Toxic effects of DMSO on cultured beating heart cells at temperatures above zero

Toxic influences of DMSO were studied on spontaneously beating rat heart cells in tissue culture. Injurious effects of DMSO on the contractility and on the individual cell survival were investigated at three different temperatures, +4 ° C, +24 ° C, and + 37 ° C. As indicated by the 50% levels of irreversible damage it may be concluded that osmotic stress during the addition and the removal of DMSO should be avoided carefully at +4 ° C.With slow introduction and slow removal of DMSO optimal survival may be obtained up to 4.7 m DMSO at +4 ° C and at +24 ° C. Although at +37 ° C osmotic stress becomes less damaging, optimal survival can only be obtained at lower concentrations (up to 3.5 m depending on the incubation program). At +37 ° C the more specific toxic properties of DMSO are the limiting factor.At +24 ° C slow removal of DMSO instead of sudden removal at the end of an incubation period improves the survival considerably.At +24 ° C a good compromise between damage by osmotic shock and by other more specific toxic properties can be obtained.     

Effects of dimethylsulfoxide (DMSO) on the digestive-lysosomal system in Paramecium caudatum

The effects of DMSO (dimethylsulfoxide) on cell growth and on the digestive-lysosomal system of axenically grown Paramecium caudatum were studied. A general protocol of exposing cells to different concentrations of DMSO at the beginning of each of the four processes in the digestive cycle enabled us to analyze the effect of DMSO at each step. Vacuole formation and the beginning of a digestive cycle were initiated by adding latex beads to the cells. Maximum cell densities at stationary phase of growth were found to be inversely proportional to DMSO between 0.5 and 1.75%, and the duration of the generation time was exponentially proportional. At 2% DMSO cellular division was completely blocked, and above 2% it was cytotoxic. P. caudatum survived for 8 h in 4% DMSO and died instantaneously in 10%. This inhibitory effect on growth was reversible, though this reversibility might depend on the duration and level of DMSO exposure. DMSO exerted a dose- and time-dependent inhibitory effect on the rate of DV formation but had little effect on the acidification-condensation and the lysosome fusion-digestion processes. The size of the DV formed was also reduced, and this effect was dose-but not time-dependent; vacuole size reduction occurred immediately with DMSO exposure, and no further reduction was observed during exposures of up to 24 h. DMSO at 3 and 4% inhibited vacuole defecation, but the cells could overcome this inhibition when exposed to DMSO for longer periods.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cryoprotection by dimethyl sulfoxide and dimethyl sulfone

Preservation of cells and tissues at low temperatures requires the presence of effective cryoprotectants with low toxicity to which cells are relatively permeable. Two similar compounds, dimethyl sulfoxide (DMSO) and dimethyl sulfone (DMSO2), exhibit both features for cryoprotectants, yet DMSO is a very effective cryoprotectant while DMSO2 is ineffective. This anomaly was investigated by relating observations on the phase behavior of DMSO and DMSO2 in aqueous solutions to the recovery of human lymphocytes frozen in the presence of these compounds. The lack of cryoprotection in the presence of DMSO2 appears to be due to the precipitation of DMSO2 from the solution at subzero temperatures. The observation of reduced cell recovery after freezing with increasing concentrations of DMSO2 implies that cell damage is related to the amount of solid DMSO2 present. Precipitation of DMSO2 occurs both intra- and extracellularly, but it is argued that intracellular precipitation of DMSO2 is the damaging phenomenon. Cryoprotective compounds are normally selected based on the criteria of low toxicity and permeability to the plasma membrane. An additional condition, solubility, must be included for interpretation of experimental data and for development of effective protocols for cryopreservation.     

Beta-carotene uptake and metabolism in human lung bronchial epithelial cultured cells depending on delivery vehicle

