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B i o c h i m i c a e t B i o p h y s i c a A c t a , 497 (1977) 615--621 © Elsevier/North-Holland Biomedical Press

BBA 28202 AN ELECTRON PARAMAGNETIC RESONANCE STUDY OF FREE RADICALS IN CELLS

ANNA LAURA SEGRE a, ARRIGO BENEDETTO b, TAMILLA EREMENKO c, PIETRO VOLPE c, ALFREDO DI NOLA d and FILIPPO CONTI d a L a b o r a t o r i o di Chimica e Tecnologia dei R a d i o e l e m e n t i C.N.R., Padova, b Centro di Virologia, O.O.R.R., R o m a , c I s t i t u t o I n t e r n a z i o n a l e di Genetica e Biofisica del C.N.R., N a p o l i and d I s t i t u t o Chimica, Universita di R o m a , R o m a (Italy)

(Received September 27th, 1976) Summary An electron paramagnetic resonance study was performed on cell lines of the following strains: HeLa, 37RC, L, FLC, NRK/RSV, 3T3/SV40. Unsynchronized and synchronized HeLa cells were studied with particular attention paid to the relation between growth and free radical concentration. Free radical levels were shown to be a function of the growth stage and different phases of the cell cycle.

Introduction Mechanisms which control growth rate in eucaryotic cells constitute a fundamental part of biology, both for their general interest as well as for their basic connection with the cancer problem. Many papers in the recent literature involve the study of free radicals as related to their chemical composition and to their cellular concentrations [1--6]. It has been suggested by Burlakova [7] t h a t in cells, reactions involving the generation of free radicals should be controlled by various inhibitors. In a steady state situation the ratio of free radical activators to these inhibitors can then be expressed as a constant K, which is equal to free radical concentration. The actual value of K then affects the mitotic cell state. It was also suggested [7] that a larger value of K, as derived, for example, by an increased rate of initiation of free radical reactions or by a lower activity of inhibitors, should constitute a trigger to stop mitosis. In this context, a reduction of free radical concentration should be reflected in a less~controlled cell growth. This problem was approached in vivo [3] by following the effect on t u m o r growth of substances able to perturb free radical reactions e.g. oxidizing or reducing agents, [8--10], and by studying lipids in diets containing oxide radicals [1,11--15].

616 In none of these studies, however, has a distinction been made as to whether the variation in radical levels observed in tumors derives from variations in initiators or in inhibitory factors present in the t u m o r cells themselves, or is due to external agents (e.g. convection of the blood, and/or external induction by the t u m o r itself). Moreover, although many EPR signals ascribed to free radicals are reported in the literature, only one at g = 2.003 is present in all living cells [ 1]. Studies on the free radical content in fractionated tissues show that the signal is present only in the mitochondria-enriched fraction and was assumed to be due to a semiquinone radical [ 16--17]. The intensity variations in this signal have been related to cancer growth [ 3]. For all these reasons, we decided to carry out a study of the free radicals in cell cultures in vitro. This system is reproducible, biologically well-characterized, and external contamination by paramagnetic materials can be carefully controlled. Materials and Methods All cell strains employed in this study were maintained as monolayers in glass bottles in Eagle minimum essential medium (from Grand Island Biological Co. U.S.A.) supplemented by 5% fetal calf serum and antibiotics (1000 Units Oxford penicillin and streptomycin 100 pg/ml). The cells were seeded at a density 4 • 103 cells/cm 2. Non,synchronized cells were detached from bottle walls with trypsin and centrifuged at 800 × g. Synchronized cells were decanted. Nutrition liquid was removed (it does not contain appreciable EPR signals) and cell strains were taken up with a pasteur pipette and transferred into quartz EPR tubes (internal diameter 3.8 mm). HeLa cells were grown in suspension [18] and synchronized with a double thymidine block [ 19]. The length of each phase of the cell life cycle was monitored as reported elsewhere [20--21]. Starting from the removal of thymidine from culture medium, at hourly intervals during the whole mitotic cycle, samples of culture suspension containing 20 • 106 cells were withdrawn and washed in a large volume of Hank's salt solution (from Grand Island Biological Co. U.S.A.). EPR tubes, containing the centrifuged or decanted cells, were quickly frozen in liquid N2 *. Samples, treated this way, and kept at liquid nitrogen temperature for one week show an EPR spectrum which does n o t change. All EPR spectra were run on a Varian E-line spectrometer operating near 9200 Mc/s. Before and after every experiment, ESR signals were calibrated by measuring the frequency and the intensity of a standard sample (Varian weak pitch EPR sample containing 10 ~3 spin/ cm). Due to the weakness of the g = 2.003 signal, computer-facilitated spectral accumulation was necessary. This was done by connecting the EPR spectro* We w i s h t o p o i n t o u t t h a t i n m a n y s t u d i e s i n v o l v i n g b i o l o g i c a l m a t e r i a l t h e h i g h l y q u e s t i o n a b l e practice of lyophilization or drying has been employed. This has often led to sample contamination presumably due to excessive manipulation. A complete review of experimental methods and d r a w b a c k s i n u s i n g l i v i n g - m a t e r i a l f o r E P R c a n b e f o u n d i n R e f . 1. I n o u r s t u d y t h e u s e o f f l a t rectangular cavities suitable for room temperature studies was impossible, due to the weakness of t h e E P R signals. We t h e r e f o r e o p e r a t e d at 77 K o n f r o z e n s a m p l e s [ 2 2 - - 2 3 ] .

