Immunological studies on peroxynitrite modified human DNA

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.