Additive effect of alpha-tocopherol and ascorbic acid in combating ethanol-induced hepatic fibrosis

Abstract

Objective

To investigate the efficacy of combined administration of alpha-tocopherol (AT) and ascorbic acid (AA) in reducing ethanol-induced hepatotoxicity.

Methods

Rats were maintained for 90 days and grouped as follows: I – control rats, II – ethanol, III – alpha-tocopherol, IV – ethanol + alpha-tocopherol, V – AA, VI – ethanol + ascorbic acid, VII – alpha-tocopherol + ascorbic acid, VIII – ethanol + alpha-tocopherol + ascorbic acid. At the end of the experimental period, markers of hepatic function, oxidative stress, and the expression of markers of inflammation and fibrosis were assayed.

Results

The markers of hepatic function, lipid peroxidation products, protein carbonyls, and the expression of nuclear factor kappa B, tumor necrosis factor alpha, transforming growth factor beta 1, cytochrome P4502E1, and collagen Type I were elevated after ethanol administration. All these parameters were reduced in the ethanol group administered AT and AA in combination. The activities of antioxidant enzymes which were reduced by ethanol administration were enhanced on combined administration of AT and AA. The reduction in hepatic fibrosis was almost 20% more in AT and AA co-administered group compared with AT and AA alone treated groups.

Discussion

Combined administration of fat soluble AT and water soluble AA was beneficial against ethanol-induced hepatotoxicity. This may be due to their different subcellular localizations.     

EFFECT OF VITAMIN E SUPPLEMENTATION AND PARTIAL SUBSTITUTION OF POLY- WITH MONO-UNSATURATED FATTY ACIDS IN PIG DIETS ON MUSCLE,AND MICROSOME EXTRACT a-TOCOPHEROL CONCENTRATION AND LIPID OXIDATION

The experiment was organized in a 3×2 factorial arrangement with three dietary fat blends and a basal (20 mg kg^?1 diet) or supplemented (220 mg kg^?1) level of α-tocopheryl acetate. Dietary vitamin E and monounsaturated to polyunsaturated fatty acid ratio (dietary MUFA/PUFA) affected muscle α-tocopherol concentration (α-tocopherol [log μg g^?1]=0.18 (±0.105)+0.0034 (±0.0003)·dietary α-tocopherol [mg kg^?1 diet] (P<0.0001)+0.39 (±0.122)·dietary MUFA/PUFA (P<0.0036)). An interaction between dietary α-tocopherol and dietary MUFA/PUFA exists for microsome α-tocopherol concentration (α-tocopherol [log μg g^?1]=1.14 (±0.169) (P<0.0001)+0.0056 (±0.00099)·dietary α-tocopherol [mg kg^?1 diet] (P<0.0001)+0.54 (±0.206)·dietary MUFA/PUFA (P<0.0131)?0.0033 (±0.0011)·dietary α-tocopherol [mg kg^?1)]×dietary MUFA/PUFA (P<0.0067)), and hexanal concentration in meat (hexanal [ng·g^?1]=14807.9 (±1489.8)?28.8 (±10.6) dietary α-tocopherol [mg·kg^?1] (P<0.01)?8436.6 (±1701.6)·dietary MUFA/PUFA (P<0.001)+24.0 (±11.22)·dietary α-tocopherol·dietary MUFA/PUFA (P<0.0416)). It is concluded that partial substitution of dietary PUFA with MUFA lead to an increase in the concentration of α-tocopherol in muscle and microsome extracts. An interaction between dietary α-tocopherol and fatty acids exists, in which at low level of dietary vitamin E inclusion, a low MUFA/PUFA ratio leads to a reduction in the concentration of α-tocopherol in microsome extracts and a concentration of hexanal in meat above the expected values.     

Regeneration of lipophilic antioxidants by NAD(P)H:quinone oxidoreductase 1

Summary.  The aim of this work was to study the activity of NAD(P)H:(quinone acceptor) oxidoreductase 1 (EC 1.6.99.2) in the regeneration of lipophilic antioxidants, alpha-tocopherol, and reduced-coenzyme Q analogs. First, we tested whether or not two isoforms of the NAD(P)H:(quinone acceptor) oxidoreductase 1 designated as “hydrophilic” and “hydrophobic” (H. J. Prochaska and P. Talalay, Journal of Biological Chemistry 261: 1372–1378, 1986) show differential enzyme activities towards hydrophilic or hydrophobic ubiquinone homologs. By chromatography on phenyl Sepharose, we purified the two isoforms from pig liver cytosol and measured their reduction of several ubiquinone homologs of different side chain length. We also studied by electron paramagnetic resonance the effect of NAD(P)H:(quinone acceptor) oxidoreductase 1 on steady-state levels of chromanoxyl radicals generated by linoleic acid and lipooxygenase and confirmed the enzyme's ability to protect alpha-tocopherol against oxidation induced with H₂O₂-Fe²⁺. Our results demonstrated that the different hydrophobicities of the isoforms do not reflect different reactivities towards ubiquinones of different side chain length. In addition, electron paramagnetic resonance studies showed that in systems containing the reductase plus NADH, levels of chromanoxyl radicals were dramatically reduced. Morever, in the presence of oxidants, alpha-tocopherol was preserved by NAD(P)H:(quinone acceptor) oxidoreductase 1, supporting our hypothesis that regeneration of alpha-tocopherol may be one of the physiologic functions of this enzyme. Received May 20, 2002; accepted September 20, 2002; published online May 21, 2003 RID="*" ID="*" Correspondence and reprints: Departamento de Biología Celular, Fisiología e Inmunología, Facultad de Ciencias, Edificio Severo Ochoa, Campus de Rabanales, Universidad de Córdoba, 14014 Córdoba, Spain.     

