Oxidative Damage of U937 Human Leukemic Cells Caused by Hydroxyl Radical Results in Singlet Oxygen Formation

Marek Rác; Michal Křupka; Svatopluk Binder; Michaela Sedlářová; Zuzana Matušková; Milan Raška; Pavel Pospíšil

doi:10.1371/journal.pone.0116958

. 2015 Mar 2;10(3):e0116958. doi: 10.1371/journal.pone.0116958

Oxidative Damage of U937 Human Leukemic Cells Caused by Hydroxyl Radical Results in Singlet Oxygen Formation

Marek Rác ¹, Michal Křupka ², Svatopluk Binder ³, Michaela Sedlářová ⁴, Zuzana Matušková ⁵, Milan Raška ², Pavel Pospíšil ^1,^*

Editor: Dariush Hinderberger⁶

PMCID: PMC4346403 PMID: 25730422

Abstract

The exposure of human cells to oxidative stress leads to the oxidation of biomolecules such as lipids, proteins and nuclei acids. In this study, the oxidation of lipids, proteins and DNA was studied after the addition of hydrogen peroxide and Fenton reagent to cell suspension containing human leukemic monocyte lymphoma cell line U937. EPR spin-trapping data showed that the addition of hydrogen peroxide to the cell suspension formed hydroxyl radical via Fenton reaction mediated by endogenous metals. The malondialdehyde HPLC analysis showed no lipid peroxidation after the addition of hydrogen peroxide, whereas the Fenton reagent caused significant lipid peroxidation. The formation of protein carbonyls monitored by dot blot immunoassay and the DNA fragmentation measured by comet assay occurred after the addition of both hydrogen peroxide and Fenton reagent. Oxidative damage of biomolecules leads to the formation of singlet oxygen as conformed by EPR spin-trapping spectroscopy and the green fluorescence of singlet oxygen sensor green detected by confocal laser scanning microscopy. It is proposed here that singlet oxygen is formed by the decomposition of high-energy intermediates such as dioxetane or tetroxide formed by oxidative damage of biomolecules.

Introduction

Reactive oxygen species (ROS) are continuously produced as byproducts of various metabolic pathways localized in the different cellular compartments. Superoxide anion radical (O₂ ^•−) is produced by electron leakage to molecular oxygen in mitochondria, endoplasmic reticulum, microbodies and cell walls [1,2]. The spontaneous and enzymatic dismutation of O₂ ^•− results in the formation of hydrogen peroxide (H₂O₂), whereas the subsequent one-electron reduction of H₂O₂ by transition metal ions such as Fe²⁺, Cu⁺ and Zn⁺ results in the formation of hydroxyl radical (HO^•) [3–7]. It is well known that ROS participate in various biochemical processes as signal transduction and defense against microbial pathogens [8,9]. Due to the highly positive redox potential of HO^•/H₂O redox couple (E ₀ ´(HO^•/H₂O) = 2.3 V, pH 7), HO^• is highly reactive towards biomolecules such as lipids, proteins and nuclei acids. To eliminate the oxidative damage of biomolecules, the non-enzymatic and enzymatic antioxidant systems are engaged. When the concentration of ROS overwhelms the capacity of antioxidant system, HO^• causes the oxidative damage resulting in the structural and function modifications of lipids proteins and nuclei acids [10].

The formation of malondialdehyde (MDA), product of lipid peroxidation, is commonly used as a marker of oxidative damage of lipids [11]. It has been previously demonstrated that hydrogen abstraction from lipids mediated by HO^• results in the formation of lipid alkyl radical during the initiation of the lipid peroxidation (S1 Fig. panel A). In the next step, the unstable lipid alkyl radical reacts with molecular oxygen forming lipid peroxyl radical. During the propagation of lipid peroxidation, the lipid peroxyl radical reacts with free fatty acid forming another lipid alkyl radical and lipid hydroperoxide. In the termination step, lipid peroxyl radical forms cyclic peroxide and subsequently cyclic endoperoxide [10] know either to decompose to MDA and other products [12] or triplet excited carbonyl ³(R = O)* and lipid hydroxide. The triplet-singlet energy transfer from ³(R = O)* to molecular oxygen forms singlet oxygen (¹O₂). Alternatively, the recombination of two peroxyl radicals forms tetroxide which can decompose to ¹O₂ via Russell mechanism [13].

The protein carbonylation is commonly used as a marker of oxidative damage of proteins [14]. It is well established that the hydrogen abstraction from proteins by HO^• brings about the formation of protein alkyl radical known to interact with molecular oxygen forming protein peroxyl radical (S1 Fig. panel B). The second hydrogen abstraction by protein peroxyl radical from proteins leads to the formation of protein hydroperoxide known to be reduced to protein alkoxyl radical by transition metals such as Fe²⁺, Cu⁺ and Zn⁺. The β-scission of protein alkoxyl radical leads to the formation of protein carbonyls and protein alkyl radical [15]. It has been assumed that the recombination of protein peroxyl radicals forms tetroxide known to decompose to ¹O₂, protein carbonyls and protein hydroxide [16].

The DNA fragmentation is commonly used as a marker of oxidative damage of DNA [17]. It has been previously demonstrated that DNA oxidation is initiated by the hydrogen abstraction by HO^• from deoxyribose forming deoxyribose radical (S1 Fig. panel C). The subsequent radical reactions of deoxyribose radical lead to the rearrangement of the deoxyribose. The scission of the deoxyribose phosphate backbone results in the formation of DNA strand breaks [18]. The oxidative damage of DNA bases results in the alternation of the bases including thymine hydroperoxide. In the presence of metal ions or metalloproteins, thymine hydroperoxide can be reduced to thyimine peroxyl radical. The recombination of two thyimine peroxyl radicals through the Russell mechanism results in the formation of ¹O₂ [16].

In this study, the oxidative damage of biomolecules caused by the addition of H₂O₂ and Fenton reagent was examined. Whereas the lipid peroxidation was initiated solely after the addition of Fenton reagent, the protein carbonylation and DNA fragmentation occurred after the addition of both H₂O₂ and Fenton reagent. Evidence is provided that oxidative damage of lipids, proteins and DNA leads to the formation of ¹O₂ as confirmed by EPR spin-trapping spectroscopy and SOSG fluorescence using confocal laser scanning microscopy.

