umu-test for rapid detection of genotoxins and genotoxic potentials of environmental samples

Mutation Research, 253 (1991) 215-222

215

© 1991 Elsevier Science Publishers B.V. All rights reserved 0165-1161/91/$03.50

MUTENV 08798

A microplate version of the SOS/umu-test for rapid detection of genotoxins and genotoxic potentials of environmental samples G. Reifferscheid a, j. Heil a, y. Oda b and R.K. Zahn a a Arbeitskreis Molekulare Mechanismen umweltbedingter Gentoxizitiit (AMMUG), Johannes Gutenberg-Uniuersitiit, Mainz and Akademie der Wissenschaften und der L,iteratur, D-6500 Mainz (F.R.G.) and b Osaka Prefectural Institute of Public Health, Osaka (Japan)

(Received 29 October 1990) (Revision received 13 May 1991) (Accepted 16 May 1991)

Keywords: UmuC; Genotoxins; Mutagenesis; Salmonella typhimurium (TA1535/pSK1002); SOS response

Summary The u m u - m i c r o t e s t is a miniaturized automated short-term test version proposed for screening of u m u C - d e p e n d e n t mutagenic potentials of chemicals relevant to environmental pollution, river water and industrial waste water. The test is based on the S O S / u m u - t e s t and has been modified in order to allow extensive testing of environmental samples. Genetically engineered Salmonella typhimurium (TA1535/pSK1002) are incubated on a microplate rotor in a sloping position for 2 h with the test samples, followed by addition of fresh culture medium to reach a 10-fold dilution of the incubation medium. 2 h later, the activity of the /3-galactosidase, which reflects u m u C induction, is determined colorimetrically. The incubation of the bacteria in the presence of the test compounds as well as the assessment of /3-galactosidase activity takes place in 96-well microplates, thus enabling simultaneous screening of large numbers of samples. Data of the genotoxic potentials are available within 8 h. Computer-controlled automation is possible by using a laboratory workstation.

Recently, a new test, the S O S / u m u - t e s t , has been developed for the detection of chemical mutagens and carcinogens (Oda et al., 1985). The test is based on Salmonella typhimurium (strain TA1535/pSK1002) carrying a l a c Z gene that has

Correspondence: Prof. Dr. Dr. h.c.R.K. Zahn, Arbeitskreis Molekulare Mechanismen umweltbedingter Gentoxizit~it,Johannes Gutenberg-Universit~it, Obere Zahlbacherstr. 63, D6500 Mainz (F.R.G.).

been fused to the u m u C operon (Shinagawa et al., 1983). There is good evidence that the u m u C gene product, as a part of the SOS pathway, is directly involved in mutagenesis as a consequence of treatment with DNA-damaging agents (Kato and Shinoura, 1977; Bagg et al., 1981). Most mutations in bacteria provoked by UV-irradiation as well as by many chemicals require the products of either the u m u D C operon or the plasmid-derived operon m u c A B , which is analogous to the u m u D C gene products in both function and regulation. It is used in various Salmonella/micro-

216 some assay tester strains to enhance mutagenesis (Ferguson et al., 1989). Recent studies indicate that umuDC gene products act as stabilizing factors for D N A polymerase III holoenzyme enabling to bypass a lesion, a process leading to mutagenic misinsertions at apurinic sites (Maenhaut, 1985; Hevroni and Livneh, 1988). A number of similar tests measuring SOS gene expression have also been published (Quillardet et al., 1982; Elespuru and Moore, 1986; Brams et al., 1987). Lately, results indicate that monitoring of umuC induction is a very sensitive and specific method, compared to other tests based on genes of the SOS regulon (Ysern et al., 1990; McDaniels et al., 1990). As a consequence of the increasing knowledge about the relation between umuC and mutagenic events, there is a great interest in the SOS/umutest. For that reason, the standard assay protocol has been modified into a microplate version, which allows - by the use of an appropriate laboratory workstation - automated computer-assisted sample preparation, dilution steps, performance of the enzyme assay and data analysis. Similar automation efforts have been made with the SOS chromotest, a comparable test system, which uses a sfiA'-'lacZ fused gene product to monitor the SOS-inducing potency of genotoxic agents (Janz et al., 1989). Our umu-test version allows for intensive screening of complex mixtures such as native water samples, river water extracts and industrial waste waters in routine testing. Materials and methods

