Earthworm uptake routes and rates of ionic Zn and ZnO nanoparticles at realistic concentrations, traced using stable isotope labeling

The environmental behavior of ZnO nanoparticles (NPs), their availability to, uptake pathways by, and biokinetics in the earthworm Lumbricus rubellus were investigated using stable isotope labeling. Zinc isotopically enriched to 99.5% in 68Zn (68Zn-E) was used to prepare 68ZnO NPs and a dissolved phase of 68Zn for comparison. These materials enabled tracing of environmentally relevant (below background) NP additions to soil of only 5 mg 68Zn-E kg–1. Uptake routes were isolated by introducing earthworms with sealed and unsealed mouthparts into test soils for up to 72 h. The Zn isotope compositions of the soils, pore waters and earthworms were then determined using multiple collector inductively coupled plasma mass spectrometry. Detection and quantification of 68Zn-E in earthworm tissue was possible after only 4 h of dermal exposure, when the uptake of 68Zn-E had increased the total Zn tissue concentration by 0.03‰. The results demonstrate that at these realistic exposure concentrations there is no distinguishable difference between the uptake of the two forms of Zn by the earthworm L. rubellus, with the dietary pathway accounting for ∼95% of total uptake. This stands in contrast to comparable studies where high dosing levels were used and dermal uptake is dominant

Zinc isotopic compositions of breast cancer tissue

This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. https://creativecommons.org/licenses/by-nc-nd/3.0/ The attached file is the published version of the article.An early diagnostic biomarker for breast cancer is essential to improve outcome. High precision isotopic analysis, originating in Earth sciences, can detect very small shifts in metal pathways. For the first time, the natural intrinsic Zn isotopic compositions of various tissues in breast cancer patients and controls were determined. Breast cancer tumours were found to have a significantly lighter Zn isotopic composition than the blood, serum and healthy breast tissue in both groups. The Zn isotopic lightness in tumours suggests that sulphur rich metallothionein dominates the isotopic selectivity of a breast tissue cell, rather than Zn-specific proteins. This reveals a possible mechanism of Zn delivery to Zn-sequestering vesicles by metallothionein, and is supported by a similar signature observed in the copper isotopic compositions of one breast cancer patient. This change in intrinsic isotopic compositions due to cancer has the potential to provide a novel early biomarker for breast cancer.This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. https://creativecommons.org/licenses/by-nc-nd/3.0/ The attached file is the published version of the article

Vitamin-D-Binding Protein Contributes to the Maintenance of α Cell Function and Glucagon Secretion

Vitamin-D-binding protein (DBP) or group-specific component of serum (GC-globulin) carries vitamin D metabolites from the circulation to target tissues. DBP is highly localized to the liver and pancreatic α cells. Although DBP serum levels, gene polymorphisms, and autoantigens have all been associated with diabetes risk, the underlying mechanisms remain unknown. Here, we show that DBP regulates α cell morphology, α cell function, and glucagon secretion. Deletion of DBP leads to smaller and hyperplastic α cells, altered Na+ channel conductance, impaired α cell activation by low glucose, and reduced rates of glucagon secretion both in vivo and in vitro. Mechanistically, this involves reversible changes in islet microfilament abundance and density, as well as changes in glucagon granule distribution. Defects are also seen in β cell and δ cell function. Immunostaining of human pancreata reveals generalized loss of DBP expression as a feature of late-onset and long-standing, but not early-onset, type 1 diabetes. Thus, DBP regulates α cell phenotype, with implications for diabetes pathogenesis.This article is freely available via Open Access. Click on the Publisher URL to access it via the publisher's site.D.J.H. was supported by MRC ( MR/N00275X/1 and MR/S025618/1 ) and Diabetes UK ( 17/0005681 ) project grants. This project has received funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (Starting Grant 715884 to D.J.H.). L.J.B.B. was supported by a Sir Henry Wellcome Postdoctoral Fellowship ( Wellcome Trust ; 201325/Z/16/Z ) and a Junior Research Fellowship from Trinity College, Oxford . P.E.M. was funded by a foundation grant from the Canadian Institutes of Health Research (grant 148451 ). G.G.L. was supported by a Wellcome Trust Senior Research Fellowship ( 104612/Z/14/Z ). N.G.M. and S.J.R. were supported by Diabetes UK ( 15/0005156 and 16/0005480 ), MRC ( MR/P010695/1 ), and JDRF ( 2-SRA-2018-474-S-B ) project grants. We thank Dr. Deirdre Kavanagh and COMPARE for microscopy assistance. Human pancreas sections were provided by the Alberta Diabetes Institute IsletCore at the University of Alberta in Edmonton, with the assistance of the Human Organ Procurement and Exchange (HOPE) program, Trillium Gift of Life Network (TGLN), and other Canadian organ procurement organizations.published version, accepted version, submitted versio

