INT. J . RADIAT. BIOL ., 1987, VOL . 51, NO . 5,.907-940

Abstracts of the 20th Annual Meeting of theEuropean Society for Radiation Biology

Pisa, 15-19 September 1986

E. New approaches in biological dosimetryF . Modifications of radiation sensitivity IG. RBE for high LET radiationH.

Radiobiological aspects of tumour therapyJ .

Cellular effects of non-ionizing radiationJa. DNA damage and repair IIK.

Cellular radiobiologyKa. Radiation-induced transformation in vitroKb . Effects on membranes and other subcellular structuresL .

Modifications of radiation sensitivity II

Abstracts of the plenary sessions and poster sessions A T) were published in theApril issue of this Journal .

Session E. New approaches in biological dosimetry

Changes in blood components as biochemical indicators for irradiation

T. BUTKOWSKYJ-WALKIW t, A. STAMM t, R. HOFMANN t, N. WILLICH t, A. SPIEGELBERG t,J. STANEK t, D. PUFAL t, W. BOGL t

t Federal Health Office, Institute for Radiation Hygiene, 8042 Neuherberg/Munich,F.R. Germany

$ Radiology Department of the University Hospital Munich, Radiotherapy Section,F.R. Germany

Our group is looking for a new approach to biological dosimetry which allows a rapid doseassessment after radiation exposure without burdening the patient unduly during sampling .With the help of biochemical indicators we hope to find a linear dose effect relationship .Therefore it seems especially interesting to try to achieve results using easily available bloodcomponents .

At the moment we are working on three different methods . The first is the change inelectrophoretic mobility (EPM) of erythrocytes after in vitro irradiation of whole blood . Ourpreliminary studies indicate a dose effect relationship in a low dose range from 3 to 5 Gy . Nowwe are trying to prove these in vitro results with radiotherapy patients. The second method isthe investigation of increasing enzyme activities of alpha-amylase in the blood of radiotherapypatients . With this simple and rapid technique we found a well established dose effectrelationship in a dose range from 0 . 5 to 10 Gy .

The third procedure which we are testing is changes of serum thymidine concentrations .Previous investigations with tumour patients show the existence of various effects . Forexample, there is one group with low initial thymidine concentrations increasing afterirradiation and another one with high initial thymidine concentrations decreasing afterirradiation . In summary we conclude that all described effects are probably due to injury of thecell membrane .

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908

ESRB Abstracts

Micronucleus test in cytokinesis-blocked (CB) human lymphocytes as a newapproach for biological dosimetry

C. CATANA t, A. MATTONI t and F . PACCHIEROTTI tt Laboratory of Dosimetry and Biophysics, ENEA, CRE-Casaccia, c.p. 2400, Roma, Italy

t Laboratory of Toxicology, ENEA, CRE-Casaccia . c .p. 2400, Roma, Italy

For over 20 years the measurement of chromosome aberration yield in peripherallymphocytes has been the only biological method of radiation dosimetry routinely used .Recently, flow cytogenetics, prematurely condensed chromosomes, and micronucleus assayoffer alternative methods for biological dosimetry purposes . Fenech and Morley (MutationRes., 147, 29 (1985)) have proposed a modified micronucleus test where micronuclei are scoredin cells with preserved cytoplasm blocked in their cytokinesis (CB cells) by cytochalasin B(Cyt-B). In this way only binucleate cells that have undergone one division are scored . Humanwhole blood was irradiated with 250 kV X-rays (dose rate 0-3 Gy/min) at single acute dosesranging from 0 . 1 to 2 Gy. Lymphocytes were separated and cultured in growth medium withphytohaemagglutinin for 72 h . At 48 h Cyt-B (3 yg/ml) was added . Micronuclei were scoredonly in CB cells . At each dose at least 3000 cells were analysed . Even at the lowest dose tested astatistically significant increase above the control value was measured . The dose-effect curvecan be described by the linear-quadratic model. As the recognition of micronuclei istechnically much easier than the direct scoring of mitotic chromosomes and is much morerapid, this new approach can be used as an alternative to the conventional cytogenetictechnique . For this purpose the response at doses lower than 0 . 1 Gy remains to be assessed .

Cellular effects of diagnostic pulsed ultrasound and heat on mouse testis :cytometric approaches

R. DE VITA t, A. CALUGI, C. CHIARANTANO 1, D. FORTE § and F . MAURO t

t Division of Physics and Biomedical Sciences, ENEA Casaccia,$ ENEA thesis, and § Clinic Obstetric and Gynecologic, University 'Tor Vergata', Roma,

Italy

Mouse spermatogenesis is a good in vivo test system for noxious agents . Cellular DNAcontent, determined by flow cytometry, of the male germinal line allows the recognition of thedifferent cell types in various stages of spermatogenesis . The system has been used to evaluatethe effect of diagnostic pulsed ultrasound (2 . 5 MHz, 4mW/cm2) administered by immersionof the mouse scrotum in a thermostated water-bath at 30°C and 36 °C and irradiated using acommercial apparatus . The same approach has been used for hyperthermic exposure .Experiments have been carried out with graded exposure up to 42°C, 60 min heat, that is, inthe dose range of therapeutic or environmental exposure . Cellular samples were obtained fromthe testis of BC3 F I mice at various intervals after treatment . A monocellular suspension wasobtained by mechanical and pepsin treatment and stained with combined mithramycin andethidium bromide ; DNA content distribution histograms were analysed in order to determinethe fraction of the different cell subpopulations . The results showed a slight reduction in thefraction of elongated and round spermatids 28 and 35 days after ultrasonic treatment . Thispreliminary result indicated possible non-thermic effects . The results, after hyperthermicexposure, showed an appreciable reduction in the fraction of elongated spermatids in thetemperature range 38° to 42°C and analysed at 14 and 28 days, reflected a cytotoxic effect onboth the dividing spermatogonia and the spermatocytes . The effect can also be evaluated interms of testis weight loss 14 and 28 days after exposure . The present results confirm thatmouse spermatogenesis analysed by flow cytometry is a reliable method for biologicaldosimetry of the effects of noxious agents.

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ESRB Abstracts

909

Use of chromosomal aberrations for biological dosimetry in cell populationsafter the first post-irradiation mitosis

M. T. DOLOY, J. L. MALARBET, G . GUEDENEY, M. BOURGUIGNON, A. LEROY andM. REILLAUDOU

C.E.A ., L.P . S.N., D .P.S ., S .P.E., C .E.N.F.A.R ., B.P.6, F92260 Fontenay aux Roses, France

From the comparison of the frequency and the distribution of dicentrics (DIC), rings (R)and acentrics (Ac) in the first or second mitosis of human lymphocytes, mathematicalformulae have been established in order to describe the modifications of these abnormalitiesduring mitosis .

The behaviour of Ac was determined: risk of loss for the two daughter cells =0 . 37,probability that the 2 strands of an Ac behave as a single unit =0 . 26 or as two independentunits =0 .74. Ac may disappear (P=0 . 53), appear as single Ac (P=0 . 32) or as double Ac(P=0. 15) .

It is possible to evaluate from this second information the proportion of DIC and R non-accompanied by Ac during the second mitosis, as a function of the observed frequency of Ac .The risk for a cell to disappear during a mitosis depends on its DIC and R content .

These predictions were tested on human lymphocytes in 48, 56, 64 and 72 h cultures fromirradiated blood (2, 3, 4, 6 Gy) . The mitotic activity estimated from the proportion of DIC andR without Ac and the Ac frequency allowed us to obtain in all these cultures an accurateevaluation of radio-induced damage .

These results show that it is possible to perform biological dosimetry on cells undergoing asecond or third post-irradiation mitosis . Particularly, this situation occurs rapidly in man afterprotracted irradiation. This biological dosimetry can be used for any dose rate and any type ofirradiation .

Irradiation-induced abnormal diploid mammalian spermatids

W. GOHDEt, U . HACKER-KLOM $, TH . HEIDEN§, J . SCHUMANN §,M. SPANO ¶ and F . MAURO ¶

j Department of Radiology, $ Department of Radiation Biology,and § Special Clinic Hornheide, University, Munster, F .R. Germany,

¶ ENEA, Rome, Italy

Following the in vivo exposure of mammalian testes to ionizing radiation, an increase ofcells with abnormal DNA content which built up a separate peak in the DNA histogram wasdetected flow cytometrically 21 days following irradiation . In order to identify these cells, theywere sorted and characterized morphologically .

Adult male mice were part-body exposed to acute fission or fast neutrons (0 .4 or 14 MeV,respectively, or 60Co-y-rays . Flow cytometry and sorting were performed with the Partec-PAS-Il flow sorter . Cells were stained with Dapi and sulphorhodamin 101 .

Fission-neutron-induced cells with abnormal DNA content were sorted and identified asdiploid spermatids . They had undergone a genome mutation . These cells were oversizedwhen compared to normal haploid spermatids but had undergone regular spermioh-istogenesis (chromatin and morphological changes) . Neutron doses > 0 .5 Gy and photondoses > 2 .5 Gy induced significant increases of diploid spermatids mainly in primaryspermatocytes . The RBE values for 14 MeV neutrons were suprisingly low (1 .2 to 1 .7 forneutron doses of 1 to 7 Gy) . Following exposure to 7 Gy of fast neutrons, or about 13 Gy ofphotons, about 30 per cent of the spermatids were diploid .

The increase of diploid spermatids was also observed following application of cytostatics,e.g. adriamycin, and might be useful as a mutagenesis test system .

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910

ESRB Abstracts

Flow cytometric analysis of the effects of low LET and neutron irradiationon the murine testis

U. HACKER-KLOM t, W. GOHDE $, M. SPANO §, R . UCCELLI §, U . HAVERKAMPand J . SCHUTZ

t Department of Radiation Biology, and Department of Radiology, University of Munster,§ ENEA, Rome, Italy

Mouse testes react very sensitively to ionizing radiation. Therefore, this biological testmodel was applied for intercomparison of different rediation qualities . A flow cytometer fromPartec AG has been used. Staining of the cellular DNA was performed with ethidiumbromide/mithramycin . Adult male mice were exposed to acute or split-dose of 200 kV X-rays,60Co-y-rays or 0 . 4 or 14 MeV neutrons .

The RBE values for the reduction of testicular weight to 50 per cent of the control value 28days following 0.4 MeV and 14 MeV neutron exposure compared to 60Co-y-rays were 7 . 0 and2 . 7, respectively . The RBE values for the reduction of haploid germ cells following 14 MeV-neutron exposure to 80, 50, 37 and 10 per cent of the control value were 6 . 8, 3 . 8, 3 . 4 and 2 .5,respectively . The RBE values for 0 . 05, 0. 1 and 0 . 25 Gy of 0 . 4 MeV neutrons were 4.0, 3 . 4, and3 . 3 . Following split-dose exposure of 60Co-y-rays (24h interval), significantly less diploidabnormal elongated spermatids were induced than following acute or split-dose (8 h interval)photon irradiation.

The spermatogenesis model is especially suitable for the lower dose range below 1 Gy ofsparsely ionizing radiation, for the determination of RBE values and low dose effects .

Automated detection of dicentric chromosomes

T. LORCH t, G. STEPHAN t and J . BILLE

t institute for Radiation Hygiene of the Federal Health Office, Ingolstadter Landstr . 1, 8042Neuherberg, F. R. Germany

$ Institute of Applied Physics I, University of Heidelberg, F .R. Germany

The scoring of structural chromosome aberrations, especially of dicentric chromosomes,in human lymphocytes can be used for dose estimation in cases of suspected radiationexposure. It is the only method for dosimetry using biological parameters that is in practicaluse. The main disadvantage of this method is the great amount of work, which at present stillprevents its becoming widespread for routine analyses .

For that reason a system was designed and built which has all the basic hardware functionsrequired for partial automation of dicentric scoring . The high processing speed necessary ismade feasible by application of the multimicroprocessor-system `POLYP' and of specialelectronics for the direct evaluation of video signals .

Simultaneous with the development of the hardware, algorithms and computer programsfor metaphase search and relocation, image acquisition and segmentation, and final classific-ation of chromosomes were developed . The metaphase finding program is already in routineuse, a false positive rate of 1 .8 per cent was found . A test of the segmentation method gave 89 . 5per cent correctly segmented chromosomes . The classification of chromosomes was devel-oped using a large learning set, the error rates are 0'-74 per cent false positive and 16 . 3 per centfalse negative . All objects classified as dicentrics will be interactively checked by the operator .

Flow cytometric detection of chromosome aberrations and micronucleusinduction in irradiated V79-cells

MICHAEL NUSSE, HANS JOACHIM EGNER and MATTHIAS KRAMER

Gesellschaft fur Strahlen- and Umweltforschung, 6000 Frankfurt/M, F . R. Germany

To detect radiation-induced chromosome aberrations, chromosome fluorescence distri-butions of irradiated V79-cells were measured with high resolution flow cytometry . The

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ESRB Abstracts

911

radiation-induced relative background area which was used as a parameter for radiation actionshowed a linear increase with dose (0.5-9 Gy) and depended on the cell cycle . phase duringirradiation. The fraction of undamaged chromosomes decreased with increasing size of thechromosomes .

To understand the amount and distribution of the radiation-induced relative backgroundarea, model calculations were performed assuming random breakage of chromosomes andincluding the different sensitivity of chromosomes with various sizes . The number ofradiation-induced chromosome aberrations per cell and the number of breaks per chromo-some could be estimated if exchange formations were neglected (about 0 . 4 aberrations per celland Gy) .

The number and DNA distribution of radiation-induced micronuclei were measured in aflow cytometer and compared with distributions of acentric fragments calculated by randombreakage of single chromosomes . The fraction of radiation-induced micronuclei per cellnucleus showed a linear increase with dose . Dicentric chromosomes were measured using aslit scanning technique. A quadratic increase of the number of large dicentrics per cell withdose was observed.