Materials Bacto tryptone was obtained from Difco Laboratories (Detroit, U.S.A.). Ampicillin, o-nitrophenyl-fl-o-galactopyranoside and /3-mercaptoethanol came from Sigma (Deisenhofen, F.R.G.). Dimethyl sulfoxide, glucose and sodium dodecyl sulfate were purchased from Merck (F.R.G.). Microplates came from Falcon (Heidelberg, F.R.G.). $9 liver homogenate from Aroclor-induced rats was purchased from Organon Teknika (Eppelheim, F.R.G.). All reagents used were of the purest grade available.

Bacterial strain Salmonella typhimurium (TA1535/pSK1002) was used as the tester strain. The multicopyplasmid pSK1002, bearing an umuC'-'lacZ gene fusion product (Shinagawa et al., 1983), was introduced into Salmonella typhimurium TA1535 (Oda et al., 1985). The umu operon is genetically regulated by the SOS genes recA and lexA. Umu-microtest The cultivation of the tester strain was a modification of the procedure published by Whong et al. (1986). Bacteria from stock ( - 80 ° C; in T G A medium containing 5 0 / x g / m l ampicillin and 10% DMSO as cryoprotective agent) were thawed and suspended in 1 ml T G A medium (1% Bacto tryptone, 0.5% NaC1, 0.2% glucose). DMSO was removed by centrifugation for 5 min at 3000 x g and discarding the supernatant. The bacterial pellet was resuspended with 1 ml TGA. Then 0.5 ml of this suspension was added to 20 ml T G A medium. This tester strain suspension was incubated overnight with slight shaking until an optical density (OD600)of 1.5 was reached. The day after, the overnight culture was diluted with fresh T G A medium and incubated for 2 h at 37 ° C in order to get log-phase cells. Preparation of concentrated water extracts 24-h representative water samples were taken from the rivers Rhine (near Mainz), Main (near Frankfurt), and Moselle. Suspended matter was separated by continuous-flow centrifugation (30,000 × g). Water samples (usually 3 1) were concentrated by adsorption to XAD-7 resin columns (2 g) using a peristaltic pump at a flow rate of 20 ml/min. Then excess water was blown out with nitrogen. Adsorbed compounds were eluted with 20 ml acetone, and the eluate was nearly reduced to dryness by vacuum rotary evaporation. Subsequently, the remaining solution was resuspended with 10% DMSO in distilled water to a final concentration corresponding to 10 1 original wat e r / m l extract (i.e., concentration factor 1: 10,000). Chemical substances Chemical substances were dissolved in distilled water at the highest concentration possible. In

217

most cases, however, on account of their poor solubility, DMSO had to be used as a solvent. Usually, 1:2 dilution series were made in triplicate using the laboratory workstation Biomekl000 (Beckman Instruments). Then 10/zl of each concentration was transferred to another microplate already containing 290 ~1 of an exponentially growing tester culture with an optical density of 0.2 (OD600). Each well was mixed several times using the multichannel pipette of the workstation. Finally, the microplate was covered with a lid. In cases where metabolic activation of test chemicals was necessary, $9 mix was added according to Ames et al. (1975), with the modification by Whong et ai. (1986) and additional modifications. The $9 concentration in the assay was 0.75% for routine testing with the pH value of the $9 mix adjusted to 7.7. Native water samples

For the test procedure, 20/xl 10-fold concentrated TGA (10% Bacto tryptone, 5% NaCI, 2% glucose, 500 /zg/ml ampicillin), 180 ~1 native water sample and 65/zl log-phase cell suspension (OD600 0.6) were pipetted into each well and extensively mixed by the multichannel pipette. For negative controls, 180 p~l distilled water was used.

