Photochemistry and Photobiology, 2013, 89: 655–664

Ambient UVA-Induced Expression of p53 and Apoptosis in Human SkinMelanoma A375 Cell Line by Quinine

Neera Yadav1, Ashish Dwivedi1, Syed Faiz Mujtaba1, Hari Narayan Kushwaha2, Shio Kumar Singh2 andRatan Singh Ray*11Photobiology Division, CSIR-Indian Institute of Toxicology Research, Lucknow, India2Pharmacokinetics and Metabolism Division, CSIR-Central Drug Research Institute, Chhattar Manzil Palace, Lucknow, IndiaReceived 15 October 2012, accepted 8 January 2013, DOI: 10.1111/php.12047

ABSTRACT

This study aimed to analyze the phototoxic mechanism andphotostability of quinine in human skin cell line A375 underambient intensities of UVA (320–400 nm). Photosensitizedquinine produced a photoproduct 6-methoxy-quinoline-4-ylm-ethyl-oxonium identified through LC-MS/MS. Generation of1O2, O2

•�, and •OH was measured and further substantiatedthrough their respective quenchers. Photosensitized Quinine(Q) caused degradation of 2-deoxyguanosine, the most sensi-tive nucleotide to UV radiation. The intracellular ROS wasincreased in a concentration-dependent manner. Significantreduction in metabolic status measured in terms of cell via-bility (54%) at 25 lg mL�1 was observed through MTTassay. Results of MTT assay accord NRU assay. Singlestrand DNA breaks and apoptosis were increased signifi-cantly (P < 0.01) as observed through comet assay andEB/AO double staining. Photosensitized quinine caused cells toarrest in G2 phase of cell cycle and induced apoptosis(5.08%) as revealed through FACS. Real-Time PCR showedupregulation of p21 (4.56 folds) and p53 (2.811 folds) genesexpression. Thus, our study suggests that generation of reac-tive oxygen species by quinine under ambient intensity ofUVA may result into deleterious phototoxic effects amonghuman population.

INTRODUCTIONUVA-induced phototoxic skin responses are post effects of theexposure of skin to photosensitized drugs. The solar radiationreaching the earth’s surface is comprised of 95% UVA (320–400 nm). Shortwave and longwave UV light has recently beenclassified as class I carcinogen by the WHO InternationalAgency for Research on Cancer Monograph Working Group (1).Drug users are normally unaware of the penetration of UVAcoming through clouds, window glass, thin clothing and mayexperience phototoxic responses. Out of total solar UVA radia-tion 50% penetrates into the dermis and excites photosensitizerspresent in the skin with subsequent generation of ROS andfinally photo-oxidative stress.

Quinine is a hydrophobic amine which acts on the K/Hantiporter of the mitochondria and halts K+ transport, inhibits

nucleic acid synthesis, protein synthesis and glycolysis to killfalciparum parasite. Its bioavailability is 76–88% in healthy adults.Drugs are absorbed either locally into the skin or via the bloodcirculation, 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 andgets photosensitized (4). Quinine is photosensitized via singletoxygen generation as well as free radical pathway (5).Sometimes it causes allergic reactions and lead to hemolysis infalciparum malaria patients. Quinine causes mast cell degranula-tion (6). Earlier studies of drug phototoxicity were performed athigher doses of UVR (7). In a report, photosensitized quinine(200 lM) generated ROS under 250 W m�2 UVA irradiation (8).The undesirable cutaneous and ocular side effects associated withquinine administration could be related to its ability to produce1O2 especially if the drug is present in low polarity microenvi-ronments in biological systems (9). The phototoxic effects ofUVA occur through ROS (10). UVA-mediated cellular responsesrevealed more photoadducts than UVB (11). UV radiation medi-ates a variety of cellular reactions such as inflammation and cellcycle regulatory events (12). Photosensitized quinine mayproduce stable phototoxic products which may lead to in vivophototoxicity (13).

Despite the existing studies on quinine phototoxicity, the basicmolecular mechanism involved in phototoxicity is still in itsjuvenile phase. Therefore, this study involved a mechanisticapproach that entails the ability of quinine under UVA irradiationfor oxidative stress through ROS generation and its effect onDNA damage, programmed cell death (PCD)/apoptosis and iden-tification of the photoproducts. Use of A375 cell line, which is ahuman immortal melanoma is an effective model (14).

