R a d i o s e n s i t i v i t y o f Escherichia coli B B a c t e r i a C o n t a i n i n g D i f f e r e n t A m o u n t s o f :Nucleic A c i d s a n d P r o t e i n s

M I L E N A V~ZDALOV2~ and B.

LI~KA

Institute of Biophysics, Czechoslovak Academy of Sciences, Brno Received November 19, 1964

A B S T R A C T . Escherichia coli B bacteria cultivated under aerobic and anaerobic conditions were irradiated

with X-rays a t different phases of growth of the culture (at the outset and end of the logarithmic phase and in the stationary phase). Changes in the nucleic acid and protein content and in the number of nuclear equivalents per cell were determined in irradiated bacteria. The extrapolation n u m b e r of the survival curves increased proportionately to the increase in the cell components. I t was not in direct agreement with the increase in any of the individual components, however. The slope of the survival curves changed simultaneously with the extrapolation number, showing t h a t radiosensitivity changes were probably of a complex character and t h a t they might be the function of at least two factors, manifested as a change in the number and size of the targets (evaluated from the aspect of the target theory).

The X-ray sensitivity of bacteria is changed during cultivation under aerobic and anaerobic conditions (Hollaender et al., 1951). This applies in a similar manner to bacteria irradiated during different phases of growth of the culture (Stapleton, 1955). Radiosensitivity changes are also accompanied b y changes in the normal nucleic acid and protein content of the cell (Billen, 1959; Moiseenko, 1960). The cause of death of the cell after irradiation is probably irreversible destruction of given molecules and structures, which the cell is incapable of replacing and which are needed in an intact state to overcome the other effects of irradiation. Since these molecules or structures - - as far as their chemical composition is concerned - could belong to the nucleic acids or proteins, the authors attempted to determine whether radiosensitivity changes (evaluated from the shape of survival curves) corresponded to changes in the nucleic acid and protein content.

MATERIALS AND METHODS

Basic culture: Escherichia coli B (Delbriick & Luria, 1942) was cultivated 24 hours at 37~ on a slanted maintenance medium containing the following ingredients: nutrient broth (L.C.Gurr) 8 g, peptone (Spofa) 5 gr, Bacto tryptose (Difco) 5 g, agar 20 g, H20 1,000 g (sterilized for 25 rain at 1 arm). The culture was always kept in a refrigerator for three weeks at + 4 ~ and was then transferred to fresh medium. Cultures for irradiation were first cultivated at 37 ~ C in the apparatus described b y Sev~ik et al. (1964). They were cultivated in minimal medium (given in the same paper), enriched with 1 g peptone (Spofa) and 1 g tryptone per litre. The bacteria were cultivated in this medium until they were in the middle of the logarithmic phase of growth. This culture was used to inoculate 1,000 ml flasks containing 500 ml enriched medium

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(roughly 5 ml), so that the number of bacteria was about 5 • 10e/ml. The flasks had a side t u b e for collecting samples during cultivation. Another tube led through the ground glass neck and down the middle of the flasks into glass frit at the bottom, through which the gas (sterilized b y filtration) in the presence of which cultivation was carried out, was conducted into flask and dispersed. In aerobic cultivation, air was dispersed through the culture. In anaerobic cultivation, a mixture of 95 % nitrogen and 5 CO2 was used; traces of oxygen were removed b y passing the mixture through three washing vessels containing a solution consisting of 100 g pyrogallol, 105 g K O H and 500 g H~O. In both sets of experiments the flow rate of gas through the culture was 200 ml/min. Growth of the culture was studied photometrically, and the number of cells was determined b y plating on nutrient agar and microscopically, b y counting in a 'Ihoma counting cell. Growth of the cultures is illustrated in Fig. 1, which also shows the phases in which samples were collected for irradiation and analysis. Samples for irradiation were collected in the required amount (not more than 50 m]) from the various growth phases, spun down, washed in phosphate buffer o

