Species Differences in Carbon Tetrachloride-Induced Hepatotoxicity: The Role of Ccl, Activation and of Lipid Peroxidationl M. I. D~AZ G~MEZ, C. R. DE CASTRO, N. D’ACOSTA, 0. M. E. C. DE FERREYRA, AND J. A. CASTRO Laboratorio


de Quimica Bio-Tonicoldgica, CITEFA, Zujiiiategui y Varela, Villa Marteili, Pcia de Buenos Aires, Argentina Received December 18,1974; accepted May 14,1975

SpeciesDifferences in Carbon Tetrachloride-Induced Hepatotoxicity: The role of CC& Activation and of Lipid Peroxidation. D~AZ G~MEZ, M. I., CASTRO; C. AND CASTRO,

R. DE, D’ACOSTA, N., FENOS, 0. M. DE, FERREYRA, E. C. DE., J. A. (1975).Toxicol. Appl. Pharmacol. 34, 102-114.Carbon

tetrachloride-inducedliver necrosiswasmore intensein the mousethan in the guineapig or the hamster,which are more susceptiblethan the rat. The chickenwasresistantto Ccl+ The extent of the irreversible binding of 14C from 14CC14to cellular componentsdecreasesin the following order: mouse= hamster> guinea pig > rat > chicken, while the intensity of the Ccl,-induced lipid peroxidation decreasedin the order rat > hamster= guinea pig > chicken = mouse.The resultsroughly suggesta better parallelism betweenintensity of the processof irreversible binding to cellular componentsand necrosisthan between lipid peroxidation and necrosis. In the caseof the mousethe necroticprocesswasobservedin absenceof lipid peroxidation during the entire 24-hr period of intoxication. It was not possibleto absolutely correlate the extent of the irreversible binding to microsomallipidsin the different species with either thecharacteristicsof the spectralchangesproduced by Ccl, by interaction with cytochrome P-450 (P-450)or with the content of P-450or that of the P-450reductaseactivity. Several workers have studied the effects of Ccl, on the liver of different animal species; a summary of the studiesperformed have been reported by Cameron and Karunaratne (1936) and by Von Oettingen (1955). Most animal speciesare very susceptibleto liver injury by Ccl, with the notable exception of the chicken and birds which may survive enormous dosesof the hepatotoxin (Hall and Schillinger, 1923).This peculiar unresponsive behavior of the chicken liver to the deleterious effect of Ccl, was considered by Slater (1966) as one of the basic questions concerning the necrogenic activity of Ccl,. In the course of the past 10yr, evidence for the hypothesis that Ccl, hepatotoxicity is related to its metabolism has accumulated; at present many authors believe that as a result of this metabolic activation *Ccl3 and *Cl are formed which becauseof their free radical nature are able to spark a lipid peroxidative process(Slater, 1966; Recknagell967; Recknagel and Glende, 1973)that is fatal to the cell. In agreementwith this 1Thiswork wassupportedby GrantsAM-13195-06from the NationalInstitutesof Health(USA) andfrom ConsejoNationaldeInvestigaciones Cientfficasy T&nicas(Argentine). Copyright 0 1975 by Academic Press. Inc. All d&s of reproduction in any form reserved. Printed in Great Britain







view, Slater (1968) showed that in vitro microsomal lipid peroxidation from chicken liver preparations is considerably less intense than that observed using rat liver. Not all authors share the view that lipid peroxidation is the key event leading to liver necrosis (Castro et al., 1972b, 1973; Klaassen and Plaa, 1969; McLean, 1967; Chopra et al., 1972). Moreover, some have even questioned its occurrence during CC& poisoning (Keller et al., 1971; Green et al., 1969; Green, 1972). This group of workers have in common an alternative hypothesis that the irreversible binding of the *Ccl, and *Cl free radicals to cellular components may be the critical event in cell death. If this alternative hypothesis is feasible it should also explain the insensitiveness of the chicken liver to Ccl,-induced damage. We have attempted to establish if there is some correlation between the extent of CCI, activation to -Ccl, in livers of different animal species and the liver microsomal content of both cytochrome P-450 (P-450) and cytochrome P-450 reductase (P-450 reductase) activity. In the course of previous studies from our laboratory we have postulated that Ccl, activation to *Ccl, is a reductive process occurring when the CC&/P-450 complex is reduced by NADPH in the catalytic presence of P-450 reductase (Castro and Diaz Gomez, 1972; Castro et al., 1972; D’Acosta et al.. 1972, 1973; Castro et al., 1973; Diaz Gomez et al., 1973). METHODS

