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Towards a "free radical theory of graying":

melanocyte apoptosis in the aging human hair
follicle is an indicator of oxidative stress
induced tissue damage
Article in The FASEB Journal August 2006
DOI: 10.1096/fj.05-4039fje Source: PubMed

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The FASEB Journal FJ Express Full-Length Article

Towards a free radical theory of graying: melanocyte

apoptosis in the aging human hair follicle is an
indicator of oxidative stress induced tissue damage
Petra Clara Arck,,1 Rupert Overall,*,1 Katharina Spatz,* Christiane Liezman,*
Bori Handjiski, Burghard F. Klapp, Mark A. Birch-Machin, and
Eva Milena Johanne Peters*1,2
*Cutaneous Neuroimmunology, Biomedical Research Center, University Medicine Charite, Virchow
and Mitte Campus; Psychoneuroimmunology, Biomedical Research Center, University Medicine
Charite, Virchow Campus; and Internal Medicine, Psychosomatics, University Medicine Charite,
Mitte Campus, Humboldt-University of Berlin, Berlin, Germany; and Dermatology, Medical School,
University of Newcastle, UK
Oxidative stress is generated by a multitude of environmental and endogenous challenges such
as radiation, inflammation, or psychoemotional stress.
It also speeds the aging process. Graying is a prominent
but little understood feature of aging. Intriguingly, the
continuous melanin synthesis in the growing (anagen)
hair follicle generates high oxidative stress. We therefore hypothesize that hair bulb melanocytes are especially susceptible to free radical-induced aging. To test
this hypothesis, we subjected human scalp skin anagen
hair follicles from graying individuals to macroscopic
and immunohistomorphometric analysis and organ culture. We found evidence of melanocyte apoptosis and
increased oxidative stress in the pigmentary unit of
graying hair follicles. The common deletion, a
marker mitochondrial DNA-deletion for accumulating
oxidative stress damage, occurred most prominently in
graying hair follicles. Cultured unpigmented hair follicles grew better than pigmented follicles of the same
donors. Finally, cultured pigmented hair follicles exposed to exogenous oxidative stress (hydroquinone)
showed increased melanocyte apoptosis in the hair
bulb. We conclude that oxidative stress is high in hair
follicle melanocytes and leads to their selective premature aging and apoptosis. The graying hair follicle,
therefore, offers a unique model system to study
oxidative stress and aging and to test antiaging therapeutics in their ability to slow down or even stop this
process.Arck, P. C., Overall, R., Spatz, K., Liezman,
C., Handjiski, B., Klapp, B. F., Birch-Machin, M. A.,
Peters, E. M. J. Towards a free radical theory of
graying: melanocyte apoptosis in the aging human hair
follicle is an indicator of oxidative stress induced tissue
damage. FASEB J. 20, E908 E920 (2006)

ABSTRACT

Key words: gray hair premature aging

It is obvious that the color of our hair has important

socio-economic implications and propels a multimilE908

lion hair product industry. Common and premature

graying (canities) are common and intriguing phenomena frequently discussed in the context of environmental or endogenous processes that lead to an accelerated
aging process such as pollution, UV-exposure, inflammation, or even psychoemotional stress (6, 28, 31, 48,
58). These and other challenges can be viewed as
stressors that affect our health by generating free
radicals, which alter proteins and nucleic acids important in the maintenance of our biological functions.
Clinical observation states that in senile canities by
the age of 50, 50% of all hair follicles in 50% of all men
have lost their pigment (see Ref 1). Premature canities
is much more striking and often lead to rapid graying
of rather young individuals. This dramatic change in
physical appearance is frequently used in the arts as
well as medical literature to demonstrate and witness
severe disease, rapid aging, psychoemotional stress experiences, and radical changes in life (see ref 2 4).
However, this condition may also serve as an indicator
of biological response to a stressor. However, the
process of graying and its link to environmental and
endogenous stressors is seldomly studied in a scientific
context.
The biological process appears to be associated with
a progressive loss of the pigment-producing cells, the
melanocytes, from the aging hair bulb and outer root
sheath with physiological aging and premature aging
syndromes (1, 57). To date, the cause of this loss has
not been satisfactorily explained, though a number of
interesting hypothesis have been brought forward (1,
6 8, 10). From animal experiments and mutationreports, we know about predetermined braking points
1

Correspondence: Biomedical Research Center, Rm. Nr.

2.0549, University Medicine Charite, Virchow Campus, Humboldt University of Berlin, Augustenburger Platz 1, Berlin
13353, Germany. E-mail: eva.peters@charite.de or frl_peters@
yahoo.com
doi: 10.1096/fj.05-4039fje
0892-6638/06/0020-0908 FASEB

in the function of the hair follicle pigmentary unit,

which is formed between the pigment-producing melanocytes, anchored to the basement membrane above
the dermal papilla, and the hair shaft producing keratinocytes in the hair bulb (c.f. 1, 6, 9).
Pigmentation braking points include the exhaustion
of growth factors and melanogenesis-related enzymes,
impairment of DNA-repair mechanisms, loss of antioxidative enzymes and cofactors, loss of telomerase, or loss
of antiapoptotic signals (1, 5, 6, 9 12). One prominent
hypothesis features decreased stem cell factor (SCF)
signaling through its tyrosinekinase receptor c-Kit,
since anti-hypothesis c-Kit treatment can produce gray
hair follicles (68). Another focuses on Bcl-2, which
scavenges oxygen radicals in the membranes of mitochondria, because its loss by knockout leads to premature graying of mice that otherwise lack a striking
phenotype (10). Analysis of the fate of melanocytes
during the murine hair cycle revealed that melanocytes
can be lost from the hair bulb by apoptosis during hair
follicle involution (catagen) (13, 14). Ultimately, this
may lead to the exhaustion of a melanocyte stem cell
pool residing in the bulge region of the hair follicle
(12, 15). However, conclusive proof that any of the
above braking points of melanocyte function are relevant to common graying has not been brought forward
to date, and it remains open whether apoptosis occurs in aging hair follicle melanocytes and what might
cause it.
Oxidative stress appears to play a key role in many of
the indicated pathways (6). Accordingly, an anecdotal
report has it that melanocytes in the graying human
hair bulb show vacuoles prior to their loss, as an
indicator of increased oxidative stress (Westerhof in
16). In this context, note that melanin-synthesis by itself
generates cellular oxidative stress (17). Several of the
steps in melanin production yield H2O2 (18, 19) and
other free radicals (20, 21). This places melanocytes
under a higher oxidative stress load than for example
keratinocytes, which is most prominent in hair follicle
melanocytes because they produce large quantities of
melanin constitutively throughout the anagen phase of
the hair cycle. Oxidative stress generated outside hair
follicle melanocytes, e.g., by UV-light induced (1, 46,
56), psychoemotional (22, 23), or inflammatory (24,
25) stress may add to this endogenous oxidative stress,
overwhelm the hair follicle melanocyte antioxidant
capacity, and speed up terminal damage accumulating
for example in the aging hair follicle.
Widely used markers to determine the degree of
cellular oxidative damage are mitochondrial DNA deletions (26 29). Deletions such as the so-called common deletion lead to defects in the respiratory chain,
more oxidative stress, hydrops, and ultimately apoptosis
of the damaged cells (30, 31). In theoretical concepts
of aging, this chain of events is known as the free
radical theory of aging (32); in analogy, we would like
to propose a free radical theory of graying. Along this
concept we attempt to answer the following questions:
1) Are decreased numbers, morphology and melaGRAYING AND OXIDATIVE STRESS

nocyte apoptosis in the hair follicles of aging individuals associated with oxidative stress in the pigmentaryunit?
2) Are melanocytes in graying hair follicles protected
from oxidative stress by endogenous scavengers such as
Bcl-2 and supported by growth factors such as SCF?
3) Do graying hair follicles have higher levels of
oxidative stress-induced permanent damage (mitochondrial DNA damage)?
4) Is exogenous oxidative stress leading to apoptosis
selectively in hair follicle melanocytes?
To address these questions we performed morphological, immunohistochemical, and rtPCR analysis of
pigmented, graying, and unpigmented human scalp
skin anagen hair follicles to determine oxidative stress
in the hair follicle pigmentary unit. We then used an in
vitro culture system (33) for human scalp skin anagen
hair follicles to analyze viability of pigmented vs. unpigmented hair follicles and the response of the hair
follicle pigmentary unit to exogenous oxidative stress as
an in vitro model of graying.

