Exploring the Extent of Phosphorus and Heavy Metal Uptake by Single Cells of Saccharomyces cerevisiae and Their Effects on Intrinsic Elements by SC-ICP-TOF-MS.

The effect of six heavy metals, namely, silver (Ag), lead (Pb), palladium (Pd), copper (Cu), nickel (Ni), and chromium (Cr), on phosphorus (P) uptake by yeast was investigated by single-cell analysis using inductively coupled plasma time-of-flight mass spectrometry (SC-ICP-TOF-MS). It was found that the P content in cells with 1.55 g L-1 P feeding after P starvation was increased by ∼70% compared to control cells. Heavy metals at 10 ppm, except Cu, had a negative impact on P accumulation by cells. Pd reduced the P content by 26% in single cells compared to control cells. Metal uptake was strongest for Ag and Pd (0.7 × 10-12 L cell-1) and weakest for Cr (0.05 × 10-12 L cell-1). Exposure to Cr markedly reduced (-50%) Mg in cells and had the greatest impact on the intrinsic element composition. The SC-ICP-TOF-MS shows the diversity of elemental content in single cells: for example, the P content under standard conditions varied between 12.4 and 890 fg cell-1. This technique allows studying both the uptake of elements and sublethal effects on physiology at a single-cell level.

Ruthenium red: a highly efficient and versatile cell staining agent for single-cell analysis using inductively coupled plasma time-of-flight mass spectrometry.

Staining of biological cells with heavy metals can increase their visibility in mass spectrometry. In this study, the potential of ruthenium red (RR) as a staining agent for single-cell analysis by inductively coupled plasma time-of-flight mass spectrometry (SC-ICP-TOF-MS) is explored using two different yeast strains and one algal species. Time-of-flight mass spectrometry allows the simultaneous detection of Ru and multiple intrinsic elements in single cells. Ru has a better correlation with Mg than with P in Saccharomyces cerevisiae (S. cerevisiae) cells. For the three tested strains, the staining efficiency of RR exceeded 96%; the staining strengths were 30-32 ag µm-2 for the yeast cells and 59 ag µm-2 for the algal cells. By deriving the cell volume of single cells from their Ru mass, the concentration of Mg and P in individual cells of S. cerevisiae can be calculated. Elemental concentrations of Mg and P were highly variable in the cell individuals, with their 25-75 percentile values of 0.10-0.19 and 0.76-2.07 fg µm-3, respectively. RR staining has several advantages: it is fast, does not affect cell viability and is highly efficient. Provided that the shape of the individual cells of a culture is similar, Ru staining allows the elemental content to be directly correlated with the cell volume to accurately calculate the intracellular concentration of target elements in single cells. Therefore, RR can be a promising cell staining agent for future application in SC-ICP-TOF-MS research.

Determination of elemental distribution and evaluation of elemental concentration in single Saccharomyces cerevisiae cells using single cell-inductively coupled plasma mass spectrometry.

Single-cell analysis using inductively coupled plasma mass spectrometry (SC-ICP-MS) is a method to obtain qualitative and quantitative information of the elemental content and distribution of single cells. Six intrinsic target elements were analyzed in yeast cells at different cell growth phases cultured in medium with different phosphorus concentrations (0, 7, 14 mM) to study its effect on cell growth and composition. SC-ICP-MS results were compared with those obtained by the acid digestion and the average ratio was 0.81. The limits of detection of this method were 0.08, 2.54, 12.5, 0.02, 0.02, and 0.08 fg cell-1 for Mg, P, K, Mn, Cu, and Zn, respectively. During the exponential growth phase, the cells exhibited higher elemental contents, wider distribution for most elements, and larger cell size in comparison to the stationary growth phase. Phosphorus-free conditions reduced the average P content in single cells of stationary growth phase from 650 to 80 fg. Phosphorus deficiency led to decreasing intracellular concentrations not only of P but also of K and Cu, and to increasing Zn concentration after 48 h. Mg maintained its concentration at ∼0.11 fg µm-3 and did not change significantly under the three investigated conditions after 48 h. Accordingly, Mg content was successfully used to estimate the intracellular concentration of other intrinsic elements in single yeast cells. SC-ICP-MS is suited to determine target elements in single yeast cells, and allows the study of heterogeneity of cell composition and effects of stressors on the elemental content, distribution, and concentrations of intrinsic elements.