The use of flow cytometry for biological dosimetryon mammalian spermatogenesis

M . SPANO t, F. PACCHIEROTTI t, R . UCCELLI t, W . GOHDE $,J . SCHUMANN $, U . HACKER-KLOM $

t ENEA, CRE Casaccia, Division of Physics and Biomedical Sciences, Rome, Italy$ Fachklinik Hornheide and Radiologische Klinik, University of Munster, F .R. Germany

Flow-cytometrically (FCM) determined DNA content of cells of the male germinal lineallows the recognition of the different cell types in various stages of spermatogenesis .Cytotoxic and cytostatic effects of noxious agents can be therefore measured by variations inthe percentage of the different cell types . In addition, mutagenic effects can be detected by theinduction of dipoid spermatids and by the increased coefficient of variation of the peak relativeto the immature spermatid cell population . A survival curve of differentiating spermatogoniacan be derived by the reduction in the number of mature spermatids calculated from FCMhistograms. Experiments carried out with X-rays or fission neutrons have shown someheterogeneity in the response of this cell population to low LET radiation ; in addition RBEvalues ranging from 3 . 3 to 4 as a function of neutron dose have been estimated . The occurrenceof an extra peak in the DNA content profile with a fluorescence value twice that of the maturespermatids has been observed after both types of radiation : the induction of diploid elongatedspermatids has been confirmed after FCM sorting of cells belonging to that peak . Recentdevelopments of this approach are: (1) the detection of S-phase cells by biparametricmeasurements after fluorescence immunochemical procedure using monoclonal antibodiesagainst bromodeoxyuridine incorporated in vivo for the labelling of cells actively synthetizingDNA; (2) the estimation of the specific organ toxicity of chemical substances tested formutagenic activity by cytogenetic methods; diethylstilbestrol, for instance, has been shown toinduce chromosome non-disjunction during meiotic divisions at doses which did not causeany significant cytotoxicity .

DNA damage induced by cadmium and X-rays measured in Chinese hamstercells by flow cytometry

M . ITEM, B. OGAREK and W. BURKARTBiology and Environment, 81/SU, EIR, CH-5303 Wurenlingen, Switzerland

DNA strand breaks may be determined in single cells by a modification of the DNAalkaline unwinding method (Ahnstrom and Erixon, Int . J . Radiat . Biol., 23, 285 (1973)). Theratio of single-stranded to double-stranded DNA can be measured with fluorescent dyeswhich bind stoichiometrically and undergo a wavelength shift from double-stranded tosingle-stranded DNA (Rydberg, Int . J. Radiat. Biol., 46, 521 (1984)) . In this way, large

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912

ESRB Abstracts

numbers of cells can be analysed for the study of strand breaks and the time-course of repair .In addition, this method offers the potential to discriminate between cells in the various stagesof the cell cycle. The effect of X-rays and cadmium was measured. Preliminary results indicatethat the primary damage to DNA, i .e. the number of strand breaks caused by X-rays orcadmium, is independent of the cell cycle stage .

Immunochemical detection of bromodeoxyuridine incorporated in intracellularDNA: Flowcytometric detection of unscheduled DNA synthesis after UV-irradiation

W. BEISKER t§¶, W. HITTELMAN t and J . W . GRAY §

t Department of Experimental Radiotherapy, and t Department of Chemotherapy Research,The University of Texas Cancer Center, M . D. Anderson Hospital & Tumor Institute,

Houston, Texas 77030, U .S.A .§Lawrence Livermore National Laboratories, Biomed Division, Livermore, CA 94550,

U.S.A .¶ Arbeitsgruppe Zytometrie der Gesellschaft fur Strahlen- and Umweltforschung GmbH,

Herrenhaeuser Str . 2, D-3000 Hannover 21, F . R. Germany

A recently developed staining method for BrdUrd/DNA analysis using histone extractionby hydrochloric acid and thermal denaturation of the intracellular DNA allows the detectionof DNA-incorporated BrdUrd at extremely low levels . For mammalian cell cultures BrdUrdincorporated (for S-phase cells) during time periods as short as 2 seconds can be measuredusing flow cytometry. The method also provides good stoichiometry, i .e . a linear relationshipbetween the number of BrdUrd molecules and the intensity of green fluorescence . This opensa wide field of new applications .

Different monoclonal antibodies against BrdUrd differ in their capability of mono- anddivalent binding against BrdUrd substituted DNA . In order to determine mono- and divalentbinding properties, DNA substituted at different BrdUrd/thymidine ratios provides an idealsubstrate for antibody binding, which can easily be measured by flow cytometry .

The kinetics of BrdUrd uptake by exponentially growing CHO cells for time periodsstarting at 2 seconds has been used to measure intracellular pool equilibrium times using flowcytometry. Pulse chase experiments with BrdUrd chased by thymidine and thymidine chasedby BrdUrd gave, as expected, approximately comparable results .

Due to the high sensitivity of the BrdUrd detection in flow cytometry, DNA repairsynthesis in GI arrested mammalian cells can be monitored after UV-irradiation at doses aslow as 0 . 1 J/m2 . Dose and time response curves can be determined easily on a single cell basis .At high doses of UV, saturation of the repair system can be observed . In the future the methodmight be extended to evaluate DNA repair synthesis after treatment of cells with alkylatingagents. Further applications may include studies of synergistic effects with UV and chemicaltreatment .

An approach to automatized screening in cytogenetic dosimetry

CARIOPEPR GROUP

INFN, Laboratori Nazionali di Frascati, Rome, and USL Roma 29, Gruppo ENEA, Rome,Italy

The goal of the present work is to develop the algorithms for automatic chromosome imageprocessing in the context of the Cariopepr program of the study of chromosome aberrationsinduced by ionizing radiations and their use in cytogenetic dosimetry . The starting point inthe work is the experience gained in processing photographs in the field of high energy physicswith the PEPR (Precision Encoding and Pattern Recongnition) device of LNF (LaboratoriNazionali di Frascati) .

The first algorithm locates and classifies objects within the elementary square, by alternatescans across this with a line-element and a spot . These functions are embedded into a specialpurpose digital processor, which uses a'local signal maximum detection' method . The second

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ESRB Abstracts

913

algorithm identifies the object as a chromosome arm through the detection of the orientationand the width . A metaphase location system is thus obtained . The results of this initialautomation level should be assessed before progressing toward fully automated dicentriccounting .

Session F. Modifications of radiation sensitivity I

Hypothermia, anaesthesia and hyperbaric oxygen

A. H . W. NIAS, P. PERRY and EILEEN SMITH

Richard Dimbleby Department of Cancer Research, United Medical and Dental Schools,St. Thomas' Hospital, London SEI 7EH

When C3H mouse mammary tumours are given single doses of radiation in hyperbaricoxygen instead of air, there is only a small decrease in TCD50 . The enhancement ratio (ER) isonly 1 . 15; but this is when no anaesthetic is used . When anaesthesia is used with hyperbaricoxygen, however, the TCD50 is significantly reduced (ER 1 . 7) . One consequence ofanaesthesia is hypothermia in the mice with a fall in temperature from 37 °C to 30°C within30 min. Previous estimation of 133Xe clearance had shown that the blood supply to suchtumours is not reduced by anaesthetic-induced hypothermia . Polarographic studies had alsoshown that oxygen tension is increased in the tumours by hyperbaric oxygen to the sameextent under anaesthetic-induced hypothermia as at normal temperature .

The question arises whether the enhancement of radiation response is attributable to thereduction in oxygen metabolism in the tumour due to hypothermia or due to the anaestheticdrug or to both . Initial studies had shown that if hypothermia was induced without usinganaesthetic there was less enhancement of radiation response but when a vasodilating drug isadded to the regime a full effect can be obtained . Whether an additional effect is obtainablewhen anaesthesia is restored to the regime remains to be seen . The effect of anaesthetic drugson tumour cells is obviously independent of hypothermia and the mechanism has beeninvestigated using the Clark Electrode system, nuclear magnetic resonance and clonogenicassays .

Radioprotective effect of WR 2721 incorporated in microspheres after oraladministration in mice

M. FATOME, F. COURTEILLE, J . D. LAVAL and V . ROMAN

Division de Radiobiologie et Radioprotection, C.R.S .S.A ., Clamart, France

Like most radioprotectors, WR 2721 has only slight activity when given orally . This maybe due to its acid hydrolysis and degradation in the gastro-intestinal tract . To prevent or lowerthis loss the compound has been incorporated in carriers . Ethylcellulose microspherescontaining WR 2721 have been prepared by the emulsion-solvent evaporation technique . Thestrong hydrophilicity of WR 2721 necessitated some protocol changes . Determinations oftotal WR 2721 content in microspheres (13 mg WR 2721/20 mg microspheres) have shown thelack of any loss or degradation of the compound during preparation . Scanning electronmicrographs show microspheres with a diameter of about 50-200 µm and the presence of WR2721 crystals adsorbed onto the microspheres . Oral administration of these microspheres,compared with that of WR 2721 in oily suspension, has led to a lowering of toxicity and tosustained and enhanced radioprotective activity, with a D .R.F. equal to 1 . 7-1 .8 over 2-3 h .This activity is as high as the best described for oral intake and has a longer duration . Thisstudy confirms the usefulness of such carriers to obtain improved radioprotective activity afteroral administration .

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914

ESRB Abstracts

The effects of whole body y-irradiation on rat haematopoietic recovery couldbe influenced by post-cyclophosphamide (CY) serum activity in vivo

N . Pujie t, N . RADOTIB t, 0. MILIt t, R. STOJANOVI6 t and A. Dupe t

t Department of Radiobiology, Institute of Nuclear Sciences, `Boris Kidric', Vinca,and $ Institute of Experimental Medicine, Military Medical Academy, Belgrade, Yugoslavia

The modulatory effect of the sera, obtained after treatment by a single, high dose of CY, onhaematopoietic regeneration, induced by y-irradiation, was investigated in vivo . Cytomorpho-logical methods were used to follow the proliferative capacity of the bone marrow and spleniccells, as well as regeneration of blood cells . Post-CY serum was collected from the 2 . 5-month-old Wistar rats injected 48 h previously with a single dose (300 mg/kg) of CY . Further, ratswere damaged by whole body y-irradiation (3 . 5 or 6 . 5 Gy) and immediately after injected with0 . 5 ml i .v. post-CY-sera (0 day). All cytomorphological parameters were followed after 1, 2, 3and 7 days. For statistical analysis, the Student t-test was used . The results obtained indicatethat after treatment with y-irradiation, post-CY serum, injected, in vivo, significantly delaysor reduces the destructive effects of irradiation .

Lymphocytic subsets of C57B1 mice after total body irradiation and applicationof poly (ADP-ribose) transferase modifying factors

H. TUSCHL

Institut fur Biologie, Forschungszentrum Seibersdorf, A-2444 Seibersdorf, Austria

C57B1 mice were treated with 3-methoxybenzamide (3-MBA) or N-N'-diacetyl-1, 6-diaminohexane (DAH) in order to investigate the effects of poly-(ADP-ribose) transferase(ADPRT) modifying factors on lymphocyte subpopulations . The capacity to performunscheduled (UDS) and replicative (RDS) DNA synthesis was measured . 3-MBA and DAHhave been shown to induce differentiation and to inhibit in vitro transformation, probably bymodifying the ADPRT activity . C57B1 mice were chosen because of the high incidence ofmalignant tumours .

Lymphocyte subpopulations were evaluated by monoclonal antibodies : anti-Lyt 2 andanti-Thy 1 .2. UDS was induced with UV irradiation of spleen cells and measured byautoradiography . Total body irradiation (TBR) was performed with a 60Co source at a dose of1 Gy. The effect of TBR on the percentage of PAN T cells and Lyt 2' cells was reversed by invivo pretreatment with 3-MBA, but only slightly with DAH . Both drugs had no effect onUDS and only a slight one on RDS .

Session G . RBE for high LET radiation

Neutron RBE at low doses:micronuclei formation in the murine eye-lens epithelium

M. COPPOLA and G. BERTONCELLO

ENEA, CRE Casaccia, C .P. 2400, 00100 Roma A.D ., Italy

Studies of the dependence of RBE on dose and radiation quality are essential for theassessment of quality factors . In this experiment the frequency of micronuclei present in theepithelial cells of the eye-lens of BC3F 1 mice was measured 25 days after irradiation withvarious doses of either fast neutrons (1 MeV and 16 MeV) or photons (250 kVp X-rays and"Co-7-rays) . Due to the high radiosensitivity of this biological end-point, an appreciableeffect could still be observed at neutron doses down to only a few mGy . The results show thatthe dose-response curve obtained with 16 MeV neutrons is consistent with a linear non-threshold model . At a neutron energy of I MeV, however, the response to doses below 10 mGyappears to be higher than expected from that model . For photons the dose dependence of the

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915

response is in good agreement with the linear-quadratic model . From the comparison of thesedata, RBE values have been determined with respect to the 60Co radiation. It is seen that for16 MeV neutrons the RBE tends to a limiting value of about 15 at low doses . For 1 MeVneutrons, however, the RBE is continuously rising, in the whole dose range of the presentexperiment, when the dose is decreased, and attains a value around 70 at 3 mGy neutron dose .The results of this study are discussed in relation with other RBE data obtained at the samelaboratories .

The radiobiological effectiveness of 24 keV neutrons

P. D . HOLT, G . R . MORGAN t and C . J . ROBERTS

Atomic Energy Research Establishment, Harwell, Didcot, OX11 ORA, U .K .j' on attachment from the MRC Radiobiology Unit, Harwell, Didcot, U .K .

A filtered beam of low-energy neutrons has been obtained from the Harwell reactorPLUTO; 80 per cent of the kerma is due to neutrons of 24 keV . The dose-rate is 0.18 Gy h- ' .Measurements of the relative biological effectiveness (RBE) of this neutron beam with respectto acute y-radiation have been made for cell-killing and the induction of chromosomeaberrations in plateau-phase cultures of Chinese hamster cells (V79/AHI ). The survival curvewas exponential and the induction of dicentrics was linear with dose over the range 0-2 .5 Gy .The RBE values, based on the initial slopes of the response curves, were 2 . 7 ± 0 . 6 and 6 . 5 ± 1 . 4,respectively . The dose-mean linear-energy transfer (LET) of the neutron spectrum was62 keV µm - I . The RBE value was in accord with values obtained for track segmentirradiations in this range of LET (Thacker et al ., Int. J. Rad. Biol ., 36, 137 (1979)) .

Mammalian cell survival studies with 24 keV neutrons

A. J . MILL

Health Physics Research Section, CEGB, Berkeley Nuclear Laboratories, Berkeley,Gloucestershire, GL13 9PB, U .K .

There is currently much interest in the biological effects of neutrons (and other high-LETradiations) and how these effects relate to the neutron quality factor . Most neutron radiationfields in and near neutron-producing installations are characterized by a high proportion ofintermediate-energy neutrons . (Intermediate-energy neutrons may be loosely defined ashaving energies from 1 eV to 100 keV .) This energy region poses particular problems withmonitoring and with the availability of suitable calibration sources . The paucity of suitableneutron sources has also meant that radiobiological experiments with intermediate-energyneutrons have not been undertaken .

However, it is possible to obtain relatively pure monoenergetic neutrons at intermediateenergies by filtering the neutrons from a high flux reactor . Such a filtered beam has beendeveloped on the PLUTO reactor at AERE, Harwell . The filter consists of iron, aluminiumand sulphur to provide a beam of 24 keV neutrons at 95 per cent purity . The dose-rate in thebeam is 0.2 Gy/h.

