J. Cell Sci. 16, 39-47 (1974) 39

Printed in Great Britain

EFFECTS OF DMSO ON VACUOLE FORMATION,

CONTRACTILE VACUOLE FUNCTION, AND

NUCLEAR DIVISION IN TETRAHYMENA

PYRIFORMIS GL

J. R. NILSSON

Institute of General Zoology, University of Copenhagen, Universitetsparken is,DK--z\ooCopenhagen 0 , Denmark

SUMMARY

Increasing concentrations of dimethyl sulphoxide (DMSO) affect vacuole formation inTetrahymena, as measured quantitatively by the uptake of carmine particles. The rate ofvacuole formation decreased to about 50% of the control value in 5 0 % DMSO (v/v) and tozero in 7 5 % . At the latter concentration, the inhibition was expressed immediately; however,the effect of i-h exposure was reversible after removal of DMSO by washing. In vivo obser-vations revealed abnormal function of the contractile vacuole in 7 5 % DMSO, while cellmotility and cell division appeared to be unaffected. Although cell division occurred there waslittle or no increase in cell number, as studied over a cell generation time. Feulgen preparationsshowed that nuclear division was inhibited and that cell division resulted in one anucleate andone nucleate daughter cell. This effect was also observed in some dividing cells at lower con-centrations of DMSO. The effect of DMSO on Tetrahymena was dependent not only on theconcentration of the compound but also on the physiological state of the cells.

INTRODUCTION

Dimethyl sulphoxide (DMSO) is commonly used as a solvent for water-insolublesubstances applied to biological material, or as a radio- or cryoprotective agent (Leake,1967a; Hwang, Davis & Alexander, 1964; Wang & Marquardt, 1966; Ashwood-Smith,1967; Jacob, Rosenbaum & Wood, 1971). Recent investigations (Lappenbusch &Willis, 1971; Friend, Scher, Holland & Sato, 1971; Sato, Friend & de Harven, 1971;Furmanski & Lubin, 1972; Bohm, 1973; Dye, 1973; Kisch, Kelley, Crissman &Paxton, 1973; Preisler, Scher & Friend, 1973) have described the effects of DMSO onvarious biological systems in order to gain a better understanding of its mode of action.

A recent study showed inhibition of food-vacuole formation in Tetrahymena withcytochalasin B, but DMSO alone, used as a solvent, had no significant effect in con-centrations up to 2-5 % (v/v) (Nilsson, Ricketts & Zeuthen, 1973). The present investi-gation shows that DMSO in higher concentrations does affect vacuole formation inTetrahymena, and that the effect of 7-5 % DMSO, which completely inhibits vacuoleformation, is reversible after removal of DMSO by washing. In addition, DMSO wasfound to influence certain other cell activities in Tetrahymena, such as the function ofthe contractile vacuole and nuclear division, while other functions, such as cell motilityand cell division, apparently are unaffected.

40 J. R. Nilsson

MATERIAL AND METHODS

Tetrahymena pyriformis GL was grown axenically at 28 °C in a 2% proteose-peptone medium(PP) enriched with o-i% liver extract and salts (Plesner, Rasmussen & Zeuthen, 1964).Exponentially multiplying cultures (3-10 x io4 cells/ml) were used; the cells either remained inthe growth medium or were transferred to an inorganic salt medium (IM) (Hamburger &Zeuthen, 1957) 1 h prior to testing.

Aliquots (2 ml) of the cell suspension were incubated at 28 °C with the test amount of di-methyl sulphoxide (DMSO) (Hopkins & Williams). Usually, the cells were incubated for 1 hbefore a 10-min exposure to a standardized carmine particle suspension in the same mediumand in the same amount of DMSO (Nilsson, 1972); after the exposure, the cells were fixed in4 % glutardialdehyde in IM. The number of carmine-containing vacuoles was counted in 100randomly chosen cells, as previously described (Chapman-Andresen & Nilsson, 1968). Thecontrol samples were treated similarly, but without addition of DMSO. The mean number ofvacuoles formed per cell in the samples with DMSO was expressed as the percentage of themean number of vacuoles per cell in the controls (100%). In the reversibility tests, DMSO wasremoved by washing the cells 3 times with either PP or IM medium using a hand centrifuge(Plesner et al. 1964).