Phytoremediation potential of twelve wild plant species for toxic elements in a contaminated soil.

Green remediation of soils highly contaminated with potentially toxic elements (PTEs) can be achieved using suitable plants. Such phytoremediation procedure often takes into consideration PTE concentrations in plants only, but not produced biomass. Phytoremediation potential of certain species of wild plants for PTEs in contaminated floodplain soils has not been assessed yet. Therefore, in this work 12 native species were tested, 3 of which (Poa angustifolia, Galium mollugo, and Stellaria holostea) to our knowledge have never been used before, in a two-year pot experiment and assessed their potential as phytoremediation species. The results showed that plant PTE concentrations were dramatically elevated for Cd and Zn in Alopecurus pratensis, Arrhenatherum elatius, Bromus inermis, Artemisia vulgaris, Achillea millefolium, Galium mollugo, Stellaria holostea, and Silene vulgaris. A. vulgaris was by far the most highly PTE absorbing plant among the 12 tested in this work, especially concerning Zn, Cd, and to a lesser degree Cu and Ni. Also, among species non-studied-before, G. mollugo and S. holostea were characterized by high Zn and Cd uptake, while P. angustifolia did not. Assessing the number of harvests necessary to decrease soil PTE to half of the initial concentrations, it was found that for Cd plants would achieve site phytoremediation within 8 (A. vulgaris) to 28 (S. holostea) and 51 (G. mollugo) harvests, while for Zn, harvests ranged from 104 (A. vulgaris) to 209 (S. holostea), and 251 (A. millefolium). A clear grouping of the tested species according to their functional type was evident. Herbaceous species were collectively more efficient than grasses in PTE uptake combined by high biomass accumulation; thus, they may act as key-species in a phytoremediation-related concept. Our approach puts phytoremediation into a practical perspective as to whether the process can be achieved within a measureable amount of time. In conclusion, A. vulgaris behaved as a hyperaccumulator plant species in our heavily contaminated soil, while never-studied-before G. mollugo and S. holostea also had a hyperaccumulator behavior, especially for Cd and Zn. Although more research is necessary for conclusive results, our study is pivotal in that it would help in assessing plant species as potential phytoremediation species in heavily contaminated soils.

Surfactant assisted extraction of incidental nanoparticles from road runoff sediment and their characterization by single particle-ICP-MS.

A surfactant assisted extraction (SAE) method was developed to extract incidental nanoparticles (INPs) in the <300 nm particle size fraction from road runoff sediments and applied to a road runoff sediment. The method was evaluated by spiking experiments of road runoff sediment with engineered nanoparticle (ENP) suspensions of gold (Au-ENPs) between 30 nm and 200 nm and platinum (Pt-ENPs) between 30 nm and 70 nm with content ranging from 40 to 800 ng/g. Suspensions were analyzed by single particle (sp-)ICP-MS. In the presence of a road runoff matrix, an increase in ENP sizes by a maximum of 8% for Au-ENPs and 9% for Pt-ENPs was observed. ENPs mass recovery was >50% for all Au-ENPs with content higher than 200 ng/g and for 30 and 50 nm Pt-ENPs at content of 160 ng/g while for lower content the recovery was 0%. For 70 nm Pt-ENPs, recovery was always >80% and increased with increasing Pt content up to 100% in the presence of road runoff matrix. Metal content of INPs in the road runoff sediment in the fraction <300 nm decreased from copper (Cu; µg/g)> zinc (Zn)> zirconium (Zr)> cerium (Ce)> lead (Pb)> cadmium (Cd) > platinum (Pt; µg/g). Over 90% of Pt-, Zn-, Cd-, Pb- and Ce-INPs are composed of particles with less than 20 fg, while Zr- and Cu-INPs are dominated by masses higher than 20 fg. The tested SAE may be applicable to determine environmental contents of INPs in sediments and possibly in soils.

The class II benzoyl-coenzyme A reductase complex from the sulfate-reducing Desulfosarcina cetonica.

