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DIACONU LAVINIA

SERIA B, GRUPA 11

CUPRINS
RADIATIILE IONIZANTE SI IMPORTANTA LOR RADIOBIOLOGICA........................................................................................2 UTILIZAREA BIOLOGICA SI MEDICALA A RADIATIILOR IONIZANTE.....................................................................................................4 NOTIUNI DE PROTECTIE IMPOTRIVA RADIATIILOR IONIZANTE....5 Elemente de radioprotectie..........................................................5 Reducerea expunerii pacientului la radiatii ionizante..................7 METODE DE DETECTARE A RADIATIILOR IONIZANTE......................8 Marimi caracteristice surselor radioactive si factori ce influenteaza masuratorile acestor surse...................................... 8 Tipuri de detectori....................................................................... 10 EFECTELE BIOLOGICE ALE IRADIERII...................................................14 Efectele iradierii asupra duratei de viata....................................15 Efectul cataractogenic al radiatiilor ionizante............................17 Carcinogeneza si leucemogeneza prin iradiere..........................18 Tipuri de cancer provocate de radiatii ionizante........................20 Efectul mutagen al radiatiilor.....................................................23 RECOMANDARI ALE COMISIEI INTERNATIONALE DE PROTECTIE IMPOTRIVA RADIATIILOR ( CIPR ) PRIVIND NIVELE DE IRADIERE PERMISE LA OM............................................................................................32 BIBLIOGRAFIE...............................................................................................35

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RADIATIILE IONIZANTE SI IMPORTANTA LOR RADIOBIOLOGICA


Prin radiatii ionizante se inteleg radiatiile electromagnetice cu lungime de unda sub 100 A, precum si radiatiile corpusculare, ca electronii, neutronii, particulele si , protonii, ionii grei accelerati si fragmentele de fisiune. Caracteristica importanta a radiatiilor ionizate este depunerea localizata a unor cantitati de energie in mediile pe care le strabat, avand ca rezultat producerea de ionizari si exercitari atomice sau moleculare. Energia disipata de radiatiile ionizante per eveniment de ionizare in materialul biologic este de aproximativ 33 eV, adica practic cu un ordin de marime mai mare decat energiile legaturilor chimice tari, cum ar fi de exemplu legatura C=C ( ~ 4,9 eV ). Diferentele dintre raspunsurile biologice la radiatiile ionizante din spectrul electromagnetic sunt seminificative. Astfel, organismul uman poate tolera foarte bine o doza totala de radiatii infrarosii care cauzeaza o crestere a temperaturii corporale cu 1 grad Celsius, in timp ce o doza de 400 rad de radiatii X, corespunzatoare unei absorbtii de energie ce ar ridica temperature corporala cu numai 0,001 grade Celsius, este letala. Efectele produse in mediile biologice traversate de radiatiile ionizante isi au originea in particularitatile fizice ale interactiunii acestor radiatii cu mediile respective. 1 Radiatiile ionizante se pot imparti in radiatii electromagnetice compuse din radiatiile X si gamma si radiatii corpusculare, formate din electroni, protoni, neutroni, particule alfa si alti nuclei grei. Ambele tipuri de radiatii produc efecte biologice prin ionizarea compusilor importanti ai celulelor in urma absorbtiei lor. Daca din punct de vedere al efectelor biologice produse si al modului de actiune sunt identice, ele totusi se deosebesc cantitativ prin puterea de penetrare si modul in care ele pierd energia in interiorul celulelor. Caracteristice fiecarui tip de radiatii sunt LET (transferul liniar de energie ) si EBR (eficacitatea biologica relativa ).

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Eficacitatea biologica relativa pentru diverse tipuri de radiatii TIPUL RADIATIEI Radiatii X si gamma Radiatii beta Neutroni termici Protoni Neutroni rapizi Radiatii alfa EBR 1 1 5 10 10 20 2 Societatea de astzi este ngrijorata cu privire la pericolele de radiaii ionizante, n special n perioada de dup explozia centralei de la Cernobl. Pe de alt parte, exist o lips generalizat de nelegere a radiaiilor biologice i radioepidemiologice - tiinele care furnizeaz date despre estimarile riscurilor au fost deviate. Ziarul Jurnalul a prezentat la un atelier de lucru "radiaiile de nivel sczut i cancer: de date i metode",intalnire ce a avut loc pe 10-11 decembrie, n Feuisberg, n apropiere de Zurich. ntlnirea a fost organizat de Institutul Social de Medicin Preventiv i de Universitatea din Zurich, sub auspiciile Biroului Federal Elvetian de Sntate Public. Obiectivele sale au fost trei. n primul rnd, pentru a da o introducere a unor fapte de baz i aspecte metodologice n fizica radiaiilor, biologie i epidemiologie. n al doilea rnd, pentru a oferi o imagine de ansamblu de disponibilitate a datelor pentru activiti de cercetare radioepidemiologice n Elveia i, n al treilea rnd, pentru a evalua posibilele strategii de cercetare n aceast ar. O list a unor noiuni i uniti utilizate n mod obinuit n tiinele radiaiilor servete o introducere la un cmp (G. Schler et al.). Folosind unitile i noiunile este important s se fac distincia intre descrierea experimentelor biologice i observaii epidemiologice de la definiiile i proieciile de risc propuse de rapoarte internaionale i organismele de consens n scopuri de radioprotectie. Documentele urmtoare ocupa, n special, anumite aspecte ale tiinelor de baz. 3

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UTILIZAREA BIOLOGICA SI MEDICALA A RADIATIILOR IONIZANTE

Cercetarea biologica si explorarea medicala utilizeaza radioizotopii ca trasori, folosind faptul ca izotopii unui element au, din cauza structurii electronice identice, proprietati chimice asemanatoare. In acest scop, se sintetizeaza molecule marcate ce au unul sau mai multi atomi radioactivi in compozitia lor, lucru care nu le schimba comportarea chimica si, deci, nu afecteaza ciclurile biochimice la care acestea participa. Tehnica moleculelor marcate a facut posibila dezvoltarea unor procedeuri analitice in biochimie (analiza prin activare, analiza prin fluorescenta X, radiocromatografia), a unor metode de studiu cinetic a compartimentelor biologice (ansambluri de molecule ce au aceeasi probabilitate de a suferi o transformare data transformare chimica sau migrare printr-o bariera), a unor metode de studiu cinetic al circulatiei pentru masurarea vitezelor si debitelor circulatorii. Moleculele marcate si radiatiile pot fi utilizate si in studii sau explorari medicale morfologice, prin tehnicile de autoradiografie, scintigrafie si radiografie. Cercetarea biologica si terapia medicala folosesc efectele produse de radiatiile ionizante, pe de-o parte pentru a clarifica mecanismele lor de actiune fie nociva, fie utila (stimularea germinatiei semintelor, de exemplu) si, pe de alta parte, in tratamentul tumorilor maligne, care are ca scop distrugerea selectiva si completa a populatiei de celule canceroase.

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NOTIUNI DE PROTECTIE IMPOTRIVA RADIATIILOR IONIZANTE


Limitarea pericolului pe care il reprezinta actiunea radiatiilor ionizante asupra organismelor vii se poate face prin 2 cai mai importante : - micsorand sau chiar remediind efectele deja produse; - reducand gradul de iradiere prin prevenirea poluarii radioactive a mediului.

ELEMENTE DE RADIOPROTECTIE
Protectia contra efectelor nocive produse de radiatiilr ionizante se poate face in mai multe feluri, in functie de mijloacele care se folosesc. Protectia fizica. Se realizeaza prin mijloace adecvate de reducere a dozei de iradiere la valoarea permisa, cum sunt : ecranarea , marirea distantei si scurtarea timpului de expunere, alaturi de ansamblul masurilor de organizare a lucrului cu surse radioactive. Se stie ca activitatea surselor () scade invers proportional cu patratul distantei (d): 1/2=d22/d21 , unde 1 este activitatea sursei la distanta d1 si 2 este activitatea surselor la distanta d2. Marirea distantei poate asigura o protectie buna pentru sursele , , in cazul iradierii externe. Acest lucru se realizeaza si prin sisteme de telemanipulare, care pot fi simple pense si clesti sau pot ajunge pana la utilizarea unor maini mecanice cu care se manevreaza sursele de mare radioactivitate. Scurtarea la minimum a timpului de expunere la radiatii poate fi realizata daca operatiile care se fac cu surse radioactive sunt minutios pregatite anterior. -5-

Ecranarea este mijlocul cel mai eficace de protectie fizica. Radiatiile se ecraneaza usor: datorita TLE mare ele au un parcurs mic, de ordinul micronilor. Manusile de cauciuc sunt suficiente pentru oprirea lor. Pentru oprirea completa a radiatiilor se folosesc ecrane cu grosime mai mare decat parcursul lor, din materiale cu procent mare de H (bachelita,materiale plastice). In niciun caz nu trebuie utilizate substante cu numar atomic Z mare (plumb), deoarece in acestea creste probabilitatea aparitiei radiatiilor de franare care sunt foarte penetrante. In cazul radiatiilor X si scopul ecranarii este de a reduce intensitatea acestor radiatii sub doza maxima permisa sau chiar pana la valoarea fondului natural de radiatii. Materialele folosite sunt de preferinta cele cu numar atomic Z mare, in care probabilitatea de interactiune cu electronii orbitali creste: plumb sau fier. Se utilizeaza si ecrane din beton sau sticla. Protectia chimica. Se realizeaza prin substante care, administrate imediat inaintea iradierii, maresc radiorezistenta organismului, producand urmatoarele modificari: - inhiba sau fixeaza radicalii radiolitici; - diminueaza oxigenul intra- si extracelular; - micsoreaza continutul de apa, mai ales in organele radiosensibile; - impiedica organele integratoare din organism sa amplifice efectele. Protectia biologica. Se face prin celule viabile care, administrate dupa iradiere, favorizeaza refacerea functiei hematopoetice, grav afectata de actiunea radiatiilor. Acest gen de protectie ridica, insa, serioase probleme de compatibilitate imunologica. 4

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REDUCEREA EXPUNERII PACIENTULUI LA RADIATII IONIZANTE

Tomografia computerizata (CT) ofera cea mai mare parte din doza de radiatii, aceasta doza fiind intr-o continua crestere. De exemplu, un articol din 2004, n Radiologie documentara subliniaza faptul ca CT a constituit aproximativ 13% din ordinele de imagistica de diagnostic pentru spitale din Statele Unite, dar cu conditia de 70% din doza. n plus, un raport din 2006 al Institutului National de Cancer a sugerat faptul ca numarul de CT efectuate la adulti si copii a crescut cu aproape 7 ori n ultimii 10 ani. Un numar de institutii medicale au evaluat recent cunostintele personalului lor medical despre doza de radiatii si riscul asociate acesteia. Multe studii au demonstrat o lipsa de cunostinte de catre personalul cu privire la doza de radiatie pentru procedurile radiologice. ntr-un studiu din 2004, la 1285-paturi de spital, doar 27,5% din medicii intervievati au fost capabili sa faca un scor de 45% sau mai mare, la 11 ntrebari despre doza de radiatie. n plus, 28% si 10% din medici intervievati n mod incorect credeau ca angiografia prin rezonanta magnetica si ultrasunete prezinta un risc de radiatiile ionizante. ntr-un studiu din 2004 de la Universitatea Yale, un centru de ngrijire tertiar, cei mai multi pacienti, medici si radiologi au fost n imposibilitatea de a stabili doza corecta a radiatiei pentru o scanare CT, comparativ cu o radiografie toracica. n plus, 78% din medici au recunoscut ca nu au explicat riscurile si beneficiile CT pentru pacienti si 93% dintre pacienti intervievati au sustinut ca nu au primit aceste informatii. Un studiu din 2007 care implica trei spitale universitare, o cercetare de spital si trei clinici n ambulatoriu a constatat ca 93% din personalul intervievati au subestimat cantitatea de doza de radiatii pentru proceduri de diagnosticare. Un articol din 2005, n Jurnalul International de Cardiologie a concluzionat ca cei mai multi medici nu poseda cunostinte despre doza de radiatii pentru proceduri de diagnosticare. Toate aceste studii recomanda o mai buna educatie a medicilor si a personalului cu privire la expunerea la radiatii si riscurile asociate acesteia. 5

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METODE DE DETECTARE A RADIATIILOR IONIZANTE


In biologie si medicina, masurarea radiatiilor ionizante este necesara in doua situatii mai importante: - cand se determina activitatea unei surse radioactive folosita ca trasor; - cand se determina efectul unui fascicul de radiatii ionizante asupra unui mediu traversat, in punctul unde se afla detectorul. Efectul fizic produs de o particula ionizanta este foarte slab. Unii detectori insa pot amplifica acest efect, rezultand un semnal detectabil. Aceste aparate capabile sa inregistreze particulele ionizante una cate una sunt numaratoarele. Cele mai utilizate tipuri sunt detectorii cu gaz si detectorii cu scintilatie. In alti detectori insa, semnalul electronic dat este prea slab si acestia nu pot fi utilizati ca numaratoare. Daca, insa, un numar mare de particule ating acest detector, semnalul electric rezultant este destul de mare pentru a fi masurat. Aceste aparate, numite dozimetre, sunt utilizate pentru o detectare globala si nu individuala a trecerii particulelor ionizante. Cel mai utilizat tip este camera de ionizare.

