title: Involvement of mitogen-activated protein kinase pathway in T-2 toxin-induced cell cycle alteration and apoptosis in human neuroblastoma cells.
abstract: T-2 toxin is the most toxic trichothecene and a frequent contaminant in many agriculture products. Dietary ingestion represents the most common route of T-2 toxin exposure in humans. T-2 toxin exposure leads to many pathological conditions like nervous disorders, cardiovascular alterations, immune depression and dermal inflammation. However, the neuronal toxicity of T-2 toxin in vitro remains unclear. In the present study, we investigated the mechanism of T-2 toxin-induced apoptosis in human neuroblastoma cells (IMR-32). T-2 toxin was cytotoxic at a low concentration of 10 ng/ml. The 50% inhibitory concentration (IC50) of T-2 toxin was found to be 40 ng/ml as assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, crystal violet dye exclusion test and lactate dehydrogenase (LDH) leakage. T-2 toxin increased intracellular reactive oxygen species generation as early as 15 min and peaked at 60 min as analyzed by flow cytometry. Annexin V + propidium iodide staining showed time-dependent increase in percent apoptotic cells. DNA gel electrophoresis showed oligonucleosomal DNA fragmentation typical of apoptotic cells. Additionally, casapse-3 activation and PARP cleavage indicated involvement of mitochondrial mediated caspase-dependent pathway of apoptosis. Cell cycle analysis revealed time-dependent increase in sub-G1 population of cells and significant up-regulation of CDK2, CDK6, cyclin A and p21 messenger RNA (mRNA) levels. Exposure to T-2 toxin induced the phosphorylation of extracellular signal-regulated kinase (ERK), p38-mitogen-activated protein kinase and c-jun N-terminal kinases (JNK). Analysis of human phospho-mitogen-activated protein kinase (MAPK) antibody array revealed time-dependent increase in phosphorylation. Upstream of ERK pathway Grb2, Ras and Raf and downstream transcription factors c-fos and c-jun were significantly up-regulated. Z-VAD-FMK and MAPK inhibitors (PD 98059, SB 203580 and ZM 336372) exposure prior to T-2 toxin treatment significantly decreased percent of apoptotic cells compared to only T-2 toxin-exposed cells. Results of the present study show that T-2 toxin at nanogram concentrations can induce apoptosis in human neuronal cells through multiple signal transduction pathways. The study provides possible leads for developing therapeutic approaches to prevent T-2 toxin-induced neurotoxicity.
authors.affiliation: Division of Pharmacology and Toxicology, Defence Research and Development Establishment, Gwalior, 474002, India.
authors.forename: Mona
authors.initials: M
authors.lastname: Agrawal
authors.forename: A S B
authors.initials: AS
authors.lastname: Bhaskar
authors.forename: P V
authors.initials: PV
authors.lastname: Lakshmana Rao
mesh_terms: D017209:Apoptosis / Q000187:drug effects* / Q000502:physiology; D002453:Cell Cycle / Q000187:drug effects*; D006801:Humans; D020935:MAP Kinase Signaling System / Q000187:drug effects*; D008928:Mitochondria / Q000187:drug effects* / Q000378:metabolism; D009447:Neuroblastoma / Q000188:drug therapy / Q000378:metabolism / Q000473:pathology*; D010766:Phosphorylation; D013605:T-2 Toxin / Q000494:pharmacology*; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism
chemical_list: D048051:p38 Mitogen-Activated Protein Kinases; D013605:T-2 Toxin
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