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Phthalate esters reveal skin-sensitizing activity of phenethyl isothiocyanate in mice
Food and Chemical Toxicology 48 (2010) 1704–1708
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Phthalate esters reveal skin-sensitizing activity of phenethyl isothiocyanate in mice Tomoko Matsuda, Takashi Maruyama, Hiromi Iizuka, Ayumi Kondo, Takuma Tamai, Kohta Kurohane, Yasuyuki Imai * Laboratory of Microbiology and Immunology and the Global COE Program, University of Shizuoka School of Pharmaceutical Sciences, Shizuoka 422-8526, Japan
a r t i c l e
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Article history: Received 3 December 2009 Accepted 30 March 2010
Keywords: Adjuvant Contact hypersensitivity DBP Isothiocyanate Vegetable
a b s t r a c t Phenethyl isothiocyanate (PEITC) is a constituent of edible cruciferous vegetables and has received attention due to its potential cancer chemopreventive activity. Although its protein-binding activity is known, PEITC has not been studied as a contact sensitizer. We previously demonstrated that phthalate esters, including dibutyl phthalate (DBP) and di-n-propyl phthalate (DPP), enhance skin sensitization in a fluorescein isothiocyanate (FITC)-induced contact hypersensitivity mouse model. In this study, we examined whether DBP and DPP enhance skin sensitization to haptens other than FITC. During this study, we tested PEITC as a contact sensitizer. BALB/c mice were epicutaneously sensitized with a suboptimal dose of a hapten with or without a phthalate ester. Sensitization was evaluated as the ear swelling response after a challenge with the respective hapten. DBP and DPP did not enhance the sensitization to two T-helper 1type (2,4-dinitrofluorobenzene and oxazolone) or three T-helper 2-type (trimellitic anhydride, methylenediphenyl 4,40 -diisocyanate, and tolulene 2,4-diisocyanate) haptens. In contrast, DBP and DPP enhanced the sensitization to two FITC analogues (eosin 5-isothiocyanate and rhodamine B isothiocyanate) as well as to PEITC. Adjuvant effects of DBP and DPP were observed in contact hypersensitivity to haptens other than FITC, including a food ingredient PEITC. Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction Phenethyl isothiocyanate (PEITC) is a constituent of many edible cruciferous vegetables, and has received attention due to its potential cancer chemopreventive activity (Antosiewicz et al., 2008; Hu et al., 2007; Mi et al., 2007; Xiao and Singh, 2007; Yin et al., 2009). Although the protein-binding activity of PEITC has been demonstrated (Mi et al., 2007), there has been no report on it as a contact sensitizer. Phthalate esters are widely used as plasticizers for plastics such as soft polyvinyl chloride toys, vinyl floorings, wall coverings, synthetic leather and blood transfusion bags. Some phthalate esters with short alkyl chains are used for cosmetics and mosquito repellents for topical use (Api, 2001; Vartak et al., 1994). It has been reported that some phthalate esters were implicated in allergic
Abbreviations: CHS, contact hypersensitivity; DBP, dibutyl phthalate; DC, dendritic cell; DNFB, 2,4-dinitrofluorobenzene; DPP, di-n-propyl phthalate; EITC, eosin 5-isothiocyanate; MDI, methylenediphenyl 4,40 -diisocyanate; PEITC, phenethyl isothiocyanate; RITC, rhodamine B isothiocyanate; TDI, tolulene 2,4-diisocyanate; Th1, T-helper 1; Th2, T-helper 2; TMA, trimellitic anhydride. * Corresponding author. Address: Laboratory of Microbiology and Immunology, University of Shizuoka School of Pharmaceutical Sciences, 52-1 Yada, Suruga-ku, Shizuoka-shi, Shizuoka 422-8526, Japan. Tel.: +81 54 264 5716; fax: +81 54 264 5715. E-mail address: [email protected] (Y. Imai). 0278-6915/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.fct.2010.03.049
symptoms in children (Bornehag et al., 2004; Kolarik et al., 2008) as well as in adults (Chowdhury and Statham, 2002). Experimentally, dibutyl phthalate (DBP) has been empirically included in a solvent system for fluorescein isothiocyanate (FITC), which is widely used as a hapten in contact hypersensitivity (CHS) mouse models (Kripke et al., 1990). We previously demonstrated that DBP exhibits an adjuvant effect during sensitization to FITC (Sato et al., 1998). We noticed that DBP itself did not show antigenicity (Sato et al., 1998). In addition to DBP, we found that diethyl phthalate (DEP), di-n-propyl phthalate (DPP), dipentyl phthalate (DPNP), dihexyl phthalate (DHXP), and diheptyl phthalate (DHPP) also exhibit adjuvant effects in the FITC system (Imai et al., 2006; Shiba et al., 2009). We also found that DBP and DPP promote the trafficking of FITC-presenting CD11c+ dendritic cells (DC) to draining lymph nodes in the sensitization phase of FITC-induced CHS (Imai et al., 2006). However, it is not clear whether the phthalate esters exhibit adjuvant effects on sensitization to various chemicals other than FITC or there are some rules in combination with antigenic haptens. There are haptens classified as T-helper 1-type (Th1) or T-helper 2-type (Th2) haptens based on helper T cell polarization tendencies. They are defined by measuring cytokine production or immunoglobulin class switches (Hopkins et al., 2005; Plitnick et al., 2005; Sun et al., 2007). Earlier studies indicated that the Th2-type response, which can be characterized by IL-4 production, is
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required for FITC-induced CHS (Dearman and Kimber, 2000; Takeshita et al., 2004; Tang et al., 1996). Consistent with these results, we found that IL-4 production in draining lymph nodes was enhanced when DBP or DPP was included in the solvent for FITC (Maruyama et al., 2007a, b). One interesting question is whether the enhancing effect of phthalate esters on hapten sensitization is general or selective for one of the types of haptens. In the present study, we examined whether the adjuvant effects of DBP and DPP are observed on haptens other than FITC. One specific question is whether the adjuvant effects of DBP and DPP depend on the helper T cell polarization tendencies with haptens that are known to induce a Th1- or Th2-type response. Another specific question is whether or not phthalate esters enhance skin sensitization by haptens containing an isothiocyanate group. Toward this end, we first tested effects of DBP and DPP on FITC analogues, rhodamine B isothiocyanate (RITC) and eosin 5isothiocyanate (EITC), which have four aromatic rings. Second, we tested a hapten with single isothiocyanate group and single aromatic ring, PEITC. During the experiments, we found the ability of PEITC to sensitize mice epicutaneously in the presence of a phthalate ester.
For sensitization with EITC and RITC, mice were sensitized on days 0 and 7 by applying 160 ll of an EITC (0.5%, w/v) and RITC (0.5%, w/v) solution, respectively. On day 14, mice were challenged by applying 20 ll of EITC (0.5%) or RITC (0.5%) in acetone/DBP on the right auricle. RITC was dissolved in DMSO before dilution in the solvent. Therefore, the RITC solution contained a final concentration of 1% DMSO. For sensitization with PEITC, mice were sensitized on days 0, 1, 7, and 8 by applying 160 ll of PEITC (2.5%, v/v). On day 14, mice were challenged by applying 20 ll of PEITC (2.5%) in acetone/DBP on the right auricle. In this particular case, PEITC was dissolved in acetone/olive oil (3:1, v/v) during sensitization under the control condition. Ear swelling at X h is defined as follows: ((thickness of the right ear at X h) (thickness of the right ear at 0 h)) ((thickness of the left ear at X h) (thickness of the left ear at 0 h)). As a control experiment, five mice were epicutaneously treated on days 0 and 7 by applying 160 ll of acetone, acetone/DBP or acetone/DPP. On day 14, mice were challenged by applying 20 ll of acetone/DBP on the left auricle. Thickness of left ear was determined before (0 h) and after (24 and 48 h) the challenge. 2.4. Statistical analysis Statistical significance was analyzed among groups with different sensitization conditions by means of one-way ANOVA. As a post-hoc test, Dunnett’s test was performed compared with an acetone control.
