Histochemical Responses of Human Skin Following Ultraviolet Irradiation1

HISTOCHEMICAL RESPONSES OF HUMAN SKIN FOLLOWING ULTRAVIOLET IRRADIATION * FARRINGTON DANIELS, JR., M.D., DORIS BROPHY, Lic. ES. SC. AND WALTER C. LOBITZ, JR., M.D.

The earliest clinical evidence of a sunburn of enzyme studies and of other special stains. reaction in human skin is the development of This paper will emphasize the effect of sunburn

upon the epidermis; a comparison of these exposure to sufficient quantity of ultraviolet changes with those produced by furocoumarin irradiation between .29 and .32 microns (1—3). (methoxsalen) photosensitization and the reThe erythema reaches its maximum at approx- covery of the epidermis from the established erythema beginning approximately 4 hours after

imately 24 hours (1), then gradually fades, being sunburn reaction as studied histochemically will supplanted by a suntan. The earliest histopatho- be dealt with in subsequent reports. We also logical change other than dilatation of dermal consider the reaction to cold quartz or germicidal

vessels is degeneration of scattered individual ultraviolet as different from sunburn. This will prickle cells in the outer one-half of the epi- be also reported in other studies and not disdermis, first evident 24 hours after exposure cussed here. (4—6). These individual cells show a shrunken, METHODS homogenized, densely-staining glassy cytoplasm, and a hyperchromie condensed pyknotic nucleus.

The appearance is somewhat that of individual cell dyskeratosis. The shrunken cytoplasm is

surrounded by intracellular edema since the cell margin with prickles is still apparent next to adjacent cells. Thirty to 48 hours after exposure, intercellular edema occurs around these altered prickle cells. In severe sunburn, clinical vesicula-

1. Ultraviolet exposure. The source of sunburn ultraviolet radiation was a water-cooled AH6 1000

watt high-pressure capillary mercury arc filtered through an interference filter with a maximum at .295 microns. Study of this light source with a Beckman DK-1 spectroreflectometer indicated that this combination isolated the .2967 and the .3131 mercury lines. Although this high-pressure

mercury arc puts out some continuous backapparently little of this passes the intertion appears at this time. The damaged outer ground, ference filter, which has a maximum transmission one-third gradually shows more homogenization and deep staining on Giemsa and other special

of less than 10 per cent. Test exposures were used to determine the individual's erythema response, stains. This area is then shed without going and exposures given to produce mild to moderate

through a granular layer. By the end of 5 days sunburn. Spectrophotometric reflectance of the a new granular layer is beginning to reform under- skin was determined at regular intervals with a neath the desquamating damaged prickle cells. Beckman DK-1 recording spectroreflectometer. In It is of interest that there is a variable interval the long run the reflectance information will of approximately 4 hours between the time of permit correlations of the degree of erythema with irradiation and the first signs of reaction in the the degree of histologic change. However, in the series of six subjects to be reported here, the dermis, when erythema and vasodilatation begin. number is too small to establish meaningful corThere is, further, a 24-hour interval between the relations. The sunburns, therefore, will be identime of irradiation and the morphologic re- tified only as mild, moderate, or severe. Mild

action of the keratinocytes in the outer half includes the minimal perceptible erythema and a of the epidermis. One of the purposes of this slightly more intense erythema. Moderate consists study was to search for earlier evidence of radiation damage by the histochemical techniques * From the Division of Dermatology, University of Oregon Medical School, Portland, Oregon. Presented at the Twenty-second Annual Meeting of The Society for Investigative Dermatology, Inc., New York, N. Y., June 27, 1961. This work was carried out under U. S. Public Health Service, N.I.H. Grants C-5052(C1) and E-3102 (Cl). 351

of more severe grades of erythema, and severe sunburn is that which includes clinical evidence of

vesiculation. This three-unit scale refers to the appearance at 24 hours, the time of the sunburn erythema maximum, rather than the appearance at the time of biopsy. The test subjects were 6 normal young men of white extraction who bad not

had ultraviolet exposure of the back for at least six months prior to test.

