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The baseline frequency of sister chromatid exchanges in pig lymphocytes detected by a one-step immunofluorescent method

Marcelo L. Larramendy^I,II; Stella J. Nylund^II; Miguel A. Reigosa^I; Sakari Knuutila^II

^ILaboratorio de Citogenética y Cátedra de Citología, Facultad de Ciencias Naturales y Museo de La Plata, Universidad Nacional de La Plata, 1900 La Plata, Argentina. Tel.: 54-21-246187, Fax: 54-21-530189. Send correspondence to M.L.L.
^IIDepartment of Medical Genetics, University of Helsinki, Helsinki, Finland

ABSTRACT

A one-step immunofluorescent method was used to measure the frequency of sister chromatid exchanges (SCEs) and analyze the proliferation rate of pig lymphocytes in culture. The method employs a low concentration of bromodeoxyuridine (3.3 pM, BUdR) for chromosome labelling during the first cell cycle in vitro (dose equivalent to a tenth of the conventional concentration), and anti-BUdR monoclonal antibody conjugated with fluorescein isothiocyanate (FITC) for detection, avoiding any sequential incubation steps with secondary antibodies. Peripheral blood was obtained from four male Duroc pigs, and whole blood and plasma leukocyte cultures were set up. Cultures were stimulated with phytohemagglutinin and allowed to grow for the first of two cell cycles in the presence of 3.3 or 33 pM of BUdR. The culture medium was then replaced with BUdR-free culture medium and the cells were cultured for an additional cell cycle. Metaphase chromosomes were denatured with distilled boiling water or formamide, and exposed to the anti-BUdR-FITC monoclonal antibody. Chromosomes were counterstained with propidium iodide and 4,6-diamino-2-phenylindole dihydrochloride (DAPI) and analyzed using a fluorescence photomicroscope with appropriate filter combinations.
The results demonstrate that the basal SCE frequency and the cell cycle progression in pig lymphocytes are independent of the concentration of BUdR in the culture medium, at least in the range of base analogue employed.

Keywords: sister chromatid; pig lymphocytes; immunofluorescent.

REFERENCES

Bianchi, M.S., Bianchi, N.O., Larramendy, M.L. and García-Heras, J. (1981). Chromosomal radiosensitivity of pig lymphocytes in relation to sampling time. Mutat. Res. 80: 313-320.

Bianchi, M.S., Larramendy, M.L. and Bianchi, N.O. (1986). Mitomycin C-induced damage and repair in human and pig lymphocytes. Mutat. Res. 160: 27-32.

Bianchi, N.O. and Lezana, E.A. (1976). Kinetics of lymphocyte division in blood cultures studied with the BrdU-Giemsa technique. Experientia 32: 1257-1258.

Brewen, J.G., Preston, R.J., Jones, K.P. and Gosslee, D.G. (1973). Genetic hazards of ionizing radiation: cytogenetics extrapolations from mouse to man. Mutat. Res. 17: 245-254.

Carrano, A.V., Thompson, L.H., Lindl, P.A. and Minkler, J.L. (1978). Sister chromatid exchange as an indicator of mutagenesis. Nature 271: 551-553.

Carrano, A.V., Minkler, J.L., Stetka, D.G. and Moore, D.H. (1980). Variation in the baseline sister chromatid exchange frequency in human lymphocytes. Environ. Mutagen. 2: 325-337.

Davidson, R.L., Kaufman, E.R., Dougherty, C.P., Quellette, A.M., DiFolco, C.M. and Latt, S.A. (1980). Induction of sister chromatid exchanges is largely independent of the BudR content of DNA. Nature 284: 74-76.

Gebhart, E. (1981). Sister chromatid exchange (SCE) and structural chromosome aberration in mutagenicity testing. Hum. Genet. 58: 235-254.

Johnson, G.D. and Nogueira Araujo, G.M.C. (1981). A simple method of reducing the fading of immunofluorescence during microscopy. J. Immunol. Meth. 43: 349-350.

Kato, H. (1977). Spontaneous and induced sister chromatid exchanges as revealed by the BUdR-labeling method. Int. Rev. Cytol. 45: 55-97.

Kligerman, A.D., Wilmer, J.L. and Erexson, G.L. (1982). Characterization of a rat lymphocyte culture system for assessing sister chromatid exchange. II. Effect of 5-bromodeoxyuridine concentration, number of white blood cells in the inoculum, and inoculum volume. Environ. Mutagen. 4: 585-594.

Korenberg, J.R. and Freedlander, E.F. (1974). Giemsa technique for the detection of sister-chromatid exchanges. Chromosoma 48: 355-360.

Lambert, B., Hansson, K., Lindstein, J., Sten, M. and Werelius, B. (1976). Bromodeoxyuridine-induced sister chromatid exchanges in human lymphocytes. Hereditas 83: 163-174.

