Antimony Impairs Nucleotide Excision Repair: XPA and XPE as Potential Molecular Targets

Antimony Impairs Nucleotide Excision Repair: XPA and XPE asPotential Molecular Targets

Claudia Grosskopf,† Tanja Schwerdtle,‡ Leon H. F. Mullenders,§ and Andrea Hartwig*,†

Fachgebiet Lebensmittelchemie und Toxikologie, Institut fur Lebensmitteltechnologie und Lebensmittelchemie,Technische UniVersitat Berlin, GustaV-Meyer-Allee 25, 13355 Berlin, Germany, Institut fur Lebensmittelchemie,Westfalische Wilhelms-UniVersitat Munster, Corrensstrasse 45, 48149 Munster, Germany, and Department of

Toxicogenetics, Leiden UniVersity Medical Center, EinthoVenweg 20, 2333 ZC, Leiden, The Netherlands

ReceiVed January 4, 2010

Trivalent antimony is a known genotoxic agent classified as a possible human carcinogen by theInternational Agency for Research on Cancer (IARC) and as an animal carcinogen by the German MAKCommission. Nevertheless, the underlying mechanism for its genotoxicity remains elusive. Because ofthe similarities between antimony and arsenic, the inhibition of DNA repair has been a promisinghypothesis. Investigations on the removal of DNA lesions now revealed a damage specific impairmentof nucleotide excision repair (NER). After irradiation of A549 human lung carcinoma cells with UVC,a higher number of cyclobutane pyrimidine dimers (CPD) remained in the presence of SbCl3, whereasprocessing of the 6-4 photoproducts (6-4PP) and benzo[a]pyrene diol epoxide (BPDE)-induced DNAadducts was not impaired. Nevertheless, cell viability was reduced in a more than additive mode aftercombined treatment of SbCl3 with UVC as well as with BPDE. In search of the molecular targets, adecrease in gene expression and protein level of XPE was found, which is known to be indispensable forthe recognition of CPD. Moreover, trivalent antimony was shown to interact with the zinc finger domainof XPA, another NER protein, since SbCl3 mediated a concentration dependent release of zinc from apeptide consistent with this domain. In the cellular system, association of XPA to and dissociation fromdamaged DNA was diminished in the presence of SbCl3. These results show for the first time that trivalentantimony interferes with proteins involved in nucleotide excision repair and partly impairs this pathway,pointing to an indirect mechanism in the genotoxicity of trivalent antimony.

1. Introduction

From a toxicological point of view only sparse attention hasbeen paid to antimony for a long time. In contrast to thecomparatively well investigated arsenic, little is known aboutthis metalloid to date, making an evaluation of its toxicitydifficult. Nevertheless, there is an urgent demand for furtherinformation since the growing industrial use of antimony as acatalyst or flame retardant in the last few decades resulted inincreased exposure. The existing toxicological data point to ahigh similarity between antimony and arsenic: Both are clas-togenic in their trivalent state, whereas the less toxic pentavalentspecies reveal no genotoxicity. In addition, both metalloids givenegative results in commonly applied mutagenicity assays (1, 2).

The question of the carcinogenicity of antimony in humansis still unsettled since coexposure with arsenic hampers aninterpretation of epidemiological data. However, based on twoinhalation studies with rats (3, 4), antimony trioxide, thecommercially most important compound, was classified as apossible carcinogen to humans by IARC (Group 2B) and as ananimal carcinogen by the German MAK Commission (Carcino-gen category 2) (5, 6). Recently, the German MAK Commissionextended their classification to antimony and its inorganiccompounds, except stibin (7).

As molecular mechanisms contributing to antimony geno-toxicity the induction of oxidative stress as well as the inhibitionof DNA repair have been proposed (8, 9). In both cases,experimental data are scarce. Concerning the impact of trivalentantimony on DNA repair results are restricted to only one study,in which cytotoxic concentrations of SbIII were shown to impairDNA double strand break repair in CHO cells (10). Incomparison, DNA repair inhibition by arsenic is much betterinvestigated, especially its effect on nucleotide excision repair(NER) (11, 12). This repair pathway is responsible for theremoval of bulky and helix distorting lesions, induced, e.g., bybenzo[a]pyrene or UVC, and comprises several steps includinglesion recognition, excision of the damaged oligonucleotide,polymerization, and ligation of the new DNA fragment (13).More than 30 proteins are involved in this process includingthe xeroderma pigmentosum group A through G proteins.Defects in these proteins result in the severe human disorderxeroderma pigmentosum, which is characterized by extreme UVsensitivity and enhanced risk for skin cancer (14).

With regard to the high affinity of trivalent antimony towardthiols and imidazoles, the metalloid might be able to interactwith proteins, also with those involved in DNA repair, via theircysteine or histidine side chains. Complexes between SbIII andglutathione via the sulfur binding site of the tripeptide havealready been confirmed (15, 16). In zinc binding proteindomains, thiols and imidazoles play an eminent role. Coordinat-ing zinc through four cysteine or histidine side chains is essentialfor the correct conformation of the protein. Zinc bindingdomains mediating protein-protein interactions and DNAbinding are frequently found in proteins maintaining genomic

* To whom correspondence should be addressed. Tel: +49-030-314-72789. Fax: +49-030-314-72823. E-mail: [email protected].

† Technische Universitat Berlin.‡ Westfalische Wilhelms-Universitat Munster.§ Leiden University Medical Center.

Chem. Res. Toxicol. 2010, 23, 1175–1183 1175

10.1021/tx100106x 2010 American Chemical SocietyPublished on Web 05/28/2010

stability, e.g., in transcription factors and DNA repair proteins,but also p53 (17–20). As the transcriptional regulator of theXPC and XPE gene, the tumor suppressor protein p53 isindirectly involved in NER (21–23).

