A glycomic approach to hepatic tumors in N-acetylglucosaminyltransferase III (GnT-III) transgenic mice induced by diethylnitrosamine (DEN): identification of haptoglobin as a target molecule of GnT-III

A glycomic approach to the identification of target molecules in glycosyltransferase gene targeting mice is a promising strategy to understand the biological significance of glycosyltransferase genes in vivo . In order to understand the biological effects of N -acetylglucosaminyltransferase III (GnT-III) on tumor formation in the liver, diethylnitrosamine (DEN) induced tumor formation in the GnT-III transgenic mice was examined. Our findings show that the incidence of hepatic tumor could be dramatically suppressed. A glycomic approach using two-dimensional gel electrophoresis followed by lectin blot analysis and sequence analysis revealed that haptoglobin, a radical scavenger molecule in serum was heavily glycosylated in hepatic tumor-bearing GnT-III transgenic mice that had been treated with DEN. Immunoprecipitation followed by E 4 -PHA lectin blot analysis also confirmed that the bisecting GlcNAc, a product of GnT-III was added to haptoglobin molecules. Since the use of DEN is known to lead to the production of lipid peroxidation products which facilitate this reaction and haptoglobin is an acute phase reactant, acting as a radical scavenger against hemoglobin or iron stimulated lipid peroxidation, a relationship between the glycosylation of haptoglobin and the suppression of hepatoma development can not be ruled out. This paper is the first report that shows a relationship between the sugar chains of glycoproteins with radical scavenger activity and hepatocarcinogenesis.     

Glycomic/glycoproteomic analysis by liquid chromatography/mass spectrometry: analysis of glycan structural alteration in cells

The alteration of glycosyltransferase expression and the subsequent changes in oligosaccharide structures are reported in several diseases. The analysis of glycan structural alteration in glycoproteins is becoming increasingly important in the discovery of therapies and diagnostic markers. In this study, we propose a strategy for glycomic/glycoproteomic analysis based on oligosaccharide profiling by LC/MS followed by proteomic approaches, including 2-DE and 2-D lectin blot. As a model of aberrant cells, we used Chinese hamster ovary cells transfected with N-acetylglucosaminyltransferase III (GnT-III), which catalyzes the addition of a bisecting N-acetylglucosamine (GlcNAc) to beta-mannose of the mannosyl core of N-linked oligosaccharides. LC/MS equipped with a graphitized carbon column (GCC) enabled us to elucidate the structural alteration induced by the GnT-III expression. Using 2-D lectin blot followed by LC/MS/MS, the protein carrying an extra N-acetylhexosamine in cells transfected with GnT-III was successfully identified as integrin alpha3. Thus, oligosaccharide profiling by GCC-LC/MS followed by proteomic methods can be a powerful tool for glycomic/glycoproteomic analysis.     

Hyperexpression of N-acetylglucosaminyltransferase-III in liver tissues of transgenic mice causes fatty body and obesity through severe accumulation of Apo A-I and Apo B

N-Acetylglucosaminyltransferase (GnT)-III catalyzes the attachment of an N-acetylglucosamine (GlcNAc) residue to mannose in beta(1-4) configuration in the region of N-glycans and forms a bisecting GlcNAc. To investigate the pathophysiological role of dysregulated glycosylation mediated by aberrantly expressed GnT-III, we generated transgenic mice hyperexpressing the human GnT-III in the liver by introducing human GnT-III cDNA under the control of mouse albumin enhancer/promoter. Total five transgenic founder mice (pGnTSVTpA-10, -14, -20, -25, and -51) expressed the human GnT-III in their livers and were characterized by molecular genetic means. The copy number of transgene integrated into the genome of these mice ranged between 1 and 3 copies per haploid genome. Northern and Western blot analyses showed that the transgene is specifically expressed in the liver but not in any other tissues tested. The triglyceride level in GnT-III transgenic mice was significantly decreased, however, no significant differences in the levels of glucose, cholesterol, or albumin were observed between transgenic and nontransgenic mice. Although glutamate oxaloacetic transaminase and glutamic pyruvic transaminase activities of transgenic mice were also higher than those of nontransgenic mice, no differences in total bililubin and total protein were observed between the two animal lines. Large amounts of apolipoprotein (Apo) A-I and Apo B were specifically detected in the intracellular liver of transgenic mice. The accumulation of Apo A-I in hepatocytes may be due to aberrant glycosylation, since glycosylated Apo A-I was not observed in transgenic mice. However, the accumulated Apo B was severely glycosylated. Therefore, it is suggested that highly expressed transgenic GnT-III allowed unknown target proteins to be glycosylated in large amounts, and the resulting target protein(s) disrupted in assembly formation of Apo A-I in the hepatocytes and cause a decrease in the release of lipoproteins and accumulations of Apo A-I and Apo B in the liver. The transgenic mice showed aberrant glycosylation by GnT-III, resulting in numerous lipid droplets in liver tissues and the obesity. These mice showed microvesicular fatty changes with abnormal lipid accumulation in the hepatocytes. Our study provides the basis for future analysis of the role of glycosylation in hepatic pathogenesis. In the transgenic mice, Apo A-I and Apo B were significantly increased compared with levels in nontransgenic liver tissues.     

