Daniel R. Dietrich

Ochratoxin A: The Continuing Enigma

Abstract The mycotoxin ochratoxin A (OTA) has been linked to the genesis of several disease states in both animals and humans. It has been described as nephrotoxic, carcinogenic, teratogenic, immunotoxic, and hepatotoxic in laboratory and domestic animals, as well as being thought to be the probable causal agent in the development of nephropathies (Balkan Endemic Nephropathy, BEN and Chronic Interstitial Nephropathy, CIN) and urothelial tumors in humans. As a result, several international agencies are currently attempting to define safe legal limits for OTA concentration in foodstuffs (e.g., grain, meat, wine, and coffee), in processed foods, and in animal fodder. In order to achieve this goal, an accurate risk assessment of OTA toxicity including mechanistic and epidemiological studies must be carried out. Ochratoxin has been suggested by various researchers to mediate its toxic effects via induction of apoptosis, disruption of mitochondrial respiration and/or the cytoskeleton, or, indeed, via the generation of DNA adducts. Thus, it is still unclear if the predominant mechanism is of a genotoxic or an epigenetic nature. One aspect that is clear, however, is that the toxicity of OTA is subject to and characterized by large species- and sex-specific differences, as well as an apparently strict structure–activity relationship. These considerations could be crucial in the investigation of OTA-mediated toxicity. Furthermore, the use of appropriate in vivo and in vitro model systems appears to be vital in the generation of relevant experimental data. The intention of this review is to collate and discuss the currently available data on OTA-mediated toxicity with particular focus on their relevance for the in vivo situation, and also to suggest possible future strategies for unlocking the secrets of ochratoxin A.

Toxicological and Pathological Applications of Proliferating Cell Nuclear Antigen (PCNA), A Novel Endogenous Marker for Cell Proliferation

A major stimulus to study cell proliferation, particularly in rodent carcinogenicity assays and human tumors, has been the belief that the quantification of this fundamental biological process will provide the toxicologist and pathologist with objective data allowing a better understanding of the mechanisms involved in the toxicity and/or carcinogenicity of certain compounds as well as guiding more effective management of patients afflicted with neoplasia. Among the markers used for cell proliferation measurement, PCNA has recently gained much attention and holds much promise as it is intricately involved in the cell replication processes. It not only could allow measurement of the replication rates without necessitating pretreatment of the animal/tissue in prospective studies, but also would allow retrospective assessment of the proliferative rates in archival tissues due to the conservation of this marker in fixed and paraffin-embedded tissues. Finally, knowledge of the function of PCNA in the cell cycle and its regulation by other factors may help us understand the advantages and limitations of PCNA as a cell proliferation marker in its application in toxicology and as a prognostic marker in human tumors.

The role of organic anion transporting polypeptides (OATPs/SLCOs) in the toxicity of different microcystin congeners in vitro: a comparison of primary human hepatocytes and OATP-transfected HEK293 cells.

Cellular uptake of microcystins (MCs), a family of cyclic cyanobacterial heptapeptide toxins, occurs via specific organic anion transporting polypeptides (OATPs), where MCs inhibit serine/threonine-specific protein phosphatase (PP). Despite comparable PP-inhibitory capacity, MCs differ greatly in their acute toxicity, thus raising the question whether this discrepancy results from MC-specific toxikokinetic rather than toxicodynamic differences. OATP-mediated uptake of MC congeners MCLR, -RR, -LW and -LF was compared in primary human hepatocytes and HEK293 cells stably expressing recombinant human OATP1B1/SLCO1B1 and OATP1B3/SLCO1B3 in the presence/absence of OATP substrates taurocholate (TC) and bromosulfophthalein (BSP) and measuring PP-inhibition and cytotoxicity. Control vector expressing HEK293 were resistant to MC cytotoxicity, while TC and BSP competition experiments reduced MC cytotoxicity in HEK293-OATP transfectants, thus confirming the requirement of OATPs for trans-membrane transport. Despite comparable PP-inhibiting capabilities, MCLW and -LF elicited cytotoxic effects at lower equimolar concentrations than MCLR and MCRR, hence suggesting congener selective transport into HEK293-OATP transfectants and primary human hepatocytes. Primary human hepatocytes appeared one order of magnitude more sensitive to MC congeners than the corresponding HEK293 -OATP transfectants. Although the latter maybe due to a much lower level of PPs in primary human hepatocytes, the presence of OATPs other than 1B1 or 1B3 may have added to an increased uptake of MCs. In view of the high sensitivity of human hepatocytes and currently MCLR-only based risk calculations, the actual risk of human MC-intoxication and ensuing liver damage could be underestimated in freshwater cyanobacterial blooms where MCLW and-LF predominate.

