The Effect of Dimethyl Sulfoxide on Rat Skin Oxygen Utilization*

Vol. 52, t'lo. 5

THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

Copyright

Printed in U.S.A.

1969 by The Williams & Wilkins Co.

THE EFFECT OF DIMETHYL STJLFOXIDE ON RAT SKIN OXYGEN UTILIZATION* CAROL A. ROTH,t RONALD B. BERGGREN, M.D4 AEO

MILTON A. LESSLER, Pn.D.

ABSTRACT 1. The respiratory activity of rat skin was measured with an oxygen electrode system following soaking of the skin either in 0.9% NaCI or one of three different concentrations of

DM50. 2. Oxygen consumption rates of the fresh rat skin samples were significantly increased after soaking in DMSO at low concentration (5% for two hours), not changed by exposure to 10% DMSO, but decreased by soaking in 20% DMSO for one or two hours as compared to the saline treated samples.

3. These alterations in cellular metabolism may contribute to DMSO's previously reported cryophylactic abilities.

Exposure to low concentrations of dimethyl sulfoxide (DM50) before liquid nitrogen storage

has been shown to increase the post-thawing Viability of mnny cells and organs, including skin (1, 2). According to recent investigations, DM50 may preserve cellular viability by protecting mitochondrial respiratory activity (3). We felt that a preliminary study was necessary to determine the effect of DM50 on fresh skin before investigating DMSO's influence on the respiratory activity of skin after freezing. For

solution containing 0.040 M sucrose, 0.005 M dex-

trose, 0.0002 M EDTA and .003 M sodium phosphate buffer. The pH of the solution was adjusted to 6.9 with KOH. Oxygen utilization was measured with a Ycllow

Springs Instrument Company Model 53 Oxygen Monitor, in the manner described by Lessler, et at. (4, 5). The oxygen electrode system was used in

preference to conventional manometrie tech-

niques because it is reliable for rapid quantitative measurement of tissue oxygen utilization (6). In

contrast to the 1—2 hours usually required for manometrie determinations, oxygen electrode can be completed within 15 minthis preliminary study, we duplicated our regular measurements utes. Gesinki ef at. (7) tested the Y.S.I. oxygen procedure for preparing rat skin grafts for liquid monitor with bone marrow samples and found that it was at least as reliable as the Kirk micronitrogen storage (2). respirometer or the conventional Warburg appaMETHODs AND MATERIALS ratus. Statistical results were tested with Love's Full-thickness skin grafts were excised from the

(6) modification of Student's Test for small sam-

shaved abdomens of 16 ether-anesthetized, 200 ples. gram female Wistar rats. The grafts were divided into four pieces, each of which was soaked for one

or two hours in either 0.154 M (0.9%) NaC1 or 0.7 M, 1.6 M, or 3.5 M DMSO at 4' C. Expressed as volume-volume percentages, these molarities represent 5%, 10%, and 20% concentrations of DMSO in 0.9% NaC1. The samples were then trimmed of subcutaneous fat and blood vessels and stored raw-side down in Petri dishes. Each sample

was blotted, rapidly weighed on a Roller-Smith balance and minced with a siliconized razor blade. The minced sample was equilibrated at 37' C in a reaction chamber holding 4 ml of a Krebs-Ringer

RESULTS AND DISCUSSION

An average oxygen consumption rate of 0.552 mieroliters 02/mg wet weight/hr was obtained for freshly excised skin. By comparison, Skoog's manometric measurements (9) showed an average value of 0.54 microliters 0,/mg dry weight/

hr for fresh rat skin. Our polarographie results were slightly higher than Skoog's manometric resulta because we measured only the initial respiratory activity of the tissue. However, the internal comparisons were nearly identical.

Supported by USPHS Grant AM-09326, NaData on different soaking times in 0.9% NaC1 tional Institute of Arthritis and Metabolic Diseases and a grant from the Roessler Foundation, and several concentrations of DMSO are deColumbus, Ohio.

Received July 5, 1968; accepted for publication

picted graphically in Figure 1. Oxygen consump-

tion rates of the samples soaked for one hour o1ogy, The Ohio State University College of in 5% or 10% DMSO approximated the values December 3, 1968.

* From the Departments of Surgery and Physi-

Medieine,t Columbus, Ohio 43210.

of the saline-soaked controls from the same rats.

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DMSO ON SKIN 02 UTILIZATION

Soaking the samples in 20% DMSO for one or two hours depressed the oxygen consumption

rate. This decrease from the control rate was significant at the 0.05 level of confidence. Two-

hour soaking in 10% DMSO gave an average oxygen consumption rate nearly identical with the two-hour control value, but samples soaked two hours in 5% DMSO showed an increase in oxygen utilization significant at the 0.002 level of confidence.

The elevated respiratory activity of the skin exposed to 5% DMSO for two hours may be a response similar to uncoupling of cellular respira-

tion. DMSO could cause such a response by forming complexes with important enzymes of oxidative phosphorylation, or their associated membranes (G. P. Brierly, Associate Professor

of Physiological Chemistry, Ohio State University, personal communication). Respiratory activity was not increased after one hour of

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exposure to 5% DMSO, suggesting that a longer Fie. 1. Relation between rate of oxygen conexposure was necessary to form the DMSO en- sumption of fresh rat skin and soaking in saline zyme complexes that resulted in increased oxygen

and various concentrations of DMSO for one

(dotted columns) and two hours (solid columns).

utilization. In the presence of greater DM50 The standard error of the mean is indicated at the concentrations, this time factor apparently was top* of each column. Increase from saline control value significant less important. High concentrations of DMSO at the 0.02% level. ** Decrease from saline control value significant may affect an increasing number of cellular en-

zymes, eventually resulting in depressed oxygen at the 5% level. utilization. At the 10% concentration, this tendency to depress respiratory activity may be bal- been exposed to 20% DMSO; Samples of these anced by the increased oxygen utilization due to grafts had the lowest oxygen utilization at the the presumptive uncoupling of respiration. time of grafting (Fig. 1). The poorest grafts were Combination of these two divergent effects may those soaked in saline, while those pretreated be responsible for the apparently normal respira- with 5% and 10% DMSO were of intermediate tory activity after exposure to 10% DMSOF for quality. The superior appearance of the DMSO-treated one to two hours. Significant depression of

respiratory activity became manifest after ex- grafts could be related to the DMSO-enzyme posure to 20% DMSO. In summary, exposure to complexes postulated to explain the respiratory 5% DMSO increased oxygen utilization, while changes previously observed. Complexing enhigher DMSO concentrations became increas- zymes with DMSO at the appropriate soaking ingly toxic with resultant decreases in respiratory time and concentration would reduce general metabolism until the cells of the autograft apactivity. Forty autografts were made from skin soaked proached an essentially dormant state. Such relafor two hours in 0.9% saline, 5%, 10% and 20% DMSO (ten iii each solution). Failures because

tively dormant cells would have less acute metabolic requirements than cells without

of technical difficulties reduced the number of DMSO-complexed enzymes and would be more grafts surviving to four to six in each of the likely to survive the relative metabolite deprivafour groups. Although these numbers are too tion which must occur during the first 36—48 small for reasonably accurate statistical analysis, hours, while the graft is ischemic. After graft when evaluated after 28 days on the basis of circulation has been re-established, the DMSO hair growth and scar pattern, the best grafts would redistribute into the total body fluids, were consistently seen in the group which had freeing the previously-complexed enzymes to re-

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THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

sume normal cellular activity and maintain the viability of the autograft. This sequence may also be responsible for the increased survival of DMSO-treated autografts following liquid nitro-

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