DMSO induces drastic changes in human cellular processes and epigenetic landscape in vitro

Abstract

Though clinical trials for medical applications of dimethyl sulfoxide (DMSO) reported toxicity in the 1960s, later, the FDA classified DMSO in the safest solvent category. DMSO became widely used in many biomedical fields and biological effects were overlooked. Meanwhile, biomedical science has evolved towards sensitive high-throughput techniques and new research areas, including epigenomics and microRNAs. Considering its wide use, especially for cryopreservation and in vitro assays, we evaluated biological effect of DMSO using these technological innovations. We exposed 3D cardiac and hepatic microtissues to medium with or without 0.1% DMSO and analyzed the transcriptome, proteome and DNA methylation profiles. In both tissue types, transcriptome analysis detected >2000 differentially expressed genes affecting similar biological processes, thereby indicating consistent cross-organ actions of DMSO. Furthermore, microRNA analysis revealed large-scale deregulations of cardiac microRNAs and smaller, though still massive, effects in hepatic microtissues. Genome-wide methylation patterns also revealed tissue-specificity. While hepatic microtissues demonstrated non-significant changes, findings from cardiac microtissues suggested disruption of DNA methylation mechanisms leading to genome-wide changes. The extreme changes in microRNAs and alterations in the epigenetic landscape indicate that DMSO is not inert. Its use should be reconsidered, especially for cryopreservation of embryos and oocytes, since it may impact embryonic development.

Figure 1

Graphical overview of experimental design combined with summary of differential entities of each analysis method. Tissue-specific information is depicted in orange for cardiac and green for hepatic. Furthermore, exposures are coloured blue and measurement platforms purple. Abbreviations: h = hours; mRNA = messenger RNA; miRNA = microRNA.

Figure 2

PCAs depicting differences between DMSO and UNTR for all measured platforms. (a) PCA of RNAs indicates clear differences in RNA expression between DMSO (triangle) and UNTR (circles). Cardiac samples (left) are more distinct from UNTR than hepatic samples (right). (b) PCA of miRNAs reveals clear separation between DMSO and UNTR in cardiac samples, while hepatic samples seem more susceptible to the duration of the exposure (as seen by colour pattern that corresponds to the specific time points, see legend). (c) PCA of promotor methylation indicates differences between DMSO and UNTR for cardiac samples but not for hepatic samples.

Figure 3

DMSO effect in the process of Gene silencing by RNAs. DEGs in biogenesis of miRNA. The complete process is divided in sub-processes (purple ovals) and depicting involved genes (blue rectangles) and detected DEGs for cardiac (orange rectangles) and hepatic (green rectangles) samples.

Figure 4

Epigenetic regulation of gene expression. (a) DEGs involved in DNA methylation. The process is divided in sub-processes (purple ovals) and depicting involved genes (blue rectangles) and detected DEGs for cardiac (orange rectangles) and hepatic (green rectangles) samples. (b) Relative enrichment of DMRs (DMSO vs UNTR, FDR 5%) within genomic features. For each feature, the number of overlapping DMRs over the total number of tested windows for the respective feature is depicted. Compared to all genomic regions, hypermethylated regions are enriched for satellites, simple repeats, and CGIs distal to promoters without known regulatory evidence and hypomethylated regions are enriched for simple repeats.