A micronucleus test is a test used in toxicological screening for potential genotoxic compounds. The assay is now recognized as one of the most successful and reliable assays for genotoxic carcinogens, i.e., carcinogens that act by causing genetic damage and is recommended by the OECD guideline for the testing of chemicals.[1] There are two major versions of this test, one in vivo and the other in vitro.
The in vivo test normally uses mouse bone marrow or mouse peripheral blood. When a bone marrow erythroblast develops into a polychromatic erythrocyte, the main nucleus is extruded; any micronucleus that has been formed may remain behind in the otherwise anucleated cytoplasm. Visualisation of micronuclei is facilitated in these cells because they lack a main nucleus. An increase in the frequency of micronucleated polychromaticerythrocytes in treated animals is an indication of induced chromosome damage.[2]
Micronuclei (B, C) and nuclear abnormalities (A, D) in peripheral blood erythrocytes of penguins Pygoscelis papua.
Micronuclei were first used to quantify chromosomal damage by H.J. Evans et al., in root tips of the Broad Bean, Vicia faba. Subsequently the in vivo assay was developed independently by W. Schmid and by J.A. Heddle and their colleagues. The mouse peripheral blood assay was developed by J.T. MacGregor and has now been adapted for measurement by flow cytometry by A. Tometsko and colleagues. The first use of micronuclei in cultured cells was by J.A. Heddle and colleagues in human lymphocytes. The assay has been improved by M. Fenech and colleagues for use in lymphocytes and other cells in culture cells.
Simple Giemsa staining was originally used for MN scoring. Later, the cytokinesis-block micronucleus (CBMN) method was established, where Cyt-B, an inhibitor of the spindle assembly, was used to prevent cytokinesis occurring after nuclear division. The CBMN method is used for the assessment of chromosomal loss, breakage, and associated apoptosis and necrosis induced by different mutagens.[3]
A micronucleus is the erratic (third) nucleus that is formed during the anaphase of mitosis or meiosis. Micronuclei (the name means 'small nucleus') are cytoplasmic bodies having a portion of acentric chromosome or whole chromosome which was not carried to the opposite poles during the anaphase. Their formation results in the daughter cell lacking a part or all of a chromosome. These chromosome fragments or whole chromosomes normally develop nuclear membranes and form as micronuclei as a third nucleus. After cytokinesis, one daughter cell ends up with one nucleus and the other ends up with one large and one small nucleus, i.e., micronuclei. There is a chance of more than one micronucleus forming when more genetic damage has happened. The micronucleus test is used as a tool for genotoxicity assessment of various chemicals. It is easier to conduct than the chromosomal aberration test in terms of procedures and evaluation. Using fluorescent in situ hybridization (FISH) with probes targeted to the centromere region, it can be determined if a whole chromosome, or only a fragment is lost.
In genetics, a mutagen is a physical or chemical agent that permanently changes genetic material, usually DNA, in an organism and thus increases the frequency of mutations above the natural background level. As many mutations can cause cancer in animals, such mutagens can therefore be carcinogens, although not all necessarily are. All mutagens have characteristic mutational signatures with some chemicals becoming mutagenic through cellular processes.
In cell biology, mitosis is a part of the cell cycle in which replicated chromosomes are separated into two new nuclei. Cell division by mitosis gives rise to genetically identical cells in which the total number of chromosomes is maintained. Therefore, mitosis is also known as equational division. In general, mitosis is preceded by S phase of interphase and is often followed by telophase and cytokinesis; which divides the cytoplasm, organelles and cell membrane of one cell into two new cells containing roughly equal shares of these cellular components. The different stages of mitosis altogether define the mitotic (M) phase of an animal cell cycle—the division of the mother cell into two daughter cells genetically identical to each other.
Cypermethrin (CP) is a synthetic pyrethroid used as an insecticide in large-scale commercial agricultural applications as well as in consumer products for domestic purposes. It behaves as a fast-acting neurotoxin in insects. It is easily degraded on soil and plants but can be effective for weeks when applied to indoor inert surfaces. Exposure to sunlight, water and oxygen will accelerate its decomposition. Cypermethrin is highly toxic to fish, bees and aquatic insects, according to the National Pesticides Telecommunications Network (NPTN). It is found in many household ant and cockroach killers, including Raid, Ortho, Combat, ant chalk, and some products of Baygon in Southeast Asia.
Nondisjunction is the failure of homologous chromosomes or sister chromatids to separate properly during cell division (mitosis/meiosis). There are three forms of nondisjunction: failure of a pair of homologous chromosomes to separate in meiosis I, failure of sister chromatids to separate during meiosis II, and failure of sister chromatids to separate during mitosis. Nondisjunction results in daughter cells with abnormal chromosome numbers (aneuploidy).
