Skip to Main Navigation
Skip to Page Content
Share This:
https://ntp.niehs.nih.gov/go/gt

Genetic Toxicology

Scientist examines DNA molecule

Study Overview

Study: Mutagenicity & cytogenetics
Species: Rats, mice, Salmonella typhimurium, Escherichia coli

Description

NTP has developed a range of techniques and testing methods to evaluate the potential for environmental and occupational substances to damage DNA. These studies involve both in vivo (laboratory animals, human subjects) and in vitro (cells in culture) testing; in vivo animal studies are conducted using rodent models.

Protocols

Ames Test: Mutagenicity in Salmonella typhimurium (S. typhimurium) or Escherichia coli (E. coli) Tester Strains

The testing protocol contains more information.

Overview

The Ames test uses bacteria cultures to determine whether a substance causes mutations. Because there is a significant overlap between substances that are mutagens and those that are carcinogens, a quick and inexpensive mutagenicity test is useful as an initial screening for possible carcinogens.

Methodology

  1. Bacteria Strains

    The Ames test relies on specific strains of S. typhimurium, namely:

    • TA97
    • TA98
    • TA100
    • TA102
    • TA104
    • TA1535
    • TA1537
    • TA1538

    In addition to the S. typhimurium strains, the test may also use the E. coli strain WP2 uvrA pKM101.

  2. Principle of Assay

    Each of the bacterial strains has a mutation that prevents it from making an amino acid (a protein building block) required for normal cell growth. The S. typhimurium strains are unable to manufacture histidine; the E. coli strain cannot manufacture tryptophan. The tester strains will not be able to grow without amino acid supplementation, unless the mutated gene is changed back to the correct DNA sequence (reversion mutation). Being exposed to a mutagenic chemical increases the chance that the mutant gene will be restored to the correct sequence, which allows the bacteria to grow and form colonies. Each substance under study is tested in a variety of bacterial strains with different types of altered DNA sequences in order to provide a comprehensive assessment of the mutagenic potential of the substance.

  3. Metabolic Conversion

    Some non-mutagenic substances are converted to mutagenic substances when metabolized by the liver; in contrast, some substances that are mutagenic in their natural state are converted to non-mutagens by the liver. All substances are tested in their natural state, as well as in the presence of liver enzymes that can produce this metabolic conversion of the test substance. Extracts of rat, hamster, or mouse liver enzymes (S9 mix) are used for this purpose.

  4. Preparing the Cultures

    Multiple sets of cultures are prepared using a range of doses and different amounts of liver enzymes for each bacterial strain. Each culture is prepared in a test tube containing a suspension of one bacterial strain and either an S9 mix or plain buffer solution. Then the test substance is added. Negative control cultures are established using the same ingredients, but without the test substance. Positive control cultures are established using known mutagens active in particular bacterial strains and metabolic conditions (for example, with or without S9). Once prepared, each culture is incubated for 20 minutes at 37° C. Agar is then added to the cultures, and the contents of each tube are poured onto the surface of a Petri dish that was prepared with standard bacterial culture medium. The plates are then incubated, usually for two days.

  5. Variations

    Depending on the substance to be tested, the exposure and incubation processes may be modified. These variations include:

    • Elimination of the 20-minute incubation period in a test tube prior to plating (this method is referred to as the standard plate incorporation method)
    • Enzymatic reduction of azo and diazo substances (this process occurs in the intestine or liver of animals) using a chemical called flavin mononucleotide (FMN) or intestinal bacterial preparations from rats
    • Testing in sealed chambers such as desiccators for volatile and gaseous chemicals

Analysis

To validate the test, a trained investigator compares the number of colonies on the chemical-treated plates to the number of colonies on the negative control plates. If the substance under test is mutagenic, the chemical-treated plates will have a much greater number of colonies than the negative control plates.

