DNA Exposure to Buckminsterfullerene (C60): Toward DNA Stability

ARTICLE pubs.acs.org/est

DNA Exposure to Buckminsterfullerene (C60): Toward DNA Stability, Reactivity, and Replication Hongjie An and Bo Jin* School of Earth and Environmental Sciences, and School of Chemical Engineering, The University of Adelaide, Adelaide SA 5005, Australia

bS Supporting Information ABSTRACT: Buckminsterfullerene (C60) has received great research interest due to its extraordinary properties and increasing applications in manufacturing industry and biomedical technology. We recently reported C60 could enter bacterial cells and bind to DNA molecules. This study was to further determine how the DNA
C60 binding affected the thermal stability and enzymatic digestion of DNA molecules, and DNA mutations. Nano-C60 aggregates and water-soluble fullerenols were synthesized and their impact on DNA biochemical and microbial activity was investigated. Our results revealed that water-soluble fullerenols could bind to lambda DNA and improve DNA stability remarkably against thermal degradation at 70
85 °C in a dose-dependent manner. DNase I and HindIII restriction endonuclease activities were inhibited after interacting with fullerenols at a high dose. Experimental results also showed the different influence of fullerenol and nano-C60 on their antibacterial mechanisms, where fullerenols contributed considerable impact on cell damage and mutation rate. This preliminary study indicated that the application of fullerenols results in significant changes in the physical structures and biochemical functions of DNA molecules.

’ INTRODUCTION Buckminsterfullerene (C60) has been studied extensively in recent decades due to its extraordinary properties and increasing applications in drug delivery, material manufacturing, and medical technology.1,2 The widespread applications of C60 have resulted in the possibility of releasing these nanoparticles residues into ecosystems, which leads to considerable hazards for the environment and human health. Pristine C60 may have no acute or subacute toxicity in biological systems due to its hydrophobic properties.3
5 However, C60 can form stable aggregates in aqueous phase, making these nanoscaled C60 particles biologically active. Previous studies have reported that nano-C60 aggregates can enter microbial cells and bind to DNA molecules, and thus alter the molecular structures and biochemical functions of DNA by adhering to lipids, inducing oxidative stress and leading to interruption of cellular respiration.6
9 The potential biomedical applications of C60 are restricted by its low solubility in water, so a variety of methods to synthesize water-soluble C60 derivatives by introducing functional groups, such as
OH,
COOH, and
NH2, have been developed.10 Water-soluble C60 derivatives perform remarkable biological activities, and are practically useful for biomedical technologies.11
13 Among these derivatives, polyhydroxylated C60, also known as fullerol or fullerenol C60(OH)x, has received enormous interest in fundamental and applied research. Fullerenol can be prepared in two ways. One is prepared in the presence of phase transfer catalysts, such as tertrabutyl-moniu hydroxide. However, this method might increase the toxicity in biological systems caused by the trace amount of organic residues. The other approach to r 2011 American Chemical Society

generate fullerenol by electrophilic addition in an acid condition can effectively avoid that concern. Fullerenol demonstrates excellent free-radical scavenging and antioxidative ability in biological systems. They have been proven to inhibit tumor cell growth,14,15 antiproliferative,16,17 and neuroprotective activities.18,19 On the other hand, these findings also implicate that fullerenols can be toxic to normal cells.20 Zakharenko et al. reported that fullerenols are not genotoxic.21 Recent findings revealed that fullerenols could bind to DNA molecules,22 thus leading to significant impact on DNA stability and DNA activities. However, there is lack of information on how C60
DNA binding interactions affect the DNA molecular structures and functional ability, such as DNA stability and biochemical activities. A few studies showed that C60 and DNA interactions caused selective photocleavage effects.13,23,24 The interaction resulted in the variations in primary structures, stability, and error functions of DNA molecules, which are sensitive to temperature change. DNA depurination can thus be induced by cleavage of the nearby phosphodiester beyond a temperature of 50 °C.25,26 From previous studies13,23 and our recent findings,27 we hypothesize that the C60
DNA interaction could alter DNA dimensional structure, biological activities, and genetic functions. In this study, we aim to experimentally describe how the C60
DNA interactions could affect the DNA thermal stability, DNase I and HindIII Received: April 12, 2011 Accepted: June 30, 2011 Revised: June 24, 2011 Published: June 30, 2011 6608

