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Late preventive effects of trifluoperazine on carbon tetrachloride-induced hepatic necrosis
TOXICOLOGY
AND
APPLIED
PHARMACOLOGY
83,287-293
(1986)
Late Preventive Effects of Trifluoperazine on Carbon Tetrachloride-Induced Hepatic Necrosis M. C. VILLARRUEL, G. FERNANDEZ, E. C. DE FERREYRA, 0. M. DE FENOS, AND J. A. CASTRO Centro de Investigaciones Toxicoldgicas (CEITOX). CITEFA/CONICET, Zufiategui 4380, 1603 Villa Marteili. Provincia de Buenos Aires, Republica Argentina
Received February 20, 1985: accepted November I, I985 Late Preventive Effects of Trifluoperazine on Carbon Tetrachloride-Induced VILLARRUEL,
M.
C., FERNANDEZ,
G.,
DE FERREYRA,
E. C., DE FENOS,
0.
Hepatic Necrosis. M.,
AND
CASTRO,
(1986). Toxic01 Appl. Pharmacol. 83, 287-293. As a very preliminary test for a possible role of calmodulin in CC&-induced hepatic injury, we studied the effects of the anticalmodulin drug trifluoperazine (TFP) on several deleterious actions of CCL on the liver. TFP administrated 30 min before or 6 or 10 hr after Ccl, significantly prevented hepatic necrosis induced by the hepatotoxin at 24 hr but not at 72 hr. TFP did not modify the Ccl, concentrations reaching the liver, or the intensity of the covalent binding of CC&-reactive metabolites to hepatic microsomal proteins or lipids or the CCL,-induced cytochrome P-450 and glucose 6 phosphatase destruction. TFP administration decreased body temperature between 0 and 1“C in controls and between 1.2 and 3.5”C in CCl&reated animals during the 24-hr observation period. When TFP-treated CCL-poisoned animals were kept normothermic, protective effectswere eliminated. One possibility is that the protective effect of TFP might be due to a nonspecific action related to decreased body temperature. Alternatively, prevention might result from TFP inhibition of a late-occurring process critical for CCld-induced cell necrosis requiring calmodulin participation. If this alternative were in operation, protective consequences of this inhibitory effect of TFP should be either canceled or counteracted in the normothermic TFP + CCL-treated animal. 0 1986 Academic PI.s, IIIC. J. A.
Intracellular calcium homeostasis has been proposed as a possible reason for liver cell death induced by CCL, and several other hepatotoxins (Chenery er al., 198 1; Farber, 198 1, 1982; Moore et al., 1976; Pencil et al., 1982; Schanne et al., 1979; Younes et al., 1983). Notwithstanding, the mechanism by which calcium causes cytotoxic effects is not known, but likely targets include enzyme systems of high relevance to cell function which are sensitive to calcium (Farber, 198 1, 1982; Pencil et al., 1982). There is considerable evidence that many cell activities mediated by calcium are probably being accomplished through calmodulin regulation, since calmodulin functions as an intracellular mediator of the effects of calcium (Means et al., 1982; Weiss et al.,
1980). A very interesting property of calmodulin is its rather specific binding to a variety of pharmacological agents (Means et al., 1982; Weiss et al., 1980). These drugs, known as calmodulin antagonists, may prove very useful in preliminary testing of a possible role of calmodulin in calcium-mediated, CC&-induced cell necrosis. The most widely used anticalmodulin pharmacological agent employed in these type of studies has been trifluoperazine (TFP), which binds the calcium-calmodulin complex with high affinity and interferes with its ability to bind the receptor protein (Means et al., 1982; Weiss et al., 1980). In the studies described in this report, we tested whether TFP administration to rats prevents several manifestations of CC&-induced liver injury includ287
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VILLARRUEL
288
ing hepatic necrosis. We also tested its effects on several parameters known to modulate the deleterious actions CClh. METHODS Chemicals. The purity of [‘4C]CC14’ (sp act 2.8 mCi/ mmol) was greater than 99% when determined by gasliquid chromatography with a column of Carbowax 400 Durapak on Porasil C at 65°C. Trifluoperazine hydrochloride’ was pharmaceutical grade. All other chemicals employed were of reagent grade. Animals and animal treatmenl. Sprague-Dawley male rats bred in our laboratory (240 to 290 g) were used throughout these experiments. Feed (Purina Chow rat diet) was withdrawn 12 to 14 hr before TFP was given. Animals did not have accessto feed during the experimental period but water was available ad libifum. Temperature in the animal room was 23 + 2°C and the relative humidity was between 35 and 65%. Light in the animal room was on from 6 AM to 6 PM. TFP was given ip in 0.9% NaCl solution at a dose of 50 mg/kg (IO mg/ml solution) 30 min before or 6 or 10 hr after CC&. Control rats received an equivalent amount of 0.9% NaCl. [‘4C]CC14 was given ip in CC& 5% (v/v) solution in olive oil (30 X IO6 dpm/ml) at a dose of 5 ml of solution/kg. CC& (I ml/kg) was given ip as a 20% (v/v) solution in olive oil in a volume of 5 ml/kg of final solution. Control rats received olive oil ip. The animals were killed by decapitation at different times after CC& administration, and their livers were rapidly removed and processed. Whenever blood samples were taken, animals were kept under light ether anesthesia. and blood was obtained with a heparinized syringe from the inferior vena cava. Rectal temperatures of the rats were recorded with a small animal thermistor probe (Telethermometer, Nihon-Kohden). In some experiments the normal body temperature of TFP + Ccl, and TFP-treated animals was maintained by keeping them at 37 f 1“C in an air-ventilated incubator. Isolation of microsomes. The hepatic microsomal fraction was isolated by the procedures described by Castro ef al. (1972). Enzymatic and chemical determinations. The in viva incorporation of radioactivity from “CC14 to microsomal proteins was determined according to procedures described by Rao and Recknagel ( 1969). A IO-mg aliquot of the purified and dried protein was dissolved in formic acid and counted as previously described (Diaz Gomez et nl., 1973). The in vivo incorporation of 14Cfrom “CCl, to microsomal lipids was determined according to a procedure described by Castro et al. (1972). Results are given in disintegrations per minute per ’ Radiochemical Centre, Amersham, England. 2 Smith. Kline & French Laboratories.
