In vivo ultraviolet radiation (290-350 nm, 800 J/m2
) in the absence of any exogenously added sensitizer induced oross-ltnkirng of DNA to protein in chromatin isolated from guinea pig epidermis.
The chemical properties and the biologic activity of epidermal
chromatin isolated from both irradiated and non-irradiated
specimens were examined. One hour post-irradiation the lesion was
no more detectable, indicating that a repair process was effective
1n the viable mammalian epidermis

A . Kornhauser

E. Zimmermann

G. Szabo

M. A. Pathak

English

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CROATICA CHEMICA ACTA CCACAA 48 (3) 385-390 (1976) CCA-946 YU ISSN 0011-1643 .577.1 :614.875 Note The in vivo Effect of Ultraviolet Irradiation (290-350 nm) on Epidermal Chromatin A . Kornhauser*, M. A. Pathak**, E. Zimmermann*** , and G. Szabo* *Harvard Scholl of Dental Medicine Boston, Massachusetts 02115, **Department of Dermatology Harvard Medical School Boston, Massach1isetts 02115 and ***University of Munster Physiological Institute 44 Munster, Germany Received March 30, 1976 In vivo ultraviolet radiation (290-350 nm, 800 J/m2) in the absence of any exogenously added sensitizer induced oross-ltnkirng of DNA to protein in chromatin isolated from guinea pig epi-dermis. The chemical properties and the biologic activity of epi-dermal chromatin isolated from both irradiated and non-irradiated specimens were examined. One hour post-irradiation the lesion was no more detectable, indicating that a repair process was effective 1n the viable mammalian epidermis. Ultraviolet (.uv) <radiation o.f wavelengths shorter than 350 nm from sunlight and artificial sources ilS kno:vn1 to have profound effects on a number of cellular functions. Deoxyrilbonucleic acid is the most sensitive ,target of uv p.hotons1. The J"elative yields o.f different types of photo-.products that are prnduced in DNA, however, oan vary depending upon 1the tyipe of organism used, 'the irradiation conditions, wavelengths, and the physical state and ·the enviTon-ment -0f the DNA dur.ing irradiation. Although the thymime dimer .iLs 1still the most thoroughly studied lesion shown to be of .great biological importance, there is an ever increasing number of observatitons being .reported which suggest tha1t there are -0ther types of uv damage responsible for !biological alteratio1ns2 • As DNA does not exist in a cell 1n pure, solution, but .i1s iln intimate contact with proteins, RNA, lipids, and other biomolecules, it •]s conceivable that photochemical heteroadducts can occur and play a role .in the radiation induced molecular and pathological lesions to cells. The ·cvoss-linking of DNA and protein in bacteria was the first in viva photochemical heteroadduct reaction to be rep°'rted3. There have been some studies reported on uv induced DNA and proteiln oross-links in mammalian cells which were mainly ba.sed on the reduced DNA extracta:bility after uv radiat,ion4•5• Evidence .that this lesion plays a si1gnHicam.t role im. the killing of uv irradiated cells has been shown for ·several experimental condLtions2 • The chemical nature of the DNA-plftotein cross-linkis is as yet not known. An in vitro photochemical Teaction between thymine and cysteine has been observed which may be one of the possible mechanisms for the covalent linking of DNA to protein in vivo6 • Furthevmore, 11 of the common amino acids have been shown to combine photochemically with uracil in different model systems7 • 386 A . KORNHAUSER ET AL. All .these findings .sug.gest that uv irradiiation can i:nduce significant alterations of the chromosomal material, the chromatin. Bearing this in mind, we foOO\Sed our investigati'Ons in the present woirk on the isolation of chro-matim. from irradiated and non-irradiated epidermal cel1s of guinea pig skin. F·or thiJS purpose the backs of 6 adult albino guim.ea pigs (700-900 g) were epilated8, a!Ild 