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

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

doi:10.1007/s00204-020-02852-w

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 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.

Highlights

It is well established that many commercially available organophosphorous compounds (OPs) used as oil additives and insecticides, inhibit the activities of serine hydrolases such as acetyl and butyryl cholinesterase (Aldridge 1954; Chambers and Levi 1992; Gupta 2006; Kamanyire and Karralliede 2004)
TCP induces a neurodegenerative condition known as OP-induced delayed neuropathy (OPIDN), which was associated with a major outbreak of Ginger Jake poisoning during the years of prohibition in the USA (Bishop and Stewart 1930; Zeligs 1938)
The onset of OPIDN is preceded by a number of early molecular lesions, such as irreversible inhibition of neuropathy target esterase (NTE), disrupted Ca2+ homeostasis, proteolytic degradation and altered phosphorylation of cytoskeletal proteins (Hargreaves 2012) in the central and peripheral nervous systems

Summary

Introduction

It is well established that many commercially available organophosphorous compounds (OPs) used as oil additives (e.g., tricresyl phosphate; TCP) and insecticides (e.g., chlorpyrifos; CPF), inhibit the activities of serine hydrolases such as acetyl and butyryl cholinesterase (Aldridge 1954; Chambers and Levi 1992; Gupta 2006; Kamanyire and Karralliede 2004). They are capable of inducing delayed neurodegenerative and/or developmental effects through covalent interactions with non-cholinesterase protein targets (Ray and Richards 2001; Hargreaves 2012; Sánchez-Santed et al 2016). A number of cell culture studies have shown that phenyl saligenin phosphate (PSP), a structural analogue of SCOTP, inhibits the outgrowth of neurites in differentiating neuroblastoma cells in association with complete inhibition of NTE, increased activation of ERK 1/2, and transient hyperphosphorylation followed by a reduction in the protein levels of neurofilament heavy chain (Hargreaves et al 2006)

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