MATERIALS AND METHODS

Chemicals and reagents. Quinine (Q), N,N-dimethyl-p-nitrosoaniline(RNO), superoxide dismutase (SOD), nitro-blue tetrazolium (NBT), fetalbovine serum (FBS), Dulbecco’s modified eagle’s medium (DMEM F-12HAM), Hank’s balanced salt solution (HBSS), acetonitrile (ACN), antibi-otic and antimycotic solution, trypsin (0.25%), L-Histidine, 3-(4,5-dimeth-ylthiazolyl-2)-2,5-diphenyl-2H-tetrazolium bromide (MTT), dimethylsulfoxide (DMSO), neutral red (NR), 1,4-diazabicyclo 2–2–2–octane(DABCO), mannitol, sodium azide (NaN3), carbonate and phosphate buf-fers, RNase, ethidium bromide (EB), propidium iodide, 2 7-dichlorodihy-drofluorescein diacetate (carboxy-H2 DCFDA), N-acetyl-cysteine (NAC),2′-deoxyguanosine (2′-dGuO), low melting point agarose (LMPA) andnormal melting point agarose (NMPA) were procured from SigmaChemical Co. (St. Louis, MO). Ferrous sulfate, ammonium acetate,

*Corresponding author email: ratanray.2011@rediffmail.com (Ratan Singh Ray)© 2013 Wiley Periodicals, Inc.Photochemistry and Photobiology © 2013 The American Society of Photobiology 0031-8655/13

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acetylacetone and formaldehyde were procured from M/s Merck Indiaand M/s Qualigens, India Ltd. Milli Q double-distilled deionized waterwas used throughout the study. All plastic wares including 96-well platesand 75 and 25 cm2 (polystyrene coated) culture flasks were purchasedfrom Nunc.

Cell culture. The human melanocyte cell line A375 (passage number37) was procured from National Centre for Cell Sciences, Pune, India. Itwas then subcultured and maintained in the cell culture facility of ourlaboratory. Cell line was cultured in DMEM F-12 HAM culture mediumsupplemented with FBS (10%) and antibiotic-antimycotic solution(1.5%) and kept in CO2 incubator (37°C, 5% CO2 and 95% relativehumidity).

Source and method of radiation exposure. The radiation sourcecomprised an array of UV-R emitting tubes (1.2 m long) manufacturedby Vilber Lourmat (France). A microprocessor-controlled RMX-3Wradiometer (Vilber Lourmat) equipped with calibrated UV-R measuringprobes was used to measure the intensity of emitted light. The spectralemission of UVA source ranged from 320 to 400 nm with a peak at365 nm. We used to carry out dosimetry at our laboratory’s roof topbetween 1.00 P.M. and 1.30 P.M. (5 days a week) to select the intensityfor exposure. For photochemical assay we used glass Petri dishes(60 9 15 mm). For cell culture experiments, 35 mm, 96- and 6-wellculture plates were used. All the UVA exposure was carried out in aradiation chamber (Temp. 25°C � 2°C). The distance between source ofradiation and samples was at least 22.0 cm to minimize the evaporationdue to heat.

Spectra of quinine. Quinine (100 lg 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 spec-trum range from 200 to 500 nm.

LC-MS/MS analysis. Stock solution (1000 lg mL�1) of quinine wasprepared in 20% ethanol and stored at 2–8°C. Working solution(5 lg mL�1) was prepared in double-distilled water. The solution wassubjected to UVA irradiation for 0, 12, 14, 18 and 20 h. Mass spectro-metric detection was performed on an API 4000 mass spectrometer(Applied Biosystems, MDS Sciex Toronto, Canada) equipped with anAPI electro spray ionization (ESI) source. Quinine was optimized by con-tinuous infusion at 10 lL min�1 using syringe pump (Model ‘11’, Har-vard apparatus). Molecular weight of quinine is 324.43. The optimizedprecursor (protonated form of analyte, M + H+) was m/z ? 325.6. Zeroair and nitrogen gas were used as source and curtain gas respectively.The optimized declustering potential for Q was 90 V. At these optimizedconditions, Q1 scan for control and test samples was performed.

