The effect of glutathione depletion on methyl mercury-induced microtubule disassembly in cultured embryonal carcinoma cells.
Microtubule (MT) assembly and stability are thought to be dependent on intracellular glutathione for the maintenance of critical sulfhydryl groups. Since methyl mercury (MeHg) is a sulfhydryl-binding toxicant, it is possible that alteration of intracellular glutathione status might enhance the toxic effects of MeHg on microtubules. The influence of MeHg on the relationship between intracellular glutathione and the structural integrity of interphase microtubules was assessed in embryonal carcinoma cells by immunofluorescence microscopy, using antibodies to tyrosinated and acetylated alpha-tubulins. Intracellular glutathione concentrations were reduced by treatment with 10 microM buthionine sulfoximine (BSO; an inhibitor of gamma-glutamyl cysteine synthetase) for 18-24 hr. BSO-treated cells displayed little change in the pattern of microtubule staining, despite reduction of glutathione levels to less than 10% of control levels. Similarly, a combination of BSO and the nonspecific glutathione-depleting agent diethylmaleimide (DEM) had little effect on microtubule networks, except at the highest concentrations of DEM where nonspecific cytotoxicity was observed. The susceptibility of microtubules to MeHg-induced disassembly was determined in normal and glutathione-depleted cells incubated with 1.0 to 7.5 microM MeHg for 2 hr. MeHg treatment alone resulted in concentration-dependent disassembly of microtubules; depletion of glutathione with BSO prior to MeHg treatment did not enhance MT damage. Further, BSO-pretreated cells exposed to MeHg still showed substantial recovery of microtubule networks following removal of MeHg from culture media, even when glutathione levels remained less than 5% of control levels. These data indicate that the integrity of interphase microtubules is largely unaffected by reductions in glutathione concentration and that susceptibility of microtubules to MeHg-induced disassembly is not directly dependent on intracellular glutathione content.
Graff
R D
RD
Department of Pharmacology and Toxicology, College of Pharmacy, Rutgers University, Piscataway, New Jersey 08855-0789.
Philbert
M A
MA
Lowndes
H E
HE
Reuhl
K R
KR
D000818:Animals; D019328:Buthionine Sulfoximine; D004622:Embryo, Mammalian / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure; D005978:Glutathione / Q000037:antagonists & inhibitors / Q000502:physiology*; D008717:Methionine Sulfoximine / Q000031:analogs & derivatives / Q000494:pharmacology; D008767:Methylmercury Compounds / Q000633:toxicity*; D051379:Mice; D008856:Microscopy, Fluorescence; D008870:Microtubules / Q000187:drug effects* / Q000648:ultrastructure; D013438:Sulfhydryl Compounds / Q000378:metabolism; D014407:Tumor Cells, Cultured
D008767:Methylmercury Compounds; D013438:Sulfhydryl Compounds; D008717:Methionine Sulfoximine; D019328:Buthionine Sulfoximine; D005978:Glutathione