The role of ultraviolet radiation and tyrosine stimulated melanogenesis in the induction of oxidative stress alterations in fair skin melanocytes
Melanocytes are producing melanin after UV irradiation as a defense mechanism. However, UV-induced damage is involved in melanoma initiation, depending on skin phototype. Melanocytes seem to be extremely susceptible to free radicals. Their main enzymatic antioxidants are superoxide dismutase and cat...
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Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України
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| Цитувати: | The role of ultraviolet radiation and tyrosine stimulated melanogenesis in the induction of oxidative stress alterations in fair skin melanocytes / I. Baldea, T. Mocan, R. Cosgarea // Experimental Oncology. — 2009. — Т. 31, № 4. — С. 200-208. — Бібліогр.: 53 назв. — англ. |
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irk-123456789-1382052018-06-19T03:03:11Z The role of ultraviolet radiation and tyrosine stimulated melanogenesis in the induction of oxidative stress alterations in fair skin melanocytes Baldea, I. Mocan, T. Cosgarea, R. Original contributions Melanocytes are producing melanin after UV irradiation as a defense mechanism. However, UV-induced damage is involved in melanoma initiation, depending on skin phototype. Melanocytes seem to be extremely susceptible to free radicals. Their main enzymatic antioxidants are superoxide dismutase and catalase. Aim: To study how melanin synthesis modulates the activity of the oxidative stress defense enzymes and cell proliferation after UV induced cell damage. Methods: Normal human melanocyte cultures from fair skin individuals were exposed to high levels of L-tyrosine and irradiated, with 20, 30, 40 mJ/cm2 UVA, and respective UVB. Proliferation was measured using a MTS assay; viability was assessed by trypan blue exclusion dye method. Spectrophotometrical methods were used to determine total melanin content, the enzymatic activity of tyrosinase, superoxide dismutase and catalase. Results: Tyrosine had a negative effect on proliferation, enhanced with time elapsed. Overall, UV irradiation decreased proliferation. UVA increased proliferation relative to UVB in the cultures exposed for a longer time to high (2 mM) tyrosine concentration. There were no proliferation differences between UVA and UVB irradiation in lower tyrosine concentration exposed melanocytes. Both, UV irradiation and tyrosine increased melanogenesis. Exposure of the melanocytes to increased levels of tyrosine in medium (0.5 mM and 1 mM) and UV irradiation enhanced the activity of superoxide dismutase and catalase. The enzymes showed a high activity rate in melanocytes while exposed for a short time to 2 mM tyrosine, but their activity was dramatically decreased with longer tyrosine exposure and UV irradiation. Conclusion: Our data indicate that in low phototype melanocytes, melanogenesis, either following UV irradiation, or tyrosine exposure, especially in high concentrations, was detrimental for the cells by reducing the activity of catalase and superoxidedismutase, the natural antioxidants. UVA was more efficient in stimulating the activity of superoxide dismutase and catalase but also in depleting the reserves of the enzymatic defense against oxidative stress, especially catalase, than UVB. This physiologic response to UV light can be considered as an adjunctive risk factor for people with low phototype for developing a melanoma, when exposed to UV irradiation. 2009 Article The role of ultraviolet radiation and tyrosine stimulated melanogenesis in the induction of oxidative stress alterations in fair skin melanocytes / I. Baldea, T. Mocan, R. Cosgarea // Experimental Oncology. — 2009. — Т. 31, № 4. — С. 200-208. — Бібліогр.: 53 назв. — англ. 1812-9269 http://dspace.nbuv.gov.ua/handle/123456789/138205 en Experimental Oncology Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України |
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Original contributions Original contributions |
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Original contributions Original contributions Baldea, I. Mocan, T. Cosgarea, R. The role of ultraviolet radiation and tyrosine stimulated melanogenesis in the induction of oxidative stress alterations in fair skin melanocytes Experimental Oncology |
| description |
Melanocytes are producing melanin after UV irradiation as a defense mechanism. However, UV-induced damage is involved in melanoma initiation, depending on skin phototype. Melanocytes seem to be extremely susceptible to free radicals. Their main enzymatic antioxidants are superoxide dismutase and catalase. Aim: To study how melanin synthesis modulates the activity of the oxidative stress defense enzymes and cell proliferation after UV induced cell damage. Methods: Normal human melanocyte cultures from fair skin individuals were exposed to high levels of L-tyrosine and irradiated, with 20, 30, 40 mJ/cm2 UVA, and respective UVB. Proliferation was measured using a MTS assay; viability was assessed by trypan blue exclusion dye method. Spectrophotometrical methods were used to determine total melanin content, the enzymatic activity of tyrosinase, superoxide dismutase and catalase. Results: Tyrosine had a negative effect on proliferation, enhanced with time elapsed. Overall, UV irradiation decreased proliferation. UVA increased proliferation relative to UVB in the cultures exposed for a longer time to high (2 mM) tyrosine concentration. There were no proliferation differences between UVA and UVB irradiation in lower tyrosine concentration exposed melanocytes. Both, UV irradiation and tyrosine increased melanogenesis. Exposure of the melanocytes to increased levels of tyrosine in medium (0.5 mM and 1 mM) and UV irradiation enhanced the activity of superoxide dismutase and catalase. The enzymes showed a high activity rate in melanocytes while exposed for a short time to 2 mM tyrosine, but their activity was dramatically decreased with longer tyrosine exposure and UV irradiation. Conclusion: Our data indicate that in low phototype melanocytes, melanogenesis, either following UV irradiation, or tyrosine exposure, especially in high concentrations, was detrimental for the cells by reducing the activity of catalase and superoxidedismutase, the natural antioxidants. UVA was more efficient in stimulating the activity of superoxide dismutase and catalase but also in depleting the reserves of the enzymatic defense against oxidative stress, especially catalase, than UVB. This physiologic response to UV light can be considered as an adjunctive risk factor for people with low phototype for developing a melanoma, when exposed to UV irradiation. |
| format |
Article |
| author |
Baldea, I. Mocan, T. Cosgarea, R. |
| author_facet |
Baldea, I. Mocan, T. Cosgarea, R. |
| author_sort |
Baldea, I. |
| title |
