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. 2024 Jan 17;388(2):613-623.
doi: 10.1124/jpet.123.001666.

Pharmacologic Inhibition of Transient Receptor Potential Ion Channel Ankyrin 1 Counteracts 2-Chlorobenzalmalononitrile Tear Gas Agent-Induced Cutaneous Injuries

Satyanarayana Achanta  1 , Narendranath Reddy Chintagari  2 , Shrilatha Balakrishna  2 , Boyi Liu  2 , Sven-Eric Jordt  1
Affiliations

Pharmacologic Inhibition of Transient Receptor Potential Ion Channel Ankyrin 1 Counteracts 2-Chlorobenzalmalononitrile Tear Gas Agent-Induced Cutaneous Injuries

Satyanarayana Achanta et al. J Pharmacol Exp Ther. .

Abstract

Deployment of the tear gas agent 2-chlorobenzalmalononitrile (CS) for riot control has significantly increased in recent years. The effects of CS have been believed to be transient and benign. However, CS induces severe pain, blepharospasm, lachrymation, airway obstruction, and skin blisters. Frequent injuries and hospitalizations have been reported after exposure. We have identified the sensory neuronal ion channel, transient receptor potential ankyrin 1 (TRPA1), as a key CS target resulting in acute irritation and pain and also as a mediator of neurogenic inflammation. Here, we examined the effects of pharmacologic TRPA1 inhibition on CS-induced cutaneous injury. We modeled CS-induced cutaneous injury by applying 10 μl CS agent [200 mM in dimethyl sulfoxide (DMSO)] to each side of the right ears of 8- to 9-week-old C57BL/6 male mice, whereas left ears were applied with solvent only (DMSO). The TRPA1 inhibitor HC-030031 or A-967079 was administered after CS exposure. CS exposure induced strong tissue swelling, plasma extravasation, and a dramatic increase in inflammatory cytokine levels in the mouse ear skin. We also showed that the effects of CS were not transient but caused persistent skin injuries. These injury parameters were reduced with TRPA1 inhibitor treatment. Further, we tested the pharmacologic activity of advanced TRPA1 antagonists in vitro. Our findings showed that TRPA1 is a crucial mediator of CS-induced nociception and tissue injury and that TRPA1 inhibitors are effective countermeasures that reduce key injury parameters when administered after exposure. Additional therapeutic efficacy studies with advanced TRPA1 antagonists and decontamination strategies are warranted. SIGNIFICANCE STATEMENT: 2-Chlorobenzalmalononitrile (CS) tear gas agent has been deployed as a crowd dispersion chemical agent in recent times. Exposure to CS tear gas agents has been believed to cause transient acute toxic effects that are minimal at most. Here we found that CS tear gas exposure causes both acute and persistent skin injuries and that treatment with transient receptor potential ion channel ankyrin 1 (TRPA1) antagonists ameliorated skin injuries.

