Mutation Research, 260 (1991) 349-367 © 1991 Elsevier Science Publishers B.V. 0165-1218/91/$03.50 ADONIS 016512189100117Q
349
MUTGEN 01679
Performance of 133 compounds in the lambda prophage induction endpoint of the Microscreen assay and a comparison with S. typhimurium mutagenicity and rodent carcinogenicity assays T.G. Rossman, M. Molina *, L. Meyer * *, P. Boone * * *, C.B. Klein, Z. Wang, F. Li t, W.C. Lin tt and P.L. Kinney Institute of Environmental Medicine, N Y U Medical Center, Long Meadow Road, Tuxedo, N Y 10987 (U.S.A.) (Received 16 October 1990) (Revision received 2l January 1991) (Accepted 22 January 1991)
Keywords: Escherichia coli; Microscreen assay; Lambda prophage induction endpoint
Summary The Microscreen assay was developed as a means of testing very small samples, as in complex mixture fractionation. It is a multi-endpoint assay which utilizes E. coli WP2s(?Q. Exposure takes place to serial dilutions of the test compound in microtitre wells (250 /tl) followed by sampling from wells in which growth has occurred ('non-toxic wells'). Although a number of different endpoints can be measured, only the prophage induction endpoint (the first one developed) has been extensively tested. Results with 133 compounds are presented. These include 111 compounds which have been tested in the S. typhimurium assay and 66 compounds for which both rodent bioassay and S. typhimurium assay data exists. The concordance for the Microscreen assay and the S. typhimurium assay was 71%. For this group of compounds, the sensitivity of the Microscreen assay in detecting carcinogens was 76% compared with 58% for the S. typhimurium assay. However, the S. typhimurium assay was somewhat more specific (69%) compared with the Microscreen (56%). The overall association between carcinogenicity and Microscreen results was statistically significant (p = 0.029), whereas for the S. typhimurium assay the association with carcinogenicity was non-significant (p = 0.086). The Microscreen assay was able to detect halogenated compounds better than the S. typhimurium assay. The Microscreen assay should prove useful in complex mixture fractionation, or in other situations where sample size is limiting.
*
* Present address: Hoffmann-La Roche, Inc., Nutley, NJ 07110 (U.S.A.). * * Present address: N.J. Department of Environmental Protection, Office of Science and Research, 401 E. State Street, Trenton, NJ (U.S.A.). * * Present address: U.S.E.P.A., Edison Field Facility, Bldg. 209, Edison, NJ 08837 (U.S.A.).
Present address: Zenith Laboratory, Inc., 140 Legrand Avenue, Northvale, NJ 07647 (U.S.A.). t t Permanent address: Department of Toxicology, Beijing Medical University, Beijing (China). Correspondence: Dr. T.G. Rossman, Institute of Environmental Medicine, NYU Medical Center, Long Meadow Road, Tuxedo, NY 10987 (U.S.A.).
350
Although first developed to test pure compounds that are available in large quantities, bacterial systems for genotoxicity are now being used in the fractionation of complex mixtures such as diesel exhaust and food mutagens, and in aiding the identification of active metabolites. The identification of genotoxic agents in complex mixtures or body fluids can help to identify potential human hazards and to pinpoint mutagenic metabolites which might prove to be ultimate carcinogens. The S. typhimurium microsuspension assay has enabled testing to be done on a smaller sample size (Kado et al., 1983). However, bacterial reversion assays have a serious drawback in screening complex mixtures. Because each tester strain reverts by a specific mechanism, at least six different tester strains are now required to cover most possible reversion mechanisms. In practice, however, because of limitations in sample size, time and personnel, only a single S. typhimurium strain is often used. In screening for chemicals with unknown genotoxic effects, an ideal test system would be
able to detect the largest number of possible mutagenic events of various kinds using only one strain of bacteria. The Microscreen assay (Fig. 1) was developed in this laboratory with this concept in mind. The original assay (Rossman et al., 1985, 1986) used only lambda prophage induction in E. coli as an endpoint. The induction of lambda prophage results from the derepression of the bacterial SOS system, a set of coordinately regulated genes that become activated when D N A is damaged (Walker, 1985). The use of lambda prophage induction in screening for mutagens is well established (Elespuru, 1984). Yet, this assay differs in some important ways from previously described prophage induction assays: (1) It utilizes an E. eoli B strain, in which prophage induction is a lower-frequency event than it is in K12 strains (Rossman et al., 1986). This allows > 99% survival of treated cultures so that other endpoints can be screened. In addition, there is evidence that E. coli B strains are more sensitive to some intercalating agents than are K12 strains, and that this difference may be due
Controls
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Endpoint Measured
Prophage Induction
Mechanism
Induction of SOS Systemby DNA Damage
Ampicillin Resistance
TRP + Reversion Base Pair Substitution
(Ochre Suppression)
Base Pair Substitution or Gene Amplification
Fig. l, Microscreen assay.
