[CANCER RESEARCH 37, 610-618, February 1977J

SUMMARY

Nine patients with malignant disease were given i.v. injections of tnitiated thymidine, 0.2 mCi/kg, for tumor cell kinetics studies. Serial plasma, urine, saliva, and air vapor sampIes were collected variously for up to 79 days, and tnitiumactivity was measured. The initial half-life of plasma activitywas rapid. After 1 day, the activity decayed with a half-life of10.8 days, indicating equilibration of activity with the totalbody water. Urine activity was over 100 times the plasmaactivity within 1 hn, with equilibration approaching theplasma activity after 2 days, and then decayed at a similarmate.Saliva and air vapor activity increased to plasma levelsand then decayed at the same mateas did plasma activity. Inthe first 24 hn, approximately one-third of the total injectedactivity was excreted in the urine. During the first 12 daysthere were 54.2% urinary and 10.6% insensible losses. Maximum lasses determined by extrapolation of observed datawere 68% urinary and 19.5% insensible losses, or a total of87.5%. Approximately 7% of the injected activity may represent material initially incorporated into DNA but later metabolizedand excreted.

The radiation dose from total body water is estimated at0.69 mad.The estimated dose absorbed by cell nuclei fromincorporated material is a maximum of 20.5 rads. Theseradiation doses would not seem to contraindicate injectionof 0.2 mCi tnitiated thymidmne per kg to patients in thisclinical and experimental setting. Measurements of activityin personnel and room aim indicate that the use of suchdoses is not hazardous if appropriate precautions are fobbowed.

INTRODUCTION

The radioactive DNA precursor [3H]thymidmne4is rapidlyand selectively incorporated into DNA (7). The accumulation of activity in DNA is essentially complete within 1 hrfollowing the i.v. ami.p. injection of [3H]thymidmne into expemimental animals (36) and humans (33). [3H]Thymidmne

I Present address: Section of Medical Oncology, University Hospital,

Boston University Medical Center, 75 E. Newton Street, Boston, Mass. 02118.To whom requests for reprints should be addressed.

2 Present address: Department of Medicine, Barnes Hospital, St. Louis,

Mo.3 Consultant in Radiation Safety to the National Cancer Institute, Be

thesda, Md.4 The abbreviations used are: [3H]thymidine, [methyl-―Hjthymidins; LI,

labeling index; PLM, percentage labeled mitosis.Received April 5, 1976; accepted November 12, 1976.

hasa maximum 1@energyof18keV and an averagerangeinnuclear emulsions of 1 to 2 @m,making possible highresolution radioautographs of labeled cell nuclei (4, 7).These attributes have resulted in the widespread and effective use of [3H]thymidine in the study of normal and tumorcell kinetics in animals (17) and in humans (10).

Direct estimates of the radiation hazard due to tnitiumactivity in the body water or in the cell DNA have primarilybeen made inrodents(7,23,29,34,41,43),withextrapolation of the results to humans. Wade and Shaw (43) estimated from mice that a single p.o. ingestion of 1.0 mCi of[3H]thymidmneby man would be required to deliver a dose of1.25mads oven a periodof 13 weeks to the nucleiof asignificant fraction of cells in the total body.

In kinetic studies of patients with beukemias and solidtumors (2,5, 6, 15,21),i.v.doses of 10 to 40 mCi of[3H]thymidmnehave been necessary for adequate madioautographs. There has been, howver, only 1 report, to ourknowledge, in which radiation hazards were directly estimated from metabolic studies of [3H]thymidine in humans.Two patients received i.v. [3H]thymidine. Measurements oftnitium activity in plasma, urine, stool, and spinal fluid wereperformed . It was estimated that oven 50% of the injecteddose was incorporated into DNA (33).

In this report, frequent collections of various body fluidsfrom 9 patients with lung or head and neck cancer wereanalyzed for tnitium activity for up to 79 days following thei.v. administration of [3H]thymidine. Quantitative 24-hr urinecollections and frequent expired water vapor specimenspermitted an accurate accounting of excreted activity. Single on serial tumor samples were obtained after[3H]thymidmne injection and were processed far madioautographic cell kinetics studies. Based on the metabolic studies in conjunction with cell kinetics parameters, we haveattempted to define more specifically the radiohazard of[3H]thymidmne to the patient. Corollary data defining theradiohazard to personnel are included.

MATERIALS AND METHODS

Patient Selection. All patients in this study were afebnileand had advanced lungcanceron head and neck cancerwith normal renal and hepatic function and had tumor adcessible for cell kinetics investigations (Table 1). Patientsreceived an undiluted i.v. injection of [3H]thymidine, 0.2mCi/kg body weight, specific activity, 1.9 Ci/mmole; andsterile pyrogen-free aqueous solution 1 mCi/mi (Schwarz/

610 CANCER RESEARCH VOL. 37

The Uptake, Excretion, and Radiation Hazards of TritiatedThymidine in Humans

Marc J. Straus,1 Stephen E. Straus,2 Lewis Battiste,3 and Susan Krezoski

CellKineticsLaboratory,NationalCancerInstitute-VeteransAdministrationHospital,Washington,D. C. 20422(M. J. S., S. K.J,and the NationalInstituteofAllergyand InfectiousDiseases,Bethesda,Maryland20014(5. E. 5.1

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PatientprofileAgePatient(yr)SexWt

(kg)DiagnosisA.

