Methyl Paraben Potentiates UV Damage 2

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  • 8/4/2019 Methyl Paraben Potentiates UV Damage 2

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    Methylparaben potentiates UV-induced damage of skinkeratinocytes

    Osamu Handaa, Satoshi Kokura

    a, ,, Satoko Adachi

    b, Tomohisa Takagi

    a, Yuji Naito

    c, Toru Tanigawa

    e,

    Norimasa Yoshida

    d

    and Toshikazu Yoshikawa

    b

    aDepartment of Biomedical Safety Science, Kyoto Prefectural University of Medicine, Kyoto 602-8566, Japan

    bDepartment of Inflammation and Immunology, Kyoto Prefectural University of Medicine, Kyoto 602-8566, Japan

    cDepartment of Medical Proteomics, Kyoto Prefectural University of Medicine, Kyoto 602-8566, Japan

    dDepartment of Molecular Gastroenterology and Hepatology, Kyoto Prefectural University of Medicine, Kyoto

    602-8566, Japan

    eDepartment of Environmental Systems Science, Faculty of Engineering, Doshisha University, Kyoto 610-0321,

    Japan

    Received 16 May 2006;

    revised 11 July 2006;

    accepted 12 July 2006.

    Available online 28 July 2006.

    AbstractFor many years, methylparaben (MP) has been used as a preservative in cosmetics. In this study, we

    investigated the effects of ultraviolet-B (UVB) exposure on MP-treated human skin keratinocytes. HaCaT

    keratinocyte was cultured in MP-containing medium for 24 h, exposed to UVB (15 or 30 mJ/cm2) and further

    cultured for another 24 h. Subsequent cellular viability was quantified by MTT-based assay and cell death was

    qualified by fluorescent microscopy and flow cytometry. Oxidative stress, nitric oxide (NO) production and cellular

    lipid peroxidation were measured using fluorescent probes. In addition, activation of nuclear factor kappa B andactivator protein-1 was assessed by electro-mobility gel-shift assay. Practical concentrations of MP (0.003%) had

    a little or no effect on cellular viability, oxidative stress, NO production, lipid peroxidation and activation of nuclear

    transcription factors in HaCaT keratinocytes. Low-dose UVB also had little or no effect on these parameters in

    HaCaT keratinocytes. However, UVB exposure significantly increased cell death, oxidative stress, NO

    production, lipid peroxidation and activation of transcription factors in MP-treated HaCaT keratinocytes. These

    results indicate that MP, which has been considered a safe preservative in cosmetics, may have harmful effects

    on human skin when exposed to sunlight.

    Keywords: Methylparaben; Ultra-violet; Transcription factor; Apoptosis; Oxidative stress

    Abbreviations: AP-1, activator protein-1; DAF-DA, 4-amino-5-methylamino-2,7-difluorescein diacetate; DCF-DA, 5-(and 6)-carboxy-2,7-dichlorodihydrofluorescein diacetate; DHR123, dihydrorhodamine-123; DMEM,

    Dulbecco's modified essential medium; DPPP, diphenyl-1-pyrenylphosphine; EMSA, electro-mobility gel shift

    assay; FBS, fetal bovine serum; FITC, fluorescein isothiocyanate; HO342, Hoechst 33432; MP, methylparaben;

    NFB, nuclear factor kappa B; NO, nitric oxide; PBS, phosphate-buffered saline; PI, propidium iodide; ROS,

    reactive oxygen species; UVA, ultra-violet A; UVB, ultra-violet B; UVC, ultra-violet C

    Article Outline

    1.Introduction

    2.Materials and methods

    2.1.Reagents

    2.2.Human normal keratinocytes

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    2.3.UVB irradiation

    2.4.Cell viability assay

    2.5.Microscopic analysis for dead cells

    2.6.Flowcytometric quantification of dead cells

    2.7.Production of reactive oxygen species (ROS) and nitric oxide (NO)

    2.8.Electro-mobility gel-shift assay (EMSA) for nuclear transcription factors

    2.9.Lipid peroxidation in HaCaT keratinocytes

    2.10.Electron paramagnetic resonance (EPR) study

    2.11.Statistical analysis

    3.Results

    3.1.Cytotoxic effects of MP on HaCaT keratinocytes

    3.2.MP-induced production of ROS and NO

    3.3.Cytotoxic effects of UVB irradiation on HaCaT keratinocytes

    3.4.MP enhances UVB irradiation-induced cell death

    3.5.MP enhances ROS and NO production in HaCaT keratinocytes induced by UVB irradiation

    3.6.MP enhances NFB and AP-1 activation of HaCaT keratinocytes induced by UVB irradiation

    3.7.MP enhances lipid peroxidation of HaCaT keratinocytes induced by UVB irradiation

    3.8.EPR study

    4.Discussion

    References

    1. Introduction

    Various additives and preservatives are used in cosmetics, foods and medicines in order to prevent deterioration.

