An endogenous metabolite of dopamine, 3,4-dihydroxyphenylethanol, acts as a unique cytoprotective agent against oxidative stress-induced injury

Original Contribution

AN ENDOGENOUS METABOLITE OF DOPAMINE,3,4-DIHYDROXYPHENYLETHANOL, ACTS AS A UNIQUE CYTOPROTECTIVE

AGENT AGAINST OXIDATIVE STRESS-INDUCED INJURY

TSUNEICHI HASHIMOTO,* MASAKAZU IBI,* KUNIHARU MATSUNO,* SHINGO NAKASHIMA,* TORU TANIGAWA,y

TOSHIKAZU YOSHIKAWA,y and CHIHIRO YABE-NISHIMURA*

*Department of Pharmacology and yFirst Department of Internal Medicine, Kyoto Prefectural University of Medicine, Kyoto, Japan

(Received 5 August 2003; Revised 17 November 2003; Accepted 5 December 2003)

Abstract—A natural compound contained in olive oil, 3,4-dihydroxyphenylethanol (DOPE), is also known as anendogenous metabolite of dopamine. The role of DOPE in oxidative stress-induced cell damage was investigatedusing differentiated PC12 cells. Superoxide (O2

-) and H2O2 induced a dose-dependent leakage of lactatedehydrogenase (LDH) and decreased cell viability denoted by MTT assay. While O2

- -induced cell damage was notaffected by DOPE, pretreatment of the cells with DOPE dose-dependently prevented the leakage of LDH inducedby H2O2. In these cells, augmented activity of catalase was demonstrated, while the levels of glutathione andglutathione peroxidase activity remained unchanged. The effect of DOPE was abolished when an inhibitor ofcatalase, 3-amino-l, 2,4-triazole, was included in the medium. DOPE also protected against cell damage induced byH2O2 and Fe2+. In the hydroxyl radical (.OH) assay using p-nitroso-N, N-dimethylaniline (PNDA), oxidation ofPNDA by .OH generated by the Fenton reaction was significantly attenuated in the presence of DOPE. By anelectron spin resonance spin trapping study that represents the direct activity of DOPE to scavenge .OH, however,limited scavenging activity was demonstrated for DOPE. Taken together, DOPE may act as a unique cytoprotectivecompound in nerve tissue subjected to oxidative stress. D 2004 Elsevier Inc. All rights reserved.

Keywords—Catalase, 3,4-Dihydroxyphenylethanol, Dopamine, Olive oil, Oxidative stress, Reactive oxygen species,PC12 cells, Free radicals

INTRODUCTION

Reactive oxygen species (ROS) cause oxidative dam-age to various biological macromolecules includingDNA, lipids, and proteins. The major ROS generatedin the cells are superoxide and the more detrimentalhydroxyl radical (.OH), the latter being derived fromhydrogen peroxide (H2O2). Cells are well equippedwith defense mechanisms against ROS. Catalase, glu-tathione peroxidase (GSH-Px), and superoxide dismu-

tase (SOD) play pivotal roles in preventing cellulardamage caused by ROS. A low steady-state level ofintracellular superoxide is maintained by SOD, andH2O2, generated by SOD is removed by catalase orGSH-Px, which also acts on lipid hydroperoxides.Under biological conditions where free iron is releasedfrom the storage pool, the toxicity of H2O2, is partlyattributed to (.OH) generated by a Fenton-type reaction[1].

3,4-Dihydroxyphenylethanol (DOPE), also calledhydroxytyrosol, is a naturally occurring phenolic com-pound found in olive oil. DOPE possesses specificbiological properties that may contribute to the preven-tion of cardiovascular and thrombotic diseases [2]. Epi-demiological studies have shown a correlation betweenincreased consumption of phenolic antioxidants in foodsand reduced risk of cardiovascular disease [3,4] as well

* Address correspondence to: Chihiro Yabe Nishimura, M.D.,

Ph.D., Department of Pharmacology, Kyoto Prefectural University of

Medicine, Kawaramachi-Hirokoji, Kamikyoku, Kyoto 602-8566,

Japan; Fax: +81-75-251-5348; E-mail: [email protected].