6 days post-epilation, i:rradiated under a 400 watt high pressure mercury lamp equipped with a replaceable yellow cellophane :fiilter which trasmi.tted wave:lengths 290- 350 mn. The back of each animal was div1ided into two equal sections prilor to irradiation. The right secti!on (3.5 X 10 cm) was irradiated, and the left half served as a non-irradiated control. Each animal .received a dose of 800 J /m 2 which was equivale.n:t to approximately 5 times the minimal erythema dose (MED)*. Animals were sacrificed immediately after irradiation, and 60 minutes after irradiation. The epidermis, iincluding the horny layeT, of -irradiated and non-irradiated skin was is olated by the stretch method8, and homogenized. Chromatin from the so obtaimed epidermal homogenates was isolated according to Bonner's procedure9, and purufied by centrufigation .in 2M sucrose solution. The isolated epidermal chromatin sho-wed an optical density ·ratio (260/230) of 0.95. Its transcription ability was studred with E. coli RNA polymerase according to Burgess' method10, 1and was found to be 18-230/o as compared to free epidermal DNA. Fractionation and di1ssociation of 1the chromosomal complex were achieyed by column chroma-tography on sepharose B4-200 and DEAE cellulose columns with 2M NaCl as the elua:nt. Optical densities at 260 and 230 nm were obtaiined for each f.rac-tion. Changes ill molecular weight of DNA isolated from uv ir.radiated and non-irradiated epidermis were examined by centri:fiugati:on of the ·isolated DNA on a sucrose density gradient (5-25°/(); SW 29 .rotor, 6 hours at 25,000 rpm), and the sedimentation profiles were compared w.ith a standard DNA of known molecular weight, (Thymus DNA, M. W. 7.2 X 106) . Figure 1 shows the fra'Ctionation profile of epidermal chromatin on a DEAE cellul·ose column. Fractron 1 represents p110tein fraction that contains niootilnamtde adenine idilnucleotide (NAD). Fract1on 2 contains protei1ns and traces of NAD and RNA. We could also detect 1similarr amounts of NAD afteir fractionation •of human skin. Fraction 3 is the DNA devoid of protein, and fraction 4 represents a nucleo--protein complex. This lia1st fractffi0111 could not be dissociated with 2M NaCl, and this finding constitutes an interesting cha-racteriistic of epidermal chromatin. Figure 2 shows a representative fractionation profile of epidermal chro-matin on sepharose B4 columns before and 1after uv irradiation. The majo.r changes 1upon irradiation are seen i n the first peak, which Tepresents epidermal DNA. I:n the noon-irradiated specimen (A), the optical density rafao {260/230) is 1.99, whereas in the lower curve the optical density ratio dropped to about 1. This drop in optical density ratio as well as the presence of :proteins (8-11°/<i) as determined by Low;ry's 'test in this fraction, indicate the cross--li:nking ·of DNA and prntein. Peak 2 represents histones and acidic pDoteins. Peak 3 is the staible DNA-pwtern complex. This fraction is apparently not affected by uv irradiation, and is presently under dnvestigation for its iphysical--chemical properties. * The MED is defined as the minimal dose of uv irradiation (290---320 .nm) that produces def.inite, but minimally perceptible redness, at 24 hours afteJ' exposure. EPIDERMAL CHROMA TIN 387 a.o FltACrtOlt ltUMIClt Figure 1. Fractionation profile of skin chromatin on a DEAE cellulose column (1.5 X 120 cm). Elution: linear gradient, 250 ml of 0.001 M sodium phosphate pH 7.2 to 2 M NaCl in sodium phosphate, pH 7.2. Fraction volume 5.0 ml/20 min. OD at 260 nm (---) OD at 230 nm (- -). Fraction 1 represents protein and nicotinamide adenine dinucleotide (1 mole NAD per 50 moles nucleotides) . Fraction 2 is protein, traces of NAD and RNA. NAD' was determined on the basis of the characteristic absorption of its reduced derivative at 340 nm. Fraction 3 is DNA; Fraction 4 is the stable DNA-protein complex. Fraction 4 contained 7-9°/o protein and had a transcription ability of 7-80/e as compared to free epidermal DNA. t .O o.s ... ... ~ :: 0 .. ~ ~o ' .. OS 0 A B tO .. ------.:: .. :: .., .. -.. ········-················-··· 40 00 '"""""' 1111nc11 Figure 2. Fractionation profile of epidermal chromatin on a sepharose B4 (2 X 40 cm) before (A) and after (BJ uv irradiation. Irradiation dose r.oo J /m', 290-350 nm. Eluant 2 M NaCl, in 0.1 M EDTA, fraction volume 2.0 ml, flow rate !