MATERIALS AND METHODS

Scalp skin samples
Temporal scalp skin containing mainly anagen VI hair follicles from disposed excess skin samples of patients undergoing
elective plastic surgery was obtained with informed consent.
The study was conducted according to Declaration of Helsinki Principles. Available donor samples included in this
study were derived from female donors between 50 72 years
of age and past menopause.
After excision, tissue was maintained in Williams E Medium
(Biochrom KG seromed, Berlin, Germany) for transportation
at 4C up to 24 h. On arrival, the samples were divided by two:
One part was immediately snap-frozen in liquid nitrogen for
immunohistochemical analysis, the second was processed for
rtPCR analysis and hair follicle culturing as described below.
Hair follicle isolation
Single human anagen VI hair follicles (34, 35) were obtained
by microdissection from human skin biopsies following, with
slight modifications, the protocol published by Philpot and
colleagues (33, 36). Briefly, after separation of epidermis and
dermis from subcutaneous (s.c.) fat, the dermis just above the
dermis/subcutis border under a binocular dissecting microscope, the proximal two-thirds of anagen hair follicles located
in the s.c. fat were isolated using watchmakers forceps and
subsequently were collected in Petri dishes containing complete hair follicle culture medium (Williams E, Biochrom KG
seromed); 1% penicillin-streptomycin (Life Technologies,
Eggenstein, Germany); 1% l-glutamine 200 mM (Life Technologies); 0.02% hydrocortisone (Sigma, Taufkirchen, Germany); and 0.1% Insulin (Sigma).
Microdissected hair follicles consisted of the hair bulb and
the distal hair follicle, up to the level where the arrector pili
muscle inserts into the bulge region (including the hair shaft,
the inner and outer root sheath, the connective tissue sheath,
and the dermal papilla). This part of the hair follicle is
constantly reconstructed during the hair-growth cycle and
contains the dermal papilla, formed between the pigment
producing melanocytes above the dermal papilla and the hair
E909

shaft producing keratinocytes (the pigmentary-unit, compare

Fig. 1), and the proximal part of the hair shaft, inner root
sheath, and outer root sheath (33). It does not contain the
constant part of the hair follicle that consists of the stem cell
niche in the bulge region of the distal outer root sheath, the
arrector pili muscle, the sebaceous gland, and the hair follicle
ostium (33).
Determination of pigmentation status
With the help of an inverted microscope, floating microdissected hair follicles were classified macroscopically as hair
follicles, which produced a pigmented hair shaft when the
dermal papilla was hidden in the strongly pigmented pigmentary unit and no pigment-dilution was evident in the hair shaft
(Fig. 1). Hair follicles were classified as graying when individual melanocytes could be distinguished in the pigmentaryunit and pigment dilution was evident in the hair shaft (Fig.
1). Hair follicles were classified as unpigmented when no
melanocytes could be detected macroscopically and the hair
shaft appeared unpigmented (Fig. 1). For simplification, hair
follicles will be termed pigmented, graying, or unpigmented
hair follicles throughout the manuscript, bearing in mind
that pigmentation occurs only in the pigmentary-unit and
hair shaft.
Only anagen VI hair follicles were included in the study
(compare Fig. 1) (34, 35). Pigmentation status was photodocumented with the help of a digital camera attached to
the ocular of the microscope (Nikon Coolpix 990, Torrance,
CA). Hair follicles were then either transferred to a fresh
24-well tissue culture dish for culture or to an Eppendorf tube
for polymerase chain reaction (PCR) analysis. For transfer,
watchmaker forceps where used and great care was applied to
touch the hair follicle only at the distal outer root sheath.
From each donor, the number of isolated pigmented,
graying, and unpigmented hair follicles was determined, and
a mean percentage of pigmented, graying, and unpigmented
hair follicles in relation to total isolated hair follicle number
as calculated (Table 1). We also assessed presence of melanocytes in the bulge region and morphology and distribution
of melanocytes in the hair bulb (for details, see Table 2).
Immunohistochemistry I: TUNEL-assay
For the analysis of TUNEL melanocytes, cryostat sections of
whole-mount scalp skin samples or of cultured hair follicles
were processed for TUNEL-labeling (apoptotic cell nuclei)
according to previously published protocols (37) adapted for
human antigens. The TUNEL assay detects DNA fragmentation by enzymatic labeling of free 3-OH ends according to
the manufacturers instructions (Apopdetect Fluorescein,
QBiogene, Heidelberg, Germany). Subsequently, sections
were incubated with mouse anti-human NKI-beteb antiserum,
a pan-melanocyte marker (Anti-Human NKI-beteb antigen,
monoclonal, mouse, Sanbio Biotechnology Research Products, Beutelsbach, Germany). Labeling was detected by a
Rhodamine-conjugated goat antimouse secondary antibody
(Ab). Cell nuclei were counterstained with 4,6-diamidin-2phenylindol-dihydrochloride (4,6-diamidino-2-phenylidole
(DAPI), Boehringer Mannheim, Mannheim, Germany) with a
concentration of 1 g/ml (1 min, room temperature). For
negative controls, the terminal deoxynucleotidyl transferaseenzyme or primary Ab step was omitted. Finally, hair bulbs were
photodocumented at 200 magnification with a Zeiss Axioscope 2 fluorescence microscope (Zeiss, Gottingen, Germany).
The number of samples derived from different donors of a
whole-mount scalp skin biopsy that contained hair follicles
with TUNEL melanocytes was determined. Also, for each
E910

Vol. 20

July 2006

donor the total number of hair follicles that allowed full

appreciation of a longitudinal-sectioned dermal papilla and
pigmentary-unit was determined as well as the number of
such hair follicles with TUNEL melanocytes.
For cultured hair follicles, TUNEL melanocytes were
counted per individual hair follicle and a mean number per
group was calculated. The following tissue compartments
were analyzed: the pigmentary unit, the proximal hair bulb,
and the outer root sheath that allowed full appreciation of a
longitudinal-sectioned dermal papilla and pigmentary unit.
Since this is very difficult to achieve, the total number of hair
bulbs analyzed was restricted to 312 hair bulbs per group.
Immunohistochemistry II: oxidative stress assays and
growth factor analysis
For analysis of oxidative stress protection, oxidative stress
damage, and growth factor support in the hair follicle pigmentary unit, we performed an immunohistochemical analysis of Bcl-2 (scavenger in mitochondrial membrane), 8-hydroxyguanosine (8OhDG) (oxidatively damaged DNA base),
and c-Kit (tyrosinekinase receptor for SCF), adapting established staining protocols (38). Briefly, 10-m-thick cryosections fixed in acetone (at 20C, 10 min) were preincubated
with 10% normal goat serum (20 min, room temperature)
and then incubated overnight at room temperature with a
monoclonal primary Ab to Bcl-2 (BD Biosciences, San Diego,
CA), 8OHdG (goat polyclonal, Acris Antibodies, Hiddenhausen,
Germany), or c-Kit (rabbit polyclonal, DAKO, Carpinteria, CA)
supplemented with 2% normal goat serum. This procedure was
followed by an incubation of 60 min at room temperature with
tetramethylrhodamine-isothiocyanate (TRITC)-conjugated F(ab)2
fragments of guinea pig anti-goat IgG (Dianova, Hamburg,
Germany) diluted 1:200 in tris-buffered saline. Incubation steps
were interspersed by three washes with tris-buffered saline (5
min each). Then sections were stained with DAPI for identification of cell nuclei and NKI-beteb for identification of melanocytes. For negative controls, slides were incubated with the
secondary Ab alone.
Hair follicle culture
Per well, three pigmented or unpigmented hair follicles were
randomly distributed and cultured in Costar 24-well plates
(Corning Life Sciences, Wiesbaden, Germany) containing
500 l of complete hair follicle culture medium per well. Per
experiment, a minimum of three wells (containing a total of
nine hair follicles) was assigned to each group and were
either left untreated to analyze growth rates of pigmented vs.
unpigmented hair follicles or supplemented with different
concentrations of hydroquinone (Sigma). Each experiment was
repeated with hair follicles from different donors (20 donors for
hair growth analysis, 4 donors for hydroquinone analysis).
Every second day, each well was photodocumented, the total
length of each hair follicle was measured, medium was replaced,
and fresh supplements were added. After 8 (hair growth analysis) or 3 (hydroquinone analysis) days hair follicles were snapfrozen in a drop of OCT Cryochrome (Shandon, Pittsburgh,
PA) and stored at 80C until cryosectioning.
PCR analysis of the common deletion
To detect mitochondrial oxidative stress damage, we performed a PCR-based assay that applied primers to a region of
mitochondrial genome unaffected by rearrangements as positive control and to the regions flanking the mutation site of
a 4977 bp DNA fragment deletion known as the common
deletion. Numbering is given as in http://www.gen.emory.

