Toxicology

Volume 227, Issues 1–2, 3 October 2006, Pages 62–72

Methylparaben potentiates UV-induced damage of skin keratinocytes

  • a Department of Biomedical Safety Science, Kyoto Prefectural University of Medicine, Kyoto 602-8566, Japan
  • b Department of Inflammation and Immunology, Kyoto Prefectural University of Medicine, Kyoto 602-8566, Japan
  • c Department of Medical Proteomics, Kyoto Prefectural University of Medicine, Kyoto 602-8566, Japan
  • d Department of Molecular Gastroenterology and Hepatology, Kyoto Prefectural University of Medicine, Kyoto 602-8566, Japan
  • e Department of Environmental Systems Science, Faculty of Engineering, Doshisha University, Kyoto 610-0321, Japan
Corresponding author contact information
Corresponding author. Tel.: +81 75 251 5505; fax: +81 75 252 3721.
Received 16 May 2006
Revised 11 July 2006
Accepted 12 July 2006
Available online 28 July 2006
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1. Introduction

2. Materials and methods

3. Results

4. Discussion

References

Abstract

For 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 and activator 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.

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;
  • NFκB, 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

Keywords

  • Methylparaben;
  • Ultra-violet;
  • Transcription factor;
  • Apoptosis;
  • Oxidative stress

Figures and tables from this article:

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

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Fig. 2. Effect of MP on cell viability 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.

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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.

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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.

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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.

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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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Fig. 7. UVB-induced NFκB activation in MP-treated HaCaT keratinocytes. Nuclear NFκB level was assessed by EMSA. The results for untreated (lane 1: control), UVB-exposed (15 mJ/cm2) (lane 2), UVB (30 mJ/cm2)-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/cm2)-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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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/cm2)-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/cm2)-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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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.

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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.

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Table 1. UVB-induced ROS and NO production in MP-treated HaCaT keratinocytes

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.

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Corresponding author contact information
Corresponding author. Tel.: +81 75 251 5505; fax: +81 75 252 3721.

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