Antimony Impairs Nucleotide Excision Repair - American Chemical

May 28, 2010 - Antimony Impairs Nucleotide Excision Repair: XPA and XPE as. Potential Molecular Targets. Claudia Grosskopf,† Tanja Schwerdtle,‡ Le...
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Chem. Res. Toxicol. 2010, 23, 1175–1183

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Antimony Impairs Nucleotide Excision Repair: XPA and XPE as Potential Molecular Targets Claudia Grosskopf,† Tanja Schwerdtle,‡ Leon H. F. Mullenders,§ and Andrea Hartwig*,† Fachgebiet Lebensmittelchemie und Toxikologie, Institut fu¨r Lebensmitteltechnologie und Lebensmittelchemie, Technische UniVersita¨t Berlin, GustaV-Meyer-Allee 25, 13355 Berlin, Germany, Institut fu¨r Lebensmittelchemie, Westfa¨lische Wilhelms-UniVersita¨t Mu¨nster, Corrensstrasse 45, 48149 Mu¨nster, 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 the International Agency for Research on Cancer (IARC) and as an animal carcinogen by the German MAK Commission. Nevertheless, the underlying mechanism for its genotoxicity remains elusive. Because of the similarities between antimony and arsenic, the inhibition of DNA repair has been a promising hypothesis. Investigations on the removal of DNA lesions now revealed a damage specific impairment of 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, whereas processing of the 6-4 photoproducts (6-4PP) and benzo[a]pyrene diol epoxide (BPDE)-induced DNA adducts was not impaired. Nevertheless, cell viability was reduced in a more than additive mode after combined treatment of SbCl3 with UVC as well as with BPDE. In search of the molecular targets, a decrease in gene expression and protein level of XPE was found, which is known to be indispensable for the recognition of CPD. Moreover, trivalent antimony was shown to interact with the zinc finger domain of XPA, another NER protein, since SbCl3 mediated a concentration dependent release of zinc from a peptide consistent with this domain. In the cellular system, association of XPA to and dissociation from damaged DNA was diminished in the presence of SbCl3. These results show for the first time that trivalent antimony 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 has been paid to antimony for a long time. In contrast to the comparatively well investigated arsenic, little is known about this metalloid to date, making an evaluation of its toxicity difficult. Nevertheless, there is an urgent demand for further information since the growing industrial use of antimony as a catalyst or flame retardant in the last few decades resulted in increased exposure. The existing toxicological data point to a high similarity between antimony and arsenic: Both are clastogenic in their trivalent state, whereas the less toxic pentavalent species reveal no genotoxicity. In addition, both metalloids give negative results in commonly applied mutagenicity assays (1, 2). The question of the carcinogenicity of antimony in humans is still unsettled since coexposure with arsenic hampers an interpretation of epidemiological data. However, based on two inhalation studies with rats (3, 4), antimony trioxide, the commercially most important compound, was classified as a possible carcinogen to humans by IARC (Group 2B) and as an animal carcinogen by the German MAK Commission (Carcinogen category 2) (5, 6). Recently, the German MAK Commission extended their classification to antimony and its inorganic compounds, except stibin (7). * To whom correspondence should be addressed. Tel: +49-030-31472789. Fax: +49-030-314-72823. E-mail: [email protected]. † Technische Universita¨t Berlin. ‡ Westfa¨lische Wilhelms-Universita¨t Mu¨nster. § Leiden University Medical Center.

As molecular mechanisms contributing to antimony genotoxicity the induction of oxidative stress as well as the inhibition of DNA repair have been proposed (8, 9). In both cases, experimental data are scarce. Concerning the impact of trivalent antimony on DNA repair results are restricted to only one study, in which cytotoxic concentrations of SbIII were shown to impair DNA double strand break repair in CHO cells (10). In comparison, DNA repair inhibition by arsenic is much better investigated, especially its effect on nucleotide excision repair (NER) (11, 12). This repair pathway is responsible for the removal of bulky and helix distorting lesions, induced, e.g., by benzo[a]pyrene or UVC, and comprises several steps including lesion recognition, excision of the damaged oligonucleotide, polymerization, and ligation of the new DNA fragment (13). More than 30 proteins are involved in this process including the xeroderma pigmentosum group A through G proteins. Defects in these proteins result in the severe human disorder xeroderma pigmentosum, which is characterized by extreme UV sensitivity and enhanced risk for skin cancer (14). With regard to the high affinity of trivalent antimony toward thiols and imidazoles, the metalloid might be able to interact with proteins, also with those involved in DNA repair, via their cysteine or histidine side chains. Complexes between SbIII and glutathione via the sulfur binding site of the tripeptide have already been confirmed (15, 16). In zinc binding protein domains, thiols and imidazoles play an eminent role. Coordinating zinc through four cysteine or histidine side chains is essential for the correct conformation of the protein. Zinc binding domains mediating protein-protein interactions and DNA binding are frequently found in proteins maintaining genomic