Determination of reactive oxygen species. Singlet oxygen (1O2): Thegeneration of 1O2 under aerobic condition was measured in aqueous solu-tion. A solution of sodium phosphate buffer (0.025 M, pH 7.0) containingRNO (0.35–0.4 9 10�5

M) and 10�2M L-Histidine (a selective acceptor

of 1O2) was prepared. The reaction mixture (10 mL) was taken in a Petridish 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 at440 nm using a spectrophotometer (Varian UV-visible- Carry-300).DABCO and NaN3, specific quenchers of 1O2 were used for the confir-mation of 1O2 generation.

Superoxide anion radical (O2•�): The measurement of O2

•� generatedby photosensitized Q is based on the principle of reactivity of O2

•� withNBT to form a blue-colored complex NBF whose absorbance was mea-sured at 560 nm. A solution of NBT (1.67 9 10�4

M) was prepared insodium carbonate buffer (0.01 M, pH 10). The reaction mixtures (10 mLeach) containing Q from 5 to 25 lg mL�1 were irradiated under UVA(1.8 J cm�2) and absorbance of NBF thus formed, was measured. Photo-chemical 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 of167 lM iron-EDTA (1:2), EDTA (0.1 mM), ascorbic acid (2 mM) anddimethyl 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 experi-mental sets ascorbic acid was replaced by Q (5–25 lg mL�1). AfterUVA exposure, 1.0 mL TCA (17.5% w⁄v) was added to the reactionmixture. Equal volumes (1.5 mL) of sample and ammonium acetate acet-ylacetone 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 specificquencher of •OH was used for the confirmation of its generation (17).

Photodegradation of 2′-deoxyguanosine (2′-dGuO): For the determina-tion of photo-oxidative degradation of 2′-dGuO by UVA-photosensitizedQ, a 10 mL reaction mixture of 2′-dGuO was prepared in carbonate buf-fer (0.01 M, pH 10.0) with or without Q (5–25 lg mL�1) and irradiatedfor different time intervals. Percent photo degradation of 2′-dGuO wasmonitored spectrophotometrically at 260 nm as a decrease in absorbance.Photodegradation was further confirmed by inhibiting the reaction withDABCO and sodium azide as specific quenchers (18).

Intracellular ROS: For measurement of intracellular ROS, cells weregrown in 96-multiwell black plates (2 9 104cells per well) and treatedwith Q (5–25 lg mL�1). Cells were then incubated for 30 min at 37°Cwith 5 lM carboxy H2-DCFDA prepared in HBSS and UVA-irradiated.On completion of exposure, the intensity of DCF fluorescence wasmeasured at 480 nm excitation and 520 nm emission wavelengthsthrough flourometer (Fluostar Omega – BMG Labtech). A parallelexperimental set was run in 35 mm culture Petri plates for qualitativedetermination of intracellular ROS. For the same purpose, cells wereexposed with Q under UVA. After completion of exposure, the cellswere photographed under fluorescent microscope. The generation ofintracellular ROS was confirmed by adding NAC (10 lM mL�1) as aspecific quencher (19).

Morphological study: Cells were seeded in 6-well culture plates up to80–90% confluence and treated with Q (5–25 lg mL�1) followed byUVA 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 9 104 cells per well) and placed in CO2 incubator. The medium wasremoved and cells were washed with HBSS. Stock Q was diluted todesired concentrations in HBSS. Cells treated with drug were incubatedin CO2 incubator for 30 min prior to UVA exposure. A basal control(untreated cells with no Q and UV exposure), dark control (Q treatedcells in dark) and light control (cells exposed to UVA only) samples andthe experimental sets were run parallel under same conditions. The cellsthus treated, were further used for MTT and NRU assay.

MTT assay: HBSS was replaced with MTT reagent (500 lg mL�1)(100 lL per well) prepared in DMEM F-12 HAM medium. The cultureplates were incubated for 4 h at room temperature. Cells were thenwashed twice with HBSS and 100 lL DMSO was added to each welland kept on rocker shaker (NuRS-60; Nulife) for 20 min to dissolve theformazan crystals. The absorbance was read at 530 nm by micro platereader (Fluostar Omega- BMG Labtech) (16).

Neutral red uptake assay: After treatment, the cells were washed withHBSS and allowed to incubate for 3 h in neutral red dye (50 lg mL�1)prepared in DMEM F-12 HAM medium followed by a quick wash withfixing solution (1% w/v CaCl2 + 0.5% v/v formaldehyde) to remove theunbound dye. A solution of 50% ethanol containing 1% acetic acid (v/v)was used to extract the accumulated dye. Plates were then kept on arocker shaker for 20 min. The absorbance was read at 540 nm by microplate reader (Fluostar Omega-BMG Labtech) (16).