T5 Resistance
All Types of
Forward Mutation
351
to different levels of DNA supercoiling in these strains (Bridges et aI., 1981). (2) The assay is run in Microtitre plates containing 96 wells, in a volume of 250 ttl/well. The culture is grown overnight in the presence of the test agent. Long-term incubation at the concentration being tested may contribute to the very small amounts of test compounds detectable. (3) Toxicity is evident by the absence of growth, and sampling is carried out only from sub-toxic wells. Thus, the toxic endpoint is determined in the same assay as the genotoxic endpoints. (4) The indicator strain cannot be easily lysogenized and therefore gives rise to clear plaques, allowing ease of scoring. The relationship between mutagenicity, as determined by short-term in vitro tests, and carcinogenicity has been studied in great detail for the last 15 years. In 1975, McCann et al. concluded that the S. typhimurium mutation assay could identify about 90% of rodent carcinogens, whereas more recent studies from the National Toxicology Program conclude that the figure is 45% for a different group of compounds (Tennent et al., 1987). The fraction of false positives (noncarcinogens which are mutagens) also appears to have increased with time, from about 10% (McCann et aI., 1975) to 14% (Tennant et al., 1987). The reasons for the apparent declining predictivity of the S. typhimurium assay have been discussed by others (e.g. Clive, 1988; Shelby et al., 1988, Brockman and DeMarini, 1988; Prival and Dunkel, 1989). It is not our purpose to enter into the debate as to whether in vitro genotoxicity can be an adequate surrogate for animal carcinogenicity testing. Nevertheless, in suggesting that the Microscreen assay could be used in bioassay-directed fractionation of complex mixtures, it behooves us to demonstrate that this assay will perform at least as well as other accepted assays. We have previously reported that a number of metal compounds are capable of inducing lambda prophage (Rossman et al., 1984), and have demonstrated use of this assay in the fractionation of organic extracts from air pollution particles (Rossman et al., 1985), in studying indoor air pollution (Boone et al., 1989), and in assessing the mutagenicity of oxidized nucleosides (Shirnamr-Mor6 et al., 1987). DeMarini and coworkers have shown
that chlorinated pesticides (Houk and DeMarini, 1987) and other chlorinated organic compounds (DeMarini et al., 1990) which are negative in the Ames test, are able to induce lambda prophage in the Microscreen assay. The Microscreen assay prophage induction endpoint was also able to detect genotoxic material from hazardous waste sites (Houk and DeMarini, 1988), from airborne particulate matter from Rio de Janeiro (Miguel et al., 1990) and from a steel plant in Beijing (Lin et al., 1987). Kojic acid, a product of Aspergillus species which is converted to the 'freshly baked' odor and flavor enhancer maltol, was found to induce prophage in the Microscreen assay (Lin et al., 1987). We have recently added two forward mutation endpoints to this assay and have shown that metabolites of benzene are mutagenic at these endpoints (Rossman et al., 1989). However, because the prophage induction endpoint was the first one developed, a large number of compounds have been assayed in the Microscreen assay using only that endpoint. In this paper, we address the question whether the Microscreen assay (using only one endpoint and one strain of bacteria) is as sensitive in detecting rodent carcinogens as the S. typhimurium assay (using all strains available) for a group of compounds that span several major chemical classes. We also address the extent to which the Microscreen and S. typhimurium assays complement one another as predictors of rodent carcinogenicity. Materials and methods
The microscreen assay (1) E. coli strains and media All strains are derivatives of E. co# B / r . WP2(Yk) is a lambda lysogen of WP2 (trpE, uvrA), isolated in our laboratory from WP2 and wild-type lambda phage, both obtained from Dr. Evelyn Witkin, Rutgers University. The indicator strain SR714 (trpE uvrD3) was obtained from Dr. Kendric Smith, Stanford University. The indicator strain was chosen for its ability to give clear plaques. Cultures were grown in medium MST, which is Minimal Broth Davis (Difco) containing 0.2% glu-
352 TABLE 1
TABLE 1 (continued)
133 CHEMICALS TESTED IN MICROSCREEN ASSAY Chemical name Chemical name
CAS number Source
Acetone Acridine Acridine orange Actinomycin D Acyclovir
67-64-1 260-94-6 65-61-2 50-76-0 59277-89-3
Baker Aldrich Sigma Sigma D. Clive
9-Aminoacridine 2-Aminoanthracene 2-Aminopurine Aniline Anthracene
52417-22-8 613-13-8 452-06-2 4165-61-1 120-12-7
Sigma Sigma Sigma Aldrich Aldrich
Anthracene-9-carboxylic acid Ascorbic acid 8-Azaguanine Barium chloride Benzamide
723-62-6 50-81-7 134-58-7 10361-37-2 55-21-0
Pfaltz and Bauer Sigma Sigma Alfa Sigma
Benz[ a ]anthracene-7,12-dione Benzene Benzoic acid Benzoin Benzo[ c]cinnoline
2498-66-0 71-143-2 65-85-0 119-53-9 230-17-1
Aldrich Sigma Sigma NTP a Aldrich
Benzo[a]pyrene Benzo[e]pyrene Benzo[ f ]quinoline Benzyl acetate Beryllium chloride
50-32-8 192-97-2 85-002-9 140-11-4 7787-47-5
Aldrich Aldrich Aldrich NTP a Fluka
2-Biphenylamine Bleomycin sulfate 5 '-Bromo-2'-deoxyuridine 5-Bromouracil Cadmium chloride
90-41-5 9041-93-4 59-14-3 51-20-7 10108-64-2
Pfaltz and Bauer Bristol Sigma Sigma Alfa
Caffeine Calcium chloride Caprolactam Carbon tetrachloride Chloramine T
58-08-2 22691-02-7 105-60-2 56-23-5 127-005-1
Sigma Fisher NTP a Aldrich Fisher
Chromium chloride Cinnamyl anthranilate Cupric chloride Cysteamine Deoxycytidine
10049-05-5 87-29-6 10125-13-0 60-23-1 951-78-0
Alfa NTP a Fisher Sigma Sigma
Deoxyuridine Diallyl phthalate Dibenz[ a, c]anthracene Dibenz[ a, h ]anthracene 3,3'-Dichlorobenzidine
951-78-0 131-17-9 215-58-7 53-70-3 71-94-1
Sigma NTP a Aldrich Aldrich Sigma
2,6-Dichloro-p-phenylenediamine Di(2-ethylhexyl)adipate
609-20-1 103-23-1
NTP a NTP a
CAS number Source
Di(2-ethylhexyl)phthalate 117-81-7 3,3'-Dimethoxybenzidine 119-90-4 7,12-Dimethylbenzanthracene 57-97-6
NTP a Fluka Fluka
Dimethylsulfoxide 2,4-Dinitrochlorobenzene 1,5-Dinitronaphthalene Diphenylphosphate Ethanol
67-68-5 97-00-7 605-71-0 838-85-7 64-17-5
Sigma Fisher Aldrich Fluka Sigma
Ethidium bromide Ethylmethane sulfonate Ferric chloride Ferrous chloride Fluoranthene
1239-45-8 62-50-0 7705-08-0 7758-94-3 206-44-0
Calbiochem Aldrich Fisher Baker Aldrich
Formaldehyde Geranyl acetate Hydrogen peroxide 9-Hydroxyfluorene Hydroxylamine HC1
50-000-0 105-87-3 7722-84-1 2443-58-5 5470-11-1
Sigma NTP a Sigma Pfaltz and Bauer Sigma
6-N-Hydroxylaminopurine 5-Hydroxymethyl-2 '-deoxyuridine 5-Hydroxymethyluracil 8-Hydroxyquinoline Hydroxyurea
5667-20-9
U.S. Biochem
5116-24-5 4433-40-3 148-24-3 120-07-1
Sigma Sigma Sigma Sigma
Hypoxanthine ICR 170 ICR 191 Lead nitrate Manganous chloride
68-94-0 146-59-8 17070-45-0 10099-74-8 10034-96-5
Sigma Polysciences Polysciences Sigma Sigma
Melamine Mercuric chloride Methotrexate 3-Methylcholanthrene Methylene chloride
108-78-1 7487-94-7 59-05 -2 56-49-5 75-09-2
NTP a Sigma Sigma Sigma Sigma
Methylglyoxal Methylmethane sulfonate N-Methyl-N '-nitroN-nitrosoguanidine Michler's ketone Mitomycin C
78-98-8 66-27-3
Sigma Aldrich
70-25-7 90-94-8 50-07-0
Sigma NTP a Sigma
Nalidixic acid 1,2-Naphthoquinone Nickel acetate 2-Nitrofluorene 4-Nitroquinoline-1 -oxide
389-08-2 524-42-5 6018-89-9 607-57-8 56-57-5
Sigma Fluka Aldrich Aldrich Aldrich
N-Nitrosodiethylamine N-Nitrosodiisopropylamine
55-18-5 601-77-4
Aldrich Aldrich
353
cose and 20 /~g/ml tryptophan. Mid-exponential phase cultures (optical density = 0.3 at 550 nm) were used for all experiments. The culture is centrifuged to eliminate free phage just before use.
