Heavy metal cations permeate the TRPV6 epithelial cation channel

TRPV6 belongs to the vanilloid family of the transient receptor potential channel (TRP) superfamily. This calcium-selective channel is highly expressed in the duodenum and the placenta, being responsible for calcium absorption in the body and fetus. Previous observations have suggested that TRPV6 is not only permeable to calcium but also to other divalent cations in epithelial tissues. In this study, we tested whether TRPV6 is indeed also permeable to cations such as zinc and cadmium. We found that the basal intracellular calcium concentration was higher in HEK293 cells transfected with hTRPV6 than in non-transfected cells, and that this difference almost disappeared in nominally calcium-free solution. Live cell imaging experiments with Fura-2 and NewPort Green DCF showed that overexpression of human TRPV6 increased the permeability for Ca(2+), Ba(2+), Sr(2+), Mn(2+), Zn(2+), Cd(2+), and interestingly also for La(3+) and Gd(3+). These results were confirmed using the patch clamp technique. (45)Ca uptake experiments showed that cadmium, lanthanum and gadolinium were also highly efficient inhibitors of TRPV6-mediated calcium influx at higher micromolar concentrations. Our results suggest that TRPV6 is not only involved in calcium transport but also in the transport of other divalent cations, including heavy metal ions, which may have toxicological implications.

Selective uptake, distribution, and redistribution of Cd-109, Co-57, Zn-65, Ni-63, and Cs-134 via xylem and phloem in the heavy metal hyperaccumulator Solanum nigrum L

The focus of this article was to explore the translocation of Cd-109, Co-57, Zn-65, Ni-63, and Cs-134 via xylem and phloem in the newly found hyperaccumulator Solanum nigrum L. Two experiments with the uptake via the roots and transport of Cd-109, Co-57, and Zn-65 labeled by roots, and the redistribution of Cd-109, Zn-65, Co-57, Ni-63, and Cs-134 using flap label in S. nigrum in a hydroponic culture with a standard nutrient solution were conducted. The results showed that Cd-109 added for 24 h to the nutrient medium of young plants was rapidly taken up, transferred to the shoot, and accumulated in the cotyledons and the oldest leaves but was not efficiently redistributed within the shoot afterward leading to a rather low content in the fruits. In contrast, Co-57 was more slowly taken up and released to the shoot, but afterward, this element was redistributed from older leaves to younger leaves and maturing fruits. Zn-65 was rapidly taken up and transferred to the shoot (mainly to the youngest leaves and not to the cotyledons). Afterward, this radionuclide was redistributed within the shoot to the youngest organs and finally accumulated in the maturing fruits. After flap labeling, all five heavy metals tested (Cd-109, Co-57, Zn-65, Ni-63, Cs-134) were exported from the labeled leaf and redistributed within the plant. The accumulation in the fruits was most pronounced for Ni-63 and Zn-65, while a relatively high percentage of Co-57 was finally found in the roots. Cs-134 was roughly in the middle of them. The transport of Cd-109 differed from that previously reported for wheat or lupin and might be important for the potential of S. nigrum to hyperaccumulate cadmium.

Ice-Core Based Assessment of Historical Anthropogenic Heavy Metal (Cd, Cu, Sb, Zn) Emissions in the Soviet Union

The development of strategies and policies aiming at the reduction of environmental exposure to air pollution requires the assessment of historical emissions. Although anthropogenic emissions from the extended territory of the Soviet Union (SU) considerably influenced concentrations of heavy metals in the Northern Hemisphere, Pb is the only metal with long-term historical emission estimates for this region available, whereas for selected other metals only single values exist. Here we present the first study assessing long-term Cd, Cu, Sb, and Zn emissions in the SU during the period 1935–1991 based on ice-core concentration records from Belukha glacier in the Siberian Altai and emission data from 12 regions in the SU for the year 1980. We show that Zn primarily emitted from the Zn production in Ust-Kamenogorsk (East Kazakhstan) dominated the SU heavy metal emission. Cd, Sb, Zn (Cu) emissions increased between 1935 and the 1970s (1980s) due to expanded non-ferrous metal production. Emissions of the four metals in the beginning of the 1990s were as low as in the 1950s, which we attribute to the economic downturn in industry, changes in technology for an increasing metal recovery from ores, the replacement of coal and oil by gas, and air pollution control.

Monitoring pollution in river Mure , Romania, Part III: biochemical effect markers in fish and integrative reflection

Along a downstream stretch of River Mure , Romania, adult males of two feral fish species, European chub (Leuciscus cephalus) and sneep (Chondrostoma nasus) were sampled at four sites with different levels of contamination. Fish were analysed for the biochemical markers hsp70 (in liver and gills) and hepatic EROD activity, as well as several biometrical parameters (age, length, wet weight, condition factor). None of the biochemical markers correlated with any biometrical parameter, thus biomarker reactions were related to site-specific criteria. While the hepatic hsp70 level did not differ among the sites, significant elevation of the hsp70 level in the gills revealed proteotoxic damage in chub at the most upstream site, where we recorded the highest heavy metal contamination of the investigated stretch, and in both chub and sneep at the site right downstream of the city of Arad. In both species, significantly elevated hepatic EROD activity downstream of Arad indicated that fish from these sites are also exposed to organic chemicals. The results were indicative of impaired fish health at least at three of the four investigated sites. The approach to relate biomarker responses to analytical data on pollution was shown to fit well the recent EU demands on further enhanced efforts in the monitoring of Romanian water quality.

Structural model of the CopA copper ATPase of Enterococcus hirae based on chemical cross-linking

The CopA copper ATPase of Enterococcus hirae belongs to the family of heavy metal pumping CPx-type ATPases and shares 43% sequence similarity with the human Menkes and Wilson copper ATPases. Due to a lack of suitable protein crystals, only partial three-dimensional structures have so far been obtained for this family of ion pumps. We present a structural model of CopA derived by combining topological information obtained by intramolecular cross-linking with molecular modeling. Purified CopA was cross-linked with different bivalent reagents, followed by tryptic digestion and identification of cross-linked peptides by mass spectrometry. The structural proximity of tryptic fragments provided information about the structural arrangement of the hydrophilic protein domains, which was integrated into a three-dimensional model of CopA. Comparative modeling of CopA was guided by the sequence similarity to the calcium ATPase of the sarcoplasmic reticulum, Serca1, for which detailed structures are available. In addition, known partial structures of CPx-ATPase homologous to CopA were used as modeling templates. A docking approach was used to predict the orientation of the heavy metal binding domain of CopA relative to the core structure, which was verified by distance constraints derived from cross-links. The overall structural model of CopA resembles the Serca1 structure, but reveals distinctive features of CPx-type ATPases. A prominent feature is the positioning of the heavy metal binding domain. It features an orientation of the Cu binding ligands which is appropriate for the interaction with Cu-loaded metallochaperones in solution. Moreover, a novel model of the architecture of the intramembranous Cu binding sites could be derived.

Efficient estimation of the total number of acini in adult rat lung

Pulmonary airways are subdivided into conducting and gas-exchanging airways. An acinus is defined as the small tree of gas-exchanging airways, which is fed by the most distal purely conducting airway. Until now a dissector of five consecutive sections or airway casts were used to count acini. We developed a faster method to estimate the number of acini in young adult rats. Right middle lung lobes were critical point dried or paraffin embedded after heavy metal staining and imaged by X-ray micro-CT or synchrotron radiation-based X-rays tomographic microscopy. The entrances of the acini were counted in three-dimensional (3D) stacks of images by scrolling through them and using morphological criteria (airway wall thickness and appearance of alveoli). Segmentation stopper were placed at the acinar entrances for 3D visualizations of the conducting airways. We observed that acinar airways start at various generations and that one transitional bronchiole may serve more than one acinus. A mean of 5612 (±547) acini per lung and a mean airspace volume of 0.907 (±0.108) μL per acinus were estimated. In 60-day-old rats neither the number of acini nor the mean acinar volume did correlate with the body weight or the lung volume.

