TOXICOLOGY
AND
APPLIED
Modification
PHARMACOLOGY
of Carbon
41,369-316
(1977)
Tetrachloride Chemicals
D. SURIYACHANAND
Hepatotoxicity
by
A. THITHAPANDHA
Department of Pharmacology, Faculty of Science, M&idol
University, Bangkok 4, Thailand
Received December 9,1976; accepted February 7, I977 Modification of Carbon Tetrachloride Hepatotoxicity by Chemicals. SURIYACHAN, D., AND A. (1977). Toxicol. Appl. Pharmacol. 41, 369-376. Cobaltous chloride (60 mg/kg, SC, daily for 2 days), which was found to effectively decrease the microsomal cytochrome P-450 content of mouse liver to approximately half of its normal value and which impaired the oxidative metabolism or hydroxylation of aminopyrine, ethylmorphine, and hexobarbital, offered no protection against CC&induced liver damage. However, this hemoprotein inhibitor had no effect on the rate of reduction of cytochrome P-450 by NADPH and exerted a slight effect on aniline hydroxylation. SKF-525A (50 mg/kg, ip) also failed to protect against Ccl, hepatotoxicity though it has been shown to inhibit the hydroxylation of a number of substrates. This inhibitor, a type I compound, was found to enhance cytochrome P450 reduction by NADPH. Further studies revealed that CCI,-induced hepatic injury could be prevented by phenazine methosulfate (2 mg/kg, ip, 5 doses at 0.5.hr intervals), which in vitro was found to inhibit NADPH-cytochrome c reductase noncompetitively. All of these findings are not satisfactorily explainable by electron transfer from NADPH-cytochrome c reductase to CCI, as the activation reaction for Ccl, but are compatible with the hypothesis previously proposed by others that cytochrome P-450 is the critical site for Ccl, activation. TH~THAPANDHA,
Although it is well documented that Ccl,-induced hepatotoxicity is probably causedby its free radicals arising during a Ccl,-activation step mediated by the microsomal drug oxidizing system (Slater, 1966; Recknagel, 1967; Glende and Recknagel, 1969), the actual site for this activation is still a subject of debate by several groups of investigators. Nayak et al. (1970) and Chopra et al. (1972) suggestedthat Ccl, might be metabolized at the early stage of the hepatic microsomal electron transport chain where NADPH is oxidized to NADP by NADPH-cytochrome c reductase. However, serious doubts as to the validity of the hypothesis that NADPH-cytochrome c reductase is the locus of carbon-chloride bond cleavage have been raised by Glende (1972). Cignoli and Castro (1971) and Castro et al. (1973) pointed out that cytochrome P-450 was likely to be the site for Ccl, activation. The reason for the discrepancy among the results of theseworkers remains unknown. The present communication reports on the results of both in vitro and in vivo studies with Ccl, and the modification of its hepatotoxicity by chemicals capable of modifying the activities of certain components along the electron transport chain of mouse liver’s drug oxidizing system. These chemicals include 2-diethylaminoethyl-2,2diphenylvalerate (SKF-525A), cobaltous chloride (CoCl, .6H,O) and phenazine methosulfate (P&IS). The role of NADPH-cytochromec reductase and cytochrome P450 on Ccl, activation has been evaluated. Copyright 0 1977 by Academic Press. Inc. All rights of reproduction in any form reserved. Printed in Great Britain
369
ISSN 004 1408X
370
SURIYACHAN
AND
THITHAPANDHA
METHODS Treatment of Animals Male mice of New Zealand strain were used throughout the study. The animals (2530 g) were fed with commercial mouse pellets, premium quality feed, Zuellig (Gold Coin Mills) Pte. Ltd., Singapore, and tap water ad libitum. Ccl, was prepared as 10% solution (v/v) in liquid paraffin and administered ip at a dose of 0.5 ml/kg. Other chemicals were prepared in physiologic saline just before use. SKF-525A (50 mg/kg, ip) was given 30 min before Ccl, intoxication. Animals were pretreated with cobaltous chloride (60 mg/kg, SC) daily for 2 days. Ccl, was then injected 24 hr after the last dose of the hepatic hemoprotein inhibitor; animals were sacrificed 3 hr after Ccl, administration. Phenazine methosulfate (2 mg/kg, ip) was given to the animals for 5 doses at 0.5-hr intervals. Ccl, was administered 10 min after the first dose of phenazine methosulfate; mice were sacrificed 3 hr after Ccl,. Preparation
of Liver Microsomes
Male mice, 25-30 g, were killed by decapitation. Livers were removed and homogenized with a motor-driven glass-Teflon homogenizer in two volumes of 1.15% KC1 containing 20 mM Tris-KC1 buffer, pH 7.4. The homogenate was centrifuged for 20 min at 9,000 g in a Sorvall refrigerated centrifuge, and the supernatant was decanted and recentrifuged for 1 hr at 105,OOOg in a Spinco model L2-65B preparative ultracentrifuge. The microsomal pellet was resuspended and washed in the Tris-KC1 buffer and recentrifuged at 105,000 g for 50 min. The washed microsomal pellet was resuspended in 0.1 M phosphate buffer, pH 7.4, and used as the sources for subsequent determinations. Enzyme Assays Serum glutamic-pyruvic transaminase (SGPT) was determined by the ReitmanFrankel method (1957), according to the description given in Sigma Technical Bulletin No. 555. Activity is expressed in Sigma-Frankel units; one such unit corresponds with the formation of 4.82 k 10e4 ~01 of glutamate per min at pH 7.5 and 25°C (Nadeau and Marchand, 1973). Aminopyrine N-demethylase and aniline hydroxylase were measured from the 9000-g supernatant of mouse liver as described by Maze1 (1971). Ethylmorphine Ndemethylase, NADPH-cytochrome c reductase, and cytochrome P-450 in a microsomal preparation were measured as described by Baron and Tephly (1969). Glucose-6-phosphate from the 9000-g supernatant was determined by the method of Swanson (1955). NADPH-P-450 reductase activities were quantitated as described in detail by Gigon et al. (1969). Protein Determination Protein content both in the supernatant and microsomal by the biuret method (Gornall et al., 1949).
preparations
was measured
Hepatic Triglyceride The concentration of triglyceride in liver was measured according to the method of van Handel and Zilversmit (1957) as modified by Butler et al. (1961). The content was expressed as milligrams per gram of liver wet weight.
