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The mutagenic activity of hydroxyurea in Chlamydomonas reinhardi
217
Mutation Research, 41 (1976) 217--224 © Elsevier/North-Holland Biomedical Press
THE MUTAGENIC ACTIVITY OF H Y D R O X Y U R E A IN CHLAM YDOMONAS REINHARDI
MONICA ADAMS * and J.R. WARR
Department of Biology, University of York, Heslington, York (England) (Received February 17th, 1976) (Revision received June 6th, 1976) (Accepted July 5th, 1976)
Summary Hydroxyurea, an inhibitor of ribonucleotide reductase, increases the frequency of streptomycin resistant mutants in liquid cultures of Chlamydomonas reinhardi after 45 h incubation. After more prolonged incubation in hydroxyurea medium the frequency of streptomycin resistant mutants declines. This m a y be due to the slower growth rate of streptomycin resistant mutants compared to wild type cells in h y d r o x y u r e a containing medium. Studies on solid medium show that b o t h the rate of forward mutation to streptomycin resistance and reverse mutation to nicotinamide independence are increased several fold by growth on hydroxyurea.
Introduction M o n o h y d r o x y u r e a is a chemotherapeutic agent which has been used effectively in the treatment of several human neoplasms (see [23] for references). The drug is c y t o t o x i c or cytostatic to a variety of cell types, maximal activity having been observed against continuously dividing cells rather than those which are n o t dividing or which have been stimulated to divide [14]. It is cytotoxic to a variety of mammalian cell types during S phase in vitro [20] and in vivo [13,15]. A cytostatic effect has also been observed on a range of mammalian cell types with cells accumulating at the G1/S boundary until the inhibitor is removed [22]. Considerable evidence from p r o - a n d eukaryotic studies suggest that h y d r o y u r e a inhibits DNA rather than RNA or overall protein synthesis [2,4,5,16]. Histone synthesis m a y also be sensitive [12] b u t this is presumably due to its tight coupling with DNA synthesis [8]. The effects of hy* Present
address: I n f e c t i o n
Control Unit, Clifton Hospital, York, U.K.
218 droxyurea on DNA synthesis cell division and chromosome structure have recently been extensively reviewed by Timson [21]. During the course of studies on the mechanism of resistance to hydroxyurea in Chlamydomonas we observed that the drug apparently induced considerable variation in colony size and pigmentation and furthermore h y d r o x y u r e a resistant mutants arose with a remarkably high frequency when selection was made on h y d r o x y u r e a containing medium. These observations suggested the possibility that h y d r o x y u r e a might be mutagenic and prompted the present studies on mutagenic activity of this substance in Chlamydomonas. Materials and methods Strains Wild type, mating type positive Chlamydomonas reinhardi (Strain bridge Collection of Algae) was used in investigations of occurrence mycin resistant mutants. A strain requiring nicotinamide (nic7-, bridge Collection of Algae) was used in investigations of reverse Strains were repurified before each experiment.
32C, Camof streptomt ÷, Cammutation.
Culture media and conditions Unsupplemented media was based on medium 1 of Sager and Granick [19], ferric chloride being replaced by 0.01 g/1 ferric citrate and 0.01 g/1 citric acid. 0.5% peptone and 1.5% Difco agar were added to unsupplemented media for use in agar plates except in the case of plates used for measuring the number of reverse mutants from nicotinamide dependence where no peptone and 1% agar were used. Medium was sterilized by autoclaving at 15 p.s.i, for 15 min. Strept o m y c i n was dissolved in sterile medium and added to autoclaved medium in appropriate amounts. H y d r o x y u r e a (Calbiochem) was dissolved in medium, sterilized by filtration through a porcelain candle filter and then added to cool sterile media. Nicotinamide was filter sterilized and added to give a final concentration of 0.75 mg/1 where necessary. Cultures were incubated at 25°C with a light intensity of 500 foot candles provided by tubular fluorescent lamps. Measurement of mutation rate in liquid cultures A late log phase culture of wild type cells was inoculated into 50 ml quantities of