Food and Chemical Toxicology 65 (2014) 105–114

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Food and Chemical Toxicology

journal homepage: www.elsevier.com/locate/foodchemtox

Dermal toxicity elicited by phthalates: Evaluation of skin absorption,

immunohistology, and functional proteomics
Tai-Long Pan a, Pei-Wen Wang a,b, Ibrahim A. Aljuffali c, Yi-Yun Hung d,e, Chwan-Fwu Lin f,⇑,
Jia-You Fang d,e,g,h,⇑
a
School of Traditional Chinese Medicine, Chang Gung University, Kweishan, Taoyuan, Taiwan
b
Research Center for Industry of Human Ecology, Chang Gung University of Science and Technology, Kweishan, Taoyuan, Taiwan
c
Department of Pharmaceutics, College of Pharmacy, King Saud University, Riyadh, Saudi Arabia
d
Pharmaceutics Laboratory, Graduate Institute of Natural Products, Chang Gung University, Kweishan, Taoyuan, Taiwan
e
Chinese Herbal Medicine Research Team, Healthy Aging Research Center, Chang Gung University, Kweishan, Taoyuan, Taiwan
f
Department of Cosmetic Science, Chang Gung University of Science and Technology, Kweishan, Taoyuan, Taiwan
g
Graduate Institute of Health Industry Technology, Chang Gung University of Science and Technology, Kweishan, Taoyuan, Taiwan
h
Chinese Medicine Research and Development Center, China Medical University Hospital, Taichung, Taiwan

a r t i c l e i n f o a b s t r a c t

Article history: The toxicity of phthalates is an important concern in the fields of environmental health and toxicology.
Received 29 August 2013 Dermal exposure via skin care products, soil, and dust is a main route for phthalate delivery. We had
Accepted 19 December 2013 explored the effect of topically-applied phthalates on skin absorption and toxicity. Immunohistology,
Available online 31 December 2013
functional proteomics, and Western blotting were employed as methodologies for validating phthalate
toxicity. Among 5 phthalates tested, di(2-ethylhexyl)phthalate (DEHP) showed the highest skin reservoir.
Keywords: Only diethyl phthalate (DEP) and dibutyl phthalate (DBP) could penetrate across skin. Strat-M mem-
Ò

Phthalate
brane could be used as permeation barrier for predicting phthalate penetration through skin. The accu-
Skin absorption
Toxicity
mulation of DEHP in hair follicles was 15 nmol/cm2, which was significantly greater than DBP and DEP.
Proteomics DBP induced apoptosis of keratinocytes and fibroblasts via caspase-3 activation. This result was con-
Biomarker firmed by downregulation of 14-3-3 and immunohistology of TUNEL. On the other hand, the HSP60 over-
expression and immunostaining of COX-2 suggested inflammatory response induced by DEP and DEHP.
The proteomic profiling verified the role of calcium homeostasis on skin inflammation. Some proteins
investigated in this study can be sensitive biomarkers for dermal toxicity of phthalates. These included
HSPs, 14-3-3, and cytokeratin. This work provided novel platforms for examining phthalate toxicity on
skin.
Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction compounds (SVOC) (Mielke et al., 2011; Weschler and Nazaroff,

Phthalates are known as plasticizers to give plasticity for rigid
materials in many products. Nowadays the industry generates bil-
lions of pounds of phthalates every year. Phthalates are wide-
spread contaminants in both indoor and outdoor environments
(Guo and Kannan, 2011). These toxicants can be delivered into
body via inhalation, dietary intake, and skin absorption (Singh
and Li, 2011; Guo et al., 2012; Bekö et al., 2013). Skin is the largest
organ of the body, which is easily exposed to a large number of
toxicants, such as bisphenol A and semi volatile organic
⇑ Corresponding authors. Address: Department of Cosmetic Science, Chang Gung
University of Science and Technology, 261 Wen-Hwa 1st Road, Kweishan, Taoyuan
333, Taiwan (C.F. Lu). Address: Pharmaceutics Laboratory, Graduate Institute of
Natural Products, Chang Gung University, 259 Wen-Hwa 1st Road, Kweishan,
Taoyuan 333, Taiwan. Tel.: +886 3 2118800; fax: +886 3 2118236 (J.Y. Fang).
E-mail address: fajy@mail.cgu.edu.tw (J.-Y. Fang).
2012). Dermal absorption occurs at a significant rate for phthalates
(Schettler, 2006). Humans can be exposed to phthalates from
building materials, household furnishing, soil, and dust. Further-
more, some personal care products provide a direct contact of
phthalates to skin. Phthalates are employed in skin care products
for the roles as stabilizers, binders, emulsifiers, and lubricants
(Koniecki et al., 2011). The products containing phthalates for cos-
metic and topical uses include skin moisturizers, mosquito repel-
lents, perfumes, deodorants, shampoos, and nail polishes
(Matsuda et al., 2010). Guo et al. (2014) recently demonstrated that
hand and body lotions are the main contributors to dermal expo-
sure, especially for diethyl phthalate (DEP). Since phthalates are
not chemically bound to most of the products, they are easily re-
leased from products to skin (Kamarei et al., 2011).
There are more than 10 articles reporting the cause of allergic
contact dermatitis by phthalates (Yanagisawa et al., 2008; Lampel
and Jacob, 2011). Takano et al. (2006) suggested that phthalates

