Copper exposure induces mitochondrial dysfunction and hepatotoxicity via the induction of oxidative stress and PERK/ATF4 -mediated endoplasmic reticulum stress

Meng Li; Shusheng Tang; Tony Velkov; Jianzhong Shen; Chongshan Dai

doi:10.1016/j.envpol.2024.124145

Copper exposure induces mitochondrial dysfunction and hepatotoxicity via the induction of oxidative stress and PERK/ATF4 -mediated endoplasmic reticulum stress

Environ Pollut. 2024 May 10:352:124145. doi: 10.1016/j.envpol.2024.124145. Online ahead of print.

Authors

Meng Li¹, Shusheng Tang¹, Tony Velkov², Jianzhong Shen¹, Chongshan Dai³

Affiliations

¹ National Key Laboratory of Veterinary Public Health and Safety, College of Veterinary Medicine, China Agricultural University, Beijing, 100193, PR China.
² Department of Pharmacology, Biodiscovery Institute, Monash University Clayton, Victoria, 3800, Australia.
³ National Key Laboratory of Veterinary Public Health and Safety, College of Veterinary Medicine, China Agricultural University, Beijing, 100193, PR China. Electronic address: daichongshan@cau.edu.cn.

PMID: 38735462
DOI: 10.1016/j.envpol.2024.124145

Abstract

Copper is an essential trace element, and excessive exposure could result in hepatoxicity, however, the underlying molecular mechanisms remain incompletely understood. The present study is aimed to investigate the molecular mechanisms of copper sulfate (CuSO₄) exposure-induced hepatoxicity both in vivo and in vitro. In vitro, HepG2 and L02 cells were exposed to various doses of CuSO₄ for 24 h. Cell viability, ROS production, oxidative stress biomarkers, mitochondrial functions, ultrastructure, intracellular calcium (Ca²⁺⁾ concentration, and the expression of proteins related to mitochondrial apoptosis and endoplasmic reticulum (ER) stress were assessed. In vivo, C57BL/6 mice were treated with CuSO₄ at doses of 10 and 30 mg/kg BW/day and co-treated with 4-PBA at 100 mg/kg BW/day for 35 days. Subsequently, liver function, histopathological features, and protein expression were evaluated. Results found that exposure to CuSO₄ at concentrations of 100-400 μM for 24 h significantly decreased the viabilities of HepG2 and L02 cells and it was in a dose-dependent manner. Additionally, CuSO₄ exposure induced significant oxidative stress and mitochondrial dysfunction in HepG2 cells, which were partially ameliorated by the antioxidant N-acetylcysteine (NAC). Furthermore, CuSO₄ exposure prominently triggered ER stress, as evidenced by the upregulation of GRP94, GRP78, phosphorylated forms of PERK and eIF2α, and CHOP proteins in livers of mice and HepG2 cells. NAC treatment significantly inhibited CuSO₄ exposure -induced ER stress in HepG2 cells. Pharmacological inhibition of ER stress through co-treatment with 4-PBA and the PERK inhibitor GSK2606414, as well as genetic knockdown of ATF4, partially mitigated CuSO₄-induced cytotoxicity in HepG2 cells by reducing mitochondrial dysfunction and inhibiting the mitochondrial apoptotic pathway. Moreover, 4-PBA treatment significantly attenuated CuSO₄-induced caspase activation and hepatoxicity in mice. In conclusion, these results reveal that CuSO₄-induced hepatotoxicity involves mitochondrial dysfunction and ER stress by activating oxidative stress induction and PERK/ATF4 pathway.

Keywords: Apoptosis; Copper sulfate; ER stress; Hepatotoxicity; Mitochondrial dysfunction.