Free Radical Biology & Medicine, Vol. 36, No. 5, pp. 555 –564, 2004Copyright D 2004 Elsevier Inc.

Printed in the USA. All rights reserved0891-5849/$-see front matter

doi:10.1016/j.freeradbiomed.2003.12.003

555

as prostate or colon cancer [5,6], for which the involve-ment of an uncontrolled free radical production waspostulated [7]. In vitro experiments denoted that DOPEefficaciously counteracted chemical oxidation of low-density lipoprotein [8], possibly by acting as a potentscavenger of superoxide radicals [9]. In human erythro-cytes and also in the Caco-2 cell line, toxic effects ofH2O2, and superoxide were eliminated by DOPE [10,11].In animal experiments, an extract of olive mill wastewa-ter, containing DOPE as the most abundant component,significantly prevented passive smoking-induced oxida-tive stress in rats [12].

In contrast to the antioxidative effects of DOPEdemonstrated in nonneural cells, the function of DOPEin nerve tissue has not been fully elucidated. Of note wasthe finding that DOPE is endogenously generated fromdopamine (DA) as an alcohol metabolite [13], and alsofrom 3,4-dihydroxyphenylacetic acid (DOPAC) byDOPAC reductase identified in the rat brain [14]. Inthe nervous system ROS are implicated in a variety ofpathological conditions including ischemia–reperfusion[15], Parkinson’s disease [16], Alzheimer disease[17,18], and amyotrophic lateral scleorosis [19,20]. Itthus appears relevant to clarify whether DOPE has aninherent role in neural cells subjected to oxidative stress.We here report that DOPE, with its unique effect on

catalase activity, may act as an endogenous cytoprotec-tive agent in dopaminergic neurons.

MATERIALS AND METHODS

Materials

PC12 cells were obtained from Dainippon Pharma-ceutical Company (Osaka, Japan). All culture media,horse serum (HS), fetal bovine serum (FBS), L-gluta-mine, and penicillin–streptomycin were purchased fromInvitrogen Corporation (Carlsbad, CA, USA). DOPEwas obtained from Cayman Chemical (Ann Arbor, MI,USA) DA, DOPAC, homovanillic acid (HVA), 3-methoxytyramine (3-MT), tyrosine, 3,4-dihydroxyphe-

Fig. 2. Effects of DOPE on H2O2-induced cell damage. PC12 cells werepretreated with various concentrations of DOPE for 6 h and exposed to150 AM H2O2. LDH leakage (A) and viability of the cells (B) weredetermined after 18 h. Each value represents the mean F SEM obtainedfrom six separate experiments. *p < .05, **p < .01, compared with theindicated group.

Fig. 1. Effects of DOPE on xanthine/xanthine oxidase (XA+XO)-induced cell damage. PC12 cells were pretreated with variousconcentrations of DOPE for 6 h and exposed to O2

- generated withxanthine (250 AM) and xanthine oxidase (5 mU/ml). Cells were furtherincubated for 18 h, and the viability of the cells was determined byMTT assay. Each value represents the mean F SEM obtained from fourseparate experiments.*p < .05, compared with control.

T. Hashimoto et al.556

nylalanine (L-DOPA), 3,4-dihydroxyphenylethylene gly-col (DPEG), and 3-methoxy-4-hydroxyphenylethanol(MOPE) were purchased from Sigma Chemical (St.Louis, MO, USA). 5,5-Dimethylpyrroline l-oxide(DMPO) was purchased from Labotec (Tokyo, Japan).3-(4,5-Dimethylthiazole-2-yl)-2,5-diphenyltetrazoliumbromide (MIT) and diethylenetriamine pentaacetic acid(DTPA) were from Dojindo Laboratory (Kumamoto,Japan). All other chemicals used were of analyticalgrade.

