PRECLINICAL EVALUATION OF INFRARED-A RADIATION AND VISIBLE LIGHT
ON MELANOGENESIS PROCESS USING AN IN VITRO AND EX VIVO MODEL
OF SKIN CULTURE
Samara Eberlin1, Stefano Piatto Clerici1, Samir Eberlin2, Ana Lúcia Tabarini Alves
Pinheiro3, Adriano Pinheiro3
1
KOLderma Clinical Trials Institute, Valinhos/SP – Brazil; 2Sante D’or Institute,
Sumaré/SP – Brazil; 3Kosmoscience Ciência e Tecnologia Cosmética Ltda,
Campinas/SP – Brazil.
Keywords: melanogenesis, endothelin-1, melanin
ABSTRACT
Skin pigmentation is dependent on both the increased number of melanocytes as the
increase on melanogenic enzymes. Exposure to sunlight, artificial tanning,
pharmaceuticals and other chemicals, as well as chronic inflammatory processes
and hormonal influences, can increase the production of factors such as melanocyte
stimulating hormone and endothelin-1, which trigger the melanization of the skin. In
addition to ultraviolet light, studies also show that the infrared radiation and visible
light promote changes in skin pigmentation. In this work we evaluated the
consequences of ultraviolet A and B (UVA/UVB), infrared-A (IR-A) and visible light
(VL) irradiation on melanin and endothelin-1 synthesis, using an in vitro model of
melanocyte and keratinocyte cultures, and ex vivo model of skin culture. All radiation
(UV, IR-A, VL and Association of radiations) produced a significant increase in
melanin production when compared to non-irradiated control, contributing to the
process of cutaneous pigmentation. UV radiation, VL and the association of all
radiation, produced a significant increase in the production of endothelin-1 when
compared to non-irradiated control. Consistent with these results, skin fragments
subjected to radiation have a higher melanin density in comparison with control
fragments. An increasing number of papers has been published describing the
harmful effects of exposures to mainly or exclusively short-wavelength IR (IR-A)
radiation in cultures of human skin cells and in human skin biopsies. In the same
way, visible light has been identified as not inert radiation and studies have
demonstrated that it increases cutaneous pigmentation, even higher than the UV
radiation. Our results corroborate previous data from literature and indicated the
need for specific photoprotection strategies which are not addressed by conventional
sunscreens available.
INTRODUCTION
Clinical signs evidenced by chronic exposure to solar radiation, such as the
formation of wrinkles and hyperpigmentation, are among the main aesthetic concerns
affecting the human being, judging by the amount of ingredients and cosmetic
products on the market with supposed action in mitigating these manifestations1-6.
Skin pigmentation is dependent on both the increased number of
melanocytes as the increase on melanogenic enzymes. After exposure of the skin to
sunlight, the melanogenesis process begins with the oxidation of the amino acid
tyrosine by the enzyme tyrosinase, producing DOPA, which is metabolized to
produce melanin7. Melanin plays an important role in protecting the skin from the
harmful effects of solar radiation, but excessive accumulation of this pigment can
become
an
aesthetic
problem8.
Exposure
to
sunlight,
artificial
tanning,
pharmaceuticals and other chemicals, as well as chronic inflammatory processes
and hormonal influences, can increase the production of factors such as melanocyte
stimulating hormone and endothelin-1, which trigger the melanization of the skin9-11.
For many years, photoaging and skin damage were associated almost
exclusively with UV radiation. However, infrared radiation (IR) and visible light (VL)
has awakened interest from the scientific community, due to induction of similar
histological changes to those induced by chronic exposure to UV radiation9-20.
OBJECTIVE
The purpose of this study was to evaluate the consequences of infrared-A
(IR-A) and visible light (VL) radiations on melanin and endothelin-1 synthesis, using
an in vitro model of melanocyte and keratinocyte cultures, and ex vivo model of skin
culture.
METHODS
Cell culture: Human epidermal melanocyte and keratinocytes cultures (Life
Technologies, NY, USA) were grown and subcultured at 37°C in 5% CO2 in a
humidified incubator. At 80–90% confluence, cells were seeded into 24-well culture
plates (4x104 cells per well) for further irradiation.
Skin explants: fragments of healthy human skin, obtained from plastic surgery
(approved by the committee of ethics), were fractionated using a number 4 punch (4
mm) and maintained in culture for subsequent exposure to radiations.
Irradiation protocol: to simulate solar stress, the culture medium was replaced by
EBSS (Gibco-Life Technologies) and the cultures were subjected to oxidative stress
by exposure to UVA/UVB, IR-A and VL, as well as the association of three radiation.
For UVA/UVB radiation (10 J/cm2 UVA) the following devices were used: UVA Cube
400, SOL500 H2 filter and UV Meter (Hönle UV America Inc, MA, USA). In relation to
the IR-A radiation (360 J/cm2) Hydrosun 750 and HBM1 devices (Hydrosun
Medizintechnik GmbH, Müllheim, Germany) were used. For visible light (VL)
radiation (480 J/cm2) an apparatus was assembled with the devices UVA Cube 400,
SOL500 H1 filter to filter out UVA radiation; Cinegel Rosco filter 3114 to filter UVB
and VLP-471RAD Radiometric Sensor (Deta Ohm Caselle di Selvazzano, Italy) to
measure VL radiance.
