Benzo[a]pyrene aggravates atopic dermatitis-like skin lesions in mice

Abstract

Background: Benzo[a]pyrene (BaP) affects the immune system and causes mutagenic and carcinogenic effects. Purpose: We aimed to evaluate the effects of systemic exposure to BaP on mite allergen–induced atopic dermatitis (AD)–like skin lesions in mice. Methods: Mite allergen (Dermatophagoides pteronyssinus; Dp) was injected intradermally into the right ears of NC/Nga male mice on eight occasions every 2–3 days. Benzo[a]pyrene was administered intraperitoneally in the equivalent doses of 0, 2, 20, 200, or 2000 μg/kg/day, once a week on four occasions. Results: AD-like skin inflammation related to mite allergen worsened by BaP exposure at 2, 20 µg/kg/day doses; this was in parallel with eosinophil and mast cell infiltration and mast cell degranulation. A trend was also observed toward increased proinflammatory molecule expression, including macrophage inflammatory protein-1 alpha, interleukin (IL)-4, IL-13, and IL-18, in the ear tissue. However, 200 or 2000 µg/kg/day BaP attenuated the enhancing effects. In the regional lymph nodes, 2 µg/kg/day BaP with Dp enhanced antigen-presenting cell and T cell activation compared with Dp alone. Conclusions: This suggests that BaP exposure can aggravate Dp-induced AD-like skin lesions through T_H2-biased responses in the inflamed sites and the activation of regional lymph nodes. Therefore, BaP may be responsible for the recent increase in AD incidence.

Keywords

Benzo[a]pyrene atopic dermatitis environmental pollutant

Introduction

Atopic dermatitis (AD), the most common chronic inflammatory skin disease, has a lifetime prevalence of up to 20%. AD substantially affects the quality of life. Environmental factors, including environmental chemicals, are involved in the increase in allergic diseases such as AD. Previous studies have shown that oral and peritoneal exposure to phthalates, including di-(2-ethylhexyl) phthalate and diisononyl phthalate, enhance mite allergen-induced AD-like skin lesions in mice.^1–3

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous in indoor and outdoor environments; hence, humans can be easily exposed. Outdoor PAH sources are as follows: vehicle emissions,⁴ forest fires, volcanoes, and industrial processes,⁵ such as aluminum smelting and coke production. Indoor sources include cooking,^6,7 heating,⁸ smoking,⁹ firewood burning,¹⁰ candle burning,¹¹ and incense burning,⁷ which form because of incomplete combustion. Therefore, humans are exposed to PAHs through several routes, of which food has been recognized as the main route of intake of PAHs.

Benzo[a]pyrene (BaP), a PAH, is a ubiquitous environmental pollutant. Benzo[a]pyrene is produced from diesel exhaust particles, cigarette smoke, industrial waste, and food products. Benzo[a]pyrene is known to be mutagenic and carcinogenic as well as immunotoxic. An intranasal administration of BaP was shown to aggravate cedar pollen-induced allergic rhinitis in a guinea pig model.¹² In ex vivo studies, intranasal and oral exposure to BaP with allergen enhanced allergen-specific immunoglobulin (Ig) E and cytokine production in allergen-restimulated splenocytes.^13,14 Chowdhury et al. reported that BaP exposure increases CD86 expression in murine bone marrow‐derived antigen‐presenting cells.¹⁵ We showed that intratracheal exposure to BaP could partly exacerbate allergic airway inflammation via the development of T_H2-type immune responses and activation/proliferation of mediastinal lymph node (LN) cells.¹⁶ More recently, our study showed that lactational exposure to BaP exerts minor effects on the allergic and nonallergic immune responses in the offspring.¹⁷ Moreover, Wang et al. showed that BaP with mite allergen facilitates airway hyperresponsiveness and lung inflammation in a murine model of allergic asthma.¹⁸ A recent study showed that the oral administration of BaP before the final allergen challenge exacerbates hapten-induced AD-like skin lesions in mice.¹⁹ Hong et al. reported that the expression of aryl hydrocarbon receptor (AhR) and AhR nuclear translocator were elevated in the skin of patients with AD compared with healthy controls.²⁰ However, whether repeated exposure to BaP can contribute to the exacerbation of AD remains unclear. Therefore, we investigated the effects of BaP exposure on allergen-induced AD-like skin lesions in a mouse model.

