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The gradient plate assay: a modified Ames assay used as a prescreen for the identification of bacterial mutagens
Genetic Toxicology
ELSEVIER
Mutation Research 341 (1995) 185-192
The gradient plate assay: a modified A m e s assay used as a prescreen for the identification of bacterial mutagens M.A. Rexroat
*, T . J . O b e r l y , B . J . B e w s e y , M . L . G a r r i o t t
Toxicology Research Laboratories, Lilly Research Laboratories, A Division of Eli Lilly and Company, Greenfield, IN 46140, USA Received 21 March 1994; revision received 18 August 1994; accepted 30 August 1994
Abstract
Bacterial test systems have been used extensively to identify the mutagenic potential of new compounds. In particular, the Ames test has gained worldwide acceptance and is required by many regulatory agencies to support product registration. The gradient plate assay (GPA) is a modification of the Ames test. It is used as a high capacity prescreen to detect the mutagenic potential of synthetic intermediates, impurities, and research compounds over a concentration gradient. Since the development of the GPA, over 4000 compounds have been tested in the assay. Selection and use of the GPA in our laboratory is due to many factors: reliability; sensitivity; capacity; timeliness of reporting results; and establishment of safety standards in the laboratory. In this manuscript, results of the GPA method are compared with results from the traditional Ames assay. To date, 113 compounds of identical lots have been evaluated in both tests, and in all but 3 instances the results are the same. Thus, the GPA is an ideal assay for use as a prescreen in determining the ability of a compound to induce bacterial mutation.
Keywords: Gradient plate assay; Concentration gradient; Prescreen; Ames
I. Introduction
The Ames test has been used extensively for testing the ability of a chemical to interact with D N A and cause mutations. It is the most commonly used assay for assessing the genotoxic potential of compounds to which humans are exposed (McCann et al., 1975a; McCann and Ames, 1977; Sugimura et al., 1977). Moreover, a correlation between in vivo carcinogenicity and bacterial
* Corresponding author. Tel. 317-277-4154; Fax 317-2774954.
mutagenicity has been established (Rinkus and Legator, 1979, 1981; Probst et al., 1981; Ames and McCann, 1981; Kier et al., 1986; Shelby, 1988; Shelby and Zeiger, 1990). In order to expedite the process of bacterial mutagenicity testing, new assays such as the Spiral Salmonella assay (Houk et al., 1989) and the Simultest (Nestmann et al., 1987) have been developed. The Spiral assay is used to eliminate the need for serial dilutions and multiple plates to obtain dose-response data. In a Spiral assay, the bacteria, compound, and $9 are dispensed on an agar plate by a spiral plater. The plater can dispense a fixed or variable dilution to the agar
0165-1218/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0 1 6 5 - 1 2 1 8 ( 9 4 ) 0 0 0 6 1 - 1
186
M.A. Rexroat et al. / M u t a t i o n Research 341 (1995) 185-192
plate thereby eliminating the need for a top agar overlay. Each spiral plate can generate dose-response information over a concentration range of approximately 15:1. Extracts, pure compounds, and complex environmental mixtures can be evaluated in the spiral assay (Houk et al., 1989; H o u k et al., 1991). The Simultest is an assay in which a mixture of the compound, _+$9, and multiple bacterial strains are placed on one plate simultaneously. The combination of bacterial strains can successfully detect mutagenic activity, however, it cannot distinguish between base-pair substitution and frameshift mutations. Positive results are confirmed by a repeat Simultest, and nonmutagens are confirmed with a replicate test using individual strains. Extracts and complex mixtures can be evaluated in the Simultest (Nestmann et al., 1987), but the testing of volatile compounds has not yet been reported. The gradient plate assay was developed at Eli Lilly and Company in 1976 as a preliminary screen for the identification of direct-acting and activation dependent mutagens (Cline and McMahon, 1977; McMahon et al., 1979; Probst et al., 1981; Thompson et al., 1980; Thompson et al., 1983; Rexroat and Probst, 1985). This procedure has, to date, been used to test over 4000 compounds from a variety of sources. Two distinct advantages of the G P A are: (1) it requires a smaller amount of the test article than the conventional Ames assay; and (2) as many as 14 compounds can be tested simultaneously. A variety of compounds including complex mixtures, viscous solutions, and volatile liquids have been evaluated in the GPA. A modification of the G P A (reverse gradient) is used to accommodate the testing of volatile liquids (McMahon et al., 1979). Similar to the Spiral assay and Simultest, the G P A is used as a preliminary screen to detect the mutagenicity of intermediates a n d / o r products in early development. Since the results from the G P A are evaluated qualitatively and not quantitatively, it is not possible to substitute the G P A for the complete Ames on submissions to regulatory agencies. Nevertheless, the G P A plays an important role in gathering preliminary information for early selection of new chemical entities in drug development.
In this report, results were compared for 113 compounds that have been tested in both the Ames assay and GPA. While more compounds had been tested in both assays, it was necessary to compare only those compounds where the same lot was used since lots varied in purity, potency, and stability. A generic list of the compounds and their chemical classes is presented. F u r t h e r m o r e , recent technical modifications which make the G P A procedure more analogous to that of the Ames assay are described.