unsupplemented media and media containing 5 × 10 -3 M hydroxyurea, to give an initial cell count of 8 × 104/ml. At the times shown in Fig. 1, 5 ml samples were removed from each flask spun down and resuspended in 2 ml of sterile medium. Of each 2 ml, 1 ml was taken and diluted for viable counts performed in triplicate and 0.1-ml samples were plated on eight 100 pg/ml streptomycin plates. Plates for viable counts were incubated for 4 days, and the strept o m y c i n plates for 14 days when the n u m b e r of resistant colonies was counted. Measurement o f mutation rate on plates 5 × 104 cells of a log phase culture of wild type were spread onto each of the unsupplemented plates and plates supplemented with hydroxyurea. The plates
219 were incubated and at appropriate time intervals nine of each series were removed. Viable counts were performed by washing off cells from three plates with 5 ml of sterile buffer per plate. 1 ml of each washing was taken and after appropriate dilutions viabilities were d e t e r m i n e d by plating in triplicate on unsupplemented media. The remaining six plates of each original set were replica plated onto 100 pg/ml streptomycin and these plates were then incubated for 12 days, after which time the number of resistant colonies was counted. Reverse mutation from nicotinamide dependence was measured in a similar fashion except that nic7- cells were plated originally on plates supplemented with nicotinamide and the cells were replica plated onto minimal media. Results Effect o f hydroxyurea on mutation rate The growth of wild type and the accumulation of streptomycin resistant cells following growth in unsupplemented media and 5 X 10 -3 M h y d r o x y u r e a is shown in Figs. 1 and 2. The growth rate of wild type cells is immediately reduced in medium containing h y d r o x y u r e a b u t little difference in the frequency of m u t a n t cells is seen up to at least 29 h growth in h y d r o x y u r e a compared to the control. However after 45 h some cell death occurs in h y d r o x y u r e a and at the same time the frequency of m u t a n t cells in h y d r o x y u r e a rises sharply from around 4 X 10 -6 to 35 X 10 -6. Rather surprisingly the frequency of m u t a n t cells declines between 70 and 90 h.
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The possibility t h a t streptomycin resistant mutants might survive hydroxyurea treatment better than sensitive cells and so form an increased proportion of the surviving cells was investigated. However, n o t only did streptomycin resistant cells show a lower growth rate compared to wild type cells in medium containing 5 X 10 -3 M h y d r o x y u r e a but they were also killed by this concentration of h y d r o x y u r e a after 46 h incubation (Fig. 3). This latter p h e n o m e n o n could explain the fall off in the frequency of m u t a n t cells in h y d r o x y u r e a medium with longer incubation times. The effects of h y d r o x y u r e a on growth rate and viable count after prolonged incubation times mean, however, that comparisons of the frequency of m u t a n t cells in unsupplemented and h y d r o x y u r e a medium at a given time are being made on cells in different phases of the growth cycle. The experiments described so far clearly demonstrate an increase in the frequency of mutants in the presence of h y d r o x y u r e a in liquid medium. However, the differences in growth rates and viabilities between streptomycin resistants and sensitives in unsupplemented and h y d r o x y u r e a containing media precluded any calculation of m u t a t i o n rates from the available data [11]. Further experiments of different design were therefore performed in order to achieve this.
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•ncubation time (h) Fig. 3. G r o w t h o f wild t y p e a n d s t r e p t o m y c i n r e s i s t a n t C h l a m y d o m o n a s in t h e p r e s e n c e a n d a b s e n c e of h y d r o x y u r e a in liquid m e d i a . (© o) Wild t y p e , u n s u p p l e m e n t e d m e d i u m ; (~ D) wild t y p e , 5 X 10 -3 M h y d r o x y u r c a ; (o e ) s t r e p t o m y c i n r e s i s t a n t , u n s u p p l e m e n t e d m e d i u m ; (A z~) s t r e p t o m y c i n r e s i s t a n t , 5 X 10 -3 M h y d r o x y u r e a .