MARIMI CARACTERISTICE SURSELOR RADIOACTIVE SI FACTORI CE INFLUENTEAZA MASURATORILE ACESTOR SURSE


O sursa radioactiva poate fi caracterizata cantitativ masurandu-i-se viteza de numarare, din care apoi se poate calcula activitatea sursei, cat si fluxul de radiatii emis de catre aceasta. 1. Activitatea este marimea principala care caracterizeaza o sursa radioactiva definita ca numarul dezintegrarilor ce au loc in unitatea de timp. Unitatea acestei marimi este dezintegrarea pe secunda ( dez/s), numita in S.I. bequerel (Bq). Deoarece aceasta unitate este mult prea mica pentru sursele uzuale, se folosesc multiplii si submultiplii altei unitati numite Curie (Ci).

Intr-o unitate nucleara unde se efectueaza lucrari de categoria I, cu surse de radiotoxicitate mare, se admit activitati de 10-2 Ci, in timp ce pentru sursele de radiotoxicitate mica se admit activitati de 10 Ci. -82. Fluxul de radiatii emis de o sursa radioactiva este o alta marime caracteristica unei surse. Pentru masurarea activitatii trebuie precizat tipul si numarul de radiatii de un anumit tip emis la o dezintegrare, care poate fi calculat prin examinarea schemelor de dezintegrare a radionuclizilor (fig.7).

Fig.7- Schemele de dezintegrare In cazul unei scheme de dezintegrare cu o singura tranzitie , sursa emite cate o particula la fiecare dezintegrare si, deci, fluxul total de radiatii (emis in tot spatiul) este egal cu activitatea . Daca,insa, sursa are o schema de dezintegrare mai complexa trebuie calculat factorul de schema (s). De exemplu, in cazul Co60 s =1 si s =2. In acest caz, fluxul de radiatii ( ) emis in toate directiile va fi = s , iar fluxul de radiatii ( ) emis in toate directiile va fi =s . 3. Viteza de numarare. O sursa radioactiva este masurata cu un detector de radiatii. Daca detectorul este plasat la distanta x fata de sursa, el se afla intr-un fascicul de radiatii a carui intensitate intr-un punct (I) se obtine raportand fluxul total la suprafata sferei de raza x, deoarece radiatiile emise de sursa radioactiva se distribuie uniform in spatiu (fig.8) : I=/4x2=s/4x2

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Fig.8- Geometria de masurare Daca detectorul prezinta la iradiere o fereastra de intrare cu suprafata a sau o sectiune transversala cu suprafata a, iar eficacitatea lui este , atunci viteza de numarare (R) este: R= a I=as/4x2

TIPURI DE DETECTORI
Detectorii folositi pentru inregistrarea radiatiilor ionizante au ca element sensibil, unde se petrece ionizarea, un mediu gazos, lichid sau solid. Detectorii cu gaz Efectul fizic utilizat in detectare este ionizarea unui gaz. Daca o particula (direct sau indirect ionizanta) interactioneaza cu mediul, va produce un anumit numar de perechi de ioni. Intr-un gaz, acesti ioni se recombina si nu se va decela niciun efect. Daca acest gaz este insa plasat intr-un camp electronic, ionii pozitivi si negativi se vor deplasa spre cei doi electrozi care vor culege

acesti ioni. Astfel, interactia unei particule cu gazul din contor se traduce printrun semnal electric cules la borne. Amplitudinea acestui semnal q ( cantitate de electricitate ) este proportionala cu numarul de ioni culesi pe electrozi (fig.9). - 10 -

Fig.9- Detectorul cu gaz Transportul unui ion pozitiv la catod si a unui electron la anod are ca rezultat global transportul unei singure sarcini prin circuit. Astfel, ionul (+) se recombina cu un electron de pe catod, si totul se petrece ca si cum un electron ar fi fost transportat de la catod la anod. Pentru un anumit tip de particula (o energie absorbita, constanta in cursul interactiunii), sarcina q culeasa variaza cu tensiunea U aplicata pe electrozi. Astfel, se pot distinge mai multe tipuri de utilizare (regimuri de functionare) a detectorilor cu gaz. Camera de ionizare Cand se aplica o tensiune mica pe electrozi, un anumit numar de ioni se recombina intre ei si culegerea pe electrozi este incompleta, dar creste proportional cu cresterea tensiunii. La o anumita valoare a tensiunii, toti ionii formati vor fi culesi. Se atinge un platou qi care corespunde domeniului de lucru al camerei de ionizare (culegere completa a ionilor, fara multiplicare). Se poate calcula numarul de ioni formati in timpul interactiunii, care este dat de raportul : qi/e ,unde e este sarcina electronului ( 1,6* 10-19C). Numarul de ioni produsi de o particula este in general prea mic pentru a produce o cantitate de electricitate detectabila ( camera de ionizare nu este, deci, un numarator de particule), dar aceste sarcini electrice cumulate in timp la nivelul

electrozilor sunt totalizate. Astfel se ajunge la doza de ioni si detectorul este utilizat ca un dozimetru ( etalonat in rntgeni ). - 11 Se poate calcula, de asemenea, si doza de energie, stiind ca fiecare pereche de ioni formati corespunde consumului de catre aer, de exemplu, a unei energii de 34 nV. Energia pierduta de radiatie (E) in volumul camerei de ionizare va fi,deci : E=qi/e*34. Si in acest caz detectorul este folosit ca un dozimetru (etalonat in razi). Contorul Geiger-Mller Cand tensiunea aplicata este suficient de mare, ionizarea primara declanseaza un fenomen de avalansa de electroni care se propaga pe electrod. Se obtine astfel un impuls de curent de amplitudine mare independenta de energia absorbita. Acesta este domeniul contorilor Geiger-Mller, care sunt foarte mult folositi din cauza ca nu necesita o amplificare mare ulterioasa a impulsului din detectori. Impulsul de curent produce un impuls de tensiune pe un rezistor R ( fig.10) care va fi inregistrat de un numar electronic de impulsuri.

Fig. 10- Detector Geiger-Mller cu instalatia de numarare auxiliara

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Detectori solizi sau lichizi Detectorul cu scintilatie este astazi cel mai utilizat tip de detector. Detectorul (sonda de scintilatie) se compune dintr-un cristal de scintilatie, un fotomultiplicator si un preamplificator. Sonda este conectata la o sursa de tensiune (1000-1500 V) si la numaratorul de impulsuri. Scintilatorul este de obicei un cristal de NaI activat cu thaliu, in care se petrec urmaatoarele doua fenomene mai importante: - energia radiatiei incidente se absoarbe in cristal producand ionizarea sau excitarea moleculelor cristalului; - moleculele care se dezexcita emit fotoni luminosi (deci scintilatii) in spectrul vizibil sau in domeniul UV apropiat. Cantitatea de lumina emisa este proportionala cu energia absorbita in cristal. 4

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EFECTELE BIOLOGICE ALE IRADIERII

Schema de ansamblu a efectelor ionizante asupra organismului

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EFECTELE IRADIERII ASUPRA DURATEI DE VIATA

In multe experiente, pe animale, s-a observat ca expunerea la iradiere accelereaza procesul de imbatranire si duce de regula la o scurtare a vietii fata de animalele de control. Procesul natural de imbatranire nu are o explicatie suficienta, dar se presupune ca implica o degenerare progresiva a tesuturilor normale, ceea ce duce la afectarea functionala si eventual la moarte. S-a sugerat ca expunerea organismului la iradiere produce o diminuare in populatiile de suse celulare si in populatiile de celule diferentiate, precum si o alterare a tesuturilor vasculoconjuctive. In realitate procesele de imbatranire a organismului intreg sunt asemanatoare cu acelea implicate in patologia modificarilor produse prin iradiere la nivelul organelor individuale si tesuturilor. Rotblat si Lindop (1961) au pus in evidenta o relatie lineara intre scurtarea vietii si doza de radiatii (fig.1).

Fig.1- Relatia dintre scurtarea procentuala a timpului de viata si varsta avuta in momentul iradierii, la soareci (Duncan si Nias,1977) - 15 Trebuie facuta o diferenta intre observatiile facute dupa iradiere cronica si cele facute dupa expunerea la doze unice, la care rata mortalitatii nu se modifica, iar animalele mor la varsta mai tanara decat martotii. Dupa iradierea cronica, rata mortalitatii la animalele expuse creste, efect descris a imbatranire accelerate. S-a observat ca la aceste animale se produc boli degenerative cu o rata mai ridicata decat la martori. Se crede ca reducerea duratei de viata ca urmare a iradierii este rezultatul unei accelerari a proceselor degenerative, ceea ce duce in plus in unele cazuri la o incidenta crescuta a bolilor specifice precum cancerul si moartea timpurie. Rotblat si Lindop (1961) au aratat ca scurtarea duratei de viata este mai mare atunci cand sunt iradiate animalele immature decat atunci cand sunt iradiate animale mature, iar efectul creste cu varsta. Fenomenele observate in imbatranirea precoce, produse ca o consecinta a iradierii seamana in mare cu procesele de imbatranire normala dar la o analiza mai profunda ele nu mai apar atata de asemanatoare. In orice caz, procesul de imbatranire radioindus prezinta urmatoarele semne: albirea parului, formarea de cataracte, atrofie pe iris, leziuni vasculare si debut al cancerului. Animalele expuse unei imbatraniri premature mor de aceleasi boli, ca si martorii neiradiati, dar debutul bolii este mai timpuriu

iar moartea, prematura. La soarece durata de viata este redusa cu circa 10% pe 100 rad la iradierea intregului corp cu radiatii X sau atat timp cat iradierea este aplicata inaintea varstei medii. Daca aplicarea se face mai tarziu efectele nu sunt atata de pronuntate. Nu exista date suficiente si certe referitoare la efectul iradierii la om in ceea ce priveste scurtarea duratei de viata. In studiile facute de radiologii americani in perioara 1945-1954, mentionate de Duncan si Nias (1977), s-a demonstra ca acestia au avut o durata redusa de viata comparative cu alte categorii de medici. S-au facut si se pot face numeroase supozitii, dar oricum exista multe rezerve in ceea ce priveste valididatea concluziei acestei cercetari. Ca raspuns la acest studio tot Duncan si Nias mentioneaza o cercetare asupra radiologilor britanici in practica pe perioada 1897-1957 in ceea ce priveste durata lor de viata, la care nu sa semnalat un efect nociv al expunerii ocupationale la iradiere. De asemenea Hutchinson (1968), mentioneaza ca nu exista date certe provenind de la supravietuitorii celor doua explozii atomice din japonica in ceea ce priveste un efect nespecific de scurtare a vietii, desi s-au semnalat o incidenta crescuta a mortalitatii prin anemie aplazica si diferite forme de cancer ( Court-Brown si Doll, 1965). In unele experiente ale lui Upton (1960), s-a observat ca atunci cand debitul dozei a fost de circa 0,1 pana la 0,5 radiatii pe zi, animalele iradiate au trait mai mult decat martorii. - 16 Explicatia acestor constatarI este departe de a fi clara. S-a presupus ca efectul se poate datora unor factori de mediu ambient fara vreo legatura cu iradierea. S-a considerat ca unele efecte produse de doze scazute de radiatii, cum ar fi cresterea activitatii sistemului limforeticular si cresterea usoara a neutrofilellor si limfocitelor pot prezenta avantaje pentru animale (Beebe, 1981).

