minocycline and diphenyleneiodonium

Minocycline is neuroprotective in animal models of a number of acute CNS injuries, neurodegenerative diseases and CNS infection. While anti-inflammatory and anti-apoptotic effects of Minocycline have been characterized, the molecular basis for the neuroprotective effects of Minocycline remains unclear. We report here that Minocycline and two classical antioxidant compounds inhibit the Japanese Encephalitis Virus (JEV)-induced free radical generation in mouse neuroblastoma. In cultures of Neuro2a (N2a) cells infected with JEV for up to 24h, the number of cells undergoing cell death was also reduced by Minocycline (20 microM). JEV infection resulted in increased oxidative stress, as revealed by an increase in the fluorescence intensity for 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate (CM-H2DCFDA), a reactive oxygen species (ROS) indicator. Minocycline at 20 microM inhibited this ROS production. Cells were moderately protected from JEV-induced death by diphenyleneiodonium (DPI), an inhibitor of flavon-containing enzyme inhibitor, whereas common antioxidants such as N-acetyl-cysteine (NAC) turned out to be ineffective. Direct antioxidant property of Minocycline and reference antioxidant compounds is evaluated by LDH assay, ROS measurement and mitochondrial membrane potential measurement. Our findings suggest that Minocycline reduces the neuronal damage seen in JEV infection in neuronal cell culture models at least in part through inhibition of oxidative stress.

Topics: Acetylcysteine; Animals; Anisotropy; Anti-Bacterial Agents; Antioxidants; Blotting, Western; Brain Neoplasms; Cell Death; Cell Line, Tumor; Encephalitis, Japanese; Enzyme Inhibitors; Free Radicals; In Situ Nick-End Labeling; L-Lactate Dehydrogenase; Membrane Fluidity; Membrane Potentials; Mice; Mice, Inbred BALB C; Minocycline; Mitochondrial Membranes; Neuroblastoma; Onium Compounds; Reactive Oxygen Species

Previous in vitro studies in our laboratory have shown that mancozeb (MZ) and maneb (MB), both widely used EBDC fungicides, are equipotent neurotoxicants that produce cell loss in mesencephalic dopaminergic and GABAergic cells after an acute 24h exposure. Mitochondrial uncoupling and inhibition were associated with fungicide exposure. Inhibition of mitochondrial respiration is known to increase free radical production. Here the mechanism(s) of neuronal damage associated with MZ exposure was further explored by determining the role that reactive oxygen species (ROS) played in toxicity. Damage to mesencephalic dopamine and GABA cell populations were significantly attenuated when carried out in the presence of ascorbate or SOD, indicative of a free radical-mediated contribution to toxicity. ROS generation monitored by hydrogen peroxide (H(2)O(2)) production using Amplex Red increased in a dose-dependent manner in response to MZ. Inhibition of intracellular catalase with aminotriazole had little effect on H(2)O(2) generation, whereas exogenously added catalase significantly reduced H(2)O(2) production, demonstrating a large extracellular contribution to ROS generation. Conversely, cells preloaded with the ROS indicator dye DCF showed significant MZ-induced ROS production, demonstrating an increase in intracellular ROS. Both the organic backbone of MZ as well as its associated Mn ion, but not Zn ion, were responsible and required for H(2)O(2) generation. The functionally diverse NADPH oxidase inhibitors, diphenylene iodonium chloride, apocynin, and 4-(2-aminoethyl)benzene-sulfonyl fluoride hydrochloride significantly attenuated H(2)O(2) production by MZ. In growth medium lacking cells, MZ produced little H(2)O(2), but enhanced H(2)O(2) generation when added with xanthine plus xanthine oxidase whereas, in cultured cells, allopurinol partially attenuated H(2)O(2) production by MZ. Minocycline, an inhibitor of microglial activation, modestly reduced H(2)O(2) formation in mesencephalic cells. In contrast, neuronal-enriched cultures or cultures treated with MAC-1-SAP to kill microglia, did not show an attenuation of ROS production. These findings demonstrate that Mn-containing EBDC fungicides such as MZ and MB can produce robust ROS generation that likely occurs via redox cycling with extracellular and intracellular oxidases. The findings further show that microglia may contribute to but are not required for ROS production by MZ.

Topics: Acetophenones; Allopurinol; Amitrole; Animals; Antioxidants; Ascorbic Acid; Catalase; Cells, Cultured; Dose-Response Relationship, Drug; Enzyme Inhibitors; Fungicides, Industrial; Hydrogen Peroxide; Maneb; Mesencephalon; Microglia; Minocycline; Molecular Structure; NADPH Oxidases; Neurons; Onium Compounds; Oxidation-Reduction; Oxidative Stress; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Structure-Activity Relationship; Sulfones; Superoxide Dismutase; Time Factors; Xanthine Oxidase; Zineb