The Ames test is a mutagenesis test that consists in the detection of mutations by a bacteria that is histidine-dependant: Salmonella typhimurium. Several strains of this bacteria, all carriers of a mutation in the his operon can be used (cfr. table 1). The strains TA 97a, TA 98, TA 100 and TA102, auxotrophs for histidine (His-) can revert spontaneously to His+, and thus grow in a histidine-free medium. This very weak spontaneous reversion can be increased by mutagens, which allows the quantification of the mutagenic potential of these substances.
Table 1. Genotypes of bacterial strains widely used in the Ames test
| Strain | amino acid marker | other relevant mutations | |||
| histidine mutation | type of mutation | main DNA target | cell-wall | DNA-repair | |
| TA 97a | hisD6610 | frameshift | GC | rfa | uvrB |
| TA 98 | hisD3052 | frameshift | GC | rfa | uvrB |
| TA 100 | hisG46 | base pair substitution | GC | rfa | uvrB |
| TA 102 | hisG428 | base pair substitution | AT | rfa | + |
Unlike mammals, these bacteria lack the necessary oxidative enzyme systems for metabolizing foreign compounds to electrophilic metabolites capable of reacting with DNA. The bacteria are therefore treated with the test compound in the presence of a post-mitochondrial supernatant ('S9' or 'microsome fraction') prepared from livers of mammals (usually rats). The metabolic activity of S9 (predominantly mono-oxygenase activity mediated via the cytochrome P-450/P-448 system) is enhanced by treating the rats with a potent inducer of drug-metabolizing enzymes before they are killed and their livers are removed. The S9 is buffered and supplemented with the essential co-factors NADP and glucose-6-phosphate to form 'S9-mix'.
Bacteria, the test compound
and S9 mix are added to molten, diluted agar ('top agar' to which
a trace of histidine has been added) which is then mixed and poured onto the surface of a
minimal-agar plate containing glucose ('bottom-agar'). The thin layer of
top-agar immediately sets. The plates are incubated at 37°C for 2-3 days.
In the first few hours, the whole population of auxotrophic bacteria grows in
the presence of the test compound and the metabolically active S9 until all the
histidine is used up. Parallel assays without S9 are included, to allow for
detection of directly-acting test compounds whose mutagenicity may be
decreased or abolished by metabolisation or by the scavenging action of S9
constituents.
After this phase of auxotrophic growth, only those cells which sustained mutations which reversed the existing histidine mutation will continue to grow to form visible colonies of prototrophs ('his+ revertants) , each colony descending from a single revertant bacteria.
An increase in revertant colonies related to increasing dose of the test compound indicates a mutagenic response.
This test can be used
for the detection of mutagenic effects of individual test substances, mixtures
and extracts.
The detection of mutagenic compounds in urine with the Ames test allows us to assess the systemic production of potentially mutagenic compounds by the body, without identification of either the mutagen, the concerned organ(s), and the in vivo genotoxic effects.
- preparation and
conservation of samples:
urinary extracts are placed in DMSO (dimethylsulfoxide) solution) and kept at
-80°C
minimal volume: 100 µl (25ml non-concentrated urine)
- used bacterial strains:
TA 98 -S9 mix: used for the detection of environmental mutagens
TA102: used for the detection of cytostatic drugs
- Ames test (De Méo et al., 1995; 1996):
The strains are grown in Oxoid Nutrient Broth No.2. After a 12h incubation period at 37°C with shaking, the following ingredients are placed in polystyrene tubes: various volumes of concentrated urine samples, 0.1 ml of the metabolic fraction (S9 mix, for TA98 only) and 0.1 ml of the 12h culture. 4 doses of urine are tested. The mixtures are incubated at 37°C for 60 min with rapid shaking. Then 2 ml volumes of melted top agar containing 0.045 mM histidine and biotin were added to the agar plates. After 48h incubation the revertants are counted on each plate with a laser colony counter equipped with a bacterial enumeration program.
The analysis of urine with the Ames test will be performed in collaboration with the Unité de Toxicologie Industrielle et Médecine du Travail of the Université catholique de Louvain (Prof. Dr. D. Lison) and the Laboratoire de Biogénotoxicologie & Mutagenèse Environmentale of the Université de la Méditerranée (Marseille, France, Dr. M.P. De Méo).
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