Factors aft'ecting radiation sensitivity (•)

}[ARCELLO QUINTlLIANl

DepartTMnl of Biologk4l Chemùtry

While the literature on factors affecting the radiation sensttlVJty of biologica! systems is par ticularly extensive, few attempts have so fa r becn madc to arrange the available data into some organic and coherent pattern.

lt was felt, therefore, that it would be timely and appropriate to make such an attempt with regard t o the lethal effect of radiation on s ingle cell sys tems, which is probably the most extensively s tudied effect in ccUuJar radiobiology.

UsuaUy an empirica! measurcment of radiation sensitivity can be easily obtained from survival curvcs, but then if one trie to describe, w bat this parameter means in terms of the biological proper ties of the irradjoted cclls, considerable difficultics are encountercd in affording a satisfnctory description.

On the one hand the features of survival curvcs strongly suggest that living Cl'lls contain non redundant sites, whose s tructural integrity is essrntial for survival, and that it is the damage to these sites which ia r esponsihle for the lethaJ effect of radiation. On the other band a large body of experimental evidencc demonstrates, beyond any doubt, that rodiation senl!itivity depends on a variety of factors, ranging from t he s t agc in the cell cycle to the oxygen pressure and from the d egrec of hydra-1 mon to thc prcsence of a variety of added substances.

The conccpt of the criticai sitcs for cell dcath and the evidence for tht' num l'rous factors affccting radiation sensitivity may not be contra· dictory, if one assumes that such s ites bave to be interprctcd as dynamic ~ntities, 'ariable according to the intrinsic and environmcn tal conditions o ( ct>Us.

( •) fn vucd paper presentcd at the 8th Annual ){ceting of the F.uropean S04"iety for H adiatiun Diology. held in Ba.,ko Poljc, Yugoslavia. ~ptcmber 1971.

·• n.n . ht. S••oer. S111ollò 11971l 1, 1H7~60

648

A~ to thc php;ico-chcmical id<'ntifi ea tion of tlw l'lt' llSJli\'C s itcs, it appearf' today that ovcrhclming t•videuc·r rxists irulieutin~ that D.:XA is thl' primary targct of racliation cfl"t·rts in Jj, ing celi ~ and that the damagt> to DNA is responsiblr for cdi inarti' ation. Thcr t>forc. Ul'<'Ording tu what ha~; bcen said in the previous parugraph, if onc RiHHliD"" that DNA h a;, a uniquc and criticai role in tl cll·rmiuin j! tht• radio8Cti'·Ìti,·it~ of ]j,-ing rdJ... on c must admit tha t the Dì\A damage which fina lly hring;, about thc• ohst'rvable biologica] efl'cct cau be afl't•t·tNI by man~ tlifl'crt•nt fn <' Lor:..

Tbt· question ari«cs. al thi~ poinl. nf tlw imporlant•t• of radioindut·t>tl li';. Ìoll!' t o otber macromoll'cuJa r ccii "'' rut·L un·-. in addition t o D'\.-\. Sue h lesion" mus t exist h t"camw of th t> raudom d<'position of th t• t' tll' rgy from ionizing radiation . and in fac t do cxi"t.

One possibility couJd b e tha t l:iuch lt ·~> iou ... onl~ cOOJWralt· in thc• fiua l expressiou of D:'IJA damagr. The structurc::- in' oh t•d. tlu·rt'furt·. nHild Le indit·ated as cooperative sites or tnrgt· l ~. Tbt· ;..t'l'ond ]'O!'sibilit~ i-. that lesions to s tructure;; othcr t han D'\ A ma y lw lt' thul in t hem,.eh c,.. l n thi s case such structun:;, would bt· trut• ult t· ruuti' t' ~itt•,. for celi kill ing. Thr latter possibility ha;.. bee11 cxten~i' t' l~ tli~ru-. ... t' d n nel H'wral llypu­thesc~s ha v t• ber n proposed. ProhaiJl y, tht· ultertll'l l i H l arget which ha,. n ccivcd mos t attention is that rt' JJT <'i>l' lll~d by biolugical mcmbrancs. Acc·urt.lingly it has bcen pos tulatt·cl (1) Ùlat tlamagl' tu •·e li mr mbram· antl or to intraccllular mt·mbran es, like nucl f'a r m<·mbran •--. mitochundrial memhratw;;, ly:,.usomnl Jll (' mhra rtl':- , euu ld irulcp t· rlll <•n tl~ lo·ufl tu n· ll dl·ath.

