Nitroxide TEMPO: A genotoxic and oxidative stress inducer in cultured cells

Abstract

Abstract2,2,6,6-Tetramethylpiperidine-1-oxyl (TEMPO) is a low molecular weight nitroxide and stable free radical. In this study, we investigated the cytotoxicity and genotoxicity of TEMPO in mammalian cells using the mouse lymphoma assay (MLA) and in vitro micronucleus assay. In the absence of metabolic activation (S9), 3 mM TEMPO produced significant cytotoxicity and marginal mutagenicity in the MLA; in the presence of S9, treatment of mouse lymphoma cells with 1-2 mM TEMPO resulted in dose-dependent decreases of the relative total growth and increases in mutant frequency. Treatment of TK6 human lymphoblastoid cells with 0.9-2.3 mM TEMPO increased the frequency of both micronuclei (a marker for clastogenicity) and hypodiploid nuclei (a marker of aneugenicity) in a dose-dependent manner; greater responses were produced in the presence of S9. Within the dose range tested, TEMPO induced reactive oxygen species and decreased glutathione levels in mouse lymphoma cells. In addition, the majority of TEMPO-induced mutants had loss of heterozygosity at the Tk locus, with allele loss of ≤34 Mbp.These results indicate that TEMPO is mutagenic in the MLA and induces micronuclei and hypodiploid nuclei in TK6 cells. Oxidative stress may account for part of the genotoxicity induced by TEMPO in both cell lines.

ROS generation and JNK activation contribute to 4-methoxy-TEMPO-induced cytotoxicity, autophagy, and DNA damage in HepG2 cells

“…Nitroxides have a single unpaired electron delocalized between a nitrogen and an oxygen, and their important biological activities have been recognized for decades (Gallez et al 2004; Guo et al 2013). Piperidine nitroxides, a class of stable organic free radicals of low molecular weight, are lipophilic and permeable through cell membranes (Soule et al 2007a; Wilcox and Pearlman 2008).…”

“…It has been reported that TEMPO (2,2,6,6-tetramethylpiperidine-1-oxyl), one of the frequently used piperidine nitroxide catalysts, resulted in intercellular reactive oxygen species (ROS) generation and DNA damage in mammalian cells (Guo et al 2015). Furthermore, TEMPO and several of its derivatives induced genotoxicity in the Ames test, the mouse lymphoma assay, and the in vitro micronucleus assay (Gallez et al 1992; Guo et al 2013; Sies and Mehlhorn 1986). One TEMPO derivative, 4-methoxy-TEMPO, is commonly employed for oxidation of alcohols, as well as in synthesis of 2-substituted benzoxazoles that are widely used as antibacterial, antifungal, antimicrobial, anti-proliferative, and antiviral agents (Chen et al 2008; Demmer and Bunch 2015).…”

“…Previously, we demonstrated that TEMPO triggered oxidative stress (Guo et al 2013), induced apoptosis and DNA damage, and activated the JNK pathway (Guo et al 2015). In the present study, we investigated the toxicity of 4-methoxy-TEMPO and the possible underlying mechanisms, focusing on the roles of ROS and DNA damage.…”

Development of a Manufacturing Process for an HCV Protease Inhibitor Candidate Molecule

“…The crude product contained approximately 5000 ppm TEMPO, which was unacceptably high since in vitro 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 33 results suggest that TEMPO is genotoxic in mammalian cells. 15 A study was carried out where a number of solvent systems were screened to determine whether solid 1 could be obtained. A solvent mixture of MTBE/heptane precipitated 1 out of solution containing 300 ppm of residual TEMPO.…”

Kinetics and thermodynamics of reversible disproportionation–comproportionation in redox triad oxoammonium cations – nitroxyl radicals – hydroxylamines

“…[6] Just like their inorganic congener -nitric oxide [7] -nitroxyl radicals at submillimolar concentrations exhibit antioxidant and cytoprotective properties, [8,9] but at concentrations ≥10 À3 M, they increase oxidative stress and exert a cytotoxic effect on tumor cells. [10][11][12] Analogously to other redox active agents, [13] nitroxyl radicals modulate the activity of clinically used anticancer drugs and may improve their chemotherapeutic properties. [14][15][16] For effective use of nitroxyl radicals in such applications, it is important to know the redox and acid-base characteristics of a wide variety of structures 1-2-3.…”