Effects of novel antioxidants on carbon tetrachloride-induced lipid peroxidation and toxicity in precision-cut rat liver slices

Previous studies in rat liver microsomes have demonstrated the effectiveness of the 21-aminosteroid, U-74,006F, the troloxamine, U-78,517G, and N,N'-diphenyl-p-phenylenediamine (DPPD) in preventing carbon tetrachloride (CCl4)-induced lipid peroxidation. Studies reported here utilized liver slices to assess whether these antioxidants could prevent lipid peroxidation and ensuing toxicity in a more complete/complex system. Liver slices prepared from Aroclor 1254-induced SD rats were incubated in Dulbecco's modified eagle media, 37 degrees C, for up to 9 hr. Slices were preincubated with test compounds for 30 min prior to addition of CCl4. Lipid peroxidation, as measured by the formation of thiobarbituric acid-reactive substances and ethane evolution, was decreased by U-74,006F (100 microM), U-78,517G (100 microM), and DPPD (1 microM). CCl4 (2.5 microliters) decreased intracellular K+ content, intracellular lactate dehydrogenase (LDH), and intracellular isocitrate dehydrogenase (ICD) activities over a 9-hr incubation period. Despite the marked effects on lipid peroxidation, U-74,006F showed no protection against K+ or LDH loss and only moderate protection against ICD loss. U-78,517G showed no protection against K+ loss but substantial protection against enzyme loss. DPPD demonstrated slight protection against K+ and marked protection against enzyme loss. All three compounds inhibited CCl4-induced lipid peroxidation; U-78,517G being most effective, followed by DPPD and U-74,006F. Inhibition of lipid peroxidation provided protection to the membrane structure as indicated by inhibition of LDH and ICD loss. The antioxidants failed to protect against CCl4-induced toxicity (K+ loss). These results suggest that CCl4-induced lipid peroxidation and toxicity may be dissociable.     

Lipid Peroxidation Induced by Adriamycin in Linolenic Acid-Loaded Cultured Hepatocytes

Abstract: Addition of more than 10 μM of adriamycin to cultured rat hepatocytes loaded with α-linolenic acid (linolenic acid-loaded hepatocytes) caused marked lipid peroxidation as measured by an accumulation of malondialdehyde during a 9 hr incubation. After addition of 50 μM of adriamycin to linolenic acid-loaded hepatocytes, malondialdehyde accumulation significantly increased at 3 hr, followed by cellular reduced glutathione decrease and lactate dehydrogenase leakage after 6 hr. Inhibition of adriamycin-induced lipid peroxidation by addition of N, N′-diphenyl-p-phenylenediamine or α-tocopherol, both lipid radical scavengers, or deferoxamine, which is a Fe ion chelator, prevented both glutathione decrease and lactate dehydrogenase leakage, indicating that lipid peroxidation caused cellular damage to linolenic acid-loaded hepatocytes exposed to adriamycin. The effect of SKF 525-A, which is a cytochrome P450 inhibitor, on adriamycin-induced lipid peroxidation and on 7-ethoxycoumarin O-deethylase activity was determined by 6 hr incubation of linolenic acid-loaded cells. Addition of SKF 525–A suppressed adriamycin-induced lipid peroxidation comparably with its 7-ethoxycoumarin 0-deethylase inhibitory activity. These results suggest that cytochrome P450 contributes to the one-electron bioreduction of adriamycin into its semiquinone radical in rat hepatocytes.     

Preventive effect of KZ-1026 on acute liver injury induced by CCl4 in rats: histochemical, enzyme-histochemical and ultrastructural studies