Materials and methods

Animals

Seven-week-old SPF NC/Nga male mice (weight, 22–25g) were purchased from Charles River Japan (Osaka, Japan) and maintained in conventional conditions for 1 week. All mice had access to food and water ad libitum and were housed in an animal facility maintained at a temperature of 22°C–26°C and humidity of 40%–69% with a 12 h light/dark cycle (7:00–19:00). All study procedures were approved by the Animal Care and Use Committee of the National Institute for Environmental Studies. The study followed the guidelines for the Care and Use of Laboratory Animals of the National Institute for Environmental Studies. All animals were treated humanely, and suffering, if any, was alleviated immediately.

Study protocol

Mice were divided into six experimental groups (11 animals/group). The Dp group was treated with mite allergen extract (Dermatophagoides pteronyssinus; Dp, Cosmo Bio LSL, Tokyo, Japan) dissolved in saline. Dp 5 µg (10 µL) or saline was administered intradermally on the ventral side of the right ears on days 1, 3, 5, 8, 10, 12, 15, and 17 under anesthesia using 4% halothane (Takeda Chemical Industries, Ltd., Osaka, Japan). Benzo[a]pyrene dissolved in olive oil was injected intraperitoneally on days -5, 2, 9, and 16 (0.1 mL). A total of five doses of BaP were administered (equivalent doses of 0, 2, 20, 200, 2000 μg/kg/day) in a 10-fold dilution series from the 10-fold concentration based on the no-observed-adverse-effect level (NOAEL, 0.21 mg/kg/day).²¹ NOAEL was calculated based on the noncancer effect by oral exposure to BaP in rats. Twenty-four hours after the intradermal administration, we measured ear thickness using a caliper (OZAKI MFG, Osaka, Japan) and evaluated clinical scores using the following characteristics: skin dryness, eruption, edema, and wound, which were graded from 0 to 3 (no symptoms, 0; mild, one; moderate, two; and severe, 3). The final clinical scores were considered as the sum of these values.

Histological evaluation

The right ears of the mice were removed 24 h after the final intradermal injection (day 18), fixed in 10% neutral buffered formalin solution (pH 7.4), and embedded in paraffin (5 animals/group). Embedded blocks were cut into 3-µm sections using a rotary microtome and stained with hematoxylin and eosin or toluidine blue (pH 4.0).

Serum collection

Blood was collected by cardiac puncture 24 h after the final intradermal injection and centrifuged at 1200 ×g for 15 min at 4°C (5 animals/group). The separated serum samples were stored at −80°C until assayed.

Protein lysate preparation from ear tissue

The right ears of mice were removed 24 h after the final intradermal injection and stored at −80°C until use (5 animals/group). The frozen tissue samples were subsequently homogenized using PHYSCOTORON NS-310E (MICROTEC Co., Ltd., Chiba, Japan). The homogenates were centrifuged at 31,000 rpm for 1 h at 4°C. The supernatant fraction was separated and stored at −80°C until further use.

Submandibular LN cell preparation and activation analysis

In a separate experiment, single-cell suspensions were prepared from submandibular LNs (6 animals/group) by straining cells via a sterile stainless wire mesh into phosphate-buffered saline (PBS) (−) (pH 7.4; Takara Bio Inc., Shiga, Japan) 24 h after the final intradermal injection. LN cells were collected after centrifugation at 400 ×g for 5 min at 20°C, followed by red blood cell lysis using ammonium chloride. After a PBS (−) wash, cells were resuspended in R10 culture medium that consisted of Gibco RPMI 1640 medium (Thermo Fisher Scientific) supplemented with 10% of heat-inactivated fetal bovine serum (MP Biomedicals Inc., Eschwege, Germany), 100 U/mL of penicillin, 100 μg/mL of streptomycin (Sigma-Aldrich), and 50 μM of 2-mercaptoethanol (Thermo Fisher Scientific). Finally, LN cells were pooled from two mice, and three cell samples per group were used for each examination. Cell viability was determined by trypan blue exclusion (Thermo Fisher Scientific).

Lymph node cells (1 × 10⁶/mL) were cultured with Dp (10 μg/mL) in 200 μL of R10 in 96-well flat-bottom plates. The cells were cultured in triplicate at 37°C in 5% CO₂/95% air atmosphere for 70 h. After that, the culture supernatant was collected and stored at −80°C until further use.

