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Mutagenesis in mammalian cells can be modulated by radiation-induced voltage-dependent potassium channels
Mutation Research Letters
ELSEVIER
Mutation Research 324 (1994) 171-176
Mutagenesis in mammalian cells can be modulated by radiation-induced voltage-dependent potassium channels A . H . S a a d *, L . Y . Z h o u , E . K . L a m b e ,
G.M. Hahn
Department of Radiation Oncology, Stanford University, Stanford, CA 94305, USA Received 30 March 1994; revision received 6 May 1994; accepted 9 May 1994
Abstract
In mammalian cells, little is known about the initial events whose ultimate consequence is mutagenesis or DNA repair. The role the plasma membrane may play as an initiator of such a pathway is not understood. We show, for the first time, that membrane voltage-dependent potassium (K +) currents, activated by ionizing radiation (Kuo et al., 1993), play a significant role in radiation mutagenesis. Specifically, we show that the frequency of mutation at the HGPRT locus is increased as expected to 37.6 + 4.0 mutations per 100000 survivors by 800 cGy of ionizing radiation from a spontaneous frequency of 1.5 + 1.5. This increase, however, is abolished if either K ÷ channel blocker, CsCI or BaCI 2, is present for 2 h following irradiation of the cells. RbC1, chemically similar to CsCI but known not to block K* channels, is ineffective in reducing the mutation frequency. Treatment of cells with CsC1 or BaCI 2 had no effect on radiation-induced cell killing.
Key words: K ÷ channel; Cs÷; Ba2+; HGPRT; Radiation
1. Introduction
We have reported on the activation of voltage dependent potassium (K ÷) channels within 3 min following exposure of cells to ionizing radiation (Kuo et al., 1993). The total induced current increased with increasing dose of irradiation and plateaued at approximately 150 cGy. Above this dose, the characteristics of the current changed from an outward delayed rectifier to a slow inactivating current (Koong et al., 1993; Saad et al.,
* Corresponding author. Tel. (415) 723-8437; Fax (415) 7237382. Elsevier Science B.V.
SSDI 0 1 6 5 - 7 9 9 2 ( 9 4 ) 0 0 0 3 3 - H
1994). Above 800 cGy, the current was mostly made up of the latter component. This current was sensitive to the blocking action of CsC1 (Koong et al., 1993) and BaC12 (as shown in this paper). All induced currents were observed to decay within 2 h after exposure to radiation. As a result, treatment of cells with K ÷ channel blockers was limited to 2 h. In this p a p e r we present results that show that treatment for 2 h of cells with compounds that block radiation-induced currents in Chinese hamster cells ( C H O ) reduces the radiation-induced mutation frequency at the H G P R T locus. This reduction in the mutation frequency by the K ÷ channel blockers is radiation dose dependent.
172
A.H. Saad et al. / Mutation Research 324 (1994) 171-176
The effectiveness of the K + channel blockers to reduce the mutation frequency is maximal at 800 cGy and diminishes with increasing dose of radiation.
2. Materials and methods
Cells and media CHO cells used in these experiments were a kind gift from Dr. J.B. Little (Harvard University School of Public Health, Boston, MA). CHO ceils were maintained in HAT medium (hypoxanthine 100 /zM, aminopterin 0.4 /xM and thymidine 16 /zM) containing McCoy's medium supplemented with 10% fetal bovine serum (FBS), to prevent the accumulation of spontaneous mutants (O'Neill et al., 1977). Irradiation 2-4 x 105 cells were plated into T75 Nunc Flasks and incubated overnight in McCoy's medium supplemented with 10% FBS. The medium was replaced 10 min prior to irradiation with fresh medium either without or with CsCI (20 mM) or RbCI (20 mM) or BaCI2 (5 mM). The cells were irradiated using a 137Cs source (J.L. Shepherd and Associates, Mark 1, Model 50) at a dose rate of 178 cGy/min. During the irradiation, the flasks were continuously rotated at a rate of 30 rpm. Mutagenesis assay Radiation induced mutations at the H G P R T locus were examined using 6-thioguanine resistance (O'Neill et al., 1977). Cells were seeded at 2-4 x 105 cells per T75 flask (Nunc, Denmark) in HAT-free medium supplemented with 10% FBS and incubated overnight at 37°C. Following irradiation, the cells were maintained in exponential growth for 8 days to allow expression of the mutations. The cells were then trypsinized and 2 x 105 cells seeded per 100 mm Petri dish (20 replicate dishes per experiment) and grown in the presence of 6-thioguanine for 10 days. At the time of selection, three replicate 60 mm Petri dishes were seeded with 100 cells each to determine plating efficiency. The mutation frequency
is expressed as the total number of 6-thioguanine-resistant colonies divided by the total number of viable cells determined from the plating efficiency.
