immunological studies on peroxynitrite modified human dna

Life Sciences 77 (2005) 2626–2642

www.elsevier.com/locate/lifescie

Immunological studies on peroxynitrite modified human DNA

Kiran Dixit, MoinuddinT, Asif Ali1

Department of Biochemistry, Faculty of Medicine, A.M.U., Aligarh 202002, India

Received 30 August 2004; accepted 9 February 2005

Abstract

Peroxynitrite (ONOO�) is a strong and potent oxidizing and nitrating agent, formed by rapid reaction of two

highly reactive, nitric oxide and superoxide anion. The action of peroxynitrite generated by synergistic action of

diethylamine NONOate (a nitric oxide donor) and 1,4-hydroquinone (a superoxide donor), on human placental

DNA was monitored by ultraviolet and fluorescence spectroscopy, melting temperature studies, S1 nuclease

digestibility and alkaline agarose electrophoresis. The peroxynitrite modified human DNA (ONOO�-DNA) was

found to be highly immunogenic in rabbits inducing high titre immunogen specific antibodies. However, the

induced antibodies exhibited appreciable cross-reactivity with various polynucleotides and nucleic acids. The data

demonstrate that the antibodies, though cross-reactive, preferentially bind ONOO�-modified epitopes on DNA.

Visual detection of immune complex formation with native and ONOO�-DNA reiterated preferential binding with

modified human DNA. DNA modified by ONOO� presents unique epitopes which may be one of the factors for

the induction of autoantibodies in cancer patients.

D 2005 Published by Elsevier Inc.

Keywords: Peroxynitrite; DNA damage; Cancer; Autoantibodies

Introduction

Peroxynitrite (ONOO�) is formed in inflamed tissue by a diffusion limited reaction of nitric oxide

(NO) and superoxide anion (O2!�) (Huie and Padmaja, 1993). The formation of excess ONOO� has been

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T Correspond

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E-mail add1 Present Ad

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see front matter D 2005 Published by Elsevier Inc.

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ing author. Department of Biochemistry, J.N. Medical College, A.M.U., Aligarh-202002, India. Tel.: +91 571

ress: [email protected] (Moinuddin).

dress: Department of Clinical Biochemistry, College of Medicine and Medical Sciences, King Khalid University,

Abha, Kingdom of Saudi Arabia.

K. Dixit et al. / Life Sciences 77 (2005) 2626–2642 2627

implicated in many pathological conditions including reperfusion injury, chronic inflammation,

atherosclerosis, neurodegenerative diseases and an increased cancer incidence in tissues under chronic

inflammatory conditions (Rachmilewitz et al., 1993; Salgo et al., 1995; Ohshima, 2003). Peroxynitrite is

a powerful oxidizing and nitrating agent that exhibits high reactivity at physiological pH. (Richeson et

al., 1998). Cellular DNA is an important molecular target for ONOO� and the radical is capable of

reacting with both the deoxyribose and nucleobases. Prominent DNA modifications induced by exposure

to ONOO� include formation of 8-nitroguanine and 8-oxyguanine as well as the induction of DNA

single-strand breaks (Szabo and Ohshima, 1997). Peroxynitrite is mutagenic in the sup F gene, inducing

primarily G to T transversions and deletions clustered at the 5Vend of the gene (Tretyakova et al., 2000;

Juedes and Wogan, 1996). The mutagenicity of ONOO� is believed to result from chemical

modifications at guanine leading to miscoding. DNA single strands generated by ONOO� leads to

activation of the nuclear enzyme poly(ADP-ribose) synthetase (PARS), its activation and subsequent

cellular processes play an important role in ONOO�-induced alteration in a variety of pathophysio-

logical conditions (Szabo and Dawson, 1998).

