APE1 modulates cellular responses to organophosphate pesticide-induced oxidative damage in non-small cell lung carcinoma A549 cells

Abstract

Monocrotophos (MCP) and chlorpyrifos (CP) are widely used organophosphate pesticides (OPPs), speculated to be linked with human pathologies including cancer. Owing to the fact that lung cells are most vulnerable to the environmental toxins, the development and progression of lung cancer can be caused by the exposure of OPPs. The present study investigates the oxidative DNA damage response evoked by MCP and CP in human non-small cell lung carcinoma A549 cells. A549 cells were exposed to MCP and CP; cytotoxicity and reactive oxygen species (ROS) generation were measured to select the non-toxic dose. In order to establish whether MCP and CP can initiate the DNA repair and cell survival signalling pathways in A549 cells, qRT-PCR and Western blotting techniques were used to investigate the mRNA and protein expression levels of DNA base excision repair (BER)-pathway enzymes and transcription factors (TFs) involved in cell survival mechanisms. A significant increase in cell viability and ROS generation was observed when exposed to low and moderate doses of MCP and CP at different time points (24, 48 and 72 h) studied. A549 cells displayed a dose-dependent accumulation of apurinic/apyrimidinic (AP) sites after 24 h exposure to MCP advocating for the activation of AP endonuclease-mediated DNA BER-pathway. Cellular responses to MCP- and CP-induced oxidative stress resulted in an imbalance in the mRNA and protein expression of BER-pathway enzymes, viz. PARP1, OGG1, APE1, XRCC1, DNA pol β and DNA ligase III α at different time points. The treatment of OPPs resulted in the upregulation of TFs, viz. Nrf2, c-jun, phospho-c-jun and inducible nitric oxide synthase. Immunofluorescent confocal imaging of A549 cells indicated that MCP and CP induces the translocation of APE1 within the cytoplasm at an early 6 h time point, whereas it promotes nuclear localization after 24 h of treatment, which suggests that APE1 subcellular distribution is dynamically regulated in response to OPP-induced oxidative stress. Furthermore, nuclear colocalization of APE1 and the TF c-jun was observed in response to the treatment of CP and MCP for different time points in A549 cells. Therefore, in this study we demonstrate that MCP- and CP-induced oxidative stress alters APE1-dependent BER-pathway and also mediates cell survival signalling mechanisms via APE1 regulation, thereby promoting lung cancer cell survival and proliferation.

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Acknowledgements

This work was supported by the BSR-startup Grant from the University Grants Commission (UGC), New Delhi, India, and the funds received under the scheme Research Seed Money (RSM) from the Central University of Punjab, Bathinda (CUPB) to A.K.M. A.K.M. also acknowledges the training in the field of BER-pathway and Redox biology under the guidance of Prof. Sankar Mitra at University of Texas Medical Branch, Galveston, Texas, USA. S.T. acknowledges the financial support in the form of Senior Research Fellowship (SRF) from the Indian Council of Medical Research (ICMR), New Delhi, India. Central Instrumentation Laboratory (CIL) facility of CUPB is thankfully acknowledged for providing confocal microscopy facility. Because of the limited focus of the article, many relevant and appropriate references could not be included, for which the authors apologize.

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Correspondence to Anil K. Mantha.

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Thakur, S., Dhiman, M. & Mantha, A.K. APE1 modulates cellular responses to organophosphate pesticide-induced oxidative damage in non-small cell lung carcinoma A549 cells. Mol Cell Biochem 441, 201–216 (2018). https://doi.org/10.1007/s11010-017-3186-7

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Keywords

  • Monocrotophos
  • Chlorpyrifos
  • APE1
  • BER-pathway
  • ROS