ARTICLE
Auteur(s) : Thibaut Saguet1, Sophie
Robin3, Laurence Nicod4, Delphine
Binda1, Céline Viennet1, François
Aubin1, Bernard Coulomb5, Philippe
Humbert1,2,*
1Laboratoire d’ingénierie et de biologie cutanées, EA
3183, IFR 133 Ingénierie cellulaire et tissulaire, Université de
Franche-Comté, 25041 Besançon cedex France Fax: (+33) 381 21 82
79
2Département de dermatologie, Centre hospitalier
régional universitaire, 2 place Saint-Jacques, 25030 Besançon
Cedex, France
3BioExigence, 8 rue Alfred de Vigny, 25000 Besançon
France
4Sciences séparatives et biopharmaceutiques, EA3924,
Université de Franche-Comté, 25030 Besançon cedex, France
5Réparation et remodelage oro-fasciaux, EA2496,
Université René Descartes-Paris 5, Faculté de chirurgie dentaire, 1
rue Maurice Arnoux, 92120 Montrouge, France
accepté le 28 Mars 2006
Skin exposure to ultraviolet B radiation (UVB) (290-320 nm), which
is a minor but active constituent of sunlight (4% of total solar UV
radiation), contributes to the development of deleterious cutaneous
damage such as sunburn, premature cutaneous photoaging or
carcinogenesis [1-5]. These effects are emphasized by the intrinsic
aging process [6] and by the increase of exposure to UVB related to
the use of UV tanning lamps. Among the mechanisms by which UV
radiation damages skin and in addition to the known direct
cytotoxicity related to genotoxic events, the generation of
reactive oxygen species (ROS), and its role in various skin
diseases have been extensively studied and reviewed in the
literature [7-9]. Indeed, Masaki and Sakurai [10] have demonstrated
that UVB irradiation generates ROS in fibroblasts in vitro. As a
consequence of the oxidative stress resulting from UVB exposure,
Morliere et al. pointed out an increase of fibroblast lipid
peroxidation, a marker of cell membrane damage [11].Several
mechanisms have been proposed to explain skin aging, and the free
radical theory is receiving particular attention because human skin
is constantly exposed to ROS coming from the environment (air,
solar radiation, ozone, and other pollutants). ROS are also
generated from cellular metabolism, both from the mitochondrial
respiratory chain, where excess electrons are provided to molecular
oxygen to generate superoxide anions [6, 12], and from oxidative
metabolism by cytochromes P450 [13].Adverse effects resulting from
oxidative stress can be counteracted by different cellular pathways
[14]. Specific enzymes like superoxide dismutase (SOD),
glutathione-peroxidase (GPx) and catalase are involved in the
detoxification of O2•- into
H2O2, and subsequently into H2O
and O2. A second major way to maintain the redox
equilibrium is the scavenging of free radicals by reduced
glutathione (GSH) and related enzymes such as glutathione reductase
(GRed).The aims of this work were to compare first the basal
antioxidant status of primary cultured human fibroblasts from child
foreskin and adult abdominal skin, and secondly the UVB-induced
cellular damage (lipid peroxidation) and antioxidant impairment
(catalase, SOD, GPx, GRed, GSH) in both cell populations. Moreover
we assessed the effects of a range of UVB doses on the apoptotic
response related to oxidative damage.
Materials and methods
Chemicals
All chemicals were obtained from Sigma-Aldrich (Saint Quentin
Fallavier, France). Fetal calf serum (FCS) was provided by Cambrex
(Vervier, Belgium) and other media and additives used for
fibroblasts isolation and culture were purchased from Invitrogen
(Cergy-Pontoise, France).
Experimental procedure
Fibroblasts isolation and culture
Primary cultures of foreskin fibroblasts (FF) and abdominal
fibroblasts (AF) were obtained from explants of healthy human
dermis (n = 5 for each). The donors’ ages ranged from 3 to 8 years
(mean = 5.0 ± 1.2) for foreskins and 26 to 37 years (mean = 33.4 ±
2.0) for abdominal skin.
Fibroblasts were grown in Dulbecco’s modified Eagle’s medium
(DMEM) supplemented with 10% FCS, streptomycin (50 U/mL) and
penicillin (50 μg/mL) at 37 °C with 5% CO2. Cells
were used between 3rd and 7th passages:
fibroblasts were seeded at 104 cells/cm2 in
75 cm2 flasks and on blades or 5.103
cells per well in 96 wells.
