Cytoplasmic Metallothionein Overexpression Protects NIH 3T3 Cells ...

THEJOURNAL OF BIOLOGICAL CHEMISTRY 0 1994 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 269, No. 21, Issue of May 27, pp. 15238-15243, 1994 Printed in U.S.A.

Cytoplasmic Metallothionein Overexpression Protects NIH 3T3 Cells from tert-Butyl Hydroperoxide Toxicity* (Received forpublication, January 25, 1994)

Margaret A. Schwa&§, John S . Lazon, Jack C. Yalowichn, Ian Reynoldsnll, Valerian E. Kagan**, Vladimir Tyurin**$$,Young-Myeong Kim§§,Simon C. Watkinsm, and Bruce R. Pita From the Wepartments of Pharmacology, $Anesthesiology and Critical Care Medicine, **Environmental and Occupational Health, $$Surgery, and llllcell Biology and Physiology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261

Metallothioneins (MT) are ubiquitous low molecular weight metal-binding proteins that may act as antioxidants. We examined the sensitivity ofNIH3T3 cells transfected with a plasmid containing mouse metallothionein-I gene (NIH3T3/MT) tothe membrane permeant oxidant, tert-butyl hydroperoxide (tBH). NIH3T3/MT cells had a 4-fold increase in intracellular metallothionein as compared to cells transfected with a plasmid containing an inverted gene (NIH3T3/TM). Newly expressed metallothionein appeared to be localized to the cytoplasm as determined by immunofluorescence and confocal microscopy. NIH3T3/MTcells were6 times more resistant than NIH3T3/TM cells to the cytotoxic effects of tBH. The antioxidant activity of NIH3T3/MT cellswas greater than NIH3T3Ll“cells, since exposure to tBH resulted in significantly less: (a) thiobarbituric acid-reactive substances and (b)fluorescence after loading cells with the oxidant-sensitivedye, 2’7’-dichlorodihydrofluorescein diacetate. Furthermore, homogenates of NIH3T3/MT cells were more capable of scavenging in vitro generated phenoxyl radicals as quantified by electron spin resonance detection. In contrast, overexpression of cytoplasmic MT did not protect against tBH-induced DNA damage, suggesting that subcellular location ofMT is important for its function and thatDNA damage is not a key determinant of cytotoxicity. These data provide direct support for an antioxidant role for MT, since physiologically relevant elevations in cytoplasmic MT interfere with tBH-induced cytotoxic peroxidation.

