Oxime-functionalized anti-insecticide fabric reduces insecticide exposure through dermal and nasal routes, and prevents insecticide-induced neuromuscular-dysfunction and mortality

Abstract

Farmers from South Asian countries spray insecticides without protective gear, which leads to insecticide exposure through dermal and nasal routes. Acetylcholinesterase plays a crucial role in controlling neuromuscular function. Organophosphate and carbamate insecticides inhibit acetylcholinesterase, which leads to severe neuronal/cognitive dysfunction, breathing disorders, loss of endurance, and death. To address this issue, an Oxime-fabric is developed by covalently attaching silyl-pralidoxime to the cellulose of the fabric. The Oxime-fabric, when stitched as a bodysuit and facemask, efficiently deactivates insecticides (organophosphates and carbamates) upon contact, preventing exposure. The Oxime-fabric prevents insecticide-induced neuronal damage, neuro-muscular dysfunction, and loss of endurance. Furthermore, we observe a 100% survival rate in rats when repeatedly exposed to organophosphate-insecticide through the Oxime-fabric, while no survival is seen when organophosphate-insecticide applied directly or through normal fabric. The Oxime-fabric is washable and reusable for at least 50 cycles, providing an affordable solution to prevent insecticide-induced toxicity and lethality among farmers.

Fig. 1. Developing an oxime-fabric that can hydrolyze insecticides to prevent insecticide-induced toxicity.

Top, acetylcholinesterase (AChE) enzyme controls neuromuscular function by hydrolyzing acetylcholine, a neurotransmitter at synapses. While spraying, agriculture workers get exposed to insecticides (methyl parathion, MPT), through dermal and inhalation routes. Upon exposure, organophosphates covalently inhibit AChE, which causes severe toxicity and mortality. bottom, In our approach, α-nucleophile, oxime-based silyl-pralidoxime is covalently attached to the cellulose of fabric to generate Oxime-fabric. Oxime-fabric can be stitched as a bodysuit and a facemask, which, upon contact, can hydrolyze organophosphates into phosphoric acid that cannot inhibit AChE. Therefore, Oxime-fabric could prevent exposure to insecticides, thereby preventing insecticide-induced toxicity and mortality.

Fig. 2. Washable and reusable Oxime-fabric deactivates MPT to prevent MPT-mediated AChE inhibition, ex vivo.

a–c Schematic of bromophenol blue (BPB) assay to quantify the amount of silyl-pralidoxime attached to the fabric (a); b photographs of Oxime-fabric and normal fabric before and after treating BPB dye, and c quantification of silyl-pralidoxime present on fabric, suggested that silyl-pralidoxime (125 μg/cm²) was present on Oxime-fabric, while it was not detectable on normal fabric (P < 0.0001; unpaired two-tailed t test). d Schematic of Franz diffusion cell. e The efficacy of Oxime-fabric to prevent methyl parathion (MPT)-induced AChE inhibition, an ex vivo assay was performed using rat blood. Active AChE was measured in unexposed native blood and 3 hr post addition of MPT. The normal fabric could not prevent MPT diffusion into the acceptor chamber, which resulted in significant inhibition of AChE activity (P < 0.0001; ordinary one-way ANOVA with Tukey’s post hoc analysis). On the contrary, Oxime-fabric could hydrolyze MPT before it diffuses, preventing the MPT-induced inhibition of AChE (P = 0.6161; ordinary one-way ANOVA with Tukey’s post hoc analysis). f–h Oxime-fabric was subjected to delicate washing cycles in a washing machine using a mild non-ionic detergent (0.1%). Photographs of Oxime-fabric after 10, 25, and 50 cycles of washing and stained with BPB dye (f), and quantification of silyl-pralidoxime after 10, 25, and 50 cycles of washing (g). h Franz diffusion assay to investigate the efficacy of multiple cycles of washed Oxime-fabric to hydrolyze MPT to prevent MPT-induced inhibition of AChE activity. Direct exposure to MPT significantly inhibited AChE activity (P < 0.0001; ordinary one-way ANOVA with Tukey’s post hoc analysis). On the contrary, multiple washing cycles did not cause the leaching of active compound from the fabric and retained its activity. Data are mean ± s.d. (n = 4, from independent experiments). c P values were determined by two-tailed Student’s t test with Welch’s correction, and for e, h, by ordinary one-way ANOVA with Tukey’s post hoc analysis, and for g, by repeated measures one-way ANOVA by GraphPad PRISM 9, and exact P values are indicated. ns not significant. Source data are provided as a Source Data file.

