ABC> Vol.2 No.3, August 2012

Measurement of DNA damage by terminal deoxynucleotidyl transferase reaction

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ABSTRACT

An improved terminal deoxynucleotidyl transferase- mediated dUTP-biotin nick end labeling method for the quantification of DNA damage in tissues and cultured cells was developed. Many reports have revealed that histochemistry of DNA damage can be visualized using immunohistochemistry for the terminal deoxynucleotidyl transferase reaction in tissue sections. However, few reports have described quantification of DNA damage in tissues or cells. In this study, to estimate the degree of DNA damage, the confirmed method for histochemistry using biotinylated dUTP and deoxynucleotidyl transferase was applied to label the cleaved DNA ends caused by DNA damage in tissues or cells. After end-labeling, avidin-conjugated peroxidase was reacted. A significant correlation was observed between numbers of cleaved DNA ends and peroxidase activity after the reaction. The obtained signals for presented method showed higher than those for ordinary method, and correlate with degree of DNA damage caused by serum deprivation and chemical dose. In addition, DNA damage caused by apoptosis in cells treated with 6-hydroxydopamine or Cu and in the tissues of rats administered a diet containing no Zn could be evaluated quantitatively using the present method.

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Cite this paper

Kurasaki, M. , Sun, Y. , Komori, M. , Miyajima, M. , Hosokawa, T. and Saito, T. (2012) Measurement of DNA damage by terminal deoxynucleotidyl transferase reaction. Advances in Biological Chemistry, 2, 243-247. doi: 10.4236/abc.2012.23030.

References

[1] Gavrieli, Y., Sherman, Y. and Ben-Sasson, S.A. (1992) Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation. Journal of Cell Biolology, 119, 493-501. HUdoi:10.1083/jcb.119.3.493U
[2] Tu, Y.F., Tsai, Y.S., Wang, L.W., Wu, H.C., Huang, C.C. and Ho, C.J. (2011) Overweight worsens apoptosis, neuroinflammation and blood-brain barrier damage after hypoxic ischemia in neonatal brain through JNK hyperactivation. Journal of Neuroinflammation, 8, 40. HUdoi:10.1186/1742-2094-8-40U
[3] Hosokawa, T., Okabe, M., Saito, S., Saito, T. and Kurasaki, M. (2000) Protective role of metallothionein on DNA damage in rat kidney caused by cis-diamminedi- chloroplatinum. Pharmacology and Toxicology, 86, 276- 282. HUdoi:10.1111/j.0901-9928.2000.860606.xU
[4] Shigenaga, M.K., Park, J.W., Cundy, K.C., Gimeno, C.J. and Ames, B.N. (1990) In vivo oxidative DNA damage: measurement of 8-hydroxy-2’-deoxyguanosine in DNA and urine by high-performance liquid chromatography with electrochemical detection. Methods in Enzymology, 186, 521-530. HUdoi:10.1016/0076-6879(90)86146-MU
[5] Maccubbin, A.E., Patrzyc, H.B., Ersing, N., Budzinski, E.E., Dawidzik, J.B., Wallace, J.C., Iijima, H. and Box, H.C. (1999) Assay for reactive oxygen species-induced DNA damage: Measurement of the formamido and thymine glycol lesions. Biochimica et Biophysica Acta, 1454, 80-88. HUdoi:10.1016/S0925-4439(99)00022-8U
[6] Spencer, S.L. and Sorger, P.K. (2011) Measuring and modeling apoptosis in single cells. Cell, 144, 926-939. HUdoi:10.1016/j.cell.2011.03.002U
[7] Jacobson, M.D., Weil, M. and Raff, M.C. (1997) Programmed cell death in animal development. Cell, 88, 347-354. HUdoi:10.1016/S0092-8674(00)81873-5U
[8] Batistatou, A. and Greene, L.A. (1991) Aurintricarboxylic acid rescues PC12 cell and sympathetic neurons from cell death caused by nerve growth factor deprivation: correlation with suppression of endonuclease activity. Journal of Cell Biology, 115, 461-471. HUdoi:10.1083/jcb.115.2.461U
[9] Kawakami, M., Inagawa, R., Hosokawa, T., Saito, T. and Kurasaki, M. (2008) Mechanism of apoptosis induced by copper in PC12 cells. Food and Chemical Toxicology, 46, 2157-2164. HUdoi:10.1016/j.fct.2008.02.014U
[10] Woodgate, A., MacGibbon, G., Walton, M. and Dragunow, M. (1999) The toxicity of 6-hydroxydopamine on PC12 and P19 cells. Brain Research Molecular Brain Research, 69, 84-92. HUdoi:10.1016/S0169-328X(99)00103-5U
[11] Nodera, M., Yanagisawa, H. and Wada, O. (2001) Increased apoptosis in a variety of tissues of zinc-deficient rats. Life Sciences, 69, 1639-1649. HUdoi:10.1016/S0024-3205(01)01252-8U
[12] Rink, A., Fung, K.M., Trojanowski, J.Q., Lee, V.M., Neugebauer, E., and McIntosh, T.K. (1995) Evidence of apoptotic cell death after experimental traumatic brain injury in the rat. American Journal of Pathology, 147, 1575-1583.
[13] Conti, A.C., Raghupathi, R., Trojanowski, J.Q. and McIntosh, T.K. (1998) Experimental brain injury induces regionally distinct apoptosis during the acute and delayed post-traumatic period. Journal of Neuroscience, 18, 5663- 5672.
[14] Walker, P.R., LeBlanc, J. and Sikorska, M. (1997) Evidence that DNA fragmentation in apoptosis is initiated and propagated by single-strand breaks. Cell Death and Differentiation, 4, 506-515. HUdoi:10.1038/sj.cdd.4400273U
[15] Linde, V., Flodgaard, H., Kastrup, J.S. and Bj?rn, S. (2000) Measurement of apoptosis by the TUNEL method using scintillating microplates. Analytical Biochemistry, 280, 186-188. HUdoi:10.1006/abio.2000.4512U

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