Antioxidative function of L‐FABP in L‐FABP stably transfected Chang liver cells

Liver fatty acid binding protein (L‐FABP) contains amino acids that are known to possess antioxidant function. In this study, we tested the hypothesis that L‐FABP may serve as an effective endogenous cytoprotectant against oxidative stress. Chang liver cells were selected as the experimental model because of their undetectable L‐FABP mRNA level. Full‐length L‐FABP cDNA was subcloned into the mammalian expression vector pcDNA3.1 (pcDNA‐FABP). Chang cells were stably transfected with pc‐DNA‐FABP or vector (pcDNA3.1) alone. Oxidative stress was induced by incubating cells with 400 μmol/L H2O2 or by subjecting cells to hypoxia/reoxygenation. Total cellular reactive oxygen species (ROS) was determined using the fluorescent probe DCF. Cellular damage induced by hypoxia/reoxygenation was assayed by lactate dehydrogenase (LDH) release. Expression of L‐FABP was documented by regular reverse transcription polymerase chain reaction (RT‐PCR), real‐time RT‐PCR, and Western blot. The pcDNA‐FABP–transfected cells expressed full‐length L‐FABP mRNA, which was absent from vector‐transfected control cells. Western blot showed expression of 14‐kd L‐FABP protein in pcDNA‐FABP–transfected cells, but not in vector‐transfected cells. Transfected cells showed decreased DCF fluorescence intensity under oxidative stress (H2O2 and hypoxia/reoxygenation) conditions versus control in inverse proportion to the level of L‐FABP expression. Lower LDH release was observed in the higher L‐FABP–expressed cells in hypoxia/reoxygenation experiments. In conclusion, we successfully transfected and cloned a Chang liver cell line that expressed the L‐FABP gene. The L‐FABP–expressing cell line had a reduced intracellular ROS level versus control. This finding implies that L‐FABP has a significant role in oxidative stress. (HEPATOLOGY 2005;42:871–879.)

[1]  B. Hasinoff.  Dexrazoxane (ICRF-187) protects cardiac myocytes against hypoxia-reoxygenation damage , 2007, Cardiovascular Toxicology.

[2]  F. Burczynski,et al.  Enhanced expression of cytosolic fatty acid binding protein and fatty acid uptake during liver regeneration in rats , 2004, Molecular and Cellular Biochemistry.

[3]  E. Stadtman.  Cyclic oxidation and reduction of methionine residues of proteins in antioxidant defense and cellular regulation. , 2004, Archives of biochemistry and biophysics.

[4]  A. McIntosh,et al.  Liver fatty acid-binding protein colocalizes with peroxisome proliferator activated receptor alpha and enhances ligand distribution to nuclei of living cells. , 2004, Biochemistry.

[5]  B. Tuchweber,et al.  Prevention of CeCl3-induced hepatotoxicity by hypolipidemic compounds , 1978, Archives of Toxicology.

[6]  D. Deplanque.  Protection cellulaire par activation des récepteurs nucléaires PPAR , 2004 .

[7]  D. Deplanque.  [Cell protection through PPAR nuclear receptor activation]. , 2004, Therapie.

[8]  S. B. Prasad,et al.  Changes in glutathione-related enzymes in tumor-bearing mice after cisplatin treatment , 2004, Cell Biology and Toxicology.

[9]  N. Kaplowitz,et al.  Mechanisms for sensitization to TNF‐induced apoptosis by acute glutathione depletion in murine hepatocytes , 2003, Hepatology.

[10]  Y. Yamaoka,et al.  Overexpression of thioredoxin prevents acute hepatitis caused by thioacetamide or lipopolysaccharide in mice , 2003, Hepatology.

[11]  Hartmut Jaeschke,et al.  Molecular mechanisms of hepatic ischemia-reperfusion injury and preconditioning. , 2003, American journal of physiology. Gastrointestinal and liver physiology.

[12]  G. Zhong,et al.  Infection of myocytes with chlamydiae. , 2002, Microbiology.

[13]  S. Storey,et al.  Expression of fatty acid binding proteins inhibits lipid accumulation and alters toxicity in L cell fibroblasts. , 2002, American journal of physiology. Cell physiology.

[14]  Á. Catala,et al.  Retinal fatty acid binding protein reduce lipid peroxidation stimulated by long-chain fatty acid hydroperoxides on rod outer segments. , 2002, Biochimica et biophysica acta.

[15]  M. F. Weiss,et al.  Cellular oxidant stress and advanced glycation endproducts of albumin: caveats of the dichlorofluorescein assay. , 2002, Archives of Biochemistry and Biophysics.

[16]  J. Manautou,et al.  Protection against acetaminophen hepatotoxicity by clofibrate pretreatment: Role of catalase induction , 2002, Journal of biochemical and molecular toxicology.

[17]  Fengli Zhang,et al.  Binding of 13-HODE and 15-HETE to Phospholipid Bilayers, Albumin, and Intracellular Fatty Acid Binding Proteins , 2001, The Journal of Biological Chemistry.

[18]  J. Veerkamp,et al.  Fatty-acid-binding proteins do not protect against induced cytotoxicity in a kidney cell model. , 2001, The Biochemical journal.

[19]  M. Schroeter,et al.  Effects of oxidative stress on the expression of antioxidative defense enzymes in spontaneously hypertensive rat hearts. , 2000, Free radical biology & medicine.

[20]  P. Burcham,et al.  Clofibrate-induced in vitro hepatoprotection against acetaminophen is not due to altered glutathione homeostasis. , 2000, Toxicological sciences : an official journal of the Society of Toxicology.

[21]  G. Belling,et al.  Clofibrate pretreatment in mice confers resistance against hepatic lipid peroxidation , 2000, Journal of biochemical and molecular toxicology.

