lucigenin: redox potential in aqueous media and redox cycling with o2 β production

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Page 1: Lucigenin: Redox potential in aqueous media and redox cycling with O2 β Production

151 1 THE PRO-OXIDATIVE ACTIVITY OF SOD AND NITROXIDE SOD-MIMICS

f?is%AA.. An elo Russo, and Amram Samuni. Hebrew UnioersihJ, @usalem,

Native SOD and synthetic SOD-mimics sometimes demonstrate an apparently anomalous bell-shaped dose-response relationship when protecting various biological systems from oxidative stress. Several mechanisms have previously been proposed to account for such an effect. In the present study, ferrocyanide and papain-thiol, which are susceptible to l-electron and 2-electron oxidation, respectively, have been subjected to a flux of superoxide, in the presence and absence of SOD or SOD-mimic. The results show that: (a) either superoxide or Ii202 alone is capable of inactivating papain, whereas, when combined, they act synergistically; (b) nitroxide SOD-mimics, but not SOD, exhibit a bell-shaped dose-response relationship ln protecting papain from oxidation; (c) SOD, which at low dose inhibits superoxide-induced oxidation of ferrocyanide, loses-this anti-oxidative effect as its concentration increases. These findings offer an additional explanation for the pro-oxidative activity of SOD and SOD-mimics. The most significant outcome of an increase in SOD level is a decrease of [superoxidelsteady state, rather than any notable elevation of [H202]steady state. As a result, the reaction kinetics of the high oxidation state of each catalyst are altered. In the presence of ultra-low [superoxidelsteady state, the oxidized form of the SOD (Cu(II),Zn-SOD) or the SOD-mimic (oxo-ammonium cation) does not react with superoxide but rather oxidizes the target molecule that it was supposed to have protected. Consequently, whether these catalysts exert an anti- or pro-oxidative effect depends on their level.

L-_T-l Lucigenin: Redox Potential in Aqueous Media and

Redox Cycling with Op Production Ivan Sossoievic, Stefan I. Liochev and Irwtn Fridovich

Department of Biochemistxy, Duke University Medical Center Durham, North Carolina 27710

Univalent reduction of lucigenin, Luc”, gives the radical Luc’. which reacts with superoxide radical, 02-s to produce chemiluminescence (CL). However, Luc+* may autooxidize i.e.reduce Oz and produce Oz.* leading to an artifactoal detection of OP [1,2]. Nevertheless, perhaps due to the high sensitivity and convenience of the CL measurements, Luc* is still used as an assay for Oz.* formation in biological systems. Moreover, in a recent work using an invalid extrapolation of an incorrect redox potential for Luc” from non-aqueous to aqueous conditions these authors attempted to disprove the possibility of the autooxidation of Luc* [3].

Now we present, for the first time, reversible cyclic voltammetty of lucigenin in aqueous medium. The redox potential obtained for Luc*, E” = -0.14 + 0.02 V vs NHE, is virtually identical to the E” reported for 02/0>

couple i.e. -0.16 V vs NHE. Our data also show that the EIn for Luc* was only moderately affected (-100 mV) by going from water to DMSO, acetonitrile or dichloromethane, rather than the 500 mV “correction” applied in the above mentioned work [3].

In addition, the Luc*-induced oxygen consumption in the NADH I xanthine oxidase (X0) system has been detected down to - 6 pM Luc* in what has been claimed to be the “non - redox cycling” region of Luc+) [4].

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Liochev, S. I., and Fridovich, I. (1998) Free Radic. Biol Med. X,926-928. Tarpey, M.. M.., white, C. R.., Suarez, E., Richardson, G., Radi, R., and Freeman, B. A. (1999) Circ. Rex 84,1203-1211. Afanasev, I. B., Ostrachovitch, E. A., and Korkina, L. G. (1999) Arch. Biochem. Biophys. 366,267.274. Li, Y., Zhu, H., Kuppusamy, P., Roubaud, V., Zweier, J. L., and Tush, M. A. (1998) J Biol Chem. 273,2015-2023.

CYTOSOLIC OR MITOCHONDRIAL CATALASE PROTECTS FROM Hz02 TOXICITY AND REVERSES THE PROLIFERATIVE INHIBlTION ASSOCIATED WITH MnSOD OVEREXPRBSSION. Ana M. Rodriguez, Pauline MCarrico and J.Andres Melendez. Department of Biochemistry & Molecular Biology, Albany Medical College, Albany, NY 12208.

Manganese superoxide dismutase (MnSOD) overexpression has been shown to reverse the malignant phenotype in a variety of tumor cell lines. The inhibition of proliferation and reversal of the malignant phenotype has been attributed to an increase in Hz02 production as a result of to the dismutation reaction. However, direct evidence in support of this hypothesis has not been forthcoming. To evaluate the contribution of Hz02 in regulating cell growth in response to MnSOD overexpression, control and MnSOD overexpressing cells were stably transfected with constructs that direct catalase to either the mitochondrial or cytosolic compartments. Overexpression of catalase in either compartment reversed the proliferative and clonogenic inhibition associated with MnSOD overexpression and increased protection from the cytotoxicity of H202. In addition, mitochondrial or cytosolic catalase enhances respiration through complex I and II in both control and MnSOD overexpressing cell lines. Thus, catalase reverses the proliferative inhibition associated with MnSOD overexpression and may also play an important role in metabolic regulation.

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REDOX REACTIONS OF LUCIGENIN Peter Wardman and Borivoj Vojnovic Gray Laboratory Cancer Research Trust, PO Box 100, Mount Vernon Hospital, Northwood, Middr HA6 2./R, UK

The use of lucigenin (LC’+, bis-N-methylacridinium) as a chemiluminescent probe for superoxide (027 is controversial. Liochev and Fridovich (1997, 1998) and Vkquez-Vivar et al. (1997) have drawn attention to superoxide production from reduction of LC’+ to LC’ and redox cycling, whilst Li ef al. (1998, 1999) and Skatchov et al. (1999) have explored the validation of LC2+ in spite of this drawback. Central to this controversy are the kinetics and position of the electron-transfer equilibrium (1):

LC’ + o* F! Lc*+ + 0s’ (1) and the reduction potential of the LC”/LC” couple, which we have characterized using Bulse radiolysis. Redox equilibrium (2) with benzyl viologen (BV +) was established in microseconds:

BV’ + LC*+ 2 BV2+ + LC-+ (2) with Kr = 40, i.e. E(LC2’iLC”) = - 0.28 V vs. NHE, and hence K, = 50. Measuring KI directly is complicated by the likely concurrent reaction of LC’+ with Or-, but the decay of LC’ in Or occurred in < 1 ms, with rate close to first-order in [02] and consistent with /cl - 3 x lo6 dm3 mol-’ s-t. These properties explain the high reactivity of LC*+ with flavoprotein reductases as well as superoxide stimulation. Common quinone radicals Q‘ react with 02 much faster than LC’+, raising the possibility that some quinones could act as redox mediators in LC*/Or redox cycling. This is a further complication in the use of LC’+. Work supported by the Cancer Research Campaign.

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