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| Proc Natl Acad Sci U S A. 2007 November 6; 104(45): 17575–17578. Published online 2007 October 29. doi: 10.1073/pnas.0704030104. | PMCID: PMC2077033 |
Copyright © 2007 by The National Academy of Sciences of the USA Chemistry Molecular mechanism for metal-independent production of hydroxyl radicals by hydrogen peroxide and halogenated quinones Ben-Zhan Zhu, *†‡ Balaraman Kalyanaraman, § and Gui-Bin Jiang **State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, People's Republic of China; †Linus Pauling Institute, Oregon State University, Corvallis, OR 97331; and §Biophysics Research Institute, Medical College of Wisconsin, Milwaukee, WI 53226 Received May 2, 2007. |
Abstract We have shown previously that hydroxyl radicals (HO•) can be produced by H2O2 and halogenated quinones, independent of transition metal ions; however, the underlying molecular mechanism is still unclear. In the present study, using the electron spin resonance secondary radical spin-trapping method, we found that tetrachloro-1,4-benzoquinone (TCBQ), but not its corresponding semiquinone anion radical, the tetrachlorosemiquinone anion radical (TCSQ•−), is essential for HO• production. The major reaction product between TCBQ and H2O2 was identified by electrospray ionization quadrupole time-of-flight mass spectrometry to be the ionic form of trichlorohydroxy-1,4-benzoquinone (TrCBQ-OH), and H2O2 was found to be the source and origin of the oxygen atom inserted into the reaction product TrCBQ-OH. On the basis of these data, we propose that HO• production by H2O2 and TCBQ is not through a semiquinone-dependent organic Fenton reaction but rather through the following mechanism: a nucleophilic attack of H2O2 to TCBQ, forming a trichlorohydroperoxyl-1,4-benzoquinone (TrCBQ-OOH) intermediate, which decomposes homolytically to produce HO•. This represents a mechanism of HO• production that does not require redox-active transition metal ions. Keywords: ESR spin-trapping, tetrachloro-1,4-benzoquinone, tetrachlorosemiquinone anion radical, Fenton reaction |
The hydroxyl radical (HO •) has been considered to be one of the most reactive oxygen species produced in biological systems. It has been shown ( 1– 4) that HO • can cause DNA, protein, and lipid oxidation. One of the most widely accepted mechanisms for HO • production is through the transition metal-catalyzed Fenton reaction (where “Me” represents a transition metal, such as iron or copper) ( 1– 4):
We reported previously ( 5, 6) that, by using the salicylate hydroxylation assay and electron spin resonance (ESR) spin-trapping methods, HO • can be produced by H 2O 2 and halogenated quinones independent of transition metal ions; however, the underlying molecular mechanism is still unclear. On the basis of our experimental results, a unique mechanism was proposed for HO • production by H 2O 2 and halogenated quinones. |
Results and Discussion TCBQ, but Not Its Corresponding Semiquinone Anion Radical, Is Essential for HO• Production. We showed previously ( 6) that the tetrachlorosemiquinone anion radical (TCSQ •−) signal was markedly decreased by the addition of H 2O 2 to TCBQ (a carcinogenic metabolite of the widely used wood preservative pentachlorophenol, here used as a model halogenated quinone compound), with concurrent formation of HO •. These data suggest that TCSQ •− may directly react with H 2O 2, reducing it to HO •, which is analogous to the Fe 2+-catalyzed decomposition of H 2O 2 to HO • ( Reaction 1). On the basis of these findings, we proposed ( 5, 6) that the mechanism for the production of HO • by H 2O 2 might be through a semiquinone-dependent organic Fenton reaction:
where TCSQ •− substitutes for ferrous iron in the classic, metal-dependent Fenton reaction. This type of reaction between semiquinone radicals and H 2O 2 was previously proposed by Koppenol and Butler ( 7), who suggested that if a quinone/semiquinone couple has a reduction potential between −330 and +460 mV, it can theoretically bring about a metal-independent Fenton reaction. However, no convincing experimental evidence has been provided for this kind of reaction. According to this mechanism, the production of HO • from H 2O 2 and TCBQ should depend on the concentration of TCSQ •−, i.e., the higher the concentration, the more HO • should be produced. Furthermore, the main product of this reaction should be TCBQ. Using the ESR secondary radical spin-trapping method, we found that DMPO/ •CH 3 and DMPO/HO • adducts can be produced by H 2O 2 and TCBQ in the presence of the spin-trapping agent 5,5-dimethyl-1-pyrroline N-oxide (DMPO) and HO • scavenger dimethyl sulfoxide (DMSO) ( Fig. 1A). One of the minor oxygen-centered radical adducts was tentatively identified as the DMPO adduct with methoxy radicals ( •OCH 3) (DMPO/ •OCH 3: aHβ = 10.7 G, aHγ = 1.32 G, and aN = 14.5 G) ( 8, 9). It should be noted that another minor carbon-centered radical adduct was also observed, but its exact nature is still unclear, and further studies are needed to identify this radical adduct. However, no DMPO/ •CH 3 or DMPO/HO • adducts were detected from H 2O 2 and tetrachlorohydroquinone (TCHQ), the reduced form of TCBQ, although high concentrations of TCSQ •− were produced during the autooxidation of TCHQ ( Fig. 1B). [The central signal in this spectrum was identified as TCSQ •−, with g value = 2.0056 ( 6), which was also observed in the absence of DMPO. It should also be noted that basically the same results were observed from a spin-trapping experiment in which everything was present except H 2O 2 under the same experimental conditions as shown in Fig. 1A.] Interestingly, if TCHQ was quickly oxidized to TCBQ with myeloperoxidase, DMPO/ •CH 3 and DMPO/HO • adducts could again be detected ( Fig. 1C), similar to those produced by TCBQ ( Fig. 1A). Furthermore, the formation of DMPO/ •CH 3 and DMPO/HO • was found to be directly dependent on the concentrations of TCBQ ( Fig. 2) and H 2O 2 (data not shown). These results strongly suggest that TCBQ, but not TCSQ •−, is essential for HO • production. Therefore, the production of HO • by TCBQ and H 2O 2 appears not to occur through a semiquinone-dependent organic Fenton reaction.  | Fig. 1.TCBQ, but not TCSQ•−, is essential for the formation of DMPO/•CH3 and DMPO/HO• adducts. Reactions were carried out in Chelex-treated phosphate buffer (100 mM, pH 7.4). All reaction mixtures contained 100 mM DMPO, 5% DMSO, (more ...) |
 | Fig. 2.The formation of DMPO/•CH3 and DMPO/HO• adducts depends on TCBQ concentration. Reactions were carried out at room temperature in 100 mM Chelex-treated phosphate buffer (pH 7.4). All reaction mixtures contained 100 mM DMPO and 10 mM H2 (more ...) |
The Major Reaction Product Between TCBQ and H2O2 Was Identified to Be the Ionic Form of Trichlorohydroxy-1,4-benzoquinone (TrCBQ-OH). UV-visible (UV-vis) spectral studies showed a direct interaction between TCBQ and H2O2, with the reaction mixture changing quickly from the original yellow color (λmax = 292 nm) to a characteristic purple color (λmax = 295 and 535 nm) in phosphate buffer (pH 7.4). The final reaction products between TCBQ and H 2O 2 were identified by electrospray ionization quadrupole time-of-flight mass spectrometry (ESI-Q-TOF-MS). The mass spectrum of TCBQ in 0.1 M CH 3COONH 4 buffer (pH 7.4) is characterized by a four-chlorine isotope cluster at m/ z 246 ( Fig. 3A) and traces of a three-chlorine isotope cluster at m/ z 227. The addition of H 2O 2 to TCBQ led to complete disappearance of the molecular ion peak clusters at m/ z 246 and dramatic increase in the peak clusters at m/ z 227 ( Fig. 3B). Similar ESI-MS results were observed when TCBQ was substituted with other halogenated quinones such as tetrabromo-, tetrafluoro-, and 2,5-dichloro-1,4-benzoquinone (data not shown). These results indicate that the major reaction product between TCBQ and H 2O 2 was probably the ionic form of TrCBQ-OH (peak clusters at m/ z 227). This was further confirmed by comparing with the authentic TrCBQ-OH synthesized according to the published method ( 10), which showed the same ESI-MS profile ( Fig. 3C) and the same retention time in HPLC (see Materials and Methods). Tandem mass spectrometric analysis showed that the peak at m/ z 225 could be fragmented to form the peak at m/ z 197 ( Fig. 3D), which suggests that the peak at m/ z 197 is solely derived from the peak at m/ z 225 ( Fig. 3C).  | Fig. 3.Mass spectral analysis. (A–C) ESI-Q-TOF-MS spectra of TCBQ (A), TCBQ with H2O2 (B), and TrCBQ-OH (C) in 0.1 M CH3COONH4 buffer (pH 7.4). TCBQ, 0.5 mM; H2O2, 100 mM; TrCBQ-OH, 0.5 mM. (D) Tandem mass spectrometric analysis showed that the peak (more ...) |