    The parabens have been widely used as preservatives in cosmetics as well as in foods and medicines for more

    than 50 years due to its broad antimicrobial function. Methylparaben (MP) is a methyl ester of p-hydroxybenzoic

    acid (Fig. 1) and is one of a homologous series of parabens (including methyl, ethyl, butyl, heptyl and benzyl

    parabens). MP is one of the most commonly used preservatives in cosmetics (Mowad, 2000). MP meets severalof the criteria for an ideal preservative; it exhibits a broad spectrum of antimicrobial activity, is colorless, odorless

    (slight burnt flavor), stable over the pH range, resistant to hydrolysis in hot and cold water (autoclaveable)

    (Alexander et al., 1978), non-volatile, has a low tendency towards absorption in commonly used plastics and

    primary packing materials, and is cheap. Moreover, MP has been shown to have low acute- and long-term

    toxicity, has no carcinogenic activity ([Rodrigues et al., 1986]and[Matthews et al., 1956]) and is not mutagenic

    (Kawachi et al., 1980). Therefore, it was concluded that practical concentrations of MP have little or no toxicity

    either in vivoor in vitro(Soni et al., 2002). Although MP has been reported to be readily and completely absorbed

    through the skin and from the gastrointestinal tract, and shows no evidence of accumulation (Soni et al., 2002),

    recent studies have reported that small amounts of MP remain unhydrolyzed in the epidermis ([Oh et al.,

    2002]and[Cross and Roberts, 2000]) and body tissue (Darbre et al., 2004). Therefore, it is possible that MP

    remaining in the epidermis influence keratinocytes.

    Full-size image(4K)

    Fig. 1. Methylparaben (MP) is a methyl ester of p-hydroxybenzoic acid.

    The skin is more or less exposed to sunlight as part of daily life, in which the effects of ultraviolet radiation are not

    negligible, and many people use cosmetics that contain MP. Although MP has been considered to be safe and is

    used widely as a preservative in cosmetics, the effects of MP on skin under the sunlight is unknown. In the

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  • 8/4/2019 Methyl Paraben Potentiates UV Damage 2

    3/13

    present study, we investigated the effects of ultraviolet-B (UVB) exposure on MP-treated human skin

    keratinocytes in vitro.

    2. Materials and methods

    2.1. ReagentsMethyl 4-hydroxybenzoate (methylparaben; MP), Hoechst 33432 (HO342) and propidium iodide (PI) were

    purchased from Sigma (St. Louis, MO, USA). Dihydrorhodamine-123 (DHR123), 5-(and 6)-carboxy-2,7-

    dichlorodihydrofluorescein diacetate (DCF-DA) and 4-amino-5-methylamino-2,7-difluorescein diacetate (DAF-

    DA) were from Molecular Probes (Eugene, OR, USA). Diphenyl-1-pyrenylphosphine (DPPP) was from Dojindo

    (Kumamoto, Japan). Ethanol and other reagents were from Wako Pure Chemical Industries, Ltd. (Osaka, Japan).

    MP was dissolved in 99.5% ethanol and added at a final concentration of 0.003%, 0.03% or 0.3% (0.197, 1.97 or

    19.7 mM, respectively) to cell culture medium (final concentration of ethanol was 0.1%, which had no effect on

    cell viability within 72 h; data not shown), as described below. As a control group, cell culture medium containing

    0.1% ethanol was used.

