The Cytoprotection By Almond Skin Extracts To Rat Hepatocytes Toxicity By Fructose And Its Two Metabolites

Dietary fructose consumption is associated with the development of obesity and hepatic steatosis. A two hit hypothesis by Day has been proposed for the pathogenesis of non-alcoholic steatohepatitis (NASH), the first hit being hepatic steatosis, and the second hit being oxidative stress. Excessive sugar intake in animal models may cause tissue damage associated with oxidative and carbonyl stress cytotoxicity as well as inflammation. AGEs (advanced glycation end products) formation is also key factors that are involved in aging and chronic diseases, including NASH (non-alcoholic steatohepatitis), NAFLD (non-alcoholic fatty liver disease) and diabetes. In addition, rats fed fructose developed insulin resistance and increased their serum methylglyoxal (MGO), a fructose metabolite. As previously reported, glyoxal (GO) induced hepatocyte toxicity could be attributed to mitochondrial toxicity. However the toxic mechanism of fructose, a glyoxal precursor has not been investigated. In present thesis, we investigate the the cytotoxicityTo test out whether hepatocyte toxicity is induced by fructose"Molecular Mechansm Removed"a cell free model was used to study the carbonyl stress caused by fructose and its metabolites (glyceraldehyde and glycoaldehyde). Isolated hepatocytes were incubated with different concentrations of fructose and its metabolites (glycolaldehyde and glyceraldehyde) . The toxicity and the molecular cytotoxic mechanisms involved were also investigated. Fructose alone did not carbonylate bovine serum albumin (BSA) in a cell free system but did so in the presence of low concentrations of FeII/H2O2 whereas CuI/H2O2 was less effective. Furthermore protein carbonylation by the fructose metabolites glyceraldehyde or glycolaldehyde were also markedly increased by FeII/H2O2 whereas CuI/H2O2 was less effective. Protein carbonylation was attributed to glyoxal formation as the glyoxal trapping agent aminoguanidine completely prevented the protein carbonylation. Glyoxal were also much more effective than most carbonyls at causing protein carbonylation. Glyoxal was rapidly formed when fructose, glycolaldehyde or glyceraldehyde were oxidized by FeII/H2O2.. Hydroxyl radical scavengers inhibited the protein carbonylation induced by fructose, glyceraldehyde or glycolaldehyde and catalysed by FeII/H2O2 but not by CuI/H2O2 suggesting that hydroxyl radicals were formed by a FeII catalysed Fenton reaction but less so by a CuI catalysed Fenton reaction. In the hepatocytes inflammation model, fructose itself was toxic at 1.5M, whereas only 10mM fructose was toxic when hepatocytes were also exposed to a non-cytotoxic dose of H2O2. The cytotoxicity was further increased in the presence of FeIII:HQ. The molecular mechanism for fructose cytotoxicity involved oxidative stress as endogenous reactive oxygen species (ROS) and H2O2 formation preceded cytotoxicity. Fructose/H2O2 cytotoxicity was markedly increased by trace amounts of FeIII: 8-hydroxyquinoline (HQ). Fructose, glyceraldehyde or glycolaldehyde cytotoxicity was also markedly increased by a non toxic H2O2 infusion generated by glucose/glucose oxidase which was further increased if the hepatocytes were loaded with non toxic 2μM FeIII:HQ. The order of the increased fold of cytotoxicity was : Fructose > glycolaldehyde > glyceraldehydes. Cytotoxicity induced by glyceraldehyde or glycolaldehyde/FeIII:HQ/H2O2 but not glyceraldehyde or glycolaldehyde/CuII:HQ/H2O2 was inhibited by hydroxyl radical scavengers. The glyoxal scavenger aminoguanidine inhibited the cytotoxicity by glyceraldehyde or glycolaldehyde /FeII:HQ/H2O2 and glyceraldehyde or glycolaldehyde /CuII:HQ/H2O2. Protein carbonylation by glyceraldehyde/FeII:HQ/H2O2 was inhibited by hydroxyl radical scavengers or the glyoxal scavenger aminoguanidine.Oxidative and carbonyl stress induced by dicarbonyls such as glyoxal or methylglyoxal, are detrimental in the pathogenesis of diabetic complications, as well as in other chronic diseases. However, this process may be decreased by dietary bioactive compounds. Almond skin is an abundant source of bioactive compounds and antioxidants, including polyphenolic flavonoids, which may contribute to the decrease in oxidative and carbonyl stress. In this study, four Almond Skin Extracts (ASEⅠ, ASEⅡ, ASEⅢ, and ASEⅣ) were prepared by different methods and evaluated for their antioxidant activity. The order of the polyphenol content (totalμM gallic acid equivalents) of the four extracts was found to be, in decreasing order of effectiveness: ASEⅠ> ASEⅢ> ASEⅣ> ASEⅡ. The order of Ferric-reducing antioxidant power (FRAP,μM FeSO4/g) value, in decreasing order was ASEⅠ(216) > ASEⅢ(176) > ASEⅣ(89) > ASEⅡ(85). The order of ASE effectiveness for decreasing protein carbonylation induced by the copper Fenton reaction was ASEⅠ> ASEⅣ> ASEⅡ> ASEⅢ. The order of antioxidant effectiveness for inhibiting tertiary-butyl hydroperoxide (TBH) induced microsomal lipid peroxidation was ASEⅠ> ASEⅣ> ASEⅡ, ASEⅢ. Also, the order of ASE effectiveness for inhibiting TBH induced hepatocyte toxicity was: ASEⅢ, ASEⅣ> ASEⅠ, ASEⅡ. Catechin also protected hepatocytes from TBH induced hepatocyte lipid peroxidation and cytotoxicity. In a cell free model, ASEⅠ,catechin or epicatechin rescued lbumin from protein carbonylation induced by glyoxal. Equimolar concentrations of catechin or epicatechin rescued serum albumin from protein carbonylation induced by methylglyoxal. ASEⅠCatechin, epicatechin and ASEⅠall decreased glyoxal induced hepatocyte toxicity and Reactive oxygen species (ROS) formation in GSH depleted hepatocytes. Catechin and epicatechin protected against GO or MGO induced hepatocyte toxicity, protein carbonylation and ROS formation. Catechin was more effective than epicatechin.Conclusion: Glyoxal is formed from fructose and its metabolites by Fenton reaction and contrbutes to cytotoxicity in a inflammatory model. Fatty liver induced by an excessive sugar diet in animal models has been proposed as the first hit for NASH whilst oxidative stress induced by the oxidation of fructose or fructose metabolites by Fenton FeII/H2O2 could be a'second hit'. A perpetual cycle of oxidative stress in hepatocytes could lead to cytotoxicity and NASH development. Bioactive almond skin constituents in the non-lipophilic polyphenol extract were the most effective at protecting hepatocytes against hydroperoxide induced hepatocyte oxidative stress and in protecting against dicarbonyl induced cytotoxicity. Catechins, the major polyphenol in the extract, were also effective at preventing GO or MGO cytotoxicity likely by trapping GO and MGO and/or rescuing hepatocytes from protein carbonylation.