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Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

This study aimed to analyze the phototoxic mechanism and photostability of quinine in human skin cell line A375 under ambient intensities of UVA (320–400 nm). Photosensitized quinine produced a photoproduct 6-methoxy-quinoline-4-ylmethyl-oxonium identified through LC-MS/MS. Generation of 1O2, O2•−, and OH was measured and further substantiated through their respective quenchers. Photosensitized Quinine (Q) caused degradation of 2-deoxyguanosine, the most sensitive nucleotide to UV radiation. The intracellular ROS was increased in a concentration-dependent manner. Significant reduction in metabolic status measured in terms of cell viability (54%) at 25 μg mL−1 was observed through MTT assay. Results of MTT assay accord NRU assay. Single strand DNA breaks and apoptosis were increased significantly (< 0.01) as observed through comet assay and EB/AO double staining. Photosensitized quinine caused cells to arrest in G2 phase of cell cycle and induced apoptosis (5.08%) as revealed through FACS. Real-Time PCR showed upregulation of p21 (4.56 folds) and p53 (2.811 folds) genes expression. Thus, our study suggests that generation of reactive oxygen species by quinine under ambient intensity of UVA may result into deleterious phototoxic effects among human population.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

UVA-induced phototoxic skin responses are post effects of the exposure of skin to photosensitized drugs. The solar radiation reaching the earth's surface is comprised of 95% UVA (320–400 nm). Shortwave and longwave UV light has recently been classified as class I carcinogen by the WHO International Agency for Research on Cancer Monograph Working Group [1]. Drug users are normally unaware of the penetration of UVA coming through clouds, window glass, thin clothing and may experience phototoxic responses. Out of total solar UVA radiation 50% penetrates into the dermis and excites photosensitizers present in the skin with subsequent generation of ROS and finally photo-oxidative stress.

Quinine is a hydrophobic amine which acts on the K/H antiporter of the mitochondria and halts K+ transport, inhibits nucleic acid synthesis, protein synthesis and glycolysis to kill falciparum parasite. Its bioavailability is 76–88% in healthy adults. Drugs are absorbed either locally into the skin or via the blood circulation, responsible for phototoxic reactions in the skin [2]. Quinine treatment may lead to sub-optimal treatment outcomes [3]. It accumulates in skin which is a rich source of melanin and gets photosensitized [4]. Quinine is photosensitized via singlet oxygen generation as well as free radical pathway [5]. Sometimes it causes allergic reactions and lead to hemolysis in falciparum malaria patients. Quinine causes mast cell degranulation [6]. Earlier studies of drug phototoxicity were performed at higher doses of UVR [7]. In a report, photosensitized quinine (200 μm) generated ROS under 250 W m−2 UVA irradiation [8]. The undesirable cutaneous and ocular side effects associated with quinine administration could be related to its ability to produce 1O2 especially if the drug is present in low polarity microenvironments in biological systems [9]. The phototoxic effects of UVA occur through ROS [10]. UVA-mediated cellular responses revealed more photoadducts than UVB [11]. UV radiation mediates a variety of cellular reactions such as inflammation and cell cycle regulatory events [12]. Photosensitized quinine may produce stable phototoxic products which may lead to in vivo phototoxicity [13].

Despite the existing studies on quinine phototoxicity, the basic molecular mechanism involved in phototoxicity is still in its juvenile phase. Therefore, this study involved a mechanistic approach that entails the ability of quinine under UVA irradiation for oxidative stress through ROS generation and its effect on DNA damage, programmed cell death (PCD)/apoptosis and identification of the photoproducts. Use of A375 cell line, which is a human immortal melanoma is an effective model [14].

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

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.

Cell culture

The human melanocyte cell line A375 (passage number 37) was procured from National Centre for Cell Sciences, Pune, India. It was then subcultured and maintained in the cell culture facility of our laboratory. Cell line was cultured in DMEM F-12 HAM culture medium supplemented with FBS (10%) and antibiotic-antimycotic solution (1.5%) and kept in CO2 incubator (37°C, 5% CO2 and 95% relative humidity).