Tissue inflammation and chronic infection lead to the overproduction of nitric oxide and superoxide

which rapidly combine to yield peroxynitrite, which is thought to be involved in both cell death and an

increased cancer risk observed for inflamed tissues (Ohshima and Bartsch, 1994). Increased levels of

circulating antibodies and autoantibodies have been reported in the serum of patients with malignancies

(Chagnand et al., 1992; Faiderbe et al., 1992; Becker et al., 1994). Elevated levels of antinuclear

antibodies have been reported in the serum of cancer patients of hepatocellular carcinoma. It has been well

established that native DNA in B-conformation is not immunogenic where as various modified forms of

DNA are immunogenic and induce cross-reactive antibodies (Jansson, 1985; Hasan and Ali, 1990; Ali et

al., 1985). Recent studies from our laboratory have reported ROS-modified DNA and NO-modified DNA

to be an effective immunogenic stimulus and the induced antibodies showed polyspecificity (Alam et al.,

1993; Ara and Ali, 1993; Ahmad et al., 1997; Ashok and Ali, 1999; Dixit and Ali, 2001).

In the present study, the immunogenicity of ONOO�-modified human DNA has been investigated.

The induced antibodies have been characterized for their fine antigenic binding specificity towards a

variety of ONOO�-modified and unmodified nucleic acid polymers and synthetic polynucleotides so as

to gain insight into the mechanism of autoantibody production in cancer. The study also involved the

comparative immunobinding of antibodies present in sera of various cancer patients, with native and

ONOO�-modified human DNA.

Methods

Human placental DNA, diethylamine NONOate (DEA-NO), 1,4-hydroquinone, nuclease S1,

methylated bovine serum albumin (MBSA), diethylenetriaminepentaacetic acid (DTPA), 1H-imidazol-

1-oxy,2-(14-carboxyphenyl)-4, 5-dihydro-4,4,5,5-tetra methyl-3-oxide (carboxy-PTIO), mannitol, su-

peroxide dismutase (SOD), desferrioxamine, anti-human and anti-rabbit IgG alkaline phosphatase

conjugates, p-nitrophenyl phosphate, ethidium bromide, coomassie brilliant blue G250 and R250,

Freund’s complete and incomplete adjuvants, synthetic polynucleotides, Tween-20, and DNA bases

were obtained from Sigma Chemical Company (U.S.A.). Protein-A Sepharose CL-4B was obtained from

Genei, India. Polystyrene flat bottom ELISA plates were from Nunc, Denmark. All other chemicals used

were of highest grade available.

K. Dixit et al. / Life Sciences 77 (2005) 2626–26422628

Plasmid DNA (pUC18) was isolated in a highly purified form through gentle lysis of bacterial cells

followed by centrifugation to remove the bulk of chromosomal DNA using plasmid isolation Mega Kit

from Qiagen, U.S.A.

DNA purification

Commercially available human placental DNA (Sigma, U.S.A.) was purified free of proteins, RNA

and single stranded regions as described earlier (Ali et al., 1985). Purity of DNAwas confirmed by A260/

A280 ratio.

Modification of human placental DNA by peroxynitrite

The experiment was carried out by incubating human DNA (50 Ag) in Na-Pi buffer, pH 7.4 containing

0.1 mM DTPA, 0.1 mM DEA-NO and 0.1 mM 1,4-hydroquinone at 37 8C for 3 h. DEA-NO and 1,4-

hydroquinone were removed by extensive dialysis against PBS, pH 7.4. All the other nucleic acid

samples and DNA bases were similarly modified.

The UV absorption characteristics of native and ONOO�-DNA were recorded on Shimadzu UV-240

spectrophotometer. Fluorescence emission spectroscopical analysis on native and ONOO�-DNAsamples (5 Ag/ml each) was undertaken using the intercalating dye, ethidium bromide, as the external

chromophore (2.5 Ag/ml). The readings for EtBr-DNA and EtBr-ONOO�-DNA were recorded on a

Shimadzu RF-5301 Spectrofluorometer at an excitation wavelength of 325 nm.

Agarose gel electrophoresis

Native and ONOO�-DNA were mixed with a 1/10 volume of sample buffer (0.125% bromophenol

blue, 30% Ficoll-400, 5 mM EDTA in 10� electrophoresis buffer). The samples were electrophoresed

for 2 h at 30 mA on 1% agarose gels. The gels were stained with ethidium bromide (0.5 Ag/ml), viewed

by illumination under UV light and photographed.

