Chemoprevention of doxorubicin-induced alopecia in mice by dietary administration of L-cystine and vitamin B6

Abstract

Chemotherapy-induced hair loss is one of the most serious and feared adverse effects of cancer therapy. Almost all traditional chemotherapeutic agents induce a more or less severe alopecia. At present, there is no effective treatment capable of preventing this damage. Several different experimental approaches, using various animal models, have been investigated over the last years, with promising results. Sulphur-containing amino acids (cystine, cysteine) are essential components for the health of normal hair. L: -Cystine is used in the treatment of various forms of alopecia. Vitamin B6 plays an important role in the development and maintenance of the skin and it is useful in reducing hair loss. In the present study, we demonstrated that the combined oral administration at high dosages of L: -cystine (1,600 or 800 mg/kg body weight/day) and vitamin B6 (160 or 80 mg/kg body weight/day) is an effective chemopreventive treatment against alopecia induced by doxorubicin treatment (1.1 mg/kg body weight intravenously) in C57BL/6 mice.

Full text

ORIGINAL PAPER
Chemoprevention of doxorubicin-induced alopecia in mice
by dietary administration of L-cystine and vitamin B6
Francesco D’Agostini •Paolo Fiallo •
Massimo Ghio •Silvio De Flora
Received: 10 February 2012 / Revised: 25 May 2012 / Accepted: 1 June 2012 / Published online: 15 June 2012
ÓSpringer-Verlag 2012
Abstract Chemotherapy-induced hair loss is one of the
most serious and feared adverse effects of cancer therapy.
Almost all traditional chemotherapeutic agents induce a
more or less severe alopecia. At present, there is no
effective treatment capable of preventing this damage.
Several different experimental approaches, using various
animal models, have been investigated over the last years,
with promising results. Sulphur-containing amino acids
(cystine, cysteine) are essential components for the health
of normal hair. L-Cystine is used in the treatment of various
forms of alopecia. Vitamin B6 plays an important role in
the development and maintenance of the skin and it is
useful in reducing hair loss. In the present study, we
demonstrated that the combined oral administration at high
dosages of L-cystine (1,600 or 800 mg/kg body weight/day)
and vitamin B6 (160 or 80 mg/kg body weight/day) is an
effective chemopreventive treatment against alopecia
induced by doxorubicin treatment (1.1 mg/kg body weight
intravenously) in C57BL/6 mice.
Keywords Mouse Alopecia Doxorubicin 
Chemoprevention L-Cystine Vitamin B6
Introduction
Chemotherapy-induced alopecia (CIA) is considered one of
the most negative factors in cancer patient care. Female
patients are particularly affected; different studies demon-
strated that 47 % patients consider CIA the most traumatic
side effect of chemotherapy, and 8 % patients would reject
chemotherapy due to fear of CIA [67,70]. Alopecia also
results in reduced social interactions in school-age children
and teenagers [10,38,93]. The negative psychological
impact of CIA may have additional undesirable biological
consequences: in fact, depression impairs immune func-
tions, leading to cancer progression [81]. A more or less
severe grade of alopecia is induced by almost all the tra-
ditional cytostatic chemotherapeutic agents (doxorubicin,
taxol, cyclophosphamide, etoposide) used in the treatment
of leukemias and breast, lung, ovarian and bladder cancer.
Alopecia is particularly severe in patients treated with the
anthracycline antibiotic doxorubicin. Clinical trials docu-
ment a consistent hair loss in these patients [12].
While multiple drugs have been developed to manage
chemotherapy-induced bone marrow suppression and gas-
trointestinal disturbances, at present no treatment appears
to be really effective in reliably preventing CIA. Various
animal models of CIA and several different experimental
approaches for its prevention have been investigated over
last years. Two comprehensive reviews on the advances in
protection against CIA have been published by Wang et al.
[94] and by Jimenez et al. [47].
