Epic Ate Chin and Its Methyl at Ed Metabolite Attenuate UVA-Induced Oxidative Damage to Human Skin Fib Rob Lasts

8/4/2019 Epic Ate Chin and Its Methyl at Ed Metabolite Attenuate UVA-Induced Oxidative Damage to Human Skin Fib Rob Lasts

1/12

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

910


2/12

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.

911Epicatechin protects against UVA damage


3/12

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.

912 S. BASU-MODAK et al.


4/12

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).



5/12

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



6/12

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.



7/12

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.



8/12

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.



9/12

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.



10/12

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.

REFERENCES

[1] Tyrrell, R. M. Ultraviolet radiation and free radical damage to

skin. Biochem. Soc. Symp. 61:4753; 1995.[2] Soter, N. A. Acute effects of ultraviolet radiation on the skin.Semin. Dermatol. 9:1115; 1990.

[3] Murphy, G. M The acute effects of ultraviolet radiation on the

skin. In: Hawk, J. L. M, ed. Photodermatology. London: Chap-

man and Hall; 1998:4352.[4] Wlaschek, M.; Tantcheva-Poor, I.; Naderi, L.; Ma, W.; Schneider,

L. A.; Razi-Wolf, Z.; Schuller, J.; Scharffetter-Kochanek, K.

Solar UV irradiation and dermal photoaging. J. Photochem. Pho-

tobiol. B 63:4151; 2001.[5] Tyrrell, R. M. UVA (320380 nm) radiation as an oxidative

stress. In: Sies, H, ed. Oxidative stress: oxidants and antioxidants.

London: Academic Press; 1991:57 83.[6] Tyrrell, R. M. Role of singlet oxygen in biological effects of

ultraviolet A radiation. Methods Enzymol. 319:290 296; 2000.[7] Pourzand, C.; Tyrrell, R. M. Apoptosis, the role of oxidative

stress and the example of solar UV radiation. Photochem. Pho-tobiol. 70:380 390; 1999.[8] Lautier, D.; Lusher, P.; Tyrrell, R. M. Endogenous glutathione

levels modulate both constitutive and UVA radiation/oxidant-

inducible expression of the human heme oxygenase gene. Carci-

nogenesis 13:227232; 1992.[9] Tyrrell, R. M. The molecular and cellular pathology of solar

ultraviolet radiation. Mol. Aspects Med 15:77; 1994.

[10] Basu-Modak, S.; Tyrrell, R. M. Modulation of gene expression by

solar ultraviolet radiation. In: Giacomoni, P. U., ed. Sun protec-

tion in man. St. Louis, MO: Elsevier Science B.V.; 2001:303320.

[11] Young, A. R. The sunburn cell. Photodermatol. 4:127134; 1987.[12] Morita, A.; Krutmann, J. Ultraviolet A radiation-induced apopto-

sis. Methods Enzymol. 319:302309; 2000.[13] Rice-Evans, C.; Miller, N. J.; Paganga, G. Structure-antioxidant

activity relationships of flavonoids and phenolic acids. Free Radic. Biol. Med. 20:933956; 1996.[14] Ross, J. A.; Kasum, C. M. Dietary flavonoids: bioavailability,

metabolic effects and safety. Annu. Rev. Nutr. 22:19 34; 2002.[15] Arts, I. C. W.; van De Putte, B.; Hollman, P. C. H. Catechin

contents of foods commonly consumed in The Netherlands. 1.

Fruits, vegetables, staple foods, and processed foods. J. Agric.

Food Chem. 48:1746 1751; 2000.[16] Arts, I. C. W.; van De Putte, B.; Hollman, P. C. H. Catechin

contents of foods commonly consumed in The Netherlands. 2.

Tea, wine, fruit juices, and chocolate milk. J. Agric. Food Chem.48:17521757; 2000.

[17] Kuhnle, G.; Spencer, J. P. E.; Schroeter, H.; Shenoy, B.; Debnam,

E. S.; Srai, S. K. S.; Rice-Evans, C.; Hahn, U. Epicatechin and

catechin are o-methylated and glucuronidated in the small intes-

tine. Biochem. Biophys. Res. Commun. 277:507512; 2000.[18] Spencer, J. P. E.; Schroeter, H.; Rechner, A. R.; Rice-Evans, C.

Bioavailability of flavan-3-ols and procyanidins: gastrointestinaltract influences and their relevance to bioactive forms in vivo. Antioxid. Redox Signal. 3:10231039; 2001.

[19] Rechner, A. R.; Kuhnle, G.; Bremner, P.; Hubbard, G. P.; Moore,

K. P.; Rice-Evans, C. The metabolic fate of dietary polyphenols in

humans. Free Radic. Biol. Med. 33:220 235; 2002.[20] Hollman, P. C. H.; Tijburg, L. B. M.; Yang, C. S. Bioavailability

of flavonoids from tea. Crit. Rev. Food Sci. Nutr. 37:719 738;1997.

