Mechanistic insights into multi-metal toxicity interactions via plasmid DNA assay

Heavy metals (Hg²⁺, Cd²⁺, Se⁴⁺, Zn²⁺) are widespread contaminants from industrialization and urbanization, posing severe ecological and human health risks due to persistence, bioaccumulation, and oxidative DNA damage. Traditional risk assessments focus on single-metal toxicity, overlooking complex interactions (synergism or antagonism) among coexisting metals, undermining hazard evaluations and risk predictions for multi-contaminant scenarios.

This is an in vitro study in which a plasmid DNA assay assessed oxidative DNA damage (via supercoiled DNA relaxation and linearization) induced by single metals, binary, ternary, and quaternary mixtures. Equipartition Ray designed binary ratios, and Uniform Design Ray optimized ternary (1:1:1 toxicity/mass concentration ratios) and quaternary (five distinct toxicity ratios) mixtures. Toxicity was quantified using TD₂₀ (dose causing 20% DNA damage), and interactions were analyzed via Toxicity Unit, Additive Index, and Mixed Toxicity Index.

Single-metal toxicity followed the hierarchy Hg²⁺ > Se⁴⁺ > Cd²⁺ > Zn²⁺, with TD₂₀ values of 0.026, 0.116, 0.911, and 1.009 mmol/L, respectively. Binary mixtures revealed synergistic effects between Cd²⁺ and Hg²⁺ (most pronounced at 1:1 molar ratio), while Zn²⁺ or Se⁴⁺ exhibited antagonistic effects with Hg²⁺ or Cd²⁺. In ternary and quaternary systems, Zn²⁺ and Se⁴⁺ mitigated Hg²⁺/Cd²⁺ toxicity; notably, 1:1:1 mass ratios showed stronger antagonism than toxicity-effect ratios, and most multi-metal combinations reduced DNA damage compared to individual metals.

Metal interactions are concentration-dependent and non-linear, with Zn^2⁺ and Se^4⁺ as modulators of Hg^2⁺/Cd^2⁺-induced oxidative DNA damage. These findings provide insights for refining multi-metal risk assessments, enabling more accurate hazard predictions, and informing remediation strategies for contaminated sites. Extrapolation of these in vitro results to in vivo systems requires further investigation.