Cells were plated on u n s u p p l e m e n t e d and h y d r o x y u r e a c o n t a i n i n g solid m e d i a and at intervals viable c o u n t s and n u m b e r s o f m u t a n t colonies were e s t i m a t e d as d e s c r i b e d in the M e t h o d s section. T h e m u t a t i o n rates were t h e n calculated a c c o r d i n g t o the f o r m u l a , m u t a t i o n rate = log 2 (M2 --M,) (N2 -- N , ) TABLE I M U T A T I O N R A T E S T O S T R E P T O M Y C I N R E S I S T A N C E A N D N I C O T I N A M I D E I N D E P E N D E N C E IN T H E A B S E N C E A N D P R E S E N C E O F 2.5 × 1 0 - 3 M H U . M U T A T I O N R A T E S C A L C U L A T E D AS D E S C R I B E D IN T H E T E X T . Mutation rates/107 cells/generation
S t r e p t o m y c i n resistance, E x p e r i m e n t 1 S t r e p t o m y c i n resistance, E x p e r i m e n t 2 Nicotinamide independence
Unsupplemented media
Hydroxyurea media
1.23 1.23 0.12
6.43 4.88 0.85
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where M1 and M2 are the numbers of m u t a n t clones and N1 and N2 are the total number of cells at times 1 and 2 respectively [1 ]. Preliminary experiments showed little or no effect on the m u t a t i o n rate in cells growing on 10 -3 M h y d r o x y u r e a and a marked growth inhibitory effect during growth on plates containing 5 X 10 -3 M hydroxyurea, greater than that observed previously in liquid media containing the same concentration (compare wild type growth curves in Figs. 1 and 3 with Fig. 4). 2.5 X 10 -3 M hydroxyurea was therefore selected as an optimal concentration for measurement of mutation rates for forward m u t a t i o n to streptomycin resistance and reverse m u t a t i o n to nicotinamide independence. As can be seen from Table I, growth on this concentration of h y d r o x y u r e a increases the mutation rate several-fold. Discussion This paper shows t h a t h y d r o x y u r e a is mutagenic in the eucaryotic alga Chlamydomonas reinhardi. In view of the very extensive literature concerning
223
the inhibitory effects of h y d r o x y u r e a on DNA synthesis in a very wide range of organisms and the evidence for chromosome aberrations following exposure to this drug [21] it is perhaps surprising that such mutagenic activity has not been reported previously. Huberman and Heidelberger [10] found no increase in the frequency of azaguanine resistant mutants in Chinese hamster cells in partially toxic concentrations of hydroxyurea. Exposure to the drug was, however, only continued for 4 h, whereas in our experiments prolonged exposure for over 29 h was necessary before any significant accumulation of mutant cells could be detected. Although any precise correlation of time periods required to induce comparable effects on two such different cell types as Chinese hamster and Chlamydomonas are clearly impossible, it does n o w seem worth re-examining the mutagenic effects of h y d r o x y u r e a in mammalian cells over prolonged periods. This is particularly the case in view of the fact that h y d r o x y u r e a is still used clinically as an antitumour drug and several workers have proposed that mutation may play a significant role in t u m o u r progression [9]. H y d r o x y u r e a treatment m a y involve a balance between preferential destruction of t u m o u r cells and accelerated t u m o u r progression due to mutagenic activity. The reason for the delay in the induction of mutations during this work has n o t been established. We may speculate that ribonucleotide reductase must be inhibited for a sufficiently long period to allow depletion of deoxyribonucleoside phosphate pools which then leads to m u t a n t induction. Drake has pointed o u t (p. 156 in [6] } that along delay in filling a position during DNA replication might favour the incorporation of incorrect nucleotides and this might explain why a number of other c o m p o u n d s which interfere with pyrimidine and purine production are mutagenic in Escherichia coli. Such c o m p o u n d s include azaserine, benzimidazole, 6 mercaptopurine, 5 aminopurine amongst others. However, Fig. 1 does show that there is considerable inhibition of growth, presumably due to inhibition of DNA synthesis, during the first 30 h growth in 5 X 10 -3 M h y d r o x y u r e a without any significant induction of mutation (Fig. 2). During the course of a study on the effects of inhibitors on recombination in Chlamydomonas, Chiu and Hastings [3] showed that 5 X 10-3M h y d r o x y u r e a over a 2-h period gave a 71% inhibition of DNA synthesis, 27% inhibition of RNA synthesis and no inhibition of protein synthesis. Although their work was carried out in a slightly different medium and at a lower temperature than ours, it does strongly suggest that DNA synthesis is rapidly inhibited in Chlamydomonas cells growing in 5 X 10 -3 h y d r o x y u r e a after a very short period. Cytoplasmic mutations have long been known to occur in Chlamydomonas reinhardi [ 18] and we should therefore consider whether the mutations studied during the present w o r k are chromosomal or cytoplasmic. The concentration of streptomycin used to select resistant mutants during this work (100 pg/ml) does select b o t h chromosomal and non-chromosomal streptomycin resistant mutations in Chlamydomonas, b u t the frequency of the former is approx. 2 X 103 fold greater than the latter on minimal medium [17]. We therefore consider it likely that the great majority of our streptomycin resistant mutations are chromosomal. The nicotinamide auxotroph used for reversion to prototrophy is a mutation in a well characterised chromosomal gene [7] and so this problem does not arise.
