Here we had little subjects to discover, like Bio-materials & Bio-compatibility, let’s start.
1) What is Genotoxicity/gene toxicity/Genetic toxicity?
Many chemical compounds might exert genotoxic effect through interactions with cellular DNA, and potentially be human carcinogens and/or mutagens which eventually lead to cancer or other heritable defects!
Genotoxicity testing evaluates gene mutations, changes in chromosomes or DNA and gene toxicities caused by by-products or compounds over an extended period of time. The International Organization for Standardization (ISO) standard 10993–3 outlines tests for genotoxicity, carcinogenicity and reproductive toxicity. The ISO guidelines for genotoxicity testing require examination of gene mutation (bacterial mutagenicity test), chromosomal aberrations (chromosomal aberration assay) and DNA effects (mouse lymphoma assay). The FDA also requires three genotoxicity tests. The bacterial reverse mutation and the in vitro mouse lymphoma tests are the same as those recommended by ISO. A third test, which some within the FDA recommend, is an in vivo test, such as the mouse micronucleus test.
The majority of toxic effects, especially due to xenobiotics, are due to specific biochemical interactions without causing recognizable damage to a cell or its organelles.
- The tissue may be completely repaired and return to normal.
- The tissue may be incompletely repaired but is capable of sustaining its function with reduced capacity.
- Death of the organism or the complete loss of a tissue or organ. In some instances, the organism can continue to live with the aid of medical treatment, for example, replacement of insulin or by organ transplantations.
- Neoplasm or cancers may result, many of which will result in death of the organism and some of which may be cured by medical treatment.
In many situations, the damage to a cell may be so severe that the cell cannot survive. Cell death occurs mainly by two methods: necrosis and apoptosis.
Genetic toxicity is determined using a wide range of test species including whole animals and plants (for example, rodents, insects, and corn), microorganisms, and mammalian cells. A large variety of tests have been developed to measure gene mutations, chromosome changes, and DNA activity.
gene mutation tests
|Microorganisms||Salmonella typhimurium and Escherichia coli are commonly used bacterial tests. The S. typhimurium assay is known as the Ames Test. Yeasts are also used to detect gene mutations in culture systems.|
|Mammalian cells||The two main cell lines are mouse lymphoma and Chinese hamster ovary (CHO) cells.|
|Fruit Flies||Drosophila melanogaster is used to detect sex-linked recessive lethal mutations.|
|Mice||The Mouse Specific-Locus Test is the major gene mutation test that employs whole animals. Exposed mice are bred and observed for hereditary changes.|
Chromosomal effects can be detected with a variety of tests, some of which utilize entire animals (in vivo) and some which use cell systems (in vitro). Several assays are available to test for chemically induced chromosome aberrations in whole animals. We list common in vivo means of testing chromosomal effects.
|Rodent chromosomal assay||Involves exposure of mice or rats to a single dose of a substance. Their bone marrow is analyzed for chromosome aberrations over a 48-hour period.|
|Dominant lethal assay||Exposed male mice or rats are mated with untreated females. The presence of dead implants or fetuses is the result of the fertilized ovum receiving damaged DNA from the sperm. This leads to the death of the embryo or fetus. The genetic defect in the sperm is thus a heritable dominant lethal mutation.|
|Micronucleus test||Mice are exposed once and their bone marrow or peripheral blood cells are examined for 72 hours for the presence of micronuclei, such as broken pieces of chromosomes surrounded by a nuclear membrane.|
|Heritable translocation assay||Exposed male Drosophila or mice are bred to non-exposed females. The offspring males (F1 generation) are then bred to detect chromosomal translocations.|
|Sister chromatid exchange assay (SCE)||Mice are exposed to a substance and their bone marrow cells or lymphocytes are examined microscopically for complete chromosomal damage. This is indicated by chromatid fragments joining sister chromatids rather than their own.|
In vitro Testing
In vitro tests for chromosomal effects involve exposure of cell cultures and followed by microscopic examination of them for chromosome damage.
The most commonly used cell lines are Chinese Hamster Ovary (CHO) cells and human lymphocyte cells. The CHO cells are easy to culture, grow rapidly, and have a low chromosome number (22), which makes for easier identification of chromosome damage.
Human lymphocytes are more difficult to culture. They are obtained from healthy human donors with known medical histories. The results of these assays are potentially more relevant to determine effects of xenobiotics that induce mutations in humans.
Two widely used genotoxicity tests measure DNA damage and repair that is not mutagenicity. DNA damage is considered the first step in the process of mutagenesis. Common assays for detecting DNA damage include:
- Unscheduled DNA synthesis (UDS) — involves exposure of mammalian cells in culture to a test substance. UDS is measured by the uptake of tritium-labeled thymidine into the DNA of the cells. Rat hepatocytes or human fibroblasts are the mammalian cell lines most commonly used.
- Exposure of repair-deficient E. coli or B. subtilis — DNA damage cannot be repaired so the cells die or their growth may be inhibited.
In the future, there will likely be additional and refined in vitro methods, and the emergence of in silico and “chip” approaches (also Induced Pluripotent Stem Cells (iPSCs)). Many current efforts are underway to refine, develop, and validate in vitro methods.