Zinc distribution within breast cancer tissue of different intrinsic subtypes.

PURPOSE: To show feasibility of laser ablation inductively coupled mass spectrometry (LA-ICPMS) for analysis of zinc content and concentration in breast cancer tissue and to correlate this with validated prognostic and predictive markers, i.e. histological grading and expression of steroid receptors (estrogen receptor, ER; progesterone receptor, PR) and human epidermal growth-factor receptor 2 (Her2). METHODS: 28 samples of human invasive ductal breast cancer tissue were subclassified into groups of four different intrinsic subtypes according to the expression of ER, PR and Her2 by immunohistological staining and then analyzed for zinc content and distribution by LA-ICPMS applying a calibration technique based on spiked polyacrylamide gels. A correlation of zinc concentration with histological grading and molecular subtypes was analyzed. RESULTS: Consistent with results of a pilot-study LA-ICPMS was feasible to show zinc accumulation in cancerous tissue, even more adjacent healthy stroma was with proportional increase of zinc. Zinc levels were most elevated in triple-positive (TPBC) and in triple-negative (TNB) breast cancers. CONCLUSION: LA-ICPMS was feasible to confirm a connection between zinc and grade of malignancy; furthermore, focusing on a correlation of zinc and intrinsic breast cancer subtypes, LA-ICPMS depicted an upwards trend of zinc for "high-risk-cancers" with highest levels in Her2-positive and in triple-negative (TNBC) disease. The currently uncommon alliance of clinicians and analytical chemists in basic research is most promising to exploit the full potential of diagnostic accuracy in the efforts to solve the enigma of breast cancer initiation and course of disease.

Zinc distribution within breast cancer tissue: A possible marker for histological grading?

PURPOSE: Focusing on the trace metal zinc as a potential biomarker for breast cancer, the literature describes bulk zinc concentrations in breast cancer tissue to be higher than in normal tissue. From a histopathological point of view, cancer cells are intermingled with normal cells of the stroma within breast cancer tissues; therefore, bulk analysis cannot reflect this situation adequately. To address this problem, analysis of zinc distribution in histological sections is the method of choice. METHODS: In the present study, nine samples of invasive ductal and lobular breast carcinoma of histological grade 1-3 were investigated, clearly differentiating between cancer and stroma areas. Zinc concentrations were determined by laser ablation inductively coupled plasma mass spectrometry applying a calibration technique based on spiked polyacrylamide gels. RESULTS: Direct comparison between hematoxylin- and eosin-stained tissues and zinc contour plots revealed that zinc is enriched in cancer tissue containing tumor cells in contrast to normal stroma. Moreover, zinc concentration in carcinomatous tissues directly correlates with the histological malignancy grade. CONCLUSIONS: Differentiation between carcinomatous tissue and stroma by determination of zinc content and the correlation of zinc concentration with the histological malignancy grade not only provides a key feature for clinical decision making for cancer therapy but also suggests the trace metal zinc as a potential biomarker for breast cancer.

Investigation of biomethylation of arsenic and tellurium during composting.

Though the process of composting features a high microbiological activity, its potential to methylate metals and metalloids has been little investigated so far in spite of the high impact of this process on metal(loid) toxicity and mobility. Here, we studied the biotransformation of arsenic, tellurium, antimony, tin and germanium during composting. Time resolved investigation revealed a highly dynamic process during self-heated composting with markedly differing time patterns for arsenic and tellurium species. Extraordinary high concentrations of up to 150 mg kg(-1) methylated arsenic species as well as conversion rates up to 50% for arsenic and 5% for tellurium were observed. In contrast, little to no conversion was observed for antimony, tin and germanium. In addition to experiments with metal(loid) salts, composting of arsenic hyperaccumulating ferns Pteris vittata and P. cretica grown on As-amended soils was studied. Arsenic accumulated in the fronds was efficiently methylated resulting in up to 8 mg kg(-1) methylated arsenic species. Overall, these studies indicate that metal(loid)s can undergo intensive biomethylation during composting. Due to the high mobility of methylated species this process needs to be considered in organic waste treatment of metal(loid) contaminated waste materials.