Single cell gel electrophoresis: Single strand DNA damage was deter-mined by alkaline single cell gel electrophoresis with slight modification(20,21). Cells were seeded in a 6-well plate. After 24 h, cells were trea-ted with different concentrations of Q (2.45, 6.125 and 12.25 lg mL�1)(LD50 12.25 lg mL�1 based on MTT assay) and irradiated under UVA(5.4 J cm�2). Cells were harvested and suspended in chilled PBS. About20 lL of cell suspension (approx. 10 000 cells) was mixed with 80 lLof 0.5% LMPA and layered on precoated slides with 200 lL normal aga-rose (1%). A third layer of 1% LMPA was prepared. Slides were thenleft for solidification for 10 min. The slides were immersed in lysingsolution (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 hori-zontal gel electrophoresis unit and slides were left for 20 min forunwinding of the DNA and then electrophoresis was carried out for20 min at 22 V (0.8 V cm�1) and 300 mA current. The slides werewashed with tris buffer (0.4 M, pH 7.5) to neutralize the alkali andstained with 100 lL EB (20 lg mL�1). Cells were then scored using animage analysis system (Komet-5.0; Kinetic Imaging, Liverpool UK)connected to fluorescent microscope (DMLB, Leica, Germany). About100 cells per concentration (50 cells per slide) were analyzed. The DNAdamage in the cells was quantified as percent tail DNA (100% headDNA) and olive tail moment (OTM).

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EB/AO double staining for morphology assay: EB/AO double stainwas used to determine the live, apoptotic (early and late) and necroticcells after the exposure of cells to photosensitized Q (22). The assay isbased on the characteristic properties of apoptotic cells such as chromo-somal condensation and fragmentation, whereas necrosis was character-ized by the ability to accumulate vital dye, leading to intense orangestaining of nuclei. The procedure is suitable for qualitative analysis ofapoptotic and necrotic cells. A mixture of EB and AO (100 lg mL�1)was prepared in PBS and added onto the cells after the exposure to Q (5,10 and 25 lg mL�1) and UVA alone or Q with UVA. AO and EB inter-calate within DNA and emit green and orange fluorescence, respectively,as viewed under fluorescent microscope.

Cell cycle analysis: Quinine (5 and 10 lg mL�1) treated cellsexposed under UVA (1.44 J cm�2) were washed twice with PBS andreplaced with fresh culture medium and incubated for 6 h. Afterincubation, cells were fixed in 70% ethanol, washed twice with PBS andsuspended in 500 lL PI solution (50 lgmL�1 PI + 0.05% TritonX-100 + 100 lgmL�1 RNaseA) and kept at 37°C for 40 min in dark. Thecells were washed with PBS (3 mL) and centrifuged. Pellet was resus-pended in PBS (500 lL) and analyzed. Chromatin content was quantifiedby flow cytometry using the Cell Quest program and Mod Fit softwaredepending upon 2n or 4n number of chromosomes in different phases ofcell cycle (11).

Quantitative Real-Time PCR analysis: Cells were treated with Q (5and 10 lg mL�1) under UVA (2.7 J cm�2) exposure. After treatment ofcells total RNA was isolated by using TRIzol reagent according to manu-facturer’s protocol (Life technologies) and RNA was quantified at260 nm by Nano-drop spectrophotometer (ND-1000 Thermo scientific).Complementary DNA was synthesized by high-capacity cDNA ReverseTranscription Kit. The relative expression of p53 and p21genes (eachsample in triplicate) was carried out with Real-Time PCR (Applied Bio-systems- 7900 HT Fast-Real-Time PCR system) using ABI – sequencedetection system (PE Applied Biosystems – Foster City – CA). The vari-

ous steps of real-time PCR consist of initial denaturation for 10 min at95°C, 40 cycles of 95°C for 15 s and 50°C for 60 s. The CT values(cycle threshold) were normalized with b actin, a housekeeping gene and2DDct method was employed to calculate the fold change in the expres-sion of genes (23).

Statistical analysis: For each parameter, at least three or four indepen-dent experiments were carried out in duplicates. Data were expressed asmean (�SE) and analyzed by one-way ANOVA and Dunnett’s multiplecomparison tests. P-value <0.01 was considered statistically significant.

