Mutation Research, 267 (1992) 183-192

183

© 1992 Elsevier Science Publishers B.V. All rights reserved 0027-5107/92/$05.00

MUT 00304

Modulation of the mutagenic response in prokaryotes Silvio D e F l o r a , A n n a C a m o i r a n o , F r a n c e s c o D ' A g o s t i n i a n d R o u m e n Balansky h~stitute of Hygiene and Prerentice Medicine, Unh~ersityof Genoa, 1-16132 Genoa (Italy) (Received 1 August 1991) (Revision received ! 8 October 1991) (Accepted 18 October 1991)

Keywords: Prokaryotes; Antimutagenicity; DNA damage; Modulation

Summary Short-term tests investigating genetic end-points in prokaryotes have been extensively used worldwide not only for risk assessment purposes but also for evaluating the modulation of the mutagenic response. In spite of some intrinsic limitations, such as the lack of cell compartmentalization or the need for an exogenous metabolic system working extracellularly, experimental systems in bacteria can provide useful preliminary indications and some information on the mechanisms involved. In the large majority of studies the putative modulator is mixed with a known mutagen and then assayed in target bacteria, with suitable controls. However, under natural conditions exposure of target cells to modulators may either precede, co-exist with, or follow exposure to mutagens. Therefore, a variety of methodological variations, involving pre-treatment, co-treatment, or post-treatment of bacteria with the putative modulator, have been designed. Application of these procedures showed that the effects of modulators can be completely upset, from inhibition to enhancement, or vice versa, by changing the experimental conditions. Use of methodological variations may provide more complete information on the spectrum of possible effects in bacteria as well as a better insight into modulation mechanisms. Several examples illustrating the flexibility of the Salmonella test in this field of research are available. On the other hand, the widespread use of these relatively simple techniques, yet requiring skillfulness and experience, may lead to some misuse or oversimplifications. A rather common inadequacy is to use excessive amounts of test mutagens, or to express the results in terms of revertants/survivors, rather than revertants/plate. In fact, in the Salmonella test the number of revertants is rather unrelated to the initial number of plated bacteria, provided a normal background lawn of bacterial growth is formed. Thus, a 50% killing of bacteria will not appreciably influence the number of revertants/plate, but expressed as revertants/survivors the effect will look twice as large.

Correspondence: Dr. S. De Flora, Istituto di Igiene e Medicina Preventiva, Universit'~ di Genova, via Pastore I, 1-16132 Genoa (italy).

There is no doubt that for many years (Clarke and Shankel, 1975) a large proportion of the continuously growing literature in the field of

le t

antimutagenesis has been generated using bacterial test systems and, most frequently, the Ames Sa|monella/microsome test. As compared to higher organisms, unicellular targets have typical intrinsic limitations. Compared to eukaryotes, they suffer frbm the lack of a compartmentalized structure of the cell. In addition, there is the need for an exogenous metabolic system, which works in an unnatural position and cannot thoroughly mimic the network and delicate balance of intraceUular biochemical pathways. It should be noted, however, that host-mediated assays using pairs of bacterial strains differing in their DNA repair capacity have also been proposed, and applied to the study of the protective effects of dietary constituents in various organs of mice (Knasmiiller et ai., 1991). in spite of some disadvantages, short-term tests in bacteria provide versatile and flexible tools for the assessment of the antimutagenic properties of both chemical compounds and complex mixtures, as well as for the identification of active components thereof. When properly used, they can also contribute to the understanding of the mechanisms involved. The rapidity, low cost and relative simplicitj of these tests can be exploited in order to explore various experimental details and to repeat the assays several times, an advantage which can be hardly achieved when using timeconsuming, expensive and complex techniques in other targets. In any case it is clear that the indications of prokaryotic test systems should preferably be coupled with the findings provided by other in vitro and in rive tests. Especially when the results are used for extrapolating antimutagenicity data to potential cancer protective effects, adequate information on pharmacokinetics, metabolism and in vivo tolerability should be made available. In the present paper we refrain from presenting an overview of the available bacterial test systems and techniques, a topic which is already covered by a broad literature (see, e.g., Maron and Ames, 1983: Hofnung and Quillardet, 1986, for reviews). Rather, we will consider both con. ceptual and methodological approaches, concerning the application of these tests in the study of the modulation of the mutagenic response,