2. Materials and methods
Five bacterial strains were used in both the Ames assay and GPA. They include four auxotrophs of S. typhimurium LT-2: TA1535, TA1537, TA100, and TA98 and a tryptophan auxotroph derived from E. coli: WP2uvrA . S. typhimurium strains were obtained from Dr. Bruce N. Ames of the University of California at Berkeley, and the E. coli strain was obtained from Dr. Brian A. Bridges of the M R C Cell Mutation Unit, Sussex, UK. The genotypes of these strains were confirmed using the procedure of Maron and Ames (1983). The overnight cultures were prepared from frozen stock ( - 1 5 0 ° C ) by inoculating 0.2 ml of each tester strain in 20 ml of 2.5% Oxoid Nutrient Broth No. 2 (Oxoid LTD). Minimal agar medium included 100 ml of 20% glucose; 80 ml of 10 x histidine-tryptophan-biotin (each his-tryp-bio, 0.5 mM); 40 ml of 25 × VogelBonner salts (VB) (Vogel and Bonner, 1956); and 280 ml Milli-Q water. The above mixture was warmed to 50°C and QS to 1 liter with melted 2.5% Difco Bacto agar. Final concentrations were 2% glucose, 1 × VB salts, and 0.04 mM of histryp-bio in 1.25% agar medium. The concentration gradient was prepared by adding a base layer containing 10 ml of minimal agar medium to a 9 x 9 cm square Petri plate tilted at a slight angle (Fig. 1). The agar was allowed to solidify at room temperature into a wedge shaped layer. A stock concentration of the test chemical was prepared at 100 m g / m l , and three 1:10 serial dilutions were performed. A concentration of 1000 # g / m l of test compound
M.A. Rexroat et aL / Mutation Research 341 (1995) 185-192
was prepared by adding 10 ml of minimal agar to 0.1 ml of a 100 m g / m l solution of compound in the appropriate vehicle. The top agar was thoroughly mixed and poured onto the appropriate base layer plate. This resulted in a ten-fold concentration gradient across the plate ranging from 100 to 1000/~g/ml. Three additional plates were prepared with concentration ranges of 10 to 100, 1 to 10, and 0.1 to 1 /.~g/ml. Therefore, the four plates prepared for each test compound resulted in a 10000-fold concentration range (0.1-1000 /~g/ml). Four plates were also prepared for each of the positive controls used in the nonactivated assay, 2-nitrofluorene and N-methyl-N'-nitro-Nnitrosoguanidine. These plates ranged in concentration from 0.01 to 100 and 0.001 to 1 0 / z g / m l , respectively, for the two compounds. Three plates were prepared for the solvent control at a concentration of 1%. The 5 bacterial strains were inoculated in duplicate by streaking the cultures across the concentration gradient of each plate using a 10-barreled micropipet. The test was also conducted with metabolic activation using a rat post-mitochondrial fraction (Aroclor 1254-induced $9) which was prepared by Lilly Research Laboratories (Cline and McMahon, 1977; Thompson et al., 1980) or obtained
from a commercial source (Molecular Toxicology, Annapolis, Maryland). The $9 was validated with appropriate positive controls prior to use. The $9 fraction with cofactors was mixed with agar and overlaid onto the chemical concentration gradient just prior to inoculating the bacterial tester strains. In the activated portion of the GPA and Ames assay, final $9 concentrations of 2.7% and 2.0%, respectively, were used. Four plates were also prepared for each positive control used in the activated assay, 2-aminoanthracene and streptozotocin. These plates ranged in concentration from 0.0005 to 5 /~g/ml and 0.1 to 1000 /zg/ml, respectively, for the two compounds. Three plates were prepared for the solvent control at a concentration of 1%. A total of 134 plates was required for testing 14 compounds in the G P A including solvent and positive controls, with and without activation. Plates were incubated for approximately 48 h at 37°C and then examined for the growth of mutant colonies within the lawn of each inoculum streak. The preparation of the gradient assay along with scoring of plates and interpretation of results have been described in detail by McMahon et al. (1979). The results of the GPA were compared to the results from the Ames test which
GRADIENT PLATE ASSAY Pour wedge of minimal agar.
Pour second wedge of minimal agar containing test compound.
Let stand to allow concentration gradient to form.
Pour layer of $9 in agar on top for "activated" plates.
J Five bacterial strains streaked in duplicate over the surface of the agar.
Fig. 1.
187
188
M.A. Rexroat et al. /Mutation Research 341 (1995) 185-192
was conducted according to the methods of Ames et al. (1975), with the necessary modification (inclusion of tryptophan, 0.04 mM) for E. coli WP2uvrA- as previously described (Green and Muriel, 1976). In the Ames assay, 5 bacterial strains, 5 test concentrations, 1 solvent control, and 1 concentration per positive control were tested in triplicate, with and without metabolic activation (210 plates). Revertant colonies were counted using an Artek 880 Automated Colony Counter (Artek Systems Corporation, Farmingdale, NY). A test article was considered to have induced a positive response for bacterial mutation when a concentration-related increase in revertants was observed in which the number of revertants exceeded the value of the vehicle control by at least two-fold (strains TA98, TA100, and W P 2 u v r A - ) or a least three-fold (strains TA1535 and TA1537), for two successive concentrations of the test article. These criteria are consistent with the prevailing attitude of the scientific community (Kier et al., 1986). Since results from the GPA were qualitative rather than quantitative, colonies were only counted when it appeared that the compound may have elicited a weakly mutagenic response. In these cases, the colonies were counted manually.
