The Stepwise Process of Chromium-Induced DNA Breakage

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Chem. Res. Toxicol. 2005, 18, 1563-1566

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The Stepwise Process of Chromium-Induced DNA Breakage: Characterization by Electrochemistry, Atomic Force Microscopy, and DNA Electrophoresis Pei-Hui Yang,† Hong-Yang Gao,‡ Jiye Cai,† Jen-Fu Chiu,§ Hongzhe Sun,‡ and Qing-Yu He*,‡ Department of Chemistry, Jinan University, Guangzhou, Department of Chemistry and Open Laboratory of Chemical Biology, and Department of Anatomy, University of Hong Kong, Pokfulam, Hong Kong, China Received May 24, 2005

DNA conformational change and breakage induced by Cr(VI)-GSH interaction were characterized by the integrated tools of electrochemistry, atomic force microscopy (AFM), and DNA electrophoresis. While electrochemistry confirmed the formation of the active species generated from Cr(VI)-GSH reduction, which causes the DNA conformational changes, AFM imaging vividly demonstrated the stepwise process of the DNA denaturation and breakage for the first time. Our DNA electrophoresis further validated that the DNA breakage occurs unevenly at both of the single strands of the molecule. A scheme was drawn based on the experimental observations to explain the phenomenon of the Cr-induced DNA cleavage.

Introduction Chromium (Cr) exists primarily in two valence states, Cr(III) and Cr(VI). While there are arguments that suggest that Cr(III) is an essential nutrient for mammals, which require it for normal carbohydrate and lipid metabolism (1-3), Cr(VI) has been proven to be toxic and carcinogenic by epidemiological, animal, and cellular studies (4-6). Cr(VI) can readily enter cells through the sulfate channel and undergoes rapid metabolic reduction in cells by glutathione (GSH),1 ascorbic acid, or cysteine (7). During Cr(VI) reduction, Cr(V) and Cr(IV) intermediates, Cr(III) species, and oxidative radicals are believed to be the sources of Cr genotoxicity, since these species interact with DNA to form Cr-DNA adducts and cause DNA cross-links and DNA strand breaks (3, 8). Isolated Cr(V) and Cr(IV) complexes of R-hydrdoxy carboxylic acids also directly induce the breakage of single- and double-stranded DNA (9, 10). Recent detailed characterization of the Cr(VI)-GSH and Cr(V)-GSH species in solution demonstrated that labile Cr(IV) intermediates formed during the reactions of Cr(VI) with GSH and model thiols (11, 12) and that Cr(V) intermediates are the active species, which cleave DNA (13). Cr genotoxicity and carcinogenicity caused by the Cr-induced DNA damage have been the focus of extensive investigations using various biochemical and biophysical methods, including EPR, NMR, electronic and X-ray absorption spectroscopy, electrospray mass spectrometry, kinetics (3, 8), and molecular biology (8, 14-18). However, the * To whom correspondence should be addressed. Tel: +852 2299 0787. Fax: +852 2817 1006. E-mail: [email protected] or [email protected]. † Jinan University. ‡ Department of Chemistry and Open Laboratory of Chemical Biology, University of Hong Kong. § Department of Anatomy,University of Hong Kong. 1 Abbreviations: AFM, atomic force microscopy; GSH, glutathione; CV, cyclic voltammogram; CD, circular dichroism; LEC, lung epithelial cells.

molecular mechanisms of Cr biological activity in cells are far from fully understood. Electrochemistry is a useful tool in detecting various oxidation states in a complex reaction. The electrochemistry of Cr(VI)-GSH interaction should therefore be informative in monitoring the formation of the active Cr intermediate species. Atomic force microscopy (AFM) is a powerful surface analytical technique that can generate high-resolution images at the single molecule level (19, 20). Since its development, AFM has been employed to study biological structures and biomolecular interactions in physiologically relevant environments. In this study, we used electrochemical methods to monitor the formation of Cr intermediates during Cr(VI) reduction and took advantage of the ability of nanoscale imaging by AFM to visualize Cr-induced DNA strand breaks. We also used DNA electrophoresis to further illustrate the DNA strand cleavage. Our observations clearly demonstrated a stepwise process of DNA denaturation and provided evidence for the formation of DNA lesions and uneven single strand breaks.

