Elsevier

Free Radical Biology and Medicine

Volume 45, Issue 9, 1 November 2008, Pages 1308-1317
Free Radical Biology and Medicine

Original Contribution
The manganese superoxide dismutase Ala16Val dimorphism modulates iron accumulation in human hepatoma cells

https://doi.org/10.1016/j.freeradbiomed.2008.08.011Get rights and content

Abstract

The Ala/16Val dimorphism incorporates alanine (Ala) or valine (Val) in the mitochondrial targeting sequence of manganese superoxide dismutase (MnSOD), modifying MnSOD mitochondrial import and activity. In alcoholic cirrhotic patients, the Ala-MnSOD allele is associated with hepatic iron accumulation and an increased risk of hepatocellular carcinoma. The Ala-MnSOD variant could modulate the expression of proteins involved in iron storage (cytosolic ferritin), uptake (transferrin receptors, TfR-1 and-2), extrusion (hepcidin), and intracellular distribution (frataxin) to trigger hepatic iron accumulation. We therefore assessed the Ala/Val-MnSOD genotype and the hepatic iron score in 162 alcoholic cirrhotic patients. In our cohort, this hepatic iron score increased with the number of Ala-MnSOD alleles. We also transfected Huh7 cells with Ala-MnSOD-or Val-MnSOD-encoding plasmids and assessed cellular iron, MnSOD activity, and diverse mRNAs and proteins. In Huh7 cells, MnSOD activity was higher after Ala-MnSOD transfection than after Val-MnSOD transfection. Additionally, iron supplementation decreased transfected MnSOD proteins and activities. Ala-MnSOD transfection increased the mRNAs and proteins of ferritin, hepcidin, and TfR2, decreased the expression of frataxin, and caused cellular iron accumulation. In contrast, Val-MnSOD transfection had limited effects. In conclusion, the Ala-MnSOD variant favors hepatic iron accumulation by modulating the expression of proteins involved in iron homeostasis.

Introduction

Alcohol consumption increases reactive oxygen species (ROS) formation in mitochondria [1], thus causing oxidative stress and mitochondrial damage [2], [3], [4]. The alcohol-induced increases in ROS and cytokines lead to cell death, fibrogenesis, and carcinogenesis [5], [6]. ROS detoxification by hepatic mitochondria relies on the successive action of superoxide dismutases and other enzymes. Manganese superoxide dismutase (MnSOD) is present in the matrix and on the inner membrane of hepatic mitochondria, while copper–zinc superoxide dismutase (CuZnSOD) is found in the mitochondrial intermembrane space [7]. MnSOD and CuZnSOD catalyze the dismutation of the superoxide anion into hydrogen peroxide, which is then detoxified into water by glutathione peroxidase 1 (GPx1) and peroxiredoxin III [8], [9], while catalase is absent in hepatic mitochondria [10]. Alcohol consumption may cause a basal imbalance between MnSOD and GPx1 activities, and this basal imbalance may be further aggravated by genetic polymorphisms, which modulate the activities of antioxidant enzymes [11]. One genetic dimorphism incorporates either an alanine (Ala) or a valine (Val) in the mitochondrial targeting sequence of human MnSOD. In this Ala16Val MnSOD dimorphism, the Ala-MnSOD variant is better imported into the mitochondria than the Val-MnSOD variant, and its mRNA is more stable, leading to a higher MnSOD activity than the Val-MnSOD variant [12]. Another genetic dimorphism encodes for either proline (Pro) or leucine (Leu) at codon 198 of human GPx1 [13]. In the presence of physiologic concentrations of selenium, the Leu-GPx1 variant exhibits a lower enzyme activity than the Pro-GPx1 variant [13]. We have previously shown that the Ala-MnSOD allele, particularly when combined with the Leu-GPx1 allele, is a major risk factor for hepatocellular carcinoma (HCC) development and death in alcoholic cirrhotic patients [14], [15]. Similarly, women who had two Ala-MnSOD-encoding alleles and, concomitantly, two Leu-GPx1-encoding alleles had an increased risk of developing breast cancer [16]. Thus, more than a specific MnSOD activity or GPx1 activity, it is the imbalance between these two antioxidant enzyme activities, which seems to increase the risk of hepatic and breast cancer, possibly by leading to high H2O2 steady-state levels and/or high hydroxyl radical formation [15], [16], [17].

