Effect of paraquat exposure on nitric oxide-responsive genes in rat mesencephalic cells.

When neural cells are exposed to paraquat, nitric oxide generation increases primarily due to an increase in the expression of the inducible isoform of nitric oxide synthase. The nitric oxide generated has controversial actions in paraquat exposure, as both protective and harmful effects have been described previously. While the actions mediated by nitric oxide in neural cells have been well described, there is evidence that nitric oxide may also be an important modulator of the expression of several genes during paraquat exposure. To better understand the actions of nitric oxide and its potential role in paraquat-induced gene expression, we examined changes in GCH1, ARG1, ARG2, NOS1, NOS2, NOS3, NOSTRIN, NOSIP, NOS1AP, RASD1, DYNLL1, GUCY1A3, DDAH1, DDAH2 and CYGB genes whose expression is controlled by or involved in signaling by the second messenger nitric oxide, in rat mesencephalic cells after 3, 6, 12 and 24h of paraquat exposure. A qPCR strategy targeting these genes was developed using a SYBR green I-based method. The mRNA levels of all the genes studied were differentially regulated during exposure. These results demonstrate that nitric oxide-related genes are regulated following paraquat exposure of mesencephalic cells and provide the basis for further studies exploring the physiological and functional significance of nitric oxide-sensitive genes in paraquat-mediated neurotoxicity.

Nitric oxide in paraquat-mediated toxicity: A review.

Paraquat, a cationic herbicide, produces degenerative lesions in the lung and in the nervous system after systemic administration to man and animals. Many cases of acute poisoning and death have been reported over the past few decades. Although a definitive mechanism of toxicity of paraquat has not been delineated, a cyclic single electron reduction/oxidation is a critical mechanistic event. The redox cycling of paraquat has two potentially important consequences relevant to the development of toxicity: the generation of the superoxide anion, which can lead to the formation of more toxic reactive oxygen species which are highly reactive to cellular macromolecules; and the oxidation of reducing equivalents (e.g., NADPH, reduced glutathione), which results in the disruption of important NADPH-requiring biochemical processes necessary for normal cell function. Nitric oxide is an important signaling molecule that reacts with superoxide derived from the paraquat redox cycle, to form the potent oxidant peroxynitrite, which causes serious cell damage. Although nitric oxide has been involved in the mechanism of paraquat-mediated toxicity, the role of nitric oxide has been controversial as both protective and harmful effects have been described. The present review summarizes recent findings in the field and describes new knowledge on the role of nitric oxide in the paraquat-mediated toxicity.

Protective effect of the glial cell line-derived neurotrophic factor (GDNF) on human mesencephalic neuron-derived cells against neurotoxicity induced by paraquat.

Paraquat is a cationic herbicide that causes acute cell injury by undergoing redox cycling. Oxidative stress is thought to be the crucial mechanism invoked by this redox-cycling compound. The cytotoxicity of paraquat was examined in an immortalized human mesencephalic neuron-derived cell line. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide reduction activity was examined as cytotoxicity indicator. Cells were seeded with densities at inoculation of 5 × 10(4)cells/ml and 10 × 10(4)cells/ml, and paraquat was added 24h later to give final concentrations from 10 to 500 µM. At 24 and 48 h of treatment, mitochondrial activity was determined with the MTT assay. To further understand the effect of paraquat exposure on human mesencephalic neuron-derived cells, the cells were differentiated and similar experiments were carried out. Supplementation of culture medium with dibutyryl cyclic AMP and GDNF significantly increased the resistance of the cultures to the paraquat-mediated cytotoxicity. These results confirm that GDNF confers protection against paraquat-mediated cytotoxicity and show that immortalized human mesencephalic neuron-derived cells are an adequate in vitro system for evaluating the cytoprotective effects of GDNF on oxidative injury caused by xenobiotics.

Paraquat exposure induces nuclear translocation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the activation of the nitric oxide-GAPDH-Siah cell death cascade.

Paraquat (PQ) is a well-known herbicide that exerts its effects by elevating intracellular levels of superoxide. It has been previously demonstrated that oxidative and nitrosative stress participate to PQ-induced cell death. Here, we document that PQ increases the levels of nitric oxide (NO) in rat mesencephalic cells and causes nuclear translocation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) to activate the NO/GAPDH/Siah cell death cascade. PQ exposure increases expression of the p300/CREB-binding protein (p300/CBP) and phosphorylation of p53 at Ser 15, which stimulates p53-dependent transactivation through increased binding with p300. Although this cascade could be inhibited by preincubation with the monoamine oxidase B inhibitor deprenyl, cell death was not prevented. Pretreatment of cells with the neuronal nitric oxide synthase inhibitor 7-nitroindazole efficiently prevented the activation of the GAPDH/NO/Siah cell death cascade, thereby protecting cells against PQ-induced toxicity. The results suggest that PQ induces this novel cell death cascade in rat mesencephalic cells, but inhibition of the pathway does not impede cell death because of an oxidative burst generated by the pesticide.

