Mutagenicity Testing

 

EBPI Analytics offers a large selection of traditional Ames tests to meet any research requirement. There are multiple options offered for these assays, and clients can choose from different bacterial strains, developed for specific sensitivities to mutation type. We also offer both Salmonella and E. coli test bacteria which allows standardization of our assays to previously established test protocols depending on client requirements. Clients can also choose between three assay methods including a standard plate assay, fluctuation test, and a modified ISO procedure. The assay choices promote flexibility depending on personal preference, budget, application, or experimental design. We also offer optional addition of S9 fraction rat liver extracts to metabolically bioactivate genotoxins, which increases the number of mutagenic compounds detected by the Ames assays.

Options for Ames Tests 

Name of Ames Test	Description of Ames Test	S9 Availability	Comments
Ames Test	Traditional Ames Test based on the Pour Plate Method	S9 +/-	Pre-exposure of the bacteria for 30-100 minutes  3 day assay Maximum sample per test is 2mL
Muta-ChromoPlate Kit	96 well Microplate format Ames Test	S9 +/-	No pre-exposure, bacteria are exposed during the duration of the assay 5-7 day assay Maximum sample per test is 17.5mL S9- 15.5mL S9+
Ames ISO	384 well Microplate format Ames Test	S9 +/-	Pre-exposure of the bacteria for 30-100 minutes 3 day assay Maximum sample per test is 2ML
Modified ISO	48 well format run in a 96 well Microplate	S9 +/-	Pre-exposure of the bacteria for 30-100 minutes 3 day assay Maximum sample per test is 2 mL

 

Test Method Options

EBPI Analytics also offers three testing methods for our mutagenicity assays that can be chosen depending on application and budget.

Standard Ames Test Procedure

For the standard Ames test, the bacterial strains are pre-exposed to the test compound for 100 minutes in the presence or absence of the S9 fraction. The reaction mixture is then poured onto an agar plate. The agar plate contains biotin and a minimal amount of histidine. The bacteria are able to undergo a few rounds of cell division before the histidine runs out. After an incubation period of 2-3 days, the colonies are counted and tabulated as the number of revertants per plate. Comparisons are made between growth in the control and exposed plates.

• Uses 2 mL of sample (small sample sizes)
• Quantification is simple (counting)
• Concentration of samples may be required
• Most widely used mutagenic test and used as the industry standard.

 

Modified ISO Procedure

This procedure uses a modification of the Ames fluctuation assay. Bacterial strains are pre-exposed to differing concentrations of the mutagenic compounds with a growth media in suspension for 100 minutes. Each dilution is run in triplicate. The samples are then transferred, mixed with a reversion solution and plated onto a 96 well plate. The samples are incubated for 2-3 days and the number of revertant colonies (individual wells) are counted by colorimetric analysis.

• Uses small sample size
• Bacterial concentration is checked by optical density (ensures log growth phase)
• Quantification is simple and fluctuation test scoring is quick and easy
• Assay is more sensitive than the plate-incorporation assay
• Utilizes pre-exposure period to enhance bacterial uptake
• More consistent exposure concentration during auxotrophic growth phases
• Concentration may be required and exposure time may be extended to increase the uptake and positive results.

 

MUTA-ChromoPlate Procedure

This is the simplest procedure of the three Ames assays and utilizes the fluctuation assay procedure. The test solution is mixed directly with the bacterial strains and the reaction mixture and the entire solution is allowed to incubate for 3 to 5 days at 37 °C. The plates are removed and revertant colonies are counted by colorimetric changes.

• Large sample sizes (17.5 mL, ideal for waste water treatments)
• No pre-exposure period, exposure and uptake occurs over the duration of the test.
• No concentration of the sample is necessary
• Assay is more sensitive than the plate incorporation assays
• Exposure concentration is kept consistent during auxotrophic growth phase

 

Ames Test Bacteria Strain Options

Salmonella typhimurium: Histidine dependence

Strain	Mutation Site (gene)	Type of Reversion Mutation	Standard Mutagens	Comments
TA 100	hisG46	Base-pair substitution (specific for G:A transitions, also detects G:T and G:C transversions)	NaN3/ NQO	uvr B repair deficient, with rfa mutation to increase permeability includes plasmid pKM 101which induces an error-proneDNA repair pathway and inhibits proper DNA replication
TA 98	hisD3052	+1 and -2 frameshifts	2NF	uvrB repair deficient, with rfa mutation to increase toxicant permeability contains plasmid pKM 101, induces an error-prone DNA repair pathway and inhibits proper DNA replication
TA 97a	his6610	-1 and +1 frameshifts	9AA	uvr B repair deficient, with rfa mutation to increase permeability includes plasmid pKM 101 which induces an error-prone DNA repair pathway and inhibits proper DNA replication contains extra mutational hotspot to detect similar mutations to TA98 strain
TA 1535	hisG46	Base-pair substitution (specific for G:A transitions, also detects G:T and G:C transversions)	NAN3	uvr B repair deficient, with rfa mutation of the cell wall to increase permeability similar mutations detected as TA100, but lacks pKM 101 plasmid

 

Escherichia coli: Tryptophan dependence

Strain	Mutation Site (gene)	Type of Reversion Mutation	Standard Mutagens	Comments
WP2 uvrA	trpE65	Base-pair substitution (G:C to A:T)	4NQO	uvr A deletion mutation eliminates accurate excision repair mechanism more DNA lesions repaired by error-proned system
WP2 uvrA pKM 101	trpE65	Base-pair substitution (G:C to A:T)	4NQO	enhances chemical and UV mutagenisis by conferring supplementary error-prone DNA repair activity in pKM 101 plasmid

 

In addition to the traditional assays offered, EBPI Analytics is also offering newly developed technologies and protocols to provide the most sensitive mutagenic assessments available. We are very excited to provide testing services utilizing our recently engineered Ames-Express bacterial strains.

