The Distribution and Fate of Bromodeoxyuridine and
The evidence that incorporation of the halogenated thymidine analog, &-bromodeoxyuridine(BUdR), into the deoxyribonucleic acid (DNA) ofmammalian cells cultured in vitro may result in anincreased susceptibility of the cells to the lethaleffects of x-radiation has recently been reviewedby Djordjevic and Szybalski (8). These authorspointed out that radiosensitization is most readilyelicited when BUdR has replaced an appreciablequantity of the thymidine in both strands ofDNA. Quantitative incorporation of BULR intoDNA of cells in vitro is markedly affected by theconcentration of BUd.R in the medium and by theduration of incubation (19). BUdR-monophosphate (BUdRP) may also be recovered from theDNA of neoplastic cells after they have been incu
* This investigation was supported by grant CY-4096
from the National Cancer Institute, National Institutes ofHealth, U.S. Public Health Service, Bethesda, Maryland.
t Onleavefromthe Institutefor TumorBiology,Karolinaka Institutet, Stockholm, Sweden.
Received for publication October 9, 1961.
bated with bromodeoxycytidine (BCCIR) (7). Thepossible clinical usefulness of BUdR or BCCIR ininducing radiosensitization of tumor tissue willdepend not only upon local factors governing theirincorporation into DNA, but also on the fate ofthese compounds in [email protected] the use of BUdRBr82 and BCd.R-Br82, a study was undertaken toinvestigate the extent to which these compoundsare incorporated into the DNA of various tissuesof the rat and mouse in vivo after parenteral administration. As contrasted to the use of H' or C'4as the isotopic label, the use of Br82convenientlypermits both the detection of minute quantities ofbromine-containing compounds in DNA after administration of tracer quantities of BUdR andBCdR and an estimation of the rate and extent oftheir dehalogenation. Observations were also madeon the conservation of the incorporated cornpounds within cells during multiplication and onthe proliferation of BUdR-treated cells in vivoafter x-radiation.
9254
Bromodeoxycytidine in the Mouse and Rat*
J OSEPH P. KRISS AND LASZL6 IUvi@szt
WITH THE TECHNICAL Assiwr@sicz o@ Lvcrz TVNG AND SUSAN Eoi@o@r
(Departments of Medicine and Radiology, Sianford Univerriiy School of Medicine, Stanford, California)
SUMMARY
Bromodeoxyuridine (BUdR) was rapidly and extensively degraded in vivo in the rat,with the concomitant formation of bromouracil and bromide ion. The liver is amajor site of dehalogenation. A portion of the administered compound escaped degradation and was incorporated into the DNA of various tissues in a pattern similar tothat of thymidine incorporation.
In the rat, bromodeoxycytidine (BCdR) was more slowly degraded than BUdR.Neither bromocytosine nor bromouracil could be detected as degradation products.Following administration of BCdR-Br82, the distribution of Br@ activity in the DNAof various organswasdifferent from that which followed injection of BUdR-Br82. Someevidence was presented which suggests that such a difference may be due to deoxycytidylate deaminase activity in particular tissues, such as the bone marrow.
Following incorporation of Br@ by Ehrlich ascites cells after intraperitoneal administration of BUdR-Br@ or BCdR-Br82 to mice, the radioactive label was conservedduring neoplastic cellular multiplication for at least four successive generations.
EhrliCh ascites cells, exposed in vivo to repeated intraperitoneal injections of BUdRand subsequently irradiated with 550 r, showed a greater inhibition of their post-treatment growth curves in vivo than did irradiated cells not exposed to BUCIR.
KRIsS AND Ri@vi@sz—Fate of BUdR and BCdR in vivo 9255
MATERIALS AND METHODS
Animals.—C57BL/Ka and CSH/Ka male mice,weighing 920—SOgm., and male Long-Evans rats,weighing approximately 9200grn., were used. Theanimals were maintained on Purina chow diet andwater ad libitum.
Tumors.—An Ehrlich ascites tumor line,1 referred to as ELD, was used. A number of cellularand growth properties of this tumor are summarizedin a recent publication (928).In this laboratory thetumor cells were carried in C57BL male mice byweekly serial transfer of 0.1 ml. ascitic fluid,diluted with sterile 0.9 per cent saline in a propertion of 1 :92.The cell concentration was determinedin an aliquot of the ascitic fluid in a hemocytometer after dilution with Turck's solution. Allintraperitoneal injections were made by a 925-gauge needle through the musculature of the lumbar region in order to avoid leakage of peritonealfluid to the outside. The multiplication of theintraperitoneally inoculated tumor cells was dotermined by a quantitative rinsing procedure (18).
