INT. J. RADIAT. BIOL ., 1974, VOL. 26, NO . 3, 251-258

Hypoxic protection at low X-ray doses : A fractionated dosestudy using Vicia faba

B. M. WINSTONt and R. J. BERRY$Research Institute, The Churchill Hospital, Oxford, U .K .

and R. OLIVERMedical Physics Department, The Royal Post Graduate Medical School,Hammersmith Hospital, London W12, U .K .

(Received 29 April 1974 ; accepted 13 June, 1974)

Inhibition of root growth in Vicia faba has been used to measure the oxygenenhancement ratio (o .e .r .) at small doses of X-rays . Measurable biologicaleffects were obtained by giving up to 11 fractions of each small dose, allowingsufficient time for intracellular repair between fractions .

No variation of o .e .r . with dose was seen . This result is in agreement withthe suggestion that the extrapolation number is unaffected by the state ofoxygenation at least down to < 100 p .p.m. 02 . It is not possible to compare theresults of Revesz and Littbrand who worked at much lower levels of oxygenation .

1. IntroductionAttention has been focused recently on the way in which oxygen modifies

the response of biological material to ionizing radiation . In most experiments,oxygen has been found to act as a simple modifying factor on the dose, thisfactor being known as the oxygen-enhancement ratio (o .e .r .) . It has beensuggested (Neary 1955, Porter 1965) that the initial slope of the survival curve,implying a single-event process, could be attributed to high-LET events, whichmight be expected to lead to a reduced o .e .r. at small dose and low dose-rates .However, Barendsen (1966) suggested that the LET in the terminal parts of theelectron tracks would not be high enough to reduce the o .e .r . Wideroe (1970)suggested that, under hypoxic conditions, the densely-ionizing high-LET` a-component ' is the only effect applicable, as the sparsely-ionizing low-LET` fl-component ' is reduced to zero . Thus he predicts that the survival curvehas an exponential shape under hypoxic conditions . Such a survival curvehas been observed by Revesz and Littbrand (1967), but only under conditionsof extreme hypoxia (< 2 p .p.m. 02) . If the survival curve is exponential underhypoxic conditions, the o .e .r. is no longer a constant but decreases with doseuntil, at low doses, oxygen acts as a protecting agent since the hypoxic survivalcurve crosses the aerobic curve .

To test this possibility directly, it is necessary to irradiate with small doses .However, small doses usually do not produce a sufficiently large biological effect-e.g. reduction in surviving fraction of cultured cells-to be measured accurately .Thus the extrapolation of a survival curve has to be estimated from the effectsof high radiation doses, which leads to inaccuracies in its estimation (Porter 1963) .

f Present address: Radioisotope Department, St. Bartholomews Hospital, London E .C.1,U.K .

I Present address : MRC Radiobiological Research Unit, Harwell, Didcot, Berks ., U.K .(N.B.-As from 1 October, 1974) .

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It is possible to obtain a large biological effect by giving multiple small doses insufficient numbers so that their equivalent single dose lies in the range which givesaccurately measurable effects . It is necessary to assume in planning such anexperiment that the shape of the survival curve is unaffected by radiation historyand that repair of sub-lethal damage is complete between fractions .

The root of the broad bean, Vicia faba, suggested itself as a suitable systemfor this investigation for the following reasons :

(1) By storing the beans at 3 .5 ° C, cell-division is almost completelyhalted (Savage and Evans 1959) . Thus the effect of a fractionateddose is not affected by cell proliferation .

(2) Repair of sub-lethal damage proceeds at this lower temperature andis 98 per cent complete within 12 hours (Hall and Lajtha 1963,Oliver 1964) .

2. Materials and methods2.1 . Culture o f the seedlings

The broad beans used were the variety ` Sutton's Prolific Long Pod' . Themethod of culture was substantially that of Hall (1961) . The beans were soakedin constantly running water at 19°C for three days to allow them to germinate,after which they were planted in moist vermiculite . After a further three days,when the roots had reached an average length of 50 mm, they were numbered,measured and transferred to the culture tanks, which were supplied with runningtap-water and were thermostatically maintained at 19°C . After one day, theroots were remeasured and those with abnormally high or low growth or lengthwere discarded . The remainder were divided into eight groups of twelve beanseach .

After irradiation, the roots were remeasured and the beans returned to the19°C culture tanks . The roots were allowed to grow for 10 days, after whichthey were remeasured and the incremental growth of each root was calculated .

2.2. DosimetryThe radiation source was a Maximar X-ray therapy unit operated at 220 kVp

and 15 mA with added filtration of 0 . 5 mm Cu and 1 mm Al (1 . 3 mm Cu h .v.l.) .An applicator was used to provide a field size of 120 mm x 100 at a f .s.d. of480 mm.

