Effects Of Oxidized Tyrosine Products On REDOX Status In HepG2Cell

The amino acid residues of protein are vulnerable to be oxidized in the processing. Thereis an increasing attention about the effect to the body after intake of the oxidized protein,while the mechanism has been rarely reported. As one part of tyrosine oxidized products,dityrosine and3-nitrotyrosine has been seen as the protein oxidative damage biomarkers,however it is rare to see a report about the influence of dityrosine and3-nitrotyrosineaccumulation to human body. Firstly, in this paper, dityrosine and3-nitrotyrosine wasincubated with bovine serum albumin, whey protein and ovalbumin for exploring the effect oftyrosine products to the protein in body and the protein in food. Secondly, HepG2cell was useto simulate body environment for exploring the effect of dityrosine and3-nitrotyrosine to thebody cells.Methods:(1) Dityrosine preparation: by comparing the dityrosine yield in Cu2+/H2O2system and Fe2+/H2O2system, determined the optimal synthesis conditions, thencharacterized the product through3D scanning fluorescence and mass spectrometry.(2)dityrosine and3-nitrotyrosine was incubated with bovine serum albumin, whey protein andovalbumin, then detected the influence of dityrosine and3-nitrotyrosine to fluorescencespectroscopy and particle size, EPR, carbonyl and sulfhydryl concentration and DPPHscavenging of albumins.(3) cytology experiment: dityrosine and3-nitrotyrosine wasincubated with HepG2cell in various concentration an time length, then detected the cellviability and ROS (O2-、H2O2) concentration, enzyme activity of antioxidant enzymes (T-SOD,CAT, GSH-Px) and MDA concentration of HepG2cell. Lastly, detected some genesexpression relates to the antioxidant system.(4) dityrosine and3-nitrotyrosine was incubatedwith HepG2cell for a long time, then detected mitochondrial membrane potential andapoptosis-related mRNA expression.Result:(1) The yield of dityrosine of Cu2+/H2O2system is significantly higher than Fe2+/H2O2system.(2) Dityrosine and3-nitrotyrosine could cause a decline of fluorescence and anincrease of particle size of all3kinds albumins with the impact trend is ovalbumin> bovineserum albumin> whey protein.(3) There was an remarkable increase in ROS concentrationwhen whey protein incubated with dityrosine and3-nitrotyrosine respectively. The modifiedalbumins were found carbonyl concentration rise, sulfhydryl concentration drop and DPPHscavenging decline.(4) dityrosine and3-nitrotyrosine could cause HepG2cell viabilitydecreased, and it is positively correlated with the incubation concentration and incubationtime.300μmol/L dityrosine incubated with HepG2cell by72h caused cell viability decreasedby50%, while3-nitrotysine only need to interacted with HepG2by48h to reach the samelevel.(5) Intracellular O2-, H2O2content increased by dityrosine and3-nitrotyrosine. 200μmol/L dityrosine incubated by12h caused a peak Intracellular ROS level, while whenincubated by72h the low concentration group reached a high ROS level as well. Comparedwith dityrosine,3-nitrotyrosine used a shorter time length and a lower concentration toachieve the same level.(6) The enzyme activity of intracellular antioxidant enzymes weresignificantly affacted by dityrosine and3-nitrotyrosine. T-SOD activity was inverselyproportional to dityrosine and3-nitrotyrosine concentration. CAT and GSH-Px activity raisedin the low concenration group of dityrosine and3-nitrotyrosine. Whereas it droped in the highconcenration group. Meanwhile intracellular MDA concentration grown because of dityrosineand3-nitrotyrosine and it is a positive correlation.(7) The low concentration group ofdityrosine and3-nitrotyrosine caused antioxidant-related gene (Nrf2, Prdx1, and SirT3)unregulated the expression. However High concentration group downregulated the expression.(8) Dityrosine and3-nitrotyrosine caused mitochondrial membrane potential depolarization,further induced apoptosis, and it was positively correlated with the concentration of dityrosineand3-nitrotyrosine.(9) The apoptosis caused by dityrosine and3-nitrotyrosine might be dueto caspase signal path activation. Fas, Bax, caspase-1, caspase-3, caspase-8, caspase-9geneswere upregulated, Bcl-2was downregulated.Summary: Tyrosine oxidation products could cause oxidative damage to albumins,crosslinking the protein, resulting in changes in their structure and function; As to the cell,tyrosine oxidation products could broke the balance of cellular redox system and causedapoptosis.