Oxidative stress has been implicated in the pathogenesis of Parkinson disease based on its role in the cascade of biochemical changes that lead to dopaminergic neuronal death. This study analyzed the role of oxidative stress as a mechanism of the dopaminergic neurotoxicity produced by the combined paraquat and maneb model of the Parkinson disease phenotype. Transgenic mice overexpressing either Cu,Zn superoxide dismutase or intracellular glutathione peroxidase and non-transgenic mice were exposed to saline, paraquat, or the combination of paraquat ؉ maneb twice a week for 9 weeks. Non-transgenic mice chronically exposed to paraquat ؉ maneb exhibited significant reductions in locomotor activity, levels of striatal dopamine and metabolites, and dopaminergic neurons in the substantia nigra pars compacta. In contrast, no corresponding effects were observed in either Cu,Zn superoxide dismutase or glutathione peroxidase transgenic mice. Similarly, the increase in levels of lipid hydroperoxides in the midbrain and striatum of paraquat ؉ maneb-treated non-transgenic mice was not detected in either Cu,Zn superoxide dismutase or glutathione peroxidase transgenic mice. To begin to determine critical pathways of paraquat ؉ maneb neurotoxicity, the functions of cell death-inducing and protective mechanisms were analyzed. Even a single injection of paraquat ؉ maneb in the non-transgenic treated group modulated several key pro-and anti-apoptotic proteins, including Bax, Bad, Bcl-xL, and upstream stress-induced cascade. Collectively, these findings support the assertion that protective mechanisms against paraquat ؉ maneb-induced neurodegeneration could involve modulation of the level of reactive oxygen species and alterations of the functions of specific signaling cascades.Parkinson disease (PD) 1 is a neurodegenerative disorder resulting, in part, from the progressive loss of dopamine (DA) neurons in the substantia nigra pars compacta (SNpc). A lack of evidence for heritability of the idiopathic form of PD has pointed to environmental risk factors as potential contributors to the disease etiology. In accord with that possibility, exposures to environmental agents have been linked repeatedly with the development of PD (1-9). Animal models, epidemiological studies, and clinical case reports have repeatedly identified exposure to the herbicide paraquat (PQ) and the fungicide maneb (MB) as potential risk factors for the PD phenotype (1, 9 -15). Our laboratories have demonstrated extensively that exposure to the combination of PQ ϩ MB produces a potentiated loss of dopaminergic neurons in the SNpc of mice compared with either compound alone (16). This model exemplifies the "multiple hit" hypothesis associated with PD. Furthermore, the neurotoxicity of PQ ϩ MB seems to be highly selective to the nigrostriatal dopaminergic system. Additionally, we have shown that exposure during critical periods of development and during advanced age as well as gender and genetic background all modify the susceptibility and response of dopaminergic...

We report the identification and characterization of a new ischemia/reperfusion-inducible protein (IRIP), which belongs to the SUA5/YrdC/YciO protein family. IRIP cDNA was isolated in a differential display analysis of an ischemia/reperfusion-treated kidney RNA sample. Mouse IRIP mRNA was expressed in all tissues tested, the highest level being in the testis, secretory, and endocrine organs. Besides ischemia/reperfusion, endotoxemia also activated the expression of IRIP in the liver, lung, and spleen. The transporter regulator RS1 was identified as an IRIP-interacting protein in yeast two-hybrid screening. The interaction between IRIP and RS1 was further confirmed in coimmunoprecipitation assays. A possible role of IRIP in regulating transporter activity was subsequently investigated. IRIP overexpression inhibited endogenous 1-methyl-4-phenylpyridinium (MPP ؉ ) uptake activity in HeLa cells. The activities of exogenous organic cation transporters (OCT2 and OCT3), organic anion transporter (OAT1), and monoamine transporters were also inhibited by IRIP. Conversely, inhibition of IRIP expression by small interfering RNA or antisense RNA increased MPP ؉ uptake. We measured transport kinetics of OCT2-mediated uptake and demonstrated that IRIP overexpression significantly decreased V max but did not affect K m . On the basis of these results, we propose that IRIP regulates the activity of a variety of transporters under normal and pathological conditions.