3. Results 2. Materials and methods
3.1. Lack of immunogenicity of phthalate esters
2.1. Mice Specific pathogen-free female BALB/c mice were purchased from Japan SLC Inc. (Shizuoka, Japan), and were used at 8 weeks of age. They were housed at 22–24 °C and 50–60% humidity under artificial lighting conditions with a 12-h light/dark cycle. They had access to food (Oriental Yeast Co., Tokyo, Japan) and water ad libitum. Animal care and experiments were performed in accordance with the guidelines for the care and use of laboratory animals of the University of Shizuoka. 2.2. Reagents Acetone, dibutyl phthalate (DBP) (CAS No. 84-74-2, EINECS# 201-557-4), 2,4dinitrofluorobenzene (DNFB) (CAS No. 70-34-8, EINECS# 200-734-3), methylenediphenyl 4,40 -diisocyanate (MDI) (CAS No. 101-68-8, EINECS# 202-966-0), trimellitic anhydride (TMA) (CAS No. 552-30-7, EINECS# 209-008-0), and tolulene 2,4-diisocyanate (TDI) (CAS No. 584-84-9, EINECS# 209-544-5) were purchased from Wako Pure Chemicals (Osaka, Japan); di-n-propyl phthalate (DPP) (CAS No. 131-16-8, EINECS# 205-015-8) from Kanto Chemicals (Tokyo, Japan); eosin 5-isothiocyanate (EITC) (CAS No. 60520-47-0), 4-ethoxymethylene-2-phenyl-2-oxazolin-5-one (oxazolone) (CAS No. 15646-46-5, EINECS# 239-713-9), rhodamine B isothiocyanate (RITC) (CAS No. 36877-69-7, EINECS# 253-248-9), and phenethyl isothiocyanate (PEITC) (CAS No. 2257-09-2, EINECS# 218-855-5) from Sigma (St. Louis, MO, USA); and dimethyl sulfoxide (DMSO) from Nacalai Tesque (Kyoto, Japan). Purities of DNFB and PEITC were more than 99%, those of DBP and DPP were more than 98%, those of MDI and TMA were more than 97%, those of TDI and EITC were more than 95%, and that of oxazolone was more than 90%.
We have previously shown that DBP itself did not exhibit antigenicity (Sato et al., 1998). We confirmed that DBP and DPP did not behave as an antigen. Mice were epicutaneously treated with acetone, with a mixture of acetone and DBP or with acetone and DPP in the absence of any other hapten. Upon challenge with acetone/DBP on the ear, increase in the ear thickness was determined. The mean values ± SEM (in lm) were as follows: 2.0 ± 2.0 (acetone), 0.6 ± 3.2 (acetone/DBP), 8.0 ± 4.8 (acetone/DPP) at 24 h after challenge; 2.2 ± 4.8 (acetone), 1.3 ± 2.7 (acetone/ DBP), 2.7 ± 6.4 (acetone/DPP) at 48 h after challenge. These results indicate essentially no change in ear thickness. 3.2. Effects of phthalate esters on sensitization to haptens that predominantly induce a Th1-type response Our previous studies indicated that DBP and DPP had adjuvant effects on sensitization to FITC, which is known to induce an immune response with a Th2-type cytokine profile (Dearman and
Table 1 Effects of phthalate esters during sensitization with a hapten that is prone to induce a Th1-type response.
2.3. Sensitization and elicitation of a contact hypersensitivity reaction Sensitization with a hapten dissolved in Mouse forelimbs were shaved using small animal clippers one day before sensitization. Mice were epicutaneously sensitized with a suboptimal dose of a hapten dissolved in acetone or a mixture of acetone and a phthalate ester (1:1, v/v). For challenge, the hapten solution was applied on the right auricle while the vehicle was applied on the left auricle as a control. For sensitization with DNFB, the sensitization schedule was based on the published protocol except that a suboptimal hapten concentration was used (Simon et al., 1994; Weiss et al., 1997). Mice were sensitized on days 0 and 1 by applying 160 ll of DNFB solution (0.04%, w/v). On day 5, mice were challenged by applying 20 ll of a DNFB solution (0.1% in acetone) on the right auricle. The left auricle was treated with 20 ll of acetone as a control. Ear thickness was measured before (0 h) and after (24, 48, and 72 h) the challenge using a dial thickness gauge (Mitsutoyo, Kanagawa, Japan). For sensitization with oxazolone (Garssen et al., 1999), mice were sensitized on day 0 by applying 160 ll of an oxazolone (0.1%, w/v) solution. On day 5, mice were challenged by applying 20 ll of an oxazolone solution (0.5% in acetone) on the right auricle. For sensitization with TMA (Lauerma et al., 1997), MDI (Thorne et al., 1987), and TDI (Karol and Kramarik, 1996), mice were sensitized on day 0 by applying 160 ll of a TMA (2.5%, w/v), MDI (0.01%, w/v), and TDI (0.05%, w/v) solution, respectively. On day 5, mice were challenged by applying 20 ll of TMA (25%), MDI (0.5%), and TDI (0.5%), respectively, dissolved in acetone on the right auricle.
Hapten
h
Acetonea Ear swelling
Acetone/DBPb Ear swelling
DNFB
24 48 72
88.7 ± 18.9d 144.0 ± 26.0 125.3 ± 26.7
37.3 ± 13.3 58.7 ± 20.6* 61.3 ± 25.0
Oxazolone
24 48 72
201.3 ± 22.5 156.0 ± 19.0 124.0 ± 14.4
132.7 ± 28.2 164.0 ± 11.3 84.0 ± 7.2
Acetone/DPPb Ear swelling
Un-sensc Ear swelling
82.0 ± 13.5 160.0 ± 15.4 134.0 ± 20.2
8.3 ± 3.7 9.4 ± 4.1 10.6 ± 8.0
197.3 ± 44.2 154.7 ± 33.7 116.0 ± 40.0
19.3 ± 5.6 22.0 ± 3.4 12.0 ± 2.3
BALB/c mice were epicutaneously sensitized with a hapten dissolved in each solvent. After challenge on auricles, the ear swelling at 24, 48 and 72 h was determined. a Mice were sensitized with a hapten dissolved in acetone. b Mice were sensitized with a hapten dissolved in acetone/phthalate ester mixture. c Mice were not sensitized but challenged with hapten. This group is not included in ANOVA. d The mean value ± SEM (n = 5) as lm ear swelling. * P < 0.05 by Dunnett’s test compared with acetone–hapten group as a control group.