EFECTUL CATARACTOGENIC AL RADIATIILOR IONIZANTE

Primele observatii prinvind efectul cataractogenic al radiatiilor ionizante sau facut pe muncitorii ce lucrau la un cyclotron ( Woods A. C., 1949). Ulterior, cercetari atente pe cazurile iradiate terapeutic ( Merrian si Focht, 1957; Worgul, 1981 ) si pe supravietuitorii bombardamentului atomic de la Hiroshima ( Dodo,

1975 ) au adus dovezi noi indicand o perioada de latenta pentru instalarea cataractei de circa 2 ani ( Cogan si colaboratorii, 1950; Albert si colaboratorii, 1968; Bendel si colaboratorii, 1978). Cunostintele referitoare la relatia dintre doza, timpul de latenta si opacifierea cristalinului la om sunt controverste in literature de specialitate. In 1979, Beebe a sistematizat datele existente in felul urmator: la leziuni clinice semnificative, produse de radiatiile cu TLE scazut pragul de aparitie este de la 600-1000 rad, respective 75-100 pentru neutronii rapizi; EBR pentru neutroni rapizi se situeaza intre 2-10 valorile inalte fiind associate cu dozele joase; dozele fractionate reduc riscul de formare a cataractei in cazul radiatiilor cu TLE scazut, dar nu si in cazul celor cu TLE mare; latenta de aparitie a cataractei este de la 2-7 ani; estimarea procentuala a riscului de aparitie a cataractei a fost de 10% la pacientii expusi la 250 rad radiatii si 75 rad iradiere cu neutroni iar la cei expusi la 1350 rad riscul a fost de 100%. Instalarea cataractei ca urmare a iradierii, este precedata de o serie de modificari biochimice si fiziologice la nivelul cristalinului, cum ar fi scaderea transportului active si cresterea permeabilitatii pentru cation. Hidratarea cristalinului camane neafectata pana la instalarea cataractei mature. S-a constatat o scadere progresiva a glutationului la nivelul cristalinului in urma iradierii indicand un posibil rol al acestuia in reglarea transportului de cation si in hidratare. Alte componente biochimice studiate ca acid ascorbic, superoxiddismutaza, H2O2 nu au prezentat modificari semnificative fata de lotul .(de control ( Matsuda si colaboratorii, 1981 1 - 17 -

CARCINOGENEZA SI LEUCEMOGENEZA PRIN IRADIERE


Estimarile riscului cancerigen este o problem foarte complex i complexitatea acesteia este cel mai bine ilustrata prin luarea n considerare a datelor cu privire la efectul radiaiilor ionizante. Aceste date sunt, eventual, exemplul cel mai extins de la un rezultat al sanatatii umane, in care un numr mare de oameni sunt expui la o gam larg de doze de radiatii X si gamma X. Aceste date permit s ia n considerare gradul de adecvare a diverselor modele statistice pentru riscul cancerigen n populaiile umane. Datele au demonstrat c exist muli factori complexi implicai n ncercarea de a prezice un risc de cancer de la un singur agent. Ipotezele care stau la baza unuia dintre aceti factori pot avea un efect profund asupra estimarilor ulterioare cantitative in orice standard de expunere sau de reglementare care este derivat din astfel de estimri. 6

Criteriile de evaluare a riscului carcinogenic sunt: - abundenta, natura si calitatea datelor epidemiologice; - certitudinea asocierilor raportate; - consistenta evidentei epidemiologice : masura in care studii epidemiologice efectuate in diferite aranjamente experimentale cu parametri experimentali diferiti conduc la rezultate similare; masura in care rezultatele sunt asemanatoare cu cele obtinute in alte studii; - temporalitatea : masura in care studiile epidemiologice pot sa evidentieze faptul ca o anumita afectiune se datoreaza unei anumite expuneri precedente; - raspunsul la doza : masura in care studiile pot sa demonstreze ca persoanele expuse in conditii mai dure prezinta un risc mai mare de afectare; - cantitatea si natura evidentei datelor de laborator; - certitudinea datelor de laborator : masura in care efectele obtinute prin experiente in vitro sau pe animale pot fi interpretate ca fiind carcinogene si pentru om; - plauzibilitatea biologica; - plauzibilitatea biofizica/biochimica. 7

- 18 Cu cateva decenii in urma, principala sursa de preocupare in domeniul radioprotectiei era prezentata de potentialul mutagen al radiatiilor ionizante. Cercetari extensive pe animale de laborator au furnizat relatii doza-efect presupuse a fi lineare in domeniul dozelor mici si utilizabile in stabilirea limitelor standard de expunere a populatiei umane la radiatii. Studii epidemiologice effectuate pe oameni si acoperand domeniul 10-100 rad au relevant o incidenta scazuta a mutagenezei radioinduse in populatiile umane, datorita probabil si numarului mic de cazuri. Din contra, carcinogeneza leucemogeneza au putut fi puse clar in evidenta la supravietuitorii bombardamentelor atomice din Japonia. Din aceasta cauza, potentialul carcinogenetic al radiatiilor ionizante reprezinta azi sursa majora de preocupare .(in determinarea efectelor pe termen lung ale expunerii la radiatii ( Todd, 1981 Aparitia neoplaziilor radioinduse depinde de : doza de iradiere, debitul .dozei, timpul de expunere, organul afectat, varsta, sex, stare fiziologica Comisia Internationala de Protectie Radiologica ( CIPR ) a estimate ca iradierea generala la populatia adulta cu 1 rad pe an duce la aparitia intr-un an a doua cazuri de leucemie si a doua cazuri de alte forme de cancer, intr-o populatie de 100 000 de indivizi. Nivelul total al radiatiei provenind din surse

naturale este de pilda in Marea Britaniei de circa 0,1 rad pe an, iar numarul de cancere produse la acest ordin de marime al dozei este foarte mic ( aproximativ 0,07%) comparative cu numarul de cazuri datorate altor cause. In ceea ce priveste leukemia s-a estimate ca 10% din cazuri pot fi datorate expunerii la .(iradiere (Duncan si Nias, 1977 Mentionam ca observatiile CIPR se bazeaza pe ipoteza unei relatii lineare doza-raspuns, iar aceasta presupunere supraestimeaza riscurile la doze sub 100 .rad Leucemiile sunt cele mai importante boli neoplazice produse de radiatiile ionizante. In prezent se apreciaza ca peste jumatate din celelalte forme de cancer sunt produse la om de agenti chimici, iar cresterea generala a incidentei datorita iradierii este foarte mica. Incidenta modificarii maligne consecutive iradierii este atat de mica incat se impun observatii multiple pe grupe mari de populatii pentru a se putea face o interpretare corecta si exacta. Deoarece perioada latenta pentru producerea cancerului este lunga (5-8 ani pentru leucemii si probabil peste 15 ani pentru celelate forme de cancer), este necesar ca toate aceste observatii sa se intinda pe foarte multi ani ( Hall si Miller, 1981). Nesiguranta referitoare la rata incidentei spontane a cancerului si leucemiei la populatia neexpusa, precum si incidenta deceselor din boli curente face ca analiza comparative a datelor provenite de la populatia expusa sa fie o operatie statistica foarte dificila. Incidenta cancerului si a leucemiei creste in mod natural odata cu varsta, si acest factor trebuie controlat cu foarte mare grija atunci cand se determina riscurile .expunerii la iradiere 21 Studiile lui Anderson si colaboratorii (1967), au aratat ca expunerea intrauterina la doze de radiatii de la 1-2 rad a dus la cresterea probabilitatii de dezvoltare a leucemiei cu 1,5 pana la 2 ori mai mult fata de cazurile neiradiate. Copiii ale caror mame au avut nevoie de exmamene radiologice in timpul .sarcinii ai reprezentat un grup cu risc mai mare la leucemie In producerea cancerului, Borek si Hall (1973) au aratat ca este importanta nu numai doza totala de radiatii, ci si debitul dozei. Exista totusi date putine referitoare la influenta debitului dozei asupra incidentei transformarii neoplazice. In masura in care datele sunt concludente se poate presupune ca .intenstitatile mai mari de doza nu cresc procentul transformarilor neoplazice Producerea leucemiei dupa iradiere reprezinta o exceptie, in sensul ca poate fi independenta de debitul dozei. S-a constatat pe animale ca administrarea in mod fractionat a unei doze de iradiere ( cunoscuta ca fiind cancerigena) nu determina o diminuare a efectului cancerigen, comparative cu iradierea acuta (Fry, 1981). Radiatiile cu transfer linear de energie ( TLE) ridicat, cum ar fi neutronii rapizi, sunt mai eficiente in producerea transformarii maligne decat .radiatiile cu TLE scazut

Procesele patologice declansate ca urmare a iradierii pot fi considerate ca fiind bazate fie pe efecte locale, fie pe efecte mediate ale iradierii asupra .tesutului implicat 1

TIPURI DE CANCER PROVOCATE DE RADIATII IONIZANTE

Cancerul tiroidian Incidenta tumorilor tiroidiene este crescuta la copiii care au fost iradiati cu scop terapeutic in regiunea gatului si capului pentru afectiuni ca: hipertrofie de timus, acne, hipertirodism, TBC al ganglionilor cervicali. Neoplasmele include carcinoame, adenoame, noduli hiperplastici care s-au dezvoltat dupa o perioada de latenta care a variat intre 10 si 20 de ani. Totusi, nici un studiu epidemiologic nu a putut cuprinde un numar suficient de pacienti pentru a indica o relatie precisa intre doza si incidenta. Duffy si Fitzgerald (1950), studiind un lot de 430 pacienti cu cancer tiroidian, au observat ca dintre acestia 28 erau sub 18 ani si au fost iradiati pe regiunea timisului la varsta de 4 si 16 luni. - 20 Latourette si Hodges (1959) pe un grup de 867 copii iradiati pentru hipertrofie de timus inainte de 1942, cu doze mai mici de 200 r, printr-o poarta de intrare de 10/10 cm, au semnalat 2 cazuri de limfoame (o leucemie) in plus fata de incidenta statistic prevazuta. La un copil a fost diagnosticat un carcinoma tiroidian. Saenger si colaboratorii (1960) au studiat 1644 persoane din 2230 care au fost iradiati in copilarie in regiunea capului, gatului si toracelui, in comparative cu 3777 martori. S-au gasit 11 carcinoame tiroidiene si un caz de leucemie la lotul studiat, fata de nici unul la lotul de control. La supravietuitorii de la Nagasaki si Hiroshima, la 1215 autopsii practicate, au fost evidentiate 21 neoplasme tiroidiene diagnosticate histologic; 7 din acestea au fost gasite la persoane care au fost expuse iradierii la o distanta de pana la 1400 m de la hipocentrul exploziei. Numarul statistic era de 3 ( in baza ipotezei ca nu ar exista o relatie intre incidenta tumorilor tiroidiene si iradiere).