Cell cultures

Cells were seeded at a density of 5–10! 105 cells/cm2

in a 24-well plate and maintained in 500 Al of RPMImedium, supplemented with 10% HS, 5% FBS, 100 U/mlpenicillin/streptomycin, and 50 ng/ml nerve growth factor(NGF). Media were exchanged on the second day afterseeding the cells. Cells cultured for 4 days in the presenceof NGF were pretreated with DOPE or other relatedcompounds for 6 h in the experiments. For experimentsusing FeCl2, RPMI medium was exchanged to the con-ditioned medium containing 135 mM NaCl, 1.3 mMCaCl2, 5.36 mM KCl, 0.45 mM MgSO4, and 5.5 mMglucose, (pH 7.4).

Enzyme assays

Activities of lactate dehydrogenase (LDH) in themedium were measured as previously described [21].LDH leakage was calculated as the percentage of LDHin the medium versus total LDH activity in the cells.Catalase activity was determined by the method ofAebi [22]. Briefly, cell lysates, prepared by sonicationin ice-cold 50 mM sodium phosphate buffer (pH 7.0),were centrifuged at 13,000g for 10 min at 4jC, andthe supernatants were used for the activity assay.Decomposition of H2O2 at 25jC was monitored bythe decrease in absorbance at 240 nm using a Bio-Spec-1600 spectrophotometer (Shimadzu Ltd, Kyoto,Japan). Activity of glutathione peroxidase (GSH-Px)was measured according to the method of Paglia andValentine [23]. Activities of catalase and GSH-Px wereexpressed relative to the amount of protein in the cellextracts determined by the method of Lowry et al.[24].

Cell viability assay

Cell viability was evaluated by the reduction of MTTas described previously [25]. After 3 h incubation withMTT (0.5 mg/ml) at 37jC, cells were lysed in dimethylsulfoxide (DMSO). The formazan crystals formed afterreduction of MTT by mitochondrial dehydrogenaseswere measured spectrophotometrically at 550 nm withbackground subtraction at 650 nm.

Quantification of GSH and cysteine

Cellular GSH and cysteine were extracted with 0.1 Mperchloric acid, and their levels were determined by

Fig. 3. Effect of dopamine (DA) and related compounds on H2O2-induced cell damage. Each compound (50 AM) was added to theincubation medium 6 h before exposure to 150 AM H2O2. Cells werefurther incubated for 18 h, and their viability was determined by MTTassay. Each value represents the meanF SEM obtained from six to eightseparate experiments compared. *p < .05, with the indicated group.

Fig. 4. Effects of DOPE on cellular GSH levels. Cells pretreated with25 AM DOPE for 6 h were exposed to 150 AM H2O2. Cells were furtherincubated for 18 h, and the levels of GSH were measured by HPLC.Each value represents the mean F SEM obtained from four to nineseparate experiments. **p < .01, compared with control.

An endogenous cytoprotective agent, 3,4-dihydroxyphenylethanol 557

high-performance liquid chromatography (HPLC) usingthe Yanaco L-5000 (YANACO Ltd, Kyoto, Japan) withECD 100 electrochemical detector and Eicompac SC-5ODS (Eicom Ltd, Kyoto, Japan) according to the

method of Honegger et al. [26]. The mobile phase was0.1 M sodium phosphate buffer (pH 2.5) containing 5mg/l EDTA, 100 mg/l 1-octanesulfonic acid, and 1%methanol. The flow rate of the mobile phase was 0.5 ml/min and detector potential was set at 550 mV against theAg/AgCl electrode.