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Table 2 Effects of phthalate esters during sensitization with a hapten that is prone to induce a Th2-type response. Hapten
h
Sensitization with a hapten dissolved in Acetonea n
TMA
MDI
TDI
24 48 72 24 48 72 24 48 72
d
5 5 5 10 10 10 11 11 11
Acetone/DBPb
Acetone/DPPb
Un-sensc
Ear swelling
n
Ear swelling
n
Ear swelling
n
Ear swelling
54.7 ± 12.2e 48.0 ± 7.9 48.0 ± 8.5 207.3 ± 18.3 181.0 ± 14.5 133.0 ± 7.9 260.6 ± 22.9 193.6 ± 19.9 149.1 ± 10.9
5 5 5 10 10 10 10 10 10
44.0 ± 12.3 28.7 ± 5.9 18.7 ± 6.0* 77.7 ± 14.5** 98.7 ± 16.2** 92.7 ± 10.1** 186.0 ± 19.1 120.7 ± 13.2** 105.7 ± 11.0*
5 5 5 10 10 10 10 10 10
64.0 ± 12.5 37.3 ± 9.1 28.0 ± 6.7 157.0 ± 21.1 115.7 ± 11.3** 111.0 ± 8.0 224.7 ± 21.7 158.7 ± 20.3 131.7 ± 12.0
5 5 5 10 10 10 10 10 10
3.3 ± 2.6 4.4 ± 2.5 3.9 ± 2.0 45.7 ± 3.4 57.0 ± 4.0 57.0 ± 6.6 51.0 ± 5.1 41.3 ± 2.8 39.7 ± 4.8
BALB/c mice were epicutaneously sensitized with a hapten dissolved in each solvent. After challenge on auricles, the ear swelling at 24, 48 and 72 h was determined. a Mice were sensitized with a hapten dissolved in acetone. b Mice were sensitized with a hapten dissolved in acetone/phthalate ester mixture. c Mice were not sensitized but challenged with hapten. This group is not included in ANOVA. d Number of mice per group. e The mean value ± SEM as lm ear swelling. * P < 0.05. ** P < 0.01 by Dunnett’s test compared with acetone–hapten group as a control group.
Kimber, 2000; Takeshita et al., 2004; Tang et al., 1996). We examined whether or not these phthalate esters have similar adjuvant effects during sensitization to haptens, which predominantly induce Th1-type responses. In this category, two chemicals, DNFB (Hopkins et al., 2005) and oxazolone (Garssen et al., 1999; Maestroni, 2004), were tested using suboptimal doses for sensitization. In our protocol, 0.04% DNFB was the minimal dose for sensitization, which stably induced ear swelling, while 0.02% was not sufficient (data not shown). Upon sensitization with 0.04% DNFB in acetone, a significant increase in ear swelling was observed that peaked at 48 h after challenge, while ear swelling was not observed in unsensitized mice. Neither DBP nor DPP enhanced the sensitization to DNFB. The ear swelling was significantly lower in the DBP group at 48 h after challenge (Table 1). We determined that 0.3% oxazolone was the optimal dose for sensitization (data not shown). Upon sensitization with 0.1% oxazolone, significant ear swelling was observed that peaked at 24– 48 h after challenge, while ear swelling was not observed in unsensitized mice. Neither DBP nor DPP affected the sensitization to oxazolone (Table 1).
much higher in the sensitized mice. Under these conditions, the ear swelling was significantly lower in the DBP group at 24, 48 and 72 h after challenge (Table 2). The ear swelling was also significantly lower in the DPP group at 48 h after challenge (Table 2). We chose 0.05% TDI for sensitization, which is a suboptimal dose compared with published results (Karol and Kramarik,
3.3. Effects of phthalate esters on sensitization to haptens that predominantly induce a Th2-type response We next examined whether phthalate esters had adjuvant effects on sensitization to haptens that are known to induce an immune response with a Th2-type cytokine profile other than FITC. In this category, three chemicals, TMA (Dearman et al., 2000; Hopkins et al., 2005), MDI (Plitnick et al., 2005), and TDI (Plitnick et al., 2005; Sun et al., 2007), were tested using suboptimal doses for sensitization. Optimal sensitization was obtained with more than 5% of TMA, with which more than 150 lm ear swelling was obtained after challenge with 25% TMA (data not shown). We chose 2.5% TMA for sensitization and 25% TMA for elicitation. Under these conditions, moderate but significant ear swelling was observed, while no swelling was observed in unsensitized mice. Neither DBP nor DPP enhanced the sensitization to TMA. The ear swelling was significantly lower in the DBP group at 72 h after challenge (Table 2). We chose 0.01% MDI for sensitization, which is a suboptimal dose compared with published results (Thorne et al., 1987). Upon challenge, MDI produced a relatively high background in ear swelling, presumably due to irritation. However, the ear swelling was
Fig. 1. Effects of phthalate esters on sensitization to EITC, RITC and PEITC. BALB/c mice (8-week-old) were epicutaneously treated with 0.5% EITC (A and B), RITC (C and D), or PEITC (E and F) dissolved in the control vehicle, or in a 1:1 mixture of acetone and DBP (A/DBP) or acetone and DPP (A/DPP) on forelimb skin on days 0 and 7 (for EITC and RITC) or on days 0, 1, 7, and 8 (for PEITC). The control vehicles are acetone for EITC and RITC, and acetone/olive oil 3:1 (AO) for PEITC. As a negative control, mice were not sensitized (Un-sens). On day 14, mice were challenged with 0.5% of the respective hapten dissolved in A/DBP on the right ear. Ear swelling at 24 and 48 h was determined. Each bar represents the mean and SEM. for each group. For EITC, n = 8 except acetone group (n = 7); for RITC, n = 8 except A/DBP group (n = 7); for PEITC, n = 9 except A/DPP group (n = 10). Statistical significance (compared with acetone control) was analyzed by means of one-way ANOVA, followed by Dunnett’s test. *P < 0.05, **P < 0.01.
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1996). Upon challenge, TDI produced a relatively high background in ear swelling, presumably due to irritation. However, the ear swelling was much higher in the sensitized mice. Under these conditions, the ear swelling was significantly lower in the DBP group at 48 and 72 h after challenge (Table 2). DPP did not affect the sensitization. 3.4. Effects of phthalate esters on sensitization to haptens that are structurally related to FITC The results described above failed to demonstrate adjuvant effects of phthalate esters on CHS to haptens other than FITC regardless of polarization in the cytokine profiles. We then examined whether the adjuvant effects were restricted to the FITC-induced CHS models. First, we tested chemicals EITC and RITC, which are structurally related to FITC. When EITC was used as a hapten, DBP as well as DPP significantly enhanced the sensitization (Fig. 1A, B). This was also the case for RITC in that DBP and DPP enhanced the sensitization (Fig. 1C, D). 3.5. Effects of phthalate esters on sensitization to phenethyl isothiocyanate We then tested the effects of phthalate esters on sensitization with much smaller haptens with a single isothiocyanate group. Due to the low immunogenicity of PEITC, we sensitized mice four times. When PEITC was used as a hapten, DBP as well as DPP significantly enhanced the sensitization (Fig. 1E, F). The results indicated that the adjuvant effects of DBP and DPP are not restricted to FITC analogues.
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ical features of haptens such as isothiocyanate group. That is, DBP and DPP enhanced sensitization to two isothiocyanates, RITC and EITC, which are FITC analogues. Because FITC, EITC and RITC share a chemical feature, i.e., an isothiocyanate group, we then tested whether or not phthalate esters enhance skin sensitization by much smaller haptens with an isothiocyanate group. The results indicated that the adjuvant effects of DBP and DPP were not restricted to FITC and FITC analogues. Specifically, we found that DBP and DPP uncover the skin-sensitizing ability of PEITC. PEITC is known as a dietary cancer preventive (Antosiewicz et al., 2008). A significant point of this study is that some ingredients of edible plants may contribute to the development of allergies under the influence of certain chemicals. Our studies have revealed the involvement of sensory neuron activation in the adjuvant effects of phthalate esters in the FITC system (Maruyama et al., 2007a; Shiba et al., 2009). PEITC may be used as a natural hapten that shares the sensitization process with FITC. Adjuvant effects of phthalate esters are not generally observed for a wide variety of happens, and we have found only four haptens that are susceptible to the enhancing effect of phthalate esters. Further studies are needed to reveal what kind of chemical features and biological processes are the bases for the adjuvant effect of phthalate esters. In conclusion, DBP and DPP were shown to enhance skin sensitization to several mono-isothiocyanate haptens using mouse models. Skin-sensitizing activity of PEITC was uncovered when DBP or DPP was applied together with the hapten. On the other hand, DBP and DPP did not always exhibit adjuvant effect during hapten sensitization. The lack of adjuvant effects was not correlated with the tendency of helper T cell polarization.