Effect of potassium channel blockers on radiationinduced mutation 10 min prior to irradiation, CHO cells seeded in T75 flasks were exposed to 20 ml of HAT-free McCoy's medium containing either CsCI (20 mM), BaCI 2 (5 mM) or RbC1 (20 mM). Following irradiation, the cells were incubated for 2 h at 37°C and the medium was then replaced with normal HAT-free McCoy's medium supplemented with 10% FBS. The cells were maintained in exponential phase for 8 days to express the induced mutants. Whole cell voltage clamping Following irradiation, the medium in which the cells were suspended was replaced with fresh serum-free medium. Glass electrodes with a tip resistance of 3-7 MI2 were pulled using a Brown-Flaming pipette puller. The electrodes were coated with sylgard to reduce artifact capacitance noise and filled with a solution (pH 7.2) containing a standard 140 mM KCI, 2 mM MgC12, 11 mM EGTA, 1 mM CaC12, 10 mM Hepes. An X-Y-Z micromanipulator was used to position the tip of the microelectrode over a cell. A test pulse of 20 mV was applied and the cell sucked up against the tip of the microelectrode to form a GO seal. Once a GO seal was achieved, currents were recorded in the whole cell model (Hamill et al., 1981). The software (Fastlab from Indec Systems, Capitola) used to control the analog/digital board (Labmaster) allowed for the simultaneous stimulation of the cell and recording of currents. All the currents were recorded in the whole cell mode. The potential of the cell under voltage clamp was stepped down from a holding potential of - 75 mV to a prepulse voltage of - 120 mV for 500 ms followed by a voltage step to the test potential of - 1 0 0 mV for 1 s. The tail currents were recorded during the post pulse at a voltage of - 7 5 mV. This cycle was repeated eight times every 5 s, each time increasing the test pulse by 25 mV. Membrane currents recorded in response
A.H. Saad et al./Mutation Research 324 (1994) 171-176
to the test pulse were filtered at 2 - 1 0 kHz using a 4-pole low pass Bessel filter b e f o r e s a m p l i n g with the 12-bit d i g i t a l / a n a l o g L a b m a s t e r b o a r d installed in a n I B M 386 A T c o m p u t e r . L e a k a g e c u r r e n t s were s u b t r a c t e d from the total c u r r e n t m e a s u r e d at each voltage after d e t e r m i n i n g the l i n e a r c o m p o n e n t from the c u r r e n t - v o l t a g e relation n e a r to the h o l d i n g p o t e n t i a l of - 7 5 mV.
2.5 nA [ _ _ A 100 ms - + 75 rnV -+ 50 rnV 0 mV -100 rnV- 25 mY
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3. Results and d i s c u s s i o n Fig. 1A shows the o u t w a r d c u r r e n t i n d u c e d in C H O cells following exposure to 800 cGy. T h e i n d u c e d c u r r e n t (Fig. 1B) was blocked by CsC1 (20 raM) a n d BaC12 (5 m M ) b u t n o t RbC1 (20 M M ) suggesting that the i n d u c e d c u r r e n t was d u e to K ÷ since Cs ÷ is a s t a n d a r d K ÷ c h a n n e l blocker (Hille, 1992). T o verify that the i n d u c e d c u r r e n t s were d u e to the o u t w a r d flow of K +, we investigated the effect of varying the c o n c e n t r a t i o n of K ÷ in the external m e d i u m o n the reverse p o t e n tial. T h e reverse p o t e n t i a l of the tail c u r r e n t s was linearly r e l a t e d to the logarithm of the c o n c e n t r a tion of extracellular K ÷ (Fig. 1C) as p r e d i c t e d by the N e r n s t e q u a t i o n . T h e data p r e s e n t e d in Fig. 1 show that exposure of C H O cells to ionizing r a d i a t i o n i n d u c e d o u t w a r d voltage d e p e n d e n t K ÷ c u r r e n t s similar to those observed in A549 cells (Kuo et al., 1993). W e investigated the effects of blocking the i n d u c e d K + c u r r e n t s in C H O cells o n cell survival following radiation. T h e c o m p o u n d s were p r e s e n t d u r i n g the i r r a d i a t i o n period a n d were r e m o v e d 2 h following radiation. Fig. 2 shows that the doses of the alkali a n d alkali-earth metals used in o u r e x p e r i m e n t s to block K ÷ c u r r e n t s did n o t affect survival following a single dose of ionizing radia-
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Fig. 1. (A) Outward current induced in CHO cells 5 min after exposure to 800 cGy. Currents were recorded in the whole cell model (Hamill et al., 1981). The potential of the cell under voltage clamp was stepped down from a holding potential of -75 mV to a prepulse voltage of - 120 mV for 500 ms followed by a voltage step to the test potential of -100 mV for 1 s. The tail currents were recorded during the post pulse at a voltage of -75 mV. This cycle was repeated eight times every 5 s, each time increasing the test pulse by 25 mV. (B) The current-voltage relationship for the radiation-induced K + current in CHO cells. The same pulse protocol described in A was used to record voltage-dependent currents with or without CsCI (20 raM) in the external medium. The figure shows the peak current recorded at each voltage as a function of the voltage. The procedure was repeated for BaCI2 (5 mM) and RbCI (20 mM) using different cells plated in different dishes. (C) The linear relationship between the reverse potential of the tail currents and the logarithm of the concentration of K + also indicated that radiation activated K + currents in CHO cells. The pulse sequence and the procedure used to generate this relationship were described earlier (Burki, 1980).