Oatp-associated uptake and toxicity of microcystins in primary murine whole brain cells

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-brain-barrier (BBB) and blood-cerebrospinal-fluid-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 (mOatp) in primary murine whole brain cells (mWBC) was investigated at the mRNA and protein level. MC transport was tested by exposing mWBCs to three different MC-congeners (MC-LR, -LW, -LF) 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 microM) and BSP (50 microM). MC-Western blot analysis demonstrated a concentration-dependent accumulation of MCs. In conclusion, the in vitro data support the assumed MC-congener-dependent uptake in a mOatp-associated manner and cytotoxicity of MCs in primary murine whole brain cells.

Environmental risk assessment of pharmaceutical drug substances--conceptual considerations.

Drugs, i.e. active ingredients of human medicinal products, may be introduced into the environment after use in patients by sewage effluent pathways and consequently are detected at low concentrations in sewage effluents and in surface waters. Legal requirements in a number of geographical regions (Europe, US, and intended in Canada) demand environmental risk assessments (ERA) for new drug substances. Existing regulatory concepts of ERA are based initially on a set of short-term ecotoxicological studies in three to four different species, environmental behavior and the application of assessment factors to correct for the ERA inherent uncertainty. Based on theoretical considerations and the experience with a very limited, but well investigated, number of examples while considering that drugs are highly biologically active compounds, the appropriateness of this risk assessment procedure for all drug substances might be questioned. Indeed, e.g. long-term effects may occur at much lower concentrations and follow different toxicodynamic mechanism than extrapolated from short-term studies., In such cases, the application of assessment factors for deriving chronic no-observed effect concentration (NOECs) appears to be problematic. Although long-term tests with a variety of organisms would provide a complete database for the evaluation of the environmental risks, this is unachievable for all drugs due to time, money and animal welfare constraints. In order to avoid unnecessary testing, a concept is presented, which makes use of pharmacological and toxicological, as well as pharmaco- and toxicokinetic information derived from mammals during drug substance development. Useful data for adoption in a case-by-case testing strategy can be obtained by evaluating (a) the pharmacological activity, which indicates specific targets in mammalian species and may allow for an analysis, whether a similar target is available in aquatic species; (b) the mammalian toxicity, which may indicate, which targets are most susceptible to adverse effects; (c) the difference between acute and chronic effects in mammals, since the magnitude of this difference may indicate, whether long-term effects are expected at significantly lower levels than acute effects; (d) the (pharmacologically and toxicologically) effective plasma levels in mammalian test organisms, which may be compared with the relevant exposure scenario for the environment. Additionally, activity classes of compounds may be established based on experience with specific substances, in order to develop an appropriate test strategy. The above preliminary considerations should support decisions on the selection of candidate substances for chronic effects studies and for the appropriate selection of test species and endpoints to monitor. Generally, ecologically relevant endpoints such as impairment of growth, development and reproduction should be used to assess the ecotoxicologic effects.

Toxin production in cyanobacterial mats from ponds on the McMurdo Ice Shelf, Antarctica

Cyanobacteria are known to produce hepatotoxic substances, the functional and ecological role of these toxins, however, remains largely unclear. Toxic properties of cyanobacteria collected in Antarctica were investigated to determine whether toxin-producing species can also be found under these environmental conditions. Samples were collected from meltwater ponds on the McMurdo Ice Shelf, Antarctica in the summers of 1997 to 1999. These ponds are colonized by benthic algae and cyanobacterial mats. Oscillatoriales, Nodularia sp., and Nostoc sp. constituted the major taxa in freshwater ponds, while Nostoc sp. was missing from brackish and saline ponds. Samples were taken from either floating, submerged or benthic mats, and extracted for in vitro toxicity testing. The presence of toxins was determined by the phosphatase-inhibition assay and by high performance liquid chromatography. The cytotoxic properties of the extracts were investigated in hepatocytes determining 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide metabolism and trypan blue dye exclusion. The results show that all cyanobacterial extracts display phosphatase-inhibiting activity, of which approximately half had significantly greater than 50% inhibiting activity. The presence of nodularin and microcystin-LR was established by high performance liquid chromatography. Cytotoxic properties, independent of the phosphatase inhibiting activity, were also detected. Toxic strains of cyanobacteria can therefore also be found in Antarctica and this finding may lead to further insight into potential ecological roles of cyanobacterial phosphatase inhibiting toxins.

Endocrine disruption: Fact or urban legend?