Sulphur-containing amino acids (cystine, cysteine) are
essential components for the production of normal healthy
hair [100]. In fact, reduced amounts of cystine-rich proteins
are associated with the structural weakness of hair shafts in
different pathological conditions, such as trichothiodys-
trophy, a genetic disorder characterized by hair fragility,
F. D’Agostini (&)S. De Flora
Section of Hygiene and Preventive Medicine,
Department of Health Sciences, University of Genova,
Genoa, Italy
e-mail: fda@unige.it
P. Fiallo
Section of Social Dermatology, Department of Health Sciences,
University of Genova, Genoa, Italy
M. Ghio
Department of Internal Medicine, S. Martino Hospital,
Genoa, Italy
123
Arch Dermatol Res (2013) 305:25–34
DOI 10.1007/s00403-012-1253-1

excessive weathering and loss of the overlapping cuticular
cells [19,36]. A decreased cystine content has been dem-
onstrated in patients with alopecia congenita [7]. L-Cystine
is currently used in the therapeutical protocols of different
forms of alopecia [20,40,72,88].
Vitamin B6 is an important nutrient, acting as a cofactor for
at least 10 enzymes. It plays a key role in skin development
and maintenance [23]. Skin lesions were the first symptoms
identified with vitamin B6 deficiency in rats [37] and seborr-
heic dermatitis was subsequently observed in vitamin B6
deficiency in humans [74]. It has been demonstrated that the
administration of vitamin B6 reduces hair loss, especially in
alopecia of telogenic pathomechanisms [16,91].
Dietary supplementation of L-cystine and B-complex
vitamins resulted in a significant improvement and nor-
malization of hair growth in healthy women with telogen
effluvium [59].
We already demonstrated that the combined oral
administration of L-cystine and vitamin B6 with the diet
resulted in a protective effect against alopecia induced by
tobacco smoke exposure in mice [25].
The goal of the present study was to evaluate whether
oral administration of L-cystine and vitamin B6 with the
diet could prevent CIA in doxorubicin-treated mice.
Materials and methods
Drugs
Doxorubicin was used in the form of an injectable commercial
product, containing 10 mg of doxorubicin chlorohydrate per
vial (Adriblastina, Pfizer Italia, Latina, Italy). The drug was
dissolved in distilled water just before injection.
The two chemopreventive agents (L-cystine and vitamin
B6) were supplied mixed as a powder (Lot. No. VE 2245,
Laboratoires Gale
´niques Vernin, Dammarie-les-Lys,
France). The proportion of L-cystine and vitamin B6 in the
mix was 10:1 (w/w). The highest doses tested were chosen
after a preliminary sub-chronic toxicity study, in order to
find the maximum tolerated dose (MTD). Signs of suffer-
ing ([10 % loss of body weight, as compared to untreated
controls) were evident in mice receiving L-cystine and
vitamin B6 at a dosage of 2,000 and 200 mg/kg b.w./day,
respectively. Therefore, we chose 1,600 and 160 mg/kg
b.w./day, respectively, as the highest dosage. The mid and
lowest dosages were chosen by halving the previous dos-
age, in order to find the minimal efficacious dose.
Animals
Fifty adult female C57BL/6 mice (Harlan Laboratories, S.
Pietro al Natisone, UD, Italy), aging 8 weeks and having an
average body weight of approximately 25 g were used. They
were housed in Makrolon
TM
cages and acclimatized for a
week before starting the treatments. The temperature of the
animal room was 23 ±2°C, with a relative humidity of
55 %, a ventilation accounting for 15–20 air renewal cycles/
h, and a 12-h day–night cycle. Standard rodent diet (Teklad
9607, Harlan Laboratories) was used. The animals received
drinking water ad libitum. Housing and all treatments were in
accordance with the Italian (D.L. No. 116/92) and European
Community (86/609/EEC Directive) guidelines.
After acclimatization, mice were divided into the fol-
lowing experimental groups, each composed of 10 animals:
(a) untreated controls
(b) doxorubicin-treated mice
(c) doxorubicin-treated mice receiving L-cystine ?vita-
min B6 (1600 ?160 mg/kg b.w./day)
(d) doxorubicin-treated mice receiving L-cystine ?vita-
min B6 (800 ?80 mg/kg b.w./day)
(e) doxorubicin-treated mice receiving L-cystine ?vita-
min B6 (400 ?40 mg/kg b.w./day).
