J. Phys. Chem. C 2009, 113, 15997–16001
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EPR Study of Visible Light-Induced ROS Generation by Nanoparticles of ZnO Anat Lipovsky,†,‡ Zeev Tzitrinovich,‡ Harry Friedmann,‡ Guy Applerot,† Aharon Gedanken,*,† and Rachel Lubart*,‡ Department of Chemistry, Kanbar Laboratory for Nanomaterials, Institute of Nanotechnology and AdVanced Materials, Bar-Ilan UniVersity, Ramat-Gan 52900, Israel, and Departments of Chemistry and Physics, Bar-Ilan UniVersity, Ramat-Gan, Israel ReceiVed: March 9, 2009; ReVised Manuscript ReceiVed: July 23, 2009
Nanoparticles of ZnO have been shown to have marked antibacterial and anticancerous activities. The toxic effect of ZnO has been found to exist due to a reaction of the ZnO surface with water. In the present work electron-spin resonance measurements revealed that aqueous suspensions of small nanoparticles of ZnO produce increased levels of reactive oxygen species, namely hydroxyl radicals and singlet oxygen. Interestingly, a remarkable enhancement of the oxy radicals, was detected when the ZnO water suspension had been irradiated with blue (400-500 nm) light. The characterization of the mechanism of light-induced oxy radicals formation by ZnO nanoparticles would contribute to its use as a sterilization agent. Introduction
Materials and Methods
In recent years, nanomaterials have been of special interest due to their higher chemical reactivity, as compared to that of similar materials in the bulk form. Of particular interest are nano-TiO2 and ZnO, which have been widely used for their bactericidal and anticancerous properties.1-4 Although there are numerous studies regarding the antibacterial effect of ZnO, little is known about the mechanism underlying its cytotoxic effect. Sawai5-7 proposed that hydrogen peroxide generation is responsible for bacterial destruction, whereas Stoimenov et al.8 suggested that the binding of ZnO particles on the bacterial surface due to electrostatic forces kills the pathogens. Regarding the anticancerous properties of ZnO, it was suggested that ROS generation plays a crucial role in nanoparticles induced cytotoxicity.2 The photodynamic properties of ZnO under UV radiation were established by Daneshvar.9 Moreover, it was assumed that even under ordinary room light with a total light intensity of 10 µW/cm-2, the intensity of UV light, which is ∼1 µW/cm-2, is sufficient to induce ROS formation.10 ROS production was detected in a few semiconductor nanoparticles, radical production depended on the size and on the band gap of these nanoparticles.11,12 In the present study, using electron paramagnetic resonance (EPR) coupled with the spin-trapping technique, we demonstrate the formation of hydroxyl radicals and singlet oxygen in a water suspension of ZnO nanoparticles. Moreover, the level of oxy radicals was found to increase considerably when the suspension was irradiated with visible light at the range of 400-500 nm. Preliminary results of this study were published by Applerot et al.13 To the best of our knowledge, there is no previous report of blue light inducing the ROS formation of a ZnO suspension in water, nor has the nature of the formed radicals been described. Oxy radicals were only found when sol-gel-derived ZnO films were illuminated by UV irradiation.14 The immediate result of the increase in the formation of oxy radicals is reflected in greatly enhanced killing of various bacterial species.
