changes in isolated liver cell plasma membranes.'0 It was suggested that an effect of galactosamine on hepatocyte membranes produces an influx of Ca++ ions that is ultimately responsible for the death of the cells. The demonstration of the killing of these same cells by galactosamine in culture allowed us to test this hypothesis directly. We evaluated the role of the extracellular Ca++ concentration in the galactosamine-induced death of cultured hepatocytes by incubating these cells with 0.5 mM galactosamine in the presence of increasing concentrations of Ca++ ions. The results of this experiment are illustrated in Textfigure 3. Immediately upon isolation the hepatocytes were incubated in Complete Williams Medium (1.8 mM CaCl2). At the end of 90 minutes, fresh medium was added to all cultures. In this case the medium was Williams E made up without CaCl2 and again containing 10% fetal calf serum. This medium is referred to as Minus Ca++ in Text-figure 3. The only Ca++ ions here were those added with the 10% fetal calf serum. The Ca++ ion concentration was 0.13-0.18 mM. In the remaining cases illus-

uridine TEXT-FIGURE 2-Conditions affecting the viability of cultured hepatocytes. Primary cultures of rat hepatocytes were treated with 0.5 mM D-galactosamine (galNH2); 0.5 mM D-galactosamine plus 3 nM uridine (galNH2 + uridine), or 0.5 mM D-glucosamine (gluNH2). Viability of the cells was assessed after 36 hours.

30


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TEXT-FIGURE 3-Effect of extracellular Ca++ concentration on galactosamine-induced cell death. Isolated hepatocytes were incubated for 90 minutes in Complete Williams Medium. The cells were then placed in Williams E made without CaCI2 and containing 10% heat-inactivated fetal calf serum, 50 jig/ml garamycin, and 0.02 U/ml insulin, referred to as Minus Ca++. This medium was then made up to either 0.9, 1.8, or 3.6 mM CaC12. In each case, the cells were incubated with (striped bars) or without 0.5 mM D-galactosamine for 18 hours, at which time viability was determined by trypan blue exclusion. The difference between the extent of galactosamine-induced cell death in "Minus Ca++" medium and 0.9 mM Ca++ is significant at P < 0.01; that between 0.9 mM and 1.8 mM and between 1.8 mM and 3.6 mM is significant at P < 0.001. The effect of galactosamine on viability in "Minus Ca++" medium is significant at P < 0.025; in 0.9 mM Ca++ at P < 0.005; and in 1.8 and 3.6 mM Ca++ at P < 0.001.

trated, CaCl2 was added to a final concentration of 0.9, 1.8, or 3.6 mM. The data in Text-figure 3 show that the loss of cell viability in the presence of 0.5 mM galactosamine is directly related to the Ca++ ion concentration in the medium. With the "Minus Ca++" medium, there was only a very slight loss of viability of the cells cultured with galactosamine. With 0.9 mM Ca++, 25% of the cells were killed by galactosamine; with 1.8 mM Ca++, 53% of the cells were killed; and with 3.6 mM Ca'+, 69% of the cells were killed. In each case, there was no loss of viability of the control cells in the absence of added galactosamine. It might be argued that extracellular Ca++ ions are necessary in order for the hepatocytes to metabolize galactosamine. That this is not the case shown previously by Hofmann et al.13 These authors incubated isolated hepatocytes in a Ca++-free Krebs-Henseleit buffer and showed a galactosamine-dependent decline in the concentrations of UTP and uridine diphosphoglucose. Uridine reversed the losses of both these compounds. This finding implies that the dependency of the galactosamine-induced cell death on extracellular Ca++ ions shown in Text-figure 3 must be rewas

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Vol. 100, No. 1 July 1980

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chlor. TEXT-FIGURE 4-Effect of chlorpromazine on the viability of galactosamine-treated hepatocytes. Cultured hepatocytes were incubated in Complete Williams E containing 1.8, 3.6, or 5.4 mM CaCJ2 to which either 0.5 mM D-galactosamine or galactosamine plus 1.4 ,uM chlorpromazine were added. The viability of the cells was assessed after 36 hours. The protective effect of chlorpromazine is significant at a P < 0.001 in 1.8 and 3.6 mM CaCI2.

