Studies on the mechanism of the protective action of 16,16-dimethyl PGE2 in carbon tetrachloride induced acute hepatic injury in the rat

We have previously demonstrated the partial protection of the rat liver by 16,16-dmPGE₂ (DMPG) against a number of hepatotoxins including carbon tetrachloride (CCl₄). However, it has not been determined whether hepatoprotection by DMPG represents a true “cytoprotective” action or if merely accomplished through inhibition of CCl₄ metabolism to reactive, toxic trichoromethyl (CCl₃·) free radicals. This report details a series of experiments in which the effects of DMPG on CCl₄ metabolism was evaluated in the rat.These data indicate that pretreatment with DMPG may reduce the hepatic concentration of the toxic CCl₃· free radicals in CCl₄ poisoned rats. Evidence is presented which suggests that this reduction in binding may have been due to a decrease in the rate of CCl₄ metabolism. However, DMPG did not affect the hepatic concentration of total microsomal cytochrome P450, the necessary enzyme in this metabolic process. On the other hand, free radical spin trapping experiments indicate that the rate of free radical formation from CCl₄ was slowed by treatment. Also, indirect evidence suggests that the metabolism of another cytochrome P450 substrate, phenobarbital, was slowed in DMPG treated rats. We conclude that the rate of CCl₄ metabolism may be reduced by pretreatment with DMPG. Furthermore, some measure of hepatic protection might be expected to occur as a result of the reduction in the rate of CCl₄ metabolism. However, we are unable to determine if this action was solely responsible for the observed hepatic protection.     

Single-flask synthesis of (C5H5)W(CO)3Cl from W(CO)6

(C₅H₅)W(CO)₃Cl has been prepared in >90% yield from W(CO)₆, cyclopentadiene, and CCl₄ in a single-flask synthesis involving the intermediates W(CO)₃(CH₃CN)₃ and (C₅H₅)W(CO)₃H.     

Factors Influencing the Formation of the Carbon Dioxide Radical Anion (CO2−) Spin Adduct of Pbn in the Rat Liver Metabolism of Halocarbons

Spin trapping techniques have been used to detect free radicals generated from the in vitro metabolism by rat liver microsomes of carbon tetrachloride (CCl₄) and bromotrichloromethane (BrCCI) under conditions of varying oxygen tension and pH. Dispersions of rat liver microsomes incubated with ¹²CCl₄, ¹³CCl₄ or Br¹²CCl₃, α-phenyl-tert-butyl nitrone (PBN) and NADPH/NADH in a phosphate buffer varying in pH from 6.6 to 8.0 under varying oxygen tensions produced various amounts of four different PBN adducts: PBN-CCl₃, PBN-L, PBN-OL and PBN-CO^?₂ where L is a carbon-centered lipid type radical and LO is an oxygen-centered lipid type radical. The relative amount of PEN-CO; increases with the absence of oxygen. With the use of ³¹P-NMR in vivo spectroscopy it was possible to detect a pH change from 7.4 to 6.8 in the livers of rats treated with CCl₄, or BrCCl₃. These results suggest that halocarbon metabolism in biological systems may depend on both oxygen tension as well as pH.     

Stimulation of arachidonic acid metabolism by CCl4 in isolated rat hepatocytes

Arachidonic acid metabolism was evaluated in isolated rat hepatocytes after CCl₄ exposure. CCl₄ induced dose-dependently the synthesis and release of prostacyclin (PGI₂) and thromboxane (TXB₂). Treatment with prostaglandin E₂ (PGE₂) 30 min after exposure to CCl₄, significantly reduced the cell damage as well as the release of TXB₂ from the cells.     