Tht• possiLi c tt'latiom,hip:- bt•twct•u nlt·mbranl ' durnng<· antl n ·pru­du cti ,·c ccii death ha\<' b ecn invcsti~atrd in yt·asl ~:. a r11l barteria hy 'arious Authors , but thc rc;;ults ba ve g••ncraUy inJicatt·J t ha t nn dt•linilt' relation ­ship couJd b e estahlish ed (2-~) . AIM> in our laboral Hr) \ H' han ,.tudil'd the possiblc rolc of primary 1esion;, to <'eli memhrant' w thl' lo,.,. of reproductin caparity of E. coli n r irrndiatcd ''ith )\. or y ray.... Tht' loss of K + r ctcntiYity at OOC wa!.' takcn n" an iudic·atur uf ;,tructural da­magc to celi membrane. Some of our n·~-o ult <.. are ... ummarized in Tnblc l. The table hows that. al thc Je vcl of 5011 o dft·ct, !>UT\ j, al i:- !>iguifìcautly more scnsitive than K~ rct cn tivity. I n addition thi:. latter. f'Ontrary to suni val which shows tht• wc li known oxyg••n efl't•t· t . j., prac tically inde­p endent of thc prcscucc of oxygcn. l 'i nali) '\" -c·thyl malt' imidc ( rEl\1) h as opposite effcct s on un' ivnl ami K "' rct cntivity: in fact. it enhancc,., the damagc respons ihle for luss of viaLility, to a gn·att•r cxtcnt in tbc abscn ce of oxygen, in agn·cm ent witb what i <~ alrcady \n·ll known from the litcrature '(6), wbercas i t rc<lucc)) thc da mago respout- iLle fo r pota!>sium r eleasc to .n~arly the samc cxtent, iu thc prest·nc:e of oxygcn and in auoxia.

Anu. / M, ,<;u)>cr. Sallilù Cl9711 7, GH~60

QUI:-iTII.IANI 649

TAJILE

Effective radiation dose for survival and K + re tentivity

OER Oxygen Enh.ancement Rntio - DMF = Dose ~Iodifying Factor

COND ITIONS l DOSE (K.rad} FOR SO % OF COI'<TROL VALUE

l Sunnval l K '" lou

.\ itrogen l 12 22

Oxygcn 5 20 (OER = 2.5) (OER = 1.10)

\ E\f. \itrogen 7 35

l (D-"IF = 0.57) (OMF - 1.60)

\' E~ f-Oxygen 4 31 (miF = o . so) (O)fF - 1.55)

Cunlrary tll ~E)'l , radiosensitizing iodino compounds show a complete parallclism in their activity on cell survival and K + r e tentivi ty, as shown in Fig. l.

100

••

IO

• Cont,ot a N•l . ,,. . , .... D IAA

IO .. ••

OJ

""'

. ...... .. ..... . ··-·

Fig. l. - Oosr-rtTect curve for K + retentivity and survival of E. coli B/ r cells irradiated wit.h X rays in the prescnce of some iodine contaioing compounds. L\A = iodoacetic acid ; IAM = iodoacetamide; lP A - iodopropionic acid.

An important contrihution to the understanding of tbe mecbanism of the action of iodine compounds has recently been made from the data publish ed by Myers (7) who studied DNA damage and its repair

650

in E. coli B 'r in the presen ce of iodid<· ione; ami ioduur('lamide (IAM). Such data are summarized in Tuhlt' 2.

TADLE 2

Efl'ect of iodide ione and iodoucetamide on tbe repair of single-straod breaks ioduced by X rays on the DNA of E. coli B r (Myers, 1971)

Nonr

lA-'l

Kl

:'I one

lA :\l

Kl

l A•u•~• f>O•hìnn , f ON4 p•• k ot n tom •

- -0-k.-.. -d-• - -~ --10 k rod•

0.79

0. 7R 0.7H

0 .78

l o,(,:;

0.53 0.5(1

20 lrool•

0.5:1

o. 13 Il. -l~

.

A••"'&• po•iuoa o! D.S <\ ~·L •• l llour

o. 77

0. 51

0. 55

SO luad •

O. H

0.38

0.39

0.75

0.37 0.37

Tbr experiments were bas<•d on thc production of sin gll··~:~ trand break:-. in tbc DNA of irradiated J~. coli cclls, u s ing the well known tccbniqut' of Mc Grath and Williams (8) . Tbc figur<' s in tbc tablr indicate the a' t·· rage position of D NA peaks in ccntrifuge tube, whi<·h is u sually takcn as an indica ti o n of t be sizc of D N A molrculC'. l n tbc uppcr p art of tbc· t ablc a re thc results of the an alysis carr ird out imrnediatt'ly nfter irradia· tion, sbowing a slight increase iu the numb<'r nf s iugl<··st rand break.., whcn IAM or l was present. In tbt· low<' r purt a re thc r csults obtaincd after onc hour incubation in nutrient broth at 300C. lt appcars that. in tbc absen cr of iodine compounds during tht• irrarliation, s inglt' ·Slrand brcaks werc almost completely rt'pnir cd by tht• incubatt•J cd ls . Thi, repair was completely inhihiud if I AM or I wcrc p re-.<· n t during th1· expo ur{' to radiation . Contro] cxperimcnt ~>howcd in fact tbat adding tbe iodine compound immediately after irrndiation did not pre\'Cnt tbt· r epair process. Tb<' data als() indicat e tbat tbt> s ligbt incn·ase in s ingll' strand breaks noticed immcdiatcly aftcr irrudiatiou , ' ' hieh doc~ not justify tbc sensit izing effect of iodine compound~::, i :. po..,-.ihly duP Lo tlu· inbibition of tbc r cpair whicb occu r ' dur ing t be irradiat ion tim.·.

