Formation of active oxygen species during autoxidation of DOPA

Chemosphere,Vol.

39, No. 3, pp. 443-453, 1999 e 1999 Elsevier Science Ltd. All rights reserved 0045-6535/99/$ - see front matter

Pergamon

PII: S0045-6535(99)00007-7 FORMATION OF ACTIVE OXYGEN SPECIES DURING AUTOXIDATION OF DOPA Irena Kruk*, Krzysztof Lichszteld*, Michel Bounias**, Aleksandra I~adna*** and Lidia Kubera-Nowakowska* * Institute of Physics, Technical Univerity of Szczecin, AI. Piast6w 48/49, 70-310 Szczecin, Poland '* Unit~ de Biomath~matique &Toxicologie, Facult6 des Sciences, Universit~ d'Avignon, F84000 Avignon, France *** Department of Human Ecology, Faculty of Natural Sciences, University of Szczecin, A1. Piast6w 40b/6, 71-065 Szczecin, Poland (Received in Germany 23 September 1998; accepted 3 December 1998)

ABSTRACT The light emission during DOPA autoxidation and the transition metal ion-catalyzed oxidation of this amine to melanins has been studied. Chemiluminescence and fluorescence spectra from both systems were measured. The light emission was inhibited by quenchers of singlet oxygen (Io2) and scavengers of superoxide anion radical (O 3 ), hydroxyl radical ( HO ) and by catalase. The generation of 10 2 was confirmed using electron spin resonance (ESR) and 2,2,6,6-tetramethylpiperidine as a trap as well as by spectrophotometry method. The formation of H O was demonstrated by the technique of ESR spin trapping with DMPO as a spin trap. ©1999 ElsevierScienceLtd. All rightsreserved KEY WORDS: DOPA, oxygen free radicals, singlet oxygen, antioxidants. INTRODUCTION Interest in 3,4-dihydroxyphenylalanine (DOPA) autoxidation process leading to the melanin formation has grown rapidly over the years, especially with regard to generation of reactive oxygen species (free radicals: O 2 , HO and singlet oxygen I o 2) [1-3]. The reaction of the reactive oxygen species with cell components including DNA can cause the most serious cellular damage leading to many disease states [3-5]. In good agreement with the oxygen active species ethiology of diseases, Wick et al. [2] showed L-DOPA toxicity to dopamine neurons in tissue culture, and Smith et al. [1 ] found a DOPA-dependent enhancement in in vivo local nigral generation of riO radicals which paralleled the rate of the catecholamine metabolism. DOPA similarly as the other biogenous catecholamines plays an important role in the genesis of neuromelanins and rheomelanins present in the human brain and blood, respectively [6, 7]. We found that DOPA greatly accelerated the generation of HO radicals from the Fenton reaction [8-10] and that the peroxidation of the amine is accompanied by the generation of electronically excited products and highly reactive species [10, 11]. 443

444 In this work we report on the chemiluminescence (CL) accompanying autoxidation of DOPA and the production of the oxygen free radicals like O5 , HO and IO2, which may be responsible, at least partially, for the subcellular damage observed. MATERIALS AND METHODS D,L-DOPA was obtained from Aldrich Chemical Co., SOD (EC. 1.15.1.1) from bovine erythrocytes protein activity, 5,5-dimethyl-l-pyrolline-N-oxide (DMPO) and 2,2,6,6-tetramethylpiperidine