(1972). On account of the short lightpath in the microplate version, however, it is not necessary to determine OD550 for correction of the light scattering caused by cell debris, in contrast to the original method of Miller. Briefly, 120 /zl B-buffer (20.18 g NazHPO 4. 2H20; 5.5 g NaHzPO 4 • H20; 0.75 g KCI; 0.25 g MgSO4"7H20; 1 g SDS; 2.7 ml /3-mercaptoethanol; H 2 0 ad 1000 ml; pH 7.0) was distributed into each well. 30-~1 aliquots of the bacterial culture were added and mixed. The enzyme reaction was initiated by addition of 30 /zl 2-nitrophenyl-/3-D-galactopyranoside solution (4.5 m g/ m l in 0.1 M phosphate buffer, pH 7.0). After mixing several times, the uncovered plate was incubated at 28° C in a small microplate incubator (Thermocult; Boehringer Mannheim) positioned on a shaker. The reaction was terminated after 30 min by adding 120/zl of 1 M Na2CO 3. Bubbles which could interfere with the vertical light path of the photometer were blown away by an air stream. The absorbance at 0D420 and OD60o was measured with the photometric tool of the workstation, including simultaneous data transfer to the calculation program. The units of/3-galactosidase activity were calculated according to the formula of Miller (1972), adapted for the microplate system.

Water extracts

Water extracts (dissolved in 10% DMSO) were diluted in 96-well microplates as outlined above. Then 22 /xl of each dilution was distributed into the microplate wells by the laboratory workstation. Subsequently, 270/zl exponentially growing tester strain suspension was added. 22/xl DMSO (10%) was used as negative control. After 2 h of incubation at 3 7 ° C on a rotor bearing a frame for the plates in a sloping position, the bacterial suspension was diluted 10-fold with warm TGA. This was followed by an additional incubation period of 2 h. Subsequently, bacterial growth was measured as turbidity at 600 nm using the photometric tool of the laboratory workstation. Enzyme assay

In the next step, the /3-galactosidase activity was determined according to the method of Miller

Results and discussion

Since

Oda

et

al.

(1985) developed

the

S O S / u m u - t e s t for the detection of the SOS-in-

ducing potency of environmental compounds or environmental carcinogens, this test has received worldwide use and acceptance. In view of the increasing importance of the umu-test, we decided to change the standard protocol, obviating the use of test tubes, and to further validate the test for use in an automated version. First, we examined the dependence of bacterial cell density on the induction of the umu operon. Fig. 1 shows the increasing sensitivity of the umu-test when fewer bacterial cells are used. At a concentration of 0.2/zmole/l the well-known potent genotoxin 4-nitroquinoline-N-oxide (4NQO) did not show significant genotoxicity when incubated with large numbers of cells. However,

218

1200.0

looi.o

800.0

I -100

x

._ --’

..I’

-75

I

0.1

02 A600

(bacterial

0.4

I

I1111

0.6

0.8

,,.*,l,

,,,“a

0.001

,.,,,.,

when the incubation period was started with an OD,, of 0.1 or less, i.e., at low bacterial counts, the induction ratio reached 6 times the negative control values, suggesting that weak genotoxic chemicals, or those rated as non-genotoxic, may turn out positive only when low cell numbers are used in the assay. In order to optimally standardize the conditions for routine testing in the microplate system, we decided to start incubation with an optical density of 0.2, corresponding to about 2 x lo8 cells/ml. In the microplate version a single concentration-response curve covers several decades with each concentration tested in triplicate. Fig. 2 shows a typical 1Cpoint concentrationresponse relationship of the genotoxic pyrolysis product 3-amino-1,4-dimethyl-SH-pyrido-(4,3-b)indole acetate (Tip-P-l). The concentration eliciting a 2-fold increase of umu gene expression is 4 x 10e8 mole/l. As shown in Fig. 3, the optimal pH value for metabolic activation of Tip-P-1 is between pH 7.6 and 7.8. Previous experiments with other precarcinogens also gave evidence that the S9 mixture used in the Salmonella/microsome assay, which is performed in solid agar medium on petri dishes, is not optimal with regard to its pH stability when adapted for liquid culture in the SOS/umu-test. Here the buffering capacity of the S9 mixture had to be increased.

