Letter to the Editor

follicle reconstitution. Each has distinct and different advantages Acknowledgements

and disadvantages, depending upon how it is to be used. The
patch assay had the advantage that low cell numbers were needed,
it was not labour intensive, and histological readouts could be
made very quickly. However, it was not the best to investigate hair
shaft quality or density. The chamber and flap approaches were
best to evaluate hair shaft and density but both were labour inten-
sive and required surgical skills. Each has advantages at manipu-
lating cells or mixing and matching donor cells to address specific
questions.
Y. Liang designed the experiments, performed the tissue culture work, analy-
sed data and wrote the manuscript. K. A. Silva and V. Kennedy performed
the surgeries and mouse injections, collected tissue samples, took gross photo-
graphs and processed samples. J. P. Sundberg designed the study, supervised
the experiments, interpreted the histopathology, immunohistochemistry and
scanning electron microscopy, took the photomicrographs, analysed data and
wrote the manuscript. The authors thank Lesley Bechtold for her technical
assistance with the scanning electron microscopy.
Conflict of interest
None.

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DOI:10.1111/j.1600-0625.2011.01353.x
www.blackwellpublishing.com/EXD
Letter to the Editor

Effects of topical application of EGCG on testosterone-induced hair

loss in a mouse model
Yoon Young Kim1, Sun up No2, Min Ho Kim2, Hei Sung Kim2, Hoon Kang2, Hyung Ok Kim2 and Young Min
Park2
1
Cha and Park Skin Clinic, Uijeonbu, Korea; 2Department of Dermatology, College of Medicine, The Catholic University of Korea, Seoul, Korea
Correspondence: Young Min Park, MD, Department of Dermatology, Seoul St. Mary’s Hospital, College of Medicine, The Catholic University of
Korea, 505, Banpo-dong, Seocho-gu, Seoul 137-040, Korea, Tel.: +82-2-2258-2867, Fax: +82-2-594-3255, e-mail: yymmpark6301@hotmail.com

Abstract: We investigated the effect of topical epigallocatechin-3-
gallate (EGCG) on testosterone (T)-induced hair loss in mice.
Marked hair loss was observed at the T-injected site, and topical
EGCG significantly reduced the hair loss (P < 0.05). TUNEL
staining showed apoptosis of follicular epithelial cells in the
T-injected groups where topical EGCG was found to significantly
diminish T-induced apoptosis (P < 0.05). Topical EGCG down-
regulated the T-induced expression of androgen receptor but did
not down-regulate 17b-hydroxysteroid dehydrogenase (HSD) and
three b-HSD expression. Analysis using liquid chromatography
tandem mass spectrometry (LC-MS ⁄ MS) on serum and tissue
samples revealed no significant difference in T and
dihydrotestosterone concentrations between the T-injected and
T + EGCG groups. Thus, we found that T injection in a mouse
model induces hair loss by apoptosis of the hair follicles rather
than through the androgen metabolic pathway and also saw that
T-induced apoptosis of hair follicles was reduced by topical
EGCG.
Key words: apoptosis – epigallocatechin-3-gallate – hair loss –
testosterone
Accepted for publication 21 July 2011