10.1021/tx100106x  2010 American Chemical Society Published on Web 05/28/2010

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stability, e.g., in transcription factors and DNA repair proteins, but also p53 (17–20). As the transcriptional regulator of the XPC and XPE gene, the tumor suppressor protein p53 is indirectly involved in NER (21–23). Another example for a zinc binding protein is xeroderma pigmentosum group A (XPA). The 31 kDa zinc metalloprotein is assumed to be involved in damage recognition/verification during NER and acts as an assembly factor for the preincision complex, thereby binding not only DNA but also several other NER factors such as RPA, ERCC1, and TFIIH (24–26). The zinc finger motif (Cys105-X2-Cys108-X17-Cys126-X2-Cys129) is located within the minimal DNA binding domain (27) and is indispensable for XPA function since replacement of one of the cysteines by serine or glycine resulted in pronounced loss of DNA repair activity (28). Within the present study, we aimed to shed more light on the mechanism responsible for antimony genotoxicity and focused on a possible inhibition of nucleotide excision repair. The impact of SbIII on the removal of UVC induced lesions as well as of DNA adducts provoked by the reactive benzo[a]pyrene metabolite (+)-anti-BPDE in A549 human lung carcinoma cells was measured, and cell survival after treatment with the respective 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 also known as DDB2 or p48. Further investigations were performed on XPA and its zinc finger as a possible target for antimony action. At the subcellular level, a short peptide, which resembles the zinc finger domain of human XPA, was applied to elucidate a potential interaction with trivalent antimony by quantification of released zinc; at the cellular level, the impact of trivalent antimony on the association and dissociation of XPA to and from 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, and G418 were from Gibco and trypsin and dimethyl sulfoxide (DMSO) from Sigma-Aldrich. Culture dishes were from Biochrom. Antimony trichloride 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 fu¨r Umweltcarcinogene (Grossharnsdorf, Germany). The XPA peptide (XPAzf) was custom-synthesized from N-Schafer (Copenhagen, Denmark) with the sequence Ac-DYVICEECGKEFMDSYLMNHFDLPTCDNCRDADDK-HK-NH2 and a purity >95%, and the PCR primers were synthesized from MWG-Biotech AG (Ebersberg, Germany). HEPES, hydrogen peroxide, and BSA were from Merck. All other chemicals were from Roth. 2.2. Cell Culture and Treatments. A549 human lung adenocarcinoma cells (ATCC) were grown in tissue culture dishes as monolayers in DMEM supplemented with FBS (10%), penicillin (100 units/mL), and streptomycin (100 µg/mL) in a humidified atmosphere with 5% CO2 at 37 °C. Fibroblasts derived from a normal individual and immortalized by telomerase transfection (VH10hTert (29)) as well as XP-E cells derived from a xeroderma pigmentosum group E patient and immortalized by Epstein-Barr virus immortalization (XP23PV (30)) were cultured under equal conditions except for DMEM additionally supplemented with G418 (25 µg/mL). After completion of at least one cell cycle, cells were treated with antimony trichloride. Stock solutions of SbCl3 were prepared in DMSO, stored at -20 °C, and further diluted with DMSO prior to 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, irradiated without medium at a wavelength of 254 nm using a germicidal