Aluminum toxicity in a tropical montane forest ecosystem in southern Ecuador

Aluminum phytotoxicity frequently occurs in acid soils (pH < 5.5) and was therefore discussed to affect ecosystem functioning of tropical montane forests. The susceptibility to Al toxicity depends on the sensitivity of the plant species and the Al speciation in soil solution, which can vary highly depending e.g., on pH, ionic strength, and dissolved organic matter. An acidification of the ecosystem and periodic base metal deposition from Saharan dust may control plant available Al concentrations in the soil solutions of tropical montane rainforests in south Ecuador. The overall objective of my study was to assess a potential Al phytotoxicity in the tropical montane forests in south Ecuador. For this purpose, I exposed three native Al non-accumulating tree species (Cedrela odorata L., Heliocarpus americanus L., and Tabebuia chrysantha (Jacq.) G. Nicholson) to increased Al concentrations (0 – 2400 μM Al) in a hydroponic experiment, I established dose-response curves to estimate the sensitivity of the tree species to increased Al concentrations, and I investigated the mechanisms behind the observed effects induced by elevated Al concentrations. Furthermore, the response of Al concentrations and the speciation in soil solution to Ca amendment in the study area were determined. In a final step, I assessed all major Al fluxes, drivers of Al concentrations in ecosystem solutions, and indicators of Al toxicity in the tropical montane rainforest in Ecuador in order to test for indications of Al toxicity. In the hydroponic experiment, a 10 % reduction in aboveground biomass production occurred at 126 to 376 μM Al (EC10 values), probably attributable to decreased Mg concentrations in leaves and reduced potosynthesis. At 300 μM Al, increased root biomass production of T. chrysantha was observed. Phosphorus concentrations in roots of C. odorata and T. chrysantha were significantly highest in the treatment with 300 μM Al and correlated significantly with root biomass, being a likely reason for stimulated root biomass production. The degree of organic complexation of Al in the organic layer leachate, which is central to plant nutrition because of the high root density, and soil solution from the study area was very high (mean > 99 %). The resulting low free Al concentrations are not likely to affect plant growth, although the concentrations of potentially toxic Al3+ increased with soil depth due to higher total Al and lower dissolved organic matter concentrations in soil solutions. The Ca additions caused an increase of Al in the organic layer leachate, probably because Al3+ was exchanged against the added Ca2+ ions while pH remained constant. The free ion molar ratios of Ca2+:Al3+ (mean ratio ca. 400) were far above the threshold (≤ 1) for Al toxicity, because of a much higher degree of organo-complexation of Al than Ca. High Al fluxes in litterfall (8.8 – 14.2 kg ha−1 yr−1) indicate a high Al circulation through the ecosystem. The Al concentrations in the organic layer leachate were driven by the acidification of the ecosystem and increased significantly between 1999 and 2008. However, the Ca:Al molar ratios in organic layer leachate and all aboveground ecosystem solutions were above the threshold for Al toxicity. Except for two Al accumulating and one non-accumulating tree species, the Ca:Al molar ratios in tree leaves from the study area were above the Al toxicity threshold of 12.5. I conclude that toxic effects in the hydroponic experiment occurred at Al concentrations far above those in native organic layer leachate, shoot biomass production was likely inhibited by reduced Mg uptake, impairing photosynthesis, and the stimulation of root growth at low Al concentrations can be possibly attributed to improved P uptake. Dissolved organic matter in soil solutions detoxifies Al in acidic tropical forest soils and a wide distribution of Al accumulating tree species and high Al fluxes in the ecosystem do not necessarily imply a general Al phytotoxicity.

Effects of PEG-Induced Water Deficit in Solanum nigrum on Zn and Ni Uptake and Translocation in Split Root Systems

Drought strongly influences root activities in crop plants and weeds. This paper is focused on the performance of the heavy metal accumulator Solanum nigrum, a plant which might be helpful for phytoremediation. The water potential in a split root system was decreased by the addition of polyethylene glycol (PEG 6000). Rubidium, strontium and radionuclides of heavy metals were used as markers to investigate the uptake into roots, the release to the shoot via the xylem, and finally the basipetal transport via the phloem to unlabeled roots. The uptake into the roots (total contents in the plant) was for most makers more severely decreased than the transport to the shoot or the export from the shoot to the unlabeled roots via the phloem. Regardless of the water potential in the labeling solution, 63Ni and 65Zn were selectively redistributed within the plant. From autoradiographs, it became evident that 65Zn accumulated in root tips, in the apical shoot meristem and in axillary buds, while 63Ni accumulated in young expanded leaves and roots but not in the meristems. Since both radionuclides are mobile in the phloem and are, therefore, well redistributed within the plant, the unequal transfer to shoot and root apical meristems is most likely caused by differences in the cell-to-cell transport in differentiation zones without functional phloem (immature sieve tubes).