This beam has been used to irradiate plateau-phase cultures of HeLa cells which werescored for loss of reproductive ability . It was found that the limiting RBE at low doses was 2 . 9when compared with 250 kVp X-rays . For this end-point, this is a relatively high value for theRBE . More recent experiments in which HeLa cells were exposed at various depths in atissue-equivalent phantom have confirmed this conclusion .

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Dosimetry and effectiveness of Auger electrons from 1251

E. POMPLUN, J . Booz, and L . E . FEINENDEGEN

Institute of Medicine, KFA Julich, P.O. Box 1913, D-5170 Julich, F . R. Germany

The quality factor recommended by the International Commission on RadiobiologicalProtection (ICRP) for electrons is set to Q = 1 . This recommendation would characterize theAuger electron emitter 12 .I as a low-LET irradiator . However, a microdosimetric approachshows that in small targets like nucleosomes 12 .I behaves like an emitter of high-LETradiation .

A comparison of the physical microdosimetric radiation quality with experimentalfindings of the effectiveness of incorporated 1251 demonstrates that even the maximum qualityfactor of Q = 20 for high-LET radiation insufficiently represents the radiation effectiveness of1251 in DNA. This fact is supported by new data on increased biological effectiveness of otherhigh-LET radiations such as fast neutrons .

On the basis of the physical radiation quality on the one hand and the biological effects onthe other, a value of Q=40 for the high-LET radiation of DNA-incorporated 125! has beenevaluated which is consistent with a recent suggestion for an increased quality factor as afunction of LET (Morstin et al ., Radiat . Prot . Dosim ., 12, 319 (1986)) .

Session H. Radiobiological aspects of tumour therapy

In vitro uptake of ['H]thymidine by tumours of the larynx

M . BALZI t, E. SCUBLA t, M . B. NINU $, O. FINI STORCHI $, E . ALAJMO $,P. BOANINI t, E . ZANIERI t, R . BONDI § and A. BECCIOLINI t

t Laboratory of Radiation Biology, Department of Clinical Physiopathology,University of Florence, Italy

$ O.R.L. Clinic, University of Florence, Italy§ Institute of Pathological Anatomy, University of Florence, Italy

The aim of the study was to determine some parameters of cell kinetics in tumours of thelarynx . The labelling index (L .I .) was correlated with : (a) TNM staging ; (b) histologicalgrading; (c) presence of nodal micrometastases in patients whose regional nodes appearedclinically negative ; and (d) prognosis through evaluation of relapses and survival .

Patients affected by supraglottic epidermoid carcinoma of the larynx were included in thestudy, the majority of them was T2-T3 , No . At surgery a piece of tumour was cut into smallfragments and incubated with [ 3H]thymidine . After autoradiography at least three fragmentswere observed and 5000 tumour cells were counted for each fragment . The differences in L .I .among fragments of the tumour were within 40 per cent whereas among the sections of thesame fragment the variability was 20 per cent . The L .I . values of all cases ranged between 7 . 30per cent and 18 . 50 per cent. No statistically significant differences were observed in patients ofdifferent ages and presently no clear relation with the TNM classification was shown . Thelowest L . I . levels were observed in patients previously treated by radiotherapy . A correlationwith the evolution of neoplasia cannot yet be made given the recent nature of the study .

Quantification of radiobiological responses of leukaemic stem cells

E. BARBIERI t, E. EMILIANI t, P. PEROCCO $, C. POLUZZI § and M . A. SANTUCCI §

Institutes oft Radiotherapy, § Hematology and $ Cancerology, University of Bologna,Policlinico S. Orsola, Via Massarenti, 9 40138 Bologna, Italy

In this study the effect of total body irradiation (TB I), usually employed as preparation forbone marrow transplantation in acute leukaemia (AL) patients, on growth pattern andclonogenic potential of leukaemia stem cells was investigated in order to answer some

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questions: (1) Do leukaemic stem cells at different degrees of commitment show differentdose-survival curves? (2) Is TBI ineffective in destroying neoplastic populations? (3) Is theselection of radioresistant clones responsible for tumour repopulation? Several humanleukaemic cells lines at various degrees of differentiation (K562, KGI, HL60, U937) wereirradiated at 5 cGy/min. The radiation damage was quantified after 5 increasing total doses(1, 3, 5, 7, 10 Gy) and dose survival curves were drawn . The various methods employed were ;(a) growth pattern in suspension culture, measured by the number of cells and their viability(trypan blue exclusion) ; (b) the distribution on the cell cycle, by monitoring of DNA syntheticrate ( 3H-TdR incorporation) and mitotic rate (MI) ; (c) the clonogenic potential, expressed bythe plating efficiency in MEC cultures ; (d) the genotypic and phenotypic features of the cellpopulation, to investigate if different radioresistant clones are selected . Our preliminaryresults suggest that dose-survival curves are different for different cell populations, but TBI isnot always able to destroy whole neoplastic populations . (Supported in part by 'CentroInterdipartimentale Ricerca Sul Cancro' and 'CNR-Progetto Finalizzato Oncologia No84.00834.44' .)

Radiotherapy of the R1H-tumour: induction of metastasis

M. BAUMANN, H.-P . BECK-BORNHOLDT, S . BECKER, T . MAURER and H . VOGLER

Institute of Biophysics and Radiobiology, University of Hamburg, Martinistr . 52,D-2000 Hamburg 20, F .R. Germany

The aim of this work was to quantify induction of metastasis during fractionatedirradiation of the RIH rhabdomyosarcoma by means of flow cytometric DNA-indexmeasurements. RIH rhabdomyosarcomas of the rat (doubling time 3 .5 days, non-immunogenic, DNA-index 3 . 6) were treated using different fractionated irradiation regi-mens. At different time intervals after the end of treatment some animals showed pronounceddyspnea. These animals were sacrificed and sectioned . In general these animals showedvarying numbers of metastases in the lungs and the mediastinum, but also in the diaphragmand in the subcutis metastases were found occasionally. The metastases and the primarytumour were excised, single cell suspensions were prepared and subjected to flow cytometricmeasurements (ICP22, Phywe, 33258 Hoechst staining) allowing determination of the DNAindex. Since the DNA-index of the clonogenic tumour cells decreases with total dose, thecomparison of the DNA-indices of metastases and primary tumours allows a calculation of thetime of metastasis induction . The results indicate an increased induction of metastasis at theend of treatment. Since the Rl H tumour does not metastasize well in untreated animals, thisfinding could be of great interest for clinical radiotherapy . (Supported by the DeutscheForschungsgemeinschaft (Ba 648/2-2) .)

Radiotherapy of the R1H-tumour: the influence of the number of fractions

H.-P . BECK-BORNHOLDT, S . BECKER, T . MAURER, H . VOGLER and F. WURSCHMIDTInstitute of Biophysics and Radiobiology, University of Hamburg,

Martinistr. 52, D-2000 Hamburg 20, F .R. Germany

The aim of this work was to study the influence of the number of fractions on tumourresponse to fractionated irradiation . The RIH rhabdomyosarcoma of the rat (doublingtime= 3 . 5 days, non-immunogenic) was treated by fractionated irradiation in the dose range of0.43 to 12 . 5 Gy per fraction . Regimens of 6, 18, 30, 42 and 126 fractions applied in 6 weekswere compared, Tumour response was assessed by three different endpoints : (a) in vitrocolony assay, determining the number of clonogenic tumour cells per tumour ; (b) tumourcontrol probability ; (c) tumour growth delay (using non-parametric statistics) . While weobserved a clear reduction of skin damage with increasing number of fractions, the response oftumours measured either by colony assay, tumour control or growth delay was dependent onlyon the total dose per week, i .e . the cellular response was the same whether the weekly dose wasgiven in 1, 3, 5, 7 or 21 fractions . These results are in contrast to what was expected on the basis

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of the acute radiation survival curve which shows a broad shoulder . As similar results havealready been reported for the Lewis lung carcinoma (Beck-Bornholdt et al . Int . J. Rad. Oncol.Biol. Phys ., 11, 1171 (1985)), the present data for the RIH tumour seem to indicate a moregeneral phenomenon . (Supported by the Deutsche Forschungsgemeinschaft (Ba 648/2-2) .)

Radiotherapy of the R1H-tumour : accelerated and hyperfractionation

H.-P . BECK-BORNHOLDT, S. BECKER, T . MAURER, M . OMNICZYNSKI and H. VOGLER

Institute of Biophysics and Radiobiology, University of Hamburg,Martinistr. 52, D-2000 Hamburg 20, F .R. Germany

The aim of this work was to study the effect of hyperfractionation, accelerated fraction-ation and accelerated hyperfractionation on the response of the R I H rhabdomyosarcoma toirradiation. The RIH rhabdomyosarcoma of the rat (doubling time =35 days, non-immunogenic) was treated by fractionated irradiation in the dose range of 0 . 43 to 2 . 67 Gy perfraction . Regimens of 30, 63 and 126 fractions were applied using intervals of 8 h, thus in anoverall treatment time of 10, 21, and 42 days respectively, were compared . Tumour responsewas assessed by two different endpoints : (a) tumour control probability, (b) tumour growthdelay (using non-parametric statistics) . Despite the increased overall treatment time, theTCD37 decreased with increasing number of fractions indicating an increased effectivenesswith increasing number of fractions . Net growth delay per Gy increased with increasingnumber of fractions, thus confirming the results obtained with the control experiment . Themost probable explanation for this effect seems to be cell cycle effects . Flow cytometricmeasurements indicated an accumulation of up to 70 per cent of tumour cells in the G2 phase .(Supported by the Deutsche Forschungsgemeinschaft (Ba 648/2-2) .)

A technique for dating initiation of metastasis by flow cytometry

HANS-PETER BECK-BORNHOLDT

Institute of Biophysics and Radiobiology, University of Hamburg,Martinistr. 52, D-2000 Hamburg 20, F .R. Germany

A crucial problem in research concerning metastasis is the dating of the initiation ofmetastasis . As has been shown by several authors, extrapolation of growth curves is a poortechnique, since the growth characteristics are irregular . Thus, there is a need for a techniqueto date metastasis . The non-immunogenic RIH tumour is highly hyperploid (DNA-index3 . 8). As was found after single dose and after fractionated irradiation, the clonogenic tumourcells of the regrowing tumours show a reduced DNA-index as compared to the untreatedcontrol . This is probably due to a micronuclei formation, which in this tumour, because of thehighly redundant DNA, does not necessarily lead to cell death . The reduction in the DNA-index is strongly dose dependent . This effect offers the possibility to date the initiation ofmetastasis in hyperploid tumours after irradiation. Since the clonogenic tumour cells have adefined DNA-index after a certain dose, the DNA-index of the metastasis can be related to thetotal dose applied up to the time of metastasis initiation . This technique might help to answersome important questions . (Supported by the Deutsche Forschungsgemeinschaft (Ba 648/2-2) .)

Skin and leg contraction in mice with multifraction radiotherapy :early and late damage

A. CIVIDALLIt, L. GALLONF and G . ARCANGELI$

t ENEA CRE Casaccia, 00060 Roma, Italy$ Istituto Medico e di Ricerca Scientifica, 00184 Roma, Italy

Damage to normal tissue is the limiting factor in the use of radiotherapy and thereforeknowledge of the time-dose relationships in multifraction radiotherapy is necessary . Leg

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contraction (the difference in the ability to extend the control and of the irradiated hind legs ofmice) (Stone, Int. J . Radiat. Oncol. Biol. Phys ., 10, 1053 (1984)) and skin contraction (thedecrease in intertattoo distances) (Hayashi and Suit, Radiology ., 103, 431 (1972)) have beeninvestigated . The right hind legs of unanaesthetized male mice were irradiated withorthovoltage X-rays. The schemes tested were : (a) Three fractions per day with 3 .5 h intervalsfor 5 consecutive days; (b) one fraction per day, 5 days a week for 3 weeks . In both experiments,the doses used were 2, 3, 4 and 5 Gy per fraction . Using the three fractions per day scheme,skin and leg contraction developed rapidly in the first weeks and then progressed slowlyreaching a maximum around the 50th day . Following this, it either decreased or remainedconstant until the 80th day when it then started to decrease slowly . The time to thedevelopment of the contraction was the same at the various doses, while the amount ofcontraction was dose-dependent . Using one fraction per day, the amount of contraction wasless than with three fractions per day, especially with fraction sizes of 2, 3 and 4 Gy .

Repopulation in mouse skin during multifractional X-ray treatment

C. MARINO, A . ROJAS, I . NINis and AND J . F. FOWLERGray Laboratory, Cancer Research Campaign, Mount Vernon Hospital,

Northwood, Middlesex, U.K .

We have investigated the degree of repopulation in a fast-proliferating normal tissue whena gap of 48 h is introduced at different times during a fractionated regime . Twelve fractions ofX-rays were given to mouse skin over a 14-day period with a gap after the third, sixth or ninthdaily fraction and compared with the response of 12 fractions given in 11 days .

Acute skin reactions were scored using an arbitrary scale for oedema, dry and moistdesquamation . The average reaction, over an equivalent period of time, was calculated foreach of the four different modalities . Extending the overall treatment from 11 to 14 daysproduced a dose sparing of 1 Gy per day which is in agreement with the report by Denekamp(Br. J . Radiol., 46, 381 (1973)). However, we found no evidence of a differential sparing if thegap was introduced at the beginning, middle or at the end of treatment .

Data using 24 fractions with a longer interfraction gap (i .e . 14 days) is also presented andcompared with the previous results .

Phototoxic effects induced by rhodamine-123 on human tumour cells in vitroafter argon laser irradiation

E. MELLONI, T . DASDIA, G . FAVA, F . ZUNINO and R. MARCHESINI

National Cancer Institute, Milano, Italy

Rhodamine-123 (Rh-123) is a fluorochrome commonly used for staining mitochondria inliving cells . This specificity appears to be related to the interaction between the cationic dyeand the negative charge of the internal side of mitochondria membrane . In addition, Rh-123was demonstrated to be selectively retained by carcinoma cells in vitro . This unusual retentionof Rh- 123 in tumour cells has been exploited to reduce clonal growth of carcinoma cells invitro. To establish whether an increase in tumour cell death could be induced by Argon laserirradiation after incubation with Rh- 123, human carcinoma cell lines (HT29 and MCF7) andhuman malignant melanomas (M14) were exposed to different radiant exposures (from 7 . 5 to30J/cm 2 ) . The evaluation of cell viability was performed by a colony forming assay . Resultsshowed that the ID 50 was 1 . 55, 3 . 03 and 2 . 05 Mg/ml Rh-123 incubation for 24h, respectivelyfor HT29, MCF7 and M14 whereas after laser irradiation at 50mW/cm 2 for 5 min, theID50 decreased to 0.25, 0.22 and 0 .80µg/ml Rh-123 . Tumour cell killing was shown to besolely due to a photochemical effect and not due to a direct (thermal) effect of the laser .(Supported by Special Project 'Oncologia', Grant n .85.020401 .44 CNR, Rome .)

