Thymine and Uracil Metabolism in the Epidermis* Howard P

THYMINE AND URACIL METABOLISM IN THE EPIDERMIS* HOWARD P. BADEN, M.D. As compared with the viable epidermis, the1.0 cm in diameter full thickness skin biopsy stratum corneuin contains very small amountsspecimens obtained at varying intervals. The biopsy specimens were heated at 50° C for 1 of both DNA and RNA (1, 2, 3, 4). This decreaseminute and the epidermis separated by gentle in nucleic acid content is correlated with thescraping with a scalpel. The epiderrnal tissue was disappearance of nuclei and ribosomes whichhomogenized at 0° C in .25 M perchioric acid, the occurs at the junction of the granular layer andhomogenate centrifuged and the precipitate washed two times with the same solvent. The stratum corneum. Enzymes necessary for thecombined supernatants (the cold acid extract) hydrolysis of these cellular organdIes have beencontained the nucleosides and their degradation found in the epidermis (5, 6, 7, 8, 9), but theproducts. The pellets were then treated with 10% purine and pyrimidine bases which are majorperchioric acid at 80° C for 15 minutes to extract the nucleic acids (2). The precipitates were washed components of nucleic acids are either absent orwith ethanol and ether (3 to 1), and ether to are present in trace quantities (1, 4). Some purineremove lipid and the pellets consisting largely of metabolites have been found in the stratumproteins were then dried. corneum (1, 4), but no data is available on the Aliquots of the hot and cold perchloric acid pyrimidines. Since certain disorders of the epi-extracts were pipetted into 1/2 ml of 1 M hyamine dermis such as psoriasis are associated withand 15 ml of dioxane with 4 gins 2, 5-diphenyl- oxazole, 250 mg 1, 4-bis-2(5-phenyloxozolyl)- abnormalities of nucleic acid turnover (10, 11,benzene and 100 gin naphthalene per liter (15) and 12, 13), it was felt that more detailed informationthe radioactivity assayed in a liquid scintillation on the metabolism of pyrimidines by the epi-counter. Correction for differences in quenching dermis would be of importance. The purpose ofwas made by the addition of an internal standard and the data expressed as cpm for two 1 cm biopsy this report is to describe the fate of thymidinespecimens. Radioactivity in the ethanol and ether and uridine incorporated into epidermal nucleicextracts was determined as above, while that in acids in vivo, and to investigate the catabolismthe protein pellet by a combustion technic (16). A of their respective pyrimidine bases by epidermissecond aliquot of the cold acid extract was neu- tralized with potassium hydroxide, centrifuged in vitro. and the supernatant concentrated on a rotary evaporator. Following the addition of carrier MATERIALS thymine and uracil ascending paper chromatogra- Methyl tritiated thymidine (8 c/mmole),phy was done in isopropyl alcohol, acetic acid and thymine-2-C'4 (11 mc/mmole) and uridine-2-C'4water (50:25:25: v/v/v) (14). The location of the (25 mc/mmole) were purchased from New England pyrimidines was identified by examining the papers Nuclear Corp., and dihydrothymine (PITT),under ultraviolet light and then the papers were dihydrouracil (DHU), B-alanine, and B-aminoiso-cut and the radioactivity eluted with water. The butyric acid (BAIB) from the California Bio-washings were concentrated and counted as de- chemical Corp. The B-ureidoisobutyric (BUlB)scribed above. and B-ureidopropionic acid (BUP) were prepared Two biopsy specimens 1.9 cm in diameter were as described by Fink et al (14). obtained from mice injected twelve days pre- viously with labelled thymidine. The epidermis METHODS was separated from one with heat while the other was stripped with cellophane tape to remove the Thymidine and uridine nietabolism in vivo stratum corneum. Radioactivity was determined in the epidermis and cellophane strippings by the One hundred c of labelled thymidine or 10 ZC of combustion technic (16). uridine were injected intraperitoneally into hairless mice (hr, Jackson Memorial Lab.) and twoDHT and DHU catabolism by epidermis in vitro Supported by Grant Number AM-08838 from National Institutes of Health, United States Guinea pig skin was epilated with wax (17) and Public Health Service. biopsy specimens 1.9 cm in diameter were obtained Received for publication April 9, 1966. with a dermal punch. Specimens containing the * From the Harvard Medical School, Depart-epidermis and a thin layer of dermis were prepared ment of Dermatology and the Dermatology Serv-from the biopsy material with a Stadie Riggs ice at the Massachusetts General Hospital, Bos-microtome (17). Slices of liver were prepared ton, Massachusetts 02114. similarly. Ten slices were incubated in 10 ml of 235 236 THE JOURNALOF INVESTIGATIVE DERMATOLOGY

DAYS FIG. 1. Radioactivity in the epidermis following the injection of labelled thymidine. 0—0 DNA, •—• cold acid extract.