Jeldis, Jablon si Ishida (1964) au diagnosticat 7 carcinoame tiroidiene la 124 autopsii la persoane expuse iradierii la o distanta de pana 1400 m de la hipocentru exploziei, fata de 3,5 prevazute statistic. Hempelmann si colaboratorii (1967) au urmarit 2878 copii pe o perioada medie de 16,5 ani si au observat 19 carcioame tiroidiene fata de 0,14 prevazute statistic. Doza medie tiroidiana a fost de aproximativ 160 rads, iar rata de inducere a cancerului tiroidian a fost de 0,4%/100 rads. Modan si colaboratorii (1974) la 10900 copii iradiati pentru micoze ale pielii capului au gasit 12 carcinoame tiroidiene fata de 2 prevazute statistic, dupa o perioada de 13-24 ani de la iradiere. In cuida faptului ca doza medie tiroidiana a fost de 6,5 rads, rata de inducere a cancerului tiroidian a fost crescuta, 1,46%/100 rads. Intr-o lucrare recenta, Wilkins (1978) semnaleaza ca in SUA peste 1 milion de oameni au primit iradiere in regiunea capului si gatului pentru diferite afectiuni benigne la varsta de copl mic. Dupa calculele autorului, din acest lot 735 persoane vor muri prin cancer tiroidian.

Cancerul cutanat Prima localizare neoplazica atribuita efectului radiatiilor ionizante a fost un carcinoma epidermoid pe mana unui radiolog in 1902. In SUA, primul pacient care a murit in urma unei tumori radioinduse a fost asistentul lui Thomas Edison, Clarance Madison Dally, in 1904, care a lucrat cu surse de radiatii X. Cazuri similare au fost observate la radiologii care si-au expus in mod repetat mainile iradierilor. La toti acestia s-au semnalat in antecedente - 23 radiodermite, iar aparitia tumorilor cutanate a urmat dupa o lunga perioada de latenta de la momentul iradierii. Majoritatea cancerelor aveau urmatoarele tipuri histologice: carcinom pavimentos spinocelular carcinoma bazocelular, fibrosarcoame. In general, se postuleaza ca incidenta cancerului este dependenta de severitatea radiodermitelor din antecedente si a fost sugerat ca expuneri prelungite la mai putin de 4 r pe zi pot culminca cu o neoplazie, semnalandu-se tumori in zoca iradiata pe pielea care parea clinic normala. 2 Cancerul mamar

Radiatii ionizante (IR) sunt un cunoscut agent cancerigen uman de sn. Desi capacitatea de mutagen al IR este recunoscut ca baza pentru actiunile sale ca substanta cancerigena, noi si altii am aratat ca IR pot determina, de asemenea, factori de crestere si de remodelare a matricei extracelulare. n consecinta, neam propus ca un factor suplimentar care contribuie la carcinogeneza IR este de ntrerupere potentialul de constrngerile critice care sunt impuse de interactiunile celulelor normale. Datele epidemiologice arata ca femeile expus la radiatii ionizante (IR), fie pentru terapie, n scopuri de diagnosticare, sau ca o consecinta de bombe atomice au un risc crescut de cancer la san. Experimentele recente sugereaza faptul ca carcinogeneza poate fi indus de interactiuni ntre celulele anormale si micromediul lor. n cazul n care expunerea la radiatii afecteaza nu numai fenotipul de stromale, dar si a celulelor epiteliale, aceste modificari ar putea promova progresia neoplazica n celulele sensibile. Pentru a testa aceasta ipoteza, am ntrebat daca dozele IR subletale perturba capacitatea HMEC sa fie supuse mamare-morfogenezei specifice ntr-un context fiziologic prin utilizarea testului 3D RBM. Pentru a reproduce o componenta cheie a stromei iradiate, TGF- a fost adaugat in unele culturi. Pentru a masura consecintele globale de iradiere, am folosit microscopul confocal si unui sistem de formare a imaginii bioinformatice pentru achizitia unei imagini integrate. Am constatat ca iradierea coloniilor HMEC a dat nastere la alta colonii n cazul n care aproape toate descendenta nu a reusit sa stabileasca polaritate bazala si a pierdut integritatea organizatorica, masurata prin mai multi parametri. 8

- 22 Alte localizari ale cancerelor radioinduse In afara de localizarile neoplazice radioinduse amintite mai sus, au fost raportate si altele, induse de asemenea de radiatiile ionizante. Trebuie mentionat insa ca dovezile in ceea ce priveste originea lor radioindusa sunt mai putin concludente decat cele pentru tumorile mai sus discutate pe larg. Dintre aceste tumori se pot aminti: limfoamele la supravietuitorii bombardamentelor atomice din Japonia si la pacientii iradiati pentru spondilita anchilopoietica; tumori faringiene la pacientii iradiati pentru spondilita, la cei tratati pentru tireotoxicoza sau alte afectiuni la nivelul gatului; tumori gastrice si pancreatice la pacientii iradiati pentru spondilita; tumori uterine la supravietuitoarele bombardamentelor atomice; colagioame si hemangioendotelioameale ficatului la pacientii injectati

intravascular cu Thorotrast; cancer ale sanului la bolnavii examinati in repetate randuri pentru TBC pulmonar si la supravietuitorii bombardamentelor atomice; tumori ale glandelor salivare la cei care au fost iradiati in regiunea capului si gatului in copilarie si alte multe neoplasme provenite din iradierea intense a diferitelor zone ale corpului. 2

EFECTUL MUTAGEN AL RADIATIILOR

Mutatiile radioincluse se clasifica dupa marimea partii afectate din genom in ;doua categorii: a). mutatiile cromozomiale .b). mutatiile genice Mutatii cromozomiale Aberatiile cromozomiale induse de radiatii sunt secundare evenimentelor radiologice primare din celula, care depend de eficienta si acuratetea sistemelor de reparare a AND. Rezulta asadar ca aberatiile cromozomiale sunt o manifestare imediata a diferentelor tipuri de afectari nereparate la nivelul AND .iradiat 25 Mutatiile radioinduse presupun doua conditii si anume: a).radiatiile trebuie sa patrunda direct in celula si b).energia lor sa fie absorbita de cromozomi. Asa cum s-a aratat in capitolul intai procesele radiochimice consecutive absorbtiei energiei radiante sunt foarte complexe si au o anumita .durata de timp Atat in cazul mutatiilor genice cat si a celor cromozomiale, aparute ca urmare a unei iradieri, exista un stadium potential reversibil asupra carui se poate actiona fie in timpul iradierii, fie dupa iradiere. Odata terminat procesul de .stabilizare al mutatiilor, acestea trec in categoria modificarilor ireversibile Extrema fragilitate a cromozomilor la actiunea radiatiilor ionizante, frecventa anomaliilor structurale si numerice au impus examenul cromozomial ca un test de dozimetrie biologica in evaluarea efectelor acute sau tardive ale iradierilor .accidentale, ocupationale si medicale

Referitor la mecanismul de producere al mutatiilor cromozomiale, este acceptate azi in radiologice ca ele se produc atat prin interactiunea directa a energiei radiante cu cromozomii, cat si prin fenomene extragenetice. Acstea din urma pot implica fie lizozomii, a caror membrane permeabilizata prin iradiere va permite eliberarea enzimelor, fie alti factori plasmatici, ca de exemplu radicalii liberi, care modificati prin radiatii sunt capabili sa induca dezordini .cromozomiale Indiferent de mecanismul prin care se instaleaza leziunile moleculare, radiatiile ionizante produc doua feluri de anomalii cromozomiale: numerice si structurale. Anomaliile cromozomiale numerice sunt nespecifice, ele putand fi provocate si de alti factori de mediu. Mutatiile structurale sau morfologice produse de radiatii asupra cromozomilor cresc in frecventa proportional cu doza .de iradiere, putand fi cromatidiene sau cromozomiale Mutatiile structurale cromatidiene sunt produse de iradierea la celulele care sunt in stadiul G2 din ciclul de diviziune. Aceasta categorie de mutatii nu reflecta fidel doza de radiatii absorbite. Un alt inconvenient al studierii lor in radiobiologie este faptul ca sunt destul de frecvente si la organismele neiradiate, .ceea ce sugereaza faptul ca la aparitia lor isi dau concursul si alte cauze Anomaliile cromozomiale sunt mai mult specifice in iradiere; ele au la baza fenomenul de fragmentare transversala a cromozomilor, urmat de reunire a fragmentelor cromozomice prin capetele lor rupte la unul din capetele rupte ale celorlalti cromozomi. Schimbarile structurale mentionate pot aparea fie in cadrul unui singur cromozom, fie la ambii membri ai unei perechi de cromozomi sau .(chiar intre cromozomi neomologi (fig.2 Prin ruperea si reunirea fragmentelor cromozomiale intr-o alta secventa decat cea initiala rezulta cromozomi aberanti cu doi centromeri sau lipsiti de centromer; mai rar se intalnesc la organismele iradiate cromozomi inelari, .tricentrici si policentrici 26 Aberatiile structurale induse de radiatii ale cromozomilor pot afecta fie numarul genelor dintr-un cromozom, fie modul de aranjare a genelor pe cromozom. In primul caz avem de a face cu doua feluri de aberatii: deletii si duplicatii. In al doilea caz intalnim ca tipuri de aberatii inversiunea (alipirea unui segment de cromozom intr-o ordine inverse) si translocatia (schimbarea .(reciproca a unor fragmente cromozomiale intre doi cromozomi neomologi Aberatiile structurale ale cromozomilor apar si in mod spontan, in natura, constituind cauza evolutiei si diversificarii vietuitoarelor, dar frecventa lor creste .semnificativ la organismele iradiate sau tratate cu agenti chimici cu rol mutagen

Fig.2-Exemple de aberatii cromozomiale structurale intalnite la pacienti in (timpul si dupa radioterapie (Amorose,1967 Primele mutatii radioinduse au fost puse in evidenta de Muller care in 1927 prezinta la al V-lea Congres Mondial ce se tinea la Berlin primele dovezi privind transmiterea artificiala a genelor Drosophila ca urmare a iradierii cu raze X. pe langa faptul ca a contribuit mult la intelegerea moderna a radiogeneticii, Muller a fost timp de aproape 30 de ani un adversat sincer al evitarii incarcarii genetice umane prin eliminarea folosirii radiatiilor, in special a razelor X, in scopuri .medicale 27 Ulterior efectul mutagenic al radiatiilor a fost descries la plante. Au fost facute aopi numeroase studii la mamifere de Brewen si Luippold (1971), Clemenger si Scott (1973), Bajerska si Liniecki (1975), iar la om de Buckton si colaboratorii (1971) si Schmid si colaboratorii (1974) care au aratat ca frecventa aberatiilor cromozomiale induse in limfocite in vitro este atat sub aspect calitativ .cat si cantitativ similar cu aberatiile observate dupa expunerile in vivo Dintre variatele tipuri de aberatii induse de radiatii in celule, cromozomii dicentrici au fost considerati ca cel mai real indicator al expunerii limfocitelor umane, deoarece acesti cromozomi pot fi usor si obiectiv numarati, aratand o variabilitate redusa printer culturile expuse in vitro si sant evenimente extreme de rare in limfocitele umane care nu au fost expuse la iradiere (frecventa

spontaba a dicentricilor a fost estimate ca fiind eproximativ de 1 la 4000 .(metafaze limfocitare Expresia matematica a producerii unor astfel de aberatii a fost data de Lea ;((1955 C+D+D2=1 unde C este frecventa aberatiilor spontane, defineste proportia aberatiilor induse de un singur eveniment de iradiere, iar defineste proportia aberatiilor de doua sau mai multe evenimente de radiatii. Un exemplu care ilustreaza relatia de .mai sus este dat de figura 3

Fig.3-Frecventa la aparitie a inelelor centrice si dicentricilor in limfocitele umane dupa o expunere in vitro la raze X (Brewen si (Luippold,1971 28 -

In limfocitele umane iradiate in vitro s-a constatat ca frecventa cromozomilor este specifica, in functie de marimea dozei si debitul dozei de iradiere. Un exemplu al efectelor produse de debitul dozei in inducerea .dicetricilor este dat in figura 4

