Int. J. Rad. Biol., 1961, Vol. 3, No. 4, 369-377

Variability in the oxygen effect observed withmicro-organisms

Part II. Escherichia coli B

TIKVAH ALPER

Medical Research Council, Experimental Radiopathology Research Unit,Hammersmith Hospital, London, W. 12

(Received 13 June 1960)

Post-irradiation conditions of culture are considerably more effective inaltering the response of E. coli B to ionizing radiation if this has been deliveredin anoxic conditions. Thus the oxygen-enhancement ratio depends onconditions of culture, and has been found to vary from 1 6 to 37. A linearrelationship exists between 37 per cent dose and oxygen-enhancement ratio.This supports the inference that radiation causes lethal damage by inflictingat least two different types of injury, one of which is much more affected by thepresence of oxygen during irradiation.

1. INTRODUCTION

The effect of oxygen during irradiation may be assessed by irradiating similarpreparations of cells in anoxic and in fully aerobic conditions. The two dose-effect curves so obtained are compared, the ' oxygen-enhancement ratio' beingdefined as the ratio of doses required to give the same effect in both cases.When dose-effect curves depend on culture conditions after irradiation, theoxygen-enhancement ratio will be independent of these only if post-irradiationmodifying agents act to the same extent on anaerobically and aerobicallyirradiated cells. X-irradiated cells of Escherichia coli Strain B may be ' rescued 'by growing them on a poor medium (Alper and Gillies 1958 a), or by treatingwith metabolic inhibitors (Gillies and Alper 1959, Alper and Gillies 1960),and rescue occurs to a greater extent when the cells have been irradiatd in anoxicconditions (Alper and Gillies 1958 b, Gillies and Alper 1959). The oxygenenhancement ratio therefore depends on how the cells are cultured after irradia-tion.

With this strain, a further method of modifying the extent of damage due toionizing radiation is by preliminary incubation at different temperatures. Alperand Gillies (1960) reported briefly that preliminary incubation of both x- andultra-violet-irradiated cells immediately after irradiation on nutrient medium at20° and 30° brought more damage to light than was observed if they were incu-bated immediately at 37°c. On the other hand, it was found that post-irradiationincubation at 45°c resulted in the rescue of x-irradiated cells, as has been reportedto be the case after ultra-violet irradiation (Stein and Meutzner 1950, Anderson1951, Buzzell 1956). This method of treating E. coli B effected even greaterdifferences in its radiation response than had previously been reported; it wastherefore possible to test and verify the prediction (Alper and Gillies 1958 b) that,if a means were found of reducing the sensitivity of E. coli B to "less than itsvalue with Difco (medium), a still greater ratio of ' oxygenated' to ' anoxic'

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radiosensitivities will be observed ". The converse has also been verified, namelythat post-irradiation methods of enhancing the radiosensitivity beyond thatpreviously observed will concurrently reduce the effect of oxygen present at thetime of irradiation.

2. MATERIALS AND METHODS

The strain Escherichia coli B was the same as has been used in several previousinvestigations (Alper and Gillies, 1958 a, b, Gillies and Alper 1959, Alper aindGillies 1960).

2.1. Media

Two commercial ' complete' agar media, namely oxoid ' Blood Agar Base 'and Difco ' Nutrient Agar ' were used. These will be referred to, for brevity,as Oxoid and Difco media.

In one experiment, Difco Nutrient Agar plus NaCl, 4 g/l, was used.

2.2. Preparation of bacterial suspensions and control of oxygen

The cells used were always in the stationary phase of growth, having beengrown unstirred in nutrient broth at 37°c for 18-20 hours before harvesting.The suspensions were always thrice washed before irradiation, as previouslydescribed (Alper and Gillies 1958 a). The methods of gas control and of samplingwere as described by Howard-Flanders and Alper (1957).

2.3. Irradiation

Some of the irradiations were carried out with a Solus-Schall 300 kv constant-potential x-ray unit, operating at 250 kv and 10 mA, with no added filtration;the dose-rate was 1050 rads/min. Others were carried out with the 8 Mevlinear accelerator of the Medical Research Council Radiotherapeutic ResearchUnit (Batchelor, Bewley, Morrison and Stevenson 1959), as previously described(Alper 1959). This had the advantage that bacterial suspensions could beirradiated under anoxia and oxygen simultaneously. Since the radiosensitivityof E. coli B may vary somewhat with the time it is held in suspension beforeirradiation, it was useful to be able to check whether the oxygen-enhancementratio, as measured in simultaneous irradiations, was the same as when it wasmeasured by irradiating aerobic and anaerobic suspensions successively. Asshown by the figures, the results of the experiments were the same, whichevermethod of irradiation was used.

2.4. Incubation at differing temperaturesBefore samples from the irradiation vessels were seeded on the surface of the

plates, these were brought to the temperature at which incubation was to becarried out; they were returned as quickly as possible to incubators kept at therequired temperatures. Plates initially incubated at 19c were held at thistemperature for periods from 6 to 18 hours, and were then incubated at 37c forsome hours, until the colonies had grown large enough to be counted. Platesinitially incubated at 45c were left at that temperature for two hours, thenincubated at 37c overnight.

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Variability in oxygen effect: E. coli B 371

3. RESULTS

3.1. Incubation at three different temperatures on two mediaFigures 1-5 show typical survival-curves from viable counts after different

conditions of growth. It should be emphasized that samples were taken fromirradiation vessels and plated in parallel on the two media, some of each thenbeing incubated at each temperature under investigation. In effect, six survival-curves could be obtained in this way from each irradiated suspension, though inpractice not every variation of medium or incubation temperature was used inevery experiment. The points from several separate experiments have beenplotted in each case. Oxoid plates could not be left for longer than about 6 hoursat 19°c, otherwise the control counts tended to be low and erratic. Even whenthe counts were satisfactorily reproducible, control counts were only about60 per cent of those observed at the other two temperatures or with the othermedium.

Figures 1-5 illustrate the dependence of dose-response on culture conditions,with E. coli B, and also the greater dependence on these when the cells have beenirradiated in the absence of oxygen. The relationship between post-irradiationculture conditions and oxygen-enhancement ratio is illustrated by figures 3, 4and 5.

A noteworthy feature of the survival-curves is the dependence of shape, aswell as slope, on conditions of culture after irradiation. These conditions, there-fore, do not act as ' dose-multiplying agents'. Curves from viable counts onOxoid medium incubated at 19°c and 37°c, and on Difco medium incubated

IC 10-

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Figure 1. E. coli B irradiated anaerobically, plated on Difco. Preliminary incubationtemperature 19 ° , O; 37° , A; 45° , [C. Open and closed symbols refer respectivelyto irradiations with 250 kv x-rays and 8 Mev electrons.

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-IC0

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Figure 2. E. coli B irradiated aerobically, plated on Difco. Symbols as in figure 1.

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Figure 3. E. coli B plated Difco, preliminary incubation temperature 45° . Open symbols,anaerobic irradiations; closed symbols, aerobic irradiations. 95 per cent confidencelimits indicated.

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Variability in oxygen effect: E. coli B

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Figure 4. E. coli B plated Oxoid, preliminary incubation temperature 45° . Open symbols,anaerobic irradiations; closed symbols, anaerobic irradiations. 95 per cent con-fidence limits indicated.

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Figure 5. E. coli B plated Oxoid, preliminary incubation temperature 19° . Open symbols,aerobic irradiations; closed symbols, anaerobic irradiations. 95 per cent confidencelimits indicated.

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Variability in oxygen effect: E. coli B

at 19°c, show evidence of a ' resistant tail '. This apparently refers to a fractionof the irradiated population which does not respond to those conditions in whichthe maximum amount of radiation damage comes to light. The heterogeneity isevidently of physiological rather than of genetic origin, since the survival-curvesfrom counts on Difco at 37°c are exponential to levels of survival which areconsiderably lower than those at which the resistant tail appears. Pairs of curvestaken in the presence and absence of oxygen demonstrate that oxygen does act asa dose-multiplying agent; except that the enhancement ratio increases withdecreasing survival, when the curves have a resistant tail.