Tbe ahoYc facts support tbc bypothcsis thnt ie~ditH' rompound ~ bring about th eir sensitiziug elfect by inducing tlnmagt' tu tht• r c·ll membrane" which result s in the inhibition of DNA rcpair. lt appears thcrcforl' tbat the celi membrane, in tbc presencc of iodine contain ing scnsitizing co m· pouods, can play tbc role of a cooperativi~ s i t <· witb D~ A in r.cll killing

' ""· ht. Sut•rr . • ~o,.i t (i l\971) 7, o~r-OOIJ

QUrNTILIANI 651

by radiaLion. No indications, however, are afforded about the poss ihility of a true alternative role of m embrane damage with respect to DNA damage.

This is just one of t he cxperimental arguments in favour of the vali­dity of the model assigning to DNA damage the primary role in celi killing by radiation. The discrepancies of such a model with the experimental data, which bave been repeatedly pointed out, can probably be explained by taking into account the concepts of the « biologically expressible damage» and that of tbc « cooperative targe ts ». The last concept has been already discussed.

The concept of tbc hiologically expressihle damage derives from the fact that after the deposition of radiation ener gy which produces primary damage to DNA and before the oecurr<'nce of cell death, many phys ico­chemical and biological events may occur to affect s uch primary damage before it can be expressed in t erms of the biologica! end-point, that is to say as the loss of prolifcrative capncity.

A t this point, an attempt can be made to vis ualize the sequence of evcnts leading from the ahsorption of radiation encrgy to tbc fìnal cffect a n d to indicate as wdl how s uch sequence can be modified by factors which affc-ct cc lJular radiation scnsiti vity. This is represcntcd in the scheme sh own in Fig. ~.

Pnmary Target DNA

Cooperative Targets CT

t INTERACTION WITH RAOlATION ENERGV • ~ Olr.Ctor lndl,..ct

l..oss ot prolo terat ìve capaci ty

MOOIFYING FACTORS

- Alt~rin g the t .. rg~t

- lntufumg wilh md~recf .-n .. ck

- Afft cling rh e t"volution of transiMt states of t i1rget

- (?)

- Affect ing DNA r epa1r

- (?)

Fig. 2 . - ..:, cnts leadiug to ccii killing b y ionizing radiation. Rndiation ùamage to tar!!ets i ~ r<'preseuted ns transient (*), per111anent (p) auù biologically l'X pre~,i ble (h).

A nn. I ~ t. Suver. Snnitio • IQ71 7 , t;t7-Cti0

652

T bc schemc pos tulatcs t h at cl'll killing j , du<' to 1):\A ùamagt·. wbich dcvelop~ i n st ep ,.. . Fir"t radiation indu<·e~;, by clirect or indireel <' ffec t. trun.,icnt s ta t N• in th•· targct, w h ida then evo h e to p ermanenl radioche ­mical damUf{t·; s uclt damag•· c<Hl Lt• repain•d more or le ~ extensively. determini n~ in thi .., "a~ th<' damagt• which i-. fìnally exprc·.., cd in biolol!ica l cfl'cct. Tbc po ibilit) i~ eO\isagt·d of damagt• to olh t· r targrt-.. which al,o de ' dop:. follo\\i ng tlw samc main s teps aud its contril.mtiou to celi killin~ takts plat•c througb thr influcnc<, on tbt· bio ltlgically r\:prc!'.siblt· damagl' of D~ \ . On tlw right of thl' fì~urt• are imlit•att•tl four matn m!'chanic;ms through which fac tors afl'ec ting radiatiun st' nt" ili,·it y ma~· oprratt•.

1'hcy are, eh•arlr, gt:neral ntt'l' hani~m~- tu \\ hirh tlw partit·ul a r moti •· of a<•tion Of a Yariet)' of iu trinSÌl' an d Cfl \ ÌfiiiiOll"lll ,tJ fal'lor- .t fl 't•t• l Ìll~ cdlular ratlio..,rn-.i tivity cnn be ref«·rr t•t l.

l n t h <' fo llowing p agt'l'o u numht•r of r eprt•..,t•n tati\ ,. t•xampf!- .., cd' d11· m ode· of action of sevcral factor ::-. li~Led unclt•r tlw apprupriatt· l!~' ll l'ral m t•chanism, wiJI be plCb ClltCÙ. Qf COlLr:-1'. il j ., llOl Jlfi'I'UOH'd that t)tl ' enli ... t<•tl fa <· tor~ covc•r the full afl•u , nor thot tlw aLLrilmtiun of' t lw uw•l t• of a<'liou i ~ iu ali cases u ncontrov<•rs iul.