The role of ultraviolet radiation and tyrosine stimulated melanogenesis in the induction of oxidative stress alterations in fair skin melanocytes |
| title_short |
The role of ultraviolet radiation and tyrosine stimulated melanogenesis in the induction of oxidative stress alterations in fair skin melanocytes |
| title_full |
The role of ultraviolet radiation and tyrosine stimulated melanogenesis in the induction of oxidative stress alterations in fair skin melanocytes |
| title_fullStr |
The role of ultraviolet radiation and tyrosine stimulated melanogenesis in the induction of oxidative stress alterations in fair skin melanocytes |
| title_full_unstemmed |
The role of ultraviolet radiation and tyrosine stimulated melanogenesis in the induction of oxidative stress alterations in fair skin melanocytes |
| title_sort |
role of ultraviolet radiation and tyrosine stimulated melanogenesis in the induction of oxidative stress alterations in fair skin melanocytes |
| publisher |
Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України |
| publishDate |
2009 |
| topic_facet |
Original contributions |
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http://dspace.nbuv.gov.ua/handle/123456789/138205 |
| citation_txt |
The role of ultraviolet radiation and tyrosine stimulated melanogenesis in the induction of oxidative stress alterations in fair skin melanocytes / I. Baldea, T. Mocan, R. Cosgarea // Experimental Oncology. — 2009. — Т. 31, № 4. — С. 200-208. — Бібліогр.: 53 назв. — англ. |
| series |
Experimental Oncology |
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2025-07-10T05:20:19Z |
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2025-07-10T05:20:19Z |
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| fulltext |
200 Experimental Oncology 31, 200–208, 2009 (December)
Melanocytes play a central role in the response of
skin to sunlight exposure. They are directly involved
in ultraviolet (UV)-induced pigmentation as a defense
mechanism [1]. However, their alteration can lead to
melanoma, a tumor that has become one of the most
rapidly increasing malignancies in the Caucasian
population [2]. Melanoma seems to be the result of
complex interactions between environmental, constitu-
tive and genetic factors [3]. Although direct evidence
is lacking, it is assumed that solar ultraviolet A (UVA)
radiation (320–400 nm) may play a significant role
relative to ultraviolet B (UVB) radiation (290–320 nm) in
melanoma etiology [2, 4]. The transformation process
whereby UV damage may result in melanoma initiation
is poorly understood, especially in terms of UV-induced
genotoxicity in pigmented cells, where melanin can act
either as a sunscreen or as a photosensitizer [1, 5, 6].
People with different skin color possess varied sensi-
tivity to ultraviolet (UV) exposure, with darker skinned
individuals being less susceptible to sun-induced skin
alterations, including cancer, than fair skinned ones
[7]. Such a difference can be explained in terms of UV
transmission of the epidermis, because the skin color
is also related to the type of melanin, the number, size,
type, distribution and degradation of melanosomes,
and the tyrosinase activity in melanocytes [8, 9]. Three
enzymes, phenylalanine hydroxylase, tyrosine hydroxy-
lase isoform I and tyrosinase are crucial for the initiation
of melanogenesis. Intracellular phenylalanine hydroxy-
lase is providing L-tyrosine through the conversion of
L-phenylalanine while the last two enzymes are using
L-tyrosine as a substrate [10, 11]. Tyrosinase catalyzes
the hydroxilation of L-tyrosine [12] and the production
of ortho-quinones from both monohydric and dihydric
phenols [13]. Tyrosine hydroxylase isoform I uses
L-tyrosine to form L-DOPA in melanosomes [14].
High levels of tyrosine are known to reduce the
proliferative effect of alpha-MSH and forskolin and
also alter melanocytes morphology [15]; tyrosine also
stimulates the activity of tyrosinase and melanogene-
sis [4, 15–18].
In the melanocytes, the dominant skin pigment
melanin and its precursors are complex redox systems,
the resultant properties of which are modified by pH,
temperature, illumination with ultraviolet and visible
light [19]. There are conflicting reports on the role
of melanin or melanin precursors in modulating the
biologic effects of UV radiation [20]. It is conceivable,
THE ROLE OF ULTRAVIOLET RADIATION AND TYROSINE
STIMULATED MELANOGENESIS IN THE INDUCTION OF OXIDATIVE
STRESS ALTERATIONS IN FAIR SKIN MELANOCYTES
I. Baldea1, *, T. Mocan1, R. Cosgarea2
1Department of Physiology, 2Department of Dermatology, University of Medicine and Pharmacy Iuliu
Hatieganu, Cluj-Napoca 400006, Romania
Background: Melanocytes are producing melanin after UV irradiation as a defense mechanism. However, UV-induced damage
is involved in melanoma initiation, depending on skin phototype. Melanocytes seem to be extremely susceptible to free radicals.
Their main enzymatic antioxidants are superoxide dismutase and catalase. Aim: To study how melanin synthesis modulates the
activity of the oxidative stress defense enzymes and cell proliferation after UV induced cell damage. Methods: Normal human
melanocyte cultures from fair skin individuals were exposed to high levels of L-tyrosine and irradiated, with 20, 30, 40 mJ/cm2
UVA, and respective UVB. Proliferation was measured using a MTS assay; viability was assessed by trypan blue exclusion dye
method. Spectrophotometrical methods were used to determine total melanin content, the enzymatic activity of tyrosinase, su-
peroxide dismutase and catalase. Results: Tyrosine had a negative effect on proliferation, enhanced with time elapsed. Overall,
UV irradiation decreased proliferation. UVA increased proliferation relative to UVB in the cultures exposed for a longer time to
high (2 mM) tyrosine concentration. There were no proliferation differences between UVA and UVB irradiation in lower tyrosine
concentration exposed melanocytes. Both, UV irradiation and tyrosine increased melanogenesis. Exposure of the melanocytes
to increased levels of tyrosine in medium (0.5 mM and 1 mM) and UV irradiation enhanced the activity of superoxide dismutase
and catalase. The enzymes showed a high activity rate in melanocytes while exposed for a short time to 2 mM tyrosine, but their
activity was dramatically decreased with longer tyrosine exposure and UV irradiation. Conclusion: Our data indicate that in low
phototype melanocytes, melanogenesis, either following UV irradiation, or tyrosine exposure, especially in high concentrations,
was detrimental for the cells by reducing the activity of catalase and superoxidedismutase, the natural antioxidants. UVA was more
efficient in stimulating the activity of superoxide dismutase and catalase but also in depleting the reserves of the enzymatic defense
against oxidative stress, especially catalase, than UVB. This physiologic response to UV light can be considered as an adjunctive
risk factor for people with low phototype for developing a melanoma, when exposed to UV irradiation.