Blood clearance.—A polyethylene cannula filledwith heparin solution (1000 USP units/mi) wasinserted and secured into one carotid artery of arat anesthetized by nembutai (45 mg/kg bodyweight). The cannula was then clamped, and 9200USP units of heparin were administered intravenously. A physiological saline solution of theradioactive substance(s) to be studied was theninjected intravenously in a volume of 0.92—1.0ml.At frequent time intervals thereafter, the carotidcannula was momentarily unclamped, and a fewdrops of blood were collected onto wax paper. Analiquot, 10 or 920Xof blood, was added to 1 ml. of
1 Obtained from the Institute for Tumor Biology, Karo
liuska Institutet, Stockholm.2 California Corp. for Biochemical Research, Los Angeles,
Calif.3 New England Nuclear Corp., Boston, Mass., 1 rnc/mmole.
tate was centrifuged, and 0.5 ml. of the supernatant was mixed with liquid phosphors for betascintillation counting. Calculations of total circulating activity were made by a table of values ofblood volume based on the body weight of theanimal (92).
Distribution of labeled compounds.—Immediately following the completion of blood sampling insome of the experiments involving blood clearancemeasurements, the rat was killed, and varyingsized portions of the liver, kidney, spleen, testis,femoral marrow, and of the mucosa of the colon,intestine, and stomach were taken for measurement of amount and radioactivity of DNA. Radioactivity was expressed as per cent of the administered activity per mg. DNA in the sample.BUCIR-Br―, 0.5—5pc., was administered to miceintravenously, intraperitoneally, or subcutaneously. At stated intervals after the injection, theanimals were weighed and sacrificed. The followingorgans were excised in toto: skin, spleen, liver, kidneys, testes, stomach, intestine, and colon. Thethree latter were evacuated of their contents bybeing rinsed with water. Each of the organs andalso both legs were weighed and placed in separatetest tubes, filled with water to a volume of 5 ml.,and their activity was counted in a well-typescintillation counter. A tube containing a Br82standard in 5 ml. water was also counted. The skinwas counted in two portions. The carcass remaining after removal of the organs was placed in a50-ml. beaker, water was added to a total volumeof 92.5ml., and the radioactivity was measured overthe crystal of the scintillation counter. A 50-mi.beaker containing a Br8' standard in 925ml. waterwas counted under similar conditions. The radioactivity in the organs was expressed as per cent ofadministered activity per gram wet tissue.
Analytic methods.—The methods used for paperchromatography and the measurement of radioactivity and DNA content of tissues were reportedin a previous study (19).
Fractionation of PCA-soluble, Br8'-containingcompounds was accomplished by passing the solution through a column containing 920-to 50-mesh,Dowex-1-Cl, anion exchange resin, which allowspassage of BUCIR and BCdR to the eluate butretains bromide. With nonradioactive BUdR,
BCCIR, and bromide-Br8' used as test substances,it was found that washing the resin columns soquentially with 92volumes each of water, N/100HC1, and N/10 HC1 resulted in essentially cornplete recovery of BUdR and BCdR in the pooledeluate as determined by spectrophotometric meas
urements. Such treatment also elutes bromouraciland bromocytosine from the resin column, whilethere is complete retention of Br82-bromide on theresin.
Descending paper chromatography of synthesized compounds, plasma samples, and PCAsoluble fractions of plasma was carried out withsolvent systems of butanol: water (86 : 14) with 5per cent ammonia and 65 per cent v/v aqueousisopropanol :92.0N HC1.
The measurement of the activity of Br82, C'4,and H@ in the same sample was carried out in aliquid phosphor count& as follows: The samplesto be counted, together with Br82, C14, and H@standards containing amounts of perchioric acidand phosphor7 equivalent to those in the samples,were first counted at a low-voltage tap setting(tap 92).At this voltage tap, the lower discriminator can be set so that C'4 and H@activity are notdetected, and only Br82 ($ energy = 0.44 Mev) iscounted. The samples and standards are thencounted at an intermediate voltage (tap 6) and ahigh voltage (tap 10). The Br82 contribution foreach sample at taps 6 and 10 can be calculatedfrom the values for the Br82 standard at the threetap settings. Subtraction of the Br82 contributionat taps 6 and 10 from the total count at thesesettings gives the activity due to C'4 and [email protected]'4 activity (@ energy= .155 Mev) is recordedmaximally at tap 6, whereas H' activity (@energy = .018 Mev) is more efficiently detected attap 10. From the values of the C'4 and H@standards at taps 6 and 10, and the total sample countsat these taps, double equations can be solved forC'4 and H@activity, the final expressions being
Th—bT'° T'°—al'c6= —andH'°= —,
1—ab 1—ab
rays were generated at 9250kv. and 15 ma. (HVL =0.34 mm. Cu). The dose rate, without added filter,at 50 cm. target-based distance, was 9200r.p.m.