    2.2. Human normal keratinocytesHaCaT keratinocytes are a spontaneously immortalized human keratinocyte line (Boukamp et al., 1988), and

    were kindly supplied by Dr. Kato of the Department of Dermatology of Kyoto Prefectural University of Medicine

    (Kyoto, Japan). HaCaT keratinocytes were grown in 80 cm2

    cell culture flasks and maintained in Dulbecco's

    modified essential medium (DMEM; Gibco-BRL, Gaithersburg, MD, USA) supplemented with 5% fetal bovine

    serum (FBS; Equitech-Bio Inc., Kerrville, TX, USA), 2 mM glutamine and 100 U/ml penicillin/streptomycin (Gibco)

    at 37 C in a humidified atmosphere containing 5% CO2. Cells were passaged every 7 days at a 1:10 split.

    2.3. UVB irradiation

    The UVB source was a bank of six fluorescent sunlamps (FL-20SE-30; Toshiba Medical Supply, Tokyo, Japan)

    with an emission spectrum of 275375 nm, mainly in the UVB range, peaking at 305 nm and including a small

    amount of UVA and UVC (energy: UVA, 30%; UVB, 54%; UVC, 0.2%). The irradiance of UVB was measured bya UV-radiometer (UVR-3036/S2; TOPCON corporation, Tokyo, Japan). In some experiments, MP-treated or -

    untreated HaCaT keratinocytes were exposed to UVB (15 or 30 mJ/cm2). Prior to UVB irradiation, medium was

    decanted and replaced with phosphate-buffered saline (PBS). After irradiation, cells were incubated in cell culture

    medium without MP at 37 C for various time periods.

    2.4. Cell viability assay

    Cell viability was measured using a MTT-based WST-1 assay kit (Dojin Laboratory, Kumamoto, Japan) according

    to the manufacturer's instructions. Briefly, HaCaT keratinocytes were cultured in 96-well plates until confluence

    and were incubated for 6 or 24 h in the absence or presence of MP. Cells were washed twice with PBS and

    WST-1 solution was added. Cells were then incubated for 1 h at 37 C and the optical density of each well was

    read at 455 nm using a micro-plate reader (MPR-A4i; Tosoh corporation, Tokyo, Japan). Cell viability wasexpressed as a percentage of untreated control cultures.

    2.5. Microscopic analysis for dead cells

    The type of cell death (apoptosis or necrosis) induced by MP and/or UVB was determined by fluorescent

    microscopy after staining with Hoechst 33342 (HO342) and propidium iodide (PI), as described by Naito et al.

    (2002). Cells were grown in 35 mm cell culture dishes until confluence. After treatment with MP for 24 h, cells

    were exposed to UVB and then incubated for a further 24 h. The medium was removed and cells were washed

    twice with PBS and incubated with 10 g/ml HO342 dye for 15 min at 37 C and with 10 g/ml PI for 10 min at

    37 C. Dual-stained cells were examined using an IX70-23FL/DIC-SP inverted fluorescence microscope

    (Olympus, Tokyo, Japan). Photographic images (MPEG format) were taken from four random fields. Live cells,

    viable cells and early stage apoptotic cells, which have cell membrane function, take up blue dye (HO342).

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    Apoptosis was characterized morphologically by condensed chromatin. Red-stained cells (PI) were considered

    late apoptotic (condensed chromatin) or necrotic cells.

    2.6. Flowcytometric quantification of dead cells

    Quantitative analysis of cell death was performed by flow cytometry. HaCaT keratinocytes were cultured in

    35 mm cell culture dishes until confluence. After treatment with MP for 24 h, cells were exposed to UVB andincubated for a further 24 h. Subsequently, medium was removed and cells were washed twice with PBS,

    collected by centrifugation and pellets were resuspended in PBS. Cells were stained with 10 g/ml PI and were

    analyzed using a FACS Calibur (Becton Dickinson and Company: Franklin Lakes, NJ, USA). PI positive cells

    include late apoptotic cells and necrotic cells.

    2.7. Production of reactive oxygen species (ROS) and nitric oxide (NO)

    HaCaT keratinocytes were cultured in 35 mm cell culture dishes until confluence. After treatment with MP for

    24 h, cells were exposed to UVB and were incubated for a further 1 h. Subsequently, medium was removed and

    cells were washed twice with PBS. Cells were then incubated with dihydrorhodamine-123 (DHR123) at a final

    concentration of 20 M for 15 min, with 5-(and 6)-carboxy-2,7-dichlorodihydrofluorescein diacetate (DCF-DA) at a

    final concentration of 50 M for 30 min or with 4-amino-5-methylamino-2,7-difluorescein diacetate (DAF-DA) at afinal concentration of 20 M for 15 min. Quantitative analysis was performed using a fluorescent micro-plate

    reader (Fluoromark; Bio-Rad Laboratories, Hercules, CA, USA) or by flow cytometry (FACS Calibur). The

    excitation/emission wavelengths for DCF-DA, DHR-123 and DAF-DA were 504/530, 505/529 and 495/515 nm,

    respectively. At the same time, fluorescent images were observed by fluorescent microscopy.