Another example for a zinc binding protein is xerodermapigmentosum group A (XPA). The 31 kDa zinc metalloproteinis assumed to be involved in damage recognition/verificationduring NER and acts as an assembly factor for the preincisioncomplex, thereby binding not only DNA but also several otherNER factors such as RPA, ERCC1, and TFIIH (24–26). Thezinc finger motif (Cys105-X2-Cys108-X17-Cys126-X2-Cys129) islocated within the minimal DNA binding domain (27) and isindispensable for XPA function since replacement of one ofthe cysteines by serine or glycine resulted in pronounced lossof DNA repair activity (28).

Within the present study, we aimed to shed more light onthe mechanism responsible for antimony genotoxicity andfocused on a possible inhibition of nucleotide excision repair.The impact of SbIII on the removal of UVC induced lesions aswell as of DNA adducts provoked by the reactive benzo[a]py-rene metabolite (+)-anti-BPDE in A549 human lung carcinomacells was measured, and cell survival after treatment with therespective compound in the presence of SbCl3 was determined.Since a lesion specific inhibition was observed by antimony,we also examined the cellular status of the XPE protein alsoknown as DDB2 or p48. Further investigations were performedon XPA and its zinc finger as a possible target for antimonyaction. At the subcellular level, a short peptide, which resemblesthe zinc finger domain of human XPA, was applied to elucidatea potential interaction with trivalent antimony by quantificationof released zinc; at the cellular level, the impact of trivalentantimony on the association and dissociation of XPA to andfrom UVC-induced DNA damage in intact cells was examined.

2. Materials and Methods

2.1. Materials. Dulbecco’s modified Eagle’s medium (DMEM),fetal bovine serum (FBS), penicillin-streptomycin solutions, andG418 were from Gibco and trypsin and dimethyl sulfoxide (DMSO)from Sigma-Aldrich. Culture dishes were from Biochrom. Antimonytrichloride was purchased from Sigma-Aldrich in high purity(99.99+ %), and (+)-anti-benzo[a]pyrene-7,8-diol 9,10-epoxide((+)-anti-BPDE) was from Biochemisches Institut fur Umweltcar-cinogene (Grossharnsdorf, Germany). The XPA peptide (XPAzf)was custom-synthesized from N-Schafer (Copenhagen, Denmark)with the sequence Ac-DYVICEECGKEFMDSYLMNHFDLPTCDN-CRDADDK-HK-NH2 and a purity >95%, and the PCR primerswere synthesized from MWG-Biotech AG (Ebersberg, Germany).HEPES, hydrogen peroxide, and BSA were from Merck. All otherchemicals were from Roth.

2.2. Cell Culture and Treatments. A549 human lung adeno-carcinoma cells (ATCC) were grown in tissue culture dishes asmonolayers in DMEM supplemented with FBS (10%), penicillin(100 units/mL), and streptomycin (100 µg/mL) in a humidifiedatmosphere with 5% CO2 at 37 °C. Fibroblasts derived from anormal individual and immortalized by telomerase transfection(VH10hTert (29)) as well as XP-E cells derived from a xerodermapigmentosum group E patient and immortalized by Epstein-Barrvirus immortalization (XP23PV (30)) were cultured under equalconditions except for DMEM additionally supplemented with G418(25 µg/mL).

After completion of at least one cell cycle, cells were treatedwith antimony trichloride. Stock solutions of SbCl3 were preparedin DMSO, stored at -20 °C, and further diluted with DMSO priorto the treatment (final DMSO concentration in the medium: 0.5%).Medium containing 0.5% DMSO was used as the negative control.For UVC exposure, cells were washed once with PBS, irradiatedwithout medium at a wavelength of 254 nm using a germicidal

lamp (Bioblock Scientific), and postincubated in the originalmedium. The exact UVC dose was verified before each treatmentby a UV-radiometer (PRC). Nonirradiated cells were treatedaccordingly but omitting irradiation.

For investigations with the reactive metabolite (+)-anti-BPDE,a stock solution of 0.5 mg/mL in water free THF/5% triethylaminewas prepared, stored at -80 °C, and diluted to a concentration of50 µM before adding to the medium (final solvent concentrationin the medium: 0.l%). Incubation time was restricted to exactly1 h, followed by dropping of the medium, and repeated washing.In the case of post-treatment with antimony, fresh medium wasadded, and cells were reincubated with SbCl3.

2.3. Cell Number and Colony Forming Ability. Logarithmi-cally growing A549 cells treated with antimony trichloride aloneor in combination with UVC (5 J/m2) or BPDE (50 nM) weretrypsinized, collected in fresh medium, and counted using a casycell counter (Scharfe Systems). Three hundred cells per dish wereseeded in triplicate into fresh medium for the determination ofcolony forming ability. After seven days, colonies were fixed withethanol, stained with Giemsa, and counted.

