Skip to main content
NCBI home page
The American Journal of Pathology logoLink to The American Journal of Pathology
. 1990 Nov;137(5):1187–1198.

Flow cytometric analysis of the mechanism of methylmercury cytotoxicity.

R M Zucker 1, K H Elstein 1, R E Easterling 1, E J Massaro 1
PMCID: PMC1877667  PMID: 2240165

Abstract

Flow cytometric analysis of murine erythroleukemic cells (MELC) exposed in vitro to 2.5 to 7.5 mumol/l (micromolar) methylmercury (MeHg) reveals a dose-dependent decrease in the rate of DNA synthesis (rate of passage through the S phase of the cell cycle), manifested as the accumulation of most of the cells in the S phase, and a modest accumulation of cells in the G2/M phase of the cycle. Light microscopy reveals a progressive increase in chromosomal damage (condensation, pulverization). At or above 10 mumol/l MeHg, progression through all the phases of the cell cycle is blocked and mitotic cells are arrested irreversibly in anaphase, with most exhibiting arrangement of chromosomes in a wreathlike ring formation. Also the cells exhibit both nuclear propidium iodide (PI) fluorescence (indicative of loss of viability) and concurrent cytoplasmic carboxyfluorescein (CF) fluorescence (viable cells exhibit CF fluorescence and exclude PI). In addition, there is a dose-dependent increase in cellular refractive index (90 degrees light scatter), an apparent decrease in cell volume (axial light loss), and progressive resistance to detergent (NP-40)-mediated cytolysis. Resistance to detergent-mediated cytolysis is indicative of fixation (protein denaturation, cross-linking, and so on) of the plasma membrane/cytoplasm complex. Our findings indicate that DNA synthesis is the primary target of MeHg cytotoxicity and that apparent targets and degree of cytotoxicity are a complex function of dose.

