Radiation sensitivity of Saccharomyces cerevisiae in relation to induced formation of intracellular catalase

Radiation

Botuny, 1965, Vol. 5, pp. 75 to 80. Pergamon

RADIATION IN RELATION

Press Ltd. Printed in Great Britain.

SENSITIVITY OF SACCHAROMZ’ES CEREVISIAE TO INDUCED FORMATION OF INTRACELLULAR CATALASE

G. B. NADKARNI,

R. JAY-, V. ‘R. NAIK, L. D. DESHPANDE and M. V. DIVFXAR Biology Division, Atomic Energy Establishment Trombay, Bombay 8 (Received

20 January

1964 and in revisedform

9 Sejtember

1964)

Abstract-Catalase activity of Saccharomyces cerevisiae increases as a function of dose of y-irradiation, This has been observed along with an exponential fall in the survival of the cells. Evidence has been presented to show that radiation induced increase in the enzyme activity was due to de novo enzyme formation. Radiation survival of the yeast cells could be modified by preinducing different levels of intracellular catalase, increase in survival being proportional to the preinduced level of the enzyme. . In spite of dependence of the radiation sensitivity on the intracellular catalase activities, it was observed that post-irradiation increase in the enzyme activity did not contribute towards counteracting the damage caused to the ability of the cells to form colonies. Metabolic implications of these observations have been discussed in relation to the processes of cell division and enzyme formation. R&um&L’activitt catalasique de Saccharomyces cerevisiae s’accroit en fonction de la dose de rayons y. Ce fait a Ctt observe en m&me temps qu’une dtcroissance exponentielle du nombre de cellules survivantes. On a mis en evidence que I’accroissement d’activitt enzymatique induite par les rayons ttait dit a la formation d’enzymes de rwvo. La survie des cell&s de levure aux radiations a pu etre modifite en induisant prtalablement des taux differents de catalase intracellulaire. L’accroissement de survie est proportionnel au niveau de l’enzyme prealablement induit. En d&pit dune dependance de la radiosensibilitt des activites catalasiques intracellulaires, on observe que l’accroissement de l’activitt enzymatique constcutif a l’irradiation ne contracarre pas la possibilite des cell&s de former des colonies. On discute les implications metaboliques de ces observations dans leurs relations avec les processus de la division ii cellulaire et de la formation des enzymes. Zusammes&ssung-Die Katalase-Aktivitit von Saccharomyces cerevisiae steigt bei Gammabestrahlung als Funktion der Do&. Gleichzeitig h&t sich eine exponentielle Abnahme des uberlebens der Zellen beobachten. Es wird gezeigt, dass der Strahleninduzierte Anstieg der Enzymaktivitat auf de-n&o-Synthese des Enzyms beruht. Das Uberleben der Hefezellen nach Bestrahlung konnte durch vorherige Einstellung verschiedener intrazellulirer Katalase konzentrationen modifiziert werden; dabei war die Erhbhung der uberleoensrate proportional zu der jeweils eingestellten Enzymkonzentration. Obwohl also die Strahlenempfindlichkeit von der intrazellularen Katalase-Aktivitiit der Enzymaktivitlt nach der abhlingt, konnte beobachtet werden, dass die Erhijhung Bestrahhmg nicht zur Verringerung des Schadens-gemessen in der Fahigkeit der Zellen zur Koloniebildungbeitrug. Die Bedeutung dieser Beobachtungen fiir Stofhvechselvorgiinge wird diskurtiertund in Beziehung gesetzt zu den Vorgangen der Zellteilung und Enzymbildung. 75

