DNA damage and somatic mutations in mammalian cells after irradiation with a nail polish dryer

Abstract

Ultraviolet A light is commonly emitted by UV-nail polish dryers with recent reports suggesting that long-term use may increase the risk for developing skin cancer. However, no experimental evaluation has been conducted to reveal the effect of radiation emitted by UV-nail polish dryers on mammalian cells. Here, we show that irradiation by a UV-nail polish dryer causes high levels of reactive oxygen species, consistent with 8-oxo-7,8-dihydroguanine damage and mitochondrial dysfunction. Analysis of somatic mutations reveals a dose-dependent increase of C:G>A:T substitutions in irradiated samples with mutagenic patterns similar to mutational signatures previously attributed to reactive oxygen species. In summary, this study demonstrates that radiation emitted by UV-nail polish dryers can both damage DNA and permanently engrave mutations on the genomes of primary mouse embryonic fibroblasts, human foreskin fibroblasts, and human epidermal keratinocytes.

Fig. 1. Overview of the overall study design.

a Primary mammalian cells, MEFs and HFFs, were expanded into 6-well plates and treated with ultraviolet light (UV) emitted from a UV-nail polish dryer for 20 min, twice a day within one single day, termed acute UV exposure. For chronic UV exposure, primary cells were exposed consecutively in three different days with each exposure lasting 20 min. Control samples were maintained in the dark in pre-warmed PBS for 20 minutes during each exposure session. After recovery and cellular selection, whether through senescence bypass or single-cell subcloning, control and irradiated cell were grown for the same number of doubling populations and subjected to bulk whole-genome sequencing. Analysis of whole-genome sequenced samples was performed using our established pipelines for mutation calling. Analysis of mutational signatures was performed using the SigProfiler suite of tools. b Primary HEKa cells were irradiated in 10-cm dishes using UV-nail polish dryer for 20 minutes for three consecutive days. Control samples were maintained in the dark in pre-warmed PBS for 20 minutes during each exposure session. HEKa cells were kept in culture for 14 days for recovery and replication, allowing around 8 doubling populations after which the cells were subjected to high coverage duplex sequencing. Analysis of duplex sequencing samples was performed using commercially established pipelines. Analysis of mutational signatures was performed using the SigProfiler suite of tools. Asterisks in (a) and (b) denote timepoints when DNA damage and other assessments were performed for each condition, including interrogation of cytotoxicity, genotoxicity, oxidative damage, and mitochondrial damage (Supplementary Table 1).

Fig. 2. Cytotoxicity and genotoxicity in mammalian cells after irradiation with a UV-nail dryer.

a Cytotoxicity assessment following exposure of primary MEFs (top panel), HFFs (middle panel), and HEKa (bottom panel) to UV radiation emitted from a UV-nail polish dryer for different timepoints, ranging from 0 to 20 min. Multiple UV-exposure sessions were tested with one hour difference between each consecutive exposure, including: gray—one exposure in a day; yellow – two exposures in a day; red—three exposures in a day. Formazan dye absorbance was measured 48 hours after treatment cessation and was normalized to the number of unirradiated control cells at timepoint 0. The results are presented as mean percentage ± standard error from at least four replicates (n = 4 for MEFs, n = 5 for HFFs, n = 4 for HEKa). Statistically significant results from FDR corrected Mann–Whitney U two-sided tests are denoted as: *q-value < 0.05. b DNA damage evaluation by immunofluorescence of Ser139-pjosphorylated histone H2Ax (γH2Ax). Primary MEFs (top panel), HFFs (middle panel) and HEKa (bottom panel), were exposed to UV radiation either acutely or chronically. DAPI is shown in blue, Phalloidin in red, and γH2Ax in green. Quantification of the number of γH2Ax foci was performed by analyzing 100 cells per condition, collected from at least 3 independent experiments, in MEF (n = 4) (c), HFF (n = 3) (d) and HEKa (n = 3) (e) cells. The bounds of the boxplots represent the interquartile range divided by the median, and Tukey-style whiskers extend to a maximum of 1.5 × interquartile range beyond the box. Statistically significant results from FDR corrected Mann–Whitney U two-sided tests are denoted as q-values.

