Increased urinary excretion of 1-methyl-2-pyridone-5-carboxamide in rats administered 2-acetylaminofluorene.

The onset of fat accumulation within CCl₄ poisoned hepatocytes, occurring as early as 1 h after treatment, is known to be provoked by a block in lipoprotein secretion. Lipoprotein secretion involves the function of the microtubular system. Several data indicate that this early block in lipoprotein secretion is not primarily the consequence of impaired protein synthesis. Therefore effects of some derivatives of lipid peroxidation, i.e. aldehydes and linoleic acid hydroperoxide were investigated.The results described in this paper shown that the above mentioned lipid peroxidation derivatives inhibit, with different activities, [³H]colchicine binding to liver high-speed supernates. Percentage binding inhibition is directly related to concentrations of aldehydes or LAHPO. LAHPO is more effective than aldehydes. Among the aldehydes tested, 4-hydroxypentenal, produced during lipid peroxidation of biological materials, was the most active.The presence of thiols, added to the incubation medium, partially protects against the inhibition of [³H]colchicine binding by aldehydes. This suggests that aldehydes act by reacting with -SH groups of tubulin. The possibility that interaction between lipoperoxidation derivatives and tubulin in vivo may contribute to the onset of fat infiltration in CCl₄ poisoning is discussed.     

Lipid peroxidation and covalent binding in the early functional impairment of liver Golgi apparatus by carbon tetrachloride

The onset of the lipoprotein secretory block provoked by CCl4 in the whole animal was monitored after purification of liver Golgi membranes. Both lipid transit through the apparatus and hexosylation of the lipoprotein are markedly inhibited 5-15 min after poisoning. Pre-treating the animal with alpha-tocopherol, shown to prevent lipid peroxidation without modifying the covalent binding due to CCl4 metabolites, affords little protection against lipid accumulation in the Golgi, but total preservation of galactosyl transferase activity. While haloalkylation therefore appears to be the major mechanism of damage in the early phases of CCl4-induced derangement of lipid secretion, lipid peroxidation is probably more involved later; this is indicated by the marked, though never complete, protection against fatty liver afforded at 24 h after CCl4 poisoning by supplementation of the membrane with alpha-tocopherol.     

Mechanism of carbon tetrachloride-induced hepatotoxicity. Hepatocellular damage by reactive carbon tetrachloride metabolites

CCl4-induced liver damage was modeled in monolayer cultures of rat primary hepatocytes with a focus on involvement of covalent binding of CCl4 metabolites to cell components and/or peroxidative damage as the cause of injury. (1) Covalent binding of 14C-labeled metabolites was detected in hepatocytes immediately after exposure to CCl4. (2) Low oxygen partial pressure increased the reductive metabolism of CCl4 and thus covalent binding. (3) [14C]-CCl4 was bound to lipids and to proteins throughout subcellular fractions. Binding occurred preferentially to triacylglycerols and phospholipids, with phosphatidylcholine containing the highest amount of label. (4) The lipid peroxidation potency of CCl4 revealed subtle differences compared to other peroxidative substances, viz., ADP-Fe3+ and cumol hydroperoxide, respectively. (5) CCl4, but not the other peroxidative substances, decreased the rate of triacylglycerol secretion as very low density lipoproteins. (6) The anti-oxidant vitamin E (alpha-tocopherol) blocked lipid peroxidation, but not covalent binding, and secretion of lipoproteins remained inhibited. (7) The radical scavenger piperonyl butoxide prevented CCl4-induced lipid peroxidation as well as covalent binding of CCl4 metabolites to cell components, and also restored lipoprotein metabolism. The results confirm that covalent binding of the CCl3* radical to cell components initiates the inhibition of lipoprotein secretion and thus steatosis, whereas reaction with oxygen, to form CCl3-OO*, initiates lipid peroxidation. The two processes are independent of each other, and the extent to which either process occurs depends on partial oxygen pressure. The former process may result in adduct formation and, ultimately, cancer initiation, whereas the latter results in loss of calcium homeostasis and, ultimately, apoptosis and cell death.     

