Effect of Di(2-ethylhexyl)phthalate on Helicobacter pylori-Induced Apoptosis in AGS Cells.

Plastic products are wildly used in human life. Di(2-ethylhexyl)phthalate (DEHP) is an essential additive in plastic manufacturing and is used as plasticizer for many products including plastic food packaging. DEHP is a teratogenic compound and can cause potent reproductive toxicity. DEHP can also cause liver damage, peroxisome proliferation, and carcinogenesis. DEHP is also strongly associated with peptic ulcers and gastric cancer; however, the underlying effect and mechanism of DEHP on the gastrointestinal tract are not entirely clear. The oral infection route of H. pylori parallels the major ingestion route of DEHP into the human body. Therefore, we wanted to study the effect of DEHP and H. pylori exposure on the human gastric epithelial cell line, AGS (gastric adenocarcinoma). The viability of the AGS cell line was significantly lower in 80 µ M-DEHP and H. pylori (MOI = 100 : 1) coexposure than DEHP or H. pylori alone. DEHP and H. pylori coexposure also induced caspase-3 activation, and increased Bax/Bcl-2 ratio and DNA fragmentation in AGS cells. These results indicate that DEHP can enhance H. pylori cytotoxicity and induce gastric epithelial cell apoptosis. Therefore, it is possible that DEHP and H. pylori coexposure might enhance the disruption of the gastric mucosa integrity and potentially promote the pathogenesis of gastric carcinogenesis.

Activation of Trim17 by PPARγ is involved in di(2-ethylhexyl) phthalate (DEHP)-induced apoptosis on Neuro-2a cells.

Di-(2-ethylhexyl) phthalate (DEHP) is widely used as a plasticizer in plastics. Its reproductive toxicity and teratogenic effects are well known. DEHP can cause liver damage and peroxisome proliferation, as well as carcinogenesis. Animal study has shown that DEHP causes neurodegeneration in rat brain. Prenatal exposure to DEHP disrupts brain development and decreases brain weight in rats. But its mechanism of action in the brain is not clear. This study used a neuroblastoma cell line, Neuro-2a cells, to investigate the toxic effect of DEHP. The results revealed that DEHP inhibits cell proliferation, activate caspase-3, induce apoptosis in a dose and time dependent manner, and activate expression of the PPARγ and Trim17 protein. Administration of the PPARγ agonist (troglitazone) enhanced DEHP-induced Trim17 protein expression and this enhancement could be reversed by the PPARγ antagonist (GW9662). These results suggest that DEHP activates the Trim17 protein via PPARγ leading to cleavage pro-caspase-3 and apoptosis. This finding may account for the central nervous system toxicity of DEHP and implies DEHP can impair fetal brain development.

Functional changes on ascending auditory pathway in rats caused by germanium dioxide exposure: an electrophysiological study.

The semiconductor element, germanium (Ge), is essential for the manufacture of modern integrated circuits. Because of its anti-tumor and immunomodulative effects, Ge-containing compounds are also used as health-promoting ingredients in food. However, some histological studies have shown the toxic effects of Ge-containing compounds on various organs, including the central nervous system. Even now, the effect of germanium on auditory system function is not completely clear. To clarify this question, brainstem auditory evoked potentials (BAEPs) were applied to examine the effect of germanium dioxide (GeO(2)) on the ascending auditory pathway. Since the voltage-gated sodium channel is important to neuron activation and nerve conduction, the effect of GeO(2) on voltage-gated sodium channels was also examined. The result revealed GeO(2) elevated the BAEPs threshold dose-dependently. GeO(2) also prolonged latencies and interpeak latencies (IPLs) of BAEPs, but the amplitudes of suprathreshold intensities (90dB) did not show any obvious change. In addition, the results of whole cell patch clamp studies indicated GeO(2) reduced inward sodium current. These results suggest the toxic effect of GeO(2) on the conduction of the auditory system, and that inhibitory effect of GeO(2) on the voltage-gated sodium channels might play a role in GeO(2)-induced abnormal hearing loss.

Germanium dioxide induces mitochondria-mediated apoptosis in Neuro-2A cells.

Germanium (Ge) is commonly used in the semiconductor industry as well as health-promoting and medical field. Biologically, germanium possesses erythropoietic, anti-microbial, anti-tumor, anti-amyloidosis, and immunomodulative effects. However, toxic effects of Ge-containing compounds on kidney, muscle, neuronal cells, and nerves have been reported. Mitochondrial dysfunction was found to be involved in the pathogenesis of GeO(2)-induced nephropathy and myopathy. Since it is well known that mitochondria play a major role in apoptosis triggered by many stimuli, an effort was made to examine whether the Ge-induced neurotoxicity occurs through mitochondria-mediated apoptosis. A mouse neuroblastoma cell line, Neuro-2A, was used in the present study. After incubating with 0.1-800microM of GeO(2) for 0-72h, the cell viability of Neuro-2A cells was inhibited in a dose- and time-dependent manner. Further analysis showed that aside from the changes in the nuclear morphology responsible for apoptosis, the release of cytochrome c, the loss of mitochondrial membrane potential, the translocation of Bax, and the reduction of Bcl-2 expression were also observed in Neuro-2A cells after GeO(2) treatment. These results indicate that the mitochondria-mediated apoptosis is involved in this in vitro model of GeO(2)-induced neurotoxicity.

