Comparative analysis of in vitro neurotoxicity of methylmercury, mercury, cadmium, and hydrogen peroxide on SH-SY5Y cells.

Mercury (Hg) and cadmium (Cd) are the major toxic heavy metals and are known to induce neurotoxicity. Although many studies have shown that several heavy metals have neurotoxic effects, the cellular and molecular mechanisms thereof are still not clear. Oxidative stress is reported to be a common and important mechanism in cytotoxicity induced by heavy metals. However, the assays for identifying toxic mechanisms were not performed under the same experimental conditions, making it difficult to compare toxic properties of the heavy metals. In this study, we investigated the mechanisms underlying neurotoxicity induced by heavy metals and H2O2, focusing on cell death, cell proliferation, and oxidative stress under the same experimental condition. Our results showed that MeHg caused lactate dehydrogenase (LDH) release, caspase activation and cell-cycle alteration, and ROS generation in accordance with decreased cell viability. HgCl2 caused LDH release and cell-cycle alteration, but not caspase activation. CdCl2 had a remarkable effect on the cell cycle profiles without induction of LDH release, caspase activation, or ROS generation. Pretreatment with N-acetyl-l-cysteine (NAC) prevented the decrease in cell viability induced by MeHg and HgCl2, but not CdCl2. Our results demonstrate a clear difference in neurotoxic mechanisms induced by MeHg, HgCl2, CdCl2 or H2O2 in SH-SY5Y cells. Elucidating the characteristics and mechanisms of each heavy metal under the same experimental conditions will be helpful to understand the effect of heavy metals on health and to develop a more effective therapy for heavy metal poisoning.

Rho-kinase and the nitric oxide pathway modulate basilar arterial reactivity to acetylcholine and angiotensin II in streptozotocin-induced diabetic mice.

Diabetes mellitus comprises a heterogeneous group of metabolic disorders with underlying hyperglycemia and secondary cardiovascular complications. Growing evidence suggests that vascular dysfunction is among the most important causes of diabetic cardiovascular disease. Therefore, we determined whether streptozotocin (STZ)-induced diabetes in mice affects blood pressure and cerebral arterial responsiveness to angiotensin (Ang) II and acetylcholine (ACh), which are important modulators of cerebrovascular autoregulation. Diabetes was induced using a single intraperitoneal injection of STZ (50 mg/kg). Blood pressure was measured in conscious mice using the indirect tail-cuff method. Functional studies of the isolated arteries' response to vasoactive substances were performed using a micro-organ-bath system at 60 days after STZ injection. Systolic, diastolic, and mean blood pressures significantly increased at days 45 and 60 in the STZ-induced diabetic mice. In the isolated basilar arteries, ACh-induced relaxation, which is dependent on nitric oxide (NO) production from endothelial cells, decreased. In contrast, Ang II-induced contraction, mediated via rho-kinase activation in the smooth muscle, increased in the diabetic mice. There was significantly greater relaxation in the precontracted isolated basilar arteries of diabetic mice that had been treated with Y27632, a rho-kinase inhibitor, than in the control mice arteries. Pretreatment with Nω-nitro-L-arginine (L-NAME), an NO synthase inhibitor, significantly enhanced Ang II-induced contraction and Y27632-induced relaxation in the control basilar arteries but not in the STZ-induced diabetic mice arteries. These results suggest that decreased NO bioavailability and enhanced rho-kinase activity in basilar arteries contribute to altered reactivity to ACh and Ang II, respectively, in STZ-induced diabetic mice.

MARCKS is involved in methylmercury-induced decrease in cell viability and nitric oxide production in EA.hy926 cells.

Methylmercury (MeHg) is a persistent environmental contaminant that has been reported worldwide. MeHg exposure has been reported to lead to increased risk of cardiovascular diseases; however, the mechanisms underlying the toxic effects of MeHg on the cardiovascular system have not been well elucidated. We have previously reported that mice exposed to MeHg had increased blood pressure along with impaired endothelium-dependent vasodilation. In this study, we investigated the toxic effects of MeHg on a human endothelial cell line, EA.hy926. In addition, we have tried to elucidate the role of myristoylated alanine-rich C kinase substrate (MARCKS) in the MeHg toxicity mechanism in EA.hy926 cells. Cells exposed to MeHg (0.1-10 µM) for 24 hr showed decreased cell viability in a dose-dependent manner. Treatment with submaximal concentrations of MeHg decreased cell migration in the wound healing assay, tube formation on Matrigel and spontaneous nitric oxide (NO) production of EA.hy926 cells. MeHg exposure also elicited a decrease in MARCKS expression and an increase in MARCKS phosphorylation. MARCKS knockdown or MARCKS overexpression in EA.hy926 cells altered not only cell functions, such as migration, tube formation and NO production, but also MeHg-induced decrease in cell viability and NO production. These results suggest the broad role played by MARCKS in endothelial cell functions and the involvement of MARCKS in MeHg-induced toxicity.