Source and method of radiation exposure

The radiation source comprised an array of UV-R emitting tubes (1.2 m long) manufactured by Vilber Lourmat (France). A microprocessor-controlled RMX-3W radiometer (Vilber Lourmat) equipped with calibrated UV-R measuring probes was used to measure the intensity of emitted light. The spectral emission of UVA source ranged from 320 to 400 nm with a peak at 365 nm. We used to carry out dosimetry at our laboratory's roof top between 1.00 P.M. and 1.30 P.M. (5 days a week) to select the intensity for exposure. For photochemical assay we used glass Petri dishes (60 × 15 mm). For cell culture experiments, 35 mm, 96- and 6-well culture plates were used. All the UVA exposure was carried out in a radiation chamber (Temp. 25°C ± 2°C). The distance between source of radiation and samples was at least 22.0 cm to minimize the evaporation due to heat.

Spectra of quinine

Quinine (100 μg mL−1) prepared in Milli Q (deionized double distilled) water was exposed to UVA radiation (2 mW cm−2) for various time periods and then scanned over the spectrum range from 200 to 500 nm.

LC-MS/MS analysis

Stock solution (1000 μg mL−1) of quinine was prepared in 20% ethanol and stored at 2–8°C. Working solution (5 μg mL−1) was prepared in double-distilled water. The solution was subjected to UVA irradiation for 0, 12, 14, 18 and 20 h. Mass spectrometric detection was performed on an API 4000 mass spectrometer (Applied Biosystems, MDS Sciex Toronto, Canada) equipped with an API electro spray ionization (ESI) source. Quinine was optimized by continuous infusion at 10 μL min−1 using syringe pump (Model ‘11’, Harvard apparatus). Molecular weight of quinine is 324.43. The optimized precursor (protonated form of analyte, M + H+) was m/z [RIGHTWARDS ARROW] 325.6. Zero air and nitrogen gas were used as source and curtain gas respectively. The optimized declustering potential for Q was 90 V. At these optimized conditions, Q1 scan for control and test samples was performed.

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) [15]. 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) [16].

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 [17].

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 [18].

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 [19].

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) [16].

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) [16].

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 [22]. 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 [11].

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 [23].

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.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Absorption and photo degradation spectra of Q

Absorption spectrum of Q showed strong absorption at 331 nm (λmax) which comes in the range of UVA. It showed time dependent photo degradation after 12 h exposure. No photodegradation was observed before 12 h exposure as depicted in Fig. 1a.

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Figure 1. (a) Photo degradation spectrum of Q irradiated for 0 min to 20 h under UVA (2 mW cm−2), (b) Schematic representation of photoproduct P1 of quinine identified through LCMS/MS.

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Photoproduct identification by LC-MS/MS

Based upon the results obtained from absorption spectra, LC-MS/MS analysis was carried out to assess the photoproducts formed, if any. LC-MS/MS (Q1 scan) analysis was performed with nonirradiated (control) (Fig. 2a) and sample irradiated for 20 h as shown in (Fig. 2b). Q1 scan of samples irradiated for 14 and 18h showed a decrease in peak of parent compound with a gradual increase in peak of photoproduct (Fig. S1). UV exposure showed the homolytic cleavage of carbon–carbon (C–C) bond leading to the formation of P1 by elimination of bicyclic product, 3vinyl-1-aza-bicyclo [2.2.2] octane. Photoproduct of Q i.e. P1 (m/z = 190.5, 6-methoxy-quinolin-4-yl methyl-oxonium) is shown in Scheme (Fig. 1b). In product ion spectra, the Q product ions formed at varying collision energy was found to be different from photoproducts (P1) of irradiated samples.

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Figure 2. LC-MS/MS Q1 scan of Q (100 μg mL−1) and product ion spectrum of the photoproduct P1[M + H] + (m/z 190.5) irradiated under UVA (a) dark control (b) after 20 h.

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Determination of ROS

Generation and quenching of singlet oxygen (1O2)

The formation of 1O2 was concentration dependent. The highest amount of 1O2 was found to be at 25 μg mL−1 and minimum at 5 μg mL−1 concentration. Quinine did not produce 1O2 in dark (Fig. 3a). Rose Bengal (5 μg mL−1) was used as a positive control. The generation of 1O2 was confirmed by inhibiting the photochemical reaction at 25 μg mL−1 under UVA (5.4 J cm−2) irradiation by its specific quenchers, sodium azide (2 and 5 mm) and DABCO (10 and 20 mm) (Fig. 3b) which were found to be 89.43% and 83.1% respectively.