617

meter to a Laben Correlatron 4096. The interface and the trigger of both systems were home-made. All spectra were accumulated 25 times. To prevent bubbling in the Dewar flask, a small flux of helium gas was bubbled into the dewar immediately over the cavity. Results a. Presence o f free radicals (g = 2.003) in cell lines Cell lines were studied in the stationary phase of growth. The presence of an EPR signal with g = 2.003 has been observed in all the cell lines studied (Table I and Fig. 1), including those from normal tissues (37 RC, NRK), embryos (L), t u m o r tissues (HeLa) and cultures transformed by RNA or DNA oncogenic viruses (NRK/RSV transformed, 3T3/SV40 transformed and erythroid Leukemia cells transformed by Friend leukemia virus). Under these conditions, the EPR signal showed a different intensity for the various cell strains. As shown in Table I, the EPR signal has a smaller intensity in t u m o r cells as compared to normal strains, especially notable in NRK cells and their transformed counterparts [ 5,24--27 ]. However, a certain a m o u n t of variation (reproducibility approx. 50%) was obtained for the same strain in different experiments, analogous to the scattering of previously described experiments performed on tissues in vivo [2]. To investigate whether signal intensity fluctuations are biologically meaningful, we carefully studied the intensity of the EPR signal at g = 2.003 as a function of the cell growth state. Here, HeLa cells were chosen as a model of human t u m o r in vitro. b. EPR g = 2.003 signal intensity and cell growth stage in HeLa cells The cells were grown in monolayers in glass bottles. The cell growth, shown in Fig. 2, is expressed by the usual exponential curve. It begins to decline at a density of 10 • 104 cells/cm 2. Beyond this value, and w i t h o u t any changes in the medium, cell death begins, as is revealed by the detachment of the cells from the monolayer. There is a different intensity in the EPR signal corresponding to a different cell density, as is shown in Fig. 2. It may be observed that the reproducibility of the data has a standard deviation of less than 10%, demonstrating that the free radical concentration is, TABLE I INTENSITY OF THE g = 2.003 EPR SIGNAL STEADY STATE PHASE

