Differentiation of erythroleukemic cells in vitro: irreversible induction by dimethyl sulfoxide (DMSO).

The inclusion of DMSO in the media of suspension cultures of Friend erythroleukemia cells results in the erythroid differentiation of these cells. The studies reported here were directed towards answering two questions. (1) How long an exposure to DMSO is necessary to induce the differentiation of these cells; and (2) What is the fate of the differentiating cells when DMSO is removed from the medium. Exposure to DMSO for less than 24 hours failed to produce any detectable evidence of erythroid differentiation. On the other hand, culture in the presence of DMSO for 24 hours followed by culture in DMSO-free medium for four additional days produced a small but detectable increment in the proportion of benzidine positive cells in the culture. Once the differentiation of an individual cell was initiated, the process continued after removal of DMSO from the medium. The cell became progressively more differentiated as evidenced by increases in the intensity of benzidine staining as well as the rate of heme synthesis and heme content. However, when cells which had been induced to differentiate by DMSO were cultured in DMSO-free medium for more than 3--4 days, they became vacuolated and apparently died. This latter phenomenon, as well as the more rapid proliferation of the undifferentiated cells in the culture, accounts for the observation that when new cultures are established from cultures which have been grown in the presence of DMSO for several days, the culture which results ultimately contains only differentiated cells.     

Early inhibition of phospholipid synthesis in dimethyl sulfoxide (DMSO) treated Friend erythroleukemia (FL) cells.

Phosphate metabolism in Friend erythroleukemia cells undergoing DMSO-induced differentiation was studied. Thirty minutes after the cells were exposed to DMSO in medium at pH 7, an inhibition of 39% in the incorporation of phosphate into phospholipids was observed. This decrease was not due to a change in the precursor pools since phosphate uptake and the phosphorylation of the organic soluble compounds were only inhibited 13%. Inhibition of phospholipid synthesis preceded inhibition of RNA and protein synthesis and reached a maximum after 24 hours of DMSO treatment. At this time, the phospholipid content of the cells was also decreased as compared to that of the control untreated cells. Phospholipid synthesis remained at a level significantly lower than in the controls over the 4-day observation period, at which time 85% of the treated cells were benzidine positive. Separation of the different phospholipids by chromatography on thin layer silicate gel plates showed that, after one hour of DMSO treatment, more than 80% of the radioactivity was in phosphatidylcholine, phosphatidylserine and phosphatidylethanolamine. Phosphatidylethanolamine was the most inhibited. Incorporation of inositol into phospholipid was also significantly decreased. However, there was little variation in the phospholipid composition of the treated and non-treated cells other than a decrease in the percent of sphingomyelin after 48 hours of DMSO treatment. These changes in phospholipid metabolism may initiate the first step in the complex differentiation process. The phospholipids are important components of membranes and the inducers are known to influence their fluidity.     

Cyclase and phosphodiesterase activity on pre-T lymphoid human cells, treated with dimethyl sulfoxide (DMSO)

Our aim is to estimate the role of the DMSO on pre-T lymphoid human cells, we have searched the cyclase and phosphodiesterase activity. We have studied the GTPspecific cyclase (G-Case) and have observed an analogous course to that one of the cAMP-PDE, where, in both cases, the differences ratio is approximately 5. For the cyclase activity values it has been found that cAMP neo formed is undeterminable in these cells, for the controls and the treated samples.     

Effect of dimethyl sulfoxide (DMSO) on radiation-induced heteroallelic reversion in diploid yeast

Dimethyl sulfoxide has cryoprotective and radioprotective properties. It is also an efficient scavenger of radicals produced by radiolysis of water. Gamma-induced reversions of diploid yeast in the presence of this chemical during irradiation have been studied. The dose-modifying factor was in the same range as for survival. When the yeast was irradiated in the frozen state the observed protection by DMSO disappeared. The results are discussed in terms of direct and indirect actions of radiations and the radical-scavenging ability of this chemical.     

Functional preservation of the mammalian kidney. IV. Functional effects of perfusion with dimethyl sulfoxide (DMSO) at normothermia

Rabbit kidneys were perfused at 37 °C with various concentrations of DMSO in a K⁺-Mg²⁺-rich perfusate. The effects of DMSO on various functional parameters of the rabbit kidney perfused for 60 min were compared with the functional effects of perfusion without DMSO under the same conditions. DMSO produced deviations in vascular resistance and perfusate flow rate from control values. In kidneys perfused with 1.4 and 2.8 m DMSO these vascular changes resulted in changes in GFR at relatively unchanged filtration fractions. The closely parallel relationship between changes in GFR and urine flow rate in all groups indicates that perfusion per se or perfusion with DMSO may shift the regulation of urine flow rate from tubular reabsorption, which obtains in the in vivo situation, to glomerular filtration. This view was supported by the relatively unchanged parameters of Na⁺ reabsorption and fractional water excretion during perfusion with all concentrations of DMSO. Additionally, DMSO perfusion resulted in significantly greater weight gains than those observed in kidneys perfused without DMSO, and significantly depressed clearances of PAH, with 2.1 and 2.8 m DMSO.     