1,0

O~ -4x 10 9

.~0 9

~ o,5

6

u.,

Z

-I0 8

0

5

10

-1ff 15

Time (hours)

Fig. 1. Growth curves of Escherichia coli cultures cultivated under aerobic (O~) and anaerobic (N2) conditions. The points marked on the curves indicate the collection of samples for irradiation a n d

analysis.

at p H 7 (4 parts 1/15M KH2PO 4 - b 6 parts 1/15 M Na~POd), respun and suspended again in phosphate buffer to give a bacterial concentration of 1--6 • • 10~ cells/ml. The bacteria were irradiated with a Chirana X-ray apparatus (180 kV, 15 mA, focal distance 25 cm, no filters, homogeneously irradiated field, dose rate 456 r / /rain.). The culture was irradiated in small Petri dishes 5 cm in diameter, covered with cellophane; the layer of bacterial suspension was about 2.5 m m i n depth. The suspensions was exposed successively to 1,140; 2,280; 4,560; 9,120; 13,700 and 18,200 r at laboratory temperature (19--24 ~ C). The irradiated suspension and the unirradiated control were suitably diluted with phosphate buffer and the number of surviving cells was determined b y plating on nutrient agar. The shape of the cells and the number of nuclear equivalents were also determined microscopically at different phases of growth of the culture. Smears from a spun bacterial suspension, fixed b y heat on slides, were hydrolysed in 1 ~ HCI at 60 ~ C for 10 minutes. They were then washed with distilled water and stained with 0.2 ml Giemsa-Romanowski staining solution (Druchema) diluted with 10 parts 1/15 ~ phosphate buffer at p H 6.4 When staining was completed, the slides were washed and dried; they were then observed in the microscope and the number of nuclear equivalents in the bacteria was counted. One hundred cells were always evaluated, b y determining the proportion of cells with different numbers of nuclei and the mean value. Preparation oI specimens /or the determination oI nucleic acids and proteins: Culture samples were collected at various phases of growth, denoted in Fig. 1, together with samples for irradiation. When determining the D N A content, bacterial suspension was collected in amounts containing 1--3 • 10z~ ceils, and when determining R N A and proteins in

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1966

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amounts containing about 1 x 10 9 cells. usual methods. The mean values of five 'Ihe number of cells was determined in experiments are given in the results; the a 'Ihoma counting cell and by seeding on statistical errors in the figures and tables nutrient agar. ~he samples were spun are the estimated standard errors of the down and resuspendedin a small amount of selective means a x (Myslivec 1957, p. 194, distilled water, in a suitable concentra- for n = number of experiments) or have tion. These samples were frozen and were been calculated from the variance about used the next day for the determination the regression line (v. Tab. 2). of nucleic acids and proteins. DNA was extracted from the cells and R E S U L T S determined by the diphenylamine reacFig. 2, 3 and 4 give the survival curves tion by Burton's method (1956), after thawing the samples at + 3 ~ C and acidi- for the same phases of growth of bacterial fying them to a final HCIO~ concentra- cultures cultivated under aerobic a n d anaerobic conditions. ~ h e y show t h a t the tion of 0.25 N. RNA and proteins were determined in the same sample. The culture was first precipitated and washed twice with cold 10~o trichloroacetie acid (TCA). I t was then resuspended in 5 ml 5% TCA and extracted 40 minutes in a water bath at 90 ~ C. After spinning, RNA was determined in the extract by Ceriotti's technique (1955) and proteins were determined in the sediment by the technique of Lowry et al. (1951). Statistical evaluation: The experiments 10-1. were repeated at least five times, independently of each other. The determinaN2 tions in the individual experiments - - as regards the number of parallel measure- s ments in the various phases of the ex- z periment - - were carried out by the Table 1. Values of exposure in kr which c o n s t a n t l y inactivates 63~o of t h e cells in t h e linear portions of t h e survival curves. Statistical errors: -4-1 standa r d error of t h e m e a n Culture eultir a t e d under aerobic conditions

Culture cultivated under anaerobic conditions

Onset of logarithmlo phase

3.20 :E 0.24

3.52 • 0.24

E n d of logarithmic phase

3.59 ~ 0.09

3.93 ~= 0.22

M a x i m u m stat i o n a r y phase

4.09 :t: 0.34

4.86 ~: 0.29

Growth stage of culture

10-2.