3. Results and discussion
Over 4000 compounds have been tested in the GPA, the majority of which have been intermediates, i.e., chemicals involved in various stages of product synthesis. However, the compounds in this report also included chemical impurities, research entities, and several marketed compounds. Results for 113 compounds (identical lots) tested in both the Gradient Plate Assay and the Ames assay are shown in Table 1. The list of compounds covers a broad spectrum of chemical classes with a variety of intended uses. The concordance of results from the two test systems is very good (97%). Conflicting results between the two assays were observed for only three compounds (phenobarbital, HC Blue 2, and an antiobesity compound; see Table 2). The
agreement between the assays would have been even greater had more conservative response criteria been utilized (Kier et al., 1986). Furthermore, one inconsistent result (HC Blue 2) may have been attributed to lower test concentrations employed in the GPA. Phenobarbital was mutagenic to strain TA1535 in the Ames assay without metabolic activation, but was negative in the GPA. Although a rodent carcinogen, phenobarbital has generally yielded negative results in a variety of bacterial mutation assays (McCann et al., 1975a; Purchase et al.; 1978; King et al., 1979). Venitt (1985) reported that phenobarbital was difficult or impossible to detect reliably in routine screening assays using S. typhimurium. While phenobarbital was reported to produce weak, but consistent, positive results in the Ames assay (Rexroat and Probst, 1985; Zieger and Haworth, 1985), the response would have been considered negative had the more conservative criteria currently in use been utilized (Kier et al., 1986). HC Blue 2 (HCB2) was mutagenic in the Ames assay with strain TA98 in the presence and absence of liver $9. The results for bacterial mutation observed in the present report show a strong correlation with that reported by the National Toxicology Program (NTP, 1985). The test concentrations producing mutagenicity in the Ames assay were equal to, or greater than, 1000 /xg/plate. At Lilly, the Ames assay has generally been used to test compounds for regulatory submission. In the absence of toxicity or precipitation, a maximum test concentration of 5000 /xg/plate has been established for the Ames assay in order to meet regulatory guidelines. In contrast, the highest test concentration used in the GPA is 1000 /xg/ml which is due to the frequently limited availability of compounds a n d / o r the fact that results are not intended to represent the sole regulatory submission. Therefore, insufficient test article concentrations may be the reason for the negative finding in the GPA. The remaining antiobesity compound produced approximately a six-fold increase in revertants in the Ames assay with strain TA1535 in the presence of liver $9 (Rexroat and Probst, 1985),
M.A. Rexroat et al. / Mutation Research 341 (1995) 185-192 Table
Table
1
Comparison gradient
of results plate
for
bacterial
mutation
in the
Ames
and
189
1 (continued)
Compound
assay
Compound
Ames
GPA
result
result
Acridine
Ames
GPA
result
result
+
+
Biphenyl 2-Aminobiphenyl
~
9-Aminoacridine Alkylating
+
+
a,/3-Blocker
agent
-
-
-
-
-
-
-
-
-
-
Indolidan
-
-
-
-
-
-
Amesergide
Triethylenemelamine
+
a
+
a
Amine -
-
Analgesic
channel
Indecainide
Analgesic/antiinflammatory
-
-
Isomazole
Picenadol
-
-
Quinpirole
Aniline
Positive
o-Toludine
additive
Narasin Narasin/nicarbasin
combination
Nicarbasin
antagonist
Tibenelast 5-1ipoxygenase
enzyme
inhibitor
HCI
HCI inotropic
-
-
Thrombosis
inhibitor
-
-
Sergolexole
maleate
-
-
-
-
Vasodilator
and
-
-
-
-
Zatosetron
-
-
antagonist
-
-
-
Antihypertensive
+
Dopamine
LTD4/E4-receptor
blocker
-
Antiasthma LTD-antagonist
antagonist)
-
Anticoccidial Anticoccidial
(5HT2
Antiischemic Calcium
a-Naphthylamine
inotropic
maleate
5HT3
rec. a
q_ a
agonist
-
-
Quinelorane
-
-
Pergolide
-
-
HCI mesylate
-
-
-
-
Dye
-
-
HC
Blue
No.
1
-
-
Antiasthma
#1
-
-
HC
Blue
No.
2
+
-
Antiasthma
#2
-
-
Lawsone
-
-
Antibiotic
cosmetic
dye
Fungicide
Cycloserine
-
-
Cilofungin
-
-
Daptomycin
-
-
Fenarimol
-
-
Dirithromycin
-
-
Nuarimol
-
-
Erythromycylamine
-
-
Actaplanin
-
-
-
-
Ancymidol
-
-
-
-
Avilamycin
-
-
-
-
Anticancer Oncolytic
potentiator
Sulofenur Anticonvulsant
Growth
promoter
Ractopamine
Anticonvulsant
Fluoxetine
HCI
Norfluoxetine
HCI
-
Benefin
-
-
-
-
2-Chloro-4-fluorobenzophenone
-
-
-
-
2,4-Dichlorobenzophenone
-
Ethalfluralin
+
Flurprimidol
-
Antiestrogen Raloxifene
-
-
Antihistamine
Herbicide
Herbicide
Methapyrilene
HC1
-
Antidepressant
analogue
a
+ -
+
+
-
-
lsoxaben
-
-
Histamine
(HI)
antagonist
-
-
Isoxazoleamine
-
Histamine
(H2)
antagonist
-
-
Oryzalin
-
-
-
-
Antipsychotic
Soybean
PGR
Olanzapine
-
-
Tebuthiuron
-
-
Antipsychotic
-
-
Herbicide
-
-
-
Antitumor
Insulin
Gemcitabine
-
-
Biosynthetic
human
C-peptide
-
Penfenamine
-
-
Biosynthetic
human
insulin
-
-
-
-
Antiulcers
Proinsulin
Nizatidine Nizatidine Aromatic
~
Cardiovascular
sulfoxide
-
-
-
-
amines
Miticides/insecticides Insecticide
fermentation
Miticide
#1
product
-
-
2-Acetylaminofluorene
+
a
"1"- a
Miticide
#2
-
-
2-Aminoanthracene
+
a
_~_ a
Miticide
#3
+
+
+
+
Nitro
compound
2-Nitrofluorene
M~4. Rexroat et al. / Mutation Research 341 (1995) 185-192
190
and comparable test concentrations were used in the GPA. No reason can be offered for the incongruent finding at this time. Recently, changes were made in the G P A procedure to make it identical to that used in the Ames assay (Ames et al., 1975; Rexroat and Probst, 1985). Prior to these changes, subtle differences existed between the two assays with respect to the preparation of bacterial cultures, solutions, and media. Specifically, the maintenance medium was changed from trypticase soy
Table 1 (continued) Compound
Ames result
GPA result
Nitrosoureas and amides
N-Ethyl-N'-nitro-N-nitrosoguanidine
Table 2 Disagreement
in test results for the A m e s a n d g r a d i e n t p l a t e
assays Compound
Ames
GPA
- $9
+ $9
- $9
+ $9
Phenobarbital HC Blue 2
+ a + b
_ + b
_ _
_ _
Antiobesity
_
+ a
_
_
Phenobarbital doses
A m e s - $9
50
24 -+ 3
100
24 + 5
500
42 + 6 a
1000
46 -I- 5 a
5000
50 + 4 a
Solvent
19 _+ 1
Solvent
17 + 4
HC Blue #2 doses
Ames - $9
312.5
32+
9
625
31+
2 4 b
81+
1250
45+
2500
57_+ 4 b
206_+ 16 b
5000
7 6 + 12 b
1 6 9 + 12 b
Solvent
17+
1
34-+
9
Solvent
22 -+ 2
32 +
5
Antiobesity doses
A m e s + $9
+
guanidine
+
+
Streptozotocin
+
+
Safrole
-
-
Benzo[a]pyrene
+ a
+ "
Pyrene
-
-
1000
121 _+ 11 a
1500
1 3 8 + 12 a
+ ~
+ a
2000
99_+
2500
53 + 10 a
Polycyclic a r o m a t i c s
Polyhalogen compound Ethidium bromide Veterinary antibiotic Apramycin
-
-
Desmycosin derivative (Tilmicosin)
-
-
Antiparasitic
+
+
Aromatase inhibitor
-
-
Avocado pit extract
-
-
Benzoin
-
-
Caprolactam
-
-
Diethylstilbestrol
-
-
Dioctyl phthalate
-
-
Ergoline
-
-
Germall II®
-
-
Hexamethylphosphoramide
-
-
Metronidazole
+
+
Omental extract
-
-
Phenobarbital sodium
+
-
Polymer TPH D8/3
-
-
Miscellaneous compound
Unsaturated
milled
sulfone
500
51+
143+
8 b
4 a
1a
Solvent
18_+ 3
Solvent
21 _+ 2
a Positive in strain T A 1 5 3 5 . b P o s i t i v e in s t r a i n T A 9 8 .