174
120
A.H. Saad et al. / Mutation Research 324 (1994) 171-176
tion of up to 750 cGy. Clearly, the radiation-induced K + currents or treatment of cells with CsC1, BaC12 or RbCI does not affect cell survival as determined by the clonogenic assay. To investigate the effects of blocking K + currents on the frequency of induced mutation, we examined the frequency of mutations induced at the H G P R T locus in C H O cells. For this locus, the frequency of induced mutations increased with increasing radiation dose (Fig. 3). A linear quadratic model appeared to fit the data points more closely than a simple linear model. This model is consistent with data in the literature for non-human cells for dose rates in excess of 50 r a d / m i n (Grosovsky and Little, 1985; Nakamura and Okada, 1981; Jostes et al., 1980; Fox, 1975; Burki, 1980; Thacker and Stretch, 1983). In the presence of CsCI, the mutation frequency observed following irradiation at doses below 800 cGy was reduced to levels that were not significantly different from the spontaneous mutation frequencies observed in control unirradiated cells (Fig. 3). A linear quadratic model appears to fit the data points. Above 800 cGy, the anti-mutagenic effect of CsC1 was decreased.
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Radiation Dose (cGy) Fig. 3. The frequency of induced mutations at the H G P R T locus increased with increasing dose of irradiation. Radiation induced mutations at the H G P R T locus were examined using 6-thioguanine resistance (O'Neill et al., 1977). For the CsCI treated cells, the blocker was added to the m e d i u m (20 mM) 5 min prior to irradiation. The cells were incubated at 37°C for 2 h and the m e d i u m replaced with one containing no CsCI. The mutation frequency is expressed as the total n u m b e r of 6-thioguanine-resistant colonies divided by the total n u m b e r of viable cells determined from the plating efficiency.
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500
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Dose (cGy) Fig. 2. T h e K ÷ channel blockers CsCI and BaCI 2 applied for 2 h post irradiation did not affect the surviving fraction of C H O cells following a single dose of ionizing radiation of up to 750 cGy.
To discount the possibility that CsC1 reduced the mutation frequency by acting as a free radical scavenger, we compared the antimutagenic effects of CsCI when it was present during the irradiation and for 2 h post irradiation to the antimutagenic effects when CsC1 was added immediately after irradiation for 2 h. CsCI was as effective in reducing the mutation frequency whether it was present or not during the irradiation period. In contrast, if the CsCI was added 8 h after 800 cGy for a duration of 2 h, the resulting frequency of mutation was 38.2 + 3.8 (n = 15). This is not significantly different from cells not exposed to CsC1 and irradiated at 800 cGy (37.6 + 4.0, n = 68). These series of experiments showed that treatment of C H O cells with CsC1 reduced the mutation frequency if the compound was present for 2 h immediately after radiation. It is during this period that K + channels are activated (Kuo et al., 1993). These results suggest
A.H. Saad et al. / Mutation Research 324 (1994) 171-176
D e s p i t e t h e effect of t h e K ÷ c h a n n e l b l o c k e r s on the frequency of induced mutation, these b l o c k e r s h a d no significant effect on survival o f i r r a d i a t e d cells at doses u p to 750 cGy. W e p r o p o s e t h a t following i r r a d i a t i o n of cells, a signal t r a n s d u c t i o n p a t h w a y is t r i g g e r e d t h a t r e q u i r e s activation o f K + c u r r e n t s a n d l e a d s to t h e i n d u c t i o n o f o n e or m o r e e r r o r - p r o n e D N A r e p a i r systems k n o w n to exist in b a c t e r i a ( H a n a walt et al., 1979). In m a m m a l i a n cells, they m a y b e inefficient in t h e sense t h a t t h e y r e a c t i v a t e only a small p e r c e n t a g e o f t h e r a d i a t e d cells. A high p e r c e n t a g e o f t h e s e cells may, however, b e m u t a t e d . A n o t h e r possibility is t h a t t h e p a t h w a y t r i g g e r e d by t h e a c t i v a t e d c h a n n e l m a y m o d u l a t e p r e m u t a g e n i c lesions t h a t a r e not p o t e n t i a l l y lethal.
1 O0
80
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>
=.
60
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=g
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175
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Fig. 4. Blockers of K + channels are anti-mutagens. 5 min prior to irradiation, CHO cells seeded in T75 flasks were exposed to 20 ml of HAT-free McCoy's medium containing either CsCI (20 mM), BaCl 2 (5 mM) or RbCI (20 mM). Following irradiation, the cells were incubated for 2 h at 37°C and the medium was then replaced with normal HAT-free McCoy's medium supplemented with 10% FBS. The cells were maintained in exponential phase for 8 days to express the induced mutants.