Key Words: melanocyte, tyrosine, ultraviolet radiation, oxidative stress, melanogenesis.
Received: August 13, 2009.
*Correspondence: E-mail: baldeai@yahoo.com
Abreviations used: AUC — area under curve; Cat — catalase;
DMEM — Dulbecco’s modified Eagle medium; FCS — fetal calf
serum; KGM — keratinocyte growth medium; MGM — melano-
cyte growth medium; MTS — (3-(4,5-dimethylthiazol-2-yl)-5-
(3carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner
salt; PMS — phenazine methosulphate; ROS — reactive oxygen
species; SOD — superoxide dismutase; UV — ultraviolet; UVA — ul-
traviolet A; UVB — ultraviolet B.
Exp Oncol 2009
31, 4, 200–208
Experimental Oncology 31, 200–208, 2009 (December) 201
however, that melanin, which may be present in large
concentration in melanocytes, may be the most impor-
tant antioxidant [4]. Melanocytes, in particular, seem
to be extremely susceptible to free radicals, either in
the activation of their physiologic role or in deleterious
effects [1, 21, 22]. The main enzymatic antioxidants in
the melanocytes are superoxide dismutase (SOD) and
catalase (Cat), considering the low glutathione peroxi-
dase activity (GSH-Px) in melanocytesn [23], so that
the SOD/Cat ratio is considered as a parameter of the
cells susceptibility to external oxidative stress [1].
The different pattern of antioxidants in melanocytes
from people with low phototype and their physiologic
response to UV light could be an adjunctive risk fac-
tor for developing a melanoma, when exposed to UV
irradiation [1, 24].
We studied the effects of UV induced oxidative stress,
using normal human melanocyte cultures from cauca-
sian individuals, phototype II, III, according to Fizpatrick
classification, as cellular models [25]. L-tyrosine was
used to selectively modulate melanin synthesis in the
melanocytes. The aim of this study was assessment of
melanogenesis, cell survival, proliferation and defense
against oxidative stress in fair skin melanocytes when
exposed to high levels of tyrosine and UVA, respective
UVB radiation. In view of the potential role of UV irradiation
in skin carcinogenesis and especially melanoma it is im-
portant to understand how melanin synthesis modulates
the activity of the oxidative stress defense enzymes, such
as SOD and Cat after UV induced cell damage.
MATERIALS AND METHODS
Melanocyte cultures. Adult human melanocytes
were grown as previously described [26, 27]. Skin
biopsies were taken from healthy skin, trimmed of ex-
cess subcutaneous tissue and dermis. Separation of
epidermis from dermis was done after overnight incuba-
tion in 2000 UI/ml colagenase (Cellsystems, Germany).
The epidermal cells were separated by trypsinisation
and the cells of the stratum basalis were collected by
gentle scraping. Recovered cells were resuspended and
seeded onto a 25 cm2 culture plates in serum free, kerati-
nocyte growth medium (KGM) (Promocell, Germany). All
cultures were fed twice weekly and incubated in a 37 °C
and 5% CO2, humidified environment. At first passage,
the melanocytes were separated from the keratinocytes
by differential trypsinization and resuspended in com-
plete melanocyte growth medium (MGM) (Cellsystems).
We used 3 primary epidermal human adult melanocytes
cultures from individuals with phototype II and III and one
epidermal human melanocyte culture from Caucasian
newborn foreskin (Promocell).
L-tyrosine media. L-tyrosine (Sigma Chemical Co.,
St. Louis, USA) was dissolved in DMEM, supplemented
with 5% FCS to prepare the media with concentra-
tions of 0.5 mM, 1 mM, 2 mM and respective 3.4 mM,
inclu ding the tyrosine already present in the medium.
Although this medium is not optimal for melanocyte
growth; it readily maintains cell survival over a short
period of time (24 h, respective 72 h) and thus allowed
for the experiments to be performed on melanocytes
without exposure to potent non-physiologic stimulants
such as phorbol esters or cholera toxin [26].
Melanocyte bioassay. All the experiments were
conducted in subdued light, in triplicate. Melanocytes
in the 3rd and 4th passage were used. In proliferation
assays the melanocytes were seeded at 104 per well in
ELISA 96 wells micro titration flat bottom plaques (TPP,
Switzerland). For the enzymatic bioassays and melanin
assessment, the cells were seeded at 2 x 104 cells per
35 mm Petri dish. After 24 h accommodation in com-
plete MGM at 37 °C, 5% CO2, humidified environment,
the cells were washed and exposed to the L-tyrosine
media for 24, respective 72 h. Untreated controls were
exposed to DMEM, supplemented with 5% FCS, con-
taining 0.397 mM tyrosine.
UV irradiation. Irradiation was conducted us-
ing a 6 W power UV lamp (Fisher Bioblock Scientific,
Belgium) with filters for UVB (312 nm), respective UVA
(365 nm). Melanocytes were washed twice in PBS and
irradiated, in PBS, with 20, 30, 40 mJ/cm2 UVA, respec-
tive UVB. The light intensity at the position of the irradi-
ated cell plates with UVA was 700 μW/cm2 and with UVB
was 680 μW/cm2. Then, the cells were incubated for
24 h in basal medium for melanocytes (Promocell).
Melanocyte proliferation/cytotoxicity assay.