  • A positive response is a reproducible, dose-related increase in mutant colonies in any single strain, with or without the addition of S9 metabolic enzymes. While there is no minimum percentage of increase required for a result to be considered positive, a twofold increase in mutant colonies in a treated plate is usually considered to be a positive (mutagenic) response.
  • An equivocal response is any increase that is not reproducible, not dose-related, or not high enough in magnitude to be considered positive.
  • A negative response occurs when no increases in mutant colonies are seen in the cultures treated with the test chemical, compared with the control.
Comet Assay

The testing protocol contains more information.

Overview

The comet assay examines the ability of substances to cause DNA damage in cells from a variety of different tissues, such as liver, stomach, lung, or brain.

There are multiple forms of DNA damage, including DNA adducts (addition of a chemical structure to a DNA base), oxidative damage to DNA, and breaks in one or both strands. The comet assay can detect single and double strand breaks, DNA adducts, and DNA crosslinks, as well as breaks along the DNA strand that are produced during the normal processes of DNA repair or replication. Thus, the comet assay provides a snapshot of the balance between DNA damage and DNA repair at the time the tissue was harvested.

The DNA damage detected by the comet assay can resolve in several ways. The damage is most often rapidly and correctly repaired. Alternately, a cell that sustains DNA damage may die without passing the damage on to daughter cells following cell division. However, if the damage is not repaired or is repaired incorrectly, the daughter cells may inherit the damage as mutations or chromosome breaks, which are found in many human diseases including cancer. The outcome of the detected DNA damage is unknown.

The comet assay provides the advantage of being able to look at chemical effects on DNA in tissues other than bone marrow, which is the main source of cells used to examine chemical-induced chromosome damage and mutation induction in animals (see Micronucleus Assay).

Methodology

Animals (rats or mice) are treated with a test substance once or multiple times, and DNA damage is usually evaluated 3–6 hours after the final treatment.

The animal tissue is rapidly minced in a buffer solution, combined with agarose, and layered onto slides. The slides are placed in a solution that removes the cellular and nuclear membranes and cellular proteins, leaving behind the nuclear DNA. The slides are then incubated in an alkaline solution (pH > 13) to convert alkali-labile sites into strand breaks and allow the DNA to unwind. Next, the slides are subjected to an electrical field in a tank filled with a buffer solution. Broken DNA, having a negative charge, migrates out of the area of the nucleus toward the positively charged anode. This forms a comet-like shape with the nucleus at the "head" and the migrated DNA as the "tail." All steps of the method are performed using ice-cold solutions and buffers. After this process, the DNA is stained with a fluorescent dye and cells are analyzed using Comet Assay IV Imaging Software.

Analysis

For each cell, DNA migration is analyzed using an automated software system attached to a microscope. The extent of damage is characterized as the percent tail DNA (how much DNA migrated to the tail, compared to the total DNA as a percentage). At least 100 cells per sample are evaluated; current guidelines for the assay recommend analysis of at least 150 cells per sample. The data is statistically analyzed to determine if there is a significant increase in percent tail DNA in chemical-exposed animals compared to the controls (animals not exposed).

Dose-related responses, as well as significant dose groups, are considered during the evaluation of the data. For a positive test, both a dose response and at least one significant dose group are required. If only one of these measures is present, the test is considered to be equivocal. The absence of either condition results in a negative test. The final conclusions for comet assays are determined by considering the results of statistical analyses, reproducibility of any observed effects, and the magnitude of the effects. Ultimately, both biological and statistical significance are considered when reaching a final conclusion.

Erythrocyte Micronucleus Assay

The testing protocol contains more information. Detailed explanations of the micronucleus assay methods and cell scoring are also available.

Overview

The erythrocyte micronucleus test examines the ability of substances to cause chromosome damage in developing red blood cells inside bone marrow. A micronucleus (literally, "little nucleus") is a biomarker of structural chromosome damage (e.g., breaks) and changes in chromosome number (e.g., chromosome loss). If a chromosome is broken or fails to migrate properly with other chromosomes during cell division, the broken piece or lost chromosome will form a small (micro) nucleus of its own in one of the two daughter cells produced. Detection of chromosomal damage is important because it has been linked to birth defects, infertility, and certain diseases, including cancer.

