Use of whole blood directly for single-cell gel electrophoresis (comet) assay in vivo and white blood cells for in vitro assay.

The present study investigated the use of whole blood from humans and rats directly for single-cell gel electrophoresis (comet) assay. As little as 20 microl of whole blood was sufficient for comet assay, and the comet images obtained from whole blood were not different from those obtained from isolated lymphocytes. The DNA remained intact up to 4 h at 4 degrees C after isolation and had no observable strand breakage, when whole blood was cryopreserved (at -80 degrees C) in 10% pre-cooled DMSO up to 60 days. To demonstrate that the whole-blood technique could be applied to in vivo studies, we injected rats with a known carcinogen Fe/NTA and measured DNA strand breaks in whole blood in comparison with isolated lymphocytes. We showed that Fe/NTA injection resulted in similar extent of DNA strand breakage in both whole blood and lymphocytes, indicating that whole-blood method can be used for in vivo genotoxic studies. One disadvantage of the whole-blood technique is that whole blood cannot be used for in vitro studies because of the interferences from red blood cell (RBC) components. However, this problem can be overcome by prior hemolysis of RBCs and a brief centrifugation to obtain white blood cells (WBCs), which can then be used for in vitro incubation with genotoxic compounds before comet assay. Overall, this whole-blood technique for comet assay is expected to provide a simple, rapid, and cost-effective alternative for the existing comet assay using isolated lymphocytes in situations such as when time and cost are limiting factors.

[1]  P. Olive,et al.  The comet assay: a comprehensive review. , 1995, Mutation research.

[2]  B. Edwards,et al.  Lymphocyte subset analysis by flow cytometry. Comparison of three different staining techniques and effects of blood storage. , 1989, Journal of immunological methods.

[3]  F P Perera,et al.  Biomarkers and molecular epidemiology in mutation/cancer research. , 1994, Mutation research.

[4]  M. Spitz,et al.  Cryopreserving whole blood for functional assays using viable lymphocytes in molecular epidemiology studies. , 2001, Cancer letters.

[5]  R. Crebelli,et al.  DNA damage by hydroquinone in human white blood cells: analysis by alkaline single-cell gel electrophoresis. , 1999, Mutation research.

[6]  R. Floyd.  The role of 8-hydroxyguanine in carcinogenesis. , 1990, Carcinogenesis.

[7]  F. Granado,et al.  Serum carotenoids and oxidative DNA damage in human lymphocytes. , 1998, Carcinogenesis.

[8]  A. Collins,et al.  In vivo supplementation with coenzyme Q10 enhances the recovery of human lymphocytes from oxidative DNA damage , 2001, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[9]  A. Ferraris,et al.  Catalase and glutathione peroxidase are equally active in detoxification of hydrogen peroxide in human erythrocytes. , 1989, Blood.

[10]  F. Kuypers,et al.  Erythrocyte defense against hydrogen peroxide: preeminent importance of catalase. , 1991, The Journal of laboratory and clinical medicine.

[11]  X. Lao,et al.  Lymphocyte DNA damage in cigarette factory workers measured by the Comet assay. , 1999, Mutation research.

[12]  T. Lindahl,et al.  Quality control by DNA repair. , 1999, Science.

[13]  P. Leanderson,et al.  Iron bound to the lipophilic iron chelator, 8-hydroxyquinoline, causes DNA strand breakage in cultured lung cells. , 1996, Carcinogenesis.

[14]  S. Duthie,et al.  Lysis of whole blood in vitro causes DNA strand breaks in human lymphocytes. , 2001, Mutagenesis.

[15]  P. Renzi,et al.  Analysis of T cell subsets in normal adults. Comparison of whole blood lysis technique to Ficoll-Hypaque separation by flow cytometry. , 1987, Journal of immunological methods.

[16]  T. Skopek,et al.  International Commission for Protection Against Environmental Mutagens and Carcinogens. Working paper no. 3. Somatic mutant frequency, mutation rates and mutational spectra in the human population in vivo. , 1994, Mutation research.

[17]  John Paul,et al.  Tissue culture: Methods and applications: Ed. by Paul F. Kruse, Jr and M. K. Patterson, Jr. 1973. New York and London: Academic Press. Pp. xxvii and 868; 264 text figs. £10.15. , 1974 .

[18]  S. Yeh,et al.  Simple Cryoprotection and Cell Dissociation Techniques for Application of the Comet Assay to Fresh and Frozen Rat Tissues , 2002, Free radical research.

[19]  W C Willett,et al.  The causes and prevention of cancer. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[20]  P. Cerutti,et al.  Pathophysiological mechanisms of active oxygen. , 1989, Mutation research.

[21]  M. Dobrzyńska,et al.  Effects in the Comet assay of storage conditions on human blood. , 1997, Teratogenesis, carcinogenesis, and mutagenesis.

[22]  R. Tice,et al.  A simple technique for quantitation of low levels of DNA damage in individual cells. , 1988, Experimental cell research.

[23]  L. Gold,et al.  Endogenous mutagens and the causes of aging and cancer. , 1991, Mutation research.

[24]  I. Benzie,et al.  Effects of Dietary Antioxidants on Human DNA Ex Vivo , 2002, Free radical research.

[25]  H. J. Phillips.  Dye Exclusion Tests for Cell Viability , 1973 .

[26]  A. Collins,et al.  The kinetics of repair of oxidative DNA damage (strand breaks and oxidised pyrimidines) in human cells. , 1995, Mutation research.

[27]  M. Athar,et al.  Attenuation of iron-nitrilotriacetate (Fe-NTA)-mediated renal oxidative stress, toxicity and hyperproliferative response by the prophylactic treatment of rats with garlic oil. , 1998, Food and chemical toxicology : an international journal published for the British Industrial Biological Research Association.

[28]  S. Okada,et al.  Nephrotoxicity and renal cell carcinoma after use of iron- and aluminum-nitrilotriacetate complexes in rats. , 1986, Journal of the National Cancer Institute.

[29]  A. Collins,et al.  The comet assay: what can it really tell us? , 1997, Mutation research.

[30]  P. McMillan,et al.  Uracil misincorporation in human DNA detected using single cell gel electrophoresis. , 1997, Carcinogenesis.

[31]  M. Green,et al.  The single cell gel electrophoresis assay (comet assay): a European review. , 1993, Mutation research.

[32]  C. Lundby,et al.  Acute hypoxia and hypoxic exercise induce DNA strand breaks and oxidative DNA damage in humans , 2001, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[33]  A R Collins.  Workshop on single cell gel electrophoresis (the 'comet assay') held as part of the UKEMS/DNA Repair Network Joint Meeting, Swansea, March 1992. , 1992, Mutagenesis.