• • * Table :l lìhow::. tbc main ft'atun's uf targt'l rnodili c·ation.

' J \OI. f' .l

1\lodifications of target

MO O F.

QUANTITATJVE ~----~

COl'\FOR.\fATIOXAL +-----­CHEMlCAL

F .\C. T Il RS

Celi cyclc

:Factor~ offtctin~ j!ruwth rate drptndtn t macromolt>culor compositiou of ctll ~

Added compouud<

Compounds rcacliuj.t witL, or nw~aho·

Jically incorporatea in ta r!ltt mole­cule~ (i. t. holop;enated DNA ba•t•)·

T hcy ma) occur in thrce fwadamental '\1.1 )':.: that i-, lo say quantita· tivc, conformational, and chcmical; aod, in principlc, may afi'crt tbc primary as well as t bc coop erative targets. As to the factors involved , tbc celi cycle and tbc fac tors affccting growtb-ratc dcpendent ma cro·

.t n n. ht. Super. Sonird (19711 7, 6&~~~

QlT\Tli.IA:-11 653

molecular composttton of cells are indicatcd as those maitùy responsihle for quantitative and conformational modifìcations of targets.

The striking variations in crlluln r rarliosen s iti vity according to tbc stage of the life cycle are wcll es tab)i.;ho·d ~'Xprrimental facts (9•10) , although the underly ing rc·asons for snch variat ions arP st ili under discn ssion. Pro­b aùly thf'rc are ~everal ('Oncomitant reason , and quantita tiYf' anù confor­mational var iations of the radiosens itive tnrgets are among them. For ins tance, it h.as Lecn sbown both in bactcrial and mammalian celio; that the lca!'t radio-scnsitivc s tagc in the celi cycle correspon<l, to thc end of tbc D~ •\ syntbcsis (11 ), whcn the et•U, bt> fore the tlivis ion , hus a doubl1• r epli ca of its gcnome.

On thc other ha nd , Fn·l'dman nnd Rruce (l2). whu hnvt· s tutlird in .\! icrocucws radindurans the influence of diO'eren t cu lture mctlia on both mttcromolccu lar eompo~ition and r.Hiiost'nsiti,· ity, hnvc found that a::. thc growtb.-r.atc anù the quuntity uf ct"llular macromukculcs incrPase, the X -ray .scmdtivity nlso t f' nds lo int•ro•asl' . Cons itlt•ring D'A. the .Autllf)rS ha ve suggest<'<l that if tlw s izc of th i-; targct inl'r Pa::; r s, thc amou nt of tlamagc ~:tnstain cd ~hould ulso iucrcase . T he creation of more damage in n larg<·r ta rgct woultl r c:sult in incrcased inacti vnt ion on ce thf' rrpair syst t> m is dam ageù or reac.lws ~a turation.

As far as the rolc of conformational f' hanges is concl:' rned, it is known. on the onc h.and, Lhat celi tUv is ion, in purticular mitotic di v i :~ion , in\ olves importanl ch.angcs in tb.e conliguration of proteins and nuclcic acids: an1l. on t b.c other . thnt in molecular modt>l sys toms the radiation scnsitivity of macromolccttles may be s t rongly aff,•ctNL by conformational chan;.;f'., (13).

1t i s thcrefore quite plaus ihl c that m the liv in g cell the ~nme situntion would occur even though i t might be clilficuh to obtain dirf'ct eYic lcnce for this .

As an cxample of a ch clllical moJifi cation of larget atfecting its radio­scn si tivity the incorporation of h alogenated analogues of D~ A bases i!> proposcd. Such proposition howcvcr is open to so me tloubt beenusc thc re is s tili discussion b ctween lhose Authors who postula te that t he sensitization rcstùt s from an incr easc in the number of radiochcmic:~l

Ics ions with.in substitutcd DNA und othcrs who pos tulate inst l'atl that the increase of DNA clamage is secondary to the interference with tho DNA r epair system (1<1) . H owevcr, it appears that at !cast th,· tlirect cffrct of radiat.ion is enhanced by halogenated base su bstitution. Ficldcn and Lillicrap (15) bave shown that 5-bromouracil acts as an energy Rink in the subs tituted DNA molccule and that t he ex citation r nergy wil1 migrate efficiently ovcr about 200 base pairs to the 5-BU molt>cule, Sllggesting that th.is fact rnay contrihute to the sens itization by BU in liv ing cella.

.• ""· l at. Su11c r. Samld 11971) 7. ~7-66!>

* • • 'l'h t• mode of int<>r<H~ lion witb spt·cir .. r e:-p on:-ihlt• fur indin·ct allack

t o lar~et $Ìte:- an d t h t• rc•la l i' 1· f:H'lor~ a re ~umma ri.t.t·d in 'l'ab le l.