A catalytically inactive beta 1,4-N-acetylglucosaminyltransferase III (GnT-III) behaves as a dominant negative GnT-III inhibitor.

beta 1,4-N-Acetylglucosaminyltransferase III (GnT-III) plays a regulatory role in the biosynthesis of N-glycans, and it has been suggested that its product, a bisecting GlcNAc, is involved in a variety of biological events as well as in regulating the biosynthesis of the oligosaccharides. In this study, it was found, on the basis of sequence homology, that GnT-III contains a small region that is significantly homologous to both snail beta 1,4GlcNAc transferase and beta1,4Gal transferase-1. Subsequent mutational analysis demonstrated an absolute requirement for two conserved Asp residues (Asp321 and Asp323), which are located in the most homologous region of rat GnT-III, for enzymatic activity. The overexpression of Asp323-substituted, catalytically inactive GnT-III in Huh6 cells led to the suppression of the activity of endogenous GnT-III, but no significant decrease in its expression, and led to a specific inhibition of the formation of bisected sugar chains, as shown by structural analysis of the total N-glycans from the cells. These findings indicate that the mutant serves a dominant negative effect on a specific step in N-glycan biosynthesis. This type of 'dominant negative glycosyltransferase', identified has potential value as a powerful tool for defining the precise biological roles of the bisecting GlcNAc structure.     

The glycomic effect of N-acetylglucosaminyltransferase III overexpression in metastatic melanoma cells. GnT-III modifies highly branched N-glycans

N-acetylglucosaminyltransferase III (GnT-III) is known to catalyze N-glycan “bisection” and thereby modulate the formation of highly branched complex structures within the Golgi apparatus. While active, it inhibits the action of other GlcNAc transferases such as GnT-IV and GnT-V. Moreover, GnT-III is considered as an inhibitor of the metastatic potential of cancer cells both in vitro and in vivo. However, the effects of GnT-III may be more diverse and depend on the cellular context. We describe the detailed glycomic analysis of the effect of GnT-III overexpression in WM266–4-GnT-III metastatic melanoma cells. We used MALDI-TOF and ESI-ion-trap-MS/MS together with HILIC-HPLC of 2-AA labeled N-glycans to study the N-glycome of membrane-attached and secreted proteins. We found that the overexpression of GnT-III in melanoma leads to the modification of a broad range of N-glycan types by the introduction of the “bisecting” GlcNAc residue with highly branched complex structures among them. The presence of these unusual complex N-glycans resulted in stronger interactions of cellular glycoproteins with the PHA-L. Based on the data presented here we conclude that elevated activity of GnT-III in cancer cells does not necessarily lead to a total abrogation of the formation of highly branched glycans. In addition, the modification of pre-existing N-glycans by the introduction of “bisecting” GlcNAc can modulate their capacity to interact with carbohydrate-binding proteins such as plant lectins. Our results suggest further studies on the biological function of “bisected” oligosaccharides in cancer cell biology and their interactions with carbohydrate-binding proteins.     

