Biologica! consequences of the photohinding of furocoumarin molecules with nucleic acids

J.. \1lS\.III

lns/111111' o( 1'/wrmflrl'll l l! ul (.IJ,•mi.~rr' _ l , ;,...,s.,,· ()( l'ml11 u. /tu /1

Furor<Huuariu~ ar1· a wt' ll·kiH: I\11 group of natu ra l 111 .,~ ut l ~t·IÌ•· sult<>taii­

Ct'l> . sonw ol' wlticiJ llè1r Ìui- ILlnt·•· p-.ural•·n (l). :-.antboto"in ( Il ), lu·rgapt•·••

(H l ). R-nwthylp t:nralf'n (TY)J 1''\.Prl !i lriJn~ photo,_,,,,,.iti;-in~ propPrlÌt"· o n

o:;o "o @

o o ~o vco ~o OCH3 CH3 II 111 IY

'arwu~ hinlogic·a l su hs trat t•;. h~ irraclia tio n al 36;) um (.'\1t.., \ ,IO. BtmtGIIJ EHil

8. ~AI'OHAJ,F., J 9:>-J.: ~11 !-AJO , ., ul.. llJ.)H : .\1 n•AJO & Hot>t <.: lll FJW. J \)h~ : PATIIAK 8.. FtT ZPATRICh., 19.)9 : P ATIIAK. FFJ, J.:\1-\'\ 8. "\lF\1\'\. ]9110:

PA'l'IIAK , \\ ORDEN & K .H lF:'\1.\ "-, 1Wt7). A.., the i1· mo,.t 1-'llldit·d rO't·c· t i..- skiu­

photosrnsi t izu t iuu , t br ac1 j, c• ;.ul. ,:tann·s wt•r.- dt>li•wtl lt y li " ., .., · ,.,kin-phn­

t nl'l<'USÌI Ì7.in g » (M t •sA.IO 8.. H tml<.: 11 1 F. HO. l Qft2) . BP:-: id,• t h•· a c· l i\ t' :miiKl au<·c·,; .

rnau y furocou mar iu d •· r iva li \ t·~ Pxist w h icl1 ari' in ar i i\ t'.

Tlw h inl u gical pho t n*'<'IH•Ì i i7.i n g p ro twrtit•>- of furo('llllllla r ill ,'- uwy lw t>xpluin f'd hy a photon·aet inn b r t 11 t' l'Il tl u•,;t • suh!<taucp;; ::111d n twl t·Ìt· a!'Ìth:.

' ' h irh wa~ r,.,·,.alcd for th t· fir:< l 1 inw in nur laboratory jj, , . ~·t•ar,. ago (i\lt ­SA.! O. H ouu:tiiF.RO &. DAu · 1\.CQI'A. l 9(,5) aud "hil'h tak••,., plal't' wlwn an

aqu coU!, !ln lu t ion of nuclei<' twid ., and photosens itizing furo t'llnnw rins •s ìrrndiat('(i with lon g wanlc·ugth u ltra\ iolr·t radiatiou.

As n n •sult of th is p hotor!'actiun, so m r fu roc oumarin mo lf'rult·!> a r r

covalc utly linJ.. c•d to tlu- n•acnllnolf'cu lf' (J\1 L!SA.IO ' ' '· n l. , 19(,(•). w itlw ut IJrra­ka~('H of tlw int ('rnucleo tid,· l•o ntl ,; (DAu:AcQUA. T .r.:nuO.JE\ !CII & BE:-;n~ -

. l nn . J sl . , .... ·,Jur. Stnn'lo ( 11llì !J) 5. :nU ·:$8.).

