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. 2016;5(2):641-650.
doi: 10.1039/C5TX00454C. Epub 2016 Jan 27.

Epigenetic Toxicity of Trichloroethylene: A Single-Molecule Perspective

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Free PMC article

Epigenetic Toxicity of Trichloroethylene: A Single-Molecule Perspective

Yi Cui et al. Toxicol Res (Camb). .
Free PMC article

Abstract

The volatile, water soluble trichloroethylene (TCE) is a hazardous industrial waste and could lead to various health problems, including cancer, neuropathy, cardiovascular defects, and immune diseases. Toxicological studies taking use of in vitro and in vivo models have been conducted to understand the biological impacts of TCE at the genetic, transcriptomic, metabolomic, and signaling levels. The epigenetic aberrations induced by TCE have also been reported in a number of model organisms, while a detailed mechanistic elucidation is lacking. In this study we uncover an unreported mechanism accounting for the epigenetic toxicity due to TCE exposure by monitoring the single-molecule dynamics of DNA methyltransferase 3a (Dnmt3a) in living cells. TCE-induced global DNA hypomethylation could be partly attributed to the disrupted Dnmt3a-DNA association. By analyzing the components of detached Dnmt3a, we found that the Dnmt3a oligomers (e.g., dimer, trimer, and high-order oligomers) dissociated from heterochromatin in a dose-dependent manner upon exposure. Thereafter the diminished DNA-binding affinity of Dnmt3a resulted in a significant decrease in 5-methylcytosine (5mC) under both acute high-dosage and chronic low-dosage TCE exposure. The resulting DNA demethylation might also be contributed by the elevated expression of ten-eleven-translocation (Tet) enzymes and reformed cysteine cycle. Besides the global effect, we further identified that a group of heterochromatin-located, cancer-related microRNAs (miRNAs) experienced promoter demethylation upon TCE exposure.

Keywords: DNA methylation; Dnmt3a; epigenetic toxicity; miRNA; single-molecule fluorescence; trichloroethylene.

Figures

Fig. 1
Fig. 1. Induced DNA demethylation in HEK293 cells by chronic, low-concentration exposure to TCE (n = 3 independent replicates).
Fig. 2
Fig. 2. Establishment of a cell model to probe Dnmt3a dynamics in single cells. (a) The endogenous Dnmt3a in HeLa cells was knocked out by the CRISPR/Cas9 system and replaced with the fusion protein Dnmt3a–EGFP. (b) Experimental design: the Dnmt3a–EGFP could rescue and restore the methylation profile in KO cells; however, aberrant DNA methylation would recur after exposure to TCE, which can be correlated with the altered Dnmt3a–EGFP dynamics, if any. (c) TCE concentration-dependent DNA demethylation in model cells was determined by immunoassay (n = 3 independent replicates). (d) Time-dependent DNA demethylation induced by TCE was also determined (n = 3 independent replicates).
Fig. 3
Fig. 3. Redistribution of localized, fiber-like Dnmt3a–EGFP due to TCE exposure revealed by confocal imaging (2 h exposure, scale bars: 5 μm). Histograms of pixel-based fluorescence intensity from single cell nucleus are provided (n = 10 cells).
Fig. 4
Fig. 4. Single-molecule FCS for monitoring intracellular Dnmt3a–EGFP. (a) The protein size and major motifs of Dnmts. (b) The mobility of unbound nuc-EGFP molecules gives rise to a standard autocorrelation function, i.e., the diffusion curve. (c) In contrast, the firmly anchored Dnmt3a–EGFP exhibits undetectable diffusion within the FCS timescale. (d) Along with the TCE exposure, some nuclear regions generate a diffusion curve (green square), indicating the detachment of Dnmt3a–EGFP. Further, the stoichiometry of detached Dnmt3a–EGFP could be inferred from the hydrodynamic size by fitting the diffusion curve.
Fig. 5
Fig. 5. Summary of all detected Dnmt3a–EGFP diffusion times by FCS measurements, corresponding to the stoichiometric states of Dnmt3a in living cells (n > 100 measurements from over 40 cells).
Fig. 6
Fig. 6. The transcriptional changes of Dnmts and Tets as cells respond to TCE (n = 4 independent replicates).
Fig. 7
Fig. 7. Changes in the promoter methylation of the heterochromatin localized, cancer-related miRNAs. (a) The general trend of changes. (b) Representative miRNAs experienced promoter demethylation.

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