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The action of dimethyl sulphoxide and DNase on the fine structure of Chironomid salivary gland cells
J. Insect Physiol., 1968, Vol. 14, pp. 847 to 854. Pergamon Press. Printed in Great Britain
THE ACTION OF DIMETHYL SULPHOXIDE AND DNASE ON THE FINE STRUCTURE OF CHIRONOMID SALIVARY GLAND CELLS S. J.
BERRY
and W.
DIETZ*
Department of Biology, Wesleyan University, Middletown, Connecticut 06457, and Marine Biological Laboratories, Woods Hole, Massachusetts (Received 5 December 1967) Abstract-Dimethyl sulphoxide (DMSO) has been shown to facilitate the entrance into cells of substances normally excluded by permeability barriers. Treatment of isolated Chironomid salivary glands with DMSO and DNase resulted in destruction of the polytene chromosomes. DNase by itself produced no observable changes in cell morphology, and it 1s assumed that it is excluded from the cell because of the size of the molecule. Light and electron microscope studies of nuclei digested in situ reveal uniformly distributed, weakly Feulgenpositive material which consisted of fibres and granules. The undigested granules in treated nuclei appear to be mainly ribosomes and messenger RNAprotein complexes. No cytoplasmic effects of DNase were detected, but DMSO produced rather extreme structural alterations. These alterations appeared to be consistent with the uptake of water, and consisted mainly of bloating of membrane-bounded structures. Evidence is presented which indicates that these physical alterations may not seriously affect functions such as RNA synthesis and respiration. INTRODUCTION
CONSIDERABLEinterest pharmacological interest
actions
generated,
has recently developed in biological and particularly of dimethylsulphoxide (DMSO). As an example of the
a symposium
publication
New York Academy of Sciences Vol. various
aspects
of the chemistry,
of over
141, article
metabolism,
600
pages
1) has recently
and pharmacological
(Annals
of the
been devoted activity
to
of this
drug. Among the interesting properties of DMSO are the ability to protect cells from radiation and freezing damage (ASHWOOD-SMITH,1967), and its ability to penetrate even such refractory tissue as human skin (KLIGMAN, 1965). Along with the ability to penetrate cellular membranes, DMSO has remarkable ability to act as a vehicle for the transport of substances into and across cells (HORITAand WEBER, 1964; JACOB et al., 1964). Although DMSO may have some long-term sideeffects which make it unsuitable for use as a routine drug (discussion by RUBIN and BARNETT, 1967), it has been shown to be non-toxic even when applied to tissues in extremely high concentration. The effects of modest doses of DMSO on the fine structure of cells have not been extensively reported.
* Present address: University sylvania.
of Pennsylvania Medical School, Philadelphia, Penn847
S. J.
848
BERRYANDu’. DIETZ
In the studies to be reported here, the ability of DMSO to facilitate the entrance into cells of protein molecules normally excluded by permeability barriers was examined. The protein chosen was DNase, and the tissue used for assay of penetration was the salivary gland of the fly Chironomus thummi thummi in which the polytene chromosomes are large enough to be seen easily by routine light microscopy. Any extensive entry of DNase into the nucleus should be followed by destruction of the DNA. Hydrolysis of chromosomal DNA should be visible at the light microscope level, although more subtle changes caused by treatment with DMSO alone might be visible at the fine-structure level.
MATERIALS
AND
METHODS
Larvae of Chironomus thummi thummi were raised in aerated spring water and fed dried nettle leaves. The developing larvae were maintained under a constant photoperiodic regime of 16 hr light and 8 hr darkness. Salivary glands were dissected from last instar larvae directly into drops of sterile insect Ringer solution (EPHRUSSI and BEADLE, 1936) on a glass microscope slide. Whole-mount preparations Isolated glands were rinsed with fresh Ringer and transferred to a second slide where they were allowed to dry just enough to cause the tissue to adhere to the glass. A solution of 10% DNase (pancreatic DNase I--once recrystallized, Worthington Company, Freehold, N. J.) in insect Ringer was pipetted directly onto the tissue, and the preparation allowed to stand for 20 min. A second group of glands was prepared in the same way, and allowed to stand in 0.2 ml of several concentrations of DMSO (Baker Reagents-assayed at 99.994 DMSO). The concentrations tested were 0.02, 0.04, 0.06, 0.08, and 0.1% in insect Ringer, and the time of exposure again was 20 min. Tissues treated in this manner and untreated glands served as controls. An experimental group was treated with solutions containing both DMSO and DNase at the concentrations described above. After treatment, the tissues were rinsed with Ringer and fixed in Zenker fluid for 20 min. The tissue, still adhering to the slides, was then stained with malachite green-pyronine as described by BAKER and WILLIAMS (1965), dehydrated and cleared, and mounted under cover-slips with Permount.
FIG. 1. Light microscope preparations. (a) Untreated salivary gland, whole-mount preparation fixed in Zenker, stained with malachite green-pyronine. x 320. (b) Whole mount after 20 min DMSO, DNase treatment. Fixed and stained as in (a). x 320. (c) Section fixed and stained with osmium-ethyl gallate, untreated control. x 800. (d) Fixed as in (c), intact gland treated with 0.06% DMSO for 20 min. x 800. (e) Sections prepared as in (c) intact gland treated with 0.06% DMSO+ 5% DNase for 20 min, stained by the Feulgen procedure. x 800. (f) Autoradiogram, intact gland incubated in Ha uridine for 20 min after 20 min incubation with 0.06% DMSO. Prepared as in (c) Nu = nucleolus. x 800.
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FIG. 2. Salivary gland mitochondria. (a) Untreated control. (b) and (c) Mitochondria of glands incubated in 0"06~o DMSO for 20 rain. No significant differences were observed in mitochondria of cells treated with DMSO + DNase. In (b) the inner compartment is enlarged, while in (c) the intecristal space shows the greatest expansion.
FIG. 3. Rough endoplasmic reticulum. (a) Untreated control. (b) and (c) Endoplasmic reticulum of cells treated with 0'06% DMSO for 20 rain. In (b) the intracisternal spaces show the greatest enlargement, while in (c) the ground substance is swollen. Note apparently intact lyosomes (L).
