Dimethyl sulphoxide: A review of its applications in cell biology

Dimethyl sulphoxide is a water miscible solvent that has wide applications in cell biology. It acts as a cryoprotective agent in a variety of cells and tissues allowing prolonged storage at subzero temperatures. The action of dimethyl sulphoxide on the stability of the liquid matrix of cell membranes appears to be responsible for its effects and this appears also to be true for related effects on membrane permeability and fusion. Dimethyl sulphoxide is also known to act as an inducer of cellular differentiation and as a free radical scavenger and radioprotectant. A review of the underlying molecular basis of all these effects of dimethyl sulphoxide is presented.     

Membrane lipid peroxidation by ultrasound: Mechanism and implications

Ultrasonic radiation produced a dose-dependent linear increase in lipid peroxidation in the liposomes membrane as reflected in the measurement of conjugated dienes, lipid hydroperoxides and malondialdehydes. Ultrasound induced malondialdehyde production could not be inhibited by any significant degree by superoxide dismutase or histidine or dimethyl furan but was very significantly inhibited by butylated hydroxytoluene, cholesterol, sodium benzoate, dimethyl sulphoxide, sodium formate and EDTA. The scavenger studies indicated the functional role of hydroxyl radicals in the initiation of ultrasound induced lipid peroxidation.     

Influence of dimethyl sulphoxide on intermediate filament proteins in human rhabdomyosarcoma cell lines: modulation at subcellular level

Summary The effects of dimethyl sulphoxide have been investigated on differentiation in human rhabdomyosarcoma cell lines obtained from typically malignant, poorly differentiated tumours. The expression of cell differentiation marker proteins (desmin and vimentin) was assessed in cell lines A-204, A-673 and RD, and the modifications in expression after 3, 8 and 24 h of induction with 1.25% dimethyl sulphoxide were recorded. Protein expression in both the cytoplasm and cytoskeleton was significantly altered by treatments lasting 8 and 24 h, the most noteworthy changes being increased desmin and decreased vimentin expression. The results clearly indicate that dimethyl sulphoxide induced changes typical of differentiation in rhabdomyosarcoma cell lines A-673 and RD; less marked changes were observed in line A-204.     

The glucan components of the cell wall of baker''s yeast (Saccharomyces cerevisiae) considered in relation to its ultrastructure

1. Commercial pressed baker's yeast, and cell walls prepared from it, were extracted in various ways and the products examined by a number of techniques, including infrared spectroscopy and electron microscopy. 2. The glucan components of the walls cannot be extracted from intact yeast cells by 3% (w/v) sodium hydroxide at 75 degrees , but at least one-third of the glucan of cell wall preparations is dissolved under these conditions, and more will dissolve after ultrasonic treatment. 3. If intact cells are given a preliminary treatment with acid the wall glucans dissolve in dilute aqueous alkali. 4. Acid conditions as mild as sodium acetate buffer, pH5.0, for 3hr. at 75 degrees are sufficient for this preliminary treatment; the glucan then dissolves in 3% sodium hydroxide at 75 degrees leaving a very small residue, which contains chitin and about 1% of the initial glucan of the wall. Dissolution is hindered by exclusion of air, or by a preliminary reduction with sodium borohydride, suggesting that some degradation of the glucan by alkali is taking place. 5. After treatment with 0.5m-acetic acid for 24hr. at 90 degrees the glucan dissolves slowly at room temperature in 3% sodium hydroxide, or in dimethyl sulphoxide. The extraction with acetic acid removes glycogen and a predominantly beta-(1-->6)-linked glucan (not hitherto recognized as a component of baker's yeast), but none of the beta-(1-->3)-glucan, which remains water-insoluble. 6. Without treatment with acid, the glucan is not significantly soluble in dimethyl sulphoxide, but can be induced to dissolve by ultrasonic treatment. 7. These results are interpreted by postulating the presence of an enclosing membrane, composed of chitin and glucan, that when intact acts as a semipermeable membrane preventing the escape of the alkali- and dimethyl sulphoxide-soluble fraction of the glucan. Mild acid treatments damage this membrane, and ultrasonic and ballistic disintegration disrupt it. 8. Some support for this hypothesis is given by the effects of certain enzyme preparations, which have been found to render a substantial part of the glucan extractable by dimethyl sulphoxide.     

Studies on horseradish peroxidase in dimethyl sulphoxide/water mixtures. The activation of hydrogen peroxide and the binding of fluoride.

