Astrocytes, the most numerous glial cells in the mammalian brain, are responsible for maintaining brain homeostasis [23]. For example, astrocytes regulate glutamatergic neurotransmission and prevent glutamate excitotoxicity by removing excess glutamate from the extracellular synaptic space through glutamate transporters [24]. Astrocyte dysfunction has been implicated in a variety of neurological diseases [25].
Based on this, it is necessary to investigate effects of DMSO on astrocytes. However, to our knowledge, this issue has not been explored. In the present study we provided evidence that DMSO has cytotoxic effects on astrocytes via disruption of mitochondrial integrity and membrane potential, resulting in oxidative stress, apoptosis and down-regulation of glutamate transporters.
This finding expands our understanding of the neurotoxic mechanisms of DMSO and provides an experimental basis to select a safe dose of DMSO in neuroscience research. DMSO at concentrations of 0. DMSO has been shown to concentration-dependently modulate the structure and properties of cell membranes [26] , [27]. The mitochondrial membrane has a typical lipid bilayer structure and is vulnerable to various injury factors [28]. It is also known that mitochondrial damage is a major cause of decreased cell viability [29].
Thus, the impairment of DMSO on astrocyte mitochondria was investigated by using the transmission electron microscopy. A Representative transmission electron micrographs showing substructural morphology of astrocytes after treatment with different concentrations of DMSO for 24 h.
The disruption of mitochondria integrity becomes more severe with increased DMSO concentrations. B Quantitation of mitochondrial cross-sectional area. The results confirmed DMSO-induced mitochondrial swelling, with a significant rightward shift in the mitochondrial area cumulative frequency curve, relative to untreated control. C The quantitative analysis showed increases in the percentage of mitochondrial vacuolization in astrocytes treated with DMSO in a dose-dependent manner.
It is also known that disruption of mitochondrial integrity results in a release of Cyt c from the mitochondrial intermembrane space into the cytosol [30]. Therefore, we investigated the effects of DMSO on mitochondrial Cyt c release in cultured astrocytes. DMSO-treated astrocytes had low levels of Cyt c in the mitochondrial fraction and high levels of Cyt c in the cytosolic fraction, compared with intact controls Figure 4C—D.
C Representative western blot bands showing expression levels of Cyt c in cytosolic and mitochondrial fractions of astrocytes treated with various concentrations of DMSO for 24 h. D The quantitative analysis of the relative optical density of cytosol and mitochondrial Cyt c showing that DMSO caused translocation of Cyt c from the mitochondria into the cytoplasm of astrocytes.
A Representative flow cytometry data showing Mito-SOX fluorescence, a highly selective indicator of superoxide in live cell mitochondria, in astrocytes treated with various concentrations of DMSO for 24 h. Thus, we examined the effects of DMSO on astrocyte apoptosis. Astrocytes were incubated with various concentrations of DMSO for 24 h. Cell nuclei counterstained with Hoechst blue.
B Quantitative analysis of astrocyte apoptosis. C Representative western blot bands showing expression levels of procaspase-3, cleaved caspase-3 and Bcl-2 in astrocytes.
Astrocytes are responsible for maintaining glutamate homeostasis in the central nervous system by glutamate transporters that are vulnerable to oxidative stress [37]. However, the biological effects of DMSO may be neglected, because a vehicle control is not always incorporated into the design of experiments.
An early study suggested that exposure of primary hippocampal cell cultures to DMSO in the range of 0. A followed study has shown that treatment with DMSO at concentrations of 0. Collectively, these results provide strong in vitro evidence for the neuropharmacological and neurotoxicological effects of DMSO.
The amphiphilic characteristic of DMSO imparts extensive actions in membrane fusion processes, cryopreservation, and membrane permeability enhancement [39] , [40]. A molecular dynamics simulation employing coarse-grained models suggested that DMSO modulates the structure and properties of cell membranes in a concentration-dependent manner [27].
The present results demonstrated that cultured mouse astrocytes exposed to 1. This result suggests that mitochondrial membrane is vulnerable to DMSO, which might be due to its relatively high membrane fluidity [28].