Possible Protective Effect of Kolaviron on CCl4-Induced Erythrocyte Damage in Rats

The possible protective effect of kolaviron on rat erythrocytes followingsimultaneous administration of kolaviron (100 mg/kg of body weight/day) withcarbon tetrachloride CCl₄ (1.195 g/kg of body weight/day) by separateintraperitoneal injections was investigated.Kolaviron, a biflavonoid fraction of the defatted alcoholic extract ofGarcinia kola seed, inhibits the accumulation of lipid peroxidationproducts in erythrocytes. A significant reduction (p>0.05) by about34% of lipid peroxidation products was observed in erythrocytes of ratstreated simultaneously with CCl₄ and kolaviron when compared to CCl4-treatedrats. Similarly, the significant increase (p>0.05) in membranecholesterol observed in CCl₄-treated rats was significantly decreased(p>0.05) in rats treated simultaneously with CCl4 andkolaviron. Therefore, there was no significant difference (p>0.05) incholesterol–phospholipid ratio (C/P) of rats treated simultaneouslywith CCl₄ and kolaviron, and the controls. Thus, kolaviron normalizes theCCl₄-induced change in erythrocyte membrane composition.In addition, kolaviron antagonizes the effect of CCl₄ on the activity ofthe membrane bound enzyme, Ca²⁺-ATPase. These results suggest thatkolaviron protects erythrocyte membranes from free radical attack, on bothlipids and proteins.     

Evaluation of carbon tetrachloride-induced stress on rat hepatocytes by P NMR and MALDI-TOF mass spectrometry: lysophosphatidylcholine generation from unsaturated phosphatidylcholines

Carbon tetrachloride (CCl₄) represents an excellent model to study oxidative injury of cells. It is widely accepted that hepatocellular injury is a consequence of the metabolic conversion of CCl₄ into highly reactive, free radical intermediates. Among the direct toxic effects of CCl₄, stimulation of lipid peroxidation and the binding of the electrophilic radicals to membrane lipids have been suggested to play important roles in the pathogenesis of irreversible cell damage. CCl₄-induced liver damage was modeled in cultures of rat hepatocytes with the focus on alterations of phosphatidylcholine (PC). The PC acyl chain composition was analyzed by ³¹P NMR spectroscopy and MALDI-TOF mass spectrometry. The content of the membrane arachidonoyl PC was decreased by almost 30% after incubation of the cells with CCl₄. This relative decrease was found to correlate with increased concentrations of the corresponding saturated lysophosphatidylcholine (LPC). It is concluded that LPC represents a useful biomarker of CCl₄-mediated damaging of hepatocytes. It is also speculated that de novo biosynthesis of PC is influenced by CCl₄.     

16,16-Dimethyl PGE2 and fatty acids protect hepatocytes against CCl4-induced damage

Summary 16,16-Dimethyl PGE₂ (dmPGE₂) has previously been shown to protect the in vivo rat liver against CCl₄-induced damage. These studies were undertaken to determine if this protection could be demonstrated in vitro where factors of absorption, secretion, and blood flow are not present. Primary hepatocyte cultures were established by perfusing rat liver with collagenase. Hepatocytes were plated at a density of 2×10⁴ cells/cm, allowed 90 min to attach, then stabilized in L15 medium for 18 h. Hepatocytes were then challenged with CCl₄ with concomitant exposure to 10⁻⁹ to 10⁻⁵ M dmPGE₂, stearic acid, oleic acid, or ethanol vehicle (0.00001 to 0.1%). After 1 h, challenge was aspirated and cells were stained with 0.04% trypan blue to determine viability. Hepatocytes in the vehicle groups took up more trypan when exposed to CCl₄ than those treated with dmPGE₂, stearic acid, or oleic acid at concentrations of 10⁻⁹ to 10⁻⁷ M. At 0.1% ethanol vehicle protected as well as all other treatments. Protection against CCl₄ by dmPGE₂, stearic, and oleic acids as well as high concentrations of ethanol may occur by altering the metabolism of CCl₄.     