Materials and Methods Calf thymus DNA, GSH (reduced form), K2Cr2O7, CH3COOH, and CH3COONa were purchased from Sigma. Glutamine, enicillin, and streptomycin were purchased from Gibco BRL (Grand Isle, NY), and newborn bovine serum was purchased from JRH Bioscience (Lenexa, KS). Cell Culture. Lung epithelial cells (LECs) were routinely grown at 37 °C in 95% air/5% CO2 using F-12 nutrient, supplemented with 2 mmol/L glutamine, 100 units/mL penicillin, 100 µg/mL streptomycin, and 10% (V/V) newborn bovine serum. DNA Extraction. DNA was extracted and purified according to the protocol described in Molecular Cloning (Sambrook and Russell, Cold Spring Harbor Laboratory Press, New York 2001). LECs (5 × 107) were collected and washed twice with ice-cold Tris-buffered saline (TBS) and then resuspended in TE (1 mL, pH 8.0). Lysis buffer [10 mM Tris-Cl, pH 8.0; 0.1 M EDTA, pH

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8.0; 0.5% (w/v) SDS; and 20 µg/mL DNase-free RNase] (10 mL) was added into the cell suspension and incubated for 1 h at 37 °C. Proteinase K (the final concentration was 100 µg/mL) was mixed with the lysate, and the mixture was incubated for 3 h at 50 °C. The mixed lysate was then extracted by phenol equilibrated with 0.1 M Tris-HCl (pH 8.0) once and by phenol: chloroform (1:1) twice. Ethanol (2 volumes) was added to the aqueous phase to precipitate the DNA, which was rewashed once by 70% ethanol and dissolved in MilliQ H2O. The concentration of the DNA solution was measured by absorbance at 260 nm. DNA Treatment with Cr(VI)-GSH. In acetate buffer solution (pH 6.5), K2Cr2O7 (2.0 × 10-3 mol/L) was mixed with GSH (1.5 × 10-2 mol/L) for 2-3 min. DNA (1.5 µg; final concentration, 2.0 × 10-4 mol/L) was added to the mixture and incubated at 37 °C for 20, 30, 40, and 60 min. The products were analyzed by 1% neutral or alkaline agarose gel electrophoresis. DNA Electrophoresis. Cr(VI)-GSH-treated DNA was mixed with 6× loading buffer [40% (w/v) sucrose, 0.25% bromophenol blue] and assayed using neutral agarose gel (1%) electrophoresis in 1× TBE (89 mmol/L boric acid, 89 mmol/L Tris, and 2 mmol/L EDTA) at a voltage gradient of 2 V/cm until bromophenol blue running to the place of three-fifths of the gel. After electrophoresis, the ethidium bromide-stained gels were illuminated and photographed in an UV transilluminator. For alkaline gel, Cr(VI)-GSH-treated DNA was mixed with 6× alkaline loading buffer (18% Ficall 400, Pharmacia), 10× alkaline buffer (500 mmol/L NaOH and 10 mmol/L EDTA), and 0.02% bromophenol blue and assayed using alkaline agarose gel (1%) electrophoresis running in 1× alkaline buffer (50 mmol/L NaOH and 1 mmol/L EDTA). The gel was then neutralized with neutral buffer (1.5 mol/L NaCl and 1 mol/L Tris‚Cl, pH 7.6) for 45 min and stained with ethidium bromide and photographed as described above. Electrochemistry. Electrochemical measurements were performed on a CHI660A electrochemistry work station (CH Instruments Co., United States). The electrochemical cell consists of a three-electrode system with a gold electrode (Φ ) 2 mm) as the working electrode, a saturated calomel electrode (SCE) as reference, and a platinum wire as counter electrode. All experimental solutions were deaerated by sparging with pure nitrogen for 15 min, and a nitrogen atmosphere was kept over the solution during measurements. The electrochemical dynamics measurements were carried out at a temperature of 25 × 0.5 °C. The cyclic voltammograms (CVs) were recorded at a scan rate of 100 mV/s. The gold electrode was first polished with abrasive paper and then with alumina (0.05 µm) slurry on microcloth pads, followed by rinsing with water and ethanol and brief cleaning in an ultrasonic bath. Circular Dichroism (CD). CD spectra were measured using a Jasco J-720 spectropolarimeter (Jasco, Japan) in a rectangular quartz cell (1.00 mm path length) at 200 nm/min scanning speed. The DNA concentration was maintained at 100 µM. AFM. AFM imaging was performed using a multimode AFM with a Nanoscopy IIIa controller (Veeco/Digital Instruments, Santa Barbara, CA). The DNA sample (10 µL) was deposited on freshly cut mica substrates and was imaged in air at room temperature. Commercially available NCH-100 silicon microcantilevers (Nanosensors, Germany) with spring constants of 21-78 N/m were used for tapping mode scanning with an E-scanner (15 µm) and a drive frequency of ∼300 kHz. Images were captured at a scan rate of 0.5 Hz in a 512 × 512 pixel format and flattened to remove the background slop.