2.4. Detection of UVC Induced DNA Lesions. A549 cells wereseeded onto coverslips and grown for 48 h before harvesting. Cellswere pretreated with SbCl3 for times as indicated for the respectiveexperiment and finally irradiated with a dose of 10 J/m2 (CPD) or20 J/m2 (6-4PP) UVC to induce clearly detectable DNA lesions.After an adequate repair time in the presence of trivalent antimony,the coverslips were washed once in cold PBS and fixed with 0.03%formaldehyde and 0.02% Triton × 100 in PBS for 30 min on ice.This was followed by two washing steps with PBS, denaturationwith 0.1 M HCl for 10 min at 37 °C, repeated washing with PBS,and blocking with BSA (0.03% in PBS) for 1 h. Afterward, thespecific primary antibody (mouse monoclonal anti-CPD, 1:1500,mouse monoclonal anti-6-4PP, 1:400; MBL) was applied andincubated at 37 °C for 1 h. Coverslips were washed 3× withwashing buffer (0.5% BSA and 0.05% Tween in PBS) andincubated for 1 h with the secondary Alexa Fluor 488-conjugatedantibody (1:500, Invitrogen). Finally, after washing and postfixationwith formaldehyde (7% in PBS), coverslips were placed onmicroscope slides with a drop of Vectashield (Vector LaboratoriesInc.). Labeled UVC lesions were visualized and recorded using anAxio Imager.M1 fluorescence microscope with AxioCam MRmcamera (Zeiss). The fluorescence intensity of at least 100 cells perslide was analyzed by the AxioVision 4.4 software (Zeiss). Onlysignals in DAPI stained nuclei were quantified. Data were correctedfor background fluorescence in nonirradiated control cells.

2.5. BPDE-DNA Adducts. BPDE-DNA adduct levels weremeasured via HPLC/fluorescence detection after the release of thecorresponding tetrols as described elsewhere (31). Briefly, treatedcells were trypsinized, washed three times with ice-cold buffer(0.0027 M KCl, 0.137 M NaCl, and 0.025 M tris-base, pH 7.4),and collected by centrifugation. DNA was isolated, washed severaltimes with 70% ethanol (Rotisol), and quantified spectrophoto-metrically by measuring the absorbance at 260 nm (PeqlabNanoDrop). After hydrolysis of adducted DNA and subsequentneutralization, tetrol I-1 was quantified via HPLC with fluorescencedetection.

2.6. Gene Expression. Real time RT-PCR was performed forquantification of the XPA and XPE mRNA level according to Nollenet al. (32). Incubated cells were harvested, washed once, andresuspended in PBS. Afterward, RNA was isolated with NucleospinRNA-Isolation Kit (Macherey-Nagel) and quantified at 260 nm byapplying a NanoDrop spectrophotometer (Peqlab). One microgramof RNA was transcribed into cDNA (qscript cDNA synthesis kit,Quanta Biosciences) and an aliquot of the product was used forreal time PCR with an iCycler (Bio-Rad) by applying the forwardprimer (XPA: 5′-CCG ACA GGA AAA CCG AGA AA-3′; XPE:5′-CCT GAA CCC ATG CTG TGA TTG-3′) and the reverse primer(XPA: 5′-TTC CAC ACG CTG CTT CTT ACT G-3′; XPE: 5′-GCT GGC TTT CCC TCT AAC CTG-3′) as well as B-RSYBRGreen Supermix (Quanta Biosciences). The specificity of theamplification products was checked by melting curve analysis. The

1176 Chem. Res. Toxicol., Vol. 23, No. 7, 2010 Grosskopf et al.

relative expression ratio R was calculated on the basis of theefficiency E, the threshold cycle (Ct), and the reference geneGAPDH.

2.7. Protein Levels. The impact of antimony on the XPA andXPE protein levels was investigated by Western blot analysis.Trypsinized cells were collected in PBS with 10% FBS, centrifuged,washed once with PBS, and sonicated in RIPA buffer (0.01 M Tris,pH 7.6, 0.15 M NaCl, 0.001 M EDTA, 0.001 M PMSF, 1% TritonX-100, 1% sodium desoxycholate, 1 µg/mL aprotinin, 1 µg/mLleupeptin, 1 µg/mL pepstatin, 1% DOC, 50 mM NaF, 0.1% SDS,and 1 mM Na3VO4). After centrifugation, the protein content ofthe supernatant was quantified (Bradford). Loading buffer and DTT(both Fermentas) were added to an aliquot of the cell extract, andthe proteins were denatured at 95 °C for 10 min. Subsequently,proteins were separated by a 12% denaturing SDS-PAGE andtransferred to a PVDF membrane (GE Healthcare) using a semidryblotter (Peqlab). The membrane was blocked (5% milk powder inPBST), incubated with the primary antibody (rabbit anti-XPApolyclonal antibody, 1:1000, Santa Cruz; goat anti-XPE polyclonalantibody, 1:2000, R&D Systems) overnight at 4 °C, and washedthree times in PBST for 5 min before the HRP-conjugated secondaryantibody (goat antirabbit polyclonal antibody, 1:2000, Santa Cruz;donkey antigoat polyclonal antibody, 1:2000, Santa Cruz) wasapplied for 1 h at room temperature. This procedure was followedby three washing steps in PBST and one in PBS. Detection wasaccomplished by enhanced chemiluminescence reaction (ECL)measured with a LAS 3000 cooled CCD camera (FujiFilm). Proteinlevels were quantified by densitometric analysis with AIDA ImageAnalyzer software (Version 4.13, raytest, Straubenhardt, Germany)and normalized to controls.

2.8. Zinc Release from XPAzf. For the studies on zinc release,a short peptide (37 amino acids) representing the zinc finger domainof human XPA was applied as described elsewhere (12). Aftersaturation with zinc buffer, the peptide (20 µM) was incubated withantimony chloride in 20 mM HEPES-NaOH buffer (pH 7.4) at 37°C for 30 min. Subsequently, zinc release was quantified spectro-photometrically via complexation with 4-(2-pyridylazo)-resorcinol(PAR, 100 µM, Riedel-de Haen) at 492 nm. Results were referredto 10 mM H2O2 (Merck) corresponding to 100% zinc release.

