title: Induction of vanadium accumulation and nuclear sequestration causing cell suicide in human Chang liver cells.
abstract: Very little is known about the modulation of vanadium accumulation in cells, although this ultratrace element has long been seen as an essential nutrient in lower life forms, but not necessarily in humans where factors modulating cellular uptake of vanadium seem unclear. Using nuclear microscopy, which is capable of the direct evaluation of free and bound (total) elemental concentrations of single cells we show here that an NH4Cl acidification prepulse causes distinctive accumulation of vanadium (free and bound) in human Chang liver cells, concentrating particularly in the nucleus. Vanadium loaded with acidification but leaked away with realkalinization, suggests proton-dependent loading. Vanadyl(4), the oxidative state of intracellular vanadium ions, is known to be a potent source of hydroxyl free radicals (OH). The high oxidative state of nuclei after induction of vanadyl(4) loading was shown by the redox indicator methylene blue, suggesting direct oxidative damage to nuclear DNA. Flow cytometric evaluation of cell cycle phase-specific DNA composition showed degradation of both 2N and 4N DNA phases in G1, S and G2/M cell cycle profiles to a solitary IN DNA peak, in a dose-dependent manner, effective from micromolar vanadyl(4) levels. This trend was reproduced with microccocal nuclease digestion in a time response, supporting the notion of DNA fragmentation effects. Several other approaches confirmed fragmentation occurring in virtually all cells after 4mM V(4) loading. Ultrastructural profiles showed various stages of autophagic autodigestion and well defined plasma membrane outlines, consistent with programmed cell death but not with necrotic cell death. Direct intranuclear oxidative damage seemed associated with the induction of mass suicide in these human Chang liver cells following vanadium loading and nuclear sequestration.
authors.affiliation: Department of Anatomy, National University of Singapore, Kent Ridge, Singapore.
authors.forename: K H
authors.initials: KH
authors.lastname: Sit
authors.forename: R
authors.initials: R
authors.lastname: Paramanantham
authors.forename: B H
authors.initials: BH
authors.lastname: Bay
authors.forename: K P
authors.initials: KP
authors.lastname: Wong
authors.forename: P
authors.initials: P
authors.lastname: Thong
authors.forename: F
authors.initials: F
authors.lastname: Watt
mesh_terms: D000643:Ammonium Chloride / Q000378:metabolism / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects*; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism*; D004247:DNA / Q000378:metabolism; D004249:DNA Damage; D005434:Flow Cytometry; D005452:Fluoresceins / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D017665:Hydroxyl Radical / Q000378:metabolism; D008099:Liver / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure; D008751:Methylene Blue / Q000378:metabolism; D008836:Micrococcal Nuclease / Q000378:metabolism; D008853:Microscopy; D013050:Spectrometry, Fluorescence; D014639:Vanadium / Q000378:metabolism* / Q000494:pharmacology*; D014965:X-Rays
chemical_list: D005452:Fluoresceins; D005456:Fluorescent Dyes; D014639:Vanadium; D000643:Ammonium Chloride; D017665:Hydroxyl Radical; C043829:2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein; D004247:DNA; D008836:Micrococcal Nuclease; D008751:Methylene Blue