Benzoyl-CoA reductases (BCRs) catalyse a key reaction in the anaerobic degradation pathways of monocyclic aromatic substrates, the dearomatization of benzoyl-CoA (BzCoA) to cyclohexa-1,5-diene-1-carboxyl-CoA (1,5-dienoyl-CoA) at the negative redox potential limit of diffusible enzymatic substrate/product couples (E°' = -622 mV). A 1-MDa class II BCR complex composed of the BamBCDEGHI subunits has so far only been isolated from the Fe(III)-respiring Geobacter metallireducens. It is supposed to drive endergonic benzene ring reduction at an active site W-pterin cofactor by flavin-based electron bifurcation. Here, we identified multiple copies of putative genes encoding the structural components of a class II BCR in sulfate reducing, Fe(III)-respiring and syntrophic bacteria. A soluble 950 kDa Bam[(BC)2 DEFGHI]2 complex was isolated from extracts of Desulfosarcina cetonica cells grown with benzoate/sulfate. Metal and cofactor analyses together with the identification of conserved binding motifs gave rise to 4 W-pterins, two selenocysteines, six flavin adenine dinucleotides, four Zn, and 48 FeS clusters. The complex exhibited 1,5-dienoyl-CoA-, NADPH- and ferredoxin-dependent oxidoreductase activities. Our results indicate that high-molecular class II BCR metalloenzyme machineries are remarkably conserved in strictly anaerobic bacteria with regard to subunit architecture and cofactor content, but their subcellular localization and electron acceptor preference may differ as a result of adaptations to variable energy metabolisms.

One-megadalton metalloenzyme complex in Geobacter metallireducens involved in benzene ring reduction beyond the biological redox window.

Reversible biological electron transfer usually occurs between redox couples at standard redox potentials ranging from +0.8 to -0.5 V. Dearomatizing benzoyl-CoA reductases (BCRs), key enzymes of the globally relevant microbial degradation of aromatic compounds at anoxic sites, catalyze a biological Birch reduction beyond the negative limit of this redox window. The structurally characterized BamBC subunits of class II BCRs accomplish benzene ring reduction at an active-site tungsten cofactor; however, the mechanism and components involved in the energetic coupling of endergonic benzene ring reduction have remained hypothetical. We present a 1-MDa, membrane-associated, Bam[(BC)2DEFGHI]2 complex from the anaerobic bacterium Geobacter metallireducens harboring 4 tungsten, 4 zinc, 2 selenocysteines, 6 FAD, and >50 FeS cofactors. The results suggest that class II BCRs catalyze electron transfer to the aromatic ring, yielding a cyclic 1,5-dienoyl-CoA via two flavin-based electron bifurcation events. This work expands our knowledge of energetic couplings in biology by high-molecular-mass electron bifurcating machineries.

Four Molybdenum-Dependent Steroid C-25 Hydroxylases: Heterologous Overproduction, Role in Steroid Degradation, and Application for 25-Hydroxyvitamin D3 Synthesis.

Side chain-containing steroids are ubiquitous constituents of biological membranes that are persistent to biodegradation. Aerobic, steroid-degrading bacteria employ oxygenases for isoprenoid side chain and tetracyclic steran ring cleavage. In contrast, a Mo-containing steroid C-25 dehydrogenase (S25DH) of the dimethyl sulfoxide (DMSO) reductase family catalyzes the oxygen-independent hydroxylation of tertiary C-25 in the anaerobic, cholesterol-degrading bacterium Sterolibacterium denitrificans Its genome contains eight paralogous genes encoding active site α-subunits of putative S25DH-like proteins. The difficult enrichment of labile, oxygen-sensitive S25DH from the wild-type bacteria and the inability of its active heterologous production have largely hampered the study of S25DH-like gene products. Here we established a heterologous expression platform for the three structural genes of S25DH subunits together with an essential chaperone in the denitrifying betaproteobacterium Thauera aromatica K172. Using this system, S25DH1 and three isoenzymes (S25DH2, S25DH3, and S25DH4) were overproduced in a soluble, active form allowing a straightforward purification of nontagged αßγ complexes. All S25DHs contained molybdenum, four [4Fe-4S] clusters, one [3Fe-4S] cluster, and heme B and catalyzed the specific, water-dependent C-25 hydroxylations of various 4-en-3-one forms of phytosterols and zoosterols. Crude extracts from T. aromatica expressing genes encoding S25DH1 catalyzed the hydroxylation of vitamin D3 (VD3) to the clinically relevant 25-OH-VD3 with >95% yield at a rate 6.5-fold higher than that of wild-type bacterial extracts; the specific activity of recombinant S25DH1 was twofold higher than that of wild-type enzyme. These results demonstrate the potential application of the established expression platform for 25-OH-VD3 synthesis and pave the way for the characterization of previously genetically inaccessible S25DH-like Mo enzymes of the DMSO reductase family.IMPORTANCE Steroids are ubiquitous bioactive compounds, some of which are considered an emerging class of micropollutants. Their degradation by microorganisms is the major process of steroid elimination from the environment. While oxygenase-dependent steroid degradation in aerobes has been studied for more than 40 years, initial insights into the anoxic steroid degradation have only recently been obtained. Molybdenum-dependent steroid C25 dehydrogenases (S25DHs) have been proposed to catalyze oxygen-independent side chain hydroxylations of globally abundant zoo-, phyto-, and mycosterols; however, so far, their lability has allowed only the initial characterization of a single S25DH. Here we report on a heterologous gene expression platform that allowed for easy isolation and characterization of four highly active S25DH isoenzymes. The results obtained demonstrate the key role of S25DHs during anoxic degradation of various steroids. Moreover, the platform is valuable for the efficient enzymatic hydroxylation of vitamin D3 to its clinically relevant C-25-OH form.