FIG. 4. Secretory border. (a) Untreated control. The microvillae and the secretory material are clearly visible at the upper right-hand corner. (b) After treatment with 0.06% D M S O . Note the invaginated secretory material deep in the cytoplasm at S, and the absence of microvillae.
Fro. 5. N u c l e a r contents. (a) U n t r e a t e d control. (b) N u c l e o p l a s m a n d a portion of a c h r o m o s o m e showing the diffuseness caused b y D M S O t r e a t m e n t . (c) Portion of t h e nucleus a n d cytoplasm of a cell treated w i t h 0.06'~;~ D M S O plus ~"i I, D N a s e for 20 min. N o t e the absence of c h r o m o s o m a l s t r u c t u r e s a n d the enlarged nuclear pores (NP).
FIG. 6. RNA-containing structures in the nucleus. (a) Untreated control. Note the extension of the nuclear envelope into the cytoplasm, and the 400 A granules clearly visible. (b) A portion of the nucleoplasm after treatment with 0"06°4, DMSO and 5~o DNase for 20 min. Note the fibrous meshwork and granules of the correct dimensions for ribosomes and mRNA-protein complexes.
DMSO AND
DNaSe
EFFECTS
ON
CHIRONOMID
SALIVARY
GLAND
CELLS
849
Sectioned material
Salivary glands were dissected as described above, but transferred to O-5 ml sterile Ringer in the wells of a porcelain spot-plate. Control tissues were treated in one case with O.O6o/oDMSO, and 5*Oo/oDNase in the other. A third group was treated with both DNase and DMSO. The final volume of fluid was adjusted to 1.0 ml in all cases. After 20 min incubation the tissue was fixed in 1% buffered osmium tetroxide (PALADE,1952), ‘developed’ in saturated ethyl gallate, and double embedded in agar and esterwax as described by WIGGLE~WORTH(1959). The remaining glands were fixed in ice-cold 6% glutaraldehyde in O*lM cacodylate buffer, pH 7.4 (SABATINI, et al., 1963), and post-fixed in 1% osmium. The intact glands were then dehydrated and embedded in a mixture of Dow epoxy resins and dodecyl succinic anhydride by the method of LOCKWOOD (1964). Light microscope sections were cut at 1 to 4 p on a rotary microtome and either stained by the Feulgen or malachite greenpyronin procedures or simply examined without further staining. Sections for electron microscopy were cut on a Sorvall MT-2 ultramicrotome, stained with uranium acetate and lead citrate, and examined in a Phillips 200 electron microscope. Autoradiography In a third series of experiments, groups of salivary glands were treated with DMSO alone, DNase alone, and both reagents together as described in the previous section, except that after the 20 min incubation period the tissue was rinsed with Ringer and transferred to a second well containing O-5 ml fresh Ringer. To each well 10 PC tritiated uridine (0.9 c/m-mole Schwartz Bioresearch, Mt. Vernon, N.Y.) were added, and the glands were incubated for an additional 20 min. After this treatment, light microscope sections were prepared as described above. These sections were coated with Kodak NTB3 nuclear track emulsion according to methods described by KOPRIWA and LEBLOND (1962). Slides were stored at - 10°C in light-tight boxes for 21 days, and the autoradiograms developed with Kodak Dektol for 90 set at 18°C. RESULTS Whole mount preparations Examination of whole mounts revealed no differences between salivary glands treated with DNase alone and controls fixed immediately after isolation. No attempt was made to compare the puffing patterns, but otherwise DNase had no DMSO by itself had little observable appreciable effect on any cell structures. effect, except that the chromosomes appeared slightly more diffuse than in controls. In both, the integrity of the chromosomes was maintained and banding patterns were clearly visible. The effect of DNase in conjunction with DMSO was striking. The contents of the nucleus were no longer visible [Fig. 1, compare (a) and (b)] after 20 min
S. J. BERRYAND W. DIETZ
850
The effect was somewhat incubation with both reagents. concentrations of DMSO (0.02 and 0.04%), but at 0*06Oh or pyronine-staining structures tions, no methyl-greennucleus. On the basis of these observations, O*O6o/oDMSO selected for all succeeding experiments.
variable at the lowest and higher concentrawere present in the was the concentration
Sectioned material Light microscope examination of sections revealed no differences between DNase-treated and untreated tissue. The diffuseness of the chromosome structure after DMSO treatment was more obvious, but the integrity of these structures was maintained [Fig. 1, compare (c) and (d)]. Malachite green and Feulgen staining of the chromosomes was unimpaired. An additional feature of DMSO treatment which was not obvious from inspection of whole mounts was considerable vacuolation of the cytoplasm. After treatment with DMSO and DNase, no malachite-green-stained structures were present in the nucleus, but Feulgen treatment revealed faint pink staining which was uniformly distributed throughout the area bounded by the nuclear envelope (Fig. le). Occasionally, diffuse osmiophilic material probably representing remnants of the nucleolus was observed. Autoradiograms Incorporation of labelled uridine was not above background in the DNasetreated controls after the relatively short incubation period. In cells treated with DMSO light, but significant, labelling (3 grains/l0 p2 over background) was observed. A similar light incorporation was observed over the cytoplasm of cells treated with DMSO plus DNase. The nuclei of cells treated with DMSO alone showed considerable overall label (1540 grains/l0 p2), while the nuclei of cells treated with both reagents showed only sparse labelling comparable to that observed in the cytoplasm. The nucleoli of cells treated with DMSO were heavily labelled, in most the number of grains was too high to count accurately. Most of the silver grains were associated with the less dense central region of the nucleolus, while little label was associated with the dense peripheral region (Fig. If). No grains were observed over the nucleolar ‘remnants’ of cells treated with both DMSO and DNase. Fine-structure
observations