Grosskopf et al. lamp (Bioblock Scientific), and postincubated in the original medium. The exact UVC dose was verified before each treatment by a UV-radiometer (PRC). Nonirradiated cells were treated accordingly but omitting irradiation. For investigations with the reactive metabolite (+)-anti-BPDE, a stock solution of 0.5 mg/mL in water free THF/5% triethylamine was prepared, stored at -80 °C, and diluted to a concentration of 50 µM before adding to the medium (final solvent concentration in the medium: 0.l%). Incubation time was restricted to exactly 1 h, followed by dropping of the medium, and repeated washing. In the case of post-treatment with antimony, fresh medium was added, and cells were reincubated with SbCl3. 2.3. Cell Number and Colony Forming Ability. Logarithmically growing A549 cells treated with antimony trichloride alone or in combination with UVC (5 J/m2) or BPDE (50 nM) were trypsinized, collected in fresh medium, and counted using a casy cell counter (Scha¨rfe Systems). Three hundred cells per dish were seeded in triplicate into fresh medium for the determination of colony forming ability. After seven days, colonies were fixed with ethanol, stained with Giemsa, and counted. 2.4. Detection of UVC Induced DNA Lesions. A549 cells were seeded onto coverslips and grown for 48 h before harvesting. Cells were pretreated with SbCl3 for times as indicated for the respective experiment and finally irradiated with a dose of 10 J/m2 (CPD) or 20 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, denaturation with 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, the specific primary antibody (mouse monoclonal anti-CPD, 1:1500, mouse monoclonal anti-6-4PP, 1:400; MBL) was applied and incubated at 37 °C for 1 h. Coverslips were washed 3× with washing buffer (0.5% BSA and 0.05% Tween in PBS) and incubated for 1 h with the secondary Alexa Fluor 488-conjugated antibody (1:500, Invitrogen). Finally, after washing and postfixation with formaldehyde (7% in PBS), coverslips were placed on microscope slides with a drop of Vectashield (Vector Laboratories Inc.). Labeled UVC lesions were visualized and recorded using an Axio Imager.M1 fluorescence microscope with AxioCam MRm camera (Zeiss). The fluorescence intensity of at least 100 cells per slide was analyzed by the AxioVision 4.4 software (Zeiss). Only signals in DAPI stained nuclei were quantified. Data were corrected for background fluorescence in nonirradiated control cells. 2.5. BPDE-DNA Adducts. BPDE-DNA adduct levels were measured via HPLC/fluorescence detection after the release of the corresponding tetrols as described elsewhere (31). Briefly, treated cells 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 several times with 70% ethanol (Rotisol), and quantified spectrophotometrically by measuring the absorbance at 260 nm (Peqlab NanoDrop). After hydrolysis of adducted DNA and subsequent neutralization, tetrol I-1 was quantified via HPLC with fluorescence detection. 2.6. Gene Expression. Real time RT-PCR was performed for quantification of the XPA and XPE mRNA level according to Nollen et al. (32). Incubated cells were harvested, washed once, and resuspended in PBS. Afterward, RNA was isolated with Nucleospin RNA-Isolation Kit (Macherey-Nagel) and quantified at 260 nm by applying a NanoDrop spectrophotometer (Peqlab). One microgram of RNA was transcribed into cDNA (qscript cDNA synthesis kit, Quanta Biosciences) and an aliquot of the product was used for real time PCR with an iCycler (Bio-Rad) by applying the forward primer (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-R SYBRGreen Supermix (Quanta Biosciences). The specificity of the amplification products was checked by melting curve analysis. The

Antimony Impairs Nucleotide Excision Repair relative expression ratio R was calculated on the basis of the efficiency E, the threshold cycle (Ct), and the reference gene GAPDH. 2.7. Protein Levels. The impact of antimony on the XPA and XPE 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% Triton X-100, 1% sodium desoxycholate, 1 µg/mL aprotinin, 1 µg/mL leupeptin, 1 µg/mL pepstatin, 1% DOC, 50 mM NaF, 0.1% SDS, and 1 mM Na3VO4). After centrifugation, the protein content of the supernatant was quantified (Bradford). Loading buffer and DTT (both Fermentas) were added to an aliquot of the cell extract, and the proteins were denatured at 95 °C for 10 min. Subsequently, proteins were separated by a 12% denaturing SDS-PAGE and transferred to a PVDF membrane (GE Healthcare) using a semidry blotter (Peqlab). The membrane was blocked (5% milk powder in PBST), incubated with the primary antibody (rabbit anti-XPA polyclonal antibody, 1:1000, Santa Cruz; goat anti-XPE polyclonal antibody, 1:2000, R&D Systems) overnight at 4 °C, and washed three times in PBST for 5 min before the HRP-conjugated secondary antibody (goat antirabbit polyclonal antibody, 1:2000, Santa Cruz; donkey antigoat polyclonal antibody, 1:2000, Santa Cruz) was applied for 1 h at room temperature. This procedure was followed by three washing steps in PBST and one in PBS. Detection was accomplished by enhanced chemiluminescence reaction (ECL) measured with a LAS 3000 cooled CCD camera (FujiFilm). Protein levels were quantified by densitometric analysis with AIDA Image Analyzer 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 domain of human XPA was applied as described elsewhere (12). After saturation with zinc buffer, the peptide (20 µM) was incubated with antimony chloride in 20 mM HEPES-NaOH buffer (pH 7.4) at 37 °C for 30 min. Subsequently, zinc release was quantified spectrophotometrically via complexation with 4-(2-pyridylazo)-resorcinol (PAR, 100 µM, Riedel-de Haen) at 492 nm. Results were referred to 10 mM H2O2 (Merck) corresponding to 100% zinc release. 2.9. XPA Association and Dissociation. Investigation on the DNA damage assembly of XPA was performed by generation of local DNA lesions in combination with immunofluorescent labeling of the protein as described before (32, 33). A549 cells, grown on coverslips, were preincubated with SbIII for 2 h and washed once in PBS before they were covered by an 8 µm pore filter (Millipore) during irradiation with a UVC dose of 30 J/m2 which corresponds to a global UVC dose of 10 J/m2. After the filter was carefully removed, cells were postincubated in the same medium for the indicated time. Subsequently, cells were fixed and treated according to the UVC lesion detection protocol except for denaturation, which was omitted. The primary antibody (polyclonal rabbit anti-XPA, 1:100) was obtained from Santa Cruz Biotechnologies and the secondary Cy3-conjugated antibody (1:500) from Jackson ImmunoResearch. Evaluation of the fluorescence intensity of at least 60 XPA spots was performed according to DNA lesion quantification with the AxioVision 4.4 software (Zeiss). Data were corrected for overall fluorescence in nonspotted nuclei.