A.52M60Epidermoid carcinoma of hypopharynx with metastasis tolymphnodesJ.

B.56M55Epidermoid carcinoma of lung,poorlydifferentiatedJ.

C.63M75Small cell carcinoma oflungH.D.76M83Adenocarcinoma metastatic to

lungK.H.51M62Small cell carcinoma oflungw.J.59M70Small cell carcinoma oflungC.P.42M68Large cell anaplastic carcinoma of

lung with metastasis to lymphnodes ofneckw.

S.66M70Epidermoid carcinomaof epiglottiswith metastasis to lymph nodesofneckP.

T.53M77Epidermoid carcinoma of buccalmucosa metastatic to skin offace andneckF.

V.49M65Epidermoid carcinoma of mouth,well differentiated

Metabolism and Dosimetry of Tritiated Thymidine

Table 1 [3H]thymidine. The door of the patient's room was closedaver 99% of the study period . Two portable Hankscraft coldwater humidifiers operated continuously starting 1 day befameinjection and delivered approximately 4 liters of watereach day. A Sears dehumidifier collected approximately 3liters of the roam vapor each day. The activity in triplicateabiquats of collected water was determined as above.

Cell Kinetics. Tumor samples were obtained by 1 of 4methods. In some cases, biopsies were performed underdirect visualization with flexible fibenoptic bnonchoscopy. Inother patients with accessible masses, the tumor was memoved by surgical biopsy, by skin punch, or by aspiration ofthe lesion through a 25-gauge needle. Solid tissue wasplaced in Baum's solution fan 24 hr and cleared with 70%ethanol. Tissue was sectioned onto gelatin-subbed slidesand avenstained with eosin . Aspirates were smeared directlyonto slides and aim dried. The slides were prepared forradioautography in a darkroom with controlled temperatureand humidity. The slides were dipped in NTB2 emulsiondiluted 1:1 with distilled water. These were dried and incubated in the dark for varying exposure durations and laterwere developed (Kodak D-19 developer) and fixed in Kodakfixer. Special procedures adapted for background analysisand grain counting will be described elsewhere.5

Analysis of radiaautography data involves the followingparameters (6, 7). The LI is defined as the fraction of apopulation of cells that incorporates detectable amounts ofradioactive label. The grain count distribution indicates thefrequency of cells with various grain counts. The graincount of a given cell is proportional to its DNA synthesismate.The PLM curve is the ratio of labeled mitotic cells tototal mitotic cells as a function oftime. PLM curves are usedto derive the time required for cells to traverse the phases ofthe cell cycle. The DNA-synthetic phase (S phase) (16) isdetermined from the PLM curve as the time between themidpoints ascending and descending the 1st wave of thelabeled mitasis curve. The generation time an mean cellcycle time (To) is defined as the time interval between theinitiation of 2 sequential mitases. T@is operationally determined by measuring the time between equivalent points ofsequential mitases in a PLM curve.

Radiation Dosimetry. The total body dose delivered duning a time interval t, to t2 is given as (18)

where

Mann, Orangebung, N. Y.). Isotope use was authorized bythe United States Food and Drug Administration, and specific informed consent was obtained from each subject.

Measurement of Activity in Plasma, Urine, Saliva, andAir Vapor. Serial blood samples were drawn from all patients and anticoagulated with EDTA. Plasma was collectedfollowing centnifugatian at 600 x g. Triplicate 0.1-mb plasmaaliquots were counted in vials containing 10 ml of scintillation cocktail [toluene, 1000 ml; 2-(4'-t-butylphenyl)-5-(4'-biphenyl)-1 ,3,4-oxadiazale, 8 g; 2-(4'-biphenyb)-6-phenylbenzoxazale, 0.5 g, (Fluoralboy TLA); and BBS-3 Bio-Sobv,10% v/w], Beckmann Instruments, Inc., Fullerton, Calif.Radioactivity was measured in the Beckmann LS-255 liquidscintillation system (Beckmann Instruments) with a counting efficiency of 40%. Quench correction was performed bythe external standards ratio method. Samples were countedto at least the 3% level of statistical accuracy. The results oftriplicate determinations were averaged and expressed as

@Ci/mb.Voided urine specimens from 8 patients were collected

fractionally for at least 2 days, and 24-hr specimens werepooled daily far at beast 12 days. Thereafter, intermittentvoided specimens were collected far up to 79 days. In 2incontinent patients, urine samples were collected by indwelling catheterization. The tnitium activity of triplicateurine aliquats were determined, as was plasma activity.

Serial saliva specimens were collected from 3 patients (D.S., W. S., F. V.). Triplicate 0.1-mbsamples were handled fordetermination of tnitium content as described above.

