[CANCER RESEARCH54, 6407-6412, December 15, 1994J

peptide ligands, its oxidation potential can be lowered to as low as0.79 V, which allows it to be oxidized by H202 (20).

In an attempt to provide evidence of Ni(II)-induced formation ofoxidants in intact cells, we have used the dye 2',7'dichlorofluorescinthat fluorescences when it is oxidized (21). It was shown previouslythat short (1—6h) treatment of intact cultured cells with water-soluble(NiC12) and water-insoluble (Ni3S2) nickel compounds resulted in anincreased intracellular fluorescence of this dye (22, 23).

If oxidative stress is involved in the molecular response to Ni(II), thenit follows that the nickel-resistant cells might also be more resistant thanthe parental cell line to oxidative stress. Here we show that nickelresistant B200 cells tolerate H202 and menadione better than wild-type3D cells. Although nickel caused glutathione levels to decrease in bothcell lines, the parental cells were more sensitive to this depletion. Themore efficient depletion ofglutathione in B200 cells with BSO comparedto 3T3 cells suggested a higher rate of glutathione turnover in thenickel-resistant cells. Cellular responsiveness to oxidative stress can beassessed with mobility shift assays using transcription factors, such asNFkB and AP-1, that are highly sensitive to oxidative stress (24, 25). Wedemonstrate lower levels of initial binding but more inducible binding ofboth transcription factors NFkB and AP-1 following oxidative stress inB200 cells compared to 3D cells. These data provide further evidencethat the nickel-resistant cells have acquired alterations in theirresponsiveness to oxidative stress.

MATERIALS AND METHODS

Cell Cultures. Nickel-resistant B200 cells were derived from BALB/c-3T3mouse cells by continuous selection in media containing increasing concentrations of NiC12 (26). The cells were routinely maintained in a-MEM supplemented with 10% fetal bovine serum (Sigma Chemical Co., St. Louis, MO),100 units penicillin, 100 g.@g/mlstreptomycin (GIBCO-BRL, Gaithersburg,MD), and 2 mM glutamine.

Determination ofCytotoxiclty. Cell survival was measured by plating 300cells into 60-mm dishes and exposing them to various concentrations of NiC12,H202, BSO, and menadione. The duration of treatments and concentrationsutilized are outlined in the figure legends. After 7—9days of incubation at37°C, colonies were fixed with methanol, stained with crystal violet, and

counted.fluorescent Measurement of Intracellular Oxidants. Cells were pre

treated with NiC12(0.3 mM)or H202 (0.2 @M)and then loaded with 50 @.tMDCF-dAC for an additional 30 mm at 37°C.Cells were washed twice withice-cold PBS, scrapped from the plate, and resuspended at 2 X 10@cells/ml for

fluorescence measurement, as described previously (22, 23).Flow Cytometry. Cell cycle analyses were carried out on EPIC-751 flow

cytometer (Coulter Corp., Hialeah, FL) using propidium iodine (excitationwavelength, 488 nm; emission wavelength, 530 nm) as the DNA fluorescentstain. Cells were collected for analysis in PBS and then fixed with 0.1%paraformaldehyde and 0.1% Triton X-100 in this same buffer. Cells werestained with 50 p@g/mlpropidium iodine, washed in PBS, and resuspended at106 cells/mI

Measurement of Glutathione Levels by a HPLC Method. HPLC anal

ysis of GSH was based on the method published by Lindroth and Mopper (29)with modifications. Cells were plated in duplicate in 100-mm dishes at adensity of 7 X i0@cells/dish. After incubation with various concentrations ofNiCI2, H202, and 0.1 mM BSO, cells were washed with saline A, and 500 ,tlof 3% perchloric acid (Aldrich Chemical Co., Milwaukee, WI) was added toeach dish. Cells were scraped from the plate, and the precipitated proteins werecollected at 4°Cusing an Eppendorf microcentrifuge at its maximal setting.

