Skip to main content
Download PDF

Nociceptive transient receptor potential ankyrin 1 (TRPA1) in sensory neurons are targets of the antifungal drug econazole

BMC Pharmacology and Toxicology volume 25, Article number: 53 (2024) Cite this article

Abstract

Background

Econazole is a widely used imidazole derivative antifungal for treating skin infections. The molecular targets for its frequent adverse effects of skin irritation symptoms, such as pruritus, burning sensation, and pain, have not been clarified. Transient receptor potential (TRP) channels, non-selective cation channels, are mainly expressed in peripheral sensory neurons and serve as sensors for various irritants.

Methods

We investigated the effect of econazole on TRP channel activation by measuring intracellular calcium concentration ([Ca2+]i) through fluorescent ratio imaging in mouse dorsal root ganglion (DRG) neurons isolated from wild-type, TRPA1(−/−) and TRPV1(−/−) mice, as well as in heterologously TRP channel-expressed cells. A cheek injection model was employed to assess econazole-induced itch and pain in vivo.

Results

Econazole evoked an increase in [Ca2+]i, which was abolished by the removal of extracellular Ca2+ in mouse DRG neurons. The [Ca2+]i responses to econazole were suppressed by a TRPA1 blocker but not by a TRPV1 blocker. Attenuation of the econazole-induced [Ca2+]i responses was observed in the TRPA1(−/−) mouse DRG neurons but was not significant in the TRPV1(−/−) neurons. Econazole increased the [Ca2+]i in HEK293 cells expressing TRPA1 (TRPA1-HEK) but not in those expressing TRPV1, although at higher concentrations, it induced Ca2+ mobilization from intracellular stores in untransfected naïve HEK293 cells. Miconazole, which is a structural analog of econazole, also increased the [Ca2+]i in mouse DRG neurons and TRPA1-HEK, and its nonspecific action was larger than econazole. Fluconazole, a triazole drug failed to activate TRPA1 and TRPV1 in mouse DRG neurons and TRPA1-HEK. Econazole induced itch and pain in wild-type mice, with reduced responses in TRPA1(−/−) mice.

Conclusions

These findings suggested that the imidazole derivatives econazole and miconazole may induce skin irritation by activating nociceptive TRPA1 in the sensory neurons. Suppression of TRPA1 activation may mitigate the adverse effects of econazole.

Peer Review reports

Background

Econazole is a broad-spectrum imidazole antifungal agent that is primarily used for treating dermatophyte infections, such as tinea pedis and ringworm. It is typically administered topically in the form of a cream. Although many patients experience a favorable cure rate of 90%, 1–4% of individuals may encounter side effects, such as pruritus, burning sensation, and erythema [1, 2].

Pain and itching are uncomfortable sensations that arise through complex pathways involving afferent sensory neurons in peripheral tissues, such as the skin. Various types of transient receptor potential (TRP) channels serve as nociceptors, which respond to chemical, thermal, and mechanical stimuli to evoke sensations of pain and itching [3]. Among these channels, TRPV1 and TRPA1 play essential roles in nociception [4]. As nonselective cation channels in sensory neurons, their activation in peripheral terminals results in the entry of Ca2+ and Na+, leading to membrane depolarization and the generation of action potentials. These action potentials transmit sensory information to the spinal cord and, subsequently, to higher nerve centers [5, 6].

Although the concept of TRPA1 functioning as a cold sensor in mammals remains a topic of debate [7], this channel is well-known for its responsiveness to various chemical stimuli, such as cinnamaldehyde and mustard oil [8]. In contrast, TRPV1 channel is recognized as a receptor for pungent compounds, such as capsaicin, as well as acidic pH and heat [9, 10]. Notably, some medications that are currently in use can stimulate nociceptive TRP channels. For instance, the widely used local anesthetic lidocaine activates both TRPA1 and TRPV1, leading to the sensation of pain [11, 12]. Flufenamic acid, which is a nonsteroidal anti-inflammatory drug (NSAID), has been reported to activate TRPA1 [13], and several NSAIDs have been linked to skin irritation [14, 15]. Propofol, which is an intravenous general anesthetic, induces intense vascular pain by activating TRPA1 [16]. Furthermore, retinoid, which is commonly used in the treatment of skin disorders, has been found to sensitize TRPA1 and cause skin irritation [17]. These studies collectively suggest the association of TRP channel activation with the adverse effects of skin irritation by certain medications.

In this study, we aimed to investigate the involvement of TRPA1 and TRPV1 channels in econazole-induced skin irritation using analytical techniques, such as calcium imaging, along with pharmacological and genetic approaches. We also investigated whether econazole causes itch and pain behaviors using cheek injection model. Moreover, to understand the structural specificity of econazole, we examined the activities of miconazole, which is another imidazole antifungal drug, and the triazole antifungal drug fluconazole.

Methods

Experimental animals

All experimental protocols on animals were approved by the Committee on Animal Experimentation of Tottori University. The number of animals used was minimized.

Isolation and culture of mouse DRG neurons

We used adult (4–12-week-old) C57BL/6 mice [wild type, TRPA1(−/−), and TRPV1(−/−)] of either sex. The TRPA1(−/−) mice were kindly provided by Dr. Julius of the University of California, whereas the TRPV1(−/−) mice were purchased from Jackson Lab (Sacramento, CA, USA).

Euthanasia was administered to the mice by CO2 gas inhalation. Mouse dorsal root ganglion (DRG) cells were isolated and cultured, as described previously [18]. In brief, DRGs were removed and minced in phosphate-buffered saline (PBS: 137 mM NaCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, and 2.7 mM KCl). Thereafter, the DRGs were enzymatically digested for 30 min at 37 °C in PBS containing 1 mg/ml of type II collagenase (Worthington, Lakewood, NJ, USA) and 1 mg/ml of DNase I (Roche Molecular Biochemical, Indianapolis IN, USA). Subsequently, the DRGs were further digested in PBS containing 10 mg/ml of trypsin (Sigma, St. Louis, MO, USA) and 1 mg/ml of DNase for 15 min at 37 °C. The digested ganglia were immersed in Dulbecco’s modified Eagle medium (Sigma) supplemented with 10% fetal bovine serum (BioSera, Cholet, France), penicillin G (100 U/ml), and streptomycin (100 µg/ml). DRG cells were obtained by gentle trituration using Pasteur pipettes. The cell suspension was centrifuged at 800 rpm for 2 min at room temperature, and the pellet was resuspended in the culture medium. Aliquots were placed onto glass cover slips coated with poly-D/L-lysine (Sigma) and cultured in a humidified atmosphere of 95% air and 5% CO2 at 37 °C. The cells were used within 24 h after the isolation.