TABLE 1 (continued) Chemical name
CAS number
Source
N-Nitrosopyrrolidine Orcinol Oxalic acid
930-55-2 504-15-4 2065-73-8
Aldrich Fluka Sigma
4,4'-Oxydianiline m-Phenylenediamine o-Phenylenediamine p- Phenylenediamine Phenanthrene
101-80-4 108-45-2 95-54-5 106-50-3 85-01-8
NTP a Sigma Sigma Sigma Aldrich
Polybrominated biphenyl Potassium chloride Potassium chromate Potassium permanganate fl-Propiolactone
59536-65-1 7447-40-7 7789-00-6 7722-64-7 57-57-8
NTP ~ Sigma Mallinckrodt Sigma Kodak
Pyrene Quercetin Rhodamine B Silver nitrate Sodium arsenite
129-00-0 6151-15-3 181-88-9 7761-88-1 7784-46-5
Sigma Sigma Kodak Sigma Fisher
Sodium Sodium Sodium Sodium Sodium
azide bisulfite hypochlorite molybdate nitrate
26628-22-8 7631-90-5 7681-52-9 7631-95-0 7631-99-4
Sigma Aldrich Aldrich Sigma Sigma
Sodium Sodium Sodium Sodium Sodium
nitrite saccharine selenate selenite sulfate
7632-00-0 81-07-2 13410-01-0 10102-18-8 7757-82-6
Sigma Sigma Sigma Sigma Sigma
Sodium tungstate Stannous chloride Streptomycin sulfate Thymidine Thymine
10213-10-2 7772-99-8 3810-74-0 50-89-5 65-71-4
Fisher Alfa Sigma Sigma Sigma
Toluene Trichloroethylene Xanthine Xylenes 2,6-Xylidine Zinc chloride
108-88-3 78-34-2 69-89-6 1330-20-7 87-62-7 7646-85-7
Aldrich Aldrich Sigma Fluka NTP a Alfa
a NTP Chemical Repository, Radian Corporation, Austin, Texas.
(2) Exposure 8 serial 2-fold dilutions of the test agent in 150 /~1 MST were made across a row in a Falcon Micro Test III tissue culture multiwell plate containing 96 wells. In the first experiment, the starting concentration was 100 /~g/well and the final concentration was 0.78/~g/well. This series should allow at least one but no more than five wells to show growth inhibition. In cases where no growth inhibition occurs, the starting concentration was increased up to the concentration at which precipitation occurs. In cases where more than 5 wells show growth inhibition, only the two subtoxic wells could be sampled, and the experiment is repeated at a lower starting concentration. The negative control consisted of growth medium containing the highest concentration of solvent used. If the test agent was not water soluble, acetone (up to 10% v / v ) was used. DMSO was avoided as older opened bottles sometimes accumulated phage-inducing material. Positive controls consisted of M N N G (6.25 /~g/ml) for direct acting agents and benzo[a]pyrene (25 /~g/ml) for metabolic activation. The wells are inoculated with 75/~1 of a mid-exponential culture of WP2(~) (OD550 =0.3), diluted 1 / 1 0 after centrifugation so that each well received approximately 2 × 107 bacteria. At this cell density, no turbidity was evident. The test plate was incubated overnight at 37°C and scored after 20 h for turbidity. Control (turbid) wells contained approximately 1.3 X 109/ml. Wells which were visually less turbid (scored as + / - ), were found to contain 1-5 × 108 cells/ml. After scoring for growth, aliquots of 5/~1 from 3 - 4 subtoxic ( + or + / - ) wells are diluted in 1 ml MST. Diluted samples (100 #1), which contain approximately 6.5 x 105 lysogens from fully turbid wells, are added to tubes containing 2.5 ml soft agar (0.65% Bacto agar, 10 mM MgSO4), held at 47°C. A mid-exponential culture of indicator strain SR714 (100/~1) is added, and the tubes are mixed and poured onto half strength Luria agar plates (Difco). After overnight incubation at 37°C, the plates are scored for plaques. All assays are run in duplicate, and all experiments have been repeated at least once. The range and variability of plaques in the Microscreen assay have been
354 TABLE 2 INDUCTION OF PROPHAGE IN MICROSCREEN ASSAY Compound
$9
Max. e n h a n c e m e n t over background
Amount at Max. a
L Polycyclic aromatics and derivatives Acridine
-
nb
8.0
Anthracene
-
3.5
12.5
+
4.8
12.5
Anthracene-9-carboxylic acid Benzanthracene-7,12-dione
-
n
+
30
127 (p) c 127 (p)
-
n
122 (p)
+
9.3
122 (p)
Benzo[ c]cinnoline
+
n n
127 127
Benzol a ] p y r e n e
+
3 10.4
Benzo[e]pyrene
+
n n
Benzo[ f ]quinoline
+ +
n 6.7 n 8
7.5 7.5 1.44 1.44
-
n
2.0
+
4
2.0
7,12-Dimethylbenzanthracene
+
n 11.5
5.0 5.0
Fluoranthene
+
n n
50 (p) 50 (p)
9-Hydroxyfluorene
+ +
n n n n
143 (p) 143 (p) 6.7 6.7
3-Methylcholanthrene
+
n 10
1,2-Naphthoquinone
+ +
n n n 4.5
3.13 12.5 25 25
+
n n
50 50
+ -
n 3.5 68 21
+ + +
73 n 10 n 8 n 7
Dibenz[a,c]anthracene Dibenz[ a, h ] a n t h r a c e n e
