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Toxicology Letters 200 (2011) 92–99

Contents lists available at ScienceDirect

Toxicology Letters

journa l homepage: www.e lsev ier .com/ locate / tox le t

sian dust storm particles induce a broad toxicological transcriptional programn human epidermal keratinocytes

yun Choia, Dong Wook Shina, Wonnyon Kimb, Seong-Jae Dohb, Soo Hwan Leec, Minsoo Nohc,∗

Bioscience Institute, AmorePacific Corporation R&D Center, Yongin, Gyeounggi-do, 446-729, Republic of KoreaDepartment of Earth and Environmental Sciences, Korea University, Seoul 136-713, Republic of KoreaCollege of Pharmacy, Ajou University, Suwon, Gyeounggi-do, 443-749, Republic of Korea

r t i c l e i n f o

rticle history:eceived 9 August 2010eceived in revised form 22 October 2010ccepted 27 October 2010vailable online 4 November 2010

eywords:sian dust storm particlesuman epidermal keratinocytesYP1A1

a b s t r a c t

Exposure to airborne dust particles originated from seasonal Asian dust storms in Chinese and Mongoliandeserts results in increased incidence of a range of diseases including asthma, contact dermatitis andconjunctivitis. The areas affected by Asian dust particles extend from East China to the west coast ofNorth America. In order to study toxicological mechanisms in human skin, we evaluated the effects ofdust particles collected during Asian dust storms (Asian dust particles) on gene expression in humanepidermal keratinocytes (HEK). In HEK, exposure to Asian dust particles significantly increased geneexpressions of cytochrome P450 1A1 (CYP1A1), CYP1A2, and CYP1B1, which is an indication of arylhydrocarbon receptor (AHR) activation. In addition, Asian dust particles increased gene transcriptionof the cytokines IL-6, IL-8, and GM-CSF, which have broad pro-inflammatory and immunomodulatory

L-6M-CSFaspase 14

properties. Asian dust particles significantly up-regulated expression of caspase 14 in HEK, suggestingthat Asian dust particles directly affect keratinocyte differentiation. We also demonstrated that proteinextract of pollen, a material frequently adsorbed onto Asian dust particles, potentially contributes tothe increased transcription of IL-6, CYP1A1, CYP1A2, and CYP1B1. Taken together, these studies suggestthat Asian dust particles can exert toxicological effects on human skin through the activation of thecellular detoxification system, the production of pro-inflammatory and immunomodulatory cytokines,

ssion

and changes in the expre

. Introduction

Asian dust storms originating from Chinese and Mongolianeserts increase the number of airborne dust particles and are aevere health concern during the dry season in affected regionsuch as Northeastern China, Korean, and Japan (Ichinose et al.,008a,b). In addition to the Asian continent, dust storms are com-on in arid or semi-arid regions throughout the world, such as the

ahara desert. Therefore, toxicological studies of airborne dust par-icles during Asian dust storms (Asian dust storm particles, ADSPs)

an be extrapolated to the effect of dust storms on affected regionsorldwide (Korenyi-Both et al., 1992; Kwon et al., 2002; Meng and

hang, 2007). For instance, sand dust from both Asia and Arizonaas a similar effect on eosinophil recruitment in the bronchial sub-

Abbreviations: ADSPs, Asian dust storm particles; AHR, aryl hydrocarboneceptor; CYP450, cytochrome P450 monooxygenase; HEK, human epidermal ker-tinocytes; PAHs, polycyclic aromatic hydrocarbons; PM, particulate matter; TiO2,itanium dioxide.∗ Corresponding author. Tel.: +82 31 219 3680; fax: +82 31 219 3675.

E-mail address: minsoo@alum.mit.edu (M. Noh).

378-4274/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved.oi:10.1016/j.toxlet.2010.10.019

of proteins essential in normal epidermal differentiation.© 2010 Elsevier Ireland Ltd. All rights reserved.

mucosa of mice (Ichinose et al., 2008b). Interestingly, relativelyfine particles originating from severe Asian dust storms can betransported across the Pacific Ocean where they may affect therespiratory health of people in the west coast of North America(Duce et al., 1980). Recent epidemiological studies have suggestedthat increased exposure to ADSPs is associated with the increasedsusceptibility to asthma, allergic rhinitis, contact dermatitis andconjunctivitis (Chen and Yang, 2005; Kwon et al., 2002; Yang et al.,2005; Yang, 2006; Im et al., 2006).