Parallel on-line detection of a methylbismuth species by hyphenated GC/EI-MS/ICP-MS technique as evidence for bismuth methylation by human hepatic cells.

Methylation of metal(loid)s by bacteria or even mammals is a well known process that can lead to increased toxicity for humans. Nevertheless, reliable analytical techniques and tools are indispensable in speciation analysis of trace elements, especially since environmental or biological samples are usually characterised by complex matrices. Here the methylating capability of hepatic cells was observed in vitro. HepG2 cells were incubated with colloidal bismuth subcitrate, bismuth cysteine and bismuth glutathione, respectively for a period of 24 h. For identification the cell lysate was ethylated by sodium tetraethyl borate under neutral conditions. After cryo focussing by purge and trap, the bismuth speciation was carried out via GC/EI-MS/ICP-MS. Colloidal bismuth subcitrate and bismuth cysteine were methylated by HepG2 cells, while no methylated bismuth species was detected after incubation with bismuth glutathione.

Methylated metal(loid) species in humans.

While the metal(loid)s arsenic, bismuth, and selenium (probably also tellurium) have been shown to be enzymatically methylated in the human body, this has not yet been demonstrated for antimony, cadmium, germanium, indium, lead, mercury, thallium, and tin, although the latter elements can be biomethylated in the environment. Methylated metal(loid)s exhibit increased mobility, thus leading to a more efficient metal(loid) transport within the body and, in particular, opening chances for passing membrane barriers (blood-brain barrier, placental barrier). As a consequence human health may be affected. In this review, relevant data from the literature are compiled, and are discussed with respect to the evaluation of assumed and proven health effects caused by alkylated metal(loid) species.

Effects of monomethylarsonic and monomethylarsonous acid on evoked synaptic potentials in hippocampal slices of adult and young rats.

Arsenite and its metabolites, dimethylarsinic or dimethylarsinous acid, have previously been shown to disturb synaptic transmission in hippocampal slices of rats (Krüger, K., Gruner, J., Madeja, M., Hartmann, L.M., Hirner, A.V., Binding, N., Mubetahoff, U., 2006a. Blockade and enhancement of glutamate receptor responses in Xenopus oocytes by methylated arsenicals. Arch. Toxicol. 80, 492-501, Krüger, K., Straub, H., Binding, N., Mubetahoff, U., 2006b. Effects of arsenite on long-term potentiation in hippocampal slices from adult and young rats. Toxicol. Lett. 165, 167-173, Krüger, K., Repges, H., Hippler, J., Hartmann, L.M., Hirner, A.V., Straub, H., Binding, N., Mubetahoff, U., 2007. Effects of dimethylarsinic and dimethylarsinous acid on evoked synaptic potentials in hippocampal slices of young and adult rats. Toxicol. Appl. Pharmacol. 225, 40-46). The present experiments investigate, whether the important arsenic metabolites monomethylarsonic acid (MMA(V)) and monomethylarsonous acid (MMA(III)) also influence the synaptic functions of the hippocampus. In hippocampal slices of young (14-21 days-old) and adult (2-4 months-old) rats, evoked synaptic field potentials from the Schaffer collateral-CA1 synapse were measured under control conditions and during and after 30 and 60 min of application of the arsenic compounds. MMA(V) had no effect on the synapse functions neither in slices of adult nor in those from young rats. However, MMA(III) strongly influenced the synaptic transmission: it totally depressed the amplitudes of fEPSPs at concentrations of 50 micromol/l (adult rats) and 25 micromol/l (young rats) and LTP amplitudes at concentrations of 25 micromol/l (adult rats) and 10 micromol/l (young rats), respectively. In contrast, application of 1 micromol/l MMA(III) led to an enhancement of the LTP amplitude in young rats, which is interpretable by an enhancing effect on NMDA receptors and a lack of the blocking effect on AMPA receptors at this concentration (Krüger, K., Gruner, J., Madeja, M., Hartmann, L.M., Hirner, A.V., Binding, N., Mubetahoff, U., 2006a. Blockade and enhancement of glutamate receptor responses in Xenopus oocytes by methylated arsenicals. Arch. Toxicol. 80, 492-501). These effects are probably not mediated by changes in cell excitability or in presynaptic glutamate release rates, since antidromically induced population spikes and paired-pulse facilitation failed to show any MMA(III) effect. The impairment of the excitatory CA1 synapse is more likely caused by the action of MMA(III) on postsynaptic glutamatergic receptors and may be jointly responsible for dysfunctions of cognitive effects in arsenic toxicity.

