Development and Characterization of a Monoclonal Antibody against Ochratoxin B and Its Application in ELISA

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

Gefährliche Kosmopoliten - Die Arbeitsgruppe von Prof. Daniel Dietrich erforscht die Gefahren von Blaualgen

Schlagzeilen wie "Algenalarm in der Ostsee", "Alles grün: Bloß nicht in der Alster baden", "Algen verfärben den Chiemsee rot" oder, "Alzheimer nach Badeurlaub in Schweden" - trüben vielerorts Jahr für Jahr die sommerlichen Badefreuden. Noch alarmierender sind Meldungen wie "Blaualgen-Nahrungsergänzungsmittelhersteller Cell Tech in Oregon nach Todesfall verklagt". Hintergrund sind meist nur wenige Mikrometer große Organismen: Cyanobakterien, auch als Blaualgen bekannt. An der Universität Konstanz beschäftigt man sich in der Arbeitsgruppe von Prof. Daniel Dietrich seit zehn Jahren intensiv mit dieser Problematik

Unravelling the ochratoxin enigma

Ochratoxins are a group of nephrotoxins produced by a variety of moulds and were first described in 1965 [1]. Dietary exposure to ochratoxins represents a serious health issue and has been associated with several human and animal diseases including porcine nephropathy, Human Endemic Nephropathies and urinary tract tumours in humans. More than 20 years ago, ochratoxin A (OTA) - the most prominent member of this toxin family - was shown to be carcinogenic in rodents and since then extensive research has been performed in order to investigate whether OTA acts by a DNA reactive mode of action. Only recently, this theory has been conclusively disproven and a non-genotoxic mechanism is currently widely accepted for renal toxicity and carcinogenicity of OTA. The work presented in this thesis contributed to this field of science in various ways. First of all, new renal in vitro models were established and existing models were improved for the investigation of renal ochratoxin toxicity. Using these models, the in vitro effects of various OTA exposure scenarios were investigated and endpoints included amongst others general cytotoxicity, cell cycle analysis, protein binding and toxin transport. In all of these studies, distinct species- and sex-differences were observed which mirrored the observed effects in vivo described in literature. Furthermore, the differential toxicity of ochratoxin group members was investigated, the results of which inferred a need for specific detection of OTA and OTB. Due to the lack of such a detection tool, a new, highly specific and sensitive OTB-ELISA was successfully developed based on an OTB-specific monoclonal antibody produced and characterised in our laboratory. This tool allows screening of OTB in food and feed products, which will further improve detection and risk assessment. All of these studies contributed to the elucidation of the underlying mechanisms of ochratoxin toxicity. Therefore, this thesis can be seen as a fundamental part of a paradigm change on our understanding of how ochratoxins exert their harmful long-term effects in humans and animals, thus elucidating the ochratoxin enigma

Primary porcine proximal tubular cells as an alternative to human primary renal cells in vitro : an initial characterization

BackgroundA 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.ResultsWe 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.ConclusionsOn 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

Comparison of two renal cell lines (NRK-52E and LLC-PK1) as late stage apoptosis models

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

Species-, Sex-, and Cell Type-Specific Effects of Ochratoxin A and B

The ubiquitous mycotoxin ochratoxin A (OTA) is associated with the development of urothelial tumors and nephropathies in laboratory animals and in humans with stark species and sex differences with respect to susceptibility in disease development. The mechanism of action remains unknown. OTA-mediated disruptions in normal cell-cycle control could be a major constituent of the mechanisms underlying both its carcinogenic and nephropathy-inducing activities. Assessment of OTA's toxic effects (sum of antiproliferative, apoptotic, and necrotic effects) in rat and porcine continuous cell lines and in primary cells from humans and pigs of both sexes, have displayed a similar sex- and species-sensitivity rank order to that observed in previous in vivo experiments. Furthermore, these toxic effects were observed at nM concentrations in the presence of serum in vitro, thus closely mimicking the in vivo situation. These effects were reversible in all cell types except in human primary epithelial cells of both sexes and did not appear to be primarily dependent on the amount of OTA taken up. Indeed, fibroblasts (NRK-49F) were insensitive to OTA-mediated cell cycle inhibition in spite of accumulating comparable amounts of OTA. The results presented here support the continued use of primary renal epithelial cells for the investigation of the mechanism of OTA-induced carcinogenesis and nephropathy and provide an as-yet preliminary data set that supports the existence of a causal relationship between OTA exposure and human nephropathy