Multiple novel prostate cancer susceptibility signals identified by fine-mapping of known risk loci among Europeans

Genome-wide association studies (GWAS) have identified numerous common prostate cancer (PrCa) susceptibility loci. We have fine-mapped 64 GWAS regions known at the conclusion of the iCOGS study using large-scale genotyping and imputation in 25 723 PrCa cases and 26 274 controls of European ancestry. We detected evidence for multiple independent signals at 16 regions, 12 of which contained additional newly identified significant associations. A single signal comprising a spectrum of correlated variation was observed at 39 regions; 35 of which are now described by a novel more significantly associated lead SNP, while the originally reported variant remained as the lead SNP only in 4 regions. We also confirmed two association signals in Europeans that had been previously reported only in East-Asian GWAS. Based on statistical evidence and linkage disequilibrium (LD) structure, we have curated and narrowed down the list of the most likely candidate causal variants for each region. Functional annotation using data from ENCODE filtered for PrCa cell lines and eQTL analysis demonstrated significant enrichment for overlap with bio-features within this set. By incorporating the novel risk variants identified here alongside the refined data for existing association signals, we estimate that these loci now explain ∼38.9% of the familial relative risk of PrCa, an 8.9% improvement over the previously reported GWAS tag SNPs. This suggests that a significant fraction of the heritability of PrCa may have been hidden during the discovery phase of GWAS, in particular due to the presence of multiple independent signals within the same regio

Novel applications of stable isotope tracing methods in biomedical and environmental sciences

The use of multiple-collector inductively coupled plasma mass spectrometry (MC-ICP-MS) for trace element stable isotope ratio analysis has been well established within geochemistry since its conception. The utilization of this high precision technique has since progressed to some environmental applications; however, the use of this tool in biological and biomedical work is limited to few studies. The aim of this thesis is to establish viable techniques to enable high precision isotope analyses of Cu and Zn in biological media, and demonstrate these methods via several collaborative pilot studies across biomedicine and ecotoxicology. For this, a new method for the separation of copper from biological materials to enable accurate and precise Cu isotope analyses (±0.10 ‰) has been developed. This has been applied to a study that, with the use of a 65Cu enriched stable isotope tracer, shows that abnormalities of the human copper metabolism exist in those with Parkinson’s disease. The first Zn isotope analyses of various engineered ZnO nanomaterials (NMs) has been performed, which in conjunction with an extensive literature survey and modelling studies, has shown that highly selective and sensitive detection of ZnO NMs can only be achieved with the use of purpose made isotopically labelled ZnO NMs. This is in part due to the high natural background levels of this element and relatively low Zn concentrations involved in ecotoxicologically relevant exposures. This concept has been successfully implemented to investigate ZnO NM uptake by an estuarine invertebrate, and has shown that Zn from ZnO nanoparticles is bioavailable to these organisms. These pilot studies have shown that there is vast scope for high precision isotope analyses of trace metals in life sciences, and support the transition of such investigations to full-scale studies

Evaluation of Stable Isotope Tracing for ZnO NanomaterialsNew Constraints from High Precision Isotope Analyses and Modeling.

This contribution evaluates two possible routes of stable isotope tracing for ZnO nanomaterials. For this we carried out the first high precision Zn isotope analyses of commercially available ZnO nanomaterials, to investigate whether such materials exhibit isotope fractionations that can be exploited for tracing purposes. These measurements revealed Zn isotopic compositions (of δ^66/64Zn = +0.28 to −0.31‰ relative to JMC Lyon Zn) that are indistinguishable from “normal” natural and anthropogenic Zn in environmental samples. Stable isotope tracing therefore requires the application of purpose-made isotopically enriched ZnO nanoparticles. A detailed evaluation identified the most suitable and cost-effective labeling isotopes for different analytical requirements and techniques. It is shown that, using relatively inexpensive ⁶⁸Zn for labeling, ZnO nanoparticles can be reliably detected in natural samples with a Zn background of 100 μg/g at concentrations as low as about 5 ng/g, if the isotopic tracing analyses are carried out by high precision mass spectrometry. Stable isotope tracing may also be able to differentiate between the uptake by organisms of particulate ZnO and Zn²⁺ ions from the dissolution of nanoparticles

Evaluation of Stable Isotope Tracing for ZnO NanomaterialsNew Constraints from High Precision Isotope Analyses and Modeling.