pate indefense against intracellularoxidants. Support for this possibility is derived from observations that: (a)MT genes are transcriptionally activated in cells (3) and tissues (4) during oxidative stress, and ( b ) in vitro, MT can scavenge free hydroxyl radicals and superoxide anions produced by xanthinexanthine oxidase reaction (51,as well as inhibit degradation of isolated DNA by hydroxyl radicals (6). Although these latter(5,6) observations are supportive of an antioxidant function forMT, its role as an antioxidant in intact cells remains unclear. Overexpression of MT with heavy metals or cytokines produces resistance to oxidant injury caused by ionizing radiation (7), hydrogen (8) and organic peroxides (91, hyperoxia (lo), and carbon tetrachloride (11).Heavy metals and cytokines are promiscuous transcriptional activators, however, and may affect other cellular proteins in addition to MT. For instance, it appears that glutathione and notMT accounts for heavy metal-associated resistance of V79 cells to cytotoxicity of hydrogen peroxide (12). Recently, it was found that expression of yeast or simian metallothionein proteins, in the presence of copper, suppressed oxidant injury inSaccharomyces cerevisiae mutants lacking copper-zinc superoxide dismutase (13). The protection afforded these mutantsby MT was limited to injury caused by the accumulation offree radicals when the yeast were grown onthe non-fermentable carbon source,lactate, and it was reported to not affect their sensitivity t o high oxygen or paraquat. Furthermore, the antioxidant effect of MT appeared limited to theCu(1)-speciated form of MT. Thus, we examined the potential antioxidant properties of MT by elevating cytoplasmic MT to a physiologically level via gene transfer in higher eukaryotes that have functionalCuZn-superoxide dismutase. We transferred themouse MT-I gene intoNIH 3T3 cells and Metallothioneins (MT)’ are low molecular mass ( 4 ‘ kDa), cysteine-rich (30 mol %) proteins for which the biological func- noted a 4-fold enhancement of MT localized to the cytoplasm. tions remain ill defined (1).One likely role for MT involves The resultingNIH 3T3 cells were almost10-fold more resistant intracellular metal ion homeostasis, since transcription of MT than control cells t o the cytotoxic effects of tert-butyl hydroperkill is induced by heavy metals suchas zinc, cadmium, andcopper; oxide (tBH), a membrane permeant oxidant thought to MT binds these same heavy metals (2). MT may also partici- mammalian cells by peroxidizing membrane lipids (14). Cells that overexpressed MT had a greater antioxidantactivity than * This work was supported in part by National Institutes of Health control cells as revealed by: (a) accumulation of less intracelGrants HL32154 and CA 61299. The costsof publication of this article lular peroxidation products upon exposure to tBH (as deterwere defrayedin part by the payment of page charges. This article mustmined using the oxidant-sensitive fluorophore dichlorodihythereforebeherebymarked“advertisement” in accordancewith18 drofluorescein diacetate and thiobarbituric acid-reactive U.S.C. Section 1734 solelyto indicate this fact. 5 Recipient of a Clinician-Investigatorshipaward fromthe American substances), and( b )greater ability toscavenge phenoxyl radiHeart Association. ‘Ib whom correspondence should beaddressed Dept. cal (as determined byelectron spin resonance (ESR)). of Pharmacology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261. Tel.: 412-648-9420; Fax: 412-648-1945. EXPERIMENTALPROCEDURES 11 Recipient of a grant-in-aid from the American Heart Association. Cell Culture-NIH 3T3 cells were purchased from American Type $$ Recipient of support from MCBN, UNESCO. Culture Collection (CRL 1658) and maintained in 10% fetal bovine The abbreviations used are: MT, metallothionein; tBH, tert-butyl hydroperoxide; DCF, dichlorofluoroscein; H2-DCF, dichlorodihydrofluo- serum (HyClone,Logan,UT),penicillinandstreptomycin,HEPES rescein; BPV, bovine papillomavirus; GRP, glucose-regulated promoter; buffer, and Dulbecco’s modified Eagle’s medium. Cells were grown at PBS,phosphate-bufferedsaline;mT, millitesla; Gy, gray; mW, milli- 37 “C in 5% CO, and were routinely found freeof mycoplasma. Tkansfection-Plasmids pBPVGRPMT and pBPVGRPTM were genwatt; BrdUrd, 5’-bromo-2’-deoxyuridine; TBAR, thiobarbituric acid-reerous gifts from Dr. Kathryn A. Morton (University of Utah, Salt Lake active substance.

