RCCl.958039.001 - OSTI.GOV

Ward 8 . Silberatdn. M.D. 19811 &en, K.D. b p n e L. Saenger, l4.D. J u e G. breiakea, Ph.D.

RCCl.958039.001

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University of Cincinnati College of Hedicine Cincinxiati, Ohio

Supported in part by the Defame Atomic Support Nancy, Cantract No. DASA 01-69-C-0131. Uuhington, D.C.; the Albertine 0. Sckoepf Research Fund of the College of Hmdicine Univermity of Cincinmti urd USW PR- . 05408 General mearch' Support Grant of the College of Wdicine University of Cincinnati.

ABSTRACT

The resu l t of radiat ion interact ing w i t h l iv ing tiraue is the depooition of energy therein. Tbim mer= trimers numerous ch.nica1 nac t iocu within tho rn1ecul.r of 'the &asget t i . s u u . We have u u u r e d fn u n the remultr of soma of theme reactions a t dome. up t o 300 rad.: i n the kine t ics of rpec i f i c human c e l l p 0 p d A t i O M ; change. in 37 biochemical coamtituents OK nema andlor urine.

p a r t i a l l y perfected but there are numerow d i a c r e p ~ n c i u in data between d i f f e ren t l a b o r a t o r i a . Etiocholanolone c.o be wed t o evaluate mnrrov in jury before the white c e l l count f a l l s below 5000 per ku. m. W a t b i o e k d c l l doairneterr evaluated gave negative or incormistent resul ts . Elowever, sa i ivarg amylase is a p?Od6ing ind ica tor of buman radiation injury from domes as low as 100 rad6.

c h r o a i o w aberratiaum; . I te ra t ions

The u t i l i z a t i o n of ehmmos- AB a biological dosimeter is

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Damage due t o ionizing radiation i n biological systems is the r e s u l t of deposition of anerglr within the t i asue of the animal irra- diated. -Y rece ive , in jury from blast and bum an well as radiat ion, and a l l th ree modalities involve the t r r m f e r of caergy t o tissue, the in- portance of finding a radii t ion-mpecific dosimeter cannot be over- Mtfpated. d g h t be r e l a t ive ly non-specific. wide va r i e ty of pathophysiologic changes induced by i r r ad ia t ing hman beings with whole o r partial body radiat ion. These have included the a l t e r a t i o n s remalting f r o m the i r rad ia t ion of nucleic-acid nucleoprotein complexes as re f lec ted in chromosome morphology; changes induced by the s ceroid metabolite etiochol.llolone on granulocyte reaemes; abnor- malities i n the l eve l of several serm and ur ine enzymes; glycoproteins. and i ron. Theac s tudies were a l l performcd i n ambulatory human subjects who c l ea r ly understood the experimental nature of t h i s study according t o the Declaration of Ilelsinki. S w e r a l of the subjec ts were tumor f ree and e s sen t i a l ly normal (follcuipg radiation-induced tumor regression) receiving prophylactic whole body radiation. The r e s t had metastat ic carcinomas which were inoperable and not amenable t o conventional chemo- therapy. Nevertheleaa, these pat ients Were a11 c l i n i c a l l y s tab le . many of then working dai ly . u w e l l as our chromosome cul ture v t h o d w i l l be found in the Appendices A and B.

Since the victim of cha explosion of a nuclear weapon

Bowever, one might expect a u r i o r i that biochemical changes Our Laboratory has looked a t a

The radiat ion technique employed i n theae pa t ien ts

L11RObKEOME ABERRATIONS

The use of chrowsome aber ra t iom a# a biological rad ia t ion dosimeter began w i t h the c l u a i c a l e x p e r l a a t . of Salts (1. 2, 3) and of Lea and Catcheside (4) on the quant i ta t ive r e l a t ion between the change i n yield of radiation-induced chrwoorome aberrations and increasing dose. these s t u d i e s were performd thirty years ago, t h i ~ we of chroworome aber ra t ions remainn a highly cont rovera id area. gene t i c i s t s who bel ieve that the k ine t ics of dose-raeponse s tudied in these ear ly experikts are inapplicable to man, because the earlier work involved plant nuclei having la rge chromosol.8 and low chromosome ntrmbers (5). while m n has 96 chrooosomer (6) of amall individual voltme.

Although

There ate today cyto-

To j u s t i f y the use of any dosimeter one a m t be ab le t o repro- duce the ca l ib ra t ion curve betVenn the dose of rad ia t ion and the response of t h e test system. Furthermore. one U t be able t o employ

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the dosimeter t o ascertain t o what extent the t es t systaa has been expoled t o radiation. That l a , one w u l d l i k e t o knw whether the b io logica l system t o b e examined hm received an h m g e n e o w dose of rad ia t ion dis t r ibuted uniformly o r sn inhomogeneow dose inter- ac t ing with a l l or par t of t h a t myate+ about the cal ibrat ion of hman lpmphowte response to ' r ad ia t ion and the problem of distinguishing vholm.fron p a r t i a l body rad ia t ioa w i t h this myatem do not permit u t i l i za t ion of radiation-induced chromosome aberration dose-rssponse curves i n a rigorous fashion t o date. Houever, there bave been several instances where chrorm- some ana lys is has been useful i n proving tha t high doses indicated by rad ia t ion badges were in f a c t not 'received by the individuals wearing them (7.8). Furthermore, chro.cuome aberration6 caused by rad ia t ion i n man are not affected by mbient temperature md pressure a s are physical dosimeters ( fo r uhich correctioru can wunl ly be made)

Continuibg disagreement

Study of radiat ion e f f ec t s on -1i.n c e l l s is corplicated by the face that radiat ion sen6 i t iv i ty of calla varies w i t h the s tage of t h e i r c a l l cycle. Repair time of chromorome breaks has been found t o be 90 minutes in the G1 (por t d t o t i c pre--6ynthetic phase) and S (DNA synthesis) p h u e but 60 minutes i n late S and G2 (post- synthet ic p r e - d t o t i c phase) (9). Furthemore. there is inter-species var ia t ion i n radiation-induced chraorome aberrat ion curves (10). vith the extra- polat ion f h o a n i ~ l 6 t o man mouewhat hazardow. This var ia t ion i o probably r e l a t ed t o differences i n c h r m m rider and voltme bttwemn species examined. One vould l i k e to w e primatea in this research, but i dea l ly one m a t f ind an animal which ham the same chromosome nmber and configuration an men i d order t o be confident of the extrapolation.

An important breakthrough occurred when i t was found tha t human ,

lymphocytes could be a t k l a t e d t o divide i n vitro (11) where the mitosis could be a r rea ted w i t h colchicine Lud then analyzed under tha microscope. Furthermore. 99.9% 0f'hucP.n lymphocytcll are i n the C growth phme (12) and t h i s is a period of uniform radiat ion sensitivi& .(13). Thus nmerous inves t iga to r s have employed thim s y a t a to obtain doae-reaponae curves (5, 14-24). The re l a t ion rh ip of i n vivo t o in vitro experiments w i l l be discrused b e l w .

Theae curvea were ruual ly derived from in vitro studies .