Mass Cytometry for Detection of Silver at the Bacterial Single Cell Level.

Background: Mass cytometry (Cytometry by Time of Flight, CyTOF) allows single-cell characterization on the basis of specific metal-based cell markers. In addition, other metals in the mass range such as silver can be detected per cell. Bacteria are known to be sensible to silver and a protocol was developed to measure both the number of affected cells per population and the quantities of silver per cell. Methods: For mass cytometry ruthenium red was used as a marker for all cells of a population while parallel application of cisplatin discriminated live from dead cells. Silver quantities per cell and frequencies of silver containing cells in a population were measured by mass cytometry. In addition, live/dead subpopulations were analyzed by flow cytometry and distinguished by cell sorting based on ruthenium red and propidium iodide double staining. Verification of the cells' silver load was performed on the bulk level by using ICP-MS in combination with cell sorting. The protocol was developed by conveying both, fast and non-growing Pseudomonas putida cells as test organisms. Results: A workflow for labeling bacteria in order to be analyzed by mass cytometry was developed. Three different parameters were tested: ruthenium red provided counts for all bacterial cells in a population while consecutively applied cisplatin marked the frequency of dead cells. Apparent population heterogeneity was detected by different frequencies of silver containing cells. Silver quantities per cell were also well measurable. Generally, AgNP-10 treatment caused higher frequencies of dead cells, higher frequencies of silver containing cells and higher per-cell silver quantities. Due to an assumed chemical equilibrium of free and bound silver ions live and dead cells were associated with silver in equal quantities and this preferably during exponential growth. With ICP-MS up to 1.5 fg silver per bacterial cell were detected. Conclusion: An effective mass cytometry protocol was developed for the detection and quantification of silver in single bacterial cells of different physiological states. The silver quantities were generally heterogeneously distributed among cells in a population, the degree of which was dependent on micro-environmental conditions and on silver applied either in ion or nanoparticle-aggregated form.

Phthaloyl-coenzyme A decarboxylase from Thauera chlorobenzoica: the prenylated flavin-, K+ - and Fe2+ -dependent key enzyme of anaerobic phthalate degradation.

The degradation of the industrially produced and environmentally relevant phthalate esters by microorganisms is initiated by the hydrolysis to alcohols and phthalate (1,2-dicarboxybenzene). In the absence of oxygen the further degradation of phthalate proceeds via activation to phthaloyl-CoA followed by decarboxylation to benzoyl-CoA. Here, we report on the first purification and characterization of a phthaloyl-CoA decarboxylase (PCD) from the denitrifying Thauera chlorobenzoica. Hexameric PCD belongs to the UbiD-family of (de)carboxylases and contains prenylated FMN (prFMN), K+ and, unlike other UbiD-like enzymes, Fe2+ as cofactors. The latter is suggested to be involved in oxygen-independent electron-transfer during oxidative prFMN maturation. Either oxidation to the Fe3+ -state in air or removal of K+ by desalting resulted in >92% loss of both, prFMN and decarboxylation activity suggesting the presence of an active site prFMN/Fe2+ /K+ -complex in PCD. The PCD-catalysed reaction was essentially irreversible: neither carboxylation of benzoyl-CoA in the presence of 2 M bicarbonate, nor an isotope exchange of phthaloyl-CoA with 13 C-bicarbonate was observed. PCD differs in many aspects from prFMN-containing UbiD-like decarboxylases and serves as a biochemically accessible model for the large number of UbiD-like (de)carboxylases that play key roles in the anaerobic degradation of environmentally relevant aromatic pollutants.

