Dietary copper induces cuproptosis and disrupts the gut-gonadal axis in zebrafish (Danio rerio)

Dietary exposure is a recognized route of metal toxicity, yet its mechanistic impacts on aquatic organisms remain poorly understood. In this study, adult zebrafish (Danio rerio, five months old, both sexes) were exposed to dietary Cu (2.4 (Control), 4.4 (Cu-L), 9.8 (Cu-M), and 54.4 (Cu-H) μg/g ww) for 28 days to elucidate tissue-specific Cu accumulation, cuproptosis-related toxicity, intestinal disruption, reproductive effects, and their potential linkage through the gut-gonadal axis. Cu concentrations significantly increased in all examined tissues of both sexes, with tissue-specific fold changes ranging from 1.7 to 7.7 in females and 1.9-2.7 in males, and significant ionic homeostatic disturbance difference observed in intestine and gonads. Transcriptomic and protein analyses revealed tissue-dependent regulation of cuproptosis-related markers, characterized by widespread suppression of mitochondrial enzymes (e.g., fdx1, dlat, DLST; 52-74% decreases in female brains and 56-66% in gonads) alongside consistent induction of the Cu uptake transporter SLC31A1 (1.6-5.4 folds), suggesting disrupted copper homeostasis favoring intracellular Cu retention. Intestinal histopathology showed villus atrophy and goblet cell loss at high Cu exposure, accompanied by sex-dependent gut microbiota dysbiosis, with females exhibiting decreased α-diversity and genus-level shifts. In addition, delayed gametogenesis, reduced estradiol (E2), elevated testosterone (T), and dysregulation of hypothalamic-pituitary-gonadal (HPG) axis-related genes were witnessed, with females being more affected. These findings indicate a mechanistic link between cuproptosis and gut-gonadal axis dysfunction. This study provides new insights into the impacts of dietary Cu exposure and underscores the ecological relevance of cuproptosis in metal-induced endocrine and reproductive disruption.