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Archives of Biochemistry and BiophysicsVol. 387, No. 2, March 15, pp. 250–256, 2001doi:10.1006/abbi.2000.2238, available online at http://www.idealibrary.com on

A Novel Glutathione Peroxidase Mimicwith Antioxidant Activity

Xiaojun Ren,*,† Liquan Yang,* Junqiu Liu,* Dan Su,* Delin You,* Chuanpen Liu,* Kun Zhang,*Guimin Luo,*,1 Ying Mu,* Ganglin Yan,* and Jiacong Shen†,‡*Key Laboratory of Molecular Enzymology and Engineering, †Key Laboratory of Supramolecular Structure andSpectroscopy, and ‡Department of Chemistry, Jilin University, Changchun 130023, People’s Republic of China

Received September 6, 2000, and in revised form November 16, 2000; published online February 16, 2001

Many diseases are associated with the overproduc-tion of hydroperoxides that inflict cell damage. Anovel cyclodextrin derivative, 6A,6B-diseleninic acid-6A*,6B*-selenium bridged b-cyclodextrin (6-diSeCD),

as synthesized to be a functional mimic of glutathi-ne peroxidase (GPX) that normally removes theseydroperoxides. The mimic had high catalytic GPXctivity of 13.5 U/mmol, which is 13.6-fold higher than

ebselen (PZ51), and was chemically and biologicallystable in vitro. Antioxidant activity was studied byferrous sulfate/ascorbate-induced mitochondria dam-age model system. These data show that the mimic hasgreat antioxidant activity. Such mimics may result inbetter clinical therapies for diseases mediated by hy-droperoxides. © 2001 Academic Press

Key Words: glutathione peroxidase; b-cyclodextrin;artificial enzyme; antioxidant; selenium.

Many diseases can be characterized as conditions inwhich the body fails to contain the overproduction of anundesired metabolic by-product. All mammalian lifecontains reactive oxygen species as the metabolic by-product of O2 supporting cellular respiration. Superox-ide radicals, which were formed by side reaction of themitochondria electron transport chain or an NADH-independent enzymes, can be converted to H2O2 and tothe powerful oxidant, the hydroxyl radical (1). Undernormal circumstances, these reactive oxygen species,including superoxide radical, H2O2, and organic perox-ides, were controlled by superoxide dismutase (SOD),glutathione peroxidase (GPX), and catalase. In certain

1

To whom correspondence and reprint requests should be ad-ressed. Fax: 86-431-8923907. E-mail: gmluo@mail.jlu.edu.cn.

50

diseases, the production of reactive oxygen species isenhanced, resulting in reactive oxygen species-medi-ated cell injury. Examples of such oxidative stress-related diseases include reperfusion injury, brain isch-emia, tumor, and various types of inflammation andphysiological aging (2, 3). GPX is a powerful enzyme toblock the production of reactive oxygen species andinhibit their damage (4).

Because of the limitations associated with enzymetherapies (solution instability, limited cellular acces-sibility, immunogenicity, short half-lives, costs ofproduction, and proteolytic digestion), many GPXmimics have been prepared and were used to studyantioxidant properties. They include catalytic anti-bodies (5), semisynthetic enzyme (6, 7), diaryl di-selenides (8), a-(phenylselenenyl)ketones (9), cyclicselenenamides (10), transition metal complex (11),organic tellurides (12, 13), ebselen (PZ51) (14).Among them ebselen is the best-known GPX mimic.Although this interesting molecule has been re-searched extendedly from pulse-radiolytic studies onradical reactivity through its biological properties inorgans to clinical setting, it has some drawbacks,such as low GPX activity and water insolubility (15).Using cyclodextrin as enzyme model, we have discov-ered a stable and active class of GPX mimics (16 –18).Here we synthesized a novel GPX mimic (6-diSeCD),which catalyzes the decomposition of H2O2 with GPXactivity of 13.5 U/mmol exceeding Ebselen (0.99U/mmol) (8). The mimic is water solubility and hasobvious advantages for pharmacological application.We also investigated oxidative damage to mitochon-dria, which are a major physiological source of reac-tive oxygen species and are protected by the mimic.