Enzyme-linked immunosorbent assay

An ELISA kit was used to quantify proteins. The following were quantified in the supernatant fraction of ear tissue homogenates: interleukin (IL)-4 (General Electric Company, Buckinghamshire, MA, USA), IL-5 (Thermo Fisher Scientific, MA, USA), IL-13 (R&D systems, Minneapolis, MN), interferon-gamma (IFN-γ) (Thermo Fisher Scientific), CCL3/macrophage inflammatory protein-1α (CCL3/MIP-1α) (R&D systems), thymic stromal lymphopoietin (TSLP) (R&D systems), and IL-18 (Medical & Biological Laboratories Co., Ltd., Nagoya, Japan). Moreover, the following were estimated in the serum: total immunoglobulin E (IgE) (BD Biosciences, San Diego, CA, USA) and histamine (Bertin Bioreagent, Montigny le Bretonneux, France). All estimations were performed as per manufacturer’s instructions. Furthermore, the Dp-specific IgG₁ level in serum was measured as previously described.²² Cell proliferation (Roche Molecular Biochemicals, Mannheim, Germany), IFN-γ (Thermo Fisher Scientific), and IL-4 (General Electric Company) were evaluated in the culture supernatants after Dp restimulation as per the manufacturer’s instructions. In brief, cell proliferation was analyzed by incorporating 5-bromo-2^′-deoxyuridine 20 h before the measurement.

Flow cytometry

Flow cytometry was used to analyze the submandibular LN cells. The following monoclonal antibodies were used for experiments: anti-major histocompatibility complex (MHC) class II I-A/I-E (2G9, Rat IgG2a κ fluorescein isothiocyanate (FITC)-conjugated; BD Biosciences), anti-CD86 (GL1, Rat IgG2a κ PE-conjugated; BD Biosciences), anti-T cell receptor (TCR) β (H57-597, Hamster IgG2 λ1 FITC-conjugated; BD Biosciences), CD69 (H1.2F3, American Hamster IgG1 λ3 PE-conjugated; BD Biosciences). The cell samples were prepared as per the manufacturers’ instructions and incubated with the antibodies for 30 min on ice. Fluorescence was then measured on FACSCalibur (Becton, Dickinson and Company, Franklin Lakes, NJ, USA), as reported previously.^1,23

Statistical analysis

Data are expressed as means ± standard errors of the mean. Differences between the groups were analyzed using one-way analysis of variance or the Kruskal–Wallis test using Ekuseru-Toukei 2010 statistical software (Social Survey Research Information Co., Tokyo, Japan). Moreover, differences between the experimental and control groups were determined using Dunnett’s multiple comparison test or Steel’s multiple comparison test. p < 0.05 was considered statistically significant.

Results

Benzo[a]pyrene aggravates AD-like skin lesions

To evaluate the effects of BaP exposure on AD-like skin lesions induced by repeated mite allergen (Dp) treatment, clinical scores and ear thickening were analyzed. The results showed that the intradermal administration of Dp significantly increased ear thickening and exacerbated clinical scores (dryness, eruption, wound, and edema) compared with saline (Figures 1(a) and (b); p < 0.05). Exposure to 2 and 20 µg/kg/day BaP with Dp significantly enhanced AD-like skin lesions compared with Dp alone, which was more prominent in the mice treated with Dp + 2 µg/kg/day (p < 0.05). In the histopathological experiment, the Dp + 2 µg/kg/day BaP and Dp + 20 µg/kg/day BaP groups showed increased mast cell and eosinophil infiltration as well as increased degranulation of mast cells (Figures 1(c) and (d) show data regarding the Dp + 2 µg/kg/day BaP group). By contrast, 200 and 2000 µg/kg/day BaP reduced Dp-induced AD-like symptoms.

Figure 1.

The effects of BaP in atopic dermatitis–like skin lesions induced by mite allergen. Ear thickness and clinical scores 24 h after administration and macroscopic features 24 h after the last intradermal Dp administration. (a) Ear thickness, (b) clinical scores, (c) hematoxylin–eosin staining, and (d) toluidine blue staining. Data are mean ± SE of five animals per group *; p < 0.05, Dp-treated groups versus Saline+Vehicle group †; p < 0.05, Dp+BaP 20 μg/kg/day group versus C ‡; p < 0.05, Dp+BaP 2 μg/kg/day group versus Dp+Vehicle group. Arrows show granulated (thin) and degranulated (thick) mast cells, respectively. BaP: benzo[a]pyrene.