Measurement of hydroxyl radical

For detection of .OH, 0.1 mM H2O2, and 0.1 mMFeCl, were mixed in a test tube, and reduction of 50 AMp-nitroso-N,N-dimethylaniline (PNDA) at 440 nm wasmonitored [27]. The spin trapping technique usingelectron spin resonance (ESR) was performed by themethod of Ando et al. [28]. Hydroxyl radicals, generat-ed in a Fenton-type reaction with 1 mM H2O2 and 50AM Fe2+ chelated with 0.1 mM DTPA in 100 mMphosphate buffer (pH 7.4), were trapped with 1.0 mMDMPO to produce stable hydroxyl radical spin adducts(DMPO-OH). These adducts were monitored by ESRspectroscopy JEOL TE 200 (JEOL, Akishima, Japan).The amount of DMPO-OH spin was quantified exactly90 s after the addition of H2O2, and intensities of thesignals of DMPO-OH were evaluated by the peak of thesecond signal of the quartet of the DMPO-OH spinadducts relative to the intensity of Mn2+ used as aninternal standard.

Fig. 6. Effects of an inhibitor of catalase on H2O2-induced cell damage. Various concentrations of an inhibitor of catalase, 3-amino-1H-1,2,4-triazole (3-AT), and 25 AM DOPE were added to the incubation medium 6 h before exposure to 150 AM H2O2. Cells were furtherincubated for 18 h, and leakage of LDH was determined. Each value represents the mean F SEM obtained from 10 to 12 separateexperiments. *p < .05, **p < .01 compared with the indicated group.

Fig. 5. Effects of DOPE on catalase activity. Cells pretreated with 25 AMDOPE for 6 h were exposed to 150 AM H2O2. Catalase activity wasdetermined 18 h after exposure. Each value represents the meanF SEMobtained from six separate experiments. #p < .05, compared with thecells exposed to H2O2.

T. Hashimoto et al.558

Statistical analysis

Values were presented as means F SEM. Significanceof differences between groups was determined by Stu-dent’s t test or ANOVA followed by Dunnett’s multiplecomparison.

RESULTS

Effects of DOPE on xanthine/xanthine oxidase-inducedcell damage

When PC12 cells were exposed to O2- generated with

xanthine (250 AM) and increasing amounts of xanthineoxidase (1–55 mU/ml), a dose-dependent increase inLDH leakage, in addition to a decrease in cell viabilitywas demonstrated. Significant reduction in cell viabilitywas observed when 5 mU/ml xanthine oxidase wasapplied in the reaction (data not shown). As shown inFig. 1, however, this decline in cell viability was unaf-fected by pretreatment of the cells with various concen-trations of DOPE for 6 h before exposure to O2

-.

Effects of DOPE on H2O2-induced cell damage

A dose- and time-dependent increase in LDH leakagewas demonstrated in PC12 cells treated with H2O2. At aconcentration greater than 150 AM, LDH leakage wassignificantly increased compared with that of nontreatedcells. In line with these findings, MTT assay showed adose-dependent decrease in cell viability. Significantreduction in viability was observed in the cells treatedwith greater than 100 AM H2O2 (data not shown). Toevaluate the effect of DOPE on H2O2-induced cell dam-age, a concentration of 150 AM was applied in thesubsequent experiments.

As shown in Fig. 2A, pretreatment of the cells withDOPE dose-dependently suppressed H2O2-inducedleakage of LDH, and these findings were furtherverified by MTT assay (Fig. 2B). Treatment of thecells with DOPE alone did not affect LDH leakageand cell viability. These results clearly indicate thatDOPE has a protective role against H2O2-inducedinjury.

Effects of dopamine and related compounds onH2O2-induced cell damage

As DOPE exits as an endogenous metabolite ofdopamine (DA), the effects of DA and related com-pounds on H2O2-induced cell damage were investi-gated. As shown in Fig. 3, neither DA, DOPAC,HVA, 3-MT, nor 3,4-dihydroxyphenylethyleneglycol(DPEG), an analogous alcoholic metabolite of norepi-nephrine, affected the H2O2-induced decrease in cellviability.

Effects of DOPE on cellular GSH levels

We next examined the effect of DOPE on the levelof cellular GSH, a major endogenous antioxidant.While the level of GSH was significantly elevated inthe cells exposed to H2O2, pretreatment with DOPEhad no effect on the increased GSH level induced byH2O2 (Fig. 4). The level of cellular cysteine wassimilarly elevated by H2O2; however, the level wasunchanged in the cells pretreated with DOPE (data notshown).