+
+
Antiobesity #1
+ ~
-
Antiinflammatory bowel disease
-
-
Antiviral
-
-
Antipsoriatic agent II
-
-
Antiobesity #2
-
-
Metabolic activation with $9 required.
7 b
105+16b
+
N-Methyl-N'-nitro-N-nitroso-
Ames + $9
broth containing 0.5% yeast extract (static 37°C incubation) to oxoid nutrient broth # 2 (shaker water bath at 37°C incubation). The test medium was changed from Difco purified agar to Difco Bacto agar; and the final concentration of histid i n e / t r y p t o p h a n was changed from 0.005 mM to 0.04 mM. It is expected that these modifications should further enhance the agreement between the assays. Re-evaluation of H C Blue 2 and the antiobesity compound could not be done due to a lack of availability of material, however; phenobarbital was re-evaluated using the new methodology in the G P A and Ames assay. Although a different lot from the previous assay, phenobarbital was
M.A. Rexroat et al. / Mutation Research 341 (1995) 185-192
found to be negative for bacterial mutation in b o t h t h e G P A a n d A m e s assay. I n s u m m a r y , t h e G P A is a n i d e a l p r e s c r e e n f o r potential bacterial mutagenicity. The distinct advantages of the GPA over the standard Ames a s s a y a r e : (1) 14 c o m p o u n d s a r e t e s t e d in t h e s a m e a m o u n t o f t i m e t h a t is r e q u i r e d f o r t e s t i n g o n e c o m p o u n d i n t h e A m e s ; (2) t h e G P A r e q u i r e s o n l y 35 m g o f t e s t a r t i c l e as c o m p a r e d t o 4 0 0 m g f o r a n A m e s assay; a n d (3) t e s t i n g o n e compound for an Ames assay requires 210 plates, w h e r e a s t e s t i n g 14 c o m p o u n d s i n t h e G P A r e q u i r e s o n l y 134 p l a t e s . S i n c e a l a r g e n u m b e r o f c o m p o u n d s c a n b e e x a m i n e d in a t i m e l y f a s h i o n , this allows for early detection of bacterial mutag e n s a n d a s s i s t s in s e t t i n g s a f e t y s t a n d a r d s i n t h e l a b o r a t o r y f o r e m p l o y e e s w h o m i g h t c o m e in c o n tact with potential genotoxins.
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M.A. Rexroat et al. / M u t a t i o n Research 341 (1995) 185-192
scheduled DNA synthesis by monosubstituted anilines. Environ. Mutagen. 5, 803-811. Venitt, S. (1985) Summary report on the performance of the bacterial mutation assays, in: J. Ashby and F.J. de Serres et al. (Eds.), Progress in Mutation Research. Elsevier, Amsterdam, Vol. 5, pp. 11-23. Vogel, H.J. and D.M. Bonner (1956) Acetylorithinase of Es-
cherichia coli: partial purification of some properties. J. Biol. Chem., 218, 97-106. Zeiger E. and S. Haworth (1985) Tests with a preincubation modification of the Salmonella/microsome assay, in: J. Ashby, F.J. de Serres et al. (Eds.), Progress in Mutation Research, Elsevier, Amsterdam, Vol. 5, pp. 187-199.
ELSEVIER
Mutation Research 341 (1995) 185-192
The gradient plate assay: a modified A m e s assay used as a prescreen for the identification of bacterial mutagens M.A. Rexroat
*, T . J . O b e r l y , B . J . B e w s e y , M . L . G a r r i o t t
Toxicology Research Laboratories, Lilly Research Laboratories, A Division of Eli Lilly and Company, Greenfield, IN 46140, USA Received 21 March 1994; revision received 18 August 1994; accepted 30 August 1994
Abstract
Bacterial test systems have been used extensively to identify the mutagenic potential of new compounds. In particular, the Ames test has gained worldwide acceptance and is required by many regulatory agencies to support product registration. The gradient plate assay (GPA) is a modification of the Ames test. It is used as a high capacity prescreen to detect the mutagenic potential of synthetic intermediates, impurities, and research compounds over a concentration gradient. Since the development of the GPA, over 4000 compounds have been tested in the assay. Selection and use of the GPA in our laboratory is due to many factors: reliability; sensitivity; capacity; timeliness of reporting results; and establishment of safety standards in the laboratory. In this manuscript, results of the GPA method are compared with results from the traditional Ames assay. To date, 113 compounds of identical lots have been evaluated in both tests, and in all but 3 instances the results are the same. Thus, the GPA is an ideal assay for use as a prescreen in determining the ability of a compound to induce bacterial mutation.