Isolation, characterization and antioxidative effect of phyllanthin against CCl4-induced toxicity in HepG2 cell line

The present study was an attempt to investigate the hepatoprotective and antioxidative property of Phyllanthus amarus (P. amarus) extract and phyllanthin. Phyllanthin, one of the active lignin present in this plant species was isolated from the aerial parts, by silica gel column chromatography employing gradient elution with hexane-ethyl acetate solvent mixture. It was obtained in high yields (1.23%), compared to reported procedures and the purity was ascertained by HPTLC and reversed-phase HPLC analysis. Characterization of phyllanthin was done by mp, UV-Visible spectrophotometry, elemental analysis, FT-IR, ¹H NMR, ¹³C NMR and mass spectral analysis. Free radical scavenging activity of P. amarus extract and phyllanthin was also examined using DPPH assay. The protective effect of P. amarus extract and phyllanthin was studied on CCl₄-induced toxicity in human hepatoma HepG2 cell line. The results indicated that CCl₄ treatment caused a significant decrease in cell viability. In addition, the toxin treatment initiated lipid peroxidation (LPO), caused leakage of enzymes like alanine transaminase (ALT) and lactate dehydrogenase (LDH) with a significant decrease in glutathione (GSH) levels. It was observed that phyllanthin effectively alleviated the changes induced by CCl₄ in a concentration-dependent manner, with much smaller strengths as compared to P. amarus extract.     

Spin-trapping of the trichloromethyl radical produced during enzymic NADPH oxidation in the presence of carbon tetrachloride or bromotrichloromethane

Utilizing the spin-trapping agent phenyl-t-butyl nitrone, a free radical has been detected which is produced from carbon tetrachloride or bromotrichloromethane during the enzymic oxidation of NADPH by rat liver microsomes. The presence of NADPH is obligatory for generation of the radical.The formation of the trichloromethyl radical-phenyl-t-butyl nitrone adduct is an enzymic process, as evidenced by the inhibition of its formation in systems containing heated microsomes and in systems containing p-hydroxymercuribenzoate. A computer-simulated ESR spectrum for the trichloromethyl adduct of phenyl-t-butyl nitrone can reproduce the essential features of the spectrum of the spin-trapped radical produced enzymically from CCl₄. A mechanism is proposed for the formation of the trichloromethyl radical from CCl₄ or BrCCl₃.     

Reaction of the trichloromethyl and halothane-derived peroxy radicals with unsaturated fatty acids: A pulse radiolysis study

The absolute rates of reaction of the trichloromethylperoxy radical, CCl₃OO, derived from carbon tetrachloride and the halothane peroxy radical, CF₃CHClOO, with oleic, linoleic, linolenic and arachidonic acids have been determined using the fast reaction technique of pulse radiolysis. In general, the rates of reaction of the radical derived from carbon tetrachloride are approximately five times greater than those for the halothane related radical. In both cases the rate constant increases with increasing unsaturation of the fatty acid in agreement with the known greater susceptibility of polyunsaturated fatty acids to peroxidative decomposition.     

Luminescent CeCl3 nanoparticles by Tris(1,1,1,5,5,5-hexafluoro-2,4-pentanedionato)cerium diglyme photolysis in chlorinated solvents

Irradiation of [Ce(hfac)₃(diglyme)] (hfac = 1,1,1,5,5,5-hexafluoro-2,4-pentanedionato and diglyme (DG) = 2,5,8,11,14-pentaoxapentadecane) in chlorinated solvents (CH₂Cl₂, CCl₄) with UV light led to luminescent colloidal CeCl₃ that was characterized by transmission electron microscopy (TEM) analysis. When a substrate, quartz or silicon was present in the reaction cell, photoluminescent films were obtained, containing either pure CeCl₃ or mixtures of CeCl₃, CeF₃ and CeOx in function of the experimental parameters of irradiation. Nanostructured and luminescent pure CeCl₃ films were obtained by irradiation of the cerium complex in CCl₄ at high intensity light for a few minutes. The films were characterized by X-ray diffraction (XRD), Energy dispersive X-ray (EDX), X-ray photoelectron spectroscopy (XPS), TEM, scanning electron microscopy (SEM) and atomic force microscopy (AFM). The kinetics of the [Ce(hfac)₃(diglyme)] solution photodegradation, followed by UV spectrophotometry and spectrofluorimetry, pointed to CeCl₃ formation by a solvent-initiated reaction, whereas the other inorganic compounds were the products of side reactions.     