The preventive effect of KZ-1026 on acute liver injury induced by CCl4 in rats was evaluated histochemically, enzyme-histochemically and ultrastructurally. Rats that received 50% CCl4 (2 ml/kg) intraperitoneally were sacrificed at 3, 7 and 24 hr. Remarkable reductions in hepatic glycogen, RNA and G-6-Pase activity were observed in the centrilobular area at 3 hr, and ballooned cells appeared in the mid-zone at 7 hr. At 24 hr, the above histochemical parameters in the hepatocytes of the centrilobular area and mid-zone were extensively reduced, while the number of ballooned cells in the mid-zone was increased. KZ-1026 (200 mg/kg) was given orally at 24 and 4 hr before, simultaneously with or 3 hr after CCl4 treatment, and each rat was sacrificed at 24 hr after CCl4 administration. Pretreatment with KZ-1026 24 hr before CCl4 administration prevented reduction of RNA, glycogen and G-6-Phase activity, as well as disruption of rER and proliferation of sER due to CCL4 toxicity. This preventive effect of KZ-1026 was reduced by posttreatment; however, only the decrease in cytoplasmic RNA was well prevented. These results suggested that KZ-1026 is protective against CCl4-induced acute liver injury.     

Effect of iron overload on spontaneous and xenobiotic-induced lipid peroxidation in vivo

To study the effect of iron-overload on hepatic lipid peroxidation, two rat models of haemochromatosis were employed: in the first model resembling secondary haemochromatosis, repeated i.p. injections with Fe-dextran led to an accumulation of Fe in Kupffer cells, while in the second model resembling hereditary haemochromatosis, iron was located mainly in periportal hepatocytes after feeding on a diet containing 3.5% Fe-fumarate for 3 weeks. In both models, total hepatic iron content was elevated four- to fivefold over controls. In vivo lipid peroxidation (ethane exhalation) was enhanced only in the second model, indicating that the hepatocytes are the main targets of Fe-induced lipid peroxidation. Low hepatotoxicity was observed in the second model. Additional treatment of the rats with hepatotoxic agents led to different results: with ethanol and bromobenzene, lipid peroxidation was only evident in both models of iron-overload, while paracetamol-induced lipid peroxidation was seen only in Fe-fumarate-fed rats. CCl4-induced lipid peroxidation was strongly enhanced in both models of haemochromatosis. Hepatotoxicity was enhanced by iron overload only in the case of CCl4-treated, Fe-fumarate-fed rats. The activities of phase I and phase II enzymes of xenobiotic metabolism were not markedly altered in livers of iron-overloaded rats. This implies that neither the bioactivation nor the detoxification of the agents studied were affected in experimental haemochromatosis.     

Evaluation of cultured, precision-cut rat liver slices as a model to study drug-induced liver apoptosis

The precision-cut liver slice culture model has been used widely to investigate drug metabolism and drug-induced necrosis. However, apoptosis, a key mediator of liver toxicity remains to be studied in this model. We evaluated apoptosis induced by thioacetamide (TAA) in rat liver slices, and in livers taken from TAA-treated rats as a control. Rat liver slices were treated with 50, 75 and 100 mM of TAA for 15 h. Histopathological examination of the liver slices revealed specific centrilobular localization of apoptotic hepatocytes at 75 mM but randomly distributed at 100 mM. Apoptosis in centrilobular hepatocytes was confirmed by appearance of cleavage products of caspase-3 and DNA fragmentation studied by TUNEL method. A concentration-dependent release of cytochrome c was observed in the slices, suggesting a role for mitochondria in the apoptosis triggered by TAA. The in vitro results were compared to the data obtained in male Sprague-Dawley rats given a single ip injection of 40 mg/kg TAA and sacrificed 1, 2, 3 and 6 h after dosing. Histopathological analyses showed specific centrilobular localization of apoptosis after 6 h treatment. Caspase-3 activation, DNA fragmentation and cytochrome c release were also observed in the liver of rats treated with TAA. Overall these data indicated that precision-cut liver slices provide a valuable in vitro system to study drug-induced liver apoptosis.     