Methodological approaches In the large majority of the studies available in the literature, modulation of genetic end-points in bacteria, e.g., reversion, forward mutation, differential lethality in repair-proficient and -deficient strains, induction of SOS repair, DNA fragmentation, induction of prophages, etc., is investigated by mixing together a known mutagen and the candidate modulator, using some kind of preincubation procedure. Thereafter, variations of the genotoxic potency are evaluated in the system used, as compared to suitable controls. In other words, bacteria are employed as biological indicators of the outcome of the interaction between two compounds, like a spectrophotometer is used for measuring the outcome of chemical or biochemical reactions. This kind of approach is acceptable ft~r largescale monitoring. For instance, techniques of this type have been used for antimutagenicity screenings of quite large numbers of natural products (Wall et ai., 1990), or for the assessment of structure-antimutagenicity relationships (De Flora et al., in preparation). However, in other cases some additional information would be desirable. A crucial point is that under natural conditions exposure of target cells to modulators may either precede, co-exist with, or follow exposure to mutagens. Situations of either type can be mimicked in bacteria by applying methodological variations of the standard procedures used in risk assessment. Table 1 provides examples of some procedures suggested for evaluating the modulation of the mutagenic response in the Salmonella reversion test, basically performed according to liquid incubation variations of the standard plate incorporation test (Maron and Ames, 19~'~3). Some technical details and general guidelines are given in the footnote to this table, Conceptually similar procedures can be adapted to other bacterial reversion test systems and to forward mutation assays as well.

Pre-treatment procedures The procedure reported in Table l, and similar ones, are designed in order to favor penetration of the modulator into bacterial cells prior to

185 TABLE 1 M E T H O D O L O G I C A L VARIATIONS OF B A C T E R I A L TEST SYSTEMS A I M E D AT EVALUATING THE MODULATION O F T H E M U T A G E N I C RESPONSE AND T H E MECHANISMS INVOI.VED Methodological approach

Possible mechanisms involved in the modulation of mutagcnicity

Outline of the suggested procedures

Intracellular reaction of the modulator with the test mutagen, or interference with cellular mechanisms leading to its genotoxicity

1

2 Co-treatment

Extracellular reaction of the modulator with the test mutagen or with components of the exogenous metabolic system

2A - Bacteria + modulator + mutagen +$9 mix (20 rain) - Wash bacteria and plate 2B - Modulator + mulagen +_$9 mix (20 rain) - Add bacteria and plate 2C - Mutagen + $9 mix (20 min) - Add the modulator (20 rain) - Add bacteria and plate

3 Post-treatment

Effect of the modulator on DNA ~epair, fixation and expression of DNA damage produced by the: test mutagen

3A - Bacteria + mutagen + $9 mix (20 rain) - Wash bacteria, add the modulator and plate 3B -" Bacteria + mutagen _+$9 mix (20 rain) - Wash bacteria and incubate with the modulator in fresh broth (30-60 rain) - Wash bacteria and plate 3C - Bacteria + mutagen _+$9 mix (20 rain) - Wash and further incubate bacteria in fresh broth (30-60 rain) - Add the modulator and plate

1

Pre-treatment

- Modulator + bacteria in fresh nutrient broth (4 h) - Wash bacteria - Bacteria + mutagen + $9 mix (20 rain) - Wash bacteria and plate

Bacteria. Procedure 1 always requires logarithmically growing bacteria. All remaining procedures may either use overnight broth cultures or logarithmically growing cells of the most suitable S. typhhnurium his- strain, or an E. colt trp or are strain, or any other bacterial strain sensitive to reversion. Conceptually similar procedures can be used with bacterial strains revealing forward mutations (e.g., rif r, hal r, aru r, etc.). Soh'etlts for t~:~t mutagens and modulutor.~. If possible 0.2 M phosphate buffered solution (PBS), pH 7.4: otherwise dimethyl sulfoxide (DMSO) or, provided no turbidity occurs, DMSO with further dilution in PBS. Check the final p14! Volumes of reagents, 100/~l for mutagen and modulator solutions, and bacterial suspensions: 5 ml for nulrient broth (Oxoid No. 2) in procedure 1; 5-10 ml PBS for washing bacteria: 0.5 ml for 59 mix incorporating 10r',~ (or another optimal concentration fi)r activating test promutagens) St) or S12 rat liver fractions (or other suitable metabolic system), Doses of reagents (selected in preliminary assays). Mutagen: doses falling in the linear part of the dose-response curve under test conditions (see 'control of the mutagen'), and in any case yielding no more than 1500 revertants/plate. Modulator: start from l0 mmol or the highest sublethal dose, and then test at least 3 additional doses diluted with a 3.3 geometric ratio (or closer dilutions in confirmation assays). Controls. Of the mutagen: replace the modulator with its solvent. Of the modulator: replace the mutagen with its .solvent. Of the metabolic system: omit $ 9 / S I 2 fractions from S9 mix. Of bacteria: replace mc~dulator and mutagen with their solvents. Incubations. All incubations at 37°C with continuous gentle mixing, e.g., in a Dubnoff water bath or in rotating tubes in a thermostat. Replicates. All assays in triplicate plates. Each e×perimcnt should be repeated at least 3 times. Scoring of results. Using a magnifying lens or, better, a stereomicroscope, check accurately the consistence of the background lawn of bacterial growth, as compared to controls of bacteria. If toxicity is suspected, the results should be discarded. Count colonies of revertant bacteria with the aid of an automatic colony counter or, better, manually (experienced reader!). Express the results (rely as revertants per plate (see text for a discussion on this point), and calculate mean_+SD. Et'aluation of results. Subtract spontaneous revertants and, at each dose of modulator, calculate a simple modulation index: revertants [mutagen + modulator] ., revertants [mutagen controls] _