MUSAJO 377

NUTO, 1968). After the photoreaction Tm value of DNA is increased (D.u­L'AcQUA & RODIGHIERO, 1966). Moreover, the photoreaction does not re­

quire the presence of oxygen (MuSAJO et al., 1966). With the aim of clarifying the reactive sit es of DNA we have examined

the behaviour of the simple components of nucleic acids by irradiating at 365, nm aqueous solutions of purine and pyrimidine bascs, nucleosides anù nucleotides in the prescnce of skin-photosensitizing furocoumarins. On!y with pyrimidine derivatives did we obscrve the formation of new compounds (MusAJO, RoniGRIERO & DALL'AcQUA, 1965; MusAJO & Ro­DTGRJEno, 1965).

A C1-cyclo-addition reaction takes piace he tween pyrimidines and furo­eoumarines with the formation of a new cyclohutane ring. Pyrimicline bases always photoreact with their 5-6 double bond. Furocoumarins on the con­trary can photoreact ei ther a) with their 4' -5' double bond or b) with their 3-4 double bond. (MusAJO et al., 1967b; MuSAJO, BOROIN & BEVILACQUA, 1967 ; K RAUCR, Kni-i.MER & WACKER, 1967). Therefore ori gin may be given to two types of c(lmpounds.

As an example of the structures of these photoadducts, we may consider those obtained frorn psoralcn (I) and thymine, they are indicatecl by the formulas V and VI for the type a), VII and VIII for the type b). The two

v

VII

VI

o ..

~:~ WoJ~o~

Ylll

fo rms for eaeh type dei:ive from the douhle po~sibility of adùition of the suh­s tances. The photocompouncls of type a) have a v iolet fluorcscc-ncc when ob­servcd in long wavelength ultraviolt~ t light, while thnse of type b) arf' not flunrcscPnt.

378

Also in nativi' DNA pyrimidint· La~>c:. art· t Ile' rPacti n• sitl'!-1, g i' ing a photo-cyclo-addition reaction analuf{ous to that wlaich occur;, witb Llw ~:oir ll­

plf' compounds . In fac t among tbf' produ f' lb ofhydroly~>il- of a sawple of DNA irradiatt·d iu

tbc presence of psoralr n an d tben hydrolyzr d by ]J('a ting iu acitlic mcdtum '' ,. bave isolated two fiuoresccut substances idt' nljcal to th t> 4'. :i'-photoadduct • of psoraleu with tbr mine and cytosinf' and a nou-fluoret.ccnt ont>, identi t•a l witb the 3,4-pho toadduct of psoralt•n '' itb thymint·, ali a lready pn·viowd~ obtained h y irradiation of tbc siml'lc compound!; (ML SAJO et a/ .. 1Y67c).

The amoun t of thr fluoresccnl pho toadducl" so oLtained "at- nearl~

thre<' timcs greatcr tban that of tbt· non -fluoresccnt ont' (BoRDJN, .MusA.IO

& BEVILACQUA, 1969).

Let u s considcr now wh cthn tlte W t'll knuw·n ),iolug ical efl'ec ts whir h are produced by the « sk.in-photo&<•nsitiziug » furocuuma rins aftrr irrat.lia­tion at 365 nm can Le explained on thf' hases of tLt• pbo torcaction with nu­cleic acids . In a second timc wc' shall considcr !)(Jrul· new biologica! comw­quences of t bis photoreaction.

I. - As I ha vr alrcady said , the more known and s ludied hiologit·nl effect of furocoumarins is s kin-photosensitization.

Concerning this effect , we ba ve dctcrmined the action speetrum of xantbotoxin anJ h ergapten for the photon·uction with n a ti vr DNA, obtaiu­ing n·sult.b which wen · in a~reemeul wiù1 those ohtaint!d h) B ucK. MAC"òUS

& P ORTEII (1960) arrd by P ATRAK (1961) in s tudying th e ac liou spcctrum of xanthotoxin for tlw produ ction of rry thcntn on hu ruan or guint·a pig sk iu : in hoth cascs tbc more effective radia t itJnl:l were thos(' lying in tht· long ultra­violct region (DALL' ACQUA, .M.AIIGIA'H & RooiGliiF.HO, 1969).