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Figure 3. (a) Amount of 1O2 generated at various concentrations of Q under different doses of UVA irradiation (2.7, 5.4, 8.1 and 10.8 J cm−2), (b) Percent photochemical quenching of 1O2 at 25 μg mL−1 Q by DABCO and NaN3 under UVA (5.4 J cm−2), (c) Amount of O2•− at various concentrations of Q under UVA (1.8 J cm−2) irradiation and quenching of O2•− produced by Q (25 μg mL−1) by SOD (25 U mL−1) simultaneously, (d) OH generation at various concentrations of Q (5, 10 and 25 μg mL−1) under UVA (5.4 J cm−2) irradiation and quenching of OH produced by Q (25 μg mL−1) by mannitol (0.5 m) simultaneously. Rose Bengal (RB) was used as a positive control. Values are mean of three observations ±SD (*< 0.05 or **< 0.01 as compared to 2.7 J cm−2 and 5 μg mL−1).

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Generation and quenching of superoxide anion radical (O2•−)

The generation of O2•− at various concentrations of Q (5, 10 and 25 μg mL−1) under UVA (1.8 J cm−2) irradiation has been summarized in Fig. 3c. The highest yield of O2•− was observed at 25 μg mL−1 while lowest at 5 μg mL−1. However, quinine was not able to generate O2•− in absence of UVA exposure. Generation of O2•− was confirmed by the incorporation of SOD (25 U mL−1) together with drug in reaction mixture. SOD was able to inhibit the reduction of NBT to NBF up to 90% by dismutating O2•− radical.

Generation and quenching of hydroxyl radical (OH)

The photochemical generation of OH under UVA (5.4 J cm−2) has been shown in Fig. 3d. Q generated OH in a concentration dependent manner. Highest generation was recorded at 25 μg mL−1 concentration. Concomitant inhibition of OH generated by Q at 25 μg mL−1 was observed through mannitol (0.5 m) and found to be significant i.e. 63.29%.

Photodegradation of 2′-dGuO

The photo-oxidative degradation of 2′-dGuO by photosensitized Q was studied at different concentrations (Fig. 4a). Highest photo degradation was observed at 25 μg mL−1 after 4 h irradiation. Quenching of 2′-dGuO photo degradation by NaN3 (5 mm) and DABCO (20 mm) showed 70% and 72% quenching respectively (Fig. 4b).

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Figure 4. (a) Photodynamic degradation of 2′-dGuO by photosensitized Q at 5, 10 and 25 μg mL−1 concentration under UVA (2.0 mW cm−2) exposure for different time durations (*< 0.05 or **< 0.01 as compared to 5 μg mL−1), (b) Percent quenching of photodynamic degradation 2′-dGuO by DABCO and NaN3 under UVA (2.0 mW cm−2) at 25 μg mL−1 Q. Values are mean of three observations ±SD.

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Cell morphology study

Morphological study of cells was carried out after 6 h incubation which revealed that cells started detaching from surface of the culture plate at 5 μg mL−1 under UVA irradiation. Untreated control and UVA alone exposed cells were normal in appearance (Fig. 5a,b) while cells treated with Q (10 μg mL−1) under UVA acquired spherical shape and clustered together forming large masses (Fig. 5c).

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Figure 5. Morphology of A375 cell line. (a) Control (b) cells exposed under UVA (5.4 J cm−2) alone (c) cells exposed with 10 μg mL−1 Q and UV-A (5.4 J cm−2).

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Intracellular ROS in cells

The ability of Q under UVA irradiation to induce intracellular ROS production in A375 cell line was assessed by measuring DCF fluorescence (Fig. 6a). A significant (< 0.01) increase in DCF fluorescence intensity was observed in all UVA and Q treated cells in a concentration dependent manner as compared with control. Significant reduction in fluorescence in presence of NAC (10 μm) confirmed ROS production in cells. In qualitative measurement, cells showed an increase in green fluorescence with concentration as observed in Fig. 6b.