Molecular evolution and expression of the CRAL_TRIO protein family in insects

CRAL_TRIO domain proteins are known to bind small lipophilic molecules such as retinal, inositol and Vitamin E and include such gene family members as PINTA, α-tocopherol transfer (ATT) proteins, retinoid binding proteins, and clavesins. In insects, very little is known about either the molecular evolution of this family of proteins or their ligand specificity. Here we characterize insect CRAL_TRIO domain proteins and present the first insect CRAL_TRIO protein phylogeny constructed by performing reciprocal BLAST searches of the reference genomes of Drosophila melanogaster, Anopheles gambiae, Apis mellifera, Tribolium castaneum, Bombyx mori, Manduca sexta and Danaus plexippus. We find several highly conserved amino acid residues in the CRAL_TRIO domain-containing genes across insects and a gene expansion resulting in more than twice as many gene family members in lepidopterans than in other surveyed insect species, but no lepidopteran homolog of the PINTA gene in Drosophila. In addition, we examined the expression pattern of CRAL_TRIO domain genes in Manduca sexta heads using RNA-Seq data. Of the 42 gene family members found in the M. sexta reference genome, we found 30 expressed in the head tissue with similar expression profiles between males and females. Our results suggest this gene family underwent a large expansion in Lepidoptera, making the lepidopteran CRAL_TRIO domain family distinct from other holometabolous insect lineages.     

Alpha-tocopheryl acetate is absorbed and hydrolyzed by Caco-2 cells comparative studies with alpha-tocopherol

Caco-2 cells were used as a model for investigating and comparing the absorption of alpha-tocopherol (Tol) and alpha-tocopheryl acetate (Tac) solubilized in micelles based on a mixture of sodium taurocholate (NaTC) and oleic acid. Surprisingly, the uptake of Tac was found to be similar to that of Tol, and in both cases, the dose-response plots suggest that protein-mediated transport processes were involved. Moreover Tol or Tac were also secreted into the basolateral medium of Caco-2 cells but Tac was mainly hydrolyzed either prior to absorption or intracellularly. The solubilization of Tol or Tac by NaTC on the apical side of the cell monolayer is a prerequisite for the uptake process, although larger amounts of the bile salt are necessary to solubilize Tac than Tol. Caco-2 cells showed hydrolytic activity on Tac, and additional cholesterol esterase may be taken up by the cells, thus increasing the rates of intracellular hydrolysis of Tac. Based on our findings, a scheme is suggested accounting for the absorption of alpha-tocopheryl acetate by enterocytes.     

The more--the better? Estimating the inhibitory activity of alpha-tocopherol towards lipid oxidation

Tocopherols are considered to be powerful antioxidants, but prooxidative effects are discussed for higher concentrations. The aim of this in vitro study was to investigate the dose-dependent inhibition of oxidation product formation caused by alpha-tocopherol, and to estimate the range of maximum antioxidant activity of alpha-tocopherol at different stages of lipid oxidation. Alpha-tocopherol was added to rapeseed oil triglycerides (ROTG, purified rapeseed oil) in concentrations ranging from 25 to 1500 micromol/kg ROTG. The inhibitory activity of alpha-tocopherol increased up to a concentration of 100 micromol/kg ROTG. A concentration of 125 micromol alpha-tocopherol/ kg ROTG did not result in an improved antioxidant effect. The formation of volatile secondary oxidation products followed the same trend, and the maximum inhibitory effect was also found for 100 micromol alpha-tocopherol/kg. Further, concentrations between 250 and 1500 micromol alpha-tocopherol/kg ROTG clearly caused increased formation of hydroperoxides during the induction period. However, compared to the control, all tested alpha-tocopherol concentrations resulted in a reduction of hydroperoxide formation and no prooxidative effects were observed.     