Determination of trimethylbismuth in the human body after ingestion of colloidal bismuth subcitrate.

Biological methylation and hydride formation of metals and metalloids are ubiquitous environmental processes that can lead to the formation of chemical species with significantly increased mobility and toxicity. Whereas much is known about the interaction of metal(loid)s with microorganisms in environmental settings, little information has been gathered on respective processes inside the human body as yet. Here, we studied the biotransformation and excretion of bismuth after ingestion of colloidal bismuth subcitrate (215 mg of bismuth) to 20 male human volunteers. Bismuth absorption in the stomach and upper intestine was very low, as evidenced by the small quantity of bismuth eliminated via the renal route. Total bismuth concentrations in blood increased rapidly in the first hour after ingestion. Most of the ingested bismuth was excreted via feces during the study period. Trace levels of the metabolite trimethylbismuth [(CH(3))(3)Bi] were detected via low temperaturegas chromatography/inductively coupled plasma-mass spectrometry in blood samples and in exhaled air samples. Concentrations were in the range of up to 2.50 pg/ml (blood) and 0.8 to 458 ng/m(3) (exhaled air), with high interindividual variation being observed. Elimination routes of bismuth were exhaled air (up to 0.03 per thousand), urine (0.03-1.2%), and feces. The site of (CH(3))(3)Bi production could not be identified in the present study, but the intestinal microflora seems to be involved in this biotransformation if accompanying ex vivo studies are taken into consideration.

Methylantimony and -arsenic species in sediment pore water tested with the sediment or fauna incubation experiment.

In this study, the speciation of arsenic (As) and antimony (Sb) across a water-sediment interface and the formation of mono-, di-, and trimethylated species overtime in a microfiltered pore water solution were examined. We used an experimental technique, known as the sediment or fauna incubation experiment (SOFIE), which enables the determination of chemical speciation across redox zones in undisturbed systems. Five different incubation experiments were run: Over a 76 day incubation period, pore water was sampled and speciated 5 times. These experiments revealed the complete methylated species pattern for arsenic and antimony in the microfiltered sediment pore water. This constitutes the first report of methylated As and Sb species in a true pore water solution of sediments. Predominant organic species were dimethylantimony (DMSb up to 2.7 microg/L) and dimethylarsenic (DMAs up to 4.3 microg/L) followed by monomethylated species (MMAs and MMSb). These data (i) indicate that methylation significantly influences the translocation of As and Sb in sediments, (ii) demonstrate good agreement between the occurrence of methylantimony and the occurrence of methylarsenic in the pore water, (iii) reveal that As transformation in sediments is faster than Sb transformation but is more susceptible to disturbances from acidification, and (iv) regarding the translocation of these elements and antimony in particular, methylation is clearly a relevant, and perhaps as yet underestimated, factor.

Role of intestinal microbiota in transformation of bismuth and other metals and metalloids into volatile methyl and hydride derivatives in humans and mice.

