Ames MPF Mutagenicity Assay:

Scientific Background

and Practical Procedure

Dr. Markus Kamber

Xenometrix AG

Gewerbestr. 25

CH-4123 Allschwil

Switzerland

Overview

• Principles of the Ames Assay

• General Features Ames MPF

• Differences Ames / Ames MPF

• Advantages

• Versions available

• Validation Studies

The Ames Assay

The most widely used bacterial test to detect mutagenic

compounds.

Required by regulatory authorities for registration of new

medicines: OECD guideline 471; ICH guidelines (Specific

Aspects of Regulatory Genotoxicity Tests for

Pharmaceuticals, S2a)

The Ames Assay (2)

Based on several auxotrophic his- Salmonella

typhimurium and trp- E. coli strains:

Strains have a defect in one of the histidine/tryptophan -

synthesizing enzymes and can not grow in the absence of

histidine/tryptophan.

Base-pair and/or frameshift mutations can correct

(=revert) these defects and allow the revertants to

synthesize histidine/tryptophan and grow under minimal

nutrient conditions.

Reversion

Mutagenic event

No growth in

minimal medium

Growth in

minimal medium

his- his+

The Ames Assay (3)

• Upon exposure to mutagenic compounds reversion to

histidine prototrophy can occur.

• Such bacteria can then grow in media/agar that does not

provide external histidine.

• Classic Ames test performed by counting colonies growing

on agar plates.

Mutagen

Minimal agar plates

The Ames Assay (4)

• Minimally run with 1 base-pair strain (e.g.TA100) and 1

frame-shift strain (e.g.TA98) (Environmental samples)

• Full analysis typically uses up to 5 strains

(pharmaceutical samples)

S9 Liver Microsome Fraction

• Added to mimic mammalian metabolism

• Compounds can be de-toxified or become mutagenic

under the action of S9 enzymes

Disadvantages / Inconveniences

of the classic Ames assay

• Amount of test substance

• Amount of plasticware

• Maintenance/Quality testing of tester strains

• Time for media preparation

• Hands-on time

• Colony counting

Ames MPF Assay

Same principle and same strains as traditional Ames

but using a liquid low volume multiwell format instead

of agar plates

• Main advantages:

-less test sample

-enables high-throughput analyses.

Practical Advantages of Ames MPF

over Agar Plate Method

• Less sample or extract necessary

• Liquid microplate format: easy handling

⇒ Multichannel pipettes, less hands-on-time,

simultaneous processing of several replicates

• Ready-to-use media, strains, S9, positive controls

• Less S9 consumption (13x)

• Higher throughput, partly automatable

• Colorimetric read-out

• Less plastic ware, reduced disposal costs

Ames Microplate Assays

Indicator

Medium

Test Compound

Culture

Stored at –80°C

Overnight

Culture Assay Preparation

37°C, 11 –15-h

250 rpm

OD600

Exposure Culture

24-Well Plate

37°C, 90 min, 250 rpm

Bacteria Culture

S9-Mix

Exposure Medium

C-

D1

D2

D3

D4

D5

D6

C+

TAxxx

A

B

C

D

C-

D1

D2

D3

D4

D5

D6

C+

A

B

C

D

C-

D1

D2

D3

D4

D5

D6

C+

384-Well Plate

48 h

3 7 Co

Ames MPF Assay

Bacterial growth

pH

Indicator Medium Indicator Medium

• Plate incorporation vs. liquid culture

Experimental Differences:

Ames MPF Test - Ames Plate Test

1 conc. per plate

8 concentrations per plate

Experimental Differences (2):

Ames MPF Test - Ames Plate Test

• Colony counting vs. colorimetry

Positive Control Negative Control

Negative Control

Positive Control

Experimental Differences (3): Hands-on-time for 96 Replicates

Traditional Ames Ames MPF (using ready-to-use agar plates)

Sample dilutions: 5 min 5 min

Top agar (preparation of tubes): 15 min -

Addition of sample, culture, S9: 25 min 10 min

Plating: 15 min -

Transfer to 384-well plates: - 15 min

Counting (by eye): 30-100 min ~8 min

e.g 1 sample at 6 concentrations, 2 strains (TA98/TA100), negative and

positive control, -/+ S9, triplicates

Total time: ~1½-2½ h ~40 min

Plate Counting Time

8

30-100 20-60 sec per plate = 1 replicate

40 sec per plate = 8 replicates

TA98 +S9

TA100 +S9

TA100 -S9

TA98 -S9

Comparison Ames Plate Incorporation and Ames MPF:

Minimum Amount of Sample and S9 Needed for Testing

Ames MPF: 3.4-fold less test compound

almost 10-fold less S9

Very important for substances in early phases of

drug development!