This contribution evaluates two possible routes of stable isotope tracing for ZnO nanomaterials. For this we carried out the first high precision Zn isotope analyses of commercially available ZnO nanomaterials, to investigate whether such materials exhibit isotope fractionations that can be exploited for tracing purposes. These measurements revealed Zn isotopic compositions (of δ^66/64Zn = +0.28 to −0.31‰ relative to JMC Lyon Zn) that are indistinguishable from “normal” natural and anthropogenic Zn in environmental samples. Stable isotope tracing therefore requires the application of purpose-made isotopically enriched ZnO nanoparticles. A detailed evaluation identified the most suitable and cost-effective labeling isotopes for different analytical requirements and techniques. It is shown that, using relatively inexpensive ⁶⁸Zn for labeling, ZnO nanoparticles can be reliably detected in natural samples with a Zn background of 100 μg/g at concentrations as low as about 5 ng/g, if the isotopic tracing analyses are carried out by high precision mass spectrometry. Stable isotope tracing may also be able to differentiate between the uptake by organisms of particulate ZnO and Zn²⁺ ions from the dissolution of nanoparticles

An inter-laboratory comparison of high precision stable isotope ratio measurements for nanoparticle tracing in biological samples

Stable isotope labeling coupled with high precision isotope ratio measurements by multiple collector-ICP-MS is effective for tracing zinc from zinc oxide nanoparticles (ZnO NPs) in complex media and against a high zinc background. Using this technique, a previous study detected a slightly enhanced uptake of Zn in the blood of human females following dermal exposure to a sunscreen containing ZnO NPs, relative to a sunscreen of the same formulation but with larger ZnO particles ('bulk' material). Until now, the results of these exposures and the accuracy of the analyses, which require the resolution of subtle differences in Zn isotope ratios, have not been independently confirmed. Here we compare data from the previous study with results obtained in new, independent analyses of the same and additional samples, that were performed using similar techniques and instrumentation but in a different laboratory. The data sets are in good agreement, with a Pearson correlation coefficient of r ≈ 0.98, thus establishing the usefulness of the methods for tracing engineered ZnO NPs (and other anthropogenic sources of Zn) in Zn-rich biological materials. Previously unanalyzed blood samples from the earlier study provide further evidence for the increased uptake of Zn from ZnO NPs relative to bulk ZnO and they confirm that uptake rates decrease once sunscreen application has ceased.7 page(s

Copper Isotope Compositions of Superoxide Dismutase and Metallothionein from Post-Mortem Human Frontal Cortex

The natural copper isotopic compositions of superoxide dismutase and metallothionein from six post-mortem human frontal cortices were determined using a combination of size exclusion protein liquid chromatography, followed by anion exchange chromatography and multiple collector inductively-coupled plasma mass spectrometry. Superoxide dismutase was enriched in the heavier 65Cu relative to the metallothionein fraction in all specimen pairs. The isotopic compositions were independent of copper content. This finding provides evidence that nitrogen ligands in protein copper binding sites will be enriched in heavy metal isotopes, and sulphur ligands will preferentially incorporate lighter isotopes in vivo. This in turn has implications for understanding isotopic distributions within different components in the body and the dominant ligands in different tissues. Differences in Cu isotope distributions between the two proteins were seen between Alzheimer’s disease and healthy control samples, when normalised for sex

Dermal absorption of Zn from ZnO particles in sunscreens applied to humans

Zinc oxide (ZnO) nanoparticles (NP) in sunscreens provide protection against UV exposure. Most dermal penetration studies of NP concluded that the NP reside near the surface of the skin. However, most of these studies have been short, used animal models or in vitro assays, and/or not taken into account the effects of UV exposure or skin flexing. Use of ZnO with a highly enriched stable 68Zn isotope (>99%), coupled with measurements of Zn isotopic ratios by multiple-collector inductively coupled plasma mass spectrometry, is highly sensitive in tracing 68Zn from 68ZnO particles in a complex matrix against a high Zn background. Using this approach, a study in Australia detected small increases of 68Zn in the blood of human females following dermal and UV exposure over 5 days to a sunscreen containing 68ZnO NP (~30 nm) compared with a sunscreen with larger 68ZnO particles (~100 nm). There was no difference in 68Zn concentrations in the blood of males exposed to either sunscreen. In an earlier pilot study over 5 days of 3 adults with minimal UV exposure, employing 68ZnO enriched to only 51% in a different formulation, small increases in 68ZnO in blood were also detected. These investigations require the resolution of subtle differences in Zn isotopic ratios. To independently confirm the above outcomes, blood samples were transferred from Australia to Imperial College, London. Data from the two studies performed 3 years apart, using similar techniques and instrumentation, but in different laboratories and analysts, show good agreement (r = 0.98, n = 13).1 page(s