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

Abstract

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.

Keywords: C. elegans; Glyphosate; Herbicide; Hydrogen peroxide; Mitochondrial inhibition; Reactive oxygen species.

Figure 1

Decreased mitochondrial respiration following treatment with TD. Following chronic treatment with TD, worms in the mid- and high-concentration groups consistently showed a statistically significant decrease in oxygen consumption. Data are presented as mean percent oxygen consumed and represent N ≥ 4 separate synchronizations. ***p < 0.001, or ****p < 0.001 compared to controls.

Figure 2

Proton gradient integrity in TD-treated worms. Following treatment with TD, ANOVA indicated a statistically significant decrease in number of red pixels associated with worms in the highest treatment group. Although not statistically significant, a trend (p = 0.0625) towards a decrease in fluorescence was observed in the 2.7% group. Data are presented as mean pixel number ± SEM and represent N ≥ 3 separate synchronizations. ***p < 0.001 compared to control.

Figure 3

Relative ATP amount following TD treatment of worms. One-way ANOVA indicated no statistically significant decrease in luminescence (A), a measure of ATP, even though viability in the same group was statistically significantly lower (B). Data are presented as mean intensity ± SEM and represent N ≥ 3 separate synchronizations. *p < 0.05 compared to control worms.

Figure 4

Use of fluorescence to assess ROS production following TD treatment of worms. In order to assess whether TD exposure could lead to increased oxidative stress, worms were exposed to DHE, to assess superoxide production (A). No statistically significant changes in DHE fluorescence was observed in groups compared to control. Chronic treatment with 5.5% or 9.8% TD resulted in a statistically significant decrease in fluorescence of AmplexRed, a probe used to detect H₂O₂ (B). Data are presented as mean intensity ± SEM and represent N ≥ 3 separate synchronizations. ****p < 0.001 compared to control.

Figure 5

Increased GST-4::GFP fluorescence following treatment with TD. Transgenic worms treated with the mid- and high-level concentrations of TD showed statistically significant increases in green fluorescence compared to control worms. Data are presented as mean intensity ± SEM and represent N ≥ 4 separate synchronizations. ***p <0.001 compared to controls.