110 | Integrative ultrastructural and molecular evidence of ferroptosis in lead-exposed zebrafish liver

Ilaria Olivito, Abdalmoiz I.m. Ahmed, Federica Talarico, Marcello Mezzasalma, Elvira Brunelli | Department of Biology, Ecology and Earth Sciences DiBEST, University of Calabria, Rende [CS], Italy.

Abstract

Lead (Pb) is a widespread environmental pollutant that endangers the health of both humans and animals. In humans it is estimated to cause around 900,000 deaths per year and there is no established safe exposure threshold [1]. Pb toxicity induces toxic systemic effects, affecting almost all organs, mainly the liver, due to its key role in metabolic and detoxification processes [2]. Despite the established link between Pb exposure and both oxidative stress [3] and iron dysregulation [4], two key factors in ferroptosis, direct evidence for the activation of this specific type of programmed cell death is still limited. In the present study, we investigated whether Pb-induced hepatotoxicity is associated with the activation of ferroptotic pathways. Adult zebrafish, used as an in vivo model, were chronically exposed to environmentally relevant doses of Pb (2.5 and 5 μg/L) and ferroptosis activation in the liver was evaluated by assessing mitochondrial damage using transmission electron microscopy, and Western blot analysis to quantify the expression of key proteins, including ACSL4, GPX4, SLC7A11, and the NRF2/KEAP1 axis. Pb exposure caused dose-dependent mitochondrial damage in hepatocytes, observed ultrastructurally as loss of cristae and membrane rupture. In line with the morphological findings, molecular analysis indicated ferroptotic profile in the liver of group exposed to Pb highlighted by increased ACSL4 expression, reduced SLC7A11 levels, dysregulation of GPX4, and impaired NRF2/KEAP1 signaling. Our data collectively indicate that Pb exposure triggers a ferroptosis-like mechanism in zebrafish hepatocytes via mitochondrial destabilization, compromised antioxidant defenses, and ACSL4-mediated lipid peroxidation.