Oxidative stress and mitochondrial dysfunctions induced by cyanobacterial microcystin-LR in primary grass carp hepatocytes

Abstract

Microcystin-LR (MC-LR), a cyclic heptapeptide produced by freshwater cyanobacteria, induces a range of liver injuries. However, the mechanisms underlying MC-LR toxicity in primary hepatocytes of aquatic organisms remains poorly understood. In this study, we investigated the effects of MC-LR on oxidative stress and mitochondrial function using primarily cultured grass carp hepatocytes. The results revealed that IC₅₀ of MC-LR on grass carp primary liver cells for 24 hours was 2.40 μmol/L. Based on 24h-IC₅₀, concentrations of 0, 0.30, 0.60, and 1.20 μmol/L were used in subsequent experiments. MC-LR exposure led to a significant reduction in cell viability, induced abnormal cell morphology, and caused plasma membrane rupture, as indicated by elevated LDH activity in a concentration-dependent manner. Additionally, MC-LR exposure induced oxidative stress, resulting in increased ROS levels and downregulation of genes associated with oxidative stress, including keap1, nrf2, cat, sod1, gpx, gst, and gr (P<0.05). Furthermore, the electron microscopy results showed that MC-LR caused damage to the ultrastructure of primary hepatocytes, including mitochondrial membrane rupture, vacuolation, and induction of mitochondrial autophagy. Moreover, MC-LR exposure elevated intracellular Ca²⁺ concentration, reduced MMP and ATP levels, and inhibited mitochondrial respiratory chain complex I activity (P<0.05). qRT-PCR analysis demonstrated that MC-LR treatment significantly decreased the transcriptional levels of genes related to mitochondrial quality control including pgc-1α, tfam, nrf1, drp1, opa1, mfn1, and mfn2 (P<0.05). Collectively, our findings highlight that MC-LR causes oxidative stress and impairs mitochondrial function, leading to further hepatocyte damage, which provides insights into the mechanisms of MC-LR-induced hepatotoxicity and offers valuable references for further investigations.