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.     

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

Abstract

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.     

Failure to detect nephrotoxicity of chronically administered dimethyl sulfoxide (DMSO) in rats.

Male and female rats received daily intraperitoneal injections of dimethyl sulfoxide (DMSO; 2 or 4 g/kg) or saline for 28 days. At the end of that time, the ability of the animals' kidneys to transport p-aminohippurate (PAH) and N-methylnicotinamide (NMN) was tested in an in vitro system.Both doses of DMSO used produced measurable toxicity of a general nature: There was some mortality, and growth of the animals was retarded. However, DMSO had no detectable nephrotoxic action. Terminal serum urea and creatinine concentrations were normal in all treatment groups, and there was no effect of DMSO on the uptake of PAH or NMN by renal cortical slices. The data suggest that DMSO does not have an important specific toxic effect on the kidneys in this species.     

Lipase-catalyzed esterification of fatty acid in DMSO (dimethyl sulfoxide) modified AOT reverse micellar systems

AOT reverse micellar system was modified with DMSO for improved esterification activity of Chromobacteriumviscosum lipase (glycerol–ester hydrolase, EC 3.1.1.3). The enzymatic activity was strongly affected by the concentration of DMSO, and maximum activity was obtained at 30–40 mM. The various relevant physical parameters such as w₀ (molar ratio of water to AOT), pH and reaction temperature that influence the activity of lipase were studied in order to obtain the best value and compared with those in simple AOT reverse micelles. The apparent activation energy decreased in the presence of DMSO. The stability of lipase entrapped in modified AOT systems was excellent, and the half-life was about 3.25 times than that observed in simple AOT systems at 25°C. A simple first-order deactivation model was considered to determine the deactivation rate constant. The thermodynamic stability of lipase in reverse micelles was measured by the Gibbs free energy. A fluorescence study was performed to provide information on structural changes in AOT reverse micelles which was accompanied by the addition of DMSO.     

Lipase-catalyzed esterification of fatty acid in DMSO (dimethyl sulfoxide) modified AOT reverse micellar systems

AOT reverse micellar system was modified with DMSO for improved esterification activity of Chromobacterium viscosum lipase (glycerol-ester hydrolase, EC 3.1.1.3). The enzymatic activity was strongly affected by the concentration of DMSO, and maximum activity was obtained at 30-40 mM. The various relevant physical parameters such as w₀ (molar ratio of water to AOT), pH and reaction temperature that influence the activity of lipase were studied in order to obtain the best value and compared with those in simple AOT reverse micelles. The apparent activation energy decreased in the presence of DMSO. The stability of lipase entrapped in modified AOT systems was excellent, and the half-life was about 3.25 times than that observed in simple AOT systems at 25°C. A simple first-order deactivation model was considered to determine the deactivation rate constant. The thermodynamic stability of lipase in reverse micelles was measured by the Gibbs free energy. A fluorescence study was performed to provide information on structural changes in AOT reverse micelles which was accompanied by the addition of DMSO.     

Myocardial function during hypoxia: Effects of dimethyl sulfoxide (DMSO) and different buffers

Stimulated rabbit left atria in various buffer solutions [bicarbonate, Tris (hydroxymethyl) aminomethane (Tris), or both] in the presence or absence of 0.84 (6%) dimethyl sulfoxide (DMSO) were subjected to mild or severe hypoxia. Contractile strength and baseline tone changes were measured and analyzed. Oxygen consumption of right and left atria was measured before and after hypoxia in the above mentioned solutions. Results indicate (1) that DMSO allows cardiac muscle to maintain contractility during hypoxia, (2) that the absence of bicarbonate during hypoxia compromises tissue function, and (3) that Tris can exhibit toxic effects which are increased in the presence of DMSO.     

Induction of umu gene expression in Salmonella typhimurium TA1535/pSK1002 by dimethyl sulfoxide (DMSO)

The genotoxicity of dimethyl sulfoxide (DMSO) was demonstrated by the umu test using Salmonella typhimurium TA1535/pSK1002 carrying the umuC-lacZ fusion gene. The level of beta-galactosidase activity which shows umu gene expression in the test system was dependent on the concentration of DMSO in the culture medium. The maximum beta-galactosidase activity was approximately 3.5 times as high as the background level with 10% of DMSO in the culture medium. The lowest concentration of DMSO required for a response of over twice the background level was approximately 5%. Four structurally related chemicals (acetone, di-n-butylsulfoxide, dimethylsulfide, methylphenylsulfoxide) did not show umu gene expression at their non-toxic doses.     

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.