Species-Specific Activation of Transient Receptor Potential Ankyrin 1 by Phthalic Acid Monoesters

Abstract

Phthalic acid (PA) diesters are widely used in consumer products, as plasticizers, and are ubiquitous environmental pollutants. There is a growing concern about their adjuvant effect on allergic diseases. Although its precise mechanism remains unknown, possible involvement of transient receptor potential ankyrin 1 (TRPA1) has been suggested. Hence, in this study, the activation of human and mouse TRPA1s by a series of PA di- and monoesters was investigated using a heterologous expression system in vitro. Consequently, it was found that monoesters activated human TRPA1, where EC₅₀ values were in the order of mono-hexyl > mono-heptyl > mono-n-octyl > mono-2-ethylhexyl > mono-isononyl and mono-isodecyl esters. Significant species differences in TRPA1 activation by PA monoesters were also discovered; PA monoesters activated human TRPA1 but not mouse TRPA1 in a concentration-dependent manner up to 50 µM. These findings suggest that PA esters may exert TRPA1-dependent adverse effects on humans, which have never been demonstrated in experimental animals.

INTRODUCTION

Phthalic acid (PA) esters are ubiquitous food and environmental contaminants.^1–3) PA diesters, such as di(2-ethylhexyl)phthalate (DEHP), are widely used as plasticizers for polyvinyl chloride (PVC).⁴⁾ They are also utilized in many consumer products, including personal care products, paints, and adhesives.⁵⁾ As PA diesters do not covalently bind with plastics, they easily leach to the surface of materials.^6,7) Consequently, humans can easily come in contact with and are commonly exposed to PA diesters.

Hydrolysis is the most important first-step metabolism of PA diesters in humans. For instance, orally exposed PA diesters, such as DEHP, are rapidly hydrolyzed to corresponding monoesters (mono(2-ethylhexyl) phthalate, MEHP) by lipases expressed in the intestinal tract.⁸⁾ Dermally exposed PA diesters (e.g., dibutyl phthalate (DBP) and benzyl butyl phthalate (BnBP)) are hydrolyzed by cutaneous esterase, converted into monoesters, and then absorbed.⁹⁾ However, PA diesters with longer alkyl side chains, such as DEHP, are retained in the skin because of its high lipophilicity.⁹⁾ In addition to hydrolysis in the human body, PA monoesters are formed by environmental hydrolysis reactions, such as those in microorganisms and on the surface of concrete materials.^10–12) In these cases, humans are directly exposed to PA monoesters.

The primary adverse effects of PA esters in laboratory animals include liver toxicity, altered immune responses, male and female reproductive effects, developmental effects, and carcinogenicity.⁴⁾ For instance, DEHP was reported to induce hepatomegaly associated with peroxisomal proliferation in rodents, adjuvant effects in sensitized rats and mice, toxic effects to male reproductive system, particularly the testis, and increased incidence of liver tumors in F344 rats and B6C3F1 mice.⁴⁾ Among these toxic effects, risk assessment of PA diesters has focused mainly on reproductive and developmental toxicities. Based on reproductive toxicity and endocrine-disrupting properties, the use of four PA diesters (DEHP, DBP, BnBP, and diisobutyl phthalate) in consumer products are restricted under Registration, Evaluation, Authorization and Restriction of Chemicals in Europe (https://echa.europa.eu/substances-restricted-under-reach).

In recent years, there is also a growing concern about their allergic effects as environmental pollutants. As reviewed by Bølling et al., relatively weak but statistically significant associations were reported between airway allergic symptoms, such as asthma and cough, and the presence of PVC materials at home, PA diester mass fractions in indoor dust, and PA diester metabolite concentrations in urine.¹³⁾

For instance, a correlation was reported between DEHP concentrations in house dust and asthma and allergic prevalence in children based on epidemiological surveys.¹⁴⁾ A correlation was also observed between maternal MEHP levels and children’s allergic reactions up to 7 years of age.¹⁵⁾ MEHP was also investigated for its adjuvant effects in a mouse inhalation model.¹⁶⁾ Orally exposed DINP causes exacerbation in a fluorescein isothiocyanate-induced contact dermatitis model.¹⁷⁾

Regarding the mechanisms of their allergic effects, it has been suggested that the activation of transient receptor potential (TRP) channels, such as TRP vanilloid 1 (TRPV1) and TRP ankyrin 1 (TRPA1), is responsible for the adjuvant effects of PA diesters.^17–19) TRPA1 functions as a pivotal sensor for both neuropathic and inflammatory pain.²⁰⁾ TRPA1 is highly expressed in sensory neurons,²¹⁾ lungs,²²⁾ immune cells,²³⁾ intestinal tract,²⁴⁾ skin keratinocytes,²⁵⁾ and pancreas.²⁶⁾ TRPA1 responds to various stimuli, including mechanical stress,²⁷⁾ cold temperature,²⁸⁾ and chemical agents, such as allyl isothiocyanate (AITC),²¹⁾ cinnamaldehyde,²⁹⁾ allicin,³⁰⁾ acrolein,³¹⁾ and formaldehyde.³²⁾

However, to date, limited information on the agonistic effects of PA esters on TRP channels is available. Therefore, in this study, the activation of human and mouse TRPA1s by a series of PA di- and monoesters was comprehensively investigated in a heterologous expression system in vitro.