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1000 Mark Irradiator; J . L. Sheppard and Associates, Glendale, CA, City, UT). Bothplasmids contain the genome of bovine papilloma virus (BPV)that is useful for maintaining multiple copies of the plasmid for 4.15 Gy/min)and were added as internal standards. Cells werelayered ; Sciences, Inc., Ann Arbor, transcription (15). In pBPVGRPMT, mouse metallothionein-I is driven on polyvinyl chloride filter (0.8 p ~ Gelman by rat glucose-regulatedpromoter of 78 kDa (GRP)and transfected cells MI) and lysed with 2% sodium dodecyl sulfate, 10 mM disodium EDTA, and 0.5 mg/ml proteinase K at pH 10.0. DNA was eluted from the filter are referred to as NIH3T3MT. In the control plasmid, pBPVGRP'I", after addition of tetrapropylammonium hydroxide (pH 12.1). Radioacmouse metallothionein-I gene was inverted in sequence and separated from its GRP promoter.Cells transfected with this plasmid are referred tivity was determined in the column effluent using standard liquid to as NIH3T3/TM. Both celltypes were co-transfected with the plasmid scintillation spectrometry andthe frequency of tBH-induced DNA pSV2ne0, which contains the aminoglycoside phosphotransferase gene strand breakage was quantified as the fraction of V4C1DNA remaining driven by the SV-40 early promoter (American Tissue Culture Collec- on the filter when 75% of internal L3H1DNA remained. A calibration tion, Rockville, MD).Co-transfectionof pBPVGRPMT or pBPVGRPTM curve for relating frequency of tBH-induced DNA strand breaks to corresponding effect of ionizing radiation using I4C-labeled cellswas obwith pSV2neo (ratio of 3:l) was performed via electroporation using a single pulse of 220 V and 950 microfarads (Genezapper, Bio-Rad). tained by plotting radiation exposure (Gy)versus ['*CIDNA retention of Forty-eight hours after transfection, cells were exposed to 200 pdml the [3HlDNAinternal standard. Determination of Intracellular Oxidants and Lipid Peroxidation G-418 (Life Technologies, Inc.). After 3 weeks, surviving cells were expanded in G-418 (100 pg/ml) and complete medium (see above). Cells Products-The fluorescent probe dichlorodihydrofluorescein(H,-DCF; were then studied over the ensuing 1-6 weeks, during which time Molecular Probes, Eugene, OR) was used to monitor net intracellular generation of-reactive oxygen species by tBH (22).NIH3T3MT and TM intracellular MT was quantified routinely (see below). In addition, we cells were grown on 31-mmglass coverslips. Cells were rinsed in HEPESalso studied cells transfected with pSV2neo alone (NIH3T3Neo) and buffered salt solution (HBSS)and loaded with 10 PM H,-DCF diacetate selected in G-418 or native NIH 3T3 cells(control). dye in HBSS supplemented with 5 mg/ml bovine serum albumin for 15 Cellular MetallothioneinDetermination-Cell lysates were prepared by repetitive freezing and thawing in Tris buffer (10 mM) containing min at 37 "C. Cells wererinsed with HBSS and mounted in arecording CdC1, (10 p ~ ) After . ultrasonic dessication, the supernatant was ana- chamber. Recordings were made at room temperature using an ACAS lyzed for protein content using a modified Bradford technique and MT 570c imaging system (Meridian Instruments, East Lansing, MI) in the illuminated with an argon laser (488 nm; 200 was measured by a modification of a '"Cd binding assay (16).Total thiol confocal mode. Cells were content in sonicated cell homogenateswas determined by a modification mW). The laser was set at 200 mW and illuminating light attenuated by of Ellman assay (17) using 5,5'-dithiobis-(2-nitrobenzoicacid) and re- passage through an acousto-optical modulator that eliminated 95% of the signal and also by passage through a 7% neutral density filter. Inpeated in the presence of glutathione peroxidase (1.94 unitdpl) and tracellular antioxidant activity was assessed by monitoring increases in cumene hydroperoxide (33 p ~ to ) determine GSH and protein thiol fluorescence due to oxidation of H,-DCF to dichlorofluoroscein(DCF). content. For MT immunolocalization,NIH3T3MT and TM cells were trans- Images were obtained prior to addition of tBH to assess autoxidation and ferred to eight-well Lab-Tek chamber slides (Nunc, Naperville, IL). At laser-induced changes in fluorescent emission of Hz-DCF. Aftera stable middle logarithmic growth phase, cells were rinsed twice with phos- base line was obtained, tBH (0.5 mM) was added following aspiration of phate-buffered saline (PBS) and fixed in 2% paraformaldehyde in PBS. the recording chamber solution. Seven additional images were then obAfter being permeabilized for 15 min with 0.1% Triton X-100 in 2% tained, 1min apart. Fluorescenceintensity was estimated by delineating paraformaldehyde and phosphate-bufferedsaline, cells wererinsed with the area of the image corresponding to NIH 3T3 cells (as corroborated PBS and nonspecificity of antibody blocked with a 0.5% bovine serum by simultaneous phase contrast imaging). albumin, 0.15% glycine,and 5% goat serum solution (bufferA)for 30 min. Thiobarbituric acid-reactive substances (TBARs) weredetermined as Cells were then incubated at room temperature for 1 h with buffer A an independent measurement of lipid peroxidation. Cells were grownto solution containing a 1:lOO dilution of a previously described(18) affin- confluence and exposed to tBH (0.5 mM; 4 h). Samples were removed ity-purified rabbit antiserum that recognizes all major MT isoforms. from medium at 20-min intervals and evaluated for malondialdehyde Chambers were then rinsed with buffer A and andCy3 Fluorolink-con- production using a spectrophotometric assay for thiobarbituric acidjugated, affinity-purified goat anti-rabbit IgG (Jackson ImmunoRe- reactive substances (23). Cells weretrypsinized and protein determined search Laboratories, Westgrove,PA) was applied. One h later,cells were as above. To determine intracellular antioxidant activity toward phenoxyl radirinsed with buffer A followed by three PBS washes and the chambers were stripped and slides mounted with Gelvatol (Monsanto, St. Louis, cals, we quantified the ability of cellular homogenates fromNIH3T3NT MO). Using confocal microscopy, which ensures single plane illumina- and TM cells to quench an in vitro generated phenoxyl radical using our recently described method(24). Briefly, peroxidative attack on the phetion, cells were evaluated for MT content and subcellular location. In a subset of these determinations, the nuclei of proliferating cells nolic moiety of etoposide by tyrosinase in air-saturated PBS (pH 7.4) were further outlined by preincubating cells for 24h with 5'-bromo-2'- results in typical signals of phenoxyl radicals as detected by ESR specdeoxyuridine(BrdUrd).After washing with PBS and fixation, cells were troscopy. The oxygen-centered phenoxyl radical exhibits a hyperfine structure similar to semiquinone radicals but is relatively more stable processed as above for MT localization while also being stained with a fluorescein-labeled anti-BrdUrd mousemonoclonal antibody (Boeh- and doesnot require additional metal ionor alkaline stabilization. Furthermore, it has a discriminating predilection for thiols. Cellular ringer Mannheim) at a 15000 dilution. With confocal microscopy and differential filtration, it was possible to assign the cytoplasmic distri- homogenates containing various reductants are capable of reducing this radical and causing a transientdisappearance of the characteristic ESR bution within cells unambiguously. Cytotoxicity-Cells (5000) were transferred to 96-well plates and ex- signal. We compared the capacity of cellular homogenates prepared from NIH3T3/MT versusTM cells to quench the continuously recorded posed to tBH in serum-free Dulbecco's modified Eagle's medium (0.1etoposide phenoxyl radical ESR signal. ESR determinations were per0.6 m~ for 1 h, followedby 23 h in serum-free Dulbecco'smodified Eagle's medium) or CdC1, (1-300 p;24 h). The viability of subconfluent formed on a JEOL-RE1Xspectrometer at 25 "Cin gas-permeableTeflon tubing. Spectra were recorded at center field = 335.5 mT, field modulacells (6-12 replicatedconcentration) was quantified using reduction of tion = 0.04 mT, receiver gain = 500, time constant = 0.03 s, and power 3-[4,5-dimethylthiazol-2-yll-2,5-diphenyl tetrazolium bromide anda multiplate reader (Titertek Multiscan Plus, Flow Laboratories, = 20 mW. Statistical Analyses-Data are summarized as mean f S.E. (unless McLean, VA) at 540 nm (19). In addition, clonogenic assays (20) were performed. Briefly, cells were plated at a density of 10,000 celld100-mm indicated otherwise). Effect of tBH or cadmium on individual groups of Petri dish. Cells were allowed toattach and, 10 h later, were exposed to cells wereanalyzed by two-wayanalysis of variance and multiple means tBH (0.005-1.0 mM) for 1 h (in triplicate). tBH was removed and re- were comparedusing a Neuman-Keul's test. Differences betweenmean placed with complete medium. Sixto seven days later, plates were values of NIH3T3MT and TM groups were tested for significance by rinsed with PBS and exposed for 1h to 0.25%crystal violet in formalin unpaired t test. Significance was established at p < 0.05 level (25). and methanol. After rinsing the plates, colonies that were greater or RESULTS equal to 20 cells were counted with the aid of a binocular microscope. The plating efficiency of untreated cells was between 5 and 10%. Metallothionein Overexpression and Subcellular LocalDNA Single-strand Breaks-tBH-induced DNA damage was as- ization-In native NIH 3T3 cells, MT content was approxisessed using a modified alkaline elution technique for high frequency single-strand breaks (21). NIH3T3MT and TM cells were labeled with mately 0.1 pg/mg protein, which was similar to that in cells [2-"C]thymidine and then exposed to tBH (0.6 mM; 1h). NIH 3T3 cells transfected with either pSV2neo alone or pBPVGRPTM (Fig. 1). In contrast, there wasa 4-fold increase in metallothionein incubated with [3Hlthymidine for 48 h and containing [3HlDNA, received 15 Gy irradiation from a 137Cssource irradiator (Gamma cell content after transfection with pBPVGRPMT. Total cellular