Antioxidant and hepatoprotective activities of low molecular weight sulfated polysaccharide from Laminaria japonica

A low molecular weight sulfated polysaccharide (LMWF) was prepared from Laminaria japonica by mild acid hydrolysis. The antioxidant activity of LMWF in vitro was studied using three kinds of oxygen free radical systems. LMWF had effective scavenging abilities on superoxide radical, hydroxyl radical and hypochlorous acid directly in vitro. The hepatoprotective effect of LMWF was studied using two acute liver injury mice models induced by carbon tetrachloride (CCl₄) and D-galactosamine (D-GalN). Addition of LMWF significantly lowered the content of serum malonaldehyde and markedly increased the activities of superoxide dismutase and glutathione peroxidase, compared with the model groups in both kinds of liver injury mice. Moreover, administration of LMWF significantly inhibited the elevation of glutamate pyruvate transaminase induced by CCl₄ and D-GalN in mice. The results suggest that the antioxidant activity of LMWF plays an important role in its hepatoprotective effect in the liver injury mice induced by CCl₄ and D-GalN.     

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.     

Direct Observation of Spin-Trapped Carbon Dioxide Radicals in Hepatocytes Exposed to Carbon Tetrachloride

Carbon dioxide radical adducts of the spin trapping agent, α-phenyl N-t-butyl nitrone (PBN), have been observed to occur in the urine and bile of rats exposed to carbon tetrachloride as well as in perfusates of liver in which the perfusion medium contained carbon tetrachloride (Connor er al., J. Biol. Chem., 261, 4542, (1986)). The carbon dioxide adduct was proven to be derived from CCI, by use of 13-C-labelled compound. These adducts were not observed in the liver itself suggesting that they might be rapidly secreted from the liver. However, using isolated hepatocytes, we have demonstrated that the carbon dioxide radical adduct can be observed directly in the liver cells as it is formed. Since this water-soluble adduct cannot be extracted by non-aqueous solvents such as chloroform or toluene, its formation in liver in vivo or in perfused livers was not detected. Lowering the oxygen tension in the system diminished the intensity of production of the carbon dioxide adduct, consistent with the adduct being produced as a result of ·OOCCl₃ generation. It is not clear the extent to which this adduct is formed as a result of the ·CO₂ radical or is produced by metabolic oxidation of the trichloromethyl radical adduct of PBN per se to the carbon dioxide radical adduct. The intensity of the signal of the carbon dioxide radical adduct suggests that adduct conversion may be the route of formation since it seems unlikely that a sufficient amount of the halocarbon could be metabolized to ·COCl or ·CO₂ radicals to generate a signal of the magnitude involved. The ·CO₂ adduct is readily observed in intact hepatocytes, but the ·CCl₃ adduct is not (although we know the ·CCl₃ adduct has been produced in these cells), indicating that the ·CO₂ adduct is present in considerable abundance compared to the ·CCl₃ adduct.     

Confirmation of assignment of the trichloromethyl radical spin adduct detected by spin trapping during 13C-carbon tetrachloride metabolism in vitro and in vivo

Rat liver microsomal incubation systems containing the free radical spin trap, phenyl-t-butyl nitrone, as well as an NADPH generating system and [¹³C]CCl₄ (90 atom % ¹³C) produce electron spin resonance spectra consistent with that expected for a trichloromethyl-phenyl-t-butyl nitrone adduct. This same spectrum is observed in a lipid extract of the liver from a rat orally administered [¹³C]CCl₄ as well as in a solution of phenyl-t-butyl nitrone and [¹³C]CCl₄ irradiated with ultraviolet light.     