Nuclease S1 digestion

ONOO�-DNA was characterized by nuclease S1 treatment followed by agarose gel electrophoresis

(Matsuo and Ross, 1987). The digesting capability of nuclease S1 under our experimental conditions

was checked using heat denatured calf thymus DNA. It was almost completely digested, whereas no

digestion was observed in the case of purified native DNA. One microgram each of native and ONOO�-DNAwas incubated with nuclease S1 (20 units/Ag DNA in acetate buffer, pH 5.0, at 37 8C for 30 min).

The reaction was stopped by adding a 1/10 volume of 0.2 M EDTA, pH 8.0 and the sample

electrophoresed on 1% agarose gel for 2 h at 30 mA. The nucleic acid bands were visualized under UV

light after staining with ethidium bromide.

Reaction of plasmid DNA with peroxynitrite

The conversion of covalently closed circular-double stranded supercoiled DNA (form I) to a

relaxed open circle (form II) was used to investigate DNA single strand breaks induced by the


combined action of DEA-NO and 1,4-hydroquinone. The experiment was carried out by

incubating plasmid PUC 18 DNA (100 Ag) in 100 mM PBS, pH 7.4 containing 0.1 mM

DTPA and either 0.1 mM DEA-NO alone or 0.1 mM 1,4-hydroquinone alone, or these two

compounds in combination at 37 8C for 1 h (final volume 10 ml). The reaction was stopped

by the addition of 2 Al sample buffer and the samples electrophoresed on 1% agarose gel for

2 h at 30 mA. The nucleic acid bands were visualized under UV light after ethidium bromide

staining.

Thermal denaturation studies

Thermal denaturation of native and ONOO�-DNA under identical conditions was evaluated by a

temperature scan from 30 8C to 95 8C at an increment of 1 8C/min on a Shimadzu UV-240

spectrophotometer equipped with a temperature programmer and controller assembly. The change in

absorbance at 260 nm was recorded and melting temperature (Tm) of the samples calculated (Ara and

Ali, 1992).

Quenching studies

The effects of various quenchers like superoxide dismutase (SOD) nitric oxide-trapping agent

(carboxy-PTIO at 10 mM), hydroxyl radical scavenger (d-mannitol at 10 mM), antioxidants (ascorbic

acid at 10 mM and uric acid at 0.2 mM) and a metal ion chelator (desferrioxamine at 10 mM) on DNA

modification were studied by incubating DNA (50 Ag) in 100 mM PBS pH 7.4 containing 0.1 mM

DTPA with 0.1 mM DEA-NO plus 0.1 mM 1,4-hydroquinone at 37 8C for 3 h. Percent quenching was

calculated from absorbance at A260 nm.

Immunization schedule

The immunization of random bred, female, New Zealand white rabbits was performed as described

previously (Hasan et al., 1991). Briefly, rabbits (n=4; two each for native and ONOO�-DNAantigens) were immunized intramuscularly at multiple sites with 50 Ag of antigen complexed with

methylated BSA in the ratio of 1:1 (w/w) and emulsified with an equal volume of Freund’s complete

adjuvant. The animals were boosted in Freund’s incomplete adjuvant at weekly intervals for 7 weeks

with the same amount of antigen. Test bleeds were performed 7 days post boost, which gave,

appropriate titre of the antibody. The animals were bled and the serum separated from the blood

(preimmune and immune) was heated at 56 8C for 30 min to inactivate complement proteins and

stored at �20 8C.

Purification of IgG

Immunoglobulin G was isolated from immune sera on Protein A-Sepharose CL-4B column. Sera

diluted (1:1) with PBS (pH 7.4) were passed through the column. After washing the unbound proteins

with PBS (pH 7.4) the bound IgG was eluted with 0.58% acetic acid and 0.85% sodium chloride (Alam

et al., 1993). To prevent the effect of acidic pH of eluting buffer on IgG, the eluate was collected in 1.0

ml of 1 M Tris–HCl buffer (pH 8.5).


