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Induction of DNA Double-Strand Breaks in the H4IIECell Line Exposed to Environmentally RelevantConcentrations of Copper, Cadmium, and Zinc, Singlyand in CombinationsRenate Haldsrud a & Åse Kr⊘kje a

a Department of Biology, Norwegian University of Science and Technology (NTNU),Trondheim, NorwayPublished online: 30 Jan 2009.

To cite this article: Renate Haldsrud & se Krkje (2009) Induction of DNA Double-Strand Breaks in the H4IIE Cell Line Exposedto Environmentally Relevant Concentrations of Copper, Cadmium, and Zinc, Singly and in Combinations, Journal of Toxicologyand Environmental Health, Part A: Current Issues, 72:3-4, 155-163, DOI: 10.1080/15287390802538964

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Journal of Toxicology and Environmental Health, Part A, 72: 155–163, 2009Copyright © Taylor & Francis Group, LLCISSN: 1528-7394 print / 1087-2620 online DOI: 10.1080/15287390802538964

155

UTEHInduction of DNA Double-Strand Breaks in the H4IIE Cell Line Exposed to Environmentally Relevant Concentrations of Copper, Cadmium, and Zinc, Singly and in Combinations

DNA DOUBLE-STRAND BREAKS IN THE H4IIE CELL LINERenate Haldsrud and Åse KrøkjeDepartment of Biology, Norwegian University of Science and Technology (NTNU), Trondheim, Norway

Xenobiotics, including heavy metals, exist in nature as complexmixtures of compounds with possible interactions. Induction ofDNA damage such as DNA strand breaks may exert detrimentalconsequences to both individuals and populations. In this study,the induction of DNA double-strand breaks was assessed using theH4IIE rat hepatoma cell line following exposure to high and envi-ronmentally relevant concentrations of chloride salts of the metalscadmium (Cd), copper (Cu), and zinc (Zn), both singly and incombination. DNA strand break analysis was performed usingagarose gel electrophoresis. Median molecular lengths were calcu-lated from fragment size distributions acquired from gel imagedata and were used as a quantitative measure of DNA double-strand break induction. Exposure to high concentrations of Cuand Cd in combination produced a significant increase in theoccurrence of DNA strand break. However, exposing cells to highconcentrations of Cu, Cd, and Zn in combination resulted insignificantly lower DNA double-strand break compared to controlcells. Addition of low Zn to the Cd/Cu mixture restored DNAdamage level back to that of the control. Environmentally rele-vant concentrations of Cd, Cu, and Zn did not appear to induceDNA strand breaks in the H4IIE cell line.

Heavy metals can exist either as pure metal mixtures, or inmixture with other types of contaminants. Metals occur naturallyin all ecosystems, but in variable concentrations. They are redis-tributed in nature through geochemical cycles, but anthropogenicactivity interferes with these natural cycles, leading to excessiveaccumulation of metals in certain compartments. This studyfocused on the metals cadmium (Cd), copper (Cu), and zinc(Zn). When metals coexist in a compartment, the speciationproperties and bioavailability of the individual metals are

affected. Chemicals also compete for uptake routes in organisms,as well as toxic target sites, transport mechanisms, and excretionroutes within organisms (Eaton & Gilbert, 2008). Copper and Znare, as opposed to Cd, essential metals (Uauy et al., 1998; Valkoet al., 2005). Copper is involved in the normal function of differentenzymes such as cytochrome c-oxidase, Cu/Zn superoxide dis-mutase, and tyrosinase, as well as in metabolic complexes (Sternet al., 2007). Zinc serves as a cofactor for a number of metal-loenzymes and is also an essential component in many proteinsinvolved in gene expression and regulation of genetic activities(Berg & Shi, 1996; Watanabe et al., 1997). Many proteinsinvolved in DNA repair mechanisms and cell signalling path-ways are Zn finger proteins (Ho, 2004). Zinc deficiency in cellsmay therefore pose a larger threat to DNA integrity than excessZn. However, essential metals have the potential to becometoxic when present in excessive amounts.

