Oxidative Damage Induced to Guanine and 8-oxoguanine by the ...

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Oxidative Damage Induced to Guanine and 8-oxoguanine by the ...

    Ni(II)-Induced DNA Damage

    Nickel(II)-catalysed oxidative guanine and DNA damage beyond 8-oxoguanine

    Michele C. Kelly, Gillian Whitaker, Blánaid White*, Malcolm R. Smyth

    School of Chemical Sciences, Dublin City University, Glasnevin, Dublin 9, Ireland. *Corresponding Author: Phone +353 1 700 8774 ; Email Address:


    Ni(II)-Induced DNA Damage

     Nickel(II)-catalysed oxidative guanine and DNA damage beyond 8-oxoguanine Abstract

    Oxidative DNA damage is one of the most important and most studied mechanisms of disease. It has been associated with a range of terminal diseases such as cancer, heart disease, hepatitis and HIV, as well as with a variety of everyday ailments. There are various mechanisms by which this type of DNA damage can be initiated, through radiation and chemical oxidation, among others, though even still, these mechanisms have yet to be fully elucidated. A HPLC-UV-EC study of the oxidation of DNA mediated by Nickel(II) obtained results that show an erratic, almost oscillatory formation of 8-oxoguanine (8-oxoG) from free guanine and from guanine in DNA. Sporadic 8-oxoG concentrations were also observed when 8-oxoG alone was subjected to these conditions. A HPLC-MS/MS study showed the formation of oxidised-guanidinohydantoin (oxGH) from free guanine at pH 11, and the formation of guanidinohydantoin (GH) from DNA at pH 5.5.

    Keywords: Nickel, HPLC-UV-EC, HPLC-MS/MS, oxidative DNA damage, guanine, 8-oxo-7,8-dihydroguanine.

    Abbreviations: HPLC-UV-EC, high performance liquid chromatography with ultra-violet and electrochemical detection; HPLC-MS, high performance liquid chromatography with


    Ni(II)-Induced DNA Damage

    mass spectrometric detection; HIV, human immunodeficiency virus; DNA,

    deoxyribonucleic acid; G, guanine; 8-oxoG, 7,8-dihydro-8-oxoguanine; 8-OH-dG, 8-hydroxy-2’-deoxyguanosine; SOD, superoxide dismutase; FapyG, formamidopyrimidine; FHIT, fragile histidine triad; LOD, limit of detection; EC, electrochemical; GH, guanidinohydantoin; oxGH, oxidised guanidinohydantoin; Sp, spiroimidodihydantoin; GF-AAS, graphite furnace atomic absorption spectroscopy; ROS, reactive oxygen species.


    DNA is subjected to thousands of oxidative hits per day.[1] Oxidative DNA damage has been implicated as a factor of cancer, neurodegeneration, and heart disease. It can cause strand breaks, base modifications and base mutations.[2] Diet, lifestyle and other environmental conditions and factors can alter the amount of oxidative stress that a body will undergo.[3-6] There is much research in this area at present, both in trying to determine the causes of oxidative stress and to elucidate the mechanisms of action of these external and internal contributory factors and to determine any roles this oxidative stress may have in various diseases.[7-9]

    Most of the research to date has been centred around guanine (G) and 2’-

    deoxyguanosine (dG), the most easily oxidised of the four DNA bases and the four nucleosides respectively.[10] Their primary oxidation products, 7,8-dihydro-8-oxoguanine (8-oxoG) and 8-hydroxy-2’-deoxyguanosine (8-OH-dG) respectively, have

    been analysed in depth to date, as they are considered to have the potential to lead to the


    Ni(II)-Induced DNA Damage

    determination of the degree of oxidative DNA damage. Analysis of oxidative DNA products can also potentially lead to the elucidation of oxidative stress mechanisms.