,,,,,,,

0.01 Trp-P-l

Fig. 1. Induction of B-galactosidase activity by 0.2 pmole 4-NQO/l in dependence on the bacterial cell concentration at the beginning of the incubation period (values shown represent the average bacterial concentration determined as optical density at 600 nm in triplicate; the units of the negative control have been subtracted).

ruwivingtraction(X)

----

0.0

densky)

,....,,

-

1 (,mol/l)

+ S-9

2. Concentration-response curve of the pyrolysis product Trp-P-l with S9 preparation added as measured in the umumicrotest (values shown represent the average + SD of triplicate determinations). (-•---_) P-galactosidase activity (units); (- - -) % surviving bacteria (SalmoneNa typhimurium TA1535/pSK1002) as compared to the controls.

Table 1 demonstrates the genotoxicity of several substances tested with the umu-microtest. With the exception of DMSO, all chemicals listed previously had been classified as ‘positive’ with at least one of the Ames tester strains. The pyrolysis products 2-amino-3-methyl-3H-imidazo-(4,5fjquinoline (IQ), Trp-P-l and 3-amino-l-methyl5H-pyrido-(4,3-6)indole acetate (Trp-P-2) are among the most potent genotoxins. Whereas some classical genotoxins like acridine orange or Nmethyl-N’-nitro-N-nitrosoguanidine (MNNG) yield induction ratios of 3 and 8 respectively, the induction level of Trp-P-l rises to 18-fold the background level, and this compound thus appears to be one of the most potent genotoxins tested so far. If metabolic activation of agents is

pH ot the

beginning

of the

incubation

period

Fig. 3. Influence of pH shifts on the genotoxic Trp-P-l

with metabolic

activation

(S9 mix).

effects

of

219

necessary, it has to be considered that the sensitivity of the test strictly depends on the pH value in the incubation mixture (Fig. 3). Even without $9 activation, all pyrolysis products tested showed at least some weak genotoxicity. In contrast to an earlier publication on the outcome with the umu-test (Nakamura et al., 1987), NaNO 2 in our hands clearly showed genotoxicity without prolonged incubation, raising

maximal /3-galactosidase induction to about 3 times above background level. Sodium azide is a potent mutagen in the Salmonella test (McCann et al., 1975). Although earlier investigations gave negative results in the SOS/umu-test (Oda et al., 1985), retesting with the microplate version showed weak induction of SOS responses at high concentrations (Fig. 4). A 2-fold increase of /3-galactosidase activity was

TABLE 1 E V A L U A T I O N O F G E N O T O X I C I T Y OF S E V E R A L CHEMICALS IN T H E umu-MICROTEST Substance

(CAS No.)

Concentration ( m o l e / l ) causing doubling of ~3-galactosidase activity

% surviving bacteria

Acridine orange 2-Aminoanthracene 2-Aminoanthracene ( + $9) 3-Amino-l,4-dimethyl-5H-pyrido(4,3-b)indole acetate (Trp-P-1) 3-Amino-l,4-dimethyl-5H-pyrido(4,3-b)indole acetate (Trp-P-1 + $9) 3-Amino-l-methyl-5 H-pyrido(4,3-b)indole acetate (Trp-P-2) 3-Amino-l-methyl-5 H-pyrido(4,3-b)indole acetate (Trp-P-2 + $9) 2-Amino-3-methyl-3 H-imidazo(4,5-f)quinoline (IQ) 2-Amino-3 -methyl-3 H-imidazo(4,5-f)quinoline (IQ + $9) Amphotericin B Azinphos-Methyl Benzylchloride Captan 1-Chlor-2,4-dinitrobenzol Cisplatin Dichlofluanid N,N-Dimethylhydrazine Dimethyl sulfoxide (DMSO) Dithianon Methyl methanesulfonate

(494-38-2) (613-13-8) (613-13-8)