Acknowledgements
strongly t h a t t h e a n t i m u t a g e n i c a c t i o n o f CsC1 r e s u l t e d f r o m its i n h i b i t o r y a c t i o n on K ÷ c h a n nels. To further demonstrate that the antimutagenic effect o f CsC1 was a c o n s e q u e n c e o f b l o c k i n g t h e i n d u c e d K ÷ c u r r e n t , we c o m p a r e d t h e a n t i m u t a genic effects o f BaC12, a K ÷ c h a n n e l b l o c k e r t h a t is c h e m i c a l l y u n r e l a t e d to CsCI, with t h a t o f RbC1 which is c h e m i c a l l y similar to CsC1 b u t i n c a p a b l e o f b l o c k i n g K ÷ c u r r e n t s . T r e a t m e n t o f C H O cells with BaCI 2 (5 m M ) d u r i n g t h e i r r a d i a t i o n a n d for 2 h p o s t i r r a d i a t i o n also r e d u c e d t h e m u t a t i o n f r e q u e n c y o b s e r v e d following e x p o s u r e to 800 c G y (Fig. 4). I n c o n t r a s t , similar t r e a t m e n t of cells with RbC1 (20 m M ) d i d n o t significantly affect t h e m u t a t i o n f r e q u e n c y o b s e r v e d following e x p o s u r e to 800 c G y (Fig. 4). O n l y t r e a t m e n t o f cells with cations that block K + currents reduced the mutation f r e q u e n c y at t h e H G P R T locus.
Burki, H.J. (1980) Ionizing radiation-induced 6-thioguanineresistant clones in synchronous CHO cells, Radiat. Res., 81, 76-84. Fox, M. (1975) Factors affecting the quantitation of dose-response curves for mutation induction in V79 Chinese hamster cells after exposure to chemical and physical mutagens, Mutation Res., 29, 449-466. Grosovsky, A.J. and J.B. Little (1985) Evidence for linear response for the induction of mutations in human cells by X-ray exposures below 10 rads, Proc. Natl. Acad. Sci. USA, 82, 2092-2095. Hamill, O.P., A. Marty, E. Neher, B. Sakmann and F.J. Sigworth (1981) Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches, Pfliigers Arch., 391, 85-100. Hanawalt, P.C., P.K., Cooper, A.K. Ganesan and C.A. Smith (1979) DNA repair in bacteria and mammalian cells, Annu. Rev. Biochem., 48, 783-801. Hille, B. (1992) Ionic channels of excitable membranes, in: M.A. Sunderland (Ed.), Ionic Channels of Excitable Membranes, Sinauer. Jostes, R.F., K.M. Bushnell and W.C. Dewey (1980) X-ray induction of 8-azaguanine-resistant mutants in syn-
W e t h a n k Dr. A m a t o J. G i a c c i a a n d Dr. J a m e s H. Elwell for i n v a l u a b l e discussions. S u p p o r t e d by t h e N I H .
References
176
A.H. Saad et al. /Mutation Research 324 (1994) 171-176
chronous Chines hamster ovary cells, Radiat. Res., 83, 146-161. Koong, A.C., A.J. Giaccia, G.M. Hahn and A.H. Saad (1993) Activation of potassium channels by hypoxia and reoxygenation in the human lung adenocarcinoma cell line A549, J. Cell. Physiol., 156, 341-347. Kuo, S.S., A.H., Saad, A.C. Koong, G.M. Hahn and A.J. Giaccia (1993) Potassium channel activation in response to low doses of gamma-irradiation involves reactive oxygen intermediates in non-excitatory cells, Proc. Natl. Acad. Sci. USA, 90, 908-912. Nakamura, N. and S. Okada (1981) Dose rate effects of gamma-ray-induced mutations in cultured mammalian cells, Mutation Res., 83, 127-135. O'Neill, J.P., P.A. Brimer, R. Machanoff, G.P. Hirsch and
A.W. Hsie (1977) A quantitative assay of mutation induction at the hypoxanthine-guanine phosphoribosyl transferase locus in Chinese hamster ovary cells ( C H O / H G P R T system): development and definition of the system, Mutation Res., 45, 91-101. Saad, A.H., S.S. Kuo, A.C. Koong, G.M. Hahn and A.J. Giaccia (1994) Modulation of potassium channels by protein tyrosine kinase inhibitors, J. Cell Physiol. in press. Thacker, J. and A. Stretch (1983) Recovery from lethal and mutagenic damage during postirradiation holding and low-dose-rate irradiations of cultured hamster cells, Radiat. Res., 96, 380-392.
