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Genetic Toxicology

Scientist examines DNA molecule

Study Overview

Study: Mutagenicity, cytogenetics, microarray
Species: Rats, mice, hamsters, drosophila, salmonella, E. coli

Description

NTP has developed a range of techniques and testing regimes for evaluating the potential of environmental and occupational substances to damage DNA. These studies are carried out primarily using rodent models and involve both in vitro and in vivo testing.

Protocols

Ames Test: Salmonella or E. coli Mutagenicity

The testing protocol has been published and contains more information.

Overview

The Ames test uses bacteria cultures to determine whether a substance causes genetic mutations. There is a significant overlap between substances that are mutagens and those that are carcinogens. For this reason, 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 Salmonella, namely:

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

    A similar test may also use E. coli WP2 uvrA pKM101. Each of these strains has a genetic defect and cannot create the amino acids it needs to grow. Because of this defect, growth will be limited unless the bacteria are mutated by the substance being tested. For the most accurate results, each substance should be tested on several strains.

  2. Metabolic Conversion

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

  3. Preparing the Cultures

    Multiple sets of cultures are prepared using a range of doses and different amounts of liver enzymes and bacteria strains. Each culture is prepared in a test tube containing a suspension of one bacterial strain and either an S9 mix or a plain buffer. Then the test substance is added. Control cultures are created with the same ingredients, but without the test substance. A positive control group is created using a known mutagen. Once prepared, each culture is incubated for 20 minutes at 37º C.

    After the first incubation period, agar is mixed into the cultures, and each tube is poured onto the surface of a Petri dish prepared with the standard medium. The plates are then incubated, usually for two days.

  4. Variations

    Depending on the substance to be tested, the process may be changed. Variations include:

    • No initial incubation (standard plate incorporation)
    • Reduction of substances using FMN or rat cecal bacteria (for azo compounds)
    • Testing in sealed Petri dishes (for volatile liquids)
    • Testing in a sealed dessicator (for gases)

Analysis

The test is only valid if the number of positive control colonies is greater than for the control cultures. Once this has been confirmed, the test cultures are compared to the control. If the substance is mutagenic, it will have caused greater bacteria growth.

  • A positive response is a reproducible, dose-related increase in any set of test cultures. There is no minimum percentage of increase to define a result as positive.
  • An equivocal response is any increase that is not reproducible, not dose-related, or not statistically significant.
  • A negative response occurs when none of the cultures tested shows more growth than the control.
Comet Assay

Overview

The in vivo comet assay examines the ability of substances to cause DNA damage in cells from a variety of different tissues in an organism, such as stomach, liver, lung, or brain. The DNA damage detected in the comet assay may be in the form of breaks or adducts, as well as transient damage resulting from normal DNA repair processes.

The DNA damage detected by this assay may be:

  • Repaired and thus, cause no permanent effects in the cell
  • Lethal to the cell
  • Converted into a persistent mutation by errors in DNA repair, which may lead to disease

DNA breaks may also lead to chromosome breaks, which are found in many human diseases including cancer.

Micronucleus

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

Overview

The micronucleus test examines the ability of substances to cause damage to chromosomes. Abnormal chromosome counts, chromosomal rearrangements, and chromosomal damage have been linked to birth defects, infertility, and cancer.

Methodology

Animals are treated with a test substance and the frequency of micronucleated cells is determined after exposure. If treated animals show significantly higher frequencies of micronucleated cells than untreated animals, the test substance is considered capable of causing chromosomal damage.

  1. Bone Marrow Assays

    The test subjects are male rodents that are exposed to the test substance either orally or by injection. One to three treatments of the test substance are administered at 24-hour intervals up to the maximum tolerated dose. Bone marrow samples are taken 24 hours after the last dose, and red blood cells are examined for the presence of micronuclei.

  2. Micronucleus Analysis

    Micronucleus tests are performed on mice being treated in 13-week toxicity studies. The test subjects are male and female rodents that are exposed to the test substance orally, dermally, or through inhalation. At the end of the 13-week exposure period, bone marrow samples are taken and red blood cells are examined for the presence of micronuclei. Samples are examined using standard slide scoring procedures and flow cytometric analysis.

Analysis

The data is analyzed to determine if there is an overall increase in the frequency of cells containing micronuclei. Samples from treated animals are compared to samples from untreated animals and any statistical differences in the data are recorded.

Factors that may affect micronucleus test data include:

  • Dose levels
  • Frequencies of micronucleated cells in the treated vs. non-treated animals
  • Number of animals per dose group
  • Number of doses administered
  • Results of the statistical analyses
  • Route of administration
  • Sample time (interval between last dosing and harvesting of cells for analysis)
  • Tissue and cell type being analyzed

The final conclusions for micronucleus tests are determined by considering the results of statistical analyses, reproducibility of any observed effects, and significance of those effects.

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.