The micronucleus test is performed in rapidly dividing cell populations, and bone marrow is a site of rapid blood cell division. Both bone marrow and peripheral blood samples can be examined for the presence of micronuclei in red blood cells.

Methodology

Animals are treated with a test substance once or daily over a period of time, and the frequency of micronucleated cells is determined 24–48 hours after the final treatment, depending on the source of the cells (bone marrow or blood) that are examined and the treatment regimen. If treated animals show significantly higher frequencies of micronucleated red blood cells than untreated animals, the test substance is considered capable of causing chromosomal damage.

  1. Bone Marrow Tests

    The test subjects are rodents (rats or mice) that are exposed to the test substance, usually by oral gavage or injection. Typically, one to three daily treatments of the test substance are administered over a range of doses; the highest dose tested is near the maximum tolerated dose. Groups of negative and positive control animals are included for each test. Bone marrow samples are obtained from all animals 24 hours after the last treatment, and red blood cells are examined for the presence of micronuclei.

  2. Peripheral Blood Tests

    Micronucleus tests are performed on male and female rodents that are exposed to the test substance orally, dermally, or by inhalation in subchronic (e.g., 28 or 90 days) or short term (e.g., 48–72 hours) toxicity studies. 24 to 48 hours after the final exposure, peripheral blood samples are obtained and red blood cells are examined for the presence of micronuclei. Samples may be examined using standard slide scoring procedures or automated flow cytometric approaches. Since 2007, NTP has routinely relied on flow cytometry to evaluate the frequency of micronucleated cells in peripheral blood.

    Peripheral blood micronucleus assays are typically integrated into all NTP toxicity studies, and they also lend themselves to serial monitoring of micronucleus frequencies over time, since blood samples can be obtained without the need to sacrifice the animals.

Analysis

Blood or bone marrow samples from treated animals are compared to samples from untreated animals. With the slide-based approach for data acquisition, 1000–2000 cells were scored per animal. Using flow cytometric procedures to evaluate blood samples, 20,000 immature red blood cells and around one million mature red blood cells are examined per animal, resulting in a marked increase in the ability to detect small changes in the frequency of micronucleated red blood cells. The data is analyzed using statistical methods to determine if there is a significant increase in the frequency of cells containing micronuclei. Both dose-related responses and the magnitude of response for each independent dose group are considered during the evaluation of the data. For a positive test, both a significant dose response (trend) and at least one significant dose group are required. If only one of these measures is present, the test is determined to be questionable. The absence of either condition results in a negative test. The final conclusions for micronucleus tests are determined by considering the results of statistical analyses, reproducibility of any observed effects, and the magnitude of the effects. Ultimately, both the biological significance as well as statistical significance is considered when reaching a final conclusion.

Chinese Hamster Ovary Cell Cytogenetics

The testing protocol has been published and contains more information. Substance evaluations are also available.

Overview

These in vitro assays were conducted to evaluate a substance's ability to cause genetic damage in cloned Chinese hamster ovary (CHO) cells. Two tests were performed using the ovary cells: Sister Chromatid Exchange (SCE) test and Chromosomal Aberration (CA) test.

Methodology

  1. SCE Test

    Two SCE tests were performed on CHO cells. These tests were performed with and with the use of the liver enzyme, S9.

    1. Testing without S9

      In the SCE tests without S9, the cells were incubated with the test substance for 26 hours in McCoy's 5A medium supplemented with fetal calf serum, L-glutamine, and antibiotics. After 26 hours, the test substance was removed and incubation continued with fresh medium with BrdU and Colcemid for an additional 2 hours. Then the cells were harvested, fixed to slides, and stained with Hoechst 33258 and Giemsa. The slides were scored to determine the frequency of SCE per cell.

    2. Testing with S9

      In the SCE tests with S9, the cells were incubated with the test substance, serum-free medium, and S9 for 2 hours. After 2 hours, the test substance was removed and incubation continued with fresh medium containing seum and BrdU for an additional 26 hours. Colcemid was added during the final 2 hours of incubation. Then the cells were harvested, fixed to slides, and stained with Hoechst 33258 and Giemsa. The slides were scored to determine the frequency of SCE per cell.