The FASEB Journal

ARCK ET AL.

Figure 1. Melanocytes disappear gradually and undergo apoptosis in the graying hair bulb. Graying differed morphologically
from depigmentation in catagen hair follicles (a). Scalp skin biopsies from graying individuals contained hair follicles with
varying degrees of depigmentation (f o). Pigmentation status ranged from full pigmentation (b e) to almost complete
depigmentation (l o). Schematic representations (c, h, m) summarize macroscopic and immunhistochemical findings and show
localization of the dermal papilla, inner and outer root sheath, and the differentiated melanocytes above Aubers line (dotted
lines). These melanocytes are attached to the basement membrane to transfer their pigment to the hair shaft producing
keratinocytes, forming the pigmentary-unit (gray areas). Hair bulbs may display any number of melanocytes in the
pigmentary-unit within the hair matrix as can be observed macroscopically on isolated hair follicles (b, f, g, k, l) or by TUNEL
(green label)/NKI-beteb (red label) double-labeling on full-thickness scalp skin biopsies (d, e, i, j, n, o). Nuclei are
counterstained with DAPI (blue fluorescence). Abbreviations: bm, basement membrane; cts, connective tissue sheath; dp,
dermal papilla; em, ectopic differenting melanocyte; es, epithelial strand; hc, hair canal; hm, hair matrix; hs, hair shaft; irs, inner
root sheath; m, differentiated dendritic melanocyte in the pigmentary-unit; ors, outer root sheath; pu, pigmentary-unit; rm,
rounded melanocyte dissociating from basement membrane. a) A catagen hair bulbs can be easily distinguished from graying
anagen hair bulbs by their characteristic morphology (compare b o). Melanocytes in the catagen hair bulb appear densely
pigmented and rounded. They have no dendrites and are arranged around the condensed dermal papilla. These features
resemble melanocytes in graying hair bulbs. However, melanocytes in catagen hair follicles are more pigmented and less
dendritic and no oligodendritic lightly pigmented melanocytes can be detected (compare f o). Note that the pigmentary-unit
has disintegrated and no longer exists and that the hair shaft production has been discontinued, while an unpigmented
epithelial strand forms between hair shaft and dermal papilla. b) In a fully pigmented hair bulb, no pigment dilution can be
observed in the hair shaft and the dermal papilla is completely hidden by pigment. Individual melanocytes cannot be
distinguished in the pigmentary-unit. c) Schematic representation of a fully pigmented anagen VI human hair bulb. Note that
all melanocytes in the pigmentary-unit in the center of the hair matrix are in close contact with the basement membrane
separating the hair follicle epithelium from the dermal papilla. d) A fully pigmented anagen hair follicle from a scalp skin
biopsies of a graying individual displays numerous NKI-beteb melanocytes in the outer root sheath (insert). e) A fully
pigmented anagen hair follicle from a scalp skin biopsies of a graying individual displays highly dendritic NKI-beteb
melanocytes (inserts) in the hair bulb, located in the pigmentary-unit above Aubers line (dotted white line). f, g) The hair shafts
of two graying hair bulbs display pigment dilution and allow to distinguish individual keratinocytes packed with melanosomes,
giving the hair shaft a spotted appearance. Melanocytes move away from the dermal papilla and appear rounded, oligodendritic,
and stubby. Individual lightly pigmented oligodendritic melanocytes occur below Aubers line (arrows). h) Schematic
representation of a graying anagen VI human hair bulb. i, j) In a graying hair follicle, numerous TUNEL apoptotic
melanocytes can be detected in the pigmentary-unit (i and inserts). Melanocytes are also detectable below Aubers line (j). k,
l) In two hair bulbs, which do not produce pigmented hair shafts, melanocytes appear triangular rather than dendritic, and small
rounded pigmented fragments can be observed. In addition, single dendritic melanocytes with very light pigmentation are
located in the most proximal hair bulb adjacent to the basement membrane surrounding the hair follicle epithelium and
separating it from the connective tissue sheath. The low number of pigmented melanocytes in the pigmentary-unit allows full
appreciation of the tear-shaped dermal papilla. m) Schematic representation of an almost depigmented anagen VI human hair
bulb. n) In a graying hair follicle a single NKI-beteb melanocyte can be detected in the outer root sheath (insert). o) In the
hair bulb of a graying hair follicle the number and dendricity of melanocytes is dramatically reduced (inserts), melanocytes can
be detected below Aubers line, and a single dendritic melanocyte is located in the most proximal hair bulb adjacent to the
basement membrane surrounding the hair follicle (insert). TUNEL cell nuclei are also detectable in the terminally
differentiating inner root sheath and serve as internal positive control (arrowheads).

GRAYING AND OXIDATIVE STRESS

E911

TABLE 1.

Hair follicles in all states of pigmentation could be isolated from aging donors

Number of pigmented hair

follicles

556 (67.45%)

Number of gray hair

follicles

Number of unpigmented hair

follicles

Total number of hair

follicles

32(3.8%)

234(28,66%)

822(100%)

Pooled data derived from hair follicles isolated from 10 representative donors. Hair follicle pigmentation status was classified with the help
of an inverted microscope at a magnification of 200 on hair follicles floating in culture medium.

edu/MITOMAP/mitoseq.html: undeleted mitochondrial

DNA region, L3108 and H3717; deleted mitochondrial DNA
by standard PCR, L8282, and H13851, forward primer: 5-ccc
ctc tag agc cca ctg taa agc-3, reverse primer: 5 gtt gag gtc tag
ggc tg tta-3; (39) internal primers to amplify deleted DNA in
a nested PCR in conjunction with the Barron primers as
control of the standard PCR, L8377, and H13484.
As the name suggests, the common deletion is the most
commonly found DNA rearrangement in the mitochondrial
genome. Flanked by two direct repeats, the common deletion removes 4977 bp from the mitochondrial chromosome,
including DNA coding for two components of the mitochondrial oxidative phosphorylation (OXPHOS) system, thereby
initiating the breakdown of the respiratory chain with subsequent cell hydrops and death. This deletion has been shown to
be a suitable marker for the overall degree of DNA damage (40).
Twenty different donors were included into the study, each
donating pigmented, graying, and unpigmented hair follicles.
For pigmented hair follicles, a total of 51, for graying of 54,
and for unpigmented of 48 hair follicles were analyzed. The
PCR cycle used contains short extension times (less then 2
min) that do not allow amplification of the full-length,
undeleted product (5.5 kb) and so selectively only amplify
deleted DNA (592 bp).
DNA was extracted as follows. The follicles were digested in
proteinase K buffer (500 mM Tris, pH 8.5; 1 mM EDTA; 0.5%
Tween-20; 200 g proteinase K) for 16 h at 37C and then
ground using a plastic pestle. Another 50 g proteinase K was
added and incubated for 2 h. The DNA was then extracted
using phenol/chloroform, the supernatant ethanol was precipitated, and the DNA was resuspended in a vol of 50 l.
TABLE 2.

