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N-Acetylcysteine Partially Reverses Oxidative Stress and Apoptosis Exacerbated by Mg-Deficiency Culturing Conditions in Primary Cultures of Rat and Human Hepatocytes

Laboratoire de Biologie Cellulaire (H.M., L.R.)
Laboratoire de Physiologie (A.B.)
Kaly-Cell (C.A.), UFR des Sciences Médicales et Pharmaceutiques, Service de Chirurgie Digestive et Vasculaire
Hôpital Jean Minjoz (B.H., G.M.), Besançon, FRANCE

Address reprint requests to: Dr. Hélène Martin, Laboratoire de Biologie Cellulaire, EA 3921, UFR des Sciences Médicales et Pharmaceutiques, Place Saint-Jacques, 25030 Besançon cedex, France. E-mail: helene.martin{at}univ-fcomte.fr

Objective: The effects of magnesium (Mg) deficiency on the rate of oxidative stress and apoptosis in primary cultures of human hepatocytes were compared to cultured rat hepatocytes. The possible reversion by N-acetylcysteine (NAC) in Mg-deficient culturing conditions was evaluated.

Methods: Incubations were conducted for up to 72 h in media containing a deficient (0–0.4 mM) or a physiological (0.8 mM) Mg concentration, and in the presence or absence of NAC after 24h of culture in these Mg concentration conditions.

Results: We obtained similar profiles in terms of apoptosis and oxidative stress in primary cultures of human hepatocytes, as compared to rat hepatocytes, i.e. a Mg concentration-dependent effect on the caspase-3 activity and GSH levels after 72h of culture, caspase-3 activity being highest and GSH levels being lowest in Mg-free cultures. The addition of NAC to culture media after the first 24h of culture increased GSH concentrations. This was accompanied in Mg-deficient cultures by a decrease in both the caspase-3 activity and the lipid peroxidation. However, when culturing hepatocytes with physiological Mg concentrations, an increase in both caspase-3 activity and lipid peroxidation was observed.

Conclusions: Our results indicate that Mg deficiency exacerbates the rate of apoptosis in cultured hepatocytes, associated with an increase in oxidative stress, the sensitivity of human hepatocytes being equivalent to that of rat hepatocytes. They also indicate a dual role of NAC and/or GSH, i.e. protective for hepatocytes placed in a Mg-deficient environment, while deleterious for hepatocytes placed in a Mg-physiological environment.

Key words: magnesium concentration, primary hepatocyte cultures, apoptosis, glutathione, lipid peroxidation, interspecies comparison, N-acetylcysteine

Abbreviations: AMC = 7-amino-4-methylcoumarin • BSA = bovine serum albumin • FCS = fetal calf serum • GSH = reduced glutathione • HH = human hepatocytes • MDA = malondialdehyde • Mg = magnesium • NAC = N-acetylcysteine • PBS = phosphate buffered saline • RH = rat hepatocytes • ROS = reactive oxygen species • TBARS = thiobarbituric acid reactive substances • WME = Glutamax Williams’E medium • WME– = Williams’E medium without magnesium sulfate

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