Jörg Hippler

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.

Methylated Bismuth, but Not Bismuth Citrate or Bismuth Glutathione, Induces Cyto- and Genotoxic Effects in Human Cells in Vitro

Bismuth compounds are widely used in industrial processes and products. In medicine, bismuth salts have been applied in combination with antibiotics for the treatment of Helicobacter pylori infections, for the prevention of diarrhea, and in radioimmunotherapy. In the environment, bismuth ions can be biotransformed to the volatile bismuth compound trimethylbismuth (Me3Bi) by methanobacteria. Preliminary in-house studies have indicated that bismuth ions are methylated in the human colon by intestinal microflora following ingestion of bismuth-containing salts. Information concerning cyto- and genotoxicity of these biomethylated products is limited. In the present study, we investigated the cellular uptake of an organic bismuth compound [monomethylbismuth(III), MeBi(III)] and two other bismuth compounds [bismuth citrate (Bi-Cit) and bismuth glutathione (Bi-GS)] in human hepatocytes, lymphocytes, and erythrocytes using ICP-MS. We also analyzed the cyto- and genotoxic effects of these compounds to investigate their toxic potential. Our results show that the methylbismuth compound was better taken up by the cells than Bi-Cit and Bi-GS. All intracellularly detected bismuth compounds were located in the cytosol of the cells. MeBi(III) was best taken up by erythrocytes (36%), followed by lymphocytes (17%) and hepatocytes (0.04%). Erythrocytes and hepatocytes were more susceptible to MeBi(III) exposure than lymphocytes. Cytotoxic effects of MeBi(III) were detectable in erythrocytes at concentrations >4 microM, in hepatocytes at >130 microM, and in lymphocytes at >430 microM after 24 h of exposure. Cytotoxic effects for Bi-Cit and Bi-GS were much lower or not detectable in the used cell lines up to a tested concentration of 500 microM. Exposure of lymphocytes to MeBi(III) (250 microM for 1 h and 25 microM/50 microM for 24 h) resulted in significantly increased frequencies of chromosomal aberrations (CA) and sister chromatid exchanges (SCE), whereas Bi-Cit and Bi-GS induced neither CA nor SCE. Our study also showed an intracellular production of free radicals caused by MeBi(III) in hepatocytes but not in lymphocytes. These data suggest that biomethylation of bismuth ions by the intestinal microflora of the human colon leads to an increase in the toxicity of the primary bismuth salt.

Parallel ICP-MS and EI-MS detection after GC separation as a unique tool for simultaneous identification and quantification of volatile heteroatomic organic compounds

The presented technique combines the quantitative benefits characteristic of element specific inductively coupled plasma mass spectrometry (ICP-MS) techniques with the advantages of highly selective and molecule specific electron ionisation mass spectrometry (EI-MS) detection by parallel hyphenation of these detectors. As proof of concept, parallel detection of four iodinated alkanes (iodomethane, iodoethane, 1-iodopropane, 2-iodopropane) with EI-MS and ICP-MS after capillary gas chromatography (GC) is demonstrated. Analytical figures of merit resulting from linear calibration are presented and compared with respect to the two different detectors. Limits of detection (0.6 pg as iodine) comparable to values reported in literature were achieved for ICP-MS detection of all investigated species.

Cross-Contamination of a UROtsa Stock with T24 Cells – Molecular Comparison of Different Cell Lines and Stocks

Background UROtsa is an authentic, immortalized human urothelial cell line that is used to study the effects of metals and other toxic substances, mostly in the context of bladder cancer carcinogenesis. Unusual properties on the molecular level of a provided UROtsa cell line stock prompted us to verify its identity. Methods UROtsa cell line stocks from different sources were tested on several molecular levels and compared with other cell lines. MicroRNA and mRNA expression was determined by Real-Time PCR. Chromosome numbers were checked and PCR of different regions of the large T-antigen was performed. DNA methylation of RARB, PGR, RASSF1, CDH1, FHIT, ESR1, C1QTNF6, PTGS2, SOCS3, MGMT, and LINE1 was analyzed by pyrosequencing and compared with results from the cell lines RT4, T24, HeLa, BEAS-2B, and HepG2. Finally, short tandem repeat (STR) profiling was applied. Results All tested UROtsa cell line stocks lacked large T-antigen. STR analysis unequivocally identified our main UROtsa stock as the bladder cancer cell line T24, which was different from two authentic UROtsa stocks that served as controls. Analysis of DNA methylation patterns and RNA expression confirmed their differences. Methylation pattern and mRNA expression of the contaminating T24 cell line showed moderate changes even after long-term culture of up to 56 weeks, whereas miRNAs and chromosome numbers varied markedly. Conclusions It is important to check the identity of cell lines, especially those that are not distributed by major cell banks. However, for some cell lines STR profiles are not available. Therefore, new cell lines should either be submitted to cell banks or at least their STR profile determined and published as part of their initial characterization. Our results should help to improve the identification of UROtsa and other cells on different molecular levels and provide information on the use of urothelial cells for long-term experiments.

