ORIGINAL ARTICLE

Impaired Activation of the Nrf2-ARE Signaling

Pathway Undermines H2O2-Induced Oxidative Stress
Response: A Possible Mechanism for Melanocyte
Degeneration in Vitiligo
Zhe Jian1,2, Kai Li1,2, Pu Song1,2, Guannan Zhu1, Longfei Zhu1, Tingting Cui1, Bangmin Liu1, Lingzhen Tang1,
Xiaowen Wang1, Gang Wang1, Tianwen Gao1 and Chunying Li1

Vitiligo melanocytes possess higher susceptibility to oxidative insults. Consistent with this, impairment of the
antioxidant defense system has been reported to be involved in the onset and progression of vitiligo. Our
previous study showed that the nuclear factor E2-related factor 2–antioxidant response element (Nrf2-ARE)
pathway and its downstream antioxidant enzyme heme oxygenase-1 (HO-1) are crucial for melanocytes to cope
with H2O2-induced oxidative damage. Here, we sought to determine whether the diminished Nrf2-ARE activity
that contributes to reduced downstream antioxidant enzymes and increased oxidative stress could be the reason
why melanocytes are more vulnerable to vitiligo. We found that vitiligo melanocytes exhibited hypersensitivity to
H2O2-induced oxidative injury because of reduced Nrf2 nuclear translocation and transcriptional activity, which
led to decreased HO-1 expression and aberrant redox balance. Moreover, we also found that the level of serum
HO-1 was significantly decreased and that of IL-2 was markedly increased in 113 vitiligo patients when compared
with healthy controls. These data demonstrate that impaired activation of Nrf2 under oxidative stress could result
in decreased expression of antioxidant enzymes and increased death of vitiligo melanocytes.
Journal of Investigative Dermatology advance online publication, 17 April 2014; doi:10.1038/jid.2014.152

INTRODUCTION accumulation of free radicals in the epidermis of affected

Vitiligo is a common skin depigmenting disorder character-
ized by the death of melanocytes from lesional epidermis
(Schallreuter et al., 2008; Spritz, 2008). Despite much
research, the etiology of vitiligo and the causes of melano-
cyte death are not yet fully understood. Recent experimental
and clinical evidence suggests that oxidative stress and
1
Department of Dermatology of Xijing Hospital, Fourth Military Medical
University, Xi’an, China
2
These authors contributed equally to this work.
Correspondence: Tianwen Gao or Chunying Li, Department of Dermatology,
Xijing Hospital, Fourth Military Medical University, Xi’an 710032, China.
E-mail: gaotw75401@hotmail.com or lichying@fmmu.edu.cn
Abbreviations: ARE, antioxidant response element; CAT, catalase; DMEM,
Dulbecco’s Modified Eagle’s Medium; DTNB, 5, 5-dithio-bis (2-nitrobenzoic)
acid; ER, endoplasmic reticulum; FRDA, Friedreich ataxia; GAPDH,
glyceraldehyde 3 phosphate dehydrogenase; GCLC, g-glutamyl cystine ligase
catalytic subunit; GCLM, g-glutamyl cystine ligase modulatory subunit; GPx,
glutathione peroxidase; GSH, reduced glutathione; GSHt, total glutathione;
GSSG, oxidized disulfide; GST, glutathione-S-transfereases; H2DCF-DA,
dihydrodichlorofluorescein diacetate; HO-1, heme oxygenase-1; Keap1,
Kelch-like ECH-associated protein; MDA, malondialdehyde; NQO1,
NAD(P)H: quinone reductase; Nrf2, nuclear factor E2-related factor 2; ROS,
reactive oxygen species; SOD, superoxide dismutase; TBA, thiobarbituric acid;
TNB, 5-thio-2-nitrobenzoic acid
Received 20 November 2013; revised 7 February 2014; accepted 25
February 2014; accepted article preview online 24 March 2014
skin leads to melanocyte degeneration (Taieb, 2000; Gauthier
et al., 2003). However, the exact disappearance mechanisms
of melanocytes lack a clear explanation. Studies have
demonstrated that melanocytes involved in vitiligo may have
inherent aberrations that make them more vulnerable to
extracellular insults such as chemical oxidants or UVB, both
of which are able to induce reactive oxygen species (ROS)
generation (Maresca et al., 1997; Jimbow et al., 2001; Kroll
et al., 2005; Lee et al., 2005). Among the great variety of ROS,
hydrogen peroxide (H2O2) has a pivotal role in the onset and
progression of vitiligo (Schallreuter et al., 2007). This point
was supported by the increased levels of H2O2 detected in the
epidermis of vitiligo patients (Schallreuter et al., 1999; Shalbaf
et al., 2008).
Vitiligo melanocytes display cellular and biological impair-
ments when compared with normal melanocytes, namely:
(a) low proliferation rate and inefficient subculture (Puri et al.,
1987), (b) dilative endoplasmic reticulum (ER) and aberrant
melanosome clustering (Boissy et al., 1991; Im et al., 1994),
(c) grossly enlarged and retracted dendrites (Iyengar and Misra,
1988), (d) defective arrangement of the membrane lipids
and altered lipid components (Dell’Anna et al., 2007), and
(e) lower levels of decay accelerating factor (CD 55) and
membrane cofactor protein (CD 46) that could render them

