Culture medium and supplements were obtained from Gibco-BRL (Carlsbad, CA, USA), with the exception of recombinant human basic fibroblast growth factor (bFGF), isobutylmethylxanthine (IBMX) and cholera toxin (CT), which were purchased from PeproTech, Inc. (Rocky Hill, NJ, USA). H₂O₂ and quercetin were purchased from Sigma-Aldrich (St. Louis, MO, USA). Mouse monoclonal anti-tyrosinase and rabbit polyclonal anti-calreticulin antibodies were purchased from Abcam (Cambridge, MA, USA), anti-β-actin mouse monoclonal antibody was from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA), Alexa Fluor^® 488 donkey anti-mouse Immunoglobulin G (IgG; heavy and light chain, H+L) and Alexa Fluor^® 594 donkey anti-rabbit IgG (H+L) from Life Technologies (Grand Island, NY, USA) and IRDye 680RD goat anti-mouse IgG highly cross adsorbed was from LI-COR Biosciences (Lincoln, NE, USA).

Human epidermal melanocytes were obtained from normal foreskins as previously described (18). Written informed consent was obtained from the patients and the study was approved by the ethics committee of the Third People’s Hospital of Hangzhou (Hangzhou, China). In brief, the samples were incubated in a solution of 0.25% trypsin and 0.2% ethylenediamine tetraacetic acid for 20 h; trypsinization was terminated by the addition of Dulbecco’s modified Eagle’s medium (Gibco-BRL) containing 10% fetal bovine serum (FBS; Gibco-BRL). Melanocytes were then scraped from the epidermis, washed with phosphate-buffered saline (PBS) and centrifuged at 100 × g for 5 min. The pellet was resuspended in Hu16 medium (F12 medium with 20 ng/ml bFGF, 20 μg/ml IBMX, 10 ng/ml CT, 50 μg/ml gentamicin and 10% FBS). Cells were incubated in a humidified 95% air/5% CO₂ atmosphere at 37°C. Geneticin was added to the medium (100 μg/ml) three days later in order to eliminate contaminating cells. Primary cultures were stored until they reached 80% confluence and then the melanocytes were detached using 0.125% trypsin/0.01 M EDTA solution, centrifuged at 100 × g for 5 min, resuspended and then seeded into culture flasks for subculture. Melanocytes used in the present study were of passage 2–3.

In order to evaluate effect of H₂O₂ on melanocytes, cultured cells were seeded at 1×10⁴ per well in 96-well plates at different H₂O₂ concentrations (0–400 μM) for 24 h. Cell viability was then assessed using an MTT assay kit (Promega, Sunnyvale, CA, USA).

In order to investigate the cytoprotective activity of quercetin, cells (2×10⁵/well) were seeded onto six-well plates. Following serum starvation for 24 h, cells were pretreated with quercetin (0–200 μM dissolved in NaOH) for 24 h, H₂O₂ was then added to each well and incubated for 24 h. Cell viabilities were determined using an MTT assay.

MTT assays were used to assess cell viability in melanocytes following H₂O₂ and quercetin treatment. Following treatment, 10 μl MTT (10 mg/ml) was added to cells seeded in 96-well plates and incubated for 4 h, then 100 μl dimethyl sulfoxide was added for 15 min to dissolve. The absorbance value was measured at 490 nm using a microplate spectrophotometer (SoftMax Pro5; Molecular Devices, LLC, Sunnyvale, CA, USA).

ROS levels were determined by measuring the oxidative conversion of 2′,7′-dichloro-fluorescin diacetate (DCFH-DA) into the fluorescent compound dichlorofluorescin (DCF) using a ROS assay kit (Beyotime Insitute of Biotechnology, Jiangsu, China). In brief, 1×10⁴ cells/well were seeded into a 96-well plate and then the medium was replaced with serum-free medium for 24 h starvation. Cells from each well were then incubated with 10 μM DCFH-DA for 20 min at 37°C. Cells were treated with quercetin or NaOH for 30 min and then H₂O₂ was added to each well, except those containing the untreated group, and the cells were incubated for 30 min. For the estimation of intracellular ROS, DCF fluorescence was determined at 485 nm excitation and 520 nm emission using a flow cytometer (BD FACSCalibur™; BD Biosciences, Franklin Lakes, NJ, USA). Three independent experiments were performed.

Cells were seeded into a six-well plate and the medium was replaced with serum-free medium for 24 h starvation. Cells were pretreated with 0.01 μM NaOH or 25 μM quercetin for 24 h at 37°C; 200 μM H₂O₂ was then added to each well, except the untreated group, and incubated for 24 h. ER configuration was observed using electron microscopy as previously described (16). In brief, cells were collected and fixed with 2.5% glutaraldehyde in 0.1 M PBS at 4°C overnight; cells were then post-fixed with 1% OsO₄ in 0.1 M PBS at 4°C for 1 h. Cells were then embedded in 5% agarose (Sigma-Aldrich) and cut into 2–3-mm² blocks, dehydrated in a graded series of ethanol and embedded in epoxy resin (West System, Bay City, MI, USA). Ultrathin sections were stained with uranyl acetate (Sigma-Aldrich) and lead citrate (Sigma-Aldrich) and were examined using a transmission electron microscope (JEM-1230; JEOL, Ltd., Tokyo, Japan).