RESULTS

Absorption and photo degradation spectra of Q

Absorption spectrum of Q showed strong absorption at 331 nm(kmax) which comes in the range of UVA. It showed time depen-dent photo degradation after 12 h exposure. No photodegradationwas observed before 12 h exposure as depicted in Fig. 1a.

Photoproduct identification by LC-MS/MS

Based upon the results obtained from absorption spectra,LC-MS/MS analysis was carried out to assess the photoproductsformed, if any. LC-MS/MS (Q1 scan) analysis was performedwith nonirradiated (control) (Fig. 2a) and sample irradiated for20 h as shown in (Fig. 2b). Q1 scan of samples irradiated for 14and 18h showed a decrease in peak of parent compound with agradual increase in peak of photoproduct (Fig. S1). UV exposureshowed the homolytic cleavage of carbon–carbon (C–C) bond

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 photoproductP1 of quinine identified through LCMS/MS.

Photochemistry and Photobiology, 2013, 89 657

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) isshown in Scheme (Fig. 1b). In product ion spectra, the Q prod-uct ions formed at varying collision energy was found to be dif-ferent from photoproducts (P1) of irradiated samples.

Determination of ROS

Generation and quenching of singlet oxygen (1O2). The forma-tion of 1O2 was concentration dependent. The highest amount of1O2 was found to be at 25 lg mL�1 and minimum at5 lg mL�1 concentration. Quinine did not produce 1O2 in dark(Fig. 3a). Rose Bengal (5 lg mL�1) was used as a positive con-trol. The generation of 1O2 was confirmed by inhibiting the pho-tochemical reaction at 25 lg 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 be89.43% and 83.1% respectively.

Generation and quenching of superoxide anion radical (O2•�).

The generation of O2•� at various concentrations of Q (5, 10 and

25 lg mL�1) under UVA (1.8 J cm�2) irradiation has been sum-marized in Fig. 3c. The highest yield of O2

•� was observed at25 lg mL�1 while lowest at 5 lg mL�1. However, quinine wasnot able to generate O2

•� in absence of UVA exposure. Genera-

tion of O2•� was confirmed by the incorporation of SOD

(25 U mL�1) together with drug in reaction mixture. SOD wasable to inhibit the reduction of NBT to NBF up to 90% by dis-mutating O2

•� radical.

Generation and quenching of hydroxyl radical (•OH). The photo-chemical generation of •OH under UVA (5.4 J cm�2) has beenshown in Fig. 3d. Q generated •OH in a concentration dependentmanner. Highest generation was recorded at 25 lg mL�1 con-centration. Concomitant inhibition of •OH generated by Q at25 lg mL�1 was observed through mannitol (0.5 M) and foundto be significant i.e. 63.29%.

Photodegradation of 2′-dGuO. The photo-oxidative degradationof 2′-dGuO by photosensitized Q was studied at different con-centrations (Fig. 4a). Highest photo degradation was observed at25 lg mL�1 after 4 h irradiation. Quenching of 2′-dGuO photodegradation by NaN3 (5 mM) and DABCO (20 mM) showed70% and 72% quenching respectively (Fig. 4b).

Cell morphology study

Morphological study of cells was carried out after 6 h incubationwhich revealed that cells started detaching from surface of theculture plate at 5 lg mL�1 under UVA irradiation. Untreatedcontrol and UVA alone exposed cells were normal in appearance

Figure 2. LC-MS/MS Q1 scan of Q (100 lg 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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(Fig. 5a,b) while cells treated with Q (10 lg mL�1) under UVAacquired spherical shape and clustered together forming largemasses (Fig. 5c).

Intracellular ROS in cells

The ability of Q under UVA irradiation to induce intracellularROS production in A375 cell line was assessed by measuringDCF fluorescence (Fig. 6a). A significant (P < 0.01) increase inDCF fluorescence intensity was observed in all UVA and Q trea-ted cells in a concentration dependent manner as compared withcontrol. Significant reduction in fluorescence in presence of NAC(10 lM) confirmed ROS production in cells. In qualitative mea-surement, cells showed an increase in green fluorescence withconcentration as observed in Fig. 6b.

