The ‘antimutagenic’ effect of cinnamaldehyde is due to a transient growth inhibition

Mutation Research, 201 (1988) 97-105 Elsevier

97

MTR04615

The 'antimutagenic' effect of cinnamaldehyde is due to a transient growth inhibition Bernhard Rutten and Elmar Gocke Biol. Pharm. Research Department, F. Hoffmann-La Roche and Co. Ltd., CH-4002 Basel (Switzerland) (Received 21 August 1987) (Revision received 20 January 1988) (Accepted 10 February 1988)

Keywords: Cinnamaldehyde; Antimutagenic effect; Growth inhibition.

Summary Addition of cinnamaldehyde to the selective medium causes a reduction in the number of revertant colonies of S. typhimurium or E. coli when the cells have been mutagenized with 4NQO but not when they have been mutagenized with M N N G . Toxicity of the cinnamaldehyde exposure depends largely on the status of growth a n d / o r nutrient supply of the cells. We present evidence that simple growth inhibition due to lack of nutrients mimics the effect of cinnamaldehyde in 4NQO- and MNNG-treated cells. This argues that the reduction of mutant colonies is due to a transient growth retardation caused by cinnamaldehyde exposure, which presumably allows the cells to repair 4NQO-induced damage - but not MNNG-induced damage - via a more error-free pathway.

Kada and coworkers (Ohta et al., 1983a) described an antimutagenic effect of cinnamaldehyde in E. coli WP2 uorA after exposure of the cells to 4-nitroquinoline-N-oxide (4NQO) but not to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). The cells were treated with the mutagens in liquid medium, then washed and plated on minimal-agar plates containing cinnamaldehyde and trace amounts of nutrients to allow expression of the induced mutations. Survival, measured on the same minimal-agar plates, was not reduced. Thus the term 'antimutagenic' seemed to be appropriate for this effect of cinnamaldehyde.

Correspondence: Dr. E. Gocke, Biol. Pharm. Research Department, F. Hoffmann-La Roche and Co. Ltd., CH-4002 Basel (Switzerland).

The reduction of mutant colonies of 4NQOtreated E. coli or S. typhimurium was also observed by us. However, we could not reproduce the 'non-toxicity' of the cinnamaldehyde treatment: in our experiments the survival of the cells, when tested on the minimal-agar plates, was at least as strongly reduced as the number of mutant colonies. Similar findings have recently been reported by De Silva and Shankel (1987). These observations prompted us to investigate the toxicity of cinnamaldehyde in more detail. The effects of cinnamaldehyde exposure were compared to the effects caused by post-treatment incubation of the cells in various liquid media and to the effect of withholding essential nutrients on the agar plates for defined periods of time. Our results indicate that cinnamaldehyde induces a transient growth delay which allows the

0027-5107/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)

98 error-free repair of DNA damage inflicted by 4NQO while the delay has no effect on the repair of damage inflicted by M N N G . Material and methods

Strains The Salmonella strains were obtained from B. Ames, the E. coli K-12/343/765 (STR d) from G. Mohn and the E. coli WP2 uvrA from t h e National College of Industrial and Marine Bacteria Ltd., Aberdeen (Scotland). The identity of the Salmonella strains has been verified by the recommended procedures (Maron and Ames, 1983). The spontaneous mutant frequency of TA102 is around 100-200 if no tetracycline is added to the overnight cultures and 300-500 if 2 /tg/ml tetracycline is added. Media SEM agar (Witkin, 1975) consisted of VBE medium (Vogel and Bonner, 1956) supplemented with 0.4% glucose, 0.016% nutrient broth (Difco) and 1.5% agar. VB agar consisted of VBE medium supplemented with 2% glucose and 1.5% agar. Histidine and biotin were added to the soft agar at the standard concentrations of 21 and 24 ktg/plate (Maron and Ames, 1983). Nutrient broth agar consisted of 2.5% nutrient broth and 1.5% agar. Cinnamaldehyde (BDH chemicals) was added to the agar shortly before pouring the plates. The experiments were performed with plates not more than 5 days old. Overnight cultures were grown in nutrient broth. For E. coli K - 1 2 / 3 4 3 / 7 6 5 PEPS broth (Mohn, 1984), containing 50 mg/1 streptomycin, was used. Mutagen treatment An overnight culture was diluted 10 times and incubated for 3 h at 37 ° C. The cells were then harvested by centrifugation, exposed to the mutagen for 1 h in phosphate buffer, centrifuged and washed thereafer and plated in 2 ml soft agar (0.7%) on the various agar plates or incubated in liquid media as described below. 4NQO was from Dr. Ashby, ICI, M N N G from Serva. The colonies were counted after 2 days incubation at 37 ° C. Background growth was examined