Pharmacological characteristics of Artemisia vulgaris L. in isolated porcine basilar artery.

ETHNOPHARMACOLOGICAL RELEVANCE: In Vietnamese traditional herbalism, there are conflicting opinions about the effect of Artemisia vulgaris L. (AVL, English name: mugwort) on hypertension. Some ethnic doctors recommend the use of AVL for treatment of hypertension, whereas others advise against it. The purpose of this study was to clarify the pharmacological characteristics of AVL in isolated arteries to explain the conflicts surrounding the use of AVL for treatment of hypertension. MATERIALS AND METHODS: We initially performed a functional study using an organ bath system to investigate the effect of AVL extract on isolated porcine basilar artery. We then measured the change in intracellular free Ca(2+) concentration elicited by AVL using cultured smooth muscle cells loaded with the Ca(2+) indicator fluo-4. Finally, using HPLC, we determined the active components in AVL. RESULTS AND DISCUSSION: AVL induced vasoconstriction at resting tension, and endothelial removal enhanced this effect significantly. Pretreatment with PD123319 (an AT2 receptor antagonist), Nω-nitro-L-arginine (a nitric oxide synthase inhibitor), or both, also enhanced this effect. AVL-induced contraction was competitively inhibited by methiothepin (a 5-HT1 and 5-HT2 receptor antagonist) in the presence of ketanserin (a 5-HT2 receptor antagonist). Removal of extracellular calcium with nifedipine (an L-type Ca(2+) channel blocker) or ruthenium red (a ryanodine receptor blocker) significantly reduced AVL-induced contraction, whereas losartan (an AT1 receptor antagonist) and diphenhydramine (a H1 receptor antagonist) had no effect on this contraction. AVL increased the intracellular free Ca(2+) concentration in cultured cells, and this increment was inhibited by methiothepin. HPLC analysis revealed that the retention time of the first peak in the AVL profile was similar to that of the 5-HT standard, and that addition of 5-HT to the AVL sample enhanced this peak. On the other hand, AVL induced endothelium-independent relaxation under precontracted conditions with 60mM KCl. Captopril (an angiotensin converting enzyme inhibitor), atenolol (a ß1 receptor antagonist) and cimetidine (a H2 receptor antagonist) had no effect on this relaxation. In Ca(2+)-free 60mM KCl-containing solution, pretreatment with AVL significantly inhibited CaCl2-induced contraction. CONCLUSION: For the first time, the present study has demonstrated that AVL has two opposite effects, contraction and relaxation, on isolated artery, which may help to explain the conflicting indications for AVL in traditional herbalism. 5-HT is a significant factor affecting artery contraction in the presence of AVL.

Methylmercury affects cerebrovascular reactivity to angiotensin II and acetylcholine via Rho-kinase and nitric oxide pathways in mice.

AIMS: Methylmercury (MeHg) exposure results in increased risk of hypertension and cardiovascular diseases. In this study, we aimed to investigate whether the in vivo exposure of MeHg in mice affects blood pressure and basilar arterial responses to angiotensin II (Ang II) and acetylcholine (ACh), which are important modulators of cerebrovascular autoregulation. MAIN METHODS: Mice were exposed to MeHg (40ppm) in drinking water for 21days. Blood pressure was measured in conscious mice by an indirect tail-cuff method. Functional studies of the isolated arteries' response to vasoactive substances were performed using a micro-organ-bath system. KEY FINDINGS: Systolic and mean blood pressures were significantly increased after 2 and 3weeks of treatment with MeHg, respectively. Ang II-induced contraction in an isolated basilar artery, which is mediated via Rho-kinase activation, was increased in MeHg-treated mice. ACh-induced relaxation, which is dependent on NO production from the endothelial cells, was decreased in MeHg-treated mice. However, alterations of vascular responses to Ang II and ACh were not observed in the isolated thoracic aorta. SIGNIFICANCE: This study demonstrated that the cerebral vasculature appears to be particularly sensitive to in vivo exposure of MeHg. Our results suggest that in vivo MeHg increases blood pressure and causes alterations in the cerebrovascular reactivity in response to Ang II and ACh through enhancement of Rho-kinase activity and inhibition of NO bioavailability, respectively.