A
B
C
D
E
F
G
H
I

The above DNA preparation (1 l) was amplified using the

total mtDNA or the 4977 primer sets described above in a 25
l reaction containing 1.5 mM MgCl2, 0.2 mM dNTP, 15
pmol of each primer, and 0.5 U Amplitaq Gold DNA polymerase (Perkin-Elmer, Rodgau - Jugesheim, Germany). Reactions were run using the following cycles. For the Total
mtDNA primers: 12 min 94C (45 s at 94C, 30 s at 51C, 1
min 72C) for 34 cycles and a final extension of 8 min at 72C.
For the 4977 primers: 12 min 94C (45 s at 94C, 30 s at
51C, 1 min 72C) for 65 cycles and a final extension of 8 min
at 72C. Half of the reaction was visualized by electrophoresis
in a 1% agarose gel containing EtBr.
Histomorphometry
TUNEL melanocyte numbers were pooled per group for
each experiment. The results among different experiments
using samples from different donors were highly comparable.
Thus, the mean scores and numbers of TUNEL melanocytes per group and from all experiments were then pooled
again and statistical differences between groups were determined by Mann-Whitney U test for unpaired samples.

RESULTS
Donor samples older than 50 years all contained
graying hair follicles
We were able to isolate varying numbers of pigmented
and unpigmented hair follicles (Fig. 1) and graying

Melanocytes disappear gradually from the graying hair follicle

Melanocytes in the bulge region (stem

cell niche)
Pigment dilution in hair shaft
Differentiated melanocytes below
Aubers line
(stem cell niche?)
Rounding of hair bulb melanocytes
Dendricity of hair bulb melanocytes
TUNEL melanocytes
Oxidative stress in pigmentary -unit
Hair shaft elongation in culture
Selective melanocyte susceptibility to
exogenous oxidative stress

Pigmented hair follicles

Gray hair follicles

Unpigmented hair follicles

0
0

()

()

Not determined
Not determined

()
0
0

Not determined

Pigmented melanocytes detectable in intact isolated hair follicles with the help of an inverted microscope (A, D, E), melanocytes labeled
with NKI-beteb (B, C, D, E) and/or TUNEL (F) or 8OHdG (G) were analyzed. Arbitrary units were assigned to the following criteria:
melanocytes in the bulge region 4 , 2 4 , 1 ; pigment dilution in hair shaft: no dilution 0 to no pigment ;
melanocytes below Aubers line: 12, 3 4, 4; rounding of melanocytes: 0 multidendritic cells, fully rounded cells;
dendricity of hair bulb melanocytes: multidendritic to 0 non-dendritic. A total of 90 hair follicles derived from 10 different donors
was included into the analysis. Presence of TUNEL or 8OhdG melanocytes in the pigmentary-unit of graying individuals (full-thickness scalp
skin biopsies of 20 different donors) was summarized as follows: present, 0 not present. Hair shaft elongation in cultured hair follicles
of the same donors (20 different donors, each donating 9 hair follicles per group) was summarized as follows: present, significantly
larger. Susceptibility of hair bulb constituting cells in pigmented hair follicles to exogenous oxidative stress (hydroquinone) was summarized
as follows: selective susceptibility of hair bulb melanocytes.

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ARCK ET AL.

hair follicles with different grades of depigmentation

from each donor (Tables 1 and 2). The number of
unpigmented hair follicles was lower (Table 1) than the
average number of unpigmented hairs reported in
donors aged 50 and older in the literature [c.f. (1)]. In
our experience microdissection selects for pigmented
hair follicles, since the unpigmented hair follicles are
difficult to locate at the dermis-subcutis border during
isolation. Also, many hair follicles that produce a
seemingly white hair still retain some melanocytes in
their hair bulb and are therefore classified as graying
under the dissection microscope.

mented hair bulbs (Fig. 1E) or in melanocytes outside

the pigmentary unit (Fig. 1D, E, J, N, O).
Immunohistochemistry confirms presence of
oxidative stress in the pigmentary unit
of graying individuals
By immunohistochemistry, the oxidatively damaged
oligonucleotide 8OHdG could be detected in the pigmentary unit of anagen hair follicles in the scalp skin of
graying individuals (Fig. 2). The strongest expression
localized to the melanocytes (Fig. 2) was in the pigmentary unit. Strong expression was also detected in the

Hair bulb melanocytes in the graying hair follicle

display features of oxidatively stressed
and apoptotic cells
In the search of morphological hints to pathways
involved in graying, we first analyzed number, appearance, and distribution of melanocytes in graying hair
follicles from aged donors. In contrast to fully pigmented anagen hair bulbs (Fig. 1B, C, E), melanocytes
in graying hair bulbs (Fig. 1FO) were reduced in
number and showed features of oxidative stress and
apoptosis by morphometric analysis (Fig. 1, Table 2).
They appeared rounded and oligodendritic and moved
away from their designated site, the basement membrane, which separates the pigmentary unit of the hair
bulb from the mesenchymal pace-maker of hair growth,
the dermal papilla (13). This process appeared to be
progressive because the fewer melanocytes we detected
above Aubers line, the more rounded and less dendritic was their appearance (Table 2) (1).
Outside the pigmentary unit, melanocytes were frequently detectable in the bulge region of the outer root
sheath of pigmented hair follicles, where melanocytic
stem cells reside (Fig. 1D; Table 2). In graying or
unpigmented hair follicles of the same donors, melanocytes were only rarely detected in this location (Fig.
1N; Table 2). Additional single, lightly pigmented oligodendritic melanocytes became detectable in the
proximal hair bulb below Aubers line. In this compartment, unpigmented and undifferentiated putative melanocyte stem cells, but not pigmented differentiated
melanocytes, are normally found. Interestingly however, comparable ectopic differentiation of melanocytes has been reported before in the graying hair
follicle bulge region (Fig. 1G, H, K, L, M, O; Table 2)
(12). In addition to the reduced number of melanocytes in the pigmentary unit, this finding indicates a
diminished and prematurely differentiated stem cell
pool selectively in graying and unpigmented hair follicles.
By immunohistochemical analysis, hair bulbs with an
intermediate number of melanocytes in their pigmentary unit occasionally displayed TUNEL-positive labeling of pigment-producing melanocytes (Fig. 1I and J,
Table 2). This finding demonstrates the loss of melanocytes from the graying hair bulb by apoptosis. Such
apoptotic melanocytes were never seen in fully pigGRAYING AND OXIDATIVE STRESS

Figure 2. Oxidative stress in the pigmentary-unit is high.

Immunohistochemistry for 8OHdG reveals strong expression
in the pigmentary-unit and melanocytes of a hair bulb in a
graying individual (b and c). Cell nuclei are counterstained
with DAPI (a and c). Arrows point at melanocytes in the
individual (a and b) and merged photomicrographs (c).