CARBON
TETRACHLORIPE
371
HEPATOTOXIClTY
Sleeping Time Hexobarbital (100 mg/kg, ip) was administered to control mice and to mice previously treated with cobaltous chloride (60 mg/kg, SC.48 and 24 hr earlier). The sleepingtime in each group was then recorded in the usual manner. All data in this report were analyzed statistically by Student’s t test. RESULTS
When cobaltous chloride (60 mg/kg, SC,daily for 2 days) was administered to mice, it exerted a marked influence on the activities of several well-known cytochrome P-450. requiring hepatic enzymes. Both the level of ethylmorphine N-demethylation, measured from a microsomal preparation, and that of aminopyrine N-demethylation, measured from the 9000-g supernatant, were reduced to 5%60% of the control (Table 1). TABLE EFFECTS OF ETHYLMORPHINE
I
COBALTOUS CHLORIDE PRETREATMENT ON HEXOBARBITAL N-DEMETHYLASE, AMINOPYRINE N-DEMETHYLASE, HYDROXYLASE ACTIVITIES~
Ethylmorphine Sleeping time Treatment
(min)
Control CoCl,d
39 & 4 66 + 10
SLEEPING AND
Aminopyrine
N-demethylationb (nmol of HCHO formed/mg of
Aniline
N-dem,.thylation* (nmo: of HCHO f,lrmed/mg of rrotein/min)
protein/min) 3.40 + 0.30 1.72 + 0.24
hydroxylaseh.’ -nmol of PAP/mg of protein/ 20 min) 6.15 + 0.26 4.95 & 0.26
3 1.76 + 2.48 20.63 + 2.05
“Ten mice in each group were pretreated with cobaltous chloride and sacrificed Control animals received equal volume of physiologic saline. Hexobarbital administered 24 hr after the last dose of CoCI,, and the sleeping time was recorded. b Each value is the mean f SE of three determinations from 10 animals. c PAP stands for p-aminophenol.
dose.
d All
values
in CoCl,-treated
group
are significantly
different
from
controls,p
TIME, ANILINE
24
hr after
the
last
(100 mg/kg. ip) was
< 0.05.
Physically, the animals exhibited signs of central nervous system depression after the first dose of this chemical. They appeared sedatedand seemedto lose appetite. None of the animals died, however. The in vivo oxidative metabolism of hexobarbital, which also requires cytochrome P-450, was significantly impaired, as evidenced by a marked prolongation of the sleeping time; 39 f 4 in 10 control mice against 66 i 10 in IO CoCl,-treated animals (Table 1). However, when animals pretreated with this dosage regimen of cobaltous chloride were challenged acutely with Ccl, (0.5 ml/kg, ip), the haloalkane was still effective in causing hepatic damage, as indicated by a rise in the activity of SGPT and an increase in hepatic triglycerides (Table 2). In essence, cobaltous chloride afforded no protection against Ccl,-induced hepatic injury. This is rather unexpected in view of the fact that hepatic drug metabolism is involved in several types of hepatic drug intoxications including Ccl, (Recknagel, 1967). Since cobaltous chloride was able to decreasethe activities of the hydroxylating enzymes (Table l), it would be expected to prevent or reduce the hepatotoxicity caused by the haloalkane. But this was not found to be the case. Thus, the role of cytochrome P-450 in Ccl, activation might be in doubt.
372
SURIYACHAN
AND
THITHAPANDHA
However, when one realizes that drug hydroxylation
and the reduction of cytochrome
P-450 may be two separate entities one could not easily rule out the involvement of the hemoprotein in Ccl, metabolism. In contrast to aminopyrine N-demethylase and ethylmorphine N-demethylase, both of which were greatly reduced by CoCl,, the TABLE EFFECTS PYRUVIC
2
OF COBALTOUS CHLORIDE PRETREATMENT ON SERUM TRANSAMINASE ACTIVITY AND HEPATIC TRIGLYCERIDE AFTER CARBON TETRACHLORIDE INJECTION“
Treatment Control COCI, ccl, CoCl, + ccl,
Triglyceride (mg/g of liver)
SGPT Unitb 37.25 40.50 96.67 94.28
GLUTAMICCONTENT
+ 9.83 (1) k 7.23 (10) +_ 15.98 (10) t 10.28 ( 10)d
10.70 12.43 23.60 25.23
k rt k &
1.48 1.27 3.65 2.95
(10) (10) (10) (lO)d
(I All values given are mean & SE, with the number of animals shown in parentheses. Animals were killed 3 hr after CCI, administration. b One unit corresponds to the formation of 4.82 f 10m4prnol of glutamate/min at pH 7.5 and at 25°C. LISignificant difference from control and CoCl,,p < 0.05. d No difference from CCI,
activity of aniline hydroxylase was slightly reduced by this hemoprotein inhibitor (Table 1). Since aniline hydroxylation also requires cytochrome P-450 for its activity, it is quite likely that the species of this cytochrome involved in Ccl, activation is not very much affected by pretreatment with cobaltous chloride. As shown in Table 3, though microsomal cytochrome P-450 content was reduced to approximately 50% of normal TABLE EFFECTS
OF COBALTOUS
Compound Control CoCI,, 0.25 mM SKF-525A, 0.05 mM
3
CHLORIDE AND SKF-525A ON CYTOCHROME HEPATIC MICROSOMES FROM MALE MICE~
P-450 REDUCTION
Cytochrome P-450 (nmol/mg of protein)
Initial rate of reduction of P-450 by NADPH (nmol reduced/min/mg of protein)
1.40 & 0.16 0.52 + 0.08b 0.96 & 0.32
8.21 k 0.74 9.00 + 1.10 18.87 k 0.52b
IN
(1Values expressed as mean & SE of four determinations, each of which was made with a microsomal preparation obtained from the pooled livers of five animals. b Significantly different from control,p < 0.05.