ADSPs are complex mixtures consisting of various environ-mental pollutants, heavy metals, and microbiological materials(Ichinose et al., 2009). Due to the known correlation of Asiandust storm with increased clinical visits for asthma and rhinitis(Chen and Yang, 2005; Kwon et al., 2002; Yang, 2006; Yang et al.,2005), the toxicological effects of ADSPs on the respiratory sys-tem have been extensively evaluated (Ichinose et al., 2008a,b; Kimet al., 2003; Yanagisawa et al., 2007). In addition to their respi-

ratory effects, airborne pollutants have also been associated withepidemics of inflammatory skin diseases, such as contact hyper-sensitivity or dermatitis (Arruda et al., 2005; Heo et al., 2001;Mastrangelo et al., 2003; Saxon and Diaz-Sanchez, 2000; Takenakaet al., 1995; Wu et al., 2003; Yamamoto and Tokura, 2003). In spite

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f a increasing public perception of the risk of dermatitis by Asianust storms, the association between ADSPs and skin diseases haseceived much less attention than the association with respiratoryiseases.

In human skin, epidermal keratinocytes play a role in the for-ation of a primary defensive skin barrier against environmental

oxicants. Epidermal keratinocytes in the basal layer of the epider-is move upward and ultimately differentiate into cornified cells in

he epidermal stratum corneum, thus forming the epidermal per-eability barrier (Elias et al., 2002; Feingold et al., 2007). Disruption

f the epidermal permeability barrier is associated with atopic der-atitis, probably due to increased penetration of environmental

oxicants through the stratum corneum to trigger inflammatorykin conditions. Human epidermal keratinocytes (HEK) respondo toxic chemicals or organic materials by secreting various pro-nflammatory autacoids and cytokines which can, in turn, affect thentegrity of the epidermal permeability barrier (Monteiro-Rivieret al., 2004; Proksch et al., 2008).

In the present study, to understand the toxicological effects ofDSPs on human skin, we systematically evaluated mRNA expres-ion profiles of epidermal permeability barrier-associated proteins,nti-microbial peptides, detoxifying enzymes, keratinocyte stemell marker proteins, caspases, and pro-inflammatory cytokines.

e found that Asian dust particle samples collected from threendependent storm events consistently increased mRNA levels ofhase I cytochrome P450 enzymes, IL-6, IL-8, GM-CSF, and cas-ase 14 in HEK, suggesting that ADSPs increase pro-inflammatoryeactions in skin and can disrupt normal epidermal differentiation.

. Materials and methods

.1. Collection of Asian dust particle samples

ADSPs were collected from the roof of a Korea University science building,ocated in the northeastern region of Seoul, Korea (37◦35′N, 127◦01′E). We collectedhree Asian dust particle samples on April 12th 2004, March 11th 2006, and April 8th006 using a high capacity air collector with a quartz filter. On the sample collectionates, the daily concentration of total suspended particulates (TSP) was >200 �g/m3,hich was ∼3 times higher than the annual mean TSP in Seoul, Korea. ADSP samplesere sterilized by incubation in ethanol for 2 days for cell culture study.

.2. Scanning electron microscope (SEM)

The composition, morphology, and size were analyzed using SEM with energyispersive X-ray spectroscope (EDS) analysis. For microscopic analysis, subsamplesdimensions 0.5 cm × 0.5 cm) were prepared from ADSP samples. The prepared sub-amples were coated with 3–5 nm sized platinum particles using an ion sputterHitachi E-1030, Japan) to identify anthropogenic or environmental componentsncluding carbon-containing materials and bio-organisms (Kim et al., 2008).

.3. Culture of human keratinocytes

Normal HEK from neonatal foreskin were purchased from Lonza (Basel,witzerland) and cultured in KBM medium containing KGM2 growth supplementsontaining insulin, human epidermal growth factor, bovine pituitary extract, hydro-ortisone, epinephrine, transferrin, and gentamicin/amphotericin B, purchased fromonza. Cells were serially passaged at 70–80% confluence and experiments wereonducted with subconfluent cells at passage two or three. HEK cells were starvedor 24 h in keratinocyte KBM medium without cortisone or transferrin, followedy stimulation with ADSP samples (25 �g/ml), Ragweed pollen extract (25 �g/ml;osmo Bio Co., LTD., Tokyo, Japan), and Mite extract (25 �g/ml; Cosmo Bio Co., LTD.,okyo, Japan) for 24 h. Titanium oxide (TiO2, 99% purity), with a particle size of1.0 �m, was purchased from Sigma Chemical Co., St. Louis, MO, USA.

