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

In this thesis, I have explored the role of three genes related to p53, namely p21, MDM2 and MDMX, in regulating ferroptosis, a form of non-apoptotic cell death. Ferroptosis, an iron-dependent mechanism that leads to cell death due to lipid peroxidation, has a large potential to be used as a cancer therapy. My results indicate that p21, the effector of p53-mediated cell cycle arrest, can suppress ferroptosis possibly through its interaction with CDKs. Further, that MDM2 and MDMX, the negative regulators of p53, can act as pro-ferroptosis agents and that this role is independent of p53. Using various approaches to alter their activity, I found that MDM2 and MDMX, likely working in part as a complex, normally facilitate ferroptotic death. They were found to alter the cellular lipid profile to prevent the cells from mounting an adequate defense against lipid peroxidation. For example, inhibition of MDM2 or MDMX lead to increased levels of FSP1 protein and a consequent increase in the levels of coenzyme Q₁₀, an endogenous lipophilic antioxidant. Moreover, I found that PPARα activity is essential for MDM2 and MDMX to promote ferroptosis. My findings also suggest that MDM2-MDMX inhibition might be useful for preventing degenerative diseases involving ferroptosis. Further, that MDM2/MDMX amplification may predict sensitivity of some cancers to ferroptosis inducers. Therefore, I believe that this thesis project has successfully identified several new regulators of ferroptosis and this knowledge can aid better design of therapies centered around ferroptosis.

Free radicals are thought to be involved in the destructive process of beta cells in Type 1 diabetes mellitus. Studies were performed to test the hypotheses (1) that malondialdehyde (MDA), a by-product of lipid peroxidation, affects $ beta$-cell function and integrity in vitro and (2) that such effects might be prevented in the BB rat (a model of spontaneous autoimmune diabetes) in vivo by administration of $ alpha$-phenyl-N-tert-butylnitrone (PBN), a free radical spin trap. First, islets of Wistar-Furth rats were studied at 12, 24 and 40 hr of culture in either 5.5, 11 or 16.5 mM glucose, and MDA at a range of concentrations ($6 times10 sp{-12}$-10$ sp{-3}$M). High concentrations of MDA inhibited glucose-stimulated insulin release without corresponding decreases in islet insulin content, suggesting that in situations with high predicted islet free radical content (e.g., autoimmune insulitis) beta cell function may be affected even before the cells are destroyed. Second, 28 diabetes-prone (BBdp) and 13 non diabetes-prone (BBn) rats were given PBN (20 mg/kg) s.c. 2x/day and 27 BBdp and 12 BBn rats received an equal volume of saline. PBN was able to decrease MDA in the absence of the autoimmune process and is remarkably non-toxic. However, it did not prevent diabetes for reasons which may include its concentration at the site of the inflammatory process or specificity to types of radicals trapped. Because it did decrease MDA, either a higher dose or a combination of PBN with other agents may hold promise for disease prevention.

A two by six factorial design was used to investigate the effect of zinc and all-trans retinoic acid (RA) on the growth of two human breast cancer cell lines differing in their expression of metallothionein (MT) and of estrogen receptors; MCF7 cells express estrogen receptors, BT-20 cells do not. Cells were treated with zinc to induce MT then treated with six concentrations of RA. Cell proliferation, lipid peroxidation, MT protein, MT mRNA and glutathione concentrations were measured. / BT-20 cells expressed higher constitutive MT concentrations than MCF7 cells. MT was significantly induced by zinc treatment in BT-20 cells but not in MCF7 cells. Low RA concentrations stimulated growth proliferation but higher concentrations inhibited cell proliferation. High RA concentrations increased lipid peroxidation. There was a significant negative correlation between lipid peroxidation and cell proliferation. Growth inhibition and lipid peroxidation were reduced by zinc in BT-20 cells but not in MCF7 cells. Glutathione did not appear to be a significant factor. / Induction of metallothionein by zinc may modulate the growth inhibitory effects of all-trans retinoic acid in human breast cancer cells. One mechanism of growth inhibition may be through increased lipid peroxidation.

No description available.

No description available.