Setup: 2 strains, 6 concentrations, ½ log dilution steps, triplicates, -/+ 30% S9 mix

Ames plate incorporation:

5 mg 2 mg 1 mg/plate

90 36 18

7.2 7.2 7.2

Ames MPF:

5 mg 2 mg 1 mg/ml

26.3 10.5 5.3

0.75 0.75 0.75

Top dose:

Compound (mg):

S9 fraction (ml):

Summary: Ames MPF Kits /Strains

• Ames II • Ames MPF 98/100

• Ames MPF 1535, 1537, E.coli

• Ames Penta I

• 98

• 100

• 1535

• 1537

• E.coli Combo

Screening

Special chemical classes

OECD 471

Water analysis • Ames MPF 98/100 AQUA

Ames MPF Kit Versions

• Ames MPF AQUA (Salmonella)

Aqua: non-concentrated water samples

(strongly polluted water bodies)

CS: Concentrated water or

pharmaceuticalsamples

(low level contaminant mixtures)

• Ames MPF CS (Salmonella, E.coli)

Ames MPF 98/100

CS and AQUA Exposure

Ames MPF CS conc. samples

1X Exposure medium

Test sample

Ames MPF AQUA Non-conc. samples

10X Exposure medium

Test sample

S9 mix

Bacteria

S9 mix

Bacteria

Test sample dilution: 1:25

Final concentration in the assay: 4%

Test sample dilution: 1:1.35

Final concentration in the assay: 74%

Summary

• Ames MPF - Ames traditional: same principle

• Same tester strains

• MPF: based on pre-incubation and fluctuation

method

• MPF: less test sample, less S9

• MPF: many practical advantages

• Many studies comparing the 2 test protocols

⇒ excellent concordances

Conclusion

• Based on the large database there is clear evidence

that the Ames MPF is as capable as the standard

Ames at detecting known mutagens. The MPF

method works excellently even with questionable to

weakly positive chemicals.

• The standard plate method requires large amounts

of test sample and is less convenient than the

liquid microplate method for testing water

samples. The MPF method is therefore a useful

alternative.

Any questions?

Thank you !

www.xenometrix.ch

Mutagenicity Testing Example: Switch from Traditional Ames to Ames MPF

Formerly: Ames plate test

• XAD-SPE extraction at pH 7 and 2

• conc. factor 25’000

• 2 mL extract ⇒ 50 L water

• 3 samples at a time

Now: Ames MPF

• smaller assay volumes ⇒ less extract necessary

• possible with Oasis-HLB-SPE cartridges ⇒ conc. factor 10’000

• 200 µL extract ⇒ 2L water

• 32 samples at a time

• enables analysis of water from at laboratory scale

• less work to perform

0

10

20

30

40

50

60

1

Lit

ers

2 L

50 L

Direct Comparison

Ames MPF - Ames Pre-incubation

• Same overnight cultures, chemicals and S9 to exclude external

variations, i.e. culture growth, chemical purity, weighing errors, S9

activity.

