Toxicology Mechanisms and Methods, 18:589–595, 2008Copyright ©c Informa UK Ltd.ISSN: 1537-6516 print; 1537-6524 onlineDOI: 10.1080/15376510802205676

Biochemical Evaluation of Human DNA-LysinePhotoadduct Treated with Peroxynitrite

Rizwan Ahmad,Zafar Rasheed,Esha Kaushal,and Divya SinghDepartment of Biochemistry,SBS Post Graduate Institute ofBiomedical Sciences andResearch, Dehradun 248161,India

Haseeb AhsanDepartment of Toxicology,Jamia Hamdard, New Delhi110062, India

ABSTRACT Peroxynitrite is a reactive oxidant produced from nitric oxide(.NO) and superoxide anion (O2

.−). It is produced by the body in responseto environmental toxins, stress, ultraviolet light, ischemia/reperfusion, inflam-mation, etc. In vivo, peroxynitrite is formed in macrophages, endothelial cells,platelets, leukocytes, and neurons. It reacts with a variety of biomoleculesincluding proteins, lipids, and DNA. We have investigated the photochemicaladdition of lysine to native DNA in view of its potential importance in thephoto-cross-linking of histones to DNA in chromatin. Lysine- and arginine-rich histone H1 in nucleosome on modification by physical, chemical, orenvironmental agents forms histone-DNA adducts. We have characterizedthe photoadducts by absorption, fluorescence, and chromatographic methods.The UV absorption spectra of the DNA-lysine photoadduct showed hyper-chromism, indicating structural distortions in DNA either due to single-strandbreaks or opening of the double helix at the site of lysine conjugation. Onperoxynitrite treatment, the melting temperature (Tm) of the DNA-lysineadduct increased by 15◦C compared to the native DNA-lysine adduct. Adecrease in the fluorescence intensity of the DNA-lysine photoadduct withrespect to the modified adduct was observed. The gel filtration profile of theperoxynitrite-modified adduct was also found to be different from that of thenative DNA and DNA-lysine photoadduct. Hence, the peroxynitrite-modifiedphotoadduct may have important implications in toxicology, mutagenesis, andcarcinogenesis.

KEYWORDS Human DNA; Lysine; Peroxynitrite; Photoadduct

INTRODUCTIONPeroxynitrite (ONOO−) is a reactive oxidant produced from the reaction between the

free radicals, nitric oxide (.NO), and superoxide anion (O2.−), which reacts with a variety of

biomolecules including proteins, lipids, and DNA. Peroxynitrite is produced by the body inresponse to a variety of toxicologically relevant molecules including environmental toxins,ischemia/reperfusion, and inflammation (Pacher et al. 2007; Szabo et al. 2007). Because ofits oxidizing nature, peroxynitrite can damage a wide range of molecules in cells, includingDNA and proteins. In vivo, peroxynitrite reacts nucleophilically with carbon dioxide toform nitrosoperoxy-carbonate (ONOOCO2

−). Nitrosoperoxycarbonate homolyzes to formcarbonate radical and nitrogen dioxide. These radicals are believed to cause peroxynitrite-related cellular damage (Szabo 2003).

Peroxynitrite levels are elevated in inflammation and infection and play an importantrole in carcinogenesis (Ohshima 2003). It damages tumor suppressor genes and enhancesexpression of proto-oncogenes. Peroxynitrite-induced DNA damage leading to mutations

Received 20 February 2008;accepted 15 May 2008.

The authors are grateful to themanagement of SBSPGI, Dehradun,for providing necessary laboratory andinstrument facilities for the study.

Address correspondence to HaseebAhsan, Department of MedicalElementology and Toxicology, Facultyof Science, Jamia Hamdard, HamdardUniversity, New Delhi 110062, India.E-mail: hahsan@rediffmail.com

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has been strongly implicated in carcinogenesis (Tretyakova et al.2000). Peroxynitrite is a mutagenic agent with the potentialto produce nitration, nitrosation, and deamination reactionson DNA bases. It reacts significantly only with guanine,which upon oxidation and nitration leads to mutagenicityand strand breaks, respectively (Wiseman and Halliwell 1996;Yermilov et al. 1995). Prominent DNA modifications inducedby exposure to peroxynitrite lead to the formation of 8-nitro-guanine and 8-oxyguanine, as well as the induction of single-strand breaks (Szabo and Ohshima 1997). The mutagenicity ofperoxynitrite is believed to result from chemical modificationsat guanine leading to miscoding. DNA single strands generatedby peroxynitrite leads to activation of the nuclear enzymepoly (ADP-ribose) synthetase (PARS), which can trigger cellularsuicidal pathway (Szabo and Ohshima 1997).