Effects of DOPE on the activities of catalase and GSHperoxidase in the cells exposed to H2O2

H2O2 is decomposed by catalase or GSH-Px, theendogenous antioxidant enzyme. We therefore examined

Fig. 7. Effects of DOPE on H2O2/Fe2+-induced cell damage. (A) Cells

were exposed to various concentrations of H2O2 in the presence orabsence of 100 AM FeCl2 for 3 h. Results are expressed as means FSEM obtained from three to eight separate experiments. *p < .05,**p < .01, compared with 0 AM H2O2.

#p < .05, ##p < .01, comparedwith FeCl2 ("). (B) Cells were incubated in medium containing variousconcentrations of DOPE or 10 mM DMTU, 100 AM H2O2, and 100 AMFeCl2. Results are expressed as means F SEM obtained from three toeight separate experiments. *p < .05, **p < .01, compared with theindicated group.

An endogenous cytoprotective agent, 3,4-dihydroxyphenylethanol 559

the effect of DOPE on catalase and GSH-Px activitiesin PC12 cells. As shown in Fig. 5, the activity ofcatalase tended to increase in the cells exposed to H2O2.DOPE alone did not affect enzyme activity, whereaspretreatment with 25 AM DOPE significantly augment-ed activity in the cells exposed to H2O2 for 18 h. Onthe other hand, cellular GSH-Px activity as unaffectedby H2O2 with or without DOPE pretreatment (data notshown).

To verify the implication of elevated catalase activityin the protective effect of DOPE against H2O2-inducedcell damage, an inhibitor of catalase, 3-amino-lH-1,2,4-triazole (3-AT), was included in the medium. As shownin Fig. 6, the effect of DOPE on LDH leakage wascompletely abolished when cells were exposed to H2O2

in the presence of 5 or 10 mM 3-AT. These findingssuggest that increased activity of catalase underlies theprotective role of DOPE against H2O2-induced celldamage.

Effects of DOPE on H2O2/Fe2+-induced cell damage

When PC12 cells were exposed to various concen-trations of H2O2 in the presence of Fe2+, leakage of LDHinto the culture medium was markedly enhanced (Fig.7A). As shown in Fig. 7B, DOPE dose-dependentlysuppressed LDH leakage from cells exposed to H2O2

and Fe2+. At the highest concentration of DOPE (100 Am),

leakage of LDH was almost completely abolished. Alongwith these findings, 10 mM N,NV-dimethylthiourea(DMTU), a scavenger of .OH, also suppressed LDHleakage induced by H2O2 and Fe2+.

Effects of DA and related compounds onH2O2/Fe

2+-induced cell damage

To elucidate the specificity of the cytoprotective role ofDOPE against H2O2 and Fe2+-induced cell damage,effects of DA and related compounds were examined.As shown in Fig. 8, DA and L-DOPA at 100 AMsignificantly suppressed the cytotoxic effects of H2O2

and Fe2+. In this experiment, the effects of L-DOPAwereobserved during the incubation period of 3 h, much soonerthan in the previous studies (24–72 h) where toxic effectsof L-DOPA on undifferentiated PC12 cells or striatalneurons were reported [29,30]. L-DOPA or DA alone didnot elicit LDH leakage from the cells (data not shown).

Among the metabolites of DA, DOPAC and HVApartially rescued the cells from H2O2- and Fe2+-induceddamage, whereas tyrosine, 3-MT, and MOPE did notaffect the increase in LDH leakage.

Trapping activity of DOPE, DA, and related compoundsfor .OH

To further characterize the protective role ofDOPE, DA, and L-DOPA in H2O2- and -Fe2+-induced

Fig. 8. Effects of DA and related compounds on H2O2 /Fe2+-induced cell damage. Cells were incubated for 3 h in medium containing

100 AM of each compound, 100 AM H2O2, and 100 AM FeCl2. Values represent means F SEM obtained from three to seven separateexperiments. **p < .01, ***p < .001 compared with (—). ##p < 0.01, compared with control.