It was done using CellTiter 96® AQueous Non-Radio-
active Cell Proliferation Assay (Promega Corporation,
U.S.A). The cells in 100 μl medium were exposed to
20 μl of MTS/PMS mixture (2 ml/100 μl), for 1–4 h.
Absorbance at 490 nm was recorded using an ELISA
plate reader (Tecan, Austria).
Morphology. Morphological aspect of the mela-
nocytes was observed by microscopic examination
(Nikon Eclipse T 100, Japan) and documented photo-
graphically. Melanocytes were released from the culture
dishes with a soft rubber cell scraper and pelleted.
Cell viability. It was assessed by trypan blue exclu-
sion dye method (Biochrom AG).
Cell lysis. Cells were lysed on ice, in Nonidet 1%
(Sigma) in PBS solution for one hour, in the pre sence
of 1% complex of protease inhibitors (Sigma). Cell
extracts were spun at 14 000 g for 30 min at 4 °C.
Supernatant was removed and a fixed volume (50 μl)
was used for determining the protein content by the
Bradford method (Biorad, USA).
Total melanin content. It was determined as pre-
viously described [28]. Remaining pellet was dissolved
in 0.1 ml 1 M NaOH, and diluted with 0.4 ml water.
Melanin content was assessed by spectrophotometric
determination (DU 730 UV VIS Beckman Coulter, USA)
of absorption at 475 nm against a standard curve of
synthetic melanin (Sigma).
Tyrosinase enzymatic activity as DOPA oxidase.
100 μl cell lysate were incubated for 30 min at 37 °C with
1000 μl DOPA (2.5 mg/ml) in 10 mM phosphate buffer,
pH 7.2. The recording L-dopacrom formation at 475 nm
was measured by spectrophotometry (DU 730 UV VIS
Beckman Coulter); absorbance was compared with a stan-
dard curve using mushroom tyrosinase (Sigma) [29, 30].
202 Experimental Oncology 31, 200–208, 2009 (December)
SOD enzymatic activity. 50 μl cell lysate were
added to 2.9 ml cytochrome c (2 μmol) from horse
heart (Sigma) in 50 mM phosphate buffer, ph 7.8,
containing 0.1 mM EDTA solution and the reaction
was started with 50 μl of freshly prepared solution of
0.2 U/ml xanthine oxidase in 0.1 mM EDTA. Absor-
bance at 550 nm was recorded by spectrophotometry
(DU 730 UV VIS Beckman Coulter), against a standard
curve using pure bovine liver SOD (Sigma) [31, 32].
Cat enzymatic activity. 20 μl of cell lysate were
mixed with 3 ml solution of 10 mM H2O2 in 50 ml potassium
phosphate buffer; absorbance at 240 nm was continu-
ously measured by spectrophotometry (DU 730 UV VIS
Beckman Coulter) at 240 nm, for 3 min. For calculation we
considered one unit of catalase as the amount of enzyme
which induces a change of 0.43 in the absorption (240 nm)
during the 3 min incubation period [31, 32].
Statistics. Data were analyzed using non-parametric
methods: Mann — Whitney U Test, Kruskall — Wal-
lis test, Spearman r calculus. Dynamic evaluations
were assessed by means of area under curve (AUC)
calculations. Specific tests (Mann — Whitney U Test,
Kruskall — Wallis test) were used to evaluate differences
between 2 or three dynamic patterns. Although chars
are presented with equal interval among moments in
time, real time scale was used when determining AUC
values and for testing AUC differences. Results were
considered significant for p ≤ 0.05. Statistical packages
SPSS 13.0 — Statistical Software Package (SPSS Inc,
Chicago, Illinois, USA) and MedCalc 8.1.0.0 were used
for data analysis.
RESULTS
Melanocyte proliferation and cytotoxicity.
Overall, tyrosine had a negative, statistically significant
dynamic effect on proliferation, as seen in Fig. 1 (overall
AUC comparison among all tyrosine concentrations,
p = 0.000). Tyrosine diminished cell proliferation com-
pared to controls, when used in lower concentrations
(AUC control versus tyrosine 0.5 mM, p = 0.000; respec-
tive AUC control vs tyrosine 1 mM, p = 0.000). However,
no significance was obtained between tyrosine 0.5 mM
vs 1 mM dynamic effect (AUC comparison, p = 0.987).
0
100
200
300
400
500
600
0 h 24 h 72 h
M
ea
n
pe
rc
en
t o
f i
ni
tia
l v
al
ue
(%
) c1 = 0.397 mM tyrosine
c2 = 0.5 mM tyrosine
c3 = 1 mM tyrosine
c4 = 2.000 mM tyrosine
c5 = 3.400 mM tyrosine
Fig. 1. Melanocyte proliferation after tyrosine exposure for dif-
ferent time periods
High tyrosine concentrations had a significant
negative effect on cell proliferation (AUC control vs
tyrosine 2 mM, p = 0.000, respective control vs ty-
rosine 3.4 mM, p = 0.000). However, no significance
was obtained and between tyrosine 2 mM vs. 3.4 mM
dynamic effect (AUC comparison, p = 0.897).
UV irradiation of the tyrosine exposed melanocytes
reduced cell proliferation. There were no significant
differences in the proliferation rates after exposure
to low concentrations of tyrosine (0.5 mM, 1 mM) ac-
cording to tyrosine concentration and UV irradiation,
compared with controls (Fig. 2).
70
120
170
220
270
320
0 h 24 h 72 h
M
ea
n
pe
rc
en
t o
f i
ni
tia
l v
al
ue
(%
)
1 = 0.5 mM tyrosine + UVA
2 = 0.5 mM tyrosine + UVB
3 = 1.0 mM tyrosine + UVA
4 = 1.0 mM tyrosine + UVB
Fig. 2. Melanocyte proliferation after UV irradiation of previ-
ously low tyrosine concentrations exposed cultures for different
periods of time
However, 24 h exposure to high tyrosine concentra-
tions (2 mM, 3.4 mM) induced an increased cell prolife-
ration rate after UVB irradiation compared to UVA, while
the 72 h tyrosine exposed cultures showed an increased
proliferation rate after UVA irradiation, compared to UVB
radiation and non irradiated cultures (Fig. 3).