IN NORMAL

AND NEOPLASTIC

CELL LINES IN THE

Cell strains

Origin

Intensity of the g = 2.003 EPR signal

37 RC NRK L HeLa FLC NRK/RSV 3T3/SV40

African Green Monkey kidney Normal Rabbit kidney Mouse embryonic fibroblast Carcinoma of human cervix F r i e n d l e u k e m i a ceils NRK transformed by Rous sarcoma virus Mouse fibroblast transformed by SV40 virus

very strong strong steong medium-weak weak weak weak

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F i g . 1. T h e free r a d i c a l s i g n a l o b s e r v e d i n H e L a c e l l s , w h i c h is c h a r a c t e r i s t i c o f all c e l l s e m p l o y e d i n t h i s s t u d y . T h e E P R s p e c t r a l c o n d i t i o n s w e r e t h e f o l l o w i n g : f i e l d s e t , 3 2 0 0 G ; f i e l d s w e e p , 4 0 0 g; s w e e p t i m e , 2 o r 4 m i n ; t i m e c o n s t a n t , 0.1 o r 0 . 3 s; m i c r o w a v e p o w e r , 5 0 roW; m o d u l a t i o n a m p l i t u d e 1 0 g; t e m p e r a t u r e , 7 7 K. F i g . 2. G r o w t h c u r v e a n d f r e e r a d i c a l c o n c e n t r a t i o n o f H e L a c e l l s as a f u n c t i o n o f t i m e T h e n o r m a l i z e d E P R s i g n a l i n t e n s i t y is m a x i m a l n e a r 3 4 h f r o m cell i m p l a n t a t i o n , w h i l e cell d e n s i t y is m a x i m a l a t 4 8 h. The experiments were repeated ten times.

indeed, a f u n ct i on of cell growth. The m a x i m u m intensity of the free radical c on cen tr atio n is obtained before the log curve reaches its half value, i.e. in the early exponential phase of growth. Thus, the subsequent loss of intensity in the EPR signal can not be at t r i but e d in any way to cell damage. Moreover, when death begins to occur, a second signal at g = 2.025 was observed. We do n o t give any interpretation to this new signal, apart from the fact t hat its value suggest R - O radicals [28--29]. Viability studies on liver tissues also show the presence o f anomalous signals [2]. The data summarized in Fig. 2 are analogous to the relationship between EPR signal intensity and cancer growth found by Emanuel [3]. T hey also d emo n s tr ate the d e p e n d e n c y of the g = 2.003 radical concent rat i on by the cell growth stage, unrelated to any factor n o t belonging to the cell population itself [7]. The free radical c o n t e n t as shown in Fig. 2 is n o t affected by fetal calf serum e m p l o y e d for cell cultivation, which does n o t contain free radicals [2] b u t which might contain inhibitors or activators of free radical reactions. In fact, the data are i n d e p e n d e n t of change of medium during the course o f the experiments. It must also n o t e d t hat Vanin et al. [30], studying a signal at g = 2.030, related to yeast growth, f ound a similar dependency. However the g = 2.030 signal, which th e y attributed to a free radical with localization of the unpaired electron on a suphur atom, was n o t generally found in cell lines and tissues. Our data can n ot demonstrate t hat the modulation of intensity of the EPR signal depends on factor(s) operating in the single cell (i.e. that, in which the free radical variation occurs). In ot he r words, the variation of the intensity of

619

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14

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F i g . 3. F r e e r a d i c a l c o n c e n t r a t i o n i n H e L a c e l l s as a f u n c t i o n o f t i m e a f t e r s y n c h r o n i z a t i o n . degree of s y n c h r o n y was 90%. E x p e r i m e n t r e p e a t e d t h r e e t i m e s . F o r a d e s c r i p t i o n see t e x t .