Reduction of the major xenoantigen on glycosphingolipids of swine endothelial cells by various glycosyltransferases

The effect of the various glycosyltransferases on glycosphingolipids was examined, using transfected swine endothelial cell (SEC) lines. The reactivity of parental SEC to normal human serum (NHS) and Griffonia simplicifolia IB(4) (GSIB4) lectin, which binds to the Gal alpha1-3 Gal beta 1-4 GlcNAc-R (alpha-galactosyl epitope), was reduced by approximately 20% by the treatment with D-PDMP (D-threo-1-phenyl-2-decan- oylamino-3-morpholino-1-propanol), suggesting that glycosphingolipids contained by SEC have a considerable amount of the alpha-galactosyl epitope. The overexpression of two different types of glycosyltransferase, N-acetylglucosaminyl transferase III (GnT-III), as well as alpha2, 6-sialyltransferase (ST6Gal I), alpha2,3-sialyltransferase (ST3Gal III), and alpha1,2-fucosyltransferase (alpha1,2FT), suppresses the total antigenicity of SEC significantly. However, the reduction in reactivities toward NHS and GSIB4 lectin in the case of GnT-III transfectants was milder than those in other transfectants. Western blot analysis indicated that the glycoproteins in all transfectants had diminished reactivity to NHS and GSIB4 lectin to approximately the same extent. Therefore, the neutral glycosphingolipids of these transfectants were separated by thin layer chromatography, followed by immunostaining with NHS and GSIB4 lectin. The levels of the alpha-galactosyl epitope in glycosphingolipids were not decreased in the GnT-III transfectants but were in the ST6Gal I, ST3Gal III, and alpha1,2FT transfectants. These data indicate that ST6Gal I, ST3Gal III, and alpha1,2FT reduced the alpha-galactosyl epitope in both glycoproteins and glycosphingolipids, while GnT-III reduced them only in glycoproteins.     

Roles of N-acetylglucosaminyltransferase III in epithelial-to-mesenchymal transition induced by transforming growth factor β1 (TGF-β1) in epithelial cell lines

The epithelial-to-mesenchymal transition (EMT) plays crucial roles in embryonic development, wound healing, tissue repair, and cancer progression. Results of this study show how transforming growth factor β1 (TGF-β1) down-regulates expression of N-acetylglucosaminyltransferase III (GnT-III) during EMT-like changes. Treatment with TGF-β1 resulted in a decrease in E-cadherin expression and GnT-III expression, as well as its product, the bisected N-glycans, which was confirmed by erythro-agglutinating phytohemagglutinin lectin blot and HPLC analysis in human MCF-10A and mouse GE11 cells. In contrast with GnT-III, the expression of N-acetylglucosaminyltransferase V was slightly enhanced by TGF-β1 treatment. Changes in the N-glycan patterns on α3β1 integrin, one of the target proteins for GnT-III, were also confirmed by lectin blot analysis. To understand the roles of GnT-III expression in EMT-like changes, the MCF-10A cell was stably transfected with GnT-III. It is of particular interest that overexpression of GnT-III influenced EMT-like changes induced by TGF-β1, which was confirmed by cell morphological changes of phase contrast, immunochemical staining patterns of E-cadherin, and actin. In addition, GnT-III modified E-cadherin, which served to prolong E-cadherin turnover on the cell surface examined by biotinylation and pulse-chase experiments. GnT-III expression consistently inhibited β-catenin translocation from cell-cell contact into the cytoplasm and nucleus. Furthermore, the transwell assay showed that GnT-III expression suppressed TGF-β1-induced cell motility. Taken together, these observations are the first to clearly demonstrate that GnT-III affects cell properties, which in turn influence EMT-like changes, and to explain a molecular mechanism for the inhibitory effects of GnT-III on cancer metastasis.     

