The human body is negatively affected by environmental pollutants such as particulate matter, diesel gas, and nicotine smoke (Beamish
Benzo[a]pyrene (B[a]P) is one of the most common environmental pollutants and is classified as a polycyclic aromatic hydrocarbon. It has been found to exert harmful cytotoxic and carcinogenic effects (Hassan
To maintain homeostasis, endogenous antioxidant enzymes in the skin act to return elevated ROS to a normal level. These enzymes include heme oxygenase-1 (HO-1) and NAD(P)H dehydrogenase [quinone] 1 (NQO1). Expression of these antioxidant genes is regulated by nuclear factor erythroid 2-related factor-2 (Nrf2), which is a central transcriptional regulator of antioxidant signaling (Kim
Resorcinol (1,3-Benzenediol or
In the present study, we investigated the protective effect of resorcinol on B[a]P-induced damage and its mechanism of action in HaCaT cells, a human keratinocyte cell line.
HaCaT, a normal human keratinocyte cell line, was cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 1% antibiotics (penicillin/streptomycin) and 10% fetal bovine serum (FBS) in a humidified 37°C and 5% CO2 incubator. The HEK293-TRPV1-luciferase stable cell line was maintained in DMEM supplemented with 10% FBS, 1% antibiotics, and 10% puromycin in a humidified incubator at 37°C and 5% CO2. All TaqMan reverse transcription polymerase chain reaction (RT-PCR) reagents (primers and probes) were obtained from Applied Biosystems (Waltham, MA, USA). Resorcinol (99% purity) was obtained from Sigma-Aldrich (St. Louis, MO, USA) and dissolved in distilled water. Antibodies including anti-AhR, anti-CYP1A1, anti-NQO1, anti-Nrf2, and anti-β-actin were obtained from Sigma-Aldrich.
ROS production was quantitatively measured with the DCFDA-cellular reactive oxygen species detection assay kit (ab113851) using a fluorescence microscope and microplate (Nikon Instruments, Inc., Melville, NY, USA). Cells were seeded on 60-mm dishes or 96-well plates. Cultured cells were irradiated with resorcinol or tert-butyl hydroperoxide (TBHP) solution as a positive control. After 24 h, cells were washed twice in PBS and stained with 25 µM DCFDA in PBS for 15 min at 37°C in the dark. After washing again, the oxidized DCFDA signal was measured at 485/535 nm (excitation/emission). Results were calculated as percentage change from control after background subtraction.
To determine the cytotoxic effect of B[a]P or resorcinol on HaCaT cell growth
ON-TARGETplus SMARTpool human siRNAs were purchased from Thermo Fisher Scientific (Waltham, MA, USA), including siRNA targeting Nrf2 (L-004018-00-0020), targeting AhR (L-004990-00-0020), and non-targeting siRNA (D-001810-10-05). Cells were transfected with 50 nM siRNAs for 24 h using the DharmaFECT transfection agent (Dharmacon Research, Lafayette, CO, USA), according to the manufacturer’s instructions.
Total cellular RNA was prepared from HaCaT cells using the TRIzol reagent (Invitrogen, Carlsbad, CA, USA). For cDNA synthesis, Moloney murine leukemia virus reverse transcriptase and random primers (Invitrogen, Carlsbad, CA, USA) were used according to the manufacturer’s protocols. Real-time RT-PCR analysis was conducted using an ABI7900HT Real-Time PCR Instrument (Applied Biosystems), and pre-designed and optimized Assays-on-Demand (Applied Biosystems) for
To assay the activity of XRE and ARE-containing promoters, cells were transfected with XRE-luciferase (XRE-Luc) (Stratagene, La Jolla, CA, USA) or ARE-luciferase (ARE-Luc) reporters (Addgene, MA, USA), and Renilla-luciferase plasmid (1 μg) (for normalization) (Promega, Madison, WI, USA) using the DharmaFECT® Duo transfection reagent (Thermo Fisher Scientific) according to the manufacturers’ protocols (Kang
An ELISA kit (Invitrogen) was used to measure interleukin-8 (IL-8) levels according to the manufacturer’s protocol. Absorbance measurements were conducted using a Labsystems Multiskan MS microplate reader (Thermo Bio-Analysis Japan, Tokyo, Japan). The results were confirmed by three independent experiments. In order to measure target protein levels, cell lysates were prepared, electrophoresed, and transferred onto polyvinylidene difluoride membranes. The membranes were probed with antibodies (anti-β-actin, anti-Nrf2, anti-CYP1A1, anti-AhR, or anti-NQO1) and imaged using an enhanced chemiluminescence system (Amersham Biosciences, Piscataway, NJ, USA). The results were verified by three independent experiments.