H2O2 Is the Source and Origin of the Oxygen Atom Inserted into the Reaction Product TrCBQ-OH. To better understand the source and origin of the oxygen atom inserted into the reaction product TrCBQ-OH, formed from the reaction between H2O2 and TCBQ, TCBQ was incubated with oxygen-18-enriched H2O2 ([18O]H2O2). The mass spectra of the molecular ion region of deprotonated TrCBQ-OH, obtained with unlabeled and labeled H2O2, demonstrated the shift of the molecular ion isotope cluster peaks of the unlabeled compound with 2 mass units, as could be expected for the incorporation of 18O (data not shown). These results indicate that H2O2 is the source and origin of the oxygen atom inserted into the reaction product TrCBQ-OH. It has also been shown ( 11) that both TCBQ and H 2O 2 are consumed with a stoichiometric ratio of ≈1:1, and H 2O 2 accelerates the rate of TCBQ decomposition by two orders of magnitude with the loss of chloride. Molecular Mechanism of Metal-Independent Production of HO• by H2O2 and Halogenated Quinones. The metal-independent production of HO • by TCBQ and H 2O 2 appears not to occur through a previously proposed semiquinone-mediated organic Fenton reaction. On the basis of the above experimental results and the fact that H 2O 2 is a better nucleophile than H 2O ( 12), a unique mechanism can be proposed for HO • production by H 2O 2 and TCBQ. A nucleophilic reaction may take place between TCBQ and H 2O 2, forming a trichlorohydroperoxyl-1,4-benzoquinone (TrCBQ-OOH) intermediate, which can decompose homolytically to produce HO • and trichlorohydroxy-1,4-benzoquinone radical (TrCBQ-O •). TrCBQ-O • may then disproportionate to form the ionic form of trichlorohydroxy-1,4-benzoquinone (TrCBQ-O −) ( Scheme 1), and the disproportionation reaction may be described as
 | Scheme 1.Proposed mechanism for HO• production by TCBQ and H2O2. A nucleophilic reaction may take place between TCBQ and H2O2, forming a TrCBQ-OOH intermediate, which can decompose homolytically to produce HO• and TrCBQ-O•. TrCBQ-O• (more ...) |
Alternatively, TrCBQ-O • may obtain an electron from TCSQ •−, which was probably produced as a result of H 2O addition to TCBQ ( 6), to form TrCBQ-O −. Further studies are needed to elucidate the exact pathway by which TrCBQ-O • was converted to TrCBQ-O −. This pathway represents a unique mechanism of HO • formation that does not require the involvement of redox-active transition metal ions and may partly explain the potential carcinogenicity of widely used biocides, including polychlorinated phenols (such as the wood preservatives pentachlorophenol and 2,4,6- and 2,4,5-trichlorophenol), hexachlorobenzene, and Agent Orange [the mixture of 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) and 2,4-dichlorophenoxyacetic acid (2,4-D)], given that these compounds can be metabolized in vivo to tetra-, di-, or monochlorinated quinones ( 6, 13, 14). It should be noted that neither TrCBQ-OOH nor TrCBQ-O• could be detected under our current experimental conditions. It may be that their half-life spans are too short or their steady-state concentrations are too low. Further studies are needed to identify these reaction intermediates. |
Materials and Methods Chemicals. TCBQ, tetrabromo-1,4-benzoquinone (TBrBQ), tetrafluoro-1,4-benzoquinone (TFBQ), 2,5-dichloro-1,4-benzoquinone (DCBQ), and DMPO were purchased from Aldrich (Milwaukee, WI). H2O2 was purchased from Sigma (St. Louis, MO). The [18O]H2O2 (91%) was purchased from Icon (Summit, NJ). The chemicals were used as received, without further purification. ESR Studies. The basic system used in this study consisted of halogenated quinones dissolved in DMSO (final DMSO concentration in the reaction mixture, 5–10%), H2O2, and the spin-trapping agent DMPO (100 mM), in 100 mM Chelex-treated phosphate buffer (pH 7.4) at room temperature. All reaction mixtures were air-saturated for the EPR experiments. ESR spectra were recorded 1 min after the interactions between H2O2 and TCBQ (or other halogenated quinones) at room temperature under normal room-lighting conditions on a Bruker (Billerica, MA) ER 200 D-SRC spectrometer operating at 9.8 GHz and a cavity equipped with a Bruker Aquax liquid sample cell. Typical spectrometer parameters were scan range, 100 G; field set, 3470 G; time