Compound-specific adaptation of hepatoma cell lines to toxic iron.

Cellular adaptation to excess iron (Fe) is a major determinant to protect tissues from toxicity. The adaptation of hepatoma cell lines following exposure to toxic levels of Fe compounds was studied. A dose- and time-dependent induction of toxicity was observed that was strictly compound-specific. Similar ranging orders of toxicity, i.e. iron chloride >iron sulfate >iron citrate, were observed in four human hepatoma cell lines. Long-term cultivation of HepG2 cells in 10 mM iron citrate resulted in a resistant cell line that displayed high proliferation rates for several months. Resistant cells showed increased viability at iron citrate concentrations ranging from 5-15 mM, while exposition to iron chloride or iron sulfate induced high rates of toxicity similar to parental cells. Resistance was not due to decreased Fe uptake/storage since high intracellular Fe levels were observed. A broad range of modulated gene expression was associated with short- and long-term iron citrate exposition; however, after weaning of resistant cells, re-exposition to Fe induced a similar level of toxicity as observed in parental cells suggesting that a transient adaptation of gene expression was mounted. The results indicate that, depending on the nature of the Fe compound, a specific level of toxicity is induced in hepatic cells which however can be overcome by establishment of resistance.

µXRF and LA-ICP-TQMS for quantitative bioimaging of iron in organ samples of a hemochromatosis model.

Hereditary hemochromatosis is the most common autosomal recessive genetic disorder of the iron metabolism. Iron accumulation in various organs, especially in liver and pancreas leads to diseases and may cause organ failure. In this study, methods for elemental bioimaging by means of quantitative micro X-ray fluorescence analysis (µXRF) and laser ablation-inductively coupled plasma-triple quadrupole mass spectrometry (LA-ICP-TQMS) were developed and applied to investigate the pathophysiological development of iron accumulation in murine tissue based on animals with an iron-overload phenotype caused by a hepatocyte-specific genetic mutation. The use of an external calibration with matrix-matched gelatin standards enables the quantification of iron by means of µXRF without the typically used fundamental parameters method or Monte Carlo simulation, which becomes more imprecise when analyzing thin tissue sections. A fast, non-destructive screening of the iron concentration and distribution with a spatial resolution of 25 µm in liver samples of iron-overload mice was developed. For improved limits of detection and higher spatial resolution down to 4 µm, LA-ICP-TQMS was used with oxygen as reaction gas. By monitoring the mass shift of 56Fe to 56Fe16O, a limit of detection of 0.5 µg/g was obtained. With this method, liver and pancreas samples of iron-overload mice as well as control mice were successfully analyzed. The high spatial resolution enabled the analysis of the iron distribution in different liver lobules. Compared to the established Prussian blue staining, both developed methods proved to be superior due to the possibility of direct iron quantification in the tissues.

Spatially resolved analysis of liquid crystal displays by laser ablation-atmospheric pressure chemical ionization-mass spectrometry.

In recent years, liquid crystal displays (LCDs) became more and more popular in industrial and consumer electronics applications. Unevenly distributed liquid crystals (LC) and the presence of organic contaminations in the LC layer, for example from the display sealant, can cause display defects and therefore lower the display quality. This work describes the development of a fast and easy screening method for display samples by laser ablation coupled to atmospheric pressure chemical ionization-mass spectrometry (LA-APCI-MS). With this ambient technique, LC components as well as sealant materials, which are one of the main sources for display contaminations, can be detected simultaneously using spatial resolutions down to 100 µm. Minimal sample preparation is needed for this method and exact masses can be acquired by using an Orbitrap mass analyzer. By LA-APCI-MS, nineteen different LCs and five different sealant materials were detected as pure substances as well as on commercially available LCDs. In addition, a principle component analysis (PCA) was used to differentiate between LCs and sealant ions in the complex data matrix.

A High-Calorie Diet Aggravates Mitochondrial Dysfunction and Triggers Severe Liver Damage in Wilson Disease Rats.