& 2014 The Society for Investigative Dermatology www.jidonline.org 1

 Z Jian et al.
Faulty Nrf2-HO-1 Signaling in Vitiligo Melanocytes
more vulnerable to autologous complement attack (van den
Wijngaard et al., 2002). In addition, recent studies have
confirmed an imbalance in the intracellular redox status in
vitiligo patients in peripheral blood, lesional epidermis, and
cultured melanocytes from the perilesional skin of vitiligo
patients (Ines et al., 2006; Arican and Kurutas, 2008; Khan
et al., 2009). Actually, several antioxidant proteins have also
been found to be altered in patients with vitiligo, such as
catalase (CAT), superoxide dismutase (SOD), glutathione
peroxidase (GPx), and vitamins C and E (Ines et al., 2006;
Arican and Kurutas, 2008). However, thus far, the reason why
vitiligo melanocytes are hypersensitive to oxidative stress has
not been elucidated. Previous studies indicate that vitiligo
keratinocytes are vulnerable to apoptosis by TNF-a treatment
because of reduced activation of NF-kB via impaired PI3K/Akt
activation (Kim et al., 2007). On the basis of these findings,
identification of signaling pathways that account for redox
system imbalance and melanocyte disappearance may
provide a better understanding of the pathomechanisms
underlying vitiligo and possibly improve the therapeutic
approach.
We recently revealed that the nuclear factor E2-related
factor 2–antioxidant response element (Nrf2-ARE) antioxidant
pathway and its downstream antioxidant enzyme heme
oxygenase-1 (HO-1) have a crucial role in the ability of
melanocytes to cope with H2O2-induced oxidative stress
(Jian et al., 2011). It is known that the key transcription
factor Nrf2 regulates the expression of phase II detoxifying
and antioxidant genes by binding to the ARE sequence
(Kensler et al., 2007). Under unstimulated conditions, Nrf2
is sequestered in the cytosol, where it is bound to Kelch-like
ECH-associated protein 1 (Keap1) (Hayes and McMahon,
2001). When cells are under oxidative stress, Nrf2 is
rapidly released from Keap1; it then translocates to the
nucleus where it binds to ARE and induces the phase II
antioxidant genes (Dinkova-Kostova et al., 2002). These genes
encode heme oxygenase-1 (HO-1), CAT, SOD, glutathione-S-
transferase (GST), GPx, NADH quinone oxidoreductase 1
(NQO1), glutamate-cysteine ligase catalytic subunit (GCLC),
and glutamyl cystine ligase modulatory subunit (GCLM)
(Zhu et al., 2005), which are important antioxidants in
melanocytes.
Given that the inability of melanocytes to deal with
oxidative stress has been implicated in vitiligo, and the
importance of the Nrf2-ARE pathway in the defense against
oxidative stress, we hypothesized that dysfunction in the Nrf2-
ARE signaling pathway may result in increased sensitivity to
H2O2-induced oxidative insult of vitiligo melanocytes.
RESULTS
Decreased expression of HO-1 and increased sensitivity of
vitiligo melanocytes to H2O2-induced oxidative stress
To investigate whether vitiligo melanocytes are more suscep-
tible to oxidative stress, we used H2O2 as a means of
administering oxidative stress and performed an MTS assay
to measure cell viability. No remarkable difference in cell
viability was found between the epidermal primary normal
human melanocytes taken from healthy controls (control) and
2 Journal of Investigative Dermatology
the perilesional skin samples taken from patients with vitiligo
(vitiligo) when H2O2 was absent, whereas the presence of
1.0 mM H2O2 caused a statistically significant (Po0.01)
reduction in vitiligo melanocyte cell viability compared with
control melanocytes (Figure 1a). These data demonstrate that
vitiligo melanocytes are more vulnerable to H2O2-induced
oxidative stress.
Our previous study confirmed that HO-1 is the main target
antioxidant gene of the Nrf2-ARE pathway in protecting
human melanocytes from H2O2-induced oxidative stress
(Jian et al., 2011). Therefore, we measured the expression of
HO-1 mRNA and protein levels in the control and vitiligo
melanocytes with or without exposure to H2O2 treatment. In
control melanocytes, H2O2 led to a greater than fivefold
increase in HO-1 mRNA, whereas in vitiligo melanocytes,
the increases in HO-1 mRNA were far smaller, with only a less
than threefold increase compared with that in the H2O2-
untreated group (Figure 1b). Similarly, a significant difference
was also observed in HO-1 protein levels (Figure 1c and d).
However, the basal expression of HO-1 in normal control
and vitiligo melanocytes showed no significant difference
(Figure 1b–d).
Abnormal cellular sublocalization of the Nrf2 transcription
factor and impaired Nrf2 transcriptional activity in vitiligo
melanocytes
Transcription of inducible antioxidant genes is chiefly con-
trolled by the Nrf2 transcription factor (Zhu et al., 2005).
Therefore, we examined the localization of Nrf2 in control
and vitiligo melanocytes by laser confocal scanning
microscopy. As shown in Figure 2a, nuclear Nrf2 localization
was mainly observed in the control melanocytes, whereas
vitiligo melanocytes primarily showed cytoplasmic staining.
Next, we performed western blot analyses of nuclei-enriched
and cytoplasmic fractions from control and vitiligo melano-
cytes. Under normal conditions, the expression of nuclear
Nrf2 in the control melanocytes was higher than that in the
vitiligo melanocytes, whereas the expression of cytoplasmic
Nrf2 in the vitiligo melanocytes was higher than that in the
control melanocytes. Moreover, in the nuclei-enriched frac-
tions of control and vitiligo melanocytes, Nrf2 content
increased in response to H2O2 (Figure 2b and c). Far smaller
Nrf2 increases were seen in the nuclei-enriched fractions of
vitiligo melanocytes (Figure 2b and c). This suggests that
nuclear translocation of Nrf2 is relatively reduced in vitiligo
melanocytes in response to H2O2-induced oxidative stress.
To compare the Nrf2-ARE transcriptional activity of control
with vitiligo melanocytes, we used a dual-luciferase reporter
assay to measure ARE luciferase activity in the presence and
absence of H2O2. PIG1 (a normal human epidermal melano-
cyte cell line) and PIG3V (a vitiligo melanocyte cell line) cells
were transiently transfected with an ARE-luciferase reporter
plasmid (pGL3-ARE) and a Renilla luciferase reporter plasmid
(pRLtk), and treated with 1.0 mM H2O2 for 6, 12, and 24 h
before the luciferase activity was measured. As shown in
Figure 2d, treatment with H2O2 increased the transcriptional
activity of Nrf2 in terms of ARE luciferase activity in a time-
dependent manner in both groups, whereas ARE luciferase
 Z Jian et al.
Faulty Nrf2-HO-1 Signaling in Vitiligo Melanocytes