6407

Altered Oxidative Stress Responses in Nickel-resistant Mammalian Cells'

Konstantin Salnikow, Mm Gao, Vika Voitkun, Xi Huang, and Max Costa2

Nelson Institute of Environmental Medicine (K. S., M. G., V. V., X. H., M. C] and The Kaplan Comprehensive Cancer Center (M. C.]. New York University Medical Center,New York@New York 10016

ABSTRACT

BALB 3T3 cells exposed to NiCl2acquired resistance to concentrationsas high as 200 @siwand retain resistance for many generations in theabsence of nickeL This resistance was not due to alterations in uptake or

to metallothionein overexpression. The nickel-resistant B200 cell line wasfound to also exhibit cress-resistance to hydrogen peroxide and menadione. These nickel-resistant cells had 1.8 times higher basal levels ofglutathioae compared to wild-type cells. Studies with the glutathionesynthesis Inhibitor buthionine sulfoxhnine showed that while glutathioneturnover was more rapid In the nickel-resistant cells, its depletion followhag NICI2 treatment of the parental BALB 3T3 cell line was greater thanIn the nickel-resistant B200 cells. The reduced level of binding of NFkBand AP-1 transcription factors to their DNA consensus sequences in B200cells compared to wild-type cells, and their more reactive response following treatment of resistant cells with H2O2 or buthionine sulfoximine,strengthens the hypothesis that nickel resistance is closely allied to oxidafive stress responses.

INTRODUCTION

Ni3 is an important environmental toxicant which has been implicated in the etiology of nasal and lung cancers in humans (1, 2).Certain nickel compounds, such as Ni3S2, are potent carcinogens atmany sites in experimental animals (3). Nickel compounds are directacting carcinogens since they transform cultured cells in vitro (4), butthey are also synergistic and potentiate the effects of other genotoxiccarcinogens (5). Nickel compounds are potent carcinogens but ratherweak mutagens in most mammalian cell systems (6—9).They enhancethe formation of ROIs in cells, and these radical species are ostensiblyimportant initiators of cellular transformation (10, 11). However, mostof the damage produced by nickel compounds occurs in geneticallyinactive heterochromatic regions (4). This observation may explainwhy potently carcinogenic and genotoxic nickel compounds are notmutagenic.

There are several lines of evidence supporting the idea that Ni(II)induces oxidative stress. First is the finding that Ni(II) enhancesoxidation of all the DNA bases in vitro (12—15). Ni(II) alsoenhances lipid peroxidation in vivo (2, 16—19).Although Ni(II) isnot as active as iron or copper in Fenton chemistry, it neverthelessis thought to generate very low levels of ROl. To our knowledgethere is no direct data demonstrating the formation of higheroxidation states of nickel in reactions of Ni(II) with DNA andproteins; however, redox cycling of nickel ions has been proposedas a possible mechanism for ROl (10, 11). The oxidation potentialof NiII@ is 1.09 V, which is too high to be catalyzed in mostbiological systems. However, following binding of NiII@to certain

Received 8/4/94; accepted 10/19/94.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby marked advertisement in accordance with18U.S.C.Section1734solelyto indicatethisfact.

I This work was supported from NIH Grants ES 00260, ES 04895, ES 04715, and ES

05512,andNationalCancerInstituteGrantCA 16087.2To whom all requests for reprints should be addressed, at Nelson Institute of

Environmental Medicine and The Kaplan Comprehensive Cancer Center, New YorkUniversity Medical Center, 550 First Avenue, New York, NY 10016.

3The abbreviation used are: Ni, nickel; ROl, reactive oxygen intermediate; BSO,buthionine sulfoximine; DCF-dAC, dichlorofluorescin diacetate; GSH, glutathione; MCB,monochlorobimane.

