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rNT.J. RADTAT. BroL.,1976,vol-. 30, No. 5, 409-+17 +3 Recovery response of dividing cells in the thymus of whole-body gamma -furadiated mic e D. SUCIV, Z.URAY and M. MANIU Oncological Institute, 3400 Cluj-Napoca, Romania (Receiaed14 June 1,976;accepted25 August 1976) Mice were irradiated with different doses of gamma-rays 30 min after the administration of s2P-orthophosphate. The dose-response curves determined at 72 hours after exposure showed an inflection point in the total activity present in the DNA in thymus and spleen. In the low dose-range, the dose-response (n:2'5) for the curves have D6:55 rad (n:2'5) for thyrnus and Do:95rud spleen. Thirty minutes after the administration of 32P-orthophosphate, the dividing cells from thymus were partially synchronized by the administration of 80 mg per kg body-weight hydroxyurea. At different time-intervals, the mice were irradiated with 80 rad, and the total activity of DNA was determined at A significant maximum of recovery was found 72 hours after synchronization. at 5 hours (S phase) after the administration of hydroxyurea. In similar conditions, the dose-response curves corresponding to the Gr, S and M phase of the division cycle were also determined. The synchronization of dividing cells induced by hydroxyurea failed in the spleen. 1. Introduction The recovery of dividing cells from radiation injury has been demonstrated in different cellular systems (Elkind and Whitmore 1967), including bonemarrow (McCulloch and TilI1962, 196+)and splenic lymphocytes (Okada 1970). However, in early investigations, it was found that thymic lymphocytes were unable to recoverfrom radiation damage(Jackson,Christensenand Bistline 1'969, ]ackson and and Christensen 1,972). It was concluded that the mechanism responsiblefor thymus cell-death is radiation-induced interphase death followed by cell lysis (Jackson, Christensen and Forkey 1968, Okada t970). More recently, from dose-fractionation studies, it was suggestedthat the mitoticallyactive cells from thymus can recover from radiation injury (Suciu, Uray and Abraham 1975). As an extensionof this study, the present work was undertaken to determine the effect of whole-body irradiation on the dividing cells from thymus and spleen, previously labelled by the administration of 3zP-orthophosphate. The retention of 32P-DNA in the thymus and spleen was taken as a measure of both the activity of DNA synthesis and the loss of dividing cells. The radiation response of dividing cells from thymus and spleen was also investigated after partial synchrony induced by hydroxyurea, an S-killing and Gr/S blocking agent (Brown 1975). The results support the assumption that the mitotically-active cells from thymus and spleen are capable of recovery from radiation injury without the loss of reproductiveintegrity. 2. Materials and methods 2.1. Radioactiaelabelling Male mice of the NN{RI strain weighing 28 t2 g were injected intraperitoneally with 25 pCi of 32P-orthophosphate(6 Ci per mmole, IFA 4i0 D. Suciu et al. Bucharest,Romania) or 25 yCi of 3H-thymidine.(5 Ci per mmole, Amersham, England). 2.2. Irradiation conditions Mice were whole-body irradiated in plastic cagesby a 60Cotherapeutic unit (Theratron 80) (FSD:80 cm, 100 rad per min). The dose-ratewas measured with a Siemens universal dosimeter. The irradiated and control mice were given a standard diet. 2.3. DNA determination Thymus and spleenwere homogenizedin cold 0.1+ N NaCl with a PotterElvehjem homogenizer. The homogenatewas treated with a solution of KOH to a final concentrationof 0'3 N, and the sampleswere incubated for 24 hours at 37"C. Perchloric acid was added-to-a final'eoncentration'of 0.2 I{, and the samples were centrifuged for 10 min at 1400x g. The supernatant fluid ri'as discarded;and the sedimentwas washed in0'2N HCIO4 and centrifugedfor 10 min at 1400x g. The sedimentwas hydrolysedin 0.2 N HCIO4 for 15 min at 100'C. Fractions of hydrolysate were used for the diphenylamine colour reaction (De France and Le Pecq 196l) and the determination of radioactivity. 