Heterologous expression in HEK293 cells

Human embryonic kidney (HEK) 293 cells were maintained in the culture medium, as described above. The cells were transfected with the expression vectors (mouse TRPA1 and mouse TRPV1) [18] using a transfection reagent (Lipofectamine 2000, Invitrogen, Tokyo, Japan), according to the manufacturer’s protocol, and used within 24 h after transfection.

Calcium imaging

The intracellular Ca2+ concentrations ([Ca2+]i) in the individual cells were measured using a fluorescent-imaging system (Aqua Cosmos, Hamamatsu Photonics, Japan), as described previously [19]. Briefly, the cells were incubated for 40 min at 37 °C with 10 µM fura-2 AM (Molecular Probes) in HEPES-buffered solution (134 mM NaCl, 6 mM KCl, 1.2 mM MgCl2, 2.5 mM CaCl2, 10 mM glucose, and 10 mM HEPES; pH 7.4). A coverslip with fura-2-loaded cells was transferred into an experimental chamber and mounted on an inverted microscope (Olympus IX71), which was equipped with image acquisition and analysis system. The cells were illuminated every 5 s with light at 340 and 380 nm, and the respective fluorescence signals at 500 nm were detected. The ratios of fluorescent signals (F340/F380) were analyzed. Calibration of fura-2 was carried out with standard Ca2+ buffer solutions (Calcium Calibration Buffer Kits, Invitrogen) containing 4 µM fura-2 (Invitrogen) with the equation reported by Grynkiewicz et al. [20]. The cells were continuously superfused with an external solution at a flow rate of approximately 2 ml/min by gravity. Drugs were applied by replacing the reflux tube with a test tube containing the drug in the HEPES-buffered solution. The 80 mM KCl solution included 80 mM KCl, 60 mM NaCl, 1.2 mM MgCl2, 2.5 mM CaCl2, and 10 mM HEPES. For the Ca2+-imaging experiments, regions of interest (ROIs) were set on spherical, neuron-like cells. Consequently, almost all selected cells responded to 80 mM KCl. Cells with significantly high resting [Ca2+]i or cells that were evidently dead were excluded from the analysis. All experiments were carried out at room temperature (22–25 °C).

Behavioral test

A cheek injection model was used to determine econazole-induced itch and pain using male wild type and TRPA1(−/−) mice (8–14 weeks old). After a 30 min habituation, 10 µl of vehicle or econazole (20 nmol) was intradermally injected into the right cheek of the mice with a 30-gauge needle syringe (BD Lo-Dose, Becton Dickinson, Fukushima, Japan). Behaviors were recorded for 30 min with a digital camera (HDR-PJ40V, Sony, Tokyo, Japan). Scratching and wiping behaviors directed toward the injection site were counted, and the former and latter was judged as itch and pain, respectively [21, 22].

Chemicals

The following drugs were used as vehicle and concentration for stock solution: allyl isothiocyanate (AITC) in 1 M dimethyl sulfoxide (DMSO) (Nakarai, Tokyo, Japan); capsaicin (Cap) in 1 mM ethanol (Sigma-Aldrich); A967079 in 0.01 M DMSO (Santa Cruz); N-(4-t-butylphenyl)-4-(3-chloropyridin-2-yl) tetrahydropyrazine-1(2 H)-carboxamide (BCTC) in 5 mM DMSO (Wako); and econazole, miconazole, and fluconazole, each in 0.1 M DMSO (Tokyo Chemical Industry, Tokyo, Japan); thapsigargin in 0.01 M DMSO (Wako). All the other drugs used were obtained from Wako.

Data analysis

The data are presented as mean ± SEM (n = number of cells). Comparison between two groups was performed by unpaired Student’s t-test. For multiple comparisons, one-way analysis of variance followed by the Tukey–Kramer test or Steel-Dwass test was used. A P value of < 0.05 was considered statistically significant.

Results

[Ca2+]i response to econazole in mouse DRG neurons

In Fig. 1A, we present the chemical structures of the three antifungal drugs used in the present study. Because nociceptive TRP channels are known for their high Ca2+ permeability [23], we used calcium imaging to examine the effects of econazole on [Ca2+]i in mouse DRG neurons. Econazole elicited [Ca2+]i increases in mouse DRG neurons that were responsive to 80 mM KCl (Fig. 1B). The amplitude of the [Ca2+]i and the percentage of neurons responding to econazole increased in a concentration-dependent manner (Fig. 1C, D). High concentrations of econazole seem to evoke Ca2+ release from intracellular stores (see in Fig. 3). To determine whether stored Ca2+ depletion was related to the saturation of [Ca2+]i responses, we examined the effect of thapsigargin, an inhibitor of endoplasmic reticulum Ca2+-ATPase, which leads the depletion of intracellular stored Ca2+ [24]. There was no difference in the magnitude of the response of econazole (100 µM) between thapsigargin (1 µM)-treated and -untreated cells (treated: 98.0 ± 5.2 nM, n = 95; untreated: 91.2 ± 4.0 nM, n = 88). Notably, econazole at 100 µM increased the [Ca2+]i in nearly all neurons and suppressed the [Ca2+]i responses to the subsequently applied KCl (Fig. 1B and C). These findings suggested that econazole stimulated sensory neurons, but it also exhibited nonspecific effects at high concentrations. Consequently, in the following experiments, we analyzed the effects of econazole at concentrations of 10 and 30 µM.

Fig. 1
figure 1

Econazole-induced changes in [Ca2+]i in mouse DRG neurons. (A) The chemical structures of econazole, miconazole, and fluconazole are shown. (B) The actual recordings of [Ca2+]i responses to econazole at 10 µM (left) and 100 µM (right) and to 80 mM KCl in wild type mouse DRG neurons. (C) The relationship between the magnitude of [Ca2+]i increase induced by econazole (Eco) and by KCl at different concentrations of econazole. (D) The percentages of the neurons responding to econazole at various concentrations are shown. Symbols or columns with vertical lines represent the mean values ± SEM for each econazole concentration (n = 193–395 from 4–6 mice). (E) The actual recording of [Ca2+]i responses to 10 µM econazole in the absence of external Ca2+ (0Ca) and KCl is shown. The 0Ca period is indicated with a grayish bar. (F) A summary of the amplitudes of [Ca2+]i responses to econazole at 10 and 30 µM in the presence (control) and absence of extracellular Ca2+ (0Ca) (n = 133–190 from three mice) is shown. **P < 0.01 vs. control, as determined by Student’s t-test

Full size image

Next, we investigated the effect of removing extracellular Ca2+ on the econazole-induced responses. Removal of extracellular Ca2+ completely eliminated the econazole-induced increase in the [Ca2+]i (Fig. 1E, F). These results indicated that the [Ca2+]i responses to econazole at 10 and 30 µM were solely dependent on the influx of extracellular Ca2+.

