ARTICLE
Auteur(s) :, Rozenn Le Gall*,
Cécile Marchand, Jean-François Rees
Laboratory of Cell Biology, Institut des Sciences de la Vie,
Université catholique de Louvain, 5 place Croix du Sud, 1348
Louvain-la Neuve, Belgium
accepté le 10 Janvier 2005
Sunlight exposure is a well-known cause of human skin alterations,
such as sunburn, premature aging, inflammatory reactions, and
cancer [1]. Many researchers are, therefore, interested in
understanding the mechanisms underlying UV-induced cell damage and
in discovering new agents to protect against it. Since the first
targets of UV radiation are epidermal and dermal cells, skin cells
from plastic surgery are often used as in vitro models. UVA
irradiation of a primary culture of skin fibroblasts leads to lipid
peroxidation [2, 3], degradation of proteins [4], DNA damage [5, 6]
and, ultimately, to cell death [7]. Using these cells, the
protective properties of different compounds against UVA-induced
damage can be studied easily. However, the use of cells from
surgical material carries a high risk of microbial contamination
because micro-organisms are invariably present on the skin [8].
When contamination occurs, cells may grow poorly or not at all,
have an abnormal morphology, die, or lose their usual
characteristics and become unsuitable for experiments. In order to
culture skin cells under the best conditions, antibiotics are often
added to the cell culture medium [9-12] to prevent bacterial,
fungal, and yeast contamination [13]. The agents most commonly used
consist of a combination of the antimicrobial drugs penicillin and
streptomycin with amphotericin B deoxycholate (amB), also known as
fungizone [7, 14].One possible drawback of this strategy is that
these compounds may interfere with the experiments later performed.
In addition to the possible effects of these drugs on cell
physiology, some drugs [15], cosmetic products and food
constituents [5] can act as photosensitizers, causing the formation
of photo-adducts with bio-molecules and generating deleterious
reactive oxygen species (ROS) [5]. Indeed, adverse cutaneous
photoreactions are frequently observed in subjects receiving drugs
before sunlight exposure [15]. Antibiotics are known also for their
capacity to absorb UVA, inducing photoreactions [16]. For example,
tetracycline and fluoroquinolones may undergo photoreactions and
lead to photodermatoses, such as polymorphic light eruption or
systemic metabolic disorder [15]. Moreover, in correlation with
clinical observations, tetracyclines, fluoroquinolones, and other
antibiotics, like nalidixic acid and amphotericin B, are proven
generators of singlet oxygen [16] or superoxide radical [17] under
conditions of UVA radiation.In order to eliminate the possible
photoreactions of antimicrobial and antifungal agents, these are
removed from the culture medium just prior to in vitro
UVA-irradiation of skin cells [11]. However, it is possible that
these agents may still be present in cells, and exacerbate
UVA-induced damage. This study analysed the impact of three
commonly used compounds in human skin cell culture, amB
deoxycholate, penicillin, and streptomycin, on UVA-irradiated
fibroblasts.
Material and methods
Chemicals, culture media
DMEM F12/ mix nut, fœtal calf serum (FCS), penicillin-streptomycin
and amphotericin B deoxycholate (fungizone) were purchased from
GibcoBrl. Phosphate-Buffered saline (PBS), dichlorofluorescein
diacetate (DCFH-DA), dichlorofluorescein (DCF), penicillin G,
streptomycin sulfate, boric acid, linoleic acid,
polyoxyethylenesorbitan monolaurate (Tween20) and 2-2’ azobis
(2-amidino propane) hydrochloride (AAPH), malondialdehyde (MDA),
thiobarbituric acid (TBA), and tricholoracetic acid (TCA) were
obtained from Sigma-Aldrich. Di-sodium hydrogen phosphate
(Na2HPO4) and sodium dihydrogen phosphate
(NaH2PO4) were purchased from Merck-Eurolab. Butanol was
from Fluka and the lactate dehydrogenase (LDH) assay kit was
purchased from Roche.