76

G. B. NADKARNI,

R. JAYARAMAN,

V. R. NAIK,

INTRODUCTION RECENTLY, attempts have been made to see if radiation sensitivity of a cell is related to its ability to destroy hydrogen peroxide formed in aqueous systems by ionizing radiation.(i*le) However, no unequivocal relationship has been obtained either in mammalian cells(ie) or in micro-organisms(i). Catalase activity and radiation sensitivity could not be correlated in Escherichia colW and in Rhodopeudomonas spheroides(0). Experiments with high catalase mutants of these organisms indicated that this enzyme was not a significant radiation protective agent.(i*sJ0J2) In genetically related respiration deficient and respiring strains of yeast, however, it was observed that radiation resistance could be linked to catalase activities(ls), but radiation resistance in them could not be altered by addition of hydrogen peroxide or crystalline catalase. Most of the observations with microorganisms are based on a comparison of catalase activities in genetically related mutants which showed different radiation viabilities. The present report describes studies on radiation sensitivity of a haploid strain of Saccharomyces cerevisiae in relation to induced formation of intracellular catalase.

METHODS The cells of Saccharomyces cerevisiae* were grown on agar slants containing glucose 2 per cent, Bacto-peptone 1 per cent, yeast extract O-3 per cent and agar 2 per cent, and were maintained on biweekly transfers. During the experiments the organism was grown on the same medium but without agar under practically anaerobic conditions as described by BHUVANESHWAFUN et a1.t4) The cells were harvested after 24 hr of growth, washed free of medium with ice-cold isotonic saline and suspended in a “non-growth” medium (pH 6.8) consisting of glucose, 1 per cent; KHsPOI, 1 per cent; MgS0,.7H,O, 0.01 per cent and CaCI,, 0.01 per cent. Cell suspensions 100-150 mg dry weight per 10 ml of this medium were used for aeration and irradiation.

*The strain of S. cerevisiae was obtained from Dr. D. V. Rege, Department of Chemical Technology, University of Bombay.

L. D. DESHPANDE,

M. V. DIVEKAR

Aeration was accomplished at 30°C by gently bubbling air through the suspension in 20 ml test-tubes. Irradiation was carried out in glass test-tubes (20 mm dia.) at room temperature with a 2.5 kc CosO-source at a dose rate of 2400 r/mm. The cell suspensions subjected to different doses of exposure to y-rays were used for determinations described below. A portion of the cell suspension was appropriately diluted with sterile water. Colony counts were made on agar medium having the same composition as described above, using pourplate technique. The plates were incubated at 37°C for 72 hr before colony counts were recorded. The cells washed free of the medium used during irradiation were resuspended in 0.25 M phosphate buffer pH 6.8. Catalase was determined using 0.002 M hydrogen peroxide as described by BHWANESHWARAN et aZ.14) The activity is expressed in “kat f” units in terms of disappearance of the substrate hydrogen peroxide per mg dry weight of cells used for irradiation and is not calculated on the basis of viable count obtained after exposure to y-rays. The enzyme assay used in these experiments, therefore, presents total intracellular catalase for the whole cell mass that was irradiated. Adenine-8-W solution (0.5 ~c/~mole/ml) was added to the 10 ml cell suspension just before or immediately after y-irradiation. The cells were processed to isolate RNA fraction as described by CHANTRENNE(~), three hours after the addition of labelled adenine. RNA content was estimated spectrophotometrically at 260 rnp using yeast RNA (Nutritional Biochemicals Corporation). The amount of radioactivity incorporated was measured on “Tricarb” liquid scintillation spectrometer (Packard Instruments Inc.). Adenine-8-C14 was obtained from the Isotope Division of our Establishment. RJEWLTS Figure 1 shows that catalase activity of the yeast cells increases with the dose of y-irradiation. The enzyme showed maximal activity, about 6-8 times that of the non-irradiated control at 200 kr above which there was no observable change. It can be seen that despite the rise in catalase activity, the ability of the cells