These data suggest that the mimic plays a primary

0003-9861/01 $35.00Copyright © 2001 by Academic Press

All rights of reproduction in any form reserved.

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251GLUTATHIONE PEROXIDASE MIMIC AND ANTIOXIDANT ACTIVITY

role in protection against oxidative stress and hasgreat application potential.

MATERIALS AND METHODS

Apparatus. The characterization of structure of the mimic wasperformed with a Varian Unity-400 NMR Spectrometer, a BrukerIFS-FT66V Infrared Spectrometer, and a Perkin–Elmer 240 DS El-emental Analyzer. The content and valence of selenium were deter-mined by means of an ESCALAB MKII X-ray Photoelectron Spec-trometer. All spectrophotometric measurements were made using aShimadzu UV-3100 Spectrophotometer interfaced with a personalcomputer. Data were acquired and analyzed using UVS SpectroscopySoftwares. The temperature for UV time courses studies were con-trolled within (6) 0.5°C by use of a Lauda Instrument Thermostatwith a circulating water bath pumped to the spectrophotometer.

Materials. b-Cyclodextrin (b-CD) was purchased from Tianjinhemical Plant, recrystallized twice from water and dried for 12 h at00°C in vacuum. 1.3-Benzenedisulfonyl chloride was obtained fromldrich. Sodium borohydride, glutathione (GSH), glutathione reduc-

ase (type III), and b-nicotinamide adenine dinucleotide phosphate(NADPH) were obtained from Sigma. Sephadex G-25 was purchasedfrom Pharmacia and DEAE-52 from Whatman. Thiobarbituric acidand ferrous sulfate was obtained from Shanghai Second ReagentPlant. Ascorbic acid was purchased from Fluka. Analytical gradepyridine was predried by refluxing over KOH for 12 h, then overextremely anhydrous BaO for 12 h and distilled just before use.Buffers were prepared with distilled, deionized water. All the othermaterials were of analytical grade and were used without furtherpurification.

Synthesis of 6A,6B-diseleninic acid-6A9,6B9-selenium bridged b-cy-clodextrin (6-diSeCD). The regiosepecific ditosylation of 6A,6B-hy-droxyl positions of b-CD was carried out according to the method

escribed by Tabushi et al. (19) to obtain 6A,6B-capped-b-CD using,3-benzenedisulfonyl chloride as a specific reagent. Sodium hy-roselenide (NaHSe) was prepared according to the reference (20).he selenolation of 6A,6B-capped-b-CD is as follows: 100 mg of

6A,6B-capped-b-CD was dissolved in 2 ml of 50 mM potassiumhosphate buffer, pH 7.0. The reaction mixture was bubbled usingure nitrogen for 20 min and then 100 ml of 1 M NaHSe solution was

added. Under the protection of pure nitrogen the mixture was keptfor 36 h at 60°C. The reaction mixture was oxidized in air and thenpurified by centrifugation and Sephadex G-25 column chromatogra-phy using distilled, deionized water as eluent. Three peaks appearedand then the first peak was collected and freeze-dried. The lyophi-lized powder was washed with acetone to give 10 mg fresh yellowproduct (6-diSeCD) with a yield of about 10%.

Characterization of 6-diSeCD. The structure of the mimic wasanalyzed by means of elemental analysis, IR, 1H NMR. The datawere shown as follows. Anal. (C84H138O70Se4 z 6H2O) C, H. C: calcd,37.48; found; 37.64. H: calcd, 5.62; found, 5.73. IR (KBr): 3311(2OH), 2935 (CH, CH2), 1610 (2OH), 1165, 1072, 1020 (-O-). 1H

MR (400 MHz, D2O) d: 4.9-4.82(1-H), 3.80-3.59 (3-, 5-, 6-H), 3.50-3.2(2-, 4-, 6-H).