3. Results 3.1. Cytotoxicity. Cell number and colony forming ability were determined to examine the immediate as well as longterm cytotoxicity of antimony and its impact on UVC- and BPDE-induced cytotoxicity. A549 cells preincubated with SbCl3 for 2 h were irradiated with 5 J/m2 UVC or treated with 50 nM BPDE, respectively, where indicated, and postincubated with SbCl3 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 in cell and colony number to less than 70%. In contrast, 90% of

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Figure 1. Impact of SbCl3 on the viability of A549 cells with and without UVC irradiation (a) or BPDE treatment (b). Cells were preincubated with antimony for 2 h, treated with 5 J/m2 UVC or 50 nM (+)-anti-BPDE for 1 h where indicated, and postincubated for 24 h in the presence of SbIII. Afterward, the cells were trypsinized and counted. Three × 300 cells were seeded into fresh medium and grown for one week before colonies were fixed, stained, and counted. Results refer to cell or colony number for the untreated solvent control. Shown are the mean values of 3 independent determinations + SD.

the cells were still viable at 250 µM SbCl3. Irradiation with a UVC 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 more pronounced effect on colony forming ability at higher SbCl3 concentrations (Figure 1a). Whereas the decrease in cell number can be ascribed to the addition of the cytotoxic effects of each component alone, it appears that SbIII impairs the colony forming ability, a parameter for long-term cytotoxicity, in a more than additive mode. Similar results were obtained in combination with BPDE (Figure 1b). Treatment with the reactive metabolite of benzo[a]pyrene only slightly decreased cell number and colony forming ability, and SbCl3 caused an additive reduction in cell number as well. Most notably, colony forming ability was also impaired in a superadditive manner by SbCl3 in concentrations above 250 µM, gaining an additional reduction of 30% at 500 µM SbCl3. 3.2. Repair of UVC-Induced DNA Lesions. Effects of trivalent antimony on the repair of UVC-induced DNA lesions were measured by immunofluorimetric detection of unprocessed photoadducts. UVC mainly induces two kinds of lesions, the cyclobutane 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, doses required to achieve sufficient detectable lesions (33). According to their fast repair, removal of 6-4PP was determined 2 h after irradiation. Within that time, almost 70% of the initially induced lesions became repaired in irradiated cells in the absence of antimony treatment corresponding to a repair efficiency of 100%. 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

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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 with formaldehyde. Detection of DNA lesions was performed by fluorescence antibody labeling. (b) Fluorescence signals within the nucleus were quantified and 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).