4

8

Exposure in

12

16

20

kiloroentgens

Fig. 2. Survival curves of Escherichia coli cultures cultivated under aerobic (02) and anaerobic (N2) conditions, a t t h e o u t s e t of t h e logarithmic g r o w t h phase. N = t h e n u m b e r of cell/ml, f o r m i n g colonies after irradiation a n d N 0 = t h e n u m b e r of cells]ml. forming colonies before irradiation.

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MILENA VfZDALOVz( AND B. L I ~ K A

culture cultivated under anaerobic conditions survived better, than t h a t under aerobic conditions. In addition to the extrapolation number, the slope of the curves also changed, with a more abrupt drop in the survival curve of the aerobic culture (which was more radiosensitive). Changes in the slope of the survival curves can be characterized by the exposure which constantly inactivated 63~ of the cells in the linear portions of the survival curves. The respective values of the exposures corresponding to these doses are given in kr in Tab. 1. In any interpretation of the survival curves as single or multi-target curves (v. Discussion), those doses also characterize the size of the targets.

Vol. I1

I t can be seen (Fig. 2, 3 and 4) t h a t only bacteria cultivated under aerobic conditions, in the stationary phase and at the end of the logarithmic phase of growth, gave single-hit, single-target sur~ viral curves. The survival curves of bacteria cultivated under anaerobic conditions and at the onset of the logarithmic phase of growth of bacteria cultivated under aerobic conditions were either of a multi-hit or a multi-target character (evaluated according to the target theory). In association with these findings, it should be noted t h a t the proportion of the cell components (Tab. 2 and 3) was lowest in bacteria which gave single-target, single-hit curves. These cells also had only one nucleus. The amount

lO"-4

10-1

2

\ N2

Ii

Z

O:

10-7 .-~

9

10-2.

O:

|

I

I

4

8

12

I

lb

20

Exposure in kiloroentgens Fig. 3. Survival curves of Escherichia coli cultures a t t h e end of the logarithmic phase. Other detailB as in Fig. 2.

1'2

,'6

2'0

Exposure in kiloroentgens Fig. 4. Survival curves of Escherichia cell cultures in stationary phase. Other details as in Fig. 2.

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Table 2. Relative content of DNA, RNA, proteins (prot.) a n d n u m b e r of nuclei per cell a n d extrapolation n u m b e r (n). Aerobically cultivated E. cell B culture. The values determined in cells from the stationary phase have been t a k e n as unit quantities. The statistical errors in determining the cell components a r e s t a n d a r d errors of the mean. The statistical error of the extrapolation n u m b e r has boon calculated with the aid of the formula of variance about the regression line V~ar y(z) (Roth et al., p. 117,1962). DNA

RNA

Prot.

Nuclei

Onset of logar, phase

2.56 -V 0.24

5.48 • 0.68

4.57 4- 0.43

3.27 4- 0.33

E n d of logar, phase.

