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No description available.

No description available.

No description available.

Oxidative degradation of lipids, not only leads to the quality deterioration in foodstuffs but also associates with a multitude of physiological processes. One of the causations involved in these damaging effects is the generation of reactive carbonyl species (RCS) in lipid peroxidation process. RCS are notorious toxins that possess reactivity towards biological nucleophiles (such as proteins and DNA) with potential functional alternation in these biomolecules. Therefore, the exogenous intervention is required to inhibit the toxicity related to RCS/lipid peroxidation. In present study, the screening for effective natural polyphenols to trap two representative RCS, acrolein (ACR) and 4-hydroxy-trans-2-nonenal (HNE), was performed with mechanism elucidation. It was found that the polyphenols from the categories of flavan-3-ols, theaflavins, cyanomaclurins and dihydrochalcones were effective scavengers of ACR/HNE. Subsequently, facilitated by HPLC, LC-MS/MS and NMR analysis, the characterization of polyphenols’ as sacrificial nucleophiles towards these two electrophiles products was accomplished. Michael addition at A ring of polyphenols’ to the C=C bond of ACR/HNE was suggested to be responsible for trapping of these two RCS and thus render their active sites unavailable to covalently modify critical biomolecules. Further investigation of phloretin’s effect to attenuate ACR-induced modification on lysine residue and proteins was carried out. Phloretin’s protective effect against ACR’s toxicity was clearly reflected by its inhibition of the formation of Nε-(3-formyl-3,4-dehydropiperidino) lysine (FDP-lysine), blocking the electrophilic site in FDP-lysine, lowering protein carbonylation in bovine serum albumin (BSA) and lessening protein oligomerization in bovine pancreas ribonuclease A. Such protection might be mediated by phloretin’s directly trapping of ACR and consequently deactivation of ACR in covalent modification of amino acids and proteins. The biological relevance of polyphenols’ trapping activity of ACR was explored in a cell culture model. Natural polyphenols including phloretin, EGCG and quercetin were proved to be active to inhibit ACR-induced cytotoxicity in human neuroblastoma SH-SY5Y cells. The cytoprotection of phloretin (as the most potent one in alleviation of ACR stress) was suggested to be achieved through the reduction of the increased cellular protein carbonyl level as revealed by Western blotting analysis. In the final part of this study, an in vitro system containing metal-catalyzed fatty acids and BSA was established to study the modification on protein induced by lipid peroxidation and possible inhibitory effects conferred by some natural polyphenols. The protective effects of these polyphenols against lipid peroxidation-induced modification on BSA was manifested by the observation of reduced levels of high-molecular-weight proteins, MDA-related fluorescent substances and protein carbonylation. However, poor correlations were found between such protection and antioxidant activities, suggesting alternative mechanisms were existed such as carbonyl-scavenging. In conclusion, findings from the present study highlighted certain kinds of natural polyphenols as promising agents in counteracting RCS/lipid peroxidation-induced toxicity in amino acid, protein and cell models. / published_or_final_version / Biological Sciences / Doctoral / Doctor of Philosophy

published_or_final_version / Biological Sciences / Master / Master of Philosophy

Aldehydic lipid peroxidation products, free radical mediated oxidative stress and antioxidant status were investigated in three in vitro plant systems with the objective of exploring the involvement of oxidative stress in plant tissue cultures. <i>Daucus carota</i> was used as a model system for the study of somatic embryogenesis and for the development of an enzyme linked immunosorbant assay (ELISA) technique, for the detection of hydroxynonenal-protein adducts in plant extracts. This study shows for the first time, in plants, that exogenously applied lipid peroxidation products hydroxynonenal (HNE) and malondialdehyde (MDA) inhibit callus proliferation and development (as somatic embryogenesis). Removal of the aldehydes led to a reversal of inhibitory effects. For the first time, HNE and MDA were measured in extracts of callus generated from different explants of <i>Ipomoea batatas</i> (an economically significant crop species). Using ELISA, HNE-protein adducts have been detected, in extracts from all three cultivars of I. batatas tested. Increases in the activities of the antioxidants, catalase and peroxidase were also observed in nodes of <i>I. batatas</i> after transfer to callus induction medium. Three callus cultures of <i>Glycine max</i>, one of which was habituated against benzylamino purine, the second contained chlorophyll (green) and the third, devoid of chlorophyll (white) were profiled for lipid peroxidation products and antioxidant activity. This is first report of the detection free HNE and MDA in <i>G. max</i> and were detected in all three callus types, with the highest concentrations for both aldehydes in the habituated callus line. HNE-protein adducts were only detected in the white callus line of <i>G. max</i>. The level of hydroxyl radical activity was found to be increased in aged callus compared to callus taken from the mid-point of the subculture cycle. Low activities of catalase, peroxidase and other antioxidants were found in the habituated callus. It is proposed that increased amounts of hydroxyl radicals, high levels of HNE and MDA and the low activity of antioxidants leads to oxidative stress in extreme conditions such as habituation in <i>in vitro </i>plant systems. This study has implications regarding the understanding of <i>in vitro</i> plant recalcitrance.