dx.doi.org/10.1021/es2012319 | Environ. Sci. Technol. 2011, 45, 6608–6616

Environmental Science & Technology restriction endonuclease digestion, and DNA replication. Molecule research tools, such as atomic force microscopy (AFM), electrophoretic assay, and restriction endonuclease digestion were used to study the systems so as to provide new insight on the contribution of C60
DNA complex to the DNA functions.

’ MATERIALS AND METHODS Preparation of Nano-C60 and Fullerenols. To prepare nanoC60, 100 mg of C60 (Bucky, USA) was dissolved in 50 mL of toluene and sonicated for 30 min. Water (200 mL) was then added drop-by-drop, followed by sonication at room temperature for 72 h. After that, toluene was removed by evaporation under reduced pressure. The solution was filtered through a 0.2-μm membrane filter. The final concentration of C60 aqueous solution was measured by UV adsorption, where the water was evaporated and the dried deposit was transferred to toluene with the absorbance recorded using a spectrophotometer. Fullerenol was synthesized by a modified electrophilic addition method reported by Chiang et al.28 and Ko et al.29 C60 (80 mg) was added to a 100-mL flask containing 30 mL of concentrated sulfuric acid and 10 mL of nitric acid. The solution was vigorously stirred at room temperature for 72 h, and was then treated with Milli-Q water (5 mL) dropwise with vigorous stirring incubated in an ice
water bath. The mixture solution was further stirred at room temperature for 1 h, followed by centrifugation at 4000g for 10 min to remove insoluble particles. Two N NaOH was added drop-wise, and solution color changed from green
yellow to dark brown
red during the neutralization. The solutions were then allowed to stand at 0 °C for 24 h with the generated salt being removed by centrifugation. Solution was condensed by gradually evaporating the water. Characterization of Fullerenols. X-ray photoelectron spectroscopy (XPS) measurements were performed using a Leybold Heraeus LHS-10 spectrometer, with a Specs XR-50 Dual Anode X-ray source. The spectra were taken using Mg Ka radiation (1253.6 eV) at an anode power of 250 W (12.5 kV, 20 mA). The dried fullerenol were grinded into fine powder and mounted onto a stub using double-sided adhesive tapes. The stub was placed in the ultrahigh vacuum analysis chamber of the spectrometer. Deconvolution of the resulting spectra was performed using Fityk V 0.9.4 software. Assignment of the spectra peaks was made using the NIST database http://srdata.nist.gov/xps/, NIST. UV
vis spectra were taken with a LIUV-201 UV/vis spectrophotometer (Lambda Scientific Pty Ltd.). All infrared spectra (IR) were recorded at room temperature with a Nicolet 6700 spectrometer (Thermo Fisher Scientific Inc.) equipped with a ZnSe attenuated total reflectance (ATR) accessory, a DTGS detector, and a KBr beam splitter. The zeta-potentials of fullerenols were measured using a Malvern light scattering system (DLS, Malvern Nano ZS90). Matrix-assisted laser desorption/ionization (MALDI) time-offlight (TOF) mass spectra were acquired using a Bruker ultraflex III MALDI TOF/TOF mass spectrometer (Bruker Daltonik GmbH) operating in reflectron positive ion mode under the control of the flexControl software (Version 3.0, Bruker Daltonik GmbH). The MS spectra obtained were analyzed using flexAnalysis software (version 3.0, Bruker Daltonik GmbH) employing smoothing, background subtraction, and peak detection algorithms. Fullerenol or Nano-C60 and DNA Binding. Commercially available lambda DNA (Biolab, 500 ng/μL) was diluted to