Determination of the Se content and the valence of 6-diSeCD. TheSe content and valence of the mimic were determined by X-rayphotoelectron spectroscopy (21). The energy of the exciting X-ray was1253.6 eV (Mg, Ka). C1s 5 285.0 eV was served as standard. Thecans were performed five times.Determination of the selenium content of 6-diSeCD. Selenium

ontent of the mimic was determined by the DTNB method (22). Theimic was treated with excess sodium borohydride in 50 mM, pH

.0, potassium phosphate buffer under nitrogen atmosphere for 30

in. The solution was treated with 1 M imidazole to quench the

xcessive reducing reagent and adjusted to pH 7.0 with 1 M HCl, and

00 ml of the solution was added to 5 mL DTNB solution (0.1 mM).he concentration of selenol in the mimic was determined from thebsorption of 3-carboxy-4-nitrobenzenethiolate at 410 nm (e 5 11400

M21 cm21).Determination of the GPX activity of 6-diSeCD. The GPX activity

of 6-diSeCD was measured according to Wilson’s method (8). Thereaction was carried out at 37°C in 500 ml of the solution containing0 mM potassium phosphate buffer, pH 7.0, 1 mM ethylenedi-minetetraacetic acid (EDTA), 1 mM sodium azide, 1 mM GSH, 1nit of GSH reductase, and 10–50 mM 6-diSeCD. The mixture was

preincubated for 10 min and then 0.25 mM NADPH solution wasadded and incubated for 3 min at 37°C. The reaction was initiated byaddition of 0.5 mM H2O2. The activity was followed by the decreaseof NADPH absorption at 340 nm. Appropriate controls were carriedout without enzyme mimic and were subtracted. The activity unit ofthe mimic is defined as the amount of the mimic that utilizes 1 mmolof NADPH per minute. The activity is expressed in U/mmol of enzyme

imic.Assay of kinetics of 6-diSeCD. The assay of kinetics of 6-diSeCDas similar to that of native GPX (23). The initial rates were mea-

ured by observing the change of NADPH absorption at 340 nm ateveral concentrations of one substrate while the concentration ofhe other substrate was kept constant. All kinetic experiments wereerformed in 0.5 ml of the reaction solution containing 50 mMotassium phosphate buffer, pH 7.0, 1 mM EDTA, 1 unit of GSHeductase, 0.25 mM NADPH, and appropriate concentration of GSH,

2O2, and 6-diSeCD. The mimic was preincubated with GSH,NADPH, and GSH reductase. The reaction was initiated by additionof appropriate concentrations of H2O2. The nonenzymatic reactionffecting the measurement of the initial rate was taken into accountnd subtracted to obtain exact kinetic values. Kinetic data werenalyzed by double-reciprocal plots and fit to Eq. [1] using a programf Clealand (24), where Vm is the maximal velocity, A and B are the

concentrations of substrate H2O2 and GSH, and KA, and KB are theMichaelis constants for H2O2 and GSH, respectively.

n0 5Vm AB

KAB 1 KB A 1 AB [1]

Ferrous sulfate/ascorbate-induced mitochondria damage. Mito-chondria were prepared from myocardium of bovine according to themethod of Lansman (25) and were suspended in 0.25 M sucrose, 10mM EDTA, and 25 mM Hepes–NaOH buffer, pH 7.4, and maintainedat 0°C. The concentration of mitochondria protein was determined byCommassie brilliant blue (26), using bovine serum albumin as thestandard. The incubation mixture consisted of 0.15 M KCl, 10 mMEDTA, 1 mM glutathione, mitochondria (0.2 mg protein/ml) andappropriate enzyme mimic in 25 mM Hepes-NaOH buffer, pH 7.4,37°C. Lipid peroxidation and swelling of mitochondria was initiatedby addition of 60 mM ascorbate and 6.4 mM ferrous sulfate. Controlexperiments have been done without enzyme mimic; blank experi-ments have been performed without enzyme mimic, ascorbate, andferrous sulfate.