Samples of the exhaled air vapor of 1 patient (W. S.) werecollected serially. Expired moisture was obtained by directing exhaled air with a Rudolph's valve into a disposable testtube set in a dry ice trap. After a collection period of 15 mm,the test tube was removed from the trap, and the activity oftriplicate aliquots of the condensed water was determined,as described above.

Room water vapor activity was determined for Patient W.S. during the 11 days following the administration of

D, (rads) = F@ ‘q@.fr)dt

F (rads/day) = (A. E.CF)/(M .100 ergs g-1 rad1)

(A)

(B)

and 1 @.tCi= 3.2 x 10@disintegratians/day, A = activity atinitial time t (disintegratians/day), E = mean energy/disintegration = 5.7 x 10@eV, M = mass under consideration (g),CF = conversion factor = 1.6 x 1012 engs/eV, r = bialogical half-life of tnitium, and q@.(r)=

Equation A gives the body dose due to the presence oftnitium activity which is decaying with a half-life equivalent

5 M. J. Straus, S. E. Straus, and A. E. Moran. A Comprehensive Cytoki

netic Analysis of 5-180 Ascites Tumor Growth, in preparation.

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to that of the body water. Energy deposited within the cellnucleus due to the incorporation of activity into DNA isgiven by Equation B (4). For cell nuclei energy, deposition isassumed to be constant until cell division, at which timeenergy deposition persists at one-half the former rate.

Data Analysis. Statistical analysis and regression formulas were calculated with a Honeywell 516 computer timesharing system (DataComp, Inc., Silver Spring, Md.).

RESULTS

Uptake and Distribution of [3H]Thymidine

Plasma. Following the i.v. administration of[3H]thymidmne (0.2 mCi/kg), the plasma activity was determined. Maximum activity was observed within 2 to 3 mm inboth patients studied during the 1st mm postinjection(Chart 1, A and B). Equal distribution of the administeredactivity throughout the total body water, assuming an average of 55% of body weight as total body water fan malepatients over age 40 (12),would result in an expected activity of 0.354 @.tCi/ml. Plasma levels approaching 3 times this

activity were observed during the 1st 5 to 7 mm. This mdidates that uniform mixing of the injected dose throughoutthe total body water was not attained until after the 1stseveral mm. Initial clearance of plasma activity was veryrapid , with a half-life of 3 to 4 mm associated with equilibration of the dose with the body water. Thereafter, clearancewas much slower and approached a uniform rate of decayby the end of the 1stday.

Plasma activity was determined at intervals during 79days following the administration of [3H]thymidmneto 8 patients in 9 individual experiments. Representative observatians of plasma activity are plotted for 3 patients in Chart 2.A least-squares regression analysis (Chart 3 A) performed

E

0

>-I-..>I—04

I—

I-

on 216 data paints collected in the 9 experiments indicatesthat mean plasma activity may be expressed during the 1stday by Equation C, although we recognize that this is acomposite expression representing a series of distinct cornponents.

Plasmaactivity (Day1) = 0.22e°33' (C)

For subsequent days, plasma activity is described by EquationD.

Plasmaactivity (day > 1) = 0.16e084' (D)

The latter equation indicates that, after the 1st day, plasmaactivity decays with a half-life of 10.8 days.

. FV0 CP

a OS

0.001

4

U)4-Ja.z

1'

DAYSPOSTINJECTION[@H]TdR

Chart 2. Decay curves of tritium activity in plasma of 3 patients, F. v.,C. P., and D. S., for 70 daysfollowing injection of tritiated thymidins ([‘HjTdR).

B

100

Urine

10.@

1 0

\ Plasmabo@0_@.o---@--o---0-

01

w.S.-14mCi[3H]TdRl.V.DAY0

, J I@ I

10

10

01

001

0001 UUI20 40 60 80 100 120

DAYS POSTINJECTION [3H]TdRChart 1. Curves of tnitium activity in the plasma and urine of 2 patients, H. D. and W. S., in the 1st 2 hr following injection of tnitiated

thymidine ([3H]TdR).

612 CANCER RESEARCH VOL. 37

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@‘-

NNjo

I I I I I 1@@―;.). I

10 20 30 40 50 60 70

(E) DAYS POSTINJECTION (3H]TdR

Chart 5. Mean (±S.E.)cumulative percentage of total dose activity excreted in the urine in 9 patients with extrapolation to Day 60, based uponregression analysis. [3H]TdR, tritiated thymidine.

Insensible Losses. Additional loss of administered activity occurred insensibly in the form of expired water vapor,sweat, saliva, and stool. The water vapor activity in 30 airsamples collected from Patient W. S. in 2 experiments demonstrated the rapid appearance of activity in expired air.The concentration of the activity remained slightly lowerthan plasma activity but, after the 1St2 to 3 days, decayed atthe same rate (Chart 6A).

I0 20 30 40

Metabolism and Dosimetry of Tritiated Thymidine

10.000

1.000

0.100

0.010

U)

-JU-zE

0

>.I-.5F-0

I-a:H

za::@z

0:1.

