Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 15 (3), 309-22

The Topical Antimicrobial Zinc Pyrithione Is a Heat Shock Response Inducer That Causes DNA Damage and PARP-dependent Energy Crisis in Human Skin Cells

Affiliations

The Topical Antimicrobial Zinc Pyrithione Is a Heat Shock Response Inducer That Causes DNA Damage and PARP-dependent Energy Crisis in Human Skin Cells

Sarah D Lamore et al. Cell Stress Chaperones.

Abstract

The differentiated epidermis of human skin serves as an essential barrier against environmental insults from physical, chemical, and biological sources. Zinc pyrithione (ZnPT) is an FDA-approved microbicidal agent used worldwide in clinical antiseptic products, over-the-counter topical antimicrobials, and cosmetic consumer products including antidandruff shampoos. Here we demonstrate for the first time that cultured primary human skin keratinocytes and melanocytes display an exquisite vulnerability to nanomolar concentrations of ZnPT resulting in pronounced induction of heat shock response gene expression and impaired genomic integrity. In keratinocytes treated with nanomolar concentrations of ZnPT, expression array analysis revealed massive upregulation of genes encoding heat shock proteins (HSPA6, HSPA1A, HSPB5, HMOX1, HSPA1L, and DNAJA1) further confirmed by immunodetection. Moreover, ZnPT treatment induced rapid depletion of cellular ATP levels and formation of poly(ADP-ribose) polymers. Consistent with an involvement of poly(ADP-ribose) polymerase (PARP) in ZnPT-induced energy crisis, ATP depletion could be antagonized by pharmacological inhibition of PARP. This result was independently confirmed using PARP-1 knockout mouse embryonic fibroblasts that were resistant to ATP depletion and cytotoxicity resulting from ZnPT exposure. In keratinocytes and melanocytes, single-cell gel electrophoresis and flow cytometric detection of gamma-H2A.X revealed rapid induction of DNA damage in response to ZnPT detectable before general loss of cell viability occurred through caspase-independent pathways. Combined with earlier experimental evidence that documents penetration of ZnPT through mammalian skin, our findings raise the possibility that this topical antimicrobial may target and compromise keratinocytes and melanocytes in intact human skin.

