DMSO : Biological Actions of Dimethyl Sulfoxide DMSO - New York Academy of Sciences

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ANNALS OF THE NEW YORK ACADEMY OF SCIENCES

VOLUME 243

BIOLOGICAL ACTIONS OF

DIMETHYL SULFOXIDE

Stanley W.Jacob Robert Herschler

PUBLISHED BY THE

NEW YORK ACADEMY OF ANYAA9

\

/

SCIENCES

243 1-508

(1975)

i

^

THE NEW YORK ACADEMY OF SCIENCES (Founded

in 1817)

BOARD OF GOVERNORS,

1975

PHILIP FEIGELSON, President PHILIP SIEKEVITZ, President-FAect Honorary Chairman of

the

Board of Governors

BORIS PREGEL

I.

Honorary Life Governors

LASKOWITZ

B.

IRVING

MARGARET MEAD HERMAN COHEN

JOEL

L.

SELIKOFF H.

Corresponding Secretary

Treasurer

GORDON Y BILLARD

PAUL MILVY

Elected

GEORGE

SIDNEY

G.

THOMAS

ROTH

1974-1976

MARIE M. DALY

I.

FUJIMOTO

G overnors-at-Large 1973-1975

CHARLOTTE FRIEND

BORIS PREGEL CHRISTINE REILLY

SEYMOUR MELMAN ETHEL TOBACH

LEBOWITZ

Recording Secretary

JOHN

J.

Vice-Presidents

C.

KAVANAGH

HERBERT J. KAYDEN

RENE DUBOS 1975-1977

J.

BURNS

ALLAN GEWIRTZ

CUYLER HAMMOND KENNETH WADE THOMPSON A. KORFF

VIRGINIAS. SEXTON

Past Presidents (Governors)

MINORUTSUTSUI N. HENRY MOSS JACOB FELD

E.

SERGE

Financial Counselors FREDERICK A. STAHL

MATTHEW B. ROSENHAUS

LLOYD MOTZ

Counselors to the Board of Governors Budget Counselor

GEORGE

B.

DAETZ

Legal Counselor

EDWARD D. BURNS

ANNALS OF THE NEW YORK ACADEMY OF SCIENCES Volume 243

EDITORIAL STAFF Executive Editor

BILL

BOLAND

Associate Editors

^cAp^^

i&n

ANNE CAHILL JONAS ROSENTHAL

-.hnirai information

-r

Center

^"The*«e company 37

/\

Street

ANNALS OF THE NEW YORK ACADE^Yjgj^SfJ},^N®:S^2-9l^^ ^ Volume 243

'

January 27, 1975

_^

BIOLOGICAL ACTIONS OF DIMETHYL SULFOXIDE* Stanley W. Jacob and Robert Herschl^r

J

f^^B

\i\

r f ru Chairman Conference

'-^

[\\

'^ CONTENTS

?. ^

I.

Mechanism

5

Dimethyl Sulfoxide

of Action of

Pharmacologic and Biochemical Considerations of Dimethyl Sulfoxide. By and Julianne Wood Physical Properties of Dimethyl Sulfoxide and

its

Function

in

Don

C.

Wood 7

Biological Systems.

By H.

Harry Szmant

20

Influence of Nonionic Organic Solutes on Various Reactions of Energy Conservation and Utilization.

Effects of F.

The

By Thomas

E.

Conover

24

Dimethyl Sulfoxide on Subunit Proteins. By

Henderson, and

J.

Margaret

S.

Thomas

R.

Henderson, Rogene

Lyndal York

Effect of Dimethyl Sulfoxide on a

38

Lysosomal Membrane. By Donald W. Misch and

Misch

54

Specific Modifications of the Sulfoxide. By Joseph D.

Na". K"-Dependent Adenosine Triphosphatase by Dimethyl

Robinson

60

Mitogenic Responsiveness of Chronic Leukemic Lymphocytes and Normal Human Lymphocytes Treated with Dimethyl Sulfoxide. By Anthony J. Dennis and

Altered

Henry

E.

Wilson

73

Cell-Mediated Immunity:

Modulation by Dimethyl Sulfoxide. By Harry Bartfeld

Its

AND Andrew Goldstein

81

Antiarthritic and Antithrombotic Effects of Topically Applied Dimethyl Sulfoxide.

Peter

Gorog and

Iren

Part

B.

II:

Biological

Hark AWAY,

91

Toxicology, Fate, and Metabolism in

Combination. By Lionel

of the Metabolites of Dimethyl and R. Snyder

Effects

By

Kovacs

Toxicity of Dimethyl Sulfoxide, Alone and

By

F.

Rubin

98

Kocsis,

S.

Mammalian Myocardium.

B\

Sulfoxide.

Pharmacological Effects of Dimethyl Sulfoxide on the Marshal Shlafer and Arvland M. Karow, Jr

J.

J.

104

1

10

The Influence of Dimethyl Sulfoxide on Cellular Ultrastructure and Cytochemistry. By 122 Edmund B. Sandborn, Heather Stephens, and Moise Bendayan Metabolism and Excretion of Dimethyl Sulfoxide in Cows and Calves after Topical and Parenteral Application. By J. Tiews, E. Scharrer, N. Harre, L. Flogel, and W.

Jochle

1975 \^i ^

LIBRARY

Opening Remarks. By Chauncey D. Leake

Part

'

139

'

The Penetration and Clearance of Dimethyl Sulfoxide from the Rat Eye After Topical Application. By W. J. Weaver, R. V. Hill, F. S. Weber, and S. W. Jacob

151

*This series of papers is the result of a conference entitled Conference on Biological Actions of Dimethyl Sulfoxide, held bv The New York Academy of Sciences on January 9, 10. and 1, 1974. 1

Part

III.

Effects of Dimettiyl Sulfoxide on

Tumor Systems

Stimulation by Dimethyl Sulfoxide of Erythroid DiflFerentiation and Hemoglobin Synthesis in Murine Virus-Induced Leukemic Cells. By Charlotte Friend and

William Scher

155

Effect of Dimethyl Sulfoxide and Dimethylformamide on the Growth and Morphology of Tumor Cells. Bv E. Borenfreund, M. Steinglass, G. Korngold, and A. Bendich '.

164

Studies on the Intracisternal A-Type Particles in Mouse Plasma Tumor Cells: Induction of Maturation of the Particles. By S. E. Stewart, G. Kasnic, Jr., C. Urbanski, M. Myers, and T. Sreevalsan Effect of Dimethyl Sulfoxide on the Hepatic Disposition of

Walter

172

Chemical Carcinogens. Bv

G. Levine

'..

185

Potentiation of Antineoplastic Compounds by Oral Dimethyl Sulfoxide in Tumor-Bearing Rats. By Joel Warren, Miriam R. Sacksteder, Harriet Jarosz, Bruce

Wasserman, and Peter

E.

Andreotti

194

Studies on the Modifying Effect of Dimethyl Sulfoxide and Other Chemicals on Experimental Skin Tumor Induction. By Frej Stenback and Humberto Garcia 209

Part IV. Dimethyl Sulfoxide Fate and Metabolism of Dimethyl Sulfoxide

Smale, Neil

J.

in

in

Agriculture

Agricultural Crops.

By Bernard C.

Lasater, and Bruce T. Hunter

228

Accumulation and Persistence of Sulfur^"^ in Peach Foliage and Fruit Sprayed with Ra237 diolabeled Dimethyl Sulfoxide. By Harry L. Keil

Use of Dimethyl Sulfoxide

to Control Aflatoxin Production.

George W. Rambo

Bv George A. Bean and 238

.'

Part V. Dimethyl Sulfoxide: Basic Considerations Current Concepts Concerning Radioprotective and Cryoprotective Dimethyl Sulfoxide in Cellular Systems. By M. J. Ashwood-Smith

The

Effect of Dimethyl Sulfoxide on Forelimb Regeneration of the Adult viridescens. By Gerald G. Slattery and Anthony J. Schmidt

Properties

of

246

Newt, Triturus 257

In vivo and in vitro Effects of Dimethyl Sulfoxide on Streptomycin-Sensitive and -Resistant Escherichia coli. By William E. Feldman, James D. Punch, and Patricia C.

Holden The

269

Effect of Dimethyl Sulfoxide on

Pneumocystis

carinii.

By John W. Smith and

Walter T. Hughes The

278

Effects of Dimethyl Sulfoxide on Neurite

Development

in vitro.

By Fred Jerrold 279

Roisen Dimethyl Sulfoxide:

A

Tool

in

the Study of

Sperm

Motility Control.

By Leonard

Nelson

297

Discussion Paper: Dimethyl Sulfoxide as a Cryoprotective Agent for Platelet Pres306 ervation by Freezing. By Mario G. Baldini

Part VI. Effects of Dimethyl Sulfoxide Production of Interferon

in

the

in

Lower Animal Models

White Mouse by Dimethyl Sulfoxide. By Michael Kunze 308

Orally Administered Dimethyl Sulfoxide: Its Effects on Blood Concentrations on Salicylic Acid, Sulfanilamide, and Warfarin. By G. Thomas Passananti, Carol A. Shively,

and Elliot

S.

311

Vesell

A Comparison

of Glycerol and Dimethyl Sulfoxide as Cryoprotective Agents for an Ex317 perimental Tumor: A Pilot Study. By William P. Graham, III

Dimethyl Sulfoxide and Hydrogen Peroxide on Tissue Gas Tensions. By M. 320 Bert Myers and William Donovan

Effect of

The

Effect of Dimethyl Sulfoxide on Hypothalamic-Pituitary-Adrenal Functions

Rat.

By John

P.

Allen and Catherine

F.

Allen

in

the

325

Part VII. Effects of Dimethyl Sulfoxide on the Central Nervous System The

Effect of Dimethyl Sulfoxide on the mission in Aplysia Ganglion Cells. By

Neuronal Excitability and Cholinergic Trans-

Masashi Sawada and Makoto Sato

337

Enhancement of Resistance of Glial Cells by Dimethyl Sulfoxide against Sonic Disrup358 tion. By Ramon Lim andSean Mullan Dimethyl Sulfoxide

Kawanaga,

in

D.

Central Nervous System Trauma. By J. C. de la Torre, H. M. C. M. Johnson, D. J. Goode, K. Kajihara, and S.

W. Rowed,

Mullan

362

,

Part VIII. Dimethyl Sulfoxide: Clinical Concepts Design of Therapeutic Trials for Dimethyl Sulfoxide. By

Thomas

The Present and

Connective Tissue Disorders. By

Potential Role of Dimethyl Sulfoxide

in

G.

Kantor

390

John Baum

391

Discussion Paper: Methodology and Techniques in the Evaluation of Dimethyl Sulfoxide 393 for Connective Tissue Disorders. By Raul Fleischmajer

Dimethyl Sulfoxide Therapy

Various Dermatological Disorders. Bv

in

Lazaro Sehtman 395

;

Experimental and Clinical Evaluation of Topical Dimethyl Sulfoxide of the Extremities. By A. Kappert

Dimethyl Sulfoxide Therapy

in

in

Venous Disorders 403

Chronic Skin Ulcers. By Rene Miranda-Tirado

408

Dimethyl Sulfoxide Therapy as Toxicity-Reducing Agent and Potentiator of Cyclophosphamide in the Treatment of Different Types of Cancer. By Jorge Cornejo GarRiDO and Raul Escobar Lagos 412

Dimethyl Sulfoxide Therapy in Severe Retardation in Mongoloid Children. By Manuel J. AspiLLAGA, Ghislaine Morizon, Isabel Avendano, Mila Sanchez, and Lucila

Capdevile

421

Dimethyl Sulfoxide Therapy in Nonmongoloid Infantile Oligophrenia. By AND Maria E. M. de Bernadou Oral

Ana Giller 432

Dimethyl Sulfoxide in Mental Retardation. By Jeanne Gabourie, Janis W. Becker, Barbara Bateman, Michael Dunn, and Stanley Jacob 449

Dimethyl Sulfoxide Therapy

in

Bronchiolitis.

By Aristides Zuniga, Rodolfo Burdach,

AND Santiago Rubio

460

Dimethyl Sulfoxide Therapy

in

Subjective Tinnitus of

Unknown

Origin.

By Aristides

Zuniga Caro

468

Evaluation of Dimethyl Sulfoxide Therapy

Chronic Respiratory Insufficiency of Bronchopulmonary Origin. By Renato EulufI Marin 475

Dimethyl Sulfoxide

in

the

in

Treatment of Retinal Disease. By Robert V. Hill

Dimethyl Sulfoxide Therapy

in Sterility

Due

to

Tubal Obstruction. By

Hugo Venegas

485 ....

494

Experience in the Use of Dimethyl Sulfoxide in the Diseases of the Supporting Motor Apparatus and General Suppurative Surgery. By Michael B. Dubinsky and Andrew A. Skager 497

The Human Toxicology of Dimethyl Sulfoxide. By Richard D. Brobyn

500

Summary

510

f^^A

of the Dimethyl Sulfoxide Conference.

^%3

y fe^

^;^-^

By Arthur

L.

Scherbel

NOTE TO READER DMSO

Although the abbreviation is not a correct definition, we decided to use it throughout this monograph, simply because dimethyl sulfoxide has been generally known as for some time, and we felt that to introduce a more correct but less well-known designation, such as MczSO, might produce some confusion. We have, however, used the full term dimethyl sulfoxide in the titles of all papers, and of the monograph itself, in order to further the concept of employing the full chemical

DMSO

name

for biological agents or substances.

Stanley

W. Jacob and Robert Herschler

This monograph was aided by contributions from:

Acoustical and Fireproofing Portland, Oregon

Amato

Brothers

Company

Mrs. Elizabeth Kinsman Portland, Oregon

Fred Meyer Foundation

Portland, Oregon

Portland, Oregon

Mr. Thomas Autzen The Autzen Foundation Portland, Oregon

Oregon

Mrs. Eleanor H. Cole New York, New York

Mrs. Eleanor Ooten City,

Oregon

Mr. William J. Polits Portland, Oregon Mrs. Harrison Ring

Mrs. Eunice L. Gaido Conroe, Texas

Portland, Oregon

Miss Mozelle M. Wilson John C. Higgins Foundation Portland, Oregon

Portland, Oregon

^

OPENING REMARKS Chauncey D. Leake University of California

San Francisco, California 94143

This

the third international conference convened during the past decade on

is

that remarkable substance, dimethyl sulfoxide

(DMSO). The

first

was a comprehen-

organized by The New York Academy of Sciences in 1966, under the direction of Drs. Stanley Jacob and Edward E. Rosenbaum, of the University of Oregon. These keen clinicians had discovered the extraordinarily useful potenin the treatment of various painful pathological conditions. tial of The second was convened in Vienna in 1966, under the auspices of Schering A. G. of West Berlin. Organized under the skilled direction of Dr. Gerhard Laudahn, this sive conference,

DMSO

second

The

DMSO conference dealt largely with clinical reports. third conference on

DMSO, now

convening here, was again arranged by Dr.

Stanley Jacob. This is to be oriented toward broad biological and clinical applications of the demonstrated and verified properties of DMSO. It will also include reports on

its clinical

usefulness.

decade since the first hesitant reports on DMSO came from Dr. Jacob and his colleagues, there have been many thousands of published reports on its biological actions and its clinical usefulness in human and veterinary medicine. These reports have come from all over the world. In the U.S.S.R., where bureaucratic control of drug preparation and use is even tighter than in the United States, the clinical usefulness of has been considered sufficiently verified to make it available for use in accordance with the In the

DMSO

judgment of physicians. The most informative publications on the reports and discussions at the

DMSO are:

New York Academy

of Sciences conference This was separately summarized by Leake. 2. the reports and discussions of the Vienna conference in 1966.^ This was sumptuously published and distributed by Schering of West Berlin. The book was dis1.

in 1966.^

AG

tinguished by fine color photographs, especially of reversible lens changes

of

in

the eyes

some experimental animals.

3. the sharp journalistic book prepared for popular distribution by Pat McGrady."* This gives much detail on the efforts of our Food and Drug Administration to justify its rulings on the use of DMSO. 4. the carefully prepared summaries of the properties, biological actions, and uses of DMSO, edited so well by Jacob and colleagues.^ 5. the cautious, officialized report of an ad hoc committee of the National

Academy

of Sciences.®

It is significant all

that there

parts of the world.

is

Many

a great deal of published material on

are listed

in

DMSO

the reports given at the

from

New York

Academy of Sciences conference in 1966. The Vienna Symposium listed 395. The volume edited by Jacob and colleagues^ lists over 2,000 references on chemistry, cryobiology, and pharmacology alone. There are probably more than this number of clinical reports on DMSO in both human and veterinary medicine. The National Academy of Sciences ex cathedra Committee referred specifically to 230 reports, and

listed a

"bibliography" of about 300. 5

.

.

Annals

6 It is

New York Academy of Sciences

increasingly difficult to obtain bureaucratic permission to study

It is difficult for

physicians to use their

own judgment any more in

new drugs.

prescribing drugs.

They are the people licensed by our governments to use drugs in accordance with their judgment as to their safety and effectiveness for individual patients, taking into account the relation of

risk to benefit in the widely differing conditions of their

different patients.

Our Food and Drug Administration, however, operates under

the terms of a Act of our Congress. This Act seems to imply that no drug is to be used by a physician unless it is demonstrated to be absolutely safe and absolutely effective. There is no such drug: not even tap water or table salt is absolutely safe and

detailed

Gaussian distribution inevitably occurs in the response to any chemical enough people are involved, it will always be found that with the same dose, strictly quantitated on a weight basis, some few persons will show practically no effect, while some few others may show a very strong response, even death. It is the effective.

agent. If

professional responsibility of physicians

in

actual practice to judge this relation of

risk to benefit for their individual patients.

In my opinion, it is the responsibility of our governments and of our drug industry to furnish physicians with the verified scientific facts about drugs: their chemical composition and physicochemical properties; their rates of absorption and distribution; the character and rates of their changes in our bodies; their rates and modes of excretion; their biological actions over the range of organizational levels of living material (from molecules, subcellular units, cells, organs, tissues, and indi-

upon single or repeated adminisuntoward reactions likely to be encountered; and the preparations available. Then it is up to the judgment of licensed physicians to decide how and for what purpose they will use them. With regard to SO, the relevant scientific facts have been clearly established by many competent scientists in many different places. This scientific information on DMSO has been well and widely applied in many appropriate clinical conditions of animals and humans. Our conference will add further detail to this scientific knowledge of DMSO and of its clinical usefulness. As I said when summarizing the first DMSO conference, *'The well-known legal principle of res ipsa loquitur might well apply to the situation involving DMSO." I concluded then, as I do now, that **rarely has a new drug come so quickly to the judgment of the members of the health professions with so much verifiable data from so many parts of the world, both experimentally and clinically, as to safety and efficacy." So, let the deliberations and discussions of this Conference result in the wisdom

viduals to societies and ecologies); their toxicities tration; the

DM

being gained that

may

allow

DMSO to finds its place in alleviating some of the pains

and pathological distress which can

afflict

us

all.

References

3.

Jacob, S. & E. E. Rosenbaum. 1967. Ann. N. Y. Acad. Sci. 141 1-67 1 Leake, C. D. 1966. Science 152: 1646-1649. Laudahn, G. & K. Gertich. 1966. DMSO Internationales Symposium, 219 pp.

4.

McGrady,

1

2.

:

Saladruck. Berlin, West Germany. p. 1973. The Persecuted Drug: The Story of

Company, 5.

6.

Inc.

Garden

City,

DMSO,

372 pp. Doubleday

&

N.Y.

Jacob, S., E. E. Rosenbaum & D. C. Wood. 1971. Dimethyl-Sulfoxide. Vol. 1. Basic Concepts, 479 pp. Marcel Dekker. New York, N.Y. National Academy of Sciences. 1973. Dimethyl sulfoxide as a therapeutic agent. Report, 1 19 p.

Part

I.

Mechanism of Action of Dimethyl Sulfoxide

PHARMACOLOGIC AND BIOCHEMICAL CONSIDERATIONS OF DIMETHYL SULFOXIDE Don

C.

Wood

and Julianne

Wood

Medlab Computer Services Lake City, Utah 84102

Salt

I DMSO

have A broad spectrum of the primary pharmacological properties of been reported. Concurrently with pharmacologic studies in laboratory experimental models, there have been evaluations of its clinical therapeutic efficacy, and an equally broad spectrum of favorable therapeutic claims have also been made. It has been perplexing to all serious students of this drug that the basic and clinical studies have produced conflicting reports. Controversial claims, however, are usually associated with the development and refinement of any new drug. In many respects the case of is unique, in that it appears to palliate such a large number of different clinical conditions and also to evidence such a wide range of pharmacological properties. As we gain more experience with DMSO, a greater degree of standardization will result, and many of the areas of controversy will be resolved. In comparing published reports, the reader should be careful to review comparable ex-

DMSO

perimental design, common stages of disease in patients, similar dosings, routes of therapy, frequency of therapy, methods of measuring results, and inherent experimental error, and so on. Few studies have been reported in which identical models have been used by different investigators to evaluate DMSO. In addition to experimental design, patient selection, and methodology, other physical or chemical characteristics may affect the results obtained in

DMSO

These factors often appear unique to DMSO, and may or may not have been published. Some DMSO investigators have "grown up" with a trial-and-error understanding of these potential experimental problems. A property of DMSO that has caused considerable difficulty in the interpretation of laboratory findings is the degree of ionization it may impose upon the constituents of an aqueous buffer system. If an aqueous phosphate buffer is prepared to a specific pH value, then is subsequently diluted with DMSO, not only will the concentration of buffer change with the dilution, but the pH value will change as well. If the DMSO-buffer mixture is not checked just before use, erroneous interpretations of data can result. Enzyme activities, metabolic studies, tissue or cell cultures, in vitro microbiology, and numerous other test systems could be seriously modified, yielding results that relate simply to a buffer pH change, rather than to an effect of DMSO on the primary physiologic or pharmacologic property being examined. Investigators have been concerned that enzymes fail to respond in a predictable fashion when mixed with DMSO solutions. There are enzymes that appear to be unaffected by high concentrations of DMSO. Other enzymes are potentiated in catalytic activity. A third group of enzymes are inhibited in relatively dilute DMSO solutions. Do these studies reflect pH changes or modifications of protein constudies.

formation, or in the case of enzyme inhibition, is the catalytic activity related to chemical changes in functional groups? This very complex matter has been discussed elsewhere.^"* It is

known

that

DMSO

is

reduced to dimethyl sulfide

vitro with glutathione or cysteine.^

DMSO

will

(DMS) when mixed

interfere with

many

procedures commonly used to measure constituents in biological blood glucose, ascorbic acid, and so on.^ The concentration of

fluids

DMSO

7

in

laboratory in

such as the test

Annals

8

New York Academy of Sciences

system is important to the degree of interference. Most in vivo experiments would probably be unaffected by these problems, but many in vitro studies are affected. Numerous other examples could be sited, with particular reference to pharmacologic and therapeutic studies in clinical testing, in which the concentration and frequency of treatment are extremely critical to success. Most clinicians who have had extensive experience with now have an understanding of this therapeutic problem. Frequency of treatment and the concentrations of used in clinical evaluations are extremely critical to the results that will be obtained. The fact that some of this information is not clear has been one of the important factors leading to differences in published experimental findings. This paper will attempt to bring together not only pharmacological laboratory experimental models, but also some clinical studies which might lend support to a better understanding of and its action. As one attempts to understand the mechanism of in a disease process, one must consider the fact that multiple pharmacological properties can be expressed in a single clinical problem. It is likely that some of the drug characteristics would be favorable in a particular clinical situation, while other properties might work to the patient's disadvantage.

DMSO

DMSO

DMSO

DMSO

DMSO

Membrane

DMSO

Penetration

varying concentrations has been shown to cross body membranes in is reversible. The integrity of most membranes is unaffected; except where extremely concentrated (90-100%) doses of come into direct animals.

in

The process

DMSO

contact with the membrane. In clinical therapeutic conditions, such concentration levels would rarely be reached. Exceptions to the rapid penetration of the membrane are nails and the enamel of the teeth, where little penetration, if any, can be demonstrated. Numerous studies on the fate and metabolism of labelled have confirmed the rapid movement and generalized distribution of the labelled component in nearly every tissue of the body. Extraction and analysis of the label or one of its metabolites. ^~*' from various tissues shows the presence of Klingman^^'^^ mixed dyes, antiperspirants, and steroids with varying concentrations of DMSO. He reported enhanced penetration through human skin, as compared with controls that did not contain DMSO, and claimed that the enhancement of penetration was not dependent on irreversible damage to the stratum corneum. Maibach and Feldman^'' reported a threefold increase in the penetration of percutaneous steroid when mixed with DMSO. Maximum urinary excretion of testosterone and hydrocortisone occurred in man within 36 hours after treatment of

DMSO

DMSO

the skin.

Sulzberger and colleagues ^^ used iron dyes, iodine, and methylene blue as visual markers of penetration following dermal application of DMSO dye mixtures. They found in biopsies that there was essentially no penetration of the marker below the stratum corneum. The stratum corneum was completely invaded, however. Stroughton^^"^* also transported hydrocortisone and hexachlorophene into the stratum corneum. He found that the deposit remained for as long as 16 days, and was resistant to washing with alcohol or soap and water. Perl man and Wolfe^^ found that solutions of allergen of low molecular weight would penetrate intact human skin when mixed with 90% DMSO. Allergens that had molecular weights of more than 3,000 failed to penetrate the skin. Smith and Hegre^° reported, however, that specific antibodies did form in rabbits treated with dermal applications of DMSO and bovine serum albumin.

Wood & Wood:

Pharmacologic and Biochemical Considerations

9

Brink and Stein^^ have suggested that DMSO-pemoline mixture, when administered intraperitoneally, resulted in higher brain concentrations of the drug than were produced when the pemoline was administered as a 0.3% tragacanth suspension.

DMSO

has been used as a vehicle for idoxuridine (IDU) and its derivatives in the treatment of viral infections. Juel-Jensen and colleagues" found that IDU in was effective in the treatment of herpes zoster. Other studies""^' have demonstrated that treatment of herpes simplex infections with mixtures of DMSO, IDU, and other agents was successful. Maloney and Kaufman, ^^ however, reported that IDU in saline was more effective than DMSO-IDU mixtures in the treatment of corneal herpes simplex. Perhaps the difference and apparent conflict in these observations is related to the nature and structure of the cornea. It seems possible that might transport IDU through the cornea into the aqueous chamber, thus being flushed past the infected site so rapidly that chemotherapy might not be very effective. Saline-IDU mixture, on the other hand, might perhaps be transported at a slower rate, thus resulting in a more effective drug level in the corneal viral lesion. Many clinical studies have been published that report the effective use of as a vehicle for fungicidal and fungistatic agents. ^^"'*^ Parasitic infestations have likewise been successfully treated with topical DMSO-thiabendazole mixtures.'''*"*® This treatment was more effective than oral administration of thiabendazole alone. The exciting and successful studies of Dachi and colleagues,''^ Elzay,''* and Garcia-Perez and colleagues,''^ in which was used as a vehicle in cancer chemotherapy models, suggests that there may be great therapeutic value in this pharmacologic property. Shklar and coUeagues^^ reported that retarded the carcinogenic effects of 9,10-dimethyl-l,2-benzanthracene. is known to be an extremely reactive compound. It would be interesting to know whether inactivation of this carcinogenic property took place before the animals were treated. MondaP^ made a similar observation with 3-methylcholanthrene. In a similar manner, the biological activities of some antibiotics and cancer chemotherapeutic agents might be modified in a manner that would reduce their therapeutic effectiveness. On the other hand, such a chemical change might potentiate the drug. It becomes difficult to determine whether the drug potentiation that has been observed after treatment is the result of potentiation of concomitantly administered drugs, or of better tissue distribution of the therapeutic agent. In most cases the latter would be suspected. Levine and colleagues'® treated 1 1 cats infected with ringworm Microsporum canis. The animals were treated with mixtures of and griseofulvin. Clinical cure was obtained in all the animals in 10 days or less. Normally a successful oral griseofulvin treatment would take 21-22 days. Extracts of the skin from these animals showed antifungal activity in vitro. Control cats treated with alone failed to cure the Microsporum canis. but extracts from the skin of these control animals also inhibited fungi.

DMSO

DMSO

DMSO

DMSO

DMSO DMSO

DMSO

DMSO

A n ti'Inflam motion Formanek and Kovak^^ produced edema in the rat paw by the injection of autologous blood and by trauma that resulted from a steel clamp. DMSO, when applied immediately after the injection of blood or induction of trauma, resulted in reduction of the edema in both test procedures.

Annals

10

New York Academy of Sciences

Gorog and Kovacs^^ developed edema in the edema in

carrageenin. They reported finding less orally or dermally with

paw by means of injections of the limb of rats that were treated

rat

DMSO.

Gorog and Kovacs^^ have studied other experimental inflammatory models: granuloma of the pouch, carrageenin-induced edema of the rat paw, adjuvantinduced pig

and

arthritis,

contact dermatitis, allergic eczema, and calcification of guinea each of these models, proved to have anti-inflamma-

DMSO

rat skin. In

tory properties.

Suckert"-" treated croton oil arthritis induced in the rabbit knee joint and in rat paws with DMSO. Results again showed that it has antiinflammatory properties. Suckert" observed that if the rat paw was treated with DMSO prior to induction of formalin or dextran edema, formation of the edema was inhibited; however, if DMSO was applied after induction of the inflammatory response, the edema was increased. postischemic edema

Preziosi and Scapagnini^* induced inflammatory response in rabbits' eyes with

mustard

oil.

They

also induced

edema

in

the rat

paw

with formalin, egg white,

dextran, and carrageenin. In each of these experimental models the animals were treated with topical, oral, or intraperitoneal without significant evidence of

DMSO

anti-inflammatory response. Burn edema in rats was unsuccessfully treated with by Ashley and colleagues.^® Weismann and colleagues^ ^^ have shown that lysosomes can be stabilized with cortisone against many types of damaging agents. When cortisone was dissolved in DMSO, the dilution of cortisone necessary to protect the lysosome was reduced might provide a vehicle that from 1:10 to 1:1000. It was suggested that makes steroids more readily available to target tissues, thus resulting in reduced inflammation as evidenced by the experimental models described above. Brown and Mackey®° found that used alone could protect erythrocytes from heat-induced hemolysis. Further, they found that it enhanced the protective eff'ect of indomethacin-like compounds.

DMSO

DMSO

DMSO

Vasodilation Pedicle flaps were raised on the backs of rats by Adamson and colleagues.*^ The experimental animals were divided into control and DMSO-treated groups. The slough was reduced significantly in the DMSO-treated group. The authors suggested this was due to increased circulation to the flap, which resulted from a histaminepossesses potent like response. Klingman^^-^^ reported earlier that histamine-liberating properties. The increased blood flow through experimental pedical flaps and the exciting studies of pial vessels reported by de la Torre and colleagues®^ confirm that vasodilation occurs. These studies do not confirm the

DMSO

mechanism of action, however. Other experiments with smooth muscle®^®'' and with the rectum and uterus*^ confirmed that there was a decrease in contractability. Histamine, nicotine, and carbachol receptors were blocked. Norepinephrine receptors were unaff"ected. Zetler and Laughof®* suggested that the failure of receptors was the result of interaction between and membrane protein structure. Perhaps, as with steroid receptors, the receptor activity described above is dependent upon the funcis known to react with the tional presence of a free sulfhydryl (SH) group. glutathione-SH group; perhaps receptor inactivation is also reated to the oxidation of receptor SH groups, resulting in a biological block.

DMSO

DMSO

1

Wood & Wood: Ketchum and

Pharmacologic and Biochemical Considerations

colleagues*^ studied vascular augmentation

in

1

clinical cases of

DMSO was found to be effective in promoting studied the effects of DMSO in healing a tympano-

pedicle grafts done by several means.

Vishwakarma*' wound. Twenty patients treated with

graft survival.

plasty

healed better than 10

who

DMSO,

applied locally to the lesion,

received absorbable gelatin sponge and oxytetracycline

treatment. While the blood supply to the healing flap or lesion is critical, other (its antibacterial capabilities) would help to keep the wound properties of free from invading organisms.

DMSO

Perhaps the significance of dilation

is

not restricted to blood vessels alone. The

principle of dilation of other anatomical sites

may have

application

in

the therapy of

disease processes. For example, Stewart, and colleagues®^ treated patients with interstitial cystitis

by intravesical administration of

50% DMSO. The

condition was

successfully controlled in over half of the 21 patients studied.

Bacteriostasis

Some of the first studies by Jacob and colleagues®^ showed that Escherichia coli, Pseudomonas, and Staphylococcus aureus were inhibited in growth when the medium contained 20% DMSO. The results of these studies have been supported and amplified by other workers. Seibert and colleagues"" studied the effects of

DMSO on the highly pleomorphic

bacteria isolated from tumors and leukemic sera.

They reported that

all

such bac-

were inhibited in DMSO concentrations of 12.5-25%. An unusual and important observation was made by Pottz,^^ who reported that tubercle bacilli resistant to 2000 ^m streptomycin isoniazid became sensitive to 10 ^m after treatment with 0.5-5% DMSO. His observations were supported by Kamiya and colleagues,"^ who found antibiotic-resistant organisms more sensitive to the drug after treatment with DMSO. They also observed that drug-sensitive organisms become more sensitive to the antibiotic after short incubations with 15% teria tested

DMSO.

DMSO alone,

at

90%

concentration, has no effect on dermatophytes

when mixed with known antifungal effective transport agent in the

agents, however,

treatment of

DMSO

becomes

clinical fungal disease (as

in vitro^'^-'^

and has been mena potent

tioned earlier). It should be kept in mind, however, that DMSO's powerful ability to transport substances into and through membranes could potentiate a hazardous condition. Should an infected lesion that contained a DMSO-resistant bacterial or viral strain be treated dermally, spread of the causative agent could produce an extension of the

Transport of microbial toxins could also potentiate a clinical risk. A rapid transport and dilution of toxin out of a local site could, however, offer clinical advantage to that site. Kunze and Klein"-* studied experimental stapphyloccocal infection

infection.

DMSO

DMSO

was mice. increased the spread and lethality of the process when administered and the microorganism was also inoculated by the intraperitoneal

in

route.

Effect on Collagen

Scherbel and colleagues'^ reported that biopsy specimens taken from the skin of scleroderma patients, before treatment and three times at weekly intervals

DMSO

2

Annals

1

during continual dissolution.

The

DMSO

New York Academy

of Sciences

treatment, showed that the treatment produced collagen remained intact. They also observed increased urine scleroderma patients treated with DMSO, but not in a

elastic fibers

hydroxyproline levels in normal control group. Gries and colleagues"^ examined rabbit skin before and after 24 hours of m vitro immersion in 100% DMSO. They reported that after treatment the neutral salt-extractable collagen fraction decreased. The authors observed a remarkable reduction in the pathologic deposition of postirradiation subcutaneous fibrosis in

DMSO

DMSO. Treatment of normal normal collagen metabolism.

patients treated with

the equilibrium of

Engle" reported that keloids treated with

tissue apparently did not

DMSO

showed

change

histologic improve-

ment.