Inhibition of rat liver microsomal lipid peroxidation by N-acyldehydroalanines: an in vitro comparative study

Captodative substituted olefins are radical scavengers which react with free radicals to form stabilized radical adducts. One of those compounds, N-(paramethoxyphenylacetyl)dehydroalanine (AD-5), may react and scavenge both superoxide anion (O-2) and alk-oxyl radicals (RO.), and in this way prevent the appearance of their mediated biological effects. Nitrofurantoin and tert-butyl hydroperoxide were used as model compounds to stimulate free radical production and their mediated lipid peroxidation in rat liver microsomes. In addition, lipid peroxidation was also initiated by exposure of rat liver microsomal suspensions to ionizing radiation (gamma rays). The microsomal lipid peroxidation induced by these chemicals and physical agents was inhibited by the addition of AD-5. These effects were dose-dependent in a millimolar range of concentration. In addition, AD-5 has no effect on microsomal electron transport, showing that NADPH-cytochrome P450 reductase activity was not modified. These data, together with the comparisons of the effects of AD-5 and some antioxidant molecules such as superoxide dismutase, uric acid, and mannitol, support the conclusion that inhibition of lipid peroxidation by AD-5 is the result of its free radical scavenger activity. In addition, the inhibitory effect of AD-5 on microsomal lipid peroxidation was dependent of the nature of the free radical species involved in the initiation of the process, suggesting that O-2 is scavenged more efficiently than RO.     

All-Trans-Retinoic Acid Modulates ICAM-1 N-Glycan Composition by Influencing GnT-III Levels and Inhibits Cell Adhesion and Trans-Endothelial Migration

Changes in the expression of glycosyltransferases directly influence the oligosaccharide structures and conformations of cell surface glycoproteins and consequently cellular phenotype transitions and biological behaviors. In the present study, we show that all-trans-retinoic acid (ATRA) modulates the N-glycan composition of intercellular adhesion molecule-1 (ICAM-1) by manipulating the expression of two N-acetylglucosaminyltransferases, GnT-III and GnT-V, via the ERK signaling pathway. Exposure of various cells to ATRA caused a remarkable gel mobility down-shift of ICAM-1. Treatment with PNGase F confirmed that the reduction of the ICAM-1 molecular mass is attributed to the decreased complexity of N-glycans. We noticed that the expression of the mRNA encoding GnT-III, which stops branching, was significantly enhanced following ATRA exposure. In contrast, the level of the mRNA encoding GnT-V, which promotes branching, was reduced following ATRA exposure. Silencing of GnT-III prevented the molecular mass shift of ICAM-1. Moreover, ATRA induction greatly inhibited the adhesion of SW480 and U937 cells to the HUVEC monolayer, whereas knock-down of GnT-III expression effectively restored cell adhesion function. Treatment with ATRA also dramatically reduced the trans-endothelial migration of U937 cells. These data indicate that the alteration of ICAM-1 N-glycan composition by ATRA-induced GnT-III activities hindered cell adhesion and cell migration functions simultaneously, pinpointing a unique regulatory role of specific glycosyltransferases in the biological behaviors of tumor cells and a novel function of ATRA in the modulation of ICAM-1 N-glycan composition.     

Structure-activity relationship of 3-methyl-1-phenyl-2-pyrazolin-5-one (edaravone)

This paper describes the discovery of a novel free radical scavenger, 3-methyl-1-phenyl-2-pyrazolin-5-one (edaravone, 1), as a potent antioxidant agent against lipid peroxidation. The structure-activity relationship of edaravone indicated that lipophilic substituents were essential to show its lipid peroxidation-inhibitory activity. In vivo studies revealed that edaravone showed brain-protective activity in a transient ischemia model.     

Beta-carotene prolongs survival, decreases lipid peroxidation and enhances glutathione status in transplantable murine lymphoma.

Carotenoids of dietary origin have recently been the subject of renewed research interest because of epidemiological evidence indicating an inverse relationship between intake of carotenoids-rich plant substances and risk of certain cancers. This study was attempted to understand the biological actions of dietary beta-carotene (BC) on Dalton's lymphoma (DL), a rapidly proliferating transplantable tumor, in effecting the survival of the lymphoma-bearing mice. The glutathione (GSH) level and the extent of lipid peroxidation in the liver, kidney and brain were monitored in BC-treated (100 mg/kg food) mice transplanted with DL. These markers showed substantial alterations during the whole length of tumor progression in lymphoma-bearing mice without BC supplementation. When treated with BC, both malondialdehyde contents (evidence of lipid peroxidation) and the GSH levels in different organs were found to be closer to normal values in the initial period of tumor progression. BC-mediated protection against lipid peroxidation was maximally found to be in hepatic tissue throughout the study following DL transplantation. This was fairly reflected in the higher BC concentration in hepatic tissue of BC-treated lymphoma group compared to untreated lymphoma control. Significantly higher survival time (51-55 days) was observed in BC-treated animals in comparison to their untreated DL counterparts (35-38 days). The prolonged survival observed in the BC-supplemented animals may be attributed to the higher resistance offered by animals receiving BC towards lipid peroxidation-related tissue injury.     