Relationships between responsiveness to econazole and TRP channel agonists in mouse DRG neurons

TRPA1 and TRPV1 are nonselective cation channels that are permeable to calcium, expressed in DRG neurons, and involved in pain and itch signaling [3]. We aimed to investigate the econazole sensitivity of a subpopulation of neurons expressing TRPA1 and/or TRPV1. Since TRP channel activators often elicit channel desensitization [25], mouse DRG neurons were applied with econazole first, followed by the selective agonists for TRPA1 (i.e., AITC, 100 µM) and TRPV1 (i.e., capsaicin, 1 µM). This order was adopted so that the TRP agonists would not affect the action of econazole. The same protocol was done with miconazole and fluconazole, which are described later. Some of the econazole-sensitive neurons responded to AITC and/or capsaicin (Fig. 2A). The Venn diagram in Fig. 2B illustrates the characteristics of all neurons. Many neurons responded to both AITC and capsaicin, likely because a significant number of neurons coexpress TRPA1 and TRPV1 [26]. At 10 µM of econazole, almost all of the econazole-sensitive neurons responded to AITC (n = 35, 94.6%), and a subset responded to capsaicin (n = 22, 59.5%). Of the DRG neurons that responded to 30 µM of econazole (n = 67), some were sensitive to AITC (n = 27, 40.2%) and capsaicin (n = 34, 50.7%). Moreover, the neurons that did not respond to either AITC or capsaicin responded to 30 µM of econazole (n = 28, 41.8%). These results suggested that econazole at 10 µM primarily increased [Ca2+]i in the DRG neurons expressing TRPA1; at 30 µM, it can also affect a different subset of neurons expressing molecules other than TRPA1 and TRPV1.

Fig. 2
figure 2

The [Ca2+]i responses to econazole and effects of TRP channel antagonists and gene deletion in mouse DRG neurons. (A) The actual recordings of [Ca2+]i responses to the sequential application of 10 µM econazole (Eco), 0.1 mM AITC, 1 µM capsaicin (Cap), and 80 mM KCl are shown. (B) The Venn diagrams of the number of neurons that responded to each stimulation with econazole at 10 µM (left) and 30 µM (right). The outer area of all the circles indicates the number of neurons that responded to KCl alone. The data are derived from three mice. (C) A summary of [Ca2+]i responses to econazole at 10 µM (left) and 30 µM (right) in the absence (control) or presence of 10 µM A967079 (A96) or 1 µM BCTC is shown. Columns with vertical lines represent the mean values ± SEM (10 µM econazole: n = 158–293, 30 µM econazole: n = 265–321 from four mice). **P < 0.01 vs. control, one-way ANOVA with Tukey–Kramer test. (D) A summary of [Ca2+]i responses to econazole at 10 µM (left) and 30 µM (right) in wild type (Wild), TRPA1 knockout (A1(−/−)), and TRPV1 knockout (V1(−/−)) mouse DRG neurons is shown. Columns with vertical lines show the mean values ± SEM (10 µM econazole: wild, n = 305; A1(−/−), n = 280; V1(−/−), n = 310 from four mice. 30 µM econazole: wild, n = 250; A1(−/−), n = 291; V1(−/−), n = 171 from four mice). *P < 0.05, **P < 0.01 vs. wild, one-way ANOVA with Tukey–Kramer test

Full size image

Effect of pharmacological and genetic inhibition of TRPA1 and TRPV1 on econazole-induced stimulation in mouse DRG neurons

Because econazole increased [Ca2+]i in neurons that were sensitive to TRP channel agonists, we examined the effect of pharmacological inhibition on the [Ca2+]i responses to econazole in mouse DRG neurons. Application of the TRPA1 antagonist A967079 (10 µM) significantly suppressed the [Ca2+]i responses to econazole at 10 µM but not at 30 µM. Conversely, treatment with selective TRPV1 antagonist BCTC (10 µM) failed to inhibit the [Ca2+]i responses to econazole (Fig. 2C).

We further assessed the effect of genetic depletion of TRPA1 and TRPV1 on the econazole-induced [Ca2+]i responses using knockout mouse DRG neurons. As shown in Fig. 2D, the econazole-induced responses were significantly diminished, although only slightly at 30 µM, in the TRPA1(−/−) mouse DRG neurons. In contrast, the magnitudes of the [Ca2+]i responses to econazole in the TRPV1(−/−) mouse DRG neurons were not significantly reduced, compared with those in wild type neurons.

These results suggested that econazole selectively activated the TRPA1 channels at 10 µM and may interact with other molecules at higher concentrations, leading to Ca2+ influx in mouse DRG neurons.

Effect of econazole on heterologous TRPA1-expressing cells

To further investigate the stimulatory action of econazole on TRPA1, we evaluated its effects on HEK293 cells with heterologous expression of TRPA1 (TRPA1-HEK). Based on our findings on nonspecific [Ca2+]i responses at high concentrations of econazole in mouse DRG neurons, we first examined the effect of econazole on naïve HEK293 cells (naïve HEK). As shown in Fig. 3 A and B, 30 µM of econazole increased [Ca2+]i in naïve HEK, and this effect was not eliminated by the removal of extracellular Ca2+. These results suggested that econazole at 10 µM did not increase [Ca2+]i, but at 30 µM, it triggered [Ca2+]i responses through Ca2+ release from intracellular stores in naïve HEK.

Fig. 3
figure 3

[Ca2+]i responses to econazole in naïve and TRPA1-expessed HEK 293 cells. (A) The actual recordings of [Ca2+]i responses to 30 µM econazole in naïve HEK293 cells (naïve HEK) in the presence (left) or absence of external Ca2+ (0Ca) (right) are shown. The 0Ca period is indicated with a grayish bar. (B) A summary of the amplitudes of [Ca2+]i responses to econazole at 10 µM (left) and 30 µM (right) in naïve HEK293 cells in the presence (control) or absence of extracellular Ca2+ (0Ca) is shown. Columns with vertical lines represent mean ± SEM (10 µM: control, n = 362; 0Ca, n = 278 from four experiments; 30 µM: control, n = 390; 0Ca, n = 340 from four experiments). (C) The actual recording of [Ca2+]i responses to 10 µM econazole in HEK293 cells expressing TRPA1 (upper, A1-HEK) and TRPV1 (lower, V1-HEK) is shown. The cells are stimulated by each selective agonist (100 µM AITC and 1 µM Cap). (D) The relationship between [Ca2+]i responses and the concentrations of econazole in A1-HEK and naïve HEK cells is shown. Symbols with vertical lines represent mean ± SEM (A1-HEK, n = 302–420; naïve HEK, n = 150–165 from four experiments). **P < 0.01 vs. naïve HEK, as determined by Student’s t-test

Full size image

Next, we measured the [Ca2+]i responses to econazole in TRPA1-HEK cells. Econazole at 10 µM evoked [Ca2+]i increases in TRPA1-HEK cells, as determined by the responsiveness to AITC (Fig. 3C). In contrast, econazole had no effect on the cells expressing TRPV1. Econazole dose-dependently increased [Ca2+]i in TRPA1-HEK cells (Fig. 3D). As mentioned earlier, econazole induced nonspecific [Ca2+]i increases at concentrations exceeding 30 µM in naïve HEK. These results clearly indicated that econazole could activate TRPA1.