The amount of food consumed daily by the animals was
measured. The chemopreventive agents were incorporated
into the food by means of a mixer, adjusting their concen-
trations according to the body weight of animals and to the
daily food consumption, in order to obtain a ‘‘calculated
daily intake’’. In comparison to oral gavage, oral dietary
admixture offers several advantages, including ease of
administration, minimal handling of animals, and elimina-
tion of the risk of stress and injury associated with intubation.
This method offers relatively precise dose administration
(even if not as good as gavage): in fact, both mean food
consumption and mean body weight for periods of several
weeks are easily measured. We used this method of admin-
istration in several of our chemoprevention studies in
rodents, and the results obtained were always satisfactory.
Treatments
Mice belonging to groups (b), (c), (d), and (e) received two
intravenous (i.v.) injections of doxorubicin in the lateral
tail vein, at the dose of 1.1 mg/kg b.w., at a one-week
interval.
Animals belonging to groups (c), (d), and (e) received
the chemopreventive agents with the diet, starting 3 days
before doxorubicin treatment.
After 8 weeks of treatment, all mice were anesthetized
with diethyl ether and killed by cervical dislocation.
Microscopy
Hair loss was evaluated microscopically, according to the
method described by Malkinson et al. [65]. The number of
26 Arch Dermatol Res (2013) 305:25–34
123

hairs in selected skin samples of standardized area (4 mm
2
)
was recorded.
Representative skin samples were fixed in formalin and
embedded in paraffin; 5-lm sections were stained with
haematoxylin and eosin for histopathological analysis.
Immunohistochemistry and apoptosis evaluation
Cell proliferation was evaluated by immunohistochemical
detection of the proliferating cell nuclear antigen (PCNA)
in hair follicles; 5-lm skin sections were stained using a
commercially available PCNA detection kit (Invitrogen,
Camarillo, CA, USA), following the manufacturer’s
instructions. Five hundred hair follicle cells per mouse
were examined and the frequency of PCNA-positive cells
was recorded.
Apoptosis was evaluated by TdT-mediated dUTP Nick
End Labeling (TUNEL) method; 5-lm skin sections were
stained using a commercially available kit (DermaTACS,
Trevigen, Gaithersburg, MD, USA), according to the
manufacturer’s instructions. Five hundred hair follicle cells
per mouse were examined and the frequency of apoptotic
cells was recorded and expressed as apoptotic index (%).
Statistical analysis
Statistical analysis regarding differences in the number of
surviving animals and mice with alopecia was made by v
2
test. Statistical analysis regarding differences in the number
of hairs and frequencies of PCNA-positive cells and
apoptotic cells was made by Student’s ttest for unpaired
data.
Results
Treatment with doxorubicin resulted in a slightly higher
mortality, as compared to untreated controls. In particular,
in group (b) one mouse died after 5 weeks and another
mouse died after 6 weeks. In group (c) one mouse died
after 4 weeks and another mouse died after 5 weeks. In
group (d) one mouse died after 4 weeks. In group (e) one
mouse died after 4 weeks. All untreated mice survived
until the end of the experiment. None of these differences
in survival was statistically significant.
Mice belonging to groups (b), (d), and (e) developed a
mild, diffuse alopecia on their back, which was already
evident 2 weeks after injection of doxorubicin and became
progressively more pronounced up to 5 weeks after treat-
ment (Fig. 1). No hair loss was macroscopically detectable
in untreated control mice or in mice belonging to group (c).
Figure 2shows the comparative appearance of doxorubi-
cin-treated mice, 6 weeks after the first injection of
doxorubicin, with or without oral administration of the
chemopreventive agents.
Table 1reports the time-course induction of alopecia, as
related to doxorubicin treatment and oral administration of
L-cystine and vitamin B6.
Table 2shows the number of hairs detected in skin samples
of standardized area (4 mm
2
). Doxorubicin treatment induced
a statistically significant decrease in the number of hairs. This
decrease was prevented by oral administration of the two
chemopreventive agents at the higher dosage (1,600 ?160
mg/kg b.w./day) and attenuated at the intermediate dosage
(800 ?80 mg/kg b.w./day). Oral administration of the two
chemopreventive agents at the lower dosage (400 ?40 mg/
kg b.w./day) exerted no protective effect.