Materials and Methods

U937 cell culture

Human leukemic monocyte lymphoma cell line U937 was used. The culture is derived from histocytic lymphoma and is frequently used as a model of monocyte/macrophage cell lineage [19,20]. U937 cells were grown in RPMI-1640 supplemented with 2 mM L-glutamine, 10% FBS, antibiotics at 37°C in humidified 5% CO₂ atmosphere. Viability of the cells was measured by Trypan Blue viability test. Subsequently, 10 μl of cell suspension was mixed with 10 μl of 0.4% Trypan Blue. Cells were counted by TC20 automated cell counter (Bio-Rad Laboratories, California, USA).

EPR spin-trapping spectroscopy

EPR spin-trapping spectroscopy was used to monitor formation of HO^• and ¹O₂ in cell suspension. Hydroxyl radical was detected by a 4-pyridyl-1-oxide-N-tert-butylnitrone (POBN)/ethanol spin-trapping system [21]. Cell suspension was treated with 5 mM H₂O₂ or Fenton reagent (5 mM H₂O₂ and 1 mM FeSO₄) in the presence of 50 mM POBN, 170 mM ethanol. Singlet oxygen was detected by hydrophilic spin trap compound TMPD (2, 2, 6, 6-Tetramethyl-4-piperidone) (Sigma) [22]. To eliminate impurity TMPD EPR signal TMPD was purified twice by vacuum distillation. Cell suspension was treated with 5 mM H₂O₂ or Fenton reagent (5 mM H₂O₂ and 1 mM FeSO₄) in the presence of 100 mM TMPD and culture medium. Cell suspension previously exposed to H₂O₂ and Fenton reagent treatments were put in a glass capillary tube (Blaubrand intraMARK, Brand, Germany) and EPR spectra were recorded using an EPR spectrometer MiniScope MS400 (Magnettech GmbH, Berlin, Germany). The mixture of 10 mM molybdic acid and 10 mM H₂O₂ was used as a positive control for ¹O₂ generation. The TEMPONE EPR spectra were recorded in the presence of 50 mM TEMP immediately after the mixture was prepared. EPR conditions were as follows: microwave power, 10 mW; modulation amplitude, 1 G; modulation frequency, 100 kHz; sweep width, 100 G; scan rate, 1.62 G s-1, gain 500. To prevent overscaling of EPR signal, the gain was decreased to 100, when EPR signal was measured 30 min after the addition of Fenton reagent to the cell suspension. Simulation of EPR spectra was done using Winsim software freely available from the website of National Institute of Environmental Health Sciences.

HPLC

The sample preparation and derivatization of malondialdehyde with 2,4-dinitrophenylhydrazine DNPH was performed as described previously [23] with some modifications. Cell suspension was treated with 5 mM H₂O₂ or Fenton reagent (5 mM H₂O₂ and 1 mM FeSO₄) for 30 min were centrifuged for 20 min at 11000 x g and supernatant was removed. Pellet was stirred up in 200 μl of phosphate buffer saline (PBS). After this, cells were disrupted by sonication for 90 seconds. The sample with disrupted cells was centrifuged at 2000 x g for 10 min. The amount of 125 μl of supernatant was taken into the eppendorf vial and 25 μl of 6 M aqueous sodium hydroxide was added. This mixture was incubated in a 60°C water bath for 30 min to achieve alkaline hydrolysis of protein bound MDA. Then, protein was precipitated adding 62.5 μl of 35% (v/v) perchloric acid. The sample was vortexed and centrifuged at 16000 x g for 10 min. A volume of 125 μl of supernatant was put into the dark eppendorf vial and mixed with 1 μl DNPH prepared as a 50 mM solution in 50% sulphuric acid. This mixture was incubated in dark for 30 min at room temperature. An aliquot of 50 μl of this mixture was injected into the HPLC system. The samples were analyzed on the HPLC system (Shimadzu LC-20A Prominence, Kyoto, Japan) with UV detection at 310 nm. A Lichrospher 100 RP-18 column (4.0 x 250 mm) with 5 μm particle size (Merck, Germany) preceded by a Lichrospher precolumn of the same material as the stationary phase (4.0 x 4.0 mm) was used. Elution was performed isocraticaly with a mixture of 25 mM triethylamine adjusted to pH 3.5 and acetonitrile (50:50, v/v) at a flow rate of 1.5 ml/min at 35°C.

Dot blot immunoassay

Proteins were isolated according to Pierce protocol with using RIPA buffer. Cell suspension treated with 5 mM H₂O₂ or Fenton reagent (5 mM H₂O₂ and 1 mM FeSO₄) for 30 min were washed twice in cold PBS. Cold RIPA buffer was added and mixture was incubated on ice for 5 min. Lysate was centrifuged at 14 000 x g for 15 min at 4°C. Supernatant containing proteins was derivatized with 400 μM DNPH for 30 min. Samples were transferred onto the PVDF membrane, using Don-blot apparatus (BioRad). Membrane was blocked in SuperBlock blocking buffer (Thermo Scientific) for 1 h and afterwards was incubated with primary antibody (anti-dinitrophenyl-KLH, rabbit IgG fraction, biotin-labeled, Molecular Probes) overnight. Than membrane was washed PBS-T buffer (PBS + Tween 20) and incubated with streptavidin-HRP conjugate in blocking buffer for 1 h. After washing, the reaction was visualized by ECL chemistry using X-ray sensitive film.

Comet assay

The DNA damage was studied by applying the comet assay. Microscope slides were firstly precoated with 1% HMP (high melting point) agarose (SERVA Electrophoresis), in distilled H₂O and then placed in a drying oven at temperature of 60°C for at least 30 min. 85 μl of 1% HMP agarose in PBS was applied on the precoated slides covered with a cover slip. The slides were then placed in a refrigerator in order to enhance gelling of the agarose. Cell suspension treated with 5 mM H₂O₂ or Fenton reagent (5 mM H₂O₂ and 1 mM FeSO₄) for 30 min was centrifuged (6 min, 1200 rpm) and cell pellet was dispersed in 20 μl of PBS and vortexed. 85 μl of 1% LMP (low melting point) agarose (Qbiogene) was added into this solution and 85 μl of this suspension was cast on the solidified agarose on the microscope slide (the cover slide was removed prior to cell inoculation on the gel) and covered by a new glass cover slip to form a thin layer and moved to the refrigerator again. After solidifying the cover slips were removed again and the microscope slides were immersed in a lysis buffer (2.5 M NaCl (Sigma-Aldrich), 100 mM EDTA (ethylenediaminetetraacetic acid) (Sigma-Aldrich), 10 mM Tris (tris(hydroxymethyl)aminomethane) (Sigma-Aldrich), 1% Triton X-100 (SERVA Electrophoresis), pH = 10) at 4°C for at least 1 h. After the lysis the slides were washed in distilled water to remove all salts and then placed in an electrophoretic tank and dipped in cool electrophoresis solution (300 mM NaOH (Sigma-Aldrich), 1 mM EDTA (Sigma-Aldrich) for 40 min. Electrophoresis was run at 0.8 V/cm and 350 mA for 20 min. After the electrophoresis the slides were rinsed 3x for 5min with neutralisation buffer (0.4 M Tris (Sigma-Aldrich), pH = 7.5) at 4°C. The samples were subsequently stained by SYBR Green (Invitrogen). Fifty randomly chosen cells from each sample was visualized using fluorescence microscope Olympus IX 70 with CCD camera. Computerized image analysis system (TriTek CometScore Freeware 1.5) was used to measure several comet parameters (tail length, tail moment, tail % DNA).