MATERIALS AND METHODS

Chemicals

In this study, all PA esters were commercially obtained and listed in Table 1. Fetal bovine serum (FBS), Dulbecco’s Modified Eagle Medium (DMEM), 200 mM L-alanyl-L-glutamine solution (GlutaMAX), and hygromycin B antibiotic were purchased from Thermo Fisher Scientific (Waltham, MA, U.S.A.). Antibiotic solutions (10000 U/mL penicillin and 10000 µg/mL streptomycin), AITC, TRPA1 antagonist A-967079, and 2-mercaptoethanol were purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan). Hanks’ Balanced Salt Solution (HBSS) was purchased from Nissui Pharmaceutical (Tokyo, Japan). Dimethyl sulfoxide (DMSO) was purchased from Sigma-Aldrich (St. Louis, MO, U.S.A.). All other commercially obtained chemicals were of the highest available grades.

Table 1. PA Esters Used in This Study

Name	Abbreviation	CAS number	Suppliera	Purity	LogPb
Di esters
Dimethyl phthalate	DMP	113-11-3	TCI	99%	1.67
Diethyl phthalate	DEP	84-66-2	Junsei	99%	2.35
Di-n-propyl phthalate	DPP	131-16-8	Wako	98%	3.32
Di-n-butyl phthalate	DBP	84-74-2	TCI	97%	4.16
Di-n-pentyl phthalate	DPenP	131-18-0	TCI	98%	4.99
Di-n-hexyl phthalate	DHexP	84-75-3	TCI	98%	5.83
Di-n-heptyl phthalate	DHepP	3648-21-3	Sigma	97%	6.66
Di-n-octyl phthalate	DNOP	117-84-0	TCI	98%	7.50
Di(2-ethylhexyl)phthalate	DEHP	117-81-7	TCI	98%	7.46
Di-isononyl phthalatec	DINP	28553-12-0	Sigma	99%	8.16
Di-isodecyl phthalatec	DIDP	26761-40-0	Kanto	98.4%	8.99
Mono esters
Monomethyl phthalate	MMP	4376-18-5	Sigma	97%	1.41
Monoethyl phthalate	MEP	2306-33-4	TRC	98%	1.75
Mono-n-propyl phthalate	MPP	4376-19-6	SCBT	98%	2.24
Mono-n-butyl phthalate	MBP	131-70-4	Sigma	97–103%	2.65
Mono-n-pentyl phthalate	MPenP	24539-56-8	TRC	98%	2.70
Mono-n-hexyl phthalate	MHexP	24539-57-9	TRC	98%	3.49
Mono-n-heptyl phthalate	MHepP	24539-58-0	TRC	97%	3.90
Mono-n-octyl phthalate	MNOP	5393-19-1	TRC	97%	4.07
Mono(2-ethylhexyl)phthalate	MEHP	4376-20-9	Wako	93%	4.30
Mono-isononyl phthalatec	MINP	106610-61-1	TRC	94%	4.65
Mono-isodecyl phthalatec	MIDP	31047-64-0	TRC	98%	5.07

a) TCI, Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan); Junsei, Junsei Chemical Co., Ltd. (Tokyo, Japan); Wako, FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan); Sigma, Sigma-Aldrich (St. Louis, MO, U.S.A.); Kanto, Kanto Chemical Co., Inc. (Tokyo, Japan); TRC: Toronto Research Chemicals (Toronto, Canada); SCBT, Santa Cruz Biotechnology, Inc. (Dallas, TX, U.S.A.). b) The lipophilicities of the compounds were calculated as n-octanol-water partition coefficients (LogP) using ChemDraw (20. 1. 1. 125). c) Mixture of isomers.

Plasmid Construction

Full-length mTRPA1 cDNA (GenBank accession number NM_177781) was obtained from GeneCopoeia (Rockville, MD, U.S.A.) and used as a PCR template. An mTRPA1 coding DNA sequence was amplified by high-fidelity PCR using Pfx DNA polymerase (Thermo Fisher Scientific), following the manufacturer’s instructions. The primer pairs were as follows: 5′-CACCATGAAGCGCGGCTTG-3′ (N-terminal forward primer with CACC sequence) and 5′-AAAGTCCGGGTGGCTAATAG-3′ (C-terminal reverse primer without a stop codon). Then, the PCR product was separated on a 1.0% agarose gel (Seakem; LONZA, Basel, Switzerland), and the band was excised and purified on spin columns (MinElute Gel Purification kit: Qiagen GmbH, Hilden, Germany). After purification, the DNA fragment was subcloned into a pENTR/D-TOPO vector (Thermo Fisher Scientific) and named mTRPA1-pENTR/D-TOPO. The plasmid constructed was confirmed by sequence analysis (Eurofins Genomics KK, Tokyo, Japan) using sequence primers (Supplementary Table S1). mTRPA1-pENTR/D-TOPO was recombined with a pEF5/FRT/V5-DEST vector (Thermo Fisher Scientific) using Gateway LR reaction clonase enzyme mix (Thermo Fisher Scientific) and named mTRPA1-pEF5/FRT/V5. After endotoxin-free purification (endotoxin-free Qiagen Maxiprep kit; QIAGEN), the plasmid was used for transfection.