ET AL milligram of microsomal protein or lipid. The 14CC14concentrations in liver were estimated according to a procedure described by Recknagel and Litteria (I 960) up to the microdiffusion step; then, the 14CC14collected in the toluene of the center well ofthe cell was transferred to a scintillation counting vial and counted. Results were corrected for loss and for quenching by the channels ratio method. Lipid peroxidation in viva was measured by conjugated diene ultraviolet absorption of the microsomal lipid extracts (Klaassen and Plaa, 1969). The results are expressed as the absorbance at 243 nm x 1000 for a solution having I mg of microsomal lipid/ ml. The hepatic microsomal cytochrome P-450 (P-450) content was determined as described by Schenkman et al. (1967). Glucose-6-phosphatase activity (G6Pase) in microsomal suspensions was measured according to Harper (1963). Activity is expressed in micrograms of inorganic phosphorus produced in I5 minutes at 37°C per milligram of protein. NADPH-linked isocitric acid dehydrogenase (ICD) activity in plasma was measured by the method of Sterkel e/ al. (1958). Activity is given in units: one unit is the amount of enzyme producing I nmol NADPH/ml plasma/hr at 25°C. Protein concentrations were estimated by Lowry’s procedure (Lowry et ol., 1951). Histological techniques. After removal of the liver, small portions from the left and central lobes were immediately fixed in Bouin’s solution, embedded in paraffin, and stained with hematoxylin and eosin. The specimens were coded to avoid bias and were evaluated histologically by at least three independent observers. To quantify the morphological changes, hepatic sections were graded for changes such as hydropic degeneration or necrosis. using an arbitrary scale: + = light (about 20 to 30% necrosis): ++ = moderate (about 50% necrosis): +++ = marked (about 75% necrosis): ++++ = very intense (about 90 to 100% necrosis). Results reported as representative for each experimental condition are the mean of observations made by all observers for a given experimental condition. Stafistics. A decision tree for selecting hypothesis testing statistical procedures described by Gad and Weil ( 1982) was applied to results from every experiment. Homogeneity of variance was established using either the Bartlett’s homogeneity of variance test or the f test for comparisons involving three or more groups or two groups, respectively. According to significance of homogeneity of variance and size of the groups involved, one of the following tests was applied: Duncan’s multiple range test; Dunnett’s test: the Student f test or the Cochran t test (Gad and Weil, 1982). Parametric or nonparametric analysis of variance testswere used for comparisons involving three or more groups of data (Gad and Weil, 1982).
RESULTS
Eflect of TFP in vivo on microsomal lipid peroxidation produced by CCL at dl@erent times after administration. TFP alone pro-
TRIFLUOPERAZINE TABLE
AND
CARBON
1
CCJNDUCED LIPID PEROXIDATION OF MICROSOMAL LIPIDS IN RATS PREVIOUSLY TREATED WITH TFP Lipid peroxidation Treatment” CO”t”Jl
CCL TFP TFP + cc14
(X i SD)*
1 hr
70’
3 hr
21 1.6 k 21.2 346. I k 4~%4~ 335.9 + 86.5’
164 159
179.8 + 49.7 333.2 k 72.3 299.6 k 66.1
185’ 167’
443.5 + 44.9d.e
209
350.4 k 103.4
195C’
’ TFP was given ip at a dose of 50 @kg. Ccl, was given ip as a 20% solution in olive oil at a dose of 5 ml/kg, 30 min after TFP administration. Eight animals per group were used in these experiments. Animals were killed I and 3 hr after CCL administration. ’ The lipid peroxidation value is expressed as A absorbance at 243 nm X 1000 for a solution having I mg of microsomal lipid/ ml. ‘a < 0.01 when compared with its respective control: Student’s 1 test. dp < 0.001 when compared with its respective control; Student’s t test. ‘The p value for the overall effect of the TFP on the CC&induced lipid peroxidation obtained by analysis of variance was p > 0.05 for all the times treated.
duced an increase of the lipid peroxidation process as measured by the diene conjugation technique at 1 or 3 hr (Table 1). TFP did not modify the intensity of the CC&induced lipid peroxidation process (Table 1). E#ect ofpretreatment with TFP on the covalent binding of 14C from [‘4C]CCld to mi-
TABLE COVALENT
BINDING
Treatment”
somal lipids and proteins. As shown in Table 2, prior administration of TFP to rats did not alter the extent of covalent binding of [‘4C]CC14 to microsomal lipids or proteins at 1 or 3 hr. Eflect of TFP on hepatic concentrations of [‘4C]CC/d. Liver content of [ “C]CC14 at different times after its administration in TFPtreated rats was not significantly different from that observed in untreated animals at 1 and 3 hr (Table 3). Eflect of TFP administration on the glucose 6-phosphatase activity and cytochrome P-450 destruction caused by CCld. TFP administration did not modify the destruction of G6Pase or P-450 3 hr after CCL administration (Table 4). Eflect of pretreatment with TFP on CC&induced decrease of body temperature. Treatment with TFP alone lowered the body temperature of the rats at 1 and 3 hr but not at 6 and 24 hr after administration. The CCLpretreated rats had a significant decrease in body temperature (p < 0.05 at 1 and 3 hr). TFP significantly increased hypothermia following CC4 injection (p < 0.05 at 1 hr, p < 0.001 at 3 hr, and p < 0.01 at 6 hr) (Table 5). Eflect of TFP on CC&-induced liver damage. Pretreatment with TFP 30 min before or 6 or 10 hr after CC4 significantly diminished the extent of CCkinduced necrosis 24 hr after ad-
2
OF CC& REACTIVE METABOLITES TO HEPATIC MICROSOMAL PROTEINS OF RATS PREVIOUSLY TREATED WITH TFP Covalent Lipid + SD 6bM-w)
binding
Protein + SD (dmUm)
of 14C from Lipid
+ TFP
167.0 k 29.3 144.3 + 30.2’
LIPIDS AND
[‘4C]CC1., f SD
Protein
(dpm/mg)
I hr [‘4c]ccl, [‘4C]CC14
289
TETRACHLORIDE
+ SD
kbm/mg) 3 hr
43.7 -c 6.7 42.5 + 8.9’
139+ I1 155 f 5.1b
39.5 + 6 36.0 + lb
a [‘4C]CC14 was given ip in CC4 5% (v/v) solution in olive oil (30 X lo6 dpm/ml) at a dose of 5 ml solution/kg, 30 min after TFP. TFP was administered as in Table I. The animals were killed at 1 or 3 hr after [‘4C]CCI, administration; their livers were processed for microsomal protein isolation. lipid extraction, and counting (see Methods for details). Eight animals per group were used in these experiments. b p > 0.05 when compared with its respective control; Student’s t test.