224
Acknowledgement We gratefully acknowledge financial assistance from the Yorkshire Cancer Research Campaign in the form of a research studentship to one of us (M.A.). References 1 Beale, G . H . , A m e t h o d f o r t h e m e a s u r e m e n t o f m u t a t i o n r a t e f r o m p h a g e s e n s i t i v i t y t o p h a g e resist a n c e in Escherichia coli, J. G e n . M i c r o b i o l . , 2 ( 1 9 4 8 ) 1 3 1 - - 1 4 2 . 2 C a m e r o n , I.L. a n d J . R . J e t e r , A c t i o n o f h y d r o x y u r e a a n d N - c a r b a m o y l o x y u r e a o n t h e cell c y c l e o f T e t r a h y m e n a , Cell Tissue K i n e t . , 6 ( 1 9 7 3 ) 2 8 9 - - 3 0 1 . 3 C h i n , S.M. a n d P . J . H a s t i n g s , P r e - m e i o t i c D N A s y n t h e s i s a n d r e c o m b i n a t i o n in Chlamydomonas reinhardi, G e n e t i c s , 7 3 ( 1 9 7 3 ) 2 9 - - 4 3 . 4 C o h e n , L.S. a n d G.P. S t u d z i n s k i , C o r r e l a t i o n b e t w e e n cell e n l a r g e m e n t a n d n u c l e i c a c i d a n f p r o t e i n c o n t e n t o f H e L a ceils i n u n b a l a n c e d g r o w t h p r o d u c e d b y i n h i b i t o r s o f D N A s y n t h e s i s , J. Cell P h y s i o l . , 69 (1967) 331--340. 5 C o y l e , M.B. a n d B. S t r a u s s , Ceil killing a n d t h e a c c u m u l a t i o n o f b r e a k s in t h e D N A o f H E p - 2 cells inc u b a t e d in t h e p r e s e n c e o f h y d r o x y u r e a , C a n c e r R e s . , 3 0 ( 1 9 7 0 ) 2 3 1 4 - - 2 3 1 9 . 6 D r a k e , J.W., T h e m o l e c u l a r b a s i s o f m u t a t i o n , H o l d e n D a y , S a n F r a n c i s c o , 1 9 7 0 . 7 E b e r s o l d , W . T . , R . P . L e v i n e , E . E . L e v i n e a n d M . A . O l m s t e d , L i n k a g e m a p s in Chlarnydomonas reinhardi, G e n e t i c s , 4 7 ( 1 9 6 2 ) 5 3 1 - - 5 4 3 . 8 Elgin, S . C . R . a n d H . W e i n t r a u b , C h r o m o s o m a l p r o t e i n s a n d c h r o m a t i n s t r u c t u r e , A n n . R e v . B i o c h e m . , 44 (1975) 725--774. 9 F a r b e r , E., C a r c i n o g e n e s i s - c e l l u l a r e v o l u t i o n as a u n i f y i n g t h r e a d , C a n c e r R e s . , 3 3 ( 1 9 7 3 ) 2 5 3 7 - - 2 5 5 0 . 1 0 H u b e r m a n , E. a n d C. H e i d e l b e r g e r , T h e m u t a g e n i c i t y t o m a m m a l i a n cells o f p y r i m i d i n e n u c l e o s i d e analogues, Mutation Res., 14 (1972) 130--132. 11 L u r i a , S . E . a n d M. D e l b r u c k , M u t a t i o n s o f b a c t e r i a f r o m v i r u s s e n s i t i v i t y t o v i r u s r e s i s t a n c e , G e n e t i c s , 28 (1943) 491--511. 1 2 M a l p o i x , P., F. Z a m p e t t i a n d M. Fievez, D i f f e r e n t i a l i n h i b i t i o n b y a c t i n o m y c i n , p u r o m y c i n a n d h y d r o x y u r e a o f p r o t e i n s y n t h e s i s in t h e n u c l e u s a n d c y t o p l a s m o f c h i c k e m b r y o e r y t h r o p l a s t s , B i o e h e m . Biophys. Acta., 182 (1969) 214--227. 1 3 Neff, G . L . a n d E . R . H o m a n , T h e e f f e c t o f close i n t e r v a l o n t h e survival o f L 1 2 1 0 l e u k e m i c m i c e treated with DNA synthesis inhibitors, Cancer Res., 33 (1973) 895--901. 1 4 Philips, F . S . , H y d r o x y u r e a . I. A c u t e cell d e a t h in p r o l i f e r a t i n g tissues i n rats, C a n c e r Res., 2 7 ( 1 9 6 7 ) 61--74. 1 5 R a j e w s k y , M . F . , S y n c h r o n i s a t i o n i n vivo: k i n e t i c s o f a m a l i g n a n t cell s y s t e m f o l l o w i n g t e m p o r a r y inh i b i t i o n o f D N A s y n t h e s i s w i t h h y d r o x y u r e a , E x p . Cell R e s . , 6 0 ( 1 9 7 0 ) 2 6 9 - - 2 7 6 . 