Niacinamide (NIA), also known as nicotinamide, is a hydrophilic amide of vitamin B3 that is an important component in various cosmetics and medicines. NIA is found in a wide array of foods such as fish, mushroom, and nuts (Damian, 2010). As a typical medicine for treating pigmentary disorders, NIA blocks the melanosome migration between melanocytes and keratinocytes and suppresses skin pigmentation (Bissett et al., 2007; Rolfe, 2014). The biological activities of NIA also include antimicrobial, photo-protection, lighting, and anti-pruritus (Wohlrab and Kewft, 2014). Moreover, as a coenzyme in the glycolysis pathway, NIA promotes the repair of DNA damage induced by UV in keratinocytes (Sousa et al., 2012; Surjana et al., 2013).

Due to excessive consumption of fossil fuels, air pollution has become a major health hazard for humans (Park et al., 2018). Both indoor and outdoor particulate matter (PM) damage human systems, such as bronchial epithelial cells (Yuan et al., 2019), the cardiovascular system (Cao et al., 2016), the central nervous system (Wang et al., 2017), and the pulmonary immune system (Li et al., 2017). In addition, PM can penetrate the skin barrier, mainly by appendageal route and stratum corneum, thereby disrupting the protective activity of the skin (Pan et al., 2015), causing wrinkling and thickening (Kim et al., 2016). PM contributes to skin aging by inducing oxidative stress and inflammation (Magnani et al., 2016). We have previously reported that PM induces oxidative stress, promotes apoptosis, and causes dysfunction of keratinocytes (Piao et al., 2018; Zhen et al., 2018, 2019).

In this study, we focused on particulate matter <2.5 μm (PM_2.5), which can attach to epidermal skin due to its small size. PM_2.5 is known to stimulate the generation of reactive oxygen species (ROS) in keratinocytes (Hyun et al., 2019b). NIA has been shown to suppress oxidative stress notably in vivo (Abdullah et al., 2018). However, there are no reports on the cytoprotective action of NIA against PM_2.5-induced oxidative stress in human keratinocytes. Therefore, in this study, we examined whether NIA can protect HaCaT cells against PM_2.5-induced damage.

Preparation of NIA and PM_2.5

NIA (Fig. 1A) was purchased from Sigma-Aldrich, Inc. (St. Louis, MO, USA) and dissolved in dimethylsulfoxide (DMSO). Diesel particulate matter NIST SRM 1650b (PM_2.5, Sigma-Aldrich) was dissolved in DMSO and stored at a concentration of 25 mg/mL (Piao et al., 2018).

Cell culture

HaCaT human keratinocytes from Cell Lines Service (Heidelberg, Germany) were cultured in Dulbecco’s Modified Eagle’s Medium (Life Technologies Co., Grand Island, NY, USA) with 10% heat-inactivated fetal calf serum at 37°C with 5% CO₂.

Cell viability

The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to test the cytotoxicity of NIA. Cells were seeded into a 24-well plate with 12.5, 25, 50, 100, or 200 μM of NIA. Then, MTT solution was added to each well and samples were incubated for 4 h. Finally, the solutions from DMSO and yield formazan crystals were detected at 540 nm using the scanning multi-well spectrophotometer (Piao et al., 2017).

Determination of intracellular ROS

To investigate anti-oxidative stress effect of NIA, we used 2′,7′-dichlorofluorescein diacetate (DCF-DA, Sigma-Aldrich) staining. ROS generation was induced using PM_2.5 (50 μg/mL). After 16 h of incubation, the cells were treated with 12.5, 25, 50, 100, or 200 μM of NIA and PM_2.5, followed by addition of 25 μM DCF-DA. The fluorescence of DCF-DA was measured using a flow cytometer (Becton Dickinson, Mountain View, CA, USA) (Piao et al., 2018). The inhibitory effect of NIA (100 μM) on PM_2.5-induced ROS was assessed by a confocal microscope (Carl Zeiss, Oberkochen, Germany).

NADP/NADPH assay

To determine the ratio of intracellular NADP and NADPH, we used NADP/NADPH assay kit (Abcam, Cambridge, UK) according to the manufacturer’s instructions.