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terminally differentiating inner root sheath keratinocytes (not shown), a compartment devoid of melanocytes and characterized by the most rapid terminal
differentiation within the hair follicle. These observations indicate high oxidative stress selectively in these
compartments.
Hair follicles in graying individuals lack oxidative
stress protection by Bcl-2 and growth factor support
by c-Kit-signaling
Immunohistochemical analysis of full mount scalp skin
biopsies of graying individuals revealed that Bcl-2 expression was weakly present in the melanocytes of the
pigmentary unit of fully pigmented hair follicles (Fig.
3ai). By contrast, melanocytes in the pigmentary unit of
graying and almost white hair follicles lacked this
expression (Fig. 3bi, ci). Bcl-2 expression in melanocytes of graying and catagen hair bulbs was located to
single, oligodendritic, and immature appearing melanocytes outside the pigmentary unit (Fig. 3: bi, d). As an
intrinsic positive control, Bcl-2 expression was always
found in melanocytes of the outer root sheath and the
epidermis (Fig. 3e, f). Interestingly, hydrochinone treatment failed to induce Bcl-2 expression in hair bulb
melanocytes (Fig. 3g).
Also, we could not detect any Kit expression in
anagen scalp hair follicles from graying individuals
(Fig. 3aii cii) or in catagen hair follicles (Fig. 3h). By
contrast, and serving as an intrinsic control, c-Kit
expression was always found in epidermal melanocytes,
in outer root sheath melanocytes and in dermal mast
cells (Fig. 3i, j). Again, hydrochinone treatment failed
to induce c-Kit expression in pigmented hair follicles
from graying scalp skin (Fig. 3k).
Both Bcl-2 and c-Kit were absent from melanocytes in
the bulge region of aging hair follicles (not shown).
As a result of increased oxidative stress, graying hair
follicles show increased mitochondrial DNA deletion
Pigmented, graying, and unpigmented hair follicles
that were harvested from the same donors were analyzed for the presence of the common deletion. A
total number of 20 different donor samples could be
included. We obtained 23 pigmented, graying and
unpigmented hair follicles from each sample (total
number was 153 hair follicles: 51 pigmented, 54 graying, 48 unpigmented). We found an increased number
of deletions in hair follicles that were macroscopically
classified as graying (8 out 20 donors40%) and in
unpigmented hair follicles (4 out of 20 donors20%)
(Fig. 4). These later hair follicles may still contain some
unpigmented, macroscopically undetectable melanocytes. In macroscopically pigmented hair follicles, we
found only one sample with a deletion (1 out of 20
donors5%) (Fig. 4). The presence of mitochondrial
DNA deletions was independent of age within the
sample group (all donors older than 50 years).
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Unpigmented hair follicles retain full viability and

grow better in culture than pigmented hair follicles
Over a culture period of 8 d, unpigmented hair follicles
showed efficient hair shaft elongation (Fig. 5). When
compared with hair shaft elongation of pigmented hair
follicles from the same donors, unpigmented hair
follicles showed significantly higher growth rates than
pigmented hair follicles (Fig. 5). Both groups maintained an anagen-like morphology of the hair bulb and
did not enter into a catagen-like stage over this culture
period (not shown).
Pigmented hair follicles show melanocyte apoptosis in
the pigmentary unit after additional exogenous
oxidative stress
Pigmented hair follicles cultured over a period of 3 d in
the presence of hydroquinone at concentrations between 103 to 107 M showed a dose dependent
increase of TUNEL apoptotic melanocytes in their
pigmentary-unit (Fig. 6). Hydroquinone (107) appeared to be without effect and showed no significant
differences to control in TUNEL melanocyte number. Hydroquinone (104 and 103) showed toxic
effects on the hair follicle, with increased apoptosis in
the hair follicle keratinocyte population in the hair
matrix and the inner root sheath (not shown). Hydroquinone (105 to 106 M) showed apoptosis of melanocytes selectively in the pigmentary-unit by TUNELlabeling (Fig. 6).

DISCUSSION
Here we provide unique evidence for oxidative stress
induced loss of melanocytes from the human hair
follicle during aging. In detail, we show for the first
time that:
1) a decreased number of viable melanocytes in the
aging hair follicle bulge and bulb and an increased
incidence of hair bulb melanocyte apoptosis in aging
individuals are associated with oxidative stress in the
pigmentary unit;
2) the aging hair follicle is characterized by the
absence of oxidative stress-protectors, such as Bcl-2, and
melanocyte growth factors, such as c-Kit;
3) a higher frequency of oxidative stress associated
mitochondrial DNA damage occurs in graying hair
follicles, while unpigmented hair follicles prove to be
not older than pigmented hair follicles;
4) melanocytes of the pigmentary unit are highly
and selectively susceptible to exogenous oxidative stress
damage.
One major route, by which oxidative stress leads to
permanent melanocyte damage, appears to pass by the
mitochondria, since their DNA is not so well protected
as genomic DNA. The accumulation of mutations,
therefore, correlates with age and is indicative of general exposure and generation of oxidative stress, (39,

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ARCK ET AL.

Figure 3. Oxidative stress protection and growth factor support is down-regulated in hair follicles from graying individuals.
Abbreviations: bm, basement membrane; D, dermis; dp, dermal papilla; e, epidermis; em, ectopic differenting melanocyte; hs, hair
shaft; irs, inner root sheath; m, differentiated dendritic melanocyte in the pigmentary-unit; ors, outer root sheath; pu, pigmentary-unit;
rm, rounded melanocyte dissociating from basement membrane. Schematic representations of hair bulbs in graying individuals
depict a fully pigmented hair bulb (a), a graying hair bulb (b), and an almost white hair bulb (c). Photomicrographs that correspond
with the respective schematic have been labeled with the same letter and (i) for Bcl-2 immunolabeled bulbs or (ii) for c-Kit
immunolabeled bulbs. Each photomicrograph is shown in triplicate showing the DAPI staining for cell nuclei on the left, the
NKI-beteb staining for melanocytes in the center and the specific staining for Bcl-2 or c-Kit, respectively, on the right as indicated
above the photomicrographs. Higher magnifications of individual regions of interest are indicated by white boxes. All photomicrograhs were taken from immunolabeled sections from full-thickness skin biopsies from graying individuals with the exception of (g)
and (k), which were taken from pigmented anagen scalp skin hair follicles treated with hydrochinone as described in Materials and
Methods. ai) Bcl-2 immunoreactivity is weak but readily detectable in melanocytes of the pigmentary-unit of a fully pigmented,
nongraying hair bulb. bi) Bcl-2 immunoreactivity is absent from melanocytes of the pigmentary-unit of a graying hair bulb. By contrast,
a single oligodendritic and weakly NKI-beteb melanocyte outside the pigmentary-unit is Bcl-2. ci) Bcl-2 immunoreactivity is absent
from an almost white hair bulb showing a single, rounded melanocyte dissociated from the basement membrane in the
pigmentary-unit (arrow). d) Single, strongly NKI-beteb polydendritic melanocytes in a catagen hair follicle retain Bcl-2-expression.
e, f) Dendritic melanocytes in the epidermis (e) and outer root sheath (f) are strongly labeled with Bcl-2 and NKI-beteb (arrowheads),
while single, nondendritic putative melanocyte stem cells in the epidermis (e) are labeled only by Bcl-2 (arrows). g) Hydrochinone
treatment induced the development of catagen-like morphology with a rounded dermal papilla in pigmented anagen hair bulb. After
the treatment, Bcl-2 expression was no longer detectable in melanocytes of the pigmentary-unit (arrowheads) and greatly diminished
in melanocytes moving out of the pigmentary-unit (arrows). aii cii) c-Kit expression is absent from melanocytes in (arrowheads) and
outside (arrows) of the pigmentary-unit in the hair bulb of a fully pigmented (aii), a graying (bii), and an almost white (cii) hair
follicle. d) Melanocytes in a catagen hair follicle are not labeled by c-Kit, neither in (arrowheads) nor outside (arrows) the
pigmentary-unit. e) Melanocytes in the epidermis (arrowheads) and outer root sheath (arrows) are c-Kit. f) A large, granular, putative
mast cell (arrows) in the dermis is labeled with c-Kit but not NKI-beteb. g) Hydrochinone treatment induced the development of
catagen-like morphology with a rounded dermal papilla in pigmented anagen hair bulb but failed to induce c-Kit expression in
melanocytes inside (arrowheads) and outside (arrows) of the pigmentary-unit.

