Two Mechanisms of Cc14-Induced Fatty Liver: Lipid Peroxidation Or Covalent Binding Studied in Cultured Rat Hepatocytes

With cultured hepatocytes it was studied whether CCl₄-induced inhibition of secretion of VLDL and HDL from liver cells is a consequence of covalent binding of CC1₄ metabolites (i.e. CO,; CC1,00) to cell constituents or of membrane damage by lipid peroxidation. Comparing the kinetics of inhibition of lipoprotein secretion with that of CCl₄-bioactivation it was found, that covalent binding of (^HC)-CC1₄ occurred at early time points (5 min) after CC1₄ administration and inhibited the lipoprotein secretion. At 100μM CC1₄ it was depressed by 53% within 60min. Incubations of CC1₄-treated cells with increasing concentrations of vitamin E blocked lipid peroxidation, but lipoprotein secretion was still inhibited. Piperonyl butoxid, a radical scavenger, protected against CCl₄-induced inhibition of lipoprotein section, lipid peroxidation and covalent binding.

These results show that during the early phases of CC1₄ poisoning fat accumulation is the consequence of covalent binding of CC1₄ metabolities to cell structures.     

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.     

Effects of some aldehydes on brain microtubular protein

4-Hydroxynonenal is one of the main breakdown products of lipid peroxidation. It has an antiproliferative effect, which may partly be the consequence of an interaction with cytoskeletal structures. Its effects on microtubular protein are compared with those of homologous aldehydes with the same number of carbon atoms, and with that of benzaldehyde. Unlike the other aliphatic aldehydes, this latter aldehyde does not impair microtubular functions at every concentration in the range. Nonanal has the greatest effect on tubulin polymerization, whereas it only slightly impairs colchicine binding activity. 2-Nonenal and 4-hydroxynonenal have less inhibiting effect on tubulin polymerization; their effect on colchicine binding activity is dose-dependent. The targets of 4-hydroxynonenal on tubulin are -SH groups; the action mechanism of other aldehydes has not yet been identified.     

Aldehyde-induced modifications of the microtubular system in 3T3 fibroblasts.

The molecular structure of aldehydes is closely related to their antimicrotubular effect. Morphological modifications of the microtubular system in living cells after incubation with certain aldehydes are consistent with biochemical alterations detected in previous research. The microtubular arrangement was visualized by an immunofluorescence technique with antitubulin antibodies, while the content of tubulin in the cells was evaluated by a colchicine binding assay. 2-Nonenal behaved similarly to 4-hydroxynonenal, a lipid peroxidation product, disorganizing microtubular network in 3T3 fibroblasts and decreasing the amounts of tubulin able to bind labelled colchicine. Nonanal did not significantly impair the tubulin characteristics in the cells, despite the fact that it has been shown to be active on the purified microtubular system; benzaldehyde was ineffective. This would appear to explain the mechanisms of interaction of aliphatic aldehydes which might be suitable for use as antimicrotubular drugs.     

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.     

Role of B-ring of colchicine in its binding to Zn(II)-induced tubulin-sheets

Colchicine-tubulin dimer comPlex, a Potent inhibitor of normal microtubule assembly undergoes extensive self-assembly in the Presence of 1 X 10^-4 M zinc sulPhate. Polymers assembled from colchicine-tubulin dimer comPlexes are sensitive to cold. Although colchicine can be accomodated within the Polymeric structure, the drug cannot bind to tubulin subunits in the intact Polymers. This is evidenced by the fact that (a) the colchicine binding activity of tubulin is lost when allowed to Polymerize with zinc sulPhate, (b) the loss in colchicine binding could be Prevented by Preincubation of tubulin with 1 X 10^-3 M CaCl₂ or 1 X 10^-5 M vinblastine sulPhate and finally (c) no loss in colchicine binding activity is found when tubulin is kePt at a concentration far below the critical concentration for Polymerization. Unlike colchicine, its B-ring analogues desacetamido colchicine (devoid of the B-ring subtituent) and 2-methoxy-5-(2′, 3′, 4′-trimethoxyPhenyl) troPone (devoid of the B-ring) can bind to tubulin subunits in the intact Polymers. Thus we conclude that the colchicine binding domain on the tubulin molecule is mostly (if not comPletely) exPosed in the Zn(II) -induced Polymers and the B-ring substituent Plays a major role in determining the binding ability of a colchicine analogue to tubulin in the intact Zn(II) -induced sheets.     