30
40
50
60
70
80
90
100
0 h 24 h 72 h
M
ea
n
pe
rc
en
t o
f i
ni
tia
l v
al
ue
(%
)
0.397 mM tyrosine + UVA
0.397 mM tyrosine + UVB
2.000 mM tyrosine + UVA
2.000 mM tyrosine + UVB
3.400 mM tyrosine + UVA
3.400 mM tyrosine + UVB
Fig. 3. Melanocyte cytotoxicity assay after UV irradiation
(30 mJ/cm2) of previously high tyrosine concentrations exposed
cultures for different periods of time
The differences between the proliferation rates of
UVA and UVB irradiated cultures previously exposed to
tyrosine were not statistically significant (AUC control
UVA, vs control UVB, p = 0.769; tyrosine 2 mM UVA
vs UVB, p = 0.825; tyrosine 3.4 mM UVA vs UVB, p =
0.854). High tyrosine concentration strongly inhibited
cell proliferation in the UVA irradiated cultures (AUC
control vs tyrosine 2 mM, p = 0.026, AUC control vs
tyrosine 3.4 mM, p = 0.019). However, no statistically
significance was obtained between AUCs of tyrosine
2 mM vs 3.4 mM, p = 0.897. Same results were re-
corded after UVB irradiation (AUC control vs. tyrosine
2 mM, p = 0.036, AUC control vs tyrosine 3.4 mM, p =
0.029, but AUC tyrosine 2 mM vs 3.4 mM, p = 0,657).
There is no statistical significance between AUCs of
tyrosine 2 mM unirradiated vs UVA, p = 0.843 respec-
tive no irradiated vs UVB, p = 0.954 (see Fig. 3).
Experimental Oncology 31, 200–208, 2009 (December) 203
Microscopic examination. The short period (24 h)
of tyrosine exposure did not produce visible alterations in
the melanocyte morphology; their aspect was similar with
untreated controls (Fig. 4, a). Melanocytes exposed to
tyrosine for the longer period (72 h) were deeply modified.
2 mM tyrosine exposed cells were heavily pigmented,
with the cell body occupied with large melanosomal com-
plexes (Fig. 4, b). Higher tyrosine concentration (3.4 mM)
severely altered the microscopic aspect; the cells were
showing mosaicism, polymorphism, with heterogeneity of
pigmentation, vacuolar degeneration, and loss of surface
adherence (Fig. 4, c) — characte ristics of a late-passage,
senescent melanocyte culture in vitro [33].
a
b
c
Fig. 4. Microscopic examination of the melanocyte cultures.
a, normal microscopic aspect of the epidermal human melano-
cytes in culture (untreated controls, photo taken through Nikon
Eclipse T 100 microscope, 40 x). b, heavily pigmented cells, with
a few, short, plump dendrites; the cell body is occupied with
large melanosomes, except the central area, where the nucleus
is still visible (melanocyte cultures exposed for 72 h to 2 mM
tyrosine, photo taken through Nikon Eclipse T 100 microscope,
40 x). c, polymorphism exhibiting large, monstrous cells, small
cells, bipolar and round cells, uneven distribution of the melanin
pigment, vacuolar degeneration, loss of surface adherence
(melanocyte cultures exposed for 72 h to 3.4 mM tyrosine, photo
taken through Nikon Eclipse T 100 microscope, 40 x)
Melanocyte viability was assessed for the
untreated controls and the higher (2 mM) tyrosine
exposed cultures (Table 1). It showed a high viability
ratio. Viability was significantly decreased with tyrosine
exposure (Table 2). Interestingly, time exposure to
tyrosine had no significant influence on melanocyte
viability. We noticed a slight viability decrease when
melanocytes were exposed to tyrosine and UVB, com-
pared with the tyrosine and UVA combination, but the
difference was not significant (Table 3).
Total melanin content. Tyrosine exposure had
a different effect on melanin synthesis according to
concentration. Lower concentrations (0.5 mM, 1 mM)
significantly increased melanin content, while the high
concentration (2 mM) decreased it (not significant),
compared to controls (see Table 1). Melanin content
was significantly increased with time exposure to
tyrosine in 1 mM tyrosine treated melanocytes (see
Table 2). Pigment production was increased with higher
UV energy irradiation; significantly after UVA irradiation
30 and 40 mJ/cm2, respective after UVB irradiation
40 mJ/cm2, compared to unirradiated controls (see
Table 3). There was a higher melanin content in the
cultures exposed to the combination of UVA and tyrosine
than the combination of UVB and tyrosine (Table 4).
Tyrosinase enzymatic activity significantly
increased with high tyrosine concentration (2 mM)
(see Table 1). Time exposure to tyrosine increased
tyrosinase activity (see Table 2).
Both UVA and UVB stimulated tyrosinase activity.
UVA irradiation determined a higher stimulation of
tyrosinase activity than UVB; the difference was signifi-
cant with lower concentrations (0.5 mM, 1 mM) (see
Table 3). Tyrosinase enzymatic activity increased with
irradiation energies, not significant (see Table 4).
SOD and Cat enzymatic activity were significantly
increased in the melanocyte cultures exposed to lower
tyrosine concentrations (0.5 mM, 1 mM) compared
to controls (see Table 1). Time exposure to tyrosine
significantly altered the activity of SOD. Cat activity dif-
fered significantly when melanocytes were exposed to
0.5 mM and 2 mM tyrosine concentration. Interes tingly,
after high tyrosine concentration exposure, SOD and
Cat showed increased activity rates after a short treat-
ment, compared to untreated controls, but their activity
was dramatically decreased with longer tyrosine expo-
sure. However, this situation was completely different
with low tyrosine concentrations (see Table 2).
UVA irradiation stimulated SOD and Cat activity
at lower tyrosine concentrations (0.5 mM) than UVB
(1 mM). UVA increased Cat activity after low tyrosine
concentration (0.5 mM) exposure, relative to UVB.