_

,-

x

100;

in this way, in case of inhibition, the dose inhibiting 50% of mutagenicity (IbDsn) can be inferred from dose-response curves and used as an indication of antimutagenic potency. More sophisticated analyses, e.g., keeping into account the slope and profile of dose-response curves, can alternatively be used.

186

exposure to the test mutagen. In such a way, it is possible to investigate intracellular reactions of the modulator (or its intraceilular derivatives) with the test mutagen, or interferences of the modulator with cellular pathways leading to genotoxieity. As suggested in Table 1, incorporation of the modulator into target cells is better achieved following a long-lasting contact with logarithmically growing cells. As is well known, penetration of both mutagens and modulators is favored by the high permeability of the cell membrane of S. typhimurium strains carrying the rfa mutation. An inconvenience is that it is rather difficult, albeit not impossible, to check the amounts of compounds incorporated into bacteria, and some residues of compounds adsorbed on the outer cell membrane cannot be ruled out even after multiple washings.

Co-treatment procedures These procedures are aimed at investigating possible reactions between modulator and mutagen occurring in the extracellular environment. In cases of promutagens requiring $9 activation, interference of the modulator with the exogenous metabolic system can also be studied. Reactions between modulator, mutagen and, if needed, $9 mix can be carried out in the presence of bacteria (procedure 2A), which may be particularly convenient when the compound or its metabolites are short-lived. Alternatively, bacteria can be added at the end of the preincubation step (procedure 2B). The parallel use of these two procedures may be useful to discriminate the possible contribution of intracellular mechanisms (procedure 2B does not involve washing of bacteria) from pure extracellular mechanisms (procedure 2A). Procedure 2C, involving addition of the modulator after metabolic activation of the promutagen, is suggested in order to check whether a possible inhibition of the activity of a promutagen may be due to blocking of electrophilic metabolites, provided they are sufficiently long lived, rather than to interference with biochemical pathways (see example below).

Post-treatment procedures When the mutagen has already been incorporated into target bacteria, thereby starting its

DNA-damaging activity, addition of the modulator can reveal possible effects on DNA repair, fixation and expression of DNA damage, lnhibitors acting at this level have been referred to as bioantimutagens by Kada et al. (1982). Of the methods reported in Table 1, the first two differ in that procedure 3A involves addition of the modulator to mutagen-treated bacteria just at the moment of plating, which implies a persistent action of the modulator throughout the 48-h growth period in soft agar. In procedure 3B the possible action of the modulator is conversely limited to a short period of incubation in broth before final washing and plating of bacteria. Procedure 3C is designed in such a way that the modulator is applied after a preliminary growth in broth (30-60 min or even longer) of mutagentreated bacteria, which may allow the detection of late effects of the modulator itself. It should be stressed that all post-treatment procedures involve growth of bacteria in soft agar in the absence of any extracellular mutagenic species.

Variability factors involved in the modulation of bacterial genotoxleity it is evident that a systematic application of a whole array of procedures, like those reported in Table 1 and discussed in the previous sections, is rather complex and time-consuming. As such, it is not suitable for screening purposes. However, the comparative use of different procedures is strongly advisable for elucidating the mechanisms of modulators as well as for evaluating the whole spectrum of possible effects on the mutagenic response under different experimental conditions.