4. Discussion Accumulating evidence suggests that phthalate esters exhibit adjuvant activity in a FITC-induced CHS mouse model (Imai et al., 2006; Shiba et al., 2009). It has not been studied whether or not phthalate esters influence skin sensitization by variety of chemical haptens. We tested several haptens that are typically used in CHS mouse models and found no enhancement in skin sensitization by DBP or DPP. It has been reported that a Th1- or Th2biased response was observed depending on the type of chemical (Dearman et al., 2003). The lack of enhanced sensitization by phthalate esters was not correlated with the tendency of helper T cell polarization. It is not straightforward to study how phthalate esters decrease the sensitization effect of haptens at present. In the case of FITC, it was easy to correlate with the enhancement of DC trafficking from skin sites to draining lymph nodes because of its fluorescence (Imai et al., 2006). In the case of non-fluorescent haptens, we have not been able to detect hapten-presenting DC in the draining lymph nodes yet. One should also consider the effects of solvent that may change the bioavailability of haptens from skin surface. Such physicochemical effects may increase or decrease the sensitization efficiency of haptens. However, it is not clear what the common features of haptens are to which sensitization efficiency was rather decreased by phthalate esters. Therefore it would be more appropriate to say that the combination of phthalate ester and antigenic hapten does not generally result in the enhanced sensitization to haptens. Two isocyanate haptens, MDI and TDI, have been shown to produce Th2-promoted responses in mice (Plitnick et al., 2005; Sun et al., 2007). The lack of enhancing effects of phthalate esters in the sensitization to these isocyanate haptens is in contrast to the enhancement of the FITC-sensitization, which is also known as a Th2-type hapten. Rather than classifying haptens based on helper T cell polarization patterns, it will be useful to focus on the chem-
Conflict of Interest The authors declare that there are no conflicts of interest. Acknowledgements This work was supported partly by a grant-in-aid (18659033, 20390041) and by research funding for the Global COE Program from the Japan Society for the Promotion of Science, and through a grant of Long-range Research Initiative (LRI) by Japan Chemical Industry Association (JCIA). References Antosiewicz, J., Ziolkowski, W., Kar, S., Powolny, A.A., Singh, S.V., 2008. Role of reactive oxygen intermediates in cellular responses to dietary cancer chemopreventive agents. Planta Med. 74, 1570–1579. Api, A.M., 2001. Toxicological profile of diethyl phthalate: a vehicle for fragrance and cosmetic ingredients. Food Chem. Toxicol. 39, 97–108. Bornehag, C.G., Sundell, J., Weschler, C.J., Sigsgaard, T., Lundgren, B., Hasselgren, M., Hagerhed-Engman, L., 2004. 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. Chowdhury, M.M., Statham, B.N., 2002. Allergic contact dermatitis from dibutyl phthalate and benzalkonium chloride in Timodine cream. Contact Dermatol. 46, 57. Dearman, R.J., Kimber, I., 2000. Role of CD4+ T helper 2-type cells in cutaneous inflammatory responses induced by fluorescein isothiocyanate. Immunology 101, 442–451. Dearman, R.J., Warbrick, E.V., Humphreys, I.R., Kimber, I., 2000. Characterization in mice of the immunological properties of five allergenic acid anhydrides. J. Appl. Toxicol. 20, 221–230. Dearman, R.J., Betts, C.J., Humphreys, N., Flanagan, B.F., Gilmour, N.J., Basketter, D.A., Kimber, I., 2003. Chemical allergy: considerations for the practical application of cytokine profiling. Toxicol. Sci. 71, 137–145. Garssen, J., Vandebriel, R.J., De Gruijl, F.R., Wolvers, D.A., Van Dijk, M., Fluitman, A., Van Loveren, H., 1999. UVB exposure-induced systemic modulation of Th1- and Th2-mediated immune responses. Immunology 97, 506–514.
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Hopkins, J.E., Naisbitt, D.J., Kitteringham, N.R., Dearman, R.J., Kimber, I., Park, B.K., 2005. Selective haptenation of cellular or extracellular protein by chemical allergens: association with cytokine polarization. Chem. Res. Toxicol. 18, 375– 381. Hu, J., Straub, J., Xiao, D., Singh, S.V., Yang, H.S., Sonenberg, N., Vatsyayan, J., 2007. Phenethyl isothiocyanate, a cancer chemopreventive constituent of cruciferous vegetables, inhibits cap-dependent translation by regulating the level and phosphorylation of 4E-BP1. Cancer Res. 67, 3569–3573. Imai, Y., Kondo, A., Iizuka, H., Maruyama, T., Kurohane, K., 2006. Effects of phthalate esters on the sensitization phase of contact hypersensitivity induced by fluorescein isothiocyanate. Clin. Exp. Allergy 36, 1462–1468. Karol, M.H., Kramarik, J.A., 1996. Phenyl isocyanate is a potent chemical sensitizer. Toxicol. Lett. 89, 139–146. Kolarik, B., Naydenov, K., Larsson, M., Bornehag, C.G., Sundell, J., 2008. The association between phthalates in dust and allergic diseases among Bulgarian children. Environ. Health Perspect. 116, 98–103. Kripke, M.L., Munn, C.G., Jeevan, A., Tang, J.M., Bucana, C., 1990. Evidence that cutaneous antigen-presenting cells migrate to regional lymph nodes during contact sensitization. J. Immunol. 145, 2833–2838. Lauerma, A.I., Fenn, B., Maibach, H.I., 1997. Trimellitic anhydride-sensitive mouse as an animal model for contact urticaria. J. Appl. Toxicol. 17, 357–360. Maestroni, G.J., 2004. Modulation of skin norepinephrine turnover by allergen sensitization: impact on contact hypersensitivity and T helper priming. J. Invest. Dermatol. 122, 119–124. Maruyama, T., Iizuka, H., Tobisawa, Y., Shiba, T., Matsuda, T., Kurohane, K., Imai, Y., 2007a. Influence of local treatments with capsaicin or allyl isothiocyanate in the sensitization phase of a fluorescein-isothiocyanate-induced contact sensitivity model. Int. Arch. Allergy Immunol. 143, 144–154. Maruyama, T., Shiba, T., Iizuka, H., Matsuda, T., Kurohane, K., Imai, Y., 2007b. Effects of phthalate esters on dendritic cell subsets and interleukin-4 production in fluorescein isothiocyanate-induced contact hypersensitivity. Microbiol. Immunol. 51, 321–326. Mi, L., Wang, X., Govind, S., Hood, B.L., Veenstra, T.D., Conrads, T.P., Saha, D.T., Goldman, R., Chung, F.L., 2007. The role of protein binding in induction of apoptosis by phenethyl isothiocyanate and sulforaphane in human non-small lung cancer cells. Cancer Res. 67, 6409–6416. Plitnick, L.M., Loveless, S.E., Ladics, G.S., Holsapple, M.P., Smialowicz, R.J., Woolhiser, M.R., Anderson, P.K., Smith, C., Selgrade, M.J., 2005. Cytokine mRNA profiles for
isocyanates with known and unknown potential to induce respiratory sensitization. Toxicology 207, 487–499. Sato, K., Imai, Y., Irimura, T., 1998. Contribution of dermal macrophage trafficking in the sensitization phase of contact hypersensitivity. J. Immunol. 161, 6835– 6844. Shiba, T., Maruyama, T., Kurohane, K., Iwasaki, Y., Watanabe, T., Imai, Y., 2009. TRPA1 and TRPV1 activation is a novel adjuvant effect mechanism in contact hypersensitivity. J. Neuroimmunol. 207, 66–74. Simon, J.C., Mosmann, T., Edelbaum, D., Schopf, E., Bergstresser, P.R., Cruz Jr., P.D., 1994. In vivo evidence that ultraviolet B-induced suppression of allergic contact sensitivity is associated with functional inactivation of Th1 cells. Photodermatol. Photoimmunol. Photomed. 10, 206–211. Sun, L.Z., Elsayed, S., Bronstad, A.M., Van Do, T., Irgens, A., Aardal, N.P., Aasen, T.B., 2007. Airway inflammation and bronchial remodelling in toluene diisocyanateexposed BALB/c mouse model. Scand. J. Immunol. 65, 118–125. Takeshita, K., Yamasaki, T., Akira, S., Gantner, F., Bacon, K.B., 2004. Essential role of MHC II-independent CD4+ T cells, IL-4 and STAT6 in contact hypersensitivity induced by fluorescein isothiocyanate in the mouse. Int. Immunol. 