Singlet oxygen detection by confocal laser scanning microscopy and image analysis

In vivo production of ¹O₂ was visualized by Singlet Oxygen Sensor Green (SOSG) reagent (Molecular Probes Inc., Eugene, OR, U.S.A.). 5mM SOSG stock solution was freshly prepared each time by adding 33 μL methanol to a 100 μg vial, and kept in darkness at +4°C until used during a half-day work session. Studied U937 cells were stained with the final concentration of 50 μM SOSG for 30 min, in darkness, at room temperature. At the beginning of incubation, cell suspension was supplemented with 5 mM H₂O₂ or Fenton reagent (5 mM H₂O₂ and 1 mM FeSO₄). Negative controls were induced in the presence of 10 mM histidine. Following incubation, U937 cells were gently washed with 20 mM K-buffer, and consequently the SOSG fluorescence was measured by confocal laser scanning microscope, Fluorview 1000 confocal unit attached to IX 80 inverted microscope (Olympus Czech Group, Prague, Czech Republic). Microphotographs were taken in the transmitted light detection module (405 nm excitation and Nomarski DIC filters) combined with the fluorescence channel, representing the SOSG fluorescence (excitation by a 488 nm line of argon laser and detection by 505–525 nm emission filter set). At the start of each experiment, the proper intensity of lasers was checked according to an unstained sample. To evaluate differential ¹O₂ production during individual treatments, the intensities of SOSG fluorescence within confocal images were analyzed using Olympus FV10-ASW 3.0 Viewer software. The average intensity of SOSG fluorescence superior to the background and the percentage of U937 cells with signals were calculated from the SOSG fluorescence channel of five to ten representative microphotographs per variant.

Statistical analysis

Statistical analysis was performed using software Statistika, version 12, (StatSoft CR s.r.o., Czech Republic). One-way ANOVA together with Post-hoc test at a significance level of 0.05 were used.

Results

Formation of hydroxyl radical detected by EPR spin-trapping spectroscopy

To study the oxidative damage of biomolecules caused by ROS, H₂O₂ and Fenton reagent (H₂O₂ and FeSO₄) were added to cell suspension. To confirm the formation of HO^• in cell suspension after the addition of H₂O₂ and Fenton reagent, EPR spin-trapping spectroscopy was used. The detection of HO^• was accomplished using POBN/ethanol spin-trapping system. It is well established that the interaction of HO^• with ethanol yields α-hydroxyethyl radical (CH(CH₃)HO^•) known to form a stable α-hydroxyethyl radical adduct of POBN (POBN-CH(CH₃)OH adduct) by the interaction with POBN [19]. When POBN/ethanol spin-trapping system was added to the control cell suspension, no POBN-CH(CH₃)OH adduct EPR spectrum was observed (Figs. 1A, trace a). The addition of H₂O₂ to the cell suspension forms small POBN-CH(CH₃)OH adduct EPR signal (Fig. 1, trace b), while pronounced POBN-CH(CH₃)OH adduct EPR signal was observed after the addition of Fenton reagent to the cell suspension (Fig. 1A, trace c). When POBN-CH(CH₃)OH adduct EPR spectra were measured 30 min after the addition of H₂O₂ to the cell suspension (Fig. 1B, trace b), POBN-CH(CH₃)OH adduct EPR signal was enhanced twice or three times as compared to POBN-CH(CH₃)OH adduct EPR signal measured immediately after the addition of H₂O₂ (Fig. 1A, trace b). Similarly, POBN-CH(CH₃)OH adduct EPR signal observed 30 min after the addition of Fenton reagent to the cell suspension increased ten times (Fig. 1B, trace c) as compared to POBN-CH(CH₃)OH adduct EPR signal measured immediately after the addition of Fenton reagent (Fig. 1A, trace c). To confirm identification of POBN-CH(CH₃)OH adduct EPR signal, simulation of experimental data was performed (Figs. 1A and B, trace c, dotted line). The best simulation of experimental data was accomplished using hyperfine coupling constants a ^N = 15.75 G, a ^H = 2.40 G known to be attributed to POBN-CH(CH₃)OH adduct [21]. These observations reveal that HO^• is formed after the addition of H₂O₂ or Fenton reagent to the cell suspension with more pronounced effect observed after the addition of Fenton reagent.

EPR spectra of POBN-CH(CH₃)OH adduct were detected 0 min (A) and 30 min (B) after the addition of H₂O₂ and Fenton reagent to the U937 cells. EPR POBN-CH(CH₃)OH adduct spectra were measured in the control (trace a), the H₂O₂-treated (trace b) and the Fenton reagent-treated (trace c) U937 cells in the presence of 100 mM POBN/170 mM ethanol system. U937 cells were treated with 5 mM H₂O₂ (b) and Fenton reagent (5 mM H₂O₂ and 1 mM FeSO₄) (c) for 0 min (A) and 30 min (B). In A and B, trace c (dotted line) shows the simulation of POBN-CH(CH₃)OH EPR adduct signal using hyperfine coupling constants a ^N = 15,75 G, a^H = 2,40 G. Experimental EPR conditions were as follows: microwave power, 10 mW; modulation amplitude, 1 G; modulation frequency, 100 kHz; sweep width, 100 G; scan rate, 1.62 G s⁻¹, gain 500. In B (trace c), to avoid overscaling of POBN-CH(CH₃)OH adduct EPR signal, 50 mM POBN/170 mM ethanol system and gain 100 was used. Bars represent 2000 relative units.

