Sodium azide induced neuronal damage in vitro: evidence for non-apoptotic cell death.

The features of neuronal damage induced by the mitochondrial toxin NaN(3) were investigated in rat primary cortical neuron cultures. Cell viability (MTT colorimetric determination) and transmembrane mitochondrial potential (J-C1 fluorescence) were concentration-dependently reduced 24 h after NaN(3); neither nuclear fragmentation by DAPI, nor Annexin V positivity by flow cytometry were detected, ruling out the occurrence of apoptosis. The loss in cell viability (to 54 +/- 2%) observed 24 h after a 10-min treatment with 3 mM NaN(3) was prevented by the NMDA glutamate receptor antagonist MK801 (1 microM), by the antioxidants trolox (100 microM) and acetyl-L-carnitine (1 mM) and by the nitric oxide synthase inhibitor, L-NAME (100 microM), but not by the guanylylcyclase inhibitor ODQ, 10 microM. The mitochondrial dysfunction induced by NaN(3) provides a common platform for investigating the mechanisms of both ischemic and degenerative neuronal injury, useful for screening potential protective agents against neuronal death.

Cisplatin-induced apoptosis in human promyelocytic leukemia cells.

Cis-diamminedichloroplatinum (II) cisplatin (CDDP) is an organometallic compound frequently used in anti-cancer therapy, in particular ovarian, testicular, and head and neck tumors. We found cisplatin was effective against human promyelocytic leukemia cell line HL-60, inhibiting cell cycle progression and inducing time- and concentration- dependent cell death. Presence of nuclear fragmentation, caspase-3 cleavage and annexin V positivity suggests cell death occurred by apoptosis, although DNA internucleosomal fragmentation was not detected. In addition, analysis of malondialdehyde (MDA) production and protein carbonylation indicated that cisplatin increased lipid peroxidation and oxidation of cell proteins. This occurrence was prevented by antioxidants such as N-acetylcysteine (N-aC) and glutathione (GSH), which, consistently, were also able to prevent CDDP-induced cell death. Collectively, these findings indicate that, besides growth inhibition, an increase of oxygen radicals and lipid degradation can account for a significant part of CDDP-induced apoptosis.

Determination of histamine in the whole blood of colon cancer patients.

The aim of the present work is to investigate whether histamine assay could be useful in detecting the presence of primary cancer. The high-performance liquid chromatographic (HPLC)-based o-phthalaldialdehyde (OPA) histamine derivatization assay was investigated with respect to several variables, dramatization reagent concentration, organic solvent requirement, derivatization time and counter-ion effect on chromatographic separation. The OPA histamine assay, in the absence of added -SH groups, was found to detect histamine in whole blood samples with relative standard deviations <14% and recoveries not less than 90%. The assay showed high selectivity towards other aminic-containing compounds and a detection limit of 18 nM of histamine was evaluated. Calibration curves in the range 50-500 nM were obtained by using histamine standards in 0.1 M HCl with a regression coefficient value (r(2)) of 0.9969. In order to assess the usefulness of this assay in primary tumor monitoring, two groups of individuals, 29 controls and 29 colon cancer patients were selected, and serum levels of histamine, carcinogen embrionary antigen (CEA), carcinogen antigen 19.9 (CA19.9), and tumor staging, were determined. A significant histamine reduction (P=0.028) between controls (180.12+/-70.4 nM) and patients (134.5+/-90.3 nM) was found, and a cut-off value of 157.5 nM was extrapolated as intercept point of sensitivity and specificity curves. Fifty percent of patients showed a histamine value below the cut-off, while 45.8 and 8.3% of patients were positive for CEA and CA19.9, respectively. No correlation was found between Tumor Node Metastasis staging and histamine amount, indicating that this marker is not related to the tumor mass. Our data suggest that histamine level, together with other classical tumor markers, could be a potentially interesting tumor marker in colon cancer monitoring.

Minocycline attenuates gentamicin induced hair cell loss in neonatal cochlear cultures.

Minocycline, a second-generation tetracycline antibiotic used against gram-negative and gram-positive bacteria, protects against a wide range of neurodegenerative disorders by inhibiting caspases, iNOS and the release of cytochrome c. Since aminoglycoside antibiotics damage sensory hair cells in the inner ear by activating caspase-mediated cell death pathways, we hypothesized that minocycline would protect against gentamicin (GM) ototoxicity. To test this hypothesis, postnatal day 3 (P3) rat, cochlear organotypic cultures were treated with GM alone or in combination with minocycline (10-500 microM). Treatment with GM induced a dose-dependent loss of outer hair cells (OHC) and inner hair cells (IHC). Addition of minocycline to the GM-treated cultures greatly reduced the amount of GM-induced hair cell damage in P3 cochlear cultures. The greatest protection was achieved with 100 microM of minocycline. Application of minocycline alone had no adverse effects on hair cell survival. The advantage of this combination therapy is that minocycline prevents GM-induced hair cell loss while helping to suppress the bacterial infection.

RNA expression induced by cisplatin in an organ of Corti-derived immortalized cell line.