INDUCED

CATALASE

AND RADIOSENSITIVI’IY

to form colonies (survival) falls exponentially. This wouId suggest that increase in catalase brought about by irradiation could be independent of radiation survival. It was observed that when irradiated cells were kept in ice-bath, there was no noticeable increase in catalase activity and incubation at room temperature for 3 hr was necessary. Therefore, catalase assay was performed 3 hr after irradiation. The appearance of catalase on aeration in anaerobically grown yeast cells has been shown unequivocally by earlier authors as due to de nova enzyme formationt 4.‘) and the inducibility of this enzyme in the strain used in the present experiments has been establishedc4). Table 1 shows

77

levels of catalase at different periods of aeration and Fig. 2 presents survival curves obtained after subjecting these cells to varying doses of y-irradiation. Thus curve A gives the survival at the minimal level of the enzyme, while curves B, C and D at the increased levels. It can be seen that enhanced catalase levels afford a significant protective effect. The magnitude of the modifying effect of catalase could be more clearly observed from the values for LD,, and the dose modification factor (DMF) obtained for every preinduced level of the enzyme (Table 1). The survival curves having been exponential DMF could be calculated as the ratio of slopes of the curves at different enzyme activities(s). The ,250

225

T -175

$

- 25 04 ’ 0

’ 50

’ 100

’ 150

’ 200

6 250 DOSE

0

I 50

I 100

I 150

I 200

I 250

IN Kr

FIG. 1. Effect of gamma irradiation on catalase activities and the survival of cerevisiae. Vertical lines represent standard deviation from the mean of average values.

Saccharomyces

Table

1.

Dose modifying

efect

of preinduced

Time of aeration (min) Catalaseactivity (units) Lb,

W

DoseModification Factor (DMF)

catalase

levelsin S. cerevisiae

A

B

0 46

20

40

121

163

37

-

47

1.48

C

75

1.60

D 60 225 95

1.84

78

G. B. NADKARNI,

R. JAYARAMAN,

V. R. NAIK,

relationship of catalase to radiation survival could be observed only with cells aerated for short time intervals as shown in Table 1, which also showed significant differences in enzyme activity. Aeration for longer periods up to 120

o.,-

50

100 DOSE

150 200 IN

250

Kr

FIG. 2. Relationship of preinduced levels of intracellular catalase and the survival of S. cerevisiae. Curves A, B, C and D are for different enzyme levels induced by aeration as shown in Table 1.

L. D. DESHPANDE,

M. V. DIVEKAR

min gave the same survival values as for cells aerated for 60 min (curve D in Fig. 2). Catalase activity also was about the same. These experiments thus bring out that preirradiation levels can affect the radiation sensitivity of S. cerevisiae, the protective effect being in keeping with the increased enzyme activity. If increase in catalase activity after y-irradiation was due to de rwvo enzyme formation, then inhibitors of protein synthesis would cause impairment in the post-irradiation increase of the enzyme activity. In the present experiments the effects of 8-azaguanine(ll), ethionine(ls) and chloramphenicol(‘*) were studied. Table 2 shows that addition of these compounds inhibits radiation induced increase in catalase by about 70 per cent. It was observed that pre-treatment of the cells with these inhibitors did not have any effect on the viability of the cells. Whether these compounds were added immediately before or after irradiation did not make any difference wii respect to inhibition caused by them. It is known that induced enzyme formation follows an accelerated turnover of RNA, caused either by formation of new specific RNA molecules or by some change in the pre-existing RNA.t4*7*8*UJ3) Catalase formation in yeast has been shown to be accompanied by an increased incorporation of the labelled bases into RNAtrts). Table 3 presents the dose effect on the incorporation of adenine-8-04 into RNA nucleotides. It can be seen that irradiation causes enhanced appearance of the labelled base into RNA, concomitant to the magnitude of the dose received by the cells. This is paralleled by the increase in

Table 2. EJect of inhibitors on radiation induced increase in catalase activities in S. cerevisiae. To 10 ml cell surpension was added 200 Kg/ml of inhibitor immediate& af!er irradiation. Cataiase activity was determined 3 hr aftcl. this addition

Non-irradiated (control) Irradiated (200 kr) +8-azaguanine ,, +ethionine 3, + chloramphenicol ,,

