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8/4/2019 Epic Ate Chin and Its Methyl at Ed Metabolite Attenuate UVA-Induced Oxidative Damage to Human Skin Fib Rob Lasts
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Original Contribution
EPICATECHIN AND ITS METHYLATED METABOLITE ATTENUATE UVA-INDUCED OXIDATIVE DAMAGE TO HUMAN SKIN FIBROBLASTS
SHARMILA BASU-MODAK,* MATTHEW J. GORDON,* LAURA H. DOBSON,* JEREMY P. E. SPENCER,
CATHERINE RICE-EVANS, and REX M. TYRRELL*
*Department of Pharmacy and Pharmacology, University of Bath, Bath, UK; and Wolfson Centre for Age-Related Diseases, GKTSchool of Biomedical Sciences, Kings College, London, UK
(Received 17 March 2003; Revised 19 June 2003; Accepted 27 June 2003)
AbstractThe ultraviolet A component of sunlight causes both acute and chronic damage to human skin. In this study
the potential of epicatechin, an abundant dietary flavanol, and 3'-O-methyl epicatechin, one of its major in vivo
metabolites, to protect against UVA-induced damage was examined using cultured human skin fibroblasts as an in vitromodel. The results obtained clearly show that both epicatechin and its metabolite protect these fibroblasts against UVA
damage and cell death. The hydrogen-donating antioxidant properties of these compounds are probably not the
mediators of this protective response. The protection is a consequence of induction of resistance to UVA mediated by
the compounds and involves newly synthesized proteins. The study provides clear evidence that this dietary flavanol has
the potential to protect human skin against the deleterious effects of sunlight. 2003 Elsevier Inc.
KeywordsUVA radiation, Flavanoids, Epicatechin, Cell death, Necrosis, Heme oxygenase-1, Free radicals
INTRODUCTION
Ultraviolet A (UVA) radiation (320380 nm), a compo-nent of the solar UV spectrum, penetrates through the
dermis and beyond to the subcutaneous tissue and affects
both the epidermal and dermal components of skin [1].
The deleterious effects of UVA are manifested in human
skin as erythema, photoaging, and skin cancer (reviewed
in [24]). At the cellular level, UVA radiation causes
significant oxidative stress due to generation of reactive
oxygen species such as singlet oxygen, hydroxyl radical,
superoxide anion, and hydrogen peroxide, and the re-
lease of free iron, but most of the cellular effects of
UVA irradiation seem to be mediated by singlet oxygen
(reviewed in [57]). Constitutive cellular defensesagainst UVA-induced damage include simple antioxi-
dant molecules (glutathione, carotenoids, ascorbate, and
-tocopherol), and proteins (ferritin, heme oxygenase,
glutathione peroxidase, superoxide dismutase, catalase).
When cellular defenses are overwhelmed, UVA-induced
oxidative damage becomes visible as depletion of intra-
cellular glutathione [8], and oxidation of nucleic acids,
proteins and membrane lipids (reviewed in [9]). Dam-
aged cells respond by inducing a variety of genes in
keratinocytes and fibroblasts that are implicated in both
acute and chronic responses to this oxidative stress (re-
viewed in [10]). Extensive cellular damage results in cell
death, which could occur either by apoptosis or necrosis
in skin cells [7,11,12].
The current approach to protection against solar UV-
induced oxidative damage to human skin relies heavily
on either avoidance of excessive sunlight or the use of
sunscreens, but dietary sources of antioxidants have the
potential to complement these strategies. Plant polyphe-
nols, especially the flavonoids, constitute an important
dietary source of antioxidants (reviewed in [13,14]). Thecatechin/flavanol families of flavonoids are major con-
stituents of green tea, wines, apples, and chocolate. Re-
cent studies that determined the flavanol content in foods
and beverages commonly consumed in the Netherlands
showed that epicatechin (EC) was the abundant flavanol
in a wide variety of fruits, vegetables, and beverages
[15,16]. After dietary intake, flavanols can undergo con-
jugation and metabolism in the small intestine and liver
involving glucuronidation, methylation, and sulphation
([17], reviewed in [18]). They also undergo colonic me-
Address correspondence to: Professor R. M. Tyrrell, Department ofPharmacy and Pharmacology, University of Bath, Claverton Down,Bath BA2 7AY, UK; Tel: 44 (1225) 386793; Fax: 44 (1225)383408; E-Mail: [email protected].
Free Radical Biology & Medicine, Vol. 35, No. 8, pp. 910921, 2003Copyright 2003 Elsevier Inc.
Printed in the USA. All rights reserved0891-5849/03/$see front matter
doi:10.1016/S0891-5849(03)00436-2
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tabolism [19]. Increased plasma concentrations of EC as
well as sulphate, glucuronide, and sulphoglucuronide
conjugates of EC and its methylated metabolite have
been observed in human volunteers after dietary intake
[20 22]. These metabolites may contribute to the pro-
tective effects of dietary flavonoids. Although it is not
known yet whether EC and its metabolite 3'-O-methylepicatechin (MeOEC) accumulate in human skin,
radioactivity derived from orally administered
[3H]()epigallocatechin gallate has been detected in the
skin of male and female mice [23], a finding which
supports the possibility that EC and MeOEC could ac-
cumulate in human skin.