Cisplatin [cis-diamminedichloroplatinum(II)] (CDDP) is an organic compound that is widely used for the treatment of a large number of tumors. Its clinical use is limited by the presence of some undesired side effects, like as oto- and nephro toxicity, whose mechanisms of action are not understood. One of the possible CDDP toxicity mechanism seems to involve the generation of reactive oxygen species (ROS), that can impair morphology and function of hair cells (HC) in the organ of Corti. To test this hypothesis we evaluated the effect of CDDP treatment on RNA steady-state levels of 15,000 genes by microarray analysis, using, as a experimental model, the OC-k3 cell line, obtained from the organ of Corti of transgenic mice and constitutively expressing the large SV40 T antigen. We have found overexpression of several genes related to arachidonate mobilization including phospholipase A2, group IV and V, phospholipase A2 activating protein and lysophospholipase I and III, as well as lipoxygenation like arachidonate 12-lipoxygenase and arachidonate 5-lipoxygenase activating protein. In addition, we found significant transcription of genes regulating cell respiration, including cyt c oxidase, as well as genes involved in xenobiotic detoxification and lipid peroxidation such as cyt P450, and other oxidases including spermine oxidase and monoamine oxidase. As a whole, overexpression of the group of different genes seems to indicate that an oxidative burst could take place during cisplatin administration. We therefore searched for evidences of superoxide anion and hydrogen peroxide by means of electron paramagnetic resonance (EPR) spectroscopy and flow cytometry, but failed to detect them. On the other hand, we found an increase of malondialdehyde (MDA) synthesis and protein carbonylation products, indicating the occurence of lipid peroxidative degradation. When we tested the effectiveness of butylated hydroxytoluene (BHT), dithiothreitol (DTT) and N-acetylcysteine (N-Ac) as cytoprotectants, all of them reduced protein carbonylation to control levels and significantly protected OC-k3 from CDDP-induced cell death, with an higher protection when using the lipophylic antioxidant BHT. The same antioxidants prevented also the onset of protein carbonylation, which extent was decreased to basal levels. These data indicate that CDDP is able to stimulate gene expression up to 12 h after the beginning of the treatment. This increase in gene transcription involves a large number of genes potentially able to increase the level of cell ROS. Consistently, cells survival is improved by cotreatment with antioxidants, in particular lipophilics.

Cisplatin cytotoxicity in organ of Corti-derived immortalized cells.

Cisplatin is an anticancer drug currently used in the treatment of genital and head and neck tumors. Its use in these and other types of tumors is narrowed by onset of chemoresistance and severe undesired side effects, like as nephro- and ototoxicity, whose mechanisms of action are only partially understood. In the present study we investigated the effects of cisplatin (cis-dichlorodiaminoplatin, CDDP) on a cell line (OC-k3) developed from organs of Corti of transgenic mice. We observed at 48 h that cell death due to cisplatin was time and concentration-dependent. The cell death displayed some morphological hallmarks of apoptosis, including nuclear fragmentation into several large nuclear fragments, surrounded by a rearranged and thickened actin cytoskeleton. No DNA laddering was detected, suggesting absence of endonuclease activity, nor annexin V positivity, suggesting absence of phosphatidylserine externalization. Several molecules protected the cells against CDDP induced cytotoxicity, including methionine, suramin and PD98059. Methionine reduced CDDP-uptake, while suramin, a polycathionic compound a specifically binding external proteins, did not. This finding suggested that suramin could exert its protective effect by acting on an intracellular transduction pathway. We tested this hypothesis by studying the effect of suramin and PD98059, a MEK inhibitor, on the mitogen activated protein kinase (MAPK) cascade. After CDDP treatment, we found an increase of phosphorylation of extracellular regulated kinases (ERK)1/2, that could be inhibited by PD98059 and suramin. These data suggest that ERK pathways can play a role in mediating the cell death induction in presence of a CDDP challenge.

Gentamicin-induced cytotoxicity involves protein kinase C activation, glutathione extrusion and malondialdehyde production in an immortalized cell line from the organ of corti.

The objective of the present study was to investigate the biochemical mechanisms underlying gentamicin cytotoxicity in OC-k3 cells derived from an immortalized cell line developed from the organ of Corti of transgenic mice. Administration of 50 microM gentamicin significantly reduced cell proliferation and viability, as well as initiating morphological changes associated with apoptosis. Protein kinase C (PKC) alpha activity was increased in gentamicin-treated cells, peaking 15 min after dosing (+138.2%). This PKCalpha increase was followed by a rise of glutathione (GSH) efflux and a concomitant 29% decrease in intracellular GSH levels at 30 min. PKCalpha-specific inhibitors blocked these cytotoxic effects. Gentamicin also increased malondialdehyde levels, while N-acetylcysteine, GSH and ascorbic acid prevented gentamicin-induced cell death. These findings suggest that gentamicin cytotoxicity involves a production of intracellular reactive oxygen species and a concomitant PKC-dependent fall of intracellular scavenging abilities (GSH), events that together drive cells to undergo apoptosis.