2.2 x 10- 5 > 1.0 x 10 -3 1.0 x 10-6

80 100 80

3.0 8.0 3.0

50 55 50

i00

18.0

52

N-MethyI-N'-nitro-N-nitrosoguanidine Mitomycin C a Nalidixic acid 4-Nitroquinoline-N-oxide N-Nitroso-butylurea 4-Nitroso-N, N-dimethylaniline N-Nitrosodiphenylamine Sodium azide a,b Sodium nitrite

Max. induction ratio c

% surviving bacteria

(68808-54-8)

4.5 × 10 -5

(68808-54-8)

4.0 x 10 - s

(72254-58-1)

1.2 x 10 4

63

2.2

50

(72254-58-1)

2.0 × 10 -6

100

8.0

50

(76180-96-6)

1.2x 10 -4

100

2.0

90

(76180-96-6) (1397-89-3) (86-50-0) (100-44-7) (133-06-2) (97-00-1) (15663-27-1) (1085-98-9) (57-14-7) (67-68-5) (3347-22-6) (66-27-3) (70-25-7) (50-07-7) (389-08-2) (56-57-5) (869-01-2) (138-89-6) (156-10-5) (26628-22-8) (7632-00-0)

4.0 x 10- 7 5.0 x 10 -4 6.0 x 10 -4 1.6 x 10- 4 3.5 x 10- 7 1.0 x 10 - 4 9.0x 10 -7 1.0x 10 5 6.0 × 10- 2 1.62 1.8 x 10-5 1.5 x 10-4 6.0x 10 -6 1.0 X 10- 8 2.4 × 10- 6 1.0 x 10- 7 1.6 x 10 - 4 5.8 x 10 - 5 3.8 x 10-4 5.4 X 10 -3 4.2X 10 -2

95 100 65 65 100 95 100 80 85 60 57 100 95 100 87 95 82 85 70 57 78

9.5 2.6 2.0 12.6 6.0 15.0 3.5 6.0 2.5 2.0 12.0 8.0 14.0 8.2 12.5 8.0 9.5 3.0 2.2 3.0

50 50 65 51 50 58 50 50 56 57 50 50 50 50 50 50 50 50 51 50

a Dissolved in distilled water. b Prolonged incubation period. c Values showing less than 50% surviving bacteria are excluded.

220 500.

400- -100 :g

m

300- -75

~

2oo:-so 100

0

-25

i 0.01

........

i 0.1 Sodium

........ azide

i I

.... ........

, u r ~ i . g f ~ c t i ~ (=) i ........ I 10 100

(mmol/I)

Fig. 4. Concentration-response curve of sodium azide as measured in the umu-microtest (values shown represent the average ± SD of triplicate determinations). ( - - e - - ) / 3 - g a l a c tosidase activity (units); (- - -) % surviving bacteria (Salmonella typhimurium TA1535/pSK1002) as compared to the controls.

observed at a concentration of 5.4 × 10 -3 mole/l. Therefore sodium azide has to be considered a weak inducer of u m u gene expression. The test performance was found to be increased when the microtiter plates carrying the incubation mixture had been put on a rotor in a sloping position, thus not only causing better mixing and increasing the metabolism, but also giving

better measuring conditions with higher sensitivity. In Germany, public health agencies are very interested in simple, rapid and automated tests for the detection of genotoxicity in water. Therefore we examined native water samples as well as XAD-7 extracts of 3 important German rivers: the Rhine, Main and Moselle. As shown in Table 2, no u m u gene expression was observed in native water samples of the 3 rivers. Nevertheless, the XAD-7 extracts showed quite variable genotoxic effects. Whereas only a slight induction of SOS response could be detected in extracts from the river Moselle (even 750-fold concentration factors did not double /3galactosidase activity), the rivers Rhine and Main - known to receive greater loads of industrial wastes - showed higher induction ratios at lower concentration factors. Our results clearly demonstrate that the microplate version of the u r n u - t e s t is a sensitive tool for the detection of genotoxins of different origins and in different mixtures. The advantages are obvious. Data concerning the mutagenic potency of single chemical substances, of mixtures or waste waters will be available within 8 h. The

