Zinc stable isotope analysis reveals Zn dyshomeostasis in benign tumours, breast cancer, and adjacent histologically normal tissue.

The disruption of Zn homeostasis has been linked with breast cancer development and progression. To enhance our understanding of changes in Zn homeostasis both inside and around the tumour microenvironment, Zn concentrations and isotopic compositions (δ66Zn) were determined in benign (BT) and malignant (MT) tumours, healthy tissue from reduction mammoplasty (HT), and histologically normal tissue adjacent to benign (NAT(BT)) and malignant tumours (NAT(MT)). Mean Zn concentrations in NAT(BT) are 5.5 µg g-1 greater than in NAT(MT) (p = 0.00056) and 5.1 µg g-1 greater than in HT (p = 0.0026). Zinc concentrations in MT are 12.9 µg g-1 greater than in HT (p = 0.00012) and 13.3 µg g-1 greater than in NAT(MT) (p < 0.0001), whereas δ66Zn is 0.17‰ lower in MT than HT (p = 0.017). Benign tumour Zn concentrations are also elevated compared to HT (p = 0.00013), but are not significantly elevated compared to NAT(BT) (p = 0.32). The δ66Zn of BT is 0.15‰ lower than in NAT(BT) (p = 0.045). The similar light δ66Zn of BT and MT compared to HT and NAT may be related to the isotopic compensation of increased metallothionein (64Zn-rich) expression by activated matrix metalloproteinase (66Zn-rich) in MT, and indicates a resultant 66Zn-rich reservoir may exist in patients with breast tumours. Zinc isotopic compositions thus show promise as a potential diagnostic tool for the detection of breast tumours. The revealed differences of Zn accumulation in healthy and tumour-adjacent tissues require additional investigation.

Zinc stable isotopes in urine as diagnostic for cancer of secretory organs.

Breast, prostate, and pancreatic cancers alter the zinc (Zn) metabolism. Combined analyses of urinary Zn concentrations [Zn] and Zn stable isotope compositions (δ66Zn) may provide a non-invasive approach for tracing malignancy-induced Zn dyshomeostasis. In this study, we measured [Zn] and δ66Zn in urine from prostate (n = 22), breast (n = 16), and from women with benign breast disease (n = 14) and compared those with age-matched healthy controls (22-49 years or 50+ years) and published data for pancreatic cancer (n = 17). Our results show that cancer-induced changes are reflected in higher urinary [Zn] and lower urinary δ66Zn for pancreatic and prostate cancer and benign breast disease when compared with healthy controls. For prostate cancer, the progression of low [Zn] and high δ66Zn for patients of low-risk disease toward high [Zn] and low δ66Zn for the higher risk patients demonstrates that [Zn] and δ66Zn in urine could serve as a reliable prognostic tool. Urinary excretion of isotopically light Zn by patients with prostatic and pancreatic cancer is probably the result of increased reactive oxygen species in cancerous cells, which limits the scavenging of hydroxyl radicals and thus facilitates the oxidation of metalloproteins with sulfur-rich ligands. Urine from breast cancer patients shows undistinguishable δ66Zn to healthy controls, implying that the expression of metalloproteins with sulfur-rich ligands is stronger in breast cancer tissues. In conclusion, urinary δ66Zn may provide a non-invasive diagnostic tool for pancreatic cancer and support disease prognosis for prostate cancer. These findings should translate to comprehensive transverse and longitudinal cohort studies in future.

Investigations on Zinc Isotope Fractionation in Breast Cancer Tissue Using in vitro Cell Culture Uptake-Efflux Experiments.

