Effects of styrene monomer on a mouse model of atopic dermatitis

Abstract Aim: Styrene monomer (SM) is a basic chemical used as a raw material for polystyrene and unsaturated polyester resins and in the production of synthetic resins, synthetic rubbers, paints, and adhesives. To date, it is unclear whether SM is associated with the aggravation of atopic dermatitis. The aim was to investigate the effects of SM on atopic dermatitis-like skin lesions induced by mite allergen in NC/Nga mice. Methods: Male mice were injected intradermally with mite allergen on their right ears. In the presence of an allergen, SM (3.5 or 350 μg/animal/week) was administered by intraperitoneal injection. We evaluated clinical scores, ear thickening, histologic findings, and the protein expressions of cytokines and chemokines. Results: Macroscopic and microscopic examinations demonstrated that exposure to SM at a dose of 3.5 μg caused an exacerbation of atopic dermatitis-like skin lesions related to mite allergen. These changes were consistent with the level of histamine in the ear tissue as an overall trend. In contrast, 350-μg SM did not show significant enhancement effects. Conclusion: These results indicate that SM exacerbated atopic dermatitis-like skin lesions at hundred-fold lower levels than the level that causes no observed adverse effects as determined by histologic changes in rodent livers. SM could be at least partly responsible for the recent increase in atopic dermatitis. Impact statement Styrene monomer (SM) is classified as an International Agency for Research on Cancer group 2B carcinogen and includes neurotoxicity and respiratory disorders. However, the effects of SM as a chemical substance on existing allergic pathophysiology have not been elucidated yet. This study demonstrated that SM exacerbated murine atopic dermatitis-like skin lesions at hundred-fold lower levels than the level that causes no observed adverse effects as determined by histologic changes in rodent livers, which was concomitant with the local level of histamine. These data hasten a need for comprehensive research to clarify the chemical pollutants’ effects of doses much lower than NOAEL on vulnerable pathophysiologies such as allergy/atopy.


Introduction
Allergic diseases, such as atopic dermatitis, asthma, and hay fever, affect more than 20% of the global population and are among the most common chronic diseases, along with lifestyle-related diseases [1]. Over the past few decades, the number of affected individuals has increased rapidly in developed and developing countries [1]. One possible cause of this increase is exposure to environmental pollution, as pollutants can interact with the immune system to increase the risks of the development or exacerbation of allergies and atopy [2].
Styrene monomer (SM) is a basic chemical used as a raw material for polystyrene and unsaturated polyester resins as well as in the production of synthetic resins, synthetic rubbers, paints, and adhesives [3]. Polystyrene resins are found in plastic products in household appliances (e.g. color televisions), office equipment (e.g. personal computers and printers), and other goods (e.g. toys). Unsaturated polyester resins are used for resin tanks, bathtubs, and floor linings.
SM is classified as group 2B by the International Agency for Research on Cancer (2002). Group 2B health hazards include neurotoxicity and respiratory disorders [3,4]. McGague et al. [5] epidemiologically demonstrated that long-term exposure to styrene could cause asthma-like conditions, such as reduced fractional exhaled nitric oxide and wheezing. In addition, we demonstrated in an animal experiment that the nanoparticles of polystyrene, a polymer of SMs, exacerbated atopic dermatitis [6]. However, the effects of SM as a chemical substance on allergic pathophysiology have not been elucidated.
The objective of our study was to experimentally examine the effects of SM on allergic conditions in the skin.

Animals
Five-week-old male atopic-prone NC/Nga mice were purchased from Charles River Japan Inc. (Kanagawa, Japan). Experiments were performed when the mice were 6-weeks old (weighing 24-27 g). Mice were given sterile distilled water and a commercial diet (CE-2; Japan Clea Co., Tokyo, Japan) ad libitum and housed in an animal facility maintained at 24 C-26 C with a 12-h light/dark cycle under conventional conditions. The Institutional Review Board of the International University of Kyoto approved all animal studies. Animals were treated humanely and with regard to alleviation of suffering.

Chemicals
SM was purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan) and injected intraperitoneally at 3.5 lg (L-SM) or 350 lg (H-SM), dissolved in 0.1 ml olive oil (Wako Pure Chemical Industries), into two groups on days À6, 1, 8, 15 and 22 (a total of five times).

Experimental design
The mice were divided into six groups as follows: (1) vehicle (control group), (2) (L-and H-) SM, (3) mite extract (Dermatophagoides pteronyssinus [Dp]; Cosmo Bio LSL, Tokyo, Japan), and (4) (L-and H-) SM þ Dp. As shown in Figure 1, the animals in the experimental groups were exposed to the allergen by subcutaneous injection of 5 mg of Dp dissolved in 10 mL of saline to the ventral side of the right ear at 2 or 3 days per week (a total of 11 times) under anesthesia with 4% isoflurane (Abboto, Tokyo, Japan). All animals were sacrificed on the last day of the experiment (day 24).