RESULTS
Clearance of Br8'-labeled BUdR and BCdR fromthe blood.—The clearance of BUCIR from the bloodof rats was studied in two experiments. In eachexperiment, thymidine (TdR) was administeredsimultaneously as a standard for comparison. Inthe first experiment each of two rats was given injections intravenously of equimolar quantities (.9292
@moles)of BUdR-Br82 and TCIR-C'4, 92 sc. and 1.4sc., respectively. Serial blood samples were obtamed via a carotid cannula during 60 minutes.The Br82 and C'4 radioactivities in the PCAsoluble fraction of 920X aliquots of each bloodsample were determined. From these values, andfrom the value for blood volume of the animals, asestimated from the respective body weightfigures, the total amount of radioactivity in thecirculation was calculated and expressed as percent of the administered radioactivity. No correction was made for the blood lost due to sampling.The results are shown in Chart 1. It will be notedthat the data obtained from the two animals arein good agreement. The clearances of Br@ and C'4differed markedly in both rats. The C'4 content ofthe blood decreased quickly, and less than 92percent remained at the conclusion of the experimentafter 1 hour. On the other hand, within the firstSminutes after injection, the Br82 activity of theblood fell to about 19 per cent. Thereafter it rosea few per cent and subsequently maintained a level
Where: T6 and T'°= total sample counts at tapS 6 and 10, respectively, less Br@2contribution
C6and C'°= O@contribution at taps 6 and 1O,'respectively, less Br@2contribution
H6 and H'°= H3 contribution at taps 6 and 10, respectively, less Br@2contribution
do@ bystandardd'4
b=@#ro, bystandardH3.
of about 920per cent until the end of the experiment.
An explanation for the disparate curves for Br82activity on the one hand and C'4 activity on theother was provided by observations which weremade in the second experiment. Equimolar quantities (.9292pinole) of BUdR-Br@ and TdRI.C'4
X-radiation.—Aliquots of ice-cooled suspensionsof ELD tumor cells were irradiated in air in a flatbottomed plastic irradiation chamber with adiameter of SO mm. and a height of 40 mm. X
6Tri-carbLiquidScintillationCounter,PackardInstrument Co., La Grange, Ill.
KEISS AND REvi@sz—Fa@e of BUdR and BCdR in vivo 9257
(50 @c.and 1.4 pc. respectively) were injectedintravenously in a mixture, and 920?@blood sampieswere taken at intervaLs for a 60-minute period. A0.5-mi. aliquot of the PCA-soluble fraction of eachsample was counted for its Br82 and C'4 activity inthe liquid scintillation counter. The remainder ofthe PCA-soluble fraction was counted for its Br8'activity in a well-type scintillation counter andthen passed through an anion exchange resincolumn. The column was eluted sequentially with92volumes each of water, N/i® HC1, and N/10HC1, and the radioactivity of the pooled eluate ofeach sample was determined.
Chart 92shows the Br82 activity in the eluates ofthe samples, expressed as per cent of the activity ofthe PCA-soluble blood fraction before resin treatment. It will be noted that the eluate containedabout 80 per cent of the radioactivity of the firstsample taken 1 minute after injection of theBUdR.Br82. The percentage recovery declinedrapidly during the first 10 minutes and thereafterat a slower rate.
Chart 3 shows the Br82 and C'4 activity of bloodsamples as determined by the liquid scintillationcounter. An early dip and a slight rebound elevation in the Br8' curve, and a fall in the C'4 curve
CHART 1.—Blood C'4 and Bra' activities after intravenousinjection of TdR-C14 and BUdR-Br― (two rats).
in a manner similar to that observed in the previous experiment, were noted (of. Chart 1). Accordlag to the information obtained from resin treatment of the corresponding PCA-soluble aliquot(cf. Chart 92),the Bra' activity, retainable or notretainable by resin, of each blood sample was calculated. During the first 5 minutes after injectiona rapid rise in Bres activity retainable by resin oc
curred, which thereafter approximated but wasslightly less than the total Br8' activity of theblood samples. The elutable Br8' activity fellrapidly for the first 10 minutes and more slowlythereafter. The rate of fall of elutable Br8' activitycorresponded closely to that observed for C'4activity.
Cn.@mr2.—Elutable Bra' activity of PCA-soluble bloodfractions after treatment with anion exchange resin, followingintravenous injection of BUdR-Br―. The radioactivity of theeluate samples is expressed as a percentage of that activitypresent before resin treatment.
0 br62 activity
Q Calculated @r82aetivity —r@tainabIeby resin
Calculated @6@activltynot retainable by resin
C@4octivity
to 20 30
Time in minutes
CHART 8.—Observed blood C'4 and Br@ activities, andcorresponding calculated Bra' activity, retainable and notretainable by anion exchange resin, after intravenous administration of TdR-C'4 and BUdR-Br@'.
Plasma samples and the PCA-soluble fractions The question arose whether the rate and/orof blood samples obtained at various intervals extent of degradation of brominated deoxyribosefrom 92to 60 minutes after the intravenous injec- nucleosides is related to their chemical structure,tion of doses of BUdR-Br82 of higher radioactivity specifically whether the presence of an amino(100-9200 ac.) were subjected to desceMing paper group on the C-4 position might increase the stabihchromatography, with the use of solvent systems ity of the molecule. An experiment was underof butanol :water :ammonia and aqueous iso- taken, therefore, to study the degradation ofpropanol :HC1' and standards of BUdR, bro- bromodeoxycytidine, BCCIR, which has such an
_________________________________ amino group. A mixture of BCdR-Br8' (30 tic., .47@@ —@----@---@—-@@ janole)andthymidine-C'4(10ac.,.64@mole)was
—::• injected intravenously into a rat prepared for- blood clearance study. Blood samples were taken
- at intervals up to 60 minutes and processed to
- determine Br8' activity in PCA-soluble fractions
- of the samples before and after resin treatment.