Keywords: Gradient plate assay; Concentration gradient; Prescreen; Ames
I. Introduction
The Ames test has been used extensively for testing the ability of a chemical to interact with D N A and cause mutations. It is the most commonly used assay for assessing the genotoxic potential of compounds to which humans are exposed (McCann et al., 1975a; McCann and Ames, 1977; Sugimura et al., 1977). Moreover, a correlation between in vivo carcinogenicity and bacterial
* Corresponding author. Tel. 317-277-4154; Fax 317-2774954.
mutagenicity has been established (Rinkus and Legator, 1979, 1981; Probst et al., 1981; Ames and McCann, 1981; Kier et al., 1986; Shelby, 1988; Shelby and Zeiger, 1990). In order to expedite the process of bacterial mutagenicity testing, new assays such as the Spiral Salmonella assay (Houk et al., 1989) and the Simultest (Nestmann et al., 1987) have been developed. The Spiral assay is used to eliminate the need for serial dilutions and multiple plates to obtain dose-response data. In a Spiral assay, the bacteria, compound, and $9 are dispensed on an agar plate by a spiral plater. The plater can dispense a fixed or variable dilution to the agar
0165-1218/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0 1 6 5 - 1 2 1 8 ( 9 4 ) 0 0 0 6 1 - 1
186
M.A. Rexroat et al. / M u t a t i o n Research 341 (1995) 185-192
plate thereby eliminating the need for a top agar overlay. Each spiral plate can generate dose-response information over a concentration range of approximately 15:1. Extracts, pure compounds, and complex environmental mixtures can be evaluated in the spiral assay (Houk et al., 1989; H o u k et al., 1991). The Simultest is an assay in which a mixture of the compound, _+$9, and multiple bacterial strains are placed on one plate simultaneously. The combination of bacterial strains can successfully detect mutagenic activity, however, it cannot distinguish between base-pair substitution and frameshift mutations. Positive results are confirmed by a repeat Simultest, and nonmutagens are confirmed with a replicate test using individual strains. Extracts and complex mixtures can be evaluated in the Simultest (Nestmann et al., 1987), but the testing of volatile compounds has not yet been reported. The gradient plate assay was developed at Eli Lilly and Company in 1976 as a preliminary screen for the identification of direct-acting and activation dependent mutagens (Cline and McMahon, 1977; McMahon et al., 1979; Probst et al., 1981; Thompson et al., 1980; Thompson et al., 1983; Rexroat and Probst, 1985). This procedure has, to date, been used to test over 4000 compounds from a variety of sources. Two distinct advantages of the G P A are: (1) it requires a smaller amount of the test article than the conventional Ames assay; and (2) as many as 14 compounds can be tested simultaneously. A variety of compounds including complex mixtures, viscous solutions, and volatile liquids have been evaluated in the GPA. A modification of the G P A (reverse gradient) is used to accommodate the testing of volatile liquids (McMahon et al., 1979). Similar to the Spiral assay and Simultest, the G P A is used as a preliminary screen to detect the mutagenicity of intermediates a n d / o r products in early development. Since the results from the G P A are evaluated qualitatively and not quantitatively, it is not possible to substitute the G P A for the complete Ames on submissions to regulatory agencies. Nevertheless, the G P A plays an important role in gathering preliminary information for early selection of new chemical entities in drug development.
In this report, results were compared for 113 compounds that have been tested in both the Ames assay and GPA. While more compounds had been tested in both assays, it was necessary to compare only those compounds where the same lot was used since lots varied in purity, potency, and stability. A generic list of the compounds and their chemical classes is presented. F u r t h e r m o r e , recent technical modifications which make the G P A procedure more analogous to that of the Ames assay are described.
2. Materials and methods
Five bacterial strains were used in both the Ames assay and GPA. They include four auxotrophs of S. typhimurium LT-2: TA1535, TA1537, TA100, and TA98 and a tryptophan auxotroph derived from E. coli: WP2uvrA . S. typhimurium strains were obtained from Dr. Bruce N. Ames of the University of California at Berkeley, and the E. coli strain was obtained from Dr. Brian A. Bridges of the M R C Cell Mutation Unit, Sussex, UK. The genotypes of these strains were confirmed using the procedure of Maron and Ames (1983). The overnight cultures were prepared from frozen stock ( - 1 5 0 ° C ) by inoculating 0.2 ml of each tester strain in 20 ml of 2.5% Oxoid Nutrient Broth No. 2 (Oxoid LTD). Minimal agar medium included 100 ml of 20% glucose; 80 ml of 10 x histidine-tryptophan-biotin (each his-tryp-bio, 0.5 mM); 40 ml of 25 × VogelBonner salts (VB) (Vogel and Bonner, 1956); and 280 ml Milli-Q water. The above mixture was warmed to 50°C and QS to 1 liter with melted 2.5% Difco Bacto agar. Final concentrations were 2% glucose, 1 × VB salts, and 0.04 mM of histryp-bio in 1.25% agar medium. The concentration gradient was prepared by adding a base layer containing 10 ml of minimal agar medium to a 9 x 9 cm square Petri plate tilted at a slight angle (Fig. 1). The agar was allowed to solidify at room temperature into a wedge shaped layer. A stock concentration of the test chemical was prepared at 100 m g / m l , and three 1:10 serial dilutions were performed. A concentration of 1000 # g / m l of test compound
M.A. Rexroat et aL / Mutation Research 341 (1995) 185-192
was prepared by adding 10 ml of minimal agar to 0.1 ml of a 100 m g / m l solution of compound in the appropriate vehicle. The top agar was thoroughly mixed and poured onto the appropriate base layer plate. This resulted in a ten-fold concentration gradient across the plate ranging from 100 to 1000/~g/ml. Three additional plates were prepared with concentration ranges of 10 to 100, 1 to 10, and 0.1 to 1 /.~g/ml. Therefore, the four plates prepared for each test compound resulted in a 10000-fold concentration range (0.1-1000 /~g/ml). Four plates were also prepared for each of the positive controls used in the nonactivated assay, 2-nitrofluorene and N-methyl-N'-nitro-Nnitrosoguanidine. These plates ranged in concentration from 0.01 to 100 and 0.001 to 1 0 / z g / m l , respectively, for the two compounds. Three plates were prepared for the solvent control at a concentration of 1%. The 5 bacterial strains were inoculated in duplicate by streaking the cultures across the concentration gradient of each plate using a 10-barreled micropipet. The test was also conducted with metabolic activation using a rat post-mitochondrial fraction (Aroclor 1254-induced $9) which was prepared by Lilly Research Laboratories (Cline and McMahon, 1977; Thompson et al., 1980) or obtained
