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.

KEYWORDS:

• Precision toxicology
• single-cell sequencing
• cell cycle arrest
• DNA methylation
• ochratoxin A

Acknowledgments

This work was financially supported by the National Special Program of Transgenic Research (Grant No. 2016ZX08011-005). As a visiting student at the University of Pennsylvania, B. Z. was partially supported by the China Scholarship Council. We sincerely thank James Eberwine for the inspiration; thank you to Yu Hou from Peking University for instructions and advice on single-cell sequencing, and Fei Wang from the National Center for Protein Sciences at Peking University for assistance with the cell sorting. We also want to thank Geek Gene Beijing for their technical support.

Disclosure statement

No potential conflict of interest was reported by the authors.

Author contributions

W.X. and K.H. conceived the study and supervised the work. B.Z. performed all the experiments and wrote the paper. L.Z. provided help during cell culture and single cell acquisition. Y.D. and H.L. provided help during library construction. Y.L. gave advices during data analysis.

Supplementary material:

Supplemental data for this article can be accessed here.

Additional information

Funding

This work was supported by the National Special Program of Transgenic Research [2016ZX08011-005].