    3. Analysis

      To analyze SCE test results, researchers conducted statistical analyses to compare exposed cells to vehicle controls. An SCE frequency of 20% above the control value was chosen as a positive result. Positive and weak positive trials were repeated.

  2. CA Test

    Two CA tests were performed on CHO cells. These tests were performed with and with the use of the liver enzyme, S9.

    1. Testing without S9

      In the CA tests without S9, the cells were incubated with the test substance for 8-14 hours in McCoy's 5A medium supplemented with fetal calf serum, L-glutamine, and antibiotics. Colcemid was added during the final 2 hours of incubation. Then the cells were harvested, fixed to slides, and stained with Giemsa. The slides were scored to determine the presence of chromosomal aberrations.

    2. Testing with S9

      In the CA tests with S9, the cells were incubated with the test substance and S9 for 2 hours. After 2 hours, the test substance was removed and incubation continued with fresh medium for an additional 10 hours. Colcemid was added during the final 2 hours of incubation. Then the cells were harvested, fixed to slides, and stained with Giemsa. The slides were scored to determine the presence of chromosomal aberrations.

    3. Analysis

      To analyze CA test results, researchers conducted statistical analyses to compare exposed cells to vehicle controls. The presence of a statistically significant increase in chromosomal aberrations above the control value was chosen as a positive result. Positive and weak positive trials were repeated.

Drosophila Melanogaster

The testing protocols for adult and larval subjects have been published and contain more information.

Overview

Drosophila melanogaster is a species of fruit fly that mates and grows quickly. This makes it ideal for testing whether substances cause germ cell mutations. Two tests were performed with these fruit flies: one for sex-linked recessive lethal mutations (SLRL) and one for reciprocal translocations (RT). The SLRL test was done first, and if the result was positive, the same route was used for the RT test.

Methodology

  1. SLRL Test

    The test subjects were Canton-S wild-type males which were exposed to the test substance. The amount of the substance used was set by preliminary tests to control for death and sterility rates.

    1. Adult Exposure

      Adult flies were given the substance in their food beginning before 24 hours of age, or injected with the substance between 24 and 72 hours of age. The treated Canton-S males were each bred to three Basc females for three days. Then they were bred to two more groups of fresh females for two days apiece, for a total of three matings over the course of a week.

    2. Larval Exposure

      Larvae were given the substance in their food, then allowed to mature. Control groups were created by treating flies with any solvents used, without the test substance. As they matured, adult males about 24 hours old were each bred to two groups of females. Each group of females contained between three and five flies. Two single-day broods were produced.

    3. Subsequent Breeding

      For both adult and larval subjects, the F1 offspring were bred with their siblings, then placed in separate vials. Daughters of the same male parent were grouped together to identify clusters. Clusters occured when a male had a spontaneous mutation which then showed in a large number of offspring. If the number of mutants from one male was much more than the predicted number, the data from that male was thrown out.

    4. Analysis

      To analyze SLRL test results, researchers compared test subjects with the current controls and with records of older control groups. Presumptive lethal mutations were identified as vials which contained less than 5% of the expected number of wild-type males after 17 days. After mutations were identified, they were retested to confirm the results.

  2. RT Test

    1. Exposure and Breeding

      To test for reciprocal translocations, the same route was used that tested positive for SLRL. The exposure protocols were the same. Canton-S male flies were then mass bred to marker females (bw;st or bw;e). The females were transferred to fresh medium every three or four days to lay six broods over the course of three weeks. The F1 males were then backcrossed to bw;st females.

    2. Analysis

      Reciprocal translocations were identified by screening for pseudolinkage. Pseudolinkage is when two genes from different chromosomes behave like they are linked. This is caused by translocation in the parent male's germ cell. If reciprocal translocations were suspected, the flies were retested to confirm the results.

Mouse Lymphoma: Mammalian Cell Mutagenicity

The testing protocol has been published and contains more information.