Fig. 3. Oxime-fabric prevents acute exposure to a lethal dose of MPT, and reduces AChE inhibition, in vivo.

a Schematic of experimental design: The dorsal coat of Sprague-Dawley (SD) rats was clipped using a hair clipper one day prior to exposure. Organophosphate, methyl parathion (MPT, 150 mg/kg) was applied on the skin either directly or in the presence of normal fabric or Oxime-fabric. b–g Before and after 72 h of exposure to MPT, active AChE in the blood (b) and internal organs such as brain (c), lung (d), liver (e), kidney (f), and heart (g) was quantified. Three days post-exposure to MPT, animals were sacrificed, tissues were collected, and the amount of active AChE was quantified using modified Ellman’s assay. Dermal exposure of MPT directly or through normal fabric significantly reduced the active AChE in blood and tissues. On the contrary, Oxime-fabric deactivated MPT and prevented MPT-induced inhibition of AChE. Data are mean ± s.d. (n = 5 rats per group). b P values were determined by two-tailed Student’s t test with Welch’s correction, and for c–g by ordinary one-way ANOVA with Tukey’s post hoc analysis by GraphPad PRISM 9, and exact P values are indicated. ns not significant. Source data are provided as a Source Data file.

Fig. 4. Oxime-fabric prevented sciatic nerve function impairment and involuntary muscle activity, in vivo.

Schematic of an alley in which rats were walked to collect the footprints (a). Fifteen Sprague-Dawley rats were randomly divided into three groups (n = 5 per group). All paws of the animal were colored with different non-toxic water colors, and trained to walk through an ally (8 cm width, 120 cm length and 10 cm height) leading to its cage. Before exposure to methyl parathion (MPT), footprints of animals in all groups were collected (b), and analyzed manually, and the Sciatic function index (SFI) was calculated using the formula reported elsewhere^,. c A single dose of MPT (150 mg/kg) was exposed dermally either directly or through normal fabric or Oxime-fabric, and 72 h post-exposure SFI was calculated. Dermal exposure of MPT directly or through normal fabric significantly reduced SFI values, which represents severe impairment of sciatic nerve function, whereas Oxime-fabric prevented such impairment. d Electromyograms (EMGs) were recorded before and 72 h post-exposure to MPT for all animals in three groups. Representative graphs are given here. MPT exposure directly and through normal fabric caused overstimulation of neuromuscular signaling and muscle spasms, while Oxime-fabric showed complete prevention of muscle spasms. Data are mean ± s.d. (n = 5 rats per group). c P values were determined by a two-tailed Student’s t test with Welch’s correction by GraphPad PRISM 9, and exact P values are indicated. ns not significant. Source data are provided as a Source Data file.

Fig. 5. Oxime-fabric prevented insecticide-induced muscular dysfunction and neuronal damage, in vivo.