DNA double-strand break formation is considered to be aserious form of DNA damage, due to destruction of templatesessential for DNA replication and transcription. Induction ofDNA double-strand breaks may lead to induction of apoptosis,mutations, chromosomal rearrangements and development ofcancer (Pfeiffer, 1998). DNA double-strand breaks may beinduced directly by ionizing radiation or radiomimetic com-pounds, or are formed during replication when the replicationfork encounters an unrepaired single-strand break (Helledayet al., 2007). Heavy metals produce DNA damage in differentways. Being a Fenton metal, Cu has the ability to participate inredox cycling, ultimately leading to formation of reactiveoxygen species (ROS) such as hydroxyl radicals (•OH) (Valkoet al., 2005). Cu also has the ability to bind directly to DNA,although the presence of hydrogen peroxide (H2O2) apparentlyis necessary for a genotoxic effect to occur (Sagripanti et al.,1991; Moriwaki et al., 2008). Cadmium does not form ROSdirectly. However, Cd was shown to be involved in indirectformation of ROS by decreasing the levels of free radical scav-engers such as glutathione, or by displacing Fenton metalsfrom proteins (Szuster-Ciesielska et al., 2000; Galán et al.,2001; Waisberg et al., 2003). Cadmium also has the ability toinhibit antioxidant enzymes such as catalase, glutathione

Financial support was obtained by the Norwegian Ministry ofForeign Affairs, organized by the Centre for International UniversityCooperation (SIU) via the project “ENLINO master programnetwork” (project CCP 03/02). The authors are grateful to ChrisBingham for his valuable assistance.

Address correspondence to Åse Krøkje, Department of Biology,Norwegian University of Science and Technology (NTNU) 7491,Trondheim, Norway. E-mail: ase.krokje@bio.ntnu.no

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156 R. HALDSRUD AND Å. KRØKJE

peroxidase (GPox), and superoxide dismutase (SOD) involvedin removal of ROS (Waisberg et al., 2003; Filipi6 & Hei, 2004;Valko et al., 2005). Mechanisms underlying Cd-induced ROSformation involve disturbance of mitochondrial electron trans-fer chains and damage to lysosomal membranes (Bradley et al.,1987; Wang et al., 2004; Fotakis et al., 2005). Cadmium hasthe ability to interfere with DNA repair mechanisms such asnucleotide excision repair, possibly by competing with Zn inZn-finger proteins (Hartwig 1998, Asmuss et al., 2000). Jinet al. (2003) found that Cd interfered with mismatch repairmechanisms, and Fatur et al. (2003) showed that the metalinhibited steps in the base excision repair pathway. Base exci-sion repair is one of the main pathways for removal of oxida-tive DNA lesions such as 8-hydroxyguanine (Evans et al.,2004). Interference with cell cycle regulation by exposure toCd was also reported by Mukherjee et al. (2004). Oliveira et al.(2008) found that the metal was capable of binding directly toDNA. However, no oxidative DNA damage associated with Cdbinding was noted in their experiment. Zinc is, as opposed toCd and Cu, associated with antioxidant effects. Bray andBettger (1990) showed that Zn protected sulfhydryl groups andcompeted with transition metals for binding to target sites fortoxicity. However, the main antioxidant property associatedwith Zn is its ability to induce synthesis of metal bindingproteins such as metallothionein (MT) (Chan & Cherian,1992). Metallothionein may protect against metal toxicity by(1) limiting uptake of metals into the cells, (2) sequesteringmetals within the cells, or (3) increasing export of metals out ofcells (Park et al., 2001). Zinc and Cd are known to be potentinducers of metallothionein, but Cu also induces the protein(Klaassen et al., 1999). Asmuss et al. (2000) found that coex-posure to Zn and Cd produced less inhibition of nucleotideexcission repair than exposure to Cd alone.