.4. Evaluation of cell viability

Cell viability was evaluated as previously reported (Lee et al., 2010). Cells

ere seeded in 60 mm plates for 24 h in the presence of 25 �g/ml ADSPs. The

ells were incubated for 2 h in a humidified atmosphere with 4-3-[4-lodophenyl]--4(4-nitrophenyl)-2H-5-tetrazolio-1,3-benzene disulfonate (WST-1; 10 �M pureolution, Roche Molecular Biochemical, Indianapolis, IN, USA). The absorbance ofhe samples (A450) was determined using an enzyme-linked immunosorbent assayeader. Absolute optical density was expressed as a percentage of the control value.

ters 200 (2011) 92–99 93

2.5. Total RNA isolation and quantitative real-time RT-PCR (Q-RT-PCR)

Total RNA was isolated using the Trizol reagent (Invitrogen, Carlsbad, CA, USA),according to the manufacturer’s instructions. The concentration of RNA was deter-mined by spectrophotometry and the integrity of the RNA was assessed using aBioAnalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA). Two microgramsof RNA were reverse-transcribed into cDNA using SuperScript TM reverse tran-scriptase (Invitrogen) and aliquots were stored at −20 ◦C. Q-RT-PCR was used fordetermining the expression levels of the selected target genes. The gene identifica-tion number for TaqMan probes (Applied Biosystems) used in the Q-RT-PCR analysiswas presented in Table 1. Human GAPDH (4333764F, Applied Biosystems) was alsoamplified to normalize variations in cDNA quantities from different samples. ThecDNA was amplified by PCR and the FAM fluorescence of each PCR cycle was mea-sured with the Corbett Research Rotor-Gene 6 Detection System (Corbett Research,Mortlake, NSW, Australia). The cycling conditions included a denaturing step at 95 ◦Cfor 10 min, and each cycle at 95 ◦C for 15 s, and annealing and elongation at 60 ◦C for1 min. Q-RT-PCR reactions were performed in triplicate or in quadruplicate. Resultsof Q-RT-PCR data were represented as Ct values, where Ct is defined as the thresh-old cycle. Quantification of relative differences in gene expressions in HEK inducedby ADSPs was calculated using equations from a mathematical model developed byPfaffl et al. (2002).

2.6. Statistical analysis

All statistical analyses were performed with MINITAB® software (Minitab Inc.State College, PA, USA). Results are expressed as the means ± standard deviation ofat least three independent experiments. Statistical analyses were performed withStudent’s t-test for comparison with control.

3. Results

3.1. Effect of ADSPs on HEK viability

In order to study cellular mechanisms of contact hypersensitiv-ity and dermatitis induced by airborne dust particles originatingfrom Asian dust storm events, we performed toxicological stud-ies with ADSPs that we have studied in a previous report (Kimet al., 2008). In order to confirm that anthropogenic or environ-mental pollutants are co-precipitated with the ADSP samples ona quartz filter in a collecting device, we performed SEM obser-vation with energy dispersive X-ray spectroscope (EDS) analysis.As shown in Fig. 1A, we observed a variety of inorganic parti-cles, including carbon-containing materials of anthropogenic originand organic particles, including pollen and microorganisms, onADSPs.

Before investigating mRNA expression profiles of functionallyimportant proteins in HEK in response to ADSPs, we first deter-mined the non-cytotoxic concentration of three ADSP samplesusing a WST-1 assay. When we treated HEK with ADSPs for 24 h,cell viability was reduced in a concentration-dependent manner(Fig. 1B). In order to exclude particulate associated non-specificeffects of ADSPs on HEK, we used titanium dioxide (TiO2) as an inertparticulate control. Coarse and find particle sized TiO2 (>100 nm) isknown as a biologically inert and used as a particulate in humantoxicological studies (Bernard et al., 1990; Hart and Hesterberg,1998). In this study, there were no cytotoxic effects of TiO2 to100 �g/ml in primary human keratinocytes. Three ADSP samplescollected during different Asian dust storm events were morecytotoxic in HEK culture than equivalent concentrations of TiO2.For expression profile studies to evaluate toxicological effects, wetreated HEK with 25 �g/ml ADSPs, a concentration that did notsignificantly affect cell viability.

