ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 288, No. 1, July, pp. 215-219, 1991

Mn(lII)-Desferrioxamine Superoxide Alternative Modes of Action Stephen M. Hahn,* *Radiation tMolecular

C. Murali

Krishna,*

Dismutase-Mimic:

Amram Samuni,? James B. Mitchell,*

and Angelo RUSSO**~

Oncology Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland Biology, Hebrew University Medical School, Jerusalem 91010, Israel

20892; and

Received October 24, 1990, and in revised form March 5, 1991

Various low-molecular-weight copper chelates have been synthesized to mimic superoxide dismutase (SOD) by catalyzing 02 dismutation. However, in the presence of cellular proteins, such chelates dissociate and thereby lose their SOD-mimetic activity. In contrast, desferrioxamine-Mn(II1) 1: 1 chelate (DF-Mn), an SOD-mimic that affords protection from oxidative damage, reportedly is stable in the presence of serum albumin. DF-Mn, unlike SOD, is reported to permeate the membrane of at least one cell type and can protect cells by detoxifying intracellular 0;. Recently DF-Mn was shown to protect hypoxic cells from H20,-induced damage. Such results suggest that DF-Mn can protect cells from OG-independent damage by alternative mechanisms. This study examines such possibilities. To avoid 0; participation in the damaging process, killing of monolayered V79 Chinese hamster cells was induced in a hypoxic environment by tbutyl hydroperoxide (t-BHP). Damage induced by t-BHP was inhibitable by DF-Mn. DF-Mn was also found to rapidly oxidize iron(bound DNA. Additionally, once DF-Mn oxidizes Fe(I1) or Cu(I), the DF moiety of DFMn dissociates and rapidly binds to Fe(III) or Cu(I1). Without excluding the possibility that DF-Mn protects cells by facilitating the removal of 0;) the present results show that this SOD-mimic can confer protection from cytotoxic processes independent of Of or of OG-derived active species. (c7 1991 Academic Press, Inc.

Superoxide dismutase (SOD)-mediated2 protection of cells exposed to superoxide-derived reactive species and 0; itself has been extensively documented (l-4). Nev1 To whom correspondence should be addressed. ’ Abbreviations used: EPR, electron paramagnetic resonance; t-BHP, t-butyl hydroperoxide; DTPA, diethylenetriamine pentaacetic acid; DF, desferrioxamine (Desferal); X0, xanthine oxidase; HX, hypoxanthine; cyt-c’n, ferricytochrome c; DF-Mn, desferrioxamine-Mn(II1); SOD, superoxide dismutase; DMPO, 5,5-dimethyl-l-pyrroline-N-oxide; PBN, N-tert-butyl-oc-phenylnitrone. 0003.9861/91 $3.00 Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.

ertheless, difficulty in elevating intra- as well as extracellular SOD levels has prompted a search for nontoxic SOD-mimics which can facilitate 0; removal from the extra-, and most importantly, intracellular compartments. Since copper, iron, and manganese can all be cofactors in the active site of native SOD, attention has focused on low-molecular-weight chelates of these ions (5-9). To be an effective agent, the SOD-mimic should be nonimmunogenic, nontoxic, and stable to dissociation or cell metabolism. Certain copper chelates were found to react rapidly with 0; but proved to be biologically ineffective because of their high rate of reaction with molecular oxygen or dissociation of the chelate. In contrast, manganese chelates were found not to dissociate and to effectively remove 0: radicals (8-10). In particular, manganesebased chelates, such as desferrioxamine-Mn(II1) (DFMn), were found to react rapidly with 0; and to protect cells from various types of oxidative insults (11-13). Recent studies showed that DF-Mn can protect mammalian cells under experimental conditions in which 0, radical participation would be very unlikely (14). This anamolous finding suggested that DF-Mn might function by mechanisms other than as an SOD mimic. In a search for alternative modes of action of DF-Mn, V79 Chinese hamster cells were used as a test system and the injury induced by t-butyl hydroperoxide (t-BHP) under hypoxia was determined. The present results clearly show that DF-Mn can protect cells from Og-independent damage. The possible mechanisms responsible for cell protection are discussed. MATERIALS