Adducts arise from the chemical modification of bases inDNA or amino acids in proteins by toxic chemicals and high-energy UV radiation. Many chemicals known to be carcinogenicin humans have been shown to form adducts. Ultravioletradiation is regarded as one of the major environmental factorsresponsible for the photoconjugation of DNA with aminoacid residues. Lysine is an amino acid of particular interestas a potential participant in DNA-protein photo-cross-linking.Nearly 60% of thymine and cytosine bases in DNA aremodified due to lysine photoaddition and on average, everyhelical turn of DNA contains one lysine molecule in thephotobound state (Islam and Ali 1998). It appears to enhancethe antigenicity of the DNA-lysine adduct, suggesting possibleroles of peroxynitrite-induced neoepitopes in damaged DNA inthe production of autoantibodies in cancer patients (Dixit et al.2005).

In the present study, we have characterized the peroxynitrite-modified DNA-lysine photoadduct by using UV absorption,fluorescence, and chromatographic methods.

MATERIALS AND METHODSIsolation of Genomic DNA from

Human BloodBlood was collected from healthy individuals with informed

consent after obtaining permission from the Institute’s Govern-ing Committee on Medical Ethics. Genomic DNA was isolatedfrom human blood by using the standard proteinase K buffermethod (Miller et al. 1988; Epplen and Lubjuhn 1999). It waschecked for purity by calculating the A260/280 ratio (1.74) andUV absorption spectra between 200 nm and 400 nm (λmax at260 nm).

Formation of DNA-Lysine AdductDNA (1 µM) was mixed with lysine (3 µM) in phosphate-

buffered saline (PBS, pH 7.4) and incubated at 4◦C overnight.The complex was subjected to UV irradiation for 30 min. NativeDNA, unirradiated DNA-lysine complex, unirradiated lysine,irradiated lysine, and irradiated DNA were used as control.The photoadduct and its controls were extensively dialyzedagainst 10 mM carbonate-bicarbonate buffer (pH 10.7) andsubsequently dialyzed against PBS (pH 7.4) in order to obtainthe samples in the working medium (Islam and Ali 1998).

Modification of DNA-Lysine Adductwith Peroxynitrite

The DNA-lysine adduct was modified by peroxynitritethrough the synergistic action of a nitric oxide donor, sodiumnitroprusside (SNP), and a superoxide donor, pyrogallol, inthe presence of a chelator, EDTA (Dixit et al. 2005). Thesamples were incubated for 3 h at 37◦C such that 0.075 mMbp DNA-lysine adduct was treated with SNP/pyrogallol/EDTAmixture, each having a concentration of 0.5 mM. The reactionmixture was extensively dialyzed against PBS (pH 7.4) to removethe salts.

UV Absorption and DifferenceSpectra

The absorbance of native DNA, DNA-lysine adduct, andmodified DNA-lysine adduct were read at 260 nm and 280 nm,with the help of double beam spectrophotometer (Shimadzu,Japan) using PBS (pH 7.4) as blank (Ahsan et al. 2002; Arjumandet al. 1997). The A260/280 ratio and change in λmax wererecorded. Difference spectra of the DNA-lysine adduct againstnative DNA and the modified DNA-lysine adduct against theDNA-lysine adduct was plotted between 200 and 400 nm.

Thermal Denaturation StudiesThermal denaturation analysis of DNA was done in order

to ascertain the degree of modification in the DNA (Dixitet al. 2003; Arif and Ali 1996). The native, adduct, andmodified samples were subjected to heat denaturation. Thesamples were heated from 30◦C to 95◦C, the absorbances atvarious temperatures were recorded at 260 nm, and the meltingtemperature, Tm, was evaluated with the help of a curve plottedfor the percent denaturation calculated as follows:

%Denaturation = [(AT − A30)/(Amax − A30)]∗100

AT = Absorbance at a temperature T◦CAmax = Final maximum absorbance on the completion of

denaturationA30 = Initial absorbance at 30◦C

Fluorescence StudiesFluorescence measurements were performed on a Hitachi-

F200 Spectrofluorimeter (Japan). The fluorescence spectra weremeasured at 25 ± 0.1◦C with a cell of 1-cm path length.The excitation and emission slits were set at 5 and 10 nm,respectively. Intrinsic fluorescence was measured by exciting theDNA solution at 260 nm and emission spectra were recorded inthe range of 300 to 400 nm (Arjumand et al. 1997; Habib et al.2006). Loss of fluorescence intensity (FI) was calculated usingthe following equation:

% Loss of FI = [(FInative DNA − FImodified DNA)/FInative DNA]∗100

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FIGURE 1 UV absorption spectra of native DNA (�), DNA-lysine photoadduct (�), and peroxynitrite-modified adduct (�).

Sephadex G-100 Gel ChromatographyGel filtration was performed on a Sephadex G-100 column

equilibrated with PBS (pH 7.4). Samples of 1 mL each ofnative DNA, DNA-lysine adduct, and peroxynitrite-modifiedadduct were applied separately onto the column. Fractions of2.5 mL were collected and absorbance monitored at 260 nm(Dixit et al. 2005).

Statistical AnalysisAll experiments were performed two or more times unless

stated otherwise. The graphical data obtained during the processof characterization depict one such representative set.

RESULTSStudies have been carried out to synthesize and characterize

the photoconjugate between positively charged amino acid,lysine, and human DNA. Native human DNA was covalentlycross-linked with lysine under ultraviolet light. The amino acidwas found to be covalently photoconjugated to human DNAand resulted in the formation of a photoadduct. The resultingDNA-lysine photoadduct was also modified with peroxynitrite,resulting in the formation of a modified photoadduct. TheDNA-lysine and peroxynitrite modified DNA-lysine adducts

were characterized by various physicochemical methods such asUV absorption and difference spectra, thermal melting profile,fluorescence spectra, and Sephadex column chromatography.The characteristic differences in the DNA-lysine and modifiedphotoadducts were obtained from the studies presented below.

UV Absorption Spectral Analysis ofDNA-Lysine and Modified AdductsUV absorption spectral characteristics of the DNA-lysine

photoadduct were analyzed in order to monitor the changesincurred in DNA as a result of photomodification. A substantialincrease in absorbance was observed over the entire UV spectralrange (200–400 nm) for the DNA-lysine photoadduct (curve2) when compared with that of native DNA and lysine ascontrol (Fig. 1). This hyperchromic effect (52%) signifies theformation of single-stranded regions due to adduct formation.The UV absorbance ratio (A260/280) of the DNA-lysine pho-toadduct decreased to 1.2 from the usual 1.74 for native DNA(Table 1).

The UV absorption spectra of the peroxynitrite-modifiedDNA-lysine photoadduct (curve 1) exhibited an 84% increasein absorbance over the entire UV range and most noticeablyat 260 nm (Fig. 1). The UV absorbance ratio (A260/280) of the

TABLE 1 Absorption and fluorescence characteristics of native DNA, DNA-lysine adduct, and modified photoadduct∗

Biochemical parameters Native DNA DNA-lysine photoadduct Peroxynitrite-modified DNA-lysine adduct

A260/280 ratio 1.74 1.20 1.01Melting temperature (◦C) 75 70 85Hyperchromicity (%) — 52 84Loss of FI (%) — 76.1 21.3

∗ The data represent mean value from two or more independent experiments.

591 Peroxynitrite-Modified DNA-Lysine Photoadduct

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FIGURE 2 UV difference spectra of native DNA (�) and DNA-lysine photoadduct (�).

modified DNA-lysine photoadduct decreased to 1.01 from theusual 1.74 for native DNA (Table 1).

UV Difference Spectral Analysis ofDNA-Lysine and Modified AdductsThe UV difference spectral analysis of the DNA-lysine

adduct against native DNA showed a hypochromic shift overthe entire spectral range (200–400 nm). The peak at 260 nm wasdisturbed in the DNA-lysine photoadduct (curve 2) in contrast

to native DNA and the maximal change occurred at 260 nm(Fig. 2).

