Arsenite induces DNAprotein crosslinks and cytokeratin expression in the WRL-68 human hepatic cell line
P. Ramírez,
L.M. Del Razo1,
M.C. Gutierrez-Ruíz2 and
M.E. Gonsebatt3
Departamento de Genética y Toxicología Ambiental, Instituto de Investigaciones Biomédicas, UNAM, AP 70228, Ciudad Universitaria, Mexico 04510 DF, México,
1 Toxicología Ambiental CINVESTAV, IPN, 07000 DF, México and
2 Laboratorio de Fisiología Celular, Departamento de Ciencias de la Salud, Division de Ciencias Biologicas y de la Salud, Universidad Autonoma Metropolitana-Iztapalapa, 09340 DF, México
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Abstract
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The induction of DNAprotein crosslinks (DPC) has been proposed as an indicator of early biological effects due to the fact that known or suspected carcinogens induce an increased proportion of proteins tightly bound to DNA. Arsenic, a human carcinogen, is reduced and methylated mainly in liver cells generating a number of intermediate reactive forms which could lead to the formation of DNAprotein crosslinks. The induction of DPC by arsenite [As(III)] was investigated in the WRL-68 human hepatic cell line, testing the possibility that cytokeratins or cytokeratin-like proteins, due to their high content of SH groups, could participate in DPC. The formation and decay of DPC was dose-related. Arsenite was the only intracellular species present since no methylated As forms could be detected. Thus, DPC can be attributed to the presence of arsenite, an important species present in liver during As exposure, whose permanence in the tissue would depend on the methylation rate of the organism. Several cytokeratins were identified by immunoblotting among the proteins crosslinked with DNA, including cytokeratin 18 (CK18), a specific liver intermediate filament. An augmented presence of CK18 was detected in treated cultures by immunoblotting of total protein PAGE. In liver cells cytokeratin synthesis is tightly correlated with differentiation programs, thus arsenite could not only be damaging DNA but also modifying differentiation patterns in this tissue.
Abbreviations: CK18, cytokeratin 18; DPC, DNAprotein crosslinks; GSH, reduced glutathione; iAs, inorganic arsenic; PBS, phosphate-buffered saline.
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Introduction
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The induction of DNAprotein crosslinks (DPC) has been proposed as an indicator of early biological effects due to the fact that known or suspected carcinogens, such as formaldehyde, ß-propiolactone and UV light, chemotherapeutic agents, such as cisplatin and mytomycin C, and some metal compounds, like nickel sulfate, arsenic oxide and potassium dichromate, among others, induce the formation of crosslinks between proteins and DNA (14). Moreover, the level of DPC appears to be directly related to the carcinogenicity of formaldehyde in target tissues such as rat nasal cells, where they seem to be the primary genotoxic effect (5,6). For these reasons DPC were used to estimate the formaldehyde effective dose (6). Another human carcinogen, arsenic, which induces skin, lung, bladder and liver cancers (79), is reduced and methylated mainly in liver cells through a series of reactions that require reduced cellular glutathione (GSH), generating a number of intermediate reactive forms (11,12) that could lead to the formation of DPC. To investigate and characterize mechanisms of As carcinogenicity, DPC were isolated from arsenite-treated WRL-68 human hepatic cells, a nontumorigenic cell line derived from fetal liver that preserves the activity of some characteristic or specific liver enzymes (13). Furthermore, since trivalent As species are thought to accumulate in tissues with a high keratin content due to its affinity for thiol groups (14), and keratins and keratin-like proteins have been identified in the nuclei of Novikoff ascites hepatoma and rat liver cells (1517), the presence of cytokeratins among the proteins bound to DNA was also investigated. To separate proteins from DNA, DPC were treated enzymatically or with reducing agents or under high salt conditions. Dose-related induction and removal of DPC were observed after arsenite treatment of confluent cell cultures. Cytokeratins were identified among the proteins crosslinked with DNA; this crosslinking was sensitive to nuclease digestion and to high salt conditions.
