Critical Choices in Predicting Stone Wool Biodurability: Lysosomal Fluid Compositions and Binder Effects.

The hazard potential, including carcinogenicity, of inhaled man-made vitreous fibers (MMVFs) is correlated with their biodurability in the lung, as prerequisite for biopersistence. Abiotic dissolution testing serves to predict biodurability. We re-analyzed the International Agency for Research on Cancer Monograph on MMVFs and found that the correlation between in vivo biopersistence and abiotic dissolution presented therein confounded different simulant fluids and further confounded evaluation of leaching vs structural elements. These are critical choices for abiotic dissolution testing, as are binder removal and the rate of the flow that removes ions during testing. Therefore, we experimentally demonstrated how fluid composition and binder affect abiotic dissolution of a representative stone wool MMVF. We compared six simulant fluids (all pH 4.5, reflecting the environment of alveolar macrophage lysosomes) that differed in organic acids, which have a critical role in their ability to modulate the formation of Si-rich gels on the fiber surfaces. Removing the binder accelerates the average dissolution rate by +104% (max. + 273%) across the fluids by suppression of gel formation. Apart from the high-citrate fluid that predicted a 10-fold faster dissolution than is observed in vivo, none of the five other fluids resulted in dissolution rates above 400 ng/cm2/h, the limit associated with the exoneration from classification for carcinogenicity in the literature. These findings were confirmed with and without binder. For corroboration, five more stone wool MMVFs were assessed with and without binder in one specific fluid. Again, the presence of the binder caused gel formation and reduced dissolution rates. To enhance the reliability and robustness of abiotic predictions of biodurability, we recommend replacing the critically influential citric acid in pH 4.5 fluids with other organic acids. Also, future studies should consider structural transformations of the fibers, including changes in fiber length, fiber composition, and reprecipitation of gel layers.

Nonaqueous capillary electrophoresis as separation technique to support metabolism studies by means of electrochemistry and mass spectrometry.

The first combination of electrochemistry (EC), NACE, and ESI-MS to mimic the metabolic fate of drugs is described. While the combination of EC, HPLC, and ESI-MS has been used for this purpose before, NACE is able to deliver valuable additional information about possible metabolites of harmane when compared to HPLC. In this paper, NACE is used as a comprehensive separation technique in metabolism studies of harmane, a naturally occurring monoaminooxidase inhibitor, since it exhibits beneficial properties for the separation of polar compounds. Harmane is known to be metabolized via the oxidative metabolism catalyzed by cytochrome P450 enzymes, which are the most important metabolizing superfamily of enzymes in the human liver. The application of HPLC and NACE enabled the detection of 37 products in total, with 14 different mass-to-charge ratios. A total of 31 products could be detected in HPLC-MS and 26 in NACE-MS analysis. The combination of both NACE and RP-HPLC allows the identification of significantly more potential metabolites than any of the separation techniques alone.

Investigating the influence of standard staining procedures on the copper distribution and concentration in Wilson's disease liver samples by laser ablation-inductively coupled plasma-mass spectrometry.

The influence of rhodanine and haematoxylin and eosin (HE) staining on the copper distribution and concentration in liver needle biopsy samples originating from patients with Wilson's disease (WD), a rare autosomal recessive inherited disorder of the copper metabolism, is investigated. In contemporary diagnostic of WD, rhodanine staining is used for histopathology, since rhodanine and copper are forming a red to orange-red complex, which can be recognized in the liver tissue using a microscope. In this paper, a laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) method is applied for the analysis of eight different WD liver samples. Apart from a spatially resolved elemental detection as qualitative information, this LA-ICP-MS method offers also quantitative information by external calibration with matrix-matched gelatine standards. The sample set of this work included an unstained and a rhodanine stained section of each WD liver sample. While unstained sections of WD liver samples showed very distinct structures of the copper distribution with high copper concentrations, rhodanine stained sections revealed a blurred copper distribution with significant decreased concentrations in a range from 20 to more than 90%. This implies a copper removal from the liver tissue by complexation during the rhodanine staining. In contrast to this, a further HE stained sample of one WD liver sample did not show a significant decrease in the copper concentration and influence on the copper distribution in comparison to the unstained section. Therefore, HE staining can be combined with the analysis by means of LA-ICP-MS in two successive steps from one thin section of a biopsy specimen. This allows further information to be gained on the elemental distribution by LA-ICP-MS additional to results obtained by histological staining.

Downregulation of hepatic multi-drug resistance protein 1 (MDR1) after copper exposure.

Copper homeostasis is strictly regulated in mammalian cells. We investigated the adaptation of hepatocytes after long-term copper exposure. Copper-resistant hepatoma HepG2 cell lines lacking ATP7B were generated. Growth, copper accumulation, gene expression, and transport were determined. Hepatocyte-like cells derived from a Wilson disease (WD) patient and the liver of a WD animal model were also studied. The rapidly gained copper resistance was found to be stable, as subculturing of cells in the absence of added copper (weaning) did not restore copper sensitivity. Intracellular copper levels and the expression of MT1 and HSP70 were increased, whereas the expression of CTR1 was reduced. However, the values normalized after weaning. In contrast, downregulation of multi-drug resistance protein 1 (MDR1), encoding P-glycoprotein (P-gp), was shown to be permanent. Calcein assays confirmed the downregulation of MDR1 in the resistant cell lines. MDR1 knockdown by siRNA resulted in increased copper resistance and decreased intracellular copper. Treatment of the resistant cells with verapamil, a known inducer of MDR1, was followed by increased copper-induced toxicity. Downregulation of MDR1 was also observed in hepatocyte-like cells derived from a WD patient after copper exposure. In addition, MDR1 was downregulated in Long-Evans Cinnamon rats when the liver copper was elevated. The results indicate that downregulation of MDR1 is an adaptation of hepatic cells after sustained copper exposure when ATP7B is non-functional. Our data add to the versatile functions of MDR1 in the hepatocyte and may have an impact on the treatment of copper-related diseases, prominently WD.