by cobaltous chloride, the rate of reduction of this hemoprotein remained unchanged. Therefore, cobaltous chloride, which could increase or decrease drug hydroxylation, might not appreciably affect the cytochrome P-450 species that catalyzes the activation of Ccl,. In support of this conclusion, SKF-525A, which is known to inhibit the hydroxylation of a number of substrates and which was found in our studies and
CARBON
TETRACHLORIDE
373
HEPATOTOXICITY
previously shown by Gigon et al. (1969) to enhance P-450 reduction (Table 3), offered no protection against the toxic actions of Ccl, (Table 4). This is presumably due to the inability of SKF-525A to bind the cytochrome P-450 species which metabolizes Ccl,. The effect of phenazine methosulfate (PMS) on CC&-induced liver damage is shown in Table 5. Glucose-6-phosphatase, a membrane-bound enzyme known to be more TABLE 4 EFFECTS OF SKF-525A PRETREATMENT ON THE ACTIVITIES OF AMINOPYRINE N-DEMETHYLASE, SERUM GLLJTAMIC-PYRLJWC TRANSAMINASE AND HEPATIC TRIGLYCERIDE CONTENT AFTER AN INTRAPERITONEALADMINISTRATIONOFCARBONTETRACHLORIDE~
Treatment
Aminopyrine N-demethylation (nmolof HCHO formed/mgof protein/30min)
SGPT Unit
30.00 + 4.28 34.50 k 8.35
ccl,
33.22 + 2.15 31.35 + 1.90 22.90 k 1.76b
SKF-525A + CC&
23.10f 0.68’
82.80 f 13.53’
Control SKF-525A
89.40 k 10.61b
Hepatictriglyceride (mg/gof liver) 12.14 + 1.03
14.21 + 0.65 27.38 k 3.35b 26.76 k 2.25'
a Values are expressed as mean + SE, with 10 mice in each group. Ccl, was given 30 mitt after Animals were killed 3 hr after Ccl, b Signiticantly different from control and SKF-525A (p < 0.05). cNot significantly different from Ccl,.
SKF-525A.
TABLE 5 EFFECTS OF CARBON TETRACHLORIDE ONTHE ACTIVITIES OF GLUCOSE-6-PHOSPHATASEAND AMINOPYRINE N-DEMETHYLASE IN MICE PRETREATED WITH PHENAZINE METHOSULFATE"
Treatment
Control PMS ccl,
PMS + Ccl,
Glucose-6-phosphatase (nmol of Pi formed/mg of protein/min) 127.22k 5.83 126.49+ 4.53 64.26 k 5.50b 108.51+ 7.45c
Aminopyrine N-demethylation (nmolof HCHO fortned/mg of protein/30min)
35.22 k 3.47 33.08 k 3.20 21.90 + 2.10b 31.20 + 1.40’
0 Each value is the mean & SE of 10 determinations from four to six mice. The animals were killed 3 hr after Ccl, administration. * Significantly different from controkg < 0.05. c Significantly different from Ccl, (p < 0.05) but not from control.
sensitive to the action of Ccl,, was unaffected by PMS treatment but its activity was greatly decreased by Ccl, administration (Table 5). However, when mice were pretreated with PMS at the dosesindicated, the haloalkane produced no change on the activity of either glucose-6-phosphataseor aminopyrine N-demethylase (Table 5). Although data are not shown, the activity of SGPT and hepatic triglyceride content were found to be the same as in controls. Thus, PMS seemsto be an effective inhibitor
374
SURIYACHAN
AND
THITHAPANDHA
of CC&-induced hepatic injury. Further, it was found that PMS inhibited NADPHcytochrome c reductase and that the inhibition was noncompetitive (Table 6). TABLE INHIBITION
OF NADPH-CYTOCHROME METHOSULFATE
Phenazine methosulfate (M)
0 1 x IO--2 1 x 10-I 1 x 10-d
6 c REDUCTASE IN MICE
Enzymatic activity” (nmol/mg of protein/min)
130.2t 14.1 60.5 2 10.4 84.2 _+ 12.2 124.0 t 15.0
BY PHENAXNE
Inhibition
(96)
0 53 35 4
“The activity of NADPH-cytochrome c reductase in microsomal preparations was measured on a Gilford Model 2000 recording spectrophotometer as the rate of increase in absorbance 550 nm produced by the reduction of cytochrome c. Each cuvette of l-cm light path contained, in a total volume of 3 ml, I.0 ~01 of sodium cyanide, 0.1 pmol of cytochrome c, 1 mg of microsomal protein, PMS at the concentrations indicated, and 0.2 M potassium phosphate buffer, pH 7.4. At zero time, 0.4 pmol of NADPH was added to start the reaction. Enzymatic activity, which was determined by using 19.7 rnr.-’ cm-’ as the millimolar difference extinction coefficient at 550 nm, was expressed as nanomoles of cytochrome c reduced per milligram of protein per minute. Each value is given as mean & SE of three experiments.