Cocaine hepatotoxicity during protein undernutrition of retrovirally infected mice.

Effects of cocaine administration on lipid peroxidation and liver damage in immunocompromised mice fed different levels of dietary proteins were investigated. Indices of lipid peroxidation and serum aminotransferases as evidence of free radical attack and liver damage were compared in mice fed a low protein (4%) or regular protein diet (20% protein) for 3 weeks and then infected with murine leukemia virus and given daily intraperitoneal injections of increasing progressive doses of 5-45 mg.kg-1.day-1 of cocaine for 11 weeks. Cocaine administration significantly increased hepatic triglycerides, serum aminotransaminases, conjugated dienes, lipid fluorescence, and malondialdehyde levels. These changes were exacerbated by retroviral infection and also by protein undernutrition. Retroviral infection additively increased indices of cocaine-induced lipid peroxidation and hepatic damage. Significant increases in indices of lipid peroxidation and greater liver injury were also detected in similarly treated mice that received the low protein diet compared with well-nourished mice. These results show that immunocompromised mice fed low levels of dietary protein form significantly increased immunogenic lipid peroxidation adducts during cocaine treatment.     

Effect of the Peroxisome Proliferator Ciprofibrate on Lipid Peroxidation and 8-Hydroxydeoxyguanosine Formation in Transgenic Mice with Elevated Hepatic Catalase Activity

Peroxisome proliferators are a group of non-genotoxic hepatic carcinogens which have been proposed to act by increasing oxidative damage in the liver. To test this hypothesis, we have produced a transgenic mouse line that has elevated catalase activity specifically in the liver. In this study, we have examined if catalase overexpression influences the induction of lipid peroxidation or oxidative DNA damage, two mechanisms which have been hypothesized to be important in the carcinogenesis by peroxisome proliferators. Transgenic mice or non-transgenic litter mates were fed either 0.01% ciprofibrate or a control diet for 21 days. The activities of fatty acyl CoA oxidase and lauric acid hydroxylase were not significantly affected by catalase overexpression, although the ratio of fatty acyl CoA oxidase to catalase was significantly decreased in transgenic animals. Hepatic lipid peroxidation was estimated by quantifying the concentrations of malondialdehyde and conjugated dienes. Ciprofibrate treatment did not affect either endpoint, but catalase overexpression increased the concentrations of malondialdehyde (in untreated mice only) and conjugated dienes (in both untreated and ciprofibrate-fed mice). Oxidative DNA damage was estimated by quantifying 8-hydroxydeoxyguanosine (8-OHdG) by high-performance liquid chromatography/electrochemical detection. Ciprofibrate treatment significantly increased hepatic 8-OHdG concentrations, in agreement with several previous studies, but catalase overexpression did not significantly affect them, although 8-OHdG concentrations were decreased 50% in untreated mice. These results imply that the metabolism of hydrogen peroxide by catalase is not an important factor in the development of hepatic lipid peroxidation. The decrease in hepatic 8-OHdG in untreated transgenic mice and the increase seen after ciprofibrate administration imply that hydrogen peroxide is important in the formation of 8-OHdG. While the lack of decreased 8-OHdG levels in ciprofibrate-treated transgenic mice does not support this conclusion, it is possible that catalase levels were not sufficiently high to affect this endpoint. Transgenic mice with higher hepatic catalase activities may be required to resolve this issue.     