Numerous with

DMSO

clinical studies

have reported that treatment of scleroderma patients

resulted in reduced pain, greater flexibility of the diseased skin, and

enhanced healing of extremity ulcers. '*"*° Tuff'anelli,*^ however, was unable to confirm these findings. One is tempted to suggest that with the improved clinical manifestations of this collagen disease, DMSO therapy does in fact modify pathologic collagen, perhaps by dissolution and vasodilation, thus restoring circulation, increasing nourishment, and resulting in healing of ulcers and better limb movement. Such a hypothesis, however, would not explain the development of new skin ulcers during DMSO therapy."*-*^ However, the DMSO concentration used and the frequency of DMSO therapy may be very critical to the successful treatment of skin ulcers. Perhaps a more aggressive approach to therapy in scleroderma would prevent ulcer formation.

Nerve Blockade

Sams and colleagues*^ immersed sciatic nerve in a solution of 6% DMSO, and demonstrated that conduction velocity was reduced 40%. It was shown that the effect could be reversed after a 1-hour buffer rinse of the nerve.

Shealy*^ studied the effects of various

small fibers after discharge.

C

fiber activity in

1

He found

min. After

fiber activity returned.

DMSO concentrations on feline peripheral 5-10% DMSO solutions would eliminate

that

DMSO

had been washed out of the system, the C this to be a good model with which

The author has suggested

to test for efficacy of analgesics.

DMSO on pain in patients with

musculoskeletal all causes of pain were relieved within one hour after treatment with 80%^ DMSO. In the case of strains and sprains, pain relief was found to be better than with standard treatment. The mechanism of this pain relief has not been clarified. Brown*'*-*^ studied the effects of

diseases (strains, sprains, acute bursitis, and tendonitis). In these studies

DMSO to thoracotomy incisions. Patients DMSO required fewer doses of narcotic. Arno to produce relief with DMSO treatment of post-

Penrod and colleagues®^ applied treated with high concentrations of

and colleagues,*" however,

partum episiotomy

failed

pain.

Rosenbaum and

colleagues** treated 37 patients suffering

from intractable pain

(seen during surgical practice). Pain had been present for over one year

in each case. Conventional therapy failed to control it, but 32 of the 37 patients reported remarkable relief after therapy. The patients included 1 1 with tic douwith postamputation phantom pain, 10 with posttraumatic intractable loureux, pain, and 5 with postoperative intractable pain.

DMSO

1

1

Wood & Wood:

Pharmacologic and Biochemical Considerations

13

Numerous clinical studies have been reported in which pain relief, though in many cases only of a palliative nature, was found to be the most significant factor. These studies include cases of acute traumatic injuries**-*^-*®"®* and cases of acute and chronic painful shoulder, as well as nontraumatic pain.^°'*''®*-*^'*®"*°2

Muscle Relaxation Birkmayer and colleagues'^^ treated patients who had headaches associated with syndrome with DMSO. Electromyographic evidence of relief of muscle spasm was observed within 60 min after topical adminiscervical disease or shoulder-hand

tration of

DMSO. Nonspecific Enhancement of Resistance

Raettig'^''

observed that mice treated with oral doses of

DMSO 8 days prior to a

compared to the inThe immune response was not

challenge with murine typhus showed an enhanced resistance, as fections

of animals not treated with

decreased

On

in

DMSO.

any of his studies.

the other hand. Smith and colleagues'^^ reported that allergens mixed with

DMSO failed to produce local find antibody after

reactivity in

dermal applications of

humans. Valer and Racz'^ also failed to DMSO and antigens. Azar and Good'°^

DMSO

found no increase in complement levels after therapy. does not demonstrate a significant delayed-hypersensitivity response. It is, however, a strong potentiator of low-molecular-weight sensitizers, because of its ability to move antigens through the skin. Numerous studies have been reported in which permits demonstration of an allergic reaction of haptenic allergens, with extremely low concentrations of antigen. '°*"''' The mechanism of this enhancement is not understood, but is probably related to vehicle transport.

DMSO

DMSO

Alterations of Activity for Concomitantly Administered Drugs

Rosen and colleagues' '^ treated mice and rats orally with quarternary ammonium salts. They observed that toxicity was increased when pentolinium tartrate and hexamethonium bitartrate were dissolved in DMSO. No toxicity was noted when the compounds were administered alone. DMSO decreased the toxic effects of decamethonium iodide in rats. It would be interesting to know whether pentolinium

hexamethomium bitartrate were simply being transported by DMSO. Perhaps reacted directly with decamethonium iodide, making it nontoxic. Kocsis and Harkaway"^ and Kocsis"'' reported that potentiated the lethal effectiveness of benzene when administered intraperitoneally with DMSO. They found a dose effect. High concentrations of were more lethal than dilute solutions when mixed with benzene. tartrate and

DMSO

DMSO

DMSO

Cholinesterase Inhibition

Sams and cardiac,

DMSO

DMSO

concentrations on depressed the response of the

colleagues*^ studied the effects of low

smooth, and skeletal muscle.

Annals

14

diaphragm

New York Academy of Sciences

to both direct electrical stimulation of

muscle and indirect electrical caused spontaneous fasciculations of the skeletal muscle. Smooth muscle of the stomach, when treated with DMSO, showed an increased response to both muscle and nerve stimulation; 6% DMSO, however, decreased the vagal threshold by 50%. It has been speculated that inhibition of cholinesterase could explain the fasciculation of skeletal muscle, increased tone of smooth muscle, and lowered vagal threshold. In vitro assays of bovine erythrocyte cholinesterase have shown that 0.8-8.0% produces a 16-18% inhibition of enzyme activity. stimulation of nerves.

It

DMSO

Diuresis

Formanek and Suckert^^* found that when experimental rats were treated five times daily with 0.5 ml 90% DMSO, their urine volume was increased tenfold over that of a control animal group. In addition to increased urine volume, there was an increase in sodium and potassium excretion.

Summary

DMSO

has multiple known pharmacological properties. In addition to those

referred to in the text of this paper, other properties are described by Jacob and colleagues.'^® These include: cryoprotective action, radioprotective effect, influence on serum cholesterol in experimental hypercholesteremia, and platelet aggregation antagonism. How the multiple properties of this chemical aff'ect therapy in clinical medicine must yet be explained. It is clear, however, that DMSO does affect biological systems, and that this influence has many clinical applications. Perhaps one of the most interesting and significant properties of DMSO is its ability to move other drugs through membranes. When mixed with DMSO, many drugs appear to be potentiated in their physiologic eff"ect; thus smaller doses are required and less toxicity is demonstrated. In cancer chemotherapy, this value already has practical use.

Some

of the studies to be reported

in this

monograph

will

describe additional,

almost unbelievable observations. Perhaps the mechanism of action of these clinical phenomena will be found in one or more of the pharmacological properties described. It would not be surprising, however, if we were to conclude with a resolution to search for new explanations of the mystery of DMSO, for it would appear that is really a new principle in medicine and cannot always be measured by

DMSO

existing standards.

References

Rammler, D. H.

&

A. Zaffaroni. 1967. Biological implications of

review of its chemical properties. Ann. N.Y. Acad. Sci. 141:

DMSO

Rammler, D. H.

1967. The effect of 141:291. Rammler, D. H. 1971. Use of Sulfoxide. Vol. 1. S. W. Jacob, E. E. Dekker, Inc. New York, N.Y.

Acad.

DMSO based on

a

13.

on several enzyme systems. Ann. N.Y.

Sci.

DMSO

Friedman, M. 1968. Chemical

in

enzyme-catalyzed reactions. In Dimethyl & D. C. Wood, Eds.: 189. Marcel

Rosenbaum

reactivities of protein functional

related solvents. Quart. Rep. Sulfur

Chem.

3: 125.

groups

in

DMSO and

Wood & Wood: 5.

Wood,

D.

C, N.

considerations 6.

Pharmacologic and Biochemical Considerations

V. Wirtk, F. S.

of dimethyl

15

Weber

sulfoxide

«fe M. A. Palmquist. 1971. Mechanism (DMSO)-lenticular changes in rabbits. J.

Pharmacol. Exp. Therap. 177: 528. D. C, D. Sweet, J. van dolah, J. C. Smith II & I. Contaxis. 1967. A study of DMSO and steroids in rabbit eyes. Ann. N.Y. Acad. Sci. 141 346. Gerhards, E. & H. GiBiAN. 1967. The metabolism of dimethyl sulfoxide and its metabolic effects in man and animals. Ann. N.Y. Acad. Sci. 141 65. Denko, C. W., R. M. Goodman, R. Miller & T. Donovan. 1967. Distribution of dimethyl sulfoxide'^'S in the rat. Ann. N. Y. Acad. Sci. 141: 77. Wood, D. C. 1971. Fate and metabolism of DMSO. In Dimethyl Sulfoxide. Vol. 1. S. W. Jacob, E. E. Rosenbaum & D. C. Wood, Eds.: 133. Marcel Dekker, Inc. New York, N.Y. KoLB, K. H., G. Jaenicke, M. Kramer & P. E. Schulze. 1967. Absorption, distribution and elimination of labeled dimethyl sulfoxide in man and animals, Ann. N. Y. Acad. Sci. 141:85. HucKER, H. B., J. K. Miller, A. Hochberg, R. D. Brobyn, F. H. Riordan & B. Calesnick. 1967. Studies on absorption excretion and metabolism of dimethyl sulfoxide (DMSO) in man. J. Pharmacol. Exp. Therap. 155: 309. Kligman, a. M. 1965. Topical pharmacology and toxicology of dimethyl sulfoxide (DMSO). Part 1. J. Amer. Med. Assoc. 193: 796. Kligman, A. M. 1965. Topical pharmacology and toxicology of dimethyl sulfoxide (DMSO). Part 2. J. Amer. Med. Assoc. 193: 923. Maibach, H. I. & R. J. Feldman. 1967. The effect of DMSO on percutaneous penetration of hydrocortisone and testosterone in man. Ann. N.Y. Acad. Sci. 141 423. Sulzberger, M. B., T. A. Cortese, Jr., L. Fishman, H. S. Wiley & P. S. PeyakoviCH. 1967. Some effects of DMSO on human skin in vivo. Ann. N. Y. Acad. Sci. 141:

Wood,

:

7.

:

8.

9.

10.

11.

12.

13.

14.

:

15.

437. 16.

Stoughton, R. B. 1965. Dimethylsulfoxide (DMSO) induction of a steroid reservoir in human skin. Arch Dermatol. 91 657. Stoughton, R. B. 1965. Percutaneous absorption. Toxicol. Appl. Pharmacol. 7: 1. Stoughton, R. B. 1966. Hexachlorophene deposition in human stratum corneum— :

17. 18.

enhancement by dimethylacetamide, dimethylsulfoxide, and methylethyl

19.

20.

21.

22.

ether. Arch. Dermatol. 94: 646. Perlman, F. & H. F. Wolfe. 1966. Dimethylsulfoxide as a penetrant carrier of allergens through intact human skin. J. Allergy 38: 299. Smith, R. E. & A. M. Hegre. 1966. Use of in allergy skin testing. Ann. Allergy 24:633. Brink, J. J. & D. G. Stein. 1968. Facilitation of learning in rats by pemoline dissolved in 100 percent dimethylsulfoxide (DMSO). Federation Proc. 27: 437. Juel-Jensen, B. E., F.O. MacCallum, A. M. R. Mackenzie & M. C. Pike. 1970. Treatment of zoster with idoxuridine in dimethyl sulphoxide. Results of two double-

DMSO

blind controlled trials. Brit. 23.

AsHTON,

Frenk

&

Med. J. 4: 776. J. Stevenson.

1971. Therapeutics XIV. Herpes simplex and idoxuridine. Brit. J. Dermatol. 84: 496. Goldman, L. «& K. W. Kitzmiller. 1965. Topical 5-iodo-2'-deoxyuridine in dimethylsulfoxide (DMSO), a new treatment for severe herpes simplex. Ohil Med. J. 61:532. Inglot, a. D. & A. Woyton. 1971. Topical treatment of cutaneous herpes simplex in humans with the non-steroid antiinflammatory drugs: Mefenamic acid and indomethacin in Dimethylsulfoxide. Arch. Immunol. Therap. Exp. 19: 555. Longson, M. 1970. Treatment of severe cutaneous herpes. Lancet 1:81. MacCallum, F. O. & B. E. Juel-Jensen. 1966. Herpes simplex virus skin infection in man treated with idoxuridine in dimethyl sulphoxide. Results of double-blind con-

H., E.

C.

virus infections

24.

25.

26. 27.

28. 29.

trolled trial. Brit. Med. J. 2: 805. Robinson, T. W. E. Personal communication. Robinson, T. W. E. & J. R. Dover. 1972. Experimental zosteriform herpes simplex

virus infection in

mouse

skin. Brit. J.

Dermatol. 86: 40.

New York Academy

Annals

16

ToMLiNSON. A. H.

31.

TuRNBULU

C.

B.

land Med.

Malontv. vest.

34.

J.

T-e e-.hancmg

196*^

5-icc

:e

;

;

effect of -ndine Nev. Zea-

\

3r.

70:

&

E. D.

& H. C. Stringer. upon the anli-Wral actions ol

M.acGr£GOR

I.

dimethvlsulfo.xide vehicle

33.

of Sciences

k F. O. MacCallum. 196S. The effect of f-iodo-Z -deoxsuridine on herpes simples virus infections in guinea-pig skin. "Brit. J. E.xp. Pathol. 49: 2".

30.

32.

"

:

.

H. E. Kaufman. 1965. Dissemmai::-

Ophthalmol.

;

:::-ei: -.erpes simplex. In-

:

4: S"2,

1*^6^.

E. III. R. S. Mathews. J. .\. Grant. W. G. Edwards k H. S. Neilsen. Loci! che~::he:ir\ maduromvcosis caused bs A/; ':;5r; ^ ;.'': ::r:: srz^'->:u'y:.

A:::.

I-:e:"^:

Buckley. C.

Mec

i:4:""4.S.

i9"0. Exp^rier.ce w;:h :he '.::i! irr!::^:::". ::" ^-. ^-.:im>cotic ir.; .-r: :^-.ed m dimethyl sulfoxide DMSOi:-. :;--::-..:- v.:::-. :-.:-;--; ::5es. Mykoser 13: .": Lemne. H. B.. J. R. H. Friedman. 19"1. Griseofuhm in dimeth>. >-::';\.^e:

HiE.vuscH.

I.

:

I

35.

M CoBB&

Penetratior.

i.-.::

£uinea-pi2 skin and clinical findings

rinsworm.

in feline

S.i:

:

_.-^-c;a

9:43. 36.

Male. O.

Enhancement oi

19(?S.

dimethylsulfo.xide 37.

PoLAY.

3S.

Rakhmanow

\'.

dermatology. Robinson. H.

Sperber.

J,.

J.

M

H.

\'

Stone. O.

Exp De:~i::'. 233:

J..

a.-.::.~\

re'v".

':^3

::;;c -£e-.:

nmancm. M\kose.i

12:

.

F.

;

\er.e:ol. 2:13.

k O. E. Graessle. 1964. .-Xntimycotic properties of Dermatol. 42: 4'9. Treatment of ::eer;".z e:_r:.:- •^::r. oraJly and topically Phares

Invest.

administered thiabendazole. 41.

antimycetic effectiveness of gnseofuhin by

the

K.lir.

L. I\ANON. N. S. Potekae\. I. S. .-Kzhgikhin. .-\. V. Iashkul" \^i?~ Dimethyl sulfoxide and aspects of its use in

Dt:rr.i:c\.

196".

a.

p.

O.

.A.

\'es:r.

thiabendazole. 40.

.^rch.

1969. Experiences with the

.\.

KoNSTANTiNON k 39.

in vitro,

E. B.

Ritchie

J.

k

Florida C,

J.

Mec

.A.ss: :

Willis. 196c

54: .;:-

Tr.-r-erc-ole

Dermatol. 93: 24! Engel. l^tb. Dimethyl sulphoxide

m

dimetr.:..>-:::\:de

for tinea nigra palmaris. .A.rch.

42.

k

Stringer. H.

G. B.

:":: -.^:;

.-.feciions.

Lancet

1:

S25. 43.

Weitgasser. H. 1969. Local tre^:~e': :: :--:-.: r^::----: -;- s ;:~r;r^:::r :f 44 I _ \ :e Z H Ge>: t > : ^ r v triamcinolone-acetonide and dime s Katz. R. & R. W. Hood. 1966. The'->c ;: ::r:-: :--r-e"-::;e - c ~e::. s-.: \ ^e for creeping eruption: Pre'.:~.:-2" report. J. Invest. Dermatol 46: 'yj^. --- T:pical thiabendazole for creeping eruption. .Arch. Katz. R. k R. \^'. Hood :'

:.".

44.

45.

46.

4".

.

^

.

:

:

.^

:

r.

-

.-.

'.

:

.k

:

;

Dermatol. 94: 643 Smith. G. C. 19(?c, Orser. ^:::r> :r :rei::rg c.:^re:-s ;-•.. ~:g-^rs J. S. Carolina Med. .\ssoc.62:2c5. Dachi. S. F.. J. E. Sanders k E. M. Urie. 196". Effects oi dimethyl sulfo.xide on dimethylbenzanthrace-.e-:-.d-ced cir::". ogeres:s :r :-.e hamster creek pouch. Cancer

51.

Res.27:llS3. Elzay. R. p. 196". Dime:-;.: >.,::":\:ce ir.z e\re::~e-:^: ::i.\ ci-::- :ge-e>:s ;- :he hamster pouch. .\rch. Pa::.:. 83: 2^3 Garcia-Perez. a.. M. Aparicio k F. J. Carapeto l-". C:~.r2::>:- :: :-.e :/.: :al :: :-FL' p.-ecedec ?> and histological effects of 5-fluorouracil (5-FUt aio.-.e 19. dimethyl-sulpho.xide (DMSO) on senile keratosis. Dermatologica 140 iSuppl. Shklar. G.. S. Turbiner k \\ Siegel. 1969. Chemical carcinogenesis oi hamster mucosa. Reaction to dimethyl sulfo.xide. .Arch. Pathol 8": ^3". Mondal. S. 19"0. Hvdrocarbon carcinogenesis in vitro, l-.iern. Cancer Congr. Proc. 1:

52.

Formanek.

48.

49.

'j.?.c.

1

50.

i;

1

.

269.

K.

ratte"r:'::e-.

53.

GOROG.

P

i:

k W Ko\ak. 1966. Die wirkung von DMSO auf experimentell erzeugte D.MSO Symposium, p. 18. Saladruck. Berlin. West German). .

:deme. Ik 1.

B.

KovACS. 196S

Effect of dimethvl sulfoxide

perimental inflammations. Curre-: T-.e:.!? Re>

k

54.

GoROG.

55.

perimental cutaneous reactions. Pharmacc. :g; 2: 3 '.}. SuCKERT. R, 1969. Effect of dim.ethyl sulfoxide en croton loint. Wien. Klin. \Vo;-er>:.-' 81: 15".

P.

I.

B.

KovACS.

1969. E.ffec:

::"

(

DMSO

i

on various ex-

li:i:-i>':

c:~e:";.. Sulfoxide

oil

iDMSOi

on ^ario-s ex-

arthritis in the rabbit

knee

.

Wood & Wood: 56.

57.

58.

Pharmacologic and Biochemical Considerations

17

SucKERT, R. 1967. Effect of various drugs on the post-ischemic edema of rat paws. Wien. Med. Wochenschr. 117: 1098. SucKERT, R. 1970. Pharmacological influence on traumatic edema in animal experiments and clinical practice. Wien. Klin. Wochenschr. 82: 281.

&

Preziosi, p.

U. Scapagnini. 1966. Action of dimethylsulfoxide on acute inflamma-

tory reactions. Current Therap. Res. 8: 26 1 59.

Ashley,

&

H.

A. N. Johnson, D. V. McConnell, D. V. Galloway, R. C. Machida Sterling. 1967. Dimethyl sulfoxide and burn edema. Ann. N. Y. Acad, Sci.

F. L.,

E.

141:463. 60.

61.

J. H. & H. K. Mackey. 1968. Further studies on the erythrocyte anti-inflammatory assay. Proc. Soc. Exp. Biol. Med. 128: 504. Adamson, J. E., C. E. Horton, H. H. Crawford & W. T. Ayers. 1966. The eff'ects of dimethyl sulfoxide on the experimental pedicle flap: A preliminary report. Plastic Re-

Brown,

construc. Surg. 37: 105. 62.

DE LA Torre,

C,

W. Rowed, H. M. Kawanaga

&

Mullan.

1973. Dimethyl Neurosurg. 38: 345. ACHARI, G. & C. N. SiNHA. 1968. Action of dimethyl sulfoxide on Finkleman preparation. Japan. J. Pharmacol. 18:86. Zetler, G. & H. J. Langhof. 1971. Dimethyl sulfoxide, a reversible inactivator of receptor-eff"ector systems in the isolated guinea-pig ileum. Arch. Exp. Pathol, Pharmakol.270:361. BONNARDEAUX, J. L. 1971. A comparison of the efl"ects of three organic solvents: Dimethyl sulfoxide, formamide, and propylene glycol, on spontaneous activity of isolated smooth muscle. Can. J. Physiol. Pharmacol. 49: 632. Ketchum, L. D., S. S. Ellis, D. W. Robinson & F. W. Masters. 1967. Vascular augmentation of pedicled tissue by combined histamine iontophoresis and hypertensive perfusion. Plastic Reconstruc. Surg. 39: 138. ViSHWAKARMA, S. K. 1968. Dimethyl sulfoxide in tympanoplasty. A preliminary report. Plastic Reconstruc. Surg. 42: 15. Stewart, B. H., A. C. Branson, C. B. Hewitt, W. S. Kiser & R. A. Straffon. 1972. The treatment of patients with interstitial cystitis, with special reference to intraJ.

D.

S.

sulfoxide in the treatment of experimental brain compression.

63.

64.

65.

66.

67.

68.

vesical 69.

70.

DMSO.

J.

J.

Urol. 107: 377.

Jacob, S. W., M. Bischel & R. J. Herschler. 1964. Dimethyl sulfoxide (DMSO): A new concept in pharmacotherapy. Current Therap. Res. 6: 1 34. Seibert, F. B., F. K. Farrelly & C. C. Shepherd. 1967. DMSO and other combatants against bacteria isolated from leukemia and cancer patients. Ann. N. Y. Acad. Sci. 141:175.

71.

PoTTZ, G. E. 1966. The use of DMSO in the staining of mycobakterium and Other microorganisms in smears and tissue sections. In DMSO Symposium, p. 40. Saladruck. Berlin,

72.

West Germany.

Wakao &

K. Nishioka. 1966. Studies on improvement of eye drops. 3. DMSO. Japan. J. Clin. Ophthalmol. 20: 143. Kligman, a. M. 1965. Dimethyl sulfoxide. Part 2. J. Amer. Med. Assoc. 193: 923. KuNZE, M. & H. J. Klein. 1971. The eff'ect of dimethyl sulfoxide upon experimental staphylococcal infection of the Swiss albino mouse. Zentr. Bakteriol. Parasitenk. Abt.

Kamiya,

S.,

T.

Bacteriological consideration of

73. 74.

I.Orig.216: 175. 75.

ScHERBEL, A.

L., L. J.

McCoRMACK & M.

J.

Poppo. 1965. Alteration of collagen

in

78.

scleroderma (progressive systemic sclerosis) after treatment with dimethyl sulfoxide. Cleveland Clinic Quart. 32: 47. Gries, G., G. Bublitz & J. Lindner. 1967. The eff'ect of dimethyl sulfoxide on the components of connective tissue (clinical and experimental investigations). Ann. N. Y. Acad. Sci. 141:630. in clinical Engel, M. F. 1967. Indications and contraindications for the use of dermatology. Ann. N. Y. Acad. Sci. 141 638. Ehrlich, G. E. & R. Joseph. 1965. Dimethyl sulfoxide in scleroderma. Penn. Med. J.

79.

Engel, M.

generalized

76.

77.

DMSO

:

68:(12):51.

J.

65:71.

F. 1972.

Dimethyl sulfoxide

in

the treatment of scleroderma. Southern

Med.

Annals

18 80.

ScHERBEL, A. sulfoxide

82.

&

L. J.

scleroderma

generalized 81.

L.

New York Academy of Sciences

(DMSO).

McCoRMACK. (progressive

Arthritis

Rheum.

1967. Collagen alterations in patients with systemic sclerosis) treated with dimethyl

10: 309

TuFFANELLi, D. L. 1966. A clinical trial with dimethyl sulfoxide in scleroderma. Arch. Dermatol. 93: 724. Sams, W. M., Jr., N. V. Carroll & P. L. Crantz. 1966. Effect of dimethylsulfoxide on isolated-innervated skeletal smooth and cardiac muscle. Proc. Soc. Exp. Biol. Med. 122: 103.

83. 84.

85.

Shealy, C. N. 1966. The physiological substrate of pain. Headache 6: 101. Brown, J. H. 1967. Clinical experience with DMSO in acute musculoskeletal conditions comparing a noncontrolled series with a controlled double blind study. Ann. N. Y. Acad. Sci. 141:496. Brown, J. H. 1966. DMSO its efficacy in acute musculoskeletal problems as

—

evaluated by a "double blind" study. Ind. Med. Surg. 35: 777. 86.

Penrod, D.

& J. Y. Templeton III. 1967. Dimethyl sulfoxide for thoracotomy: Preliminary report. Ann. N. Y. Acad. Sci. 141:

Bacharach

S., B.

incisional pain After

493. 87.

Arno,

I.

relief

88.

C,

P.

Rosenbaum, W. M., dimethylsulfoxide

89.

90.

&

M. Wapner

I.

E.

Brownstein.

1967. Experiences with

DMSO

in

of postpartum episiotomy pain. Ann. N. Y. Acad. Sci. 141: 403. E.

E.

(DMSO)

Rosenbaum

&

S.

W. Jacob.

1965.

The use of

for the treatment of intractable pain in surgical patients.

Surgery 58: 258. ISAAK, P. G. 1965. The use of (dimethyl sulfoxide). Alaska Med. 7: 35. John, H. G. Laudahn. 1967. Clinical experiences with the topical application of in orthopedic diseases: Evaluation of 4180 cases. Ann. N. Y. Acad. Sci. 141:

DMSO

&

DMSO

506. 91.

LocKiE, L. M.

&

B.

M. Norcross.

1967.

A

clinical

study on the effects of dimethyl

sulfoxide in 103 patients with acute and chronic musculoskeletal injuries and inflam-

94.

mations. Ann. N. Y. Acad. Sci. 141: 599. Dubinskii, M. B. & A. A. Skager. 1970. Use of dimethylsulfoxide (DMSO) in patients with affection of the support-motor apparatus. Ortop. Travmatol. Protez. 31: 74. treatment of approximately 1000 Day, P. L. 1966. Evaluation of the results of cases in orthopedic practice. In Dimethyl-Sulfoxyd. G. Laudahn & K. Gertich, Eds.: 107. Internationales Symposium am 8./9. Saladruck. Berlin, West Germany. Paul, M. M. 1967. Interval therapy with dimethyl sulfoxide. Ann. N. Y. Acad. Sci. 141:

95.

Goldman,

92.

93.

DMSO

DMSO

586. J.

superficial

96.

97.

98.

99.

100.

1967.

A

brief

resume of

clinical

observations

in

the treatment of

burns, trigeminal neuralgia, acute bursitis, and acute musculoskeletal

trauma with dimethyl sulfoxide. Ann. N. Y. Acad. Sci. 141: 653. a., A. dal Monte & M. Montanaro. 1965. On the therapeutic action of dimethyl sulfoxide (RE 421) in sports-related traumas and in osteoarticular atlopathies. Gazz. Intern. Med. Chir. 70: 1605. Stewart, G. & S. W. Jacob. 1965. Use of dimethyl sulfoxide (DMSO) in the treatment of post-amputation pain. Amer. Surg. 31:460. Paul M. M. 1966. Comparison of dimethyl sulfoxide and conventional treatment of sports injuries. In Dimethyl Sulfoxyd. G. Laudahn & K. Gertich, Eds.: 101. DMSO Internationales Symposium am 8./9. Saladruck. Berlin, West Germany. Parsons, J. L., W. L. Shepard & W. M. Fosdick. 1967. DMSO as an adjuvant to physical therapy in the chronic frozen shoulder. Ann. N. Y. Acad. Sci. 141: 569. Becker, D. P., H. F. Young, F. E. Nulsen & J. A. Jane. 1969. Physiological effects of

Venerando,

dimethyl sulfoxide on peripheral nerves: Possible role

in

pain

relief.

Exp. Neurol. 24:

272. 101.

Rosenbaum,

E. E., R.

J.

culoskeletal disorders. 102.

Rosenbaum,

E. E.

&

S.

Herschler & S. W. Jacob. 1965. Dimethyl sulfoxide in musAmer. Med. Assoc. 192: 309. W. Jacob. 1964. Dimethyl sulfoxide (DMSO) in acute muscu-

J.

loskeletal injuries and inflammations. sitis.

Northwest Med. 63:

167.

I.

Dimethyl sulfoxide

in

acute subdeltoid bur-

Wood & Wood: 103.

Pharmacologic and Biochemical Considerations

19

DMSO

W .. W. Damelczyk & H. Werner. 1966. bei Spondy-longenen Symposium. Vienna. G. LauNeuropathien. Herausgegeben von Priv.-Doz. In dahn & K. Gertich. Eds.: 134. Saladruck. Berlin. West Germany. in der e.xperimentellen immunologie. In Raettig. H. 1966. Moglichkeiten des Symposium, p. 5 1. Saladruck. Berlin. West Germany. in allergy skin testing. .Ann. Smith. R. E. & A. M. Hegre. Jr. 1966. Use of .Allergy 24:633. Valer. M. & I. Racz. 1970. Examinations relating to the sensibilizating ability of trivalent chromium salts. I. Relation between the epidermal penetrability of trivalent chromium salts and the reactivity of epicutaneous tests. Borgyogyaszati Venerol. Szemle 46:8. .AzAR, M. M. & R. .A. Good. 1971. The inhibitory effect of vitamin .A on complement levels and tolerance production. J. Immunol. 106: 241. Heise, H., a. Mattheus & H. Flegel. 1969. Experimental investigations of DMSOapplication to the chloro-dinitrobenzene eczema of the guinea-pig. .Arch. Klin. Exp. Dermatol. 234: 133. Kligman, -A. M. 1966. The identification of contact allergens by human assay. II. Factors influencing induction and measurement of allergic contact dermatitis. J. Invest. Dermatol. 47: 375. Kligman, A. M. 1966. The SLS provocative patch test in allergic contact sensitization. J. Invest. Dermatol. 46: 573. Rupec, M., H. Geissler & F. Vakjlzadeh. 1968. Effect of dimethyl-sulfoxide on experimental eczema. Sensitization with otherwise subliminal DNCB concentrations. Z. Haut- Geschlechtskrankh. 43: 673. Rosen. H.. .A. Blumenthal, R. Panascltch & J. McCallum. 1965. Dimethyl sulfoxide (DMSO) as a solvent in acute toxicity determinations. Proc. Soc. Exp. Biol. Med. 120:511. Kocsis, J. J. & S. Harkaway. 1966. Hypertaurinuria induced by benzene and other aromatic compounds. Pharmacologist 8(2): 218. Kocsis, J. J. 1967. Biological effects of dimethyl sulfoxide (DMSO), dimethylsulfone (DMSO2), and dimethylsulfide (DMS Pharmacologist 9(2): 67. Formanek. K. & R. SucKERT. 1966. Diuretische wirkung von DMSO. In SymBiRKMAVER,

DMSO

104.

DMSO

DMSO

105.

106.

107.

108.

109.

1

10.

111.

112.

113.

114.

DMSO

).

115.

116.

117.

DMSO

posium, p. 21. Saladruck. Berlin. West Germany. Jacob, S. W. 1971. Pharmacology of DMSO. 'in Dimethyl sulfoxide. Vol. 1. S. W. Jacob, E. E. Rosenbaum & D. C. Wood, Eds.: 99. Marcel Dekker, Inc. New York,

N.Y. Weissmann.

G.. G. Sessa & V. Bevans. 1968. Effect of DMSO on the stabilization of lysosomes by cortisone and chloroquine in vitro. Ann. N."!'. Acad. Sci. 141 326. :

PHYSICAL PROPERTIES OF DIMETHYL SULFOXIDE AND ITS FUNCTION IN BIOLOGICAL SYSTEMS H. Harry Szmant

Department of Chemistry University of Detroit Detroit, Michigan 48221

The unique capability of dimethyl sulfoxide (DMSO) to penetrate living tissues without causing significant damage is most probably related to its relatively polar nature, its capacity to accept hydrogen bonds, and its relatively small and compact structure. This combination of properties results in the ability of to associate with water, proteins, carbohydrates, nucleic acid, ionic substances, and other constituents of living systems. Of foremost importance to our understanding of the possible functions of in biological systems is its ability to replace some of the water molecules associated with the cellular constituents, or to affect the structure of the omnipresent water. The work discussed in this paper addresses itself to the latter point, and is based on the study' of the liquid system water-DMSO by means of the spin-lattice relaxation and chemical shift behavior of both the water and protons. The binary system was investigated over a relatively large temperature range, and the relaxation times (fi) and chemical shifts were determined as a function of the concentration of the two components and of their deuterated analogues. The proton spin-lattice relaxation time is a measure of the energy decay of an array of excited hydrogen nuclei. The predominant mechanism of the dissipation of energy involves magnetic dipole-dipole interactions between the nuclei of neighboring protons. Since this principal relaxation path is very sensitive to the distance that separates the interacting protons (a sixth-power relationship applies), the experimentally observed changes in the t^ values reflect the dynamic changes that occur in the stereochemical environment of the protons under consideration as one varies the composition and the temperature of the system. Changes in the structure of the liquid can be inferred to have occurred from the experimentally determined t^ values, since the reciprocal of ^i is directly proportional to the molecular correlation time (tc), which in turn represents the time required for a complete rotation of the molecular moiety that contains the protons under consideration. Thus maxima in the plots of 1/?! versus composition, for example, reveal an increased structuring of the system, which may involve attractions between the molecular moieties that contain the interacting protons, hence prolonging their correlation times. It is of interest to note that the relaxation of deuteron nuclei results from the interaction not of nuclear dipoles, but rather of nuclear quadrupoles, and consequently the deuteron relaxation mechanism involves only intramolecular interactions. One can take advantage of this difference in the behavior of protons and deuterons by examining mixtures of the analogous isotopic substances; in this way one can separate the intra- and intermolecular contributions to the relaxation process. It stands to reason that a decrease in the concentration of the protonated species dissolved in its deuterated counterpart gradually eliminates the intermolecular contributions to the total relaxation time, and that extrapolation of the relaxation times to an infinitely dilute solution of the protonated species, dissolved in the magnetically relatively inert counterpart, allows one to evaluate the intramolecular ^i or Tc values.