@ Chart 4 shows that the percentage recovery of_J Br8'activityintherespectiveresineluatesafter
@ BCdR-Br8' injection decreased very slowly and: did not reach a value below 70 per cent even after.
. 60 minutes. After BUdR injection the recovery of
activity fell to about 4 per cent during the same@ period (cf. Chart 92). Chart 5 shows the C'4 and.
@1Br8'activitiesofthebloodsamples,expressedasI I I@ I
50 60
C0
0:‘UC-UC
0@0
00
t@60
@ 40 -@ -
C
@ 20 -C-
C
>-‘ 0
>C
C-0
0@\
0 20 30 110Time in minutes
CHART 4.—Elutable Br― activity of PCA-soluble bloodfractions after treatment with anion exchange resin, followingintravenous injection of BCdR-Br@'. The radioactivity of thecleats samples is expressed as a percentage of that activitypresent before resin treatment.
mouracil, BCdR, bromocytosine, and bromideBr82. Two minutes after injection of BUdR-Br8'the plasma contained BUdR-Br82, bromide-Br82,and bromouracil-Br82 in the approximate propertions 92:92: 1, respectively. At 4 minutes post-injection, BUdR-Br82 and bromouracil-Br8' activitieshad declined to about 920and 14 per cent of the total plasma radioactivity, respectively. Bromouracil-Br82 was not detected in the samples obtained 6minutes or more after administration of the labeledBUdR. The bromide-Br8' component of the bloodsamples was removable by treatment of the sampiewith exchange resin. The Br82 activity retained bythe resin can be considered, therefore, to representbromide-Br82, and elutable Br82 activity representsBUdR and bromouracil. The results shown inChart 3 would thus indicate that a rapid degradation of part of the administered BUdR occurred.Cells being impermeable to bromide, the bromideBr8' liberated would mix within the extracellularfluid (15). A rapid extracellular distribution of Br@would account for the relative constancy of thevalues for the total and resin-retainable Br8'activity of the blood samples after 10 minutes.
20Timein minutes
CHART 5.—Blood C―and Bra' activities after intravenousinjection of TdR-C'4 and BCdR-Br@'.
per cent of the administered activity in the circulation. The rate of disappearance of C'4 activityfrom the blood was of a similar pattern as thatobserved in the previous experiments describedabove (cf. Charts 1, 8). However, the rate of disappearance of Br8' activity after the injection ofBCdR-Br@' was different from that observed proviously after the injection of BUCIR-Br82. Although
KRIss AND Ri@v@sz—Fate of BUdR and BUdil in vivo 9259
the initial values were in the same range, a continuous fall in total Br8' activity after BCdR-Br@administration was noted, as contrasted to theearly dip and rebound elevation in Br8' activityobserved with BUdR-Br82 (cf. Charts 1, 8). By theend of 60 minutes, only 8 per cent of the administered Br82 activity was in the circulationafter BCdR injection, whereas about 920per centremained circulating after the same period whenBUdR was given. The values for elutable Br8'activity were calculated from the values for totalBr8' activity and the percentage of Br8' activityelutable from resin and are also shown in Chart 5.These values are only slightly lower than those oftotal Br8' activity. The clearance curve of elutableBr8' differs from that of C's, in contrast to thedata obtained when BUd.R-Br8' and thymidine-C'4were administered (Chart 8).
Plasm@a samples and the PCA-soluble fractionsof blood samples obtained at intervals of from 92to60 minutes after the intravenous or intraperitonealinjection of doses of BCdR-Br8' of higher activity( 100 j@ic.) into other rats were subjected to descend
ing paper chromatography with the use of thesolvent systems and standards employed in theparallel experiments with BUdR-Br82. The samplesobtained within the first 92and 5 minutes afterintravenous and intraperitoneal injection, respectively, contained Br8' activity corresponding onlyto BCdR. At progressively later time intervals increasing amounts of Br8' activity were found in theposition corresponding to bromide. However, evenat the end of 60 minutes the major proportion ofthe total radioactivity was found to representBCdR. Bromocytosine or bromouracil radioactivity was not detected. As would be expected,bromide-Br8' activity of the sample was removableby resin treatment. Elutable Br8' activity wasfound to represent BCdR. The clearance curve ofelutable Br8' shown in Chart 5, therefore, mdicates the clearance curve of BCdR.
Di4itri&ution of Br8' activity in different ti@sueaafter administration of labeled BUdR and BCdR,—One hour after the intravenous administration ofan equimolar amount of BUCIR-Br82 and thymidine-C'4 to each of two rats (.9292@imolewith 92and .9292@molewith 1.4 ac., respectively), and afterserial blood samples were taken (cf. Chart 1), therats were killed, and the content and radioactivityof DNA were determined in portions of varyingsizes of the organs. The Br8' and C'4 activities inthe DNA of liver, kidney, spleen, testis, femoralmarrow, and of the mucosae of colon, intestine,and stomach of each animal were expressed as percent of the administered activity per rng. DNAand are shown in Chart 6. It will be noted that the
activity of C'4 and Br8' per mg. DNA varied considerably in the various tissues. Each activity wasgreatest in the colon and relatively high in intestine, stomach, bone marrow, and spleen. Relatively little activity was found in testis, liver, and kidney. With the exception of the kidney, the ratio ofC'4/Br8' concentrations in the DNA varied littleand had a mean value of 92.35 (Chart 6).