Benzo[a]pyrene elevates the protein expression of inflammatory molecules in the skin

The expression of inflammatory molecules in the supernatant fraction of ear tissue homogenates is shown in Figure 2. Dp administration increased the expression of IL-4, IL-13, MIP-1α, and eotaxin compared with saline. A trend was observed toward an increase in MIP-1α, TSLP, IL-4, and IL-18 levels in the Dp + 2 µg/kg/day BaP and Dp + 20 µg/kg/day BaP groups compared with the Dp group. Moreover, IL-4 was found to be increased in the Dp + 20 µg/kg/day BaP group compared with that in the Dp + Vehicle group. Although the IFN-γ level was lower in Dp-administrated mice than in vehicle-administrated mice, no change was observed following exposure to BaP (data not shown).

Figure 2.

The effects of benzo[a]pyrene on protein expression of inflammatory molecules in the supernatant fraction of ear tissue homogenates. Protein levels in the ear homogenates were analyzed using ELISA 24 h after the last intradermal Dp administration. (a) MIP-1α, (b) eotaxin, (c) TSLP, (d) IL-4, (e) IL-13, and (f) IL-18. Data are the mean ± SE of five animals per group *; p < 0.05 versus Saline+Vehicle group **; p < 0.01 versus Saline+Vehicle group #; p < 0.05 versus Dp+Vehicle group.

Benzo[a]pyrene increases Ig and histamine in serum

Ig production in serum was examined to determine the adjuvant activity of BaP. Total IgE was higher in the Dp + 2 µg/kg/day BaP and Dp + 20 µg/kg/day BaP groups than in the Saline + Vehicle group (Figure 3(a)). In addition, Dp administration significantly increased Dp-IgG₁ production compared with saline (Figure 3(b); p < 0.01). Exposure to 2–200 µg/kg/day BaP revealed a further increase in Dp-specific IgG₁ compared with vehicle exposure in Dp-induced AD mice. By contrast, Dp-specific IgG₁ in the Dp + 2000 µg/kg/day BaP group decreased at the same level as those in the Dp + Vehicle group. Moreover, we measured histamine levels in serum (Figure 3(c)), and the results showed that BaP exposure increased serum histamine levels compared with vehicle in Dp-treated mice. In particular, this result was remarkable in the Dp + 20 µg/kg/day BaP (p < 0.05) and Dp + 2000 µg/kg/day BaP groups (p < 0.01).

Figure 3.

The effects of benzo[a]pyrene on immunoglobulin production and histamine release in serum. (a) Total IgE, (b) Dp-specific IgG₁, and (c) histamine in serum were measured using ELISA 24 h after the last intradermal Dp administration. Data are the mean ± SE of five animals per group *; p < 0.05 versus Saline+Vehicle group **; p < 0.01 versus Saline+Vehicle group #; p < 0.05 versus Dp+Vehicle group.

Benzo[a]pyrene enhances regional LN cell activation

To elucidate the mechanism of exacerbation of AD-like skin lesions caused by BaP exposure, we examined the activation of submandibular LN cells in the Dp + 2 μg/kg/day BaP group, which demonstrated the most remarkable aggravation of dermatitis symptoms. Exposure to Dp + 2 μg/kg/day BaP significantly elevated the cell number (Figure 4(a)) and cell proliferation (Figure 4(b)) compared with vehicle. However, no statistical differences were found between the Dp and Dp + 2 μg/kg/day BaP groups. The activation of antigen-presenting cells (APCs) and T cells was also analyzed using fluorescence-activated cell sorting. MHC II⁺CD86⁺ (Figure 4(c)) and TCRβ⁺CD69⁺ (Figure 4(d)) cells were higher in the Dp + 2 μg/kg/day BaP group than in the Saline + Vehicle and Dp + Vehicle groups. Dp + 2 μg/kg/day BaP increased the production of IFN-γ (Figure 4(e)) and IL-4 (Figure 4(f)) in cell culture supernatant after Dp restimulation compared with Dp alone, but no significant changes were found.

