Dietrich, Daniel R.
Pitfalls in microcystin extraction and recovery from human blood serum
2014, Heussner, Alexandra H., Altaner, Stefan, Kamp, Lisa, Rubio, Fernando, Dietrich, Daniel R.
Microcystins (MCs) contaminate water bodies due to cyanobacterial blooms all over the world, leading to frequent exposure of humans to MCs through consumption of meat, fish, seafood, blue-green algal products and water, accidental ingestion of contaminated water and scum during recreational activities and inhalation of cyanobacterial aerosols. For monitoring of human exposure, sensitive screening methods are needed. However, during the analytical process of various matrices, such as human serum, some problems appear to regularly occur during sample preparation and storage, leading to MC loss and thus to underestimation of the true MC concentration. The aim of the current study was therefore to assess the pitfalls of the MC-extraction method from human serum with more detail.pi
Comparison of two renal cell lines (NRK-52E and LLC-PK1) as late stage apoptosis models
2013, Heussner, Alexandra H., Dietrich, Daniel R.
Renal cell lines are frequently used models in toxicology. The aim of the experiments described here was to investigate the suitability of two of those renal cell lines, namely NRK-52E and LLC-PK1, as models for mid to late stage apoptosis under standard cell culture conditions; the latter means that testing was performed in the presence of serum in the culture media. Seven known inducers of apoptosis already positively tested by other investigators were chosen as test substances using chromatin condensation (Hoechst staining) as endpoint. These substances were cadmium chloride (CdCl2), dithiothreitol (DTT), sodium chloride (NaCl), mercuric chloride (HgCl2), tributyltin oxide (TBT-O), tributyltin chloride (TBT-Cl) and staurosporine. From these, only TBT-O, TBT-Cl and staurosporine induced morphological features typical of apoptosis in LLC-PK1 cells. Morphologically discerned apoptosis was confirmed by DNA fragmentation (DNA laddering assay) analysis. LLC-PK1 cells, but not NRK-52E cells, were shown to be suitable models of mid to late stage apoptosis under the conditions employed. TBT-O, TBT-Cl and staurosporine were shown to be suitable positive controls for apoptosis in renal cells in vitro.
Development and Characterization of a Monoclonal Antibody against Ochratoxin B and Its Application in ELISA
2010, Heussner, Alexandra H., Ausländer, Simon, Dietrich, Daniel R.
A monoclonal antibody specific to ochratoxin B (OTB) was employed for the development of an indirect competitive OTB-ELISA. The optimized OTB-ELISA resulted in a limit of detection (LOD) for OTB of 3 μg/L (8 nM), a limit of quantification (LOQ) of 3.7 μg/L (10 nM), and a 50% inhibitory concentration (IC50) of 150 nM. Due to very low cross-reactivity to OTA (2.7%) and structurally related molecules (0%), this OTB-ELISA was found to be suitable to detect OTB with excellent precision in different matrices, i.e., beer, coffee and wine. Therefore, this OTB-ELISA will allow screening of OTB in food and feed products.
Effects of repeated ochratoxin exposure on renal cells in vitro
2007, Heussner, Alexandra H., O'Brien, Evelyn, Dietrich, Daniel R.
In the present study an in vitro model of subchronic repeated exposure to OTA and OTB was employed to generate ochratoxinderived subpopulations of human and porcine proximal tubular cells (HKC, IHKE, PKC, LLC-PK1). These cell subpopulations were subsequently used to investigate effects on cell proliferation rates, expression of marker proteins (cytokeratins, vimentin) and the acute cytotoxicity of OTA and OTB (MTT reduction, neutral red uptake, cell number). The hypothesis was tested whether repeated exposure at moderate concentrations of these toxins could provide for a reduced sensitivity of selected cell subpopulations to subsequent toxin exposure. Despite the observed increased cell population doubling times and the reduced sensitivity toward OTA and OTB exposure of some cell types, with the exception of the primary human epithelial cells, no overt changes in the expression of cytokeratin and vimentin
could be determined. The presented data, however suggest that repeated exposure of renal epithelial cells to ochratoxins A or B will provide for a subpopulation of cells with reduced ochratoxin-sensitivity and alterations in growth characteristics.
Comparison of two ELISA-based methods for the detection of microcystins in blood serum
2014, Heussner, Alexandra H., Winter, Isabell, Altaner, Stefan, Kamp, Lisa, Rubio, Fernando, Dietrich, Daniel R.