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Influence of X-rays on adrenergic reactions of human and rat urinary bladder

M. CH. MICHAILOV, K. TEMPEL, H . HbLZL, J. KNOWLES and A. Luz

Institut fur Pharmakologie and Toxikologie, Strahlenbiologisches Institut,Universitat Munchen ; Gesellschaft fur Strahlen- and Umweltforschung

Neuherberg/Munchen, F .R. Germany

The possible role of the adrenergic system in acute and chronic functional disturbances ofthe urinary bladder after gynaecological and urological radiotherapy has been studied . Iso-lated detrusor strips from human (surgical material) and Sprague-Dawley rats were kept in aspecial organ bath and their mechanical activity was recorded (Strahlentherapie, 155, 294) . Allpreparations reacted to adrenaline, isoprenaline and fenoterol (10 nM-I µM) with a relaxationwhich was antagonized by propranolol (fl-adrenergic stimulation) . X-irradiation in vitro(50 kV) with doses of 2 . 5-10 Gy for human and 200 Gy for rat detrusor (dose-rate30-60 Gy/min) reduced the adrenergic relaxing effect and at higher doses even inverted it sothat contraction was caused . To investigate chronic effects the urinary bladder of anaesthizedrats was locally irradiated with 50 Gy (300 kV, 4 Gy/min; total radiotherapuetic doses areusually 50-100 Gy). At 1 day after X-irradiation the relaxing effect of adrenaline was 2 timesweaker than in control animals, after 7 days 3 times, after 14 days 4 times, after 35 days 8 timesweaker (total n=32) than in the controls (n=28) . No histological changes were seen up to 2weeks after X-irradiation . After 4-6 weeks an infiltration of lymphocytes as well as asubepithelial and interstitial fibrosis were observed . It is concluded that the X-irradiation invitro or in vivo inhibited the fl-adrenergic receptors in human and rat detrusor . This could befollowed by disturbances in cell metabolism which are related to pathophysiological andpathomorphological changes in the urinary bladder after radiotherapy .

On the pharmacophysiological analysis of the detrusor-reaction after X-irradiation

E. NEU, I . PRECHTER, J . KNOWLES, N. BREITER, U. WELSCHER and M . CH . MICHAILOV

Strahlenbiologisches Institut der Universitat Munchen,Gesellschaft fur Strahlen- and Umweltforschung Neuherberg, Munchen, F.R. Germany

In connection with functional disturbances of the urinary bladder (hypertonia, changes inmicturition) after gynaecological or urological radiotherapy (Strahlentherapie, 149, 602)pharmacophysiological analyses of the acute (reversible and persistent) contractile reactionsof human and guinea-pig detrusor preparations have been carried out (Strahlentherapie, 147,290 and 155, 294) . The human preparations were about 50 times more radiosensitive(threshold dose 1 Gy at dose-rate 30 Gy/min) than those of the guinea-pig . y-Adrenergic andcholinergic blocking agents (phentolamine, atropine; 1 pM) as well as the neurotropic drugtetrodotoxin (1 pM) and the anaesthetic procain (100µM) had no effect on the X-ray reaction .The prostaglandin blocking drug Indometacin (1 µM), the phosphodiesterase blocking drugtheophylline (10µM), cAMP (100µM) and vasopressin (10 lOOmU/ml) all inhibited the X-ray-reactions . NaCl-free (replaced by Tris) and KCI-deficient solutions had a similar effect,while tetraethylammonium (100µM) and cooling (37--i25°C) potentiated the X-ray-inducedreactions . It is concluded that the contractile responses of human and guinea-pig detrusor toX-irradiation are of myogenic (and not neurogenic) origin and that y-adrenergic andcholinergic mechanisms do not participate in the reaction . Further it is suggested thatdisturbances of metabolism of the excitable detrusor cells are related to the cAMP-system andthat prostaglandins are of importance in the pathophysiological reactions after X-irradiation .The ionic effects are possibly non-specific and due to inhibition of ionic-dependent enzymaticreactions .

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Problems of hypoxyradiotherapy

K. NEUMEISTER

Clinic for Radiology, DDR-9003 Karl-Marx-Stadt, German Democratic Republic

In co-operation between the G .D .R. and the U .S.S.R. experimental data for clinical use ofhypoxyradiotherapy (HRT) has been obtained since 1974 . These investigations proved indifferent species of animal and different organs, as well as in the field of metabolism, that byusing hypoxia at the time of irradiation an obvious protection of normal tissue can be obtained(DMF 1 .2-1 . 4). On the basis of these results, we have started the clinical testing of HRT . Asystem was developed producing a respiratory gas mixture with 10 per cent oxygen. There isan interdisciplinary treatment team : radiologist, anesthesiologist, neurologist, biologist,biochemist and physicist . After exclusion of contraindications, clinical and paraclinicalinvestigations are carried out to estimate the tolerance of hypoxia . If these investigations yieldno contraindications, a test hypoxia is made with the patient (10 per cent, 15 min) withsimultaneous control of different functions. If this test agrees with the patient, HRT ispossible. With the aim of protection of normal tissue during radiotherapy, HRT was carriedout in patients with different tumours . Results of a phase-I-study demonstrated that HRT iswithout acute and late toxicity for patients .

The effect of cytostatics and radiation on the development andgrowth of Yoshida sarcoma

N. PETROVI~, K . SAVOVSKI, O . MILIE, N. RADOTIU and D. STEPANOVItDepartment of Radiobiology, Institute for Nuclear Science `Boris Kidric',

Vinca, Yugoslavia

In this paper the combined effect of cytostatics and radiation on the survival of rats withimplanted Yoshida sarcoma is considered . The development and growth of the tumour wasalso followed . Female Wistar rats, aged 2 .5 months, weighed 200-220g were used . 2 x 10'cells per ml of Yoshida ascites sarcoma (YAS) were implanted i .p . to all rats . On the third dayafter YAS implantation the animals were divided in 5 experimental groups and treated in thefollowing way: group I, received cytostatic therapy with Methotrexate (MTX)6 . 5 mg/kg, 5 fluorouracil (5 FU) 7 . 5 mg/kg, and after 24 h cyclophosphamide (CY) 50 mg/kg(PTC); group II, irradiated with 3 Gy ; group III, received PCT and after 15 days wasirradiated with 3 Gy ; group IV, irradiated with 3 Gy and after 15 days again with 3 Gy ; groupV, treated only with YAS (control). The survival rate, the growth and development of YASwere followed for 45 days. In the V (control) group no survival was obtained . The 100 percent mortality was reachedhed after 9 days of YAS implantation . The results show that astatistically significant increase of survival till 45 days from the YAS implantation wasobtained in all four groups . In group I the survival was about 70 per cent on the 45th day aftertreatment, but the sarcoma reappeared in 30 per cent of animals . In group I I the survival wasabout 70 per cent and in 45 per cent of them sarcoma reappeared up to 45 days . In group I IIthe survival was 60 per cent and sarcoma did not reappear . In group IV, the survival was 70 percent and 60 per cent of them had sarcoma. The total time of observation was 45 days .

Radiotherapy of the RIH-tumour: sublethal damage repair

R. SCHELP, H.-P . BECK-BORNHOLDT, S . BECKER, T . MAURER and H . VOGLERInstitute of Biophysics and Radiobiology, University of Hamburg,

Martinistr. 52, D-2000 Hamburg 20, F .R. Germany

The aim of this work was to quantify the effect of sublethal damage repair during frac-tionated irradiation on the response of the RIH rhabdomyosarcoma to irradiation. The RI Hrhabdomyosarcoma of the rat (doubling time= 3 . 5 days, non-immunogenic) was treated byfractionated irradiation in the dose range 1 . 23 to 2 . 67 Gy per fraction . A standard fractionation

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regimen of 30 fractions given in 6 weeks was compared to a split-dose-fractionation regimen,where the daily fraction was split into two fractions with a time interval of 2 h . Tumourresponse was assessed by tumour growth delay (using non-parametric statistics) . In addition,skin damage was scored to allow a comparison with normal tissue damage . There was asignificant decrease in growth delay in the split-dose fractionation treatment . Also skindamage was reduced . There was a tendency for more dose sparing of skin damage than oftumour response, however this difference was not significant . (Supported by the DeutscheForschungsgemeinschaft (Ba 648/2-2) .)

Parameters of cell kinetics in human oral cavity carcinomas after irradiation

E. SCUBLA t, M. BALZI t . P. BOANINI t, M. LADDAGA $ . M . G . FABRINI $, A. CHIAVACCI §,D. CREMONINI t and A. BECCIOLINI t

t Laboratory of Radiation Biology, Department of Clinical Physiopathology,University of Florence, Italy

$ Institute of Radiology, University of Pisa, Italy§ Radiotherapy, U .S.L. 10/D of Florence, Italy

The response to ionizing radiations in solid tumours, even of the same histologicalclassification shows a high variability . The reasons for this are not yet completely understood .Therefore the evaluation of proliferate parameters may be essential to better recognizing thegrowth features of the tumour. Moreover cell kinetics can influence therapy and radiation ordrug dose administration schedules. The study deals with the use of labelling index (L . I .) todetermine: (1) the presence of a relation between pretreatment and post 9-10 Gy values ; (2) ifradiation response is related to the evolution of the neoplasia .

Biopsies of patients with oral cavity carcinoma were performed before treatment,after a total dose of about 10 Gy and, if possible, later during the therapy . The fragments wereincubated in TCM containing [ 3H]thymidine for 60 min at 37 °C in 5 per cent COZ .Autoradiography has been performed on histological sections and L .I . and mitotic index(M. I .) have been determined by counting at least 5000 cells per fragment of the tumour . Threefragments for each case have been studied . Moreover cell density has been scored to evaluatecell destruction . The data of currently available cases show L . I . values ranging from 12 to 24per cent. After 10 Gy cell death is infrequent in tumour tissue ; L .I . is 20-30 per cent lowerthan pretreatment levels, whereas M .I . increases significantly . It is worth noting thatnumerous mitoses show an irregular morphology . At higher doses cell destruction becomesprogressively more evident and the remaining cells show severe morphological alterations . Atthis point L .I . and cell density decrease significantly .

Serum tissue polypeptide antigen (TPA) and plasma amylase activity as earlybiochemical indicators of radiation injury to salivary glands

M. S . TOMMASI t, S. PORCIANI t, B. FANTAPPIE $, E. CELLAI § and A . BECCIOLINI t

t Radiation Biology Laboratory, $ Nuclear Medicine, and § Radiation Therapy Unit,Department of Clinical Physiopathology, University of Florence, Italy

The need to quantify tissue injury from radiation therapy and to be able to predict long-term effects has prompted the study of biochemical indicators of radiation damage . Theresults of this study make evident this characteristic of TPA and y-amylase for patients withtumours localized in the head and neck, for whom the volume of irradiation included thesalivary glands. When different fractionation schedules were employed, at the first day oftreatment the higher the dose the greater the increase both in serum y-amylase and TPA .

The correlation between the total daily dose and the effect obtained on the first day oftreatment was highly significant for TPA (r=0 .971, P<0 .01) and for y-amylase (R=0 . 999,P<0-001). The concentration of the molecules returned to normal 3-4 days after thebeginning of the treatment, according to the irradiation schedules .

The curves of both molecules are analogues whether considered for each single patient orfor the different fractionation groups and both seem to affect radiation injury to the salivary

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glands, in particular of the duct cells for TPA, and of the acinous cells for y-amylase . The totalduration of the phenomenon makes it clear that TPA and y-amylase are early markers ofdamage, although their validity is limited to the first days of therapy .

Cerebral radionecrosis after treatment of Gliomas: a radiobiological analysis

E. VASARIO t, R. SOFFIETTI $, M . T. GIORDANA :, G. L. NEGRI fi,G. L. SANNAZZARI t and D . SCHIFFER

f Department of Radiotherapy, and $ II Neurological Clinic,University of Torino, Italy

Delayed radionecrosis represents the most serious hazard following irradiation of the CNSfor intracranial tumours and only few data are available concerning the risk factors . Weretrospectively analysed the treatment modalities of 107 cases of gliomas who have recieved atleast 45 Gy and had a minimum follow-up of 3 years . Biologically equivalent doses fortreatment schedules of all cases were calculated using the NSD, ED and btu formulas .Radionecrosis with minimal or no persistent tumour was pathologically documented in 3 outof 30 autopsied patients. Tumour doses of cases of radionecrosis were higher than 55 Gy .Below values of NSD=1666, ED=1253 and btu=1078 (^_-60Gy/35 fractions/49 days) nonecrosis was found . Isodose curve reconstruction on planes corresponding to the coronalhistological sections of the brain showed that doses absorbed by areas of necrosis were higher(12-25 per cent) than the calculated mid-plane doses in two instances . Conclusions: (1) thedifferent isoeffect formulas seem to discriminate equally well the risk of radiation damage afterfractionated treatments with total doses < 60 Gy and fractions of not more than 2 Gy ; (2) theactual dose distribution within the brain has always to be calculated to obtain reliable dose-effect correlations .

Radiotherapy of the R1H-tumour: kinetics of cellular inactivation

HUBERT VOGLER and HANS-PETER BECK-BORNHOLDT

Institute of Biophysics and Radiobiology, University of Hamburg, Martinistr . 52,D-2000 Hamburg 20, F .R. Germany

The kinetics of cellular inactivation by fractionated irradiation in the R1H rhab-domyosarcoma of the rat (doubling time= 3 . 5 days, non-immunogenic) was studied in thedose range of 1 .07 to 12 .5 Gy per fraction . Regimens of 1, 3, 5, 7 and 10 fractions per week for 3to 10 weeks were compared . The number of clonogenic tumour cells was determined using anin vitro colony assay (Stephens et al ., Br .J . Cancer, 38, 573 (1978)). Under the experimentalconditions used, the fraction of tumour cells inactivated per week was dependent only on thetotal dose per week, i .e . the cellular response was the same whether the weekly dose was givenin 1, 3, 5, 7 or 10 fractions . A simultaneous fit of all data of the different fractionation regimensindicates that tumour cells are inactivated with a Do =4 .3 ±0 . 2 Gy and surviving tumour cellsrepopulate the tumour with a doubling time of 9 ± 2 days during the course of the treatment .After the end of the treatment repopulation continued at a low rate for about one weekfollowed by an accelerated repopulation with a doubling time of clonogenic tumour cells of 1 . 5days. (Supported by the Deutsche Forschungsgemeinschaft (Ba 648/2-2) .)

