Overdosage of Methylparaben Induces Cellular Senescence In Vitro and In Vivo  Hwa Jun Cha, Seunghee Bae, Karam Kim, Seung Bin Kwon, In-Sook An, Kyu Joong Ahn, Junghwa Ryu, Hey-Sun Kim, Sang-Kyu Ye, Byung-Hak Kim, Sungkwan An  Journal of Investigative Dermatology  Volume 135, Issue 2, Pages 609-612 (February 2015) DOI: 10.1038/jid.2014.405 Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 MP induces CS through c-Jun-dependent ROS modulation in nHDFs. (a) The growth capability measured by the population doubling time assay was repressed in the methylparaben (MP) (0, 0.1, 0.2, and 0.4 mg ml-1)-treated normal human dermal fibroblasts (nHDFs) in a dose-dependent manner. (b) MP elevated SA-β-gal staining in nHDFs. Scale bar=100 μm. (c) MP elevated the levels of intracellular reactive oxygen species (ROS) measured by dichlorofluorescein diacetate (DCF-DA) staining in nHDFs. (d) MP (0.4 mg ml-1)-induced cellular senescence (CS) in nHDFs was reduced by treatment with NAC (1 mM). Scale bar=100 μm. (e) Expression of antioxidant enzymes in the MP-treated nHDFs. (f) MP (0.4 mg ml-1) repressed the activity of the antioxidant response elements (AREs) in the antioxidant gene promoter. (g) ARE binding of transcription factors measured using western blotting (left) and quantitative real-time PCR (right). (h) MP repressed the activity of the TPA-responsive element in the c-Jun promoter. (i) MP-dependent repression of GPX1 and SOD1 levels was restored by overexpression of c-Jun. (j) MP-induced ROS levels were restored by overexpression of c-Jun. (k) MP (0.4 mg ml-1)-induced CS was restored by c-Jun overexpression. nHDFs were treated with 0–0.4 mg ml-1 MP for 48 hours; results represent the mean±SD, and the symbol denotes a significant difference compared with the untreated nHDFs (*P<0.05) and compared with the MP-treated nHDFs (#P<0.05). Scale bar=100 μm. Journal of Investigative Dermatology 2015 135, 609-612DOI: (10.1038/jid.2014.405) Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 MP induces CS and alters ECM components in vitro and in vivo via the GR-c-Jun-ROS pathway. (a) Methylparaben (MP) (4 mg ml-1) treatment resulted in the translocation of the GRα from the cytosol to the nucleus of normal human dermal fibroblasts (nHDFs) . (b) MP increased the transcriptional activity of the GR element. (c, d) Knockdown of the GRα restored the MP-induced levels of c-Jun mRNA (c) and protein (d) in nHDFs. (e) Knockdown of the GRα blocked the MP-induced reactive oxygen species (ROS) levels in nHDFs. (f) Knockdown of the GRα reduced the percentage of senescent cells. Scale bar=100 μm. (g) The macroscopic images of mouse backs were represented (upper), and cellular senescence (CS) assessment of the mice skin was performed using SA-β-gal (blue) and nuclear fast red, NRF, (red) double staining (bottom) in response to MP (400 mg ml-1) and NAC (1 M). De, dermal layer; Ep, epidermal layer. (h) Mmp1 mRNA levels increased (left), whereas those of Col1a1 decreased (right) in MP-treated mouse skin compared with the control skin, analyzed using quantitative real-time PCR. Results are shown as mean±SD of three independent experiments. *P<0.05 compared with vehicle-treated group; #P<0.05 compared with MP-treated group. (i) The MP-induced CS pathway. Journal of Investigative Dermatology 2015 135, 609-612DOI: (10.1038/jid.2014.405) Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions