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2-methoxyestradiol (2-ME) is a physiological anticancer compound, metabolite of 17β-estradiol. Previously, our group evidenced that from mechanistic point of view one of anticancer mechanisms of action of 2-ME is specific induction and nuclear hijacking of neuronal nitric oxide synthase (nNOS), resulting in local generation of nitro-oxidative stress and finally, cancer cell death. The current study aims to establish the substantial mechanism of generation of reactive nitrogen species by 2-ME. We further achieved to identify the specific reactive nitrogen species involved in DNA-damaging mechanism of 2-ME. The study was performed using metastatic osteosarcoma 143B cells. We detected the release of biologically active (free) nitric oxide ( • NO) with concurrent measurements of peroxynitrite (ONOO - ) in real time in a single cell of 143B cell line by using • NO/ONOO - sensitive microsensors after stimulation with calcium ionophore. Detection of nitrogen dioxide ( • NO 2 ) and determination of chemical rate constants were carried out by a stopped-flow technique. The affinity of reactive nitrogen species toward the guanine base of DNA was evaluated by density functional theory calculations. Expression and localization of nuclear factor NF-kB was determined using imaging cytometry, while cell viability assay was evaluated by MTT assay. Herein, we presented that 2-ME triggers pro-apoptotic signalling cascade by increasing cellular reactive nitrogen species overproduction - a result of enzymatic uncoupling of increased nNOS protein levels. In particular, we proved that ONOO - and • NO 2 directly formed from peroxynitrous acid (ONOOH) and/or by auto-oxidation of • NO, are inducers of DNA damage in anticancer mechanism of 2-ME. Specifically, the affinity of reactive nitrogen species toward the guanine base of DNA, evaluated by density functional theory calculations, decreased in the order: ONOOH > ONOO -  >  • NO 2  >  • NO. Therefore, we propose to consider the specific inducers of nNOS as an effective tool in the field of chemotherapy., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)