/ml/min. OD ratio 260/230 of .peak 1 in A= 1.99, In B 1.0. Number 2, fraction 50-65 represents histones and acidic proteins. Number 3 is the stable DNA-protein complex. The fractionation profile of chromatin isolated from epidermis one hour after uv irradiation is shown in Figure 3. The optical density ratio of peak 1 is back to 1.99, the original control level, indicating that an active repair process has resulted in the elimination of the DNA-protein cross-linkage. The chromatin pattern ii.n peak 3 did not show any -significant changes, again suggesting that thiis DNA-protein complex is unaffected by uv radiation. 388 .. .. !:! 1.0 ~ 0.5 a •o A. KORNHAUSER ET AL. f {\ ; : fl f \ ! .................. . ...... ........ 20 30 40 Figure 3. Fractiona tion profile on sepharose B4 of epidermal chromatin 60 minutes post-irradi-ation, (regeneration phase). Peak number 1 represents DNA with a 260/ 230 ratio 1.99 ; number 2, fraction 52-67, is histones and acidic proteins; nu!'Jlber 3, DNA-protein complex. The phy-sical properties and the transcription ability of the chromatin material isolated 60 minutes postirradiation corresponded to the chromatin obtained from the non-irradiated control specimen. With our techniques we are able to ·obtai:n 4-5 mg of ex<tractatble DNA free of pI10'tein from 1 gram of wet epidemna1 tissue. Immediately 1after uv radiation the yield "Of this extractable DNA is reduced by 20-300/o, ' presu-mable as a .result of DNA to protein cross-linking, and possibly to DNA strmd breakage. This later observation i;s consi•stent · wi·th our previously reported fi:ndings8, as well as with the reported findings of single and double strand breakage of DNA upon uv (> 290 nm) irradiation16• We also 1suggested that the epidermal DNA is the primary chromophore. for uv absorption. Its main lesiio.ns are the result of direct absorption of uv ·by sk.in DNA, and apparently do not proceed substantially through a photosensitized reaction by triplet energy transfer from some other molecules11 . A compa,dson of the sucrose gradient centrifigation prof.iles of the uv irradiated epidermal chromatin aind the non-irradiated epidermal DNA, i'Il.clu-ding a calf thymus DNA (molecular weight 1.3 X 106) as an intemal standard showed: a) a significant breakdown -0f high molecular weight DNA fracti"Dn and the presence of small molecular weight DNA fragments o.n the top of the sucrose .gradient after uv irradiation; and b) an increment of the high molecular DNA after 60 minutes post-irradiat1on (regeneratiO'Il. or repair phase). The described experirrnents were performed 3 times using 2 antmals per eX'periment. The above results can be summairized as follows: 1) uv irradiation in physi·ologic dos.es (5 MED) of 290-350 'nm, decreaised the actual amount of dissociable chromosomal DNA by 20-300/o as a result o.f cross-linkilng of DNA to pmtein and DNA strand breakage; 2) a comparison of the corresponding elutiion priofiles from sepharose columns of the dtssociable DNA isolated from uv irradiated and :non-irradiated epidermal specimens indicated cross-linking of protein to DNA; 3) uv irradiation caused a significant breakdown ·of high molecular weight DNA that was isolated upon irradiation, and 4) in the regeneration phase there was an active repair ·process operating i:n the viable cells of the epidermis. The mechaniism of this repair process is presently under investigation. The .role of NAD in skin is beiing