MT as an Antioxidamt in NIH 3T3 Cells

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TABLE I Thiol content and VP-16 radical formationin NIH 3T3 cells Thiol contents given as nmol/0.4 x lo6 cells. Data are mean f standard deviation, n = 3.

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5.9 0.3' 3.5 f 0.3' 2.4 f 0.3 * 1.0"

4.4 * 0.2 2.3 2 0.2 2.1 f 0.2 14.0 f 0.5

Significantly different from NIH3T3lTM,p < 0.05.

ample is shown in Fig. 6. Prior to tBH,no difference was seen in theDCF signal of NIH3T3PI" (Fig. 6 A )or NIH3TB/MT (Fig. 6C). A rapid increase in intracellularoxidant levels was noted in both cell types after 0.5 mM tBH as assessed by a n increase Control Neo LIT TM in DCF fluorescence, but the oxidant burden was greater in FIG.1. Metallothionein content is increased in NIH 3T3 cells NIH3T3PTM (Fig. 6 B ) than NIH3TB/MT (Fig. 6 D ) after tBH after direct gene transfer with plasmid pBPVGRPMT. Values are exposure. Asummary of DCF fluorescence (normalized to premeans f S.E. of metallothionein content in whole cell lvsates from wild tBH levels) in NIH3T3MT andTM cells exposed to 0.5 m~ tBH is shown in Fig. 7. There was significantly less antioxidant activity in NIH3T3/TM cells than MT cells as assessed by DCF fluorescence. and protein thiols were significantly greater in NIH3T3MT The antioxidant properties of MT against tBH were further than TM cells, but glutathionelevels weresimilar in either cell (Table I). The increase inMT content was confirmed by a quan- assessed by measuring theemergence of TBARs. Within 2 hof in titatively greater immunoreactive MT present in NIH3T3MT tBH (0.5 mM) exposure, there were TBARs present (Fig. 2B) than NIH3T3PTM (Fig. 2 A ) cells. Moreover, the in- NIH3T3PTM but not in NIH3T3MTcells (Fig. 8). The delay in crease in metallothionein appeared tobe primarily localized to TBARs formation in NIH3T3PTM cells lasted approximately 1 the ,cytoplasm as ascertained by confocal microscopy (Fig. 2). h. At all times measured, NIHBT3Ll" cells consistently had Because of a previous report (26) of MTnuclear translocation in more TBARs in the medium compared to NIHBTBIMT cells. hepatocytes during S phase, we examined the distribution of There was no significant appearance of TBARs in the medium MT in cells transgressing S phase using BrdUrd labeling and in theabsence of tBH treatment (data notshown). To probe further thefunctional antioxidant properties of MT, a n anti-BrdUrd antibody. No evidence of nuclear MT was seen in BrdUrd-labeled or unlabeled cells (Fig. 2). BrdUrd treatment we examined the ESR spectra of etoposide phenoxyl radical did not affect MT subcellular localization when compared to generated by tyrosinase-catalyzed etoposide oxidation in the presence of NIH3T3RM (Fig. 9A) and NIH3T3NT (Fig. 9B) cells not treated with BrdUrd (data not shown). Cellular Sensitivity to tBH-Overexpression of MT produced cell homogenates. The appearanceof the ESR signal with chara phenotype that was resistant both to CdC1, (Fig. 3 A ) and tBH acteristic features of etoposide phenoxyl radical was apparent (Fig. 3B). The concentrationsof CdCl, or tBH required topro- before addition of cell homogenate, and each cell type immediduce 50% decrease in cell viability in NIH3T3MT cells was ately quenched the signal. The kinetics of the regeneration of 2-3-fold greater than thatneeded with NIH3T3ll" cells ( p < the phenoxyl radical hyperfine ESR signal caused by cellular 0.05). Thesensitivity of untransfectedNIH 3T3 cells and homogenates provides an estimate of endogenous reductants. That the overexpression of MT produces a functional increase pSV2neo alone cells to CdC1, or tBH was similarNIH3T3/'I" to cells (data notshown), indicating that thetransfection process in intracellular antioxidant activity was demonstrated by the did not affect cellular sensitivity. While colorometric assays differential lag period in ESR signal regeneration (see Fig. 9 provide a rapid method for determining cellular sensitivity to and Table I) between NIH3T3D" cells (14.0 2 0.5 min) versus toxins, they are frequentlyinsensitive. Thus, t o quantify more NIH3T3MT cells (22.0 2 1.0 min; p < 0.05; n = 6 ) . precisely differences in the sensitivity of the transfectedcells to DISCUSSION oxidants, we next performed clonogenic assays on NIH3T3fI" Oxidative stress is the result of a n imbalance between the and MT cells after a brief exposure to tBH. Based on the concentration of tBHrequiredto reduce t o 50% survival of production of reactive oxygen species including superoxideanNIH3T3ll" (0.03 mM) and NIH3T3/MT (0.18 n " ) cells (Fig. 41, ion, hydrogen peroxide and hydroxyl radical, and cellular anoverexpression ofMT enhancedresistance 6-fold. Untrans- tioxidant defense mechanisms.Reactive oxygen species are procellular a duced duringrespiration,inflammation,andother fected or pSV2neo transfected alone were sensitive to tBH in metabolic events; in many cases, the metal-catalyzed production manner similar to NIH3T3D" cells (data not shown). DNADamage-We performed alkaline elution measure- of hydroxyl radical is considered criticaltoward oxidant damage ments for DNA integrity 1h after cellular exposure t o a range to DNA, proteins, and lipids. Cells have developed elaborate of tBH concentrations t o determine if metallothionein overex- networks to deal with potentially damaging radicalsincluding pression decreased sensitivity to DNA damage. In Fig. 5, we enzymatic (superoxidedismutase, catalase, glutathioneperoxishow that 0.6 mM tBH caused detectable DNA strand scissions dase) andnon-enzymatic antioxidant systems. In this latter rewithin 1 h, but the damageto NIH3T3fl" cells caused by 0.6 gard, thenon-protein thiol,glutathione (GSH), has been studied mM tBH was no greater than that to NIH3T3/MT cells. Aver- extensively (27). Much less attention hasbeen directed to proaging results from three independent experiments, radiation tein thiols, such as metallothionein. Metallothionein is particuequivalent DNA damage induced by 0.6 mM tBH was 5.09 2 152 larly interestingbecause expression of MT is readily inducible (2). Transcriptional activation occurs via heavy metals, as well versus 6.19 68 ( p = 0.54; not significant) Gy for NIH3T3D" as cytokines, hormones,and stress; thus, metallothionein levels compared to MT cells, respectively. conditions known to accomAntioxidant Activity-Antioxidant activity of cells was as- increase incells and tissues during sessed by changes in Hz-DCF fluorescence, and a typical ex- pany elevations in reactive oxygen species (4).

