Article pubs.acs.org/est
Hydroxyl Radical Generation Mechanism During the Redox Cycling Process of 1,4-Naphthoquinone Yu Shang,†,‡,# Chenyong Chen,†,# Yi Li,§,* Jincai, Zhao,∥ and Tong Zhu†,* †
State Key Laboratory for Environmental Simulation and Pollution Control, College of Environmental Sciences and Engineering, Peking University, Beijing 100871, China ‡ Institute of Environmental Pollution and Health, School of Environmental and Chemical Engineering, Shanghai University, Shanghai 200444, China § Chinese Academy of Meteorological Sciences, Beijing 100081, China ∥ Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, China S Supporting Information *
ABSTRACT: Airborne quinones contribute to adverse health effects of ambient particles probably because of their ability to generate hydroxyl radicals (·OH) via redox cycling, but the mechanisms remain unclear. We examined the chemical mechanisms through which 1,4-naphthoquinone (1,4-NQ) induced ·OH, and the redox interactions between 1,4-NQ and ascorbate acid (AscH2). First, ·OH formation by 1,4-NQ was observed in cellular and acellular systems, and was enhanced by AscH2. AscH2 also exacerbated the cytotoxicity of 1,4-NQ in Ana-1 macrophages, at least partially due to enhanced ·OH generation. The detailed mechanism was studied in an AscH2/H2O2 physiological system. The existence of a cyclic 1,4-NQ process was shown by detecting the corresponding semiquinone radical (NSQ·−) and hydroquinone (NQH2). 1,4-NQ was reduced primarily to NSQ·− by O2·− (which was from AscH2 reacting with H2O2), not by AscH2 as normally thought. At lower doses, 1,4-NQ consumed O2·− to suppress ·OH; however, at higher doses, 1,4-NQ presented a positive association with ·OH. The reaction of NSQ·− with H2O2 to release ·OH was another important channel for OH radical formation except for Haber-Weiss reaction. As a reaction precursor for O2·−, the enhanced ·OH response to 1,4-NQ by AscH2 was indirect. Reducing substrates were necessary to sustain the redox cycling of 1,4-NQ, leading to more ·OH and a deleterious end point.
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(8-OHdG), a biomarker for oxidative stress in human body.17 1,2-naphthoquinone is reported to cause concentrationdependent contraction of tracheal smooth muscle in guinea pigs, which may result in asthma exacerbation.18 Acenaphthenequinone is found to induce cyclooxygenase-2 gene expression, a key enzyme in inflammatory process.19 Quinone redox cycling is a process with reduction of quinones (Q) to semiquinone anion radicals (Q·−) or hydroquinones. Quinones undergo one-electron reduction to form Q·−, catalyzed by cellular reductases, such as NADPH-cytochrome P-450 reductase, or reducing agents (e.g., GSH).20 Q·− then reduce O2 to O2·−, accompanied by an oxidization to quinones, thus completing the cycle. Two-electron reduction to hydroquinones has been suggested to lead to detoxification of the quinones, because hydroquinones can be conjugated to nontoxic sulfates or glucuronides.21 Along with the enzymatic or nonenzymatic redox cycling of quinones, ROS are generated progressively.
INTRODUCTION Associations between exposure to ambient particulate matter (PM) and cardiopulmonary diseases are widely demonstrated in epidemiological and toxicological studies.1−3 Because of the highly complex and heterogeneous properties of PM,4 the underlying mechanisms remain poorly understood. Oxidative stress and reactive oxygen species (ROS) generation are considered as key factors in harmful effects caused by particles.5−7 Quinones are diketones with a fully conjugated cyclic dione structure, which are present both in gas and particle phase in air. Quinones are hypothesized to contribute to adverse health effects of PM because of their ability to generate ROS via redox cycling.8−11 Most quinones found in PM are polycyclic aromatic quinones, such as anthraquinone, naphthoquinone, and phenanthrenequinone.12,13 These quinones are primarily from combustion process of fossil fuels and/or atmospheric photochemical conversions from polycyclic aromatic hydrocarbons (PAHs) and phenols.14 Reported concentration levels are in pg/m3 and the lower ng/m3 range.13 In traffic-related particles, the contents of anthraquinone and naphthoquinone are even higher than benzo(a)pyrene.15,16 Personal exposure to anthraquinone (AQ) in fine particles is correlated with urinary 8-hydroxy-2′-deoxyguanosine © 2012 American Chemical Society