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Figure 6. Intracellular ROS generation. (a) Percent DCF fluorescence at 5, 10 and 25 μg mL−1 concentration under UVA (5.4 J cm−2) and simultaneous quenching of intracellular ROS by N-acetyl-l-cysteine (10 μm) at 25 μg mL−1 (b) Images of cells showing increase in DCF fluorescence at various concentrations under UVA. Values are mean of three observations ±SD (*< 0.05 or **< 0.01 as compared to 5 μg mL−1).

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Photocytotoxicity and cell viability (MTT and NRU) assay

Photosensitizing effect of Q under UVA (5.4 J cm−2) at various concentrations (5, 10 and 25 μg mL−1) was recorded as percent cell viability by MTT (Fig. 7a) and NRU assay (Fig. 7b). As concentration increased, percent cell viability was reduced significantly. Cytotoxicity with different concentrations was assessed in dark also. l-His (50 μg mL−1) and CPZ (5 μg mL−1) were used as negative and positive controls, respectively, in each set of experiments. The highest decrease in percent cell viability (MTT assay) by photosensitized Q was observed to be 54.4% at 25 μg mL−1 concentration as compared to negative control (< 0.05 or < 0.01). The results obtained from NRU assay were found to be comparatively similar with that of MTT assay. Fig. 8.

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Figure 7. Photocytotoxic effect of Q (5, 10 and 25 μg mL−1) on A375 cell line as percent cell viability by (a) mitochondrial dehydrogenase activity (MTT assay) under, UVA (5.4 J cm−2) exposure and Dark (b) NRU assay. Chlorpromazine (5 μg mL−1) and l-Histidine (50 μg mL−1) were used as positive and negative controls respectively. Three independent experimental data were summarized as Mean ± SD (*< 0.05 or **< 0.01 as compared to dark control).

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Figure 8. Photogenotoxic effect of Q on A375 cells under UVA 5.4 J cm−2. (a)% tail DNA, (b) Olive Tail Moment (OTM). Three independent experimental data were summarized as Mean ± SD (*< 0.05 or **< 0.01 as compared to untreated control). Single cells showing DNA damage after exposure to Q and UVA (5.4 J cm−2), (c) Dark control, (d) treated with Q under UVA.

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Single strand DNA damage

The DNA damage in single cells was measured as% tail DNA and olive tail moment (OTM) in the control as well as photosensitized Q exposed cells. The DNA of the exposed cells migrated toward the anode more rapidly at the maximum concentration than lowest one during electrophoresis. The cells treated with different concentrations of Q under UVA showed significantly higher (< 0.01) DNA damage than control samples. Highest DNA damage was recorded at 12.25 μg mL−1 (Fig. 10a,b). Figure 10c shows the untreated control cells having intact DNA while Fig. 10d shows the cell treated with photosensitized Q showing damaged DNA in the form of a tail.

Apoptosis and necrosis

Apoptotic cells induced by photosensitized Q under UVA irradiation were viewed by fluorescence microscope. Based on the fluorescence and the chromatin condensation in the nucleus, four types of cells were observed (1) viable cells with green nucleus having intact chromatin (Fig. 9a); (2) early apoptotic cells having light-orange nucleus and initiation of chromatin condensation; (3) late apoptotic cells having orange nucleus with condensed chromatin; (4) necrotic cells stained with both AO and EB due to membrane damage hence, uniformly orange to red nucleus observed with condensed chromatin(Fig. 9b).

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Figure 9. Viable and apoptotic cells after Q (10 μg mL−1) with or without UVA (2.7 J cm−2) exposure. (a) Live cells (b) Apoptotic cells.

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Cell cycle

The total number of cells in different phases of cell cycle was studied through Flow Cytometry using propidium iodide (PI) as a dye of choice for staining the DNA. One set of cells treated with Q (10 μg mL−1) was exposed to UVA (1.44 J cm−2) irradiation and another set was kept in dark. Result thus obtained showed that number of cells at G2 phase increased significantly than dark (control) cells. The increase occurred with simultaneous decrease in number of cells in S-phase. An increase in number of cells in G0 phase is an indicator of progression of apoptosis due to quinine phototoxicity (Fig. 10a–d).