3.2. Effects of Asian dust particles on mRNA expression profiles ofgenes regulating the physiological function of HEK

To determine toxicological effects of ADSPs on HEK, we usedQ-RT-PCR to measure mRNA levels of genes regulating a variety ofphysiological processes in HEK. The genes included those associ-ated with cellular detoxification and pro-inflammatory responses,

94 H. Choi et al. / Toxicology Letters 200 (2011) 92–99

Table 1Effects of ADSPs on HEK gene transcription.

Gene category TaqMan® assay ID # Gene (gene symbol) Fold change(mean ± SD, n = 3)

Cellular detoxifying system Hs00907314 m1 Aryl hydrocarbon receptor (AHR) 1.5 ± 0.3Hs01054797 g1 Cytochrome P450, family 1, subfamily A, polypeptide 1 (CYP1A1) 50.1 ± 9.1Hs01070374 m1 Cytochrome P450, family 1, subfamily A, polypeptide 2 (CYP1A2) 9.7 ± 0.9*

Hs00164383 m1 Cytochrome P450, family 1, subfamily B, polypeptide 1 (CYP1B1) 8.3 ± 1.2*

Hs00156308 m1 Catalase (CAT) 0.7 ± 0.1Hs02516751 s1 Glutathione peroxidase 1 (GPX1) 0.5 ± 0.1Hs01045994 m1 NAD(P)H dehydrogenase, quinone 1 (NQO1) 1.6 ± 0.5Hs00602020 mH Peroxiredoxin 1 (PRDX1) 1.3 ± 0.2Hs00428488 g1 Peroxiredoxin 2 (PRDX2) 1.1 ± 0.1Hs00705355 s1 Peroxiredoxin 6 (PRDX6) 1.1 ± 0.1Hs00167309 m1 Superoxide dismutase 2 (SOD2) 2.0 ± 0.6

Interleukins Hs00174097 m1 Interleukin 1, � (IL1B) 1.9 ± 0.2Hs99999032 m1 Interleukin 6 (IL6) 4.1 ± 0.4**

Hs00174103 m1 Interleukin 8 (IL8) 6.5 ± 0.6**

Hs01038788 m1 Interleukin 18 (IL18) 1.2 ± 0.1Hs99999044 m1 Granulocyte-macrophage colony stimulating factor (GM-CSF, CSF2) 5.3 ± 0.4**

Hs99999041 m1 Interferon gamma (IFNG) 1.2 ± 0.1

Anti-microbial peptide Hs00189038 m1 Cathelicidin anti-microbial peptide (CAMP, LL37) 0.8 ± 0.3Hs00175474 m1 �4-Defensin (DEFB4) 1.0 ± 0.2Hs00218678 m1 �-Defensin 103B (DEFB103B) 0.9 ± 0.1

Epidermal permeability barrier Hs00196158 m1 Keratin 1 (KRT1) 2.1 ± 0.5Hs01699178 g1 Keratin 6A (KRT6A) 1.2 ± 0.4Hs01043110 g1 Keratin 10 (KRT10) 1.5 ± 0.6Hs00856927 g1 Filaggrin (FLG) 1.2 ± 0.1Hs00846307 s1 Involucrin (IVL) 1.4 ± 0.3Hs01894962 s1 Loricrin (LOR) 1.4 ± 0.5Hs01070316 m1 Transglutaminase 1 (TGM1) 1.0 ± 0.2Hs01096681 m1 Transglutaminase 2 (TGM2) 2.1 ± 0.3Hs00154599 m1 Cystatin M precursor (CST6) 1.4 ± 0.1Hs00160066 m1 Elafin preproprotein (PI3) 1.8 ± 0.4Hs00268204 m1 Secretory leukocyte peptidase inhibitor (SLPI) 1.0 ± 0.1Hs00199260 m1 Serine peptidase inhibitor, Kazal type 5 isoform (SPINK5) 1.2 ± 0.1

Caspases Hs00234387 m1 Caspase 3 (CASP3) 0.7 ± 0.1Hs00236278 m1 Caspase 8 (CASP8) 0.7 ± 0.1Hs00609641 m1 Caspase 9 (CASP9) 1.1 ± 0.1Hs00201637 m1 Caspase 14 (CASP14) 30.0 ± 0.7*

Keratinocyte stem cell marker proteins Hs00173952 m1 Integrin alpha chain, alpha 6 isoform (ITGA6) 0.6 ± 0.2*

Hs00174609 m1 Transferrin receptor (TFRC, CD71) 1.2 ± 0.1

ADSPs collected at March 11 2006 (25 �g/ml) were used (n = 3).

