Neuronal in vitro models for the estimation of acute systemic toxicity.

The objective of the EU funded integrated project "ACuteTox" is to develop a strategy in which general cytotoxicity, together with organ-specific endpoints and biokinetic features, are taken into consideration in the in vitro prediction of oral acute systemic toxicity. With regard to the nervous system, the effects of 23 reference chemicals were tested with approximately 50 endpoints, using a neuronal cell line, primary neuronal cell cultures, brain slices and aggregated brain cell cultures. Comparison of the in vitro neurotoxicity data with general cytotoxicity data generated in a non-neuronal cell line and with in vivo data such as acute human lethal blood concentration, revealed that GABA(A) receptor function, acetylcholine esterase activity, cell membrane potential, glucose uptake, total RNA expression and altered gene expression of NF-H, GFAP, MBP, HSP32 and caspase-3 were the best endpoints to use for further testing with 36 additional chemicals. The results of the second analysis showed that no single neuronal endpoint could give a perfect improvement in the in vitro-in vivo correlation, indicating that several specific endpoints need to be analysed and combined with biokinetic data to obtain the best correlation with in vivo acute toxicity.

Evaluation of acute antiapoptotic effects of Li+ in neuronal cell cultures.

Li(+) exerts protective effect against several neurotoxins in neuronal cell preparations. Here we examined the antiapoptotic effects of GSK3beta in cerebellar granule neurons (CGNs) in the presence of several neurotoxins. Acute treatment with Li(+) protected neurons against nocodazole and serum/potassium (S/K) deprivation, but were ineffective against kainic acid and MPP(+). Li(+) 5 mM also decreased caspase-3 activation induced by nocodazole and S/K deprivation as measured by Ac-DEVD-p-nitroaniline and the breakdown of alpha-spectrin. All the neurotoxins used in the present study activated GSK3beta, evaluated with a specific antibody phospho-GSK-3beta (Ser9) by Western-blot and immunocytochemistry and were always inhibited by Li(+) 5 mM. Our results implicate Li(+) in the regulation of apoptosis mediated by caspase activation (Type I). Furthermore inhibition of GSK3beta by acute treatment with Li(+) 5 mM is not an indicator of neuroprotection. The acute antiapoptotic function of Li(+) is discussed in terms of its inhibition of Type I pathway, the intrinsic (mitochondrial) apoptotic pathway in cerebellar granule cells.

Potential eye irritation of some "biodegradable" liquid scintillation cocktails determined in vitro.

The potential eye irritation of a range of liquid scintillation cocktails has been evaluated by an in vitro method using the chorioallantoic membrane as an alternative to the Draize rabbit test. All the cocktails studied are potentially moderately to strongly irritant. The only slightly irritant product was Solventgreen, which is a solvent used in some of the cocktails. There is a correlation between the concentration of the cocktail and the potential eye irritation induced. Manufacturers of chemicals including liquid scintillation cocktails should accept responsibility for disclosing data about the composition and toxicity of their products with regard to waste disposal and safety of laboratory staff.

(+/-)-huprine Y, (-)-huperzine A and tacrine do not show neuroprotective properties in an apoptotic model of neuronal cytoskeletal alteration.

Acetylcholinesterase inhibitors (AChEI) are among the drugs most widely used in the treatment of Alzheimer's disease. They increase the levels of acetylcholine and thus improve the cognitive symptoms that are impaired. We tested whether specific AChEI show additional neuroprotective properties against colchicine-induced apoptosis in cerebellar granule neurons (CGNs), a well established apoptotic model mediated by neuronal cytoskeleton alteration. Colchicine-induced apoptosis is due to an increase in the activity of GSK-3beta and CDK5, two enzymes involved in cytoskeletal alteration. Furthermore, the intrinsic apoptotic pathway is activated by colchicines, as revealed by cytochrome c release and Bax translocation. Tacrine, (-)-huperzine A and (+/-)-huprine Y, the AChEI tested in the study, did not reverse the loss of neuronal viability induced by colchicine. Moreover, the increase in apoptotic features induced by colchicine treatment, as measured by flow cytometry and nuclear chromatin condensation, was not prevented by these AChEI. Although some of these drugs are of interest to treat Alzheimer's disease, their lack of efficacy in the prevention of colchicine-induced apoptosis in CGNs suggests that they cannot prevent neuronal loss due to cytoskeleton alteration.