DMSO

DMSO

DMSO

20

Szmant: Physical Properties and Biological Function The

21

on work' with four Hquid systems: water and finally, mixbehavior of each kind of proton was examined separately by means of the saturation recovery spectrometer was used. method; a modified Varian A60A The addition of DMSO to water, or vice versa, raises the I//1 values of both the water and DMSO protons, and both protons exhibit maximum values at ca. A'h20 = results discussed here are based

DMSO; water and DMSO-^g; deuterium oxide and DMSO; tures of DMSO, DMSO-Je, and deuterium oxide. The relaxation and

NMR

0.65.

30%

The

effect of the

admixture of the other component, however,

is

approximately

larger in the case of the protons of water, even though a priori iht absolute l/t^

values of the water protons are about twice as high as the 1//, values of DMSO. These results imply that a molar ratio of water and DMSO of approximately 2:1 produces the greatest restriction on the rotational freedom of the components, and that such a restriction is more pronounced in the case of water, even though the latter is presumably a more highly structured liquid than DMSO. Naturally, a decrease in the thermal agitation of the molecular system causes the 1//, maxima to develop to a higher degree, but it is noteworthy that the I//1 maxima are discernible even at the highest temperature employed here (41.4''C), and again they are more so in the case of the water protons. The structural implications of these observations can be stated as follows. It appears that DMSO induces a more intensive structuring of water than vice versa, and the concentration dependence of this phenomenon suggests that three DMSO molecules are very effective in producing a highly structured cluster of six water molecules. The decision to translate the 2:1 molar ratio to structural units that involve six water and three DMSO molecules is based on the well-recognized^ structural unit of ice I, which consists of six water molecules arranged in a chairlike conformation. Since similar ice-Hke clusters of water are believed to represent, at least in part, the highly structured portion of liquid water, the above-mentioned results of the relaxation measurements suggest that DMSO molecules are able to "lock in" the hexameric water clusters, and thus to increase their concentration above and beyond that present in neat water at a given temperature.

The replacement of

DMSO by

DMSO-^e

does not have an appreciable effect on

the relaxation times of the water protons except at the lowest temperatures

em-

ployed (18.7°C and 9.8° C), and then only in the water-rich mixtures. This observation is consistent with the structural model described above, since it places the burden of relaxation on interactions among the water protons, and allows for a high degree of rotational freedom of the molecules, even when they function to

DMSO

"lock in" the water clusters. We shall turn our attention for a moment to those proton relaxation results that have a bearing on the Hquid structure of DMSO. The replacement of water by deuterium oxide has a relatively large effect on the relaxation of the protons, and this effect persists over the whole temperature range of 9.8-41. 4° C, although it is more pronounced in the water-rich mixtures. On the basis of these observations one

DMSO

may draw

the conclusion that, apart from the intramolecular proton interactions,

the relaxation

mechanism of liquid

DMSO involves intermolecular

proton interac-

promote the These conclusions are consistent with the concept that DMSO has a predominantly liquid structure in which pairs of methyl groups of neighboring DMSO molecules point away from each other, regardless of whether the molecules are associated in the form of chains or large rings. ^ The introduction of water molecules disrupts this arrangement and finally brings about the proposed DMSO-stabilized, ice-like water clusters, in which the DMSO molecules that are hydrogen-bonded to the same cluster (or aggregate of clusters) can bring their methyl groups into close tions to a relatively latter.

minor extent, and the presence of water

is

able to

22

Annals

New York Academy

of Sciences

DMSO proton relaxation times in mixtures of and 0,0 allows one to separate the intra- and intermolecular found that in the temperature range of 29.9-41.4' C, the

proximity. The determination of the

DMSO. DMSO-^e. contributions.

It

is

intramolecular interactions contribute nearly twice as much as the intermolecular interactions between molecules, and the presence of D.O has relatively little effect on these contributions. On the other hand, in the lower temperature range of 9.8-18."' C. the magnitude of the intermolecular interactions approaches that of the intramolecular ones, and this happens to the greatest extent when .Yd20= 0.6. Unfortunately, because of the ver\ rapid hydrogen-deuterium exchange in mixtures of water and deuterium oxide, it was impossible to separate the intra- and intermolecular contributions to the relaxation of the water protons. The chemical shifts of the water protons in the presence of were determined over a temperature range from -54.2'C to 41.4'C. The relatively greater chemical shift (downfield) of the water protons observed in a water-rich environment suggests that this circumstance is conducive to the formation of linear

DMSO

DMSO

hydrogen bonds of polar character. The latter are associated with the highly structured, ice-like water, and thus it is not surprising that the chemical shifts also increase as the temperature is lowered. Since the chemical shifts of the water protons at the other extreme condition of a water molecule in a DMSO environment are also known, it is possible to calculate the chemical shifts that would result from simple, statistical contributions of both proton environments in accord \^ith the composition of the mixture. Any deviation of the observed chemical shifts from the calculated shifts indicates preferential structuring of the liquid, and the direction of a given deviation indicates the typ)e of liquid structure that

preferred.

is

The

ap-

plication of such a treatment to the chemical shifts of water protons in the presence

of

DMSO

chemical

shifts in the direction of the

is

values;

relatively temperature-insensitive,

it

At low concentrations of

reveals the existence of two such deviations.

water, there is

a deviation of the

DMSO

and reaches

a

DMSO-rich

maximum when

the

molar ratio of 1:2. This result reveals that in relatively DMSO-rich mixtures, the water molecules tend to become doubly hydrogen-bonded to DMSO. The formation of 1:2 water-DMSO complexes has been reported previously.^ and is probably of little interest in biological systems, because lO^c water. Another deviation in it represents a mixture that contains only ca. chemical shifts was found, however, in the range of high concentrations of water. The direction of this deviation was toward the chemical shift values of neat water; it was rather sensitive to the temperature of the liquid system, and it exhibited a maximum when the water and DMSO molecules were at a molar ratio of 3:1. This obser\'ation again agrees >Aith the previously proposed effect of DMSO on the structure of water. Even though the maximum deviation in the chemical shift corresponds to a mixture that contains only ca. 41 ^c water, the buildup of the structure water and

molecules are

at a

that gives rise to this deviation begins as soon as

DMSO

is

introduced into water.

seems that even low concentrations of DMSO are able to promote the accumulation of ice-like water clusters, and that this tendency becomes more intense at

Thus

it

the lower

temperatures.

On

the basis of chemical

shifts,

the

DMSO

DMSO-induced

are at a molecular climax when water and ratio of 6:2. while the relaxation results suggest a molecular ratio of 6:3. This discrepancy can be resolved by considering the accuracy of the basic data and the sensitivity of both criteria to structural changes: the preference falls on the molecular ratio of 6:2 (this is deduced from the chemical shift measurements). The latter molecular ratio happens to coincide with the composition of a compound that melts at -62' C, which has been detected^ in the phase diagram of the water-DMSO

structuring of water

svstem.

comes

to a

Szmant: Physical Properties and Biological Function Eight years ago

The New York Academy of Sciences sponsored

23

a conference on

the subject of forms of water in biological systems,^ and the structure of water, the

changes induced

in its

structure by the presence of different solutes, and the bio-

water were of great concern to the parthat stabilizes ice-like water clusters, and that it may therefore be capable of displacing the equilibrium between the less and more highly structured water, in favor of the latter. Since the hydration of cell constituents and the activity of water in general are not necessarily the same may exert an indirect effect on in the different states of water, it follows that biological systems by virtue of the changes that it causes in the liquid structure of water. Among the more important biological consequences of this indirect effect of DMSO, one can mention changes in the conformations and associations of proteins and other molecules. More direct biological effects caused by DMSO, without a profound change in its chemical identity, may include changes in ion-pairing equilibria and in the specific solvation of hydrogen-bond donors. logical implications of the different states of ticipants.

The evidence presented here suggests

DMSO

DMSO

References 1.

ToKUHiRO,

2.

EiSENBERG, D.

Menafra & H. H. Szmant. 1974. J. Chem. Phys. In press. & W. Kauzmann. 1969. The Structure and Properties of Water.

T., L.

University Press, Inc. 3.

4. 5.

Oxford

New York, N.Y.

Szmant, H. H. 1971. In Dimethyl Sulfoxide. S. W. Jacob, E. Wood, Eds. Marcel Dekker Inc. New York, N.Y. Rasmussen, D. H. & A. P. Mackenzie. 1968. Nature 220: 1315. Whipple, H. E., Ed. 1965. Ann. N.Y. Acad. Sci. 125(2): 249.

E.

Rosenbaum

&

D. C.

INFLUENCE OF NONIONIC ORGANIC SOLUTES ON VARIOUS REACTIONS OF ENERGY CONSERVATION AND UTILIZATION Thomas

E.

Conover

Department of Biological Chemistry Hahnemann Medical College Philadelphia, Pennsylvania 19102

Unlike many of the papers to be included in this monograph, this paper will not elaborate on the various unique properties of dimethyl sulfoxide; rather it will discuss some of the effects that it shows in common with a broader class of compounds, the water-soluble, organic solvent. In apology, I must confess that the main

work was not toward dimethyl sulfoxide at all, although the effects of have been studied. The major prototype chosen in this work was glycerol; I hope, however, to indicate the various similarities in their effects. It might be mentioned here that although most of the compounds studied can be called solvents, they have been studied in aqueous solutions, and therefore will be referred to as thrust of this this solvent

solutes.

The study of enzymic reactions in aqueous solutions of organic solvents goes back to the work of Nelson and Schubert,^ whose studies on the effect of alcohol on the hydrolysis of sucrose catalyzed by "invertase" established the role of water in this enzymic reaction in 1928. Numerous other workers have studied purified singleenzyme systems by this means. ^-^ In such systems, the effect of solvent can often be attributed to its effect of decreasing the water concentration in the reactions that involve water as a participant.^ The problem of applying such an approach to a system as complex and ill-understood as that of mitochondrial energy conservation is obviously a more precarious it is a primary product of ATP synthesis, must be intimately involved in these processes. Indeed, Mitchell's chemiosmotic theory"* notwithstanding, a major reason, if not the primary one, for the vital importance of the integrity of the mitochondrial membrane in the processes of energy conservation lies in the need to exclude water, both environmental water and, perhaps, that produced at the sites of energy coupling. In spite of such reservations, this problem was examined. At its initiation, this investigation was guided by the fond hope that interference with the active water concentrations by highly soluble, biologically innocuous solutes might lead to a stabilization of high-energy intermediates and to a possible increase in the P:0 ratio and the rate of energy-linked reactions. task, although water, since

Effect

of Glycerol on Respiration and Oxidative Phosphorylation Submitochondrial Particles

in

In these studies, submitochondrial particles prepared from bovine heart mitochondria by sonication were used.^ Such preparations have the advantage of good stability, and do not present the problem of osmotic effects due to high solute concentration, which might be anticipated with intact mitochondria. When such a preparation was incubated with varying concentrations of glycerol in aqueous me-

24

Conover: Influence of Nonionic Organic Solutes

2

4

6

25

8

GLYCEROL CONCENTRATION, MOLAR Figure

1.

Effect of glycerol on respiration and oxidative phosphorylation in submitochon-

drial particles.

The

reaction

MgCl2, 4 /xmol ATP,

1

medium contained 20 ^mol triethanolamine-HCl (pH 7.4), 8 ^mol EDTA, 20 ^imo\ '^P-orthophosphate (10* cpm/^mol, pH 7.4); 24

/xmol

mg hexokinase, 2 mg bovine serum albumin, and 1.5-3 mg submitochondrial volume of 2.0 ml. The substrates were 20 ^mol succinate with 2 /imol malonate in one case, and 40 /xmol DL-/8-hydroxybutyrate with 0.5 /xmol NAD+ in the other. The glycerol concentration was as indicated. Oxygen consumption was followed polarographically, and the '^P-orthophosphate esterification was determined after deproteinization, as described elsewhere.^ The oxygen content of mixtures of glycerol and water was determined from values given in the International Critical Tables.^ The temperature was 25° C. /xmol glucose, 0.2 particles in a

dium, and the oxygen uptake and the inorganic phosphate esterification in the presence of a hexokinase-glucose coupling system were followed, the results shown in Figure 1 were obtained. With a very active substrate such as succinate, a linear decrease in the oxygen consumption was observed as the concentration of glycerol was increased. With the NAD+-linked substrate /S-hydroxybutyrate, which is oxidized at a considerably lower rate than succinate, little effect was seen until the concentration of glycerol exceeded 3.0 M. This concentration of glycerol decreased succinate oxidation to a rate similar to that of the jS-hydroxybutyrate oxidation. At concentrations of glycerol above 3.0 M, the rate of respiration with ^hydroxybutyrate showed a linear decline of activity with the increasing glycerol concentration, parallel to that of succinate. It is presumed that the rate-limiting step in jS-hydroxybutyrate oxidation is the dehydrogenase step; and inhibition of glycerol did not become apparent until the respiratory chain was sufficiently inhibited to become limiting. The P:0 ratio was affected in the opposite manner. The ratio with /3hydroxybutyrate was rapidly decreased at low concentrations of glycerol, presumably because phosphate esterification was being inhibited while oxygen

consumption was not. With succinate, the P:0 ratio declined less sharply, since succinate oxidation was also being inhibited at these concentrations. It

appears, therefore, that electron transport

relation with increasing concentrations of glycerol.

is

No

inhibited in a simple linear simple relation in the case of

Annals

26

New York Academy of Sciences

P:0 ratio was apparent, however, nor was there any apparent stabilization of energy coupling which it had been hoped would be observed with the increased glycerol concentrations. It may be pointed out, however, that at least a portion of the phosphate esterification reaction seemed to be quite resistant to glycerol inhibition. Whether this might be due to a higher stability of one specific site of phosphate esterification or to a nonspecific resistance of the process of phosphate esterification to high concentrations of glycerol is not clear.

the

Effect

of Organic Solutes on Electron Transport

in

Submitochondrial Particles

Tyler and Estabrook^ have studied the electron transport chain in similar systems by means of spectrophotometric measurement of the components of the chain, for which they used a dual-wavelength recording spectrophotometer. Since the rate of reduction of each individual component may be followed in this manner, it is possible to determine both the steady-state level of reduction of the component and the rate of substrate oxidation. In Figure 2 the values they obtained for both cytochrome c -\- Ci components (551-540 nm) and cytochrome a -\- a^ components (605-625 nm) are shown. It can be seen that the inhibition of oxidation was linear with glycerol concentration, giving 50% inhibition at about 3.0 M. This is similar to the data shown in Figure 1 for succinate oxidation. It is most interesting, however, that at all concentrations of glycerol the steady-state reduction level of the components did not vary, but acted as if they were frozen in place. This is very different from the situation that appears with the usual site-specific inhibitors, such as antimycin A or cyanide, which produce marked changes in the steady-state level of reduction, depending on the site of inhibition.® This constant level of reduction at steady state must result from a nonspecific inhibition of all components, which apparently affects all components in a quantitatively similar manner. This, then, might be termed a nonspecific inhibition, and must affect some element common to all components and their reactions.^ Figure 3 shows the effect of various other organic solutes, including dimethyl sulfoxide, on the oxidation of by submitochondrial particles. One of these solutes, sucrose, has been previously reported to inhibit electron transport and oxidative phosphorylation at higher concentrations.*"*^^ It may be seen that although the concentration required to produce a 50% inhibition of respiration varied widely, sucrose being the most effective and ethylene glycol the least, the inhibitions all showed a simple linear relationship with concentration up to at least 60 or 70% inhibition. Furthermore, these inhibitions were all reversible, and all appeared to be of

NADH

NADH

the

same

nonspecific type.*

has been suggested that one effect of glycerol might be to increase the viscosity of the medium, thereby perhaps hindering the complex interaction of components. '^ The data of Tyler and Estabrook* would clearly eliminate such a consideration, since there would appear to be little relation between viscosity and the degree of inhibition. For instance, dimethyl sulfoxide proved to be an effective nonspecific inhibitor, but solutions of dimethyl sulfoxide do not show much of an increase in viscosity, at least compared to solutions of sucrose or glycerol.* Since our concern here is with the modification of the water content of the medium by the solutes, the inhibitions may better be expressed in relation to the volume of water or solute as a percentage of the total solvent volume. In Figure 4, the data of Figure 3 have been replotted in terms of the percentage of solute, on a volume/ It

Conover: Influence of Nonionic Organic Solutes

27

100

% Inhibition /Molarity

*/•

Reduction in Steady State

55l-540n»/4

#

•

605-625 m/A

1^

20 Glycerol

3.0

4.0

5.0

Concentration Molar

NADH

Figure 2. Effect of glycerol on the oxidase and the steady-state reduction of endogenous cytochrome components. Reaction mixtures contained 0.1 potassium phosphate buffer (pH 7.2), 0.18 NADH, and 6 mg submitochondrial particles. The glycerol concentration was as indicated. The temperature was 22° C. The kinetics of cytochrome reduction were recorded in a dual-wavelength spectrophotometer, set at 551-540 mn (cytochrome c -\Ci) or 605-625 m/i (cytochrome a + Gz). Two plots of percentage inhibition versus molarity are shown, which were based on the degree of inhibition calculated from the spectrophotometric traces obtained at the two pairs of wavelength settings used. The percentage steady-state reduction of cytochrome was calculated as the absorbance change at steady state relative to the overall change between the fully oxidized and the fully reduced condition. For more details, see the original paper. (From Tyler and Estabrook.* Reproduced by permission of the Journal of Biological Chemistry.)

mM

M

volume

basis, which would be one way of expressing this relationship. Tyler and Estabrook used a g/g basis, which did not always appear to give as good a fit for sucrose in our results.^ It may be seen that with all of these compounds, a congruent relation now appears to exist between the inhibition of electron transport and the water content. One might note that dimethyl sulfoxide is perhaps the most effective inhibitor among these compounds, which suggests that it may either have other effects or may be in some way more effective in reducing the active water content.

Nevertheless, the conclusion is implied that the nonspecific inhibitory effect of the various solutes was related to a reduction in the water content of the reaction mixtures,

and was independent of the chemical nature of the substance used.

DMFA EtGI

Molarity

Figure

3.

Of

NADH

Relation between solute inhibition of

reaction mixture contained 0.1

Solvent oxidase and solute molarity. The mg submitochondrial

M potassium phosphate (pH 7.2) and 5.5

were as indicated. Sue = sucrose; DMSO = dimethyl sulfoxide; EtGI = ethylene glycol. The temperature was 22° C. (From Tyler and Estabrook.* Reprinted by permission of the Journal of Biological Chemistry.) particles. Solute concentrations

Gly = glycerol;

DMFA = dimethylformamide;

100

o GLYCEROL

A ETHYLENE GLYCOL D SUCROSE • DIMETHYL SULFOXIDE

A n-PROPYL ALCOHOL

10

30

20

%

SOLUTE

Figure 4. Relation between solute inhibition of The data of Figure 3 have been replotted

solute.

solute inhibition of

40

50

v/v

NADH to

oxidation and the volume percent emphasize the relationship between the

NADH oxidase and the water content of the reaction media. 28

Conover: Influence of Nonionic Organic Solutes of Organic Solutes on Energy-linked Reactions

Effect

When we

in

29

Submitochondrial Particles

compounds on energy-conservation reacbecomes more complex, as is shown in Figure 5. In this figure the effects of glycerol on a number of reactions related to energy conservation are shown. These are oxidative phosphorylation, the ^^Pj-ATP exchange reaction, ATPase activity, and the succinate-linked reduction of NAD^ driven by ATP. The exchange reaction and the ATPase activity represent what is thought to be a partial reversal of the phosphate esterification reaction. The succinate-linked reduction of turn to the effects of these

tions, the situation

NAD^

represents the total reversal of oxidative phosphorylation, in that a reversal

of electron transport driven by the hydrolysis of

O 3 Q •

ATP

is

measured.

It

can be seen

P:0 RATIO NAD^- REDUCTION

ATPase ACTIVITY Pr>'^-MP

Vo glycerol

—

EXCHANGE

v/v

Figure 5. The effect of glycerol on related reactions of oxidative phosphorylation in submitochondrial particles. The reaction medium for oxidative phosphorylation was as described in the legend to Figure 1, with succinate as substrate. The reaction medium for the '2Pi-ATP exchange contained 16 mhioI ATP, 16 /imol MgC^, 1 Mmol EDTA, 40 ^mol ^^p-orthophosphate (10" cpm/^mol, pH 7.4), and 1 mg submitochondrial particles, in a final volume of 1.0 ml. The reaction medium for the ATPase assay contained 50 ^mol triethanolamine-HCl (pH 7.4), 3.0 Mmol MgCU, 6.0 ^niol ATP, 0.8 ^mol 2,4-dinitrophenol, 5.0 //mol phosphoenolpyruvate, 0.05 mg pyruvate kinase, and 0.4 mg submitochondrial particles, in a volume of 1.0 ml. The reaction medium for the energy-linked reduction of NAD"^ by succinate contained 50 ^mol triethanolamine-HCl (pH 7.4), 10 /xmol MgClj, 1 Mmol ATP, 0.5 ^mol NAD"^, 10 Mmol succinate, 1 Mmol Na2S, 0.5 Mmol EDTA, 1 mg bovine serum albumin, and 0.4 mg submitochondrial particles, in a final volume of 2.0 ml. The glycerol concentration was as indicated in each case. The temperature was 25° C for all assays, o o = P:0 ratio (activity without glycerol, 0.88); • • = "Pj-ATP exchange reaction (activity without glycerol, 0.14 Mmol/mg/min); • • = ATPase activity (activity without glycerol, 0.65 Mmol/mg/min); • • = energy-linked NAD+ reduction by succinate (activity without glycerol, 0.027 Mmol/mg/min). (From Conover.* Reproduced by permission of the Journal of Biological Chemistry.)

Annals

30

New York Academy of Sciences

do not show the linear inhibition relative to concentration observed with electron transport, but rather a complex family of sigmoid curves. Low

that these reactions

concentrations of glycerol often actually stimulated activity, as is shown here for NAD+ reduction. This phenomenon was commonly observed with the ATPase activity, and occasionally even with the exchange reaction as well. This was followed by a rather sharp drop in activity as the concentrations increased. It is possible that the sigmoidicity may result from the observed stimulation by the solute and a simultaneously occurring linear inhibition. On the other hand, oxidative phosphorylation as measured by the P:0 ratio declined rather less rapidly, as we noted earlier. This was undoubtedly due in part to the simultaneous inhibition of both respiration and phosphate esterification. However, when the actual rate of orthophosphate-^^P esterification during both oxidative phosphorylation and the ^^PjATP exchange under similar conditions was determined (Figure 6), it can be seen that the exchange was considerably more sensitive to inhibition by the organic solute. Although the rate of the exchange was more rapid in the absence of glycerol, it actually dropped below that of oxidative phosphorylation at higher concentrations of solute. This contrast

to

is

possibly related to the fact that the ^^Pj-ATP exchange reaction in

esterification in both

reactions

phosphorylation involves the reactions of phosphate forward and reverse directions. Indeed, as mentioned, all the

oxidative

shown

phosphorylation.

in

Figure

It is

5 require the reversal

of these reactions, except oxidative

possible that the reverse reaction

is

somewhat more

sensitive

to this inhibition by organic solutes.

o SUCCINATE OXIDATION » /3-HYDROXYBUTYRATE OXIDATION

EXCHANGE

Vo glycerol

—

v/v

Figure 6. A comparison of the rates of orthophosphate -"? esterification during oxidative phosphorylation and the "P, -ATP exchange reaction in the presence of glycerol. The conditions for the determination of these rates were those described in the legends to Figures 1 and • = oxidative 5. o o = Oxidative phosphorylation with succinate as substrate;* • ="Pi -ATP exchange phosphorylation with /9-hydroxybutyrate as substrate; • reaction. (From Conover." Reproduced by permission of the Journal of Biological Chemistry.)

Conover: Influence of Nonionic Organic Solutes

O INITIAL (0 Mg"^"*") 3 lOmM Mg"*"*" • Q5mMATP + IOmM

20

10

®/o

Figure

7.

The

effect

of glycerol

submitochondrial particles. The 7.4), 0.5

Mmol NADP+,

0.1

initial

on

40

30

glycerol the

—

Mg-^-^

50

v/v

energy-linked

transhydrogenase activity of

reaction contained 50 /xmol triethanolamine-HCl

^mol NAD+,

31

(pH

EDTA, 85 /xmol ethanol, 0.03 0.4 mg submitochondrial parti-

2 /xmol NazS, 0.5 /xmol

mg bovine serum albumin, and volume of 2.0 ml. After the initial rate of NADP"^ reduction had been recorded, 20 /imol MgCla were added, and the rate was recorded again. Finally, 1 /xmol ATP was added and o = initial rate; the rate was recorded again. The temperature was 25° C. o 9 = rate after addition of MgClz; • • = rate after addition of ATP. (From 9 Conover." Reproduced by permission of the Journal of Biological Chemistry.) mg

alcohol dehydrogenase,

1

cles, in a

Figure

shows another possible example of

this: the eff"ects of glycerol on the energy-driven transhydrogenase activity of submitochondrial particles. This rather unusual reaction of mitochondria shows an inhibition by organic solutes that is logarithmic with solute concentration, for reasons that are not under-

7

NADH-NADP"^

stood at this time. Among the reactions of energy conservation in mitochondria, this one frequently shows rather unique properties. ^^ It can be seen that the eff'ect of glycerol on the initial rate, in the absence of ATP, was less marked than its effect on the rate after the addition of Mg++ and ATP to drive the reaction. That we are still talking about a nonspecific inhibition similar to that observed with electron transport can be seen in Figures 8 and 9, which demonstrate the similarity of the inhibitions by a variety of the organic solutes. Figure 8 shows the effects on the ^^Pj -ATP exchange. The polyhydroxyl compounds glycerol, ethylene glycol, and sucrose all showed excellent agreement when the results were plotted by percent volume against concentration. Dimethylformamide, however, was somewhat more inhibitory, for unexplained reasons. Dimethyl sulfoxide again appeared to give an inhibition similar to the others, although as with electron transport, it seemed to be somewhat more effective than the polyols. By contrast, A2-propyl alcohol was shown to be a much more inhibitory compound, producing a marked, irreversible inhibition of these reactions. This is presumably related to the more asymmetric polarity of primary alcohols and their possible effects on membrane

100

o GLYCEROL

A ETHYLENE GLYCOL D SUCROSE

• DIMETHYL SULFOXIDE

DIMETHYLFORMAMIDE X n-PROPYL ALCOHOL

10

20

%

30 SOLUTE

40

50

v/v

Figure 8. The effect of various organic solutes on the ^^Pj-ATP exchange reaction of submitochondrial particles. The conditions for the assay of the ^^Pj -ATP exchange reaction were those described in the legend to Figure 5. o o=Glycerol; a A = ethylene = sucrose; • glycol; A = dimethylformamide; • = dimethyl sulfoxide; a X X = ^-propyl alcohol.

o GLYCEROL

A ETHYLENE

GLYCOL D SUCROSE • DIMETHYL SULFOXIDE

A DIMETHYLFORMAMIDE X

n-PROPYL ALCOHOL

Vo solute - v/v 9. The effect of various organic solutes on the ATPase activity of submitochondrial The reaction mixture in each case contained 80 ^mol triethanolamine-HCl (pH 7.4), 8 ^imol MgClz, 8Mmol ATP, 2 /xmol 2,4-dinitrophenol, and 0.2 mg submitochondrial particles, in a total volume of 4.0 ml. The concentrations of solute were as indicated. The temperao o= glycerol; a n = sucrose; ture was 25° C. a = ethylene glycol; n

Figure

particles.

•

• =dimethyl sulfoxide;

A=dimethylformamide; x

32

x = /7-propyl alcohol.

Conover: Influence of Nonionic Organic Solutes

33

Figure 9 shows a similar study of the ATPase activity, which produced same picture. Clearly, there again appears to be a common relation-

structure.

very nearly the

ship between the extent of inhibition by these organic solutes and the decrease water content of the reaction mixture.

Reversibility

A

the

of Solute Inhibition of Energy-linked Reactions

second indication of the nonspecific nature of

Figure

in

this inhibition is its reversibility.

shows the reversal of the glycerol inhibition of the ^^Pj-ATP exchange reaction. This experiment was done by diluting each incubation mixture tenfold before starting the assay; hence the small decline in activity was possibly due to the small quantity of glycerol remaining. Experiments performed by washing particles with 0.25 sucrose appeared to support this contention, as shown in Table 1. Inhibition of the ^^Pj-ATP exchange by the other solutes was also reversible; however, in the case of dimethylformamide, part of the inhibition was not reversed at higher concentrations of solute, even after the particles had been washed. The inhibition with /7-propyl alcohol was almost totally irreversible, as previously noted. 10

M

100

20 30 40 Vo glycerol v/v Figure 10. The reversal of the glycerol inhibition of the ^^I^ -ATP exchange reaction in submitochondrial particles. The conditions for the assay of the ^^Pj -ATP exchange reaction were those described in the legend to Figure 5. • • = submitochondrial particles mixed for 2 min at 0°C with glycerol at the concentrations indicated, and added to an assay medium that contained glycerol at the same concentration; o o = submitochondrial particles mixed for 2 min at 0°C with glycerol at the concentrations indicated, and added to an assay = submitochondrial particles inmedium that contained no glycerol (dilution, 1:10); o cubated for 10 min at 25° with glycerol at the concentrations indicated, and added to an assay medium that contained no glycerol (dilution, 1:10). (From Conover.^ Reproduced by permission of the Journal of Biological Chemistry.)

Annals

34

New York Academy of Sciences Table

1

Reversal of Solute Inhibition on the ^^Pi-ATP Exchange Reaction ^^P, Esterified

Solute

With Solute

40%, v/v

After Washing*

nmol/rrig/min

None Glycerol Ethylene glycol Sucrose

Dimethyl sulfoxide Dimethylformamide

142

128

8

126

9

132

7

123

5

115

3

98

w-Propyl alcohol

18

*Submitochondrial particles, incubated 5 min at 25°C with solute, were diluted with MgCl2 and 0.001 ATP, and were centrifuged sucrose, which contained 0.01 at 100,000 X g for 40 min. The assay was made as described in the legend to Figure 5, on particles resuspended in the sucrose medium.

0.25

M

M

M

Relation of Solute Inhibition to Substrate Concentration

Although the submitochondrial particle is very crude in the sense that it is a complex multienzyme system, an attempt was made to study the effect of glycerol on the binding of ATP, Mg+^, and orthophosphate in the ^^p.-ATP exchange reaction. Reciprocal plots of some of the data are shown in Figures 1, 12, and 13. 1

Caution should perhaps be exercised

in the interpretation

of such plots; however,

it

10

i/Catp]-m ^Tj-ATP exchange reaction by glycerol, as a function of The conditions of assay were those described in the legend to Figure 5. The values for v on the ordinate are expressed as Mmol AT^T formed/mg/min. # = 20% glycerol (V/ -o=10% glycerol (K/K); • o = Without glycerol; o o V). (From Conover.^ Reproduced by permission of the Journal of Biological Chemistry.) Figure

ATP

11.

The

inhibition of the

concentration.

Conover: Influence of Nonionic Organic Solutes

35

200

100

l/[Mg++J-M"' Figure 12. The inhibition of the ^^P^-ATP exchange reaction by glycerol, as a function of orthophosphate concentration. The conditions of the assay were those described in the legend to Figure 5. The values for v on the ordinate are expressed as /xniol AT^^P formed/mg/min. o o = without glycerol; » •=20% glycerol (V/ me

solutions of 4

ml

diluted at zero time to 0.4

in 0.01

buffer

^pH

S.Oi

were prepared

mg

/

proidn.

Henderson

et al.

:

Effects on Subunit Proteins

41

46

10

20

15

25

PER CENT DMSO Figure 2. Reciprocal effects of DMSO on glulamate and alanine dehydrogenase activities. The GDH assay was as described in the legend for Figure the alanine dehydrogenase (ADH) assay mixture was similar, but contained 150 ^mol L-alanine instead of glutamate. GDH and 1

ADH

activities in the

absence of

culated relative to this.

GDH

ADH

;

DMSO were arbitrarily set at 1.0, and other values were calabsence of DMSO was approximately 2% of the

activity in the

activity.

an equilibrium between an active and inactive conformation. The active

monomer

appears to associate linearly to form rods of indefinite length. The strong stabilizing effects of D2O on the polymeric form and the stabilization of against denaturation by dilution suggest that bound water plays a significant role in maintaining the structure of this enzyme and in facilitating conformational-type changes. ^'^

GDH

The

effects of

DMSO that

we have detected on

this

enzyme include

dissociation

of the polymeric forms, shifting of the equilibrium between the active and inactive

monomers

(to favor the inactive

monomer), and

either stabilization or denaturation

DMSO concentration and temperature.* The effect of DMSO on GDH that was detectable at the lowest DMSO levels was protection of of the enzyme, depending on the the

enzyme from

inactivation by dilution at low temperatures.

GDH

was appreciably

These

effects result primarily

of

stabilized at

5°C

Figure

1

shows that

even by 5% DMSO. on the hydrophilic groups

in dilute solutions,

from the effects of DMSO and bound water, since hydrophobic bonding is minimized at low temperaIndependent evidence for this interpretation was shown by the similar stabi-

GDH

tures.

GDH against inactivation by dilution in a D2O environment.^ DMSO was added to the medium during GDH assay, the GDH activity diminished as the DMSO concentration was increased (Figure 2). The sigmoid

lization of

When

shape of the curve suggests that as

is

this

was due

activity also suggests that there

dehydrogenase the active

activity

monomer.

is

to a cooperative type of inhibition, such

The increase

in alanine dehydrogenase were conformational changes, since the alanine

often seen with allosteric modifiers.

considered to be associated with the inactive rather than

New York Academy of Sciences

Annals

42

When the effects of DMSO on the inhibition of GDH activity in H2O and D2O were compared, DjO appeared to exert a synergistic effect with DMSO in inhibiting GDH (Figure 3). This can be interpreted as an apparent increase in the concenmonomer. D2O facilitates the reaction of the enzyme with but does not change the maximal slope of 2*7; this indicates that the inactive monomer may have more exchangeable groups on the surface of the protein tration of the inactive

DMSO,

DMSO or D2O than does the active monomer. DMSO in inhibiting GDH were reversible by the allosteric effector ADP, in a competitive manner (Figure 4). This further suggests that DMSO shifts form hydrogen bonds with

that can

The

effects of

monomers, thus stabilizing the inactive form. ADP supby stabilizing the active monomer, and thus shifting the active inactive monomer equilibrium in the opposite direction from DMSO. levels above Although the effects of on are reversible, at ca. 30%, denaturation-type effects occur that are no longer reversed by dilution of the or addition of ADP. This is evidence that when it is present in higher concentrations, may denature subunit proteins by rupturing both hydrophilic and hydrophobic bonding. In order to study the effects of on the maintenance of an active conformation in a nonallosteric-type subunit protein, we have used as a model the lysosomal enzyme /^-glucuronidase (jS-D-glucuronohydrolase, EC 3.2.1.31). This enzyme is widely distributed in animal tissues, and can readily be assayed by measurement of the rate of hydrolysis of phenolphthalein j(3-glucuronide.*° Under ordinary conditions this is a relatively stable enzyme, but it has been reported ^®'^° that highly purified jS-glucuronidase can be dissociated by dilution into inactive subunits, which can be reassociated and reactivated nonspecifically by incubation the equilibrium between

posedly affects

—

GDH

DMSO

DMSO

GDH

DMSO

DMSO

DMSO

with polycations.*®'^"

+ 1.2

Figure

3.

