Chun-Fa Huang

Arsenic induces pancreatic β-cell apoptosis via the oxidative stress-regulated mitochondria-dependent and endoplasmic reticulum stress-triggered signaling pathways

Arsenic (As), a ubiquitous toxic metal, is an important environmental and industrial pollutant throughout the world. Inorganic As (iAs) is usually more harmful than organic ones and with a high risk of diabetes incidence by exposure. However, the toxicological effects of iAs on growth and function of pancreatic β-cells still remain unclear. Here, we found that iAs significantly decreased insulin secretion and cell viability, and increased ROS and MDA formation in pancreatic β-cell-derived RIN-m5F cells. iAs also induced the increases in sub-G1 hypodiploids, annexin V-Cy3 binding, and caspase-3 activity in RIN-m5F cells, indicating that iAs could induce β-cell apoptosis. Moreover, iAs induced MAPKs activation, mitochondria dysfunction, p53 up-regulation, Bcl-2 and Mdm-2 down-regulation, PARP, and caspase cascades, which displayed features of mitochondria-dependent apoptotic signals. In addition, exposure of RIN-m5F cells to iAs, could trigger ER stress as indicated by the enhancement in ER stress-related molecules induction (such as GRP78, GRP94, CHOP, and XBP1), procaspase-12 cleavage, and calpain activation. The iAs-induced apoptosis and its-related signalings could be effectively reversed by antioxidant N-acetylcysteine. We next observed that exposure of mice to iAs in drinking water for 6 consecutive weeks significantly decreased decreased the plasma insulin, elevated glucose intolerance and plasma lipid peroxidation, and induced islet cells apoptosis, which accompanied with arsenic accumulation in the whole blood and pancreas. N-acetylcysteine effectively antagonized the iAs-induced responses in mice. Taken together, these results suggest that iAs-induced oxidative stress causes pancreatic β-cells apoptosis via the mitochondria-dependent and ER stress-triggered signaling pathways.

Cadmium induces apoptosis in pancreatic β-cells through a mitochondria-dependent pathway: the role of oxidative stress-mediated c-Jun N-terminal kinase activation.

Cadmium (Cd), one of well-known highly toxic environmental and industrial pollutants, causes a number of adverse health effects and diseases in humans. The growing epidemiological studies have suggested a possible link between Cd exposure and diabetes mellitus (DM). However, the toxicological effects and underlying mechanisms of Cd-induced pancreatic β-cell injury are still unknown. In this study, we found that Cd significantly decreased cell viability, and increased sub-G1 hypodiploid cells and annexin V-Cy3 binding in pancreatic β-cell-derived RIN-m5F cells. Cd also increased intracellular reactive oxygen species (ROS) generation and malondialdehyde (MDA) production and induced mitochondrial dysfunction (the loss of mitochondrial membrane potential (MMP) and the increase of cytosolic cytochrome c release), the decreased Bcl-2 expression, increased p53 expression, poly (ADP-ribose) polymerase (PARP) cleavage, and caspase cascades, which accompanied with intracellular Cd accumulation. Pretreatment with the antioxidant N-acetylcysteine (NAC) effectively reversed these Cd-induced events. Furthermore, exposure to Cd induced the phosphorylations of c-jun N-terminal kinases (JNK), extracellular signal-regulated kinases (ERK)1/2, and p38-mitogen-activated protein kinase (MAPK), which was prevented by NAC. Additionally, the specific JNK inhibitor SP600125 or JNK-specific small interference RNA (si-RNA) transfection suppressed Cd-induced β-cell apoptosis and related signals, but not ERK1/2 and p38-MAPK inhibitors (PD98059 and SB203580) did not. However, the JNK inhibitor or JNK-specific si-RNA did not suppress ROS generation in Cd-treated cells. These results indicate that Cd induces pancreatic β-cell death via an oxidative stress downstream-mediated JNK activation-triggered mitochondria-regulated apoptotic pathway.