Nanoparticles. ZnO was synthesized by an ultrasonic method, which is described in a previous paper,13 yielding particles of 6.8 ( 2 as calculated using the Debye-Scherer formula and 11.6 nm measured from TEM. Specific surface area (SSA) was 25.8 (m2 g-1), as calculated using the BrunauerEmmett-Teller (BET) method.13 Spin Trapping Measurements Coupled with EPR Spectroscopy. Detection of •OH (hydroxyl), •O2- (superoxide anion), and 1O2 (singlet oxygen) Radicals in a Water Suspension of ZnO Nanoparticles. In order to detect •OH, •O2-, and 1O2, we used the EPR-spin trapping technique coupled with the spin traps 5,5-dimethyl-1-pyrroline-N-oxide (DMPO, 0.02 M) (Sigma, St. Louis, MO), 5-(diethoxyphosphoryl)-5-methyl-1-pyrrolineN-oxide (DEPMPO, 0.01 M) (Almog Diagnostics, Israel), and 2,2,6,6-Tetramethylpiperidine (TEMP, 0.1M) (Sigma, St. Louis, MO). Aqueous suspensions (10 mg/mL) of ZnO (10 nm) and the appropriate spin trap were drawn by a syringe into a gaspermeable Teflon capillary (Zeus Industries, Raritan, NJ) and inserted into a narrow quartz tube that was kept open at both ends. The tube was then placed in the EPR cavity and the spectra were recorded before and after illumination through the EPR cavity, on a Bruker EPR 100d X-band spectrometer. The EPR measurement conditions were as follows (unless otherwise stated). Frequency, 9.74 GHz; microwave power, 20 mW; scan width, 65 G; resolution, 1024; receiver gain, 2 × 105; conversion time, 82 ms; time constant, 655 ms; sweep time, 84 s; scans, 2; modulation frequency, 100 kHz. After acquisition, the spectrum was processed using the Bruker WIN-EPR software version 2.11 for baseline correction. The peak intensity was calculated by double integration of the peak signals, and the intensity was expressed in arbitrary units. Simulation of the recorded spectra was performed using an algorithm provided in the WINSIM program, which is available from NIEHS (National Institutes of Health, web site: http:// epr.niehs.nih.gov/pest_mans/winsim.html). Purification and Characterization of the Spin Traps. DMPO. DMPO is a common spin probe that detects •OH to give the DMPO-OH spin adduct, which has a quartet EPR signal. The DMPO can also trap O2- to produce the DMPO-OOH
* To whom correspondence should be addressed. E-mail: gedanken@ mail.biu.ac.il. † Instute of Nanotechnology and Advanced Materials, Bar-Ilan University. ‡ Departments of Chemistry and Physics, Bar-Ilan University.
10.1021/jp904864g CCC: $40.75 2009 American Chemical Society Published on Web 08/12/2009
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Figure 1. Simulation spectra of DEPMPO-OH (gray line) and DEPMPO-OOH (black line). The simulation shows the ESR spectrum of the DEPMPO-OH adduct radical with a(N)) 14.11 G, a(H) ) 13.33 G, a(P) ) 49.46 G; and the ESR spectra of DEPMPO-OOH with a(N) )13.00 G, a(H) )11.7 G, a(P) ) 50.6 G.
spin adduct (consisting of a sextet EPR signal).15 Nevertheless, since the latter is unstable, it decomposes to a DMPO-OH adduct.16,17 DMPO was purified in the dark in ddH2O, with activated charcoal. After about 30-60 min, the solution was filtered and its concentration was determined spectrophotometrically using ε227 nm ) 8.0 mM-1 · cm-1. The solution was stored at -20 °C for no longer than 2 weeks. DEPMPO. DEPMPO reacts with hydroxyl radicals to produce DEPMPO-OH or with superoxide anion radicals to produce the DEPMPO-OOH spin adduct.18 Although both adducts have characteristic EPR spectra of 18 lines, they differ in the field of 3335-3365 G, as demonstrated in Figure 1. TEMP. TEMP reacts with 1O2. The reaction of 1O2 with TEMP leads to the free radical 2,2,6,6-tetramethyl-4-piperidoneN-oxyl (TEMPO) with a characteristic EPR spectrum comprised of three lines of equal intensity. Measurement of ZnO Absorption. A powder composed of ZnO nanoparticles was suspended in ddH2O at a final concentration of 50 µg/mL, and the absorbance in the visible range was measured using a Carry 300 Scan UV-visible spectrophotometer (Varian, Palo Alto, CA). Illumination. Light Sources. A number of homemade broadband tungsten-halogen lamps (400-800 nm) with filters for the UV and IR wavelengths were used in this study. The intensity of the light devices was ranged between 40 and 400 mW/cm2. In some experiments, color dichroic filters (Edmund Optics) were used.
Figure 2. Hydroxyl and superoxide radical formation in a water suspension of ZnO nanoparticles (10 nm particles, 10 mg/mL) in the presence of the spin trap DMPO.