It is obvious from inspection of electron micrographs that DMSO, by itself, produced ‘considerable alteration in the fine structure of the tissue. Mitochondria were tremendously swollen [Fig. 2, compare (a) with (b) and (c)]. The area of longitudinal sections of mitochondria were determined by planimetry and were approximately three times that of controls. In some the inner compartment was enlarged, and in others the outer compartment and intercristal spaces were swollen. In most the length of the cristae was preserved in spite of the distortion of other elements. The rough endoplastic reticulum showed comparable swelling, with the cistemae enlarged in some cells and the ground substance in others
DMSO AND DNaSe
EFFECTS ON CHIRONOMID
SALIVARY GLAND CELLS
851
[Fig. 3, compare (a) with (b) and (c)l. The secretory border is characteristically lined with microvillae, but after treatment with DMSO these structures were completely abolished by swelling. Some of the secretory product appeared to become trapped by the lateral expansion of the microvillae, and can be seen as extensions of dense material invaginated deep into the swollen cytoplasm (Fig. 4a, b). There were no obvious effects of DNase plus DMSO over DMSO alone in the cytoplasm. Ribosomes appear normal, and the occasional microtubules encountered were apparently unaltered by either treatment. One consistent feature of all DMSO-treated cells was that although membrane-bounded organelles were considerably enlarged, the membranes themselves appeared to be unbroken. The fine structure of the nucleus was altered somewhat by DMSO, the chromosomes were more diffuse, as light microscope observations suggested, and the nuclear pores enlarged, although the double-membrane structure was otherwise unaltered [Fig. 5, compare (a) with (b) and (c)l. The nucleus was considerably altered after treatment with DMSO and DNase together. The chromosomes were no longer visible, and in their place was a fine network of fibrils and granules of various sizes (Fig. 6b). The most prominent granules in the treated nuclei fell into two categories: one group approximately 150 A dia., the size of ribosomes, and a second group approximately 400 A dia. It has been suggested by other workers (BEERMANand BAHR, 1954 ; STEVENSand SWIFT, 1966) that the larger granules represent a form of messenger RNA complexed with protein. The appearance of these structures in untreated tissue is shown in Fig. 6(a). DISCUSSION Both light and electron microscope observations show that DMSO can facilitate the entry of DNase into intact cells. The penetration of living cells by the relatively small RNase molecule (mol. wt. about 13,000) and the destruction of cellular RNA in situ have been demonstrated by Brachet and other workers (LANSING and ROSENTHAL,1952; BRACHET,1954; BRACHETand LEDOUX, 1955; KAUFMANand DAS, 1955). The larger DNase molecule mol. wt. 40,000 to 60,000 (LASKOWSKI,1959) does not readily enter intact cells except under special circumstances. LANDMANand SPIEGELMAN(1955) have studied its entry into lysozymetreated protoplasts of &Z&&G meguteriwn. ANDERSON(1953) and ALLFREY(1954) have demonstrated the destruction of chromosomes by DNase in isolated nuclei of liver and thymus cells respectively. The DMSO-facilitated entry of DNase into intact cells suggests that it may be possible to study the physiology of cells in which the genetic apparatus has been disrupted. Any studies of this sort would have to take into account any deleterious effects of DMSO on living cells. In the observations recorded here, besides demonstrating the facilitation of DNase entry into cells, we have attempted to assess some of the immediate effects of modest doses of DMSO on living cells. Our results indicate that some disagregation of chromosomal structure may be induced by DMSO, but the fact that labelled uridine can still be incorporated into the nucleolus and other nuclear regions suggests that such treatment does not seriously impair the RNA-synthetic
852
S. J. BERRYANDW. DIETZ
The heavy concentration of label in the central capacities of the chromosomes. region of the nucleolus is entirely consistent with the rapid labelling of newly synthesized ribosomal RNA, since it has been shown by KALNINS et al. (1964) that the chromosome passes through this region. The cytoplasmic labelling observed in both situations when DMSO was used can presumably be attributed to insufficient washing of the tissue in conjunction with DMSO-facilitated penetration of the uridine, and probably does not represent actual RNA synthesis. RNase digestion of the cytoplasm was ineffective, although most of the nuclear label was removed. The dissociation of isolated Cecropia ribosomes has been reported (APPLEBAUMet al., 1966), but the effective concentration was enormously higher (80 per cent DMSO) than that employed in the present studies. The observation of normal-appearing cytoplasmic ribosomes and 150 and 400 A particles in the nucleus after treatment with DNase and DMSO further supports the contention that modest doses of DMSO do not seriously disrupt RNA-containing structures. Electron micrographs suggest that the water content of the cells is considerably increased by DMSO. The remarkable swelling of such structures as the mitochondria, the cisternae and ground substance of the endoplasmic reticulum, and the secretory border is consistent with the uptake of water. Although the membranebounded structures are considerably bloated, the membranes are apparently still intact and uninterrupted. Preliminary determinations (COFFIN, unpublished) indicate that oxygen uptake is similar in treated and untreated, isolated glands, except that endogenous substrates seem to be more rapidly depleted in DMSOtreated tissue. Thus, although the mitochondria are grossly expanded, they appear to retain their respiratory function. It is interesting to note that the mitochondrial cristae, thought to be the site of highly organized respiratory assemblies, are often not appreciably stretched or disturbed even though the inner compartment and occasionally the intercristal spaces are enlarged. It would seem that the critical spacing of the respiratory assemblies may be maintained even though the outer membrane and stretches of the inner membrane between cristae are considerably lengthened. The mechanism by which DMSO is able to overcome permeability barriers is not well understood. It appears that the induced changes are not caused by poisoning of active transport (FRANZ and VANBRUGGEN,1967), but involve changes in the structure of the membranes. These structural changes may result in the accumulation of DMSO-complexed compounds in the cell. PUIG-MUSET and MARTIN-ESTBVE (1965) suggest that DMSO affects the membrane lipids directly, causing conformational changes by the formation of peroxides. Other workers have been impressed by the afhnity of DMSO for polar molecules and suggest that membrane proteins may be affected (RAMMLER and ZAFFARONI,1967). It has been assumed in the present studies that the hydrolysis of chromosomes after treatment with both reagents was entirely caused by permeability changes, allowing the entry of DNase previously excluded. MONDER (1967) has shown that DMSO activates DNase in w&o and increases the efficiency of hydrolysis. It