ª 2011 John Wiley & Sons A/S, Experimental Dermatology, 20, 1011–1037 1015
 Letter to the Editor
Background
The effects of green tea, such as anti-cancer and anti-oxidant
properties, are primarily mediated by epigallocatechin-3-gallate
(EGCG; 1,2). EGCG has effects on the epidermis, such as accelera-
tion of keratinocyte differentiation, and protection of hair follicles
from radiation. Interestingly, EGCG selectively induces apoptosis
of tumor cells, but not the normal human epidermal keratinocytes
(3). EGCG inhibits apoptosis of normal cells by suppressing many
genes coding for pro-apoptosis factors. EGCG up-regulates heat-
shock protein 70 and phosphorylation of Erk and Akt, but down-
regulates c-myc, and increases the ratio of Bcl-2 ⁄ Bax (3–5). EGCG
also has several effects on androgen metabolism; EGCG inhibits
5a-reductase (6,7) and androgen action by repressing the tran-
scription of the androgen receptor (AR) gene (8).
Androgens, such as testosterone (T) and dihydrotestosterone
(DHT), are believed to act on the hair follicles (9,10). In human
hair follicles, androgen exerts its effect either directly or after con-
version by an enzyme, 5a-reductase, to DHT (11) that binds to
ARs in hair follicles (12,13). DHT is known to cause hair loss
(12), but there are only a few reports about T-induced apoptosis
in hair (14), and little is known about the influence of T on hair
growth. Moreover, there are only a few experiments on apoptosis
of hair follicles using a mouse model (15).
Questions addressed
The aim of this study was to examine the effect of topical EGCG
on hair loss caused by T injection.
Experimental design
Sixty B6CBACF1 ⁄ J female mice were injected intra-dermally on
the upper dorsal skin with T and ⁄ or topical EGCG for 12 weeks
(Appendix S1).
Results
Effects of T injection and topical EGCG
The T-injected group had approximately 10% hair loss compared
with their hair density before the experiment, which was counted
using a folliscope on the fifth week of injection. However, in the
T + EGCG group, 10% hair loss was noted after the sixth week of
injection. Compared with the T-injected group, the T + EGCG
group had marked hair density in the 12th week of experi-
ments (Fig. 1a–f), which was also found in histology (P < 0.05;
(a) (b) (c) (g) (h) (i)
(d) (e) (f) (j) (k) (l)
Figure 1. Photographs of T-induced hair loss (a–f) and hair re-growth after
epilation (g–l) in T-injected B6CBAF1 ⁄ j mice with the effect of topical EGCG after
12 weeks. Topical application of 10% EGCG (a, g), testosterone (T)’s vehicle
injection (b, h), T injection (c, i), T injection + topical application of EGCG vehicle
(ethanol)) (d, j), T injection + topical application of 10% cyproterone acetate (e, k)
and T injection + topical application of EGCG (f, l). The circled area at the upper
dorsal skin is the site of T injection (a–f). The boxed area of the mice is the
epilated area, and the circled area is the site of the T injection (g–l). The inlet is
taken by Folliscope on day 16 after epilation.
1016
(a) (b) (g) (h)
(c) (d) (i) (j)
(e) (f) (k) (l)
Figure 2. Histologic findings of the upper dorsal skin stained with haematoxylin
and eosin (a–f), and TUNEL staining (g–l). Topical application of 10% EGCG (a, g),
testosterone (T)’s vehicle injection (b, h), T injection (c, i), T injection + topical
application of EGCG vehicle (ethanol) (d, j), T injection + topical application of
10% cyproterone acetate (e, k) and T injection + topical application of EGCG (f, l).
[(a–f) · 100, (g–l) · 400].
Fig. 2a–f). The T + cyproterone acetate (CA) group showed no
significant hair loss compared with the T + EGCG group. CA has
strong anti-androgenic effects (16) and has been used in other
T-induced alopecia experiments (17,18). On the histologic exami-
nation, T-injected group showed a decrease in the number of hair
follicles, compared with the T’s vehicle-injected group. To see
whether the hair loss induced by T injection is mediated by the
apoptotic effect, TUNEL staining was performed. The TUNEL
staining demonstrated that the T + vehicle group had 50–70%
staining intensity in follicular epithelial cells whereas the T +
EGCG group had <10% of staining in follicular epithelial cells
(P < 0.05; Fig 2g–l; Table S1).
Next, to examine the effect of EGCG on hair growth, epilation
was performed. Hair growth was first observed at the epilation site
by Folliscope (·50, Folliscope; Lead M Co, Seoul, Korea) 11 days
after epilation in the T + EGCG group, while the T-injected group
had hair growth 13 days after epilation. The T + EGCG group
showed a significantly higher density of hair than the T-injected
group, 16 days after epilation (P < 0.05; Table S2).
These results suggest that T injection in this mouse model can
induce hair loss through apoptosis of hair follicles. EGCG can
reduce the T-induced hair loss by down-regulating the apoptosis
of follicular epithelial cells and even stimulate hair re-growth.
Effect of T injection and EGCG on AR, 17b- and
3b-hydroxysteroid dehydrogenase (HSD) expression of
hair follicles
We examined the effect of T on AR expression by IHC staining.
IHC of AR demonstrated that the T + vehicle group had 50–70%
staining intensity in follicular epithelial cells as well as sebaceous
glands, whereas the T + EGCG group had <10% staining in follic-
ular epithelial cells (Figure S1 and Table S1). IHC staining for
17b- and 3b-HSD was also performed to determine whether there
were changes in the androgen pathway by T or EGCG. However,
there was no difference in 17b- and 3b-HSD expression among
the T-injected and T + EGCG groups (unpublished data). These
results demonstrate that EGCG can down-regulate T-induced AR
expression of hair follicular cells but has no effect on 17b- and
3b-HSD expression.
ª 2011 John Wiley & Sons A/S, Experimental Dermatology, 20, 1011–1037
 Letter to the Editor