Results and Discussion In acetate buffer solution (pH 6.5), GSH alone has no signal in CV (Figure 1A, curve 1). However, Cr(VI) alone shows an irreversible 3e- reduction at 0.10 V (vs SCE) (Figure 1A, curve 2). When Cr(VI) was mixed with GSH, the peak at 0.10 V disappeared and a new reduction signal at 0.24 V emerged (Figure 1A, curve 3), signifying a Cr(VI)-GSH interaction. The Cr(VI)-GSH transient shows quasi-reversible behavior with a peak-to-peak

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Figure 1. (A) CV of potassium dichromium in acetate buffer solution at pH 6.5. Scan rate ) 100 mv/s. Key: 1, [GSH] ) 1.0 × 10-3mol/L; 2, [K2Cr2O7] ) 2.0 × 10-4 mol/L; 3, [K2Cr2O7] ) 2.0 × 10-4mol/L + [GSH] ) 1.0 × 10-3mol/L. (B) Peak current of potassium dichromium solution as a function of time after addition of GSH. Lines 1-6: 0, 10, 20, 30, 60, and 90 min.

Figure 2. Electrochemical kinetics of DNA denaturizing. (A) CV of DNA solution with gold electrode. [DNA] ) 2.0 × 10-5 M + [K2Cr2O7] ) 1.0 × 10-4 mol/L + [GSH] ) 5.0 × 10-4 mol/L. (B) Peak currents of denaturing DNA changed with incubation time; T1/2 ) 25 min.

separation of ∼110 mV, confirming the reported results that Cr(VI) forms characterizable complexes, which are short-lived but detectable (11, 12). As the reaction proceeded, the reduction peak of 0.24 V shifted to 0.19 V (Figure 1B, curves 1-3) and then further shifted to ∼0.1 V (Figure 1B, curves 4-6), with the peak current initially increasing and then decreasing. Only one redox process was observed for each cycle throughout the entire reaction within the potential range scanned. This phenomenon clearly indicates subsequent intermediates during the interaction, which may represent products of the reduction of the Cr(VI)-GSH complex followed by sequential reduction to Cr(V) and Cr(III) species through transient Cr(IV) intermediates (12, 13, 21, 22). The reduction of Cr(VI) to Cr(III) by GSH was completed within 90 min (Figure 1B), consistent with the kinetics data of t1/2 ) 60 min for Cr(VI)-GSH interaction (23). In light of the time frame of the Cr intermediate formation, DNA (calf thymus) conformational changes induced by the Cr(VI)-GSH interaction can be followed by electrochemical kinetics, as shown in Figure 2. In the presence of DNA, the CV spectrum of the Cr(VI)-GSH reaction changes dramatically, with the redox potentials shifting from 0.24/0.35 to 0/0.20 V (Figure 2A) accompanied by changes in the CD spectrum (Figure 3), indicating conformational changes in the DNA. Further shifting of the reduction peak during the reaction reflects the participation of the multiple Cr intermediates generated during the Cr(VI) reduction. The CV spectra with 3

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Figure 3. CD spectra of DNA showing DNA conformational change in the presence of Cr(VI)-GSH interaction.

min scan intervals contain multiple peaks, implying the simultaneous presence of a number of redox-active species. Figure 2B shows the plots of the peak currents increasing with time. Curve fitting of the plots with a first-order equation yielded k ) 0.04 min-1 or t1/2 ) 25 min for both oxidation and reduction. Given the fact stated above that the Cr(VI) reduction by GSH is a successive process with a t1/2 ) 60 min, the smaller t1/2 ) 25 min for the DNA conformational changes suggests that the Cr(V) or Cr(IV) intermediate species may play major roles in the conformational changes (13). As shown in Figure 3, calf thymus DNA exhibits a typical B-form conformation (double helix) as evidenced by a pair of conservative CD bands including a positive band at 278 nm and a negative band at 246 nm (24, 25). When both Cr(VI) and GSH were added to the DNA solution, the conservative CD bands were significantly perturbed, along with the disappearance of the positive band at 220 nm. After 40 min, the CD spectrum further altered with the paired CD bands at 246 and 278 nm red shifting, indicating that the DNA double helix conformation was substantially changed. Investigation with AFM revealed that the Cr-induced DNA conformational changes were subsequently accompanied by DNA strand breakage. As shown in Figure 4, AFM images captured at successive intervals demonstrate the entire process of DNA conformational changes, denaturation, and strand breaking induced by Cr(VI)GSH interaction. Figure 4A shows a single DNA molecule with some initial binding of Cr species. The interaction between DNA and Cr species caused a number of prominences on the DNA strand in 20 min (Figure 4B). The DNA double strand conformational changes can be visualized in 30 min (Figure 4C), accompanied by partial opening of the double-stranded molecule. The image taken at 40 min clearly showed the denatured doublestranded DNA and the break site in a single strand (Figure 4D). It appears from Figure 4D that the DNA denaturation may be limited to the small regions in the neighborhood of Cr-DNA complexes at the single strand breaks. These phenomena are consistent with the observations from the electrochemical and CD experiments described above. The high-resolution AFM images display the formation of Cr-DNA adducts (Figure 4B), DNA conformational changes (Figure 4C), and DNA strand breaks (Figure 4D). To our knowledge, this is the first time that the entire process of Cr(VI)-induced DNA

