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Mutation Research, 41 (1976) 217--224 © Elsevier/North-Holland Biomedical Press

THE MUTAGENIC ACTIVITY OF HYDROXYUR 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 r ibonucleotide reductase, increases the fre- quency of s t reptomycin resistant mutants in liquid cultures of Chlamydomonas reinhardi after 45 h incubation. After more prolonged incubation in hydroxy- urea medium the frequency of s t reptomycin resistant mutants declines. This may be due to the slower growth rate of s t reptomycin resistant mutants com- pared to wild type cells in hydroxyurea containing medium. Studies on solid medium show that both the rate of forward mutat ion to s t reptomycin resis- tance and reverse mutat ion to nicotinamide independence are increased several fold by growth on hydroxyurea.

Int roduct ion

Monohydroxyurea is a chemotherapeutic agent which has been used effec- tively in the t reatment of several human neoplasms (see [23] for references). The drug is cy to toxic or cytostat ic to a variety of cell types, maximal activity having been observed against continuously dividing cells rather than those which are not dividing or which have been stimulated to divide [14]. It is cyto- toxic to a variety of mammalian cell types during S phase in vitro [20] and in vivo [13,15] . A cytostat ic effect has also been observed on a range of mam- malian cell types with cells accumulating at the G1/S boundary until the inhibi- tor is removed [22] . Considerable evidence from p r o - a n d eukaryotic studies suggest that hydroyurea inhibits DNA rather than RNA or overall protein syn- thesis [2,4,5,16]. Histone synthesis may also be sensitive [12] but this is pre- sumably 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.

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droxyurea on DNA synthesis cell division and chromosome structure have re- cently 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 hydroxyurea resis- tant mutants arose with a remarkably high frequency when selection was made on hydroxyurea containing medium. These observations suggested the possibil- ity that hydroxyurea 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 32C, Cam-

bridge Collection of Algae) was used in investigations of occurrence of strepto- mycin resistant mutants. A strain requiring nicotinamide (nic7-, mt ÷, Cam- bridge Collection of Algae) was used in investigations of reverse mutation. Strains were repurified before each experiment.

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. Strep- tomycin was dissolved in sterile medium and added to autoclaved medium in appropriate amounts. Hydroxyurea (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 con- centration 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 quanti-

ties 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 per- formed in triplicate and 0.1-ml samples were plated on eight 100 pg/ml strepto- mycin plates. Plates for viable counts were incubated for 4 days, and the strep- tomycin plates for 14 days when the number of resistant colonies was count- ed.

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

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were incubated and at appropriate time intervals nine of each series were re- moved. 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 de te rmined by plating in triplicate on un- supplemented media. The remaining six plates of each original set were replica plated onto 100 pg/ml s t reptomycin and these plates were then incubated for 12 days, after which time the number of resistant colonies was counted.

Reverse mutat ion 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 s t reptomycin resistant cells following growth in unsupplemented media and 5 X 10 -3 M hydroxyurea is shown in Figs. 1 and 2. The growth rate of wild type cells is immediately re- duced in medium containing hydroxyurea bu t little difference in the frequency of mutant cells is seen up to at least 29 h growth in hydroxyurea compared to the control. However after 45 h some cell death occurs in hydroxyurea and at the same time the frequency of mutant cells in hydroxyurea rises sharply from around 4 X 10 -6 to 35 X 10 -6. Rather surprisingly the frequency of mutant cells declines between 70 and 90 h.

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5 X 10 -3 M hydroxyurea. (o o) Unsupplemented medium; (o o) medium containing 5 × 10 -3 M hydroxyurea.

220

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Fig. 2. A c c u m u l a t i o n o f s t r e p t o m y c i n res i s tant m u t a n t s f o l l o w i n g g r o w t h in 5 X 10 -3 M h y d r o x y u r e a . (o o) Cells g r o w n in m i n i m a l m e d i u m ; (o 8) cells g r o w n in m e d i u m s u p p l e m e n t e d w i t h 5 × 10 -3 M h y d r o x y u r e a .

The possibility that streptomycin resistant mutants might survive hydroxy- urea t reatment better than sensitive cells and so form an increased proportion of the surviving cells was investigated. However, not only did streptomycin re- sistant cells show a lower growth rate compared to wild type cells in medium containing 5 X 10 -3 M hydroxyurea but they were also killed by this concentra- tion of hydroxyurea after 46 h incubation (Fig. 3). This latter phenomenon could explain the fall off in the frequency of mutan t cells in hydroxyurea me- dium with longer incubation times. The effects of hydroxyurea on growth rate and viable count after prolonged incubation times mean, however, that com- parisons of the frequency of mutant cells in unsupplemented and hydroxyurea 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 fre- quency of mutants in the presence of hydroxyurea in liquid medium. However, the differences in growth rates and viabilities between streptomycin resistants and sensitives in unsupplemented and hydroxyurea containing media precluded any calculation of mutat ion rates from the available data [11]. Further experi- ments of different design were therefore performed in order to achieve this.