Pharmacological properties of dibenzo[a,c]cyclooctene derivatives isolated from Fructus Schizandrae chinensis III. Inhibitory effects on carbon tetrachloride-induced lipid peroxidation,metabolism and covalent binding of carbon tetrachloride to lipids

Fructus Schizandrae, a traditional Chinese tonic, has been shown to lower the elevated serum glutamic pyruvic transaminase (SGPT) levels of patients with chronic viral hepatitis and several of its components decrease the hepatotoxicity of carbon tetrachloride (CCl₄) in animals. This paper deals with the mechanism of protection against CCl₄-hepatotoxicity of these compounds as well as of DDB, a synthetic analogue of Schizandrin (Sin) C. Of the seven components, Sin B and C, Schizandrol (Sol) B, Schizandrer (Ser) A and B, as well as dimethyl-4,4′-dimethoxy-5,6,5′,6′-dimethylenedioxy-biphenyl-2,2′-dicarboxylate (DDB) were shown to inhibit CCl₄-induced lipid peroxidation and [¹⁴C]Cl₄ covalent binding to lipids of liver microsomes from phenobarbital(PB)-treated mice. The compounds also decreased carbon monoxide (CO) production and cofactor (NADPH, oxygen) utilization during CCl₄ metabolization by liver microsomes. It may be postulated, therefore, that the hepatoprotective effect of certain components isolated from Fructus Schizandrae as well as DDB is due to their inhibitory effect on CCl₄-induced lipid peroxidation and the binding of CCl₄-metabolites to lipids of liver microsomes.     

Podophyllum hexandrum aqueous extract as a potential free radical scavenger

Abstract

The present study was undertaken to evaluate the effect of the aqueous extract of Podophyllum hexandrum against free radical-mediated damage and also explore its anticancer activity. The extract exhibited significant activity in scavenging 1, 1-diphenyl-2-picryl-hydrazyl radicals, ^?OH radical-mediated DNA damage, and lipid peroxide production in rat liver microsomes. The extract was also tested for its reducing abilities. The activity of liver marker enzymes and antioxidant defense enzymes in rat liver homogenate was assessed in control and carbon tetrachloride (CCl₄)-treated animals. It was observed that CCl₄-induced changes viz., increases in the activities of aspartate aminotransferase, alanine aminotransferase, and lactate dehydrogenase, a decrease in reduced glutathione as well as decreases in the activities of superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase, and glutathione-S-transferase. All these parameters showed reversal when pretreated with aqueous extract of P. hexandrum. Podophylotoxin and etoposide are the two known anticancer agents derived from P. hexandrum and interestingly the aqueous extract of P. hexandrum showed a typical DNA ladder formation in HL-60 cells confirming its role as an inducer of apoptosis. The results obtained suggest that the plant extract exhibits inhibition of and free radical production and lipid peroxidation, increase in antioxidant enzyme activities, revealing its antioxidant properties, and is also able to show potent anticancer activity as depicted by its ability to cause fragmentation of DNA.     

Effect of mandur bhasma on lipolytic activities of liver,kidney and adipose tissue of albino rat during CCl4 induced hepatic injury

‘Mandur bhasma’, an ayurvedic preparation of iron is used in traditional medicine against hepatitis. In the present study the hepatoprotective property of this drug was tested in albino rats during CC1₄ induced hepatic injury. The effect of mandur bhasma on the activities of the lipolytic enzymes of rat liver, kidney and adipose tissue were studied during hepatitis induced by CCl₄. The activities of acid lipase, alkaline lipase, lipoprotein lipase and hormone sensitive lipase exhibited significant alterations during CCl₄ induced hepatic injury, indicating a role for these enzymes in the mobilization of fat from adipose tissue and accumulation of fat in liver and kidney. Simultaneous treatment with mandur bhasma prevented the paraffin mediated and CC1₄ mediated changes in the enzyme activities. These results suggest the hepatoprotective role of mandur bhasma during CCl₄ induced hepatic injury.     

