Stowell (2) has demonstrated that the nucleolar volume in actively proliferating cells

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1 THE JOURNAL OF JNvgsnoATvE DERMATOLOGY Copyright i565 by The Williams & Wilkins Co. Vol. 45, No. 2 Printed in U.S.A. AN ULTRASTRUCTURAL STUDY OF NUCLEOLAR ENLARGEMENT FOLLOWNG ULTRAVOLET RRADATON OF HUMAN EP]DERMS* THOMAS E. NX, JR., M.D., ROBERT E. NORDQUST, BA., J. RANDOLPH SCOTT, B.A. ANO MARK ALLEN EVERETT, M.D. The nucleolus has been studied intensively in many species and has been found to change markedly in response to physiologic and patbologic stimuli. Tewari and Bourne (1) have recently reviewed the biochemical evidence indicating that size of the nucleolus is related to the synthetic activity of the cell. nvestigative studies indicate that nucleolar volume is usually largest before or during an increase in nucleolar ribonucleic acid content; this, in turn, precedes most active protein synthesis by the cell. Stowell (2) has demonstrated that the nucleolar volume in actively proliferating cells of embryonic mouse pancreas is two to three times that of adult mouse pancreatic nucleoli. Caspcrsson (3) reported that the cytoplasmic nucleotides and proteins were considerably reduced in pancreatic acinar cells after pilocarpine injection but were rapidly resynthesized during restoration of zymogen granules. Extending this work, Stowell measured nucleolar volumes and found them to increase during the resynthesis of zymogen granules. Castro and Foraker (4) studied nucleolar mass in normal rat livers as well as in regenerating livers of partially hepatectomized rats. By interference microscopy, the nucleoli of regenerating liver cells were found to have a larger mean mass than those from normal liver. Although a number of investigations have been concerned with the effects of ultraviolet light on the nucleolus in tissue culture cells (5, 6, 7, 5, 9, 1, 11, 12), the effects on the nucleolus in the intact animal have not previously been studied. This report concerns ultraviolet lightinduced nucleolar changes in human epidermis studied Received for publication September 25, * From the Department of Dermatology, University of Oklahoma Medical Center, Oklahoma City, Oklahoma. This work was supported by a grant from the Division of Ceneral Medical Sciences of the U.S. Public Health Service. 114 by electron microscopy. The resolution obtained with this instrument permitted precise measurements of nucleolar size as well as observations of qualitative changes. MATEaAL AND METHODS Four times the minimal erythema dose of medium pressure, mercury vapor irradiation filtered through 2 cm of water was administered to the left lumbar area of a normal 24 year old white male. Biopsy specimens were obtained without anesthesia from the irradiated site and symmetrical control site at intervals of 1, 3, 12, 72 hours and 1 week. Tissues were fixed immediately in cold, 1%, phosphatebuffered (ph 7.3) osmium tetroxide solution, postfixed in 1% neutral buffered formalin, and dehydrated in graded alcohols. Specimens were embedded in Maraglas epoxy resin (13) followed by curing at 6 degrees C for 48 hours. Sections 2 to 5 A thick were cut on a PorterBlum microtome and placed on copper grids. After staining with uranyl acetate, they were studied in an RCA EMU3F electron microscope at 5 KV. Stratum spinosum nucleolar areas (area = rab) were computed from micrographs of epidermis in the 12 and 72 hour and 1 week specimens. The longest axis (A) of a given nucleolus was measured and the second measurement (B) was of an axis perpendicular to the longest axis and at the midpoint of the longest axis (Figure 2). Measurements were carefully restricted to include only nucleoli in cells distal to the stratum basale aod proximal to the stratum granulosum. Tbe aim was to exclude influences on nucleolar size resulting from mitosis (stratum basale) or other nuclear transformations (stratum granulosum). No measurements were taken of nucleoli unless they were well delineated and clearly separate from the nuclear membrane and nucleolusassociated chromatin. Thick sections (1 to 3 y) were cut from the same specimens and prepared for study by light microscopy. The thick sections were treated with an epoxy solvent (14) for one minute. After rinsing with water, they were stained for one minute in a crystal violet solution (1% in 5% aqueous sodium bicarbonate). RESULTS Careful examination of many crystalviolet stained sections by light microscopy revealed