Photocytotoxicity and cell viability (MTT and NRU) assay

Photosensitizing effect of Q under UVA (5.4 J cm�2) at variousconcentrations (5, 10 and 25 lg mL�1) was recorded as percentcell viability by MTT (Fig. 7a) and NRU assay (Fig. 7b). Asconcentration increased, percent cell viability was reduced signif-icantly. Cytotoxicity with different concentrations was assessedin dark also. L-His (50 lg mL�1) and CPZ (5 lg mL�1) wereused as negative and positive controls, respectively, in each setof experiments. The highest decrease in percent cell viability(MTT assay) by photosensitized Q was observed to be 54.4% at25 lg mL�1 concentration as compared to negative control(P < 0.05 or P < 0.01). The results obtained from NRU assaywere found to be comparatively similar with that of MTT assay.Fig. 8.

Single strand DNA damage

The DNA damage in single cells was measured as% tail DNAand olive tail moment (OTM) in the control as well as photosen-sitized Q exposed cells. The DNA of the exposed cells migrated

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) Per-cent photochemical quenching of 1O2 at 25 lg mL�1 Q by DABCO and NaN3 under UVA (5.4 J cm�2), (c) Amount of O2

•� at various concentrationsof Q under UVA (1.8 J cm�2) irradiation and quenching of O2

•� produced by Q (25 lg mL�1) by SOD (25 U mL�1) simultaneously, (d) •OH genera-tion at various concentrations of Q (5, 10 and 25 lg mL�1) under UVA (5.4 J cm�2) irradiation and quenching of •OH produced by Q (25 lg mL�1)by mannitol (0.5 M) simultaneously. Rose Bengal (RB) was used as a positive control. Values are mean of three observations �SD (*P < 0.05 or**P < 0.01 as compared to 2.7 J cm�2 and 5 lg mL�1).

Figure 4. (a) Photodynamic degradation of 2′-dGuO by photosensitizedQ at 5, 10 and 25 lg mL�1 concentration under UVA (2.0 mW cm�2)exposure for different time durations (*P < 0.05 or **P < 0.01 as com-pared to 5 lg mL�1), (b) Percent quenching of photodynamic degrada-tion 2′-dGuO by DABCO and NaN3 under UVA (2.0 mW cm�2) at25 lg mL�1 Q. Values are mean of three observations �SD.

Photochemistry and Photobiology, 2013, 89 659

toward the anode more rapidly at the maximum concentrationthan lowest one during electrophoresis. The cells treated withdifferent concentrations of Q under UVA showed significantlyhigher (P < 0.01) DNA damage than control samples. HighestDNA damage was recorded at 12.25 lg mL�1 (Fig. 10a,b). Fig-ure 10c shows the untreated control cells having intact DNAwhile Fig. 10d shows the cell treated with photosensitized Qshowing damaged DNA in the form of a tail.

Apoptosis and necrosis

Apoptotic cells induced by photosensitized Q under UVA irradi-ation were viewed by fluorescence microscope. Based on the flu-orescence and the chromatin condensation in the nucleus, fourtypes of cells were observed (1) viable cells with green nucleushaving intact chromatin (Fig. 9a); (2) early apoptotic cells havinglight-orange nucleus and initiation of chromatin condensation;

(3) late apoptotic cells having orange nucleus with condensedchromatin; (4) necrotic cells stained with both AO and EB dueto membrane damage hence, uniformly orange to red nucleusobserved with condensed chromatin(Fig. 9b).

Cell cycle

The total number of cells in different phases of cell cycle wasstudied through Flow Cytometry using propidium iodide (PI) asa dye of choice for staining the DNA. One set of cells treatedwith Q (10 lg mL�1) was exposed to UVA (1.44 J cm�2) irra-diation and another set was kept in dark. Result thus obtainedshowed that number of cells at G2 phase increased significantlythan dark (control) cells. The increase occurred with simulta-neous decrease in number of cells in S-phase. An increase innumber of cells in G0 phase is an indicator of progression ofapoptosis due to quinine phototoxicity (Fig. 10a–d).

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 lg mL�1 Q and UV-A(5.4 J cm�2).

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

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Gene expression

The photosensitized Q induced expression of p21 and p53 genesin A375 cell line which was quantified through real-time PCR.

