By utilizing ultraviolet (UV)–visible absorption spectroscopy, circular dichroism (CD), resonance light scattering (RLS), and transmission electron microscope (TEM) techniques, the toxic interaction of methyl violet (MV) with calf thymus DNA (ctDNA) was investigated at the molecular level. The UV–visible absorption spectra results showed that MV intercalated into ctDNA base pairs at low MV concentrations, while MV was attached to the negative charged phosphate groups at higher concentrations and the binding constant (<i>K</i><sup>θ</sup><sub>25°C</sub>) was 1.80 × 10<sup>3</sup> L mol<sup>–1</sup>. The absorbency results showed that the genotoxic interaction of MV with ctDNA is under the electrostatic binding mode. MV had strong toxic interaction with ctDNA at 4.0 × 10<sup>–5</sup> mol L<sup>–1</sup> MV and pH 6.5. The toxic interaction of MV with ctDNA can result in the formation of massive aggregates and change of the ctDNA conformation, which showed a significant linear dose–response relationship.

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