8-Hydroxyquinoline
Phenanthrene Pyrene
12.5 (p) 12.5 (p) 25 (p) 25 (p)
50 50
I1. Nitro aromatics and nitroso compounds Benzamide 1,5-Dinitronaphthalene 2-Nitrofluorene 4-Nitroquinoline-l-oxide N-Nitrosodiethylamine N-Nitrosodiisopropylamine N-Nitrosopyrrolidine
15.5 15.5 15.5 25 0.16 600 600 300 300 225 225
355 T A B L E 2 (continued)
Compound
$9
Max. e n h a n c e m e n t over background
Amount at Max. a
Ill. Aromatic amines Aniline 2-Biphenylamine
Acridine orange 9-Aminoacridine
-
n n
0.8 62.5
+
3.9
62.5
-
20 n
3,3 ' - D i m e t h o x y b e n z i d i n e
-
n
Ethidium bromide
+ -
n 13
9.4 3.1 60 60 15.6
Melamine
-
7
78
4,5 ' - O x y d i a n i l i n e
+ -
4 n
78 6.25
+
4.6
6.25
m- P h e n y l e n e d i a m i n e o-Phenylenediamine
-
19 20
175 125
p-Phenylenediamine 2,6-Xylidine
-
11 n
125 39
+
3
39
IV. Halogenated organic compounds Carbon tetrachloride Chloramine T 3,3 '- D i c h l o r o b e n z i d i n e 2,6-Dichloro-p-phenylenediamine
2,4-Dinitrochlorobenzene M e t h y l e n e chloride Polybrominated biphenyl Trichloroethylene
-
n
200
+
6.5
200
+ + +
3 9.6 n n n n
255 255 125 125 250 250
-
+ -
n
20 19 n
+
5.7
-
n
+
5
25 8250 (2.5~) 8250 (2.5~) 3.1 3.1 18 18
V. Antimetabolites and antibiotics Actinomycin D 8-Azaguanine B l e o m y c i n sulfate Hydroxyurea
n 7.5 27 27
Methotrexate Mitomycin C Nalidixic acid S o d i u m azide
14 16 100 +
S t r e p t o m y c i n sulfate
8 17 n
15.6 37.5 0.00625 0.92 3.9 0.00078 0.4 36.5 36.5 1.1
V1. Bases, nucleosides and analogs Acyclovir 2-Aminopurine
n 20
375 75
356 T A B L E 2 (continued) Compound
$9
Max. enhancement over background
5 '-Bromo-2 '-deoxyuridine 5-Bromouracil Deoxycytidine Caffeine Deoxyuridine 5-Hydroxymethyl-2 '-deoxyuridine 5- Hydroxymethyluracil 6-N-Hydroxylaminopurine Hypoxanthine Thymidine Thymine Xanthine
Amount at Max. a
8 n n
125 675 675
n n 52 7 5
938 675 125 125 132
n n n n
35.6 125 125 125
VII. Small direct-acting alkylating agents 77 15 33 11
Ethylmethane sulfonate Methylmethane sulfonate N-Methyl-N '-nitro-N-nitrosoguanidine fl-Propiolactone
3.125 0.39 1.56 0.026
VIIL Inorganic compounds Barium chloride Beryllium chloride C a d m i u m chloride Calcium chloride C h r o m i u m chloride Cupric chloride Ferric chloride Ferrous chloride Hydrogen peroxide Hydroxylamine Lead nitrate Manganous chloride Mercuric chloride Nickel acetate Potassium chromate Potassium chloride Potassium permanganate Silver nitrate Sodium arsenite Sodium bisulfite Sodium Sodium Sodium Sodium Sodium
hypochlorite molybdate nitrate nitrite selenate
Sodium selenite Sodium sulfate Sodium tungstate
--
n
-
n
-
n
-
n
-
6
--
n
-
3.5
-
3.9
-
4.5
--
n
-
10
-
32
-
n 5 28
-
n
-
3
-
n
--
n
--
n
-
33.6 3.5
-
8
-
4.6
-
9.3
-
< 3-10.4
-
n
-
4
38 100 3.5 278 33.3 2.15 0.041 0.05 0.5 37.5 27.3 77.3 0.012 11 2.4 600 7.9 0.01 104 275 103 103 132.5 12.5 25 25 275 412
357 TABLE 2 (continued)
Compound
$9
Max. enhancement over background
Amount at Max. a
Stannous chloride Zinc chloride
-
9.8 3
50 21.3
+ + +
n n n n n n
10% 10% 5% 5% 10% 10% 10% 10%
IX. Solvents Acetone Benzene Dimethylsulfoxide
Ethanol Toluene Xylenes
-
n
+
n
-
n
0.5%
+
n
0.5%
-
n
1%
+
n
1%
X. Miscellaneous Ascorbic acid Benzoic acid Benzoin
n n n
+ Benzyl a c e t a t e + Caprolactam
n
+ Cinnamyl anthranilate + Cysteamine Diallylphthalate
7 n n n
+ Di(2-ethylhexyl)adipate + Di(2-ethylhexyl)phthalate + Diphenylphosphate Formaldehyde +
Geranyl acetate
n n n 6.8 n
349
MUTGEN 01679
Performance of 133 compounds in the lambda prophage induction endpoint of the Microscreen assay and a comparison with S. typhimurium mutagenicity and rodent carcinogenicity assays T.G. Rossman, M. Molina *, L. Meyer * *, P. Boone * * *, C.B. Klein, Z. Wang, F. Li t, W.C. Lin tt and P.L. Kinney Institute of Environmental Medicine, N Y U Medical Center, Long Meadow Road, Tuxedo, N Y 10987 (U.S.A.) (Received 16 October 1990) (Revision received 2l January 1991) (Accepted 22 January 1991)
Keywords: Escherichia coli; Microscreen assay; Lambda prophage induction endpoint