lated to some effect of Ca++ subsequent to the metabolism of galactosamine. This effect of Ca++ is dependent upon an influx of these ions, as indicated by the data in Text-figure 4. In this experiment the hepatocytes were treated with galactosamine alone or with galactosamine and chlorpromazine in the presence of increasing extracellular Ca++ concentrations from 1.8 to 5.4 mM. Chlorpromazine has a number of effects on biologic membranes.'4 In particular, it has been shown to inhibit the flux of Ca++ ions across several different membranes.'4 This action has been used to explain the ability of chlorpromazine to inhibit a number of physiologic phenomena felt to be dependent on increased Ca++ flux."'20 This inhibitory effect of chlorpromazine is overcome by raising the extracellular Ca ion concentration.17'19 Addition of 1.4 ,uM chlorpromazine to the tissue culture medium significantly reduces the extent of the galactosamine-induced death of cultured hepatocytes (left-hand panel in Text-figure 4). This protective effect can be overcome by progressively raising the extracellular Ca++ concentration to 3.6 mM (middle panel) or to 5.4 mM CaCl2. These data imply that the dependency of galactosamine-induced hepatocyte death on extracellular Ca++ ions is related to an increased flux of these ions into the cells. The ability of chlorpromazine to protect hepatocytes from the lethal effects of galactosamine can also be demonstrated in the intact animal. Text-figure 5 shows the effect on the content of total liver cell Ca++ of the treatment of rats with chlorpromazine (25 mg/kg) 2 hours after the ad-

32


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TEXT-FIGURE 5-Effect of chlorpromazine on Ca++ content in galactosamine-intoxicated rat liver cells. All animals were treated with 400 mg/kg D-galactosamine. Two hours later, half of the animals received 20 mg/kg chlorpromazine (broken line); the other half received saline (solid line). At the times indicated, the animals were killed and the total content of liver Ca++ determined as described in Materials and Methods. Results are the mean ± SD of five separate determinations on each of 5 animals. The effect of chlorpromazine is significant at 4, 8, 12 and 18 hours at a P < 0.01.

ministration of 400 mg/kg galactosamine. Our previous studies showed that 2 hours after galactosamine administration the liver cells manifest changes in their plasma membranes and slight increases in total liver cell Ca". However, the liver cells are not necrotic at this point. Text-figure 5 confirms our previous observation that the Ca++ content continues to rise between 2 and 8 hours and does not return to normal until some 24-36 hours later. Chlorpromazine given 2 hours after galactosamine prevents any further rise in total liver cell Ca++ content for at least 48 hours. At the same time, chlorpromazine prevents the appearance of liver cell necrosis. Figure 1 illustrates the appearance of a representative liver lobule 12 hours after the administration of galactosamine alone. Figure 2 illustrates a similar liver lobule in a rat given galactosamine and then chlorpromazine 2 hours later. With galactosamine alone there are numerous isolated necrotic liver cells accompanied by a mild inflammatory infiltrate. With galactosamine plus chlorpromazine, the liver is essentially normal.

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Discussion

Galactosamine has been a valuable tool with which to explore the mechanisms of toxic liver cell necrosis., " The results of the present study demonstrate that the use of this agent can be extended to an in vitro system employing primary cultures of rat hepatocytes. The data presented allow several conclusions with regard to the killing of such cells by galactosamine. The effect of galactosamine on cultured hepatocytes reproduces many features of the action of this compound in the intact animal. Isolated hepatocytes metabolize galactosamine, with the resultant depletion of uridine nucleotides and UDP-hexoses.'3 Such changes characterize the action of galactosamine on intact liver cells.2' Uridine will reverse both deficiencies in vivo 21 and in vitro."3 Uridine will also prevent the death of galactosamine-intoxicated liver cells in vivo.21'23 The data in Text-figure 2 indicate a similar action of uridine in vitro. Amines of hexoses other than galactose do not produce liver cell necrosis in vivo.'2 Text-figure 2 indicates that glucosamine is similarly not toxic to cultured hepatocytes. Galactosamine induces in liver cells in the intact animal a disturbance in intracellular Ca++ homeostasis that cannot be dissociated from the development of liver cell necrosis.'0 Doses of galactosamine that injure but do not kill the liver cells do not perturb the level of Ca++ ions in these cells.'0 Pretreatment with uridine prevents the increases in liver cell Ca++ content and the development of liver cell necrosis.'0 Treatment with uridine after initial manifestations of galactosamine-induced liver cell injury will restore normal liver Ca++ homeostasis and prevent liver cell necrosis.'0'2123 The similar effect of uridine on the manifestations of plasma membrane injury suggested that there are at least three steps in the mechanism by which galactosamine kills liver cells.22 First, the metabolism of galactosamine leads to an accumulation of UDP-galactosamine, much of which is not N-acetylated as is all of the normally occurring UDP-hexosamines. At the same time, a deficiency of uridine nucleotides and UDPhexoses results. Second, and in some manner as yet poorly defined, these metabolic abnormalities result in an alteration in the plasma membranes of the liver cells. Third, this damage is associated with an increased influx of Ca++ ions down the steep electrochemical gradient that exists between the inside and the outside of the cells. The increased Ca++ content mediates or at least initiates mechanisms ultimately responsible for the death of the cells. The Ca++ dependency of the galactosamine-induced death of cultured hepatocytes demonstrated in the present report supports this mechanism.