    8-oxoG and 8-OH-dG are also subjected to oxidative attack, and are even more susceptible to oxidative damage, as their oxidation potentials are lower than that of their precursors. This means that there is also a spectrum of potential further oxidation products to investigate, giving an even more in-depth view of the full picture of oxidation of DNA.

    The main causes of oxidative DNA damage are irradiation, chemical reactions and oxidation by reactive oxygen species (ROS).[8] One of the most investigated ROS is ?OH.[11,12] One of the methods from which it is produced is the Fenton reaction,[13] where a transition metal is oxidised to a higher oxidised state, by donating an electron to a hydrogen peroxide species, resulting in the formation of a hydroxyl radical and a hydroxyl ion.[13,14] ?OH is one of the most studied reactive biological radicals,[15] and

    has been implicated in reactions with the nucleic acid bases of DNA [16]. ?OH reacts

    preferentially with the -bonds of DNA bases, but can also interact with the sugar units by hydrogen abstraction.[17] ?OH is known to react with each of the four DNA bases,

    resulting in mutagenic lesions.

    ?OH attacks the guanine moiety at the C4, C5 or the C8 position. The addition of the radical to the C4 position is in greater yield (60%) than the C8 position (25%). The formation of radicals is seen initially. On C8 oxidation, the resulting 8-hydroxy-7,8-dihydroguanyl radical is redox ambivalent, i.e., it can be oxidised or reduced to form

    oxidation product, 8-oxoG, or reduction product, formamidopyrimidine (Fapy-G)

     respectively.[18] The 4- or 5- OH-guanine radicalcan be dehydrated to form a further


    Ni(II)-Induced DNA Damage

    oxyl radical which is then converted to final oxidation products, imidozolone and oxazolone derivatives. These can also decay to reform guanine, in what can be considered as an “auto-repair” mechanism. 8-oxoG is the most abundant oxidation

    product of ?OH oxidation of guanine, with a 50% yield. Fapy-G had a reported yield of 20%. Cadet et al. also implicated the ?OH in tandem DNA base damage.[11]

    This damage via the Fenton reaction can be mediated in vivo by labile transition

    metals, such as iron (Fe), copper (Cu) and nickel (Ni).[19] In a study of the effect of carcinogenic nickel compounds, Kawanishi et al. looked at the effects of NiSOinduced 4

    oxidative DNA damage. The formation of 8-OH-dG was monitored over time with samples taken at lengthy intervals (2, 4, 16 and 24 hours). Ni was found to induce damage to DNA.[20] Ni is an abundant transition metal in the environment. It is a trace element, present in some chocolate, nuts, oatmeal, beans and pulses, with daily dietary intake varying from about 100 to 900 g/day.[21-23] Ni has been found in its highest

    ] concentrations in the lungs, kidneys and in some hormone-producing tissues.[24-25

    Some Ni compounds are known carcinogens, e.g. nickel subsulfide and nickel

    carbonyl have been reported to cause lung and nasal cancer and have been labelled as Group A and Group B2 carcinogens respectively. Metallic nickel can also cause skin irritations and dermatitis and is a Group C carcinogen.[26-27]

    Both soluble and insoluble forms of Ni damage genetic material. Examples of such damage include: DNA strand breaks, mutations, chromosomal damage, cell transformation, and disrupted DNA repair.[11, 28, 29] Ni has also been reported to damage other cellular factors such as the tumour suppressor genes p53 and FHIT (fragile histidine triad) via protein damage.[30]