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FIG.2. Immunolocalizationofmetallothionein in NIH3T3fl" (panel A) and NIH3T3/MT (panel E ) cells. TransfectedNIH 3T3cells were fixed, permeabilized, and incubated with a rabbit anti-human metallothionein antiserum and then a Cy3 Fluorolink-conjugated, anti-rabbit secondary antibody (see "Experimental Procedures"). Cells were also simultaneously labeled with mouse monoclonal antibodyto BrdUrd and BrdUrd colocalized with anti-mouse-Ig-fluorescein antibody. Cells were examined with aconfocal microscope, and fluorescence intensity was displayed by pseudocolor imaging.

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FIG.4. NIH 3T3 cells that overexpress metallothionein after gene transfer (NIH3T3MTyopen squares) are leas sensitive to the cytotoxic effects of brief exposures to tBH than control (NI3T3/l"MYopen diamonds) cells. Clonogenic assays (see "Experimental Procedures") were performed on three separate occasions on NIH3T3MT and NIH3T3PTM cells after exposure to tBH (0.03-0.30 m ~for ) 1 h. Determinations were performedin triplicate, normalized to control, and mean * standard deviation plotted uersus [tBHl. There were significant differences between NIH3T3LMT and NIH3T3lTM cells a t all concentrations of tBH (*, p e 0.05).

MT TM

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0.2

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Fro. 3. NIH 3T3 cells that overexpress metallothionein after gene transfer(NIH3T3/MT, open squares)are less sensitivethan control (NIH3T3/", open diamonds) cells to the acute toxic effects of CdCI, (panel A) and tBH (panel B ) . Cell viability was ascertained by colorimetric assay (see "Experimental Procedures") and normalized to percent of control. InpaneZA,values are mean 2 standard deviation of t h e e separate experiments (or mean of two experiments). In panel B , values are mean 2 S.E. of 10 separate experiments (each performed in triplicate). Significant ( p e 0.05) differencesbetween NIH3T3rrXlI and NIH3TS" are indicated by an asterisk.

Most previous studies addressing the antioxidant properties of metallothionein have used inducers such as zinc or other heavy metals, which are promiscuous, affectinga host of genes (28). For example, Chubatsu and Meneghini (8, 12) suggested the protection against HzOz afforded by cadmium andor zinc pretreatment is associated with heavy metal effects on other