Mter this . wr tricd to ascertain whethcr a parallclism exist s b et wrt•n the ability of the furocoumariu s to pho toreact in vitro with tbr nucleic acicls and thc ability of tb e same suhstanccs t o photosensitize human nr gui11ea-pig skin.

I r ecall tbat from the IJcginniug of our rcsf'arch on th<' photosc•nsitizing propcrties of these suLstances, we have trit·d t o obtain a quantitative f' va­luation of this propcrty . W c achievcd it b y determinirg for each substancc the m inimum irradiution time n ecessary to obtain the outcome of erytherna, operating of course in standard conditions .

Our early test (MusAJO, RoorGHIERO & CAPO HALE, 1954 ; MusAJO & RoDJGHIEno, 1962) consisted in plncing 5 !Lg of substancc· for cm2 of skin (the backs of human volunt<•ers were used), in irradiating a t 365 nm and in determining the minimum irradiation tÌm(' necessary for the appearance of erytbema. Consiclering thc activity of psoralen (thc parent compound

Attu . l Hl. ioitt}Jer. Satt(l il (1969) 6, 376·3H6.

MUSAJO 379

in this group) as equal to 100, the relative activities of the other suhstances were calculatcd.

Recently we have modified our prcvious test making \t more suitahle for assaying thc very active methyl-derivatiives of psoralen (CAPORALE

et al., 1967); guinea-pig skin was used, reducing the quantity of suhstance applied (2.5 ~gfcm2) but always determining the minimum irradiation time

TABLE l.

Relative photoreactlvlty (365 nm) wlth native DNA and wlth yeast RNA and relative skin·photosensltlzing actlvlty (365 nm) of some furocoumarlns.

:-lative 01\A (") Ycast RI\A (" ") Relative lrrMllt•tion lrradl~tlon Kkin·pboto-tlmo nocea· Rcll\tlvo timo nf\Co8- Rell\tivo ~cnHitlzing MlU'Y tor O. sary tor o. Furocouma rìna 20 •t. linknge photo-reao· 10"/o llnknge photorcao- nctlvlty on of furocou- tlvlty (P80· ot furooou· tivlty {pso· guinca-pig

marin to ralcn - 100) marin to rtùcn - 100 (PS01'6len ONA HN.\

(II<_'COUds) (sooonds) - 100)

8-methylpsorulen 90 373 126 190.4 540

5-methylpsorolen , ,~4 233 360 66.6 450

Psorolen _ 336 100 240 100.0 100

Xanthotoxin 558 60 371l0 6A 71 (8· metbox y· psoralen)

Bergapten (5-methoxy-psoralen)

1026 33 4440 5.1 61

Angelicin . (isopsoralen)

2160 15 1800 13.3 12 (***)

Xanthotoxol - - - - inactive (8· h ydrox y· psoralen) l (•) Aqueous 0.1 % solutions of native calf-thymus DNA containing IO fLgfml of

3H-furocoumnrin were irrodia ted at 22oC (The incident radiation on 2 mi of solution was

equivalent to 2.9 X 1018 quantajsec.).

(*•) Aqucous 0.1 % solutions of yeast RNA containing 3 (J.g/ml of 3ll-furocoumarin were irradiated at 22oc. l ncident radiation as in (•).

( .. *) T he low skin-photosensitizing activity of angelicin did not allow a correct deter· mination on guinea·pig skin, which has a !esser sensitivity than human skin (CAPOR.UE

el al., 1967). Therefore this ùatum is referred to the tes t on human skin (MiiSAJO, RODI·

GniERO & CA PORALE, 1954).

(in standard conditions) necessary to obtain erythema. The relative skin· photoscnsitizing activities of the various furocoumarins reportcd in Table l are ohtained in these last conditions.

. lnn. 1.~1. Stt.j)Cr. S<tnità ( l969) 5, 376-385.