>-H>F-0

:DF-a:F-

Urine activity (all days)

= 8.0e282' + 0.033e°47' + 0.17e°°64'

The cumulative renal excretion far the 1st 12 days wasdetermined by measuring the activity of the total 24-hr urineoutput collected. Mean cumulative urinary losses for 9 patients are plotted in Chart 5. Data points are fitted by integration of Equation E. A mean of 54.2 ±2.4% (S.E.) of thetotal administered dose was excreted in the urine in the 1st12 days.IntegrationofEquationE predictsthecumulativeurinary loss of 67.4% of the injected dose within 60 dayswith a theoretical limit of 68% cumulative loss. Mast of theinjected dose, then, is last by renal excretion.

50

wza:

zaUiI-Uia:0xUi

0:1.

>-F->I-04

I-a:I-

60 70 80

DAYSPOSTINJECTION(“H]TdR

DAYS POSTINJECTION (5H)TdRChart 4. Decay curves of tritium activity in urine of 3 patients, F. V.,

C. P. and D. S. , for 70 days following injection of tritiated thymidine [3H]-TdR.

LU

LL@@

LU

2wI— I-4 LU_J@:, 0

:D LU(_) >-

@ I-

cii+1 I—

C-)z << _jLU 4

@ I—0I-

B

o@@'TtT@@

DAYSPOSTINJECTION[‘HJTdR

Chart 3. A, curves of tritium activity decay in plasma fitted from all patients. Most of the data points in the 1st 24 hr were not included because of alack of sufficient space on the graph. B, curves of tritium activity decay inurine fitted from all patients. [3HJTdR,tritiated thymidine.

Urine. Within 2 to 3 mm after i.v. administration of[3H]thymidine, tnitium activity was detected in urine specimens (Chart 1, A and B). Urine activity rapidly increasedtoa concentration as high as 115times plasmaactivity withinthe 1st60 mm. Tnitiumactivityexcretedinthe urineup to79days postinjection was determined for 9 patients in 10 expeniments. Representativeobservations of urinary activityfan 3 patients are shown in Chart 4. The beast-squaresregression analysis of 224 data points from all patientsindicates that the urine activity may be described by Equation E (Chart 3B).

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‘IA instantaneous total body water activity predicted in Equa

bOO -t —BLOOD P ASMA tions C and D indicates that the minimum of 1 .4% of the

SALIVA L administered dose is lost insensibly during the 1st day,

‘ AIR VAPOR 10.6% loss during the 1st 12 days, and 19.5% ultimate loss.

@ 00@@ Total Excreted Activity. Total lasses, then, include those

\•\ acounted for by urinary and insensible excretion. During

@ the 1st 12 days, 54.2% urinary and 10.6% insensible losses

-.. . -..-. — .-. -. - . —. — — account for 64.8% of the administered dose. Integration of

0 JO@@ -@ —.- Equations C, D, and E predicts a total of 68% urinary and

. 19.5% insensible losses, am 87.5% of the initial dose.

w S -i4mCi (‘H)TdRI V. DAY0 It has been demonstrated that the incorporation of

. :@@@ I I I I I I@@ [3H]thymidmne into cell DNA predominantly occurs within

0.0.@ 2 3 4 5 6 7 8 9 0 II 2 the 1st several mm following i.v. administration and is es

[JAYS POSTINJECTION [3H]TdR sentially complete by the end of the 1st hr (33, 36). Residual

activity in the body water at the end of the 1st day is,therefore, unavailable for incorporation into DNA and willeventually be excreted. Table 2 presents the plasma activi

F.V.- l3mCl [3H]TdR IV. DAY 0 ties and cumulative urine losses in the study group at the

end of the 1st and 12th days. The total body water activity,as reflected by the mean plasma activity of 0.164 @Ci/mlafter the 1st day, represents a pool of 45.1% of the administered dose which will eventually be excreted. During the 1st

.—.—.— URINE day, 33.7% urinary and 1 .4% insensible losses were ob

— BLOOD PLASMA served . Therefore, the sum of the observed excretion during

SALIVA the 1st day and the eventual excretion of total body water

activity enable us to account for 80.2% of the injectedactivity. However, a total of 87.5% excretion was observed.

The difference between the 80.2 and 87.5% losses can beaccounted for by the available data. The lass of bodywater activity between Days 1 and 12 determined by thedecrease in mean plasma activity (Table 2) from 0.164 to0.080 @Ci/mlconstitutes 23.1% of the administered dose.Observed urinary and insensible losses for the same periodrepresent 29.6% of the injected dose (6.5% difference).Furthermore, projected urinary and insensible bosses foblowing Day 12 account for the excess lass of an additional______ 0.8%ofinjectedactivitybeyondthatanticipatedfromthetotal body water concentration. The activity balance would

DAYS POSTINJECTION [3HJTdR indicate that, particularly during the 1st 12 days, more tnt

0.0 10

0.005

Plasmaactivity and cumulativeurinelosses[3H]ThyPlasma activity (@Ci/Cumulative urine ac

midine administeredml)tivity

excreted(mCi)Patient(mCi)Day

1 Day 12Day 1 Day12R.

A.120.165 0.1014.835.82J.C.150.1730.0783.255.76H.