As shown in Figs. 3and 4, histopathological analysis of
selected skin samples, collected after killing of animals at
the end of the experiment, did not reveal any significant
alteration other than a decrease in the number of hair fol-
licles, in all doxorubicin-treated mice, as compared to
controls.
Fig. 1 Macroscopic appearance of alopecia induced by doxorubicin
(1.1 mg/kg b.w., i.v.), 5 weeks after treatment
Arch Dermatol Res (2013) 305:25–34 27
123

The frequency (%) of PCNA-positive cells in hair fol-
licles was 4.6 ±1.03 (mean ±SE) in controls, 5.0 ±1.41
in group (b), 4.2 ±0.80 in group (c), 4.8 ±0.97 in group
(d), and 5.2 ±0.74 in group (e). None of these differences
was statistically significant.
The apoptotic index (%) in hair follicle cells was
1.1 ±0.55 in controls, 1.4 ±0.75 in group (b), 1.2 ±0.58
in group (c), 1.5 ±0.51 in group (d), and 1.2 ±0.74 in
group (e). Again, all treatments did not induce any statis-
tically significant variation among experimental groups.
Fig. 2 Appearance of C57BL/6 mice, 6 weeks after treatment with
doxorubicin and receiving a diet containing L-cystine and vitamin B6
at three different dosages. aDoxorubicin (1.1 mg/kg b.w., i.v.).
bDoxorubicin (1.1 mg/kg b.w., i.v.) ?L-cystine ?vit. B6
(1,600 ?160 mg/kg b.w./day). cDoxorubicin (1.1 mg/kg b.w.,
i.v.) ?L-cystine ?vit. B6 (800 ?80 mg/kg b.w./day). dDoxorubi-
cin (1.1 mg/kg b.w., i.v.) ?L-cystine ?vit. B6 (400 ?40 mg/kg
b.w./day)
Table 1 Number of mice ( %) with alopecia as related to treatment and time of the experiment (week)
Week Treatment
Controls Doxorubicin (DOX)
(1.1 mg/kg b.w., i.v.)
DOX (1.1 mg/kg b.w., i.v.) ?
L-cystine ?vit. B6
(1,600 ?160 mg/kg b.w./day)
DOX (1.1 mg/kg b.w., i.v.) ?
L-cystine ?vit. B6
(800 ?80 mg/kg b.w./day)
DOX (1.1 mg/kg b.w., i.v.) ?
L-cystine ?vit. B6
(400 ?40 mg/kg b.w./day)
00 0 0 0 0
10 0 0 0 0
2 0 2/10 (20) 0 3/10 (30) 2/10 (20)
3 0 4/10 (40)* 0 5/10 (50)** 4/10 (40)*
4 0 8/10 (80)*** 0 9/9 (100)*** 7/9 (78)***
5 0 9/9 (100)*** 0 9/9 (100)*** 9/9 (100)***
6 0 8/8 (100)*** 0 9/9 (100)*** 9/9 (100)***
Values in parentheses are expressed in %
Statistical analysis: * P\0.05, ** P\0.01 and *** P\0.001, as compared to controls
28 Arch Dermatol Res (2013) 305:25–34
123

Discussion
The goal of the present study was to investigate the ability
of two chemopreventive agents, L-cystine and vitamin B6,
administered with the diet, to inhibit hair loss induced by
doxorubicin treatment in C57BL/6 mice.
Doxorubicin is responsible for several damages of the
skin. This drug causes severe atrophy of all epidermal
layers [26]. In addition, it induces a large number of dif-
ferent alterations both in the mesenchymal and epithelial
compartment of the hair follicle [76]. Apoptosis in matrix
cells has been proposed as the major cause of CIA [14,18].
This form of programmed cell death could be related to
different mechanisms, such as altered expression of the p53
gene [15], downregulation of beta-catenin expression [75],
glutathione depletion [32,87], mitochondrial alterations
[34,57] or generation of reactive oxygen species [89].
