Induced Rat Liver S9 Fraction: A Metabolic Activation System for In Vitro Genotoxicity and Mutation Tests

Keywords: OECD 471, OECD 473, OECD 476, OECD 487, Mutation Test, Genotoxicity, Genetic Toxicity, Induced Rat Liver S9, Ames Test, Mini Ames Test, Chromosomal Aberration, Micronucleus, HPRT/HGPRT Assay, TK Assay

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Introduction

Induced rat liver S9 is a key component in assessing the genetic toxicity potential of chemicals, especially in regulatory toxicology. It is commonly used in combination with in vitro assays, such as the Ames test and mutation tests, to evaluate the mutagenic properties of compounds. By providing enzymatic system rich in cytochrome P450 (CYP450), the induced rat liver S9 fraction plays a crucial role in simulating the metabolic processes that occur in the liver, helping to determine whether chemicals can potentially cause genetic mutations or cancer.

Induced Rat Liver S9

The liver S9 fraction refers to a post-mitochondrial supernatant obtained from rat liver homogenates after a series of centrifugation steps. The term "induced" refers to the treatment of rats with specific compounds that enhance the activity of liver enzymes, particularly the cytochrome P450 enzymes (CYP450), which are responsible for the metabolism of a wide range of xenobiotic.

The induced rat liver S9 fraction contains a variety of enzymes that are involved in the phase I and phase II metabolism of chemicals. This includes enzymes such as cytochrome P450 monooxygenases, which facilitate oxidative biotransformation of substrates, thereby mimicking human liver metabolism.

CYP450 Activity and Metabolic Activation

The cytochrome P450 enzyme family (CYP450) plays a pivotal role in the metabolism of a wide variety of substances, including many pharmaceuticals, environmental chemicals, and carcinogens. The induced rat liver S9 fraction contains these enzymes and is essential for evaluating chemicals that may only become genotoxic after metabolic activation.

Many compounds are initially non-toxic but can become toxic after metabolism in the liver. These pro-mutagens (which require metabolic activation to become mutagenic) and pro-carcinogens (which require activation to cause cancer) can only be detected through assays that include an S9 metabolic activation system. Without the induction of metabolic enzymes like CYP450, these substances may appear to be harmless in standard genotoxicity tests.

By adding induced rat liver S9 to in vitro assays, researchers can assess how a substance interacts with the CYP450 enzymes. This is particularly important for pharmaceuticals, as metabolism by CYP450 enzymes is a critical step in the pharmacokinetics of many drugs. 

Applications of Induced Rat Liver S9

1. Ames Test (OECD 471)

The Ames test, outlined in Guideline OECD 471, is one of the most well-known and widely used methods for assessing mutagenicity. It involves exposing strains of Salmonella bacteria to a test chemical to see if it induces mutations that cause the bacteria to revert to a histidine-independent state.

To simulate the human metabolic process, induced rat liver S9 is often added to the test system. The S9 fraction provides the necessary enzymes, including CYP450, which may be required to metabolize the compound into a more reactive form that could cause mutations in the bacterial DNA. This combination of S9 with the Ames test allows for a more comprehensive assessment of mutagenic potential by mimicking both direct and metabolically activated mutagenic mechanisms.

2. Mutation Tests (Chromosomal Aberration Test, Micronucleus Test, and Other In Vitro Assays)

In addition to the Ames test, mutation tests are commonly used to evaluate the genotoxic potential of chemicals. These tests are more comprehensive in assessing a range of genetic damage, including chromosomal mutations, gene mutations, and the formation of micronuclei. Induced rat liver S9 is utilized in mutation tests for the following reasons:

Micronucleus Test (OECD 487)

This test detects the formation of micronuclei in cells, which are small, extranuclear bodies containing fragments of chromosomes or whole chromosomes that have not been incorporated into the nucleus during cell division. The micronucleus test can detect clastogenic (chromosome-breaking) and aneugenic (affecting chromosome number) effects. Induced rat liver S9 is used here to activate the test substance metabolically, as some chemicals only cause chromosomal damage once they have been metabolized by liver enzymes. The S9 fraction enhances the sensitivity of the test by simulating the metabolic activation that may occur in vivo.