Rare earth elements and their release dynamics under pre-definite redox conditions in a floodplain soil.

For the first time, the impact of pre-definite redox conditions on the release dynamics of rare earth elements (REEs) and the determining factors pH, iron (Fe), manganese (Mn), aluminum (Al), dissolved organic carbon (DOC), dissolved inorganic carbon (DIC), and sulfate (SO42-) in a floodplain soil was elucidated using an advanced, highly sophisticated automatic biogeochemical microcosm apparatus. The redox potential (EH) ranged between +82 and + 498 mV during the experiment. The systematic increase of EH caused a decreasing pH from 6.6 to 4.6 which resulted in an enhanced mobilization and release of REEs along with Fe, Al, and Mn under oxic and acidic conditions. Also, organic matter seems to contribute to the mobilization of REEs under changing redox conditions. A factor analysis identified that the REEs form one group with EH, Fe, Al, and Mn what indicates that REEs and sesquioxides have a similar geochemical behavior. The pH, DOC, and DIC are together in another cluster which demonstrates that their behavior substantially differs from the other group. The sequential extraction procedure revealed that the majority of the REEs were bound in the residual fraction, followed by the reducible, the oxidisable and the water soluble/exchangeable/carbonate bound fraction. Future studies should further elucidate the specific release kinetics of REEs, the controlling factors on the release dynamics and the underlying mobilization processes in highly dynamic wetland soils around the globe.

Redox-controlled release dynamics of thallium in periodically flooded arable soil.

To our knowledge, this is the first work to mechanistically study the impact of the redox potential (EH) and principal factors, such as pH, iron (Fe), manganese (Mn), dissolved organic carbon (DOC), dissolved inorganic carbon (DIC), chlorides (Cl-) and sulfates (SO42-), on the release dynamics of thallium (Tl) in periodically flooded soil. We simulated flooding using an automated biogeochemical microcosm system that allows for systematical control of pre-defined redox windows. The EH value was increased mechanistically at intervals of approximately 100 mV from reducing (-211 mV) to oxidizing (475 mV) conditions. Soluble Tl levels (0.02-0.28 µg L-1) increased significantly with increases in EH (r = 0.80, p < 0.01, n = 30). Thallium mobilization was found to be related to several simultaneous processes involving the gradual oxidation of Tl-bearing sulfides, reductive dissolution of Fe-Mn oxides and desorption from mineral sorbents. Manganese oxides did not appear to have a considerable effect on Tl retention under oxidizing conditions. Before conducting the microcosm experiment, Tl geochemical fractionation was assessed using the modified BCR sequential extraction procedure. The BCR revealed a majority of Tl in the residual fraction (77.7%), followed by reducible (13.3%) and oxidizable fractions (5.9%). By generating high levels of Tl toxicity at low doses, Tl released under oxidizing conditions may pose an environmental threat. In the future, similar studies should be conducted on various soils along with a determination of the Tl species and monitoring of the Tl content in plants to achieve more detailed insight into soluble Tl behavior.

Biogeochemistry of Ni and Pb in a periodically flooded arable soil: Fractionation and redox-induced (im)mobilization.