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TreatmentGSH(% control)3'@aGSH

(% control)B200―Nia2

(0.5mM)59.5 ±5.586.8 ±12.7NiCl2

(1.0 miu)31.6 ±2.963.6 ±0.7NiCl2(1.0 msi) + H202 (50 ELM)37.7 ±5.437.2 ±4.2H202(50 mM)92.2 ±12.179.6 ±0.7BS0

(0.1 mM)3 h78.3 ±3.348.2 ±2.6BSo(0.1 mM)6 h20.6 ±4.010.5 ±1.9a

Each value is the mean of four determinations ±SD.

0 20 40 60Menadione Concentration (riM)

ADAPTATION TO NICKEL-INDUCEDOXIDATIVE STRESS

The supernatants were stored at —20°Cfor further analysis. Protein concentration in cell pellets was determined using the Bio-Rad protein detection kit(Bio-Rad Laboratories, Hercules, CA). Immediately prior to performing theassay, the supernatants were thawed, neutralized with 2N KOH, and reactedwith o-phtaldialdehyde. Total glutathione (GSH + oxidized GSH) levels were

determined with a Waters HPLC system (Waters Chromatography, Inc., Milford, MA) equipped with gradient capability and a fluorescence detector. TheHPLC column used was an Adsorbsphere OPA HR (4.6 X 150 mm) (AlltechDeerfield, IL). A 50-s.d aliquot of the derivatized sample was injected into thecolumn and a mobile phase A [100 mMNa acetate (pH 5.6)-3% 2-isopropanol]was maintained for 10 mlii, followed by a linear gradient from 0 to 60% ofsolvent B (100% methanol-l.5% of 2-isoproponol) for 30 mm. Ser-Phen(Sigma) was used as internal standard for derivatization. The Millennium 2010

program (Waters Chromatography, Inc.) was used to process the data.

Measurement of Reduced Glutathione Using Monocifiorobimane.

MCB (Molecular Probes, Inc. Eugene, OR) was used as a specific stain for themeasurement of the reduced form of glutathione (26). 3T3 and B200 cells werecultured in flat-bottommed 24-well plates (Falcon, Lincoln Park, NJ). After 24h, the cells were treated with various concentrations of NiCI2,H202, and BSO.MCB (40 @M)was added to cells and they were incubated for an additional 15mm at room temperature. The fluorescence was detected at 460 am in responseto excitation at 395 am with a Cytofluor 2300 plate reader (Millipore, Inc.).

Mobifity Shift Assay. Nuclear extracts were prepared using a modificationof the procedure of Dignam et al. (28). Double stranded consensus oligonucleotide sequences for AP-1, Oct-i, and NFkB were purchased from Promega(Madison, WI) and were end labeled using [‘y-32PJATPand T4 polynucleotidekinase. The labeled oligonucleotides were incubated with 2—4 @gof nuclearextract, and the mixture was subjected to electrophoresis in a 6% nondenaturing polyacrylamide gel. The gel was dried and exposed to X-OMAT AR 2 film

(Eastman Kodak Co., Rochester, NY).Northern Blot. Twenty @tgof total RNA was separated in a 1% formal

dehyde agarose gel and then transferred to a nylon Nytran membrane(Schleicher and Schuell, Keene, NH) in 20X SSC (1 X SSC = 3 M sodiumchloride, 0.3 M sodium citrate, pH 7.0) buffer. Hybridization was carried out asrecommended by the manufacturer. Ethidium bromide staining of the gel wasused to assess RNA loading.

Statistical Evaluation of the Data. Data was evaluated for statisticalsignificance using Macintosh 8.0 Minitab (Student's t test, SD, etc.).

