Why is rat liver extract, specifically the S9 (supernatant 9) fraction, used in the Ames test?

Rat Liver Extract (S9 Fraction) in the Ames Test

Rat liver extract, specifically the S9 fraction, is used in the Ames test to provide metabolic activation of potential mutagens that require enzymatic conversion to become genotoxic, thereby simulating mammalian metabolism in this bacterial mutagenicity assay. 1

What is the S9 Fraction?

The S9 fraction is a post-mitochondrial supernatant prepared from rat liver homogenate. It contains:

• Cytochrome P450 enzymes
• Other metabolic enzymes necessary for phase I and phase II biotransformation
• Cofactors required for enzymatic activity

Purpose in the Ames Test

The S9 fraction serves several critical functions in the Ames test:

1. Metabolic Activation: Many chemicals are not directly mutagenic but become mutagenic after metabolic activation. The S9 fraction provides the enzymatic machinery to convert these "procarcinogens" into active "carcinogens" 2

2. Simulation of Mammalian Metabolism: Bacteria used in the Ames test (Salmonella typhimurium strains) lack the metabolic capabilities of mammalian cells. The S9 fraction bridges this gap by providing mammalian metabolic enzymes 3

3. Standardized Approach: Using rat liver S9 provides a consistent and reproducible method for metabolic activation across laboratories 4

Preparation and Enhancement

The S9 fraction is typically prepared from rats pretreated with enzyme inducers to enhance metabolic capacity:

• Common Inducers: Phenobarbital and 5,6-benzoflavone (PB/BF) or other combinations like β-naphthoflavone 5
• Enzyme Induction: This pretreatment increases cytochrome P450 activity, enhancing the S9 fraction's ability to metabolize test compounds 6

Importance in Genotoxicity Testing

The use of S9 in the Ames test is critical because:

• It allows detection of compounds that require metabolic activation to exert mutagenic effects
• Without S9, many carcinogens would yield false negative results
• The metabolic activation step more closely mimics what happens in the human body

Specific Applications in Mutagenicity Testing

The S9 fraction is particularly important for detecting the mutagenicity of:

• Polycyclic aromatic hydrocarbons (e.g., benzo[a]pyrene)
• Heterocyclic amines
• Nitrosamines
• Aromatic amines
• Other procarcinogens that require metabolic activation 2, 6

Limitations and Considerations

• Species Differences: Rat S9 metabolism may differ from human metabolism for certain compounds
• Concentration Effects: The standard concentration is typically 10% S9, though 30% is sometimes used for specific applications 4
• Quality Control: Enzyme activity levels must be monitored to ensure consistent performance 3

Alternative Approaches

While rat liver S9 is the standard, other options include:

• Human S9: May provide more relevant metabolism for human exposure scenarios but typically has lower enzymatic activity than induced rat S9 3
• Hamster S9: Used in some protocols as an alternative metabolic activation system 4
• Hepatocytes: Primary hepatocytes can sometimes be used as an alternative to S9 fractions 5

The rat liver S9 fraction remains the gold standard for metabolic activation in the Ames test due to its reproducibility, standardization, and high enzymatic activity, making it an essential component in the screening of chemicals for mutagenic potential.