16, 685– 695. Tang, A., Judge, T.A., Nickoloff, B.J., Turka, L.A., 1996. Suppression of murine allergic contact dermatitis by CTLA4Ig. Tolerance induction of Th2 responses requires additional blockade of CD40-ligand. J. Immunol. 157, 117– 125. Thorne, P.S., Hillebrand, J.A., Lewis, G.R., Karol, M.H., 1987. Contact sensitivity by diisocyanates: potencies and cross-reactivities. Toxicol. Appl. Pharmacol. 87, 155–165. Vartak, P.H., Tungikar, V.B., Sharma, R.N., 1994. Comparative repellent properties of certain chemicals against mosquitoes, house flies and cockroaches using modified techniques. J. Commun. Dis. 26, 156–160. Weiss, J.M., Sleeman, J., Renkl, A.C., Dittmar, H., Termeer, C.C., Taxis, S., Howells, N., Hofmann, M., Kohler, G., Schopf, E., Ponta, H., Herrlich, P., Simon, J.C., 1997. An essential role for CD44 variant isoforms in epidermal Langerhans cell and blood dendritic cell function. J. Cell Biol. 137, 1137–1147. Xiao, D., Singh, S.V., 2007. Phenethyl isothiocyanate inhibits angiogenesis in vitro and ex vivo. Cancer Res. 67, 2239–2246. Yin, P., Kawamura, T., He, M., Vanaja, D.K., Young, C.Y., 2009. Phenethyl isothiocyanate induces cell cycle arrest and reduction of a- and b-tubulin isotypes in human prostate cancer cells. Cell Biol. Int. 33, 57–64.
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Phthalate esters reveal skin-sensitizing activity of phenethyl isothiocyanate in mice Tomoko Matsuda, Takashi Maruyama, Hiromi Iizuka, Ayumi Kondo, Takuma Tamai, Kohta Kurohane, Yasuyuki Imai * Laboratory of Microbiology and Immunology and the Global COE Program, University of Shizuoka School of Pharmaceutical Sciences, Shizuoka 422-8526, Japan
a r t i c l e
i n f o
Article history: Received 3 December 2009 Accepted 30 March 2010
Keywords: Adjuvant Contact hypersensitivity DBP Isothiocyanate Vegetable
a b s t r a c t Phenethyl isothiocyanate (PEITC) is a constituent of edible cruciferous vegetables and has received attention due to its potential cancer chemopreventive activity. Although its protein-binding activity is known, PEITC has not been studied as a contact sensitizer. We previously demonstrated that phthalate esters, including dibutyl phthalate (DBP) and di-n-propyl phthalate (DPP), enhance skin sensitization in a fluorescein isothiocyanate (FITC)-induced contact hypersensitivity mouse model. In this study, we examined whether DBP and DPP enhance skin sensitization to haptens other than FITC. During this study, we tested PEITC as a contact sensitizer. BALB/c mice were epicutaneously sensitized with a suboptimal dose of a hapten with or without a phthalate ester. Sensitization was evaluated as the ear swelling response after a challenge with the respective hapten. DBP and DPP did not enhance the sensitization to two T-helper 1type (2,4-dinitrofluorobenzene and oxazolone) or three T-helper 2-type (trimellitic anhydride, methylenediphenyl 4,40 -diisocyanate, and tolulene 2,4-diisocyanate) haptens. In contrast, DBP and DPP enhanced the sensitization to two FITC analogues (eosin 5-isothiocyanate and rhodamine B isothiocyanate) as well as to PEITC. Adjuvant effects of DBP and DPP were observed in contact hypersensitivity to haptens other than FITC, including a food ingredient PEITC. Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction Phenethyl isothiocyanate (PEITC) is a constituent of many edible cruciferous vegetables, and has received attention due to its potential cancer chemopreventive activity (Antosiewicz et al., 2008; Hu et al., 2007; Mi et al., 2007; Xiao and Singh, 2007; Yin et al., 2009). Although the protein-binding activity of PEITC has been demonstrated (Mi et al., 2007), there has been no report on it as a contact sensitizer. Phthalate esters are widely used as plasticizers for plastics such as soft polyvinyl chloride toys, vinyl floorings, wall coverings, synthetic leather and blood transfusion bags. Some phthalate esters with short alkyl chains are used for cosmetics and mosquito repellents for topical use (Api, 2001; Vartak et al., 1994). It has been reported that some phthalate esters were implicated in allergic
Abbreviations: CHS, contact hypersensitivity; DBP, dibutyl phthalate; DC, dendritic cell; DNFB, 2,4-dinitrofluorobenzene; DPP, di-n-propyl phthalate; EITC, eosin 5-isothiocyanate; MDI, methylenediphenyl 4,40 -diisocyanate; PEITC, phenethyl isothiocyanate; RITC, rhodamine B isothiocyanate; TDI, tolulene 2,4-diisocyanate; Th1, T-helper 1; Th2, T-helper 2; TMA, trimellitic anhydride. * Corresponding author. Address: Laboratory of Microbiology and Immunology, University of Shizuoka School of Pharmaceutical Sciences, 52-1 Yada, Suruga-ku, Shizuoka-shi, Shizuoka 422-8526, Japan. Tel.: +81 54 264 5716; fax: +81 54 264 5715. E-mail address: [email protected] (Y. Imai). 0278-6915/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.fct.2010.03.049
symptoms in children (Bornehag et al., 2004; Kolarik et al., 2008) as well as in adults (Chowdhury and Statham, 2002). Experimentally, dibutyl phthalate (DBP) has been empirically included in a solvent system for fluorescein isothiocyanate (FITC), which is widely used as a hapten in contact hypersensitivity (CHS) mouse models (Kripke et al., 1990). We previously demonstrated that DBP exhibits an adjuvant effect during sensitization to FITC (Sato et al., 1998). We noticed that DBP itself did not show antigenicity (Sato et al., 1998). In addition to DBP, we found that diethyl phthalate (DEP), di-n-propyl phthalate (DPP), dipentyl phthalate (DPNP), dihexyl phthalate (DHXP), and diheptyl phthalate (DHPP) also exhibit adjuvant effects in the FITC system (Imai et al., 2006; Shiba et al., 2009). We also found that DBP and DPP promote the trafficking of FITC-presenting CD11c+ dendritic cells (DC) to draining lymph nodes in the sensitization phase of FITC-induced CHS (Imai et al., 2006). However, it is not clear whether the phthalate esters exhibit adjuvant effects on sensitization to various chemicals other than FITC or there are some rules in combination with antigenic haptens. There are haptens classified as T-helper 1-type (Th1) or T-helper 2-type (Th2) haptens based on helper T cell polarization tendencies. They are defined by measuring cytokine production or immunoglobulin class switches (Hopkins et al., 2005; Plitnick et al., 2005; Sun et al., 2007). Earlier studies indicated that the Th2-type response, which can be characterized by IL-4 production, is
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required for FITC-induced CHS (Dearman and Kimber, 2000; Takeshita et al., 2004; Tang et al., 1996). Consistent with these results, we found that IL-4 production in draining lymph nodes was enhanced when DBP or DPP was included in the solvent for FITC (Maruyama et al., 2007a, b). One interesting question is whether the enhancing effect of phthalate esters on hapten sensitization is general or selective for one of the types of haptens. In the present study, we examined whether the adjuvant effects of DBP and DPP are observed on haptens other than FITC. One specific question is whether the adjuvant effects of DBP and DPP depend on the helper T cell polarization tendencies with haptens that are known to induce a Th1- or Th2-type response. Another specific question is whether or not phthalate esters enhance skin sensitization by haptens containing an isothiocyanate group. Toward this end, we first tested effects of DBP and DPP on FITC analogues, rhodamine B isothiocyanate (RITC) and eosin 5isothiocyanate (EITC), which have four aromatic rings. Second, we tested a hapten with single isothiocyanate group and single aromatic ring, PEITC. During the experiments, we found the ability of PEITC to sensitize mice epicutaneously in the presence of a phthalate ester.