Although H2O2 is already reactive by itself, it can react with Fe2+to form the hydroxyl radical, via the Fenton reaction. The highly reactive hydroxyl radical can then damage lipids, proteins, and DNA, to cause somatic DNA mutations and cancer [15]. Thus, the deleterious consequences of H2O2 formation could be amplified by concomitant iron accumulation. Interestingly, iron stores can be increased in alcoholic liver disease, suggesting a possible synergistic role of iron in hepatotoxicity and fibrosis [18]. Furthermore, in a cohort of alcoholic cirrhotic patients prospectively followed up from the time of the first diagnosis of cirrhosis until the time of HCC occurrence or death, we have found that those patients who might have high H2O2 levels in view of their mitochondrial antioxidant enzyme genotypes also frequently had qualitative hepatic iron accumulation on the initial liver biopsy [15]. This iron accumulation mainly affected the hepatocytes, and was a risk factor for HCC development in this cohort [15]. Taken together, these results led us to hypothesize that a genetically determined imbalance in antioxidant enzymes could influence hepatocarcinogenesis, not only by possibly modulating the steady-state levels of H2O2 but also by contributing to hepatic iron accumulation.

The present study was undertaken to test the concept that the expression of the high activity-associated Ala-MnSOD variant could favor hepatic iron accumulation. We first determined the hepatic iron score on a Perls' stain, as well as the Ala/Val-MnSOD genotype, in 162 alcoholic patients with cirrhosis. We then developed an in vitro model focused on mitochondrial antioxidant system modulation in an attempt to mimic the clinical situation. For this purpose, we transfected Huh7 human hepatoma cells with an empty plasmid or an Ala-MnSOD-or Val-MnSOD-encoding plasmid, and cultured these transfected cells for 48h with iron citrate (50μM). In the alcoholic patients, we found that the hepatic iron score increased progressively with the number (0, 1, or 2) of Ala-MnSOD-encoding alleles. In Huh7 cells, incubation with iron decreased transfected MnSOD activities. With or without iron, however, MnSOD activity was higher after Ala-MnSOD transfection than after transfection with the Val-MnSOD or the empty plasmid. Transfection with Ala-MnSOD in Huh7 cells modulated the expression of several proteins involved in the uptake, extrusion, storage, and cellular distribution of hepatic iron, and led to intracellular iron accumulation.

Section snippets

MnSOD genotyping, hepatic Perls' stain, and hepatic iron score in alcoholic cirrhotic patients

This study is based on a previously described cohort of 162 alcoholic cirrhotic patients prospectively followed up for HCC development according to their MnSOD genotypes and liver iron overload [15]. These patients fulfilled the following inclusion criteria: (1) biopsy-proven hepatic cirrhosis; (2) daily alcohol intake of 80 grams or more; (3) no other cause of liver disease and no infection by the human immunodeficiency virus, hepatitis C or hepatitis B virus; (4) no evidence of HCC at the

MnSOD genotypes and hepatic iron accumulation in alcoholic cirrhotic patients

Hepatic iron was assessed on Perls' stains using the Deugnier's iron score in 162 alcoholic cirrhotic patients whose MnSOD genotype was determined [15]. The Deugnier's iron score (mean ± SEM) increased progressively according to the number of Ala-MnSOD allele(s): 0.9 ± 0.3 (median, 0; 25th percentile, 0; 75th percentile, 1) in Val/Val patients (patients with two Val-MnSOD alleles and no Ala-MnSOD allele), 1.7 ± 0.3 (median, 0.5; 25th percentile, 0; 75th percentile, 3) in Ala/Val patients (one

Discussion

The present study shows that the overexpression of MnSOD through transfection leads to iron accumulation in Huh7 human hepatoma cells cultured in the presence of iron citrate (50 μM). Iron accumulation is mild or absent in cells exposed to iron citrate after transfection with the Val-MnSOD-encoding plasmid, but marked in cells exposed to iron after transfection with the Ala-MnSOD-encoding plasmid (Fig. 1, Fig. 2, Fig. 5). This observation is confirmed by both electron microscopy (Fig. 2) and by

Acknowledgments

The work was supported in part by grants from AFEF (Association Française pour l'Etude du Foie) and Université Paris 13. Pierre Nahon was supported by a fellowship from AFEF (Association Française pour l'Etude du Foie). The authors thank Thomas Chaigneau and Oualid Haddad for technical help.

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