    2.8. Electro-mobility gel-shift assay (EMSA) for nuclear transcription factors

    HaCaT keratinocytes were cultured in 35 mm cell culture dishes until confluence. After treatment with MP for

    24 h, cells were exposed to UVB and were incubated for a further 2 h. Subsequently, medium was removed and

    cells were washed twice with PBS. Nuclear protein from HaCaT keratinocytes was extracted as described

    previously (Cepinskas et al., 2003). For EMSA, 3 g of total nuclear proteins was incubated with 1.0 pmol of

    double-stranded [32P] ATP end-labeled oligonucleotides containing consensus binding sequences for NFB

    (sense strand 5-AGGGACTTTCCGCTGGGGACTTTCC-3) or for activator protein-1 (AP-1; sense strand 5-

    CGCTTGATGAGTCAGCCGGAA-3) in binding buffer (10 mM Hepes, pH 7.9, 80 mM NaCl, 3 mM MgCl2, 0.1 mM

    EDTA, 1 mM DTT, 1 mM PMSF and 10% glycerol) for 30 min. After electrophoresis under non-denaturing

    conditions (0.5 TBE buffer), gels were dried and radioactive bands were visualized on X-ray films.

    2.9. Lipid peroxidation in HaCaT keratinocytes

    HaCaT keratinocytes were cultured in 96-well plates until confluence. After treatment with MP for 24 h, cells were

    exposed to UVB and were incubated for a further 1 h. Subsequently, medium was removed and cells were

    washed twice with PBS. Cells were then incubated with diphenyl-1-pyrenylphosphine (DPPP) at a final

    concentration of 20 M for 15 min. Cell fluorescence was quantified using a f luorescent micro-plate reader. The

    excitation/emission wavelengths for DPPP were 352/380 nm.

    2.10. Electron paramagnetic resonance (EPR) study

    EPR was performed using an FR-80 ESR Spectrometer (JEOL, Akishima, Japan) equipped with a UV-irradiation

    system (RUVF-203S, Radical Research, Tokyo, Japan). The UV spectrum consists of UVB and UVA, and total

    UV power was around 100 mW/cm2. Methylparaben was dissolved in PBS at 0.15% (w/v). Dimethylpyrroline-N-

    oxide (Dojindo, Kumamoto, Japan) at 1 M was used as a spin trapping agent. Samples were introduced into a flat

    quart EPR cuvette (Radical Research, Tokyo, Japan) and were placed in the EPR cavity. UV was introduced to

    the cavity via optical fiber. EPR conditions were as follows: microwave frequency, 9.4 GHz; microwave power,

    5 mW; modulation width, 0.1 mT; sweep rate, 10 mT/min; response time, 0.03 s.

    2.11. Statistical analysis

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    Results are presented as means S.E.M. Data were compared by two-way analysis of variance. Differences

    were considered significant if the P-value was less than 0.05 based on Fisher's protected least significant

    difference tests. Statistical analysis was performed using Stat View 5.0-J (Abacus Concepts, Berkeley, CA).

    3. Results

    3.1. Cytotoxic effects of MP on HaCaT keratinocytes

    MP at 0.003% had no effect on cellular viability of HaCaT keratinocytes within 24 h, whereas higher

    concentrations of MP caused significant reductions in cellular viability within 6 h, as assessed by MTT-based

    WST-1 assay (Fig. 2). To evaluate the type of cell death in HaCaT keratinocytes, we employed a double-staining

    method using Hoechst 33342 (HO342) and propidium iodide (PI). The obtained images indicated that small basal

    amounts of apoptosis were present and that lower concentrations of MP had no effect on cell death, whereas

    higher concentrations of MP caused significant increases in necrotic cells but not apoptotic cells within 6 h (Fig.

    3).

