Acute exposure to a Mn/Zn ethylene-bis-dithiocarbamate fungicide leads to mitochondrial dysfunction and increased reactive oxygen species production in Caenorhabditis elegans

Abstract

Mn/Zn ethylene-bis-dithiocarbamate (Mn/Zn-EBDC) fungicides are among some the most widely-used fungicides in the world. Although they have been available for over 50 years, little is known about their mechanism of action in fungi, or their potentially toxic mechanisms in humans. To determine if exposure of Caenorhabditis elegans (C. elegans) to a representative fungicide (Manzate; MZ) from this group inhibits mitochondria or produces reactive oxygen species (ROS), we acutely (30 min) exposed worms to various MZ concentrations. Initial oxygen consumption studies showed an overall statistically significant decrease in oxygen consumption associated with addition of Complex I- and/or II-substrate in treatment groups compared to controls (*p < 0.05). In order to better characterize the individual complex activity, further studies were completed that specifically assessed Complex II or Complex IV. Data indicated that neither of these two complexes were targets of MZ treatment. Results from tetramethylrhodamine ethyl ester (proton gradient) and ATP assays showed statistically significant reductions in both endpoints (*p < 0.05, **p < 0.01, respectively). Additional studies were completed to determine if MZ treatment also resulted in increased ROS production. These assays provided evidence that hydrogen peroxide, but not superoxide or hydroxyl radical levels were statistically significantly increased (*p < 0.05). Taken together, these data indicate exposure of C. elegans to MZ concentrations to which humans are exposed leads to mitochondrial inhibition and concomitant hydrogen peroxide production. Since mitochondrial inhibition and increased ROS are associated with numerous neurodegenerative diseases, we suggest further studies to determine if MZ catalyzes similar toxic processes in mammals.

Cell Biology of the Mitochondrion

“…Studying whole nematodes, Fitsanakis and colleagues showed that Mn ++ exposure led to defective mitochondrial complex I function, with an increase in hydrogen peroxide concentrations (Bailey et al 2016;Todt et al 2016). In response to the increase in hydrogen peroxide, the expression of a glutathione S-transferase reporter (Pgst-4::gfp) was also increased in the exposed animals.…”

Chronic exposure to a glyphosate-containing pesticide leads to mitochondrial dysfunction and increased reactive oxygen species production in Caenorhabditis elegans

“…Protocols for synchronization, treatment, and endpoint assays used in our lab were previously published in detail (Bailey et al , 2016; Todt et al , 2016). Briefly, C. elegans were grown at 20°C on 8P plates (51.3 mM NaCl, 25.0 g bactoagar/L, 20.0 g bactopeptone/L, 1 mM CaCl 2 , 500 µM KH 2 PO 4 (pH 6), 13 µM cholesterol (95% ethanol), and 1 mM MgSO 4 ) with a lawn of NA22 E. coli (grown in 16 g tryptone/L, 10 g yeast extract/L, 85.5 mM NaCl) until gravid.…”

“…All endpoint assays have been previously described in detail (Todt et al , 2016). Briefly, all treatment groups were standardized to yield 10,000 live worms/mL/treatment concentration.…”

“…Afterwards, worms were incubated for 15 min with the luciferin-based ATP probe. Luminescence was measured as previously reported (Todt et al , 2016).…”

Exposure of Drosophila melanogaster to Mancozeb Induces Oxidative Damage and Modulates Nrf2 and HSP70/83

“…This condition may lead to oxidative damage to biomolecules such as proteins, lipids, and nucleic acids [ 9 ] and is implicated in the etiology of several diseases, such as tumors and neurodegenerative disorders [ 10 – 12 ]. A possible cause of the increment of ROS levels by MZ is the inhibition of the mitochondrial complexes activity, resulting in an inefficient transfer of electrons, leading to the formation of oxygen radicals [ 13 , 14 ]. Also, induction of NADPH oxidase and xanthine oxidase by MZ leading to augmented H 2 O 2 has been reported [ 15 ], and modulation of antioxidant enzyme activity, as published recently by our research group, has been reported in carp brain [ 16 ].…”