Enzyme linked immunosorbent assay (ELISA)

Antibodies against native and ONOO�-DNA were detected and quantitated by ELISA performed

on flat bottom 96-wells, polystyrene immunoplates (Nunc, maxisorp) as described previously (Ali

and Alam, 2002). Briefly the plates were coated with 100 Al of antigen (2.5 Ag/ml) for 2 h at room

temperature and overnight at 4 8C. After washing three times with TBS-T (20 mM Tris, 2.68 mM

KCl, 150 mM NaCl, pH 7.4 containing 0.05% Tween-20), unoccupied sites were blocked with 150

Al of 1.5% BSA in TBS (10 mM Tris, 150 mM NaCl, pH 7.4) for 4–6 h at room temperature. The

test serum serially diluted in TBS-T (100 Al/well) was absorbed for 2 h at room temperature and

overnight at 4 8C. Bound antibodies were assayed with anti-rabbit IgG alkaline phosphatase

conjugate using p-nitrophenyl phosphate as conjugate. The absorbance of each well was monitored

at 410 nm on an automatic microplate reader. Proper controls were included in each set of

experiments.

Competition ELISA

Antibody specificity was ascertained by competitive binding assay (Dixit and Ali, 2001). Varying

concentrations of inhibitors (0–20 Ag/ml) were allowed to interact with a constant quantity of antibody

(50 Ag/ml IgG or 1/10 diluted serum) for 2 h at room temperature and overnight at 4 8C. The mixture

was added to antigen coated plates and the bound antibody was diluted as described in the direct binding

ELISA. Inhibition was expressed as the amount of inhibitor required for 50% elimination in the antibody

binding to the solid phase antigen.

Band shift assay

For the visual detection of antigen–antibody interaction and the formation of immune complexes,

band shift assay was performed on 1% agarose (Dixit and Ali, 2004). Varying amounts (0–60 Ag) of IgGwere incubated with a fixed amount (0.5 Ag) of antigen for 2 h at 37 8C and overnight at 4 8C. Theresulting immune complex was electrophoresed on 1% agarose for 2 h at 30 mA in 40 mM Tris-acetate-9

mM EDTA buffer (TAE), pH 8.0. The gel was stained with ethidium bromide and photographed under

UV illumination.

Results

Human placental DNA purified free of proteins, RNA and single stranded regions (Ali et al., 1985)

was exposed to peroxynitrite generated by DEA-NO plus 1,4-hydroquinone. In order to optimize the

dose of DEA-NO and 1,4-hydroquinone for peroxynitrite generation, the reaction was carried out with

varying concentrations (0.1 mM–0.5 mM) of both the chemicals. The results were analyzed by UV-

absorption spectra (data not shown). DNA subjected to 0.5 mM of DEA-NO and 0.5 mM of 1,4-

hydroquinone showed moderate modification, whereas at lower concentrations of 0.4 to 0.1 mM the

modification was found increased. DNA subjected to 0.1 mM DEA-NO and 0.1 mM 1,4 hydroquinone

showed maximum modification. However, when the concentrations were reduced below 0.1 mM, the

results were not consistent vis-a-vis DNA damage. All the experiments were run in duplicates. Based on


the above results we have chosen the DNA modified by 0.1 mM DEA-NO plus 0.1 mM 1,4-

hydroquinone for further studies.

Characterization of ONOO�-DNA

The ultraviolet absorption characteristics of ONOO�-DNA, when compared with native human

DNA, exhibited around 49% increase in absorbance over the entire UV range and most noticeably at

260 nm (Fig. 1). In UV-difference spectral analysis the maximal difference between the modified

and unmodified DNA species occurred at 280 nm (Fig. 1, inset). The corresponding controls, i.e.

DNA incubated individually with DEA-NO (0.1 mM) alone and DNA treated with 1,4-hydroquinone

(0.1 mM) alone showed non-significant changes in their UV absorption spectroscopical

characteristics.

Fluorescence spectra of the DNA samples (EtBr-DNA and EtBr-ONOO�-DNA) exhibited a decrease

in the fluorescence intensity in case of the modified DNA signifying perturbations in the double helix as

a result of ONOO� modification (Fig. 2).