Genotoxic compounds represent a major environmentalchallenge because they may lead to impaired reproduction,accelerated aging processes, and tumor induction in exposedindividuals. Genetic damage is also transmitted to offspring(Würgler & Kraemers, 1992). Development of methods that

predict genotoxic impact of exposure to environmental con-taminants before serious effects on the ecosystem level occuris, therefore, required. Several methods such as the alkalineunwinding assay, comet assay, and agarose gel electrophoresiswere previously applied for detection of DNA strand breaks incells and tissues (Shugart, 2000).

One possible strategy of biomarker development involvesmeasurements of adverse and genotoxic effects of pollutantsin isolated cell cultures or cell lines. Hepatoma cells are rele-vant study objects, due to the important role the liver playsin detoxification, bioactivation, and excretion of xenobioticsas well as endogenous compounds. Metal concentrationsmeasured in liver of different organisms are given in Table 1.The H4IIE rat hepatoma cell line has primarily been used foranalysis of CYP1A1 induction, owing to its low basal levelof this enzyme (Whyte et al., 2004). However, the cell linewas chosen for the present study because of its previous use inexperiment involving genotoxic endpoints such as DNA strandbreaks (Grant et al., 2001, Wätjen et al., 2005) and cell deathmechanisms (Kim et al., 2003). The aim of the present studywas to evaluate induction of DNA double-strand breaks as abiomarker of exposure to heavy metals in the H4IIE cell line,following exposure to both high and lower, environmentallyrelevant, concentrations of Cd, Cu and Zn. These three metalsfrequently occur together in nature, and obtaining knowledgeabout how the metals behave in mixtures is of ecotoxicologi-cal relevance. DNA double-strand break induction wasanalyzed by using a technique involving cell lysis andrelease of DNA within agarose gel plugs prior to agarose gelelectrophoresis.

MATERIALS/METHODS

ChemicalsFetal bovine serum (FBS) (heat inactivated) (10108-157),

L-glutamine medium supplement (200 mM) (25030-032),penicillin–streptomycin (5000 U/ml) (15070-063), and trypsin

TABLE 1 Levels of Cd, Cu, and Zn Measured in Liver Samples From Organisms at Different Sampling Sites

Species Location Cd (μg/g) Cu (μg/g) Zn (μg/g) Reference

Yellow perch (Perca flavescens) Quebec, Canada 30.8a 101.68a 167.37a Kraemer et al., 2005Brown trout (Salmo trutta) Røros, Norway 5.48b 103.42b 47.99b Olsvik et al., 2001Ringed seal (Pusa hispida) Holman, Canada 6.65b 9.25b 43.99b Dehn et al., 2005Loggerhead turtle (Caretta caretta) Mediterranean Sea, Italy 3.36b 7.69b 29.3b Storelli et al., 2005Caribou (Rangifer tarandus groenlandicus) Akia, Greenland 0.695b 21.8b 29.5b Aastrup et al., 2000Moose (Alces alces) Yukon, Canada 4.94b 40.32b 34.87b Gamberg et al., 2005Glaucous gull (Larus hyperboreus) Bear Island, Norway 6.06a 19.7a 111a Savinov et al., 2003Black-footed albatross (Diomedea nigripes) North Pacific, Japan 22b 5.1b 69b Ikemoto et al., 2004

Note. Superscripts denote: a, dry weight; b, wet weight.