In vivo observations were made using Reichert anoptral optical system. Feulgen preparationswere made from smears of cells fixed in methanol-formalin-glacial acetic acid (Bohm, Sprenger,Schliiter & Sandritter, 1968), hydrolysed for 10 min in 1 N HC1 at 60 °C, and processed furtheraccording to the procedure described by Culling & Vassar (1961) using ordinary Schiff reagent.

Mixing of DMSO with water is exothermic (von Martin, Weise & Niclas, 1967), and additionof DMSO to 2 ml of cell suspension, to give a final concentration of 7-5 %, resulted in an increasein temperature of about 2 °C. Upon addition of DMSO the cells become wrinkled and flattenedin response to the osmotic shock, but the body shape is largely restored within a few minutes.

RESULTS

DMSO was added to Tetrahymena populations to give the following final concentra-tions in the media: 0-5, i-o, 2-5, 5-0, 6-o, 7-5, and io-o% (v/v). The rate of vacuoleformation in the presence of DMSO was measured quantitatively; after i-h exposurethe carmine particle suspension was added for a 10-min period before fixation of thecells. The data were expressed as the percentage of the control value from cellswithout DMSO; the results are shown in Fig. 1 A. A slight difference in response wasfound between cells in the growth (PP, • ) and the starvation (IM, x) media. At thelower concentrations (0-5-2-5 %) of DMSO, vacuole formation occurred at the normalrate, as shown previously (Nilsson et al. 1973); if anything, a slight increase wasobserved. In 5-0% DMSO vacuole formation was reduced to about half the controlvalue and the vacuoles usually contained only a few carmine particles. In 7-5 % and1 o-o % DMSO no vacuoles were formed and the cells were rounded due to an enlargedcontractile vacuole.

The time required for initiation of inhibition of vacuole formation was determinedusing 7-5% DMSO. When the cells were exposed simultaneously to DMSO andcarmine particles, some cells formed a single vacuole with a few carmine particles, butmost cells showed no uptake. Cells tested at later times during the i-h exposure to7-5% DMSO showed no uptake of carmine particles, as shown in Fig. IB.

Furthermore, the possible reversibility of the effect of i-h exposure to 7-5 % DMSOwas tested. The cells were exposed to carmine particles at intervals after removal ofDMSO by washing; the results are shown in Fig. ic . While full recovery was found,

Effects of DMSO on Tetrahymena 41

with cells in PP after about 1 h, the vacuole-forming capacity of the starved cells inIM did not return to the control value.

In vivo observations were made of cells in PP with 7-5 % DMSO. Cell motility andcell division appeared unaffected even after 3-h exposure. No vacuoles were seen to beformed at the cytostome, in agreement with the quantitative studies; the cells contained

o 100

•5 50

oo

100

50

DMSO concentration, %10 0 30 60 0

7-5% DMSO, min60

After 1 h in 7-5%DMSO, min

Fig. 1. Effects of DMSO (dimethyl sulphoxide) on vacuole formation in Tetrahymenaas determined by the uptake of carmine particles during a 10-min period, A, afteri-h exposure to increasing concentrations of DMSO. B, during i-h exposure to7 5 % DMSO. C, reversibility after 1 h in 7-5% DMSO. (PP, proteose-peptone(growth) medium, # ; IM, inorganic salt (starvation) medium, to which the cells aretransferred 1 h prior to testing, x .) Each point represents a mean of from 2 to 7experiments, each of which involves counts of 100 cells. The vertical bars indicate thestandard deviation of the mean. (In Fig. B each point is the mean of 4 experiments.)