Cell Culture and the Development of mTRPA1-Flp-In 293 Cell Line

An Flp-In 293 cell line (a derivative of HEK-293 cell line) was obtained from Thermo Fisher Scientific. The development of the hTRPA1 stably expressing cell line, hTRPA1/Flp-In 293, was described in a previous study.³³⁾ To generate the mTRPA1-Flp-In 293 cell line, Flp-In 293 cells were cotransfected with mTRPA1-pEF/FRT/V5 and pOG44 vectors (Thermo Fisher Scientific) using lipofectamine 3000 (Thermo Fisher Scientific). Then, stable clones expressing mTRPA1 were selected using hygromycin B antibiotics, and colonies were cultured to produce a large stock of mTRPA1-expressing cells. During the initial cultivation of cell clones, a reduced hygromycin concentration of 50 µg/mL was used instead of a typical 200 µg/mL. All cell lines were cultured in high-glucose DMEM containing 10% FBS, antibiotic solution (100 U/mL penicillin and 100 µg/mL streptomycin), and 2 mM GlutaMAX. The cells were maintained in a humidified atmosphere containing 5% CO₂ and 95% air at 37 °C.

Western Blotting

Cells (1.0 × 10⁶ cells) were seeded in a 60-mm dish (IWAKI, Shizuoka, Japan) and precultured for 24 h. Then, the cells were washed with ice-cold phosphate-buffered saline (PBS) and incubated with 350 µL of radio-immunoprecipitation assay (RIPA) buffer (FUJIFILM Wako Pure Chemical Corporation) for 30 min on ice. The lysates were centrifuged at 12000 × g for 10 min at 4 °C. The supernatant was mixed with sodium dodecyl sulfate (SDS) sample buffer with 2-mercaptoethanol and boiled for 5 min. Then, the samples were separated by electrophoresis on a 7.5% SDS-polyacrylamide gel (7.5% Mini-PROTEAN TGX Precast Protein Gels; Bio-Rad, Hercules, CA, U.S.A.) and transferred to polyvinylidene difluoride membranes (ATTO, Tokyo, Japan). The membrane was incubated in a blocking buffer (Tris-buffered saline containing 5% nonfat dry milk and 0.05% Tween 20) for 1 h, followed by incubation with anti-V5-HRP antibodies (Thermo Fisher Scientific) in the blocking buffer (1 : 2500 dilution) for 1 h at ambient temperature. After washing, the membranes underwent chemifluorescent detection using an enhanced chemiluminescence kit (Pierce ECL Plus; Thermo Fisher Scientific), following the manufacturer’s instructions, and scanned using an Amersham Typhoon scanner 5 system (Cytiva, UT, U.S.A.).

Intracellular Ca²⁺ Measurement

The cells were plated at 80–90% confluence on 96-well, Poly-D-lysine black-walled, clear-bottomed plates (Greiner bio-one, Frickenhausen, Germany) 24 h prior to initiating experiments. The cells were incubated for 2 h at 37 °C in HBSS buffer with 20 mM N-(2-hydroxyethyl)piperazine-N′-2-ethanesulfonic acid (HEPES) (pH 7.4) containing a FLIPR calcium 6 assay reagent (Molecular Devices, Sunnyvale, U.S.A.), followed immediately by fluorescence measurement using FlexStation 3 (excitation at 485 nm and emission at 525 nm) and SoftMax Pro 7.1.2 software (Molecular Devices). The test compound was prepared in DMSO and added to the HBSS buffer (final DMSO concentration, 0.1%). EC₅₀ values were determined using a Prism 8 software (GraphPad Software, LaJolla, CA, U.S.A.).

Statistical Analysis

Each experiment was repeated at least thrice. All values are expressed as mean ± standard deviation (S.D.) unless otherwise stated. The statistical significance of differences between sample mean values was assessed using one-way ANOVA with Dunnett’s post hoc test. The correlation coefficient between EC₅₀ and LogP was determined using Pearson’s chi-square test. Data analyses were performed using Prism 8 software.

RESULTS

The expression of human and mouse TRPA1s in Flp-In 293/hTRPA1 and Flp-In 293/mTRPA1 cell lines was confirmed by Western blot analysis (Fig. 1A). Two specific bands were observed in Flp-In 293/hTRPA1 and Flp-In 293/mTRPA1 cell lines between 100 and 150 kDa, which might be corresponding to intact and glycosylated TRPA1, as previously reported.³⁴⁾ In addition, AITC, a known TRPA1 agonist, had an increased intracellular fluorescent intensity in a dose-dependent manner, indicating a TRPA1-dependent Ca²⁺ influx in the cell lines (Fig. 1B). The estimated EC₅₀ values in Flp-In 293/hTRPA1 and Flp-In 293/mTRPA1 cell lines were 2.5 and 9.2 µM, respectively, in accordance with previous reports using the FLIPR assay.^35,36) Since some TRPA1 ligands were reported to show species-specific effects,^37–39) the TRPA1 activation potency of PA esters, including diesters and monoesters, was determined, and the values obtained from the two species were compared.