DISCUSSION
In recent years, evidence hasbeen accumulated in a number of laboratories indicating that the severity of Ccl,-induced liver damage is directly proportional to the activity of the microsomal drug oxidizing or cytochrome P-450 system @later, 1966; Recknagel, 1967; Sasameet al., 1968). In newborn and hepatectomized rats, Ccl, has been shown to be less toxic than in normal adult animals (Dawkins, 1963; Pani er al., 1975), presumably because the drug oxidizing system in these animals is functionally depressed.On the other hand, phenobarbital, a microsomal enzyme inducer, increases the toxicity of Ccl, (Garner and McLean, 1969). Thus, the toxic effects of Ccl, may be prevented by lowering its metabolism; this can be effected by modifying the functional state of the drug oxidizing system. For example, cystamine and cysteamine have been reported to prevent several structural and biochemical alterations caused by the haloalkane by inhibiting Ccl, activation taking place at the hemoprotein, cytochrome P-450 (Castro et al., 1973). Recently, it has been found that Ccl,-induced liver necrosis was also prevented by lead; however, the exact mechanismby which this heavy metal interferes with the activity of the cytochrome P450 system is still not known (Pani et al., 1975). In our studies,we have attempted to modify the functional state of the drug oxidizing system by three chemicals: CoCl,, SKF-525A, and phenazine methosulfate. When mice were pretreated with CoCI, (60 mg/kg, SC, daily for 2 days) and subsequently challenged with a dose of Ccl, (0.5 ml/kg, ip), poisoning with the haloalkane was not prevented. The time course of Ccl,-induced hepatic damage, as indicated by a rise in
CARBON
TETRACHLORIDE
HEPATOTOXICITY
375
the activity of SGPT and an increase in hepatic triglyceride, was unaltered, and these effects were evident as early as 3 hr or sooner (Table 2). With this dosage schedule of CoCI, pretreatment, microsomal cytochrome P-450 was reduced to about half (Table 3), in parallel with a reduction in the activity of a drug oxidizing enzyme such as aminopyrine N-demethylase or ethylmorphine N-demethylase, which requires cytochrome P-450 for its activity (Table 1). These findings are similar to those previously reported in rats by Tephly and Hibbeln (1971). However, although it seems possible on the basis of these findings that this cytochrome may not be involved in the activation of the haloalkane, it is still premature to draw such a conclusion. This is because it is possible that the species of cytochrome P-450 that is involved in Ccl, metabolism may not be affected by CoCl, treatment. Actually, this possibility is considered highly probable in view of the fact that the activity of aniline hydroxylase, another P-450-requiring enzyme, was decreased by only 20% with this regimen of CoCl, treatment (Table 1). Further, the results obtained from experiments with SKF525A seem to support this view. SKF-525A, which has been found to inhibit the hydroxylation of several drugs, was ineffective against Ccl, hepatotoxicity (Table 4). Moreover, SKF-525A was found to enhance the reduction of cytochrome P-450 (Table 3). A more likely explanation for these SKF-525A findings is that it does not under these conditions inhibit the species of cytochrome P-450 that is involved in the activation of Ccl,. Our conclusion that cytochrome P-450 is a site for Ccl, activation is similar to that reported independently by Glende (1972) and by Castro et al. (1973); the former has shown that development of resistance to Ccl, is directly related to depressionof the drug oxidizing system and a significant reduction of hepatic cytochrome P-450 content, whereas the latter found that protection of Ccl, hepatotoxicity by cysteamine was due to the ability of this compound to inhibit the reduction of cytochrome P-450 by P-450 reductase. After analyzing the results with phenazine methosulfate, we were led to consider at least two possibilities: (1) PMS acts as an electron scavenger, interacting with the free radical forms generated during the reduction of cytochrome P-45O/CCl, complex by P450 reductase; and (2) NADPH-cytochrome c reductase may be another site for CCI, activation. Inhibition of this enzyme by PMS would then inhibit Ccl, activation. This second possibility, which has been favored by several investigators (Nayak et al., 1970; Chopra et al.. 1972), does not seemlikely since the concentrations of PMS required to inhibit the enzyme were relatively high (Table 6). Recently, Glende (1972) has presentedevidence to exclude the participation of NADPH-cytochrome c reductase as a site for Ccl, activation. Although we have not been able to directly show the involvement of a cytochrome P450 speciesin Ccl, metabolism, all of our experimental findings are in accord with the view that this hemoprotein is the central figure for the haloalkane activation. If it is assumedthat this cytochrome is required for Ccl, activation, then phenazine inhibition of NADPH-cytochrome c reductase would also inhibit cytochrome P-450-mediated activation of Ccl, since this flavoprotein is necessary for P-450 reduction and subsequentchemical transformations mediated by this hemoprotein. Thus, our results together with a bulk of evidence presented by Glende (1972) strongly suggesta critical role of cytochrome P-450 in the activation of Ccl,.
376
SURIYACHAN
AND
THlTHAPANDHA
REFERENCES BARON, J., AND TEPHLY, T. R. (1969). Effect of 3-amino-1,2,4triazole on the stimulation of hepatic microsomal heme synthesis and induction of hepatic microsomal oxidases produced by phenobarbital. Mol. Pharmacol. 5, 10-20. BUTLER,W. M., MALING, H. M. HORNING,M. G., AND BRODIE,B. B. (1961). The direct determinationof liver triglycerides.J. Lipid Res. 2, 95-96. CASTRO,J. A., DE FERREYRA, E. C., DE CASTRO,C. R., GOMEZ, M. I. D., D’ACOSTA, N., AND DE FENOS,0. M. (1973). Studieson the mechanism of cystaminepreventionof severalliver structural and biochemical alterations caused by carbon tetrachloride. Toxicol. Appl. Pharmacol. 24, l-19. CHOPRA,P., ROY,S., RAMALINGASWAMI, V., ANDNAYAK, N. C. (1972).Mechanismof carbon tetrachloridehepatotoxicity. Lab. Invest. 26, 7 16727. CIGNOLI,E. V., ANDCASTRO,J. A. (1971).Effect of inhibitorsof drug metabolizingenzymeson carbontetrachloridehepatotoxicity. Toxicol. Appl. Pharmacol. l&625-637, DAWKINS,M. J. R. (1963).Carbontetrachloridepoisoningin the liver of newbornrat. J. Pathol. Bacterial. 85, 189-196. GARNER,R. C., ANDMCLEAN,A. E. M. (1969).Increasedsusceptibilityto carbontetrachloride poisoningin the rat after pretreatmentwith oral phenobarbitone.Biochem. Pharmacol. 18, 645-650.