TADI.I. 4

lntcractiou \\ ith iutlia•t:t:l uli:Hck

.MODE

Ra!lical scavcugìup;

Formutiuu of secuudar ~

dantugìnp; radica ! ~

FAt.'fOH!:i

'\ utural Cl'll Ctllnpoueul·

(aOecl rd l" c l'Il t· ~ ck autl dìfT rrrul

factor• or uddNI cnmtwtuui· )

Acld~:tl comrt>urul-( H uon ~ll-pr11tl'l' lt1r-. wrlìru• .. ru•ÌlÌZt!r•. l'Il,.)

Two possiblc modcs of a ct ion han· Lec·n indicau·d: radi ca! ~> t ' a\ t>ngin g

an d th1· formation of secondary damagi n g radicnJ .. ; t IH'M' <'Orr<'"I'Orl<L r ei>pt'l'li,·el). t o protec tion or sensitiza t iou with rcgard lo tlw damugc

lo targN s i t cs. As a matt(' r of fa l' l , it shiJultl lw poi nt cd o nt that tlw,.,e arc l\\U a,.pcct s of the same fundamental mc·chani~m in "hich tbc racli­cal formt'Ù by intt•ra<·tiou of radiation with walt•r. iu'l tt'arl uf n ·uc ting

din·<· LI w i t h thc targct ~; t;ructun•;;, r<•nr t '' ith othcr rnolt·cul t·~ prt·M•nt in th t• immt'diatt· cn vironmcnt. In thi ,. ,,·a) M'Contlar~ r :ulicul -. ar~· for­

m ecl al th1• I'XPI' Il '><' of " Uc h molt'culc>-. Tb,.,..,. racli1·ul,.;. tJwu. may d1•l'ay

cau:-ing lillle or no damag t· t o tlw targct ,.; trncturp,.,: or d><l' tlw~ ma y attnrk :- u r h AtruN urcs rnon· cfTi ciently or more· ~ pt·t·iflctdl~· thau 1 h 1· wa lt•r rauiraJ .. tht·m seh {'~. Thi;,. of COIIrH'- t• rltin·l~ dt·pcrHJ, 011 t be Ila t nn·

an d rrneti vity of ,;t•condary rudiral>-. ''h i eh in tl~t·ir 1 urn , ar~· obviuu:-; ly

d cp cndt>nt on tht' na turt• of the co mpound pn .. t•u t. Thc fu ctor s invol\'cd in such a medtani ... m art• polt•n tiull~ vcry

num r rous: in fac t the only <·onditio n for tlw mt•chuni ::o m to OJw ratc i~ for

tbc intNacting compound Lo b e pn·scnl in th,, up Ji ruprinl <' <·o urt•ntrn tion at thc proper :.i le. Tlw fa c t or~ <'an b t> intrin ~ i l·. in ulh,·r \\Ord-<. ju-1

tbc ccii componen t s, or compoundo; ade led I'XIPrna ll y t n tlw !-<) Oo\ LI'm. Il nppearA t b r r cfor c tbat e, ·ery changc in thc cdi rompo:-i tion. may ÙP r'"'l"­

' an t. a s wdl a .. eH•ry fartor in 111<' ccii <'11' ironmcnt. ;\ key question for c,· a l uating the importanN· 11f tht• m rchani .. m urH.Ier

c l i sc u:~~ion i" thc r elath c ~ignificarH'l' of dirct·L and indirc·rt action in Cf· ll inartiva tion. This is s tiU a rath t> r cont ro' rr~ia l IJIIe;, t ion, h owc' rr th t•

majo rity of autlwrs agre<>, at t b t· prt>sent tim r, tb ot in mo,.. t t. ituation-.

QUINTII. IANI 655

botb direN and indirect artions are important in producing celi killings. In E . coli B cells s uspendcd in dis tillcd water, both in tbe pre!>en ce and absen ct' of Yarious compouncls, Sanner and P hil (16) havc shown that tbc indirc·ct action r ould account for approximately 50°:, of the lctha l t·lfcct ob:sen t' d undcr anoxia. The Authors a l.,o showed t ba t there \\a no eorrl'iation LctwPen tbC' firs t ordcr rate cons tants of \'arious clt'ctro n scavengcrs ami thri r radio·protective clfect, whcreas a good correlation cuuld be o bs<.>rn•d for OH sca,•e ngers .

