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Journal of the American College of Nutrition, Vol. 25, No. 5, 363-369 (2006)
Published by the American College of Nutrition

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

Hélène Martin, PhD, Catherine Abadie, PhD, Bruno Heyd, MD, PhD, Georges Mantion, MD, PhD, Lysiane Richert, PhD and Alain Berthelot, PhD

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


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 ACKNOWLEDGMENTS
 REFERENCES
 
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{oplus} = Glutamax Williams’E medium • WME– = Williams’E medium without magnesium sulfate


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 ACKNOWLEDGMENTS
 REFERENCES
 
Magnesium (Mg) is an essential nutrient to maintain cellular homeostasis and a normal development/a good health in animals and humans. The abundant literature dealing with the identification of Mg deficiency-related effects, evidences that Mg has pleiotropic effects, ranging from its contribution to the functionality of numerous body organs and different cell types in each organ, to its regulatory role on cellular pathways. Mg deficiency has been reported to exacerbate the risk of heart diseases, hypertension, total stroke, diabetes and atherosclerosis (for review, see [1]). Taken together, these results should have a strong impact on human health recommendations but surprisingly, marginal Mg intake and chronic hypomagnesemia are commonplace in most industrialized countries. As an example, a French population-based epidemiologic study has revealed that 77% of women and 72% of men out of a cohort of 5448 subjects have a dietary Mg intake below the reference intakes [2].

In a previous work we have studied the effects of extracellular Mg concentration on the rate of apoptosis in rat hepatocytes in primary cultures [3]. The liver was chosen as targeted tissue for this analysis since it plays a major role in Mg homeostasis [4] and it regulates many metabolic pathways, including glucose, xenobiotics and steroid hormone metabolisms. Moreover, only few studies have reported on the impact of Mg deficiency on liver [5–9]. We found an induction of apoptosis in Mg-deficient cultures, most probably due to an oxidative stress-related mechanism, as suggested by a decrease in reduced glutathione (GSH) concentration and an increase in lipid peroxidation. Moreover, we recently demonstrated by cDNA microarray study that cellular homeostasis was impaired in rat liver after a chronic nutritional Mg deficiency, mostly as a result of an increased oxidative stress (Martin et al., article in submission). We now aim to address the following questions:

i) Is the influence of extracellular Mg concentration on oxidative stress and apoptosis equivalent in rat and human primary hepatocyte cultures? The extrapolation of data from animals to the humans is indeed sensitive and could have hazardous consequences on human health when misinterpreted. In this context, human hepatocytes in primary cultures are now recognized as the "gold standard" in vitro model for evaluating the impact of drugs and other xenobiotics on human liver, as evidenced by the abundant literature using this model in the pharmacotoxicology research and development area [10–14].

ii) Could a GSH supplementation counteract Mg deficiency-induced hepatocyte oxidative stress and apoptosis? Our observation of a decrease in GSH concentration in rat hepatocytes cultured in the presence of low Mg concentration [3] is in line with several publications reporting a reduction in GSH content associated with Mg deficiency in liver, red blood cells and cultured endothelial and cortical cells of both animal [8,15–19] and human [20] origin. Here we added N-acetylcysteine (NAC), a GSH synthesis precursor after its de-acetylation to cysteine [21], to culture media. NAC has been used therapeutically in several disorders related to oxidative stress and is widely used for the treatment of hepatic failure due to paracetamol overdose [22].


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 ACKNOWLEDGMENTS
 REFERENCES
 
Chemicals
Collagenase type IV, Glutamax® Williams’E medium (WME{oplus}), Williams’E medium without magnesium sulfate (WME–), HBSS, phosphate buffered saline (PBS), fetal calf serum (FCS), glutamine were purchased from InVitrogen (Paisley, UK). Hydrocortisone hemisuccinate, insulin, bovine serum albumin (BSA) and NAC were from Sigma Chemicals (St Louis, MO, USA). Magnesium sulfate was from Prolabo (Fontenay/bois, France). All other reagents were of analytical grade.