1.31 4- 0.16

1.42 -P 0.18

1.37 4- 0.10

1.35 4- 0.15

n

I

I

3.80 4- 1.30

1.00 -P 0.09

I J

Stationary phase

1.00 4- 0.11

1.00 • 0.12

of the cell components contained in bacteria cultivated under aerobic conditions, in the stationary phase of growth, was therofor~ taken as the basic unit amount in relation to the amount contained in the other samples of bacteria, which were larger and multinuclear. Since the increase in the nucleic acid and protein content was accompanied by an increase in the extrapolation number of the survival curves, the relationship between the increase in the amount of cell components per cell and the increase in the extrapolation number was determined. This relationship is illustrated in Tab. 4 and 5. I f the standard errors are taken into account (only zx is given in the tables), it is seen t h a t the quotient given in Tab. 4 and 5 does not differ

1.00 -4- 0.10

1.00 • 0.05

]

f

1.00 • 0.17

demonstrably, in most cases, from 1 and in the other cases is in the region of 1. This means t h a t the extrapolation number rose proportionately to the increase in the amount of the cell components. This increase in the extrapolation number, which (from the aspect of the target theory), under given conditions, represents the number of targets, d!d not however, conform directly to the increase in the amount of a n y of the individual components. DISCUSSION

One of the questions to which constant attention is paid in radiation biophysics is the type of cell structures and molecules which are injured by radiation in such

Table 3. E. cell B culture, cultivated under anaerobic conditions. The values determined in cells from the s t a t i o n a r y phase of the culture, grown under aerobic conditions, have boon t a k e n as unit quantities. Other details as in Table 2 DNA

RNA

Prof.

Nuclei

n

2.71 q- 0.21

4.93 -4- 0.52

3.96 • 0.46

3.01 =k 0.40

4.00 • 1.20

E n d of logar, phase

1.78 4- 0.12

2.08 ~: 0.18

1.66 ~'- 0.09

Stationary phase

2.11 4- 0.23

1.52 4- 0.18

1.42 • 0.12

1.58 ~ 0.17

2.40 • 0.35

Onset of logar, phase

1

26 M I L E N A VfZDA_LOVX A N D 13. L I ~ K A

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Table 4. Relationship between increase in content of cell components a n d increase o f t h e extrapolation n u m b ) r in culture c u l t i v a t e d u n d e r aerobci c o n d i t i o n s . The values of the c o n t e n t o f cell components a n d n used for calculation a n d t h e explanation of the symbols are presented in Table 2 I)NA : n

RNA : n

I

]

Prot. : n

l

I

I

Nuclei : n

1 $

Onset of logar, phase

E n d of logar, phase

Stationary phase

0.67 :k 0.29

1.44 q- 0.67

1.20 • 0.16

'1.31 :k 0.28

1.42 • 0.22

1.37 q- 0.22

1.00 :k 0.28

1.00 ~ 0.29

1.00 =L 0.27

[

0.86 • 0.38

1.35 -4- 0.16

1

1.00 -t- 0.22

I concentration of one substance, b u t also to the other changes which take place in such cells. I t ensues from the target theory that different factors, such as (1) an increase in the essential number of hits on one target, (2) an increase in the number of interchangeable targets and (3) a change in the size of the target, affect the change in the shape of survival curves in different ways. Variations in radioresistance cannot therefore be evaluated only from the change in survival (e.g. only for a single radiation dose), but changes in the slope of the survival curves, the extrapolation number and the shape of the survival curves must also be taken into account, especially in the portion where the curve passes over to an approximately straight line. It is not always possible, on the basin

a manner as to cause the death of the cell. The target theory provides a possibility of verifying hypotheses on the decisive role of certain targets from the course of survival curves. An association was already found between the amount of certain substances - - particularly deoxyribonucleic acid - - in the cell and survival (Billen, 1959; Moiseenko, 1960). Kaplan and Moses (1964) recently submitted an interesting elaboration of a number of these data. An increase in the content of cysteine (Vinter, 1960) and other substances also raises radioresistance, however. Furthermore, in multinuclear cells, the concentration of other components per single multinuclear cell increases together with the DNA content. Attention should therefore be paid not only to changes in the

Table 5. Relationship between increase in content of cell components a n d increase of t h e extrapolation n u m b e r in culture, cultivated under anaerobic conditions. The values of the content of cell components and n used for calculation are presented in Table 3. The explanation of the symbols a n d other details as in Table 2 DBIA : n

