Neurite outgrowth inhibitory levels of organophosphates induce tissue transglutaminase activity in differentiating N2a cells: evidence for covalent adduct formation

Almami, Ibtesam S. and Aldubayan, Maha A. and Felemban, Shatha G. and Alyamani, Najiah and Howden, Richard and Robinson, Alexander J. and Pearson, Tom D. Z. and Boocock, David and Algarni, Alanood S. and Garner, A Christopher and Griffin, Martin and Bonner, Philip L. R. and Hargreaves, Alan J (2020) Neurite outgrowth inhibitory levels of organophosphates induce tissue transglutaminase activity in differentiating N2a cells: evidence for covalent adduct formation. Archives of Toxicology. ISSN 1432-0738

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Abstract

Organophosphate compounds (OPs) induce both acute and delayed neurotoxic effects, the latter of which is believed to involve their interaction with proteins other than acetylcholinesterase. However, few OP-binding proteins have been identified that may have a direct role in OP-induced delayed neurotoxicity. Given their ability to disrupt Ca homeostasis, a key aim of the current work was to investigate the effects of sub-lethal neurite outgrowth inhibitory levels of OPs on the Ca -dependent enzyme tissue transglutaminase (TG2). At 1-10 µM, the OPs phenyl saligenin phosphate (PSP) and chlorpyrifos oxon (CPO) had no effect cell viability but induced concentration-dependent decreases in neurite outgrowth in differentiating N2a neuroblastoma cells. The activity of TG2 increased in cell lysates of differentiating cells exposed for 24 h to PSP and chlorpyrifos oxon CPO (10 µM), as determined by biotin-cadaverine incorporation assays. Exposure to both OPs (3 and/or 10 µM) also enhanced in situ incorporation of the membrane permeable substrate biotin-X-cadaverine, as indicated by Western blot analysis of treated cell lysates probed with ExtrAvidin peroxidase and fluorescence microscopy of cell monolayers incubated with FITC-streptavidin. Both OPs (10 µM) stimulated the activity of human and mouse recombinant TG2 and covalent labelling of TG2 with dansylamine-labelled PSP was demonstrated by fluorescence imaging following SDS-PAGE. A number of TG2 substrates were tentatively identified by mass spectrometry, including cytoskeletal proteins, chaperones and proteins involved protein synthesis and gene regulation. We propose that the elevated TG2 activity observed is due to the formation of a novel covalent adduct between TG2 and OPs.

Item Type: Article
Additional Information: ** From PubMed via Jisc Publications Router ** History: received 19-01-2020; accepted 14-07-2020.
Identification Number: https://doi.org/10.1007/s00204-020-02852-w
Date: 4 August 2020
Uncontrolled Keywords: Covalent adduct, Neurite outgrowth, Organophosphate toxicity, Tissue transglutaminase
Subjects: C100 Biology
Divisions: Faculty of Health, Education and Life Sciences > School of Health Sciences
SWORD Depositor: JISC PubRouter
Depositing User: JISC PubRouter
Date Deposited: 19 Aug 2020 09:14
Last Modified: 19 Aug 2020 09:14
URI: http://www.open-access.bcu.ac.uk/id/eprint/9702

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