The cytotoxic effects of dimethyl sulfoxide in mouse preimplantation embryos: a mechanistic study

Abstract

Rationale: Dimethyl sulfoxide (DMSO) is commonly used as a solvent for water-insoluble substances, a vehicle for drug therapy, and a cryoprotectant for cultured cells. DMSO induced embryonic defects and its mechanism of action remains unclear. The rationale is based on the assumption that DMSO supplementation should induce long-term negative effects on both pre- and post-implantation embryo development.Methods: DMSO induced oxidative stress, ER stress, autophagy, mitophagy, signaling responsible genes and proteins were determined by RT-qPCR, Western blotting, immunofluorescence, and confocal microscopy. DMSO induced mitochondrial dysfunction was measured by transmission electron microcopy and JC-1 assay. Apoptosis was estimated using TUNEL and comet assay. Post-implantation embryo developmental capability was estimated by implantation site and fetus numbers.Results: Exposure to DMSO induced an early oxidative stress response within 0.5 to 2 h in 1-cell zygotes by disrupting the balance of pro- and anti-oxidants. Notably, DMSO-treated 2-cell embryos showed increased expression of unfolded protein response genes such as Hspa5, Hsp90b1, Ddit3, Atf4, and Xbp1. As a result, the development of many embryos is arrested at the 2-cell, 4-cell, or morula stages in a dose-dependent manner. Further, DMSO-induced endoplasmic reticulum stress increased mitochondrial Ca2+ levels, induced mitochondrial depolarization/dysfunction, and induced apoptotic cell death via the JNK/ATF2-dependent pathway. Consequently, treatment with DMSO increased the expression of autophagy initiation-, phagophore elongation-, and autophagosome formation-related genes, as well as localization of PINK1/Parkin, which are the main mediators of mitophagy, in mitochondria. Interestingly, DMSO causes cytotoxic effects in preimplantation embryos by inducing extensive mitophagy and autophagy. Especially, DMSO treatment decreased the inner cell mass and trophectoderm cell numbers as well as mRNA expression of B3gnt5 and Wnt3a in developed blastocysts, which decreased the implantation and developmental rates of full-term offspring after being transferred into pseudopregnant mice.Conclusion: These results provide a significant contribution to finding effective protective agents to combat DMSO mediated reproductive toxicity for application in human embryos in the near future.

BiP Inducer X: An ER Stress Inhibitor for Enhancing Recombinant Antibody Production in CHO Cell Culture

“…Chemical additives such as dimethyl sulfoxide (DMSO) and sodium butyrate (NaBu), which have been used for enhancing mAb production in rCHO cell cultures, may also activate ER stress. DMSO has been observed to induce ER stress in mouse embryonic cells . When cells are exposed to ER stress, an unfolded protein response (UPR) is activated to restore stress or to adapt to stress conditions in order to maintain homeostasis and cellular function .…”

Effects of DMSO on the Pluripotency of Cultured Mouse Embryonic Stem Cells (mESCs)

“…However, DMSO may have side effects on cell culture that are usually overlooked. For example, it has already been shown that DMSO can affect cell cycle [ 6 – 10 ], mitochondrial function [ 5 , 11 ], and the epigenetic landscape of various cell types [ 12 – 14 ]. More importantly, DMSO can prime the differentiation of various types of stem cells [ 7 , 15 – 18 ], and it is able to sustain mESC culture pluripotency in the absence of LIF [ 20 ].…”

“…However, DMSO is also a hydrogen-bound disrupter, an intercellular electrical uncoupler, and a hydroxyl radical scavenger, among other properties [ 1 , 3 ], and could conceivably affect cell culture, depending on the cell type and the concentration used, issues that are usually overlooked. For instance, it has already been shown that apart from inducing cytotoxicity [ 4 – 6 ], DMSO can affect cell cycle [ 6 – 10 ] and mitochondrial function [ 5 , 11 ], as well as the epigenetic landscape of different cell types [ 12 – 14 ], and influence the differentiation of various types of stem cells [ 7 , 15 – 18 ]. Indeed, in 2012, Pal and colleagues reported that when exposing differentiating human embryonic stem cells (hESCs) to three different concentrations of DMSO, there was a dose-dependent downregulation of pluripotency markers and that the lowest concentration biased the differentiation process by enhancing mesodermal differentiation [ 17 ].…”

Cryoprotective agents affect amino acids incorporation into total proteins in cells of lymphoid organs and liver of experimental animals

“…Modern data show that even in low concen-trationstheCPAsaffectsomaticandreproductive cells [8][9][10]. In this regard, the question of the CPAs influen ceonmetabolicprocessesincellsandtissues is getting relevant, especially in view of the development of such areas in medicine as cell and tissue therapy as well as assisted reproductive technologies [8][9][10].…”

“…Modern data show that even in low concen-trationstheCPAsaffectsomaticandreproductive cells [8][9][10]. In this regard, the question of the CPAs influen ceonmetabolicprocessesincellsandtissues is getting relevant, especially in view of the development of such areas in medicine as cell and tissue therapy as well as assisted reproductive technologies [8][9][10]. Moreover, recently many authors have demanded the re-considering of using the CPAs in reproductive technologies, as modern experimental results demonstrate that CPAs within a wide range ofconcentrationshaveatoxiceffectatthegenome level, which leads to irreversible impairments in structure and functions of cells [8][9][10].…”