However, higher concentrations of tyrosine exposure
prior to UV irradiation changed this effect. After 1 mM
tyrosine concentration exposure, the effects of UVA
and UVB on Cat activity were similar, while at high
tyrosine concentration (2 mM) UVA decreased Cat ac-
tivity compared to UVB and controls (see Table 3). The
enzymatic activity of both SOD and Cat was increased
with the energy of irradiation (see Table 4).
204 Experimental Oncology 31, 200–208, 2009 (December)
SOD activity was directly correlated (r = 0.732, p =
0.000) with the enzymatic activity of tyrosinase, when the
melanocytes were treated with 1 mM tyrosine (Fig. 5).
y = 61.016x + 1061.9
R2 = 0.5857
0
500
1000
1500
2000
2500
3000
3500
4000
4500
0 10 20 30 40 50
Tyrosinase enzymatic activity (U)
SO
D
(U
/m
g
pr
ot
ei
n)
Fig. 5. Correlation between SOD and tyrosinase enzymatic
activity in the melanocyte cultures exposed to 1 mM tyrosine
and irradiated
However, when the melanocytes were exposed to
2 mM tyrosine, SOD and enzymatic tyrosinase activity
were indirectly correlated (p = 0.011. r = –0.488) (Fig. 6).
y = –13.571x + 1998.6
R2 = 0.1109
0
500
1000
1500
2000
2500
3000
3500
4000
4500
5000
0 20 40 60 80 100 120
Tyrosinase enzymatic activity (U)
SO
D
(U
/m
g
pr
ot
ei
n)
Fig. 6. Correlation between SOD and tyrosinase enzymatic
activity in the melanocyte cultures exposed to 2 mM tyrosine
and irradiated
Table 1. Melanocyte bioassay: effects of different tyrosine concentrations on cell viability, total melanin content and the enzymatic activity of tyrosinase,
SOD and Cat
Tyrosine
concentration (mM)
Total melanin content
(μg/culture)
Enzymatic activity
of tyrosinase (U)
Superoxide dismutase
(U/mg protein) Catalase (U/mg protein) Viability (%)
Median/range Median/range Median/range Median/range Median/range
0.397 3.39/4.7 18.44/57.52 1363.91/3618.66 27.335/43.04 88.965/15.71a
0.5 2.64/3.1b 20.08/50.16a 2022.13/2355.80b 37.086/231.54b
1 2.38/2.1b 19.86/32.44a 2247.57/2724.52b 69.15/190.54b
2 3.4/5.1a 73.54/91.44b 1649.58/4421.74c 37.165/81.46a 91.86/8.93b
ap > 0.05, not statistically significant, bp < 0.05, statistically significant, cp = 0.166, marginally significant, compared with the untreated control, represented
by the melanocyte cultures treated with DMEM with 5% FCS, tyrosine concentration 0.397 mM.
Table 2. Melanocyte bioassay: effects of different periods of time exposures to tyrosine on cell viability, melanin content and the enzymatic activity of tyrosinase,
SOD, Cat
Tyrosine
concentration mM
Exposure time to
tyrosine (h)
Total melanin
content (μg/culture)
Enzymatic activity of
tyrosinase (U)
Superoxide dismutase
(U/mg protein)
Catalase (U/mg
protein) Viability (%)
Median/range Median/range Median/range Median/range Median/range
0.397 24 3.3/3.5 13.46/29.52 1855.47/3367.49 30.16/20.18 88.825/14.59
72 3.79/4.5a 27.44/56.80a 939.43/1840.54b 20.285/43.04a 91.475/10.78a
0.5 24 2.76/3.1 18.56/50.16 1698.97/2282.54 34.10/28.54
72 2.42/3.6a 25.04/35.08c 2580.84/2105.83b 87.37/230.32b
1 24 1.96/2 19.36/29 1982.14/2724.52 56.43/170.68
72 2.4/1.6b 24.84/27.84a 2620.61/1943.48b 81.61/190.54a
2 24 3.45/2.4 15.50/45.04 2695.47/2861.13 70.85/50 41.94/61.31
72 3.2/3.9a 87.12/41.28b 941.68/2779.75b 20.55/62.62b 20.285/45.73b
ap > 0.05, not statistically significant, bp < 0.05, statistically significant, cp = 0.158, marginally significant, effects of 24 h compared to 72 h time of tyrosine
exposure.
Table 3. Melanocyte bioassay: comparison between the effects of UVA vs UVB irradiation after previous tyrosine exposure on cell viability, melanin
content and the enzymatic activity of tyrosinase, SOD, Cat
Tyrosine
concentration (mM) Radiation type
Total melanin
content (μg/culture)
Enzymatic activity of
tyrosinase (U)
Superoxide dismutase
(U/mg protein)
Catalase (U/mg
protein) Viability (%)
Median/range Median/range Median/range Median/range Median/range
0.397 UVA 3.75/4.4 28.44/57.52 1274.84/3618.66 27.07/17.49 88.825/14.59
UVB 3.39/4.7a 13.46/31.08a 1378.5/3618.66a 30.425/43.04a 91.475/10.78a
0.5 UVA 3.165/2.3 29.35/37.48 2720.90/1699.31 79.84/218.05
UVB 2.875/2.5a 37.96/41.48b 1365.85/1200.0b 27.10/21.63b
1 UVA 2.57/1.7 19.86/20.14 2127.48/1793.78 61.325/184.63
UVB 2.215/1.8b 30.74/17.44b 3404.78/1300b 68.67/176.59a
2 UVA 4.75/2.2 46.44/79.92 407.145/1581.42 24.32/51.46 88.96/7.39
UVB 4.2/3.9a 28.44/79.2 a 384.455/2504.43a 36.285/43.04a 88.15/12.94a
ap > 0.05, not statistically significant, bp < 0.05, statistically significant, UVA compared to the UVB effects on the previously tyrosine exposed cultures.
