LAUSR

Aims: Long‐term treatment of Isoniazid (INH) in tuberculosis (TB) patients can lead to anti‐tuberculosis drug‐induced hepatotoxicity. To understand the mechanism of hepatotoxicity, an attempt has been made to elucidate the role of Nrf2, a transcription factor induced by oxidative stress, in INH induced apoptosis liver cancer cell lines. Materials and methods: Cytotoxicity was evaluated by MTT assay. Apoptosis and reactive oxygen species (ROS) generation was performed by flow cytometry. mRNA and protein expression of various genes involves in INH induced toxicity was evaluated via Real‐time PCR and western blot analysis respectively. Differential protein expression was performed by two‐dimensional gel electrophoresis followed by identification using MALDI TOF/TOF. Key findings: INH induced ROS and apoptosis in HepG2 as well as THLE‐2 cells. Nuclear damage was also observed by INH treatment in HepG2 cells. Expression of apoptotic (Cytochrome C and Caspase 9) and antioxidative (Keap1 and Nrf2) genes were observed to increase. INH induced PKC&dgr; phosphorylation and released Nrf2 from its inhibitor Keap1 in the cytoplasm of HepG2 cells. However, over‐expression of Nrf2 did not affect nuclear Nrf2 protein level as well as its downstream target NQO1. Nrf2 importer, Karyopherin &bgr;1 level was observed to decrease in HepG2 as well as THLE‐2 cells following INH treatment. Significance: These findings suggest that INH prevented Nrf2 translocation into the nucleus by inhibiting its importer Karyopherin &bgr;1. Therefore Nrf2 might not able to bind ARE sequences from inducing antioxidative response for protecting the cells undergoing apoptosis. Graphical abstract: Figure. No caption available. Abbreviations: Nrf2: nuclear factor erythoid 2‐like 2; INH: isoniazid; DNA: deoxyribonucleic acid; Annexin V FITC: annexin V fluorescein isothiocyanate; DAPI: 4,6‐diamino‐2‐phenylindole; FACS: fluorescence‐activated cell sorting; PI: propidium iodide; Cyt C: cytochrome C; Keap1: Kelch‐like ECH‐associated protein1; PKC&dgr;: protein kinase C&dgr;; qRT‐PCR: quantitative real time polymerase chain reaction; KPNB1: karyopherin &bgr;1; ICC: immunocytochemistry; NQO1: NAD(P)H dehydrogenase (quinone 1); ROS: reactive oxygen species; mRNA: messenger ribonucleic acid; ARE: Antioxidative response element; IC50: inhibitory concentration 50; DMSO: dimethyl sulphoxide; OD: optical density; DMEM: Dulbecco's modified eagles medium; H2DCFDA: 2′,7′‐dicholorofluorescin diacetate; UV: ultra violet; PBS: phosphate buffered saline; cDNA: complementary deoxyribonucleic acid; FP: forward primer; RP: reverse primer; PBST: phosphate buffered saline tween‐20; GFP: green fluorescence protein; ECL: enhanced chemiluminiscence; t‐BHQ: tertiary‐butylhydroquinone; 5‐FU: 5‐fluorouracil; 2DE: 2 dimensional gel electrophoresis; IPG: immobilized pH gradient; IEF: isoelectric focussing; CBB: coomassie brilliant blue; MALDI‐TOF: matrix‐assisted laser desorption/ionisation time of flight; ACN: acetonitrile; RT: room temperature; ANOVA: analysis of variance.