2009, Ernst, Bernhard, Höger, Stefan J., O´Brien, Evelyn, Dietrich, Daniel R.

Regular occurrences of the cyanobacterium Planktothrix rubescens have been observed in several lakes that have undergone recent re-oligotrophication, e.g. Lake Ammersee. Planktothrix species are known to produce microcystins, potent phosphatase inhibitors that have been associated with morbidities and mortalities in humans and animals. The aim of this study was to characterise the temporal and spatial abundance and toxicity of P. rubescens in Lake Ammersee.
P. rubescens cell densities and biovolumes were calculated via fluorescence image analyses. P. rubescens was present during the entire observation period from 1999 to 2004, albeit at different cell densities. Maximum biovolumes of 45 cm³ m‾² were observed in May 2001. Filaments were regularly distributed over the entire water column during winter and stratified in distinct metalimnic layers during summer, reaching maximum cell densities of ≤15,000 (winter) and ≤77,000 cells ml‾¹ (summer). The results demonstrate that P. rubescens abundance is strongly influenced by water transparency, i.e. illumination in the metalimnion. Moreover, the P. rubescens abundance appears to result from regular phosphate depletion in the epilimnion. possibly additionally benefiting from high nitrogen loads.
Microcystin (MC) was detectable in 27 and 38 of 54 seston samples via HPLC and Adda-ELISA measurements, respectively. The main microcystin congeners in the seston samples were [Asp³]-MC-RR and [Asp³,Dhb7]-MC-RR. Microcystin concentrations correlated significantly with the respective phycoerythrin (PE)-concentrations. The variation in the MC/PE-ratios was low suggesting that the microcystin production of P. rubescens in Lake Ammersee is consistent and indicates that the appearance of P. rubescens coincides with measurable microcystin levels. Moreover, the observation of pronounced metalimnic oxygen depletions appears to be causally related to recurring high P. rubescens abundance.
In conclusion the results suggest that aquatic organisms such as indigenous fish populations (e.g. coregonids) are regularly confronted with potentially adverse P. rubescens densities, which might provide a possible explanation for the often observed impaired health and growth retardation of coregonid populations in P. rubescens containing pre-alpine lakes.

2008, Kreke, Nicola, Dietrich, Daniel R.

Selective serotonin reuptake inhibitors (SSRIs) are among the pharmaceutical compounds frequently detected in sewage treatment plant effluents and surface waters, albeit at very low concentrations, and have therefore become a focus of interest as environmental pollutants. These neuroactive drugs are primarily used in the treatment of depression but have also found broader use as medication for other neurological dysfunctions, consequently resulting in a steady increase of prescriptions worldwide. SSRIs, via inhibition of the serotonin (5-hydroxytryptamine, 5-HT) reuptake mechanism, induce an increase in extracellular 5-HT concentration within the central nervous system of mammals. The phylogenetically ancient and highly conserved neurotransmitter and neurohormone 5-HT has been found in invertebrates and vertebrates, although its specific physiological role and mode of action is unknown for many species. Consequently, it is difficult to assess the impact of chronic SSRI exposure in the environment, especially in the aquatic ecosystem. In view of this, the current knowledge of the functions of 5-HT in fish physiology is reviewed and, via comparison to the physiological role and function of 5-HT in mammals, a characterization of the potential impact of chronic SSRI exposure on fish is provided. Moreover, the insight on the physiological function of 5-HT strongly suggests that the experimental approaches currently used are inadequate if not entirely improper for routine environmental risk assessment of pharmaceuticals (e.g., SSRIs), as relevant endpoints are not assessed or impossible to determine

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.

2009, Feurstein, Daniel, Holst, K., Fischer, Andreas, Dietrich, Daniel R.