Measurement of lipid peroxidation. Lipid peroxidation in ferroussulfate/ascorbate-treated mitochondria was analyzed by thiobarbitu-ric acid (TBA) assay. In this assay, TBA reacts with malonaldehyde(MDA) and/or other carbonyl by-products of free-radical-mediatedlipid peroxidation to give 2:1 (mol/mol) colored conjugates (27). Thereaction was terminated by adding 0.1 mL of 0.5% (W/W) trichloro-acetic acid. Finally, the MDA in mitochondria membrane was mea-sured using tetraethoxypropane as the standard. In plotting lipidperoxide formation in the graphs, the absorbance reading has been

graphed directly rather than by means of a fixed conversion to MDAand/or other carbonyl byproducts equivalents.

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252 REN ET AL.

Assays for mitochondria swelling. Swelling of mitochondria wasassayed as described by Hunter et al. (28). Changes in light scatter-ing are correlated with mitochondria swelling. The swelling of mito-chondria was measured as the decrease in turbidity of the reactionmixture at 520 nm. The decrease of the absorbance indicates anincrease in the mitochondria swelling and a decrease in the mito-chondria integrity.

RESULTS

Synthesis and Characterization of 6-diSeCD

The selenol groups were introduced on 6-positions ofb-CD by selective ditoslation of 6-hydroxyls on theprimary side of b-CD with regiosepecific reagent 1,3-benzenedisulfonyl chloride. The nucleophilic substitu-tion of 6A,6B-capped b-CD gave diselenol-b-CD, then itwas oxidized in air to obtain 6A,6B-diseleninic acid-6A9,6B9-selenium bridged b-CD (6-diSeCD). The syn-thetic route of 6-diSeCD is shown in Scheme 1.

The selenium content of the mimic was measured byDTNB method and found to be 3.90 6 0.15 mol sele-nium/mol mimic. The ratio of C/Se was also given byX-ray Photoelectron Spectroscopy as follows: C/Se,20.5:1 (calculated 21:1), which shows that 1 mole of themimic contains 3.91 mole of selenium. The Se3d elec-tron binding energy of 6-diSeCD was 54.9ev and 55.3ev,respectively. The 54.9ev approaches the binding energyof SeCyss of 55.1ev. This result is consistent with ourprevious work (16) in which the GPX mimic 6-SeCDcontained diselenide bridge, indicating that the seleniumin 6-diSeCD was partly presented as the form of di-selenide bridge (-Se-Se-). The 55.3ev was assigned to bethe binding energy of seleninic acid, which is in agree-ment the studies about X-ray Photoelectron Spectroscopyof GPX (21), seleninic acid occurred in native GPX (29),and semisynthetic enzyme (30, 31).

The GPX Activity of 6-diSeCD

The GPX activity of the 6-diSeCD-catalyzed the re-

SCH

duction of H2O2 was listed in Table I and the GPXb

activities of other species were included for compari-son. The GPX activity of 6-diSeCD for the reduction ofH2O2 by GSH was found to be 13.5 U/mmol, indicatingthat 6-diSeCD displays a higher GPX activity thanebselen (8), 6-SeCD (16), DisecysCD (17), and 2-SeCD(18). To gauge the catalytic efficiency of 6-diSeCD, wecompared 6-diSeCD with model compound ebselen, awell-studied GPX mimic (8, 14, 15). At 37.0°C and pH7.0, the initial rate of the reduction of H2O2 (0.5 mM)by GSH (1 mM) in the presence of 11 mmol 6-diSeCD is2.9 3 1024 M min21, but only 3.6 3 1027 M min21 in theabsence of enzyme mimic under similar conditions,when 2 mmol ebselen was used as the catalyst, theinitial rate is only 4.1 3 1026 M min21. These data showthat 6-diSeCD is 13-fold more efficient than ebselen.