31ill tA(."JOII' AFFlc.CT~C Ct.l.l.lt.AH PllOTO!-hl'l>I\IJ)

1 n order tu t e&l tl11• connectinn IH'1" l'l'Il this proprrt ~ an d tbc phot u­

n •ut tion " ·ith nurlrir ucid~ , wt: havr chu~<rn a numbr r uf furucuumarin dc·n­va tivt·s from amoug tl111sc· which ha vr eitlwr a very bif.!ll artivity or nnl~ a ruo{kratc activity or from amonf.! thost• which are inactivt·. Ali of them wen· lahelled with tritiu111 ancl their photon·artivity was evaluated studyin~t th• ralt'l- of photoreactions witb native D l\A h) ìrradiatinf.! al 363 nm aqut-ou ' !-lolut ions of th1• Rul,staners for difl'c• r1·u t periods. operuting always in tlw samt· eonditions, an d dNcrmining aftt' r ca eh JWriod of irrsuliat io n Lll f' amou111 '

of furncoumarins linhd t o DNA (.Homc mF.HO f• t al .. 196()). Thr results so obtuineJ ar1· repnrt ed iu Fig. J. It i;. ,., irlrnl that thl' 'a ­

riou;. furoroumuriu~ ha w a vcry di0't'ren1 photorearti' i t ~ with D A.

BO

il 60 ~

.§

·= ... "' ~ 40 o .... o ... :;;J

o 20 ~

5 lO 20

8-methyl-psoralen

5-methyl-psoralen

xanthotox1n

angel lctn _____.

4', 5'-d 1hydro-psoraten xanthotoxol

30 50

lime or ir r ad1atlon m1nutes

Fi~. l . - Pb(ttorcactiou~ brtwt•eu soute furo<·outu ll riub aud uutivt• 1)[1\A h) irradiatiuu ot 365 um. Percentug~" of furoromnnrin~ (rl'fcrrcd to UH• omount initially prt'­senl) linked lo D A os a funrtion of tlw period of irracfio tiou ore repor trd.

ln urder to definl' in numhcrs the pho toreacti' il y of rach compouud, W<' ha vt' calculatl·d l'rum tht' data obtaint'd the time nf irradiation which wa~:~ nccessary tu give a Jinkage to DNA corn'sponding to 20% of thc amnunl of furocoumarin initially present. On the basis of the data so obtaÌHI:' t~ ,

consiùering thc photorcartivity of pso ral••n as equal t o l 00 the rclati-.e plw­toreactivities of tiH' compounds ha,·e bren calculated.

Thc rcsults an· rl'portt·d in Tahl<· 1 togt·tlwr with tlw n·lative skin-pho­lO~t'nPitizing activity of the various subs tances obtainPd in thf' test on gui­nea-pig skin. As it appcars, tlw t\\"O activitics, in vivo aml in vitro, are almosl

para Ilei.

, 11111. Jsl . ::iiiJitr • .'im1iltl 11969) !'t , :li6<1";,,

MUSAJO 381

Considering that furocoumarins may pbotoreact also with RN A, tbc cxpcriments wcre rcpcatcd with this type of nucleic acid. The results are reported also in Tahle l. In this case wc can see much less agreement het­ween the in vitro photoreactivity and the in vivo skin-photosensitizing pro­perties.

I think that the rrsults now presented offer a clear support for a close r orrelation bet,•rrn the capacity of furocoumarins to photoreact with nucleic acids and that of provoking skin-erythema. DN seems to be more directly involved than R A.

But a lways conc<'rning this topic, I recall another s tudy worked out wry recently by PATIIAK and coworkers (personal communicatioD). Aftcr ha ving applied a tritiatc<.l furocoumarin to the skin of a guinea-pig an d irra­cliated at 36S nm, thc ;\uthors observed that both DNA and RNA extracted from the t reated portion of skin showed an evidcnt radioactivity, whilc uo trace of radioactivity was shown by various protcin fractions, also extractcd frurn the same portion of ;.;kin.