Anyway, apoptosis may not be the only form of cellular
damage within the epithelial compartment of doxorubicin-
treated hair follicles. Selleri et al. [76] proposed oncosis,
another form of cell death distinct from apoptosis, as an
alternative mechanism of cell depletion. Doxorubicin has
Table 2 Number of hairs in 4-mm
2
skin samples of C57BL/6 mice, as related to treatment
Treatment No. of animals No. of hairs (mean ±SE)
Controls 10 173.6 ±11.36
Doxorubicin (DOX) (1.1 mg/kg b.w., i.v.) 8 101.3 ±10.78
a
DOX (1.1 mg/kg b.w., i.v.) ?L-cystine ?vit. B6 (1,600 ?160 mg/kg b.w./day) 8 169.5 ±8.37
b
DOX (1.1 mg/kg b.w., i.v.) ?L-cystine ?vit. B6 (800 ?80 mg/kg b.w./day) 9 121.4 ±1.40
a,b
DOX (1.1 mg/kg b.w., i.v.) ?L-cystine ?vit. B6 (400 ?40 mg/kg b.w./day) 9 99.1 ±5.50
a
The results are mean ±SE of the results obtained in all surviving animals per group
Statistical analysis:
a
P\0.001, as compared to controls;
b
P\0.05, as compared to DOX-treated mice
Fig. 3 Histopathological appearance of the skin collected, at the end
of the experiment, from C57BL/6 mice. H&E, original magnification
940. aControl mice. bMice treated with doxorubicin (1.1 mg/kg
b.w., i.v). cMice treated with doxorubicin ?L-cystine ?vit. B6
(1,600 ?160 mg/kg b.w./day). dMice treated with doxorubicin ?L-
cystine ?vit. B6 (400 ?40 mg/kg b.w./day)
Arch Dermatol Res (2013) 305:25–34 29
123

been reported to induce oncosis in different cell types [56,
58,64]. Glycogen metabolism is very important in the
epidermis and, in particular, within the hair follicle [96]. It
has been suggested that glucose transport may represent an
early target for doxorubicin [43], thus causing a reduction
in the amount of glycogen in outer root sheath cells, which
leads to oncosis. Doxorubicin also shows a marked ability
to form covalent adducts and crosslinks with DNA [80,
101] and this feature strongly correlates with its cytotoxic
activity. It has also been demonstrated that doxorubicin
causes significant increases in nitric oxide (NO) synthesis
and works partially through a NO-dependent mechanism
[50,61]. Doxorubicin induced platelet cytotoxicity through
generation of reactive oxygen species, decreased glutathi-
one levels and thiol depletion [52]. Doxorubicin also exerts
a strong toxic effect on the hair follicle blood vessel net-
work, causing inhibition of angiogenesis. This damage may
play a major role in hair dystrophy and alopecia [2,3]. Last
but not least, doxorubicin treatment induces regression or
even complete disappearance of sebaceous glands [77].
Normal development and function of the sebaceous glands
appear to be very important for correct hair growth and
cycling. Abnormal sebaceous gland function has been
clearly associated with hair loss in several animal models
of alopecia [73,82]. It is therefore very likely that the
doxorubicin-induced damage of the sebaceous gland also
plays a role in CIA.
Taking into account all the above-mentioned damaging
mechanisms exerted by doxorubicin, it is not surprising that
only few protective agents against doxorubicin-induced
alopecia have been identified so far in animal models of CIA:
they include prostaglandins [65], liposome-entrapped
monoclonal antibodies [8], and CDK inhibitors [28].
L-Cystine shows several protective mechanisms which
could counteract the noxious activity of doxorubicin.