Figure 4.

The effects of BaP exposure on the activation of regional LN cells. Submandibular LN cells were prepared 24 h after the last intradermal Dp administration and evaluated by flow cytometry and ELISA. Each examination was performed on three cell samples pooled from two mice in each group. (a) Total cell number, (b) cell proliferation (ABS), (c) MHC class II⁺CD86⁺cells, (d) TCRβ⁺CD69⁺cells, (d) IFN-γ, and (f) IL-4. Data are the mean ± SE of three samples per group *; p < 0.05 versus Saline+Vehicle group **; p < 0.01 versus Saline+Vehicle group #; p < 0.05 versus Dp+Vehicle group. LN: lymph node; BaP: benzo[a]pyrene.

Discussion

We examined whether continuous exposure to BaP worsens Dp-induced AD-like skin lesions in mice. In the Dp + 2 µg/kg/day BaP and Dp + 20 µg/kg/day BaP groups, a marked enhancement of Dp-induced dermatitis was observed compared with the Dp + Vehicle group. Moreover, cytokine and chemokine expression in the inflamed site and antibody secretion in serum were elevated. Further, histamine levels increased in a BaP dose-dependent manner. Finally, Dp + 2 µg/kg/day BaP promoted the activation of APCs and T cells in regional LN cells.

Benzo[a]pyrene, a ubiquitous indoor and outdoor environmental pollutant, affects immune and allergic responses and causes carcinogenic and mutagenic effects. Repeated intraperitoneal exposures to BaP were found to enhance Dp-induced AD-like skin lesions in our murine model in the present study. In particular, these results were observed in lower doses (2 and 20 µg/kg/day BaP). These doses were comparable to 1/10^th and 1/100^th of NOAEL, respectively. In a previous study, dermal exposure to BaP (0.5 ppm/mice/day, 5 days) in allergen-patched mice led to epidermal thickening and the migration of Langerhans cells to regional LNs.²⁰ In addition, Tajima et al. reported that a single oral administration of high-dose BaP (0.3, 1, 3 mg/kg) before the final allergen challenge worsens hapten-induced ear swelling and scratching in a mouse model.¹⁹ To our knowledge, the present study reported for the first time the effects of repeated systemic exposure to low-dose BaP in AD mice. Humans, except smokers, are mainly exposed to PAHs, including BaP, through foods. BaP has been detected in smoked foods,²⁴ bread,²⁵ aquatic products,²⁶ and charcoal-grilled meat.^27,28 A review showed that the dietary intake of BaP for adults and toddlers is 472 ng/day and 11–268 ng/day, respectively.²⁹ Yu et al. reported that the calculated dietary intake of BaP was 0.609–4.69 ng/kg/day among individuals in Beijing, whereas inhalation intake was 0.087 ng/kg/day.³⁰ Human dietary intake of total PAHs ranged from 0.004 to 0.063 μg/kg/day (children), 0.002 to 0.028 μg/kg/day (adolescents), 0.01 to 0.017 μg/kg/day (males), 0.002 to 0.027 μg/kg/day (females), and 0.002 to 0.025 μg/kg/day (senior citizens).³¹ Therefore, an additional study is needed to investigate the effects of human exposure to lower doses of BaP.

Cytokines and chemokines play crucial roles in mediating and modulating the immune system in AD pathogenesis. In the present study, there was a trend toward higher levels of MIP-1α, IL-4, TSLP, and IL-18 in the inflamed site after exposure to BaP in Dp-induced AD-like mice. Moreover, BaP with Dp treatment activated LN cells and enhanced IFN-γ and IL-4 levels in cell culture after Dp restimulation. Atopic diseases are generally associated with cytokine overproduction (e.g., IL-4, IL-5, and IL-13), blood eosinophilia, and increased serum IgE levels. IL-4 induces isotype switching toward IgE production in human B cells. IL-18 belongs to the IL-1 superfamily and is expressed in various cell types, including macrophages and keratinocytes. IL-18 participates in developing relapsing dermatitis, accumulating T_H2 cytokines, overproducing IgE, and increasing histamine levels.³² MIP-1a, a C-C chemokine family member, is mainly produced by CD8⁺ lymphocytes; its expression in peripheral blood mononuclear cells is increased in patients with AD.³³ In a previous study, a transdermal administration of BaP significantly increased IL-5 and IL-13 levels in allergen-restimulated LN cells from AD mice.²⁰ Another report showed that BaP exposure increased IL-5 and IL-13 levels in regional LN cell culture and TSLP in inflamed tissue in T_H2-type hapten-induced AD mice.¹⁹ Moreover, the infiltration of T cells, B cells, and dendritic cells in regional LNs following BaP administration was observed. Taken together, repeated exposure to BaP may contribute toward the induction of T_H2-biased responses and activate regional LN cells, thereby aggravating AD symptoms.