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But which "rrsponse" should be studied? AB noted above. rad ia t ion u w e ~ ekrowmooe breakage i n nucleoprotein or poly- nucleotide chaixu (25) which may o r may not be followed by recombination betwean broken ends (1-3). An a l te rna t ive wch- anism ham been proposed by R e v e l l , the so-called "exchanp- first" hypothesis (26). end. do not r e jo in perfectly) are of two types. c h r m t i d and chrorosorc a b e r r a t i o ~ . whsn chromsoncs a r e in the C pre-DNA synthesis phase. a r e the types observed i n the l p b o c y t e rys t ao with which we are concerned. Breakagr and recolPbination occurring in C w i l l be duplicated during the 8 phase BO that both chromahd. of a chromoswc vi11 show the abnormalities when viewed i n metaphase. Therefore, abnormalities in jwt one of A pa i r of chromatids are unrelated t o radiat ion in the system. the CoIPYIn c h n o s ~ aberrations. Chromosomes.with more than two centrorcres have been observed. are far euier t o score than Che more s u b t l e tr8nBlocation and inversion typu wherr s l i g h t changes i n centromere posi t ion may be the only h i n t of preceding bredcage. Furthemore. In one study t h e s m of a l l abcr ra t iom apar t from dicentric. made up 60 t o 70% of the d icen t r i c y ie ld (27). With each d i cen t r i c one should a l s o ident i fy an acent r ic be l o s t a f t e r one division. one is indeed obsendng the f i r s t post-phytohemagglutinin induced mitosia. ~%UB one may w e f a r less experienced labor- a tory personnel fo r the chomooora analysis and it - be performed f a r -re rapidly than a search fo r synmoetric 8berrat ions would allow. containing the chromosome aberrat ion are not dividing it may be expected tha t even the unutablr d i cen t r i c and ring aberrationa may p e r s i s t for a COnBidBYlbh period i f the c e l l s containing them are not ac t iva ted by an endogenow mitogen (28). aad i f they are long-lived lymphocytes u opposed t o the lymphocyte population w i t h a br i e f l i f e span (29.36).

Th. aberrat ions produced ( i f broken

Incrementa i n the latter. which occur

Flgures 1 and 2 indicate

me dicentr ic and r ing f o m

fragment whidbwould Its presence k evidence tha t

Since v i r t u a l l y a l l of t h r lymphocytes

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For the purpose of dosimetry however, 1y.phocyte cul tures shoyld be made (u 'aoon a8 poaaible. There d o u not appear t o be a difference t n aberrat ion yield over 24 hours pmt- i r radiat ion (5,?,31). 48 to 72 hours .the lylaphocyte count i n an i r rad ia tad human has usually f a l l e n so lat tha t finding enough mitoses to count (SO t o 200) I s more d i f f i c u l t . in peripheral leukocytes remained re la t ive ly constant for t h e f i r s t throe or fojrr weeks a f t e r radiation" (22). l og ica l dosimcter bear carefu l cornideration because of the current controversies surrounding t h e dose-response relationship. The rings and d lcen t r i c s we measure a r e each formed from two separate breaks i n one or two chromosomes respectively. (Figure 2). These aberrations. when induced by sparsely ionizing x-rays o r g- rays should therefore usually require tvo separate photons t o in t e rac t with the environment of t h e chromosome, and thus the aberrat ion frequency should increase w i t h the square of the rad ia t ion dose, while aberrations requiring b u t a s i n g l e interact ion should increase in l i n e a r re la t ionship t o the radi- a t i o n Jose. Only an occasional. well-placed single photon should lead t o two repara te chromasome breakr. high l i n e a r energy t r ans fe r (L.E.T.) such aa neutrons produce a much l a r g e r volume of ionizat ions per pa r t i c l e , and therefore for high L.E.T. rad ia t ion d icent r ics and rings incresse as a linear function of dose (32). I n other words, the neutron and other pa r t i c l e s with high l i n e a r energy t r ans fe r genera1ly.produce more than one chromosome break per neutron. Hcuever, for sparsely ionizing radiation. one m i g h t an t i c ipa t e t h e y le ld of rings and d icent r ics t o be represented by the quadratic equation Y = c + aD + bDn where Y ia the y ie ld of d icent r ics and rings a f t e r 48 hours of cul ture , c is the sponteneoua frequency fo r rings and d i cen t r i c s (approximately one in 5000 normal cellm). a the coef f ic ien t of aberrat ion for a s i n g l e photon inducing rings end d icent r ics , b t h e coe f f i c i en t for tvo h i t exchanges, D t h e dose of radiat ion i n rad8 and n e q u a l t o appmxlmately 2 (33). O r i f one believes t h a t two separata ionizing events are always neceasary fo r the production of d icent r ics 'and r ings. and s ince these abnormalities are esmantially absent i n a

After

Hwever, Bender haa r t a t e d tha t "aberration l eve l s measured

The k ine t ics of t h i s bio-

Radiation containing pa r t i c l e s w i t h

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"normal" population, one may try t o f i t the observed data t o Y - bDn assuming n w i l l be close t o 2. Tablee 1 and 2 s-rize avai lab le data f o r c e l l s analyzed a f t e r 44 t o 52 hours of cul ture tin for r i n g s plus d i cen t r i c s o r dicentr ica alone (since the r a t i o of dicentr ics t o ringa 1s usually About 5 to 1). Studies performed on c e l l s cultured f o r longer periods of time (for example, reference 14') a re excluded despi te t h e i r h i s t o r i c a l and, s c i e n t i f i c significance. (See paragraph 2 below).

explained? The discrepancies i n theme r e s u l t s a re s t r ik ing . How can they be

We may o f f e r several teaions fo r the var ia t ions i n data.

1. There remains uncertainty i f the chromosome response t o sparsely-ionizing radiat ion (x-ray and gama ray) follows l i nea r , quadratic, o r power-lnr (dose- squared) k ine t i c s , so t h a t there cannot ye t be agrec- ment on a ca l ibra t ion curve a f t e r 8 years of experimentation. The data may be f i t t e d by several axperiorntal models.

2. Technical differences do not appear to be a t the hear t of the matter i f lymphocytes are cultured for no more than 52 hours before f ixa t ion , a t a t i m e when they are i n the f i r s t mitotic d iv is ion (13). vi11 lead t o errora i n counting by l o s s of BOW d icen t r i c and r ing chroeoromes during second mitot ic divis ion and t h e formation of "artefactual" d icent r ics from chrourt id anmal i e s which s e l f rep l ica te pr ior t o the second mi to t ic division. theore t ica l ly could decrease the d e r of dicent r ics observed a t 50 rads, d o u not seem t o b e a problem & - v i t r o a t doses up t o 500 rad (13.36). Temperature variation8 of as l i t t l e as 1 degree centigrade may have marked e f f ec t s on influencing the r a t e of c e l l response t o phytohemagglutin (37). whose most recmt r e s u l t s diverge widely wed idan- t i c a l techniques. h k e v e r (17, 19).

Waiting longer

Mitotic delay, which

Wo laborator ies

One n u t obviously be

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whole body irradiation mame patientr an above, cells taken 24 brs . after

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careful of observer bias i n analyzing slide. and so one should count only d i c m t r l u , In c e l l a which a l so contain an acentr ic fragment u noted above. indicat ing chat one is indeed observing the f i r s t poet-radiation mitosis. If one comts more sub t l e aberrations such as breaks. trsns- locations, and inversions. there is arch greater danger of erroneous subject ive in te rpre ta t ion . Data on gamna o r x- i r rad ia ted cul tures ( 5 , 17, 34) give lower pouer functions and higher coef f ic ien ts for rings and dicentr ics than data on i r rad ia ted whole blood ( 2 4 ) .

