Omics Analysis On Mechanisms Of Glutathione And Zinc Ion In Relieving Ochratoxin A-induced Toxicity

As a secondary metabolite, Ochratoxin A (OTA) is a mycotoxin produced by fungi of two genera: Aspergillus. sp and Penicillium. sp. Mechanism of ochratoxin A toxicity has been a focus for researchers and results have shown that:OTA is nephrotoxic, hepatotoxic, genetoxic, phytotoxic, etc. However, details of the mechanism is still unclear. Accumulated reactive oxygen species and activated oxidative stress might account for part of toxicity. Based on studied from physiological, proteomic and transcriptional perspectives previously, we found that both reduced glutathione and zinc ions could relieve the OTA-induced oxidative stress. Herein, proteomics and transcriptome study, combined with real-time quantitative PCR are done to analysis the effect of fluctated GSH metabolism on the OTA toxicity effect and the proteomic changes by zinc ions on the OTA-treated HepG2cell, to reveal the possible mechanisms.In this thesis, the main results are as follows:(1) Two-dimensional electrophoresis (2DE) analysis of Arabidopsis thaliana detached leaves treated by buthionine sulfoximine (BSO), OTA and BSO+OTA showed12protein spots were identified differentially expressed, mainly involved in GSH synthesis, GSH metabolism, energy metabolism, etc. Under OTA stress, GSH synthesis inhibition by BSO will reactive the metabolic process of GSH catalyzed by GSTs, promote the degradation of the ATP synthase and inhibit the formation of photosynthesis complexes.(2) RNA-seq analysis through Illumina HiSeqTM2000sequencing of Arabidopsis thaliana wild type (WT), glutathione peroxidases (GPX) mutant gpx2and the OTA-treated group of both kinds of seedlings show that, there are637differentially expressed genes between the WT and WT (+) group,448between gpx2vs gpx2(+),159between WT vs gpx2and196between WT (+) vs gpx2(+). Gene ontology (GO) analysis shows that differentially expressed genes are mainly involved in the biological processes of cell apoptosis, cell metabolism, cell component biosynthesis, respond to stimuli, etc. OTA fluctates GSH metabolism in the WT and gpx2seedlings and promotes the GSH metabolic pathways catalyzed by GST and ascorbate-glutathione (ASC-GSH) cycle. Decreased expression of GPX2induces blocked GSH metabolism and up-regulated expression of genes encoding the tau family of GSTs. OTA, at the transcriptional level, restrains arabidopsis elongation of main root and differentiation of lateral root and root hair by promoting the expression of basipetal auxin transporter gene ABCB4and ZIFL1, down-regulating the DWARF1expression which encoding regulating protein on the auxin transport and reducing extensin protein-coding gene expression, etc.(3) Low concentration of dehydroascorbic acid (DHA) and N-acetyl cysteine (NAC) have a protective effect on HepG2cell viability, and can effectively relieve the OTA toxicity on the cell viability. OTA inhibites the SOD enzyme activity and1mM DHA or NAC pretreatment for12h have a raised effect. But BSO could aggravate the OTA toxicity of decreasing CAT enzyme activity with the DHA pretreatment has an up-regulating effect.(4) Two-dimensional electrophoresis analysis of Zn2+, OTA and OTA+Zn2+treated groups of HepG2 cells for24h detected25,47, and41differentially expressed proteins, compared with control, respectively. A total of51points are identified unambiguously and the proteins are mainly involved in the processes of primary metabolism,(carbohydrate metabolism, amino acid metabolism, lipid metabolism, etc.), the celluar structure and cytoskeleton, stress response, cell apoptosis regulation, nucleotide metabolism, etc. Analysis on the proteomics show that Zn2+treatment can down regulate the amino acid metabolism, increase the carbohydrate hydrolysis and protein synthesis. On some extent, Zn2+induces apoptosis and the imbalance of intracellular metal ions homeostasis. OTA induces pro-apoptosis protein accumulation, activates carbohydrates degradation by pentose-phophate pathway, changes expressions of endoplasmic reticulum stress related protein and destroies the cytoskeleton. Zn2+can alleviate OTA-induced decrease of the cell viability and the significant difference begins from12h. In addition, Zn2+inhibits OTA-induced early apoptosis. Both OTA and zinc ions can activate the endoplasmic reticulum stress response. At12h, OTA most significantly activates the genes expression of unfolded protein response and corresponding pro-apoptosis genes, but Zn2+can inhibit apoptotic signals by down regulating DDIT3expression, and BBC3, FAS etc. in p53pathway. Also, Zn2+can induce the MAPK cascade signal itself at the same time.