UVB irradiation
Fibroblast cultures were rinsed with phosphate buffer salt (PBS),
and irradiated with UVB (290-320 nm). The UVB source was a Waldmann
Bridge equipped by fourteen F8T5 UVB Tubes (Reichstett, France).
Doses applied were 20, 500, 1000 mJ/cm2. The emitted
radiation was checked using a UV radiometer IL-1700 with UVB filter
(Dexter Industrial Green, Newburyport, Massachusetts). After UVB
exposure, cells were maintained in DMEM, supplemented as
previously, for 2 or 24 h until further analysis.
Fibroblast homogenate preparation
Irradiated fibroblasts in 75 cm2 flasks were washed
with cold homogenate buffer [Tris-HCl 50 mM, KCl 150 mM, EDTA 2
mM]. Cells were scrapped in 1.5 mL homogenate buffer and
sonicated for 15 s. Homogenates were aliquoted and frozen at
– 80 °C until biochemical analysis. Homogenates protein
content was assayed using the bicinchoninic acid method [15].
Cytological analysis
Morphological analysis
Cells colonizing blades were fixed in 4% paraformaldehyde (PFA) and
stained with May-Grünwald-Giemsa (MGG). Cell morphology was
analyzed by light microscopy.
Apoptosis
Apoptotic cells were assessed by flow cytometry as fractions with
sub G1 DNA content [16]. Cells were harvested by trypsinization.
Pellets of cells were fixed in 70% (v/v) cold ethanol and further
washed with cold PBS before the addition of 1 mg/mL RNaseA
Dnase-free and 0.1 mg/ml propidium iodide.
Cells were analyzed on an Epics® Altra flow cytometer
(Beckman Coulter, Villepinte, France) with 488 nm excitation laser.
A minimum of 20,000 events was analyzed for each sample. The
analyses, performed on a gated cell population in order to discard
cellular debris and doublets, were quantified with Wincycle
software (Phoenix Flow Systems, San Diego, CA).
Biochemical analysis
GSH content was measured in homogenates according to the
5,5’-dithiobis (2-nitrobenzoic acid) (DNTB) recycling procedure
described by Griffith et al. [17] and modified by Allen and Arthur
[18]. Briefly, 40 μL homogenate was deproteinized by addition
of 25% 5-sulfosalicylic acid; supernatant was mixed (v/v) with DNTB
(0.6 mM) and absorbance was measured at 405 nm immediately after
mixing. Results were expressed as nmol/mg cell proteins.
GRed activity was determined in homogenates, according to the
procedure described by Carlberg et al. [19] and modified by Bellomo
et al. [20], by measuring the disappearance of NADPH (0.1 mM) at
340 nm, in the presence of oxidized glutathione (1 mM). Results
were expressed as nmol NADPH oxidized/min/mg cell proteins.
GPx activity was measured as described by Lawrence and Burk [21]
in the presence of GSH (2 mM), GRed (0.05 U/mL), NADPH (0.2 mM),
NaN3 (2.5 mM) and cumene hydroperoxide (4 mM). GPx
activity was determined spectrophotometrically by following the
oxidation of NADPH at 340 nm. Results were expressed as nmol NADPH
oxidized/min/mg cell proteins.
Catalase activity was evaluated, according to the method of Aebi
[22], by spectrophotometric analysis (at 240 nm) of the rate of
hydrogen peroxide decomposition in PBS. Homogenates were mixed
(v/v) with hydrogen peroxide (15 mM). Enzyme activity was expressed
as U/mg cell proteins.
SOD activity was analyzed according to the method of McCord and
Fridovich [23] modified by Flohe and Otting [24]. SOD activity was
assessed in homogenates as the inhibition of ferricytochrome c
reduction by superoxide anions generated from a xanthine-xanthine
oxydase system. Ferricytochrome c reduction was monitored
spectrophotometrically at 550 nm, with 1 U of SOD corresponding to
a 50 % decrease in ferricytochrome reduction. Enzyme activity
was expressed as U/mg cell proteins.
Lipid peroxidation was evaluated in homogenates by the method of
Okhawa et al. [25] with slight modifications. Briefly
thiobarbituric acid reactive substances (TBARS) were measured using
a fluorometric procedure. Malonaldehyde (MDA) standards were
prepared from tetraethoxypropane and results were calculated as
pmol of TBARS/mg proteins.