Epigallocatechin gallate, the most abundant flavanol
in green tea, has been studied extensively in vivo in both
rodents and humans, and has been found to have protec-
tive effects against UVB-induced inflammatory re-
sponses, immune-suppression, intracellular generation of
hydrogen peroxide, and skin carcinogenesis ([24,25],
reviewed in [26,27]). Such studies have not employed
EC. However, in in vitro studies, pretreatment with EC
was found to protect cultured human skin fibroblasts,
primary murine cortical neurons, and a hepatocyte cell
line against oxidative damage induced by hydrogen per-
oxide [28 30], as well as primary striatal neurons against
oxidative stress induced by oxidized LDL [31]. Interest-
ingly, the in vivo metabolite, MeOEC, was shown to be
equally protective against oxidative stress in fibroblasts
and neurons, suggesting that the mechanism does not
involve conventional antioxidant activity [29,31]. In con-
trast, the glucuronidated compounds elicited no cytopro-
tective effect.
In the current study the ability of EC and one of its
major in vivo metabolites, MeOEC, to protect against
UVA-induced oxidative damage to cultured human skin
fibroblasts was examined. Pretreatment with either of
these compounds protects these cells against UVA-in-
duced cell damage and cell death. This increased resis-
tance to UVA-induced damage requires protein synthesis
and seems to be largely independent of the antioxidant
properties of EC.
MATERIALS AND METHODS
Materials
Routine tissue culture reagents were purchased from
Gibco Invitrogen Ltd. (Paisley, UK). Fetal calf serum
Gold was purchased from PAA Laboratories (Somerset,
UK). Fibroblast growth medium and Minimum Essential
Medium with Earls salts (without L-leucine and L-
glutamine) were purchased from PromoCell (Heidelberg,
Germany). LightCycler DNA Master SYBR Green I and
Annexin-V-Fluos were purchased from Roche Molecular
Biochemicals (Lewes, UK). TRIZOL reagent was pur-
chased from Invitrogen Ltd. (Paisley, UK). MTT, neutral
red, propidium iodide, ()- epicatechin and puromycin
were purchased from Sigma (Poole, UK). Epicatechin
was further purified by preparative HPLC. L-[4, 5-3H]
Leucine (specific activity 120 190 Ci/mmol) was pur-
chased from Amersham (Buckinghamshire, UK). 3'-O-methyl epicatechin was synthesized as described previ-
ously [29].
Cell culture
Normal human skin fibroblasts FEK4 [32] were
cultured routinely at 37C in 5 % CO2
in Minimum
Essential medium with Earles salts (EMEM) supple-
mented with 2 mM L-glutamine, 50 u/ml penicillin, 50
g/ml streptomycin, 0.2% sodium bicarbonate, and
15% FCS as described previously [33]. For all exper-
iments, cells were used between passages 11 and 15.
Cell treatments were carried out at 37C in a humid-ified CO
2incubator.
Treatment of cells with flavanols and UVA irradiation
Stock solutions of the flavanols were prepared in 50%
methanol and stored at 80C until use. Aliquots were
thawed only once for use in order to avoid degradation of
the compound. FEK4 cells were seeded in 3 cm dishes (5
104 cells/dish) in complete EMEM and approximately
30 h later the medium was replaced with Fibroblast
Growth medium (FGM) containing 5 g/ml insulin, 1
ng/ml basic fibroblast growth factor, 50 ng/ml ampho-
tericin B, and 50 g/ml gentamicin. The cell monolayers
were incubated in this medium (2.4 ml/dish) for 24 h
before addition of either EC or MeOEC. For treatment,
the volume of the conditioned medium was reduced to 1
ml and the excess medium was placed at 37C. The
compounds were added to the required final concentra-
tion such that the vehicle never exceeded 0.1% in the
medium in order to avoid cellular effects of the vehicle
itself. The highest concentration of EC and MeOEC that
could be used was dictated by the solubility of the
compounds in the vehicle. Control cells were pretreated
cells with the vehicle alone. Cells were incubated withthe compound for approximately 18 h and then irradiated
with 500 kJm2 UVA (unless specified) in Ca2/Mg2
PBS (1 ml/dish containing 0.5 g/ml each of CaCl2 and
MgCl2
) using a broad spectrum UVA lamp (350 450
nm, Sellas, Germany) as described previously [34]. After
irradiation the conditioned medium was added back (1
ml/dish) and the monolayers were incubated further for
either 30 min (for MTT and Neutral red assays) or 18 h
(for flow cytometry) and then analyzed. The compounds
were not included during irradiation and postirradiation.
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MTT assay
The MTT assay [35,36] is a commonly used sensitive,
quantitative, and reliable assay for measuring viability of
cells. This assay is often considered as an indicator of
mitochondrial function, but it has been established [37]
that most of the MTT reducing activity is present in the
endoplasmic reticulum that separates as the microsomal
fraction after cell fractionation.