Measurement of intracellular superoxide levels by dihydroethidium (DHE) oxidation

To detect superoxide levels in cells, we measured the DHE oxidation. Cells were treated with NIA and PM_2.5 and incubated with DHE (10 μM) for 30 min. After incubation, the fluorescence intensity was analyzed by a confocal microscope.

Lipid peroxidation assay

Lipid oxidative stress was investigated using two methods. The cells were dyed with 5 μM diphenyl-1-pyrenylphosphine (DPPP, Sigma-Aldrich) and fluorescence was analyzed using the confocal microscope. Harvested cells were subjected to analysis using the 8-Isoprostane ELISA kit (Cayman Chemical, Ann Arbor, MI, USA) according to the manufacturer’s instructions (Zhen et al., 2019).

Protein carbonylation assay

The harvested cells were used for detecting protein oxidation with an Oxiselect^TM Protein Carbonyl ELISA kit (Cell Biolabs, San Diego, CA, USA) according to the manufacturer’s instructions.

Detection of 8-oxoguanine (8-oxoG)

To quantify oxidative DNA damage, we determined the level of 8-oxoG through the Bioxytech 8-OHdG ELISA kit (OXIS Health Products, Portland, OR, USA) according to the manufacturer’s instructions. For image analysis, the cells were fixed on a chamber slide and stained with avidin-tetramethylrhodamine isothiocyanate (TRITC) (1:200) conjugate (Sigma-Aldrich), and fluorescence images were obtained using a confocal microscope (Piao et al., 2011).

Single cell gel electrophoresis (Comet assay)

The comet assay was used to detect DNA damage induced by PM_2.5. Harvested cells were dispersed in low-melting agarose (1%). Then, the mixtures were solidified on microscopic slides, and the slides were immersed in lysis buffer (2.5 M NaCl, 100 mM Na-EDTA, 10 mM Tris, 1% Trion X-100, and 10% DMSO, pH 10) for 1 h at 4°C. After electrophoresis, the slides were stained with ethidium bromide, and the percentage of the comet tail fluorescence and the tail length (50 cells per slide) was determined using a fluorescence microscope equipped with an image analysis software (Kinetic Imaging, Komet 5.5, Liverpool, UK) (Park et al., 2017).

Quantification of Ca²⁺ level

To determine the intracellular Ca²⁺ in viable cells, the cells were co-cultured with 10 μM fluo-4-acetoxymethyl ester (Fluo-4-AM, Sigma-Aldrich) dye for 30 min, and green fluorescence in the confocal micrographs was quantified.

Mitochondrial membrane potential (Δψ_m) analysis

To visualize the changes in membrane potential, the cells were stained with 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylben zimidazolylcarbocyanine iodide (JC-1, Invitrogen, Carlsbad, CA, USA) and were analyzed by confocal microscopy.

Hoechst 33342 staining

To observe apoptotic bodies, we used the nuclear-specific dye, Hoechst 33342 (Sigma-Aldrich). Cells were pre-treated with 100 μM NIA, treated with PM_2.5 for 24 h, and then stained with Hoechst 33342 (10 μM). Nuclei were visualized under a fluorescence microscope equipped with a Cool SNAP-Pro color digital camera (Media Cybernetics, Silver Spring, MD, USA) (Han et al., 2019).

Statistical analysis

Data are shown as mean ± standard error, and were analyzed using the Tukey’s test and analysis of variance (ANOVA) by the Sigma Stat (v12) software (SPSS, Chicago, IL, USA). p-values <0.05 were considered statistically significant.

NIA showed anti-oxidative effect

Here, we used PM_2.5 at a concentration of 50 μg/mL for ROS induction. This concentration was selected based on the findings of our recent study investigating whether PM_2.5 induced oxidative stress. In that study, we measured ROS generation at various concentrations (25–100 μg/mL) of PM_2.5, and 50 μg/mL PM_2.5 was found to be optimal concentration to clearly observe oxidative stress-induced cell damage and inflammatory response (Piao et al., 2018; Ryu et al., 2019). In the MTT assay, NIA, up to 100 μM, showed no toxicity toward human keratinocytes, but cytotoxicity was observed at 200 μM (Fig. 1B). In addition, NIA dose-dependently inhibited the ROS generation induced by PM_2.5 (Fig. 1C). To further investigate the anti-oxidative effect of NIA, we used 100 μM NIA as the optimal concentration for subsequent experiments.