2.9. XPA Association and Dissociation. Investigation on theDNA damage assembly of XPA was performed by generation oflocal DNA lesions in combination with immunofluorescent labelingof the protein as described before (32, 33). A549 cells, grown oncoverslips, were preincubated with SbIII for 2 h and washed oncein PBS before they were covered by an 8 µm pore filter (Millipore)during irradiation with a UVC dose of 30 J/m2 which correspondsto a global UVC dose of 10 J/m2. After the filter was carefullyremoved, cells were postincubated in the same medium for theindicated time. Subsequently, cells were fixed and treated accordingto the UVC lesion detection protocol except for denaturation, whichwas omitted. The primary antibody (polyclonal rabbit anti-XPA,1:100) was obtained from Santa Cruz Biotechnologies and thesecondary Cy3-conjugated antibody (1:500) from Jackson Immu-noResearch. Evaluation of the fluorescence intensity of at least 60XPA spots was performed according to DNA lesion quantificationwith the AxioVision 4.4 software (Zeiss). Data were corrected foroverall fluorescence in nonspotted nuclei.

3. Results

3.1. Cytotoxicity. Cell number and colony forming abilitywere determined to examine the immediate as well as long-term cytotoxicity of antimony and its impact on UVC- andBPDE-induced cytotoxicity. A549 cells preincubated with SbCl3

for 2 h were irradiated with 5 J/m2 UVC or treated with 50 nMBPDE, respectively, where indicated, and postincubated withSbCl3 for 24 h (Figure 1). With respect to antimony alone,pronounced cytotoxic effects were limited to 500 µM SbCl3,the highest tested concentration, which caused a reduction incell and colony number to less than 70%. In contrast, 90% of

the cells were still viable at 250 µM SbCl3. Irradiation with aUVC dose of 5 J/m2 reduced cell viability only slightly by 10%.The combination of UVC irradiation with antimony treatment,however, resulted in a reduction of cell viability with a morepronounced effect on colony forming ability at higher SbCl3

concentrations (Figure 1a). Whereas the decrease in cell numbercan be ascribed to the addition of the cytotoxic effects of eachcomponent alone, it appears that SbIII impairs the colony formingability, a parameter for long-term cytotoxicity, in a more thanadditive mode. Similar results were obtained in combinationwith BPDE (Figure 1b). Treatment with the reactive metaboliteof benzo[a]pyrene only slightly decreased cell number andcolony forming ability, and SbCl3 caused an additive reductionin cell number as well. Most notably, colony forming abilitywas also impaired in a superadditive manner by SbCl3 inconcentrations above 250 µM, gaining an additional reductionof 30% at 500 µM SbCl3.

3.2. Repair of UVC-Induced DNA Lesions. Effects oftrivalent antimony on the repair of UVC-induced DNA lesionswere measured by immunofluorimetric detection of unprocessedphotoadducts. UVC mainly induces two kinds of lesions, thecyclobutane pyrimidine dimers (CPD) and 6-4 photoproducts(6-4PP), which were quantified after irradiation with 10 J/m2

in the case of CPD and 20 J/m2 in the case of 6-4PP, dosesrequired to achieve sufficient detectable lesions (33). Accordingto their fast repair, removal of 6-4PP was determined 2 h afterirradiation. Within that time, almost 70% of the initially inducedlesions became repaired in irradiated cells in the absence ofantimony treatment corresponding to a repair efficiency of100%. Preincubation with antimony for 2 h (data not shown)and 24 h had no effect on the removal (Figure 2a and b).Nevertheless, SbCl3 impaired the removal of the CPD, which

Figure 1. Impact of SbCl3 on the viability of A549 cells with andwithout UVC irradiation (a) or BPDE treatment (b). Cells werepreincubated with antimony for 2 h, treated with 5 J/m2 UVC or 50nM (+)-anti-BPDE for 1 h where indicated, and postincubated for 24 hin the presence of SbIII. Afterward, the cells were trypsinized andcounted. Three × 300 cells were seeded into fresh medium and grownfor one week before colonies were fixed, stained, and counted. Resultsrefer to cell or colony number for the untreated solvent control. Shownare the mean values of 3 independent determinations + SD.

Antimony Impairs Nucleotide Excision Repair Chem. Res. Toxicol., Vol. 23, No. 7, 2010 1177

were detected 24 h after irradiation due to their slow repair(Figure 2a). In the absence of antimony, about two-thirds ofthe dimers were removed, whereas 2 h pre- and 24 h postin-cubation with antimony resulted in larger amounts of remainingCPD even at noncytotoxic concentrations (Figure 2b). At 250µM SbCl3, the fraction of nonrepaired lesions increased to nearly50%. The number of still remaining CPD in the presence of500 µM SbIII after 24 h was even twice as high as compared tothat of untreated cells, consistent with a repair inhibition of morethan 50%.

3.3. Repair of BPDE-DNA Adducts. The impact of trivalentantimony on nucleotide excision repair was also investigatedfor the removal of BPDE-DNA adducts. The main metaboliteof benzo[a]pyrene, (+)-anti-BPDE, causes specific DNA ad-ducts at the N2 position of guanine, from which tetrol I-1 canbe generated during hydrolysis with HCl. Analysis of the tetrolcontent by HPLC fluorescence detection reflects the number ofDNA adducts. Determination of the adduct levels was performedat two time points, immediately (0 h) and 8 h after incubationwith 50 nM BPDE to examine the induction of adducts as wellas their repair. Preincubation of A549 cells with SbCl3 for 2 hdid not affect the formation of BPDE adducts (Figure 3). Onlya marginal reduction in the adduct level was found at 500 µMSbCl3. No effect was also observed on the repair after 8 h inthe presence of antimony. The efficiency of adduct removal wasabout 30% in the absence or presence of SbCl3. Extending thepreincubation time with SbCl3 to 24 h did not affect the adductlevel either (data not shown). Thus, trivalent antimony appearsto compromise neither BPDE-DNA adduct formation norBPDE-DNA adduct removal.

