Research ArticleNEUROPHYSIOLOGY

Prevention of pesticide-induced neuronal dysfunction and mortality with nucleophilic poly-Oxime topical gel

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Science Advances  17 Oct 2018:
Vol. 4, no. 10, eaau1780
DOI: 10.1126/sciadv.aau1780
  • Fig. 1 Nucleophilic poly-Oxime gel to deactivate pesticides to limit toxicity.

    (A) Dermal penetration of pesticide (MPT) leads to the inhibition of AChE, which plays a pivotal role in biological functions including neuronal signaling and neuromuscular coordination (NMC). MPT-mediated inhibition of AChE leads to severe toxicity including neuromuscular dysfunction, loss of endurance, and locomotor function. In our approach, the presence of α-nucleophile (an oxime) attached polymer (poly-Oxime) formulated as a topical gel could deactivate organophosphorus ester, MPT, through hydrolysis. This limits MPT penetration into the skin, which leads to the reduction of pesticide-induced toxicity. (B) Synthesis of poly-Oxime using pyridine-2-aldoxime connected to chitosan through acetamide linker. With humectants like glycerin, propylene glycol, and carbopol 940, topical gel was prepared. Two types of topical gels, poly-Oxime–containing active gel and, as a control, unfunctionalized chitosan–containing inactive sham gel, were prepared. rt, room temperature; DMF, N,N′-dimethylformamide.

  • Fig. 2 Poly-Oxime gel deactivates MPT to limit MPT-mediated AChE inhibition ex vivo.

    (A) Schematic of Franz diffusion cell: A thin layer of either poly-Oxime gel or sham gel was applied on a dialysis membrane, which was placed between donor and acceptor chambers. (B to E) Concentration of MPT and pNP (hydrolytic degradation product of MPT) in donor and acceptor chambers was measured using UFLC. The presence of sham gel did not prevent the diffusion of MPT into the acceptor chamber and could not hydrolyze MPT to generate pNP, whereas poly-Oxime actively hydrolyzed to limit the penetration of toxic MPT into the acceptor chamber. (F and G) An ex vivo assay to demonstrate the ability of poly-Oxime to limit MPT-induced assay AChE inhibition using rat blood. AChE containing rat blood was placed in the acceptor chamber, and MPT was added in the donor chamber in the presence of either poly-Oxime or sham gel. Active AChE was measured in the blood before and 3 hours after addition of MPT. In the absence of poly-Oxime gel, MPT diffused into an acceptor chamber and significantly inhibited AChE activity. However, poly-Oxime gel could hydrolyze MPT before diffusion, therefore limiting the MPT-induced inhibition of AChE. Data are means ± SD (n = 3, performed at least twice); P values were determined by one-way analysis of variance (ANOVA). ****P < 0.0001. ns, not significant.

  • Fig. 3 Poly-Oxime gel limits AChE inhibition after exposure to a lethal dose of MPT in vivo.

    (A) Coat on the dorsal side of rats was clipped 1 day before the exposure. A dose of MPT (150 mg/kg) was applied on the skin either directly or in the presence of the sham gel (220 mg per animal) or poly-Oxime gel (220 mg per animal) layer. (B) Active AChE in the blood was quantified using Ellman’s assay. Direct exposure of MPT significantly reduced the active AChE in the blood. Sham gel could not limit MPT-induced AChE inhibition, while poly-Oxime gel deactivated MPT before entering into the skin, therefore reducing the MPT-induced AChE inhibition. (C to F) On day 5, rats were sacrificed, tissue was collected, and the amount of active AChE was quantified. Dermal exposure of MPT either directly on the skin or in the presence of sham gel led to the decrease in the active AChE in all tissues such as brain, heart, liver, and lung, while poly-Oxime gel significantly reduced this MPT-mediated deactivation of AChE. Data in (B) to (F) are means ± SD (n = 6 rats per group); P values were determined by one-way ANOVA with Tukey post hoc test. *P < 0.05, **P < 0.01, ****P < 0.0001.

  • Fig. 4 Either daily or a single application of poly-Oxime gel prevented mortality during repeated exposure of MPT in vivo.