Fig. 1. Ultraviolet absorption spectra of native human DNA (a) and human DNA modified with 0.1 mM DEA-NO (b), 0.1 mM

1,4-hydroquinone (c) and 0.1 mM DEA-NO plus 1,4-hydroquinone (d). Inset: Ultraviolet difference spectra of native and

ONOO�-human DNA. Native DNAwith reference to PBS, pH 7.4 (—),ONOO�-DNAwith reference to native DNA (– – – –).

Fig. 2. Fluorescence emission spectra of ethidium bromide, 2.5 Ag/ml (–d d –), EtBr-DNA, 5.0 Ag/ml (—) and EtBr-ONOO�-

DNA, 5.0 Ag/ml (– – –). Excitation wavelength was 325 nm.

Fig. 3. Thermal denaturation profile of native human DNA (o) and ONOO�-human DNA (.).


Table 1

Ultraviolet and thermal denaturation characteristics of native and ONOO�-modified human DNA

Parameter Native DNA Modified DNA

Absorbance ratio (A260/A280) 1.76 1.4

Hyperchromicity (%) at 95 8C 36.5 24

Melting temperature (Tm), 8C 86.0 71

Onset of duplex melting, 8C 78 31.5


The melting profile of native and ONOO�-DNA (Fig. 3) was analyzed between the temperature range

30 8C and 95 8C. Increase in absorbance at 260 nm was taken as a measure of helix denaturation. The

process was characterized by determining the percent DNA in denatured state as a function of increasing

temperature and then computing the melting temperature (Tm). The Tm for ONOO�-DNAwas found to

be 71 8C and that of its unmodified form 86 8C indicating a net decrease of 15 8C in the Tm for modified

DNA. Moreover, the melting curve for modified DNA indicated a progressive and heterogeneous local

melting with the increase in temperature as compared to more homogeneous global melting in the

control. Physicochemical characteristics of native and ONOO�-DNA are summarized in Table 1.

To further evaluate the effect of peroxynitrite on human DNA, agarose gel electrophoresis was

performed. The electrophoretic pattern of DNA modified individually by DEA-NO and 1,4-

hydroquinone as well as by their combined action is shown in Fig. 4. DNA modified by ONOO�

migrated as a discrete band minus the characteristic stretch exhibited by the native DNA. Whereas DNA

treated individually to DEA-NO and 1,4-hydroquinone showed similar migration as native DNA.

Fig. 4. Agarose gel-electrophoresis of native and ONOO�-human DNA. Lanes: (1) Native DNA; (2) DNA modified with 0.1

mM DEA-NO; (3) DNA modified with 0.1 mM 1,4-hydroquinine; (4) DNA modified with 0.1 mM DEA-NO plus 1,4-

hydroquinone. DNA concentration was 0.5 Ag/ml in all the lanes.

Fig. 5. Nuclease S1 sensitivity of native and ONOO�-human DNA. Lanes: (1) Native DNA; (2) nuclease S1 treated native

DNA; (3) ONOO�-DNA; (4) nuclease S1 treated ONOO�-DNA. DNA concentration was 0.5 Ag/ml in all the lanes.


The generation of single strand breaks in ONOO�-DNA was demonstrated by nuclease S1

digestibility followed by agarose gel electrophoresis. The controls were native and modified DNA

without digestion with nuclease S1. S1 treated ONOO�-DNA showed no fluorescence, probably

reflecting extensive degradation of the modified DNA. However, such a loss of fluorescence signal was

not found in the case of S1 treated native DNA (Fig. 5). Agarose gel electrophoresis of plasmid pUC 18

reiterate the generation of single-strand breaks induced by ONOO� (Fig. 6). Incubation of the plasmid

DNAwith either 0.1 mM DEA-NO or 0.1 mM 1,4-hydroquinone resulted in non-significant conversion

Fig. 6. Agarose gel-electrophoresis of native and ONOO� treated pUC 18 DNA. Lanes: (1) Native DNA; (2) DNA modified

with 0.1 mM DEA-NO; (3) DNA modified with 0.1 mM 1,4-hydroquinine; (4) DNA modified with 0.1 mM DEA-NO plus 1,4-

hydroquinone. DNA concentration was 0.5 Ag/ml in all the lanes.


of covalently closed circular-double stranded supercoiled DNA (form I) to a linear form (form II),

compared with that of the native plasmid. However, when the plasmid was incubated with DEA-NO plus

1,4-hydroquinone, form I completely disappeared where as there was an almost quantitative conversion

to linear form (form II) suggesting the conversion of form I to form II as a result of generation of strand

breaks in the DNA upon ONOO� modification.