Animal treatments: 14CC14 (27.5 mCi/mmol) was obtained from the Radiochemical Centre Amersham, England. All other chemicals used were of analytical grade. All experiments were performed with fasted (12-14 hr) animals of the following species: male rat (Sprague-Dawley) 220-250 g; male mouse (A/J strain) 24-28 g wt; male guinea pig (General Purpose; Romanelli Laboratories, Buenos Aires, Argentina); male Syrian Golden Hamster (Argentinian Atomic Energy Commission) 80-I 10 g; chicken (Leghorn, Romanelli Laboratories, Buenos Aires, Argentina) 1.5-l .6 kg. Ccl4 was given ip either as a 20 % or as a 5 % (v/v) (as stated) solution in olive oil at a dose of 5 ml of solution/kg. 14CC14of sp act 27.5 mCi/mmol was dissolved in olive oil to give a solution containing 23 nmol/ml (1.4 x IO6 dpm/ml) and given ip at a dose of 5 ml of the olive oil solution per kilogram. Control animals received the equivalent amount of olive oil ip. The animals were sacrificed by decapitation at different times after CCI, or 14CC14administration. Their livers were rapidly removed, weighed, and processed. Enzymatic and chemical determinations: The isolation of the microsomal fractions for the different studies has been previously described (Castro et al., 1972b). The incorporation in vivo of 14C from 14CC14into microsomal lipids was determined according to the procedure described by Castro et al. (1972b). The results were expressed in disintegrations per minute per milligram of microsomal lipid. The incorporation in vitro of 14CC14 into microsomal lipids was carried out in incubation mixtures (3 ml) of 0.05~ Tris/HCl, pH 7.4/0.15 M KC1 buffer containing magnesium chloride 5mM; sodium isocitrate 8 mM; NADP 0.33 m&r; isocitric acid dehydrogenase 0.36 IU/ml and microsomal protein 8 mg/ml. 14CC14 was dissolved in ethanol 95% to give a solution producing 2 x lo7 dpm/ml; 5 ~1 of 14CC14solution were added per milliliter of incubation mixture. Ice cold-NADPH generating system and microsomal suspensions were bubbled with deoxygenated Nz before placing into gas tight vials closed with a rubber septum. Vials were further gassed with the deoxygenated N2 for 5 min before “CCI,





addition. After acclimation to temperature for 5 min before 14CC1, addition at 37°C in a Dubnoff shaker the 14CC1, solution was added and incubation was allowed to proceed for 15 min. At the end of the incubation, the samples were processed for irreversible binding to lipids as previously described (Castro et al., 1972b). The results were expressed in disintegrations per min per milligram of lipid per milligram of microsomal protein per milliliter. The quantitative estimation of the lipid peroxidation in vivo was determined by conjugated diene ultraviolet absorption of lipid extracts of the microsomal fractions as described by Klaassen and Plaa (1969). The results were expressed as the change in absorbance at 243 nm x 1000 for a solution having 1 mg of microsomal lipid/ml. The microsomal P-450 content was determined as described by Schenkman et al. (1967). P-450 reductase activity was measured as described by Gigon et al. (1969). The procedure followed to determine Ccl, and 14CC14content in liver was that of Recknagel and Litteria (1960). Results were corrected for loss as indicated in the method. Histological techniques.After removing the liver, small portions from the left and central lobes were immediately fixed in Bouin’s solution, embedded in paraffin, and stained with hematoxylin-eosin. Statistics. The significance of the difference between two mean values was assessed by Student’s t test (Bancroft, 1960). RESULTS

Cytochrome P-450 Content and NADPH Cytochrome P-450 ReductaseActivity in Different Animal Species

The data revealed the following decreasing order of P-450 content in the species studied: guinea pig > hamster > rat > mouse 9 chicken. A similar analysis of the results of liver microsomal P-450 reductase activity led to the following sequence: hamster > guinea pig > mouse > rat $ chicken (Table 1). Spectral Binding Constant of Ccl, in Diflerent Animal Species