Although it has not yet been reported in the literature a
clear capability of L-cystine to inhibit apoptosis, many
studies demonstrate that thiol depletion cause apoptosis [5,
13,44,55,83,98,99]. It is well known that glutathione
(GSH) plays important roles in antioxidant defense and
regulation of key cellular events, such as gene expression,
DNA synthesis, cell proliferation, and apoptosis. GSH and
cysteine-containing proteins are critical for many cell
functions, chromosome consolidation, DNA protection
from oxidative stress and protection of DNA-binding pro-
teins. Furthermore, GSH deficiency contributes to oxida-
tive stress. A comprehensive review of all these aspects is
reported in Go and Jones [33]. Enteral or parenteral cystine
is an effective precursor of cysteine for GSH synthesis
[97]. The balance cysteine/cystine modulates many
Fig. 4 Hair follicles in the skin of C57BL/6 mice either untreated (a), treated with doxorubicin alone (b), or treated with doxorubicin and
receiving L-cystine ?vit. B6 with the diet. H&E, original magnification 9200
30 Arch Dermatol Res (2013) 305:25–34
123

relevant cellular events, including cell proliferation [48],
and resistance to apoptosis [46]. The cystine/cysteine redox
cycle is an important node in the circuitry for redox sig-
naling and control [49]. It regulates susceptibility to cell
death and must be viewed as a major regulator of cell
survival [9]. While cystine did not show any protective
effect against cytogenetical damage [25,85], it has dem-
onstrated its ability to inhibit the toxicity of paraquat, a
pesticide exerting genotoxic effects on different organs [53,
84]. Cystine also displayed antimutagenic properties in
bacterial in vitro systems [22,78] and showed a protective
effect against induction of hydrogen-adduct radicals [17].
Cystine exerted a high protective effect against thallium
poisoning in rats, reducing alopecia induced by chronic
thallotoxicosis and decreasing the mortality rate of acute
thallium exposure [35]. Dietary cystine was found to exert
beneficial effects against glycogen intracellular reduction
induced by cyanide [27]. Oral administration of L-cystine
to rats also caused an increase in the levels of liver gly-
cogen [41]. Cystine is a key component of the vascular
endothelial growth factor (VEGF), a family of hormones
and extracellular signaling molecules essential for angio-
genesis [42,92]. It is therefore likely that its administration
plays a role in maintaining blood vessel network.
Vitamin B6 has been considered for many years to act only
as an enzymatic cofactor. However, it became recently clear
that this compound is of very high importance for cellular
well-being. In fact, besides playing a primary role as a cofactor
for a large number of essential enzymes, which catalyze
important steps in cellular metabolism, this vitamin is now
regarded as a very promising protective factor. A recent
review [69] provides a detailed overview on the metabolic
aspects and protective properties of this compound. Vitamin
B6 also shows several protective activities able to fight the
doxorubicin damaging mechanisms. In particular, the differ-
ent forms of this vitamin may play a crucial role in protecting
cells from oxidative stress and damage induced by reactive
oxygen species (ROS), as demonstrated by several recent
studies, both in vitro and in vivo [4,30,51,62,63,68,95]. The
anti-apoptotic properties of vitamin B6 have been investigated
and elucidated in recent years.In particular, mice fed a vitamin
B6-depleted diet showed abnormal apoptosis in the thymus
[71], while deprivation of vitamin B6 in the culture media
decreased cell proliferation and increased apoptosis in
hybridoma cells [45]. Vitamin B6 compounds were effective
in preventing apoptosis in yeast cells under oxidative stress
[21], in bovine endothelial cells treated with homocysteine
and copper [31], in cultured human monocytic cells treated
with 2-deoxy-D-ribose [6], and in radiation-exposed intestinal
cultured cells or small intestine epithelial cells in vivo [86].
Vitamin B6, in its different forms, also showed antimutagenic
and antigenotoxic properties in in vitro systems, as well as in
animal models [11,29,79]. Different anticarcinogenic effects
of vitamin B6 were investigated and related to various
mechanisms [54,66]. An essential role of vitamin B6 in gly-
cogen metabolism and mobilization has also been demon-
strated [39].
Taking into account all the noxious mechanisms of
doxorubicin (induction of apoptosis and oncosis, mainly
via oxidative stress, damage of ionic pumps on the plasma
membranes, alteration of the mitochondrial activity, inter-
ference with the glycogen metabolism, genotoxic activity,
toxic effect on angiogenesis), and the related protective
properties of L-cystine and vitamin B6 discussed above, it
is arguable that the combined administration of the two
agents could result in a protective effect against doxoru-
bicin-induced hair loss.