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The data indicate a causal role for extracellular Ca++ ions. In the absence of a sufficient extracellular Ca++ concentration, galactosamine did not kill the liver cells. The simplest explanation of this requirement for extracellular Ca++ ions is the necessity for a sufficient concentration to drive Ca++ ions into the injured cells. However, even in the presence of sufficient extracellular Ca++ ions both in vivo and in vitro, liver cells can be protected from the lethal effects of galactosamine with chlorpromazine. Chlorpromazine is known to inhibit Ca++ fluxes across cellular membranes."4 That chlorpromazine is so acting to prevent galactosamine-induced liver cell death is implied by the data (Text-figure 4) showing that, at least in vitro, the protective effect can be overcome by the simple raising of the extracellular Ca++ concentration. This raising of the concentration most probably simply restores a sufficient gradient of Ca++ to again drive these ions into the cells. The results of the present study extend the list of those agents that have been shown to be dependent upon extracellular Ca++ in order to kill cultured liver cells. All of previously employed toxins did not need metabolic activation by the liver cells and were all capable of directly interacting with cellular membranes.'"2 With galactosamine, an agent has been added that presumably does not directly interact with cellular membranes and that needs to be metabolized by the cells. However, this metabolism does produce membrane alterations.'0 There is, then, a close parallel between the effects of galactosamine in vitro and the action of the previously studied toxins. It seems very likely that the roles of extracellular Ca++ ions are similar in all cases. With each agent an influx of calcium across a damaged plasma membrane and down a steep electrochemical gradient may represent a final common pathway for the toxic death of the cultured hepatocytes. The present study also suggests that this same mechanism operates in the intact animal. Chlorpromazine protects hepatocytes both in vitro and in vivo from the lethal effects of galactosamine. The protective effect of chlorpromazine in vitro could be overcome by the simple raising of the extracellular Ca++ concentration. It is likely that the ability of chlorpromazine to block a lethal influx of Ca++ ions is the mechanism of its protective action in vivo as well. A final comment should be addressed to a different implication of the results of the present study. There have been data published recently suggesting that the death of galactosamine-injured liver cells is triggered by extrahepatic mechanisms that lead to an activated complement system of endotoxin."26 The present study has established conditions sufficient for galactosamine-induced cell death that do not necessarily include a source

