Diquat Induces Cell Death and dopamine Neuron Loss via Reactive Oxygen Species Generation in Caenorhabditis elegans

Abstract

Diquat (DQ), a contact herbicide extensively utilized in both agricultural and nonagricultural domains, exhibits a high correlation with neuronal disorders. Nevertheless, the toxicity and underlying mechanisms associated with exposure to environmental concentrations of DQ remain ambiguous. Here, we report dose-dependent cellular neurotoxicity of DQ in Caenorhabditis elegans. First, DQ significantly compromised the development and brood size of worms, shortened the lifespan, and caused epidermal abnormalities. An unbiased transcriptomic analysis disclosed several pathways related to cell death and peroxisome homeostasis underlying this organismal-level toxicity. Moreover, exposure of DQ to C. elegans led to a notable increase of embryonic cell death. Concurrently, DQ exposure specifically caused the loss of dopamine neurons but not two other types of neurons in adulthood, which is in accordance with DQ-induced muscle-related defects such as pharyngeal pumping, body bends, and head thrashes. Mechanistically, DQ exposure induces the generation of reactive oxygen species (ROS) and enhances glutathione-related ROS scavenging pathway. Protein levels and activities of mitochondrial electron transport chain complexes were specifically impaired in the DQ-treated worms. Collectively, this study suggests an ROS-mediated cell death pathway involving the neuronal and behavioral toxicity of DQ, which offers a novel mitochondria-related perspective to elucidate the general toxicity caused by a widely distributed herbicide, DQ, at near-environment concentrations.

Keywords: C. elegans; ROS; cell death; diquat; oxidative damage.

Figure 1

Effects of DQ on the nervous system of C. elegans. (A) Dopamine neuron in F0; (B) serotonin neuron and glutamate neuron in F0; (C) head thrashes of F0–F2; (D) body bends of F0–F2; (E) pharyngeal pumping of F0–F2; and (F) AChE activity in F0. (C–E) Data are normalized to control groups. Error bars indicate ± SEM. p values are determined by the one-way ANOVA test and are compared with the control group. * Statistical significance at p < 0.05 and ** Statistical significance at p < 0.01.

Figure 2

Effects of DQ on transcriptional levels of C. elegans. (A) Numbers of up- or down-regulated genes; (B) KEGG signaling pathway diagram; and (C) RT-qPCR validation. C: Data are normalized to control groups. Error bars indicate ± SEM. p values are determined by t-test and are compared with the control group. * Statistical significance at p < 0.05 and ** Statistical significance at p < 0.01.

Figure 3

Effects of DQ on ectopic cell death in C. elegans embryos. (A,B) Number of cell corpses in embryos with indicated developmental stages and treatments. N2 strain are shown in A, while engulfment defect strain ced-1 (e1735) are shown in B. (C) Fluorescence images of apoptotic cells. (D) Quantification of apoptotic cell fluorescence, as shown in C. elegans. Error bars indicate ± SEM. * Statistical significance at p < 0.05 and ** Statistical significance at p < 0.01 by a one-way ANOVA test.

Figure 4

Effect of DQ on the reproduction of C. elegans. (A) Brood size of F0–F2; (B) embryo size of F0–F2; (C,D) gonad size in F0; (E) number of oocytes in uterus in F0; and (F) percentage of eggs laid in 3 h of F0. A, B, and E: Data are normalized to control groups. The treatment conditions are labeled in the graphs. Error bars indicate ± SEM. p values are determined by one-way ANOVA test and are compared with the control group. * Statistical significance at p < 0.05 and ** Statistical significance at p < 0.01.

Figure 5

Effects of DQ on oxidative stress in C. elegans. (A) Representative images of C. elegans that were stained for ROS or with a transgenic allele that expresses GST-4p:GFP; (B) quantification of ROS fluorescence in A; (C) quantification of GST-4p:GFP expression using the CL2166 reporter strain; (D) SOD activity; (E) CAT activity; (F) T-AOC; (G) GR activity; (H) GSH-Px activity; and (I) GSH/GSSG ratio. The treatment conditions are labeled in the graphs. Error bars indicate ± SEM. p values are determined by a one-way ANOVA test and are compared with the control group. * Statistical significance at p < 0.05 and ** Statistical significance at p < 0.01.

Figure 6

Effects of DQ on aging-like cellular damages in C. elegans. (A) Representative images of worms stained for lipofuscin; (B) quantification of lipofuscin fluorescence; (C) MDA levels; (D) protein carbonyls; and (E) 8-OHdG levels. Error bars indicate ± SEM. p values are determined by a one-way ANOVA test and are compared with the control group. * Statistical significance at p < 0.05 and ** Statistical significance at p < 0.01.

Figure 7

Effects of DQ on mitochondria in C. elegans. (A) Production of the mitochondrial superoxide anion (mitochondrial O^2̇–) was measured using MitoSOX Red with fluorescence microscopy. Red: MitoSOX Red and blue: DAPI (nucleus); (B) quantitation of the mitochondrial O^2˙– level based on the fluorescence intensity; (C) transmission electron microscopy images of mitochondria in C. elegans with indicated treatment; (D) MMP; (E) mitochondrial complex I activity; (F) Mitochondrial complex III activity. (G,H) Effect of DQ on the protein levels of mitochondrial ETC complex I–V in the human kidney epithelial HEK293 cell line. The bands corresponding to ETC complex I (NDUFB8), complex II (SDHB), complex III (UQCRC2), complex IV (COX II), and complex V (ATP5A) are labeled by indicated arrows. B: Data are normalized to control groups. Quantification of band intensities is shown in F. Error bars represent SEM, while statistical differences are determined by one-way ANOVA. *p < 0.05 and **p < 0.01.

Figure 8

Mitigation of DQ-induced ROS by antioxidant reagents. (A) ROS levels; (B–D) heat stress. (B) C. elegans simultaneously exposed to a mixture of DQ and antioxidants for 72 h; (C) C. elegans first exposed to DQ for 36 h, followed by exposure to antioxidants for 36 h; and (D) C. elegans first exposed to antioxidants for 36 h, followed by exposure to DQ for 36 h. Error bars indicate ± SEM p values are determined by a one-way ANOVA test and are compared with control group. * Statistical significance at p < 0.05 and ** Statistical significance at p < 0.01.