Fig.4- Frecventa dicentricilor in limfocitele umane iradiate cu radiatia a 137Cs, la debite de doza de 400 rad/ora si 10 rad/ora (dupa (Purrott si Reeder,1976 Numeroasele lucrari mentioneaza aberatiile cromozomiale intalnite in culturile de limfocite provenite de la persoanele iradiate ( medical, accidental sau occupational). Printre aceste date cele provenite prin iradierile medicale sunt cele mai accesibile pentru studiul aberatiilor cromozomiale radioinduse la om. In culture de limfocite provenite de la pacienti iradiati cu 20 de ani in urma pentru spondilita ankilopoetica s-au pus in evidenta cromozomi dicentrici si inelari. In 1962 Stewart si Sanderson descriu la un pacient cu sindromul Klinefelter, la care s-au luat probe de limfocite la 8 ore de la iradiere, existenta dicentricilor in 2 celule din cele 31 analizate. Horman si colaboratorii sai in 1964 au examinat 5138 de limfocite la un numar de 10 lucratori de spital ce primisera in medie 88 rem intr-un ritm de 1-3 rem pe an si au pus in evidenta 7 .cromozomi discentrici 29 -

La un grup de 36 de lucratori care primisera o doza anualat de 1,46 rem sau gasit 14 dicentrici in 14 836 de cellule analizate. Mouriquand (1971) mentioneaza prezenta cromozomilor discentrici in limfocitele unor radiologi. Imreh si colaboratorii sai intr-un studio pe personalul din unitatile de radiology si de medicina nucleara semnaleaza existenta unor aberatii cromozomiale de .tipul dicentricilor si inelarilor

Aberatii cromozomiale de tipul celor amintite s-au semnalat si la personalul afectat tehnologiilor nucleare, precum si la supravietuitorilor bombardamentului atomic de la Heroshima si Nagasaki. Intr-un studio de persoane iradiate accidental cu neutroni si radiatii gamma la Oak-Ridge, Tennessee, in 1958, s-au evidentiat leziuni genetice cromozomiale de tipul: dicentrici, inelari, translocatii, inversiuni etc. Brondon si colaboratorii sai (1978) au evidentiat in limfocitele minerilor de la minele de uranium din Colorado un efect citogenetic la dozele premise occupational. Recent, Vladescu (1982), intr-un model experimental, au iradiat sobolani prin administrare de 59Fe, la doza de 5 rem pe an. Aceasta doza a fost administrate fractionat si acut. In ambele cazuri s-au gasit in maduva hematogena aberatii cromozomiale, dar frecventa lor a fost mai mare la iradierea .acuta Intr-un studio privind evaluarea aberatiilor cromozomiale in culture de limfocite provenite de la 122 de persoane ce traiesc si muncesc intr-o regiune cu fond ridicat de radiatii din Austria ( Badgastein, din Alpii centrali), Ruling si Fischer (1978) au gasit pana la o doza de 300 mrad pe an o crestere lineara a .aberatiilor cromozomiale Curba doza-efect privind radiosensibilitatea in inducerea discentricilor in culture de limfocite aflate in conditii indentice (Muamatsus si Matsuoka, 1976) .(la diverse specii de mamifere prezinta un character taxonomic (fig.5 Semnificatia biologica a aberatiilor cromozomiale din celulele somatice ridica o serie de probleme pentru radiobiologi. Una din acestea ar fi semnificatia clinica a lor. Intr-un studiu pe supravietuitorii de la Hiroshima si Nagasaki, Awa (1975) a raportat ca dupa 25 de ani de la iradiere persista in culturile de .(limfocite aberatiile cromozomiale (fig.6 Autorul amintit conchide in studiul sau : se poate afirma in momentul de fata ca aberatiile cromozomiale intalnite la supravietuitorii puternici nu au fost associate cu manifestari postclinice. Studiile pe un grup de copii nascuti din parinti expusi in timpul bombardamentului si pe un alt grup de parinti neexpusi nu au aratat diferente semnificative in leziunile cromozomiale intre cele doua .grupe Detectarea aberatiilor cromozomiale radioinduse se bucura de un interes oractic considerabil, deoarecei indicatiile obtinute sunt superioare celor oferite de dozimetria fizico-chimica si chiar examenul hemato-logic, care este sensibil .la doze mult mai mari 30 Din aceste considerente, dozimetria biologica, bazata pe studiul aberatiilor cromozomiale, merita sa fie utilizata sistematic ca mijloc de depistare a nivelului de iradiere la subiectii care, prin natura preocuparilor sau din motive terapeutice, .intra in contact cu surse de radiatii

Fig.5- Comparatia intre relatiilr doza-efect care leaga doza de iradiere cu raze X de frecventa dicentricilor, la diferite specii de mamifere (Muramatsus si Matsuoka,1976)

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Fig.6- Frecventa de aparitie a celulelor cu aberatii cromozomiale in culturi de limfocite preluate de la supravietuitorii bombardamentelor de la Hirosima si Nagasaki la 25 de ani de la expunerea la radiatii (Awa,1975) Mutatii genice Tipurile de mutatii si alterari cromozomiale care se produc in spermatozoizi si ovule sunt in esenta similare acelor care apar in celulele somatice, dar afectand genele sunt mai greu de depistat si pentru faptul ca se transmit si la urmasi au fost numite mutatii genetice. Modificari genetice ca rezultate al iradierii celulelor reproducatoare nu au fost observate in populatiile umane, fapt pentru care in prezent toate informatiile provin din lucrari experimentale pe animale. Anumite tipuri de modificari cromozomiale sunt caracteristice numai celulelor care sufera meioza (gameti), din cauza diferentelor din organizarea si dispunerea cromozomilor. Diferentele esentiale din mutatia somatica si mutatia genetica provin si din alte considerente fundamentale. In primul rand efectele mutatiei in celulele somatice pot fi facute ineficare sau annihilate de catre celulele sanatoase, normale, din organism, in timp ce un embrion, care pleaca cu un complement diploid de cromozomi, isi replica mutatia in toate celulele 32 -

rezultate din diviziunile ulterioare, neexistand posibilitatea compensarii din .partea altor celule In al doilea rand mutatiile somatice dispar odata cu organismul purtator, in timp ce mutatiile genetice pot fi transmise indefinite. Din aceste considerente potentialul nociv sal unei mutatii genetice poate fie enorm in comparative cu .acela al unei mutatii somatice Aberatiile genetice, ca produse finale ale evenimentelor de afectare si reparare, impugn o depistare intr-un stadiu mult mai timpuriu. In acest scop, s-a pus problema daca exista un prag efectiv al expunerii la o doza de radiatii sub care san u mai apara mutatii. Rezultatul cercetarilo sunt foarte mult mascate de capacitatea distemelor enzimatice de a repara modificarile de baza ale AND, de tendinta fragmentelor cromozomiale de a suferi un process de refacere, precum si de faptul ca exista o frecventa normala a mutatiilor. Din aceste considerente s-a apreciat ca o cale utila de evidentiere a efectelor genetice ale radiatiilor este conceptual de doza de dublare (Duncan si Nias, 1977). Prin doza de dublare se intelege acea doza de radiatii care dubleaza frecventa normala a mutatiilor genetice. Pentru om, o doza de dublare este de 15-30 rem cand iradierea se face .acut si in jur de 100 rem cand iradierea se face cronic Studii de radiogenetica la mamifere au fost efectuate indepand din 1950 de W. L. Russell. Folosind un numar imens de soareci, Russell si echipa sa (1951, 1952) au studiat frecventa mutatiilor in spermatogenii, spermatozoizi si oocitelor. Aceste cercetari au evidentiat ca dependenta frecventei mutatiilor de debitul doze este mai mare la femelele de soareci, ceea ce pledeaza pentru o sensibilitate mai mare, la doze mici de radiatii, a oocitelor comparative cu spermatogenii. La spermatozoizi nu s-a pus in evidenta un efect al debitului dozei de iradiere, capacitatea lor de refacere fiind redusa sau absenta. O mare variabilitate in radiosensibilitatea diferentelor locusuri genetice a fost .demonstrate de Russell atat la gametii masculi, cat si la cei femeli Faptul ca oocitul are o insemnata capacitate de refacere in urma iradierii a ridicat unele probleme pentru practica radiological curenta. In radiologie se prefera efectuarea examinarilor la femeie in perioada de procreare, dupa menstruatie si inainte de ovulatie pentru a evita iradierea unui embrion radiosensibil. Din punctual de vedere al radiogeneticianului, momentul este considerat ca cel mai neindicat deoarece un oocit iradiat si cu mutatii poate fi fertilizat la urmatoarea ovulatie, fara a avea timpul necesar de refacere a leziunii genetice. 1

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RECOMANDARI ALE COMISIEI INTERNATIONALE DE PROTECTIE IMPOTRIVA RADIATIILOR ( CIPR ) PRIVIND NIVELE DE IRADIERE PERMISE LA OM
Una din cele mai controversate probleme in domeniul radioprotectiei o constituie stabilirea nivelului maxim permis de iradiere a organismului, adica valoarea debitului dozei pe care omul adult normal sa-l poata primi continuu fara a se provoca vreo afectiune nociva constatabila medicala pe parcursul vietii sale. In anul 1934, CIPR a fixat limita de iradiere ocupationala la 1 R pe saptamana, sau 50 R/an pentru a preveni aparitia efectelor acute, cum ar fi inrosirea pielii. Conceptul care a stat la baza stabilirii acestei limite a fost existenta unui prag de doza pentru producerea efectelor radiobiologice nocive, valoarea de 1 R/saptamana fiind descrisa ca doza de toleranta. In 1950, CIPR a recomandat o limita de iradiere de 0,3 rem/saptamana, pentru a se proteja asa-zisele organe critice ( sangele, maduva osoasa, gonadele, ochii ). Din aceasta formulare, rezulta o doza maxima permisa anuala de 15 rem. In anul 1959, in urma admiterii existentei unei relatii lineare intre doza de iradiere si marimea efectului radiologic, CIPR a redus doza anuala de expunere a intregului organism pentru persoanele ocupate profesional la 5 rem, limitand totodata varsta de incepere a iradierii ocupationale la 18 ani. Pentru celelalte parti ale corpului s-au stabilit urmatoarele nivele maxime permise: Gonade,maduva osoasa, cristalin......................5 rem/an Piele, tiroida.......................................................30 rem/an Extremitati (maini,antebrate,...............................75 rem/an picioare si glezne Alte organe...........................................................15 rem/an - 34 -

In cazul indivizilor tineri din populatia obisnuita neexpusa profesional, nivelul maxim permis de iradiere a intregului organism a fost fixat la 0,5 rem/an, cu limitarea suplimentara ca doza genetica acumulata in 30 de ani nu trebuie sa depaseasca 2 rem. Pentru restul populatiei obisnuite, dozele maxime admise au fost fixata la valori reprezentand 1/10 din valorile acceptate pentru expunerea ocupationala. In anul 1977, CIPR, pastrand limita expunerii ocupationale la 5 rem/an, a adus urmatoarele precizari: - fiecare organ are o contributie in stabilirea riscului la nivelul intregului organism; - contributia fiecarui organ este proportionala cu doza de iradiere primita de organul respectiv. Pe baza datelor epidemiologice, a fost atasata o pondere contributiei fiecarui organ la riscul estimat pentru intregul organism, in conditiile iradierii uniforme a acestuia. Valorile ponderilor corespunzatoare diferitelor organe sunt: Gonade..........................................................0,25 Piept..............................................................0,15 Maduva osoasa..............................................0,12 Plamani..........................................................0,12 Tiroida...........................................................0,03 Suprafetele oaselor........................................0,03 Alte organe....................................................0,30 1,00 In 1978, a fost recomandata o pondere de 0,01 pentru riscul cancerelor de piele rezultand din expunerea la iradiere a grupurilor mari de populatie. Sistemul actual de limitare a dozelor de iradiere se bazeaza pe principiul ca riscul este acelasi, indiferent daca iradierea intregului corp este uniforma sau nu. Utilizand factorii de pondere, expunerea neuniforma a corpului trebuie limitata astfel incat riscul total sa nu depaseasca riscul unei iradierii a intregului corp de 5 rem/an. - 35 -