Fig. 3. Oxidative damage in mammalian cells after irradiation with a UV-nail dryer.

a Assessment of oxidative DNA damage by live imaging of CellROX green reagent in control and irradiated MEF (top panel), HFF (middle panel), and HEKa (bottom panel) cells. DAPI is shown in blue while CellROX is shown in green. b HEKa cells were challenged with 2mM N-acetyl-L-cysteine (NAC) for 1 hour prior to irradiation. Quantification of the number of CellROX foci was performed by analyzing at least 100 cells per condition and comparing cells with and without NAC pre-treatment, collected from triplicates. The bounds of the boxplots represent the interquartile range divided by the median, and Tukey-style whiskers extend to a maximum of 1.5 × interquartile range beyond the box. Oxidative DNA damage measurement of 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxo-dG) by ELISA in MEF (c), HFF (d), and HEKa (e) cells. Measurements of 8-oxo-dG were performed at different timepoints (0, 4, 24 hours post-treatment). Measurements of 8-oxo-dG were also performed on a negative control (NC), consisting of water only, and on three positive controls, namely, hydrogen peroxide (H₂O₂), ultraviolet C radiation (UVC), and potassium bromate (KBrO₃). In all cases, data are presented as a mean value ± standard deviation from n = 3 independent biological replicates. For all panels: statistically significant results from FDR corrected Mann–Whitney U two-sided tests are denoted as: *q-value < 0.05; **q-value < 0.01; ***q-value < 0.001; and ****q-value < 0.0001.

Fig. 4. Mitochondrial disruption after irradiation with a UV-nail dryer in HEKa cells.

a Evaluating the mitochondrial membrane potential after irradiation of HEKa cells with a UV-nail dryer. DAPI is shown in blue, MitoTracker in green, and tetramethylrhodamine dye (TMRM) in red. Yellow corresponds to overlaps between MitoTracker and TMRM. Images collected from three independent experiments. b Examining mitochondrial ROS production in HEKa cells. DAPI is shown in blue, MitoTracker in green, and MitoSOX in red. Yellow corresponds to overlaps between MitoTracker and MitoSOX. In both (a) and (b), MitoTracker green reagent is used to localize mitochondria regardless of mitochondria membrane potential. Images collected from three independent experiments. c Quantification of the number of MitoSOX foci in 100 cells per condition in HEKa, examined over 4 independent replicates. The bounds of the boxplots represent the interquartile range divided by the median, and Tukey-style whiskers extend to a maximum of 1.5 × interquartile range beyond the box. Statistically significant results from FDR corrected Mann–Whitney U two-sided tests are denoted as q-values.

Fig. 5. Mutations found in the genomes of MEFs and HFFs irradiated by a UV-nail dryer.

a Mutation count per megabase (Mb) detected in the different conditions, represented in colors, in MEFs and HFFs. Data is presented as a mean value ± standard error from n = 5 independent biological replicates. Statistically significant results from FDR corrected Mann–Whitney U two-sided tests are denoted as: *q-value<0.05; **q-value < 0.01. b Fold increase of single base substitutions in UV-treated clones compared to controls. Fold increase is expressed as mean fold-change ± SE (standard error) from n = 5 independent replicates. c Spearman’s rank correlations between the number of C > A substitutions and the number UV exposures in the five independent UV-treated clones in MEFs (top panel) and HFFs (bottom panel). Acute and chronic exposures correspond to 2 and 3 exposures, respectively. P-value is calculated based on a two-sided t-distribution with n-2 degrees of freedom. Gray bands represent the 95% confidence intervals. d Number of small insertions and deletions (indels) in MEF and HFF clones for each irradiation condition.

Fig. 6. Mutation analysis of HEKa cells irradiated by a UV-nail dryer.

a Mutation count per megabase (Mb) detected in the different conditions, represented in colors, in HEKa cells. Data is presented as a mean value ± standard error from n = 3 independent biological replicates. b Fold increase of single base substitutions in UV-treated clones compared to controls. Fold increase is expressed as mean fold-change ± SE (standard error) from n = 3 independent replicates. c Spearman’s rank correlations between the number of C > A substitutions and the number UV exposures in each of the 3 biological replicates. P-value is calculated based on two-sided t-distribution with n-2 degrees of freedom. Gray bands represent the 95% confidence intervals. d Number of small insertions and deletions (indels) in HEKa cells. Statistically significant results from FDR corrected Mann–Whitney U tests are denoted as: *q-value < 0.05; **q-value < 0.01; ***q-value < 0.001; and ****q-value < 0.0001.

Fig. 7. Somatic Mutations due to COSMIC signatures SBS18/36 in cells and cancer samples.

a The patterns of mutational signatures COSMIC SBS18 and SBS36. b Number of mutations per megabase attributed to COSMIC SBS18/36 in UV exposed MEF clones, per condition, extracted from n = 5 biological replicates per condition. c Number of mutation per megabase attributed to COSMIC SBS18/36 in UV exposed HFF clones, per condition, extracted from n = 5 biological replicates per condition. d Mutational patterns of irradiated (upper track) and control (lower track) primary HEKa cells. e COSMIC signatures assignment of UV-irradiated HEKa cells (upper track) and control (lower track). f Pearson’s correlation of C > A and C > T mutations in n = 144 skin cancer samples from the PCAWG project. P-value is calculated based on two-sided t-distribution with n-2 degrees of freedom. The bounds of the boxplots represent the interquartile range divided by the median, and Tukey-style whiskers extend to a maximum of 1.5 × interquartile range beyond the box.