The dose-rate at the centre of the jig was measured with a Baldwin Farmerdosemeter, held in a Perspex insert which filled the centre section and replacedthe usual water. The dose-rate was checked at regular intervals and nevervaried by more than 2 per cent from the average value, which was 47.0 rads /min .The variation in dose-rate over the area in which the root-tips might lie was± 2 per cent, and the drop in dose-rate between the front and back of the rootcompartment was estimated to be 4 per cent . Thus the dose received byindividual beans would be expected to vary by up to ± 4 per cent .

2.3. Method o f irradiationThe jig in which the roots were irradiated was slightly modified from the

design of Hall (1961) to allow water and air to be supplied continuously to thebeans . The beans were placed in the jig, so that the root-tips were closetogether, certainly within a circle of 40 mm diameter ; to achieve this beans with

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shorter roots were placed at the centre of the jig and those with longer roots atthe sides . The thickness of the root compartment was 3 mm.

During irradiation, the jig was placed with its face flush with the front ofthe applicator to provide an easily reproducible set-up .

In all the fractionation experiments, the beans were kept at 3 .5°C forseven days . The jigs containing the beans were placed in a large refrigeratedwater-tank, with pumped circulating water through each jig . The flow-ratein each jig was about 100 ml/min . To avoid the shock of sudden transfer to thecold, the tank was filled with tap-water, the jigs were placed in it and then therefrigerator was switched on so that the beans were cooled over a period of a fewhours . Similarly, the beans were allowed to warm up slowly after removal fromthe cold before being replaced in the 19°C culture tanks . In all eight jigs wereused, enabling three doses to be given under standard conditions and three dosesunder test conditions with one control group for each set . Air was suppliedcontinuously to the jigs by four aquarium pumps, each serving two jigs . Theuncooled air supply did not produce a measurable increase in temperature .When hypoxic conditions were required during irradiation, the air and watersupplies were disconnected and nitrogen of ' white spot ' purity, supplied bythe British Oxygen Company, was bubbled through the jig at a flow-rate ofabout 1 litre/min for 35 min before the irradiation and also during it . Theeffluent gas was monitored with a Hersch cell, and it was found possible toreduce the oxygen concentration at this point to less than 100 p .p.m .

No attempt was made to keep the beans cold while they were transportedto and from the irradiation room, or during the irradiation itself, since theradiosensitivity has been shown to be virtually independent of temperature .The rate of heating of the water in the jig was measured and found to be0 .4°C/min while air or nitrogen was bubbled through in the irradiation room .Even for the longest exposure (about 10 .5 min for 500 rads) the temperaturewould not have exceeded 7 .5°C and in the majority of cases would not haveexceeded 5°C.

No irradiations were carried out within 24 hours of the beans beingtransferred to the cold or restored to room temperature, thus allowing five daysfor the irradiations . The interval between irradiations was 12 hours ; to ensurethat repair of sub-lethal damage was complete, the interval should have beenabout 20 hours but this would have restricted severely the number of fractionsthat could be given . As the half-time for decay of dose-equivalent of sub-lethaldamage is two hours at 3 .5°C (Oliver 1964), 12 hours is sufficient to reduce thedose-equivalent of 1/64 of its original value ; we therefore felt justified inirradiating at 12-hour intervals . The maximum number of fractions possiblewith these limitations was 11, and experiments were carried out with 1, 2, 5 and 11fractions . The fractions were timed so that the last fraction was always given24 hours before the beans were removed from the cold, i .e. on day 6. Initially,it was intended that each test condition (i .e . a certain number of fractionsdelivered in air or nitrogen) would be compared with a single dose deliveredunder aerobic conditions. On reviewing the results of the initial experiments,it was noted that the results of the single fraction experiments varied ratherwidely from the average in some cases, and more consistent results could beobtained by considering the result of each test condition separately. After thisdecision, experiments were therefore carried out with irradiation under hypoxic

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B. M. Winston et al .

conditions compared with irradiation under aerobic conditions for the samenumber of fractions .

The control groups were sham-irradiated under the same conditions as thecorresponding test groups . The procedure followed for sham-irradiations wasprecisely the same as for actual irradiations . The beans were gassed asappropriate, taken into the irradiation room and the X-ray set energized . Theonly difference between the handling of control roots and irradiated roots wasthat in the former case the shutter on the X-ray set was not opened . Thegrowth of the two control groups in each experiment were compared with eachother by means of an unpaired t test. In no experiment was the difference ingrowth of the two control groups significant at the 95 per cent level andaccordingly the growths of all the control roots were pooled within eachexperiment .