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** P < 0.01.* P < 0.05.

hose essential for the development of the epidermal permeabil-ty barrier, and those encoding keratinocyte stem cell markersTable 1). We first examined mRNA levels of aryl hydrocarboneceptor (AHR), cytochrome P450 enzymes (CYP), peroxidases, anderoxiredoxins to assess transcriptional changes in the cellularetoxification system. ADSPs significantly increased mRNA levels ofYP1A1, CYP1A2, and CYP1B1, whereas gene transcription of AHR,key transcription factor regulating cytochrome P450 enzyme

xpression in NHEK, was not affected. In addition, ADSPs had noignificant effect on gene transcription of catalase, peroxiredox-ns, superoxide dismutase, and NAD(P)H quinine dehydrogenasen HEK (Table 1).

HEK produce pro-inflammatory cytokines and growth factorsn response to various environmental stimuli (Bashir et al., 2009;

aeda et al., 2009). We evaluated whether ADSPs affected mRNAevels of cytokines known to be expressed in HEK such as IL-1�, IL-6,L-8, IL-18, interferon gamma (IFN�), and granulocyte macrophageolony-stimulating factor (GM-CSF). ADSPs significantly increased

RNA levels of IL-6, IL-8, and GM-CSF in HEK. Gene transcription

f IL-18 and IFN� were not significantly affected by treatment withDSPs (Table 1).

In addition, we evaluated whether ADSPs can affect the tran-criptional profile of proteins pivotal in the development of the

epidermal permeability barrier such as filaggrin, loricrin, cytok-eratins, and transglutaminases. Abnormal development of theepidermal permeability barrier plays a role in the etiology ofatopic dermatitis and other inflammatory dermatological condi-tions (Elias and Schmuth, 2009; Elias et al., 2008). However, asshown in Table 1, the mRNA levels of most proteins related to epi-dermal permeability development were unaffected by treatmentwith ADSPs. Although mRNA levels of keratin 1 and transgluat-minase 2 in HEK were increased about 2-fold by treatment withADSPs, these changes were not statistically significant. Caspase-14(CASP14) is expressed in terminally differentiating keratinocytesand can regulate filaggrin metabolism in the epidermis. There-fore, CASP14 in HEK is important in regulating skin hydrationand cornification of the stratum corneum (Demerjian et al., 2008;Denecker et al., 2008). ADSPs significantly increased CASP14 mRNAexpression while other apoptosis-related cysteine proteases likecaspase-3, -8, and -9 were unaffected (Table 1).

Transferrin receptor (CD71) and �6-integrin can be used as

markers to isolate and enrich epidermal stem cell populations using(Tani et al., 2000). In HEK culture, ADSPs collected at March 11th2006 decreased mRNA levels of �6-integrin whereas CD71 genetranscription was unchanged compared with that of the control(Table 1).

H. Choi et al. / Toxicology Letters 200 (2011) 92–99 95

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ig. 1. Characterization of Asian dust storm particles. (A) SEM analysis with EDS aarticles. Based on EDS-analysis of materials, particles were composed of organiculfur). (B) Cytotoxicity caused by ADSPs in HEK. HEK cells were cultured and theniability was measured using the WST-1 assay, as described in Section 2. Values rep

.3. Sample variations of the response profile to ADSPs in HEK

We next evaluated whether samples of ADSPs obtained from dif-erent Asian dust storm events differed in their ability to regulateene transcription in culture HEK (Fig. 2). We measured the mRNAxpression of genes whose expression was significantly changed byhe ADSP sample that was harvested on March 11th 2006 (Table 1).he response profile of CYP1A1, CYP1A2, and CYP1B1 showed thathese cytochrome P450 enzymes were significantly up-regulatedy the treatment of HEK with each of the three ADSP samplesFig. 2A–C). Although we observed an increase in IL-1� gene tran-cription in response to the ADSP sample collected on March 11th006, the other two ADSP samples did not significantly affect IL-� mRNA expression (Fig. 2D). In contrast, IL-6, IL-8, and GM-CSFere consistently up-regulated in HEK by all three ADSP samples

Fig. 2E–G). CASP14 mRNA expression was significantly increasedn HEK by all three ADSPs (Fig. 2H), suggesting that the ADSPs canegulate the development of the epidermal permeability barrier.ignificant down-regulation of �6-integrin was only detected fol-owing treatment of HEK with one of the three ADSPs (Fig. 2I).herefore, although there is minor variation in the mRNA expres-ion profile of HEK in response to different ADSP samples, the mRNAxpression of cytochrome P450 enzymes, IL-6, IL-8, GM-CSF, andASP14 is significantly affected by ADSPs obtained from three dif-

erent Asian dust storm events.