The spectral dissociation constant Ksp of CCI, in different animal species showed a high degree of variation. In effect, the highest value, which was observed in the chicken TABLE CYT~CHROME P-450 CONTENT REDUCTASE ACTIVITY



P-450 content (nmol/mg protein rk SD)


0.70 * 0.09


0.61 f 0.10


0.76 + 0.12 1 .OO f 0.08 0.13 + 0.04

Guinea pig Chicken

P-450 reductase (nmol/min/mg protein I!ZSD) 0.80 1.05 3.15 2.09 0.21

+ 0.18 + 0.14 + 0.56 _+ 0.26 & 0.12

’ Fasted (12-14 hr) animals of the different species were employed. Results from mice, hamsters and chicken were obtained with five animals per group and those from rats and guinea pigs with eight animals per group.






is about 8.6 times greater than the smallest one which was that obtained for the guinea pig. The decreasing order of variation was : chicken > rat > mouse > hamster > guinea pig. The maximal spectral change in contrast varied within more narrow limits, the highest value being that observed for the rat and the smallest one observed in the hamster. The decreasing sequence for the maximal spectral changes values was : rat r mouse z chicken > guinea pig > hamster (Table 2). TABLE SPECTRAL





Rat Mouse Hamster Guinea pig Chicken


Maximal spectral change (Abs/mg/ml x 10-s)

KSV X 10-4)

20 10 3.3 2.9 25

+ f 2 + *


1.8 0.8 0.3 0.3 2.0

2.86 2.72 0.81 1.38 2.60

+ + f + +

0.18 0.16 0.04 0.06 0.25

a Liver microsomes were suspended in 0.025 M Tris-HCI buffer/ 0.15 M KCl, pH 7.5,2 mg protein/ml. Ccl4 (as a Tris-KC1 saturated solution) was added to one cuvette and buffer was added in equal volumes to the reference cuvette. The total volume of additions was 300 ~1 of Ccl,-saturated solution. After each addition spectra were recorded and the differences in absorbance at 430 nm minus that at 490 nm versus concentration were graphed as double reciprocal plots according to Schenkman et al. (1967) to obtain the spectral constants. The results are the mean f SD of three determinations obtained with pooled microsomal suspensions from livers of three animals per species. TABLE In Vitro LIVER


Species Rat

Mouse Hamster Guinea pig Chicken



Irreversible binding (dpm/mg lipid/mg


14CC14 TO



of 14C from 14CCl, protein/ml + SD)

394 L- 30 1040 * 75 3OOf22 366 f 27 161 + 30

“The incorporation in vitro of 14C from WCl., into microsomal lipids was tested in incubation mixtures containing: 0.05 M Tris/HCI, 0.15 M KCl buffer, pH 7.4; magnesium chloride 5 mM; sodium isocitrate 8 mu; NADP 0.33 mu; isocitric acid dehydrogenase, 0.36 IU ml ; 5 pi/ml of a solution of ‘%X1, in 96% ethanol (2 x 10’ dpm/ml) and microsomal protein 8 mg/ml. Incubations were performed for 15 min at 37°C in a Dubnoff shaker under anaerobic conditions.





In vitro Irreversible Binding of 14C from 14CC14 to Liver Microsomal Direrent Animal Species

Lipids from

The activity in vitro of the Ccl,-activating system as measured through the irreversible binding of 14C from 14CC14to microsomal lipids is as much as two to three times greater in mouse than in the rat, the guinea pig, or the hamster (Table 3). The activity of the Ccl,-activating system in the chicken is about half of that found in the hamster which in turn was the least active Ccl,-activating system among those of all the mammals tested. In vivo Irreversible Binding of 14C from 14CC14 to Liver Microsomal 14CC14 Concentrations in Livers from D@erent Animal Species

Lipids and free

The ratio between the irreversible binding of 14C from 14CC14 to liver microsomal lipids and the free 14CC14concentrations in liver is an expression of the CCI, activating ability of the liver microsomes under given experimental conditions (Castro and Diaz Gomez, 1972, Diaz Gomez et al., 1973). The value of this ratio was: mouse > hamster > guinea pig > rat > chicken (Table 4). TABLE 4 In









Species” Rat Mouse Hamster Guinea pig Chicken

Irreversible binding of 14Cfrom ‘“CC1 (dpm/g lipid + SD”,

14CC14 concentration in liver (dpm/g liver _+SD)