Cholesterol is one of the most abundant lipids in the body, and like all unsaturated lipids, it can be oxidized by a variety of reactive oxygen species (ROS). Lipid peroxidation is one of the main pathways by which ROS induce oxidative damage, and has been linked to neurodegenerative and cardiovascular diseases. In 2003, Wentworth et al. detected both 3β-hydroxy-5-oxo-5,6-secocholestan-6-al (secosterol-A) and its intramolecular aldolization product 3β-hydroxy-5β-hydroxy-B-norcholestane-6β-carboxaldehyde (secosterol-B) in human atherosclerotic plaques – compounds which, at the time, were only known to be formed by cholesterol ozonolysis. However, our group has shown that cholesterol 5α-hydroperoxide, which is the product of the reaction of cholesterol with singlet oxygen, can undergo acid-catalyzed Hock fragmentation to generate secosterol-A and -B as well. Nevertheless, cholesterol 5α-hydroperoxide readily rearranges to a more thermodynamically stable cholesterol 7-hydroperoxide. Herein we show that cholesterol 7-hydroperoxide, the main product of cholesterol autoxidation, can also undergo acid-catalyzed Hock fragmentation that gives rise to electrophilic species with similar chromatographic characteristics to those that were allegedly identified as secosterol-A and -B. We also proposed to prepare authentic products of the Hock fragmentation of cholesterol 7-hydroperoxide by subjecting Δ⁶’⁷-cholesterol to ozonolysis. Herein, we explore the limitations and complications of Δ⁶’⁷-cholesterol ozonolysis as well as cholesterol 7-OOH Hock fragmentation which both resulted in unexpected (unprecedented) products.

Heat-induced hepatotoxicity accompanying hyperthermic liver perfusion was studied in the isolated, haemoglobin-free perfused rat liver. Trypan blue uptake, a sensitive indicator of cell death, was used to examine the relationship between the efflux of oxidized glutathione (oxidative stress), the appearance of cytosolic enzymes in the perfusate and cell death. Livers were perfused at 37, 42, 42.5 and 43°C. The efflux of total glutathione (GSH) and oxidized glutathione (GSSG) increased with time and temperature. Differences between temperature groups were significant for both parameters for 37 versus 42, 42.5 and 43°C (p < 0.05). Temperature-related differences in GSH levels appeared at 15 min for 37 versus 42 °C and in GSSG levels at 30 min for 37 versus 42 and 42.5°C. Biliary excretion of total GSH increased from 72 nmol at 37°C to 144 nmol at 42°C, 160 nmol at 42.5°C and 124 nmol at 43°C, which was significant for 37 versus 42 and 42.5°C (p < 0.05). The release of allantoin into the perfusate, a measure of purine catabolism and flux through xanthine oxidase, was increased at 42, 42.5 and 43°C compared to 37°C (p < 0.05). Liver injury was assessed by measuring the release of asportate aminotransferase (AST) and lactate dehydrogenase (LDH) and uptake of trypan blue after perfusion at each temperature. There was a pronounced release of LDH and AST into the perfusate after 60 min of perfusion at 42, 42.5 and 43°C, the levels of which were significantly different from the 37°C mean level. There was no uptake of trypan blue after 60 min perfusion at 37°C. Perfusion at 42, 42.5 and 43°C resulted in the uptake of trypan blue in the pericentral areas, but the dye uptake was significant (p < 0.05) compared to 37°C at 42.5 and 43°C only. These data show that heat-induced pericentral cell death is minimal after 60 min at 42-43°C, and that the biochemical processes which occurred during this period suggest 'oxidative stress' as a causative factor in hyperthermic hepatotoxicity. In addition, this liver toxicity is probably related to xanthine oxidase activity or the depletion of GSH as the initiating event which leads to lipid peroxidation and cellular damage.

No description available.