Cell culture and treatment prior to irradiation
Normal human skin fibroblasts (passage 5-15) isolated during breast
plastic surgery (one donor who was 35 years old), were purchased
from 4C biotech (Belgium) and cultured (37 °C, 5%
CO2) in DMEM F12/nutrient mix with glutamax (GicoBrl)
supplemented with 10% FCS with no other additives. When required,
2.5 μg/mL amphotericin B deoxycholate (GibcoBrl) and/or 100 U/mL
penicillin and 100 μg/mL streptomycin were added.
For all experiments, fibroblasts were seeded at 300 000 cells on
plastic Petri dishes (3.8 cm2) 24 hours before
irradiation.
UVA irradiation
Just before irradiation, the cells were washed twice with 1 mL PBS
and left in 500 μL PBS during irradiation with a BIO-SUN (Vilbert
and Lourmat, France) apparatus.
The spectrum was centred on 365 nm. Cells were irradiated from
the bottom at a light intensity of 4 mW/cm2 (5-30
J/cm2) as determined by an integrated radiometer. This
is comparable to the maximum solar UVA that reaches the earth (97
W/m2 and 35 J/cm2 per hour) [18].
Sham-irradiated control cells were similarly treated but were
placed under aluminium foil in the irradiator.
Estimation of cell survival
Cell mortality was evaluated by measuring the release of lactate
dehydrogenase (LDH) in the culture medium. LDH was quantified by a
colorimetric assay using a cytotoxic detection Kit (Roche).
Briefly, 100 μL of each sample was mixed with 100 μL of reagent
solution in a 96-well plate, and the optical density was measured
at 492 nm (Spectra Max 190, Molecular Devices) after 15 minutes in
the dark (25 °C).
In each experiment, references consisted of supernatants of
non-irradiated cells and non-irradiated cells lysed with 500 μL of
1% Triton X-100 (100% mortality). Results were expressed as
percentage of LDH released.
Quantifying ROS production
A dichlorofluorescein assay was used to evaluate cellular
production of ROS. Just after irradiation, PBS was removed and
cells were incubated for 30 minutes with 100 μM DCFH-DA in PBS in a
cell culture incubator.
DCFH-DA loaded cells were placed in a fluorescence microplate
reader (Fluoroskan Ascent FL, Labsystem) with temperature
maintained at 37 °C. Fluorescence from each well was captured with
excitation and emission filters at 485 nm and 555 nm,
respectively. Controls consisted of DCFH-DA dissolved in cell-free
wells and DCFH-DA loaded cells before incubation. DCF release was
calculated from a standard curve established with DCF in PBS.
Cellular and acellular lipid peroxidation assay
The extent of lipid peroxidation was determined by quantifying the
amount of Thiobarbituric Acid Reactive Substances (TBARS), mainly
malondialdehyde (MDA).
In cellular tests, skin fibroblast lipid peroxidation was
induced by 30 J/cm2 UVA irradiation and the amount of
MDA released in the supernatant of cells was estimated just after
irradiation.
In acellular tests, the peroxidation of a micellar solution of
TWEEN 20 and linoleic acid (0.16 mM) in 50 mM phosphate buffer, pH
7.4 was induced by the addition of 2 mM AAPH (37 °C), and the MDA
content was measured after 3 hours. For this, 30 μL of sample was
added to 24 μL TCA (15%) and 48 μL of TBA (0.67%) in a 96-well
plate and then heated at 95 °C for 30 minutes in an oven. After
cooling, 100 μL of butanol was added to samples and the plate was
centrifuged (5 minutes, 2 000 rpm, 4 °C). The fluorescence of the
MDA-TBA chromogen (excitation: 515 nm; emission: 555 nm) was
measured on a microplate fluorimeter. A standard curve was
constructed with MDA and results expressed as percentage of
inhibition of MDA production. Control tests indicated that
antibiotics did not interfere with the assay.
UV-absorption spectrum of the different agents
Penicillin, streptomycin, and amB were solubilised in saline buffer
and transferred into a 96-well plate. Their UV absorption spectra
were obtained measuring the optical density between 300 and 400 nm
(Spectra Max 190, Molecular Devices).
Statistical methods
A one-way analysis of variance (ANOVA) followed by a Tukey test was
used to assess the significance of difference between treatments.