46,200+ 7,400 81,000+ 11,000 125,000_+14,000 77,000+ 16,400 34,000+ 900

460 rt: 114 91+ 22 178+ 59 350&Y 74 649 + 217

Rb lOO+ 849f 734f 204+ 55+

18 53 127 36 14

0 Animals were injected ip with a solution of 14CC1, (27.5 mCi/mmol) in olive oil (1.5 x lo6 dpm/ml) at a dose of 5 ml of solution/kg and were sacrificed 3 hr after 14CClq administration. The results are the mean of the values derived from : 8 animals for the rat, hamster, andguineapig; 5 animalsin thecaseof the chickenandfrom 4 pooled samples ( 4 livers each) for the mouse. b R is the ratio between the values of irreversible binding and its respective 14CC1, concentration and is expressed in g liver/g lipid 4 SD.

Ccl,-Induced Liver Microsomal Lipid Peroxidation in DifSerent Animal Species

Lipid peroxidation in vivo as evidenced by the change in the ultraviolet absorption of lipid extracts of the liver microsomal fractions was only observed in the rat, hamster, and guinea pig. The ultraviolet absorption of the lipid extracts derived from chicken and mouse liver microsomes of Ccl,-treated animals was not significantly different from that derived from control animals. This fact is particularly remarkable for the mouse since observations were made at periods up to 24 hr after Ccl, administration (Table 5).






TABLE 5 CC14-I~~~c~~


In vivo lipid peroxidation & SD 3 hr 6 hr

Species Rat Mouse Hamster Guinea pig Chicken

Control cc14 Control CCL Control cc14 Control cc14 Control CCL

216? 18 355+ 12b 324+ 56 323f. 30 180+ 13 249t- 33b 101 + 18 1362 gb 393 +103 465+ 79

252+21 401?48' 185 +12 199 + 37

24 hr 124+ 25 225+5gb 118+ 52 103 & 34

aCCI, 20% (v/v) in oliveoil wasgivenip at a doseof 5 ml of solution/kg. Controlsreceived olive oil. Animals were sacrificed 3, 6, or 24 hr after administration. The lipid peroxidation value is expressed as A absorbance at 243 nm x 1000 for a solution having 1 mg of microsomal lipid/ml. Ten animals per group were used in the experiments except for the chicken when 5 animals/group were employed. b p < 0.001 when compared to its respective control.

The intensity of the Ccl, effect in relation to its respective control was: rat > hamster 2 guinea pig > chicken = mouse.

Ccl, Concentrations in Livers of Animals of Different Species 3 hr After its Administration As shown in Table 6, CC& concentrations in liver are not markedly different in the rat, hamster, and chicken while those found in the mousewere significantly 1ower:and the onesin the guinea pig were significantly higher. TABLE 6 Ccl., CONCENTRATIONS IN LIVER OF ANIMALS OF DIFFERENT SPECIES 3 HR AFTER ITS ADMINISTRATION’

Speciesb Rat Mouse

Hamster Guinea pig Chicken

Ccl, concentration in liver (fig/g liver, meank SD) 497 +- 99 189f 85 535 + 189

1,48831255 685 + 121

’ CCL 20% (v/v) in olive oil was given ip at a dose of 5 ml of solution, kg. The animals were sacrificed 3 hr after Ccl* administration. b Ten animals per group were used for the rat, mouse and hamster; and five animals per group were used for the guinea pig and chicken.





CC&Induced Damage in Livers of Animals of DifSerent Species Twenty-four hours after the ip administration of a single injection of Ccl, 20 % v/v at the dose of 5 ml of solution/kg, centrilobular necrosis of the liver was observed in all species except the chicken. The necrogenic response of the liver was marked in the rat, mouse, guinea pig, and hamster, and none in the chicken. The high intensity of the necrosis at this dosage regime did not allow establishing differences in the liver response TABLE 7 EFFECT OF Ccl, 24 hr AFTER ADMINISTRATION OF PLASMA ISOCITRIC DEHYDROGENASE


Species and treatment’ Rat Control cc14 Mouse Control CCI, Hamster Control CCI, Guinea pig Control CCI, Chicken Control CCI,



ICD activity (U/ml -CSD)

278& 44,700 k

126 6,037"

96f 134,402+

8 16,071b

489+ 52,200&

93 8,900b

2,900+ 380 79,800 f 14,007b 360& 417+

60 170

4 Ccl, was given as a 5 % v/v solution in in olive oil (5 ml of solution/kg) ip. Controls received olive oil. The animals were sacrificed 24 hr after CC& administration. Blood samples were taken and plasma assessed for ICD activity. Five animals per group were used. bp guinea pig = hamster > rat > chicken.