Comparison of the

of DMSO on and D2O. The assay conditions were as described in the

inhibitory

GDH

in

eflfects

H2O

legend for Figure -.3

+.1

I

log

+.3

[DMSO]

+ .5

1,

except that

was done in both D2O and H2O, and the data were plot-

the assay

-2.4

ted in a Hill plot.

Henderson I

Effects on Subunit Proteins

et a!.:

'

1

1

1

^

c>^y

-^

.4 -

/^

-h.3--

^^^

D Q

o-

0% 1

125

Figure

1,

.175

.15

VmM/

DMSO inhibition by ADP.

Reversal of

I07o

o

.075

.05

as described in the legend for

-

-

[ADP] 4.

^

o

1

Figure

-

A

o

.025

20 7o

DMSO

^A

•

^

43

Routine assays of

GDH were performed

with the addition of the indicated amounts of ADP.

DMSO

One of the more striking effects of on jS-glucuronidase activity was a lowering of the temperature of inactivation when the enzyme was incubated in the absence of substrate (Figure 5). In the absence of DMSO, the enzyme activity in

M

KH2PO4 was stable at 37° C, and a 30-min heating period at 56° C was necessary for 90% inactivation. In the presence of 20% DMSO, the rate of inactivation of /^-glucuronidase was slow at 25° C, but it proceeded at a faster rate at 0.01

37° C.

Replacement of

H2O

by

D2O

in

the incubation

medium had

only a minimal

DMSO could also be readily demonstrated in tissue slices or homogenates, if the DMSO concentrates were 50% effect.

The

rapid inactivation of /^-glucuronidase by

or higher.

DMSO was greatly influenced by the pH,

The

inactivation of /^-glucuronidase by concentration, and concentration.

DMSO

salt

DMSO

It

required almost twice as

much

comparable rates) at 25° C as at 37° C. The enzyme was most stable at pHs between 4.5 and 5.2, and at 37° C the stability of the enzyme in 20% DMSO was markedly reduced at pHs above and below these values. The enzyme was most stable in 0.04 KH2PO4 in the presence of 20-40% DMSO, and its stability diminished as the KH2PO4 concentration was elevated. No method has been found that will reverse the inactivation of /^-glucuronidase by DMSO. Neither overnight dialysis at 5°C nor dilution of the enzyme when to inactivate the

enzyme

(at

M

dissolved in

DMSO

solutions resulted in any appreciable reactivation.

to the assay mixture of

1

mg/ml

DNA or serum

The

addition

albumin, which has been reported

form of the enzyme, ^^-^^ had no effect. Thus the available evidence suggests that the major effect of exposure of /^-glucuronidase to in the absence of substrate was an irreversible denaturation of the enzyme. to reactivate the inactive

DMSO

In the presence of substrate,

dase

in

a reversible

moderate

manner (Figure

6).

DMSO

levels of inhibited jS-glucuroniSince the kinetics of inhibition became ir-

r

44

New York Academy of Sciences

Annals

— — — ————— I

I

T

I

I

I

I

I

I

I

960

o 840 Q_

o>

E

"^ (/>

720

c ZD «

.

>1—

>

600

»— C_)

0.6

I

/[NaCI],

DMSO

,

I

0.8

^

I

1.0

mM

2

I

Log [NaCI],

mM

Figure 2. Effect of on the activation of Na+, K+- ATPase by NaCI. The enzyme was KCl, and the ATP, 10 MgClj, 3 incubated with 50 histidine-Tris (pH 7.8), 3 concentrations of NaCI shown, in the absence (•) or presence (o) of 10% DMSO. In the lefthand panel data are presented in a Lineweaver-Burk plot, and in the right-hand panel in a Hill plot. For comparison, effects of two other lipophilic agents are shown: 2% (v/v) 1-propanol (n) and 0.3% (wt/v) Lubrol®-W (). (From Robinson.^ Reproduced by permission of Biochimica

mM

et

Biophysica Acta.)

mM

mM

mM

64

Annals 14

New York Academy of Sciences

I-

•

/

°

// //

I

/^

/./

/a

9o

//^

U' iy a

/

1

1

,

I

[KCI].

'/

mM

I

Log [KCI],

mM

Figure 3. Effects of DMSO on the activation of Na+, K+-ATPase by KCI. The experiments were performed as described in the legend to Figure 2, except that KCI was varied in the presence of 90 NaCl. Data are presented as in Figure 2 for control incubations (•) and incubations with DMSO (o), propanol (n), and Lubrol (). (From Robinson.^ Reproduced by permission of Biochimica et Biophysica Acta.)

mM

Na+ since V^^ is also reduced (Figure 2), suguncompetitive inhibition toward Na+, just as there is toward MgATP (Figure 1). Whereas the affinity for the activator is given by /:_,//:+ „ A^o.s is a function of (A:_i) + (A:+2)/A:+i, and thus an inhibitor that decreases k^.2 would reduce both Vmax and A^osRecently an alternative approach to the estimation of affinities has been described: the measurement of dissociation constants (A^^) of the cation from the enzyme in terms of cation- modified rates of inactivation of the ATPase by certain irreversible inhibitors.^-^^"^^ This approach not only minimizes certain ambiguities in is

an increase

in

the affinity for

gesting that there

is

Table

DMSO

Effect of

2

on the Response to NaCl*

Conditions

Kinetic Parameter

^d

A^o.5

mM Control plus

lO'^

DMSO

5.0

2.3

3.6

2.1

*The concentration of Na"^ for half-maximal activation (Aqs) was determined from the experiments described in the Legend to Figure 2. The dissociation constant for Na''"(A'j) was determined from measurements of the effects of NaCl on the rate of inactivation of the enzyme by DCCD. These experiments were performed in the absence of ATP, MgCl2, and KCI; the control value for K^ (2.3 mM) is close to the Aoj for the ATPase reaction 8.25 in the presence of low ATP and KCI concentrations

Robinson: Modifications of Na"*^,K^-Dependent ATPase Table Effect of

DMSO

65

3

on the Response to KCP

Conditions

Kinetic Parameter f^d

^0.5

mM Na-'.K-'-ATPase: control plus

10%

0.81

DMSO

1.3

K '"-phosphatase: control plus

10%

DMSO

2.6

1.4

2.1

0.7

0.09

0.06

0.2

0.1

K "^-phosphatase with CTPandNaCl: control plus

10%

DMSO

*The /Cos of K."^ for the ATPase and for the phosphatase were determined from the experiments described in the legends to Figures 3 and 4 respectively. The Kq^ of K"*" for the phosphatase in the presence of CTP and NaCl, which represent the phosphatase properties in the presence of the acyl phosphate form of the enzyme, were determined in experiments analogous to those of Figure 4, except that the incubation media also contained 0.3 CTP and 10 NaCl. The ^jfor K"*" was determined from measurements of the effects of KCl on the rate of inactivation of the enzyme by BeCl2. The inactivating media contained 50 ^M BeCl2, 30 histidine-Tris, 3 MgCl2, and various concentration of KCl, in the absence and presence of (these are analogous to the

mM

mM

mM

mM

DMSO

K "^-phosphatase conditions);

mM

same media containing, in addition, 10 NaCl, BeCl2 instead of 50 fxM (these are analogous to the K"^phosphatase conditions with CTP and NaCl). Values for the ^0.5 ^^^ ^d *" ^^^ presence of CTP and NaCl are close to the values for the Kqs with the Na"^, K"'"-ATPase at comparable concentrations of nucleotide and NaCl.^''^^ 225

mM

CTP, and 250

or in those

^M

methods of conventional kinetic studies (above), measurements of Kd under a variety of ligand states, including

the estimation of affinities by the

but

it

also permits

which conventional kinetic studies are impossible (for example, absence of Kjs for Na+ may be estimated from the slowing by Na+ of the rate of inactivation of the ATPase by dicyclohexylcarbodiimide (DCCD),^* and the K^ for (Table 2). It Na^ when measured in this fashion is essentially unchanged by thus seems that does not markedly influence the affinity of the ATPase for Na"^, but it achieves uncompetitive inhibition by affecting stages in the reaction sequence subsequent to the binding of Na"^ (see below),^-^ in essence by influencing k+2NaCl, the K0.5 is 0.8 MgATP and 90 For K+ in the presence of 3 (Figure 3, Table 3);^ » " 19% increases the K0.5 to 1.3 mM. This reduction by of the apparent affinity for K+ is in accord with the above formulation of an action subsequent to the binding of Na+, since the conventional reaction sequence proposes that K+ activates hydrolysis after Na+ has activated enzyme phosthose

in

substrate).

DMSO

DMSO

mM

mM

mM

DMSO

DMSO

phorylation.

Activation of the Phosphatase by

Although the K+-phosphatase

activity of the

the terminal hydrolytic steps of the overall

enzyme

ATPase

K^ is

studied as a reflection of

reaction, the apparent affinity

66

Annals

New York Academy of Sciences

14

12

i

-

10 -

,1

y

8

/

/

;/

6

4

2

/

/ V. J

E xO

i

v3o o

'A

1

I

7[KCI]. Figure

4.

Effect of

mM

Log [KCI],

DMSO on the activation of K+-phosphatase by

mM

KCI. The enzyme was

mM histidine-Tris (pH 7.8), 3 mM NPP, 3 mM MgCIj, and the concentra-

incubated with 50 tions of KCI shown,

Figure

2; also

in

the absence (•) or presence (o) of

10%

DMSO.

Data are presented

as in

included, as in that figure, are experiments with propanol (a) and Lubrol ().

(From Robinson.^ Reproduced by permission of Biochimica et Biophysica Acta.)

\

mM

considerably less than that of the ATPase: the /Tq.s is 2.6 (Figure 4, The /Tq.s for K"^ is markedly reduced, however, in the presence of certain nucleotides and Na"^ (that is, under conditions that lead to enzyme phosfor K-^

is

Table

3).'

phorylation), "•'^•^o In the presence of 0.3

mM

mM

CTP and 10 NaCl, the /To.s for reduced to 0.09 (Table 3), a value close to the A^o.s of the ATPase for K"^ in the presence of comparable nucleotide and Na"^ concentrations.*" This hypothesis that moderate- affinity sites for K"^ are transformed to highaffinity sites after enzyme phosphorylation is supported by measurements of the K for K*^ in the specific ligand states that represent free enzyme and the enzyme-phosphate intermediate.®'^®" In these cases the Kd for K+ is measured in terms of K"*^dependent inactivation of the enzyme by Be^"*" or by F~. For the free enzyme that corresponds to the conditions of the phosphatase reaction (experiments performed (Table 3). in the presence of buffer, K+, Mg^+, and inactivator), the K^ is 1.4 By contrast, for the enzyme-phosphate intermediate that corresponds to K'^-binding conditions in the overall ATPase reaction, or in the phosphatase reaction in the presence of nucleotide plus Na"^ (experiments performed in the presence of buffer, (Table 3).»*« K+, Mg2+, Na+, nucleotide, and inactivator), the K^ is 0.06 The effects of are also in accord with this formulation: the /To.sS for K"^ (Table 3). of the phosphatase activity and of the free enzyme are reduced by By contrast, the A^o.sS for K+ are increased in the ATPase reaction and in the phosphatase reaction with nucleotide plus Na"^: pathways that include the acyl phosphate intermediate. Correspondingly, the K^ for K"^ is increased under ligand conthus influences the affinity of the ditions that lead to phosphorylation. ATPase for K"^ toward an intermediate value, between the moderate affinity of the free enzyme and the high affinity of the enzyme-phosphate intermediate. K+

is

mM

mM

mM

DMSO

DMSO

DMSO

Robinson: Modifications of Na"^,K'^-DependentATPase

67

Reaction Mechansim of the Na"^, K"^-ATPase

These considerations may now be incorporated into a reaction scheme for the ATPase. Following the precedent of a charge-relay system for hydrolysis,^' this reaction scheme has been proposed (Figure 5):'^ an acyl phosphate intermediate, (now shown to be on an aspartyl residue of the enzyme^^) is first formed, and the phosphate group is then transferred to a serine residue, which is labilized by the charge-relay system so that

it

readily attacked by water.

is

that in the presence of the entire

complement of

(The

ligands that

possibility exists

promote hydrolysis,

the reaction proceeds in a concerted fashion without the formation of a quasi-stable acyl phosphate; this is indicated by the bracketed arrow in Figure 5.) Although firm evidence exists for the formation of the acyl phosphate under specific ligand conditions,'-*'^^ evidence for the successive steps in this reaction is tentative;^^ nevertheless, there is no contradictory evidence, and the data obtained in experiments with '®0-labeling^* are consistent with this mechanism.

Given

this

ion transport.

mechanism, we still have the problem of relating ATP hydrolysis to Strong circumstantial evidence supports this plausible assumption

that the ions activating the that bears the

enzyme:

ATPase

Na"*"

are the ions transported across the

outward and

membrane

K"^ inward, against their concentration

gradients.® Describing the biochemistry of this active transport thus involves re-

and release to the progress of the hydrolytic sequence. Most schemes pump describe Na'^-sites as being transformed to K+sites and back again to Na"^-sites with each reaction cycle;^ " however, considerable evidence is being accumulated against this scheme, in favor of a transport system in which there are separate, coexisting sites for Na"^ and K"^. Support for models that include separate, coexisting sites comes not only from the studies cited above,®-^®"^* in which the K^ for Na"*^ and K"^ is seen as a function of the ligand states that reprelating binding

that account for the Na"^/K"^

from kinetic studies both of enzyme by these cations"" and of the actual transport of these

sent successive steps in the reaction sequence, but also

the activation of the

cations.^*""

Na"^

is

required for an

enzyme, and

little

^'o

change

in

initial

the

step in the sequence, phosphorylation of the

K^

®o-p-o-///

for Na"^

is

observed when the enzyme passes

^-2c

>2b)

—-^2d(

>2b)

V

Kd forK''

mM Phosphatase:

1.4

0.06

4a

?^4c

1.4

>2c

-

>

'I

p

H

-^

K^forK"^

mM

—

0.06

p

—

0.1

(NPP)

-P:

p

1.4

p

Ar

Reaction scheme for Na+, K+-ATPase and K+-phosphatase using the "half-ofscheme the two catalytic sites of the dimeric enzyme are represented by the two horizontal strokes of the letter E; - P and P' refer to the acyl and ester phosphates, as in Figure 6. The reaction sequence is described in the text.

Figure

7.

the-sites active*' formulation. In this

•

in

which a nucleotide stimulates the phosphatase reaction while

itself

occupying the

active site.

Summary

DMSO

inhibits the Na"^, K'^-ATPase, but stimulates the associated K"^-phosphatase activity. For the ATPase, acts as an uncompetitive inhibitor toward both ATP and Na"^, whereas it increases the A^o.s for K"^. From measurements of the dissociation constant (K^) of these ions in the ligand states that correspond to the ATPase reaction, it can be shown that has little effect on the affinity for Na"^, but decreases the affinity for K+ of the enzyme-phosphate intermediate (the form that has the highest affinity for K+). By contrast, decreases the K^ for the phosphatase substrate (nitrophenyl phosphate) without affecting the K^axMoreover, decreases the A^o.s for K+ and also the Kj for K"*^ in the ligand states that correspond to the phosphatase reaction (which have only a moderate affinity for K+, since the acyl phosphate intermediate is absent in this pathway).

DMSO

DMSO

DMSO

DMSO

.

Robinson: Modifications of Na"*',K+- Dependent ATPase

71

These data may be incorporated into a reaction mechanism for the Na"^, K+ATPase. Initially the enzyme is phosphorylated to form an acyl phosphate intermediate, in steps that require Na"^ and Mg^"^. At this stage the affinity for K"^ is markedly increased (from the moderate affinity seen in the "free" enzyme and the phosphatase reaction). When K+ is bound, the phosphate group is transferred to the hydrolytic site where P, is ultimately released. DMSO acts at the point at which the acyl phosphate group or the phosphatase substrate enters the hydrolytic site, inhibiting one and facilitating the other. At this stage the affinity for K+ is also changing, and DMSO apparently selects an enzyme conformation of intermediate affinity. Ion transport may occur by a gate mechanism in an overall system that operates on a half-of-the-sites active enzyme pattern in which ATP hydrolysis may alternate between the dimeric subunits of the enzyme.

References 1.

2. 3.

4.

Skou, Skou,

C. 1957. Biochim. Biophys. Acta 23: 394-401. C. 1965. Physiol. Rev. 45: 596-617. Albers, R. W. 1967. Ann. Rev. Biochem. 36: 727-756. Post, R. L., S. Kume, T. Tobin, B. Orcutt A. K. Sen. 1969. J.

J.

&

J.

Gen. Physiol. 54:

306S-326S. 5.

6. 7. 8.

9.

10.

11. 12. 13.

14. 15.

16. 17. 18.

Mayer, M. & Y. Avi-Dor. 1970. Biochem. J. 116:49-54. Albers, R. W. & G. J. Koval. 1972. J. Biol. Chem. 247: 3088-3092. Robinson, J. D. 1972. Biochim. Biophys. Acta 274: 542-550. Robinson, J. D. 1967. Biochemistry 6: 3250-3258. Robinson, J. D. 1974. Ann. N. Y. Acad. Sci. In press. Veloso, D., R. W. Guyunn, M. Oskarsson & R. L. Veech. 1973. J. Biol. Chem. 248: 4811-4819. Neufeld, A. H. & H. M. Levy. 1969. J. Biol. Chem. 244: 6493-6497. Kanazawa, T., M. Saito & Y. Tonomura. 1970. J. Biochem. (Tokyo)67: 693-71 1. N0RBY, J. G. & J. Jensen. 1971. Biochim. Biophys. Acta 233: 104-1 16.

&

Hegyvary, C. R. L. Post. 1971. J. Biol. Chem. 246: 5234-5240. Y. Eilam. 1973. Proc. Nat. Acad. Sci. U. Stein, W. D., W. R. Lieb, S. J. D. Karlish S. 70: 275-278. Repke, K. R. H. R. SCHON. 1973. Acta Biol. Med. Ger. 31 19-30.

&

&

1973. J. Biol.

iNTURRisi, C. E.

Robinson, Robinson, Robinson,

23.

F.

Hackney

8l J. F.

Perdue.

& E. Titus. 1968. Mol. Pharmacol. 4: 591-599. D. 1971. Mol. Pharmacol. 7: 238-246. J. D. 1974. Biochim. Biophys. Acta 341: 232-247. J. D. 1969. Biochemistry 8: 3348-3355. Hexum, T., F. E. Samson, Jr. R. H. Himes. 1970. Biochim. Biophys. Acta 212: 322-

19.

21.

J.

Chem. 248: 2593-2605.

20.

22.

:

Kyte, J. 1971. J. Biol. Chem. 246: 4157-4165. HoKiN, L. E., J. L. Dahl, J. D. Deupree, J. D. Dixon,

J.

&

331.

29.

Squires, R. F. 1965. Biochem. Biophys. Res. Commun. 19: 27-32. Robinson, J. D. 1970. Arch. Biochem. Biophys. 139: 17-27. Robinson, J. D. 1973. Arch. Biochem. Biophys. 156: 232-243. Robinson, J. D. 1973. Biochim. Biophys. Acta 321 662-670. Robinson, J. D. 1974. FEBS Letters 38: 325-328. Rega, a. F., p. J. Garrahan & M. I. Pouchan. 1968. Biochim. Biophys. Acta 150: 742-

30.

YosHiDA, H., K. Nagai, T. Ohashi

24.

25. 26. 27. 28.

:

744.

&

Y.

Nakagawa.

1969. Biochim. Biophys.

171:178-185. 31 32.

33.

Hartley, B. S. 1970. Phil. Trans. Roy. Soc. London Ser. B 257 77-86. Robinson, J. D. 1971. Nature 233: 419-421. Post, R. L. & S. Kume. 1973. J. Biol. Chem. 248: 6993-7000. :

Acta

72 34. 35. 36. 37. 38.

39.

Annals

New York Academy of Sciences

Dahms, a. S., T. Kanazawa & P. D. Boyer. 1973. J. Biol. Chem. 248: 6592-6595. Hoffman, P. G. & D. C. Tosteson. 1971. J. Gen. Physiol. 58: 438-466. Garay, R. p. & P. J. Garrahan. 1973. J. Gen. Physiol. 231 297-325. Garrahan, P. J. & R. P. Garay. 1974. Ann. N. Y. Acad. Sci. In press. Rega, a. F., p. J. Garrahan & M. I. Pouchan. 1970. J: Membrane Biol. 3: 14-25. Levitzki, a., W. B. Stallcup & D. E. Koshland, Jr. 1971. Biochemistry 10: 3371 :

3378. 40. 41.

42.

Whittam, R. & A. R. Chipperfield. 1973. Biochim. Biophys. Acta 307: 563-577. Robinson, J. D. 1970. Arch. Biochem. Biophys. 139: 164-171. Robinson, J. D. 1971. Biochem. Biophys. Res. Commun. 42: 880-885.

ALTERED MITOGENIC RESPONSIVENESS OF CHRONIC LEUKEMIC LYMPHOCYTES AND NORMAL HUMAN LYMPHOCYTES TREATED WITH DIMETHYL SULFOXIDE Anthony

J.

Dennis

Biomedical Sciences Section Columbus Laboratories Columbus, Ohio 43201

Battelle's

Henry

E.

Wilson

Division of Hematology and Oncology The Ohio State University College of Medicine

Columbus, Ohio 43210

Introduction All critical interactions that occur between the milieu are mediated through the

mammalian

plasma membrane. As the

cell

and the external

result of a disease

membrane may be sufficiently altered to reduce the functional response of the cell. The phenomenon of the mutation of membrane integrity through neoplasia probably accounts for a portion of the broad spectrum of abnormal processes observed in malignancies. Certainly aberrations such as the loss of contact inhibition noted in cells of solid tumor origin and the depressed responsiveness of leukemic lymphocytes to stimulation by a variety of plant mitogens are direct results of changes in the plasmalemma. Modification of the membranes of malignant cells, both in vivo and in vitro, has been intended either to afifect the malignant cell detrimentally, or to manipulate the surface configuration of the neoplastic cell in order to produce greater antigenic distinction from the host tissue of its origin. Because of the immunological implications of antigenic modulation at the membrane level, the lymphomatous proliferative diseases are of particular interest. This is because in lymphocyte neoplasias, the pivotal cell type involved in a great majority of immunological functions, namely the lymphocyte, is also involved in the malignant proliferation. Lymphocytes isolated from the peripheral blood of patients with chronic lymphocytic leukemia (CLL) exhibit a reduced and delayed response in vitro to the mitogen phytohemagglutinin (PHA).^'^ It has recently been demonstrated that the CLL peripheral lymphocyte population consists of both a normal, mitogen-reactive population of cells and an abnormal, mitogen-insensitive population.^'' The leukemic lymphocyte has been shown to be defective in other membrane-dependent metabolic processes, such as cytidine deaminase activity^ and pokeweed mitogen process, the cell

reactivity.* It appears that several impairments of membrane function are involved in the diminution of reactivity of PHA. Kornfeld^ has shown that compared to normal lymphocytes, the CLL cell has approximately one-half to one-third the number of available PHA surface receptors. Several reports have linked this decrease in PHA reactivity to an increased fragility of the lysosomal membranes of CLL lymphocytes.^® It is apparent from these studies on the availability of mitogen receptors and from other studies of changes in surface immunoglobulin structures'^ that the CLL lymphocyte has a significantly altered plasma membrane (and lyso-

73

Annals

74

New York Academy of Sciences

somal membranes). These changes in membrane structure probably account for the observed decreases in membrane function. On the basis of the data on this inactivity, it appeared proper to attempt to alter the lowered responsiveness of the leukemic cell by manipulation of the cell membrane. The present investigation details the effect of the membrane-active agent dimethyl sulfoxide (DMSO) upon the PHA responsiveness of CLL lymphocytes. has been used as a cryopreservative (8.3%, v/v) in the freezestorage of the CLL lymphocytes, and was shown to have minimal cytocidal effects in exposures of short duration. ^^ Schrek and colleagues, ^^ however, have reported (v/v) is preferentially cytotoxic for CLL lymphocytes in longer exthat 2% posures (24-48 hours) of cells in culture.

DMSO

DMSO

Materials and Methods Leukemic lymphocytes were obtained by leukopharesis of three CLL patients, whom had proved resistant to multiple maintenance doses of both chlorambucil and cyclophosphamide, and had been removed from drug therapy several months before the experiment. The peripheral cells were washed and the mononuclear cells removed by density gradient centrifugation on a Ficoll®-hypaque cushion (buoyant density = 1.077). The cells were removed from the density interface, washed three times in incomplete RPMI-1640, and resuspended in complete RPMI1640 plus 10% fetal calf serum. The cells were cultured in the complete medium in a 5% CO2 atmosphere at 37° C. A standard concentration of 1 x 10® lymphocytes was used in each culture, and 5.0 ^1 PHA was the standard mitogen dose per culture. The cultures were pulsed with 1.0 /iCi tritiated thymidine 24 hours before the end of all

of

the experiment.

DMSO

at concentraSeparate aliquots of complete medium, which contained and 2.0% (v/v), were prepared before the experiment. were incubated for the duration (1, 3, or 7 Those cells that were treated with days) of the experiment in this complete, DMSO-containing medium. Other cells were incubated in these media for 3 hours, and then were washed 3 times and subsequently refed with medium that did not contain DMSO, in order to determine the reversibility of any effect attributable to the DMSO. Following incubation, the cells were processed by the standard method outlined by Caspary and Hughes, ^^ and the samples counted in a Packard Tri-Carb liquid scintillation spectrometer. Normal lymphocytes were obtained from three normal laboratory personnel by means of venipuncture, and were processed as outlined

tions of 0.0, 0.1, 0.25, 0.5, 1.0,

DMSO

above.

Results In keeping with the findings of Schrek and colleagues, '^ both normal and CLL lymphocytes were treated with varying concentrations of DMSO, ranging from 0.1% to 2.0% (v/v) per aliquot of complete medium. Determinations of cell viability by the trypan blue exclusion technique confirmed these authors' observation that 2.0% DMSO is preferentially toxic to CLL lymphocytes, as compared with normal lymphocytes. Viability counts of lymphocytes, both normal and leukemic, indicated that DMSO had no preferential cytotoxic effect up to and including a concentration of 1.0% (the viability counts for normal and leukemic cells were over 86% at 7 days for DMSO concentrations of 0.1%, 0.25%, 0.5%, and 1.0%).

Dennis

& Wilson:

Leukemic

& Normal Human Lymphocytes

75

After these preliminary observations, lymphocytes from both normal and leukemic donors were placed in culture as previously described, and were examined for incorporation of tritiated thymidine following stimulation with PHA in a medium that contained varying concentrations of DMSO, according to the following schema. Normal lymphocytes were cultured in complete medium that contained 0.1%, (v/v) for periods of 1, 3, and 7 days. The cul0.25%, 0.5%, 1.0%, or 2.0% tures were pulsed with tritiated thymidine on days 0, 2, and 6, in order to allow 24 of cells underhours for the incorporation of the labeled nucleotide into the going replication. The graphs in Figure 1 (note that the cpm scale is expanded to allow for better visualization of the graphic material) indicate that the amount of radioactive nucleotide incorporated in unstimulated normal lymphocytes was concentration employed. Statistical analysis of constant, regardless of the these values, in which Student's / test was employed, revealed that none of these values differed significantly from the values observed in the untreated cell control cultures (the lowest P value was greater than 0.30). Therefore did not appear to induce a nonspecific uptake of the labeled thymidine.

DMSO

DNA

DMSO

DMSO

DMSO

in a fashion identical to that Normal lymphocyte cultures treated with described above were stimulated by the addition of 5 ^1 of the nonspecific mitogen per culture. The DMSO-treated normal cells were stimulated to incorporate

PHA

% DMSO

by

Volume

- O.O(Control)

TIME (DAYS) Figure

1.

Incorporation of tritiated thymidine

treated with the indicated doses of

DMSO.

in

cultures of normal

human lymphocytes

76

Annals

l-'l

New York Academy of Sciences

VoDMSO

by Volume

0.0 (Control) -• 0.25

1

1

TIME (DAYS) Figure

2.

Incorporation of tritiated thymidine

treated simultaneously with the indicated doses of

in

cultures of normal

human lymphocytes

DMSO and a constant quantity of PHA.

to four times more tritiated thymidine than is reported to be routinely incorporated by untreated normal lymphocytes exposed to PHA^* (Figure 2). The mean cpm for untreated, PHA-stimulated normal cells (eight samples were used, in duplicate experiments) was 33,351 after 3 days of culture. The cpm of similar cultures, which contained 0.5% DMSO, was over 1 10,000. This same pattern held for all the concentrations tested; the test samples gave significantly higher counts {P < 0.01 for all value comparisons) than did the untreated, PHA-stimu-

from three

DMSO

lated controls.

Exposure of cultures of lymphocytes from patients with

CLL

to

DMSO in con-

centrations of 0.1%, 0.25%, 0.5%, 1.0%, and 2.0% produced a response similar to that described for the normal cell cultures (Figure 3). CLL lymphocytes treated only with for periods of 1, 3, and 7 days incorporated labeled thymidine at a

DMSO

The only exceptions were the cultures treated with 2.0% DMSO; as previously noted, this was due to the loss of cell viability. (The expanded cpm scale in Figure 3 is necessary for the graphic representation of data). CLL lymphocytes simultaneously treated with and stimulated with PHA rate statistically identical to that observed for the untreated leukemic cells.

DMSO

showed a 6(Figure 4). for

PHA

to 6.5-fold increase in the incorporation of tritiated thymidine at 3 days It

should be recalled that the normal pattern reported and reproduced is one of a delayed response, in which the

stimulation of CLL lymphocytes

Dennis

& Wilson:

Leukemic

& Normal Human Lymphocytes

77

maximal incorporation of nucleotide occurs at 5 to 7 days. However, cells cultured DMSO responded maximally at 3 days, a pattern similar to that noted for normal lymphocytes, and the cpm of the stimulated cultures was over 13,000, as compared with 2,000 for the untreated cells. Again, the 2.0% concentration of DMSO was toxic to CLL lymphocyte cultures and abrogated all response. Cell responses at days and 7 did not differ significantly from the response of the control cultures. Other experiments were performed with both normal and CLL lymphocyte cultures, in which DMSO was applied for 3 hours and then was removed from the cultures by three vigorous washings with fresh complete medium. After this, the cultures were prepared in the same way as the normal and leukemic cells in the previous groups. The cpm values for normal and CLL lymphocytes at all test periods and over the whole range of DMSO concentrations showed that the removal of DMSO reversed all counts to values that were essentially those recorded for unin

1

treated cultures.

Discussion These data raise two major questions on the mitogenic stimulation of CLL lymphocytes: (1) What effect does exert upon the cell (both normal and leukemic), to produce the strongly increased blastogenic response to PHA? (2) Is

DMSO

%

DMSO

by

Volume

AGO -D

(Control)

0.1

-• 0.25

1

1

TIME (DAYS) Figure

3.

Incorporation of tritiated thymidine

the indicated doses of

DMSO.

in cultures

of CLL lymphocytes treated with

Annals

78

1% ^

New York Academy of Sciences

OMSO

—^ny A

j

I^P

I3n !2— 12II-

•

^

">^

by Volume

—\ A

^ (Co f* ^ 0.0 ntrol) t n V.I /^

1

1

0.25 0.5 1.0

1

lO

O

10-

—

5-

VL

Im f»

H _ 3 8z 2 7-

{2

w\Vk

/»

Ui

6-

Vv;

ImI

9-

(T uj Q.

VvV

yj/

X

\\ \\

W \^

u w

Im

\

II

\\\

1 it

A \^

II

w\i

jM

v^

If

u a

z s

iP


6

7

+

(+)

+ +

4-

+ +

-

-

-

-

-

P

> O.Ol);

5 -f

+ =

significant

Gbrog

&

Kovacs: Effects

in Arthritis

I 9

^^^^^F/^l

^^^K?

^^^

& Thrombosis

93

BWyv^^^B

Si HH

^^^Kb'i'jS

1 ^H^^^l

1

_l

o 1—

2

o o

o 2 Q

3-

^ + o CO 2 o

SO •8.S

2*8

00

o

U

^

uu

d H

Annals

94

New York Academy

of Sciences

was noticeable between the effectiveness of 2% hydrocortisone ointment and 0.2% hydrocortisone dissolved in DMSO. Because of the small size of the x-ray film, six animals were chosen at random from each group; radiographs of their treated legs are to be seen in Figure 2. The findings confirm that radiographs are needed for evaluation, since in some cases a considerably increased volume was not associated with the degeneration of periarticular tissues or with osteolytis bone changes. In other cases, again, significant bone changes were found in conjunction with relatively slight increases of volume. Changes of volume found in untreated hind paws during the period of treatment are shown in Figure 3. The curves reveal that local treatment in itself did not produce a significant systemic effect; this is evidenced by the absence of any considerable difference between the volume of untreated legs and of controls. The most marked systemic effect resulted from treatment with hydrocortisone ointment; the systemic effect of hydrocortisone dissolved in DMSO was milder than that of hydrocortisone ointment. is destined for local use, thanks to its physicochemical properties. There are numerous reports that various skin reactions have been eflPiciently inhibited by local therapy in humans." DMSO proved to be effective in the inhibition of various skin reactions in animal experiments.* DMSO, as a carrier for other drugs, was also tested first in the treatment of skin reactions. Synergy of DMSO and triamcinolone has also been rep>orted in the therapy of dermatoses. At the beginning, the antiarthritic effect was the most widely debated of the antiinflammator>' and other activities of DMSO. After several experiments with inadequate controls, a convincing study was performed by the Japanese Rheumatism proved to be effective in Association in a large series under proper control; the treatment of arthritis.' Being aware of the problems and diflPiculties liable to arise in appraising the effectiveness of a new compound for the treatment of rheumatoid arthritis, '° we difference

DMSO

DMSO

DMSO

DMSO

i.O

CONTROL

DMSO

3.0

DMSO^H

H 2.0

1.0

7

U

21

28

35

42

49

56

DAYS AFTER 21.fTDAY FROM ADJUVANT INJECTION

Figure

3.

Changes of volume of untreated hind paws during the period of treatment. The in the legend to Figure 1.

abbreviations arc explained

GorCg

& Kovacs:

Effects in Arthritis

Table

& Thrombosis

95

2

DMSO

Treatment on the "Plasma Inflammation Units," Determined from the Blood of Arthritic Rats at the End of Treatment

Effect of Topical

_

Inflammation

.

Treatment^

»

mean Nonarthritic controls Arthritic controls

DMSO DMSO

thought

it

db

SE

82.4

=b

20.4

865.6

±

80.4

/o

±62.8 341.2 ±40.8 358.8 ±37.6 510.4

+ hydrocortisone

Hydrocortisone ointment

Arbitrary

, uu-^Inhibition

n * Units*

45.4

67.0 64.7

units of turbidimetry.

worthwhile to perform further studies with

tablished adjuvant disease in rats.