In vivo production of ethylene from 2-keto-4-methylthiobutyrate in mice

The use of 2-keto-4-methylthiobutyric acid (KMB), the alpha-keto analog of methionine, was studied as a potential means of detecting free radical generation in vivo. KMB-dependent ethylene production (presumably from free radical interception), and ethane production from in vivo lipid peroxidation, were monitored simultaneously by measuring the rate of exhalation of these hydrocarbons by mice. Injection of KMB (1 g/kg) into mice resulted in an 8-fold increase in ethylene production above endogenous levels seen in saline-injected controls (1.47 +/- 0.35 vs 0.17 +/- 0.02 nmoles/100 g/hr respectively). Administration of CCl4 (3.0 g/kg) to initiate hepatic lipid peroxidation, 20 min prior to KMB injection, augmented the production of ethylene (2.37 +/- 0.10 nmoles/100 g/hr). Lipid peroxidation following injection of CCl4 was monitored via the increased exhalation of ethane. Pretreating the mice with vitamin E (100 mg/kg daily for 3 days), an inhibitor of lipid peroxidation, did not result in a significant change in ethylene production from KMB by itself or after prior injection of CCl4. However, vitamin E did suppress ethane production initiated by CCl4. Similar results were obtained with mouse liver slices studied in vitro. Metyrapone (150 mg/kg), an inhibitor of hepatic mixed function oxidase activity, also suppressed significantly the CCl4-stimulated production of ethane, but not the CCl4-stimulated production of ethylene from KMB. It appears that ethylene production from KMB does not derive from free radicals generated during in vivo lipid peroxidation since suppression of lipid peroxidation by vitamin E or metyrapone did not suppress ethylene production.     

Cisplatin nephrotoxicity: In vitro studies with precision-cut rabbit renal cortical slices

Severe nephrotoxic side effects limit the use of cisplatin, a potent anticancer drug. In this study, precision-cut renal cortical slices from rabbits were evaluated as a cisplatin nephrotoxicity model. Cortical slices accumulated approximately 180 ppm (195 ppm Pt = 10(-3) M) of platinum(II) after 18 hr of incubation in medium containing 10(-3) M cisplatin. Dose- and time-dependent toxic responses for clinically relevant concentrations of cisplatin (10(-3)-10(-5) M) were apparent using leakage of intracellular K+, ATP, and lactate dehydrogenase (LDH) to determine cell damage. Histopathologic changes were also produced. Intracellular ATP levels dropped significantly after 6 hr of incubation in 10(-3) M cisplatin, and after 12 hr with 10(-4) M cisplatin. Similarly, intracellular K+ levels decreased significantly by 6 hr of incubation with 10(-3) M cisplatin but remained at control levels for 18 hr in the presence of 10(-4) M cisplatin. Decrements in intracellular LDH levels were not seen until after 12 hr of incubation in 10(-3) M cisplatin. The noncytotoxic isomer transplatin at 10(-3) M was not accumulated by slices; however, intracellular ATP levels were depressed. Of the viability parameters evaluated, intracellular K+ and ATP were found to be optimal indicators. Other active platinum analogs, carboplatin and iproplatin, also caused dose- and time-dependent leakage of intracellular K+ and ATP from renal cortical slices. The ranking of nephrotoxicity of the platinate compounds within this system at concentrations adjusted to approximate equivalent therapeutic activity was similar to that observed in vivo (cisplatin = iproplatin greater than carboplatin greater than transplatin). These results suggest that precision-cut renal cortical slices comprise a viable in vitro model for platinum-induced nephrotoxicity studies.     

Time course of the carbon tetrachloride-induced decrease in mitochondrial aldehyde dehydrogenase activity

Hepatic microsomal enzymes like cytochrome P-450 and glucose 6-phosphatase are inhibited after exposure to CCl4 in vivo. Since comparatively less is known about the effects of CCl4 on nonmicrosomal enzymes, we investigated the rapidity by which CCl4 inhibits the low Km mitochondrial aldehyde dehydrogenase (ALDH) isozyme, an enzyme known to be inhibited 24 hr after CCl4 treatment. The activity of this ALDH isozyme was significantly lowered 6 and 12 hr after a single 1 ml/kg intragastric dose of CCl4. The mitochondrial low Km ALDH specific activities exhibited a similar pattern of destruction/inhibition to the documented target enzyme microsomal cytochrome P-450 in that lowest values were observed 6 hr after CCl4. These values were 44 and 37% of control for cytochrome P-450 content and the low Km ALDH activity, respectively. Alcohol dehydrogenase activity, expressed as activity per gram liver, was depressed 12 hr after CCl4 dosing. Finally, the activity of the low Km cytosolic ALDH, the isozyme that metabolizes malondialdehyde at low concentrations, was not affected by CCl4 treatment. The CCl4-induced decline in the activity of the matrix ALDH isozyme occurs earlier than previously reported mitochondrial damage. The study of sensitive enzymes like the low Km ALDH may provide valuable information by which it may be possible to determine the relationship of the truly rapid biochemical effects of CCl4 such as microsomal lipid peroxidation with later effects on nonmicrosomal components.     