Radiotherapy of the RIH-tumour: influence of overall treatment time

H. VOGLER, H.-P . BECK-BORNHOLDT, M . OMNICZYNSKI, E . THEIS and F . WURSCHMIDTInstitute of Biophysics and Radiobiology, University of Hamburg, Martinistr . 52,

D-2000 Hamburg 20, F .R. Germany

The aim of this work was to study the influence of the overall treatment time on tumourresponse to fractionated irradiation . R1 H rhabdomyosarcomas of the rat (doubling time= 3 . 5days, non-immunogenic) were treated by fractionated irradiation in the dose range of 1 . 50 to

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ESRB Abstracts

2 . 67 Gy per fraction. Regimens of 30 fractions applied in 10, 19, 30, 39 and 67 days werecompared. Tumour response was assessed by three different endpoints : (a) in vitro colonyassay determining the number of clonogenic tumour cells per tumour ; (b) tumour controlprobability; (c) tumour growth delay (using non-parametric statistics) . The results obtainedby colony assay indicate a slow repopulation of the tumour cells in the course of treatment witha doubling time of 9±2 days . Tumour control experiments indicate a slow increase of theTCD37 with overall treatment time indicating slow proliferation during treatment . Nochange in growth delay per Gy was found for the different regimens . This is due to the fact thatproliferation during treatment does not influence the growth delay measured . (Supported bythe Deutsche Forschungsgemeinschaft (Ba648/2-2) .)

Net growth delay : a novel parameter derived from tumour growth curves

FLORIAN WURSCHMIDT, H . VOGLER and H.-P . BECK-BORNHOLDT

Institute of Biophysics and Radiobiology, University of Hamburg, Martinistr . 52,D-2000 Hamburg 20, F .R. Germany

Growth delay not only reflects the treatment effect on the tumour parenchymal cellsbut also on the stroma (Tumour bed effect). Due to this effect, the extent of growth delaydetermined from tumour growth curves is highly dependent on the end point chosen . It wasaimed to minimize the influence of the tumour bed effect on the growth delay calculated bychoosing a smaller size and essentially an earlier time for regrowth . `Net' growth delay wasdefined as the interval between the time at which the regrowing tumour reaches twice itsminimal volume after passing the nadir and the time at which the tumour had the same volumebefore irradiation . Experiments were performed on the rhabdomyosarcoma R1H of the rat .Tumours were locally irradiated with X-rays. Net growth delay data were related to tumourcontrol data. The net growth delay data correlate well with the tumour control data andcolony assay data, whereas significant deviations are observed for the conventional growthdelay results as was expected due to the influence of the tumour bed effect . Net growth delay isa novel parameter derived from the tumour growth curves, allowing a better quantification ofthe tumour cell response to irradiation . (Supported by the Deutsche Forschungsgemeinschaft(Ba 648/2-2) .)

Toward the optimization of dose distribution in curietherapy

G. ZONCA, S. STEFANINI, M . BORRONI, U. CERCHIARI and A . E. SICHIROLLO

Istituto Nazionale Tumori, Servizio Fisica Sanitaria, Via Venezian 1,1-20133 Milano, Italy

With a minicomputer, it is possible to calculate for an interstitial implant or anintracavitary therapy in gynaecology, dose values in a three-dimensional grid of points . Thebasic equation for dose calculation is the Sievert integral which is evaluated using a three-point Gauss quadratic formula and the reconstruction of the radioactive implant andanatomical points is made from two orthogonal films or by 2 X-ray stereographs using a Str-3roentgenstereo comparator . From such a matrix of values isodose curves can be displayed insingle planes using a plotter but can also be viewed at the same time on multiple planes alongwith the radioactive implant and anatomical structures by projecting them on a coloured videodisplay . Better representation of the isodose surface is obtained using a hue where the intensityis decreasing with depth . It is possible to rotate the treatment planning, to change the videoscale and/or the origin and/or the length of axes, to choose isodoses to be displayed, to calculatethe distance between two points of a selected plane and to obtain a stereoscopic view .

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925

Vascularization and proliferation in human colorectal carcinomasafter irradiation

D. VAN BEUNINGEN, C . STREFFER, M .-L . MLYNEX and E . GROSS

Universitatsklinikum Essen, 4300 Essen 1, F .R. Germany

Vascularization in human colorectal carcinomas was measured . Endothelia of capillariesand arterioles were stained with triamino-tritolyl-methane-chloride (alkaline phosphatasestaining) . Some of the patients were preoperatively irradiated . Additionally, flow cytometricDNA measurements were performed to determine the amount of S-phase cells and DNAindex. A further parameter was the measurement of micronuclei (a relative measure for cellloss). Both parameters could describe cell turnover (S-phase cells over micronucleusfrequencies). The best parameter to characterize the vascularization was the relation oftumour vessels to mucosa vessels (vascularization index) . The amount of S-phase cells and themicronucleus frequencies were independent of vascularization . This means that proliferationas well as cell loss are independent of vascularization . From these results we conclude that it isnot possible to predict radiosensitivity on the basis of these parameters . However, if wecompare these three parameters before and after radiation a clear relationship can be observed .The higher the vascularization the lower the increase of the ratio of S-phase cells overmicronucleus frequencies . Some exceptions exist. These exceptions had a local relapse .

Session J. Cellular effects of non-ionizing radiation

Laser beam hazard:experimental determination of a laser retinal lesion threshold

D . COURANT, L. COURT, G . GUENEAU and V . BAILLE

C.E.A ., I .P .S .N ., D.P.S./S .P.E., Centre d'Etudes Nucleaires, B .P. no . 6,92265 Fontenay aux Roses Cedex, France

The risks of the use of lasers, particularly ocular hazards, have called for the definition ofexposure limits . Our investigations involved the retinal effects of the laser beam in the visiblespectrum and were directed at establishing a basis for the development of safety limits bydefining the thresholds of retinal damages in experiments carried out on the rabbit . The pulseduration was 600 ns, the wavelength was 593 nm and the retinal image diameter varied from 30to 570 µm. A direct ophthalmoscopic observation and a method with fluorescein angiographywere used, associated with a histological study with light and electronmicroscopy . The energycorrelates of retinal lesions were statistically analysed by a probit method . The results showedthat the determination threshold values are chiefly a function of the investigation techniqueused and the delay of observation after the exposure . Our results obtained from rabbit retinawith flourescein angiography extrapolated to the human eye support the exposure limitestablished for intrabeam viewing but not for an extended source laser . The present exposurelimits related to the experimental retinal spot size of 285 and 570 µm correspond to a damageprobability of 5 and 35 per cent, respectively .

Histopathology and ultrastructure of the retinal lesions induced inrabbits by single and low energy laser irradiations

G . GUENEAU, V. BAILLE, D. COURANT, M . Dunos and L . COURT

C.R.S.S.A., I bis rue du Lt. Raoul Batany, 92141 Clamart, France

The aim of this histological study is to specify retinal lesions induced by low-energy laserirradiations carried out in the visible spectrum, and to determine the threshold energies up towhich morphological damage is done to the retina .

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The experimental animals are adult rabbits . The laser source consists of a pulsed dye laser .The wavelength is 593 nm, pulse duration is 600 ns, and the retinal image diameter is 570 µm .Five energies, expressed in retinal irradiance, have been studied : 118, 45, 26, 16 and12 mJ cm -2. In order to follow the healing phenomena, observations with light and electronmicroscopy have been carried at different time intervals: 24h, 3, 7 and 42 days afterillumination .

When the energy delivered at the retina is less than 45 mJ cm -2 the lesion remainslocalized under the outer limiting membrane. With the lowest energy studied, the damage stillremains severe but concerns exclusively the pigment epithelium and the outer segments of thephotoreceptors. Two facts are of importance in the healing phenomena : the proliferation ofnew epithelial cells and the progressive reorganization or `repair' of the outer segments .

Radiation-induced mating type switching in yeast

J. LI'GGEN-H6LSCHER and J . KIIFER

Strahlenzentrum der Justus-Liebig-t'niversitiit Giessen, F.R. Germany

The mating type switch frequently found in homothallic strains of the yeast Saccharomycescerevisiae is assumed to be an intrachromosomal gene conversion between silent loci (HMR,HML) and the mating type locus, initiated by a double strand cut in the latter .

In heterothallic strains, the switch frequency is only about 10 - 5 , but it is enhanced after X-irradiation. To elucidate the underlying processes, switching rates were determined afterexposure to 300 kV X-rays, 313 nm and 254 nm UV light . The oxygen effect and photoreac-tivation were also investigated .

X-ray-induced mating type interconversion shows a clear oxygen effect . The averageinduction rates were, in 02, 0.7/10 6 survivors, in N 2 , 0 . 4/106 survivors per Gy, yielding anoxygen enhancement ratio of 1 .8, the same as for survival .

Switching can also be induced by 254nm UV-irradiation, but in contrast to survival, noeffect of photoreactivation is found . If the induced switching rates are plotted versus survivingfraction, no difference is seen between the different kinds of radiation in the initial part of theinduction curves (survival > 50 per cent) .

Microwave effect on growing seeds

G . SINIGAGLIA, G. MALTONI-GIACOMELLI, G. DREI and D . PIOTTI

Dipartmento di Fisica dell'Universita, Via Irnerio, 46-40126 Bologna, Italy

Seeds of Triticum vulgare were exposed during germination to a microwave powerintensity ranging 10-100 nW cm -2 . Frequencies of 9 . 7 and 8-4 GHz (y = 3 . 1 and 3 . 6 cm) wereused. Different schedules of exposure (pulsed and continuous irradiation) were compared . Astrong retarding effect on the growth of shoots was observed for continuous irradiation at lowintensity. Less retarding effect was noted at higher intensity but equal applied doses, withpulsed or discontinuous radiation. This seems to exclude pure thermal effects . Similar testsperformed on Heliantus annus seeds gave similar results . Tests performed at 30 and 300 MHzelectric field indicate little or no effect even at higher field intensity (up to 1200 V/m) .

Session Ja . DNA damage and repair II

Effect of the SOS response on the repair of psoralen-damaged plasmid DNA

C. BAULUZ . J. M . PARAMIO and R . DE VIDANIA

Radiobiology Section, Junta de Energia Nuclear, 28040 Madrid, Spain

We have studied the effect of SOS response on the repair of psoralen-induced damage toplasmid DNA. pBR322 treated with 8-methoxypsoralen plus UVA light was used to

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927

transform several E. coli strains differing in their repair capacities (wild type, uvrA6, recA13,umuC36, recA430 and uvrA6recAl 3); plasmid mutagenesis and survival were scored. Sincewe observed that psoralen-damaged plasmid was unable, by itself, to induce the SOSresponse, we promoted it by preirradiation of the host cells with 254 nm light . Preinduction ofthe SOS response, in recA+ cells, produced a high increase in plasmid survival andmutagenesis . The recA gene product, although required for the SOS response, did not appearto play any important role in the repair of the lesions by recombination . Plasmid mutagenesiswas higher in uvrA6 than in wild-type cells, whereas plasmid recovery was higher in theexcision-repair proficient cells . In the uvrA6 strain, mutagenesis appeared to be responsiblefor the observed plasmid recovery ; however, this mutagenic repair did not allow crosslinkremoval and only some fraction of monoadducts seemed to be repaired . In excision-repairproficient cells, increase in mutagenesis did not appear to account for the observed plasmidrecovery, suggesting that an error-free uvrA-dependent repair mechanism had been induced .In these cells, plasmid recovery involved the repair of monoadducts and some fraction ofcrosslinks . In conclusion SOS response seemed to have a dual involvement in the repair ofpsoralen-damaged plasmid : (1) producing an enhancement of the accurate excision repairmechanism; (2) being required, together with a functional uvrA gene product, for the removalof crosslinks .

In vitro mutagenesis of lambda phage induced by y-irradiation

HEIDI BERTRAM and CLAUDIA WINKLER

Institut fur Strahlenbiologie, Gesellschaft fur Strahlen- and Umweltforschung,D-8042 Neuherberg, F .R. Germany

Ionizing radiation is a widely used and studied mutagenic agent, but the primar lesionsresponsible for the induction of mutagenesis have not been indentified . Exposure to ionizingradiation produces structural damage such as single and double strand breaks (ssb, dsb) andalkali-labile sites (als) in the DNA . To examine the role of these lesions in mutagenesis, DNAisolated from lambda phage was irradiated with 60Co-y and packaged in vitro into lambdaphage particles. These particles were used in infect E . coli host cells that had previously beeninduced for SOS-functions by ultraviolet irradiation. Mutations of the three c-genes oflambda phage DNA were scored by the frequency ratio of clear plaques to normal turbidplaques. By DNA sedimentation the various amounts of strand breaks after y-irradiation ofDNA-solutions were determined . The data were related to the survival of in vitro packagedlambda phage particles . The results show that ssb and als interfere more with survival thanmutagenesis . Dsb do not interfere with survival since only full-length DNA molecules can bepackaged into phage particles . Irradiated phage shows ten times less mutagenesis than in vitropackaged phage with irradiated DNA .

A bacterial plasmid (pR) discriminates between damage induced by differentmutagens in mammalian cells

R . ELLI, A. ANTONELLI, P. PETRINELLI, R. BosI, F . GIGLIANI and L . MARCUCCIDepartment of Human Biopathology, Medical School, University of Rome, Italy

The best characterized inducible pathways of DNA repair are the SOS response (Littleand Mount, Cell, 29, 11 (1982)) and the adaptive response to alkylating agents (Cairns et al .,Prog. Nucleic Acid Res. Mol. Biol., 26, 237 (1981)) . The R46 plasmid and its derivatives(pK101, pR) protect bacteria against lethal effects of ultraviolet (UV) radiation and a numberof chemicals and, concomitantly, increase the mutagenicity of these agents, interacting withthe SOS pathway . In particular, the products of pKM101 muc genes protect bacteria againstmonofunctional and not bifunctional agents (Cupido and Bridges, Mutation Res ., 145, 49(1985)) . The pR plasmid enhances UV survival both in E . coli C600 and in LTA mouse cells,through the function of two regions, corresponding to the rep region and to the mucAB genesof pKM101 (Marcucci et al. Mol. Cell . Biol ., 6, 586 (1986); Elli et al ., Nucleic Acid Res ., 11,3679 (1983) ; Gigliani et al., Nucleic Acid Res ., 9, 623 (1981)). Here we report the effects of

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ESRB Abstracts

mitomycin C, cis-diamminedichloroplatinum, bleomycin and M-methyl-N-Nitro-N-nitrosoguanidine in comparison with UV and 4-nitroquinoline-1 -oxide (4NQO) in mousecells transformed by pR plasmid or UVpR mutants . The results show that, while pR protectsthe mouse cells against the damage induced by UV and 4NQO adducts, it renders the samecells more sensitive to interstrand crosslinks and single- and double-strand breaks . The pRcould therefore be a sensitive molecular probe to discriminate among different types ofdamage in mammalian cells .