0278-6915/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.fct.2013.12.033
106 T.-L. Pan et al. / Food and Chemical Toxicology 65 (2014) 105–114
may be responsible for recent increment of dermatitis in the devel-
oped countries. Another concern is that babies and children are
more exposed as compared to adults (Wormuth et al., 2006;
Kolarik et al., 2008). Sathyanarayana et al. (2008) have demon-
strated that P7 phthalates are detected in urines of 81% infants
after using baby care products. Although it is approved that skin
exposure is a major source of phthalate toxicity, the mechanism
and severity of the toxicity have not been conclusively identified.
The absorption level of phthalates via topical route also has not
been systemically examined. We aimed to establish permeation
profiles of a series of phthalates in this study. These included
DEP, dibutyl phthalate (DBP), di(2-ethylhexyl)phthalate (DEHP),
diisononyl phthalate (DINP), and trioctyl trimellitate (TOTM). This
work was also designed to explore possible mechanisms governing
phthalate toxicity on skin.
To facilitate risk assessment of phthalates, the skin permeation
and adverse effects of phthalates should be determined by using
validated and relatively new methodologies. The dermal absorp-
tion of phthalates was evaluated by Franz cell assembly. The skin
acts as a signaling interface between xenobiotics and the body.
Any stress may contribute to alteration of genome conformation
and protein expression (Huang et al., 2005). Functional proteomics
can serve as a potent tool to elucidate cellular process at protein
level and identify target proteins involved in the process. We
examined proteomics of skin with phthalate treatment. Different
responses such as inflammation, differentiation, and apoptosis
are accompanied with skin disorders. Another purpose of this
study was to seek some biomarkers which show significant change
of expression after topical phthalate application. We had utilized
immunohistology and Western blotting as platforms for searching
biomarkers.
2. Materials and methods
2.1. Materials
DEP, DBP, DEHP, DINP, and TOTM were all commercially available from Sigma–
Ò
Aldrich (St. Louis, MO, USA) with a purity of >99%. Strat-M membrane for transder-
mal diffusion test was purchased from Merck Millipore (Darmstadt, Germany).
2.2. Animals
Female nude mouse (ICR-Foxn1nu) aged eight weeks were provided by National
Laboratory Animal Center (Taipei, Taiwan). Specific pathogen-free (SPF) pigs of one
week old were supplied by Animal Technology Institute Taiwan (Miaoli, Taiwan).
The protocol for animals was reviewed and approved by Institutional Animal Care
and Use Committee of Chang Gung University (approval number: CGU-10-030).
The committee confirmed that the animal experiment followed the guidelines as
set forth by the Guide for Laboratory Factlines and Care. Ethical issues with animal
experiments complied with Directive 86/609/EEC from European Commission. Ani-
mals were housed in cages in a room with controlled temperature (25 ± 1 °C) and
relative humidity (60 ± 5%). A laboratory diet and water were given ad libitum.
2.3. In vitro skin absorption
Full-thickness skin on dorsal region was excised from mice or pigs after sacri-
fice. The skin was mounted between donor and receptor of Franz cell with stratum
corneum (SC) facing upwards into donor side. The receptor medium (5.5 ml) con-
tained 40% ethanol in pH 7.4 buffer for maintaining sink condition. The donor med-
ium was 5.4 mM phthalates in 40% ethanol/pH 7.4 buffer. The diffusion area
between compartments was 0.785 cm2. The stirring rate of the stirrers in receptor
was 600 rpm. The temperature of receptor was maintained at 37 °C. A 300-ll ali-
quot was taken from receptor at determined intervals and immediately replaced
with an equal volume of medium. The sample number in this experiment was four.
The skin was removed from the cell after a 12-h permeation. The skin was
washed and weighed, cut with scissors, and positioned into a glass homogenizer
containing methanol. The duration for homogenization was 5 min. Then the result-
ing mixture was centrifuged at 10,000 rpm for 10 min. The supernatant was col-
lected for quantifying phthalate content by high-performance liquid
chromatography (HPLC).
2.4. HPLC setup
The HPLC system was Hitachi 7-series (Tokyo, Japan) with ultraviolet detector.
Ò
A C18 column was employed as stationary phase (LiChrospher , Merck, Darmstadt,
Germany). The mobile phase consisted of methanol and water. The ratio of metha-
nol and water was 70:30, 90:10, 95:5, 100:0, and 100:0 for DEP, DBP, DEHP, DINP,
and TOTM, respectively. Flow rate and wavelength was set to 1 ml/min and 220 nm,
respectively. The limit of detection (LOD) for DEP, DBP, DEHP, DINP, and TOTM was
10, 5, 15, 20, and 40 ng/ml, respectively.
2.5. Phthalate content in hair follicles
The skin removed from Franz cell was stripped by adhesive tape 20 times to ab-
late SC. Subsequent to stripping, follicular cast was prepared by pipetting a drop of
superglue (ethyl cyanoacrylate 7004T, 3M, Taipei, Taiwan) on a glass slide. This
slide was pressed onto surface of the skin. The cyanoacrylate polymerized, and
the slide was removed with quick movement after 5 min. The superglue remaining
on slide was scrapped off, subsequently placed in a test tube with 2 ml methanol.
The tube was shaken for 3 h, and then evaporated methanol by vacuuming. The mo-
bile phase was used to dilute the samples for HPLC determination.
2.6. Cell viability test
Human keratinocytes (HaCaT) and foreskin fibroblasts (Hs68) were seeded in
96-well plates by appropriate culture medium for 24 h. Phthalates in dimethyl sulf-
oxide (DMSO) were added to wells to make final concentrations of 0.5, 1, and 5 mM.
DMSO was used as the control (100% viability). After a 24-h incubation, tetrazolium
salt in isopropanol was added to the wells. The optical density of the dissolved
material was measured by spectrophotometer for calculating viability. The number
of replications was three.
2.7. Determination of caspase-3 and poly(ADP ribose) polymerase (PARP) in cells
After pretreatment of phthalates with cells at 6 and 12 h, the proteins were iso-
lated using cell lysis buffer and measured by Bradford protein assay kit (Amresco,
Solon, OH, USA). Total proteins were separated with 10% sodium dodecylsulfate–
polyacrylamide gel electrophoresis (SDS–PAGE). A Western blot analysis was per-
formed using caspase-3 and PARP. The levels of GADPH expression was used as a
gel loading control.
2.8. In vivo topical administration
Phthalates (5.4 mM) in 40% ethanol/pH 7.4 buffer at a volume of 0.6 ml was
spread on a nonwoven polyethylene cloth (1.5  1.5 cm2), and then applied to dor-
Ò
sal region of nude mouse. The cloth was fixed by Tegaderm adhesive dressing (3M,
Ò
St. Paul, MN, USA) and Fixomull stretch adhesive tape (Beierdorf AG, Hamburg,
Germany). The cloth was changed by a new one every 24 h. After 7 days, the cloth
was withdrawn, and the treated skin area was cleaned for the following physiolog-
ical examination, immunohistology, and proteomic profiling. There were six ani-
mals tested for each group.
2.9. Physiological examination of nude mouse skin
The skin treated by phthalates was examined by physiological parameters such
Ò
as transepidermal water loss (TEWL) and skin surface pH. A Tewameter (TM300,
Courage and Khazaka, Köln, Germany) was employed for recording TEWL (g/m2/
Ò
h). The pH of skin was detected by Skin-pH-Meter pH 905 (Courage and Khazaka).
An adjacent untreated skin was used as a baseline standard for each examination.
The sample number was six for each parameter.
2.10. Immunohistology
The skin excised from nude mouse treated by phthalates was fixed in a 10% buf-
fered formaldehyde at pH 7.4. The samples were dehydrated with ethanol and
embedded by paraffin wax. The skin was vertically cut to slices with a thickness
of 3 lm. For cyclooxygenase (COX)-2, proliferating cell nuclear antigen (PCNA),
and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining,