3. Lymphocytes a r e inhomogeneoua with respect t o function and l i f e span, only about 20% of t h e m having a l i f e span of 7 2 t o 96 hours, many l iv ing much longer (30 .39 .40) . mi&t hypothesize t h i s as a cause of a igni f icnnt var ia t ion between donor lymphocyte aberrations i n response to radiat ion Injury and to s t imulat ion by phytohemagglutinin. However, the var ia t ion i n response between individual blood donors does seem qui te small i n v i t r o (14.27) and -- i n vivo (31) when cul tures are begun within 24 hours of i r rad ia t ion . Thus. although there may be s ign i f i can t differences i n varieties of lymphocyte populations between individuals. these populations a l l seem t o respond s imi la r ly to radiat ion injury.

Dose r a t e s vary widely i n the experiments noted i n the table . Our own dose r a t e is ra ther low, 3.5 t o 6 rads per minute. aberration yield v u c lea r ly lower w i t h dose r a t e s of 0.04 to 0.55 rads per minute than with races of 17.5 to 230.5 reds per a inute i n a recent series of experiments ( 5 . 27) employing the same techniques i n a l l cul tures . The yie ld i n the range from 17.5 to 230.5 rads per minute WUI essen t i a l ly ident ica l

Thus. one

4. The

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5. The r e l a t ive biological e f f ec t (R.B.E.) variea because d i f fe ren t w d a l i t i e a of radiat ion t r m r f e r d i f fe ren t mounts of energy t o the c e l l a i r rad ia ted . Therefore, d i f f e ren t ca l ibra t ion curvea would seem neceesary fo r high and low mer# panma or r r q s end for neutron,. It is seneral ly agreed tha t neutroru giw a l i n e a r dose respome became of t h e i r high l i n u r energy t rensfer . The R.B.E. of 14.1 mill ion electron v u l t neutrona compared to 250 kVp x-rays hue been net a t 1.9 (41) while fo r 0.7 MeV neutron, an amrage value of 3 t o 3.S v u found by a d i f f e rea t laboratory (23,321. (17) when compared t o 250 kVp x-rays. The problem of chromosome biologic doa imtry w i t h d r r d radiat ion from reactom o r nuclear weaponry thm becomea increaeingly complex.

found in en hypoxic environment ( 4 2 ) . people wi th r e l a t ive ly low arterial oxygen aaturat ion might have mre radio-re- s f s t a n t chroaoclospca than those with ndrmol a r t e r i a l oxygen tension.

Another probles with the chromoame aystem is tha t of sa tura t ion of ava i l . b l a . s i t ea where breaka can occur a t higher rad ia t ion doaea (24,43.44). dose-reaponae k ine t i c s have indicated aaturat ion I n d icen t r i c and cen t r i c ring y i e l d . ( 2 4 ) .

b a t ' o f the ca l ibra t ion curvea published t o date have bean from -.__ i n v i t r o s tud ies with few exceptiom, and the in vivo radiat ion h a been with &rea of 50 rads or under (7.35). cu l tu re ti* which we ncw know is too long, tha t l a 72 t o 90 houra ( 4 5 ) . i r r ad ia t ion of 100 rads and three a t 200 rada are i l l u s t r a t e d i n the follawing Figure8 3 and 4. I n vivo w e find a much lower aberrat ion rerponre than h u been seen in v i t ro . a11 h a w wtu ta t ic , inoperable carcinou and are c l i n i c a l l y a rab le although they are hematologically noma1 excopt fo r

R.B.E. of Cobalt-60 g- rays WM found t o be 0.8

6. Because fewer chromosome aberrations a f t e r i r r ad ia t ion a r e

7.

Thus. a t doses over 500 rads in v i t r o the

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Many use a

Our own preliminary data from one pat ient given whole body

(Our pa t ien ts

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frequent mild anemia. culture technique appear in Appendicu A and B. are all ambulatory and some of them are disease f ree , receiving prophylactic i r rad ia t ion) . In r l v n damped lymphocytu may be rapidly removed from the circulat ion. and since our radiat ion t i m e may extend over an hour, ve mi@t .ism seeing rmny c e l h con- taining chr-some aberrations. obtained from the pat ien t jwt before and i nwdia t a ly a f t e r i r rad ia t ion . Our dose rate, 3 t o 6 rads per minute of Cobalt-60 gama i r r ad ia t ion is lwcr than tha t employed i n the other i n vivo da ta (7,35). Perhape there may be grcatar res is tance t o radia- tion-induced chromoeome breakage in vivo than i n i n v i t r o or more rapid r epa i r of chromosome breaks i n vivo. In vivo-in- - v i t r o comparisons have b k n made largely from p a r t i a l body o r inhomogeneous i r r ad ia t ion which h a been extrapolated to whole body equivalents (22.40, 47) with mderately good agreement.

The radiat ion technique and chroloaow The pa t i en t s

However, our blood saaples are

9 . Even i f all the d i f f i c u l t f e e i n cal ibrat ion l i s t e d above can be over-, we are still l e f t with the problem of inhomo- pen- exposure which is always d i f f i c u l t t o quant i ta te exactly. yield fewer aberrations than the same dose given over t h e whole body. The n d e n of rings and d icent r ics found w i l l , be re la ted both t o the volume of tissue i r rad ia ted and to the nrmber of lymphocytee contained wi th in tha t t i a w e . For ex- ample, there are f a r more lymphocytu i n a 125 gram spleen than i n a 1400 gram hman brain. The man residence t ime of the h m m lymphocyte in the blood has been calculated at 4.7 t o 7.5 minutes by one estimate (45). culate very rapidly back i n t o the intravsacular couparcpant, then blood from a person with p a r t i a l body radiat ion delivered over so1y s ign i f i can t period of ti- ni&t shw the same in- cidence o t chromosome aberrationa aa i f i t had been delivered over the whole body. delivered homogeneowly wet a tvo hour period t o the lover ha l f of the body (31).

Other agent., such u viruses and drugr,mmy cause chrmsome aberrations. In addition to t h e non-specificity of the test system, there is always the poss ib i l i ty t ha t the individual has received p r i o r rad ia t ion of which there is no record.

A hetarogeneow dose of radiat ion w i l l obviously

I f .lymphocytes a l l c i r -

This doen not occur when 300 rads is

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The kinet ic8 of &oe-reopolue a n q u i t e important i n in te rpre t ing inhomoganeow expmuro. For r ing le broak aberration0 one night thoorire tha t 1.000 rad. diotr ibuted o w r 10% of the body r i g h t g i w the 0- c h r o w a aberration r aporue (after t h o f o r co.pleto d x h g of i r rad ia ted and mir rodia ted celh) u 100 rad. t o t he whole body, u a u i n g of ceuroo t h a t the dir t r ibu- t i o n of lymphocyteo t o the bo+ La everyuhera the rame. Xarever, f o r ringo and dieantriu th io logic doe0 not hold i f one u o m e o doae-oquarod kinet ics . For i f one d n e o the e f f ec t of 1.OOO rad0 0y.r 10% of the body i n term of the d icent r ic and ring yie ld , OM rhould find ten t h o M many rings and dicentrics u r ingle break abe r ra t i no. n i o i o becawe tbc r a t i o of ( 1 , ~ ) 0 ) 2

of radiated with unitradiated ce l lo a f t o r an a p o r u r e of 1,000 rads t o 10% of the body, there is di lu t ion of the ratio of dicentric-ring to oingle break &erratiOM from 100 t o 1 to 10 t o one. Thio log ic hao been employed t o confirm inho.ogenrour dia t r ibu t ion i n a radi-

d i a t r ibu t ion of dicentric. a t a given radiat ion done (24) t o construct h io t0gr .u t o pradict tho f r ac t ion of the body irradiated, although our preliminary data with ex- cellent do8iPwtry fo r p a r t i a l body red ia t ion & not confirm h i s theoret ical dircwoion (311, u tho aberration frequency appean to be uch lover.