Mitochondria are the center of cell metabolism and energy transformation; and their malfunction decreases cell viability [29] , [41]. In agreement with this notion, we demonstrated that DMSO inhibits astrocyte viability in a dose-dependent manner, accompanied with mitochondrial structural and functional disruption. Astrocytes exposed to DMSO at concentrations of 1.
Furthermore, the present results indicated that DMSO dose-dependently increases mitochondria-derived ROS production, which is consistent with the notion that degenerated mitochondria are the primary site of ROS production [32]. Taken together, these results reveal that mitochondrial impairment is a primary event in the astrocyte toxicity of DMSO. Glutamate, the major excitatory amino acid neurotransmitter in the brain, can become potentially toxic when it over-accumulates in the synaptic space [42].
As mentioned earlier, astrocytes are responsible for maintaining brain glutamate homeostasis via glutamate transporters [24]. Oxidative stress inhibits glutamate transporter expression and function, which has been implicated as a main pathogenesis for glutamate excitotoxicity in a variety of pathological conditions, including brain ischemia [43] , [44] , traumatic brain injury [45] , epilepsy [46] and neurodegeneration [47] , [48].
In addition to increased ROS production, decreased cell viability and mitochondrial dysfunction may impair glutamate transporter synthesis by astrocytes. High concentration of DMSO has been shown to degrade membrane structure and disturb secondary protein structures within membrane proteins [26] , [27]. Ethanol and methanol showed non-toxic effects on those cell lines at the concentrations of 1. Some toxicity was tolerable when a control sample with solvent alone was used in experiments.
This work is licensed under a Creative Commons Attribution 4. Copyright Biomedpress. A partner of Crossref. View Download Statistics from Dimensions. Nguyen, S. Comparative cytotoxic effects of methanol, ethanol and DMSO on human cancer cell lines. Biomedical Research and Therapy , 7 7 , Cell index CI of cell lines when treated with solvents. Two thousand cells were added into the wells of E-plates. Cells were then cultured in the incubator for the next 3 days.
Download figure Enlarge figure. The slope of the cell index CI when treated with solvents. Two thousand cells of each cell line were added into the wells of E-plates. Horita A. Life Sci. Curr Ther Res Clin Exp. Cartwright C. View Article Google Scholar M. Timm Considerations regarding use of solvents in in vitro cell based assays.
Because slow cooling still induces damage due to extracellular ice crystals, cryopreservation of human oocytes and embryos for in vitro fertilization IVF is accomplished by vitrification, in which higher concentrations of cryoprotectants are used to prevent ice formation not only in the cells, but in the entire solution. Furthermore, DMSO readily crosses most tissue membranes of lower animals and man 9. DMSO is also able to enhance the permeability of other low molecular weight compounds, thereby making them pass membranes or going deeper into a tissue as they normally would 12 , a property highly useful in topological therapies.
This relatively high concentration of DMSO in topological applications is necessary because the skin is harder to penetrate than cell membranes.
Most insights into the molecular effects of DMSO were obtained last century often using high doses. In the meantime, biomedical science has evolved towards more sensitive high-throughput techniques and towards new areas of research, including epigenome modifications and microRNA-mediated gene silencing.
Considering its wide use in many biological fields, we analyzed a relatively low dose of DMSO 0. For this, we exposed in vitro 3D microtissues a maturing iPSC-derived cardiac model and a mature hepatic model to 0.
Our analysis clearly demonstrated that DMSO cannot be considered biologically inert but induces large alterations in microRNAs miRNA and epigenetic landscape, especially in the maturing cardiac model. Human 3D microtissues MTs of a maturing cardiac model and a mature hepatic model were exposed to culture medium with or without 0.
The proteome approximately 2, measured proteins , the full transcriptome including miRNAs and whole-genome methylation were measured on material obtained from the same sample. Figure 1 contains a graphical overview of the experimental design. Numbers of differentially changed entities differed between the tissue types, with cardiac samples showing a larger effect of DMSO than hepatic, with the exception of mRNAs.