Stroke is one of the leading causes of death in major industrial countries. Many factors contribute to the cellular damage resulting from ischemia/reperfusion (I/R). Experimental data indicate an important role for oxidative stress and the inflammatory cascade during I/R. We are testing the hypothesis that the mechanism of protection against I/R damage observed in transgenic mice overexpressing human antioxidant enzymes (particularly intracellular glutathione peroxidase) involves the modulation of inflammatory response as well as reduced sensitivity of neurons to cytotoxic cytokines. Transgenic animals show significant reduction of expression of chemokines, IL-6, and cell death-inducing ligands as well as corresponding receptors in a focal cerebral I/R model. Reduction of DNA binding activity of consensus and potential AP-1 binding sites in mouse Fas ligand promoter sequence was observed in nuclear extracts from transgenic mice overexpressing intracellular glutathione peroxidase compared with normal animals following I/R. This effect was accompanied by modulation of the c-Jun N-terminal kinase/stress-activated protein kinase pathway. Cultured primary neurons from the transgenic mice demonstrated protection against hypoxia/reoxygenation injury as well as cytotoxicity after TNF-α and Fas ligand treatment. These results indicate that glutathione peroxidase-sensitive reactive oxygen species play an important role in regulation of cell death during cerebral I/R by modulating intrinsic neuronal sensitivity as well as brain inflammatory reactions.

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Abstract-In response to endotoxemia induced by administration of lipopolysaccharide, a complex series of reactions occurs in mammalian tissues. During this inflammation response, cells produce different mediators, such as reactive oxygen species, a number of arachidonic acid metabolites, and cytokines. The reactive oxygen species thus generated have been suggested to produce tissue injury as a result of macromolecular damage or by interfering with regulatory processes. They may also act as important signaling molecules to induce redox-sensitive genes. We report here that transgenic mice overexpressing 2 major forms of human glutathione peroxidases (GPs), intra-and extracellular GP, are able to modulate host response during endotoxemic conditions. We show that these animals have a decreased hypotension and increased survival rate after administration of a high dosage of lipopolysaccharide. Overexpression of GPs alters vascular permeability and production of cytokines (interleukin-1␤ and tumor necrosis factor-␣) and NO, affects arachidonic acid metabolism, and inhibits leukocyte migration. These results suggest an important role for peroxides in pathogenesis during endotoxemia, and GPs, by regulating their level, may prove to be good candidates for antioxidant therapy to protect against such injury. 2 There is experimental evidence that suggests oxidative damage in pathogenesis of endotoxemia. 3 To evaluate the role of glutathione peroxidase (GP) in protection against endotoxemia in vivo, we have used transgenic mice overexpressing human extracellular (GPxP) and intracellular (GPx1) GP. In the present study, we report that these animals show an increased rate of survival after administration of a large dosage of LPS. Mice with human GPs are able to modulate levels of lipid peroxidation and inflammatory mediators. These transgenic animals represent a new model system for investigation of the role of GPs and reactive oxygen species (ROS) in endotoxemia and other inflammatory disorders. Materials and Methods Animals and TreatmentsHeterozygous transgenic mice 5 to 6 months old in a C57BL/ 6ϫCBA/J background overexpressing human GPxP (strain 17) and human GPx1 (strain 23) and their normal littermates were bred in our facility. We described the generation of these mice previously. 4 For survival studies, the mice were injected with LPS at a dose of 25 mg/kg (Escherichia coli serotype 0111:B4; Sigma). Arterial blood pressure and heart rate were measured using the Kent tail blood pressure system. For characterization of cytokine production and leukocyte distribution, blood was collected from the retro-orbital cavity of a group of animals dosed with LPS (16 mg/kg of body weight). Plasma was analyzed for cytokine (tumor necrosis factor-␣ [TNF-␣] and interleukin-1␤ [IL-1␤]) production with ELISA kits from R&D Systems. Vascular permeability was determined by assessing tissue accrual of Evan's Blue, as described previously. 5 Sixteen hours after PBS or LPS injection, the animals were administered 25 mg/kg Evan's Blue by tail ...