Ceftriaxone-mediated upregulation of the glutamate transporter GLT-1 contrasts neurotoxicity evoked by kainate in rat organotypic spinal cord cultures.

Excitotoxicity is a major pathological trigger of neurodegenerative diseases like amyotrophic lateral sclerosis. This process is caused by excessive release of the transmitter glutamate that overwhelms the capacity of astroglia transporters to maintain a low extracellular level of this aminoacid and strongly stimulates neurons. Using an in vitro model of rat organotypic spinal slice culture, we explored if the excitotoxicity caused by the potent glutamate analogue kainate, widely employed as a paradigm to evoke neurotoxicity in the central nervous system, was prevented by the antibiotic ceftriaxone known to enhance glutamate transporter expression. We also tested if excitotoxicity was made worse by inhibiting glutamate uptake with dl-threo-ß-benzyloxyaspartate (TBOA). These experiments were aimed at clarifying the relative contribution to neurotoxicity by kainate-activation of glutamate receptors or kainate-mediated release of glutamate. Neither ceftriaxone nor TBOA alone had adverse effects. Ceftriaxone (10µM; 3days) significantly decreased delayed cell death induced by kainate (100µM; 1h) and limited neuronal damage especially to motoneurons. This effect was associated to stronger astrocytic immunostaining of the glutamate transporter GLT-1. Conversely, pharmacological inhibition of glutamate uptake with TBOA was per se unable to induce neurotoxicity, yet it intensified cell death evoked by kainate. These data indicate that kainate-mediated glutamate release was critical to damage neurons, an effect prevented by up regulating glutamate uptake. These data suggest that modulating glutamate uptake is an important strategy to preserve neuronal networks.

Delayed application of the anesthetic propofol contrasts the neurotoxic effects of kainate on rat organotypic spinal slice cultures.

Excitotoxicity due to hyperactivation of glutamate receptors is thought to underlie acute spinal injury with subsequent strong deficit in spinal network function. Devising an efficacious protocol of neuroprotection to arrest excitotoxicity might, therefore, spare a substantial number of neurons and allow later recovery. In vitro preparations of the spinal cord enable detailed measurement of spinal damage evoked by the potent glutamate analogue kainate. Any clinically-relevant neuroprotective treatment should start after the initial lesion and spare networks for at least 24h when cell damage plateaus. Using this strategy, we have observed that the gas anesthetic methoxyflurane provided strong, delayed neuroprotection. It is unclear if this beneficial effect was due to the mechanism of action by methoxyflurane, or it was the consequence of anesthetic depression. To test this hypothesis, we investigated the effect by propofol (commonly injected i.v. for general anesthesia) after kainate excitotoxicity induced on organotypic spinal slices. At 5µM concentration, propofol significantly attenuated cell death, including neuronal losses and, especially, damage to the highly vulnerable motoneurons. The action by propofol was fully prevented when co-applied with the GABAA antagonist bicuculline, indicating that neuroprotection required intact GABAA receptor function. Although bicuculline per se was not neurotoxic, it largely enhanced the lesional effects of kainate, suggesting that GABAA receptor activity could limit excitotoxicity. Our data might offer an explanation for the beneficial clinical outcome of neurosurgery performed as soon as possible after spinal lesion: we posit that general anesthesia contributes to this outcome, regardless of the type of anesthetic used.

Genotoxic potential of selected cytostatic drugs in human and zebrafish cells.

Due to their increasing use, the residues of anti-neoplastic drugs have become emerging pollutants in aquatic environments. Most of them directly or indirectly interfere with the cell's genome, which classifies them into a group of particularly dangerous compounds. The aim of the present study was to conduct a comparative in vitro toxicological characterisation of three commonly used cytostatics with different mechanisms of action (5-fluorouracil [5-FU], cisplatin [CDDP] and etoposide [ET]) towards zebrafish liver (ZFL) cell line, human hepatoma (HepG2) cells and human peripheral blood lymphocytes (HPBLs). Cytotoxicity was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and acridine orange/ethidium bromide staining. All three drugs induced time- and dose-dependent decreases in cell viability. The sensitivity of ZFL and HepG2 cells towards the cytotoxicity of 5-FU was comparable (half maximal inhibitory concentration (IC50) 5.3 to 10.4 µg/mL). ZFL cells were more sensitive towards ET- (IC50 0.4 µg/mL) and HepG2 towards CDDP- (IC50 1.4 µg/mL) induced cytotoxicity. Genotoxicity was determined by comet assay and cytokinesis block micronucleus (CBMN) assay. ZFL cells were the most sensitive, and HPBLs were the least sensitive. In ZFL cells, induction of DNA strand breaks was a more sensitive genotoxicity endpoint than micronuclei (MNi) induction; the lowest effective concentration (LOEC) for DNA strand break induction was 0.001 µg/mL for ET, 0.01 µg/mL for 5-FU and 0.1 µg/mL for CDDP. In HepG2 cells, MNi induction was a more sensitive genotoxicity endpoint. The LOEC values were 0.01 µg/mL for ET, 0.1 µg/mL for 5-FU and 1 µg/mL for CDDP. The higher sensitivity of ZFL cells to cytostatic drugs raises the question of the impact of such compounds in aquatic ecosystem. Since little is known on the effect of such drugs on aquatic organisms, our results demonstrate that ZFL cells provide a relevant and sensitive tool to screen genotoxic potential of environmental pollutant in the frame of hazard assessment.