16 R o s e n k r a n z , H . S . , H . S . C a r r a n d R . D . P o l l a c k , S t u d i e s w i t h h y d r o x y u r e a . VI. E f f e c t s o f h y d r o x y u r e a o n t h e m e t a b o l i s m o f sensitive a n d r e s i s t a n t s t r a i n s o f Escherichia coli, B i o c h i m . B i o p h y s . A c t a , 1 4 9 (1976) 228--245. 1 7 S a g n e r , R., S t r e p t o m y c i n as a m u t a g e n f o r n o n - c h r o m o s o m a l g e n e s , P r o c . N a t l . A c a d . Sei. (U.S.), 4 8 (1962) 2018--2026. 1 8 S a g e r , R . , C y t o p l a s m i c g e n e s a n d o r g a n e l l e s , A c a d e m i c Press, N e w Y o r k , 1 9 7 2 . 1 9 S a g e r , R . a n d S. G r a n i c k , N u t r i t i o n a l c o n t r o l o f s e x u a l i t y in Chlamydomonas reinhardi, J. G e n . P h y s . iol., 3 7 ( 1 9 5 4 ) 7 2 9 - - 7 4 2 . 2 0 S i n c l a i r , W . K . , H y d r o x y u r e a ; d i f f e r e n t i a l l e t h a l e f f e c t s o n c u l t u r e d m a m m a l i a n cells d u r i n g t h e cell cycle, Science, 150 (1965) 1729--1731. 21 T i m s o n , J., H y d r o x y u r e a , M u t a t i o n R e s . 3 2 ( 1 9 7 5 ) 1 1 5 - - 1 3 2 . 2 2 T o b e y , R . A . a n d H . A . C r i s s m a n , Use o f f l o w m i c r o f l u o r o m e t r y in d e t a i l e d a n a l y s i s o f e f f e c t s o f c h e m ical a g e n t s o n c e l l - c y c l e p r o g r e s s i o n , C a n c e r R e s . , 3 2 ( 1 9 7 2 ) 2 7 2 6 - - 2 7 3 2 . 2 3 W r i g h t , J . A . a n d W.H. L e w i s , E v i d e n c e o f a c o m m o n site o f a c t i o n f o r t h e a n t i t u m o u r a g e n t s , h y d r o x y u r e a a n d g u a n a z o l e , J . Ceil P h y s i o l . , 8 3 ( 1 9 7 4 ) 4 3 7 - - 4 4 0 .
Mutation Research, 41 (1976) 217--224 © Elsevier/North-Holland Biomedical Press
THE MUTAGENIC ACTIVITY OF H Y D R O X Y U R E A IN CHLAM YDOMONAS REINHARDI
MONICA ADAMS * and J.R. WARR
Department of Biology, University of York, Heslington, York (England) (Received February 17th, 1976) (Revision received June 6th, 1976) (Accepted July 5th, 1976)
Summary Hydroxyurea, an inhibitor of ribonucleotide reductase, increases the frequency of streptomycin resistant mutants in liquid cultures of Chlamydomonas reinhardi after 45 h incubation. After more prolonged incubation in hydroxyurea medium the frequency of streptomycin resistant mutants declines. This m a y be due to the slower growth rate of streptomycin resistant mutants compared to wild type cells in h y d r o x y u r e a containing medium. Studies on solid medium show that b o t h the rate of forward mutation to streptomycin resistance and reverse mutation to nicotinamide independence are increased several fold by growth on hydroxyurea.
Introduction M o n o h y d r o x y u r e a is a chemotherapeutic agent which has been used effectively in the treatment of several human neoplasms (see [23] for references). The drug is c y t o t o x i c or cytostatic to a variety of cell types, maximal activity having been observed against continuously dividing cells rather than those which are n o t dividing or which have been stimulated to divide [14]. It is cytotoxic to a variety of mammalian cell types during S phase in vitro [20] and in vivo [13,15]. A cytostatic effect has also been observed on a range of mammalian cell types with cells accumulating at the G1/S boundary until the inhibitor is removed [22]. Considerable evidence from p r o - a n d eukaryotic studies suggest that h y d r o y u r e a inhibits DNA rather than RNA or overall protein synthesis [2,4,5,16]. Histone synthesis m a y also be sensitive [12] b u t this is presumably due to its tight coupling with DNA synthesis [8]. The effects of hy* Present
address: I n f e c t i o n
Control Unit, Clifton Hospital, York, U.K.