from a commercial source (Molecular Toxicology, Annapolis, Maryland). The $9 was validated with appropriate positive controls prior to use. The $9 fraction with cofactors was mixed with agar and overlaid onto the chemical concentration gradient just prior to inoculating the bacterial tester strains. In the activated portion of the GPA and Ames assay, final $9 concentrations of 2.7% and 2.0%, respectively, were used. Four plates were also prepared for each positive control used in the activated assay, 2-aminoanthracene and streptozotocin. These plates ranged in concentration from 0.0005 to 5 /~g/ml and 0.1 to 1000 /zg/ml, respectively, for the two compounds. Three plates were prepared for the solvent control at a concentration of 1%. A total of 134 plates was required for testing 14 compounds in the G P A including solvent and positive controls, with and without activation. Plates were incubated for approximately 48 h at 37°C and then examined for the growth of mutant colonies within the lawn of each inoculum streak. The preparation of the gradient assay along with scoring of plates and interpretation of results have been described in detail by McMahon et al. (1979). The results of the GPA were compared to the results from the Ames test which
GRADIENT PLATE ASSAY Pour wedge of minimal agar.
Pour second wedge of minimal agar containing test compound.
Let stand to allow concentration gradient to form.
Pour layer of $9 in agar on top for "activated" plates.
J Five bacterial strains streaked in duplicate over the surface of the agar.
Fig. 1.
187
188
M.A. Rexroat et al. /Mutation Research 341 (1995) 185-192
was conducted according to the methods of Ames et al. (1975), with the necessary modification (inclusion of tryptophan, 0.04 mM) for E. coli WP2uvrA- as previously described (Green and Muriel, 1976). In the Ames assay, 5 bacterial strains, 5 test concentrations, 1 solvent control, and 1 concentration per positive control were tested in triplicate, with and without metabolic activation (210 plates). Revertant colonies were counted using an Artek 880 Automated Colony Counter (Artek Systems Corporation, Farmingdale, NY). A test article was considered to have induced a positive response for bacterial mutation when a concentration-related increase in revertants was observed in which the number of revertants exceeded the value of the vehicle control by at least two-fold (strains TA98, TA100, and W P 2 u v r A - ) or a least three-fold (strains TA1535 and TA1537), for two successive concentrations of the test article. These criteria are consistent with the prevailing attitude of the scientific community (Kier et al., 1986). Since results from the GPA were qualitative rather than quantitative, colonies were only counted when it appeared that the compound may have elicited a weakly mutagenic response. In these cases, the colonies were counted manually.
3. Results and discussion
Over 4000 compounds have been tested in the GPA, the majority of which have been intermediates, i.e., chemicals involved in various stages of product synthesis. However, the compounds in this report also included chemical impurities, research entities, and several marketed compounds. Results for 113 compounds (identical lots) tested in both the Gradient Plate Assay and the Ames assay are shown in Table 1. The list of compounds covers a broad spectrum of chemical classes with a variety of intended uses. The concordance of results from the two test systems is very good (97%). Conflicting results between the two assays were observed for only three compounds (phenobarbital, HC Blue 2, and an antiobesity compound; see Table 2). The
agreement between the assays would have been even greater had more conservative response criteria been utilized (Kier et al., 1986). Furthermore, one inconsistent result (HC Blue 2) may have been attributed to lower test concentrations employed in the GPA. Phenobarbital was mutagenic to strain TA1535 in the Ames assay without metabolic activation, but was negative in the GPA. Although a rodent carcinogen, phenobarbital has generally yielded negative results in a variety of bacterial mutation assays (McCann et al., 1975a; Purchase et al.; 1978; King et al., 1979). Venitt (1985) reported that phenobarbital was difficult or impossible to detect reliably in routine screening assays using S. typhimurium. While phenobarbital was reported to produce weak, but consistent, positive results in the Ames assay (Rexroat and Probst, 1985; Zieger and Haworth, 1985), the response would have been considered negative had the more conservative criteria currently in use been utilized (Kier et al., 1986). HC Blue 2 (HCB2) was mutagenic in the Ames assay with strain TA98 in the presence and absence of liver $9. The results for bacterial mutation observed in the present report show a strong correlation with that reported by the National Toxicology Program (NTP, 1985). The test concentrations producing mutagenicity in the Ames assay were equal to, or greater than, 1000 /xg/plate. At Lilly, the Ames assay has generally been used to test compounds for regulatory submission. In the absence of toxicity or precipitation, a maximum test concentration of 5000 /xg/plate has been established for the Ames assay in order to meet regulatory guidelines. In contrast, the highest test concentration used in the GPA is 1000 /xg/ml which is due to the frequently limited availability of compounds a n d / o r the fact that results are not intended to represent the sole regulatory submission. Therefore, insufficient test article concentrations may be the reason for the negative finding in the GPA. The remaining antiobesity compound produced approximately a six-fold increase in revertants in the Ames assay with strain TA1535 in the presence of liver $9 (Rexroat and Probst, 1985),
M.A. Rexroat et al. / Mutation Research 341 (1995) 185-192 Table
Table
1
Comparison gradient
of results plate
for
bacterial
mutation
in the
Ames
and
189
1 (continued)
Compound
assay
Compound
Ames
GPA
result
result
Acridine
Ames
GPA
result
result
+
+
Biphenyl 2-Aminobiphenyl
~
9-Aminoacridine Alkylating
+
+
a,/3-Blocker
agent
-
-
-
-
-
-
-
-
-
-
Indolidan
-
-
-
-
-
-
Amesergide
Triethylenemelamine
+
a
+
a
Amine -
-
Analgesic
channel
Indecainide
Analgesic/antiinflammatory
-
-
Isomazole
Picenadol
-
-
Quinpirole
Aniline
Positive
o-Toludine
additive
Narasin Narasin/nicarbasin
combination
Nicarbasin
antagonist
Tibenelast 5-1ipoxygenase
enzyme
inhibitor
HCI
HCI inotropic
-
-
Thrombosis
inhibitor
-
-
Sergolexole
maleate
-
-
-
-
Vasodilator
and
-
-
-
-
Zatosetron
-
-
antagonist
-
-
-
Antihypertensive
+
Dopamine
LTD4/E4-receptor
blocker
-
Antiasthma LTD-antagonist
antagonist)
-
Anticoccidial Anticoccidial
(5HT2
Antiischemic Calcium
a-Naphthylamine
inotropic
maleate
5HT3
rec. a
q_ a
agonist
-
-
Quinelorane
-
-
Pergolide
-
-
HCI mesylate
-
-
-
-
Dye
-
-
HC
Blue
No.