Site and patterns of interaction of modulators with mutagens As a demonstration of the importance of the latter point, there are several examples of compounds which appear to inhibit bacterial mutagenicity under certain conditions, but become ineffective or even co-mutagenic by changing the site and patterns of interaction with mutagens. For instance, studies by Balansky et al. (1983, and unpublished data) provided evidence that caffeine inhibited the mutagenicities of both methyl-

187 TABLE 2 E F F E C T O F C A F F E I N E ON MNU, MNNG A N D AFB I M U T A G E N I C I T Y IN T H E SALMONELLA TEST Procedure

Pre-treatment Co-treatment Post-treatment

Test mutagen MNU

MNNG

AFB 1

= ~~ ~ J,

T 1' ~~ l

-= NT

= , no change; 1', slight or 1' 1', marked enhancement; J,, slight or ~ ~, marked inhibition; NT, not tested.

nitrosourea (MNU) and N-methyl-N'-nitro-Nnitrosoguanidine (MNNG) when assayed in cotreatment and post-treatment procedures but, by contrast, enhanced MNNG mutagenicity and had no effect on MNU mutagenicity when assayed in pre-treatment procedures (Table 2). Sodium selenite enhanced the mutagenicities of both these alkylating agents in strain TA1535 of S. typhimurium when given in a pre-treatment procedure, inhibited them when given in a co-treatment procedure and had no effect when given in a post-treatment procedure (Table 3). Neither caffeine nor selenite affected the S9-mediated mutagenicity of aflato~in BI (AFB~) when assayed in pre-treatment procedures (Tables 1 and 2). Thiols, such as reduced glutathione (GSH) and N-acetylcysteine (NAC) also appear to play a dual role in the bacterial genotoxicity of MNNG. This was shown in our laboratory with both the reversion test with S. typhimurium TAI00 and a DNA repair test in E. coil, evaluating the differential lethality of MNNG in strain WP2 (repairproficient) and in the triple mutant CM871, lackTABLE 3 E F F E C T OF SELENIUM ON MNU, MNNG AND AFB~ M U T A G E N | C I T Y IN T H E SALMONELLA TEST Procedure

Pre-treatment Co-treatment Post-treatment

Test mutagen MNU

MNNG

AFBj

1' 1' ~ =

1' +~ =

= -NT

= , no change; T, slight or T 1', marked enhancement; ~, slight or ~, J,, marked inhibition; NT, not tested.

ing the uvrA, recA and lexA DNA repair mechanisms. In fact, both GSH and NAC eliminated MNNG genotoxicity when using a co-treatment procedure. Incidentally, a similar inhibition was recorded in neutral and acidic environments, a technical aspect which is worthy of being investigated in cases of modulators that are expected to interact with mutagens in the gastric environment. On the other hand, MNNG genotoxicity in both reversion and DNA repair tests was enhanced by using a pre-treatment procedure, and pre-treatment of bacteria with the GSH depletor diethyl maleate resulted in a decreased MNNG genotoxicity (Camoirano et al., 1988). These findings are consistent with the view that the intraceliular reaction of MNNG with sulfhydryl groups leads to formation of a carbonium ion which could methylate DNA, thereby enhancing MNNG genotoxicity and carcinogenicity, e.g., in the glandular stomach (Margison and O'Connor, 1979). This is also supported by a greater resistance of GSH-deficient bacterial mutants to MNNG mutagenicity and toxicity (Mohn et al., 1983). Outside the cells, e.g., in the gut, where enterobacteria can export significant amounts of GSH (Owens and Hartman, 1986), the intermediate resulting from the aforementioned reaction is conversely expected to react with water to form methanol, with consequent detoxification of MNNG.

Dose of modulators Another critical point, affecting the outcome of the interaction between a mutagen and a modulator, is the dose of both reactants. The dose of mutagen will be considered below. As to the amount of modulator, a linear dose-effect relationship would be expected to occur when an individual mechanism is responsible for modulation, at least up to saturation of the process involved. However, diphasic or multiphasic effects can be observed when two or more mechanisms are responsible for modulation, and the outcome depends on their prevalence and coordination. Therefore, it is important that dose-effect relationships are investigated in antimutagenicity studies. An example is provided by thiols, such as NAC, which exhibited a diphasic dose.related effect on the activity of several promutagens, e.g.,

188

E -1

E o ex_

"E e~t -t

Blocking of elec~ophilic melaboliles

Dose of thiol Fig. 1. Patterns and postulated mechanisms of modulation of the mutagenic potency of promutagens in the Salmonella reversion lest, as related to the dose of thiol.