Results
Primary cultures of skin fibroblasts cultured in medium containing
10% FCS, exposed to 30 J/cm2 UVA showed no signs of
mortality 24 hours after irradiation. Indeed, the rate of LDH
released (5%) was similar to that of sham-irradiated cells ( (figure 1) ). However,
using cells grown in a medium containing the mixture of antibiotics
and antimycotic, washed with saline before irradiation and
irradiated in saline, there was significant cell mortality from 5
J/cm2, increasing in a dose-dependent fashion ( (figure 1) ).
In order to determine which of the drugs was responsible for
this effect, the antibiotics were applied separately. Cells were
grown in four different culture conditions: (a) Cells cultured in
medium containing 10% FCS (Control), (b) Cells cultured in
FCS-medium to which the mixture of streptomycin-penicillin had been
added as previously described (Pen-strep). (c) Cells grown in
medium with amB, and (d) Cells grown in FCS-medium containing the
mixture of antibiotics and antimycotics (Mix). Cells were left in
drug-free PBS during irradiation and washed twice with PBS
beforehand. Like the sham-irradiated control cells, the irradiated
control cells and the irradiated Pen-strep cells had a low
mortality (2%) 24 hours after irradiation ( (figure 2) ). Cells grown
with amB had a much higher mortality (50% at 30 J/cm2).
However, the amB-treated cells suffered a significantly lower
mortality at all UVA doses when the antibiotic agents were also
present during the culture ( (figure 2) ). Lipid
peroxidation of fibroblasts was also estimated. The amount of MDA
released in the supernatant of cells was measured just after 30
J/cm2 irradiation. Sham-irradiated cells, irradiated
control cells, and irradiated pen-strep cells presented only 1
nmol/mg protein of MDA whereas cells cultured in the presence of
amB deoxycholate ( (figure 3) ) had a higher
rate of lipid peroxidation (2 nmol/mg protein of MDA).
The above experiments indicate that although amB was removed
prior to irradiation, its presence in the culture medium markedly
affected the survival and the lipid peroxidation of UVA-irradiated
cells. This suggests that amB may have remained associated with the
cells and not been rejected or washed away before irradiation.
Therefore, amB-treated fibroblasts were cultured in medium
without amphotericin B deoxycholate for 24, 48, 72, and 178 hours
before being washed with saline and irradiated. The UVA-induced
mortality of those cells was compared to that of fibroblasts never
exposed to the antimycotic agent and to that of fibroblasts
cultured with amB until irradiation ( (figure 4) ). Only the
latter treatment provoked an increased susceptibility to UVA
irradiation, indicating that the culture of cells for 24 hours in
amB-free medium before irradiation prevents the occurrence of
amB-induced UVA-susceptibility.
In order to verify that the amB effects were related to the
occurrence of some photosensitization process, we evaluated
UVA-induced ROS production by cells cultured with and without amB.
Fibroblasts were loaded with DCFH-DA just after irradiation and
incubated for 30 minutes. Within cells, DCFH-DA reacts with ROS and
generates fluorescent DCF [19, 20]. Results are presented in (
figure 5 ). UVA
irradiation in amB-free saline resulted in a double production of
ROS when cells had been previously cultured in amB-containing
medium. This effect was totally suppressed when amB was removed
from the culture medium at least 24 hours before irradiation.
Whereas the above results suggest that amB acts as a
photosensitizer, they also indicate some protection of cells by
penicillin and streptomycin. When TBARS was measured, no
significant differences were observed between cells irradiated in
the presence of the amB + Pen-strep and in the presence of amB
alone, but MDA levels were lower in the former conditions. One
mechanism for this finding could be related to some scavenging of
ROS by the antibiotics, thus acting as antioxidants. Therefore, the
possible antioxidative properties of penicillin and streptomycin
were evaluated in a cell-free lipid peroxidation system. AAPH, a
free radical generator, was used to induce lipid peroxidation of a
micellar solution of linoleic acid. Production of MDA was measured
with TBA with and without antibiotics. Results ( (figure 6) ) showed that
the mixture of penicillin-streptomycin at 100 U/mL reduced the
peroxidation of linoleic acid. A 20%-inhibition of MDA production
was observed, in both the presence and absence of amB. When
penicillin and streptomycin were tested separately, only
streptomycin (100 μg/mL) decreased the rate of lipid peroxidation,
to a similar extent as the mixture. This suggests that the
decreased mortality of irradiated cells cultured in a medium
containing streptomycin-penicillin could be related to some
chain-breaking property of streptomycin.
