Chemicals and reagents
Quinine (Q), N,N-dimethyl-p-nitrosoaniline (RNO), superoxide dismutase (SOD), nitro-blue tetrazolium (NBT), fetal bovine serum (FBS), Dulbecco's modified eagle's medium (DMEM F-12 HAM), Hank's balanced salt solution (HBSS), acetonitrile (ACN), antibiotic and antimycotic solution, trypsin (0.25%), l-Histidine, 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyl-2H-tetrazolium bromide (MTT), dimethyl sulfoxide (DMSO), neutral red (NR), 1,4-diazabicyclo 2–2–2–octane (DABCO), mannitol, sodium azide (NaN3), carbonate and phosphate buffers, RNase, ethidium bromide (EB), propidium iodide, 2 7-dichlorodihydrofluorescein diacetate (carboxy-H2 DCFDA), N-acetyl-cysteine (NAC), 2′-deoxyguanosine (2′-dGuO), low melting point agarose (LMPA) and normal melting point agarose (NMPA) were procured from Sigma Chemical Co. (St. Louis, MO). Ferrous sulfate, ammonium acetate, acetylacetone and formaldehyde were procured from M/s Merck India and M/s Qualigens, India Ltd. Milli Q double-distilled deionized water was used throughout the study. All plastic wares including 96-well plates and 75 and 25 cm2 (polystyrene coated) culture flasks were purchased from Nunc.
Determination of reactive oxygen species
Singlet oxygen (1O2): The generation of 1O2 under aerobic condition was measured in aqueous solution. A solution of sodium phosphate buffer (0.025 m, pH 7.0) containing RNO (0.35–0.4 × 10−5 m) and 10−2 m l-Histidine (a selective acceptor of 1O2) was prepared. The reaction mixture (10 mL) was taken in a Petri dish with or without Q and irradiated under UVA (2.7–10.8 J cm−2) . The production of 1O2 was measured as a decrease in absorbance at 440 nm using a spectrophotometer (Varian UV-visible- Carry-300). DABCO and NaN3, specific quenchers of 1O2 were used for the confirmation of 1O2 generation.
Superoxide anion radical (O2•−): The measurement of O2•− generated by photosensitized Q is based on the principle of reactivity of O2•− with NBT to form a blue-colored complex NBF whose absorbance was measured at 560 nm. A solution of NBT (1.67 × 10−4m) was prepared in sodium carbonate buffer (0.01 m, pH 10). The reaction mixtures (10 mL each) containing Q from 5 to 25 μg mL−1 were irradiated under UVA (1.8 J cm−2) and absorbance of NBF thus formed, was measured. Photochemical generation of O2•− was further confirmed by dismutating O2•− by SOD (25 U mL−1) .
Hydroxyl radical (•OH): •OH radical was measured in the form of HCHO by ascorbic acid-iron-EDTA method. A reaction mixture of 167 μm iron-EDTA (1:2), EDTA (0.1 mm), ascorbic acid (2 mm) and dimethyl sulfoxide (33 mm) was prepared in potassium phosphate buffer (100 mm, pH 7.4) in a final volume of 3.0 mL and irradiated. In experimental sets ascorbic acid was replaced by Q (5–25 μg mL−1). After UVA exposure, 1.0 mL TCA (17.5% w⁄v) was added to the reaction mixture. Equal volumes (1.5 mL) of sample and ammonium acetate acetylacetone reagent (2.0 m ammonium acetate + 0.05 m acetic acid + 0.02 m acetylacetone) were mixed and incubated at 37°C for 40 min. The absorbance was measured at 412 nm. Mannitol (0.5 m), a specific quencher of •OH was used for the confirmation of its generation .
Photodegradation of 2′-deoxyguanosine (2′-dGuO): For the determination of photo-oxidative degradation of 2′-dGuO by UVA-photosensitized Q, a 10 mL reaction mixture of 2′-dGuO was prepared in carbonate buffer (0.01 m, pH 10.0) with or without Q (5–25 μg mL−1) and irradiated for different time intervals. Percent photo degradation of 2′-dGuO was monitored spectrophotometrically at 260 nm as a decrease in absorbance. Photodegradation was further confirmed by inhibiting the reaction with DABCO and sodium azide as specific quenchers .
Intracellular ROS: For measurement of intracellular ROS, cells were grown in 96-multiwell black plates (2 × 104cells per well) and treated with Q (5–25 μg mL−1). Cells were then incubated for 30 min at 37°C with 5 μm carboxy H2-DCFDA prepared in HBSS and UVA-irradiated. On completion of exposure, the intensity of DCF fluorescence was measured at 480 nm excitation and 520 nm emission wavelengths through flourometer (Fluostar Omega – BMG Labtech). A parallel experimental set was run in 35 mm culture Petri plates for qualitative determination of intracellular ROS. For the same purpose, cells were exposed with Q under UVA. After completion of exposure, the cells were photographed under fluorescent microscope. The generation of intracellular ROS was confirmed by adding NAC (10 μm mL−1) as a specific quencher .
Morphological study: Cells were seeded in 6-well culture plates up to 80–90% confluence and treated with Q (5–25 μg mL−1) followed by UVA exposure and UVA alone. Cells were then incubated in CO2 incubator for 6 h and photographed using phase-contrast microscope (Olympus, Japan).
Cell viability assay: A375 cells were seeded in 96-well plates (2 × 104 cells per well) and placed in CO2 incubator. The medium was removed and cells were washed with HBSS. Stock Q was diluted to desired concentrations in HBSS. Cells treated with drug were incubated in CO2 incubator for 30 min prior to UVA exposure. A basal control (untreated cells with no Q and UV exposure), dark control (Q treated cells in dark) and light control (cells exposed to UVA only) samples and the experimental sets were run parallel under same conditions. The cells thus treated, were further used for MTT and NRU assay.