Wc may c·onl' ludc· that the photoreaction b N\\C<'D furocoumarins and nucleic acids may takc piace not only i" vitro but also in vivo and, more ~CD<'rally , that it is nn thc basis of the <' ffccts produced oD the skin.

Il. - Furucnumarins may also produce othcr biologica! effects. Among thPse I recall the lethal t' lfects produced oD bacteria cultures aftcr irradia­Lion at 3o5 run f" LUdicù by OG!NSKY et al. (1959) aod the mutagenic effecls on Sarcina lutea culturc•s observed by MATHEWS (1963) and referrcd to a tlamage to DNA.

Thc photosc•nfl itizcd rnutagenic cffccts of fu.rocoumarins wcre studied also on Drosophila mt>Lanogaster by N I COLETTJ & TRIPPA (1967) . .:\foreovt'r, CoLOMBO & LEVI S ( 1965) obscrved thc formatioo of gian t cells after irradia­tioD of mammalian cells in vitro grown in the presencc of psoralPn. CoLOMBO ( 1968) also studi cd t hl' t• ffpcts of the irradiation of sca-urchin sperm in thf' pr<'fWDce of xanthotoxin 011 the development of sca-nrchiD eggs ferti lized by the irradiatcd sperm : anomalies were obscrvcd that might be clrarJy attributed to a damagc to DNA.

Hl. - Morcovc•r Wl' have stuùied the effects on viruses, ubserving the c•on1ple te inactivation of so me DNA-v iruscs aftcr irradiatioD in the prest·ncc uf psoralen, while ~oo m<' R~A viruses were more rcsistant ()l usAJO et ul.. 1905).

Reccntly two ONA-viruses were studied mo re in detail, that is an ade·

110virus, which is lacking lipoprotcic cnvclope, and pseu(ÙJrabies t•irus be· longing to thc IH·r[wtic group, which has such an <'llvPiope (MugA,JO, P ETf:K

& BACC!CFTETTI, uupublishcd results) .

. Jnn . / .il. SlltJt'r. S rmilri ( IHG!)) 5. :rifi ·:U-1.'1.

31l:! ~i\C'TOR• 1\tt' L I.Tll't. I.ILLILAII f'JtOIU:-t:l''l\1'1 \

Tbc suspen~<icm~-> of tlw~c· t wo \Jru ~<f•;. lo;.l c·omplt·tl' l~ t IH'i r infPc' l i' i 1'

ufler irradiatiou for ft •w miiLUlt•;. witlt lon ~-: ullr:niol1·1 111 tlw prt>sf' ll t't' o (

1:!-methyl-psural<·u at a cmH·c·u lratiou of JO iJ.;.( · IIII.

The ademlf'irus (2 À 10; inf• ·<· tiou i- unit-- pc· r mi) irradia lt'd in ti ... ,,.

conditions for 60' c·xertPd an ti~PllÌI' a (' lÌ \' ÌI ~. t hat j.., i t "a~ ahlt• to pr(ltlu c·• ·

antiuodic·s a t high ·1 ill'r in 1·abiJib .

Pseudombi.es lll'rpetic 1•irus (.:l 101; infectiou:: uni t - jH'r mi) irradialt·d

ul;.o for 60' in thc· bUmc· cmHIItion~< and iuot'ulatrd in mi<·t· produt't'd an an l l·

hudy respomw of moderai P irllensÌI ). llowe \ t•r, thi :-; i ~ a rtolt' \\'ttrlh) n·s•dl .

consideri n~ the Wl'ak irnruunis iug power n l' ]l('r petÌ(' 'aceinc·" iua!'l i' a tt•d

"ith othrr mcthod~.

IV. - .Furlill'rmor!' l f- Ilali rnporl 011 ou1 1-' Ludit•i- on uwus•· Ehrlidt

aAcites tumur celi;, : tlw cfl't•ct whieh "a~· ohlniued h " irraclialiou a t 3(,,j 11111

of a SUS(H"nsion nf tlll':,e celi ~ in tlw pr«"SCll Cf' of \Try Slllall antonnl~ o f s l-m ­

photnsensi ti:r.ing fu roeoumnri11~ wa~; l l w los~ of the traus mittiug tumor capa­

c it y of tiH' t•t•lls, wlwn tbr~ wt·n· inj('c tc·d in miee ( \'ft SAJO et al .. l96id). St·t· 'J'ahlc 2.