Zinc (Zn) accumulates in breast cancer tumors compared to adjacent healthy tissue. Clinical samples of breast cancer tissue show light Zn isotopic compositions (δ66Zn) relative to healthy tissue. The underlying mechanisms causing such effects are unknown. To investigate if the isotopic discrimination observed for in vivo breast cancer tissue samples can be reproduced in vitro, we report isotopic data for Zn uptake-efflux experiments using a human breast cancer cell line. MDA-MB-231 cell line was used as a model for triple receptor negative breast cancer. We determined Zn isotope fractionation for Zn cell uptake (Δ66Znuptake) and cell efflux (Δ66Znefflux) using a drip-flow reactor to enable comparison with the in vivo environment. The MDA-MB-231 cell line analyses show Zn isotopic fractionations in an opposite direction to those observed for in vivo breast cancer tissue. Uptake of isotopically heavy Zn (Δ66Znuptake = +0.23 ± 0.05‰) is consistent with transport via Zn transporters (ZIPs), which have histidine-rich binding sites. Zinc excreted during efflux is isotopically lighter than Zn taken up by the cells (Δ66Znefflux = -0.35 ± 0.06‰). The difference in Zn isotope fractionation observed between in vitro MDA-MB-231 cell line experiments and in vivo breast tissues might be due to differences in Zn transporter levels or intercellular Zn storage (endoplasmic reticulum and/or Zn specific vesicles); stromal cells, such as fibroblasts and immune cells. Although, additional experiments using other human breast cancer cell lines (e.g., MCF-7, BT-20) with varying Zn protein characteristics are required, the results highlight differences between in vitro and in vivo Zn isotope fractionation.

Urine metallomics signature as an indicator of pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest types of cancer. Its high mortality rate is attributed largely to the difficulty of early diagnosis. Analysis of urine is an excellent non-invasive approach to trace changes in biochemical reactions due to cancer development. Here we show remarkable differences in concentration of several essential metals: significantly lower levels of urinary calcium and magnesium and increased levels of copper and zinc in PDAC when compared to healthy controls, and demonstrate that a combined analysis of these essential metals are accurate indicators (sensitivity = 99.5%) for metal dyshomeostasis in PDAC. In addition, natural stable zinc isotope composition (δ66/64Zn) in urine reveals the preferential excretion of isotopically light zinc in PDAC (δ66/64Znmedian = -0.15‰) compared to healthy controls (δ66/64Znmedian = +0.02‰), likely supporting the dysregulation of metalloproteins. These findings demonstrate for the first time that metallomics is a promising approach for discovery of biomarkers for detection of patients with PDAC, completely non-invasively, using urine samples.

Assessment of coupled Zn concentration and natural stable isotope analyses of urine as a novel probe of Zn status.

Zinc is a common trace metal in the human body, present in about 10% of proteins. Despite numerous roles of Zn in health and disease, there is still a need for a robust biomarker of Zn status. Many parameters have been proposed, with varying levels of success, with plasma Zn often favoured. This study investigates if Zn status can be assessed from the natural stable Zn isotope composition of urine. To this end, 60 urine samples were analysed from ten healthy participants. Remarkably, samples with lower Zn concentrations are systematically enriched in heavy Zn isotopes. Most of the low-Zn urine originated from individuals who omitted dairy, meat or both from their diets. When data for blood serum from age-matched, healthy individuals are compared with the urine results, the former plot at the extension of the urine trend at higher Zn concentrations and lighter isotope compositions. The observed co-variation of Zn isotope compositions with concentrations is indicative of an isotope fractionation system where both properties are controlled by the same processes. It is interpreted as arising from filtration and/or reabsorption processes within the kidney, which are associated with absorbed dietary Zn. The data suggest that the Zn in blood serum that is bound to low molecular weight molecules has an isotope composition distinct from total serum, due to the different affinities of molecular Zn-binding residues to heavy and light Zn isotopes. This technique provides additional information into an individual's Zn status compared to urine or plasma Zn levels alone.

Determination of major and trace element variability in healthy human urine by ICP-QMS and specific gravity normalisation.