the antibodies (Santa Cruz, CA, USA) were used. The procedures for immunostaining
were performed according to a previous report (Lin et al., 2009).
2.11. Two-dimensional gel electrophoresis (2DE) and image analysis
The tissue samples (250 lg) extracted from phthalate-treated skin were thawed
and diluted in immobilized pH gradient sample buffer containing 7 M urea, 2 M
thiourea, 2% 3-[3-(cholamidopropyl) dimethylammoniol]-1-propanesulfonate
(CHAPS), 65 mM dithiothreitol, and 1% IPG buffer to a volume of 350 ll. After rehy-
dration for 12 h at 30 V, isoelectrofocusing (IEF) was automatically conducted with
a total of 75 kV h. Following IEF separation and equilibration, electrophoresis was
 T.-L. Pan et al. / Food and Chemical Toxicology 65 (2014) 105–114
carried out on 12% acrylamide gels at 40 mA. Proteins were visualized by silver
staining and then scanned using an image scanner (Amersham Bioscience, Bucking-
hamshire, UK). Protein spots were quantified using Prodigy SameSpots software
(Nonlinear Dynamics, Newcastle, UK). The amount of each protein spot was pre-
sented as its volume. Spot volumes were normalized as a percentage of the total
volume of all spots appearing in a gel. The average value of each protein spot was
calculated from three independent experiments. All experiments were repeated
three times to confirm the reproducibility of the protein profiles.
2.12. In-gel-digestion of proteins and MALDI-time of flight (TOF)/MALDI-TOF-TOF mass
spectroscopy
Silver-stained spots were excised and in-gel-digested with trypsin according to
procedures described previously (Wang et al., 2006). Briefly, gels were destained
with 1% potassium ferricyanide and 1.6% sodium thiosulfate. Then the proteins
were reduced with 25 mmol/l NH4HCO3 containing 10 mM dithiothreitol at 56 °C
for 30 min and alkylated with 55 mM iodoacetamide at room temperature for
30 min. The proteins were digested with 20 lg/ml trypsin at 37 °C overnight. After
digestion, the tryptic peptides were acidified with 0.5% TCA and loaded onto an MTP
AnchorChip™ 600/384 TF (Bruker-Daltonik, Billerica, MA, USA). The MALDI-TOF MS
Ò increase in skin uptake was detected for DEHP as compared to
analysis was performed on an Ultraflex MALDI-TOF MS spectrometer (Bruker-Dal-
tonik). Monoisotopic peptide masses were assigned and used for database searches
with the MASCOT search engine (http://www.matrixscience.com) (Matrix Science,
London, UK). Search parameters were set as follows: a maximum allowed peptide
mass error of 50 ppm, and consideration of 1 incomplete cleavage per peptide.
For TOF/TOF, the 3 most intense precursor ions with a signal/noise ratio of >25 were
selected after exclusion of common background signals. The TOF/TOF mode was
operated at 1 keV, and products of metastable decomposition at the elevated laser
power were detected. TOF data were acquired with close external calibration and
TOF/TOF data using default instrument calibration (Lubec et al., 2005).
2.13. Western blotting
Proteins were separated on 12% denatured gel and transferred to polyvinylidene
difluoride membrane. The membrane was incubated for 1 h with blocking solution
of 5% non-fat milk-TBST solution, followed by immersion in the same solution with
a respective antibody against HSP27 (1:5000), HSP60(1:8000), 14-3-3 (1:2000),
cytokeratin (1:5000), and GADPH (1:8000) overnight at 4 °C. After washing with
TBST, peroxidase-labeled anti-rabbit IgG was added and incubated for 2 h. After
washing in TBST for 8 times, enhanced chemiluminescence was used for protein
detection.
2.14. Statistical analysis
Statistical analysis of differences between different treatments was evaluated
using the non-parametric Kruskal–Wallis test with Dunn’s post-test. A 0.05 level
of probability was taken as the level of significance. An analysis of variance (ANO-
VA) was also used for evaluation if necessary.
3. Results
3.1. In vitro skin absorption
Phthalates are esters with different alkyl chain types and num-
bers. Among these, DEHP and DINP possess the branched chains.
The molecular weight of phthalates studied in this report increased
in the order of TOTM > DINP > DEHP > DBP > DEP (Table 1). The
predicted partition coefficient (Alog P) measured by molecular
Ò
modeling (Discovery Studio , Accelys, San Diego, CA, USA) also
showed the same trend, with DEP and TOTM revealed the lowest
Table 1
Skin accumulation (nmol/mg) and flux (nmol/cm2/h) of phthalates (5.4 mM) after in vitro skin absorption.
Compound MWa Alog Pb Skin accumulation (nmol/mg)
Nude mouse
DEP 222 2.24 1.37 ± 0.26
DBP 278 4.20 10.8 ± 0.51
DEHP 391 7.57 12.0 ± 4.82
DINP 419 8.35 5.32 ± 2.67
TOTM 547 10.9 1.32 ± 0.53
The data represent the mean ± SD (n = 4).
a
MW, molecular weight.
Ò
b
Alog P, partition coefficient calculated by Discovery Studio version 2.0.
107
and highest lipophilicity, respectively. TOTM has three alkyl chains,
which is more than the other phthalates (two chains). The extra
chain contributes to great lipophilicity of TOTM. The skin perme-
ation of phthalates was determined through Franz diffusion cell.
Both dermal reservoir and transdermal flux were examined. In this
in vitro condition, the permeant maintained in skin reservoir indi-
cates skin deposition, whereas the permeant in receptor implies
in vivo availability into systemic absorption. Both nude mouse
and pig skins were utilized as permeation barriers. As shown in
Table 1, skin accumulation of phthalates increased following the
increase of chain length. The maximum skin uptake was achieved
by DEHP. However, the further extension of chain length (DINP and
TOTM) had decreased skin accumulation. The value of DEHP accu-
mulation within nude mouse skin was statistically similar
(p > 0.05) to the value of DBP. Skin deposition of DEHP was 9-fold
higher than that of DEP. With respect to pig skin, a 2- and 6-fold
DBP and DEP.
Phthalate flux which was calculated from the slope of pene-
trated amount versus time profiles is summarized in Table 1. The
flux of DEP and DBP across nude mouse skin was comparable
(p > 0.05). Contrary to this result, DEP flux was 2-fold statistically
higher (p < 0.05) as compared to DBP flux via pig skin. No permeant
was found in receptor after treatment of DEHP, DINP, and TOTM for
12 h. Instead of using animal skin, we detected phthalate amount
Ò
in receptor by employing Strat-M . This membrane is an alterna-
tive for transdermal diffusion testing developed by Merck Milli-
pore. As depicted in Fig. 1A, DEP gives the highest penetration,
followed by DBP. DEHP showed a negligible accumulation in recep-
tor during 12 h. This result was consistent with transdermal flux
Ò
across pig skin, demonstrating a good substitute of Strat-M for
animal skin. Hair follicles may be essential pathways to be in-
volved in topically-applied permeants. The amount of phthalates
in follicles was quantified for the selected phthalates including
DEP, DBP, and DEHP. As shown in Fig. 1B, the recovery of phtha-
lates from follicular cast is significantly increased (p < 0.05) as
the chain length increased. DEHP exhibited a follicular deposition
of 15 nmol/cm2. There was no deposition in follicles for DEP.
3.2. Cell viability test
The effect of phthalates upon cell viability of keratinocytes and
skin fibroblasts is determined as shown in Fig. 2A and B, respec-
tively. Various concentrations from 500 lM to 5 mM were tested.
Phthalates generally evoked a concentration-dependent behavior
of cell viability. At higher dose (5 mM), the cell death increased fol-
lowing the decrease of carbon chain length, with DEP showed the
lowest viability. DEP at different concentrations presented a bipha-
sic pattern on cell death. The lower doses of DEP (500 lM and
1 mM) did not affect keratinocyte and fibroblast proliferation.
DBP and DEHP demonstrated more statistically potent death
(p < 0.05) on cells as compared to DEP at both concentrations.
Flux (nmol/cm2/h)
Pig Nude mouse Pig
0.13 ± 0.01 96.5 ± 7.97 26.9 ± 6.82
0.41 ± 0.07 94.7 ± 6.49 11.9 ± 3.20
0.82 ± 0.22 0 0
0.36 ± 0.16 0 0
0.35 ± 0.19 0 0
108 T.-L. Pan et al. / Food and Chemical Toxicology 65 (2014) 105–114