Microcystins (MCs) are naturally occurring cyclic heptapeptides that exhibit hepato-, nephro- and possibly neurotoxic effects in mammals. Organic anion transporting polypeptides (rodent Oatp/human OATP) appear to be specifically required for active uptake of MCs into hepatocytes and kidney epithelial cells. Based on symptoms of neurotoxicity in MC-intoxicated patients and the presence of Oatp/OATP at the blood-brainbarrier (BBB) and blood-cerebrospinal-f1uid-barrier (BCFB) it is hypothesized that MCs can be transported across the BBB/BCFB in an Oatp/OATP-dependent manner and can induce toxicity in brain cells via inhibition of protein phosphatase (PP). To test these hypotheses. the presence of murine Oatp (rnOatp) in primary murine whole brain cells (mWBC) was invt'stigated at the mRNA and protein level. MC transport was tested by exposing mWBCs to three different MC-congeners (MC-LR, -LW, -LFJ with/without co-incubation with the OATP/Oatp-substrates taurocholate (TC) and bromosulfophthalein (BSP). Uptake of MCs and cytotoxicity was demonstrated via MC-Western blot analysis, immunocytochemistry, cell viability and PP inhibition assays. All MC congeners bound covalently and inhibited mWBC PP. MC-LF was the most cytotoxic congener followed by -LW and -LR. The lowest toxin concentration significantly reducing mWBC viability after 48 h exposure was 400 nM (MC-LF). Uptake of MCs into mWBCs was inhibited via co-incubation with excess TC (50 and 500 pM) and BSP (50 pM). MC-Western blot analys is demonstrated a concentration-dependent accumulation of MCs. In conclusion, the in vieTO data support the assumed MC-congener-dependent uptake in a mOatp-associated manner and cytotoxicity of MCs in primary murine whole brain cells.

2008, Hoeger, Birgit, Dietrich, Daniel R., Schmid, Daniela, Hartmann, Andreas, Hitzfeld, Bettina C.

The detection of low levels of pharmaceuticals in aquatic environments has lately raised concerns regarding possible adverse effects of these highly active substances on aquatic organisms. The non steroidal anti inflammatory drug diclofenac (DCF) is one of the pharmaceutical substances regularly detected in surface waters and has lately been demonstrated to elicit adverse effects in salmonid species at environmentally relevant concentrations. The aim of the present study was to investigate the distribution of DCF in indigenous brown trout (Salmo trutta f. fario) following intraperitoneal (i.p.) injection of a single dose of 14C labelled DCF. A distribution kinetic over 36 h provides information on possible accumulation of DCF in different organs as well as on DCF detoxification in trout, possibly enabling identification of sites of preferential toxicity. Approximately 57% of the total single DCF dose appeared in the bile 6 h after i.p. application. Subsequently, DCF was observed to undergo enterohepatic cycling with an amount of 14C activity comparable to the 6 h bile values reappearing in bile 36 h after application. Results for 14C activity in intestine and pylori support the observation of enterohepatic cycling with a small peak in intestine at 3 h post i.p. injection and a low peak in intestine and pylori at 6 h post i.p. injection, reflecting presence of the drug substance in bile. The highest activity in intestine was found 24 h post injection coinciding with low levels in bile, followed by a gradual decrease of activity in intestine mirroring the re uptake of DCF into bile. The finding of enterohepatic cycling of DCF in brown trout is suggestive of a prolonged retention of DCF in brown trout

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.

2008, Feurstein, Daniel, Fischer, Andreas, Dietrich, Daniel R.

Contamination of natural waters by cyanobacterial blooms is a worldwide problem, resulting in serious water pollution and health hazards for humans and livestock. The cyanobacterial micro- cystins (MCs) represent a group of >80 cyclic heptapeptide toxin congeners, known to induce hepato-, nephro-, and potentially neu- rotoxic effects via protein phosphatase (PP)-inhibition. Present evidence suggests that organic anion transporting polypeptides (rodent Oatps/human OATPs) are required for active uptake of MCs into hepatocytes and kidney epithelial cells. Based on the presence of Oatps/OATPs at the blood–brain-barrier (BBB) and blood–cerebrospinal fluid-barrier (BCFB) it was hypothesized that MCs can be transported across the BBB/BCFB and into neurons in an Oatp/OATP-dependent manner and will induce neurotoxic effects. To test this hypothesis, primary murine neurons (Cere- bellar Granule Cells, mCGC) were analyzed for the presence of mCGC Oatps (mRNA level). Subsequently, the uptake, localization and neurotoxic effects of MC-LR, -LW, and -LF were investigated using MC-immunoblotting, confocal microscopy of immunostained neurons, PP-inhibition assay, TNF- ELISA and caspase 3/7 activ- ity assay. RT-PCR demonstrated the presence of six murine Oatps (Oatp1c1, 1a5, 3a1, 1a1, 1b2 and 6d1) in mCGC. MC-LR-specific immunodetection demonstrated a concentration-dependent accu- mulation of this congener and covalent binding to PP-1 and -2A. mCGC PP activity was reduced by 20% following 48 h exposure to ≥300 nM MC-LR, -LW and -LF concurrent with a congener and concentration-dependent up regulation of TNF- expression and caspase 3/7 activity. In conclusion, the data suggest an MC congener-dependent uptake and neurotoxicity in primary mouse CGC.

