Evidence that mouse brain neuropathy target esterase is a lysophospholipase

Abstract

Neuropathy target esterase (NTE) is inhibited by several organophosphorus (OP) pesticides, chemical warfare agents, lubricants, and plasticizers, leading to OP-induced delayed neuropathy in people (>30,000 cases of human paralysis) and hens (the best animal model for this demyelinating disease). The active site region of NTE as a recombinant protein preferentially hydrolyzes lysolecithin, suggesting that this enzyme may be a type of lysophospholipase (LysoPLA) with lysolecithin as its physiological substrate. This hypothesis is tested here in mouse brain by replacing the phenyl valerate substrate of the standard NTE assay with lysolecithin for an "NTE-LysoPLA" assay with four important findings. First, NTE-LysoPLA activity, as the NTE activity, is 41-45% lower in Nte-haploinsufficient transgenic mice than in their wild-type littermates. Second, the potency of six delayed neurotoxicants or toxicants as in vitro inhibitors varies from IC50 0.02 to 13,000 nM and is essentially the same for NTE-LysoPLA and NTE (r2 = 0.98). Third, the same six delayed toxicants administered i.p. to mice at multiple doses inhibit brain NTE-LysoPLA and NTE to the same extent (r2 = 0.90). Finally, their in vivo inhibition of brain NTE-LysoPLA generally correlates with delayed toxicity. Therefore, OP-induced delayed toxicity in mice, and possibly the hyperactivity associated with NTE deficiency, may be due to NTE-LysoPLA inhibition, leading to localized accumulation of lysolecithin, a known demyelinating agent and receptor-mediated signal transducer. This mouse model has some features in common with OP-induced delayed neuropathy in hens and people but differs in the neuropathological signs and apparently the requirement for NTE aging.

Fig. 1.

Structures of OP diagnostic inhibitors and delayed toxicants or neurotoxicants.

Fig. 2.

Proposed biochemical lesion induced by OP delayed toxicants or neurotoxicants involving inhibition of NTE-LysoPLA, which normally hydrolyzes lysolecithin as its physiological substrate. The classical substrate for NTE is phenyl valerate, based on ease of assay (4, 12) rather than mechanistic considerations. NTE-LysoPLA and NTE in brain are resistant to paraoxon but sensitive to mipafox and other delayed toxicants or neurotoxicants.

Fig. 3.

Relationship between in vitro sensitivities of brain NTE-LysoPLA and NTE activities. NTE-LysoPLA and NTE were assayed with lysolecithin and phenyl valerate, respectively. Compound structures are shown in Fig. 1. (A) The data for mouse NTE-LysoPLA for all compounds and mouse NTE for paraoxon, (S)-octyl-BDPO, and DSF are from this study with Swiss–Webster mice. Other data for mouse NTE are from this laboratory (14, 16) or Veronesi et al. (26) for o-tolyloxy-BDPO. The hen NTE data are from Wu and Casida (13, 15, 27), Johnson (11), and Veronesi et al. (26). (B) Correlation for sensitivity of NTE-LysoPLA from mouse brain versus NTE from mouse brain (•) and hen brain (▵). Correlation coefficients (r²) do not include tabulated values for paraoxon (>10⁵ nM) or those in parentheses, which were determined by a different investigator (0.05 nM for mouse NTE) or a different laboratory (130 nM for hen NTE).