Onset of logar, phase

0.68•

E n d of logar, phase

0.77 4- 0.18

Stationary phase

0.88 -4- 0.22

RBTA : n

1

Prof. : n

/ffuelei : n

0.99 :]: 0.41

0.754-0.30

0.91 ~: 0.23

0.72 -4- 0.16

0.85 i 0.23

0.63 4- 0.17

0.59 -4- 0.13

0.66 • 016

1.23 ~ 0.50

[

i

I

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of statistical errors, to differentiate a given group of survival curves corresponding to widely differing combinations of the number of targets and hits (Zimmer, 1962). 9 he fact that the total effect of radiation, culminating in the death of the cell, is composed of a number of factors (Powers, 1962), should also be taken into account. ~[he cause of a change in radiosensitivity cannot therefore be demonstrated without verifying which factors participate in the total effect. ~he results of study of survival curves can, however, confirm some hypotheses on the causes of changes in radiosensitivity. I f the survival curves obtained in the present experiments are evaluated from this aspect, they could be regarded, on the basis of the statistical errors, as single-hit, single- or multi-target curves; the size of the target as well as the number of targets (expressed b y the extrapolation number) changes, according to the target theory equation: .N -- 1 - - 1 (1--eVD)"

NO

in which N = the number of living cells per ml after irradiation, N o = the number per ml before irradiation, e = 2 , 7 1 8 . . . , V = the volume of the target in co, D = the number of processes (ionizations, excitations) in co, which can destroy the target, and n = the number of targets. I t can also be assumed that cells with a larger number of nuclei and cell components will also have a larger number of targets (macromolecules or structures) capable of independent function. The concept of the "target" should not be understood only statically, b u t the dynamic concept, as formulated b y HerSik (1946, p. 331), should also be taken into account. ~he results of these experiments are in agreement with the above views. 9 he increase in the concentration of the cell components was accompanied b y approximately the same increase in the number of targets. ~he proportionality of

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this increase is especially noticeable if it is borne in mind that the increase in the concentration of the various components attained three- to fivefold values at the onset of the logarithmic growth phase. Pronounced differences might therefore have occurred in the ratio of the number of targets to the increase in the concentration of the cell components. Fig. 3 and 4, however, show that the increase in the number of targets was in relatively good agreement with the increase in the amount of the cell components. The increase in the number of targets did not conform directly to the increase in the concentration of any of the individual substances studied. ~Ihe statistical errors of this determination are also relatively high and do not preclude the possibility of differences from the measured means with the appropriate probability. It must therefore be stressed that the results of these experiments do not demonstrate that the relationship between the increase in the number of nuclei or in the amount of one or other of the cell components and the increase in the extrapolation number had definitely the quotient 1. [[hey only indicate the possibility of this relationship. [Ihe only significant determination was a change in the shape of the survival curves, characterized (1) b y a change in the slope of the curves and (2) b y a change in the extrapolation number, which can be attributed to an increase in the number of targets and a change in their size in cells in which the DNA, R N A and protein concentration and the number of nuclei increased. Detailed mathematical evaluation of the interrelationship of these parameters would need much more extensive experimental material. 9 he shape of survival curves (and hence the extrapolation number) can also be influenced by the presence of protective substances in the cell (Powers, 1962); this would be manifested in the shape of the survival curves as a progressive increase in the size of the target during irradiation. The presence of a given amount of pro-

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MILENA u

AND B. L I ~ K A

cursors injured b y radiation and their incorporation into a structure which is itself a target, can have the opposite effect on the course of hit curves. Although the authors evaluate the findings given in Fig. 3 and 4 with some reserve, it is absolutely clear, however, that the extrapolation numbers rise with the increase in the nucleic ,acids and protein content of the cell. In this respect, the results of these experiments are in agreement with those of Sargent (1961) in irradiation of mono- and multinuclear Escherichia coli B/r cells. From the data given b y Sargent, it is evident that the survival curves of multinuclear cells have a higher extrapolation number than the curves for mononuclear cells. On the basis of his results, Sargent also stated that multinuclearity of the cells was induced b y an acid reaction of the medium in anaerobic cultivation. In the present experiments, the slope of the linear portion of the survival curves changed together with the extrapolation number. With reference to the above hypotheses, this could be explained as a change in the size of the target, or as a decrease in the number of sub-targets.