Full text

PDF
1187

Images in this article

1195Figure 12
on p.1195

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Abe T., Haga T., Kurokawa M. Blockage of axoplasmic transport and depolymerisation of reassembled microtubules by methyl mercury. Brain Res. 1975 Mar 28;86(3):504–508. doi: 10.1016/0006-8993(75)90904-x. [DOI] [PubMed] [Google Scholar]
  2. Atchison W. D. Effects of activation of sodium and calcium entry on spontaneous release of acetylcholine induced by methylmercury. J Pharmacol Exp Ther. 1987 Apr;241(1):131–139. [PubMed] [Google Scholar]
  3. Bienvenue E., Boudou A., Desmazes J. P., Gavach C., Georgescauld D., Sandeaux J., Seta P. Transport of mercury compounds across bimolecular lipid membranes: effect of lipid composition, pH and chloride concentration. Chem Biol Interact. 1984 Jan;48(1):91–101. doi: 10.1016/0009-2797(84)90009-7. [DOI] [PubMed] [Google Scholar]
  4. Brown D. L., Reuhl K. R., Bormann S., Little J. E. Effects of methyl mercury on the microtubule system of mouse lymphocytes. Toxicol Appl Pharmacol. 1988 Jun 15;94(1):66–75. doi: 10.1016/0041-008x(88)90337-7. [DOI] [PubMed] [Google Scholar]
  5. Cambier J. C., Monroe J. G. Flow cytometry as an analytic and preparative tool for studies of neuroendocrine function. Methods Enzymol. 1983;103:227–245. doi: 10.1016/s0076-6879(83)03015-3. [DOI] [PubMed] [Google Scholar]
  6. Costa M., Cantoni O., de Mars M., Swartzendruber D. E. Toxic metals produce an S-phase-specific cell cycle block. Res Commun Chem Pathol Pharmacol. 1982 Dec;38(3):405–419. [PubMed] [Google Scholar]
  7. Crissman H. A., Steinkamp J. A. Rapid, simultaneous measurement of DNA, protein, and cell volume in single cells from large mammalian cell populations. J Cell Biol. 1973 Dec;59(3):766–771. doi: 10.1083/jcb.59.3.766. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Dean P. N., Jett J. H. Mathematical analysis of DNA distributions derived from flow microfluorometry. J Cell Biol. 1974 Feb;60(2):523–527. doi: 10.1083/jcb.60.2.523. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Fiskesjö G. The effect of two organic mercury compounds on human leukocytes in vitro. Hereditas. 1970;64(1):142–146. doi: 10.1111/j.1601-5223.1970.tb02283.x. [DOI] [PubMed] [Google Scholar]
  10. Goodman D. R., Fant M. E., Harbison R. D. Perturbation of alpha-aminoisobutyric acid transport in human placental membranes: direct effects by HgCl2, CH3HgCl, and CdCl2. Teratog Carcinog Mutagen. 1983;3(1):89–100. doi: 10.1002/1520-6866(1990)3:1<89::aid-tcm1770030110>3.0.co;2-o. [DOI] [PubMed] [Google Scholar]
  11. Gruenwedel D. W., Glaser J. F., Cruikshank M. K. Binding of methylmercury(II) by HeLa S3 suspension-culture cells: intracellular methylmercury levels and their effect on DNA replication and protein synthesis. Chem Biol Interact. 1981 Sep;36(3):259–274. doi: 10.1016/0009-2797(81)90070-3. [DOI] [PubMed] [Google Scholar]
  12. Howard J. D., Mottet N. K. Effects of methylmercury on the morphogenesis of the rat cerebellum. Teratology. 1986 Aug;34(1):89–95. doi: 10.1002/tera.1420340112. [DOI] [PubMed] [Google Scholar]
  13. Koerker R. L. The cytotoxicity of methylmercuric hydroxide and colchicine in cultured mouse neuroblastoma cells. Toxicol Appl Pharmacol. 1980 May;53(3):458–469. doi: 10.1016/0041-008x(80)90358-0. [DOI] [PubMed] [Google Scholar]
  14. Leblanc R. M., Joly L. P., Paiement J. pH-dependent interaction between methyl mercury chloride and some membrane phospholipids. Chem Biol Interact. 1984 Feb;48(2):237–241. doi: 10.1016/0009-2797(84)90124-8. [DOI] [PubMed] [Google Scholar]
  15. Miura K., Imura N. Mechanism of methylmercury cytotoxicity. Crit Rev Toxicol. 1987;18(3):161–188. doi: 10.3109/10408448709089860. [DOI] [PubMed] [Google Scholar]
  16. Miura K., Inokawa M., Imura N. Effects of methylmercury and some metal ions on microtubule networks in mouse glioma cells and in vitro tubulin polymerization. Toxicol Appl Pharmacol. 1984 Apr;73(2):218–231. doi: 10.1016/0041-008x(84)90327-2. [DOI] [PubMed] [Google Scholar]
  17. Miura K., Suzuki K., Imura N. Effects of methylmercury on mitotic mouse glioma cells. Environ Res. 1978 Dec;17(3):453–471. doi: 10.1016/0013-9351(78)90048-8. [DOI] [PubMed] [Google Scholar]
  18. Olson F. C., Massaro E. J. Effects of methyl mercury on murine fetal amino acid uptake, protein synthesis and palate closure. Teratology. 1977 Oct;16(2):187–194. doi: 10.1002/tera.1420160213. [DOI] [PubMed] [Google Scholar]
  19. Peckham N. H., Choi B. H. Surface charge alterations in mouse fetal astrocytes due to methyl mercury: an ultrastructural study with cationized ferritin. Exp Mol Pathol. 1986 Apr;44(2):230–234. doi: 10.1016/0014-4800(86)90073-0. [DOI] [PubMed] [Google Scholar]