D.160.180 0.0995.769.13K.H.12NA― NA6.768.14C.P.140.120 0.0604.697.22D.S.90.100 0.0562.854.88w.S.―14

140.1080.074

0.122 0.0785.177.51

6.348.62PT.150.225NA3.73NAF.V.130.280

0.0992.485.80Mean

ac 0.1640.080tivity

0

I—

>

I-04

F-

F—

.@I

50 60 70

Chart 6. A, tritium activity in plasma, urine, saliva, and air vapor in apatient monitored 12 days; B, tritium activity in plasma, urine, and saliva in apatient monitored over 60 days. [3H]TdR, tritiated thymidine.

0.00I0 20 30 40

M. J. Straus et a!.

E,...

0

>-I->I—04

F-a:F-

Table 2

Seventy-one saliva (and/or sputum) specimens were abtamed from 3 patients at various times following[3H]thymidmneinjection. Tnitium activity in the saliva is plotted for 2 patients (Chart 6, A and B). The data demonstratethe equilibration of saliva activity with the total body waterduring the 1st day.

Pleural fluid was obtained by thomacocentesis from Patient D. S. Activity in the malignant effusion 14 days after iv.administration of [3H]thymidine was determined to be 0.050@.&i/mb,identical to the plasma activity of the same day.

Reasonable estimates of insensible losses can be madeassuming that such losses occur with an activity equal tothat of blood plasma, an assumption well supported by thecollective expired air, saliva, and pleural fluid data. Conservatively, a volume equivalent to 2.8% of the total bodywater (1078 mI/day for a 70-kg man) is lost insensibly eachday in a nonfebnibe individual. Integration of 2.8% of the

a NA, data not available.“Dose administered on 2 occasions.

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Metabolism and Dosimetry of Tritiated Thymidine

ium is being excreted than could be accounted for by theactivity present in total body water.

Incorporation of [3H]Thymidine into Cell DNA. Serialbiopsies taken at intervals following i.v. administration of[3H]thymidmne were analyzed by radiaautogmaphy. PLMcurves, LI's, and grain count distributions were obtained.Detailed analysis of these results will be the subject of afuture publication.6 Data obtained from aspirates of 3 tumorsites of 1 representativepatient (C. P.) with large cell anaplastic carcinoma of the lung are presented here to permitestimates of the incorporation of labeled DNA precursorsinto rapidly proliferating cell populations.

The LI of samples obtained by aspiration at 1, 4, 54, and150 hr after injection ranged between 6.9 and 11 .3%, with amean of approximately 10%. The mean grain count was thehighest at 1 hr pastinjection (36.9 grains/cell). The PLMcurve permitted calculation of an S-phaseduration of 20 hr,and a T@of 80 to 90 hr. These kinetic parameters are typicalof those observedin vivo fanhuman solid tumors (2, 5, 15).

The efficacy of our radioautagraphy technique was determined by comparison of the specific activity of extractedcell DNA to mean grain counts in experiments with Sarcoma180 ascites tumors in BALB/c x DBA2 F1 mice.5 The observed efficiency varied from 24 to 28%. Assuming a 25%efficiency for similarly prepared radioautagraphs of the human biopsy material, estimates of cellular incorporation ofIabeledthymidmnecan be made. Fanthe 1-hrsample (PatientC. P.), a mean of 36.9 grains/cell were counted following an8-day exposure, i.e., 1.3 x 10_2 dpm/celb. Estimating thetotal burden in Patient C. P. to be 0.5 kg (38), an LI of 10%6and an assumed mean cell weight of 10@ g, roughly 5 x1010 tumor cells are labeled.

The estimations of the uptake of [3H]thymidmne into thebone marrow and intestinal cells require several assumptions. The number of nucleated bone marrow cells in humans has been determined to be 2 x 10@ cells/kg bodyweight (11). The LI of erythroid and myeboid cells determined by in vitro incubation in [3H]thymidine is 20 to 25%(39). For a 70-kg man, approximately 3.0 x 10@'bone marmowcells would be labeled. It has been estimated that thereare approximately 6.8 x 10'°proliferating intestinal cryptcells in a 70-kg man (A. Hagemann, personal communication). Assuming an LI of 10% (25) far these cells, a total of3.8 x 10@@cells in the tumor, bone marrow, and intestinalcrypt cells are labeled. Therefore, 2.2 mCi are estimated tobe incorporated into the DNA of these cell populations. Ascalculated above, a maximum of 19.8% of the injected activity could have been incorporated into cell DNA. For a 70-kgman, this represents2.7 mCi.A large portion of the incarporated activity thus can be accounted for in tumor, bonemarrow, and gastrointestinal epithelium cells.