The average

the signal in one cell might be caused by factors inhibiting or activating the radical reactions, belonging to neighboring cells which are in a different growth stage. Moreover, another question which arises is whether the free radical is always present in the various states of the living cell. To answer these questions, we studied the intensity of the EPR signal as a function of the cell cycle in synchronized cultures.

c. Free radical study o f synchronized cultures We have studied the intensity of the g = 2.003 EPR signal in HeLa cells synchronized by thymidine, at 1-h intervals from the beginning of S phase t h r o u g h o u t the 20 h cycle. As is seen in Fig. 3, a sharp increase of the signal occurs at the end of the S phase, followed by an approximatively linear decrease in the G2 phase. A second m a x i m u m is present which coincides with the m a x i m u m of mitosis, M phase, which is followed by a continuous decrease in the G1 phase. The m a x i m u m observed in the log phase of Fig. 2 is in good agreement with that observed at the eleventh hour of Fig. 3, both being related to cell duplication [7]. Note that the free radical c o n t e n t in synchronized cells, even when at a m a x i m u m , is lower by a factor of approx. 2 compared to the values observed in non-synchronized cells. To date, we do n o t have any explanation for this observation which may be caused either by the experimental procedures in the synchronization process (thymidine in excess) or by the growing in a spinner condition in a CO2~nriched atmosphere. Discussion Our data, in agreement with previous work [1--3,7], indicates a relation between free radical c o n t e n t and mitotic activity in cell populations. Moreover, this relation is independent of extra-cellular factors and the mechanism which regulates free radical c o n t e n t operates also in the cell where the signal is found. The g = 2.003 signal is present in every period of the living cycle, although with a variable intensity. These studies, however, cannot find any direct

620

demonstration of a relation between radical reactions and inhibition of mitosis. In fact, the variation in free radical content might reflect only a quantitative variation in one of cell components, (for instance, some mitochondrial enzyme) not directly bound to the cell multiplication mechanism. Data of the synchronized cultures show two points of maximum signal variation, respectively coincident with the end of phase S (S-G1 junction) and with phase M (M-G2 junction). These data are related to the data found in the unsynchronized cultures. In fact, the first derivative of the growth curve (dN/cm2)/dt is a m a x i m u m in mid,exponential phase, at which point the number of dividing cells is at a maximum; therefore, at that m o m e n t , the number of cells in the M phase is at a maximum. As mentioned above, there is a maxim u m of free radical content during the mitosis, although this m a x i m u m is only a relative one, the absolute one being at the end of S phase. The maximum number of the cells in S phase precedes the maximum of mitosis by 5 h. Due to the fact that, in unsynchronized cultures, the free radical content is the sum of free radicals in all the various stages, we would expect that the free radical content in unsynchronized cultures would reach its maximum value somewhere between the mid-exponential phase point and 5 h before. This is, in fact, the case showing the complementarity of results obtained on synchronized and unsynchronized cells. At present, we can only advance tentative suggestions on the biological meaning of the g = 2.003 signal: (1) Maximum content in free radical, as observed at the end of the S phase and during the mitosis, respectively, might represent the switch-off signal for nuclear DNA duplication and the switch regulation for cell mitosis. On the other hand, this relation may be inverted, meaning that the end of phase S and the mitosis, respectively, represent a trigger for an increase in free radicals. If this is true, the free radical variation would represent only an epiphenomenon in the biological process related to cell duplication. (2) Since the g = 2.003 is located in the mitochondria, the highest values in free radical c o n t e n t in the cell cycle might reflect parameters related to mitochondrial duplication. On this point it has been reported that in the cell cycle, there are two points at which mitochondrial DNA duplication occurs [31]. One of the maxima is at the 5th h of the S phase, the other one near the end of the G2 phase. These two points might be related to subsequent increase in free radical content, as if free radical increase signifies the end of the duplication process of Mit-DNA acting as a switch-off signal. In this case also the reverse relation is possible. At present, we do not have evidence to clarify these suggestions; other mechanisms are possible, related for instance to respiration only, and n o t to duplication. In any case, we point out t h a t the highest free radical content is coincident with metabolic minima, such as the overall macromolecolar rate of synthesis during the cell cycle (DNA plus RNA plus protein synthesis) [31--32]. Whatever the mechanism operating, the EPR signal modulation points to the most striking m o m e n t s in the life of the cell, the beginning of the phases G2 and G1 which are the well-known stages in which mammalian cell duplication can be arrested [33--35].