were detected 24 h after irradiation due to their slow repair (Figure 2a). In the absence of antimony, about two-thirds of the dimers were removed, whereas 2 h pre- and 24 h postincubation with antimony resulted in larger amounts of remaining CPD even at noncytotoxic concentrations (Figure 2b). At 250 µM SbCl3, the fraction of nonrepaired lesions increased to nearly 50%. The number of still remaining CPD in the presence of 500 µM SbIII after 24 h was even twice as high as compared to that of untreated cells, consistent with a repair inhibition of more than 50%. 3.3. Repair of BPDE-DNA Adducts. The impact of trivalent antimony on nucleotide excision repair was also investigated for the removal of BPDE-DNA adducts. The main metabolite of benzo[a]pyrene, (+)-anti-BPDE, causes specific DNA adducts at the N2 position of guanine, from which tetrol I-1 can be generated during hydrolysis with HCl. Analysis of the tetrol content by HPLC fluorescence detection reflects the number of DNA adducts. Determination of the adduct levels was performed at two time points, immediately (0 h) and 8 h after incubation with 50 nM BPDE to examine the induction of adducts as well as their repair. Preincubation of A549 cells with SbCl3 for 2 h did not affect the formation of BPDE adducts (Figure 3). Only a marginal reduction in the adduct level was found at 500 µM SbCl3. No effect was also observed on the repair after 8 h in the presence of antimony. The efficiency of adduct removal was about 30% in the absence or presence of SbCl3. Extending the preincubation time with SbCl3 to 24 h did not affect the adduct level either (data not shown). Thus, trivalent antimony appears to compromise neither BPDE-DNA adduct formation nor BPDE-DNA adduct removal. 3.4. XPE Gene Expression and Protein Level. The NER protein XPE is known to be a discriminating factor for the recognition of the CPD. In contrast to 6-4PP, the removal of the pyrimidine dimers is impaired profoundly in the absence of

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

functional XPE (21, 34). Thus, the cellular XPE status was analyzed in A549 cells by determining gene expression by RTPCR and protein level by Western Blot. Analysis of the transcript level revealed that SbCl3 did not affect XPE gene transcription after an incubation time of 10 h. During this incubation time, it also did not impair the induction of XPE expression by UVC, which in preliminary experiments was shown to reach its maximum 8-10 after irradiation. Two hours pre- and 8 h postincubation with SbCl3 resulted in an equal increase of XPE transcripts of more than 70% (Figure 4a). Moreover, no effect of SbCl3 was observed on the XPE protein content up to 6 h after irradiation with UVC (data not shown). However, SbCl3 caused a concentration dependent decline in the XPE mRNA level after 24 h, a time period which corresponds to the overall treatment with SbCl3 in the studies on CPD and 6-4PP removal (Figure 4b). Significant reduction

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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 after 10 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 isolated from collected cells and rewritten into cDNA before real time RT-PCR was performed. XPE transcript number was normalized to GAPDH mRNA levels. Shown are the mean values of 4 determinations + SD. (b) mRNA level of XPE in A549 cells after 24 h of treatment with SbCl3. Shown are 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 where indicated, and postincubated in the continued presence of SbCl3 for 24 h. Afterward, protein was extracted from collected cells, quantified according to Bradford, and analyzed for XPE level by Western blot. Shown is one representative blot out of three independent determinations as well as the densitometric quantification of all three blots (mean values + SD). Significantly different from the corresponding control cells without SbCl3 as determined by the t-test (**P < 0.01, ***P < 0.001).

by almost one-third was observed at noncytotoxic 250 µM SbCl3, whereas at 500 µM SbCl3, the transcript number was diminished by half. This effect on gene expression was also detectable at the protein level. Twenty-four hours of incubation with trivalent antimony resulted in up to 50% reduction in XPE protein content (Figure 4c and d). In combination with UVC irradiation, the decline was even more pronounced. Irradiation with 5 J/m2 UVC alone increased the amount of XPE by more than 40% after 24 h, whereas preincubation with SbCl3 for 2 h and postincubation for 24 h after irradiation resulted in a diminished increase at 100 µM and 250 µM SbCl3 and in even no increase at 500 µM SbCl3. Compared to irradiated cells not treated with antimony, only one-third of the XPE protein content was found in the presence of 500 µM SbCl3 pointing to an additional impairment of UVC-induced induction of XPE. 3.5. BPDE-DNA Adduct Formation and Removal in XPE Deficient Cells. The involvement of XPE in the recognition of CPD and 6-4PP is well investigated, while only little is known in the case of BPDE-induced lesions. To examine the role of the protein in the repair of this type of lesion, XPE deficient (XP-E cells) and XPE proficient (VH10 cells) human fibroblasts were compared with respect to their adduct levels at different times of repair (Figure 5). The number of adducts clearly declined in both cell lines displaying more than 50% removal after 72 h. Nevertheless, XPE proficient cells appear to start with repair immediately after (or even during) incubation with BPDE (50 nM, 1 h), while the adduct level in XPE deficient cells remained unchanged for at least 8 h after treatment with the diol epoxide. Sixteen hours later, the adduct number declined even in the absence of functional XPE, indicating that finally repair had taken place with similar efficiency. However,

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

decomposition of adducts derived by mechanisms other than NER cannot be excluded. 3.6. Zinc Release from XPAzf. An interaction of trivalent antimony with the repair protein xeroderma pigmentosum group A was investigated by measuring zinc release from XPAzf, a 37 amino acid containing peptide saturated with Zn2+, which represents the zinc finger domain of human XPA. Deliberated zinc 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 than equimolar ratio of 10 µM SbCl3 (Figure 6). In great excess (500-1000 µM SbCl3), the compound was almost as effective as 10 mM H2O2. Antimony itself displayed no interaction with