Each survival-curve may be represented by a characteristic form of equation,as shown by table 1: the symbolf is used to represent the fraction of cells formingcolonies after dose D. The exponent 1/Do is a measure of radiosensitivity, and itwas required to establish the value of Do, with each set of conditions, for anaerobicand aerobic irradiation, and hence the value of m, the oxygen-enhancement ratio.Do was calculated directly in every case from the fraction surviving each dose,the method of calculation used in each set of conditions being indicated in table 1.In the case of the sigmoid survival curves, the method used gave Do for the finalslopes, whereas for survival-curves with a ' resistant tail ', the Do calculated wasfor the larger and more sensitive fraction, i.e. for the initial slope of the curve.Table 2 shows the values of Do), and the corresponding values of m, determinedby taking arithmetic means of all values calculated from individual observations.Standard errors were calculated in the conventional way.

Preliminary Number of Do (kilorads) NumberMedium incubation observa- Ratio m ofpoint,

temperature tions used Anaerobic Aerobic figure 6figure 6

Difco 45 15 120 ±03 325 010 367 015 IOxoid 45 14 900+034 271 +0-06 3-32+0-13 2Difco 37 14 872 +0-17 301 +0-13 290 +±0-14 3Difco +

NaC 4 g/l 36 4 638 013 234 004 2'73 + 008 4Oxoid 37 6 247+ 0-08 122 + 005 202 021 5Difco 19 8 211 +011 118±+005 179±+011 6Oxoid 19 9 0.98 0-04 057 + 003 1.63 +±0-11 7

Table 2. Values of Do and of oxygen enhancement ratios for different culture conditions.

4. DISCUSSION

It has previously been suggested (Alper and Gillies 1958 a, b) that the variableresponse of E. coli B to irradation may arise from the fact that the overall responseis a summation of lesions occurring in at least two different sites (or differentbiochemical pathways), which may be called A and B. The cell fails to giverise to viable daughters if A is damaged, and it also fails to divide if B alone isdamaged, provided conditions are such that damage in B comes to light. Ingeneral, this happens when growth conditions are optimal. The cell survivesdamage to B when growth is slowed by poverty in the medium, or when synthesisis inhibited by chloramphenicol, or other metabolic inhibitors (Gillies and Alper1959, Alper and Gillies 1960). These facts suggest that damage to B is lethal

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when some other biochemical pathway is allowed to proceed, with a resultingimbalance in the growth of the cell (cf. Barner and Cohen 1956). Gillies (1961)has shown that, after uv, the pathway which brings the B lesion to light is con-cerned with active amino-acid metabolism; i.e. the B lesion inflicted by uv occursin some other pathway, probably concerned with nucleic acid synthesis.

The increased response to radiation which follows on preliminary incubationat 19°c would, on this hypothesis, occur because the reduced temperature actsdifferentially on the two metabolic pathways which get out of step when B isinjured, i.e. because there is some pathway less inhibited at 19°c than thebiochemical pathway in which lesion B is involved; conversely, incubation at45°c acts as a rescue mechanism because at this temperature the pathway con-cerned is relatively more inhibited than the metabolic system involving lesion B.

On the hypothesis that radiation effects on E. coli B are due to lesions in oneor the othei of at least two sites, the variability in the oxygen-enhancement ratiocan be explained if oxygen participates to a different extent in the metionicreaction (Alper 1957) which takes place in these sites. The data given here makeit possible to estimate the oxygen enhancement ratio for site B. Definingradiosensitivity as the exponent /Do in the equations to the survival-curves wemay call the sensitivities for sites A and B respectively AA and AB, in anoxicconditions, and mAAB and mBAB in aerobic conditions.

