Molecular Mechanisms For The Regulation Of Mitochondrial Function By Hazardous Food Product Acrylamide And Functional Food Factor Sesamol

Mitochondrion is a kind of double-membranous organelle in most eukaryote. The most prominent metabolic process carried out by mitochondria is oxidative phosphorylation(OXPHOS) to generate ATP, the universal energy currency. Aside from their role as intracellular power plants, mitochondria play fundamental roles in regulating multiple cellular processes such as apoptosis, redox status, and cell signaling.The impairment of mitochondrial function is associated with metabolic syndrome, obesity, cardiovascular diseases, neurodegerative dieseases, and tumors. It is known that the dietary safety and nutrition have highly impact on human health. However, the molecular mechnisms of most hazardous food factors and functional food factors are unclear, while mitochondrion could play a pivotal role in expressing biological effects of these food factors in cells.This study targets acrylamide, a hazardous product of food processing, and sesamol which is served as a functional food factor:(1) Acrylamide is produced during high-temperature process in high-strach food. It is reported that acrylamide has neurotoxicity and reproduction toxicity upon long term exposure. However, the regulating mechnisms of acrylamide on the mitochondrial energy metabolism and signaling pathways are unclear;(2) Sesamol is a liposoluble lignans extraction and prominent fragrance component in sesame oil. The bioactivities of sesamol include anti-bacterial, anti-oxidantive, anti-tumor, etc. As a new functional food factor, sesamol has potential application in the food industry. The methods and results are as following:1. Microglial cytotoxicity of acrylamide, a toxin generated during high temperature food producing process(1) This study examined the apoptotic and inflammatory properties of ACR in an immortalized mouse microglia cell line BV2. The exposure of BV2 cells to ACR reduced cell viability and induced apoptosis in a concentration-dependent manner. ACR also induced apoptosis in primary microglia. After ACR treatment, mitochondrial dysfunction was associated with a decrease of mitochondrial membrane potential and the Bcl-2/Bax ratio, thus resulting in activation of mitochondrion-driven apoptotic signaling. This was accompanied by(a) the modulation of redox-sensitive signaling: suppressed of Akt activation and activation JNK and p38 cascades and(b) increased expression of NF B and downstream inducible nitric oxide synthase(i NOS), as well as nitric oxide generation, thus supporting indirectly a pro-inflammatory effect of ACR. Nrf2 expression, but not its translocation to the nucleus, was also increased.(2) The effects of ACR on mitochondria respiration and cellular redox status and the protective effect of N-acetyl-L-cysteine(NAC) were evaluated. ACR impaired cellular energy metabolism by decreasing mitochondrial respiration, anaerobic glycolysis, and lowering the expression of complex I, III, and IV subunits. The electrophilic attack of ACR on GSH resulted in substantial loss of GSH/GSSG ratio. These changes in the cell’s redox status elicited by ACR resulted in increased H2O2 formation. The changes in mitochondrial functionality and complex subunit expression caused by ACR were reversed by NAC. Likewise, NAC restored the cell’s redox status by increasing GSH levels with concomitant attenuation of H2O2 generation; these effects resulted in decreased apoptotic cell death and inflammatory responses.Results from(1) and(2) indicated that ACR-mediated mitochondrial dysfunction along with a more oxidized redox status seems to be critical events leading to the activation of intrinsic apoptotic pathway and inflammatory responses.2. Mechnisms of sesamol on regulating mitochondrial functions and apoptosis of hepatoma cells(1) The mitochondria-dependent intrinsic apoptotic effects of sesamol in human liver cancer cell line Hep G2 were studied. The exposure of Hep G2 cells to sesamol reduced cell viability and induced apoptosis in a concentration-dependent and time-dependent manner. Sesamol induced mitochondria dysfuction with a decrease of the mitochondrial membrane potential and Bcl-2/Bax ratio, thus resulting in the activation of mitochondrion-driven apoptotic signaling. This was accompanied by the modulation of redox-sensitive signaling: suppressed of Akt activation and activation of JNK and p38 pathways. Moreover, sesamol showed nuclear localization in Hep G2 as visualized by confocal microscopy.(2) The inhibitory effects of sesamol on autophagy and mitophagy in Hep G2 cells were investigated. Sesamol treatment suppressed the PI3 K Class III/Beclin 1 signaling and the expression of downstream autophagic indicator protein LC3, which indicated that sesamol inhibited autophagy in Hep G2 cells. Moreover, sesamol suppressed mitophagy as visualized by fluorenscent microscopy. Rapamycin, an autophagy stimulus, could inhibit the apoptotic effects of sesamol. Metabolic flux assay demonstrated that sesamol suppressed mitochondrial energy metabolism and elevated the anaerobic glycolysis in Hep G2 cells.(3) Multiple measurements including ultraviolet and visible(UV/Vis), fluorescence, circular dichroism(CD), fourier transform infrared(FT-IR) spectroscopic techniques, and molecular modeling techniques were employed in this study to demonstrate the hitherto unknown interaction mechanism between sesamol and ct DNA. We have confirmed that fluorescence quenching mechanism of sesamol by ct DNA was a static quenching type and the hydrogen bond and hydrophobic interaction force may play a major role in the interaction. CD spectrum implied that changes in the ct DNA secondary structure may be the result of the formation of a complex between ct DNA and sesamol, and the binding mode was not the intercalation. FT-IR spectrum further suggested that both the phosphate groups and the bases of DNA react with sesamol. Together these findings show that sesamol can bind with DNA mainly by minor groove interaction, but not intercalation interaction. Furthermore, results obtained from molecular docking corroborate the experimental results acquired from spectroscopic investigations.Overall, data in this study unveil that sesamol impaired Hep G2 cells energy metabolism impairments through the inhibition of autophagy. The declined mitophagy might increase the accumulation of sesamol-induced damaged mitochondria, thus resulting in the stimulation of intrinsic apoptosis in Hep G2 cells. Moreover, the sesamol can interact with DNA in a minor groove mode.