Effects of repeated ochratoxin exposure on renal cells in vitro

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 vimentincould 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

In vitro investigation of individual and combined cytotoxic effects of ochratoxin A and other selected mycotoxins on renal cells

Hundreds of mycotoxins are known to date and many of them are of great interest with regard to human and animal health since they are detected frequently in plant-derived products. Various mycotoxins may occur simultaneously, depending on the environmental and substrate conditions. Considering this coincident production, it is very likely, that humans and animals are always exposed to mixtures rather than to individual compounds. Therefore, future risk assessments should consider mixture toxicity data. This is particularly true for ochratoxin A (OTA), ochratoxin B (OTB), citrinin (CIT) and occasionally for patulin (PAT) as they are all produced by a number of Penicillium and Aspergillus species. Therefore, these four toxins were chosen to study the interactive effects in vitro, using the well-established porcine renal cell line LLC-PK1 and the MTT reduction test as a cytotoxicity endpoint. By application of a step-wise approach to test combination toxicity, using various full factorial as well as a central composite experimental designs, the interactive (synergistic) cytotoxic effects of the these four toxins were assessed. The results obtained in this study confirm a potential for interactive (synergistic) effects of CIT and OTA and possibly other mycotoxins in cells of renal origin

Species- and sex-specific variations in binding of ochratoxin A by renal proteins in vitro

The mycotoxin ochratoxin A (OTA) is a potent renal carcinogen in rodents and induces renal fibrosis in pigs. Furthermore, OTA has been associated with the development of renal tumors and nephropathies in humans. Large species- and sex-differences are observed in sensitivity toward OTA-mediated toxicity and carcinogenicity, yet neither the mechanism(s) resulting in OTA toxicity nor the reasons for the observed species- and sex-specificities are known. This paper investigated variations in OTA handling viz binding to renal proteins which could possibly explain the observed differences in OTA susceptibility in vivo and in vitro. The results obtained via a modification of a standard receptor-binding assay demonstrated the presence of at least one homogeneous binding component in renal cortical homogenates from pig, mouse, rat and humans. This component was shown to bind OTA in a specific and saturable manner. A range of compounds selected for their affinity for steroid receptors and/or for various known organic anion transporters were employed in a competition assay to answer the question whether this homogenous OTA binding component represents a steroid-like receptor component or one of the known organic anion transporters of the kidney. Although many of the compounds were able to compete with OTA for protein-binding, the competition patterns displayed a distinct species specificity and did not correspond to the competition patterns associated with presently known organic anion transporters of the kidney in the mouse, rat or human. The data thus suggests the presence of a new organic anion transporter or more likely, a cytosolic binding component of unknown function with high affinity and capacity for OTA binding in humans, rats, mice and possibly pigs

Ochratoxin A : Comparative pharmacokinetics and toxicological implications (experimental and domestic animals and humans)

The causal factors for the species- and sex-differences associated with ochratoxin-mediated toxicity remain unclear. Variations in kinetic parameters may play a major role in explaining these differences, however, discrepancies and inaccuracies in the toxicokinetics reported in the literature for various species, make comparison and hence the extrapolation to the human situation impossible. The one- and two-compartment open models currently proposed may be insufficient to enable an accurate representation of the actual situation in vivo. It is likely that at least three if not four compartments must be assumed to account for the reported effects. The application of such models to existing raw data would most likely provide for a more accurate base set of toxicokinetic data and contribute to a more accurate human risk assessment. Possible explanations for the reported inconsistencies and their impact on the proposed mechanism(s) of action of OTA and risk assessment are discussed