621

Acknowledgements This work was supported by the C.N.R. We wish to thank Dr. D. Cordischi and Mrs. T. Menna, C. Buono and A. Petroni for experimental help and useful suggestions. We wish also to thank Dr. J. Peisach for revising this manuscript. The FLC cells were a kind gift from Dr. G.B. Rossi. NRK/RSV and 3T3/SV40 strains were kindly given by Dr. J. McPherson. References 1 S w a r t z , H.M. ( 1 9 7 2 ) in Biological A p p l i c a t i o n of E l e c t r o n Spin R e s o n a n c e (Swartz, H.M., B o l t o n , J . R . a n d Borg, D.C., eds.) p p . 1 5 5 - - 1 9 5 , Wiley I n t e r s c i e n c e , New Y o r k 2 S w a r t z , H.M., A m b e g a o n k a r , S., A n t h o l i n e , W., K o n i e c z n y , M. a n d Mailer, C. ( 1 9 7 3 ) A n n . N.Y. A c a d . Sci. 2 2 2 , 9 8 9 - - 1 0 0 9 3 E m a n u e l , N.M. ( 1 9 7 3 ) A n n . N.Y. A c a d . Sci. 2 2 2 , 1 0 1 0 - - 1 0 3 0 4 S w a r t z , H.M. ( 1 9 7 2 ) Adv. C a n c e r Res. 1 5 , 2 2 7 - - 2 5 2 5 V i t h a y a t h i l , A.J., T e r n b e r g , J . L . a n d C o m m o n e r , B. ( 1 9 6 5 ) N a t u r e 2 0 7 , 1 2 4 6 - - 1 2 4 9 6 Passwater, R. ( 1 9 7 3 ) Int. L a b . J u l y / A u g u s t , 1 0 - 1 9 7 B u r l a k o v a , Ye.B. ( 1 9 6 7 ) Biofizika 12, 8 2 - - 8 8 S A z h i p a , Ya.I., K a y u s h i n , L.P. a n d Nikishkin, Ye.I. ( 1 9 6 6 ) Biofizika 1 1 , 7 1 0 - - 7 1 3 9 Henrici-Oliv6, G. a n d Olivd, S. ( 1 9 7 4 ) A n g e w . C h e m . Int. Edit. 13, 2 9 - - 3 8 1 0 Fallab, S. ( 1 9 6 7 ) A n g e w . Chem. Int. Edit. 6 , 4 9 6 - - 5 0 7 11 H o r g a n , V.I. a n d P h i l p o t , J. ( 1 9 6 5 ) Int. J. R a d . Biol. 8 , 1 6 5 - - 1 7 6 1 2 Saprin, A.N., K l o c h k o , F.V., K r u g l y a k o v a , K.Ye., C h i b r i k e u , A. a n d E m a n u e l , N.M. ( 1 9 6 6 ) Dokl. A k a d . N a u k . SSSR 1 6 7 , 2 2 2 - - 2 2 4 13 Wolfson, N., Wilburh, K.M. a n d B e r n h e i m , F. ( 1 9 5 6 ) E x p . Cell Res. 1 0 , 5 5 6 - - 5 5 8 1 4 B r z h e v s k a y a , O., K a y u s h i n , L.P., K o n d r a s h o v a , M., Nedelina, O. a n d S h e k s h e y e v , E., ( 1 9 6 6 ) Biofizika 11, 1 0 7 6 - - 1 0 8 2 1 5 Dillard, C.J. a n d Tappel, A.L. ( 1 9 7 1 ) Lipids 6 , 7 1 5 - - 7 2 1 16 Chetverikov, A.G., K a l m e n s o n , A.E., K h a r i t o n e n k o v , I.G. a n d Blynmenfeld0 L.A. ( 1 9 6 4 ) Biofizika 9, 18--24 17 K a l m a n s o n , A.E., L i p c h i n a , L.P. a n d C h e t v e r i k o v , A.G. ( 1 9 6 1 ) Biofizika 6 , 4 1 0 - - 4 2 2 1 8 Volpe, P. a n d E r e m e n k o , T. ( 1 9 7 0 ) E u r . J. B i o c h e m . 