A structure-activity relationship linking non-planar PCBs to functional deficits of neural crest cells: new roles for connexins.

Migration of neural crest cells (NCC) is a fundamental developmental process, and test methods to identify interfering toxicants have been developed. By examining cell function endpoints, as in the 'migration-inhibition of NCC (cMINC)' assay, a large number of toxicity mechanisms and protein targets can be covered. However, the key events that lead to the adverse effects of a given chemical or group of related compounds are hard to elucidate. To address this issue, we explored here, whether the establishment of two overlapping structure-activity relationships (SAR)-linking chemical structure on the one hand to a phenotypic test outcome, and on the other hand to a mechanistic endpoint-was useful as strategy to identify relevant toxicity mechanisms. For this purpose, we chose polychlorinated biphenyls (PCB) as a large group of related, but still toxicologically and physicochemically diverse structures. We obtained concentration-dependent data for 26 PCBs in the cMINC assay. Moreover, the test chemicals were evaluated by a new high-content imaging method for their effect on cellular re-distribution of connexin43 and for their capacity to inhibit gap junctions. Non-planar PCBs inhibited NCC migration. The potency (1-10 µM) correlated with the number of ortho-chlorine substituents; non-ortho-chloro (planar) PCBs were non-toxic. The toxicity to NCC partially correlated with gap junction inhibition, while it fully correlated (p < 0.0004) with connexin43 cellular re-distribution. Thus, our double-SAR strategy revealed a mechanistic step tightly linked to NCC toxicity of PCBs. Connexin43 patterns in NCC may be explored as a new endpoint relevant to developmental toxicity screening.

Switching from astrocytic neuroprotection to neurodegeneration by cytokine stimulation.

Astrocytes, the largest cell population in the human brain, are powerful inflammatory effectors. Several studies have examined the interaction of activated astrocytes with neurons, but little is known yet about human neurotoxicity under such situations and about strategies of neuronal rescue. To address this question, immortalized murine astrocytes (IMA) were combined with human LUHMES neurons and stimulated with an inflammatory (TNF, IL-1) cytokine mix (CM). Neurotoxicity was studied both in co-cultures and in monocultures after transfer of conditioned medium from activated IMA. Interventions with >20 drugs were used to profile the model system. Control IMA supported neurons and protected them from neurotoxicants. Inflammatory activation reduced this protection, and prolonged exposure of co-cultures to CM triggered neurotoxicity. Neither the added cytokines nor the release of NO from astrocytes were involved in this neurodegeneration. The neurotoxicity-mediating effect of IMA was faithfully reproduced by human astrocytes. Moreover, glia-dependent toxicity was also observed, when IMA cultures were stimulated with CM, and the culture medium was transferred to neurons. Such neurotoxicity was prevented when astrocytes were treated by p38 kinase inhibitors or dexamethasone, whereas such compounds had no effect when added to neurons. Conversely, treatment of neurons with five different drugs, including resveratrol and CEP1347, prevented toxicity of astrocyte supernatants. Thus, the sequential IMA-LUHMES neuroinflammation model is suitable for separate profiling of both glial-directed and directly neuroprotective strategies. Moreover, direct evaluation in co-cultures of the same cells allows for testing of therapeutic effectiveness in more complex settings, in which astrocytes affect pharmacological properties of neurons.

Reverse-transcription quantitative PCR directly from cells without RNA extraction and without isothermal reverse-transcription: a 'zero-step' RT-qPCR protocol.

We describe an ultra-rapid and sensitive method to quantify gene expression levels in cultured cells. The procedure is based on reverse-transcription quantitative PCR (RT-qPCR) directly from cells, without RNA extraction and without an isothermal reverse-transcription step. Human neurons (Lund human mesencephalic cells) were lysed at different stages of differentiation, and the lysates were used directly as template for the combined RT-qPCR reaction. We detected a down-regulation of a proliferation marker and an up-regulation of neuronal dopaminergic genes expression. We were able to detect the reference gene target from as few as a single cell, demonstrating the application of the method for efficient amplification from small cell numbers. The data were fully in line with those obtained by the standard two-step RT-qPCR from the extracted total RNA. Our 'zero-step' RT-qPCR method proved to be simple and reliable with a total time from cell lysis to the end of the qPCR as short as 1.5 h. It is therefore particularly suitable for RT-qPCRs where large numbers of samples must be handled, or where data are required within short time.