Clearly much more i n pivo data are required vith good doaimtry. rad ia t ion dosea up to 250 rads with wen higher doors planned with the oupport of ~ r r w autotranafuolw and laminar flow *voterile" mar. body i r r a d i a t i o n i o aloo being perfotnvd t o learn mro

t o (100) s i o 100 t o 1 0 0 tha t ewo after t o t a l miring

a t ion accidUit (22). Dolphin (%) hM wed the POirsOn

We are purruing thia goal at whole body

Large voluw p a r t i a l

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about the efficacy of chronoaou mberrations u a ra- d ia t ion d o e i r t e r i n the more frequent a i tua t fon of inhollogeneow expoaure. we hope t o atudy the effecte of various dose r a t e s a v i r0 A. w e l l .

With e l i nea r accelerator

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E T I ~ O ~ O L O N E STUDIES OF -1: RESERVBS IN IBRADIATED PATIENTS.

The abeolute lymphocyte count of the peripheral blood drops U c h more rapidly than do the comts of the ce l l8 produced i n the erythroid, myeloid and megakaryocytic eerfcm. Similarly. am noted ebove. lymphocyte chrowearue may be analyzed immadfAtely a f t e r rad ia t ion for radiation-induced aberratfone. Chromoeome~ of t h e myelofd and erythroid ae r i e s pry be analyzed ~ l s o . the 6 i t u t i o n i e even mora complex than vf th the lymphocyte ae r i e r becawe boaa arrow cel l8 a re i n a l l etagee of the c e l l cycle (

tAtfOn of ohaerved abe=atfOM i e even more f r a q h t with error than i n the lymphocyte s y s t a et present. Bar thm can ve evaluate f a i l u r e of the myeloid eer ie6 soon after i r red ia t ion? Our Laboratory h u c h w m t o examine bone marrow granulocyte rememe f o r t h i e purpme.

adrenal And gonadal origin l e a potent 8tipU1m t o leukocytoeie i n man (49 , 50). The increment 18 eolaly in cell. of the g r n - ulocyt ic aerie., largely neutrophilic polywrphonuclear granulocytes (51). A t a doae of 0.10 milligram per kilogram of body weight given intr .murcularly the normal average wri- granulocyte incream within 24 hours is 5,850 2 770 par cubic millimeter i n e n end 6,700 2 1.400 pe r cubic d l l f m t a r in woman. The lover limit of the n o d granulocyte incramat f e 2.600 per cubic dll imter: .In a series of 151 in jec t ions of etiocholanolone we r e c e n t l y . d y z e d , the Uxiur increment occurred a t 16 hours ia 46%. 20 hOWA in 32%. and a t 24 houra i n 22% of tho injection.. We g f w the:etiocholanolone at 1600 houra and obtain blood couate

Here. however

,S.G2,M) w h i l e 99.9% of the lymphocyte a r e i n Gl(12). Since ra 9 i a t i o n e c a r i t f v i t y var ie r during the c e l l cycle, interprc-

Etiocholanolona. A naturally occurring s t e r o i d m t a b o l i t s of

..

, .* b0

-17-

at 0800, 1200, and 1600 houtl the foll@*ng day.

The increment in tho peripheral blood count a f t e r etio- cholanolone is the rosu l t of =b i l l r a t ion of granulocytea . from t he bone urrw reoervoa (531, and is na t ~ c r o l y the rmult of rediatr ibut lon of oxtramedullaxy cells from u r g i - d pools to the c i rcu la t ing granulocyte pool. Endotoxin hrr also been wed t o aval tuto bone marrow reserves (53, 54, 55). P y r a d , 8n endotoxin of Salmonella obortw , baa been withdrawn from tho b ' r i u n morket. but a Pseudownru product, Pixuun. i n avail- ab10 f o r t h i s p u r p a e (54) . However, this mote r id mst be in jec ted intravenously; o n d o t o x d a m y t r igger i n t r avucu la r coagulation, elthouph this r i d e effect hao not ye t beon reported with Piro-n. We a l so profor ot iocholanolon~ becarus ita only s i d e effects are local in f l ama t ion a t the in jec t ion si te and occa- sional fever never exceeding 2. Centigrade. Aloo. i n t r w c u l a r r a the r th8n intravenous in jec t ion allows a t rchnicirn to administer t h i s s t e ro id without resor t ing t o venepuncture. A. oppooad t o endotoxin, repeated d a e s of otiochol.nolone can be given without cawing reticuloendothelial blockade, an t igenic (49 ) .

Our pa t ien t population hem been described above. Whoa on8 is deal ing with pat ien ts vho are not m t i r e l y "noozp.l", the question arises UI t o whothor this pat ien t population c m be compared t o norm81 controls studied by o t h e a . tiwever, the . fact t h a t t h e average xi- granulocyte incrlmant pr ior to rad ia t ion in our pat ients WM 6,200 cubic nillimetea (normal average maxinu i n c r m n t 5,850-6,700) indicate. t ha t the N o groups are indeed canparable. d imin iehv i th ago. whole o r p a r t i a l body radiat ion doses (me Appendix B for technique).

Fif teen pat ients have been studied (Table 3). In only 1 d i d the madmum granulocyte i n c r w n t become subnormal a f t e r the white blood c e l l count had f a l l an b o l w 5000 (by 2 days). In 10 of the 15 the f a l l in d m m granulocyte increment to eubnoraal level. pre- ceded any vhite blood c o l l count drop.

and tha latter i r a l s o non-

Thir rut- increment does not These pa t ieu ts recoived 100 t o 300 rad8

In 6 of these 10 whose white comts - t d l y d i d go under M O O per CU. P., tha a b n o r u l i t y in etiocholanolone response pre- codod the 10ukop.n~o by an averago of 12 days, suggesting tha t t h i r b io logica l .doshe tor may be of s i p i f i c m t r r s i a t m c e in giving varning of an irponding leukopenia. In 4 of the 15 the uximum

. ., .- ..,.:.

~ 1 o c H O ~ u m L As A ? l m x m or L m

D ~ Y or rirrt

undar 2600 per cu. m.

Day of uhlte blood ttiocholanolono trrporue c o a t fal l ing under

5000 (or by 1000 rattent w r (white blood oo\kc por i f control count 5000)

(kdr) CC- I O11 that day (whit. blood count per in paronthrri~) cu. m. i n patentheaia)

.. . .. . , * . 6 (3700) . . 6 (3700) . , .

099 250 ; UBI*- '

095 200 6 (b300) . . ' 6 (4100)

..i .i , .. . .

.. .

. . c

-a=- . .

W *

c

granulocyte increment dropped under 2600 per CU. I. on the same day t h a t the white count f s l l under MOO. cholanolone respolue generally occurred on days 6 t o 9 a f t e r radi- a t ion. One hrmdred rad. of whole body radiat ion v u ' a u f f i c i e n t t o c a u e the t e a t t o become a b n o d u w m 150 rad. to approximately 40% of the marrow wlw.

Ihe rllbnonul et io-

The etiochol.nolom reaponre v u leas helpful in predict ing

The etio- recowry. becoming w-1 before the white count had returned t o normal l eve la in ha l f the patienta who had leukopenia. cholanolone reaponre returned t o n o d in all patient# by 9 week# a t t h e latest (range 6-62 day.).