    (A) Sprague-Dawley rats (10 weeks, males) were randomized in three groups (n = 6 rats per group): (i) direct exposure of MPT, (ii) sham gel + MPT, and (iii) poly-Oxime gel + MPT. Either sham gel or poly-Oxime gel (220 mg per rat per day) was applied every day before the MPT exposure (100 mg/kg per day for 4 days). Organs were collected either immediately after mortality or on day 30. AChE activity in blood and organs was quantified using modified Ellman’s assay. (B) MST for direct exposure of MPT and MPT received in the presence of sham gel was 4 and 6 days, respectively, while poly-Oxime gel–applied group did not show mortality. (C) Blood AChE activity dropped markedly in direct MPT and sham gel groups, while inhibition was reduced in poly-Oxime gel–applied animals. (D) Rats were divided into two groups (n = 6 rats per group). On day 0, 220 mg of sham gel and poly-Oxime gel was applied dermally on group 1 and 2 animals, respectively. Without further applying any gel, MPT (100 mg/kg per day) was given daily for 4 days. Organs were collected either immediately after mortality or on day 21. (E) MST for sham gel was 5 days, while poly-Oxime gel–applied group did not show mortality. (F) Blood AChE activity dropped markedly in group 1, while the inhibition was reduced in group 2. P values were determined by Mantel-Cox test (B and E) and one-way ANOVA with Tukey post hoc test (C and F). **P < 0.01, ****P < 0.0001.

  • Fig. 5 Poly-Oxime gel prevented loss of endurance, NMC, nerve function impairment, and uncontrolled muscle activity in vivo.

    (A) Rotarod was used to study endurance and NMC in MPT animals either directly or in the presence of poly-Oxime gel. (B) Latency to fall was measured by measuring the time the animal stayed on rotarod at a constant speed of 20 rpm, and it was normalized to the day of exposure. Directly exposed MPT animals showed significant reduction endurance that was prevented by poly-Oxime cream. (C) When animals were subjected to increasing speed from 2 to 60 rpm, the rpm reached before falling was taken as a measurement to assign NMC score, and data were normalized to the day of exposure. Poly-Oxime cream showed complete rescue of loss of NMC, which was observed with direct exposure MPT animals. (D and E) Paw prints of animals were exposed either directly or in the presence of poly-Oxime gel before and after exposure. Prints were used to calculate SFI. Direct exposure animals showed greatly reduced SFI that represents impairment of sciatic nerve function, which was again rescued by poly-Oxime cream. (F) EMGs were recorded under 2.5% isoflurane anesthesia and (G) when animals were awake. Poly-Oxime cream showed complete prevention of muscle spasm.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/4/10/eaau1780/DC1

    Fig. S1. Rheological analysis of poly-Oxime and sham gels.

    Fig. S2. Ex vivo Franz diffusion assay with commercial organophosphates.

    Fig. S3. Limiting MPT-induced AChE inhibition in blood using poly-Oxime and poly-methoxyOxime.

    Fig. S4. Change in body weight and body temperature following acute exposure.

    Fig. S5. Brain AChE and body weight decrease in repeated exposure of MPT with daily application of poly-Oxime gel.

    Fig. S6. Brain AChE and body weight decrease in repeated exposure of MPT with single application of gel.

    Fig. S7. Ex vivo Franz diffusion assay with various barrier creams to measure AChE inhibition.

    Fig. S8. Ex vivo Franz diffusion assay with poly-Oxime gel before and after exposure to UV light.

    Table S1. Pseudo–first-order rate constants for hydrolysis of organophosphates by poly-Oxime polymer.

    Supplementary Methods

  • Supplementary Materials

    This PDF file includes:

    • Fig. S1. Rheological analysis of poly-Oxime and sham gels.
    • Fig. S2. Ex vivo Franz diffusion assay with commercial organophosphates.
    • Fig. S3. Limiting MPT-induced AChE inhibition in blood using poly-Oxime and poly-methoxyOxime.
    • Fig. S4. Change in body weight and body temperature following acute exposure.
    • Fig. S5. Brain AChE and body weight decrease in repeated exposure of MPT with daily application of poly-Oxime gel.
    • Fig. S6. Brain AChE and body weight decrease in repeated exposure of MPT with single application of gel.
    • Fig. S7. Ex vivo Franz diffusion assay with various barrier creams to measure AChE inhibition.
    • Fig. S8. Ex vivo Franz diffusion assay with poly-Oxime gel before and after exposure to UV light.
    • Table S1. Pseudo–first-order rate constants for hydrolysis of organophosphates by poly-Oxime polymer.
    • Supplementary Methods

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