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(2.5%) (15090-046) were obtained from Gibco. Culturemedium RPMI 1640 was obtained from EuroClone (ECB9006L) and Sigma (R0883). Dimethyl sulfoxide (DMSO)(99.9%, spectrophotometric grade) (K21387950) was pur-chased from Merck. Ethidium bromide (EB) (10 mg/ml)(161-0433), high-strength analytical-grade agarose (ultrapureDNA grade) (162-0126), low-melt preparative grade agarose(ultra pure DNA grade) (162-0019), and sodium dodecyl sulfate(SDS) (electrophoresis grade) (161-0301) were obtained fromBio-Rad. λ DNA/HindIII digest (500 μg/ml) (#SM0101) andloading dye solution (6×) (R0611) were purchased fromFermentas. Thiazolyl blue tetrazolium bromide (MTT) (98%TLC) (M2128-1G) and proteinase K (39 U/mg) (P-2308) wereobtained from Sigma. Cadmium chloride dihydrate(CdCl2·2H2O) was purchased from Fluka Chemika (20906).Copper chloride dihydrate (CuCl2·2H2O) was obtained fromSigma (C-6641). Zinc chloride (ZnCl2) was purchased fromMerck (8816). All other chemicals were of analytical grade,and were obtained from Sigma, Merck, and Fluka Chemica.

Cell CultivationThe H4IIE rat hepatoma cell line was developed from the

Reuber Hepatoma H-35 cell line (Whyte et al., 2004). TheH4IIE cell line used in this experiment was supplied by theCentre for Applied Microbiology and Research (CAMR,ECACC, Salisbury, Wiltshire). The cells were grown in RPMI1640 medium, supplemented with fetal bovine serum (FBS)(5%), L-glutamine (200 mM) (1%), and penicillin–streptomycin(5000 U/ml) (1%) in a humid atmosphere of 5% CO2 in air at37°C. The cells were grown in 75-cm2 culture flasks(Sarstedt), and subcultured every 3–4 d after harvesting withtrypsin (0.0025%) and ethylenediamine tetraacetic acid(EDTA) (0.01%) in phosphate-buffered saline solution (PBS)and diluted 3–4 times. Cells were used at 75% monolayerconfluency.

MTT Cell Viability AssayThe 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium

bromide (MTT) assay (Mosmann, 1983) was performedaccording to Kim et al. (2003). The cells were plated in a 96-well plate (Costar) with 200 μl medium at a density of 105 cellsper well and incubated at 37°C in a humid atmosphere of 5%CO2 in air for 24 h prior to metal exposure. The stock solutionsof the metals (Cu, Cd, and Zn) were prepared in autoclaveddistilled water. The metal solutions were diluted in growthmedium to the desired concentrations, and 200 μl of the testexposure medium was applied to each well. When exposing thecells to individual metals, 6 CdCl2 concentrations in the range10-4–10 μM, 7 CuCl2 concentrations in the range 0.01–1000 μM,and 9 ZnCl2 concentrations in the range 0.01–500 μM wereused. When exposing the cells to combinations of metals, onehigh concentration and one low concentration were chosen foreach metal. These concentrations of metals are listed in Table 2.

For each exposure condition, a minimum of two parallels wereused, most conditions were tested with more than five paral-lels. At first, differences in cell viability were tested after 12,24, 36, or 48 h (data not shown). After these preliminary exper-iments, the cells were incubated with test solution for 36 hbefore determination of cell viability. After incubation, theexposure medium was removed, and the cells were incubatedfor another 4 h in medium containing MTT (0.5 mg/ml). Themedium was then removed, and to each well 200 μl dimethylsulfoxide (DMSO) was added to solubilize the produced for-mazan crystals. Relative concentration of formazan wasmeasured spectrophotometrically at 550 nm. Cell viability wasgiven as relative survival of exposed cells compared tountreated control cells, and was presented as percent of control.