Cell viability

To test the effect of H₂O₂ and Fenton reagent addition to the U937 cells, the cell viability was counted using automated cell counter. Fig. 2 shows the viability of the cells 30 min after the addition of H₂O₂ or Fenton reagent compared to the control U937 cells. The results show that there is insignificant decrease in the cell viability in both samples indicating that almost all the cells are still alive after the treatment.

Analysis of lipid peroxidation by MDA HPLC assay

Malondialdehyde, a product of lipid peroxidation, was detected to monitor oxidative damage of lipids caused by the addition of H₂O₂ and Fenton reagent to the cell suspension. An adaptation of a very rapid and simple isocratic reversed-phase HPLC separation of DNPH-MDA complex with absorption at 310 nm was used to examine the level of lipid peroxidation [23]. Fig. 3A shows the chromatogram of DNPH-MDA complex measured in the control (trace a), the H₂O₂-treated (trace b) and the Fenton reagent-treated (trace c) cell suspension. To estimate the retention time of DNPH-MDA complex, the chromatogram of DNPH-MDA standard was measured (Fig. 3B). The HPLC analysis of MDA-DNPH complex showed that the addition of H₂O₂ to the cell suspension caused no enhancement in peak area as compared to the control U937 cells. The peak area for the Fenton reagent-treated U937 cells was found three times higher compared to the peak area for the control and the H₂O₂-treated U937 cells. To quantify the concentration of MDA, standard calibration curve was obtained by plotting the peak area at 310 nm for various MDA concentrations (Fig. 3B, insert). The level of MDA in the control U937 cells and H₂O₂-treated U937 cells is 0.029±0.003 and 0.030±0.003 nmol ml⁻¹, respectively, while in the Fenton reagent-treated U937 cells the level of MDA is 0.09±0.02 nmol ml⁻¹. These observations reveal no difference in lipid peroxidation between the control and the H₂O₂-treated U937 cells while there is significant increase in lipid peroxidation in the Fenton reagent-treated U937 cells compared to the control U937 cells (p < 0.05).

The chromatogram of DNPH-MDA complex in U937 cells (A) and DNPH-MDA standard (B). In A, chromatogram of DNPH-MDA complex was measured in the control (trace a), the H₂O₂-treated (trace b) and the Fenton reagent-treated (trace c) U937 cells. The U937 cells were treated with 5 mM H₂O₂ (b) and Fenton reagent (5 mM H₂O₂ and 1 mM FeSO₄) (c) for 30 min. After the treatment, lipids were separated from proteins and DNPH was added to lipids. In B, the chromatogram of DNPH-MDA standard shows the retention time of 3 min 50 s. The insert shows the dependence of average peak area on the concentration of DNPH-MDA standard. Based on the calibration curve, the concentrations of DNPH-MDA complex determined from calibration curve were as following: 0.029±0.003 nmol ml⁻¹ (control), 0.030±0.003 (H₂O₂) and 0.09±0.02 nmol ml⁻¹ (Fenton reagent). The coefficient of determination R² was determined as 0.9997. Data are presented as mean values and standard deviations. The mean value represents the average value from at least three measurements.

Analysis of protein carbonylation by dot blot immunoassay

To monitor the oxidative damage of proteins caused by the addition of H₂O₂ and Fenton reagent, the protein carbonyl levels were detected using 2,4-dinitrophenylhydrazine dot blot immunoassay. It is well established that carbonyl groups (aldehydes and ketones) are formed on the protein side chains during the oxidation of amino acid such as Pro, Arg, Lys and Thr. Due to their stability, protein carbonyl level is the most suitable marker of oxidative damage of proteins. The identification of protein carbonyl has been facilitated by the derivatization of the carbonyl group with DNPH forming DNP product and by the binding of specific anti-DNP antibodies allowing their detection by imunoblotting analysis. Fig. 4 demonstrates dot blot membrane obtained in the control (A), the H₂O₂-treated (B) and the Fenton reagent-treated (C) U937 cells. The addition of H₂O₂ to the U937 cells 30 min prior to the analysis attenuated protein carbonyl level (Fig. 4B) as compared to the control U937 cells (Fig. 4A) (p < 0.05). The most significant increase in protein carbonyl level was observed in the Fenton reagent-treated U937 cells (Fig. 4C) (p < 0.05). The quantification of dot blot membrane performed using densitometry shows that the enhancement in protein carbonyl level observed in the H₂O₂-treated and the Fenton reagent-treated U937 cells is twice and three times as compared to the control U937 cells, respectively. These observations indicate that the addition of H₂O₂ and Fenton reagent to the U937 cells resulted in the oxidative damage of proteins with more pronounced effect observed after the addition of Fenton reagent.

Dot blot membrane in the control (A), the H₂O₂-treated (B) and the Fenton reagent-treated (C) U937 cells. The U937 cells were treated with 5 mM H₂O₂ (B) and Fenton reagent (5 mM H₂O₂ and 1 mM FeSO₄) (C) for 30 min. After the treatment, the supernatant containing proteins were pipetted on the membrane and incubated with primary antibody (anti-dinitrophenyl-KLH, rabbit igG fraction, biotinilated) overnight. Furthermore the membrane was incubated with streptavidin-HRP conjugate for 1 h, developed with ECL and chemiluminescence signal was detected on X-ray film. The densitometry results are 0.5±0.2 (control), 1.4±0.2 (H₂O₂-treated), and 2.3±0.3 (Fenton reagent). Data are presented as mean values and standard deviations. The mean value represents the average value from at least three measurements.

Analysis of DNA fragmentation by comet assay

In order to examine the oxidative damage of DNA caused by the addition of H₂O₂ and Fenton reagent to the cell suspension, the DNA fragmentation was measured by comet assay (single cell electrophoresis). In this method, the comet tail which represents the fragmented DNA from U937 cells migrates towards the anode. The visualization of DNA from tail and head stained by SYBR Green was performed by fluorescence microscopy. Fig. 5 shows the comet assay images of the control (A), the H₂O₂-treated (B) and the Fenton reagent-treated (C) U937 cells. In the control U937 cells, intact comet heads were observed, whereas heads and tails were distinguished in the H₂O₂-treated and the Fenton reagent-treated U937 cells. Table 1 summarizes the parameters of comet, head and tail determined by the image analysis in the control, the H₂O₂-treated and the Fenton reagent-treated U937 cells. Statistical analysis reveals that most of the parameters are significantly different in the H₂O₂-treated and the Fenton reagent-treated U937 cells compared to the control U937 cells (p < 0.05), whereas no significant differences were observed between the H₂O₂-treated and the Fenton reagent-treated U937 cells. These observations indicate that the addition of both H₂O₂ and Fenton reagent to U937 caused the fragmentation of DNA with non-significantly higher effect observed after the addition of Fenton reagent.

