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Biochemical and Molecular Actions of Nutrients

Magnesium Deficiency Induces Apoptosis in Primary Cultures of Rat Hepatocytes

Hélène Martin^*, Lysiane Richert and Alain Berthelot^*,1

^* Laboratoire de Physiologie, et Laboratoire de Biologie Cellulaire, UFR des Sciences Médicales et Pharmaceutiques, Besançon, France

¹To whom correspondence should be addressed. E-mail: alain.berthelot{at}univ-fcomte.fr.

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.

KEY WORDS: • primary rat hepatocyte cultures • extracellular magnesium concentration • apoptosis

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