GIGON,P. L., GRAM, T. E., ANDGILLETTE,J. R. (1969). Studieson the rate of reductionof hepatic microsomal cytochrome P-450 by reduced nicotinamide adenine dinucleotide phosphate:Effect of drug substrates. Mol. Pharmacol. 5, 109-122. GLENDE,E. A. (1972). Carbon tetrachloride-inducedprotection againstcarbon tetrachloride toxicity. Biochem. Pharmacol. 21, 1697-1702. GLENDE,E. A., ANDRECKNAGEL, R. 0. (1969). Biochemicalbasisfor the in vitro prooxidation actionof carbontetrachloride.EXR. Mol. Pathol. 11, 172- 185. GORNALL,A. G., BARAWILL,C. J., ANDDAVID, M. M. (1949).Determinationof serumprotein bv meansof biuret reaction.J. Biol. Chem. 177. 75 l-766. MABEL,P. (1971). In Fundamentals of Drug Metabolism and Drug Disposition (B. N. Ladu, H. G. Mandel, andE. L. Way, eds.),pp. 546-582. Williams& Wilkins, Baltimore,Maryland. NADEAU,D., ANDMARCHAND,C. (1973).Importanceof the route of administrationof Ccl, in the protective effect of promethazine.Biochem. Pharmacol. 22, 1250-1252. NAYAK, N. C., CHOPRA, P., AND RAMALINGASWAMI,V. (1970). The role of liver ccl endoplasmicreticulum and microsomalenzymesin carbon tetrachloridetoxicity: An in vim study. Life Sci. 9, 1431-1439. PANI, P., CORONGIU, F. P., SANNA,A., AND CONGIU,L. (1975). Protection by lead nitrate againstcarbontetrachloridehepatotoxicity. Drug Metab. Disp. 3, 148-154. RECKNAGEL, R. 0. (1967).Carbontetrachloridehepatotoxicity. Pharmacol. Rev. 19, 145-208. REITMAN,S., ANDFRANKEL,S. (1957). A calorimetricmethodfor the determinationof serun glutamicoxalaceticandglutamicpyruvic transaminases. Amer. J. C/in. Pathol. 28,56-63. SASAME,H. A., CASTRO,J. A., ANDGILLETTE,J. R. (1968). Studieson the destructionof live microsomalcytochromeP-450 by carbontetrachlorideadministration.Biochem. Pharmacoi 17, 1759-1768. SLATER,T. F. (1966). Necrogenicaction of carbon tetrachloride in the rat; A speculativt mechanismbasedon activation.Nature (London) 209,36-40. SWANSON, M. A. (1955). Glucose-6-phosphatase from liver. In Methods in Enzymology (S. P COLOWICK and N. 0. KAPLAN,eds.).Vol. 2, pp. 541-543. AcademicPress,New York. TEPHLY,T. R., ANDHIBEELN,P. (1971). The effect of cobalt chlorideadministrationon th synthesisof hepatic microsomalcytochromeP-450. Biochem. Biophys. Res. Commun. 4; 589-595.
VANHANDEL.E., ANDZILVERSMIT,D. B. (1957). Micromethodfor the direct determinationc serumtriglycerides.J. Lab. Clin. Med. 50, 152-157.
AND
APPLIED
Modification
PHARMACOLOGY
of Carbon
41,369-316
(1977)
Tetrachloride Chemicals
D. SURIYACHANAND
Hepatotoxicity
by
A. THITHAPANDHA
Department of Pharmacology, Faculty of Science, M&idol
University, Bangkok 4, Thailand
Received December 9,1976; accepted February 7, I977 Modification of Carbon Tetrachloride Hepatotoxicity by Chemicals. SURIYACHAN, D., AND A. (1977). Toxicol. Appl. Pharmacol. 41, 369-376. Cobaltous chloride (60 mg/kg, SC, daily for 2 days), which was found to effectively decrease the microsomal cytochrome P-450 content of mouse liver to approximately half of its normal value and which impaired the oxidative metabolism or hydroxylation of aminopyrine, ethylmorphine, and hexobarbital, offered no protection against CC&induced liver damage. However, this hemoprotein inhibitor had no effect on the rate of reduction of cytochrome P-450 by NADPH and exerted a slight effect on aniline hydroxylation. SKF-525A (50 mg/kg, ip) also failed to protect against Ccl, hepatotoxicity though it has been shown to inhibit the hydroxylation of a number of substrates. This inhibitor, a type I compound, was found to enhance cytochrome P450 reduction by NADPH. Further studies revealed that CCI,-induced hepatic injury could be prevented by phenazine methosulfate (2 mg/kg, ip, 5 doses at 0.5.hr intervals), which in vitro was found to inhibit NADPH-cytochrome c reductase noncompetitively. All of these findings are not satisfactorily explainable by electron transfer from NADPH-cytochrome c reductase to CCI, as the activation reaction for Ccl, but are compatible with the hypothesis previously proposed by others that cytochrome P-450 is the critical site for Ccl, activation. TH~THAPANDHA,
Although it is well documented that Ccl,-induced hepatotoxicity is probably causedby its free radicals arising during a Ccl,-activation step mediated by the microsomal drug oxidizing system (Slater, 1966; Recknagel, 1967; Glende and Recknagel, 1969), the actual site for this activation is still a subject of debate by several groups of investigators. Nayak et al. (1970) and Chopra et al. (1972) suggestedthat Ccl, might be metabolized at the early stage of the hepatic microsomal electron transport chain where NADPH is oxidized to NADP by NADPH-cytochrome c reductase. However, serious doubts as to the validity of the hypothesis that NADPH-cytochrome c reductase is the locus of carbon-chloride bond cleavage have been raised by Glende (1972). Cignoli and Castro (1971) and Castro et al. (1973) pointed out that cytochrome P-450 was likely to be the site for Ccl, activation. The reason for the discrepancy among the results of theseworkers remains unknown. The present communication reports on the results of both in vitro and in vivo studies with Ccl, and the modification of its hepatotoxicity by chemicals capable of modifying the activities of certain components along the electron transport chain of mouse liver’s drug oxidizing system. These chemicals include 2-diethylaminoethyl-2,2diphenylvalerate (SKF-525A), cobaltous chloride (CoCl, .6H,O) and phenazine methosulfate (P&IS). The role of NADPH-cytochromec reductase and cytochrome P450 on Ccl, activation has been evaluated. Copyright 0 1977 by Academic Press. Inc. All rights of reproduction in any form reserved. Printed in Great Britain
369
ISSN 004 1408X
370
SURIYACHAN
AND
THITHAPANDHA