Synthesis of bent titanocene metalloligands with the (diphenylphosphino)tetramethylcyclopentadienyl moiety. X-ray structure of [(η-C5Me4 2)2TiCl2]Mo(CO)4

The reactions of lithium(diphenylphosphino)tetramethylcyclopentadienide with CpTiCl₃ and secondly with TiCl₃ followed by CCl₄ oxidation lead to the formation of two titanocene phosphines: (η⁵-C₅H₅)[η⁵-C₅Me₄P(C₆H₅)₂]TiCl₂ (2) and [η⁵-C₅Me₄P(C₆H₅)₂]₂TiCl₂ (3), respectively. The metalloligand 3 reacts readily with Mo(CO)₄cod, Mo(CO)₅THF and Mo(CO)₆ to give in each case [(η⁵-C₅Me₄ o(CO)₄ (6) as a sole product. The structure of 6 has been determined by X-ray diffraction. Crystal data: P , a = 11.716(1), b = 11.753(2), c = 16.110(2) Å, α = 99.06(1), β = 92.61(1), γ = 104.20(1)°, Z = 2. The molybdenum-titanium distance of 5.194(1) Å rules out any metal-metal interaction. The chlorine substitution reactions by CO in 2 and 3 and by thiolate group (pH₃C-C₆H₄-S) in 16 are reported.     

Synthesis and crystal structure of bis(tetramethylammonium) aquotetra-fluorodioxouranate(VI) dihydrate, [(CI3)4 N]2[UO2 F4(H2 O)]·2H2O

The title compound has been synthesized and subjected to crystal structure analysis. M_r = 548.50, m.p. 108.1 °C (decom.), orthorhombic, Im2m,a = 7.006(2), b = 8.938(2), c = 13.619(2) Å V = 852.8(3) ų, Z = 2, D_x = 2.136, D_m, (flotation in CCl₄/CH₂I₂) = 2.128 g cm^?3, λ(Mo-Kα) = 0.71069 Å, μ = 90.79 cm^?1, F(000) = 519.89, T = 295 K, final R_F = 0.036 and R_G = 0.044 for 566 observed reflections. The discrete [UO₂F₄(H₂0)]^2? anion has site symmetry m2m, its virtually linear uranyl moiety being surrounded by fluoro and aquo ligands occupying the vertices of a pentagon in the equatorial plane. Watet molecules serve to link the complex anions by hydrogen bonds into layers, between which the organic cations are accommodated.     

Lisosan G, a powder of grain, does not interfer with the drug metabolizing enzymes and has a protective role on carbon tetrachloride-induced hepatotoxicity

Lisosan G is a powder of grain registered as an alimentary integrator. The treatment of rats for 4 days with 0.5 g Lisosan G/kg had no effect on various drug metabolizing enzymes. Experiments in vitro showed that Lisosan G had radical scavenger activity. A confirmation of the antioxidative property of Lisosan G was also confirmed when it was administered in vivo to carbon tetrachloride (CCl₄)-intoxicated rats. The toxicity caused by CCl₄-treatment of rats was restored to the control levels when the rats were given Lisosan G for 4 days before CCl₄. Lisosan G thus does not interfer with drug metabolizing system but has antioxidant properties and protects against CCl₄-induced hepatotoxicity.     

Hepatoprotective potential of new steroid against carbon tetrachloride-induced hepatic injury