Another study was conducted to investigate thedistribution of radioactivity in the DNA of varioustissues of two rats 1 hour after the simultaneousadministration of BCCIR-Br82, TCIR-C14, and H@labeled deoxycytidine-5'-monophosphate (CdRP
CHART 6.—Distribution of C―and Brn activities in theDNA of various rat tissues 1 hour after the intravenousinjection of TdR-C'4 and BUdTh.Br@(two rats).
H'), each in amount of .692j.imole, with 100 zc.,4@ and 50 tic., respectively. The results inChart 7 show the C'4, Br82, and H@activities in percent/mg DNA in the various organs. Br8' concentration was highest in the marrow, less in the intestine, and, in contrast to that observed afterBUdR-Br8' administration, was relatively low inall the other organs. With the exception of a lowvalue for the spleen, the C14 concentrations of theother organs were of the same order of magnitudeas in a previous experiment (cf. Chart 6). H@concentrations, like those of Br82, were highest in themarrow, somewhat lower in intestine, but, in contrast to Br82, were also relatively high in the colonand stomach. In contrast to the relative constancyof the ratios of C'4/Br8' concentrations observedafter thymidine-C'4 and BUCIR-Br8' injection (cf.
I 76
Meant S.E. 2.35±15@ (e@@/@k/a
0246&1012 2Li6Bl0l2% of administered octivity/mc@DNAxIO2
Chart 6), the calculated C'4/Br8' ratios in this experirnent ranged between 1.1 (liver) and 928.6( stomach) . Furthermore, no consistent relation
ship was found between the C'4 and H@concentrations or between the H' and Br8' concentrations ofthe different organs.
Distribution of Br82 after injection of BUdR-Br8'to CSH mice.—In another experiment, each of 492
CHART 7.—Distribution of C'4, Bra', and H1 activities in the
DNA of various rat tissues 1 hour after the intravenous injection of TdR-C'4, BCdR-Br@', and CdRP-H' (two rats).
CSH mice was given injections intraperitoneallyof 92.7 @c.BUdR-Br'2. One hour after injection,and at various intervals thereafter up to 171hours, groups of four or five animals were killed,the organs excised, and the mean weight and meanradioactivity for each organ were calculated. Theradioactivity, expressed as per cent of the activityof the respective organ 1 hour after injection, isshown in Chart 8. It will be noted that the concentration of radioactivity varied considerably inthe different tissues. All organs showed a progressive loss of activity with time, the greatest lossoccurring in the spleen and intestine.
Role of the liver in degradation of BUdR.—Inorder to evaluate the possible role of the liver inthe in vivo degradation of BUCIR, a clearancestudy was done in a rat in which a porto-lumbarvein shunt and ligation of the coeliac artery hadbeen performed. Such a procedure, carried outaccording to the technic of Bernstein and Cheiker,abolishes the arterial and portal venous blood supply to the liver, and blood can reach the organ onlyby reflux through the hepatic vein (3). An intravenous injection of 925 zc. (.11 @mole)BUdR-Br@was given, and, subsequently, serial blood samples
were collected through a carotid cannula until theanimal's death 927minutes later. The PCA-soiublefraction of blood aliquots was counted before andafter treatment with anion exchange resin. Theradioactivity in the eluate was persistently greaterthan 60 per cent of the activity which was observed in the corresponding samples before resintreatment (Chart 9). The percentage of the intravenously administered activity recovered in theliver at the end of the experiment was less thanone-tenth of that percentage which is found in theliver of control animals with an intact hepaticblood supply.8
The data in the previous experiment suggestedthat the liver plays a major role in the degradationof BUdR. Another experiment was designed to testthe capacity of an isolated liver to accomplishthis degradation. With the apparatus and technic of Burton et a!. (4), the freshly excised liver
C>
0
4-
4-U00
C-
C-
CCC-0
CHART 8.—Bra' activity of organs of @SHmice at variousintervals after intraperitoneal injection of BUdR-Br@. Theorgan activities are expressed as per cent of the respectiveactivities 1 hour after injection. The activities at 1 hour, cxpressed as per cent of the administered dose/gm wet tissue,are shown in the insert. Each point represents the mean valuefrom four or five animals.
of a 310-gm. Long-Evans rat was continuouslyperfused with 30 ml. of hepariized blood obtainedfrom several animals of the same strain.@ After 90minutes of perfusion, BUdR-Br82, 60 sic. (1 jsrnole),in 92.7ml. saline, was added to the perfusing blood.During the next 60 minutes serial 100 X sampleswere taken from a reservoir, which collected blood
KRIsS AND Ri@vi@sz—Fate of BUd@ and BCdR in vivo 9261
both perfusing and bypassing the liver. During thesame period, the total bile produced by the liverwas collected separately. The Br82 activity in aliquots of the PCA-soluble fraction of the bloodsamples varied considerably during the first 10minutes, indicating that mixing of the administered BUdR with the blood was incomplete. Thereafter, the fluctuation of activity of the samples wassmall, suggesting that mixing was complete. Theper cent of Br8' activity recovered in the eluatesafter resin treatment of the PCA-soluble fractionsis illustrated in Chart 9. It can be seen that, duringthe first 10 minutes, while the BUCIR and bloodwere incompletely mixed, 60—90per cent of theactivity was recoverable in the eluate. Thereafter,the percentage recovery decreased at a rate slightly less than that observed to occur in the intactanimal (Chart 9). At the end of the experiment,the bile contained 1 per cent of the administeredBr8' activity, of which 84 per cent was retainedon the resin.