METHODS Treatment of Animals Male mice of New Zealand strain were used throughout the study. The animals (2530 g) were fed with commercial mouse pellets, premium quality feed, Zuellig (Gold Coin Mills) Pte. Ltd., Singapore, and tap water ad libitum. Ccl, was prepared as 10% solution (v/v) in liquid paraffin and administered ip at a dose of 0.5 ml/kg. Other chemicals were prepared in physiologic saline just before use. SKF-525A (50 mg/kg, ip) was given 30 min before Ccl, intoxication. Animals were pretreated with cobaltous chloride (60 mg/kg, SC) daily for 2 days. Ccl, was then injected 24 hr after the last dose of the hepatic hemoprotein inhibitor; animals were sacrificed 3 hr after Ccl, administration. Phenazine methosulfate (2 mg/kg, ip) was given to the animals for 5 doses at 0.5-hr intervals. Ccl, was administered 10 min after the first dose of phenazine methosulfate; mice were sacrificed 3 hr after Ccl,. Preparation
of Liver Microsomes
Male mice, 25-30 g, were killed by decapitation. Livers were removed and homogenized with a motor-driven glass-Teflon homogenizer in two volumes of 1.15% KC1 containing 20 mM Tris-KC1 buffer, pH 7.4. The homogenate was centrifuged for 20 min at 9,000 g in a Sorvall refrigerated centrifuge, and the supernatant was decanted and recentrifuged for 1 hr at 105,OOOg in a Spinco model L2-65B preparative ultracentrifuge. The microsomal pellet was resuspended and washed in the Tris-KC1 buffer and recentrifuged at 105,000 g for 50 min. The washed microsomal pellet was resuspended in 0.1 M phosphate buffer, pH 7.4, and used as the sources for subsequent determinations. Enzyme Assays Serum glutamic-pyruvic transaminase (SGPT) was determined by the ReitmanFrankel method (1957), according to the description given in Sigma Technical Bulletin No. 555. Activity is expressed in Sigma-Frankel units; one such unit corresponds with the formation of 4.82 k 10e4 ~01 of glutamate per min at pH 7.5 and 25°C (Nadeau and Marchand, 1973). Aminopyrine N-demethylase and aniline hydroxylase were measured from the 9000-g supernatant of mouse liver as described by Maze1 (1971). Ethylmorphine Ndemethylase, NADPH-cytochrome c reductase, and cytochrome P-450 in a microsomal preparation were measured as described by Baron and Tephly (1969). Glucose-6-phosphate from the 9000-g supernatant was determined by the method of Swanson (1955). NADPH-P-450 reductase activities were quantitated as described in detail by Gigon et al. (1969). Protein Determination Protein content both in the supernatant and microsomal by the biuret method (Gornall et al., 1949).
preparations
was measured
Hepatic Triglyceride The concentration of triglyceride in liver was measured according to the method of van Handel and Zilversmit (1957) as modified by Butler et al. (1961). The content was expressed as milligrams per gram of liver wet weight.
CARBON
TETRACHLORIPE
371
HEPATOTOXIClTY
Sleeping Time Hexobarbital (100 mg/kg, ip) was administered to control mice and to mice previously treated with cobaltous chloride (60 mg/kg, SC.48 and 24 hr earlier). The sleepingtime in each group was then recorded in the usual manner. All data in this report were analyzed statistically by Student’s t test. RESULTS
When cobaltous chloride (60 mg/kg, SC,daily for 2 days) was administered to mice, it exerted a marked influence on the activities of several well-known cytochrome P-450. requiring hepatic enzymes. Both the level of ethylmorphine N-demethylation, measured from a microsomal preparation, and that of aminopyrine N-demethylation, measured from the 9000-g supernatant, were reduced to 5%60% of the control (Table 1). TABLE EFFECTS OF ETHYLMORPHINE
I
COBALTOUS CHLORIDE PRETREATMENT ON HEXOBARBITAL N-DEMETHYLASE, AMINOPYRINE N-DEMETHYLASE, HYDROXYLASE ACTIVITIES~
Ethylmorphine Sleeping time Treatment
(min)
Control CoCl,d
39 & 4 66 + 10
SLEEPING AND
Aminopyrine
N-demethylationb (nmol of HCHO formed/mg of
Aniline
N-dem,.thylation* (nmo: of HCHO f,lrmed/mg of rrotein/min)
protein/min) 3.40 + 0.30 1.72 + 0.24
hydroxylaseh.’ -nmol of PAP/mg of protein/ 20 min) 6.15 + 0.26 4.95 & 0.26
3 1.76 + 2.48 20.63 + 2.05
“Ten mice in each group were pretreated with cobaltous chloride and sacrificed Control animals received equal volume of physiologic saline. Hexobarbital administered 24 hr after the last dose of CoCI,, and the sleeping time was recorded. b Each value is the mean f SE of three determinations from 10 animals. c PAP stands for p-aminophenol.
dose.
d All
values
in CoCl,-treated
group
are significantly
different
from
controls,p
TIME, ANILINE
24
hr after
the
last
(100 mg/kg. ip) was
< 0.05.
Physically, the animals exhibited signs of central nervous system depression after the first dose of this chemical. They appeared sedatedand seemedto lose appetite. None of the animals died, however. The in vivo oxidative metabolism of hexobarbital, which also requires cytochrome P-450, was significantly impaired, as evidenced by a marked prolongation of the sleeping time; 39 f 4 in 10 control mice against 66 i 10 in IO CoCl,-treated animals (Table 1). However, when animals pretreated with this dosage regimen of cobaltous chloride were challenged acutely with Ccl, (0.5 ml/kg, ip), the haloalkane was still effective in causing hepatic damage, as indicated by a rise in the activity of SGPT and an increase in hepatic triglycerides (Table 2). In essence, cobaltous chloride afforded no protection against Ccl,-induced hepatic injury. This is rather unexpected in view of the fact that hepatic drug metabolism is involved in several types of hepatic drug intoxications including Ccl, (Recknagel, 1967). Since cobaltous chloride was able to decreasethe activities of the hydroxylating enzymes (Table l), it would be expected to prevent or reduce the hepatotoxicity caused by the haloalkane. But this was not found to be the case. Thus, the role of cytochrome P-450 in Ccl, activation might be in doubt.