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of endotoxin. In addition, it is unlikely that complement activation independent of endotoxin was occurring in our in vitro system, since the fetal calf serum was heat-inactivated before its addition to the cultures. Therefore, there may be explanations other than endotoxin-mediated, complement-directed cell injury that explain the observations referred to above.24-26 References 1. Schanne FAX, Kane AB, Young EE, Farber JL: Calcium dependence of toxic cell death: A final common pathway. Science 1979, 206:700-702 2. Kane AB, Young EE, Schanne FAX, Farber JL: Calcium dependence of phalloidininduced liver cell death. Proc Natl Acad Sci USA 1980, 77:1177-1180 3. Reynolds ES: Liver parenchymal cell injury: I. Initial alterations of the cell following poisoning with carbon tetrachloride. J Cell Biol 1963, 19:139-157 4. Reynolds ES: Liver parenchymal cell injury: II. Cytochemical events concerned with mitochondrial dysfunction following poisoning with carbon tetrachloride. Lab Invest 1969, 13:1457-1470 5. Smuckler EA: Studies on carbon tetrachloride intoxication: IV. Effect of carbon tetracnloride on liver slices and isolated organelles in vitro. Lab Invest 1966, 15:157166 6. Thiers RE, Reynolds ES, Vallee BL: The effect of carbon tetrachloride poisoning on subcellular metal distribution in rat liver. J Biol Chem 1960, 235:2130-2133 7. Moore L, Davenport GR, Landon EJ: Calcium uptake of a rat liver microsomal subcellular fraction in response to in vivo administration of carbon tetrachloride. J Biol Chem 1976, 251:1197-1201 8. Gallagher CH, Gupta DN, Judah JD, Rees KR: Biochemical changes in liver in acute thioacetamide intoxication. J Pathol Bacteriol 1956, 72:193-201 9. Rees KR, Sinha PK, Spector WG: The pathogenesis of liver injury in carbon tetrachloride and thioacetamide poisoning. J Pathol Bacteriol 1961, 81:107-118 10. El Mofty SK, Scrutton MC, Serroni A, Nicolini C, Farber JL: Early reversible plasma membrane injury in galactosamine-induced liver cell death. Am J Pathol 1975, 79:579-596 11. Farber JL, Abrams J, Glazer D: Unpublished observations 12. Keppler D, Lesch R, Reutter W, Decker K: Experimental hepatitis induced by Dgalactosamine. Exp Mol Pathol 1968, 9:279-290 13. Hofmann F, Wilkening J, Nowack J, Decker K: Response of isolated rat hepatocytes to D-galactosamine and uridine. Hoppe Seylers Z Physiol Chem 1976, 357:427433 14. Seeman P: The membrane actions of anesthetics and tranquilizers. Pharmacol Rev 1972, 24:583-655 15. Godfraind T, Kaba A: Blockade or reversal of the contraction induced by calcium and adrenalin in depolarized arterial smooth muscle. Br J Pharmacol 1969, 36:549560 16. Jaanus SD, Miele E, Rubin RP: The analysis of the inhibitory effect of local anesthetics and propanolol on adrenomedullary secretion evoked by calcium or acetylcholine. Br J Pharmacol 1967, 31:319-330 17. Seeman P, Chen SS, Chau-Wong M, Staiman A: Calcium reversal of nerve blockade by alcohols, anesthetics, tranquilizers, and barbiturates. Can J Physiol Pharmacol 1974, 52:526-534

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18. Gardos G, Lassen UV and Pape L: Effect of antihistamines and chlorpromazine on the calcium-induced hyperpolarization of the Amphiuma red cell membrane. Biochim Biophys Acta 1976, 448:599-606 19. Schreiner GF, Unanue ER: The disruption of immunoglobulin caps by local anesthetics. Clin Immunol Immunopathol 1976, 6:264-269 20. Williams JA, Poulsen JH, Lee M: Effects of membrane stabilizers in pancreatic amylase release. J Membrane Biol 1977, 33:185-195 21. Decker K, Keppler D: Galactosamine-induced liver injury. Prog Liver Dis 1972, 4:183-199 22. Farber JL, El-Mofty SK: The biochemical pathology of liver cell necrosis. Am J Pathol 1975, 81:237-250 23. Farber JL, Gill G, Konishi Y: Prevention of galactosamine-induced liver cell necrosis by uridine. Am J Pathol 1973, 72:53-62 24. Grun M, Liehr H, Grun W, Rasenack U, Brunswig D: Influence of liver-RES on toxic liver damage due to galactosamine. Acta Hepatogastroenterol (Stuttg) 1974, 21:5-15 25. Grun M, Liehr H, Rasenack U: Significance of endotoxemia in experimental "Galactosamine-Hepatitis" in rats. Acta Hepatogastroenterol (Stuttg) 1977, 24:64-81 26. Liehr H, Grun M, Seelig H-P, Seelig R, Reutter W, Heine W-D: On the pathogenesis of galactosamine hepatitis: Indications of extrahepatocellular mechanisms responsible for liver cell death. Virchows Arch [Cell Pathol] 1978, 26:331-344 27. Snedecor GW, Cochran WG: Statistical Methods. 6th edition. Ames, Iowa, Iowa State University Press, 1967, p 100

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