For sensitization with EITC and RITC, mice were sensitized on days 0 and 7 by applying 160 ll of an EITC (0.5%, w/v) and RITC (0.5%, w/v) solution, respectively. On day 14, mice were challenged by applying 20 ll of EITC (0.5%) or RITC (0.5%) in acetone/DBP on the right auricle. RITC was dissolved in DMSO before dilution in the solvent. Therefore, the RITC solution contained a final concentration of 1% DMSO. For sensitization with PEITC, mice were sensitized on days 0, 1, 7, and 8 by applying 160 ll of PEITC (2.5%, v/v). On day 14, mice were challenged by applying 20 ll of PEITC (2.5%) in acetone/DBP on the right auricle. In this particular case, PEITC was dissolved in acetone/olive oil (3:1, v/v) during sensitization under the control condition. Ear swelling at X h is defined as follows: ((thickness of the right ear at X h) (thickness of the right ear at 0 h)) ((thickness of the left ear at X h) (thickness of the left ear at 0 h)). As a control experiment, five mice were epicutaneously treated on days 0 and 7 by applying 160 ll of acetone, acetone/DBP or acetone/DPP. On day 14, mice were challenged by applying 20 ll of acetone/DBP on the left auricle. Thickness of left ear was determined before (0 h) and after (24 and 48 h) the challenge. 2.4. Statistical analysis Statistical significance was analyzed among groups with different sensitization conditions by means of one-way ANOVA. As a post-hoc test, Dunnett’s test was performed compared with an acetone control.
3. Results 2. Materials and methods
3.1. Lack of immunogenicity of phthalate esters
2.1. Mice Specific pathogen-free female BALB/c mice were purchased from Japan SLC Inc. (Shizuoka, Japan), and were used at 8 weeks of age. They were housed at 22–24 °C and 50–60% humidity under artificial lighting conditions with a 12-h light/dark cycle. They had access to food (Oriental Yeast Co., Tokyo, Japan) and water ad libitum. Animal care and experiments were performed in accordance with the guidelines for the care and use of laboratory animals of the University of Shizuoka. 2.2. Reagents Acetone, dibutyl phthalate (DBP) (CAS No. 84-74-2, EINECS# 201-557-4), 2,4dinitrofluorobenzene (DNFB) (CAS No. 70-34-8, EINECS# 200-734-3), methylenediphenyl 4,40 -diisocyanate (MDI) (CAS No. 101-68-8, EINECS# 202-966-0), trimellitic anhydride (TMA) (CAS No. 552-30-7, EINECS# 209-008-0), and tolulene 2,4-diisocyanate (TDI) (CAS No. 584-84-9, EINECS# 209-544-5) were purchased from Wako Pure Chemicals (Osaka, Japan); di-n-propyl phthalate (DPP) (CAS No. 131-16-8, EINECS# 205-015-8) from Kanto Chemicals (Tokyo, Japan); eosin 5-isothiocyanate (EITC) (CAS No. 60520-47-0), 4-ethoxymethylene-2-phenyl-2-oxazolin-5-one (oxazolone) (CAS No. 15646-46-5, EINECS# 239-713-9), rhodamine B isothiocyanate (RITC) (CAS No. 36877-69-7, EINECS# 253-248-9), and phenethyl isothiocyanate (PEITC) (CAS No. 2257-09-2, EINECS# 218-855-5) from Sigma (St. Louis, MO, USA); and dimethyl sulfoxide (DMSO) from Nacalai Tesque (Kyoto, Japan). Purities of DNFB and PEITC were more than 99%, those of DBP and DPP were more than 98%, those of MDI and TMA were more than 97%, those of TDI and EITC were more than 95%, and that of oxazolone was more than 90%.
We have previously shown that DBP itself did not exhibit antigenicity (Sato et al., 1998). We confirmed that DBP and DPP did not behave as an antigen. Mice were epicutaneously treated with acetone, with a mixture of acetone and DBP or with acetone and DPP in the absence of any other hapten. Upon challenge with acetone/DBP on the ear, increase in the ear thickness was determined. The mean values ± SEM (in lm) were as follows: 2.0 ± 2.0 (acetone), 0.6 ± 3.2 (acetone/DBP), 8.0 ± 4.8 (acetone/DPP) at 24 h after challenge; 2.2 ± 4.8 (acetone), 1.3 ± 2.7 (acetone/ DBP), 2.7 ± 6.4 (acetone/DPP) at 48 h after challenge. These results indicate essentially no change in ear thickness. 3.2. Effects of phthalate esters on sensitization to haptens that predominantly induce a Th1-type response Our previous studies indicated that DBP and DPP had adjuvant effects on sensitization to FITC, which is known to induce an immune response with a Th2-type cytokine profile (Dearman and
Table 1 Effects of phthalate esters during sensitization with a hapten that is prone to induce a Th1-type response.