372
SURIYACHAN
AND
THITHAPANDHA
However, when one realizes that drug hydroxylation
and the reduction of cytochrome
P-450 may be two separate entities one could not easily rule out the involvement of the hemoprotein in Ccl, metabolism. In contrast to aminopyrine N-demethylase and ethylmorphine N-demethylase, both of which were greatly reduced by CoCl,, the TABLE EFFECTS PYRUVIC
2
OF COBALTOUS CHLORIDE PRETREATMENT ON SERUM TRANSAMINASE ACTIVITY AND HEPATIC TRIGLYCERIDE AFTER CARBON TETRACHLORIDE INJECTION“
Treatment Control COCI, ccl, CoCl, + ccl,
Triglyceride (mg/g of liver)
SGPT Unitb 37.25 40.50 96.67 94.28
GLUTAMICCONTENT
+ 9.83 (1) k 7.23 (10) +_ 15.98 (10) t 10.28 ( 10)d
10.70 12.43 23.60 25.23
k rt k &
1.48 1.27 3.65 2.95
(10) (10) (10) (lO)d
(I All values given are mean & SE, with the number of animals shown in parentheses. Animals were killed 3 hr after CCI, administration. b One unit corresponds to the formation of 4.82 f 10m4prnol of glutamate/min at pH 7.5 and at 25°C. LISignificant difference from control and CoCl,,p < 0.05. d No difference from CCI,
activity of aniline hydroxylase was slightly reduced by this hemoprotein inhibitor (Table 1). Since aniline hydroxylation also requires cytochrome P-450 for its activity, it is quite likely that the species of this cytochrome involved in Ccl, activation is not very much affected by pretreatment with cobaltous chloride. As shown in Table 3, though microsomal cytochrome P-450 content was reduced to approximately 50% of normal TABLE EFFECTS
OF COBALTOUS
Compound Control CoCI,, 0.25 mM SKF-525A, 0.05 mM
3
CHLORIDE AND SKF-525A ON CYTOCHROME HEPATIC MICROSOMES FROM MALE MICE~
P-450 REDUCTION
Cytochrome P-450 (nmol/mg of protein)
Initial rate of reduction of P-450 by NADPH (nmol reduced/min/mg of protein)
1.40 & 0.16 0.52 + 0.08b 0.96 & 0.32
8.21 k 0.74 9.00 + 1.10 18.87 k 0.52b
IN
(1Values expressed as mean & SE of four determinations, each of which was made with a microsomal preparation obtained from the pooled livers of five animals. b Significantly different from control,p < 0.05.
by cobaltous chloride, the rate of reduction of this hemoprotein remained unchanged. Therefore, cobaltous chloride, which could increase or decrease drug hydroxylation, might not appreciably affect the cytochrome P-450 species that catalyzes the activation of Ccl,. In support of this conclusion, SKF-525A, which is known to inhibit the hydroxylation of a number of substrates and which was found in our studies and
CARBON
TETRACHLORIDE
373
HEPATOTOXICITY
previously shown by Gigon et al. (1969) to enhance P-450 reduction (Table 3), offered no protection against the toxic actions of Ccl, (Table 4). This is presumably due to the inability of SKF-525A to bind the cytochrome P-450 species which metabolizes Ccl,. The effect of phenazine methosulfate (PMS) on CC&-induced liver damage is shown in Table 5. Glucose-6-phosphatase, a membrane-bound enzyme known to be more TABLE 4 EFFECTS OF SKF-525A PRETREATMENT ON THE ACTIVITIES OF AMINOPYRINE N-DEMETHYLASE, SERUM GLLJTAMIC-PYRLJWC TRANSAMINASE AND HEPATIC TRIGLYCERIDE CONTENT AFTER AN INTRAPERITONEALADMINISTRATIONOFCARBONTETRACHLORIDE~
Treatment
Aminopyrine N-demethylation (nmolof HCHO formed/mgof protein/30min)
SGPT Unit
30.00 + 4.28 34.50 k 8.35
ccl,
33.22 + 2.15 31.35 + 1.90 22.90 k 1.76b
SKF-525A + CC&
23.10f 0.68’
82.80 f 13.53’
Control SKF-525A
89.40 k 10.61b
Hepatictriglyceride (mg/gof liver) 12.14 + 1.03
14.21 + 0.65 27.38 k 3.35b 26.76 k 2.25'
a Values are expressed as mean + SE, with 10 mice in each group. Ccl, was given 30 mitt after Animals were killed 3 hr after Ccl, b Signiticantly different from control and SKF-525A (p < 0.05). cNot significantly different from Ccl,.
SKF-525A.
TABLE 5 EFFECTS OF CARBON TETRACHLORIDE ONTHE ACTIVITIES OF GLUCOSE-6-PHOSPHATASEAND AMINOPYRINE N-DEMETHYLASE IN MICE PRETREATED WITH PHENAZINE METHOSULFATE"
Treatment
Control PMS ccl,
PMS + Ccl,
Glucose-6-phosphatase (nmol of Pi formed/mg of protein/min) 127.22k 5.83 126.49+ 4.53 64.26 k 5.50b 108.51+ 7.45c
Aminopyrine N-demethylation (nmolof HCHO fortned/mg of protein/30min)
35.22 k 3.47 33.08 k 3.20 21.90 + 2.10b 31.20 + 1.40’
0 Each value is the mean & SE of 10 determinations from four to six mice. The animals were killed 3 hr after Ccl, administration. * Significantly different from controkg < 0.05. c Significantly different from Ccl, (p < 0.05) but not from control.