    Full-size image(13K)

    Fig. 2. Effect of MP on cell viabili ty of HaCaT keratinocytes. Cellular viability was measured by MTT-based WST-

    1 assay. Values represent mean (% of control) S.E.M. of three experiments.#P< 0.05 compared with MP-

    untreated control group.

    Full-size image(150K)

    Fig. 3. MP-induced cell death in HaCaT keratinocytes. HaCaT keratinocytes were stained with HO342 (blue) and

    PI (red). Blue stained cells have intact function of cell membrane. Red stained cells are necrotic cells. Images are

    representative of three independent experiments.

    3.2. MP-induced production of ROS and NO

    It is known that excessive amounts of ROS and NO production are a major cause of cell death (Naito, 2002).

    Therefore, we assessed the MP-induced production of ROS and NO using two oxidant-sensitive fluorescent

    probes (dihydrorhodamine-123; DHR123 and 5-(and 6)-carboxy-2,7-dichlorodihydrofluorescein diacetate; DCF-

    DA) and one NO-sensitive fluorescent probe (4-amino-5-methylamino-2,7-difluorescein diacetate; DAF-DA). In

    general, DHR123 and DCF-DA exhibit no fluorescence without ROS, and become fluorescent (as an oxidized

    form; rhodamine 123 and DCF, respectively) when interacting with ROS. DAF-DA exhibits no fluorescence

    without NO and becomes fluorescent as a nitrated form, DAF, when interacting with NO. In the present study,

    fluorescent microscopy revealed that incubation of HaCaT keratinocytes in DMEM containing 0.003% or 0.03% of

    MP resulted in a little oxidation of either DHR-123 or DCF-DA, as well as a little nitration of DAF-DA within 24 h

    compared to control (data not shown). These findings were further confirmed using a fluorescent micro-platereader (Fig. 4). Note that fluorescent intensity did not change, irrespective of whether MP was added to HaCaT

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    keratinocytes. HaCaT keratinocytes were also stimulated with hydrogen peroxide (H2O2: 100 M) for 20 min as a

    positive control, and HaCaT produced significant amount of ROS and NO, respectively (data not shown).

    Full-size image(35K)

    Fig. 4. MP-induced oxidative stress and NO production in HaCaT keratinocytes. ROS production was measured

    based on fluorescent intensity of DHR123 and DCF-DA oxidation. NO production was measured based on

    fluorescent intensity of DAF-DA nitration. Values represent mean (% of control) S.E.M. of three

    experiments.*P< 0.05 compared with MP-untreated control group.

    3.3. Cytotoxic effects of UVB irradiation on HaCaT keratinocytes

    We then focused on the effects of UVB irradiation on HaCaT keratinocytes. Cells were exposed to UVB (15 or

    30 mJ/cm2) and the type of cell death was evaluated by double staining with HO342 and PI fluorescent dye. As

    shown inFig. 5, UVB irradiation at 15 mJ/cm2

    did not induce cell death, whereas UVB irradiation at

    30 mJ/cm2induced a small amount of apoptosis (mainly late apoptosis). This was consistent with the results

    obtained by flow cytometry. As shown inFig. 6, UVB irradiation at 30 mJ/cm2, but not UVB irradiation at

    15 mJ/cm2, induced a small amount of late apoptosis and necrosis (PI positive cells).

    Full-size image(155K)

    Fig. 5. UVB-induced apoptosis in MP-treated HaCaT keratinocytes. HaCaT keratinocytes were stained with

    HO342 (blue) and PI (red). Blue stained cells have intact function of cell membrane. Cells with condensed

    nucleus are early apoptotic cells (blue) and late apoptotic cells (red). Images are representative of three

    independent experiments.

    Full-size image(25K)

    Fig. 6. Quantification of PI-positive cells in UVB-exposed and MP-treated HaCaT keratinocytes. HaCaT

    keratinocytes were stained with PI and fluorescent intensity was evaluated by flowcytometer. Each value

    represents the mean S.E.M. of three experiments.*P< 0.05 compared with MP-untreated and UVB-unexposed

    control group.

    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    3.4. MP enhances UVB irradiation-induced cell death

    As described above, UVB irradiation itself induced little or no necrosis in HaCaT keratinocytes and practical

    concentrations of MP (0.003%) alone had no effect on HaCaT cell viability. However, 0.003% MP significantly

    enhanced UVB-induced cell death of HaCaT keratinocytes as assessed by immunocytochemistry (Fig. 5) and

    flow cytometry (Fig. 6).