with a stereomicroscope at 40 x magnification. In some instances photographs were taken with a Zeiss microscope with a 16 x objective and size and number of microcolonies were compared on the photographs. Protein determination The protein content was determined by centrifuging and washing an aliquot (1 ml) of the culture, dissolving the cells with 200 ILl 1 N N a O H and, after neutralizing with 2 ml 2% Na2CO 3, following the Lowry procedure. Bovine serum albumin was used as standard (Lowry et al., 1951). Results

E. coli WP2 uvrA and S. typhimurium TA102 were exposed in phosphate buffer for 1 h to 4NQO at concentrations of 2 or 6/~g/ml, respectively. After treatment, the cells were centrifuged, the pellets resuspended in buffer and plated on agar plates containing various concentrations of cinnamaldehyde. The plates used for selection of mutants (Fig. 1A) contained trace amounts of nutrients: for E. coli semi-enriched minimal agar medium (SEM, Witkin, 1975) and for S. typhimurium Vogel Bonner E medium plus histidine/ biotin (Maron and Ames, 1983) was used. Fig. 1A shows that cinnamaldehyde reduced the number of mutant colonies in a dose-dependent fashion. To test for survival, appropriately diluted cell suspensions were plated either on identical minimal medium (Fig. 1B), on nutrient broth agar (Fig. 1C) or on minimal agar containing additionally ca. 108 filler cells (Fig. 1D). Fig. 1B shows that the number of surviving colonies was even more reduced than the number of mutant colonies when minimal-agar plates were used for plating. However, if the cells were plated on rich agar (Fig. 1C), no cell killing effect was observable, suggesting that either cinnamaldehyde was inactivated by the broth or that the cells were not as sensitive to the toxic effect due to better growth status on the rich medium. To simulate the high cell density existing on the plates used for mutant selection, 108 living, but not colony-forming, E. coli 343/765 (STR d) cells were plated together with the diluted sample of the 4NQO-treated cells on minimal agar. No toxic

99

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Fig. 1. (A) Relative mutant colony numbers on cinnamaldehyde-containing agar plates after 4NQO treatment of WP2 uvrA and TA102 (1 h at 2 or 6/~g/ml respectively). The number of colonies in the absence of cinnamaldehyde was 740 for WP2 uvrA and 550 for TA102. (B, C, D) Relative survival of the 4NQO-treated cells plated at low density (B, C) or together with ca. 108 filler cells (D). The trace amounts of tryptophan or histidine/biotin in the minimal medium allow the cells to form small but clearly visible colonies, so that survival can be checked on the same plates as used for mutant selection. As filler cells E. coli 343/765 were used (Mohn, 1984). These cells require streptomycin for growth and thus are not able to form colonies on the agar plates used in this experiment.

effect of c i n n a m a l d e h y d e was obvious u n d e r these c o n d i t i o n s (Fig. 1D). Essentially a n a l o g o u s results, c o n c e r n i n g m u t a n t c o l o n y n u m b e r s as well as survival, were o b s e r v e d with o t h e r S. typhimurium strains (e.g., T A 1 0 0 (uorB, p K M 1 0 1 +) a n d TA1535 (uvrB, p K M 1 0 1 - ) ) . Based on these results, o n e has to a c c e p t that c i n n a m a l d e h y d e r e d u c e d the frequencies of m u t a n t colonies a p p a r e n t l y w i t h o u t affecting survival on the plates used for m u t a n t selection. E x a m i n a t i o n of the b a c k g r o u n d g r o w t h corr o b o r a t e d that n o r e d u c t i o n of m i c r o c o l o n y n u m b e r s was obvious o n these plates. A s has also b e e n r e p o r t e d b y K a d a a n d colleagues ( O h t a et al., 1983) a n d D e Silva a n d S h a n k e l (1987), c i n n a m a l d e h y d e d i d n o t reduce the n u m b e r of m u t a n t colonies after M N N G t r e a t m e n t (Fig. 2). S p o n t a n e o u s m u t a n t colonies were likewise n o t reduced. T h u s the ' a n t i m u t a -