Species variation in the toxic response to chemicals have been long recognized as a valuable tool for mechanistic studies (International Symposium Comparative Pharmacology, 1967). In the case of Ccl,, most species except the chicken and birds are



FIG. 2. Liver from a guinea pig 24 hr after administration of 5% CCI.+ There is a centrilobular necrosis and an intense fatty infiltration. Hematoxylin and eosin. x 197.

susceptible to its deleterious action (Cameron and Karunaratne, 1936; Hall and Schillinger, 1923). One group of workers believe that CC&-induced lipid peroxidation is the key alteration in the chain of events ending in cellular necrosis (Recknagel, 1967; Recknagel and Glende, 1973). Others are more inclined to believe that the irreversible binding of the *Ccl, and *Cl free radicals to cellular components is crucial to liver injury (Castro et al., 1972b, 1973; Gillette, 1973). The resistance of the chicken to Ccl, hepatotoxicity could be due to a very decreased CC],-induced lipid peroxidative process or to a less marked irreversible binding of Ccl, to cellular constituents. Our present observations show that in agreement with previous observations of Slater (1968), the CC],-induced in vivo microsomal lipid peroxidation in chicken liver preparations is not different than in controls. We also observed that the irreversible binding of Ccl., to lipids is less in the chicken than in the other species tested and consequently a discrimination of the contribution of each of both factors is not possible. Very useful for discrimination between both possibilities were our experiments on the other species, since we found that the necrogenic response of their livers to Ccl, decreased in the order mouse > guinea pig = hamster > rat B chicken, while Ccl,-






FIG. 3. Liver from a rat 24 hr after administration of 5 yOCC&. The liver cells of the periportal zones are well preserved. Hematoxylin and eosin. x 197.

induced lipid peroxidation decreased in the order rat > hamster = guinea pig > chicken >, mouse and the intensity of in vivo CCI, activation decreased in the order mouse N hamster > guinea pig > rat > chicken. That is, there is correlation between intensity of CCL, activation and necrosis while there is no correlation between lipid peroxidation and necrosis. The behavior of the mouse liver is particularly striking. No lipid peroxidation was detectable during the complete period of intoxication (24 hr), while it was without doubt the most susceptible of all the species tested to CC&-induced liver damage. This particular experiment shows either that lipid peroxidation is not necessary for Ccl,-induced necrosis or that the changes in ultraviolet absorption occurring after CC], administration in microsomal lipids are not necessarily related to lipid peroxidation as most authors believe (Recknagel, 1967; Recknagel and Glende, 1973). We are more inclined to believe the former possibility but further experiments would be necessary to understand these facts which no doubt may constitute a serious drawback to the lipid peroxidation theory. It should be pointed out that differences in concentrations of Ccl, in the liver cannot explain the different species susceptibility. Similar CCI, content was