benzo[a]pyrene, 2-aminofluorene, cyclophosphamide, aflatoxin B~, the pyrolysis product Trp-P-2, and a cigarette smoke condensate, in the Salmonella reversion test (De Flora et al., 1984, 1985). $9 mix containing liver preparations from either untreated or variously induced rats were used as metabolic activation system in these studies. A typical profile of a dose-response curve is represented in Fig. 1. Using a co-treatment procedure (2B, Table 1), NAC enhanced the mutagenie response of all promutagens throughout a broad range of doses. Only at high but sublethal doses did the thiol inhibit mutagenicity, an effect which also was observed using procedure 2C, i.e., adding NAC after the metabolic activation step. This suggests that antimutagenicity depends on blocking of reactive metabolites of promutagens by NAC, which is an efficient nucleophile. Conversely, potentiation of mutagenicity at lower NAC doses was no longer observed when procedure 2C was used, which suggests that NAC enhances the metabolic activation of test compounds. This conclusion is also supported by parallel in rive studies, showing the ability of NAC to stimulate arylhydrocarbon hydroxylase activity in rat liver (De Flora et al., 1991a). On the whole, modulation of the activity of promutagens by thiols appears to be a two-step process, a stimulation of their metabolic activation being coordinated with blocking of the resulting metabolites. A mechanism of this type is likely to provide the best wa:.' for detoxifying promutagens and favoring their excretion from the organism, since it avoids the accumulation of

unmetabolized xenobiotics. In fact, in vivo experiments showed that NAC efficiently prevents the formation of carcinogen-DNA adducts in rats treated with promutagens, i.e., 2-acetylaminofluorene (De Flora et al., 1991a) and benzo[a]pyrene, and also inhibits the clastogenicity of this polycyclic aromatic hydrocarbon (De Flora et al., 1991b). Certainly, a more superficial screening of modulation by thiols of the activity of promutagens in bacteria would only have revealed the potentiation mechanism, which does not correlate with the overall protective effects produced in rive and detected, with suitable procedures, also in bacterial test systems. Studies which are now in progress in our laboratory indicate that certain phenolic compounds, such as caffeic acid, also exhibit a diphasic modulation of the S9-mediated mutagenicity of cigarette smoke, i.e., potentiation at lower doses and inhibition at higher doses. These results are obtained by exposing to mainstream cigarette smoke, for a short period, the surface of agar plates incorporating bacteria (S. typhimurium TA98), varying doses of modulator, and $9 mix. Inhibition of the genotox|city of reactive oxygen species in bacterial reversion and DNA repair tests

Another demonstration of the fexibility of bacterial test systems in the field of modulation is their application for investigating scavenging of genotoxic oxygen species. Using the hypoxanthine (HX)/xanthine oxidase (XO) system, which had been so far applied in other experimental systems in order to generate superoxide anion ( 0 2 ) via monoeleetronic reduction of molecular oxygen, we obtained a reproducible mutagenic response in the Ames reversion assay, exclusively by using strain TAI04. Acceleration of dismutation by superoxide dismutase (SOD) resulted in a further enhancement of mutagenicity (Fig. 2), due to formation of hydrogen peroxide (H202), which could be inhibited by catalase (De Flora et al., 1989). In any case, as shown in Fig. 2, addition of thiols to this system markedly inhibited the mutagenicity of reactive oxygen species and, interestingly enough, also reduced the "spontaneous" mutagenicity in strain TA104. NAC also inhibited

189

t-~

~

NAC

Spontaneous

o]

nolhiol GSH MPG

LWIUWm

[HX + XO]

H202

IHX+ XO+ SOD]

i

[[~llllIHlllll~llllltl[llllllllUIn]lllrllll~llllllll~-~

0

200

400

600

ao0

1000

Revertants per plate (m -+ SD) Fig. 2. Mutagenicity in strain TAI04 of S. typhimurium, either spontaneous or induced by reactive oxygen species generated

in the H X / X O / S O D system, and its inhibition by the thiols N-acetylcysteine (NAC), glutathione (GSH) and a-mercaptopropionylglycine (MPG). Data derived from De Flora et al. (1989).