The Interactive Toxicity of CHCl3 and BrCCl3 in Precision-Cut Rat Liver Slices

The interactive toxicity of two nontoxic concentrations of chloroform(CHCl₃and bromotricchloromethane (BrCCl₃ was examined in precision-cutrat liver slices. Liver slices were prepared from male Sprague-Dawleyrats (220–250 g) pretreated with phenobarbital for 4 days.Toxicants were administered 1 hr apart. Intracellular K⁺ levelswere similar to untreated controls in slices treated with 0.2mM CHCl₃ or 0.125 µ1 (0.25 mg, 1.26 µmol) BrCCl₃alone, indicating that these concentrations were nontoxic. However,addition of both toxicants, irrespective of order, resultedin a time-dependent loss of intracellular K⁺ which was significantat 9 hr following administration. This was interpreted as evidenceof synergistic toxicity. Cytochrome P450 loss was significantas early as 3 hr following exposure to BrCCl₃ alone or whenadded with CHCl₃ This loss may be attributed to BrCCl₃ suicideinactivation of cytochrome P450. Centrilobular hepatocytes maybe more susceptible to the interactive toxicity of CHCl₃ andBrCCl₃ Activity of enzymes found predominantly in this areawas significantly decreased in slices exposed to both toxicantsrelative to controls. Conversely, activity of enzymes foundpredominantly in the periportal region was similar to that ofuntreated and treated controls. Inter active toxicity of BrCCl₃and CHCl₃ was not a consequence of increased lipid peroxidationor depletion of slice glutathione content. Further studies needto be conducted to elucidate the mechanisms mediating the interactivetoxicity of BrCCl₃ and CHCl₃.     

Lipid peroxidation and irreversible cell damage: synergism between carbon tetrachloride and 1,2-dibromoethane in isolated rat hepatocytes

The combination of 1,2-dibromoethane (DBE) with carbon tetrachloride (CCl4) in the isolated rat hepatocyte model produces a significant potentiation of both lipid peroxidation and plasma membrane damage induced by the latter compound. The increase in malondialdehyde production precedes the hepatocyte damage, evaluated in terms both of lactate dehydrogenase leakage and trypan blue exclusion. When hepatocytes are isolated from vitamin E pretreated rats, both the prooxidant and the cytotoxic effects of CCl4 are prevented. Also the synergism between CCl4 and DBE on lipid peroxidation disappears completely while that on cell damage is strongly reduced. The increased lipid peroxidation appears to be one of the mechanisms of the observed synergism between CCl4 and DBE on hepatocyte damage. Regarding the antioxidant status of the hepatocyte challenged with CCl4 and DBE, an early and significant consumption of vitamin E is observed only in the presence of the mixture of these xenobiotics. Total nonprotein thiol content is not significantly modified by CCl4 poisoning while DBE, alone and in association with CCl4, markedly decreases it. Vitamin E supplementation does not prevent but moderately delays total nonprotein thiol depletion due to DBE or to the mixture. Finally, glutathione transferase activity is significantly reduced by CCl4 treatment and not by DBE, and vitamin E supplementation totally prevents such inhibition. The increased prooxidant effect of CCl4 plus DBE compared to CCl4 alone seems related to the shift in DBE metabolism consequent to the CCl4-dependent inactivation of glutathione transferase.     