Curcumin exposure induces expression of the Parkinson's disease-associated leucine-rich repeat kinase 2 (LRRK2) in rat mesencephalic cells.

Turmeric (curry powder), an essential ingredient of culinary preparations of Southeast Asia, contains a major polyphenolic compound known as curcumin or diferuloylmethane. Curcumin is a widely studied phytochemical with a variety of biological activities. In addition to its anti-inflammatory and antimicrobial/antiviral properties, curcumin is considered as a cancer chemopreventive agent as well as a modulator of gene expression and a potent antioxidant. Since oxidative stress has been implicated in the degeneration of dopaminergic neurons in the substantia nigra in Parkinson's disease (PD), curcumin has been proposed to have potential therapeutic value for the treatment of neurodegenerative diseases such as PD. Following age, a family history of PD is the most commonly reported risk factor, suggesting a genetic component of the disease in a subgroup of patients. The LRRK2 gene has emerged as the gene most commonly associated with both familial and sporadic PD. Here, we report that exposure of rat mesencephalic cells to curcumin induces the expression of LRRK2 mRNA and protein in a time-dependent manner. The expression of other PD-related genes, such alpha-synuclein and parkin, was not affected by exposure to curcumin, and PTEN-induced putative kinase 1 (PINK1) was not expressed in rat mesencephalic cells. As LRRK2 overexpression is strongly associated with the pathological inclusions found in several neurodegenerative disorders, further studies are needed to evaluate the effects of curcumin as a therapeutic agent for neurodegenerative diseases.

[Genetic screening for the LRRK2, G2019S and R1441 codon mutations in Parkinson's disease patients from Extremadura, Spain]. / Prevalencia de las mutaciones G2019S y R1441 de LRRK2 en pacientes con enfermedad de Parkinson en Extremadura, España.

INTRODUCTION: LRRK2 mutations have been described as a common cause of Parkinson's disease (PD) in patients from northern Spain. Here we investigated the prevalence of these mutations in a cohort of Spanish PD patients (n = 96) from Extremadura, a region in southwestern Spain. SUBJECTS AND METHODS: To evaluate the rate of the G2019S and R1441G/C/H LRKK2 mutations in PD patients and healthy controls (n = 163). RESULTS: Here we show that the G2019S mutation is present at a low prevalence in our Spanish cohort, while the R1441G/C/H mutation, which has been reported to be common in northern Spain, was not observed in the PD patients or in the controls. CONCLUSION: LRRK2 mutations do not appear to be a common cause of Parkinson's disease in Extremadura, Spain.

Prevalencia de las mutaciones G2019S y R1441 de LRRK2 en pacientes con enfermedad de Parkinson en Extremadura, España / Genetic screening for the LRRK2, G2019S and R1441 codon mutations in Parkinson’s disease patients from Extremadura, Spain

Introducción. Las mutaciones en el gen que codifica para la proteína LRRK2 se han asociado frecuentemente con la enfermedad de Parkinson (EP) en pacientes en el norte de España. En este artículo se determina la prevalencia de algunas de las mutaciones más frecuentes en una cohorte de pacientes con EP (n = 96) en Extremadura, región situada en el suroeste de España. Sujetos y métodos. Se pretende determinar la presencia de las mutaciones G2019S y R1441G/C/H de LRRK2 en pacientes con EP y en individuos sanos (n = 196). Resultados. La mutación G2019S se presenta con una baja prevalencia en nuestra muestra, mientras que las mutaciones R1441G/C/H, frecuentes en el norte de España, no se han detectado en la muestra de enfermos ni en los controles. Conclusión. Las mutaciones en LRRK2 no parecen ser una causa frecuente de la EP en Extremadura (AU)

Introduction. LRRK2 mutations have been described as a common cause of Parkinson’s disease (PD) in patients from northern Spain. Here we investigated the prevalence of these mutations in a cohort of Spanish PD patients (n = 96) from Extremadura, a region in southwestern Spain. Subjects and methods. To evaluate the rate of the G2019S and R1441G/C/H LRKK2 mutations in PD patients and healthy controls (n = 163). Results. Here we show that the G2019S mutation is present at a low prevalence in our Spanish cohort, while the R1441G/C/H mutation, which has been reported to be common in northern Spain, was not observed in the PD patients or in the controls. Conclusion. LRRK2 mutations do not appear to be a common cause of Parkinson’s disease in Extremadura, Spain (AU)