218 droxyurea on DNA synthesis cell division and chromosome structure have recently been extensively reviewed by Timson [21]. During the course of studies on the mechanism of resistance to hydroxyurea in Chlamydomonas we observed that the drug apparently induced considerable variation in colony size and pigmentation and furthermore h y d r o x y u r e a resistant mutants arose with a remarkably high frequency when selection was made on h y d r o x y u r e a containing medium. These observations suggested the possibility that h y d r o x y u r e a might be mutagenic and prompted the present studies on mutagenic activity of this substance in Chlamydomonas. Materials and methods Strains Wild type, mating type positive Chlamydomonas reinhardi (Strain bridge Collection of Algae) was used in investigations of occurrence mycin resistant mutants. A strain requiring nicotinamide (nic7-, bridge Collection of Algae) was used in investigations of reverse Strains were repurified before each experiment.
32C, Camof streptomt ÷, Cammutation.
Culture media and conditions Unsupplemented media was based on medium 1 of Sager and Granick [19], ferric chloride being replaced by 0.01 g/1 ferric citrate and 0.01 g/1 citric acid. 0.5% peptone and 1.5% Difco agar were added to unsupplemented media for use in agar plates except in the case of plates used for measuring the number of reverse mutants from nicotinamide dependence where no peptone and 1% agar were used. Medium was sterilized by autoclaving at 15 p.s.i, for 15 min. Strept o m y c i n was dissolved in sterile medium and added to autoclaved medium in appropriate amounts. H y d r o x y u r e a (Calbiochem) was dissolved in medium, sterilized by filtration through a porcelain candle filter and then added to cool sterile media. Nicotinamide was filter sterilized and added to give a final concentration of 0.75 mg/1 where necessary. Cultures were incubated at 25°C with a light intensity of 500 foot candles provided by tubular fluorescent lamps. Measurement of mutation rate in liquid cultures A late log phase culture of wild type cells was inoculated into 50 ml quantities of unsupplemented media and media containing 5 × 10 -3 M hydroxyurea, to give an initial cell count of 8 × 104/ml. At the times shown in Fig. 1, 5 ml samples were removed from each flask spun down and resuspended in 2 ml of sterile medium. Of each 2 ml, 1 ml was taken and diluted for viable counts performed in triplicate and 0.1-ml samples were plated on eight 100 pg/ml streptomycin plates. Plates for viable counts were incubated for 4 days, and the strept o m y c i n plates for 14 days when the n u m b e r of resistant colonies was counted. Measurement o f mutation rate on plates 5 × 104 cells of a log phase culture of wild type were spread onto each of the unsupplemented plates and plates supplemented with hydroxyurea. The plates
219 were incubated and at appropriate time intervals nine of each series were removed. Viable counts were performed by washing off cells from three plates with 5 ml of sterile buffer per plate. 1 ml of each washing was taken and after appropriate dilutions viabilities were d e t e r m i n e d by plating in triplicate on unsupplemented media. The remaining six plates of each original set were replica plated onto 100 pg/ml streptomycin and these plates were then incubated for 12 days, after which time the number of resistant colonies was counted. Reverse mutation from nicotinamide dependence was measured in a similar fashion except that nic7- cells were plated originally on plates supplemented with nicotinamide and the cells were replica plated onto minimal media. Results Effect o f hydroxyurea on mutation rate The growth of wild type and the accumulation of streptomycin resistant cells following growth in unsupplemented media and 5 X 10 -3 M h y d r o x y u r e a is shown in Figs. 1 and 2. The growth rate of wild type cells is immediately reduced in medium containing h y d r o x y u r e a b u t little difference in the frequency of m u t a n t cells is seen up to at least 29 h growth in h y d r o x y u r e a compared to the control. However after 45 h some cell death occurs in h y d r o x y u r e a and at the same time the frequency of m u t a n t cells in h y d r o x y u r e a rises sharply from around 4 X 10 -6 to 35 X 10 -6. Rather surprisingly the frequency of m u t a n t cells declines between 70 and 90 h.