Chromosomal Aberration Test (OECD 473)

This test evaluates whether a substance can cause structural damage to chromosomes by inducing breaks, deletions, translocations, or other types of aberrations. Like the micronucleus test, the chromosomal aberration test can be applied with or without metabolic activation. Induced rat liver S9 is added to the test system to mimic the metabolic processes in the liver, allowing the assessment of substances that might not cause chromosomal damage in their original form but could do so after liver metabolism.

Table 1. Comparative Considerations of Mutation Tests

3. Gene Mutation Test 

HPRT/HGPRT Assay (OECD 476)

In the HPRT/HGPRT Assay, cultures of Chinese hamster (CHO or V79) or human lymphoblastoid (TK6) cells are exposed to the test chemical in the presence of an induced rat liver S9 metabolic mix, which supplies CYP450 enzymes required to convert pro‑mutagens into DNA‑reactive species. After a brief treatment and a seven‑day expression period, cells are challenged with 6‑thioguanine; only clones bearing loss‑of‑function mutations in the hypoxanthine‑guanine phosphoribosyltransferase gene survive. By comparing mutant colony counts to overall viability, researchers determine a mutation frequency that, if reproducibly elevated over both concurrent and historical controls, indicates genotoxic potential.

TK assays (OECD 490 & OECD 476)

The TK Assay employs either L5178Y mouse lymphoma cells (OECD 490) or CHO cells (OECD 476) engineered heterozygous at the thymidine kinase locus. Following chemical exposure ± S9 mix, cells recover and are plated in medium containing trifluorothymidine, which kills TK‑proficient cells. Surviving TK⁻ mutants form colonies over 10–14 days, with small‑colony mutants often reflecting chromosomal events and large‑colony mutants reflecting point mutations. As in the HPRT/HGPRT Assay, stringent cytotoxicity controls and positive mutagen benchmarks ensure that observed increases in mutation frequency reliably reflect a compound’s genotoxic risk.

4. Cancer Risk Assessment

Induced rat liver S9 is also used in studies aimed at assessing the cancer-causing potential of substances. By simulating the metabolic processes that occur in the human liver, the S9 fraction can help identify compounds that might lead to the formation of reactive metabolites capable of binding to DNA and causing mutations that could lead to cancer.

Enhanced Ames Test with Induced Hamster Liver S9

According to the latest guidelines issued by the European Medicines Agency (EMA), the traditional Ames test may not be sensitive enough to detect the mutagenic potential of certain n-nitrosamine impurities, especially n-nitrosodimethylamine (NDMA), among others. Therefore, the Enhanced Ames Test, developed by the National Center for Toxicological Research (NCTR), a division of the U.S. Food and Drug Administration (FDA), has been recommended as a more reliable alternative. The enhanced Ames test introduced adding of induced hamster liver s9, which performed in containing 30% rat liver S9 and 30% Hamster Liver S9. Rat and hamster desmosomal supernatants (S9s) should be prepared from rodent livers treated with Cytochrome P450 Enzyme-inducing substances. By using induced hamster liver S9, this enhanced test better simulates human metabolism and increases the reliability of the results.

Conclusion

Induced rat liver S9 serves as a critical tool in genotoxicity testing, helping researchers and regulatory agencies assess the mutagenic and carcinogenic potential of chemicals. By providing a metabolic activation system, it enhances the ability of assays like the Ames test, mutation tests, and CYP450 activity studies to detect substances that require metabolic activation to become genotoxic. Its applications are essential for ensuring the safety of new drugs, chemicals, and consumer products. With its role in simulating human liver metabolism, the induced rat liver S9 fraction continues to be an indispensable part of modern toxicology and regulatory safety assessments.