The same as DEP, DINP and TOTM did not inhibit cell proliferation These results give evidence that phthalate-induced cell death
at 500 lM and 1 mM. was mediated by cascades of caspase-3 and PARP.
We also investigated caspase-3 activation and PARP cleavage,
which are the hallmarks of apoptosis. During process of apoptosis, 3.3. Physiological examination of nude mouse skin
caspase-3 would be activated to generate subunit. As shown in
Fig. 3, caspase-3 precursor but not subunit is present in non-treat- The change of skin physiology including TEWL and skin surface
ment control at 6 and 12 h. Caspase-3 activity was detectable at 6 h pH is examined as summarized in Table 2. The D values (value of
for only DBP. At 12 h, three phthalates exhibited caspase-3 subunit, treated site minus value of the untreated site) were calculated.
with DBP showing the highest amount. PARP is cleaved by caspase- As compared to vehicle-control group, three phthalates signifi-
3 activation during cell death. A PARP fragment of 89 kDa is ap- cantly increased (p < 0.05) DTEWL. The DTEWL values of phtha-
peared by caspase-3 proteolysis. After 6 and 12 h, a significant lates were comparable to each other (p > 0.05). Phthalate
PARP cleavage was observed for DBP. A lower cleavage was de- exposure slightly but significantly (p < 0.05) shifted skin pH to
tected by DEP and DEHP treatments rather than DBP treatment. alkaline condition. The DpH of DBP was 1.76, which was greater
than those of DEP and DEHP.

