Toxicol Appl Pharmacol. 2010 Mar 15;243(3):323-31. Epub 2009 Dec 16.
Inorganic mercury causes pancreatic beta-cell death via the oxidative stress-induced apoptotic and necrotic pathways.
Chen YW, Huang CF, Yang CY, Yen CC, Tsai KS, Liu SH.
Mercury is a well-known highly toxic metal. In this study, we characterize and investigate the cytotoxicity and its possible mechanisms of inorganic mercury in pancreatic beta-cells. Mercury chloride (HgCl2) dose-dependently decreased the function of insulin secretion and cell viability in pancreatic beta-cell-derived HIT-T15 cells and isolated mouse pancreatic islets. HgCl2 significantly increased ROS formation in HIT-T15 cells. Antioxidant N-acetylcysteine effectively reversed HgCl2-induced insulin secretion dysfunction in HIT-T15 cells and isolated mouse pancreatic islets. Moreover, HgCl2 increased sub-G1 hypodiploids and annexin-V binding in HIT-T15 cells, indicating that HgCl2 possessed ability in apoptosis induction. HgCl2 also displayed several features of mitochondria-dependent apoptotic signals including disruption of the mitochondrial membrane potential, increase of mitochondrial cytochrome c release and activations of poly (ADP-ribose) polymerase (PARP) and caspase 3. Exposure of HIT-T15 cells to HgCl2 could significantly increase both apoptotic and necrotic cell populations by acridine orange/ethidium bromide dual staining. Meanwhile, HgCl2 could also trigger the depletion of intracellular ATP levels and increase the LDH release from HIT-T15 cells. These HgCl2-induced cell death-related signals could be significantly reversed by N-acetylcysteine. The intracellular mercury levels were markedly elevated in HgCl2-treated HIT-T15 cells.
Taken together, these results suggest that HgCl2-induced oxidative stress causes pancreatic beta-cell dysfunction and cytotoxicity involved the co-existence of apoptotic and necrotic cell death.
Uncontrolled industrialization has resulted in a very wide segment of the human population being exposed to agents that have the potential to cause or exacerbate disease. Mercury (Hg) is widespread and persistent in the environment. Mercury has become an important public health concern of our day because of growing evidence of its presence in some components of the human food chain. There are seeds, foodstuffs, disinfectant, disk batteries and dental amalgam (Aschner and Walker, 2002; Clarkson et al., 2003).
Mercury is a notorious heavy metal and has serious toxicity in various mammalian cells and organ systems (Guo et al., 1998; Silbergeld et al., 2000). Shaffi (1981) has reported that mercuric compounds, about 200 tons, were introduced into the environment annually as effluents from industries. A cross-sectional analysis has also shown that people in the highest quartile of fish consumption had median mercury levels 1.82 times above the levels in the lowest quartile (Latshaw et al., 2006).
In past experiments using mice and fish, it has found that HgCl2 altered intracellular Ca 2+ homeostasis and decreased insulin secretion in pancreatic β-cells or islets (Bloom et al., 1972; Liu and Lin-Shiau, 2002). However, the cytotoxic mechanism of HgCl2 on the pancreatic β-cells still remain unclear.
Reactive oxygen species (ROS) has been implicated in a wide variety of undesirable biological reaction and functional cell damage, including the induction of pancreatic β-cell dysfunction or death by autoimmune attack in type 1 diabetes (Finkel and Holbrook, 2000; Hotta et al., 2000). Mercury has been shown to induce toxic effects by oxidative stress induction that caused the alteration of cellular function and eventually resulted in cell death and pathological injury, which were accompanied with the damage of antioxidant enzymes (Sarafian, 1999; Valko et al., 2005). It has been shown that methyl mercury triggers the early acute necrosis and delayed apoptosis in murine peritoneal neutrophils (Kuo and Lin-Shiau, 2004). An apoptotic death of pancreatic β-cells has also been found to be induced by methyl mercury (Chen et al., 2006).
A recent in vivo study has also shown that inorganic mercury could induce the apoptosis and proliferative reactions in renal cells (Fouda et al., 2008).