Evaluation of skin disease and ear thickness
We determined clinical atopic dermatitis-like skin lesion (ADSL) scores 48 h after each subcutaneous injection (n ¼ 12 to 15 in each group), using a modification of the previously described method [6]. We scored symptoms of skin dryness and dander as follows: 0, no symptoms; 0.5, mild symptoms; and 1, moderate symptoms. The presence of erythema was scored as follows: 0, no symptoms; 0.5, very mild symptoms; and 1, mild symptoms. Crusting and erosion were scored as follows: 0, no symptoms; 0.5, very mild symptoms; 1, mild symptoms; 2, moderate symptoms; 3, severe symptoms; and 4, most severe symptoms. To measure ear thickness, we used a gauge (Ozaki Mfg., Tokyo, Japan). The total clinical severity score was defined as the sum of the individual scores of the measured symptoms.

Histopathological evaluation
We fixed the ears in 10% phosphate-buffered formalin (pH 7.4). Tissue sections were then embedded in paraffin and cut into 4-lm-thick slices (n ¼ 4 or 5 in each group). To evaluate eosinophil and lymphocyte infiltration in the ear, histological specimens were stained with hematoxylin and eosin. Toluidine blue staining was used to evaluate mast cells, and Luna's stain was used to evaluate eosinophils. We examined the entire area of each specimen and used an AdvanCam-U3 G/microscope (Advan Visions, Tokyo, Japan) to assess histological changes (n ¼ 4 to 5 in each group).

Quantitation of cytokines and chemokines in ear tissues
The ears of the mice were removed after sacrifice and then homogenized and centrifuged as previously described [6]. After centrifugation, supernatants were stored at À80 C until assay for cytokine profiles and other parameters (n ¼ 8 to 10 in each group). We used enzyme-linked immunosorbent assays (ELISA) to determine the cytokine and chemokine protein levels in the supernatants, following the respective manufacturer protocols. Interleukin (IL)-4 was measured using an ELISA kit from GE Healthcare Japan. IL-13, and interferon (IFN)-c were measured using an ELISA kit from Thermo Scientific (Rockford, IL, USA). Monocyte chemotactic protein (MCP)-1 and macrophage inflammatory protein (MIP)-1a were measured using an ELISA kit from R&D Systems Inc. (Minneapolis, MN, USA).

Quantification of histamine levels in ear tissues/sera
Blood was sampled by cardiac puncture (n ¼ 12 to 15 in each group). After centrifuge, serum was collected and stored at -80 C until use. We used ELISA to determine the histamine level in the ear tissue homogenates (as prepared in 2. 6.: n ¼ 8-10 in each group) and sera (n ¼ 12 to 15 in each group) by using an ELISA kit from Enzo Life Sciences (NY, USA).

Quantification of total and allergen-specific immunoglobulin in sera
Total immunoglobulin (Ig) E (IgE) antibodies in the sera (as prepared in 2.7.) were measured using a mouse IgE ELISA kit (Shibayagi Co., Shibukawa, Japan; n ¼ 12 to 15 in each group).

Statistical analysis
Data were presented as mean ± standard error (SE) for each experimental group (n ¼ 4-15). The differences were analyzed using the Tukey test or Tukey-Kramer test (Excel Statistics, Social Survey Research Information Co. Ltd., Tokyo, Japan). A p value of < 0.05 was considered statistically significant.

Sm enhances the symptoms of ADSLs
To evaluate the effects of SM on ADSLs induced by Dp, we examined clinical scores (Figure 2(A)) and ear thickening (Figure 2(B)). As compared with no treatment, treatment with Dp increased clinical scores, including dryness, eruption, wound, edema, and ear thickening, from day 9. Clinical scores and ear thickening were significantly greater in the SM þ Dp groups than in the corresponding SM groups. Also, they were significantly greater in the L-SM þ Dp group than in the Dp group (from days 18, 21, 23). Hematoxylin and eosin staining ( Figure 3) and Toluidine blue staining ( Figure 4) demonstrated that in the Dp-treated groups, the dermis and subcutaneous tissue were infiltrated with leukocytes, including eosinophils, lymphocytes, and mast cells. In particular, the infiltration was worsened in the presence of L-SM, although there was no statistical change. Additionally, we confirmed macroscopic features demonstrating that SM aggravated AD-like skin lesions related to Dp (Supplementary Figure 1).

Effects of SM on the protein expression of Th cytokines and chemokines related to Dp in the skin
To elucidate the impact of SM on local inflammatory status, we examined the protein levels of cytokines and chemokines related to allergy in the homogenates of the ears ( Figure 5). As compared with the saline-treated groups, treatment with Dp increased the expressions of IL-4, IL-13, MCP-1, and MIP-1a (p < 0.05 for Dp vs. vehicle in IL-4, MCP-1, and MIP-1a; p < 0.01 for Dp vs. vehicle in IL-13, L-SM þ Dp vs. L-SM in all molecules, and H-SM þ Dp vs. H-SM in IL-13). Exposure to L-SM plus Dp slightly increased the expressions of these molecules as compared with Dp. In contrast, H-SM significantly decreased IL-4, MCP-1, and MIP-1a, in the presence of allergen (p < 0.05). The IFN-c values were comparable among the experimental groups.