activity of PIG3V was much lower than that of PIG1 in the
presence of H2O2. These data clearly indicate that Nrf2-driven
transcriptional activation of ARE is aberrant in vitiligo
melanocytes.
a Control
100
Cell viability (% of control cells)
Upregulation of Nrf2 restores the antioxidative ability of vitiligo
melanocytes
Our previous study has shown that transfection with pCMV6-
XL5-Nrf2 increases the expression of Nrf2 and HO-1 in
melanocytes. To investigate whether the upregulation of
Nrf2 expression can restore the cell viability of vitiligo
Vitiligo
melanocytes under oxidative stress, PIG3V cells were trans-
fected with the Nrf2 over-expression plasmid pCMV6-XL5-

Nrf2. Next, we used the MTS assay to investigate the cell

75 viability in vitiligo melanocytes in response to exogenous
* oxidative stress induced by H2O2. In Figure 3, H2O2 caused
an B40% decrease in cell viability when PIG3V cells were
50 treated at a concentration of 1.0 mM H2O2 for 24 hours.
Compared with the normal group, upregulation of Nrf2
expression by cells transfected with pCMV6-XL5-Nrf2 led to
25 reduced cell death (o15%) under oxidative stress, whereas
such changes were not observed in the mock transfection
group (transfected with pCMV6-XL5) (36%). These results
0 suggested that upregulation of Nrf2 enhanced the ability of
0.0 1.0
vitiligo melanocytes to cope with H2O2-induced cytotoxicity.
H2O2 concentration (mM)

b High oxidative stress levels and low antioxidant enzyme

7 Control activities in vitiligo melanocytes
Vitiligo * In order to evaluate the status of the oxidative stress and
6
antioxidant defense system, we detected ROS levels, malon-
HO-1 mRNA level (fold)

5 dialdehyde (MDA) levels, and the activities or contents of

major antioxidant enzymes, including SOD, GPx, CAT, and
4 glutathione (GSH), in primary normal human melanocytes
*# taken from healthy controls (control) and primary melanocytes
3
from perilesional skin samples of vitiligo patients (vitiligo). As
2 shown in Figure 4a and b, vitiligo melanocytes exhibit a
significantly higher intracellular ROS level (54.6±7.5 in
1
vitiligo and 14.2±4.9 in control melanocytes, Po0.001).
0 Moreover, a higher MDA level was detected in vitiligo
H2O2: – + – + melanocytes (20.3±2.1 in vitiligo and 6.1±1.3 in control
melanocytes, Po0.01) (Figure 4c), further supporting the
c Control Vitiligo
hypothesis that the oxidative stress level is higher in vitiligo
melanocytes.
HO-1 32 kDa

β-Actin 43 kDa

H2O2: Figure 1. Detection of cell viability and heme oxygenase-1 (HO-1) expression
– + – +
after treatment with or without H2O2 in control and vitiligo melanocytes.
(a) Cell viability was determined with an MTS assay 24 hours after exposure to
d
1.00 Control 1.0 mM H2O2. Viability was calculated as the percentage of living cells in
*
Vitiligo treated compared with untreated control cells. *Po0.01 when compared with
H2O2-treated control melanocytes. (b) Modulation of HO-1 mRNA expression
levels was detected by real-time PCR in control and vitiligo melanocytes with
(/β-actin expression)