(TEMP), nitroblue

tetrazolium (NBT), catalase from bovine liver were purchased from Sigma; glutathione from Reanal (Hungary). Compounds used as antioxidants, imidazole and p-nitrosodimethylalanine(RNO) were obtained from Merck. Other reagents were analytical grade from POCH Gliwice (Poland). All solutions were prepared immediately prior to use in redistilled water. Measurements were carried out in 0.1 M.1-1 carbonate buffer. Chemiluminescence kinetics were measured using an M12FQC51 photomultiplier with $20 cathode (Germany) operating jointly with a Zeiss K-200 recorder (Germany). Reagents were introduced to a thermostated glass cuvette placed in a light-tight camera through polyethylene pipes from semi-automatic syringes. The CL spectra were measured using a set of calibrated cut--off filters (GOST 9411-66) and an EMI 9203B photomultiplier [11 ]. The absorption spectra were measured with a Zeiss SPECORD UV VIS 32-G33b spectrophotometer. The spectrophotometric determination of Io2 was performed according to the method given by Kralij~ and Moshni [12]. The method is based on bleaching of RNO by an intermediate product of the reaction of IO2 with imidazole (A), i.e. by transannular peroxide AO2 + RNO ----).-RNO + products The bleaching of RNO was detected as the decrease in absorbance at 440 nm. Fluorescence spectra were measured using a Hitachi MPF-3 spectrofluorimeter. Electron spin resonance (ESR) spectra were recorded at room temperature using a standard X-band spectrometer operating at 9.3 GHz with a 100 kHz modulation of the steady magnetic field. The spectra were recorded at a microwave power level of 2 mW and a modulation amplitude of 0.1 Gs using a quartz flat cell with the optical path length of 0.25 mm. All experiments were performed at least in triplicate except the CL spectra, which were recorded ten times within the recording time of 10 s in the wavelength range 400-750 nm with the resolution of 10 nm. RESULTS AND DISCUSSION Aqueous solutions of DOPA saturated with air or molecular oxygen undergo autoxidation to melanins. This process strongly depends on the pH value and the presence of transition metal ions [6]. The mechanism of the DOPA degradation leading to the melanins formation have been studied in a number of recent papers by several groups working in this field [6, 13, 14]. The following main steps may be distinguished in the transformation of DOPA to melanins: (1) formation of the open-chain quinone 3,4-quinone-phenylalanine;(2)

445 cyclization the quinone to 2,3-dihydro-5,6-quinone-2-carboxylic acid (dopachrome); (3) conversion of the latter compound into 5,6-dihydroxyindole and 5,6-dihydroxyindole-2-carboxylic acid, and (4) formation of melanins as the end products. The process of melanins formation is easily observable spectrophotometrically (Fig. 1).

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Fig. 1. The absorption spectra of an air-saturated solution of 1 mM DOPA (part A) and the metal-catalyzed autoxidation of this amine (parts B and C) at pH 9.9 for various times of the reactions course. Conditions: 0.5 mM CUSO4"5 HzO (B), 0.5 mM (NH4)/SOa-FeSO4'6H20 (C), T=293 K, 1 cm cell. We can observe three chromophoric stages in visible region: the first with an absorbance maximum at about 460 nm corresponding to dopachrome; the second with an absorbance maximum at 540 nm which is due to a low polymer or a mixture of polymers formed from the indole-5,6-quinone [6]; the product formed in the third stage with general absorption is considered to be a melanin. The results obtained indicate that formation of the melanin is strongly accelerated by the presence of Cu and Fe ions. These results are in agreement

446 with data obtained by Wilczok et al [15, 16] for other catecholamines which reported that adrenaline was oxidized to adrenochrome one hundred times faster in the presence of Cu 2÷ ions in comparison to metal free solution of the amine. The activating effect of the tested metal ions on the conversion of DOPA to melanin is associated with the formation of metal-DOPA complexes. Autoxidation of DOPA alone in carbonate buffer and in the presence of Cu 2+ and Fe3+ (without H202) is also accompanied by CL. The emission was pH-dependent and increased with the DOPA concentration (data not shown). No light emission was detected when the value ofpH is below 8.3, although the process of melanin formation was observed at this value of pH and lower, but much lower. It is clearly related to the state of ionization of the DOPA molecule and agrees with optimal values of the dissociation constants of hydroxyl groups. The introduction of the metal ions decreases the light intensity by a factors of about 8 and 15 for Cu 2÷ and Fe3+, respectively. Based on our previous works dealing with CL from catecholamines oxidation using H202 as an oxidant [8-10], the obtained data seem to confirm suggestions that DOPA forms complexes with Cu and Fe ions. In an attempt to determine the origin of the excited light emitting species, measurements of spectra of the CL and fluorescence (FL) were performed (Fig. 2).

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Wavelength [nm] Fig. 2. Chemiluminescence spectra from the DOPA autoxidation in the presence of Cu ions (blackened area) and metal free solution (bright area) bubbled with air 20 min. after the mixing of the reagents.. The size of the bars approximates the standard deviation. Dashed line is the fluorescence spectrum (~,e×e=335 nm, 0.1 mM DOPA). Under the same Condition as in Fig. 1