GRAYING AND OXIDATIVE STRESS

E915

Figure 4. The common deletion is highly present in graying

hair follicles. Pigmented, graying, and unpigmented anagen
hair follicles from scalp skin biopsies of graying individuals
were analyzed for the presence of the common deletion as
indicated in Materials and Methods. The graph shows data
pooled from 20 different donors. The photomicrograph
shows a representative example of the PCR analysis of one
donor. Each donors sample was analyzed in triplicate.

Figure 6. Oxidative stress induces apoptosis of melanocytes in

the pigmentary-unit. Pigmented anagen hair follicles from
scalp skin biopsies of graying individuals were cultured in the
presence of hydroquinone and immunohistochemical analysis was performed on the third day in culture. TUNEL
(green fluorescence) apoptotic melanocytes (red fluorescence) were counted per hair follicle harvested from 4
different donors (3 to 9 hair follicles per donor) and mean
and sem were calculated. Hair follicles treated with 105 M
hydroquinone showed significantly more apoptotic melanocytes in the pigmentary-unit (P0.05 by Mann-Whitney U
analysis for two independent samples). Abbreviations: hm,
hair matrix; dp, dermal papilla.

Figure 5. Hair shaft-elongation is higher in

unpigmented than in pigmented hair follicles.
Anagen hair follicles from scalp skin biopsies of
graying individuals were separately cultured in
two groups: pigmented hair follicles and unpigmented hair follicles. Examples of individual
unpigmented and pigmented hair follicles in
culture are given (hair follicles A and B). On
day 8 in culture difference in hair shaft elongation is significant (P0.05 by Mann-Whitney U
analysis for two independent samples) between
unpigmented and pigmented hair follicles. Data
was pooled from 20 independent experiments/
individuals. Each experiment included 9 hair
follicles per group.

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ARCK ET AL.

40) caused for example by psychoemotional stress,

inflammation, UV-light, and others. In summary, our
findings support the proposed hypothesis of a free
radical theory of graying and suggest that melanocytes
in the hair follicle are highly susceptible to endogenous
oxidative stress. Exogenous oxidative stress can trigger
and hasten this process and provoke permanent damage selectively and prematurely in hair bulb melanocytes.
Clinical observations reported in the literature support the premature loss of hair bulb melanocytes by
oxidative stress. Smokers for example gray early (41,
42) and in alopecia arreata, a disease characterized by
high psychoemotional and inflammatory stress, hair
frequently grows back depigmented (43). The morphological features we observed in melanocytes of aging
and graying hair follicles further support this notion.
Such features have been described before in melanocytes under oxidative stress for example in culture or in
vitiligo (6, 44, 45). Consequently, we could show that
melanocytes in the graying hair bulb undergo apoptosis
and show oxidative stress damage by 8OHdg labeling
(28).
We also observed ectopic differentiation of melanoblasts in the hair bulb below Aubers line. This observation can be interpreted as an oxidative stress damageresponse, as has been shown in the epidermis for
example after UV-light (1, 46). Such ectopic melanogenesis in the aging hair follicle has been described
before in the bulge region, (12) hosting melanocytic
stem cells in the mouse (15). It was therefore suggested
that untimely ectopic differentiation of melanocyte
stem cells in the aging hair follicle diminishes the stem
cell pool (6). However, this work did not consider
oxidative stress as a cause.
The first model that established stem cells in the hair
follicle utilized bromodeoxyuridine to detect labelretaining cells (64). These studies focused on the bulge
region of the hair follicle and did not differentiate,
what type of cell maintained the label. They rather
assumed that all label-retaining cells were keratinocytes, since they represent the majority of hair follicle
constituting cells. Only years later it was shown that the
label-retaining cell population in the bulge also hosted
melanocytic stem cells (15). Recently, data were published that show label-retaining putative stem cells in
the outer root sheath, close to the outer basement
membrane of the hair bulb (65) and in the hair follicle
matrix (66). We hypothesize that these newly defined
stem-cell reservoirs of the hair follicle also contain
melanocytic in addition to keratinocytic stem cells. This
hypothesis is further supported by the observation that
the outer root sheath and lateral hair bulb contain
amelanotic, undifferentiated melanocytes that cannot
be detected by most immunohistochemical labeling
methods. However, these cell populations are evident
from ultrastructural analysis by electronmicroscopy in
the mouse (38) and await analysis by label retention.
Ectopic differentiation of melanocytes in these niches
may add to the progressive disability of the aging hair
GRAYING AND OXIDATIVE STRESS

follicle to revive the pigmentary unit with every hair

cycle.
The progressive loss of melanocytes from aging human hair follicles was previously characterized by decreased numbers of melanocytes immunoreactive to
pigmentation-related markers, such as pMel-17, vimentin, and MITF, during anagen, telogen, and catagen of
young vs. old donors. Expression of these markers in
the ostium and sebaceous glands of young vs. old hair
follicles did not differ (5, 12). Our findings basically
confirm this progressive loss of melanocytes from the
hair follicle bulge and bulb during aging. However, by
using the pan-melanocyte marker NKI-beteb, we could
show that the presence of hair bulb melanocytes may
greatly vary even within one donor and that the decrease of hair bulb melanocytes corresponds with the
disappearance of the oxidative stress protector Bcl-2.
Moreover, the aging hair follicle, but not the aging
epidermis, contains only melanocytes that lack expression of the SCF receptor c-Kit and thereby lose the
ability to respond to SCF stimulation during migration
and pigmentation. In addition, expression of NKIbeteb, Bcl-2, and c-Kit did not differ in the hair follicle
ostium and sebaceous gland epithelium between pigmented and unpigmented hair follicles of the same
donors (not shown).
Together, these observations emphasize the specific
and permanent loss of melanocytes from the pigmentproducing unit during graying, including its stem cell
populations while the epidermal reservoirs and pigmented hair follicles appear to remain relatively untouched. However, we were unable to differentiate
between pigmented and graying hair follicles in telogen and catagen, because these follicles are characterized by their low to nil expression of melanocytes
independent of their pigmentation status and could
therefore not be categorized in terms of their pigmentation status.
Increase in oxidative stress damage has been correlated with age in a variety of species (32, 47, 48) and
8Ohdg-labeling was detected alongside increased levels
of the common deletion in the aging epidermis (28).
We were able to detect TUNEL-immunoreactivity and
8Ohdg-labeling in association with the common deletion in graying individuals, indicating permanent
oxidative stress damage in the aging hair follicle. However, pigmented and unpigmented hair follicles of the
same donors showed less frequent occurrence of the
deletion, and in culture, we found that unpigmented
hair follicles of the same donors showed increased hair
shaft elongation. Together these findings indicate that
the hair bulb melanocytes acquire permanent damage
first, while the hair shaft producing keratinocytes retain
their ability to actively and effectively produce a hair
shaft.
To our knowledge, this is first report that proves the
long-suspected and often anecdotally described accelerated growth of white scalp hair (1). The relative
protection from the aging process may also be due to
their higher antioxidant enzyme activities as compared
E917