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cell damage, competition of these compounds in .OHtrapping activity of PNDA was explored. PNDArapidly reacts with .OH, and bleaching of PNDAcan be monitored spectrophotometrically [27]. Asshown in Fig. 9A, reaction of PNDA with .OH wassignificantly attenuated in the presence of DOPE, DA,or L-DOPA. On the other hand, DOPAC partiallysuppressed the reaction, while no effect was demon-strated for 3-MT (Fig. 9B). Addition of 25 mMDMTU to the reaction completely suppressed bleach-ing of PNDA (data not shown). These results corrob-orated the preceding findings, where DOPE, DA, andL-DOPA effectively ameliorated H2O2- and Fe2+-in-duced cell damage.

Lastly, direct scavenging activities of .OH for DOPE,L-DOPA, and DOPAC were determined using ESRspectroscopy. In the presence of L-DOPA or DOPAC,

Fig. 9. Effects of DOPE, DA, and related compounds on the reaction of.OH with PNDA. (A) Oxidation of PNDA by .OH was attenuated in thepresence of DOPE, DA, or L-DOPA. (B) DOPAC but not 3-MT partiallysuppressed the reaction. The decrease in absorbance of PNDA at 440 nmwas plotted. Each value represents the meanF SEM obtained from threeseparate experiments.

Fig. 10. ESR spectra demonstrating trapping of .OH by (A) L-DOPA,(B) DOPE, and (C) DOPAC. Typical spectra of a DMPO-OH spinadduct in the H2O2/FeCl2 system are shown in the presence of variousconcentrations of each compound.

An endogenous cytoprotective agent, 3,4-dihydroxyphenylethanol 561

signal intensities of DMPO-OH in the Fenton-type .OH-generating system were markedly attenuated. Comparedwith L-DOPA and DOPAC, however, limited scavengingactivity was demonstrated for DOPE (Fig. 10). Noapparent effect of DMSO was demonstrated up to 500AM (data not shown).

DISCUSSION

The present findings clearly demonstrate the protec-tive role of DOPE against oxidative stress-induced celldamage in dopaminergic neurons. Increased LDH leak-age and decreased viability in differentiated PC12 cellsexposed to H2O2 in the presence or absence of Fe2+ wassignificantly attenuated by pretreatment with DOPE.Although the endogenous DOPE identified in the sub-stantia nigra (f0.24 AM) [14] was less than the concen-trations applied in this study, higher cellular levels canbe attained by external intake of DOPE due to itsnonpolar nature. In contrast, the concentrations ofH2O2 used in this study were in the range of thosereached under pathophysiological conditions such asduring transient cerebral ischemia [31]. Among otherrelated phenolic compounds including DPEG, an analo-gous alcoholic metabolite of norepinephrine, DOPE wasspecific in that it significantly prevented H2O2-inducedcell damage.

The protective effect of DOPE against H2O2-inducedcell injury was attributed to the augmented activity ofcatalase. When the activities of catalase and GSH-Pxthat decompose H2O2 were determined in the cellspretreated with DOPE, increased activity of catalasewas observed without any effect on GSH-Px activity.As for the major cellular antioxidant and its precursor,the levels of GSH and cysteine elevated by H2O2 werenot affected by DOPE pretreatment. The effect ofDOPE was completely abolished when an inhibitor ofcatalase 3-AT was included in the medium, suggestingthat the effect of DOPE on H2O2-induced cell injury ismediated by increased activity of catalase. It wasreported that the H2O2-induced decline in cell viabilitywas accompanied by reduced activities of catalase andGSH-Px in undifferentiated PC1 2 cells [32]. Thepresent findings are in contrast with those of a previousstudy, in that catalase activity was instead elevated inPC12 cells exposed to H2O2. Possible explanations forthese conflicting results lie in the use of differentiatedor undifferentiated cells applied in these experiments.We used differentiated PC12 cells treated with NGF,and NGF is known to protect PC12 cells from oxidativestress by increasing the activity of cellular catalase [33].Accordingly, the cellular response to H2O2 may bealtered in differentiated PC12 cells cultured in thepresence of NGF.

