Effect of Ultraviolet Light on Some Dehydrogenase Activities in the Epidermis of the Rhesus Monkey (mac Ac a Mulatta)*

Vol. 52, No. 4

Tua JOURNAL OF INVESTIGATIVE DERMATOLOGY

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Printed in U.S.A.

1965 by The Williams & Wilkins Co.

EFFECT OF ULTRAVIOLET LIGHT ON SOME DEHYDROGENASE ACTIVITIES IN THE EPIDERMIS OF THE RHESUS MONKEY (MACACA MULATTA)* MICHAEL J. C. TM, PuD.

Wohigemuth and Ikebata (1) showed that ultraviolet light and X-ray irradiation cause a depression in glyeolysis of the skin. X-ray and ultraviolet light also seem to inhibit enzymes (2—4). A single dose of 800 Roentgens of X-ray caused an initial decrease, which was followed

MATEBrAL5 AND METHODS

Samples of skin were obtained from 3 rhesus monkeys, 1½ years old. After the back of each monkey had been irradiated, the animals were kept in chairs during the one-week experiment to prevent their scratching the irradiated sites. Six spots 2.5 cm in diameter on one side of the

by an increase in the glucose incorporation into back of each animal received TO erythema doses glyeogen in the skin at 24 hours after irradia- of ultraviolet light. Symmetrical areas on the tion (5), and minimum erythema does (2 >< 10 other side of the back served as controls. The ergs/em°) of ultraviolet light produce an ac- source of ultraviolet light was Aero-Kromayer cumulation of glyeogen in the basal cell layer (Model 222 lA—from Hanovia Lamp Division, New Jersey), where the intensity for the of the epidermis after 12 hours (6). Such ac- Newark, spectral region 3130 A to 1850 A was 70,000 cumulations of glycogen may be due to an microwatts/cm°. According to the manufacturer, imbalance in the activities between phosphoryl- erythema time was one second; this was conase and glyeogen synthetase (5). Coffey et al. firmed by irradiating the flexor surfaces of the (3) reported that UV light irradiation inhibited forearms of several human beings in this laboratory. hexokinase activity in the epidermis which Biopsy specimens, 1 >< 1 cm, were taken from brought about reductions in glucose oxidation the irradiated and the control skin on the 6th, 12th, and oxygen consumption one hour after expo- 24th, 48th, 72nd, and 168th (1 week) hour after irradiation. Tissues were quickly frozen in liquid sure. It has been shown in vitro that UV light nitrogen, cut into sections 24 p thick at —20°, inactivates tyrosinase, ATPase, acid phospha- and dried at —5 in vacuum for 6 to 12 hours. tase, sueeinie dehydrogenase, and oxidative Frozen dried tissues were kept in a vacuum at —20° until the enzymes were analyzed. Irradiated phosphorylation (4). and control samples of epidermis were dissected

With the mieroenzyme assay methods of out from the frozen dried sections under the microscope, weighed out 0.5 to 2.0 pg on a tabolism in epidermal tissue injured by UV quartz fiber balance, and transferred into the test (2.5 X 25 mm) for the subsequent enzyme light irradiation. In this study the possible tube analyses. alterations in glucose catabolism in the irPreviously determined optimal analytical conLowry, it is now possible to study glucose me-

ditions of GAPDH, G6PDH, 6PGDH, and LDH some of the key enzymes in each metabolic cycle (7—9) were used in this study. The assay condition of IGDH was: isocitrate, 4 mas; Tris buffer, of glucose. The enzymes chosen were glucose-fl- 0.1 u, ph 8.1; NADP4, 1 mu; MnCI2, 025 mu; phosphate dehydrogenase (EC 1 .1.1 .49), 6- albumin, .05%; and incubation time, 60 mm. at phosphogluconate dehydrogenase (EC 1 . 1 . 144), 37°. That of MDH was: oxaloaeetie acid, 1 mu; buffer, 0.1 at, pH 8.2; NADH, 2 mu; albumin, glyceraldehyde-3-phosphate dehydrogenase (EC Tris 0.05%; and 30 mm. incubation at 37°. Pryidine 1.2.1.12),laetatedehydrogenase (EC1.l.1.27), nncleotide, stoichiometrieally reduced or oxidized isocitrate dehydrogenase (EC 1.1.1.42), and by enzyme reaction, was measured as described before, after the fluorescence of the reduced form malate dehydrogenase (EC 1.1.1.37).

radiated epidermis were studied by analyzing

had been stabilized in 0.1 at carbonate buffer,

Publication No. 369 from the Oregon Regional pH 10.5 (10) or after the fluorescence of the Primate Research Center, supported in part by oxidized form had been produced by a treatment AM 08445 and AM 05512 and Grant FR 00163 of with strong alkali (6.6 u) (9). Blank and standard the National Institutes of Health. tubes were run simultaneously; the reaction Received November 21, 1967; accepted for pub- mixture without tissue was used as blank for each lication September 30, 1968. * From the Department of Cutaneous Biology, enzyme assay. It was also confirmed that no reacOregon Regional Primate Research Center, Bea- tion resulted when the substrates were omitted from the respective reagent mixtures. DNA eonverton, Oregon 97005. 329

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tents in the microdissected samples were measured responding irradiated epidermis. These results by the method of Kissane and Robbins (11). clearly show that the epidermis responded to