So

far the

DMSO,

compounds found

in

the therapy of es-

effective in this test

have proved to be potent antirheumatic agents. The results obtained have confirmed our previous findings, as well as clinical rewhen used alone sults which show that arthritic reactions are inhibited by (Table 2). It is held to be important that the systemic effect of topical should be minimal. This may be explained by the general experience that exerts its therapeutic effect at high concentrations (70-100%). When used topically, presumably does not reach an effective concentration in distant tissues. Hydrocortisone, on the other hand, when absorbed from locally applied ointment, exerts a significant systemic effect, which is much slighter when the drug is added to DMSO. Our results show that the local antiarthritic effect of hydrocortisone is increased tenfold when is used as a carrier. Synergy of such magnitude may be highly significant in avoiding the side effects and risks connected with the clinical use

DMSO

DMSO DMSO

DMSO

DMSO

of glucocorticoids.

Antithrombotic Effects

When

investigating the effect of various anti-infiammatory agents on platelet ag-

we have found" that topically applied DMSO was thrombi induced by topically applied adenosine diphosphate the microcirculation of the hamster's cheek pouch. In the meantime we have de-

gregation

in vitro

and

in vivo,

effective against platelet in

veloped a effect of

new technique'^

that

made

it

possible to reinvestigate the antithrombotic

DMSO. He-Ne laser irradiation

in the presence of an energy-absorbing dye causes a selective endothelial injury in the irradiated small vessel. The mesoappendix of the pentobarbital-anesthetized rat (60 mg/kg, subcutaneous) was prepared for microcirculatory studies as described by Zweifach.*^ The thin mesenteric membrane was spread over a Lucite® block and was continuously bathed in Ringer's solution. With the aid of a three-way stopcock, one can change from normal Ringer's to

10% DMSO. The experimental conditions used for the formation of thrombi in the selected blood vessel by means of an optically focused He-Ne laser have been described in detail elsewhere.** The selected blood vessel was irradiated for 5 seconds in every minute, and the time from the first irradiation until the flow ceased was measured and described as flow-stop time. Five minutes before the first irradiation, 20 mg/kg Evans blue was injected intravenously. The blood level of the dye was unchanged during the first 30 min after the injection. On this Ringer's that contains

New York Academy of Sciences

Annals

96

Table Effect of Topically Applied

3

DMSO

on Laser-Induced Thrombosis

Arterioles

Number

P experiment Treatment

Venules

of

/-

Cases

m

\

^.^-^^^ pj^^.

.^

Time

did not

-

30;

\:n

Stop

min

=b

60

r> Cases m which Flow •

of

Experiment (Exp. 40-

^^

_,.

f

Number

pio^^.s^^p

'

^

ment

u

xt

•

Flow-Stop

Time

did not

n Recover

^\ ^m0)

min ± SE

SE Antithrombotic Effect

8.6± 1.56 ±2.13*

Control 10°,

DMSO

13.67

11.16±

30 26

11

12.35

1.34

±1.90

Thrombolytic Effect Control 10°„

DMSO

*Difference from the control

is

significant

{P



0.07

M; 37X

negative

papillary muscle

Dog

trabeculae carneae

concentration-dependent;

0.05).

'^

significantly shortened the half-life of the *'fast

decay, from about 95 sec without

presence of

The

DMSO. The data

are

component" of tension

DMSO to about one-half that value (49 sec) in the summarized

in

Table

2.

of the fast component of tension decline of atria placed in a solution free of Ca^"^ and agreed well with values which are descriptive of a rapidly exchanging fraction of myocardial Ca^"^, directly responsible for regulating the half-life

DMSO

cardiac contractile strength.

It

thus appears that

DMSO exerted a significant effect

myocardial Ca^+, and that the magnitude of this effect was independent of the concentration over the range of 0.14 to 1.41 M. This observation also suggests that the magnitude of the effect may not be a purely osmotic phenomenon, due to the wide range of osmolalities of the DMSO solutions, unless a threshold to trigger this action was reached at an osmolality between that of the control solution (316 mOsm) and the 0.14 solution ( « 450 mOsm). also accelerated the rate of loss of the contractile tension of rabbit atria transferred into a solution that contained half the original [Ca^"^], and accelerated the increase in the gain of tension of this preparation when suddenly exposed to higher levels of Ca^"^. to hasten the loss of Ca^"^

from a

specific pool of

DMSO

M DMSO

DMSO

Effects on Other Cardioactive Drugs

DMSO

Low concentrations of can potentiate the pharmacologic actions of other drugs that are used clinically for their ability to stimulate the heart. Melville and colleagues^'* reported that doses of digitalis glycosides which were too low to produce cardiac arrhythmias when injected alone into anesthetized cats, did so when accompanied by a single intravenous injection of 50% DMSO-saline solution (0.5 ml/kg, i.v.). They suggested that this interaction to alter cardiac automaticity might be due to digitalis-like properties of DMSO, or possibly due to a histamine-releasing

)

New York Academy of Sciences

Annals

118

2100

mM Co 000 M DMSO 000

10

k

B

x\^ n=

o

1

8

:\^ NvX= 1749X10-3 sec

\

Xr,/2»396

\

Isec

\o X«l3 455XI0-^ec"' \

T,/2=5l5sec

\

\ *60

OOOmM 14

/V/

300 2"*" Ca

DMSO

600

900

1800

2100

TIME(sec)

Figure 3. Kinetic analysis of the contractile tension decay of rabbit left atria transferred from medium containing 2.10 Ca^"^ and 0.00 DMSO to Ca^'^-free medium with no DMSO (B), at 30° C. (Modified from Shlafer et.al. '^ By peradded DMSO (A), or with 0. 14 mission of the European Journal of Pharmacology.

mM M

M

&

Shlafer

Karow: Effects on Mammalian Myocardium

1

19

Table 2* Half-Lives for Fast Component of Tension Decay of Rabbit Atria Placed IN

Mean

of

Calcium-Free Medium, With or Without

DMSOt

DMSO M

n

0.00

16

0.14

8

51.51

0.42

8

0.84

7

48.49 42.69

1.41

8

51.54

Tabular Half-Life sec

94.74

DMSO group

48.56t

fThe tabular values of half-lives are mean values obtained from the contractile tension decay curves of n-atria in each group, as shown in Figure 3. (/* < 0.01). group at 0.00 IThis is a significant difference from the * Modified from Shlafer et al}^

DMSO

action of curve for

In

DMSO,

M

although Spilker'' was unable to alter the inotropic dose- response

DMSO in guinea pig atria with triprolidine, an antihistamine. vitro, low concentrations of DMSO (0.14 M) potentiated

the

positive

inotropic responses of isolated rabbit atria to isoproterenol, a /9-adrenergic receptor

and increased the resistance of this receptor to jS-adrenergic blockade.'^ concentrations of DMSO (0.14 to 0.42 M) also reduced the threshold [Ca^+] necessary to significantly increase the incidence of spontaneous pacemaker

agonist,

Low

activity in electrically driven rabbit left atria treated with isoproterenol.^^

Effects in Clinical Cardiology

The

functional and structural cardiac effects produced by

DMSO

occur over a

wide range of drug concentrations.

The

often irreversible inhibitory effects of high concentrations of

DMSO

are of

primary relevance to programs that involve heart preservation by freezing; in this concontext, viable heart freezing and thawing presently requires the use of centrations which approach or even exceed concentrations that produce signs of cardiotoxicity. While it is inconceivable that such concentrations would ever be utilized in vivo, we must develop an understanding of these effects so that the toxicity problem can be resolved or completely avoided in the in vitro organ. However, reconcencent insight into the cryoprotective efficacy of combinations of low trations with other cryoprotectants,^* if applicable to the heart, would make the use

DMSO

DMSO

of such high

DMSO concentrations unnecessary.

The demonstrated cardiac

function

cardioactive agents, the

ability of

significantly, is

DMSO

very low concentrations of and to interact with other

alone to alter

clinically

useful

a necessary and sufficient reason to pursue in greater detail

mechanisms of these actions and

interactions.

SimpHstically, these low concentrations can produce potentially beneficial or

adverse cardiac effects mechanisms of action,

in

an

in

vivo

situation.

Pending through studies of

its

DMSO could prove to be useful as a cardiac stimulant when

1

20

Annals

New York Academy of Sciences

used alone, as an inexpensive vehicle to potentiate rently available therapeutic agents or agents yet expensive or too scarce to be used alone in effective surgery to enable the myocardium to tolerate or posed on it.

the cardiac actions of other curto be discovered, which are too

doses, or as an adjunct in cardiac recover better from stresses im-

Its successful application in these and other situations may, however, be limited by an action of that enhances the toxic manifestations of other drugs, aggravates latent or overt cardiac pathology, or produces the unpleasant side effects already reported in clinical trials. It is vitally important that we acquire data indicating the in vitro response to and tolerance of the human myocardium to low and moderate concentrations of (0.01 to 0.70 M). Underlying all the hypothesized future applications of DMSO, of course, is the necessity to understand from a purely mechanistic viewpoint how causes its various effects, and how it can be used as a useful pharmacological tool.

DMSO

DMSO

DMSO

References 1.

Leake, C. D.

(Ed.). 1967. Biological actions of dimethyl sulfoxide.

Ann. N.Y. Acad.

Sci.

141:1-671. 2.

Jacob,

S.

W., E. E.

Basic Concepts of 3.

Shlafer, M.

&

A.

Rosenbaum

DMSO.

&

Wood

D. C.

Marcel Dekker.

M. Karow,

(Eds.). 1971.

Dimethyl Sulfoxide.

I.

New York, N.Y.

Jr. 1971. Ultrastructure-function correlative studies for

11.

Hearts perfused with dimethyl sulfoxide (DMSO). Cryobiology 8: 280-289. Sams, W. M., Jr. 1967. The effect of dimethyl sulfoxide on nerve conduction. Ann. N.Y. Acad. Sci. 141:242-247. Pribor, D. B. & A. Nara. 1969. Toxicity and cryoprotection by dimethylsulfoxide and by glycerol in isolated frog sciatic nerves. Cryobiology 5: 355-365. Shlafer, M. & A. M. Karow, Jr. Unpublished data. Feuvray, D. & J. DE Leiris. 1973. Effect of short dimethylsulfoxide perfusions on ultrastructure of the isolated rat heart. J. Mol. Cell. Cardiol. 5: 63-70. Sams, W. M., Jr., N. V. Carrol Sl P. L. Crantz. 1966. Effects of dimethylsulfoxide on isolated-innervated skeletal, smooth, and cardiac muscle. Proc. Soc. Exp. Biol. Med. 122:103-107. Gandiha, a. & I. G. Marshall. 1972. Some actions of dimethylsulphoxide at the neuromuscular junction. J. Pharm. Pharmacol. 25:417-419. Spilker, B. 1970. Inotropic actions of dipolar aprotic solvents. J. Pharmacol. Exp. Therap. 175:361-367. Spilker, B. 1972. Pharmacological studies on dimethyl sulphoxide. Arch. Intern.

12.

Karow, A. M.,

cardiac

4.

5.

6. 7.

8.

9.

10.

cryopreservation.

I.

Pharmacodyn. 200: 153-167. tors. J. 13.

(DMSO)

Jr. 1972. Dimethylsulphoxide

effect

on myocardial /8-recep-

Pharm. Pharmacol. 24: 419-421.

Shlafer, M.,

L.

J.

Matheny

& A. M.

Karow,

Jr. 1974. Cardiac inotropism of dimethyl

sulphoxide: osmotic effects and interactions with myocardial calcium ion. Eur.

J.

Pharmacol. 28:276-287. 14.

Farrant, Physiol.

15.

BiCKis,

J.

1965. Permeability of guinea-pig

smooth muscle

to nonelectrolytes. J.

(London) 178: 1-13.

I. J.,

K. Kazaks,

J. J.

Finn

&

I.

W.

D.

Henderson. 1967. Permeation kinetics of hepatoma ascites cells. Cryobiology 4:

glycerol and dimethyl sulphoxide in Novikoff 1-10. 16.

Bunch, W. W.

1968.

The

the barnacle muscle cell 17.

Bunch, W. W.

&

single barnacle

C.

effect of

Edwards.

muscle

DMSO on

membrane.

cell. J.

J.

1969.

Physiol.

the permeation of nonelectrolytes through

Cellular

Comp.

Physiol. 72: 49-54.

The permeation of non-electrolytes through (London) 202: 683-697.

the

&

Shlafer

18.

Koch-Weser,

J.

Karow: Effects on Mammalian Myocardium

1963. Influence of osmolarity of perfusate on contractility of

malian myocardium. Amer. 19.

WiLDENTHAL, K. and

20.

&

J.

J.

muscle mechanics

in vivo

(London) 203: 50-5 IP.

Leiris. 1971. Effect of dimethylsulfoxide on isolated rat heart and

DE

lacticodehydrogenase release. European

&

mam-

Physiol. 204:957-962.

1969. Effects of hyperosmolality on cardiac

in vitro. J. Physiol.

Feuvray, D.

121

J.

Pharmacol. 16:8-13.

23.

CoRABOEUF. 1965. Etude comparative de I'ultrastructure du myocarde Chez le Rat etleCobaye.Compt. Rend. Soc. Biol. 159:2118-2121. Karow, A. M., Jr. & O. Carrier, Jr. 1969. Effects of cryoprotectant compounds on mammalian heart muscle. Surg. Gynecol. Obstet. 128: 571-583. Karow, A. M., Jr., O. Carrier, Jr. & W. C. Holland. 1967. Toxicity of high

24.

Karow,

25.

Farrant,

21.

22.

Denoit,

F.

E.

dimethylsulfoxide concentrations in rat heart freezing. Cryobiology 3: 464-468. W. R. Webb. 1965. Toxicity of various solute moderators used A. M., Jr. hypothermia. Cryobiology 1: 270-273.

&

J.,

C. A.

Walter

&

J.

Armstrong.

in

1967. Preservation of structure and

function of an organized tissue after freezing and thawing. Proc. Roy. Soc. (London) 26.

Ser. 8.168:293-310. Jacob, S. W., M. Bischel

permeability of biologic

& R. J. Herschler. 1964. Dimethylsulfoxide: Effects on the membranes (preliminary report). Current Therap. Res. 6: 193-

198. 27.

28.

Franz, T. J. & J. T. van Bruggen. 1967. Ann. N.Y. Acad. Sci. 141: 302-309.

Rammler, D. H.

30.

31.

32.

possible

mechanism of action of

DMSO.

DMSO

1967. The effect of on several enzyme systems. Ann. N.Y. 141:291-229. Chang, C.-Y. & E. Simon. 1968. The effect of dimethyl sulfoxide (DMSO) on cellular systems. Proc. Soc. Exp. Biol. Med. 128: 60-66. BuRGES, R. A., K. J. Blackburn & B. Spilker. 1969. Effects of dimethyl sulphoxide, dimethyl formamide, and dimethyl acetamide on myocardial contractility and enzyme activity. Life Sci. 8: 1325-1335. Henderson, T. R., R. F. Henderson & G. E. Johnson. 1969. The effect of dimethyl sulfoxide on the allosteric transitions of glutamic dehydrogenase. Arch. Biochem. Biophys. 132:242-248. Finney, J. W., H. C. Urschel, G. A. Balla, G. J. Race, B. E. Jay, H. P. Pingree, H. L. DoRMAN & J. T. Mallams. 1967. Protection of the ischemic heart with alone or with hydrogen peroxide. Ann. N.Y. Acad. Sci. 141:231-241. BiNG, O. H. L., W. W. Brooks & J. V. Messer. 1973. Heart viability during hypoxia: Protective effect of acidosis. Science 180: 1297-1298. Melville, K. L, B. Klinger & H. E. Shister. 1968. Effects of dimethyl sulfoxide (DMSO) on cardiovascular responses to ouabain, proscillaridin, and digitoxin. Arch. Intern. Pharmacodyn. Therap. 174: 277-293. Spilker, B. 1970. Comparison of the inotropic response to glucagon, ouabain, and noradrenaline. Brit. J. Pharmacol. 40: 382-395. Robinson, D. M. & P. K. Schork. 1971. Toxicity of cryoprotective agents. II. Effects on cardiac microsomal adenosine triphosphatase. Cryobiology 8: 377-378. Shlafer, M., J. L. Matheny & A. M. Karow, Jr. 1975. Manuscript in preparation. Ashwood-Smith, M. J. This monograph. Hausler, G. & U. Jahn. 1966. Untersuchungen zur Pharmakologie von Dimethylsulfoxyd (DMSO). Arch. Intern. Pharmacodyn. 159: 386-400. Karow, A. M., Jr., O. Carrier, Jr. & B. R. Clower. 1968. Toxicity of cryoprotective agents at 30° C. J. Pharm. Pharmacol. 20: 297-301.

Acad.

29.

A

Sci.

DMSO

DMSO

33.

34.

35.

36.

37. 38. 39.

40.

THE INFLUENCE OF DIMETHYL SULFOXIDE ON CELLULAR ULTRASTRUCTURE AND CYTOCHEMISTRY* .

Edmund

B.

Sandborn, Heather Stephens,! and Moise Bendayan

Department of Anatomy Universite de Montreal Montreal, Quibec

Introduction Previous studies of rapid penetration of tissues by the perfusion of a combination of aldehydes in the fixative solution* demonstrated that filaments and cytoplasmic microtubules exist in many animal tissues. The instances of apparent points of contact between these and other established organelles led us to suggest that a microcirculatory system could be a feature of all cells. ^ The lack of consistent findings in this respect made it difficult to prove this concept from electron-microscopic observations.

The published reports on

(DMSO)

and

the unique solvent properties of dimethyl sulfoxide

do studies on comparison with controls has allowed an assessment of the short-term influence of dimethyl sulfoxide on the biological ultrastructure and cytochemistry of perfused animals. its

influence on the permeability of tissues^"* led us to

tissues fixed in the presence of this chemical.

A

Material and Methods

Human tissues, fixed by immersion in phosphate-buff'ered 6.25% glutaraldehyde, were compared with those from other blocks of similar size in which DMSO, to a concentration of 5%, was added to the fixative solution. In newborn rats, cadmiumfree horse ferritin, an inert tracer, was injected into the stomach, and 3 hours later the intestine was fixed in 6.25% glutaraldehyde, 2% acrolein, and 5% DMSO solution for the examination of sections in the electron microscope. Other blocks of intestine were fixed briefly in the aldehyde-DMSO solution and immersed in 15% DMSO. The blocks were then frozen in liquid Freon®, fractured, and etched. ^'^ Replicas were made of the fractured surfaces, in order to obtain more knowledge of the interrelationship in three dimensions of organelles within the cell. In other experiments, after a perfusion with lactate Ringer's solution that contained 0.5% sodium nitrite (to prevent spasm of the renal arteries), aldehyde fixatives were circulated through the cardiovascular system of the whole anesthetized animal, in order to bring the fixative as uniformly near to each cell as (by volume) was included in the possible. In still other experiments, 5% lactate Ringer's solution, which was employed for the replacement of the blood before the introduction of the fixative into the left ventricle of the heart or into a selected artery of the laboratory animal. A very brief period of fixation by perfusion (4 to 10 min) with the aldehyde-DMSO solution was found to be adequate for transmission and scanning electron microscopy, and for cytochemical studies. For the

DMSO

This work was supported by fMiss. H. Stephens

is

grants from the Muscular Dystrophy Association of Canada.

a Fellow of the Muscular Dystrophy Association of Canada.

122

Sandborn

et al.\ Cell

Ultrastructure and Cytochemistry

123

body on the swim bladder of the eel the fixative was introduced through a cannula in the preretal artery. For all transmission electron microscopy, small blocks of the aldehyde-fixed tissues were washed in isotonic buffer and then postfixed in Veronal ^-buffered 2% osmic acid solution. The tissues were dehydrated through graded ethanols and were embedded in Epon 812. Thin sections were stained with lead and uranyl solutions before being examined. For scanning electron microscopy, the aldehyde-fixed tissues were removed and sectioned in a tissue chopper in order to expose the interior of the cell. These thick sections were dried through graded ethanols and Freons by the critical point method in order to prevent shrinkage. Various physical and chemical methods were employed to remove background matrix, in order to prevent it from obscuring the organelles. The most profitable were digestion with diastase and selective removal by exposure to combinations of gamma or proton and ultraviolet irradiation or to ozone. The surfaces were shadowed with gold. For cytochemistry the tissues were fixed in 4% glutaraldehyde and 5% for 5 to 7 min, sectioned on the tissue chopper, and incubated by the Wachstein Meisel method, for the demonstration of glucose-6-phosphatase with either glucose-6-phosphate or a-glycerophosphate as capillaries of the red

DMSO

substrates.

Results

DMSO

In all tissues the addition of hardening of the tissue. The addition of

general increase in

membrane

to the fixative resulted in a very rapid

this

chemical to the fixative resulted

in

a

structure within the cytoplasm of hepatocytes and a

sharper delineation of the endoplasmic reticulum (Figures 1 and 2). In a section from the mucosa of the ileum of the newborn rat that was fed horse ferritin as an inert label (Figure 3), the fine granules of the tracer could be seen by transmission electron microscopy in a tubule that gave alternating dilatations and constrictions. The tubule leads deep into the cytoplasm. The frozen etched specimen of a cell in the ileal epithelium of the newborn animal showed a tubule that led from the apical surface to the level of the endoplasmic reticulum (Figure 4). In the perfusion experiments the rapidity of the onset of convulsions in the animal fixed with the aldehyde-DMSO solution is quite remarkable in comparison to animals that receive the aldehyde alone. Similarly, the hardening of the tissues appears much more rapidly. In the studies of the ultrastructure of the thyroid gland, apical vesicles generally appear as round or oval when is not included in the perfused fixative (Figure 5), and the granules could rarely be seen opening into the lumen. When was added to the perfused fixative, the opening into the lumen of the thyroid follicle could be clearly shown to be a tubule that led from a granule to the lumen (Figure 6). The granules in the apex of the cells were often interconnected (Figure 7), again giving the appearance of tubules with alternating dilatations and constrictions. In addition an unusual amount of densely stained content

DMSO

DMSO

was retained

in

these tubules.

In perfused animals in which

DMSO was

added to lactate Ringer's solution but

not to the fixative, the rate of fixation was increased but the

number of interconnec-

between granules did not appear to be so numerous. In the experiments in which was included in both the Ringer's solution and the following fixative, the most rapid rigidity of the organs was obtained. By experience it was found that 2 min of perfusion with the Ringer's and was sufficient to remove the blood and prevent blockage of the small vessels by blood clots. After the perfusion with tions

DMSO

DMSO

124

Annals

New York Academy of Sciences

and 2. Figure 1 is of a glutaraldehyde, block-fixed human hepatocyte, showing endoplasmic reticulum (er), and mitochondria (m) near a bile canaliculus (be), (x 36,000.) Figure 2 shows a glutaraldehyde-DMSO, block-fixed human hepatocyte from the same biopsy specimen, otherwise prepared as in Figure I. Note the increase in the smoothsurfaced endoplasmic reticulum, (x 31,000)

Figures

glycogen

1

(gl),

Sandborn

Figures

3

and

in dilatations in

4.

et

al

Newborn

:

Cell Ultrastructure and Cytochemistry

rat ileum. In

Figure

3,

horse

ferritin

125

granules (arrows) are seen

a tubule that leads into the apex of an absorptive epithelial

cell, fixed in

DMSO.

(x 88,000.) In Figure 4, a fractured and etched surface of a frozen epithelial cell shows a tubule (arrows) that leads from the level of the base of the microvilli (mv) to the endoplasmic reticulum (er). ( x 57,000.) (From Sandborn.* By permission glutaraldehyde, acrolein, and

of Academic Press.)

Ringer's, 5 to 10

min of

fixation by the

firm fixation of whole organs.

The

DMSO-aldehyde

solution produced a very

ultrastructural preservation

was

excellent,

and

the extremely short period of fixation allowed these tissues to be processed for

cytochemistry with a minimum of inhibition of enzymatic reactions by the fixative. After the perfusion of animals or organs with in both the Ringer's and the fixative solutions, electron micrographs of sections frequently showed a continuity of membrane structure between organelles. In striated muscle cells of the rat, continuity was demonstrated between the outer mitochondrial membrane and the sarcoplasmic reticulum (Figures 8 and 9). In scanning electron microscopy, mitochondria and the sarcoplasmic reticulum form a network about the myofibrils Figures 10 and 11). The mitochondria appear to be supported by a distinct structural network (Figures 12 and 13). In the arteriolar capillary of the red body in the eel, numerous tubules that show series of dilatations intrude deeply into the cytoplasm from both the luminal and basal surfaces (Figure 14). Some of these tubules showed a branching pattern. Occasional tubules extended from the plasma membrane to the compartment between the outer and inner nuclear membranes (Figure 15). The outer mitochondrial membrane was also seen to be continuous with the membrane of the endoplasmic reticulum (Figure 16). In the shell membrane-secreting gland cells of the oviduct in the pigeon, long tubular stems project out from the granules (Figures 17 and 18). In most cases the dense content of the granules extended into these prolongations. Numerous smaller granules appeared to be interconnected by filaments (Figure 19). Photographs in

DMSO

Annals

126

New York Academy of Sciences

Figures 5, 6, and 7. Figure 5 shows a rat thyroid follicular epithelial cell fixed by perfusion with glutaraldehyde-acrolein solution. Apical vesicles are mostly spherical or oval in appearance, (x 60,000.) In Figure 6, the rat thyroid was fixed in glutaraldehyde, acrolein, and following perfusion by in lactate Ringer's solution. A tubule (arrow) interconwas nects the apical vesicles to the luminal surface of the cell, (x 92,000.) In Figure 7, included in the glutaraldehyde fixative (by perfusion) but not in the Ringer's. A number of

DMSO

DMSO

DMSO

The dense content is included in the constricted portion (arx 51,000.) (Figures 5 and 7 from Sandborn.^ By permission of Academic

apical vesicles are interconnected.

row) of the tubule. Press.)

(

Sandborn

et air. Cell

DMSO

Ultrastructure and Cytochemistry

127

Figures 8, 9, and 10. With in both the Ringer's and in a glutaraldehyde-acroleinhydroxyadipaldehyde perfusate, the outer mitochondrial membrane appears continuous (arrows) with the membrane of the sarcoplasmic reticulum in striated, skeletal muscle fibers, in Figures 8 ( x 66,000) and 9 ( x 70,000.) Figure 10 shows glutaraldehyde-acrolein-DMSO fixed skeletal muscle as seen in the scanning electron microscope after the cell has been fractured. Some myofibrils are fractured, but others are seen in surface view (mf). Bands of tubules surround each myofibril. The larger organelles are mitochondria (m). (x 4,000.)

)

128

Annals

New York Academy of Sciences

Figures 11, 12, and 13. Figure 1 1 is an enlargement of the surface view of myofibrils (mf) and the related organelles. The mitochondria (m) appear to be associated with the underlying tubes of sarcoplasmic reticulum by short tubular stems (arrows), (x 27,000.) Figure 12 shows rat cardiac muscle. The mitochondria (m) between the sarcolemma (s) and the myofibrils (mf appear to be suspended by interconnecting stems. There was DMSO in both the Ringer's and the aldehyde solutions, (x 10,000.) In Figure 13, the mitochondria between two myofibrils appear as larger structures interconnected by narrow stems (arrows). There was DMSO in both the Ringer's and the aldehyde solutions, (x 20,000.)

Sandborn

et al.:

Cell Ultrastructure and Cytochemistry

129

v2-.

Figures 14, 15, and 16. The arteriolar endothelium of the red body of the eel. The fixation was by 5% DMSO in Ringer's followed by glutaraldehyde-acrolein-DMSO, introduced into the preretal artery. In Figure 14, long tubules (ts) invaginate into the cytoplasm from both the luminal (L) and basal surfaces of the endothelial cell. The tubular systems are distinguished by alternate dilatations and constrictions. Some of these show a branching pattern (arrow), (x 38,000.) In Figure 15, one of the tubules opens onto the luminal surface as well as into the nuclear envelope (arrow), (x 43,000.) In Figure 16, the mitochondrial outer membrane (arrow) extends out to become continuous with the smooth endoplasmic reticulum. ( x 28,(XX).)

1

Annals

30

New York Academy of Sciences

II

Figures

17, 18,

and

19.

In the membrane-secreting gland of the pigeon, microtubular stems

with densely stained content project out from secretory granules. Smaller dense granules ap-

pear

in a chain,

interconnected by filaments. There was

X 37,500; Figure

18,

DMSO

in

The photographs were taken by x 45,500; Figure 19, x 39,000.)

glutaraldehyde-acrolein solutions.

the Ringer's and in the T. Makita. (Figure 17,

1

Sandborn

et al.

Cell Ultrastructure and Cytochemistry

:

stereo confirmed that larger tubules that lead

from

1

3

apical granules to the luminal

membrane that secretes gland cells in phenomena (Figures 20 and 21). Scanning

duck oviduct were microscopy three dimensions, the interconnections between granules (Figures

surface of the cell in the

the

not overlap

electron

demonstrated, in 22 and 23). After fixation by glutaraldehyde and acrolein perfusion, one occasionally finds stacks of cisternae of the smooth endoplasmic reticulum in dendrites and axons of

DMSO

is Purkinje's cells in the cerebella of various species (Figure 24). When added to the perfusion solutions, the number of these membrane stacks and cisternal units within them is markedly increased (Figures 25 and 26). Results from cytochemical incubations show the hydrolytic reaction product of the enzyme, glucose-6-phosphatase, localized in its characteristic sites in the endoplasmic reticulum of the Purkinje's cell perikaryon (Figure 27) and dendrite (Figures 28 and 29).

Neurons

fixed with

tion than

do those

DMSO

in

the fixative

show more

fixed without; although there

precision of

might be

enzyme more

slightly

localiza-

reaction

product observed over cellular organelles when the fixative contains no DMSO, there also appears to be some random, nonspecific precipitation on plasma membranes, neurotubules, mitochondria, and the cell matrix (Figures 28 and 29). The tissue is not as well preserved, and is swollen, when fixed without the presence of for the brief period utilized in order to avoid enzyme inhibition.

DMSO

Discussion In previous studies on the fixation of tissues for electron microscopy, a fixative (acrolein) that has the ability to penetrate tissues quite rapidly

was added

to stan-

dard glutaraldehyde solutions. ^-^ The rapidity of penetration by the vapors of acrolein is evidenced by its lacrimogenic effect. A more rapid fixation of the tissues by this combination of fixatives resulted in an increase in the amount of membranes preserved. In our published reports fixatives,

on the influence of

we described an improvement

in

DMSO

membrane

the evidence of continuity between organelles.*

DMSO

A

on the action of aldehyde

preservation and an increase

in

study of the effects both of by Kalt and Tandler.^ They ob-

acrolein and of in the fixative has been made served that a greater amount of membranes was retained in the cytoplasm of embryonic liver cells. This would indicate that, with other methods, a certain amount of

membrane the

is

lost during the

plasma membrane of the

prolonged periods required for the fixative to traverse cell.

The evidence of channels

that open to the surface in intestinal epithelium, in

thyroid follicular cells, and in endothelial cells suggests that with the

DMSO

treatment the fixative would be able to enter the cell without traversing a membrane barrier. From our observations that apical granules in the thyroid are interconnected and that they open by tubular stems into the lumen of the follicle, it becomes evident that there is no need for transport by isolated vesicles in this cell. It has been demonstrated by autoradiography^" that the movement of the content of these small vesicles is toward the lumen of the follicle. Therefore the tube seen between the vesicle and the lumen must be either an existing tube or a potential tube that has opened and has been kept open during the fixation procedure. It is difficult to imagine that the tube could be extended out ahead of the vesicle to fuse with the plasma membrane and then open on the surface. A similar situation exists in the secreting cells of the oviduct in the fowl.

132

Annals

New York Academy of Sciences

Figures 20, 21, 22, and 23. Figures 20 and 21 show the shell membrane-secreting gland in duck oviduct, with stereoscopic views of tubules which extend toward the luminal (L) surface from apical granules. The Ringer's and aldehyde fixatives both contained DMSO. (Figure 20, x 51,000; Figure 21, x 55,000.) Figures 22 and 23 are scanning electron microscope views of secretory granules in the duck oviduct, with interconnections (arrows) between several granules. The bird was fixed by perfusion with DMSO in the Ringer's and the glutaraldehyde-acrolein solutions, and sections were digested with diastase after fracturing on the tissue chopper. (Figure 22, x 4,000; Figure 23, x 35,000.) the

Sandborn

et al.

:

Cell Ultrastructure

and Cytochemistry

1

33

ser *^a>^

^^S

I

^^*"««

25 Figures 24 and

-0!^'

ik...

Figure 24 shows a transverse section of a Purkinje cell dendrite in the by perfusion of the animal with glutaraldehyde-acrolein solutions. Some layers of smooth endoplasmic reticulum (ser), a multivesicular body (mvb), microtubules, and mitochondria (m) are seen against a lightly stained background, (x 61,500.) Figure 25: in another rat fixed by perfusion with 4% glutaraldehyde and 5% following the lactate 25.

rat cerebellum, fixed

DMSO

Ringer's containing

5% DMSO,

sectioned Purkinje dendrite.

membranes, (x 50,000.)

stacks of

membranes

The cytoplasm appears

are found throughout a longitudinally to

have a much greater content of

134

Annals

New York Academy of Sciences

^m^^'-^

7%^'

,2-7.

J

4ii..^.

Figures 26 and

27. Figure 26 shows a Purkinje dendrite of the hamster, fixed as in Figure The number of membrane stacks is even more remarkable. The dense content is demonstrated in subunits of the multivesicular body. ( x 34,000.) In Figure 27, the rat was fixed as in Figure 25, and incubated with glucose-6-phosphate as substrate. The cytochemical reaction product is localized precisely over the smooth and the rough endoplasmic reticulum 25.

as well as the envelope of the nucleus (N), but not in the Golgi apparatus (G) of the Purkinje

cell.(x 22,500.)

Sandborn

et al.

:

Cell Ultrastructure

1^

and Cytochemistry

135

1^»

Figures 28 and

Figure

product after glucose-6-phosphate incusmooth endoplasmic reticulum of the Purkinje dendrite fixed with glutaraldehyde alone, (x 21,000.) In Figure 29, a-glycerophosphate was used as substrate. The stacks of smooth endoplasmic reticulum as well as the smaller tubules of the endoplasmic reticulum display the reaction product in the dendrite of the rat fixed with 4% glutaraldehyde and 5% DMSO, following lactate Ringer's containing 5% 29. In

bation appears to be

DMSO.(x

63,000.)