.4. Effects of biological materials commonly adsorbed to Asian

ust particles on gene transcription of HEK

Among various environmental pollutants adsorbed to ADSPs, wevaluated whether biological contaminants such as proteins andicroorganisms contribute to the transcriptional response of HEK.

is was carried out to identify the composition, morphology, and size of individualrials (e.g., pollen and microorganism) and inorganic materials (e.g., iron, ash andated with ADSPs in a dose-dependent manner (25, 50 and 100 �g/ml) for 24 h. Cellt the means ± S.D. of three independent measurements, * P ≤ 0.05 and ** P ≤ 0.01.

It is practically impossible to isolate and purify biological materi-als adsorbed on ADSPs and, for this reason, we used commerciallyavailable protein extracts from ragweed pollen (Ambrosia artemisi-ifolia) and mite (Dermatophagoides pteronyssinus) both of which areincluded in the class of biological molecules potentially adsorbedto ADSPs (Fig. 1A) (Ichinose et al., 2009). Protein extract for rag-weed pollen significantly increased gene transcription of CYP1A1,CYP1A2, CYP1B1, and IL-6 in HEK and decreased expression of �6-integrin mRNA (Fig. 3). In contrast to the effects of ADSPs on genetranscription in HEK, mRNA levels of IL-8, GM-CSF, and CASP14were unaffected by treatment with the pollen extract (Fig. 3). Pro-tein extract from mite up-regulated gene transcription of IL-6 inHEK and down-regulated that of �6-integrin (Fig. 3E and I). How-ever, gene transcription of cytochrome P450 enzymes, IL-1�, IL-8,GM-CSF, and CASP14 was unchanged by the mite extract (Fig. 3).The increase of IL-6 in HEK by ADSPs may be due to the con-tribution by both pollen and mite adsorbed on ADSPs. However,neither pollen nor mite protein extracts affected gene transcrip-tion of IL-1�, IL-8, GM-CSF, and CASP14. These results suggest thatthe transcriptional changes in HEK by ADSPs may result from thesum of multiple effects caused by various contaminants adsorbedto the dust particles.

4. Discussion

In this study, we discovered that ADSPs significantly increasegene transcription of IL-1�, IL-6, IL-8, GM-CSF, CASP14, and

cytochrome P450 enzymes including CYP1A1, CYP1A2, and CYP1B1in HEK, while they decrease �6-integrin gene transcription(Table 1). Among a variety of environmental pollutants or bio-organic materials adsorbed onto Asian dust particles, we evaluatedhow protein components of pollen and mite contribute to the tran-

96 H. Choi et al. / Toxicology Letters 200 (2011) 92–99

Fig. 2. Expression of CYPs and pro-inflammatory cytokines in HEK following exposure to ADSPs. HEK cells were exposed to each of the three ADSP samples (25 �g/ml) for2 CYP1( aluesG

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4 h. Total RNA was extracted from these cells, and Q-RT-PCR was performed for (a)i) �6-integrin. Vehicle (ethanol) treated samples were indicated as no treatment. VAPDH expression (n = 3). * P ≤ 0.05 and ** P ≤ 0.01.

criptional change in HEK in response to ADSPs. As shown in Fig. 3,he pollen extract may play a role in the regulation of transcrip-ion of CYP1A1, CYP1A2, CYP1B1, IL-6, and �6-integrin in HEK byDSPs. In contrast, mite protein only affected transcription of IL-6nd �6-integrin, suggesting that the contribution of mite proteino the effect of Asian dust particles on HEK is likely to be limitedFig. 3).

ADSPs contain various environmental pollutants such as metals,rganic materials, and inorganic toxicants from urban or industrialmissions (Ichinose et al., 2009; Lee et al., 2009; Park et al., 2007).n addition, ADSPs are mixed with locally generated environmental

ollutants and may promote direct contact of these pollutants toxposed skin regions (Im et al., 2006; Kim et al., 2008). Many chem-cal pollutants, which may be adsorbed on ADSPs, such as dioxinsnd nitrobenzanthrones, can regulate mRNA expression of the tran-cription factor AHR (Im et al., 2006; Mimura and Fujii-Kuriyama,

A1, (b) CYP1A2, (c) CYP1B1, (d) IL-1�, (e) IL-6, (f) IL-8, (g) GM-CSF, (h) CASP14, andrepresent the mean expression ± S.D. of mRNA levels of the gene relative to human