However, the effect of inorganic mercury on pancreatic β-cell survival still remains unclear.
Taken together, in the current study, we try to explore the cytotoxic effect and mechanism of inorganic mercury (HgCl2) on pancreatic β-cells. To this aim, we attempt to investigate the in vitro effects of HgCl2 on ROS generation, insulin secretion and apoptotic and necrotic cell death in pancreatic β-cell-derived HIT-T15 cells and isolated mouse islets. Moreover, the potential protective effects of antioxidant N-acetylcysteine on the pancreatic β-cell dysfunction and death in vitro and in vivo were also investigated.
Many studies have reported that mercuric compounds potently induce oxidative stress, which played a key role for cascade activation during mercury-induced cellular injury (Gatti et al., 2004; Shenker et al., 2002; Yin et al., 2007). The deleterious effect of ROS has been found to be induced in the progression of glucose toxicity-triggered pancreatic β-cell dysfunction under diabetic condition (Kajimoto and Kaneto, 2004; Robertson et al., 2007).
Despite several studies showing that mercury could induce ROS with subsequent oxidative damage in several kinds of cells and organs, the precise action and mechanism of inorganic mercury HgCl2-induced oxidative stress on the pancreatic β-cell dysfunction and cell death is still unclear, especially to discriminate between HgCl2-induced apoptosis and necrosis and elucidate the underlying signaling pathways involved in these processes.
In the present work, we investigate the HgCl2-induced cytotoxicity and its cellular mechanism on pancreatic β-cells. We found that exposure to low concentration of HgCl2 significantly decreases cell viability and insulin secretion and markedly increase ROS generation in HIT-T15 cells or isolated mouse islets. HgCl2-induced β-cell cytotoxicity could be reserved by antioxidant NAC. Therefore, these findings indicate that oxidative stress is involved in the HgCl2-induced pancreatic β-cell cell death and dysfunction.
ROS generation can serve as a trigger for cell death, which causes oxidative modification of DNA and gene mutation in a lot of cell types (Buttke and Sandstrom, 1995; Inoue et al., 2004). Oxidative stress could also trigger the inductions of apoptosis-related proteins (for examples, c-Jun-N-terminal kinase (JNK), extracellular signal-regu- lated kinase-1/2 (ERK-1/2) and p38 mitogen-activated protein kinase (MAPK) cascades), Bcl-2 family of pro- and antipoptosis proteins, p53, mitochondria-associated proteins (for examples, cytochrome c, Apaf-1 and apoptosis-inducing factor), cleavage of poly (ADP-ribose) polymerase (PARP) and caspase cascades (Kim et al., 2009; Kroemer et al., 1997; Ueda et al., 2002). PARP has been shown to be activated at an intermediate stage of apoptosis and is then cleaved and inactivated at a late stage by apoptotic proteases, namely caspase-3/CPP-32/ Yama/apopain and caspase-7 (Decker and Muller, 2002). However, several reports have also mentioned that PARP is involved in either necrosis and subsequent inflammation or apoptosis (Gobeil et al., 2001; Decker and Muller, 2002).
Furthermore, the important role of ROS in death of several kinds of cells induced by mercury has been documented; its deteriorated effects included the DNA damage, DNA and RNA synthesis inhibition and disruption of mitochondrial functions (Baskin et al., 2003; Stohs and Bagchi, 1995; Xiang and Shao, 2003).
Mitochondria are a sensitive organelle to the effects of oxidative stress (Kowaltowski et al., 1996 and 2001; Orrenius et al., 2003).
Recent studies have demonstrated that organic mercury could induce cell death by activating mitochondrial caspase- dependent apoptotic pathway in several types of cells (Humphrey et al., 2005; Nishioku et al., 2000; Sutton and Tchounwou, 2006).
There were a few reports showing that inorganic mercury (4–100 μM HgCl2) could cause cytotoxicity by ROS generation in lymphocytes, macrophages, and renal proximal tubule cells (Kim and Sharma, 2004; Sarmento et al., 2004; Sutton and Tchounwou, 2007); however, the precise action and cellular mechanism of HgCl2-induced cytotoxic effects on pancreatic β-cell cells are still unclear.