a few small vacuoles (remnants of vacuoles formed prior to the exposure) and resem-bled cells starved for the same length of time. All cells had oversized contractilevacuoles (Figs. 3, 4, 6, 7), but these functioned at long intervals. Before expulsion, thecontractile vacuole often reached a size corresponding to 1-5 times that of the nucleus,i.e. a diameter of about 15 /«m; systole then lasted for about 10 s. In certain cases thediameter of the contractile vacuole was twice that of the nucleus (Fig. 7). During thefirst 45 min of exposure to 7-5 % DMSO, a single cell showed the following expulsionintervals: 35, 80, 140, 45, 55, 45, 45, 720, 125, and 1485 s. The diameter of the con-tractile vacuole varied according to the length of diastole, i.e. it was small with shortintervals and larger with longer intervals between systoles. The diameter of anormal-sized contractile vacuole was reached 20—30 s after systole, which is close tothe normal frequency of 22 s in untreated cells (N = 50). A diameter correspondingto that of the nucleus was reached after about 2 min.

The function of the contractile vacuole was affected more in the starved cells in IMthan in the cells in PP; thus in the former cells the vacuole reached a very large sizebefore expulsion. When DMSO was removed after 1 h, the function of the contractilevacuole appeared normal within 5-10 min. In both DMSO-treated and normal cells,

42 J. R. Nilsson

the cytoplasmic surface of the contractile vacuole was coated with mitochondria, asmay be seen in Figs. 2-7.

Cell division occurred at all concentrations of DMSO in PP medium, apart fromthe 10 % samples. The division rate in i-o and 7-5 % was the same as in the control,while in 2-5 and 5-0 % it was only half that value (5 experiments); this deviation fromconcentration dependency cannot so far be explained. After 1 h in io-o % DMSO afewlarge round cells appeared with 2 nuclei; these cells were probably in late divisionstages at the start of the exposure and the observation indicates that 10 % DMSO maycause a regression of the division furrow. No cell divisions were observed in the cellsin the starvation medium (IM).

Even though the division rate appeared normal in 7-5 % DMSO in PP medium, thecell number did not increase much over a 3-h period, i.e. the doubling time for thecontrol cells. In vivo observations often revealed immotility and subsequent death ofthe anterior daughter cell, while the posterior daughter appeared unaffected. Feulgenpreparation revealed abnormal nuclear division in 94% of the dividing cells (4 experi-ments). The majority of these cells showed no nuclear division, while grossly unequalnuclear division occurred in 12% of the cells (Fig. 9); in all cases the entire or themajor part of the nucleus was positioned in the posterior daughter cell (Figs. 9, 10);little or no nuclear elongation was observed (Fig. 8). Abnormal nuclear division wasalso observed at lower concentrations of DMSO, thus with 1, 2-5, and 5 % DMSO itoccurred in 10, 22, and 90 % of the dividing cells, respectively. In any sample, less than1 % anucleate cells were observed which indicates short survival of these cells.

The effect of 7'5% DMSO was more pronounced in starved cells in the IMmedium, than in cells in the growth medium (PP). Thus, in the former cells the sizeof the contractile vacuole was larger before expulsion and cell mortality was higherthan that seen in cells in PP. Furthermore, cells early in the exponential growth phasewere more affected by DMSO than cells later in this growth phase (about io6 cells/ml).Similarly, there was a difference in the size of the contractile vacuole before expulsion,but in addition the cells early in the exponential growth phase showed only 10%survival after 20 h in DMSO, while the cells later in this growth phase showed littlemortality even after 40-h exposure, but by this time all cells were small and resembledcells starved for the same length of time, apart from the enlarged contractile vacuole.

DISCUSSION

Dimethyl sulphoxide (DMSO) is an unusual solvent owing to its physicochemicalproperties. It has some properties similar to those of water with which it is fullymiscible, but it is a dipolar aprotic solvent which has a tendency to accept rather thanto donate protons (Rammler & Zaffaroni, 1967). The hydrogen bonds which existbetween DMSO and water are stronger than those existing between water molecules(Cowie & Toporowski, 1961). In DMSO the stereoisomery of fatty acids is modified(Muset & Martin-Esteve, 1965), and the conformation of proteins is reversibly altered(Rammler & Zaffaroni, 1967; Rammler, 1967, 1971).

