TRPA1 channel mediates organophosphate-induced delayed neuropathy

Abstract

The organophosphate-induced delayed neuropathy (OPIDN), often leads to paresthesias, ataxia and paralysis, occurs in the late-stage of acute poisoning or after repeated exposures to organophosphate (OP) insecticides or nerve agents, and may contribute to the Gulf War Syndrome. The acute phase of OP poisoning is often attributed to acetylcholinesterase inhibition. However, the underlying mechanism for the delayed neuropathy remains unknown and no treatment is available. Here we demonstrate that TRPA1 channel (Transient receptor potential cation channel, member A1) mediates OPIDN. A variety of OPs, exemplified by malathion, activates TRPA1 but not other neuronal TRP channels. Malathion increases the intracellular calcium levels and upregulates the excitability of mouse dorsal root ganglion neurons in vitro. Mice with repeated exposures to malathion also develop local tissue nerve injuries and pain-related behaviors, which resembles OPIDN. Both the neuropathological changes and the nocifensive behaviors can be attenuated by treatment of TRPA1 antagonist HC030031 or abolished by knockout of Trpa1 gene. In the classic hens OPIDN model, malathion causes nerve injuries and ataxia to a similar level as the positive inducer tri-ortho-cresyl phosphate (TOCP), which also activates TRPA1 channel. Treatment with HC030031 reduces the damages caused by malathion or tri-ortho-cresyl phosphate. Duloxetine and Ketotifen, two commercially available drugs exhibiting TRPA1 inhibitory activity, show neuroprotective effects against OPIDN and might be used in emergency situations. The current study suggests TRPA1 is the major mediator of OPIDN and targeting TRPA1 is an effective way for the treatment of OPIDN.

Keywords: OPIDN; TOCP; TRPA1; malathion; organophosphate.

Figure 1

Malathion activates TRPA1 channels. (a) Time course of fluorescence signals induced by 10 μM malathion (Mala) or 10 μM AITC in HEK293 cells expressing mTRPA1. The fluorescence signals are scaled as F/F₀ (n=5). EC indicates extracellular solution. (b) Dose–response curve showing malathion-induced activation of mTRPA1 (n=8–10). (c) Whole-cell currents of a HEK293 cell expressing hTRPA1 in calcium-free extracellular solution. HC030031 (HC, 10 μM) was applied to validate the hTRPA1-mediated currents. The currents measured at −100 mV (green circles) and +100 mV (orange circles) during each ramp were plotted as a function of time. (d) The current-voltage (I–V) relationships of malathion-induced currents. The blue, purple and red lines were measured at the time points α, β and γ (shown in c), respectively. (e) Dose–response curve showing the malathion-induced currents of hTRPA1 (n=8–10). (f) The current densities of human, mouse, rat and chicken TRPA1 channels induced by 30 μM malathion (n=5). (g) The current density of hTRPA1 induced by AITC or the indicated OPs (30 μM, n=5–7). (h) Heat map showing the time course of the 10 μM malathion-induced fluorescence signals of TRPA1, TRPV1, TRPV3, TRPV4, TRPM8, TRPC4 and TRPC5 channels. The statistical data are presented as the mean±s.e.m.

Figure 2

The mechanism of malathion-mediated TRPA1 activation. (a) The I–V plots of WT and mutant hTRPA1 currents activated by 30 μM Mala. (b) Comparison of the responses of WT and mutant hTRPA1 channels with 30 μM malathion and 30 μM flufenamic acid (FA) (n=5–9). (c) Whole-cell currents of the mutant K710R in the presence of 100 μM AITC and 100 μM malathion. HC030031 (HC, 10 μM) was applied to validate the hTRPA1-mediated currents. (d) Schematic representation of the structure of hTRPA1 and the essential residues for malathion activity. The statistical data are presented as the mean±s.e.m. *P<0.05, **P<0.01 and ***P<0.001.