Neurotoxicological mechanism of methylmercury induced by low-dose and long-term exposure in mice : Oxidative stress and down-regulated Na+/K+-ATPase involved

Methylmercury (MeHg), a potent neurotoxicant, easily passes through the blood-brain barrier (BBB), accumulates in the brain regions and causes severe irreversible damage. However, the neurotoxic effects and action mechanisms of MeHg are still unclear, especially in low-dose and long-term exposure. In this study, we attempted to explore the toxic effects of low-dose MeHg (0.05 mg/kg/day), which was the possible exposed dose by ingestion in MeHg-contaminated areas, on the time course of changes in locomotor activities and auditory brainstem response (ABR) system after administration for 7 consecutive weeks in mice. The results showed that the retention time on the rotating rod (60 rpm) was preferentially decreased after 1-week oral administration with MeHg. The locomotor activities parameters of ambulatory distances and stereotype-1 episodes were significantly increased and vertical-plane entries were progressively decreased after MeHg exposure in 3 consecutive weeks. Gradually progressive abnormality of ABR (increase in hearing thresholds, prolonged absolute and interwave latencies) was found during 4-6 weeks administration of MeHg. These impairments correlated with significant Hg accumulation and biochemical alterations in brain regions and/or other tissues, including the increase of lipid peroxidation (LPO) production, influence of Na+/K(+)-ATPase activities and nitric oxide (NO) levels were found. These findings provide evidence that the signaling of oxidative stress/Na+/K(+)-ATPase/NO plays a role in the underlying mechanisms of the neurotoxic effects induced by low-dose and long-term exposure of MeHg.

Arsenic induces cell apoptosis in cultured osteoblasts through endoplasmic reticulum stress.

Osteoporosis is characterized by low bone mass resulting from an imbalance between bone resorption by osteoclasts and bone formation by osteoblasts. Therefore, decreased bone formation by osteoblasts may lead to the development of osteoporosis, and rate of apoptosis is responsible for the regulation of bone formation. Arsenic (As) exists ubiquitously in our environment and increases the risk of neurotoxicity, liver injury, peripheral vascular disease and cancer. However, the effect of As on apoptosis of osteoblasts is mostly unknown. Here, we found that As induced cell apoptosis in osteoblastic cell lines (including hFOB, MC3T3-E1 and MG-63) and mouse bone marrow stromal cells (M2-10B4). As also induced upregulation of Bax and Bak, downregulation of Bcl-2 and dysfunction of mitochondria in osteoblasts. As also triggered endoplasmic reticulum (ER) stress, as indicated by changes in cytosolic-calcium levels. We found that As increased the expression and activities of glucose-regulated protein 78 (GRP78) and calpain. Transfection of cells with GRP78 or calpain siRNA reduced As-mediated cell apoptosis in osteoblasts. Therefore, our results suggest that As increased cell apoptosis in cultured osteoblasts and increased the risk of osteoporosis.

Arsenic Inhibits Myogenic Differentiation and Muscle Regeneration

Background The incidence of low birth weights is increased in offspring of women who are exposed to high concentrations of arsenic in drinking water compared with other women. We hypothesized that effects of arsenic on birth weight may be related to effects on myogenic differentiation. Objective We investigated the effects of arsenic trioxide (As2O3) on the myogenic differentiation of myoblasts in vitro and muscle regeneration in vivo. Methods C2C12 myoblasts and primary mouse and human myoblasts were cultured in differentiation media with or without As2O3 (0.1–0.5 μM) for 4 days. Myogenic differentiation was assessed by myogenin and myosin heavy chain expression and multinucleated myotube formation in vitro; skeletal muscle regeneration was tested using an in vivo mouse model with experimental glycerol myopathy. Results A submicromolar concentration of As2O3 dose-dependently inhibited myogenic differentiation without apparent effects on cell viability. As2O3 significantly and dose-dependently decreased phosphorylation of Akt and p70s6k proteins during myogenic differentiation. As2O3-induced inhibition in myotube formation and muscle-specific protein expression was reversed by transfection with the constitutively active form of Akt. Sections of soleus muscles stained with hematoxylin and eosin showed typical changes of injury and regeneration after local glycerol injection in mice. Regeneration of glycerol-injured soleus muscles, myogenin expression, and Akt phosphorylation were suppressed in muscles isolated from As2O3-treated mice compared with untreated mice. Conclusion Our results suggest that As2O3 inhibits myogenic differentiation by inhibiting Akt-regulated signaling.