to melanocytes (49). The improved growth of unpigmented hair follicles may be due to lower overall
oxidative stress and lower calcium, which is normally
transferred to the keratinocytes with the melanosomes
and may lead to earlier terminal differentiation of
pigmented hair follicles for example in beard hair
follicles (50).
Progressive premature graying and loss of melanocytes from aging hair follicles has also been shown in
mice deficient in Bcl-2 (12, 51, 52). Bcl-2 is a molecule
protecting mitochondria from oxidative stress and subsequent cell death (5355). Thus, progressive and
selective loss of melanocytes from Bcl-2 deficient mouse
hair follicles as opposed to melanocytes in the sebaceous gland, and in the absence of general features of
premature aging, also supports that oxidative stress is a
major cause of hair follicle melanocyte apoptosis.
Along this line of evidence one study in mice has shown
that topical application of an antioxidant enzyme can
protect hair follicles from UV-light induced graying
(56).
Premature graying is a process with dramatic effect
on appearance, socio-cultural status and self-esteem of
the affected individual. Oxidative stress damage in hair
follicle melanocytes appears to be at the tip of the
iceberg and can explain the rapid graying process often
reported in individuals experiencing environmental
and endogenous challenges that generate oxidative
stress (67) such as psychoemotional (57, 58) or inflammatory disease-induced stress(24). Oxidative stress generated by such challenges may add to the endogenous
oxidative stress-load of the hair bulb melanocyte. Upon
exposure to such stressors, the highly susceptible hair
follicle melanocytes disappear and visibly mark their
disappearance (Fig. 7).
In the light of our findings the graying process may
be understood as an easily accessible and quite obvious
indicator of the overall oxidative stress an individual is
exposed to and of its redox-capacities (28). This indicator function is relevant to other organ-systems. Accordingly, premature graying was named as a risk factor
and indicator in age- and increased oxidative stressrelated diseases such as coronary heart disease (59) or
osteoporosis (60, 61). Moreover, the common deletion
was determined in hair follicles to monitor aging (62)
and end-stage renal disease (63). However, these later
studies failed to determine hair follicle pigmentation
status in the follicles used for their study.
The graying hair follicle therefore offers a unique
modelsystem to study oxidative stress effects and aging
and to test antioxidants and other antiaging therapeutics in their ability to slow down or even stop this
process. For example, protection from oxidative stressinduced apoptosis by stimulation of the endogenous
redox-capacity or treatment with exogenous antioxidants can be tested in hair follicle culture. Also, in this
model-organ the activation and maintenance of the
follicular melanocyte stem cell pool can be easily assessed. Moreover, in clinical trails the fate of hair
follicle melanocytes can be monitored as a measure for
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Figure 7. Hypothetical scenario indicating the involvement of

oxidative stress in the graying process. Melanocytes in pigmented anagen hair follicles in young and healthy subjects
can deal with the endogenous oxidative stress caused by
melanin-production. In the old and diseased subject, however, stressors induce alterations in pigment-producing and
antioxidative enzymes and cofactors and the production of
endogenous antioxidants and repair-enzymes as well as
growth factors, etc. decreases. This results in a breakdown of
the hair follicle melanocyte redox-capacity and subsequent
deleterious oxidative stress damage.

oxidative stress-tissue damage and effectiveness of antiaging and antioxidant therapeutics. Alongside with the
protection from endo- and exogenous oxidative stressors, this provides us with a plethora of tools to be used
in the prevention of excessive oxidative stress in the
hair follicle pigmentary-unit and other tissues.
This study was supported by a research grant of the
University-Medicine Charite (UFF) to Dr. Peters as well as by
the German Research Foundation (Pe890 4-1, Ar 232 14-2).
Many thanks are owed to Dr. Hoting, Schwarzkopf, Hamburg,
Germany; as well as Profs. Schallreuter and Tobin, Dept. of
Biomedical Sciences, Bradford, UK, for introducing the senior author to the world of hair follicle pigmentation. Many

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ARCK ET AL.

thanks go also to our colleagues in plastic surgery that have

generously supported our work with the supply of scalp skin
biopsies: Dr. Johannes Bruck, Clinica Vita, Berlin, Germany,
and Drs. Meyburg, Klinik fur kosmetische Chirurgie, Berlin,
Germany.

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The FASEB Journal

Received for publication June 2, 2005.

Accepted for publication January 17, 2006.

ARCK ET AL.

The FASEB Journal FJ Express Summary

Towards a free radical theory of graying: melanocyte

apoptosis in the aging human hair follicle is an
indicator of oxidative stress induced tissue damage
Petra Clara Arck,,1 Rupert Overall,*,1 Katharina Spatz,* Christiane Liezman,*
Bori Handjiski, Burghard F. Klapp, Mark A. Birch-Machin, and
Eva Milena Johanne Peters*,2
*Cutaneous Neuroimmunology, Biomedical Research Center, University Medicine Charite, Virchow
and Mitte Campus; Psychoneuroimmunology, Biomedical Research Center, University Medicine
Charite, Virchow Campus; and Internal Medicine, Psychosomatics, University Medicine Charite,
Mitte Campus, Humboldt-University of Berlin, Berlin, Germany; and Dermatology, Medical School,
University of Newcastle, UK
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-4039fje
SPECIFIC AIMS
Graying is a spectacular and intriguing phenomenon
often discussed in the context of stress and aging. In
our society, fixed on eternal youth, the loss of pigmented hair has great socio-economic as well as psychosomatic impact. Despite these pressing interests, the
phenomenon has not yet been satisfactorily explained
and a scientifically sound therapeutic strategy remains
missing. From animal experiments and mutation-reports we know about predetermined breaking points in
the function of the hair follicle pigmentary-unit. This
unit, formed between the pigment-producing melanocytes and the hair shaft-producing keratinocytes of the
hair bulb, produces melanin continuously over many
years. Melanin-synthesis generates H2O2 and other free
radicals, placing hair follicle melanocytes under high
oxidative stress. Moreover, oxidative stress appears to
be a common theme in melanocyte dysfunction and
results in impairment of growth factors, antioxidative
enzyme and cofactor functions, DNA-repair mechanisms, and antiapoptotic signals such as BclII. Ultimately, this may lead to permanent melanocyte damage
and the exhaustion of a melanocyte stem cell pool
residing in the bulge region of the hair follicle. Psychoemotional and inflammatory-induced oxidative stress
may add to the endogenous oxidative stress, overwhelm
the hair follicle melanocyte antioxidant capacity, and
speed up terminal damage to hair bulb melanocytes.
However, conclusive proof that any of the above braking points of melanocyte function are active in and
relevant to graying has not been brought forward to
date. This encouraged us to propose a free radical
theory of graying in analogy to the free radical theory
of aging. In support of this theory, we performed
experiments to answer the following questions: 1) Are
decreased numbers, morphology and melanocyte apo0892-6638/06/0020-1567 FASEB

ptosis in hair follicles of aging individuals associated

with oxidative stress in the pigmentary-unit? 2) Do
graying hair follicles have higher levels of oxidative
stress induced permanent damage (e.g., mitochondrial
DNA damage)? 3) Is oxidative stress leading to apoptosis preferably in hair follicle melanocytes?
PRINCIPAL FINDINGS
1.1 Morphological and immunohistochemical analysis
indicate accumulating and deleterious oxidative stress
damage in hair follicle melanocytes of graying
hair follicles
We microdissected pigmented, graying and unpigmented human scalp skin hair follicles from healthy
aging donors (elective face-lift) under sterile conditions and analyzed melanocyte presence, morphology
and distribution by macroscopic (inverted microscopy
on free-floating isolated hair follicles) and immunohistochemical (NKI-beteb for melanocytes; TUNEL for
apoptotic nuclei) analysis. We found, that melanocytes
in graying hair bulbs, when compared to pigmented
and unpigmented hair follicles harvested from the
same donors, were reduced in number and showed
features of oxidative stress and apoptosis by morphometric analysis (Table 1). We also found the occasional
hair bulb with TUNEL-positive labeling of pigmentproducing melanocytes in graying individuals but never
1

These authors contributed equally to this work.

Correspondence: Biomedical Research Center, Rm. Nr.
2.0549, University Medicine Charite, Virchow Campus, Humboldt University of Berlin, Augustenburger Platz 1, Berlin
13353, Germany. E-mail: eva.peters@charite.de or frl_peters
@yahoo.com
doi: 10.1096/fj.05-4039fje
2

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Table 1.