Beta-carotene (BC) could have a protective or pro-carcinogenic role in lung cancer, and cell culture systems are important to evaluate it. Nevertheless, the delivery of the hydrophobic BC to cells is difficult. Different vehicles have been used such as liposomes, tetrahydrofuran and serum lipoproteins, but presenting different problems. Water dispersible beadlets containing BC are a good choice and can produce the greatest BC uptake when compared to the above vehicles, but other beadlet components could alter the results. Dimethylsulfoxide (DMSO) could be a good alternative since it has low toxicity and it enhances the penetration of substances across biologic membranes. We aimed to characterize an appropriate model for delivering all-trans-BC to lung cells in culture and knowing its metabolism. All-trans-BC 5 microM was administered to BEAS-2B cells in beadlets or DMSO, and medium and cell samples were taken at different times. The levels of BC and its main isomers and metabolites were determined by HPLC. All-trans-BC reached the same levels in the medium (about 3.5 microM) either when supplied in beadlets or in DMSO, and, with beadlets, 13-cis-BC was also detected. However the amount of all-trans-BC taken up by the cells was the triple when delivered by DMSO. With both vehicles, intracellular all-trans-BC levels reached its maximum after 24 h of treatment, remaining equal after 72 h. The 9-cis and 13-cis isomers of BC, and oxidized metabolites, were also detected in the cells although in smaller proportion than all-trans-BC, especially with DMSO. An LDH assay did not suggest toxicity of beadlets, DMSO or BC itself. In conclusion, DMSO seems the most appropriate vehicle for delivering BC to lung cells in vitro, and we present a model that allows studying the effects of BC and its metabolism in the lung human BEAS-2B cell line.     

Dimethyl sulfoxide induces both direct and indirect tau hyperphosphorylation

Dimethyl sulfoxide (DMSO) is widely used as a solvent or vehicle for biological studies, and for treatment of specific disorders, including traumatic brain injury and several forms of amyloidosis. As Alzheimer's disease (AD) brains are characterized by deposits of β-amyloid peptides, it has been suggested that DMSO could be used as a treatment for this devastating disease. AD brains are also characterized by aggregates of hyperphosphorylated tau protein, but the effect of DMSO on tau phosphorylation is unknown. We thus investigated the impact of DMSO on tau phosphorylation in vitro and in vivo. One hour following intraperitoneal administration of 1 or 2 ml/kg DMSO in mice, no change was observed in tau phosphorylation. However, at 4 ml/kg, tau was hyperphosphorylated at AT8 (Ser(202)/Thr(205)), PHF-1 (Ser(396)/Ser(404)) and AT180 (Thr(231)) epitopes. At this dose, we also noticed that the animals were hypothermic. When the mice were maintained normothermic, the effect of 4 ml/kg DMSO on tau hyperphosphorylation was prevented. On the other hand, in SH-SY5Y cells, 0.1% DMSO induced tau hyperphosphorylation at AT8 and AT180 phosphoepitopes in normothermic conditions. Globally, these findings demonstrate that DMSO can induce tau hyperphosphorylation indirectly via hypothermia in vivo, and directly in vitro. These data should caution researchers working with DMSO as it can induce artifactual results both in vivo and in vitro.     

Precommitment states induced during HL-60 myeloid differentiation: possible similarities of retinoic acid- and DMSO-induced early events

The possible relationship of the pathways by which two inducers, retinoic acid and DMSO, cause myeloid differentiation of HL-60 promyelocytic leukemia cells was studied. HL-60 cells were first exposed to retinoic acid and then washed free of it. As reported previously, this brief exposure results in no subsequent G0 growth arrest or phenotypic differentiation. When these cells were subsequently exposed to DMSO, onset of G1/0 growth arrest but not phenotypic differentiation occurred within 24 h. Since in these cells retinoic acid or DMSO normally requires 48 h of continuous exposure for onset of significant G0 growth arrest and phenotypic differentiation, it appears that retinoic acid and DMSO induce similar early cellular events needed for subsequent G0 growth arrest but not for phenotypic differentiation. While onset of growth arrest and differentiation occur together when the cells are exposed for 48 h to retinoic acid, the present results indicate that their occurrence can be uncoupled by this split dosage to inducers. The results are discussed in terms of a previously hypothesized model of cellular response to the inducers.     

Ion transport through dimethyl sulfoxide (DMSO) induced transient water pores in cell membranes

It is well known that dimethyl sulphoxide (DMSO) increases membrane permeability, which makes it widely used as a vehicle to facilitate drug delivery across biological membranes. However, the mechanism of how DMSO increases membrane permeability has not been well understood. Recently, molecular dynamics simulations have demonstrated that DMSO can induce water pores in biological membranes, but no direct experimental evidence is so far available to prove the simulation result. Using FluxOR Tl? influx assay and intracellular Ca2? imaging technique, we studied the effect of DMSO on Tl? and Ca2? permeation across cell membranes. Upon application of DMSO on CHO-K1 cell line, Tl? influx was transiently increased in a dose-dependent manner. The increase in Tl? permeability induced by DMSO was not changed in the presence of blockers for K? channel and Na?-K? ATPase, suggesting that Tl? permeates through transient water pores induced by DMSO to enter into the cell. In addition, Ca2? permeability was significantly increased upon application of DMSO, indicating that the transient water pores induced by DMSO were non-selective pores. Furthermore, similar results could be obtained from RAW264.7 macrophage cell line. Therefore, this study provided experimental evidence to support the prediction that DMSO can induce transient water pores in cell membranes, which in turn facilitates the transport of active substances across membranes.     