Communicated by J.B. Little
ELSEVIER
Mutation Research 324 (1994) 171-176
Mutagenesis in mammalian cells can be modulated by radiation-induced voltage-dependent potassium channels A . H . S a a d *, L . Y . Z h o u , E . K . L a m b e ,
G.M. Hahn
Department of Radiation Oncology, Stanford University, Stanford, CA 94305, USA Received 30 March 1994; revision received 6 May 1994; accepted 9 May 1994
Abstract
In mammalian cells, little is known about the initial events whose ultimate consequence is mutagenesis or DNA repair. The role the plasma membrane may play as an initiator of such a pathway is not understood. We show, for the first time, that membrane voltage-dependent potassium (K +) currents, activated by ionizing radiation (Kuo et al., 1993), play a significant role in radiation mutagenesis. Specifically, we show that the frequency of mutation at the HGPRT locus is increased as expected to 37.6 + 4.0 mutations per 100000 survivors by 800 cGy of ionizing radiation from a spontaneous frequency of 1.5 + 1.5. This increase, however, is abolished if either K ÷ channel blocker, CsCI or BaCI 2, is present for 2 h following irradiation of the cells. RbC1, chemically similar to CsCI but known not to block K* channels, is ineffective in reducing the mutation frequency. Treatment of cells with CsC1 or BaCI 2 had no effect on radiation-induced cell killing.
Key words: K ÷ channel; Cs÷; Ba2+; HGPRT; Radiation
1. Introduction
We have reported on the activation of voltage dependent potassium (K ÷) channels within 3 min following exposure of cells to ionizing radiation (Kuo et al., 1993). The total induced current increased with increasing dose of irradiation and plateaued at approximately 150 cGy. Above this dose, the characteristics of the current changed from an outward delayed rectifier to a slow inactivating current (Koong et al., 1993; Saad et al.,
* Corresponding author. Tel. (415) 723-8437; Fax (415) 7237382. Elsevier Science B.V.
SSDI 0 1 6 5 - 7 9 9 2 ( 9 4 ) 0 0 0 3 3 - H
1994). Above 800 cGy, the current was mostly made up of the latter component. This current was sensitive to the blocking action of CsC1 (Koong et al., 1993) and BaC12 (as shown in this paper). All induced currents were observed to decay within 2 h after exposure to radiation. As a result, treatment of cells with K ÷ channel blockers was limited to 2 h. In this p a p e r we present results that show that treatment for 2 h of cells with compounds that block radiation-induced currents in Chinese hamster cells ( C H O ) reduces the radiation-induced mutation frequency at the H G P R T locus. This reduction in the mutation frequency by the K ÷ channel blockers is radiation dose dependent.
172
A.H. Saad et al. / Mutation Research 324 (1994) 171-176
The effectiveness of the K + channel blockers to reduce the mutation frequency is maximal at 800 cGy and diminishes with increasing dose of radiation.
2. Materials and methods
Cells and media CHO cells used in these experiments were a kind gift from Dr. J.B. Little (Harvard University School of Public Health, Boston, MA). CHO ceils were maintained in HAT medium (hypoxanthine 100 /zM, aminopterin 0.4 /xM and thymidine 16 /zM) containing McCoy's medium supplemented with 10% fetal bovine serum (FBS), to prevent the accumulation of spontaneous mutants (O'Neill et al., 1977). Irradiation 2-4 x 105 cells were plated into T75 Nunc Flasks and incubated overnight in McCoy's medium supplemented with 10% FBS. The medium was replaced 10 min prior to irradiation with fresh medium either without or with CsCI (20 mM) or RbCI (20 mM) or BaCI2 (5 mM). The cells were irradiated using a 137Cs source (J.L. Shepherd and Associates, Mark 1, Model 50) at a dose rate of 178 cGy/min. During the irradiation, the flasks were continuously rotated at a rate of 30 rpm. Mutagenesis assay Radiation induced mutations at the H G P R T locus were examined using 6-thioguanine resistance (O'Neill et al., 1977). Cells were seeded at 2-4 x 105 cells per T75 flask (Nunc, Denmark) in HAT-free medium supplemented with 10% FBS and incubated overnight at 37°C. Following irradiation, the cells were maintained in exponential growth for 8 days to allow expression of the mutations. The cells were then trypsinized and 2 x 105 cells seeded per 100 mm Petri dish (20 replicate dishes per experiment) and grown in the presence of 6-thioguanine for 10 days. At the time of selection, three replicate 60 mm Petri dishes were seeded with 100 cells each to determine plating efficiency. The mutation frequency
is expressed as the total number of 6-thioguanine-resistant colonies divided by the total number of viable cells determined from the plating efficiency.
