toxicity assays

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The Role of Laboratory and Field Leaching Tests in Hazard Identification for Solid Materials

Uuve Kirso, Natalya Irha, Janek Reinik, Gary Urb and Margit Laja

The use of various in vitro toxicity assays for testing environmental solid samples is dependent on the availability of reliable methods for the sampling and pretreatment of the material. This study focuses on the evaluation of leaching behaviour as a first step in the context of the toxicity testing of solid environmental matter. Spent shale, from oil shale retorting, was chosen as a suitable example of deposited solid waste material. For the generation of leachate in the laboratory setting, a standard two-stage batch–leaching test was applied to the samples of technogenic waste. In the field, a new type of lysimeter, which does not disturb the surface, was used for in situ leachate collection. The chemical composition of water extracts was found to be different under field conditions, as compared with the laboratory experiments. Thus, the hazard identification of a solid technogenic waste by in vitro toxicological tests applied to laboratory leachates would not be the best solution. The content of hazardous ingredients could be underestimated if only laboratory tests are used. For risk assessment concerned with solid waste materials, the generation of leachate by using field lysimeters is recommended.
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Long-term, Repeated Dose In Vitro Neurotoxicity of the Glutamate Receptor Antagonist L-AP3, Demonstrated in Rat Hippocampal Slice Cultures by Using Continuous Propidium Iodide Incubation

Bjarne W. Kristensen, Morten Blaabjerg, Jens Noraberg and Jens Zimmer

Most in vitro models are only used to assess the short-term effects of test compounds. However, as demonstrated here, hippocampal slice cultures can be used for long-term studies. The test compound used was the metabotropic glutamate receptor antagonist, L(+)-2-amino-3-phosphonopropionic acid (L-AP3), which is known to be toxic in vivo after subchronic, but not acute, administration. Degenerative effects were monitored by measuring the cellular uptake of propidium iodide (PI; continuously present in the medium) and lactate dehydrogenase (LDH) leakage, and by using a panel of histological stains. Hippocampal slices, derived from 2–3 day old rats and grown for 3 weeks, were subsequently exposed for the next 3 weeks to 0, 10 or 100μM L-AP3, with PI (2μM) in the culture medium. Exposure to 100μM L-AP3 induced severe toxicity after 4–6 days, as shown by massive PI uptake, LDH leakage, and changes in MAP2 and GFAP immunostaining and in Nissl and Timm staining. In contrast, 10μM L-AP3 did not induce detectable neuronal degeneration. Treatment with the NMDA receptor antagonist, MK-801, or the AMPA/KA receptor antagonist, NBQX, together with 100μM L-AP3, reduced neurodegeneration to close to control values. It is concluded that the continuous incubation of hippocampal slice cultures with PI is technically feasible for use in studies on inducible neuronal degeneration over time.
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The Use of Organotypic Hippocampal Slice Cultures to Evaluate Protection by Non-competitive NMDA Receptor Antagonists Against Excitotoxicity

Avi Ring, Rita Tanso and Jens Noraberg

There is great interest in testing neuroprotectants which inhibit the neurodegeneration that results from excessive activation of N-methyl-D-aspartate (NMDA) receptors. As an alternative to in vivo testing in animal models, we demonstrate here the use of a complex in vitro model to compare the efficacy and toxicity of NMDA receptor inhibitors. Organotypic hippocampal slice cultures were used to compare the effectiveness of the Alzheimer’s disease drug, memantine, the Parkinson’s disease drug, procyclidine, and the novel neuroprotectant, gacyclidine (GK11), against NMDA-induced toxicity. All three drugs are non-competitive NMDA receptor open-channel blockers that inhibit excitotoxic injury, and their neuroprotective capacities have been extensively investigated in vivo in animal models. They have also been evaluated as potential countermeasure agents against organophosphate poisoning. Quantitative densitometric image analysis of propidium iodide uptake in hippocampal regions CA1, CA3 and DG, showed that, after exposure to 10μM NMDA for 24 hours, GK11 was the most potent of the three drugs, with an IC50 of about 50nM and complete protection at 250nM. When applied at high doses, GK11 was still the more potent neuroprotectant, and also the least cytotoxic. These findings are consistent with those from in vivo tests in rodents. We conclude that the slice culture model provides valuable pre-clinical data, and that applying the model to the screening of neuroprotectants might significantly limit the use of in vivo tests in animals
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