RESULTS

Nickel-resistant CeHs Exhibit Cross-resistance to Menadioneand Hydrogen Peroxide. The B200 cells, selected for resistance to200 @.LMNiCl2, were found to also be more resistant to menadione andH202. Fig. 1 shows survival curves obtained for 3T3 and B200 cellsexposed to different concentrations of menadione for 2 h. The menadione 50% lethal concentration for the parental cell line is 32 p.M,

Survival 60(% of Control)

40

Survival

(% of Control)

20 40 60 80 100H202 Concentration (@M)

Fig. 2. Survival of wild-type and nickel-resistant cells exposed to H202. Cell cultureswere treated with the indicated concentrations of hydrogen peroxide for 2 h at 37°C.Points, mean of six determinations; bars, SD. At 20 @xMH202, resistant cells (0) differedfrom control in a statistically significant fashion (P < 0.05 student's, t test). At higherconcentrations, the statistical significance was greater (P < 0.001, student's t test).

Table 1 GSH levels in wild-type3T3 and in nickel-resistant B200 cells followingexposure to NiCl2. H202, and BSO

Absolute values of GSH were 6.4 ±1.6 pg/mg of protein in 3T3 cells and 11.9 ±1.3g.tg/mg of protein in B200 cells.

whereas it was 53 p@Mfor the nickel-resistant B200 cells. Fig. 2 showscell survival curves obtained for these cells exposed to H202 for 2 h.The 50% lethal concentration for the parental cell line was 47 @Mbutincreased to 84 p@Mfor the nickel-resistant cells.

Depletion of Cellular GSH by NiC12. Since glutathione levelsmay depend on the proliferative status of a cell population, wecontrolled for differences in proliferation by seeding and collectingcells at specific densities. Glutathione levels were measured by twodifferent methods. An HPLC method determines total glutathione,because discrimination between reduced and oxidized forms of glu

tathione after derivatization with o-phtaldialdehyde under reducingconditions are problematic (29). It is important to note, however, thatthe level of oxidized glutathione is low in living cells because oxidized GSH is actively transported out of cells (30). Thus, totalglutathione measurements approximate measurements of reduced glutathione. Glutathione levels as measured by HPLC in B200 Ni(II)resistant cells were 1.8 times higher than wild-type cells. The comparative changes in GSH levels in 3T3 and B200 cells treated with

NiC12,BSO, or H202 for 6 h are presented in Table 1. Treatment ofcells with 0.5 or 1 mM NiCl2 for 6 h resulted in a concentrationdependent depletion of GSH levels in both cell lines. Almost one-halfof GSH was depleted in 3T3 cells following exposure to 0.5 mMNiCl2for 6 h. In the nickel-resistant B200 cells, however, the levels of GSHwere higher following this treatment. Treatment of 3D cells with 50p.M of H202 did not alter GSH levels, whereas H202 treatment was

slightly more efficient in depleting GSH in the nickel-resistant B200

80 cells.BSO is a potent inhibitor of ‘y-glutamylcysteinesynthetase, and has

been used to study GSH turnover (31). BSO treatment resulted inmore pronounced depletion of GSH in B200 cells after 3 and 6 hcompared to wild-type cells (Table 1), suggesting that the turnover

80

20

Fig. 1. Survival of wild-type and nickel-resistant cells to menadione. Cells wereexposed to the indicated concentrations of menadione for 2 h at 37°C.Points, mean for atleast 6 determinations; bars, SD. The B200 cells (0) differed from wild-type cells (U) ina statistically significant manner at 30 @LMmenadione (P@ 0.01, student's t test).

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Table2 ToxicityofNiCl2and menadionefollowingdepletionofGSHCellswere incubated in the absence or presence of 0.1 mt@iBSO for 6 h prior to a5-hincubation

with NiCl2 or menadione. Each value is the mean of triplicatedeterminationsintwo independent experiments. Basal levels of GSH in 3T3 and B200 cells are giveninthelegendto Table1.Survival

(%control)Treatment

3T3B200NiC12(100 p.M) 77.6 ±6.1― 90.2 ±3.9°NiC12(350@ 493 ±4.0 92.1 ±2.1BSO

71.3±5.187.8±4.1BSO+ Ni (100 @.u@i) 36.6 ±3.8 68.8 ±5.2BSO+ Ni (350 @xsi) 20.7±4.9 61.9±4.8

Met' (30 tiM) 42.1 ±4.1 92.8 ±3.3Me(40 @.LM) 26.7 ±6.2 68.1 ±6.5BSO

+ Me(30 j.LM) 0.5±0.9 14.6±2.9BSO+ Me (40 @isi) 0 6.3 ±2.2a

Each value is the mean of six determinations ±SD.bMe, Menadione.