2500 25 *
DEP
*
Permeated amount (nmol/cm2)

DBP

Amount in follicles (nmol/cm2)

DEHP
2000 20
(A) (B)
1500 15

1000 10

500 5
*

0 0
0 2 4 6 8 10 12 DEP DBP DEHP
Time (h)
Ò
Fig. 1. In vitro permeation of the cumulative amount-time profiles of the transdermal phthalate penetration in the receptor by using Strat-M membrane as diffusion barrier
(A) and phthalate content in hair follicles by using nude mouse skin as diffusion barrier (B). All data are presented as the mean of four experiments ± SD. Indicates a
significant difference (p < 0.05) between the data of two groups.

120 0.5 mM 1 mM 5 mM 120 0.5 mM 1 mM 5 mM

(A) (B)
100 100
* *
80 *
Cell viability (%)

80
Cell viability (%)

* * *
*
60 60
*
*
* *
40 40

* *
20 * 20

0 0
DEP DBP DEHP DINP TOTM DEP DBP DEHP DINP TOTM

Fig. 2. Cell viability (%) of cultured keratinocytes (A) and skin fibroblasts (B) after treatment with different concentrations of phthalates. Each value represents the mean and
SD (n = 3). Indicates a significant difference (p < 0.05) as compared to the data of 0.5 mM.

Table 2
In vivo skin irritation examination by Dtransepidermal water loss (TEWL) and DpH
after a 6-day application of topically applied phthalates (5.4 mM) in 40% ethanol/pH
7.4 buffer.

Compound DTEWL (g/m2/h) DpH

Control 3.16 ± 2.68 0.24 ± 0.28
DEP 10.78 ± 3.46 1.06 ± 0.61
DBP 7.10 ± 4.74 1.76 ± 0.44
Fig. 3. Caspase-3 activation and PARP cleavage of cultured keratinocytes after DEHP 10.14 ± 6.77 0.55 ± 0.35
treatment with phthalates at 6 and 12 h. C indicates the control group (treatment of
40% ethanol/pH 7.4 buffer without phthalates). The data represent the mean ± SD (n = 6).
 T.-L. Pan et al. / Food and Chemical Toxicology 65 (2014) 105–114 109

3.4. Immunohistology
We evaluated phthalate-treated skin stained by COX-2, PCNA,
and TUNEL as shown in Fig. 4A–C, respectively. COX-2 was ex-
pressed at inflammatory regions. As seen in the left side of
Fig. 4A, skin histology of untreated control indicates an intact
structure. COX-2 expression is a product of the phthalates selected.
Both DEP and DEHP treatments upregulated epidermal COX-2. On
the other hand, DBP did not alter COX-2 expression. Immunohisto-
chemical results of PCNA demonstrated increased level of the three
phthalates tested (Fig. 4B). There was an accumulation of PCNA
expression near follicular regions. The expression of TUNEL is an
indicator of apoptosis. As shown in Fig. 4C, the number of nuclear
TUNEL-positive cells (brown color in red circle) is much greater for
DBP compared to DEP and DEHP. This tendency was in accordance
with the results of caspase-3 activation and PARP cleavage.
ysis to characterize protein changes in skin. Fig. 5A indicates a typ-
ical protein profile of silver-stained gels of the control. We further
identified target proteins using in-gel trypsin digestion and peptide
mass fingerprinting. Database searches with peptide masses re-
sulted in positive identification of 14 different proteins associated
with DEP, DBP, and DEHP (Fig. 5B–D). Phthalate exposure affected
protein amount in the skin. The results and multiples of change of
the spectrometric analyses are summarized in Table 3. These iden-
tified proteins are involved in various biological functions includ-
ing inflammation, differentiation, calcium binding, and oxidative
stress, etc. We observed that the amount of 5 proteins underwent
changes corresponding to >2.5-fold. These included translationally
controlled tumor protein (TCTP), myosin regulatory light chain 2
(MLRS), 14-3-3 sigma, glucose-regulated protein (GRP)78, and
TBB5. The other proteins which showed differences of <2.5-fold
were not further discussed.

3.5. 2DE and image analysis 3.6. Western blotting

To examine changes in protein expression following treatment Immunoblotting of skin samples examines expression of spe-
of nude mouse skin with phthalates for 7 days, we used a 2DE anal- cific biomarkers after in vivo phthalate treatments of nude mouse

(A)
C DEP DBP DEHP

(B)
C DEP DBP DEHP

(C)
C DEP DBP DEHP

Fig. 4. Immunohistochemical examination of nude mouse skin stained with COX-2 (A), PCNA (B), and TUNEL (C) after treatment with phthalates for 7 days. Untreated skin is
shown at the left panel of the figure for comparison. C Indicates the control group (treatment of 40% ethanol/pH 7.4 buffer without phthalates).
110 T.-L. Pan et al. / Food and Chemical Toxicology 65 (2014) 105–114

11
13
12
14

10

9
8
4
3
7
2
1
6

11 11 11

13 12 13 12 13 12
14 14 14

10 10 10

9 9 9
8 8 8
4 4 4
3 3 3
7 7 7
2 2 2
1 1 1
6 6 6

5 5 5

Fig. 5. Comparative two-dimensional electrophoretic (2DE) patterns of nude mouse skin treated by control vehicle (30% ethanol) (A), DEP (B), DBP (C), and DEHP (D). The
protein lysate (250 lg) was focused on a pH 4–7 linear IPG strip before being separated on a 12% polyacrylamide gel. The numbers in this figure indicate the proteins
extracted from skin samples: 1, TCTP; 2, GDIR2; 3, APRV1; 4, annexin A5; 5, MLRS; 6, MYL1; 7 PRDX2; 8, 14-3-3 protein sigma; 9, annexin A8; 10, serpin B5; 11, GRP78; 12,
PDIA3; 13, HSP60; 14, TBB5. The detailed information of these spots is shown in Table 3. C indicates the control group (treatment of 40% ethanol/pH 7.4 buffer without
phthalates).

