Comparative Genotoxicity of TEMPO and 3 of Its Derivatives in Mouse Lymphoma Cells

Abstract

TEMPO (2, 2, 6, 6-tetramethylphiperidine-1-oxyl) and its derivatives are stable free radical nitroxides widely used in the field of chemistry, biology, and pharmacology. TEMPO was previously found to be mutagenic and to induce micronuclei in mammalian cells. In this study, we investigated and quantified the genotoxicity of 4 structurally similar nitroxides, TEMPO and 3 of its derivatives (4-hydroxy-TEMPO, 4-oxo-TEMPO, and 4-methoxy-TEMPO), using the mouse lymphoma assay (MLA) and Comet assay in L5178Y Tk+/- cells. The results showed that all tested nitroxides were cytotoxic and mutagenic in the MLA, both in the presence and absence of S9, with metabolic activation significantly enhancing the cytotoxicity and/or mutagenicity. In addition, the 4 nitroxides caused DNA-strand breakage. The mutagenicity and DNA damaging dose-responses of the test articles were compared using the PROAST benchmark dose software package. The potency ranking of the 4 nitroxides for mutagenicity was different from the ranking of the DNA damaging effects. The mode of action analysis by a multi-endpoint DNA damage pathway assay classified all 4 nitroxides as clastogens. In addition, the majority of the induced Tk mutants showed loss of heterozygosity at the Tk and D11Mit42 loci (ie, chromosome damage <31 Mbp). These results suggest that TEMPO and its 3 derivatives are cytotoxic and mutagenic in mouse lymphoma cells through a mechanism that involves strand breakage and large alterations to DNA. The potency rankings indicate that the different TEMPO derivatives vary in their mutagenic and DNA damaging potential.

Figure 1.

Relative viability of mouse lymphoma cells exposed to TEMPO and its derivatives. Cells were exposed to various concentrations (0.5–10 mM) of 4 piperidine nitroxides for 4h and cell viability was measured using the ATP, Titer Blue, and MTS assay. (A) TEMPO; (B) 4-hydroxy-TEMPO; (C) 4-oxo-TEMPO; (D) 4-methoxy-TEMPO. The data points represent the mean ± SD from 3 independent experiments with 4 parallel replicate wells per concentration in each experiment. * p < .05 and ** p < .01 compared with the vehicle control.

Figure 2.

Cytotoxicity and genotoxicity induced by TEMPO and its derivatives in MLA. Cells were treated with various concentrations (0.1–10 mM) of 4 nitroxides for 4 h in the presence or absence of metabolic activation S9. (A) TEMPO (data from Guo et al., 2013); (B) 4-hydroxy-TEMPO; (C) 4-oxo-TEMPO; (D) 4-methoxy-TEMPO. The data points represent the mean ± SD from 3 independent experiments. * indicates positive response in the MLA, exceeding the GEF of 126 × 10⁻⁶.

Figure 3.

TEMPO and its derivatives cause DNA-strand breaks in mouse lymphoma cells. Following a 4-h treatment, DNA single- and double-strand breaks induced by 4 nitroxides were evaluated by Comet assay. (A) TEMPO; (B) 4-hydroxy-TEMPO; (C) 4-oxo-TEMPO; (D) 4-methoxy-TEMPO. The data points represent the mean ± SD from 3 independent experiments. * p < .05 compared with the vehicle control.

Figure 4.

Mutational spectra evaluated by LOH analysis in induced Tk mutants at 5 heterozygous loci along mouse chromosome 11. Following 11 days incubation, the mutants were collected from TFT-selective plates in the MLA. The data were the weighted sum of mutation percentages from large and small colony mutants. The “–” indicates the LOH extends from the Tk locus to either D11Mit42, D11Mit36, D11Mit20, or D11Mit74. (A) Treatment without S9; (B) Treatment with S9; (C) The loci that were analyzed for LOH are marked on the chromosome 11.

Figure 5.

MoA of TEMPO and its derivatives evaluated by GEF for multiflow DNA damage assay. Data are expressed as fold-increase over concurrent solvent control and plotted for each chemical. Each dot represents a different concentration of the chemical. Dash line represents the GEF for each endpoint.

Figure 6.

Clastogenic probabilities of TEMPO and its derivatives. The probabilities for an clastogen classification were calculated using an ensemble approach containing 3 machine learning algorithms, multinomial logistic regression, random forest, and artificial neural network learners. Each dot represents a different concentration of the chemical.

Figure 7.

Comparison of BMD values for the mutagenicity and DNA damaging effects induced by TEMPO and its derivatives. The BMD (BMD₅, BMD₁₀, BMD₅₀, BMD₁₀₀, and BMD₂₀₀) estimates producing a 5%, 10%, 50%, 100%, or200% increase in the background responses were calculated using Hill model of PROAST with covariate analysis approach. Top panel, treatments without S9 in the MLA; middle panel, treatments with S9 in the MLA; bottom panel, data from the Comet assay. The bars represent the calculated lower and upper 95% CI of each value.