DMSO has been widely applied as a drug carrier in clinical work because of the high

Effects of DMSO on Tetrahymena 43

rate of its penetration through dermal tissue. However, reports of severe eye damageas a result of DMSO administration has resulted in caution in its use (Leake, 19676;Waser & Benz, 1966). The fact that DMSO can be metabolized (Gerhards & Gibian,1967; Wood, 1971) suggests that the physicochemical properties of DMSO cannotalone be responsible for its action on biological material, but full understanding of themechanisms is still lacking.

The present study has shown that DMSO, in relatively low concentrations ascompared to those used in clinical work, affects at least 3 cellular functions in Tetra-hymena. Most pronounced is the effect on vacuole formation, but the function of thecontractile vacuole and the nuclear division are also affected.

The vacuole-forming capacity of the cells decreases progressively with increasingconcentrations of DMSO above 2-5% (v/v). Total inhibition of vacuole formationoccurs in 7-5 % DMSO, and DMSO acts immediately upon addition; with time thisconcentration causes starvation of the cells, but the effect is reversible after removal ofDMSO, as shown in Fig. 1. Several factors influence vacuole formation in Tetrahymenaand it may be fully inhibited by suitable concentrations of calcium, strontium, EDTA,dinitrophenol, and cytochalasin B (Nilsson, 1972, 1973, unpublished results; Nilssonet al. 1973); in addition, unpublished results indicate that the effect of glycerol onvacuole formation is similar to that of DMSO. It is not likely that the action of thesecompounds is identical, but it is conceivable that they all interfere with some propertiesof the cell membrane.

Several studies have indicated that DMSO alters the properties of the cell membraneand causes permeability changes. Thus, the conduction velocity of isolated frogsciatic nerves is slowed down by 6 % DMSO (Sams, 1967), and the electrical potentialdecreases 50-75 % in frog skin treated with 2- 5% DMSO (Franz & van Bruggen, 1967).The latency of acid phosphatase from isolated lysosomes decreases in about 2 %DMSO (Lee, 1970) when sucrose is present; however, when the substrate /?-glycero-phosphate is used as non-penetrating solute much higher concentrations of DMSO areneeded to produce the same decrease in latency (Lee, 1971); thus, the action of DMSOon membranes seems to depend upon the solutes present. That DMSO, as well asglycerol, may alter the properties of membranes has been demonstrated by Mclntyre,Gilula & Karnovsky (1974); both reagents induce redistribution of intramembranousparticles in mouse lymphocytes and the effect is reversible after removal of the agents.With respect to the effect of DMSO on endocytosis, Walters, Papadimitriou & Shilkin(1967) noted that their material (mammalian tissue) which normally shows pinocyticactivity, exhibited few pinocytic vesicles when treated with DMSO.

The function of the contractile vacuole is disturbed in Tetrahymena treated with7'5 % DMSO; in all cells the vacuole was much enlarged (compare Figs. 3, 4, 6, 7 withFigs. 2, 5), and expulsions occurred at irregular intervals. In view of the reports thatDMSO causes permeability changes, one might ask the question: Does the enlargedcontractile vacuole reflect an increased cell permeability? This, however, does notappear to be a likely explanation, because: (1) the varying expulsion intervals werecorrelated with a varying diameter of the contractile vacuole; and (2) the time elapsingfrom systole until the vacuole had reached normal size corresponded to the time

44 J- R- Nilsson

interval between expulsions in untreated cells. Furthermore, a volume correspondingto that of the nucleus was reached after about 2 min, which is close to the expected time(92-116 s) if a constant filling rate is assumed (diameter of the contractile vacuole:5 /tm; and of the nucleus: 8-9 /im). These calculations suggest that the filling rate (andthe amount expelled) per unit time of the contractile vacuole is the same in DMSO-treated cells as in normal cells, thus the cell permeability of Tetrahymena in 7-5 %DMSO cannot be greatly altered. DMSO seems to act at the level of the poremechanism of the contractile vacuole.