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Materials and methods
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Hepatic cell line culture and treatment
The human hepatic cell line WRL-68 has a morphological structure similar to hepatocytes and hepatic primary cultures. Derived from fetal liver, WRL-68 cells secrete
-fetoprotein and albumin, preserve the activity of some characteristic or specific liver enzymes (i.e. alanine aminotransferase, aspartate aminotransferase,
-glutamyl transpeptidase and alkaline phosphatase) and exhibit a cytokeratin pattern similar to other hepatic cultures, providing an in vitro model to study the toxic effects of xenobiotics (13). Cells were grown and maintained in Dulbecco's modified Eagle's medium supplemented with 8% fetal bovine serum (Gibco), 1% non-essential amino acids, 100 U/ml penicillin and 100 µg/ml streptomycin. Cells were incubated at 37°C in an atmosphere of 95% air, 5% CO2. Approximately 2x106 cells/plaque were plated in 6-well culture plates and left to reach confluence. Arsenite treatments were initiated in this phase to avoid the generation of DPC during S phase. Cells were treated over 16 h with different concentrations of arsenite (sodium salt; Sigma, St Louis, MO): 1, 10, 102 and 104 nM. Untreated cells and cells treated with 103 nM K2Cr2O7 (Sigma) served as negative and positive controls, respectively. After treatment cells were scraped and washed with phosphate-buffered saline (PBS), pH 7.2. To determine the cytoxicity of the doses employed, cell viability was determined by the trypan blue exclusion method in each experiment; cell viability was always >75%.
Intracellular As determination
The amount of intracellular As species was determined by atomic absorption spectrometry using hydride generation after column chromatographic separation of inorganic arsenic (iAs) and its metabolites. Quantification is based on the measurement of light absorbed at 193.7 nm by ground state atoms of As from an electrodeless discharge lamp source (10).
GSH determination
Determination of GSH was performed by a modification of the method described by Hissin and Hilf (18). Aliquots of 2x106 cells were cultured and treated with arsenite as described before. The cells were scraped and resuspended in phosphate/EDTA buffer solution (0.1 M sodium phosphate, 0.005 M EDTA buffer, pH 8.0). Total protein was precipitated using 2.5% perchloric acid solution. The samples were then centrifuged for 10 min at 4°C. A volume of 0.1 ml of the supernatant plus 1.8 ml PBS/EDTA, pH 8.0, and 0.1 ml of 1% o-phthalaldehyde (Sigma) were mixed and incubated in a glass tube for 15 min in the dark at room temperature. The solution was transferred to a quartz cuvette. Fluorescence at 420 nm was determined with activation at 350 nm.
DPC isolation
DPC precipitation was performed as described by Zhitkovich and Costa (3). Briefly, after viability determination, cells were lysed with a solution containing 2% SDS and then frozen at 70°C. To initiate the isolation of DPCs, the samples were thawed at 37°C and the DNA was sheared by passage of the lysates through a 21 gauge needle. The lysates were expelled into a tube by applying high pressure. A volume of 0.5 ml of 100 mM KCl containing 20 mM TrisHCl, pH 7.5 (solution A) was added and mixed by vortexing. The samples were then incubated at 65°C for 10 min, inverted and then placed on ice for 5 min to form the KClSDSproteinDNA precipitates. The precipitates were collected by centrifugation at 6000 g for 5 min at 4°C. The supernatant was removed and the pellet resuspended in solution A. Samples were heated and washed three times as described above. DNA was detected using Hoechst 33258 dye. Fluorescence was assessed by excitation at 365 nm and the emitted light was measured at 450460 nm. A bovine serum albumin solution was used as the standard blank. To evaluate the persistence of DPC after treatment, control and treated confluent cells were washed with PBS and incubated in fresh medium for 2, 6, 12 and 24 h at 37°C in an atmosphere of 95% air, 5% CO2, after which they were washed with PBS and scraped from the bottles. Cell viability (7580%) was determined by the trypan blue exclusion method. DPC isolation was performed as described above.
Protein identification
The isolated DPCs were nuclease digested for 1 h at 37°C with nuclease (25 µg/ml DNase I, sp. act. 1872 U/mg) (17) in preparation for SDSPAGE. The nuclease-digested samples were electrophoresed as described by Laemmli (19). Four percent stacking gels and 10% running gels under reducing conditions [25 mM ß-mercaptoethanol (Bio-Rad) or 10 mM dithiotreitol (Bio-Rad)] were used. The proteins were visualized using either the Coomassie blue method or by western blotting. Monoclonal anti-Pan cytokeratin antibodies recognizing human cytokeratins 1, 46, 8, 10, 13, 18 and 19 (Sigma) and monoclonal antibodies against human cytokeratin 18 (CK18) (Neomarkers, Lakeside, NY) were used. Densitometry analysis of blots was performed using an AMBIS Optical Image System densitometer (Scanalytics). The software used was RSLPSCAN v.2.1.
NaCl treatment
Isolated DPC were washed with 2.5 M NaCl (final concentration) and resuspended in 100 mM KCl, 100 mM EDTA, 20 mM TrisHCl, then dialyzed using a molecular pore size 12 00014 000 dialysis membrane against PBS, pH 7.2, for 24 h. The dialyzed DPC were digested and prepared for SDSPAGE with and without DNase treatment as described before.