Table 4. Melanocyte bioassay: comparison between the effects of the UVA respective UVB irradiation energies after previous tyrosine exposure on
melanin content and the enzymatic activity of tyrosinase, SOD, Cat
Radiation type Doses of radiation
(mJ/cm2)
Total melanin content
(μg/culture)
Enzymatic activity of
tyrosinase (U)
Superoxide dismutase
(U/mg protein) Catalase (U/mg protein)
Median/range Median/range Median/range Median/range
Unirradiated cultures 0 2.4/1.6 12.24/91.44 1363.91/4422.4 20.66/61.95
UVA 20 2.53/2.5a 17.64/45.8a 1778.3/1157.77a 38.41/107.36b
30 2.7/2.7b 19.4/53.24a 2735.02/3032.05a 33.34/203.35a
40 3.76/2.7b 36.71/79.92a 2560.02/3192.75a 43.31/243.42b
UVB 20 2.42/2.5a 26.77/64.24a 1848.4/1826.41a 32.39/93.16c
30 2.4/4.8a 29.52/79.2a 2022.13/3698.8a 26.98/138.9a
40 3.36/4b 38.9/82.76a 2393.95/2342.12a 37.19/170.86b
ap > 0.05, not statistically significant, bp < 0.05, statistically significant, cp = 0.109, marginally significant, compared with the unirradiated cultures for UVA
irradiation, respective for UVB irradiation.
Experimental Oncology 31, 200–208, 2009 (December) 205
SOD activity was also directly correlated (r = 0.227,
p = 0.099) with the total melanin content of the cultures
treated with 1 mM tyrosine (Fig. 7).
y = 196.97x + 1969.7
R2 = 0.0175
0
500
1000
1500
2000
2500
3000
3500
4000
4500
0 1 2 3 4
Total melanin content (μg/culture)
SO
D
(U
/m
g
pr
ot
ei
n)
Fig. 7. Correlation between SOD enzymatic activity and total
melanin content in the melanocyte cultures treated with 1 mM
tyrosine and irradiated
After exposure to 2 mM tyrosine SOD activity was
indirectly correlated with melanin content (p = 0.022,
r = –0.446) (Fig. 8). There were no correlations be-
tween SOD and tyrosinase activity, respective melanin
content in the cultures treated with 0.5 mM tyrosine.
y = –516.1x + 3081.3
R2 = 0.2902
0
500
1000
1500
2000
2500
3000
3500
4000
4500
5000
0 2 4 6 8
Total melanin content (μg/culture)
SO
D
(U
/m
g
pr
ot
ei
n)
Fig. 8. Correlation between SOD enzymatic activity and total
melanin content in the melanocyte cultures treated with 2 mM
tyrosine and irradiated
y = –0.1969x + 40.266
R2 = 0.1316
0
10
20
30
40
50
60
70
80
90
0 20 40 60 80 100 120
Tyrosinase enzymatic activity (U)
Ca
t (
U/
m
g
pr
ot
ei
n)
Fig. 9. Correlation between Cat and tyrosinase enzymatic
activity in the melanocyte cultures treated with 2 mM tyrosine
and irradiated
Cat indirectly correlated with tyrosinase activity
when the melanocytes were treated to 2 mM tyrosine
(p = 0.045, r = –0.397), but not with melanin produc-
tion (Fig. 9). There were no correlations between the
activity of Cat and tyrosinase when the cultures were
exposed to lower tyrosine concentrations.
DISCUSSION
We observed the effects of oxidative stress on the
rate of proliferation, melanogenesis, using normal
human melanocyte cultures as cellular models. There
were no significant differences in reaction between
the adult and newborn epidermal melanocytes in the
studied conditions. In our experiments, irradiation of
the melanocytes was expected to increase oxidative
stress defense enzymes directly and by triggering
melanogenesis. This effect was modulated by tyrosine
exposure before UV irradiation [4, 15–18, 34].
Proliferation studies showed a nonlinear decrease
of proliferation with concentration, enhanced by time
exposure to tyrosine, as previously described [15].
Exposure to lower concentrations of tyrosine
(0.5 mM, 1 mM) discretely diminished the proliferation
rate of the melanocytes, while the higher concentra-
tions (2 mM, 3.4 mM) proved to be toxic for the cells.
The cells were still viable, as shown by the trypan blue
staining, but they had no ability to proliferate.
Only small doses of UVA respective UVB (20, 30 and
40 mJ/cm2) were used in our experiments, comparable
to physiologic sun exposure. There were no important
differences of the irradiation time with UVA vs UVB,
as the intensities of the light generated by the lamp
were similar.
UVB decreased proliferation relative to UVA when
melanocytes were exposed for 72 h to high tyrosine
(2 mM, 3.4 mM), but not in cultures exposed to lower
tyrosine concentration (0.5 mM, 1 mM). These dif-
ferences can be due to the different UV light action
mechanism depending on wavelength. Most of the
biologic effects of UVA radiation in the epidermal
melanocytes are mediated by reactive oxygen species
(ROS). ROS seem to activate growth factors’ receptors
and in particular those of epidermal growth factor and
initiate multiple signaling responses associated with
mitogenesis and cell growth regulation [35–37].
UVB radiation has been established as the main cause
of nonmelanoma skin cancer, particularly squamous cell
carcinoma, due to direct DNA damage [38].
ROS generated in the cells by UVA irradiation and
in a lower degree UVB, did not destroy the plasma
membrane integrity, but decreased cell proliferation.
This effect was enhanced by previous tyrosine exposure
for both UVA and UVB irradiation. This is consistent with
the research done by others, who showed an increased
sensitivity of the skin type I melanocytes to UVA after
increasing their melanin content, with tyrosine exposure
[18]. This suggests that UVA irradiated cultured mela-
nocytes are photosensitized by their own synthesized
chromophores: melanin and pheomelanin [18].
We used melanocytes from individuals with low skin
phototype (II, III). They do not present an intense tanning
response after UV irradiation [25]. These melanocytes
synthesize eumelanin and pheomelanin, in contrast
to Negroid individuals who synthesize only eumelanin
[9]. Melanin in light skin could contribute to sunlight-
induced genotoxicity and maybe — to melanocyte
transformation [34]. Eumelanin is capable of scav-
206 Experimental Oncology 31, 200–208, 2009 (December)
enging the superoxide anion and hydrogen peroxide,
whereas pheomelanin acts as a photosensitizing agent
[39]. It has been shown that melanocytes in culture are
protected against UVB-induced direct DNA damage
by increased melanin synthesis [40]. However, several
experiments with normal and displastic nevi melano-
cytes or melanoma cells have failed to demonstrate
that melanin in melanocytes protects them significantly
against UV-induced direct DNA damage [41, 42].