Effect of T injection and EGCG on T and DHT concentra-
tions in serum and hair
Analysis using LC-MS ⁄ MS on serum and hair samples was per-
formed to determine the difference in the T and DHT concentra-
tions in each group. We found no difference in the T and DHT
concentrations between the T-injected and T + EGCG groups
(unpublished data).
Conclusion
We report the first in vivo experiment showing the influence of T
injection and EGCG on hair follicles. Our results suggest that T
injection in a B6CBAF1 ⁄ j mouse model induces hair loss by apop-
tosis of hair follicles rather than through the androgen metabolic
pathway. Furthermore, it was shown that EGCG can prevent hair
loss by reducing T-induced apoptosis of follicular epithelial cells
and provoke hair re-growth after epilation.
The best animal model of androgen-induced alopecia is the
stump-tailed macaque, but because of its large size and high cost,
researchers favour a more easily handled murine model (19). The
B6CBAF1 ⁄ j mice used in this study were first introduced by
Matias et al. (17) as a T-induced androgenic alopecia mice model.
The B6CBAF1 ⁄ j mouse is not a perfect androgen-induced alopecia
model in that androgen is needed to be given continuously, and
once androgen supplementation ceases, the mouse regains its hair
within 3 months. Because B6CBAF1 ⁄ j mice are convenient, cost-
effective and suitable with respect to the time course of results,
B6CBAF1 ⁄ j mice were used in this study.
Even though there was no difference in the expression of 17b-
and 3b-HSD between the T-injected and T + EGCG groups, we
cannot exclude the possibility of the effect of EGCG on androgen
metabolism of hair follicles because EGCG application down-regu-
lated T-induced AR expression of hair follicles. To resolve this
question, further functional in vitro studies using hair follicle cul-
ture will be needed.
From our study, we propose topical EGCG as a promising ther-
apeutic tool in the management of androgen-related hair loss.
Acknowledgements
This study was supported by the Stiefel Research Grant year of 2008
through the Korean Dermatological Association. Yoon Young Kim, Sun up
No and Min Ho Kim performed the research, Yoon Young Kim, Hoon
Kang, Hyung Ok Kim and Young Min Park designed the research study,
Yoon Young Kim, Hoon Kang and Young Min Park analysed the data and
Yoon Young Kim, Hoon Kang, Young Min Park and Hei Sung Kim wrote
the article.
Conflict of interest
The authors declare no conflict of interests.

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Supporting Information
Additional Supporting Information may be found in the
online version of this article:
Figure S1. Immunohistochemical histology of the
upper dorsal skin stained with androgen receptor (a–f).
Topical application of 10% EGCG (a), testosterone (T)’s
vehicle injection (b), T injection (c), T injection + topi-
cal application of EGCG vehicle (ethanol)) (d), T injec-
tion + topical application of 10% cyproterone acetate
(e), and T injection + topical application of EGCG (f)
(·400).
Table S1. Number of hair follicles counted by Folli-
scope on day16 after epilation.
Table S2. Comparison of the intensity of the apopto-
sis by TUNEL staining and immunohistochemical stain-
ing of androgen receptor of the treated groups.
Appendix S1. Materials and methods.
Please note: Wiley-Blackwell are not responsible for
the content or functionality of any supporting materials
supplied by the authors. Any queries (other than missing
material) should be directed to the corresponding author
for the article.

DOI:10.1111/j.1600-0625.2011.01361.x
www.blackwellpublishing.com/EXD
Letter to the Editor

The secretory clear cell of the eccrine sweat gland as the probable
source of excess sweat production in hyperhidrosis
Douglas L. Bovell1, Alison MacDonald2, Barbara A. Meyer2, Alistair D. Corbett1, William M. MacLaren1,
Susan L. Holmes2 and Mark Harker3
1
Department of Biological and Biomedical Sciences, Glasgow Caledonian University, Glasgow, UK; 2Department of Dermatology, Glasgow Royal
Infirmary, Glasgow, UK; 3Unilever Research & Development, Port Sunlight, Bebington, UK
Correspondence: Mark Harker, PhD, Unilever R&D, Quarry Road East, Bebington CH63 3JW, UK, Tel.: +44 151 641 3992, Fax: +44 151 641 1854,
e-mail: mark.harker@unilever.com

ª 2011 John Wiley & Sons A/S, Experimental Dermatology, 20, 1011–1037 1017