Quenching studies

Fig. 7 shows the modification of human DNA in the presence of different quencher to shut off the

effect of one species and let the other modify the macromolecule. As evident, the nitric oxide trapping

agent (carboxy-PTIO) strongly (N90%) inhibited the modification by DEA-NO plus 1,4-hydroquinone,

whereas antioxidant enzyme (SOD) which is scavenger of O2!� was less effective (~50% inhibition).

Effects of various scavengers of hydroxyl radical (like d-mannitol), antioxidants (ascorbic acid, uric

acid) and metal ion chelator (desferrioxamine) on DNA modification were also studied. High

concentrations of ascorbic acid and desferrioxamine (100 fold molar excess) inhibited the DNA

modification by 48% and 54%, respectively, whereas 0.2 mM uric acid inhibited it by 90%.

Fig. 7. Effects of various scavengers of free radicals (mannitol, SOD); antioxidants (ascorbic acid, uric acid); NO trapping

agent (carboxy-PTIO) and metal ion-chelator (desferrioxamine) on DNA modification induce by 0.1 mM DEA-NO plus 0.1

mM 1,4-hydroquinone.


Antigenicity of ONOO�-DNA

Peroxynitrite modified-DNA was found to be highly immunogenic, inducing high titre (N1:12800)

antibodies in rabbits (Fig. 8). The direct binding ELISA of antiserum from rabbits immunized with

native human DNA showed negligible binding to its respective immunogen. Immunoglobulin G was

isolated from preimmune and immune rabbit serum on Protein-A Sepharose CL-4B column. The

purified IgG was found to elute as a single symmetrical peak. Direct binding ELISA of the anti-ONOO�-DNA IgG showed a strong reactivity with the immunogen while the preimmune IgG showed negligible

binding (data not shown).

Competition ELISA was performed using immunogen, nucleic acids, synthetic polynucleotides and

bases as inhibitors to ascertain the antigenic binding specificity of induced antibodies. A maximum of

88.2% inhibition of antibody binding to solid phase bound antigen was observed with the immunogen as

inhibitor. The induced antibodies were highly specific for ONOO�-DNA as only 1.5 Ag/ml inhibited

50% antibody activity. Various hetero/homo polymers, modified/unmodified DNA bases, chromatin, ss-

DNA etc were used as inhibitors for the binding of anti-ONOO�-DNA IgG. The results have been

summarized in Table 2.

Band shift assay

The formation of immune complex by ONOO�-DNA and IgG was visualized by band shift assay. An

increase in the amount of IgG caused a proportional increase in the formation of high molecular weight

immune complexes resulting in retarded mobility and increased fluorescence intensity of the immune

Fig. 8. Direct binding ELISA of anti-ONOO�-human DNA antisera (o) and preimmune sera (.). The microtitre plates were

coated with ONOO�-human DNA (2.5 Ag/ml).

Table 2

Antigenic specificity of anti-ONOO�-human DNA IgG

Inhibitor Maximum % inhibition

at 20 Ag/ml

Concentration for 50%

inhibition, Ag/ml

Percent relative

affinity

ONOO�-DNA 88.2 1.5

Native human DNA 45 –a

Native calf thymus DNA 33.4 –

ONOO�-calf thymus DNA 52.5 16 9.4

Native chromatin 39.3 –

ONOO�-chromatin 49

ss-human DNA 48.2 –

ROS-human DNA 36 –

O2!� human DNA 38.4 –

Guanine 43 –

O2!� guanine 44.3 –

NO-guanine 49.6 –

ONOO�-guanine 59 9.4 16

Cytosine 26

ONOO�-cytosine 39

Adenine 19

ONOO�-adenine 37

Poly(dC) 28.1

Poly(dT) 21.3

Poly(dA) 17

The microtitre plates were coated with ONOO�-human DNA (2.5 Ag/ml).a 50% inhibition was not achieved.


complex (Fig. 9a). The retardation in mobility of the immune complex was appreciably low in the case

of native DNA as compared to that observed with ONOO�-DNA (Fig. 9b).