3.4. XPE Gene Expression and Protein Level. The NERprotein XPE is known to be a discriminating factor for therecognition of the CPD. In contrast to 6-4PP, the removal ofthe pyrimidine dimers is impaired profoundly in the absence of

functional XPE (21, 34). Thus, the cellular XPE status wasanalyzed in A549 cells by determining gene expression by RT-PCR and protein level by Western Blot. Analysis of thetranscript level revealed that SbCl3 did not affect XPE genetranscription after an incubation time of 10 h. During thisincubation time, it also did not impair the induction of XPEexpression by UVC, which in preliminary experiments wasshown to reach its maximum 8-10 after irradiation. Two hourspre- and 8 h postincubation with SbCl3 resulted in an equalincrease of XPE transcripts of more than 70% (Figure 4a).Moreover, no effect of SbCl3 was observed on the XPE proteincontent up to 6 h after irradiation with UVC (data not shown).However, SbCl3 caused a concentration dependent decline inthe XPE mRNA level after 24 h, a time period whichcorresponds to the overall treatment with SbCl3 in the studieson CPD and 6-4PP removal (Figure 4b). Significant reduction

Figure 2. Impact of SbIII on the removal of UVC-induced DNA lesions. (a) A549 cells were incubated with SbCl3 for 24 h (6-4PP) or 2 h (CPD)prior to UVC irradiation and postincubated with SbIII for the times indicated in the figure. Afterward, the cells were washed in PBS and fixed withformaldehyde. Detection of DNA lesions was performed by fluorescence antibody labeling. (b) Fluorescence signals within the nucleus were quantifiedand refer to the amount of induced lesions in the solvent control corresponding to 100%. Shown are the mean values from 3 determinations + SD.Significantly different from the control as determined by the t-test (**P < 0.01, ***P < 0.001).

Figure 3. BPDE-DNA adduct level 0 and 8 h after treatment with (+)-anti-BPDE in the presence of SbCl3. A549 cells were preincubatedwith SbCl3 for 2 h, treated with 50 nM (+)-anti-BPDE for 1 h, andpostincubated with the antimony compound for the times as indicated.DNA adducts were quantified via the tetrol by HPLC-fluorescencedetection. Shown are the mean values of 3 independent determinationsperformed in triplicate + SD.


by almost one-third was observed at noncytotoxic 250 µMSbCl3, whereas at 500 µM SbCl3, the transcript number wasdiminished by half. This effect on gene expression was alsodetectable at the protein level. Twenty-four hours of incubationwith trivalent antimony resulted in up to 50% reduction in XPEprotein content (Figure 4c and d). In combination with UVCirradiation, the decline was even more pronounced. Irradiationwith 5 J/m2 UVC alone increased the amount of XPE by morethan 40% after 24 h, whereas preincubation with SbCl3 for 2 hand postincubation for 24 h after irradiation resulted in adiminished increase at 100 µM and 250 µM SbCl3 and in evenno increase at 500 µM SbCl3. Compared to irradiated cells nottreated with antimony, only one-third of the XPE protein contentwas found in the presence of 500 µM SbCl3 pointing to anadditional impairment of UVC-induced induction of XPE.

3.5. BPDE-DNA Adduct Formation and Removal inXPE Deficient Cells. The involvement of XPE in the recogni-tion of CPD and 6-4PP is well investigated, while only little isknown in the case of BPDE-induced lesions. To examine therole of the protein in the repair of this type of lesion, XPEdeficient (XP-E cells) and XPE proficient (VH10 cells) humanfibroblasts were compared with respect to their adduct levels atdifferent times of repair (Figure 5). The number of adductsclearly declined in both cell lines displaying more than 50%removal after 72 h. Nevertheless, XPE proficient cells appearto start with repair immediately after (or even during) incubationwith BPDE (50 nM, 1 h), while the adduct level in XPE deficientcells remained unchanged for at least 8 h after treatment withthe diol epoxide. Sixteen hours later, the adduct number declinedeven in the absence of functional XPE, indicating that finallyrepair had taken place with similar efficiency. However,

decomposition of adducts derived by mechanisms other thanNER cannot be excluded.

3.6. Zinc Release from XPAzf. An interaction of trivalentantimony with the repair protein xeroderma pigmentosum groupA was investigated by measuring zinc release from XPAzf, a37 amino acid containing peptide saturated with Zn2+, whichrepresents the zinc finger domain of human XPA. Deliberatedzinc was quantified by complexation with 4-(2-pyridylazo)-resorcinol (PAR) and spectrometric measurement at 492 nm.SbIII significantly provoked zinc release from XPAzf (20 µM)in a concentration dependent manner starting at a less thanequimolar ratio of 10 µM SbCl3 (Figure 6). In great excess(500-1000 µM SbCl3), the compound was almost as effectiveas 10 mM H2O2. Antimony itself displayed no interaction with

Figure 4. Gene expression and protein levels of XPE in A549 cells after treatment with SbCl3 or SbCl3 and UVC. (a) mRNA levels of XPE after10 h of treatment with SbCl3 or 2 h of pretreatment with SbCl3, irradiation with 5 J/m2 UVC, and 8 h posttreatment with SbCl3. RNA was isolatedfrom collected cells and rewritten into cDNA before real time RT-PCR was performed. XPE transcript number was normalized to GAPDH mRNAlevels. Shown are the mean values of 4 determinations + SD. (b) mRNA level of XPE in A549 cells after 24 h of treatment with SbCl3. Shownare the mean values of at least 3 determinations + SD. Significantly different from control as determined by the t-test (***P < 0.001). (c and d)Protein level of XPE in A549 cells after treatment with SbIII and UVC. Cells were pretreated with SbCl3 for 2 h, irradiated with 5 J/m2 UVC whereindicated, and postincubated in the continued presence of SbCl3 for 24 h. Afterward, protein was extracted from collected cells, quantified accordingto Bradford, and analyzed for XPE level by Western blot. Shown is one representative blot out of three independent determinations as well as thedensitometric quantification of all three blots (mean values + SD). Significantly different from the corresponding control cells without SbCl3 asdetermined by the t-test (**P < 0.01, ***P < 0.001).