In the present study, AD pathogenesis was enhanced by lower BaP doses (2 or 20 µg/kg/day); however, this effect was attenuated at higher doses (200 or 2000 µg/kg/day). As one of the mechanisms, the induction of AhR by BaP may contribute to the development and suppression of AD-like skin lesions. AhR, a nuclear receptor, modulates the xenobiotic metabolism of environmental pollutants, such as BaP. It plays a key role in modulating the immune response; however, researchers reported that AhR exhibits both proinflammatory and anti-inflammatory effects. Poulain-Godefroy et al. reported that AhR activation by PAHs is proinflammatory in nature, which induces mucus hypersecretion and APC dysregulation that worsen asthma symptoms.³⁴ In addition, BaP exposure induces Langerhans cell migration and increases cytokine production (IL-5, IL-13, and IL-17) in allergen-restimulated LN cells; these effects were attenuated in AhR-defective mice.²⁰ Moreover, oral exposure to BaP directly activates keratinocytes and T cells via AhR activation, resulting in worsened allergic dermatitis.¹⁹ By contrast, AhR activation in dendritic cells blocked the production of proinflammatory T cells or shifted toward anti-inflammatory M2 macrophages.³⁴ Coal tar exposure increased filaggrin (a major skin barrier protein) expression through AhR activation in keratinocytes.³⁵ Moreover, the induction of filaggrin after coal tar treatment was observed in skin biopsies from patients with AD, which ameliorated skin barrier defects in AD. Taken together, BaP exposure may induce bidirectional responses through AhR activation, resulting in modified AD-like skin lesions.

Mast cell degranulation by allergen-specific or nonspecific stimulation induces several chemical mediator releases, including histamine. In patients with AD, increases in histamine levels were reported.^36,37 In the present study, BaP dose-dependently increased serum histamine in Dp-treated AD mice. Moreover, mast cell degranulation was enhanced after exposure to BaP with Dp. Finally, Kepley et al. found that BaP and BaP-quinones enhance IgE-mediated histamine release as well as IL-4 production in human basophils.³⁸ BaP exposure can enhance mast cell degranulation and the subsequent histamine release, aggravating AD-like symptoms. In conclusion, BaP exposure can worsen mite allergen–induced AD-like skin lesions by enhancing T_H2-biased responses in inflamed sites and activating regional LNs. Moreover, Ig production and histamine release in serum are enhanced. BaP may be responsible for the recent increase in AD incidence.

Footnotes

Acknowledgments

The authors thank Naoko Ueki, Miho Sakurai, and Satomi Abe for their technical assistance and Animal Care Co., Ltd. for their animal breeding. The authors would like to thank Enago () for the English language review.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by a grant from the National Institute for Environmental Studies.

ORCID iD

Rie Yanagisawa

References

Koike

Yanagisawa

Sadakane

, et al. Effects of diisononyl phthalate on atopic dermatitis in vivo and immunologic responses in vitro. Environ Health Perspect 2010; 118: 472–478. DOI: 10.1289/ehp.0901255.

Sadakane

Ichinose

Takano

, et al. Effects of oral administration of di-(2-ethylhexyl) and diisononyl phthalates on atopic dermatitis in NC/Nga mice. Immunopharmacol Immunotoxicol 2014; 36: 61–69. DOI: 10.3109/08923973.2013.866678.

Takano

Yanagisawa

Inoue

, et al. Di-(2-ethylhexyl) phthalate enhances atopic dermatitis-like skin lesionsin mice. Environ Health Perspect 2006; 114: 1266–1269. DOI: 10.1289/ehp.8985.

Dubowsky

Wallace

Buckley

. The contribution of traffic to indoor concentrations of polycyclic aromatic hydrocarbons. J Expo Sci Environ Epidemiol 1999; 9: 312–321. DOI: 10.1038/sj.jea.7500034.