TABLE 2

UmuC G E N E E X P R E S S I O N I N D U C E D BY N A T I V E W A T E R S A M P L E S A N D E X T R A C T S O F G E R M A N R I V E R SAMPLES O F D I F F E R E N T O R I G I N IN T H E u m u - M I C R O T E S T ( W I T H O U T M I C R O S O M A L A C T I V A T I O N ) Water sample

Control (distilled water) River Main near Frankfurt a Control (distilled water) River Rhine near Mainz b Control (distilled water) River Moselle a

N u m b e r of samples

fl-Galactosidase activity (units) native water sample (0.68 I/1)

% surviving bacteria

6

49

100

6

49

100

16

51

100

16

46

100

15 15

46 45

100 100

a Samples were collected at 3 different places. b Samples were collected at 1 place. Negative control units were 49 ± 5.7 for D M S O (10%). Negative control units for distilled water as indicated.

Activity doubling of /3-galactosidase at X A D - 7 concentration factors of (range) 169 (92-375) 390 (180-750) > 750

% surviving bacteria

95 95 100

221

amount of genotoxins as well as the sample sizes of chemical wastes needed for testing have been drastically reduced in this test procedure. Because of their easy handling, SOS tests in general are highly suitable for automation of all the essential, time-consuming steps in the assay procedure, as was recently shown for the SOS chromotest (Janz et al., 1989). This guarantees high reproducibility at small standard deviations and allows for screening of large numbers of environmental samples at moderate costs. In contrast to Janz et al., however, we think that the introduction of an additional 1:10 dilution step during the incubation period greatly improves the discrimination between genotoxic and cytotoxic effects and reduces potential bias induced by colored compounds. The SOS induction pathway includes approximately 20 genes in Escherichia coli; many of these genes are known to code for proteins involved in DNA repair, mutagenesis and recombination (Walker, 1984). Recent studies indicate the existence of DNA-damage-inducible genes not only in bacteria, but also in yeast - where up to 11 inducible genes have been described (Robinson et al., 1986; Ruby and Szostak, 1985) and in mammalian cells. Fornace et al. (1988) found many mammalian cDNA transcripts, which show similarities to damage-inducible genes of yeast and bacteria, namely low abundance and rapid 2-10-fold induction as a consequence of UV-irradiation and treatment with base-damaging agents. Recently, Little et al. (1989) mentioned that the dependence upon pKM101 (which contains mucAB, a plasmid-derived operon analogous in both structure and function to umuDC) in the detection of mutagens in the Ames test is a valid indication of mammalian genotoxicity, as a comparison with sister-chromatid exchange demonstrates. So, the induction of umuC during the SOS response surely reflects genetically relevant events, which should be regarded as an alert for possible carcinogenic activity.

Acknowledgements This work was supported by the Ministerium fiir Umwelt und Gesundheit des Landes Rhein-

land-Pfalz (Department of Environment and Health), Mainz, and the German Academy of Sciences and Literature. We thank Beckman Instruments for generously providing us with a laboratory workstation Biomekl000.