Summary The Microscreen assay was developed as a means of testing very small samples, as in complex mixture fractionation. It is a multi-endpoint assay which utilizes E. coli WP2s(?Q. Exposure takes place to serial dilutions of the test compound in microtitre wells (250 /tl) followed by sampling from wells in which growth has occurred ('non-toxic wells'). Although a number of different endpoints can be measured, only the prophage induction endpoint (the first one developed) has been extensively tested. Results with 133 compounds are presented. These include 111 compounds which have been tested in the S. typhimurium assay and 66 compounds for which both rodent bioassay and S. typhimurium assay data exists. The concordance for the Microscreen assay and the S. typhimurium assay was 71%. For this group of compounds, the sensitivity of the Microscreen assay in detecting carcinogens was 76% compared with 58% for the S. typhimurium assay. However, the S. typhimurium assay was somewhat more specific (69%) compared with the Microscreen (56%). The overall association between carcinogenicity and Microscreen results was statistically significant (p = 0.029), whereas for the S. typhimurium assay the association with carcinogenicity was non-significant (p = 0.086). The Microscreen assay was able to detect halogenated compounds better than the S. typhimurium assay. The Microscreen assay should prove useful in complex mixture fractionation, or in other situations where sample size is limiting.
*
* Present address: Hoffmann-La Roche, Inc., Nutley, NJ 07110 (U.S.A.). * * Present address: N.J. Department of Environmental Protection, Office of Science and Research, 401 E. State Street, Trenton, NJ (U.S.A.). * * Present address: U.S.E.P.A., Edison Field Facility, Bldg. 209, Edison, NJ 08837 (U.S.A.).
Present address: Zenith Laboratory, Inc., 140 Legrand Avenue, Northvale, NJ 07647 (U.S.A.). t t Permanent address: Department of Toxicology, Beijing Medical University, Beijing (China). Correspondence: Dr. T.G. Rossman, Institute of Environmental Medicine, NYU Medical Center, Long Meadow Road, Tuxedo, NY 10987 (U.S.A.).
350
Although first developed to test pure compounds that are available in large quantities, bacterial systems for genotoxicity are now being used in the fractionation of complex mixtures such as diesel exhaust and food mutagens, and in aiding the identification of active metabolites. The identification of genotoxic agents in complex mixtures or body fluids can help to identify potential human hazards and to pinpoint mutagenic metabolites which might prove to be ultimate carcinogens. The S. typhimurium microsuspension assay has enabled testing to be done on a smaller sample size (Kado et al., 1983). However, bacterial reversion assays have a serious drawback in screening complex mixtures. Because each tester strain reverts by a specific mechanism, at least six different tester strains are now required to cover most possible reversion mechanisms. In practice, however, because of limitations in sample size, time and personnel, only a single S. typhimurium strain is often used. In screening for chemicals with unknown genotoxic effects, an ideal test system would be
able to detect the largest number of possible mutagenic events of various kinds using only one strain of bacteria. The Microscreen assay (Fig. 1) was developed in this laboratory with this concept in mind. The original assay (Rossman et al., 1985, 1986) used only lambda prophage induction in E. coli as an endpoint. The induction of lambda prophage results from the derepression of the bacterial SOS system, a set of coordinately regulated genes that become activated when D N A is damaged (Walker, 1985). The use of lambda prophage induction in screening for mutagens is well established (Elespuru, 1984). Yet, this assay differs in some important ways from previously described prophage induction assays: (1) It utilizes an E. eoli B strain, in which prophage induction is a lower-frequency event than it is in K12 strains (Rossman et al., 1986). This allows > 99% survival of treated cultures so that other endpoints can be screened. In addition, there is evidence that E. coli B strains are more sensitive to some intercalating agents than are K12 strains, and that this difference may be due
Controls
Controls
Q.)
Test Agents
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OVERNIGHT INCUBATION -
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Endpoint Measured
Prophage Induction
Mechanism
Induction of SOS Systemby DNA Damage
Ampicillin Resistance
TRP + Reversion Base Pair Substitution
(Ochre Suppression)
Base Pair Substitution or Gene Amplification
Fig. l, Microscreen assay.