Comet assay of the control (A), the H₂O₂-treated (B) and the Fenton reagent-treated (C) U937 cells. The U937 cells were treated with 5 mM H₂O₂ (B) and Fenton reagent (5 mM H₂O₂ and 1 mM FeSO₄) (C) for 30 min. After the treatment, U937 cells were stained by SYBR Green.

Table 1. Analysis of the medians of comet, head, and tail parameters in the control, the H₂O₂-treated and the Fenton reagent-treated U937 cells.

Comet
	Length [μm]	Height [μm]	Area [μm²]	Intensity [r.u.]	Mean Intensity [r.u.]
Control	20 ± 2	18 ± 2	301 ± 71	71506 ± 18571	51 ± 5
H₂O₂	44 ± 2	24 ± 2	762 ± 66	99182 ± 7767	27 ± 1
Fenton	51 ± 6	25 ± 3	301 ± 71	123451 ± 18571	51 ± 5
Head
	Diameter [μm]	%DNA	Area [μm²]	Intensity [r.u.]	Mean Intensity [r.u.]
Control	18 ± 2	91 ± 4	262 ± 48	65320 ± 16978	52 ± 7
H₂O₂	13 ± 1	30 ± 2	178 ± 15	27219 ± 1385	31 ± 5
Fenton	10 ± 1	21 ± 10	141 ± 32	24705 ± 7742	31 ± 2
Tail
	Length [μm]	%DNA	Area [μm²]	Intensity [r.u.]	Mean Intensity [r.u.]
Control	2 ± 1	8 ± 4	31 ± 21	5888 ± 3993	42 ± 9
H₂O₂	30 ± 3	69 ± 3	524 ± 36	64238 ± 9192	26 ± 1
Fenton	39 ± 8	78 ± 10	659 ± 161	94581 ± 32988	29 ± 2

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Data are presented as mean values and standard deviations. The mean value represents the average value from at least three measurements.

Formation of singlet oxygen detected by EPR spin-trapping spectroscopy

To study whether oxidative damage of biomolecules generates ¹O₂, EPR spin-trapping technique was used to monitor the formation of ¹O₂ in the H₂O₂-treated and the Fenton reagent-treated cell suspension. The spin-trapping was accomplished by utilizing the oxidation of hydrophilic diamagnetic TMPD by ¹O₂ know to yield paramagnetic 2, 2, 6, 6-tetramethyl-4-piperidone-1-oxyl (TEMPONE). The addition of TMPD to the control cell suspension results in the appearance of negligible TEMPONE EPR signal (Fig. 6A). Fig. 6B shows that negligible TEMPONE EPR signal was stable over the whole measured period. When TEMPONE EPR spectra were measured after the addition of H₂O₂ to the cell suspension, TEMPONE EPR signal was observed (Fig. 6C). Fig. 6D shows that TEMPONE EPR signal increases within 10 min followed by gradual decrease. The gradual decrease of TEMPONE EPR signal is likely caused by the oxidation of TEMPONE resulting in the formation of EPR silent oxidized TEMPONE. These observations indicate that ¹O₂ is formed after the addition of H₂O₂ and Fenton reagent to the cell suspension. When TEMPONE EPR spectra were measured after the addition Fenton reagent, significant TEMPONE EPR signal was observed (Fig. 6E). Fig. 6F shows that TEMPONE EPR signal increases within 10 min followed by steady-state level. Comparison of TEMPONE EPR signals showed TEMPONE EPR signals measured after the addition of Fenton reagent is higher compared to TEMPONE EPR signals measured after the addition of H₂O₂. Statistical analysis of the data revealed there is significant difference between control and treated samples after 5 min of treatment (p < 0.05). The results from control, H₂O₂ treated and Fenton treated samples obtained 10–30 min of treatment were found to be significantly different (p < 0.05). Fig. 6 (C and E) show the simulation of TEMPONE EPR signal using hyperfine coupling constants a ^N = 16 G, known to be attributed to TEMPONE [24]. These findings reveal the formation of ¹O₂ after the addition of H₂O₂ or Fenton reagent to the cell suspension with more pronounced effect observed after the addition of Fenton reagent.

TEMPONE EPR spectra were measured in the control (A and B), the H₂O₂-treated (C and D) and the Fenton reagent-treated (E and F) U937 cells in the presence of 100 mM TEMPD. U937 cells were treated with no addition (A), 5 mM H₂O₂ (C) and Fenton reagent (5 mM H₂O₂ and 1 mM FeSO₄) (E) for a period indicated in Fig. In C and E, top traces show the simulation of TEMPONE EPR signal using hyperfine coupling constants a ^N = 16 G. Chemical source of ¹O₂ (10 mM molybdic acid + 10 mM H₂O₂ measured right after the preparation) was used as a positive control (C,E). Bar graphs represent the hight of the middle peak of TEMPONE EPR signal in the control (B), the H₂O₂-treated (D) and the Fenton reagent-treated (F) U937 cells. Experimental EPR conditions were as follows: microwave power, 10 mW; modulation amplitude, 1 G; modulation frequency, 100 kHz; sweep width, 100 G; scan rate, 1.62 G s-1, gain 500. Bars represent 4000 (A) and 8000 (C and E) relative units. Data are presented as mean values and standard deviations. The mean value represent the average value from at least three measurements.

