2017 年 40 巻 11 号 p. 2010-2013
Dibutyl phthalate (DBP) is a plasticizer used for many consumer products including cosmetics. Potential health concerns regarding DBP include reproductive and developmental toxicity, endocrine disruption and neurotoxicity. DBP is a high priority chemical as to reduction of exposure of children to it. Through reproductive toxicity studies, monobutyl phthalate (MBP) has been proposed to be the active metabolite derived from DBP. We previously demonstrated that DBP activates transient receptor potential ankyrin 1 (TRPA1) cation channels expressed on sensory neurons. We have also shown that DBP enhanced skin sensitization in a fluorescein isothiocyanate (FITC)-induced contact hypersensitivity (CHS) mouse model. Through MBP formation by esterase in the skin, it is possible that MBP exerts a major effect on the biological activity we observed. To test this possibility, we directly compared DBP and MBP. A more than 40-fold higher concentration of MBP as compared with DBP was required for activation of TRPA1 in vitro. Unlike DBP, MBP did not enhance skin sensitization to FITC. These results demonstrated that DBP directly, i.e., not through its metabolite MBP, activates TRPA1 and enhances FITC-CHS. It is noteworthy that butyl benzoate, a related compound, activated TRPA1 and enhanced FITC-CHS.
Di-n-butyl phthalate (DBP) is widely used as a plasticizer for consumer products such as paint, adhesives, paper coatings, printing ink and cosmetics.1–5) Regarding children’s health concerns, DBP has become one of the highest priority chemicals as to reduction of exposure of children to it in consumer products.6) The health concerns include reproductive and developmental toxicity, endocrine disruption and neurotoxicity.6–8)
We have been studying DBP from an immuno-toxicological perspective. We employ a fluorescein isothiocyanate (FITC)-induced contact hypersensitivity (CHS) mouse model, in which DBP is empirically added to the solvent for FITC.9) We found that DBP acts as an adjuvant during skin sensitization to FITC.10) We also demonstrated that DBP had the ability to stimulate transient receptor potential ankyrin 1 (TRPA1) cation channels, which are expressed on sensory neurons.11) We then compared several types of phthalate esters regarding adjuvant effects and TRPA1 stimulation. The immunological and nervous activation effects caused by phthalate esters were found to be well correlated.11,12)
In studies on reproductive toxicity, the role of monobutyl phthalate (MBP) as an active metabolite derived from DBP has been investigated.13,14) Although studies on reproductive toxicity involve systemic administration of DBP, with which MBP is more likely to be produced in the body, topical application of DBP may yield MBP through esterase in the skin. Thus, there is a possibility that MBP has biological effects as an active metabolite. To examine this idea, we investigated whether MBP is more active than DBP regarding TRPA1 activation or enhancement of FITC-CHS. We also tested a related compound, monobutyl benzoate (BBz), from these two angles. Structures of DBP, MBP, phthalic acid and BBz are shown in Fig. 1.
Di-n-butyl phthalate (DBP), mono-n-butyl phthalate (MBP), phthalic acid, and mono-n-butyl benzoate (BBz) were used in this study.
Acetone, DBP and dimethyl sulfoxide (DMSO) were purchased form Wako Pure Chemical Industries, Ltd. (Osaka, Japan); MBP, phthalic acid and BBz from Tokyo Chemical Industry (Tokyo, Japan); and FITC and Fluo 4-AM from Dojindo Laboratories (Kumamoto, Japan).
CellsChinese hamster ovary (CHO) cells expressing human TRPA1 (TRPA1-CHO) were established and maintained as described.11,15,16) As a control, T-REx CHO cells not expressing TRPA1 were used.
AnimalsSpecific pathogen-free female BALB/c mice were purchased from Japan SLC Inc. (Shizuoka, Japan) at 7 weeks of age. Animal care and experiments were performed as described previously in accordance with the guidelines for the care and use of laboratory animals published by the Ministry of Education, Culture, Sports, Science and Technology of Japan, and those of the University of Shizuoka.16) The plans for animal experiments were reviewed and approved by the Institutional Animal Care and Use Committee of the University of Shizuoka (approval numbers: 146135, 166199 and 176237).