References Ames, B.N., J. McCann and E. Yamasaki (1975) Methods for detecting carcinogens and mutagens with the Salmonella/ mammalian-microsome mutagenicity test, Mutation Res., 31,347-364. Bagg, A., C.J. Kenyon and G.C. Walker (1981) Inducibility of a gene product required for UV and chemical mutagenesis in Escherichia coli, Proc. Natl. Acad. Sci. (U.S.A.), 78, 5749-5753. Brams, A., J.P. Buchet, M.C. Crutzen-Fayt, C. De Meester, R. Lauwerys and A. L6onard (1987) A comparative study, with 40 chemicals, of the efficiency of the Salmonella assay and the SOS chromotest (Kit procedure), Toxicol. Lett., 38, 123-133. Elespuru, R.K., and S.G. Moore (1986) Micro-BIA, a colorimetric microtiter assay of lambda prophage induction, Mutation Res., 164, 31-40. Ferguson, L.R., W.A. Denny and S.M. O'Rourke (1989) Mutagenic activity of nitracrine derivatives in Salmonella typhimurium: relationship to drug physicochemical parameters, and to bacterial m,rB and recA genes and plasmid pKM101, Mutation Res., 223, 13-22. Fornace, A.J., I. Alamo and M.Ch. Hollander (1988) DNA damage-inducible transcripts in mammalian cells, Proc. Natl. Acad. Sci. (U.S.A.), 85, 8800-8804. Hevroni, D., and Z. Livneh (1988) Bypass and termination at apurinic sites during replication of single-stranded DNA in vitro: a model for apurinic site mutagenesis, Proc. Natl. Acad. Sci. (U.S.A.), 85, 5046-5050. Janz, S., G. Wolff, T. Huttunen, F. Raabe and H. Storch (1989) Quantitation of the relationship between test cell number inoculated and the SOS-inducing potency of 4nitroquinoline-l-oxide (4-NQO) in an automated version of the SOS chromotest, J. Basic Microbiol., 29, 403-411. Kato, T., and Y. Shinoura (1977) Isolation and characterization of mutants of E. coli deficient in induction of mutations by UV light, Mol. Gen. Genet., 156, 12t-131. Little, C.A., D.J. Tweats and R.J. Pinney (1989) Relevance of plasmid pKM101-mediated mutagenicity in bacteria to genotoxicity in mammalian cells, Mutagenesis, 4, 371-376. Maenhaut, M.G. (1985) Mechanisms of SOS-induced targeted and untargetet mutagenesis in E. coli, Biochimie, 67, 365-369. McCann, J., E. Choi, E. Yamasaki and B.N. Ames (1975) Detection of carcinogens as mutagens in the Salmonella/microsome test: assay of 300 chemicals, Proc. Natl. Acad. Sci. (U.S.A.), 72, 5135-5139. McDaniels, A.E., A.L. Reyes, L.J. Wymer, C.C. Rankin and G.N. Stelma Jr. (1990) Comparison of the Salmonella (Ames) test, umu tests, and the SOS Chromotests for detecting genotoxins, Environ. Mol. Mutagen., 16, 204-215.

222 Miller, J.H. (1972) Assay of fl-galactosidase, in: Experiments in Molecular Genetics, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, pp. 322-355. Nakamura, S., Y. Oda, T. Shimada, I. Oki and K. Sugimoto (1987) SOS-inducing activity of chemical carcinogens and mutagens in Salmonella typhimurium TA1535/pSK1002: examination with 151 chemicals, Mutation Res., 192, 239246. Oda, Y., S. Nakamura, J. Oki and T. Kato (1985) Evaluation of the new system (umu-test) for the detection of environmental mutagens and carcinogens, Mutation Res., 147, 219-229. Quillardet, P., O. Huisman, R. D'Ari and M. Hofnung (1982) SOS chromotest, a direct assay of induction of an SOS function in Escherichia coli K-12 to measure genotoxicity, Proc. Natl. Acad. Sci. (U.S.A.), 79, 5971-5975. Robinson, G.W., C.M. Nicolet, D. Kalainov and E.C. Friedberg (1986) A yeast excision-repair gene is inducible by

DNA damaging agents, Proc. Natl. Acad. Sci. (U.S.A.), 83, 1842-1846. Ruby, S.W., and J.W. Szostak (1985) Specific Saccharomyces cerevisiae genes are expressed in response to DNA-damaging agents, Mol. Cell. Biol., 5, 75-84. Shinagawa, H., T. Kato, T. Ise, K. Makino and A. Nakata (1983) Cloning and characterization of the umu operon responsible for inducible mutagenesis in Escherichia coli, Gene, 23, 167-174. Walker, G.C. (1984) Mutagenesis and .inducible responses to DNA damage in E. coli, Microbiol. Rev., 48, 60-93. Whong, W.-Z., Y. Wen, J. Steward and T. Ong (1986) Validation of the SOS/umu-test with mutagenic complex mixtures, Mutation Res., 175, 139-144. Ysern, P., B. Clerch, M. Castafio, I. Gilbert, J. Barb~ and M. Llagostera (1990) Induction of SOS genes in Escherichia coli and mutagenesis in Salmonella typhimurium by fluoroquinolones, Mutagenesis, 5, 63-66.