After the postirradiation incubation of 30 min, cell
monolayers were washed twice with warm PBS and 500 l
of serum-free-EMEM containing 0.5 mg/ml MTT was
added to each dish. Cells were incubated with the substrate
for 2.5 h at 37C in a CO2
incubator. The substrate-con-
taining medium was removed at the end of the incubation
and 500 l of DMSO was added per dish to dissolve the
formazan crystals. The dishes were agitated on a rocking
platform for at least 10 min at ambient temperature, after
which the absorbance of 100 l aliquots was measured
against DMSO at 550 nm in a microplate reader (MR5000Dynatech Laboratories, West Sussex, UK) and considered
as the MTT reduction activity. The absorbance of the irra-
diated samples was expressed as a percent of the corre-
sponding vehicle/compound-treated shams and plotted as
percent activity in graphs. This activity was also used for
estimation of fold increase in cytoprotection with com-
pound treatment by assigning the activity in vehicle-treated
samples a value of 1.
Neutral red assay
NR is a water soluble, weakly basic dye that passes
across the plasma membrane passively and accumulatesin the lysosomes of live cells [38,39]. In damaged cells
the dye is no longer retained by the lysosomes and is lost
from the cells, as the plasma membrane does not act as a
barrier. As lysosomal membranes are also damaged in
UVA-irradiated cells, the NR assay was chosen as an
alternative assay for measuring damage in irradiated
cells.
After the 30 min postirradiation incubation, the con-
ditioned medium in each dish was replaced with 500 l
of neutral red medium (50 g/ml neutral red dye in fresh
EMEM) and cells were placed for 1.5 h in a CO2
incu-
bator at 37C. The neutral red medium was prepared onthe day of the experiment, incubated at 37C for 30 min,
and centrifuged at 3000 rpm for 10 min to clear any
precipitate. This medium was then filter sterilized and
added to the cells. After incubation, the neutral red
medium was removed and cell monolayers were fixed for
1 min with 500 l of fixing solution (10% CaCl2, 0.4%
formaldehyde). The dye was extracted from the cells by
addition of 500 l of extraction solution (19% acetic
acid, 50% ethanol) and 10 min agitation on a rocking
platform at ambient temperature. The absorbance of 200
l aliquots was measured at 550 nm in a microplate
reader (MR5000 Dynatech Laboratories). The absor-
bance in the irradiated samples was expressed as a per-
cent of the corresponding vehicle/compound-treated
shams and the value for percent retention was considered
as an indicator of cell viability, as the dye is taken up
actively into the lysosomes of live cells. The fold in-crease in cytoprotection was calculated as described for
the MTT assay.
Flow cytometry
This technique allows quantification of live, apopto-
tic, and necrotic cells on a single-cell basis in cell pop-
ulations. Distinction between necrotic and apoptotic cell
death can be achieved reliably by combining a specific
marker of apoptosis with a DNA stain. Fluorescein-
conjugated Annexin V (AV) and the DNA stain pro-
pidium iodide (PI) were employed as markers of apopto-
sis and necrosis. AV, a Ca2
-dependent phospholipidbinding protein with high affinity for phosphatidyl serine
(PS), stains specifically externalized PS in the plasma
membrane of apoptotic cells (reviewed in [40,41]). The
DNA stain PI enters cells only when the plasma mem-
brane is damaged.
FEK4 cells were incubated for 18 h after irradiation
and the medium was collected to harvest detached cells.
Cell monolayers were trypsinized with 0.25% trypsin/0.6
mM EDTA (0.5 ml/dish). After inactivation of trypsin
with an equal volume of complete EMEM, the cell
suspensions from two dishes were pooled with the cor-
responding reserved medium to constitute a sample.Cells were stained with Annexin-V-Fluos (AV) and pro-
pidium iodide (PI) as described in the manufacturers
protocol (Roche Molecular Biochemicals) with slight
modification as follows. Cell pellets were washed twice
with incubation buffer, resuspended in 100 l of the
same buffer, and 2 l of AV was added. Staining was
carried out in the dark at ambient temperature for 15 min,
after which 400 l of incubation buffer containing 5
g/ml propidium iodide was added. Flow cytometry was
performed in a Becton Dickinson FACS Vantage
(Cellquest version 3.3 software, Belgium) calibrated
with Fluoresbrite beads and set up for electronic com-pensation of the emission spectra. Samples were ana-
lyzed using 488 nm excitation and detection in FL1
channel for AV (520 nm bandpass filter for fluorescein)
and FL3 channel ( 620 nm longpass filter) for PI. Live
(AV/PI), apoptotic (AV/PI), and necrotic (total
PI) cell populations were determined in a total of
10,000 cells and expressed as percent. The apoptotic and
necrotic cells in sham samples were subtracted from the
corresponding populations in irradiated samples in order
to exclude the low levels of cell death unrelated to UVA.
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RNA extraction
Cell monolayers were lysed in the culture dishes (1 ml
TRIZOL reagent/dish) at 4 h postirradiation and lysates
from 3 dishes were pooled to constitute a sample. Gly-
cogen was added to the lysates at a final concentration of
50 g/ml in order to increase RNA yields. Total cellular
RNA was extracted according to the manufacturers pro-
tocol (Invitrogen Life Technologies).