NIA blocked ROS generation via NADPH oxidase activity

Evaluation of the NADPH oxidase (NOX) activity (NADP/NADPH ratio) showed that PM_2.5 increased the oxidation of NADPH, which was reversed by NIA pre-treatment (Fig. 2A). In the DCF-DA staining assay, NIA inhibited PM_2.5-induced fluorescence in the cells, demonstrating that NIA protected cells from PM_2.5-induced ROS generation (Fig. 2B). Similarly, DHE staining revealed that superoxide generation induced by PM_2.5 was blocked by NIA (Fig. 2C). These results further indicated that NIA exerted antioxidant effects in keratinocytes.

NIA inhibited PM_2.5-induced lipid peroxidation

The detection of phosphine oxide (DPPP oxide) using the DPPP probe indicated that PM_2.5 significantly induced lipid peroxidation, and pretreatment with NIA decreased the fluorescence emitted by the probe (Fig. 3A). Moreover, the levels of 8-isoprostane, a specific indicator of lipid peroxidation, revealed that NIA suppressed PM_2.5-induced peroxidation (Fig. 3B). These results showed that NIA rescued cells from PM_2.5-induced lipid oxidation.

NIA suppressed PM_2.5-induced oxidative stress on proteins

The levels of protein carbonylation, a specific indicator of protein oxidation, revealed that NIA significantly inhibited PM_2.5-induced oxidative protein modification (Fig. 4). These results showed that NIA rescued cells from PM_2.5-induced protein carbonylation.

NIA protected cells from PM_2.5-induced DNA damage

The 8-OHdG assay was used to assess oxidative damage on DNA caused by PM_2.5 (Fig. 5A). Notably, NIA reduced 8-OHdG generation induced by PM_2.5. Furthermore, confocal microscopy showed that the level of 8-oxoG in PM_2.5-exposed cells was the highest, suggesting severe DNA lesions formed via avidin-TRITC binding. Consistent with other results, NIA ameliorated the DNA lesions (Fig. 5B). The protective effect of NIA on DNA damage was also observed in the comet assy. Length of comet tails and the percentage of tail fluorescence were increased by PM_2.5, but decreased following NIA pretreatment (Fig. 5C). Collectively, these results illustrated that NIA protected against DNA damage induced by PM_2.5.

NIA lowered excessive intracellular Ca²⁺, balanced membrane potential, and suppressed apoptosis induced by PM_2.5

Intracellular Ca²⁺, tracked by Fluo-4-AM, was detected by confocal microscopy. The image analysis revealed that PM_2.5 stimulated excessive Ca²⁺, which could be reduced by NIA treatment (Fig. 6A). JC-1 staining was used to determine Δψ_m, with red and green fluorescence representing polarization and depolarization, respectively. The images obtained from confocal microscopy showed that Δψ_m polarization and depolarization were decreased and increased by PM_2.5 treatment, respectively; however, they were reversed by NIA pretreatment (Fig. 6B). PM_2.5 also promoted apoptotic bodies, when observed using Hoechst 33342 staining, and NIA pretreatment reduced their numbers (Fig. 6C). These results proved that PM_2.5 disrupted the homeostasis of intracellular Ca²⁺ levels and accelerated cell apoptosis, but NIA exerted cytoprotective effects against these PM_2.5-induced damages.

The skin is the outermost organ and acts as the first protective layer from air pollution. Current studies indicate that air pollutants damage skin via two main routes. The first route is from the outside to inside, whereby PM penetrates the skin (including keratinocytes), and the second route is from the inside to the outside, whereby toxic effects in the lung subsequently influence the skin (Krutmann et al., 2014). In addition, PM_2.5 stimulates inflammation by disrupting the cytokine network, thereby causing epidermal hyperplasia (Kim et al., 2017). Moreover, PM is known to cause oxidative damage to keratinocytes by promoting ROS generation (Romani et al., 2018). NIA is a well-known skin whitening agent (Hakozaki et al., 2002) and is known to improve skin barrier function and decrease sebum secretion (Draelos et al., 2005). In the present study, we mainly focused on the potential protective effect of NIA on PM_2.5-induced oxidative damage in keratinocytes. NIA pretreatment at all tested concentrations inhibited PM_2.5-induced ROS generation in cells.