The perturbations occurring in the DNA-lysine photoadductas a consequence of peroxynitrite modification were alsoanalyzed by UV difference spectroscopy (Fig. 3). The differ-ence spectra of the peroxynitrite-modified DNA-lysine adductrevealed a characteristic 260-nm peak (curve 1) and broadeningof the spectral curve in the region of 250 to 320 nm with respectto the DNA-lysine photoadduct. The difference spectra showeda substantial hyperchromic shift over the entire UV spectralrange (200–400 nm).

FIGURE 3 UV difference spectra of DNA-lysine photoadduct (�) and peroxynitrite-modified photoadduct (�).

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FIGURE 4 Thermal melting profile of native DNA (�), DNA-lysine photoadduct (�), and peroxynitrite-modified photoadduct (�).

Thermal Helix-Coil TransitionThe melting profile of native DNA, the DNA-lysine pho-

toadduct, and the peroxynitrite-modified DNA-lysine pho-toadduct was analyzed between the temperature ranges of 30◦Cand 95◦C (Fig. 4). Increase in absorbance at 260 nm was taken asa measure of helix denaturation. The process was characterizedby determining the percent DNA in the denatured state as afunction of increasing temperature and then computing themelting temperature (Tm). The helix-coil thermal denaturationof native DNA was used as control and did not exhibit anysignificant denaturation from 30◦C to 65◦C. However, fullydenatured preparation was obtained at 90◦C. On the otherhand, heating of the DNA-lysine photoadduct (curve 1) showed

progressive denaturation starting at 65◦C, which persisted until85◦C. The Tm value of the DNA-lysine photoadduct was foundto be 70◦C. The result shows a net decrease of 5◦C in the Tmvalue of the DNA-lysine photoadduct when compared to nativeDNA (75◦C). The Tm for the peroxynitrite-modified DNA-lysine adduct (curve 3) was found to be 85◦C, indicating a netincrease of 15◦C in the Tm of the peroxynitrite-modified DNA-lysine adduct as compared to the DNA-lysine adduct (Fig. 4).

Fluorescence Spectral StudiesAs shown in Figure 5, the fluorescence emission intensity

(FI) was highest for native DNA (curve 1) and least for the

FIGURE 5 Fluorescence spectra of native DNA (top curve), DNA-lysine photoadduct (botton curve), and peroxynitrite-modified adduct(middle curve).

593 Peroxynitrite-Modified DNA-Lysine Photoadduct

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FIGURE 6 Column chromatographic profile of native DNA (�), DNA-lysine photoadduct (�), and peroxynitrite-modified adduct (�).

DNA-lysine photoadduct (curve 3). However, on peroxynitritemodification there was a change in the emission intensity, asseen in the figure (curve 2). A decrease in FI of 45.2% for theDNA-lysine photoadduct in comparison to the peroxynitrite-modified DNA-lysine adduct was observed from fluorescencespectroscopy measurements (Table 1). Loss of FI of 21.3% inthe peroxynitrite-modified adduct with respect to native DNAis indicative of the loss of structural integrity in DNA andgeneration of single-strand regions.

Sephadex G-100 Gel ChromatographyEqual volumes of native DNA, DNA-lysine adduct, and

peroxynitrite-modified adduct were analyzed on a SephadexG-100 column. The column chromatographic profile of nativeDNA showed peaks at fractions 2, 7, 14, and 16. TheDNA-lysine photoadduct (curve 1) also gave similar peakscorresponding to the same fraction numbers (Fig. 6). Onperoxynitrite treatment, two minor peaks were observed atfraction numbers 3 and 11 and two significant peaks wereobserved at fractions 15 and 19. The elution profile of theperoxynitrite-modified DNA-lysine adduct (curve 3) showselimination of the peak at fraction number 7. Eliminationof a peak (fraction 7) and observation of two different peaks(fractions 15 and 19) reiterate the modification of the DNA-lysine adduct by peroxynitrite. Elution of the peroxynitrite-modified DNA-lysine adduct corresponding to peaks 15 and19 and shifting of peaks may be attributed to the modificationof DNA backbone and nitrogenous bases by peroxynitrite.

Hence, the above study highlights the differences inthe DNA-lysine and peroxynitrite-modified DNA-lysine pho-toadduct, obtained through changes in absorption and flu-orescence spectra, melting temperature, and column chro-matography. These characteristic features of the native andmodified photoadducts will be helpful in their identificationand characterization and may have important implications intoxicology, carcinogenesis, and mutagenesis.