Catalase activity (units)

Increase over non-irradiated control (per cent)

53+ 18 230 + 30 123537 141& 25 113k27

434 132 166 110

Inhibition of radiation induced increase in the enzyme activity (per cent) 69.6 61.7 74.6

INDUCED

CATALASE

AND

Tabk 3. Dose effecton th incorporation of aahine43-C14 into RNA fraction of S. cerevisiae

Dose of irradiation (W

0 50 100 200

Adenine-8-P incorporation into RNA cpm/mg of RNA Adenine added before irradiation

Adenine added after irradiation

1160 6700 7400 10,100

1160 3962 4424 5565

cpm added to 10 ml cell suspension were l-4 x 106. The cells were deproteinised with per&lo& acid 3 hr after addition of labelled adenine solution (see text). catalase activity as shown in Fig. 1. No changes in RNA content of the cells were observed after irradiation. It was interesting to note that adenine-8-Cl4 incorporation was quantitatively more when it was added to the cell suspension just before irradiation than when it was added immediately after. Since in this system no external inducer such as aeration has been used, the apparent changes in the extent of incorporation of the labelled base could be ascribed as due to the dose of irradiation only. DISCUSSION It has been observed above that by increasing the levels of intracellular catalase the radiation sensitivity of S. cerevisiae could be modified. Catalase addition to lysogenic strains of some bacteria brought about increase in their radiation survival though this effect was not observed with non-lysogenicstrainsoftheseorganisms. (%17) Since there were no differences in the intracellular catalase levels of lysogenic and nonlysogenic bacteria, it was suggested that the site of catalase protection could be around the bacterial surface. The results of the present studies bring out the fact that unlike in the above cited bacteria, the survival of the yeast cell is determined by the intracellular levels of this enzyme. This would explain why addition of crystalline catalase (as high as 700 units/ml) or HsO, to cell suspensions prior to or during irradiation did not alter radiation resistapce of

RADIOSENSITIVITY

79

genetically related strains of yeast(rs). Since there is a uniform distribution of the enzyme in the yeast cell@), it is unlikely that there are any particular sites for catalase action. It would be of interest to note that increase in catalase activity by irradiation is accompanied by simultaneous decrease in the viability of the yeast cells. This indicates that it is only the initial content of intracellular catalase that determines the radiation sensitivity. Damage to the mechanisms of cell division is not counteracted by post-irradiation increase of catalase activities. The mechanisms of cell division are known to be extremely sensitive to radiation(r*) and the survival index obtained by the plating technique would merely indicate the ability of the cell to divide and form colonies. ARONSONet al.@) had earlier observed similar increase in intracellular yeast catalase activity after Xand +-radiation. These workers explained it as due to alteration in the physical state of the enzyme similar to that reported by KAPLAN et al. (le) It was presumed that in yeast, catalase exists as a RNA-complex and radiation would release the enzyme from this complex. the possibilities of new enzyme However, synthesis could not be ruled out. Using Xirradiated yeast cells, CHANTRENNE and Dx.vaxux(s) showed that aeration could induce as much catalase in the irradiated cells as in the non-irradiated control. These observations indicated that the integrity of the induction mechanisms remained intact even after irradiation. The increased catalase activities after y-irradiation observed in the present studies could be arrested by 8-azaguanine, ethionine and chloramphenicol. Though these compounds differ in their mode of action, they bring about the same effect, namely, inhibition of protein synthesis.(lr~1sJ6) It was also accompanied by stimulated incorporation of adenine-8-Cl* into RNA. It would, therefore, appear that the increased enzyme activities were due to de novo catalase formation rather than liberation of the enzyme from the RNA-complex. In the cells in which maximal catalase induction was accomplished by aeration, no appreciable increase in the enzyme activity was seen after irradiation (unpublished observations). In bacterial systems catalase induction process was affected as much as the ability of the cells to form colonies.(e*ls)

80

G. B. NADKARNI,

R. JAYARAMAN,

V. R. NAIK,

However, in these organisms increased activities of catalase after irradiation as observed in yeast did not occnr.