The m!aaur(PDant of the maximum granulocyte increment poat- r ad ia t ion al lova one t o predict vhether an i r rad ia ted individual w i l l experience leukopenia. H0Ipev.r. nei ther the timing, granu- locyte count nor granulocyte increment in the initial drop in the etiocholanolone reapocue ind ica tu how sevare the eventual leuko- penia w i l l be.

granulocyte r e a e r w s h u not y e t been determined and thi. i a under study noy. It l a s ign i f i can t ly lover t h ~ t ha t in a atudy employ- ing endotoxin where it v u fotmd t h a t a t l o u t 60% of the marrow mst be i r r ad ia t ed t o dosage levels o w r 1MO 1 before the endotoxin raapocua became abnormal ( 5 4 ) .

The lover l i m i t of the eensitivity of thia teat of M-

The timing of recovery of P=OY reaervea ruemblu t h a t noted in pat ien ts with Hodgkin's d i a e u e given a t c n s i v e frac- t ionated radiotherapy (56).

Deoxycytidine

Following the report of a radiation-indocad i n c r e u e in urinary excretion of deoxycytidine (Cdo) in the ra t . our laboratory has examined deoxycytidinutie in man u a biological doaimeter (5.9. 59, 60, 81). that t h e urinary and blood deoxycytidine levels of rats were elevated about ai. hours a f t e r X-irradiation, reaching a uxiwm in blood a t 9 hours, urine a t 12 houra.

Using 8 S e U 8 i t i ~ e C O l O f l m O t f l C techaiq- ( 5 9 ) W e found

Deoxycytidine excretion i n the rat w u proportional to the mount of r ad ia t ion up t o 200 R. Approximatelt. 8 eix-fold increase from the average p r c i r r a d i a t i o n value of 0.7 .(I per 24 hour urine followed 200 R.

.

c I I. .

-20-

W With tritiated CdR h b e l l e d on carbon-5 of cytonine, rat. i r rad ia ted v i t h 200 R excreted Z l f of the t o t a l radioact ivl ty while only 13% of rad ioac t iv i ty v u excreted by u n i r r d i a t e d rats (61, Pig.5). s p e c i f i c rad ioac t iv i ty of CdR inolated from the urine of i r rad ia ted rat. d e c r e a r d 2 t o 6 fold an eosparsd with t h a t of CdR in the urine of cmirradiated ram, the frw CdR pool nire v u i n c r e u e d in the i r r ad ia t ed rat (Table 4). Thin radiation-induced Cdlt excretion could be rupprenned w i t h rerotonin nnd I-cyntdna, the renpectiva dose re- duction facto- being 1.7 and 1.5 for thin variable . '

b

Since

c.

'r

Man, however, excrete. a mch smaller quantity of CdR in h i n Urine than doen tha rat. SB of CdR per 24 hour urine v u found in m (Tnble 5) M coapared to 0.7 mg in t he r a t . The lw CdR sscretim, in am say be CAWLd by the a c t i v i t y of a CdR d e d n a n e in h w n plasma (31) and liver (82). Hwcver. the range in our cancer pqtientn w u 3.9 t o 26 microgram f o r 24 hours (Table 6). Pollotting whole o r pa r t i a l body rad ia t ion at dose of 150 rads o r above CdR excretion me. 50 t o 350% w day one port I r radiat ion. re-

c. turning t o normal an day two (Table 6). However, this is not a dosimeter npec i f ic t o radiation injuzy. A pat ient with lymphoepitheliomn and eewral burned pa t imtn alno rhowed abnormally h i & urinary CdR excretion in proportion t o the severity m d extant of the burn (Table 7). Thus, deoxycytidinuria appearn to be related t o general t i s n u e c8 tabol im from n e w r a l catmen, including radiation. Other problem in w i n g urinary CdR include variFtionn in excretion dum t o race (57) and age (92) .

An awra- pro-irradiation value of 0.007

Creatine

8.diation-induced crea t inur ia in rati (61) f o l l w r a f a i l u r e of m a c l e t i s n r u t o u t i l i z e creat ine nynthenired at A n o d r a t e by l i v e r and m n c l e (62 ) . a f t e r a s i n g l e whole body &eo in the rat, at lrut up t o 650 9. Bow- war, we have been tmabln t o d-trat. increased amount* of creat ine in the ur ine of our p a t i m t n pont- i r radht ion as r u u r e d by the in- c r e u c d c rea t ine t o creatininn r a t i o (83). Rune ntudien were done n t done8 of 100 rad. o r l u n of whole body rad ia t ion hwever, p.rhap. below the n m e i t i v l t y of thin donimnter in MU.

It is done-dapnndant, r u c h i n g a peak over 4 dayn

Beta-Aoi~iorobutyr ic Add (MM)

Another n u d d c acid r t a b o l i t e , be t a -dno inobu ty r i c acid, a product of thymidim a tabol iam has b o a focmd in i n c r e u e d m m t n in i r r i d i a t e d hrrunm, w i t h d i n a g M O Y l l t u t o the proportional increase i n BAIBA with &#orbed radiation &m (63, 80). Hwevar, the i n c r e u e han not been coruisteut in our patientn (Figurer 6.7).

L

. -21-

l i p u m 5

+--A unitradiated frradiuted (200R)

' TIME AFTER IWECTION (HOURS) Fffeci of lrrodi tior. ( OOA at 1) Excretion

of L t r t f Cdk in t o w

L ir

Y

c

lbt No.

1

2

3

-22- Tabla i

%-Cdl EXCRETED I N 22 HOUR URINE IN RATS

?re- t r r Ad iA t ion

192.000

179.000

lC1,WO

0.60

1.04

1.93

POW-irradiation

Specif ic Radioact iv i ty

.( cpdmg )

61,000

31,000

83,000

Totnl excreted

( m8ld.y)

5 . 1 3

7.16

6.50

c.

c

-23- Tabla S

C ~ R ? b r n I N ~ S t : ACTIVITY IN SERW AND cdn c o r n IW URINE FROM

bLLeC'fED HUMAN CONTROLS. . . .

c

ir

c

M e sex activity (units / q protein) hour urine (1111)

1. 6 M 2. 8 n 3. 9 n 4. 9 ?L

5. 12 F 6. 22 F 7. 23 M 8. 25 ?4

9 28 F 10. 20 n 11. 29 n 12. 30 n 13. 5 1 H

0.46 0.96 1.02 0.65 0.71 0.37 0.38 0.71 0.94 0.51 0.43 0.59 0.98

5.7 12 .o 13.1 4.0 2.4 5.0 8.4

4. 8 ' 4 . 0

2.0 6 .O

12.0 7.5

Average 0.67 5 0.24 6.68 2 3.67

t

-24-

Tablo 6 .

U l 1. ch. Urim of Irradhtod htfionck

C48 in 22 how urlne h - i r r d i . r t o o PO.( 1rra4tat:on + days.

