Investigation On Proteomics And Trx1-mediated Oxidative Stress Of DBDCT-induced Hepatotoxicity

Objective: To investigate di-n-butyl-di-(4-chlorobenzohydroxamato) tin (IV)(DBDCT) induced hepatotoxic mechanism using proteomics methods. To study thedifferentially expressed protein-Trx1mediated oxidative stress signaling pathway. Tosearch the potential biomarkers for the early diagnosis of hepatotoxicity.Methods:(1) SD rats were treated with DBDCT intraperitoneally, and the bodyweight, liver weight ratio, plasma biochemistry, liver histopathology and total Snwere recorded and determined.(2)2-DE-TOF-TOF-MS was performed to analyzeand identify the differentially expressed proteins between control and DBDCT-treatedgroups in vivo.(3) Cytotoxicities of DBDCT to HL-02cells were assessed using MTTmethod. The morphology was examined under transmission electron microscope. Theextracellular level of LDH was determined using colorimetric method. The cellapoptosis, cell circles and the intracellular level of ROS were detected by flowcytometrye.(4) The total protein of HL02cell line was separated by2-DE, and thedifferentially expressed proteins between DBDCT-treated and untreated groups wereidentified by MALDI-TOF-TOF-MS. Meanwhile, the expression and function of twoidentified proteins were verified by Western blot and RT-PCR methods.(5) Theexpression of ASK1, JNK and P38, the downstream factors of Trx1-medited oxidativestress signaling pathway, were determined in DBDCT-treated group using Westernblot and RT-PCR methods. The cell viability, the level of ROS and the expression ofabove factors were detected after the treatment of antioxidant NAC and DBDCT.Results:(1) The body weight was increased followed with ascites, lethargy anddecreased spontaneous activity in DBDCT treated groups, and several animals weredied in high-dose group. The liver weight ratio was decreased in DBDCT-treatedgroups with an obvious does-effect relationship. The levels of AST，AKP and ACPwere increased significantly in middle and high-dose treated groups. However, the ALT level was decreased. The liver cells eosinophilic change and karyopyknosis wereobserved in middle and high-dose treated groups. The atomic absorption resultsshowed that there was a certain accumulation of tin in rat liver and observed in a dosedependent manner.(2) Sixteen proteins were indentified according to the2-DE-TOF-TOF-MS, including glyceraldehyde-3-phosphate dehydrogenase(RGD1565368). Phosphatidylethanolamine-binding protein1(Pebp1), Isoamylacetate-hydrolyzing esterase1homolog (Iah1), S-adenosylmethionine synthaseisoform type-1(Mat1a), Glutamate dehydrogenase1(Glud1), Eukaryotic translationinitiation factor5A-1(Eif5a), Isoform2of Adenylate kinase2(KAD2), Proteindisulfide-isomerase A3(PDIA3), Succinate dehydrogenase (DHSA), Aldehydedehydrogenase (ALDH2), Glutathione S-transferase Mu2(GSTM2), Heat shockprotein HSP90-beta (HS90B), Dimethylglycine dehydrogenase (M2GD),4-hydroxyphenylpyruvate dioxygenase (HPPD), Ornithine aminotransferase (OAT).The identified proteins were participated in the process of oxidoreduction/oxidativestress, electronical transmission of mitochondrial respiratory chain, enzymaticmetabolism and synthesis of amino acid.(3) The IC50value of DBDCT to HL-7702cells was5.77μM at24h. The morphology showed that the cell contour becameblurred gradually, the nuclear membrane was fractured followed with the highexpression of extracellular LDH in DBDCT-treated group. Compared with the controlgroup, DBDCT could obvious increase the cell apoptosis, the release of ROS andcould induce the cycle arrest. The results indicated that cell apoptosis, oxidative stressand cycle arrest might be the major hepatotoxic mechanism.(4) Nine proteins wereindentified according to the2-DE-TOF-TOF-MS in vitro, including Thioredoxin1(Trx1), Histidine triad nucleotide-binding protein1(HINT1), Protein DJ-1(PARK7),Cytochrome c oxidase subunit5A (COX5A), Galectin-1(Gal-1), Peroxiredoxin-2(PRDX2), Superoxide di (SODM), Isoform1of3-hydroxyacyl-CoA dehydrogenasetype-2(HCD2), and NADH dehydrogenase [ubiquinone] iron-sulfur protein8(NDUS8). The expression of Trx1and PARK7were checked by Western blot andRT-PCR methods to gain the identical results with2-DE. The identified proteins wereparticipated in the process of oxidoreduction/oxidative stress, electronical transmission of mitochondrial respiratory chain, regulation of microtubule, cellapoptosis, proliferation and differentiation.(5) The study on Trx1-mediated oxidativestress pathway showed that the expression of Trx1, DJ1, ASK1, JNK and P38wereincreased significantly with an obvious dose and time dependent manner on bothprotein and mRNA levels. However, after the co-treated with NAC, the cell viabilitywas increased followed with the decreased expression of ROS. Meanwhile, the levelsof Trx1, DJ1, JNK and P38were decreased significantly. According to the results, wepresumed that oxidative stress was the major hepatotoxic mechanism. DBDCT couldinduce the high expression of free radical, then Trx1was dissociated from ASK1toresist the redundant free radical, regulated the JNK/P38signaling pathway resultingin cell apoptosis. Meanwhile, as a chaperonin of oxidative stress, DJ1was also highexpressed to bind with Daxx which was integrated with ASK1in normal cells.Therefore, ASK1was liberated to activate the JNK/P38signaling pathway.Conclusions:(1) DBDCT presented obvious hepatotoxicity in vivo and in vitro.(2)DBDCT could evoke the high expression of ROS to activate Trx1-mediated oxidativestress signaling pathway to result in the cell apoptosis and necrosis. Therefore,oxidative stress played an important role in DBDCT-induced hepatotoxicity.(3)Antioxidant NAC could decrease the ROS level to intercept Trx1-mediated oxidativestress signaling pathway to resist DBDCT-induced hepatotoxiciy.(4) Trx1might be apotential biomarker for the early diagnosis of hepatotoxicity induced by DBDCT.