title: Molecular mechanisms of 3,3'-dichlorobenzidine-mediated toxicity in HepG2 cells.
abstract: 3,3'-Dichlorobenzidine (DCB) (CAS 91-94-1), a synthetic, chlorinated, primary aromatic amine, is typically used as an intermediate in the manufacturing of pigments for printing inks, textiles, paints, and plastics. In this study, we found that DCB could significantly inhibit the cell viability of HepG2 cells in a concentration-dependent manner. Flow cytometry revealed that DCB induced G2/M-phase arrest and apoptosis in HepG2 cells. DCB treatment dramatically induced the dissipation of mitochondrial membrane potential (Δψm ) and enhanced the enzymatic activities of caspase-9 and caspase-3 whilst hardly affecting caspase-8 activity. Furthermore, Western blotting indicated that DCB-induced apoptosis was accompanied by the down-regulation of Bcl-2/Bax ratio. These results suggested that DCB led to cytotoxicity involving activation of mitochondrial-dependent apoptosis through Bax/Bcl-2 pathways in HepG2 cells. Furthermore, HepG2 cells treated with DCB showed significant DNA damage as supported by the concentration-dependent increase in olive tail moments as determined by the comet assay and by concentration- and time-dependent increase in histone H2AX phosphorylation (γ-H2AX). Two-dimensional-difference gel electrophoresis (2D-DIGE), combined with mass spectrometry (MS), was used to unveil the differences in protein expression between cells exposed to 25 µM or 100 µM of DCB for 24 hr and the control cells. Twenty-seven differentially expressed proteins involved in DNA repair, unfolded protein response, metabolism, cell signaling, and apoptosis were identified. Among these, 14-3-3 theta, CGI-46, and heat-shock 70 protein 4 were confirmed using Western blot assay. Taken together, these data suggest that DCB is capable of inducing DNA damage and some cellular stress responses in HepG2 cells, thus eventually leading to cell death by apoptosis.
authors.affiliation: Department of Nutrition, I-Shou University, Kaohsiung City, Taiwan, Republic of China.
authors.forename: Lei-Chin
authors.initials: LC
authors.lastname: Chen
authors.forename: Jong-C
authors.initials: JC
authors.lastname: Wu
authors.forename: Yen-Fan
authors.initials: YF
authors.lastname: Tuan
authors.forename: Yi-Kuan
authors.initials: YK
authors.lastname: Tseng
authors.forename: You-Cheng
authors.initials: YC
authors.lastname: Hseu
authors.forename: Ssu-Ching
authors.initials: SC
authors.lastname: Chen
mesh_terms: D015101:3,3'-Dichlorobenzidine / Q000009:adverse effects*; D017209:Apoptosis / Q000187:drug effects*; D014408:Biomarkers, Tumor / Q000378:metabolism; D015153:Blotting, Western; D002273:Carcinogens / Q000494:pharmacology*; D006528:Carcinoma, Hepatocellular / Q000188:drug therapy / Q000378:metabolism / Q000473:pathology*; D053148:Caspase 3 / Q000378:metabolism; D053181:Caspase 8 / Q000378:metabolism; D053453:Caspase 9 / Q000378:metabolism; D002453:Cell Cycle / Q000187:drug effects*; D049109:Cell Proliferation / Q000187:drug effects*; D015180:Electrophoresis, Gel, Two-Dimensional; D006801:Humans; D008113:Liver Neoplasms / Q000188:drug therapy / Q000378:metabolism / Q000473:pathology*; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014407:Tumor Cells, Cultured
chemical_list: D014408:Biomarkers, Tumor; D002273:Carcinogens; D015101:3,3'-Dichlorobenzidine; D053148:Caspase 3; D053181:Caspase 8; D053453:Caspase 9
keywords: 2D-DIGE; 3; HepG2 cells; apoptosis; comet assay; ′-dichlorobenzidine