The redox-induced (im)mobilization of nickel (Ni) and lead (Pb) under pre-definite redox conditions and their binding forms were studied in a periodically flooded, slightly acidic arable soil enriched with serpentine minerals at the Velika Morava River valley, Serbia. The total contents of Ni and Pb were 152 and 109 mg kg-1, respectively. Geochemical fractionation of Ni, combined with mineralogical analysis, confirmed its geogenic origin in the soil. Potentially mobile fractions were the dominating binding forms of Pb; thus, indicating anthropogenic sources as prevailing. Risk assessment indicated a low risk of Ni and Pb transfer from soil to other environmental constituents. However, the results imply that geogenic metals might pose higher environmental risk than those from anthropogenic origin, in dependence of their total concentrations and contents in the specific solid-phase fractions. Flooding of the soil was simulated in an automated biogeochemical microcosm system, which allows a control and a continuous measurements of redox potential (EH) and pH. Subsequently, the EH was increased in steps of approximately 100 mV from anoxic to oxic conditions. Concurrently, the concentrations of soluble Ni, Pb, iron (Fe), manganese (Mn), dissolved organic carbon (DOC), and sulfates were measured. The EH was brought from low to high values (-220 to 520 mV) and correlated negative with soluble Ni, Pb, Fe, Mn and DOC. Soluble Ni ranged from 125 to 228 µg l-1 while Pb ranged from 3.0 to 21.4 µg l-1. Concentrations of both metals in solution were high at low EH and decreased with increasing EH. Nickel immobilization may be attributed to sorption to or co-precipitation with re-oxidized Fe-Mn (hydr)oxides, whereas Pb, in addition, might be immobilized via precipitation with inorganic ligands, such as carbonates and phosphates. The results imply that Ni and Pb solubility might also be related to the formation of metal-DOC complexes. The detected dynamic and mechanisms might be useful in providing critical information for assessing the potential environmental risk and creating appropriate environmental management strategies for agricultural areas enriched with Ni and Pb.

Cadmium toxicity investigated at the physiological and biophysical levels under environmentally relevant conditions using the aquatic model plant Ceratophyllum demersum.

Cadmium (Cd) is an important environmental pollutant and is poisonous to most organisms. We aimed to unravel the mechanisms of Cd toxicity in the model water plant Ceratophyllum demersum exposed to low (nM) concentrations of Cd as are present in nature. Experiments were conducted under environmentally relevant conditions, including nature-like light and temperature cycles, and a low biomass to water ratio. We measured chlorophyll (Chl) fluorescence kinetics, oxygen exchange, the concentrations of reactive oxygen species and pigments, metal binding to proteins, and the accumulation of starch and metals. The inhibition threshold concentration for most parameters was 20 nM. Below this concentration, hardly any stress symptoms were observed. The first site of inhibition was photosynthetic light reactions (the maximal quantum yield of photosystem II (PSII) reaction centre measured as Fv /Fm , light-acclimated PSII activity ΦPSII , and total Chl). Trimers of the PSII light-harvesting complexes (LHCIIs) decreased more than LHC monomers and detection of Cd in the monomers suggested replacement of magnesium (Mg) by Cd in the Chl molecules. As a consequence of dysfunctional photosynthesis and energy dissipation, reactive oxygen species (superoxide and hydrogen peroxide) appeared. Cadmium had negative effects on macrophytes at much lower concentrations than reported previously, emphasizing the importance of studies applying environmentally relevant conditions. A chain of inhibition events could be established.

Determination of moderately polar arsenolipids and mercury speciation in freshwater fish of the River Elbe (Saxony, Germany).

Arsenic and mercury are frequent contaminants in the environment and care must be taken to limit their entrance into the food chain. The toxicity of both elements strongly depends upon their speciation. Total amounts of As and Hg as well as their species were analyzed in muscle and liver of 26 fishes of seven freshwater fish species caught in the River Elbe. The median concentrations of As were 162 µg kg(-1) w.w. in liver and 92 µg kg(-1) w.w. in muscle. The median concentrations of total Hg were 241 µg kg(-1) w.w. in liver and 256 µg kg(-1) w.w. in muscle. While this level of Hg contamination of the freshwater fish in the River Elbe is significantly lower than 20 years ago, it exceeds the recommended environmental quality standard of 20 µg Hg kg(-1) w.w. by a factor of 5-50. However, the European maximum level of 500 µg Hg kg(-1) for fish for human consumption is rarely exceeded. Arsenic-containing fatty acids and hydrocarbons were determined and partially identified in methanolic extracts of the fish by HPLC coupled in parallel to ICP-MS (element specific detection) and ESI-Q-TOF-MS (molecular structure detection). While arsenobetaine was the dominant As species in the fish, six arsenolipids were detected and identified in the extracts of liver tissue in common bream (Abramis brama), ide (Leuciscus idus), asp (Aspius aspius) and northern pike (Esox lucius). Four arsenic-containing fatty acids (AsFA) and two arsenic-containing hydrocarbons (AsHC) are reported in freshwater fish for the first time. With respect to mercury the more toxic MeHg(+) was the major species in muscle tissue (>90% of total Hg) while in liver Hg(2+) and MeHg(+) were of equal importance. The results show the high relevance of element speciation in addition to the determination of total element concentrations to correctly assess the burden of these two elements in fish.