CHART 9.—Bra' activities in resin eluates of PCA-soluble
fractions of blood at various intervals after injection of BUdRBr@into a rat with a portolumbar vein shunt and ligation of thecoeliac artery, and after introducing BUdR-Br@' into the bloodperfusing an isolated rat liver. Analogous data of a previousexperiment performed in an animal with a normal hepaticblood supply are also shown.
Cellular conservation of Br82 activ@ityafter admini4ration of labeled BUdR and BCdR.—To dotermine to what extent BUdR-Br8' and BCdRBr8' incorporated into cells in vivo are conservedduring successive multiplications, an experimentwas designed with the use of ELD ascites tumorcells. Such cells have been shown to incorporateBr8' into DNA on incubation with BUCIR-Br― int'itro (19), and their multiplication can be deter
mined quantitatively in vivo (18). An intraperitoneal inoculum of 920X 10@ascites tumor cellswas given to each of three C57BL mice. Three
days later the animals were given injections intraperitoneally of equimolar (.4 j@mole) and equipotent (92.56 ic.) amounts of KBr-Br82, BUdRBr82, and BCCIR-Br82, respectively, dissolved in0.8 ml. saline. The peritoneal fluid of each animal
TABLE 1
RECOVERY OF BRn AcnvrrY FROM ASCITES TUMOR CELLs18 HouRs AFTER INJECTiON WITH KBR-BR@',
BUDR-BR―, AND BCDR.BRn
was harvested quantitatively 18 hours later, andthe peritoneal cavity rinsed several times with 0.9per cent saline. The volume of collected asciticfluid, together with corresponding rinsings of eachanimal, was measured, and the cell concentrationwas determined. An aliquot of the cell suspension
was used for radioactivity measurements. Another aliquot was subsequently inoculated intonew untreated hosts.
The total radioactivity of each of the cell suspensions was calculated from activity measure
ments in 92-ml. portions. These aliquots were subsequently centrifuged, the supernatant discarded,the cells resuspended in 0.9 per cent saline andrecentrifuged. The radioactivity of the washedcellular sediment was measured, and the radioactivity confined to the neoplastic cell populationof the respective animals calculated (Table 1,column 5). From this value and the specific activity of the bromine, the intracellular concentrationof brominated material was calculated (column 6).The concentration was about 50 per cent higherafter BCdR treatment than after treatment withBUdR.
Groups of three previously untreated C57BLmice were given inoculations intraperitoneally of92-ml. aliquots (containing 925 X 106 cells) ofthe suspension collected from the KBr@Br8t,BUdR-Br8'-, and BCdR-Br8@-treated animaLs, respectively. At various intervals up to 90 hoursafter inoculation, the ascitic fluid of one animal in
each group was collected quantitatively. The concentration and the radioactivity of the sedimentedcells were measured, and the total neoplastic cellpopulation and its radioactivity calculated foreach animal. The increase of the tumor cell population in the animals is shown as the number ofdoublings of the cells in the inoculum (Chart 10).The total cellular activity is expressed in per
I@ I
CHART 10.—Conservation of Bra' incorporated into ELDascites cells after exposure in vivo to BUdR-Br@ and BCdRBra, d@th@neoplastic cellular multiplication
centage of the activity contained in the inoculatedcells. It can be seen that the BUdR- and BCdRtreated cells collectively retained nearly all theirlabel during three and four cell generations, respectively.
Effect of x-radialion on the muliiplicaLion of ELDascite3 cells trealed with BUdR.—A group of tenCS7BL male mice were given inoculations intraperitoneally of 920X 10@ELD ascites cells. Beginning on the 3d day after inoculation, at 6-hourintervals, each of five mice received twelve intraperitoneal injections of 0.5 mg. BUdR dissolved in0.92 ml. of sterile saline. At the same intervals, thefive remaining animals of the group were used ascontrols and given injections intraperitoneally of0.92 ml. saline. Six days after the inoculation oftumor cells the animaLs were killed, and the asciticfluid of BUdR-treated and control mice was collected and pooled in separate flasks. The cell concentration in both pools was adjusted to 920X 10@cells per ml. by the addition of an appropriateamount of saline. The BUdR-treated and controlmaterials, in 8 ml. of diluted ascitic fluid, wereeach irradiated with 150 r, 350 r, and 700 r, respectively. Subsequently, 92 X 10@ unirradiated
cells and cells irradiated with the different doseswere inoculated intraperitoneally into respectivegroups, each consisting of sixteen C57BL malemice. At various intervals after inoculation thetumor cell number was determined quantitativelyin pairs of mice taken from each group. Theanimals were chosen at random at intervals up to8 days. After this period, only mice with ab.dominal distension were selected.