[21] Baba, S.; Osakabe, N.; Yasuda, A.; Natsume, M.; Takizawa, T.;

Nakamura, T.; Terao, J. Bioavailability of ()-epicatechin upon

intake of chocolate and cocoa in human volunteers. Free Radic.

Res. 33:635 641; 2000.[22] Warden, B. A.; Smith, L. S.; Beecher, G. R.; Balentine, D. A.;



11/12

Clevidence, B. A. Catechins are bioavailable in men and women

drinking black tea throughout the day. J. Nutr. 131:17311737;2001.

[23] Suganuma, M.; Okabe, S.; Oniyama, M.; Tada, Y.; Ito, H.; Fujiki,

H. Wide distribution of [3H]()-epigallocatechin gallate, a cancer

preventive tea polyphenol, in mouse tissue. Carcinogenesis 19:

17711776; 1998.[24] Katiyar, S. K.; Matsui, M. S.; Elmets, C. A.; Mukhtar, H. Poly-

phenolic antioxidant (

)-epigallocatechin-3-gallate from greentea reduces UVB-induced inflammatory responses and infiltrationof leukocytes in human skin. Photochem. Photobiol. 69:148 153;1999.

[25] Katiyar, S. K.; Afaq, F.; Perez, A.; Mukhtar, H. Green tea poly-

phenol ()-epigallocatechin-3-gallate treatment of human skin

inhibits ultraviolet radiation-induced oxidative stress. Carcino-

genesis 22:287294; 2001.[26] Katiyar, S. K.; Bergamo, B. M.; Vyalil, P. K.; Elmets, C. A.

Green tea polyphenols: DNA photodamage and photoimmunol-

ogy. J. Photochem. Photobiol. B 65:109 114; 2001.[27] Katiyar, S. K.; Elmets, C. A. Green tea polyphenolic antioxidants

and skin photoprotection. Int. J. Oncol. 18:13071313; 2001.[28] Spencer, J. P. E.; Schroeter, H.; Crossthwaithe, A. J.; Kuhnle, G.;

Williams, R. J.; Rice-Evans, C. Contrasting influences of glucu-ronidation and o-methylation of epicatechin on hydrogen perox-

ide-induced cell death in neurons and fibroblasts. Free Radic. Biol. Med. 31:1139 1146; 2001.

[29] Spencer, J. P. E.; Schroeter, H.; Kuhnle, G.; Srai, S. K. S.; Tyrrell,

R. M.; Hahn, H.; Rice-Evans, C. Epicatechin and its in vivo

metabolite, 3'-O-methyl epicatechin, protect human fibroblastsfrom oxidative-stress-induced cell death involving caspase-3 ac-

tivation. Biochem. J. 354:493500; 2001.[30] Roig, R.; Cascon, E.; Arola, L.; Blade, C.; Salvado, M. J. Pro-

cyanidins protect Fao cells against hydrogen peroxide-induced

oxidative stress. Biochim. Biophys. Acta 1572:2530; 2002.[31] Schroeter, H.; Spencer, J. P. E.; Rice-Evans, C.; Williams, R. J.

Flavonoids protect neurons from oxidized low-density-lipopro-

tein-induced apoptosis involving c-Jun N-terminal kinase (JNK),

c-Jun and caspase-3. Biochem. J. 358:547557; 2001.[32] Tyrrell, R. M.; Pidoux, M. Quantitative differences in host cell

reactivation of ultraviolet-damaged virus in human skin fibro-

blasts and epidermal keratinocytes cultured from the same fore-skin biopsy. Cancer Res. 46:26652669; 1986.

[33] Basu-Modak, S.; Lusher, P.; Tyrrell, R. M. Lipid metabolite

involvement in the activation of the human heme oxygenase-1

gene. Free Radic. Biol. Med. 20:887 897; 1996.[34] Tyrrell, R. M.; Basu-Modak, S. Transient enhancement of heme

oxygenase 1 mRNA accumulation: a marker of oxidative stress to

eukaryotic cells. Methods Enzymol. 234:224 235; 1994.[35] Twentyman, P. R.; Luscombe, M. A study of some variables in a

tetrazolium dye (MTT) based assay for cell growth and chemo-

sensitivity. Br. J. Cancer 56:279 285; 1987.[36] Mosmann, T. Rapid colorimetric assay for cellular growth and

survival: application to proliferation and cytotoxicity assays.