290
VILLARRUEL TABLE
3
“CC14 CONCENTRATIONS IN LIVER AT DIFFERENT TIMES AFTER ADMINISTRATION TO RATS PRETREATEII WITH TFP
Treatment” ‘TC14 “CC14 “Tel, “‘CCL,
+ TFP + TFP
Time (W 1 1 3 3
YC14 concentration in liver (dpm/g + SD) 185,140 193,460 118.228 135,400
f + + k
48,480 53,630’ 18,350 52,420b
% of control 100 104 100 114
a “CC14 and TFP were given as indicated in Tables 2 and I, respectively. Five rats per group were used in these experiments. * p > 0. I: Student’s t test.
ministration, as evidenced by determinations of ICD in plasma (Table 6). Hepatic necrosis was also assessed by histological examination. TFP alone did not significantly alter the normal hepatic architectural pattern at 24 hr. Administration of CCL+ to animals produced an intense centrolobular necrosis at 24 hr (Table 6). TFP markedly decreased the extent of the necrosis produced by CC& 24 hr after administration when administered 30 min before or 6 or 10 hr after the hepatotoxin (Table 6). However, liver necrosis in both CC&- and TFP + CC&-treated animals was very intense (++++) when assessed72 hr after CC&. In this experiment eight animals per group were employed and the reported results are the mean of the observations made, as detailed under Methods. No ICD values are given in this case because at 72 hr after CCL, administration, they returned to normal in spite of the histologically observable damage (Marzi et al., 1980). If animals treated with TFP at 10 hr after Ccl4 were maintained at 37°C in an incubator, TFP did not have any protective action. In this experiment, animals were not placed in an incubator immediately after CC& since they would not survive for 24 hr. To increase survival body temperature regulation in incubator was started at 10 hr after CCld. In this case 1 out of 10 animals survived. Survivors in the TFP + CCL, group
ET
AL.
had intense liver necrosis (++++) as did the CCL, group (ICD values: 111, 982 units for eight animals in CCL+ group and 97,500 units for five animals in the TFP + CC& group; p > 0.05). DISCUSSION Our studies on ICD activity in plasma and the histological evaluation of the livers of TFPtreated animals receiving CC& revealed that this drug significantly delayed the onset of CC&-induced liver necrosis when given 30 min before the hepatotoxin. It is particularly interesting that TFP-delaying effects were still observable when the drug was given 6 or 10 hr after CCL. Moreover, beneficial effects of TFP at this late stage of the intoxication process were more pronounced than when the drug was given 30 min prior to CC&. This behavior suggests that events occurring late in the pathway followed by the CC&-injured liver cell were modulated by TFP and not those events predominating at early periods of in-
TABLE
4
EFFECT OF CC& ADMINISTRATION ON THE CYTOCHROME P-450 CONTENT AND GuJ~~~E-~-PH~~PHATAsE ACTIVITY OF RATS PREVIOUSLY TREATED WITH TFP
P-4506
Treatment” Control CC-14 TFP TFP + Ccl,
0.32 0.22 0.35 0.23
f 0.02 f 0.03d + 0.01 57 0.03”’
G6Pasec 47.0 32.8 55.5 35.9
+ * i t
6.0 3.8d 5.5 3.4”’
’ CC& and TFP doses were given ip as in Table I. Ccl4 was administered 30 min after TFP. and the animals were killed 3 hr after CC& administration. Eight animals per group were used in these experiments. *P-450 values are given in nanomoles per milligram of protein + SD. ’ GhPase activity is expressed in micrograms of inorganic phosphorus produced in I5 min/mg of microsomal protein at 37°C & SD. dp < 0.001 when compared with its respective control; Student’s I test. ’ The p value of the overall effect of TFP on the CCL effects obtained by analysis of variance was >O.O I.
TRIFLUOPERAZINE
291
AND CARBON TETRACHLORIDE TABLE 5
EFFECTSOF CARBON TETRACHLORIDE AT DIFFERENT TIMES AFTER ADMINISTRATION ON BODY TEMPERATURE OF RATS PREVIOUSLY TREATED WITH TFP Body temperature (“C + SD)* Treatment’ Control CC-14
TFP TFP + CC&
I hr 36.2 + 0.7 35.0 f 0.4' 33.7 + I.ld 33.8 k I.Oc.d
3 36.3 35.3 33.8 33.2
hr t * k k
0.4 0.3' 1.6' 0.7'
6
hr
36.4 k 0.4 36.7 k 0.4 35.3 + 1.2 34.4 f 1.2d
24 36.8 36.2 37.2 32.7
f + * -+
hr 0.5 0.6 0.5 3.7'ld
a CC& and TFP were given as indicated in Table I. Five rats per group were used in these experiments. b The p value for the overall effect of TFP on the CC&-induced decrease in body temperature obtained by analysis of variance was p > 0. I for 1, 3, and 6 hr. and p < 0.05 for 24 hr. 'p < 0.05 for CCL, vs control at 1 and 3 hr. for TFP vs control at 3 hr. for TFP + Ccl4 vs TFP at 24 hr and for TFP + Ccl, vs CC& at 1 hr; Student’s t test. dp < 0.01 for TFP vs control at 1, 6, and 24 hr. and for TFP + CCL, vs CCL, at 6 hr; Student’s t test. 'p < 0.001 for TFP + CC& vs control and for TFP + Ccl., vs CC& at 3 hr: Student’s t test.