genic' effect of c i n n a m a l d e h y d e is specific to 4 N Q O - e x p o s e d cells. To obtain information on possible transient toxic (i.e., g r o w t h - d e l a y i n g ) effects of the c i n n a m a l d e h y d e t r e a t m e n t , p r o t e i n c o n t e n t a n d cell titers of liquid cultures were d e t e r m i n e d . Fig. 3 shows as an e x a m p l e that c i n n a m a l d e h y d e severely i n h i b i t e d the p r o t e i n synthesis in strain T A 1 0 0 g r o w i n g in m i n i m a l m e d i u m s u p p l e m e n t e d with histidine/biotin. At a cinnamaldehyde concentration of 50 # g / m l p r a c t i c a l l y n o increase of the p r o t e i n c o n t e n t o c c u r r e d after 6 h i n c u b a t i o n . However, after overnight i n c u b a t i o n the p r o t e i n c o n t e n t s of t r e a t e d a n d u n t r e a t e d cultures r e a c h e d similar levels. A n a l o g o u s curves were o b t a i n e d for the cell titers ( d a t a n o t shown). F r o m these e x p e r i m e n t s , however, it c a n n o t be a n s w e r e d w h e t h e r all o f the cells finally o v e r c a m e a transient g r o w t h i n h i b i t i o n or w h e t h e r a f r a c t i o n

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containing cinnamaldehyde at the indicated concentrations. An OD of 0.1 corresponds to 45 #g protein per ml culture, as determined with the Lowry method. The cultures were inoculated at a cell density of 107/ml. The minimal medium contained histidine/biotin at concentrations of 10 and 1 #g/ml, respectively.

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60

cinnamaldehyde(ug/ml) Fig. 2. (A) Relative mutant colony numbers of untreated or MNNG-treated cells on cinnamaldehyde-containing agar plates. The colony numbers in the absence of cinnamaldehyde were 100 for TA102 untreated (no tetracycline added to the overnight culture), 10 for WP2 uvrA untreated and 320 for WP2 uvrA (MNNG, 1 h, 2 #g/ml). (B) Relative survival on minimal-agar plates or nutrient broth agar plates containing cinnamaldehyde (see Fig. 1B, C).

o f the i n i t i a l l y e x p o s e d cells d i e d a n d t h e rem a i n i n g s u r v i v o r s g r e w u p to t h e f i n a l d e n s i t y . I n f o r m a t i o n c o n c e r n i n g this p o i n t c a n b e o b tained from the background lawn on the minimal-agar plates used for mutant selection. As m e n t i o n e d a b o v e , t h e n u m b e r o f b a c k g r o u n d colo n i e s o n t h e c i n n a m a l d e h y d e - c o n t a i n i n g p l a t e s was n o t r e d u c e d n o r w a s t h e i r size c h a n g e d w h e n c o m p a r e d to t h e c o n t r o l plates. A s a n e x a m p l e , p h o t o g r a p h s (Fig. 4) o f the b a c k g r o u n d g r o w t h are s h o w n of p l a t e s w h e r e c i n n a m a l d e h y d e w a s s p o t t e d in a c e n t r a l well o f a m i n i m a l - a g a r p l a t e c o n t a i n i n g 4 N Q O - t r e a t e d S a l m o n e l l a cells. N o

Fig. 4. Background growth on a minimal-medium agar plate on which 4NQO-treated TA102 cells were plated and which contained in a central well 500 #g of cirmamaldehyde. (A) Background growth in the outermost ring where no reduction of revertant colonies was observed (the dark spot is a macrocolony). (B) Background growth in a more central ring where no revertant colonies were present, i.e., where cinnamaldehyde exerted an 'antimutagenic' effect.

101

70( MUTANTS 60( 50( 40( --~30( ~20( ~E 10(

difference in the appearance of the microcolonies is seen between a central area where cinnamaldehyde reduced the number of mutant colonies and a more distal area where cinnamaldehyde concentration was too low to exert 'antimutagenic' action. This argues that the numbers of microcolony-forming cells did not decrease and therefore it suggests that the toxic effect of cinnamaldehyde observed above is limited to a transient growth inhibition. In order to examine the influence of such a transient growth delay on mutant colony formation the following simulation experiment was performed: after treatment of E. coli WP2 uvrA with either 4 N Q O or M N N G (at doses producing approximately equal numbers of mutants) the cells were washed and plated on minimal agar (VB) without any addition of trace nutrients. At various times a second soft-agar layer containing the same amount of nutrient broth as present in SEM was poured on top of the agar layer containing the

