Metallothionein as a biomarker of aquatic contamination in fish: An in silico and in vitro approach using zebrafish as experimental model organism

Abstract

Human activities contaminate aquatic ecosystems with chemicals like metals and pesticides. Fish, sensitive to pollution, are key toxicological models. Metallothionein (Mt) expression, a biomarker for metal contamination, varies depending on the chemical exposure. This study investigated differences in metal affinity for Zn²⁺ binding sites of proteins and Mt induction by the insecticides dichlorvos (DDPV) and deltamethrin (DTM) in Danio rerio. First, D. rerio Zn-binding protein structures with different cell functions were used to evaluate the difference between the binding scores of five metals with the binding site with highest affinity for Zn²⁺ through molecular docking and from there to infer the most potent inducers. Cadmium ion was found to have the highest binding score mean for the selected proteins (Cd²⁺>Cu²⁺>Pb²⁺>Mn²⁺>Cu⁺>Hg²⁺), and, thus, cadmium chloride (CdCl₂) was used as a positive control for Mt induction in D. rerio larvae. D. rerio embryos were exposed to sublethal concentrations of Cd (100 μg L⁻¹), DDPV (1 mg L⁻¹), and DTM (0.01 μg L⁻¹) up to 96 h post-fertilization (hpf). Larvae exposed to Cd and DDPV showed increased Mt levels, whereas DTM exposure had no effect. Proteomic analyses suggest that Mt induction in D. rerio larvae exposed to Cd follows a distinct mechanism from DDPV exposure. Enrichment analysis supports a possible link between DDPV exposure and oxidative stress-induced Mt expression. In contrast, Cd-induced Mt expression likely involves metal transcription factor activation by Zn²⁺. These differences in responsiveness highlight the need for careful consideration when using Mt as a biomarker of metal contamination.