2.3. Sensitization and elicitation of a contact hypersensitivity reaction Sensitization with a hapten dissolved in Mouse forelimbs were shaved using small animal clippers one day before sensitization. Mice were epicutaneously sensitized with a suboptimal dose of a hapten dissolved in acetone or a mixture of acetone and a phthalate ester (1:1, v/v). For challenge, the hapten solution was applied on the right auricle while the vehicle was applied on the left auricle as a control. For sensitization with DNFB, the sensitization schedule was based on the published protocol except that a suboptimal hapten concentration was used (Simon et al., 1994; Weiss et al., 1997). Mice were sensitized on days 0 and 1 by applying 160 ll of DNFB solution (0.04%, w/v). On day 5, mice were challenged by applying 20 ll of a DNFB solution (0.1% in acetone) on the right auricle. The left auricle was treated with 20 ll of acetone as a control. Ear thickness was measured before (0 h) and after (24, 48, and 72 h) the challenge using a dial thickness gauge (Mitsutoyo, Kanagawa, Japan). For sensitization with oxazolone (Garssen et al., 1999), mice were sensitized on day 0 by applying 160 ll of an oxazolone (0.1%, w/v) solution. On day 5, mice were challenged by applying 20 ll of an oxazolone solution (0.5% in acetone) on the right auricle. For sensitization with TMA (Lauerma et al., 1997), MDI (Thorne et al., 1987), and TDI (Karol and Kramarik, 1996), mice were sensitized on day 0 by applying 160 ll of a TMA (2.5%, w/v), MDI (0.01%, w/v), and TDI (0.05%, w/v) solution, respectively. On day 5, mice were challenged by applying 20 ll of TMA (25%), MDI (0.5%), and TDI (0.5%), respectively, dissolved in acetone on the right auricle.
Hapten
h
Acetonea Ear swelling
Acetone/DBPb Ear swelling
DNFB
24 48 72
88.7 ± 18.9d 144.0 ± 26.0 125.3 ± 26.7
37.3 ± 13.3 58.7 ± 20.6* 61.3 ± 25.0
Oxazolone
24 48 72
201.3 ± 22.5 156.0 ± 19.0 124.0 ± 14.4
132.7 ± 28.2 164.0 ± 11.3 84.0 ± 7.2
Acetone/DPPb Ear swelling
Un-sensc Ear swelling
82.0 ± 13.5 160.0 ± 15.4 134.0 ± 20.2
8.3 ± 3.7 9.4 ± 4.1 10.6 ± 8.0
197.3 ± 44.2 154.7 ± 33.7 116.0 ± 40.0
19.3 ± 5.6 22.0 ± 3.4 12.0 ± 2.3
BALB/c mice were epicutaneously sensitized with a hapten dissolved in each solvent. After challenge on auricles, the ear swelling at 24, 48 and 72 h was determined. a Mice were sensitized with a hapten dissolved in acetone. b Mice were sensitized with a hapten dissolved in acetone/phthalate ester mixture. c Mice were not sensitized but challenged with hapten. This group is not included in ANOVA. d The mean value ± SEM (n = 5) as lm ear swelling. * P < 0.05 by Dunnett’s test compared with acetone–hapten group as a control group.
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Table 2 Effects of phthalate esters during sensitization with a hapten that is prone to induce a Th2-type response. Hapten
h
Sensitization with a hapten dissolved in Acetonea n
TMA
MDI
TDI
24 48 72 24 48 72 24 48 72
d
5 5 5 10 10 10 11 11 11
Acetone/DBPb
Acetone/DPPb
Un-sensc
Ear swelling
n
Ear swelling
n
Ear swelling
n
Ear swelling
54.7 ± 12.2e 48.0 ± 7.9 48.0 ± 8.5 207.3 ± 18.3 181.0 ± 14.5 133.0 ± 7.9 260.6 ± 22.9 193.6 ± 19.9 149.1 ± 10.9
5 5 5 10 10 10 10 10 10
44.0 ± 12.3 28.7 ± 5.9 18.7 ± 6.0* 77.7 ± 14.5** 98.7 ± 16.2** 92.7 ± 10.1** 186.0 ± 19.1 120.7 ± 13.2** 105.7 ± 11.0*
5 5 5 10 10 10 10 10 10
64.0 ± 12.5 37.3 ± 9.1 28.0 ± 6.7 157.0 ± 21.1 115.7 ± 11.3** 111.0 ± 8.0 224.7 ± 21.7 158.7 ± 20.3 131.7 ± 12.0
5 5 5 10 10 10 10 10 10
3.3 ± 2.6 4.4 ± 2.5 3.9 ± 2.0 45.7 ± 3.4 57.0 ± 4.0 57.0 ± 6.6 51.0 ± 5.1 41.3 ± 2.8 39.7 ± 4.8
BALB/c mice were epicutaneously sensitized with a hapten dissolved in each solvent. After challenge on auricles, the ear swelling at 24, 48 and 72 h was determined. a Mice were sensitized with a hapten dissolved in acetone. b Mice were sensitized with a hapten dissolved in acetone/phthalate ester mixture. c Mice were not sensitized but challenged with hapten. This group is not included in ANOVA. d Number of mice per group. e The mean value ± SEM as lm ear swelling. * P < 0.05. ** P < 0.01 by Dunnett’s test compared with acetone–hapten group as a control group.
Kimber, 2000; Takeshita et al., 2004; Tang et al., 1996). We examined whether or not these phthalate esters have similar adjuvant effects during sensitization to haptens, which predominantly induce Th1-type responses. In this category, two chemicals, DNFB (Hopkins et al., 2005) and oxazolone (Garssen et al., 1999; Maestroni, 2004), were tested using suboptimal doses for sensitization. In our protocol, 0.04% DNFB was the minimal dose for sensitization, which stably induced ear swelling, while 0.02% was not sufficient (data not shown). Upon sensitization with 0.04% DNFB in acetone, a significant increase in ear swelling was observed that peaked at 48 h after challenge, while ear swelling was not observed in unsensitized mice. Neither DBP nor DPP enhanced the sensitization to DNFB. The ear swelling was significantly lower in the DBP group at 48 h after challenge (Table 1). We determined that 0.3% oxazolone was the optimal dose for sensitization (data not shown). Upon sensitization with 0.1% oxazolone, significant ear swelling was observed that peaked at 24– 48 h after challenge, while ear swelling was not observed in unsensitized mice. Neither DBP nor DPP affected the sensitization to oxazolone (Table 1).
much higher in the sensitized mice. Under these conditions, the ear swelling was significantly lower in the DBP group at 24, 48 and 72 h after challenge (Table 2). The ear swelling was also significantly lower in the DPP group at 48 h after challenge (Table 2). We chose 0.05% TDI for sensitization, which is a suboptimal dose compared with published results (Karol and Kramarik,
3.3. Effects of phthalate esters on sensitization to haptens that predominantly induce a Th2-type response We next examined whether phthalate esters had adjuvant effects on sensitization to haptens that are known to induce an immune response with a Th2-type cytokine profile other than FITC. In this category, three chemicals, TMA (Dearman et al., 2000; Hopkins et al., 2005), MDI (Plitnick et al., 2005), and TDI (Plitnick et al., 2005; Sun et al., 2007), were tested using suboptimal doses for sensitization. Optimal sensitization was obtained with more than 5% of TMA, with which more than 150 lm ear swelling was obtained after challenge with 25% TMA (data not shown). We chose 2.5% TMA for sensitization and 25% TMA for elicitation. Under these conditions, moderate but significant ear swelling was observed, while no swelling was observed in unsensitized mice. Neither DBP nor DPP enhanced the sensitization to TMA. The ear swelling was significantly lower in the DBP group at 72 h after challenge (Table 2). We chose 0.01% MDI for sensitization, which is a suboptimal dose compared with published results (Thorne et al., 1987). Upon challenge, MDI produced a relatively high background in ear swelling, presumably due to irritation. However, the ear swelling was
Fig. 1. Effects of phthalate esters on sensitization to EITC, RITC and PEITC. BALB/c mice (8-week-old) were epicutaneously treated with 0.5% EITC (A and B), RITC (C and D), or PEITC (E and F) dissolved in the control vehicle, or in a 1:1 mixture of acetone and DBP (A/DBP) or acetone and DPP (A/DPP) on forelimb skin on days 0 and 7 (for EITC and RITC) or on days 0, 1, 7, and 8 (for PEITC). The control vehicles are acetone for EITC and RITC, and acetone/olive oil 3:1 (AO) for PEITC. As a negative control, mice were not sensitized (Un-sens). On day 14, mice were challenged with 0.5% of the respective hapten dissolved in A/DBP on the right ear. Ear swelling at 24 and 48 h was determined. Each bar represents the mean and SEM. for each group. For EITC, n = 8 except acetone group (n = 7); for RITC, n = 8 except A/DBP group (n = 7); for PEITC, n = 9 except A/DPP group (n = 10). Statistical significance (compared with acetone control) was analyzed by means of one-way ANOVA, followed by Dunnett’s test. *P < 0.05, **P < 0.01.