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Figure 6. Effect of SbIII on zinc release from XPAzf after 30 min of incubation at 37 °C. Deliberated zinc was quantified spectrophotometrically via complex formation with PAR. Results refer to zinc release provoked by 10 mM H2O2 (corresponding to 100%). Shown are the mean 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 DNA lesions. A549 cells were pretreated with SbCl3 for 2 h, irradiated through a pore filter with 30 J/m2 UVC, and postincubated with the chemical for the times as indicated. Subsequently, the cells were fixed, and XPA spots were detected via fluorescent antibody labeling. Spot intensity correlates with the association of the protein to damaged DNA. Results refer to the 10 min UVC solvent control. Shown are the mean values of 3 determinations + SD. Significantly different from the corresponding control (10 or 60 min) as determined by the t-test (*P < 0.05, **P < 0.01, ***P < 0.001).

PAR, and DMSO (final concentration 1%), used as the solvent for SbCl3, also did not affect zinc release (data not shown). 3.7. XPA Association and Dissociation. The association of XPA to damaged DNA sites was examined in the absence or presence of trivalent antimony. To measure XPA association, local DNA lesions were induced by irradiating A549 cells with 30 J/m2 UVC through a porous filter corresponding to a global irradiation with 10 J/m2 and detection of XPA by fluorescentlabeled antibodies. XPA foci were detectable as bright distinct spots within the nucleus. Spot intensity as an indicator for association was measured at two time points, 10 and 60 min after DNA damage induction. Within this time, XPA migrates to the lesion and dissociates from the repair complex again. In cells preincubated for only 2 h with SbCl3, a decrease in XPA spot intensity by up to 20% was measurable at the 10 min time point indicating a reduced association of the protein (Figure 7). Sixty minutes after irradiation, spot intensity declined to almost 50% in the absence of antimony. However, the spot intensity in antimony treated cells declined to a considerably lesser extent indicating a delayed or disturbed dissociation of the protein after 60 min (Figure 7). Association and dissociation of the NER protein XPC, assembling to the DNA lesion before XPA, was also investigated. In contrast to XPA, no effects on the association of XPC were observable after 2 h of preincubation with antimony (data not shown). 3.8. XPA Gene Expression and Protein Level. To elucidate the impact of trivalent antimony on the XPA protein level, we determined the transcription of the corresponding gene after

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

treatment with antimony alone as well as in combination with UVC. After preincubation with SbCl3 for 2 h, irradiation with UVC, and postincubation for 8 h in the presence of the antimony, compound cells were harvested, and mRNA was analyzed by real time RT-PCR. In the range of 100-500 µM, trivalent antimony itself did not significantly alter the mRNA level of XPA in A549 cells within this time (Figure 8a). This was also the case for long time incubation with SbCl3 of 24 h (data not shown). Irradiation of the cells with a UVC dose of 5 J/m2 alone also did not affect XPA expression after 8 h. Likewise, the combination of antimony treatment and UVC irradiation had no impact on the level of XPA transcripts as well. Western blot analysis confirmed these results. No changes in the protein level of XPA were observable after 24 h of incubation with 100-500 µM SbCl3 (Figure 8b). This was also the case for an additional UVC treatment: neither UVC irradiation 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 genotoxic metals such as cadmium, cobalt, and arsenic (35). However, in the case of antimony experimental evidence has been missing. Our results now show that SbIII exerted a pronounced impact on nucleotide excision repair as demonstrated for the removal of 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 of remaining CPD increased in the presence of SbCl3, starting at noncytotoxic concentrations. The unequal impact on these photolesions may be explained by the faster and thus preferred