This difference is especially noticeable for miRNAs and genome methylation. Because proteomics data was least informative due to its partial nature, these results were included in Supplementary Data.
Furthermore, principal component analysis PCA , using averages of triplicates, for each platform Fig. 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. Cardiac samples left are more distinct from UNTR than hepatic samples right. To study the molecular effects of DMSO, affected cellular processes were analysed using the full transcriptome. Thereafter, tissue-specific effects of DMSO on regulation of gene expression were investigated by analysing changes in miRNAs and genome-methylation.
The clear separation between UNTR and 0. A summary containing the highest hierarchical pathway levels from now on referred to as clusters is included in Table 1 for cardiac and Table 2 for hepatic MTs.
There was substantial overlap between the tissue types, with 60 pathways and 15 clusters found in both. Although there were differences in magnitude of DMSO effect between tissue types, the affected biological processes by DMSO do not appear to be tissue-specific. DMSO effects in this cluster were mainly detected in processes related to Golgi-mediated protein transport and secretion. Since the most significantly affected pathways were found in both tissue types, this indicates robust actions of DMSO.
Pathways related to regulation of gene expression and translation were already detected during the pathway analysis of cardiac MTs Table 3. Though these pathways were not overrepresented in hepatic MTs, the amounts of DEGs detected indicated that DMSO was able to influence these processes and could induce biological alterations to the cell model.
Furthermore, the PCA plot Fig. Furthermore, the PCA plot not only revealed a clear separation between miRNAs of the treatment groups, but indicated also a time-dependent effect. To investigate the source of tissue-specific difference, gene expression changes in the process of miRNA biogenesis were investigated in more detail Fig.
In cardiac MTs, polymerase II contained 1 upregulated and 8 downregulated genes. In hepatic MTs, 5 genes were downregulated and 1 was upregulated. Though fewer genes were downregulated in hepatic MTs, the fold changes were larger compared to cardiac MTs.
The changes in this process could induce differences in the cells miRNA content and therefore affect their regulatory function. 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. The obtained gene targets were used to visualize overrepresented pathways using the Reactome Pathway Browser Supplementary Fig.
Unexpectedly, overrepresented pathways were located in the same clusters for both tissue types. However, extreme deregulation of cardiac MTs and limited information about MTIs is making any downstream analysis on putative affected mRNA irrelevant. Pathway analysis of cardiac gene expression revealed deregulation of DNA methylation pathways. Upregulation of epigenetic writers and downregulation of erasers after DMSO treatment pointed towards genomic hypermethylation, which potentially reduced transcriptional activity.
In contrast, in hepatic pathway analysis, deregulation of DNA methylation was not observed. Note that, in both tissue types, transcriptional evidence for deregulation of other related epigenetic mechanisms were observed, such as histone methylation, where 16 genes and 13 genes were differentially expressed in cardiac and hepatic MTs respectively.
Epigenetic regulation of gene expression. 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. 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.
These alterations affected 1. The effect of DMSO on the upregulation of DNMTs and subsequent hypermethylation of repeat sequences was in line with previous findings in mouse embryoid bodies As expected, hypermethylated regions were also highly enriched in CGIs without regulatory evidence, e. The odds ratio of these regions over the genome was 5. While CGIs in general had an odds ratio of 3. Just as promoter regions, other features such as exons, introns, and TFBS were affected by hypermethylation at approximately the odds of the captured genomic background Fig.
Although we found specific examples where promoter hypermethylation co-occurs with transcriptional repression, no global correlation between differential promoter methylation and gene expression was observed. Only of the 1, genes The induced alterations affected preferentially satellites and simple repeats, as well as CGIs distal to promoters without known regulatory evidence.
The expression of neighboring genes was not significantly correlated to methylation differences. We therefore conclude that transcriptional repression by promoter hypermethylation was not the primary regulatory effect of the altered methylation marks, but the findings indicate a global disruption of DNA methylation mechanisms. While high doses of DMSO were initially investigated for medical applications, lower doses are now commonly used as solvent for toxicology or for cryopreservation of cell cultures, oocytes and embryos.