28, the reaction

more randomly dispersed

in

the

^

1

Annals

36

New York Academy of Sciences

In the intestinal absorptive cells, the transport is from lumen toward the base of the cell, and the ferritin tracer studies indicate that the long tubules that invaginate deep into the cytoplasm are the route of this transport. Since alternate di-

and constrictions appear

in the tubule that contains the ferritin, one must be peristaltic waves. Since the studies of the liver, the intestine, and the thyroid were made in animals in which the was added only to the fixative, its influence must be simultaneous with that of the fixative. The fixation commences immediately, and to all intents and purposes it is complete within a very few minutes (as compared with one hour or more by ordinary glutaraldehyde fixation). Since this is such a rapid fixation procedure, it is doubtful that the

latations

realize that these

may

DMSO

membranous tubular interconnections could be artifacts produced by the method. It more likely that a deterioration and breakdown of existing structures, allowed by

is

the slower methods,

somewhat

is

is

prevented. That the tubular system

in

the cell could be dilated

possible, but in the study of the interrelationship of organelles this

would be a desirable feature, since the larger the interconnections between more frequently they are encountered in sections. Any section through a dilatation in a tubular system that does not intersect the dilatation and the constriction in perfect orientation at the exact depth gives the appearance of an

organelles, the

isolated vesicle. Since the osmolarity of the solution

is considerably increased if by approximately 180 mOsm), it is unlikely that a dilatation would occur under the influence of DMSO. Since Bone and Denton '^ reported, however, that it is the osmolarity of only the mineral salts that is of significance, this feature of the may not play a role in either constriction or dilatation. The increase in the incidence of tubular stems seen when was added to the lactate Ringer's for 2 min could be the result of a dilatation of the system and then an even more rapid acceptance of the fixative by the cell. The reported increase in the permeability of tissues under the influence of supports this possibility. The wide-open channels in the arteriolar capillary endothelium of the red body of the swim bladder are in sharp contrast to the vesicles that had been demonstrated previously. ^^ These vesicles had been credited with a role in the special function of this endothelium, as a hairpin countercurrent multiplication system for the increase of lactic acid in the blood that enters the swim bladder wall.'^*'* Since it is evident that the tubules lead from the nuclear envelope to the surface and are even continuous with the mitochondrial membranes, any breakdown and refusion of vesicles becomes unnecessary and improbable. The continuity between mitochondria and sarcoplasmic reticulum seen in striated muscle, both in thin section and in scanning electron microscopy, eliminates the need for calcium and other ions to make complicated transfers across membranes in order to pass from one compartment to another. Continuity of membrane structure between mitochondria and endoplasmic reticulum has been demonstrated in axons* and in mesothelial

DMSO

is

added (1%

DMSO increases osmolarity

DMSO

DMSO

DMSO

cells.

The oviduct of

the laying fowl

is

ideal for the study of secretion, since specific

types of protein are secreted by various parts of the organ.

The evidence

that tubular

stems exist on many of the granules and their visualization in three dimensions by scanning electron microscopy again demonstrate that a fusion process between vesicles is not necessary in the transfer of the products of synthesis to the lumen. Meldolesi and colleagues*^ demonstrated that the membranes of the pancreatic exocrine cell are chemically and functionally distinct, and they do not mix with one another during the transport of secretory products. The observations recorded here can offer an explanation of how content can be passed from one organelle to the other without the aid of ion pumps and the highly improbable fusion and breakdown required in the pinocytosis-exocytosis theory that has been advanced. This theory has

Sandborn

et al.

:

Cell Ultrastructure and Cytochemistry

1

37

been developed on the strength of negative evidence, produced firstly by light microscopy in which the interconnections are far beyond the limits of resolution of the instrument, and secondly by electron microscopy after methods of preparation that leave very much to be desired. Our studies and those of Kalt and Tandler^ indicate that most methods of fixation allow the breakdown of a considerable amount of membrane structure. If, as it would appear from the work of Guidotti, membranes contain actomyosin-like proteins, one would expect a vesiculation of tubules while this breakdown is occurring. The breakdown of tubules into vesicles with certain fixatives was shown to occur by Rosen bluth.'* The improvement in the precision of localization of the reaction product by cytochemical procedures after the addition of to the fixative*^ may be dependent on several factors: 1. The brief period of time required for adequate fixation probably reduces enzyme inhibition by excessive exposure to the fixative. 2. The rapid penetration of the fixative could prevent diffusion of the enzyme. 3. Any substrate and capturing agent may more readily enter the cell, which could be of importance for substrates that are not lipid-soluble and of high molecular weight or of high polarity. In summary, the study of the influence of on cell membranes has allowed us to demonstrate that the internal organelles of the cell probably form an interconnected network. We postulate that a possible action of the drug is to open potential pores in the cell membrane, thus allowing the cell to take up materials or chemicals that the membrane barrier would ordinarily block. The pores actually open directly into channels that probably represent a part of a system in most cells. It is suggested that the current concepts of transport across membranes and within the cell should undergo a thorough reevaluation. Ling** calculated that the consumption of energy by the Na+, Ca+"^, and Mg+"^ pumps alone would be far in excess of the total energy available to the cell. In addition, a great deal more energy would be required to operate the membrane breakdown and reconstruction involved in the pinocytosisexocytosis phenomenon. Given the continuity of membrane structure demonstrated by the use of in this study, it becomes evident that the need for many of these energy-dependent processes may be considerably less than has been surmised. The simplest of all methods of transport, propulsion through a tubular system, probably accounts for a major part of the process.

DMSO

DMSO

DMSO

Acknowledgements The authors express their appreciation for the excellent technical assistance of Miss C6cile Venne.

References Sandborn,

E. B., P. F.

Koen,

tubules in

mammalian

cells. J.

Sandborn,

J.

E. B. 1966. Electron

ments. Can.

J.

D.

McNabb &

G.

Moore.

1964. Cytoplasmic micro-

Ultrastruct. Res. 11: 123-38.

microscopy of the neuron membrane systems and

fila-

Physiol. Pharmacol. 44: 329-38.

Franz, T. J. & J. T. van Bruggen. 1967. A possible mechanism of action of DMSO. Ann. N. Y. Acad. Sci. 141: 302-10. Jacob, S. W., M. B. Bischel & R. J. Herschler. 1964. Dimethyl sulfoxide: Effects on the permeability of biologic

193-8.

membranes (preliminary

report). Curr. Therap. Res. 6:

—

1

Annals

38

New York Academy of Sciences

8.

H. & K. Muhlethaler. 1963. Fine structure in frozen-etched yeast cells. J. Cell 17:609-28. Steere, R. L. 1957. Electron microscopy of Structural detail in frozen biological specimens. J. Biophys. Biochem. Cytol. 3:45-60. LuFT, J. H. 1961. Improvements in epoxy resin embedding method. J. Biophys. Biochem. Cytol. 9: 409- 14. Sandborn, E. B. 1970. Cells and Tissues by Light and Electron Microscopy. Vols. I and

9.

Kalt, M. R.

5.

Moor, Biol.

6.

7.

II.

Academic

Press,

&

B.

New

electron microscopy. 10.

Nadler, N.

11.

Bone, Q. Biol. 49:

12.

Dorn,

&

B. A.

J.,

thyroglobulin

J.

1971. A study of fixation of early amphibian Ultrastruct. Res. 36: 633-45.

Young,

the thyroid

in

E. J.

York, N.Y.

Tandler.

Denton.

C. P.

follicle.

1971.

Leblond

&

B.

Mitmaker.

embryos

for

1964. Elaboration of

Endocrinology 74: 333.

Osmotic

effects of electron

microscope

fixatives. J. Cell

571-81.

E.

Uber den Feinbauder Schwimmblase von Anguilla

1961.

vulgaris

L.

Z.

Zellforsch. 55: 849-912. 13.

14.

Anguilla vulgaris. J. B. 1963. The physiology of the swimbladder in the eel The mechanism of gas secretion. Acta Physiol. Scand. 59: 221-41. Bendayan, M., E. B. Sandborn & E. Rasio. 1974. The capillary endothelium in the

Steen,

rete

swimbladder of the eel {Anguilla anguilla); functional and ultrastructural aspects. Can. J. Physiol. Pharmacol. 52:613-623. Meldolesi, J., J. D. Jamieson & G. E. Palade. 1971. Composition of cellular membranes in the pancreas of the guinea pig. III. Enzymatic activities. J. Cell Biol. 49: mirabile

15.

III.

of the

109-58. 16.

RosENBLUTH

J.

1963. Contrast between

osmium

fixed

and permanganate fixed toad

spinal ganglion. J. Cell Biol. 16: 143-157. 17.

Sandborn,

E. B., T.

Makita

&

K. Lin. 1969.

The use of dimethyl

sulfoxide as an ac-

celerator in the fixation of tissues for ultrastructural and cytochemical studies and in 18.

19.

freeze etching of cells. Anat. Record 163: 255. Ling, G. N. 1969. A new model for the living cell: A summary of the theory and recent experimental evidence in its support. Intern. Rev. Cytol. 26: 1-61. GuiDOTTi, G. 1972. Discussion paper: Membrane proteins. Ann. N.Y. Acad. Sci. 195:139-141.

METABOLISM AND EXCRpTION OF DIMETHYL SULFOXIDE IN COWS AND CALVES AFTER TOPICAL AND PARENTERAL APPLICATION J.

Tiews, E. Scharrer, N. Harre, and L. Flogel

Department of Animal Physiology and Animal Nutrition University of Munich Munich, Federal Republic of Germany

W.

JOchle

International Veterinary Section

Syntex Research Palo Alto, California 94304

The therapeutic success experienced with dimethyl

sulfoxide

(DMSO)

in

man,

horses, and dogs raised questions as to whether this drug could feasibly be used for the treatment of disease conditions in the food-producing bovine and porcine species. Prerequisites for the therapeutic

are knowledge of

its

employment of the drug

metabolism and excretion patterns

possibilities that exist for tissue residues of

DMSO

in

or

in cattle

or swine

these species, and of the its

more important me-

tabolites.

The studies reported here deal with DMSO metabolism and excretion in cattle. They show that the bovine species metabolizes and excretes DMSO and its two main metabolites, DMS and DMSO2, fast and effectively. This is not surprising, since DMSO2 has long been known to occur in cattle blood and adrenal glands, ^ and DMS and DMSO2 are components of the cow's milk.^~^ ^

Materials and Methods

Three male dairy calves, 2 to 4 weeks of age, that weighed 50 to 65 kg (Trials 1 to and two three-year old dairy cows (Holstein) that produced 10 kg milk (Trial 4) and 16 kg milk (Trial 5) daily, were used. The calves were housed in metabolic cages at room temperature, and were fed a commercial milk-replacer diet. Urine and feces were collected separately. The cows were kept in stalls, and received hay ad lib. and 4 kg concentrate daily. Cows were milked by hand, and each quarter was milked separately. Feces were not collected from cows, but urine was collected thus: indwelling catheters were inserted into the bladder and emptied into plastic 3),

containers.

Routes of application of [^''CJDMSO, the amounts of DMSO, and the specific employed are given in Tables 1 and 2. Determination of ^^C-Activities in Urine Samples. Three ml fresh urine were combined with 10 ml scintillation liquid (4 g 2,5-diphenyloxazole [PPO] and 100 mg dimethyl- l,4-/7Z5-2-[5-phenyloxazolyl]-benzene [dimethyl-POPOP] in liter toluol) and 10 ml Triton® X-100, and were placed in scintillation tubes. The well-shaken tubes were placed in a liquid scintillation spectrometer and were measured three times. An internal standard ([^^C]toluol; liquid scintillation spectrometer model 314 Ex, Packard Instruments) or external standard (liquid scintillation spectrometer, model 3375, Packard Instruments) were used to make quench corrections.*"^" activities

1

139

140

Annals

O

New York Academy of Sciences

-o

vn

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8

a:

O

c o

T3 15

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CO CJ

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03

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Friend

& Scher:

Murine Virus-Induced Leukemic Cells Table

161

2

Comparison of the Survival Times of Mice Inoculated with Virus Recovered FROM THE Spleens of Control and DMSO-Treated* FLV-Inoculated Mice Time After

c Virus Source offi/

\/

i

days

Untreated micet DMSO-treated micet

.

LD50I >10"'*

14

>10"'*

21

DMSO-treated mice

21

Untreated mice

28

DMSO-treated mice

28

10"^' >10~'* 10"^''^

>10~'*

7-day course of treatment was followed by intermittent

..

Mean

c

Surviva

i

days

14

Untreated mice

*A

_. Titer

• m FLV Inoculation

rest

73.4

56.0 101.2

52.2

99.0 87.6

periods and continued

treatment.

tMice were inoculated with 0.2 ml 10~ dilution of FLV. tLD5o per 0.2 ml FLV was injected intraperitoneally.

leukemic cell system. Our hope is to find an ideal chemotherapeutic agent that would act selectively in "programming" neoplastic cells for death, as has been suggested by Basile and colleagues.^'' lating differentiation in the

References Friend, C, M. C. Patuleia & E. de Harven. 1966. Erythrocytic maturation in vitro of murine (Friend) virus-induced leukemic cells. Nat. Cancer Inst. Monograph 22: 505522.

Friend, C, H. D. Preisler & W. Scher. 1974. Studies on the control of differentiation of murine virus-induced erythroleukemic cells. In Current Topics in Developmental Biology. A. Monroy & A. A. Moscona, Eds.: 81-101. Academic Press Inc. New York,

N.Y. Friend, C. & J. Haddad. 1960. Tumor formation with transplants of spleen or liver from mice with virus-induced leukemia. J. Nat. Cancer Inst. 25: 1279-1289. Rossi, G. B. & C. Friend. 1970. Further studies on the biological properties of Friend virus-induced leukemic cells differentiating along the erythrocytic pathway. J. Cell. Physiol. 76: 159-166.

&

v., M. Furusawa H. Sugano. 1973. Erythrocyte membrane-specific antigens Friend virus-induced leukemia cells. In Unifying Concepts of Leukemia. Bibliotheca Haematologica No. 39. R. M. Dutcher and L. Chieco-Bianchi, Eds.: 955-967. Verlag S. Karger. Basel, Switzerland.

Ikawa, in

Ostertag, W., H. Melderis, G. Steinheider, N. Kluge & S. Dube. 1972. Synthesis of mouse haemoglobin and globin mRNA in leukemic cell cultures. Nature New Biol. 239: 231-234. J. G. Holland & C. Friend. 1971. Hemoglobin synthesis in murine virusinduced leukemic cells in vitro: Partial purification and identification of hemoglobins. Blood 37: 428-437.

Scher, W.,

BoYER, S. H., K. D. Wuu, A. N. Noyes, R. Young, W. Scher, C. Friend, H. D. Preisler & A. Bank. 1972. Hemoglobin biosynthesis in murine virus-induced leukemic cells in vitro: Structure and amounts of globin chains produced. Blood 40: 823-825. Barski, G. & J. K. Youn. 1966. Protective effect of specific immunization in Rauscher leukemia. Nat. Cancer Inst. Monograph 22: 659-670.

Annals

162 10.

YosHiKURA,

H., Y.

New York Academy of Sciences

HiROKAWA, M. Yamada

&

H. SuGANO. 1968. Interference

in

Friend

virus infection in vitro. J. Virol. 2: 85-86. 11.

ScHLOM,

J.,

J.

B.

Moloney &

V.

Groupe.

1971. Evidence for the rapid decrease in

leukemogenic potential of Rauscher leukemia virus

in

celLculture.

Cancer Res. 31 260:

264. 12.

N., W. Scher & C. Friend. 1973. Evidence for the rapid decrease in leukemogenic potential of Friend leukemia virus in cell culture. Federation Proc. 32: 1020

Gabelman, (Abstr.).

13. 14.

Gabelman, N. & C. Friend. To be published. Klement, v., M. O. Nicolson & R. J. Huebner. forming virus from rat non-productive Biol.

15.

16. 17.

18.

19.

20.

lines

197 1 Rescue of the genome of focus by 5'-bromodeoxyuridine. Nature New .

234: 12-14.

DE Harven, E. & C. Friend. 1966. Origin of the viremia in murine leukemia. Nat. Cancer Inst. Monograph 22: 79-105. Friend, C. & B. Pogo. Unpublished data. Friend, C. & G. B. Rossi. 1968. Transplantation immunity and the suppression of spleen colony formation by immunization with murine leukemia virus preparations (Friend). Intern. J. Cancer 3: 523-529. Friend, C. 1973. Immunologic studies with leukemogenic and nonleukemogenic strains of murine leukemia virus (FLV). In Virus Tumorigenesis and Immunogenesis. W. Ceglowski & H. Friedman, Eds.: 387-391. Academic Press Inc. New York, N.Y. Friend, C, W. Scher, J. G. Holland & T. Sato. 1971. Hemoglobin synthesis in murine virus-induced leukemic cells in vitro. Stimulation of erythroid differentiation by dimethyl sulfoxide. Proc. Nat. Acad. Sci. U.S. 68: 378-382. Scher, W., H. D. Preisler & C. Friend. 1973. Hemoglobin synthesis in murine virusinduced leukemic cells in vitro: Effects of 5-bromo-2'-deoxyuridine, dimethylformamide

21.

and dimethyl sulfoxide. J. Cell. Physiol. 81: 63-70. Sato, T., C. Friend & E. de Harven. 1971. Ultrastructural changes in Friend erythroleukemia cells treated with dimethyl sulfoxide. Cancer Res. 31: 1402-1417.

22.

Till,

&

23.

E. A. McCulloch. 1961. A direct measurement of the radiation sensitivity mouse bone marrow cells. Radiation Res. 14: 213-222. Singer, D., M. Cooper, G. Maniatis, P. A. Marks & R. H. Rifkind. 1973. Effect of

24.

Friend,

25.

Friend,

J.

E.

of normal

(DMSO) on

Friend leukemia cells. J. Cell Biol. 59: 320(a). D. Preisler J. G. Holland. 1973. Studies on erythroid differentiation of Friend virus-induced murine leukemic cells. In Unifying Concepts of Leukemia. Bibliotheca Haematologica No. 39. R. M. Dutcher and L. Chieco-Bianchi, Eds.: 916-922. Verlag S. Karger. Basel, Switzerland.

dimethylsulfoxide

&

C, W. Scher, H.

&

C, W. Scher

H. D. Preisler. 1974. Hemoglobin biosynthesis in murine in vitro. Ann. N.Y. Acad. Sci. 241. In press. Preisler, H. D., W. Scher & C. Friend. 1973. Polyribosome profiles and polyribosome-associated RNA of Friend leukemia cells following DMSO-induced virus-induced leukemia cells

26.

differentiation. Differentiation 1: 27-37. 27.

Williamson,

RNA"

in

R.,

G.

Lanyon

&. J.

Paul. 1969. Preferential degradation of "messenger Nature

reticulocytes by ribonuclease treatment and sonication of polysomes.

223:628-630. 28.

Ikawa,

Y., J.

Ross, K. Hayashi,

P.

Ebert,

J.

Gielen

&

P.

Leder. 1973. Kinetics of

erythrodifferentiation of cultured leukemia cells. In Proceedings of the Sixth International

Symposium on Comparative Leukemia Research. Nagoya and Ise-Shima, Japan.

In press.

&

J., Y. Kiawa P. Leder. 1972. Globin messenger-RNA induction during erythroid differentiation of cultured leukemia cells. Proc. Nat. Acad. Sci. U.S. 69:

29.

Ross,

30.

Preisler, H. D., D. Housman, W. Scher & C. Friend. 1973. The effects of 5-bromo-2'deoxyuridine on the production of globin in dimethyl sulfoxide-stimulated Friend leukemia cells. Proc. Nat. Acad. Sci. U.S. 70: 2956-2959.

3620-3623.

mRNA

31.

Sato, T., E. de Harven & C. Friend. 1973. Increased virus budding from Friend erythroleukemic cells treated with dimethyl sulfoxide, dimethyl formamide and/or

Friend

32.

& Scher:

Murine Virus- Induced Leukemic Cells

163

bromodeoxyuridine in vitro. In Proceedings of the Sixth International Symposium on Comparative Leukemia Research. Nagoya and Ise-Shima, Japan. In press. Lin, S.-Y. & A. D. Riggs. 1972. Lac operator analogues: Bromodeoxyuridine substitution in the lac operator affects the rate of dissociation of the lac repressor. Proc. Nat.

U.S. 69: 2574-2576. M. R. Sacksteder, B. M. Ellis & R. D. Schwartz. 1973. Enhancement of viral oncogenicity by the prior administration of dimethyl sulfoxide. Cancer Res. 33: 618-622. Basile, D. v., H. N. Wood & A. C. Braun. 1973. Programming of cells for death under defined experimental conditions. Proc. Nat. Acad. Sci. U.S. 70: 3055-3059.

Acad.

33.

34.

Sci.

Warren,

J.,

EFFECT OF DIMETHYL SULFOXIDE AND

DIMETHYLFORMAMIDE ON THE GROWTH AND MORPHOLOGY OF TUMOR CELLS* E. Borenfreund,

M.

Steinglass, G. Korngold,

and A. Bendich

Laboratory of Cell Biochemistry

Memorial Sloan-Kettering Cancer Center New York, New York 10021

Introduction The morphological pattern in which cells grow in culture has often been taken as an indication either of their malignancy or nontumorigenicity. Unlike their normal counterpart, tumor cells grow in disarray in culture, piling up and migrating past each other in a manner that resembles their behavior in tumors. We have been interested to see whether reversal of the pattern of growth back to that characteristic of nontumor cells could be achieved, and whether this would also be accompanied by loss of malignancy. '^he reported decrease in the malignancy of Friend erythroleukemic cells and the marked enhancement of their differentiation along the erythroid pathway after their treatment with the simple solvent dimethyl sulfoxide (DMSO)^~^ stimulated our study of the effect of this agent, and of the closely related solvent dimethylformamide (DMF), on various malignant and normal cell lines used in our laboratory. An alteration in the pattern of growth of tumor cells resulted after treatment with these agents. This effect is mimicked by that of the totally unrelated agent 5bromodeoxyuridine (BrdU).'*~®

Materials and Methods Cell Lines.

The mouse tumor

cells

used included the melanoma

tablished in our laboratory, Ehrlich ascites cells adapted to

grow

line

Mel B16, es3T3 cells

in vitro,

TCMK-SV40, a virus-transformed obtained from the Tissue Type Culture Association. Most of the experiments were carried out with QUA, a cell line established by Dr. June Biedler (Sloan-Kettering Institute) from a tumor induced in C57/B1/6 mice by methylcholanthrene.^ Cells were grown in Eagle's minimum essential medium, supplemented with 10% fetal bovine serum, streptomycin, and penicillin, and were maintained in a humidified atmosphere of 5% CO2 and 95% air at 37° C. Cultures were frequently monitored for the presence of mycoplasma, and were found to be free of contamination. Chemicals. Reagent-grade were obtained from Fisher and transformed by polyoma virus (3T3-Py), and

mouse kidney

cell line

DMSO

Scientific

DMF

Company, New York, thymidine and BrdU from Schwarz/Mann,

Orangeburg,

New

from Upjohn, New York, New York, and Baker, Dagenhew, England, concanavalin A and

Jersey, cycloheximide

ethidium chloride from

May

*This work was supported by National Cancer Commission contract AT[1 l-i]-3521.

164

Institute grant

CA 08748 and Atomic Energy

Borenfreund

et ai: Effect

on Growth of Tumor Cells

165

wheat germ lipase from Calbiochem, San Diego, California, puromycin from Lederle Lab, Pearl River, New York, and actinomycin from Merck Sharp and Dohme, Rahway, New Jersey. [^H]fucose (1.8 Ci/mM) and ['*C]fucose (160 mCi/ mM) were purchased from Amersham/Searle, Arlington Heights, Illinois, and [^H]2-deoxyglucose (7.2 Ci/mM) from New England Nuclear Corporation, Boston, Massachusetts. plastic Petri dishes, Assays. An inoculum of 250 cells was seeded onto 60 and the various agents were added to the medium 24-48 hours later. Concentrations to 3 Mg/ml, of DMSO varied from 1 to 2%, DMF from 0.5 to 1% and BrdU from depending on the sensitivity of the cell lines studied. In other experiments, chemicals were added only after the typically random-piling individual colonies had been established. For mass culture studies, 2 x 10^ cells were inoculated into either milk dilution bottles or plastic Petri dishes, to which media that contained the agents were

mm

1

then added.

Studies that involved the incorporation of radioactive fucose into surface glyco-

Buck and Figure 1), and the digested "trypsinates" were chromatographed on Sephadex® G-50 columns. Radioactivity in chromatographic fractions was determined in a Packard-Tri-Carb liquid scintillation counter. Agglutination studies with wheat germ lipase and concanavalin A were carried out as described by Burger and Goldberg^ and Inbar and Sachs. ^° Growth curves and replication cycles were determined by seeding a series of replicate dishes and counting cells at given time intervals. Viability was assessed by the neutral red exproteins were carried out according to the procedure described by

colleagues* (see the legend to

clusion test.

mm

To measure contained

1

the uptake of 2-deoxyglucose, duplicate 60 x 10^ cells were washed with prewar med, glucose- free

and incubated for 10 min (7.2

at 39°

C

dishes

Hanks

that

solution

with 2.0 ml 0.25 ^lC\ [^H]2-deoxyglucose per ml

Ci/mM). Hanks without glucose was used

as

described by

Martin and

colleagues." After four rapid washes with ice-cold glucose-free Hank, cells were

scraped into 0.5 ml water and disaggregated in a vortex mixer, and aliquots were taken for scintillation-counting in a toluene-Triton® mixture and for protein determination.'^

Results

A

marked

alteration in the

randomly

piling,

non-contact-inhibited growth pat-

QUA tumor cell line occurred when the normal growth medium was supplemented by DMSO at final concentrations of 1-2%, by DMF at 0.5-1%, or BrdU

tern of the at 1-3

Mg/ml. Instead of the piling pattern, monolayers of cells in regular parallel formed that were typical of nonmalignant fibroblasts (Figure 1). The same drastic changes were observed when the agents were added, even after colonies of piled-up cells characteristic of this tumor line had been allowed to form. Accordingly, this reversion of colonial morphology was not due to a selection of cells from within the total population. This was borne out by a study of the replication cycle, which showed that although there was an increase in generation time, there was no cell death. The doubling time was about 30% longer in the presence of the agents. Incubation with or for about 3 days, and with BrdU for about 5-6 days, was required before morphological changes were manifested. These changes could be maintained indefinitely by growth in the continuous presence of the chemical agents. Reversion to the original growth pattern could be effected in about orientation

DMF

DMSO

Annals

166

New York Academy

of Sciences

'^'r^--^ DMF on the in vitro growth pattern of tumor cells, (a) QUA tumor cells grown for 8 days in presence of DMSO; (c) colony of QUA tumor cells; (d) colony of QUA tumor cells grown for 7 days in

Figure Colony of

2%

QUA

presence of

grown

The

1.

effect of

tumor

DMSO

cells; (b)

and

colony of

1% DMF;

for 10

photographed

(e) colony of Melanoma B16 cells; (0 colony of Melanoma B16 cells presence of 0.5% DMF. (a and b x 32, Giemsa stain; c to f x 200, living state, with Nomarski optics.)

days in

in

removal of DMSO or DMF, or in 6 days after withdrawal of BrdU from the medium. Thymidine (but not uridine) prevented the BrdU-induced effect if added to the growth medium at 5 times the molar concentration of BrdU, but no such interference with the observed DMF or DMSO effect occurred. 5-Bromouridine at concentrations of 1-10 Mg/ml did not mimic the effect induced by BrdU. The tumor lines QUA, Mel B16, 3T3-Py, and TCMK-SV,o behaved similarly when 3 days after

treated with the agents under investigation.

The changes described above could not be induced by treatment of cells

with dibutyryl cyclic

AMP,

the

tumor

with or without theophylline (although such

Borenfreund

et ai: Effect

on Growth of Tumor Cells

changes had been observed by other investigators cytochalasin

mold metabolite

a

B,

microfilament function.

BrdU,

cell

To

that

167

in different cell lines'^'")

interferes

with

cell

study the mechanism of action of

or by

movement and

DMSO, DMF,

and

cultures that contained these agents were treated with inhibitors of the

biosynthesis of protein, cytoplasmic

RNA,

or mitochondrial

DNA. The

inhibitors

days of growth in the presence of the agents are required before morphological changes are manifested. Under these conditions puromycin (0.2 ^g/ml), cycloheximide (0.05 Mg/ml), and ethidium chloride (10"' M) failed to interfere with the observed effects. Since the membrane properties of the cells appeared to be affected after incubation with the agents under investigation, their effect on the agglutinability of the cell with wheat germ lipase and concanavalin A was examined. We observed a considerable decrease in agglutinability after the cells were grown in the presence of DMSO, DMF, or BrdU. This prompted us to examine the surface glycoproteins by a double-label experiment in which we exposed cells to ['''C]- or [^H-]fucose before and after incubation with the chemical agents. Chromatographic analysis of

had to be used

at subtoxic concentrations, since several

pronase-digested trypsinates of the surface membrane glycoproteins showed that or brought about a decrease in the high-moleculartreatment with weight fucose-containing fraction and increased the low-molecular-weight fraction of the tumor cells, as compared with these fractions in the untreated tumor cells (Figure 2). Incubation with BrdU, however, did not significantly alter the elution

DMSO

DMF

profile of the glycopeptide fractions of these cells.

GLYCOPEPTIDES

FROM TUMOR CELLS WITHOUT DMF WITH

DMF

X-FUCOSE 'H-FUC05E

100

4>Re6

50

75

100

125

150

FRACTION NUMBER (Sephadex G-50)

DMF

2. Effect of on glycopeptide production of QUA tumor cells. 2 x 10® QUA were grown in each of 7 Blake flasks with 50 ml Eagle's minimum essential media and Hank's salts, supplemented with 10% fetal bovine serum and [**C]fucose to a final concen-

Figure

cells

mCi/mM). After 24 hours, 25 ml medium (without additional fueach flask and the cells were grown for an additional 48 hours. Cells that in the presence of DMSO, DMF, or BrdU were incubated in media that contained the respective agents and 0.4 ^Ci [^H]fucose/ml medium (1.8 Ci/mM). After incubation, cells were washed 4 times with 0.01 Tris buffer (pH 7.4), trypsinized, digested with pronase, and chromatographed on Sephadex G-50 as described elsewhere.* Fractions were counted in a Pad d-Tri Carb liquid scintillation counter. tration of 0.2 /xCi/ml (160

was added had been grown cose)

to

M

Annals

168

New York Academy of Sciences Table

Effect of Cytochalasin B on

1

QUA Tumor

AND After Treatment with

Cells Before

DMSO*

Nun-iber of Nuclei per Cell Treatment 2

1

4

3

>5

5

/o

92 90

Control

2%

DMSO for

Cytochalasin

13 days

B

2%DMSOfor

2

13

4 28

22

1

1

1

1

6

3

13 days, then

B BrdU(lMg/ml)forlOdays BrdU(Ug/ml)forlOdays, cytochalasin B cytochalasin

*Two hundred

5

4 28

cells

were counted

in

16

70

12

2

83

12

2

2

1

22

40

22

15

2

each group. Cells were exposed for 4 days to

1

/xg

cytochalasin B/ml.

Further indication that the cells to

DMSO treatment induced a reversion

more normal state came from experiments and untreated tumor cells to cytochalasin B (1 a

of the tumor

that involved exposure of

/ig/ml) for 4 days. It had been reported^^-^^ that exposure of normal cells to cytochalasin B resulted in cells that predominantly contained one or two nuclei, whereas in tumor cells so exposed, a high percentage of cells that had more than two nuclei appeared. About 40% of the tumor cells exposed to cytochalasin B alone have one or two nuclei, and as many as 60% are highly multinucleated (Table 1). Cells pretreated with DMSO, on the other hand, behave more like normal cells, in that 86% of these cells have one or more nuclei after growth in the presence of cytochalasin B, and only 14% show a more abnormal morphology and multinucleation. BrdU appears to be less effective than in causing reversion to a more normal pattern. Several laboratories have reported that there is an increased uptake of 2-deoxyglucose by virus-transformed cells, as compared to their normal counterparts.'^'^ This is presumably due to increased phosphorylation of the sugar by the tumor treated

DMSO

Table

2

Uptake OF [^H]2-Deoxyglucose BY Tumor Cells AFTER Incubation with DMSO, DMF, OR BrdU Treatment

Length of Treatment

BrdU(lMg/ml)

30 min 30 min 30 min

Control

DMSO(l%) DMF(1%) BrdU(lMg/ml)

>

707

Control

DMSO(l%) DMF(1%)

CountJ per ng Protein 791

694 561 1,195

42 days 13 days 27 days

982 1,158

919

*After short-term or long-term incubation of the tumor cells with the agents studied, the monolayers were washed with glucose-free Hanks, then incubated with 0.25 ^Ci [^H]2deoxyglucose (7.2 Ci/mM) for 10 min at 39°C (see text for details). Aliquots were removed for counting and protein determination.

Borenfreund^/ '^

cells.

fl/.;

Effect on

Growth of Tumor

Uptake of 2-deoxyglucose by the untreated parent

line

169

Cells

of tumor cells was

not significantly altered after treatment with DMSO, DMF, or BrdU. Similar results were obtained regardless of whether the agents were present in the cultures for 30 min or for several days (Table 2). These findings are at variance with a report that there

is

a decrease in the uptake of 2-deoxyglucose after treatment of

3T6

fibro-

blasts with BrdU.''

Discussion It has been shown that DMSO, DMF, and BrdU affect the morphology and growth pattern of tumor cells in culture, inducing a drastic change from the randomly piling, non-contact-inhibited growth characteristic of tumor cells to one that more closely resembles the parallel, oriented growth of normal cells. This effect was readily reversed after withdrawal of the chemical from the medium. Despite a similarity of action, the three agents exert their effects by different mechanisms and on different targets within the cell. BrdU has been reported to suppress cytodifferentiation,''^ melanin production, and tumorigenicity®^^ of cells in vitro, and

to preferentially inhibit the synthesis of tyrosine aminotransferase in cells.

'^

These

effects of

BrdU,

reversed with excess thymidine; this was not so for the tures.

BrdU may

the other hand,

DMSO- or

interact directly with the genetic material.

seem

to affect

hepatoma

as well as those observed in our studies, could be

membrane components,

DMF-treated

DMSO

and

cul-

DMF,

on

or their assembly, thereby

leading to structural changes or spatial rearrangements of the glycoproteins at the

The observations by Buck and colleagues^ and Warren and colleagues^^ amounts of high-molecular-weight fucose-containing glycopeptides are presefit at the surfaces of virus-transformed cells than at those of their normal controls and the observation by Bosman and colleagues^' that there is a marked incell surface.

that larger

crease of glycosyl and galactosyl transferases that

DMSO

and

DMF

exemplified by the

may exert DMSO-induced

their effects

in

virus-transformed

on cellular

cells

suggest

differentiation. This

erythroid differentiation of

tumor

cells,'

is

the in-

crease of hydroxyproline concentration in human fibroblasts,^^ and the extrusion and budding of virus particles from virus-infected cells. ^^^'^ also reversibly

DMSO

by mouse neuroblast cells in vitro.^^ and DMF, which are both very stable, highly polar substances, with dielectric constants greater than that of water, are known to enhance cell perinhibits neurite extension

DMSO

meability. ^^-^^ It

is

possible that these properties and the influence these solvents

may exert on the hydration and solvation shells around the membrane components may account for the various effects on cells, including the increased uptake of RNA isolated from Mengo virus. ^*

An enhanced rate of uptake of 2-deoxyglucose by transformed cells has been reported by a number of laboratories.'^'®-^® This could reflect either membrane alterations during the process of transformation or enhanced phosphorylation of the sugar by intracellular kinases.'® Whatever the effects of or on cell

DMSO

membranes may be, they did not the tumor cells in our studies.

significantly affect the

DMF

uptake of 2-deoxyglucose by

It is tempting to speculate that BrdU may act at the gene level, where due to its incorporation into DNA, it exerts an influence on transcription, and thus on the synthesis of some of the gene products characteristic of the state of differentiation of these cells. ^°-^' and DMF, on the other hand, may stimulate tumor cells to

DMSO

differentiate.