2008, Donohue, Joyce, Orme-Zavaleta, Jennifer, Burch, Michael, Dietrich, Daniel R., Hawkins, Belinda

The Risk Assessment Work Group focused on six charge questions related to CHABS, cyanobacteria and their toxins. The charge questions covered the following topics: Research needed to reduce uncertainty in establishing health based guidelines. Research that minimize the cost and maximize the benefits of various regulatory approaches. Exposure pathways for receptors of concern. Data available to support the derivation of health-based guideline values for harmful cyanobacterial algal blooms. Ecological services that guidelines or regulations should protect? A framework for making risk management determinations that incorporates consideration of the characteristics of CHABs, the risk for human health, ecosystem viability, and the costs and benefits of CHABs detection and management? The Work Group concluded that there is a considerable amount of human case-study data and information from animal studies to demonstrate that cyanobacterial toxins pose a hazard to humans, domestic animals, wildlife, and the ecosystem. However, the data on dose-response are limited and confounded by a lack of sufficient pure toxin to conduct most of the toxicological studies that will be needed in order to answer remaining questions on risk, and to provide the data for quantitative dose-response analysis. The Work Group recommended that research on purification or synthesis of pure toxin must be accomplished before the large scale studies to establish dose-response relationships will be possible. As the necessary-pure toxins become available, the Work Group recommended that studies be prioritized by the impact that they will have on reducing the uncertainty in the risk assessment in order to minimize the research costs and maximize the risk assessment benefits. Use of quantitative structure activity relationships (QSAR) and toxicity equivalency factor studies are also recommended as approaches for filling dose-response data gaps. The Work Group recognized that CHABs rarely introduce single toxins into the water supply. Under CHAB conditions, affected water is likely to contain a variety of toxins in varying concentrations that may change over the duration of the bloom. Accordingly, research on cyanotoxin interactions is needed, along with the development of risk assessment approaches for CHAB mixtures. The development of simple, accurate analytical methods that can be utilized by most analytical laboratories or used in the field was recognized as a major data need for establishing exposure potential and monitoring bloom conditions. Most currently available methods are time-consuming and/or costly. Human exposure to cyanobacterial toxins can occur through ingestion of contaminated drinking water, plus dermal contact and/or inhalation of aerosols while bathing and showering in tap water. Treatment can reduce the concentrations of both the toxins and the bacteria in the treated water but there is still much to be learned about the effectiveness of most treatment technologies on cyanobacteria and toxin removal. Human exposure to cyanobacteria and their toxins also occurs through incidental ingestion, dermal contact, and inhalation of aerosols during recreational use of surface waters, ingestion of contaminated fish and other foods of aquatic origin, and/or BGAS supplements. Establishing intakes and duration parameters for these exposure scenarios will facilitate the application of risk assessment approaches to these situations.

2007, Stemmer, Kerstin, Ellinger-Ziegelbauer, Heidrun, Ahr, Hans-Jürgen, Dietrich, Daniel R.

Eker rats heterozygous for a dominant germline mutation in the tuberous sclerosis 2 (Tsc2) tumor suppressor gene were used as a model to study renal carcinogenesis. Eker and corresponding wild-type rats were exposed to genotoxic aristolochic acid (AA) or non-genotoxic ochratoxin A (OTA) to elucidate early carcinogen-specific gene expression changes and to test whether Eker rats are more sensitive to carcinogen-induced changes in gene expression. Male Eker and wild-type rats were gavaged daily with AA (10 mg/kg body weight) or OTA (210 µg/kg body weight). After 1, 3, 7, and 14 days of exposure, renal histopathology, tubular cell proliferation, and Affymetrix gene expression profiles from renal cortex/outer medulla were analyzed. AA-treated Eker and wild-type rats were qualitatively comparable in all variables assessed, suggesting a Tsc2-independent mechanism of action. OTA treatment resulted in slightly increased cortical pathology and significantly elevated cell proliferation in both strains, although Eker rats were more sensitive. Deregulated genes involved in the phosphatidylinositol 3-kinase-AKT-Tsc2-mammalian target of rapamycin signaling, among other important genes prominent in tumorigenesis, in conjunction with the enhanced cell proliferation and presence of preneoplastic lesions suggested involvement of Tsc2 in OTA-mediated toxicity and carcinogenicity, especially as deregulation of genes involved in this pathway was more prominent in the Tsc2 mutant Eker rat.