Inhibition of poly(ADP-ribose) polymerase-1 attenuates the toxicity of carbon tetrachloride

Carbon tetrachloride (CCl₄) is routinely used as a model compound for eliciting centrilobular hepatotoxicity. It can be bioactivated to the trichloromethyl radical, which causes extensive lipid peroxidation and ultimately cell death by necrosis. Overactivation of poly(ADP-ribose) polymerase-1 (PARP-1) can rapidly reduce the levels of β-nicotinamide adenine dinucleotide and adenosine triphosphate and ultimately promote necrosis. The aim of this study was to determine whether inhibition of PARP-1 could decrease CCl₄-induced hepatotoxicity, as measured by degree of poly(ADP-ribosyl)ation, serum levels of lactate dehydrogenase (LDH), lipid peroxidation, and oxidative DNA damage. For this purpose, male ICR mice were administered intraperitoneally a hepatotoxic dose of CCl₄ with or without 6(5H)-phenanthridinone, a potent inhibitor of PARP-1. Animals treated with CCl₄ exhibited extensive poly(ADP-ribosyl)ation in centrilobular hepatocytes, elevated serum levels of LDH, and increased lipid peroxidation. In contrast, animals treated concomitantly with CCl₄ and 6(5H)-phenanthridinone showed significantly lower levels of poly(ADP-ribosyl)ation, serum LDH, and lipid peroxidation. No changes were observed in the levels of oxidative DNA damage regardless of treatment. These results demonstrated that the hepatotoxicity of CCl₄ is dependent on the overactivation of PARP-1 and that inhibition of this enzyme attenuates the hepatotoxicity of CCl₄.     

Free radical scavenging and hepatoprotective actions of Quercus aliena acorn extract against CCl4-induced liver

In the present study, we investigated the protective effect of Quercus aliena acorn extracts against CCl₄-induced hepatotoxicity in rats, and the mechanism underlying the protective effects. Aqueous extracts of Quercus aliena acorn had higher superoxide radical scavenging activity than other types of extracts. The Quercus aliena acorn extracts displayed dose-dependent superoxide radical scavenging activity (IC₅₀ = 4.92 μg/ml), as assayed by the electron spin resonance (ESR) spin-trapping technique. Pretreatment with Quercus aliena acorn extracts reduced the increase in serum aspartate aminotransferase (AST) and serum alanine aminotransferase (ALT) levels. The hepatoprotective action was confirmed by histological observation. The aqueous extracts reversed CCl₄-induced liver injury and had an antioxidant action in assays of FeCl₂- ascorbic acid induced lipid peroxidation in rats. Expression of cytochrome P450 2E1 (CYP2E1) mRNA, as measured by RT-PCR, was significantly decreased in the livers of Quercus aliena acorn-pretreated rats compared with the livers of the control group. These results suggest that the hepatoprotective effects of Quercus aliena acorn extract are related to its antioxidative activity and effect on the expression of CYP2E1.     

Lipoperoxidation after carbon tetrachloride poisoning in rats previously treated with antioxidants

Propyl gallate (PG), reduced glutathione (GSH) and N,N′-diphenyl-p-phenylenediamine (DPPD), administered to rats prior to carbon tetrachloride, protect against hepatic fat infiltration until the fourth hour after poisoning. This effect does not seem to be mediated by a block in lipid mobilization from depot fat.A preliminary treatment with DPPD succeeds in inhibiting the double bond shifting in liver microsomal lipids within 30 min after dosing with CCl₄. The early peroxidative alteration occurs at the normal rate after the administration of either GSH or PG. The amount of lipid-bound radiocarbon and of ¹⁴CO₂ exhaled within 2 h after intragastric ¹⁴C-labelled carbon tetrachloride is not affected by the preliminary protection with the antioxidants.CCl₄ metabolites and/or lipoperoxides impair the in vitro combination of serum apoprotein with lipid. No changes are observed when lipoperoxidation is inhibited by antioxidants.These findings are interpreted as a support for the hypothesis that the possible contribution given by the enhancement of lipid peroxidation to the pathogenesis of CCl₄-induced fatty liver could depend on further structural and functional alterations occurring in the cytoplasmic environment of hepatocytes rather than the early radical attack onto the unsaturated lipids of liver microsomes. The functional integrity and the supply of the protein carrier for the triglyceride secretion mechanisms could be considered a target of the hepatotoxic action of CCl₄, at the molecular level.     