Effect of potassium channel blockers on radiationinduced mutation 10 min prior to irradiation, CHO cells seeded in T75 flasks were exposed to 20 ml of HAT-free McCoy's medium containing either CsCI (20 mM), BaCI 2 (5 mM) or RbC1 (20 mM). Following irradiation, the cells were incubated for 2 h at 37°C and the medium was then replaced with normal HAT-free McCoy's medium supplemented with 10% FBS. The cells were maintained in exponential phase for 8 days to express the induced mutants. Whole cell voltage clamping Following irradiation, the medium in which the cells were suspended was replaced with fresh serum-free medium. Glass electrodes with a tip resistance of 3-7 MI2 were pulled using a Brown-Flaming pipette puller. The electrodes were coated with sylgard to reduce artifact capacitance noise and filled with a solution (pH 7.2) containing a standard 140 mM KCI, 2 mM MgC12, 11 mM EGTA, 1 mM CaC12, 10 mM Hepes. An X-Y-Z micromanipulator was used to position the tip of the microelectrode over a cell. A test pulse of 20 mV was applied and the cell sucked up against the tip of the microelectrode to form a GO seal. Once a GO seal was achieved, currents were recorded in the whole cell model (Hamill et al., 1981). The software (Fastlab from Indec Systems, Capitola) used to control the analog/digital board (Labmaster) allowed for the simultaneous stimulation of the cell and recording of currents. All the currents were recorded in the whole cell mode. The potential of the cell under voltage clamp was stepped down from a holding potential of - 75 mV to a prepulse voltage of - 120 mV for 500 ms followed by a voltage step to the test potential of - 1 0 0 mV for 1 s. The tail currents were recorded during the post pulse at a voltage of - 7 5 mV. This cycle was repeated eight times every 5 s, each time increasing the test pulse by 25 mV. Membrane currents recorded in response
A.H. Saad et al./Mutation Research 324 (1994) 171-176
to the test pulse were filtered at 2 - 1 0 kHz using a 4-pole low pass Bessel filter b e f o r e s a m p l i n g with the 12-bit d i g i t a l / a n a l o g L a b m a s t e r b o a r d installed in a n I B M 386 A T c o m p u t e r . L e a k a g e c u r r e n t s were s u b t r a c t e d from the total c u r r e n t m e a s u r e d at each voltage after d e t e r m i n i n g the l i n e a r c o m p o n e n t from the c u r r e n t - v o l t a g e relation n e a r to the h o l d i n g p o t e n t i a l of - 7 5 mV.
2.5 nA [ _ _ A 100 ms - + 75 rnV -+ 50 rnV 0 mV -100 rnV- 25 mY
•
3500 S
=
cT"'°l
3000
i
c7 "° "S (")
2500 ¸
2000 ¸ 4J
///
I/
1500-
173
3. Results and d i s c u s s i o n Fig. 1A shows the o u t w a r d c u r r e n t i n d u c e d in C H O cells following exposure to 800 cGy. T h e i n d u c e d c u r r e n t (Fig. 1B) was blocked by CsC1 (20 raM) a n d BaC12 (5 m M ) b u t n o t RbC1 (20 M M ) suggesting that the i n d u c e d c u r r e n t was d u e to K ÷ since Cs ÷ is a s t a n d a r d K ÷ c h a n n e l blocker (Hille, 1992). T o verify that the i n d u c e d c u r r e n t s were d u e to the o u t w a r d flow of K +, we investigated the effect of varying the c o n c e n t r a t i o n of K ÷ in the external m e d i u m o n the reverse p o t e n tial. T h e reverse p o t e n t i a l of the tail c u r r e n t s was linearly r e l a t e d to the logarithm of the c o n c e n t r a tion of extracellular K ÷ (Fig. 1C) as p r e d i c t e d by the N e r n s t e q u a t i o n . T h e data p r e s e n t e d in Fig. 1 show that exposure of C H O cells to ionizing r a d i a t i o n i n d u c e d o u t w a r d voltage d e p e n d e n t K ÷ c u r r e n t s similar to those observed in A549 cells (Kuo et al., 1993). W e investigated the effects of blocking the i n d u c e d K + c u r r e n t s in C H O cells o n cell survival following radiation. T h e c o m p o u n d s were p r e s e n t d u r i n g the i r r a d i a t i o n period a n d were r e m o v e d 2 h following radiation. Fig. 2 shows that the doses of the alkali a n d alkali-earth metals used in o u r e x p e r i m e n t s to block K ÷ c u r r e n t s did n o t affect survival following a single dose of ionizing radia-
U
1000-
500-
01 -I O 0
~-~-40--20
0
2o
40
60
80
v o l t a g e (my)
C
0
~"
-20
E
-4O 0 >
0 o iT-
-60
-8O
~
-100
In [k+]
asumd
Theoretical
Fig. 1. (A) Outward current induced in CHO cells 5 min after exposure to 800 cGy. Currents were recorded in the whole cell model (Hamill et al., 1981). The potential of the cell under voltage clamp was stepped down from a holding potential of -75 mV to a prepulse voltage of - 120 mV for 500 ms followed by a voltage step to the test potential of -100 mV for 1 s. The tail currents were recorded during the post pulse at a voltage of -75 mV. This cycle was repeated eight times every 5 s, each time increasing the test pulse by 25 mV. (B) The current-voltage relationship for the radiation-induced K + current in CHO cells. The same pulse protocol described in A was used to record voltage-dependent currents with or without CsCI (20 raM) in the external medium. The figure shows the peak current recorded at each voltage as a function of the voltage. The procedure was repeated for BaCI2 (5 mM) and RbCI (20 mM) using different cells plated in different dishes. (C) The linear relationship between the reverse potential of the tail currents and the logarithm of the concentration of K + also indicated that radiation activated K + currents in CHO cells. The pulse sequence and the procedure used to generate this relationship were described earlier (Burki, 1980).