Fig. 1. Human and Mouse TRPA1 Proteins Stably Expressed in Flp-In 293 Cell Line Detected by Western Blotting

(A) The band stands for TRPA1 protein fused with V5-epitope protein. The fusion protein was detected in fluorescent by anti-V5-HRP antibody. TRPA1 has a molecular mass of 128 kDa. (B) AITC activated human and mouse TRPA1s in a concentration-dependent manner. Hill slope values for AITC were 1.9 and 2.1 for human and mouse. Solid line: Flp-In 293/hTRPA1 cell line, dot-line: Flp-In 293/mTRPA1 cell line. Data are the mean ± S.D. of at least three separate experiments.

First, we examined the ability of DEHP and MEHP, which are commercially dominant PA esters and its hydrolyzed metabolite, in activating human and mouse TRPA1 expressed in Flp-In 293 cell line using a FlexStation-based calcium influx assay. Surprisingly, among 2-ethylhexyl-possessing PA esters, MEHP and not DEHP showed an increase in Ca²⁺ influx in Flp-In 293/hTRPA1 but not in Flp-In 293/mTRPA1 and nontransfected Flp-In 293 cell lines (Fig. 2A). Furthermore, an MEHP-dependent increase in intracellular fluorescent intensity was diminished dose-dependently by the preaddition of A-967079, a TRPA1 antagonist, indicating that MEHP induced a Ca²⁺ influx via the TRPA1 channel (Fig. 2B). Compared with AITC, MEHP was much more potent (EC₅₀ = 2.5 µM for AITC; 0.65 µM for MEHP; n = 3).

Fig. 2. Species-Specific Effects of DEHP and MEHP

(A) Structure and activity of DEHP and MEHP. MEHP evoked Ca²⁺ influx in Flp-In 293 cell line expressing human TRPA1 and had no effect on mouse TRPA1, as represented by an increase in fluorescence signals (RFU, relative fluorescence unit). The asterisk indicates a significant difference (** p < 0.01) from the control as determined using one-way ANOVA and Dunnett’s post hoc test. (B) MEHP (10 µM)-induced Ca²⁺ influx was blocked by preincubation (10 min) of A-967079 (0, 125, or 1000 nM).

Likewise, a series of PA diesters and monoesters (50 µM) were examined in both Flp-In 293/hTRPA1 and Flp-In 293/mTRPA1 cell lines using a FlexStation-based calcium influx assay. Among them, nine PA diesters and monoesters (DPP, DNOP, MPenP, MHexP, MHepP, MNOP, MEHP, MINP, and MIDP) increased Ca²⁺ influx in the Flp-In 293/hTRPA1 cell line, whereas only MNOP, MINP, and MIDP slightly affected the Flp-In 293/mTRPA1 cell line (Fig. 3). The order of EC₅₀ values were as follows: MHexP > MHepP > MNOP > MEHP > MINP and MIDP (Fig. 4, Table 2). These values showed a good correlation with LogP (Pearson’s correlation coefficient = −0.9318, p = 0.0068) (Fig. 5).

Fig. 3. Ca²⁺ Increases When Stimulated by 50 µM PA Monoesters (A) and Diesters (B)

(A) PA monoesters 50 µM (C1; MMP, C2; MEP, C3; MPP, C4; MBP, C5; MPenP, C6; MHexP, C7; MHepP, Cn8; MNOP, C8; MEHP, C9; MINP, C10; MIDP). (B) PA diesters 50 µM (C1; DMP, C2; DEP, C3; DPP, C4; DBP, C5; DPenP, C6; DHexP, C7; DHepP, Cn8; DNOP, C8; DEHP, C9; DINP, C10; DIDP). The asterisk indicates a significant difference (* p < 0.05 and ** p < 0.01) from the control as determined using one-way ANOVA and Dunnett’s post hoc test.

Fig. 4. Dose–Response Curves of Human and Mouse TRPA1 Activation by PA Monoesters Based on the Calcium Assay

The fitted sigmoidal dose–response curves of EC₅₀ were calculated using GraphPad Prism 8. Human TRPA1-activating potency of phthalic acid monoesters. y-Axis: relative activity to 100 µM AITC (%). Ca²⁺ response by PA monoesters in TRPA1-expressing cells. x-Axis: agonist concentration (Log M); Each data point represents mean ± S.D., n = 3.

Table 2. EC₅₀ Values of Human TRPA1 Activation by PA Monoesters

Name	Human TRPA1 EC50 (µM)
MMP	—
MEP	—
MPP	—
MBP	—
MPenP	—
MHexP	9.93
MHepP	3.15
MNOP	1.60
MEHP	0.65
MINP	0.47
MIDP	0.44

Fig. 5. hTRPA1 Activation by PA Esters Correlates with Compound Lipophilicity

Relationship between the EC₅₀ of hTRPA1and LogP across PA monoesters. Pearson’s chi-square test was used to determine the correlation coefficient (Pearson’s correlation coefficient=−0.9318; p = 0.0068). It was judged to be significant when the p-value was lower than 0.01.

