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

Ibtesam S. Almami, Maha A. Aldubayan, Shatha G. Felemban, Najiah Alyamani, Richard Howden, Alexander J. Robinson, Tom D.Z. Pearson, David Boocock, Alanood S. Algarni, A. Christopher Garner, Martin Griffin, Philip L.R. Bonner, Alan J. Hargreaves*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review


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 Ca2+ homeostasis, a key aim of the current work was to investigate the effects of sub-lethal neurite outgrowth inhibitory levels of OPs on the Ca2+-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.

Original languageEnglish
Pages (from-to)3861-3875
Number of pages15
JournalArchives of Toxicology
Issue number11
Early online date4 Aug 2020
Publication statusPublished - 1 Nov 2020

Bibliographical note

This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit


  • Covalent adduct
  • Neurite outgrowth
  • Organophosphate toxicity
  • Tissue transglutaminase


Dive into the research topics of 'Neurite outgrowth inhibitory levels of organophosphates induce tissue transglutaminase activity in differentiating N2a cells: evidence for covalent adduct formation'. Together they form a unique fingerprint.

Cite this