DMSOANDDNa9e EFFECTSON CHIRONOMID SALIVARY GLANDCELLS
853
seems unlikely that any significant amount of the observed hydrolysis of chromatin reflects activation of intracellular DNase, but some of the diffuseness of chromosomes after treatment with DMSO alone may be attributable to such activation. Lysosomes appear normal after DMSO treatment (Fig. 3b, c), although some of the contents might leach out without affecting the density of the contents appreciably. The presence of faintly Feulgen-positive material in nuclei in which the chromosomes had been digested suggests that the DNA had not been completely The fibrous material observed in electron microdegraded to deoxynucleotides. graphs of digested nuclei may thus represent short lengths of DNA attached to nucleoprotein. These intranuclear fibres resemble in structure those prepared from isolated chromatin by Rrs (1966). It is not clear from the present work whether tissue isolated and treated with modest doses of DMSO would recover and survive if transplanted into host animals, but it seems likely since other tissues survive treatment with doses of up to 80% DMSO administered in situ. If tissues will indeed survive the treatment, the fate of biologically active molecules such as DNase can be studied in cells from which they are normally excluded by permeability barriers. Acknowledgements-We wish to thank Dr ALAN BELL for his assistance in taking the electron micrographs and Dr. HANS LAUFER for providing Chironomid eggs. The investigations have been supported by funds from the National Science Foundation and the National Institutes of Health. REFERENCES ALLFREY V. (1954) Amino acid incorporation by isolated thymus nuclei-I. The role of deoxyribonucleic acid in protein synthesis. Proc. nat. Acad. Sci. Wash. 40, 881-885. hDJ3RSON N. G. (1953) Studies on isolate cell components--VI. The effects of nucleases and proteases on rat liver nuclei. Exp. Cell Res. 5, 361. APPLEBAUMS. W., EBSTEINR. P., and WYAT-~G. R. (1966) Dissociation of ribosomal ribonucleic acid from silkmoth pupae by heat and DMSO: evidence for specific cleavage points. J. mol. Biol. 21, 29-41. ASXWOOD-SMITHM. J. (1967) Radioprotective and cryoprotective properties of dimethyl sulfoxide in cellular systems. Amt. N. Y Acad. 5%. 141, 45-62. BAKERJ. R. and WILLIAMS E. G. M. (1965) The use of methyl green as a histochemical agent. Quart.J. v&r. Sci. 106, 3-14. BEERMANW. and BAHR G. F. (1954) The submicroscopic structure of the Balbiani ring. Exp. Cell. Res 6, 195. BRACHETJ. (1954) Effects of ribonuclease on the metabolism of living root-tip cells. Nature, Lond. 174, 836-877. BRACHETJ. and LEDOUX L. (1955) L’action de la ribonuclease sur la division des o&s d’amphibiens-2. Etude cytologique et cytochimique des effects de la ribonuclease chea le Pleurodele. Ex#. Cell Res. (Suppl. 3), 27-39. EPHRU~~IB. and BEADLBG. (1936) A technique of transplantation for Drosophila. Am. Nat. 70, 218-225. FRANZ T. J. and VAN BRUGGENJ. T. (1967) A possible mechanism of action of DMSO. Ann. N.Y. Acad. Sci. 141, 302-309. HORITAA. and WEB= J_ (1964) Skin penetrating property of drugs dissolved in dimethyl sulfoxide (DMSO) and other vehicles. Life Sci. 3, 1389.
854
S. J. BERRY ANDW. DIETZ
JACOBS. W., BISH~L M., and HERXHLER B. S. (1964) Dimethyl sulfoxide (DMSO): a new concept in pharmacotherapy. Cur*. therapeut. Res. 6, 134-135. KALNINS V. I., STICH H. F., and BENCOSMES. A. (1964) Fine structure of the nucleolar organizer of salivary gland chromosome of Chironomids. J. Ultrastruct. Res. 11, 282291. KAUFMANB. P. and DAS N. K. (1955) The role of ribonucleoproteins in the production of mitotic abnormalities. Chromosoma 7, 19-38. KLICMANA. M. (1965) Topical pharmacology and toxicology of dimethyl sulfoxide. J. Am. med. Ass. 193,796-804,923-928. KOPRIWAB. and LEBLONDC. P. (1962) Improvements in the coating technique of autoradiography. J. Histochem. Cytochem. 10, 269-286. LANDMAN 0. E. and SPIEGELMANS. (1955) Enzyme formation in protoplasts of Bacillus megaterium. Proc. nat. Acad. Sci. U.S. 41, 698-704. LANSINGA. I. and ROSENTHAL T. B. (1952) The relation between ribonucleic acid and ionic transport across the cell surface. J. cell. camp. Physiol. 30, 337-345. LA~KOWSKIM. (1959) Deoxyribonucleases. In The Enzymes (Ed. by LARDY H. and MYRBACKD.) 5,123-148. Academic Press, New York. LOCKWOOD W. R. (1964) A reliable and easily sectioned epoxy embedding medium. Anat. Rec. 150,129-140. MONDERC. (1967) Effect of DMSO on enzyme activity. Ann. N.Y. Acad. Sci. 141, 300-301. PALADEG. E. (1952) A study of fixation for electron microscopy. J. exp. Med. 95, 285-298. PUIG-MUSETP. and MARTIN-ESTEVEJ. (1965) Physiological cell permeability and pharmacological action of DMSO. Experientia 21, 649-650. RAMMLERD. H. and ZAFFARONIA. (1967) Biological implications of DMSO based on a review of its chemical properties. Ann. N. Y. Acad. Sci. 141, 13-23. RIS H. (1966) Electron microscope studies on nucleohistone fibres from interphase nuclei. J. Cell Biol 31, 134A. RUBIN L. F. and BARNETTK. C. (1967) Ocular effects of oral and dermal application of dimethyl sulfoxide in animals. Ann. N. Y. Acad. Sci. 141, 333-345. SABATINID. S., BENSCHK., BARRNE~ R. G. (1963) Cytochemistry and electron microscopy, the preservation of cellular ultrastructure and enzymatic activity by aldehyde fixation. J. Cell Biol. 17, 1958. STEVENSB. J. and SWIFT H. (1966) RNA transport from nucleus to cytoplasm in Chironomus salivary glands. J. Cell Biol. 31, 55. WIGGLESWORTH V. B. (1959) A simple method for cutting sections in the 0.5 to 1 p range, and for sections of chitin. Quart.J. micr. Sci. 100, 315-320.