the ability of H 2 0 2 to produce a differential killing in the E. coli strains WP2 and CM871 (De Flora, 1984). Singlet oxygen (~AgO2), an electronically excited form of oxygen, reverted neither TAI04 (De Flora et al., in preparation) nor 26 S. typhimurium his- strains other than TA104 (Dahl et al., 1988). However, we recently applied a system in which the genotoxicity of volatile oxy. gen species can be detected in a battery of DNA repair-deficient E. coil strains, the most sensitive of which is WP100 (uvrA - recA-). By changing the experimental conditions, it is possible to mainly obtain electron transfer reactions, generating O~ and H 2 0 z, or energy transfer reactions, generating IAgOz. Incorporation of NAC in the system resulted in a dose-dependent elimination of genotoxicity (Camoirano et al., in preparation). Therefore, bacterial test systems appear to be quite suitable to detect the genotoxicity of various oxygen species as well as specific antigenotoxic effects. Some common artifacts and technical inadequacies

The possible occurrence of artifacts is a general problem in any research methodology, and

short-term tests in bacteria are no exception. Certainly, their occurrence tends to be enhanced by the use of modulation procedures which deviate from standardized and well validated techniques. An additional problem may arise from some technical inadequacies which appear to be rather common in antimutagenicity studies.

Dose of test mutagen For instance, a rather common drawback is the use of excessive amounts of test mutagen. As reported in the footnote to Table 1, it is advisable to use doses of mutagen falling in the linear part of the dose-response curve, to be assessed in preliminary assays. In any case, controls of test mutagen should yield no more than 1500 revertants/plate. Higher numbers of bacterial colonies tend to overlap and cannot be accurately read (incidentally, manual counting is preferable to automatic colony counting). Moreover, over certain levels a further enhancement in the number of revertants can be hardly appreciated, and inhibition of mutagenicity becomes more evident by lowering the dose of mutagen. Composition and pH of the reaction medium Attention should be paid to variations of pH following mixture with the candidate modulator, and suitable controls should be included, as suggested in the footnote to Table 1. The composition of buffers and media is also important. For instance, as shown in Fig. 3 for MNU, the mutagenicity in S. typidmurium of the aikylating agents MNU and MNNG is considerably enhanced following a short preincubation step in phosphate buffered solution containing potassium, as compared to a similar buffer, having the same pH and ionic strength, but containing sodium (Balans~ and Bryson, 1985). The carbon source used can also influence the results, as shown, e.g., by the considerable decrease of 9-aminoacridine mutagenesis in S. typhimurium strains carrying the hisC3076 frameshift marker when glucose is used as the carbon source (Kopsidas and MacPhee, 1991). Expression of results One of the main sources of artifacts results from killing of bacteria by certain modulators.

190

Therefore, as suggested in the footnote to Table 1, suitable controls of the modulator, without test mutagen, should also be included. However, toxicity phenomena could also ensue from additive effects of mutagen and modulator, or from their interaction. Therefore, the absence of toxicity should be carefully checked by observing the background lawn of bacterial growth, possibly by means of a stercomicroscopc, or by other methods measuring the number of viable bacteria grown in top agar rather than simply the number of bacteria surviving before plating. In our opinion, the results obtained at doses of modulator a n d / o r mutagen yielding toxic phenomena should be discarded. We are against the use of normalizing the revertants to viable bacteria, an approach which is not infrequent in mutagenicity studies and is even more common in antimutagenicity studies. Expression of results as mutagens/survivors is quite correct in other genotoxicity test systems, e.g., in eukaryotes, but is not correct in bacterial reversion assays. In fact, the number of revertants is rather unrelated

2200

Revertants /plate

Revertants / 108 bacteria

':°i [

0 , , , ....

1,oo Isp°"ta"e°"~( , , , i

2000!

~

,oool

':::f 4000I

ii!ii i l

i ..........

l 4NQ0~0"4 Pg ..........

t

/0 t 5000

,o.

;:

11:00

........... jt:oo: ooo `0°°

o 40 ~o 12o lso ~oo o 40 so 12o 15o 200 Bacterial inoculam (IJ.I) Fig. 4. Relationships between the density of the bacterial population (S. typhimurium TAI00) and mutagenicity, either spontaneous or induced by 4-nitroquinoline I-oxide (4NQO) or benzo[a]pyren¢ (BP). The results are expressedas number of revertants (mean + SD of triplicates) either per plate or per 10" bacteria (corresponding to an inoculum of 100 #l/plate).

2000 180o •~,

16o0 14oo

"

t

~ "/

pf

1200 looo eoo 600t 400[ OL

I

2"

3

4.