Figure 4. AFM images showing the process of DNA denaturation and strand breakage induced by Cr(VI)-GSH interaction. (A-D) Images were taken with incubation times of 0, 20, 30, and 40 min, respectively. Arrows indicate the sites of Cr-DNA binding (A) and DNA strand breaks (D).

Figure 5. DNA electrophoresis for calf thymus DNA under neutral (A) and alkaline (B) conditions and for LECs DNA under neutral (C) and alkaline (D) conditions. Lanes 1-5: DNA treatment with Cr(VI)-GSH for 0, 20, 30, 40, and 60 min.

breakage has been directly demonstrated and visualized through AFM imaging. Confirmation experiments were carried out by using DNA electrophoresis under neutral and alkaline conditions for analyzing double- and single-stranded DNA, respectively. Figure 5 presents the results for both calf thymus DNA and LEC DNA, the latter extracted from Cr(IV)-treated LEC cells. Because commercially available calf thymus DNA contains molecules of different lengths, it displays a smear DNA ladder in the neutral gel (Figure 5A, lane 1). This DNA ladder did not change under neutral conditions even after the interaction with Cr(VI)-GSH (Figure 5A). The pure LEC DNA showed a single band in the neutral gel during the entire process of Cr(VI)-GSH interaction (Figure 5C). These results from the DNA neutral electrophoresis suggest that the DNA molecules remained intact in size although uneven

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Scheme 1. Model for Explaining the Occurrence of DNA Breakage Induced by Cr(VI)-GSH Interaction

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imaging demonstrated the stepwise process of the DNA conformational changes, denaturation, and breakage vividly. These results add new information for better understanding the molecular mechanisms involved in Cr(VI)-induced neoplasia.

Acknowledgment. This work was partially supported by Hong Kong Research Grants Council Grants HKU 7227/02M (to Q.Y.H.) and HKU 7395/03M (to J.F.C.) and University Grant 200411159054 (to Q.Y.H.), China National Grant Programs 2001CB510101, 30230350, 021190, and 2003Z3-D2041, the Department of Chemistry, and the Areas of Excellence scheme of Hong Kong University Grants Committee.

References

breakages may occur on the single strands. Under alkaline conditions, this intact DNA was completely unwound to single strand molecules, which present in a variety of sizes due to uneven breakages. Figure 5D clearly demonstrated the gradient of the single strand DNA bands, indicating the increasing breakage of pure LEC DNA with increasing time of treatment with Cr(VI)-GSH. For calf thymus DNA, the large smear band (Figure 5B, lane 1) from the distribution of molecular sizes is present. However, the Cr-induced DNA breakage can still be distinguished by carefully comparing the alkaline gels at different reaction times (Figure 5B). These breakages may be captured by AFM imaging as described above. On the basis of these results, we can draw a scheme to explain how DNA breakage occurs in the presence of Cr(VI)-GSH interaction (Scheme 1). The active species generated from Cr(VI)-GSH interaction forms adducts with DNA (Figure 4A,B), which cause DNA conformational changes and cleavage unevenly at some sites of both of the single strands (Figure 4C,D). The partially strand broken DNA remains a single molecule that migrates in a single band in neutral gel (Figure 5A,C). Alkaline buffer completely unwinds the double-stranded DNA resulting in single strands of different sizes. We therefore observed the smear bands in the alkaline gel after the interaction (Figure 5B,D). In conclusion, our current data confirmed the process of double-stranded DNA conformational changes and cleavage induced by the Cr(VI)-GSH interaction and further validated the occurrence of uneven breakages on the single strands of the denatured DNA. Consistent with previous studies, the electrochemical experiments suggested that Cr(V) intermediates may play a major role in the DNA cleavage. In particular, the present AFM

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