Results Hydroxyl and Superoxide Radical Generation in a Water Suspension of ZnO Nanoparticles. To characterize ROS formation by ZnO nanoparticles, hydroxyl, and superoxide anion radicals, were first measured using EPR spin-trapping coupled with the DMPO spin trap. A water suspension of ZnO (10 nm, 10 mg/mL) was introduced into the EPR cavity, as described in the Material and Methods section, and the EPR spectrum was recorded. As shown in Figure 2, a characteristic DMPO-OH spin adduct with a hyperfine splitting constant (HFSC), giving rise to four resolved peaks (AN ) AHβ ) 14.9 g), was obtained. These HFSC values suggest that HO• was generated from ZnO suspended in water. Since DMPO can also trap O2- to produce the spin adduct DMPO-OOH, which is unstable and decomposes to a DMPO-OH adduct,16,17 O2- formation could not be excluded. Distinguishing between HO• and O2- Production. In order to determine whether the spectrum presented in Figure 2 arises from the production of superoxide or/and •OH, we used DMSO to scavenge •OH. As shown in Figure 3, the addition of DMSO abolished the quartet signal, thus suggesting that only OH radicals are produced by ZnO nanoparticles. The new peaks in Figure 3 (indicated by
Figure 3. ROS formation in suspensions of ZnO (10 nm, 10 mg/mL) nanoparticles with DMSO: (A) Simulation spectrum of DMPO-OH in the presence of DMSO. (B) Experimental spectrum of ZnO and 10% DMSO in the presence of the spin trap DMPO. (C) Experimental spectrum of ZnO in the presence of the spin trap DMPO. The asterisks indicate new peaks formed by 10% v/v DMSO, and the quartet formed by DMPO-OH is marked by arrows.
an asterisk) result from a DMPO-CH3 adduct, which is formed according to the equations below (eqs 1 and 2).
(CH3)2SO + •OH f •CH3 + CH3SO2H
(1)
DMPO + •CH3 f DMPO - CH3
(2)
To verify the absence of the superoxide anion, we also used the EPR spin trap, DEPMPO. In Figure 4, different spectra of the DEPMPO spin trap are shown; spectrum A from the trapping of •O2- obtained from the reaction of xanthine and xanthine oxidase, while spectra B
EPR Study of Visible Light-Induced ROS Generation
Figure 4. EPR spectra of DEPMPO (0.01M) with (A) xanthine and xanthine oxidase system, (B) Fenton reaction, and (C) ZnO.
was obtained from trapping the OH radical from the Fenton reaction. In Figure 4C, a spectrum of DEPMPO with nanoZnO is depicted. The similarity of the spectra obtained by measuring the nano-ZnO suspension (C) with that of the DEPMPO-OH (B) confirms that only hydroxyl radical was formed. The addition of superoxide dismutase, a superoxide oxide anion scavenger, to the suspension, had no effect on the spectrum obtained in Figure 4C (results not shown), further confirming that •OH is the only oxy radical generated in the suspension in the absence of light. Visible Light-Induced OH in Water Suspensions of ZnO. We next studied the effect of visible light illumination on OH production by ZnO nanoparticles. Nonilluminated samples served as controls. Suspensions of ZnO were illuminated with white light (40 mW/cm2), and the EPR spectra were measured after 1 min of illumination. The spectra in Figure 5 show that illumination caused an elevation in OH production. Production of •OH by Nanosized ZnO as a Function of Illumination Time. The kinetics of •OH production were then studied. Suspensions of ZnO were illuminated with white light (40 mW/cm2), and EPR spectra were taken at 0′ before illumination, and 1, 2, 3, and 5 min after illumination. Illumination was performed within the EPR cavity. As demonstrated in Figure 6, the signal amplitude of the ESR signal of the DMPO-OH spin adduct increased in a timedependent manner in the first 3 min of illumination, reaching a steady state, probably due to recombination of the various ROS. Production of •OH by Nanosized ZnO as a Function of the Illumination Wavelength. To examine the amount of hydroxyl radicals formed from suspensions of nano-ZnO as a function of the wavelength of illumination, we employed broadband visible illumination coupled with blue or red color filters (Edmond Optics). The EPR spectrum of each sample was measured before and after illumination (400 mW/cm2) for 1 min with white light or with an appropriate color filter, yielding blue/ red light with an intensity of 120 mW/cm2 each. It can be seen from Figure 7b and c that the same enhancement of the EPR signal was observed following illumination with blue (400-500 nm) and white light, the percent of change in peak intensity for blue and white light illumination being 167% ( 64% and 70% ( 15%, respectively. Figure 7a reveals no increase of the signal intensity following red light illumination. Singlet Oxygen Production by ZnO Nanoparticles in the Presence of Visible Light Irradiation. We also studied singlet oxygen production by water suspensions of nano-ZnO before and after illumination using EPR spectroscopy and TEMP as a spin trap.