0.75
*#
HO-1 expression

or without 24-h H2O2 treatment. Data are shown as ratios of gene expression
in treated cells to that in untreated controls after normalization on the basis of
the expression of the GAPDH housekeeping gene. *Po0.01 compared with
0.50
the control group (H2O2 untreated). *#Po0.05 compared with the control
group (H2O2 treated). (c) HO-1 protein levels were measured by western blot.
Representative gel blots depicting HO-1 protein by using a HO-1-specific
0.25
antibody. b-Actin was used as an internal control. (d) The intensity of each
band was quantified by densitometry analysis. All protein expression was
normalized to that of b-actin. *Po0.05 compared with the control group
0.00 (H2O2 untreated). *#Po0.01 compared with the control group (H2O2 treated).
H2O2: – + – +
Error bars represent means±SD across three independent cultures (n ¼ 3).

www.jidonline.org 3
 Z Jian et al.
Faulty Nrf2-HO-1 Signaling in Vitiligo Melanocytes

a 100 Untreated pCMV6-XL5

Anti-Nrf2 DAPI Merge
Normal pCMV6-XL5-Nrf2
*#
Control

Cell viability (% of control cells)

75
1 2 3 * *
50 μm
Vitiligo

50

4 5 6

b c 25
Control Vitiligo 3 N-Nrf2
Protein expression (/lamin
B1 or β-actin expression)

C-Nrf2
Nuclear Nrf2 57 kDa
2
Cytosolic Nrf2 57 kDa 0
Δ ∇
# 1.0 mM H2O2 (24 h)
Lamin B1 68 kDa 1
*
Figure 3. Determination of cell viability in vitiligo melanocytes pretreated
β-Actin 43 kDa
0 with pCMV6-XL5-Nrf2 at 48 hours after exposure to 1.0 mM H2O2. The
H2O2 – + – + H2O2 – + – + immortalized vitiligo melanocytes PIG3V were pretreated with pCMV6-XL5-
Control Vitiligo
Nrf2 or pCMV6-XL5. Cell viability was determined with an MTS assay 24 hours
after exposure to 1.0 mM H2O2. Cell viability was calculated as the percentage
d 15.0 Control
# of living cells in the treated compared with the untreated cells. Data are
Vitiligo
12.5 expressed as means±SD of three independent experiments. *Po0.01 when
Luciferase activity (fold)

10.0
7.5
5.0
# The SOD and GPx activities in vitiligo melanocytes were
2.5
*
0.0
Untreated 6h
1.0 mM H2O2
Figure 2. Nrf2 location, amount, and dual-luciferase reporter assays of
antioxidant response element (ARE) activity in control and vitiligo patient
melanocytes under normal and oxidative stress conditions. (a) Nrf2
localization in control and vitiligo melanocytes under normal conditions was
observed by laser confocal scanning microscopy after 24 hours of culture.
Nuclear Nrf2 translocation occurred in control melanocytes, but not in vitiligo
melanocytes. Scale bar ¼ 50 mm. (b) Western blots of nuclear and cytoplasmic
fractions from control and vitiligo melanocytes under normal conditions (lanes
1, 3) or after H2O2 treatment (lanes 2, 4). Representative gel blots depicting
nuclear and cytosolic Nrf2 proteins by using specific antibodies. Fraction purity
was assessed by labeling with b-actin (cytoplasm) and lamin B1 antibody
(nuclei). (c) The intensity of each band was quantified by densitometry analysis.
Nuclear protein expression was normalized to that of lamin B1 and
cytoplasmic protein expression was normalized to that of b-actin. N-Nrf2:
nuclear Nrf2, C-Nrf2: cytosolic Nrf2. *Po0.01 compared with N-Nrf2 in
control group (H2O2 untreated). #Po0.05 compared with C-Nrf2 in control
group (H2O2 untreated). DPo0.001 compared with N-Nrf2 in control group
(H2O2 treated). ,Po0.01 compared with C-Nrf2 in control group (H2O2
treated). Error bars represent means±SD across three independent cultures
(n ¼ 3). (d) PIG1 and PIG3V cells were co-transfected with an ARE-luciferase
reporter plasmid (pGL3-ARE) and a Renilla luciferase reporter plasmid (pRLtk)
for 24 hours and treated with 1.0 mM H2O2 for 6, 12, and 24 hours before
luciferase activity was measured. Firefly luciferase activity in relative light units
per second (RLU s  1) was normalized to Renilla luciferase activity and was
expressed as x-fold multiples of the control, a ratio of the experimental to the
control (control cells without treatment of H2O2). *Po0.05 compared with
control cells (H2O2 treated for 6-, 12-, and 24-hour groups, respectively).
#
Po0.01 compared with control cells (H2O2 untreated).
compared with the untreated group. *#Po0.05 compared with normal (mock
transfection group treated with H2O2).
*
#
*
comparatively lower than in control melanocytes (Figure 4d).
12 h 24 h Interestingly, there was no significant difference in CAT
activity between the two groups. In the meanwhile, low total
GSH and GSH content was detected in the vitiligo group
(Figure 4e). Many studies suggested that the GSH/GSSG
ratio may act as an indicator of oxidative stress. As presented
in Figure 4f, the GSH/GSSG ratio in the normal group was
higher than in the vitiligo group. These results suggest that
the antioxidant enzyme system is impaired in vitiligo
melanocytes.
Decreased serum HO-1 levels are correlated with increased
serum IL-2 levels in patients with vitiligo
Besides abnormalities in redox balance, several biochemical
abnormalities are commonly found in patients with vitiligo.
We tested the serum levels of HO-1 and IL-2 in 113 patients
with non-segmental vitiligo and 113 healthy control subjects
by using ELISA. Serum HO-1 levels were significantly lower in
patients with vitiligo compared with those in healthy controls
(Figure 5a). However, the overall level of serum IL-2 in the
patients with vitiligo was obviously higher than that of healthy
controls (Figure 5b). In both progressive and stable stages of
vitiligo, the levels of IL-2 and HO-1 were significantly different
from the levels in healthy controls. As HO-1 might suppress
IL-2 production by regulating T-cell activation (Pae et al.,
2004), we examined the relationship between serum HO-1
levels and IL-2 levels in vitiligo patients. We found that the
level of serum HO-1 was inversely correlated with that of
serum IL-2 in vitiligo patients (r ¼  0.4482, P ¼ 0.0001,
n ¼ 113; Figure 5c).