Protective Effect of Cornuside against Carbon Tetrachloride-Induced Acute Hepatic Injury

This study elucidated the effects of cornuside on carbon tetrachloride (CCl₄)-induced hepatotoxicity. Rats were treated intraperitoneally with 0.5 mL/kg of CCl₄. Sixteen h after CCl₄ treatment, the levels of serum aminotransferases, tumor necrosis factor-α (TNF-α), and lipid peroxidation were significantly elevated, whereas the hepatic antioxidative enzyme activities were decreased. These changes were attenuated by cornuside. Histological studies also indicated that cornuside inhibited CCl₄-induced liver damage. Furthermore, the contents of hepatic nitrite, inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2) were elevated after CCl₄ treatment, while cytochrome P450 2E1 (CYP2E1) expression was suppressed. Cornuside treatment inhibited the formation of liver nitrite, and reduced the overexpression of iNOS and COX-2 proteins, but restored the liver CYP2E1 content as compared with the CCl₄-treated rats. Our data indicate that cornuside protects the liver from CCl₄-induced acute hepatotoxicity, perhaps due to its ability to restore the CYP2E1 function and suppress inflammatory responses, in combination with its capacity to reduce oxidative stress.     

Mechanism of in vivo carbon tetrachloride-induced liver microsomal cytochrome P-450 destruction.

Prior administration of aminotriazole (3-amino-1,2,4-triazole) or pyrazole to rats resulted in a significant prevention of the CCl₄-induced decrease in the liver microsomal P-450 content. In A/J mice the CCl₄ activation and P-450 destruction occurred in absolute absence of lipid peroxidation as determined by uv absorption. The data suggest that P-450 destruction is mainly mediated by direct attack of CCl₄ metabolites rather than by CCl₄-induced lipid peroxidation.     

Effects of Schisandrin B and α-tocopherol on lipid peroxidation, in vitro and in vivo

Effects of Schisandrin B (Sch B) and -tocopherol (-TOC) on ferric chloride (Fe³⁺) induced oxidation of erythrocyte membrane lipids in vitro and carbon tetrachloride (CCl₄) induced lipid peroxidation in vivo were examined. While -TOC could produce prooxidant and antioxidant effect on Fe³⁺-induced lipid peroxidation, Sch B only inhibited the peroxidation reaction. Pretreatment with -TOC (3 mmol/kg/day × 3) did not protect against CCl₄-induced lipid peroxidation and hepatocellular damage in mice, whereas Sch B pretreatment (0.3 mmol/3.0 mmol/kg/day × 3) produced a dose-dependent protective effect on the CCl₄-induced hepatotoxicity. The ensemble of results suggests that the ability of Sch B to inhibit lipid peroxidation, while in the absence of pro-oxidant activity, may at least in part contribute to its hepatoprotective action.Abbreviations ALT alanine aminotransferase - CCl₄ carbon tetrachloride - Fe³⁺ ferric chloride - MDA malondialdehyde - Sch B Schisandrin B - TBA 2-thiobarbituric acid - TBARS thiobarbituric acid reactive substances - -TOC dl--tocopherol     

Carbon tetrachloride activation in liver microsomes from rats induced with 3-methylcholantrene

The irreversible binding of¹⁴C from¹⁴CCl₄ to microsomal lipids is decreased in animals treated with 3-methylcholantrene (3-MC), while it is increased in animals induced with phenobarbital (PB). CCl₄-induced lipid peroxidation in 3-MC treated rats is as intense as in controls. Destruction of glucose 6-phosphatase (G6P-ase) by CCl₄ is smaller in 3-MC treated rats than in controls. Destruction of total cytochrome P-450 (P-450 + P₁-450) by CCl₄ is smaller in 3-MC treated than in PB treated rats but similar to that obtained in controls. Results would indicate that P-450 would participate in CCl₄ activation much more effectively than P₁-450.     