447 The CL spectra revealed one broad band in, the 400-570 nm range and bands with maxima at 580-590, 630-640 and 700 nm. Neither the reaction time nor added catalyst changed the position of the bands. The FL emission spectrum recorded during ~,ex¢=265-340nm covers the range from 350-600 nm and shows the maximum at 440 nm. The band was decreasing slowly with the reaction course, which means that certain fluorescent products are formed and subsequently consumed during the DOPA degradation. DOPA alone in water (pHil6.8) reveals an ultraviolet FL with maximum at 315 nm, thus the emission observed during autoxidation of the amine should be described to its oxidation products. Comparing the CL spectrum with the FL spectrum one can notice that a product showing the FL emission at 440 nm can be only partialy identified as the emitter of the observed CL. No new emission band was detected when ~-excwas increased up to 420 nm. A significant difference was seen between FL during autoxidation and oxidation of DOPA using H202 as an oxidant. In the case of the DOPA oxidation with H202 we found [8] that the FL spectrum showed the strong maximum at 405 nm and very weak one at 460 nm, when Lexc=320 nm. When the FL was measured using ~,exc=410nm a new band with a maximum at 505 nm appeared. From the previously reported data dealing with the FL of DOPA [8] and taking into account the mechanism of the amine degradation [6] the most likely explanation for the 440 nm emission appear to be IS ~lSo transition in excited 5,6dihydroxyindole molecule. The observed CL in the spectral region of 400-570 nm may originate mainly from the electronically excited form of a semiquinone and from the formation of carbonate and bicarbonate radicals and their recombination according to Stauff et al. [17]. Emission bands at 580, 620-640 and 700 nm can be explained by the formation of excited (02-02) dimers as a result of an collision of two molecules of oxygen in the singlet states (lAg). The band at 580 nm can be attributed to the 202[IAg] --~ 20213~ g ] simultaneous transition with the vibrational quantum number of (1,0), whereas the bands with maxima at 640 and 700 nm correspond to the above mentioned transition with vibrational quantum number of (0,0) and (0,1) for 640 and 700 nm, respectively [18]. In order to elucitate the participation of reactive oxygen species to the observed CL the effect of some compounds known as scavengers of O~ , HO, quenchers of IO 2 and catalase on the CL during autoxidation of DOPA was studied (Fig. 3). As can be seen from the figure the addition of catalase (decomposes H202) exerted a very strong quenching effect. This means that H202 is generated during the DOPA autoxidation. Important biological antioxidants such as: captopril, thiourea, cimetidine, carnosine, myoglobin compounds known from the literature [19] to be efficient scavengers of HO radicals strongly decrease the CL. It is worthwhile to mention that majority of tested compounds show multifunctional properties against a cell damage. For example, camosine, the histidine-containingdipeptide in the basic medium undergoes oxidation and this process is primarily due to interaction with HO radicals [19]; myoglobin (hemoprotein) present in skeletal muscles of vertebrates decomposes H202, similarly as does catalase, and also reacts with

448 HO" and O 5 species; captopril reacts with HO at diffusion-controlled rate and the thiol groups participate in the radical scavenging RSH + H O ~ RS" + H 2 0

(1)

The thiol radical RS' may also form complexes with semiquinones and quinoic metabolites of the DOPA oxidation thus decreasing the amine degradation. The next found quenchers of CL such as methionine, histidine, tryptophan, besides being good radical scavengers, are known as efficient 102__quencher s [20]. It is interesting, that the examined compounds exerted the quenching effect both when they were introduced to the reaction mixture before the start of the DOPA autoxidation as well as during its course, which confirms their multifunctional antioxidant properties. These results show the important role of O 5 and HO as precursors of 10 2 in the CL observed during formation of melanin from DOPA.

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Fig. 3. The effect of 0 2 , HO scavengers, lO 2 quenchers and catalase on the chemiluminescence intensity of DOPA alone autoxidation and the metal ions catalyzed autoxidation. Concentration of inhibitors were 5 mM except of myoglobine and NBT, which were 0.1 mM and 1 mM, respectively. Catalase was 100 U/ml, SOD - 180 U/ml. T=310 K. Remaining conditions as in Fig. 1. In addition, we report ESR evidence using the nitrone spin trap such as DMPO for formation of HO' during autoxidation of DOPA. The addition of 0.05 M DMPO to the reaction system produced four spectral lines (Fig. 4 A), which are attributed to DMPO-OH adduct on the basis of hyperfine splittings constants AN=AH=14.96 Gs [21].