DISCUSSIONPeroxynitrite is a relatively long-lived oxidant that may serve

as an important cytotoxic agent. Its biological effects are dueto its reactivity toward a large number of molecules includinglipids, amino acids, and nucleic acids. It is involved in tissuedamage in a number of pathophysiological conditions such asneurodegenerative diseases, cardiovascular disorders, etc. (Szabo2003; Pacher et al. 2007; Szabo et al. 2007).

A change in the structure of DNA could either be due toradiation or due to interaction with different free radicals (Ahsanet al. 2003). Since there are many polybasic compounds in thevicinity of DNA, there exists a possibility of their interactionwith DNA on exposure to radiation or free radicals. Lysine-and arginine-rich histones in nucleosomes on modificationby environmental agents form histone-DNA adducts, makingit immunogenic. It appears that the pathogenic anti-DNAautoantibodies are generated through some modified epitopeson nucleic acids (Ahsan et al. 2002; Dixit et al. 2003; Habib etal. 2006). Prominent DNA modifications induced by exposureto peroxynitrite include the formation of 8-nitro-guanine and8-oxyguanine, as well as the induction of single-strand breaks(Ohshima and Bartsch 1994; Szabo and Ohshima 1997).Peroxynitrite reacts significantly only with guanine, which uponoxidation and nitration leads to mutagenicity and strand breaks,respectively. Peroxynitrite also damages DNA by covalent bondformation and removal of DNA bases (Yermilov et al. 1995).

In the present study, we have investigated the photochemicaladdition of lysine to native DNA in view of its potentialimportance in the photo-cross-linking of histones to DNA inchromatin. The C-2 carbon atom of thymine in DNA undergoesa covalent photoaddition reaction with the ε-amino group oflysine on UV irradiation to form a DNA-lysine photoconjugateor photoadduct (Islam and Ali 1995).

The UV spectroscopic analysis of the DNA-lysine pho-toadduct showed hyperchromism, indicating either the for-mation of single-stranded breaks in DNA or “breathing” ofa double-stranded polymer at the site of lysine conjugation.Peroxynitrite caused substantial damage to the DNA-lysine

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adduct as evident from the hyperchromicity of the spectralcurve, which could be attributed to the generation of strandbreaks. On peroxynitrite modification, the hypochromicityincreased, which may be due to the shielding effect of lysine,limiting the sites for peroxynitrite action. Hypochromicitymay also be attributed to the extensive cross-linking betweenperoxynitrite and the DNA-lysine adduct (Ahsan et al. 2002).

Difference spectral analysis of the DNA-lysine photoadductagainst native DNA showed hypochromic shift due to photo-modification. Elimination of the characteristic 260-nm peakand broadening of the spectral curve in the region of 250 to320 nm in the case of the peroxynitrite-modified DNA-lysineadduct are indicative of the loss in the double helical structure ofDNA. It may also be attributed to the substantial destabilizationof the double helix due to the single-stranded regions in theDNA-lysine adduct as a result of peroxynitrite modification.

The melting profile of the DNA-lysine photoadduct revealsthat the ultraviolet radiation induced covalent incorporation oflysine into the native DNA. The photo addition of lysine toDNA might have obliterated the favorable A = T and G = Cpairing interaction of double helical native DNA (Islam and Ali1998), thus decreasing the duplex melting temperature (Tm)by 5◦C relative to the fully paired parent native DNA. Inthe present study, on peroxynitrite treatment, the Tm of theDNA-lysine adduct increased by 15◦C with respect to the nativeDNA-lysine photoadduct. This may be due to shielding of theavailable sites for peroxynitrite action by lysine. Hence, moreenergy would be needed to break the covalent bonding betweenlysine and the DNA bases in order to denature the double helix.

Alteration of DNA resulting from photomodification orperoxynitrite could lead to the development of antibodiesor mutations to modified DNA. Therefore, the DNA-lysinephotoadduct and modified photoadduct could have importantimplications in various pathophysiological conditions such astoxicology, carcinogenesis, and autoimmune phenomena.

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Dixit, K., Ahsan, H., and Ali, A. 2003. Polydeoxyribonucleotide Cphotoconjugated with lysine or arginine present unique epitopesfor human anti-DNA autoantibodies. Hum. Immunol. 64(9):880–886.

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