Received: Revised: Accepted: Published: 2935
August 31, 2011 December 5, 2011 January 30, 2012 January 30, 2012 dx.doi.org/10.1021/es203032v | Environ. Sci. Technol. 2012, 46, 2935−2942
Environmental Science & Technology
Article
ROS consist primarily of superoxide anion radicals (O2·−), hydrogen peroxide (H2O2), and hydroxyl radical (·OH). ·OH is the strongest ROS and triggers extensive cellular damage.22 Quinones are able to produce O2·− and H2O2 at a cellular level, ultimately leading to ·OH formation. However, the exact mechanisms remain unclear. It is generally accepted that ·OH is generated through O2·− reacting with H2O2 (the Haber-Weiss reaction). Thermodynamic calculations indicate that semiquinones with a reduction potential between −330 and 460 mV can, theoretically, bring about one-electron reduction of H2O2, resulting in ·OH generation.23 Persistent paramagnetic signals of semiquinone radicals on particles are observed,24 indicating that they may be an important source for ·OH. The redox cycling of quinones is dependent on reducing equivalents, thus determining whether ·OH radicals are formed.25,26 Semiquinone radicals are considered to be essential active intermediates. However, the interrelations between quinone recycling and reducing substrates (such as ascorbate acid, AscH2, a reducing agent in lung fluid) are complex and chemical mechanisms for ·OH formation are not investigated in detail.27,28 The aim of this work was to study the ability of the 1,4naphthoquinone (1,4-NQ) and AscH2 association to cause ·OH generation and its roles in cytotoxicity in vitro. This is important because AscH2 is an antioxidant present in lung fluid and is thought to facilitate ·OH generation induced by iron absorbed on particle surfaces.29,30 Further, the related chemical pathways for ·OH formation in both cellular and acellular systems were studied. 1,4-NQ is widely detected in a variety of particle samples,31 and was used as a model.
Figure 1. Experimental flowchart.
system to examine the chemical mechanism(s) of 1,4-NQ to generate ·OH. The blank control consisted of the same physiological system with no 1,4-NQ. All reactions took place in a 37 °C water bath and were terminated 30 min (experiments showed products reached almost highest after 30 min, data not shown) later by the addition of 125 mM mannitol. Following the reaction, a 1 mL aliquot of the reaction suspension was filtered through polypropylene Acrodisc syringe filters (0.22 μm) and was analyzed later. All of the vitreous containers used were immersed in 10% nitric acid for 24 h to remove adsorbed iron. Recycling Process for 1,4-NQ. Semiquinone anion radicals (NSQ·−) and corresponding hydroquinone (NQH2) were measured to elucidate the recycling process. Redox Interactions with AscH2. Three different reaction systems were constructed in a phosphate milieu (pH 7.4): 1,4NQ/AscH2/H2O2, 1,4-NQ/AscH2, and 1,4-NQ/H2O2. ·OH and semiquinone radicals were detected in each to examine the interactions between 1,4-NQ and AscH2, and their roles in ·OH generation. Methods. Cell Culture and Cytotoxicity Assay. Cells were cultured with RPMI 1640 with 10% FBS, 100 units/mL penicillin and streptomycin in an atmosphere of 5% CO2 and 100% relative humidity at 37 °C. For viability assay, cells were plated in 96-well plates with a density of 5 × 103 cells per well and cultured 24 h to allow cell adhesion. Medium was removed and cells were treated with 1,4-NQ (0−250 μM) for 8 h. Cells that were not treated with 1,4-NQ were control, and culture medium was background (BD). After treatment, cells were incubated with 100 μL of freshly prepared MTT (1 mg/mL) in medium for 4 h in dark at 37 °C. Formazan crystals formed in the cells were solubilized by the addition of 100 μL DMSO. Finally, absorbance values (OD) were read using a microplate enzyme-linked immunosorbent assay (ELISA) reader (Biotek ELx 800, U.S.) at a wavelength of 570 nm.