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Biopsy specimens were obtained from normal skin as controls, and from the irradiated areas

without anesthesia with a high-speed rotary

TABLE I Effect of sunburn on glycogen and mitotic activity

punch. Biopsy specimens were obtained from two

subjects every 12 hours for 5 days; one was hiopsied at 24 hours, another at 48 bours, and two had a series of intensities of exposure biopsied at

Per Cent Basal Cells Having Mitoses per 1005 Cells Hours After Radiation

GJycngen

RB.

L. C.

RB.

L.C.

12 hours.

Histochemical technics were selected with the hope of demonstrating chemical and/or structural changes in the stratum corneum and in the cyto-

0 4

plasm and nuclei of the prickle cells at various levels within the epidermis and within the epidermal basal cells. These included Giemsa (1:50

12

"4 24 36 48 60 72 84 106 118 120

dilution) in water, hematoxylin-phloxine, periodic acid Schiff reaction (7) controlled with diastase for

glycogen, Gram stain (8) and Toluidine Blue buffered at pH 5.0. Disulfide and sulfhydryl groups were demonstrated by the method of Barrnett and Seligman (9). Enzyme systems studied were non-specific csterascs (10), alkaline

0 3

0

53 61 15

70

0 0 0 0 1

10

4 0

3 0 0 0

0.0 0.32 0.9 9.7 11.0

2.2

21.8

4.5 1.3

0

0

1

.42

0 0

1.4

3.5 2.0

phosphatase (11), and succinic dchydrogenase (12). In some samples, other stains and procedures

were carried out which are not reported in this article. RESULTS

The percentage of glyeogen-containing basal cells then gradually decreased over the next 24 hours. The course of basal cell glycogen is given

in Table I. The basal cells that were involved With one exception, there were no obvious in glycogen accumulation activity do not have histochemical alterations demonstrated in the any of the morphologic characteristics of melanoepidermis by any of these procedures before the

cytes.

24-hour morphologic changes were evident. Figure 1 shows the morphologic picture occurring at 24 hours in a mild sunburn (Subject Once these morphologic changes, "degeneration", have appeared in the outer half of the L. C.). Individual cell degeneration is clearly epidermis, subsequent sequential histochemical

shown in this Giemsa-stained section. Figure 2

changes that occurred could be related to is a PAS stain of nonirradiated skin in the same known processes associated with recovery subject. The glycogen accumulation in the basal from injury, and not necessarily the results of layer 24 hours following radiation is indicated the ultraviolet irradiation itself. Succinic de- in the PAS stained slide in Figure 3. hydrogenase accumulations in the epidermis did Since the amount of glycogen was so striking not differ from the control specimens at any at 12 hours, two additional subjects svere given time during the entire five-day experiments. mild erythema doses consisting of 120 seconds Stains for alkaline phosphatase failed to increase in intensity in the dermal blood vessels before,

exposure to the mercury are lamp filtered through

the .295 micron maximum interference filter.

during or after the development of mild to They were also given exposures of 60, 30, and

moderate clinical sunburn. There were no evident changes in the stains for disulfide and sulfhydryl linkages prior to morphological damage. The striking finding which preceded morphologic evidence of epidermal damage was accumulation of glycogen in the cytoplasm of the epidermal basal cells. Glycogen began to appear in some basal cells 4 hours after ultraviolet irradiation, and involved the maximum of 60 to 70 per cent of the basal cells by 12 hours after irradiation leading to mild or moderate sunburn.

12 seconds. In both subjects, at 24 hours, the 120 seconds showed a mild erythema and the 60 seconds an extremely faint, barely detectable

erythema. The shorter exposures showed no erythema. The appearance of glyeogen at 12 hours paralleled the erythema; there was no basal cell glyeogen at 12 and 30 seconds, a small

amount at 60 seconds, and at 120 seconds an amount comparable to that shown in Figure 3. The two subjects studied at the single 48 hour point, 24 hour point and the two subjects studied

