Methyl p-hydroxybenzoate causes pain sensation through activation of TRPA1 channels

Abstract

Background and purpose: Parabens are commonly added in pharmaceutical, cosmetic and food products because of their wide antibacterial properties, low toxicity, inertness and chemical stability, although the molecular mechanism of their antibacterial effect is not fully understood. Some agonists of the transient receptor potential (TRP) A1 channels are known to have strong antibacterial activities. Therefore, a series of experiments was conducted to find out the effects of parabens on TRP channels expressed in sensory neurons, particularly the TRPA1 channels.

Experimental approach: Effects of parabens, especially of methyl p-hydroxybenzoate (methyl paraben) on TRP channel activities were examined using Ca(2+)-imaging and patch-clamp methods. In addition, an involvement of methyl paraben in the development of pain-related behavior in mice was investigated.

Key results: Methyl paraben specifically activated TRPA1 in both HEK293 cells expressing TRPA1 and in mouse sensory neurons with an EC(50) value of 4.4 mM, an attainable concentration in methyl paraben-containing products. Methyl paraben caused pain-related behavior in mice similar to that caused by allyl isothiocyanate, which was blocked by the TRP channel blocker, ruthenium red.

Conclusions and implications: Our data indicate that methyl paraben is able to activate TRPA1 channels and can cause pain sensation. As such, methyl paraben provides a useful tool for investigating TRPA1 function and development of antinociceptive agents acting on TRPA1 channels.

Figure 1

Chemical structure of parabens, where R=methyl (CH₃), ethyl (C₂H₅), propyl (C₃H₇), butyl (C₄H₉).

Figure 2

Increase in cytosolic Ca²⁺ concentration ([Ca²⁺]_i) mediated by the activation of TRPA1 channels by methyl paraben in HEK293 cells. (a) Methyl paraben (MpBZ) caused an increase in cytosolic Ca²⁺ concentration ([Ca²⁺]_i) in HEK293 cells expressing TRPA1 channels in the presence of extracellular Ca²⁺ (n=15), but not in cells expressing TRPV1 in the presence of extracellular Ca²⁺ (n=15) or cells expressing TRPA1 channels in the absence of extracellular Ca²⁺ (n=15). Control stimulus indicates cinnamaldehyde (500 μM) for TRPA1 or capsaicin (1 μM) for TRPV1 in the presence of extracellular Ca²⁺. Horizontal bars indicate the duration of applied stimulus. (b) MpBZ increased [Ca²⁺]_i in HEK293 cells expressing TRPA1 channels (n=24), but not in cells expressing TRPV1 (n=15), TRPV2 (n=130), TRPV3 (n=12), TRPV4 (n=48), TRPM8 (n=122) or vector alone (n=24). ^**P<0.01. (c) Effects of MpBZ (1 mM, n=24), ethyl paraben (EpBZ, 1 mM, n=52), propyl paraben (PpBZ, 1 mM, n=21) or butyl paraben (BpBZ, 0.5 mM, n=24) on [Ca²⁺]_i in HEK293 cells expressing TRPA1 channels. ^**P<0.01. (d) MpBZ caused an increase in [Ca²⁺]_i in HEK293 cells expressing TRPA1 channels in the presence of extracellular Ca²⁺ at a constant temperature of 34°C (n=19). Lower panel shows bath temperature. Control stimulus indicates cinnamaldehyde (500 μM). Horizontal bars indicate the duration of applied stimulus.

Figure 3

Methyl paraben increases cytosolic Ca²⁺ concentration ([Ca²⁺]_i) in sensory neurons. (a) Methyl paraben (MpBZ) caused an increase in [Ca²⁺]_i in mouse DRG neurons, which also responded to allyl isothiocyanate (AITC) and capsaicin (CAP) (n=16). (b) MpBZ-induced increases in [Ca²⁺]_i were inhibited by ruthenium red (RR) in mouse DRG neurons (n=14).

Figure 4

Methyl paraben activates TRPA1 channels in HEK293 cells. (a) A representative whole-cell current trace activated by methyl paraben (MpBZ, 1 mM) or cinnamaldehyde (CA, 500 μM) in the presence of extracellular Ca²⁺. V_h=−60 mV. Horizontal bars indicate the duration of test compound application. (b) A representative current trace in response to MpBZ or capsaicin (CAP, 1 μM) in the presence of extracellular Ca²⁺. (c) A representative current trace activated by MpBZ showing desensitization in the presence of extracellular Ca²⁺. (d) A representative current trace activated by MpBZ in the absence of extracellular Ca²⁺. (e) A dose-dependent profile of currents through TRPA1 channels activated by MpBZ. EC₅₀ value was 4.4 mM; Hill coefficient was 2.6. Membrane currents were normalized to the current activated by 10 mM MpBZ in each cell (n=6). (f) and (g) Representative MpBZ-activated current traces inhibited by ruthenium red (RR, 10 μM, f) or camphor (5 mM, g) in the absence of extracellular Ca²⁺. (h) A representative I–V curve of MpBZ-activated current showing an outward rectification.

Figure 5

Methyl paraben activates inward currents in mouse DRG neurons. (a) A representative whole-cell current trace activated by methyl paraben (MpBZ, 5 mM), allyl isothiocyanate (AITC, 20 μM) or capsaicin (CAP, 1 μM). V_h=−60 mV. Bars indicate duration of the compound application. (b) A representative current trace in response to AITC, MpBZ or CAP. (c) A representative MpBZ-activated current trace inhibited by ruthenium red (RR, 10 μM). (d) A representative I–V curve of MpBZ-activated currents showing an outward rectification. To maintain giga-ohm seals we performed patch-clamp experiments on DRG neurons in the presence of extracellular Ca²⁺ for all experiments, except those with RR.

Figure 6

Pain-related behavior produced by methyl paraben injection into mouse hind paws. (a) Time course of licking an biting induced by methyl paraben (MpBZ, 100 μM) or allyl isothiocyanate (AITC, 10 μM) with or without ruthenium red (RR, 100 μM). The time spent licking and biting the injected paw was measured for 1 min durations up to 10 min after subcutaneous injection of the compounds into the hind paw. n=10 each. ^{*, **}P<0.05, P<0.01 vs control (10% DMSO used for dilution of MpBZ and AITC). (b) Dose dependency of MpBZ effects on pain-related behavior in mice. n=10 each ^*P<0.05, ^**P<0.01. (c) Effects of ruthenium red (RR) on time spent licking and biting for 10 min. n=10 each. ^**P<0.01 vs treatment with RR.