174
120
A.H. Saad et al. / Mutation Research 324 (1994) 171-176
tion of up to 750 cGy. Clearly, the radiation-induced K + currents or treatment of cells with CsC1, BaC12 or RbCI does not affect cell survival as determined by the clonogenic assay. To investigate the effects of blocking K + currents on the frequency of induced mutation, we examined the frequency of mutations induced at the H G P R T locus in C H O cells. For this locus, the frequency of induced mutations increased with increasing radiation dose (Fig. 3). A linear quadratic model appeared to fit the data points more closely than a simple linear model. This model is consistent with data in the literature for non-human cells for dose rates in excess of 50 r a d / m i n (Grosovsky and Little, 1985; Nakamura and Okada, 1981; Jostes et al., 1980; Fox, 1975; Burki, 1980; Thacker and Stretch, 1983). In the presence of CsCI, the mutation frequency observed following irradiation at doses below 800 cGy was reduced to levels that were not significantly different from the spontaneous mutation frequencies observed in control unirradiated cells (Fig. 3). A linear quadratic model appears to fit the data points. Above 800 cGy, the anti-mutagenic effect of CsC1 was decreased.
Contr01
100
U. 0 0 e- o
60
2cff
20
0 ,
,
0
i
200
-
•
400
•
, 600
, 8OO
.
= 1000
1200
Radiation Dose (cGy) Fig. 3. The frequency of induced mutations at the H G P R T locus increased with increasing dose of irradiation. Radiation induced mutations at the H G P R T locus were examined using 6-thioguanine resistance (O'Neill et al., 1977). For the CsCI treated cells, the blocker was added to the m e d i u m (20 mM) 5 min prior to irradiation. The cells were incubated at 37°C for 2 h and the m e d i u m replaced with one containing no CsCI. The mutation frequency is expressed as the total n u m b e r of 6-thioguanine-resistant colonies divided by the total n u m b e r of viable cells determined from the plating efficiency.
10 0
10"1 C
o 3
U. 10"2
¢J) C -g
7
~...l.=
10"3
10"4 0
250
500
750
1000 1250
1500
1750 2000
Dose (cGy) Fig. 2. T h e K ÷ channel blockers CsCI and BaCI 2 applied for 2 h post irradiation did not affect the surviving fraction of C H O cells following a single dose of ionizing radiation of up to 750 cGy.
To discount the possibility that CsC1 reduced the mutation frequency by acting as a free radical scavenger, we compared the antimutagenic effects of CsCI when it was present during the irradiation and for 2 h post irradiation to the antimutagenic effects when CsC1 was added immediately after irradiation for 2 h. CsCI was as effective in reducing the mutation frequency whether it was present or not during the irradiation period. In contrast, if the CsCI was added 8 h after 800 cGy for a duration of 2 h, the resulting frequency of mutation was 38.2 + 3.8 (n = 15). This is not significantly different from cells not exposed to CsC1 and irradiated at 800 cGy (37.6 + 4.0, n = 68). These series of experiments showed that treatment of C H O cells with CsC1 reduced the mutation frequency if the compound was present for 2 h immediately after radiation. It is during this period that K + channels are activated (Kuo et al., 1993). These results suggest
A.H. Saad et al. / Mutation Research 324 (1994) 171-176
D e s p i t e t h e effect of t h e K ÷ c h a n n e l b l o c k e r s on the frequency of induced mutation, these b l o c k e r s h a d no significant effect on survival o f i r r a d i a t e d cells at doses u p to 750 cGy. W e p r o p o s e t h a t following i r r a d i a t i o n of cells, a signal t r a n s d u c t i o n p a t h w a y is t r i g g e r e d t h a t r e q u i r e s activation o f K + c u r r e n t s a n d l e a d s to t h e i n d u c t i o n o f o n e or m o r e e r r o r - p r o n e D N A r e p a i r systems k n o w n to exist in b a c t e r i a ( H a n a walt et al., 1979). In m a m m a l i a n cells, they m a y b e inefficient in t h e sense t h a t t h e y r e a c t i v a t e only a small p e r c e n t a g e o f t h e r a d i a t e d cells. A high p e r c e n t a g e o f t h e s e cells may, however, b e m u t a t e d . A n o t h e r possibility is t h a t t h e p a t h w a y t r i g g e r e d by t h e a c t i v a t e d c h a n n e l m a y m o d u l a t e p r e m u t a g e n i c lesions t h a t a r e not p o t e n t i a l l y lethal.
1 O0
80
¢_> G
>
=.