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.     

Differences in the carbon tetrachloride-induced damage to components of the smooth and rough endoplasmic reticulum from rat liver

There is a higher activity of ethyl morphine N-demethylase (EM-ase) and cytochrome P-450 (P-450) reductase as well as higher P-450 content in the smooth endoplasmic reticulum (SER) than in the rough endoplasmic reticulum (RER). The extent of the irreversible binding of the¹⁴C from¹⁴CCl₄ to lipids and proteins, as well as the CCl₄-induced destruction of P-450 is more intense in SER than in RER while the opposite was found for glucose 6-phosphatase (G6P-ase) destruction. CCl₄-induced lipid peroxidation is as intense in SER as is in RER.¹⁴C from¹⁴CCl₄ gets irreversibly bound to ribosomal proteins.     

Effect of dietary antioxidants and phenobarbital pretreatment on microsomal lipid peroxidation and activation by carbon tetrachloride.

The effect of the dietary antioxidants, vitamin E and selenium, and the effect of phenobarbital pretreatment on $\text{in}$$\text{vitro}$ NADPH-dependent microsomal lipid peroxidation and the activation of microsomal lipid peroxidation by CCl₄ were studied. The rate of microsomal lipid peroxidation decreased as a function of dietary anti-oxidant, while the degree of CCl₄ activation increased. Phenobarbital pretreatment diminished the antioxidant inhibition of microsomal lipid peroxidation found with microsomes from rats fed high levels of antioxidant. Phenobarbital pretreatment lowered the extent of lipid peroxidation as measured by malonaldehyde production but had little effect on the rate of lipid peroxidation as measured by oxygen uptake. The kinetics of lipid peroxidation and the stoichiometry of the reaction were assessed as a function of dietary antioxidant.The findings suggest that at low microsomal antioxidant concentrations, the lipid peroxidation reaction occurs at a maximal rate dependent upon some rate-limiting step, such as the reduction of Fe⁺³, which is unaffected by CCl₄ addition. Conversely, at high microsomal antioxidant concentrations, the antioxidant termination reactions appear to determine the overall reaction rate.     

Synthesis,biological evaluation and molecular docking studies of aminochalcone derivatives as potential anticancer agents by targeting tubulin colchicine binding site

A series of aminochalcone derivatives have been synthesized, characterized by HRMS, ¹H NMR and ¹³C NMR and evaluated for their antiproliferative activity against HepG2 and HCT116 human cancer cell lines. The most of new synthesized compounds displayed moderate to potent antiproliferative activity against test cancer cell lines. Among the derivatives, compound 4 displayed potent inhibitory activity with IC₅₀ values ranged from 0.018 to 5.33 μM against all tested cancer cell lines including drug resistant HCT-8/T. Furthermore, this compound showed low cytotoxicity on normal human cell lines (LO2). The in vitro tubulin polymerization assay showed that compound 4 inhibited tubulin assembly in a concentration-dependent manner with IC₅₀ value of 7.1 μM, when compared to standard colchicine (IC₅₀ = 9.0 μM). Further biological evaluations revealed that compound 4 was able to arrest the cell cycle in G2/M phase. Molecular docking study demonstrated the interaction of compound 4 at the colchicine binding site of tubulin. All the results indicated that compound 4 is a promising inhibitor of tubulin polymerization for the treatment of cancer.     

Synthesis of 2-anilinopyridine dimers as microtubule targeting and apoptosis inducing agents

A series of 2-anilinopyridine dimers have been synthesized and evaluated for their anticancer potential. Most of the compounds have showed significant growth inhibition of the cell lines tested and compound 4d was most effective amongst the series displaying a GI₅₀ of 0.99 μM specifically against the prostate cancer cell line (DU145). Studies to understand the mechanism of action of 4d indicates that it disrupts microtubule dynamics by inhibiting tubulin polymerization thereby arresting the cell cycle in G2/M phase. Competitive colchicine binding assay suggests that 4d binds into colchicine binding site of the tubulin. Further from some detailed biological studies like mitochondrial membrane potential, caspase-3 assay, DNA fragmentation analysis and Annexin V-FITC assay it is evident that 4d induces apoptosis. Molecular modeling studies provide an insight into the binding modes of 4d with colchicine binding site of tubulin and the data obtained correlates with the antiproliferative activity.     