J. Phys. Chem. C, Vol. 113, No. 36, 2009 15999 A characteristic EPR triplet spectrum of TEMPO was detected (Figure 8B), proving the production of singlet oxygen, when an aqueous solution of ZnO was irradiated in the presence of TEMP. The addition of sodium azide (C) abolished appearance of the triplet, thus further confirming the production of singlet oxygen in the irradiated ZnO suspension. In nonirradiated samples of ZnO (A), no triplet signal was detected. On the other hand, a weak triplet signal (marked with arrows) was observed in a water suspension of ZnO in the presence of TEMP and DMPO (Figure 9), proving that singlet oxygen in minute amounts is generated in water suspensions of ZnO even in the absence of light. The characteristic spectrum of DMPO-OH, monitoring OH production, is also shown in Figure 9 (marked with asterisks). In view of these results, we suggest that singlet oxygen in small amounts is produced in nonilluminated ZnO, but its signal is decreased by the reaction of TEMPO with the hydroxyl radical produced by ZnO (Figure 1). Figure 10 shows a Fenton reaction in the presence of commercial TEMPO demonstrating a reduction of the triplet intensity. We describe this phenomenon in more detail in the Discussion section. Production of Singlet Oxygen by Nanosized ZnO as a Function of the Illuminated Wavelength. To examine the wavelength dependence of visible light-induced singlet oxygen in suspensions of nano-ZnO, we illuminated the suspension using a blue filter (Edmond Optics). The EPR spectrum of each sample was measured before and after illumination (400 mW/ cm2) for 5 min with white light or with the same light coupled with a filter yielding 120 mW/cm2 blue light (data not shown). The intensity of the EPR signals obtained following illumination with white light were the same as those obtained for blue light, suggesting that only the blue part (400-500 nm) of the visible spectrum is responsible for singlet oxygen formation by ZnO nanoparticles. Absorption Spectra of ZnO. The absorption spectrum of ZnO was measured in an attempt to explain ROS production following illumination by blue light. The absorption spectra showed that ZnO nanoparticles have an absorption peak in the UV-A at 384 nm with a marked absorption at the blue region (results not shown). Discussion The present study describes the ability of an aqueous suspension of ZnO nanoparticles to produce ROS in the presence or absence of visible light illumination. The formation and amount of ROS was monitored by using the EPR spin trapping technique employing three spin traps, DMPO, DEPMPO, and TEMP. We show that hydroxyl radicals, as well as singlet oxygen, are generated in ZnO water suspensions. No formation of superoxide anion radicals was detected either with or without light illumination. We demonstrated the formation of hydroxyl radicals in water suspensions of ZnO (Figure 2) by using the DMPO spin trap. Since DMPO can trap superoxide anion as well, DMSO was added to the sample. As can be seen from Figure 3, the relatively high quartet signal of DMPO-OH monitoring •OH formation is totally abolished by DMSO, suggesting that only hydroxyl radicals are produced. The new peaks (marked by asterisk in Figure 3 are DMPO-CH3 adducts, as explained in eqs 1 and 2). To verify the absence of superoxide anion, we also used the DEPMPO spin trap. DEPMPO can trap •OH and -O2 radicals, yielding somewhat different spectra (Figure 1). We measured the spectra of superoxide and hydroxyl radicals generated by
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Figure 5. OH production by ZnO nanoparticles before (black) and after (bold black) broadband visible light illumination using DMPO as a spin trap.
Figure 6. Effect of illumination time on ROS production in suspensions of nano-ZnO (10 nm, 10 mg/mL) using the spin trap DMPO.
Figure 8. Singlet oxygen production in (10 nm, 10 mg/mL) nanoZnO water suspensions: (A) Nonilluminated ZnO, (B) ZnO after illumination (400-800 nm, 40 mW/cm2, 5 min), and (C) ZnO after illumination in the presence of sodium azide (10%).
Figure 9. Singlet oxygen production in suspensions of nano-ZnO in the presence of TEMP and DMPO (the triplet of TEMPO signal is marked with arrows, DMPO-OH marked with asterisks).