Real time RT-PCR. A two-step RT-PCR approach was
used to analyze HO-1 mRNA accumulation.
cDNA synthesis. Total RNA (1 g/sample) was reverse
transcribed using SuperScript first-strand synthesis kit
(Invitrogen Life Technologies) according to the manu-
facturers protocol. Random hexamers were used for
priming the reaction to obtain a better representation of
the RNA population in the cDNA samples. A 1:10 dilu-
tion of each cDNA was freshly prepared in PCR-gradewater before each run and 2 l was used in each PCR
reaction.
Primers. The Heme oxygenase-1 (HO-1) primer pair
[42] 5'-AAG AGG CCA AGA CTG CGT TC-3' (for-
ward) and 5'-GGT GTC ATG GGT CAG CAG C-3'
(reverse) gave an amplicon size of 74 bp in the human
HO-1 cDNA sequence. Glyceraldehyde-3-phosphate de-
hydrogenase (GAPDH) was used as a reference gene and
the primer pair [43] 5'-GAC ATC AAG AAG GTG GTG
AA-3' (forward) and 5'-TGT CAT ACC AGG AAA
TGA GC-3' (reverse) had an amplicon size of 178 bp inthe human GAPDH cDNA. Desalted primers were ob-
tained from Life Technologies, Paisley, UK and used for
PCR reactions without further purification.
Real time PCR. Real time PCR reactions were carried out
in the LightCycler (Roche Molecular Biochemicals) us-
ing the fluorescent dye SYBR Green I. The optimal
MgCl2
concentration determined for both primer sets
was 3 mM and each primer pair was used at a final
concentration of 0.5 M. The HO-1 and GAPDH mR-
NAs were analyzed in separate runs as amplification
conditions optimized for the two primer sets wereslightly different due to the amplicon sizes. PCR reac-
tions were carried out in a volume of 20 l and the
experimental protocol consisted of predenaturation, am-
plification, melting curve analysis, and cooling programs
(LightCycler 3 Run version 5.32) with the following
parameters for HO-1: the predenaturation step was a
single cycle of 30 s at 95C. The amplification step
consisted of 40 cycles of 0 s at 95C, 5 s at 55C, and 4 s
at 72C, with fluorescence measurement at the end of each
cycle. The melting curve analysis comprised of 1 cycle of
0 s at 95C, 15 s at 65C, followed by a gradual increase to
95C (transition rate of 0.1C) with continuous fluorescence
acquisition. The default parameters of the cooling program
were used, and unless specified, temperature transition rate
of 20C/s was used in all the programs. The GAPDH
experimental protocol was essentially the same except for
the difference in the elongation time in the amplificationcycle, which was 8 s at 72C.
The standard curve for quantification of PCR samples
was generated as follows. FEK4 cells were seeded in 10
cm dishes (6 105 /dish) in complete EMEM and ap-
proximately 72 h later these were irradiated with 250
kJm2 UVA. After irradiation the conditioned medium
was added back to the culture dishes and these were
incubated further for variable times up to 8 h in a 37 C
CO2
incubator. Real time PCR analysis of this time
course of HO-1 mRNA accumulation showed that max-
imum levels were reached between 3 and 5 h postirradi-
ation. Total RNA from 3, 4, and 5 h time point sampleswere reverse transcribed (4 g/sample) as mentioned
above, and the cDNA samples were pooled to use as a
standard. Serial dilutions in the range 1:10 to 1:3000 of
this cDNA pool with arbitrary concentration values of 70
ng 0.23 g were used to generate separate standard
curves for HO-1 and GAPDH. Each standard curve con-
sisted of 6 dilution steps with a range of 3 orders of
magnitude in the dilution series. Triplicates of each di-
lution were used for the standard curve runs and the data
from these runs were used to create a coefficient file in
the Relative Quantification software (Roche Molecular
Biochemicals).A large batch of cDNA with a relatively high HO-1
signal was diluted 1:10 and used as calibrator for all
experiments. Each sample run consisted of one set of
experimental cDNAs and duplicates of the calibrator.
The data file of each run was exported and analyzed in
the Relative Quantification software using the dual mode
with efficiency correction. The normalized HO-1 mRNA
values in the irradiated samples were expressed as fold
increase of the corresponding controls.
Absorption spectra
EC was diluted to a final concentration of 30 M in
Ca2/Mg2-PBS and irradiated at ambient temperature
with 500 kJm2 UVA in plastic disposable cuvettes with
stoppers. The sham samples were left at room tempera-
ture for the time of irradiation. The absorption spectrum
was measured immediately between 200 and 350 nm at
50 nm/min scan speed in a Kontron Instruments spectro-
photometer (Uvikon 922, Milan, Italy). Samples were
scanned in quartz cuvettes against Ca2/Mg2-PBS con-
taining 0.06% methanol (vehicle).
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Protein synthesis
FEK4 cells were seeded in 3 cm dishes and prepared
for inhibitor treatment as described in an earlier section
for EC treatment. Cells were treated in 1 ml with either
50 g/ml puromycin or with 0.06% methanol (vehicle)
for 3 h at 37C, after which the medium was aspirated,
cell monolayers were washed once with leucine-freemedium (EMEM without L-leucine, but supplemented
with L-glutamine, penicillin and streptomycin, and 0.5%
FCS) and incubated in 1 ml of leucine-free medium for
1 h at 37C to deplete cells of leucine. After this deple-
tion step, the medium was replaced with 500 l of
labeling medium (20 Ci of 3H-Leucine/ml of leucine-
free medium) and incubated further for 2 h at 37C.