Figures

Fig. 1
Fig. 1
Gene expression array analysis of ZnPT-treated human skin keratinocytes. Differential gene expression in HEKs exposed to ZnPT (500 nM; 24 h) or left untreated was analyzed using the RT2 Human Stress and Toxicity Pathway FinderTM PCR Expression Array performed in three independent repeat experiments and analyzed using the two-sided Student’s t test. Changes in cycle threshold (Ct) for genes of interest relative to GAPDH for untreated control (x-axis) versus ZnPT-treated (y-axis) cells are displayed as scatter blot. Upper and lower lines represent the cutoff indicating 3-fold up- or downregulated expression, respectively. The arrows specify the ten genes with statistically significant (p < 0.05) ZnPT-induced up- or downregulation of expression by at least 3-fold as summarized in the table
Fig. 2
Fig. 2
Upregulation of HSPA6, HSP70, and HO-1 protein levels in ZnPT-treated human skin keratinocytes. a Induction of HSPA6 protein expression in ZnPT-treated HEKs was determined by ELISA analysis at 24-h exposure. b and c Western blot analysis of ZnPT-induced HSP70 and HO-1 upregulation was performed in HEKs. b Time course of HSP70 and HO-1 upregulation induced by ZnPT. c Dose response of ZnPT-induced HSP70 and HO-1 upregulation analyzed at 24-h exposure
Fig. 3
Fig. 3
Antiproliferative and cell death-inducing activity of ZnPT in human skin keratinocytes. a Dose response relationship of ZnPT-induced inhibition of cell proliferation. After 72 h of exposure to increasing concentrations of ZnPT and ZnSO4 (1 µM), proliferation was examined by cell counting and expressed as % of untreated control (mean ± SD, n ≥ 3). Representative light microscopy pictures were taken after 72 h of exposure; I control, II ZnPT (250 nM), III ZnPT (400 nM). b Dose response of ZnPT-induced cell death. Cells were exposed to ZnPT (24 h) or left untreated, and viability was assessed by flow cytometric analysis of AV-FITC/PI-stained cells. c Time course of ZnPT-induced cell death. Cells were exposed to ZnPT or left untreated, and viability was assessed by flow cytometric analysis. d Induction of cell death upon extended exposure (24 h) to ZnPT in the absence or presence of the pan-caspase inhibitor zVADfmk (42 µM), NAC (10 mM), or DTPA (60 µM) was assessed by flow cytometric analysis. The numbers indicate viable cells (AV, PI, lower left quadrant) in percent of total gated cells (mean ± SD, three independent experiments). Flow cytometric panels depict one representative experiment
Fig. 4
Fig. 4
PARP-dependent ATP depletion in ZnPT-treated human skin keratinocytes and mouse embryonal fibroblasts. a Time course of cellular ATP depletion in HEKs exposed to ZnPT in the absence or presence of PARP inhibitors (3-ABA, 4 mM; PJ-34, 0.5 µM; added 1 h before ZnPT). b Cellular ATP depletion in HEKs exposed to ZnPT (1 µM, 6 h) in the absence or presence of DTPA (60 µM; added 1 h before ZnPT). c Cellular glutathione levels in HEKs exposed to a dose range of ZnPT (100–1,000 nM, 6 h). Changes in cellular glutathione were not significant (n.s.). d Time course of cellular ATP depletion in PARP−/− versus wild-type MEFs exposed to ZnPT (mean ± SD, n = 3). e Immunoblot detection of PARP-1 protein in PARP−/− versus wild-type MEFs. f Western blot analysis of ZnPT-induced HSP70 upregulation in PARP−/− versus wild-type MEFs as a function of exposure time. g Western blot analysis of ZnPT (500 nM)-induced HSP70 and HO-1 upregulation in HEKs as a function of exposure time and PJ-34 pretreatment (0.5 µM). h Induction of cell death in PARP−/− versus wild-type MEFs upon exposure to ZnPT (24 h) as assessed in Fig. 1b–d. The numbers indicate viable cells (AV, PI, lower left quadrant) in percent of total gated cells (mean ± SD, three independent experiments). Flow cytometric panels depict one representative experiment. i Formation of poly(ADP-ribose) polymer (PAR)-modified cellular proteins as detected by immunoblotting of cellular extracts obtained from HEKs exposed to ZnPT (5 µM) as a function of exposure time. Left panel Ponceau-stained nitrocellulose membrane confirming equal protein loading; right panel PAR immunoblot
Fig. 5
Fig. 5
Impairment of genomic integrity in ZnPT-treated human skin keratinocytes. a Average comet tail moments. HEKs were exposed to ZnPT (100–500 nM; 1 to 12 h), and DNA damage was detected using the alkaline comet assay. As a positive control, cells were exposed to H2O2 (100 µM, 30 min). b Representative comets as visualized by fluorescence microscopy. C untreated control, H2O2 positive control; right panels ZnPT. c Induction of g-H2A.X in ZnPT-treated HEKs (1 µM, 1 and 6 h of exposure, left panel). As a positive control, g-H2A.X was detected in UVB-treated (240 mJ/cm2) cells (1 and 6 h after UV, right panel). d Assessment of oxidative DNA damage using the Fpg-endonuclease modified comet assay. Cells were exposed to ZnPT (500 nM; 3 h) or H2O2 (100 µM; 20 min). Fold increase in mean tail moment of nuclei after incubation in buffer plus Fpg versus buffer only was calculated as specified in “Materials and methods”
Fig. 6
Fig. 6
ZnPT-induced impairment of proliferation, genomic integrity, and ATP homeostasis with upregulated HSP70 expression in human skin melanocytes. a Dose response relationship of ZnPT-induced inhibition of cell proliferation. After 72 h of exposure to increasing concentrations of ZnPT, proliferation was determined as specified in Fig. 3a. b Dose response of ZnPT-induced HSP70 protein upregulation by immunoblot analysis (24 h of exposure). c Cellular ATP depletion induced by ZnPT (1 h) in the absence or presence of 3-ABA (4 mM, added 1 h before ZnPT). d Average comet tail moment induced by ZnPT (1 h exposure) and H2O2 (100 µM, 30 min exposure) performed as detailed in Fig. 5a. IIV depict representative comets as visualized by fluorescence microscopy: I untreated control, II H2O2, III ZnPT (100 nM), IV ZnPT (500 nM)

Similar articles

See all similar articles

Cited by 21 articles

See all "Cited by" articles

Publication types

MeSH terms

Feedback