    3.5. MP enhances ROS and NO production in HaCaT keratinocytes induced by UVB irradiation

    As shown inTable 1, UVB irradiation alone induced little or no increase in ROS and NO production in HaCaT

    keratinocytes as assessed by flow cytometry. Although MP alone induced no significant or very small amount of

    ROS and NO production, MP significantly enhanced UVB-induced ROS and NO production in HaCaT

    keratinocytes.

    Table 1.

    UVB-induced ROS and NO production in MP-treated HaCaT keratinocytes

    UVB (mJ/cm2)

    () 15 30

    (A) Fluorescent intensity of DHR123

    MP

    () 100.0 39.3 109.8 29.5 137.7 50.8*

    0.003% 101.6 18.0 123.0 60.7*

    100.0 72.1

    0.03% 109.8 44.3 145.9 55.7*

    762.3 136.1*

    (B) Fluorescent intensity of DCF-DA

    MP

    () 100.0 5.2 124.6 6.2 170.4 11.2*

    0.003% 114.4 3.1 179.8 4.9*

    184.2 5.5*

    0.03% 120.8 9.3* 187.7 8.9* 213.8 14.4*

    (C) Fluorescent intensity of DAF-DA

    MP

    () 100.0 12.0 100.5 22.4 120.4 10.2*

    0.003% 97.5 20.0 105.6 12.4 136.1 21.0*

    0.03% 97.7 35.5 145.8 30.1* 155.5 10.8*

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    ROS production was measured based on fluorescent intensity of DHR123 and DCF-DA oxidation using flow

    cytometry. NO production was measured by fluorescent intensity of DAF-DA nitration using flow cytometry. Each

    value represents the mean (% of control) S.E.M. of three experiments.*P< 0.05 compared with MP-untreated

    and UVB-unexposed control group.

    3.6. MP enhances NFB and AP-1 activation of HaCaT keratinocytes induced by UVB irradiation

    Redox-sensitive nuclear transcription factors, such as NFB and AP-1, play an important role in cell signal

    transduction (Handa et al., 2004). As shown inFig. 7andFig. 8, UVB alone induced a little or no activation of

    NFB and AP-1 in HaCaT keratinocytes as assessed by electro-mobility gel-shift Assay (EMSA). Although MP

    alone induced no significant NFB and AP-1 activation, MP significantly enhanced UVB-induced NFB and AP-1

    activation.

    Full-size image(39K)

    Fig. 7. UVB-induced NFB activation in MP-treated HaCaT keratinocytes. Nuclear NFB level was assessed by

    EMSA. The results for untreated (lane 1: control), UVB-exposed (15 mJ/cm2) (lane 2), UVB (30 mJ/cm

    2)-exposed

    (lane 3), MP (0.003%)-treated (lane 4), MP (0.003%)-treated and UVB (15 mJ/cm2)-exposed (lane 5), MP

    (0.003%)-treated and UVB (30 mJ/cm2)-exposed (lane 6), MP (0.03%)-treated (lane 7), MP (0.03%)-treated and

    UVB (15 mJ/cm2)-exposed (lane 8), MP (0.03%)-treated and UVB (30 mJ/cm

    2)-exposed (lane 9), are shown. A

    representative result (EMSA) of three experiments is shown in the lower panel and quantitative result

    (densitometry) is shown in the upper panel. Each value represents the mean S.E.M. (% of control).*P< 0.01

    compared with MP-untreated corresponding group.#P< 0.01 compared with MP (0.003%)-treated corresponding

    group, respectively.

    Full-size image(41K)

    Fig. 8. UVB-induced AP-1 activation in MP-treated HaCaT keratinocytes. Nuclear AP-1 level was assessed by

    EMSA. The results for untreated (lane 1), UVB-exposed (15 mJ/cm2) (lane 2), UVB (30 mJ/cm

    2)-exposed (lane

    3), MP (0.003%)-treated (lane 4), MP (0.003%)-treated and UVB (15 mJ/cm2)-exposed (lane 5), MP (0.003%)-

    treated and UVB (30 mJ/cm2)-exposed (lane 6), MP (0.03%)-treated (lane 7), MP (0.03%)-treated and UVB

    (15 mJ/cm2)-exposed (lane 8), MP (0.03%)-treated and UVB (30 mJ/cm

    2)-exposed (lane 9), are shown. A

    representative result (EMSA) of three experiments is shown in the lower panel and quantitative result

    (densitometry) is shown in the upper panel. Each value represents the mean S.E.M. (% of control).*P< 0.01

    compared with MP-untreated corresponding group.#P< 0.01 compared with MP (0.003%)-treated corresponding

    group, respectively.