Morphology and cytochrome P450 isoforms expression in precision-cut rat liver slices

Precision-cut liver slices are a widely accepted in vitro system for the examination of drug metabolism, enzyme induction, or hepatotoxic effects of xenobiotics. The maintenance of the distinct lobular expression and induction pattern of phase I biotransformation enzymes, however, has not been examined systematically so far. Thus, in the present study, both longitudinal and transversal sections of male rat liver slices were investigated morphologically, as well as immunohistochemically for the expression of different cytochrome P450 (CYP) isoforms after prolonged incubation or after exposure to typical inducers. Histopathological examinations revealed an increasing vacuolization of the periportal hepatocytes mainly in the middle of the slices from 6 h of incubation on, paralleled by a loss of glycogen in the respective cells. After 24 h, mainly in the center of the slices, necroses of cells occurred. After 48 h of incubation, typically a central band of coagulative necrosis flanked by superficial layers of viable cells was observed. Freshly prepared slices displayed a CYP subtypes expression as normal liver specimen, a very low centrilobular CYP 1A1 immunostaining, but a strong CYP 2B1 and 3A2 expression predominantly in the central and intermediate lobular zones. From 2 h on, the immunostaining for CYP 2B1 and 3A2 was to some extent reduced. After 24 h of incubation with beta-naphthoflavone, the CYP 1A1 and 2B1 expression was induced mainly in the viable cells around central veins, around some portal fields with bigger vessels and in the cell layers close to the slice surface. At the same sites, phenobarbital led to an increased CYP 2B1 and 3A2 expression and dexamethasone to an elevated CYP 3A2 immunostaining. These results show, that an in vitro induction of phase I enzymes in precision-cut liver slices can be demonstrated also immunohistochemically.     

Comparative cytotoxicity of N-substituted N'-(4-imidazole-ethyl)thiourea in precision-cut rat liver slices

In order to more rationally design thiourea-containing drugs and drug candidates, specifically thiourea-containing histamine H3 receptor antagonists, it is necessary to develop structure-toxicity relationships (STRs). For this purpose, the cytotoxicity of a series of thiourea-containing compounds was tested in precision-cut rat liver slices. A concentration of 1000 microM of N-p-bromophenyl, N'-(4-imidazole-ethyl)thiourea (8) or N-p-nitrophenyl, N'-(4-imidazole-ethyl)thiourea (9) was found to cause cytotoxicity, evidenced as LDH leakage, resulting in more than 95% LDH leakage after 6h. N-p-Methoxyphenyl, N'-(4-imidazole-ethyl)thiourea (6) caused 40.6 +/- 19.7% LDH leakage after 6h. Control levels of cell death (1% methanol as control vehicle) were below 20% in 6h. After 6h of exposure, N-p-chlorophenyl, N'-(4-imidazole-ethyl)thiourea (7), 8, and 9 were already found to cause significant cytotoxicity at a concentration of 100 microM. At 200 microM, 9 was found to cause significantly more cytotoxicity than 7 and 8. N-Naphthyl, N'-(4-imidazole-ethyl)thiourea (12) was found to cause significant cytotoxicity towards precision-cut rat liver slices after 6h of exposure to a concentration of 500 microM. All other N-substituted, N'-(4-imidazole-ethyl)thiourea tested in this study were not found to be cytotoxic towards precision-cut rat liver slices within the 6h of exposure up to a concentration of 1000 microM. Intracellular glutathione (GSH) content and mitochondrial MTT reduction activity were also examined after exposure of slices to N-substituted, N'-(4-imidazole-ethyl)thiourea. Both of these markers, however, were not found to provide additional information regarding the possible mechanisms of cytotoxicity, i.e. GSH depletion or reduced mitochondrial activity since these markers did not clearly precede LDH leakage. A correlation was found between cytotoxicity towards precision-cut rat liver slices and Vmax/Km values for the formation of sulfenic acids from N-substituted N'-(4-imidazole-ethyl)thiourea by hepatic rat flavin-containing monooxygenases (FMO). The compound with the highest Vmax/Km value for the formation of sulfenic acids, 9, was also the most cytotoxic. Compounds with a significantly lower Vmax/Km value, 7, 8, and 12, were less cytotoxic than 9. Compounds with a Vmax/Km value for the formation of sulfenic acids lower than 0.0788 ml/(minmg) were found not to be cytotoxic towards precision-cut rat liver slices for concentrations up to 1000 microM at an exposure time of 6h. It is concluded, from this study, that N-phenyl substituted N'-(4-imidazole-ethyl)thiourea-containing electron-withdrawing p-substituents are cytotoxic towards precision-cut rat liver slices. Cytotoxicity is increased with increasing electron-withdrawing capacity of the p-substituent. A correlation was found to exist between Vmax/Km value for the formation of sulfenic acids by rat liver FMO enzymes and cytotoxicity.     