60
tL o
=g
40
z~ 20
0
-6
~
÷ ÷
175
÷
Fig. 4. Blockers of K + channels are anti-mutagens. 5 min prior to irradiation, CHO cells seeded in T75 flasks were exposed to 20 ml of HAT-free McCoy's medium containing either CsCI (20 mM), BaCl 2 (5 mM) or RbCI (20 mM). Following irradiation, the cells were incubated for 2 h at 37°C and the medium was then replaced with normal HAT-free McCoy's medium supplemented with 10% FBS. The cells were maintained in exponential phase for 8 days to express the induced mutants.
Acknowledgements
strongly t h a t t h e a n t i m u t a g e n i c a c t i o n o f CsC1 r e s u l t e d f r o m its i n h i b i t o r y a c t i o n on K ÷ c h a n nels. To further demonstrate that the antimutagenic effect o f CsC1 was a c o n s e q u e n c e o f b l o c k i n g t h e i n d u c e d K ÷ c u r r e n t , we c o m p a r e d t h e a n t i m u t a genic effects o f BaC12, a K ÷ c h a n n e l b l o c k e r t h a t is c h e m i c a l l y u n r e l a t e d to CsCI, with t h a t o f RbC1 which is c h e m i c a l l y similar to CsC1 b u t i n c a p a b l e o f b l o c k i n g K ÷ c u r r e n t s . T r e a t m e n t o f C H O cells with BaCI 2 (5 m M ) d u r i n g t h e i r r a d i a t i o n a n d for 2 h p o s t i r r a d i a t i o n also r e d u c e d t h e m u t a t i o n f r e q u e n c y o b s e r v e d following e x p o s u r e to 800 c G y (Fig. 4). I n c o n t r a s t , similar t r e a t m e n t of cells with RbC1 (20 m M ) d i d n o t significantly affect t h e m u t a t i o n f r e q u e n c y o b s e r v e d following e x p o s u r e to 800 c G y (Fig. 4). O n l y t r e a t m e n t o f cells with cations that block K + currents reduced the mutation f r e q u e n c y at t h e H G P R T locus.
Burki, H.J. (1980) Ionizing radiation-induced 6-thioguanineresistant clones in synchronous CHO cells, Radiat. Res., 81, 76-84. Fox, M. (1975) Factors affecting the quantitation of dose-response curves for mutation induction in V79 Chinese hamster cells after exposure to chemical and physical mutagens, Mutation Res., 29, 449-466. Grosovsky, A.J. and J.B. Little (1985) Evidence for linear response for the induction of mutations in human cells by X-ray exposures below 10 rads, Proc. Natl. Acad. Sci. USA, 82, 2092-2095. Hamill, O.P., A. Marty, E. Neher, B. Sakmann and F.J. Sigworth (1981) Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches, Pfliigers Arch., 391, 85-100. Hanawalt, P.C., P.K., Cooper, A.K. Ganesan and C.A. Smith (1979) DNA repair in bacteria and mammalian cells, Annu. Rev. Biochem., 48, 783-801. Hille, B. (1992) Ionic channels of excitable membranes, in: M.A. Sunderland (Ed.), Ionic Channels of Excitable Membranes, Sinauer. Jostes, R.F., K.M. Bushnell and W.C. Dewey (1980) X-ray induction of 8-azaguanine-resistant mutants in syn-
W e t h a n k Dr. A m a t o J. G i a c c i a a n d Dr. J a m e s H. Elwell for i n v a l u a b l e discussions. S u p p o r t e d by t h e N I H .
References
176
A.H. Saad et al. /Mutation Research 324 (1994) 171-176
chronous Chines hamster ovary cells, Radiat. Res., 83, 146-161. Koong, A.C., A.J. Giaccia, G.M. Hahn and A.H. Saad (1993) Activation of potassium channels by hypoxia and reoxygenation in the human lung adenocarcinoma cell line A549, J. Cell. Physiol., 156, 341-347. Kuo, S.S., A.H., Saad, A.C. Koong, G.M. Hahn and A.J. Giaccia (1993) Potassium channel activation in response to low doses of gamma-irradiation involves reactive oxygen intermediates in non-excitatory cells, Proc. Natl. Acad. Sci. USA, 90, 908-912. Nakamura, N. and S. Okada (1981) Dose rate effects of gamma-ray-induced mutations in cultured mammalian cells, Mutation Res., 83, 127-135. O'Neill, J.P., P.A. Brimer, R. Machanoff, G.P. Hirsch and
A.W. Hsie (1977) A quantitative assay of mutation induction at the hypoxanthine-guanine phosphoribosyl transferase locus in Chinese hamster ovary cells ( C H O / H G P R T system): development and definition of the system, Mutation Res., 45, 91-101. Saad, A.H., S.S. Kuo, A.C. Koong, G.M. Hahn and A.J. Giaccia (1994) Modulation of potassium channels by protein tyrosine kinase inhibitors, J. Cell Physiol. in press. Thacker, J. and A. Stretch (1983) Recovery from lethal and mutagenic damage during postirradiation holding and low-dose-rate irradiations of cultured hamster cells, Radiat. Res., 96, 380-392.
Communicated by J.B. Little