G-values for DNA single (ssb) and double strand break (dsb) induction

KLAUS HEMPEL and ERIKA MILDENBERGER

Institute of Medical Radiation Research of the University,Versbacher Str. 5, D-8700 Wurzburg, F .R. Germany

Covalently closed circular double-stranded DNA (cc) of native plasmids was used todetermine the yield of ssb and dsb in consequence of X-ray irradiation . One ssb transformsDNA of the CC form to the nicked circular form (NC), whereas one dsb produced either in adirect manner or from coincidence of single-strand breaks transforms DNA of the CC as wellas of the NC form to linear DNA molecules (LI form) .

DNA (30-800µg/ml) was irradiated in oxygen-saturated 29 mM sodium phosphatebuffer. The different forms of DNA were separated by gel electrophoresis and their amountswere measured fluorometrically using ethidium bromide . From the quantitative changes ofeach conformation, D 37 values of ssb and dsb were calculated as a function of the DNAconcentration . Finally G-values (strand breaks produced per 100 eV of energy absorbed) weredetermined by competition plot .

The following yields were measured : G(ssb) 0 . 4, and G(dsb) 0 . 003 . G(dsb) refers only tothose dsb produced without further treatment by heat or alkali . With these treatments G-values could well be larger. The maximal distance between two independently introduced ssbin opposite strands leading to a dsb varied between 40 to 50 base pairs .

R46 and pKM101 plasmid-mediated resistance to ionizing radiationin Escherichia coli

F. HERNADI, I. FRANCIA and P. KovAcs

Institute of Pharmacology, Department of Chemotherapy, University Medical School,H-4012 Debrecen, P .O. Box 12, Hungary

The ability of two drug-resistant plasmids to modify 60Co-sensitivity of E . coli wasstudied. R46 R factor increased the survival of the wild type strain and had no effect on thesurvival of recA - and recA - recB- double mutants . An easily detectable increase in 60Co-resistance was found in the recB - mutant harbouring R46 factor . 5-Fluorouracil (1 mg/ml)eliminated R46 R factor from the parent and its rec - mutant strains and they lost not only theantibiotic resistance coded by R46 R factor but also their radioresistance . pKM101 plasmidalso enhanced bacterial survival in the wild-type strain and its recB - recC - and recF`mutants but had no effect on the survival of recA - mutant. Thus the effects proved to bedependent on recA + genotype but not on recB + , recB + , recC + , and recF + genotypes .

Poly(ADP) ribose and the enhancement of DNA repair by nicotinamide

EMANUEL RIKLIS, RACHEL MARKO and RINA KOL

Nuclear Research Center-Negev, Radiobiology Department, Beer-Sheva, Israel

The involvement of the chromosomal enzyme poly(ADP) ribose transferase in excisionrepair has been suggested by several authors, with disagreement about the mode of action,whether on ligation or on endonucleolytic activities . We have investigated the influence of the

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929

addition of nicotinamide on the post-irradiation cellular level of NAD + and on the DNArepair capacity of several cell lines . Using our PUVA method for measurement of repairsynthesis by incorporation of [ 3H]thymidine into cells in which normal synthesis was fullyinhibited by cross-linking the DNA by psoralen + near UV light, we have previouslydetermined the repair capacities of various cell lines . DNA repair synthesis increasedmarkedly upon the addition of a low concentration (3 mM) of nicotinamide to repair-proficient cells . The highest increase was noted in y-irradiated HEp-2 cells and KD fibroblastsbut no increase was noted in repair-deficient XP cells when exposed to UV-C light . At higherconcentrations NA caused a decrease of repair synthesis . The level of NAD +, noted over aperiod of 4 h after exposure decreased sharply in the repair-proficient cells but not in thenonrepairing XP cells, except after y-irradiation to which XP cells did show a repaircapability . Thus, the positive effect on repair of a low concentration of NA is dependent on afunctional repair system . This seems to indicate direct involvement of poly(ADP) ribose inrepair functions .

Inhibition of repair of X-ray induced DNA damage by hyperthermia

A. W. T . KONINGS, J . B. M . JORRITSMA and H. H . KAMPINGADepartment of Radiopathology, Bloemsingel 1, 9713 BZ Groningen, The Netherlands

Hyperthermic treatment of cells may lead to radiosensitization. The most plausibleexplanation for this heat effect on the cellular level is the inhibitory action of hyperthermia onthe repair of radiation-induced DNA damage .

Heat partially inactivates cellular DNA polymerase a and P . Heat also leads to an enhancedbinding of non-histone/chromatin proteins (NHCPs) to nuclear structures . We are currentlyinvestigating the possible role of these heat-induced alterations in the cell in the process ofrepair inhibition and radiosensitization . The work presented is concerned with the role ofDNA polymerase a in the repair process and the possibility that heat inhibits DNA repair viathe partial inactivation of DNA polymerase a . It is found that aphidicolin (inhibitor of DNApolymerase a) inhibits the repair of X-ray-induced DNA damage . This inhibition is howevernot fully competitive with the inhibition caused by hyperthermia . It is concluded that theinactivation of DNA polymerase a by heat is not the sole cause of the observed cellularradiosensitization by hyperthermia .

The less-than-additive effects of heat and aphidicolin on the repair of DNA damage maypossibly be explained by an accessibility of the damaged DNA to the repair enzymes as a resultof heat-induced protein binding. This binding gradually decreases after the heat treatment,enabling more DNA to be repaired and giving aphidicolin the opportunity to exhibit more ofits inhibitory action .

Repair of DNA lesions in heavily damaged cells

KARL J. JOHNSON and OLLE OSTLING

Department of Radioecology, Swedish University of Agricultural Sciences P .O. Box 7031,S-750 07 Uppsala, Sweden

DNA damage was analysed in individual cells using a new method-DNA microelectro-phoresis (Ostling et al . Biochem. Biophys. Res. Commun ., 123, 291 (1984)) . Briefly, cells areembedded in agarose gel and after lysis exposed to a weak electric field . After staining withacridine orange, the migration of DNA is quantified using a microscope photometer .

Cells with damaged DNA migrate more than cells . After irradiation with very high dosesof 60Co-y-rays cells were left for various repair periods . The DNA migration showed a veryheterogeneous pattern-some cells seemed to repair almost all DNA damage while others stillappeared to have heavily damaged DNA. The relevance of mean values of a cell population canthus be questioned .

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Radiation-induced transsulphuration in the system DNA-methionine

O. MERWITZ f, W. KOHNLEIN $ and P . OHNESEIT $

t Institut fur Chemie der Kernforschungsanlange Julich GmbH, D-5170 Julich, and$ Institut fur Strahlenbiologie der Universitat Munster, D-4400 Munster, F .R. Germany

Alkylthio-substituted DNA is a product of the reaction of mustard gas with DNA(Lawley, Prog. Nucl. Acids Res. Mol. Biol ., 5, 89 (1966)) . We studied radiation-inducedtranssulphuration in the system DNA-methionine using [35S]-methionine . The DNA con-centration was 100 pg/ml, and that of methionine was 6 . 25 pg/ml (specific activity92 .5 kBq/ml). After irradiating with 60Co, volatile breakdown products were removed,together with water, by freeze-drying . The residue was dissolved in the same volume of water,and DNA separated from methionine by liquid chromatography (Sephadex G25 columns) .From the elution pattern, a dose-dependent increase of the 35S-activity bound to DNA wasobserved. The extent of transsulphuration in the dose range of 9 . 6 to 87 Gy was 0.4 to 1 . 4 percent related to the total activity of methionine . These results indicate a new pathway ofradiation carcinogenesis in biological systems . The scavenging capacity of methionine for OHradicals reduces enormously the number of DNA strand breaks, as shown by gel electrophore-tic analysis of DNA-methionine solutions . The implication of these findings on thetranssulphuration of DNA is of importance in radiological research .

Repair mechanisms involved in the removal of lesions induced by UVA and8-methoxypsoralen plus UVA on plasmid DNA

J. M. PARAMIO, B . ARIAS, C . BAULUZ and R . DE VIDANIA

Radiobiology Section, Junta de Energia Nuclear, 28040 Madrid, Spain

We present a comparative study of the lethal effects of UVA and 8-methoxypsoralen plusUVA (PUVA) on plasmid DNA . pBR322 DNA was irradiated with increasing fluences of360nm-light (0-390kJm -2 ) either in the absence or presence of 0.34µM 8-methoxypsoralen(8-MOP) (0 .01 pg 8-MOP/pg DNA) . In order to determine the repair pathways involved inthe removal of plasmid damage, several strains of E . coli having different repair capacities(wild type, recA13, uvrA6, uvrA6, recA13, recA430, umuC36) were transformed with thetreated plasmids . UVA-induced lesions seemed to be mainly repaired by the excision pathwayand to a lesser extent by recombination . Repair of PUVA-induced lesions appeared to show agreater requirement for the excision pathway and a minor involvement of the recombinationmechanism . Plasmid lesions by themselves did not appear to significantly induce the SOSfunctions. When survival curves of PUVA-treated plasmids were corrected for the effects dueto UVA alone, it appeared that the contribution of 8-MOP to the observed lethality iscomparatively small. Some fraction of psoralen-induced lesions was removed mostly by theexcision pathway . It also appeared that the 8-MOP-induced lesions remain constant from arelatively low fluence of UVA light and do not increase on further irradiation . We concludethat excision repair is the mechanism mainly involved in removing PUVA-induced plasmidlesions, whereas lesions induced during UVA irradiation could be responsible by themselvesfor the partial involvement of the recombination pathway . Therefore, studies on psoralengenotoxicity should be carefully interpreted taking into account the possible effect of the360 nm-light irradiation .

DNA repair in gamma and ultraviolet irradiated vascular endothelial cells

E. RIKLIS, A . PRAGER, M . GREEN, M . MINTSBERG, I. VLODAVSKY and A . ELDOR

Nuclear Research Center-Negev, Beer-Sheva, and Hadassah University Hospital,Jerusalem, Israel

The response of cells of the vascular system to radiation is a limiting factor in radiotherapyprotocols. Morphological and functional changes have been noted, but less attention is given

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931

to biochemical and metabolic alterations involved in the expression of damage induced byradiation in the vessel wall . One expression of such damage is a decrease, in irradiated cells inculture, in the capacity to synthesize prostacycline (PGI 2 ) from the arachidonic acid cascadeand a large release of PGI 2 from the cells to the bathing medium . Studies on the effects ofradiation on cell viability and on cellular capacity to repair damage to DNA were undertaken .DNA repair capacity was measured in gamma or ultraviolet irradiated cultured endothelialcells, previously treated with psoralen + near UV light, enabling determination of DNArepair synthesis while the normal synthesis is fully inhibited . There is a distinct difference insurvival and DNA repair synthesis between `young' (3 day culture) subconfluent logarithmi-cally growing cells and 'old' (14 days) confluent contact-inhibited cells when exposed togamma or UV (254nm) radiation . Dose-dependent repair capacity is much higher in theconfluent than in the 'young' cells . It is clear that the capacity to repair damage to DNA isincreasing with age in culture and is not dependent on passage number . This may explain theobservation that young cells are more sensitive to radiation also in vivo and that time isrequired before the start of repopulation of cells lining an irradiated aorta .

Relative importance of monoadducts and crosslinks in the inactivation of8-methoxypsoralen-damaged plasmids

R . DE VIDANIA, C . BAULUZ and J. M . PARAMIORadiobiology Section, Junta de Energia Nuclear, 28040 Madrid, Spain

Since 8-methoxypsoralen (8-MOP) photoreacts with DNA yielding monadducts andcrosslinks, we were interested in discriminating between their lethal effects . As a firstapproach to study the possible differences in their lethality, we have performed a physicalassay to determine the extent of crosslinking in pBR322 DNA treated with increasingconcentrations of 8-MOP and a fixed UVA irradiation . The fraction of crosslinked moleculeswas calculated as the percentage of DNA able to renature after heat-denaturation and rapiddecrease of the temperature . Experimental results were fitted by numerical minimizationusing an empirical parametrization which allowed us to know the theoretical fraction ofcrosslinked molecules in the assayed 8-MOP concentration range . Consequently, we knew thefraction of plasmid molecules without crosslinks (NCF) which would represent the minimumexpected survival if crosslinks were the only lethal defect . Surviving fractions higher thanNCF would indicate that crosslinks had been repaired, whereas results lower than NCF wouldindicate the lethal effect of monoadducts . We have also estimated, by an independent assay,the total number of defects (TND) induced by 8-MOP (crosslinks and monoadducts) .Surviving fractions not higher than TND would indicate that both monoadducts andcrosslinks were completely lethal . Experimental surviving fractions were obtained bytransforming several E . coli strains (wild type, recAl 3, uvrA6, uvrA6recA13, recA430 andumuC36) with the modified plasmid . Survival results suggest : (1) crosslinks were completelylethal in all the strains ; (2) monoadducts were also found to be lethal although, to some extent,they were removed by the excision pathway .

Session K . Cellular radiobiology

Inactivation of Chinese hamster V79 cells by high LET proton beams

M. BELLI t#, R. CHERUBINI §, S . FINOTTO §, G. MOSCHINI §¶, O . SAPORA t,G. SIMONE 11 and M. A . TABOCCHINI t:

t Istituto Superiore di Sanity, Roma; $ INFN Sezione Sanita, Roma ; § LaboratoriNazionali di Legnaro dell' INFN, Padova ; ¶ Dipartmento di Fisica University di Padova ;

11 Istituto di Fotochimica e Radiazioni di Alta Energia del CNR, Bologna, Italy

Low-energy proton beams can give useful information about the action of high linearenergy transfer (LET) radiations at the cellular level . The results we obtained at the Legnaro

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irradiation facility on V79 cells have shown that 25 keV/µm protons are more effective inproducing DNA double strand breaks than a-particles of comparable LET used by otherauthors.

In this work we have studied the survival of V79 cells irradiated with proton beams withthree different LET, namely 10, 25 and 35 keV/µm . The survival curves obtained in the doserange 0 . 5-4 Gy were fitted by the linear-quadratic model and compared with the curveobtained with 200 kV X-rays . The coefficients found for 10 keV /µm protons are a =0 .31 Gy- 'and #= 0-094 Gy-2 . The experimental data for 25 keV/µm protons are well fitted by a simplelinear model, with a=0 .89. The a/a x ratios, which give the RBEs at low doses, are 2 . 8 and 8,respectively. These values are considerably higher than those reported for heavier-particles ofcomparable LET, for which the highest a/a r ratio (ranging from 7 to 9) was obtained around100 keV/µm . On the other hand, we found that increasing the LET of protons from 25 to35 keV/µm does not significantly increase their RBE . These results may indicate that theRBE-LET relationship for protons is shifted to lower LET values, compared with heavierparticles .