?LtIPcfS DOIL

C u e no. Soxlraco1.m mumria ( W a 1 awraio 1 2

IN148 c. 1- I76 kcal* 6.6 16.7 -- -- 0.92 TlW60 Ca I rebat 130 ?. 'hit 5.8 2 1.1 10.0 8.1 --

-- -- -- T l U l 5 4 C. bront 200 loc.1H 8.5 16.5 -- --- . . t. -1.

b. 15 TlVI60 C. Ov.11r loo?. l a n r 5.4 2 1.1 6 .8 b . 1 6.0 0.72 Tlu161 L W - a t . Iwor 8. t 15.2 ? . 7 -- 0.81 U I ~ I L P C. &Ion #o ?. l a n r 8.1 2 1.1 16.4 1.0 --

h.b 3 .4 -- 0.81 T l U l 5 2 kmchqomlc 106 c01.1 4.2 0.2 0.8b F I Y I 5 7 8 t m e h q n l c 100 t.c.1 3.9 + 0.9 b.5 4.0 -- 0.83 T l U l 7 8 C. 8 t . Y t 100 t o w 1 5.5 9.3 6.5 b.88 t1w154 c. 1- 150 t o t r l 8.4 17.8 b.4 - 0.1? TlUlb3 & ?h.r)u 200 toc.1 . 6.9 2 0.b lJ.9 12.2 14.3 0.78 n1n155 8raebasaolc m tot.1 10.4 2 4.3 31.0 31.0 -- 0.87 tlulll M m ' a tsmor zoo t0t.l 3.9 + 1.1 5.8 4.6 --

a. 79 ~ I U I J O C. 8 r a n t 106 tot.1 21.0 2 5.2 39.0 25.0 11.1

1 ::

-25-

Tablo 7

Pat ients , .. Sex Ago Diagnosis

.

So- CdR d o m i n u ; Act iv i ty

@nitr / Protein) ..

CdR DUnINASE ALTIVITY I N SERLW AN0 CdR CON'TLXT

IU URINE FROM PATIEMTS WXfH VARIOUS DISEASES.

1. !4 14 LI.phoeptthelioaa 2. H 30 Chondrorsrcms (shoulder)

3. ? 76 Carcinoma (bladder) I . F 62 Carcinoma (brcnsc)

5. It 61 Lyaphou

6. F 42 Sronchopnouwnis 7 . x 19 ~ ~ r a p i 0 8 i a

8. I4 16 Hyuthonia 6 r J V i S

9. F 67 Rheuntoid a r t h r i t l r

10. J4 28 Diabetes mellitus 11. X 12 Body burna (55%)

12. X 16 Body burn8 (72%)

13. F. 28 Pregnant (3 m o t h s ) 16. F 1 7 Pregnant (8 months)

--

~ .~

1.20

' 0.5h5"' .+ 0.66

0.60

0.20 0.13 0.76 '

1.12 0 . s

0.36 0.90 1.21 0.64

0.72

I .

cdl i n 24

hour urine '111)

38.0 14.0

20.0 18.0

18.0 6.1 - 3.9

11.5

IO. 0

5.9 32.6

-*- " - * C I . .- . a&- 6 *

I URIN&? EXCRETION OF AWN0 lSoeUTYRlC ACID I L BEfOh'E AND AFTER TOTAL BODY RADIATION 'r

I

7 10

0 I. A I I 1 . 4

r w

'O1 I

L 100 RAO I _ -

6OCF

D A C S BEFORE I& , D A Y S A F T t R

. 9 . + '1 i ! i.

. . . ..:. . - ..: . . . .

.. I .

c

. .

v b

I I .

. .. ..

-27- P i lo r . I

URICIIIRY E%CR€TKWJ O f AMINO ISOSUTVRIC ACID 8EIcocK AN0 AFTER TWAL W O Y RAOIATION

W

70 -1

I

I 0' . I . . I I . I I I 1 I

-28-

W 1c.

Another p r o b l a in Interpret ing ur ine BAIBA data i a that ttere is an hereditary uymptolvt ic condition of high excretion

~ 1 0 % of people examined) ( 6 4 ) . This compound, too, is a non-specific sign of t ieaua breakdown.

Taurine and Other Amino Acid8

Another .&no ac id , taurine, is excreted by the r a t and dog. but not the guinea pig, in axcesa amounts a f t e r whole body radiat ion ( 6 5 , 66 . 67 , 68, 6 9 ) . A second radiat ion exposure in about a veek following the f i r s t do- not elicit a a imi la r excretion of taur ine ( 6 7 ) . Previow work h a sham that an a i r done grea te r than 450 R

required to ralae urinary taurine a f t e r hmmn whole body i r rad- i a t i o n (65). Then v u no s ignf f icant increase in the taurine ex- c re t ion amom' our pat ients up to a dose of 200 rads. Nor w.8 there any a lgn i f i can t change in excretion of any of the urinary &no acids examined (Table 8 ) . after surgery or in fec t ion and are therefore also a wn-specif ic s ign of t iaaue breakdown (84). evaluat ion of urimrp mino acid levels in general , and taurine s p e c i f i c a l l y , is t h a t the levels of most d n o acid. in the urine are grea t ly influenced by dietary protein intake. In s-ry, our laboratory h u been h l s t o confirm that there are any s igni f icant difference. behreao the mounts of there urinarp d n o acids ex- creted by cancer pa t ien ts before o r a f t e r whole body i r r ad ia t ion a t 1evela O f 50 t o 200 rad8 (81, 83).

Glycoprotein8 Including Traneferrin; Seem Iron

Urinary,taurine levels have been found in children

Another var iable d i f f i c u l t t o control in

Serrn glycoprotein leveh have been reported t o have prognostic s ign i f icance a f t e r rad ia t ion injury (70 ) . Uslng pre- i r radiat ion levels as controlr the concentration of t o t a l protein-bound carbo- hydrates. pa r t i cu la r ly t ranafer r in end haptoglobin (71) are found t o rise t o high levels in mice, rats and dogs who have died a f t e r rad i - a t i o n arposure, while s u r u i v o a ahowad little change. invaa t iga tor reported no s ign i f i can t difference between the man serum 8lycoprotein concentration in rats' blood 88 t o 90 hourr a f t e r 600 It of X-irradiation 8nd t h e i r unir tadiated control# (72) but t h i s my w e l l be due t o the expetiuntd d u i g n .Iployed (69) . The masurement of t o t a l iron binding capacity ( t ransfer r in) showed no s igni f icant rise a f t e r i r r ad ia t ion in our pat ien ts (Table?). p a t l e n t s aunr iwd their treatment this l a a t lwt consis tent with Evans' predictlonn t h a t lack of glycoprotein rise correlates with aur- Vivrl .

Harever. another

.Since dl our

c -29-

Table 8

The Urinary Amino Adds Analyzed w i t h No Change af ter Radiation:

Glutamic acfd Serine

., Glycine 1 Threonine A1 ani ne

If 4 Glutamine Be ta-a1 ani ne

Y - 2 :

>

Valine Phenylalanine T a d ne Pro1 i ne Beta-aml noi tobutyrl c'

Lysine Cystine Tyrotfne Methyl h i t t l d i ne Leucine Ethanolani ne

H i s ti dine Aspartic acid acid

Y

-30- Tabls 9

CIIANGES IN TOTAL IRON 8INDfNC CAPACITY (TRANSFERRIN) FOLLOUlNC

H W IKMDIATION

Change in

(percent)

Duae total iron (rilds) binding capacity

c.

% .

f "'L

i w.

Day of

change post radiation

M X i O U r

. -E% I

- UriR .--- OR 7 200 -1 32 9

09 1 200 -16X 9

0 098 200 -23% . .

09 3 150 -29% 9

086 150 +I% 3

-- wen. --- - UHR

- WBR

wait -

096 100 6% 6

102 ZOO +bt 4

092 1 SO -8Z 2

089 2 50 -122 9

09L 1 so -1bZ 6

UP R

?(-Pee

5-P

CBR***

-

.i L..