Krebs' Ringer phosphate with either .02M DHTpapers were removed and counted in a scintillation or DHU at 370 C with constant shaking for 2 hours. counter (18). The controls used were heated tissue with substrate, tissue with no substrate and substrate RESULTS without tissue. DHT and DHTJ were used instead of thymine and uracil since they have been shown Following the injection of mice with labelled to be better substrates for measuring the de-thymidine there is a peak of radioactivity in DNA gradative pathways of the pyrimidine bases (14). The incubation mixture was adjusted to pH 5,at 24 hours, and then a slow decrease to 50% of heated for 5minutesin a boiling water bath andthe maximum value in about six days (Figure 1). homogenized at 0° C. The suspension was clarified The radioactivity in the cold acid extract drops by centrifugation and the supernatant dried in amore rapidly and remains low even while the rotary evaporator. This residue was extracted with 6N hydrochloric acid in acetone (1/20) andactivity in DNA continues to decrease. Radio- ascending chromatography done as describedactivity in the cold acid extract of blood which above. DHT, BUlB, BAIB, DHU, BUP andrepresents catabolites of the pyrimidines formed B-alanine were identified as described by Finkin the liver (19) shows the same decrease with et al (14), The amount present was estimatedtime as the epidermis. Some of the radioactivity visually on an arbitrary scale by the intensity of color developed with a known aliquot of thein the cold acid extract of epidermis may have sample. resulted from these catabolites having been deposited in the epidermis. Chromatography of the Decarboxylation of thymine by epidermis in vitroepidermal cold acid extracts showed that la- Epidermis was separated from four 1.0 cm inbelled thymine accounted for 25 to 50% of the diameter biopsy specimens with collagenase (17),total radioactivity. The remainder of the radio- and incubated in 3 ml of Krebs' Ringer phosphateactivity occurred in a number of different posi- with 5 uc of thymine-2-C14 for 2 hours at 37° C. The reaction was carried out in sealed Warburgtions on the chromatogram, but could not be flasks with filter paper saturated with 1 N po-identified more precisely because of the low tassium hydroxide in the center well to collect thelevels of radioactivity. The amount of radio- evolved C"02. Four 1.0 cm in diameter slices ofactivity found in fat and protein was less than liver prepared with a Stadie Riggs microtome were similarly incubated. The reaction wasthat in the cold acid extract at all time intervals. stopped by addition to the incubation mixture of The distribution of radioactivity in the epi- .2 ml of 1 N sulfuric acid from the side well. Thedermis 12 days after injection (Table I) indicates THYMINE AND URACIL METABOLISM 237 that most is found in the viable epidermis, while TABLE III that in the stratum corneum represents less than Catabolism of DHU by epidermis and liver 5% of that originally incorporated into DNA. Following the injection of hairless mice with BUP $-ALANINE labelled uridine, the pattern of labelling in the Liver Slices + DIIU 10+ 10+ hot and cold perchloric acid extracts is similarLiver Slices (heated) + 1+ 0 to that observed in the thymidine experiments DHU (Figure 2). Skin Slices + DHU 1+ 0 Chromatography of extracts from epidermisSkin Slices (heated) + 1+ 0 showed no thymine, uracil, DHT, DHU, BUlB, DHU BlIP, BAIB, or B—alanine. As shown in TablesDill 1+ 0 II and III there is no evidence of catabolism ofSkin Slices 0 0

TABLE IV Decarboxylation of thymine-P-C14

C"O evolved (cpm)

Skin Slices 0 Skin Slices (heated) 0 Liver Slices 2,000 Liver Slices (heated) 100

DHT or Dill following the incubation with epidermis. Furthermore, there is no decarboxylation