THE ACTION OF DIMETHYL SULPHOXIDE AND DNASE ON THE FINE STRUCTURE OF CHIRONOMID SALIVARY GLAND CELLS S. J.
BERRY
and W.
DIETZ*
Department of Biology, Wesleyan University, Middletown, Connecticut 06457, and Marine Biological Laboratories, Woods Hole, Massachusetts (Received 5 December 1967) Abstract-Dimethyl sulphoxide (DMSO) has been shown to facilitate the entrance into cells of substances normally excluded by permeability barriers. Treatment of isolated Chironomid salivary glands with DMSO and DNase resulted in destruction of the polytene chromosomes. DNase by itself produced no observable changes in cell morphology, and it 1s assumed that it is excluded from the cell because of the size of the molecule. Light and electron microscope studies of nuclei digested in situ reveal uniformly distributed, weakly Feulgenpositive material which consisted of fibres and granules. The undigested granules in treated nuclei appear to be mainly ribosomes and messenger RNAprotein complexes. No cytoplasmic effects of DNase were detected, but DMSO produced rather extreme structural alterations. These alterations appeared to be consistent with the uptake of water, and consisted mainly of bloating of membrane-bounded structures. Evidence is presented which indicates that these physical alterations may not seriously affect functions such as RNA synthesis and respiration. INTRODUCTION
CONSIDERABLEinterest pharmacological interest
actions
generated,
has recently developed in biological and particularly of dimethylsulphoxide (DMSO). As an example of the
a symposium
publication
New York Academy of Sciences Vol. various
aspects
of the chemistry,
of over
141, article
metabolism,
600
pages
1) has recently
and pharmacological
(Annals
of the
been devoted activity
to
of this
drug. Among the interesting properties of DMSO are the ability to protect cells from radiation and freezing damage (ASHWOOD-SMITH,1967), and its ability to penetrate even such refractory tissue as human skin (KLIGMAN, 1965). Along with the ability to penetrate cellular membranes, DMSO has remarkable ability to act as a vehicle for the transport of substances into and across cells (HORITAand WEBER, 1964; JACOB et al., 1964). Although DMSO may have some long-term sideeffects which make it unsuitable for use as a routine drug (discussion by RUBIN and BARNETT, 1967), it has been shown to be non-toxic even when applied to tissues in extremely high concentration. The effects of modest doses of DMSO on the fine structure of cells have not been extensively reported.
* Present address: University sylvania.
of Pennsylvania Medical School, Philadelphia, Penn847
S. J.
848
BERRYANDu’. DIETZ
In the studies to be reported here, the ability of DMSO to facilitate the entrance into cells of protein molecules normally excluded by permeability barriers was examined. The protein chosen was DNase, and the tissue used for assay of penetration was the salivary gland of the fly Chironomus thummi thummi in which the polytene chromosomes are large enough to be seen easily by routine light microscopy. Any extensive entry of DNase into the nucleus should be followed by destruction of the DNA. Hydrolysis of chromosomal DNA should be visible at the light microscope level, although more subtle changes caused by treatment with DMSO alone might be visible at the fine-structure level.
MATERIALS
AND
METHODS
Larvae of Chironomus thummi thummi were raised in aerated spring water and fed dried nettle leaves. The developing larvae were maintained under a constant photoperiodic regime of 16 hr light and 8 hr darkness. Salivary glands were dissected from last instar larvae directly into drops of sterile insect Ringer solution (EPHRUSSI and BEADLE, 1936) on a glass microscope slide. Whole-mount preparations Isolated glands were rinsed with fresh Ringer and transferred to a second slide where they were allowed to dry just enough to cause the tissue to adhere to the glass. A solution of 10% DNase (pancreatic DNase I--once recrystallized, Worthington Company, Freehold, N. J.) in insect Ringer was pipetted directly onto the tissue, and the preparation allowed to stand for 20 min. A second group of glands was prepared in the same way, and allowed to stand in 0.2 ml of several concentrations of DMSO (Baker Reagents-assayed at 99.994 DMSO). The concentrations tested were 0.02, 0.04, 0.06, 0.08, and 0.1% in insect Ringer, and the time of exposure again was 20 min. Tissues treated in this manner and untreated glands served as controls. An experimental group was treated with solutions containing both DMSO and DNase at the concentrations described above. After treatment, the tissues were rinsed with Ringer and fixed in Zenker fluid for 20 min. The tissue, still adhering to the slides, was then stained with malachite green-pyronine as described by BAKER and WILLIAMS (1965), dehydrated and cleared, and mounted under cover-slips with Permount.
FIG. 1. Light microscope preparations. (a) Untreated salivary gland, whole-mount preparation fixed in Zenker, stained with malachite green-pyronine. x 320. (b) Whole mount after 20 min DMSO, DNase treatment. Fixed and stained as in (a). x 320. (c) Section fixed and stained with osmium-ethyl gallate, untreated control. x 800. (d) Fixed as in (c), intact gland treated with 0.06% DMSO for 20 min. x 800. (e) Sections prepared as in (c) intact gland treated with 0.06% DMSO+ 5% DNase for 20 min, stained by the Feulgen procedure. x 800. (f) Autoradiogram, intact gland incubated in Ha uridine for 20 min after 20 min incubation with 0.06% DMSO. Prepared as in (c) Nu = nucleolus. x 800.
4 L .................. ~ii!i
~i~ !!i¸il iii i!i ~ii~i~ i~ i,!~!!
FIG. 2. Salivary gland mitochondria. (a) Untreated control. (b) and (c) Mitochondria of glands incubated in 0"06~o DMSO for 20 rain. No significant differences were observed in mitochondria of cells treated with DMSO + DNase. In (b) the inner compartment is enlarged, while in (c) the intecristal space shows the greatest expansion.
FIG. 3. Rough endoplasmic reticulum. (a) Untreated control. (b) and (c) Endoplasmic reticulum of cells treated with 0'06% DMSO for 20 rain. In (b) the intracisternal spaces show the greatest enlargement, while in (c) the ground substance is swollen. Note apparently intact lyosomes (L).