Microcystins (MCs) are cyanobacterial toxins which place the public at risk via exposure to MC contaminated water, food or algal food supplements. Subsequent to the fatal intravenous exposure of dialysis patients in Caruaru, Brazil, several techniques (LC–MS, GC–MS and ELISA) were adapted to detect MCs in human serum. As patients chronically exposed to low concentrations of MCs also present with very low MC serum levels, only LC–MS methodology would appear to allow detection of these MC levels. However, LC–MS detection depends on the availability of respective MC congener standards and the levels of non-covalently bound MC in the sample. In contrast, immunological techniques, e.g. MC-ELISA potentially could detect even covalently bound MC, provided the MC-antibody was raised against an epitope found in nearly all of the MC congeners. As the Adda-side-chain moiety is present in nearly all of the MC congeners known to date, the anti-Adda antibodies, when applied in Adda-ELISAs, could represent a relatively simple and robust technique for the qualitative and quantitative determination of MC in human serum.
The aim of the current study was to determine whether commercially available Adda-ELISAs and their respective sample preparation methods would allow MC quantification in human serum. The Adda-ELISA (polyclonal antibody) and the Adda-ELISA (monoclonal antibody) kit for serum (Serum-ELISA) were used for determination of the concentration-dependent recovery of MCs in MC-spiked serum.
Human serum samples were spiked with varying concentrations of MCs (MC-LR, -YR, -RR, -LA, -LW, -LF and defined MC mixtures) and extracted using two different methods. MC-spiked bovine serum and standard cell culture medium containing 10% FBS served to investigate potential matrix effects. Inter-laboratory comparison was performed allowing identification of potential sources of error.
The results suggest that both ELISAs are suitable tools for the analysis of MCs in human blood serum although both also displayed some weaknesses notably the time needed for sample preparation or the overestimation of some specific MC congener concentrations. Based on the ELISA detection ranges, sample concentration and/or MC spiking may be required for detection of low levels of MCs in human blood.
MeOH, methanol; SPE, solid phase extraction; MC, microcystin; LOD, limit of detection; FBS, fetal bovine serum; RT, room temperature; DMEM, Dulbecco’s modified Eagle medium; PPIA, protein phosphatase inhibition assay; HPLC, high performance liquid chromatography; LC–MS, liquid chromatography–mass spectrometry; ELISA, enzyme-linked immunosorbent assay; PP, polypropylene; LDPE, low-density polyethylene; HDPE, high-density polyethylene
Primary porcine proximal tubular cells as an alternative to human primary renal cells in vitro : an initial characterization
2013, Heussner, Alexandra H., Dietrich, Daniel R.
A good in vitro model should approximate an in vivo-like behavior as closely as possible in order to reflect most likely the in vivo situation. Regarding renal physiology of different species, humans are more closely related to pigs than to rodents, therefore primary porcine kidney cells (PKC) and their subsequent cell strain could be a valid alternative to primary human cells for renal in vitro toxicology. For this PKC must display inherent characteristics (e.g. structural organization) and functions (e.g. transepithelial transport) as observed under in vivo conditions within the respective part of the kidney.
We carried out a comprehensive characterization of PKC and their subsequent cell strain, including morphology and growth as well as transporter expression and functionality. The data presented here demonstrate that PKC express various transporters including pMrp1 (abcc1), pMrp2 (abcc2), pOat1 (slc22a6) and pOat3 (slc22a8), whereas pMdr1 (abcb1) and pOatp1a2 (slco1a2) mRNA could not be detected in either the PKCs or in the porcine cortical tissue. Functionality of the transporters was demonstrated by determining the specific PAH transport kinetics.
On the basis of the presented results it can be concluded that PKC and to some extent their subsequent cell strain represent a valuable model for in vitro toxicology, which might be used as an alternative to human primary cells.
Investigation of Microcystin Congener-Dependent Uptake into Primary Murine Neurons
2010, Feurstein, Daniel, Kleinteich, Julia, Heussner, Alexandra H., Stemmer, Kerstin, Dietrich, Daniel R.
Contamination of natural waters by toxic cyanobacteria is a growing problem worldwide, resulting in serious water pollution and human health hazards. Microcystins (MCs) represent a group of > 80 cyclic heptapeptides, mediating cytotoxicity via specific protein phosphatase (PP) inhibition at equimolar concentrations (comparable toxicodynamics). Because of the structure and size of MCs, active uptake into cells occurs via organic anion-transporting polypeptides (OATP/Oatp), as confirmed for liver-specific human OATP1B1 and OATP1B3, mouse Oatp1b2 (mOatp1b2), skate Oatp1d1, and the more widely distributed OATP1A2 expressed, for example, at the blood-brain barrier. Tissue-specific and cell-type-specific expression of OATP/Oatp transporters and specific transport of MC congeners (toxicokinetics) therefore appear prerequisite for the reported toxic effects in humans and other species upon MC exposure. Beyond hepatotoxicity induced by the MC-LR congener, the effects of other MC congeners, especially neuronal uptake and toxicity, are unknown.
Bioavailability and potential carcinogenicity of polycyclic aromatic hydrocarbons from wood combustion particulate matter in vitro
2013-11-25, Gauggel-Lewandowski, Susanne, Heussner, Alexandra H., Steinberg, Pablo, Pieterse, Bart, van der Burg, Bart, Dietrich, Daniel R.
