The sunless tanning agent dihydroxyacetone induces stress response gene expression and signaling in cultured human keratinocytes and reconstructed epidermis

Abstract

Sunless (chemical) tanning is widely regarded as a safe alternative to solar UV-induced skin tanning known to be associated with epidermal genotoxic stress, but the cutaneous biology impacted by chemical tanning remains largely unexplored. Chemical tanning is based on the formation of melanin-mimetic cutaneous pigments ('melanoidins') from spontaneous amino-carbonyl ('glycation') reactions between epidermal amino acid/protein components and reactive sugars including the glycolytic ketose dihydroxyacetone (DHA). Here, we have examined the cutaneous effects of acute DHA-exposure on cultured human HaCaT keratinocytes and epidermal reconstructs, profiled by gene expression array analysis and immunodetection. In keratinocytes, DHA-exposure performed at low millimolar concentrations did not impair viability while causing a pronounced cellular stress response as obvious from rapid activation of phospho-protein signal transduction [p-p38, p-Hsp27(S15/S78), p-eIF2α] and gene expression changes (HSPA6, HMOX1, CRYAB, CCL3), not observable upon exposure to the non-ketose, tanning-inactive DHA-control glycerol. Formation of advanced glycation end products (AGEs) from posttranslational protein-adduction was confirmed by quantitative mass spectrometric detection of N-ε-(carboxyethyl)-l-lysine (CEL) and N⁷-carboxyethyl-l-arginine, and skin cells with CRISPR-Cas9-based elimination of the carbonyl stress response gene GLO1 (encoding glyoxalase 1) displayed hypersensitivity to DHA-cytotoxicity. In human epidermal reconstructs a topical use-relevant DHA-dose regimen elicited a comparable stress response as revealed by gene expression array (HSPA1A, HSPA6, HSPD1, IL6, DDIT3, EGR1) and immunohistochemical analysis (CEL, HO-1, p-Hsp27-S78). In DHA-treated SKH-1 hairless mouse skin IHC-detection revealed epidermal occurrence of CEL- and p-Hsp27-epitopes. For comparison, stress response gene expression array analysis was performed in epidermis exposed to a supra-erythemal dose of solar simulated UV (2 MEDs), identifying genes equally or differentially sensitive to either one of these cutaneous stimuli [DHA ('sunless tanning') versus solar UV ('sun-induced tanning')]. Given the worldwide use of chemical tanners in consumer products, these prototype data documenting a DHA-induced specific cutaneous stress response deserve further molecular exploration in living human skin.

Keywords: Dihydroxyacetone; Glycation; Phosphoprotein signaling; Reconstructed human epidermis; Stress response gene expression; Sunless tanning.

Fig. 1

DHA attenuates cell viability, proliferation and cell cycle progression in human keratinocytes. (a) Impairment of cellular viability in response to acute DHA exposure (≤50 mM; 1 h in PBS followed by 24 h in growth medium) as assessed by flow cytometry (annexin V-PI staining). Numbers in quadrants indicate viable cells (AV-negative, PI-negative) in percent of total gated cells; bar graph summarizes numerical values (mean ± SD). (b) DHA-induced impairment of cellular proliferation [≤ 50 mM, continuous exposure (72 h) in growth medium; n = 3]. (c) DHA-induced cell cycle alteration [≤ 30 mM, continuous exposure (48 h) in growth medium] as assessed by flow cytometry of PI-stained cells; bar graph summarizes numerical values (mean ± SEM, n = 3). (d) Representative cell cycle histograms per treatment group.

Fig. 2

Array analysis reveals early stress response gene expression in human keratinocytes receiving acute DHA exposure. (a) HaCaT keratinocytes underwent short term DHA exposure (20 mM; 1 h in PBS followed by 5 h in growth medium). Stress response gene expression was then assessed by RT² Profiler™ Gene Expression Array analysis (volcano blot; p < 0.05). (b) Table summarizes numerical values (n = 3, p < 0.05). (c) Confirmatory single RT-qPCR analysis (HMOX1, mean ± SEM). (d) Immunoblot analysis profiling stress response protein expression (≤30 mM DHA, ≤ 24 h); bar graphs summarize densitometric analysis of antigens (mean ± SEM).

Fig. 3

Acute DHA exposure induces dose- and time-dependent phospho-protein stress signaling in human keratinocytes. Protein phosphorylation in response to acute DHA exposure was profiled by immunoblot analysis: (a) dose-response relationship (≤30 mM DHA in PBS; 1 h); (b) time course (20 mM DHA in PBS; ≤ 2 h); bar graphs summarize densitometric analysis of antigens (mean ± SEM).

Fig. 4

Glycation stress and AGE-formation in DHA-exposed human keratinocytes. (a) DHA-induced (≤40 mM, 2 h; mean ± SEM) intracellular oxidative stress as assessed by flow cytometric analysis of DCF-stained cells. (b) DHA-induced activation of DNA damage response as detected by flow cytometric analysis of γ-H2AX staining. (c) DHA-induced (20 mM) oxidative DNA damage as detected by comet assay with FPG-digestion for 8-oxo-dG detection [mean comet tail moment ± SEM; representative images (left) with dot blot depiction of quantitative analysis (right)]. (d) Glycerol-induced (≤30 mM; 1 h in PBS) phosphoprotein signaling as assessed by immunoblot analysis; bar graphs summarize densitometric analysis of antigens (mean ± SEM). (e) AGE-type posttranslational modification detected in total protein extracts from DHA-treated keratinocytes (≤20 mM, 1 h in PBS, followed by 5 h in growth medium, n = 6, mean ± SEM) by LC-MS/MS analysis. (f) DHA-modulation (≤20 mM) of GLO1 mRNA expression in HaCaT keratinocytes determined by RT-qPCR. (g) GLO1 mRNA expression in A375_WT and GLO1_KO cells as determined by RT-qPCR; insert depicts representative immunoblot analysis of GLO1 protein levels. (h) GLO1 enzymatic activity compared between A375_WT and GLO1_KO cells. (i) DHA-induced impairment of cell viability (A375_WT versus A375-GLO1_KO) as determined in Fig. 1a; bar graph summarizes numerical values (mean ± SD).

Fig. 5

Differential stress response gene expression analysis in human reconstructed epidermis exposed to topical DHA or acute solar simulated UV. (a) After treatment [DHA (10% in Vanicream™) versus carrier control; 24 h], organotypic human epidermal reconstructs were imaged for colorimetric analysis; left: representative images; right: bar graph summarizing colorimetric values (mean ± SD). (b–c) After treatment [DHA (10% in Vanicream™) versus carrier control; 6 h], organotypic human epidermal reconstructs were analyzed for gene expression changes: (b) Individual RT-qPCR analysis (HSPA1A, HSPA6; mean ± SD; DHA: 1 or 10%). (c) Gene expression changes as determined using the Human Stress and Toxicity PathwayFinder™ PCR Array technology comparing DHA- and carrier-exposed epidermal reconstructs depicted as a volcano plot and summarized numerically in Table 1 (d-e) Comparative gene expression array analysis of solar simulated UV-exposed epidermal reconstructs [240 mJ/cm² UVB versus unexposed carrier control; 6 h after UV) displayed as a volcano plot (d), versus DHA (in Venn diagram depiction with total number of genes per group) (e), and summarized numerically in Table 1. (f) Immunohistochemical analysis of epidermal reconstructs [prepared as in (a)]. (g) Immunohistochemical analysis of SKH-1 mouse skin 3 d after treatment [DHA (10% in Vanicream™) versus carrier control].