The al tack by secondary radicals has no t r eceived so fa r a grt•at dcal of uttention. Tt has ht•en s hown to be, vcry likely, thc mode uf ac tion of ioc.line <'ontainiog st·ns itizing compounds. su <'h ns iodint' ions. iodoacct a mide. ioduacetic nnd iodopropionic a cid ( 17• 1 ~) . In this paper T ha,·c already discn>--,etl th1• n:periml·ntal data indicating that the artÌ\ l'

int<•rmediates forntl·d from iodine eompounds rnay attack ~ome <'noprra· ti' l' targ<.> l in bac tl'rial m e mbrane impairing in this wny tbl' r t•pair of tlama~t:> tu 0:\'.\. ft cannot lw t•xc hull' rl that o thl'r st•n!li tizing compound~"

ma) art wi th :-nmr analogou:. m t•t•hanism ami thi, po~,ihilit) ,huulcl pr()hahly h c more· t·x t cns i n ly in\'1':- ligated .

• • • Tubl t:> ,; tlcal>~ with fnctor;. intcrf1·ring with th1· eYn lutinn uf t ran:-it'nt

:, taLcs in thc targ<.> t.

loter ference with tbe evolution of trans i4'nt states in the target

li ODE

lle~ titution h)' electron or h ydrogen donation

::i tab iliza tion of a tran~ient

lesi o n

F'.\ CTORS

'\" atural ct•ll compontnls (affcctt•d by celi ryclc onrt tlifft•rt>nt factor~ or added rumpound•)

Added compounds

L (SH/nun ' R ·prntrctor•. O,, \ E\f, T.\~ , t'le.)

T h t> m cchanjs rns indicatcd, like t bose involwd in tbc interac t io n wi t h intlin•c t attaek, ha \'1' bcen pron~d tn occur in .. imple molccular -,y .. tt•m,, an tl tlwi r undcn.tanding hm, bcPn gr.-utly s timulatcd by u;,e of such tceh· niq•w" as 1'11'1' 1 rnn ..,pin rNwn tm et' :- pl' <' lrometr y nnd p tt l"e radiuly~ i s .

T'\ o mai n ruodt•, of action han• h<'I:'D indicat t'd corrt·..,pomlin~ lu t h t· t\\ o opposi t e una! l'lfccts Oll Larg<.>t s: und they a re the rl''ttitution by drc· tron nr hydrogcn do1tatiou , or th e !' tabiliz<tti on of a t rnn,.icnl lcs ion in 1 ht· target.

65(·

Again 011 thi:- Ot'l':t,..il)n we rnay prubaùl~ ,.. tatP that thc t\1"0 mode~'

of action un· two a ~-. pect;. of tht• :>ame funùant~•ntal mrchani l'l m in that

tltt ') art• t he rel'ult of cncrgy tramfer p hc nomcna in irra riiat<'tl biologica(

S)hlrm,. By t he;,c ph<'n o nwna tht· raùintion c>rll'r!!~ alrntd~- a ccumul.ttt·d

in tlw targ<'l s tru cturc;, b ecomc, tran .,fN cÙ lO t'OIIIJIOUntb prP.·H' JI t in

tht• immetliat .- viri n il y of s uch l' tructurt:,. anJ i n t bi ... prt.H'e~" 1 :h' re:.t o­

r alion u r thc s t abiliza llort of thc damagl' may O<'<" llr . Once again a v arietr of compound!-l can b r in ' oh cd in l'l llC h prot•t•,;,e,, provicled that t h t·y

po~-.st· ~o;. th•· prOpl'r n ·acti ' Ìt) an d an: in ;,uflH'ÌI'nl cu nct•ntral ion an rl al

thl' righ t piace . urh <' o mpuundl' ma} h l' Cf'll <·o mponcnts a" \\'C II a' addccl co mpountl,, . ,\Il f al'LOrs atl't·rtiug <'d i t' ompo,.i tiun art• tlwrt•ft~n•

r ekvnnt t11 thc prcsent m i'chani -. nt.

Evidt•nci' that SH corn pounch, ma) rf';;;t itnl t' fn·t· radica!:- h) b~·drogcu

transft•r ha;, lwcu c·learly di'mon.,tratt•d in moùt· l :.y~ tcm-< b ) th<' t cchtlÌ<JUC

of pul:l<' rodioly&iF (19) and incli c·utions ha-.·•· bt·cn ohtaitwd tha t tlù~ m e·

ch ani;,m ma~ opcrat<• in hac·tc•ri a i rradiat<' cl in froun ... tatP (!l') . \\ ith r('gard to tbc s tahiJization of a tran><it•nt lc"•Ìon , it ha« to b i'

point<·d oul in tlw fir~t p larr•, 1 ha t ;, udt m ecba ni 'lm i-; ;.urpo'l{'d Lo play

an importanl rol c iu t h c scn!'itizntion by o,·y~··n. The fo rmation of perox) -radica l-. i;. <'Ommonly indicat<'d U" tht• IÌr~<~ t n ·uC'tion in t hl' pro<'c·~s (~1 )

ami ('' idC' Il('l' thnt tla i, n·ac ti ou a l'tuall\ uct tu" •n Ji,·ing ")'~t<'m,.. h a ... hc·t•n obtuin<'d b~ E Jt ~-> p e<·trumNry nf irrnd iult·tl Lactt>ria at ii0 h. e~l -