Histological evidence for dissociation of lipid peroxidation and cell necrosis in bromotrichloromethane hepatotoxicity in the perfused rat liver

Bromotrichloromethane (CBrCl3)-induced hepatic lipid peroxidation and cell necrosis were studied histologically and biochemically, using isolated perfused livers from phenobarbital-pretreated rats. Lipid peroxidation was assessed by fuchsin staining of the liver slices and release of thiobarbituric acid reactive substances (TBARS) into the perfusate; necrosis was assessed by trypan blue uptake and lactate dehydrogenase (LDH) leakage. A good correlation was observed between the Schiff-positive reaction and TBARS release under various experimental conditions, supporting the validity of the fuchsin staining method for histological detection of lipid peroxidation. Lobular localization of lipid peroxidation and necrosis was as follows: Under high oxygen supply (95% O2-saturated buffer), infusion of CBrCl3 caused the Schiff-positive reaction in the pericentral to midzonal hepatocytes, irrespective of the direction of perfusion, but did not produce necrosis. Under low oxygen supply (20% O2) with retrograde perfusion, dissociation of lipid peroxidation and necrosis was observed, i.e., trypan blue uptake in the periportal zones and Schiff-positive staining in the pericentral hepatocytes. Thus, lipid peroxidation by itself may have a relatively minor role in the development of CBrCl3-induced acute hepatic cell death.     

Suppressive effect of tritoqualine on enzyme leakage induced by D-galactosamine in rat primary-cultured hepatocytes

Tritoqualine (TRQ) was previously reported to suppress enzyme leakage and lipid peroxidation induced by carbon tetrachloride in isolated hepatocytes. In the present study, we investigated the effect of TRQ on enzyme leakage from rat primary-cultured hepatocytes using D-galactosamine (GalN) which causes hepatic injury without lipid peroxidation. Leakage of GPT and GPT was significantly increased at 18 hr after GalN addition, being saturated at 42-50 hr. This enzyme leakage was suppressed dose-dependently by TRQ at 42 hr, but not by vitamin E. These results suggest that TRQ shows a suppressive effect on enzyme leakage from hepatocytes independently of its inhibitory action on lipid peroxidation.     

Metabolic viability and pharmaco-toxicological reactivity of cryopreserved human precision-cut renal cortical slices.

We have tested the suitability of cryopreserved human precision-cut renal cortical slices for metabolic and pharmaco-toxicological studies. The viability of these slices and their pharmaco-toxicological reactivity were assessed using intracellular ATP and protein contents, lactate dehydrogenase (LDH) leakage, lactate and glutamine metabolism and the ammoniagenic effect of valproate. Despite a decrease in ATP and protein contents when compared with those of fresh slices, cryopreserved slices did not show any LDH leakage and retained the capacity to metabolize glutamine and lactate. Glutamine removal and ammonia, lactate and alanine production were similar in fresh and cryopreserved slices; by contrast, cryopreserved slices accumulated more glutamate as a result of decreased flux through glutamate dehydrogenase which catalyses an oxygen-dependent reaction. Valproate markedly and similarly stimulated glutamine metabolism in fresh and cryopreserved slices. Cryopreservation did not alter lactate removal but inhibited lactate gluconeogenesis. In conclusion, these results demonstrate that, although their mitochondrial oxidative metabolism seems to be diminished, cryopreserved human precision-cut renal cortical slices remain metabolically viable and retain the capacity to respond to the ammoniagenic effect of valproate. Thus, this experimental model may be helpful to optimize the use of human renal tissue for metabolic and pharmaco-toxicological studies.     