Chart 11 illustrates the in vivo growth curves.Unirradiated cells of either BUdR-treated or con@trol material grew similarly. However, irradiationwith 150 r and 350 r, respectively, inhibited themultiplication of BUdR-treated cells to a progressively greater extent than that of the controls.Irradiation with 700 r inhibited the progressivemultiplication of both kinds of cells.
Animals without abdominal distension totaled81 mice in all groups by the 16th day. The distribution of the mice in the different groups isshown in Table 92.Animals failed to develop tumors in greater number after inoculation of BUdR.'treated, x-radiated cells than did mice which received x-radiated control inocula. This difference
I I I
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10 20 30 40 50 60 70 80 90 I00@Time in hours
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CHART 11.—Growth curves of ELD ascites cells in rico,after their exposure to intraperitoneally administered BUdRand subsequent cellular irradiation in vitro.
without abdominal distension 16days after inoculation1103413
:Kniss AND Ri@v@sz—Fate of BUdR and BCdR in vivo 9263
is especially noticeable between the groups whichreceived 150 r or 300 r. The mice without appareattumors, together with six previously untreatedmice of the same strain, were then given inoculations of 920X 10@ascites cells which were collectedfrom mice used in the routine transplantation ofELD. Except for one mouse which was inoculatedwith BUdE-treated cells given 700 r, the reinoculated animals survived 14 days without any signof tumor growth. The six previously untreatedhosts succumbed to progressively growing tumorsbetween the 9th and 14th day.
DISCUSSIONThese results indicate that when bromodeoxy
Mridine (BUdR) is administered parenterally tothe rat, the compound undergoes a dual fate. Alarge part of the compound undergoes a rapid dogradation in the liver, whereas that which escapesdegradation diffuses into cells, where it may besubsequently incorporated into DNA. The degradation of BUdR in vivo appears, at least inpart, to involve the formation and subsequent dobromination of bromouracil. According to theamount of the bromide-Br8' remaining in thecirculation 1 hour after the administration ofBUdR-Br82, the extent of debromination must belarge and is estimated to be at least 80 per cent ofthe administered dose. That the liver plays amajor role in the degradation of BUdR is not surprising. This organ is known to be responsible forthe reductive catabolism of pyrimidines in animals(5, 10). Pohl et al. (9292)have obtained evidencethat, in the human, following administration of@5-bromouracil, there is extensive reduction of thepyrimidine ring with liberation of HBr and subsequent cleavage of the ring. Barrett and West (1)studied the in vivo dehalogenation of a number ofhalogenated compounds in the rat, and these authors postulated a metabolic reduction of the 5:6double bond with the formation of dihydropyrimidines with the elimination of HBr, with or without ring hydrolysis. In the case of both thymineand uracil, the reductive steps require reducedtriphosphopyridine nucleotide (TPNH) (5). Ofconsiderable interest is the observation that, whenthe catabolic capacity of the liver was exceeded bygiving injections of thymidine every hour for 5hours, the thymidylic kinase activity of liver andkidney was greatly increased (16). This observation suggests the possibility that a similar series ofinjections of thymidine to an animal might augment the incorporation into DNA of a subsequentinjection of BUdR.
The degradation of BUdR in vivo appears to beanalogous to the degradation observed after ad
ministration of the iodinated thymidine analog,5-iododeoxyuridine (IUdR). Within 924hours following the parenteral administration of IUdR-I'@1(and KI to prevent thyroidal accumulation of1131) to mice, rats, or humans, deiodination of the
major portion of the dose, as determined bymeasurement of urinary excretion of iodide-I's',has been reported by a number of investigators(9, 11, 13, 923). In contrast to iodide, bromide iseliminated from the body relatively slowly byurinary excretion, so that short-term measurementof the cumulative excretion of bromide would notbe a reliable indicator of the extent of degradationof BUd.R. A more accurate estimate of the quantityof BUdR degraded would depend only to a smallextent upon the measurement of urinary bromideand to a large extent on the measurement of the
TABLE@
FREQUENCY OF FAiLuRE To DEVELOP GRoss ASCITES PUMOR AFI@ERINJECTION OF X.RADIATED, BUDR.
TREATEDASCITESTUMORCsLisEach group consisted of sixteen animals
total amount of bromide in the extracellular space(independently measured) after a sufficient timehas elapsed for the anion to equilibrate in thebody.
Owing to the extensive degradation, only aminor part of the intravenously administeredBUdR dose is available for incorporation into thecells of various tissues. Once incorporated, BUdRwas shown to be retained by the cells of the ELDascites tumor in the course of four to five successive generations in vivo. Such a conservation ofBUdR confirms the expectation that it has beenincorporated into a metabolically stable cell constituent and is forwarded to daughter cells withcell division. In a previous study (927), with another ascites tumor labeled with adenine-8-C'4 orgiycine-92-C'4, an inverse relationship was foundbetween the total cell number and the specificactivity of the DNA. The close parallelism between the growth curve and the dilution of theradioactive label indicates a high metabolic stabil
ity of DNA, implying that no exchange of theDNA molecule takes place in resting cells and thateach cell division is correlated with the formationof a new DNA set per cell, without elimination orpartial replacement of the old.