4 Journal of Investigative Dermatology

 Z Jian et al.
Faulty Nrf2-HO-1 Signaling in Vitiligo Melanocytes

a Negative control

100
b #
60

50

DCF-H fluorescence
Counts 100 101 102 103 104
100 Control Vitiligo 40

30

20
*
10
100 101 102 103 104 100 101 102 103 104
0
FL1-H NC Control Vitiligo

c d 60 Control
25
MDA level (μmol per mg protein)

Activity (unit per mg protein)

Vitiligo
* 50
20
40
15
30
10
20 *
5 10
*
0 0
Control Vitiligo SOD GPx CAT

e f
GSH/GSSG content (μmol per mg

60 * 17.5
Content (μmol per mg protein)

Control
* 15.0 *
50 Vitiligo
12.5
40
protein)

10.0
30
7.5
20
5.0
10 2.5
0 0.0
Total GSH GSH GSSG Control Vitiligo

Figure 4. Antioxidant enzyme activities or content, reactive oxygen species (ROS) and malondialdehyde (MDA) levels in control and vitiligo melanocytes.
(a) Representative results for ROS production. (b) Bar graphic representations of the fluorescence intensity in control and vitiligo melanocytes. Bar graphic
representations of (c) MDA, (d) SOD, GPx, and CAT, (e) GSH and GSSG, (f) GSH/GSSG in control and vitiligo melanocytes. Error bars represent means±SD across
three independent cultures (n ¼ 3). *Po0.05 compared with control melanocytes. #Po0.01 when compared with NC (negative control).

DISCUSSION Recent evidence, including our findings, indicated that

In this study, we provided a rational explanation for the nuclear transcription factor Nrf2 mainly regulates HO-1
hypersensitivity of vitiligo melanocytes to H2O2-induced expression, and is involved in protecting human melanocytes
oxidative damage. We found that vitiligo melanocytes under against H2O2-induced oxidative stress (Surh, 2003; Jian et al.,
H2O2-induced oxidative stress conditions showed lower HO- 2011). Thus, we focused on the expression, localization, and
1 levels, resulting in the decline of antioxidative ability to transcriptional activity of Nrf2. Interestingly, although we
cope with abnormally high levels of H2O2. These data support did not observe any difference in Nrf2 expression between
the views that melanocytes from vitiligo subjects show the control and vitiligo melanocytes (data not shown), Nrf2
increased susceptibility to high levels of epidermal ROS nuclear localization and transcriptional activity are
production (Maresca et al., 1997; Jimbow et al., 2001; decreased in vitiligo melanocytes. Despite severe oxidative
Boissy and Manga, 2004; Kroll et al., 2005). stress that should have induced activation of Nrf2 and

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 Z Jian et al.
Faulty Nrf2-HO-1 Signaling in Vitiligo Melanocytes

a *** Total contributing to melanocyte dysfunction and/or loss. Similar

*** PV
results were found by Paupe et al. (2009) in Friedreich ataxia
70 *** SV
Control (FRDA) cultured fibroblasts. Moreover, lower ARE luciferase
60 activity of PIG3V cells was detected by a dual-luciferase
50 reporter assay with or without H2O2 stimulus. These data
HO-1 (pg ml–1)

strongly suggested that the Nrf2-ARE pathway is dysfunctional

40
in vitiligo melanocytes and provides an explanation for the
30 impaired induction of antioxidants seen in these cells in
response to H2O2-induced oxidative stress.
20
Another interesting finding from our study was that upre-
10 gulation of Nrf2 could restore the antioxidant capability of
0 PIG3V cells, which allowed them to cope with H2O2-induced
Total PV SV Control cytotoxicity. This was accompanied by significant upregula-
tion of the Nrf2 target antioxidant genes, including HO-1,
b *** Total
*** PV GST, GCLC, GCLM, and NQO1. This effect has been
** SV
150
Control
confirmed in human melanocytes by research from our group
(Jian et al., 2011) and that by Marrot et al. (2008). In fact,
125
many studies have found that activation of the Nrf2-ARE
100 pathway with different means has an important protective role
IL-2 (pg ml–1)