Isolation, Culture And Treatment Of Hepatocytes
Rat hepatocytes were isolated as previously described [3]. Human liver samples were obtained from macroscopically healthy surgical waste after tumor resection (for details, see Table 1). Human hepatocytes were isolated by a two-step collagenase perfusion, following the recently published protocol of harvest and transport of liver resections and isolation of hepatocytes [14].


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  		Table 1. Characteristics of Human Liver Samples

 
Isolated rat and human hepatocytes were seeded onto collagen-coated six-well plates (1 x 106 viable cells/well) in WME{oplus} supplemented with FCS (10%), insulin (4 mg/L), hydrocortisone (10–5 M) and gentamicin (50 mg/L) and cultured at 37°C in humidified 5% CO2/95% air. After overnight attachment, the medium was changed to a serum-free medium, consisted of WME– supplemented with glutamine (2 mM), insulin (4 mg/L), hydrocortisone (10–5 M) and gentamicin (50 mg/L). At this time point (referred as T0), cultures were exposed to various concentrations of magnesium sulfate (Mg: 0 to 0.8 mM). Subsequently the medium was changed every 24h. Moreover some hepatocyte cultures were treated with NAC (various concentrations of magnesium sulfate + NAC 5 mM) for 24 h.

Oxidative Stress Evaluation
At the appropriate time points following treatment, hepatocyte homogenates were prepared as previously described [23] and were frozen at –80°C until analysis. GSH and thiobarbituric acid reactive substances (TBARS) concentrations were determined from hepatocyte homogenates as previously described [24]. GSH concentration was determined from the standard curve for GSH and expressed as nmol per mg protein. TBARS concentrations were calculated relative to a standard preparation of 1,1,3,3-tetra-ethoxypropane and expressed as nmol malondialdehyde (MDA) per mg protein.

Apoptosis Evaluation
At the appropriate time points following treatment, hepatocyte monolayers were washed once with ice-cold PBS. Cells from two wells per group were scraped in PBS and pelleted at 70 x g for 3 min at 4°C. Cell pellets were frozen and stored at –80°C until use. Caspase-3 activity was then measured according to the manufacturer’s instructions. Caspase-3 activity was measured with a commercially available kit (Interchim, Montluçon, France). Standard curves were obtained using 7-amino-4-methylcoumarin (AMC). Caspase-3 activity was determined using a fluorescence spectrophotometer (excitation at 340 nm and emission at 450 nm) and expressed as pmol AMC per min per mg protein.

Protein Determination
The protein concentration was determined by the bicinchoninic acid protein assay kit, according to the manufacturer’s instructions (Sigma Chemicals, St Louis, MO, USA) and BSA was used as a standard.

Statistical Analysis
Statistical comparisons among experimental animal groups were performed by one-way analysis of variance, using Tukey’s test to adjust for the multiple comparisons of each mean with each other mean. The level of statistical significance was set at 0.05.


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
ACKNOWLEDGMENTS
 REFERENCES
 
Comparison of the Effects of Mg Concentrations on Oxidative Stress and Apoptosis in Cultured Hepatocytes from Rat and Human Origin
Among the sensitive markers of apoptosis we used in our previous publication dealing with the effect of Mg on rat hepatocytes [3], caspase-3 activity was chosen as an indicator of apoptosis in the present study based on the scarcity of human material available for these analyses. As shown in Fig. 1A, time course of this activity in the human hepatocyte culture HH1 treated either with deficient or with the standard concentrations of Mg was quite similar to that we obtained in rat hepatocytes [3]. No differences were observed for up to 48h of culture with the different Mg concentrations, but after 60h of culture, caspase-3 activity was significantly higher in Mg-free cultures, as compared to the activity in hepatocytes exposed to 0.2, 0.4 and 0.8 mM Mg, which was similar. After 72h of culture, a Mg concentration-dependent effect on the activity was observed in this culture, caspase-3 activity being highest in Mg-free culture conditions. The increase of activity with time in culture obtained in physiological Mg condition reflects spontaneous apoptosis occurring in hepatocyte in primary culture. Of particular interest is therefore that we demonstrated an accentuation of this effect in Mg-deficient culturing conditions. This was confirmed in two additional human hepatocyte cultures (HH2 and HH5) after 72h of treatment (Fig. 1B). The profiles of a Mg concentration-dependent effect on apoptosis in human hepatocytes were equivalent to that obtained in rat hepatocyte cultures (RH, Fig. 1B).