a Schematic of rotarod and Gait analysis done over time post-exposure to methyl parathion (MPT). Rotarod was used to study muscle function endurance in animals that were exposed to MPT (150 mg/kg) through normal fabric or Oxime fabric. b 10 Sprague-Dawley rats were trained on a rotarod for four days, and latency to fall was measured by measuring the time the animal stayed on the rotarod at a constant speed of 20 rpm. The latency to fall obtained on day 4 was considered as 100%, and ten rats were randomly divided into two groups and exposed to MPT through either normal fabric or Oxime-fabric. Post-exposure to MPT, latency to fall has significantly dropped in the normal fabric group, indicating muscle dysfunction and loss of endurance, whereas Oxime-fabric prevented loss of endurance. c On day 4, before MPT exposure, the sciatic functional index (SFI) was measured for animals in both groups. Subsequently, after exposing to MPT (150 mg/kg) through either normal fabric or Oxime-fabric, SFI was measured as a function of time. A significant drop in SFI values when exposed to MPT through the normal fabric was observed, indicating neuronal damage, which Oxime-fabric prevented. Data are mean ± s.d. (n = 5 rats per group). c P values were determined by Repeated Measures one-way ANOVA by GraphPad PRISM 9, and exact P values are indicated. ns = not significant. Source data are provided as a Source Data file.

Fig. 6. Oxime-fabric prevented mortality during repeated exposure of ethyl-paraoxon (EPx), in vivo.

a Sprague-Dawley rats (10 weeks, males) were randomized in three groups (n = 6 rats per group); (i) direct exposure of EPx, (ii) normal fabric + EPx, and (iii) Oxime-fabric + EPx. On day 0, 50 mg/kg/day of EPx and 25 mg/kg/day of EPx were applied dermally for five days. b Median survival time (MST) for direct exposure of EPx and EPx received through the normal fabric was 1 and 4 days, respectively, while the Oxime-fabric group did not show mortality (P < 0.0001; Mantel-Cox test). c–h Active AChE in blood and organs was quantified using a modified Ellman’s assay. Blood AChE activity dropped significantly in direct EPx and normal fabric groups, while inhibition of AChE was prevented in Oxime-fabric group animals (c). Organs were collected either immediately after mortality (for group i and ii animals) or on day 14 (group iii animals), and the amount of active AChE was quantified using Ellman’s assay in brain (d), lung (e), liver (f), kidney (g), and heart (h). Dermal exposure of EPx directly or through normal fabric significantly reduced the active AChE in tissues. On the contrary, Oxime-fabric has deactivated EPx and prevented EPx-induced inhibition of AChE. Data are mean ± s.d. (n = 6 rats per group). b P values were determined by Mantel-Cox test, and for c by two-tailed Student’s t test with Welch’s correction, and for d–h, by ordinary one-way ANOVA with Tukey’s post hoc analysis by GraphPad PRISM 9, and exact P values are indicated. ns not significant. Source data are provided as a Source Data file.

Fig. 7. Oxime-fabric prevents methyl parathion (MPT) exposure through inhalation route, in vivo.

a Schematic of experimental design: MPT aerosols (0.4 mg/L for 1 h) were generated through a nebulizer that was connected to a closed chamber, where three groups of animals were restrained and inhaled MPT aerosols for 1 h. Fifteen Sprague-Dawley rats were randomly divided into three groups. Group 1 animals inhaled aerosols directly, while group 2 and 3 animals inhaled aerosols through normal and Oxime-fabric, respectively. b Active AChE was quantified before and after 72 h of post-exposure, and data showed that MPT aerosol exposure either directly or through normal fabric did not prevent MPT-induced AChE inhibition, while Oxime-fabric prevented inhibition of AChE. c–g Three days post-exposure to MPT aerosols, animals were sacrificed, tissues were collected, and the amount of active AChE was quantified in brain (c), lung (d), liver (e), kidney (f), and heart (g). Nasal exposure of MPT directly or through normal fabric significantly reduced the active AChE in tissues. On the contrary, Oxime-fabric has deactivated the aerosol form of MPT and prevented MPT-induced inhibition of AChE. Data are mean ± s.d. (n = 5 rats per group). b P values were determined by two-tailed Student’s t-test with Welch’s correction, and for c–g by ordinary one-way ANOVA with Tukey’s post hoc analysis by GraphPad PRISM 9, and exact P values are indicated. ns = not significant. Source data are provided as a Source Data file.