Study On Mechanism Of Copper Mediated Green Tea EGCG Oxidation Under The Effect Of Theanine

Green tea polyphenol,with the most abundant bioactive compound(-)-epigallocatechin-3-gallate(EGCG),is a plant-based prescription drug approved by the American Food and Drug Administration(FDA).EGCG has a variety of beneficial effects,such as anti-oxidation,prevention of cancer,cardiovascular disease,regulation of glucose and lipid metabolism.Some study has revealed that the biological effects of EGCG are mostly associated with its anti-oxidant or pro-oxidant properties,depending upon the dose levels and the biological circumstances.Clinical experiments reported that hepatotoxicity,nausea and abdominal pains were owing to high dose intake of EGCG in some individuals,which could ascribe to EGCG pro-oxidation.Reactive oxygen species(ROS),such as superoxide anion(·O2-),hydrogen peroxide(H2O2),generated via EGCG auto-oxidation;while transition metals,such as copper and iron ions,can promote EGCG oxidation and moreover,have Fenton reaction with H2O2,and producing hydroxyl radicals(·OH-),more toxic than the other two ROS.Meanwhile,EGCG transformed to quinone,subsequently attack the free thiol group of cysteine residues of proteins,and covalently bind to protein to form quinoprotein(QP).Copper is the third most abundant trace element in human body,with the highest concentration in liver,serve as co-factor of diverse enzymes.With exceptionally high redox activity,excessive copper can harm human body and cause protein dysfunction,hence intracellular free copper concentrations is well regulated to keep their quantities in line with the cellular necessary,whose limit pools estimated to be far less than a single free copper ion per cell.Compounds of dithiocarbamates(DTC)family are widely used in agricultural,industrial and therapeutic domains therefore high occurrence for human exposure to DTC.Disulfiram(DSF)along with its metabolized diethyldithiocarbamate(DEDTC),with strong capacity of metal-chelating,are able to inhibit the activity of variety metalloenzymes,such as Cu/Zn-superoxide dismutase(SOD)via chelating copper ions and forming copper complex(Cu(DEDTC)2).DEDTC is lipophilic and can act as a copper ionophore to transport copper into the liver cells,causing hepatocellular copper overload.And we found in this study that Cu(DEDTC)2 maintained redox activity.Liver is the major organ site of EGCG oxidative toxicity,once inhibiting the activity of SOD,a kind of ROS scavenger,and enrichment of redox-activity copper in liver in the meantime,the toxicity of EGCG intensively promoted as a consequence.A research previously showed that,EGCG and DEDTC,all at pharmacological dose,both intraperitoneal injected to mice simultaneously and evoked severe injury and lethality consequence mainly due to the amplified of EGCG oxidizing toxicity.We elaborated in this investigation the mechanism of the injury: 1.the level of EGCG auto-oxidation raised from weakened SOD activity along with enriched redox-activity copper in liver,resulting of EGCG consumption accelerating;2.the amplified EGCG oxidizing toxicity provoking the expression of hepatocellular inflammation and apoptosis gene and protein;3.QP robustly formation;4.lipid peroxidation.L-Theanine(L-T),a unique non-protein amino acid in tea plants,contributes to green tea infusion the umami taste,and has been demonstrated to have many beneficial physiological effects such as to improve cognition and mood.High-dose intake of EGCG can trigger oxidative stress and lead to hepatic toxicity.In current study,we investigated the effect of EGCG oxidation under the influence of L-T,and the regulation in vivo.The result in vitro shown that L-T bound to Cu including Cu ions and ceruloplasmin,a main Cu-containing protein,which lead to EGCG pro-oxidation and triggered DNA damage and QP formation,and we applied mice model for EGCG induced sub-acute hepatotoxicity via EGCG 55 mg/kg intraperitoneal injecting once daily for 5 days,and the results showed that L-T protected mice from EGCG caused elevated transaminase level,unregulated p53 genes,oxidative stress,DNA injury and QP formation in liver,whereas raised Nrf2 genes were down regulated by administration of L-T,hence we deduced that,L-T can inhibit EGCG auto-oxidation directly.These results indicated that L-T can efficaciously suppress EGCG oxidation caused toxicity,and should be of great significance for safe utilization of healthy functions of catechins.In conclusion,copper enhances EGCG toxicity via promoting EGCG auto-oxidation.DEDTC inhibited SOD activity,and transported the captured copper into liver cells,consequently EGCG oxidation enhancing,and reduced the safety threshold of EGCG or other catechins.L-T,derived from tea plant with EGCG,can effectively bind with copper and inhibit redox-activity copper along with Cp,main copper containing polyphenol oxidase,on promoting EGCG oxidation,therefore raise the safety threshold of catechins.