The index of damage used was reduction in growth in 10 days (G 10) . Thisis here defined as :

Incremental growth of one irradiated root in 10 daysG10 Mean incremental growth of control roots in 10 days

2.4 . Choice of dosesThe experimental doses which were given as multiple fractions were decided

on the best available three-parameter survival curve (Oliver 1964) .S=exp (-D/190)[1-{1-exp (-D/70)} 2]

Hall's experiments had shown that the range of single doses between 100 and200 rads would give a satisfactory range of G1 0i i .e. not too small while giving astraight line on the plot of G10 versus log dose . The intermediate dose waschosen as 141 rads to give a uniform separation between the doses on a log plot .On the assumption that the shape of the survival curve is unaffected by anyprevious number of fractions, the dose required in N fractions of d rads to beequivalent to a single dose of D rads is given by :exp (-D/190)[1 - {1 - exp (- D/70) }2]= exp (- d/190)[1 - {1-exp (- d/70) JI] N

This equation cannot be solved explicitly and so was solved by an iterativeprocedure using a WANG 700 programmable desk calculator . Doses at or nearto the predicted values were given in the experiments except for the first of thetwo fraction experiments in which the doses were 141, 200 and 288 rads . Alldoses given under hypoxic conditions were 2 . 5 times the corresponding dosegiven under aerobic conditions .

3. ResultsFigure 1 shows values of the mean G10 of a group plotted against the logarithm

of the dose . All the values of G 10 lie in the normal range 0 .2-0 .8 which hasbeen found to give a straight line in this type of plot . Thus the results areconsistent with the chosen parameters of the survival curve and the model forresponse to fractionated doses . The single dose-response in air was determinedin a large number of experiments, and therefore not all the points are shown forreasons of clarity ; instead the means and ranges for the values of G 10 are plotted .Straight lines were first fitted separately to each set of points by regressionanalysis . Lines having a common slope were then fitted and a variance ratio test

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Hypoxic protection at low doses

1 . 3

0. 8

0. 2

11 Fractions

IIIIII

100

200

300

500 700 900Dose (raos)

Figure 1 . Growth in ten days (G lo ) plotted against log dose showing the effect of 1, 2, 5 and11 fractions . The lines are all parallel and are fitted by regression analysis . Forsingle doses under aerobic conditions the mean and range of all experiments is shown .For the other cases, each point represents the mean G,o of a group and the errorbars indicate one standard error of the mean .

Table 1 . Experimental variation of o.e .r. with number offractions (95 per cent confidence limits shown in brackets) .

showed that the variance ratio was insignificant at the 95 per cent level . Fromthe parallel fitted lines, the o .e .r. and associated 95 per cent confidence limitswere calculated for each number of fractions and the values are shown in table 1 .The statistical calculations were based on the method described by Finney (1952) .

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Number of fractions o.e .r .

1 2 . 53(2 . 42-2 . 64)

2 2 .46(2 . 33-2 . 59)

5 2 .67(2 . 53-2 . 81)

11 2 .37(2 . 25-2 . 50)

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4. DiscussionThe possible theoretical variations of o .e .r. with dose may be considered

in relation to the 3-parameter survival curve equation previously discussed :S=exp (-D/D1)[1-{1-exp (-D/D„)}]

Wideroe's model leads to the result that the survival curve for cells irradiatedunder hypoxia would have an exponential form . (Model A .)

It is also possible that both processes are equally affected by hypoxia so thatthe survival curve has the same shape under all conditions of oxygenation(Model B) . Two further possibilities may be considered (Oliver, personalcommunication) . The single event process (cx-effect) may be unaffected byoxygen tension (Model C) or it may display an o .e.r. in between unity and theobserved high dose value of 2 .5 (Model D) . In either case, it is necessary toassume that the multitarget process is reduced in the exact proportion that theo.e .r. at high doses remains at a value of 2 . 5 . It is hard to visualize a physicalreason why this should happen .

Table 2 . Survival curve parameters predicted by fourdifferent theoretical models (see text) .