Immunofluorescence
WRL-68 cells were grown on sterile glass coverslips until semi-confluent. The cells were treated with arsenite in the concentration range 1104 nM for 16 h. After incubation, the cells on coverslips were washed twice with PBS, pH 7.2, then fixed and permeabilized for 90 min in 95% ethanol at 0°C. They were then washed in PBS twice for 5 min and blocked for 30 min with 3% (w/v) bovine serum albumin in PBS, followed by incubation for 1 h with anti-CK18 monoclonal antibodies. The coverslips were then washed twice for 5 min and incubated for a further 1 h with FITC-conjugated anti-IgG, diluted 1:100 in blocking buffer. Finally, the cells were washed three times for 10 min in PBS, mounted in glycerol and viewed with a Nikon E 400 epifluorescent microscope and with an Odissey Noran 3.2 confocal microscope. The cells were photographed using ASA 400 Hyperfilm and the fluorescence intensity was determined using MetaMorph system 1.3. Negative controls were prepared using culture medium instead of monoclonal antibodies.
Data analysis
Average, standard deviation and standard error were calculated from quantitative data obtained for at least three replicate experimental conditions. Statistical analyses were performed using one way ANOVA and Dunnett's test or the Student NewmanKeuls test for multiple comparisons, with the level of significance set at 5%. Simple linear correlations were used to study doseresponse effects.
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Results
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Arsenite induced dose-dependent DPCs in confluent cell cultures of WRL-68 cells treated for 16 h with different concentrations of arsenite (Figure 1A
). The proportion of DPC correlated linearly with intracellular concentration of trivalent iAs (Figure 1B
). Although the presence of methylated forms of As could not be detected in the cells, induction of GSH synthesis was observed (P = 0.0267, Figure 2
). To separate crosslinked proteins, DPC were nuclease digested and then electrophoresed under reducing conditions with ß-mercaptoethanol or dithiotreitol. Similar, although less dense, band patterns were observed in PAGE of DPC proteins from untreated cells compared with arsenite- and chromate-treated cells (Figure 3
). When arsenite was removed from the cultures, we observed a dose-related decay of DPC. The fastest disappearance of DPC was observed at the lowest doses tested (Figure 4
) and 24 h after treatment all cultures showed background levels of DPC, except for those treated with 1 nM arsenite (P < 0.05).

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Fig. 1. DPC induced by sodium arsenite. (A) WRL-68 cells were treated for 16 h in complete medium with arsenite. After scraping and washing, cells were subjected to SDSKCl precipitation of DPC. Significant effects were observed at all doses (ANOVA, F = 13.7, P = 0.0025; Dunnett's post hoc test P < 0.05; simple linear correlation coefficient r = 0.9527, P < 0.05). (B) Correlation between DPC and intracellular iAs(III) (r = 0.9684, P < 0.05). The intracellular arsenite concentration was determined by atomic absorption spectrometry after cell lysis (ANOVA H = 12.9, P = 0.0011).
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Fig. 2. GSH induction by arsenite in WRL-68 cells. Aliquots of 2x106 cells were cultured and treated with different concentrations of arsenite. Cells were then lysed with perchloric acid and resuspended in PBS/EDTA, pH 8.0, buffer. The GSH concentration was determined using 1% o-phthalaldehyde. Fluorescence at 420 nm was determined with activation at 350 nm. (Simple linear correlation coefficient r = 0.9522, P < 0.05; ANOVA H = 16.2, P = 0.00276).
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Fig. 3. Electrophoresis of DPC proteins. DPC were DNase treated and separated by SDSPAGE under reducing conditions using ß-mercaptoethanol. One thousand micrograms of protein were loaded in the lanes. The gel was stained with Coomassie blue. The lane numbers correspond to proteins isolated from DPC induced by: (1) 103 nM K2Cr2O7 ; (2) control; (3) 1 nM arsenite; (4) 10 nM arsenite; (5) 102 nM arsenite; (6) 104 nM arsenite; (7) 30 µg of proteins from lysed WRL-68 cells.
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Fig. 4. DPC persistence. WRL-68 cells were treated for 16 h in complete medium with arsenite. After the treatment period the culture medium with As was removed and the cultures were incubated for 2, 6, 12 and 24 h at 37°C in 5% CO2. Then the cells were scraped, washed and subjected to SDSKCl precipitation of DPC. Empty bars, untreated cultures; diagonal bars, 1 nM arsenite; full bars, 100 nM; horizontal bars, 104 nM. *Significantly different (P < 0.05) from untreated cells at each recovery time (ANOVA, F = 8.66, P = 0.00512; Dunnett's post hoc test P < 0.05).