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Time [min] Fig. 4. Part A: ESR spectrum of the 50 mM DMPO+DOPA+Cu(II)+ HO- system detected after 20 min from the beginning of the reaction and time course of the ESR signal seen from the above system. Part B: ESR spectrum of the 25 mM TEMP+DOPA+Cu(II)+ HO- system measured after 120 min from the start of the reaction and time course of the ESR signal seen from the above system. Remaining conditions as in Fig. 1. The present study reveals two basic aspects, namely, a) generation of products in the electronically excited states, and b) formation of reactive oxygen radicals during oxidative transformation of DOPA to melanins. One of emitters of the observed light emission is 10 2 , which is believed to be a product of the interaction between the oxygen intermediates such as O~ , HO' and H202. The oxygen species can be generated according to the scheme DOPA + HO- -~ DOPA- + H 2 0

(2)

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450 of the reaction i.e. transformation of the amine to the amine quinone and with ring closure resulting in the formation of dopachrome and 5,6-dihydroxyindole compounds [6]. In addition, it is well documented that catechols form a free radical complexes with transition metals (Me n* e.g. Cu >, Fe3.) by the coordination of the metal ion with two ortho hydroxyl groups at the 3 and 4 position of the catechol ring [22]: Me n+ + DOPA --~ Me (n+l)+ ... DOPA-

(6)

Such complexes are good electron donors [23]. The arising free radical can abstract hydrogen atom from biomolecules, and promote their peroxidation. The DOPA-Men+-o2 complexes may catalyze the oxidation of DOPA to its o-semiquinone as well as participate in one-electron reduction of dopachrome forming osemiquinone. Oxidation of Me n+ to Me ~n+l)+may involve reduction of o-semiquinones to o-hydroquinones. These reactions result in the formation of active oxygen species [24]. Additionally, metal ions may act as the 02 carriers as well as their presence in the reaction mixture causes the redox cycling reactions in the presence of orto-hydroquinone: SQ' + Me n+ -+ HQ + Me (n+l)+

(7)

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It is well known that the light emission observed during oxidation of catecholamines is proportional to the rate of aminochromes disappearance. The decrease in the CL intensity observed especially in the case of the Fe3+ presence may result in formation of Fe3+-DOPA complex, which according to the paper of Chaix et al. [25] is not autoxidizable. The complex shows the light absorption at 480-500 run masking the amount of dopachrome formation (Fig. 1). Another possible explanation for the light intensity diminution is an increased H202 generation in the presence of the metal ions. It has been reported that H202 reacts with osemiquinones preventing their autoxidation to quinones [24]. Since the role of carbonate and bicarbonate radicals in elementary reactions generating emitters of the CL in the spectral region of 400-570 nm is important, interaction of buffer molecules with metal ions or with free radicals generated in the systems containing the metal ions might also limit the emission [26]. The presence of the transition metal ions and H202 in a solution is a source of HO radicals produced by the Fenton mechanism [27]: Me n+ + H202 --~ Me (n+l)+ + H O + HO-

(10)

The O j interaction with H202 (Haber-Weiss reaction) also gives HO radical and additionally 102 : O 5 +H202--+102 + H O + H O -

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and could be the transition metal ion catalyzed. The H O would scavenger O 5 as follows: H O +O~ -+102 + H O and thus may be considered also as an source of 102 .

(12)

451 The formation of 102 using TEMP as a spin trap is shown in Fig. 4B for the DOPA + Cu(II) + HOsystem. The three lined ESR signal detected after mixing the reagents is characteristic of a stable nitroxide free radical resulting from the reaction of I o 2 with TEMP. The product of the reaction is expected to be 2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPO) on the basis of hyperfine splittng constants of 16.0 Gs [28]. The accumulation of the nitroxide radicals with time was monitored by measuring the intensity of the central line of the signal. The concentration of NO-radicals increases in the oxidation time obtaining maximum after 10-12 min from the beginning of the reaction. This means that the rate of 10 2 formation is the greatest at this time. The ESR spectra for the remaining systems were almost the same and similar to that observed for TEMPO. We have also checked the participation of l o 2 in the DOPA autoxidation process using the spectrophotometric method [12]. Figure 5 shows the bleaching of RNO in the RNO + imidazole + DOPA + HOand RNO + imidazole + DOPA + metal ion + HO- systems. 0.4

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Time [min] Fig. 5. Time-course of the bleaching of RNO in the RNO+imidazol+DOPA+ HO- and RNO+imidazol+ DOPA+metal ion+ HO- system measured at pH 9.9 and pH 10.8. All samples contained 0.45 mM RNO and 60 mM imidazol. All other conditions as in Fig 1. As can be seen from the figure, the bleaching of RN0 is strongly dependent on the presence of the metal ions and its kind, and was greater at a higher pH's value. In conclusion, our findings strongly favour the role of potentially cytotoxic reactive oxygen species generated during autoxidation and metal-accelerated DOPA oxidation in the oxidative stress which has been reported to play a key role in aging and another neurodegenerative diseases.