toxication, such as lipid peroxidation or covalent interactions of CC&-reactive metabolites with cellular constituents (CB). In fact, our present results support this hypothesis, since no significant effects of TFP treatment on CB or lipid peroxidation were observed. Furthermore, delaying effects can not be attributed to interference by TFP in the ability of CC& to reach the liver, since CC4 concentrations in livers of TFP-treated and in untreated animals were not significantly different. The above pattern of TFP explains why it does not prevent CC&-induced hepatic microsomal P-450 destruction or prevent glucose-6-phosphatase activity depression. In fact, the deleterious effects of CC& on these parameters are mediated by either CB or lipid peroxidation or both (Recknagel, 1983; Slater, 1982; Castro, 1984). These parameters remain unchanged during TFP treatment of CC& poisoning. Concerning the nature of the late-occurring event modulated by TFP having delaying effects in the course of CCLinduced hepatic necrosis, there are several possible hypotheses. One might be that TFP blocks a critical event involving calmodulin participation as intracellular mediator of the deleterious effects of calcium accumulation. Another possibility might be that TFP exerts a nonspecific delay-
ing effect mediated by its ability to decrease body temperature, similar to that previously reported for chlorpromazine (Marzi et al., 1980). It is known that decreases in body temperature, caused by hypothermia or cordotomy, delay the CC&-induced hepatic necrosis (Larson and Plaa, 1963, 1965; Rice and Plaa, 1968), and that preventive effects observed in those cases and in that of chlorpromazine at 24 hr after CC& are gone by 72 hr (Marzi et al., 1980). In the case of TFP, disappearance of delaying effects at 72 hr was also observed. Moreover, we also found that TFP-delaying effects at 24 hr after CC& were no longer observable if TFP + CCktreated animals were kept normothermic. This behavior was also observed in the case of chlorpromazine (Marzi et al., 1980). These findings appear to favor the latter hypothesis. However, both mechanisms might be operating in conjunction or simultaneously. In effect, temperature may cancel or produce changes counteracting calmodulin-mediated effects. The preliminary nature of these studies does not allow one hypothesis to be selected over the other. Irrespective of the mechanism of the TFP preventive effects, however, the fact remains that this drug modulates the intensity of late stages of CC&-induced hepatic injury and might give
292
VILLARRUEL
ET AL.
TABLE 6
REFERENCES
EFFECTSOF TFP ADMINISTRATION ON CC&-INDUCED HEPATIC NECROSISAT 24 HR AFTER ADMINISTRATION TO RATS
Treatment’
ICDb (units + SD)
Degree of histologically observable necrosis’
30 min before Control ccl,
160 + 35 178.500 * 32.900
TFP + CCL,
15,180 140 _+ + 70 11,300‘+
+++s +++ -
6 hr after Control cc14
180 + 50 200,400 f 17,300
++++
CASTRO,J. A. ( 1984). Mechanistical studies and prevention of free radical cell injury. Proc. IX Intern. Congress Pharmacol.. Vol. 2, pp. 243-270. Macmillan & Co., London. CASTRO, J. A., CIGNOLI, E. V., DE CASTRO, C. R., AND DE FENOS, 0. M. (1972). Prevention by cystamine of liver necrosis and early biochemical alterations induced by carbon tetrachloride. Biochem. Pharmacol. 21, 4957. CHENERY, R.. GEORGE, M., AND KRISHNA, G. (1981). The effectof ionophore A23 I87 and calcium on carbon tetrachloride-induced toxicity in cultured rat hepatocytes. Toxicol.
Appl. Pharmacol.
TFP + CC&
27,000 135 f+ 60 16,800”’
+ to- ++
10 hr after Control ccl,
155 + 45 189,280 f 39,030
TFP + Ccl,
13, 150 570 ff 708,930”’
++++ +to++ -
’ Ccl, and TFP were administered as indicated in Table I. TFP was administered 30 min before CC& or 6 or IO hr after the hepatotoxin. The animals were killed 24 hr after Ccl,. Seven animals per group were used in these experiments. b Isocitric acid dehydrogenase: I unit of enzyme is the amount required to form I nmol NADPH/ml plasma/hr at 25°C. ’ + = slight (20-30% necrosis): ++ = moderate (50% necrosis); +++ = marked (75% necrosis); ++++ = very intense (90-100% necrosis). d The p value for the overall effect of TFP on the CC&induced increase in ICD obtained by two-way analysis of variance wasp < 0.001 at 30 min, 6- and lo-hr values. ep < 0.001 for TFP + Ccl, vs CCL; Student’s t test.
an insight into the nature of the events involved. ACKNOWLEDGMENT This work was supported by Grant AM- 13 195- 16 from the National Institutes of Health (USA).
Appl. Pharmacol.
60,241-252.
DfAZ G~MEZ, M. I., CASTRO, J. A., DE FERREYRA,E. C., D’ACOSTA, N., AND DE CASTRO, C. R. (1973). Irreversible binding of “CC& to liver microsomal lipids and proteins from rats pretreated with compounds altering microsomal mixed-function oxygenaseactivity. To.xicol. 25, 534-541.
FARBER, J. L. (I 98 1). The role of calcium in cell death. LifeSci. 29, 1289-1295. FARBER, J. L. (1982). Calcium and mechanism of liver necrosis. Prog. Liver Dis. 7, 347-360. GAD, S. C., AND WEIL, C. S. (1982). Statistics for toxicologists. In Principles and Methods of Toxicology (A. W. Hayes, ed.), pp. 273-320. Raven Press. New York. HARPER, A. E. (1963). Methods oj’Enzvmatic Analysis, pp. 788-792. Academic Press, New York. KLAASSEN, C. D.. AND PLAA, G. L. (1969). Comparison of the biochemical alterations elicited in livers from rats treated with carbon tetrachloride. chloroform, 1, I ,2trichloroethane and 1,I, 1-trichloroethane. Biochem. Pharmacol. l&2019-2027. LARSON, R., AND PLAA, G. L. (1963). Spinal cord transection and Ccl., toxicity. Experientia 19, 604. LARSON, R., AND PLAA, G. L. (1965). A correlation of the effect of cervical cordotomy, hypothermia and catecholamines on carbon tetrachloride-induced hepatic necrosis. J. Pharmacol. Exp. Ther. 147, 103-l 13. LOWRY, 0. H., ROSEBROUGH.N. J.. FARR, A. I., AND RANDALL, R. J. (195 1). Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265-275. MARZI, A.. DE TORANZO, E. G. D., AND CASTRO, J. A. ( 1980). Mechanism of chlorpromazine prevention of carbon tetrachloride-induced liver necrosis. Toxicol. .4ppl. Pharmacol.
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MEANS, A., TASK, J. S., AND CHAFOUELEAS,J. G. ( 1982). Physiological implications of the presence, distribution and regulation of calmodulin in eukaryotic cells. Physiol. Rev. 62, I-39. MOORE, L., RODMAN-DAVENPORT, G., AND LANDON. E. J. (1976). Calcium uptake of a liver microsomal subcellular fraction in response to in vivo administration of carbon tetrachloride. J. Biol. Chem. 251, 1 I97- 120 I.
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AND CARBON
PENCIL. S. D., GLENDE, E. A., AND RECKNAGEL, R. 0. ( 1982). Loss of calcium sequestration capacity in endoplasmic reticulum of isolated hepatocytes treated with carbon tetrachloride. Res. Commun. Chem. Pathol. Pharmacol.