The in vivo antioxidant and antifibrotic properties of green tea (Camellia sinensis, Theaceae).

The in vivo antioxidant and antifibrotic properties of green tea (Camellia sinensis, Theaceae) were investigated with a study of carbon tetrachloride (CCl(4))-induced oxidative stress and hepatic fibrosis in male ICR mice. Oral administration of green tea extract at doses of 125, 625 and 1250 mg/kg for 8 weeks significantly reduced (p<0.05) the levels of thiobarbituric acid-reactive substances (TBARS) and protein carbonyls in the liver by at least 28% compared with that was induced by CCl(4) (1 mL/kg) in mice. Moreover, green tea extract administration significantly increased (p<0.05) the activities of catalase, glutathione peroxidase (GSH-Px) and glutathione reductase (GSH-Rd) in the liver. Our study found that oral administration of green tea extract prevented CCl(4)-induced hepatic fibrosis, as evidenced by a decreased hydroxyproline level in the liver and a reduced incidence of hepatic fibrosis by histological observations. These results indicate that green tea exhibits potent protective effects against CCl(4)-induced oxidative stress and hepatic fibrosis in mice by inhibiting oxidative damage and increasing antioxidant enzyme activities.

Neurotoxicological effects of low-dose methylmercury and mercuric chloride in developing offspring mice.

Mercury is a well-known toxic metal and potently induces severe neurotoxicological effects, especially in infants and children. The purpose of this study was to explore the underlying mechanisms of neurotoxic effects of mercurial compounds on the different stages of developing mice. Low-doses (the probability of human exposure in mercury-contaminated areas) of methylmercury (MeHg) (M, 0.02mg/kg/day) and mercury chloride (HgCl(2)) (H, 0.5mg/kg/day) were administered to mice of the following groups: (1) treatment with distilled water for 7 consecutive weeks after weaning (control-vehicle (CV)); exposure to mercurial compounds at different stages; (2) for 7 consecutive weeks after weaning (control-MeHg (CM) and control-HgCl(2) (CH)); (3) only during perinatal and weaning stages (MeHg-vehicle (MV) and HgCl-vehicle (HV)); and (4) in all experimental stages (MeHg-MeHg (MM) and HgCl(2)-HgCl(2) (HH)). Results revealed the neurobehavioral defects (increased locomotor activities, motor equilibrium impairment, and auditory dysfunction) that correlated with increasing Hg accumulation in CM and CH groups. However, it revealed a decrease and an increase in locomotor activities in MV and HV groups, respectively; these became more severe in MM and HH groups than in MV and HV groups. Motor equilibrium performance in MV and HV groups remained normal, while that in MM and HH groups was decreased. The most severe auditory defects (altered auditory brainstem response, ABR test) found in MM and HH groups than those in the respective CM and CH, MV and HV, including absolute wave III delays and interwave I-III latencies, which suggested that the irreversible auditory dysfunction caused by mercurial compounds. Furthermore, the alteration of lipid peroxidation (LPO), Na(+)/K(+)-ATPase activities, and nitric oxide (NO(x)) in the brain tissues contributed to the observed neurobehavioral dysfunction and hearing impairment. These findings provide evidence that fetuses were much more susceptible to the effects of mercurial compounds with regard to inducing severely neurotoxicological injuries as that found in human beings. The signaling of ROS/Na(+)-K(+)-ATPase/NO(x) plays a crucial role in the underlying mechanism for mercurial compound-induced toxic effects in offspring.