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The possibility t h a t streptomycin resistant mutants might survive hydroxyurea treatment better than sensitive cells and so form an increased proportion of the surviving cells was investigated. However, n o t only did streptomycin resistant cells show a lower growth rate compared to wild type cells in medium containing 5 X 10 -3 M h y d r o x y u r e a but they were also killed by this concentration of h y d r o x y u r e a after 46 h incubation (Fig. 3). This latter p h e n o m e n o n could explain the fall off in the frequency of m u t a n t cells in h y d r o x y u r e a medium with longer incubation times. The effects of h y d r o x y u r e a on growth rate and viable count after prolonged incubation times mean, however, that comparisons of the frequency of m u t a n t cells in unsupplemented and h y d r o x y u r e a medium at a given time are being made on cells in different phases of the growth cycle. The experiments described so far clearly demonstrate an increase in the frequency of mutants in the presence of h y d r o x y u r e a in liquid medium. However, the differences in growth rates and viabilities between streptomycin resistants and sensitives in unsupplemented and h y d r o x y u r e a containing media precluded any calculation of m u t a t i o n rates from the available data [11]. Further experiments of different design were therefore performed in order to achieve this.
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•ncubation time (h) Fig. 3. G r o w t h o f wild t y p e a n d s t r e p t o m y c i n r e s i s t a n t C h l a m y d o m o n a s in t h e p r e s e n c e a n d a b s e n c e of h y d r o x y u r e a in liquid m e d i a . (© o) Wild t y p e , u n s u p p l e m e n t e d m e d i u m ; (~ D) wild t y p e , 5 X 10 -3 M h y d r o x y u r c a ; (o e ) s t r e p t o m y c i n r e s i s t a n t , u n s u p p l e m e n t e d m e d i u m ; (A z~) s t r e p t o m y c i n r e s i s t a n t , 5 X 10 -3 M h y d r o x y u r e a .
Cells were plated on u n s u p p l e m e n t e d and h y d r o x y u r e a c o n t a i n i n g solid m e d i a and at intervals viable c o u n t s and n u m b e r s o f m u t a n t colonies were e s t i m a t e d as d e s c r i b e d in the M e t h o d s section. T h e m u t a t i o n rates were t h e n calculated a c c o r d i n g t o the f o r m u l a , m u t a t i o n rate = log 2 (M2 --M,) (N2 -- N , ) TABLE I M U T A T I O N R A T E S T O S T R E P T O M Y C I N R E S I S T A N C E A N D N I C O T I N A M I D E I N D E P E N D E N C E IN T H E A B S E N C E A N D P R E S E N C E O F 2.5 × 1 0 - 3 M H U . M U T A T I O N R A T E S C A L C U L A T E D AS D E S C R I B E D IN T H E T E X T . Mutation rates/107 cells/generation
S t r e p t o m y c i n resistance, E x p e r i m e n t 1 S t r e p t o m y c i n resistance, E x p e r i m e n t 2 Nicotinamide independence
Unsupplemented media
Hydroxyurea media
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where M1 and M2 are the numbers of m u t a n t clones and N1 and N2 are the total number of cells at times 1 and 2 respectively [1 ]. Preliminary experiments showed little or no effect on the m u t a t i o n rate in cells growing on 10 -3 M h y d r o x y u r e a and a marked growth inhibitory effect during growth on plates containing 5 X 10 -3 M hydroxyurea, greater than that observed previously in liquid media containing the same concentration (compare wild type growth curves in Figs. 1 and 3 with Fig. 4). 2.5 X 10 -3 M hydroxyurea was therefore selected as an optimal concentration for measurement of mutation rates for forward m u t a t i o n to streptomycin resistance and reverse m u t a t i o n to nicotinamide independence. As can be seen from Table I, growth on this concentration of h y d r o x y u r e a increases the mutation rate several-fold. Discussion This paper shows t h a t h y d r o x y u r e a is mutagenic in the eucaryotic alga Chlamydomonas reinhardi. In view of the very extensive literature concerning
223