Antimony Impairs Nucleotide Excision Repair

repair of the 6-4PP, which cause larger helix distortions and, if unrepaired, exhibit a higher mutagenic potential than the CPD (36, 37). Damage recognition within global genomic repair of the nontranscribed DNA strand is assumed to be mainly responsible for differences in their removal. While most lesions are directly recognized by XPC, the subtle helix distortion induced by CPD is assumed to require a more sensitive recognition process. This seems to be accomplished by an additional sensor, the UV-DDB complex, which consists of two subunits, DDB1/p127 and DDB2/XPE/p48, and specifically binds UV-damaged DNA with high affinity (38, 39). Mutations in the XPE gene were shown to be responsible for the clinical symptoms of xeroderma pigmentosum complementation group E, which is characterized by only partial repair inhibition and modest sensitivity toward UV light (30, 40). It was demonstrated that XPE is necessary for the recruitment of XPC to CPD but not to 6-4PP (41) and enhances global genomic repair of pyrimidine dimers, while the repair of 6-4PP was less affected in the absence of functional XPE (21, 29, 34, 42). In the present study, quantification of the XPE protein level revealed a decline of cellular XPE after incubation with even noncytotoxic SbCl3 concentrations especially in combination with UVC, resulting in up to 67% less protein. Thus, XPE protein might become limiting for the recognition of CPD after antimony treatment since the protein is tightly regulated. XPE is degraded via the proteasome shortly after UV irradiation but also induced several hours later by its transcriptional regulator p53 (21, 23, 42, 43). Considering our observation that incubation with SbIII for 24 h affects XPE already at the transcriptional level, it may well be that the expression of the XPE gene by p53, regulating also the basal level of this protein, is impaired. In contrast, short incubation times did not alter the XPE status even in combination with UVC, which indicates a rather late impact on the removal of CPD. In addition to UVC-induced lesions, the effect of SbCl3 on the 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 long time (24 h) pretreatment with trivalent antimony followed by continued treatment after BPDE-incubation. To elucidate the role of XPE in the repair of this type of lesion, adduct removal in XPE deficient and proficient cells was compared. Both cell lines were able to repair BPDE adducts, but the repair kinetic differed considerably. Up to 8 h after BPDE treatment, XPE deficient cells displayed almost no adduct removal, while at later time points, repair efficiency appeared to be similar to that of XPE proficient cells. Although it can not be completely excluded that the reduction of lesions might be due to a decomposition of BPDE-DNA adducts by alternative mechanisms, such as hydrolysis of the N-glycosidic bond, or due to the fact that some differences may be due to the comparison of nonisogenic cells, the results appear to be best explained by an accelerated removal of these lesions in the presence of XPE, which is, however, not indispensable for their repair. This characteristic had been also demonstrated for the removal of the 6-4PP after low doses of UVC pointing to a stimulated recognition of a limited number of lesions by the UV-DDB complex (29). With respect to our results and in contrast to completely XPE deficient cells, the amount of XPE after treatment with SbCl3 might have been still sufficient to efficiently recognize the relatively small number of BPDE-DNA adducts, considering the fact that the number of lesions induced by 50 nM BPDE is several magnitudes lower than the number of CPD and 6-4PP induced by 10 or 20 J/m2 UVC.

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Despite the fact that removal of the BPDE-induced lesions was not impaired by antimony, a pronounced reduction in cell viability was observed. In combination with high SbCl3 concentrations, colony forming ability declined more than additive after treatment with BPDE, while this effect was not detectable in the cell number after 24 h. Nearly identical observations were also made for lesions induced by a noncytotoxic UVC dose when cotreated with the same concentrations of trivalent antimony. Surprisingly, the extent of the reduction in colony number was very similar for both, although far more lesions were induced by 5 J/m2 UVC as compared to 50 nM BPDE. Thus, trivalent antimony appears to affect the cellular response to NER lesions generally and independent from the type of lesion, which seems to contradict our results on a selective impairment of lesion removal. However, an impact of SbCl3 on the metabolism of BPDE, which is known to play a major role in the toxicity of this compound, cannot be ruled out. Moreover, the test system applied quantifies adduct levels and their incision, while late steps in NER such as polymerization/ligation may be affected as well. In any case, loss of functional XPE is known to have only mild effects on cell viability after irradiation with UVC, which is probably due to the fact that unrepaired CPD seem not to trigger cell death because these lesions can be bypassed by polymerase η (14, 44, 45). Thus, it is most likely that a further defect is responsible for the impact of SbCl3 on cell viability after mutagen treatment. Several observations on XPA, an indispensable component of the NER damage recognition complex, suggest that according to its expression levels as well as to modifications in its sequence preferential repair of certain lesions may also occur (46–49). Concerning these data, an impact on XPA by trivalent antimony might deliver a further explanation for lesion specific repair inhibition. XPA contains a zinc binding motif, which consists of 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 been previously shown that SbIII causes the loss of almost all proteinbound zinc from rat liver metallothionein, in which zinc is tetrahedrally coordinated to four cysteines similar to XPA (50). Within the present study, we demonstrated the ability of antimony to release zinc from the zinc finger domain of human XPA in a concentration dependent manner starting at equimolar concentrations. In previous studies, several metals, including cadmium or cobalt, caused zinc release from this peptide as well (12, 51, 52). Compared to the data on arsenite, antimony is even more effective in releasing zinc from XPA, yielding 50% zinc release at 10 times lower concentrations (12). To elucidate a potential impact on XPA function in living cells, the association of XPA to sites of DNA damage was examined by immunofluorescent labeling after local irradiation of A549 cells with UVC. Declined spot intensity of up to 20% in cells pretreated with SbCl3 for 2 h revealed a diminished assembly of XPA shortly after UVC irradiation, whereas 20% higher spot intensity after 1 h was observed. This can be explained either by pronounced delayed association or by both disturbed association and dissociation. Which alternative applies to antimony requires further investigations, for example, by applying photobleaching experiments. Keeping in mind that XPA protein is usually present in excess and has to be diminished to levels of less than 10% to be rate limiting for NER (53), these results are quite remarkable. In contrast, the association of the NER protein XPC, which contains no zinc