in several types of cells and diseases. For instance, studies in

75 neurodegenerative disease have found that activation of Nrf2
protects against H2O2-induced PC12 cell death and apoptosis
50
(Tanaka et al., 2010; Tusi et al., 2010). In addition, Beyer et al.
25 (2007) demonstrated that upregulation of Nrf2 had an anti-
inflammatory role in skin wounds, and that activation of this
0 pathway protects skin from carcinogenesis induced by
Total PV SV Control
chemicals.
c 200 An increasing number of studies have demonstrated that
r = –0.4482
p = 0.0001 melanocyte impairment in vitiligo may be related to increased
n = 113 oxidative stress. Excess production of ROS or insufficient
150 antioxidant enzymes may cause oxidative damage in mela-
Serum IL-2 (pg ml–1)

nocytes. Recent studies using lesional epidermis and blood

100
50
0 increased and that these antioxidant systems are insufficient in
0 10 20 30 40 50
Serum HO-1 (pg ml–1)
Figure 5. Serum HO-1 and interleukin (IL)-2 levels in healthy controls
(n ¼ 113) and patients with vitiligo (n ¼ 113) and correlation between serum
HO-1 and IL-2 in patients with vitiligo. (a) Decreased serum HO-1 levels and
(b) increased serum IL-2 levels in patients with vitiligo. **Po0.01 and
***Po0.001 as determined by the unpaired Mann–Whitney U-test. Control,
healthy controls; PV, progressive vitiligo; SV, stable vitiligo; Total, total
patients. (c) Association between serum HO-1 and IL-2 levels. Serum HO-1
levels were inversely correlated with serum IL-2 levels in vitiligo patients
(r ¼  0.4482, P ¼ 0.0001, n ¼ 113, Spearman’s rank correlation test). The
solid line represents the regression line.
upregulation of its downstream gene products, vitiligo
melanocytes showed progressive reduction of nuclear Nrf2
content and a markedly diminished Nrf2 activation. This
suggests that in vitiligo melanocytes, some process may
block the activity of Nrf2 nuclear translocation, potentially
(serum) of vitiligo patients have found that an imbalance in
redox status is a possible pathogenic clue in vitiligo (Taieb,
2000; Ines et al., 2006; Arican and Kurutas, 2008; Khan et al.,
2009). SOD, CAT, GSH, and GPx are a group of antioxidant
enzyme systems that scavenge ROS to prevent cell damage. In
this study, we confirmed that the levels of ROS and MDA are
60 70
vitiligo melanocytes.
Previous studies have shown that the serum, plasma, and
vitiliginous tissue samples from different types of vitiligo
patients have higher MDA levels, which is in concordance
with our findings (Yildirim et al., 2003; Koca et al., 2004;
Yildirim et al., 2004; Arican and Kurutas, 2008). However, our
study identified higher levels of MDA in vitiligo melanocytes.
There are many conflicting studies on SOD activity in patients
with vitiligo. Some studies show higher levels of SOD
(Dell’Anna et al., 2001; Yildirim et al., 2003; Agrawal et al.,
2004; Yildirim et al., 2004; Hazneci et al., 2005; Shajil and
Begum, 2006; Arican and Kurutas, 2008), whereas others
show lower levels of SOD in vitiligo patients (Arican and
Kurutas, 2008; Khan et al., 2009). GPx activity in patients with
vitiligo has been found to be normal (Picardo et al., 1994;
Passi et al., 1998) in some studies and lower in others (Beazley
et al., 1999; Shajil and Begum, 2006; Khan et al., 2009). In
fact, we failed to find any significant difference in CAT activity
between normal and vitiligo melanocytes in the present study.

6 Journal of Investigative Dermatology

 Z Jian et al.
Faulty Nrf2-HO-1 Signaling in Vitiligo Melanocytes

a b
Normal Nrf2-ARE/HO-1 pathway Aberrant Nrf2-ARE/HO-1 pathway

H 2O 2

ROS ROS
Cytoplasm

Epidermis Epidermis

Melanocytes Melanocytes

Dissociation Dissociation
Keap1 Keap1
Lymphocytes Keap1 Nrf2 Nrf2 Keap1
IL-2 levels Lymphocytes
Increased nuclear Decreased nuclear IL-2 levels
translocation translocation

Transcriptional Transcriptional
upregulation P P P P downregulation
Nrf2 Nrf2
HO-1 HO-1
Increase ARE ARE Decrease

Nuclear

Melanocyte Melanocytes degeneration

Normal (control) Vitiligo

Figure 6. Schematic proposal of the Nrf2-ARE/HO-1 pathway in normal (control) human melanocytes and vitiligo melanocytes under H2O2-induced oxidative
stress. (a) Under H2O2-induced oxidative stress conditions in normal (control) human melanocytes, Nrf2 escapes from Kelch-like ECH-associated protein (Keap1)
and phosphorylated Nrf2 translocates to the nucleus where it binds to antioxidant response element (ARE) and transcriptionally upregulates HO-1 gene expression.
HO-1 can further protect human melanocytes from H2O2-induced oxidative damage and may decrease IL-2 levels by inhibiting T-cell activation that maintains
skin homeostasis. (b) In vitiligo melanocytes, impaired activation of the Nrf2-ARE/HO-1 pathway (reduced Nrf2 nuclear translocation, decreased transcriptional
activity, and aberrant antioxidant defense system) leads to excessive ROS generation and decreased HO-1 induction, which fails to inhibit H2O2-induced oxidative
stress and IL-2 production. This may eventually result in melanocyte degeneration and cause vitiligo.