Even if different animal models (monkey, dog, cat) have
been proposed in the past, there is no doubt that rodents
represent the more commonly used and best suited models to
study various types of CIA. Although several differences do
exist between rodents and humans, the mouse is an excellent
model system for studying the hair cycle. In fact, the first two
cycles of the mouse hair follicle are synchronized and the
whole hair cycle takes about 3 weeks. This short synchro-
nized hair cycle allows hair follicles to be harvested and
examined at specific time points very easily. Furthermore, all
the stages of the hair cycle in the mouse have been well
described and characterized. C57BL/6 mice are particularly
prone to develop hair loss than other strains and therefore
better suited to study this type of disorders.
In the present study we chose an experimental protocol
similar to that we had already used in our previous chemo-
prevention studies [24], in which doxorubicin is adminis-
tered intravenously to mice without any preliminary
depilation. This protocol has the advantage that the admin-
istration route is the same as used in humans, and the dosage
of the drug is comparable to the therapeutical doses for
treatment of human malignancies. As we demonstrated in
some preliminary studies, dietary administration of L-cystine
or vitamin B6 alone did not exert any satisfactory protective
effect in doxorubicin-treated mice (data not shown). There-
fore, in this study we decided to administer the two drugs in
combination. A full chemopreventive effect of the L-cystine/
vitamin B6 mix was observed only at the highest dosage
(1,600 ?160 mg/kg b.w./day), while a partial protective
effect was obtained by administering the two agents at the
intermediate dosage (800 ?80 mg/kg b.w./day). Therefore,
it could hypothesized that only the highest dosage warrants a
plasmatic concentration sufficient to counteract the damag-
ing effects of doxorubicin. We cannot say whether the pro-
tective effect observed is the result of a synergy or simply a
sum of the two single effects. Further studies are needed in
order to clarify these aspects.
The lack of differences in the histopathological aspect of
skin, or in the proliferative and apoptotic index amongst the
Arch Dermatol Res (2013) 305:25–34 31
123

experimental groups could be ascribed to the fact that, after
8 weeks, the normalization process of the skin was already
completed and the only detectable alteration was thedecrease
in the number of hairs. It is well known that doxorubicin
causes several damages to the skin and hair follicles; none-
theless, these lesions are typical of the early stages of treat-
ment and are fully reversible, as confirmed by the regression of
alopecia, after the conclusion of treatment, in humans
undergoing chemotherapy. Indeed, in future studies it will be
of great interest to analyze histopathological alterations in skin
samples collected from L-cystine/vitamin B6-treated mice
immediately after doxorubicin administration, in order to
obtain information about hair follicle damage and recovery.
Even if the results obtained in the present study indicate
a clear chemopreventive activity of a high-dosage admin-
istration of L-cystine and vitamin B6 against doxorubicin-
induced alopecia in mice, it should be stressed that the
results obtained in animal models cannot be automatically
extrapolated to humans, taking also into account the dif-
ferent hair growth patterns in humans and rodents. Further
studies are needed to validate this protocol. In particular, it
will be of interest to investigate the effects of doxorubicin
on different elements of hair follicle, such as nestin-
expressing pluripotent stem cells, which have been found
in the bulge area and in the dermal papilla. These cells are
essential for the development of the new hair follicle
structure, as well as for their ability to form new blood
vessels during skin angiogenesis [1,60,90]. In addition, it
will be necessary to investigate whether the systemic oral
administration of L-cystine and vitamin B6 at high dosages
does not impair or interfere with the antitumoral thera-
peutical activity of doxorubicin. Furthermore, clinical trials
in humans should be performed, in order to evaluate the
efficiency of the two chemopreventive agents and to
establish their right dosages. Therefore, at present it is not
easy to recommend a specific dose for use in humans.
Anyway, from the results of the present study, it could be
concluded that the oral combined administration of L-cys-
tine and vitamin B6 represent an interesting and promising
protective strategy against hair loss induced by chemo-
therapeutic drugs. In particular, several studies have
already demonstrated protective effects of various agents
(CDK inhibitors, cyclosporin A, dexamethasone, vitamin
D
3
, etc.) against alopecia induced by cyclophosphamide or
etoposide [47,94]. It will be also of interest to test the same
ability of L-cystine and vitamin B6 against these drugs.
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