Larramendy, M.L. and Reigosa, M.A. (1986). Variation in sister chromatid exchange frequencies between human and pig whole blood, plasma leukocyte, and mononuclear leukocyte cultures. Environ. Mutagen. 8: 543-554.

Larramendy, M.L., Reigosa, M.A. and Bianchi, M.S. (1990). Erythrocytes modulate the baseline frequency of sister-chromatid exchanges and the kinetics of lymphocyte division in culture. Mutat. Res. 232: 63-70.

Larramendy, M.L., Reigosa, M.A. and Knuutila, S. (1993). Pig plasma modulates cell cycle kinetics but not the baseline frequency of sister chromatid exchanges in human lymphocytes. Mutat. Res. 292: 165-173.

Latt, S.A. (1974). Sister chromatid exchanges, indices of human chromosome damage and repair: detection by fluorescence and induction by mitomycin C. Proc. Natl. Acad. Sci. (USA) 71: 3162-3166.

Latt, S.A., Schreck, R.R., Loveday, K.S., Dougherty, C.P. and Shuler, C.F. (1980). Sister chromatid exchanges. Adv. Hum. Genet. 10: 267-331.

Léonard, A., Gerber, G.B., Papworth, D.G., Decat, G., Léonard, E.D. and Deknudt, G. (1976). The radiosensitivities of lymphocytes from pig, sheep, goat and cow. Mutat. Res. 36: 319-332.

Lezana, E.A., Bianchi, M.S. and Bianchi, N.O. (1978). Kinetics of division in PHA-stimulated pig lymphocytes. Experientia 34: 30-31.

McClintock, B. (1938). The production of homozygous deficient tissues with mutant characteristics by means of the aberrant mitotic behavior of ring-shaped chromosomes. Genetica 23: 315-376.

Morgan, W.F. and Wolff, S. (1984). Effect of bromodeoxyuridine on induced sister chromatid exchanges. In: Sister Chromatid Exchanges (Tice, R.T. and Hollaender, A., eds.). Plenum Press, New York, pp. 281-292.

Natarajan, A.T., Tates, A.D., Meijers, M., Neuteboom, I. and Vogel, N. (1983). Induction of sister-chromatid exchanges (SCEs) and chromosomal aberrations by mitomycin C and methyl methanesulfonate in Chinese hamster ovary cells. Mutat. Res. 121: 211-223.

Natarajan, A.T., Rotteveel, A.H.M., van Pieterson, J. and Schliermann, M.G. (1986). Influence of incorporated 5-bromodeoxyuridine on the frequencies of spontaneous and induced sister-chromatid exchanges, detected by immunological methods. Mutat. Res. 163: 51-55.

Ockey, C.A. (1981). Methyl methanesulphonate (MMS) induced SCEs are reduced by BrdU used to visualize them. Chromosoma 84: 243-256.

Perry, P. (1980). Chemical mutagens and sister chromatid exchange. In: Chemical Mutagens (De Serres, F.J. and Hollaender, A., eds.). Plenum Press, New York, pp. 1-39.

Perry, P. and Wolff, S. (1974). New Giemsa method for the differential staining of sister chromatids. Nature 251: 156-158.

Pinkel, D., Thompson, L.H., Gray, J.W. and Vanderlaan, M. (1985). Measurement of sister chromatid exchanges at very low bromodeoxyuridine substitution levels using monoclonal antibody in Chinese hamster overy cells. Cancer Res. 45: 5795-5798.

Popescu, N.C., Amsbaugh, S.A. and DiPaolo, J.A. (1980). Reduced N-methyl-N'-nitro-N-nitrosoguanidine sister chromatid exchange induction in Chinese hamster V79 cells pre-exposed to 5-bromodeoxyuridine. Chromosoma 76: 329-338.

Sandberg, A.A. (1982). Sister chromatid exchanges in human states. In: Sister Chromatid Exchange. Progress and Topics in Cytogenetics (Sandberg, A.A., ed.). Alan R. Liss Inc., New York, pp. 619-651.

Tucker, J.D., Christensen, M.L., Strout, C.L. and Carrano, A.V. (1986). Determination of the baseline sister chromatid exchange frequency in human and mouse peripheral lymphocytes using monoclonal antibodies and very low doses of bromodeoxyuridine. Cytogenet. Cell. Genet. 43: 48-42.

Wilmer, J.L., Erexson, G.L. and Kligerman, A.D. (1983). Implications of an elevated sister-chromatid exchange frequency in rat lymphocytes cultured in the absence of erythrocytes. Mu to t. Res. 109: 231-248.

Wolff, S. and Perry, P. (1974). Differential Giemsa staining of sister chromatids and the sudy of sister chromatids exchanges without autoradiography. Chromosoma 48: 341-353.