Effect of miconazole on [Ca2+]i in mouse DRG neurons and heterologous TRPA1-expressing cells

We also examined the [Ca2+]i responses to miconazole, which is a structurally related antifungal drug to econazole [2, 27], in mouse DRG neurons. At concentrations of > 3 µM, miconazole increased [Ca2+]i in a concentration-dependent manner. At a high concentration (100 µM), miconazole triggered nonspecific [Ca2+]i responses and suppressed KCl responses (Fig. 4A). Removal of extracellular Ca2+ reduced the [Ca2+]i responses to miconazole (Fig. 4B). Of the mouse DRG neurons that responded to 10 µM of miconazole (n = 30), those that responded to AITC alone, capsaicin alone, both, or neither were 30% (n = 9), 13.3% (n = 4), 20% (n = 6), and 36.7% (n = 11), respectively (Fig. 4C). The TRPA1 blocker significantly inhibited the responses to miconazole at 10 µM but not at 30 µM, whereas no inhibition was observed with the TRPV1 blocker (Fig. 4D). Moreover, the responses to 10 µM of miconazole were significantly lower in TRPA1(−/−) mouse DRG neurons than in wild type neurons (Fig. 4E). There were no differences in the [Ca2+]i responses to miconazole between the wild type and TRPV1(−/−) mouse DRG neurons.

Fig. 4
figure 4

The [Ca2+]i responses to miconazole and effects of TRP channel antagonists and gene deletion in mouse DRG neurons. (A) The relationship between the magnitude of [Ca2+]i increase induced by miconazole (Mico) and by KCl at different concentrations of miconazole. Symbols with vertical lines represent mean ± SEM (n = 132–231 from 3–6 mice). (B) A summary of the amplitudes of [Ca2+]i responses to 10 and 30 µM of miconazole in the presence (control) and absence of extracellular Ca2+ (0Ca) is shown (10 µM: control, n = 103; 0Ca, n = 127; 30 µM: control, n = 106; 0Ca, n = 93 from three mice). Some vertical lines are obscured by symbols. (C) The Venn diagram shows the number of neurons that responded to 10 µM miconazole (Mico), 0.1 µM AITC, 1 µM capsaicin (Cap), and 80 mM KCl. The outer area of all the circles indicates the number of neurons that responded to KCl alone. Data are from three mice. (D) A summary of [Ca2+]i responses to miconazole at 10 µM (left) and 30 µM (right) in the absence (Cont) or presence of 10 µM A967079 (A96) or 1 µM BCTC is shown. Columns with vertical lines represent the mean values ± SEM (10 µM miconazole, n = 120–201; 30 µM miconazole, n = 142–199 from four mice). **P < 0.01 vs. control, one-way ANOVA with Tukey–Kramer test. (E) A summary of [Ca2+]i responses to miconazole at 10 µM (left) and 30 µM (right) in wild type (Wild), TRPA1 knockout (A1(−/−)), and TRPV1 knockout (V1(−/−)) mouse DRG neurons is shown. Columns with vertical lines show the mean values ± SEM (10 µM miconazole: wild, n = 215; A1(−/−), n = 201; V1(−/−), n = 198 from four mice. 30 µM miconazole: wild, n = 223; A1(−/−), n = 198; V1(−/−), n = 220 from four mice). *P < 0.05 vs. wild, one-way ANOVA with Tukey–Kramer test

Full size image

Miconazole increased [Ca2+]i in naïve HEK cells (Fig. 5A). After removal of extracellular Ca2+, the responses to miconazole at 10 µM were mitigated but were only partially attenuated at 30 µM (Fig. 5B). Miconazole increased [Ca2+]i in TRPA1-HEK but not in TRPV1-HEK cells (Fig. 5C). The concentration–response relationship of the [Ca2+]i responses to miconazole was significantly more pronounced in TRPA1-HEK than in naïve HEK cells (Fig. 5D).

Fig. 5
figure 5

[Ca2+]i responses to miconazole in naïve and TRPA1-expessed HEK 293 cells. (A) The actual recordings of [Ca2+]i responses to 30 µM miconazole in the presence (left) or absence of extracellular Ca2+ (0Ca) (right) in naïve HEK 293 cells are shown. The 0Ca period is indicated with a grayish bar. (B) A summary of the amplitudes of [Ca2+]i responses to miconazole at 10 µM (left) and 30 µM (right) in naïve-HEK cells in the presence (control) or absence (0Ca) of extracellular Ca2+ is shown. Columns with vertical lines represent the mean ± SEM (miconazole 10 µM: control, n = 103; 0Ca, n = 127; miconazole 30 µM: control, n = 106; 0Ca, n = 93 from three experiments). **P < 0.01 vs. control, as determined by Student’s t-test (C) The actual recordings of [Ca2+]i responses to 10 µM miconazole and 100 µM AITC in TRPA1-expressed HEK293 cells (upper, A1-HEK) and to 1 µM capsaicin (Cap) in TRPV1-expressed HEK cells (lower, V1-HEK) are shown. (D) The relationship between [Ca2+]i responses and the concentrations of miconazole in A1-HEK and naïve HEK cells is shown. Symbols with vertical lines represent the mean ± SEM (A1-HEK, n = 54–134; naïve-HEK, n = 298–333 from 3–4 experiments). **P < 0.01 vs. naïve-HEK, as determined by Student’s t-test

Full size image

These results suggested that miconazole activated TRPA1, although less pronounced than that induced by econazole in mouse DRG neurons.