DISCUSSION

In this study, human and mouse TRPA1 activation was investigated using 11 kinds of PA diesters and corresponding monoesters. Results showed that PA monoesters activated human TRPA1, while PA diesters did not. A significant species-specific difference was also observed between human and mouse TRPA1 activation by PA monoesters. The EC₅₀ values of human TRPA1 activation by PA monoesters were in the order of MHexP > MHepP > MNOP > MEHP > MINP and MIDP. The affinity for hTRPA1 was observed to increase as the lipophilicity of alkyl side chains increases (Fig. 5, Table 2). Similar lipophilicity dependences were reported for TRPA1 activation by alkylphenols,⁴⁰⁾ primary alcohols,⁴¹⁾ and parabens.⁴²⁾ Therefore, the correlation between EC₅₀ and LogP values suggests that lipophilicity is an important factor that determines the affinity for TRPA1. However, lipophilic interactions between ligand and TRPA1 could not explain the species-specific activation by PA monoesters.

Two mechanisms were proposed on TRPA1 activation. First, TRPA1 is thought to be activated through covalent modification of three critical cysteine residues in the N-terminal region of the channel. The other is a mechanism that a less reactive ligand activates TRPA1 by noncovalent binding. Since the chemical reactivity of PA monoesters seems to be relatively weak, it is unlikely that the former mechanism is involved. In addition, three highly reactive cysteine residues were conserved between human and mouse, which cannot explain the large species-specific differences observed in TRPA1 activation by PA monoesters. On the other hand, menthol^37,38) and nicotine⁴³⁾ are ligands that activate TRPA1 noncovalently. Among noncovalent ligands, menthol activates hTRPA1 in a sigmoidal fashion, whereas it bimodally activates mTRPA1 at low concentrations but exhibits an inhibitory effect at higher concentrations.³⁸⁾ This different response between species was shown to be due to the substitution of a single amino acid residue between human and mouse (human 875 V, mouse 878G) located within the fifth transmembrane domains.³⁸⁾ However, in PA monoesters, this bimodal fashion was not observed, suggesting that the interaction between PA monoesters and human and mouse TRPA1s occur at an amino acid residue other than 875 V/878G. Further studies are needed to clarify the structural differences between human and mouse TRPA1s to better understand the toxic effects of PA esters.

Few reports dealt with the activation of hTRPA1 by PA diesters. Shiba et al. carried out a comparative study on hTRPA1 activation by PA diesters with one to nine carbon atoms in alkyl side chains (DMP, DEP, DPP, DBP, DPenP, DHexP, DHepP, DEHP, and DINP).⁴⁴⁾ They found that PA diesters with six or less carbon atoms (DEP, DPP, DBP, DPenP, and DHexP) activated hTRPA1. In contrast, as shown in Fig. 3, only two PA diesters (DPP and DNOP) activated hTRPA1 among the 11 PA diesters tested (DMP, DEP, DPP, DBP, DPenP, DHexP, DHepP, DNOP, DEHP, DINP, and DIDP). Regarding these controversial results, there is still no explanation, except for the difference in heterologous expression systems (Shiba et al. used Chinese Hamster Ovary cells, whereas we used HEK293-derived cells) and concentration ranges applied (up to 1 mM by Shiba et al., whereas we used a concentration of up to 50 µM because of solubility limitation). Recently, Kang et al. reported the possible involvement of TRPA1 in the adjuvant effects of DINP in an allergic contact dermatitis model.¹⁷⁾ Orally administered DINP to Balb/c mice seems to undergo intestinal hydrolysis, and the resulting MINP might participate in the observed adjuvant effects. Therefore, the results presented here may help explain the mechanisms involved in the allergic effects of DINP.

TRPA1 is expressed in various tissues and cells and plays an important role in various biological and immune responses. TRPA1 is abundant in primary afferent nociceptive fiber terminals, especially in nociceptive C and Aδ fibers of the dorsal root ganglion and trigeminal ganglion. It is also involved in the induction of pain sensation.^45,46) In addition, TRPA1 is expressed in bronchi and pulmonary vagus nerves. Hence, it is also involved in respiratory diseases, such as asthma, chronic obstructive pulmonary disease, and chronic cough.^47–52) In addition, a recent study showed that TRPA1 is expressed in mature sperms and testes, which is one of the major target tissues of PA esters.⁵³⁾ Although it is currently unknown whether TRPA1 is also expressed in human sperms, these results suggest that humans may be more susceptible to testicular toxicity caused by PA esters.

CONCLUSIVE REMARKS

In this study, we identified TRPA1 as a new target protein for PA monoesters. We also found a species-specific difference between humans and mice, where hTRPA1 was at least 100-fold more susceptible to PA monoesters than mTRPA1. An important finding was that the EC₅₀ value of hTRPA1 activation by MEHP was within the range reported in previous epidemiological studies (maximum serum levels of 39–1700 ng/mL).^54,55) These findings imply that PA esters may exert TRPA1-dependent adverse effects on humans, which have never been demonstrated in experimental animals. Further studies will contribute to the risk assessment of PA esters.