Differentiation induced by ionizing radiations :effects of different schedules of dose administration on Friend leukaemia cells

F . FRACIOLINI, A. CIANFEROTTI and A . BECCIOLINI

Radiation Biology Laboratory, Department of Clinic Physiopathology,University of Florence, Italy

It is well-known that Friend leukaemia cells (FLC) differentiate under a variety ofchemical substances and physical agents such as UV and ionizing radiation . Differentiationdegree is nevertheless different, so that ionizing radiations are put into the class of weakinducers . FLC continuous cultures were studied in order to evaluate either injury ordifferentiation induced in cancer cells by different exposure conditions . For this purpose FLCwere exposed to y-rays from a 60Co source and irradiated with single and fractionated dosesranging from I to 30Gy total dose . Single doses and two fractions, the second of which %%asadministered after 24h, were used . Parameters studied were : growth and cellular death,fraction of surviving cells, mitotic index and the percentage of multinucleated and the largestdiameter cells . For each experiment 4000-6000 cells were scored . Single exposures evidencedpartial or total inhibition of growth beginning from the second day after irradiation, and thenumber of multinucleated and largest cells increased with increasing doses . Similar behaviourwas observed when cells were irradiated with the fractionated doses . Moreover, the resultsshowed a certain degree of early differentiation .

Autosynchronization loss in postirradiation colony growth of CHO fibroblasts

GERD K. HAGEMANN and HEINRICH EVERS

Medizinische Hochschule Hannover, 3000 Hannover 61, F .R. Germany

Population kinetics of colony growth have been measured in the past by observations ofgrowth rates, colony sizes and by time-lapse studies . The resolution of the growing cellnumbers increases with observation frequency and the significance of the results with thenumber of samples . We have combined growth rate measurements with a frequency of 3 1/3 /hand simultaneous observations in 12 Petri dishes with 1 colony/dish and incubation times upto 120 h . The 12 Petri dishes were incubated in a special sample changer and observed by amicroscope with a TV-camera set up . The camera was connected on line with an interactiveimage analysis system (Kontron) . Developed programs allow counting of the number ofcells/colony in time intervals of 90 s and to store the values up to evaluation of a run .

Numerous runs have shown that the cell numbers/colony increased periodically with timeup to about 1000 cells/colony . The length of every period corresponded to the cycle time of12 h. This phenomenon of autosynchronization decreased but did not vanish with radiationdoses up to 3 Gy . Differentiation of the curve of log (cells/colony) for the incubation time

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yields the relative mitotic rate . Comparison of the cycle time with the doubling time gives thecell loss as a consequence of heritable lethal radiation damage . (Supported by the DeutscheForschungsgemeinschaft .)

Session Ka. Radiation-induced transformation in vitro

Effect of dose rate on C3H/10T2 cell transformation by X-rays

ELIZABETH K. BALCER-KUBICZEK and GEORGE H . HARRISON

University of Maryland School of Medicine, Department of Radiation Oncology,Baltimore, Maryland 21201, U .S .A .

We measured the lethality and frequency of oncogenic transformation in C3H/10T2cells using a constant exposure time technique in which the dose rate is proportional to thetotal dose . Cell transformation and survival data were analysed using the linear-quadraticrelation. Experiments were performed at 37°C using asynchronous, actively growing cultures .The dose-response curves obtained at the exposure times of 1, 3, 5 h were compared with thatobtained for acute (less than 1 min) exposure. Throughout the range of total doses examined(0 . 25 to 2 Gy), the protracted exposure of C3H/lOT2 cells resulted in a significant reductionin transformation frequency, relative to the frequency from an acute exposure to the samedose. A qualitatively similar reduction was measured for cell lethality .

The reduction of the effect (cell killing or induction of oncogenic transformation) was afunction of total exposure time, indicating that time for the repair of sub-effective damagerather than the dose rate per se is an important factor in relating the oncogenic transformationor cell lethality after brief and protracted exposure to low-to-moderate doses of low-LETradiation. (Supported by PHS Grant CA 32729 awarded by the National Cancer Institute,DHSS .)

Split-dose effects in C3H/10T2 exposed to low LET radiations

D. BETTEGA, P . CALZOLARI and L. TALLONE LOMBARDI

Dipartmento di Fisica dell `University di Milano, Via Celoria, 16, 201333 Milan, Italy

A systematic study of the oncogenic potential of 31 MeV proton beams,LET=1 .83±0 .02keV/µm in tissue, YD =3.8±0.3, keV/pm has been carried out in ourlaboratory using the in vitro technique . C3H/10T2 line cells have been irradiated in the doseinterval 0 . 25-7 Gy and growth, survival and transformation dose-effect curves have beendetermined .

Fractionation effects have been studied with total dose of 0 . 5, 1 and 7 Gy and interfractiontime intervals of 2- 5, 5, and 10 h . It was found that the fractionation of 7 Gy results in anapproximately 2-fold increase in survival and 3-fold reduction in transformants/survivor ;complete recovery from sublethal and subtransformant damage occurs within the first 2 . 5 h .Fractionation of 1 Gy has no appreciable effect on cell survival as expected because of thehigh survival level at this dose, nor on transformation . At the contrary a significant decreasefollowing split-dose is present at 0 . 5 Gy where a factor of more than 3 between single and split-dose has been evaluated .

Quantification of radiation transformation frequencies

C. B. SEYMOUR and CARMEL MOTHERSILL

Saint Luke's Hospital, Rathgar, Dublin 6, Ireland

The relationship between the administered dose of irradiation and the frequency oftransformation in vitro remains to be clarified . Several authors have detected 'bell-shaped' or

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plateau-type dose response curves, where the transformation frequency either reaches aconstant level or falls off at high doses, even after an allowance is made for cell kill .

We have recently been studying the development of transformation in primary thyroidcultures and found a characteristic bell-shaped curve when our results were corrected forinitial cell kill using a clonogenic assay . However, we also noticed while trying to isolateimmortal clones in serial subcultures that the plating efficiency of cells contained in irradiatedsurvivor colonies in a variety of cell types including C3H/IOTZ and thyroid cells wasconsiderably below normal . The effect is dose dependent and is significant out to the thirdsubculture. This means that using the first survival curve will lead to a considerableoverestimate of the number of surviving cells and a consequent underestimate of transform-ation frequency. Because of the problem of senescence of primary thyroid cells it is not easy toquantify the degree to which the effect alters the observed transformation frequency but whenthe effect is taken into consideration using C3H/10TZ cells it raises the observed transform-ation frequency based on the initial surviving fraction considerably and converts thecharacteristic plateau type C3H/1 OTZ transformation dose response curve to a curvilinear orlinear response . The results have disturbing implications for the use of in vitro transformationdata in the assessment of carcinogenic risk .

Oncogenic transformation of C311/10V cells by differenttypes of ionizing radiation

L . HIEBER, G. PONSEL, S . FENN, E . FROMKE and A. M . KELLERER

Institute fur Medizinische Straglenkunde der Universitat Wurzburg, F .R. Germany

In this study, C3H/lOT1 cells were used to compare the transformation frequencies bysparsely ionizing y-rays, by densely ionizing a-particles (LET= 147keV/µm), and by heavyions from oxygen to uranium (LET from 300 to 15700keV/µm) .

Alpha particles are substantially more efficient both for cell inactivation and for oncogenictransformation. The relative biological effectiveness for cell inactivation and for oncogenictransformation ranges from 3 to 10 and from 4 to 10, respectively .

In contrast, heavier charged particles of very high LET appear less effective for bothendpoints at specified doses . On a per particle basis the heavier ions are more effective withregard to cell inactivation . For transformation, however, they are least effective, even on a perparticle basis . The data are still preliminary, but the observation is important . It would implythat extremely densely ionizing heavy ions, for example in space radiation, are much less of ahazard than frequently postulated. It would also be in line with a recent proposal of the twoInternational Radiological Commissions (ICRU 40), that the equality factor should decreaseat LET values in excess of a few hundred keV/µm .

Session Kb . Effects on membranes and other subcellular structures

Membrane conductivity changes in erythrocytes and white ghostsfollowing y-irradiation

C . BALLARIO t, A . BONINCONTRO t, C. CAMETTI t, A. Rosi I and L . SPORTELLI §

t Dipartmento di Fisica, University di Roma `La Sapienza', Roma, Italyt Istituto Superiore di Sanity, Viale Regina Elena, 259 Roma, Italy

§ Dipartimento di Fisica, Gruppo di Biofisica Molecolare, University della Calabria, Cosenza,Italy

The membrane conductance GM and the membrane capacitance CM of intact humanerythrocytes and white ghosts have been estimated by means of conductivity measurementscarried out in the frequency range 1 kHz to 100 kHz . The erythrocyte cells and ghosts inphysiological saline solution (Hct = 50 per cent) were irradiated with a 60Co y-ray source in thedose range 2 x 103 to 50x 10 3 Gy (dose rate 37 .2 Gy/min) .

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The results show a marked decrease of GM up to a dose of about 10 kGy, whereas noappreciable effect on CM is observed. Moreover, between unirradiated and irradiated whiteghosts, both electrical parameters remain unchanged . On the contrary, an intermediatebehaviour is observed when haemoglobin (at a concentration of 4 . 5 g/dl) is added to ghostsuspensions . Our results suggest that haemoglobin is responsible for the alteration in thepermeation and transport mechanism occurring in the membrane induced by y-irradiation .Further support for the role of haemoglobin was found from electron spin resonancemeasurements carried out on erythrocytes and ghosts labelled with 5-, 12-, 16-nitroxilstearicacid irradiated in the same dose range . These labels probe the external monolayer at threedifferent penetration levels and indicate an increase of the membrane rigidity in erythrocytesand haemoglobin-riched ghosts, whereas no significant effects are observed in white ghosts .

Effects of hyperthermia and vitamin A on human tumour cells in culture :an NMR study

L . GUIDONI f $, G. MARIUTTI t$, G. M . RAMPELLI f, A. Rosi t$, V. VITI t$f Laboratorio di Fisica, Istituto Superiore di Sanita ;

and $ INFN Sezione Sanita, Roma, Italy

Human colon adenocarcinoma cell lines HCT-8R and LS-174T have been studied bymeans of 31P, 'H and 13C NMR spectroscopy. Both cell lines show the presence of mobilephospholipids, possibly related to membrane structures (L . Guidoni et al .) . In fact, peaksobserved on cell samples and extracts obtained by mild perchloric acid treatment are visible ataround Op .p.m. (t>20°C) in the 31 P NMR spectra and are not perturbed by depletion ofoxygen and cell metabolism. Very intense signals from the fatty acid chains are also visible inthe 'H NMR spectra . Hyperthermic treatment, alone or associated with vitamin A is likely toinduce membrane modifications, possibly affecting the phospholipid structures revealed byhigh resolution NMR . The spectral parameters (peak intensities, line shape and relaxationtimes T1 and T2 ) are affected to some extent by single and combined treatments .

Hyperthermia and pH variation effects on extracted lipids and membranes fromE. coli K-1060: A 31P NMR study

F . IANZINI t, L . GUIDONI f, M . T. SANTINI t, G. SIMONE $,V. VITI t and M. B . YATVIN§

t Laboratorio di Fisica, Istituto Superiore di Sanita, Rome Italy$ Istituto di Fotochimica e Radiazione d'Alta Energia, Bologna, Italy

§ Radiobiology Research Laboratory, University of Wisconsin, Madison, U .S.A.

An understanding of how heat kills cells could result in improved cancer therapy . It hasbeen suggested that cell sensitivity to heat is influenced by membrane order . Different fattyacids were used to modify the membrane lipid composition of an unsaturated fatty-acid-requiring auxotroph of E . coli . 31P NMR spectra of extracted lipids from the inner and outermembrane fraction show changes in bilayer-organization depending on pH variation (pH6-7 . 4). More difficult is the interpretation of the 31P NMR spectra of the inner and outermembrane fraction . For example, the NMR spectra of oleic acid-growth cells at pH 6 and37°C indicate that there is a lack of bilayer structure . However, when the temperature is raisedto 43°C at the same pH, a bilayer structure is in evidence . In contrast, at pH 7 . 4, the bilayerphase was equally prominent at both temperatures . Furthermore, if one assumes that thebilayer phase provides stability (i .e. increasing cell survival), cell survival is not alwayspositively correlated with the 31P NMR data.

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Ferrous ion-ascorbate and X-ray irradiation effects on multilamellarliposomes of phosphatidylglycerols

FIORENZA IANZINI t$ and MILTON B. YATVIN tt Radiobiology Research Laboratory, University of Wisconsin, Madison, U.S.A ., and

$ Laboratorio di Fisica, Istituto Superiore di Sanita, Rome, Italy

The effect of ferrous ion-ascorbate or X-ray radiation on synthetic membranes was studiedusing multilamellar lipsomes composed of unsaturated and saturated phosphatidylglycerols(PAPG=I-palmitoyl 1-2-arachidonoyl-phosphatidylglycerol; DMPG=L-a-phosphatidyl-D,L-glycerol, dimiristoyl ; DSPG=L-ot-phosphatidyl-D,L-glycerol, distearoyl) . Thethiobarbituric acid (TBA) method was used to measure peroxidation . With ferrous ion-ascorbate, peroxidation was time- and temperature-dependent for liposomes composed ofpolyunsaturated lipids (PAPG), whereas no TBA reactive substances were formed forlipsomes composed of saturated lipids (DMPG and DSPG) . When DSPG was added toPAPG, liposomes (molar ratio 1 :9 or 1 :1 at 37°C and 60 °C), ferrous ion-ascorbate peroxid-ation was prevented. When X-ray irradiation was used as the source of oxidation, DSPG hadno antioxidant role . These results suggest that the composition of liposomes plays animportant role in the extent to which peroxidation occurs when a chemical oxidant is used . Onthe other hand, our results show that the influence of lipid composition on peroxidation isnegligible when the oxidant employed is ionizing radiation . One explanation is that thechemical oxidant is prevented from having easy access to the readily oxidable double bond bythe lipid composition (in the mixed lipid liposomes) .