sensitive to the action of Ccl,, was unaffected by PMS treatment but its activity was greatly decreased by Ccl, administration (Table 5). However, when mice were pretreated with PMS at the dosesindicated, the haloalkane produced no change on the activity of either glucose-6-phosphataseor aminopyrine N-demethylase (Table 5). Although data are not shown, the activity of SGPT and hepatic triglyceride content were found to be the same as in controls. Thus, PMS seemsto be an effective inhibitor
374
SURIYACHAN
AND
THITHAPANDHA
of CC&-induced hepatic injury. Further, it was found that PMS inhibited NADPHcytochrome c reductase and that the inhibition was noncompetitive (Table 6). TABLE INHIBITION
OF NADPH-CYTOCHROME METHOSULFATE
Phenazine methosulfate (M)
0 1 x IO--2 1 x 10-I 1 x 10-d
6 c REDUCTASE IN MICE
Enzymatic activity” (nmol/mg of protein/min)
130.2t 14.1 60.5 2 10.4 84.2 _+ 12.2 124.0 t 15.0
BY PHENAXNE
Inhibition
(96)
0 53 35 4
“The activity of NADPH-cytochrome c reductase in microsomal preparations was measured on a Gilford Model 2000 recording spectrophotometer as the rate of increase in absorbance 550 nm produced by the reduction of cytochrome c. Each cuvette of l-cm light path contained, in a total volume of 3 ml, I.0 ~01 of sodium cyanide, 0.1 pmol of cytochrome c, 1 mg of microsomal protein, PMS at the concentrations indicated, and 0.2 M potassium phosphate buffer, pH 7.4. At zero time, 0.4 pmol of NADPH was added to start the reaction. Enzymatic activity, which was determined by using 19.7 rnr.-’ cm-’ as the millimolar difference extinction coefficient at 550 nm, was expressed as nanomoles of cytochrome c reduced per milligram of protein per minute. Each value is given as mean & SE of three experiments.
DISCUSSION
In recent years, evidence hasbeen accumulated in a number of laboratories indicating that the severity of Ccl,-induced liver damage is directly proportional to the activity of the microsomal drug oxidizing or cytochrome P-450 system @later, 1966; Recknagel, 1967; Sasameet al., 1968). In newborn and hepatectomized rats, Ccl, has been shown to be less toxic than in normal adult animals (Dawkins, 1963; Pani er al., 1975), presumably because the drug oxidizing system in these animals is functionally depressed.On the other hand, phenobarbital, a microsomal enzyme inducer, increases the toxicity of Ccl, (Garner and McLean, 1969). Thus, the toxic effects of Ccl, may be prevented by lowering its metabolism; this can be effected by modifying the functional state of the drug oxidizing system. For example, cystamine and cysteamine have been reported to prevent several structural and biochemical alterations caused by the haloalkane by inhibiting Ccl, activation taking place at the hemoprotein, cytochrome P-450 (Castro et al., 1973). Recently, it has been found that Ccl,-induced liver necrosis was also prevented by lead; however, the exact mechanismby which this heavy metal interferes with the activity of the cytochrome P450 system is still not known (Pani et al., 1975). In our studies,we have attempted to modify the functional state of the drug oxidizing system by three chemicals: CoCl,, SKF-525A, and phenazine methosulfate. When mice were pretreated with CoCI, (60 mg/kg, SC, daily for 2 days) and subsequently challenged with a dose of Ccl, (0.5 ml/kg, ip), poisoning with the haloalkane was not prevented. The time course of Ccl,-induced hepatic damage, as indicated by a rise in
CARBON
TETRACHLORIDE
HEPATOTOXICITY
375
the activity of SGPT and an increase in hepatic triglyceride, was unaltered, and these effects were evident as early as 3 hr or sooner (Table 2). With this dosage schedule of CoCI, pretreatment, microsomal cytochrome P-450 was reduced to about half (Table 3), in parallel with a reduction in the activity of a drug oxidizing enzyme such as aminopyrine N-demethylase or ethylmorphine N-demethylase, which requires cytochrome P-450 for its activity (Table 1). These findings are similar to those previously reported in rats by Tephly and Hibbeln (1971). However, although it seems possible on the basis of these findings that this cytochrome may not be involved in the activation of the haloalkane, it is still premature to draw such a conclusion. This is because it is possible that the species of cytochrome P-450 that is involved in Ccl, metabolism may not be affected by CoCl, treatment. Actually, this possibility is considered highly probable in view of the fact that the activity of aniline hydroxylase, another P-450-requiring enzyme, was decreased by only 20% with this regimen of CoCl, treatment (Table 1). Further, the results obtained from experiments with SKF525A seem to support this view. SKF-525A, which has been found to inhibit the hydroxylation of several drugs, was ineffective against Ccl, hepatotoxicity (Table 4). Moreover, SKF-525A was found to enhance the reduction of cytochrome P-450 (Table 3). A more likely explanation for these SKF-525A findings is that it does not under these conditions inhibit the species of cytochrome P-450 that is involved in the activation of Ccl,. Our conclusion that cytochrome P-450 is a site for Ccl, activation is similar to that reported independently by Glende (1972) and by Castro et al. (1973); the former has shown that development of resistance to Ccl, is directly related to depressionof the drug oxidizing system and a significant reduction of hepatic cytochrome P-450 content, whereas the latter found that protection of Ccl, hepatotoxicity by cysteamine was due to the ability of this compound to inhibit the reduction of cytochrome P-450 by P-450 reductase. After analyzing the results with phenazine methosulfate, we were led to consider at least two possibilities: (1) PMS acts as an electron scavenger, interacting with the free radical forms generated during the reduction of cytochrome P-45O/CCl, complex by P450 reductase; and (2) NADPH-cytochrome c reductase may be another site for CCI, activation. Inhibition of this enzyme by PMS would then inhibit Ccl, activation. This second possibility, which has been favored by several investigators (Nayak et al., 1970; Chopra et al.. 1972), does not seemlikely since the concentrations of PMS required to inhibit the enzyme were relatively high (Table 6). Recently, Glende (1972) has presentedevidence to exclude the participation of NADPH-cytochrome c reductase as a site for Ccl, activation. Although we have not been able to directly show the involvement of a cytochrome P450 speciesin Ccl, metabolism, all of our experimental findings are in accord with the view that this hemoprotein is the central figure for the haloalkane activation. If it is assumedthat this cytochrome is required for Ccl, activation, then phenazine inhibition of NADPH-cytochrome c reductase would also inhibit cytochrome P-450-mediated activation of Ccl, since this flavoprotein is necessary for P-450 reduction and subsequentchemical transformations mediated by this hemoprotein. Thus, our results together with a bulk of evidence presented by Glende (1972) strongly suggesta critical role of cytochrome P-450 in the activation of Ccl,.
376
SURIYACHAN
AND
THlTHAPANDHA
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