Metal Exposure for DNA Double-Strand Break Assessment

The cells were seeded in 12-well plates (Costar), and incu-bated at 37ºC and in a humid atmosphere of 5% CO2 in air for24 h prior to metal exposure. After incubation, the growthmedium was replaced by 3 ml of fresh medium containing themetal for exposure. The metal concentrations used arepresented in Table 2. The cells were exposed to metals for 36 hat 37ºC and in a humid atmosphere of 5% CO2 in air. Afterexposure, the exposure medium was removed, and the cellswere washed with PBS (pH 7.4). The cells were harvested withtrypsin (0.0025%) and EDTA (0.01%) in PBS. The cell pelletobtained after centrifugation (5 min, 1200 × g) of the cellsuspension was dissolved in 500 μl TE-buffer (10 mM Tris, 1 mMEDTA, pH 8) prior to agarose plug preparation.

Agarose Plug PreparationThe agarose plugs for electrophoresis were prepared according

to the procedure described by Theodorakis et al. (1994) andKrøkje et al. (2006) with modifications. The cells obtainedafter harvesting cells were suspended in TE buffer, incubatedat 37°C, and mixed with an equal volume of premelted solutionof 1% low-melting-point-agarose at 37ºC. From this solution,agarose plugs were made by loading 20 μl of the low-melting-point agarose cell suspension into wells of a 1.5% standard

TABLE 2 High and Low Concentrations of the Metals Cadmium,

Copper, and Zinc Used for MTT Assay and DNA Strand Break Analyses in the H4IIE Cell Line After Exposure of the Metals

Singly and in Combination

Metal High concentration (μM) Low concentration (μM)

Cu 100 0.01Cd 1 0.0001Zn 10 0.01

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agarose gel, and chilling the gel mold at 4°C for 1 h. The plugswere then cut from the mould, placed in 500 μl digestion buffer(10 mM Tris, 25 mM EDTA, 100 mM NaCl, 0.5% SDS, pH 8)with proteinase K (1 mg/ml), and incubated at 55°C overnight.After incubation, the plugs were chilled, loaded into the wellsof an agarose gel (0.6% agarose in TBE buffer (90 mM Trisbase, 90 mM boric acid, 2 mM EDTA, pH 8), and sealed with1% low-melting-point agarose prior to electrophoresis.

DNA Double-Strand Break AssayDNA from the exposed cells embedded in low-melting-point

agarose plugs was electrophoretically separated. Lambda-DNAHindIII digest was used as a molecular size marker. The sameTBE buffer used for gel preparation was used as running bufferfor electrophoresis. The electrophoresis was run at 1.5 V/cmfor 14 h, followed by staining of the gel with EB for 2 h.Gel image data was acquired using the BioRad Gel Doc 2000system for quantification of DNA double-strand breaks.Calculation of median molecular length (MML) of DNAfragments in the gel was performed, using densitometric dataobtained from gel image analysis. The DNA fragment sizedistribution determined from gel image data was comparedto a DNA size marker (Hind III digested λ-phage DNA) runon the same gel. Calculated MML values were used as thequantitative measure of the level of damage due to DNAdouble-strand breaks.

Statistical AnalysisStatistical differences between control cells and cells

exposed to different mixtures and concentrations of metalswere calculated using the nonparametric Mann–Witney test.The statistical tests were performed using MINITAB Release14. The criterion for significance was set at p < .05.

RESULTS

MTT Cell Viability AssayFor all metal concentrations analyzed, the cell viability was

expressed as percent formation of formazan crystals in exposedcells compared to controls. The preliminary experimentsperformed to assess differences in cell viability as a function ofexposure time indicated that no differences in survival as afunction of exposure time existed (data not shown). In furtherexperiments 36-h exposures were chosen.

The results from exposure of the cells to single metals inincreasing concentrations are presented in Figure 1. Whenexposing the H4IIE cells to CdCl2 concentrations in the rangeof 10-4–10 μM for 36 h, a decline in cell viability was detectedat 10 μM Cd (32.6% cell survival) (Figure 1A). After expos-ing the same cell line to CuCl2 concentrations in the range of0.01–1000 μM for 36 h, a fall in cell viability was detected at1000 μM Cu (39.4% survival) (Figure 1B). When exposing the

H4IIE cells to ZnCl2 concentrations in the range of 0.01–500 μMfor 36 h, the cell viability showed a marked decrease at 100 μMZn (34.8% survival) (Figure 1C).