Effect of fluconazole on mouse DRG neurons and HEK 293 cells with heterologous expression of TRPA1

Unlike the imidazole antifungals econazole and miconazole, fluconazole belongs to the triazole class of antifungals. Fluconazole did not increase [Ca2+]i in mouse DRG neurons, even at a concentration of 100 µM, regardless of the expression of TRPA1 and/or TRPV1 (Fig. 6A). Furthermore, [Ca2+]i responses were not observed when fluconazole (100 µM) was applied to TRPA1-HEK and TRPV1-HEK cells (Fig. 6B). These data showed that TRPA1 and TRPV1 were not activated by fluconazole.

Fig. 6
figure 6

[Ca2+]i responses to fluconazole in mouse DRG neurons and HEK293 cells expressing TRPA1. (A) The actual recordings of [Ca2+]i responses to the sequential application of 100 µM fluconazole, 0.1 mM AITC, 1 µM capsaicin (Cap), and 80 mM KCl in mouse DRG neurons depict four patterns of responses. (B) The representative changes in [Ca2+]i induced by 100 µM fluconazole in HEK293 cells expressing TRPA1 (left: A1-HEK) and TRPV1 (right: V1-HEK). To determine the expression of each channel, a specific agonist was applied as follows: 0.1 mM ATIC for TRPA1 and 1 µM capsaicin (Cap) for TRPV1. Similar results are obtained in the two additional experiments

Full size image

Involvement of TRPA1 in econazole-induced itch and pain

Finally, to examine that econazole actually elicited itch and pain in mice, econazole was intradermally injected into the cheeks of wild type and TRPA1(−/−) mice, and subsequent scratching and wiping behaviors were counted. Econazole induced scratching and wiping behaviors in wild type mice, but the time course of these reactions differed among animals (Fig. 7A, B). The total number of 30-minute behaviors was significantly greater in the econazole-injected group than in the vehicle-injected one. In contrast, the number of both behaviors was significantly lower in TRPA1(−/−) mice than in wild type mice. Since scratching and wiping behavior reflect itch and pain behavior, respectively [21, 22], these data suggest that econazole causes itch and pain in mice through the activation of TRPA1.

Fig. 7
figure 7

Econazole induces scratching and wiping behaviors in mice. (A, B) The time courses of scratching (A) and wiping behaviors (B) every 5 min for 30 min after the intradermal injection of econazole (50 nmol/10 µl per site) into the right cheeks of wild type (Wild) and TPA1 knockout (A1(−/−)) mice. (C, D) Total number of scratching (C) and wiping behaviors (D) induced by econazole (Eco) during 30 min in wild type and TRPA1(−/−) mice. Vehicle was administrated into wild type mice. Columns with vertical lines represent mean ± SEM for 4 mice per group. *P < 0.05, with Steel-Dwass test

Full size image

Discussion

Econazole is a broad-spectrum antifungal drug that is used for various diseases, such as dermatomycosis. However, its typical adverse effect of skin irritation often hinders effective utilization [1, 2]. In this present study, we aimed to elucidate the mechanisms of econazole-induced irritation by investigating its effects on sensory neurons. Econazole-induced behavioral responses were also determined. Moreover, we examined the effects of miconazole, which is a structural analog of imidazole antifungal drugs, and fluconazole, which is a triazole antifungal drug.

Econazole dose-dependently increased the [Ca2+]i in mouse DRG neurons. At 100 µM, nearly all neurons responded and inhibited KCl-induced responses. Consequently, high concentrations of econazole may exhibit nonspecific actions and potentially cause cellular damage in mouse DRG neurons. Indeed, econazole has been documented to be cytotoxic in various cell types and holds promise as a potential antitumor drug. In one study, econazole increased the lethality of rat thymocytes [28]. In ADR breast cancer cells, econazole reduced cell proliferation and induced strong toxicity, including apoptosis [29]. Furthermore, econazole significantly decreased the viability of human prostate cancer PC3 cells [30]. Similarly, our results showed that miconazole appeared to exert cytotoxic effects (Fig. 4A), consistent with the results of previous studies [31, 32].

Econazole at concentrations of 10 and 30 µM increased [Ca2+]i, and these effects were mitigated upon removal of extracellular Ca2+. Furthermore, nearly all DRG neurons that responded to econazole at 10 µM were found to be sensitive to AITC, suggesting the potential activation of TRPA1 by econazole. The econazole-induced increase in [Ca2+]i was significantly attenuated by a TRPA1 antagonist; and in TRPA1(−/−) mouse DRG neurons, the [Ca2+]i responses to econazole were substantially reduced. Moreover, econazole at 10 µM activated the heterologously expressed TRPA1. While econazole exceeding 30 µM induced [Ca2+]i release in naïve HEK, consistent with previous reports [33, 34]. These findings supported the conclusion that econazole stimulated TRPA1 activation. Topical econazole cream or powder is typically administered as 1% nitric acid, with approximately 10% penetrating through the stratum corneum [35, 36]. Moreover, econazole applied at 1% to human skin has been reported to be estimated 20 µg/ml (equivalent to 44.9 µM) in epidermal tissue [37]. Therefore, the doses used in clinical practice may be sufficient to activate TRPA1. In contrast, its cytotoxicity may not occur at clinical concentrations, since it does not appear to reach concentrations above 100 µM (Figs. 1C and 4A).

Some of the neurons that responded to econazole also exhibited sensitivity to capsaicin. However, we did not obtain direct evidence on the action of econazole on TRPV1 in HEK 293 cells (Fig. 3C) and this was consistent with the results of a previous report [34]. Notably, econazole at 30 µM partially increased the [Ca2+]i in TRPA1(−/−) mouse DRG neurons. The [Ca2+]i responses to this concentration of econazole may be attributed to the Ca2+ influx through other channels, as evidenced by the reduced response upon extracellular Ca2+ removal. Previous studies have indicated that econazole-induced Ca2+ influx can be attenuated by La3+ in MDCK cells [38] and by L-type calcium channel blockers in human PC3 cells [30]. Recent report shows that store-operated Ca2+ entry, extracellular signaling pathway (ERK) 1/2, protein kinase C and phospholipase A2 are involved in the [Ca2+]i responses to econazole in human oral cancer cell lines [39]. Therefore, it is suggested that econazole may stimulate many molecules in addition to TRPA1 identified in this study. Since TRPA function is regulated by PKC [40], indirect action via PKC may also be involved in TRPA1 activation by econazole. The nonspecific [Ca2+]i increases in naïve HEK cells were not abolished by the removal of extracellular Ca2+. It has been reported that econazole induces Ca2+ mobilization from intracellular Ca2+ stores, similar to thapsigargin, an endoplasmic reticulum Ca2+-ATPase inhibitor [30, 34, 38]. Therefore, this mechanism was considered to elicit in the present study.