Endocrine disruptors (EDs) are substances that cause adverse health effects via endocrine-mediated mechanisms in an intact organism or its progeny or (sub) populations. Purported EDCs in personal care products include 4-MBC (UV filter) or parabens that showed oestrogenic activity in screening tests, although regulatory toxicity studies showed no adverse effects on reproductive endpoints. Hormonal potency is the key issue of the safety of EDCs. Oestrogen-based drugs, e.g. the contraceptive pill or the synthetic oestrogen DES, possess potencies up to 7 orders of magnitude higher than those of PCP ingredients; yet, in utero exposure to these drugs did not adversely affect fertility or sexual organ development of offspring unless exposed to extreme doses. Additive effects of EDs are unlikely due to the multitude of mechanisms how substances may produce a hormone-like activity; even after uptake of different substances with a similar mode of action, the possibility of additive effects is reduced by different absorption, metabolism and kinetics. This is supported by a number of studies on mixtures of chemical EDCs. Overall, despite of 20 years of research a human health risk from exposure to low concentrations of exogenous chemical substances with weak hormone-like activities remains an unproven and unlikely hypothesis.

Physiological Endpoints for Potential SSRI Interactions in Fish

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.

Determination of vitellogenin kinetics in male fathead minnows (Pimephales promelas)

A lack of knowledge persists concerning the combination of kinetics on protein and mRNA levels of the most commonly used biomarker for estrogenic influences-vitellogenin (VTG). Consequently, male fathead minnows were exposed to 17alpha-ethinylestradiol (EE(2)) for 35 days, followed by an equally long depuration period in a flow-through system. VTG mRNA levels reached a plateau after 3 days of exposure, which remained stable until 3 days after EE(2) removal. Control levels were re-attained within 7 days of the depuration phase. VTG protein accumulated in the plasma following a two-phased model. The first phase depicting an exponential increase lasted 15 days and was followed by a saturation phase approaching a plateau of approximately 47 mg VTG/ml plasma. Clearance kinetics could be described by a two-compartment open model, with half-lives of 2.17 and 21.32 days for the alpha- and beta-phases, respectively. In addition, a high VTG protein synthesis rate seemed to adversely affect fitness and mortality of the fish.

Effects of endocrine modulating substances on reproduction in the hermaphroditic snail Lymnaea stagnalis L.

Various man-made agents like pesticides, industrial chemicals and some natural substances have the potential to alter hormonal pathways that regulate reproductive processes in certain species of wildlife. Until now, only a few investigations have been undertaken to determine the effects of these substances on reproductive capacities (fecundity and fertility) in exposed invertebrate aquatic species. In this study one of the most abundant mollusc of European limnic systems, the hermaphroditic gastropod Lymnaea stagnalis was investigated to determine the effects of endocrine modulating substances on reproductive parameters. Known endocrine modulating substances were tested using the following nominal concentrations; Tributyltin (TBT in ng Sn/l) and beta-sitosterol at 1, 10 and 100 ng/l, respectively, and 4-nonylphenol (4-NP) at 1, 10 and 100 microg/l. In addition, experiments were carried out with 1, 10 and 100 ng/l of t-methyltestosterone. All the testing was carried out on recently matured adults of Lymnaea. Fifteen to twenty snails per treatment were exposed for between 7 and 12 weeks in a semi-static test with a weekly exchange of testwater. Shell height and weight and mortality of the adults, egg production, hatching rate of the eggs, and histopathology of the adult snails were analysed. The same parameters were investigated on F(1) generation snails from exposed parents at two dates (1 week after exposure and at the end of the exposure duration). Treatments with TBT and 4-NP had only slight effects on the egg production of the adults and hatching rate of the eggs. However, increased histopathological changes were observed in epithelial tissues of the adult snails, e.g. lung and foot also characterised by extreme inflammatory processes. While beta-sitosterol and t-methyltestosterone had no effect on the shell height and weight or the mortality of adult snails nor on the egg production or ensuing egg hatching rate, beta-sitosterol treated snails presented a distinct atrophy of the albumen gland and so did t-methyltestosterone, albeit with a lower degree of atrophy. The observed histopathological effects due to exposure to tributyltin or 4-NP are suggested to lead to long-term adverse reproductive effects mediated by an impairment of the fitness of the snails. In the experiments the steroid-dependant (beta-sitosterol and t-methyltestosterone) degeneration of the albumen gland caused no obvious adverse effects on the fecundity nor fertility of the adults or on F(1)-generation. However, the impact on fertility following a prolonged exposure to high concentrations of the phytoestrogen cannot be predicted.