Spatial investigation of the elemental distribution in Wilson's disease liver after d-penicillamine treatment by LA-ICP-MS.

At present, the copper chelator d-penicillamine (DPA) is the first-line therapy of Wilson's disease (WD), which is characterized by an excessive copper overload. Lifelong DPA treatments aim to reduce the amount of detrimental excess copper retention in the liver and other organs. Although DPA shows beneficial effect in many patients, it may cause severe adverse effects. Despite several years of copper chelation therapy, discontinuation of DPA therapy can be linked to a rapidly progressing liver failure, indicating a high residual liver copper load. In order to investigate the spatial distribution of remaining copper and additional elements, such as zinc and iron, in rat and human liver samples after DPA treatment, a high resolution (spotsize of 10µm) laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) imaging method was applied. Untreated LPP-/- rats, an established animal model for WD, appeared with a high overall copper concentration and a copper distribution of hotspots distributed over the liver tissue. In contrast, a low (>2-fold decreased) overall copper concentration was detected in liver of DPA treated animals. Importantly, however, copper distribution was highly inhomogeneous with lowest concentrations in direct proximity to blood vessels, as observed using novel zonal analysis. A human liver needle biopsy of a DPA treated WD patient substantiated the finding of an inhomogeneous copper deposition upon chelation therapy. In contrast, comparatively homogenous distributions of zinc and iron were observed. Our study indicates that a high resolution LA-ICP-MS analysis of liver samples is excellently suited to follow efficacy of chelator therapy in WD patients.

Tracing gadolinium-based contrast agents from surface water to drinking water by means of speciation analysis.

In recent decades, a significant amount of anthropogenic gadolinium has been released into the environment as a result of the broad application of contrast agents for magnetic resonance imaging (MRI). Since this anthropogenic gadolinium anomaly has also been detected in drinking water, it has become necessary to investigate the possible effect of drinking water purification on these highly polar microcontaminats. Therefore, a novel highly sensitive method for speciation analysis of gadolinium is presented. For that purpose, the hyphenation of hydrophilic interaction liquid chromatography (HILIC) and inductively coupled plasma-mass spectrometry (ICP-MS) was employed. In order to enhance the detection power, sample introduction was carried out by ultrasonic nebulization. In combination with a novel HILIC method using a diol-based stationary phase, it was possible to achieve superior limits of detection for frequently applied gadolinium-based contrast agents below 20pmol/L. With this method, the contrast agents Gd-DTPA, Gd-DOTA and Gd-BT-DO3A were determined in concentrations up to 159pmol/L in samples from several waterworks in a densely populated region of Germany alongside the river Ruhr as well as from a waterworks near a catchment lake. Thereby, the direct impact of anthropogenic gadolinium species being present in the surface water on the amount of anthropogenic gadolinium in drinking water was shown. There was no evidence for the degradation of contrast agents, the release of Gd(3+) or the presence of further Gd species.

Element bioimaging of liver needle biopsy specimens from patients with Wilson's disease by laser ablation-inductively coupled plasma-mass spectrometry.

A laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) method is developed and applied for the analysis of paraffin-embedded liver needle biopsy specimens of patients with Wilson's disease (WD), a rare autosomal recessive disorder of the copper metabolism causing various hepatic, neurological and psychiatric symptoms due to a copper accumulation in the liver and the central nervous system. The sample set includes two WD liver samples and one negative control sample. The imaging analysis was performed with a spatial resolution of 10 µm. Besides copper, iron was monitored because an elevated iron concentration in the liver is known for WD. In addition to this, both elements were quantified using an external calibration based on matrix-matched gelatine standards. The presented method offers low limits of detection of 1 and 5 µg/g for copper and iron, respectively. The high detection power and good spatial resolution allow the analysis of small needle biopsy specimen using this method. The two analyzed WD samples can be well differentiated from the control sample due to their inhomogeneous copper distribution and high copper concentrations of up to 1200 µg/g. Interestingly, the WD samples show an inverse correlation of regions with elevated copper concentrations and regions with high iron concentrations.

Elemental bioimaging and speciation analysis for the investigation of Wilson's disease using µXRF and XANES.

A liver biopsy specimen from a Wilson's disease (WD) patient was analyzed by means of micro-X-ray fluorescence (µXRF) spectroscopy to determine the elemental distribution. First, bench-top µXRF was utilized for a coarse scan of the sample under laboratory conditions. The resulting distribution maps of copper and iron enabled the determination of a region of interest (ROI) for further analysis. In order to obtain more detailed elemental information, this ROI was analyzed by synchrotron radiation (SR)-based µXRF with a beam size of 4 µm offering a resolution at the cellular level. Distribution maps of additional elements to copper and iron like zinc and manganese were obtained due to a higher sensitivity of SR-µXRF. In addition to this, X-ray absorption near edge structure spectroscopy (XANES) was performed to identify the oxidation states of copper in WD. This speciation analysis indicated a mixture of copper(i) and copper(ii) within the WD liver tissue.