Effect of irradiation on structure and function of angiotensin, anoligopeptide transmitter

L. SCHACHINGER, G. WERNER et al .GSF, Institut fur Strahlenbiologie, D-8042 Neuherberg, F .R. Germany

Angiotensin (A°), which contains eight amino acids including phenylalanine and tyrosine,shows increased optical density and slightly reduced fluorescence after irradiation . Botheffects point to a hydroxylation of the aromatic amino acids mentioned . The physiologicaleffect of All was measured by the contractile response of rabbit aorta to concentrations between5 x 10 -10 and 10 -6 mol dm-3 . All was irradiated in a 10 -4 mol dm-3 solution with X-rays,60 kV, 8 Gy min -1 and diluted for the experiment. Compared to the control, the con-centration of irradiated A ll at which contraction started no difference up to a dose of 350 Gy,then a sharp decrease of the physiological effect took place . Concerning the highestcontraction reached at the same concentration as the control, irradiated A « showed anincreased effect up to a dose of 250 Gy, and a sharp decrease at doses higher than 350 Gy asmentioned before . Diminishing of the physiological effect of A ° could be caused bydestruction of the active groups due to complete hydroxylation or by splitting of the molecule .An inhibitory effect as in the case of irradiated cAMP does not take place .

Session L. Modifications of radiation sensitivity II

Difluoromethylornithine: a potentiator of radiation response

A . COURDI, G. MILANO, J . GIOANNI and A . COSTA

Centre A. Lacassagne, 36 Voie Romaine, 06054 Nice, France

Substances that inhibit polyamine biosynthesis such as alpha-difluoromethylornithine(DFMO), a specific and irreversible inhibitor of ornithine decarboxylase (ODC), have beenshown to be potentiators of drug-induced cytotoxicity in animal tumour models . DFMO wasadded at 1 mm or 10 mm to exponentially growing CAL 18A cells-a human breast carcinoma

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cell lin and irradiated I h or 24 h thereafter . Another polyamine inhibitor : MDL 72 .175 wasalso used in preliminary experiments, ODC activity in DFMO-treated cells was reduced toless than 10 per cent its original value. Maximum radiosensitization was obtained at 10 mMand 1 h incubation, with a sensitizer enhancement ratio (SER) of 1 . 88 at 0 . 1 survival . Plateauphase cells were used to investigate potentially lethal damage repair (PLDR) after irradiationin the presence of DFMO . The recovery ratio of treated cells was 1 . 14 versus 2 . 03 in controlcells, suggesting inhibition of PLDR . However, SER for plateau phase cells was lower thanthat of exponential cells, indicating that DFMO was more effective on actively growing cells .MDL 72.175 was toxic at low concentrations and did not sensitize the cells . DFMOradiosensitization may not act through polyamine depletion .

Oxygen enhancement ratio and spatial distribution of cellular glutathione

MARGARETA R. EDGREN

Department of Tumor Biology II, Karolinska Institute, Stockholm, Sweden

Oxygen enhancement ratios (OER), determined with DNA strand breaks as the end-pointof the radiation effect, were lower for cells with a genetically determined glutathione (GSH)deficiency than for cells depleted of GSH artificially to a corresponding degree by treatmentwith buthionine sulphoximine (BSO)(Edgren et al., Int . J . Radiat . Oncol . Biol . Phys ., 12,1147(1986)) . The possible persistence of GSH at a location close to the critical target in the BSO-treated cells was considered as an explanation of the discrepancy . To test this possibility, GSHwas measured in whole cells and separated nuclei from Chinese hamster (V79-379A) cellcultures after treatment with BSO in different concentrations and for varying periods. Theconcentration- and time-dependent depletion of GSH was, in all cases, less in the nuclei thanin the whole cells . The OER of the BSO-treated cells was linearly related to the GSH contentin the nucleus, but not to that in the whole cells . This observation can be regarded assupporting the idea that GSH remaining after BSO treatment in a close proximity to thecritical DNA target functions in the radical competition processes which define OER . Extra-nuclear GSH may not play any major role in the processes .

Does t-butanol protect y-irradiated cells by OH scavenging or by aninfluence on enzymatic repair?

J. W. HULSEWEDE and D. SCHULTE-FROHLINDE

Max-Planck-Institut fur Strahlenchemie, Stiftstrasse 34-36,4330 Mulheim a.d. Ruhr, F.R. Germany

The survival of various E. coli K12 strains has been measured with and without addition oft-butanol or isopropanol as radioprotectors in aerated solution . Four arguments lead to theproposal that the protection by t-butanol and isopropanol is mainly the result of an influenceon enzymatic repair. Firstly, the dose dependence of protection by t-butanol is predominantlyof zero order for the E. coli strains AB 1157 and their genetically closely-relatedrecombination-deficient mutants. This means that Dq is increased by the addition of t-butanolbut not Do. The zero order is not compatible with free radical scavenging as a protectionmechanism. Secondly, the degree of protection is large only for strains with an impact rec .ABC recombination system (the protection by t-butanol is 20 times larger for the strain AB1157(Dq =140 Gy) than for the strain AB 2463 (D q = 7 Gy)) . Thirdly, under delayed platingconditions and after pretreatment of the cells with chloramphenicol, which prevents thesynthesis of proteins, a much smaller protection by t-butanol is observed in the wild-type AB1157. Fourthly, the addition of t-butanol was found to induce a prolongation of the lag phaseof growth. This suggests that the protection may partly be due to a longer time available forenzymatic repair under growth conditions before the replication of the genome begins .

The results indicate that from the protective effect by scavengers like alcohols alone, noclear conclusion can be made concerning the contribution of the indirect effect to cell killingafter ionizing irradiation .

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Radioprotective effects of inhibitors of neutral proteinases

M . KORBELIK t, M . OSMAK t, R. ARE2INA t, J . SKRK T, A. SUHAR§ and V . TURK §t Boskovic Institute, Zagreb ; t The Institute of Oncology, Ljubljana ; and

§J. Stefan Institute, Ljubljana, Yugoslavia

Inhibitors of intracellular proteinases can act as anticarcinogens in suppressing inductionof neoplastic transformation, mutagenesis and sister chromatid exchanges by radiation andchemical carcinogens . Some of them are also effective inhibitors of potentially lethalradiolesions . Moreover, the ability of some proteinase inhibitors to act as radioprotectiveagents has also been discussed (Troll and Wiesner, In Radioprotectors and Anticarcinogens,edited by Nygaard and Simic (New York: Academic Press)(1983)) .

Exponentially growing Chinese hamster V79 cells were y-irradiated with or without thepresence of the proteinase inhibitor under study, and survival (colony formation) andmicronuclei induction were determined . Results indicate that inhibitors of neutral proteinases(e .g . bovine pancreas and soybean trypsin inhibitors, antipain) can act as radioprotectors, butconcentrations much higher than those required for inhibition of serine proteinases areneeded. Inhibitors of cysteine and aspartic proteinases do not seem to have radioprotectivecapacities .

Induction of DNA damage in mammalian cells by the nitroimidazole, RSU-1069

O. SAPORA 1, T. J . JENNER t, T. C. JENKINS t, P. O'NEILL t and E. M . FIELDEN tt MRC Radiobiology Unit, Chilton, Didcot, U.K .

$ Istituto Superiore Sanita, Rome, Italy

RSU-1069, 1-(2-nitro-l-imidazolyl)-3-(1-aziridinyl)-2-propanol, is more efficient thanmisonidazole as a radiosensitizer and chemopotentiator . The induction of DNA strandbreakage by RSU-1069 and misonidazole in mammalian cells after incubation for 2 h at 37°Cunder both hypoxic and aerobic conditions has been investigated using concentrations of theagents comparable to those used for cytotoxicity studies . The time dependence of strandbreakage in cellular DNA is also dependent upon both temperature and the concentration ofthe compound . Double strand breaks are produced predominantly under hypoxic conditions .

From these observations the following points will be emphasized : (1) the yield of singlestrand breaks (determined under alkali conditions) induced by both misonidazole and RSU-1069 is substantially increased in hypoxia ; (2) in air, RSU-1069 is much more effective thanmisonidazole at causing strand breaks; whereas (3) under hypoxic conditions, RSU-1069 is- 10 3 -fold more effective than misonidazole (on a concentration basis) at inducing DNAbreakage .

These findings are consistent with the mechanisms proposed for drug action wherebyRSU-1069 acts as a monofunctional alkylating agent (via the aziridine moiety) under aerobicconditions, as witnessed from the increased strand breakage by this compound . Further, it isinferred that the greatly increased efficiency of the nitroimidazoles under hypoxic conditionsis due to bioreduction of the compounds to form reactive metabolites . In the case of RSU-1069,such cellular bioreduction results in a metabolite which has bifunctional character .

Interaction of thiyl free radicals with oxygen :radiation chemical and biological studies

G. SIMONE t, M . TAMBA t, J . C. M. BREMNER t, B . R. GUERRAand M . QUINTILIANI t

t Institute FRAE (CNR), Bologna and t Institute TBM (CNR), Roma, Italy

Thiyl radicals RS- are reported to react with oxygen with large variations in rate constant(from 10 9 M -1 s- 1 for CyS • and GS- to 10' for PenS •) leading, ultimately, to sulphur oxidationproducts, probably via the formation of sulphur peroxy intermediates . The occurence of such

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a reaction can be demonstrated either indirectly or directly. The reaction of RS- radicals withoxygen was followed by the build-up of a transient absorption with maximum at 540 nm,whose formation kinetics were in good agreement with the rate of disappearance of thiylradical absorption at 320-330 nm . From the formation of the signal at 540 nm, ascribed to thesulphur radical RS00 •, experimental support was gained for an equilibrium reaction, whosekinetic constants have been measured .

It has been known for many years that radiation-induced enzyme inactivation shows an`oxygen effect' in the presence of thiol compounds . This greater inactivation in the presence ofoxygen could be attributed to the inactivating effect of thiol peroxy radicals . Studies onenzyme inactivation have been carried out on two macromolecules : lysozyme and trypsin.

Effects of low-dose rate soft X-irradiation on iododeoxyuridine-labelled CHO cells

SYNNOVE SUNDELL-BERGMAN, KARL J . JOHANSON and SVEN RICHTERDepartment of Radioecology, Swedish University of Agricultural Sciences

P.O. Box 731, S-750-07 Uppsala, Sweden

Iododeoxyuridine (IUdR), a thymidine analogue is incorporated into DNA of dividingcells. Irradiation with low-energy photons initiates K-shell vacancies in iodine resulting inemission of Auger electrons . Maximum effect is obtained at energies slightly above the K-edge for iodine (33 .2keV). Damage caused by Auger electrons within DNA has a very lowprobability of being repaired which is similar to that found after high LET-radiation .

Photonactivation of IUdR by soft X-rays delivered at moderately high dose-ratesenhances the killing of CHO cells (Fairchild et al ., Invest . Radiol . 17, 407 (1982)) and impairsthe repair of DNA strand breaks .

In this study we have investigated the survival of IUdR-labelled cells after irradiation withsoft X-rays at low dose rates, 0 .04 or 0.004 Gy min -1 respectively . The effects of protractedlow dose-rate exposures were found to be much less in control cells than for IUdR-labelledcells . At a dose rate of 0 . 04 Gy min-1 the Do-values were calculated to be 2 Gy for IUdR-labelled cells and 3 Gy for control cells . This yields a dose modifying factor (DMF) of 1 . 5 . Thecontrol cells showed an explicit shoulder compared to IUdR-labelled cells and the Dq wasestimated to be 0 .96 Gy .

At the lower dose rate , 0 .004 Gy min 1 , the Do was 1 . 4 Gy for IUdR-labelled cells and4. 1 Gy for control cells yielding a DMF of 2 . 9 .

Synthesis and testing of new radiosensitizing compounds :4-substituted-3-nitro-quinoline derivatives

L. P. VARGA t and E. BERfNYIt 'FJC' National Research Institute for Radiobiology and Radiohygiene, and

$ EGIS Pharmaceuticals, Budapest, Hungary

Several types of 4-substituted-3-nitro-quinoline derivatives were synthetized and testedfor radiosensitizing ability. N(3-nitro-4-quinolyl)-morpholino-carboxamidine proved to bean effective hypoxic cell sensitizer since this drug is able to decrease the mean lethal dose (D 0 )and to inhibit the repair of radiation-induced damage (shoulder-removing effect) . Effects ofthis compound were tested on mammalian cells cultured in vitro (CHO and Hela), and in vivoon the Lewis lung carcinoma tumour implanted into BDF-1 mice . The sensitizing propertiesof the drug were compared to those of misonidazole and metronidazole under indenticalconditions. Enhancement ratios (ER) of 3 . 5-3 . 9 were obtained for hypoxic cells treated withthe drug (0 .5-2mM) for lh at 37 ° C and subsequently exposed to X-rays, while formisonidazole (5 mm) this ratio was 1 .95. The oxygen enhancement ratio (OER) of 2 .76. Theshoulder region of the radiation survival curves disappeared upon treatment with the drug .Similarly encouraging results were observed when tumour-bearing mice were given sensitizeri .v. in an amount of 60 pg/g body wt followed by local irradiation with 10 Gy . Using theregrowth analysis, values for SER of 1 . 5-2 .1 were calculated depending on the experimental

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conditions . These results indicate that the 3-nitro-quinolines are potentially efficientradiosensitizers .

Isoptin reduces DNA damage by radiation and radiomimetic drugs

P. OHNESEIT and W. KOHNLEINInstitut fur Strahlenbiologie der Universitat Munster, D-4400 Munster, F .R. Germany

Isoptin (Verapamil), a common antiarrhythmic and vasodilatory drug, enhances theefficiency of peplomycin in Hela and mouse FM3A cells (Mizuno, Biochem. Biophys . Res .Commun., 107, 1021 (1982)) and the production of chromosome aberrations of peplomycin(PEP) and bleomycin (BLM) in human lymphocytes (Scheid et al . Experientia, 40, 746(1986)) . These results stimulated investigations of the combined effect of isoptin withradiomimetic drugs (PEP, BLM) and ionizing radiation at both the molecular and cellularlevel . The paper disc method was used to measure bacteriostatic effects in B . subtilis . Strandbreak production was measured by the conversion of supercircular Col El DNA into opencircular and linear forms, employing agarose gel electrophoresis . The spot tests indicate noinfluence of isoptin on the germination and growth inhibition effect of BLM and PEP .Production of single and double strand breaks, however, was considerably reduced in thepresence of isoptin (1 . 25 nmol dm -3 final) . Reduction factors of 5-9 for BLM and 3-5 for PEPwere found . Irradiation of Col El DNA in the presence of isoptin (0 . 14 and 0 .7 mmol dm -3 )yields protection factors of 4 .5 and 21 . The action of isoptin at the molecular level can beexplained by its OH-radical scavenging capacity . At the cellular level the isoptin-inducedmembrane modification apparently leads to a higher drug concentration within the nucleusand thus enhances chromosomal damage .

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