Assessment of neurotoxic effects of tri-cresyl phosphates (TCPs) and cresyl saligenin phosphate (CBDP) using a combination of in vitro techniques.
Environmental exposures to tri-cresyl phosphates (TCPs) and the possible formation of toxic metabolites (e.g. cresyl saligenin phosphate; CBDP) may cause a variety of neurotoxic effects in humans. As reported for other organophosphorus compounds (OPs), the inhibition of acetylcholine esterase (AChE) has also been proposed as the underlying mechanism for TCP neurotoxicity. The ortho-isomer, ToCP and its metabolite CBDP are also known to affect neuropathy target esterase (NTE) leading to organophosphate-induced delayed neuropathy (OPIDN). Recently, in vitro testing has led to the identification of other molecular targets and alternative mechanisms of ToCP toxicity. The metabolite CBDP and other isomers, as well as commercial mixtures have not been tested for such additional modes of actions. Accordingly, the present study investigates alterations of neurobiological correlates of central nervous processes using different in vitro techniques. The three symmetric TCP isomers - ToCP, TpCP, and TmCP - that contain a methyl group at the ortho-, para-, or meta-position of the aromatic ring system, respectively, together with a commercial TCP mixture, and CBDP were all tested using concentrations not exceeding their cytotoxic concentrations. Isolated cortical neurons were kept in culture for 6days followed by 24h incubation with different concentrations of the test compounds. Thus, all endpoints were assessed after 7days in vitro (DIV 7), at which time cell viability, neurite microstructure, and the function of glutamate receptors and voltage-gated calcium cannels (VGCC) were measured. While the cytotoxic potential of the TCP isomers and their mixture were comparable (IC50≥80μM), CBDP was more cytotoxic (IC50: 15μM) to primary cortical neurons. In contrast, CBDP (up to 10μM) did not compromise the microstructure of neurites. Ten μM of ToCP significantly reduced the size and complexity of neurite networks, but neither TmCP and TpCP nor the mixture affected this second endpoint of neurotoxicity assessment. TCPs and their mixture significantly reduced the Ca2+influx in response to glutamate and KCl stimulation in concentrations of 10μM. Only ToCP showed a specific effect on glutamate receptors with 100nM reducing the evoked Ca2+influx. The effects of CBDP on the provoked Ca2+influx were much weaker than those observed for TCPs. These results confirmed that ToCP has a unique mode of action on glutamate receptors that are not observed with the metabolite CBDP and the other symmetric TCP isomers. In addition, the TmCP isomer seems to have the lowest potency with respect to inducing neurotoxic effects. CBDP did not affect the neurospecific endpoints investigated in this study. Therefore, the specific affinity of CBDP for NTE and the reported general cytotoxicity might be the most relevant modes of action of this toxic metabolite in the context of ToCP-induced neurotoxicity, including OPIDN.
IfADo-Leibniz Research Center for Working Environment and Human Factors, 44139 Dortmund, Germany. Electronic address: hausherr@ifado.de.
Vanessa
V
Hausherr
Ruhr-University Bochum, Department of Animal Physiology, 44801 Bochum, Germany. Electronic address: nicole.schoebel@gmx.de.
Nicole
N
Schöbel
IfADo-Leibniz Research Center for Working Environment and Human Factors, 44139 Dortmund, Germany. Electronic address: liebing@ifado.de.
Julia
J
Liebing
IfADo-Leibniz Research Center for Working Environment and Human Factors, 44139 Dortmund, Germany. Electronic address: thriel@ifado.de.
Christoph
C
van Thriel
D000818:Animals; D002118:Calcium / Q000378:metabolism; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000166:cytology; D004305:Dose-Response Relationship, Drug; D004338:Drug Combinations; D020128:Inhibitory Concentration 50; D051379:Mice; D016501:Neurites / Q000187:drug effects; D009474:Neurons / Q000166:cytology / Q000187:drug effects*; D009498:Neurotoxins / Q000633:toxicity*; D009943:Organophosphorus Compounds / Q000633:toxicity*; D011189:Potassium Chloride / Q000494:pharmacology; D014317:Tritolyl Phosphates / Q000633:toxicity*
D004338:Drug Combinations; D009498:Neurotoxins; D009943:Organophosphorus Compounds; D014317:Tritolyl Phosphates; C000516:2-(2-cresyl)-4H-1-3-2-benzodioxaphosphorin-2-oxide; D011189:Potassium Chloride; D002118:Calcium
Aerotoxic syndrome; Cresyl saligenin phosphate (CBDP); Glutamate signaling; Neurotoxicity; Tri-cresylphosphate; Tri-ortho-cresyl phosphate