Data obtained when exposing the H4IIE cells to differentcombinations of metals (concentrations given in Table 2) arepresented in Figure 2. When the cells were exposed to highconcentrations of Cu and Cd in combination for 36 h, a signifi-cant decrease in cell viability was observed. None of the othermetal combinations induced any significant change in cellviability.

DNA Double-Strand Break AssayMedian molecular length (MML) values for the DNA frag-

ments in the different exposure categories were calculatedbased on the size separation of DNA fragments in the agarosegel and presented in Figure 3. Cells exposed to high concentra-tions of all three metals (Cu, Cd, and Zn) in combination hadMML values that were significantly higher than control values,indicating a lower frequency of DNA double-strand breaks inthese cells. The cells exposed to high concentrations of Cu andCd in combination had significantly lower MML values thanboth control and cells exposed to high concentrations of allmetals in combination, indicating a higher frequency of DNAdouble-strand breaks in these cells. All the other exposureconditions tested did not yield MML values that differedsignificantly from the control. In a separate experiment, thefollowing mixtures were tested: high concentrations of Cu andCd with low concentration of Zn, low concentrations of Cu andCd, low concentration of Cu with high concentration of Cd,and high concentration of Cu with low concentration of Cd.None of these mixtures yielded MML values different fromcontrol values (data not shown).

DISCUSSIONThe aim of the present study was to evaluate induction of

DNA double-strand breaks in the H4IIE cell line as a biomarkerof exposure to environmentally relevant concentrations ofmetals Cu, Cd, and Zn. These metals are common environmentalpollutants that often are found together in nature, related tomining runoffs or emissions from smelting industries.

H4IIE Cell Viability After Exposure to Cd, Cu, and ZnFor assessment of cell viability, the H4IIE cells were

exposed to the metals Cd, Cu, and Zn both singly and in com-binations. When exposing the cells to a range of increasing Cdconcentrations, a marked decline in cell viability was observedbetween 1 and 10 μM Cd. In the same cell line, Kim et al.(2003) also reported a decrease in cell viability after exposureto 0.3 μM Cd for 12 h, and after exposure to concentrationsbetween 1 and 2 μM Cd (Son et al., 2001), which are in agree-ment with our findings. However, in the experiments by Kimet al. (2003), the Cd concentration producing a decline in cell

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DNA DOUBLE-STRAND BREAKS IN THE H4IIE CELL LINE 159

viability was lower than the findings in our study. This maypossibly be explained by the fact that the H4IIE cells in theexperiments by Kim et al. (2003) had been starved for 12 hprior to exposure. Cadmium cytotoxicity has been associated withthe metal interfering with mitochondrial membrane potentials,leading to ROS formation and lipid peroxidation (Pourahmad &O’Brien, 2000).

When exposing the H4IIE cells to a range of Cu concen-trations, a fall in cell viability was found to occur between500 and 1000 μM Cu. Rau et al. (2004) found cell survival of75% after exposing H4IIE cells to 500 μM of Cu for 12 h.This indicates lower cell viability than the findings in ourexperiment. In a study with another cell line, Hep G2, Sethet al. (2004) showed a decline in cell viability after exposureto 50 μM Cu after both 24 and 48 h of exposure, indicatingthat the H4IIE cell line is relatively tolerant to Cu exposure

compared to the Hep G2 cell line. Copper bound to metal-lothionein enters cellular lysosomes. Copper cytotoxicity wasshown to arise from production of ROS in lysosomes, withfollowing lysosomal membrane damage and release of lysosomalproteases and phospholipases into the intracellular matrix(Pourahmad et al., 2001).

