Detection And Mechanism Of Oxidative Modification Of Sulfhydryl Proteins In Carbon Nanomaterials Induced-hepatotoxicity

Carbon nanomaterials（CNMs）are widely used in cellular imaging and drug delivery due to their excellent optical properties and large specific surface area.However,CNMs are difficult to be metabolized and degraded,and easy to accumulate in the liver,causing irreversible damage to the liver and other organs.For example,CNMs can lead to intracellular REDOX imbalance by inducing the production of reactive oxygen species（ROS）.However,less attention has been paid to the process of protein oxidative modification in hepatocytes.Oxidative modification of proteins is the main type of protein post translational modification（PTMs）,which is caused by the reaction of amino acid residues with ROS or reactive nitrogen species.It may be an important switch behind the events of hepatocyte damage.As a kind of sulfhydryl-containing protein,Glutaredoxin-1（GLRX1）has an important effect on maintaining cellular redox homeostasis.Previous studies mostly focus on the effects of increased ROS on the expression of GLRX1.However,the potential mechanisms involved in the oxidative modification of GLRX1 induced by ROS needed further exploration.In this study,a nanoprobe that specifically recognizes cysteine sulfinic acid and cysteine sulfonic acid was developed.The probe was able to image the spatial-temporal oxidative modifications of proteins in liver cells exposed to CNMs.Based on proteomic analysis information,the mechanism of molecular interaction can be analyzed.Furthermore,the toxicological mechanism of liver injury induced by CNMs were revealed by establishing the association between oxidative regulation of key proteins and liver injury.The main research contents are as follows:1.We evaluated the toxicological effects of carbon quantum dots（CDs）,carbon nanotubes（CNTs）,and graphene oxide（GO）on normal human liver cells（HL-7702）,and found that two main types of ROS（H₂O₂and·OH）level and cytotoxicity of GO group were higher,while the ROS（H₂O₂ and·OH）level and cytotoxicity of CNTs or CDs group were relatively lower.Mass spectrometry characterization showed that the excessive H₂O₂ induced by GO caused sulfonation modification at the active sites（Cys78 and Cys82）of GLRX1.Moreover,the active sites（Cys22and Cys25）of GLRX1 were further oxidative modified to sulfonation via·OH induced by GO.Such irreversible oxidative modification could break the cross-linking with the Apoptosis Signal-regulating Kinase,leading to the activation of the JNK/p38 signaling pathway and ultimately hepatocyte apoptosis.On the contrary,in HL-7702 cells incubated with CNTs or CDs,lower levels of ROS generation acted as signaling molecules,resulting in the upregulation of GLRX1 instead of sulfonation modification of its active sites.The highly expressed GLRX1enabled it to effectively remove intracellular ROS and maintain the cellular redox homeostasis,thus causing no evident toxic effect to HL-7702 cells.This implied that GO induced hepatotoxicity can be attributed to GLRX1 oxidative modification.2.In this study,small molecule fluorescent probes Z-1 and H-1,which specifically recognize sulfinic acid and sulfonic acid,were self-assembled with Bovine Serum Albumin（BSA）to construct a nanoprobe BSA-Z-H for simultaneous imaging of cysteine sulfination and sulfonation modifications.BSA-Z-H exhibited strong sulfinic and sulfonic acid-dependent absorption at 522 nm and 471 nm,which enabled high sensitivity for simultaneous fluorescence detection of cysteine sulfinic and cysteine sulfonic modifications.The probe played an important role in assessing REDOX status and early diagnosis of disease.Therefore,this work provided a good tool for noninvasive,sensitive,and real-time of cysteine sulfinic acid and sulfonic acid.