A Multimodal Approach to Identify Metallothionein Metal Inducers in Nile Tilapia: Insights From Molecular Docking and Hepatocyte Exposure

Many human activities contribute to the pollution of aquatic ecosystems, primarily through agricultural, industrial, mining, and domestic discharges into water bodies. Fish, being highly sensitive to environmental changes, serve as valuable models for monitoring the health of these ecosystems. Metallothionein (Mt), a biomarker for metal contamination, shows variable expression depending on the metal involved. Transcription of the mt gene is regulated by intracellular metal concentrations and mediated by interactions between metal‐responsive transcription factors (Mtf) and metal response elements (MRE) in the mt promoter. Zinc plays a key role by binding to Mtf, activating it, and enabling interaction with MRE sequences to initiate transcription. In this context, this study aimed to identify the most potent inducers of mt expression in Oreochromis niloticus . Initially, zinc‐binding proteins from O. niloticus were modeled to assess differential binding scores of various metals using molecular docking, which suggested the potency ranking Cd 2+ > Cu 2+ > Mn 2+ > Hg 2+ > Pb 2+ . These predictions were validated using primary hepatocytes exposed to concentrations 10 times higher than the maximum allowed for effluent discharge under Brazilian law. The expression of both mt mRNA and Mt protein was evaluated in hepatocytes. Both in silico and in vitro results identified cadmium as the most potent inducer of Mt. Other metals did not induce mt expression under the tested conditions. These findings underscore the importance of understanding how Mt expression varies by metal and tissue, as differences in responsiveness can influence the interpretation of Mt levels in teleost fish used for water quality monitoring and environmental toxicology.