Ethanol potentiation of carbon tetrachloride hepatotoxicity: possible role for the in vivo inhibition of aldehyde dehydrogenase

A potentiation of CCl4-induced hepatotoxicity was observed in rats pretreated with ethanol 18 hr prior to CCl4 exposure. Hepatic microsomal aldehyde dehydrogenase (ALDH) was significantly inhibited in animals sacrificed 1 hr following the sequential exposure, however, no more so than in those animals receiving CCl4 alone. The animals receiving ethanol alone had ALDH activity similar to vehicle treated controls. Twenty-four hours following a potentiating dose of ethanol and CCl4 an 81 and 57% decline in NAD+-dependent microsomal and mitochondrial ALDH activity was observed, respectively. Similar results were observed for microsomal and mitochondrial NADP+-dependent ALDH activity. The decline in membrane-bound ALDH was greater in potentiated animals than in those receiving CCl4 alone. A relatively smaller decline in cytosolic ALDH activity was observed in CCl4 treated rats with or without ethanol pre-exposure. The data suggest that inhibition of membrane bound ALDH may be one of the major mechanisms of in vivo potentiation of CCl4-induced hepatotoxicity by ethanol.     

Effects of oxygen deficiency and calcium omission on carbon tetrachloride hepatotoxicity in isolated perfused livers from phenobarbital-pretreated rats

The effect of oxygen concentration and Ca2+ omission on CCl4-induced hepatotoxicity was studied in a non-recirculating and hemoglobin-free liver perfusion system using phenobarbital-pretreated rats. With 95% O2-saturated perfusate, infusion of 0.5 mM CCl4 caused an instantaneous increase of thiobarbituric acid reactive substances (TBA-RS) in the effluent perfusate, accompanied by only a slight leakage of K+ and lactate dehydrogenase (LDH). CBrCl3 produced a far greater increase in the TBA-RS level, but again with slight K+ and LDH leakage. With 20% O2-saturated perfusate, CCl4 caused a marked LDH leakage, which was preceded by an early and considerable increase in K+ leakage coupled with Na+ uptake, Ca2+ uptake was initially slight, being enhanced concurrently with the LDH leakage. The TBA-RS level changed biphasically with an initial moderate and a succeeding greater increase coupled with LDH leakage. N,N"-Diphenyl-p-phenylenediamine and promethazine suppressed the TBA-RS production, but improved neither K+ nor LDH leakage. Omission of the Ca2+ from the perfusate reduced the initial K+ leakage as well as the later TBA-RS release, and markedly delayed the LDH leakage. In retrograde perfusion under low oxygen supply with Ca2+, CCl4 produced essentially the same toxic manifestations as those observed in the anterograde perfusion. Hepatocytes of the periportal and pericentral areas were not stained with trypan blue in the antero- and retrograde perfusion systems respectively. Thus, oxygen deficiency, rather than lipid peroxidation by itself, and the essential role of extracellular Ca2+ may be important for CCl4-induced hepatic cell necrosis, in which plasma membrane permeability change may be an early and critical event.     

In vivo metabolism of CCl4 by rats pretreated with chlordecone, mirex, or phenobarbital

