Impact of an SLC30A8 loss-of-function variant on the pancreatic distribution of zinc and manganese: laser ablation-ICP-MS and positron emission tomography studies in mice.

Introduction: Common variants in the SLC30A8 gene, encoding the secretory granule zinc transporter ZnT8 (expressed largely in pancreatic islet alpha and beta cells), are associated with altered risk of type 2 diabetes. Unexpectedly, rare loss-of-function (LoF) variants in the gene, described in heterozygous individuals only, are protective against the disease, even though knockout of the homologous SLC30A8 gene in mice leads to unchanged or impaired glucose tolerance. Here, we aimed to determine how one or two copies of the mutant R138X allele in the mouse SLC30A8 gene impacts the homeostasis of zinc at a whole-body (using non-invasive 62Zn PET imaging to assess the acute dynamics of zinc handling) and tissue/cell level [using laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) to map the long-term distribution of zinc and manganese in the pancreas]. Methods: Following intravenous administration of [62Zn]Zn-citrate (~7 MBq, 150 µl) in wild-type (WT), heterozygous (R138X+/-), and homozygous (R138X+/+) mutant mice (14-15 weeks old, n = 4 per genotype), zinc dynamics were measured over 60 min using PET. Histological, islet hormone immunohistochemistry, and elemental analysis with LA-ICP-MS (Zn, Mn, P) were performed on sequential pancreas sections. Bulk Zn and Mn concentration in the pancreas was determined by solution ICP-MS. Results: Our findings reveal that whereas uptake into organs, assessed using PET imaging of 62Zn, is largely unaffected by the R138X variant, mice homozygous of the mutant allele show a substantial lowering (to 40% of WT) of total islet zinc, as anticipated. In contrast, mice heterozygous for this allele, thus mimicking human carriers of LoF alleles, show markedly increased endocrine and exocrine zinc content (1.6-fold increase for both compared to WT), as measured by LA-ICP-MS. Both endocrine and exocrine manganese contents were also sharply increased in R138X+/- mice, with smaller increases observed in R138X+/+ mice. Discussion: These data challenge the view that zinc depletion from the beta cell is the likely underlying driver for protection from type 2 diabetes development in carriers of LoF alleles. Instead, they suggest that heterozygous LoF may paradoxically increase pancreatic ß-cell zinc and manganese content and impact the levels of these metals in the exocrine pancreas to improve insulin secretion.

Role of the luminal composition on intestinal metal toxicity, bioavailability and bioreactivity: An in vitro approach based on the cell line RTgutGC.

The bioavailability of metal complexes is poorly understood. To evaluate bioavailability and toxicity of neutral and charged complexes as well as free metal ions, Visual Minteq, a chemical equilibrium model, was used to design media containing different metal species. Two non-essential (silver and cadmium) and two essential (copper and zinc) metals were selected. The rainbow trout (Oncorhynchus mykiss) gut cell line (RTgutGC) was used to investigate bioavailability, bioreactivity and toxicity of the different metal species. Toxicity was measured using a multiple endpoint cytotoxicity assay, bioavailability by measuring intracellular metal concentration, and bioreactivity by quantification of mRNA level of the metal responsive genes, metallothionein (MT), glutathione reductase (GR) and zinc transporter 1 (ZnT1). Speciation calculations showed that silver and cadmium preferentially bind chloride, copper phosphate and bicarbonate, and zinc remained primarily as a free ion. Cysteine avidly complexed with all metals reducing toxicity, bioavailability and bioreactivity. Silver and copper toxicity was not affected by inorganic metal speciation, whereas cadmium and zinc toxicity was decreased by chloride complexation. Moreover, reduction of calcium concentration in the medium increased toxicity and bioavailability of cadmium and zinc. Bioavailability of silver and zinc was reduced by low chloride while cadmium bioavailability was increased by low chloride and in presence of bicarbonate. Copper bioavailability was not affected by the medium composition. Cadmium and silver were more bioreactive, independently from the medium composition, in comparison to copper and zinc (i.e., higher induction of MT and GR). Cadmium was the only metal able to induce MT in presence of cysteine. ZnT1 was induced by cadmium in low-chloride, by zinc in low-chloride low-calcium and by cadmium and copper in the bicarbonate media. Overall, this study demonstrates that metal complexation alone is not sufficient to explain metal toxicity, and that anion exchange mechanisms play a role in metal uptake and bioreactivity.

A Systematic Analysis of Metal and Metalloid Concentrations in Eight Zebrafish Recirculating Water Systems.

Metals and metalloids are integral to biological processes and play key roles in physiology and metabolism. Nonetheless, overexposure to some metals or lack of others can lead to serious health consequences. In this study, eight zebrafish facilities collaborated to generate a multielement analysis of their centralized recirculating water systems. We report a first set of average concentrations for 46 elements detected in zebrafish facilities. Our results help to establish an initial baseline for trouble-shooting purposes, and in general for safe ranges of metal concentrations in recirculating water systems, supporting reproducible scientific research outcomes with zebrafish.

Loss of SDHB Promotes Dysregulated Iron Homeostasis, Oxidative Stress, and Sensitivity to Ascorbate.