Scopul normelor actuale de iradiere permisa este de a face ca riscul unei persoane ocupate in activitati ce presupun contactul cu radiatiile ionizante sa nu depaseasca riscul intalnit in alte domenii de activitate recunoscute ca avand un inalt grad de siguranta. Astfel de activitati sunt considerate cele in care cifra mortalitatii medii nu depaseste 10-4/an de munca. 1

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BIBLIOGRAFIE
1. N. Gheorghe, C. Vladescu, M. Apetroae Radiatiile ionizante si viata, Ed. Academiei Republicii Socialiste Romania, Buc., 1984 2.Institutul Oncologic Cluj-Napoca, Directia Sanitara a Judetului Bihor Cancerul si mediul ambiant, Ed. Materiale de documantare si indrumare metodologica, Cluj-Napoca, 1980 3. Schler G., Gutzwiller F.- Low dose ionizing radioation and cancer: findings and methods. Report of a meeting and consequences for Switzerland, PubMed 4. D. G. Margineanu, M. I. Isac, C. Tarba Biofizica, Ed. Didactica si Pedagogica, Buc, 1980 5. Angela Bruner, William Sutker, Gail Maxwell - Minimizing patient exposure to ionizing radiation from computed tomography scans, PubMed 6. David G. Hoel Models for Ionizing Radiation, PubMed 7. Mihail Zamfirescu, Gheorghe Sajin, Ion Rusu, Maria Sajin, Eugenia KovacsEfecte biologice ale radiatiilor electromagnetice de radiofrecventa si microunde, Ed. Medicala, Buc, 2000 8. Catherine C. Park, Rhonda L. Henshall-Powell, Anna C. Erickson, Rabih Talhouk, Bahram Parvin, Mina J. Bissell, Mary Helen Barcellos-Hoff Ionizing radiation induces heritable disruption of epithelial cell interactions,PubMed

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Minimizing patient exposure to ionizing radiation from computed tomography scans


Angela Bruner, PhD, DABR, William Sutker, MD, and Gail Maxwell, MS, RN, FACHE

Computed tomography (CT) is a powerful tool. It allows very quick creation of x-ray images of the body with high-resolution cross-sectional imaging. The quick, detailed result has made CT very valuable, especially in the emergency department (ED). In a very short time period, high-quality, cross-sectional images are available that help to define the medical status of the patient. This has increased our ability to provide the correct care in a timely manner. Although CT scans are not the most frequently used diagnostic study in the hospital, they provide the majority of the radiation dose to the patient population (1)and this dose level is steadily increasing. For example, a 2004 article in Radiology documented that CT scans constituted approximately 13% of the diagnostic imaging orders for hospitals in the United States but provided 70% of the dose (2). In addition, a 2006 National Cancer Institute report suggested that the number of CT scans ordered in adults and children has increased by about 7 times over the last 10 years (3). This trend of increased use has also been seen at Baylor University Medical Center (BUMC) when a second CT scanner was added to the ED during the recent remodel of the department.

LACK OF KNOWLEDGE ABOUT RADIATION DOSE A number of medical institutions have recently assessed their medical staff's knowledge of radiation dose and its associated risk. Many studies have demonstrated a lack of knowledge by staff about radiation dose for common radiological procedures. In a 2004 study at a 1285-bed hospital, only 27.5% of the physicians surveyed were able to score 45% or greater on 11 questions about radiation dose (4). In addition, 28% and 10% of the physicians surveyed, respectively, incorrectly believed that magnetic resonance angiography and ultrasound posed a risk from ionizing radiation. In a 2004 survey at a Yale University tertiary care center, most patients, ED physicians, and radiologists were unable to determine the correct radiation dose of a CT scan versus a chest radiograph (2). In addition, 78% of ED physicians at this facility admitted that they did not explain the risks and benefits of the CT scan to patients, and 93% of the surveyed patients claimed that they did not receive this information. A 2007 study involving three university hospitals, a research hospital, and three outpatient clinics found that 93% of staff surveyed underestimated the amount of radiation dose for diagnostic procedures (5). A 2005 article in the International Journal of Cardiology concluded that most physicians do not possess knowledge about the radiation dose for the diagnostic procedures they prescribe (6). These articles all recommended better education of physicians and staff about radiation exposure and its associated risks. In November 2007, the New England Journal of Medicine published an article by Brenner and Hall entitled Computed tomographyan increasing source of radiation exposure (7). The authors acknowledged that most CT scans have a positive risk-to-benefit ratio but commented that physicians often view CT studies in the same light as other radiological procedures, even though radiation doses are typically much higher with CT than with other radiological procedures. They summarized a survey of pediatric physicians that found about one third of all CT studies could be replaced with other studies or not performed at all. Brenner and Hall suggested a threefold method for addressing these issues: 1) alter the CT scan protocols to minimize the dose for each study; 2) when possible, replace the CT scan with other diagnostic imaging tools that provide a lower ionizing radiation dose or none at all; and 3) decrease the number of CT studies prescribed. The article raised awareness about the recent increase in use of CT and its consequences and risks. Not every conclusion made in the New England Journal of Medicine article is indisputable, and a variety of responses have poured in from many organizations, including the Food and Drug Administration (8), the American Association of Physicists in Medicine (9, 10), the American College of

Radiology (11), the Society for Pediatric Radiology (12), the Health Physics Society (13), the Radiological Society of North America (14), and the American Council on Science and Health (15). However, the article also sparked several radiation reduction initiatives, including the Image Gently Campaign to reduce CT dose in pediatric patients (16) as proposed by the Alliance for Radiation Safety in Pediatric Imaging. BUMC has also started an initiative to reduce our patients' radiation exposure from CT scans.

BUMC'S CT DOSE REDUCTION INITIATIVE The BUMC Radiology Imaging Department has invested in CT technology with equipment and software purchases and made efforts to reduce patient dose through protocol changes. To some extent, we have been successful in lowering the dose per study. However, for the CT dose reduction initiative to be successful, the medical staff who order the scans must also contribute to the effort. BUMC's Patient Safety Committee, chaired by Dr. William Sutker, recommended the creation of a task group to examine the issue of CT radiation dose at BUMC. This group, led by Gail Maxwell, Baylor Health Care System's vice president of administration, consisted of ED physicians, trauma surgeons, radiologists, and other key personnel. The goal is to reduce the radiation dose to patients at BUMC by 1) minimizing the dose for each CT scan ordered and 2) providing information to the physicians who order imaging studies so that they can make educated decisions about the CT scans that they prescribe. Data on prescribing habits will be reviewed over a 6-month period to determine if the physician education efforts lead to a decrease in the number of CT scans ordered.

Three of the physician education componentsthe cancer risk from radiation, equating risk from different types of radiological studies, and considerations for choice of imaging studyare reviewed below.

RADIATION AND CANCER RISK Cancer induction from radiation exposure occurs after a latent period of about 7 to 50 years after the initial exposure. Much of what is known about radiation risk and cancer comes from the US government's 50year follow-up studies of survivors of the 1945 atomic bombs in Hiroshima and Nagasaki, Japan. From these data and smaller studies of other radiation exposures (such as cancer patients undergoing radiation therapy treatments and radiation workers), we have a better understanding of the risk of inducing cancer from radiation exposure. The most recent comprehensive estimate of radiation risk appears in the Biologic Effects of Ionizing Radiation (BEIR) VII report (17), published by the National Academies' Committee to Assess the Health Risks from Exposure to Low Levels of Ionizing Radiation. This committee is chaired by Richard Monson of the Harvard School of Public Health and has members from many prominent universities worldwide and from the National Cancer Institute. The BEIR VII report's lifetime risk model predicts that there is a 1% increase in risk of developing cancer (solid tumor or leukemia) after a radiation exposure of 10,000 mrem (which is equivalent to approximately 2500 anteroposterior chest radiographs or two to three CT abdomen scans for a larger patient). This statistical chance of developing a cancer is based on a linear model and thus can be scaled up or down depending on the radiation dose. For example, a dose of 5000 mrem

would correspond to a 0.5% increase in cancer risk, and a 20,000 mrem dose, a 2% increase. BEIR VII also estimated that there is a 42% chance of getting cancer from reasons other than radiation; however, we can better control the radiation exposure risk.

EQUATING RISK FROM DIFFERENT TYPES OF RADIOLOGICAL STUDIES It becomes more difficult to equate radiation risk based on unit values of mrem (or mGy, mrad, or mSv), since these units are not commonly understood by most medical professionals or patients (4). To simplify the message, we created a table of common diagnostic procedures from CT, nuclear medicine, radiography, and fluoroscopy in units of mrem and estimated the number of corresponding anteroposterior chest radiographs (Table). Bar charts further compare the radiation dose of CT and radiographs (Figure). The mrem units were chosen because they represent the whole-body effective dose, which is a better estimate of radiation risk. (mSv is an equivalent unit in standard international units.) Table Total-body effective dose and comparable number of chest xrays for radiological studies in a patient with a normal body mass index Figure Comparison of radiation dose (mrem) for computed tomography (CT) and radiographic studies.

The doses listed in the Table are for patients with a normal body mass index (BMI) and may substantially increase (depending on the study) for a larger patient. For most studies, the CT scanner automatically adjusts the output of the x-ray tube to properly penetrate the patient's body for optimum image quality. In smaller patients, the dose is decreased with this method. However, for larger patients, the dose increase can be significant. For example, patient data acquired in the BUMC ED during a recent survey showed that the patient dose for a single abdomen pelvis CT without contrast (renal study) varied from 446 to 2567 mrem (equivalent to 112 to 642 anteroposterior chest radiographs), depending on patient size and composition. For a single abdomen pelvis CT with contrast, the dose varied from 433 to 3687 mrem (equivalent to 108 to 922 chest radiographs). A single chest CT with contrast for pulmonary embolism would provide a dose range of 109 to 1762 mrem (equivalent to 27 to 441 anteroposterior chest radiographs). A few of these studies in a larger patient could easily approach 10,000 mrem and the 1% chance of radiation-induced cancer in the future. Many of these CT studies also affect individual organs that are more sensitive to radiation, such as bone marrow, eyes, thyroid, breast, and gonads. For example, a chest CT scan provides a localized dose to the breast of approximately 2100 mrad for a patient with normal BMI (compared with 9 mrad for an anteroposterior chest radiograph) (18). An abdomen and pelvis CT scan in a normal BMI patient will directly irradiate the gonads and uterus to approximately 2300 and 800 mrad, respectively (compared with 120 and 290 mrad for abdomen and pelvis radiographs). A CT scan to the head will irradiate the bone marrow, thyroid, and eyes to approximately 270, 190, and 2500 mrad, respectively (compared with a head radiograph, with bone marrow and thyroid doses of 20 and 40 mrad).

ADDITIONAL CONSIDERATIONS Other imaging studies Before ordering a CT scan, physicians may want to consider other imaging modalities. Some imaging studies have a lower radiation dose, such as a lower-radiation-dose radiograph or a zero-radiation-dose ultrasound. Magnetic resonance imaging does not expose the patient to ionizing radiationbut it is not readily available in the ED. The Statscan device in the ED can also be used as a low-dose, quick assessment whole-body imaging tool. The goal is to educate physicians about radiation dose so that they can consider it during the assessment of study risk versus benefit.

