title: Magnesium deficiency induces apoptosis in primary cultures of rat hepatocytes.
abstract: The effects of extracellular magnesium (Mg) concentration on the rate of apoptosis in rat hepatocytes in primary culture were examined. After overnight attachment, incubations were conducted for up to 72 h in serum-free media containing low (0-0.4 mmol/L), physiological (0.8 mmol/L) or high (2 and 5.6 mmol/L) Mg concentrations. At 72 h, we observed numerous rounded hepatocytes on top of a shrunken cell monolayer at extracellular Mg concentrations < 0.8 mmol/L. These morphological features were associated with Mg-dependent differences in the total protein levels. The various Mg concentrations did not affect DNA synthesis; however, at a concentration < 0.8 mmol/L, the susceptibility of cultured rat hepatocytes to oxidative stress was increased as shown by the reduced glutathione concentration (10.6 +/- 2.8 vs. 37.3 +/- 4.1 nmol/mg protein with 0 and 0.8 mmol/L, respectively; P < 0.05) and increased lipid peroxidation (0.36 +/- 0.03 vs. 0.21 +/- 0.01 nmol malondialdehyde/mg protein with 0 and 0.8 mmol/L, respectively; P < 0.05). Fluorescence microscopy after Hoechst dye staining revealed numerous apoptotic figures in Mg-free monolayers compared with 0.8 and 5.6 mmol/L Mg conditions. These observations were confirmed quantitatively by flow-cytometric analysis after propidium iodide staining. The proportion of subdiploid cells decreased with increasing Mg concentration; for example, it was greater at 72 h in Mg-free cultures (76%) than in cultures containing 0.8 mmol/L or 5.6 mmol/L Mg (28%; P < 0.05). Caspase-3 was highly activated in Mg-free cultures after 48 h of treatment compared with 0.8 and 5.6 mmol/L conditions (P < 0.05). Overall, these results show that extracellular Mg deficiency has a negative effect on the survival of cultured rat hepatocytes by inducing apoptosis; however, supplementation of extracellular Mg did not reduce the spontaneous apoptosis that occurred over time in rat hepatocyte cultures.
authors.affiliation: Laboratoire de Physiologie, UFR des Sciences Médicales et Pharmaceutiques, Besançon, France.
authors.forename: Hélène
authors.initials: H
authors.lastname: Martin
authors.forename: Lysiane
authors.initials: L
authors.lastname: Richert
authors.forename: Alain
authors.initials: A
authors.lastname: Berthelot
mesh_terms: D000818:Animals; D017209:Apoptosis; D002470:Cell Survival; D002478:Cells, Cultured; D004247:DNA / Q000096:biosynthesis; D022781:Hepatocytes / Q000166:cytology / Q000502:physiology*; D007425:Intracellular Membranes / Q000378:metabolism; D008274:Magnesium / Q000378:metabolism; D008275:Magnesium Deficiency / Q000503:physiopathology*; D008297:Male; D009994:Osmolar Concentration; D018384:Oxidative Stress; D051381:Rats; D017208:Rats, Wistar
chemical_list: D004247:DNA; D008274:Magnesium