353

RESPONSE OF HUMAN SKIN TO ULTRAVIOLET IRRADIATION

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at 12 hours did not show mitotic activity. The ever, no mitotic response. The present sunburn two subjects in whom the serial biopsy specimens studies show both a glycogen accumulation and were taken for 5 days showed mitotie activity the mitotic response, but with different time which reached its maximum of 21.8 mitoses per relationships than have been consistently found thousand cells at 72 hours in subject LC with a with stripping injuries. The maximum mitotie mild sunburn, whereas subject RB, with a activity occurring in the epidermal basal cell moderate sunburn, showed only 2.2 per thousand at 72 hours in studies with biopsies at every 12 mitoses at 73 hours. The time course for mitotic hours can be considered to be approximately counts is given in Table I. 68 hours following the onset of dermal erythema or 48 hours following the appearance of the DIscussIoN morphologic changes in the outer part of the It is of interest to compare the glyeogen ac- stratum spinosum. Thus the glycogen accumulacumulation in the cytoplasm of the epidermal tion and the mitotic activity are not automatibasal cells and the burst of mitotic activity in cally related in a timed sequence of repair. If these cells in the present study to glyeogen ac- mitotic activity is on a biological clock mecha-

cumulation and mitotic activity previously nism with a 48 hour delay from the initial injury studied in strip injury of the skin. In strip injury the basal cells were maximally iavolved with glycogen accumulation 12 hours after stripping (13, 14). Mitotic activity was always maximal 40 to 48 hours after the stripping injury. During studies (15) in which mid-dermis injury was pro-

stimulus, in sunburn the clock is started by the morphologic injury rather than the ultraviolet exposure. Both this time sequence and the middermal injury findings are against teleologic interpretations of the glycogen accumulation in terms Qf' "preparation" for mitotic activity.

duced selectively by mechanical cutting, glyeogen

The cytoplasmie glycogen accumulation in the

also accumulated in the epidermal basal cells epidermal basal cells reaching a maximum at 12 with a maximum accumulation evident at 12 hours after irradiation is the earliest histochemical hours. In mid-dermal injury there was, how- change detected and the only one so far found

RESPONSE OF HUMAN SKIN TO ULTRAVIOLET IRRADIATION

preceding the onset of morphologic change with individual cell degeneration.

355

the upper third of the prickle cell layer (1, 2). The morphologic findings in our stained sections

The degenerated cells have the cytoplasm are consistent with this concept. It seems most condensed around the nucleus. In Giemsa and likely, therefore, that the glycogen, as well as

Gram stained sections at 24 hours the cell out- the erythema, are indirect responses at a distance line with prickles still connecting to surrounding from the site of the primary ultraviolet injury. cells is readily identified. The degenerated cell is described by Hamperl (5) as having so-called SUMMAEY

colloid degeneration. The outline is generally

Sunburn has been produced by wavelengths rounded, suggesting that the intracellular edema of radiation between .29 and .32 microns. Histois under some pressure. No intercellular edema is logical and a number of histochemical changes evident at 24 hours. It is difficult to be certain of small differences in enzyme activity between have been observed at intervals from 4 hours to adjacent cells by histochemical techniques. How- 5 days. Suceinic dehydrogenase, alkaline phosever, the degenerated cells appear to stain more phatase, sulfhydryl and disulfide bistochemical deeply with the stain for both —SH and —S-if--S procedures showed no evident alteration in the — groups. At 48 hours the darker staining in — absence of extensive cellular damage. Glycogen SH and —S—S— groups was again seen in the appeared at 12 hours in the basal cell layer with individually degenerating cells, but again these radiation sufficient to produce erythetha. Epi-

dermal morphologic changes were evident at 24 hours and are similar to those described in the older literature. Mitotic activity reached a peak at approximately 72 hours. This contrasts with the 48 hour interval between injury and mitotie activity produced by strip injuiy.

changes are small and it is difficult to be sure of their significance without more data. The paucity of enzyme changes need not surprise us when we compare ultraviolet radiation to ionizing radiation, where doses of tens or even hundreds of thousands of roentgens are needed to produce immediate morphologic change in cells or to inactivate enzymes. This contrasts, of course, with the great sensitivity of the nucleus of cells to ionizing radiation (16, 17). In addition to the general lack of evidence for enzyme alterations prior to cellular degeneration, there was a notable absence of inflammatory cell migration from blood vessels into the dermis or epidermis. This is a striking finding, in view of the other evidences of inflammation which the sunburned skin shows. It is not clear why our sections should differ from the classical

The ultraviolet lamp based on the AH6 high pressure mercury are was designed and con-

description of sunburn. The most reasonable

York, McGraw-Hill, 1955. 3. Lorni'icz, A. L. : Physiological and pathological

explanation is that we produce less intense sun-

burn. Another alternative explanation is that we used radiation confined to the region between .29 and .32 microns.