The 6-diSeCD-catalyzed the reduction of H2O2 by GSHas followed at several pH values from 4.0 to 12, then the

ptimum pH was determined to be 9.0. The optimumemperature was found to be 45–57°C by determinationf the GPX activity of 6-diSeCD-catalyzed the reductionf H2O2 at varying of temperature from 25–65°C.

The Kinetics of 6-diSeCD

The initial velocities for the reduction of H2O2 byGSH were determined as a function of substrate con-centration at 37°C and pH 7.0, varying one substrate

E 1

TABLE I

The GPX Activity of 6-diSeCD and Other Species

Species Hydroperoxide Activity (U/mmol)

bselen H2O2 0.99DisecysCD H2O2 4.136-SeCD H2O2 4.22-SeCD H2O2 7.46-diSeCDa H2O2 13.5 (0.2)b

a Reactions were carried out in 50 mM potassium phosphate

uffer, pH 7.0, at 37°, 1 mM GSH, 0.5 mM H2O2.

b Standard deviation is shown in parentheses.

b

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253GLUTATHIONE PEROXIDASE MIMIC AND ANTIOXIDANT ACTIVITY

concentration while the other was fixed. Saturationkinetics were observed for both H2O2 and GSH. Doublereciprocal plots of the initial velocity versus the con-centration of substrates yield a family of parallel lines(Fig. 1), consistent with a Ping-Pong mechanism in-volving at least one covalent enzyme intermediate. Theapparent kinetic parameters could be calculated fromthe double-reciprocal plots. The pseudo-first-order rateconstant k cat (H2O2) and the apparent Michaelis con-stant Km (H2O2) at 1 mM GSH, for example, werecalculated to be 10.81 min21 and 2.43 3 1024 M, respec-tively, and the apparent second-order rate constant k cat

(H2O2)/Km (H2O2) was determined to be 4.44 3 104 M21

min21. This mechanism can also be described as Eq. [2]y Dalziel’s parameters (32).

@E#0 FGSH FH2O2

FIG. 1. Double-reciprocal plots for the reduction of H2O2 by GSH catalh), 2.0 mM (E), and 3.0 mM (‚). (B) [E]0/v0 (min) vs 1/[GSH] (mM21)

n05 F0 1

@GSH#1

@H2O2#, [2]

sulfate/ascorbate-induced mitochondria damage; (b) 3 mM of 6-diSeCD proton the concentration of 6-diSeCD during mitochondria lipid peroxidation.

where FGSH equals to 2.53 3 1025 M min and FH2 O2

equals to 2.20 3 1025 M min.

Antioxidant Activity of 6-diSeCD Compound

Figure 2A indicates the extent of protection af-forded by 6-diSeCD by prolonging the lag phase be-fore the onset of peroxidation. The lag phase in con-trol experiment was remarkably prolonged in thepresence of 3 mM 6-diSeCD. The 6-diSeCD (3 mM)decreased the maximal level of MDA accumulatedafter 50 min to about 55% and also the slope of therapid phase of MDA accumulation. The results fromFig. 2B show that 6-diSeCD could greatly inhibitlipid peroxidation of mitochondria and the inhibitionwas considerably dependent on 6-diSeCD concentra-tion. The amount of MDA was decreased with theincrease in 6-diSeCD concentration. When the 6-

d by 6-diSeCD. (A) [E]0/v0 (min) vs 1/[H2O2] (mM21) at [GSH] 5 1.0 mMH2O2] 5 0.5 mM (h), 1.0 mM (E), and 2.0 mM (‚).

diSeCD concentration was 6.2 mM in the incubation

FIG. 2. (A) Dependence of extent of MDA accumulation on 6-diSeCD. The absorbance intensity reading represents MDA equivalents. (a) Ferrous

ected against mitochondria damage. (B) Dependence of MDA formationContent of MDA in the damage group is defined as 100%.

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254 REN ET AL.

mixture, the MDA content was only 25% of the dam-age group, indicating that 75% of MDA productionwas inhibited. When the concentration of 6-diSeCDwas more than 6.2 mM, the protection afforded by6-diSeCD was not notably increased.