9.

Acknowledgement-The authors wish to express their gratefulness to Dr. A. R. G~PAL-A~NCAR for his keen interest and valuable suggestions in this work.

10.

1. 2.

3. 4.

5.

6.

7.

8.

REFERENCES ADLER H. I. (1963) Catalase, hydrogen peroxide and ionising radiation. Radiation Research Sup@. 3, 110-129. ALEXANDER P. (1960) Protection of macromolecules in vitro against damage by ionising radiation. pp. 3-10. In A. HOLLANDER (ed.), Radiation Protection and Recovery. Pergamon Press. ARONSON D. L., FRASER M. J. and S~H C. (1956) Enzyme alteration by ionising radiation. Radiation Research 5, 225-237. BHUVANESWARAN C., SREE~~~AN A. and REGE D. V. (1961) The relationship of induced catalase synthesis to ribonucleic acid metabolism in yeast. Enzymologia 23,185-193. CALDAs L. R. (1959) Cell restoration after ionizing and non-ionizing radiation. Inter. Am. Symp. on the Peace&l Application of Nuclear Energy, 2nd, Buenos Aires, 53-64. CHANTRENNE H. (1944) Recherche sur des particles cytoplasmique de dimension, macromoleculaires riches en acid pentose nucleique, II. Relations avec les ferments respirator&. EnzymoZogia l&213-221. CHANTREM H. (1956) Metabolic changes in nucleic acids during the induction of enzymes by oxygen in resting yeast. Arch. Biochem. Biophys. 65, 414-426. CHANTRENNE H. and DEWREXJX S. (1959) Formation induite de catalase et metabolism des acides

Il. 12.

13. 14.

15. 16. 17. 18. 19.

L. D. DESHPANDE,

M. V. DIVEKAR

nucleique chez la levure. B&him. et Biophys. Acta 31,134-141. CLAYTON R. K. and ADLER H. I. (1962) Protein synthesis and viability in X-irradiated Rkodopseudomonas spkeroides. Biochim. et Biophys. Acta 56, 251-261. CLAYTON R. K. and RUDOLPH P. S. (1962) The lethal effects of alpha radiation on wild type and on high catalase mutant of Rhodopseudomonas spkeroides. Radiatian Research 17, 839-846. CREASER E. H. (1956) The effect of 8-azaguanine upon enzyme formation in Staphylococcus aureus. Biochem.3.64,539-545. ENGEL M. S. and ADLER H. I. (1961) Catalase activity, sensitivity to hydrogen peroxide and radiation response in the genus Escherichia. Radiation Research 15,269-275. GALE E. F. and FOLKES J. P. (1953) Action of antibiotics on nucleic acids and protein synthesis in Staphylococcusaureus. Biockem. 3. 53,493-498. GONZALES E. L. and BARRON E. S. G. (1956) The X-irradiation of haploid and diploid strains of yeast and its action on cell division -and metabolism. Biochim. et Biophys. Acta 19,425-432. HALVOR~ON H.O.and SPEIGELMANS. (1952)The inhibition of enzyme formation by amino acid analogues. 3. Bacterial. 64,207-22 1. KAPLAN J. G. and PAIK W. K. (1956) The action of ultraviolet radiation on yeast catalase. 3. Gen. Physiol. 46, 147-169. LATARJET R. and CALDAS L. R. (1952) Restoration induced by catalase in irradiated microorganisms. 3. Gen. Physiol. 35, 455-470. O’BRIEN R. T. (1961) Radiation sensitivity studies on related fermenting and respiring yeasts. Radiation Botany 1, 61-68. THOMSON J. F. (1963) Possible role of catalase in radiation effects of mammals, Radiation Research supp1. 3, 93-109,