For having a di rcct eoutirm that this biologica! t·fl't·c· t i;, dut• ln a linl-agt ·

of the furocoumarin to DNA , Wl' huv<· t•xlrar tc•d UNA from a poni of Ehrlic h

8!>Cill'S llllllOr cdii" pn•vÌOU!ol )' irradia tc·d iu lht• prCS{'!H't ' of tri i Ìa t t•d p::.orall•n.

We ha\'t' found at firs t that after irraclintion, and only iu this concli1ion, p;.;ora ­

lell was linkl'd IO DNA ; mort'O\t'r , aftt· r· h ydrol) ;,i::; of tlw t'XIrartc·d DI\ A , wt·

ha"e idt"ntified a l'.ubs tanc·t· which \\a" iclentieal lo thc• fhwresrt·n t phottH' OIIl­

pound aln•ady ohtaincd fror11 psorakn :wcl thyruiuf' ( l\1t :--AJO <'l u/ .. 1<)(,7u). Tlll' phot oinac tivatt' d b) p~oralo ·n Ehrlich m;c·itc·~ l11111or t'l'Il~< i't'l'lllt'cl

l'O""hcha' !' a l-o thr untrt•a tc·d tumor r t•IJ ,.. towards tht' \\ ' righ t 's liquicl. lr~ pali

~ue arul in the '\ arburg',. apparatu~o .

V. - Anotlwr iu vf's tigation was carri l'd out with lhl' a1n1 lo asc<·rtain

wbetlwr thc Ehrlich ascitcfo tumor ct· lls photo iuactiv:ttt'd with skin-phnto­

scnsitizing furoconmarius had thc: cnpaeily t o protf'ct Lh<' ani111als again~ t

ihls""tumor. Numc' rOII.S t'XJWrinwntf; ha\'\' hN·n pl'rfnrrul'd by ll !-1 in tiH' pa~<t r;;;;r· years u sing Cl' lls inactivated by irradialion in t IH' presc' n<·t• of , ·ariou!>

compounds (bcrgapten , xanthotoxin.. psorall'n , 8-m cthyl-psnrnlt·n. an d 4,

4 ', 1:!-trinH!thylpsorult·n). 'l'lw following n·sult s, obtained u sing p~orakn and ~ethylpsoralen, ar!' reported as th t· mos l s ig nificant ones (:\1USAJO, \ r­

SENTI NI & BACCICIIETTJ, unpuhlishPd results).

Thc ccll suspcmsions w crc prep ared by diluting thr a~cites fluiù in such a mann er that each ml containcd 2 X 107 crlls, addin g JO iJ.g-ml of pHornlt·n

or 8-methyl-psorall'n and irradiating it for 30 minutt'!', as prcvinu~l~· descri­

bed (MUSAJO et al. , l967d). The innctivated cclls wcrc inj('cted into th e r <·ri­

toneum in doses of approximatcly l 07 cellsj m ou se : a) as a single injcction,

..tlnn. 1.~1. SUJirr. S ottitlr ( I!Jfi!l) 5, 3Hi ·~~:..