The inhibition of nuclear division, but not of cell division, in the presence ofDMSO is of interest since it suggests interference with a particular component. In7-5 % DMSO, but also at lower concentrations, cell division occurred in the absenceof nuclear elongation (Figs. 8-10), a process in which microtubules are involved(Tamura, Tsuruhara & Watanabe, 1969). The formation of neurites (Furmanski &Lubin, 1972) and the outgrowth of tissue culture cells (Dye, 1973) are inhibited byDMSO, in these processes microtubules may also play a role. The possible effect ofDMSO on microtubules could be an interference with their stability, with theirassembly process, or with the synthesis of their subunits. Since 10% DMSO is usedin a stabilization medium for isolated microtubules (Filner & Behnke, 1973), andsince microtubules in blood platelets, in plasma with 10% DMSO, respond readilyto cooling and rewarming by depolymerizing and repolymerizing, respectively(O. Behnke, personal communication), a possible action of DMSO could be at thesynthetic level.

Several reports have shown that DMSO interferes with the metabolic activity oftreated tissue (cells). Within the range of concentrations used in the present study,DMSO has been shown to suppress or to inhibit growth in general (Berliner & Ruh-mann, 1967; Hellman, Farrelly & Martin, 1967; Friend et al. 1971; Furmanski &Lubin, 1972; Bohm, 1973; Dye, 1973), or more specifically, the synthesis of DNA(Ashwood-Smith, 1967; Hellman et al. 1967; Hagemann & Evans, 1968), of RNA(Hellman et al. 1967; Hagemann, 1969), or of protein and lipids (Ashwood-Smith,1967). Furthermore, a marked decrease in oxygen consumption was found in frogskin treated with DMSO (Franz & van Bruggen, 1967); a similar effect is observed inDMSO-treated Tetrahymena (Skriver & Nilsson, 1974). In most of the studiesmentioned the effect of DMSO was reversible after removal of the agent.

It is difficult to draw a common conclusion on the action of DMSO in Tetrahymenaon the basis of the present study. However, one important factor may be that theeffects of DMSO are always more pronounced in rapidly growing cells than in cellslate in the exponential growth phase, i.e. cells with lower synthetic activity. Themajor effect of DMSO on Tetrahymena is that of inhibiting food-vacuole formationand if this occurred alone it could be interpreted as a membrane effect. However, theinterference with the function of the contractile vacuole and with nuclear elongationindicates additional and intracellular actions of the agent. It is likely that DMSO actsby changing the conformation of proteins in general whereby their function may bealtered.

Further information on the sites of action of DMSO in Tetrahymena may be obtained

Effects of DMSO on Tetrahymena 45

by electron microscopy; an investigation along these lines is in progress and will bereported later.

My sincere thanks to Dr Cicily Chapman-Andresen for critical reading of the manuscript andto Mrs Karen Meilvang for skilled technical assistance. The financial support of the CarlsbergFoundation is gratefully acknowledged.

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{Received 18 February 1974)

In all micrographs, the cells are oriented with the anterior end towards top of thepage.Figs. 2-7. 7n uiuo micrographs of Tetrahymena in PP medium, illustrating the differencein size of the contractile vacuole (arrows) in normal cells (Figs. 2, 5) and 7-5 % DMSO-treated cells (Figs. 3, 4, 6, 7). n, macronucleus. x 1000.Figs. 8-10. Feulgen preparations of Tetrahymena treated for 1 h with 7-5 % DMSOin PP medium, illustrating the absence of nuclear elongation during cell division(Fig. 8); this results either in grossly unequal nuclear division (Fig. 9, arrow indicatesthe nuclear fragment in the anterior daughter cell), or in anucleation of the anteriordaughter cell (Fig. 10). n, macronucleus. x 900.

Effects of DM SO on Tetrahymena

V 0

m n n

10