  20. Pollack A., Moulis H., Block N. L., Irvin G. L., 3rd Quantitation of cell kinetic responses using simultaneous flow cytometric measurements of DNA and nuclear protein. Cytometry. 1984 Sep;5(5):473–481. doi: 10.1002/cyto.990050507. [DOI] [PubMed] [Google Scholar]
  21. Ramel C. Methylmercury as a mitosis disturbing agent. Nihon Ishikai Zasshi. 1969 May 1;61(9):1072–1076. [PubMed] [Google Scholar]
  22. Rodier P. M., Aschner M., Sager P. R. Mitotic arrest in the developing CNS after prenatal exposure to methylmercury. Neurobehav Toxicol Teratol. 1984 Sep-Oct;6(5):379–385. [PubMed] [Google Scholar]
  23. Rotman B., Papermaster B. W. Membrane properties of living mammalian cells as studied by enzymatic hydrolysis of fluorogenic esters. Proc Natl Acad Sci U S A. 1966 Jan;55(1):134–141. doi: 10.1073/pnas.55.1.134. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Sager P. R., Doherty R. A., Olmsted J. B. Interaction of methylmercury with microtubules in cultured cells and in vitro. Exp Cell Res. 1983 Jun;146(1):127–137. doi: 10.1016/0014-4827(83)90331-2. [DOI] [PubMed] [Google Scholar]
  25. Sager P. R. Selectivity of methyl mercury effects on cytoskeleton and mitotic progression in cultured cells. Toxicol Appl Pharmacol. 1988 Jul;94(3):473–486. doi: 10.1016/0041-008x(88)90288-8. [DOI] [PubMed] [Google Scholar]
  26. Sager P. R., Syversen T. L. Differential responses to methylmercury exposure and recovery in neuroblastoma and glioma cells and fibroblasts. Exp Neurol. 1984 Aug;85(2):371–382. doi: 10.1016/0014-4886(84)90147-x. [DOI] [PubMed] [Google Scholar]
  27. Slotkin T. A., Bartolome J. Biochemical mechanisms of developmental neurotoxicity of methylmercury. Neurotoxicology. 1987 Spring;8(1):65–84. [PubMed] [Google Scholar]
  28. Slotkin T. A., Kavlock R. J., Cowdery T., Orband L., Bartolome M., Whitmore W. L., Bartolome J. Effects of neonatal methylmercury exposure on adrenergic receptor binding sites in peripheral tissues of the developing rat. Toxicology. 1986 Oct;41(1):95–106. doi: 10.1016/0300-483x(86)90107-1. [DOI] [PubMed] [Google Scholar]
  29. Traxinger D. L., Atchison W. D. Comparative effects of divalent cations on the methylmercury-induced alterations of acetylcholine release. J Pharmacol Exp Ther. 1987 Feb;240(2):451–459. [PubMed] [Google Scholar]
  30. Verschaeve L., Kirsch-Volders M., Hens L., Susanne C. Comparative in vitro cytogenetic studies in mercury-exposed human lymphocytes. Mutat Res. 1985 Aug-Sep;157(2-3):221–226. doi: 10.1016/0165-1218(85)90119-3. [DOI] [PubMed] [Google Scholar]
  31. Verschaeve L., Kirsch-Volders M., Susanne C., Groetenbriel C., Haustermans R., Lecomte A., Roossels D. Genetic damage induced by occupationally low mercury exposure. Environ Res. 1976 Dec;12(3):306–316. doi: 10.1016/0013-9351(76)90040-2. [DOI] [PubMed] [Google Scholar]
  32. Vogel D. G., Margolis R. L., Mottet N. K. The effects of methyl mercury binding to microtubules. Toxicol Appl Pharmacol. 1985 Sep 30;80(3):473–486. doi: 10.1016/0041-008x(85)90392-8. [DOI] [PubMed] [Google Scholar]
  33. Vogel D. G., Rabinovitch P. S., Mottet N. K. Methylmercury effects on cell cycle kinetics. Cell Tissue Kinet. 1986 Mar;19(2):227–242. doi: 10.1111/j.1365-2184.1986.tb00733.x. [DOI] [PubMed] [Google Scholar]
  34. Zucker R. M., Elstein K. H., Easterling R. E., Massaro E. J. Flow cytometric analysis of the cellular toxicity of tributyltin. Toxicol Lett. 1988 Oct;43(1-3):201–218. doi: 10.1016/0378-4274(88)90029-x. [DOI] [PubMed] [Google Scholar]
  35. Zucker R. M., Elstein K. H., Easterling R. E., Massaro E. J. Flow cytometric comparison of the effects of trialkyltins on the murine erythroleukemic cell. Toxicology. 1989 Oct 2;58(2):107–119. doi: 10.1016/0300-483x(89)90001-2. [DOI] [PubMed] [Google Scholar]
  36. Zucker R. M., Elstein K. H., Easterling R. E., Massaro E. J. Flow cytometric discrimination of mitotic nuclei by right-angle light scatter. Cytometry. 1988 May;9(3):226–231. doi: 10.1002/cyto.990090307. [DOI] [PubMed] [Google Scholar]
  37. Zucker R. M., Elstein K. H., Easterling R. E., Ting-Beall H. P., Allis J. W., Massaro E. J. Effects of tributyltin on biomembranes: alteration of flow cytometric parameters and inhibition of Na+, K+-ATPase two-dimensional crystallization. Toxicol Appl Pharmacol. 1988 Nov;96(2):393–403. doi: 10.1016/0041-008x(88)90097-x. [DOI] [PubMed] [Google Scholar]

Articles from The American Journal of Pathology are provided here courtesy of American Society for Investigative Pathology

RESOURCES