Figure 7. Effect of different wavelengths on light-induced hydroxyl radical production by illuminated ZnO (10 nm, 10 mg/mL) in the presence of the spin trap DMPO: Bold black, after illumination; black, before illumination. (a) Illumination with 500-800 nm, (b) illumination with 400-500 nm (blue light), and (c) illumination with 400-800 nm (white light).
xanthine/xanthine oxidase system and the Fenton reaction, respectively (Figure 4A, B). A resemblance between the experimental spectrum (C) obtained from nano-ZnO suspensions with DEPMPO and the spectrum obtained via the Fenton reaction suggests the conclusion that no -O2 is generated by nano-ZnO. The addition of SOD (super oxide dismutase, a
specific quencher of superoxide anion) to the reaction of ZnO and DEPMPO had no effect on the spectra, further supporting the production of OH radicals only (data not shown). Irradiating nano-ZnO water suspension with visible light resulted in an increase in the quartet signal in the EPR spectrum (Figure 5). To the best of our knowledge, this is the first time that such light-induced ROS formation has been reported. Furthermore, it can be concluded from Figure 6 that the OH radical production following illumination is time dependent, reaching a steady state after ∼3-5 min of illumination, probably due to self-quenching. By using illumination with limited wavelengths of light, we showed that the blue part of the visible spectrum is responsible for ROS production (Figure 7). The ability of nano-ZnO to generate OH radicals by blue light illumination is explained by its light absorption above 400 nm. Light absorption in nano-ZnO water suspensions peaked at 384
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Figure 10. EPR signal of commercial TEMPO (gray) and of commercial TEMPO together with Fenton reaction (black).
nm, followed by residual absorption in the blue range, which is in agreement with the results shown by Gupta et al.19 For studying singlet oxygen formation, we used the TEMP spin trap. Singlet oxygen is an important anticancer agent20). Figure 8B demonstrates that ZnO produces singlet oxygen after illumination with light in the visible range. On the other hand, when the aqueous suspension of ZnO was not illuminated (Figure 8A), no triplet signal of TEMPO was detected; this result suggested that singlet oxygen might be formed only upon illumination by visible light. However, the addition of DMPO, which traps •OH, resulted in a weak triplet signal (Figure 9). OH radicals are known to react with the nitroxyl groups of TEMPO (Figure 10) (see eq 3, below), giving rise to the production of a silent EPR signal (eqs 4 and 5).21 The removal of OH radicals by DMPO enabled the detection of a weak triplet signal monitoring singlet oxygen formation in nonilluminated ZnO suspension.
The fact that OH radicals masked singlet oxygen detection in nonilluminated suspensions was also verified by the addition of 10% ethanol, a known OH radical scavenger. The addition of ethanol resulted in a triplet EPR spectrum (data not shown). It is important to note that singlet oxygen can be generated by several pathways: (1) Dismutation of a superoxide anion to form hydrogen peroxide and singlet oxygen 22 • + 1 H•O2 + O2 + H f O2 + H2O2
(6)
(2) Fenton-type reaction: H2O2+ •O2- f 1O2 + OH• +OH-23 (3) Direct formation of singlet oxygen from ZnO, similar to the mechanism of singlet oxygen production found in TiO2.24 We reject pathways 1 and 2 as possible mechanistic steps for the formation of singlet oxygen, since we have demonstrated that superoxide anion is not generated by ZnO, although hydrogen peroxide has been detected as a product of ZnO in aqueous suspensions.5-7 Thus, we are left with pathway 3, in