3-Amino thioacridone, a selective cyclin-dependent kinase 4 inhibitor, attenuates kainic acid-induced apoptosis in neurons.

The mechanisms underlying selective neuronal cell death in kainic acid-mediated neurodegeneration are not fully understood. We have recently demonstrated that in cerebellar granule neurons, kainic acid induces the expression of proteins associated with cell-cycle progression. In the present study we show that 3-amino thioacridone (3-ATA), a selective cyclin-dependent kinase 4 inhibitor, attenuates kainic acid-induced apoptosis in cerebellar granule neurons. When neurons were pre-treated with 3-ATA 10 microM for 24 h, they were less susceptible to damage induced by kainic acid 500 microM, since the number of dead cells decreased significantly. In flow cytometry studies using propidium iodide staining, 3-ATA also reduced the ratio of apoptotic cells induced by kainic acid. Moreover, 3-ATA decreased the proportion of cells with a condensed nucleus from 55% to 22%. Our data suggest that the cell cycle pathway is involved in the mechanism of apoptosis mediated by kainic acid and that cyclin-dependent kinase 4 plays a prominent role in this process. 3-ATA may to prevent the apoptosis associated with neurodegenerative disorders without the over-activation of excitatory amino acid receptors.

C-phycocyanin: a biliprotein with antioxidant, anti-inflammatory and neuroprotective effects.

Phycocyanin (Pc) is a phycobiliprotein that has been recently reported to exhibit a variety of pharmacological properties. In this regard, antioxidant, anti-inflammatory, neuroprotective and hepatoprotective effects have been experimentally attributed to Pc. When it was evaluated as an antioxidant in vitro, it was able to scavenge alkoxyl, hydroxyl and peroxyl radicals and to react with peroxinitrite (ONOO(-);) and hypochlorous acid (HOCl). Pc also inhibits microsomal lipid peroxidation induced by Fe(+2)-ascorbic acid or the free radical initiator 2,2' azobis (2-amidinopropane) hydrochloride (AAPH). Furthermore, it reduces carbon tetrachloride (CCl(4))-induced lipid peroxidation in vivo. Pc has been evaluated in twelve experimental models of inflammation and exerted anti-inflammatory effects in a dose-dependent fashion in all of these. Thus, Pc reduced edema, histamine (Hi) release, myeloperoxidase (MPO) activity and the levels of prostaglandin (PGE(2)) and leukotriene (LTB(4)) in the inflamed tissues. These anti-inflammatory effects of Pc can be due to its scavenging properties toward oxygen reactive species (ROS) and its inhibitory effects on cyclooxygenase 2 (COX-2) activity and on Hi release from mast cells. Pc also reduced the levels of tumor necrosis factor (TNF-alpha) in the blood serum of mice treated with endotoxin and it showed neuroprotective effects in rat cerebellar granule cell cultures and in kainate-induced brain injury in rats.

C-phycocyanin protects cerebellar granule cells from low potassium/serum deprivation-induced apoptosis.

We tested the potential cytoprotective role of C-phycocyanin in rat cerebellar granule cell cultures. Cell death was induced by potassium and serum (K/S) withdrawal. Cell viability was studied using the neutral red assay and laser scanning cytometry with propidium iodide as fluorochrome. C-phycocyanin (1-3 mg/ml) showed a neuroprotective effect against 24 h of K/S deprivation in cerebellar granule cells. After 4 h K/S deprivation this compound (3 mg/ml) inhibited formation of reactive oxygen species, measured as 2',7'-dichlorofluorescein fluorescence, showing its scavenger capability. Pre-treatment with C-phycocyanin reduced thymidine incorporation into DNA below control values and reduced dramatically apoptotic bodies as visualized by propidium iodide, indicating inhibition of apoptosis induced by K/S deprivation. Flow cytometry studies, using propidium iodide in TritonX100 permeabilized cells, indicated that 24 h K/S deprivation acts as a proliferative signal for cerebellar granule cells, which show an increase in S-phase percentage and cells progressed into the apoptotic pathway. C-phycocyanin protected cerebellar granule cells from the apoptosis induced by deprivation. These results suggest that C-phycocyanin prevents apoptosis in cerebellar granule cells probably through the antioxidant activity. It is proposed that K/S deprivation-induced apoptosis could be due, in part, to an alteration in the cell cycle mediated by an oxidative stress mechanism.