The observed

inhibition by

BrdU

of the

DMSO-induced

erythroid

Annals

170

New York Academy of Sciences

leukemia cells^^ suggests that the use of other agents that provide further support for the argument that cancer is a disease of

differentiation of Friend affect cells

may

differentiation. ^^^^t

[note added in proof: After submission of this manuscript, a paper by Kisch and colleagues^* that described the reversible, phenotypic DMSO-induced reversion of polyoma virus-transformed BHK-21 cells came to our attention.]

References 1.

2.

Friend, C, W. Scher, J. G. Holland & T. Sato. 1971. Hemoglobin synthesis in murine virus-induced leukemic cells in vitro: Stimulation of erythroid differentiation by dimethylsulfoxide. Proc. Nat. Acad. Sci. U.S. 68: 378-382. Scher, W., H. D. Preisler & C. Friend. 1973. Hemoglobin synthesis in murine virusinduced leukemic cells in vitro. J. Cell. Physiol. 81 63-70. Preisler, H. D., W. Scher & C. Friend. 1973. Polyribosome profiles and polyribosome associated RNA of Friend leukemia cells following DMSO-induced differentiation. Differentiation 1:27-39. Coleman, A. W., J. R. Coleman, D. Kankel & I. Werner. 1970. The reversible control of animal cell differentiation by the thymidine analog, 5-bromodeoxyuridine. Exp. Cell Res. 59: 319-328. BiscHOFF, R. & H. HoLTZER. 1970. Inhibition of myoblast fusion after one round of DNA synthesis in 5-bromodeoxyuridine. J. Cell. Biol. 44: 134-150. SiLAGi, S. & S. A. Bruce. 1970. Suppression of malignancy and differentiation in melanotic melanoma cells. Proc. Nat. Acad. Sci. U.S. 66: 72-78. BiEDLER, J. L. & R. H. F. Peterson. 1973. Reduced tumorigenicity of syngeneic mouse sarcoma cells resistant to actinomycin D and ethidium bromide. Proc. Amer. Assoc. :

3.

4.

5.

6.

7.

8.

Cancer Res. 14: 72. Buck, C. A., M. C. Click trol

9.

10.

11.

12.

13.

14.

&

Burger, M. M.

&

Warren.

1971. Glycopeptides from the surface of conScience 172: 169-171. A. R. Goldberg. 1967. Identification of a tumor specific determinant surfaces. Proc. Nat. Acad. Sci. U.S. 57: 359-366.

and virus-transformed

L.

cells.

on neoplastic cell Inbar, M. & L. Sachs. 1969. Structural difference in sites on the surface membrane of normal and transformed cells. Nature 223: 710-712. Martin, G. S., S. Venuta, M. Weber & H. Rubin. 1971. Temperature dependent alterations in sugar transport in cells infected by a temperature sensitive mutant of Rous sarcoma virus. Proc. Nat. Acad. Sci. U.S. 68: 2739-2741. LowRY, O. H., H. J. RosEBROUGH, A. L. Farr & R. J. Randall. 1951. Protein measurement with the folin phenol reagent. J. Biol. Chem. 193: 265-275. HsiE, A. H. & T.T. Puck. 1971. Morphological transformation of Chinese hamster cells by dibutyryl adenosine cyclic 3':5'-monophosphate and testosterone. Proc. Na. Acad. Sci. U.S. 68: 358-361. Johnson, G. S., R. M. Friedman & I. Pastan. 1971. Restoration of several morphological characteristics of normal fibroblasts in nosine-3':5'-cyclic

monophosphate and

its

sarcoma

cells treated

derivatives. Proc. Nat.

Acad.

with ade-

Sci.

U.S. 68:

425-429.

QUA

tAlthough the tumor cells used in these studies uniformly grew as tumors upon their inoculation into mice,^-^* pretreatment of the cells with failed to influence their tumorigenicity significantly. The time needed for the appearance of tumors would have been quite

DMSO

malignant growth pattern, however, if the cells had been kept in culture for this protracted period. Recipient mice were therefore pretreated for several days with DMSO, and were maintained on this agent after the DMSO-treated cells had been injected. Although there was inhibition of tumorigenicity in a few instances, the results were quite variable (unpublished data). sufficient to restore the

in

the absence of

DMSO

Borenfreund 15.

Bader,

et al: Effect

on Growth of Tumor Cells

171

1973. Virus induced transformation without cell division. Science 180: 1069-

J. P.

1070. 16.

17.

18.

19.

&

deoxyglucose and cycloleucine in 3T6 fibroblasts. Cancer Res. 33: 1326-1330. Rothschild, H. & P. H. Black. 1973. Investigations of the mechanism of decreased tumorigenicity of cells grown in BrdU. J. Cell. Physiol. 81: 217-224. Stellwagen, R. H. & G. M. Tomkins. 1971. Preferential inhibition by 5-bromodeoxyuridine of the synthesis of tyrosine aminotransferase in hepatoma cell cultures. J. Mol. 167-182.

Biol. 56:

20.

&

C. Colby. 1973. SV^o virus transformation of mouse 3T3 cells does not specifically enhance sugar transport. Science 179: 1238-1240. TsuBOi, A. R. Beserga. 1973. Effect of 5-bromo-2-deoxyuridine on transport of

Romano, A. H.

Warren,

L., D.

Critchley & I. MacPherson. 1972. Surface glycoproteins and glycoliembryo cells transformed by a temperature-sensitive mutant of Rous

pids of chicken

21.

sarcoma virus. Nature 235: 275-278. BosMANN, B. H., A. Hagopian & E. H. Eylar. 1968. Membrane glycoprotein biosynthesis: Changes in levels of glycosyl transferases in fibroblasts transformed by oncogenic viruses.

22.

23.

24.

25.

26.

27.

28.

J. Cell.

Physiol. 72: 81-88.

Stenchever, M. A., A. L. Hopkins & J. Sipes. compounds. Some effects on human fibroblasts

1968. Dimethyl sulfoxide and related

in vitro. Proc. Soc. Exp. Biol. Med. 126:270-273. Sato, T., C. Friend & E. de Harven. 1971. Ultrastructural changes in Friend erythroleukemia cells treated with dimethylsulfoxide. Cancer Res. 31: 1402-1417. Steward, S. E., G. Kasnic & C. Draycott. 1972. Activation of viruses in human tumors by 5-iododeoxyuridine and dimethylsulfoxide. Science 175: 198-199. FuRMANSKi, P. & M. LuBiN. 1972. Effects of dimethylsulfoxide on expression of differentiated functions in mouse neuroblastoma. J. Nat. Cancer Inst. 48: 1355-1360. on connective tissue. In Dimethylsulfoxide. Basic Gries, G. 197 Some effects of Concepts of DMSO. Vol. 1. S. W. Jacob, E. E. Rosenbaum «& D. C. Wood, Eds.: 325335. Marcel Dekker. New York, N.Y. Jacob, S. W., M. Bischel «fe R. J. Herschler. 1964. Dimethylsulfoxide effects on the permeability of biologic membranes. Current Therap. Res. 6: 193-198. TovEL, D. R. & J. S. Colter. 1967. Observations on the assay of infectious viral ribonucleic acid. Effects of and DEAE dextran. Virology 32: 84-92. Hatanaka, M. & H. Hanafusa. 1970. Analysis of a functional change in membrane in the process of cell transformation by Rous sarcoma virus; alteration in the characteristics of sugar transport. Virology 41 647-652. Schulte Holthausen, H., S. Chacko, E. A. Davidson & H. Holtzer. 1969. Effect of 5-bromodeoxyuridine expression of cultured chondrocytes grown in vitro. Proc. Nat. Acad. Sci. U.S. 63: 864-870. Bischoff, R. 1971. Acid mucopolysaccharide synthesis by chick amnion cell cultures. Exp. Cell Res. 66: 224-236. Preisler, H. D., D. Housman, W. Scher & C. Friend. 1973. Effects of 5-bromo-2'deoxyuridine on production of globin messenger in dimethyl sulfoxide-stimulated Friend leukemia cells. Proc. Nat. Acad. Sci. U.S. 70: 2956-2959. Markert, C. L. 1968. Neoplasia, a disease of cell differentiation. Cancer Res. 28: 19081

DMSO

.

DMSO

29.

:

30.

31.

32.

RNA

33.

1914. 34.

Pierce, G. B. 1970. Differentiation of normal and malignant 1248-1254.

35.

Bendich, a.,

Borenfreund

E.

&

E.

cells.

Federation Proc. 29:

H. Stonehill. 1973. Protection of adult mice

tumor challenge by immunization with irradiated adult skin or embryo cells. J. Immunol. 111:284-285. Kelly, F. & J. Sambrook. 1973. Differential effect of cytochalasin B on normal and transformed mouse cells. Nature New Biol. 242: 217-219. against

36.

37.

O'Neill, F.

38.

cells. J. Nat. Cancer Inst. 52: 653-657. KiscH, A. L., R. O. Kelley, H. Crissman & L. Patton. 1973. Dimethyl sulfoxideinduced reversion of several features of polyoma transformed baby hamster kidney

B-treated

cells

J.

1974. Limitation of nuclear division by protease inhibitors in cytochalasin

tumor

(BHK-21).

J. Cell. Biol.

57: 38-53.

STUDIES ON THE INTRACISTERNAL A-TYPE PARTICLES IN MOUSE PLASMA CELL TUMORS: INDUCTION OF MATURATION OF THE PARTICLES S. E. Stewart, G. Kasnic, Jr., C. Urbanski,

M. Myers, and T.

Sreevalsan

Departments of Pathology and Microbiology Schools of Medicine and Dentistry Georgetown University Washington, D.C. 20007

Introduction

Two types of "virus-like" particles designated as A-type particles have been described in mouse tissue. One is the intracytoplasmic A particle that is generally associated with the mouse mammary tumor virus, and is in no way associated with any intracellular organelle. This particle is believed to be the precursor of the B-type virus (the

mammary tumor

virus).

This A-type particle

is

believed to develop into

the B-type virus as the particle migrates from the cytoplasm to the plasma membrane of the cell from which it buds. This immature B particle, which acquires

an additional membrane from the cell, also acquires an electron-dense nucleoid and becomes a mature B particle. ^-^ The other A-type particle is the one observed in the cisternae of the endoplasmic reticulum of many mouse tumors.^'* Both A-type particles measure 70-90 nm in diameter and consist of two concentric shells that surround an electron-lucent core. Biological activity has not been demonstrated in either A-type particle. This may be because at this stage they are incomplete or immature. Maturation or formation of a complete virus with an electron-dense nucleoid has not been described for the in-

A particle. Kuff and colleagues^"^ have carried out extensive biochemical studies with the intracisternal A-type particles from plasma cell tumors and neuroblastoma cell lines of the mouse. They were successful in demonstrating the presence of a particle-specific antigen, but found no high-molecular-weight nucleic acid of the type found in oncogenic viruses, although they reported that isolated particles contained about 5% (by specific assay) and showed that at least some of this was due to contamination with microsomal RNAs. They reported finding a major structural protein component with a molecular weight of about 73,000 daltons, which is antigenically unrelated to the oncogenic C-type viruses. Recently Yang and WiveP have reported that by means of a more sensitive technique (namely, labeling with high-specific-activity radioactive ribonucleosides), heavy-molecular-weight can be detected, even though it is present in small quantities and the particles do not exhibit an internal spherical component. In our studies with human tumor cell lines we have reported the activation by 5iododeoxy uridine (IDU) of an intracisternal virus^ that in the immature form resembles the intracisternal A-type particle of the mouse. We later demonstrated the maturation of many of these particles to particles with electron-dense nucleoids by further exposing the culture to dimethyl sulfoxide (DMSO).'°-'' In the present study we wish to report on the effects of IDU-DMSO treatment of the tissue-cultured cell lines of mouse plasma cells that carry intracisternal particles. tracisternal

RNA RNA

RNA

RNA

172

Stewart

e/ a/.:

Intracisternal

A-Type

173

Particles

Materials and Methods Chemicals and Isotopes. DMSO and unlabeled deoxynucleotides were obtained from Sigma Chemical Co., St. Louis, Missouri. Synthetic templates, poly(rA)-(dT) and poly(dA)-(dT), were purchased from Collaborative Research Inc., Waltham, Massachusetts. '2~^* Tritium-labeled uridine, (119 mCi/mg), adenosine (64 mCi/ mg), guanosine (44 mCi/mg), and cytidine (24 mCi/mg) were purchased from Amersham/Searle Corp., Illinois. Tritiated thymidine 5'-triphosphate (TTP, 46 Ci/ mM) was obtained from Schwarz/Mann Research. IDU, dithiothreitol (DTT), and bovine serum albumin (BSA) were purchased from Calbiochem, California. Cultures. Two established tissue culture cell lines, from plasma cell tumors derived from Balb/C mice, were used. One, designated as #265A, was obtained through the courtesy of Dr. Phillip Periman, George Washington University School of Medicine, Washington, D.C., and the other, designated as LPCl, through the courtesy of Mr. David Trisler, National Cancer Institute, Bethesda, Maryland. Human rhabdomyosarcoma cells were cultivated according to methods described elsewhere.^

The mouse tumor cell lines were maintained in nutrient mixture F-12 with glutamine (Grand Island Biological Co., Grand Island, New York); to which 5% heatinactivated fetal bovine serum, tylocine (60 fig/m\) and kanamycin (100 ^lg/Tn\) were added.

The cells were divided twice weekly, at the time the medium was changed. This was done by transferring one-half of the medium (with the suspended cells) into new flasks, leaving the attached cells, and adding fresh medium to both flasks up to the original volume.

IDU-DMSO

Treatment.

IDU from

a stock solution

was added

to freshly

divided cultures, to give a concentration of 20 Mg/ml. The cultures were then incubated at 37° C for 3 days in the dark, after which the medium was removed and fresh

medium

with

2%

DMSO was added. The medium that contained the IDU was

detached were recovered by centrifugation at 500 rpm for 10 in the medium with and incubated at 37° C in fresh flasks. Two days later the cells were harvested by scraping and centrifugation to form cell pellets. Experiments were also carried out in which cell cultures were incubated with alone for 2 days, without prior treatment with IDU. The pelleted cells were used for the following experiments: (1) electron microscopy; (2) recovery of virus for infectivity tests in vitro; (3) biochemical studies to determine the enzymes present and to check for the presence of heavy-molecularweight RNA. Electron Microscopy. Pelleted cells from both treated and untreated cultures were fixed in 2.5% glutaraldehyde in phosphate buff"er, postfixed in chromeosmium, dehydrated with increasing concentrations of ethanol, and embedded in Epon® without the use of propylene oxide. Sections were cut and stained with uranyl magnesium acetate, then with lead citrate, and were then examined for virus. Infectivity Studies. For in vitro studies, cell debris and extracts from ruptured pelleted cells were inoculated on monolayered cultures of 3T3 mouse cells (obtained from Dr. Peter Fishinger, National Cancer Institute, Bethesda, Maryland). The inoculum was prepared as follows: all procedures were carried out at 0-5° C. Cells were collected by centrifugation and washed twice in Hanks balanced salt solution. The washed cell pellet was resuspended in 9 vols ice-cold buff'er (0.01 Trishydroxymethylaminomethane, 0.002 magnesium chloride, pH 7.8) and allowed to swell for 10 min, then was homogenized with a tight-fitting bounce homogenizer

removed, and

cells that

DMSO

min. The cells were resuspended

DMSO

TM

M

M

K

Annals

174

New York Academy of Sciences

(20 strokes). Cell breakage was determined by light microscopy and by culture. The homogenized sample was centrifuged at 2,500 rpm for 5 min in an International Re-

The resulting supernatant was then cenSpinco centrifuge and the pellet was suspended in phosphate-buffered saline (PBS) and passed through a 25-gauge hypodermic needle to break up the aggregates (5-10 passages). The sample was then diluted in PBS to a final concentration of one-tenth the original volume and 0.1 ml was inoculated on to 24 plates of 3T3 monolayered cells, cultured in plastic dishes 3 inches in diameter. Before inoculation the cultures were treated with DEAE-dextran (15 Mg/ml) for 15 min, then were washed three times in serum-free medium. The inoculated plates were incubated 1 hour, then 3 ml Eagle's minimum essential medium, which contained glutamine, 10% heat-inactivated fetal calf serum, and antibiotics (as above) were added. The cultures were incubated at 37° C in a humidified incubator in a 5% CO2 atmosphere. Uninoculated control 3T3 cultures Were treated in a like manner. All cultures were changed twice weekly. After 2 weeks' incubation, both IDU-DMSO-treated and untreated cultures were examined for virus. Examination was repeated after the third week. XC Tests. The rat cell line that carried the Rous virus genome used for the XC tests was obtained from Dr. Janet Hartley, National Institutes of Health, Bethesda, Maryland. Tests were carried out with both plasma cell tumors, to determine whether sufficient C-type virus was present to give a positive test. The procedure was that described by Rowe and colleagues. ^^ Concentration and Partial Purification of the Particles from the Cells. IDUtreated cultures were labeled with [^H]adenosine, [^H]uridine, [^H]cytidine, and [^H]guanosine (25 /zCi each) at the time of addition of DMSO. An additional 25 ^Ci of each isotope was added the following day. The cells were allowed to incorporate radioactivity for 48 hours. Noninduced cells were similarly labeled. For each experiment labeled cells were removed by scraping from four culture bottles that contained approximately 5.5 x 10' cells each, and were combined with the supernatant fluid. They were pelleted at 350 x g and washed once in cold PBS. The cell pellet was resuspended in 5 ml RSB (0.01 Tris-HCl, pH 7.4, 0.01 KCl, and 0.0015 MgCla) and allowed to swell for 15 min at 4°C. The cell suspension was then passed twice through a 25-gauge needle and centrifuged for 10 min at 800 x g to remove nuclei and debris. The postnuclear fraction was pelleted at 38,000 rpm in a Spinco-type 40 rotor for 1 hour. The 38K pellet was resuspended in 5 ml 0.01 Tris HCl (pH 7.4) and 0.01 sodium ethylenediaminetetraacetate (EDTA), which contained 0.2% Triton^ X-100, and was passed serially through 19-, 22-, and 25gauge needles. The sample was centrifuged at 38,000 rpm for 1 hour and the pellet was resuspended in 0.5 ml Tris-EDTA buffer without Triton X-100. Unlabeled cells were fractionated in an identical manner for use in enzymatic and electron microfrigerated centrifuge to

trifuged at 30,000

rpm

remove the for 30 min

nuclei. in a

M

M

M

M

M

scopic analyses.

Sucrose Density Gradient Analysis. Resuspended

from the second 38 20-60% (w/w) suEDTA. Cenand 0.0001

pellets

centrifugation were immediately layered onto a preformed linear

crose gradient that contained 0.01 trifugation

was

M Tris HCl (pH 7.4)

for 17 hours at 40,000

rpm,

in

a

M

Beckman SW-65

rotor. Fractions

were collected from the top of the gradient and aliquots were analyzed for acidradioactivity and density. The methods used for this have been described elsewhere.'^ For analysis of RNA, peak fractions of radioactivity were pooled and diluted in TES buffer (0.01 NaCl, and 0.001 Tris-HCl, pH 7.4, 0.1 EDTA) that contained 1% sodium dodecylsulfate (SDS). After incubation at 37° C for 15 min, the sample was layered on a linear 15-30% sucrose gradient that contained TES with 0.25% SDS. Gradients were centrifuged at 40,000 rpm in a SWprecipitable

M

M

M

Stewart

et ai:

Intracisternal

A-Type

175

Particles

65 rotor for 2 hours at 23° C. Fractions were collected from the top and analyzed for acid-precipitable radioactivity.

Poly(dT)polymerase activity''' was measured in Tris-HCl buffer (pH 8.3), 2.5 KCl, 0.01 % (v/v) Nonidet® P-40, 19% (v/v) glycerol, MgC^, 250 DTT, 10 350 /ig/ml BSA, 100 Mg/ml poly(rA).(dT),2_,8, 5 /iCi [^H] TTP (46 Ci/mM), and enzyme sample. Reactions were carried out for 60 min at 37° C, and were stopped according to the technique of Wilson and Kuff.*'' DNA polymerase activity was tested in 100 /xl reaction mixtures that contained DTT, 60 Tris-HCl buffer (pH 8.3), 6 NaCl, 0.4 50 MgCU, 20 unlabeled dATP, dCTP, dGTP, 5 ^Ci ['H] TTP (46 Ci/mM), and enzyme sample. 5 Mg oligo(dT) was added to some reaction mixtures. When synthetic template-primers, poly(rA)-(dT)i2_i8 and poly(dA)-(dT)i2_i8, were used, they were added at a concentration of 5 /xg per reaction, and unlabeled nucleoside triphosphates were excluded from the reaction mixture. Incubation was carried out at 37° C for 30 min, and reactions were stopped by transfer of the tubes to an ice bucket and addition of 100 /ig BSA and 3 ml 10% TCA solution that contained 0.25 sodium pyrophosphate. After 15 min at 0°C, acidinsoluble material was collected on Millipore filters and washed with 30 ml cold 5% TCA. Filters were dried and the radioactivity was determined by methods described

Assay for Enzymatic

100

/il

Activity.

reaction mixtures that contained 50

mM

mM

mM

mM

mM mM

mM

mM

mM

M

elsewhere.'^

Results Electron Microscopic Studies. Figure control cultures. Only

immature

1

shows the virus as seen

in

untreated

intracisternal A-type particles, as described by

other investigators along with a rare C-type virus, were found in the cells examined. In our hands the intracisternal A particles were approximately 90 nm in diameter.

Figure 2 shows

intracisternal A-type particles in a culture treated with

FiGURE 1. Cells from an untreated culture of murine plasma A-type particles are present in most cells, x 5,040.

cisternal

cell

tumor.

Many

IDU-

intra-

Annals

176

New York Academy of Sciences

^^

-^^

;-

^m,,^Fr

Figure 2. Immature intracisternal A-type particles from IDU-DMSO-treated murine plasma cell tumor cultures. Arrows indicate budding particles, x 52,000. Inset: note fuzz on the surface of the particles and the electron-dense shell adjacent to the outer membrane, x 91,000.

•

Figure treatment.

3.

Intracisternal particles in a murine

The

particle at 2

may

be

in

plasma

cell in

culture after

an immature particle, while the particle at 3 transition from immaturity to maturity, x 76,000.

particle at

1

is

is

IDU-DMSO mature. The

Stewart

et al.

:

Intracisternal

A-Type

Particles

177

Mature intracisternal particles from murine plasma cell culture after Note the thin electron-lucent halo around the membrane of the nucleoid, and the dense area between this and the outer membrane. In Figure 4 the particle shows a break in its outer membrane, and the nucleoid appears to be retracted from its covering membrane. In Figure 5 the particle at the arrow has apparently been sectioned almost tangentially to the outer surface of the nucleoid, and the outer membranes of both particles are not as distinct as in Figure 4. x 12,000. Figures 4 and

IDU-DMSO

5.

activation.

1

particle was surrounded by a double membrane approximately 5 nm whose outer surface generally showed a fuzzy material. Closely applied to the inner aspect of this membrane was an electron-dense shell approximately 5 nm thick. A moderately electron-dense area separated this outer dense shell from an in-

DMSO. The

wide,

nm

which surrounded an electron-lucent and also after treatment with DMSO alone, the structure of the immature particles was not altered, but particles with an electron-dense nucleoid were now found within the cisternae of the endoplasmic reticulum, largely in cells that exhibited moderate to advanced degeneration. The stages of maturation are shown in Figure 3. This presumably mature particle was approximately 90 nm in diameter, and was surrounded by an outer membrane 5 nm thick. The electron-dense nucleoid was also contained within a double membrane of like dimension. Between these two membranes two separate zones were seen. An electron-lucent area of variable dimension surrounded the nucleoid and its membrane, and an electron-dense zone was seen between this and the outer membrane (Figures 4 and 5). Only about 5% of the intracisternal virus particles dener dense shell, approximately 5 nucleoid. After

thick,

IDU-DMSO activation,

veloped electron-dense nucleoids. Comparison of the intracisternal A particle in the plasma cell tumor with that found in human tumor cell lines after IDU-DMSO activation, by identical methods of preparation and microscopy, showed three distinct differences in structure

(Figures 6 and

tumor

7): (1)

intracisternal

there appeared to be

A

little

or no surface fuzz on the

particles; (2) the electron-dense shell

human

beneath the outer

membrane present in the mouse A-type particle was absent in the human tumor Atype particle; and (3) there was a difference in the size of the two particles. The average total diameter of the mouse intracisternal A particle was 90 nm, whereas it was 100 nm in the human particle. The mature particles found in the plasma cell tumors after IDU-DMSO treatment was also from 10 to 15 nm smaller in diameter than the mature forms in the treated human tumors. Also, in this study we have not

New York Academy

Annals

178

of Sciences

Figure

6.

Immature

intracisternal

A-

in

an

IDU-

DMSO-treated human tumor

cell

culture

type

particles

as

found

(rhabdomyosarcoma). Note the absence of a dense shell adjacent to the outer

membrane, x 140,000. Compare with Figure 2.

the

inset in

in the mouse A particles that stage of maturation that was occasionally seen in human A-type particles, in which the entire area between the nucleoid and the outer membrane (intermediate zone) became electron-lucent and the total diameter of the particle increased to as much as 125 nm through an expansion of the inter-

seen

the

mediate zone. Differences were also found in the antigenicity of the

human and mouse

in-

tracisternal particles. Kuff '^ has tested a Triton X-100-resistant particulate fraction

from IDU-DMSO-treated tissue culture cells from one of our human tumor cell would be expected to contain A-particle antigen if the human particle acted like a mouse particle), and has not found reactivity in complement

lines (a fraction that

made against the mouse antigen. The XC test was negative for cultures from both plasma

fixation tests with antiserum

Results on the cell tumors.

XC test.

Figure 7. Mature intracisternal particle in IDU-DMSO-treated human tumor cell culture (rhabdomyosarcoma), x 140,000. Compare with Figures 4 and

5.

Stewart

et al.:

Intracisternal

A-Type

Particles

179

%:

A-type particles in a pellet obtained from the second 38K centrifugation. The arrow may represent a transitional stage in maturation. (Compare with particle Figure 3.) x 140,000.

Figure

8.

particle at the

2

in

Results on the

in

Vitro Infectivity Tests. Examination of the control 3T3 cells by

electron microscopy (4 hours of searching) failed to reveal viruses or viruslike particles in the cell line used. This was also true of the cultures examined 2 and 3 weeks

from the disrupted plasma treatment. After IDU-DMSO

after inoculation with the concentrated supernatant fluid cells

that

had been subjected to

IDU-DMSO

treatment, however, the intracisternal A-type particle was found in both the control and the inoculated cultures. These particles thus appear to be latent in this 3T3 cell line, and to be susceptible to activation by IDU-DMSO treatment.

p-r

1^ M

Figure 000.

9.

Mature A-type

particle (arrow); seen in the

same

pellet as in

Figure

8.

x 140,

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New York Academy of Sciences

Results on Isolation of Particles and Analysis ofRNA and Enzymes Associated with the Purified Particles. The efficacy of the fractionation procedure for the purification of particles from induced cells was checked by examining the pellet from the second centrifugation at 38K. Figures 8 and 9 indicate that the structures that sediment at 38K consist of a relatively homogeneous mixture of particles, which showed little contamination with cellular debris (organelles and so on). Particles that contained electron-dense and electron-lucent cores were both seen; the latter were the predominant species. The results on the biochemical characterization of the particles in both the IDUDMSO-treated cells and the control cells, determined by the procedure described in Materials and Methods, were as follows: the sedimentation patterns on sucrose density gradients of typical preparations from IDU-DMSO treated and from untreated control cells are shown in Figures 10 and 11 respectively. With the activated cells, the sucrose density profile (Figure 10) of the labeled material was found in a fairly homogenous peak in the 1.2 density region. The sedimentation pattern of

the sucrose gradient of a typical preparation from the controls 11. In

is

shown

in

Figure

the unactivated control cells the majority of radioactivity sedimented at a

density of 1.185. tivated cells

The

from acwas examined. Peak fractions from

nucleic acid present in the particles that were isolated

and sedimented

at a density of 1.2

20-60% sucrose gradient were analyzed Materials and Methods. From the gradient

the

for

RNA

content, as described in

Figure 12, it can species in these preparations sedimented at a rate of be seen that the major about 2 IS. A minor peak was seen in the high-molecular-weight region of the gradient, with a sedimentation rate of about 64S. profile presented in

RNA

Figure

Sucrose density gradient centrifu-

10.

gation of radiolabeled particles from activated

plasma

cells.

The

pellet

obtained after the second

cycle of centrifugation of the Triton X-treated

material (38,000 in

Tris-EDTA

60%

linear

rpm

buffer

sucrose

for 60 min) was suspended and then layered on a 20-

gradient

in

Tris-EDTA

was for 16 hours at 40,000 Fractions were collected and the

buffer. Centrifugation

rpm,

at 4''C.

acid-insoluble

radioactivity

contained

in

each

was determined by methods similar those described elsewhere.'^ The method used fraction

to to

determine the density of the fractions has been Fractions

described elsewhere.

Stewart

et al.

:

Intracisternal

A-Type

5

Particles

181

12

13

12

\

4

Figure

11.

centrifugation

Sucrose density gradient of radiolabeled

from control plasma

cells.

determined as described

Figure

300

200

-

particles

The data were

in

10

20

30

40

Top

the legend to

10.

Bottom Fractions

w

Figure 12. Analysis of the labelled nucleic from partially purified particles isolated from activated plasma cells. Particles that acid

contained ^H-labeled adenosine, uridine, cytosine

100

l\

and guanosine, and sedimented at a density of 1.20 (Figure 10), were used. Centrifugation was performed on a 15-30% sucrose density gradient

70S 1

in

vw

50

30

Top

40

Bottom Fractions

TES

buffer that contained

0.25% SDS,

at 40,-

000 rpm and 23° C for 2 hours. Fractions were collected and the acid-insoluble radioactivity present was determined. The arrows represent the position at which labeled RNA from

v.

[^H]uridine-labeled

sindabis

virus

or

avian

myoblastosis virus sediments under similar conditions of centrifugation.

Annals

182

New York Academy of Sciences

Figure 13. Analysis of the labelled nucleic from partially purified particles isolated from control plasma cells. Particles that acid

contained

^H-labeled

adenosine,

uridine,

cy-

and guanosine, and sedimented at a density of 1.185 (Figure 11), were used. Centrifugation was performed on a 15-30% sucrose tosine,

density gradient in

TES

buffer that contained

0.25% SDS, at 40,000 rpm and 23° C for 2 hours. Fractions were collected and the acid-insoluble radioactivity present was determined. The arrows represent the position at which labeled RNA from [^HJuridine-labeled sindabis or avian myoblastosis virus sediments under similar conditions of centrifugation.

Fractions

The nature of the nucleic acid present in the particles that were isolated from control cells and sedimented at a density of 1.185, analyzed as above, is shown in Figure

be noted that the bulk of radioactivity sedimented in a broad peak A minor peak was also detected at about 60S. Preliminary Results on Polymerase Activity Associated with the Virus Particles. Samples of unlabeled activated cells were tested for enzymatic activity at various stages of purification. These included a crude cytoplasmic extract, the resuspended pellet from the second 38 K centrifugation, and the sucrose density gradient fractions in the 1.2 density region. Enzymatic analysis included tests for the poly(dT)synthesizing enzyme reported by Wilson and Kuff,''' polymerase without added template, and polymerase with added template primers. Both poly(rA)-oligo(dT) and poly(dA)-oligo(dT) were used. The endogenous reaction in

13. It will

the gradient, at a rate of about 26S.

DNA

DNA

was also tested with addition of oligo(dT). Preliminary experiments indicated that no endogenous or exogenous [stimulated by synthetic templates such as poly(dA)'(dT)i2 or poly(rA)-(dT),2_i8] DNA polymerase activity was associated with the particles isolated from the control or activated cells. It was also observed that the particles from the control and activated cells possessed very low levels of poly(dT)polymerase activity (as measured by the method of Wilson and Kuff '^).

Discussion There has been considerable speculation on the nature and the role of the inmouse tissues. It has not been proved whether they are true virus entities that fail to mature under natural conditions. This same tracisternal A-type particles found in

Stewart

et al.\

Intracisternal

A-Type

Particles

183

type of particle is very widespread in various species of animals. Intracisternal Atype particles, with an occasional mature form, have been described in leukemic cells from guinea pigs^^ '^ and in cat mammary tumors,'** and they are believed by some investigators to be the etiological agent in guinea pig leukemias.'^ Since we

have been in the

able,

by

in vitro

mouse plasma

particle

may

cell

play a role

in

procedures, to demonstrate a morphological maturation it is possible that under certain conditions this oncogenesis.

tumors,

be difficult to demonstrate that this particle is infective, since many mouse cells carry the virus. The 3T3 cells used in our experiments have a latent A-type intracisternal particle, which becomes apparent on activation with IDU-DMSO. IDU It will

not only activates latent C-type particles, but, as we have shown in earlier work with cell lines, it is also possible to activate intracisternal particles with this

human tumor agent. ^~''

It is

therefore not surprising that the intracisternal A-type particle of the

mouse can be similarly activated. The failure to demonstrate the presence of virus in unactivated 3T3 cells inoculated with preparations from the murine intracisternal A-type particle

may

be interpreted

in

two ways:

either the cells are not susceptible to

superinfection, or the virus prepared by the procedure described lost It is

its infectivity.

necessary to carry out further tests before any conclusions can be drawn on

its

biological activity.

Although we have been able to demonstrate by in vitro procedures that there is a morphological maturation of the intracisternal A-type particles in mouse plasma tumor cells, no distinct new peaks of virus-specific particles were seen in sucrose density gradients. It is possible that the difference in density observed in control cells (1.185) and induced cells (1.2) reflects the difficulty of separating mature and immature particles. Analysis of the present in the particles in induced cells does show that the ratio of heavy-molecular-weight to the predominant species (2 IS) is much greater in induced cells than in control cells. The inability to find detectable levels of enzyme activity in the particle preparations may be a result of too low a concentration of particles.

RNA

RNA

Summary Maturation of the intracisternal A-type particle found in two mouse plasma cell tumors was induced by treating the cells in culture with IDU-DMSO or with DMSO only. Morphologically, the mature particles with electron-dense nucleoids closely resembled the mature particles described in human tumor cell lines treated in a like manner. They also closely resembled the virus that has been described in guinea pig leukemias. It was not possible to demonstrate infectivity of the mature particle, as latent intracisternal A-type particles induced by IDU were found in the mouse cells presumed to be free of virus.

