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.

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Uncovering cellular responses from heterogeneous genomic data is crucial for molecular medicine in particular for drug safety. This can be realized by integrating the molecular activities in networks of interacting proteins. As proof-of-concept we challenge network modeling with time-resolved proteome, transcriptome and methylome measurements in iPSC-derived human 3D cardiac microtissues to elucidate adverse mechanisms of anthracycline cardiotoxicity measured with four different drugs (doxorubicin, epirubicin, idarubicin and daunorubicin). Dynamic molecular analysis at in vivo drug exposure levels reveal a network of 175 disease-associated proteins and identify common modules of anthracycline cardiotoxicity in vitro, related to mitochondrial and sarcomere function as well as remodeling of extracellular matrix. These in vitro-identified modules are transferable and are evaluated with biopsies of cardiomyopathy patients. This to our knowledge most comprehensive study on anthracycline cardiotoxicity demonstrates a reproducible workflow for molecular medicine and serves as a template for detecting adverse drug responses from complex omics data.

Doxorubicin (DOX) is a chemotherapeutic agent of which the medical use is limited due to cardiotoxicity. While acute cardiotoxicity is reversible, chronic cardiotoxicity is persistent or progressive, dose-dependent and irreversible. While DOX mechanisms of action are not fully understood yet, 3 toxicity processes are known to occur in vivo: cardiomyocyte dysfunction, mitochondrial dysfunction and cell death. We present an in vitro experimental design aimed at detecting DOX-induced cardiotoxicity by obtaining a global view of the induced molecular mechanisms through RNA-sequencing. To better reflect the in vivo situation, human 3D cardiac microtissues were exposed to physiologically-based pharmacokinetic (PBPK) relevant doses of DOX for 2 weeks. We analysed a therapeutic and a toxic dosing profile. Transcriptomics analysis revealed significant gene expression changes in pathways related to "striated muscle contraction" and "respiratory electron transport", thus suggesting mitochondrial dysfunction as an underlying mechanism for cardiotoxicity. Furthermore, expression changes in mitochondrial processes differed significantly between the doses. Therapeutic dose reflects processes resembling the phenotype of delayed chronic cardiotoxicity, while toxic doses resembled acute cardiotoxicity. Overall, these results demonstrate the capability of our innovative in vitro approach to detect the three known mechanisms of DOX leading to toxicity, thus suggesting its potential relevance for reflecting the patient situation. Our study also demonstrated the importance of applying physiologically relevant doses during toxicological research, since mechanisms of acute and chronic toxicity differ.

Monitoring the DNA-Damage Response (DDR) activated pathway in multicellular tumor spheroid models is an important challenge as these 3D models have demonstrated their major relevance in pharmacological evaluation. Herein we present DDR-Act-FP, a fluorescent biosensor that allows detection of DDR activation through monitoring of the p21 promoter p53-dependent activation. We show that cells expressing the DDR-Act-FP biosensor efficiently report activation of the DDR pathway after DNA damage and its pharmacological manipulation using ATM kinase inhibitors. We also report the successful use of this assay to screen a small compound library in order to identify activators of the DDR response. Finally, using multicellular spheroids expressing the DDR-Act-FP we demonstrate that DDR activation and its pharmacological manipulation with inhibitory and activatory compounds can be efficiently monitored in live 3D spheroid model. This study paves the way for the development of innovative screening and preclinical evaluation assays.