Ames-Express

Mechanistically, many known toxicants rely on bioactivation pathways to produce deleterious effects. The incorporation of metabolic enzyme systems into mutagenicity assays has significantly increased the biological relevance and sensitivity of these test systems to multiple classes of carcinogens. Previously, metabolic enzymes from other sources like rat liver extract were used to convey this functionality and broaden the applications of the Ames assay. EBPI Analytics still offers this testing option, and we provide S9 fraction addition with all of our traditional Ames assays.

At EBPI, we have significantly improved this bioactivation process for bacterial assays by engineering the expression of two human metabolic enzymes (CYP 450 1A2 and GST T1-1) into our Ames test bacteria which eliminates the addition of rat liver enzymes (S9 fraction) to the experiment. Our systems have demonstrated increased sensitivity, as bioactivation takes place within the bacteria, eliminating suppression due to toxicant sequestration in the S9 components, and minimizing diffusion distance of the reactive metabolite to its DNA target. Furthermore, these assays are more relevant to human health since they employ human enzymes. They have also shown incredible promise in detecting unique classes of environmental contaminants within different sample matrices.

For more information on these new Ames-Express strains, please click here or visit www.biotoxicity.com.

 

Ames Test Information 

Bacterial mutagenicity testing was pioneered in the late 1960s and early 1970s by Bruce Ames and Phil Hartman, who worked on histidine regulation in Salmonella typhimurium using histidine auxotrophic mutants. In 1972 Bruce Ames published one of the first papers on mutagenesis in bacteria (Ames et al. 1972) and used modified precursor strains that have stood the test of time and are still applied today.

The mechanistic basis of mutagenicity testing is the exposure of a compound to a set of bacterial strains, defective in histidine synthesis, but sensitive to chemical- or radiation–induced mutagenesis that results in a reversion to prototrophy i.e., independence from histidine supplementation.

Histidine auxotrophic Salmonella bacteria are unable to proliferate unless they are provided with histidine. They can, however, undergo mutagenesis that reverts the bacteria to the genetic wild type. Sensitive sites for mutational reversion (hot spots) were selected from three genes on the his operon: hisC, coding for the histidinol phosphate aminotransferase, hisD, a bifunctional histidinal and histidinol dehydrogenase, and hisG, which codes for a histidinol-phosphate aminotransferase.

The test design involves a pre-exposure period followed by plating the bacteria on an agar plate that lacks histidine. This design has been used extensively in mutagenicity studies and is still the basis for current mutagenicity testing with some procedural modifications. The bacteria that undergo reverse mutation to a protrophic state following test compound exposure can grow, and the revertant mutants can simply be counted. Another endpoint for this assay measures the expression of a colorimetric enzyme system from the growing bacteria.

Further modifications to these original strains have been shown to increase the sensitivity to chemical mutagens, as well as the applicability of the original Ames test. 

The uvrB deletion mutation eliminates an accurate excision repair mechanism, and promotes DNA lesion repair by error-prone mechanisms. However, this deletion also affects a gene responsible for biotin synthesis, therefore the bacteria are biotin dependant and require it in the growth media.

The rfa mutation leads to a defective lipopolysaccharide (LPS) layer that coats the bacterial surface. The permeability of the bacterial cell wall is affected and is more susceptible to entry from bulky toxicants.

Plasmid pKM101 enhances chemical and UV-induced mutagenesis via an increase in the recombination DNA repair pathway. The plasmid also confers ampicilline resistance, which provides a convenient marker to detect its presence. It induces an error-prone DNA repair pathway and inhibits proper DNA replication, further sensitizing the bacteria to mutagenic insult.

S9 Fractions are used in conjunction with the bacterial strains to confer metabolic activation to the mutagenic assays. Rat liver homogenate is collected by centrifugation and is largely composed of the microsomes which contain CYP 450 enzymes. This enzyme family is responsible for bioactivating many chemical mutagens, and increases the positive responses to the standard Ames test by mimicking normal metabolic processes in mammalian systems. Should you require S9 activation with your traditional Ames test, check the required box on the COC and EBPI Analytics will run a sample of your compound with and without S9 addition for comparison purposes.

According to the OECD guidelines for the testing of chemical substances, usage of at least five test strains (TA 1535, TA 1537 or TA 97, TA 98, TA 100 and TA 102) is recommended for comprehensive detection of chemical mutagens. An alternative for TA 102 is the repair proficient E. coli WP2 strain. A combination of TA 98 and TA 100 strains is used under conditions of reduced assay time, or necessity of labor efficient testing, and covers an extensive range of mutagens. The ISO standard for water testing (ISO 16240) uses this reduced strain combination.

Please specify to EBPI Analytics the bacterial strains you would like to use for your assays as positive results may depend on the type of mutation created as well as the content of your samples. Furthermore, depending on the destination of the information collected, specific strains may be required to validate the results according to international standards.