Genotoxicity is the property of chemical agents that damage the genetic information within a cell causing mutations, which may lead to cancer. While genotoxicity is often confused with mutagenicity, all mutagens are genotoxic, but some genotoxic substances are not mutagenic. The alteration can have direct or indirect effects on the DNA: the induction of mutations, mistimed event activation, and direct DNA damage leading to mutations. The permanent, heritable changes can affect either somatic cells of the organism or germ cells to be passed on to future generations. Cells prevent expression of the genotoxic mutation by either DNA repair or apoptosis; however, the damage may not always be fixed leading to mutagenesis.
Cytochalasin B, the name of which comes from the Greek cytos (cell) and chalasis (relaxation), is a cell-permeable mycotoxin. It was found that substoichimetric concentrations of cytochalasin B (CB) strongly inhibit network formation by actin filaments. Due to this, it is often used in cytological research. It inhibits cytoplasmic division by blocking the formation of contractile microfilaments. It inhibits cell movement and induces nuclear extrusion. Cytochalasin B shortens actin filaments by blocking monomer addition at the fast-growing end of polymers. Cytochalasin B inhibits glucose transport and platelet aggregation. It blocks adenosine-induced apoptotic body formation without affecting activation of endogenous ADP-ribosylation in leukemia HL-60 cells. It is also used in cloning through nuclear transfer. Here enucleated recipient cells are treated with cytochalasin B. Cytochalasin B makes the cytoplasm of the oocytes more fluid and makes it possible to aspirate the nuclear genome of the oocyte within a small vesicle of plasma membrane into a micro-needle. Thereby, the oocyte genome is removed from the oocyte, while preventing rupture of the plasma membrane.
Sudan I, is an organic compound, typically classified as an azo dye. It is an intensely orange-red solid that is added to colourise waxes, oils, petrol, solvents, and polishes. Sudan I has also been adopted for colouring various foodstuffs, especially curry powder and chili powder, although the use of Sudan I in foods is now banned in many countries, because Sudan I, Sudan III, and Sudan IV have been classified as category 3 carcinogens by the International Agency for Research on Cancer. Sudan I is still used in some orange-coloured smoke formulations and as a colouring for cotton refuse used in chemistry experiments.
Micronucleus is the name given to the small nucleus that forms whenever a chromosome or a fragment of a chromosome is not incorporated into one of the daughter nuclei during cell division. It usually is a sign of genotoxic events and chromosomal instability. Micronuclei are commonly seen in cancerous cells and may indicate genomic damage events that can increase the risk of developmental or degenerative diseases. Micronuclei form during anaphase from lagging acentric chromosome or chromatid fragments caused by incorrectly repaired or unrepaired DNA breaks or by nondisjunction of chromosomes. This incorrect segregation of chromosomes may result from hypomethylation of repeat sequences present in pericentromeric DNA, irregularities in kinetochore proteins or their assembly, dysfunctional spindle apparatus, or flawed anaphase checkpoint genes. Micronuclei can contribute to genome instability by promoting a catastrophic mutational event called chromothripsis. Many micronucleus assays have been developed to test for the presence of these structures and determine their frequency in cells exposed to certain chemicals or subjected to stressful conditions.
A clastogen is a mutagenic agent that disturbs normal DNA related processes or directly causes DNA strand breakages, thus causing the deletion, insertion, or rearrangement of entire chromosome sections. These processes are a form of mutagenesis which if left unrepaired, or improperly repaired, can lead to cancer. Known clastogens include acridine yellow, benzene, ethylene oxide, arsenic, phosphine, mimosine, actinomycin D, camptothecin, methotrexate, methyl acrylate, resorcinol and 5-fluorodeoxyuridine. Additionally, 1,2-dimethylhydrazine is a known colon carcinogen and shows signs of possessing clastogenic activity. There are many clastogens not listed here and research is ongoing to discover new clastogens. Some known clastogens only exhibit clastogenic activity in certain cell types, such as caffeine which exhibits clastogenic activity in plant cells. Researchers are interested in clastogens for researching cancer, as well as for other human health concerns such as the inheritability of clastogen effected paternal germ cells that lead to fetus developmental defects.
The ciliates are a group of alveolates characterized by the presence of hair-like organelles called cilia, which are identical in structure to eukaryotic flagella, but are in general shorter and present in much larger numbers, with a different undulating pattern than flagella. Cilia occur in all members of the group and are variously used in swimming, crawling, attachment, feeding, and sensation.