TreatmentSurvival

(%control)3T3B200Control100100GSH

(2.5mM)91.8 ±4.781.2 ±1.7NiC12(2.5 mM)4.2 ±0.864.8 ±6.1NiCl2(2.5 mM)+ GSH (2.5 msi)7.8 ±1.854.9 ±2.1Complexa11.8

±2.652.4 ±4.9

ADAPTATION TO NICKEL-INDUCED OXIDATIVE STRESS

tive conditions as determined by flow cytometry. The transcriptionfactors AP-1 and NFkB were found to bind less to consensussequences in B200 cells compared to 3T3 cells (Figs. 4 and 5).When the same nuclear extracts were used to examine the bindingof Oct- 1, a transcription factor that does not depend on redox statusof the cell, binding to its consensus sequence was found to besimilar in both cell lines (Fig. 6). Since the AP-1 transcriptionfactor usually is heterodimeric and composed of two nuclear proteins, fos and fun, we also measured the basal level of expressionof these two proto-oncogenes using Northern analysis (Fig. 7). Thelow levels of expression of c-los and c-jun in the nickel-resistantcells are in good agreement with the observed low levels of AP-1binding in these cells. Oxidative stresses (H202 or BSO) inducedthese transcription factors more in the B200 nickel-resistant cellscompared to wild-type cells (Fig. 4, Lanes 6, 7, and 8).

DISCUSSION

The interactions of Ni(II) with proteins and other intracellularmolecules represents a likely pathway for its cytotoxic effects. Ni(II)@can form square planar, tetrahedral, or octahedral complexes depending on the ligand(s) and the environment. Peptide ligands containingsuithydryl groups, imidazole nitrogens, and carboxylate oxygens offeropportunities for such coordination (32). When Ni(II)@ is bound tosuch ligands, its oxidation potential is lowered such that it can beoxidized to Ni(III)@or to a Ni(IV)@oxocomplex, and it can participate in Fenton-derived oxygen radicals (32).

The increased resistance of B200 cells to oxidative stress providesfurther evidence that Ni(II) toxicity may be mediated by ROl, whichrepresents, however, only one level of Ni(II)-induced oxidative stress.Another level of Ni(II)-induced oxidative stress is the depletion ofintracellular free radical scavengers such as GSH. GSH can be depletedby eitheroxygenradicalsor by thedirectinteractionof Ni(II)with GSH to create nickel-GSH complexes. A third possible level foroxidative stress involves Ni(II) inhibition of the transcriptional ortranslational antioxidant response, which includes suppression of theactivity of antioxidant enzymes such as catalase (18, 33). The involvement of GSH metabolic enzymes is also likely to be an important partof the observed cellular resistance to nickel in B200 cells.

We have shown previously that exposure of Chinese hamster ovarycells to soluble and insoluble nickel salts caused production of intracellular oxidants as detected by an increase in DCF-dAC fluorescence(22, 23). Here we show an increase of DCF-dAC fluorescence inmouse 3D cells treated with 0.3 m@ NiCl2. This increased level of

fluorescence(Fold Control)

Fig. 3. Oxidant response to NiCl2 or H202. Oxidant levels in 3T3 and B200 cells weredetected by a dichlorofluorescein assay as described in “Materialsand Methods.―Cellswere pretreated with NiC12(0.3 mM)or H202 (0.2 nm@)and then loaded with 50 gu@iofdichlorofluorescin diacetate for an additional 30 mm. Columns, mean of two independentexperiments.