Effects of SM on histamine synthesis/release
We measured the serum and local (ear) histamine levels to further investigate the effects of SM on allergic physiology in the atopic dermatitis model. We found that blood histamine levels were comparable among the experimental groups (Supplementary Figure 2), but the concentration of local histamine was significantly higher in the SM þ Dp groups than in the vehicle, SM or Dp group ( Figure 6).

Effects of SM on IgE production in the sera
To evaluate the adjuvant property of SM for Ig production, we measured the total IgE serum levels. Intradermal injection of Dp significantly increased the levels of total IgE. The value was greater (but nonsignificant) in the H-SM þ Dp group than in the Dp group (Figure 7).

Discussion
Some epidemiological studies have shown a positive association between styrene exposure and asthma [5,7,8], suggesting that styrene has adjuvant properties on allergic response and pathophysiology. Other studies have reported skin problems among workers in fiberglass-reinforced plastic factories, who often use styrene in the workplace [9]. In the present in vivo study, repetitive exposure to styrene significantly exacerbated Dp-evoked allergic skin inflammation, which, at least in part, provides biological evidence supporting the aforementioned human studies.
Using the same protocol as described above, we have previously shown that polystyrene nanoparticles exacerbate allergic skin inflammation [6]. That study demonstrated that three different-sized polystyrene nanoparticles (25, 50, and 100 nm) aggravated ADSLs related to mite allergen, paralleled by the local protein levels of cytokines/chemokines such as IL-4, MCP-1, MIP-1a, and MIP-1b. Furthermore, these effects increased as the polystyrene nanoparticle size decreased, suggesting that exposure to polystyrene nanoparticles under conditions of skin barrier defect/dysfunction can exacerbate ADSLs related to mite allergens in a size-  dependent manner, supporting the concept of 'hazardous nanoparticles'. Thus, it is possible that polystyrene nanoparticles exacerbate allergic skin inflammation through both particle size and the characteristics of styrene. The precise pathways of styrene's exacerbatory effects on atopic dermatitis in the mouse model remain unclear. Confounding pathophysiology results, such as clinical scores and ear thickness, were not precisely concomitant with the local expression of cytokines and chemokines related to allergy/inflammation, although these parameters showed moderate tendencies. These discrepant data may arise from differences in peak points between the pathology and cytokine production/release in the ears. Interestingly, aggravation of dermatitis, particularly in the L-SM þ Dp group, closely paralleled infiltration and activation, such as degranulation of mast cells in the dermis of the ear, followed by local histamine levels. Thus, this chemical may predominantly promote inappropriate mast cell expansion and activation. Future studies are needed to clarify the mechanism.
With regard to the dosage of SM applied in the present study, the doses of H-SM and L-SM were 350 and 3.5 lg, respectively. On the basis of previous reports, the smallest no-observed-adverse-effect level (NOAEL) of SM is 12 mg/kg in rats. This dose can be extrapolated to approximately 1.93 mg/kg in humans. In turn, 3.5 lg is equivalent to 0.12 mg/kg in mice, which corresponds to 0.01 mg/kg in humans. Accordingly, the L-SM dose we applied in the present study was 1/100th of the NOAEL of this chemical, whereas the H-SM dose was comparable with the NOAEL. In the present study, L-SM significantly exacerbated ADSL scores, whereas H-SM did not but rather tended to restrain   them (an inverted U-shaped dose-response pattern). Similar results have been shown for the effects of endocrine-disrupting chemicals. Octylphenol and 4-n-nonylphenol are important alkylphenolic compounds used widely in industry and appear to possess intrinsic estrogenic activity [10]. Exposure to these environmental chemicals reportedly exhibits an inverted U-shaped dose-response in the number of unshelled embryos of snails [11]. In addition, bisphenol A, an environmental endocrine disruptor, dose-dependently increases the mRNA level for aryl hydrocarbon receptor in mouse gonads at a dose of 0.02-200 lg/kg/day but decreases it at higher doses [12]. Furthermore, we have observed the phenomenon in di-2-ethylhexyl phthalate-enhanced atopic dermatitis in mice [13]. In view of predisposing/sensitive pathological states, in any case, probably, the NOAELs of chemical pollutants are not applicable to subjects with allergic/atopic susceptibilities. There is a need for comprehensive research to clarify the effects of doses much lower than NOAEL on vulnerable pathophysiologies such as allergy/atopy.
In conclusion, a dose of SM much lower than NOAEL aggravated the murine model of ADSL related to mite allergen. SM may be one of the deteriorating environmental factors for subjects with allergic disorders including atopic dermatitis.