The present study shows that feces samples of 14 human volunteers and isolated gut segments of mice (small intestine, cecum, and large intestine) are able to transform metals and metalloids into volatile derivatives ex situ during anaerobic incubation at 37 degrees C and neutral pH. Human feces and the gut of mice exhibit highly productive mechanisms for the formation of the toxic volatile derivative trimethylbismuth [(CH(3))(3)Bi] at rather low concentrations of bismuth (0.2 to 1 mumol kg(-1) [dry weight]). An increase of bismuth up to 2 to 14 mmol kg(-1) (dry weight) upon a single (human volunteers) or continuous (mouse study) administration of colloidal bismuth subcitrate resulted in an average increase of the derivatization rate from approximately 4 pmol h(-1) kg(-1) (dry weight) to 2,100 pmol h(-1) kg(-1) (dry weight) in human feces samples and from approximately 5 pmol h(-1) kg(-1) (dry weight) to 120 pmol h(-1) kg(-1) (dry weight) in mouse gut samples, respectively. The upshift of the bismuth content also led to an increase of derivatives of other elements (such as arsenic, antimony, and lead in human feces or tellurium and lead in the murine large intestine). The assumption that the gut microbiota plays a dominant role for these transformation processes, as indicated by the production of volatile derivatives of various elements in feces samples, is supported by the observation that the gut segments of germfree mice are unable to transform administered bismuth to (CH(3))(3)Bi.

Subcellular distribution of inorganic and methylated arsenic compounds in human urothelial cells and human hepatocytes.

Epidemiological studies have indicated that exposure of humans to inorganic arsenic in drinking water is associated with the occurrence of bladder cancer. The mechanisms by which arsenic induces this malignancy are still uncertain; however, arsenic metabolites are suspected to play a pivotal role. The aim of the present study was the investigation of uptake capabilities of human urothelial cells (UROtsa) compared with primary human hepatocytes (phH) as well as the intracellular distribution of the arsenic species. Additionally, we were interested in the cyto- and genotoxic potential (comet assay, radical generation) of the different arsenic compounds in these two cell types. Our results show that UROtsa cells accumulate higher amounts of the arsenic species than the phH. Differential centrifugation revealed that the arsenic compounds are preferentially distributed into nuclei and ribosomes. After 24-h exposure, arsenic is mainly found in the ribosomes of UROtsa cells and in the nuclei and mitochondria of phH. In contrast to the pentavalent arsenic species, the trivalent species induced a 4- to 5-fold increase of DNA damage in hepatocytes. Radical generation, measured by thiobarbituric acid reactive substances, was more pronounced in hepatocytes than in urothelial cells. In summary, the uptake of arsenic compounds appears to be highly dependent upon cell type and arsenic species. The nonmethylating urothelial cells accumulate higher amounts of arsenic species than the methylating hepatocytes. However, cyto- and genotoxic effects are more distinct in hepatocytes. Further studies are needed to define the implications of the observed accumulation in cellular organelles for the carcinogenic activity of arsenic.

Effects of dimethylarsinic and dimethylarsinous acid on evoked synaptic potentials in hippocampal slices of young and adult rats.

In this study, the effects of pentavalent dimethylarsinic acid ((CH(3))(2)AsO(OH); DMA(V)) and trivalent dimethylarsinous acid ((CH(3))(2)As(OH); DMA(III)) on synaptic transmission generated by the excitatory Schaffer collateral-CA1 synapse were tested in hippocampal slices of young (14-21 day-old) and adult (2-4 month-old) rats. Both compounds were applied in concentrations of 1 to 100 micromol/l. DMA(V) had no effect on the amplitudes of evoked fEPSPs or the induction of LTP recorded from the CA1 dendritic region either in adult or in young rats. However, application of DMA(III) significantly reduced the amplitudes of evoked fEPSPs in a concentration-dependent manner with a total depression following application of 100 micromol/l DMA(III) in adult and 10 micromol/l DMA(III) in young rats. Moreover, DMA(III) significantly affected the LTP-induction. Application of 10 micromol/l DMA(III) resulted in a complete failure of the postsynaptic potentiation of the fEPSP amplitudes in slices taken both from adult and young rats. The depressant effect was not reversible after a 30-min washout of the DMA(III). In slices of young rats, the depressant effects of DMA(III) were more pronounced than in those taken from adult ones. Compared to the (absent) effect of DMA(V) on synaptic transmission, the trivalent compound possesses a considerably higher neurotoxic potential.

Uptake of nickel from 316L stainless steel into contacting osteoblastic cells and metal ion interference with BMP-2-induced alkaline phosphatase.