An c· l<'gant mod(•l tc1 rxp lain t !te auo x it· ... CII"Ì liza tion hy ~F:M aud other clf'c t ron-affinic or~anit· CO ill(HJUncl'. inc luding uns aturatPd tli <' l ('r -.

a nd dikl'lo compound:,. inchmc• triorll'. <1uinonc,, and u th <• r ... , j-. that pro· poscd by A.tlam;. whit•b po,.tu latc•;. an c·lt•\'lron tran-.fc·r mt·chanit-m from

a chargt•d intcrm .-dintc, prmlut•f'd by di rect c nergy a hsorption in turgl't moleculc:,. and thc hf'n,itizPr:- (!a). Tlw pruct'"" of electron t ransft' r 111

molecnlcs of high clc·<·tron-affinit y ha" b r(• n c·on \ i nrint!l~- dl'mon .. tral t•cl by Aclam, a nel <·oworkt•r;: in mod t• l >'\ ,.. lt•m -. u,..in~ clifl',·r•·nl <"o m pountJ ,_

inc ll•ding D'ì \ ba,,., ancl ~'>c·n:-i t iY.t'r" lil.. c• '\fr'r and mc·nadion.· {21).

Anotlwr peri irwnl t· xamplt· of tito• tn •·c·hani-.m li!Jtl••r t'tl ll"idt· ralinu i:- thal of t h t· :>t' ll'-Ìtiza tio n by orga ni t· nitri)XÌÙI' fr,•f• rntlic·al-<. tlu · lw-. t

known of wbich j ., triaccl oUl·amin t' 1\'"-oxyl (T \ N). Thest• t'llllll"'und-.

h a ve been :.hown ttl s;<•n , iti"c anoxir bact<'ri al anJ mammnlian rl' ll ' (~;·20 ). Pul e radiol p;i cxprrimcnt~> haYc d c mon ... tral<·d Lhat the udduc t of l>~\

witb OH' intcrar ts , ·ery rapidJy with TAN (27). ancl furth1•r inn<~ ti~ations

ha ve indicatrd tba t s u eh intcrartion involvr!< thf' C' CJ\":tll'nt binclin g o [ T A l'l to DXA (18) .

4 "" 1• 1 ~UIJI'T • .'illlli ftl 1\971 ) 1, ftH- 1\1\11

QUINTILIANJ

• • •

657

Table 6 lis ts the main t ypes of factors affecting radiation sensitivity by interfering with DNA repair.

TABLE 6

Facton which afrect radiation sensitivity by interference with DNA repair

GENETIC F ACTORS

GROWTH-PBASE DEPESDENT FACTORS

POST-IRRADIATION CULTURE CONDITIONS

POST-IRRADIATION SENSITIZERS

Mutntions

Growth stage

Compo~ition of Lhe med.ium, tempera­t ure, etc.

Poo<oyl LO<<o.,, ohlmomphoWool, Aoti- l nomicyn D, etc.

No mod cs of nction have been indicated, as in the prcvious tables, because in many cases such modes have not, so far, been established.

Under the present mechanism should be included only those factors which directly affect the ability of cella to repair DNA damage, and not through the agency of some of the other mechanisms previoulsly discussed. This is the case, for instance, of the radio-sensitization by iodine com­pounds, in which secondary radicals are supposed to inactivate some m embrane component involved in DNA repair.

Tbe involvement of repair mechanisms in genetJc variations of radio· sensitivity was first r eported in the now classica} paper by Mc Grath and Williams (8), who showed that the same nnmher of single-strand brcaks were produced in t he radio-rel!istant s train E. coli Bjr and in the radio-sensitive one B,_ 1 , while the two strains differ ed in their ability to repair the lesione. In fact, in E. coli Bjr strain there were only few breaks r emaining after 40 min of r eincubation, whereas in B,_ 1 no r epair was seen during ihis time. Very recently Town, Smith and Kaplan ( 28) have shown that a D NA-polymerase defìcient mutant of E. coli K 12 was highly radio-sensitive in comparison with its progenitor s train and was not ahle to operate the rapid repair of single-strand breaks, suggesting that the gene involved in D N A-polymerase d eficiency controls a system for the rapid r epair of single-strand breaks similar to that described for Micrococcus radiodurans.

Growth·phase dependent variations in radiosensitivity in bacteria have been known for a long time (•). A recent paper, also by Town, Smith and Knplan (So), gives evidence that such differences may be account· ed for to a large extent by difrerences in s trand rejoining and in the cont rol of post-irradiation DNA degradation.

