National Key Laboratory of Veterinary Public Health and Safety, College of Veterinary Medicine, China Agricultural University, Beijing, 100193, PR China.
Department of Pharmacology, Biodiscovery Institute, Monash University Clayton, Victoria, 3800, Australia.
Environ Pollut. 2024 Jul 1;352:124145. doi: 10.1016/j.envpol.2024.124145. Epub 2024 May 10.
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 (CuSO) exposure-induced hepatoxicity both in vivo and in vitro. In vitro, HepG2 and L02 cells were exposed to various doses of CuSO for 24 h. Cell viability, ROS production, oxidative stress biomarkers, mitochondrial functions, ultrastructure, intracellular calcium (Ca 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 CuSO 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 CuSO 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, CuSO exposure induced significant oxidative stress and mitochondrial dysfunction in HepG2 cells, which were partially ameliorated by the antioxidant N-acetylcysteine (NAC). Furthermore, CuSO 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 CuSO 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 CuSO-induced cytotoxicity in HepG2 cells by reducing mitochondrial dysfunction and inhibiting the mitochondrial apoptotic pathway. Moreover, 4-PBA treatment significantly attenuated CuSO-induced caspase activation and hepatoxicity in mice. In conclusion, these results reveal that CuSO-induced hepatotoxicity involves mitochondrial dysfunction and ER stress by activating oxidative stress induction and PERK/ATF4 pathway.
铜是一种必需的微量元素，过量暴露可能导致肝毒性，但其潜在的分子机制尚不完全清楚。本研究旨在探讨硫酸铜（CuSO）暴露诱导的体内外肝毒性的分子机制。在体外，将 HepG2 和 L02 细胞暴露于不同剂量的 CuSO 中 24 小时。评估细胞活力、ROS 产生、氧化应激生物标志物、线粒体功能、超微结构、细胞内钙（Ca 浓度以及与线粒体凋亡和内质网（ER）应激相关的蛋白质的表达。在体内，用 CuSO 以 10 和 30mg/kg BW/天的剂量处理 C57BL/6 小鼠，并以 100mg/kg BW/天的剂量共同处理 4-PBA 35 天。随后评估肝功能、组织病理学特征和蛋白质表达。结果发现，暴露于 100-400μM CuSO 24 小时会显著降低 HepG2 和 L02 细胞的活力，且呈剂量依赖性。此外，CuSO 暴露会在 HepG2 细胞中引起明显的氧化应激和线粒体功能障碍，抗氧化剂 N-乙酰半胱氨酸（NAC）可部分改善这种情况。此外，CuSO 暴露会显著引发内质网应激，这表现为肝脏和 HepG2 细胞中 GRP94、GRP78、磷酸化 PERK 和 eIF2α 和 CHOP 蛋白的上调。NAC 处理可显著抑制 HepG2 细胞中 CuSO 暴露引起的内质网应激。通过与 4-PBA 和 PERK 抑制剂 GSK2606414 联合治疗以及 ATF4 基因敲低来抑制内质网应激，可部分减轻 HepG2 细胞中 CuSO 诱导的细胞毒性，减少线粒体功能障碍并抑制线粒体凋亡途径。此外，4-PBA 处理可显著减轻 CuSO 诱导的小鼠中 caspase 激活和肝毒性。总之，这些结果表明，CuSO 诱导的肝毒性通过激活氧化应激诱导和 PERK/ATF4 途径涉及线粒体功能障碍和内质网应激。