Acknowledgments

This study was supported by a Health and Labour Sciences Research Grant from the Ministry of Health, Labour, and Welfare of Japan (Grant Numbers. H27-Kagaku-Ippan-009 and H30-Kagaku-Shitei-002).

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

This article contains Supplementary Materials.

REFERENCES

• 1) Bergé A, Cladière M, Gasperi J, Coursimault A, Tassin B, Moilleron R. Meta-analysis of environmental contamination by phthalates. Environ. Sci. Pollut. Res. Int., 20, 8057–8076 (2013).
• 2) Takeuchi S, Tanaka-Kagawa T, Saito I, Kojima H, Jin K, Satoh M, Kobayashi S, Jinno H. Differential determination of plasticizers and organophosphorus flame retardants in residential indoor air in Japan. Environ. Sci. Pollut. Res. Int., 25, 7113–7120 (2018).
• 3) Giuliani A, Zuccarini M, Cichelli A, Khan H, Reale M. Critical review on the presence of phthalates in food and evidence of their biological impact. Int. J. Environ. Res. Public Health, 17, 5655 (2020).
• 4) ATSDR (US Agency for Toxic Substances and Disease Registry). 2022. “Toxicological profile for di(2-ethylhexyl)phthalate (DEHP).”: ‹https://www.atsdr.cdc.gov/ToxProfiles/tp9.pdf›, accessed 5 August, 2022.
• 5) Pagoni A, Arvaniti OS, Kalantzi OI. Exposure to phthalates from personal care products: urinary levels and predictors of exposure. Environ. Res., 212 (Pt. A), 113194 (2022).
• 6) Petersen JH, Breindahl T. Plasticizers in total diet samples, baby food and infant formulae. Food Addit. Contam., 17, 133–141 (2000).
• 7) Heudorf U, Mersch-Sundermann V, Angerer J. Phthalates: toxicology and exposure. Int. J. Hyg. Environ. Health, 210, 623–634 (2007).
• 8) Albro PW, Thomas RO. Enzymatic hydrolysis of di-(2-ethylhexyl)phthalate by lipases. Biochim. Biophys. Acta, 306, 380–390 (1973).
• 9) Sugino M, Hatanaka T, Todo H, Mashimo Y, Suzuki T, Kobayashi M, Hosoya O, Jinno H, Juni K, Sugibayashi K. Safety evaluation of dermal exposure to phthalates: metabolism-dependent percutaneous absorption. Toxicol. Appl. Pharmacol., 328, 10–17 (2017).
• 10) Nakamiya K, Takagi H, Nakayama T, Ito H, Tsuruga H, Edmonds JS, Morita M. Microbial production and vaporization of mono-(2-ethylhexyl) phthalate from di-(2-ethylhexyl) phthalate by microorganisms inside houses. Arch. Environ. Occup. Health, 60, 321–325 (2005).
• 11) Wakayama T, Ito Y, Sakai K, Miyake M, Shibata E, Ohno H, Kamijima M. Comprehensive review of 2-ethyl-1-hexanol as an indoor air pollutant. J. Occup. Health, 61, 19–35 (2019).
• 12) Boll M, Geiger R, Junghare M, Schink B. Microbial degradation of phthalates: biochemistry and environmental implications. Environ. Microbiol. Rep., 12, 3–15 (2020).
• 13) Bølling AK, Sripada K, Becher R, Beko G. Phthalate exposure and allergic diseases: review of epidemiological and experimental evidence. Environ. Int., 139, 105706 (2020).
• 14) Bornehag CG, Sundell J, Weschler CJ, Sigsgaard T, Lundgren B, Hasselgren M, Hagerhed-Engman L. The association between asthma and allergic symptoms in children and phthalates in house dust: a nested case-control study. Environ. Health Perspect., 112, 1393–1397 (2004).
• 15) Ait Bamai Y, Miyashita C, Araki A, Nakajima T, Sasaki S, Kishi R. Effects of prenatal di(2-ethylhexyl) phthalate exposure on childhood allergies and infectious diseases: the Hokkaido Study on Environment and Children’s Health. Sci. Total Environ., 618, 1408–1415 (2018).
• 16) Hansen JS, Larsen ST, Poulsen LK, Nielsen GD. Adjuvant effects of inhaled mono-2-ethylhexyl phthalate in BALB/cJ mice. Toxicology, 232, 79–88 (2007).
• 17) Kang J, Ding Y, Li BZ, Liu H, Yang X, Chen MQ. TRPA1 mediated aggravation of allergic contact dermatitis induced by DINP and regulated by NF-kappa B activation. Sci. Rep., 7, 43586 (2017).
• 18) Shiba T, Maruyama T, Kurohane K, Iwasaki Y, Watanabe T, Imai Y. TRPA1 and TRPV1 activation is a novel adjuvant effect mechanism in contact hypersensitivity. J. Neuroimmunol., 207, 66–74 (2009).