endogenous nonprotein thiol antioxidants. In the current study we used direct gene transfer in NTH 3T3 cells (Fig. 1) to enhance metallothionein expression and generate a population of cells resistant to the cytotoxicity (Figs. 3B and 4) of the membrane permeant oxidant tert-butyl hydroperoxide. This oxidant is particularly attractive as a model because ita cellular actions and fate are well studied (29). tert-Butyl hydroperoxide forms alkoxy1 and peroxyl radicals through an iron-catalyzed process that cause both cytoplasmicand nuclear damage leading to cell death (14).In hepatocytes, tBH lethality has been linked to lipid peroxidation (14) rather than to DNA single-strand lesions (30). Similarly, we saw no protection against the DNA single-strand breaks with metallothionein overexpression, although cells wereclearly protected against the cytotoxicactions of tBH. Thus, metallothionein appears to interfere with the duration of oxidants or the subsequent propagation of free radical reactions rather than the production of primary oxidants or their inactivation. We used three methods to demonstrate an increase in antioxidant activity in the transfected population: (a)spectrophotometric detection of thiobarbituric acid-reactive substances released in thecell culture medium, ( b )fluorescenceimaging of Hz-DCF oxidation in live cells,and (c) cell homogenate quenching of phenoxy1 radicals estimated by ESR. The thiobarbituric acid assay employed detects free malondialdehyde and is routinely used as an estimate of lipid peroxidation. We noted a clear decrease in appearance of " B A R S (Fig. 8) in NIH3T3MT

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FIG.5. Typicalresult of DNA scission strand break in cells over(NIH3T3/MT,open circks)or control expressing metallothionein (NM3T3/TM, closed circles) cells after exposure to 0.6 m~ tBH. WithouttBH addedto the medium,there was no detectableDNAscission FIG.6. Effect of tBH on DCF fluorescence in NIH3T3/TM and strand breakage (Control1. DNA damage to either cell type was similar after tBH as shown by the comparison of intact [l4C1DNAremaining to NIH3T3/MT cells. Subcultures of NIH3T3fl" and NIH3T3IMT cells were grown on 31-mm coverslips, loaded with H2-DCF, and exposed to internal standard (e.g. cSHI)DNA standard after irradiation. 0.5 m~ tBH. Upper panels (A and B ) and lower panels (C and D )are from NIH3T3PTM and NIH3T3IMT cells, respectively. Pseudocolor images of DCF fluorescence are shown before and at 2 min after tBH versus NIH3T3PTM cells treated with tBH. Despitethe obvious exposure. Relatively diffise increases in DCF fluorescence were noted differences in TBARs, malondialdehyde canbe generated from in all cells after tBH, but the intensity was greater in NIH3T3RM non-lipid sourcesand other endogenous aldehydes also canin- uersus NIH3T3/MT cells.

terfere with this assay (34). Moreover, the assay is relatively insensitive and considerable incubation time is required for detectable levels to appear even in cell culture. Therefore, we used an additional highly sensitive technique to detect intracellular oxidants by quantifying the fluorescent changes in DCF after tBH treatment. We noted that tBH causeda prompt and uniform increase in intracellular oxidants in both NIH3T3/MT and NIH3T3/TM cells (Fig. 61, but the antioxidant activity was greater in NIH3T3IMTcells than NIH3T3PTM cells since the relative changes in fluorescence were significantly less in the former (Fig. 7). Moreover, the magnitude of the DCF signal displayed little intercellular heterogeneity (Fig. 6).Thus, DCF and TBAR are highly suggestive of an antioxidant role for metallothionein. Although the mechanism by which metallothionein may act as an antioxidant is unclear, an attractive hypothesis is that metallothionein servesas anexpendabletarget for free radicals (31). Thus, we used a well defined phenoxylradical generating system (tyrosinase and etoposide) coupled with ESR to evaluate quenching of phenoxyl radicals by homogenates enriched with metallothionein. Thelag time before reappearance of the signature ESR signal forphenoxyl radical waspreviously shown to be independent of the presence of transition metals (24) but was well correlated withtotal intracellular thiols and poorly correlated to either cell number ortotal cellular protein (data not shown). In our study homogenates of NIH3T3/MT cells were significantly more competent than NIH3T3D" cells in directly quenching phenoxyl radicals (Fig. 9; Table I). Thus, the collective results of three distinct assays indicate a functional antioxidant role for metallothionein in NIH 3T3 cells. Recent studies have revealedan antioxidant functionality for metallothionein in lower eukaryotes. S. cereuisiae strains lacking CuZn-superoxidedismutase fail to grow on agar containing the respiratory carbon chain source, lactate, because of their enhanced sensitivity to oxidative injury (13). Expression of either yeast or simian metallothionein after DNA transfer in the presence of copper protectsthese mutants against oxygen free radical injury (13). Further support for an antioxidant role for copper metallothionein was provided by demonstrating that purified Cu(1)- metallothionein but not other metal species of

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4

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Time (min)