Remodeling of the major pig xenoantigen by N-acetylglucosaminyltransferase III in transgenic pig

We have been successful in generating several lines of transgenic mice and pigs that contain the human beta-d-mannoside beta-1,4-N-acetylglucosaminyltransferase III (GnT-III) gene. The overexpression of the GnT-III gene in mice and pigs reduced their antigenicity to human natural antibodies, especially the Galalpha1-3Galbeta1-4GlcNAc-R, as evidenced by immunohistochemical analysis. Endothelial cell studies from the GnT-III transgenic pigs also revealed a significant down-regulation in antigenicity, including Hanganutziu-Deicher antigen, and dramatic reductions in both the complement- and natural killer cell-mediated pig cell lyses. Changes in the enzymatic activities of other glycosyltransferases, such as alpha1,3-galactosyltransferase, GnT-IV, and GnT-V, did not support cross-talk between GnT-III and these enzymes in the transgenic animals. In addition, we demonstrated the effect of GnT-III in down-regulating the xenoantigen of pig heart grafts, using a pig to cynomolgus monkey transplantation model, suggesting that this approach may be useful in clinical xenotransplantation in the future.     

Inhibitory effects of echinoisoflavanone and sophoraisoflavanone D in Sophora chrysophylla SEEM on lipid peroxidation of mice brain homogenate by interaction of ferrous ion and hydrogen peroxide, in vitro

We present the results of an in vitro investigation of the inhibitory effects of echinoisoflavanone and sophoraisoflavanone D isolated from Sophora chrysophylla SEEM on lipid peroxidation of mice brain homogenate by interaction of ferrous ion and hydrogen peroxide, in vitro. They inhibited lipid peroxidation. The order of inhibitory effects of these isoflavanones and mannitol as a hydroxy radical scavenger was echinoisoflavanone > mannitol > sophoraisoflavanone D. The results suggest that some isoflavanones may be of use in cases where oxidative stress is present.     

Loss and recovery of Mgat3 and GnT-III Mediated E-cadherin N-glycosylation is a mechanism involved in epithelial-mesenchymal-epithelial transitions

Background

N-acetylglucosaminyltransferase-III (GnT-III) is a glycosyltransferase encoded by Mgat3 that catalyzes the addition of β1,4-bisecting-N-acetylglucosamine on N-glycans. GnT-III has been pointed as a metastases suppressor having varying effects on cell adhesion and migration. We have previously described the existence of a functional feedback loop between E-cadherin expression and GnT-III-mediated glycosylation. The effects of GnT-III-mediated glycosylation on E-cadherin expression and cellular phenotype lead us to evaluate Mgat3 and GnT-III-glycosylation role during Epithelial-Mesenchymal-Transition (EMT) and the reverted process, Mesenchymal-Epithelial-Transition (MET).

Methodology/Principal Findings

We analyzed the expression profile and genetic mechanism controlling Mgat3 expression as well as GnT-III-mediated glycosylation, in general and specifically on E-cadherin, during EMT/MET. We found that during EMT, Mgat3 expression was dramatically decreased and later recovered when cells returned to an epithelial-like phenotype. We further identified that Mgat3 promoter methylation/demethylation is involved in this expression regulation. The impact of Mgat3 expression variation, along EMT/MET, leads to a variation in the expression levels of the enzymatic product of GnT-III (bisecting GlcNAc structures), and more importantly, to the specific modification of E-cadherin glycosylation with bisecting GlcNAc structures.

Conclusions/Significance

Altogether, this work identifies for the first time Mgat3 glycogene expression and GnT-III-mediated glycosylation, specifically on E-cadherin, as a novel and major component of the EMT/MET mechanism signature, supporting its role during EMT/MET.     

Introduction of bisecting GlcNAc into integrin alpha5beta1 reduces ligand binding and down-regulates cell adhesion and cell migration