FIG. 4. Secretory border. (a) Untreated control. The microvillae and the secretory material are clearly visible at the upper right-hand corner. (b) After treatment with 0.06% D M S O . Note the invaginated secretory material deep in the cytoplasm at S, and the absence of microvillae.
Fro. 5. N u c l e a r contents. (a) U n t r e a t e d control. (b) N u c l e o p l a s m a n d a portion of a c h r o m o s o m e showing the diffuseness caused b y D M S O t r e a t m e n t . (c) Portion of t h e nucleus a n d cytoplasm of a cell treated w i t h 0.06'~;~ D M S O plus ~"i I, D N a s e for 20 min. N o t e the absence of c h r o m o s o m a l s t r u c t u r e s a n d the enlarged nuclear pores (NP).
FIG. 6. RNA-containing structures in the nucleus. (a) Untreated control. Note the extension of the nuclear envelope into the cytoplasm, and the 400 A granules clearly visible. (b) A portion of the nucleoplasm after treatment with 0"06°4, DMSO and 5~o DNase for 20 min. Note the fibrous meshwork and granules of the correct dimensions for ribosomes and mRNA-protein complexes.
DMSO AND
DNaSe
EFFECTS
ON
CHIRONOMID
SALIVARY
GLAND
CELLS
849
Sectioned material
Salivary glands were dissected as described above, but transferred to O-5 ml sterile Ringer in the wells of a porcelain spot-plate. Control tissues were treated in one case with O.O6o/oDMSO, and 5*Oo/oDNase in the other. A third group was treated with both DNase and DMSO. The final volume of fluid was adjusted to 1.0 ml in all cases. After 20 min incubation the tissue was fixed in 1% buffered osmium tetroxide (PALADE,1952), ‘developed’ in saturated ethyl gallate, and double embedded in agar and esterwax as described by WIGGLE~WORTH(1959). The remaining glands were fixed in ice-cold 6% glutaraldehyde in O*lM cacodylate buffer, pH 7.4 (SABATINI, et al., 1963), and post-fixed in 1% osmium. The intact glands were then dehydrated and embedded in a mixture of Dow epoxy resins and dodecyl succinic anhydride by the method of LOCKWOOD (1964). Light microscope sections were cut at 1 to 4 p on a rotary microtome and either stained by the Feulgen or malachite greenpyronin procedures or simply examined without further staining. Sections for electron microscopy were cut on a Sorvall MT-2 ultramicrotome, stained with uranium acetate and lead citrate, and examined in a Phillips 200 electron microscope. Autoradiography In a third series of experiments, groups of salivary glands were treated with DMSO alone, DNase alone, and both reagents together as described in the previous section, except that after the 20 min incubation period the tissue was rinsed with Ringer and transferred to a second well containing O-5 ml fresh Ringer. To each well 10 PC tritiated uridine (0.9 c/m-mole Schwartz Bioresearch, Mt. Vernon, N.Y.) were added, and the glands were incubated for an additional 20 min. After this treatment, light microscope sections were prepared as described above. These sections were coated with Kodak NTB3 nuclear track emulsion according to methods described by KOPRIWA and LEBLOND (1962). Slides were stored at - 10°C in light-tight boxes for 21 days, and the autoradiograms developed with Kodak Dektol for 90 set at 18°C. RESULTS Whole mount preparations Examination of whole mounts revealed no differences between salivary glands treated with DNase alone and controls fixed immediately after isolation. No attempt was made to compare the puffing patterns, but otherwise DNase had no DMSO by itself had little observable appreciable effect on any cell structures. effect, except that the chromosomes appeared slightly more diffuse than in controls. In both, the integrity of the chromosomes was maintained and banding patterns were clearly visible. The effect of DNase in conjunction with DMSO was striking. The contents of the nucleus were no longer visible [Fig. 1, compare (a) and (b)] after 20 min
S. J. BERRYAND W. DIETZ
850
The effect was somewhat incubation with both reagents. concentrations of DMSO (0.02 and 0.04%), but at 0*06Oh or pyronine-staining structures tions, no methyl-greennucleus. On the basis of these observations, O*O6o/oDMSO selected for all succeeding experiments.
variable at the lowest and higher concentrawere present in the was the concentration
Sectioned material Light microscope examination of sections revealed no differences between DNase-treated and untreated tissue. The diffuseness of the chromosome structure after DMSO treatment was more obvious, but the integrity of these structures was maintained [Fig. 1, compare (c) and (d)]. Malachite green and Feulgen staining of the chromosomes was unimpaired. An additional feature of DMSO treatment which was not obvious from inspection of whole mounts was considerable vacuolation of the cytoplasm. After treatment with DMSO and DNase, no malachite-green-stained structures were present in the nucleus, but Feulgen treatment revealed faint pink staining which was uniformly distributed throughout the area bounded by the nuclear envelope (Fig. le). Occasionally, diffuse osmiophilic material probably representing remnants of the nucleolus was observed. Autoradiograms Incorporation of labelled uridine was not above background in the DNasetreated controls after the relatively short incubation period. In cells treated with DMSO light, but significant, labelling (3 grains/l0 p2 over background) was observed. A similar light incorporation was observed over the cytoplasm of cells treated with DMSO plus DNase. The nuclei of cells treated with DMSO alone showed considerable overall label (1540 grains/l0 p2), while the nuclei of cells treated with both reagents showed only sparse labelling comparable to that observed in the cytoplasm. The nucleoli of cells treated with DMSO were heavily labelled, in most the number of grains was too high to count accurately. Most of the silver grains were associated with the less dense central region of the nucleolus, while little label was associated with the dense peripheral region (Fig. If). No grains were observed over the nucleolar ‘remnants’ of cells treated with both DMSO and DNase. Fine-structure
observations