5,

6,

MNU (raM) Fig. 3. Influence of 4 mM potassium ~K+), present in the phosphate buffered solution, pit 7,4, as compared to a potassium-free buffer(K-) on the mutagenicityof varyingconcen-

trations of MNU in S. typhimuriumTA1535.MNU was preincubated for 20 min at 37°C in the buffer containingbacteria before plating. Data derived from Balansky and Bryson (1985).

to the density of the population of plated bacteria, as shown by several investigators using the Salmonella reversion test (e.g., Salmeen and Durisin, 1981; Kazmer et al., 1983; White et al., 1983; Paes, 1984). Likewise, expression of results as revertants/survivors is erroneous in the reversion assay in trp- E. coil strains, and has been referred to as "distilled water effect or how to make every experiment a positive" (Green e Id Muriel, 1976). A demonstration of such an assumption is shown in Fig. 4, reporting spontai~eous and induced mutagenicity data in S. lyphimurium TA100, as related to the density of bacterial inoculum, varying over a 10-fold range, i.e., from 20 to 200 /zl/plate. It can be seen that the number of spontaneous revertants per plate is only very weakly affected by the amounts of bacteria plated. The reason for this is that, when

191

fewer bacteria are plated, each of them will have more histidine available for growth and will undergo more replication cycles in the soft agar, until roughly attaining the same final number of cells as that generated by a greater bacterial inoculum. Expression of results as revertants/108 bacteria (corresponding to an inoculum of about 100 /~l) will simultte an evident mutagenic response. In other words, a two-fold dilution of the initial inoculum, corresponding to a 50% killing of bacteria by a mutagen and/or modulator, will not affect the number of spontaneous revertants/ plate but when expressed as revertants/survivors the effect will look twice as large. Fig. 4 also shows that, although not as striking as in the case of spontaneous revertants, the mutagenic response induced by both direct-acting mutagens (4-nitroquinoline 1-oxide) and S9-requiring agents (bcnzo[a]pyrene) is improperly amplified when the results are expressed as revertants/bacteria plated rather than as revertants/plate. Conclusions In conclusion, short-term tests in prokaryotes are relatively simple, yet they require skillfulness and understanding. The information they can provide in mutation research as well as in antimutation research is important to such an extent that technical inadequacies and oversimplifications should be carefully avoided, both in the performance of the experiments and in their interpretation. It should be stressed again that a reliable assessment of antimutagenic and anticarcinogenic factors requires the convergence of multiple methodological approaches, both in vitro a:,,d in vivo, from prokaryotes to humans. Keeping this in mind, it is undisputable that well-done studies in bacterial test systems can give a valuable contribution for evaluating the modulation of the mutagenic response and understanding the mechanisms involved. Acknowledgement R. Balansky is recipient of a EACR Italian fellowship from AIRC (Associazione Italiana per la Ricerca sul Cancro). Preparation of this article was partly supported by MURST (40%).