In the meantime, biological effects of DMSO have been forgotten or considered negligible. With the evolution of biomedical science towards more sensitive high-throughput techniques and towards new areas of research, including epigenome modifications and microRNA-mediated gene silencing, we evaluated the biological effects of DMSO exposure on the transcriptome and the epigenome.
Differences between the investigated tissue types may relate to the fact that the cardiac model contains iPSC-derived human cardiomyocytes that are still maturing, while the hepatic model contains mature human hepatocytes.
Currently, it is not yet possible for iPSC cells to reach an adult-like phenotype 20 , 21 and the concise review on iPSC-derived human cardiomyocytes of Robertson et al. Our observations of DMSO effects, notably the extreme epigenetics modifications in fetal-like cardiac cells, are particularly relevant for cryopreservation during assisted reproductive technology embryos, oocytes and sperm cells.
Another difference to be considered between our two cell models is the number of donors. Indeed, while the cardiac MTs are composed of IPSC-derived cardiomyocytes from a single donor co-cultured with fibroblasts also from 1 donor , hepatic MTs contain mature hepatocytes from 10 donors co-cultured with Kupffer cells of 1 donor.
While a single-donor model is more comparable to an in vivo situation then a multi-donor model, single-donor cardiac MTs might be more susceptible for bias due to genotype sensitivity than our multi-donor hepatic MTs that have the ability to even out these effects. DMSO effects on cellular processes were analyzed using full transcriptome data. This indicates robust actions of DMSO that may possibly be extrapolated to other tissues. Observed changes in cellular processes include changes in mitochondrial pathways linked to ROS production and cellular ATP generation.
DMSO is a known radical scavenger Different downstream effects are relevant depending on the biological application of DMSO. For instance, for cell assays and toxicological research, changes in the amount of ROS or free energy in the cell impact on the capability of the cell to deal with induced stresses, thereby potentially leading to erroneous interpretation of results from in vitro assays.
Within assisted reproductive technology, the ATP content is a good predictor of embryo viability. The DMSO-induced ATP decrease, especially in the cleavage-stage, can induce downstream effects that may disrupt cellular function, implantation ability and fetal development 25 , DMSO-induced reduction in implantation rates and pregnancy losses were already observed from animal models Next to overlap in DMSO-affected processes between the tissue types, pathway analysis also revealed tissue-specific differences.
The higher amount of affected processes detected in cardiac MTs may indicate increased susceptibility of cardiac MTs to the effects of DMSO, though there might also be a statistical bias due to different amount of DEGs used in the pathway analysis. Cardiac MTs also show more effects in other processes that regulate gene expression. Genome-wide methylation effects upon DMSO treatment show large tissue-specific variation.
While there is no significant difference with respect to hepatic MTs, strong effects are observed with cardiac MTs, in particular with respect to specific repetitive sequences such as satellites and simple repeats. Changes in the methylation status of repeats after DMSO treatment might destabilize the genome, implying phenotypic and pathological consequences.
Indeed, recently, methylation changes of repetitive sequences, in particular satellite repeat elements, have been found in cardiomyopathic heart tissue compared to normal heart tissue Furthermore, it is also possible that DMSO acts as a methyl donor to induce hypermethylation However, it has to be kept in mind that there is an interplay between DNA methylation and chromatin configuration, which is regulated by more processes than just DNMTs. Overall, we speculate the effects of DMSO on methylation changes to be a global deregulation characterized by a genome wide hypermethylation.
Cell mechanisms in charge of removing detrimental methylations at important regions, such as TFBS, may then have removed the methylation changes with a negative outcome on cell survival, leaving an over-representation of DMR in the repetitive elements. This is in analogy to mutation rates, which are higher for repetitive elements since they are less subjected to natural selection and DNA repair mechanisms Gagne, and C. Kim, Y.
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