Bone cells contacting nickel (Ni)-containing implant materials may be affected by Ni species via disturbed signaling pathways involved in bone cell development. Here we analyze effects of the Ni-containing steel 316L and major metal constituents thereof on bone morphogenetic protein-2 (BMP-2)-induced alkaline phosphatase (ALP) of MC3T3-E1 cells. While cells grew normally on 316L, cellular Ni content increased 10-fold vs. control within 4 days. With respect to the major components of 316L, Ni2+ (3-50 microM) was most inhibitory to BMP-2-induced ALP, whereas even 50 microM Fe3+, Cr3+, Mo5+, or Mn2+ had no such effect. In line with this, BMP-2-induced ALP was significantly reduced in cells on 316L. This effect was not prevented by the metal ion chelator diethylenetriaminepentaacetic acid (DTPA). Instead, DTPA abolished the stimulatory effect of BMP-2 on ALP, pointing to chelatable metal ions involved. Zn2+, as one possible candidate, antagonized the Ni2+ inhibition of BMP-2-induced ALP in both MC3T3-E1 and human bone marrow stromal cells. Results show that cells contacting 316L steel are exposed to increased concentrations of Ni which suffice to impair BMP-2-induced ALP activity. Zn2+, as a competitor of this inhibition, may help to restore normal osteoblastic function and bone development under these conditions.

Speciation of alkylated metals and metalloids in the environment.

The analytical methodology for speciation of metals and metalloids associated with alkyl groups and biomacromolecules is critically reviewed. Alkylated metals and metalloids are not only known to be produced by microbial methylation within most anaerobic compartments in the environment, but also in the course of enzymatic transformations during human metabolism. Because of the toxicological relevance of these compounds present in trace to ultratrace concentrations, firm species identification and exact quantification are essential. While many instrumental techniques coupling chromatography (GC, HPLC, CE, GE) with plasma mass spectrometry (ICP-MS) are available for quantification, methods used for structural identification often suffer from inadequate sensitivity (EI-MS, ESI-MS, MALDI-MS, FT-ICRMS). Other problems encountered are sample derivatisation artefacts, lack of suitable standards for quantification, lack of equilibrium between spikes and sample, and the integrity of metal-protein association during separation, in particular during SDS-PAGE. Selected application examples with respect to mercury and arsenic speciation will be discussed critically.

Blockade and enhancement of glutamate receptor responses in Xenopus oocytes by methylated arsenicals.

Pentavalent and trivalent organoarsenic compounds belong to the major metabolites of inorganic arsenicals detected in humans. Recently, the question was raised whether the organic arsenicals represent metabolites of a detoxification process or methylated species with deleterious biological effects. In this study, the effects of trivalent arsenite (AsO(3) (3-); iA(III)), the pentavalent organoarsenic compounds monomethylarsonic acid (CH(3)AsO(OH)(2); MMA(V)) and dimethylarsinic acid ((CH(3))(2)AsO(OH); DMA(V)) and the trivalent compounds monomethylarsonous acid (CH(3)As(OH)(2), MMA(III)) and dimethylarsinous acid ((CH(3))(2)As(OH); DMA(III)) were tested on glutamate receptors and on voltage-operated potassium and sodium channels heterologously expressed in Xenopus oocytes. Membrane currents of ion channels were measured by conventional two-electrode voltage-clamp techniques. The effects of arsenite were tested in concentrations of 1-1,000 micromol/l and the organic arsenical compounds were tested in concentrations of 0.1-100 micromol/l. We found no significant effects on voltage-operated ion channels; however, the arsenicals exert different effects on glutamate receptors. While MMA(V) and MMA(III) significantly enhanced ion currents through N-methyl-D: -aspartate (NMDA) receptor ion channels with threshold concentrations <10 micromol/l, DMA(V) and DMA(III) significantly reduced NMDA-receptor mediated responses with threshold concentrations <0.1 micromol/l; iA(III) had no effects on glutamate receptors of the NMDA type. MMA(III) and DMA(V) significantly reduced ion currents through alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)-receptor ion channels with threshold concentrations <10 micromol/l (MMA(III)) and <1 micromol/l (DMA(V)). MMA(V) and iA(III) had no significant effects on glutamate receptors of the AMPA type. The effects of MMA(V), MMA(III), DMA(V) and DMA(III )on glutamate receptors point to a neurotoxic potential of these substances.