2.4. Radioaetiztemeasurement Fractions of hydrolysatewere dried on filter-paper discs, and the s2P-radioactivity was counted with a mica end-window Geiger-Muller counter (Typ. YAZ) connected to a VA-M-16D Vakutronik counting system. The 3H-thymidine radioactivity was counted in a liquid scintillation spectrometer (Betaszint BF-5003), as previously described(Suciu et al. 1975). From these measurements,the total activity (".p.-. per DNA per organ) and specific activity (..p.-. per mg DNA) of DNA in thymus and spleenwere determined. The valuesgiven in figures are means of the results obtained from 8 to 16 mice ( t SEM). Two hours after the administration of 25 p.Ci per mouse 3H-thymidinethe specificactivity was1.6'2+1.1mpcCiper mg DNA in thymus and 54'0 t 3'6 mpcCiper mg DNA in the spleen. In similar conditions,after the administration of 3zP-orthophosphatethe specific activity in thymus was 4'l t 0'3 mpcCiper mg DNA and 7'2 t0'7 m1.cCiper mg DNA in the spleen. These values were obtained after correction of the quenching effect. 3. Results 3.1. DNA labelling After the administrationof 3H-thymidine or s2P-orthophosphate, the specific activity of DNA was determinedat different time-intervalsin thymus and spleen (figure 1). As has previously been shown (Nygaard and Potter 1959, Pierucci 1967),the incorporation of labelled thymidine into thymus and spleenis almost complete within 2 hours after injection. In the following 6 days, a continuous decreaseof the specific activity of DNA can be observedin both thymus and spleen (figure 1) (Nygaard and Potter 1960, Sanders,Dalrymple and Robinette 196+). It was assumed that reincorporation of labelled thymidine released through breakdown of cells is negligible (Nygaard and Potter 1960). Thus, the loss of labelled DNA within the period of severaldays after labelling has been equatedwith the loss of labelled cells from the tissue(Nygaard and Potter 1960, Responseofdiz.idingcellstnuhole-ooa|'guff.nlu-.'tL.u.uoww''.U"- t I I rso \ N Q q Q q f, roa U t F \q S, tq 5 0 t00 ts ;e o . f 2 5 4 5 6 DPYS 0 . r 2 5 4 5 6 DNYS specific activity of DNA in tl Figure 1. Time-course of the 3H-thymidine 32P-ort of .(tl :t^ after admi"it""iit" in $ 2'3' (o) analysis *u."p"tfotmed as described expressed as p Thannhauser^(19a5) ( x )' Results 14 per cent' of tnt label' SBV' was less than admi"i;;;;i;" after hours 2 at values d'erset al' 1964'Suciu et aI' Gerber, Gerber, Altman ar lg75). However, the ass negligible in thYmu* "1q of t'ut-tudR-labelled DN 1975 (no* and Prusofi 1965,Myers and Feinendegen t'p-pNA increasesup to 2 days 32Pinto :;;"ifi; activity og oiio continuousincorporationof in th" principl., In 1). (figure label \ L \ R (J q \,, R R s I 200 300 too DOSE t nod t 4m +12 D. Suciu et al. DNA can be comparedwith the in aitro method of continuouslabelling by the addition of labelledthymidine into the culture medium (Mak and Till 1963, Yamadaand Puck 1961). As the purpose of this study is to investigatethe radiation-recoveryresponseof dividing cells in thymus and spleen,we used the 3zP-orthophosphate-labelling of DNA as a measureof both the continuous activity of DNA synthesisand the loss of previously-labelled cells (figures2,3, 5 and 6). However, further work is necessaryto establishthe mechanismof continuouslabelling of DNA after the in vioo administrationof 8zP-orthophosphate. \ s80 Y N t 6 0 *R 40 P N z o 200 400 { mg/kg HYDRoYYUREF, Figure 3. Total activity of DNA administration of different administered 30 min before percentage of total activity of sao 600 tooo .t200 body werghf ) in thymus (o) and spleen (o) at 24 hours after 3zP-orthophosphate was doses of hydroxyurea. injection of hydroxyurea. Results expressed as DNA at 24 hours after administration of the label. I \ N (, 8 0 .L--r--f\ c - I ! t 6 0 s rT -'k' ' . , I- ' $ 4 0 8 01 HOURS \ a 3 EF:FER s 7 I ADHNISTRAflOH Figure 4. Specific activity of DNA in thymus intervals