Overview

This mammalian mutagenicity assay tested a substance's ability to mutate cultured mouse lymphoma cells. It tested both point mutations and changes to the chromosome. Changes were measured by testing the dosed cells' resistance to trifluorothymidine (TFT), which was caused by forward mutation.

Methodology

  1. Test Groups

    A typical test used four solvent control cultures and three positive control cultures. It included two to three test cultures for each of the five to six concentrations tested. The highest concentration was chosen using cell toxicity, substance solubility, or a hard upper limit of 5 mg/ml.

  2. Preparing the Cultures

    The cells used in the test were mouse lymphoma L5178Y TK+/- cells. They were kept at 37° C as suspension cultures in Fischer's medium, with an additional 2 mM l-glutamine, 110 ug/mL sodium pyruvate, 0.05% luronic F68, antibiotics, and heat-inactivated horse serum. Their cycling time was about 10 hours. To limit random mutations, cultures were treated with thymidine, hypoxanthine, methotrexate, and glycine.

    Each culture contained 6 x 106 cells and 10 mL of medium dosed with the test substance. The cultures were incubated for 4 hours. Then the medium was removed and fresh medium without the test substance was added. The cultures were incubated for two more days. Samples of 3 x 106 cells were taken from each culture and plated. The medium they were plated in contained TFT to select for TFT-resistant cells (TK-/-). Another 3 x 106 cells from each culture were plated in medium without TFT to check cloning efficiency. All plates were incubated for ten to twelve days in 5% CO2 before cell colonies were counted.

  3. Metabolic Conversion

    Some non- mutagenic substances will create mutagenic substances when processed by the liver. Naturally mutagenic are harmless after being metabolized. Any substance that did not test positive in its natural state was treated to mimic metabolism. For this purpose, extracts of rat liver enzymes (S9) were added to the samples.

Analysis

For the test to be valid, control cultures and test cultures had to show cloning efficiency within a certain range. Each dose set had to produce at least two valid cultures. If the test chemical precipitated out of the medium, the data was thrown out.

  • For a positive result, both cultures had to show significant induced TFT resistance.
  • If one showed a significant response, the result was marked as questionable.
  • If there was no trend of increasing response and no peak, the test was negative.

Rodent Cytogenetics

The testing protocol has been published and contains more information. A detailed explanation of the statistical analyses is available.

Overview

These in vivo assays determined the effects of toxic substances on chromosomes in the bone marrow of mice. Two tests were performed: the Chromosomal Aberration (CA) test and the Sister Chromatid Exchange (SCE) test.

Methodology

  1. CA Test

    The test subjects were males B6C3F1 mice which were exposed to the test substance either orally or by injection. During testing, the mice were implanted with a BrdU to detect any defects on the chromosomes. 17-36 hours after substance exposure, the animals were euthanized, and bone marrow samples were harvested. Then the cells were examined by microscope and scored to determine the presence of chromosomal aberrations.

    1. Preparing the Slides

      The cells were treated with a hypotonic salt solution, fixed, and placed on chilled slides. After 24 hours, the slides were stained with Giemsa and scored.

    2. Analysis

      Types of aberrations were recorded separately for each animal. Chromosomal aberrations may include: gaps, breaks, or rearrangements. The mean total number and the mean percentage of cells with aberrations were determined. The data was then analyzed for statistical significance.

  2. SCE Test

    The test subjects were males B6C3F1 mice which were exposed to the test substance either orally or by injection. The mice were implanted with a BrdU during testing. 23-42 hours after substance exposure, the animals were euthanized, and bone marrow samples were harvested. Then the cells were examined by microscope and scored to determine the frequency of SCE per cell.

    1. Preparing the Slides

      The cells were treated with a hypotonic salt solution, fixed, and placed on chilled slides. After 24 hours, the slides were stained with fluorescence-plus-Giemsa and scored.

    2. Analysis

      To analyze SCE test results, researchers conducted statistical analyses to compare exposed cells to vehicle controls.