the inhibitory effects of h y d r o x y u r e a on DNA synthesis in a very wide range of organisms and the evidence for chromosome aberrations following exposure to this drug [21] it is perhaps surprising that such mutagenic activity has not been reported previously. Huberman and Heidelberger [10] found no increase in the frequency of azaguanine resistant mutants in Chinese hamster cells in partially toxic concentrations of hydroxyurea. Exposure to the drug was, however, only continued for 4 h, whereas in our experiments prolonged exposure for over 29 h was necessary before any significant accumulation of mutant cells could be detected. Although any precise correlation of time periods required to induce comparable effects on two such different cell types as Chinese hamster and Chlamydomonas are clearly impossible, it does n o w seem worth re-examining the mutagenic effects of h y d r o x y u r e a in mammalian cells over prolonged periods. This is particularly the case in view of the fact that h y d r o x y u r e a is still used clinically as an antitumour drug and several workers have proposed that mutation may play a significant role in t u m o u r progression [9]. H y d r o x y u r e a treatment m a y involve a balance between preferential destruction of t u m o u r cells and accelerated t u m o u r progression due to mutagenic activity. The reason for the delay in the induction of mutations during this work has n o t been established. We may speculate that ribonucleotide reductase must be inhibited for a sufficiently long period to allow depletion of deoxyribonucleoside phosphate pools which then leads to m u t a n t induction. Drake has pointed o u t (p. 156 in [6] } that along delay in filling a position during DNA replication might favour the incorporation of incorrect nucleotides and this might explain why a number of other c o m p o u n d s which interfere with pyrimidine and purine production are mutagenic in Escherichia coli. Such c o m p o u n d s include azaserine, benzimidazole, 6 mercaptopurine, 5 aminopurine amongst others. However, Fig. 1 does show that there is considerable inhibition of growth, presumably due to inhibition of DNA synthesis, during the first 30 h growth in 5 X 10 -3 M h y d r o x y u r e a without any significant induction of mutation (Fig. 2). During the course of a study on the effects of inhibitors on recombination in Chlamydomonas, Chiu and Hastings [3] showed that 5 X 10-3M h y d r o x y u r e a over a 2-h period gave a 71% inhibition of DNA synthesis, 27% inhibition of RNA synthesis and no inhibition of protein synthesis. Although their work was carried out in a slightly different medium and at a lower temperature than ours, it does strongly suggest that DNA synthesis is rapidly inhibited in Chlamydomonas cells growing in 5 X 10 -3 h y d r o x y u r e a after a very short period. Cytoplasmic mutations have long been known to occur in Chlamydomonas reinhardi [ 18] and we should therefore consider whether the mutations studied during the present w o r k are chromosomal or cytoplasmic. The concentration of streptomycin used to select resistant mutants during this work (100 pg/ml) does select b o t h chromosomal and non-chromosomal streptomycin resistant mutations in Chlamydomonas, b u t the frequency of the former is approx. 2 X 103 fold greater than the latter on minimal medium [17]. We therefore consider it likely that the great majority of our streptomycin resistant mutations are chromosomal. The nicotinamide auxotroph used for reversion to prototrophy is a mutation in a well characterised chromosomal gene [7] and so this problem does not arise.
224
Acknowledgement We gratefully acknowledge financial assistance from the Yorkshire Cancer Research Campaign in the form of a research studentship to one of us (M.A.). References 1 Beale, G . H . , A m e t h o d f o r t h e m e a s u r e m e n t o f m u t a t i o n r a t e f r o m p h a g e s e n s i t i v i t y t o p h a g e resist a n c e in Escherichia coli, J. G e n . M i c r o b i o l . , 2 ( 1 9 4 8 ) 1 3 1 - - 1 4 2 . 2 C a m e r o n , I.L. a n d J . R . J e t e r , A c t i o n o f h y d r o x y u r e a a n d N - c a r b a m o y l o x y u r e a o n t h e cell c y c l e o f T e t r a h y m e n a , Cell Tissue K i n e t . , 6 ( 1 9 7 3 ) 2 8 9 - - 3 0 1 . 3 C h i n , S.M. a n d P . J . H a s t i n g s , P r e - m e i o t i c D N A s y n t h e s i s a n d r e c o m b i n a t i o n in Chlamydomonas reinhardi, G e n e t i c s , 7 3 ( 1 9 7 3 ) 2 9 - - 4 3 . 4 C o h e n , L.S. a n d G.P. 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K . , H y d r o x y u r e a ; d i f f e r e n t i a l l e t h a l e f f e c t s o n c u l t u r e d m a m m a l i a n cells d u r i n g t h e cell cycle, Science, 150 (1965) 1729--1731. 21 T i m s o n , J., H y d r o x y u r e a , M u t a t i o n R e s . 3 2 ( 1 9 7 5 ) 1 1 5 - - 1 3 2 . 2 2 T o b e y , R . A . a n d H . A . C r i s s m a n , Use o f f l o w m i c r o f l u o r o m e t r y in d e t a i l e d a n a l y s i s o f e f f e c t s o f c h e m ical a g e n t s o n c e l l - c y c l e p r o g r e s s i o n , C a n c e r R e s . , 3 2 ( 1 9 7 2 ) 2 7 2 6 - - 2 7 3 2 . 2 3 W r i g h t , J . A . a n d W.H. L e w i s , E v i d e n c e o f a c o m m o n site o f a c t i o n f o r t h e a n t i t u m o u r a g e n t s , h y d r o x y u r e a a n d g u a n a z o l e , J . Ceil P h y s i o l . , 8 3 ( 1 9 7 4 ) 4 3 7 - - 4 4 0 .