The biochemical

show distinct new peaks of virus-specific partiwhen the particles in the treated cells were comof the untreated cells. There was a difference in the density

studies did not

cles in sucrose density gradients

pared with the particles of the particles observed in the induced cells (1.2) and those of the control cells (1.185). This may reflect the difficulty of separating mature and immature particles. Analysis of the present in the particles showed that the ratio of heavymolecular-weight in activated cells to the predominant species (2 IS) is much greater than that in control cells. Detectable levels of enzyme activity were not found in the induced particles. This could be due to too low a concentration of parti-

RNA RNA

cles in the preparations.

.

Annals

184

New York Academy of Sciences References

1

2. 3.

4. 5.

6.

Imai, T., H. Okano, a. Matsumoto & A. HoRis. 1966. Cancer Res. 26: 443-453. Smith, G. H. 1967. Cancer Res. 27: 2179-2196. Dalton, A. J., M.Potter & R. M. Merwin. 1961. J. Nat. Cancer Inst. 26: 1227-1267. Dalton, A. J. & M. Potter. 1968. J. Nat. Cancer Inst. 40: 1375-1385. KUFF, E., N. WiVEL & K. LuEDERS. 1968. Cancer Res. 28: 2137-2143. Klff, E., K. Lleders, H. Ozer & N. Wivel. 1972. Proc. Nat. Acad. Sci. U. S. 69: 218-

223. 7.

8. 9.

10. 11.

12. 13.

14. 15. 16.

Wivel, N., K. Lleders & E. Kuff. 1973. J. Virol. 11: 329-334. Yang, S. & N. Wivel. 1973. J. Virol. 11: 287-298. Stewart, S., G. Kasnic & C. Draycott. 1972. J. Nat. Cancer Inst. 48: 273-277. Stewart, S.,G. Kasnic & C. Draycett. 1972. Science 175: 198-199. Stewart, S., G. Kasnic & C. Draycott. 1972. In Perspectives in Virology— Persistent Infections. Vol. 8. M. Pollard, Ed. Academic Press Inc. New York, N.Y. RowE, W., W. PuGH & J. Hartley. 1970. Virology 42: 136-1 139. RosEMOND, H. & T. Sreevalsan. 1973. J. Virol. 11: 399-416. Wilson, S. & E. Kuff. 1972. Proc. Nat. Acad. Sci. U. S. 69: 1531-1536. Kuff, E. Personal communication. Nadel, E., W. Banfield, S. Burstein & A. J. Tousimis. 1967. J. Nat. Cancer Inst. 38: 1

979-981. 17. 18.

Feldman, D. G. Feldman, D. G.

& &

L. L.

Gross. 1970. Cancer Res. 30: 2702-271 1. Gross. 1971. Cancer Res. 31: 1261-1267.

EFFECT OF DIMETHYL SULFOXIDE ON THE HEPATIC DISPOSITION OF CHEMICAL CARCINOGENS* Walter G. Levine

Department of Pharmacology Albert Einstein College of Medicine Yeshiva University Bronx, New York 10461

Introduction

I The present study on the

influence of dimethyl sulfoxide

(DMSO) on

the hepatic

binding of chemical carcinogens arose from a series of investigations that we have carried out on the relationship between the metabolism and the biliary excretion of ^""^

compounds, including a number of polycyclic hydrocarbon carcinogens. has been demonstrated that these carcinogens, for example, 3,4-benzpyrene (BP), 3-methylcholanthrene (MC), and 7,12-dimethylbenzanthracene (DMBA), are converted by the liver to a large group of metabolites such as epoxides, dihydrodiols, phenols, ketones, and quinones.^"'^ Conjugated derivatives of these metabolites are highly polar and have molecular weights in excess of 350; these characteristics are usually associated with appreciable biliary excretion in the rat.'^ It is not surprising, therefore, that the bile is the principle route of excretion for these sub^""'^^"^^ Studies in our laboratory have shown that after they are stances. intravenously injected into the rat, these carcinogens are taken up very rapidly by the liver and are bound to various particulate fractions within the cell."^^ They are subsequently metabolized by the microsomal drug- metabolizing enzymes and pass foreign It

is followed by rapid biliary excretion. Pretreatment with agents such as phenobarbital, MC, and BP, which induce the microsomal enzymes, accelerates biliary excretion, while pretreatment with agents such as SKF-525A, piperonyl butoxide, emetine, and metyrapone, which inhibit these enzymes, retards biliary excretion. '~'*'2^'2'' These results suggest that metabolism may be the rate-limiting step in the overall biliary excretion mechanism for these

into the cytosol ("soluble") fraction; this

carcinogens. In view of the poor solubility of these hydrocarbons, it had been our custom to administer them as suspensions in 1% albumin, to provide as "physiological" an en-

DMSO

vironment as possible. has been used extensively in many laboratories, however, to incorporate highly insoluble compounds such as steroids and polycyclic hydrocarbons into biological systems. We therefore measured the biliary excretion of metabolites after the carcinogen had been injected as a solution in DMSO. Much to our surprise, the rate of biliary excretion greatly exceeded that seen after the injection of as a suspension in 1% albumin. The present study was undertaken to investigate this phenomenon more thoroughly and to shed light on its mechanism.

MC

MC

*This work was supported by grant National Institutes of Health.

CA- 14231 from

185

the National Cancer Institute of the

Annals

186

New York Academy of Sciences

Biliary Excretion

Experiments

Female Wistar rats that weighed approximately 200 g were anesthetized with urethane (1 g/kg), and their bile ducts were cannulated. pHjMC, as a suspension in solution, was injected intravenously (2.5 mg/kg) and bile 1 % albumin or as a samples were collected every 15 min for 90 min. Since only metabolites appear in the bile,^ samples were counted to determine their biliary excretion rates. The results (Figure 1) show that metabolite excretion was more rapid after the injection of a solution of [^H]MC than after the injection of a 1% albumin suspension of alone was injected prior to the 1% albumin suspension of MC. When 0.2 ml MC, no change in the rate of excretion of metabolites was evident. This ruled out the possibility that any alteration of the excretory mechanism was due to an independent effect of DMSO. In a parallel experiment, [^H]BP was injected into rats as a solution or as an albumin suspension and the biliary excretion rates of [^H]BP metabolites were determined. The results (Figure 2) are similar to those obtained for [^H]MC, although the difference between the two vehicles was less dramatic.

DMSO

DMSO

DMSO

DMSO

Experiments on Hepatic Binding

We next determined the hepatic subcellular distribution of [^H]MC after its intravenous injection into rats as an albumin suspension or as a solution. The carcinogen was injected in a dose of 2.5 mg/kg. The livers were removed 5 min

DMSO

3-METHYLCHOLANTHRENE

Figure

1.

BiUary excretion of

metabolites of i.v.

injection

DMSO ]% Albumin

DMSO solution (o) or as a suspen-

1%

of experiments, 0.2 ml

sion in

Albumin

preceded by

DMSO

1% albumin

30

45

60

75

MINUTES AFTER INJECTION

90

(o). In

one

set

DMSO was

min before the albumin suspension of [^H]MC (•). The ordinate represent the total rainjected 5

dioactivity 15

[^HJMC after the [^H]MC as a

of

recovered

in

each 15-

sample. Each point is the mean for 4 or more animals. Standard errors are indicated.

min

bile

Levine: Hepatic Disposition of Chemical Carcinogens

187

3,4-BENZPYRENE

Figure

Biliary

2.

[^H]BP

tabolites of

of [^H]BP as a

excretion

after the

i.v.

of

me-

injection

DMSO solution (•) or as a

suspension in 1% albumin (o). The ordinate represents the total radioactivity recovered in each 15-min bile sample.

Each point

is

the

mean

for

4 or more ani-

15

and homogenized

45

30

60

75

90

MINUTES AFTER INJECTION

mals. Standard errors are indicated.

M

sucrose; these were centrifuged at 600 x and 100,000 x g for 60 min respectively. The 600 X g and 10,000 x g fractions were washed twice and the 100,000 x g fraction once with sucrose. The final pellets were suspended in sucrose and samples of the particulate and cytosol (100,000 x g supernatant) fractions were counted in a scintillation spectrometer. As we have reported elsewhere,"" 5 min after injection most of the later

g

for 10 min, 10,000 x

cold 0.25

in 3 vols

g

for 10 min,

radioactivity within the liver

is

bound

to particulate fractions.

The small amount of [^H]MC. The salient

activity within the cytosol principally represents metabolites of

point here lies in the difference in the binding of radioactivity to the microsomes (100,000 X g fraction), the site of metabolism of foreign compounds. The binding after injection of [^HjMC in is nearly twice that seen after injection of

DMSO

[^H]MC

albumin (Table 1). This may possibly explain the more rapid rate of metabolism and hence the more rapid rate of biliary excretion seen with DMSO soluin

tions of the carcinogen.

The greater

affinity

of

DMSO solutions of [^H]MC

for the

microsomal fraction is even more apparent from in vitro experiments in which the carcinogen was added directly to a liver homogenate, which was subsequently fractionated by differential centrifugation. The addition of [^H]MC to homogenate was carried out at 5°C to minimize metabolism. Therefore these results probably reflect most accurately the relative affinity of [^H]MC for the liver fractions under the conditions of this experiment. Here the binding to microsomes is seven times that seen when an albumin suspension is used (Table 1). The hepatic subcellular binding of [^H]BP was determined in a similar manner. It is evident from the results (Table 2) that [^H]BP, when added to liver homogenate as a DMSO solution, has a far greater affinity for microsomes than it does when added as an albumin suspension. In view of the reported effect of DMSO on membranes, ^^-^^ the possibility was considered that the apparent differences in binding to microsomes might actually reflect altered sedimentation characteristics of hepatic organelles, due to the

DMSO

presence of in the liver. We found, however, that the amount of protein recovered in each fraction was not influenced by the addition of to the homogenate prior to centrifugation (Table 3). Therefore the sedimentation charac-

DMSO

Annals

188

New York Academy of Sciences Table

1

Hepatic Intracellular Distribution of Radioactivity after the Injection of [^H]MC in vivo or its Addition in vitro to Liver Homogenates Conditions of Measurement

Vehicle

DMSO

5

1% albumin

5

DMSO 1% albumin

Percentage of Total Liver Radioactivity 10,000 x g 100,000 x g Cytosol

600 x ^

after injection

18

46

22

after injection

24

61

12

1.2

in vitro

17

51

4.7

in vitro

30

23 53

min min

Table Effect of

8

7.5

0.5

2

DMSO

on the Hepatic Intracellular Distribution OF [^H]BP Added to Liver Homogenates in Homogenate 1% Albumin

Percentage of Total Radioactivity Fraction

DMSO

xg

18

59

10,000 X g 100,000 xg

24 25

18

Cytosol

13

3

600

Table Effect of

6

3

DMSO

on Distribution of Protein During Differential Centrifugation of Liver Homogenates

Fraction

with

Total Protein Recovered without

DMSO

DMSO

xg

131

121

10,000 X g 100,000 X g

101

96

83

81

Cytosol

161

162

600

Table 4 14, Binding of [''*C]DMS0 to Subcellular Fractions after Addition to Liver Homogenates in vitro

Fraction

Percentage of Total Radioactivity Added

xg

2.6

10,000 X g 100,000 X g

0.4

600

Cytosol

its

3.6

80

I

Levine: Hepatic Disposition of Chemical Carcinogens

Table

189

5

DMSO

on the Hepatic Intracellular Distribution of [^H]MC Following Injection of the Carcinogen as an Albumin Suspension or its Addition to Liver Homogenates in vitro Effect of

Percentage of[ ^H]MC Added to Horn Dgenates in vitro with with out

Percentage Df Injected Dose (

Fraction

of[

with

DMSO

600xg

18

10,000 X g 100,000 xg

45

Cytosol

teristics

^H]MC without

DMSO

DMSO

29 52

15

.

DMSO

38

32 54

8.4

7.7

4.7

4.3

0.9

0.7

0.6

0.5

of hepatic organelles seem not to be altered by in binding to particulate fractions is valid.

DMSO,

and the observation

of differences

DMSO

itself might bind to microsomes, and The next consideration was that thereby enhance the binding of carcinogen. To test this possibility a study of hepatic binding in vitro was carried out in the usual manner, except that a solution of unla-

MC in

[''^CJDMSO was added to the homogenate. After differential centrifuwas determined in each fraction. The results (Table 4) indicate that almost all the added radioactivity was recovered in the cytosol, and there was almost no binding to the microsomes. Obviously this could not explain the enhanced binding of MC to microsomes in the presence of DMSO. It was found that altered binding of MC to liver fractions occurred only when the carcinogen was injected in vivo or was added in vitro as a DMSO solution. In these experiments 0.2 ml DMSO was injected, and was followed 5 min later by [^H]MC (2.5 mg/kg) in albumin. Alternatively, DMSO was added directly to a liver homogenate from an untreated animal, and was followed in 5 min by 300 ^g [^HjMC in albumin. The results (Table 5) indicate that prior injection or addition of beled

gation, radioactivity

DMSO did not affect the binding pattern of [^H]MC in albumin, in vivo or in vitro. In another series of experiments, 600 x g, 10,000 x g, and 100,000 xg fractions were prepared from the livers of untreated animals. To each of these fractions 50 /ig [^H]MC was added in the cold, as either a solution or an albumin suspension. The fractions were then centrifuged and washed twice with cold 0.25 sucrose, and the washings from each fraction were pooled. The radioactivity was then determined for the resuspended fractions and for the washings. From the results shown in Table 6, it is apparent that when added as an albumin suspension, [^H]MC binds tightly to all particulate fractions and only a small proportion of the radioactivity can be washed out. In contrast, when [^H]MC was added as a DMSO solution, approximately 50% of the radioactivity appeared in the washings of the 600 x g and 10,000 x g fractions. Affinity for the 100,000 xg (microsomal) fraction appeared to be nearly equal to that of [^H]MC in albumin, since very little ra-

DMSO

M

Table 6 Effect of

DMSO

on the Binding of [^H]MC to Liver Fractions Percentage of Total Radioactivity Recovered

Fraction

600 x ^ 10,000

xg

100,000 X g

DMSO

DMSO

1% Albumin

1% Albumin

Pellet

Washings

Pellet

Washings

46 92

8

96 97 98

4

51

54 49

3

2

Annals

190

New York Academy of Sciences Table

Effect of

DMSO

7

on the Binding of

[

H]BP to Liver Fractions

Percentage of Total Radioactfvity Recovered

DMSO

DMSO

1% Albumin

1% Albumin

Pellet

Washings

Pellet

Washings

xg

53

10,000 X g 100,000 X g

49 76

25 29

Fraction

600

was released by washing. Thus DMSO markedly diminished the affinity for the 600 x g and 10,000 x g fractions, while allowing for tight binding to the microsomal fraction. In a parallel set of experiments, [^H]BP was added to prepared liver fractions as a DMSO solution or as a suspension in 1% albumin. Here too we found that

dioactivity

of

[^H]MC

carcinogen in albumin binds tightly to all three particulate fractions, while carcinogen in has considerably less affinity for the 600 xg and 10,000 x g fractions than for the 100,000 x g (microsomal) fraction (Table 7). From these results it has been inferred that after injection or addition in vitro of solution of carcinogen, binding to microsomes occurs more readily in view a of the diminished binding to other cellular organelles. Consequently, the carcinogen is metabolized and excreted in the bile more rapidly than when it is administered as an albumin suspension. From the foregoing results it appears that is effective in altering the binding of carcinogen only when the carcinogen is dissolved in this vehicle. This suggested that a DMSO-carcinogen complex may form that has binding characteristics different from those of the carcinogen itself. To investigate this possibility, samples of BP in benzene and in were spotted on silica gel thin-layer plates and were chromatographed with benzene as solvent. The chromatograms were viewed under uv light; the results are illustrated in Figure 3. BP spotted from a benzene solution

DMSO

DMSO

DMSO

DMSO

e

I2J

SOLVENT FRONT

3. Thin-layer chromatogram viewed under uv light. On the left BP was spotted from a benzene solution and appeared as a fluorescent spot at the

Figure

of

BP

solvent front (A). In the middle,

spotted from a

DMSO

BP was

solution and ap-

peared as two fluorescent spots, one at the solvent front (A) and a second

more

intensely fluorescent spot that trailed the first (B).

BP

was

DMSO BP

BENZENE

SPOT

B

BENZENE

On

eluted

the right

from

is

the

the spot B that chromatogram,

redissolved in benzene, and rechromatographed. The silica gel thin-layer plates were obtained from Brinkman Instruments.

•

Levine: Hepatic Disposition of Chemical Carcinogens

191

«

'

c -o •-

2 u

j=

H —g ^OQ o >^ a. c/;

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spc .

CNI

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iphen ution:

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sol

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—

front.

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M~ cr

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DMSO yl)-benzene-2,5-d

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\ i

z Ui

corresponding

s s •^

oc.

t

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>z

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hen

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solvent

or

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at

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phenyloxazo

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->

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^^ 1

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CNI

5

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['H co

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of

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A

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and

radioactivit

benzene

strips

6

the

mm

"J

5

i

,

matogram

u

red

a "^

solut

O 2

Z

most

n

^^ T3

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iiat

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10

nd

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0- "3 := oa

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radio

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reared

ither

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29

fidative phosphorylation.^' One known uncoupling and anti-inflammator> agent is DNP; however, it failed to show any effect in these studies. The concept of pretreatment. originally suggested by Mottram^-^" and refuted by Berenblum and Shubik.-"^ proposed that it might be possible to render the tissues more susceptible to the action of the initiating agent. Mottram's views were later supported by others. ^''"^- These studies, however, failed to show that pretreatment had an effect on skin tumor induction with with DNP or and croton studies,

is

a strongly

DMBA

DMSO

oil.

CPZ on skin tuand promotion, as such an effect has been recorded in other systems. ^^~''* Fujita^^ observed the inhibitory effect of CPZ on the development of hepatic tumors in rats fed 4-dimethylaminoazobenzene, and commented that CPZ might delay "transition from the precancerous to the cancerous stages." Hoshi and colleagues'*^ report that various psychotropic drugs, including CPZ. had synergistic effects with cyclophosphamide on sarcoma 18-ascites in rats. It has been suggested that the stabilization of the lysosomal and other Efforts were also

morigenesis. both

made

to study the possible inhibitor, effect of

in initiation

membranes by CPZ may be the chemical's mechanism of antineoplastic action. ^••^^ The promotion of chemical carcinogenesis in the hamster cheek pouch by membrane-labilizing agents, encountered in previous studies.-^ '^ has been tentaagents on cell membranes or lysosomes, and consequently on nuclear activity.-'"-^ Polliack and Levij- suggested that the inhibitory effects of CPZ may be related to a decreased permeability of cellular and subcellular membranes, resulting in decreased penetration of the carcinogen into the cellular structures. It has been shown that CPZ inhibits the incorporation of tritiated thymidine into DNA.^' On the other hand, it is known that in certain tumors there is preferential accumulation of CPZ. which may produce toxic effects on the metabolism of the cells concerned.'** -- Thus it is also possible that CPZ may cause a specific metabolic action on tumors, resulting in the inhibition of tumor growth. The reasons for the lack of tively attributed to the effect of these

effect of

CPZ

on tumor induction

in

these studies remain unclear.

The

possibilities

that there are dose-related effects on the specificity of tissues (skin versus liver) or specific

biochemical and metabolic aspects of

DMB.A

and croton

oil

must

vet be set-

tled.

Acknowledgments The authors gratefully acknowledge the editorial assistance of Mrs. Mardelle Susman. the photographic assistance of Messrs. Walter Williams and .Andrew V^'ashington, and the technical assistance of Mrs. C. Mackiewicz.

References 1.

Berenbllm.

I.

&L

P.

SHLBik. 194".

stages of chemical carcinogenesis

in

.A

approach to the study of the Bni J. Cancer 1: 383-391.

ne\^. quaniiiative

the mouse's skin.

.

& Garcia:

Stenback 2.

Berenblum,

I.

&

P.

Effects on Skin

Tumor

Shubik. 1947. The role of croton

single painting of a carcinogen, in

oil

225

Induction

applications, associated with a

tumour induction of the mouse's

skin. Brit. J.

Cancer

1:379-382. 3.

Berenblum,

I.

&

Shubik. 1949.

P.

An

experimental study of the initiation stage of cell mutation theory of cancer. Brit.

carcinogens, and a re-examination of the somatic J.

4.

Cancer 1:109-118.

Nakahara, W.

&

F.

FuKOKA.

1960.

Summation

of carcinogenic effects of chemically

unrelated carcinogens, 4-nitroquinoline-A^-oxide and 20-methylcholanthrene.

Gann

51:

125-137. 5.

Nakahara, W.

1961. Critique of carcinogenic mechanisms. In Progress in Experimental Research. Vol. 2: 158-202. Verlag S. Karger A.-G. Basel, Switzerland. Baba, T., D. Aoki & M. ISHii. 1967. Relation between the so-called two-phase theory and summation theory in carcinogenesis. Gann 58: 161-166. Druckrey, H. 1954. Beitrage zum Mechanismus der Carcinogenese. Acta. Uni. Intern. Contra. Cancrum 10: 29-43.

Tumor

6.

7.

8.

9.

10.

11.

12.

& D. Schmahl. 1960. Die Summationswirkung. Med. Klin. (Munich) 55: 648-655. RiSKA, E. B. 1959. On the "solvent effect" in experimental chemical carcinogenesis. Acta Pathol. Microbiol. Scand. Suppl. 114: 1-1 10. RiSKA, E. B. 1960. The effect of the solvent concentration on skin tumorigenesis following painting of mouse skin with 9,10-dimethyl-l,2-benzanthracene solubilized by Tween 20, Tween 60, and Tween 81. Acta Pathol. Microbiol. Scand. 48: 217-220. RuscH, H. P., G.C. Mueller & B. E. Kline. 1945. The effect of the autooxidizability of lipoidal solvents on sarcogenesis by 3,4-benzpyrene. Cancer Res. 5: 565-571. Druckrey, H.

Peacock,

P. R.

&

S.

Beck, 1938. Rate of absorption of carcinogens and

reaction as factors influencing carcinogenesis. Brit.

J.

local tissue

Exp. Pathol. 19: 315-319.

14.

P. R., S. Beck & W. Anderson. 1949. The influence of solvents on tissue response to carcinogenic hydrocarbons. Brit. J. Cancer 3: 296-305. Dickens, F. 1957. Influence of solvent on carcinogenic response. Brit. Med. Bull. 4: 348-

15.

Bock,

13.

Peacock,

354. F. G. 1963. Early effects of hydrocarbons on Res. 4: 126-168.

mammalian

16.

Leake, C. D. 1966. Dimethylsulfoxide. Science 152: 1646.

17.

Sweeney, T. M., A.

18.

Stenback,

skin. Progr. Exp.

Tumor

&

M. Downes A. G. Matoltsy. 1966. The effect dimethylsulfoxide on the epidermal water barrier. J. Invest. Dermatol. 46: 300-305.

of

The solvent effect on dimethylsulfoxide in experimental skin Med. Exp. Biol. Fenniae 48: 63-65. Levij, I. S., J. W. RwoMUSHANA & A. POLLIACK. 1969. Enhancement of chemical carcinogenesis in the hamster cheek pouch by prior topical application of Vitamin A F.

1970.

carcinogenesis. Ann.

19.

21

Dermatol. 53: 228-231. A. Polliack. 1971. Inhibition of chemical carcinogenesis in the hamster cheek pouch by topical chlorpromazine. Nature 228: 1096-1097. Baserga, R. & P. Shubik. 1954. The action of cortisone on transplanted and induced tu-

22.

Plaut, a.

palmitate.

20.

Levij,

mors

I.

S.

in

J.

Invest.

&

mice. Cancer Res. 14: 12-16.

&

H. SoBEL. 1949.

Human serum

as vehicle for methylcholanthrene.

Cancer

Res. 9: 294^296. J. & R. Hatchek. 1943. tjber die Beinflussbarkeit der Krebsbildung nach Benzpyrenpinselung. Z. Krebsforsch. 54: 26-38.

23.

RosiCKY,

24.

Srait, L. a.,

M. K. Hrenoff

&

K. B.

the effectiveness of carcinogenic agents. 25.

26.

27.

28.

De Ome.

1948.

Cancer Res.

The

influence of solvents

upon

8: 231-240.

Simpson, W. L., C. Carruthers & W. Cramer. 1945. Loss of carcinogenic activity when methylcholanthrene is dissolved in anhydrous lanolin. Cancer Res. 5: 1-4. MiRvisH, S. 1968. The carcinogenic action and metabolism of urethan and Nhydroxyurethan. In Advances in Cancer Research. Vol. 11: 1-46. Academic Press Inc. New York, N.Y. KoTiN, P., H. L. Falk & A. Miller. 1962. Effect of carbon tetrachloride intoxication on metabolism of benzo(a)pyrene in rats and mice. J. Nat. Cancer Inst. 28: 725-732. Metsala, p. 1971. Effect of dimethylsulfoxide (DMSO) on cytoplasmic barrier of ma-

.

Annals

226

29.

30.

31.

32.

New York Academy of Sciences

lignanl epidermal cells. An investigation in skin tumor resistant mice. Acta Radiol. Suppl.307: 1-122. StenbXck, F., H. Garcia & P. Shlbik. 1973. Studies on the influence of ultraviolet light on initiation in skin tumorigenesis. J. Invest. Dermatol. 61< 101-104. StenbXck, F. & P. Shlbik. 1973. Carcinogen-induced skin tumorigenesis in mice: Enhancement in inhibition by ultraviolet light. Z. Krebsforsch. 79: 234-240. DoNiACH, I. & J. C. MoTTRA.M. 1940. On the effect of light upon the incidence of tumours in painted mice. Amer. J. Cancer 39: 234-240. Epstein, J. H. 1965. Comparison of the carcinogenic and cocarcinogenic effects of ultraviolet light on hairless mice. J. Nat. Cancer Inst. 34: 741-745.

&

C. A. Wallick. 1954. Spectrometric studies of the persis-

33.

MoODiE. M. M.. C. Reid

34.

tence of fluorescent derivatives of carcinogens in mice. Cancer Res. 14: 367-371. Miller, E. C. 1951. Studies on the formation of protein-bound derivatives of 3,4-

35.

Adams,

36.

MoTTRAM,

benzpyrene salicylates

the epidermal fraction of

in

&

S.

R. Cobb.

1958.

A

mouse

skin.

Cancer Res.

11: 100-108.

possible basis for the anti-inflammatory activity of

and other non-hormonal anti-rheumatic drugs. Nature 181: 773.

J.

1944.

A

developing factor

1944.

A

sensitising factor in experimental blastogenesis. J. Pathol. Bacte-

in

e.xperimental blastogenesis.

Amer.

J.

Pathol.

56: 183-187. 37.

MoTTR-AM, riol.

38.

J.

56: 391-402.

& J. R. Bell. 1962. The influence of croton oil stimulation on tumour urethane in mice. Brit. J. Cancer 16: 690-695. Pound, A. W. & H. R. Withers. 1963. The influence of some irritant chemicals and scarifications of tumour initiation by urethan in mice. Brit. J. Cancer 17: 460-470. Pound, A. W. 1966. Further observations concerning the influence of preliminary stimulation by croton oil and acetic acid on the initiation of skin tumours in mice by urethane. Brit. J. Cancer 20: 385-398. PoLND, A. W. initiation by

39.

40.

41.

42.

43.

Ritchie, A., J. V. Frei & H. Shinozuka. 1963. The duplication of deoxyribonucleic acid and epidermal carcinogenesis. Acta Unio Intern. Contra Cancrum 19: 579-58 1 Hennings, H., G. T. Bowden & R. K. Boutwell. 1969. The eff"ect of croton oil pretreatment on skin tumor initiation in mice. Cancer Res. 29: 1 173-1780.

&

Becker, G. A.

A. V. Pisciotta. 1967. Potentiation of hemolytic plaque formation by

incubation of immunized spleen cells

in

phenothiazine derivatives. Proc. Soc. Exp. Biol.

Med. 124:764-767. 44.

45.

46.

47.

48.

49.

50.

51.

52.

Belkin, M. & W. G. Hardy. 1957. Eff'ect of reserpine and chlorpromazine on sarcoma 37. Science 25: 233-234. Chorazy, M. 1959. Effect of chlorpromazine on Crocker sarcoma and Ehrlich ascites carcinoma. Nature 184: 200-201. Cranston, E. M. 1958. Effects of some tranquilizers on a mammary adenocarcinoma in mice. Cancer Res. 18: 897-899. Gottlieb, L. S., M. Haze, S. Broitman & N. Zamcheck. 1960. Effects of chlorpromazine on a transplantable mouse mastocytoma. Federation Proc. 19: 181. van Woert, M. H. & S. H. Palmer. 1969. Inhibition of the growth of mouse melanoma by chlorpromazine. Cancer Res. 29: 1952-1955. FujiTA, A., S. IwASE, T. Ito & M. Matsuyama. 1958. Inhibiting effect of chlorpromazine on the experimental production of liver cancer. Nature 181: 401. HosHi, A., F. Kanzawa & K. Kuretani. 1969. Antitumor activity of psychotropic drugs and the synergic action with cyclophosphamide. Chem. Pharm. Bull. (Tokyo) 17: 848-850. GuTH, p. S. & M. A. Sprites. 1964. The phenothiazine tranquilizers: Biochemical and biophysical actions. Intern. Rev. Neurobiol. 7: 231-278. GuTH, P. S., J. Amaro, O. Z. Sellinger & L. Elmer. 1965. Studies in vitro and in vivo of the effects of chlorpromazine on rat liver Ivsosomes. Biochem. Pharmacol. 14: 769775.

53.

Levij,

I.

S.,

J.

carcinogenesis Palmitate.

J.

W. Rwomushana in

&

A. Polliack. 1969. Enhancement of chemical

the hamster cheek pouch by prior topical application of Vitamin

Invest.

Dermatol. 53: 228-231.

A

Stenback 54.

55.

56.

& Garcia:

Effects on Skin

Tumor

Induction

227

I. S. & A. PoLLiACK. 1971. Inhibition of chemical carcinogenesis in the hamster cheek pouch by topical chlorpromazine. Nature 228: 1096 1097. Allison, A. C. & L. Malluci. 1964. Lysosomes in dividing cells with special reference to lymphocytes. Lancet 2: 1371-1373. Allison, A. C. & G. R. Paton. 1965. Chromosome damage in human diploid cells following activation of lysosomal enzymes. Nature 207: 170-1 173. Hirschhorn, R., G. Kaplan, K. Hirschhorn & G. Weissman. 1964. Appearance of lysosomes before mitosis induced in human lymphocytes by pytohemagglutinin. Clin. Res. 12:449. Lucy, J. A. & J. T. Dingle. 1964. Fat-soluble vitamins and biological membranes.

Levij,

1

57.

58.

59.

60.

61.

62.

Nature 204: 156-160. PoLLiACK, A. & L S. Levij. 1972. Antineoplastic effect of chlorpromazine in chemical carcinogenesis in the hamster cheek pouch. Cancer Res. 32: 1912-1915. ZiEBERT, A. P., J. E. Hinz, J. Rydlewicz & A. V. PisciOTTA. 1966. The in vivo effect of chlorpromazine on the generation of DNA synthesizing enzymes in regenerating rat liver. J. Lab. Clin. Med. 64: 1021-1022. PisciOTTA, A. V. 1965. Studies on agranulocytosis. VIL Limited proliferative potential of CPZ-sensitive patients. J. Lab. Clin. Med. 65: 240-247. Blois, M. S. 1965. On chlorpromazine binding in vivo. J. Invest. Dermatol. 45: 475.

Part

IV.

Dimethyl Sulfoxide

in

Agriculture

FATE AND METABOLISM OF DIMETHYL SULFOXIDE IN AGRICULTURAL CROPS Bernard C. Smale, Neil

J.

Lasater,

and Bruce T. Hunter

Crown Zellerbach Central Research Camas. Washington 98607

Dimethyl sulfoxide (DMSO) has interested plant scientists and agrichemical companies since its unique solvent and membrane-penetrating properties were initially reported by Jacob and colleagues in 1963.^ By. 1965 crop specialists had studied and published reports on the adjuvant role of DMSO in the uptake of nutrients and pesticides by the foliage and roots of a number of agronomic crops. ^ Interest in these early years, however, was for the main part directed toward the discovery of new applications, and observed benefits were not followed by studies on mechanism, metabolism, or residues. During the past five years the major portion of our research efforts with DMSO has been directed toward an extensive study on the comparative absorption, translocation, and metabolism of DMSO in a number of agronomic crops. Primary analytical methods for these studies, in which ^^S-labeled DMSO was used almost exclusively, were scintillation spectrometry, gas chromatography, and thin layer chromatography. Precision of radiochemical methods allowed the detection and identification of [^^S] DMSO, dimethyl sulfone ([^^SJDMSOz) and dimethyl sulfide ([^^S]DMS) in the ppb range. Some of the data reported in this paper were used to obtain the approval of the Food and Drugs Administration for the use of DMSO in crop production. Absorption of nutrients, water, gases, and pesticides may occur in plants through most, if not all, surfaces.^ Roots, anatomically and morphologically suited to the absorption of nutrients and water, will readily absorb a number of pesticides, and in some cases will accumulate levels higher than those in the soil medium. The ability of roots to absorb DMSO from soil was initially studied with corn and soybean as test plants in the greenhouse. [^^S]DMSO was applied before emergence at a rate of lb/acre to soil in 4-inch pots, previously planted with corn and soybean. Four weeks later the corn and soybean plants were harvested by cutting at the soil line. The total ^^S levels in the stems and leaves of the crop plants reflect the rates of root absorption and translocation of [^^S]DMSO or a ^^S metabolite of it. The ^S levels in the corn stems and leaves after four weeks were approximately three times greater than in the soybean (Table 1). Accumulation of ^^S continued through 10 1

weeks; the maximum concentration (120 ppm) occurred in the tassel. It is of interest to note that corn is characterized by a dominantly carbohydrate metabolism, as is soybean, during the first half of the crop growth. At about midmaturity, the soybean (but not corn) shifts away from the carbohydrate metabolism to one dominated by fatty acids, and protein storage is initiated. The 28 ppm ^^S residue detected in the soybean pod and seed at 15 weeks, compared with less than 0.5 ppm in the whole corn ear, reflects the changed soybean metabolism. Separation of seeds and pods of field-grown soybeans treated before emergence with [^^S]DMSO showed that the major portion of ^^S was contained by

228

Smale et

al.

:

Metabolism Table

in

Agricultural Crops

1

Accumulation and Distribution of Translocated ^^S Soybean Plants after Pre-Emergence Treatment with [^^S]

Stem

229

DMSO

in

Leaves

Corn and

in

[^^S]

DMSO*

Fresh Tissue

Whole Ear or Pod

Tassel

ppm Corn at

4 weeks

weeks at 1 5 weeks Soybean at 4 weeks at 10 weeks at 15 weeks at 10

48

64

12

40

120

abs| 16