Discussion

Under normal physiologic conditions endothelial nitric oxide, a free radical, provides local anti-

thrombotic actions and regulation of vascular tone. However, the actions of NO can be severely altered

in oxidative stress (Vallance and Moncada, 1994). Of particular importance is the interaction of NO with

superoxide anion radical (Pryor and Squadrito, 1995) forming a highly potent oxidant, peroxynitrite

(Beckman et al., 1990). It is a relatively long-lived oxidant (Murphy, 1999) that may serve as an

important cytotoxic agent (Guzik et al., 2002). Peroxynitrite is a fascinating molecule and presents

multiple facets that account for its complex chemistry. Its biological effects are due to its reactivity

towards a large number of molecules including lipids, amino acids, nucleic acids and antioxidants

(Ducroiq et al., 1999). The oxidant is a mediator of toxicity and is involved in tissue damage in a number

of pathophysiological conditions such as inflammation, neurodegenerative diseases and cardiovascular

disorders (Dedon and Tannenbaum, 2004).

DEA-NO (a nitric oxide donor) generates nitric oxide in presence of DTPA (Yoshie and Ohshima,

1997).1,4-hydroquinone, a polyhydroxy aromatic compound autooxidize easily to form semiquinone

radicals that react with dioxygen to generate superoxide (Zang et al., 1995). Nitric oxide reacts rapidly

Fig. 9. Band shift assay of anti-ONOO�-human DNA IgG binding to (a) ONOO�-human DNA; (b) native human DNA. DNA

(0.5 Ag ) was incubated with buffer (lane 1) and 30, 40, 50, 60 Ag of IgG through lanes 2–5, respectively, for 2 h at 37 8C and

overnight at 4 8C. Electrophoresis was performed on 1% agarose gel for 2 h at 30 mA.


with superoxide anion to form peroxynitrite (Beckman et al., 1990). ONOO� caused substantial damage

to human placental DNA as evident from the hyperchromicity of the spectral curve of ONOO�-DNA in

comparison to the native DNA. This could be attributed to the generation of strand breaks. No change

was observed in DNA on incubation with either DEA-NO or 1,4 hydroquinone alone. Elimination of

characteristic 260 nm peak and broadening of the spectral curve in the region of (250–320 nm) in case of

the ONOO�-DNA in UV-difference spectroscopy is indicative of the loss in the double helical structure

of human DNA and substantial destabilization of the double helix due to the single stranded regions in

the DNA as a result of ONOO�-induced modification. A decrease in the fluorescence intensity of the

EtBr-ONOO�-DNA compared to EtBr-DNA in the fluorescence emission spectra also confirms the

destruction of structural integrity of DNA and generation of single strand breaks (which decreases the


efficiency of ethidium bromide intercalation). The alterations in the human DNA are also evident from

the decrease in melting temperature primarily due to generation of single strand breaks and due to altered

hydrogen bonding between base pairs.

Single strand breaks generated by ONOO� can arise from two processes—i.e. either from direct sugar

damage, which involves abstraction of hydrogen leading to formation of sugar radical (Szabo and

Ohshima, 1997) or indirectly via base damage which rapidly depurinates to generate abasic sites finally

culminating in single strand breaks. Thus, it can be concluded that the above modifications (sugar damage

and base modifications) are responsible for generation of single strand breaks in human DNA.