1
-
-
Antiasthma
#1
-
-
HC
Blue
No.
2
+
-
Antiasthma
#2
-
-
Lawsone
-
-
Antibiotic
cosmetic
dye
Fungicide
Cycloserine
-
-
Cilofungin
-
-
Daptomycin
-
-
Fenarimol
-
-
Dirithromycin
-
-
Nuarimol
-
-
Erythromycylamine
-
-
Actaplanin
-
-
-
-
Ancymidol
-
-
-
-
Avilamycin
-
-
-
-
Anticancer Oncolytic
potentiator
Sulofenur Anticonvulsant
Growth
promoter
Ractopamine
Anticonvulsant
Fluoxetine
HCI
Norfluoxetine
HCI
-
Benefin
-
-
-
-
2-Chloro-4-fluorobenzophenone
-
-
-
-
2,4-Dichlorobenzophenone
-
Ethalfluralin
+
Flurprimidol
-
Antiestrogen Raloxifene
-
-
Antihistamine
Herbicide
Herbicide
Methapyrilene
HC1
-
Antidepressant
analogue
a
+ -
+
+
-
-
lsoxaben
-
-
Histamine
(HI)
antagonist
-
-
Isoxazoleamine
-
Histamine
(H2)
antagonist
-
-
Oryzalin
-
-
-
-
Antipsychotic
Soybean
PGR
Olanzapine
-
-
Tebuthiuron
-
-
Antipsychotic
-
-
Herbicide
-
-
-
Antitumor
Insulin
Gemcitabine
-
-
Biosynthetic
human
C-peptide
-
Penfenamine
-
-
Biosynthetic
human
insulin
-
-
-
-
Antiulcers
Proinsulin
Nizatidine Nizatidine Aromatic
~
Cardiovascular
sulfoxide
-
-
-
-
amines
Miticides/insecticides Insecticide
fermentation
Miticide
#1
product
-
-
2-Acetylaminofluorene
+
a
"1"- a
Miticide
#2
-
-
2-Aminoanthracene
+
a
_~_ a
Miticide
#3
+
+
+
+
Nitro
compound
2-Nitrofluorene
M~4. Rexroat et al. / Mutation Research 341 (1995) 185-192
190
and comparable test concentrations were used in the GPA. No reason can be offered for the incongruent finding at this time. Recently, changes were made in the G P A procedure to make it identical to that used in the Ames assay (Ames et al., 1975; Rexroat and Probst, 1985). Prior to these changes, subtle differences existed between the two assays with respect to the preparation of bacterial cultures, solutions, and media. Specifically, the maintenance medium was changed from trypticase soy
Table 1 (continued) Compound
Ames result
GPA result
Nitrosoureas and amides
N-Ethyl-N'-nitro-N-nitrosoguanidine
Table 2 Disagreement
in test results for the A m e s a n d g r a d i e n t p l a t e
assays Compound
Ames
GPA
- $9
+ $9
- $9
+ $9
Phenobarbital HC Blue 2
+ a + b
_ + b
_ _
_ _
Antiobesity
_
+ a
_
_
Phenobarbital doses
A m e s - $9
50
24 -+ 3
100
24 + 5
500
42 + 6 a
1000
46 -I- 5 a
5000
50 + 4 a
Solvent
19 _+ 1
Solvent
17 + 4
HC Blue #2 doses
Ames - $9
312.5
32+
9
625
31+
2 4 b
81+
1250
45+
2500
57_+ 4 b
206_+ 16 b
5000
7 6 + 12 b
1 6 9 + 12 b
Solvent
17+
1
34-+
9
Solvent
22 -+ 2
32 +
5
Antiobesity doses
A m e s + $9
+
guanidine
+
+
Streptozotocin
+
+
Safrole
-
-
Benzo[a]pyrene
+ a
+ "
Pyrene
-
-
1000
121 _+ 11 a
1500
1 3 8 + 12 a
+ ~
+ a
2000
99_+
2500
53 + 10 a
Polycyclic a r o m a t i c s
Polyhalogen compound Ethidium bromide Veterinary antibiotic Apramycin
-
-
Desmycosin derivative (Tilmicosin)
-
-
Antiparasitic
+
+
Aromatase inhibitor
-
-
Avocado pit extract
-
-
Benzoin
-
-
Caprolactam
-
-
Diethylstilbestrol
-
-
Dioctyl phthalate
-
-
Ergoline
-
-
Germall II®
-
-
Hexamethylphosphoramide
-
-
Metronidazole
+
+
Omental extract
-
-
Phenobarbital sodium
+
-
Polymer TPH D8/3
-
-
Miscellaneous compound
Unsaturated
milled
sulfone
500
51+
143+
8 b
4 a
1a
Solvent
18_+ 3
Solvent
21 _+ 2
a Positive in strain T A 1 5 3 5 . b P o s i t i v e in s t r a i n T A 9 8 .