Formation of singlet oxygen detected by confocal laser scanning microscopy

To visualize the formation of ¹O₂ in U937 cells after the addition of H₂O₂ and Fenton reagent to the cell suspension, the SOSG fluorescence was measured by the confocal laser scanning microscopy (Fig. 7A). In order to quantify the ¹O₂ formation, the intensity of SOSG fluorescence and the percentage of cells showing the SOSG fluorescence were evaluated (Fig. 7B). Negligible SOSG fluorescence was recorded in 5% of the control U937 cells. In order to confirm that the SOSG fluorescence origins from the interaction of SOSG with ¹O₂, the effect of histidine on the SOSG fluorescence was measured. No difference in SOSG fluorescence was found in the control U937 cells incubated in the presence of histidine while the number of cells with SOSG fluorescence was 10%. No changes in cell morphology were found in the control U937 cells. The addition of H₂O₂ to the U937 cells resulted in a pronounced SOSG fluorescence observed in 60% of cells. SOSG fluorescence was localized in cytoplasm, nucleus and probably mitochondria. U937 cells were changed in shape, increased volume and became strongly vacuolized. The effect of H₂O₂ was significantly decreased in the presence of histidine (3 times lower signal in 53% of cells) (p < 0.05). The addition of Fenton reagent to the U937 cells caused SOSG fluorescence in all cells, though the signal intensity was comparable to that in the H₂O₂-treated cells. The volume and shape of cells were dramatically changed, some of them ruptured. Percentage of cells producing SOSG fluorescence was diminished by a histidine addition to 36%, whereas the intensity of SOSG fluorescence was slightly lower than in the H₂O₂-treated U937 cells in the presence of histidine.

The SOSG fluorescence from U937 cells was measured after the addition of 5 mM H₂O₂ and Fenton reagent (5 mM H₂O₂ and 1 mM FeSO₄) by confocal laser scanning microscopy. 50 μM SOSG was added 30 min prior to the data collection to the U937 cells. In A, left column shows the combination of Nomarski DIC and SOSG fluoresencence (λ_em = 505–525 nm) channels and right column the integral distribution of SOSG fluorescence. In B, the intensity of SOSG fluorescence (upper graph) and the percentage of cells showing the SOSG fluorescence (lower graph) are shown. Data are presented as mean values and standard deviations. The mean value represents the average value from five to ten representative microphotographs per variant.

Discussion

In this study, the oxidation of biomolecules such as lipids, proteins and nuclei acids by HO^• was studied after the addition of H₂O₂ and Fenton reagent to the cell suspension. The experimental data provided showed that hydrogen abstraction by HO^• initiates a cascade of oxidative reactions leading to lipid peroxidation, protein carbonylation and DNA fragmentation (Figs. 3–5). In these processes, hydrogen abstraction by HO^• from biomolecules forms alkyl radical known to from peroxyl radical by the interaction with molecular oxygen. Peroxyl radical abstracts hydrogen from another biomolecule resulting in the formation of hydroperoxide and alkyl radical [10]. Experimental evidence obtained by EPR spin-trapping spectroscopy (Fig. 6) and SOSG fluorescence using confocal laser scanning microscopy (Fig. 7) showed that the oxidative damage of lipids, proteins and nucleic acids leads to the formation of ¹O₂. The decomposition of high-energy intermediates such as dioxetane or tetroxide is proposed as a plausible mechanism for ¹O₂ formation caused by the oxidative damage of biomolecules.

Hydroxyl radical formation

EPR spin-trapping data showed that the addition of H₂O₂ (Figs. 1A and B, trace b) and Fenton reagent (Figs. 1A and B, trace c) to the cell suspension caused formation of HO^• via Fenton reaction. In this reaction, a reduced metal ion reacts with H₂O₂ forming an oxidized metal ion and HO^• [25]. Whereas after the addition of H₂O₂ to the cell suspension HO^• is formed by the reaction of H₂O₂ with endogenous metal ions, in Fenton reagent, HO^• is formed by the reaction of H₂O₂ with exogenous iron ions. Several types of endogenous metal ions such as iron, copper, manganese, zinc, chromium, cobalt, nickel and vanadium have been shown to reduce H₂O₂ to HO^•. It is well known that endogenous iron ions are coordinated to active enzyme site in metalloproteins or stored in a ubiquitous protein called ferritin [25]. Due to the lower concentration of free endogenous metal ions compared to the concentration of iron in the Fenton reagent, the formation of HO^• after the addition of H₂O₂ is significantly lower than after the addition of Fenton reagent (Fig. 1). Due to the highly positive redox potential of HO^•/H₂O redox couple (E ₀ ´(HO^•/H₂O) = 2.3 V, pH 7), HO^• is highly reactive towards lipids, proteins and nuclei acids. As the high reactivity of HO^• restricts diffusion of HO^• from the site of its formation, HO^• formed by the reaction of H₂O₂ with endogenous metal ions oxidizes biomolecules solely in the close proximity to the site of its formation [25]. Contrary, the oxidation of biomolecules by HO^• formed by the reaction of H₂O₂ with exogenous iron ions is not site-specific and therefore HO^• can react with a broader range of biomolecules in the cell.

Oxidative damage initiated by hydroxyl radical

Lipid peroxidation. The HPLC analysis of MDA-DNPH complex showed that the addition of H₂O₂ to the cell suspension has no effect on lipids, whereas the addition of Fenton reagent to the cell suspension caused pronounced lipid peroxidation in the U937 cells (Fig. 3). The observation that no lipid peroxidation was observed after the addition of H₂O₂ to the cell suspension reveals that H₂O₂ has no capability to directly initiate lipid peroxidation [26]. Due to the fact that no metal ions are bound to lipids, the formation of HO^• by reduction of H₂O₂ by endogenous metals via Fenton reaction is unlikely [10]. The observation that lipid peroxidation was detected after the addition of Fenton reagent to the cell suspension indicates that HO^• initiates lipid peroxidation by the abstraction of hydrogen from lipid and the formation of lipid alkyl radical. In the propagation step, lipid alkyl radical reacts with molecular oxygen forming peroxyl radical known to react with another lipid resulting in the formation of another lipid alkyl radical. In the termination step, lipid peroxidation is terminated either by the cyclization of peroxyl radical to cyclic endoperoxide decomposing to MDA or by the recombination of two peroxyl radicals producing tetroxide decomposing to ¹O₂ via Russell mechanism [13].