The molecular mechanisms underlying the augmentedactivity of catalase in H2O2-exposed cells pretreated withDOPE remain unclear. A recent study using culturedastrocytes demonstrated that H2O2 led to an increase incatalase mRNA levels. Transfection studies with a re-porter plasmid containing an upstream region of thecatalase gene suggested the involvement of posttranscrip-tional regulation in this process [34]. We also observedincreased levels of catalase mRNA in PC12 cells ex-posed to H2O2. However, the level of catalase mRNAwas not affected by pretreatment with DOPE (data notshown). The effect of DOPE may therefore be mediatedby posttranscriptional or posttranslational modification ofthe enzyme. In fact, the protective effect of DOPEagainst H2O2-induced cell injury was completely abol-ished when an inhibitor of protein synthesis, cyclohex-imide, was included in the medium (data not shown). Thecrucial question yet left unanswered is the precise role ofDOPE in the regulation of cellular catalase activity.

The cytoprotective role of DOPE was also shown inthe cells exposed to H2O2 and Fe2+. In the presence ofFe2+, .OH generated by the Fenton reaction appeared toelicit higher levels of LDH leakage than H2O2 itself. Incontrast with the role of DOPE against H2O2-inducedcell damage, the effect of DOPE against .OH generatedby the Fenton reaction was not specific. Among otherDA-related compounds, L-DOPA and DA similarly pro-tected the cells against H2O2 and Fe2+-mediated injury.Complex effects of L-DOPA and DA on H2O2 and Fe2+-mediated injury were previously reported [35]. Thepresent experimental findings suggest that reaction ofPNDA with .OH is significantly attenuated in the pres-ence of not only DOPE, but also L-DOPA and DA. Theprotective roles of these compounds in H2O2 and Fe2+–induced cell damage thus appeared to depend on theircommon catechol moieties. In fact, catechol wasreported to form a complex with iron at around pH7.4, which may hinder the reaction with H2O2 togenerate .OH [36]. On the other hand, ESR spin trappingmethods denoted that DOPE, unlike L-DOPA or DA,had limited scavenging activity for .OH. It is known thatthere are two types of antioxidants that act against .OH.One suppresses their generation, and the other scavenges.OH. In light of the present findings, it can be postulatedthat DOPE may sequester iron from reacting with H2O2

to generate .OH, rather than directly scavenging .OH.The functional role of DOPE against .OH turned out tobe distinct from such catechol-containing compounds asL-DOPA and DA.

CONCLUSION

DOPE may serve as a cytoprotective agent in thecellular defense mechanism against various oxidative

T. Hashimoto et al.562

stimuli. Due to the unique effects of DOPE as anendogenous metabolite of DA, the present study mayaid the development of new therapeutic interventionstrategies that can halt various pathological conditionslinked to oxidative stress.

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ABBREVIATIONS

3-AT—3-amino-1H-1,2,4-triazoleDA—dopamineDMPO—5,5-dimethylpyrroline l-oxideDMTU—N,N V-dimethylthiourea

An endogenous cytoprotective agent, 3,4-dihydroxyphenylethanol 563

DOPAC—3,4-dihydroxyphenylacetic acidDOPE—3,4-dihydroxyphenylethanolDPEG—3,4-dihydroxyphenylethylene glycolGSH-Px—glutathione peroxidaseHVA—homovanillic acidLDH—lactate dehydrogenaseMOPE—3methoxy-4-hydroxyphenylethanol

3-MT—3-methoxytyramineMTT—3(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazo-lium bromideNGF—nerve growth factorPNDA—p-nitroso-N,N-dimethylanilineROS—reactive oxygen speciesSOD—superoxide dismutase

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