Succinate dehydrogenase is a key enzyme in the tricarboxylic acid cycle and the electron transport chain. All four subunits of succinate dehydrogenase are tumor suppressor genes predisposing to paraganglioma, but only mutations in the SDHB subunit are associated with increased risk of metastasis. Here we generated an Sdhd knockout chromaffin cell line and compared it with Sdhb-deficient cells. Both cell types exhibited similar SDH loss of function, metabolic adaptation, and succinate accumulation. In contrast, Sdhb-/- cells showed hallmarks of mesenchymal transition associated with increased DNA hypermethylation and a stronger pseudo-hypoxic phenotype compared with Sdhd-/- cells. Loss of SDHB specifically led to increased oxidative stress associated with dysregulated iron and copper homeostasis in the absence of NRF2 activation. High-dose ascorbate exacerbated the increase in mitochondrial reactive oxygen species, leading to cell death in Sdhb-/- cells. These data establish a mechanism linking oxidative stress to iron homeostasis that specifically occurs in Sdhb-deficient cells and may promote metastasis. They also highlight high-dose ascorbate as a promising therapeutic strategy for SDHB-related cancers. SIGNIFICANCE: Loss of different succinate dehydrogenase subunits can lead to different cell and tumor phenotypes, linking stronger 2-OG-dependent dioxygenases inhibition, iron overload, and ROS accumulation following SDHB mutation.

Across the spectrum: integrating multidimensional metal analytics for in situ metallomic imaging.

To know how much of a metal species is in a particular location within a biological context at any given time is essential for understanding the intricate roles of metals in biology and is the fundamental question upon which the field of metallomics was born. Simply put, seeing is powerful. With the combination of spectroscopy and microscopy, we can now see metals within complex biological matrices complemented by information about associated molecules and their structures. With the addition of mass spectrometry and particle beam based techniques, the field of view grows to cover greater sensitivities and spatial resolutions, addressing structural, functional and quantitative metallomic questions from the atomic level to whole body processes. In this perspective, I present a paradigm shift in the way we relate to and integrate current and developing metallomic analytics, highlighting both familiar and perhaps less well-known state of the art techniques for in situ metallomic imaging, specific biological applications, and their use in correlative studies. There is a genuine need to abandon scientific silos and, through the establishment of a metallomic scientific platform for further development of multidimensional analytics for in situ metallomic imaging, we have an incredible opportunity to enhance the field of metallomics and demonstrate how discovery research can be done more effectively.

Lead Speciation in Microorganisms.

The biogeochemical cycles of lead (Pb) have been largely affected by anthropogenic activities as a result of its high natural abundance and use over the centuries [1]. At sites more strongly impacted by urbanization [2] and mining [3], Pb is found at high nano to low micromolar concentrations in surface waters, and can be significantly higher in soil and sediment [4]. Microorganisms are found everywhere and their responses to Pb exposure can range from resistant to highly sensitive [5, 6]. These varying levels of toxicity can be attributed to the cellular handling of Pb, making it important to understand the role of intracellular Pb speciation for more accurate toxicity predictions.

Impact of chronic lead exposure on metal distribution and biological effects to periphyton.

Chronic Pb exposure microcosm studies were carried out on two different periphyton communities over the course of 3 weeks to link Pb distribution to biological effects in periphyton. We show that three-week exposures of periphyton to 20.6 ± 0.4 µM PbT (330 nM Pb(2+)) did not have observable biological effects on photosynthesis, respiration, extracellular enzymatic activities, or biomass accrual. Metal distribution studies showed that the majority of Pb was associated with the operationally defined sorbed and non-EDTA-exchangeable fractions, and relatively little with extracellular polymeric substances (EPS). No significant effects of Pb on Fe and Mn distributions were observed, whereas higher Cu accumulation occurred from increased free Cu(2+) in the exposure medium. High Fe:C and Mn:C ratios indicated the presence of inorganic Fe and Mn material associated with the non-EDTA-exchangeable fraction, which likely sequesters Pb and explains the absence of measurable biological effects. Although no toxic effects of Pb were observed on the periphytic organisms themselves, periphyton can be a significant source of Pb to grazing organisms in aquatic ecosystems.

Conference report CH analysis 2013.

Two PhD students reflect on their experience and impressions of the latest CH analysis 2013 conference, held at Beatenberg this past November.

Characterization of extracellular polymeric substances (EPS) from periphyton using liquid chromatography-organic carbon detection-organic nitrogen detection (LC-OCD-OND).

A protocol was developed to extract, fractionate, and quantitatively analyze periphyton extracellular polymeric substances (EPS), which obtains both information on the molecular weight (M r) distribution and protein and polysaccharide content. The EPS were extracted from freshwater periphyton between July and December 2011. Organic carbon (OC) compounds from different EPS extracts were analyzed using liquid chromatography-organic carbon detection-organic nitrogen detection (LC-OCD-OND), and total protein and polysaccharide content were quantified. Four distinct OC fractions, on the basis of M r, were identified in all extracts, corresponding to high M r biopolymers (≥80-4 kDa), degradation products of humic substances (M r not available), low M r acids (10-0.7 kDa), and small amphiphilic/neutral compounds (3-0.5 kDa). Low C/N ratios (4.3 ± 0.8) were calculated for the biopolymer fractions, which represented 16-38 % of the measured dissolved organic carbon (DOC), indicating a significant presence of high M r proteins in the EPS. Protein and polysaccharide represented the two major components of EPS and, when combined, accounted for the measured DOC in extracts. Differences in specific OC fractions of EPS extracts over the course of the study could be quantified using this method. This study suggests that LC-OCD-OND is a new valuable tool in EPS characterization of periphyton.