Chromothripsis is a mutational process by which up to thousands of clustered chromosomal rearrangements occur in a single event in localised and confined genomic regions in one or a few chromosomes, and is known to be involved in both cancer and congenital diseases. It occurs through one massive genomic rearrangement during a single catastrophic event in the cell's history. It is believed that for the cell to be able to withstand such a destructive event, the occurrence of such an event must be the upper limit of what a cell can tolerate and survive. The chromothripsis phenomenon opposes the conventional theory that cancer is the gradual acquisition of genomic rearrangements and somatic mutations over time.
Cancerous micronuclei is a type of micronucleus that is associated with cancerous cells.
The murine local lymph node assay (LLNA) is an in vivo test for skin sensitisation.
The association between obesity, as defined by a body mass index of 30 or higher, and risk of a variety of types of cancer has received a considerable amount of attention in recent years. Obesity has been associated with an increased risk of esophageal cancer, pancreatic cancer, colorectal cancer, breast cancer, endometrial cancer, kidney cancer, thyroid cancer, liver cancer and gallbladder cancer. Obesity may also lead to increased cancer-related mortality. Obesity has also been described as the fat tissue disease version of cancer, where common features between the two diseases were suggested for the first time.
Mixed lymphocyte reaction (MLR) is a test used by pharmaceutical and biotech organizations to show the safety of a drug or implantable material. It is commonly used as part of the FDA clearance process. Put simply, it is mixing populations of T-lymphocytes together, and measuring the reaction that occurs. Technically, it is an ex-vivo cellular immune assay that occurs between two allogeneic lymphocyte populations. In a one-way MLR, only one lymphocyte population can respond or proliferate. In a two-way MLR, both populations can proliferate. MLR’s are performed to assess how T-cells react to external stimuli. T cells are a type of white blood cell that scans for cellular abnormalities and infections. They are essential to human immunity.
Institute for genetic engineering and biotechnology, also known as INGEB, is public Bosnian scientific institution, member Sarajevo University (UNSA), Affiliate center of International Centre for Genetic Engineering and Biotechnology – ICGEB. ICGEB was established as a special project of the United Nations organization for industrial development.
Cyclotriol is a synthetic estrogen which was studied in the 1990s and was never marketed. It is a derivative of estriol with a bridge between the C14α and C17α positions. The drug has 40% of the relative binding affinity of estradiol for the human ERα. It showed an absolute bioavailability of 40% with high interindividual variability and an elimination half-life of 12.3 hours in pharmacokinetic studies in women.
Pig-a gene mutation assay is a flow cytometry-based method for detecting mammalian cells that have inactivating mutations in the endogenous X-linked reporter gene called phosphatidyl inositolglycan class A gene. PIG-A is involved in the synthesis of glycosylphosphatidylinositol (GPI), an anchor molecule that tethers multiple protein marker molecules at the surface of the cells. When the sample containing wild-type and PIG-A mutant cells is labeled with fluorescent antibodies raised against GPI-anchored protein markers the wild-type cells will fluoresce and PIG-A mutant cells will not. The fraction of non-fluorescent PIG-A mutant cells in the antibody-labeled sample can be efficiently determined on any of the modern high throughput flow cytometers. The PIG-A mutant frequency can be determined with high accuracy within minutes by processing samples containing a total of a million cells or more.
The somatic mutation and recombination tests (SMARTs) are in vivo genotoxicity tests performed in Drosophila melanogaster. These fruit fly tests are a short-term test and a non-mammalian approach for in vivo testing of putative genotoxins found in the environment. D. melanogaster has a short lifespan, which allows for fast reproductive cycles and high-throughput genotoxicity testing. D. melanogaster also has around 75% functional orthologs of human disease-related genes, making it an attractive in vivo model for human research. The tests identify loss of heterozygosity for the specified genetic markers in heterozygous or trans-heterozygous adults using phenotypically observable genetic markers in adult tissues. Although diverse events like point mutations/deletions, nondisjunction, and homologous mitotic recombination might theoretically cause this loss of heterozygozity, nondisjunction processes are generally not relevant for most of the examined chemicals. SMARTs are two different tests that use the same genetic foundation, but target different adult tissues and are named accordingly: the wing-spot test and the eye-spot test.
Diana Anderson is a British biomedical scientist who is a professor at the University of Bradford. Her research has focussed on early cancer detection and genome stability. She was appointed an Order of the British Empire in 2022 for her services to cancer detection.
This page is based on this Wikipedia article Text is available under the CC BY-SA 4.0 license; additional terms may apply. Images, videos and audio are available under their respective licenses.