MTT assay: HBSS was replaced with MTT reagent (500 μg mL−1) (100 μL per well) prepared in DMEM F-12 HAM medium. The culture plates were incubated for 4 h at room temperature. Cells were then washed twice with HBSS and 100 μL DMSO was added to each well and kept on rocker shaker (NuRS-60; Nulife) for 20 min to dissolve the formazan crystals. The absorbance was read at 530 nm by micro plate reader (Fluostar Omega- BMG Labtech) .
Neutral red uptake assay: After treatment, the cells were washed with HBSS and allowed to incubate for 3 h in neutral red dye (50 μg mL−1) prepared in DMEM F-12 HAM medium followed by a quick wash with fixing solution (1% w/v CaCl2 + 0.5% v/v formaldehyde) to remove the unbound dye. A solution of 50% ethanol containing 1% acetic acid (v/v) was used to extract the accumulated dye. Plates were then kept on a rocker shaker for 20 min. The absorbance was read at 540 nm by micro plate reader (Fluostar Omega-BMG Labtech) .
Single cell gel electrophoresis: Single strand DNA damage was determined by alkaline single cell gel electrophoresis with slight modification [20, 21]. Cells were seeded in a 6-well plate. After 24 h, cells were treated with different concentrations of Q (2.45, 6.125 and 12.25 μg mL−1) (LD50 12.25 μg mL−1 based on MTT assay) and irradiated under UVA (5.4 J cm−2). Cells were harvested and suspended in chilled PBS. About 20 μL of cell suspension (approx. 10 000 cells) was mixed with 80 μL of 0.5% LMPA and layered on precoated slides with 200 μL normal agarose (1%). A third layer of 1% LMPA was prepared. Slides were then left for solidification for 10 min. The slides were immersed in lysing solution (100 mm Na2-EDTA, 2.5 m NaCl, 10 mm Tris pH 10 with 1% Triton X-100 and 10% DMSO added fresh) and kept at 4°C for 12 h. Fresh ice cold alkaline electrophoresis buffer (1 mm Na2-EDTA, 300 mm NaOH and 0.2% DMSO, pH 13.5) was poured into the chamber of horizontal gel electrophoresis unit and slides were left for 20 min for unwinding of the DNA and then electrophoresis was carried out for 20 min at 22 V (0.8 V cm−1) and 300 mA current. The slides were washed with tris buffer (0.4 m, pH 7.5) to neutralize the alkali and stained with 100 μL EB (20 μg mL−1). Cells were then scored using an image analysis system (Komet-5.0; Kinetic Imaging, Liverpool UK) connected to fluorescent microscope (DMLB, Leica, Germany). About 100 cells per concentration (50 cells per slide) were analyzed. The DNA damage in the cells was quantified as percent tail DNA (100% head DNA) and olive tail moment (OTM).
EB/AO double staining for morphology assay: EB/AO double stain was used to determine the live, apoptotic (early and late) and necrotic cells after the exposure of cells to photosensitized Q . The assay is based on the characteristic properties of apoptotic cells such as chromosomal condensation and fragmentation, whereas necrosis was characterized by the ability to accumulate vital dye, leading to intense orange staining of nuclei. The procedure is suitable for qualitative analysis of apoptotic and necrotic cells. A mixture of EB and AO (100 μg mL−1) was prepared in PBS and added onto the cells after the exposure to Q (5, 10 and 25 μg mL−1) and UVA alone or Q with UVA. AO and EB intercalate within DNA and emit green and orange fluorescence, respectively, as viewed under fluorescent microscope.
Cell cycle analysis: Quinine (5 and 10 μg mL−1) treated cells exposed under UVA (1.44 J cm−2) were washed twice with PBS and replaced with fresh culture medium and incubated for 6 h. After incubation, cells were fixed in 70% ethanol, washed twice with PBS and suspended in 500 μL PI solution (50 μgmL−1 PI + 0.05% TritonX-100 + 100 μgmL−1 RNaseA) and kept at 37°C for 40 min in dark. The cells were washed with PBS (3 mL) and centrifuged. Pellet was resuspended in PBS (500 μL) and analyzed. Chromatin content was quantified by flow cytometry using the Cell Quest program and Mod Fit software depending upon 2n or 4n number of chromosomes in different phases of cell cycle .
Quantitative Real-Time PCR analysis: Cells were treated with Q (5 and 10 μg mL−1) under UVA (2.7 J cm−2) exposure. After treatment of cells total RNA was isolated by using TRIzol reagent according to manufacturer's protocol (Life technologies) and RNA was quantified at 260 nm by Nano-drop spectrophotometer (ND-1000 Thermo scientific). Complementary DNA was synthesized by high-capacity cDNA Reverse Transcription Kit. The relative expression of p53 and p21genes (each sample in triplicate) was carried out with Real-Time PCR (Applied Biosystems- 7900 HT Fast-Real-Time PCR system) using ABI – sequence detection system (PE Applied Biosystems – Foster City – CA). The various steps of real-time PCR consist of initial denaturation for 10 min at 95°C, 40 cycles of 95°C for 15 s and 50°C for 60 s. The CT values (cycle threshold) were normalized with β actin, a housekeeping gene and 2ΔΔct method was employed to calculate the fold change in the expression of genes .
Statistical analysis: For each parameter, at least three or four independent experiments were carried out in duplicates. Data were expressed as mean (±SE) and analyzed by one-way ANOVA and Dunnett's multiple comparison tests. P-value <0.01 was considered statistically significant.