skin as shown in Fig. 6. Two heat shock proteins (HSP27 and
HSP60) were elucidated. DEP and DEHP elicited a slight increase
in HSP27 expression as compared to the control. Contrary to this
result, HSP27 level was reduced by DBP. DEP and DEHP signifi-
cantly upregulated HSP60. This protein was slightly increased by
DBP. The Western blot data of 14-3-3 showed a downregulation
by DBP exposure. This result was consistent with 14-3-3 sigma
expression in proteomic mapping. Cytokeratin level was reduced
after treatments of all phthalates tested, with DBP demonstrated
the lowest expression.
4. Discussion
The goal of this work was to systemically investigate phthalate
toxicity on skin. We also tried to establish a platform and find some
indicators for risk assessment of phthalates. Infants and children
are populations with high risk when exposed to phthalates because
of their developing skin. Moreover, baby care products, cosmetics,
and lotions are products which contain high phthalate levels
(Lampel and Jacob, 2011; Guo et al., 2014). It is reported that der-
mal uptake was a predominant source of DEP and DBP for infants
and teenagers (Wormuth et al., 2006). The epidermis of infants is
found to have 4.3 ± 0.7 cell layers (Telofski et al., 2012), which
was similar to the cell layers of nude mouse epidermis (3–4 cell
layers). Facial skin is a predominant site for administering skin care
products. Nude mouse skin with greater permeability than human
skin is feasible as an alternative for facial skin since the permeabil-
ity of face is higher than that of the other sites by 4-fold (Benson
et al., 2005). Nude mouse skin represents baby and facial skin in
this study. On the other hand, pig skin can be regarded as a model
mimicking adult human skin in the present work. Both skins are
acceptable in terms of a similar SC morphology and hair sparseness
to human skin (Lee et al., 2012).
DEP is extensively used as a stabilizer in cosmetics and per-
fumes. Skin exposure is the major pathway of body uptake for
DEP (Guo and Kannan, 2011). The concentration of DEP in cosmetic
formulations can be varied from 0.1% to 50% (Koo and Lee, 2004).
DBP is found in skin care products at concentration of >5%, espe-
cially nail polishes (15%) (Janjua et al., 2007). Both DEP and DBP ex-
ist in transdermal drug patch as a plasticizer with a very high
concentration of 30% (Limpongsa and Umprayn, 2008). DEHP is
the most dominant phthalate in the indoor dust and diet (Kolarik
et al., 2008; Langer et al., 2010; Guo et al., 2012). TOTM is currently
used in cable and medical devices such as blood infusion sets.
Our in vitro skin accumulation experiment showed that phtha-
lates could be absorbed into skin reservoir. DBP and DEHP demon-
strated higher reservoir as compared to the others. DBP could act
as a penetration enhancer by damaging barrier function of SC
(Dearman et al., 1996). Some phthalates are used as skin moistur-
izers for softening SC. The TEWL data confirmed SC disruption by
phthlates, thus increasing skin permeation. Skin partitioning is
 T.-L. Pan et al. / Food and Chemical Toxicology 65 (2014) 105–114 111

Table 3
List of identified spots in Fig. 5.

Spot Protein Accesion Mwa pIb Matched Sequence Ratios to control Biological function
no. no (kDa) peptides coveragec
DEP DBP DEHP
SCORE (%)
1. TCTP P63028 19,564 4.76 10 115 (44%) 2.5 1.5 2.0 Involved in calcium binding and microtubule stabilization
2. GDIR2 (Rho Q61599 22,894 4.97 6 83 (47%) 2.0 2.4 1.5 Regulates the GDP/GTP exchange reaction of the Rho proteins
GDP- by inhibiting the dissociation of GDP from them, and the
dissociation subsequent binding of GTP to them
inhibitor 2)
3. APRV1 Q09PK2 37,436 5.74 10 123 (51%) 2.0 1.5 1.2 Highly expressed in stratified epithelia in skin, tongue,
esophagus, forestomach and vagina. Also expressed in trachea,
urinary bladder and thymus. Undetectable in simple epithelia.
Within the epidermis, expressed exclusively in the granular
layer (at protein level). Levels are elevated in benign skin
tumors but are down-regulated in squamous cell carcinomas
4. Annexin A5 P48036 35,787 4.83 21 210 (65%) 1.6 1.4 2.0 This protein is an anticoagulant protein that acts as an indirect
inhibitor of the thromboplastin-specific complex, which is
involved in the blood coagulation cascade.
5. MLRS (myosin P97457 19,057 4.82 19 246 (88%) 3.0 3.4 2.5 This chain binds calcium
regulatory
light chain 2)
6–1. MYL1 P05977 20,695 4.98 13 128 (68%) 1.3 1.6 1.5 Regulatory light chain of myosin. Does not bind calcium
6–2. PRDX2 Q61171 21,936 5.20 8 70 (45%) 1.3 1.6 1.5 Involved in redox regulation of the cell. Reduces peroxides
with reducing equivalents provided through the thioredoxin
system. It is not able to receive electrons from glutaredoxin.
May play an important role in eliminating peroxides
generated during metabolism. Might participate in the
signaling cascades of growth factors and tumor necrosis
factor-alpha by regulating the intracellular concentrations of
H2O2
7. 14-3-3 protein O70456 27,803 4.72 11 143 (52%) 2.1 2.5 1.8 Adapter protein implicated in the regulation of a large
sigma spectrum of both general and specialized signaling pathways.
Binds to a large number of partners, usually by recognition of a
phosphoserine or phosphothreonine motif. Binding generally
results in the modulation of the activity of the binding partner.
When bound to KRT17, regulates protein synthesis and
epithelial cell growth by stimulating Akt/mTOR pathway
9. Annexin A8 O35640 36,929 5.68 20 203 (68%) 2.7 2.3 2.4 This protein is an anticoagulant protein that acts as an indirect
inhibitor of the thromboplastin-specific complex, which is
involved in the blood coagulation cascade.
10. Serpin B5 P70124 42,484 5.55 15 155 (62%) 2.5 1.5 2.8 Tumor suppressor. It blocks the growth, invasion, and
metastatic properties of mammary tumors. As it does not
undergo the S (stressed) to R (relaxed) conformational
transition characteristic of active serpins, it exhibits no serine
protease inhibitory activity.
11. GRP78 P20029 72,492 5.07 35 300 (51%) 3.4 3.7 2.2 Probably plays a role in facilitating the assembly of multimeric
protein complexes inside the ER
12. PDIA3 P27773 57,099 5.88 26 277 (51%) 2.1 1.5 2.0 Catalyzes the rearrangement of -S-S- bonds in proteins.
13. HSP60 P63038 61,088 5.91 16 149 (42%) 2.2 1.2 3.2 Implicated in mitochondrial protein import and
macromolecular assembly. May facilitate the correct folding of
imported proteins. May also prevent misfolding and promote
the refolding and proper assembly of unfolded polypeptides
generated under stress conditions in the mitochondrial matrix
14. TBB5 Q8WUC1 50,095 4.78 30 270 (74%) 2.6 3.7 1.2 Tubulin is the major constituent of microtubules. It binds two
moles of GTP, one at an exchangeable site on the beta chain
and one at a non-exchangeable site on the alpha chain
a
Mw: molecular weight.
b
pI: isoelectric point.
c
The sequence coverage is the percentage of the protein sequence where matching peptides have been identified. The larger the sequence coverage is, the more certain is
the characterization and the more of the sequence is confirmed by the experimental data.