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At least five cytokeratins were identified by western blotting among the proteins crosslinked with DNA. At the lower doses (1, 10 and 100 nM) of arsenite four cytokeratins with molecular masses of ~46, 48, 50 and 54 kDa were observed, while at the highest concentration evaluated (104 nM) and in the positive control an extra band with a lower molecular mass of ~45 kDa was detected (Figure 5
). As shown in Figure 6
, CK18 was identified by immunoblotting among the proteins isolated from DPC. The amount of CK18 crosslinked with DNA increased with dose, being 45% higher than controls at the lowest (1 nM) treatment dose (Figure 6
). When isolated DPC were not digested with DNase, even after ß-mercaptoethanol or dithiotreitol treatment, CK18 protein could not be detected until high salt conditions were employed (Figure 7
). Also, an increase in CK18 expression was observed by densitometry analysis of immunoblots of total cell protein content. This effect was not dose-related (Figure 8
). A similar result was observed when cells were immunostained with monoclonal antibodies against CK18. Higher immunofluorescence was determined by confocal analysis in treated cultures, indicating overexpression of CK18 (Figure 9
). Filaments were located in the cytoplasm, around the nuclear envelope and inside the nucleus. Images also showed disruption or alteration of the thread-like pattern of CK18 in treated cells (Figure 9
).

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Fig. 5. Immunoblot to identify the presence of cytokeratins in DPC. (A) DPC were isolated by SDSKCl precipitation. DPC were DNase treated and separated by SDSPAGE under reducing conditions using ß-mercaptoethanol. Seventy micrograms of protein were loaded in the lanes. Gel proteins were then electrotransferred to a nitrocellulose membrane. The lane numbers correspond to DPC proteins isolated from cultures treated with: (1) untreated cultures; (2) 1 nM arsenite; (3) 10 nM arsenite; (4) 102 nM arsenite; (5) empty lane; (6) 104 nM arsenite; (7) 103 nM K2Cr2O7. (B) Bars represent average densitometry values ± SE obtained in analysis of immunoblots from three replicate treatments, one of which is shown in (A) (F = 955.9, P < 0.001). *The percentage is expressed with respect to values obtained in untreated cultures.
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Fig. 6. Identification of CK18 by immunoblotting. (A) A duplicate of the gel shown in Figure 5 was electrotransferred to a nitrocellulose membrane and then incubated with anti-CK18 monoclonal antibodies. The lane numbers correspond to DPC proteins isolated from cultures treated with: (1) untreated; (2) 1 nM arsenite; (3) 10 nM arsenite; (4) 102 nM arsenite; (5) 104 nM arsenite. (B) Bars represent average densitometry values ± SE obtained in analysis of immunoblots from three replicate treatments, one of which is shown in (A) (F = 299.5, P < 0.001). *The percentage is expressed with respect to values obtained in untreated cultures.
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Fig. 7. High salt treatment of DPC. Isolated DPC were or were not washed with 2.5 M NaCl, resuspended in KCl/EDTA/TrisHCl solution then dialyzed in PBS buffer, pH 7.6. Seventy micrograms of protein were loaded on a polyacrylamide gel under reducing conditions. The separated proteins were electrotransferred and anti-CK18 monoclonal antibodies used to identify CK18. The lane numbers correspond to DPC isolated from cultures treated with: (1) 103 nM K2Cr2O7; (2) untreated; (3) 1 nM arsenite; (4) 10 nM arsenite; (5) 102 nM arsenite without NaCl wash; (6) 104 nM arsenite without NaCl wash; (7) untreated without NaCl wash.
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Fig. 8. Densitometry analysis of immunoblots using anti-CK18 antibodies of (A) total cell protein PAGE and (B) DPC protein PAGE. Points represent average densitometry values ± SE obtained in analysis of immunoblots from three replicate treatments. *The percentage is expressed with respect to values obtained in untreated cultures.
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Fig. 9. Immunostaining of CK18 in WRL-68 cells. Cells were cultured on glass coverslips and treated with NaAsO2. Indirect immunostaining of the cells was done using anti-CK18 monoclonal antibodies and anti-mouse IgGFITC antibodies. Analysis of the samples was done using fluorescence and confocal microscopy. Cells were treated with: (A) untreated; (B) 1 nM arsenite; (C) 10 nM arsenite; (D) 104 nM arsenite. Fluorescense intensity is given with respect to the intensity emitted in the untreated cultures.