6409

Table3 EffectofextracellularGSHon the toxicityofNiCI2GSHand/orNiCl2wereaddedto themediafor2 h. Basallevelsof GSHin 3T3and

B200 cells are given in the legend to Table 1.

a A complex of NiC12and GSH in equimolar concentration (2.5 msi) was added to theextracellular media cells. Each value is the mean of six determinations ±SD.

rate for GSH is higher in B200 cells. To verify that the oxidized formof glutathione did not contribute significantly to the GSH levelsmeasured by HPLC, these experiments were repeated with monochlorobimane. The changes in GSH levels as measured with MCB were inagreement with the results obtained with the HPLC method (data notshown).

Effect of GSH Levels on Survival of 3T3 and B200 Cells. Table2 shows the survival of 3T3 and B200 cells pretreated with 0.1 mrs@BSO for 6 h and subsequently exposed to 100 or 350 p.Mof NiCl2,respectively. Although nickel treatment was more toxic followingBSO treatment, the survival of the nickel-resistant B200 cells wasdecreased to a lesser extent than that of the parental cells treated withBSO and NiC12. The depletion of GSH also resulted in a higher levelof toxicity for menadione in 3T3 cells, whereas this combination wasless toxic in nickel-resistant cells. This suggests that GSH is alsoinvolved in protection against menadione toxicity.

To evaluate the involvement of extracellular GSH in cellular resistance to Ni(II), a GSH-Ni(II) complex was synthesized. The addition of GSH to the extracellular media slightly increased the viabilityof the 3T3 cells but decreased the viability of the B200 cells. Theaddition of the nickel-glutathione complex to the cells resulted inhigher survival of 3T3 cells but did not affect the survival of B200cells. This shows that extracellular glutathione does not significantlycontribute to nickel toxicity (Table 3).

Level of Oxidants in Wild-typo and Nickel-resistant Cells. Theendogenous levels of intracellular oxidants measured by DCF-dACwere comparable in both cell lines. Following NiC12 treatment,3T3 cells showed a significant increase in fluorescence, whereasB200 cells did not (Fig. 3). Hydrogen peroxide (0.2 mM) slightlyincreased the fluorescence of DCF-dAC in 3T3 cells but not inB200 cells (Fig. 3).

Comparison of Nuclear Extract Binding to AP-1 and NFkBConsensus Sequences. For the mobility shift assays, nuclear extracts were prepared from cells maintained in the same prolifera

Ni02@@ mM) H@O2@ 0.2mM)

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ADAPTATION TO NICKEL-INDUCED OXIDATIVE STRESS

Fig. 4. AP-1 DNA-binding activity in wild-type and nickel-resistant cells.Wild-type or nickel-resistant B200 cells were treated with NiCl2 6 h, H2022 h, and BSO 6 h using the concentrations indicated in the figure. AP-1

DNA-binding activity was determined as described in “MaterialsandMethods.

oxygen radicals resulted in the depletion of GSH, an importantcellular antioxidant.

Cross-resistance to assorted oxidative stresses has been previouslydemonstrated for other cells that were selected for resistance to heavymetal compounds. It was shown that Chinese hamster V79 cells selectedfor resistance to high concentrations (up to 220 @M)of cadmium werealso resistant to H202 (34). Chinese hamster fibroblast HA-i selected for

resistance to hydrogen peroxide also acquired resistance to cisplatin (35),cadmium(fl), and mercury(ll) (36). The resistance of murine leukemiacells to cisplatinum was shown to correlate with increased levels of GSHand modulation of GSH metabolism in these cells, as well as changes incomponents of the cellular antioxidant defense systems (37). Our study is

the first demonstration that nickel can also induce cross-resistance tooxidative stress.