Either the vehicle or the antibiotic was included during
the depletion and the labeling steps. Thus the total incu-
bation time with the antibiotic was 6 h. After labeling,
the medium was removed and cell monolayers were
washed once with ice-cold PBS and lysed in 300 l oflysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 10
mM EDTA, 1% Triton X-100, 1% SDS). Lysates (100
l/sample) were spotted on Whatman filter paper and air
dried completely before TCA precipitation. Filters were
incubated in 10% TCA for 10 min at 4C and then boiled
in 5% TCA for 10 min. After this, they were incubated in
5% TCA at ambient temperature for 10 min, transferred
to absolute ethanol at ambient temperature for 2 min and
air dried at 37C for 1 h. TCA precipitable counts were
measured in a liquid scintillation counter (Rack Beta
1209, LKB Wallace, Turku, Finland). The radioactivity
incorporated into control samples was considered as100% and that in antibiotic-treated samples was ex-
pressed as a percent of the controls.
Statistical analysis
Data were expressed as mean SD. Comparison of
means of two groups of data was made using the un-
paired, two-tailed Students t-test. Means of more than
two groups of data in a graph were compared using
one-way analysis of variance (ANOVA) with Tukeys
honestly significant difference (HSD) post hoc test in
SPSS 10 for Windows software. Graphs of EC and
MeOEC treatment were compared using univariate anal-ysis of variance with Tukeys HSD post hoc test. Statis-
tical significance was determined at p .05.
RESULTS
Photostability of EC and MeOEC
Control experiments on the effects of UVA were
undertaken. In vitro irradiation of 30 M EC or MeOEC
solutions in Ca2/Mg2-PBS with 500 kJm2 UVA did
not change the absorption spectrum or the HPLC analyt-
ical profile indicating that the compounds are photostable
(data not shown).
Attenuation of UVA-induced cell damage
EC and MeOEC were initially assessed for their effect
on cellular damage in irradiated cells. Irradiation of
FEK4 cells with 500 kJm2
UVA decreased the MTTactivity to approximately 40% that of control cells (Fig.
1A). Pretreatment with a range of concentrations (150
M) of either EC or MeOEC attenuated this loss in
irradiated cells. The protective effect of EC was already
observed at 1 M and reached a plateau at 10 M,
whereas pretreatment with MeOEC resulted in a concen-
tration-dependent increase in protection reaching steady
state at 30 M. Although both compounds increased the
cellular resistance to UVA, the effect of EC was more
pronounced than that of MeOEC at 1 and 10 M. Overall
comparison of the two curves using univariate ANOVA
revealed that the increase in cellular resistance to UVAconferred by EC treatment was significantly higher than
by its metabolite MeOEC. Treatment with EC or
MeOEC for 18 h had no effect on the MTT activity in
sham irradiated cells, indicating that the compounds did
not induce this enzyme activity (data not shown). The
increased resistance to UVA observed in irradiated cells
suggested cytoprotection by the compounds.
The magnitude of cytoprotection is quantified as a
fold increase over control MTT values in Fig. 1B (1.27
0.12 and 1.84 0.18 fold with 1 and 30 M EC,
respectively). In order to confirm that this reflected an
increase in viable cells, the neutral red (NR) assay wasalso used and the fold increase in cytoprotection result-
ing from EC treatment was similar to that obtained with
the MTT assay (Fig. 1B). Treatment with MeOEC did
not result in cytoprotection at 1 M in either assay (Fig.
1C), whereas at 30 M a significant increase in resis-
tance by 1.6 0.08 and 1.91 0.41 fold was observed
in the MTT and NR assays, respectively.
The results in Fig. 1 show that both EC and MeOEC
attenuate UVA-induced oxidative damage to cultured
human skin fibroblasts.
Cell death in UVA-irradiatedfibroblasts
UVA-induced apoptotic cell death has been observed
in superficial dermal fibroblasts of human skin recon-
structed in vitro [44]. Although it is generally observed
that most cell death is necrotic in UVA-irradiated FEK4
cells [7], both types of cell death were monitored in this
study using AV and PI staining. The cell population was
visualized as a dot plot (Fig. 2A). Live cells are not
stained for either PI or AV (lower left quadrant of Fig.
2A) because the plasma membrane is intact and PS is
completely absent from the outer leaflet. Apoptotic cells
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are AV but PI (lower right quadrant ofFig. 2A), as the
asymmetry of the lipid bilayer of the plasma membrane
is disturbed during the early stages of apoptosis and PS
Fig. 1. Attenuation of UVA-induced oxidative damage. (A) FEK4 cellswere treated with various concentrations of EC or MeOEC and irradi-
ated with UVA. The MTT activity in irradiated samples was expressedas a percent of the corresponding sham and plotted against concentra-tion of compound. Data are means of 6 samples from two independent
experiments. EC and MeOEC graphs were compared using UnivariateAnalysis of Variance with Tukeys HSD post hoc test and statisticalsignificance (*) was determined at p .05. (B,C) The percent MTTactivity obtained in control cells irradiated with UVA was consideredas 1 and the activity in 1 and 30 M treated samples was expressed as
a fold increase over controls. For the NR assay, the dye retention inirradiated samples was expressed as a percent of the correspondingsham. The fold increase in cytoprotection was calculated as for the
Fig. 2. Kinetics of UVA-induced cell death. FEK4 cells were irradiated
with UVA and analyzed for cell death by flow cytometry at varioustimes postirradiation. (A) Dot plot showing typical categorization of
live, apoptotic, and necrotic cell populations in irradiated samples. (B)The percent apoptotic and necrotic population was determined in10,000 cells, corrected for background cell death and plotted against
time. Data are means of 4 samples from independent experiments. Thetwo types of cell populations at the various time points were compared
to that obtained at 0 h using one-way ANOVA with Tukey s HSD posthoc test and statistical significance (*) was determined at p .05.