FIG.7. Change in DCF fluorescence after tBH in NIH 3T3 cells. DCF fluorescence was quantified hom laser imaging at time zero and again 1 min prior to administration of tBH. Images were then obtained every minute for a 7-min interval, and changes in fluorescence were normalized to the initial determination.A total of 8-10 celldfield were analyzed in this fashion, and six fields from six differentcoverslips were collectively analyzed. Data are mean f standard error.

metallothionein (see below), inhibited the in vitro autoxidation of 6-hydroxydopamine. Interestingly, metallothionein did not substitute for superoxide dismutase in protecting these mutants against either hyperoxia or paraquat (13), so the precise mechanism responsiblefor protection against the oxidant damage causedby lactate growth has not been established. Metallothioneinmay exist in the cytoplasmand/or the nucleus (261, and its subcellular distribution may be critical in determining its antioxidant function. In the current study, we describe a population of cells that have exclusively elevated cytoplasmic expression of metallothionein after gene transfer (Fig. 2 A ) . These cells were resistant to the cytotoxic (Fig. 4) but not the nucleus-damaging (Fig.5) effects of tBH. The dissociation between cytoplasmic and nuclear effects of tBH have previously beendemonstrated (9,30), and we assume metallothionein must be present within the nucleus to protect against DNA single-strand breaks by tBH. This concept is supportedby the recent observationthat a clone of V79 cells that had a high nuclear level of metallothionein wereresistant to H,O,. In this same study (32), antisense oligonucleotides were usedto lower nuclear metallothionein and restore the sensitivity of cells to

15243

MT as an Antioxidant in NIH 3T3 Cells 1.6

--E"

MT

T

time (minutes)

FIG.8. Thiobarbituric acid-reactive substances (TBARS) appear later and in lesser amounts &er exposure to 0.6 m~ tBH (4 h) in NIHSTS/MT (open .squares) than NIHSTS/TM (open diam o n d s ) cells. Values are mean + standard deviation of three determinations in either N I H 3 T 3 m or NIH3T3MT cells. VP-16 + NROSINASE

A

important in its potential as an antioxidant. Cu-MT was singularly protective against oxidant stress in mutantyeast lacking Can-superoxide dismutase (13).Others have noted that cadmiudzinc metallothionein can actually induce DNA strand breaks in vitro (35). Since we have little information about the metal speciation of metallothionein after gene transfer, this unknown factor could influence the antioxidant function of this protein thiol in our studies. In conclusion, this report shows an antioxidant role for metallothionein in mammalian cells replete with their normal antioxidant defense mechanisms. Depletion or destruction of superoxide dismutase (13) or GSH levels (36)are not mandatory for physiological amounts of metallothionein to protect cells against free radicals produced by exposure to the membrane permeant tBH. Despite a demonstrable decrease in oxidant burden measured by fluorescence imaging and spectrophotometric detection of thiobarbituric acid-reactive species, the level of single-strand DNA lesions was unaffected and failed to relate to cytotoxicity. Thus, the subcellular location of metallothionein may be important for its antioxidant role. Acknowledgments-We acknowledge contributions by Dr. Weili Weng and the excellent technical assistance of William Allan and Joseph DeFilippo. We are also grateful to Dr. Kathryn A. Morton (Department of Radiology, University of Utah School of Medicine, Salt Lake City, U T ) for the generous gift of pBPVGRPMT and pBPVGRPTM. REFERENCES

2 rnh

2 rnin

FIG.9. ESR spectra (A) and time course( B ) of VP-16 phenoxy1 radicals generatedby tyrosinase-catalyzedVP-16 oxidation in the presence of cell homogenate6 from NIH3T3/TM or NIH3T3IMT cells. Incubation conditions: VP-16(600 p ~ ) tyrosinase , (2.5 unitdpl), deferoxamine (100 p ~ in) 50 m~ phosphate buffer, 100 m~ NaCl, pH 7.4, at 25 "C. Homogenates prepared from 0.4 x 10' cells were added to the incubation medium. ESR conditions: center field = 335.5 mT, sweep width = 5 mT, gain = 500, field modulation = 0.04 mT, time constant = 0.03 s, and power = 20 mW.

the DNA-damagingeffects of H,O,. Conversely, further increases in nuclear metallothionein expression with zinc enhanced the resistance of this clone to H,O,-mediated nuclear damage. Failure to see protection against H,O, cytotoxicity in Chinese hamster ovary cells after transfer of the human metallothionein-IIa gene might be due to low cytoplasmic localization in thesecells (33).We do not understand the factors that direct metallothionein into different subcellular compartments. As noted above, metal speciation of metallothionein may be

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