Figure 5. BPDE adduct removal in XPE deficient (XP-E) and XPEproficient (VH10) cells. Treatment with 50 nM BPDE for 1 h wasfollowed by different repair times after which cells were harvested andthe adduct level was quantified with HPLC-fluorescence detection.Shown are the mean values of 2 independent determinations performedin triplicate + SD. Significantly different adduct level as determinedby the t-test (*P < 0.01, ***P < 0.001).


PAR, and DMSO (final concentration 1%), used as the solventfor SbCl3, also did not affect zinc release (data not shown).

3.7. XPA Association and Dissociation. The association ofXPA to damaged DNA sites was examined in the absence orpresence of trivalent antimony. To measure XPA association,local DNA lesions were induced by irradiating A549 cells with30 J/m2 UVC through a porous filter corresponding to a globalirradiation with 10 J/m2 and detection of XPA by fluorescent-labeled antibodies. XPA foci were detectable as bright distinctspots within the nucleus. Spot intensity as an indicator forassociation was measured at two time points, 10 and 60 minafter DNA damage induction. Within this time, XPA migratesto the lesion and dissociates from the repair complex again. Incells preincubated for only 2 h with SbCl3, a decrease in XPAspot intensity by up to 20% was measurable at the 10 min timepoint indicating a reduced association of the protein (Figure7). Sixty minutes after irradiation, spot intensity declined toalmost 50% in the absence of antimony. However, the spotintensity in antimony treated cells declined to a considerablylesser extent indicating a delayed or disturbed dissociation ofthe protein after 60 min (Figure 7). Association and dissociationof the NER protein XPC, assembling to the DNA lesion beforeXPA, was also investigated. In contrast to XPA, no effects onthe association of XPC were observable after 2 h of preincu-bation with antimony (data not shown).

3.8. XPA Gene Expression and Protein Level. To elucidatethe impact of trivalent antimony on the XPA protein level, wedetermined the transcription of the corresponding gene after

treatment with antimony alone as well as in combination withUVC. After preincubation with SbCl3 for 2 h, irradiation withUVC, and postincubation for 8 h in the presence of theantimony, compound cells were harvested, and mRNA wasanalyzed by real time RT-PCR. In the range of 100-500 µM,trivalent antimony itself did not significantly alter the mRNAlevel of XPA in A549 cells within this time (Figure 8a). Thiswas also the case for long time incubation with SbCl3 of 24 h(data not shown). Irradiation of the cells with a UVC dose of 5J/m2 alone also did not affect XPA expression after 8 h.Likewise, the combination of antimony treatment and UVCirradiation had no impact on the level of XPA transcripts aswell. Western blot analysis confirmed these results. No changesin the protein level of XPA were observable after 24 h ofincubation with 100-500 µM SbCl3 (Figure 8b). This was alsothe case for an additional UVC treatment: neither UVCirradiation alone nor UVC irradiation in the presence of SbCl3

affected the XPA protein level after 24 h.

4. Discussion

Inhibition of DNA repair is a common feature of genotoxicmetals such as cadmium, cobalt, and arsenic (35). However, inthe case of antimony experimental evidence has been missing.Our results now show that SbIII exerted a pronounced impacton nucleotide excision repair as demonstrated for the removalof UVC-induced DNA damage.

Two kinds of helix distorting lesions, the CPD and the 6-4PP,are predominantly generated by UVC irradiation. Nonetheless,antimony only impaired the repair of one of these lesions.Whereas the removal of 6-4PP was not affected, the number ofremaining CPD increased in the presence of SbCl3, starting atnoncytotoxic concentrations. The unequal impact on thesephotolesions may be explained by the faster and thus preferred

Figure 6. Effect of SbIII on zinc release from XPAzf after 30 min ofincubation at 37 °C. Deliberated zinc was quantified spectrophoto-metrically via complex formation with PAR. Results refer to zinc releaseprovoked by 10 mM H2O2 (corresponding to 100%). Shown are themean values of at least 3 independent determinations ( SD (**P <0.01, ***P < 0.001) as determined by the t-test.

Figure 7. Impact of SbIII on the association of XPA to local DNAlesions. A549 cells were pretreated with SbCl3 for 2 h, irradiated througha pore filter with 30 J/m2 UVC, and postincubated with the chemicalfor the times as indicated. Subsequently, the cells were fixed, and XPAspots were detected via fluorescent antibody labeling. Spot intensitycorrelates with the association of the protein to damaged DNA. Resultsrefer to the 10 min UVC solvent control. Shown are the mean valuesof 3 determinations + SD. Significantly different from the correspond-ing control (10 or 60 min) as determined by the t-test (*P < 0.05, **P< 0.01, ***P < 0.001).