MTT assay. Data are means of 5 samples from two independentexperiments. The fold increase in cytoprotection in 1 and 30 M
treated samples was compared to the controls (0 M) using one-wayANOVA with Tukeys HSD post hoc test and statistical significance(*) was determined at p .05.
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accumulates in the outer leaflet, whereas the plasma
membrane remains intact. Necrotic cells are AV and
PI, as in the late stages of apoptosis in vitro the plasma
membrane becomes permeable to vital dyes. Usually this
population is localized in the upper right quadrant of a
dot plot. However, extensive membrane damage caused
by UVA rapidly renders the plasma membrane perme-
able to vital dyes, resulting in a population of cells
distributed in both the upper two quadrants (Fig. 2A).
Therefore the total PI cells in the upper two quadrants
were considered as necrotic. In sham-irradiated samples,
the cell population consisted of approximately 90% live,
8% necrotic, and 2% apoptotic cells (data not shown),
indicating that incubation in PBS and the staining pro-
cedure did not cause extensive cell death. The back-
ground of necrotic or apoptotic cell death was subtracted
from the corresponding irradiated samples in all experi-
ments. Because the value subtracted amounts to approx-
imately 10%, the total of cells scored in irradiated sam-
ples never exceeds 90%.
Cell death was quantified in FEK4 cells at differenttime points after irradiation with 500 kJm2 UVA. Im-
mediately after irradiation, 26 2% of the cells are
identified as necrotic (Fig. 2B). This population in-
creased significantly to a maximum of 49 4% at 6 h
post irradiation followed by a small decline to 40% at
24 h. The 5% of the population identified as apoptotic did
not change significantly up to 24 h after irradiation.
Thus, cell death induced in FEK4 cells by UVA irradi-
ation was mainly necrotic. Because the level of cell death
was more or less constant between 6 and 24 h, the 18 h
time point was chosen to study the effect of EC and
MeOEC on UVA-induced cell death.
Modulation of UVA-induced cell death by EC and
MeOEC
The effect of pretreatment with 1 and 30 M EC or
MeOEC on levels of live, necrotic, and apoptotic cells
was examined in irradiated samples. Irradiation with 500kJm2 UVA resulted in death of approximately two-
thirds of the cell population (Fig. 3A and B). Treatment
with 1 and 30 M EC increased the live cell population
from 28 3% to 41 3 and 71 0.5%, respectively
(Fig. 3A). A corresponding decrease from 63 3% to 47
4 and 19 4% was observed in the necrotic cell
population. These changes were significant for both the
live and the necrotic cell populations. No effect of
MeOEC was observed at 1 M (Fig. 3B), but 30 M led
to significant protection against cell death of an order
similar to that observed for EC. The low level of the
apoptotic cell population was unaffected in both cases.Thus pretreatment with the higher concentration of EC or
MeOEC increased the cellular resistance to UVA by
2.52.8-fold.
Kinetics of EC-mediated cytoprotection
The correlation between time of treatment and appear-
ance of resistance to UVA was investigated. FEK4 cells
were treated with 30 M EC for 1, 3, and 6 h, irradiated
with 500 kJm2 of UVA radiation and then analyzed for
cell death at 18 h postirradiation. The live cell population
Fig. 3. Modulation of UVA-induced cell death. FEK4 cells were treated with EC (A) or MeOEC (B), irradiated with UVA and analyzed
by flow cytometry at 18 h postirradiation. The percentages of apoptotic and necrotic cell populations were corrected for background
cell death. Data are means of 3 samples from two independent experiments. Each type of cell population at 1 and 30 M was comparedto its corresponding control (0 M) using one-way ANOVA with Tukeys HSD post hoc test and statistical signi ficance (*) wasdetermined at p .05.
916 S. BASU-MODAK et al.
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increased from 15 5% to 26 11%, 37 5%
(2.5-fold) and 48 9% (3.3-fold) with 1, 3, and 6 h
pretreatments, respectively (Fig. 4). This was accompa-
nied by a corresponding decrease in the necrotic popu-
lation (data not shown). The live and necrotic cell pop-
ulations remained unchanged with variable time of
vehicle treatment in control samples. These results are
consistent with the idea that EC was inducing a protec-
tive response in irradiated cells.