found in livers of rats, hamster, and chicken; the mouse which showed smaller CCL, concentrations in liver than any other species exhibited the highest degree of necrosis. The higher concentrations of Ccl., found in the livers of the guinea pig than in the hamsters may help to explain why necrosis in the guinea pig liver is similar to that observed in the hamster. If only differences in the irreversible binding to cellular components were to explain differences in the extent of liver necrosis, this process should be more intense in the hamster than in the guinea pig. However, necrosis is equally intense in the hamster and in the guinea pig. The reason could be that higher concentrations of CCI, found in livers of the guinea pig than those in hamsters could compensate for a decreased ability to activate Ccl,. It is important to remember, however, that different susceptibilities of the livers of the different animal species at stages of the intoxication process other than either Ccl, activation or lipid peroxidation may also occur and their contribution has not been weighed in the present work. Although there were species differences in the magnitude of the spectral binding constant and maximal spectral change, these were not related to the species differences in the extent of either in vivo or in vitro irreversible binding. Moreover, there is no good correlation between the extent of the in vivo or the in vitro irreversible binding of 14CC14 to lipids and the microsomal P-450 content or with the activities of the microsomal P-450 reductase. These apparently contradictory observations to our previous hypothesis that CC& is activated during the reduction of the CCl,/P-450 complex by NADPH catalyzed by the P-450 reductase (Castro and Diaz G6mez 1972; Castro et al., 1972a; D’Acosta et al., 1972,1973; Castro et al., 1973; Diaz G6mez et al., 1973) should not be surprising, since many investigators have repeatedly pointed out that there are many instances in which the overall hydroxylase activity is not directly proportional to the content of any particular single component of the electron transport chain (Gillette, 1971). This is understandably so if one considers that the overall kinetics of the activation reaction is very complex and depends on a number of factors including (a) the proportion of P-450 and its chemical structure (surely different in each species); (b) the equilibrium constant for the formation of the complex between Ccl, and the active portion of all the P-450 population present in microsomes; (c) the rate of reduction of the P-45O/CCl, complex; and (d) the steady state constant for the dissociation of the reduced P-450/CC14 complex. In view of the complexities of the system it should not be surprising to see that changes in the overall activity of the system measured as the ability to catalyze the irreversible binding of 14C from 14CC14 to microsomal lipids either in vivo or in vitro do not correlate with any given component or property of the system. In addition to these difficulties when in vivo results are under consideration the precise concentration of NADPH and oxygen at the microsomal site of Ccl, activation in the different species is not known. This is particularly important since we have recently established that both factors decisively control the extent of the irreversible binding to cellular components (unpublished observations). REFERENCES BANCROFT, H. (1960). Zntroduccidna la bioestudisticcz, pp. 205-211. Eudeba, Buenos Aires. CAMERON, C., AND KARUNARATNE, W. (1936). Carbon tetrachloride cirrhosis in relation to

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CASTRO,J. A., DE CASTRO, C. R., DEFENOS, 0. M., DEFERREYRA, E. C., D~AZG~MEZ,M. I., ANDD’ACOSTA,N. (1972a).Effect of cystamineon the mixed-function oxygenasesystem from rat liver microsomesand its preventive effect on the destructionof cytochrome P 450 by carbon tetrachloride. Pharmacol. Res. Commun. 4, 185-190. CASTRO, J. A., CIGNOLI,E. V., DE CASTRO, C. R., ANI~ DE FENOS, 0. M. (1972b)Prevention by cystamineof liver necrosisandearly biochemicalalterationsinducedby carbon tetrachloride. Biochem. Pharmacol. 21,49-51. CASTRO, J. A., AND D~AZ G~MEZ, M. I. (1972).Studieson the irreversiblebinding of 14C-CC14 to microsomallipids in rats under varying experimentalconditions. Toxicol. Appl. Pharmacol.23,541-552. CASTRO, J. A., DE FERREYRA, E. C., DE CASTRO, C. R., D~AZ G~MEZ, M. I., D’ACOSTA, N., and DE FENOS, 0. M. (1973).Studieson the mechanismof cystamineprevention of several liver structural and biochemicalalterations causedby carbon tetrachloride. Toxicol. Appl. Pharmacol. 24, 1-19. CHOPRA, P., ROY, S., RAMALINGASWARM, V., AND NAYAK, N. (1972).Mechanismof carbon tetrachloride hepatotoxicity. An in vivo study of its molecularbasisin rats and monkeys. Lab. Incest. 26, 716-724. D’ACOSTA, N., CASTRO, J. A., DE FERREYRA, E. C., D~AZ G~MEZ, M. I., AND DE CASTRO, C. R. 1972). Pyrazole blockade of carbon tetrachloride activation and liver necrosis. Res. Commun. Chem. Pathol. Pharmacol. 4,641-650. D’ACOSTA, N., CASTRO, J. A., D~AZ G~MEZ, M. I., DE FERREYRA, E. C., and DE CASTRO, C. R.

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Species differences in carbon tetrachloride-induced hepatotoxicity: the role of CCl4 activation and of lipid peroxidation.

ToXICOLOGYANDAPPLIEDPHARhfACOLOOY34,102-114(1975) Species Differences in Carbon Tetrachloride-Induced Hepatotoxicity: The Role of Ccl, Activation and...
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