    Ni(II)-Induced DNA Damage

    Due to its ability to vary oxidation states, Ni in certain complexes with natural ligands can also participate in redox reactions at physiological pH and may well be able, therefore, to generate strong oxidising species in its reaction with hydrogen peroxide (HO).[31] Available nickel in vivo should, therefore, be able to cause oxidative DNA 22

    damage by the production of noxious hydroxyl radicals and other types of ROS by this reaction. The formation of 8-oxoG from reactions involving DNA and Ni has been reported before, though in relatively low levels. Damage to DNA by different Ni compounds and the enhancement of Ni oxidation by biological ligands resulted in 8-oxoG formation.[19,32-36] It is also noted that there is an association between Ni concentration and the amount of oxidative lesions in urine.[34] The formation of oxidative lesions in DNA bases found in urine over time has not been mapped extensively with min. by min. sampling. Such min. by min. sampling would allow for a more detailed insight into the mechanisms of Ni(II) -mediated damage to DNA bases. Because of its carcinogenic properties and its redox abilities, nickel was chosen for this research. We therefore chose to investigate the mechanisms of oxidative damage to DNA caused by nickel compounds.

    This research is focused on the elucidation of the mechanism of in vitro oxidation

    of G; both free in solution and in the DNA backbone, by a Ni(II)-mediated reaction. The methods used in this study were HPLC-UV-EC for the determination of 8-oxoG and G and HPLC-MS/MS for the determination of 8-oxoG and structural determination of its further oxidation products.


    Ni(II)-Induced DNA Damage

    Materials and Methods


    All chemicals including the DNA bases guanine (G0381, ?99%), adenine (A8626,

    ?99%), thymine (T0376, ?99%), cytosine (C3506, ?99%), and uracil (U0750, ?99%),

    7,8-dihydro-8-oxoguanine (R288608), calf thymus DNA sodium salt (D1501, Type I, fibres) [2,000 av. base pairs, 41.2% G/C] and nickel sulphate hexahydrate (22,767-6, ACS reagent, 99%) were purchased from Sigma-Aldrich (Tallaght, Dublin, Ireland). Ethanol, methanol and HPLC-MS grade methanol were purchased from Labscan Ltd. (Dublin, Ireland). Deionised water was purified using a MilliQ system to a specific resistance of greater than 18 M;.cm. All HPLC buffers and mobile phases were filtered through a 47mm, 0.45 m polyvinylidene fluoride (PVDF) micropore filter prior to use. Fresh solutions of all standards were prepared weekly.

    Incubation of G, 8-oxoG and DNA with Ni(II) and HO 22

    For HPLC-UV-EC, a 10 mM solution of G, prepared in 0.1 M NaOH, was

    oincubated at 37 C with 1.5 mM NiSO.6HO and 0.5 M solution of hydrogen peroxide 42

    (HO). A 2.4 mM 8-oxoG standard, also prepared in 0.1 M NaOH, and a 2 mg/ml 22

    standard of DNA in 50 mM ammonium acetate buffer pH 5.5 were analysed similarly.

    Incubations were carried out from 0-30 min., with duplicate sampling of 100 l at

    o1 min. intervals. The reaction was quenched in 1 ml of cold ethanol (cooled to 18 C).

    The solution was then dried under nitrogen and refrigerated until analysis by HPLC. G and 8-oxoG samples were reconstituted in 10% 0.1 M NaOH, 90% 50 mM ammonium acetate, 85 mM acetic acid buffer, pH 5.5 to 1 ml. DNA was hydrolysed with formic acid to release DNA bases and then reconstituted with 50 mM ammonium acetate, 85 mM


    Ni(II)-Induced DNA Damage

    acetic acid buffer, pH 5.5 prior to analysis. For mass spectrometric analysis, samples were prepared in 10 mM NaOH and reconstituted in 100 l 10 mM NaOH and 900 l 50

    mM ammonium acetate. All samples were filtered through a 4.5 m micropore filter prior

    to injection.

HPLC-UV-EC analysis of 8-oxoG formation.