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RAO, S.. AND RECKNAGEL, R. 0. ( f 969). Early incorporation of carbon tetrachloride into rat liver particulate lipids and proteins. Exp. Mol. Parhol 10, 2 19-228. RECKNAGEL, R. 0. (I 983). Carbon tetrachloride hepatotoxicity status quo and future prospects. Trends Pharmacol. Sci.. I29- I3 1. RECKNAGEL,R. 0.. AND LITTERIA, N. ( 1960). Biochemical changes in carbon tetrachloride fatty liver concentration of carbon tetrachloride in liver and blood. dtner. J. Pathol. 36, 521-531.
RICE. A. J.. AND PLAA, G. L. (1968). Effect of hypophysectomy and spinal cord transection on carbon tetrachloride-induced changes in the hemodynamics of isolated perfused rat liver. Toxicol. Appl. Phartnacol. 12, 194-20 1. SCHANNE. F., KANE, A.. YOUNG, E., AND FARBER,J. L. (I 979). Calcium dependence of toxic cell death: A final common pathway. Science (Washington. D.C.) 206. 700-702.
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SCHENKMAN. J. B., REMMER, H., AND ESTABROOK, R. W. (1967). Spectral studies of drug interaction with hepatic microsomal cytochrome. Mol. Pharmacol. 3, 113-123.
SLATER, T. F. (1982). Activation of carbon tetrachloride: Chemical principles and biological significance. In Free Radical, Lipid Pero.xidulion and Cancer (D. C. F. McBrien and T. F. Slater, eds.). pp. 243-270. Academic Press. New York. STERKEL, R., SPENCER,J., WOLFSON, S., AND WILLIAMSASHMAN, H. ( 1958). Serum isocitric dehydrogenase activity with particular reference to liver disease. J. Lab. Clin. Med.
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WEISS. B.. PROZIALECK, W.. CIMINO, M., BARNETTE, M. S.. AND WALLACE, T. L. (1980). Pharmacological regulation ofcalmodulin. .4nn. N. Y .4cud. Sci. 356,3 19345.
YOUNES, M., ALBRECHT, M.. AND SIEGERS,C. P. (1983). Interrelationship between in vivo lipid peroxidation, microsomal Ca’+-sequestration activity and hepatotoxicity in rats treated with carbon tetrachloride, cumene hydroperoxide or thioacetamide. Rex Cotnmun. Chetn. Pathol.
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40, 405-4
15.
AND
APPLIED
PHARMACOLOGY
83,287-293
(1986)
Late Preventive Effects of Trifluoperazine on Carbon Tetrachloride-Induced Hepatic Necrosis M. C. VILLARRUEL, G. FERNANDEZ, E. C. DE FERREYRA, 0. M. DE FENOS, AND J. A. CASTRO Centro de Investigaciones Toxicoldgicas (CEITOX). CITEFA/CONICET, Zufiategui 4380, 1603 Villa Marteili. Provincia de Buenos Aires, Republica Argentina
Received February 20, 1985: accepted November I, I985 Late Preventive Effects of Trifluoperazine on Carbon Tetrachloride-Induced VILLARRUEL,
M.
C., FERNANDEZ,
G.,
DE FERREYRA,
E. C., DE FENOS,
0.
Hepatic Necrosis. M.,
AND
CASTRO,
(1986). Toxic01 Appl. Pharmacol. 83, 287-293. As a very preliminary test for a possible role of calmodulin in CC&-induced hepatic injury, we studied the effects of the anticalmodulin drug trifluoperazine (TFP) on several deleterious actions of CCL on the liver. TFP administrated 30 min before or 6 or 10 hr after Ccl, significantly prevented hepatic necrosis induced by the hepatotoxin at 24 hr but not at 72 hr. TFP did not modify the Ccl, concentrations reaching the liver, or the intensity of the covalent binding of CC&-reactive metabolites to hepatic microsomal proteins or lipids or the CCL,-induced cytochrome P-450 and glucose 6 phosphatase destruction. TFP administration decreased body temperature between 0 and 1“C in controls and between 1.2 and 3.5”C in CCl&reated animals during the 24-hr observation period. When TFP-treated CCL-poisoned animals were kept normothermic, protective effectswere eliminated. One possibility is that the protective effect of TFP might be due to a nonspecific action related to decreased body temperature. Alternatively, prevention might result from TFP inhibition of a late-occurring process critical for CCld-induced cell necrosis requiring calmodulin participation. If this alternative were in operation, protective consequences of this inhibitory effect of TFP should be either canceled or counteracted in the normothermic TFP + CCL-treated animal. 0 1986 Academic PI.s, IIIC. J. A.