Figure 1
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  		Fig. 1. Effects of extracellular magnesium on caspase-3 activation. (A) Time course analysis in a primary culture of human hepatocytes (HH1) exposed to 0, 0.2, 0.4 and 0.8 mM magnesium. (B) Concentration-dependent analysis in three representative primary cultures of human hepatocytes (HH1, HH2 and HH5) and cultures of rat hepatocytes (RH, expressed as means + SEM, n = 3 different hepatocyte cultures, means without a common letter (a, b, c) differ, p ≤ 0.05) exposed to 0, 0.2, 0.4 and 0.8 mM magnesium for 72 h. Caspase-3 activities in the 0.8 mM condition are 191.8, 90.9, 243.1 and 9.1 pmol AMC/min/mg protein for HH1, HH2, HH5 and RH, respectively.

 
In the present study we confirm a decrease in GSH concentration in rat hepatocytes cultured in the presence of low Mg concentration (RH, Fig. 2), in accordance with our previous observations [3] and several publications both in animal organs and in different types of isolated cells [8,15–20]. We also measured GSH concentration in different human hepatocyte preparations (HH2, HH3, HH4) cultured either in Mg-deficient conditions (0, 0.2 or 0.4 mM Mg) or in physiological Mg concentration condition (0.8 mM Mg) (Fig. 2). In the three cultures tested, a Mg concentration-dependent effect in GSH concentration was obtained, the lowest concentration being obtained in Mg-free cultures as compared to the other conditions. These profiles were very similar to that obtained in rat hepatocyte cultures (RH), GSH concentrations being lowest in Mg-free cultures (Fig. 2).


Figure 2
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  		Fig. 2. Effects of extracellular magnesium on GSH concentration. Concentration-dependent analysis in three primary cultures of human hepatocytes (HH2, HH3 and HH4) and cultures of rat hepatocytes (RH, expressed as means + SEM, n = 3 different hepatocyte cultures, means without a common letter (a, b, c) differ p ≤ 0.05) exposed to 0, 0.2, 0.4 and 0.8 mM magnesium for 72 h. GSH concentrations in the 0.8 mM condition are 48.7, 66.5, 30.4 and 23.8 nmol/mg protein for HH2, HH3, HH4 and RH, respectively.

 
Taken together, our results show that Mg deficiency induces apoptosis in hepatocytes from both rat and human origin, with identical profiles and associated with decreases in GSH concentrations, demonstrating that no species-difference exists in the sensitivity of hepatocytes to Mg deficiency.

Implication of GSH on Mg Deficiency-Induced Apoptosis in Hepatocytes
In an attempt to precise the role of GSH in the observed increase in apoptosis in cultured hepatocytes placed in Mg-deficient conditions, we added NAC to culture medium which has been shown to allow hepatocytes to enhance de novo synthesis of GSH [21]. We used Mg concentrations which clearly affected GSH concentration in rat and human cultures, i.e. 0 and 0.2 mM Mg, and compared with hepatocytes cultured in standard conditions (0.8 mM Mg). Since we found that the deleterious effect of Mg deficiency did not occur within the first 24h of culture, both in rat (see Fig. 5 in [3]) and human hepatocyte cultures, NAC was added to the culture media after a 24h period of culture and hepatocytes were cultured for additional 24h with the same Mg concentration as before, but in the presence of NAC. Fig. 3 illustrates the effect of NAC supplementation in Mg-deficient and Mg standard cultures of rat hepatocytes. As expected, and in line with literature data [25–31], the addition of NAC to culture media significantly increased GSH concentrations. Both caspase-3 activity and lipid peroxidation in hepatocytes cultured with 0 and 0.2 mM Mg were significantly decreased when NAC was added, as compared with cultures without NAC (p ≤ 0.05). In contrast, and surprisingly, NAC addition significantly increased (p ≤ 0.05) both caspase-3 activity and lipid peroxidation in hepatocytes cultured in standard conditions, i.e. in the presence of 0.8 mM Mg.


