Ames试验

The Bacterial Reverse Mutation Assay (Ames Test): A Cornerstone of Mutagenicity Screening

Introduction and Purpose: The Bacterial Reverse Mutation Assay, universally known as the Ames Test after its principal developer Dr. Bruce Ames, is a fundamental in vitro assay employed globally for the rapid, sensitive, and cost-effective detection of chemical mutagens. Its primary purpose is to identify substances capable of inducing genetic mutations in bacterial DNA. As many mutagens are also carcinogens, this test serves as a crucial initial screen in predicting the potential carcinogenic hazard of chemicals, pharmaceuticals, food additives, pesticides, industrial compounds, and environmental contaminants. It plays a vital role in safety assessment and regulatory toxicology.

Scientific Principle: The test exploits specific strains of the bacterium Salmonella typhimurium (and sometimes Escherichia coli) that possess defined mutations in genes essential for histidine biosynthesis. These mutations render the bacteria auxotrophic for histidine – meaning they cannot synthesize this essential amino acid and thus cannot grow and form visible colonies on a culture medium lacking histidine.

• Reverse Mutation (Revertant): When a mutagenic chemical interacts with the DNA of these bacteria, it can induce a second mutation ("back mutation" or "reversion") precisely at the site of the original mutation (or sometimes suppressing it). This reverse mutation restores the functional capability of the gene.
• Detection of Mutagens: Bacteria that undergo this reverse mutation regain the ability to synthesize histidine. Consequently, they can grow and form visible colonies on a minimal agar medium lacking histidine. The significant increase in the number of these revertant colonies (histidine-independent colonies) in plates treated with the test chemical compared to untreated control plates provides clear evidence of the chemical's mutagenic potential.

Key Components:

1. Bacterial Tester Strains: Specific strains are used, each sensitive to different types of genetic damage:

   • Base-pair substitution mutations: Strains like TA100, TA1535.
   • Frameshift mutations: Strains like TA98, TA1537, TA1538.
   • Enhanced Sensitivity: Strains carry additional mutations:
     • rfa mutation (defective lipopolysaccharide layer): Increases permeability to large molecules.
     • uvrB mutation (deficient in excision repair): Prevents repair of DNA damage, increasing sensitivity.
     • Plasmid pKM101 (in some strains like TA98, TA100): Enhances error-prone DNA repair.

2. Metabolic Activation System (S9 Mix):

   • Many chemicals are not mutagenic themselves (procarcinogens) but become biologically active mutagens (ultimate carcinogens) only after metabolic transformation within a mammalian system (e.g., liver).
   • To simulate mammalian metabolism in vitro, a post-mitochondrial supernatant fraction (denoted S9) is prepared from the livers of rodents (typically rats) pre-treated with enzyme-inducing agents (like Aroclor 1254 or phenobarbital/β-naphthoflavone).
   • This S9 fraction, supplemented with cofactors (NADPH, glucose-6-phosphate, etc.), is mixed with the bacteria and test chemical to provide metabolic activation. Testing is always performed both with and without S9 mix.

3. Test Article: The chemical or mixture under investigation, dissolved or suspended in a suitable solvent.

4. Culture Media:

   • Minimal Glucose Agar Plates: Lack histidine (and tryptophan for E. coli strains). Only revertant bacteria can grow.
   • Nutrient Broth: For growing bacterial cultures.
   • Top Agar: A soft agar containing trace histidine/biotin, poured onto minimal plates. The trace histidine allows limited bacterial growth (forming a background lawn) but not enough for colony formation unless a reversion event occurs.

Standard Procedure:

1. Strain Preparation: Overnight cultures of the selected tester strains are prepared.
2. S9 Mix Preparation (if applicable): S9 fraction is combined with cofactors immediately before use.
3. Dosing and Incubation:
   • The test chemical (at various concentrations), a small aliquot of bacterial culture (typically 0.1 ml containing ~10^8 cells), and either buffer (for -S9) or S9 mix (for +S9) are combined.
   • This mixture is added to approximately 2 ml of molten top agar (maintained at 45°C), mixed gently, and poured immediately onto the surface of a minimal glucose agar plate.
   • The top agar solidifies quickly.
4. Incubation: Plates are inverted and incubated in the dark at 37°C for approximately 48-72 hours.
5. Counting Revertant Colonies: After incubation, the number of revertant colonies growing on each plate is counted manually or using automated systems.

Interpretation of Results:

1. Positive Control: Mutagenic reference chemicals (e.g., sodium azide for TA100 -S9, 2-nitrofluorene for TA98 -S9, 2-aminoanthracene for TA98/TA100 +S9) must produce a significant increase in revertants, confirming strain sensitivity and S9 activity.
2. Negative (Solvent) Control: The solvent used must not significantly increase revertants above the spontaneous background level for each strain.
3. Spontaneous Revertant Count: Each strain has a characteristic range of spontaneous revertant colonies observed on negative control plates (background mutation rate).
4. Test Article Assessment: For the test article to be considered positive for mutagenicity:
   • Significant Increase: There must be a statistically significant and dose-dependent increase in the mean number of revertant colonies per plate compared to the concurrent solvent control.
   • Reproducibility: The increase should be observed in at least one strain, with or without S9 activation, and ideally reproducible.
   • Threshold: Often, a doubling of revertants over the control (or exceeding historical control ranges significantly) is a common benchmark, though rigorous statistical analysis is essential.

Applications and Significance:

• Early Screening: First-tier test for mutagenicity in drug discovery, chemical development, and product safety evaluation.
• Regulatory Requirement: Mandated by international guidelines (e.g., OECD 471, EPA, ICH) for the safety assessment of pharmaceuticals, pesticides, industrial chemicals, and food additives.
• Structure-Activity Relationship (SAR): Identifying structural features associated with mutagenicity.
• Environmental Monitoring: Screening environmental samples (water, soil, air extracts) for potential mutagens.
• Mechanistic Studies: Understanding mechanisms of chemical carcinogenesis.

Critical Considerations and Limitations:

• False Positives/Negatives: Can occur. Cytotoxicity can mask mutagenicity or sometimes cause artifactual increases. Highly reactive or volatile compounds may pose challenges.
• Bacterial vs. Mammalian Systems: While S9 mix simulates metabolism, it's not identical to intact mammalian cells. Differences in uptake, detoxification, DNA repair, and chromosome structure exist. A positive Ames test indicates potential mammalian genotoxicity/carcinogenicity, requiring further investigation (e.g., mammalian cell assays, in vivo studies). A negative result does not guarantee safety but significantly reduces concern.
• Strain Specificity: Different strains detect different mutation types. Using multiple strains is crucial for broad detection.
• Non-Genotoxic Carcinogens: Carcinogens acting through non-genotoxic mechanisms (e.g., hormonal, cytotoxic promotion) will not be detected.
• Dose Selection: Testing appropriate concentrations, including cytotoxic levels (where reduction in background lawn is visible), is critical. Overly toxic doses can invalidate results.
• Compound Characteristics: Poor solubility or reactivity can complicate testing.

Conclusion:

The Ames Test remains an indispensable tool in genetic toxicology. Its simplicity, speed, sensitivity, and strong correlation with carcinogenic potential make it the gold standard initial screen for detecting mutagenic agents. While it has limitations inherent to its bacterial model, its results provide critical information for identifying potential genetic hazards, guiding the development of safer chemicals and pharmaceuticals, and informing regulatory decisions to protect human health and the environment. A positive finding necessitates further investigation, while a rigorously negative result provides significant reassurance regarding a compound's mutagenic potential.