carefully investigated, as this molecule could serve as a chromophore that transfers ener.gy Lnto an enzyme(s) or other cell constituent for which it is a cofacto-r, leading to the alteration of EPIDERMAL CHROMATIN 389 that enzyme. Recently it was shown that ·reduced nicotinamide adenine dinu-cleottde (NADH) is highly resi:stant to uv (300-350 nm) in vitro12. Hitherto the cross-linking ·of DNA to piroteins was reported 101I1ly in che-mical model systems6,13, bacterial systems3, or in vitro systems of ttssue culture4,5, and was not exami:ned in a mammalian sy1Stem in vivo. Furthermore, this lesion, as our experiments show, already occurs under physio1o,g.ic con-ditions when skin is i.rradiated with moderate doses of uv light that are nor-mally encountered by man. It is known that diseases and disorders resu1ting from .repeated exposure of human skin to sunlight .are manifo1'ci14, and include basal and squamO'Uis carcinomas, skin agemg, actinic elastosi:s, and many photo-sensitive reactions. So far, no evidence for the cellular repair of DNA-'protein heteroadducts has been f.ou:nd. Hain15, in a recent work on DNA-p.rotein cm.ss1inking in synchmnized HeLa cells ex;pooed to uv (254 nm), reports that no recovery from this damage was .observed. Our in vivo findings, however, suggest that cross-linking of DNA to protein in mammalian skin, ·induced by wavelengths 290-350 nm is repairable. Acknowledgments. We thank Professo·r Sarah Elgtn of Harvard University for, helpful discussions. This work was supported by U.S. National Cancer Institute Grnnt # 5 ROI Ca 05003-I4 and N. I. H. Grant #: 5 ROI DE OI766. REFERENCES 1. K. C. Smith, in: T. B. Fitzpatrick (Ed.), Sunlight and Man, University of Tokyo Press (I974) 57. 2. K. C. Smith, in S. Y. Wang (Ed.), Photochemistry and Photobiology of Nucleic Acids, Academic Press, New York (in press). 3. K. C. S mi th, Biochem. Biophys. Res. Comm. 34 (I969) 354. 4. P. A 1 ex and er and H. Moros on, Nature 194 (I962) 882. 5. V. Ha b a z i m and A. Han., Int. J. Radiat. Biol. 17 (I970) 569. 6. K. C. Smith, Biochem. Biophys. Res. Comm. 39 (I970) 1011. 7. T. J e 11 in e k and R. B. Johns, Photochem. and Photobiol. 11 (I970) 349. 8. D. M. Kram er, M. A. Path a k, A. Korn h au s er, and A. W i s k em an n , J. Invest. Derm. 62 (I974) 388. 9. K. Mau rs hi g e and J. Bonner, J. Mol. Biol. 15 (I966) I60. IO. R.R. Burges •s, A. A. Travers, J. J. Dunn, and lK. F. Bautz, Nature 221 (I969) 43. Il. A. Kornhauser and M. A. Pathak, Z. Naturforsch. 27b (I972) 550. I2. T. V. Ram ab had ran, Photochem. Photobiol. 22 (I975) 117. I3. A. B r au n and B. Me r r i ck, Photochem. and Photobiol. 21 (I975) 243. I4. T. B. Fitzpatrick, M. A. Pathak, L. C. Harber, M. Se i j i, and A. Kuk it a, in: T. B. Fitzpatrick (Ed.), Sunlight and Man, University of Tokyo Press (I974) 3. I5. A. Han, M. Korb e 1 i k and J. Ban, Int. J. Radiat. Biol. 27 · (1975) 63. 16. B. E. Zierenberg, D. M. Kramer, M. G. Geisert, and R. G. Kir-st e, Photochem. and Photobiol. 14 (I97I) 515. 390 A. KORNHAUSER ET AL. SAZETAK Djelovanje ultravioletnog ozracivanja (290-350 nm) na kromatin epiderma in vivo A . Kornhauser*, M. A . Pathak**, E. Zimmermann*** i G. Szabo* Ozraeenje ultrav.ioletnim svijetlom {290- -350 .nm; 3 x 105 erg/cm2) dovelo je do umrezenja DNA i proteina u kromatinu izoliranom iz epidermisa zamorca. Jedan sat_ nakon ozracenja, ta se lezija vise nije mogla dokazati, ilto govori u prilog tomu, da u kozi sisavca postoji aktivni mehanizam oporavka, koji prepoznaje i eliminira kovalentnu vezu DNA-protein. *HARVARD SCHOOL OF DENTAL MEDICINE BOSTON, MASSACHUSETTS 02115 ••DEPARTMENT OF DERMATOLOGY HARV ARD MEDICAL SCHOOL BOSTON, MASSACHUSETS 02115 ***UNIVERSITY OF MONSTER PHYSIOLOGICAL ' INSTITUTE 44 MONSTER, GERMANY Prispjelo 30. ofojka 1976. 

The in vivo Effect of Ultraviolet Irradiation (290-350 nm) on Epidermal Chromatin

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