• 19) Shiba T, Tamai T, Sahara Y, Kurohane K, Watanabe T, Imai Y. Transient receptor potential ankyrin 1 activation enhances hapten sensitization in a T-helper type 2-driven fluorescein isothiocyanate-induced contact hypersensitivity mouse model. Toxicol. Appl. Pharmacol., 264, 370–376 (2012).
• 20) Nassini R, Materazzi S, Benemei S, Geppetti P. The TRPA1 channel in inflammatory and neuropathic pain and migraine. Rev. Physiol. Biochem. Pharmacol., 167, 1–43 (2014).
• 21) Jordt SE, Bautista DM, Chuang HH, McKemy DD, Zygmunt PM, Hogestatt ED, Meng ID, Julius D. Mustard oils and cannabinoids excite sensory nerve fibres through the TRP channel ANKTM1. Nature, 427, 260–265 (2004).
• 22) Luostarinen S, Hamalainen M, Hatano N, Muraki K, Moilanen E. The inflammatory regulation of TRPA1 expression in human A549 lung epithelial cells. Pulm. Pharmacol. Ther., 70, 102059 (2021).
• 23) Matsuda K, Arkwright PD, Mori Y, Oikawa MA, Muko R, Tanaka A, Matsuda H. A rapid shift from chronic hyperoxia to normoxia induces systemic anaphylaxis via transient receptor potential ankyrin 1 channels on mast cells. J. Immunol., 205, 2959–2967 (2020).
• 24) Emery EC, Diakogiannaki E, Gentry C, Psichas A, Habib AM, Bevan S, Fischer MJ, Reimann F, Gribble FM. Stimulation of GLP-1 secretion downstream of the ligand-gated ion channel TRPA1. Diabetes, 64, 1202–1210 (2015).
• 25) Atoyan R, Shander D, Botchkareva NV. Non-neuronal expression of transient receptor potential type A1 (TRPA1) in human skin. J. Invest. Dermatol., 129, 2312–2315 (2009).
• 26) Cao DS, Zhong L, Hsieh TH, Abooj M, Bishnoi M, Hughes L, Premkumar LS. Expression of transient receptor potential ankyrin 1 (TRPA1) and its role in insulin release from rat pancreatic beta cells. PLOS ONE, 7, e38005 (2012).
• 27) Sotomayor M, Corey DP, Schulten K. In search of the hair-cell gating spring elastic properties of ankyrin and cadherin repeats. Structure, 13, 669–682 (2005).
• 28) Karashima Y, Talavera K, Everaerts W, Janssens A, Kwan KY, Vennekens R, Nilius B, Voets T. TRPA1 acts as a cold sensor in vitro and in vivo. Proc. Natl. Acad. Sci. U.S.A., 106, 1273–1278 (2009).
• 29) Bandell M, Story GM, Hwang SW, Viswanath V, Eid SR, Petrus MJ, Earley TJ, Patapoutian A. Noxious cold ion channel TRPA1 is activated by pungent compounds and bradykinin. Neuron, 41, 849–857 (2004).
• 30) Macpherson LJ, Geierstanger BH, Viswanath V, Bandell M, Eid SR, Hwang S, Patapoutian A. The pungency of garlic: activation of TRPA1 and TRPV1 in response to allicin. Curr. Biol., 15, 929–934 (2005).
• 31) Bautista DM, Jordt SE, Nikai T, Tsuruda PR, Read AJ, Poblete J, Yamoah EN, Basbaum AI, Julius D. TRPA1 mediates the inflammatory actions of environmental irritants and proalgesic agents. Cell, 124, 1269–1282 (2006).
• 32) Macpherson LJ, Xiao B, Kwan KY, Petrus MJ, Dubin AE, Hwang S, Cravatt B, Corey DP, Patapoutian A. An ion channel essential for sensing chemical damage. J. Neurosci., 27, 11412–11415 (2007).
• 33) Ohkawara S, Tanaka-Kagawa T, Furukawa Y, Jinno H. Methylglyoxal activates the human transient receptor potential ankyrin 1 channel. J. Toxicol. Sci., 37, 831–835 (2012).
• 34) Egan TJ, Acuna MA, Zenobi-Wong M, Zeilhofer HU, Urech D. Effects of N-glycosylation of the human cation channel TRPA1 on agonist-sensitivity. Biosci. Rep., 36, e00390 (2016).
• 35) Doihara H, Nozawa K, Kawabata-Shoda E, Kojima R, Yokoyama T, Ito H. Molecular cloning and characterization of dog TRPA1 and AITC stimulate the gastrointestinal motility through TRPA1 in conscious dogs. Eur. J. Pharmacol., 617, 124–129 (2009).
• 36) Ko MJ, Ganzen LC, Coskun E, Mukadam AA, Leung YF, van Rijn RM. A critical evaluation of TRPA1-mediated locomotor behavior in zebrafish as a screening tool for novel anti-nociceptive drug discovery. Sci. Rep., 9, 2430 (2019).
• 37) Karashima Y, Damann N, Prenen J, Talavera K, Segal A, Voets T, Nilius B. Bimodal action of menthol on the transient receptor potential channel TRPA1. J. Neurosci., 27, 9874–9884 (2007).