As with the epidermal response to stripping and mid-dermal cutting, the accumulation of glyeogen in the basal cell at a vertical distance from the site of injury is of great interest. Information on the penetration of ultraviolet of

ACKNOwLEDGMENTS

structed by Mr. Henry Kaiser of Research Products, Inc. of Cowallis, Oregon.

REFERENCES 1. BLITM, H.: The physiological effects of sunlight

on man. Physiol. Rev., 25: 483—538, 1945. H.: Sunburn. Chapt. 13, 487—528 in Hollaender, A. (ed.) Radiation Biology II.

2. BLUM,

Ultraviolet and related radiations. New changes in skin from sunburn and suntan. JAMA, 173: 1227—31, 1960. 4. Ros'r, G. A. AND KELLER, P.: Die Wirkungen

des Lichtes auf die gesunde und kranke Haut. V. Part 2, p. 1—163, Jadassohn's H andbuch der Haut-und Geschlechts-Kranksheiten, (Histology pp. 66—77). Berlin.

Springer. 1929. 5. HAMPERL, H., HEN5CHKE, V. AND SeHuLzE, R.: Vergleich der Hautreaktionen beim Bestrah-

lungserythem and bei der direkten Pigmentierung. Virehow Arch. Path. Anat.,

these wavelengths is quoted in some references,

but the difficulty of the methods makes us re-

luctant at present to state whether the basal

6.

304: 19—33, 1939. MIE5CHEE, G.: Zur Histologie der lichtbeding-

ten Reaktionen. Dermatologica, 115: 345—

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cells are hit directly at all by these wavelengths. However, the classic interpretation of sunburn

7. MCMANU5, J. F. A.: Histological and histochemical uses of periodic acid. Stain Techn.,

confines the primary photochemical events to

8. LILLIE, R. D.: The Gram stain. I. A quick

23: 99—108, 1948.

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method for staining Gram-positive organ-

isms in the tissues. Arch. Path., 5: 828, 1928.

9. BARRNRTT, II. J. AND SELIOMAN, A. M.: Histochemical demonstration of protein-bound sulfhydryl

1952. 10. Goiuorn,

groups. Science, 116: 323—327,

G.: Microscopic Histochemistry; Principles and Practice, p. 207. Chicago,

University of Chicago Press, 1958. 11. Goiwortr, G.: Microscopic Ristochemistry; Principles and Practice, p. 185. Chicago, University of Chicago Press, 1958. 12. SELIGMAN, A. M.: Cytoehemieal demonstration of succinie dehydrogenase by the use of a new p-nitrophenyl substituted ditetra-

zole. J. Histoehem. Cytochem., 5: 420—436, 1957.

13. LOBITZ, W. C., JR. AND H0LY0KR, J. B.: The

histoehemieal response of the human epidermis to controlled injury, glyeogeo. J.

Invest. Derm., 22: 189—198, 1954. 14. BROPHY, D. AND LORITZ, W. C., JR.: Injury

and reinjury to the human epidermis. II. Epidermal basal cell response. J. Invest. Derm. 32: 495—503, 1959.

15. LOBITz, W. C., JR., HOLYOKE, J. B. AND BaOPHY, D.: Unpublished data. 16. GRAY, L. H.: Cellular Radiobiology, Radiation Research. Supplement I, page 73, 1959. 17. STOCKRN, L. A.: Enzyme Inactivation. Radiation Research. Supplement I, page 53, 1959.