Intracellular calcium homeostasis has been proposed as a possible reason for liver cell death induced by CCL, and several other hepatotoxins (Chenery er al., 198 1; Farber, 198 1, 1982; Moore et al., 1976; Pencil et al., 1982; Schanne et al., 1979; Younes et al., 1983). Notwithstanding, the mechanism by which calcium causes cytotoxic effects is not known, but likely targets include enzyme systems of high relevance to cell function which are sensitive to calcium (Farber, 198 1, 1982; Pencil et al., 1982). There is considerable evidence that many cell activities mediated by calcium are probably being accomplished through calmodulin regulation, since calmodulin functions as an intracellular mediator of the effects of calcium (Means et al., 1982; Weiss et al.,
1980). A very interesting property of calmodulin is its rather specific binding to a variety of pharmacological agents (Means et al., 1982; Weiss et al., 1980). These drugs, known as calmodulin antagonists, may prove very useful in preliminary testing of a possible role of calmodulin in calcium-mediated, CC&-induced cell necrosis. The most widely used anticalmodulin pharmacological agent employed in these type of studies has been trifluoperazine (TFP), which binds the calcium-calmodulin complex with high affinity and interferes with its ability to bind the receptor protein (Means et al., 1982; Weiss et al., 1980). In the studies described in this report, we tested whether TFP administration to rats prevents several manifestations of CC&-induced liver injury includ287
0041-008x/86
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VILLARRUEL
288
ing hepatic necrosis. We also tested its effects on several parameters known to modulate the deleterious actions CClh. METHODS Chemicals. The purity of [‘4C]CC14’ (sp act 2.8 mCi/ mmol) was greater than 99% when determined by gasliquid chromatography with a column of Carbowax 400 Durapak on Porasil C at 65°C. Trifluoperazine hydrochloride’ was pharmaceutical grade. All other chemicals employed were of reagent grade. Animals and animal treatmenl. Sprague-Dawley male rats bred in our laboratory (240 to 290 g) were used throughout these experiments. Feed (Purina Chow rat diet) was withdrawn 12 to 14 hr before TFP was given. Animals did not have accessto feed during the experimental period but water was available ad libifum. Temperature in the animal room was 23 + 2°C and the relative humidity was between 35 and 65%. Light in the animal room was on from 6 AM to 6 PM. TFP was given ip in 0.9% NaCl solution at a dose of 50 mg/kg (IO mg/ml solution) 30 min before or 6 or 10 hr after CC&. Control rats received an equivalent amount of 0.9% NaCl. [‘4C]CC14 was given ip in CC& 5% (v/v) solution in olive oil (30 X IO6 dpm/ml) at a dose of 5 ml of solution/kg. CC& (I ml/kg) was given ip as a 20% (v/v) solution in olive oil in a volume of 5 ml/kg of final solution. Control rats received olive oil ip. The animals were killed by decapitation at different times after CC& administration, and their livers were rapidly removed and processed. Whenever blood samples were taken, animals were kept under light ether anesthesia. and blood was obtained with a heparinized syringe from the inferior vena cava. Rectal temperatures of the rats were recorded with a small animal thermistor probe (Telethermometer, Nihon-Kohden). In some experiments the normal body temperature of TFP + Ccl, and TFP-treated animals was maintained by keeping them at 37 f 1“C in an air-ventilated incubator. Isolation of microsomes. The hepatic microsomal fraction was isolated by the procedures described by Castro ef al. (1972). Enzymatic and chemical determinations. The in viva incorporation of radioactivity from “CC14 to microsomal proteins was determined according to procedures described by Rao and Recknagel ( 1969). A IO-mg aliquot of the purified and dried protein was dissolved in formic acid and counted as previously described (Diaz Gomez et nl., 1973). The in vivo incorporation of 14Cfrom “CCl, to microsomal lipids was determined according to a procedure described by Castro et al. (1972). Results are given in disintegrations per minute per ’ Radiochemical Centre, Amersham, England. 2 Smith. Kline & French Laboratories.
ET AL milligram of microsomal protein or lipid. The 14CC14concentrations in liver were estimated according to a procedure described by Recknagel and Litteria (I 960) up to the microdiffusion step; then, the 14CC14collected in the toluene of the center well ofthe cell was transferred to a scintillation counting vial and counted. Results were corrected for loss and for quenching by the channels ratio method. Lipid peroxidation in viva was measured by conjugated diene ultraviolet absorption of the microsomal lipid extracts (Klaassen and Plaa, 1969). The results are expressed as the absorbance at 243 nm x 1000 for a solution having I mg of microsomal lipid/ ml. The hepatic microsomal cytochrome P-450 (P-450) content was determined as described by Schenkman et al. (1967). Glucose-6-phosphatase activity (G6Pase) in microsomal suspensions was measured according to Harper (1963). Activity is expressed in micrograms of inorganic phosphorus produced in I5 minutes at 37°C per milligram of protein. NADPH-linked isocitric acid dehydrogenase (ICD) activity in plasma was measured by the method of Sterkel e/ al. (1958). Activity is given in units: one unit is the amount of enzyme producing I nmol NADPH/ml plasma/hr at 25°C. Protein concentrations were estimated by Lowry’s procedure (Lowry et ol., 1951). Histological techniques. After removal of the liver, small portions from the left and central lobes were immediately fixed in Bouin’s solution, embedded in paraffin, and stained with hematoxylin and eosin. The specimens were coded to avoid bias and were evaluated histologically by at least three independent observers. To quantify the morphological changes, hepatic sections were graded for changes such as hydropic degeneration or necrosis. using an arbitrary scale: + = light (about 20 to 30% necrosis): ++ = moderate (about 50% necrosis): +++ = marked (about 75% necrosis): ++++ = very intense (about 90 to 100% necrosis). Results reported as representative for each experimental condition are the mean of observations made by all observers for a given experimental condition. Stafistics. A decision tree for selecting hypothesis testing statistical procedures described by Gad and Weil ( 1982) was applied to results from every experiment. Homogeneity of variance was established using either the Bartlett’s homogeneity of variance test or the f test for comparisons involving three or more groups or two groups, respectively. According to significance of homogeneity of variance and size of the groups involved, one of the following tests was applied: Duncan’s multiple range test; Dunnett’s test: the Student f test or the Cochran t test (Gad and Weil, 1982). Parametric or nonparametric analysis of variance testswere used for comparisons involving three or more groups of data (Gad and Weil, 1982).
RESULTS
Eflect of TFP in vivo on microsomal lipid peroxidation produced by CCL at dl@erent times after administration. TFP alone pro-
TRIFLUOPERAZINE TABLE
AND
CARBON
1
CCJNDUCED LIPID PEROXIDATION OF MICROSOMAL LIPIDS IN RATS PREVIOUSLY TREATED WITH TFP Lipid peroxidation Treatment” CO”t”Jl
CCL TFP TFP + cc14
(X i SD)*
1 hr
70’
3 hr
21 1.6 k 21.2 346. I k 4~%4~ 335.9 + 86.5’
164 159
179.8 + 49.7 333.2 k 72.3 299.6 k 66.1
185’ 167’
443.5 + 44.9d.e
209
350.4 k 103.4
195C’
’ TFP was given ip at a dose of 50 @kg. Ccl, was given ip as a 20% solution in olive oil at a dose of 5 ml/kg, 30 min after TFP administration. Eight animals per group were used in these experiments. Animals were killed I and 3 hr after CCL administration. ’ The lipid peroxidation value is expressed as A absorbance at 243 nm X 1000 for a solution having I mg of microsomal lipid/ ml. ‘a < 0.01 when compared with its respective control: Student’s 1 test. dp < 0.001 when compared with its respective control; Student’s t test. ‘The p value for the overall effect of the TFP on the CC&induced lipid peroxidation obtained by analysis of variance was p > 0.05 for all the times treated.