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    3.7. MP enhances lipid peroxidation of HaCaT keratinocytes induced by UVB irradiation

    As cell death is strongly associated with lipid peroxidation of cell membrane, we assessed lipid peroxidation of

    HaCaT keratinocytes by fluorescent probe DPPP. UVB alone induced slight lipid peroxidation in HaCaT

    keratinocytes. Although MP alone also induces slight lipid peroxidation, MP significantly enhanced UVB-induced

    increase of lipid peroxidation of HaCaT keratinocyte (Fig. 9). In order to determine whether the effect of MP on

    UVB-induced lipid peroxidation is additive or mutually potentiating effect, the mean fluorescent value of UVB-

    and MP- treated groups subtracted by UVB-treated and MP-untreated group was statistically compared with

    the mean value of UVB-untreated corresponding group. As a result, in UVB-treated and MP (0.03%)-treated

    group, the effect of MP on UVB-induced lipid peroxidation was considered to be more than additive one.

    Full-size image(16K)

    Fig. 9. UVB-induced lipid peroxidation in MP-treated HaCaT keratinocytes. Lipid peroxidation was measured

    using a fluorescent probe, DPPP. Each value represents the mean (% of control) S.E.M. of three

    experiments.*P< 0.05 compared with MP-untreated and UVB-unexposed control group.

    #P< 0.05 compared

    with UVB-unexposed corresponding group, respectively.

    3.8. EPR study

    In the absence of MP, a four-line signal for DMPO-OH was observed, which suggests the generation of hydroxyl

    radicals (Fig. 10a). This was confirmed by the addition of dimethylsulfoxide (DMSO, 1.4 M), a potent hydroxyl

    radical inhibitor, which inhibited the four-line signal and gave a six-line signal (Fig. 10b), considered to

    correspond to the methyl radical-adduct of DMPO. In the presence of MP, DMPO-OH signal intensity was slightly

    reduced and new signals were observed (Fig. 10c). The new signals were assigned to the DMPO-H and DMPO

    adducts of carbon-centered radicals. Addition of DMSO also inhibited DMPO-OH in the paraben-containing

    solution, but had no influence on DMPO-H (Fig. 10d), thus, suggesting that the generation of DMPO-H is not

    dependent on hydroxyl radicals.

    Full-size image(21K)

    Fig. 10. EPR spectra of UV-induced radical adducts of DMPO. (a) DMPO + UV; (b) (a) + DMSO; (c) (a) + MP; (d)

    (c) + DMSO. The inverted signals at either side are for the Mn2+

    in MnO, which was used as an internal marker.

    4. Discussion

    For many years, MP has been used as a safety preservative in cosmetics, however in the present study, we

    showed detrimental potential of MP on UVB-exposed human normal skin keratinocytes in vitro.

    The use of parabens in cosmetic products up to a maximum concentration of 0.8% (w/w), calculated as p-hydroxybenzoic acid, is permitted by the Danish and EEC regulations ([Howlett, 1992]and[Rastogi et al., 1995])

    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  • 8/4/2019 Methyl Paraben Potentiates UV Damage 2

    10/13

    and typical concentrations of MP in cosmetics are less than 0.32% (Rastogi et al., 1995). In our preliminary study,

    approximately 1% of the applied MP reaches the basal layer of the skin epidermis of Yucatan mini pigs (data not

    shown). Together with reported data that small amounts of MP remain unhydrolyzed in the epidermis (Cross and

    Roberts, 2000), we employed MP at concentrations of 0.30.003% in the present study.

    We first examined the cytotoxic effects of MP on HaCaT keratinocytes. As has been reported previously

    (Pomerat and Leake, 1954), we found that practical concentrations of MP (0.003%) have no effect on cellular

    viability of HaCaT keratinocytes, while higher concentrations of MP decrease cellular viability within 6 h, thus,

    indicating that the detrimental potential of MP is dose and time dependent.