another parameter determining phthalate absorption. A permeant
in vehicle is initially partitioned and accumulated into lipophilic
SC, following is a passive diffusion to deeper strata (Lin et al.,
2012). It can be expected that skin uptake was increased with
the increase of phthalate lipophilicity. However, it is not a univer-
sal rule since DINP and TOTM showed a reduced skin accumulation
although their Alog P was relatively high (8.35 and 10.86). The
extremely lipophilic permeants do not permeate easily into skin
since the primary resistance may shift from SC to aqueous viable
strata (Doan et al., 2010; Pan et al., 2010). Another possibility of
the low accumulation of DINP and TOTM was larger molecular
volume. However, it should be noted that the effect of molecular
size on phthalate penetration was controversial. Elsisi et al.
(1989) demonstrated that diisodecyl phthalate with a molecular
weight of 447 Da could be found in urine after topical administra-
tion. Further study is needed to explore the role of molecular size
on skin permeation. Our results demonstrated that the skin could
be a significant route for phthalate intake, which was in line with
previous studies on in vitro skin permeation (Scott et al., 1987;
Frasch and Barbero, 2005). DEP and DBP initially form a reservoir
in skin (Payan et al., 2001), subsequently they diffused out of skin
into receptor. This indicates the possibility of entering systemic
circulation. Janjua et al. (2008) demonstrated that DEP and DBP are
systematically absorbed and excreted in urine via topical application.
112 T.-L. Pan et al. / Food and Chemical Toxicology 65 (2014) 105–114
Fig. 6. The Western blotting of HSP27, HSP60, 14-3-3, and cytokeratin in nude
mouse skin after treatment with phthalates for 7 days. GAPDH was used as a control
for different groups. C indicates the control group (treatment of 40% ethanol/pH 7.4
buffer without phthalates).
Guo et al. (2011) also showed a high level of DEP and DBP metabolites
Ò
in urine after human exposure. Phthalate release via Strat-M
membrane gave a good correlation with transdermal flux via skin,
indicating the feasibility of using this membrane for predicting skin
penetration behavior of phthalates without the use of animals.
The result provides evidence that hair follicles played a main
role for DEHP absorption. This effect was insignificant for DEP
and DBP. DBP can induce allergic dermatitis via a specific uptake
of appendageal routes (Simonsson et al., 2012). The severity of skin
sensitization may be augmented by topically-applied DEHP. Load-
ing follicle-related products such as shampoo, hair spray, and after
shave lotion with DEHP should be done with caution. Follicles offer
a long-term reservoir for permeants with prolonged deposition
(Lin et al., 2013).
DEP showed a biphasic inhibition behavior on skin cell prolifer-
ation. DEP at 5 mM revealed higher inhibition than the other
phthalates. On the other hand, DEP at lower concentrations
(500 lM and 1 mM) did not cause any cell death (viability  100%).
A dose-dependent inhibition on cell growth was demonstrated for
other phthalates. Keratinocytes represent an important function on
skin barrier persistence. In vivo TEWL evaluation is an indicator of
SC and epidermal barrier damage. The TEWL results showed that
DEP, DBP, and DEHP elicited significant damage of skin barrier
due to TEWL enhancement, giving evidence of decreased keratino-
cyte viability. Although keratinocytes can continuously differenti-
ate and proliferate for maintaining barrier function, exposure to
phthalates disrupted these procedures and led to damage of barrier
performance. A subsequent increase of skin permeability to phtha-
lates and other ingredients in the formulations could be occurred
following this damage.
According to biomarkers examined by immunohistology, pro-
teomics, and Western blotting, we may suggest that inflammation,
differentiation, apoptosis, and calcium binding are involved in skin
toxicity induced by phthalates. COX-2 is highly elicited by inflam-
matory stimulation. COX-2 may stimulate the synthesis of prosta-
glandins, which play a role in such functions like inflammation,
pain, and wound repair (Rundhaug and Fischer, 2008). Our immu-
nohistology suggested an upregulated expression of COX-2 by DEP
and DEHP. A previous study (Yanagisawa et al., 2008) demon-
strates that DEHP can induce skin inflammation. Exposure of skin
to DEHP initiates atopic dermatitis-like lesion. No COX-2 upregula-
tion was observed for DBP, indicating that DBP may elicit skin reac-
tions through mechanisms different from other phthalates tested.
TCTP is a proinflammatory marker (Bommer and Thiele, 2004).
TCTP in proteomic mapping also showed a more-significant in-
crease by DEP and DEHP than by DBP. The synthesis of HSPs is en-
hanced by a variety of stresses. Among all HSPs, HSP27 can be
served as a marker of skin irritation and allergic hypersensitivity
(Yusuf et al., 2009). HSP60 is an indicator of COX-2 and prostaglan-
dins (Billack et al., 2002), which is found in mitochondria. The
upregulation of HSP27 and HSP60 in DEP- and DEHP-treated skin
confirmed a significant inflammation response.
DBP decreased HSP27 level in skin. HSP27 is also an effective
marker of keratinocyte differentiation (Sur et al., 2008). The dam-
aged skin by environmental stress would reconstitute to normal
condition via skin repair and restoration. Both inflammation and
differentiation are activated during wound repair stage. It suggests
that the wound healing process after DBP treatment would be
slower than that of DEP and DEHP. This hypothesis can be ap-
proved by cytokeratin blotting. Cytokeratin is a protein involved
in skin integrity maintenance and epidermal differentiation (Sivan
et al., 2002). Although cytokeratin was downregulated by all
phthalates tested, DBP showed the least expression. The differenti-
ation procedure was retarded by DBP. The loss of cytokeratin con-
tributes to disruption of epidermal integrity (Rugg and Leigh,
2004). This was the reason of increased TEWL by phthalate expo-
sure. Cytokeratin loss also leads to cytolysis (Porter et al., 1996).
The clinical appearance of cytokeratin loss is skin aging (Oender