If most of the administered BUdR is rapidlydestroyed before it can reach a tissue anatomicallyremote from the site of its injection, the resultingconcentration of BUdR in the DNA of such atissue may be too low to render the cells hypersensitive to x-rays. Such an explanation may account for the failure to induce sensitization of asubcutaneously transplanted mouse tumor, Hepatoma 134, to x-ray by daily intraperitoneal BUdRdoses of 9200mg/kg for 8 days (17), and the failureto induce increased sensitivity of mice to totalbody irradiation (921).
When multiple doses of BUdR were givenintraperitoneally over a 3-day period to micebearing the ELD ascites tumor, it was possible toshow that the subsequent intraperitoneal growthof BUdR-treated cells treated with 350 r was inhibited to a greater extent than that of salinetreated control cells. This indicates that increasedradiosensitivity of ascites tumor cells could beachieved if a sufficient local concentration ofBUdR was repeatedly made available to the neoplastic cells over a sufficiently long interval. Themice which did not develop tumors after the transplantation of irradiated cells were proved to berefractory also against viable cells at a subsequentchallenge. The development of such resistance is inconformity with previous observations on experimental systems in which, as in ours, immunogenetic differences are bound to exist betweentumor and host (924, 925). In such tumor-hostcombinations, irradiated cells bolster the homegraft reaction (926), the strength of which can besuperimposed upon the inherent radiosensitivityof the graft. Regression itself is, therefore, arather unreliable criterion of the radiosensitivityin such a case. The radiosensitivity of a hometransplanted tumor may be better reflected by thequantitative assessment of the multiplication of irradiated cells during the first few (1—10) daysfollowing transplantation, before host resistancedevelops.
The extensive degradation, before any sufficientcellular incorporation can occur, presents a majorobstacle in using BUdR for radiosensitization invivo. Our results indicate that the debrominationof bromodeoxycytidine (BCdR) proceedsat a comparatively slower rate than that of BTJdR. A considerable advantage of BCdR over BUdR as aradiosensitizing agent in vivo may be anticipatedfrom this finding and from the following observa
tions : (a) after intravenous injection of BCdRBr82, the radioactive label is detected in the DNAof a variety of tissues; (b) BCdR is incorporated,in the form of 5-bromo-92'-deoxyuridylic acid(BUdRP), into the DNA of nooplastic cells inplace of an equivalent amount of thymidine (7);(c) ascites tumor cells incubated with BCdR-Br@'incorporated Br8' in larger amounts than didascites cells incubated with an equimolar amountof BUdR-Br8' (Table 1) ; (d) the Br8' incorporatedby ascites cells after incubation with BCdR-Br8'was conserved by the cell population during atleast four cell generations in a way similar to thecellular retention of the label after BUdR-Br8'incorporation (Fig. 10) ; (e) BCdR is as effectiveas BUdR in inducing radiosensitization in hamstercells in vitro.9
After the simultaneous injection of equimolaramounts of thymidine-C'4 and BUdR-Br82, arather consistent ratio with a mean value of 92.35was observed in the C'4/Br8' concentrations in theDNA of different tissues which indicated, in conformity with observations in vitro, a preference forthymidine to BUdR incorporation (19). No consistent relationship was found between the concentration of the radioactive labels in the DNA ofdifferent organs when thymidine-C'4 and BCdRBr8' were administered in equimolar amounts. Inthis case the C'4/Br8' ratios varied widely between1 and 923.If deamination of BCLRP to BUdRP, reported by Cramer et a!. (7), is a necessary stepfor incorporation into DNA, the inconsistent C14:Br8' relationship may be interpreted as indicating the oxistence of considerable differences in the particulardeaminase activity of different organs. Accordingly,the relatively large Br8' concentration observed inthe DNA of bone marrow may be the result of aparticularly high deaminase activity in this tissue.Such a conclusion is supported by observationsreported by Maley and Maley (920) that the bonemarrow and thymus are the sole tissues of theadult rat which have an appreciable content ofdeaminase capable of converting deoxycytidylicacid (CdRP) to deoxyuridylic acid (UdRP). Thedisproportionately large H' concentration in bonemarrow DNA after CdRP-H@ administration maybe also explained by the deaminase activity of thisorgan, if one considers two metabolic pathwaysfor the introduction of the label. After phosphorylation CdRP-H@ may be incorporated as cytosineH' (6, 192). In the presence of an appropriatedeaminase, an additional amount of CdRP-H@may be doaminated to UdRP-H@, which is subsequently converted to thymidylic acid and incorporated as thymidine-H' (920).Deoxycytidylic deam
KRISS AND R@v@sz—Fate of BUdR and BCdR in vivo 9265
inaso has also been found in relatively largeamounts in embryonic and neoplastic tissue (920).It will be important, therefore, to investigate theability of malignant tissue to metabolize andutilize BCdR, since the selective concentration ofthis compound or its metabolic products in certaintissues may be of special practical importance inradiotherapy.
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