AND

METHODS

Chemicals. Desferrioxamine (DF) was a gift from Ciba Geigy; hypoxanthine (HX) was purchased from Calbiochem; MnOz was purchased from Aldrich Chemical Co.; xanthine oxidase (EC 1.1.3.22, xanthine: oxygen oxidoreductase) (X0) grade III from buttermilk, superoxide dismutase, t-butyl hydroperoxide, and ferricytochrome c (cyt-c”‘) were obtained from Sigma; H,Oz was bought from Fisher. Xanthine oxidase was further purified on a G25 Sephadex column. All chemicals were prepared and used without further purification. The 1:l green complex 215

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of DF-Mn(II1) (final concentration IO-35 mM) was prepared with minor modifications as previously reported (9, 10). Briefly, 10% excess Mn02 and DF were mixed in water and stirred overnight. The green DF-Mn solution was centrifuged and stored at 4OC. The complex was assayed spectrophotometrically using ?Onrn = 109 MS’ cm-’ and e320”m= 1570 Mm’ cm-‘. EPR spectroscopy was used to assay the Mn(I1) content in the DF-Mn preparation, whereas the total manganese content was determined after complete reduction by excess ascorbate. Distilled-deionized water was used throughout all experiments. Hydrogen peroxide assay. H202 was assayed using a YSI Model 21 industrial analyzer (Yellow Springs Instruments) equipped with a selective electrode for HzOz. Electron paramagnetic resonance. EPR spectra were recorded with a Varian E9 X-band spectrometer, with field set at 3357 G, modulation frequency of 100 kHz, nonbroadening modulation amplitude, and nonsaturating microwave power. For aerated experiments, samples (0.050.1 ml) of solutions were drawn by a syringe into a gas-permeable teflon capillary of 0.8 mm inner diameter and 0.05 mm wall thickness (Zeus Industrial Products, Inc., Raritan, NJ). Each capillary was folded twice, inserted into a narrow quartz tube which was open at both ends (25 mm i.d.), and then placed horizontally into the EPR cavity. For hypoxic experiments, the reagents were made hypoxic using argon gas in separate arms of a two-arm H-shaped sealed test-tube. The test tube was connected to a flat EPR cell. To detect short-lived transients, either 0.1 M DMPO or 75 mM PBN spin traps were included in the reaction mixture. The EPR spectrophotometer was interfaced to an IBM PC through an analog-to-digital converter and a data translation hardware (DT2801), and the spectra were digitalized by using commercial acquisition software, enabling subtraction of background signals. Cell survival analysis. Survival of Chinese hamster V79 cells in tissue culture was assessed by clonogenic assay (14). Cells were incubated in complete F-12 medium 16-24 h prior to experimental procedures and then exposed to t-BHP. Following treatment, cells were trypsinized, rinsed, counted, and plated in triplicate for macroscopic colony formation. Following appropriate plating periods, colonies were fixed, stained, and lastly counted with the aid of a dissecting microscope. For hypoxic experiments the cells were plated into specially designed, glass flasks sealed with soft rubber stoppers. Nineteen-gauge needles were pushed through to act as entrance and exit ports for humidified gas mixture of 95% N2/5% CO, (Matheson Gas Products). Each flask was also equipped with a ground glass side arm vessel which when rotated and inverted could deliver 0.2 ml of medium containing t-BHP. Stoppered flasks were connected in series and mounted on a reciprocating platform and gassed at 37°C for 45 min prior to and throughout the experiment. After 45 min the effluent gas phase was

Mn(III)-desferrioxamine superoxide dismutase-mimic: alternative modes of action.

Various low-molecular-weight copper chelates have been synthesized to mimic superoxide dismutase (SOD) by catalyzing O2-. dismutation. However, in the...
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