Quantification of mediators: after 48 hours irradiation, the concentration of melanin
was measured in a spectrophotometer at 405 nm in cell lysate cultures of
melanocytes and compared based on a standard curve with known concentrations of
melanin. The endothelin-1 concentration was measured by sandwich ELISA assay
using commercially available kit (Uscn Life Science Inc., Houston, TX, USA). The
reading of absorbance was performed on monochromator Multiskan GO (Term
Scientific, Finland).
Histological evaluation: after 48 hours irradiation, the fragments were fixed in 10%
buffered formalin, embedded in paraffin blocks and submitted to serial sections in
microtome about 5μm thick. The sections were stained with hematoxylin and eosin
associated with Fontana-Masson staining technique. The slides were photographed
under a light microscope (Nikon Eclipse at 10X magnification) using the Image-Pro
software.
Statistical Analysis: for statistical evaluation ANOVA test was used to compare data
between all the groups. We applied the Tukey post-test, which strengthened and
made the result presented in the ANOVA more precise (GraphPad Prism v6). A level
of 5% significance was used.
RESULTS
All radiation (UV, IR-A, VL and Association) produced a significant increase of
8.2, 5.3, 6.0 and 10.6 %, respectively, in melanin production when compared to nonirradiated control (P<0.001), contributing to the process of cutaneous pigmentation
(figure 1). The induction of melanogenesis was significantly higher (P<0.05) in
melanocytes cultures stimulated with UV radiation, in relation to IR-A and VL groups.
The association of radiation produced a further increase in melanin synthesis
compared to UV, IR-A and VL radiations applied alone.
* * * P < 0 .0 0 1 , in r e la t io n to c o n t r o l;
#
&
0 .8 7 5
P < 0 . 0 5 , in r e la tio n t o I R - A a n d V L ;
P < 0 . 0 5 , in r e la t io n t o U V , a n d P < 0 .0 0 1 ,
in r e la t io n to IR - A a n d V L .
&
M e la n in (m g /m L )
0 .8 5 0
***
Figure 1. Effects of ultraviolet (UV),
infrared-A (IR-A) and visible light
#
(VL) radiations on melanin synthesis
***
0 .8 2 5
in human melanocytes cultures. Data
***
0 .8 0 0
***
represents mean ± standard
deviation of 3 replicates in each
0 .7 7 5
0 .7 5 0
C o n tr o l
UV
I R -A
VL
A s s o c i a ti o n
UV radiation, VL and the association of all radiation, produced a significant
increase of 53.27, 63.81 and 57.81 %, respectively, in the production of endothelin-1
when compared to non-irradiated control (figure 2). Despite IR-A have shown a trend
in increased synthesis of endothelin-1, it did not produce a statistically significant
result.
* * * P < 0 .0 0 1 , in r e la t io n to c o n t r o l.
20
***
E n d o th e lin -1 (p g /m L )
***
***
Figure 2. Effects of ultraviolet (UV),
infrared-A (IR-A) and visible light
15
#
(VL) radiations on endothelin-1
synthesis in human melanocytes
10
cultures. Data represents mean ±
standard deviation of 3 replicates in
5
0
C o n tr o l
UV
I R -A
VL
A s s o c i a ti o n
Consistent with the results presented in Figure 1, skin fragments subjected to
radiations have a higher melanin density in comparison with control fragments (figure
3).
Control
100 µm
UV
IR-A
VL
Association
Figure 3. Histological evaluation of melanin pigmentation in human skin culture subjected to ultraviolet
(UV), infrared-A (IR-A) and visible light (VL) radiations (photos on the left side – 10x magnification,
photos on the right side – manually amplified).
DISCUSSION AND CONCLUSIONS
An increasing number of papers has been published describing the harmful
effects of exposures to mainly or exclusively short-wavelength IR (IR-A) radiation in
cultures of human skin cells and in human skin biopsies. In the same way, visible
light has been identified as not inert radiation and studies have demonstrated that it
increases cutaneous pigmentation, even higher than the UV radiation21-22. In high
doses, VL is capable of provoking erythema and hyperpigmentation. Using a light
source that emits 98.3 VL%, 1.5% IR-A and 0.19% UV-A radiation, Mahmoud et al
(2010)23 have shown that skin phototype plays an important role in intensity, time to
onset of erythema and melanogenesis. In skin phototype IV-VI, VL (480 J/cm2) was
capable of inducing a higher erythematous halo and increase the synthesis of
melanin, when compared to skin phototype II. These authors propose that VL
induces a reaction inside the chromophore, which leads to heat generation and
stimulates eumelanin synthesis, promoting the appearance of erythema and
vasodilation.
There are several factors that contribute to skin photoaging and there is no
evidence of traditional sunscreens capable of preventing physically or chemically
against the infrared radiation and visible light. In this regard, our results corroborate
previous data from literature and indicated the need for specific photoprotection
strategies which are not addressed by conventional sunscreens available. The
finding of adjacent mechanism to those related to absorption, reflection and
scattering of incident light is an important challenge to be overcome which may
provide a broader sunscreen and enhance the prevention of photodamage caused
by infrared and the visible light radiation.
Conflict of interest
The study was supported by KOLderma Clinical Trials Institute Kosmoscience Group.
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