constant, 200 ms; scan time, 100 s; modulation amplitude, 1.0 G; modulation frequency, 100 kHz; receiver gain, 1.25 × 104; and microwave power, 9.8 mW. UV-vis Spectral Analysis. The interaction between halogenated quinones and H2O2 was monitored in a UV-vis spectrophotometer (DU-640; Beckman Coulter, Fullerton, CA), using Chelex-treated phosphate buffer (100 mM, pH 7.4) at room temperature. ESI-Q-TOF-MS. Analysis by ESI-Q-TOF-MS of the reaction products between TCBQ and H2O2 was performed on a Micromass (Manchester, U.K.) Q-TOF mass spectrometer. MS and MS/MS spectra were acquired in negative-ion mode. The reactions between TCBQ (0.5 mM) and H2O2 (100 mM) were conducted in 0.1 M CH3COONH4 buffer (pH 7.4) containing 10% DMSO at room temperature, and the sample solutions were directly introduced 1 min after the reaction at 30 μl/min, using a Z-spray ESI source. Capillary and sample cone voltages were 2.5 kV and 30 V, respectively; source and desolvation temperatures were 80° and 200°C, respectively. Nitrogen was used as the drying gas. The collision gas was argon at a pressure of 5.0 × 10−5 Torr (1 Torr = 133.322 Pa), and the collision energy was 10 V. Both the high and low resolution for mass filter were set at 5.0 V. The pressure in the TOF cell was <3.0 × 10−7 Torr. Full-scan spectra were recorded in profile mode. The range between m/z 50 and 450 was recorded at a resolution of 5,000 (FWHM), and the accumulation time was 1 s per spectrum. For MS/MS studies, the quadrupole was used to select the parent ions, which were subsequently fragmented in a hexapole collision cell by using argon as the collision gas and an appropriate collision energy (typically 10–40 eV). Data acquisition and analysis were carried out by using Masslynx software (version 4.0) (Waters, Milford, MA). Synthesis of the Reaction Product TrCBQ-OH. TrCBQ-OH was synthesized as described in the literature ( 7) by dissolving solid TCBQ (30 mg) in an ice-cold 2 M sodium hydroxide solution. The purity and identity of the synthesized compound were assessed by HPLC and ESI-MS analysis (see above). Only one peak was observed in the HPLC analysis, and a three-chlorine isotope cluster was evident at m/ z 227 [(M-H) − ion of TrCBQ-OH] for ESI-Q-TOF-MS analysis. Source and Origin of the Oxygen Atom Inserted into the Reaction Product TrCBQ-OH. To investigate the source and origin of the oxygen atom inserted into the TrCBQ-OH, formed from the reaction between H2O2 and TCBQ, 0.5 mM TCBQ was incubated with 100 mM [18O]H2O2 in a final volume of 0.1 ml of 0.1 M CH3COONH4 buffer (pH 7.4) containing 10% DMSO. A control experiment with unlabeled H2O2 was also performed. After 1 min of incubation, the samples were analyzed by ESI-Q-TOF-MS. HPLC Conditions. Samples were analyzed with an HPLC apparatus equipped with a model 2996 photodiode array detector (2695 XE; Waters). Samples were transferred directly into minivials and immediately injected onto a SUPELCOSIL LC-18 C18 column (4.6 × 250 mm, pore size 60 μm; Sigma). Elution was performed at 1 ml/min, starting with 100% solvent A (1% acetic acid in water) and then maintaining 100% of this solvent for 1 min, followed by a linear gradient to obtain 100% methanol in 25 min. The absorbance was monitored between 200 and 700 nm. Sample retention times and absorption maxima were compared with those of standard compounds. The HPLC standard TrCBQ-OH was distinguished by its absorption maxima of 295 nm. |
Acknowledgments We thank Drs. Hong-Tao Zhao, Jing Wang, and Hao Zhang, and Ms. Rui Guo, for technical help. This work was supported by Hundred-Talent Project, Chinese Academy of Sciences, Chinese National Natural Science Foundation Grants 20777080 and 20747001; National Institutes of Health Grants ES11497, RR01008, and ES00210 (to B.-Z.Z.); and Chinese National Natural Science Foundation Grant 20621703 (to G.-B.J.). |
Abbreviations | DMPO | 5,5-dimethyl-1-pyrroline N-oxide | | ESI-Q-TOF-MS | electrospray ionization quadrupole time-of-flight mass spectrometry | | MPO | myeloperoxidase | | TCBQ | tetrachloro-1,4-benzoquinone | | TCHQ | tetrachlorohydroquinone | | TCSQ•− | tetrachlorosemiquinone anion radical | | TrCBQ-OH | trichlorohydroxy-1,4-benzoquinone | | TrCBQ-OOH | trichlorohydroperoxyl-1,4-benzoquinone | | UV-vis | UV-visible. |
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