Implication of nNOS in the enlargement of AChR aggregates but not the initial aggregate formation in a novel coculture model.

The aggregation of nicotinic acetylcholine receptors (AChRs) is an early hallmark of the formation of neuromuscular junction (NMJ), and nitric oxide is recently known to play an important role. In many NMJ studies, nerve-muscle coculture model was used, and NG108-15 cells, a neuroblastoma x glioma hybrid cell line, were the most frequently used nerve cells. However, possible contributions from glial cells could not be excluded. In this study, Neuro-2a neuroblastoma cells were used instead of [corrected] coculture with myotubes, and the relationship between AChR aggregation and spatiotemporal expression and activation of nNOS (neuronal nitric oxide synthase) was examined. Upon coculture, AChR aggregates were observed by FITC-conjugated alpha-bungarotoxin, and double labeling of AChRs and neurofilament showed that the neurites of a Neuro-2a cell innervated several myotubes. After treating the cocultures with single dose of L-NAME at the end of 1-day [corrected] coculturing, only slight effect on AChR aggregation could be found indicating that nNOS is not related to the initial formation of AChR aggregates. In contrast, when L-NAME treatment was given at the end of a 3-day coculturing, the day just before reaching the maximum extent of AChR aggregation, new AChR aggregates were hardly formed and the preformed AChR aggregates were even dispersed indicating that the enlargement of AChR aggregates is highly dependent on the nNOS activity. Double-labeling study of nNOS and AChR further showed that the coupling of membranous nNOS to regions nearby the AChR aggregates was essential for the enlargement of AChR aggregates. These results not only revealed the spatiotemporal relationship between AChR aggregation and nNOS activity but also verified the feasibility and usefulness of using Neuro-2a cells in a coculture model.

Neurobehavioral changes in Taiwanese lead-exposed workers.

OBJECTIVE: The dysfunction of the central nervous system (CNS) caused by lead exposure was evaluated. METHODS: Workers who have been exposed to lead for at least 3 years and have been informed of having elevated blood lead levels (BLLs) were recruited. According to their current BLLs, 33 and 28 males were assigned to the medium (40-80 microg/dL) and low (<40 microg/dL) BLLs groups, respectively. Sixty-two nonexposed healthy men served as the control group. Their neurobehaviors were examined by a computerized evaluation system. RESULTS: Significantly impaired neurobehavioral functions were shown in the medium BLLs group, which included slow performance of psychomotor tasks, impaired processing of visual-spatial information, reduced memory and learning functions, low performance accuracy, slow execution of responses, and poor attentional control. CONCLUSIONS: Subtle CNS dysfunction could be detected from lead-exposed workers who have no obvious neurologic and cognitive deficits.

Lead-induced attenuation in the aggregation of acetylcholine receptors during the neuromuscular junction formation.

Lead (Pb2+) toxicity is more common in children and is associated with cognitive deficits, which may reflect lead-induced changes in central synaptic development and function. Aside from neurotoxicity, lead exposure may also impact mature neuromuscular junction (NMJ) and cause muscle weakness. NMJ is known as a peripheral cholinergic synapse and its signaling cascades responsible for development are similar to those for the central synapses. However, the effect of lead exposure on the formation of NMJ in mammals is unclear. In the present study, a NG108-15/C2C12 coculture model was used to measure the acetylcholine receptor (AChR) aggregates formed on the myotubes which was an early hallmark for the NMJ formation. AChR aggregates were identified by alpha-bungarotoxin under fluorescent microscope. Single dose of lead acetate with final concentrations at 10(-3), 10(-1), or 10 microM was applied to dishes at the beginning of coculturing. Following 3-day exposure, although NG108-15 cells could extend long neurites to nearby myotubes, obvious dose-dependent attenuation in AChR aggregation was shown. The averaged area of an AChR aggregate, the averaged number of AChR aggregates per myotube, and the total area of AChR aggregates per myotube were all significantly decreased. In addition, the distribution percentages of various sizes of AChR aggregates showed that almost half of the AChR aggregates were formed with a size of 2-5 microm2 regardless of lead exposure. After treating 10 microM of lead acetate, significantly more AChR aggregates ranged from 2 to 20 microm2 were formed and significantly less AChR aggregates larger than 20 microm2 were formed. These results indicated that lead exposure reduced the extent of AChR aggregation concerning both the size and number of AChR aggregates and large AChR aggregates could hardly be formed after acute high-level lead exposure. No significant change was found in the total amount of AChRs on the myotubes after lead exposure, which indicated that the attenuation of AChR aggregation was not caused by reducing the synthesis of AChRs but by remaining dispersed pattern of AChRs on the myotubes. These data suggest that lead exposure exerts detrimental effects on the formation of NMJ.