1 2 , 1 9 5 - - 2 0 0 19 P u c k , T.T., ( 1 9 6 4 ) Cold Spring H a r b o r S y m p o s i u m o n Q u a n t i t a t i v e Biology 29, 1 6 7 - - 1 7 6 20 Volpe, P. a n d E r e m e n k o , T. ( 1 9 7 0 ) E x p . Cell. Res. 6 0 , 4 5 6 - - 4 5 8 21 Volpe, P. a n d E r e m e n k o , T. ( 1 9 7 3 ) in M e t h o d s in Cell Biology, (Prescott, D.M., ed.) Vol. 4, p p . 1 1 3 - 1 2 6 , A c a d . Press, New Y o r k 22 V a n i n , A . F . , B l y n m e n f e l d , L.A. a n d Chetverikov, A.G. ( 1 9 6 7 ) Biofizika 12, 8 2 9 - - 8 3 8 23 Sagkisi, I. a n d O k a b e , T. ( 1 9 6 4 ) K h i t a t i K h e r o n 4 6 , 1 9 5 7 - - 1 9 6 5 24 C o m m o n e r , B. a n d T e r n b e r g , J. ( 1 9 6 9 ) P r o c . Natl. A c a d . Sci. U.S. 4 7 , 1 3 7 4 - - 1 3 8 4 25 K a l o m i t s e v a , I., L ' V o v , K. a n d K a y u s h i n , L. ( 1 9 6 0 ) Biofizika 5, 6 3 6 - - 6 3 7 26 Mallard, J. a n d K e n t , M. ( 1 9 6 4 ) N a t u r e 2 0 4 , 1 1 9 2 27 Pavlova, N.I. a n d Livenson, A . R . ( 1 9 6 5 ) Biofizika 10, 1 6 9 - - 1 7 1 28 L u n s f o r d , J.H. ( 1 9 7 3 ) Catal. Rev. 8 , 1 3 5 - 1 5 7 29 C o p e l a n d , E.S. ( 1 9 7 5 ) J. Magn. Res. 2 0 , 1 2 4 - - 1 3 2 30 V a n i n , A.F. a n d N a l b a n d y a n , R.M. ( 1 9 6 6 ) Biofizika, 1 1 , 1 7 8 - - 1 7 9 31 Volpe, P. ( 1 9 7 6 ) in H o r i z o n s in B i o c h e m i s t r y a n d B i o p h y s i c s (Quagliariello, E., Palmieri, F. a n d Singer, T.P., eds.) Vol. 2, pp. 2 8 5 - - 3 4 0 , Addison-Wesiey, R e a d i n g 3 2 Mitchison, J.M. ( 1 9 6 9 ) in Cell Cycle (Padilla, G.M., ed.) p p . 3 6 1 - - 3 7 2 A c a d e m i c Press, New Y o r k 33 E p i f a n o v a , O.I. ( 1 9 7 3 ) in Studies o n the C o n t r o l Mechanism of the Mitotic Cycle using I n h i b i t o r s of T r a n s c r i p t i o n a n d T r a n s l a t i o n in t h e Cell Cycle ( E p i f a n o v a , D.I., ed.), p p . 7 2 - - 1 0 3 N a u k a , Moscow 34 G e l f a n t , S. ( 1 9 6 2 ) E x p . Cell R e s e a r c h 2 6 , 3 9 5 - - 4 0 3 3 5 Bichel, P. ( 1 9 7 1 ) Eur. J. C a n c e r 7 , 3 4 9 - - 3 5 5