TRPA1 Activation in VitroMeasurement of TRPA1 activation was performed using TRPA1-CHO cells after labeling with a calcium-ion sensitive fluorescent probe, Fluo-4 AM, as described previously.15,16) After addition of a test sample to Fluo-4-loaded cells, the intracellular Ca2+ level was automatically measured over time with a fluorometric imaging plate reader, FLEXstation II (Molecular Devices, Sunnyvale, CA, U.S.A.), at 37°C. The maximal Ca2+ level was determined with 5 µM ionomycin (Life Technologies, Carlsbad, CA, U.S.A.).
FITC-Induced CHSMouse contact hypersensitivity experiments involving FITC as a hapten were performed as described previously.12,16) BALB/c mice were epicutaneously sensitized with 0.5% FITC on shaved forelimbs on days 0 and 7. FITC was dissolved in one of the following solvents: acetone alone, or acetone containing 2% (v/v) DBP, 2% (w/v) MBP or 2% (v/v) BBz. On day 14, a 0.5% FITC solution in acetone–DBP (1 : 1) was applied on the right auricle to elicit inflammation, while acetone–DBP alone was applied on the left auricle as a control. To distinguish the effect on the sensitization phase from elicitation phase, all mice were challenged with FITC solution in acetone–DBP (1 : 1) as described before.12,16) This allows comparison of the effect only on the sensitization phase for various chemicals. It should be noted that if challenge was done in the absence of DBP, only a weak ear-swelling response was observed (unpublished results). Ear thickness was measured before (0 h), and 24, 48 and 72 h after elicitation by means of a dial thickness gauge (Mitutoyo, Kanagawa, Japan). Ear swelling at X h is defined as follows: (thickness of the right ear−thickness of the left ear) at X h−(thickness of the right ear−thickness of the left ear) at 0 h.
StatisticsMultiple comparisons were performed using one-way ANOVA followed by Dunnett’s test with Graphpad Prism 5 (version 5.02; Graphpad Software, San Diego, CA, U.S.A.).
Our previous studies involving the test of various phthalate esters suggested a correlation between TRPA1 agonistic activity and the adjuvant effect on FITC-CHS.11,12) We first investigated whether MBP activates TRPA1. DBP, a positive control, induced an intracellular Ca2+ increase in TRPA1-CHO cells, while no effect on T-REx CHO cells was observed without TRPA1 expression (Fig. 2A). For MBP, a TRPA1-dependent Ca2+ increase was also observed but higher concentrations were needed (Fig. 2B). The 50% effective concentration (EC50) values for DBP and MBP were 7.3 and 313 µM, respectively. In contrast, phthalic acid did not cause a TRPA1-dependent response, while a non-specific Ca2+ increase was seen at the highest concentration, 1 mM (Fig. 2C). Regarding MBP (Fig. 2B), the response at the highest concentration tested (1 mM) was almost on the plateau level; i.e., 90% of the maximal Ca2+ level. Data fitting using the variable slope model, Hill slope was calculated to be 2.0. From the same data fitting, the plateau level was estimated to be 96%, which is close to the actual data at 1 mM (90%). We calculated EC50 using this estimated plateau level of 96%.
TRPA1-CHO (open circles) or control T-REx CHO (open triangles) cells were treated with various concentrations (abscissa) of DBP (A), MBP (B), phthalic acid (C), or BBz (D). The intracellular Ca2+ level was monitored over time. The percentages of the maximal Ca2+ level (ionomycin) are shown on the ordinate. Each datum is the mean±range (duplicate determination). Error bars underneath the symbols are not visible. Reproducibility was confirmed by two experiments.
We tested the adjuvant effect of MBP during sensitization to FITC. Mice were epicutaneously sensitized with FITC in the presence of DBP or MBP. After FITC had been applied on the ear auricles of sensitized mice, ear-swelling responses were measured over time (Fig. 3). Mice sensitized in the presence of 2% DBP exhibited significant increases in ear swelling as compared with the acetone controls at every time point. In contrast, there was no difference in the ear-swelling between mice sensitized with FITC in the presence of 2% MBP and those sensitized with FITC in acetone alone.
BALB/c mice were epicutaneously sensitized with 0.5% FITC in acetone containing 2% DBP, 2% MBP or 2% BBz, or in acetone alone on days 0 and 7. On day 14, the mice were challenged on the right auricle with 0.5% FITC in 50% DBP in acetone, while 50% DBP in acetone was applied to the left auricle. The ear-swelling responses at 24 h (A), 48 h (B), and 72 h (C) after challenge in each individual mouse under the acetone alone (n=12), acetone–DBP (n=12), acetone–MBP (n=13), or acetone–BBz (n=14) conditions are shown. Horizontal bars represent the mean for each group. *** p<0.001, n.s. (not significant) compared with the acetone alone conditions. The results of two independent experiments were combined.