The profile of miconazole-induced increase in [Ca2+]i closely resembled that of econazole in wild type mouse DRG neurons. Miconazole elicited Ca2+ influx in neurons that were insensitive to AITC and capsaicin. Notably, the use of a TRPA1 antagonist reduced the miconazole-induced responses. Furthermore, the [Ca2+]i increases induced by miconazole at 10 µM but not at 30 µM were diminished in TRPA1(−/−) mouse DRG neurons. Intriguingly, miconazole triggered the activation of TRPA1 when expressed in HEK 293 cells. This suggests that miconazole may affect other molecules involved in Ca2+ influx more than TRPA1 in mouse DRG neurons. Consequently, unlike econazole, miconazole may possess the capability to activate multiple targets, including TRPA1. Chemical structure of miconazole differs from econazole in terms of the additional single chlorine atom (Fig. 1). The stronger antifungal activity of miconazole than econazole [27, 41] may explain the distinct activities of these molecules. These differences could be helpful in further elucidating the mechanisms of TRPA1 activation.

In general, TRPA1 agonists can be categorized as electrophile or nonelectrophile compounds [8, 42]. Econazole and miconazole belong to the nonelectrophile compound category. The anti-inflammatory drug fenamic acid, which is also classified as a nonelectrophile, has been suggested to activate TRPA1 through protein modification and allosteric binding [14]. These compounds generate reactive oxygen species in the cytosol [42], thereby, indirectly leading to TRPA1 activation [43]. Moreover, econazole is recognized as an inhibitor of TRPM2 channels [44, 45] that contributes to the pathogenesis of inflammatory and neuropathic pain [46]. Similarly, fenamic acid inhibits TRPM2 [44]. There may be a common structural element contributing to the similar effects of econazole and fenamic acid on both TRPA1 and TRPM2. In addition, among TRP channels, econazole has also been reported to inhibit TRPV5 [47] and TRPV6 [48].

Fluconazole, even at a concentration of 100 µM, failed to induce any [Ca2+]i response in mouse DRG neurons and HEK cells expressing TRPA1 and TRPV1. The structural differences between fluconazole and econazole/miconazole are considerably significant (Fig. 1). Moreover, compared with imidazole compounds, fluconazole has less ability to produce reactive oxygen species, antifungal activity, and cytotoxicity [49]. In contrast, econazole is known to induce apoptosis in certain cell types [50,51,52]. Therefore, caution should be exercised when using econazole in experiments, because it exerts diverse effects on various cell functions, including TRPA1 activation and TRPM2 inhibition.

Finally, to examine whether econazole elicits itch and pain. the mouse cheek injection model was used. Intradermally injection of econazole into the cheeks elicited scratching and wiping behaviors in wild type mice, but significantly less in TRPA1(−/−) mice. Scratching behaviors reflect itch, while wiping behaviors indicate pain [21, 22]. These results suggested that econazole actually induced adverse effects; itch and pain, in mice through the activation of TRPA1.

In summary, econazole acts as an activator of TRPA1 in sensory neurons, resulting in skin irritation. Blocking TRPA1 activation may offer a potential strategy to mitigate the skin adverse effects induced by econazole.

Data availability

No datasets were generated or analysed during the current study.

Abbreviations

AITC:

Allyl isothiocyanate

ANOVA:

Analysis of variance

Cap:

Capsaicin

[Ca2+]i :

Intracellular Ca2+ concentration

DMSO:

Dimethyl sulfoxide

DRG:

Dorsal root ganglia

DW:

Distilled water

HEK 293:

Human embryonic kidney 293

HEPES:

N-2-hydroxyethyl-piperazine-2-ethanonesufonic acid

PBS:

Phosphate-buffered saline

TRPA1:

Transient receptor potential ankyrin 1

TRPV1:

Transient receptor potential vanilloid 1

TRPA1-HEK:

HEK293 cells expressing TRPA1

References

  1. Heel RC, Brogden RN, Speight TM, Avery GS. Econazole: a review of its antifungal activity and therapeutic efficacy. Drugs. 1978;16:177–201.

    Article  CAS  PubMed  Google Scholar 

  2. Firooz A, Nafisi S, Maibach HI. Novel drug delivery strategies for improving econazole antifungal action. Int J Pharm. 2015;495:599–607.

    Article  CAS  PubMed  Google Scholar 

  3. Moore C, Gupta R, Jordt SE, Chen Y, Liedtke WB. Regulation of pain and itch by TRP channels. Neurosci Bull. 2018;34:120–42.

    Article  CAS  PubMed  Google Scholar 

  4. Dai Y. TRPs and pain. Semin Immunopathol. 2016;38:277–91.

    Article  CAS  PubMed  Google Scholar 

  5. Gees M, Colsoul B, Nilius B. The role of transient receptor potential cation channels in Ca2+ signaling. Cold Spring Harb Perspect Biol. 2010;2:a003962.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Jardín I, López JJ, Diez R, Sánchez-Collado J, Cantonero C, Albarrán L, Woodard GE, Redondo PC, Salido GM, Smani T, Rosado JA. TRPs in pain sensation. Front Physiol. 2017;8:392.

    Article  PubMed  PubMed Central  Google Scholar 

  7. Zhang H, Wang C, Zhang K, Kamau PM, Luo A, Tian L, Lai R. The role of TRPA1 channels in thermosensation. Cell Insight. 2022;1:100059.

    Article  PubMed  PubMed Central  Google Scholar 

  8. Talavera K, Startek JB, Alvarez-Collazo J, Boonen B, Alpizar YA, Sanchez A, Naert R, Nilius B. Mammalian transient receptor potential TRPA1 channels: from structure to sisease. Physiol Rev. 2020;100:725–803.

    Article  CAS  PubMed  Google Scholar 

  9. Caterina MJ, Leffler A, Malmberg AB, Martin WJ, Trafton J, Petersen-Zeitz KR, Koltzenburg M, Basbaum AI, Julius D. Impaired nociception and pain sensation in mice lacking the capsaicin receptor. Science. 2000;288:306–13.

    Article  CAS  PubMed  Google Scholar 

  10. Tominaga M. Nociception and TRP channels. Handb Exp Pharmacol. 2007;179:489–505.

    Article  CAS  Google Scholar 

  11. Leffler A, Fischer MJ, Rehner D, Kienel S, Kistner K, Sauer SK, Gavva NR, Reeh PW, Nau C. The vanilloid receptor TRPV1 is activated and sensitized by local anesthetics in rodent sensory neurons. J Clin Invest. 2008;118:763–76.

