title: Quantitative analysis of macrophage apoptosis vs. necrosis induced by cobalt and chromium ions in vitro.
abstract: The potential toxicity of metal ions in tissues surrounding metal-metal hip replacements is a cause for concern. Previous studies conducted in our laboratory demonstrated that Co(2+) and Cr(3+) induce TNF-alpha secretion in macrophages, as well as cell mortality. However, the degree of apoptosis and necrosis remained to be investigated. The aim of the present study was to quantify the rate of macrophage mortality by apoptosis vs. necrosis induced by Co(2+) and Cr(3+). J774 mouse macrophages were incubated in growth medium containing 0-10 ppm Co(2+) and 0-500 ppm Cr(3+) for 24 and 48 h under conventional cell culture conditions. Transmission electron microscopy, flow cytometry (Annexin-V fluorescein isothiocyanate/propidium iodide assay) and a specific cell death detection ELISA were used to illustrate cell death and differentiate between apoptotic and necrotic cells. Cell culture exposed to low concentrations of Co(2+) (0-6 ppm) revealed a low degree of mortality. In contrast, at the highest concentrations (8-10 ppm), late apoptosis occurred within 24 h. After 48 h, however, there was a clear evidence for an increase in the rate of necrosis while apoptosis occurred at much lower rate. Macrophages exposed to Cr(3+) demonstrated a predominance of apoptosis after 24h. At concentrations lower than 250 ppm, early and late apoptosis occurred at the same rate. At higher concentrations (250-500 ppm), the number of early apoptotic cells decreased in favor of late apoptosis. After 48 h, lower concentrations of Cr(3+) (150 ppm) induced a higher degree of early apoptosis than after 24 h, and some necrosis. At higher concentrations, the percentage of early apoptotic cells decreased, while necrosis became predominant over late apoptosis. In conclusion, this study demonstrates that macrophage mortality induced by metal ions depends on the type and concentration of metal ions as well as the duration of their exposure. Overall, apoptosis was predominant after 24 h with both Co(2+) and Cr(3+) ions, but high concentrations induced mainly necrosis at 48 h. These results point to the potential for these ions of inducing tissue damage by necrosis if present in large concentrations in vivo.
authors.affiliation: Department of Biomedical Engineering, McGill University, Duff Medical Building, 3775 University Street, Montreal, QC, Canada H3A 2B4.
authors.forename: Isabelle
authors.initials: I
authors.lastname: Catelas
authors.forename: Alain
authors.initials: A
authors.lastname: Petit
authors.forename: Hojatollah
authors.initials: H
authors.lastname: Vali
authors.forename: Cathy
authors.initials: C
authors.lastname: Fragiskatos
authors.forename: Réal
authors.initials: R
authors.lastname: Meilleur
authors.forename: David J
authors.initials: DJ
authors.lastname: Zukor
authors.forename: John
authors.initials: J
authors.lastname: Antoniou
authors.forename: Olga L
authors.initials: OL
authors.lastname: Huk
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D001672:Biocompatible Materials / Q000008:administration & dosage; D002460:Cell Line; D002857:Chromium / Q000008:administration & dosage*; D003035:Cobalt / Q000008:administration & dosage*; D004305:Dose-Response Relationship, Drug; D008264:Macrophages / Q000187:drug effects* / Q000473:pathology*; D008422:Materials Testing; D051379:Mice; D008807:Mice, Inbred BALB C; D009336:Necrosis / Q000473:pathology
chemical_list: D001672:Biocompatible Materials; D002857:Chromium; D003035:Cobalt