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Figure 10. Cell cycle arrest and induction of apoptosis under UVA (1.44 J cm−2). (a) Dark control, (b) Q10 (μg mL−1) + dark, (c) UVA, (d) Q (10 μg mL−1) under UVA.

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Gene expression

The photosensitized Q induced expression of p21 and p53 genes in A375 cell line which was quantified through real-time PCR. UVA and Q (5 and 10 μg mL−1) treated cells were kept for 6 h incubation and then analyzed. Upregulation of p21 and p53 was found to be 4.56-fold (Fig. 11a) and 2.81-fold (Fig. 11b), respectively, while concomitantly in dark the expression was at the basal level (i.e. 1.0-fold). Each sample was normalized with β actin, a housekeeping gene. β actin was used as an endogenous control in all samples and it was found uniformly, which confirmed that mRNA maintained its integrity during the study.

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Figure 11. Upregulation of (a) p21 (b) p53 genes. mRNA expression was measured by real-time PCR. Three independent experimental data were summarized as Mean ± SD (*< 0.05 or **< 0.01 as compared to dark control).

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Skin phototoxicity is a common health problem, caused by the interaction of drugs with sunlight [24]. The selection of different intensities of UVA irradiation proves our viewpoint that higher intensities of UVA during peak hours in sunlight would be more deleterious [17]. We have examined thoroughly the process of UVA-induced Q photo degradation, identification of photoproducts, oxidative stress determination, cell-cycle arrest as well as apoptotic cell death in A375 cells at environmentally relevant doses. Photosensitization of Q may cause generation of 1O2 and other reactive oxygen species. UVA caused the formation of Q photoproduct at 12, 14, 18 and 20 h irradiation, which was further identified and confirmed by LC-MS/MS (Q1 scan). In an earlier study, UVA exposed Q formed unidentified photoproducts [8].

Phototoxicity induced by drug is associated with the formation of toxic photoproducts or the generation of short-lived intermediates, increased levels of ROS in the skin and may induce skin diseases [25]. Photosensitization may appear the main cause of phototoxicity of quinolones, particularly in older patients with long-term use [26]. UV exposed clinafloxacin increased photoproducts fluorescence from BAY y3118 [27]. Quinine together with UVA can enhance more cases of skin disorders which may result into tumors. UVA-induced AKT signaling and PTEN expression resulted in an increased carcinogenic potential in human keratinocytes [28]. Photosensitive drug under sunlight revealed direct photolysis as well as self-sensitized photolysis via OH and 1O2 [29]. The process of photosensitization may follow either Type I-photodynamic reaction resulting to ROS such as O2•−, OH, H2O2 or Type II-photodynamic reaction producing singlet oxygen or both [30, 31]. Photosensitized quinine generates 1O2, O2•− and OH through both Type I- and Type II-photodynamic reactions. The use of respective quenchers inhibited the photochemical reaction at a particular reaction step so there is no more transfer of energy or electron and hence, less/no ROS is generated.

The cell viability assays such as MTT and NRU exhibited concentration dependent reduction in the number of viable cells. The results of both assays differ only slightly which may be due to differences in principles of cytotoxicity [32] because, MTT assay is based on the mitochondrial membrane integrity and activity of mitochondrial membrane-bound enzyme succinate dehydrogenase while NRU assay is based on lysosomal activity of the cells. The antipsychotic drug chlorpromazine is a well-known source of ROS generation which damages the cell membrane via lipid peroxidation at clinically relevant low doses of UVA [33]. In this study, CPZ has been used as a positive control. As cells show high reduction in their number due to reduced proliferation under UVA exposure, it could be expected that sunlight may be even more phototoxic due to combined effect of all the radiations contained. Hence, the measurement of ambient intensity and a total dose, is an important factor for phototoxicity assessment. The quinine-induced phototoxicity in A375 cells was also obvious by changes in morphological appearance of the cells. Cells collapse and formed clusters under UVA which might be the result from loss of function of some membrane proteins, important for cell integrity and cell to cell contact under normal conditions.

