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.

2007, Heussner, Alexandra H., Möller, Ines, Day, Billy W., Dietrich, Daniel R., O'Brien, Evelyn

Monoclonal antibodies against ochratoxin B (OTB) were generated by immunizing Balb/c mice with OTB conjugated to keyhole limpet hemocyanin (KLH) via carbodiimide reactions with CHMC and EDAC. A stable hybridoma cell line 2F1.E10 was produced by fusion of murine splenocytes and myeloma cells. The obtained antibodies were characterized using an indirect competitive ELISA. The detection limit was calculated (27 ± 2 nM OTB) and 50% binding inhibition was reached at 500 nM free OTB. A low cross-reactivity to ochratoxin A (OTA) of 3.3% and no cross-reactivities to either coumarin or dl-phenylalanine were observed, suggesting a highly specific OTB antibody. The antibody type was identified as IgG class 1 with the light chain being of the κ configuration.
These antibodies can be used in an indirect competitive ELISA to detect OTB in the nanomolar to micromolar concentration range and may be useful for the analysis of contaminated food items.

2006, Ernst, Bernhard, Höger, Stefan J., O'Brien, Evelyn, Dietrich, Daniel R.

The microcystin-producing cyanobacterium Planktothrix is one of the most widespread genera amongst toxin producing cyanobacteria in European lakes. In particular, the metalimnic blooms of Planktothrix rubescens have been associated with growing problems in the professional freshwater fishery as a decrease in yearly yields in the important coregonids fishery often coincides with the appearance of P. rubescens. P. rubescens is a cyanobacterial species known to produce toxic compounds, e.g. microcystins. Although microcystins have been reported to affect fish health, behaviour, development and growth and have also been associated with feral fish kills, there is currently no specific information on the effects of toxic Planktothrix filaments in fish and especially coregonids. Therefore, the aim of this study was to investigate the effects of an environmentally relevant dose of P. rubescens filaments orally applied to coregonids and to discuss the findings in the context of microcystin toxicity previously reported in carp and trout.
A single dose of P. rubescens culture, at a density of 80,000 cells per 120 μl, was applied to coregonids thus corresponding to 0.6 0.9 μg microcystin-LRequiv./kg body weight. Behavioural changes and opercular beat rates, growth, hepatosomatic index, condition and plasma glucose were determined. Liver, kidney, gill and the gastrointestinal tract were assessed histopathologically and immunhistologically. Exposed fish showed behavioural changes, increased opercular beat rates and elevated plasma glucose levels, possibly representing a physiological stress response. Histopathological alterations in liver, gastrointestinal tract and kidney, also immunopositive for microcystin suggested causality of tissue damage and the in situ presence of microcystins.
The observed combination of stress and organ damage may explain the frequently reduced weight and thus the fitness noted in coregonids subjected to regular occurrences of stratified and dispersed P. rubescens blooms, e.g. in lake Ammersee, Bavaria, Germany.

2006, Dietrich, Daniel R., Hitzfeld, Bettina C., O'Brien, Evelyn

2008, Huljic, Susanne, Bruske, Ellen Inga, Pfitzenmaier, Norbert, O'Brien, Evelyn, Dietrich, Daniel R.

Differences in toxicity and carcinogenicity of the nephrotoxic compound aristolochic acid between rodents and humans suggest a species-dependent mechanism of action. The goal of this study was to investigate constitutive differences in the susceptibility of renal cortex cells originating from human, rat and porcine origin in vitro. Effects of 24 and 48 h AA exposure on cell number and MTT reduction were studied. Furthermore, using the effective concentrations causing 20 and 50 % reduction (cell number), cell cycle, 3H-thymidine incorporation and DNA damage analyses were conducted. AA cytotoxicity was observed in all cell types in a time- and concentration dependent manner with species-specific differences, with porcine cells being the most sensitive. AA had a comparable effect on the cell cycle in primary human and porcine cells and the rat NRK-52E cell line following 48 h exposure, also corroborated by the reduced 3H-thymidine incorporation in NRK-52E cells. In addition, DNA unwinding, suggestive of enhanced DNA damage, was observed in primary porcine cells. These results provide an initial insight into the sensitivity and suitability of different in vitro-systems and suggest that primary porcine renal cortex cells could be a valuable in vitro-system to study AA toxicity

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.