After exposing the H4IIE cells to a range of increasing Znconcentrations, a decrease in cell viability was observedbetween 10 and 100 μM. No comparable experiments assess-ing cell viability after Zn exposure in the H4IIE cell line wasfound in the literature. Devergnas et al. (2004) showed adecline in cell viability in the HeLa cell line at concentrationsof 150 μM and higher, which may indicate that the H4IIE cellline is more sensitive to Zn exposure than HeLa cells.

Exposing H4IIE cells to combinations of the three metalsgave variable results. The cells exposed to a combination of

FIG. 1. Cell viability in the H4IIE cells given as percent of control values after 36 h of exposure to (A) CdCl2, where each point represents the mean ± SD of 5parallels for each concentration; (B) CuCl2, where the circular points represents the mean ± SD for 7 parallels, the triangular point the mean ± SD for 2 parallels,and the square point the mean ± SD for 5 parallels; and (C) ZnCl2 where the circular points represent the mean ± SD for 2 parallels, the triangular points the mean± SD for 3 parallels, and the square points the mean ± SD for 5 parallels.

µM CdCl2

10–5 10–4 10–3 10–2

(a) (b)

(c)

10–1 100 101 102

µM CuCl2

10–3 10–2 10–1 100 101102 103 104

µM ZnCl2

10–3 10–2 10–1 100 101 102 103

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1 μM Cd and 100 μM Cu showed a significant reduction in cellviability compared to control. This is in agreement with Fotakisand Timbrell (2006), who found a decrease in cell viability whencoexposing the HTC cell line and the HepG2 cell line to Cu

and Cd, as measured by the neutral red (NR) assay. However,in the same study, coexposure to Cu and Cd showed no effectson lactate dehydrogenase (LDH) leakage, and the authors sug-gested that Cu affected the lysosomal stability in these cell

FIG. 2. Cell viability in the H4IIE cells given as percent of control after 36 h of exposure to combinations of the metals Cd, Cu, and Zn (given in Table 2). Eachbar represents the mean ± SD for each exposure category. The control bar is based on 29 parallels, with the bars representing high concentrations of Cu and Cdcombined, and low concentrations of Cu and Cd combined are based on 13 parallels. The rest of the bars are based on 6 parallels.

FIG. 3. DNA fragment size distributions given as MML values in H4IIE cells exposed to the metals Cd and Cu singly, and to Cd, Cu, and Zn in differentcombinations (concentrations are given in Table 2). In each box, the top line represents the 75% fractile and the bottom line represents the 25% fractile. The solid-drawn line inside the box represents the median value, and the dotted line represents the mean. The bars above the box represent the 90% fractile, and the barsbelow the box represent the 10% fractile.

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lines. Urani et al. (2005) found that preexposure of the HepG2cell line to non-cytotoxic concentrations of Zn prior to Cdexposure decreased the cytotoxicity of Cd, possibly due tometallothionein induction by Zn. None of the other metal com-binations tested were significantly different from control. Withthe exception of cells exposed to high concentrations of Cu andCd in combination, the findings in our study indicated no addi-tive effects or synergistic interactions between the metalsoccurred.

DNA Double-Strand Break InductionAssessment of DNA double-strand break induction in the