et al., 2008). Proliferation is also an important process for skin
recovery to normal status. The markers which enhance keratino-
cyte proliferation can be considered as factors prohibiting differen-
tiation. PCNA is a marker associated with epidermal proliferation
(Sivan et al., 2002). The combination of PCNA and cytokeratin re-
sults had suggested the significant proliferation and limited differ-
entiation of phthalate-treated skins after a 7-day administration.
Although DEP and DEHP may produce differentiation based on
HSP27 blotting, this overexpression was not very significant.
The limited differentiation by phthalates can be confirmed by
14-3-3 proteins, which are predominantly synthesized by differen-
tiated but not proliferating keratinocytes (Kilani et al., 2008). The
findings of proteomics and Western blotting of 14-3-3 indicated
a significant inhibition by DBP. The change of 14-3-3 expression
by DEP and DEHP was limited. Downregulation of 14-3-3 enhances
apoptosis by Bax sequestration (Medina et al., 2007), which is an
apoptosis regulator promoting apoptosis via binding to and antag-
onizing Bcl-2 protein. The keratinocyte and fibroblast viability
study demonstrated that DBP greatly suppressed cell growth at
higher concentrations. DBP induced apoptosis through caspase-3
activation and PARP cleavage. With respect to the in vivo exposure
of DBP for 7 days, both TUNEL and PCNA were expressed at a high
level. This may demonstrate that both apoptosis and proliferation
occurred after DBP treatment. TCTP is a protein which prevents
apoptosis by calcium binding (Bommer and Thiele, 2004). The
2DE profiles exhibited that DBP-treated skin expressed lower TCTP
as compared to DEP- and DEHP-treated skins. Inflammation is
usually accompanied with increase in calcium concentration
(Lansdown, 2002). A previous study (Ghosh et al., 2010) also
indicates that DEHP exposure in hepatocytes triggers intracellular
calcium level by a dose-dependent fashion. There were some pro-
teins related to calcium interaction in our 2DE results. Calcium
triggers binding to MLRS. Phthalates increased MLRS expression
by a 3-fold, indicating the increase of calcium concentration
although DBP also upregulated MLRS to a certain level. Calcium
homeostasis alteration and inflammation contributes to stress
response of endoplasmic reticulum (ER). GRP78 is an ER protein
highly expressing during ER stress (Rao et al., 2002). The three
phthalates, including DBP, showed GRP78 overexpression.
Although DBP did not cause significant inflammation and TCTP
level, the GRP78 enhancement was high (3.7 folds). This may be
due to that GRP78 is not only an indicator of calcium homeostasis
change, but also acts as regulator of apoptosis, oxidative stress, and
chemical toxicity (Rao et al., 2002; Pan et al., 2010).
Another concern of phthalate exposure on skin is the possibility
of carcinogenesis. Some proteins examined in this work are bio-
markers of epidermal malignancy, including HSP27 and cytokera-
tin. HSP27 overexpression suggests tumorigenesis of squamous
cell carcinoma (Huang et al., 2005). Marked increase of cytokeratin
indicates the process of skin adnexal carcinoma (Sun et al., 2009;
 T.-L. Pan et al. / Food and Chemical Toxicology 65 (2014) 105–114
Mahalingam et al., 2010). The negligible change or significant
downregulation of these proteins by phthalates demonstrated that
phthalates may not cause skin carcinogenesis after a consecutive
exposure of 7 days. The experimental results of this report indicate
a different response on skin toxicity by DBP and DEP/DEHP. The
skin reaction of DBP was mediated by modulating apoptosis and
proliferation without a significant inflammation. DEP and DEHP
produced inflammatory responses and a moderate differentiation.
DEP and DEHP showed a comparable change on most of the bio-
markers tested. Since skin deposition of DEP was much lower than
that of DEHP, this suggested a more-prominent toxicity of DEP
compared to DEHP. The report is limited to local skin behavior after
topical phthalate application. The limitation of this study was the
insufficient replicates (n = 6) in the in vivo experiment, resulting
in the deficiency for valid statistics. Another concern is the use of
animal but not human skin in this study. According to the report
by Beydon et al. (2010), the prediction of percutaneous absorption
of phthalates in human by extrapolation from animal data should
be done carefully. Further study on a large group for animals or hu-
mans is necessary to validate the findings.
5. Conclusions
The toxicity caused by phthalates is an issue with increasing
importance. Consumers receiving skin care products and cosmetics
are always exposed to more than one phthalate. This may result in
a synergistic or additive effect of skin damage. The research of skin
toxicity elicited by phthalates was conducted in this study. We had
used validated methodologies for evaluating phthalate toxicity on
skin. Our results demonstrated that different phthalates revealed
different absorption level, with DEHP showed the greatest accumu-
lation into skin. There was also no common trend of alteration of
skin protein expression by exposure to different phthalates. A sig-
nificant apoptosis was observed for DBP but not DEP and DEHP. On
the other hand, both DEP and DEHP exhibited greater inflamma-
tory stimulation than DBP. The experimental results presented in
this work possessed clinical relevance for finding effective bio-
Ò Koniecki, D., Wang, R., Moody, R.P., Zhu, J., 2011. Phthalates in cosmetics and
markers for phthalate toxicity. Strat-M could be helpful to predict
phthalate penetration across skin. HSPs, 14-3-3, and cytokeratin
may serve as sensitive biomarkers for differentiating skin toxicity
of various phthalates. Proteomic mapping supplied information
about the effect of phthalates on skin toxicity, especially calcium
homeostasis. This report is helpful to establish the knowledge
about skin toxicity elicited by phthalates.
6. Conflict of Interest
The authors declare that there are no conflicts of interest.
Transparency document associated with this article can be
found, in the online version, at http://dx.doi.org/10.1016/
j.fct.2013.12.033.
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