BACKGROUND & AIMS: In Wilson disease, ATP7B mutations impair copper excretion into bile. Hepatic copper accumulation may induce mild to moderate chronic liver damage or even acute liver failure. Etiologic factors for this heterogeneous phenotype remain enigmatic. Liver steatosis is a frequent finding in Wilson disease patients, suggesting that impaired copper homeostasis is linked with liver steatosis. Hepatic mitochondrial function is affected negatively both by copper overload and steatosis. Therefore, we addressed the question of whether a steatosis-promoting high-calorie diet aggravates liver damage in Wilson disease via amplified mitochondrial damage. METHODS: Control Atp7b+/- and Wilson disease Atp7b-/- rats were fed either a high-calorie diet (HCD) or a normal diet. Copper chelation using the high-affinity peptide methanobactin was used in HCD-fed Atp7b-/- rats to test for therapeutic reversal of mitochondrial copper damage. RESULTS: In comparison with a normal diet, HCD feeding of Atp7b-/- rats resulted in a markedly earlier onset of clinically apparent hepatic injury. Strongly increased mitochondrial copper accumulation was observed in HCD-fed Atp7b-/- rats, correlating with severe liver injury. Mitochondria presented with massive structural damage, increased H2O2 emergence, and dysfunctional adenosine triphosphate production. Hepatocellular injury presumably was augmented as a result of oxidative stress. Reduction of mitochondrial copper by methanobactin significantly reduced mitochondrial impairment and ameliorated liver damage. CONCLUSIONS: A high-calorie diet severely aggravates hepatic mitochondrial and hepatocellular damage in Wilson disease rats, causing an earlier onset of the disease and enhanced disease progression.

ALK3 undergoes ligand-independent homodimerization and BMP-induced heterodimerization with ALK2.

The bone morphogenetic protein (BMP) type I receptors ALK2 and ALK3 are essential for expression of hepcidin, a key iron regulatory hormone. In mice, hepatocyte-specific Alk2 deficiency leads to moderate iron overload with periportal liver iron accumulation, while hepatocyte-specific Alk3 deficiency leads to severe iron overload with centrilobular liver iron accumulation and a more marked reduction of basal hepcidin levels. The objective of this study was to investigate whether the two receptors have additive roles in hepcidin regulation. Iron overload in mice with hepatocyte-specific Alk2 and Alk3 (Alk2/3) deficiency was characterized and compared to hepatocyte-specific Alk3 deficient mice. Co-immunoprecipitation studies were performed to detect the formation of ALK2 and ALK3 homodimer and heterodimer complexes in vitro in the presence and absence of ligands. The iron overload phenotype of hepatocyte-specific Alk2/3-deficient mice was more severe than that of hepatocyte-specific Alk3-deficient mice. In vitro co-immunoprecipitation studies in Huh7 cells showed that ALK3 can homodimerize in absence of BMP2 or BMP6. In contrast, ALK2 did not homodimerize in either the presence or absence of BMP ligands. However, ALK2 did form heterodimers with ALK3 in the presence of BMP2 or BMP6. ALK3-ALK3 and ALK2-ALK3 receptor complexes induced hepcidin expression in Huh7 cells. Our data indicate that: (I) ALK2 and ALK3 have additive functions in vivo, as Alk2/3 deficiency leads to a greater degree of iron overload than Alk3 deficiency; (II) ALK3, but not ALK2, undergoes ligand-independent homodimerization; (III) the formation of ALK2-ALK3 heterodimers is ligand-dependent and (IV) both receptor complexes functionally induce hepcidin expression in vitro.

High spatial resolution LA-ICP-MS demonstrates massive liver copper depletion in Wilson disease rats upon Methanobactin treatment.

Wilson disease (WD) is a rare genetic disorder of the copper metabolism leading to systemic copper accumulation, predominantly in the liver. The therapeutic approach in WD patients is the generation of a negative copper balance and the maintenance of copper homeostasis, currently by the use of copper chelators such as D-penicillamine (D-PA). However, in circumstances of delayed diagnosis, poor treatment compliance, or treatment failure, mortality is almost certain without hepatic transplantation. Moreover, even after years of D-PA treatment, high liver copper levels are present in WD patients. We have recently suggested the use of the bacterial peptide Methanobactin (MB), which has an outstanding binding affinity for copper, as potentially efficient and patient-friendly remedy against copper damage in WD. Here we substantiate these findings considerably, by demonstrating a significant removal of copper from liver samples of WD rats upon short, one week only, MB treatments. Using laser ablation-inductively coupled plasma-mass spectrometry with a spatial resolution down to 4 µm, we demonstrate that only small copper hotspots remain in MB treated animal livers. We further demonstrate in WD rat liver, seven weeks after the stopped MB treatment, a lower liver copper concentration as compared to untreated control animals. Thus, MB highly efficiently depletes liver copper overload with a sustained therapeutic effect.