Abdel-Shafy

Hussein

I Mansour

Mona

SM . A review on polycyclic aromatic hydrocarbons: Source, environmental impact, effect on human health and remediation. Egyptian Journal of Petroleum 2016; 25: 107–123.

Zhu

Wang

. Sources and patterns of polycyclic aromatic hydrocarbons pollution in kitchen air, China. Chemosphere 2003; 50: 611–618. DOI: 10.1016/S0045-6535(02)00668-9.

Koo

Matsushita

JHC

, et al. Carcinogens in the indoor air of Hong Kong homes: levels, sources, and ventilation effects on 7 polynuclear aromatic hydrocarbons. Environ Tech 1994; 15: 401–418. DOI: 10.1080/09593339409385445.

Moriske

Drews

Ebert

, et al. Indoor air pollution by different heating systems: coal burning, open fireplace and central heating. Toxicol Lett 1996; 88: 349–354. DOI: 10.1016/0378-4274(96)03760-5.

Mitra

Ray

. Patterns and sources of polycyclic aromatic hydrocarbons and their derivatives in indoor air. Atmos Environ 1995; 29: 3345–3356. DOI: 10.1016/1352-2310(95)00214-J.

10.

Rogge

Hildemann

Mazurek

, et al. Sources of fine organic aerosol. 9. pine, oak, and synthetic log combustion in residential fireplaces. Environ Sci Tech 1998; 32: 13–22. DOI: 10.1021/es960930b.

11.

Lau

Fiedler

Hutzinger

, et al. Levels of selected organic compounds in materials for candle production and human exposure to candle emissions. Chemosphere 1997; 34: 1623–1630. DOI: 10.1016/S0045-6535(97)00458-X.

12.

Mizutani

Nabe

Ohtani

, et al. Polycyclic aromatic hydrocarbons aggravate antigen-induced nasal blockage in experimental allergic rhinitis. J Pharmacol Sci 2007; 105: 291–297.

13.

Kadkhoda

Pourfathollah

Pourpak

, et al. The cumulative activity of benzo(a)pyrene on systemic immune responses with mite allergen extract after intranasal instillation and ex vivo response to ovalbumin in mice. Toxicol Lett 2005; 157: 31–39.

14.

Kadkhoda

Pourpak

Akbar Pourfathallah

, et al. The ex vivo study of synergistic effects of polycyclic aromatic hydrocarbon, benzo(a)pyrene with ovalbumin on systemic immune responses by oral route. Toxicology 2004; 199: 261–265.

15.

Chowdhury

Kitamura

Honda

, et al. Synergistic effect of carbon nuclei and polyaromatic hydrocarbons on respiratory and immune responses. Environ Toxicol 2017; 32: 2172–2181. DOI: 10.1002/tox.22430.

16.

Yanagisawa

Koike

Win-Shwe

, et al. Low-dose benzo[a]pyrene aggravates allergic airway inflammation in mice. J Appl Toxicol 2016; 36: 1496–1504. DOI: 10.1002/jat.3308.

17.

Yanagisawa

Koike

Win-Shwe

, et al. Effects of lactational exposure to low-dose BaP on allergic and non-allergic immune responses in mice offspring. J Immunotoxicol 2018; 15: 31–40.

18.

Wang

Liu

, et al. Benzo(a)pyrene facilitates dermatophagoides group 1 (Der f 1)‐induced epithelial cytokine release through aryl hydrocarbon receptor in asthma. Allergy 2019; 74: 1675–1690.

19.

Tajima

Tajiki‐Nishino

Watanabe

, et al. Direct activation of aryl hydrocarbon receptor by benzo[ a ]pyrene elicits T‐helper 2‐driven proinflammatory responses in a mouse model of allergic dermatitis. J Appl Toxicol 2019; 39: 936–944.

20.

Hong

Lee

, et al. Benzopyrene, a major polyaromatic hydrocarbon in smoke fume, mobilizes Langerhans cells and polarizes Th2/17 responses in epicutaneous protein sensitization through the aryl hydrocarbon receptor. Int Immunopharmacol 2016; 36: 111–117.

21.

Ministry of the Environment Government of Japan . Benzo[a]pyrene. https://www.env.go.jp › report › pdf › chpt1 (2007, accessed 10 May 2021).