This is in accordance with a previous report that detected no
difference in CAT levels in the erythrocytes isolated from
normal and vitiligo patients by Ines et al. (2006), whereas
other studies revealed lower CAT activity in vitiligo
melanocytes (Schallreuter et al., 1991; Dell’Anna et al.,
2001; Arican and Kurutas, 2008). Our results showed lower
activities of SOD and GPx, as well as diminished levels of
GSH in vitiligo melanocytes when compared with normal
melanocytes. We believe that these varying results may be
due to the differences in the innate levels of these enzymes
in the different samples that were studied (the serum,
erythrocytes, melanocytes, and epidermis). Besides, they can
be influenced by the duration and activity of disease as well as
the laboratory techniques. Notably, the limited sample size in
our study should be taken into consideration as well, and a
larger sample size is still needed to confirm our findings.
It has been reported that T cells in vitiligo patients are
activated (Le et al., 1993), and that the increased levels of IL-2
present in these patients (Khan et al., 2012) is a sign of T-cell
activation (Rubin and Nelson, 1990). Moreover, HO-1 may
suppress IL-2 production by inhibiting T-cell activation
(Pae et al., 2004). Consistently, in this study, we found that
the IL-2 levels in patients with vitiligo were significantly higher
than those in the healthy control group, and the decreased
serum HO-1 level was inversely correlated with serum IL-2
levels. A previous study found that HO-1/CO induced
suppressive effects on T-cell proliferation and IL-2 secretion,
possibly via its inhibition of the ERK MAP kinase
pathway (Pae et al., 2004). Thus, the increased levels of IL-2
observed in the serum of patients with vitiligo may result from
the decreased levels of HO-1. This phenomenon may be the
result of the dysfunction of the Nrf2-ARE redox signaling
pathway.
Until now, no study has examined the reason why vitiligo
melanocytes are more vulnerable to oxidative stress. Our
findings identified impairment in the antioxidant system in
vitiligo melanocytes, and showed that ROS-mediated damage
in vitiligo leads to melanocyte degeneration. Nrf2 regulates
the expression of various antioxidant enzymes, including CAT,
GPx, and SOD, by using GSH as their substrate (Kim and
Vaziri, 2010). Therefore, decreased antioxidant enzyme
activity and increased ROS may be associated with impair-
ment of the Nrf2-ARE pathway in vitiligo melanocytes. As the
Nrf2-ARE pathway has a vital role in the protection against
oxidative stress–induced cellular injury (Harvey et al., 2009),
dysfunction of the pathway can lead to excessive
ROS generation and low levels of antioxidant enzymes,
which is evident in the death of vitiligo melanocytes. Our
current understanding is summarized in the scheme in
Figure 6.