Requirement of protein synthesis for the EC response
The question as to whether this protective response
was dependent on de novo protein synthesis was inves-
tigated. Cells were pretreated with 30 M EC for 6 h,followed by irradiation with 500 kJm2 of UVA and
then flow cytometric analysis was undertaken at 18 h
postirradiation. Puromycin, an inhibitor of protein syn-
thesis, was tested on FEK4 cells. Incubation with a range
of concentrations of puromycin (0 50 g/ml) for 6 h
before irradiation had no significant effect on the total
dead cell population in irradiated samples (data not
shown). The highest concentration of puromycin (50
g/ml) decreased protein synthesis to 6 7% in unir-
radiated cells as revealed by 3H-leucine incorporation in
total proteins (data not shown). Incubation of cell mono-
layers with this concentration of puromycin for 6 h hadno effect on either live or total dead cell populations in
irradiated samples (Fig. 5A). Pretreatment with 30 M
EC increased the resistance to UVA by 1.7-fold and an
absence of protein synthesis during the EC-treatment had
no effect on this increase. Protein synthesis was mea-
sured in cells 18 h after removal of the inhibitor, which
corresponded to the time point of flow cytometric anal-
ysis. 3H-leucine incorporation was 49 6% (data not
shown), indicating a partial reversal of protein synthesis
inhibition. Therefore, in a further set of experiments, cell
monolayers were treated with EC for 6 h and puromycin
was added only after UVA irradiation. Presence of the
Fig. 4. Kinetics of the EC effect. Cells were treated with 30 M EC for
various times, irradiated with UVA and analyzed for cell death at 18 h
postirradiation by flow cytometry. Control cells were treated with theappropriate concentration of vehicle. Data are means of 3 samples fromthree independent experiments. The level of the live cell population atthe various time points was compared to that at 0 h using one-way
ANOVA with Tukeys HSD post hoc test and statistical significance(*) was determined at p .05.
Fig. 5. Effect of inhibition of protein synthesis on EC-mediated cytoprotection. (A) Cells were treated with 30 M EC and 50 g/mlpuromycin for 6 h, irradiated with UVA and then incubated for a further 18 h without compound or inhibitor. Flow cytometry was
performed and data plotted as means of 6 samples from three independent experiments. (B) Cells were treated with 30 M EC for 6 hand irradiated with UVA. After irradiation 50 g/ml puromycin was added back to the appropriate samples for the 18 h postirradiation
incubation. Samples were analyzed by flow cytometry and data expressed as means of 4 samples from two independent experiments.Each type of treatment in the two cell populations was compared to its corresponding control using one-way ANOVA with Tukey sHSD post hoc test and statistical significance (*) was determined at p .05.
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protein synthesis inhibitor during the postirradiation in-
cubation before flow cytometry abolished the protective
effect of EC (Fig. 5B). Taken together, these results
indicate that the cytoprotective effect of EC was depen-
dent on protein synthesis.
UVA-induced heme oxygenase-1 mRNA accumulation
in EC-treatedfibroblasts
Heme oxygenase-1 (HO-1), the rate-limiting enzyme
in heme catabolism, is a well-known example of an
oxidant-inducible gene [45]. UVA radiation activates
this gene via singlet oxygen generation [46] and cellular
levels of HO-1 mRNA are used widely as a measure of
cellular oxidative stress status. Pretreatment of cells with
EC for 18 h before irradiation had no effect on the
UVA-induction of HO-1 mRNA up to 250 kJm2 (Fig.
6), indicating that singlet oxygen scavenging was an
unlikely mechanism of protection. At higher doses there
is a decline in HO-1 mRNA levels due to suppression of
transcription [46]. EC treatment attenuated this decline
significantly (Fig. 6), presumably because it prevented
the suppression of transcription by high doses of UVA.
DISCUSSION
These findings report protective effects of EC and one
of its in vivo metabolites, MeOEC, against UVA-in-
duced oxidative damage. The results clearly demonstrate
that pretreatment of cultured human skin fibroblasts with
either compound induces resistance to UVA-induced cell
damage and cell death. The time dependence for devel-
opment of the protective response and the requirement
for protein synthesis, together with the observation of a
similar protection using both EC and its methylatedmetabolite, support the notion that this is an adaptive
response largely independent of the hydrogen-donating
antioxidant properties of EC.
The flow cytometry measurements undertaken in this
study confirm that UVA radiation causes primarily ne-
crotic cell death in human skin fibroblasts and that apo-
ptosis is only a minor pathway of cell death. It has been
proposed that damage generated by singlet oxygen in
UVA-irradiated cells causes them to undergo apoptosis
rapidly so that they are already in the secondary necrosis
stage during irradiation [47]. Oxidative damage results in
depletion of glutathione and ATP as well as extensive
peroxidation of lipids, and these changes would promote
onset of mitochondrial permeability transition, a com-
mon event in both types of cell death [48]. However,
such depletion favors mitochondrial permeability transi-
tion towards necrotic rather than apoptotic cell death
(reviewed in [49,50]). Furthermore an increased pro-
oxidant state of cells would favor inactivation of
caspases (reviewed in [50]). These factors are all consis-
tent with the predominantly necrotic cell death actually
observed in UVA-irradiated fibroblasts.