In the current study, we found that HgCl2 was capable of inducing apoptosis by triggered mitochondrial membrane depolarization and cytochrome c release in β-cell-derived HIT-T15 cells, which could be prevented by treatment of cell with antioxidant NAC. In addition, we also found that 5 μM HgCl2 caused a significant degradation of 116-kDa PARP, and it was associated with the activation of caspase 3 protease.
These results implicate that HgCl2 induced an oxidative stress-regulated β-cell apoptosis through a mitochondria- ependent apoptotic pathway. The characterizations of apoptosis are cell shrinkage, chromatin condensation, compaction of organelles and systematic DNA cleavage, which is genetically regulated (Arends andWyllie, 1991; Bursch et al., 1992). In contrast, necrosis is characterized by cell swelling and ultimate rupture of cells in which plasma membrane is the major site of damage (Schwartz et al., 1993;Wyllie et al., 1980). Oxidative stress has been shown to be capable of inducing both apoptotic and necrotic cell death in several kinds of cells (Higuchi, 2004; Tan et al., 1998).
Several studies have also shown that mercury-induced cell death was through both apoptosis and necrosis inmacrophages, neutrophils and proximal tubular cells (Kim and Sharma, 2004; Kuo and Lin-Shiau, 2004; Lash et al., 2007). In the present study, we performed dual staining with fluorescence probes of acridine orange (cell-permeant) and ethidium bromide (cell-impermeant) for discriminating apopto tic fromnecrotic cell death by flowcytometry (Lecoeur, 2002; Johnson et al., 2005). In early stage of apoptosis, cells were impermeable to ethidium bromide and their nuclei stained green, which had condensed and/or fragmented nuclei; this ability was lost and their nuclei stained red during the later stage of apoptosis. Necrotic cells appeared a red nuclear stain, which was no nuclear condensation.
Viable cells were impermeable to ethidium bromide and their nuclei stained green. Here, our results showed that exposure to HgCl2 (5 and 20 μM) effectively induced both apoptosis and necrosis in HIT-T15 cells. On the other hand, it has been suggested that high ATP release accompanied by the decrease in the ATP levels under the cell plasma membrane damage are responsible for the cell death by necrosis (Lopez et al., 2003; Kim et al., 2003; Kon et al., 2004). Moreover, LDH release from cells has been shown to be a marker of cell membrane damage and cell death due to necrosis (Mangipudy1 and Vishwanatha1, 1999; De La Peña et al., 2007). In the present work, we found that treatment with HgCl2 for 4 and 24 h remarkably caused the intracellular ATP depletion and LDH release increase in HIT-T15 cells, respectively. These results indicated that the necrotic pathway is involved in the HgCl2- riggered pancreatic β-cell damage.
Taken together, these findings indicate that HgCl2 triggers pancreatic β-cell damage through both apoptotic and necrotic pathway. GSH is the most abundant intracellular thiol-based antioxidant in living cells. GSH has been shown to function as a direct hydrogen peroxide scavenger (Nordberg and Arner, 2001). NAC is a low- molecular-weight thiol and a precursor of glutathione. Our unpublished data have shown that the glutathione levels in β-cells treated with HgCl2 (5 and 20 μM) are not decreased at 4 h, but slightly decreased at 16 h (about 10–20% inhibition) and markedly decreased at 24 h (about 40–60% inhibition). Therefore, the antagonized effect of NAC on insulin secretion inhibition in β-cells by short-term (4 h) HgCl2 exposure may be caused by its thiol reducing action but not glutathione formation action. Nevertheless, the antagonized effect of NAC on cell viability inhibition in β-cells by HgCl2 treatment for 24 h may be caused by its glutathione formation action or both of glutathione formation action and thiol reducing action.
In conclusion, from this in vitro study, we found that HgCl2 is capable of inducing the ROS-related insulin secretion suppression and cell death in pancreatic β-cells. The further evidences indicate that HgCl2 enters β-cells and triggers oxidative stress to induce cell death through both apoptotic and necrotic pathways.