[22]  J. Thompson,et al.  Liver fatty acid binding protein: species variation and the accommodation of different ligands. , 1999, Biochimica et biophysica acta.

[23]  B. Halliwell.  Antioxidant defence mechanisms: from the beginning to the end (of the beginning). , 1999, Free radical research.

[24]  M Dietel,et al.  Increased expression of epidermal fatty acid binding protein, cofilin, and 14‐3‐3‐σ (stratifin) detected by two‐dimensional gel electrophoresis, mass spectrometry and microsequencing of drug‐resistant human adenocarcinoma of the pancreas , 1999, Electrophoresis.

[25]  E. Stadtman,et al.  Methionine residues may protect proteins from critical oxidative damage , 1999, Mechanisms of Ageing and Development.

[26]  E. Stadtman,et al.  Methionine sulfoxide reductase in antioxidant defense. , 1999, Methods in enzymology.

[27]  T. Yoshikawa,et al.  Oxidative stress in patients with hepatitis, cirrhosis, and hepatoma evaluated by plasma antioxidants. , 1998, Biochemical and biophysical research communications.

[28]  D. Cistola,et al.  Fatty acid binding proteins reduce 15-lipoxygenase-induced oxygenation of linoleic acid and arachidonic acid. , 1997, Biochimica et biophysica acta.

[29]  L. Banaszak,et al.  Intracellular lipid-binding proteins and their genes. , 1997, Annual review of nutrition.

[30]  E. Stadtman,et al.  Methionine residues as endogenous antioxidants in proteins. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[31]  K. Baba,et al.  Rat liver fatty acid-binding protein: identification of a molecular species having a mixed disulfide with cysteine at cysteine-69 and enhanced protease susceptibility. , 1996, Journal of biochemistry.

[32]  G. J. van der Vusse,et al.  Cellular fatty acid-binding proteins: their function and physiological significance. , 1996, Progress in lipid research.

[33]  N. Copeland,et al.  Chromosomal localization of the mammalian peptide-methionine sulfoxide reductase gene and its differential expression in various tissues. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[34]  J. Popp,et al.  Effect of the hepatocarcinogenic peroxisome proliferator Wy-14,643 in vivo: no increase in ethane exhalation or hepatic conjugated dienes. , 1995, Toxicology and Applied Pharmacology.

[35]  J. A. Thomas,et al.  Protein sulfhydryls and their role in the antioxidant function of protein S-thiolation. , 1995, Archives of biochemistry and biophysics.

[36]  Á. Catala,et al.  Inhibition of microsomal chemiluminescence by cytosolic fractions containing fatty acid binding protein. , 1995, Archives of physiology and biochemistry.

[37]  P Grasso,et al.  Hepatic peroxisome proliferation in rodents and its significance for humans. , 1993, Food and chemical toxicology : an international journal published for the British Industrial Biological Research Association.

[38]  U. Stenius,et al.  Peroxisome proliferation and resistance to hydrogen peroxide in rat hepatocytes: is development of resistance an adaptation to cytotoxicity? , 1992, Carcinogenesis.

[39]  R. Thurman,et al.  Induction of peroxisomes by treatment with perfluorooctanoate does not increase rates of H2O2 production in intact liver. , 1992, Toxicology letters.

[40]  U. Muller-eberhard,et al.  Localization of the heme‐binding protein in the cytoplasm and of a heme‐binding protein‐like immunoreactive protein in the nucleus of rat liver parenchymal cells: Immunocytochemical evidence of the subcellular distribution corroborated by radioimmunoassay and immunoblotting , 1990, Hepatology.

[41]  T. Watanabe,et al.  Long-term effects of hypolipidemic peroxisome proliferator administration on hepatic hydrogen peroxide metabolism in rats. , 1990, Carcinogenesis.

[42]  M. Rao,et al.  Oxidative DNA damage caused by persistent peroxisome proliferation: its role in hepatocarcinogenesis. , 1989, Mutation research.

[43]  H. Raza,et al.  Specific high affinity binding of lipoxygenase metabolites of arachidonic acid by liver fatty acid binding protein. , 1989, Biochemical and biophysical research communications.

[44]  F. Spener,et al.  Compartmentation of hepatic fatty-acid-binding protein in liver cells and its effect on microsomal phosphatidic acid biosynthesis. , 1989, Biological chemistry Hoppe-Seyler.

[45]  D. Das,et al.  Free radical scavenging by myocardial fatty acid binding protein. , 1989, Free radical research communications.

[46]  P. Tsichlis,et al.  Liver fatty acid binding protein is the mitosis-associated polypeptide target of a carcinogen in rat hepatocytes. , 1987, Proceedings of the National Academy of Sciences of the United States of America.

[47]  C. Elcombe,et al.  Lack of DNA damage or lipid peroxidation measured in vivo in the rat liver following treatment with peroxisomal proliferators. , 1987, Carcinogenesis.

[48]  R. Melnick,et al.  In vitro steady-state levels of hydrogen peroxide after exposure of male F344 rats and female B6C3F1 mice to hepatic peroxisome proliferators. , 1986, Carcinogenesis.

[49]  H. Shinozuka,et al.  Suppression of choline-deficient diet-induced hepatocyte membrane lipid peroxidation in rats by the peroxisome proliferators 4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio(N-beta-hydroxyethyl)- acetamide and di(2-ethylhexyl)phthalate. , 1986, Cancer research.

[50]  J. Reddy,et al.  Peroxisome proliferation and lipid peroxidation in rat liver. , 1986, Cancer research.

[51]  R. Ockner,et al.  Utilization of long chain fatty acids by rat liver: studies of the role of fatty acid binding protein. , 1979, Gastroenterology.