It is suggested that accountable losses of 87.5%,. whichwere in excess of the 80.2% available for lass from the bodywater (7.3% difference), are due to the breakdown of Iabeled DNA by cell turnover, particularly in the rapidly proliferating populations. The bone marrow and crypt cell poolsdo not change significantly in mass. With each crypt cell

6 M. J. Straus and R. E. Moran. Cell Cycle Parameters in Human Solid

Tumors, submitted for publication.

division, 1 daughter cell must either be sloughed off to belost in the stool or degraded with reuptake of activity intothe body water where it may be excreted on neutilizedthrough salvage pathways (7, 37). Similarly, division of Iabeled bone marrow cells may result in release of differentiated cells into the blood stream where they may remainprior to degradation for hours or months. There may also becell destruction with reutilization amexcretion of incorporated activity (13). Similar processes may coexist in tumorsdespite the gradual accrual of tumor mass (15). Transit ofthe cell cycle often implies death far a significant fraction ofthe proliferating cells. In general, the greater the growthfraction, i.e., the percentage of cells in the proliferativecompartment, the greater the death rate.

Of the cells in the proliferative cycle, the T@ordinarilymanges from 1 to 5 days. The T@of granubapoietic anderythrapaietic precursor cells is 1 to 2.5 days (9). The T@ofthe tumor in our Patient C. P. was less than 4 days. On thebasis of this kinetic parameter, many of the labeled cellswould be expected to replicate within 12 days. Hence, thisaccounts far the excretion of a significant fraction of the 2.2mCi estimated to be incorporated into the DNA of theserapidly proliferating populations.

Therefore, 7.3% of the initial dose is excreted within the1st 60 days, essentially infinite time in terms of the biobogicab half-life of water. Up to 19.8% of the activity is initiallyincorporated into cell DNA, but a substantial fraction of thisis last within the 1st several days because of continueddivision of the most rapidly proliferating cells. During thefollowing weeks, gradual cell turnover in the more slowlydividing cells results in the excretion of an additional smallcomponent of activity.

Radiation Delivered to the Body. The radiation dose received by patients administered i.v. [3H]thymidine is calcubatedin 2 parts, the dose from total body water and the dosefrom uptake in cell DNA. The dose to the total body duringthe 1st day is estimated rather conservatively, assuming thatall injected activity not incorporated into DNA irradiates thebody for the entire day. For a 70-kg man given 14 mCi, thepresence in the body water of 80.2% or 11.23 mCi of tnitiumwith a mean energy of 5.7 keV deposits 0.047 madin 1 day.After the 1st day, activity is lost from the total body waterwith a half-life of 10.8 days. Integrating Equation A from Day1, at which time the mean plasma concentration of tnitiumwas 0.164 @Ci/ml(Table 2), to infinite time, calculates thedose of the total body water to be 0.64 mad.The total dosefor all days is at mast 0.69 mad.

The dose to the cells is more difficult to determine andrequires additional assumptions. The mean nuclear volumeis 300 @m(30), the radius is 4.2 @m,and the nuclear densityis assumed to be 1.0. If the energy deposited within a sphereof this radius is approximately 72% of the total availableenergy (4), the dose to an individual cell nucleus would be4.1 rads/day (Equation B). If the entire incorporated activityis uniformly distmibute.d, with an activity of 1.2 x 102 dpm/cell, a total of 4.7 x 10@cells would be labeled, representing a nuclear mass of 141 g. This number of labeled cells,when comparing the previously estimated 3.8 x 10@'tumor,marrow, and intestinal cells, suggests that, as expected,most of the tnitium is incorporated into rapidly proliferating

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cells. The dose to this mass depends upon the rate at whichcell doubling causes the dilution of incorporated activity.Estimates can be made for some of the proliferating tissues.Assuming a T@of the proliferative cells of 1 to 5 days, asabove, with each division the loss of one-half of the daughten cells and, hence, the loss of one-half of the activityoccurs to retain constant mass so that between 8.2 and 41.0rads are ultimately deposited. The tumor mass of Patient C.P. with a T@of, at most, 90 hr increases in mass with eachdivision. Assuming no cell loss, atatal of 20.5 rads would bedeposited within the tumor mass.

The DNA synthetic time of bone marrow precursor cells isapproximately 13 hr (28), and the T@of 1 to 2.5 days (9)indicates that the radiation hazard to these cells is less.Furthermore, most of these cells go through only a fewdivisions, mature, and are removed from the population.The issue of radiation to the hematapoietic stem cell cannotbe resolved, since these cells have not been identified.

Radiation Exposure to Personnel. Estimates of radiationexposure to other patients and health care personnel havealso been made. No activity was detected in the blood orurine of medical, nursing, and technical personnel whoregularly associated with study group patients. Patientssharing rooms with the study group patients were cantinuously exposed to an atmosphere containing tnitiated water(HTO). Pinson and Langham (32) showed that, during suchexposure, activity is effectively taken up by inhalation andby direct absorption through the skin in equal amounts.Patient J. B., occupying the bed adjacent to Patient W. S.,was oliguric. The room door was kept closed aver 99% ofthe time. Plasma samples obtained from Patient J. B. duringthe 1st 2 days following the injection of 14 mCi of[3Hjthymidmne into Patient W. S. were found to contain amaximum of 1 x 10@ @Ci/ml,or a total body water contentof approximately 3.9 MCi. This amount is equivalent toroughly 0.5% of the total HTO activity lost in sweat and inexpired air by Patient W. S. Twenty-four-hr urine collectionsfrom Patient J. B. contained up to 2.4 x 10_2 MCi. Suchestimates are necessarily crude because of the inaccuracyof extrapolating few counts above background to total bodywater content.