Protein carbonylation. The addition of H₂O₂ and Fenton reagent to the cell suspension resulted in the oxidative damage of proteins in the U937 cells with significantly higher effect observed after the addition of Fenton reagent (Fig. 4). These results correlate with the EPR spin-trapping data which showed significantly higher formation of HO^• in the cell suspension treated with Fenton reagent. It is well known that protein carbonylation occurs both on the protein side chain and the protein backbone. On the protein side chain, the carbonylation of protein proceeds via direct oxidation or chain reaction. The direct oxidation of protein side chains (especially of Pro, Arg, Lys, and Thr) results in the formation of carbonyl groups [27]. In the chain reaction, the hydrogen abstraction from an amino acid residue by HO^• results in the formation of protein alkyl radical. Protein alkyl radical can subsequently react with molecular oxygen forming protein peroxyl radical. The recombination of two peroxyl radicals gives a raise to unstable tetroxide, which decomposes to two alkoxyl radicals. Once the alkoxyl radical is formed, it can undergo through rearrangement of electron resulting in the formation of carbonyl radical and carbonyl compound on the protein side chain. On the protein backbone, the formation of alkoxyl radical can also result in the oxidative cleavage of protein backbone by either the α-amidation pathway or by oxidation of glutamyl side chains leading to the formation of carbonyl groups [15].

DNA fragmentation. The addition of both H₂O₂ and Fenton reagent to the cell suspension caused the fragmentation of DNA in U937 cells with non-significantly higher effect observed after the addition of Fenton reagent (Fig. 5). It was previously reported that no DNA strand breaks were formed during the interaction of H₂O₂ and isolated DNA [28], whereas HO^• formed via Fenton reaction forms DNA strand breaks [29,30]. At least five main classes of oxidative damage caused by HO^• have been proposed including oxidized bases, abasic sites, DNA-DNA intra-strand adducts, DNA strand breaks and DNA-protein cross-links. It has been proposed that the extent of DNA strand breaking by HO^• is governed by the accessible surface areas of the hydrogen atoms of the DNA backbone [31].

Singlet oxygen formation caused by oxidative damage of U937 cells

In this study, EPR spin-trapping spectroscopy was used to monitor the formation of ¹O₂ in U937 cells caused by HO^•. When spin-trapping was performed by the oxidation of lipophilic 2,2,6,6-tetramethylpiperidine/ethanol (TEMP/ethanol) system by ¹O₂ resulting in the formation of 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO), significant TEMPO EPR signal was detected (data not shown). Based on the observation that H₂O₂ was found to oxidize TEMP/ethanol system, the use of lipophilic TEMP/ethanol system was feasible. To prevent the oxidation of TEMP/ethanol system by H₂O₂, the spin-trapping was accomplished by utilizing the oxidation of hydrophilic TMPD by ¹O₂ which yields 2, 2, 6, 6-tetramethyl-4-piperidone-1-oxyl (TEMPONE). The analysis of TEMPONE EPR spin-trapping data showed that ¹O₂ was detected after the addition of H₂O₂ and Fenton reagent to U9370 cells (Fig. 6). The kinetics of ¹O₂ formation revealed that the formation of ¹O₂ is predominantly during the first 10 min in both the H₂O₂-treated (Fig. 6D) and the Fenton reagent-treated (Fig. 6F) cell suspension. The slow decay in the H₂O₂-treated cell suspension was most probably caused by the oxidation of TEMPONE resulting in the formation of EPR silent TEMPONE. The instability of TEMPONE limits the accurate quantification of ¹O₂ formation over the longer time period. The overall formation of ¹O₂ in the Fenton reagent-treated sample was double compared to the H₂O₂-treated sample. In order to visualize the formation of ¹O₂ in the U937 cells, the SOSG fluorescence was measured (Fig. 6). It has been previously demonstrated that SOSG is unable to penetrate into the CNE2 cells [32]. However it has been demonstrated that SOSG can penetrate HeLA cells if cultivated in a special medium lacking proteins [33]. Our study showed that SOSG can penetrate through the plasma membrane of U937 cells with no such kind of limitations. The formation of ¹O₂ within the U937 cells was about the same in the H₂O₂-treated and the Fenton reagent-treated cell suspension, while the amount of cells in which ¹O₂ was produced was double in the Fenton reagent-treated cell suspension compared to the H₂O₂-treated cell suspension.

The formation of ¹O₂ detected both by EPR spin-trapping spectroscopy and SOSG fluorescence using confocal laser scanning microscopy occurs in the parallel to lipid peroxidation, protein carbonylation and DNA fragmentation. These observations indicate that the formation of ¹O₂ is closely associated with damage of lipids, proteins and DNA. Several lines of evidence have been provided that ¹O₂ is formed by decomposition of high-energy intermediates such as dioxetane and tetroxide formed during the oxidative damage of lipids, proteins and DNA [16,34].

Singlet oxygen by dioxetane decomposition. Dioxetane is formed via two different mechanisms comprising the cycloaddition of ¹O₂ to biomolecules or the cyclization of peroxyl radical [35]. Here, it is proposed that the formation of dioxetane takes place during the physiological and pathological conditions since the concentration of peroxyl radical is unlikely high enough for the formation of tetroxide via recombination of peroxyl radicals. Unstable dioxetane subsequently undergoes the decomposition to ³(R = O)* and organic hydroxide. The excitation energy from ³(R = O)* is transferred to molecular oxygen resulting in the formation of ¹O₂ [36]. It is suggested here that the decomposition of dioxetane is responsible for the ¹O₂ formation shortly after the addition of H₂O₂ and Fenton reagent to the cell suspension.

Singlet oxygen by tetroxide decomposition. It was previously reported that lipid and DNA hydroperoxides can be decomposed to peroxyl radical in the presence of metal ions, cytochrome c, peroxynitrite, chloroperoxide, and hypochlorous acid [37,38]. It has been previously suggested that the recombination of two peroxyl radicals occurs mainly in chemical system or under high oxidative damage conditions [16]. The recombination of two peroxyl radicals forms unstable tetroxide which decompose either to ¹O₂, carbonyl and organic hydroxide or molecular oxygen, ³(R = O)* and organic hydroxide. Furthermore the ³(R = O)* can react with the molecular oxygen forming ¹O₂ [16]. It has been established in the chemical system that the formation of ¹O₂ by the recombination of peroxyl radicals is 3–4 magnitude higher compared to the formation of ³(C = O)* formed the recombination of peroxyl radicals [39]. It is suggested here that the decomposition of hydroperoxides takes place later after the addition of H₂O₂ and Fenton reagent to the cell suspension due to the long lifetime of organic hydroperoxides.

Supporting Information

S1 Fig. The formation of markers of oxidative damage to biomolecules.