Methylated arsenic, antimony and tin species in soils.

Methylated species of antimony, arsenic and tin were examined in urban soils of the Ruhr basin, near the cities of Duisburg and Essen, Germany. The main aim of this study was to investigate the occurrence of mono-, di- and trimethylated species of these elements in urban soils. The influence of historical and present land use upon the species content was examined. The distribution of inorganic As, Sb and Sn and their methylated species along the profile depth was investigated. As, Sb and Sn speciation was performed by pH-gradient hydride generation purge and trap gas chromatography, followed by inductively-coupled plasma mass spectrometry (HG-PT-GC/ICP-MS). Species' structures were confirmed by GC-EI/MS-ICP-MS. Monomethylated Sb and As were the dominant species detected: the concentration of these metal(loid) species varied between <0.07-56 microg kg(-1) per dry mass. All dimethylated species and monomethyltin concentrations were between <0.01-7.6 microg kg(-1) per dry mass, and for the trimethylated species of all examined elements, concentrations between <0.001-0.63 microg kg(-1) per dry mass were detected. The highest organometal(loid) concentrations were observed in agricultural soils and garden soils; lower concentrations were found in the soils of abandoned industrial sites (wasteland, primary forest and grassland) and a flood plain soil of the Rhine. This result can be ascribed to both the cultivation and the increased biological activity of the agricultural soils, and the generally higher contamination, the disturbed structure and the artificial substrates (deposits from industrial sources) of the abandoned industrial soils. Due to periodical sedimentation, the flood plain profile was the only one where no depth dependence of organometal(loid) species concentration was detected. The other soil profiles showed a decrease of species content with increasing depth; this was particularly noticeable in soils with a clear change from a horizon with an organic character towards a mineral horizon, i.e. decreasing vitality from profile top to bottom. It is not as yet clear whether the organometal(loid) species are formed in the mineral horizons of the profiles or whether they are displaced from the organic, biologically-active horizons towards the mineral horizons. Field studies revealed that soil parameters like pH, water content or temperature did not correlate significantly with the degree of biomethylation observed. In contrast to the lower in vitro biomethylation efficiency of Sb vs. As in microbial incubations, we consistently detected higher proportions of transformed Sb compounds in situ in soil samples. These data may indicate a need to re-examine the currently accepted model of Sb biogeochemical cycling in the real environment.

Determination of siloxanes, silicon, and platinum in tissues of women with silicone gel-filled implants.

Silicone [poly(dimethylsiloxane)] gel used in breast implants has been known to migrate through intact silicone elastomer shells, resulting in the clinically observable "gel bleed" on the implant surface. Although silicon concentrations in capsular tissues of women with silicone prostheses have been measured with element-specific silicon analyses, no silicone-specific investigation of these tissues has been performed as yet.A combination of element-specific inductively coupled plasma high-resolution isotope dilution mass spectrometry (ICP-HR-IDMS) and species-specific gas chromatography coupled mass spectrometry (GC-MS) was used to analyze silicon, platinum, and siloxanes in prosthesis capsule, muscle, and fat tissues of women (n=3) who had silicone gel-filled breast implants and in breast tissue of non-augmented women (n=3) as controls.In all tissues of augmented women, siloxanes, in particular octamethylcyclotetrasiloxane (D4), decamethylcyclopentasiloxane (D5), and dodecamethylcyclohexasiloxane (D6) were identified. Depending on the siloxane species and type of tissue analyzed, siloxane levels in the range of about 10-1,400 ng g(-1) were detected; total silicon was found in all tissue samples in the range of about 8,900-85,000 ng g(-1). Higher platinum levels ranging from 25-90 ng g(-1 )were detected in fibrin layer and fat tissue of two patients with prostheses. No siloxanes were detected in control breast tissue samples. This investigation of human tissues by a combination of element-specific and species-specific analytical techniques clearly demonstrates for the first time that platinum and siloxanes leak from prostheses and accumulate in their surrounding tissues.