Due to increasing energy demand and limited fossil fuels, renewable energy sources have gained in importance. Particulate matter (PM) in general, but also PM from the combustion of wood is known to exert adverse health effects in human. These are often related to specific toxic compounds adsorbed to the PM surface, such as polycyclic aromatic hydrocarbons (PAH), of which some are known human carcinogens. This study focused on the bioavailability of PAHs and on the tumor initiation potential of wood combustion PM, using the PAH CALUX® reporter gene assay and the BALB/c 3T3 cell transformation assay, respectively. For this, both cell assays were exposed to PM and their respective organic extracts from varying degrees of combustion. The PAH CALUX® experiments demonstrated a concentration–response relationship matching the PAHs detected in the samples. Contrary to expectations, PM samples from complete (CC) and incomplete combustion (IC) provided for a stronger and weaker response, respectively, suggesting that PAH were more readily bioavailable in PM from CC. These findings were corroborated via PAH spiking experiments indicating that IC PM contains organic components that strongly adsorb PAH thereby reducing their bioavailability. The results obtained with organic extracts in the cell transformation assay presented the highest potential for carcinogenicity in samples with high PAH contents, albeit PM from CC also demonstrated a carcinogenic potential. In conclusion, the in vitro assays employed emphasize that CC produces PM with low PAH content however with a general higher bioavailability and thus with a nearly similar carcinogenic potential than IC PM.
Toxin content and cytotoxicity of algal dietary supplements
2012-12-01, Heussner, Alexandra H., Mazija, Lorena, Fastner, Jutta, Dietrich, Daniel R.
Blue-green algae (Spirulina sp., Aphanizomenon flos-aquae) and Chlorella sp. are commercially distributed as organic algae dietary supplements. Cyanobacterial dietary products in particular have raised serious concerns, as they appeared to be contaminated with toxins e.g. microcystins (MCs) and consumers repeatedly reported adverse health effects following consumption of these products. The aim of this study was to determine the toxin contamination and the in vitro cytotoxicity of algae dietary supplement products marketed in Germany. In thirteen products consisting of Aph. flos-aquae, Spirulina and Chlorella or mixtures thereof, MCs, nodularins, saxitoxins, anatoxin-a and cylindrospermopsin were analyzed. Five products tested in an earlier market study were re-analyzed for comparison. Product samples were extracted and analyzed for cytotoxicity in A549 cells as well as for toxin levels by (1) phosphatase inhibition assay (PPIA), (2) Adda-ELISA and (3) LC-MS/MS. In addition, all samples were analyzed by PCR for the presence of the mcyE gene, a part of the microcystin and nodularin synthetase gene cluster. Only Aph. flos-aquae products were tested positive for MCs as well as the presence of mcyE. The contamination levels of the MC-positive samples were ≤ 1 μg MC-LR equivalents g(-1) dw. None of the other toxins were found in any of the products. However, extracts from all products were cytotoxic. In light of the findings, the distribution and commercial sale of Aph. flos-aquae products, whether pure or mixed formulations, for human consumption appear highly questionable.
Propiverine-induced accumulation of nuclear and cytosolic protein in F344 rat kidneys : Isolation and identification of the accumulating protein
2008, Dietrich, Daniel R., Heussner, Alexandra H., O'Brien, Evelyn, Gramatté, Thomas, Runkel, Michael, Rumpf, Silke, Day, Billy W.
Male and female F344 rats but not B6C3F1 mice exposed for 104 weeks to propiverine hydrochloride (1-methylpiperid-4-yl 2,2-diphenyl-2-(1-propoxy)acetate hydrochloride), used for treatment of patients with neurogenic detrusor overactivity (NDO) and overactive bladder (OAB), presented with an accumulation of proteins in the cytosol and nuclei of renal proximal tubule epithelial cells, yet despite this, no increased renal tumor incidence was observed. In order to provide an improved interpretation of these findings and a better basis for human health risk assessment, male and female F344 rats were exposed for 16 weeks to 1000 ppm propiverine in the diet, the accumulating protein was isolated from the kidneys via cytosolic and nuclear preparations or laser capture microdissection and analyzed using molecular weight determination and mass spectrometry. The accumulating proteinwas found to be D-amino acid oxidase (DAAO), an enzyme involved in amino and fatty acid metabolism. Subsequent reanalysis of kidney homogenate and nuclear samples as well as tissue sections using western blot and DAAO-immunohistochemistry, confirmed the presence and localization of DAAO in propiverine-treated male and female F344 rats. The accumulation of DAAO only in rats, and the limited similarity of rat DAAO with other species, including humans, suggests a rat-specific mechanism underlying the drug-induced renal DAAO accumulation with little relevance for patients chronically treated with propiverine.