Figure 3 shows that the mitochondria were greatlyswelled by ferrous sulfate/ascorbate-induced mitochon-dria damage and the swelling of mitochondria wasdecreased by addition of 6-diSeCD. The absorbency at520 nm for the control group was basically constant,whereas the damage group decreased considerablywith time, indicating that the mitochondria swellingwas considerably increased. But the swelling for theprotection group, which contained a certain concentra-tion of 6-diSeCD, was apparently decreased.

DISCUSSION

Cyclodextrin are cyclic oligosaccharides consisting of ahydrophobic cavity with which many complexes can beformed via host–guest chemistry. The 2-, 3-, and 6-hy-droxyl positions of cyclodextrin can be selectively modi-fied, respectively, and a variety of catalytically activegroups can be introduced on the different hydroxyl posi-tions of cyclodextrin (33). Therefore, cyclodextrin as en-zyme model have been extendedly studied (34). What anenzyme does is to bind, and thus stabilize the transitionstate for a particular reaction. Enzymes must first recog-nize and bind their substrates to set up the correct geom-etry. The binding process is a major problem in the de-velopment of enzyme mimic (35). Cyclodextrin bindsGSH, and the binding constant is 1.01 3 102 M (17).

ased on cyclodextrin as enzyme model, several GPXimics have been prepared (16–18). The GPX activity of

-diSeCD-catalyzed the reduction of H O by GSH is

FIG. 3. Dependence of swelling of mitochondria on 6-diSeCD. (‚)ontrol; (E) damage 1 7.7 m M of 6-diSeCD; (h) damage.

2 2

remarkably higher than that of ebselen (8), 6-diSeCD

(16), DisecysCD (17), and 2-SeCD (18), thus we are veryinterested in the 6-diSeCD. The reason that 6-diSeCDexhibits high GPX activity needs to be further studied.

The kinetic pattern of the native GPX is character-ized as Ping-Pong mechanism with indefinite Michae-lis constants, indefinite maximum velocities and neg-ligible, if any, product inhibition (36). It can be de-scribed as the simple Dalziel equation Eq. [3].

@E#0

n05

FGSH

@GSH#1

FH2O2

@H2O2#[3]

n native enzyme catalytic mechanism, the fact thatcat for H2O2 at infinite concentration and Km for H2O2

are linear functions of the concentration of GSH meansthe native GPX could not be saturated by GSH and F0

is not present in Dalziel Eq. [3]. The 6-diSeCD-cata-lyzed the reduction of H2O2 by GSH followed Ping-Pongmechanism and F0 is not equal to zero, which means6-diSeCD could not be saturated by GSH. Under iden-tical conditions, these F parameters could be com-pared. The F parameters for native GPX are FH2 O2 50.93 3 10210 M min and FGSH 5 2.13 3 1028 M min (23),but, under similar conditions, these for 6-diSeCD areFH2 O2 5 2.20 3 1025 M min and FGSH 5 2.53 3 1025 Mmin. GPX is believed to have evolved near optimalefficiency for the decomposition of H2O2, having anapparent second-order rate constant for the reactionbetween H2O2 and enzyme of the order of 108 M21

min21. The 6-diSeCD falls far short of this idea, givingan equivalent rate constant for H2O2 breakdown ofapproximately 4500 M21 min21. Thus, the catalytic ef-

ciency of 6-diSeCD is lower than that of the nativePX. The catalytic efficiency of 6-diSeCD-catalyzed the

eduction of H2O2 by GSH is higher than that of othercyclodextrin-derived GPX mimics, as evidenced fromthe comparison of kinetic data. For example, the pseu-do-first-order rate constant k cat (H2O2) for 6-SeCD, theapparent Michaelis constant Km (H2O2) for 6-SeCD,and the apparent second-order rate constant k cat

(H2O2)/Km (H2O2) for 6-SeCD at 1 mM GSH, were de-termined to be 5.5 min21, 3.5 3 1024 M, and 1.53 3 104

M21 min21, respectively (16). Under identical condi-tions, the equivalent parameters for 6-diSeCD are k cat

(H2O2) 5 10.81 min21, Km (H2O2) 5 2.43 3 1024 M, andk cat (H2O2)/Km (H2O2) 5 4.44 3 104 M21 min21.