Figure 8. Gene expression and protein levels of XPA in A549 cellsafter treatment with SbCl3 or SbCl3 and UVC. (a) mRNA level of XPAin A549 cells after treatment with SbIII and UVC. Cells were pretreatedwith SbCl3 for 2 h, irradiated with a UVC dose of 5 J/m2 whereindicated, and postincubated in the presence of antimony for 8 h. RNAwas isolated from collected cells and rewritten into cDNA before realtime RT-PCR was performed. XPA transcript number was normalizedto GAPDH mRNA levels. Shown are the mean values of 4 determina-tions + SD. (b) Protein level of XPA in A549 cells after treatmentwith SbIII and UVC. Cells were pretreated with SbCl3 for 2 h, irradiatedwith 5 J/m2 UVC where indicated, and postincubated for 24 h.Afterward, protein was extracted from collected cells, quantifiedaccording to Bradford, and analyzed for XPA level by Western blot.Shown is one representative blot out of at least three independentdeterminations.


repair of the 6-4PP, which cause larger helix distortions and, ifunrepaired, exhibit a higher mutagenic potential than the CPD(36, 37). Damage recognition within global genomic repair ofthe nontranscribed DNA strand is assumed to be mainlyresponsible for differences in their removal. While most lesionsare directly recognized by XPC, the subtle helix distortioninduced by CPD is assumed to require a more sensitiverecognition process. This seems to be accomplished by anadditional sensor, the UV-DDB complex, which consists of twosubunits, DDB1/p127 and DDB2/XPE/p48, and specificallybinds UV-damaged DNA with high affinity (38, 39). Mutationsin the XPE gene were shown to be responsible for the clinicalsymptoms of xeroderma pigmentosum complementation groupE, which is characterized by only partial repair inhibition andmodest sensitivity toward UV light (30, 40). It was demonstratedthat XPE is necessary for the recruitment of XPC to CPD butnot to 6-4PP (41) and enhances global genomic repair ofpyrimidine dimers, while the repair of 6-4PP was less affectedin the absence of functional XPE (21, 29, 34, 42). In the presentstudy, quantification of the XPE protein level revealed a declineof cellular XPE after incubation with even noncytotoxic SbCl3

concentrations especially in combination with UVC, resultingin up to 67% less protein. Thus, XPE protein might becomelimiting for the recognition of CPD after antimony treatmentsince the protein is tightly regulated. XPE is degraded via theproteasome shortly after UV irradiation but also induced severalhours later by its transcriptional regulator p53 (21, 23, 42, 43).Considering our observation that incubation with SbIII for 24 haffects XPE already at the transcriptional level, it may well bethat the expression of the XPE gene by p53, regulating also thebasal level of this protein, is impaired. In contrast, shortincubation times did not alter the XPE status even in combina-tion with UVC, which indicates a rather late impact on theremoval of CPD.

In addition to UVC-induced lesions, the effect of SbCl3 onthe removal of BPDE-DNA adducts was also quantified.Immediately (0 h) as well as 8 h after incubation with BPDE,the adduct levels were not affected by short (2 h) or even longtime (24 h) pretreatment with trivalent antimony followed bycontinued treatment after BPDE-incubation. To elucidate therole of XPE in the repair of this type of lesion, adduct removalin XPE deficient and proficient cells was compared. Both celllines were able to repair BPDE adducts, but the repair kineticdiffered considerably. Up to 8 h after BPDE treatment, XPEdeficient cells displayed almost no adduct removal, while at latertime points, repair efficiency appeared to be similar to that ofXPE proficient cells. Although it can not be completely excludedthat the reduction of lesions might be due to a decompositionof BPDE-DNA adducts by alternative mechanisms, such ashydrolysis of the N-glycosidic bond, or due to the fact that somedifferences may be due to the comparison of nonisogenic cells,the results appear to be best explained by an accelerated removalof these lesions in the presence of XPE, which is, however, notindispensable for their repair. This characteristic had been alsodemonstrated for the removal of the 6-4PP after low doses ofUVC pointing to a stimulated recognition of a limited numberof lesions by the UV-DDB complex (29). With respect to ourresults and in contrast to completely XPE deficient cells, theamount of XPE after treatment with SbCl3 might have beenstill sufficient to efficiently recognize the relatively small numberof BPDE-DNA adducts, considering the fact that the numberof lesions induced by 50 nM BPDE is several magnitudes lowerthan the number of CPD and 6-4PP induced by 10 or 20 J/m2

UVC.

Despite the fact that removal of the BPDE-induced lesionswas not impaired by antimony, a pronounced reduction in cellviability was observed. In combination with high SbCl3

concentrations, colony forming ability declined more thanadditive after treatment with BPDE, while this effect was notdetectable in the cell number after 24 h. Nearly identicalobservations were also made for lesions induced by a noncy-totoxic UVC dose when cotreated with the same concentrationsof trivalent antimony. Surprisingly, the extent of the reductionin colony number was very similar for both, although far morelesions were induced by 5 J/m2 UVC as compared to 50 nMBPDE. Thus, trivalent antimony appears to affect the cellularresponse to NER lesions generally and independent from thetype of lesion, which seems to contradict our results on aselective impairment of lesion removal. However, an impactof SbCl3 on the metabolism of BPDE, which is known to playa major role in the toxicity of this compound, cannot be ruledout. Moreover, the test system applied quantifies adduct levelsand their incision, while late steps in NER such as polymeri-zation/ligation may be affected as well. In any case, loss offunctional XPE is known to have only mild effects on cellviability after irradiation with UVC, which is probably due tothe fact that unrepaired CPD seem not to trigger cell deathbecause these lesions can be bypassed by polymerase η(14, 44, 45). Thus, it is most likely that a further defect isresponsible for the impact of SbCl3 on cell viability aftermutagen treatment.