Melanocytes disappear gradually from the graying hair follicle

Pigmented hair follicles

A Melanocytes in the bulge region (stem cell niche)

B Pigment dilution in hair shaft
C Differentiated melanocytes below Aubers line
(stem cell niche?)
D Rounding of hair bulb melanocytes
E Dendricity of hair bulb melanocytes
F TUNEL melanocytes
G Oxidative stress in pigmentary unit
H Hair shaft elongation in culture
I Selective melanocyte susceptibility to exogenous
oxidative stress

Gray hair follicles

Unpigmented hair follicles

0
0

()

()

Not determined
Not determined

()
0
0

Not determined

Pigmented melanocytes detectable in intact isolated hair follicles with the help of an inverted microscope (A, D, E), or melanocytes labeled
with NKI-beteb (BE) and/or TUNEL (F ) or 8OHdG (G) were analyzed. Arbitrary units were assigned to the following criteria: melanocytes
in the bulge region 4 , 2 4 , 1 ; pigment dilution in hair shaft: no dilution 0 to no pigment ; melanocytes below
Aubers line: 12, 3 4, 4; rounding of melanocytes: 0 multidendritic cells, fully rounded cells; dendricity of hair
bulb melanocytes: multidendritic to 0 non-dendritic. A total of 90 hair follicles derived from 10 different donors was included into
the analysis. Presence of TUNEL or 8OhdG melanocytes in the pigmentary unit of graying individuals (full-thickness scalp-skin biopsies of
20 different donors) was summarized as follows: present, 0 not present. Hair shaft elongation in cultured hair follicles of the same donors
(20 different donors, each donating 9 hair follicles per group) was summarized as follows: present, significantly larger. Susceptibility
of hair bulb constituting cells in pigmented hair follicles to exogenous oxidative stress (hydroquinone) was summarized as follows:
selective susceptibility of hair bulb melanocytes.

in samples from nongraying individuals. Compared to

other hair follicles within the same donor, hair follicles
with apoptotic melanocytes consistently contained an
intermediate number of melanocytes in their pigmentary-unit and were thus classified as graying (Table 1).
Hair follicles with maximal melanocyte numbers never
contained TUNEL melanocytes, while pigment-producing melanocytes were absent from unpigmented hair
bulbs. This observation demonstrates loss of melanocytes
by apoptosis selectively from the graying hair bulbs. Moreover, outside the pigmentary-unit, melanocytes in the
stem cell niche in the bulge region of the outer root
sheath were only rarely detected in graying or unpigmented hair follicles, which indicates a diminished stem
cell pool selectively in these hair follicles (Table 1).
1.2 Immunohistochemistry confirms presence of
oxidative stress in the pigmentary-unit of graying
individuals
The oxidatively damaged oligonucleotide 8OHdG
could be detected in the pigmentary-unit of anagen
hair follicles in the scalp skin of graying individuals
(Table 1). In the hair bulb, strongest expression localized to melanocytes, indicating high oxidative stress
selectively in this cell population.
2.1 As a result of increased oxidative stress, graying
hair follicles show increased mitochondrial DNA
deletion
Mitochondrial deletions such as the so called common deletion are the result of excessive cellular oxidative stress and lead to defects in the respiratory chain,
more oxidative stress, hydrops, and ultimately apoptosis
of the damaged cells. We found an increased number
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July 2006

of deletions in hair follicles that were macroscopically

classified as graying (8 out 20 donors40%) and in
unpigmented hair follicles (4 out of 20 donors20%)
harvested from the same donors. These later hair
follicles may still contain some unpigmented, macroscopically undetectable melanocytes. In macroscopically pigmented hair follicles, we found only one sample with a deletion (1 out of 20 donors5%). The
presence of mitochondrial DNA deletions was independent of age within the sample group (all donors older
than 50 yr) and only depended on pigmentation-status.
2.2 Unpigmented hair follicles retain full viability and
grow better in culture than pigmented hair follicles
Over a culture period of 8 d unpigmented hair follicles
showed significantly higher hair shaft elongation as a
measure of in vitro hair growth than pigmented hair
follicles (Table 1). Both groups maintained an anagenlike morphology of the hair bulb and did not enter into
a catagen-like stage over this culture period.
3.1 Pigmented hair follicles show melanocyte
apoptosis in the pigmentary-unit on additional
exogenous oxidative stress
Pigmented hair follicles cultured over a period of 3 d in
the presence of exogenous oxidative stress (hydroquinone at concentrations between 103 to 107 M)
showed a dose dependent increase of TUNEL apoptotic melanocytes in their pigmentary-unit (Table 1).
107 M hydroquinone appeared to be without effect
and showed no significant differences to control in
TUNEL melanocyte number. 104 and 103 M hydroquinone showed toxic effects on the hair follicle,
with increased apoptosis in the hair follicle keratinocyte

The FASEB Journal

ARCH ET AL.

population in the hair matrix and the inner root

sheath. 105 to 106 M hydroquinone showed apoptosis of melanocytes selectively in the pigmentary-unit by
TUNEL-labeling (Table 1).

CONCLUSION
Here we provide unique evidence for oxidative stress
induced loss of melanocytes from the human hair
follicle during aging. In detail, we show for the first
time that: 1) A decreased number of viable melanocytes
in the aging hair follicle bulge and bulb and an
increased incidence of hair bulb melanocyte apoptosis
in aging individuals are associated with oxidative stress
in the pigmentary-unit; 2) a higher frequency of oxidative stress associated permanent mitochondrial DNA
damage occurs in graying hair follicles, while unpigmented hair follicles retain their growth capacity and
proof to be not generally older than pigmented hair
follicles; and 3) melanocytes of the pigmentary-unit are
highly and selectively susceptible to additional exogenous oxidative stress-damage.
Together, we provide evidence that the major route,
by which oxidative stress leads to permanent and selective melanocyte damage, passes by the mitochondria.
Mitochondrial DNA is not so well protected as genomic
DNA. The accumulation of mutations therefore correlates with age and is indicative of general oxidative
stress levels, caused for example by psycho-emotional
stress, inflammation, UV-light, etc. The highly susceptible and easily accessible hair follicle melanocytes can
thus be utilized to indicate general oxidative stressexposure and -damage, later to be expected in other
cell-types and organs.
Moreover, to our knowledge, this is first report that
proves the long-suspected and often anecdotally described accelerated growth of white scalp hair. Interestingly, keratinocytes and fibroblasts reportedly have
higher antioxidant enzyme activities than melanocytes.
This observation further supports that keratinocytes
and fibroblasts in unpigmented hair follicles are fully
viable and that melanocytes have aged selectively. Clinical observations support the premature and selective
loss of hair bulb melanocytes by oxidative stress as an
indicator for overall oxidative stress levels (Fig. 1).
Future investigations may reveal whether interindividual differences in hair graying derive from genetic
differences in antioxidative capacity, as suggested by
the premature graying of mice with BclII deficiency in
the absence of general features of premature aging.
The graying hair follicle offers a unique modelsystem
to study oxidative stress effects and aging and to test
antioxidants and other anti-aging pharmaceuticals in
their ability to slow down or even stop this process.
Supporting this line of thought, one study in mice has
already shown, that topical application of an antioxidant enzyme can protect hair follicles from UV-light
induced graying.
In summary, our findings support the proposed
GRAYING AND OXIDATIVE STRESS

Figure 1. Hypothetical scenario indicating the involvement of

oxidative stress in the graying process. Melanocytes in pigmented anagen hair follicles in young and healthy subjects
can deal with the endogenous oxidative stress caused by
melanin-production. In the old and diseased subject, however, stressors induce alterations in pigment-producing and
antioxidative enzymes and cofactors and the production of
endogenous antioxidants and repair-enzymes as well as
growth factors etc. decreases. This results in a breakdown of
the hair follicle melanocyte redox-capacity and subsequent
deleterious oxidative stress damage.

hypothesis of a free radical theory of graying and

suggest, that melanocytes in the hair follicle are highly
susceptible to endogenous oxidative stress. Psychoemotional stress and inflammatory stress may add to the
endogenous oxidative stress-load of the hair bulb melanocyte (Fig. 1). Oxidative stress damage in hair
follicle melanocytes may be viewed as the tip of the
iceberg and can be used as an indicator and model for
the systematic search for oxidative stress-reducing strategies. These strategies will involve endo- and exogenous
protection from oxidative stress-induced apoptosis by
stimulation of the endogenous antioxidative capacity or
by treatment with exogenous antioxidants.
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