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EXPERIMENTAL SECTION Materials. The 1,4-NQ was from Chem Service GmbH (Worms, Germany) with a purity >99.7%. The 1,4-naphthoquinol (NQH2) was reduced from 1,4-NQ using BaBH4. 2′,7′dichlorodihydrofluorescein diacetate (H2DCFDA) was from Molecular Probes, Inc. (Eugene, OR). Sodium dodecyl sulfate (SDS, purity >99%) and trihydroxymethyl aminomethane (Tris, purity >99%) were from Amresco Company (Solon, OH, U.S.). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoli bromide (MTT), 2,3-dihydrobenzoic acid (2,3-DHBA, purity >99%), 2,5DHBA (purity >99%), catechol (purity >99%), and salicylic acid (SA) were from Sigma Chemicals Company (Poole, Dorset, UK). The murine macrophage cell line (Ana-1) was from Maisha Biological Company (Shanghai, China). Fetal bovine serum (FBS) and RPMI 1640 culture medium were from Gibco (U.S.). All other reagents were obtained from Beijing Chemical Reagent Co. (China) and were of analytical grade. Milli-Q water was used in all experiments. Experiment Design (Figure 1). Ability of 1,4-NQ to cause ROS and ·OH, leading ultimately to cell death or cell survival, was examined in Ana-1 macrophage cells. Additionally, the exact chemical mechanism(s) of ·OH was studied in physiological solution. Cytotoxicity, ROS, and ·OH in Cells Induced by 1,4-NQ. Viability, ROS, and ·OH were measured in Ana-1 cells after treatment with 1,4-NQ. Enhancement of ROS, ·OH and Cell Death by AscH2. Cellular ROS, ·OH, and viability were investigated in Ana-1 cells after treatment with 1,4-NQ and AscH2. Physiological System. The physiological system consisted of AscH2 and hydrogen peroxide (H2O2) in 10 mM NaH2PO4− Na2HPO4 buffer (pH 7.4). We developed this system to study how PM induced ·OH generation.29,30 We used this cell-free
cell viability% = ((OD1,4 − NQ − ODBD) /(ODcontrol − ODBD)) × 100%
Intracellular ROS Detection. The compound H2DCFDA is a fluorescent dye that diffuses through cell membranes and is deacetylated by intracellular esterases to nonfluorescent 2′,7′dichlorodihydrofluorescin (DCFH). In the presence of ROS, DCFH is oxidized to highly fluorescent 2′,7′-dichlorodihydro2936
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fluorescein (DCF). Briefly, Ana-1 cells were treated with different concentrations of 1,4-NQ for 1 h and H2DCFDA (5 μM) was added in the dark 30 min before the termination of drug treatment. Cells were then washed in Hanks, trypsinized, and resuspended in 1 mL of Hanks. Green fluorescence intensity was immediately read with flow cytometry (BD FACSCalibur, U.S.). Results are expressed as arbitrary units of fluorescence per 106 cells. Intracellular ·OH Evaluation. DCFH can be oxidized by hydrogen peroxide, superoxide radicals, and ·OH. Thus, the fluorescence intensity represents oxidation due to the sum of the three ROS. Mannitol is a specific quencher of ·OH. The fluorescence reduction caused by mannitol pretreatment could be attributed to the contribution from ·OH. Briefly, 10 mM of mannitol was added to the cells and they were cultured for 10 min before being treated with H2DCFDA. Other procedures were identical to that described above. The fluorescence intensity (FI) for ·OH was calculated from
Figure 2. Cytotoxic effect of 1,4-NQ associated with AscH2 in Ana-1 macrophage cells measured by the MTT assay. Cells were treated for 8 h and an MTT assay was performed immediately. 1,4-NQ concentrations were 0, 5, 20, 60, 125, 250, and 500 μM. AscH2 concentrations were 0, 0.1, 0.5, 1.0, and 2.5 mM. The box indicated the concentration range, under which AscH2 exacerbated the cytotoxicity of 1,4-NQ. Experiments were repeated at least three times; error bars are not shown to make the figure easier to read.
FI(·OH) = FI(ROS) − FI(mannitol pretreated)
Free Radical ProcessElectron Spin Resonance (ESR). For ESR studies, 1,4-NQ, prepared freshly in DMSO, was mixed with other reactants in 10 mM NaH2PO4−Na2HPO4 buffer (pH 7.4). Ten mM MgCl2 was added to stabilize the semiquinone radical. The final concentration of DMSO was less than 5% (v/v). Reaction was initiated by the addition of 1,4NQ. ESR spectra were recorded at room temperature on a Bruker ESP 300 E spectrometer (Bruker, Germany) operating at the X band (9.78 GHz) with 100-kHz frequency modulation. The instrumental settings were microwave field 3480 G, sampling time 81.92 ms, microwave frequency 9.78 GHz, microwave power 0.010 W, receiver gain 60, and time constant 32.68 s. The sample tube had an internal diameter of 1 mm (Spectrosil). ·OH MeasurementsCapillary Electrophoresis (CE). SA reacts with ·OH to generate 2,3-DHBA, 2,5-DHBA, and catechol. The sum of products was assumed to represent ·OH level. CE (HP3D, Agilent, Germany) was used to measure SA, DHBAs and catechol concentrations, as reported previously.29,30 1,4NQ and NQH2 could also be detected with CE. All products were detected by monitoring the absorbance at 200 nm with a diode array detector in CE. Detailed information for CE measurements was shown in Supporting Information (SI). Data Analysis. Data were analyzed with SPSS software (ver. 11.5). All data are expressed as means ± standard deviation. Statistical tests was performed using one-way analysis of variance (ANOVA). Probability values 0.05). Relationships between cytotoxic effects of 1,4-NQ and AscH2 appeared quite complex. AscH2 is reported to have a crossover effect, acts as an antioxidant or a pro-oxidant, depends on its concentration.32 In literature, a wide range of concentration has 2937
dx.doi.org/10.1021/es203032v | Environ. Sci. Technol. 2012, 46, 2935−2942
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These results indicated that AscH2 could significantly enhance the effect of 1,4-NQ on ·OH production in cells. These data suggested a synergistic increase in cell killing with 1,4-NQ and AscH2 was at least partially due to ·OH production. Chemical Mechanism(s) of 1,4-NQ-Induced ·OH Generation. The chemical mechanism of 1,4-NQ-induced ·OH generation in the presence of AscH2 is a complex process; therefore, we simplified the experimental conditions. Instead of conducting cellular experiments, we investigated the chemical mechanism in a physiological system. Combined Impacts of 1,4-NQ and AscH2 on ·OH in Physiological System. The amount of ·OH was measured in a AscH2/H2O2 physiological system with 0−1500 μM of 1,4NQ added (Figure 5). Experiments were repeated for three
Figure 3. Combined effects between 1,4-NQ and AscH2 on ROS generation in Ana-1 cells. ROS detection in cells induced by AscH2 (A) and AscH2 with 5 μM 1,4-NQ (B) or 10 μM 1,4-NQ (C). Controls were vehicle-treated cells with no 1,4-NQ or AscH2.