DV light irradiation by an increase in enzyme activities by 48 to 72 hours' after irradiation Histological changes occur in epidermal cells and this increase was maintained until the end in human (6) and rhesus monkey (12) when of the experiment (one week). exposed to ultraviolet light: i.e. 24 hours after It appears that the dehydrogenase activities irradiation there is some degeneration; after (except LDH activity) in the irradiated epi40 hours, intercellular edema; and after 70 dermis tend to decrease within 24 to 48 hours hours, increased mitotic activity and new after the irradiatiun, although these decreases cellular layers under the desquamating prickle were not conspicuous when compared with cells. These morphological changes are easily the drastic increases in the later recovery phase seen in the unstained frozen dried sections of the irradiation. used in this study. In Figure 1, the changes in 1M5CUS5ION DNA content in the epidermis are summarized. The DNA content of the cell is known to reProliferation of epidermal cells is suppressed main constant even during changes in cellular temporarily during the early periods after DV volume (13). For this reason, it is used as a light irradiation. This suppression is followed reference substance in terms of which the by a hyperplasia and increased mitetie activichemical composition of a tissue is expressed ties (12). The present study presents the en(14). Therefore, the enzyme activities in this zymatic aspects for these histological changes, study are expressed in terms of the DNA con- The decreased enzyme activities within the tent. The enzyme activities produced by ultra- first 48 hours may reflect the suppression of violet light and the enzymatic changes during cellular activities in the epidermis. The greatest the recovery phase are outlined in Figure 2. decrease in glyeolytic enzyme activities coinThe nonirradiated epidermis on the opposite cided with the greatest glyeogen accumulations side also appeared to be affected by the irradia- in human epidermal cells (15). The increase in tion. Within the first 48 hours after irradiation, activities in a later stage ef recovery can also the nonirradiated tissues generally showed be regarded as a burst of cellular functions, ingreater activity of all 6 enzymes than the eor- cluding synthetic as well as mitotie activities RESULTS

20 j- DNA CONTENT

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az DL

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I

0

1

I

I

2

3

4

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DAYS AFTER UV LIGHT IRRADIATION

FIG.

1. DNA content in epidermis. Changes in DNA content in irradiated (—) and

nonirradiated (—--—) epidermis were computed from data on three animals.

331

UVL AND DEHYDROGENASE ACTIVITIES

GOPDH

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GAPD6

0.6 2.5 0.5

0.4

0.5

/ IJ,-/----

2.0

-

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1.0

0.2

0.5

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2. Effect of ultraviolet irradiation on epidermal dehydrogenases. The enzyme activities are expressed as moles/hr/g tissue DNA in the ordinate. Each value is an average of 18 determinations (6 determinations each from 3 animals) on each enzyme -activity in the FIG.

and nonirradiated (— — —) epidermis. The dark band represents the limit of 5% confidence that was computed from 30 determinations on normal epidermis obtained from 5 different animals.

irradiated (—)

(6, 12). These increased enzyme activities provide a reasonable explanation for the glucose path during the tissue repair; i.e., that glucose is utilized by the actively proliferating tissue not only to produce energy (APT) through the Embden-Meyerhof pathway but also to provide certain basic substances for nucleic acid and

fatty acid metabolism through the pentose pathway.

It may be significant that nonirradiated symmetrical areas 2 to 3 inches away from the irradiated skin also responded to UV light irradiation, showing a fluctuation in enzyme activities, at least within the first 48 hours, but to a lesser degree than that seen in irradiated skin. The effect of TJV light on nonirradiated epidermis has also been observed in relation to glycogen formation after UV light injury (15).

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skin and the effect of X-rays. J. Invest.

SUMMARY

Fluorometric microenzyme assay with 0.5—2 g

tissue sample in 10—15 d of an appropriate

Derm., 37:

381, 1961.

6. Daniels, F., Jr., Brophy, D. and Lobitz, W. C., Jr.: Histochemical responses of human skin following ultraviolet irradiation. J. Invest.

reaction mixture provided a sufficient measurement of enzyme activities in the limited area of irradiated epidermis. The epidermis of the hack skin irradiated by 10 erythema doses of ultra-

7. Tm, M. J. C., Yamasawa, S. and Adachi, K.: Quantitative histochemistry of the primate skin. III. Glycecaldehyde-3-phosphate de-

violet light responded with initial slight de-

8.

creases in all of the 6 enzymes within the first 48 hours followed by a 2-fold increase 72 hours after irradiation. The nonirradiated epidermis

seemed to be affected by the irradiation of

Derm.,37: 351, 1961.

hydrogenase. J. Invest. Derm., 47: 35, 1966.

Im, M. J. C. and Adachi, K.: Quantitative

histochemistry of the primate skin. V. Glucose-6-phosphate dehydrogenasa and 6-phosphogluconate dehydrogenase. J. Invest. Derm.,47: 121, 1966.

9. Tm, M. J. C. and Adaehi, K.: Quantitative histochemistry of the primate skin. VI. Lac-

tate dehydrogenase. J. Invest. Derm., 47: 286, 1966. ties that fluctuated during the recovery from 10. Adnchi, K. and Yamasawa, S.: Quantitative

symmetrical hack skin, showing enzyme activi-

the radiation "injury," hut to lesser degree than the irradiated epidermis.

histochemistry of the primate skin. I. Hexokinase.

11.

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