UVA and Q (5 and 10 lg mL�1) treated cells were kept for 6 hincubation and then analyzed. Upregulation of p21 and p53 wasfound to be 4.56-fold (Fig. 11a) and 2.81-fold (Fig. 11b), respec-tively, while concomitantly in dark the expression was at thebasal level (i.e. 1.0-fold). Each sample was normalized with bactin, a housekeeping gene. b actin was used as an endogenouscontrol in all samples and it was found uniformly, which con-firmed that mRNA maintained its integrity during the study.

DISCUSSIONSkin phototoxicity is a common health problem, caused by theinteraction of drugs with sunlight (24). The selection of differentintensities of UVA irradiation proves our viewpoint that higherintensities of UVA during peak hours in sunlight would be moredeleterious (17). We have examined thoroughly the process ofUVA-induced Q photo degradation, identification of photoprod-ucts, oxidative stress determination, cell-cycle arrest as well asapoptotic cell death in A375 cells at environmentally relevantdoses. Photosensitization of Q may cause generation of 1O2 andother reactive oxygen species. UVA caused the formation of Qphotoproduct at 12, 14, 18 and 20 h irradiation, which wasfurther identified and confirmed by LC-MS/MS (Q1 scan). In anearlier study, UVA exposed Q formed unidentified photoproducts(8).

Phototoxicity induced by drug is associated with the formationof toxic photoproducts or the generation of short-lived intermedi-ates, increased levels of ROS in the skin and may induce skindiseases (25). Photosensitization may appear the main cause ofphototoxicity of quinolones, particularly in older patients withlong-term use (26). UV exposed clinafloxacin increased photo-products fluorescence from BAY y3118 (27). Quinine togetherwith UVA can enhance more cases of skin disorders which mayresult into tumors. UVA-induced AKT signaling and PTENexpression resulted in an increased carcinogenic potential in

Figure 7. Photocytotoxic effect of Q (5, 10 and 25 lg mL�1) on A375cell line as percent cell viability by (a) mitochondrial dehydrogenaseactivity (MTT assay) under, UVA (5.4 J cm�2) exposure and Dark (b)NRU assay. Chlorpromazine (5 lg mL�1) and L-Histidine (50 lg mL�1)were used as positive and negative controls respectively. Three indepen-dent experimental data were summarized as Mean � SD (*P < 0.05 or**P < 0.01 as compared to dark control).

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 experi-mental data were summarized as Mean � SD (*P < 0.05 or **P < 0.01 as compared to untreated control). Single cells showing DNA damage afterexposure to Q and UVA (5.4 J cm�2), (c) Dark control, (d) treated with Q under UVA.

Photochemistry and Photobiology, 2013, 89 661

human keratinocytes (28). Photosensitive drug under sunlightrevealed direct photolysis as well as self-sensitized photolysis via�OH and 1O2 (29). The process of photosensitization may followeither Type I-photodynamic reaction resulting to ROS such asO2

•�, �OH, H2O2 or Type II-photodynamic reaction producingsinglet oxygen or both (30,31). Photosensitized quinine generates1O2, O2

•� and �OH through both Type I- and Type II-photody-namic reactions. The use of respective quenchers inhibited thephotochemical reaction at a particular reaction step so there is nomore transfer of energy or electron and hence, less/no ROS isgenerated.

The cell viability assays such as MTT and NRU exhibited con-centration dependent reduction in the number of viable cells. Theresults of both assays differ only slightly which may be due todifferences in principles of cytotoxicity (32) because, MTT assayis based on the mitochondrial membrane integrity and activity of

mitochondrial membrane-bound enzyme succinate dehydrogenasewhile NRU assay is based on lysosomal activity of the cells. Theantipsychotic drug chlorpromazine is a well-known source of ROSgeneration which damages the cell membrane via lipid peroxida-tion at clinically relevant low doses of UVA (33). In this study,CPZ has been used as a positive control. As cells show highreduction in their number due to reduced proliferation under UVAexposure, it could be expected that sunlight may be even morephototoxic due to combined effect of all the radiations contained.Hence, the measurement of ambient intensity and a total dose, isan important factor for phototoxicity assessment. The quinine-induced phototoxicity in A375 cells was also obvious by changesin morphological appearance of the cells. Cells collapse andformed clusters under UVA which might be the result from loss offunction of some membrane proteins, important for cell integrityand cell to cell contact under normal conditions.