That indicates that the changes in the cells were of a complex character and that they did not simply involve an increase in the number of targets. Since changes occurred simultaneously in the number and size of the targets, they were manifested in changes in the "general radiosensitivity" of cells from different phases of growth of the culture. The results in the aerobic culture were basically in agreement with those of Stapleton (1955), b u t unlike the aerobic culture, the irradiated culture cultivated under anaerobic conditions was most resistant in the stationary phase. Differences in the resistance of the anaerobic culture at the onset and end of the logarithmic phase were relatively small. It cannot therefore be said that radiosensitivity to the doses which more than 2 ~ of the cells survived was in this case proportionate to the nucleic acid and protein concentrantion in the cell. ~he authors are aware that the concept of the "more or less radiosensitive culture" is very vague, since with larger doses the curves would probably intersect each other (as indicated b y their slopes) and their sequence would therefore also change.

References Billen, I).: Alterations in the radiosensitivity of Escherichia coli through modification of cellular maeromolecular components. Bioehim. biophys. Acta 34 : 110, 1959. Burton, K. : A study of the conditions and mechanism of the diphenylamine reaction for the colorimetric estimation of deoxyribonucleic acid. Biochem. J. 62 : 315, 1956. Ceriotti, G. : Determination of nucleic acids in animal tissues. J. biol. Chem. 2 1 4 : 5 9 , 1955. Delbriick, M., Luria, S. E.: Interference between two bacterial viruses acting upon the same host and the mechanism of virus growth. Arch. Biochem. 1 : 111, 1942. IterSik, F.: .From the atom to life. P r a h a 1946. Hollaender, A., Stapleton, G. E., Martin, F. L.: X - r a y sensitivity orE. eoli as modified by oxygen tension, l~ature 167:103, 1951. Kaplan, I-[. S., Moses, L. E.: Biological complexity and radioseusitivity. Science 145 : 21, 1964. Lowry, H. O., Rosebrough, 1~. J., Farr, A. L., Randall, R. J. : Protein measurement with the folin ohenol reagent. J. biol. Chem. 193:265, 1951.

Moiseenko, E. V.: Nucleic acid content and radioresistance in Escheriehia coli. Biophyzika 5 : 176, Myslivee, V.: Statistical methods in agricultural and forestry research. Praha 1957. Powers, E. L.: Considerations of survival curves and target theory. Phys. Med. Biol. 7 : 3 , 1962. Roth, Z., Josifko, M., Mal~, V., TrSka, V.: Statistical methods in experimental medicine. P r a h a 1962. Sargent, T.: Effects of p H and anoxia on the cell morphology and X-ray sensitivity of Escherichia coli. Rad. Res. 14:323, 1961. Stapleton, G. E.: Variations in the sensitivity of E. eoli to ionizing radiations during the growth cycle. J. Baeteriol. 70:357, 1955. ~evSLk, F., Liw B., ]:Io~ek, B.: Apparatus for automatic inoculation and recording of growth curves of bacteria. Fol. mierobiol. 8 : 125, 1964. Vfilter, V.: Spores of microorganisms. V I I I . Th~ synthesis of specific calcium and cystine-containing structures in sporulating cells of Bacilli. Fol. microbiol. 5 : 217, 1960. Zimmer, K. G.: The problems o] quantitative radiobiology Moskva 1962.