A previous study showed that ROS is involved in various biological processes, including oxygen sensing, cell growth, cell differentiation, and cell death (Touyz et al., 2019). NOX plays a vital role in intracellular superoxide and hydrogen peroxide production (Cachat et al., 2015) and can be induced by particle exposure in keratinocytes (Kampfrath et al., 2011; Sticozzi et al., 2012). NOXs are widely distributed in mammalian tissues and, thereby, serve various functions (Touyz et al., 2019). The six homologs of the cytochrome subunit of the NOX (NOX1, NOX3, NOX4, NOX5, DUOX1, and DUOX2) are predominantly responsible for ROS production in mammalian cells (Tarafdar and Pula, 2018). NADPH is critical for the antioxidant system and reductive biosynthesis, and is required by enzymes involved in the pathologies of metabolic disorders due excessive ROS generation (Gomez-Sanchez et al., 2008; Legeza et al., 2013). Therefore, we determined the NADP/NADPH ratio, and the results showed that PM_2.5 increased the oxidized NADP ratio, while NIA decreased the this NADP ratio. In addition, NIA cleared intracellular ROS induced by PM_2.5.

Next, we examined the effect of PM_2.5 on the three main molecules in cells, namely lipids, proteins, and DNA. ROS is known to damage lipids and proteins via lipid peroxidation and protein carbonylation, respectively (Hyun et al., 2019a), as well as damaging DNA stability. Lipid peroxidation is regarded as a general oxidative process, and is related to cell death. Furthermore, ROS accumulation, induced by stimuli such as tobacco smoke, can directly damage lipids (Ayala et al., 2014). Our results showed that PM_2.5 exposure damaged lipids, while NIA protected the lipids from peroxidation caused by PM_2.5. Oxidative stress also increased protein carbonylation, which is associated with cell damage and cell disease and is considered one of the most harmful irreversible protein modifications (Fedorova et al., 2014). NIA showed preventive action against protein oxidation induced by PM_2.5. Previous study noted that PM_2.5 caused early arrest of cell cycle, resulting from induced DNA damage, and 8-oxoG, the oxidative stress biomarker, is the predominant adduct of ROS-induced oxidative changes (Abbas et al., 2019). NIA has been shown to improve DNA repair following damage induced by ultraviolet radiation (Park et al., 2010; Snaidr et al., 2019). In our study, we evaluated three indices of DNA stability (8-OHdG levels, 8-oxoG intensity, and DNA tails), and all results showed that NIA protected cells from PM_2.5-induced DNA lesions. Thus, NIA protects cells from PM_2.5-induced damage.

Calcium plays a key role in cell survival, as well as it can improve apoptosis. Ca²⁺ mediated pro-apoptotic action is a response to many endogenous organelles, including mitochondria (Hajnoczky et al., 2003). Ca²⁺ from endoplasmic reticulum plays an important role in mitochondria-related metabolic activity (Sarasija et al., 2018). The increased intracellular Ca²⁺ affects Δψ_m and the disruption of mitochondrial membrane permeability is related to cell death (Kroemer et al., 2007; Zorova et al., 2018). During the process of apoptosis, mitochondria receive pro-apoptotic signals and release proapoptotic proteins (Orrenius et al., 2015). Notably, PM_2.5 can induce cell apoptosis via various pathways, such as disruption of mitochondrial polarization, DNA fragmentation, and expression of pro-apoptotic proteins (Peixoto et al., 2017). All our results demonstrated that PM_2.5 increased intracellular Ca²⁺ levels, disrupted the balance of mitochondrial potential, and promoted apoptosis, NIA treatment could inhibit these effects.

Taken together, our findings show that PM_2.5 notably aggravated skin cell damage by inducing ROS generation, disrupting cellular components, and activating apoptotic pathways. However, cells pre-treated with NIA were protected from the ROS-induced lipid peroxidation, protein carbonylation, and DNA damage (Fig. 7). NIA also inhibited PM_2.5-induced apoptosis by maintaining both Ca²⁺ levels and mitochondrial membrane potential in a steady state. These results suggest that NIA can protect against PM_2.5-induced skin damage.

The authors declare that there are no conflicts of interest.

This work was supported by grant from the Basic Research Laboratory Program (NRF-2017R1A4A1014512) by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIP). This research was supported by the 2019 scientific promotion program funded by Jeju National University.