A nn. h t. Suocr. Sanitd (1971) 7, 647~60

658

Also thc rescuing proccdurcs a re CX JWrimcntall y wrll known in bach·­ria, as th<• influen cc of post -irradiation trcatnwnts ou 1:'. coli sun•i, al was reportcd b y Staple ton and cow. in 195!; (31). ln ~t·ncra l , i t ha!> b ccn obscrvcd in ruicrool'ganis rn H, a H well UR in mammalia u CPII ;;;, that tlw environmental condition!> optimal for ct'll divi:-ion an' antagon.is tic to rcco,·cry from pott>ntially lrthal damap: t• and \ icncr..,a. Hnwevrr t h i 'l rath cr crude gcn eralization givcs probahly much too -,impl t• an idea of a problr m which Ì!; in!;t tad ' cr) complrx; with man~ d ifrt•rcn t aspcct", not yet fully unders toocl. act·ording t o tht• partirular mt•Labol.ic and gcnetic churact «.>ristit·s of th e d ifl'er t•nt t'PII" con ... idcn·d. IJo,,«.>wr, r ,·en if the r t> lationships bet,,een p os t-irrailiatiou treatrncnt<~ and D'lA r epair processrs cannot be gen l' rali~rd in few s trnight fonHird ..,tat t•meut,... it appcars that th t• final r e!>ult of their interplay ma~ afft•t• t tlw anwunt of the D""\A hiolo­gically ex:pr es,..ible dama~e .

A :<imil ar t ) p <' of rea~-.oning ma) a ppl ) t o tht· '-Il callt•d po:ot-i rradiation sen i> ttlzers . As a rule they are metnboli t• inhibit ur~. lik<'. for instanec. pantoy l lactonc, which int r <'UAC" tbc· -. un ivnl of irracliated E. roli K-J 2, as sho,~n by Adlrr and Hardigrct• (32), und a<.'tillom) ein O "hich, according to E lkind and cow. (33) grratly r eduCt•;.. t)w !HlT\'Ì\ a l of 1-ynchronized mam­malian cells if addctl imrnt•diau·ly aftc r irrntliation, hut is complct ely inelfectiYe if gi' cn two ho uri> Jatr r , :.uggt•stin ~ t hat 1w intt•rac tion would ocNJ r if cclls w cre allowN1 to repai1· ~uhkt hai damagt'.

Eviden('c that th<· cnhanc•r mt>nl of t h• ~ letal cfft·ct of ir radia ti o n is rdated to the r rc;titution of D~ A damagr ha~ bc<'n prorlncrd in a nuwbr r of case>'. l"nr Ìll >. laiH'I'. [)l'an , l.iltl«' :wd St'rinnni ~:11 1 h ave i->hown

that chloramphrnicol (CAP) leadf- tu a pro~r<• ... ;;i\ c lu;..'- of Yi ahility of J\1. rudiodurans cdJs expused l o radiatinu dn-;•"• 'dùch an· nonnall y non let hal. <\t tbr sarnr tim <> they obl:le n rd t ha t , in tb c• prt•.,t•nee of C.\ P . tbr JW!-. t irradia tion degradation of l)'\ \. \1 hi l·h norma li) ... top~ in 60-70 mino;;, procceds unint<'rrupted o, ·er man~ hour .... nntil mor <' than 80 ~>, of DI\\ has b cen r <'nderrd aci d solublc·. ( \P ud, o n l} in conc·(•ntration-. which inhibit pro tein synthr~is. sug~rsting t hat th<' lerm ination of tht· pror ess of DN \ hrt'akdown implie~< ;;;omt· proiPin which hus tu b e l;yn · thNized al the appropriate time in sittt. I n fad, othf'r contl.ition;, in which protein synthcsis Ì;, inlùbitcd, lik'• inC'uhation in nutricnt m edi um containing actinomycin D or in v t,ronttl buffer -t x l O ! )[, pJI 8.5, ah·n induce cxcessive D:\A d cgrndation and loc:, of ' iaLility with s uble thal radiation doses .

Another pcrtint>nt exarupl~ is o!ff'red in n rccent paper b~· Kapp and Smitb (:W) . The Authors bave s howu thnt an unkno'' n factor contained in certain chromatographically impmr surnpl cs of hydroxynrca irrcvcrs­ibly preHnts the r cpair of X- rny-induC'<'d l?inglc-stra nd breaks in D NA

Ann. Z.t. SupeT. Stttlllil (!9i1J 7, fH7-tillO

of ree+ cells of E. coli K-12 whcn udded after irradiation, and ~eatly r ecluces the viability of the same cells, while it does not at ali affcct th&

viability of irradiated cells of the mutant s trains which are de6cient in their nbility to repair single-s trnnù brcaks.

Of coursc, tbe nccessity of keeping the present paper to a rcasonable size on ly allowrd a ùiscussioo of tbe t ype which shows just that the approach used cao b e vnlid. This howevcr , was esseotially the aim of" the papcr: to open n discussion along certain lines of reasooing and to show tbat i l wotùd Le worthwhile to develop it further.

AcknowltdgPmenl : [ am plrased to ucknowledge Lhc help of Dr. A. Kelly in reading the manu~ript.

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