The present study was designed to evaluate the hepatoprotective effects of newly isolated stigmast-4, 20 (21), 23-trien-3-one (STO) against carbon tetrachloride-induced hepatic injury in Wistar albino rats. Hepatotoxicity was induced by the administration of a single intraperitoneal dose of CCl₄ (0.5 mL/kg CCl₄ in olive oil) in experimental rats. Three different doses (2.5, 5.0, and 10 mg/kg, p.o) of STO was administered to the test groups during whole experimental protocol. Changes in the activity of serum ALT, AST, ALP, TB, and TP, anti-oxidant enzymes like SOD, CAT, GPx, GST, and LPO were studied in CCl₄-induced hepatocellular carcinogenesis. The altered levels of serum ALT, AST, ALP, TB, and TP restored toward normalization significantly by STO in a dose dependant manner. The biochemical observations were supplemented with histopathological examination of rat liver sections. Meanwhile, it also produced a significant and dose-dependent reversal of CCl₄-diminished activity of anti-oxidant enzymes like SOD, CAT, GPx, GST, and the reduced CCl₄-elevated level of LPO. STO significantly prevented the increased levels of serum markers, also suppressed the free radical processes by scavenging hydroxyl radicals. It also modulates the levels of LPO and markedly increases the endogenous anti-oxidant enzymes level in CCl₄-induced hepatic injury.     

Prevention of Carbon Tetrachloride-Induced Lipid Peroxidation in Liver Microsomes from Dehydroepiandrosterone-Pretreated Rats

Dehydroepiandrosterone (DHEA), a lipid soluble steroid, administered to rats (100 mg/kg b.wt) by a single intraperitoneal injection, increases to twice its normal level in the liver microsomes. Microsomes so enriched become resistant to lipid peroxidation induced by incubation with carbon tetrachloride in the presence of a NADPH-regenerating system: also the lipid peroxidation-dependent inactivation of glucose-6-phosphatase and gamma-glutamyl transpetidase due to the haloalkane are prevented. Noteworthy, the liver microsomal drug-metabolizing enzymes and in particular the catalytic activity of cytochrome P₄₅₀IIE1, responsible for the CCl₄-activation, are not impaired by the supplementation with the steroid. Consistently, in DHEA-pretreated microsomes the protein covalent binding of the trichloromethyl radical (CCl₃°), is similar to that of not supplemented microsomes treated with CCl₄. It thus seems likely that DHEA protects liver microsomes from oxidative damage induced by carbon tetrachloride through its own antioxidant properties rather than inhibiting the metabolism of the toxin.     

锌7与镉7－金属硫蛋白清除羟自由基的比较

分离及纯化兔肝金属硫蛋白.制备去金属金属硫蛋白、锌₇与镉₇金属硫蛋白．在不同pH条件下,比较后二者清除羟自由基能力;在pH6条件下,比较锌₇－金属硫蛋白与有关蛋白和无机锌盐清除羟自由基效果．结论是在近生理pH条件下锌₇－金属硫蛋白清除羟自由基能力远强于镉₇－金属硫蛋白．金属硫蛋白清除羟自由基的能力主要来源于蛋白中处于还原态的流基．     

Transformation of 1,1,1-trichloroethane in an anaerobic packed-bed reactor at various concentrations of 1,1,1-trichloroethane, acetate and sulfate

Biotransformation of 1,1,1-trichloroethane (CH₃CCl₃) was observed in an anaerobic packed-bed reactor under conditions of both sulfate reduction and methanogenesis. Acetate (1 mM) served as an electron donor. CH₃CCl₃ was completely converted up to the highest investigated concentration of 10 μM. 1,1-Dichloroethane and chloroethane were found to be the main transformation products. A fraction of the CH₃CCl₃ was completely dechlorinated via an unknown pathway. The rate of transformation and the transformation products formed depended on the concentrations of CH₃CCl₃, acetate and sulfate. With an increase in sulfate and CH₃CCl₃ concentrations and a decrease in acetate concentration, the degree of CH₃CCl₃ dechlorination decreased. Both packed-bed reactor studies and batch experiments with bromoethanesulfonic acid, an inhibitor of methanogenesis, demonstrated the involvement of methanogens in CH₃CCl₃ transformation. Batch experiments with molybdate showed that sulfate-reducing bacteria in the packed-bed reactor were also able to transform CH₃CCl₃. However, packed-bed reactor experiments indicated that sulfate reducers only had a minor contribution to the overall transformation in the packed-bed reactor. Received: 22 January 1997 / Received revision: 12 May 1997 / Accepted: 19 May 1997