:ltUSAJO 383

TAULE 2.

l nfluence of lrradlatlon (30 mln at 365 nm, uslng a PhiUps HPW 125 lamp at a dJstance ot 15 cm; lrradlatlon ln tenslty: 4.2 x 101' h v/ sec/ cm ' ) in the presence of furocou­marlns on the tumor-produclng capaclty of Ehrllch ascltes tumor cella. After lrra­dlatlon, the cells were lntraperHoneally tran splantcd In mlce.

l )io. o t

l )lorlallty

Furocoumnl'in conc'<lotrotlon (1-g/ 16• rclle trcnlod l mioo X o. l .,.

l

Psoralen (•kin-act ive): l . (controls) 168 l 168 100

o.ooos 8 6 75 0.0025 8 3

l 37

0.005 8 o o 0.025 8 o o 0.05 28 o

l o

0.25 110 o o o.;; IO o

l o

l . 60 o o Xauthoto,in (ekin-activr):

- (('ontrols) 50 50 100 0.0025 8 Il 100 0.005 8 2 75 0.025 l! o o 0.05 8 o o 0. 15 24 o o 0.25 8 o o

Brrgupten (skin-a<·ti ,·e):

(controls):

l 50

l .)0

l IUO

0.0025 8 B 100 n.oos 8 Il l 100

l 0.02'> 8 l

l 12

0.05 20 7 '15 U.IS H o l o 0.20 Il o

l o

l Xnnthotoxol (skin-inactive):

l l

- (contro!~) IO IO 100 l 11.25 IO IO 100

l l l _j

b) as two injections, the se.cond two weeks after the fìrst, c) as four iujcctions, passt•d at wt'ekly intervals . Tl1c challt'ngc was perfornwtl by injecting 2 v L03

untrf'uted tumor cl' lls iu f'ach rnou>;f'. This dose causcd death in ahout 70 % of ron trnls und in mos t of thr rast'S i t uccurred within nO days from the tran-

381 ~ ACTUH,.. >~. n' l i l l 'l· l ~. l.! . l L l lt 1'1111'1 U~L i'-'11 Il \

;.pian t . Tlu· challt·nw· ' ' a,.. acl1it·\ c·d : m t bt· ('a..,,· ... a ) a n d b) 1 bn·t· 11 Pt·l -. an d in tlw c a st· c) fuur ll (' t · k ~ aftt.,. t lll' fir~t inj t•et iou of ina<' I ÌI :t lt•d cdi;.. I n ali

rxpt•rimrnt-. tiH' n tortal rt .\ ratt- "'a"' c ~tlc · u la t t•d at tlw t•Jtd oJ ~ uruntlt ... oh­sr r vation from tb «' inoC'u lation .

Tlte dat a o1 .) t•xpt' rÌmrnt ... 11 11rk t·d n u t !-Ìtnnltan Pntt!-1\ 11it h t lw rr la t i~t · contro);., a rt• l"Urn JnarÌz t'd in Ta h lt· :L

' 1 .\111 l 3.

Mortallty obsc rvctl arter clmlle ngc with 2 x JO u nt rcatcd Ehrlich a sci tel. tumo•· cclls in contro! mie~> ;md in mice previo us ly in .lccted wlth Ehrlich asci tes ltun or celi:. pho:oinac tiva ted In thc t>l'escn cc of l'u rocoumarins.

E x pf•·

l'illlt'll t

~u.

2

Fu t·•~efHIUI .,J·i•l

P Miralt>n

P borulcu

- - --- ----

)11•·•· .... (l·a111

' d L

----'\('l .

3 8- nw t h y iJ"Orall'n . ' \ CL

- ---- -4 P sor ulen ~\.\Ì ... ~

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(•) l' valu!',. eal!'ula ted lty Ch i "'J lHtrt· .

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i llj (•t•I ICIII ..

or iualha · tt••l ('t•ll ... l l'ol'l l lllkt , ...

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Thr prescn t rt•sults Sf:f•m 1u indi<"al t> tlt at c<·lls photoin ae ti\'a lf'tl in tlw

prcsencc of p soralt·n and 8-mt•t hyl-p;.oral f• n hy long wa vrlt·nth ultra , ·iolt· t Ìrradia tion ind uce a pro l t•ct ion against tnmor in mos t of tlw treatt·d ntt rl' .

l am very gruteful to ali 111 y CO-\\ orl..er~. iu p a rt irular In Prof. G. R od iJ!hieru.

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