+WBR - Whole body radlrclon +*M-P - Xanubttum to pubts

***UBR - Nadiation of 'the hal f , of ,tho body above chr rlphoid

****LBR - Radiation nf the half of the body belou the xiphoid

****e - UWcc count always over WOO per CU. mm.

'W

I.

u -31-

Radiation-induced damage t o the e ry th rob lu ta in bone marrow should lead t o a t ire in s e m iron am exythropoiesis decreaaee. Serlnr i r o n level. roe. over the f i r s t 9 days poat i r r ad ia t ion i n only 20% of our pa t i en t s studied, and che average change ove ra l l V.6 en i n s ign i f i can t drop of 17 dcrograzm per 100 ml. eerm (Table 10). Thus. our material does not confirm som promising animal work (73. 7 4 ) . The diurnal var ia t ion in rerum iron and the var ia t ion in the levels when the ec ru is taken at the s a m tin daily (er we did) is sig- n i f i can t (75, 76) and diminishes the value o f the serum i ron as a dosimeter. The total iron binding capacity ( t ransfer r in) docs not have such diurnal var ia t ion apparently (75).

Absolute Uonocyte and Lyrmphocyte Counts

Changes i n c e l l counts fo l l a r ing radiat ion a re the r e s u l t of damage at the molecular level . penia a t 48 hours h a been re la ted to the sever i ty of the rad ia t ion in ju ry (77). rada whole body r ad ia t ioa s t i l l have absolute lymphocyte count8 grea te r than 1,000 per cubic millimeter. 11 pa t i en t s receiving 200 rads would have been c l a s s i f i ed rn having sus t r ined “very eevere“ injury, 3 “sewre“ injury a t 48 hours (See Table 11). both of these carrying grave p r o p a t i c rignificance. Per- haps t h i s concept needs revision.

The degree of port-radiation lympho-

Yet a t 72 hours. 5 of 11 of our pat ients receiving 200

By Andrews’ criteria (77) 4 of the

The muse monocyce is apparently qui te radiosensitive. Four .hundred rad. of whole body x-ray reducar the mouae abSOlUt8 monocyte count to ona-tenth of the pre-irradiation level v i t h i n 24 houra (78). Our data indicated no c l e a r downward trend in humn monocyte l eve l s u n t i l about day 15 folloving 200 rads whole body radiat ion (see Table 12). Thur, the monocyte count h a l i t t l e value in the period following i r r a d i a t i o n and the absolute lpphocyte count somewhat less than h a previously been s t a t ed .

Urinary Gonadotrophiru

A yet unexplored a rea of p i tu i t a rp hormonal response t o radia- cion ham recent ly been spotlighted in a report t ha t urinary gonado- t rophins may rise u ear ly u the 7th day a f t e r acute I r r ad ia t ion (79). The response did not 10.0 dare re lated in the pat ien ts rtudied. We are cur ren t ly axamlning t h i s finding i n our mm pat icntr .

.

W ”

i

W u”

-

v c

r

-32- Tabla 10

C W E S IN SERUM IRON ARLU HulM IRRMIATION

b x i a w charqa from prc-rndiation l evulu ,ent nose of serum i r o a v i t h i n nine days poor

(Rads1 radintion (percent) 250 -31% UBR*

r)r J 200 -592 VBR

VIR

WER

WllR

VllR

WBR

VBR

I---

c 081 --- 2 0 0 +782 - - 091 - 200 -332

o9n 200 +152X

ri9 3 150 -60%

088 150 -162

096 100 -62

102 200 -352

09 2 150 -562

089 2 0 0 -582

O K 150 +9t

100 300 - 7 s x

- ---- --

- -

-.- n-p** - .X-P

;m***

-- CBR

LBR****

- -. -

W B R - Whole body rAdiAcion ***L'BR - h d i a t t o n on t h e half of tha

body above the xiphoid

4W-P - Hanubrium t o publn ***4LBR - Radfation of the half of

the body b e l w the xiphoid

I -

t

I 1 I

..

-33-

* . I'

role

Y

l o 2 0 YO21

L

.. -34- '

Tablo 12

0,AY

- I

RI

+ I

* 2

+ 3

* 5

' 8

' 12 *I5

'18

21

* 24

* 27

e 3 0

t 33

r 3 6

* 40

* 54

1 59

740

- 460

1224

627

368

6 12

845

583

297

207

210

108

I32

I20

- 713

1250

- 1378

780

71 I

1092

1152

87 I

969

546

- I10

- - i

rxpind

P 023

692

- 1083

I276

6313

867

I076

547

- 1176

402

- - 287

484

657

10 I

P 024

I16

- 60s

876

664

550

3 24

2 56

I96

2 24

, 96

I28

-

80

67

sot

( cells per cu. m m )

t 053

1404

1573

976

loo9

318

657

1140

427

' 165

1 I I.

114

26

orpiroc

L 077

372

489

IO50

215

206

453

829

I407

128

213

226

- -

*wire(

078

750

503

5 25

274

2 40

258

459

5 2 0

56 I

284

90

105

45

I 3 6

- 396

418

9 I6

,. L.

I

w

. -35-

W " Anylane

F:lcvnted serm amylase levels (88) a f t e r p;~rorid rta1iV;iry

Tlie'amytnse rose t o B peak value at 2 4 t o VJ hours gliind r d i a t l u i i have heen noted w l t h doses as lou as 100 W (tmvr done) ( 8 5 ) . iiftcr I r rudla t ton , then declined t o normal over anorlier o w 1,r twu days. The response appears t o h e organ spec i f ic (31. 8 5 , 8 6 ) nnd liyperamylsvemia dnes not resu l t when the patient receives doses L O the pancreas (the usual source of sensa amylase) coaparabte'to those causing enzyme release from the salivary glands. h e mylase re- leased from the damaged (rlands is a t l e a s t grossly related t o the absorbed dose of radiat ion (85, 86).

Our data (Table 13, Figures 8,9) confirm these findings for whole and p a r t i a l body radiat ion in the hman. and ur ine leve ls i n pat ients receiving radiation to the sal ivary glands. The least s t r i k i n g of the three serum responses is i n J pat ien t whose parotid glands were not i n the radiat ion f i e l d . Onty the pa t ien t receiving the highest cumulative radiat ion dose shows elevated urine l eve l s of t he enzyme, however. suggesting chat t h i s is 4 less sensitive test than a e r m amylase. responses are prompt, however, and both dlminish as the cumulative rad ia t ion dose destroys glandular function. Table 11 indicates chat t h e s e n s i t i v i t y of t h i s biochemical dosimeter is ac l e a s t 100 rads. Radiation below the neck does not increase serum amylase . i n doses up t o 300 rads over the pancreas (not on char t ) . laboratory is current ly performing agar gel electrophoresis on the sefm of i r r ad ia t ed pat ients to separate the isoenzymes of amylase.

Other Wuman Urine and Serwa Protein Studies

The graphs show serum

Both urinary and serum

Our

A nmber of other hman senm and ur ine proteins have been examined f o r t h e i r response t o whole body radiation up t o 250 rads and half body radiat ion up to 300 rads. These a r e l i s t e d i n Table 1 4 . These tests have been found t o show absent or inconsistent changcs Eolloving i r rad ia t ion .

L c

Y

- 36-

Table 13

'W

- 3R-

L Y

c

I

W

-39- Table 14

-40-

. W

c

APPENDIX A

P I M T W L FOR MR0K)SOME CULTURE

L

1. A 10 m l . syringe is moistened with 0.1 ml. heparin (1 m l . = 5,000 U.S.P. uni t s containing 1% benzyl alcohol) and 10 ml. venous blood is aspirated under steri le conditions.