Organometal(loid) compounds associated with human metabolism

Biomethylation of metals and metalloids is a well-known process ubiquitously occurring in the environment, which leads to the formation of chemical species with significantly higher mobility and altered toxicology. There are only a few historical reports, e.g. about “bismuth breath” or “Gosio gas” dealing with the association of humans with methylated metal(loid)s. Although the toxicity of the latter [later identified as trimethyl arsine (Challenger 1945)] has not been conclusively demonstrated, this gas produced by fungi in wet wallpaper was considered to be the reason for the illness of people living there (Gosio 1897). Amongst other observations, dimethyltellurium in “bismuth breath” of mine workers, dimethylselenium in the upper ng/m3 range in human breath, as well as the detection of at least twenty-two different organometal(loid) species in human urine are indications for the methylation of metal(loid)s occurring in humans (Cai et al. 1995; Feldmann et al. 1996; Kresimon et al. 2001).

Trace Metal(loid)s (As, Cd, Cu, Hg, Pb, PGE, Sb, and Zn) and Their Species

Advances in instrumental analytical techniques have now enabled scientists not only to quantify even ultra-trace constituents in aquatic systems, such as platinum-group elements (PGEs) in ecosystems, or heavy metals, such as lead or antimony, at anthropogenically not-influenced lowest background levels, but also to perform speciation analysis as discussed for the metal(loid)s arsenic, antimony, and mercury. With respect to the latter, particularly, the remarkable physical chemical properties (especially mobility and amphiphilicity) of low-molecular-weight organometal(loid) species underline their importance in environmentally and toxicologically relevant processes. In reviewing speciation, the concept of hard and soft acids and bases, colloids, dissolved organic matter, and empirical as well as theoretical speciation methods are mentioned, and discussed in brief for cadmium, copper, and zinc as well. For natural waters (especially groundwater), geochemical and local background concentrations are presented based on published literature data. Compared to these levels, geogenic (e.g., arsenic in deep aquifers in West Bengal and Bangladesh) and anthropogenic contaminations of surface and groundwater are reviewed (arsenic in areas of mining, mercury in gold mining, and mercury in mine calcines). In particular, attention is focused on certain scenarios (Minamata, Florida Everglades, reservoir flooding, and estuaries) showing increased mercury methylation, eventually leading to mercury-contaminated fish, because critical parameters of natural ecosystems have been changed anthropogenically. Environmental effects of metal mining (in particular, those generating acid mine drainage) are discussed by presenting selected examples from all over the world (Spain, Sweden, Finland, Mexico, Alaska, China, and Korea). Environmental and toxicological impact of PGEs is discussed, and palladium has been identified to represent the most critical element in this group. However, it is mentioned that environmental-risk evaluations will only be possible after speciating the chemical form of these elements in different environmental compartments.

Toxicity of volatile methylated species of bismuth, arsenic, tin, and mercury in Mammalian cells in vitro.

The biochemical transformation of mercury, tin, arsenic and bismuth through formation of volatile alkylated species performs a fundamental role in determining the environmental processing of these elements. While the toxicity of inorganic forms of most of these compounds are well documented (e.g., arsenic, mercury) and some of them are of relatively low toxicity (e.g., tin, bismuth), the more lipid-soluble organometals can be highly toxic. In the present study we investigated the cyto- and genotoxicity of five volatile metal(loid) compounds: trimethylbismuth, dimethylarsenic iodide, trimethylarsine, tetramethyltin, and dimethylmercury. As far as we know, this is the first study investigating the toxicity of volatile metal(loid) compounds in vitro. Our results showed that dimethylmercury was most toxic to all three used cell lines (CHO-9 cells, CaCo, Hep-G2) followed by dimethylarsenic iodide. Tetramethyltin was the least toxic compound; however, the toxicity was also dependend upon the cell type. Human colon cells (CaCo) were most susceptible to the toxicity of the volatile compounds compared to the other cell lines. We conclude from our study that volatile metal(loid) compounds can be toxic to mammalian cells already at very low concentrations but the toxicity depends upon the metal(loid) species and the exposed cell type.

A four dimensional separation method based on continuous heart-cutting gas chromatography with ion mobility and high resolution mass spectrometry

A two-dimensional GC (2D-GC) method was developed and coupled to an ion mobility-high resolution mass spectrometer, which enables the separation of complex samples in four dimensions (2D-GC, ion mobilility spectrometry and mass spectrometry). This approach works as a continuous multiheart-cutting GC-system (GC+GC), using a long modulation time of 20s, which allows the complete transfer of most of the first dimension peaks to the second dimension column without fractionation, in comparison to comprehensive two-dimensional gas chromatography (GCxGC). Hence, each compound delivers only one peak in the second dimension, which simplifies the data handling even when ion mobility spectrometry as a third and mass spectrometry as a fourth dimension are introduced. The analysis of a plant extract from Calendula officinales shows the separation power of this four dimensional separation method. The introduction of ion mobility spectrometry provides an additional separation dimension and allows to determine collision cross sections (CCS) of the analytes as a further physicochemical constant supporting the identification. A CCS database with more than 800 standard substances including drug-like compounds and pesticides was used for CCS data base search in this work.

Volatile mercury species in environmental gases and biological samples

Mercury is emitted into the atmosphere from a number of natural as well as anthropogenic sources. Anthropogenic emissions of mercury are thought to be of the same order of magnitude as those from natural sources (Ebinghaus et al. 1998). In addition to elementary mercury in the atmosphere, dimethyl and diethylmercury, as well as particle-bound mercury have been detected in the very low pg/l concentration range (Kaiser and Tolg 1980).