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Fig. 6. The effect of various liquid holding conditions in the presence and absence of cinnamaldehyde (50 pg/ml) on mutant colony numbers (A, C, E, G) and cell growth (B, D, F, H). An overnight NB culture of E. coil WP2 uvrA was diluted 10 times and incubated in NB or S E M + 100 p g / m l tryptophan for 3 h, then washed and treated for 1 h with 2 p g / m l 4NQO, washed again and resuspended with 1/20 of the pre-treatment density in the indicated media. After the various holding periods 200 VI of the cultures were plated on SEM agax for the determination of mutants and appropriate dilutions were plated on NB agar for determination of survival. The pre- and post-treatment media are indicated in the figures. Since fewer cells are plated on the selective plates the mutant colony numbers are lower than in the other experiments (e.g., Fig. 1).

102

cells. Fig. 5 shows that the growth delay caused by the lack of essential nutrients had no influence on the number of MNNG-induced and on the spontaneous mutants. However, the number of 4NQO-induced mutants decreased rapidly. After 5 h holding the mutant colony number was reduced by almost 90% and thus shows a similar reduction as effected by cinnamaldehyde. This finding suggests that the growth delay caused either by lack of nutrients or by the toxic effect of cinnamaldehyde might mediate the 'antimutagenic' effect. Fig. 6 gives a more detailed analysis of the relationship between liquid holding under different growth conditions and the cinnamaldehyde effect. The E. coli uorA cells were grown overnight, diluted into NB (or SEM + trp) and incubated for 3 h to obtain exponentially growing cultures. The cells were exposed to 4NQO in buffer, washed and resuspended at 1 / 2 0 of the pretreatment density in different media containing or lacking cinnamaldehyde. After various incubation periods the mutant colony numbers and the cell titers were determined. When NB was employed as pre- and post-treatment medium the mutant colony number (Fig. 6A) of the cinnamaldehyde-free culture increased rapidly. The culture containing cinnamaldehyde showed a declining mutant colony number for the first 2 h (decrease by 62%); thereafter the number increased probably due to division of the established mutants. The effect of cinnamaldehyde on the cell titer was relatively small under these growth conditions: at 2 h there was a reduction by 50% but after 4 h both cultures showed similar densities as the stationary growth phase was approached (Fig. 6B). When SEM + trp was employed as pre- and post-treatment medium (Fig. 6C) the mutant colony number of the cinnamaldehyde-containing culture decreased by 73% for the first 4 h. Fig. 6D shows that cinnamaldehyde had a stronger growth-retarding effect in this medium than in NB. Even more prominent was the reduction of mutant colony number and cell growth when the cells were transferred from NB to SEM medium (Fig. 6E,F). Under this condition the number of mutants of the cinnamaldehyde-containing culture decreased by 92% for the full 6 h of liquid holding.

Even the cinnamaldehyde-free culture showed a declining mutant colony number for the first 2 h, probably as a result of the growth delay effected by the shift of incubation medium. When the ceils were held in phosphate buffer after 4NQO treatment (Fig. 6G) the mutant colony number decreased independently of the presence or absence of cinnamaldehyde for the full holding period. This series of experiments thus emphasizes the observation that the number of potential mutants after 4NQO treatment decreases when the cells are held under no-growth conditions. The longer the division delay the lower the number of mutant colonies. Whether the growth delay is caused by withholding essential nutrients, by a change of medium or the presence of cinnamaldehyde seems to be of little importance. Finally it was attempted to determine the time course of fixation of the mutant phenotype on SEM agar in the absence of cinnamaldehyde (Fig. 7). For this purpose the 4NQO-treated cells were plated on SEM agar and after various periods cinnamaldehyde was added in a second soft-agar layer. If cinnamaldehyde was added very shortly after plating of the cells, the mutant colony number was reduced by 99%, but if cinnamaldehyde addition was delayed, the mutant colony numbers increased rapidly and after 2 h reached the level of EFFECTS OF VARIOUS HOLDING CONDITIONS

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Fig. 7. The effect of various holding conditions on the mutant colony numbers of 17. coli WP2 uvrA. The cells were treated with 2 ~ g / m l 4NQO, washed and plated on agar plates as described for Fig. 5 in a first soft-agar layer containing either SEM, cinnamaldehyde (50 / t g / m l of total plate volume) or only buffer. A second soft-agar layer containing further ingredients (SEM, cinnamaldehyde, or only DMSO) was added at later times. The various ingredients of the first agar layer (e.g., SEM at 0) and of the second agar layer (e.g., C I N N delayed) are indicated for each curve in the figure.