Both EC and its methylated metabolite clearly protect
against cell damage induced by UVA radiation as judged
by simple cell damage assays (MTT and NR) in cultured
human skin fibroblasts. This is broadly in agreement with
results reporting hydrogen peroxide-induced oxidative
stress in the same cell type [29] as well as murine cortical
neurons [28]. Both compounds also protect against
UVA-induced necrotic cell death in skin fibroblasts,
whereas in the same cell type and in primary striatal
neurons, they have been shown to protect against apo-
ptotic cell death induced by hydrogen peroxide [28,29].
This would indicate that the mechanism of protection
involves an early step common to both cell death path-
ways, such as prevention of initial damage.
The protection against UVA-induced cell death couldrelate to the antioxidant properties of the compounds.
However, this is inconsistent with the similar effects seen
with both EC and its methylated derivative, because the
latter shows much lower antioxidant activity [29]. Fur-
thermore, the time dependence of the development of
EC-mediated protection is indicative of an induction
mechanism that eventually leads to the manifestation of
an adaptive response. Consistent with this, de novo pro-
tein synthesis is required for the development of the
protection. Interestingly, UVA irradiation of skin fibro-
Fig. 6. Modulation of UVA-induced HO-1 mRNA accumulation by
EC. FEK4 cells were treated with 30 M EC and irradiated with 50,100, 250, and 500 kJm2 UVA. Total cellular RNA was isolated at 4 hpostirradiation and HO-1 mRNA was quantified using two-step RT-PCR. GAPDH mRNA levels were determined in all samples and used
for normalizing HO-1 mRNA levels. Data were expressed as a foldincrease over corresponding shams and means of 3 samples from threeindependent experiments were plotted against UVA dose. The means of
the two groups at 500 kJm2 was compared using the unpaired,two-tailed Students t-test and statistical significance (*) was deter-mined at p .05.
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blasts in culture leads to an adaptive protection against
subsequent oxidative membrane damage and this re-
sponse is mediated through HO-1 [51]. High levels of
HO-1 gene expression have since been observed in sev-
eral pathological and inflammatory conditions (reviewed
in [52]) and in vitro and in vivo studies clearly show that
this enzyme mediates a protective response against celland tissue injury [53]. The significantly higher levels of
HO-1 mRNA observed at a UVA dose of 500 kJm2 in
EC-treated cells compared to the corresponding control
are consistent with the possibility that this gene may be
involved in the cytoprotection, but to examine this would
require further testing in a HO-1 deficient model. The
gene targeted HO-1 mouse model [54] could prove use-
ful for such studies.
In addition to the similarity of the protective effects of
EC and MeOEC, two other sets of experiments indicate
that protection is not due to antioxidant properties of the
compound. Firstly, cellular uptake of EC and MeOECoccurs within 2 h of treatment in FEK4 cells and the
uptake does not change with 18 h treatment [28]. A direct
antioxidant effect would be expected to result in a pro-
tective response that remained unchanged between 2 and
18 h EC treatment, and this was not the case in these
experiments. Secondly, the dose-dependent accumula-
tion of HO-1 mRNA, a widely used marker of oxidative
stress [34], was used to test the involvement of an anti-
oxidant mechanism. Because the dose-dependent accu-
mulation of HO-1 mRNA was not suppressed in EC
pretreated cells up to an intermediate dose of 250 kJm2,
this compound seems not to be acting by the preventionof the generation of active oxygen species or their inter-
action with critical targets. However, it should be noted
that in an earlier study from this laboratory, epigallocat-
echin, another green tea flavanol, was shown to decrease
significantly the UVA induction of HO-1 at 250 and 400
kJm2 in FEK4 cells [55]. Certain structurally related
differences between the compounds, such as the superior
ability of epigallocatechin to chelate iron (CRE, unpub-
lished studies), could influence the response of this early
marker of oxidative stress to UVA.
Conclusions
This study reveals a protective role of EC, an abun-
dant dietary flavanol, and one of its major in vivo me-
tabolites (MeOEC) against UVA-induced damage and
cell death in cultured human skin fibroblasts. The pro-
tection involves an adaptive response dependent on pro-
tein synthesis and is not mediated by photo-degradation
products. Given the potential of this flavanol as an agent
for enhancing the protection of human skin against acute
UVA damage, it would be of value to probe further into
the mechanism of EC-induced resistance.
Acknowledgements This research was supported by a EuropeanFifth Framework RTD programme Grant (Grant no. QLK4-1999-
01590) in which C.R.-E. and R.M.T. are contracting partners.
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ABBREVIATIONS
ANOVAAnalysis of varianceAVAnnexin-V-Fluos
DMSODimethyl sulfoxide
ECEpicatechin
EDTAEthylenediaminetetracetic acid
EMEMMinimum essential medium with Earles salts
FCSFetal calf serum
FGMFibroblast growth medium
GAPDHGlyceraldehyde phosphate dehydrogenase
HO-1Heme oxygenase-1
HSDHonestly significant difference
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LDLLow-density lipoprotein
MeOEC3'-O-methyl epicatechin
MTT3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyl tetra-
zolium bromide]
NRNeutral red
PBSPhosphate-buffered saline
PIPropidium iodide
PSPhosphatidyl serine
RT-PCRReverse transcriptase-polymerase chain reac-
tion
TCATrichloroacetic acid
UVAUltraviolet A (320 380 nm) radiation
921Epicatechin protects against UVA damage