Structural basis of enzymatic benzene ring reduction.

In chemical synthesis, the widely used Birch reduction of aromatic compounds to cyclic dienes requires alkali metals in ammonia as extremely low-potential electron donors. An analogous reaction is catalyzed by benzoyl-coenzyme A reductases (BCRs) that have a key role in the globally important bacterial degradation of aromatic compounds at anoxic sites. Because of the lack of structural information, the catalytic mechanism of enzymatic benzene ring reduction remained obscure. Here, we present the structural characterization of a dearomatizing BCR containing an unprecedented tungsten cofactor that transfers electrons to the benzene ring in an aprotic cavity. Substrate binding induces proton transfer from the bulk solvent to the active site by expelling a Zn(2+) that is crucial for active site encapsulation. Our results shed light on the structural basis of an electron transfer process at the negative redox potential limit in biology. They open the door for biological or biomimetic alternatives to a basic chemical synthetic tool.

Exploring LA-ICP-MS as a quantitative imaging technique to study nanoparticle uptake in Daphnia magna and zebrafish (Danio rerio) embryos.

The extent and the mechanisms by which engineered nanoparticles (ENPs) are incorporated into biological tissues are a matter of intensive research. Therefore, laser ablation coupled to inductively coupled plasma mass spectrometry (LA-ICP-MS) is presented for the detection and visualization of engineered nanoparticles (Al2O3, Ag, and Au) in ecotoxicological test organisms (Danio rerio and Daphnia magna). While ENPs are not taken up by the zebrafish embryo but attach to its chorion, incorporation into the gut of D. magna is clearly visible by a 50-µm spot ablation of 40-µm-thick organism sections. During laser ablation of the soft organic matrix, the hard ENPs are mobilized without a significant change in their size, leading to decreasing sensitivity with increasing size of ENPs. To compensate for these effects, a matrix-matched calibration with ENPs of the same size embedded in agarose gels is proposed. Based on such a calibration, the mass of ENPs within one organism section was calculated and used to estimate the total mass of ENPs per organism. Compared to the amount determined after acid digestion of the test organisms, recoveries of 20-100% (zebrafish embryo (ZFE)) and of 4-230% (D. magna) were obtained with LODs in the low ppm range. It is likely that these differences are primarily due to an inhomogeneous particle distribution in the organisms and to shifts in the particle size distribution from the initial ENPs to those present in the organism. It appears that quantitative imaging of ENPs with LA-ICP-MS requires knowledge of the particle sizes in the biological tissue under study.

Quantification of Al2O3 nanoparticles in human cell lines applying inductively coupled plasma mass spectrometry (neb-ICP-MS, LA-ICP-MS) and flow cytometry-based methods.

In order to quantify and compare the uptake of aluminum oxide nanoparticles of three different sizes into two human cell lines (skin keratinocytes (HaCaT) and lung epithelial cells (A549)), three analytical methods were applied: digestion followed by nebulization inductively coupled plasma mass spectrometry (neb-ICP-MS), direct laser ablation ICP-MS (LA-ICP-MS), and flow cytometry. Light and electron microscopy revealed an accumulation and agglomeration of all particle types within the cell cytoplasm, whereas no particles were detected in the cell nuclei. The internalized Al2O3 particles exerted no toxicity in the two cell lines after 24 h of exposure. The smallest particles with a primary particle size (xBET) of 14 nm (Alu1) showed the lowest sedimentation velocity within the cell culture media, but were calculated to have settled completely after 20 h. Alu2 (xBET = 111 nm) and Alu3 (xBET = 750 nm) were calculated to reach the cell surface after 7 h and 3 min, respectively. The internal concentrations determined with the different methods lay in a comparable range of 2-8 µg Al2O3/cm2 cell layer, indicating the suitability of all methods to quantify the nanoparticle uptake. Nevertheless, particle size limitations of analytical methods using optical devices were demonstrated for LA-ICP-MS and flow cytometry. Furthermore, the consideration and comparison of particle properties as parameters for particle internalization revealed the particle size and the exposure concentration as determining factors for particle uptake.