Antiinflammatory activity of some extracts from plants used in the traditional medicine of north-African countries (II).

Aqueous, ethanol and chloroform extracts from Corrigiliola telephiifolia, Echinops spinosus, Kundmannia sicula, Tamarindus indica and Zygophyllum gaetulum were evaluated for antiinflammatory properties in mice (ear oedema induced by arachidonic acid) and rats (subplantar oedema induced by carrageenan) after topical or i.p. administration, respectively. Our results showed that all the plants exhibit antiinflammatory activity, since at least one extract from each plant was active in one of the experimental models. Whereas all the extracts of Corrigiliola telephiifolia and Echinops spinosus were highly active on all the experimental models assayed (values of inflammation inhibition well above 50%), poorer activity profiles were recorded in Kundmannia sicula, Tamarindus indica and Zygophyllum gaetulum. These results support the traditional uses for these plants but indicate that the active principles in the chloroform extracts are probably more active and/or are contained in larger concentrations than the principles in the polar extracts used in the traditional medicine of North-African countries.

Protective effects of C-phycocyanin against kainic acid-induced neuronal damage in rat hippocampus.

The neuroprotective role of C-phycocyanin was examined in kainate-injured brains of rats. The effect of three different treatments with C-phycocyanin was studied. The incidence of neurobehavioral changes was significantly lower in animals receiving C-phycocyanin. These animals also gained significantly more weight than the animals only receiving kainic acid, whereas their weight gain did not differed significantly from controls. Equivalent results were found when the neuronal damage in the hippocampus was evaluated through changes in peripheral benzodiazepine receptors (microglial marker) and heat shock protein 27 kD expression (astroglial marker). Our results are consistent with the oxygen radical scavenging properties of C-phycocyanin described elsewhere. Our findings and the virtual lack of toxicity of C-phycocyanin suggest this drug could be used to treat oxidative stress-induced neuronal injury in neurodegenerative diseases, such as Alzheimer's and Parkinson's.

[Effect of peripheral parenteral nutrition on the immediate postoperative period in surgery of the aortic sector]. / Efecto de la nutrición parenteral periférica en el postoperatorio inmediato de la cirugía del sector aórtico.

There is not an extensive literature about nutritional value in Vascular Surgery. In this study, efficacy and cost/benefit relation in the application of Peripheric Parenteral Nutrition (PPN) in postoperative of aortic surgery, are investigated through two randomized groups, in patients with aortoiliac obstructive arteriopathy and who needed an aortobifemoral bypass.

Comparative study of the gastrointestinal tolerability of tolmetin and 4'-acetamidophenyl-2-(5'-p-toluyl-1'-methylpyrrole) acetate (AU-8001).

The comparative gastrointestinal (GI) tolerability of 4'-acetamidophenyl-2-(5'-p-toluyl-1'-methylpyrrole) acetate (AU-8001), tolmetin and placebo have been studied in rat through a radioactive quantification method with Fe-59 of the GI microbleeding. The drugs were given orally at equimolecular doses of AU-8001 and sodium tolmetin equivalent to 100 mg/kg/day of tolmetin acid. Four days administration of sodium tolmetin induced significant increases of fecal radioactivity (p less than 0.005) whereas no difference was observed between animals treated with equimolecular doses of AU-8001 and control animals. The fecal elimination of radioactivity in the animals treated with sodium tolmetin was also significantly higher than in animals receiving AU-8001 (p less than 0.02).