which singlet oxygen is produced directly from defects in nanocrystalline ZnO. To study the wavelength dependence of singlet oxygen production, blue-filtered light was used. Similar to blue lightinduced OH formation, singlet oxygen generation was enhanced only by blue light. From the results of this study, it is clear that oxy radicals are generated in nano-ZnO suspensions. Furthermore, we have shown that blue light stimulates oxy radical production. As was previously described, ZnO has been widely reported to have antibacterial activity, but the mechanism has not yet been clarified. The results of our study strongly suggest that the production of OH and singlet oxygen radicals by ZnO are responsible for the cellular damage leading to its bactericidal activity. We believe that our characterization of the mechanism of light-induced ROS formation by ZnO nanoparticles would contribute to its use as a sterilization agent. The fact that ROS are generated following a blue light irradiation and not only by UV opens the possibility to use ZnO np combined with blue light which is not dangerous, for sterilization purposes. References and Notes (1) Nair, S.; Sasidharan, A.; Divya Rani, V. V.; Menon, D.; Nair, S.; Manzoor, K.; Raina, S. J. Mater. Sci. Mater. Med. 2008. (2) Hanley, C.; Layne, J.; Punnoose, A.; Reddy, K. M.; Coombs, I.; Coombs, A.; Feris, K.; Wingett, D. Nanotechnology 2008, 19, -. (3) Li, Q. L.; Mahendra, S.; Lyon, D. Y.; Brunet, L.; Liga, M. V.; Li, D.; Alvarez, P. J. J. Water Res. 2008, 42, 4591–4602. (4) Thevenot, P.; Cho, J.; Wavhal, D.; Timmons, R. B.; Tang, L. Nanomedicine 2008, 4, 226–36. (5) Sawai, J.; Kawada, E.; Kanou, F.; Igarashi, H.; Hashimoto, A.; Kokugan, T.; Shimizu, M. J. Chem. Eng. Jpn. 1996, 29, 627–633. (6) Sawai, J.; Kojima, H.; Igarashi, H.; Hashimoto, A.; Shoji, S.; Takehara, A.; Sawaki, T.; Kokugan, T.; Shimizu, M. J. Chem. Eng. Jpn. 1997, 30, 1034–1039. (7) Sawai, J.; Shoji, S.; Igarashi, H.; Hashimoto, A.; Kokugan, T.; Shimizu, M.; Kojima, H. J. Ferment. Bioeng. 1998, 86, 521–522. (8) Stoimenov, P. K.; Klinger, R. L.; Marchin, G. L.; Klabunde, K. J. Langmuir 2002, 18, 6679–6686. (9) Daneshvar, N.; Rasoulifard, M. H.; Khataee, A. R.; Hosseinzadeh, F. J. Hazard. Mater. 2007, 143, 95–101. (10) Fujishima, A.; Rao, T. N.; Tryk, D. A. J. Photochem. Photobiol. C: Photochem. ReV. 2000, 1, 1–21. (11) Konovalova, T. A.; Lawrence, J.; Kispert, L. D. J. Photochem. Photobiol. A: Chem. 2004, 162, 1–8. (12) Ischenko, V.; Polarz, S.; Grote, D.; Stavarache, V.; Fink, K.; Driess, M. AdV. Funct. Mater. 2005, 15, 1945–1954. (13) Applerot, G.; Lipovsky, A.; Dror, R.; Perkas, N.; Nitzan, Y.; Lubart, R.; Gedanken, A. AdV. Funct. Mater. 2009, 19, 842–852. (14) Asakuma, N.; Fukui, T.; Toki, M.; Awazu, K.; Imai, H. Thin Solid Films 2003, 445, 284–287. (15) Kuppusamy, P.; Zweier, J. L. J. Biol. Chem. 1989, 264, 9880– 9884. (16) Finkelstein, E.; Rosen, G. M.; Rauckman, E. J. Arch. Biochem. Biophys. 1980, 200, 1–16. (17) Finkelstein, E.; Rosen, G. M.; Rauckman, E. J. Mol. Pharmacol. 1982, 21, 262–265. (18) Frejaville, C.; Karoui, H.; Tuccio, B.; Lemoigne, F.; Culcasi, M.; Pietri, S.; Lauricella, R.; Tordo, P. J. Med. Chem. 1995, 38, 258–265. (19) Gupta, A.; Bhatti, H. S.; Kumar, D.; Vermaa, N. K.; Tandonb, R. P. Digest J. Nanomater. Biostruct. 2006, 1, 1–9. (20) DeRosa, M. C.; Crutchley, R. J. Coord. Chem. ReV. 2002, 233, 351–371. (21) Herrling, T.; Fuchs, J.; Rehberg, J.; Groth, N. Free Radic. Biol. Med. 2003, 35, 59–67. (22) Corey, E. J.; Mehrotra, M. M.; Khan, A. U. Biochem. Biophys. Res. Commun. 1987, 145, 842–846. (23) Misra, B. R.; Misra, H. P. J. Biol. Chem. 1990, 265, 15371–15374. (24) Janczyk, A.; Krakowska, E. b.; Stochel, G. y.; Macyk, W. J. Am. Chem. Soc. 2006, 128, 15574–15575.
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