2003; Stiborova et al., 2008). AHR plays an important role in theregulation of xenobiotic metabolism by, for example, controllingthe expression of cytochrome P450 enzymes, but it is also impor-tant in cellular development and differentiation (Monk et al., 2003).Interestingly, transgenic expression of constitutively activated AHRin epidermal keratinocytes resulted in severe inflammatory skinlesions in mice (Tauchi et al., 2005). In addition, gene transcrip-tion of AHR, CYP1A1, NQO1, glutathione peroxidase (GPX), IL-1�,IL-18, anti-microbial peptide defensins, and keratin 1 was signifi-cantly up-regulated in the skin of AHR transgenic mice comparedwith that of wild type mice (Tauchi et al., 2005). In this study, the

mRNA levels of CYP1A1, CYP1A2, and CYP1B1 were significantlyup-regulated in HEK (Table 1, Fig. 2), suggesting that increased AHRactivation by ADSPs may be one of reasons for increased dermati-tis during Asian dust storms. In spite of the significant changes ingene transcription of various proteins regulating keratinocyte dif-

H. Choi et al. / Toxicology Letters 200 (2011) 92–99 97

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ig. 3. Differential gene expression patterns of CYPs and pro-inflammatory cytokinere exposed to 25 �g/ml of pollen protein, mite protein, or ADSPs obtained on AYP1A1, (b) CYP1A2, (c) CYP1B1, (d) IL-1�, (e) IL-6, (f) IL-8, (g) GM-CSF, (h) CASP14he gene relative to human GAPDH expression (n = 3). * P ≤ 0.05 and ** P ≤ 0.01.

erentiation in the AHR transgenic mice (Tauchi et al., 2005), such asQO1, GPX, IL-18, �4-defensin, and keratin 6A, there was no signif-

cant change in gene transcription of these genes in HEK in responseo ADSPs (Table 1). The reason for this difference in gene expressionetween HEK treated with ADSPs and the skin from AHR transgenicice may be related to differences in the duration of AHR signal-

ng. In our experimental system, HEK were treated with ADSPs for4 h, whereas the expression profile in AHR transgenic mouse skinesults from chronic activation of AHR. In addition, the pollen pro-ein extract increased gene transcription of CYP1A1, CYP1A2, andYP1B1 in HEK (Fig. 3). Pollens are major contaminants adsorbedo ADSPs (Ichinose et al., 2009). Our results suggest that the pollenxtract can increase mRNA levels of cytochrome P450 enzymes inEK, although we cannot exclude the possibility that environmen-

al chemicals able to activate AHR are present as contaminants inhe ragweed pollen used in this study. This possibility should beddressed in a future study. In AHR transgenic mice, activation ofhe AHR signaling pathway alone is sufficient to initiate inflam-

atory skin disorders similar to contact dermatitis, with extensive

EK after treatment with protein extracts from pollen and mite, or ADSPs. HEK cells2th 2004 for 24 h. Total RNA was extracted, and Q-RT-PCR was performed for (a)(i) �6-integrin. Values represent the mean expression ± S.D. of the mRNA levels of

cell infiltration and itching (Tauchi et al., 2005). Further investi-gation is necessary to fully understand the association betweenAHR activation and primary inflammatory skin disorders inducedby ADSPs.

ADSPs increase transcription of the pro-inflammatory cytokinesIL-6 and IL-8 in HEK (Table 1 and Fig. 2). IL-6 and IL-8 can triggerthe onset of inflammatory skin diseases such as contract dermati-tis and atopic dermatitis, and they also promote the progression ofthese diseases (Lee et al., 2008; Reich et al., 2003). IL-6 stimulatesproliferation of HEK and inflammatory responses in HEK such asup-regulation of prostaglandin E2 (PGE2) synthesis and promotionof acute phase responses important in the pathogenesis of der-matitis (Chedid et al., 1994; Ishihara and Hirano, 2002; Svobodovaet al., 2009). In lesional skin of psoriasis, atopic dermatitis, and

other dermatological conditions, IL-8 levels are up-regulated, lead-ing to enhanced recruitment of neutrophils and T lymphocytes,and amplification of the inflammatory mechanisms of these dis-eases (Bonifati and Ameglio, 1999). In this study, we demonstratedthat protein extracts from pollen or mite are associated with the

