An in vitro attempt at precision toxicology reveals the involvement of DNA methylation alteration in ochratoxin A-induced G0/G1 phase arrest

Abstract

Precision toxicology evaluates the toxicity of certain substances by isolating a small group of cells with a typical phenotype of interest followed by a single cell sequencing-based analysis. In this in vitro attempt, ochratoxin A (OTA), a typical mycotoxin and food contaminant, is found to induce G0/G1 phase cell cycle arrest in human renal proximal tubular HKC cells at a concentration of 20 μM after a 24h-treatment. A small number of G0/G1 phase HKC cells are evaluated in both the presence and absence of OTA. These cells are sorted with a flow cytometer and subjected to mRNA and DNA methylation sequencing using Smart-Seq2 and single-cell reduced-representation bisulfite sequencing (scRRBS) technology, respectively. Integrated analysis of the transcriptome and methylome profiles reveals that OTA causes abnormal expression of the essential genes that regulate G1/S phase transition, act as signal transductors in G1 DNA damage checkpoints, and associate with the anaphase-promoting complex/cyclosome. The alteration of their DNA methylation status is a significant underlying epigenetic mechanism. Furthermore, Notch signaling and Ras/MAPK/CREB pathways are found to be suppressed by OTA. This attempt at precision toxicology paves the way for a deeper understanding of OTA toxicity and provides an innovative strategy to researchers in the toxicology and pharmacology field.

In vitro and in vivo evaluation of AFB1 and OTA-toxicity through immunofluorescence and flow cytometry techniques: A systematic review

“…Fluorescence analysis of DNMT1 showed alterations in global DNA methylation, thus correlating OTA-nephrotoxicity with epigenetic changes. The genome-wide hypermethylation may contribute to OTAmediated the inhibition of cell cycle progression (Zhang et al, 2020).…”

“…In the same in vitro model, OTA may exert deleterious effects by inducing DNA damage, disrupting DNA repair-related processes and arresting the cell cycle at phase G 2 /M. DNA damage was detected by the over-expression of DSB gene, γ-H2AX, and DNA repair-related genes, BRCA1 and RAD51 (Zhang et al, 2020).…”

Selective Activation of Endoplasmic Reticulum Stress by Reactive-Oxygen-Species-Mediated Ochratoxin A-Induced Apoptosis in Tubular Epithelial Cells

“…Zhang B et al showed an OTA-induced cell cycle G0/G1 phase arrest in human proximal tubular cells via DNA methylation alteration [33]. Current evidence suggests that cell cycle arrest is a hallmark of senescence induced by ROS production in renal tubular cells.…”

“…The phenomenon could be attenuated by NAC, suggesting that OTA induced ER-stress-mediated apoptosis through ROS-dependent pathways. Zhang B et al showed an OTA-induced cell cycle G0/G1 phase arrest in human proximal tubular cells via DNA methylation alteration [33]. Current evidence suggests that cell cycle arrest is a hallmark of senescence induced by ROS production in renal tubular cells.…”

Ochratoxin A-Induced Nephrotoxicity: Up-to-Date Evidence

“…Some of the gene was related to G0/G1 cell cycle arrest, but only increased expression of PTEN due to OTA-induced hypomethylation of PTEN which involved in G1/S phase transition. G0/G1 phase cell cycle arrest and inhibition of the Notch and Ras/MAPK/CREB signaling pathway after OTA exposure would lead to renal toxicity [ 58 ].…”