452 REFERENCES 1. T. Spencer Smith, W.D. Parker, Jr and J.P. Bennett Jr, L-DOPA increases nigral production of hydroxyl radicals in vivo: potential L-DOPA toxicity? Neuro Report 5, 1009-1011 (1994). 2. M.M.Wick, L.Byres and E.Frei IlL L-DOPA: selective toxicity for melanoma cells in vitro. Science 197, 468-469 (1977). 3. I.Kruk, Environmental Toxicology and Chemistry of Oxygen Species. The Handbook of Environmental Chemistry. Volume 2. Reaction and Processes, Part I (O.Hutzinger ed) Springer, (1998). 4. B.Halliwell and J.M.C.Gutteridge, Role of the free radicals and catalytic metal ions in human disease: An interview. Methods E ~ m o l . 186, 1-85 (1990). 5. B.Halliwell, The role of oxygen radicals in human disease, with particular reference to the vascular system. Haemostasis 23 (suppl 1), 118-126 (1993). 6. G.A.Swan, Structure, Chemistry, and Biosynthesis of Melanins, in: Progress in the Chemistry of Organic Natural Products. Vien-Springer Verlag-New York, 22 (1974). 7. Z.L.Hegedus, S.H.Kuttab and M.D.Altschule, Studies on rheomelanins. VI The apparent lipofuscin characteristics of rheomelanins. Arch Int Physiol Biochim. 88, 265 (1980). 8. I.Kruk, T.Michalska, K.Lichszteld and M.Bounias, Luminescence in the reaction of DOPA with hydrogen peroxide. Zphys Chemie Leipzig 271(4): 771-780 (1990). 9. J.K|adny, I.Kruk, K.Lichszteld and T.Michalska, Luminescence accompanying peroxidation of DOPA in the presence of iron salts, haemin and cytochrome c. Toxicol Environm Chem 47, 223-234 (1995). 10. M.Bounias, J.Kladny, I.KnLk and T.Michalska, Hydroxyl radical generation during oxidation of catecholamines and DOPA. Toxicol Environm Chem 60, 163-169 (1997). 11. I.Kxuk, K.Lichszteld and T.Michalska, Evidence for the generation of singlet molecular oxygen during DOPA and dopamine peroxidation. Z Naturforsch 44C, 39-44 (1989). 12. I.Krailji6 and S.EI.Mohsni, A new method for the detection of singlet oxygen in aqueous solutions. Photochem Photobio128, 577-580 (1978). 13. H.S.Mason and C.J.Wricht, Chemistry of melanin. V. Oxidation of dihydroxyphenylalanine by tyrosinase. JBiol Chem 180, 235-247 (1949). 14. T.Sarna, Fotobiologiczne wta~ciwo~ci melanin i reakcje upigmentowanych kom6rek na ~wiatto. Zagadnienia Biofizyki Wsp6tczesnej 12, 119-132 (1987). 15. J.Wilczok, K.St~piefi and T.Wilczok, Model rheomelanins III. The effect of metal ions on the kinetics of adrenaline. Adrenochrome and adrenolutin oxidation. Studia Biophysica 131(3), 195-203 (1989). 16. K.St~piefl, B.Biliflska and T.Wilczok, Model rheomelanins II. Conversion of adrenaline-copper complexes to melanin polymers. Studia Biophysica 122(1-3), 181-189 (1987). 17. J.Stauff, U.Sander and W.Jaeschke, Chemiluminescence of perhydroxyl and carbonate radicals. Chemilumin Rep Int Conf1972, Plenum New York, N.Y, 131-141 (1973).

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26. B.R.Van Dyke, D.A.Clopton and P.Saltman, Buffer-induced anomalies in the Fenton chemistry of iron and copper, lnorg Chem 242, 57-61 (1996). 27. F.Haber and J.Weiss, The catalytic decomposition of hydrogen peroxide by iron salts. Proc Roy Soc Serd. 147, 332-351 (1934).

28. J.Moan and E.Wold, Detection of singlet oxygen production by ESR. Nature 279, 450-451 (1979).