• Parallel tests with most responsive strains of the NTP database

• Each test repeated at least once

• 87% concordance (13/15)

• Excellent concordance for questionable to weak positive chemicals

• Demonstrates validity and robustness of the liquid microplate format

Table 5: Overall test results with selected strains

Chemical Name Strain(s) S9 mix MPF

method

Preinubation

method

Average of

NTP database*

Anthracene TA100 ≥20% w+ w+ eq

1,2,3-Benzotriazole TA1535 10-30% neg pos w+ (10% S9)

Carminic acid TA100 no S9 neg neg w+

Danthron TA100, TA1537 30% pos pos eqa

1,2-Dichloropropane TA1535 no S9 neg neg eq

2,3-Dideoxyadenosine A:T strains 30% eq b eq

b pos

c

1,3-Diphenylguanidine TA1535, TA1537 -/+10% pos neg eq

Emodin TA100, TA1537 10-30% pos pos w+a

Epinephrine TA100, TA1537 no S9 w+ w+ w+

Glutaraldehyde TA100, A:T strain -/+10% pos d: pos

d: w+

e

Maltol TA1535 10-30% pos w+ w+

2-Nitroethanol TA98, TA100, WP2 [pKM101] no S9 eq eq w+

Orthanilic acid TA98 no S9 neg neg eq

Phenanthrene TA100 30% pos w+ pos

Pyrene TA1537 10-30% pos pos w+

neg, negative; eq, equivocal; w+, weak positive; pos, positive

Validation Studies: Ames II vs. classic

Ames

• P. Gee et al., Mutation Res. 412, p115-130.(1998):

24 compounds

Concordance 79 %

• V. Gervais et al. (Servier) EEMS 2003 Poster

42 compounds

Concordance 83 %

• Flückiger et al., Mutation Res. 558, p. 181-187. (2004)

19 compounds

Concordance 84%

Inter-laboratory consistency 89.5%

Validation Studies: Ames II vs. classic

Ames

Kamber M., Flückiger S., Engelhardt G., Jaeckh R. and

Zeiger E.. Mutagenesis (2009) 24, p359-366:

Comparison of the Ames II and traditional Ames test

responses with respect to mutagenicity, strain specificities,

need for metabolism and correlation with rodent

carcinogenicity.

84% agreement between the two procedures in identifying

mutagens and non-mutagens, which is equivalent to the intra- and

interlaboratory reproducibility of 87% for the traditional test

Validation Studies: Ames II vs. classic

Ames

Umbuzeiro Gde A, Rech CM, Correia S, Bergamasco

AM, Cardenette GH, Flückiger-Isler S, Kamber M.

Environ Mol Mutagen. (2010) 51,31-8:

Comparison of the Salmonella/microsome microsuspension

assay with the new microplate fluctuation protocol for

testing the mutagenicity of environmental samples

The mutagenic potencies... correlated well when tested in both

assays. Because the Ames MPF assay is easier to perform..., it

seems to be an interesting and valid alternative to the

microsuspension assay especially when a large number of

samples have to be tested, such as in monitoring programs and

EDA studies

Validation Studies: Ames MPF™

• S. Flückiger and M. Kamber, EEMS 2006 poster:

Ames II vs. Ames MPF: 14 compounds

Concordance 93 % (13/14)

• S. Flückiger and M. Kamber, SOT 2007 poster:

Ames MPF (TA98; TA100, TA1535, TA1537)

vs.published data, 24 compounds

Concordance ≥ 89 %

Ames II Salmonella typhimurium

Strains

TA98 Frame shift mutations rfa uvrB pKM101

TAMix Base pair substitutions rfa uvrB pKM101

TA7001 A:T G:C …

TA7002 T:A A:T ...

TA7003 T:A G:C ...

TA7004 G:C A:T ...

TA7005 C:G A:T ...

TA7006 C:G G:C ... rfa increased permeability for bulky chemicals

uvrB deficiency in DNA excision repair pKM101 plasmid that allows for error-prone DNA repair

Ames MPF S. typhimurium and E. coli strains

TA98 hisD3052 Frameshift mutations rfa uvrB pKM101

TA100 hisG46 Base-pair substitutions rfa uvrB pKM101

TA1537 hisC3076 Frameshift mutations rfa uvrB -

TA1535 hisG46 Base-pair substitutions rfa uvrB -

E. coli wp2 strains:

uvrA trpE65 Base-pair substitutions - uvrA -

pKM101 trpE65 Base-pair substitutions - - pKM101

rfa increased permeability for bulky chemicals

uvrB/uvrA deficiency in DNA excision repair

pKM101 plasmid that allows for error-prone DNA repair