DAYS of thymine-2-C'4 (20) by guinea pig epidermis FIG.2. Radioactivity in the epidermis follow-(Table IV). ing the injection of labelled uridine. 0—0 RNA, •—• cold acid extract. DISCUSSION Autoradiographs of skin from animals and TABLE I humans injected with radioactive thymidine Localization of radioactivity in the epidermis show that the label is incorporated first into the nuclei of the basal layer (10, 11, 21). As cell divi- Percent of radioactivity sion occurs, the radioactivity is observed in the T0 a Cpmpresent in DNA at 24 hours upper layers of the viable epidermis, but never appears in the stratum corneum (21). This sug- Strippings 75 4% gests that the nucleoside, or the free pyrimidine, Whole Epidermis 456 25% is not retained in cells as the latter ascend from the maiphigian to horny layer. Since the deposi- tion of silver grains depends on the level of radio- TABLE II activity within a given site, there is one possible Catabolism of DHT by epidermis and liver way thymine or thymidine still could be present BUlB BAIB in the stratum corneum and not be detected. This involves the redistribution of the labelled Liver Slices + DHT 10+ 10+ compounds following hydrolysis of nucleic acids. Liver Slices (heated) + 1+ 0 The data in this study indicate that this hypothe- DIIT sis is not tenable. The cold acid extract of epi- Skin Slices + DHT 1+ 0 dermis which would contain labelled thymine Skin Slices (heated) + 1+ 0 and thymidine shows no increase in radioactivity DHT during the phase when radioactivity in DNA is DHT 1+ 0 Skin Slices 0 0 decreasing. The presence of several unidentified radioactive 238 THEJOURNALOF INVESTIGATIVE DERMATOLOGY 0 0 0 L OH HN('?C—CH3 H2 HN CH-CH3 H20 H2N H20 CH-CH3 I II I cjHCH3 O=CCH 0C CH H H H H

Thymlne Dihydrothymine /3—Ureidoisobutyric ,4—Aminoisobutyrc acid acid FIG.3. Catabolism of thymine spots in the cold acid extracts could result fromreabsorption of purines or purine catabolites circulating metabolites deposited in the epidermiswould explain an increased pool of uric acid. or epidermal metabolism of thymine. The cata- Although it appears that the principle way bolic pathways for thyrnine are shown in Figurepyrimidines are lost by the epidermis is reabsorp- 3; those for uracil are similar but involve thetion into the circulation, a small amount may formation DHU, BlIP and B-alanine. Failure tobe shed with the stratum corneum. This is indi- demonstrate any of these pathways suggest thatcated by the presence of radioactivity in cello- no metabolism of thymine or uracil occurs in thephane strippings of the epidermis. Since this epidermis. represents less than 5% of radioactivity origi- There are several alternate hypotheses for thenally incorporated into DNA it must be regarded handling of pyrimidines by the epidermis: (1)as a minor pathway. They are recycled into nucleic acids and remain trapped in the viable epidermis. (2) They diffuse SUMMABY rapidly through the stratum corneum and are Hairless mice have been injected with radio- lost at the surface of the skin. (3) They are reab-active thymidine and uridine and the pattern of sorbed into the systemic circulation. labelling in the epidermis investigated. The data The phenomenon of recycling of thymine andsuggest that pyrimidine bases released following uracil is unknown in other tissues. The amount ofthe hydrolysis of nucleic acids are reabsorbed radioactivity in nucleic acids would not haveinto the systemic circulation. This is confirmed dropped in the in vivo experiments if the pyrimi-by the failure to observe any degradation of dines had been reutilized for nucleic acid syn-thymine and uracil by epidermal tissue. Small thesis. Furthermore, the half life of DNA (T )amounts of these bases may be lost in the stratum correspondsclosely to that of epidermal cells ascorneum, but this represents a minor pathway. measured by other techniques (10, 21). Rapid diffusion of pyrimidines through the stratum corneum seems most unlikely consider- REFERENCES ing the known low permeability of this structure. 1. Hodgson, C.: Nucleic acids and their decom- Other epidermal metabolites such as uric acid, position products in normal and pathological horny layers. J. Invest. Derm., 89: 69, 1962. amino acids, and urocanic are not lost in this 2. Santoianni, P. and Rothman, S.: Deoxyribo- manner (22). nucleic acid microdetermination in human epidermis. J. Invest. Derm., 40: 317, 1963. The most tenable hypothesis to explain our 3. Santoianni, P. and Ayala, M.: Fluorometric observations is that the pyrimidines are reab- ultramicroanalysis of deoxyribonucleic acid sorbed into the circulation. There is good evi- in human skin. J. Invest. Derm., 45:99, 1965. 4. Wheatley, V. R. and Farber, E. M.: Chemistry dence for the reabsorption of purine bases or their of psoriatic scale: Further studies of nucleic catabolites from the epidermis. It has been shown acids and their catabolites. J. Invest. that patients with active psoriasis have an in- Derm., 89: 79, 1962 5. Block, W. D. and Johnson, D. W.: Studies of creased pool size and urinary excretion of uric the enzymes of purine metabolism of skin. acid (23). The magnitude of the uric acid pool J. Invest. Derm., 28: 471, 1954. follows the amount of scaling and returns to 6. DeBersaques, J.: Nucleosidases in human epidermis and in normal and abnormal normal when the disease is in remission. In these scales. J. Invest. Derm., 38: 133, 1962. patients the number of cells lost per day, and 7. Liss, M. and Lever, W. F.: Purification and hence the amount of nucleic acids metabolized, characterization of ribonuclease from psori- astic scales. J. Invest. Derm., 89: 529, 1962. may be 50 times normal. A markedly increased 8. Santoianni, P. and Rothman, S.: Nucleic acid THYMINE AND URACIL METABOLISM 239

