No abstract

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 24 h 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 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. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. NIH Public Access Author ManuscriptNeurotoxicology. Author manuscript; available in PMC 2008 November 1. The findings further show that microglia may contribute to but are not required for ROS production by MZ.