Transcript levels of AtMRPs after cadmium treatment: induction of AtMRP3

In yeasts, the ABC-type transporters are involved in vacuolar sequestration of cadmium. In plants, transport experiments with isolated vacuoles indicate that this is also true. In order to know more about the response of AtMRPs, a subclass of Arabidopsis ABC transporters, to cadmium, their expression pattern was analysed using the microchip technology and semi-quantitative reverse transcriptase-polymerase chain reaction. From 15 putative sequences coding for AtMRPs, transcript levels were detected for 14. All were expressed in the roots as well as in the shoots, although at a different level. In 4-week-old Arabidopsis, transcript levels of four AtMRPs were up-regulated after cadmium treatment. In all cases up-regulation was exclusively observed in the roots. The increase of transcript levels was most pronounced for AtMRP3. A more detailed analysis revealed that induction of AtMRP3 could also be observed in the shoot when leaves were cut and cadmium allowed to be taken up in the shoot. In young plantlets, a far higher portion of Cd2+ was translocated in the aerial part compared with adult plants. Consequently, AtMRP3 transcript levels increased in both root and shoot of young plants. This suggests that 7-day-old seedlings do not exhibit such a strict root–shoot barrier as 4-week-old plants. Expression analysis with mutant plants for glutathione and phytochelatin synthesis as well as with compounds producing oxidative stress indicate that induction of AtMRP3 is likely due to the heavy metal itself.

Biochemical changes in barley plants after excessive supply of copper and manganese

The present study was undertaken to identify changes in some important proteins involved in CO2 fixation (Rubisco, Rubisco activase (RA), Rubisco binding protein (RBP)), NH4+ assimilation (glutamine synthetase (GS) and glutamate synthase (GOGAT)), using immunoblotting, and in the antioxidative defense as a result of Cu or Mn excess in barley leaves (Hordeum vulgare L. cv. Obzor). Activities and isoenzyme patterns of superoxide dismutase (SOD), ascorbate peroxidase (APX), guaiacol peroxidase (GPX) and catalase (CAT), as well as the levels of ascorbate (ASC), non-protein sulfhydryl groups, hydrogen peroxide and oxidative damage to proteins were determined. Data were correlated to the accumulation of Cu or Mn in the leaves after 5 days supply of heavy metal (HM) excess in the nutrient solution. In the highest Cu excess (1500 μM), Rubisco LS and SS were reduced considerably whereas under the highest Mn concentrations (18,300 μM) only minor changes in Rubisco subunits were detected. The RBP was diminished under the highest concentrations of both Cu or Mn. The bands of RA changed differently comparing Cu and Mn toxicity. GS decreased and GOGAT was absent under the highest concentration of Cu. At Mn excess Fd-GOGAT diminished whereas GS was not apparently changed. The development of toxicity symptoms corresponded to an accumulation of Cu or Mn in the leaves and to a gradual increase in protein carbonylation, a lower SOD activity and elevated CAT and GPX activities. APX activity was diminished under Mn toxicity and was not changed under Cu excess. Generally, changes in the isoenzyme profiles were similar under both toxicities. An accumulation of H2O2 was observed only at Mn excess. Contrasting changes in the low-molecular antioxidants were detected when comparing both toxicities. Cu excess affected mainly the non-protein SH groups, while Mn influenced the ASC content. Oxidative stress under Cu or Mn toxicity was most probably the consequence of depletion in low-molecular antioxidants as a result of their involvement in detoxification processes and disbalance in antioxidative enzymes. The link between heavy metal accumulation in leaves, leading to different display of oxidative stress, and changes in individual chloroplast proteins is discussed in the article.

High depth-resolution laser ablation chemical analysis of additive-assisted Cu electroplating for microchip architectures

Laser ablation/ionisation mass spectrometry with a vertical resolution at a nanometre scale was applied for the quantitative characterisation of the chemical composition of additive-assisted Cu electroplated deposits used in the microchip industry. The detailed chemical analysis complements information gathered by optical techniques and allows new insights into the metal deposition process.

Characterization of a Novel Composite Staphylococcal Cassette Chromosome mec in Methicillin-Resistant Staphylococcus pseudintermedius from Thailand.

A novel staphylococcal cassette chromosome mec (SCCmec) composite island (SCCmecAI16-SCCczrAI16-CI) was identified in Staphylococcus pseudintermedius. Four integration site sequences for SCC subdivided the 60,734-bp island into 41,232-bp SCCmecAI16, 19,400-bp SCCczrAI16, and 102-bp SCC-likeAI16 elements. SCCmecAI16 represents a new combination of ccrA1B3 genes with a class A mec complex. SCCczrAI16 contains ccrA1B6 and genes related to restriction modification and heavy metal resistance. SCCmecAI16-SCCczrAI16-CI was found in methicillin-resistant S. pseudintermedius sequence type 112 (ST112) and ST111 isolated from dogs and veterinarians in Thailand.