Contents lists available at ScienceDirect Archives of Biochemistry and Biophysics journal homepage: www.elsevier.com/locate/yabbi Genotoxic effect and antigen binding characteristics of SLE auto-antibodies to peroxynitrite-modified human DNA Md Asad Khan a,* , Khursheed Alam c , Syed Hassan Mehdi b , M. Moshahid A. Rizvi b a Department of Biochemistry, Faculty of Dentistry, Jamia Millia Islamia, New Delhi, India b Department of Biosciences, Jamia Millia Islamia, New Delhi, India c Department of Biochemistry, Jawaharlal Nehru Medical College, A.M.U., Aligarh, India ARTICLE INFO Keywords: Genotoxicity Human placental DNA Peroxynitrite Autoimmunity Comet assay ABSTRACT Systemic lupus erythematosus (SLE) is an inflammatory autoimmune disease characterized by auto-antibodies against native deoxyribonucleic acid after modification and is one of the reasons for the development of SLE. Here, we have evaluated the structural perturbations in human placental DNA by peroxynitrite using spectro- scopy, thermal denaturation and high-performance liquid chromatography (HPLC). Peroxynitrite is a powerful potent bi-functional oxidative/nitrative agent that is produced both endogenously and exogenously. In experi- mental animals, the peroxynitrite-modified DNA was found to be highly immunogenic. The induced antibodies showed cross-reactions with different types of DNA and nitrogen bases that were modified with peroxynitrite by inhibition ELISA. The antibody activity was inhibited by approximately 89% with its immunogen as the in- hibitor. The antigen-antibodies interaction between induced antibodies with peroxynitrite-modified DNA showed retarded mobility as compared to the native form. Furthermore, significantly increased binding was also observed in SLE autoantibodies with peroxynitrite-modified DNA than native form. Moreover, DNA isolated from lymphocyte of SLE patients revealed significant recognition of anti-peroxynitrite-modified DNA im- munoglobulin G (IgG). Our data indicates that DNA modified with peroxynitrite presents unique antigenic de- terminants that may induce autoantibody response in SLE. 1. Introduction Systemic lupus erythematosus (SLE) is a multisystem, inflammatory autoimmune disorder of connective tissues with an unknown etiology [1]. It involves both humoral and cell mediated immune response of the innate and acquired immune system and is demonstrated by the pre- sence of auto-antibodies [2] directed against components of cell nu- cleus, cytoplasm, cell membranes and others [3] in the sera of patients. Autoantibodies against double-stranded DNA (dsDNA) [4], modified self-antigens [5,6], self-proteins that cross-react with native DNA [7,8] and anti-nucleosome antibodies are among the pathogenic antibodies found in SLE patients. It is known that native dsDNA is non-im- munogenic [8,9] and several studies have shown that reactive oxygen species (ROS) and reactive nitrogen species (RNS)-modified DNA and polynucleotides are immunogenic in experimental animals and reveal SLE like autoantibody characteristics [8,10]. Generation of nitric oxide in vivo via nitric oxide synthase mediated arginine oxidation is one of the common cellular pathways [11,12] and nitric oxide (NO) serves as secondary messenger and mediator for in- flammatory response [13]. Frequently, NO combine with superoxide (O 2 _ ) to form peroxynitrite (ONOO ─ ) leading to DNA damage [14]. Several studies have shown that peroxynitrite reacts with DNA causing nitration, mutation, strand break and structural changes leading to the induction and progression of autoimmune diseases [15,16]. Peroxyni- trite induces nitrosative stress, modifies DNA and induces mutations in human lymphoblastoid cell lines [17] suggesting that cytotoxicity and mutagenicity associated with inflammation process may lead to auto- immune diseases [17–20]. In the present study, human placental DNA was damaged by per- oxynitrite formed by the experimentally combination of nitric oxide and superoxide anions. Peroxynitrite mediated alterations in DNA were investigated by physicochemical techniques, spectroscopy, melting profile and HPLC. The antibodies induced against peroxynitrite-mod- ified DNA were used as a probe for the diagnosis of neo-epitopes on damaged DNA and circulating antibodies in SLE patients. The induced antibodies were also evaluated for antigenic epitope between and DNA isolated from SLE patients and induced antibodies against peroxyni- trite-modified DNA in experimental animals. http://dx.doi.org/10.1016/j.abb.2017.10.008 Received 3 November 2016; Received in revised form 12 October 2017; Accepted 13 October 2017 * Corresponding author. E-mail address: asad1amu@gmail.com (M.A. Khan). Archives of Biochemistry and Biophysics 635 (2017) 8–16 Available online 16 October 2017 0003-9861/ © 2017 Elsevier Inc. All rights reserved. MARK