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

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

662 Neera Yadav et al.

DCFH-DA is a sensitive nonfluorescent compound, whichafter internalization into the cells gets hydrolyzed by intracellularesterase enzymes to nonfluorescent dichlorodihydrofluorescein(DCFH), which reacted with intracellular ROS to form highlyfluorescent product dichlorofluorescein (DCF) with characteristicabsorption and emission spectra. The presence of diacetate,attached to DCFH makes it nonpolar to enter the lipid bilayereasily. The increase in DCF fluorescence confirms the intracellu-lar ROS generation by photosensitized quinine because nonfluo-rescent DCF reacts with various ROS generated inside the cellduring oxidative stress to produce fluorescence. As oxidativestress increases, ROS generation also increases and hence anincrease in fluorescence also intensifies. UVA and Q treated cellscaused dose-dependent increase in ROS generation. The genera-tion of ROS was quenched by NAC which resulted in significantdecrease in fluorescence. The role of NAC may be that of anantioxidant. The fluorescent green color appeared to be moreintense toward the periphery rather than the center of cellbecause concentration of molecular oxygen is more toward theperiphery due to presence of a large number of mitochondriathere. Deoxycholate under UVA irradiation generates intracellu-lar ROS in human skin fibroblasts culture (34).

Photosensitized Q caused single strand DNA breaks whichwere observed clearly in the form of comet, typical of SCGE. Thesingle strand breaks may be a result of either direct interaction ofphotosensitized Q or interaction of ROS (indirect) with DNAbases. Apoptotic cells were observed by double staining with AOand EB. The mode of entry of AO and EB into the cell is differ-ent. AO enters passively and appears green on fluorescence whileEB gets entry only when there is damage in the membrane, there-fore, chromatin stained differently. Study by FACS analysis dem-onstrates that photosensitized quinine (10 lg mL�1) caused arrestof the cell cycle in G2 phase with decreasing cell proliferation and

hence, reduction in number of cells in S-phase. UVA exposedethyl 1,4-dihydro-8-nitro-4-oxoquinoline-3-carboxylate damagedDNA and caused cell cycle arrest in G0/G1 and G2/M phases inL1210 cells through free radicals mediated mitochondrial pathwayfor apoptotic cell death (35).

Photosensitized Q produced ROS in mitochondria, leading tofree radicals attack on membrane phospholipids, loss of mito-chondrial membrane potential and activation of procaspases andfinally formation of apoptosome resulting in apoptosis of the cell(36). Apoptotic cells have some characteristic features like cellshrinkage, nuclear membrane blebbing, chromatin cleavage andcondensation (37,38) as observed in this study. UVA exposed 8-MOP caused apoptosis in various cell types (39). Oral applica-tion of lomefloxacin and 8-methoxypsoralen induced photocar-cinogenic skin tumors in mice on solar UVA exposure (40).Solar UVA generates more pyrimidine dimers than oxidativeDNA damage (41). Some photo labile fluoroquinolone antibioticscaused phototoxic and photocarcinogenic effects (42).

UVA-irradiated quinine caused upregulation of p53 gene andinduced p21 gene activity. Upregulation of p21 is indicative ofcytotoxicity which suggests that p21 might be required to main-tain normal integrity and structure of the cell (43). The genes,p21 and p53 are important in cell cycle regulation, DNA repairand 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 vari-ous intracellular ROS such as 1O2, O2

�� and �OH either via TypeI or Type II photosensitizing mechanism under normal UVAexposure, which lead to DNA damage, cell cycle arrest andfinally cell death. UV exposure of patients during quinine intakemay result in skin phototoxicity and disorders. Therefore, sun-light exposure should be avoided by use of protective clothing,sunglasses etc. Since, quinine formed a photoproduct underUVA exposure, which may also contribute to phototoxicity.Therefore, phototoxic response of its photoproduct is also a mat-ter of further investigation to understand the total health impactof quinine on human beings.

Acknowledgements—The authors thank the Director, CSIR-IITR for hisvaluable support in this study. This work is supported by UGC, NewDelhi, India and Council of Scientific and Industrial Research, NetworkProject NWP 34, New Delhi, India.

SUPPORTING INFORMATIONAdditional Supporting Information may be found in the onlineversion of this article:

Figure S1. Product ion spectrum of the photoproduct P1[M + H]+ (m/z 190.5) of Q (m/z 325.6) after UVA exposure.(2c) 14 h (2d) 18 h.

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