• 38) Xiao B, Dubin AE, Bursulaya B, Viswanath V, Jegla TJ, Patapoutian A. Identification of transmembrane domain 5 as a critical molecular determinant of menthol sensitivity in mammalian TRPA1 channels. J. Neurosci., 28, 9640–9651 (2008).
• 39) Nagatomo K, Kubo Y. Caffeine activates mouse TRPA1 channels but suppresses human TRPA1 channels. Proc. Natl. Acad. Sci. U.S.A., 105, 17373–17378 (2008).
• 40) Startek JB, Milici A, Naert R, Segal A, Alpizar YA, Voets T, Talavera K. The agonist action of alkylphenols on TRPA1 relates to their effects on membrane lipid order: implications for TRPA1-mediated chemosensation. Int. J. Mol. Sci., 22, 3368 (2021).
• 41) Komatsu T, Uchida K, Fujita F, Zhou Y, Tominaga M. Primary alcohols activate human TRPA1 channel in a carbon chain length-dependent manner. Pflugers Arch., 463, 549–559 (2012).
• 42) Fujita F, Moriyama T, Higashi T, Shima A, Tominaga M. Methyl p-hydroxybenzoate causes pain sensation through activation of TRPA1 channels. Br. J. Pharmacol., 151, 134–141 (2007).
• 43) Talavera K, Gees M, Karashima Y, Meseguer VM, Vanoirbeek JA, Damann N, Everaerts W, Benoit M, Janssens A, Vennekens R, Viana F, Nemery B, Nilius B, Voets T. Nicotine activates the chemosensory cation channel TRPA1. Nat. Neurosci., 12, 1293–1299 (2009).
• 44) Shiba T, Maruyama T, Kurohane K, Iwasaki Y, Watanabe T, Imai Y. TRPA1 and TRPV1 activation is a novel adjuvant effect mechanism in contact hypersensitivity. J. Neuroimmunol., 207, 66–74 (2009).
• 45) Story GM, Peier AM, Reeve AJ, Eid SR, Mosbacher J, Hricik TR, Earley TJ, Hergarden AC, Andersson DA, Hwang SW, McIntyre P, Jegla T, Bevan S, Patapoutian A. ANKTM1, a TRP-like channel expressed in nociceptive neurons, is activated by cold temperatures. Cell, 112, 819–829 (2003).
• 46) Kobayashi K, Fukuoka T, Obata K, Yamanaka H, Dai Y, Tokunaga A, Noguchi K. Distinct expression of TRPM8, TRPA1, and TRPV1 mRNAs in rat primary afferent neurons with a delta/c-fibers and colocalization with trk receptors. J. Comp. Neurol., 493, 596–606 (2005).
• 47) Geppetti P, Patacchini R, Nassini R, Materazzi S. Cough: the emerging role of the TRPA1 channel. Lung, 188 (Suppl. 1), S63–S68 (2010).
• 48) Long L, Yao H, Tian J, Luo W, Yu X, Yi F, Chen Q, Xie J, Zhong N, Chung KF, Lai K. Heterogeneity of cough hypersensitivity mediated by TRPV1 and TRPA1 in patients with chronic refractory cough. Respir. Res., 20, 112 (2019).
• 49) Wallace H. Airway pathogenesis is linked to TRP channels. Neurobiology of TRP Channels. (Emir TLR ed.) CRC Press, Boca Raton, FL, pp. 251–264 (2017).
• 50) Yang H, Li S. Transient receptor potential ankyrin 1 (TRPA1) channel and neurogenic inflammation in pathogenesis of asthma. Med. Sci. Monit., 22, 2917–2923 (2016).
• 51) Brozmanova M, Mazurova L, Ru F, Tatar M, Kollarik M. Comparison of TRPA1-versus TRPV1-mediated cough in guinea pigs. Eur. J. Pharmacol., 689, 211–218 (2012).
• 52) Nassenstein C, Kwong K, Taylor-Clark T, Kollarik M, Macglashan DM, Braun A, Undem BJ. Expression and function of the ion channel TRPA1 in vagal afferent nerves innervating mouse lungs. J. Physiol., 586, 1595–1604 (2008).
• 53) Saha S, Sucharita S, Majhi RK, Tiwari A, Ghosh A, Pradhan SK, Patra BK, Dash RR, Nayak RN, Giri SC, Routray P, Kumar A, Kumar G P, Goswami C. TRPA1 is selected as a semi-conserved channel during vertebrate evolution due to its involvement in spermatogenesis. Biochem. Biophys. Res. Commun., 512, 295–302 (2019).
• 54) Li Y, Yao Y, Xiao N, Liu Y, Du Y, Liu M, Zhang Q, Zhao H, Zhang T, Zhang H, Wang L, Luo H, Zhang Y, Sun H. The association of serum phthalate metabolites with biomarkers of ovarian reserve in women of childbearing age. Ecotoxicol. Environ. Saf., 242, 113909 (2022).
• 55) Li Z, Wu D, Guo Y, Mao W, Zhao N, Zhao M, Jin H. Phthalate metabolites in paired human serum and whole blood. Sci. Total Environ., 824, 153792 (2022).