2 ULTRAVOLET NDUCED NUCLEOLAE ENLARGEMENT 115 c "AarnJYSP"aW %1 Wis SiZ' 4 4 ' S ' p t i j ' r } / s. 7 4 a, FG. 1. Epidermis (stratum spinosum), 72 hours postirradiation. Nucleus (N). Nucleolus (No). Large particle portion of nucleolus (Lp). Small particle portion of nucleolus (Sp). Ribosomes (R). Mitochondrion (M). Melanin granules (Me). Dense body (Db). Tonofibrlls (Tf). Golgi vesieles (G). Vacuole (V). X 13,3. *,Tf.T? V * w.c. #_,; V that nucleolar size was increased in the 72 hour postirradiation skin when compared with control skin. However, accurate measurement of nucleolar diameters in the thick sections used for light microscopy was impossible. There was no increase in the number of nucleoli per nucleus, and nuclear size did not seem altered. Nucleoli in our specimens had the two principal structural components described by previous investigators (15) (Figs. 1 and 2). One zone contained particles of fairly uniform size, measuring in diameter from 145 A to 289 A (mean of 25 A). These were similar in size and character to particles in the nucleoplasm and cytoplasm which have been identified as ribonucleoprotcin particles (ribosomes). The other major portion of the nucleoli was composed of zones of smaller, less well resolved particles of variable density. Figures 1 and 2 depict the structure of the nucleolus in the 72 hour postirradiation epidermis. The irradiated nucleoli exhibited no readily apparent morphologic differences from the unirradiatcd nucleoli except for increase in size. A summary of nucleolar measurement data is given in Table. The nucleoli were roughly spherical to ellipsoidal in shape. Thickness of stratum corneum was essentially normal in the 12 hour specimen but was markedly increased in the 72 hour and 1 week postirradiation specimens (Fig. 3). STATSTCAL ANALyss Since the variances of the different biopsy specimens were heterogeneous, nucleolar measurement data were analyzed using a weighted

3 116 THE JOUENAL OF NVESTGATVE DEEMATOLOGY,,: ij''j,. 'Spi:' :. FG. 2. Epidermis (stratum spinosum), 72 hours postirradiation. Nucleus (N). Nucleolus (No). Large particle portion of nucleolus (Lp). Small particle purtion of nucleolus (Sp). Ribosomes (R). Mitochondrion (M). Arrow A indicates longest axis of nucleolus. Arrow B indicates axis perpendicular to midpoint of longest axis. )< 3,685. TABLE Measurements of nucleoli (N = number of nucleoli measured. Mean expressed in 5cb) Time Treatment N Mean Std.Error 95% Conf. Limit 12 hrs rrad hrs Unirrad. 58 ' ' to '1.183 to hrs rrad hrs Unirrad. 9'l to to week rrad to week Unirrad '1.799 to analysis of variances of a completely randomized design. The weighted analysis yielded an J? ratio which was significant with p <.1. Because of the heterogeneity of the variances and the disproportionate sample sizes, further tests were not possible. Ninetyfive percent confidence limits were computed for the means of each of the biopsy specimens and these are depicted in Figure 4. The only group which is clearly different is the 72 hour postirradiation specimen. Variation in the width of the confidence limits is an indicator of the heterogeneity of the variances. DSCUSSON The specific function of nucleoli in cells has not yet been definitely ascertained. t has been well established, however, that the cell nucleus