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1996). Upon challenge, TDI produced a relatively high background in ear swelling, presumably due to irritation. However, the ear swelling was much higher in the sensitized mice. Under these conditions, the ear swelling was significantly lower in the DBP group at 48 and 72 h after challenge (Table 2). DPP did not affect the sensitization. 3.4. Effects of phthalate esters on sensitization to haptens that are structurally related to FITC The results described above failed to demonstrate adjuvant effects of phthalate esters on CHS to haptens other than FITC regardless of polarization in the cytokine profiles. We then examined whether the adjuvant effects were restricted to the FITC-induced CHS models. First, we tested chemicals EITC and RITC, which are structurally related to FITC. When EITC was used as a hapten, DBP as well as DPP significantly enhanced the sensitization (Fig. 1A, B). This was also the case for RITC in that DBP and DPP enhanced the sensitization (Fig. 1C, D). 3.5. Effects of phthalate esters on sensitization to phenethyl isothiocyanate We then tested the effects of phthalate esters on sensitization with much smaller haptens with a single isothiocyanate group. Due to the low immunogenicity of PEITC, we sensitized mice four times. When PEITC was used as a hapten, DBP as well as DPP significantly enhanced the sensitization (Fig. 1E, F). The results indicated that the adjuvant effects of DBP and DPP are not restricted to FITC analogues.
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ical features of haptens such as isothiocyanate group. That is, DBP and DPP enhanced sensitization to two isothiocyanates, RITC and EITC, which are FITC analogues. Because FITC, EITC and RITC share a chemical feature, i.e., an isothiocyanate group, we then tested whether or not phthalate esters enhance skin sensitization by much smaller haptens with an isothiocyanate group. The results indicated that the adjuvant effects of DBP and DPP were not restricted to FITC and FITC analogues. Specifically, we found that DBP and DPP uncover the skin-sensitizing ability of PEITC. PEITC is known as a dietary cancer preventive (Antosiewicz et al., 2008). A significant point of this study is that some ingredients of edible plants may contribute to the development of allergies under the influence of certain chemicals. Our studies have revealed the involvement of sensory neuron activation in the adjuvant effects of phthalate esters in the FITC system (Maruyama et al., 2007a; Shiba et al., 2009). PEITC may be used as a natural hapten that shares the sensitization process with FITC. Adjuvant effects of phthalate esters are not generally observed for a wide variety of happens, and we have found only four haptens that are susceptible to the enhancing effect of phthalate esters. Further studies are needed to reveal what kind of chemical features and biological processes are the bases for the adjuvant effect of phthalate esters. In conclusion, DBP and DPP were shown to enhance skin sensitization to several mono-isothiocyanate haptens using mouse models. Skin-sensitizing activity of PEITC was uncovered when DBP or DPP was applied together with the hapten. On the other hand, DBP and DPP did not always exhibit adjuvant effect during hapten sensitization. The lack of adjuvant effects was not correlated with the tendency of helper T cell polarization.
4. Discussion Accumulating evidence suggests that phthalate esters exhibit adjuvant activity in a FITC-induced CHS mouse model (Imai et al., 2006; Shiba et al., 2009). It has not been studied whether or not phthalate esters influence skin sensitization by variety of chemical haptens. We tested several haptens that are typically used in CHS mouse models and found no enhancement in skin sensitization by DBP or DPP. It has been reported that a Th1- or Th2biased response was observed depending on the type of chemical (Dearman et al., 2003). The lack of enhanced sensitization by phthalate esters was not correlated with the tendency of helper T cell polarization. It is not straightforward to study how phthalate esters decrease the sensitization effect of haptens at present. In the case of FITC, it was easy to correlate with the enhancement of DC trafficking from skin sites to draining lymph nodes because of its fluorescence (Imai et al., 2006). In the case of non-fluorescent haptens, we have not been able to detect hapten-presenting DC in the draining lymph nodes yet. One should also consider the effects of solvent that may change the bioavailability of haptens from skin surface. Such physicochemical effects may increase or decrease the sensitization efficiency of haptens. However, it is not clear what the common features of haptens are to which sensitization efficiency was rather decreased by phthalate esters. Therefore it would be more appropriate to say that the combination of phthalate ester and antigenic hapten does not generally result in the enhanced sensitization to haptens. Two isocyanate haptens, MDI and TDI, have been shown to produce Th2-promoted responses in mice (Plitnick et al., 2005; Sun et al., 2007). The lack of enhancing effects of phthalate esters in the sensitization to these isocyanate haptens is in contrast to the enhancement of the FITC-sensitization, which is also known as a Th2-type hapten. Rather than classifying haptens based on helper T cell polarization patterns, it will be useful to focus on the chem-
Conflict of Interest The authors declare that there are no conflicts of interest. Acknowledgements This work was supported partly by a grant-in-aid (18659033, 20390041) and by research funding for the Global COE Program from the Japan Society for the Promotion of Science, and through a grant of Long-range Research Initiative (LRI) by Japan Chemical Industry Association (JCIA). References Antosiewicz, J., Ziolkowski, W., Kar, S., Powolny, A.A., Singh, S.V., 2008. Role of reactive oxygen intermediates in cellular responses to dietary cancer chemopreventive agents. Planta Med. 74, 1570–1579. Api, A.M., 2001. Toxicological profile of diethyl phthalate: a vehicle for fragrance and cosmetic ingredients. Food Chem. Toxicol. 39, 97–108. Bornehag, C.G., Sundell, J., Weschler, C.J., Sigsgaard, T., Lundgren, B., Hasselgren, M., Hagerhed-Engman, L., 2004. 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. Chowdhury, M.M., Statham, B.N., 2002. Allergic contact dermatitis from dibutyl phthalate and benzalkonium chloride in Timodine cream. Contact Dermatol. 46, 57. Dearman, R.J., Kimber, I., 2000. Role of CD4+ T helper 2-type cells in cutaneous inflammatory responses induced by fluorescein isothiocyanate. Immunology 101, 442–451. Dearman, R.J., Warbrick, E.V., Humphreys, I.R., Kimber, I., 2000. Characterization in mice of the immunological properties of five allergenic acid anhydrides. J. Appl. Toxicol. 20, 221–230. Dearman, R.J., Betts, C.J., Humphreys, N., Flanagan, B.F., Gilmour, N.J., Basketter, D.A., Kimber, I., 2003. Chemical allergy: considerations for the practical application of cytokine profiling. Toxicol. Sci. 71, 137–145. Garssen, J., Vandebriel, R.J., De Gruijl, F.R., Wolvers, D.A., Van Dijk, M., Fluitman, A., Van Loveren, H., 1999. UVB exposure-induced systemic modulation of Th1- and Th2-mediated immune responses. Immunology 97, 506–514.
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