103 the control cultures not exposed to cinnamaldehyde. Thus the pathway towards error-prone fixation of most of the premutational lesion was irreversibly entered within the first 2 h of incubation on the SEM agar plates. Contrasting to this curve, Fig. 7 also shows the decline of the number of mutant colonies upon holding the cells on VB plates without tryptophan and adding SEM soft agar after the various holding periods. As the 2 curves show an almost reciprocal course it is obvious that the 2 processes the fixation of the mutant phenotype on SEM agar and the disappearance of mutant colonyforming cells under no-growth conditions - possess very similar kinetics.

Discussion

The description of cinnamaldehyde as an antimutagen by Kada and colleagues (Ohta et al., 1983) rested on the observation of decreased mutant colony numbers on SEM plates and no apparent decrease of colonies when aliquots of the cells were plated on identical plates at low cell densities for estimation of survival. In our initial experiments, however, we found that survival, when checked on SEM plates, was at least as much affected as mutant colony numbers. Thus our initial results only indicated a toxic but not an antimutagenic effect. No lethal effects of cinnamaldehyde were, however, observed when survival was determined by plating the cells on a richer agar. Because of either different cell density or different nutrient supply both these plating conditions do not fully resemble the conditions existing on the plates used for mutant selection. Therefore we investigated survival on minimal plates in the presence of a high number of streptomycin-dependent filler cells, which were alive but not able to form colonies. Under these conditions no killing of the mutagen-treated cells was observed. Thus it could be concluded that cinnamaldehyde reduced the number of mutant colonies without reducing survival on the plates used for mutant selection. Therefore a general lethal effect cannot be the cause of the reduction of the mutant colony numbers although selective killing of potential mutants

cannot be excluded from these results. Experimentally such a selective killing is very difficult to determine. However, it seems unlikely that the toxic action of cinnamaldehyde should specifically act on potential trp ÷ revertants of E. coli as well as potential his ÷ revertants of S. typhimurium only when the lesions were induced by 4NQO and not when induced by M N N G . Clearly a general growth and protein synthesis retardation is demonstrable after cinnamaldehyde exposure. It is therefore suggestive that holding of the mutagen-treated ceils under no-growth conditions (by omitting essential nutrients; Fig. 5) closely mimics the reduction of mutant colony numbers for the 4NQO-treated cells and has no effect on the MNNG-treated cells. The more elaborate experiment (Fig. 6) shows that upon liquid holding of the cells under no-growth conditions the mutant frequency declines rapidly and with similar kinetics irrespective of whether the growth delay is caused by a lack of essential nutrients or the presence of cinnamaldehyde. Even a transient growth delay caused by a change of the medium (NB to SEM) induces a reduction of the mutant colony numbers. We therefore argue that the decrease of the mutant frequency after cinnamaldehyde exposure is mediated by the transient division delay which allows an error-free repair system to remove the pre-mutational lesions before they become fixed by the error-prone repair mechanism. The cells containing the 4NQO-induced premutational lesions become committed on SEM agar towards the error-prone pathway and on no-growth medium towards the error-free pathway with roughly similar kinetics (Fig. 7): within the first 2 h repair of most of the lesions has entered the respective pathways. This scheme involves some sort of regulation of the action of the different repair enzymes. It might be that the error-free system has precedent over the error-prone system as long as the cells are not 'forced' to divide. Or the induction of the errorprone repair enzymes specific for the 4NQO damage might be inhibited under no-growth conditions caused by a lack of nutrients or cinnamaldehyde exposure. Mutations following 4NQO damage are reported to be predominantly produced through the

104 TABLE 1 SURVIVAL OF 4NQO-TREATED E. coli WP2 uvrA CELLS ON DIFFERENT MEDIA (NB OR SEM) CONTAINING OR LACKING CINNAMALDEHYDE (80 /tg/ml) OR AFTER A 4-h HOLDING PERIOD ON THE PLATES LACKING ESSENTIAL NUTRIENTS BEFORE SEM WAS ADDED IN A SECOND SOFT-AGAR OVERLAY The colony count of the non-treated cells on cinnamaldehydefree NB plates is taken as 100% survival. Cells plated on NB NB + Cinn. SEM SEM (after 4 h holding) SEM + CINN