Several observations on XPA, an indispensable componentof the NER damage recognition complex, suggest that accordingto its expression levels as well as to modifications in its sequencepreferential repair of certain lesions may also occur (46–49).Concerning these data, an impact on XPA by trivalent antimonymight deliver a further explanation for lesion specific repairinhibition. XPA contains a zinc binding motif, which consistsof four cysteines and thus represents a possible target for SbIII

displaying a high affinity toward thiols. As mentioned before,the zinc finger is part of the minimal DNA binding domain (27)and is indispensable for XPA function during NER. It had beenpreviously shown that SbIII causes the loss of almost all protein-bound zinc from rat liver metallothionein, in which zinc istetrahedrally coordinated to four cysteines similar to XPA (50).Within the present study, we demonstrated the ability ofantimony to release zinc from the zinc finger domain of humanXPA in a concentration dependent manner starting at equimolarconcentrations. In previous studies, several metals, includingcadmium or cobalt, caused zinc release from this peptide aswell (12, 51, 52). Compared to the data on arsenite, antimonyis even more effective in releasing zinc from XPA, yielding50% zinc release at 10 times lower concentrations (12).

To elucidate a potential impact on XPA function in livingcells, the association of XPA to sites of DNA damage wasexamined by immunofluorescent labeling after local irradiationof A549 cells with UVC. Declined spot intensity of up to 20%in cells pretreated with SbCl3 for 2 h revealed a diminishedassembly of XPA shortly after UVC irradiation, whereas 20%higher spot intensity after 1 h was observed. This can beexplained either by pronounced delayed association or by bothdisturbed association and dissociation. Which alternative appliesto antimony requires further investigations, for example, byapplying photobleaching experiments. Keeping in mind thatXPA protein is usually present in excess and has to bediminished to levels of less than 10% to be rate limiting forNER (53), these results are quite remarkable. In contrast, theassociation of the NER protein XPC, which contains no zinc


finger motif in its structure, was not impaired under equalconditions pointing to a specific effect on XPA function.Because of preincubation with SbCl3 for only 2 h, it can alsobe ruled out that loss of XPE is the primary cause since effectson gene expression and protein level were restricted to latertime points. An altered expression of the XPA gene or anenhanced degradation of the corresponding protein can beexcluded as well since real time RT-PCR as well as Westernblot analysis revealed no inhibitory effect by trivalent antimony.

The fact that antimony alters the association at the lesionsbut not the total cellular amount of XPA protein indicates animpaired function of XPA within the damage recognition/incision complex. It is well established that the interaction withRPA enhances the affinity of XPA for UV damaged DNA(54, 55). Interestingly, binding of XPA to the 70 kDa subunitof RPA is supposed to be mediated also via its zinc fingerdomain (56, 57). Thus, an interaction of SbIII with this domaincould affect the formation of a complex and might explain theretarded association of XPA to sites of UV damage. With respectto in Vitro experiments displaying a higher affinity of purifiedrecombinant XPA to 6-4-PP than to CPD (58, 59), an impairedinteraction of XPA with RPA could preferentially affect thebinding of XPA to pyrimidine dimers. It is also worth mention-ing that RPA also contains a Cys4-zinc finger motif within the70 kDa subunit, which is responsible for the redox regulatedDNA binding activity of the protein (60).

Besides RPA, an interaction of XPA with ATR via itsminimal DNA binding domain and its phosphorylation by ATRseems to be also important for the recognition of CPD.Preventing phosphorylation by ATR diminished only the repairof the CPD after irradiation with UVC, whereas removal of6-4PP was not impaired. Independent from XPA phosphoryla-tion, the interaction with ATR was also shown to be necessaryfor nuclear localization and consequently association of XPAto DNA damage as well (47). Therefore, trivalent antimonymight also act by disturbing the interaction with ATR.

Although the data presented in this article imply an interactionwith XPA, the underlying mechanism remains speculative. Theinterference with the zinc finger domain as demonstrated forthe corresponding peptide is at present the most promisingtheory. Different processes can be responsible for the observedzinc release. Cadmium or cobalt, for example, were shown toreplace the zinc in the complex, whereas arsenite oxidized thethiols to disulfides (52, 61). Moreover, the metal species playsa decisive role too. Monomethylarsonous acid (MMA(III)), ametabolite of arsenite, formed a mixture of single and doublemonomethylarsinated XPAzf as well as fully oxidized XPAzf(61). However, in each case the structure of the zinc fingerwould be impaired resulting, more or less, in a loss of XPAfunction and loss of NER capacity. In the case of trivalentantimony, further investigations are needed to reveal themechanism.

The consequences of an impairment of NER capacity are veryserious. Cells are constantly exposed to DNA damaging agentssuch as UV radiation or benzo[a]pyrene. If lesions are removedmore slowly or only partly, the frequency of mutations wouldbe expected to rise resulting in an increased cancer risk. This isalarming since antimony is ubiquitously disseminated, andexposure is magnified by several antimony-containing materialssuch as fabrics or plastics.

In conclusion, these results support the theory that antimonyinterferes with DNA repair, thereby implying an indirectmechanism of SbIII genotoxicity and perhaps carcinogenicity.A crucial step within might be the impairment of XPE gene

expression and the interaction with zinc finger domains of keyproteins such as XPA. Regarding the abundance of zinc bindingdomains, e.g., in p53, an impairment of proteins outsidenucleotide excision repair is also conceivable, creating the imageof a multitarget mode of action. Therefore, more research isrequired to achieve a more precise understanding of the effectscaused by antimony.

Acknowledgment. This work was supported by DFG GrantNumbers Ha 2372/3-4, Ha 2372/5-1, and Schw 903/3-2. Theauthors declare that there are no conflicts of interest.

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