Effect of dimethyl sulfoxide on cytosolic ionized calcium concentration and cytoskeletal organization of hepatocytes in a primary culture

The addition of dimethyl sulfoxide (DMSO) to a chemically defined, serum free medium prolonged hepatocytes survival in primary culture. DMSO exposure had a remarkable effect on morphological change and F-actin filaments distribution of hepatocytes. When hepatocytes were cultured in a medium containing 2% DMSO, the cells showed a compact and cubical shape and intracellular F-actin filaments were mainly observed in a ring-like fashion around the intercellular space. After exposure to DMSO, fibronectin fibers in the interspace between cell and substratum were not apparent. Exposing the hepatocytes to DMSO also caused a sharp increase in cytosolic free ionized calcium ([Ca2+]). The initial increase in [Ca2+]i following the addition of DMSO was not attenuated by the chelation of extracellular Ca2+ with EGTA. The Ca2+ signal in the absence of extracellular Ca2+ was transient and returned to the basal levels within 1-2 min, while it was maintained at a high steady state in the presence of extracellular Ca2+. These results suggest that DMSO may be able to increase [Ca2+]i by two mechanisms, by the release of the ion from intracellular pools and, by the stimulation of influx across the plasma membrane. The increase in [Ca2+]i induced by DMSO treatment may play a role in prolonging hepatocyte survival in culture, since [Ca2+]i is one of the most important dynamic second messengers in various cellular metabolic processes.     

Influence of dimethyl sulfoxide on freezing resistance of lettuce seeds

DTA of lettuce seeds was used to study the cryoprotective mode of action of DMSO. Lettuce seeds were imbibed for 16 hr in 5, 10, 15 or 20% aqueous solutions of DMSO either with or without a 6 hr preimbibition in water. Seeds were frozen to postsecondary exotherm temperatures; exotherms were recorded; and germination was tested after freezing. DMSO had at least a twofold effect on the lettuce seeds: it increased the degree of supercooling, and it imparted a capacity to survive following freezing beyond secondary exotherm levels.     

二甲基亚砜诱导人食管癌细胞分化的实验研究

The fact that treatment of leukemia (Acute Promyelocytic Leukemia) with ATRA (All-Trans Retinoic Acid) was so succeeded that it was considered as a good example for tumor therapy. In the treatment of solid tumors by means of induced differentiation, however, has not been yet so broken-through. DMSO (Dimethylsulfoxide) was a common and simple organic compound, which comprised a variety of biological activities. For example, DMSO induced differentiation of leukemia in many reports. However, the effect of DMSO on solid tumors was to be explored further. In the present study, DMSO was used to human esophageal cancer cell lines in vitro in comparison with the classical inducer ATRA. From the view of morphology, cell cycle, growth inhibition, cytokeratin 4 expression, dye transfer and tumorigenecity, the results demonstrated that DMSO as well as ATRA could induce differentiation of human esophageal cancer cells. Interestingly, DMSO was confirmed to be more effective in inducing differentiation of esophageal cancer cells than ATRA. It suggests that DMSO showed some good prospects for the treatment of solid tumors.     

二甲基亚砜诱导人食管癌细胞分化的实验研究

维甲酸诱导分化治疗白血病取得巨大成功,为治疗肿瘤开辟了新思路,但诱导分化治疗实体瘤目前尚无突破性进展。二甲基亚砜有多种生物学作用如诱导白血病细胞分化,对实体瘤细胞的作用研究却不多。本文将二甲基亚砜试用于体外培养的人食管癌细胞株,同时设维甲酸作参照,从形态和细胞周期改变、角蛋白表达及成瘤性等多方面证实二甲基亚砜和维甲酸一样可诱导食管癌细胞分化,并且发现二甲基亚砜的作用优于维甲酸,强烈提示二甲基亚砜对实体瘤治疗的可能应用前景。     