References Balansl~, R., and L. Bryson (1985) Stimulatory effect of potassium ions on MNU- and MNNG-inc'uced mutagenesis in Salmonella typhimarium TA1535 and TAI00, Neoplasma, 32, 313-321. Balansky, R., P. Blagoeva and Z. Mircheva (1983) The ~nfluence of selenium and caffeine on chemical carcinogenesis in rats, mutagenesis in bacteria and unscheduled DNA synthesis in human lymphocytes, Biol. Trace Element Res., 5, 331-343. Camoirano, A., G.S. Badolati, P. Zanacchi, M. Bagnasco and S. De Flora (1988) Dual role of thiols in N-methyI-N'nitro-N-nitrosoguanidine genotoxicity, Life Sci. Adv. Exp. Oncol., 7, 21-25. Clarke, C.H., and D.M. Shankel (1975) Antimutagenesis in microbial systems, Bacteriol. Rev., 39, 33-53. Dahl, T.A., W.R. Midden and P.E. Hartman (1988) Pure exogenous singlet oxygen: nonmutagenicity in bacteria, Mutation Res., 201, 127-136. De Flora, S. (1984) Detoxification of genotoxic compounds as a threshold mechanism limiting their carcinogenicity, Toxicol. Pathol., 12, 337-343. De Flora, S., C. Bennicelli, P. Zanacchi, A. Camoirano, A. Morelli and A. De Flora (1984) hz citro effects of Nacetylcysteine on the mutagenicity of direct-acting compounds and procarcinogens, Carcinogenesis, 5, 505-510. De Flora, S., C. Bennicelli, A. Camoirano, D. Serra. M. Romano, G.A. Rossi, A. Morelli and A. De Flora (1985) hi z'ico effects of N-acetylcysteine on glutathione metabolism and on the biotransformation of carcinogenic and/or mutagenic compounds, Carcinogenesis, 6, 17351745. De Flora, S., C. Bennicelli, P. Zanacchi, F. D'Agostini and A. Camoirano (1989) Mutagenicily of active oxygen species in bacteria and its enzymatic or chemic:d inhibition, Mutation Res., 214, 153-158. De Flora, S., A. Camoirano, A. lzzotti, P. Zanacchi, M. Bagnasco and C,F. Cesarone (1991a) Antimutagenic and anticareinogenic mechanisms of aminothiols, in: O.F. Nygaard (Ed.), Anticarcinogenesis and Radiation Protection Ili, Plenum Press, New York, lap. 275-285. De Flora, S., F. D'Agostini, A. lzzotti and R. Balansky (1991b) Prevention by N-acetylcysteine of benzo[a]pyrene clastogenicity and DNA adducts in rats, Mutation Res., 250, 87-93. Green, M.H.L, and W.J. Muriel (1976) Mutagen testing using trp* reversions in Escherichia coil, Mutation Res., 38, 3-32. Hofnung, M., and P. Quillardet (1986) Recent developments in bacterial short-term tests for the detection of genotoxic agents, Mutagenesis, I, 319-330. Kada, T., T. lnoue and N. Namiki (1982) Environmental desmutagens and antimt,,agens, in: E.J. Klekowski (Ed.), Environmental Mutagenesis and Plant Biology, Praeger, New York. pp. 137-151. Kazmer, S., M. Katz and D, Weinstein (1983) The effect of culture conditions and toxicity on the Ames Salmo-

192 nella/mierosome agar incorporation mutagenicity assay, Environ. Mutagen., 5, 541-551. Knasmiiller, S., W. Huber and R. Schulte Hermann (1991) Prevention of genotoxic effects by dietary constituents in various organs of mice treated with nitrosam~,nes (Abstract), Third International Conference on Mechanisms of Antimutagenesis and Anticarcinogenesis (ICMAA-III), II Ciocco, Lucca (Italy), May 5-10, p. 117. Kopsidas, G,, and D.G. MacPhee (1991) The antimutagenic effect of glucose on 9-aminoacridine-induced mutagenesis in Salmonella typhimurium (Abstract), Third International Conference on Mechanisms of Antimutagenesis and Anticarcinogenesis (ICMAA-Ill), I! Ciocco, Lucca (Italy), May 5-10, p. 23. Margison, G.P., and P.J. O'Connor (1979) Nucleic acid modification by N-nitroso compounds, in: P.L. Grover (Ed.), Chemical Carcinogens and DNA, Vol. I, CRC Press, Boca Raton, FL, pp. 111-159. Moron, D.M., and B.N. Ames (1983) Revised methods for the Salmonella mutagenicity test, Mutation Res., 113, 173-215. Mohn, (3., P. de Knijff and A. Baars (1983) Cellular glu-

tathione content and sensitivity to alkyl-nitrosoguanidineinduced lethality and mutagenesis in Escherichia coil KI2, Mutation Res., 111, 25-31. Owens, R.A., and P.E. Hartman (1986) Glutathione: a protective agent in Salmonella typhimurium and Escherichia coil as measured by mutagenicity and by growth delay assays, Environ. Mutagen., 8, 659-673. Paes, D.J. (1984) Microbial mutagenicity of selected hydrazines (Misuse of data), Mutation Res., 136, 89-90. Salmeen, I., and A.M. Durisin (1981) Some effects of bacterial population on quantitation of Ames Salmonella-histidine reversion mutagenesis assays, Mutation Res., 85, 109-118. Wall, M.E., M.C. Wani, T.J. Hughes and H. Taylor (1990) Plant antimutagens, in: Y. Kuroda, D.M. Shankel and M.D. Waters (Eds.), Antimutagenesis and Anticarcinogenesis Mechanisms II, Plenum, New York, pp. 61-78. White, G.L., D.T. Beranek and R.H. Heflich (1983) Effect of bacterial concentration on reversions induced in Salmonella typhimurium TA1538 by N-hydroxy-2-acetylaminofluorene, Environ. Mutagen., 5, 565-575.

Modulation of the mutagenic response in prokaryotes.

Short-term tests investigating genetic end-points in prokaryotes have been extensively used worldwide not only for risk assessment purposes but also f...
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