Review of recent images Another consideration is the avoidance of unnecessary repeat tests. Baylor's Picture Archive and Communications System (PACS) provides a network of digital images and reports that are available 24 hours a day, 7 days a week at all Baylor hospitals. Dedicated workstations are located at various locations in each Baylor hospital, and the system can be accessed through the Internet from any Baylor workstation and, with proper log-ins and passwords, from any computer through the Baylor physician portal. Radiological tests done at other Baylor hospitals can also be accessedat either dedicated workstations in the BUMC ED or

through the Radiology Imaging Department's reading room. Currently, images from Baylor Medical Center at Waxahachie and Baylor Medical Center at Garland can be viewed in the BUMC ED on dedicated workstations, and images from Baylor Plano and Baylor Grapevine will be added soon. Any physician can ask for assistance in accessing these images in the radiology ED reading room, if they do not have access to the dedicated workstations in the ED. Over the next few years, PACS should be enterprise-wide, with information on patients from all Baylor Health Care System facilities. The Radiology Imaging Department at BUMC is also working to facilitate use of image CDs or DVDs from other facilities. Because special software is often required to open the images, the CDs and DVDs may appear to be unreadable. A system is being developed in the ED where the original CD or DVD can be dropped off, uploaded to PACS, and available at a workstation. An order will also be generated for a radiologist to read the images and provide a written report. It is helpful to check the PACS for the availability of images. For example, a patient might return to the ED several times with the same symptoms and therefore receive the same CT scan repeatedly. After the CT exam, the radiology staff is aware of the repeated scans because PACS automatically brings up the historical image for comparison. However, the referring physician, without looking first on PACS, might not realize the scan had already been done prior to ordering the study. In some cases, the repeat exam is entirely justified, but this should be at the discretion of the physician.

CONCLUSIONS Only a physician is properly trained to evaluate the benefit versus risk of a particular radiological study. Radiation dose and associated risk should be considered in addition to medical necessity. In addition, it might be possible to replace a CT order with an imaging study that has a lower radiation dose or to eliminate the study altogether. The latter could be considered, for example, when recent archived images could be reviewed. At BUMC, our goal is to do our best to reduce our patients' radiation exposure.

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Ionizing radiation induces heritable disruption of epithelial cell interactions


Catherine C. Park,* Rhonda L. Henshall-Powell,* Anna C. Erickson,* Rabih Talhouk, Bahram Parvin, Mina J. Bissell,* and Mary Helen Barcellos-Hoff* ABSTRACT Ionizing radiation (IR) is a known human breast carcinogen. Although the mutagenic capacity of IR is widely acknowledged as the basis for its action as a carcinogen, we and others have shown that IR can also induce growth factors and extracellular matrix remodeling. As a consequence, we have proposed that an additional factor contributing to IR carcinogenesis is the potential disruption of critical constraints that are imposed by normal cell interactions. To test this hypothesis, we asked whether IR affected the ability of nonmalignant human mammary epithelial cells (HMEC) to undergo tissue-specific morphogenesis in culture by using confocal microscopy and imaging bioinformatics. We found that irradiated single HMEC gave rise to colonies exhibiting

decreased localization of E-cadherin, -catenin, and connexin-43, proteins necessary for the establishment of polarity and communication. Severely compromised acinar organization was manifested by the majority of irradiated HMEC progeny as quantified by image analysis. Disrupted cellcell communication, aberrant cellextracellular matrix interactions, and loss of tissue-specific architecture observed in the daughters of irradiated HMEC are characteristic of neoplastic progression. These data point to a heritable, nonmutational mechanism whereby IR compromises cell polarity and multicellular organization. Epidemiologic data indicate that women exposed to ionizing radiation (IR) for either therapy (1, 2), diagnostic purposes (3), or as a consequence of atomic bombs (4) have an increased risk of breast cancer. The action of IR as a DNA-damaging agent, and consequently as a mutagen, is widely considered to be the basis for its action as a carcinogen (5). However, tissue response to radiation, and hence risk, is a composite of genetic damage and epigenetic events, such as altered intercellular communicaton (6). Recent experimental models suggest that carcinogenesis can be driven by abnormal interactions between cells and their microenvironment (reviewed in refs. 7 and 8). We have shown that irradiated mammary stroma promotes tumorigenesis of unirradiated mammary epithelial cells (9), and that transforming growth factor 1 (TGF-) activation mediates cellular and tissue response to IR (1012). Thus, in addition to causing DNA damage, radiation exposure alters key regulators of cell phenotype that affect, directly or indirectly, the ability of normal tissue to suppress abnormal cell growth (13). Epithelial cells depend on signals from the microenvironment to establish the requisite polarity for functional differentiation (14). Release from these constraints has profound consequences on tumorigenesis,

progression, and metastasis (reviewed in refs. 6, 8, and 15). Tumorigenic and nontumorigenic human mammary epithelial cells (HMEC) are nearly indistinguishable when cultured as monolayers, but readily diverge in terms of morphogenesis in an appropriate microenvironment, which is evident in a three-dimensional reconstituted basement membrane (3D rBM) assay that we developed (16). In this assay, nonmalignant HMEC arrest growth and form lumen-containing acini similar to those found in situ, whereas breast cancer cells continue to proliferate and aggregate, rather than organize. Formation of acini requires expression and appropriate localization of proteins involved in the establishment of tissue structure and polarity (17). HMEC colonies that develop into phenotypically normal acini exhibit among other markers, E-cadherin at the interface between cells, basolateral 1-integrin, and basal 6-integrin (18). In contrast, breast cancer colonies exhibit disorganized, decreased, or aberrant expression of these markers, similar to what is observed in primary breast cancer. If radiation exposure affects not only the phenotype of stromal but also of epithelial cells, such alterations could potentially promote neoplastic progression in susceptible cells. To test this hypothesis, we asked whether sublethal IR doses perturbed the ability of HMEC to undergo mammary-specific morphogenesis in a physiological context by using the 3D rBM assay. To replicate a key component of the irradiated stroma (1012), TGF- was added to some cultures. To measure the global consequences of irradiation, we used confocal microscopy and an imaging bioinformatics system for integrated image acquisition, annotation, and hierarchical image abstraction to register localization and expression information of targets along with positional references and morphological features (19).

We found that irradiated single HMEC gave rise to colonies where nearly all progeny failed to establish basal polarity and lost organizational integrity as measured by several parameters. As shown by quantitative image analysis, these changes were shared by the majority of the population. This finding is inconsistent with a radiation-induced mutational mechanism, which was confirmed by the absence of measurable changes in the population genome analyzed by comparative genomic hybridization analysis. Moreover, because the phenotype is exhibited by the daughters of individually irradiated cells, these data suggest that radiation causes a heritable alteration in pathways affecting cell adhesion, extracellular matrix (ECM) interactions, epithelial polarity, and cellcell communication. Thus, epigenetic events after radiation exposure disrupt multicellular organization, which we postulate will override the positive influence of tissue architecture that usually impedes neoplastic progression. METHODS Cell Culture. HMT-3522-S1 human mammary epithelial cells (S1; passages 5360) were grown as described (18). Although phenotypically normal and nonmalignant, the S1 are an established cell line that have a number of chromosomal changes and an extended life span in culture (20). S1 cell monolayers were grown until 70% confluent before trypsinization, and single cells (8 105 cells per ml) were embedded into Matrigel (Collaborative Research) with or without 400 pg/ml recombinant human TGF1 (R & D Systems) and irradiated within 5 h by using 60Co radiation at a dose rate of 90 cGy/min to a total dose of 2 Gy. Dosimetry was determined by using a Victoreen ionization chamber. Control plates were sham irradiated. Media were changed on alternate days. Cells were grown in the presence of epidermal growth factor for 6 days, and harvested at 10 days. For immunocytochemistry cultures were embedded in Tissue-Tek compound (Sakura Finetek, Torrance, CA), and frozen in a dry ice/ethanol bath. Blocks were stored frozen until time of sectioning.

Immunofluorescence. Cryosections (20 m) were cut at 30C onto gelatin-coated coverslips. Sections were fixed by using methanol/acetone (1:3) at 20C for 10 min or 4% paraformaldehyde for E-cadherin. Nonspecific sites were blocked by using the supernatant from a 0.5% casein/PBS (pH 7.4) solution for 1 h at room temperature (RT). Sections were incubated in primary antibody diluted in blocking buffer for 1 h at RT in a humidified chamber. Antibodies used were rat anti-mouse CD29 (Pharmingen) to integrin 1 chain monoclonal antibody, rat anti-human CD49f monoclonal antibody (Pharmingen) to integrin 6 chain, and mouse monoclonal antibody to E-cadherin (BD Transduction Laboratories). Sections were washed in PBS containing 0.1% BSA, before incubating in secondary antibody conjugated to Alexa Fluor 488 (Molecular Probes) for 1 h at RT in a dark humidified chamber, washed, and counterstained with TO-PRO-3 iodide (Molecular Probes), before mounting with Vectasheild mounting medium (Vector Laboratories, Burlingame, CA). Image Acquisition, Processing, and Analysis. Dual immunofluorescence confocal images were acquired by using a Zeiss LSM 410 inverted laser scanning confocal microscope equipped with an external argon/krypton laser. Confocal images were captured at 0.5-m intervals as 8-bit images by using a Zeiss Fluor 40 (1.3 numerical aperture) objective. Images were standardized by comparing only images stained with the same antibodies in the same experiment, captured with the same parameters at the same times, and scaled and displayed identically. Relative intensity of images was scaled by using
SCILIMAGE

(TNO Institute of Applied Physics, Delft, The Netherlands),

which was used to define a standard sized region of the TO-PRO-3 iodide image (nuclei slice) without reference to the Alexa Fluor 488 images. Statistical significance of the mean fluorescence intensity for

each region of interest (n = 20 colonies) and standard error for each treatment group was determined by using the unpaired Student's t test (PRISM, GraphPad, San Diego). The displayed images were those closest to the mean intensity for the treatment group. Segmentation of nuclei was used to determine acinar organization at the colony midsection (21). This model-based approach assumes that the projection of each nucleus is quadratic in the image space. Instead of grouping step and roof edges, the segmentation is initiated from a representation that corresponds to the zero crossings of the image. The zero crossing image is then filtered with geometrical and illumination constraints to form binarized clump of nuclei, which is then partitioned into several nuclei through a process that is called centroid transform. Protein Extraction and Immunoblotting. Cells in the 3D rBM assay were isolated by ice-cold PBS/EDTA (0.01 M sodium phosphate, pH 7.2, containing 138 mM sodium chloride and 5 mM EDTA) (18) and lysed in buffer as described (18). Equal amounts of protein lysates were run on reducing SDS/PAGE and then immunoblotted and detected by using a Pierce Super-Signal system (Pierce). Blots were also probed for -actin to assess equal loading of protein. Exposed films were scanned and subjected to densitometric analysis for the determination of relative amount. Comparative Genomic Hybridization. Array comparative genomic hybridization was performed at the University of California, San Francisco, Cancer Center as described (22). Briefly, 1 g each of test and reference genomic DNAs were fragmented by DPNII digestion, labeled by random priming with CY3- and CY5-dUTP (Amersham Pharmacia), respectively, coprecipitated with 80 g of human cot-1 DNA (Life Technologies), and resuspended in 20 l of hybridization buffer (50% formamide/10% dextran sulfate/2 SSC/2% SDS/200 g of yeast

tRNA). This mixture was denatured at 75C for 10 min followed by 60 min at 37C. Just before hybridization, array slides were processed following the manufacturer's recommendations (Surmodics, Eden Prairie, MN). A frameseal frame was placed around each array, hybridization mix was added, and the slide was placed in a plastic slide holder, prewarmed to 37C, containing 200 l of wash buffer (50% formamide/2 SSC) to prevent evaporation. Hybridization was carried out at 37C for 4872 h on a gently rocking platform. After hybridization, slides were immersed for 15 min at 48C in wash buffer, followed by washes at 48C in 2 SSC, 0.1% SDS for 30 min, and 0.1 M sodium phosphate buffer containing 0.1% Nonidet P-40, pH 8.0, at RT for 10 min. Slides were then rinsed in 2 SSC and dried by centrifugation. RESULTS Progeny of Irradiated Cells Exhibit Perturbed CellECM and Cell Cell Adhesion. To determine whether IR alters the ability of epithelial cells to functionally interact with the microenvironment, we used the 3D rBM assay of morphogenesis in a laminin-rich basement membrane where changes in tissue structure can be quantified (16). Single HMT3522 S1 human mammary epithelial cells were cultured, with and without the addition of TGF-, and irradiated with a dose of 2 Gy, except where noted, 35 h after plating in Matrigel. Surviving cells, 80% (not shown), formed multicellular colonies over 57 days and then underwent morphogenesis into hollow spheres that recapitulate mammary acini by day 10. Immunofluorescence of 1 and 6 integrins and -catenin at the colony mid-section were analyzed by using confocal microscopy .HMEC colonies express basolateral 1-integrin and basal 6-integrin, which are critical for acinar organization (23). HMEC colonies arising from

