Abstract
Methylmercury (MeHg), a long-lasting organic pollutant, is known to induce cytotoxic effects in mammalian cells. Epidemiological studies have suggested that environmental exposure to MeHg is linked to the development of diabetes mellitus (DM). The exact molecular mechanism of MeHg-induced pancreatic β-cell cytotoxicity is still unclear. Here, we found that MeHg (1-4 μM) significantly decreased insulin secretion and cell viability in pancreatic β-cell-derived RIN-m5F cells. A concomitant elevation of mitochondrial-dependent apoptotic events was observed, including decreased mitochondrial membrane potential and increased proapoptotic (Bax, Bak, p53)/antiapoptotic (Bcl-2) mRNA ratio, cytochrome c release, annexin V-Cy3 binding, caspase-3 activity, and caspase-3/-7/-9 activation. Exposure of RIN-m5F cells to MeHg (2 μM) also induced protein expression of endoplasmic reticulum (ER) stress-related signaling molecules, including C/EBP homologous protein (CHOP), X-box binding protein (XBP-1), and caspase-12. Pretreatment with 4-phenylbutyric acid (4-PBA; an ER stress inhibitor) and specific siRNAs for CHOP and XBP-1 significantly inhibited their expression and caspase-3/-12 activation in MeHg-exposed RIN-mF cells. MeHg could also evoke c-Jun N-terminal kinase (JNK) activation and reactive oxygen species (ROS) generation. Antioxidant N-acetylcysteine (NAC; 1mM) or 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (trolox; 100 μM) markedly prevented MeH-induced ROS generation and decreased cell viability in RIN-m5F cells. Furthermore, pretreatment of cells with SP600125 (JNK inhibitor; 10 μM) or NAC (1 mM) or transfection with JNK-specific siRNA obviously attenuated the MeHg-induced JNK phosphorylation, CHOP and XBP-1 protein expression, apoptotic events, and insulin secretion dysfunction. NAC significantly inhibited MeHg-activated JNK signaling, but SP600125 could not effectively reduce MeHg-induced ROS generation. Collectively, these findings demonstrate that the induction of ROS-activated JNK signaling is a crucial mechanism underlying MeHg-induced mitochondria- and ER stress-dependent apoptosis, ultimately leading to β-cell death.
Highlights
Heavy metals are significant environmental pollutants with hazardous effects on mammal [1]
We examined whether cell apoptosis was involved in MeHg-induced pancreatic β-cell cytotoxicity
To determine whether MeHg-induced β-cell apoptosis is mediated by a mitochondrialdependent pathway, we examined the effects of MeHg on membrane potential (MMP) and cytochrome c release
Summary
Heavy metals are significant environmental pollutants with hazardous effects on mammal [1]. Many studies have reported that heavy metal-induced deleterious effects are positively correlated with the development of human diseases, including cancer, cardiovascular disease, neurological disorders, and diabetes mellitus (DM) [1,2]. Methylmercury (MeHg), one of the most toxic heavy metals, is a widely distributed, highly poisonous environmental toxicant generated by the methylation of inorganic mercury, one which has become a ubiquitous pollutant causing adverse effects in humans [3]. Growing studies have pointed out a potentially strong association between MeHg and disease development including DM [3,6]. In patients with diagnosed Minamata disease (MeHg poisoning), an increased incidence of DM was observed [7]. Epidemiological studies have indicated a positive correlation between higher mercury levels in hair/blood and diagnosed DM, in individuals with higher blood levels of mercury [8–10].
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