    Samples were separated by reversed phase HPLC using a Varian ProStar HPLC system with Varian ProStar 230 Solvent Delivery Module and Varian ProStar 310 UV-VIS Detector. The eluent composition was 10% methanol, 90% 50 mM ammonium acetate, 85 mM acetic acid buffer through a Restek reverse phase Ultra C18 5 m 4.9 x

    250 mm column, equipped with Ultra C18 4 x 10 mm guard column. The separation was carried out at 1.0 ml/min. isocratic elution and the run time for the separation was 6 min. G and uracil were detected using UV detection at 254 nm and any 8-oxoG formed was detected by electrochemical (EC) detection, using a CC-4 electrochemical cell comprising of glassy carbon working electrode, stainless steel auxiliary electrode and Ag/AgCl reference electrode at a detection potential 550 mV. EC chromatograms were generated using a Shimadzu integrator. UN-SCAN-IT digitising software was used to digitise integrator chromatograms, which were then imported into SigmaPlot 8.0 or MS Office Excel.

    HPLC-MS/MS Analysis of Further Oxidation Products.

    Incubated samples were analysed by HPLC-MS-MS using an Agilent 1100 HPLC System with diode array detection coupled to a Bruker Daltonics Esquire 3000 LC-MS. Reconstituted samples were separated by HPLC using gradient elution through a Supelco


    Ni(II)-Induced DNA Damage

    Supelcosil LC-18 reversed phase column 5 m 2.1 mm x 250 mm. Eluent A consisted of

    10 mM ammonium acetate buffer pH 5.5. Eluent B was 50/50 methanol/water. A flow rate of 0.2 ml/min. was used with a linear gradient of 0-10% B from 0-22 min., 10-0% B from 22-25 min. DNA bases and oxidation products were also detected by UV detection at 210, 254 and 280 nm. Mass Spectrometric analysis was carried out at an ionisation

    otemperature of 300 C and at an ionisation potential of +15 V unless otherwise stated.

Controlled Experiments

     Controlled incubations were performed, with both G and 8-oxoG. Each of the oxidation reagents was replaced with deionised water, first singly, to determine whether one of the reagents could generate oxidative damage alone, and then both reagents were replaced to measure how much, if any, artifactual oxidation was caused by the reaction conditions themselves.

     A graphite furnace atomic absorption spectroscopy study of nickel sulphate was performed using glassware washed in 20% nitric acid and ultra-pure deionised water. The GF-AAS was calibrated using a pre-mix method with prepared standards. A 17 mM

    sample of the NiSO.6HO was analysed for iron content. 42


    Determination of 8-oxoG formation over time

    Controlled experiments

    A number of control experiments were undertaken on G, to ensure that all results were due to Ni/HO induced oxidative DNA damage, and not due to artifactual oxidation 22


    Ni(II)-Induced DNA Damage

    from the methodology involved or from residual contaminants such as iron in any of the reactants. EC detection was applied in order to determine the concentration of 8-oxoG production.

    A residual concentration of 8-oxoG was generated by the addition of HO alone 22

    (the same amount of 8-oxoG as was measured in untreated DNA, 0.06 0.02 μM).

    However, there was no increase in the concentration of 8-oxoG present as the incubation time with HOincreased and the concentration of 8-oxoG did not fluctuate with 22

    increasing incubation time. Neither was there any consumption of free G as the incubation time increased, nor any fluctuation in its concentration.

    Significant concentrations of 8-oxoG were not detected in any of the remaining controls, indicating that the oxidative damage caused was by the Ni(II)- HO reaction. 22

    There was no 8-oxoG detected when G was dried under nitrogen with no incubation performed. G concentration was mapped with UV detection and both G and 8-oxoG concentrations remained constant during all these controlled incubations.

    As a further control, graphite furnace atomic absorption spectroscopy (GF-AAS) studies carried out on nickel sulphate samples indicated that there was less than 0.0001% iron in these samples. This means for the 1.5 mM Ni(II) used in the experiments there was less than 0.0000075 mM (7.5 nM) of Fe present. As illustrated in the control experiments, concentration of iron was not responsible for the oxidative damage observed below.

    G Incubations

    The incubation of G with Ni(II) and HO showed an oscillatory concentration of 22

    8-oxoG over the 30 min. incubation period. In the Ni-HO mix, the highest oscillations 22


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