It is obvious from inspection of electron micrographs that DMSO, by itself, produced ‘considerable alteration in the fine structure of the tissue. Mitochondria were tremendously swollen [Fig. 2, compare (a) with (b) and (c)]. The area of longitudinal sections of mitochondria were determined by planimetry and were approximately three times that of controls. In some the inner compartment was enlarged, and in others the outer compartment and intercristal spaces were swollen. In most the length of the cristae was preserved in spite of the distortion of other elements. The rough endoplastic reticulum showed comparable swelling, with the cistemae enlarged in some cells and the ground substance in others
DMSO AND DNaSe
EFFECTS ON CHIRONOMID
SALIVARY GLAND CELLS
851
[Fig. 3, compare (a) with (b) and (c)l. The secretory border is characteristically lined with microvillae, but after treatment with DMSO these structures were completely abolished by swelling. Some of the secretory product appeared to become trapped by the lateral expansion of the microvillae, and can be seen as extensions of dense material invaginated deep into the swollen cytoplasm (Fig. 4a, b). There were no obvious effects of DNase plus DMSO over DMSO alone in the cytoplasm. Ribosomes appear normal, and the occasional microtubules encountered were apparently unaltered by either treatment. One consistent feature of all DMSO-treated cells was that although membrane-bounded organelles were considerably enlarged, the membranes themselves appeared to be unbroken. The fine structure of the nucleus was altered somewhat by DMSO, the chromosomes were more diffuse, as light microscope observations suggested, and the nuclear pores enlarged, although the double-membrane structure was otherwise unaltered [Fig. 5, compare (a) with (b) and (c)l. The nucleus was considerably altered after treatment with DMSO and DNase together. The chromosomes were no longer visible, and in their place was a fine network of fibrils and granules of various sizes (Fig. 6b). The most prominent granules in the treated nuclei fell into two categories: one group approximately 150 A dia., the size of ribosomes, and a second group approximately 400 A dia. It has been suggested by other workers (BEERMANand BAHR, 1954 ; STEVENSand SWIFT, 1966) that the larger granules represent a form of messenger RNA complexed with protein. The appearance of these structures in untreated tissue is shown in Fig. 6(a). DISCUSSION Both light and electron microscope observations show that DMSO can facilitate the entry of DNase into intact cells. The penetration of living cells by the relatively small RNase molecule (mol. wt. about 13,000) and the destruction of cellular RNA in situ have been demonstrated by Brachet and other workers (LANSING and ROSENTHAL,1952; BRACHET,1954; BRACHETand LEDOUX, 1955; KAUFMANand DAS, 1955). The larger DNase molecule mol. wt. 40,000 to 60,000 (LASKOWSKI,1959) does not readily enter intact cells except under special circumstances. LANDMANand SPIEGELMAN(1955) have studied its entry into lysozymetreated protoplasts of &Z&&G meguteriwn. ANDERSON(1953) and ALLFREY(1954) have demonstrated the destruction of chromosomes by DNase in isolated nuclei of liver and thymus cells respectively. The DMSO-facilitated entry of DNase into intact cells suggests that it may be possible to study the physiology of cells in which the genetic apparatus has been disrupted. Any studies of this sort would have to take into account any deleterious effects of DMSO on living cells. In the observations recorded here, besides demonstrating the facilitation of DNase entry into cells, we have attempted to assess some of the immediate effects of modest doses of DMSO on living cells. Our results indicate that some disagregation of chromosomal structure may be induced by DMSO, but the fact that labelled uridine can still be incorporated into the nucleolus and other nuclear regions suggests that such treatment does not seriously impair the RNA-synthetic
852
S. J. BERRYANDW. DIETZ
The heavy concentration of label in the central capacities of the chromosomes. region of the nucleolus is entirely consistent with the rapid labelling of newly synthesized ribosomal RNA, since it has been shown by KALNINS et al. (1964) that the chromosome passes through this region. The cytoplasmic labelling observed in both situations when DMSO was used can presumably be attributed to insufficient washing of the tissue in conjunction with DMSO-facilitated penetration of the uridine, and probably does not represent actual RNA synthesis. RNase digestion of the cytoplasm was ineffective, although most of the nuclear label was removed. The dissociation of isolated Cecropia ribosomes has been reported (APPLEBAUMet al., 1966), but the effective concentration was enormously higher (80 per cent DMSO) than that employed in the present studies. The observation of normal-appearing cytoplasmic ribosomes and 150 and 400 A particles in the nucleus after treatment with DNase and DMSO further supports the contention that modest doses of DMSO do not seriously disrupt RNA-containing structures. Electron micrographs suggest that the water content of the cells is considerably increased by DMSO. The remarkable swelling of such structures as the mitochondria, the cisternae and ground substance of the endoplasmic reticulum, and the secretory border is consistent with the uptake of water. Although the membranebounded structures are considerably bloated, the membranes are apparently still intact and uninterrupted. Preliminary determinations (COFFIN, unpublished) indicate that oxygen uptake is similar in treated and untreated, isolated glands, except that endogenous substrates seem to be more rapidly depleted in DMSOtreated tissue. Thus, although the mitochondria are grossly expanded, they appear to retain their respiratory function. It is interesting to note that the mitochondrial cristae, thought to be the site of highly organized respiratory assemblies, are often not appreciably stretched or disturbed even though the inner compartment and occasionally the intercristal spaces are enlarged. It would seem that the critical spacing of the respiratory assemblies may be maintained even though the outer membrane and stretches of the inner membrane between cristae are considerably lengthened. The mechanism by which DMSO is able to overcome permeability barriers is not well understood. It appears that the induced changes are not caused by poisoning of active transport (FRANZ and VANBRUGGEN,1967), but involve changes in the structure of the membranes. These structural changes may result in the accumulation of DMSO-complexed compounds in the cell. PUIG-MUSET and MARTIN-ESTBVE (1965) suggest that DMSO affects the membrane lipids directly, causing conformational changes by the formation of peroxides. Other workers have been impressed by the afhnity of DMSO for polar molecules and suggest that membrane proteins may be affected (RAMMLER and ZAFFARONI,1967). It has been assumed in the present studies that the hydrolysis of chromosomes after treatment with both reagents was entirely caused by permeability changes, allowing the entry of DNase previously excluded. MONDER (1967) has shown that DMSO activates DNase in w&o and increases the efficiency of hydrolysis. It