To examine the effects of fat composition and supplemental vitamin E (Vit E) and selenium (Se) on in vivo lipid peroxidation, diet-induced hypercholesterolemia, and glutathione (GSH) metabolism, male Syrian hamsters were fed for three weeks butter fat (BF-) or fish oil- (FO-)based diets supplemented with Vit E and/or Se. The effect of supplemental Vit E and Se on tissue lipid peroxidation (LPO), glutathione peroxidase (GSH-Px) activity and GSH concentrations differed between heart and liver and also was affected by dietary fat. The reduced glutathione/oxidized glutathione (GSH/GSSG) ratio was more consistently associated with tissue lipid peroxidation than was tissue Vit E content. Plasma lipids were lowered with supplemental Se and Vit E. Se supplementation, however, exerted a more potent hypolipidemic effect than Vit E. A pro-oxidative action of Se in hearts of FO-fed hamsters was noted, which was inhibited by supplemental Vit E. Hence, the combination of Vit E and Se may offer the most benefit against diet-induced oxidative stress and hyperlipidemia.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / The protective role of dimethylsulfoxide (DMSO), lipoic acid (LA) and ternatin (TN), known free radical scavengers and cell membrane protectants were evaluated in an experimental model of testis ischemia/reperfusion. One hundred and twenty young male Wistar rats, mean weight 172.5Â15g, were randomized in five equal groups (GS - Sham, GC â Control, GD â Dimethylsulfoxide, GA â Lipoic acid and GT/D â Ternatin/DMSO). Rats of each group were distributed into four subgroups (T-0, T-1, T-3 and T-6), each comprising six animals. All surgical procedures were performed under inhalatory ether anesthesia. Experimental rats received intraperitoneal (i.p.) injections of 3% aqueous solution of DMSO (10 ml/kg, GD), LA (10 mg/kg, GA) or TN/DMSO (12 mg/kg, suspended in 3% aqueous solution of DMSO), GT/D) 24, 12 and 4 hours before cord detorsion. Last dose was given 1 hour before ischemia induction or second Sham operation. Sham (GS) and Control (GC) rats received NaCl 0.9% 10 ml/Kg i.p. Thiobarbituric acid reactive substances (TBARS) and reduced glutathione (GSH) levels (Âmol/g wet tissue) were assayed in testis. Total Antioxidant Power (TAP) (ÂM) was measured in blood plasma. Data were first analyzed by Kolmorogov-Smirnov test to assess differences in the general shapes of distribution. Comparisons between groups were made using the t test for normally distributed data and the Mann-Whitney U test for non-normally distributed data. P values of <0.05 were considered to indicate statistical significance. Comparisons between antioxidant-treated and saline-treated rats (Control group, GC) were made using Dunnettâs test. Spermatic cord torsion induced significant decrease in testis MDA levels during ischemia and reperfusion in DMSO (GD) and LA (GA) and in TN/DMSO (GT/D) treated rats during reperfusion, compared with respective controls. MDA levels were significantly increased in GC compared with Sham (GS) rats at the end of the ischemia and during reperfusion as well as one hour after detorsion (T-1) in GC rats compared with T-0, in the same group, indicating additional damage imposed by the afflux of oxygenated blood (reperfusion) to the ischemic tissues. GSH levels decreased significantly in saline-treated (GC) compared with Sham-operated rats and increased significantly in DMSO and LA treated rats at the end of ischemia and during reperfusion. The model utilized in this study did not induce systemic effects. TAP was significantly increased during reperfusion (T-1) in LA (GA) and TN/DMSO (GT/D) pretreated rats, compared with respective controls. The results of the present study reinforce the hypothesis that ischemia and reperfusion are free radicals generating processes. The different antioxidants studied here have demonstrated great efficacy in cell membrane protection by decreasing lipid peroxidation in all experiments. Increased reduced glutathione levels in DMSO and LA treated rats support the view that these substances can exert a protective effect against testis oxidative stress injury caused by ischemia/reperfusion in rats subjected to torsion of the spermatic cord lasting three hours. TN protective effects in reducing lipoperoxidation were more pronounced in ischemic tissues. / O efeito protetor do dimetilsulfÃxido (DMSO), do Ãcido lipÃico (LA) e da ternatina (TN), conhecidos seqÃestradores de radicais livres e protetores da membrana celular foram avaliados utilizando um modelo experimental de isquemia/reperfusÃo do testÃculo. Cento e vinte ratos Wistar jovens com