Panel A: Formation of MDA during lipid peroxidation. In the first step, HO^• abstracts the hydrogen atom from lipid resulting in the formation of alkyl radical (reaction 1). Subsequent reaction of alkyl radical with molecular oxygen give raise to the peroxyl radical (reaction 2). Peroxyl radical undergoes cyclization to form cyclic peroxide (reaction 3) and consequently cyclic endoperoxide (reaction 4) known to decompose to alkyl radical and MDA (reaction 5). Panel B: Formation of protein carbonyl by β-scission of protein alkoxyl radical. The abstraction of hydrogen from carbonyl by HO^• results in the formation of protein alkyl radical (reaction 1) known to interact with molecular oxygen forming protein peroxyl radical (reaction 2). The second hydrogen abstraction by protein peroxyl radical from proteins leads to the formation of protein hydroperoxide (reaction 3) known to be reduced to protein alkoxyl radical by Fe²⁺ (reaction 4). The β-scission of protein alkoxyl radical leads to the formation of protein carbonyls and protein alkyl radical (reaction 5). Panel 3: DNA strand break initiated by HO^•. Hydrogen abstraction from deoxyribose forms deoxyribose radical (reaction 1) resulting in the instability of the deoxyribose phosphate backbone leading to the strand break (reaction 2).

(TIF)

Click here for additional data file.^{(250.9KB, tif)}

Acknowledgments

We would like to thank Prof. Pavel Anzenbacher for his support with respect to the HPLC and Dr. Ankush Prasad for discussion of results.

Data Availability

All relevant data are within the paper.

Funding Statement

This work was supported by the Ministero degli Affari Esteri, Direzione Generale per la Promozione e la Cooperazione Culturale (Significant bilateral project between Italy and Slovak) (http://www.esteri.it/MAE/IT/Politica_Estera/CooperScientificaTecnologica/Progetti_grande_rilevanza.htm). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Fig. The formation of markers of oxidative damage to biomolecules.

(TIF)

Click here for additional data file.^{(250.9KB, tif)}

Data Availability Statement

All relevant data are within the paper.

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[pone.0116958.ref009] 9. Britigan BE, Coffman TJ, Adelberg DR, Cohen MS (1988) Mononuclear phagocytes have the potential for sustained hydroxyl radical production—use of spin-trapping techniques to investigate mononuclear phagocyte free-radical production. J Exp Med 168: 2367–2372. [DOI] [PMC free article] [PubMed] [Google Scholar]

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[pone.0116958.ref011] 11. Wu TY, Rifai N, Roberts LJ, Willett WC, Rimm EB (2004) Stability of measurements of biomarkers of oxidative stress in blood over 36 hours. Cancer Epidem Biomar 13: 1399–1402. [PubMed] [Google Scholar]

[pone.0116958.ref012] 12. Gutteridge JMC (1995) Lipid-peroxidation and antioxidants as biomarkers of tissue-damage. Clin Chem 41: 1819–1828. [PubMed] [Google Scholar]

[pone.0116958.ref013] 13. Russell GA (1957) Deuterium-isotope Effects in the Autoxidation of Aralkyl Hydrocarbons. Mechanism of the Interaction of Peroxy Radicals. J Am Chem Soc 79: 3871–3877. [Google Scholar]

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[pone.0116958.ref019] 19. Schlesinger JJ, Brandriss MW (1980) Antibody-mediated Enhancement of yellow-fever virus (YFV) replication in a macrophage-like cell-line, U937—role of the FC and viral receptors. Clin Res 28: A644–A644. [PubMed] [Google Scholar]

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[pone.0116958.ref021] 21. Pou TE, Murphy OJ, Young V, Bockris JO, Tongson LL (1984) Passive films on iron—the mechanism of breakdown in chloride containing solutions. J Electrochem Soc 131: 1243–1251. [Google Scholar]

[pone.0116958.ref022] 22. Moan J, Wold E (1979) Detection of singlet oxygen production by ESR. Nature 279: 450–451. [DOI] [PubMed] [Google Scholar]

[pone.0116958.ref023] 23. Pilz J, Meineke I, Gleiter CH (2000) Measurement of free and bound malondialdehyde in plasma by high-performance liquid chromatography as the 2,4-dinitrophenylhydrazine derivative. J Chromatogr B 742: 315–325. [DOI] [PubMed] [Google Scholar]

[pone.0116958.ref024] 24. Wei SH, Zhou JH, Huang DY, Wang XS, Zhang BW, et al. (2006) Synthesis and Type I/Type II photosensitizing properties of a novel amphiphilic zinc phthalocyanine. Dyes Pigments 71: 61–67. [Google Scholar]

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PERMALINK

Oxidative Damage of U937 Human Leukemic Cells Caused by Hydroxyl Radical Results in Singlet Oxygen Formation

Marek Rác

Michal Křupka

Svatopluk Binder

Michaela Sedlářová

Zuzana Matušková

Milan Raška

Pavel Pospíšil

Roles

Abstract

Introduction

Materials and Methods

U937 cell culture

EPR spin-trapping spectroscopy

HPLC

Dot blot immunoassay

Comet assay

Singlet oxygen detection by confocal laser scanning microscopy and image analysis

Statistical analysis

Results

Formation of hydroxyl radical detected by EPR spin-trapping spectroscopy

Figure 1. Detection of hydroxyl radical by EPR spin-trapping spectroscopy.

Cell viability

Figure 2. Determination of the U937 cell viability.

Analysis of lipid peroxidation by MDA HPLC assay

Figure 3. Detection of lipid peroxidation product malondialdehyde by HPLC analysis.

Analysis of protein carbonylation by dot blot immunoassay

Figure 4. Detection of protein carbonyl compounds by dot blot immunoassay.

Analysis of DNA fragmentation by comet assay

Figure 5. Analysis of DNA strand breaks by Comet assay.

Table 1. Analysis of the medians of comet, head, and tail parameters in the control, the H2O2-treated and the Fenton reagent-treated U937 cells.

Formation of singlet oxygen detected by EPR spin-trapping spectroscopy

Figure 6. Detection of singlet oxygen by EPR spin-trapping spectroscopy.

Formation of singlet oxygen detected by confocal laser scanning microscopy

Figure 7. Singlet oxygen imaging by confocal laser scanning microscopy.

Discussion

Hydroxyl radical formation

Oxidative damage initiated by hydroxyl radical

Singlet oxygen formation caused by oxidative damage of U937 cells

Supporting Information

Acknowledgments

Data Availability

Funding Statement

References

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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Table 1. Analysis of the medians of comet, head, and tail parameters in the control, the H₂O₂-treated and the Fenton reagent-treated U937 cells.