We studied the variation in spectra and in reactivity towards H2O2 of solutions of horseradish peroxidase in dimethyl sulphoxide/water mixtures, obtained by diluting stock solutions of the enzyme in either water or dimethyl sulphoxide, and assayed the enzyme activity and studied the binding of F- by the peroxidase in 65% (v/v) dimethyl sulphoxide. A broadly similar pattern of changes is observed whether one starts from water or from dimethyl sulphoxide; the changes are essentially reversible, though hysteresis is observed. When the dimethyl sulphoxide content of the solvent mixture is increased, the peroxidase retains its ability to activate H2O2 up to 74% (v/v) dimethyl sulphoxide. The peroxidase in 65% (v/v) dimethyl sulphoxide binds F- together with a proton (or the equivalent loss of HO-), as already established for aqueous solutions. We point out that the occurrence in such solutions of both the ability to activate H2O2 and the inability to bind F- without taking up H+ or losing HO- supports the proposed mechanism for activating H202, whereby the protein binds the substrate in the form of the much more reactive HO2-.     

Flow cytometry is a new method for the characterization of intestinal plasma membrane.

The highly differentiated plasma membrane of rabbit enterocytes constitutes an interesting model for membrane studies. Flow cytometry allows combined measurements of the size of membrane vesicles by light-scatter and fluorescence polarization at a single-particle level. The degree of fluorescence polarization of 1,6-diphenylhexa-1,3,5-triene was determined at 4, 18 and 37 degrees C in the brush-border and basolateral plasma membranes. The fluorescence polarization was considerably higher in brush-border than in basolateral membranes. After incubation with dimethyl sulphoxide, the membrane fluidity decreased in both types of membranes. Moreover, a time-effect study of dimethyl sulphoxide showed changes in fluorescence polarization. Only in brush-border membrane a temporary fluid phase was observed. The different properties of the two membrane domains in relation to the lipid-protein dynamics of enterocytes are discussed.     

Fatty acid utilization during development of the rat

The effects of dimethyl sulphoxide and glycerol on ox brain microsomal Na(+)+K(+)-stimulated adenosine triphosphatase (EC 3.6.1.3), K(+)-stimulated p-nitrophenyl phosphatase and K(+)-dependent muscle pyruvate kinase (EC 2.7.1.40) were studied. Dimethyl sulphoxide at concentrations below 20% (v/v) was found to stimulate the p-nitrophenyl phosphatase and pyruvate kinase by increasing their affinity for K(+) but to inhibit the Na(+)+K(+)-stimulated adenosine triphosphatase. The latter enzyme activity was also inhibited by glycerol, which like dimethyl sulphoxide, stimulated the K(+)-activated p-nitrophenyl phosphatase at a wide range of concentrations. The solvent effects were promptly reversed by dilution. Similarity was found between glycerol and dimethyl sulphoxide, on one hand, and ATP, on the other, in their stimulatory effect and their ability to increase the ouabain- and oligomycin-sensitivity of the K(+)-stimulated p-nitrophenyl phosphatase. However, only the solvents, not the ATP, increased the binding of K(+) by the microsomes. From the above findings it is suggested that solvents may act on K(+)-dependent enzymes by altering the state of solvation of the activating cation as well as by changing the enzyme structure.     

Effect of dimethyl sulphoxide on the crystal structure of porcine pepsin

The structure of porcine pepsin crystallized in the presence of dimethyl sulphoxide has been analysed by X-ray crystallography to obtain insights into the structural events that occur at the onset of chemical denaturation of proteins. The results show that one dimethyl sulphoxide molecule occupies a site on the surface of pepsin interacting with two of its residues. An increase in the average temperature factor of pepsin in the presence of dimethyl sulphoxide has been observed indicating protein destabilization induced by the denaturant. Significant increase in the temperature factor and weakening of the electron density have been observed for the catalytic water molecule located between the active aspartates. The conformation of pepsin remains unchanged in the crystal structure. However, the enzyme assay and circular dichroism studies indicate that dimethyl sulphoxide causes a slight change in the secondary structure and complete loss of activity of pepsin in solution.     

Studies on haemin in dimethyl sulphoxide/water mixtures.

The nature of the complexes and equilibria shown by solutions of protohaemin in dimethyl sulphoxide/water mixtures and in the presence of acid and base were studied by u.v.-visible spectrophotometry. In neutral solutions containing from 40 to 100% dimethyl sulphoxide, haemin is present as a monomeric complex in which the Cl-ion is not coordinated. Only a single pH-dependent equilibrium pK12 is observed over the range 40-80% dimethylsulphoxide, corresponding to formation of the mu-oxo dimer. As the dimethyl sulphoxide content is lowered below 35%, so the single equilibrium (pK12) is replaced by two equilibria (pK1 and pK2); with solutions of 5 microM-haemin, pK1 decreases (from pK12 7.55 in 65% dimethyl sulphoxide to pK1 approx. 1.5 in 0.01% dimethyl sulphoxide), whereas pK2 hardly changes (from pK12 7.55 in 65% to pK2 approx. 7.5 in 0.01%).     

Lipid peroxidation associated protein damage in rat brain crude synaptosomal fraction mediated by iron and ascorbate

In crude synaptosomal fractions from rat brain exposed to iron and ascorbate, enhanced lipid peroxidation (more than 3-fold compared to control), loss of protein thiols up to the extent of 40% compared to control, increased incorporation of carbonyl groups into proteins (more than 4.5-fold compared to control) and non-disulphide covalent cross-linking of membrane proteins have been observed. The phenomena are not inhibited by catalase or hydroxyl radical scavengers like mannitol or dimethyl sulphoxide. However, chain breaking antioxidants like alpha-tocopherol and butylated hydroxytoluene prevent both lipid peroxidation and accompanying protein oxidation. It is suggested that in this system lipid peroxidation propagated by the decomposition of preformed lipid hydroperoxides by iron and ascorbate is the primary event and products of the peroxidation process cause secondary protein damage. In view of high ascorbate content of brain and availability of several transition metals, such ascorbate mediated oxidative damage may be relevant in the aetiopathogenesis of several neurodegenerative disorders as well as ageing of brain.     

Low concentrations of dimethyl sulphoxide accelerate conjugation and incorporation of glycine and glucosamine intetrahymena

Summary Dimethyl sulphoxide at relatively low comentrations, 0.01 to 1 mM, enhanced the conjugation and cell-to-cell adhesion of complementary strains of matingTetrahymena thermophila. The time required to form stable conjugates was reduced by dimethyl sulphoxide. This chemical stimulated the uptake of glycine and glucosamine from the suspending media. Incorporation of 2-¹⁴C-glycine and 6-³H-D-glucosamine into protein and glycoprotein was enhanced in whole cells, surface membrane and cilia. Incorporation of glucosamine into the microsomal fraction was increased in the dimethyl sulphoxidetreated cells while there was little change in glycine incorporation. There were no detectable changes in glycine and glucosamine incorporation into the nuclear fractions isolated from conjugatingTetrahymena exposed to dimethyl sulphoxide.     

Effect of cryoprotectors on the structural state of liposomes cooled to minus 25 degrees C

Cryoprotectors (propylene glycol), ethylene glycol, polyethylene glycol-1500 and dimethyl sulphoxide) are studied for their effect on permeability of liposomes for incorporated molecules of 5,5-dithiobis-2-nitrobenzoic acid (DTNB) under cooling within a temperature range from 0 degree C to -25 degrees C. A similarity is found in the way of ethylene glycol and propylene glycol, dimethyl sulphoxide and polyethylene glycol-1500 effect on the liposome permeability way. Cooling in the presence of ethylene glycol and propylene glycol causes changes in liposome permeability with a local maximum at -18 degrees C. In the medium with 2M NaCl and ethylene glycol, liposomes were resistant to cooling. Dimethyl sulphoxide and polyethylene glycol-1500 induced a two-phase kinetics of changes in liposome permeability, the first phase being within the 0 = -9 degrees C and the second--within -9--25 degrees C temperature ranges. The found differences are supposed to be associated with the effect of the cryoprotective compounds on the lipid crystallization in a lower-temperatures range.     

An approach towards understanding the genesis of sunlight-induced skin cancer

The molecular basis of the sunlight-induced skin carcinogenesis has been elucidated. Of the two ultraviolet components of sunlight that reach the earth's surface the UV-B is known to be carcinogenic but the mode of action of UV-A, the predominant component of sunlight, is ill understood. Using the liposomes as a model system, it has been shown here that UV-A causes dose-dependent lipid peroxidation as estimated by measurements of conjugated dienes, lipid hydroperoxides, malondialdehydes and the fluorescent adducts (Schiff bases) produced by the reaction of MDA with glycine. Direct exposure to sunlight has also been shown to cause dose-dependent lipid peroxidation. The UV-A induced lipid peroxidation has also been shown to be dependent on dose rate. While the sodium formate, dimethyl sulphoxide, superoxide dismutase and EDTA do not have any significant effect, sodium azide, histidine, beta-carotene and dimethylfuran were shown to inhibit significantly the UV-A induced lipid peroxidation, thereby providing significant evidence of the involvement of singlet oxygen (1O2) as the initiating agent. The use of D2O in place of H2O as the liposome dispersing medium enhanced to great extent the UV-A induced lipid peroxidation, thereby lending additional support to the finding that singlet oxygen was the initiating agent. The possible mode of formation of 1O2 on exposure to UV-A was discussed. This study also highlighted the role of environmental factors on the sunlight-induced cutaneous damage. Finally, the relation between lipid peroxidation, DNA damage and carcinogenesis has been discussed in a way to suggest the possible link between sunlight exposure and causation of skin cancer.     

Solution structures of ferrihaem in some dipolar aprotic solvents and their binary aqueous mixtures

1. Conductivity and u.v. and visible spectroscopic techniques were used to investigate the solution structure of the prosthetic group of the ferric haemoproteins (ferrihaem) in dimethyl sulphoxide, NN-dimethylacetamide, NN-dimethylformamide and sulpholane, and certain of their aqueous mixtures. 2. In neutral or acid dimethyl sulphoxide, chlorohaemin is monomeric and completely dissociated into Cl⁻ion and a ferrihaem species with dimethyl sulphoxide molecules in the fifth and sixth co-ordination positions on iron. 3. In neutral NN-dimethylacetamide and NN-dimethylformamide chlorohaemin is monomeric but is largely undissociated, giving different spectra from that of chlorohaemin in dimethyl sulphoxide. On acidification, dissociation occurs and the dimethyl sulphoxide type of spectrum results. 4. Studies in a fourth solvent, sulpholane, indicate that solvent co-ordinating power (ligand strength) rather than bulk dielectric constant is responsible for dissociation of chlorohaemin. 5. In neutral dimethyl sulphoxide–water mixtures chlorohaemin remains monomeric and completely dissociated, and spectra are independent of mixture composition, except at high water concentrations, when precipitation occurs. In alkaline dimethyl sulphoxide–water mixtures, where the complete solvent mixture range is accessible, ferrihaem is polymeric (probably dimeric) and spectra are dependent on solvent composition. A quantitative analysis indicates that the spectral changes are due to replacement by water of one molecule of co-ordinated dimethyl sulphoxide per ferrihaem aggregate, and do not involve a two-molecule replacement as has been suggested for the alkaline pyridine–water system.     

Osmotically inactive volume, hydraulic conductivity, and permeability to dimethyl sulphoxide of human mature oocytes

Controlled ovarian stimulation during an in vitro fertilization cycle usually produces large numbers of oocytes and, consequently, it is likely that more embryos will be generated than can be transferred in a given cycle. It is desirable to freeze-bank surplus oocytes before insemination to avoid the ethical and legal complications of disposing of or storing embryos. Although many attempts have been made to cryopreserve human oocytes, to date, post-thaw survival has been poor, and viable pregnancies after in vitro fertilization have been rare. A possible explanation for the lack of success is that the freezing methods have been adapted from animal studies but have not been optimized for the human oocyte. In this study, video microscopy was used to determine the volumetric responses of mature human oocytes to changes in osmolarity during preparation for freezing. A Boyle van't Hoff plot of data collected in static experiments with fresh human oocytes gave a value of 0.19 +/- 0.01 (mean +/- SEM) for the osmotically inactive volume. Dynamic measurements during exposure to dimethyl sulphoxide at room temperature (22 degrees C) were analysed by a two-parameter transport model and produced values of 1.30 x 10(-6) cm atm-1 s-1 for the hydraulic conductivity of the plasma membrane and 3.15 x 10(-5) cm s-1 for dimethyl sulphoxide permeability (chi-squared = 0.43, df = 20) of fresh human oocytes. Oocytes that had failed to fertilize had a slightly lower hydraulic conductivity and dimethyl sulphoxide permeability and, after exposure to 1.5 mol dimethyl sulphoxide l-1, these cells appeared to become permeable to normally impermeable solutes. These permeability properties have been used to design a protocol for the addition and removal of dimethyl sulphoxide to control the magnitude of volumetric changes.     

A water molecule participates in the secondary structure of hyaluronan.

The structure of hyaluronan was investigated in water/dimethyl sulphoxide mixtures by using high-field n.m.r. and space-filling molecular models. The secondary structure previously established in detail in 'dry' dimethyl sulphoxide [Heatley, Scott & Hull (1984) Biochem. J. 220, 197-205] undergoes changes on addition of water, compatible with the incorporation of a water bridge between the uronate carboxylate and acetamido NH groups. Molecular models show that such a configuration is highly probable, and saturation-transfer experiments yield rates of NH proton exchange that support this proposed structure. The existence of two distinct stable configurations for hyaluronan, in water-rich and water-poor conditions respectively, may have biological implications, e.g. during its biosynthesis in cell membranes. There are extensive hydrophobic regions in both forms, which may be important for interactions with e.g., membranes, proteins and itself.     

Dimethyl sulphoxide protects cells against polypeptide toxins and poliovirus.

The effect of dimethyl sulphoxide and other cryoprotective compounds on the sensitivity of cells to polypeptide toxins and to poliovirus was tested. In the presence of these compounds, which all affect membrane fluidity, the cells were protected against the toxic proteins and against poliovirus. The large protection obtained was not due to reduced binding and endocytosis of the toxins. Apparently, the cryoprotective compounds interfere with the entry of toxins and of the poliovirus genome across the cell membrane.     

Experimental study and analysis of mechanical properties of frozen rabbit aorta by fracture mechanics approach

In order to investigate fracture problems associated with cryopreservation of aorta, experiments were carried out to study the effects of temperature, freezing rate and cryoprotective agent on the fracture mechanics properties of frozen rabbit aorta and a test method for crack criterion of frozen rabbit aorta was developed. Results showed that as temperature decreases, the fracture modes of frozen rabbit aorta changed from typical ductile fracture to typical brittle fracture, the crack was more difficult to grow as temperature decreased, and its resist-fracture ability weakened remarkably from -20 to -80 degrees C. However, freezing rates had no effects on the fracture modes when sample was cooled to -50 degrees C, while the resist-fracture ability was stronger when the sample was cooled at a higher freezing rate. Due to the hydration action of dimethyl sulphoxide (DMSO), the rabbit aorta permeated by 10% (v/v) DMSO presented typical ductile fracture when it was cooled to -50 degrees C, so its resist-fracture ability was enhanced obviously. Compared to the axial sample, the circumferential sample's resist-fracture ability is better because of its larger deformation energy available.     

Effect of capsaicin and dimethyl sulphoxide (DMSO) on ion transport in isolated skin of frog (Rana esculenta L.)

The effects ofcapsaicin, dimethyl sulphoxide and pH changes on transport of sodium and/or chlorine ions in an isolated frog skin, were studied using electrophysiological methods, adapted to evaluation of ionic currents occurring in the epithelial tissues and organs. The experiment consisted in measuring potential difference (PD in mV) of an isolated skin of the aquatic frog, Rana esculenta L., placed in a Ussing apparatus. The ionic transport processes were modified through incubation of the tissue in Ringer solution and in Ringer solution supplemented with amiloride, bumetanide, and also with dimethyl sulphoxide. The direct effect of capsaicin and dimethyl sulphoxide (DMSO) on frog skin was assessed while these compounds were added to the Ussing chamber with a pipette and a peristaltic pump. Adaptive reactions of the tissue were assessed following at least 60-min exposure to those compounds. It has been demonstrated that amiloride-inhibited sodium ion transport and acidification of the incubation medium (pH 6.4) inhibited mechanically induced epithelium reactions. Both compounds, capsaicin and DMSO modified ionic transport processes depending on the mechanical stimulation.     

Inhibition of distinct steps in the adipocyte differentiation pathway in 3T3 T mesenchymal stem cells by dimethyl sulphoxide (DMSO)

Abstract. The process of adipocyte differentiation in murine 3T3 T mesenchymal stem cells involves three well-defined steps: 1 predifferentiation growth arrest; 2 nonterminal (reversible) differentiation and 3 terminal differentiation associated with the irreversible loss of proliferative potential. To further investigate these processes, the effects of dimethyl sulphoxide (DMSO), an agent that affects differentiation in several other cell systems, was tested. The results show that DMSO modulates two distinct steps of adipocyte differentiation. The first effect is evident when growing 3T3 T cells are cultured in differentiation-inducing medium in the presence of DMSO. Therein the expression of adipocyte phenotype is inhibited because the cells fail to growtharrest at the predifferentiation growth arrest state. Instead in the presence of DMSO, cells growth-arrest at a biological state that does not support differentiation. The second effect is evident if nonterminally differentiated adipocytes are cultured in terminal differentiation-inducing medium containing DMSO. Therein the terminal step in differentiation is inhibited. These inhibitory effects occur in a dosage-dependent manner; maximum inhibition of differentiation requires 2% DMSO. Therefore, whereas DMSO typically promotes differentiation in other cell systems, DMSO inhibits multiple steps in the process of adipocyte differentiation. These observations support the conclusion that a single pharmacological agent can have markedly different effects on specific cell types. Even more important, the data establish that DMSO can now be used as a tool to study the molecular mechanisms involved in the multistep process of adipocyte differentiation.