    PubMed  PubMed Central  Google Scholar 

  12. Leffler A, Lattrell A, Kronewald S, Niedermirtl F, Nau C. Activation of TRPA1 by membrane permeable local anesthetics. Mol Pain. 2011;7:62.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Guinamard R, Simard C, Del Negro C. Flufenamic acid as an ion channel modulator. Pharmacol Ther. 2013;138:272–84.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Hu H, Tian J, Zhu Y, Wang C, Xiao R, Herz JM, Wood JD, Zhu MX. Activation of TRPA1 channels by fenamate nonsteroidal anti-inflammatory drugs. Pflugers Arch. 2010;459:579–92.

    Article  CAS  PubMed  Google Scholar 

  15. Liu DJ, Collaku A, Dosik JS. Skin irritation and sensitization potential of fixed-dose combination of diclofenac 1% and menthol 3% topical gel: results of two phase I patch studies. Drug Res (Stuttg). 2017;67:119–26.

    CAS  PubMed  Google Scholar 

  16. Fischer MJ, Leffler A, Niedermirtl F, Kistner K, Eberhardt M, Reeh PW, Nau C. The general anesthetic propofol excites nociceptors by activating TRPV1 and TRPA1 rather than GABAA receptors. J Biol Chem. 2010;285:34781–92.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Yin S, Luo J, Qian A, Du J, Yang Q, Zhou S, Yu W, Du G, Clark RB, Walters ET, Carlton SM, Hu H. Retinoids activate the irritant receptor TRPV1 and produce sensory hypersensitivity. J Clin Invest. 2013;123:3941–51.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Shimada T, Takahashi K, Tominaga M, Ohta T. Identification of molecular targets for toxic action by persulfate, an industrial sulfur compound. Neurotoxicology. 2019;72:29–37.

    Article  CAS  PubMed  Google Scholar 

  19. Ohta T, Imagawa T, Ito S. Novel gating and sensitizing mechanism of capsaicin receptor (TRPV1): tonic inhibitory regulation of extracellular sodium through the external protonation sites on TRPV1. J Biol Chem. 2008;283:9377–87.

    Article  CAS  PubMed  Google Scholar 

  20. Grynkiewicz G, Poenie M, Tsien RY. A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem. 1985;260:3440–50.

    Article  CAS  PubMed  Google Scholar 

  21. Kittaka H, Uchida K, Fukuta N, Tominaga M. Lysophosphatidic acid-induced itch is mediated by signalling of LPA5 receptor, phospholipase D and TRPA1/TRPV1. J Physiol. 2017;595:2681–98.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Yamanoi Y, Kittaka H, Tominaga M. Cheek Injection Model for Simultaneous Measurement of Pain and Itch-related behaviors. J Vis Exp. 2019;151.

  23. Mickle AD, Shepherd AJ, Mohapatra DP. Nociceptive TRP channels: sensory detectors and transducers in multiple pain pathologies. Pharmaceuticals (Basel). 2016;9:72.

    Article  PubMed  Google Scholar 

  24. Thastrup O, Cullen PJ, Drøbak BK, Hanley MR, Dawson AP. Thapsigargin, a tumor promoter, discharges intracellular Ca2+ stores by specific inhibition of the endoplasmic reticulum Ca2+-ATPase. Proc Natl Acad Sci U S A. 1990;87:2466–70.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Gordon-Shaag A, Zagotta WN, Gordon SE. Mechanism of Ca2+-dependent desensitization in TRP channels. Channels. 2008;2:125–9.

    Article  PubMed  Google Scholar 

  26. Gouin O, L’Herondelle K, Lebonvallet N, Le Gall-Ianotto C, Sakka M, Buhé V, Plée-Gautier E, Carré JL, Lefeuvre L, Misery L, Le Garrec R. TRPV1 and TRPA1 in cutaneous neurogenic and chronic inflammation: pro-inflammatory response induced by their activation and their sensitization. Protein Cell. 2017;8:644–61.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Hu Z, Zhang J, Cheng X. Antifungal efficiency of miconazole and econazole and the interaction with transport protein: a comparative study. Pharm Biol. 2015;53:251–61.

    Article  CAS  PubMed  Google Scholar 

  28. Matsui H, Sakanashi Y, Oyama TM, Oyama Y, Yokota S, Ishida S, Okano Y, Oyama TB, Nishimura Y. Imidazole antifungals, but not triazole antifungals, increase membrane Zn2+ permeability in rat thymocytes: possible contribution to their cytotoxicity. Toxicology. 2008;248:142–50.

    Article  CAS  PubMed  Google Scholar 

  29. Dong C, Chen Y, Ma J, Yang R, Li H, Liu R, You D, Luo C, Li H, Yang S, Ke K, Lin MC, Chen C. Econazole nitrate reversed the resistance of breast cancer cells to adriamycin through inhibiting the PI3K/AKT signaling pathway. Am J Cancer Res. 2020;10:263–74.

    CAS  PubMed  PubMed Central  Google Scholar 

  30. Huang JK, Liu CS, Chou CT, Liu SI, Hsu SS, Chang HT, Hsieh CH, Chang CH, Chen WC, Jan CR. Effects of econazole on Ca2+ levels in and the growth of human prostate cancer PC3 cells. Clin Exp Pharmacol Physiol. 2005;32:735–41.

    Article  CAS  PubMed  Google Scholar 

  31. Jung HJ, Seo I, Jha BK, Suh SI, Baek WK. Miconazole induces autophagic death in glioblastoma cells via reactive oxygen species-mediated endoplasmic reticulum stress Oncol let. 2021;21:335.

  32. Lam PL, Wong MM, Hung LK, Yung LH, Tang JC, Lam KH, Chung PY, Wong WY, Ho YW, Wong RS, Gambari R, Chui CH. Miconazole and terbinafine induced reactive oxygen species accumulation and topical toxicity in human keratinocytes. Drug Chem Toxicol. 2022;45:834–8.

    Article  CAS  PubMed  Google Scholar 

  33. Chang HT, Liu CS, Chou CT, Hsieh CH, Chang CH, Chen WC, Liu SI, Hsu SS, Chen JS, Jiann BP, Huang JK, Jan CR. Econazole induces increases in free intracellular Ca2+ concentrations in human osteosarcoma cells. Hum Exp Toxicol. 2005;24:453–8.

    Article  CAS  PubMed  Google Scholar 

  34. Mathivanan S, de la Torre-Martinez R, Wolf C, Mangano G, Polenzani L, Milanese C, Ferrer-Montiel A. Effect of econazole and benzydamine on sensory neurons in culture. J Physiol Pharmacol. 2016;67:851–8.