Figure 5. Combined effects of 1,4-NQ and AscH2 on ·OH generation after addition to the physiological solution (pH 7.4). Trapped ·OH represented the sum of 2,5-DHBA, 2,3-DHBA, and catechol, three major products of ·OH reacting with SA. The concentrations of 1,4NQ were 0, 20, 100, 200, 500, 1000, and 1500 μM; those of AscH2 were 0.5, 1, and 2 mM. The concentration of H2O2 was 3.6 mM. Reactions took place in a 37 °C water bath and lasted 30 min. Experiments were repeated three times and each experiment was carried out in triplicate. The values are expressed as means ± SEM (n = 9). One data point at 20 μM 1,4-NQ with AscH2 of 0.5 mM was discarded due to experimental mistakes.
without statistic significance. At 1 mM, ROS decreased to control level again. Under this condition, the addition of AscH2 reactivated reductases and recovered the oxidative stress in cells, leading to additional ROS release. ·OH in Ana-1 Cells. Figure 4 shows the ·OH response (calculated as described in Methods) to 1,4-NQ with or
concentrations of AscH2 (0.5, 1, and 2 mM). In the absence of 1,4-NQ, AscH2 may react with H2O2 to generate O2·−, followed by ·OH formation through Haber-Weiss reaction. At each AscH2 level, increased 1,4-NQ caused the level of ·OH first decreased and then increased dose-dependently, until to the final concentration. Figure 5 also showed that AscH2 markedly enhanced the amount of ·OH at each dose of 1,4-NQ, except 20 μM. Additionally, the turning doses for 1,4-NQ’s two-sided effects on ·OH were inversely correlated with AscH2. A higher concentration of AscH2 corresponded to a lower turning point. These results showed that AscH2 could potentiate the promoting effects of 1,4-NQ on ·OH and indicated the interrelationships between 1,4-NQ and AscH2. Chemical Process Analysis. To determine why 1,4-NQ first decreased and then increased ·OH, the chemical process was investigated thoroughly. The assumed pathways for ·OH generation in the 1,4-NQ/AscH2/H2O2 system are shown in Figure S2 (SI). As generally accepted, 1,4-NQ was reduced to a radical NSQ·− directly by AscH2, and simultaneously the redox cycling process between 1,4-NQ and NSQ·− was started. During this process, O2·− was generated through NSQ·− reacting with O2, and ·OH was finally produced via the Haber-Weiss
Figure 4. ·OH generated in Ana-1 cells treated with 1,4-NQ with or without AscH2. The value less than zero may be due to experiment error.
without AscH2. A positive association between 1,4-NQ and ·OH levels was observed. The amount of ·OH was significantly higher after AscH2 was added, but it was not dose-dependent. 2938
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Table 1. Impacts of AscH− and/or H2O2 on NSQ·− and ·OH Detection in a Physiological System Contained 250 μM 1,4-NQa
reaction. To demonstrate this and verify the detailed chemical pathways, the redox process of 1,4-NQ and the interactions between 1,4-NQ and AscH2 were examined. Determination of Redox Cycling for 1,4-NQ. The existence of a redox cyclic process was shown by determining NSQ·− and the two-electron reduced product, NQH2. (a). ESR Measurements. ESR signals for NSQ·− and ascorbyl radicals (Asc·−) were detected in 1,4-NQ/AscH2/H2O2 aqueous solution (Figure 6), confirming that NSQ·− and Asc·− were
AscH2 (mM)
H2O2 (mM)
trapped ·OH (mM)
0 0 1 1
0 3.6 0 3.6
ND ND 0.040 ± 0.001 0.076 ± 0.009
1,4-NQ transformation ratio (%) NSQ·− Asc·− 0 0