Free carboxyl group on the benzene ring might be the reason for the low biological activity of MBP. An alternative possibility is that two butyl alcohol esters are required for the activity. To examine these possibilities we tested BBz, which lacks a free carboxyl group but has one butyl alcohol ester. BBz showed specific activation of TRPA1 (Fig. 2D). The EC50 value was 86.5 µM. Sensitization in the presence of 2% BBz enhanced FITC-induced ear-swelling responses as compared with under the acetone alone conditions (Fig. 3).
TRPA1 activation with various phthalate esters as well as an alternative plasticizer, diisopropyl adipate, was found to be a good predictor of enhanced sensitization in FITC-CHS.11,16) Furthermore, several natural products known as TRPA1 agonists also enhanced skin sensitization to FITC.15) Thus, we initially compared TRPA1 activation in vitro. MBP could specifically activate TRPA1 but high concentrations were required compared to DBP. The EC50 was more than 40-fold higher than that of DBP. For phthalic acid, no specific activation of TRPA1 was observed. Thus, we did not carry out further experiments using phthalic acid regarding FITC-CHS.
Consistent with our previous studies,16) 2% DBP enhanced skin sensitization to FITC as compared with under the acetone alone conditions. Ear swelling was more evident at 24 h after challenge with FITC than those at 48 or 72 h (Fig. 3). This time course is consistent with our previous studies under the 2% DBP conditions.16) Such a time course of the ear swelling was also seen for the group under the 2% BBz conditions (Fig. 3), suggesting that it is shared among this CHS model. Lack of significant ear swelling at any time point under the MBP conditions indicated that MBP did not exhibit an adjuvant effect at 2% concentration.
These two findings demonstrated that DBP itself gave rise to both TRPA1 activation and FITC-CHS enhancement, but not through its metabolite MBP.
There are two possibilities for the reduced activity of MBP as compared with DBP. One is the exposure of a free carboxyl group on the benzene ring and the other is a loss of a butyl alcohol side chain from the benzene ring. To examine these possibilities, we tested BBz. BBz also specifically activated TRPA1. The activity level was between those of DBP and MBP, thus, the EC50 was 12-fold higher than that of DBP. In contrast, 2% BBz unequivocally enhanced FITC-CHS sensitization to a similar level to that with 2% DBP. A possible explanation is that hydrophobic BBz might facilitate skin penetration of the FITC hapten more effectively than anionic MBP in vivo. In other words, two butyl alcohol esters are not essential for the enhanced skin sensitization to FITC. In an aqueous environment in vitro, the presence of two butyl alcohol side chains may play some positive role in TRPA1 activation.
We mentioned the effect on skin penetration of FITC hapten as a possible additional mechanism for the enhanced sensitization with coexisting chemicals. Although we have not compared DBP and related compounds from this angle, this is an interesting subject for further studies. Skin environment is different from the aqueous one used for cell-based assays in that the former is abundant in hydrophobic sebum. More hydrophobic BBz might have an advantage in the delivery of FITC through the skin than MBP, which has a free carboxylic acid group. There is a report demonstrating that DBP induced specific uptake of FITC via the pilosebaceous unit in the skin.17) Thus, the effect of chemicals on the bioavailability of allergic hapten may be another important issue.
TRPA1 agonistic activity of BBz was weaker than that of DBP in vitro. Nevertheless, the adjuvant effect of BBz on FITC-CHS was on a similar level to that of DBP. We do not argue that the ability of TRPA1 activation is the single quantitative determinant that predicts the adjuvant effect of chemicals in FITC-CHS. We may also need to uncover the roles of other mechanisms involved in the adjuvant effect and biological activity of chemicals based on experimental evidences. At present, we propose that TRPA1 activation is one of the useful features for the prediction of adjuvant activity of chemicals in FITC-CHS.
This work was supported by a Grant-in-Aid for Challenging Exploratory Research (Grant number 15K14990) from the Japan Society for the Promotion of Science to YI. We thank Ms. Yukina Endo, Ms. Narumi Mochizuki, Mr. Akimasa Orii and Ms. Mutsumi Nose for technical assistance. We thank Mr. Nicholas J. Halewood for the language editing service.
The authors declare no conflict of interest.