T5 Resistance
All Types of
Forward Mutation
351
to different levels of DNA supercoiling in these strains (Bridges et aI., 1981). (2) The assay is run in Microtitre plates containing 96 wells, in a volume of 250 ttl/well. The culture is grown overnight in the presence of the test agent. Long-term incubation at the concentration being tested may contribute to the very small amounts of test compounds detectable. (3) Toxicity is evident by the absence of growth, and sampling is carried out only from sub-toxic wells. Thus, the toxic endpoint is determined in the same assay as the genotoxic endpoints. (4) The indicator strain cannot be easily lysogenized and therefore gives rise to clear plaques, allowing ease of scoring. The relationship between mutagenicity, as determined by short-term in vitro tests, and carcinogenicity has been studied in great detail for the last 15 years. In 1975, McCann et al. concluded that the S. typhimurium mutation assay could identify about 90% of rodent carcinogens, whereas more recent studies from the National Toxicology Program conclude that the figure is 45% for a different group of compounds (Tennent et al., 1987). The fraction of false positives (noncarcinogens which are mutagens) also appears to have increased with time, from about 10% (McCann et aI., 1975) to 14% (Tennant et al., 1987). The reasons for the apparent declining predictivity of the S. typhimurium assay have been discussed by others (e.g. Clive, 1988; Shelby et al., 1988, Brockman and DeMarini, 1988; Prival and Dunkel, 1989). It is not our purpose to enter into the debate as to whether in vitro genotoxicity can be an adequate surrogate for animal carcinogenicity testing. Nevertheless, in suggesting that the Microscreen assay could be used in bioassay-directed fractionation of complex mixtures, it behooves us to demonstrate that this assay will perform at least as well as other accepted assays. We have previously reported that a number of metal compounds are capable of inducing lambda prophage (Rossman et al., 1984), and have demonstrated use of this assay in the fractionation of organic extracts from air pollution particles (Rossman et al., 1985), in studying indoor air pollution (Boone et al., 1989), and in assessing the mutagenicity of oxidized nucleosides (Shirnamr-Mor6 et al., 1987). DeMarini and coworkers have shown
that chlorinated pesticides (Houk and DeMarini, 1987) and other chlorinated organic compounds (DeMarini et al., 1990) which are negative in the Ames test, are able to induce lambda prophage in the Microscreen assay. The Microscreen assay prophage induction endpoint was also able to detect genotoxic material from hazardous waste sites (Houk and DeMarini, 1988), from airborne particulate matter from Rio de Janeiro (Miguel et al., 1990) and from a steel plant in Beijing (Lin et al., 1987). Kojic acid, a product of Aspergillus species which is converted to the 'freshly baked' odor and flavor enhancer maltol, was found to induce prophage in the Microscreen assay (Lin et al., 1987). We have recently added two forward mutation endpoints to this assay and have shown that metabolites of benzene are mutagenic at these endpoints (Rossman et al., 1989). However, because the prophage induction endpoint was the first one developed, a large number of compounds have been assayed in the Microscreen assay using only that endpoint. In this paper, we address the question whether the Microscreen assay (using only one endpoint and one strain of bacteria) is as sensitive in detecting rodent carcinogens as the S. typhimurium assay (using all strains available) for a group of compounds that span several major chemical classes. We also address the extent to which the Microscreen and S. typhimurium assays complement one another as predictors of rodent carcinogenicity. Materials and methods
The microscreen assay (1) E. coli strains and media All strains are derivatives of E. co# B / r . WP2(Yk) is a lambda lysogen of WP2 (trpE, uvrA), isolated in our laboratory from WP2 and wild-type lambda phage, both obtained from Dr. Evelyn Witkin, Rutgers University. The indicator strain SR714 (trpE uvrD3) was obtained from Dr. Kendric Smith, Stanford University. The indicator strain was chosen for its ability to give clear plaques. Cultures were grown in medium MST, which is Minimal Broth Davis (Difco) containing 0.2% glu-
352 TABLE 1
TABLE 1 (continued)
133 CHEMICALS TESTED IN MICROSCREEN ASSAY Chemical name Chemical name
CAS number Source
Acetone Acridine Acridine orange Actinomycin D Acyclovir
67-64-1 260-94-6 65-61-2 50-76-0 59277-89-3
Baker Aldrich Sigma Sigma D. Clive
9-Aminoacridine 2-Aminoanthracene 2-Aminopurine Aniline Anthracene
52417-22-8 613-13-8 452-06-2 4165-61-1 120-12-7
Sigma Sigma Sigma Aldrich Aldrich
Anthracene-9-carboxylic acid Ascorbic acid 8-Azaguanine Barium chloride Benzamide
723-62-6 50-81-7 134-58-7 10361-37-2 55-21-0
Pfaltz and Bauer Sigma Sigma Alfa Sigma
Benz[ a ]anthracene-7,12-dione Benzene Benzoic acid Benzoin Benzo[ c]cinnoline
2498-66-0 71-143-2 65-85-0 119-53-9 230-17-1
Aldrich Sigma Sigma NTP a Aldrich
Benzo[a]pyrene Benzo[e]pyrene Benzo[ f ]quinoline Benzyl acetate Beryllium chloride
50-32-8 192-97-2 85-002-9 140-11-4 7787-47-5
Aldrich Aldrich Aldrich NTP a Fluka
2-Biphenylamine Bleomycin sulfate 5 '-Bromo-2'-deoxyuridine 5-Bromouracil Cadmium chloride
90-41-5 9041-93-4 59-14-3 51-20-7 10108-64-2
Pfaltz and Bauer Bristol Sigma Sigma Alfa
Caffeine Calcium chloride Caprolactam Carbon tetrachloride Chloramine T
58-08-2 22691-02-7 105-60-2 56-23-5 127-005-1
Sigma Fisher NTP a Aldrich Fisher
Chromium chloride Cinnamyl anthranilate Cupric chloride Cysteamine Deoxycytidine
10049-05-5 87-29-6 10125-13-0 60-23-1 951-78-0
Alfa NTP a Fisher Sigma Sigma
Deoxyuridine Diallyl phthalate Dibenz[ a, c]anthracene Dibenz[ a, h ]anthracene 3,3'-Dichlorobenzidine
951-78-0 131-17-9 215-58-7 53-70-3 71-94-1
Sigma NTP a Aldrich Aldrich Sigma
2,6-Dichloro-p-phenylenediamine Di(2-ethylhexyl)adipate
609-20-1 103-23-1
NTP a NTP a
CAS number Source
Di(2-ethylhexyl)phthalate 117-81-7 3,3'-Dimethoxybenzidine 119-90-4 7,12-Dimethylbenzanthracene 57-97-6
NTP a Fluka Fluka
Dimethylsulfoxide 2,4-Dinitrochlorobenzene 1,5-Dinitronaphthalene Diphenylphosphate Ethanol
67-68-5 97-00-7 605-71-0 838-85-7 64-17-5
Sigma Fisher Aldrich Fluka Sigma
Ethidium bromide Ethylmethane sulfonate Ferric chloride Ferrous chloride Fluoranthene
1239-45-8 62-50-0 7705-08-0 7758-94-3 206-44-0
Calbiochem Aldrich Fisher Baker Aldrich
Formaldehyde Geranyl acetate Hydrogen peroxide 9-Hydroxyfluorene Hydroxylamine HC1
50-000-0 105-87-3 7722-84-1 2443-58-5 5470-11-1
Sigma NTP a Sigma Pfaltz and Bauer Sigma
6-N-Hydroxylaminopurine 5-Hydroxymethyl-2 '-deoxyuridine 5-Hydroxymethyluracil 8-Hydroxyquinoline Hydroxyurea
5667-20-9
U.S. Biochem
5116-24-5 4433-40-3 148-24-3 120-07-1
Sigma Sigma Sigma Sigma
Hypoxanthine ICR 170 ICR 191 Lead nitrate Manganous chloride
68-94-0 146-59-8 17070-45-0 10099-74-8 10034-96-5
Sigma Polysciences Polysciences Sigma Sigma
Melamine Mercuric chloride Methotrexate 3-Methylcholanthrene Methylene chloride
108-78-1 7487-94-7 59-05 -2 56-49-5 75-09-2
NTP a Sigma Sigma Sigma Sigma
Methylglyoxal Methylmethane sulfonate N-Methyl-N '-nitroN-nitrosoguanidine Michler's ketone Mitomycin C
78-98-8 66-27-3
Sigma Aldrich
70-25-7 90-94-8 50-07-0
Sigma NTP a Sigma
Nalidixic acid 1,2-Naphthoquinone Nickel acetate 2-Nitrofluorene 4-Nitroquinoline-1 -oxide
389-08-2 524-42-5 6018-89-9 607-57-8 56-57-5
Sigma Fluka Aldrich Aldrich Aldrich
N-Nitrosodiethylamine N-Nitrosodiisopropylamine
55-18-5 601-77-4
Aldrich Aldrich
353
cose and 20 /~g/ml tryptophan. Mid-exponential phase cultures (optical density = 0.3 at 550 nm) were used for all experiments. The culture is centrifuged to eliminate free phage just before use.