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8 H. Choi et al. / Toxicolo

DSP-induced up-regulation of IL-6, but not IL-8 (Fig. 3). Generally,he individual effect of pollen or mite on IL-6 expression in HEKas lower than that of ADSPs (Fig. 3). Other environmental pol-

utants or organic contaminants may contribute to the increasedL-8 gene transcription in HEK by ADSPs. It has also been reportedhat exposure to fine dust particles (Particulate matter whose aer-bic diameter equal or less than 2.5 �m, PM2.5) can increase theevels of IL-6 and IL-8 in various cell types, such as respiratorypithelial cells, after exposure to dust particles (Mathiesen et al.,004; Monn and Becker, 1999; Monn et al., 2003; van Eeden et al.,001). Therefore, we cannot exclude the possibility that fine dustarticles (PM2.5) in ADSPs are major contributors to ADSP-inducedoxicity. In addition to regulating expression of pro-inflammatoryytokines IL-6 and IL-8, ADSPs also significantly up-regulate mRNAevels of GM-CSF (Table 1 and Fig. 2), an important immunomodu-atory cytokine involved in Langerhans cell (LC) maturation in skinJux et al., 2009). Therefore, the regulation of expression of bothro-inflammatory and immunomodulatory cytokines by ADSPs ishighly plausible mechanism to explain the increased prevalencef skin diseases such as contact dermatitis following Asian dusttorms.

Proliferating HEK undergo differentiation in the basal layer ofpidermis to generate the epidermal permeability barrier in thetratum corneum (Houben et al., 2007). In the basal layer, ker-tinocyte stem cells undergo self-renewal and also give rise toifferentiated transit amplifying cells (Houben et al., 2007). Twoell surface biomarkers, �6-integrin and CD71 (transferring recep-or), have been widely used to analyze keratinocyte stem cellsnd their progeny cells. In flow cytometry, the keratinocyte stemell-enriched population is characterized by high expression of6-integrin and low expression of CD71, while transit amplify-

ng cells are determined by high expression of both �6-integrinnd CD71 (Tani et al., 2000). In this study, exposure to proteinxtracts from ragweed pollen and mite led to decreased �6-integrinene transcription in HEK, and had no effect on CD71 mRNAxpression (Fig. 3). This result suggests that ragweed pollens andites frequently observed in ADSP samples may have an effect

n keratinocyte stem cell function and differentiation. In addition,omozygous mutations of �6-integrin are associated with blister-

ng skin diseases like junctional epidermolysis bullosa (Ruzzi et al.,997).

ADSPs increase gene transcription of CASP14, a unique cys-einyl aspartate-specific protease that is involved in formationf the epidermal permeability barrier (Denecker et al., 2007).ASP14 is reported as a protective factor against UVB irradiation-

nduced skin damage and water loss, and it also plays a role inro-filaggrin processing into natural moisturizing factor (NMF)Denecker et al., 2008). Besides CASP14, there was no significanthange in gene transcription of filaggrin and other cornified cellnvelope-associated proteins essential to the generation of thepidermal permeability barrier (Table 1). Because filaggrin also reg-lates aggregation of the keratin cytoskeleton during epidermalifferentiation to generate a condensed structure prior to conver-ion into NMF, the relative expression level of CASP14 and filaggrinay have an impact on the normal differentiation of HEK. There-

ore, the results suggest that ADSPs may cause dysfunction in thepidermal permeability barrier, leading to aggravated toxicologi-al outcomes in human skin. It usually takes more than a week tobserve significant changes in gene transcription of some of the epi-ermal differentiation markers in cultured HEK (Kolly et al., 2005),

t will be interesting to examine the association between ADSPs

nd dysfunction of epidermal permeability barrier in more detailn future studies.

In conclusion, the present study elucidates potential mecha-isms of Asian dust particle-induced cellular toxicity in human skin.xposure of human keratinocytes to ADSPs was associated with

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increased AHR activation, up-regulation of pro-inflammatory andimmunomodulatory cytokines IL-6, and IL-8, increase in LC matura-tion by GM-CSF, and CASP14 up-regulation that may affect normalepidermal differentiation. These changes in the HEK transcriptionprofile in response to ADSPs might contribute to the pathogenesisof inflammatory skin diseases caused by Asian dust storms.

Conflict of interest

Authors Hyun Choi and Dong Wook Shin are employees ofAmorePacific Co.

Acknowledgments

This study was supported by a grant of the Korea Healthcaretechnology R&D project, Ministry for Health, Welfare & Fam-ily Affairs, Republic of Korea (Green Cosmetic Research Center,A092055).

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