minocycline and acetovanillone

Topics: Acetophenones; Drug Delivery Systems; Humans; Microglia; Minocycline; Nervous System Diseases; Plant Preparations

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

Blood-brain barrier (BBB) disruption after stroke can worsen ischemic injury by increasing edema and causing hemorrhage. We determined the effect of microglia on the BBB and its primary constituents, endothelial cells (ECs) and astrocytes, after ischemia using in vivo and in vitro models.. Primary astrocytes, ECs, or cocultures were prepared with or without added microglia. Primary ECs were more resistant to oxygen-glucose deprivation/reperfusion than astrocytes. ECs plus astrocytes showed intermediate vulnerability. Microglia added to cocultures nearly doubled cell death. This increase was prevented by minocycline and apocynin. In vivo, minocycline reduced infarct volume and neurological deficits and markedly reduced BBB disruption and hemorrhage in mice after experimental stroke.. Inhibition of microglial activation may protect the brain after ischemic stroke by improving BBB viability and integrity. Microglial inhibitors may prove to be an important treatment adjunct to fibrinolysis.

Topics: Acetophenones; Animals; Antioxidants; Astrocytes; Blood-Brain Barrier; Brain; Brain Ischemia; Cell Death; Cells, Cultured; Cerebral Hemorrhage; Cerebral Infarction; Coculture Techniques; Endothelial Cells; Glucose; Hydrogen Peroxide; Hypoxia; Male; Mice; Mice, Inbred C57BL; Microglia; Minocycline; Nervous System Diseases; Superoxides; Tumor Necrosis Factor-alpha