Culture cytogenetics

Cohen, M. M. (1963), The specific effects of streptonigrin activity on human chromosomes in culture. Cytogenetics 2, 271-274. 

Wolman, S. R., Hirschhom K., and Todaro, G. J. (1964), Early chromosomal changes in SV infected human fibroblast cultures. Cytogenetics 3, 45-61. 

B. Cytogenetic analysis in cultured Chinese hamster or human cells... 

Pehn, K., Hirvonen, A., Taavirsainen, M., and Linnainmaa, K. (1995). Cytogenetic response to asbestos fibers in cultured human primary mesothclial cells from 10 different donors. Mutat. Res. 3,34, 22.5-233. 

Several groups of drugs that bind to tubulin at different sites interfere with its polymerization into microtubules. These drugs are of experimental and clinical importance (Bershadsky and Vasiliev, 1988). For example, colchicine, an alkaloid derived from the meadow saffron plant Colchicum autumnale or Colchicum speciosum), is the oldest and most widely studied of these drugs. It forms a molecular complex with tubulin in the cytosol pool and prevents its polymerization into microtubules. Other substances such as colcemid, podophyllotoxin, and noco-dazole bind to the tubulin molecule at the same site as colchicine and produce a similar effect, albeit with some kinetic differences. Mature ciliary microtubules are resistant to colchicine, whereas those of the mitotic spindle are very sensitive. Colchicine and colcemid block cell division in metaphase and are widely used in cytogenetic studies of cultured cells to enhance the yield of metaphase plate chromosomes. 

Singh S, Eehmann-Grube B, Boedde HW. 1984. Cytogenetic effects of paraoxon and methyl-parathion on cultured human lymphocytes SCE, clastogenic activity and cell cycle delay. Int Arch Occup Environ Health 54 195-200. 

The major Impetus to the development of methods for the prenatal detection of genetic disorders derives. In historical terms, from the roughly simultaneous development of three major techniques (11-14). One was the technique, and the willingness to use It, for obtaining samples of amnlotlc fluid early In gestation. The second was the development of techniques for the culture of human cells in vitro, and the third was the development of better techniques for cytogenetic analysis. As will be described below, with the availability of these three techniques It became possible first to work out methods for the examination of fetal chromosomes, and then, by extension, to devise ways of determining other characteristics of the fetus. 

Cytogenetic Analysis and Sex Determination. Since amnlotlc fluid cells are fetal In origin, they have the chromosome complement of the fetus Itself Therefore, the chromosomes of the fetus can be examined by karyotyping the cultured amnlotlc fluid cells There are two principal reasons for examining fetal chromosomes One, the numerically less frequent reason. 

Weitberg, A.B., Weitzman, S.A., Destrempes, M., Latt, S.A. and Stossel, T.P. (1983). Stimulated human phagocytes produce cytogenetic changes in cultured mammalian cells. N. Engl. J. Med. 308, 26-30. 

Lee, T.C., M. Oshimura, and J.C. Barrett. 1985. Comparison of arsenic-induced cell transformation, cytotoxicity, mutation and cytogenetic effects in Syrian hamster embryo cells in culture. Carcinogenesis 6 1421-1426. 

The in vitro cytogenetic assay is a short-term mutagenicity test for detecting chromosomal damage in cultured mammalian cells. 

For monolayer cultures, the cultures are set up the day before BrdU treatment so that the cells will be in exponential growth before the addition of BrdU or the test compound. After BrdU addition the cells are allowed to undergo the equivalent of two cell cycles before cell harvest. A spindle inhibitor such as colchicine or colcemid is introduced for the final 1-2 h of culture to arrest cells in metaphase, after which the cells are harvested and chromosome preparations are made by routine cytogenetic techniques. 

Peripheral blood cultures are established in medium containing BrdU and PHA. Cocemid is added 1-2 h before harvest and the cells are harvested between 60 and 70 h post-PHA stimulation. Cell harvest and slide preparations are conducted according to routine cytogenetic methods. 

Natarajan, A.T. and Obe, G. (1982). Mutagenicity testing with cultured mammalian cells cytogenetic assays. In Mutagenicity, New Horizons in Genetic Toxicology, (Heddle, J.A., Ed.). Academic Press, New York, pp. 172-213. 

OSHIMURA, M., HESTERBERG, T.W., TSUTSUI, T, AND Barrett, J.C. (1984). Correlation of asbestos-induced cytogenetic effects with cell transformation of Syrian hamster embryo cells in culture, Cancer Res. 44, 5017. 

Marx. M.P., Smith, S., Heyns, A.P van Tonder, I.Z. (1983) Fanconi s anemia a cytogenetic study on lymphocyte and bone marrow cultures utilizing l,2 3,4-diepoxybutane. Cancer Genet. Cytogenet., 9, 51-60... 

Sasaki, M., Sugimura, K., Yoshida, M.A. Abe, S. (1980) Cytogenetic effects of 60 chemicals on cultured human and Chinese hamster cells. Kromosome II, 20, 574-584... 

In the more recent cytogenetic studies in occupationally exposed populations, increases in chromosomal aberrations (two studies), micronuclei (one study) and of DNA strand breaks (one study) have been described. These effects have also been observed in rats and mice in some studies and in cultured mammalian cells. DNA adducts have not been detected. 

Sofuni, T., Hayashi, M., Matsuoka, A., Sawada, M., Hatanaka, M. Ishidate, M., Jr (1984a) Cytogenetic effects of gaseous and volatile chemicals on mammalian cells in vitro and in vivo. 1. Chromosome aberration tests in cultured mammalian cells. Eisei Shikenjo Hokoku, 10, 77-83 (in Japanese)... 

Various types of cells can be scored for chromosomal aberrations. Spermatogonial cells, spermatocytes, oocytes, early embryos, bone marrow cells, and lymphocytes have been used successfully after in vivo treatments of an animal. For short-term cytogenetic analysis, however, an in vitro cell-culture assay is often chosen. Such in vitro assays usually use a peripheral lymphocyte system or a monolayer cell-culture system. 

Like other cytogenetic tests, SCE tests can be performed on cells treated in culture or in vivo. For the in vivo method, cells from treated animals can be excised and cultured in vitro in the presence of BrdUrd or the animal itself can be given an injection or infusion of BrdUrd so that SCE can be observed in bone marrow or spermatogonial cells directly. The latter method is one of the simplest and least expensive ways to observe the effects of chemicals in gonads. The test is not limited to mammalian cells, but has been successfully carried out in plants, insects, and fish. 

Many chromosomal alterations can be detected in plants,149 and, in addition to studies of mitotic cells, methods using microsporocytes (i.e., pollen mother cells) have permitted the study of meiotic cells.263 Although basic research in plant cytogenetics continues, cytogenetic tests in plants have been replaced to some extent in genetic toxicology by mammalian cell-culture tests and tests in whole mammals. 

This in vitro cytogenetic test is a clastogenicity test system for the detection of chromosomal aberrations in cultured mammalian cells or primary cultures. Chromosomal aberrations may be either structural or numerical. However, because cytogenetic assays are usually designed to analyze cells at their first posttreatment mitosis and numerical aberrations require at least one cell division to be visualized, this type of aberration is generally not observed in a routine cytogenetic assay. The best estimate of aberration frequency is the first cell division after the start of treatment. Structural aberrations are of two types chromosome or chromatid aberrations. 

---