splitting enzymes in human epidermis and16. Baden, H. P.: Improved technique for deter- their possible role in keratinization. J. mination of C'4 and H by flask combustion. Invest. Derm., 87:489,1961. Anal. Chem., 80:960,1964. 9. Tabachnick, J.: Studies in the biochemistry17. Baden, H. P. and Peariman, C.: The effect of of epidermis. II. Some characteristics of ultraviolet light on protein and nucleic deoxyribonucleases I & II of albino guinea acid synthesis in the epidermis. J. Invest. pig epidermis and saline extracts of hair. J. Derm., 48:71,1964. Invest. Derm., 42:471,1964. 18. Buhler, D. R.: A simple scintillation counting 10. Epstein, W. L. and Maibach, H. I.: Cell re- technique for assaying C'40, in a Warburg newal in human epidermis. Arch. Derm. flask. Anal. Biochem., 4: 413, 1962. (Chicago), 92: 462, 1965. 19. Rubini, J. H., Cronkite, E. P., Bond, V. P. 11. Kaku, H., Igarashi, Y. and Fujita, S.: Cyto- and Fliedner, J. M.: The metabolism and kinetic analysis of the human skin in vivo fate of tritiated thymidine in man. J. Clin. in normal and pathologic conditions: A 3H Invest., 89:909,1960. thymidine autoradiographic study. Arch.20. Fink, K., Cline, R. E., Henderson, R. B. and Histol. Jap., 24: 457 1964. Fink, R. M.: Metabolism of thymine 12. Weinstein, G. D. and 'Jan Scott, E. J.: Auto- (methyl-C14 or -2-C') by rat liver in vitro. radiographie analysis of turnover times of J. Biol. Chem., 221:425,1956. normal and psoriatic epidermis. J. Invest.21. Fukuyama, K. and Bernstein, I. A.: Auto- Derm., 45:257,1965. radiographic studies of the incorporation 13. Van Scott, E. J. and Ekel, T. M.: Kinetics of thymidine-H' into deoxyribonucleic acid of hyperplasia in psoriasis. Arch. Derm. in the skin of young rats. J. Invest. Derm., (Chicago), 88:373,1963. 80:211,1961. 14. Fink, R. M., McGaughey, C., Cline, R. E.22. Tabachnick, J.: Studies on the biochemistry and Fink, K.: Metabolism of intermediate of epidermis. I. The free amino acids, am- pyrimidine reduction products in vitro. J. monia, urocanic acid and nucleic acid con- Biol. Chem., 218:1,1965. tent of normal albino guinea pig epidermis. 15. Takahashi, H. and Bunji, M.: Liquid scintilla- J. Invest. Derm., 32:563,1959. tion counting of biological compounds in23. Eisen, A. Z. and Seegmiller, J. A.: Uric acid aqueous solution. Anal. Chem., 85:1982, metabolism in psoriasis. J. Clin. Invest., 1963. 40:1486,1961.