DCFH-DA is a sensitive nonfluorescent compound, which after internalization into the cells gets hydrolyzed by intracellular esterase enzymes to nonfluorescent dichlorodihydrofluorescein (DCFH), which reacted with intracellular ROS to form highly fluorescent product dichlorofluorescein (DCF) with characteristic absorption and emission spectra. The presence of diacetate, attached to DCFH makes it nonpolar to enter the lipid bilayer easily. The increase in DCF fluorescence confirms the intracellular ROS generation by photosensitized quinine because nonfluorescent DCF reacts with various ROS generated inside the cell during oxidative stress to produce fluorescence. As oxidative stress increases, ROS generation also increases and hence an increase in fluorescence also intensifies. UVA and Q treated cells caused dose-dependent increase in ROS generation. The generation of ROS was quenched by NAC which resulted in significant decrease in fluorescence. The role of NAC may be that of an antioxidant. The fluorescent green color appeared to be more intense toward the periphery rather than the center of cell because concentration of molecular oxygen is more toward the periphery due to presence of a large number of mitochondria there. Deoxycholate under UVA irradiation generates intracellular ROS in human skin fibroblasts culture [34].

Photosensitized Q caused single strand DNA breaks which were observed clearly in the form of comet, typical of SCGE. The single strand breaks may be a result of either direct interaction of photosensitized Q or interaction of ROS (indirect) with DNA bases. Apoptotic cells were observed by double staining with AO and EB. The mode of entry of AO and EB into the cell is different. AO enters passively and appears green on fluorescence while EB gets entry only when there is damage in the membrane, therefore, chromatin stained differently. Study by FACS analysis demonstrates that photosensitized quinine (10 μg mL−1) caused arrest of the cell cycle in G2 phase with decreasing cell proliferation and hence, reduction in number of cells in S-phase. UVA exposed ethyl 1,4-dihydro-8-nitro-4-oxoquinoline-3-carboxylate damaged DNA and caused cell cycle arrest in G0/G1 and G2/M phases in L1210 cells through free radicals mediated mitochondrial pathway for apoptotic cell death [35].

Photosensitized Q produced ROS in mitochondria, leading to free radicals attack on membrane phospholipids, loss of mitochondrial membrane potential and activation of procaspases and finally formation of apoptosome resulting in apoptosis of the cell [36]. Apoptotic cells have some characteristic features like cell shrinkage, nuclear membrane blebbing, chromatin cleavage and condensation [37, 38] as observed in this study. UVA exposed 8-MOP caused apoptosis in various cell types [39]. Oral application of lomefloxacin and 8-methoxypsoralen induced photocarcinogenic skin tumors in mice on solar UVA exposure [40]. Solar UVA generates more pyrimidine dimers than oxidative DNA damage [41]. Some photo labile fluoroquinolone antibiotics caused phototoxic and photocarcinogenic effects [42].

UVA-irradiated quinine caused upregulation of p53 gene and induced p21 gene activity. Upregulation of p21 is indicative of cytotoxicity which suggests that p21 might be required to maintain normal integrity and structure of the cell [43]. The genes, p21 and p53 are important in cell cycle regulation, DNA repair and apoptosis mechanisms [44]. The p21 gene mediates DNA-damage-induced cell cycle arrest in normal cells [45].

In conclusion, the study suggests that quinine generates various intracellular ROS such as 1O2, O2 and OH either via Type I or Type II photosensitizing mechanism under normal UVA exposure, which lead to DNA damage, cell cycle arrest and finally cell death. UV exposure of patients during quinine intake may result in skin phototoxicity and disorders. Therefore, sunlight exposure should be avoided by use of protective clothing, sunglasses etc. Since, quinine formed a photoproduct under UVA exposure, which may also contribute to phototoxicity. Therefore, phototoxic response of its photoproduct is also a matter of further investigation to understand the total health impact of quinine on human beings.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

The authors thank the Director, CSIR-IITR for his valuable support in this study. This work is supported by UGC, New Delhi, India and Council of Scientific and Industrial Research, Network Project NWP 34, New Delhi, India.

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  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information
FilenameFormatSizeDescription
php12047-sup-0001-FigureS1.tifimage/tif150KFigure S1. Product ion spectrum of the photoproduct P1[M + H]+ (m/z 190.5) of Q (m/z 325.6) after UVA exposure. ([2]c) 14 h ([2]d) 18 h.

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