    CAS  PubMed  Google Scholar 

  35. Plempel M. Pharmacokinetics of imidazole antimycotics. Postgrad Med J. 1979;55:662–6.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Hänel H, Raether W, Dittmar W. Evaluation of fungicidal action in vitro and in a skin model considering the influence of penetration kinetics of various standard antimycotics. Ann N Y Acad Sci. 1988;544:329–37.

    Article  PubMed  Google Scholar 

  37. Schaefer H, Stüttgen G. Absolute concentrations of an antimycotic agent, econazole, in the human skin after local application. Arzneimittelforschung. 1976;26:432–5.

    CAS  PubMed  Google Scholar 

  38. Jan CR, Ho CM, Wu SN, Tseng CJ. Multiple effects of econazole on calcium signaling: depletion of thapsigargin-sensitive calcium store, activation of extracellular calcium influx, and inhibition of capacitative calcium entry. Biochim Biophys Acta. 1999;1448:533–42.

    Article  CAS  PubMed  Google Scholar 

  39. Wang JL, Jan CR, Chen MH. Action of econazole on Ca2+ levels and cytotoxicity in OC2 human oral cancer cells. J Dent Sci. 2023;18:1280–7.

    Article  PubMed  PubMed Central  Google Scholar 

  40. Sánchez JC, Muñoz LV, Galindo-Márquez ML, Valencia-Vásquez A, García AM. Paclitaxel regulates TRPA1 function and expression through PKA and PKC. Neurochem Res. 2023;48:295–304.

    Article  PubMed  Google Scholar 

  41. Qin F, Wang Q, Zhang C, Fang C, Zhang L, Chen H, Zhang M, Cheng F. Efficacy of antifungal drugs in the treatment of vulvovaginal candidiasis: a bayesian network meta-analysis. Infect Drug Resist. 2018;11:1893–901.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Macpherson LJ, Dubin AE, Evans MJ, Marr F, Schultz PG, Cravatt BF, Patapoutian A. Noxious compounds activate TRPA1 ion channels through covalent modification of cysteines. Nature. 2007;445:541–5.

    Article  CAS  PubMed  Google Scholar 

  43. Viana F. TRPA1 channels: molecular sentinels of cellular stress and tissue damage. J Physiol. 2016;594:4151–69.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Chen GL, Zeng B, Eastmond S, Elsenussi SE, Boa AN, Xu SZ. Pharmacological comparison of novel synthetic fenamate analogues with econazole and 2-APB on the inhibition of TRPM2 channels. Br J Pharmacol. 2012;167:1232–43.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Hill K, McNulty S, Randall AD. Inhibition of TRPM2 channels by the antifungal agents clotrimazole and econazole. Naunyn Schmiedebergs Arch Pharmacol. 2004;370:227–37.

    Article  CAS  PubMed  Google Scholar 

  46. Haraguchi K, Kawamoto A, Isami K, Maeda S, Kusano A, Asakura K, Shirakawa H, Mori Y, Nakagawa T, Kaneko S. TRPM2 contributes to inflammatory and neuropathic pain through the aggravation of pronociceptive inflammatory responses in mice. J Neurosci. 2012;32:3931–41.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Hughes TET, Lodowski DT, Huynh KW, Yazici A, Del Rosario J, Kapoor A, Basak S, Samanta A, Han X, Chakrapani S, Zhou ZH, Filizola M, Rohacs T, Han S, Moiseenkova-Bell VY. Structural basis of TRPV5 channel inhibition by econazole revealed by cryo-EM. Nat Struct Mol Biol. 2018;25:53–60.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Neuberger A, Nadezhdin KD, Sobolevsky AI. Structural mechanisms of TRPV6 inhibition by ruthenium red and econazole. Nat Commun. 2021;12:6284.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Kobayashi D, Kondo K, Uehara N, Otokozawa S, Tsuji N, Yagihashi A, Watanabe N. Endogenous reactive oxygen species is an important mediator of miconazole antifungal effect. Antimicrob Agents Chemother. 2002;46:3113–7.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Choi EK, Park EJ, Phan TT, Kim HD, Hoe KL, Kim DU. Econazole induces p53-dependent apoptosis and decreases metastasis ability in gastric cancer cells. Biomol Ther (Seoul). 2020;28:370–9.

    Article  CAS  PubMed  Google Scholar 

  51. Ho YS, Wu CH, Chou HM, Wang YJ, Tseng H, Chen CH, Chen LC, Lee CH, Lin SY. Molecular mechanisms of econazole-induced toxicity on human colon cancer cells: G0/G1 cell cycle arrest and caspase 8-independent apoptotic signaling pathways. Food Chem Toxicol. 2005;43:1483–95.

    Article  CAS  PubMed  Google Scholar 

  52. Dong C, Yang R, Li H, Ke K, Luo C, Yang F, Shi XN, Zhu Y, Liu X, Wong MH, Lin G, Wang X, Leung KS, Kung HF, Chen C, Lin MC. Econazole nitrate inhibits PI3K activity and promotes apoptosis in lung cancer cells. Sci Rep. 2017;7:17987.

    Article  PubMed  PubMed Central  Google Scholar 

Download references

Funding

This work was supported, in whole or partly, by the JSPS KAKENHI (Grant No. JP22K06001 to TO).

Author information

Authors and Affiliations

  1. Department of Veterinary Pharmacology, Faculty of Agriculture, Tottori University, Tottori, 680-8553, Japan

    Kaoru Kasuya, Kenji Takahashi & Toshio Ohta

  2. Department of Veterinary Pharmacology, Joint Graduate School of Veterinary Sciences, Tottori University, Tottori, 680-8553, Japan

    Kenji Takahashi, Miho Hashimoto & Toshio Ohta

Authors
  1. Kaoru Kasuya
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Kenji Takahashi
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Miho Hashimoto
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Toshio Ohta
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

T.O. designed the study. K.K. and M.H. conducted the experiments. K.K., K.T., and T.O. conducted data analyses and wrote the manuscript.

Corresponding author

Correspondence to Toshio Ohta.

Ethics declarations

Ethics approval and consent to participate

This study makes use of mice, and the experimental protocol for the use of animals was approved (Approved number: 21-T-8) by the Committee on Animal Experimentation of Tottori University, Tottori, Japan. No specific consent was needed for the current investigation. All methods were performed following the relevant guidelines and regulations.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Kasuya, K., Takahashi, K., Hashimoto, M. et al. Nociceptive transient receptor potential ankyrin 1 (TRPA1) in sensory neurons are targets of the antifungal drug econazole. BMC Pharmacol Toxicol 25, 53 (2024). https://doi.org/10.1186/s40360-024-00779-x

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1186/s40360-024-00779-x

Keywords

BMC Pharmacology and Toxicology

ISSN: 2050-6511

Contact us