Reactive oxygen species, such as superoxide anion,hydrogen peroxide, and organic peroxide, are toxicby-products of various metabolic reactions and areproduced in response to various stimuli. Moreover, ithas recently been revealed that reactive oxygen spe-cies regulate the physiological state of cell and influ-ence cell death (4, 37). Mitochondria are a major

physiological source of reactive oxygen species,

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255GLUTATHIONE PEROXIDASE MIMIC AND ANTIOXIDANT ACTIVITY

which can be generated during mitochondria respi-ration. Among these reactive oxygen species, neithersuperoxide anion nor H2O2 can directly initiate lipidperoxidation, while hydroxyl radical is the source ofa more reactive ultimate oxidant (38, 39). The mem-brane lipid of mitochondria is the primary target.Therefore, we chose the mitochondria as the model ofoxidative damage.

Naturally accruing oxidative damage can be mim-icked by exposing mitochondria in vitro to redox activexenobiotics. The reaction for ferrous sulfate/ascorbateinduced mitochondria damage could be proposed:

oxidative ascorbate 1 2H1 1 2O2•2 3 H2O2

1 reducative ascorbate [4]

Fe21 1 H2O2 3 Fe31 1 OH2 1 •OH [5]

•OH 1 LH 3 H2O 1 L• [6]

L• 1 O2 3 LOO• [7]

LOO• 1 LH 3 L• [8]

here L represents lipid compounds. Lipid peroxida-ion would be initiated by hydrogen abstraction of annsaturated lipid (Eq. [6]) (3). Subsequent chain prop-gation steps (Eqs. [7] and [8]) would generate lipidydroperoxides (LOOHs), with accompanying disrup-ion of membrane structure and function. In order tonhibit lipid peroxidation, H2O2 and hydroxyl radical

must be scavenged, as evidenced by the mechanismproposed. In many mitochondria, catalase is lack (40).Therefore, GPXs including cGPX and PHGPX play animportant role in scavenging hydroperoxides (Eqs. [9]and [10]) (4). GPX and GPX mimics can scavenge hy-droperoxides, block the lipid peroxidation, and protectmitochondria against oxidative damage (12–14).

H2O2 1 2GSHO¡GPX

H2O 1 GSSG [9]

NADPH 1 H1 1 GSSGO¡

Glutathione reductase

NADP1

1 2GSH [10]

In ferrous sulfate/ascorbate-induced mitochondriadamage and swelling model system, 6-diSeCD pro-longed the lag phase before the onset of peroxidation

and decreased the maximal level of MDA accumulation

1

and also the slope of rapid phase of MDA accumulation.The inhibition of lipid peroxidation by 6-diSeCD isdependent upon a dose manner. The MDA accumula-tion was decreased with the increase of the amount of6-diSeCD. The reason that 6-diSeCD inhibited lipidperoxidation can be explained by 6-diSeCD acting as aGPX mimic, which effectively scavenged hydroperox-ides and protected against oxidative damage.

In summary, our results demonstrate that 6-diSeCDis an excellent GPX mimic, as evidenced by enzymaticnature. The 6-diSeCD has apparent antioxidant activ-ity in model of ferrous sulfate/ascorbate-induced mito-chondria damage. These results show these moleculesmay have potential for curing reactive oxygen species-related diseases, which include chronic inflammation,cardiovascular disease, cancer, and cataract.

ACKNOWLEDGMENTS

We thank the financial support from the National Key Project onFundamental Research and Development (973, G2000078102) andthe National Natural Science Foundation of China (20072010).

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