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Pendimethalin exposure disrupts mitochondrial function and impairs processes related to implantation
in Reproduction*(M Kim and J Park contributed equally to this work)
In brief
Pendimethalin as a dinitroaniline herbicide is used to eliminate weeds during the cultivation of various crops such as grains, fruits, and vegetables. This study reveals that pendimethalin exposure at various concentrations led to disruption in Ca2+ homeostasis and mitochondrial membrane potential, as well as dysregulation of the mitogen-activated protein kinase signaling pathway and implantation-related genes in porcine trophectoderm and uterine luminal epithelial cells.
Abstract
The use of herbicides is a major control method in agriculture. Pendimethalin (PDM) has been increasingly used as a herbicide for approximately 30 years. PDM has been reported to cause various reproductive problems, but its toxicity mechanism in the pre-implantation stage has not been investigated in detail. Herein, we studied the effects of PDM on porcine trophectoderm (pTr) and uterine luminal epithelial (pLE) cells and identified a PDM-mediated anti-proliferative effect in both cell types. PDM exposure generated intracellular reactive oxygen species, induced excessive Ca2+ influx into mitochondria, and activated mitogen-activated protein kinase signaling pathway. Ca2+ burden resulted in the dysfunction of mitochondria and eventual disruption of Ca2+ homeostasis. Further, PDM-exposed pTr and pLE cells showed cell cycle arrest and programmed cell death. In addition, a decrease in migration ability and dysregulated expression of genes related to the functioning of pTr and pLE cells was evaluated. This study provides insight into time-dependent transitions within the cell environment after PDM exposure and elucidates a detailed mechanism of induced adverse effects. These results imply that PDM exposure can potentially cause toxic effects on the implantation-related process in pigs. Moreover, to the best of our knowledge, this is the first study to describe the mechanism by which PDM induces these effects, enhancing our understanding of the toxicity of this herbicide.
Supplementary Materials
- Supplementary Figure 1. FACS gating strategies of cell cycle analysis using PI staining. [A] pTr and [B] pLE cells, excluding debris, were gated into the P5 group. Gated cells were confirmed by measurement to be single cells.
- Supplementary Figure 2. FACS gating strategies and analysis of apoptosis using annexin V/PI. [A] The gating strategy used for pTr and pLE cells. Cells were gated without debris to faciliate analysis of apoptosis. [B] Absence of autofluorescence in pTr and pLE cells was confirmed via detection of cells without staining. [C] To compensate for potential interference between PI and FITC fluorescence, single staining with PI or annexin V dye was performed.
- Supplementary Figure 3. FACS gating strategies and results of JC-1 mitochondrial membrane potential analysis. [A-B] Strategies used to compensate for interference between JC-1 polymer and monomer fluorescence in [A] pTr and [B] pLE cells. Compensation strategy was adjusted using histograms created for each fluorescence wavelength. [C] Gating strategies were used to exclude debris for both cell types. [D] Analysis of undyed cells confimed the absence of autofluorescence
- Supplementary Figure 4. FACS gating strategies and undyed controls used in the analysis of Rhod-2, Fluo-4, and ROS. [A-B] Gating strategies and autofluorescence were confirmed in the Rhod-2 assay. [A] pTr and [B] pLE were gated to exclude debris, and no autofluorescence was detected. [C-D] In the Fluo-4 staining assay, both cell types were gated without debris. Autofluorescence was not identified in either [C] pTr or [D] pLE cells. [E-F] Gating strategies for ROS analysis were identified. Autofluorescence was not present in either undyed [E] pTr or [F] pLE cells.
- Supplementary Figure 5. Original images of western blotting. [A-B] Changes in MAPK signaling molecules after PDM exposure illustrated at different PDM concentrations in [A] pTr and [B] pLE cells. Bands of target proteins are presented with size markers. [C-D] Crosstalk between MAPK signaling molecules identified via co-treatment of PDM and MAPK signaling inhibitors in [C] pTr and [D] pLE cells. [E-F] The expression of β-actin, the housekeeping proteins was identified in [E] pTr and [F] pLE cells. Size markers are shown on the left of each image.
- Supplementary Figure 6. Images of the wound healing assay. Wound area decreased owing to cell migration over time. Quality of Figure 2A was improved to better visualize cells.
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