221

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Fig. 3. G r o w t h of wild t y p e and s t r e p t o m y c i n res is tant C h l a m y d o m o n a s in the presence and absence of h y d r o x y u r e a in l iquid m e d i a . (© o) Wild t ype , 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 res is tant , u n s u p p l e m e n t e d m e d i u m ; (A z~) s t repto- m y c i n res is tant , 5 X 10 -3 M h y d r o x y u r e a .

Cells were pla ted 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 con ta in ing solid media and at intervals viable coun t s and number s o f m u t a n t colonies were es t imated as descr ibed in the Methods sect ion. The m u t a t i o n rates were then calculated accord ing to the fo rmula ,

m u t a t i o n rate = log 2 (M2 - - M , ) (N2 -- N, )

T A B L E I

M U T A T I O N R A T E S TO 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 OF 2.5 × 1 0 - 3 M HU. 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 .

Muta t i on r a t e s /107 ce l l s /genera t ion

U n s u p p l e m e n t e d med ia H y d r o x y u r e a med ia

S t r e p t o m y c i n res is tance , E x p e r i m e n t 1 S t r e p t o m y c i n res is tance , E x p e r i m e n t 2 N ico t inamide i n d e p e n d e n c e

1.23 1 .23 0 .12

6 . 4 3 4 . 8 8 0 . 8 5

222

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Tncubation t ime (h) Fig. 4. G r o w t h of wild t ype C h l a m y d o m o n a s on solid m e d i u m s u p p l e m e n t e d wi th var ious c o n c e n t r a t i o n s of h y d r o x y u r e a . (o o) U n s u p p l e m e n t e d m e d i u m ; (o o) m e d i u m con ta in ing 10 -3 M h y d r o x y - urea ; (D D) m e d i u m con ta in ing 2.5 )< 10 -3 M h y d r o x y u r e a ; (~ ~) m e d i u m con ta in ing 5.0 X

10 -3 M h y d r o x y u r e a .

where M1 and M2 are the numbers of mutant 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 mutat ion rate in cells growing on 10 -3 M hydroxyurea 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 (com- pare wild type growth curves in Figs. 1 and 3 with Fig. 4). 2.5 X 10 -3 M hy- droxyurea was therefore selected as an optimal concentration for measurement of mutat ion rates for forward mutat ion to streptomycin resistance and reverse mutat ion to nicotinamide independence. As can be seen from Table I, growth on this concentration of hydroxyurea increases the mutat ion rate several-fold.

Discussion

This paper shows that hydroxyurea is mutagenic in the eucaryotic alga Chlamydomonas reinhardi. In view of the very extensive literature concerning

223

the inhibitory effects of hydroxyurea 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 now seem worth re-examining the mutagenic effects of hydroxyurea in mammalian cells over prolonged peri- ods. This is particularly the case in view of the fact that hydroxyurea is still used clinically as an ant i tumour drug and several workers have proposed that mutat ion may play a significant role in tumour progression [9]. Hydroxyurea t reatment may involve a balance between preferential destruction of tumour cells and accelerated tumour progression due to mutagenic activity.

The reason for the delay in the induction of mutations during this work has not been established. We may speculate that r ibonucleotide reductase must be inhibited for a sufficiently long period to allow depletion of deoxyribonucleo- side phosphate pools which then leads to mutant induction. Drake has pointed out (p. 156 in [6] } that a long delay in filling a position during DNA replication might favour the incorporation of incorrect nucleotides and this might explain why a number of other compounds which interfere with pyrimidine and purine production are mutagenic in Escherichia coli. Such compounds include azaser- ine, benzimidazole, 6 mercaptopurine, 5 aminopurine amongst others. How- ever, Fig. 1 does show that there is considerable inhibition of growth, presum- ably due to inhibition of DNA synthesis, during the first 30 h growth in 5 X 10 -3 M hydroxyurea wi thout any significant induction of mutat ion (Fig. 2). During the course of a s tudy on the effects of inhibitors on recombination in Chlamydomonas, Chiu and Hastings [3] showed that 5 X 10-3M hydroxyurea 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 Chlamydo- monas cells growing in 5 X 10 -3 hydroxyurea after a very short period.

Cytoplasmic mutat ions have long been known to occur in Chlamydomonas reinhardi [ 18] and we should therefore consider whether the mutat ions studied during the present work are chromosomal or cytoplasmic. The concentration of s t reptomycin used to select resistant mutants during this work (100 pg/ml) does select both chromosomal and non-chromosomal s treptomycin resistant mutat ions in Chlamydomonas, but the frequency of the former is approx. 2 X 103 fold greater than the latter on minimal medium [17]. We therefore con- sider it likely that the great majority of our s t reptomycin resistant mutat ions are chromosomal. The nicotinamide auxotroph used for reversion to proto- t rophy is a mutat ion in a well characterised chromosomal gene [7] and so this problem does not arise.

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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.).

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