Epithelial-Mesenchymal Transition Induced by a Metal Mixture in Liver Cells With Antioxidant Barrier Decreased

Abstract

Occupational exposure to arsenic (As), cadmium (Cd), and lead (Pb) affects many sectors, necessitating research to understand their transformation mechanisms. In this study, we characterized the process of epithelial-mesenchymal transition (EMT) in a rat hepatic epithelial cell line with decreased expression of catalase and glutamate cysteine ligase catalytic (GCLC) subunit that was exposed to a mixture of As, Cd, and Pb at equimolar occupational exposure concentrations. We evaluated the expression of genes and proteins involved in EMT. Our findings revealed that cells with a decreased antioxidant barrier showed a decreased expression and abundance of epithelial genes when exposed to a mixture of metals. Additionally, we observed alterations in the expression of transcription factors (TFs) associated with EMT and an increase in the expression and abundance of mesenchymal genes. Specifically, we found that E-cadherin expression decreased by ~50% at both the gene and protein levels. In contrast, the expression of vimentin, α-smooth muscle actin, and N-cadherin genes increased by ~70%, whereas their corresponding protein levels increased by nearly 100%. Furthermore, the TFs zinc finger e-box binding homeobox 1 and snail family transcriptional repressor 1 showed a 30% increase in gene expression and an ~80% increase in protein expression. These changes enable the cells to acquire migratory capabilities. Our results confirmed that exposure to this mixture of As, Cd, and Pb can induce EMT in cells with a decreased antioxidant barrier.

Keywords: catalase and GSH abatement; epithelial–mesenchymal transition; metals mixture; wound healing.

Figure 1

Sequences of the shRNAs designed based on the messenger RNA of the target sequence: ShRNA1 (AGGTCACCCACGATATTA) hybridizes from nucleotide 389–406 of catalase mRNA, shRNA2 (TGTGAATGTCCAGAGTTA) hybridizes to nucleotide 1900–1917 of GCLC mRNA and structure of plasmid pLVx-shRNA. GCLC, glutamate cysteine ligase catalytic.

Figure 2

Transfection effectiveness. Downregulation of catalase and GCLC expression in C9 hepatic rat cells was measured 72 h after hairpin transfection. Student t test. ⁣^∗p < 0.05, ⁣^∗∗p < 0.01. GCLC, glutamate cysteine ligase catalytic.

Figure 3

Viability measurement. For the measurement of viability, the dual fluorescein diacetate [3′,6′-diacetylfluorescein] (FDA) and ethidium bromide (EtBr) method was used. Viability was measured every 6 days until 36 days.

Figure 4

Changes in EMT-related genes. Changes expression in CDH1, ZEB1, SNAI1, VIM, α-SMA, and CDH were evaluated in C9 cells with a decreased antioxidant barrier. One-way ANOVA. ⁣^∗p < 0.05, ⁣^∗∗∗p < 0.001. ANOVA, analysis of variance; EMT, epithelial–mesenchymal transition; VIM, vimentin.

Figure 5

Immunofluorescence staining. A representative image of the cell population under immunofluorescence for each protein is shown in (A) (10X) and a more detailed image in (B) (40X). Images were taken under a Carl Zeiss fluorescence microscope.

Figure 6

Fluorescence intensity quantification. The fluorescence intensity was determined using software ImageJ (NIH, USA). First, each image was split by channels, considering green channel as positive stain and red channel as background. The images were divided into four panels and the results were presented as the subtraction between positive stain and background divided by quantification area. Statistical differences among multiple groups were determined by one-way ANOVA and Tukey test as post hoc analysis. ⁣^∗p < 0.05, ⁣^∗∗p  < 0.01, ⁣^∗∗∗p  < 0.001. ANOVA, analysis of variance.

Figure 7

Evaluation of wound test. The evaluation of % wound closure was performed by quantifying the area with ImageJ software (NIH, USA). The percentage was measured at 12 and 24 h. Student's t test. ⁣^∗p < 0.05.