THE MECHANISM OF ACTION OF COLCHICINE : Colchicine Binding Properties of Sea Urchin Sperm Tail Outer Doublet Tubulin

The thermal depolymerization procedure of Stephens (1970. J. Mol. Biol. 47:353) has been employed for solubilization of Strongylocentrotus purpuratus sperm tail outer doublet microtubules with the use of a buffer during solubilization which is of optimal pH and ionic strength for the preservation of colchicine binding activity of chick embryo brain tubulin. Colchicine binding values were corrected for first-order decay during heat solubilization at 50°C (t_½ = 5.4 min) and incubation with colchicine at 37°C in the presence of vinblastine sulfate (t_½ = 485 min). The colchicine binding properties of heat-solubilized outer doublet tubulin were qualitatively identical with those of other soluble forms of tubulin. The solubilized tubulin (mol wt, 115,000) bound 0.9 ± 0.2 mol of colchicine per mol of tubulin, with a binding constant of 6.3 x 10⁵ liters/mol at 37°C. The colchicine binding reaction was both time and temperature dependent, and the binding of colchicine was prevented in a competitive manner by podophyllotoxin (K_i = 1.3 x 10^-6 M). The first-order decay of colchicine binding activity was substantially decreased by the addition of the vinca alkaloids, vinblastine sulfate or vincristine sulfate, thus demonstrating the presence of a vinca alkaloid binding site(s) on the outer doublet tubulin. Tubulin contained within the assembled microtubules did not decay. Intact outer doublet microtubules bound less than 0.001 mol of colchicine per mol of tubulin contained in the microtubules, under conditions where soluble tubulin would have bound 1 mol of colchicine per mol of tubulin (saturating concentration of colchicine, no decay of colchicine binding activity). The presence of colchicine had no effect on the rate of solubilization of outer doublet microtubules during incubation at 37°C. Therefore, the colchicine binding site on tubulin is blocked (not available to bind colchicine) when the tubulin is in the assembled outer doublet microtubules.     

Resistance of Rosa microtubule polymerization to colchicine results from a low-affinity interaction of colchicine and tubulin

The inhibition of the polymerization of tubulin from cultured cells of rose (Rosa. sp. cv. Paul's scarlet) by colchicine and the binding of colchicine to tubulin were examined in vitro and compared with data obtained in parallel experiments with bovine brain tubulin. Turbidimetric measurements of taxol-induced polymerization of rose microtubules were found to be sensitive and semiquantitative at low tubulin concentrations, and to conform to some of the characteristics of a nucleation and condensation-polymerization mechanism for assembly of filamentous helical polymers. Colchicine inhibited the rapid phase of polymerization at 24°C with an apparent inhibition constant (K _i) of 1.4·10^-4 M for rose tubulin and an apparent K _i=8.8·10^-7 M for brain tubulin. The binding of [³H]colchicine to rose tubulin to form tubulin-colchicine complex was mildly temperature-dependent and slow, taking 2–3 h to reach equilibrium at 24°C, and was not affected by vinblastine sulfate. The binding of [³H]colchicine to rose tubulin was saturable and Scatchard analysis indicated a single class of low-affinity binding sites having an apparent affinity constant (K) of 9.7·10² M^-1 and an estimated molar binding stoichiometry (r) of 0.47 at 24°C. The values for brain tubulin were K=2.46·10⁶ M^-1 and r=0.45 at 37°C. The binding of [³H]colchicine to rose tubulin was inhibited by excess unlabeled colchicine, but not by podophyllotoxin or tropolone. The data demonstrate divergence of the colchicine-binding sites on plant and animal tubulins and indicate that the relative resistance of plant microtubule polymerization to colchicine results from a low-affinity interaction of colchicine and tubulin.Abbreviations MT microtubule - TC tubulin-colchicine complex     

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.     

Synthesis and biological evaluation of N-alkyl-N-(4-methoxyphenyl)pyridin-2-amines as a new class of tubulin polymerization inhibitors

Based on our prior antitumor hits, 32 novel N-alkyl-N-substituted phenylpyridin-2-amine derivatives were designed, synthesized and evaluated for cytotoxic activity against A549, KB, KB_VIN, and DU145 human tumor cell lines (HTCL). Subsequently, three new leads (6a, 7g, and 8c) with submicromolar GI₅₀ values of 0.19–0.41 μM in the cellular assays were discovered, and these compounds also significantly inhibited tubulin assembly (IC₅₀ 1.4–1.7 μM) and competitively inhibited colchicine binding to tubulin with effects similar to those of the clinical candidate CA-4 in the same assays. These promising results indicate that these tertiary diarylamine derivatives represent a novel class of tubulin polymerization inhibitors targeting the colchicine binding site and showing significant anti-proliferative activity.     

