Glyphosate-containing herbicides are among the most widely-used in the world. Although glyphosate itself is relatively non-toxic, growing evidence suggests that commercial herbicide formulations may lead to increased oxidative stress and mitochondrial inhibition. In order to assess these mechanisms in vivo, we chronically (24h) exposed Caenorhabditis elegans to various concentrations of the glyphosate-containing herbicide TouchDown (TD). Following TD exposure, we evaluated the function of specific mitochondrial electron transport chain complexes. Initial oxygen consumption studies demonstrated inhibition in mid- and high-TD concentration treatment groups compared to controls. Results from tetramethylrhodamine ethyl ester and ATP assays indicated reductions in the proton gradient and ATP levels, respectively. Additional studies were designed to determine whether TD exposure resulted in increased reactive oxygen species (ROS) production. Data from hydrogen peroxide, but not superoxide or hydroxyl radical, assays showed statistically significant increases in this specific ROS. Taken together, these data indicate that exposure of Caenorhabditis elegans to TD leads to mitochondrial inhibition and hydrogen peroxide production.

Glyphosate-based herbicides, such as Touchdown (TD) and Roundup, are among the most heavilyused herbicides in the world. While the active ingredient is generally considered non-toxic, the toxicity resulting from exposure to commercially-sold formulations is less clear. In many cases, cell cultures or various model organisms exposed to glyphosate formulations show toxicity and, in some cases, lethality. Using Caenorhabditis elegans, we assessed potential toxic mechanisms through which a highly-concentrated commercial formulation of TD promotes neurodegeneration. Following a 30-minute treatment, we assayed mitochondrial electron transport chain function and reactive oxygen species (ROS) production. Initial oxygen consumption studies indicated general mitochondrial inhibition compared to controls (*p < 0.05). When Complex II activity was further assessed, inhibition was observed in all TD-treated groups (*p < 0.05). Complex IV activity, however, was not adversely affected by TD. This electron transport chain inhibition also resulted in reduced ATP levels (*p < 0.05). Furthermore, hydrogen peroxide levels, but not other ROS, were increased (*p < 0.05). Taken together, these data indicate that commercially-available formulations of TD may exert neurotoxicity through Complex II (succinate dehydrogenase) inhibition, decreased ATP levels, and increased hydrogen peroxide production.

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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.