peso mÃdio de 174 g foram distribuÃdos ao acaso em cinco grupos (GS - Simulado, GC â Controle, GD â DimetilsulfÃxido. GA â Ãcido lipÃico e GT/D â Ternatina/DMSO) numericamente iguais e posteriormente redistribuÃdos em quatro subgrupos (T-0, T-1, T-3 e T-6), com seis animais cada. Todos os procedimentos cirÃrgicos foram realizados sob os efeitos da anestesia geral inalatÃria. Os animais receberam injeÃÃes intraperitoniais (i.p.) de soluÃÃo aquosa de DMSO a 3% (10 ml/kg, GD), LA (10 mg/kg, GA) ou TN+DMSO (12 mg/kg, GT/D, em soluÃÃo aquosa de DMSO a 3%) 24, 12 e 4 horas antes da destorÃÃo do cordÃo espermÃtico. A Ãltima dose foi administrada uma hora antes da induÃÃo da isquemia ou da segunda operaÃÃo simulada (Sham). Os ratos do grupo Simulado (GS) e do grupo Controle (GC) receberam de NaCl 0,9% (10 ml/Kg) i.p. Foram determinadas as concentraÃÃes de substÃncias reativas ao Ãcido tiobarbitÃrico (TBARS) e glutationa reduzida (GSH) no tecido (testÃculo), e a capacidade antioxidante total (CAP) do plasma. Para determinaÃÃo do padrÃo de distribuiÃÃo das amostras utilizou-se o teste de Kolmorogov-Smirnov. As comparaÃÃes entre os grupos foram feitas utilizando-se o t teste ou o teste U de Mann-Whittney quando assim indicado. Para as comparaÃÃes dos grupos tratados com antioxidantes ao grupo Controle (GC) utilizou-se o teste de Dunnett. Valores de p<0,05 foram considerados significantes. A torÃÃo do cordÃo espermÃtico induziu uma reduÃÃo significante das concentraÃÃes de MDA, no tempo mÃximo de isquemia e na reperfusÃo, em tratos prÃ-tratados com DMSO (GD) e LA (GA) e durante a reperfusÃo naqueles prÃ-tratados com TN/DMSO (GT/D), comparados com seus respectivos controles. Os nÃveis de MDA estavam significantemente elevados nos ratos do grupo GC comparados aos animais do grupo GS, no tempo mÃximo de isquemia e durante a reperfusÃo, bem como uma hora apÃs a destorÃÃo (T-1) comparada ao T-0, demonstrando o dano adicional decorrente do afluxo de sangue oxigenado (reperfusÃo) ao tecido isquÃmico. As concentraÃÃes de GSH diminuÃram nos ratos prÃ-tratados com soluÃÃo salina e aumentaram significativamente nos ratos prÃ-tratados com DMSO ou LA, no tempo mÃximo de isquemia e durante a reperfusÃo. O modelo isquÃmico utilizado nÃo foi capaz de gerar efeitos sistÃmicos. A capacidade antioxidante total aumentou significantemente durante a reperfusÃo (T-1) nos ratos prÃ-tratados com LA (GA) e TN/DMSO (GT/D) comparados aos respectivos controles. Esses resultados fortalecem a hipÃtese de que a isquemia e reperfusÃo sÃo processos geradores de radicais livres. Os diferentes antioxidantes estudados mostraram grande eficiÃncia na proteÃÃo da membrana celular, reduzindo a peroxidaÃÃo lipÃdica, em todos os experimentos. O aumento dos nÃveis de glutationa reduzida, nos ratos prÃ-tratados com DMSO e LA, mostra que estes antioxidantes exercem uma proteÃÃo eficaz contra o estresse oxidativo induzido pela isquemia/reperfusÃo em ratos submetidos à torÃÃo do cordÃo espermÃtico por trÃs horas. Os efeitos protetores da ternatina se manifestam com maior intensidade no tecido isquÃmico, no controle da peroxidaÃÃo lipÃdica.

The nonapoptotic form of regulated cell death known as ferroptosis is an attractive target for combating numerous diseases. Ferroptosis is an iron-dependent death of cells by lipid peroxidation. Pharmacological inhibition of anti-ferroptotic pathways is a promising therapeutic avenue for treatment of cancer, and death by ferroptosis has been implicated in numerous neurodegenerative and ischemia-reperfusion-driven diseases. Therefore, demystifying the dynamics of lipid peroxidation in this cell death process opens a window to understanding disease processes and how to treat them. This dissertation makes use of ferroptosis-modulating compounds as chemical probes to elucidate the roles of different subcellular membranes in ferroptotic lipid peroxidation. Chapters two and three explore the structure-activity-distribution relationship of fatty acids and the ferroptosis inducer FINO2, respectively, and together demonstrate the endoplasmic reticulum as a driver of lipid peroxidation in ferroptosis. Chapter two makes use of stimulated Raman scattering imaging, while chapter three uses confocal fluorescence imaging. Chapter four shifts gears to focus on development of FINO2 as a drug lead, performing structure activity relationship analysis to increase the potency and pharmacological properties of the analogs. Altogether, this work answers questions about how cells die by ferroptosis, and provides footwork for how we can better modulate ferroptosis against cancer and other illnesses.