ARTICLE

100 ng/μL with TE buffer (10 mM Tris
HCl, 1 mM ethylenediaminetetraacetic acid (EDTA), pH 8.0). Lambda DNA of 15 ng/μL was mixed with nano-C60 or fullerenols in a 200-μL vial. Concentrations of nano-C60 and fullerenol were adjusted to 0, 0.1, 0.5, 1, 5, 10, 50, 100, and 500 ng/μL. The solutions were adjusted to a total volume of 30 μL with TE buffer and then incubated at temperatures of 37, 50, 60, 65, 70, 75, 80, 85, and 90 °C for 1 h individually. Gel-shift assays were conducted on 0.8% agarose gel and stained with SYBR Safe DNA Gel Stain. Gel imaging was recorded by a Syngene imaging system. DNase I and HindIII Restriction Enzyme Digestion. Fullerenols and nano-C60 were diluted to the final concentration of 0, 0.1, 0.5, 1, 5, 10, 50, 100, and 500 ng/μL using DNase I reaction buffer (10 mM Tris
HCl, pH 7.6, 2.5 mM MgSO4, 0.5 mM CaCl2) and incubated with 15 ng/μL lambda DNA in a 200-μL PCR vial at room temperature for 30 min. Twenty units of DNase I (M0303S, New England Biolabs) solution was added to the mixture solutions, and the samples were incubated at 37 °C for another 1 h, followed by the addition of 5 μL of 20 mM EDTA to stop the reaction. The stability and morphology change of plasmid DNA were quantified by 0.8% agarose gel electrophoresis. HindIII (R0104S) was purchased from New England Biolabs. HindIII digestion of 0.45 μg of lambda DNA was also performed in the presence of fullerenols or nano-C60 at series concentrations as described above. DNA was adjusted to a final concentration of 15 ng/μL in a 200-μL PCR vial. HindIII (200 units) was added and NEB buffer 2 was used to adjust the volume up to 30 μL. These solutions were incubated at 37 °C for 2 h and the HindIII was deactivated at 65 °C for 20 min. Atomic Force Microscopy (AFM). AFM visualization was performed using a scanning probe microscope(Multimode AFM Nanoscope IV, Veeco, Santa Barbara, CA) which was equipped with an E scanner. Silicon cantilevers (NSC-11, MikroMasch) with spring constant of 48 N/m were used. Images were collected by Tapping mode AFM in air at room temperature under relative humidity of 25
35%. For AFM imaging, DNA solutions were deposited onto the fleshly cleaved mica surface pretreated by 10 mM nickel nitrate. Nickel nitrate solution (20 μL) was pipetted to a parafilm membrane surface covered by freshly cleaved mica. Three min later, the mica was repeatedly washed by doubledistilled water, and blown dry by N2. The suspensions (10 μL) of the DNA with and without nano-C60/fullerenol were deposited onto these pretreated mica surfaces and blown dry by N2 before AFM visualization. Fullerenol or Nano-C60 Affects DNA Replication in vivo. Strain E. coli cells transformed with plasmid of pMOL21 were incubated with fullerenes directly as described in previous work.30,31 The pMOL21 contained ampicillin resistance (Apr) and rpsL genes which encodes the small ribosomal protein S12. Protein S12 can bind streptomycin. The mutations in rpsL gene make the host cells resistant to streptomycin. The infected cells were cultivated in batch Luria
Bertani broth (LB) medium overnight with fullerenol or nano-C60. The cells were diluted and spread on ampicillin (100 mg/L) and streptomycin (80 mg/L) plates to determine the mutant number of rpsL. The cells were also transferred and spread on ampicillin plates to determine the total number of cells. The colony mutation frequency and error rate were calculated based on the ratio of rpsL mutants and the total number of colonies according to our previous article.32 Electron Microscopy Characterization. Samples for transmission electron microscopy (TEM) were prepared according to the following procedure. After overnight incubation, the cells 6609

dx.doi.org/10.1021/es2012319 |Environ. Sci. Technol. 2011, 45, 6608–6616

Environmental Science & Technology

ARTICLE

Figure 1. Gel-shift analysis of dose-dependent effect of fullerenol on lambda DNA after incubation at various temperatures. The final concentrations of fullerenol in lanes 1
9 were: 0, 0.1, 0.5, 1, 5, 10, 50, 100, and 500 ng/μL, respectively. Lanes M for markers (lambda DNA/HindIII plus marker).