Appreciable evidence for the generation of strand breaks in the human DNA as a consequence of

ONOO� modification was gathered by nuclease S1 digestibility and plasmid nicking approach. DNA

strand breaks are induced synergistically when plasmid DNA is incubated in presence of both DEA-NO

and 1,4 hydroquinone. DEA-NO or 1,4 hydroquinone alone induced no appreciable change. Our results

are consistent with the observations that peroxynitrite, rather than NO is responsible for DNA strand

breakage (Yoshie and Ohshima, 1997).

Studies by various radical scavenging species indicate that single strand breaks and modification in

DNA induced by NO plus 1,4 hydroquinone can be inhibited by NO trapping agent (carboxy-PTIO) and

O2!� scavenger (SOD), suggesting that concurrent generation of NO and O2

!� is necessary for DNA

modification. Native DNA is a poor immunogen (Isenberg et al., 1994); however, conformational

isomers of the B-form have proved to be highly immunogenic (Jansson, 1985; Hasan and Ali, 1990; Ali

et al., 1985). The ONOO�-DNA was a potent immunizing stimulus inducing high titre antibodies in

rabbits which were highly immunogen specific but showing cross-reactivity with the native DNA.

Modification of DNA by ONOO� might have generated potential epitopes against which antibodies are

raised. The cross-reactivity with native human DNA, native calf thymus DNA may be due to recognition

of the common antigenic determinant, possibly the sugar phosphate backbone. Gel retardation data

further reiterated the preferential recognition of ONOO�-DNA over native DNA. Native calf thymus

DNA and native chromatin on peroxynitrite modification were more inhibitory than their unmodified

counterparts. The immune IgG was relatively more specific to peroxynitrite modified guanine, cytosine

and adenine. Peroxynitrite reacts significantly only with guanine which upon oxidation and nitration

leads to mutagenicity and strand breaks, respectively (Vermilov et al., 1995; Kennedy et al., 1997). In

addition, the induced antibodies showed recognition of various synthetic polynucleotides. Analysis of

the data indicates that anti-ONOO�-DNA IgG is immunogen specific and the varying degree of cross-

reactivity is due to sharing of common antigenic determinants which might be the modified bases or

peroxynitrite-induced lesions.

It has been proposed that tissue inflammation, immunostimulation (due to chronic infection) and

oxidative stress lead to the overproduction of nitric oxide and superoxide (Tretyakova et al., 2000).

These two species rapidly combine to yield peroxynitrite which in turn reacts with proteins (Beckman,

1996) resulting in their denaturation/modification or it could react with exposed nucleic acids leading to

single strand break generation which are potent signals for apoptosis (Levine, 1997). The subsequent

release of the altered DNA during apoptosis may enable it to act as an antigen inducing antibodies cross-

reactive with native DNA (Ahsan et al., 2003). The ONOO�-DNA thus acts as an autoantigen against

which anti-DNA antibodies could be generated.

Carcinogenesis is induced by altered DNA or tissue damage, mutations and chromosomal aberrations

(Gordon and Weitzman, 1990; Ames et al., 1993) Peroxynitrite levels are elevated in inflammation and

infection, and play an important role in carcinogenesis (Vamvakas and Schmidt, 1997; Ohshima, 2003).


Peroxynitrite induced DNA damage leading to mutations has been strongly implicated in carcinogenesis

(Tretyakova et al., 2000; Dedon and Tannenbaum, 2004). If these mutations relate to critical genes,

initiation, and/or progression of cancer can occur. Our preliminary studies using antibodies from cancer

sera showed higher inhibition by ONOO�-DNA in comparison to native DNA when the immobilized

antigen was ONOO�-DNA. Studies on the binding specificity of cancer autoantibodies clearly

demonstrated that ONOO�-modified DNA is a better inhibitor of naturally occurring antibodies in most

of the cancer sera studied. Peroxynitrite appears to enhance the antigenicity of native DNA, thus

suggesting possible roles of ONOO�-induced neo-epitopes in the damaged DNA in production of

autoantibodies in cancer patients.

Acknowledgements

The authors are grateful to the University Grants Commission, Government of India for providing

financial assistance for this study vide grant no. F.3-16/2001(Sr-II). The authors are also grateful to Prof.

Rashid Ali for providing research facilities and for the critical analysis of the manuscript.

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