duced an increase of the lipid peroxidation process as measured by the diene conjugation technique at 1 or 3 hr (Table 1). TFP did not modify the intensity of the CC&induced lipid peroxidation process (Table 1). E#ect ofpretreatment with TFP on the covalent binding of 14C from [‘4C]CCld to mi-
TABLE COVALENT
BINDING
Treatment”
somal lipids and proteins. As shown in Table 2, prior administration of TFP to rats did not alter the extent of covalent binding of [‘4C]CC14 to microsomal lipids or proteins at 1 or 3 hr. Eflect of TFP on hepatic concentrations of [‘4C]CC/d. Liver content of [ “C]CC14 at different times after its administration in TFPtreated rats was not significantly different from that observed in untreated animals at 1 and 3 hr (Table 3). Eflect of TFP administration on the glucose 6-phosphatase activity and cytochrome P-450 destruction caused by CCld. TFP administration did not modify the destruction of G6Pase or P-450 3 hr after CCL administration (Table 4). Eflect of pretreatment with TFP on CC&induced decrease of body temperature. Treatment with TFP alone lowered the body temperature of the rats at 1 and 3 hr but not at 6 and 24 hr after administration. The CCLpretreated rats had a significant decrease in body temperature (p < 0.05 at 1 and 3 hr). TFP significantly increased hypothermia following CC4 injection (p < 0.05 at 1 hr, p < 0.001 at 3 hr, and p < 0.01 at 6 hr) (Table 5). Eflect of TFP on CC&-induced liver damage. Pretreatment with TFP 30 min before or 6 or 10 hr after CC4 significantly diminished the extent of CCkinduced necrosis 24 hr after ad-
2
OF CC& REACTIVE METABOLITES TO HEPATIC MICROSOMAL PROTEINS OF RATS PREVIOUSLY TREATED WITH TFP Covalent Lipid + SD 6bM-w)
binding
Protein + SD (dmUm)
of 14C from Lipid
+ TFP
167.0 k 29.3 144.3 + 30.2’
LIPIDS AND
[‘4C]CC1., f SD
Protein
(dpm/mg)
I hr [‘4c]ccl, [‘4C]CC14
289
TETRACHLORIDE
+ SD
kbm/mg) 3 hr
43.7 -c 6.7 42.5 + 8.9’
139+ I1 155 f 5.1b
39.5 + 6 36.0 + lb
a [‘4C]CC14 was given ip in CC4 5% (v/v) solution in olive oil (30 X lo6 dpm/ml) at a dose of 5 ml solution/kg, 30 min after TFP. TFP was administered as in Table I. The animals were killed at 1 or 3 hr after [‘4C]CCI, administration; their livers were processed for microsomal protein isolation. lipid extraction, and counting (see Methods for details). Eight animals per group were used in these experiments. b p > 0.05 when compared with its respective control; Student’s t test.
290
VILLARRUEL TABLE
3
“CC14 CONCENTRATIONS IN LIVER AT DIFFERENT TIMES AFTER ADMINISTRATION TO RATS PRETREATEII WITH TFP
Treatment” ‘TC14 “CC14 “Tel, “‘CCL,
+ TFP + TFP
Time (W 1 1 3 3
YC14 concentration in liver (dpm/g + SD) 185,140 193,460 118.228 135,400
f + + k
48,480 53,630’ 18,350 52,420b
% of control 100 104 100 114
a “CC14 and TFP were given as indicated in Tables 2 and I, respectively. Five rats per group were used in these experiments. * p > 0. I: Student’s t test.
ministration, as evidenced by determinations of ICD in plasma (Table 6). Hepatic necrosis was also assessed by histological examination. TFP alone did not significantly alter the normal hepatic architectural pattern at 24 hr. Administration of CCL+ to animals produced an intense centrolobular necrosis at 24 hr (Table 6). TFP markedly decreased the extent of the necrosis produced by CC& 24 hr after administration when administered 30 min before or 6 or 10 hr after the hepatotoxin (Table 6). However, liver necrosis in both CC&- and TFP + CC&-treated animals was very intense (++++) when assessed72 hr after CC&. In this experiment eight animals per group were employed and the reported results are the mean of the observations made, as detailed under Methods. No ICD values are given in this case because at 72 hr after CCL, administration, they returned to normal in spite of the histologically observable damage (Marzi et al., 1980). If animals treated with TFP at 10 hr after Ccl4 were maintained at 37°C in an incubator, TFP did not have any protective action. In this experiment, animals were not placed in an incubator immediately after CC& since they would not survive for 24 hr. To increase survival body temperature regulation in incubator was started at 10 hr after CCld. In this case 1 out of 10 animals survived. Survivors in the TFP + CCL, group
ET
AL.
had intense liver necrosis (++++) as did the CCL, group (ICD values: 111, 982 units for eight animals in CCL+ group and 97,500 units for five animals in the TFP + CC& group; p > 0.05). DISCUSSION Our studies on ICD activity in plasma and the histological evaluation of the livers of TFPtreated animals receiving CC& revealed that this drug significantly delayed the onset of CC&-induced liver necrosis when given 30 min before the hepatotoxin. It is particularly interesting that TFP-delaying effects were still observable when the drug was given 6 or 10 hr after CCL. Moreover, beneficial effects of TFP at this late stage of the intoxication process were more pronounced than when the drug was given 30 min prior to CC&. This behavior suggests that events occurring late in the pathway followed by the CC&-injured liver cell were modulated by TFP and not those events predominating at early periods of in-
TABLE
4
EFFECT OF CC& ADMINISTRATION ON THE CYTOCHROME P-450 CONTENT AND GuJ~~~E-~-PH~~PHATAsE ACTIVITY OF RATS PREVIOUSLY TREATED WITH TFP
P-4506
Treatment” Control CC-14 TFP TFP + Ccl,
0.32 0.22 0.35 0.23
f 0.02 f 0.03d + 0.01 57 0.03”’
G6Pasec 47.0 32.8 55.5 35.9
+ * i t
6.0 3.8d 5.5 3.4”’
’ CC& and TFP doses were given ip as in Table I. Ccl4 was administered 30 min after TFP. and the animals were killed 3 hr after CC& administration. Eight animals per group were used in these experiments. *P-450 values are given in nanomoles per milligram of protein + SD. ’ GhPase activity is expressed in micrograms of inorganic phosphorus produced in I5 min/mg of microsomal protein at 37°C & SD. dp < 0.001 when compared with its respective control; Student’s I test. ’ The p value of the overall effect of TFP on the CCL effects obtained by analysis of variance was >O.O I.