4NQO treatment (15 min) 0/~g/ml 100 105

10 ~g/ml 25 52

20 ttg/ml 6 18

101

39.5

15

103

52

20

0

0

0

u m u / m u c - d e p e n d e n t error-prone SOS repair (Kato and Nakano, 1981). Ohta et al. (1983b) showed that cinnamaldehyde does not interfere with the SOS functions of filamentous growth and prophage induction. Of interest is also their finding that cinnamaldehyde increased the survival of the E. coli WP2 uvrA cells after 4 N Q O treatment. We also observed such a trend when the cells were plated on nutrient broth agar but not when they were plated on SEM plates, as in the latter case the toxicity of cinnamaldehyde was dominant (Table 1). Again a holding period of 4 h under no-growth conditions gave a similar increase of survival as cinnamaldehyde exposure. These observations are more easily reconciled with the notion that growth delay (by cinnamaldehyde or lack of nutrients) does not reduce the overall repair capacity by inhibiting errorprone repair but rather that it enhances the repair capacity by supporting an error-free repair of 4NQO-induced lesions. For M N N G - i n d u c e d lesions the cells do not seem to have an option of using alternative repair pathways with diverse fidelities under different growth conditions. The findings of declining revertant colony numbers and enhanced survival after holding

mutagenized cells under no-growth conditions share some phenomenological similarities to M F D (mutation frequency decline) and L H R (liquid holding recovery): UV-induced mutagenesis is decreased and survival is increased as a consequence of post-treatment incubations that cause metabolic imbalances, e.g., by holding the cells in defined growth media without a required amino acid (Witkin, 1956, 1975; Green et al., 1977; Tang and Smith, 1980). However, contrary to the effects reported here the processes of M F D and L H R depend on an intact excision repair. Doubleday et al. (1975) reported that plating of UV-irradiated E. coli cells in the presence of pantoyl lactone reduced the mutant frequency and increased survival. These effects were observed in an excision-repair-defective strain. However, transient incubation in liquid medium containing pantoyl lactone and then plating on pantoyl lactonefree medium gave the full yield of mutations. Thus while transient incubation in cinnamaldehydecontaining medium appears to cause a fairly rapid loss of premutational 4NQO-induced lesions this does not seem to happen with the UV-induced lesions in pantoyl lactone-containing medium. Another difference is that pantoyl lactone repressed several SOS functions. From our observations we infer that the effect of cinnamaldehyde is a very indirect one, based on a transient toxic effect, which gives the cells time to repair 4NQO-inflicted D N A damage without producing errors (i.e., mutations). Zeiger and Pagano (1984) studied a variety of compounds which suppressed mutant colony formation when mixed with mutagens. They attributed the effects to toxic activity during the first cell divisions which, however, did not manifest itself in a visible reduction of the background growth. Mechanistically, such toxic effects should reduce the number of mutant colonies independently of the type of mutagen. In most cases this was found to be true. However, in some instances, the suppressive effects were specific to one or several mutagens. The investigation presented here gives one possible interpretation as to the basis for these findings. Zeiger and Pagano (1984) used the term 'suppressive' and avoided the term 'antimutagenic' in their study. To us also it seems debatable whether the latter term is appropriate for such an indirect

105 effect, which is caused essentially b y a n o n - l e t h a l b u t toxic action. T h e d r a m a t i c differences i n toxicity of c i n n a maldehyde, d e p e n d i n g o n p l a t i n g m e d i u m a n d cell density, d e m o n s t r a t e the need for detailed analysis of toxic action, n o t only for investigation of the a c t i o n m e c h a n i s m s of m u t a t i o n - m o d u l a t i n g comp o u n d s b u t also for tests where m u t a n t frequencies are estimated b y p l a t i n g different cell densities a n d / o r using different p l a t i n g media. It is c o n c e i v a b l e that the fraction of p o t e n t i a l l y c o l o n y - f o r m i n g cells d e t e r m i n e d b y colony n u m bers o n the survival plates is u n d e r e s t i m a t e d a n d thus a false-positive response is generated. The presented system (Fig. 1D) using streptomycin-dep e n d e n t cells as 'filler' cells seems to be a simple alternative to the more elaborate scheme proposed by, e.g., Maliniack et al. (1986).

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