Methods for determining the efficacy of radical-trapping antioxidants

Hydrocarbon autoxidation is the free radical chain reaction primarily responsible for the oxidative degradation of organic materials, including those that make up cells, tissues, and organs. The identification of compounds that slow this process (antioxidants) and the quantitation of their efficacies have long been goals of academic and industrial researchers. Antioxidants are generally divided into two types: preventive and radical-trapping (also commonly referred to as chain-breaking). Preventive antioxidants slow the rate of initiation of autoxidation, whereas radical-trapping antioxidants slow the rate of propagation by reacting with chain-propagating peroxyl radicals. The purpose of this review is to provide a comprehensive overview of different approaches to measure the kinetics of the reactions of radical-trapping antioxidants with peroxyl radicals, and their use to study the inhibition of hydrocarbon (lipid) autoxidation in homogeneous solution, as well as biphasic media (lipid bilayers) and cell culture. Direct and indirect approaches are presented and advantages and disadvantages of each are discussed in order to facilitate method selection for investigators seeking to address particular questions in this immensely popular field.     

Exhaustive exercise affects fluidity and alpha-tocopherol levels in brain synaptosomal membranes of normal and vitamin E supplemented rats

Alpha-tocopherol level and fluidity were studied in the neuronal membrane of rat brain after exhaustive exercise. The order parameter, 5-doxyl-stearic acid (5-DS), which is utilized for assessing the fluidity of the lipid bilayer closer to the hydrophilic face of the membrane, decreased in the pons-medulla oblongata, and the motion parameter, 16-doxyl-stearic acid (16-DS) for the core of the lipid bilayer, decreased in the cortex, hippocampus, hypothalamus and striatum, whereas it increased in the cerebellum after exercise. The w/s ratio of n-(1-oxyl-2,2,6,6-tetramethyl-4-piperidinyl)-maleimido (maleimido-TEMPO) for the conformation of SH-protein also decreased in the hippocampus and midbrain after exercise. These changes were not observed in alpha-tocopheryl acetate supplemented rats after exercise. Although the levels of 5-DS, 16-DS and maleimido-TEMPO were affected by alpha-tocopheryl acetate in rat neuronal membranes, fluidity changes were reversible with exercise.     

Neutrophil superoxide-anion generating capacity in chronic smoking: Effect of long-term α-tocopherol therapy

We investigated whether long-term alpha-tocopherol therapy in chronic smoking affects superoxide generating capacity of neutrophils ex vivo. To this purpose, we randomly assigned 128 male chronic smokers (37 +/- 21 pack years of smoking) to treatment with placebo (n = 64) or alpha-tocopherol (400 IU dL-a-tocopherol daily, n = 64). After two years of therapy, we measured phorbol 12-myristate 13-acetate-induced superoxide production of isolated neutrophils and of diluted whole blood by monitoring reduction of ferricytochrome c and luminol-enhanced peroxidase-catalyzed chemiluminescence. Plasma lipids and lipoproteins were not different between the two treatment groups. As expected, concentrations of alpha-tocopherol in plasma and in low-density lipoproteins were markedly elevated in the supplemented group compared to the placebo group (+ 120%, P < 0.0001 and + 83%, P < 0.0001, respectively). Consequently, resistance to in vitro oxidation of low-density lipoproteins (reflected by lag time of conjugated diene formation) was higher in the supplemented group than in the placebo group (+ 22%, P < 0.0001). Superoxide generating capacity of neutrophils and superoxide production in diluted whole blood did not differ between alpha-tocopherol and placebo group. It is concluded that in chronic smoking long-term supranormal alpha-tocopherol intake does not reduce neutrophil superoxide-anion generating capacity, despite large increases in the concentrations of alpha-tocopherol in plasma and in low-density lipoproteins.     

Retinoid metabolism and its effects on the vasculature

Retinoids, the metabolically-active structural derivatives of vitamin A, are critical signaling molecules in many fundamental biological processes including cell survival, proliferation and differentiation. Emerging evidence, both clinical and molecular, implicates retinoids in atherosclerosis and other vasculoproliferative disorders such as restenosis. Although the data from clinical trials examining effect of vitamin A and vitamin precursors on cardiac events have been contradictory, this data does suggest that retinoids do influence fundamental processes relevant to atherosclerosis. Preclinical animal model and cellular studies support these concepts. Retinoids exhibit complex effects on proliferation, growth, differentiation and migration of vascular smooth muscle cells (VSMC), including responses to injury and atherosclerosis. Retinoids also appear to exert important inhibitory effects on thrombosis and inflammatory responses relevant to atherogenesis. Recent studies suggest retinoids may also be involved in vascular calcification and endothelial function, for example, by modulating nitric oxide pathways. In addition, established retinoid effects on lipid metabolism and adipogenesis may indirectly influence inflammation and atherosclerosis. Collectively, these observations underscore the scope and complexity of retinoid effects relevant to vascular disease. Additional studies are needed to elucidate how context and metabolite-specific retinoid effects affect atherosclerosis. This article is part of a Special Issue entitled: Retinoid and Lipid Metabolism.