The propensity of chlordecone (CD) to potentiate hepatotoxic and lethal effects of CCl4 is well established. Mirex (M), a close structural analogue of CD, or phenobarbital (PB), powerful inducers of hepatic microsomal drug metabolizing enzymes, are much weaker potentiators of CCl4 toxicity. The purpose of this study was to test the possibility that CD potentiates the toxicity of CCl4 by increasing the metabolism of CCl4 to a greater degree than either PB or M. We compared the in vivo metabolism of CCl4 in rats pretreated with CD, M, or PB, by measuring the hepatic content of 14CCl4, the expiration of 14CCl4, expiration of 14CCl4-derived 14CO2, and lipid peroxidation. Male Sprague-Dawley rats (250-270 g) were pretreated with a single oral dose of CD (10 mg/kg), M (10 mg/kg), or corn oil vehicle (1 ml/kg). PB pretreatment consisted of an ip injection of sodium PB (80 mg/kg) in saline (0.9%) for 2 successive days. Twenty-four hours later, 14CCl4 (0.1 ml/kg; sp act: 0.04 mCi/mmol) was administered ip in corn oil and the radioactivity present in the expired air was collected for 6 hr. Excretion of the parent compound as represented by the 14C label in the toluene trap was unchanged by any of the pretreatments. Expiration of 14CO2 measured during the 6 hr after CCl4 administration was increased in animals pretreated with PB or CD. In vivo lipid peroxidation measured as diene conjugation in lipids extracted from the livers was increased to a similar extent in animals pretreated with PB and CD, whereas the serum transaminases (ALT, AST) were significantly elevated only in animals pretreated with CD.M did not affect 14CO2 production and was without a significant effect on the lipid peroxidation. The radiolabel present in the liver at 6 hr showed no difference in hepatic content of free 14CCl4 among the groups, but the covalently bound label present in the lipid fractions of the livers pretreated with PB was elevated in comparison to CD and M treatments. These data indicate that a single oral administration of CD (10 mg/kg) 24 hr prior to CCl4 administration (100 microliter/kg) enhances the oxidative metabolism of CCl4 but to a lesser extent than PB (80 mg/kg, ip, twice), which is in inverse relationship to the potentiation of the hepatotoxic and lethal effects of CCl4 associated with these pretreatments.     

Suppressive effect of tritoqualine on lipid peroxidation and enzyme leakage induced by carbon tetrachloride in rat hepatocytes

Since tritoqualine (TRQ) is effective in suppressing the increase of serum transaminases in acute hepatic injured rats induced by some hepatotoxins, protection of the hepatocyte membrane is suggested to be one of the pharmacological effects of TRQ. In the present study, we investigated the effects of TRQ on lipid peroxidation and enzyme leakage caused by carbon tetrachloride (CCl4) exposure in isolated hepatocytes and the liver in vivo, compared with vitamin E. The results were as follows: Hepatocytes isolated from TRQ-administered rats showed less enzyme leakage than those from control rats after CCl4 addition. TRQ displayed strong inhibition of lipid peroxidation in isolated hepatocytes. In comparison with vitamin E, TRQ showed almost the same inhibitory action on lipid peroxidation, but a stronger suppression of enzyme leakage. Vitamin E showed a weaker protection from increase of glutamic oxaloacetic transaminase than TRQ, in spite of its stronger inhibition of lipid peroxidation in vivo. From these results, it is suggested that the membrane protecting action of TRQ is partially derived from its suppression of lipid peroxidation, but "another action" may also play an important role in protecting the fragile membrane.     

Histological detection of lipid peroxidation following infusion of tert-butyl hydroperoxide and ADP-iron complex in perfused rat livers

Lipid peroxidation was assessed histologically and biochemically in hemoglobin-free perfused rat livers using two different types of stimulators. The Schiff reaction of fuchsin with cellular aldehydes was used as a histological index for lipid peroxidation. t-Butyl hydroperoxide (BHP, 0.8 mM) infusion caused a rapid and sustained release of thiobarbituric acid reactive substances (TBARS) into the effluent perfusate for up to 60 min, which was accompanied by lactate dehydrogenase (LDH) leakage after 30 min. The Schiff positive foci were initially restricted to periportal zones and spread with time to whole areas, accompanied by periportal necrosis. Coinfusion of diphenyl-p-phenylenediamine suppressed the TBARS release, with negative fuchsin staining, but the LDH leakage was unaffected. Under retrograde perfusion, BHP produced pericentral staining and necrosis. With 2.5 mM ADP-100 microM Fe3+, little TBARS was released up to 60 min, even though the hepatic TBARS levels increased considerably by this time. By 90 min, marked TBARS release occurred, but LDH leakage remained low. Irrespective of the direction of perfusion, pericentral hepatocytes became Schiff positive after 30 min. The fuchsin staining method may be useful for detecting peroxidized zones of the liver lobules.