Blood is then transfarred t o a r te r l le screw cap tube . and the fed cells are allowed to sacliment while under refr igerat ion for one hour.

2. I t is mixed gently-

3. 0.5-1.0 ml. leukocyte r ich p l a n a is added t o 4 steri le, dlsposablc flasks containing 1-5 m l . of the folluving media: 15% Fata l Bovine S e w . 2.4% phytohemagglutinin, 1.2% 1-glutamine, and 1% pei i lc i l l in and streptomycin.

Incubate t h e cul ture at 37.C fo r 46-50 hours a t ambient p02. added to the system.

80% Minimum Essentiol Media.*

4. No C 0 2 i s

5 . Then 0.1 m l . 0.0004X colchicine i n Hank’s** balonced s a l t solut ion is added t o each cu l ture which is returned t o incubator f o r 1-2 hours.

The f lasks a r e agi ta ted and contents emptied i n t o a serologic t u b e which is centrifuged f o r 2 minutes i n an Adam Sero-Fuga.

6 .

b L 7 . One then asp i ra tes the supernatant and discards it. The cel ls a r e washed with 37. C 0.7% s o d i m citrare and placed i n a 37.C water bath fo r 1-7 minutes. then centrifuged as i n Step 6 .

8. One next asp i ra tes the supernatant and discards i t . the cel l button, one adds 1-2 m l . Carnoy’s f ixa t ive (3 par t s Methanol: 1 par t Glacial Acetic Acid) and allows the button t n stand 30 minutes.

Without dfsturhing

9. Then one resuspends the cells and centrifuges as before.

10. Steps 8 and 9 may be repeated as.needed, usually twice.

11. Next we add su f f i c i en t Carnoy’s f ixa t ive LO obtain an opalescent appearance, about 0.5 m l .

Sl ides are prepared by dropping 3-5 drops of cel l suspension on clean s l i d e s which has been wet In cold d i s t i l l e d water, ign i t ing them momentarily i n an alcohol burner.

They are s ta ined with 1 : l O Giemsa s t a fn f o r 14 Pinutes.

1 2 .

13.

-41-

b-

c.

c

14. Next we rime w i t h acetone Mea, acetone: xylol (1:1) once and then place the mlidem in 100% ~ 1 0 1 until all are rudy to mount.

Wa -t by adding 1 drop*** Pm-t and 8 covers l ip . 15.

*Gibcos Grand I m l d Biological Co., Grand Imland. R.w yo*.

**Difco Pluruceuticalm, Datroit Wch1g.n.'

***Fisher Scimtif ic , P d r l u a , Ww Jarsay.

c

c

-4 2-

APPENDIX B

DOSIHETRY:

b

b

I-

C.

1. Total Body Radiation

CobAlt - 60:

?he radiation is delivered by a Cobalt 60 T e l e t h e r a p y U n i t under the following exposure conditions:

The radiat ion beam is directed horizontally at a wall 338 cm. away w i t h t h e pa t ien t mldline at 282 CP. from the source. 50 percent isodose curve a t the pat lent mldline distance is a square approx- imately 72 cm. by 72 cm. (Figure 10). posi t ion with legs raised and head t i l t e d s l i gh t ly f o n a r d . The i r r ad ia t ion is given by del iver ing half the specified apoaure l a t e r a l l y through one s i d e of the pa t ien t . l ivered l a t e r a l l y through the other side.

The beam &rea f o r t h e

The patient is placed i n a s i t t i n g

The pat ient is then turned and the other half exposure de-

The variat ion of air exposure with distance f r m the source was deter- mined with a Victoreen 25r chamber. The resu l t s indicated no departure from the inverse square l r r d a t i o n s h i p fo r distances used i n the study. no correct ion WY required fo r a possible dose contribution t o the pa t ien t due t o backscatto? I m m the w a l l .

Therefore,

Preliminary measurements were made in a masonite phantom using dosimeters placed on lateral surfaces and a t the d d l i n e of the head, trunk and knee portions of the phantom. If the d d l i n e doses t o the trunk, head. and knees are compared, the maximum variat ion in these doses is about 1 6 per- cent.

The expowre to the pat ient waa etermlned M follows. The percentage depth dose a t di f fe ren t depths f o r a 400 c 3 f i e l d area and a source-skin d is tance of 80 cm is given by H.E. Johns. "The Physics of Radiology." Charles C. Thomas, Springfield, I l l i n o i s . The depth dose at the greater source-skin distances used f o r t he pat ients WM found by multiplying the depth doses a t 80 cm. by t h e "F" fac tor postulated by Wyneord and tamerton ( B r i t . 5 . Radiol. 14:255, 1941).

C.

L

! f ' Figura 10

c mi ! I

Isodose Curves f o r Aadtation Technique Employed

141th Fatlent M l d l i n e at 282 c m . From Source

:. '" -44-

t

W b

F -

El + d f 2

c

where fl and f 2 are aource-skin distances and d l a the depth.

ha l f lateral dimension of the trunk) and a conversion factor of 0.97 rads per roentgen for Cobalt gama radiation. the surface dose and midline o i r exposure required t o give a desired midline absorbed d w e i n rads was calculated.

By w i n g the corrected depth dose a t t he pat ient midline (cne-

A d i r e c t comparison of the calculated and measured (phantom) doseR vas made f o r one pat ient who had the same l a t e r a l trunk dimenaions as the phantom. Is given in Figure 11. m d e in the phantom and compare qu i t e well with the calculated doses. The combined dose of the NO radiation f i e l d s l a ala0 given i n t h i s f iaurc and shows a good hoaogenew dose d is t r ibu t ion through t h i s pat5ent. va r i a t ion i n lateral h e dis t r ibu t ion was + 1 3 percent f o r one patient having a lateral trunk dimcnaion of 36 cm.

The r e l a t ive depth dose f o r each l a t e r a l exposure t o t h i s patient The doses indicated by crosses arc masurements

The maximum b L

c

c

Partial Body radiation:

In the individuala receiving p a r t i a l body radiation, the teletherapy coll imator is rued to restrict the beam. 'he isodose curves for t h i s la t ter case is shown i n Figure 12. shown in the Figure. luminescent dosimeters. t h e boundary. and Hwland (The Miological Effects of Upper Body X-irradiation of Bcagles, VR 580, University of Rocheater A t d c Energy Project, 1960).

The relative dose f o r upper body radiation i s These phantom mcaauremcnta were determined with thermo-

For p a r t i a l body radiation. t he xiphoid w a s used a t Thir technique l a a i d l a r t o that described by Hanse. Michaelsoo,

. Relative Depth Dose for Each 1.ateral Radiation

Exposure

RELATIVE DOSf

* - I

'k

i I

-46-

Figure 12

-4 7-

REFERENCES

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2. Sax. K.. Ihe ti- factor in x-ray production of chrowsome aberrat ions, Proc. Nat. Acad. Sci.. Wash. 2: 397. 1939.

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v c

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! .f .I

t i ! ,

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c

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42. Effect of o%yRen on t h e frequency of

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b

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I ' I . ,

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k

72. O l e t s k l l , E. The e f fec t of t o t a l x-radiation on the glyco- protein content of blood #arm in n o m 1 4nd hypothyroid animala, Byul. Elupeti.. Blol. i. Ned. g: 54-56. 1965, quoted by . E v ~ M , A. in Ref. 70.

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76. Bwie. E., Tame. W.. Sjoberg, W'., Y ~ ~ ~ g u c h i , li. D a i l y

Iron-59

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