In our experiments, tyrosine exposure stimulated
melanogenesis, as previously described [4, 15–18].
This effect was enhanced with the time of exposure
and tyrosine concentration. UVB stimulated pigmenta-
tion more than UVA after previous tyrosine exposure
with lower concentrations (0.5 mM and 1 mM), as
expected, considering that UVB is three to four times
more effective per unit physical dose (J/cm2) than
UVA in inducing erythema, DNA damage, tanning
and skin cancer in mice [43]. However, this situa-
tion was changed at high levels of tyrosine (2 mM) in
medium.
Exposure to tyrosine, even in low concentrations,
was stressful for the cells, which exhibited high levels
of SOD and Cat activities. This was consistent with the
work of others who found that tyrosine-induced melano-
genesis in melanocytes was accompanied by increased
production of ROS and decreased concentration of
intracellular glutathione [42]. It also increased early
induction of heme oxygenase 1 gene, a typical response
to oxidative stress, after UVA irradiation [34].
Considering that UVA acts mainly through the ROS
generation, physiological antioxidants play a crucial
role in the skin photoprotection [44].
Lower tyrosine concentrations (0.5 mM, 1 mM)
stimulated the enzymatic activity of SOD and Cat, but
the high tyrosine concentration (2 mM) after an initial
increase at 24 h, decreased their activity dramatically
with time exposure to tyrosine. That correlated with
the proliferation assay, which demonstrated that this
concentration of tyrosine was toxic for the melanocytes
through deleterious effect on the oxidative stress de-
fense of the cells.
In cultures exposed to tyrosine, UVA was a more ef-
ficient stimulus for the induction of the stress enzymes
than UVB. This could be explained through ROS genera-
tion. UVA irradiation enhanced SOD and Cat activity after
less tyrosine stimulation than with UVB, but depleted the
enzymatic reserves, especially Cat, after lower tyrosine
exposure relative to UVB. Overall, UVA was more effec-
tive than UVB in inducing impairment in Cat activity, as
shown also in previous studies [45, 46].
Our data showed that the levels of SOD and Cat ac-
tivity in the cultures after 2 mM tyrosine exposure were
low, regardless of the irradiation type. Low levels of Cat
activity were previously observed in different cutaneous
experimental models and they were always associated
with a stress-prone status [1, 24, 47–49]. In melano-
cytes, the role of Cat is critical because it is the major
enzyme responsible for the neutralization of H2O2 [23],
a byproduct of the melanogenic pathway [50]. Cat oxi-
dative damage is detrimental, because when damaged
it recovers slowly [45, 51]. This results in accumulation
of H2O2 in the cell and damages of several constituents,
including Cat [22, 51] and tyrosinase [11].
Enhanced proliferation at high tyrosine concentra-
tion following UVA compared with UVB increased the
number of melanocytes that exhibited imbalances of
the normal antioxidant mechanisms, common in hu-
man melanoma cells [52]. Although viability was not
significantly altered, cells experienced further oxida-
tive stress and were depleted of antioxidant enzymatic
defenses.
The hypothesis of melanocyte carcinogenesis
states that an essential part of melanocytes’ malig-
nant transformation is a change in the redox state of
melanin from a mostly antioxidant state to a prooxidant
state [20]. This is supported by data that show that
melanoma cells have a remarkably abnormal content
of antioxidants, including vitamin E, polyunsaturated
fatty acids, and catalase [4, 44, 53]. Also, on the
clinical level, displastic nevi, recognized precursors of
melanoma, suffer from chronic oxidative stress, even
without the influence of UV radiation, due to increased
pheomelanin synthesis [42].
In the cultures exposed to 1 mM tyrosine concen-
tration the increased pigment production was directly
correlated with the enzymatic activity of SOD. This is a
very good indicator that melanogenesis itself directly
produced oxidative stress in the cells.
The highest tyrosine concentration (2 mM) used for
the melanocyte bioassay is 35–40 times higher than
the physiological one and exerted a strong proliferation
inhibition of the melanocytes, while modulating melano-
genesis [5, 15]. In these cultures, increase of melano-
genesis was correlated with the decrease of SOD and
Cat activities that depleted the defense mechanisms
against oxidative stress and proved to be damaging for
the cells. This effect was enhanced by longer tyrosine
exposure, which stimulated melanogenesis and trig-
gered early senescent aspect of the melanocytes. Ex-
hausting of the cell defense mechanisms rendered the
melanocytes incapable of neutralizing the free radicals
generated by UV exposure and melanin production.
Also, synthesis of pheomelanin consumes cysteine and
this may limit the capacity of the cellular antioxidative
defense [42]. This hypothesis is sustained by the dy-
namics of the oxidative stress defense enzymes activity
and the proliferation rate of the melanocytes.
The few types of melanocytes examined do not allow
us to draw general conclusions. Our data indicate that in
low phototype melanocytes, pigment formation, either
following UV irradiation, or stimulated by tyrosine expo-
sure is inducing oxidative stress defence mechanisms
activation. In the studied conditions, UVA was more effi-
cient in stimulating the activity of the stress enzymes but
also in depleting enzymatic defenses against oxidative
stress (especially Cat) compared with UVB. Pigment
formation was detrimental for the cells, when exposed
to high tyrosine concentrations, by reducing the activity
of Cat and SOD, the natural antioxidants.
Experimental Oncology 31, 200–208, 2009 (December) 207
The physiologic response to UV light may be an
adjunctive risk factor for people with low phototype
for developing a melanoma, when exposed to UV ir-
radiation.
How these findings relate to an enhanced skin car-
cinogenesis in low phototype individuals needs further
investigation using long-term irradiation experiments.
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