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Cell Biology and Toxicology

, Volume 9, Issue 1, pp 77–83 | Cite as

Flow cytometric analysis of the cell-cycle distribution of spleen lymphocytes isolated from Fischer 344 rats exposed to ethyl nitrosourea

  • Suzanne M. Morris
  • Olen E. Domon
  • Lynda J. McGarrity
  • Anane Aidoo
  • Ralph L. Kodell
  • Daniel A. Casciano
Article

Abstract

Current studies in our laboratory are designed to determine the frequency of genotoxic responses induced in lymphocytes isolated from Fischer 344 rats. To evaluate the effect of a model compound, N-ethyl-N-nitrosourea (ENU), on the cell-cycle distribution of spleen lymphocytes, 8-week old, female Fischer 344 rats were injected i.p. with ENU and sacrificed 1, 2, 4, and 6 weeks afterexposure. Four replicate cultures per dose per exposure period were established and cells were cultured for 66 hr. Colcernid, an agent which blocks cells in mitosis and induces an accumulation of cells in the G2 + M peak, was added to two of the four cultures as a positive control. After a 3 hr incubation, the cells were harvested, the nuclei stained with propidium iodide, and the DNA content of the individual nuclei was quantified by flow cytometry. As expected, exposure to Colcemid resulted in an accumulation of cells in the G2 + M phase of the cell cycle, which was accompanied by a decrease in the Go + GI population. The increase in the G2 + M population was significant (p < 0.05) in cultures of lymphocytes assayed at 4 and 6 weeks after exposure. The eflect of increasing ENU concentratiorl was an increase in the percentage of Sphase cells (p =0.05) and a decrease (p < 0.02) in the percentage of G0 + G1 cells. This finding was observed only in those lymphocytes isolated 1 week after exposure. These findings indicate that flow cytometric analysis of the distribution of cells within the cell-cycle may provide insight into the eflects of toxicant exposure on mamnzalian cells.

Key Words

cell-cycle analysis Colcemid ENU rat lymphocyte DNA flow cytometry 

Abbreviations

BRdU

bromodeoxyuridine

ENU

N-ethyl-N-nitrosourea

FCM

flow cytometry

PHA

phytohemagglutinin

PI

propidium iodide

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References

  1. AIDOO, A., MORRIS, S.M., DOMON, O.E., McGARRITY, L.J., KODELL, R.L., and CASCIANO, D.A. (1989). “Modulation of SCE induction and cell proliferation by 2-mercaptoethanol in phytohemagglutinin-stimulated lymphocytes.” Cell Biol. Tox. 5: 237–248.Google Scholar
  2. AIDOO, A., LYN-COOK, L.E., MITTELSTAEDT, R.A., HEFLICH, R.H., and CASCIANO, D.A. (1991). “Induction of 6-thioguanine-resistant lymphocytes in Fischer 344 rats following in vivo exposure to N-ethyl-N-nitrosourea and cyclophosphamide.” Environ. Molec. Mutagen. 17: 141–151.Google Scholar
  3. ANSON, J.F., HINSON, W.G., SCHOL, H., and PIPKIN, J.L. (1983). “The use of primary spleen cell cultures in evaluating cell cycle response to toxicant insult.” J. Tiss. Cult. Methods 8: 13–16.Google Scholar
  4. BAGWELL, C.B., HUDSON, J.L., and IRVIN, G.L. (1979). “Nonparametric flow cytometry analysis.” J. Histochem. Cytochem. 27: 293–296.Google Scholar
  5. DEGAN, P., MONTESANO, R., and WILD, C.P. (1988). “Antibodies against 7-methyldeoxyguanosine: Its detection in rat peripheral blood lymphocyte DNA and potential applications to molecular epidemiology.” Cancer Res. 48: 5065–5070.Google Scholar
  6. GERSON, S.L. (1988). “Regeneration of O6-alkylguanine-DNA alkyltransferase in human lymphocytes after nitrosourea exposure.” Cancer Res. 48: 5368–5373.Google Scholar
  7. MORRIS, S.M., AIDOO, A., DOMON, O.E., MCGARRITY, L.J., KODELL, R.L., SCHOL, H.M., HINSON, W.G., PIPKIN, J.L., and CASCIANO, D.A. (1990). “Effect of interleukin-2 on cell proliferation, sister-chromatid exchange induction, and nuclear stress protein phosphorylation in PHA-stimulated Fischer 344 rat spleen lymphoyctes.” Environ. Molec. Mutagen. 15: 10–18.Google Scholar
  8. MORRIS, S.M., DOMON, O.E., McGARRITY, L.J., KODELL, R.L., and CASCIANO, D.A. (1991). “Flow cytometric evaluation of cell-cycle progression in ethyl methanesulfonate and methyl methanesulfonate-exposed P3 cells. Relationship to the induction of sister-chromatid exchanges and cellular toxicity.” Environ. Molec. Mutagen. 18: 139–149.Google Scholar
  9. SCOLLAY, R.G., BUTCHER, E.C., and WEISSMAN, I.L. (1980). “Thymus cell migration. Quantitative aspects of cellular traffic from the thymus to the periphery in mice.” Eur. J. Immunol. 10: 210–218.Google Scholar
  10. SAS User's Guide: Statistics, Version 5 Edition (1985). SAS Institute Inc., Cary, NC.Google Scholar
  11. SHAPIRO, H.M. (1988). Practical Flow Cytometry. 2nd ed., A.R. Liss, New York, NY.Google Scholar
  12. VINDELOV, L.L. (1977). “Flow microfluorometric analysis of nuclear DNA in cells from solid tumors and cell suspension.” Virchow's Arch. [Cell Pathol.] 24: 227–242.Google Scholar

Copyright information

© Kluwer Academic Publishers 1993

Authors and Affiliations

  • Suzanne M. Morris
    • 1
  • Olen E. Domon
    • 1
  • Lynda J. McGarrity
    • 1
  • Anane Aidoo
    • 1
  • Ralph L. Kodell
    • 1
  • Daniel A. Casciano
    • 1
  1. 1.Department of Health and Human ServicesUS Public Health Service Food and Drug Administration National Center for Toxicological Research Division of Genetic ToxicologyJefferson

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