TABLE 1 (continued) Chemical name
CAS number
Source
N-Nitrosopyrrolidine Orcinol Oxalic acid
930-55-2 504-15-4 2065-73-8
Aldrich Fluka Sigma
4,4'-Oxydianiline m-Phenylenediamine o-Phenylenediamine p- Phenylenediamine Phenanthrene
101-80-4 108-45-2 95-54-5 106-50-3 85-01-8
NTP a Sigma Sigma Sigma Aldrich
Polybrominated biphenyl Potassium chloride Potassium chromate Potassium permanganate fl-Propiolactone
59536-65-1 7447-40-7 7789-00-6 7722-64-7 57-57-8
NTP ~ Sigma Mallinckrodt Sigma Kodak
Pyrene Quercetin Rhodamine B Silver nitrate Sodium arsenite
129-00-0 6151-15-3 181-88-9 7761-88-1 7784-46-5
Sigma Sigma Kodak Sigma Fisher
Sodium Sodium Sodium Sodium Sodium
azide bisulfite hypochlorite molybdate nitrate
26628-22-8 7631-90-5 7681-52-9 7631-95-0 7631-99-4
Sigma Aldrich Aldrich Sigma Sigma
Sodium Sodium Sodium Sodium Sodium
nitrite saccharine selenate selenite sulfate
7632-00-0 81-07-2 13410-01-0 10102-18-8 7757-82-6
Sigma Sigma Sigma Sigma Sigma
Sodium tungstate Stannous chloride Streptomycin sulfate Thymidine Thymine
10213-10-2 7772-99-8 3810-74-0 50-89-5 65-71-4
Fisher Alfa Sigma Sigma Sigma
Toluene Trichloroethylene Xanthine Xylenes 2,6-Xylidine Zinc chloride
108-88-3 78-34-2 69-89-6 1330-20-7 87-62-7 7646-85-7
Aldrich Aldrich Sigma Fluka NTP a Alfa
a NTP Chemical Repository, Radian Corporation, Austin, Texas.
(2) Exposure 8 serial 2-fold dilutions of the test agent in 150 /~1 MST were made across a row in a Falcon Micro Test III tissue culture multiwell plate containing 96 wells. In the first experiment, the starting concentration was 100 /~g/well and the final concentration was 0.78/~g/well. This series should allow at least one but no more than five wells to show growth inhibition. In cases where no growth inhibition occurs, the starting concentration was increased up to the concentration at which precipitation occurs. In cases where more than 5 wells show growth inhibition, only the two subtoxic wells could be sampled, and the experiment is repeated at a lower starting concentration. The negative control consisted of growth medium containing the highest concentration of solvent used. If the test agent was not water soluble, acetone (up to 10% v / v ) was used. DMSO was avoided as older opened bottles sometimes accumulated phage-inducing material. Positive controls consisted of M N N G (6.25 /~g/ml) for direct acting agents and benzo[a]pyrene (25 /~g/ml) for metabolic activation. The wells are inoculated with 75/~1 of a mid-exponential culture of WP2(~) (OD550 =0.3), diluted 1 / 1 0 after centrifugation so that each well received approximately 2 × 107 bacteria. At this cell density, no turbidity was evident. The test plate was incubated overnight at 37°C and scored after 20 h for turbidity. Control (turbid) wells contained approximately 1.3 X 109/ml. Wells which were visually less turbid (scored as + / - ), were found to contain 1-5 × 108 cells/ml. After scoring for growth, aliquots of 5/~1 from 3 - 4 subtoxic ( + or + / - ) wells are diluted in 1 ml MST. Diluted samples (100 #1), which contain approximately 6.5 x 105 lysogens from fully turbid wells, are added to tubes containing 2.5 ml soft agar (0.65% Bacto agar, 10 mM MgSO4), held at 47°C. A mid-exponential culture of indicator strain SR714 (100/~1) is added, and the tubes are mixed and poured onto half strength Luria agar plates (Difco). After overnight incubation at 37°C, the plates are scored for plaques. All assays are run in duplicate, and all experiments have been repeated at least once. The range and variability of plaques in the Microscreen assay have been
354 TABLE 2 INDUCTION OF PROPHAGE IN MICROSCREEN ASSAY Compound
$9
Max. e n h a n c e m e n t over background
Amount at Max. a
L Polycyclic aromatics and derivatives Acridine
-
nb
8.0
Anthracene
-
3.5
12.5
+
4.8
12.5
Anthracene-9-carboxylic acid Benzanthracene-7,12-dione
-
n
+
30
127 (p) c 127 (p)
-
n