after administration of 80 mg per were killed 30 min after administration of as percentage of specific activity values administration of the label. lt 13 t5 /7 OF HYDROXYUPEN (o) and spleen ( x ) at different timekg body-weight hydroxyurea. Animals 32P-orthophosphate. Results expressed of untreated controls at 30 min af,ter Responseof diz'iding cells in whole-bodygamma-irradiated mice +t3 I \ 7 5 N (, q { 5 4 s P N 2 s 2 44 6 8 HOURS AFTER NDHN ISTRNTION OF HYDROXYUREA Figure 5. Total activity of DNA in thymus (o) and spleen (o) at 72 hours after a2P-orthophosphate administration of 80 mg per kg body-weight hydroxyurea. was administered 30 min before injection of hydroxyurea. At differerrt ti-"intervals the animals received 80 rad. Control group was irradiated with 80 rad at 30 min after administration of the label. Results eipressed as percentage of total activity DNA in the control group. \ L i- R (, q s P * so ";:? , ::3 , 'oo Figure 6. Dose-response curves of the total activity of DNA in thymus. szp-orthophosphate was administered 30 min before injection of 80 -g p", kg body-weight hydroxyurea. Animals were given different doses of radiation at t hour (.) (dr), 5 hours (o) (S) and 6 hours ( x ) (M) after administration of hydroxyurea. Control group was unirradiated. Mice were killed 72 hours after administration of hydroxyurea. Results expressed as percentage of total activity of DNA in the control group. SEM was less than 8 per cent. 3.2. Dose-response curoes Mice were whole-body irradiated with different doses 30 min after the administrationof 32P-orthophosphate. The total activity of DNA wasdetermined in thymus and spleen 72 hours after irradiation. The dose-responsecurves have a shoulderin the low dose-rangecorrespondingto so and i^ (figure 2). The inflectionpoint (Elkind and Whitmore 1967)indicatesthat a fractioi of the thymus and spleenpopulation is representedby cells with an increasedradioresistancein the high dose-range(curve TB and ,SB from figure 2). The +t+ D. Suciuet al. parametersof the dose-responsecurves are included in the table. The survival parameters of spleen cells as determined by other methods ur, 5o from 47 to 78 rad and n from 1'8 to 3'3 (Okada 1970). rI I bs,ralue (rad) Tt Tn Sl Ss Extrapolation number fr' f) 3o0t 95 2sot significance of Ta, ?n, se and ss is given in figure 2 and $ 3.2. t Determined by extrapolation (figure 2)' Parametersof the dose-responsecurves shown in figure 2' 3.3. Effect of hydroxyureaon the dioidingcellsfrom thymusand spleen fft! a.pietion of szP-DNA in thymus and spleenat 24 hours after administration of different doses of hydroxyurea is shown in figure 3. It was demonstratedthat the S-killing effect of hydroxyureais common for all cell systems with high rates of cellular proliferation (Sinclair,1965, Philips, Sternbetg,Schwartz, Cronin, Sodergrenand Vidal 1'967,Whithers 1975). As the utility of hydroxyureain this investigationis only to induce the synchrony of dividing celis, we decidedto limit the dose of drug to 80 mg per kg bodyweight (fi-gures+-6). The effect of this dose on the incorporation of s2P-lorthophosphate into the thymus and spleen DNA is shown in figure 4. The pulsl labelling experiment (figure 4) suggeststhe G, block induced by hydroxyurea,as deierminedby the decreaseof DNA synthesisfew hous after administrationof the drug. 3.4. Radiosensitizationof diaiding cells in thymus and spleenafter administration of hydroxyurea Mice-were given 80 mg per kg body-weight hydroxyurea30 min after the administrationof 3zP-orthophosphate.At different time-intervalsthe animals were irradiated with 80 rad (figure 5). The total activity of DNA was determinedin thymus and spleen^t 72 hours aftersynchronization. If irradiation was carried out at 5 hours after administrationof hydroxyureathe radioactivity of thymus DNA was significantlyincreased(p <0'05) comparedrvith the vaiue detlrmined for the animals irradiated 6 hours after synchronizationThe f,ecoverymaximum (figure 5) correspondsto