TRIFLUOPERAZINE
291
AND CARBON TETRACHLORIDE TABLE 5
EFFECTSOF CARBON TETRACHLORIDE AT DIFFERENT TIMES AFTER ADMINISTRATION ON BODY TEMPERATURE OF RATS PREVIOUSLY TREATED WITH TFP Body temperature (“C + SD)* Treatment’ Control CC-14
TFP TFP + CC&
I hr 36.2 + 0.7 35.0 f 0.4' 33.7 + I.ld 33.8 k I.Oc.d
3 36.3 35.3 33.8 33.2
hr t * k k
0.4 0.3' 1.6' 0.7'
6
hr
36.4 k 0.4 36.7 k 0.4 35.3 + 1.2 34.4 f 1.2d
24 36.8 36.2 37.2 32.7
f + * -+
hr 0.5 0.6 0.5 3.7'ld
a CC& and TFP were given as indicated in Table I. Five rats per group were used in these experiments. b The p value for the overall effect of TFP on the CC&-induced decrease in body temperature obtained by analysis of variance was p > 0. I for 1, 3, and 6 hr. and p < 0.05 for 24 hr. 'p < 0.05 for CCL, vs control at 1 and 3 hr. for TFP vs control at 3 hr. for TFP + Ccl4 vs TFP at 24 hr and for TFP + Ccl, vs CC& at 1 hr; Student’s t test. dp < 0.01 for TFP vs control at 1, 6, and 24 hr. and for TFP + CCL, vs CCL, at 6 hr; Student’s t test. 'p < 0.001 for TFP + CC& vs control and for TFP + Ccl., vs CC& at 3 hr: Student’s t test.
toxication, such as lipid peroxidation or covalent interactions of CC&-reactive metabolites with cellular constituents (CB). In fact, our present results support this hypothesis, since no significant effects of TFP treatment on CB or lipid peroxidation were observed. Furthermore, delaying effects can not be attributed to interference by TFP in the ability of CC& to reach the liver, since CC4 concentrations in livers of TFP-treated and in untreated animals were not significantly different. The above pattern of TFP explains why it does not prevent CC&-induced hepatic microsomal P-450 destruction or prevent glucose-6-phosphatase activity depression. In fact, the deleterious effects of CC& on these parameters are mediated by either CB or lipid peroxidation or both (Recknagel, 1983; Slater, 1982; Castro, 1984). These parameters remain unchanged during TFP treatment of CC& poisoning. Concerning the nature of the late-occurring event modulated by TFP having delaying effects in the course of CCLinduced hepatic necrosis, there are several possible hypotheses. One might be that TFP blocks a critical event involving calmodulin participation as intracellular mediator of the deleterious effects of calcium accumulation. Another possibility might be that TFP exerts a nonspecific delay-
ing effect mediated by its ability to decrease body temperature, similar to that previously reported for chlorpromazine (Marzi et al., 1980). It is known that decreases in body temperature, caused by hypothermia or cordotomy, delay the CC&-induced hepatic necrosis (Larson and Plaa, 1963, 1965; Rice and Plaa, 1968), and that preventive effects observed in those cases and in that of chlorpromazine at 24 hr after CC& are gone by 72 hr (Marzi et al., 1980). In the case of TFP, disappearance of delaying effects at 72 hr was also observed. Moreover, we also found that TFP-delaying effects at 24 hr after CC& were no longer observable if TFP + CCktreated animals were kept normothermic. This behavior was also observed in the case of chlorpromazine (Marzi et al., 1980). These findings appear to favor the latter hypothesis. However, both mechanisms might be operating in conjunction or simultaneously. In effect, temperature may cancel or produce changes counteracting calmodulin-mediated effects. The preliminary nature of these studies does not allow one hypothesis to be selected over the other. Irrespective of the mechanism of the TFP preventive effects, however, the fact remains that this drug modulates the intensity of late stages of CC&-induced hepatic injury and might give
292
VILLARRUEL
ET AL.
TABLE 6
REFERENCES
EFFECTSOF TFP ADMINISTRATION ON CC&-INDUCED HEPATIC NECROSISAT 24 HR AFTER ADMINISTRATION TO RATS
Treatment’
ICDb (units + SD)
Degree of histologically observable necrosis’
30 min before Control ccl,
160 + 35 178.500 * 32.900
TFP + CCL,
15,180 140 _+ + 70 11,300‘+
+++s +++ -
6 hr after Control cc14
180 + 50 200,400 f 17,300
++++
CASTRO,J. A. ( 1984). Mechanistical studies and prevention of free radical cell injury. Proc. IX Intern. Congress Pharmacol.. Vol. 2, pp. 243-270. Macmillan & Co., London. CASTRO, J. A., CIGNOLI, E. V., DE CASTRO, C. R., AND DE FENOS, 0. M. (1972). Prevention by cystamine of liver necrosis and early biochemical alterations induced by carbon tetrachloride. Biochem. Pharmacol. 21, 4957. CHENERY, R.. GEORGE, M., AND KRISHNA, G. (1981). The effectof ionophore A23 I87 and calcium on carbon tetrachloride-induced toxicity in cultured rat hepatocytes. Toxicol.
Appl. Pharmacol.
TFP + CC&
27,000 135 f+ 60 16,800”’
+ to- ++
10 hr after Control ccl,
155 + 45 189,280 f 39,030
TFP + Ccl,
13, 150 570 ff 708,930”’
++++ +to++ -
’ Ccl, and TFP were administered as indicated in Table I. TFP was administered 30 min before CC& or 6 or IO hr after the hepatotoxin. The animals were killed 24 hr after Ccl,. Seven animals per group were used in these experiments. b Isocitric acid dehydrogenase: I unit of enzyme is the amount required to form I nmol NADPH/ml plasma/hr at 25°C. ’ + = slight (20-30% necrosis): ++ = moderate (50% necrosis); +++ = marked (75% necrosis); ++++ = very intense (90-100% necrosis). d The p value for the overall effect of TFP on the CC&induced increase in ICD obtained by two-way analysis of variance wasp < 0.001 at 30 min, 6- and lo-hr values. ep < 0.001 for TFP + Ccl, vs CCL; Student’s t test.
an insight into the nature of the events involved. ACKNOWLEDGMENT This work was supported by Grant AM- 13 195- 16 from the National Institutes of Health (USA).
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AND CARBON
PENCIL. S. D., GLENDE, E. A., AND RECKNAGEL, R. 0. ( 1982). Loss of calcium sequestration capacity in endoplasmic reticulum of isolated hepatocytes treated with carbon tetrachloride. Res. Commun. Chem. Pathol. Pharmacol.
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