Figure 3
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  		Fig. 3. Effect of NAC supplementation in Mg deficient and Mg standard cultures of rat hepatocytes, on reduced glutathione (GSH), caspase-3 activity and thiobarbituric acid reactive substances (TBARS) concentrations. {blacksquare} control cultures and {square} cultures treated with NAC (5 mM). Values are means ± S.E.M. (n = 3 different hepatocyte cultures). For control cultures, means without a common letter (a, b, c) differ (p ≤ 0.05). For NAC cultures, means without a common letter (a', b', c') differ (p ≤ 0.05). *Different from control cultures without NAC in the corresponding Mg treatment condition (p ≤ 0.05).

 
Our results thus suggest that NAC could act as an anti-oxidant/anti-apoptotic compound in Mg-deficient culture conditions but not in physiological Mg culture conditions. In fact, publications dealing with the effect of NAC on the incidence of hepatocyte apoptosis are controversial, with some studies describing a beneficial effect [26–28,30,32–34], others a deleterious effect [31,32] or no protection [35] on apoptosis. More important, the role of GSH per se as well as its precursors including NAC, seems to be dual, since it has been suggested that they can either act as antioxidants and as pro-oxidants [30,36–40]. Since the effects of NAC on apoptosis and lipid peroxidation did not strictly correlate with intracellular GSH concentration (Fig. 3), we cannot exclude that the effects of NAC are, at least partly, unrelated to its role as GSH precursor.

Thus, in agreement with previous results of Shaikh et al. [41] showing a NAC protection against cadmium-induced lipid peroxidation without a significant modulation of GSH levels, we can hypothesise that the protective effect of NAC in Mg deficient conditions may be related:

i) to its antioxidant/radical scavenger properties [28,42], since Mg deficiency induced an increase in reactive radicals [43,44] and an impairment of the antioxydant enzyme system [20],

ii) to its capacity to modulate the redox states of cysteine residues of target proteins involved in stress response/apoptosis pathway, as recently described [45–47]. Indeed, Bussière et al. [48] reported an induction of genes encoding for this class of proteins in the Mg-deficient animals compared to the controls,

iii) to an anti-inflammatory effect [49,50] since several authors reported an inflammatory response in rats following Mg deficiency [51–54].

As important is however the observation that an excess of NAC and/or GSH could also be deleterious to cells in a physiological environment. Further investigation is needed to understand the exact role of NAC in Mg related apoptotic processes.

In conclusion, the present study shows that Mg deficiency induced oxidative stress and apoptosis in hepatocytes from both rat and human origin, with identical profiles, demonstrating that no species-difference exists in the sensitivity of hepatocytes to Mg deficiency. Finally, NAC per se and/or as a GSH precursor could counteract the Mg deficiency-induced hepatocyte apoptosis, and the related oxidative stress environment. However, an excess of NAC and/or GSH could be deleterious to hepatocytes when placed in a Mg-physiological environment.


   ACKNOWLEDGMENTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 ACKNOWLEDGMENTS
REFERENCES
 
The authors wish to thank Alexandre Bonet for his excellent technical assistance.

Received March 6, 2005. Revised January 17, 2006.
   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 ACKNOWLEDGMENTS
 REFERENCES
 

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