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.     

Biochemical determination of tubulin-microtubule equilibrium in cultured cells.

The relative amount of free and microtubule-associated tubulin in tissue culture cells was determined by colchicine binding. Both microtubules and tubulin were stabilized in a dilute homogenate containing 50% glycerol and 5% dimethylsulfoxide. Microtubules were separated by sedimentation at 100,000g for 10 min in a benchtop ultracentrifuge and then depolymerized to tubulin. Colchicine binding to free tubulin could be performed only after dilution of the organic solvents present to prevent a 70% reduction in apparent affinity of tubulin for colchicine. Tubulins purified from rat brain, human skin fibroblasts, and rat GH₃ cells were each homogeneous and similar in molecular weight, affinity for DEAE-cellulose, and apparent affinity for colchicine. Microtubules contained 34–41% of tissue culture cell tubulin. Colchicine (10^?6 to 10^?5m) and incubation at 4°C reduced microtubule-derived tubulin to less than 6% of expected.     

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.     

New Combretastatin Analogs as Anticancer Agents: Design,Synthesis, Microtubules Polymerization Inhibition,and Molecular Docking Studies

A new series of 4-(4-methoxyphenyl)-5-(3,4,5-trimethoxyphenyl)-4H-1,2,4-triazole-3-thiol derivatives were synthesized as analogs for the anticancer drug combretastatin A-4 ( CA-4 ) and characterized using FT-IR, ¹H-NMR, ¹³CNMR, and HR-MS techniques. The new CA-4 analogs were designed to meet the structural requirements of the highest expected anticancer activity of CA-4 analogs by maintaining ring A 3,4,5-trimethoxyphenyl moiety, and at the same time varying the substituents effect of the triazole moiety (ring B ). In silico analysis indicated that compound 3 has higher total energy and dipole moment than colchicine and the other analogs, and it has excellent distribution of electron density and is more stable, resulting in an increased binding affinity during tubulin inhibition. Additionally, compound 3 was found to interact with three apoptotic markers, namely p53, Bcl-2, and caspase 3. Compound 3 showed strong similarity to colchicine , and it has excellent pharmacokinetics properties and a good dynamic profile. The in vitro anti-proliferation studies showed that compound 3 is the most cytotoxic CA-4 analog against cancer cells (IC₅₀ of 6.35 μM against Hep G2 hepatocarcinoma cells), and based on its selectivity index (4.7), compound 3 is a cancer cytotoxic-selective agent. As expected and similar to colchicine , compound 3 -treated Hep G2 hepatocarcinoma cells were arrested at the G2/M phase resulting in induction of apoptosis. Compound 3 tubulin polymerization IC₅₀ (9.50 μM) and effect on V_max of tubulin polymerization was comparable to that of colchicine (5.49 μM). Taken together, the findings of the current study suggest that compound 3 , through its binding to the colchicine-binding site at β-tubulin, is a promising microtubule-disrupting agent with excellent potential to be used as cancer therapeutic agent.     

Interaction of anthelmintic benzimidazoles with AscarisSuum embryonic tubulin

The ability of mebendazole and fenbendazole to bind to tubulin in cytosolic fractions from 8-day Ascaris suum embryos was determined by inhibition studies with [³H]colchicine. Colchicine binding in the presence of 1·10^?6 M mebendazole was completely inhibited during a 6 h incubation period at 37°C. Inhibition of colchicine binding to A. suum embryonic tubulin by mebendazole and fenbendazole appeared to be noncompetative. The inhibition constants of mebendazole and fenbendazole for A. suum embryonic tubulin were 1.9·10^?8 M and 6.5·10^?8 M, respectively. Mebendazole and fenbendazole appeared to be competitive inhibitors of colchicine binding to bovine brain tubulin. The inhibition constants of mebendazole and fenbendazole for bovine brain tubulin were 7.3·10^?6 M and 1.7·10^?5 M, respectively. These values are 250–400 times greater than the inhibition constants of fenbendazole and mebendazole for A. suum embryonic tubulin. Differential binding affinities between nematode tubulin and mammalian tubulin for benzimidazoles may explain the selective toxicity. The importance of tubulin as a receptor for anthelmintic benzimidazoles in animal parasitic nematodes is discussed.