0

3.0

1.5

n (1)

Figure 2 . Variation of o .e .r . with dose. The four curves refer to the theoreticalmodels A, B, C and D (see text) . The cross-hatched areas refer to the experimentalresults for 1, 2, 5 and 11 fractions . The horizontal spread indicates the range ofdoses given in each number of fractions and the vertical spread indicates the95 per cent confidence limits,

D„rads

n Dlrads

Darads

Aerobic 70 2 190 51

HypoxicModel A 1 190 190Model B 175 2 475 128Model C 391 2 190 128Model D 209 2 333 128

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The comparison of these models was made by considering a survival curvewith the parameters deduced by Oliver (1964), where Dn = 70 rads, n = 2,D I =190 rads under aerobic conditions . The four models discussed lead topredicted parameters for anoxic conditions as shown in table 2 and the o .e .r .at different dose levels for each model was calculated from these parameters ; thecalculated values are plotted against dose in figure 2 . Experimental valuesobtained from the fractionation experiments are superimposed on the theoreticalcurves .

5. ConclusionsIt is clear that both models which predict no change in single event slope

with anoxia (Models A and C) may be excluded since they both lead to a fall ino.e .r. at small doses which is not observed experimentally . Of the remainingtwo models, model B (o .e .r.=2.5 for both processes) gives a constant o .e .r . at alldoses which provides the best fit to the experimental data . However, it is notpossible to exclude completely model D (single event process showing an o.e .r .of 1 .75) since the experimental data do not extend to sufficiently low doses .These results are consistent with Barendsen, and with Hall, Bedford and Porter(1966), who suggested that the reduction in o .e .r. seen at low dose-rates is causedby the suppression of repair of sub-lethal damage under hypoxia and not by themechanism of damage .

Although the oxygen concentration was measured only in the effluent gas,so that it is possible only to state an upper limit for the residual oxygen levelduring the ' hypoxic ' irradiation, it seems likely that this level was significantlyhigher than the < 2 p .p .m. 02 obtained by Revesz and Littbrand. It is thereforenot possible to draw conclusions about the validity of their finding of anextrapolation number of 1 under hypoxia from our present results .

On a utilise l'inhibition de la croissance radiculaire de Vicia Faba pour mesurer l'effetoxygen en utilisant de petites doses de rayons X . On a obtenu des effets biologiquesmesurables en administrant jusqu'a onze fractions de chaque petite dose et en laissantassez de temps entre les fractions pour la restauration intracellulaire .

On n'a pu observer aucune variation de l'effet oxygene avec la dose . Ce resultatconcorde avec la suggestion que le chiffre d'extrapolation n'est pas affecte par 1'etatd'oxygenation, du moins jusqu'a < 100 p .p.m. 0 2 . Il n'est pas possible de comparer lesresultats de Revesz et Littbrand qui ont travaille a des niveaux d'oxygenation beaucoupplus bas .

Die Reduktion des Wurzelwachstums in Vicia faba wurde benutzt, urn denSauerstoffverstarkungsfaktor bei Bestrahlung mit kleinen Dosen zu messen . McBbarebiologische Wirkungen wurden erhalten, wenn bis zu 11 Fraktionen von jeder kleinen Dosisgegeben wurden, wobei die Zeiten zwischen jeder Dosis so gewahlt wurden, daB Erholungvon subletalem Strahlenschaden moglich war .

Der Sauerstoffverstarkungsfaktor war unverandert bis zur kleinsten Dosis ; diesesResultat stimmt mit dem Befund iiberein, dal die Extrapolationszahl sich bis zuSauerstoffkonzentrationen < 100 p .p.m. 02 nicht verandert. Es ist nicht moglich, dieseErgebnisse mit den Arbeiten von Revesz and Littbrand zu vergleichen, da diese Autorensehr viel kleinere Sauerstoffkonzentrationen benutzten .

REFERENCES

BARENDSEN, G. W ., 1966, Br.Y. Radiol., 39, 155 .FINNEY, D. J ., 1952, Statistical Method in Biological Assay (London : Charles Griffin) .

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HALL, E. J., 1961, Br. J. Radiol ., 34, 314 .HALL, E . J ., and LAJTHA, L . G., 1963, Radiat . Res ., 20,187.HALL, E. J ., BEDFORD, J. S ., and PORTER, E. H ., 1966, Br. J. Radiol., 39, 958 .NEARY, G. J., 1955, In Progress in Radiobiology, Proc. IVth Int. Cong. Radiobiol .

(Edinburgh: Oliver & Boyd), 355 .OLIvER, R ., 1964, Int. Y. Radiat . Biol., 8, 475 .PORTER, E. H., 1963, Br . J . Radiol ., 36, 372 ; 1965, Ibid ., 38, 607 .REv sz, L., and LITTBRAND, B ., 1967, Conf. Radiation Biol . and Cancer, Kyoto, 1966

Publ. Rad. Soc. Japan, 1967, 67 .SAVAGE, J . R . K., and EvANs, H. J ., 1959, Exptl Cell. Res., 16, 364 .WIDEROE, R ., 1970, Radiol . Clin., 39, 20 .

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