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Discussion
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Trivalent iAs was not oxidized to pentavalent species in the cells, which was proved by speciation analysis. The results of As metabolism experiments indicated that WRL-68 hepatic cells accumulate arsenite from the culture medium but do not convert arsenite to methylated forms. DPC formation was linearly related to intracellular As, indicating that they represent the effect of the intracellular dose of As(III) (Figure 1A and B
). Methylation of iAs is an important step in the elimination of As because pentavalent methylated arsenicals are rapidly excreted and not retained (20,21). However, iAs accumulation also plays an important role in the toxic effects caused by As since metabolism of As is affected by the dose (2224). High doses can saturate or inhibit As methylation (25), increasing the presence of trivalent As in the tissue. The liver has been proposed to be the main site of As methylation; it is plausible that this organ accumulates As. Hepatic WRL-68 cells offer a good opportunity for the study of the toxic effects of iAs without a contribution of the toxic effects attributable to putative more toxic trivalent intermediates.
The removal or decay of DPC after treatment was dose-related (Figure 4
). Cultures treated with the lowest dose (1 nM) showed the fastest decay, maintaining significantly lower levels of DPC than untreated cultures, even after 24 h, an effect that is not observed at higher arsenite concentrations. The efficient removal of DPC at low doses suggests the induction of repair mechanisms. Increased amounts of CK18 were detected in treated cells by immunoblotting and immunostaining. While this effect was not dose-related, crosslinking of this protein to DNA was augmented with dose (Figure 8
), strengthening the idea that the formation of DPC is dependent on the presence of arsenite and could be used to estimate effective dose and early damage.
Trivalent As is known to induce the synthesis of many stress proteins and growth factors (26,27), so the higher amount of CK18 could be explained by a similar effect on cytokeratin expression. Several cytokeratins were identified among the proteins crosslinked with DNA, CK18, which is a specific liver intermediate filament, among them (28) (Figures 5 and 6
). Crosslinks between DNA and CK18 were sensitive to high salt conditions, similar to those required to isolate DNA from nuclear matrix proteins (Figure 7
) (28). Other keratins of ~39, 49 and 52 kDa were previously identified in the nuclei of Novikoff ascites hepatoma cells, as were keratin-like proteins in the nuclei of rat liver cells (15,16,29), suggesting that these proteins are part of the nuclear matrix and in crosslinking distance of DNA. Confocal microscopic analysis of immunostained cells with monoclonal anti-CK18 antibodies showed the presence of these filaments in the cytoplasm, around the nuclear envelope and inside the nucleus, thus DNA could interact with perinuclear or nuclear cytokeratin filaments to originate DPC. Also, alterations of the CK18 thread-like pattern observed in controls were seen in arsenite-treated (Figure 9AD
) and chromate-treated (data not shown) cells. Disruption of the polymerization of microtubules, another important cytoskeletal component rich in thiol groups, has been observed in human lymphocytes treated with sodium arsenite (30). Arsenite disruption of microtubules and micro and intermediate filaments could explain the in vitro loss or gain of chromosomes (aneugenic effect) and the increased micronucleus frequencies observed in buccal and bladder cells from humans exposed to As in the drinking water (9,30,31). Increased immunofluorescence (Figure 9
) coincided with an augmented presence of CK18 detected by immunoblotting of total protein PAGE (Figure 8
).
Cytokeratin synthesis is tightly correlated with differentiation programs of various epithelial cell types, among them liver cells (28), thus As could not only be damaging DNA but also modifying differentiation patterns in tissues where it accumulates (12). Interestingly, hyperkeratinization of stratified epithelia, the skin of the palms and soles of the feet, is a clinical manifestation of As poisoning (12).
Arsenite-induced DPC could impair DNA replication, leaving unreplicated stretches that might result in chromosome and chromatid-type aberrations and generating aneuploid cells through the disruption of cytoskeletal proteins observed at the concentrations employed in this work (31). DPC is a type of DNA damage that could be used as a biomarker of As exposure related to primary or early lesions of the carcinogenic process. Also, the fact that thiol-rich proteins such as cytokeratins appear strongly bound to DNA could indicate that trivalent forms of As could be directly involved in binding of the proteins.
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Acknowledgments
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We would like to thank Tzype Govezensky for technical assistance in the densitometry analysis. Isabel Perez Montfort corrected the English version of the manuscript. This work was partially sponsored by PAPIIT IN207196 and by the Programa de Apoyo para Estudios de Posgrado (PAEP). Patricia Ramírez received a scholarship form CONACYT and DGAPA.
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Notes
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3 To whom correspondence should be addressed Email: margen{at}servidor.unam.mx 
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Received June 8, 1999;
revised November 2, 1999;
accepted November 29, 1999.