Nuclear fractions were isolated to confirm the translocation of transcription factors by western blotting. NE-PER Nuclear and Cytoplasmic Extraction reagents (78833, Thermo Fisher Scientific) were used according to the manufacturer’s protocol.
All data are presented as mean ± standard deviation (SD). To compare the control and the treated groups, one-way analysis of variance (ANOVA) and Tukey’s multiple-comparison tests were applied using GraphPad Prism (5.0) (GraphPad, La Jolla, CA, USA). A
To examine the effect of resorcinol (Fig. 1A) on B[a]P-induced damage to human keratinocytes, we performed XRE-Luc reporter assays, Western blotting, and real-time PCR analyses for CYP1A1 in HaCaT cells. As shown in Fig. 1B, resorcinol suppressed B[a]P-induced activation of the XRE reporter in a concentration-dependent manner. In addition, B[a]P-induced expression of
To investigate whether resorcinol modifies B[a]P-induced ROS production, we performed a DCFDA cellular ROS detection assay. As shown in Fig. 2A, B[a]P increased cellular levels of ROS, but this effect was reduced by resorcinol co-treatment, as evidenced by ROS image analysis (Fig. 2A) and fluorescence intensity assay (Fig. 2B). These data indicate that resorcinol is protective against B[a]P-induced ROS production. In addition, we examined the effects of resorcinol on B[a]P-induced production of proinflammatory cytokines. As shown in Fig. 2C, B[a]P induced IL-8 production, but this effect was reduced by resorcinol co-treatment. In addition, knock-down of AhR by siRNA, and treatment with N-acetyl cysteine, an antioxidant and reducing agent, also reduced B[a]P-induced IL-8 production (Fig. 2C). AhR siRNA was confirmed to successfully knock-down AhR protein in HaCaT cells when compared with control siRNA (Fig. 2D). These data indicate that resorcinol has antioxidant and anti-inflammatory effects, and that the effect of resorcinol on B[a]P-induced IL-8 production may be mediated through reduction of cellular ROS levels.
In the previous experiments, we found that resorcinol suppresses B[a]P-mediated oxidative and inflammatory effects in human keratinocytes. We next examined whether resorcinol also affects the expression of antioxidant genes. First, we performed an ARE-Luc reporter assay. As shown in Fig. 3A, resorcinol increased ARE reporter activity in a concentration-dependent manner. In addition, resorcinol treatment upregulated Nrf2 protein and mRNA levels (Fig. 3B, 3C). As expected, the expression of NQO1 and HO-1, which are Nrf2 target genes, also increased with resorcinol treatment (Fig. 3B, 3C). Western blotting showed that resorcinol treatment also increased the nuclear translocation of Nrf2. (Fig. 3D). Furthermore, we found that resorcinol-induced ARE activation is mediated by Nrf2. Knock-down of Nrf2 using siRNA attenuated the effect of resorcinol on ARE activation (Fig. 4A) and upregulation of NQO1 and HO-1 genes (Fig. 4B, 4C). Nrf2 siRNA was confirmed to successfully knock-down Nrf2 protein in HaCaT cells in comparison with control siRNA, which did not show such an effect (Fig. 4D). These data indicate that resorcinol induces ARE-dependent antioxidant gene expression by activating Nrf2.
Some molecules have been reported to induce Nrf2-mediated upregulation of HO-1 via AhR activation (Shin
This study demonstrates the protective effects of resorcinol on B[a]P-induced damage to human epidermal keratinocytes. Resorcinol suppressed AhR activity, as shown through suppression of B[a]P-induced XRE reporter activation,
AhR is a xenobiotic chemical sensor highly expressed in epidermal keratinocytes (Furue
Nrf2 is also abundantly expressed in epidermal keratinocytes and is a master transcription factor regulating expression of antioxidant genes (Kim
Paradoxically, in addition to inducing oxidative stress, AhR has been reported in several recent studies to contribute to antioxidative and protective signaling in response to many ligands, including herbal medicines, azoles, and flavonoids (Furue
Nrf2-null mice are reported to have reduced HO-1 expression and a significantly stronger and longer-lasting sunburn reaction to UVB compared with wild-type mice (Kawachi
Resorcinol has been reported to possess anti-melanogenic, anticancer and antibacterial activities. In this study, we demonstrated that resorcinol is protective against B[a]P-induced damage to human epidermal keratinocytes and has antioxidative effects. These effects of resorcinol were mediated by inhibiting AhR and activating Nrf2. Our results suggest that resorcinol could be used as an agent for ameliorating the symptoms induced by B[a]P in the skin.
This research was supported by a grant (code#B0080507000574) from ‘Advanced biomaterials commercialization support’ Program funded by Ministry of Trade, Industry & Energy (MOTIE), Korea Testing Laboratory (KTL) and a grant from Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science and Technology Information and Communication (Grant No. 2020R1F1A1067731).
The authors declare no potential conflicts of interest.