www.jidonline.org 7
 Z Jian et al.
Faulty Nrf2-HO-1 Signaling in Vitiligo Melanocytes
In summary, we found that vitiligo melanocytes exhibit
a paradoxical reduction in Nrf2 activation leading to
decreased activation of downstream antioxidant molecules.
We have provided a rational explanation for the
hypersensitivity of vitiligo melanocytes to H2O2-induced
oxidative stress. Accordingly, the impairment of the Nrf2-
ARE signaling pathway in vitiligo melanocytes, shown here,
contributes to the severity of oxidative stress and the failure
of antioxidant induction in response to oxidative stress.
It also renders the intracellular environment prone to ROS-
mediated toxicity and aberrant redox homeostasis. Future
studies are needed to determine how Nrf2 localization is
regulated and why it is aberrant in vitiligo melanocytes. By
further understanding this crucial pathway and its alteration in
vitiligo progression, more clues about how melanocytes lose
their endogenous protection may be presented, leading to
potential strategies to restore this function before melanocyte
loss.
MATERIALS AND METHODS
A detailed overview of the experimental procedure is given in the
Supplementary Information online.
Specimens of patients and controls
After obtaining informed written consent, full-thickness skin biopsies
were taken from the perilesional skin (for melanocyte culture) of six
patients (three men and three women; age range, 21–57 years; mean
age, 37.5 years) affected by active (on the basis of the progression or
appearance of lesions in the last 3 months) non-segmental symme-
trical vitiligo. Samples were also obtained from six age- and sex-
matched normal controls. Perilesional skin is defined as skin along
the edge of the white patch. All biopsies were taken from sun-
unexposed areas. To measure HO-1 activity and IL-2 levels, blood
was obtained from 113 vitiligo patients with non-segmental disease
(64 men and 49 women; 29 in active and 84 in stable; age range,
18–57 years; mean age, 33 years) and from 113 age- and sex-matched
control subjects. None of the patients underwent any treatment (either
systemic or topical) 3 months before the biopsies and blood samples
were obtained. Autoimmune diseases or familiarity were not noted in
any vitiligo patient. The local ethics committee of Xijing Hospital
approved the study, which was performed in strict compliance with
the principles of the Declaration of Helsinki.
Cell culture and treatment
Skin specimens obtained from the perilesional skin of vitiligo patients
and normal controls were used for cell culture. The epidermis was
separated from the dermis after treatment with 2.4 U ml  1 dispase
(Roche, Mannheim, Germany) at 4 1C overnight. The epidermal
sheets were treated with 0.05% trypsin for 10 minutes to prepare a
suspension of individual epidermal cells. The cells were resuspended
in Medium 254 conditioned medium (Cascade Biologics/Invitrogen,
Portland, OR) supplemented with a human melanocyte growth
supplement (Cascade Biologics/Invitrogen), 5% fetal bovine serum
(Invitrogen, San Diego, CA), and penicillin–streptomycin antibiotic
mix (Invitrogen) at 37 1C in the presence of 5% CO2, and analyzed
during the subconfluent phase at passages 0–2. Three days after
confluence, the primary cultures were trypsinized, and the cell
suspension (4  104 cells per cm2) was cultured in the melanocyte
8 Journal of Investigative Dermatology
growth medium. Melanocytes in their third or fourth in vitro passage
were used for the experiments. An immortalized normal human
epidermal melanocyte cell line (PIG1) and a vitiligo melanocyte cell
line (PIG3V; both gifts from Dr Caroline Le Poole, Loyola University
Chicago, Maywood, IL) were similarly cultured in Medium 254
supplemented with human melanocyte growth supplement, 5% fetal
bovine serum, and a penicillin-streptomycin antibiotic mix at 37 1C
in the presence of 5% CO2. Normal human epidermal melanocytes
were isolated from neonatal foreskin of a Caucasian infant, and
vitiligo melanocytes were isolated from nonlesional skin of a 34-year-
old female Caucasian vitiligo patient. These cell lines were immorta-
lized by retroviral introduction with the HPV16 E6 and E7 genes
(Le et al., 1997, 2000). Cell treatment was induced by adding H2O2
(analytical pure grade; Xi’an Chemical Reagent Factory, Xi’an, China)
at 1.0 mM in serum-free, antibiotic-free DMEM (Invitrogen) for
24 hours.
Cell viability testing (MTS assay)
The MTS assay was performed as described (Jian et al., 2011) using
Cell Titer 96AQUeous One Solution Cell Proliferation Assay
(Promega, Madison, WI).
Real-time PCR
Real-time PCR was performed as described (Jian et al., 2011) with
SYBR Premix Ex Taq II (TaKaRa, Ohtsu, Japan) on a Chromo4
continuous fluorescence.
Western blot analysis
Western blots were performed as described (Jian et al., 2011) using
anti-Nrf2, anti-HO-1, anti-Lamin B1, and anti-b-Actin antibodies
(Santa Cruz, CA).
Transient transfection of Nrf2
Transient transfection of Nrf2 was performed as described (Jian et al.,
2011) using the mammalian expression plasmid pCMV6-XL5
(OriGene, MD).
Transient transfection and dual-luciferase reporter assay
The dual-luciferase reporter assay were performed as described
(Shunsuke et al., 2007) using Lipofectamine 2000 (Invitrogen) with
a pGL3-ARE reporter plasmid containing three copies of ARE (Kindly
donated by Dr Guodong Yang, Forth Military Medical University,
Xi’an, China) and pRLtk plasmid (Promega).
Laser scanning confocal immunofluorescence microscopy
Laser scanning confocal microscopy was performed as described
(Shunsuke et al., 2007).
Measurement of intracellular ROS levels
The ROS levels were determined as described (Lluis et al., 2007)
using the ROS assay kit (Beyotime, Jiangsu, China).
Biochemical assays
The contents of malondialdehyde (MDA), total glutathione (GSHt),
reduced glutathione (GSH), and oxidized disulfide (GSSG), and the
activity of SOD, GPx, and catalase were measured using a commer-
cially available assay kit (Beyotime) according to the manufacturer’s
instructions.

Analysis of serum HO-1 activity and IL-2 levels
Serum activity of HO-1 and levels of IL-2 were measured by human
HO-1 or IL-2ELISA kits (Xi Tang Biotechnology, Shanghai, China)
according to the manufacturer’s instructions.
Statistical analysis
The data represent the means±SD for at least three independent
experiments. Statistical significance was assessed with the unpaired
two-tailed Student’s t-test. The differences of serum HO-1 and IL-2
levels between patients with vitiligo and healthy controls were
analyzed using the Mann–Whitney U-test. All statistical analyses
were performed by GraphPad Prism (GraphPad Software 3.0;
San Diego, CA) and P values o0.05 were considered statistically
significant.
CONFLICT OF INTEREST
The authors state no conflict of interest.
ACKNOWLEDGMENTS
The work described in this article was supported by the National Natural
Science Foundation of China (no. 81130032 and no. 81373844). We thank Dr
Caroline Le Poole for providing the immortalized human epidermal melano-
cyte cell line PIG1 and vitiligo melanocyte cell line PIG3V and thank Dr Hua
Wang and Yu Ye for assistance with the flow cytometer analyses.
SUPPLEMENTARY MATERIAL
Supplementary material is linked to the online version of the paper at http://
www.nature.com/jid
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