DISCUSSION DR. STEPHEN ROTHMAN (Chicago, Illinois): I

was very much interested in the findings of increased amounts of glycogen in the epidermis 12—24 hours after ultraviolet irradiation followed

by an outburst of mitotic activity. Dr. Adaehi did not mention in his presentation on glycogen synthesis that we performed experiments not only of

on the effect of x-rays but also on effects

ultraviolet light. In two out of four experi-

ments we found a substantial increase of glucose

incorporation into glycogen 24 hours after a threshold erythema dose was given with ultraviolet light, while in two experiments there was

zymatic changes were noted eventually, that is, in a matter of days after the exposure. DR. HOWARD P. BADRN (Boston, Massachu-

setts): As part of a study on the incorporation of C14 methyl methionine into epidermal proteins the effect of ultraviolet light was investigated. Animals were irradiated on one side with the other side as a control, and from 0 to 26 hours later injected intravascularly with the isotope. The animals were sacrificed at 4 hours and the specific activity in the epidermis of the control and irradiated sites determined. There was marked suppression in the soluble and in-

no effect. Apparently much depends on the soluble fractions. dosage and the time sequence of events and This is direct evidence that some enzymes are it is a matter of chance whether we catch the effected by ultraviolet light, even though the brief phase of enhanced glycogen synthesis or not.

enzymes you studied showed no difference.

Both Daniels' and our own findings support DR. RICHARD B. STOTJGHTON (Cleveland, Ohio): the assumption that after irradiation there is a Some years ago, we were interested in trying to phase in which enzymatic disturbance leads to produce blisters in vitro as well as in vivo. We heightened glycogen synthesis, and that subse- used ultraviolet light and ionizing radiation, quently glycogenolysis supplies the energy needed and we didn't create blisters in this way in vitro. for mitotic division

We did observe that the nucleoproteins lost I believe that we should cease to say that almost all of their stainability after exposure to

sunburn is elicited by rays between 2,900 and 3,200 Angstroms. When working with Dr. Peter Beal (J. Invest. Dermat. 11: 415—433, 1948) we repeatedly projected the strong 3131 A line of the mercury arc on human skin without ever causing sunburn reaction while the 3020 and 2967 lines applied with the same energy clearly did. This is in conformity with all earlier ob-

ultraviolet light and ionizing radiation.

I wonder if Dr. Daniels would comment on this specific point. Dn. FARRINGTON DANIELS, JR. (in closing):

I thank the discussors for their comments. If we were to answer their questions completely and if we knew the answers, it would take quite

servations. The upper limit of the sunburn a while. Fortunately for time, we don't know spectrum is lower than 3200 A and in all prob- many of them.

I have been interested in Dr. Rothman's ability lower than 3100 A. Dr. JOHN E. EPSTEIN (San Francisco, Cali- group's findings on the similarity between ultrafornia): I enjoyed Dr. Daniels' paper very much. violet and ionizing radiation. The more I see I was interested whether any dermal or en- of these sections of skin and the more I read

RESPONSE OF HUMAN SKIN TO ULTRAVIOLET IRRADIATION

about ionizing radiation, the more impressed I am with the fact that sunburn is probably not completely different from the effects of ionizing radiation. In regard to the wave lengths, our source is pure 2967 A as far as the sunburn goes, but our filters let through some of the 3131 A line also.

357

methionine, the histochemieal stains are probably not the most subtle means of evaluating enzyme action and suppression of incorporation of the substrate is probably a much more sensitive method than the staining technics. Dr. Stoughton's comments, concerning the loss of nucleoprotein staining is extremely inIn regard to Dr. Epstein's question on the teresting in view of what we think is going on in dermal changes, these were unimpressive but ultraviolet damage. I won't give my next paper perhaps there are subtle changes that we have now, but I have been struck with the fact that overlooked so far. We didn't find the changes of all of these cells which show the individual senile elastosis occurring temporarily, as I damage are cells which still retain their DNA on a Feulgen stain. These are all cells which thought we might. Later on, the enzymes, particularly the sulfhy- still have their nucleus. The ones around them dryl and the SS stains, do show an increase in which have already lost their nucleus are apthe individually dyskeratotie cells. I don't know parently unaffected. how accurate we can consider ourselves to be It appears to us at present as if the DNA, or in examining the histoehemical section and pick something associated with an intact nucleus out one cell and say, "This cell has greater ac- may be the target rather than the tryosine or tivity than the one next to it;" but it appears tryptopban containing protein. Therefore, your that some of these enzymes do increase as the comments are of great interest to us, in that you cells become homogenized and shrunken. were able to observe something along the same

Concerning Dr. Baden's discussion about line.

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94

linolenic acid extract. Arch. This pdf is a scanned copy UV of irradiated a printed document.

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