    Solar radiation, particularly UVA and UVB, ranging from 290 to 400 nm is accountable for numerous biological

    effects in the skin. UVB radiation may only reach the epidermis and upper dermis, whereas UVA is a more

    penetrating radiation that elicits its effects in the dermis. In this study, we focused mainly on the effects of UVB

    irradiation on skin keratinocytes, the main cell forming epidermis that is the front line against external injurious

    stress. The UVB doses employed in this study, 15 and 30 mJ/cm2, are similar to the average UVB dose delivered

    by 1 min of sunlight exposure on a fine day on January and that by 30 s of sunlight exposure on a fine day in

    August, respectively, in Europe(Lebert et al., 2002).

    UV irradiation of skin has been shown to increase levels of ROS and NO in cells and to result in oxidative

    damage to lipids, proteins and DNA ([Cunningham et al., 1985],[Vile and Tyrrell, 1995],[Hattori et al.,

    1996]and[Sander et al., 2004]). In addition, the pro-inflammatory and redox-sensitive transcription factor, NFB

    (Flohe et al., 1997), has been identified among the primary molecules targeted during signal transduction initiated

    by UV irradiation of human skin (Fisher et al., 1996). As a result, UV stimulates the expression of wide variety of

    pro-inflammatory genes and causes inflammatory skin responses, photo-aging and skin cancer depending on the

    amount of UV exposure. In the present study, low-dose UVB irradiation alone and practical concentrations of MP

    alone did not induce cell death in HaCaT keratinocytes. Moreover, MP or UVB alone induced little or no ROS and

    NO production and NFB and AP-1 activation in HaCaT keratinocytes. However, practical concentrations of MP

    significantly enhanced ROS and NO production, NFB and AP-1 activation, and apoptosis of UVB exposed

    HaCaT keratinocytes. Because ROS and NO production has been reported to modulate transcription factors

    (Handa et al., 2004), UVB irradiation of MP-treated HaCaT keratinocytes may activate these transcription factors

    via ROS and NO production, thus inducing production of various cytokines and chemokines. This, in turn, could

    result in a vicious circle of inflammation, ultimately resulting in cell death.

    UVB irradiation can induce apoptotic, necrotic and differentiation pathways in human normal keratinocytes. In the

    present study, very little degree of apoptosis was induced in UVB-irradiated cells. This finding is consistent with a

    previous study byHenseleit et al. (1996). In contrast, Mammone et al. reported that low-dose UVB (520 mJ/cm2)

    increased apoptosis, while high-dose UVB (>20 mJ/cm2) induced necrosis in HaCaT keratinocytes (Mammone et

    al., 2000). This discrepancy may be the result of differences in the sunlamps employed in each study. The

    sunlamp used in the present study emitted a broad band of UV, including UVB (ranging from 280 to 315 nm) as

    well as small amounts of UVA (315400 nm) and the more harmful UVC (200280 nm). However, the FS40

    sunlamps (Westinghouse Corp., Pittsburgh, PA) used by Mammone et al. emit a smaller UV range (250360 nm)

    than those used in our study (275375 nm). Therefore, the sunlamps used by Mammone et al. emitted moreUVC than those used in the present study. In addition, the amount of UVA emitted in our study (6.96 and

    13.92 mJ/cm2

    for UVB irradiation at 15 and 30 mJ/cm2, respectively) was almost a 100 times smaller than the

    level reported to affect cellular viability.

    Although the precise mechanisms by which cell death was induced are unknown in this study, lipid peroxidation

    (Nagano et al., 2005), or ROS and NO production (Naito, 2002) has been proposed as one of the mechanisms of

    cell apoptosis. Together with our findings that MP pre-treatment significantly enhanced the UVB-induced lipid

    peroxidation, ROS and NO production, and cell death, it might be possible that the enhanced effect of UVB on

    cell death in MP pre-treated HaCaT keratinocytes is oxidative stress-dependent event.

    EPR study indicated that MP might be a source of free radicals when irradiated with UVB. Hydrogen atoms

    (=hydrogen radicals), carbon-centered radicals and hydroxyl radicals were observed and the former two were

    MP-dependent. Hydrogen atoms may scavenge other biologically toxic radicals, and thus may act as a protectivefactor. However, little is known about the biological significance of this radical to date. Hydroxyl radicals are

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