J. Immunol. Methods 65:55 63; 1983.[37] Berridge, M. V.; Tan, A. S. Characterization of the cellular

reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium

bromide (MTT): subcellular localization, substrate dependence,and involvement of mitochondrial electron transport in MTT

reduction. Arch. Biochem. Biophys. 303:474 482; 1993.[38] Lasarow, R. M.; Isseroff, R. R.; Gomez, E. C. Quantitative in

vitro assessment of phototoxicity by a fibroblast-neutral red assay. J. Invest. Dermatol. 98:725729; 1992.

[39] Bulychev, A.; Trouet, A.; Tulkens, P. Uptake and intracellular

distribution of neutral red in cultured fibroblasts. Exp. Cell Res.115:343355; 1978.

[40] Vermes, I.; Haanen, C.; Reutelingsperger, C. Flow cytometry of

apoptotic cell death. J. Immunol. Methods 243:167190; 2000.[41] Tyurina, Y. Y.; Shvedova, A. A.; Kawai, K.; Tyurin, V. A.;

Kommineni, C.; Quinn, P. J.; Schor, N. F.; Fabisiak, J. P.; Kagan,

V. E. Phospholipid signaling in apoptosis: peroxidation and ex-

ternalization of phosphatidylserine. Toxicology 148:93101;2000.

[42] Trekli, M. C.; Riss, G.; Goralczyk, R.; Tyrrell, R. M. Beta-

carotene suppresses UVA-induced HO-1 gene expression in cul-

tured FEK4. Free Radic. Biol. Med. 34:456 464; 2003.[43] Jordan, N. J.; Kolios, G.; Abbot, S. E.; Sinai, M. A.; Thompson,

D. A.; Petraki, K.; Westwick, J. Expression of functional CXCR4

chemokine receptors on human colonic epithelial cells. J. Clin.

Invest. 104:10611069; 1999.[44] Bernerd, F.; Asselineau, D. UVA exposure of human skin recon-structed in vitro induces apoptosis of dermal fibroblasts: subse-quent connective tissue repair and implications in photoaging.

Cell Death Differ. 5:792 802; 1998.[45] Tyrrell, R. M. Redox regulation and oxidant activation of heme

oxygenase-1. Free Radic. Res. 31:335340; 1999.[46] Basu-Modak, S.; Tyrrell, R. M. Singlet oxygen. A primary effec-

tor in the UVA/near visible light induction of the human heme

oxygenase gene. Cancer Res. 53:4505 4510; 1993.[47] Godar, D. E. Singlet oxygen-triggered immediate preprogrammed

apoptosis. Methods Enzymol. 319:309 330; 2000.[48] Lemasters, J. J.; Nieminen, A.-L.; Qian, T.; Trost, L. C.; Elmore,

S. P.; Nishimura, Y.; Crowe, R. A.; Cascio, W. E.; Bradham,

C. A.; Brenner, D. A.; Herman, B. The mitochondrial permeabil-

ity transition in cell death: a common mechanism in necrosis,

apoptosis and autophagy. Biochim. Biophys. Acta 1366:177196;1998.

[49] Lemasters, J. J.; Qian, T.; Bradham, C. A.; Brenner, D. A.;

Cascio, W. E.; Trost, L. C.; Nishimura, Y.; Nieminen, A.-L.;

Herman, B. Mitochondrial dysfunction in the pathogenesis of

necrotic and apoptotic cell death. J. Bioenerg. Biomembr. 31:

305319; 1999.[50] Chandra, J.; Samali, A.; Orrenius, S. Triggering and modulation

of apoptosis by oxidative stress. Free Radic. Biol. Med. 29:323333; 2000.

[51] Vile, G. F.; Basu-Modak, S.; Waltner, C.; Tyrrell, R. M. Heme

oxygenase 1 mediates an adaptive response to oxidative stress in

human skin fibroblasts. Proc. Natl. Acad. Sci. USA 91:26072610; 1994.

[52] Agarwal, A.; Nick, H. S. Renal response to tissue injury: lessons

from heme oxygenase-1 gene ablation and expression. J. Am. Soc.

Nephrol. 11:965973; 2000.[53] Abraham, N. G.; Alam, J.; Nath, K. Heme oxygenase in biology

and medicine. New York: Kluwer Academic/Plenum Publishers;

2002.

[54] Poss, K. D.; Tonegawa, S. Heme oxygenase 1 is required for

mammalian iron reutilization. Proc. Natl. Acad. Sci. USA 94:

10919 10924; 1997.[55] Soriani, M.; Rice-Evans, C.; Tyrrell, R. M. Modulation of the

UVA activation of haem oxygenase, collagenase and cyclooxy-

genase gene expression by epigallocatechin in human skin cells.

FEBS Lett. 439:253257; 1998.

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



12/12

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


Documents

Epic Ate Chin and Its Methyl at Ed Metabolite Attenuate UVA-Induced Oxidative Damage to Human Skin Fib Rob Lasts