Figure 3

Malathion elicits inward currents and induces action potential firing in small-diameter DRG neurons via TRPA1 channels. (a) Changes in the fluorescence ratio in DRG neurons from WT (left) and Trpa1^−/− mice (right) after sequential application of AITC and Mala. Capsaicin (Cap) was used to verify the functionality of the Trpa1^−/− DRG. The green, blue and purple bars indicate the AITC, malathion and capsaicin application periods, respectively. (b) Summary of fluorescence ratio changes elicited by 100 μM of the indicated OPs in WT DRG neurons (n=5–7). (c) Percentages of TRPA1- or TRPV1-positive DRG neurons from WT mice. (d) Inward currents induced by 100 μM AITC or malathion in DRG neurons. Note that malathion-induced inward currents were blocked by 10 μM HC030031 (HC) and abolished in Trpa1^−/− mice (n=7). (e) Dose-dependent effects of malathion on DRG neurons from WT mice (n=3–4). (f) Summary of current density induced by the indicated OPs (100 μM) in WT DRG neurons (n=3–4). (g) Action potential firing induced by 100 μM AITC or malathion in DRG neurons (n=4). Note that malathion-induced action potential firings were blocked by 10 μM HC030031 and eliminated in Trpa1^−/− mice. The data represent the mean±s.e.m.

Figure 4

TRPA1-KO or blockage alleviates malathion-induced nociception and nerve injury in mice. (a) Schematic diagram of the mouse tests. AITC (10 μl, 5%) or malathion (10 μl, 30%) were given by intraplantar route, followed by behavioral test and TEM imaging. (b) Time course of licking in mice treated with 5% AITC (red triangles, n=10). Mice that were pretreated with HC030031 (HC, 200 mg kg⁻¹, per oral, blue triangles, n=9) licked for significantly shorter periods than AITC-treated mice. (c) Quantification of the AITC response binned into different phases. Phase I (0–10 min); Phase II (10–60 min). (d) As in (b) but also including a malathion (Mala)-treated Trpa1^−/− group, n=8–10 per group. (e) Quantification of the malathion response during the different phases for the experimental groups. (f–j) Ultrastructural image of local nerve fibers from WT mice in the vehicle (f), 5% AITC (g), 30% malathion (h), malathion pretreated with HC030031 (i) and Trpa1^−/− mice treated with 30% malathion (j). For the panel h, two types of malathion-induced neuropathies were displayed, that is, loss of the myelin sheath (left) and avulsion (right). Scale bar is 0.5 μm for the vehicle, AITC and malathion groups and 1 μm for the malathion+HC and malathion (Trpa1^−/−) groups. The data represent the mean±s.e.m. ***P<0.001.

Figure 5

Pharmacological inhibition of TRPA1 alleviates OPIDN in hens. (a) Schematic representation of the administration of TOCP and malathion to hens. (b) Whole-cell currents of a HEK293 cell expressing cTRPA1 channels elicited by 100 μM TOCP in calcium-free external solution (upper) and the dose–response curve of the TOCP activation effect (lower) along with the structure of TOCP (n=4–8). (c) Time course of the changes of OPIDN scores following vehicle (black, n=6), TOCP (750 mg kg⁻¹, red, n=8), TOCP+HC030031 (100 mg kg⁻¹) from day 0 (blue, n=8) and from day 11 (purple, n=8). (d) Time course of the changes of the neuropathy scores of hens following vehicle (black, n=6), malathion (75 mg kg⁻¹, red, n=8), malathion+HC030031 (100 mg kg⁻¹) from day 0 (blue, n=8) and from day 11 (purple, n=8). The clinical symptoms of the delayed neuropathy was estimated in hens daily for 21 days using a five-point scale. (e) TEM image of hen spinal cords (upper row) and sciatic nerves (lower row) from the various groups (10 000×). For panels (c) and (d), two-way ANOVA was used for statistical analysis, *P<0.05, **P<0.01, ***P<0.001.

Figure 6

Duloxetine and ketotifen screened by high-throughput system alleviate OPIDN in hens. (a) The route diagram of TRPA1 inhibitor detection. (b) Scatter statistics of TRPA1 channel inhibitory activity of the drug collection library. Duloxetine (Dul) and ketotifen (Ket) are displayed in red dot. (c) Representative currents showing the inhibitory effects of 30 μM Dul or 30 μM Ket on the TOCP-induced cTRPA1 currents. (d) Dose–response curves of Dul (left) and Ket (right) on cTRPA1 channels (n=4–8). (e) Time course of OPIDN scores in hens following the indicated treatments. TOCP was administered in the same manner as described in the panel Figure 5a. Dul (100 mg kg⁻¹) and Ket (100 mg kg⁻¹) were administered daily by gavage from day 0. n=8 for each group. For panel (e), two-way ANOVA was used for statistical analysis, ***P<0.001.