Fig.5. NFkB DNA-bindingactivityin wildtype or nickel-resistant cells. Cells were treated for6 hwithNiCl2orBSOandfor2hwithH2O2usingthe concentration shown in the figure. NFkB DNAbinding activity was determined as described in“Materialsand Methods.―

1 234 5678910

123456 7 8 9 101112136410

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.@:@

00c@) 00

ADAPTATION TO NICKEL-INDUCEDOXIDATWE STRESS

of binding of these transcription factors, suggesting that higher levelsof GSH in B200 cells may be the reason for their lower bindingactivity. We have found that the GSH level in B200 cells was 1.8times higher than in 3T3 cells. The dependence of AP-1 binding onOSH levels was shown previously in hepatoma HepG2 cells (39).When the level of GSH was decreased in B200 cells by BSO treat

A c-Fos c-Jun

4

@.

i

000

c'Ji:@@c@)0

c'J@2cy)

.,.1@

!i@@P

B c-Fos c-Jun123Fig. 6. Analysis of Oct-i DNA-binding in wild-type and nickel-resistant cells. Oct-i

DNA-binding activity was determined as described in “Materialsand Methods.―Lane 1,freeprobe;Lane2, 3T3cells;Lane3, B200cells.

At physiological pH, Ni(II) can bind to GSH, creating stablecomplexes (38). In the experiments described, the depletion of GSHby Ni(II) markedly enhanced the sensitivity of 3T3 cells to oxidantssuch as hydrogen peroxide and menadione. We have also comparedthe depletion of GSH levels following the exposure of 3T3 cells toequitoxic concentrations of hydrogen peroxide and NiCl2. Exposureof 3T3 cells to hydrogen peroxide only slightly lowered GSH levels,but Ni(II) was more efficient in the depletion of GSH. Nickel-GSHcomplexes may be oxidatively active, contributing to the depletion ofOSH. In nickel-resistant B200 cells, GSH was less readily depleted byNi(II), and experiments with BSO showed GSH to be more rapidlyturned over than in 3T3 cells.

The exposure of mammalian cells to a variety of oxidants results inthe induction of an elaborate antioxidant defense system, including theexpression of genes encoding enzymes that detoxify ROl. EnhancedAP-1 and NFkB transcriptional factor binding to consensus DNAsequences signals oxidative stress. These transcription factors areregulated by distinct pathways (24). NFkB behaves as a primaryresponse factor, whereas AP-1 displays more complex regulation. Theactivation of AP-1 can be achieved at both the transcriptional andpost-transcriptional levels. This activation is responsible for the second round of response by coordination of the expression of differentgenes (24, 39, 40). At the post-transcriptional level, AP-1-bindingactivity is mediated by nuclear factor(s) sensitive to reduction andoxidation (41) and phosphorylation (42).

The depletion of GSH as a result of BSO treatment slightly decreased AP-1 and NFkB binding in 3T3 cells. The depletion of GSHlevels in B200 cells by BSO, however, resulted in a striking increase

Fig. 7. Levels of c-fos or c-jun mRNA in wild-type and nickel-resistant cells. (A) Basallevels of c-fos or c-jun mRNA. (B) Ethidium bromide staining oftotal RNA to standardizethe laneloading.

6411

‘4@4

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ADAPTATION TO NICKEL-INDUCED OXIDATIVE STRESS

ment, AP-1-binding activity rose sharply. Thus, in spite of efficientdepletion of GSH by Ni(II), no increase of transcription factor-bindingactivity was observed as the result of Ni(II) treatment. Ni(II) maydirectly prevent binding oftranscriptional factors to DNA by polypeptide misfolding. The decrease in binding of proteins to DNA in thepresence of Ni(II) has been reported previously (43).

Collectively, these data demonstrate complex alterations in oxidative stress responses in nickel-resistant cells. These cells exhibit ahigher basal level of GSH but more rapid GSH turnover. They alsoshow lower basal responses of DCF-dAC fluorescence and oxidativetranscription factor binding than 3T3 cells but have a more efficientinducible response to oxidative stress.

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