were centrifuged at 4000g for 10 min to remove the medium. The pellet was resuspended in phosphate-buffered saline (PBS) with 4% sucrose. The cells were prefixed with 4% paraformaldehyde/ 1.25 glutaraldehyde (EM grade) for overnight. Postfix was made in 2% osmium tetroxide (OsO4) for 1 h on a rotator. Dehydrate was made by subsequent rinse with serial ethanol concentrations. Cells were embedded and sectioned, then mounted on 200-mesh copper grids, and stored in a desiccator before microscopic examination. Subsequently, the ultrathin sections were imaged at 100 kV using a Philips CM 100 TEM. Combined Staining Procedures. E. coli was cultured in LB medium (tryptone 10 g/L, yeast extract 5g/L, NaCl 10 g/L) to 108 cell/mL. The cells were harvested by centrifugation at 4000g for 10 min, and then resuspended in PBS and diluted to 106 cell/mL. C60 nanoparticles were added to the aliquots of 106 cell suspensions at final concentrations of 0, 0.05, 0.5, 5, and 50 mg/L. After 1 h incubation, the cells were pelleted and resuspended in PBS. A 10-μL aliquot of propidium iodide (PI) from stock (1 mg/mL) was added to 1-mL cell suspensions and incubated in dark for another 15 min.33 The cell suspensions were further treated by 2 mg/L DAPI (40 ,6-diamidino-20 -phenylindole) for 20 min in dark at room temperature. The suspensions were centrifuged and washed with PBS with cell images monitored by a fluorescence microscope (OLYMPUS BX51, Japan).

’ RESULTS AND DISCUSSION Physicochemical Properties of Fullerenol and Nano-C60. We synthesized nano-C60 and fullerenols, which were characterized by TEM, XPS, DLS, IR, and mass spectroscopy. The detailed experimental data are available in the Supporting Information (Figures S1
S5). The zeta-potential of fullerenol was around
12.5 mV at pH 7.5, and the zeta-potential of nanoC60 was about
20 mV. Our results indicated the negative charges on surface, which is consistent with the experimental

finding reported by Jiao et al.34 TEM images showed that nanoC60 was around 100 nm. The IR spectra of fullerenol showed peaks at 3233, 1720, 1612, 1563, 1354, and 1091 cm
1, suggesting that the synthesized fullerenol contained the structures of
C
O
H and hemiketal band. XPS data analysis indicated 15 carbons in one fullerene were in oxidation state. The MALDI TOF mass spectrum shows a peak centered at m/z = 720 corersponding to the mass of C60. Other peaks with the framework of fullerenol at 940.762, 1008.994, 1059.891, 1078.085, 1095.025, 1111.879, and 1128.873 were assigned to C60(OH)13, C60(OH)17, C60(OH)20, C60(OH)21, C60(OH)22, C60(OH)23, and C60(OH)24. Fullerenol or Nano-C60 Binding to DNA and Effect on DNA Thermal Stability. Results from electrophoresis through 0.8% agarose gel showed that fullerenol performed a dose-dependent effect on DNA thermal stability against DNA degradation (Figure 1). Retardation of lambda DNA migration corresponded to the DNA interaction with fullerenol (5
500 ng/μL) from 50 to 85 °C. The DNA retained in the gel well increased with fullerenol concentration. It is worthwhile to note that DNA thermal stability could be improved significantly when DNA was interacted with fullerenol at a very low dose. Lambda DNA treated with fullerenol at 1 ng/μL was found to be degraded gradually as temperature increased (60
85 °C). The DNA in the control trial was degraded over 60 °C (as indicated by a diffused smear). However, the amount of DNA incubated with 0.5 ng/μL fullerenol decreased gradually up to 70 °C and disappeared at 75 °C. In addition, a high dose of fullerenol (5
500 ng/μL) resulted in improving DNA’s thermal stability up to 80
85 °C, and DNA molecules were degraded substantially at 90 °C. Figure 2 shows the binding ability of nano-C60 to DNA and their impact on DNA thermal stability. Nano-C60 at a concentration