+
+
Antiobesity #1
+ ~
-
Antiinflammatory bowel disease
-
-
Antiviral
-
-
Antipsoriatic agent II
-
-
Antiobesity #2
-
-
Metabolic activation with $9 required.
7 b
105+16b
+
N-Methyl-N'-nitro-N-nitroso-
Ames + $9
broth containing 0.5% yeast extract (static 37°C incubation) to oxoid nutrient broth # 2 (shaker water bath at 37°C incubation). The test medium was changed from Difco purified agar to Difco Bacto agar; and the final concentration of histid i n e / t r y p t o p h a n was changed from 0.005 mM to 0.04 mM. It is expected that these modifications should further enhance the agreement between the assays. Re-evaluation of H C Blue 2 and the antiobesity compound could not be done due to a lack of availability of material, however; phenobarbital was re-evaluated using the new methodology in the G P A and Ames assay. Although a different lot from the previous assay, phenobarbital was
M.A. Rexroat et al. / Mutation Research 341 (1995) 185-192
found to be negative for bacterial mutation in b o t h t h e G P A a n d A m e s assay. I n s u m m a r y , t h e G P A is a n i d e a l p r e s c r e e n f o r potential bacterial mutagenicity. The distinct advantages of the GPA over the standard Ames a s s a y a r e : (1) 14 c o m p o u n d s a r e t e s t e d in t h e s a m e a m o u n t o f t i m e t h a t is r e q u i r e d f o r t e s t i n g o n e c o m p o u n d i n t h e A m e s ; (2) t h e G P A r e q u i r e s o n l y 35 m g o f t e s t a r t i c l e as c o m p a r e d t o 4 0 0 m g f o r a n A m e s assay; a n d (3) t e s t i n g o n e compound for an Ames assay requires 210 plates, w h e r e a s t e s t i n g 14 c o m p o u n d s i n t h e G P A r e q u i r e s o n l y 134 p l a t e s . S i n c e a l a r g e n u m b e r o f c o m p o u n d s c a n b e e x a m i n e d in a t i m e l y f a s h i o n , this allows for early detection of bacterial mutag e n s a n d a s s i s t s in s e t t i n g s a f e t y s t a n d a r d s i n t h e l a b o r a t o r y f o r e m p l o y e e s w h o m i g h t c o m e in c o n tact with potential genotoxins.
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Maron, D.M. and B.N. Ames (1983) Revised methods for the Salmonella mutagenicity test. Mutation Res., 113, 173-215. McCann, J. and B.N. Ames (1977) The Salmonella/microsome mutagenicity test: predictive value for animal carcinogenicity, in: H.H. Hiatt, J.D. Watson and J.A. Winsten (Eds.), Origins of Human Cancer, Cold Spring Harbor Laboratory, NY, pp. 1431-1450. 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. (USA), 72, 5135-5139. McMahon, R.E., J.C. Cline and C.Z. Thompson (1979) The assay of 855 test chemicals in ten tester strains using a new modification of the Ames test for bacterial mutagens. Cancer Res., 39, 682-693. National Toxicology Program (1985) Toxicology and Carcinogenesis Studies of HC Blue No. 2 in F344/N rats and B6C3F~ mice, U.S. Department of Health and Human Services, Tech. Rep. Ser., No. 293. Nestmann, E.R., R.L. Brillinger, M.F. McPherson and K.L. Maus (1987) The simultest: a new approach to screening chemicals with the Salmonella reversion assay. Environ. Mol. Mutagen., 10, 169-181. Probst, G.S., R.E. McMahon, L.E. Hill, C.Z. Thompson, J. Epp and S.B. Neal (1981) Chemically-induced unscheduled DNA synthesis in primary rat hepatocyte cultures: a comparison with bacterial mutagenicity using 218 compounds. Environ. Mutagen., 3, 11-32. Purchase, I.F.H., E. Longstaff, J. Ashby, J.A. Styles, D. Anderson P.A. Lefevre and F.R. Westwood (1978) An evaluation of six short-term tests for detecting organic chemical mutagens. Br. J. Cancer 37, 873-959. Rexroat M.A. and G.S. Probst (1985) Mutation tests with Salmonella using the plate-incorporation assay, in: J. Ashby et al. (Eds.), Progress in Mutation Research, Elsevier, Amsterdam, pp. 201-212. Rinkus, S.J. and M.S. Legator (1979) Chemical characterization of 465 known or suspected carcinogens and their correlation with mutagenic activity in the Salmonella typhimurium system. Cancer Res. 39, 3289-3318. Shelby, M.D. (1988) The genetic toxicity of human carcinogens and its implications. Mutation Res. 204, 3-16. Shelby, M.D. and Zeiger (1990) Activity of human carcinogens in the Salmonella and rodent bone-marrow cytogenetics tests. Mutation Res. 234, 257-261. Sugimura, T., T. Kawachi, T. Matsushima, M. Nagao, S. Sato and T. Yahagi (1977). A critical review of submammalian systems for mutagen detection, in: Progress in Genetic Toxicology (D. Scott, B. A. Bridges and F.H. Sobels, Eds.), Elsevier/North-Holland, New York, pp. 125-140. Thompson, C.Z., G.W. Whitaker and R.E. McMahon (1980) Effect of Aroclor 1254 dose upon S-9 activation of aromatic amine bacterial mutagens. Mutation Res. 77, 361366. Thompson, C.Z., L.E. Hill, J.K. Epp and G.S. Probst (1983) The induction of bacterial mutation and hepatocyte un-
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