the maximum of DNA synthesis(figure 4). The ,..orr.ty effectwas absentin the spleen(figure 5). Accordingly' DNA synthesiswas dlcreasedfor a relativelylong period after the administrationof (figure 4). hydroxyurea curves The precedingdata allowedthe determinationof the dose-response szP-orthophosphate 30_minbeforethe shownin figure 6. The animalsreceived administration of 80 mg per kg body-weight hydroxyurea. Irradiation was carriedout at I hour, 5 hoursand 6 hoursafter the partial synchronization. The gamma-irradiatedmice of ditiding cellsin whole-body Response +15 total activity of thymus DNA was determined72 hours after the administration of hydroxyurea. As the meancell-cycletime for dividing thymocyteswasfound to be in the rangeof 6 to 8 hours (Metcalf and Wiadrowski 1966),it is probable that the time of irradiation correspondsto the Gt (1 hour), S (5 hours) and G, and M phases(6 hours) of the division cycle. The Gt curve (figure 6) was exponentiallyrelated to dose, indicating that no recoveryoccurs for this cell population(Do:115 rad). For the S curvewe found Do:85 rad and n:l'2. lf irradiationwasperformedat 6 hours(G, and M phase)afterthe administration of hydroxyurea,about 50 per cent of the total activity of DNA was lost at a dose of 40 rad. K >' 4. Discussion The data reported in this work are based on the assumption that the DNA in the thymus and spleenof irradiated depletionof the previously-labelled animalscorrespondsto the loss of cellswith labelledDNA (Nygaardand Potter 1960, Sanderset al. 196+,Gerber et al. 1963,Suciu et al. 1975). The thymic populationis representedby the mitotically-activemedium and large thymocytesand the non-dividing small thymocytes(Borum 1973,Metcalf and Wiadrowski 1966). The mean cell-cycletime of dividing thymocyteswas estimatedto be in the range of 6 to 8 hours. The dividing fraction of cells representsabout 10 per centfrom the wholelymphoid population. The rnedium and large thymocytes are continuously transformed into non-dividing small thymocytes. The processof cell renewalof the small size cells requiresabout is continuously 3 days(Metcalf and Wiadrowski 1,966). As 3zP-orthophosphate incorporated(figure 1) but only into the DNA of the dividing fraction of cells (Ord and Stocken1956),the labellingof DNA in the whole populationrequires about 2 or 3 days. The incorporationof labelledprecursorsinto the DNA of thymus cellsby other routesthan cell divisionis improbable(Borum 1973). The dose-responsecurves determined at 72 hours after irradiation (figure 2 and table) support the assumptionthat in the low dose-rangethe dividing cellsfrom thymus are capableof recoveryfrom radiation injury. Due to the short cellcycletime, it is probablethat 72 hours after irradiation,when the processof cell the remainingfractionof depletionis terminated(Jacksonand Christensen1,972), thymic cellsrepresentsurvivingcells(Fabrikant 1972). The experimentsshown in figure 4 suggestthat the Gt/S blocking effect of 80 mg per kg body-u'eight hydroxyureais limited to about 3 hours. Then, the DNA synthesisincreases and the radiation recoveryresponseis clearly evident (figures5 and 6). The low dose-rangeof the dose-responsecurves (figure 6) correspondingto a representativefraction of in ztioo synchronizedthymus cells indicates that the S phaseof the division cycleis the most resistantto radiation. These findings are in agreementwith previousobservationson the radiosensitivityand recovery response of other mammalian cell systems after synchrony induced by hydroxyurea(Brown 1,975). In the splenic lymphoid population both the large and small size cells are capableof division (Fliedner,Kesse,Cronkiteand Robertson1964,Hanna 796+, Post and Hoffman 1970). On the other hand, it was suggestedthat the spleen lymphoid cells consist of at least two fractions with different levels of radiosensitivity(Nygaard and Potter 1960),Gerber and Altman 1970, Raff 1973, Streffer 197+),and probable different cell-cycletimes (Fliedner et al. 1,96+. +r6 D. Suciu et