Copper excess induces autophagy dysfunction and mitochondrial ROS-ferroptosis progression, inhibits cellular biosynthesis of milk protein and lipid in bovine mammary epithelial cells

Abstract

Excessive copper (Cu) has the potential risk to ecosystems and organism health, with its impact on dairy cow mammary glands being not well-defined. This study used a bovine mammary epithelial cell (MAC-T) model to explore how copper excess affects cellular oxidative stress, autophagy, ferroptosis, and protein and lipid biosynthesis in milk. Results showed the increased intracellular ROS, MDA, and CAT (P < 0.05), alongside decreased T-SOD and GSH in CuSO₄-treated cells (P < 0.05). Transmission electron microscopy and Ad-mCherry-GFP-LC3B assays revealed significant autophagosome accumulation in CuSO₄ exposed cells (P < 0.05). Additionally, CuSO₄ exposure modulated autophagy markers, evidenced by upregulation of genes such as LC3, ATG5, JNK1, and Beclin1, and downregulation of genes such as ATG4B, and p62 (P < 0.05). CuSO₄ also led to notable mitochondrial changes, including size reduction, membrane rupture, and cristae loss, and reduced expression of the ferroptosis inhibitor GPX4 (P < 0.05). The expression of mTOR, HIF-1α and β-catenin signaling pathway were inhibited in differentiated MAC-T cells by CuSO₄ exposure (P < 0.05), activated autophagy through activation of the AMPK-mTOR pathway which in turn affected downstream levels of genes related to milk protein and lipid. In conclusion, excessive copper induces oxidative stress in MAC-T cells, promoting autophagy through JNK-Bcl2, Beclin1-Vps34 and AMPK-mTOR pathways, leading to cell ferroptosis, as well as inhibits the cellular biosynthesis of milk protein and lipid.