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Figures

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Graphical abstract
Fig. 1.
Fig. 1.
Effects of TRPA1 antagonists on CS tear gas agent–induced cutaneous inflammation. (A) CS tear gas exposure and treatment paradigm. The right ears of C57BL/6 male mice were exposed to CS (200 mM, 20 μl) and the left ears to DMSO (solvent for CS, 20 μl). At 0.5 and 4 hours after CS exposure, mice were treated with vehicle (0.5% methylcellulose), HC-030031 (HC), or A967079 (A96) intraperitoneally (i.p.). At 4 hours after CS exposure, mice were injected with IRDye 800CW contrast agent intravenously (i.v.), and in vivo imaging was performed at 5.5 hours after CS exposure. At 6.5 hours after CS exposure, mice were euthanized, ear thickness was measured, and ear punch biopsies were collected. (B–F) Ear thickness, ear punch biopsy weights, and proinflammatory cytokine markers in mice exposed to CS tear gas agent that received either vehicle (0.5% methylcellulose) or TRPA1 antagonist (HC or A96). Data were analyzed by one-way ANOVA with Tukey’s post hoc multiple comparison test. Data are presented as mean ± S.E.M., n = 5 per group. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ns = nonsignificant.
Fig. 2.
Fig. 2.
Effect of TRPA1 antagonists on decreasing CS tear gas agent–induced cutaneous vascular leakage. The right ears of C57BL/6 male mice were exposed to CS (200 mM, 20 μl) and the left ears to DMSO (solvent for CS, 20 μl). At 0.5 and 4 hours after CS exposure, mice were treated with vehicle (0.5% methylcellulose), HC-030031 (HC), or A967079 (A96) intraperitoneally (i.p.). At 4 hours after CS exposure, mice were injected with IRDye 800CW contrast agent intravenously (i.v.), and in vivo imaging was performed at 5.5 hours after CS exposure using MousePOD fitted on Li-COR Odyssey CLX. Representative scan profiles of mice showing profound vascular leakage in the control group (A) and decreased vascular leakage in the treated groups (B and C). Photograph showing MousePOD fitted with Li-COR Odyssey CLX (D). Bar graph showing the quantification of IRDye 800CW contrast agent that leaked into the ear subcutaneous tissue (E). Data were analyzed by one-way ANOVA with Tukey’s post hoc multiple comparison test. Data are presented as mean ± S.E.M., n = 3 to 4 per group. *P ≤ 0.05.
Fig. 3.
Fig. 3.
Effects of TRPA1 antagonists on CS tear gas agent–induced histopathology features. Right ears of C57BL/6 male mice were exposed to CS (200 mM, 20 μl) and left ears to DMSO (solvent for CS, 20 μl). At 0.5 and 4 hours after CS exposure, mice were treated with vehicle (0.5% methylcellulose), HC-030031 (HC), or A967079 (A96) intraperitoneally (i.p.). At 6.5 hours after CS exposure, mice were euthanized and ear punch biopsies were collected for histopathological analysis. Representative H&E-stained histopathologic sections from (A) DMSO (solvent for CS), (B) CS + vehicle, (C) CS + HC030031, and (D) CS + A967079 groups are presented at 20× magnification. Black arrow heads = infiltration of leukocytes. d, dermis; e, epidermis; ec, elastic cartilage; hf, hair follicle.
Fig. 4.
Fig. 4.
Persistent skin injuries after CS tear gas agent exposure and effects of TRPA1 antagonists on skin inflammation in an extended mouse observation model. (A) CS tear gas exposure and treatment. Right ears of C57BL/6 male mice were exposed to CS (200 mM, 20 μl) and left ears to DMSO (solvent for CS, 20 μl). At 0.5, 4, 24, and 48 hours after CS exposure, mice were treated with vehicle (0.5% methylcellulose), HC-030031 (HC), or A967079 (A96) intraperitoneally (i.p.). (B–E) Ear thickness, ear punch biopsy weights, and cytokine markers in mice receiving vehicle or TRPA1 antagonist after exposure to CS tear gas agent. Data were analyzed by one-way ANOVA with Tukey’s post hoc multiple comparison test. Data are presented as mean ± S.E.M., n = 5 per group. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ns = nonsignificant.
Fig. 5.
Fig. 5.
Histologic images of skin injuries after CS tear gas agent exposure and treatment with TRPA1 antagonists. Right ears of C57BL/6 male mice were exposed to CS (200 mM, 20 μl) and left ears to DMSO (solvent for CS, 20 μl). At 0.5, 4, 24, and 48 hours after CS exposure, mice were treated with vehicle (0.5% methylcellulose), HC-030031 (HC), or A967079 (A96) intraperitoneally (i.p.). Representative H&E-stained histopathologic sections from (A) DMSO, (B) CS + vehicle, (C) CS + HC-030031, and (D) CS + A967079 groups are presented at 20× magnification. Black arrow heads = infiltration of leukocytes; red arrow = epidermal thickening. d, dermis; e, epidermis; ec, elastic cartilage; hf = hair follicle.
Fig. 6.
Fig. 6.
Effects of advanced TRPA1 antagonists on CS tear gas agent–induced calcium influx. HEK293T cells were transfected with mouse (A and B) and human (C and D) TRPA1 plasmid. (A and C) Show dose-response curve of CS tear gas [mouse: EC50 = 0.47 μM, 95% confidence intervals (CI) = 0.32–0.83 μM; human: EC50 = 0.016 μM, 95% CI = 0.01–0.024 μM]. (B and D) TRPA1 antagonists [A967079 (A96), AMG0902, or GDC0334] inhibited the calcium influx response elicited by CS tear gas in mouse and human TRPA1-transfected cells. IC50 and 95% CI values are presented in tables under Fig. 6, B and D. Data are presented as mean ± S.E.M. Each experiment was performed with a minimum of three replicates.
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