The Single Cell Gel (SCG) Assay: An Electrophoretic Technique for the Detection of DNA Damage in Individual Cells
Techniques which permit the sensitive detection of DNA damage are needed to evaluate the genotoxic potential of biologically reactive intermediates. Since the effects of many reactive intermediates are tissue and cell-type specific, it is important to utilize techniques which can directly detect DNA damage in individual cells. Cytogenetic techniques, while providing information at the level of the individual cell, are largely limited to proliferating cell populations. Furthermore, these techniques require the processing of DNA damage into microscopically visible lesions. Biochemical techniques, such as alkaline elution and nucleoid sedimentation, circumvent these difficulties in that DNA damage can be evaluated directly in almost any cell population. However, the resulting data do not provide any information about the distribution of damage or repair among individual cells. Recently, an electrophoretic technique capable of detecting DNA single-strand breaks and alkali labile sites in individual cells was developed (Singh et al., 1988). Eukaryote cells are embedded in an agarose gel on a microscope slide, lysed by detergents and high salt at pH 10, and then electrophoresed for a short time under alkaline conditions. Cells with increased DNA damage display increased migration of the DNA from the nucleus towards the anode. The importance of this technique lies in its ability to detect intercellular differences in DNA damage in virtually any eukaryote cell population, and in its requirement for extremely small cell samples (from 1 to 10,000 cells). Data from three recent pilot studies, involving (i) the exposure of human lymphocytes to hydrogen peroxide, (ii) the incubation of isolated mouse hepatocytes with cyclophosphamide, and (iii) the treatment of mice with acrylamide, will be used to demonstrate the sensitivity and utility of this technique for evaluating intercellular differences in DNA damage.
KeywordsHuman Peripheral Blood Lymphocyte Mouse Hepatocyte Electrophoretic Technique Primary Mouse Hepatocyte Alkaline Elution
Unable to display preview. Download preview PDF.
- Ashby, J., Lefevre, P.A., Burlinson, B., and Penman, M.G. (1985). An assessment of the in vivo rat hepatocyte DNA-repair assay. Mutat. Res. 156, 1.Google Scholar
- Butterworth, B.E., Ashby, J., Bermudez, E., Casciano, D., Mirsalis, J., Probst, G., and Williams, G. (1987). A protocol and guide for the in vitro rat hepatocyte DNA-repair assay. Mutat. Res. 189, 113.Google Scholar
- Margolin, B.H. and Risko, K.J. (1987). The statistical analysis of in vivo genotoxicity data. Case studies of the rat hepatocyte UDS and mouse bone marrow micronucleus assays, In: “Evaluation of Short-Term Tests for Carcinogens. Report of the International Programme on Chemical Safety’s Collaborative Study on In vivo Assays”, J. Ashby, F.J. deSerres, M.D. Shelby, B.H. Margolin, M. Ishidate, Jr., and G.C. Becking, eds., Cambridge Univ. Press, Cambridge, U.K., Vol I, pp. 29.Google Scholar
- Mitchell, A.D., Casciano, D.A., Meltz, M.L., Robinson, D.E., San, R.H.C., Williams, G.M., and von Halle, E.S. (1983). Unscheduled DNA synthesis test: A report of the Gene-Tox Program. Mutat. Res. 123, 363.Google Scholar
- Singh, N.P., McCoy, M.T., Tice, R.R. and Schneider, E.L. (1988). A simple technique for quantitation of low levels of DNA damage in individual cells. Exp. Cell Res. 175, 184.Google Scholar
- Singh, N.P., Danner, D.B., Tice, R.R., McCoy, M.T., Collins, G.D.,and Schneider, E.L. (1989). Abundant alkali-labile sites in human and mouse sperm DNA.Exp.Cell Res.184, 461.Google Scholar
- Williams, G.M., Laspia, M.F., and Dunkel, V.C. (1982). Reliability of the hepatocyte primary culture/DNA repair test in testing of coded carcinogens and non/carcinogens. Mutat. Res. 97, 359–370.Google Scholar
- Biological Reactive Intermediates IV, Edited by C.M. Witmer et al. Plenum Press, New York, 1990 Google Scholar