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finger motif in its structure, was not impaired under equal conditions pointing to a specific effect on XPA function. Because of preincubation with SbCl3 for only 2 h, it can also be ruled out that loss of XPE is the primary cause since effects on gene expression and protein level were restricted to later time points. An altered expression of the XPA gene or an enhanced degradation of the corresponding protein can be excluded as well since real time RT-PCR as well as Western blot analysis revealed no inhibitory effect by trivalent antimony. The fact that antimony alters the association at the lesions but not the total cellular amount of XPA protein indicates an impaired function of XPA within the damage recognition/ incision complex. It is well established that the interaction with RPA enhances the affinity of XPA for UV damaged DNA (54, 55). Interestingly, binding of XPA to the 70 kDa subunit of RPA is supposed to be mediated also via its zinc finger domain (56, 57). Thus, an interaction of SbIII with this domain could affect the formation of a complex and might explain the retarded association of XPA to sites of UV damage. With respect to in Vitro experiments displaying a higher affinity of purified recombinant XPA to 6-4-PP than to CPD (58, 59), an impaired interaction of XPA with RPA could preferentially affect the binding of XPA to pyrimidine dimers. It is also worth mentioning that RPA also contains a Cys4-zinc finger motif within the 70 kDa subunit, which is responsible for the redox regulated DNA binding activity of the protein (60). Besides RPA, an interaction of XPA with ATR via its minimal DNA binding domain and its phosphorylation by ATR seems to be also important for the recognition of CPD. Preventing phosphorylation by ATR diminished only the repair of the CPD after irradiation with UVC, whereas removal of 6-4PP was not impaired. Independent from XPA phosphorylation, the interaction with ATR was also shown to be necessary for nuclear localization and consequently association of XPA to DNA damage as well (47). Therefore, trivalent antimony might also act by disturbing the interaction with ATR. Although the data presented in this article imply an interaction with XPA, the underlying mechanism remains speculative. The interference with the zinc finger domain as demonstrated for the corresponding peptide is at present the most promising theory. Different processes can be responsible for the observed zinc release. Cadmium or cobalt, for example, were shown to replace the zinc in the complex, whereas arsenite oxidized the thiols to disulfides (52, 61). Moreover, the metal species plays a decisive role too. Monomethylarsonous acid (MMA(III)), a metabolite of arsenite, formed a mixture of single and double monomethylarsinated XPAzf as well as fully oxidized XPAzf (61). However, in each case the structure of the zinc finger would be impaired resulting, more or less, in a loss of XPA function and loss of NER capacity. In the case of trivalent antimony, further investigations are needed to reveal the mechanism. The consequences of an impairment of NER capacity are very serious. Cells are constantly exposed to DNA damaging agents such as UV radiation or benzo[a]pyrene. If lesions are removed more slowly or only partly, the frequency of mutations would be expected to rise resulting in an increased cancer risk. This is alarming since antimony is ubiquitously disseminated, and exposure is magnified by several antimony-containing materials such as fabrics or plastics. In conclusion, these results support the theory that antimony interferes with DNA repair, thereby implying an indirect mechanism of SbIII genotoxicity and perhaps carcinogenicity. A crucial step within might be the impairment of XPE gene

Grosskopf et al.

expression and the interaction with zinc finger domains of key proteins such as XPA. Regarding the abundance of zinc binding domains, e.g., in p53, an impairment of proteins outside nucleotide excision repair is also conceivable, creating the image of a multitarget mode of action. Therefore, more research is required to achieve a more precise understanding of the effects caused by antimony. Acknowledgment. This work was supported by DFG Grant Numbers Ha 2372/3-4, Ha 2372/5-1, and Schw 903/3-2. The authors declare that there are no conflicts of interest.

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