H4IIE cells after metal exposure was performed by comparingMML values calculated from the agarose gel image dataobtained from electrophoretic analysis of the differentiallyexposed cell cultures. A significant reduction in MML valueswas found in cells exposed to 1 μM Cd and 100 μM Cu in com-bination. Both Cu and Cd are known ROS inducers (Waisberget al., 2003; Valko et al., 2005). Cadmium has, as previouslymentioned, the ability to participate in ROS formation throughdisturbances of mitochondrial electron transfer chains (Wanget al., 2004) and processes involving lysosomal membranedamage (Fotakis et al., 2005), while Cu produces ROS throughthe Fenton mechanism (Valko et al., 2005). Cadmium has beenassociated with formation of hydrogen peroxide, and superox-ide radicals, which are converted to hydrogen peroxide bySOD. Hydrogen peroxide is catalyzed to reactive hydroxyl rad-icals via the Fenton reaction (Szuster-Ciesielska et al., 2000;Shih et al., 2004; Bertin & Averbeck, 2006). An increased pro-duction of hydroxyl radicals when coexposing the cells to Cuand Cd might explain the decrease in cell viability and increasein DNA double-strand break formation that was observed inour study. ROS induce single-strand breaks directly by attack-ing deoxyribose, or indirectly as intermediate steps in the baseexcision repair pathway for repair of oxidized DNA bases. Thisfurther leads to induction of DNA double-strand breaks duringreplication (Evans et al., 2004; Helleday et al., 2007; Hedgeet al., 2008). The ability of Cd to inhibit DNA repair mecha-nisms (Dally & Hartwig, 1997; Asmuss et al., 2000; Jin et al.,2003) might also contribute to the observed effect.

When cells were exposed to 1 μM Cd, 100 μM Cu, and 10 μMZn in combination, the DNA fragment sizes had significantlyhigher values than both control cells and cells exposed to highconcentrations of Cu and Cd in combination without Znpresent. Zinc is an essential element that plays a critical role inantioxidant enzymes such as Cu/Zn-SOD and in Zn fingerproteins involved in cell signal pathways, DNA repair mecha-nisms, and transcription factors (Ho et al., 2004). The SigmaRPMI 1640 medium formulation does not include Zn salts,which may explain why the DNA damage level in our study islower than in control cells when high concentrations of thiselement are present. Hormesis is a favorable biologicalresponse to low levels of toxins, and is characterized by induction

of stress proteins such as heat-shock protein 70 (Hsp70) andmetallothionein (Damelin et al., 2000). All the metals in ourstudy are known inducers of Hsp70, and both Cd and Zninduce metallothionein (Murata et al., 1999). As previouslymentioned, both Cu and Cd are known to induce ROS. Zincwas found to protect sulfhydryl groups against oxidativedamage (Bray & Bettger, 1990), compete with toxic metals forcellular binding sites, and induce metallothionein (Kägi &Schäffer, 1988). Metallothionein is present in the H4IIE cellline at detectable levels (Son et al., 2001). The binding affinityto metallothionein for the metals in our study is as follows:Cu > Cd > Zn (Kägi & Schäffer, 1988). The induction poten-cies of the metals and the binding affinity taken togethermight offer an explanation for our results, with Zn inducingmetallothionein, which in turn binds Cu and Cd. Fotakis andTimbrell (2006) found a decrease in Cd uptake in the HepG2and HTC hepatoma cell lines when Zn was present and pro-posed competition for uptake routes as a possible mecha-nism. The same mechanism was suggested to be involved inan in vivo study concerning Cd-induced hepatotoxicity andZn protection in male LE rats (Takamure et al., 2006).Fotakis and Timbrell (2006) also reported an increase in Cduptake in the HTC cells when Cu was present, while theuptake of Cd decreased when Zn was present. Increased Cduptake in the hepatoma cell line with the presence of Cu mayperhaps help explain both the decrease in cell viability andincrease in DNA double-strand break levels found in ourexperiment, whereas the decreased uptake shown by coexpo-sure to zinc might explain some of the protective effect ofthis metal. When coexposing HeLa cells to Cd and Zn,Szuster-Ciesielska et al. (2000) showed that the metalsmutually influenced and decreased the penetration of themetals into the cells.

The other exposure conditions tested did not differ signifi-cantly from controls. Environmentally relevant concentrationsof the metals Cd, Cu, and Zn did not appear to produce anelevated level of DNA double-strand breaks in exposed H4IIEcell line. However, extrapolation of data obtained in vitro to invivo conditions is not a straightforward matter. Samplesobtained from the environment also, in addition to the metalsin question, contain a wide range of other components with theability to affect the response of the cultured cells.

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