al. Hanna 1.964, Post and Hoffman L970). It is therefore conceivable that the attempt to synchronizethe population of dividing cells failed (figures 4 and 5). The radiation-recovery response of the lymphoid population from spleen is represented by the low dose-range of the dose-responsecurve reported in figure 2. It may be assumedthat for both thymus and spleen the high doserange of the dose-response curves (figure 2) reflects the radiation response of some non-lymphoid cellular systems. In conclusion,our data support the view that the thymic and splenic dividing cells are capable of recovery from radiation injury. However, as the recovery processwas studied almost exclusivelyin cell-survival experiments (Elkind and Whitmore 1967, Okada 1,970)our results appear as indirect evidences. ' AcKNowLEDGMENT We are indebted to Mr. C. Donogany for valuable technical assistance. Les souris ont 6t6 irradides avec des doses diff6rentes, 30 minutes aprds l'administration du 32P-orthophosphate. Conform6ment aux analyses faites 72 heures aprds l'exposition, les courbes dose-r6ponse de l'activit6 totale de I'ADN dans le thymus et la rate pr6sentaient un point d'inflexion. Dans le domaine des doses basses les courbes dose-rdponse ont D0:55 rad (n:2,5) pour le thymus et Do:95 rad (n:2,5) pour la rate. Dans une autre exp6rience, 30 min aprds l'administration du 3zP-orthophosphate, les cellules en division ont 6tE partiellement synchronis6es par I'administration de 80 mg per kg corps. A des intervalles de temps diff6rents les souris ont 6t6 irradides avec hydroxyur6e. 80 rad et 72 heures aprds la synchronisation on a determin6 I'activit6 totale de I'ADN. On ) trouv6 un maximum de r6tablissement 5 heures (phase S) aprds I'administration de Dans des conditions similaires, on a d6termin6 aussi les courbes doseI'hydroxyur6e. r6ponse correspondant d la phase Gr, ,S et M du cycle cellulaire. La synchronisation des cellules en division par la hydroxyurlert'a pas eu lieu dans la rate. Miiuse wurden 30 min nach Verabreichung von 3zP-Ortophosphat mit verschiedenen Dosen bestrahlt. 72 Stunden nach der Bestrahlung konnte festgestellt werden, dass die Dosis-Effekt-Kurven der DNS-Gesamtaktivitet in Thymus und Milz einen Inflexionspunkt aufweisen. Im Bereich der niedrigen Dosen zeigen die Dosis-Effekt-Kurven Do:55 rad In einem anderen Experiment (n:2,5) fiir Thymus und Do:95 rad (n:2,5) fiir Milz. wurden 30 min nach der Verabreichung von 82P-Ortophosphat die sich teilenden Zellen Kcirpergewicht partiell durch Gaben von 80 mg Hydroxyharnstoff/kg aus Thymus synchronisiert. In verschiedenen Zeitabstdnden wurden die Mhuse mit 80 rad bestrahlt vnd 72 Stunden nach der Synchronisierung die Gesamtaktivitiit der DNS bestimmt. Ein bedeutsames Erholungsmaximum bei 5 Stunden (Phase S) nach der Verabreichung von Hydrohyharnstoff wurde gefunden. IJnter gleichartigen Bedingungen wurden auch In der die Dosis-Effekt-kurven, fiir die Phasen Gr, S und M des Zellzyklus bestimmt. Milz fand die Synchronisierung durch Hydroxyharnstoff der sich teilenden Zellen nicht statt. RBrBnnNcps Bonuvt, K,7973, CeIl Tissuel{inet.,6, 545. BnowN, J. M., 7975,Radiology,114,789. Path.8io1.,9,2347. Dp FneNcr,P., and Le Pecq,J. 8., 1,961, Er-xrNo, M. M., and WrutMoRE, G. F., 7967, The Radiobiologyof Cultured Mammalian Cells (New York, London, Paris: Gordon & Breach, SciencePublishers),p.7. FesnrKANt,J. I., 7972,Radiobiology(Chicago:Medical Publisher Inc.), p.75. FrrcoNen,T. M., Krssn, M., CnoNxtte, E. P., and RosnnrsoN,J. S., 1.964,Ann N.y. Acad. Sci., 113, 578. I I of diaiding cellsin whole-bodygamma-irradiatedmice Response +17 Fox, B. W., and PnusorF,W. H., 1965,Cancer Res',25r 234' Gennrn, G. n., and AlrvreN, K. J.,7970, Radiation Biochemistry,Yo| II (New York: Academic Press),P. 62. L. H., 7963,Int' J' Radiat' Gnnnnn,G. B., Grnnnn, G., A"t*eN, K. J., and HEvTpELMANN, Biol., 6' 77. ( 972, Radiat. Res.,51' 391. |tsrr-INn,R. W., 1969,Radiat. Res., 38, 560. ioRKnY,D. 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