Sixty five urine samples obtained during one or two non-consecutive days from 10 healthy individuals were analysed for major (Na, Mg, K, Ca) and trace (Co, Cu, Zn, As, Rb, Sr, Mo and Pb) element concentrations. Following microwave digestion, the analyses were carried out using ICP-QMS (inductively coupled plasma quadrupole mass spectrometry) incorporating a collision/reaction cell. Repeat analyses of quality control samples show that the procedure produces unbiased results and is well suited for routine urinalysis of the investigated elements. Concentrations were normalised using specific gravity (SG) and the resultant decrease in variability supports previous conclusions that SG-normalisation appropriately corrects for differences in urine dilution. The elemental concentrations of the individual urine samples show large differences in dispersion. Most variable are As, Co and Zn, with CVs (coefficients of variation) of >75%. The major elements as well as Rb, Sr and Mo display intermediate variability, whilst Cu and Pb have the least elemental dispersion with CV values of about 30%. A detailed assessment shows that the overall elemental variability is governed both by differences between individuals and variations for a single individual over time. Spot urine samples exhibit elemental concentrations that, on average, resemble the daily mean values to within about 30% for all elements except K and Rb. Diet-related changes in urinary element concentration are most prominent for Mg, K, Co, Rb and Pb. The concentrations of Co, As and Rb appear to vary systematically with gender but this may primarily reflect co-variance with specific diets.

High Precision Zinc Stable Isotope Measurement of Certified Biological Reference Materials Using the Double Spike Technique and Multiple Collector-ICP-MS.

Biological reference materials with well-characterised stable isotope compositions are lacking in the field of 'isotope biochemistry', which seeks to understand bodily processes that rely on essential metals by determining metal stable isotope ratios. Here, we present Zn stable isotope data for six biological reference materials with certified trace metal concentrations: fish muscle, bovine muscle, pig kidney, human hair, human blood serum and human urine. Replicate analyses of multiple aliquots of each material achieved reproducibilities (2sd) of 0.04-0.13‰ for δ66/64Zn (which denotes the deviation of the 66Zn/64Zn ratio of a sample from a pure Zn reference material in parts per 1000). This implies only very minor isotopic heterogeneities within the samples, rendering them suitable as quality control materials for Zn isotope analyses. This endorsement is reinforced by (i) the close agreement of our Zn isotope data for two of the samples (bovine muscle and human blood serum) to previously published results for different batches of the same material and (ii) the similarity of the isotopic data for the samples (δ66/64Zn ≈ -0.8 to 0.0‰) to previously published Zn isotope results for similar biological materials. Further tests revealed that the applied Zn separation procedure is sufficiently effective to enable accurate data acquisition even at low mass resolving power (M/ΔM ≈ 400), as measurements and analyses conducted at much higher mass resolution (M/ΔM ≈ 8500) delivered essentially identical results.

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 (68)Zn ((68)Zn-E) was used to prepare (68)ZnO NPs and a dissolved phase of (68)Zn for comparison. These materials enabled tracing of environmentally relevant (below background) NP additions to soil of only 5 mg (68)Zn-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 (68)Zn-E in earthworm tissue was possible after only 4 h of dermal exposure, when the uptake of (68)Zn-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.

Can we use high precision metal isotope analysis to improve our understanding of cancer?

High precision natural isotope analyses are widely used in geosciences to trace elemental transport pathways. The use of this analytical tool is increasing in nutritional and disease-related research. In recent months, a number of groups have shown the potential this technique has in providing new observations for various cancers when applied to trace metal metabolism. The deconvolution of isotopic signatures, however, relies on mathematical models and geochemical data, which are not representative of the system under investigation. In addition to relevant biochemical studies of protein-metal isotopic interactions, technological development both in terms of sample throughput and detection sensitivity of these elements is now needed to translate this novel approach into a mainstream analytical tool. Following this, essential background healthy population studies must be performed, alongside observational, cross-sectional disease-based studies. Only then can the sensitivity and specificity of isotopic analyses be tested alongside currently employed methods, and important questions such as the influence of cancer heterogeneity and disease stage on isotopic signatures be addressed.

Cardiac ferroportin regulates cellular iron homeostasis and is important for cardiac function.