irradiated cells exhibited increased 1-integrin immunoreactivity that was distributed throughout the cytoplasm .In contrast, the immunoreactivity of 6-integrin, which partners with 4 integrin, was decreased in colonies generated from irradiated cells. A collagen IV-containing basement membrane was observed in all treatment groups, indicating that changes in integrin expression were not caused by the lack of appropriate ligand for this ECM receptor (not shown). Treatment with TGF- did not alter 1 integrin localization but did reduce 6 integrin immunoreactivity further. -catenin, which is involved in cellcell adhesion via the cytoskeleton and E-cadherin, was localized to the lateral cell borders in colonies from nonirradiated cells. -catenin immunoreactivity was decreased in colonies derived from irradiated cells. Disrupted Tissue-Specific Morphogenesis and the Irradiated Phenotype as Quantified by Image Analysis Reveal a Global HMEC Response. The use of morphogenesis as a readout of cellular function requires systematic analysis of colony organization and protein localization to classify the degree of response. It is therefore desirable to conduct population studies and correlate features measured from images of cells with their treatment. The acinar-like organization of colonies was analyzed by using the relative nuclear position in confocal optical midsection as described in Methods .The degree of acinar organization around a central lumen was determined by fitting the nuclei to an ellipse . Acinar organization was significantly (P < 0.0001) reduced in colonies arising from irradiated cells that were cultured with TGF-. The number of cells per midsection was also significantly increased ( P < 0.001) in irradiated, TGF-treated HMEC colonies in comparison to colonies from control cells or those exposed to single agents. The assembly of cells into tissue-specific structures requires the interaction of different cell adhesion systems. E-cadherin is a crucial

epithelial adhesion molecule that links cells via an homophilic extracellular domain and is anchored intracellularly to the cytoskeleton via dynamic interactions with the catenins (24). Low E-cadherin immunoreactivity in breast cancer is associated with poor prognosis (25), whereas restoration of E-cadherin reverts the invasive phenotype of cancer cells (26). We localized E-cadherin by using immunofluorescence, confocal microscopy and image analysis (Colonies from irradiated cells cultured in the presence of TGF showed a significant (P < 0.0001) loss of E-cadherin immunoreactivity compared with control cells. The unlikely possibility that the colonies surviving treatment were selected from a previously existing population was addressed by examining the distribution of individual colonies within each treatment group in comparison to control colonies. A representative analysis is shown for E-cadherin, indicating that the dual treated colonies form a distinct population .To determine whether the effect on cell interactions was sensitive to radiation dose, we performed a dose response . E-cadherin immunoreactivity was significantly decreased in colonies arising from cells exposed to as little as 25 cGy, a dose that does not result in appreciable cell kill. To determine whether radiation exposure and TGF- treatment resulted in significant changes in the genomic sequence, we performed comparative genomic hybridization as described in Methods. This analysis did not reveal any significant differences between the untreated and double-treated populations (data not shown), which supports the global population response revealed by quantitative image analysis. There is an intricate relationship between cellECM and cellcell adhesion in glandular tissues. To determine whether other cellcell adhesion molecules also change, we measured connexin 43, a member of a family of proteins that assemble into gap junctions and modulate the

transfer of molecules between cells. Breakdown of gap junctional complexes is induced by tumor promoters (27) and correlate with breast cancer metastatic potential (28, 29). Connexin 43 is also associated with the function and signaling of E-cadherin (30, 31). In S1 HMEC acinar colonies, connexin-43 was localized as distinct aggregates between cells. The number of connexin 43 foci per colony decreased after radiation exposure, regardless of TGF- exposure (When normalized to the number of cells per colony, the frequency of connexin foci decreased >3-fold in the daughters of irradiated cells (2.0 0.46, n = 8) compared with those from unirradiated cells (6.9 1.1, n = 18). Decreased E-Cadherin and -Catenin Localization Are Not a Function of Protein Abundance. E-cadherin localization can be modified by the degree of association with the cytoskeleton via the catenins. -catenin and E-cadherin partner to link cells and the cytoskeleton via the adherens junction (32). To test whether decreased immunolocalization was caused by a change in the compartmentalization of these adhesion molecules, sections were detergent extracted to remove the soluble fraction. Detergent extraction before fixation did not alter the pattern or intensity of E-cadherin in dual-treated samples (data not shown). Consistent with this finding, immunoblotting total protein extracts showed that both E-cadherin and -catenin levels were decreased in TGF--treated colonies (1 integrin protein abundance, on the other hand, increased in irradiated samples regardless of TGF- exposure, which is consistent with the increased cytoplasmic staining.In contrast, only the progeny of irradiated cells showed a decrease in Ecadherin immunolocalization. These data suggest that decreased Ecadherin immunoreactivity at the cell junctions in the dual treated colonies reflects both a TGF--induced decrease in protein levels and a

radiation-induced change in localization, suggesting a change in complex formation at the cell surface. Together, these data indicate that IR can generate a persistent phenotype in daughter cells characterized by increased cytoplasmic 1 integrin, decreased 6 integrin, radically decreased cell surface localization of E-cadherin and -catenin, and loss of connexin 43. The cumulative epigenetic changes in phenotype results in a loss of tissuespecific architecture that is indicative of malignant progression. DISCUSSION In this study we show that irradiated single HMEC gave rise to colonies that had more cells, failed to establish tissue-specific organization, and expressed significantly less E-cadherin, -catenin, and connexin-43. It is remarkable that the phenotype was exhibited by progeny of individually irradiated cells, suggesting that IR causes heritable alterations in pathways affecting cell adhesion, ECM interactions, epithelial polarity, and cellcell communication. Release from cellcell interactions, as demonstrated by experimentally induced loss and restitution of Ecadherin (26, 33), has profound consequences for breast cancer tumorigenesis, progression, and metastasis. The features of individual colonies measured by quantitative image analysis showed that these changes were present in the majority of the population, a finding inconsistent with the frequency of radiation-induced mutations and confirmed by the absence of measurable changes in the population genome. Thus epigenetic mechanisms initiated by irradiation of HMEC result in a malignant-like phenotype in progeny generations after IR exposure.

Intercellular and extracellular signals are critical to the suppression of neoplastic cellular behavior. Disruption of cellcell interactions are implicated, if not required, in neoplastic progression (7, 8, 34). Radiation exposure alters the expression of many genes involved in tissue processes such as proteases, growth factors, cytokines, and adhesion proteins, which supports the view that carcinogenesis could compromise tissue integrity by altering the flow of information among cells (35, 36). Indeed, our recent experimental studies demonstrate that multicellular architecture can be dominant over genomic change in terms of malignant cellular behavior (18, 37, 38). In these studies, breast cancer cells treated with 1 integrin function-blocking antibodies revert from disorganized colonies to organized acinar-like colonies that are characterized by restoration of cytoskeletal organization, cellcell and cellECM interactions, and reduced tumorigenecity (18). Small molecule inhibitors can also be used to cooperatively block aberrant signaling and revert tumorigenic behavior (37, 38). These data, and others in hematopoetic cancers (39), suggest that cancer can be controlled by reestablishing appropriate contacts from the ECM and stroma via outside-in signaling. Although radiation can acutely regulate E-cadherin and -catenin levels (40), as well as integrin expression (41), in our studies the phenotype is exhibited by the daughters of irradiated cells several generations after radiation exposure. The redistribution of 1 integrin in daughters of irradiated cells was accompanied by increased protein determined by immunoblotting .In contrast, even though TGF- treatment decreased Ecadherin and -catenin protein levels localization of E-cadherin and catenin immunoreactivity was only affected in double-treated 3D rBM colonies .Immunostaining can reveal protein access or conformation as well as protein abundance. Preliminary studies suggest that the cell-

adhesion proteins of irradiated cells have altered cytoskeletal associations (A.C.E. and M.H.B.-H., unpublished data). Based on studies in mouse mammary gland, we have proposed that the action of radiation as a carcinogen is augmented by its ability to compromise signaling from the stromal microenvironment (42). A functional test of this concept is provided by our experiments showing that tumorigenesis is increased 4-fold when unirradiated preneoplastic mammary epithelial cells are transplanted to an irradiated mammary stroma (9). One of the most rapid and sensitive responses in the irradiated tissue is the activation of TGF- (43). TGF- has a paradoxical effect during carcinogenesis in that it suppresses tumorigenesis but promotes neoplastic progression (4446). Overexpression of active TGF can also induce an epithelial-mesenchymal phenotypic transition during progression in vivo (47). In culture, this phenotype is characterized by loss of E-cadherin, acquisition of mesenchymal cytoskeletal features, and increased cell motility and invasion (48). In our experiments, this effect of TGF- appears to be augmented by preirradiation of the cells. Similarly, the loss of E-cadherin after very low IR doses may further compromise this essential mediator of cellcell adhesion in preneoplastic breast cells that already have less E-cadherin (49, 50), and could promote progression. The loss of cell polarity and multicellular organization exhibited by the progeny of irradiated cells suggest that radiation exposure could promote malignant progression by pathways initially independent of mutational mechanisms. Consistent with this postulate is the observation that colonies from irradiated HMEC contain more cells, indicating that decreased cellcell communication resulted in loss of contact inhibition and greater proliferation. The events leading to disrupted multicellular organization in the progeny of irradiated HMEC could also contribute to

genomic instability. Radiation-induced genomic instability evidenced by increased frequency of mutation and cell death occurs in the progeny of irradiated bone marrow (51, 52) and epithelial cell culture (53). The disruption of cell contacts could permit abnormal cells to persist (54) or dysregulate genome stability functions. Inappropriate mammary expression of an activated metalloprotease in transgenic mice that disrupts cellECM interactions and cleaves E-cadherin leads to genomic instability (D. Radisky and M.J.B., unpublished data) and mammary tumors (55, 56). Here we show that IR can promote phenotypic progression by affecting pathways that inhibit the ability of daughter cells to interact with each other and the microenvironment. Agents designed to protect irradiated tissue from disruption of cellcell communication (57), or those that can reverse the irradiated phenotype, could provide a means of impeding its downstream carcinogenic potential.

ACKNOWLEDGMENTS We thank Shraddha Ravani for technical assistance and William Chou for preparation of figures. We thank Drs. Joe Gray and Wen-Lin Kuo and the University of California, San Francisco, Cancer Center for CGH analysis and support to C.C.P. This research was supported by the Low Dose Radiation Program, Office of Biological and Environmental Research, Department of Energy (R.L.H.-P. and M.H.B.-H.), National Aeronautics and Space Administration Specialized Center Of Research and Training in Radiation Health (M.H.B.-H. and C.C.P.), Office of Biological and Environmental Research Contract No. DE-AC-0376SF00098, Department of Energy and Department of Defense/Breast

Cancer Research Program Innovator Award (to M.J.B.), and Department of Defense/Breast Cancer Research Program Postdoctoral Training Grant 17-00-0224 (to A.C.E.). NOTES This paper was submitted directly (Track II) to the PNAS office. Abbreviations: IR, ionizing radiation; TGF-, transforming growth factor 1; ECM, extracellular matrix; HMEC, human mammary epithelial cells; 3D rBM, 3D reconstituted basement membrane.

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