22.

Sadakane

Ichinose

Takano

, et al. Murine strain differences in airway inflammation induced by diesel exhaust particles and house dust mite allergen. Int Arch Allergy Immunol 2002; 128: 220–228. DOI: 10.1159/000064255.

23.

Koike

Inoue

Yanagisawa

, et al. Di-(2-ethylhexyl) phthalate affects immune cells from atopic prone mice in vitro. Toxicology 2009; 259: 54–60. DOI: 10.1016/j.tox.2009.02.002.

24.

Tsutsumi

Adachi

Matsuda

, et al. Concentrations of polycyclic aromatic hydrocarbons in smoked foods in Japan. J Food Prot 2020; 83: 692–701.

25.

Eslamizad

Kobarfard

Javidnia

, et al. Determination of Benzo[a]pyrene in traditional, industrial and semi- industrial breads using a modified QuEChERS extraction, dispersive SPE and GC-MS and estimation of its dietary intake. Iranian J Pharm Res 2016; 15: 165–174.

26.

Wang

Chu

Peng

, et al. Contribution of aquatic products consumption to total human exposure to PAHs in Eastern China: the source matters. Environ Pollut 1987; 266: 115339.

27.

Fatma Aygün

Kabadayi

. Determination of benzo[a]pyrene in charcoal grilled meat samples by HPLC with fluorescence detection. Int J Food Sci Nutr 2005; 56: 581–585. DOI: 10.1080/09637480500465436.

28.

Terzi

Celik

Nisbet

. Determination of benzo[a]pyrene in Turkish doner kebab samples cooked with charcoal or gas fire. Irish J Agr Food Res 2008; 47: 187–193.

29.

Harrad

. Spatiotemporal analysis and human exposure assessment on polycyclic aromatic hydrocarbons in indoor air, settled house dust, and diet: a review. Environ Int 2015; 84: 7–16.

30.

Wang

, et al. Polycyclic aromatic hydrocarbon residues in human milk, placenta, and umbilical cord blood in Beijing, China. Environ Sci Tech 2011; 45: 10235–10242. DOI: 10.1021/es202827g.

31.

Udowelle

Igweze

Asomugha

, et al. Health risk assessment and dietary exposure of polycyclic aromatic hydrocarbons (PAHs), lead and cadmium from bread consumed in Nigeria. Rocz Panstw Zakl Hig 2017; 68: 269–280.

32.

Konishi

Tsutsui

Murakami

, et al. IL-18 contributes to the spontaneous development of atopic dermatitis-like inflammatory skin lesion independently of IgE/stat6 under specific pathogen-free conditions. Proc Natl Acad Sci USA 2002; 99: 11340–11345.

33.

Hatano

Katagiri

Takayasu

. Increased levels in vivo of mRNAs for IL-8 and macrophage inflammatory protein-1α (MIP-1α), but not of RANTES mRNA in peripheral blood mononuclear cells of patients with atopic dermatitis (AD). Clin Exp Immunol 1999; 117: 237–243.

34.

Poulain-Godefroy

Bouté

Carrard

, et al. The aryl hydrocarbon receptor in asthma: friend or foe?. Int J Mol Sci 2020; 21: 8797. DOI: 10.3390/ijms21228797.

35.

van den Bogaard

Bergboer

JGM

Vonk-Bergers

, et al. Coal tar induces AHR-dependent skin barrier repair in atopic dermatitis. J Clin Invest 2013; 123: 917–927. DOI: 10.1172/JCI65642.

36.

Ruzicka

Glück

. Cutaneous histamine levels and histamine releasability from the skin in atopic dermatitis and hyper-IgE-syndrome. Arch Dermatol Res 1983; 275: 41–44. DOI: 10.1007/Bf00516553.

37.

Ring

Plasma histamine concentrations in atopic eczema. Clin Exp Allergy 1983; 13: 545–552. DOI: 10.1111/j.1365-2222.1983.tb02636.x.

38.

Kepley

Lauer

Oliver

, et al. Environmental polycyclic aromatic hydrocarbons, benzo(a) pyrene (BaP) and BaP-quinones, enhance IgE-mediated histamine release and IL-4 production in human basophils. Clin Immunol 2003; 107: 10–19. DOI: 10.1016/S1521-6616(03)00004-4.