The enzyme beta1,4-N-acetylglucosaminyltransferase III (GnT-III) catalyzes the addition of a bisecting GlcNAc residue to glycoproteins, resulting in a modulation in biological function. Our previous studies showed that the transfection of the GnT-III gene into B16 melanoma cells results in a suppression of invasive ability and lung colonization. The suppression has been postulated to be due to an increased level of E-cadherin expression on the cell surface, which in turn leads to the up-regulation of cell-cell adhesion. In this study, we report on the effects of overexpression of GnT-III on cell-matrix adhesion. The overexpression of GnT-III, but not that of an enzymatic inactive GnT-III (D323A), inhibits cell spreading and migration on fibronectin, a specific ligand for integrin alpha(5)beta(1), and the focal adhesion kinase phosphorylation. E(4)-PHA lectin blot analyses showed that the levels of bisecting GlcNAc structures on the integrin alpha(5) subunit as well as alpha(2) and alpha(3) subunits immunoprecipitated from GnT-III transfectants were substantially increased. In addition, the affinity of the binding of integrin alpha(5)beta(1) to fibronectin was significantly reduced by the introduction of the bisecting GlcNAc, to the alpha(5) subunit. These findings suggest that the modification of N-glycan of integrin by GnT-III inhibits its ligand binding ability, subsequently leading to the down-regulation of integrin-mediated signaling.     

The effect of chronic alcohol feeding on lipid peroxidation in microsomes: lack of relationship to hydroxyl radical generation

Chronic alcohol feeding causes microsomal induction including increased generation of hydroxyl radicals. Ethanol induced liver injury may be mediated by lipid peroxidation for which hydroxyl radicals have been proposed as major mediators. Ethanol promotes lipid peroxidation when given acutely but also may serve as a hydroxyl radical scavenger. Therefore, we studied the acute and chronic effects of alcohol on microsomal lipid peroxidation and hydroxyl radical generation. Chronic alcohol feeding in rats increased microsomal generation of hydroxyl radicals but lipid peroxidation of endogenous lipid was inversely related to hydroxyl radical generation. Ethanol (50mM) had a slight inhibitory effect on hydroxyl radical production in peroxidizing microsomes, no effect on endogenous lipid peroxidation and enhanced the lysis of RBCs added as targets of peroxidation. Enhanced microsomal generation of hydroxyl radicals following chronic alcohol feeding is not an important mediator of lipid peroxidation.     

Role of p53 tumor suppressor gene in the toxicity of TCDD, endrin, naphthalene, and chromium (VI) in liver and brain tissues of mice

It has been postulated that tumor suppressor genes are involved in the cascade of events leading to the toxicity of diverse xenobiotics. Therefore, we have assessed the comparative effects of 0.01, 0.10, and 0.50 median lethal doses (LD(50)) of 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD), endrin, naphthalene, and sodium dichromate (VI) [Cr(VI)] on lipid peroxidation, DNA fragmentation, and enhanced production of superoxide anion (cytochrome c reduction) in liver and brain tissues of p53-deficient and standard C57BL/6NTac mice to determine the role of p53 gene in the toxic manifestations produced by these diverse xenobiotics. In general, p53-deficient mice are more susceptible to all four xenobiotics than C57BL/6NTac mice, with dose-dependent effects being observed. Specifically, at a 0.50 LD(50) dose, naphthalene and Cr(VI) induced the greatest toxicity in the liver tissue of mice, and naphthalene and endrin exhibited the greatest effect in the brain tissue. At this dose, TCDD, endrin, naphthalene, and Cr(VI) induced 2.3- to 3.7-fold higher increases in hepatic lipid peroxidation and 1.8- to 3.0-fold higher increases in brain lipid peroxidation in p53-deficient mice than in C57BL/6NTac mice. At a 0. 10 LD(50) dose, TCDD, endrin, naphthalene, and Cr(VI) induced 1.3- to 1.8-fold higher increases in hepatic lipid peroxidation and 1.4- to 1.9-fold higher increases in brain lipid peroxidation in p53-deficient mice than in C57BL/6NTac mice. Similar results were observed with respect to DNA fragmentation and cytochrome c reduction (superoxide anion production). For example, at the 0.10 LD(50) dose, the four xenobiotics induced increases of 1.6- to 3. 0-fold and 1.5- to 2.1-fold in brain and liver DNA fragmentation, respectively, and increases of 1.5- to 2.3-fold and 1.4- to 2.5-fold in brain and liver cytochrome c reduction (superoxide anion production), respectively, in p53-deficient mice compared with control C57BL/6NTac mice. These results suggest that the p53 tumor suppressor gene may play a role in the toxicity of structurally diverse xenobiotics.