Iron is essential to the cell. Both iron deficiency and overload impinge negatively on cardiac health. Thus, effective iron homeostasis is important for cardiac function. Ferroportin (FPN), the only known mammalian iron-exporting protein, plays an essential role in iron homeostasis at the systemic level. It increases systemic iron availability by releasing iron from the cells of the duodenum, spleen, and liver, the sites of iron absorption, recycling, and storage respectively. However, FPN is also found in tissues with no known role in systemic iron handling, such as the heart, where its function remains unknown. To explore this function, we generated mice with a cardiomyocyte-specific deletion of Fpn. We show that these animals have severely impaired cardiac function, with a median survival of 22 wk, despite otherwise unaltered systemic iron status. We then compared their phenotype with that of ubiquitous hepcidin knockouts, a recognized model of the iron-loading disease hemochromatosis. The phenotype of the hepcidin knockouts was far milder, with normal survival up to 12 mo, despite far greater iron loading in the hearts. Histological examination demonstrated that, although cardiac iron accumulates within the cardiomyocytes of Fpn knockouts, it accumulates predominantly in other cell types in the hepcidin knockouts. We conclude, first, that cardiomyocyte FPN is essential for intracellular iron homeostasis and, second, that the site of deposition of iron within the heart determines the severity with which it affects cardiac function. Both findings have significant implications for the assessment and treatment of cardiac complications of iron dysregulation.

Zinc isotopic compositions of breast cancer tissue.

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.

Stable isotope tracer to determine uptake and efflux dynamics of ZnO Nano- and bulk particles and dissolved Zn to an estuarine snail.

Zinc oxide nanoparticles (ZnO NPs) are among the most commercialized engineered nanomaterials. Their biological impact in aquatic organisms has been associated with dissolution, but there is also evidence of nanospecific effects. In this study the waterborne uptake and efflux kinetics of isotopically labeled (68)ZnO NPs (7.8 ± 1.2 nm), in comparison to aqueous (68)Zn and (68)ZnO bulk particles (up to 2 µm), were determined for the estuarine snail Peringia ulvae following a 7 d exposure (nominally 20 µg (68)Zn L(-1)) and 28 d depuration. Detection of the (68)Zn label was achieved by high precision multiple-collector ICP-MS (MC-ICP-MS). Previous characterization in artificial estuarine water revealed that the NPs underwent initial aggregation and solubilized up to 60% within 1-2 days. Bulk and aqueous forms were significantly more bioavailable than (68)ZnO NPs (p < 0.05), but after correcting for dissolution, aqueous (0.074 L(-1) g(-1) d(-1)) and NP (0.070 L(-1) g(-1) d(-1)) uptake rate constants were highly comparable. The rate constant of loss for (68)Zn aqueous (0.012 ± 0.005 d(-1)) and (68)ZnO NPs (0.012 ± 0.007 d(-1)) were identical. These results strongly suggest that in this exposure scenario the bioaccumulation of Zn from ZnO NPs is primarily dependent upon solubility.

Tracing bioavailability of ZnO nanoparticles using stable isotope labeling.

Zinc oxide nanoparticles (ZnO NPs) are widely used in commercial products and knowledge of their environmental fate is a priority for ecological protection. Here we synthesized model ZnO NPs that were made from and thus labeled with the stable isotope (68)Zn and this enables highly sensitive and selective detection of labeled components against high natural Zn background levels. We combine high precision stable isotope measurements and novel bioimaging techniques to characterize parallel water-borne exposures of the common mudshrimp Corophium volutator to (68)ZnO NPs, bulk (68)ZnO, and soluble (68)ZnCl(2) in the presence of sediment. C. volutator is an important component of coastal ecosystems where river-borne NPs will accumulate and is used on a routine basis for toxicity assessments. Our results demonstrate that ionic Zn from ZnO NPs is bioavailable to C. volutator and that Zn uptake is active. Bioavailability appears to be governed primarily by the dissolved Zn content of the water, whereby Zn uptake occurs via the aqueous phase and/or the ingestion of sediment particles with adsorbed Zn from dissolution of ZnO particles. The high sorption capacity of sediments for Zn thus enhances the potential for trophic transfer of Zn derived from readily soluble ZnO NPs. The uncertainties of our isotopic data are too large, however, to conclusively rule out any additional direct uptake route of ZnO NPs by C. volutator.

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(66/64)Zn = +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 (68)Zn 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 Zn(2+) ions from the dissolution of nanoparticles.