Discussion
In this study, we demonstrated that amB exacerbates the sensitivity
of cells to UVA. Indeed, the presence of amB in the culture medium
of human skin fibroblasts leads to a marked increase in lipid
peroxidation and in mortality induced by UVA radiation even when
cells are washed with amB-free saline prior to irradiation.
DCF-mortality based experiments indicate that this surge in
mortality is accompanied by an increased production of ROS in
UVA-irradiated fibroblasts, pointing to a photosensibilizing effect
of amB deoxycholate. This action of the antimycotic agent is not
unexpected as Pandley [16] demonstrated, in acellular assays, that
amphotericin B is an efficient generator of superoxide anion and
singlet oxygen under UVA radiation. In addition, the UV-spectrum of
amB confirmed this hypothesis, as it was the only antibiotic to
absorb in UVA-spectrum ( (figure 7) ).
However, the persistence of this photosensitizing effect after
washed cells are exposed to amB-free saline during irradiation is
surprising and suggests that amB remains associated to the cells.
The deoxycholate salt of amphotericin B is a macrolide polyene
known to interact with sterols in the plasma membrane of cells.
This interaction between amB and sterols perturbs the plasma
membrane, inducing an efflux of potassium ions from cells and,
ultimately, cell death. It interacts especially with ergosterol
[21], which is mainly present in fungal cell plasma membranes,
hence the higher susceptibility of these cells. As cholesterol is
one of the components of mammalian cell membranes, it has been
suggested that amB, present in the culture medium of human skin
fibroblasts, could be integrated into their membranes [22]. This
factor could explain the persistence of its photosensitising action
even when it is not present in saline during irradiation.
Interestingly, this effect can be totally suppressed by removing
amB from the culture medium at least 24 hours before
irradiation.
This suggests that cells can dispose of amB, and that the drug
does not affect resistance mechanisms against UVA-induced damage,
apart from its photosensitising effect. Vertut-Doi [22]
demonstrated that amphotericin B can be internalised by mammalian
cells (CHO) through endocytosis and enzymatically degraded into
lysosomes also occur in skin cells like fibroblasts.
Our work also shows that antibiotics affect the UVA-sensitivity
of fibroblasts. Indeed, streptomycin significantly reduced the
susceptibility of cells to amB-related UVA-induced mortality. Lipid
peroxidation levels were lower although not significantly so. This
effect could be linked to the observed inhibition of lipid
peroxidation by streptomycin in an acellular test. Such a property
has not been reported previously. Although limited, it is possible
that this could significantly affect cellular antioxidant defence
mechanisms in long-term cell cultures. Indeed, it has been
demonstrated that supplementation of cell culture medium with
antioxidants like vitamin C [23], β-carotene [23, 24], or selenium
[25] modulated endogenous antioxidant enzyme activity and/or
expression (glutathione peroxidase, glutathione reductase…).
Streptomycin could also act directly on cellular ROS
production.
In conclusion, this work demonstrates that the use of
antibacterial and antifungal agents can markedly influence
UVA-induced damage in skin fibroblasts. The deoxycholate salt of
amphotericin B increases cellular susceptibility whereas
streptomycin has an opposite effect. One would thus recommend not
using these antibiotics before cells are evaluated for their UVA
sensitivity or utilised in experimental studies into the mechanisms
underlying the physiological and pathological effects of UVA in
cells. For less fundamental studies, e.g., testing the effects of
new photoprotective agents, the removal of these agents just prior
to irradiation is not sufficient, and a period of 24 hours without
the agents is appropriate to prevent noticeable interactions.
Acknowledgements
We gratefully acknowledge the support of the Regional Government of
Wallonia (DGTRE) and the Fonds National de la Recherche
Scientifique (FNRS).
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