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5 118 THE JOURNAL OF NVESTGATVE DERMATOLOGY is a primary site of ribonucleic acid synthesis. Much evidence has accumulated which suggests that the nucleolus plays a predominant role in RNA synthesis in the nucleus (16, 17, 18). Particles aggregated in the nucleoli in our specimens measured 145 A to 289 A in diameter (mean 25 A). This is consistent with the observations on nuclear particle size by Allfrcy (18) who suggested that particles of this size were nuclear or nueleolar ribosomcs. t is necessary to determine whether the nucleolar enlargement observed in our preparations is a sign of (1) increased metabolic activity, (2) metabolic block with accumulation of materials in the nucleoli, or (3) nucleolar degeneration. Since nucleolar morphology seems normal except for the increase in size, there is no evidence of degeneration in the organelle. The increased thickness of the stratum corneum observed in the 72 hour and 1 week postirradiation specimens is believed to be due to enhanced synthesis of keratin, the fibrous protein which is the end preduct of epidermal cellular differentiation. The temporal coincidence of kcratin synthesis with nueleolar enlargement suggests to us that the increase in nuelcolar size is associated with increased metabolic activity and protein synthesis rather than with metabolic blocking. However, observations will have to be made to correlate these morphological changes with biochemical or cytochemical evidence of increased nuclcolar RNA synthesis in order to substantiate this conclusion. SUMMARY Biopsy specimens of normal human epidermis irradiated with ultraviolet light were studied by electron microscopy. A marked increase in size of nucleoli in the spinous layer of the epidermis in the 72 hour postirradiation specimens was observed. Statistical analysis of the size of the 72 hour irradiated and control nucleoli showed this increase to be significant. Nucleolar enlargement correlated with stratum corneum thickening suggests that nuelcolar enlargement is associated with increased protein synthesis by the epidermis. REFEHENCES 1. Tewari, H. B. and Bourne, G. H.: The histochemistry of the nucleus and nucleolus with reference to nueleoeytoplasmic relations in the spinal ganglion neuron of the rat. Acta Histoehem., 13: 323, Stowell, H. E.: The relationship of nucleolar mass to protein synthesis. Exp. Cell Res. Suppl., 9: 164, Caspersson, T., LandstroemHyden, H. and Aquilonius, L.: Cytoplasmanukleotide in liweissproduzierenden Druesenzellea. Chromosoma 2: 111, Castro, S. and Foraker, A. G.: Nueleolar mass in rat livers. Arch. Path. (Chicago), 74: 495, Carlson, J. C. and Hollaender, A.: mmediate effects of low doses of ultraviolet radiation of wave length 2537 A on mitosis in the grasshopper neuroblast. J. Cell Comp. Physiol., 23: 157, Montgomery, P. O'B., Jr. and Bonner, W. A.: A new technique for ultraviolet microbearn irradiation of living cells. Arch. Path. (Chicago), 66: 418, Errera, M., Ficq, A., Logan, H., Skreb, Y. and Vanderhaeghe, F.: Nucleocytoplasmic relationslups ia irradiated cells. Exp. Cell Hes. Suppl., 6: 268, Gaulden, M. E.: The nucleolus and mitotic activity in the cell nucleus. n The Cell Nucleus (J. S. Mitchell, ed.), pp London, Butterworths, Perry, H. P. and Errera, M.: The influence of nucleolar ribonucleic acid metabolism on that of the nucleus and cytoplasm. n The Cell Nucleus (J. S. Mitchell, ed.), pp London, Butterworths, Perry, H. P., Hell, A. and Errera, M.: The role of the nucleolus in ribonucleic acid and protein synthesis. ncorporation of cytidine into normal and nucleolar inactivated HeLa Cells. Biochem. Biophys. Acta, 49: 47, Errera, M., Hell, A. and Perry, H. P.: The role of the nucleolus in ribonueleic acid and protein synthesis. Amino acid incorporation into normal and nucleolar inactivated HeLa Cells. Biochem. Biophys. Aeta, 49: 58, Montgomery, P. O'B.: Experimental approaches to nucleolar function. Exp. Cell Res., 9: 17, Spurlock, B.., Kattine, V. C. and Freeman, J. A.: Technical modifications in Maraglas embedding. J. Cell Biol., 17: 23, Mayor, H. D., Hampton, J. C. and Hosario, B.: Simple method for removing the resin from epoxy embedded tissues. J. Biophys. Biochem. Cytol., 9: 99, Porter, K. H.: Problems in the study of nuclear fine structure. n V nternat. Kongr. f. Eleetronenmikroskopie, Bd., pp Berlin, Springer, Busch, M., Muramatsu, M., Adams, H., Steele, W. J., Liau, M. and Smetana, K.: solation nucleoli. Exp. Cell Hes. Suppl., 9: 15, Sirlin, J. L. and Jacob, J.: Function, development and evolution of the nucleolus. Nature, 195: 114, Allfrey, V. C.: Nuclear ribosomes, messenger RNA and protein synthesis. Exp. Cell Hes. Suppl., 9: 183, 1963.