122 (p)
+
9.3
122 (p)
Benzo[ c]cinnoline
+
n n
127 127
Benzol a ] p y r e n e
+
3 10.4
Benzo[e]pyrene
+
n n
Benzo[ f ]quinoline
+ +
n 6.7 n 8
7.5 7.5 1.44 1.44
-
n
2.0
+
4
2.0
7,12-Dimethylbenzanthracene
+
n 11.5
5.0 5.0
Fluoranthene
+
n n
50 (p) 50 (p)
9-Hydroxyfluorene
+ +
n n n n
143 (p) 143 (p) 6.7 6.7
3-Methylcholanthrene
+
n 10
1,2-Naphthoquinone
+ +
n n n 4.5
3.13 12.5 25 25
+
n n
50 50
+ -
n 3.5 68 21
+ + +
73 n 10 n 8 n 7
Dibenz[a,c]anthracene Dibenz[ a, h ] a n t h r a c e n e
8-Hydroxyquinoline
Phenanthrene Pyrene
12.5 (p) 12.5 (p) 25 (p) 25 (p)
50 50
I1. Nitro aromatics and nitroso compounds Benzamide 1,5-Dinitronaphthalene 2-Nitrofluorene 4-Nitroquinoline-l-oxide N-Nitrosodiethylamine N-Nitrosodiisopropylamine N-Nitrosopyrrolidine
15.5 15.5 15.5 25 0.16 600 600 300 300 225 225
355 T A B L E 2 (continued)
Compound
$9
Max. e n h a n c e m e n t over background
Amount at Max. a
Ill. Aromatic amines Aniline 2-Biphenylamine
Acridine orange 9-Aminoacridine
-
n n
0.8 62.5
+
3.9
62.5
-
20 n
3,3 ' - D i m e t h o x y b e n z i d i n e
-
n
Ethidium bromide
+ -
n 13
9.4 3.1 60 60 15.6
Melamine
-
7
78
4,5 ' - O x y d i a n i l i n e
+ -
4 n
78 6.25
+
4.6
6.25
m- P h e n y l e n e d i a m i n e o-Phenylenediamine
-
19 20
175 125
p-Phenylenediamine 2,6-Xylidine
-
11 n
125 39
+
3
39
IV. Halogenated organic compounds Carbon tetrachloride Chloramine T 3,3 '- D i c h l o r o b e n z i d i n e 2,6-Dichloro-p-phenylenediamine
2,4-Dinitrochlorobenzene M e t h y l e n e chloride Polybrominated biphenyl Trichloroethylene
-
n
200
+
6.5
200
+ + +
3 9.6 n n n n
255 255 125 125 250 250
-
+ -
n
20 19 n
+
5.7
-
n
+
5
25 8250 (2.5~) 8250 (2.5~) 3.1 3.1 18 18
V. Antimetabolites and antibiotics Actinomycin D 8-Azaguanine B l e o m y c i n sulfate Hydroxyurea
n 7.5 27 27
Methotrexate Mitomycin C Nalidixic acid S o d i u m azide
14 16 100 +
S t r e p t o m y c i n sulfate
8 17 n
15.6 37.5 0.00625 0.92 3.9 0.00078 0.4 36.5 36.5 1.1
V1. Bases, nucleosides and analogs Acyclovir 2-Aminopurine
n 20
375 75
356 T A B L E 2 (continued) Compound
$9
Max. enhancement over background
5 '-Bromo-2 '-deoxyuridine 5-Bromouracil Deoxycytidine Caffeine Deoxyuridine 5-Hydroxymethyl-2 '-deoxyuridine 5- Hydroxymethyluracil 6-N-Hydroxylaminopurine Hypoxanthine Thymidine Thymine Xanthine
Amount at Max. a
8 n n
125 675 675
n n 52 7 5
938 675 125 125 132
n n n n
35.6 125 125 125
VII. Small direct-acting alkylating agents 77 15 33 11
Ethylmethane sulfonate Methylmethane sulfonate N-Methyl-N '-nitro-N-nitrosoguanidine fl-Propiolactone
3.125 0.39 1.56 0.026
VIIL Inorganic compounds Barium chloride Beryllium chloride C a d m i u m chloride Calcium chloride C h r o m i u m chloride Cupric chloride Ferric chloride Ferrous chloride Hydrogen peroxide Hydroxylamine Lead nitrate Manganous chloride Mercuric chloride Nickel acetate Potassium chromate Potassium chloride Potassium permanganate Silver nitrate Sodium arsenite Sodium bisulfite Sodium Sodium Sodium Sodium Sodium
hypochlorite molybdate nitrate nitrite selenate
Sodium selenite Sodium sulfate Sodium tungstate
--
n
-
n
-
n
-
n
-
6
--
n
-
3.5
-
3.9
-
4.5
--
n
-
10
-
32
-
n 5 28
-
n
-
3
-
n
--
n
--
n
-
33.6 3.5
-
8
-
4.6
-
9.3
-
< 3-10.4
-
n
-
4
38 100 3.5 278 33.3 2.15 0.041 0.05 0.5 37.5 27.3 77.3 0.012 11 2.4 600 7.9 0.01 104 275 103 103 132.5 12.5 25 25 275 412
357 TABLE 2 (continued)
Compound
$9
Max. enhancement over background
Amount at Max. a
Stannous chloride Zinc chloride
-
9.8 3
50 21.3
+ + +
n n n n n n
10% 10% 5% 5% 10% 10% 10% 10%
IX. Solvents Acetone Benzene Dimethylsulfoxide
Ethanol Toluene Xylenes
-
n
+
n
-
n
0.5%
+
n
0.5%
-
n
1%
+
n
1%
X. Miscellaneous Ascorbic acid Benzoic acid Benzoin
n n n
+ Benzyl a c e t a t e + Caprolactam
n
+ Cinnamyl anthranilate + Cysteamine Diallylphthalate
7 n n n
+ Di(2-ethylhexyl)adipate + Di(2-ethylhexyl)phthalate + Diphenylphosphate Formaldehyde +
Geranyl acetate
n n n 6.8 n