DMSO regulates osteoclast development in vitro

Dimethyl sulfoxide (DMSO) is routinely used in the laboratory as a solvent and vehicle for organic molecules. Although it has been used in previous studies involving myeloid cells and macrophages, we are unaware of data demonstrating the effects of DMSO alone on osteoclast development. Recently, we were using DMSO as a vehicle and included a non-vehicle control. Surprisingly, we observed a marked change in osteoclast development, and therefore designed this study to examine the effects of DMSO on osteoclast development. Osteoclasts were generated from two sources: bone marrow macrophages and an osteoclast progenitor cell line. Cells were cultured with DMSO for various durations and at differing concentrations and mature, multinucleated (>3 nuclei) TRAP(+) cells were assessed in terms of cell number, cell surface area, and number of nuclei/cell. Osteoclast surface area increased in 5 μM DMSO to a mean of 156,422 pixels from a mean of 38,510 pixels in control culture, and subsequently decreased in 10 μM DMSO to a mean of 18,994 pixels. With serial addition of DMSO over 5 d, a significant increase in mean surface area, and number of nuclei/cell was also observed, while the opposite was true when DMSO was serially removed from culture. These findings show that DMSO exerts a marked effect on osteoclast differentiation. Since many investigators use DMSO to solubilize compounds for treatment of osteoclasts, caution is warranted as altering DMSO concentrations may have a profound effect on the final data, especially if osteoclast differentiation is being assessed.     

Dimethyl sulfoxide: reversible inhibitor of pollen tube growth

Five percent dimethyl sulfoxide (DMSO) completely inhibited tube initiation, stopped tube growth and suppressed the high respiration associated with tube growth of lily pollen. The effect of DMSO on respiration was indirect because uncoupling concentrations of 2,4-dinitrophenol abolished the inhibition of respiration. Five percent DMSO did not inhibit rapid starch synthesis during the first 30 minutes of incubation, nor did DMSO inhibit the period of high respiration associated with rapid starch synthesis. DMSO did not cause permanent damage to the cells since normal pollen tube growth occurred after its removal. Dimethyl sulfoxide is not a general inhibitor of pollen metabolism, but it may be a specific inhibitor of a process required for tube growth.     

Synthesis of DNA and alpha-fetoprotein in the monolayer culture of mouse hepatocytes

Relation between processes of proliferation and synthesis of the embryonal serum protein alpha-fetoprotein (AFP), the influence on these processes of polyelectrolyte dextran sulfate (DS) and dimethyl-sulfoxide (DMSO) has been studied in the monolayer culture of mouse hepatocytes. In control cultures the correlations between the time of appearance and the level of DNA and AFP synthesis were observed. DS and DMSO were found to inhibit both processes. Cell proliferation could be reestablished by addition of epidermal growth factor. In case of the influence of DMSO, it wasn't followed by the induction of AFP synthesis. This the processes of DNA and AFP synthesis in monolayer cultures of mouse hepatocytes can be separated. The elongated incubation of hepatocytes with collagenase during their obtaining, abolished the effects of DS. This shows that surface components of hepatocytes, lost upon enzyme degradation, may be involved in the mechanism of DS effect.     

Completion of first meiosis by sperm penetration in vitro of bovine oocytes inhibited at metaphase-I with dimethylsulphoxide

The effects of dimethylsulphoxide (DMSO) on immature oocytes during maturation in culture and following penetration by spermatozoa were examined. Germinal vesicle breakdown (GVBD) was observed in all oocytes cultured in the maturation medium supplemented with 2, 4 and 8% DMSO. When the oocytes were cultured in medium with 8% DMSO, 95% (57 60 ) of them were inhibited at prometaphase-I. Cumulus cells were significantly (P<0.05) beneficial for resumption of oocyte nuclear maturation during further culture in the maturation medium for 4, 8 and 24 h after DMSO treatment. When the oocytes were additionally cultured for 4 and 8 h in the maturation medium after DMSO treatment, the proportions of oocytes reaching metaphase-II were significantly (P<0.05) higher in those cultured with spermatozoa than without (68 vs 49% and 84 vs 56%, respectively). These results indicate that 8% DMSO does not affect GVBD of oocytes, but conversely it inhibits oocytes at prometaphase-I, and that cumulus cells are important for recovery from DMSO inhibition and for the resumption of nuclear maturation of oocytes. Sperm penetration was also found to stimulate the completion of meiotic maturation of oocytes inhibited at metaphase-I with 8% DMSO.     