In any given set of culture conditions, the observed anoxic sensitivity will begiven by

A = AA + A,

A' depending on the extent to which the lesion in B comes to light. The observedoxygen-enhancement ratio, m, will be given by

MA = mAAB3 + mBA,i.e.

m = A(m A -m) + m~~~~mA

Since A is the inverse of Do,

m = AA (mA - mB) Do+ mA.

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FigureA6. Interdependence of oxygen-enhancement ratio and DJ. 95 per cent confidencelimits on both are indicated.

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Variability in oxygen effect: E. coli B

Thus m will be linearly related to D0 , and mB will be given by the intercept of thecurve on the m axis.

The values for m and Do shown in the table have been plotted in figure 6.The linear relationship is seen to hold, and mB, the oxygen-enhancement ratiofor the lesion to B, is about 1-5.

It is not possible to assign values to A, and mA, since it is not known whetherany damage to B is manifested even in those conditions in which AA is minimaland mA maximal. It can only be said that mA is not less than 37.

In earlier papers on the variability of the oxygen effect with this strain(Alper and Gillies 1958 b, Gillies and Alper 1959), it was inferred that the lesionto B was considerably less oxygen-sensitive than the lesion to A, and it wasimplied that there might indeed by effectively no oxygen enhancement for lesionB, i.e. that mB might equal one. The more accurate analysis made possible bythe present data leads to the conclusion that mB is somewhat greater than one,but it is so much less than the oxygen-enhancement ratio observed for the otherlesion or lesions as to suggest a metionic reaction of a very different chemicalnature.

Les conditions de culture apres irradiation influencent la survie de E. coli Souche Bbien d'avantage lorsque 'irradiation a t6 faite en anaerobiose. L'effet oxygene dpenddonc de ces conditions. I y a une correlation lin6aire entre la dose 37 pour cent et lefacteur par lequel l'oxygene modifie la rponse (la valeur de ce facteur variant de 1,6 3,7).Ces constatations supportent la these selon laquelle une irradiation est 16thale par au moinsdeux mecanismes, l'un de ceux-ci etant d'avantage implique lorsque l'oxygene est present.

Die Empfindlichkeit von E. coli B. Bakterien gegen ionisierende Strahlen wird sehrstark durch Wachstumsbedingungen nach der Bestrahlung beeinflusst. Die Beeinflussing istwesentlich stairker ausgeprigt, wenn in Abwesenheit von Sauerstoff bestrahlt word. Dierelative Wirkungssteigerung durch Sauerstoff hingt daher von Wachstumsbedingungen ab.Werte zwischen 1,6 und 3,7 wurden beobachtet. Die Wirkungssteigerung durch Sauerstoffund die 37 prozent Dosis hingen linear voneinander ab. Diese Befunde unterstuitzen dieVorstellung, dass ionisierende Strahlung ihre letale Schadigung durch wenigstens zweiverschiedene Arten von Schaden erzeugen. Eine dieser Arten wird durch Sauerstoffwahrend der Bestrahlung wesentlich mehr beeinflusst als die andere.

REFERENCES

ALPER, T., 1957, Radiat. Res., 5, 573; 1959, Int. . Rad. Biol., 1, 414.ALPER, T., and GILLIES, N. E., 1958 a, Y. gen. Microbiol., 18, 461; 1958 b, Nature, Lond.,

181, 961; 1960, J. gen. Microbiol., 22, 113.ANDERSON, E. H., 1951, J. Bacteriol., 61, 389.BARNER, H. D., and COHEN, S. S., 1956, J. Bacteriol., 71, 149.BATCHELOR, A., BEWLEY, D. K., MORRISON, R., and STEVENSON. J. A., 1959, Brit. J. Radiol.,

32, 332.BUZZELL, A., 1956, Arch. biochem. biophys., 62, 97.GILLIES, N. E., 1961, Int. J. Rad. Biol. 3, 379.GILLIES, N. E., and ALPER, T., 1959, Nature, Lond., 183, 237.HOwARD-FLANDERS, P., and ALPER, T., 1957, Radiat. Res., 7, 518.STEIN, W., and MEUTZNER, I., 1950, Naturwissenschaften, 37, 167.

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