Total uptake of tnitium by other patients and health camepersonnel was never above background counts and therefore did not approach the permissible levels for an unrestnicted area as outlined in Title 10, Code of Federal Aegulatians, Part 20 (10.CFA.20) (40). Insensible losses of HTOwere considerable, and an atmospheric activity exceedingpermissible bevels (5 x 106 @Ci/mlof air, 10.CFA.20, Appendix B) would have been generated had there been noventilation of the roam. Since the hospital ventilation system was operative, the exposure to HTO was minimal,namely, the plasma and urine activity of Patient J. B.

DISCUSSION

The iv. administration of a single dose of [3H]thymidmneresults in the rapid equilibration of activity with the totalbody water. Aubini et a!. have reported 2 components withhalf-lives of 0.2 and 1.0 mm which were attributed to therapid clearance of plasma activity into other body water

compartments (33). Our results indicate an initial half-life of3 to 4 mm, but sampling at very early points was inadequatefar a more definitive appraisal of the initial events followingdose administration.

Tnitiated thymidine is metabolized rapidly. The availabilityof activity for incorporation into DNAis limited to the 1st hrafter i.v. administration (33, 36). Aubini et al. (33) havedetermined that, after 10 mm, most body water activity ispresent as volatile (HTO) and nonvolatile (other than thymidine) components (33). Nonvolatile activity observed afterthe 1st hr is largely in organic metabolites of thymidineincluding thymine and /3-ammnoisobutymicacid (14). Following the 1st day, total body water activity decayed with a halflife of 10.8 days, indicating that the predominant farm ofactivity was in HTO. It is evident, however, that nonvolatileactivity continues to comprise a significant portion of totalactivity. HTO is excreted at the same concentration as itexists in the body water (3, 32, 35). The clearance of activityin urine in a higher concentration and with a mane rapidhalf-life than plasma activity is, therefore, attributed to anonvolatile component. Urine tnitium activity of up to 115times plasma activity indicates that the renal handling oflabeled nonvolatile metabolites of thymidine includes a gbmerular filtration and/or secretion but without extensivereabsorption.

Activity in expired air (HTO) was less than that of plasmafar the 1st 2 days. A delayed equilibration of tnitium activityin saliva samples with that of plasma activity was similarlynoted. Plasma and total body water compartments equilibrate rapidly. If the tnitium activity in plasma were solely inthe form of HTO, the expired water vapor and saliva concentratian would have been equivalent to plasma activity in amuch shorter period of time (3). It is suggested, therefore,that a substantial fraction of plasma activity is initially nonvolatile but that the proportion of this component diminishes during the 1st 2 days until activity is primarily in theform of HTO.

Most of the administered dose was excreted. Urinary andinsensible excretion during the 1st 12 days accounted for64.8% of the administered activity. The eventual loss wasprojected to be 87.5% of the initial dose. Cumulative urinarylosses for 21 and 24 days were examined by Lisca et a!. (27)for 2 patients given 0.1 mCi/kg. A total of 65 and 47.5% ofthe injected doses were excreted , in general agreementwith our observations.

As indicated, the excretion data also include the activityfrom the metabolic turnover of [3H]thymidine initially incorporated into DNA. Approximately 20% of the injected doseof [3H]thymidine is incorporated into cell DNA within the 1sthr after i.v. administration, and the remaining 80% of thedose is excreted as HTO and as nonvolatile metabolites ofthymidine. Of this 20%, it is suggested that 7.3% of theinitial dose activity incorporated into DNA was metabolizedand excreted during the study period. This would explainthe observation that the cumulative tritium excretion exceeded the loss of activity from the total body water.

Our result of the percentage incorporation of[3H]thymidine into DNA is substantially different from that ofRubini et a!. (33) wherein 2 terminal patients were administered i.v. [3H]thymidmne (0.14 to 0.31 mCi/kg). Plasma,

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Metabolism and Dosimetry of Tritiated Thymidine

urine, and stool activities were followed primarily duringthe 1st 2 hr after dose administration. Their estimates of incorporation of [3H]thymidmneinto cell DNA were based uponan analysis of volatile and nonvolatile components, ratherthan upon detailed balance studies. It was observed thatless than 50% of the injected activity was converted to HTOand nonvolatile metabolites of thymidine. They concludedthat at least 50% of the administered dose was retained invivo and was presumably incorporated into cell DNA.

These results are in accord with studies of [3H]thymidineincorporation in rodents. Wade and Shaw (43) observedthat, 24 hr after the p.o. ingestion of [3H]thymidmneby mice,51% of the initial dose remained in the body, with 8%present as nonvolatile activity. Kisieleski (22) found 6% of[3H]thymidmneactivity incorporated into DNA at 1 hr after i.p.injection. Lambert and Clifton (24) reported 14% of activityincorporated into DNA following i.p. injections of[3H]thymidmne into rats. The incorporation into DNA as exernplified by these reports is somewhat less than theamount observed here for humans. The route of administration could relate significantly to this difference.