Physicochemical conjugation with deoxycholic acid and dimethylsulfoxide for heparin oral delivery

Heparin, as therapeutic medications, cannot be administered orally because of its hydrophilic and high molecular weight. Here, we present a new technology to enhance the absorption of heparin in the intestine through its chemical conjugation with deoxycholic acid (DOCA) that can interact with bile acid transporter in the intestine. For the ampiphilic property and complete dissolution, the modified heparin was physically complexed with dimethylsulfoxide (DMSO). The DOCA-conjugated heparin could form nanoparticles in aqueous solution, whereas it was completely dissolved when treated with above 10% DMSO solution. Molecular dynamics computation study and two-dimensional homonulcear (1)H nuclear overhauser effect spectroscopy (NOESY) NMR spectra demonstrated that one heparin molecule was chemically conjugated with two DOCA molecules that were physically interacted with six DMSO molecules within 4 ? via hydrophobic interactions and partly via hydrogen bonding. Its therapeutic efficacy was also pharmaceutically analyzed. When the DMSO-bound DOCA-conjugated heparin was orally administered into mice, its therapeutic efficacy was enhanced according to the amount of bound DMSO. Also, after oral administration of fluorescence-labeled DMSO-bound DOCA-conjugated heparin, it was circulated in the whole body for above 2 h. However, the DOCA-conjugated heparin without DMSO binding was fast eliminated after oral absorption. This study demonstrates that the interaction of structural constraints, DOCA and DMSO, with heparin can serve as a platform technology for potential macromolecule oral delivery.     

Biological effects of dimethyl sulfoxide on yeast

The effects of dimethyl sulfoxide (DMSO) on yeast cells were investigated. It was determined that while exposure of yeast to increasing concentrations of DMSO resulted in decreasing cell viability, it did not cause cell lysis or protein leakage from the cells. The inclusion of DMSO in growth medium resulted in the conversion of yeast cultures to respiratory deficient petites. This mutagenic effect requires cell growth for its expression.     

ON THE EFFECTS OF DMSO IN CATION TRANSPORT BY EXCISED BARLEY ROOTS

Dimethyl sulfoxide (DMSO) in concentrations of up to 10% by volume stimulates the uptake of zinc by excised barley roots. In the same concentration it severely depresses uptake of sodium and of rubidium. It does not seem to affect the permeability of the membrane since roots treated with desorption solutions which were 10% in DMSO did not lose more of the preferred ion than did roots desorbed in solutions not containing DMSO. Oxygen utilization (measured in the Warburg respirometer) was reduced when DMSO was present. It is suggested that DMSO is a poisoning agent which interferes with cation transport by attacking some aspect of metabolism and not by influencing the permeability of the membrane.     

The effect of cryopreservation on the cytogenetic characteristics of a cell subline of skin fibroblasts from the Indian muntjac

After thawing cells, previously cryopreserved in the presence of dimethyl sulfoxide (DMSO), a decrease in their viability and increase in unscheduled DNA synthesis was observed. In 7 days, these parameters restored to the control level. Cryopreservation without DMSO resulted in the decrease in both cell viability and replicative and unscheduled DNA synthesis. In 14 days, these characteristics were seen to return to the normal level. Cryopreservation of cells without DMSO and their preservation in liquid nitrogen induced the frequency of chromosomal aberrations, mostly chromosomal breaks. The frequency of chromosomal aberrations increased with the duration of cell preservation in liquid nitrogen. The normal level was achieved following 7 days after cell thawing. Cells treated with DMSO only (without cryopreservation) display an increased number of chromosomal and chromatid breaks and translocations. Nonrandom distribution of chromosomal aberrations was observed, with particular chromosomes being involved in the appearance of dicentrics and translocations. The data obtained indicate that cryoprotective activity of DMSO is probably associated with the cell repair systems. The detected antimutagenic and mutagenic activity of DMSO may presumably reflect various conditions for its interaction with cells (with or without cryopreservation), as well as it may be specific for the muntjac cell line used in the present work.     

Osteogenic and adipogenic capacity of fibroblast-like progenitor cells derived frow human fetal liver

The study of differentiation potential of multiponent stromal progenitor cells (PCs) in embryogenesis is a crucial issue for understanding their biology and role in tissue regeneration of an adult organism. In this study in monolayer culture there were investigated osteogenic and adipogenic capacities of fibroblast-like PCs derived from human fetal liver of 8-11 gestation weeks before and after exposure to cryoprotectant dimethyl sulphoxide (DMSO). It was shown that the primary suspension of human fetal liver cells included immature stromal fibroblast-like PCs which were able to be induced into osteogenic and adipogenic differentiation. A short-time exposure of freshly isolated human fetal liver cells to cryoprotectant DMSO led to altering properties of the fibroblast-like PCs. Under subculture conditions, it was found an increase in the number of fibroblast-like PCs which were able to be induced to osteogenic differentiation in vitro. The established fact of DMSO influence on the differentiation capacity of fetal fibroblast-like PCs is necessary to take into consideration while developing cryopreservation methods for stem cells.