Cytotoxicity evaluation
Fibroblast viability was assayed 2h and 24h after UVB irradiation
in 96 well plates using the 3-(4,5-dimethylthiazol-2-yl)-2,5
diphenyltetrazolium bromide (MTT) dye reduction assay [26]. Briefly
15 μL of MTT (5 mg/mL) was added to each well and plates were
further cultured for 4 h. 100 μL extraction buffer [10 %
(w/v) sodium dodecyl sulphate in 0.5 M dimethylformamide] was then
added to each well and plates were incubated overnight at
37 °C. Absorbance was assessed spectrophotometrically at 510
nm. Results were expressed as viability percentages, non irradiated
cell controls representing 100% of cell viability.
Statistical analysis
Data were expressed as means with standard errors (m ± sem).
Statistical significance was determined using a two-way variance
analysis with UVB doses and cell populations as main factors.
Differences between groups were considered as significant for
p-values < 0.05.
Results
Morphological analysis
MGG staining was performed in unexposed AF and FF or after
irradiation with the highest UVB dose (1000 mJ/cm2) (
(figure 1) ).
Controls AF or FF and cells exposed to 20 to 500 mJ/cm2
(data not shown) were fusiform, whereas cells adopted a stellate
phenotype after 1000 mJ/cm2. AF and FF densities
decreased after 500 mJ/cm2 and more, as well 2h as 24h
after irradiation.
UVB-induced apoptosis
The apoptotic cell fraction was assayed by flow cytometry. Control
FF apoptotic fractions were significantly higher than those of AF
(p < 0.05) at 24 h.
No significant difference could be observed in FF and AF 2h
after UVB treatment compared with respective controls (data not
shown). By contrast we observed a significant (p < 0.05)
increase of apoptotic fraction for both FF and AF (23.0% ± 3.6
versus 12.2 ± 2.2 for FF; 29.4% ± 7.7 versus 3.8 ± 1.96 for AF) 24h
after UV irradiation with 1000 mJ/cm2( (figure 2) ) compared with
respective controls.
UVB-induced cytotoxicity
No cytotoxic effect was detected 2 h after UV exposure ( (figure 3A) ). The
viability was significantly (p < 0.05) lower 24h after exposure
in both cell populations treated either by 500 or by 1000
mJ/cm2 compared to respective controls (respectively
81.7% ± 3.7 after 500 mJ/cm2 and 68.2% ± 4.3 after 1000
mJ/cm2 for FF, and 84.1% ± 7.1 after 500
mJ/cm2 and 81.8 ± 9.6 after 1000 mJ/cm2 for
AF).
Lipid peroxidation
Lipid peroxidation levels tended to be higher in AF than in FF for
all the UVB doses assayed. TBARS levels of irradiated FF were
similar to controls 2h and 24h after exposure whatever the UVB dose
applied was. By contrast, lipid peroxidation appeared significantly
increased in AF 2h after 1000 mJ/cm2 (p < 0.01)
compared with control cells ( (figure 4) ).
SOD activity
SOD activity was markedly higher in control FF compared to AF
(2 h: p < 0.001 and 24 h: p < 0.05) (( figure 5 )A,B). SOD
activity was not modified by UVB as well in FF as in AF.
Catalase activity
Catalase presented a significantly higher activity in AF compared
to FF 2 h after irradiation with doses lower than 1000
mJ/cm2 and 24h after irradiation with 500 and 1000
mJ/cm2 (( figure 5 )C,D).
Glutathione redox system
GPx
Baseline levels of GPx activity were 39.1 ± 7.5 and 34.9 ± 7.4
nmol/min/mg proteins at 2 h, and 32.7± 5.0 and 36.2 ± 5.1
nmol/min/mg proteins at 24 h for FF and AF respectively.
Exposure of AF to 1000 mJ/cm2 UVB induced a significant
increase of GPx activity compared to 2 mJ/cm2 (data not
shown) and 20 mJ/cm2 24h after treatment (p < 0.05)
(( figure 6
)A,B).
GRed
No significant difference in GRed activity could be observed as
well between FF and AF as in response to UVB exposure (( figure 6 )C,D).