Inhibitory Effects of Ebselen on Lipid Peroxidation in Rat Liver Microsomes

The effects of ebselen(2-pheny1-1,2-benzoisoselenazol-3(2H)-one), a synthetic seleno-organic compound with glutathione peroxidase-like activity were investigated on lipid peroxidation in rat liver microsomes. Ebselen inhibited malondialdehyde production coupled to the lipid peroxidation stimulated by either ADP-iron-ascorbate or CC1₄. The inhibitory activity of ebselen on each system was strongly increased by a 5-min preincubation with liver microsomes; the IC₅₀ values against ADP-Fe-ascorbate-stimulated and CC1₄-stimulated lipid peroxidation were 1.6/jM and 70 μM respectively. Ebselen also inhibited the endogenous lipid peroxidation with a NADPH-generating system, but it slightly stimulated the endogenous activity of ADP-Fe-ascorbate-stimulated lipid peroxidation (without a NADPH-generating system). Furthermore, ebselen inhibited oxygen uptake coupled to the lipid peroxidation by ADP-Fe-ascorbate and NADPH-ADP-iron; the IC₅₀ values were 2.5μM AND 20.3 μM respectively. Ebselen also prolonged the lag-time of onset of ADP-Fe-ascorbate-stimulated lipid peroxidation significantly, but not that observed with NADPH-ADP-Fe-stimulated lipid peroxidation.     

Effects of α-Tocopherol on Carbon Tetrachloride Metabolism in Rat Liver Microsomes

α-tocopherol is the major lipid-soluble radical-scavenging antioxidant in rat liver. It has long been used as a putative protective agent in CC1₄ induced liver injury but with variable results. We have used a-tocopherol loaded rat liver microsomes to study the effect of this vitamin on CC1₄ metabolism in vitro. As expected, a-tocopherol inhibits CC1₄-dependent microsomal lipid peroxidation and, at a very high concentration, will inhibit the covalent binding of CC1,- to microsomal protein by up to 50%. No inhibitory effect was observed towards CC1₃ production as measured by the electron spin resonance technique of spin-trapping but this apparent discrepancy may represent a limitation of the technique. The high levels required to inhibit covalent binding probably preclude the likelihood of a-tocopherol significantly affecting that phenomenon at endogenous concentrations but may be relevant to other experiments employing high doses of a-tocopherol as an experimental hepatoprotective agent.     

Inhibition of hepatic mitochondrial aldehyde dehydrogenase by carbon tetrachloride through JNK-mediated phosphorylation

The aim of this study was to investigate the mechanism of inhibition of mitochondrial aldehyde dehydrogenase (ALDH2) by carbon tetrachloride (CCl₄). CCl₄ administration caused marked hepatocyte ballooning and necrosis in the pericentral region. CCl₄ also inhibited hepatic ALDH2 activity in a time-dependent manner without altering the protein level, suggesting ALDH2 inhibition through covalent modifications such as phosphorylation by JNK. To demonstrate phosphorylation, the isoelectric point (pI) of ALDH2 in CCl₄-exposed rats was compared to that of untreated controls. Immunoblot analysis revealed that immunoreactive ALDH2 bands in CCl₄-exposed rats were shifted to acidic pI ranges on two-dimensional electrophoresis (2-DE) gels. Incubation with alkaline phosphatase significantly restored the suppressed ALDH2 activity with a concurrent alkaline pI shift of the ALDH2 spots. Both JNK and activated JNK were translocated to mitochondria after CCl₄ exposure. In addition, incubation with catalytically active JNK led to significant inhibition of ALDH2 activity, with an acidic pI shift on 2-DE gels. Furthermore, immunoprecipitation followed by immunoblot analysis with anti-phospho-Ser–Pro antibody revealed phosphorylation of a Ser residue(s) of ALDH2. These results collectively indicate a novel underlying mechanism by which CCl₄ exposure activates JNK, which translocates to mitochondria and phosphorylates ALDH2, contributing to inhibition of ALDH2 activity accompanied by decreased cellular defense capacity and increased lipid peroxidation.