Chloroacetic acid induced neuronal cells death through oxidative stress-mediated p38-MAPK activation pathway regulated mitochondria-dependent apoptotic signals

Chloroacetic acid (CA), a toxic chlorinated analog of acetic acid, is widely used in chemical industries as an herbicide, detergent, and disinfectant, and chemical intermediates that are formed during the synthesis of various products. In addition, CA has been found as a by-product of chlorination disinfection of drinking water. However, there is little known about neurotoxic injuries of CA on the mammalian, the toxic effects and molecular mechanisms of CA-induced neuronal cell injury are mostly unknown. In this study, we examined the cytotoxicity of CA on cultured Neuro-2a cells and investigated the possible mechanisms of CA-induced neurotoxicity. Treatment of Neuro-2a cells with CA significantly reduced the number of viable cells (in a dose-dependent manner with a range from 0.1 to 3mM), increased the generation of ROS, and reduced the intracellular levels of glutathione depletion. CA also increased the number of sub-G1 hypodiploid cells; increased mitochondrial dysfunction (loss of MMP, cytochrome c release, and accompanied by Bcl-2 and Mcl-1 down-regulation and Bax up-regulation), and activated the caspase cascades activations, which displayed features of mitochondria-dependent apoptosis pathway. These CA-induced apoptosis-related signals were markedly prevented by the antioxidant N-acetylcysteine (NAC). Moreover, CA activated the JNK and p38-MAPK pathways, but did not that ERK1/2 pathway, in treated Neuro-2a cells. Pretreatment with NAC and specific p38-MAPK inhibitor (SB203580), but not JNK inhibitor (SP600125) effectively abrogated the phosphorylation of p38-MAPK and attenuated the apoptotic signals (including: decrease in cytotoxicity, caspase-3/-7 activation, the cytosolic cytochrome c release, and the reversed alteration of Bcl-2 and Bax mRNA) in CA-treated Neuro-2a cells. Taken together, these data suggest that oxidative stress-induced p38-MAPK activated pathway-regulated mitochondria-dependent apoptosis plays an important role in CA-caused neuronal cell death.

Antidiabetic Effects of Pterosin A, a Small-Molecular-Weight Natural Product, on Diabetic Mouse Models

The therapeutic effect of pterosin A, a small-molecular-weight natural product, on diabetes was investigated. Pterosin A, administered orally for 4 weeks, effectively improved hyperglycemia and glucose intolerance in streptozotocin, high-fat diet–fed, and db/db diabetic mice. There were no adverse effects in normal or diabetic mice treated with pterosin A for 4 weeks. Pterosin A significantly reversed the increased serum insulin and insulin resistance (IR) in dexamethasone-IR mice and in db/db mice. Pterosin A significantly reversed the reduced muscle GLUT-4 translocation and the increased liver phosphoenolpyruvate carboxyl kinase (PEPCK) expression in diabetic mice. Pterosin A also significantly reversed the decreased phosphorylations of AMP-activated protein kinase (AMPK) and Akt in muscles of diabetic mice. The decreased AMPK phosphorylation and increased p38 phosphorylation in livers of db/db mice were effectively reversed by pterosin A. Pterosin A enhanced glucose uptake and AMPK phosphorylation in cultured human muscle cells. In cultured liver cells, pterosin A inhibited inducer-enhanced PEPCK expression, triggered the phosphorylations of AMPK, acetyl CoA carboxylase, and glycogen synthase kinase-3, decreased glycogen synthase phosphorylation, and increased the intracellular glycogen level. These findings indicate that pterosin A may be a potential therapeutic option for diabetes.

Residual thermal strain in thick GaN epifilms revealed by cross-sectional Raman scattering and cathodoluminescence spectra

Strain can significantly alter the physical properties of a solid. We demonstrate that in a thick GaN epilayer there exists a residual thermal strain along the growth direction. This result is clearly revealed by cathodoluminescence spectra, in which the band gap of the GaN film decreases with distance away from the epifilm?substrate interface. This result is further confirmed by Raman scattering spectra in which the phonon modes show a red shift along the growth direction. Our finding is important for the understanding and application of nitride semiconductors.