GSH
GSH contents of control FF and AF were respectively 14.7 ± 1.8 nmol
and 14.9 ± 4.8 nmol at 2 h, and 26.4 ± 3.3 nmol and 17.9 ± 4.7
nmol at 24 h ( (figure 6E,F) ). UVB
irradiation did not alter GSH levels at any of the doses applied
after 2 h. By contrast, irradiation induced significant
decreases of GSH 24h after 500 mJ/cm2 and more for FF,
and 1000 mJ/cm2 for AF.
Discussion
In the present study, we first compared the basal rate of apoptosis
and antioxidant status from child foreskin fibroblasts (FF) and
adult abdominal fibroblasts (AF).
It is noticeable that FF presented a higher basal apoptotic
level than AF which could be considered as a result of both the
difference between anatomic areas of cutaneous biopsies and the
higher cellular turnover in the morphogenesis of young tissues
[27]. In the same way, Jelaska and Korn [28] have previously
reported differences in the apoptotic response of such cellular
populations: adult fibroblasts appeared more susceptible to
anti-Fas antibody-induced apoptosis than young foreskin
fibroblasts. These authors suggested that (i) the process of aging
and differentiation might result in the reduction or elimination of
some fibroblast subpopulations and also in the observed difference,
and that (ii) FF and AF could present different degrees of
dependence from growth factors.
Lipid peroxidation in the absence of treatment tended to be
higher in AF than in FF even though no statistical difference was
observed. This could be explained by the different ages of the two
groups of donors as previously described in rat skin [29] and in
fibroblast diploid cultures [30].
In the present study, no difference in GSH content or GPx and
GRed activities was observed between FF and AF. Nevertheless SOD
activity appeared to be three times lower in control AF than in FF,
in accordance with the results of Allen et al. [31] who showed a
significant decrease of SOD-1 activity with age. On the contrary,
Balin et al. [32] showed that Mn-SOD, but also GPx activities were
lower in fetal cells than in adult fibroblasts.
Catalase activity was found two times lower in FF than in AF, in
accordance with Balin et al. [32]. Sinha also noticed that catalase
gene expression was lower in dermal fibroblasts from young donors
than from 50 year olds [33].
As a consequence of the previously described reshaping of
antioxidant defences, aging reduces the capacity of primary human
dermal fibroblasts to respond to oxidative stress [34].
Concurrently we assessed the effects of UVB irradiation on FF
and AF viability, antioxidant status and apoptosis.
UV radiations react with various molecules such as lipids,
proteins and nucleic acids and also weaken skin endogenous
enzymatic and non-enzymatic antioxidants [35]. Actually recent
studies have reported that in vitro exposure of human skin
fibroblasts to UV causes hydroperoxide, superoxide and hydroxyl
radical production [11, 36, 37], and stimulates immediate
protective antioxidant mechanisms [38]. Thus, it is of primary
importance to better characterize cellular oxidative impairment,
and particularly the alterations of endogenous enzymatic and
non-enzymatic defences in order to understand their role in
UVB-induced injury.
We have also compared the antioxidant response of FF and AF to
UVB exposure. Several reports [7-9] describe antioxidant status in
human skin but the present study is, to our knowledge, the first
one to assess in vitro the antioxidant impairment induced by UVB
radiation simultaneously in these two commonly used fibroblast
populations. Moreover, established dermal cell lines are often used
to study UV-induced cellular damage in vitro, but Leccia et al.
[39] showed that the sensitivity of such cell lines was different
from that of normal cells.
The wide range of UVB doses was chosen in terms of effects in
humans in vivo and according to the literature related to oxidative
stress in fibroblasts [40, 41]. The amount of solar UVB reaching
the surface of the earth approximates to 18 to 30
mJ.cm–2.min–1[42]. We used doses up to 1000
mJ/cm2 UVB light, representing a maximal dermal exposure
of a day of sunbathing at sea level [43], taking into account the
epidermis filtration. So the irradiation doses employed mimicked an
acute exposure.
The response to UVB radiation was assessed 2 h and
24 h following fibroblast exposure. These two time courses
were chosen since the short period (2 h) post-UVB is
representative of immediate intracellular deleterious effects or an
eventual transient up-regulation of enzyme activities, whereas
24 h post-UVB could reflect a later up-regulation and adaptive
changes to modulate oxidative stress.
The morphological study showed a loss of cellular density with
the highest UVB doses (500 and 1000 mJ/cm2). Cells
adopted a stellate phenotype following 1000 mJ/cm2.