The detailed balance studies reported here permit a moreaccurate appraisal of the biological hazard of [3H]thymidmnein humans than was previously possible. The radiation doseto the total body water of patients receiving i.v.[3Hjthymidmne, 0.2 mCi/kg, is roughly 0.69 mad.The calculated dose to the cell nucleus is a maximum of 4.1 rads/day.The total energy deposited in any given cell depends uponthe cell cycle time and the time to cell death. Rapidly prolifenating cells dilute the incorporated activity with each celldivision by distribution of equal parts of the parental gename to the daughter cells. Cells with prolonged cell cycletimes are far less likely to incorporate [3H]thymidine, butenergy deposition will continue as long as tnitium activity ispresent in those few cells that have incorporated label intotheir DNA. PeIc (31), however, observed the significant turnover of label, even when incorporated into the so-called“nondividing―tissues of the adult mouse, including brainand muscle, thereby limiting the potential dose. The mechanism of this was not apparent and could involve DNA repairmechanisms.

Similar estimates of the dose to the cell nucleus followingthe incorporation of [3H]thymidine have been reported previousby. Van de Aiet and Shaw (41) calculated that 8.3 @Ciadministered p.o. to mice cause the average cumulativedose delivery of 4 to 10 rads in 8 days. Wade and Shaw (43)reported a 12-madcumulative dose to the cell nucleus inmice that were fed [3H]thymidmne,0.4 mCi/kg.

The relative biological effectiveness of tnitium /3decay istaken as 1.0, indicating that radiation from intranuclean[3H]thymidmne is no more efficient than X-imradiation in producing biological effect (4). At the cellular bevel, irradiationby @3decay of tnitium causes strand breaks in DNA. As withX-imradiation, most of these breaks are repaired rapidly, sothat genetic lesions are inefficiently produced (8). Johnsonand Cronkite (19) evaluated the effect of [3H]thymidmne onmouse spermatocyte count and concluded that doses of upto 1 mCi/kg body weight were comparable to 2 rads/day of yirradiation. Vennart (42) estimated that, in rodents, 1 to 5mCi of [3Hjthymidine per kg are equivalent to 30 nadswhole

body X-imradiation. In tissue culture cells, killing is observed(dose that effectively destroys 37% of the cells) at an Xirradiation dose of 200 to 750 nads on when grown in thepresence of 0.07 to 0.21 @Ciof [3Hjthymidmneper ml, whichyielded a mean grain count of oven 100 times that observedin Patient C. P. (assuming madioautograph exposure of 8days) (4). In our studies in Sarcoma 180 ascites tumor, 0.8mCi of [3H]thymidmne per kg had no effect on tumor cellcount, compared with controls.5

It is unlikely that toxic effects of 0.2 mCi/kg, as used here,would be observable at the organism level . Baserga et a!. (1)and Lisco et al. (26) observed a significant increase in tumorrate in both young and old mice receiving [3Hjthymidine, 1mCi/kg. Johnson and Cronkite (20), however, have observed no significant increase in either tumor incidence orin mortality rate in mice given 1 or 5 mCi of [3H]thymidmneper kg. The madiobiobogical effects of incorporated[3H]thymidmne have been extensively reviewed far in vitroand animal models (4, 29). Few reports, however, have dealtwith human studies. Lisca et a!. (27) failed to detect chromasamal abnormalities in the lymphocytes of 4 patientswho received [3H]thymidine, 0.1 mCi/kg.

The radiation dose to individuals exposed to the patientstudy group was determined by urine and plasma collections from other patients and personnel. Activity was barelydetectable in only 1 obiguric patient shaming a room with apatient given tnitium. In all other cases, there were no measunable levels of activity. Unpermissible levels of exposurecould not have occurred by any manner of contact withlabeled patients except by accidental ingestion or injectionof labeled material.

We have demonstrated that several. factors limit the radiation dose in a person given injections of [3H]thymidine tobevelsonly a few times greaten than those commonly used indiagnostic radiology. First, most of the dose is rapidly excreted. Second, only 20% of i.v. [3H]thymidine is incomporated into DNA. Third, most of the activity resides in rapidlyproliferating tissues which, because of cell division anddeath, results in limited energy deposition. Fourth, prolonged retention of activity would be expected for only arare, mature, slowly dividing cell, thus allowing only a minimal chance of long-term somatic effects.

In our opinion, the potential importance of delineatingcell kinetic parameters in patients with cancers justifies theuse of the doses of [3H@thymidinewe have administered.The hazard of the [3H]thymidmne may be marginal at most.These kinetic data may provide insight into more effectiveand national uses of therapeutic modalities which are usually highly toxic. It is not our thesis, however, that thepresent data necessarily justify the use of these doses of[3H]thymidmne in other experimental situations. It is hopedthat these data can provide a mane reasonable foundationupon which appropriate determinations can be made ineach given experimental situation.

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1977;37:610-618. Cancer Res   Marc J. Straus, Stephen E. Straus, Lewis Battiste, et al.   Thymidine in HumansThe Uptake, Excretion, and Radiation Hazards of Tritiated

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