DMSOANDDNa9e EFFECTSON CHIRONOMID SALIVARY GLANDCELLS
853
seems unlikely that any significant amount of the observed hydrolysis of chromatin reflects activation of intracellular DNase, but some of the diffuseness of chromosomes after treatment with DMSO alone may be attributable to such activation. Lysosomes appear normal after DMSO treatment (Fig. 3b, c), although some of the contents might leach out without affecting the density of the contents appreciably. The presence of faintly Feulgen-positive material in nuclei in which the chromosomes had been digested suggests that the DNA had not been completely The fibrous material observed in electron microdegraded to deoxynucleotides. graphs of digested nuclei may thus represent short lengths of DNA attached to nucleoprotein. These intranuclear fibres resemble in structure those prepared from isolated chromatin by Rrs (1966). It is not clear from the present work whether tissue isolated and treated with modest doses of DMSO would recover and survive if transplanted into host animals, but it seems likely since other tissues survive treatment with doses of up to 80% DMSO administered in situ. If tissues will indeed survive the treatment, the fate of biologically active molecules such as DNase can be studied in cells from which they are normally excluded by permeability barriers. Acknowledgements-We wish to thank Dr ALAN BELL for his assistance in taking the electron micrographs and Dr. HANS LAUFER for providing Chironomid eggs. The investigations have been supported by funds from the National Science Foundation and the National Institutes of Health. REFERENCES ALLFREY V. (1954) Amino acid incorporation by isolated thymus nuclei-I. The role of deoxyribonucleic acid in protein synthesis. Proc. nat. Acad. Sci. Wash. 40, 881-885. hDJ3RSON N. G. (1953) Studies on isolate cell components--VI. The effects of nucleases and proteases on rat liver nuclei. Exp. Cell Res. 5, 361. APPLEBAUMS. W., EBSTEINR. P., and WYAT-~G. R. (1966) Dissociation of ribosomal ribonucleic acid from silkmoth pupae by heat and DMSO: evidence for specific cleavage points. J. mol. Biol. 21, 29-41. ASXWOOD-SMITHM. J. (1967) Radioprotective and cryoprotective properties of dimethyl sulfoxide in cellular systems. Amt. N. Y Acad. 5%. 141, 45-62. BAKERJ. R. and WILLIAMS E. G. M. (1965) The use of methyl green as a histochemical agent. Quart.J. v&r. Sci. 106, 3-14. BEERMANW. and BAHR G. F. (1954) The submicroscopic structure of the Balbiani ring. Exp. Cell. Res 6, 195. BRACHETJ. (1954) Effects of ribonuclease on the metabolism of living root-tip cells. Nature, Lond. 174, 836-877. BRACHETJ. and LEDOUX L. (1955) L’action de la ribonuclease sur la division des o&s d’amphibiens-2. Etude cytologique et cytochimique des effects de la ribonuclease chea le Pleurodele. Ex#. Cell Res. (Suppl. 3), 27-39. EPHRU~~IB. and BEADLBG. (1936) A technique of transplantation for Drosophila. Am. Nat. 70, 218-225. FRANZ T. J. and VAN BRUGGENJ. T. (1967) A possible mechanism of action of DMSO. Ann. N.Y. Acad. Sci. 141, 302-309. HORITAA. and WEB= J_ (1964) Skin penetrating property of drugs dissolved in dimethyl sulfoxide (DMSO) and other vehicles. Life Sci. 3, 1389.
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JACOBS. W., BISH~L M., and HERXHLER B. S. (1964) Dimethyl sulfoxide (DMSO): a new concept in pharmacotherapy. Cur*. therapeut. Res. 6, 134-135. KALNINS V. I., STICH H. F., and BENCOSMES. A. (1964) Fine structure of the nucleolar organizer of salivary gland chromosome of Chironomids. J. Ultrastruct. Res. 11, 282291. KAUFMANB. P. and DAS N. K. (1955) The role of ribonucleoproteins in the production of mitotic abnormalities. Chromosoma 7, 19-38. KLICMANA. M. (1965) Topical pharmacology and toxicology of dimethyl sulfoxide. J. Am. med. Ass. 193,796-804,923-928. KOPRIWAB. and LEBLONDC. P. (1962) Improvements in the coating technique of autoradiography. J. Histochem. Cytochem. 10, 269-286. LANDMAN 0. E. and SPIEGELMANS. (1955) Enzyme formation in protoplasts of Bacillus megaterium. Proc. nat. Acad. Sci. U.S. 41, 698-704. LANSINGA. I. and ROSENTHAL T. B. (1952) The relation between ribonucleic acid and ionic transport across the cell surface. J. cell. camp. Physiol. 30, 337-345. LA~KOWSKIM. (1959) Deoxyribonucleases. In The Enzymes (Ed. by LARDY H. and MYRBACKD.) 5,123-148. Academic Press, New York. LOCKWOOD W. R. (1964) A reliable and easily sectioned epoxy embedding medium. Anat. Rec. 150,129-140. MONDERC. (1967) Effect of DMSO on enzyme activity. Ann. N.Y. Acad. Sci. 141, 300-301. PALADEG. E. (1952) A study of fixation for electron microscopy. J. exp. Med. 95, 285-298. PUIG-MUSETP. and MARTIN-ESTEVEJ. (1965) Physiological cell permeability and pharmacological action of DMSO. Experientia 21, 649-650. RAMMLERD. H. and ZAFFARONIA. (1967) Biological implications of DMSO based on a review of its chemical properties. Ann. N. Y. Acad. Sci. 141, 13-23. RIS H. (1966) Electron microscope studies on nucleohistone fibres from interphase nuclei. J. Cell Biol 31, 134A. RUBIN L. F. and BARNETTK. C. (1967) Ocular effects of oral and dermal application of dimethyl sulfoxide in animals. Ann. N. Y. Acad. Sci. 141, 333-345. SABATINID. S., BENSCHK., BARRNE~ R. G. (1963) Cytochemistry and electron microscopy, the preservation of cellular ultrastructure and enzymatic activity by aldehyde fixation. J. Cell Biol. 17, 1958. STEVENSB. J. and SWIFT H. (1966) RNA transport from nucleus to cytoplasm in Chironomus salivary glands. J. Cell Biol. 31, 55. WIGGLESWORTH V. B. (1959) A simple method for cutting sections in the 0.5 to 1 p range, and for sections of chitin. Quart.J. micr. Sci. 100, 315-320.