2006, Dietrich, Daniel R., O'Brien, Evelyn, Hoffmann, Sebastian, Balaguer, Patrique, Nicolas, Jean-Claude, Seinen, Willem, Depledge, Michael

2007, Ernst, Bernhard, Höger, Stefan J., O'Brien, Evelyn, Dietrich, Daniel R.

Planktothrix rubescens belongs to the most ubiquitous cyanobacterial species in mesotrophic and oligotrophic lakes in the pre-alpine regions. In most of these lakes, coregonids are among the dominant species of the ichthyofauna with great importance for the professional fishery. A possible link between the occurrence of toxic Planktothrix blooms and the recurrent slumps in coregonid yields has been suggested. Indeed, acute toxic effects of microcystins and other cyanobacterial toxins have been shown for various fish species. However, chronic exposure scenarios appear to be more common and thus more environmentally realistic than acute intoxications. The aim of this study was therefore to investigate the physiological stress response and organ pathology in coregonids sub-chronically exposed to ambient water containing low, medium and high P. rubescens densities, known to be typical of pre-alpine lakes. Coregonid hatchlings were exposed in four tanks containing 0 (sham-control) and approximately 1500 (low), 15,000 (medium) and 55,000 (high) P. rubescens cells/ml for up to 28 days. Temperature, oxygen concentration, pH-value, P. rubescens cell density and microcystin concentration were recorded and the fish were observed for behavioural changes and examined for parasite infestations. Gill ventilation rates, general condition factors and mortalities were determined and liver, kidney, gut and gill were assessed histopathologically and immunhistologically.
Depending on the cell density, exposed fish showed behavioural changes, including increased ventilation rates possibly representing a physiological stress response. Susceptibility to ectoparasitic infestation and increased mortality in exposed fish suggested P. rubescens associated effects on fish fitness. Histopathological alterations in liver, gastrointestinal tract and kidney, which were also immunopositive for microcystin suggested causality of tissue damage and the presence of microcystins. In contrast, observed gill pathology appeared to result primarily from mechanical abrasion and irritation due to ectoparasitic infestation. The current exposure experiment confirmed the hypothesis that subchronic and chronic exposure to low cyanobacterial cell densities and hence microcystins can exacerbate physiological stress and sustained pathological alterations in exposed coregonids. The study therefore supports the theory that P. rubescens blooms may be causal to the observed weight reduction and hence fitness of coregonids in pre-alpine lakes such as Lake Ammersee (Germany).

2006, Heussner, Alexandra H., Dietrich, Daniel R., O'Brien, Evelyn

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.

2006, Ernst, Bernhard, Neser, Stephan, O'Brien, Evelyn, Höger, Stefan J., Dietrich, Daniel R.

Cyanobacteria occur in surface waters worldwide. Many of these produce peptides and/or alkaloids, which can present a risk for animal and human health. Effective risk assessment and management requires continuous and precise observation and quantification of cyanobacterial cell densities. In this respect, quantification of filamentous Planktothrix species is problematic. The aim of this study was to develop an automated system to count filamentous Planktothrix rubescens using image processing. Furthermore, this study aimed to assess optimum sample volumes and filament density for measurement precision and to validate image processing measurement of P. rubescens for an effective risk assessment.
Three environmental samples and one cultured sample of P. rubescens were collected by filtration onto nitrocellulose filters. Filament lengths were determined using fluorescence microscopy combined with an image processor. Cell density could be calculated from the resulting images. Cyanobacteria could easily be discriminated from algae via their fluorescence properties. The results were found to be independent of the mode of image acquisition. The precision of total filament length determination was dependent on the total filament length on the filter, i.e. analyses of highest precision could be expected for filters containing 2000 20,000 μm filaments per mm2. When using suitable filtration volumes, the detection limits of the described method are sufficient for an effective risk assessment. To summarise, this procedure is a fast, easy and accurate method to determine cell densities of filamentous P. rubescens in water samples without costly and tedious manual handling.