In order to examine the co-localization of tyrosinase and calreticulin, cells were grown in six-well plates containing coverslips, with various treatments. Cells were then fixed with 4% formaldehyde in PBS for 30 min. Anti-tyrosinase and anti-calreticulin antibodies were used at 10 μg/ml in buffer (0.5% BSA in PBS) and incubated with the coverslips for 1 h. Cells were then incubated with the secondary antibodies, Alexa Fluor^® 488 donkey anti-mouse IgG (H+L) for tyrosinase and Alexa Fluor^® 594 donkey anti-rabbit IgG (H+L) for calreticulin, at a dilution of 1:500 in PBS for 30 min at room temperature. Cover slips were washed three times in PBS for 5 mins each, mounted using a mounting medium and observed with confocal laser scanning microscope (TCS SP2; Leica Microsystems, Wetzlar, Germany).

Total proteins were isolated from cells and separated on a 10% polyacrylamide gel. Proteins were measured using western blot analysis with mouse antibodies against tyrosinase, followed by incubation with IRDye 680RD goat anti-mouse IgG highly cross adsorbed (1:10,000) for 1 h. β-actin was used as the internal control. An infrared imaging system, Licor Odyssey (LI-COR Biosciences), was used to visualize the protein bands and the relative intensities of bands were quantified using Image Studio for Licor Odyssey CLx and Classic (LI-COR Biosciences).

Values are presented as the mean ± standard deviation. Comparisons among groups were analyzed by a one-way analysis of variance using SPSS Version 13.0 (SPSS Inc., Chicago, IL, USA). P<0.05 and P<0.01 were considered to indicate a statistically significant difference.

Human melanocytes and H₂O₂ were used as a model for oxidative stress in order to investigate cell viability using an MTT assay. Cells were treated with different concentrations of H₂O₂ (0–400 μM) for 24 h, and cell viability was measured. As shown in Fig. 1, the viability of H₂O₂-treated cells significantly decreased in a dose-dependent manner. Following treatment with 200 μM H₂O₂, cell viability was reduced by ~50%, this was considered to be the optimum concentration of H₂O₂ and was used for subsequent experiments.

In order to investigate the effect of quercetin on H₂O₂-induced oxidative stress, H₂O₂-treated cells were pretreated with different concentrations of quercetin. In contrast to the H₂O₂-treated cells, quercetin was observed to have a minor, but not significant, protective effect at concentrations of 6.25 and 12.5 μM; however, quercetin concentrations of 25–200 μM exhibited significant protective effects against H₂O₂-induced cell death (Fig. 2). Therefore, 25 μM quercetin was selected to be used for following experiments.

ROS production was evaluated in melanocytes following exposure to quercetin or NaOH in the presence or absence of H₂O₂. As shown in Fig. 3, H₂O₂ and NaOH/H₂O₂ treatments resulted in an 0.5 fold increase in ROS production compared with that of the untreated control levels (P<0.01); however, no significant difference was identified between these two groups (P=0.123). In addition, ROS levels in the quercetin/H₂O₂ group demonstrated a significant decrease compared with those of the untreated control group (P=0.022).

ER modality was analyzed using electron microscopy in order to observe the effects of different conditions on melanoctyes. As shown in Fig. 4, dilated ER was observed in H₂O₂- and NaOH/H₂O₂-treated cells. In untreated cells, ER configuration was shown to be normal. Notably, normal ER configuration was also observed in quercetin/H₂O₂-treated cells. This therefore suggested that quercetin was able to prevent H₂O₂-induced ER dilation.

Confocal laser scanning microscopy was performed in order to assess the co-localization of tyrosinase, the rate-limiting enzyme for melanin synthesis, and calreticulin, an ER marker protein. In untreated cells and quercetin/H₂O₂-treated cells, tyrosinase fluorescence was beyond the fluorescence marked by calreticulin, which indicated that tyrosinase was effectively exported from the ER. However, in H₂O₂- and NaOH/H₂O₂-treated cells, the distribution of tyrosinase fluorescence was pronounced in ER marked by calreticulinin, which suggested that the export of tyrosinase from the ER was disordered (Fig. 5); this therefore indicated that H₂O₂ interfered with the functional export of tyrosinase from the ER.

Western blot analysis was used to determined the expression levels of tyrosinase in melanocytes. As shown in Fig. 6, tyrosinase expression was significantly increased in the quercetin/H₂O₂ group compared with that of the untreated group (P=0.046). By contrast, tyrosinase expression was significantly decreased in the H₂O₂ (P<0.01) and NaOH/H₂O₂ groups (P<0.01) compared with that of the control; however, no significant differences were observed between these two groups (P=0.185). This therefore indicated that quercetin attenuated the H₂O₂-induced inhibition of tyrosinase expression.