Uitto et al. [44] have hypothesized in photoaged skin fibroblasts
that this stellate phenotype could be due to an extension of rough
endoplasmic reticulum, indicating an increased biosynthetic
activity in response to intracellular perturbations.
The increased cytotoxicity observed 24 h after 1000
mJ/cm2 UVB irradiation appeared to be related to an
increase of apoptotic fractions and could be visualized through a
loss of cell density. These results are consistent with those of
Shindo and Hashimoto [45] who reported an increased DNA
fragmentation, as a marker of cell death, in irradiated dermal
fibroblasts 16 h after exposure (250 mJ/cm2).
ROS may play an essential role in the progression of the various
pathways leading to apoptosis since antioxidants have been reported
to provide protection against apoptosis [46]. ROS have both
intrinsic and extrinsic origins, and cells are protected by
multiple levels of antioxidant defences. Among them, GSH represents
a major antioxidant. Tyrrell and Pidoux [47] found, using
butylsulfoxide-induced GSH depleted cells, that endogenous GSH
protected human skin fibroblasts against UVA or UVB-induced
cytotoxicity. In our study, irradiation with the highest UVB doses
significantly decreased the intracellular GSH levels in both FF and
AF, especially 24 h after exposure. This observation suggests
that GSH was directly consumed as an antioxidant compound by
irradiated cultured fibroblasts. However, depletion of GSH might
disrupt many cellular functions and particularly reduce the
capacity to scavenge ROS. In our study, GRed activity, which is
responsible for the recycling of GSH stocks in the GSH redox
system, remained constant in both irradiated and non-irradiated
fibroblast cultures, and thus was not able to counter the decrease
of GSH levels. It could thus be hypothesized that UVB, by lowering
the level of GSH without changing the GRed activity, led to an
imbalance of antioxidant defences, making cells unable to face up
to oxidative processes and resulting in an increased cell death for
high UVB doses. Moreover, the decreased GSH concentration in UVB
irradiated cells could reflect an impairment of up-regulation of
γ-glutamylcysteine synthetase, the rate-limiting enzyme of GSH
synthesis [48]. Besides the fact that GSH protects cells by
reacting directly with ROS, it also acts as a substrate for
reducing enzymes such as GPx. Consequently the decrease in GSH
content might be related to the observed increase of GPx activity,
which was showed significant in AF submitted to the highest UVB
dose compare to lower doses (2 and 20 mJ/cm2; data not
shown). Thus this study showed that the glutathione redox system
acts slightly differently in AF and FF.
It is well accepted that lipid peroxidation can be induced in
cultured human skin fibroblasts by both UVA and UVB radiations
[11]. In the present study we showed a significant enhancement of
lipid peroxidation levels 2 h after 1000 mJ/cm2 UVB
exposure, probably responsible for the loss of viability observed
in cells 24 h after irradiation. In fact lipid peroxides might
be involved in intracellular pathways which activate antioxidant or
apoptotic mechanisms [49]. One GPx isoform, phospholipid
hydroperoxyde peroxidase [50], could partly contribute to the low
TBARS levels. So the increase of GPx activity in UVB-irradiated AF,
24 h after 1000 mJ/cm2, could explain in our
experiments the return of TBARS to levels comparable to controls.
Moreover the very low TBARS levels observed in irradiated FF
compared to AF, could be explained by the higher SOD activity in
FF, which scavenges a higher amount of cytotoxic
O2•-. These results were in accordance with
those of Shindo et al. [51].
Our data suggest that FF and AF, two cell populations coming
respectively from foreskin of young donors and abdominal skin of
adult donors, do not respond to UVB by the same pathways: in
immature cells such as FF, the elevated activity of SOD results in
a high generation of H2O2. This ROS would be
scavenged by catalase in first line and GPx in second line,
according to Masaki et al. [10, 52] and Shindo et al. [35].
To conclude, the present study showed that abdominal and
foreskin human dermal fibroblasts are supplied with different basal
antioxidant equipment which could explain their different
behaviours towards UVB. Our results suggest that AF show a greater
sensitivity to oxidative stress than FF, presumably because of a
chronic exposure related to the way of life and age of the
patients. That is why particular attention should be paid in the
choice of the most relevant model for each study.
Acknowledgments
The authors wish to thank Aude Nappez for her technical assistance.
They also thank plastic and urological surgeons from the University
Hospital Centre, the Clinique St Vincent and the Polyclinique de
Franche-Comté of Besançon.
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