Differential mechanisms of Ca2+ responses in glial cells evoked by exogenous and endogenous glutamate in rat hippocampus.

The mechanisms of Ca2+ responses evoked in hippocampal glial cells in situ, by local application of glutamate and by synaptic activation, were studied in slices from juvenile rats using the membrane permeant fluorescent Ca2+ indicator fluo-3AM and confocal microscopy. Ca2+ responses induced by local application of glutamate were unaffected by the sodium channel blocker tetrodotoxin and were therefore due to direct actions on glial cells. Glutamate-evoked responses were significantly reduced by the L-type Ca2+ channel blocker nimodipine, the group I/II metabotropic glutamate receptor antagonist (S)-alpha-methyl-4-carboxyphenylglycine (MCPG), and the N-methyl-D-aspartate (NMDA) receptor antagonist (+/-)2-amino-5-phosphonopentanoic acid (APV). However, glutamate-induced Ca2+ responses were not significantly reduced by the non-NMDA receptor antagonist 6-cyano-7-nitro-quinoxaline-2,3-dione (CNQX). These results indicate that local application of glutamate increases intracellular Ca2+ levels in glial cells via the activation of L-type Ca2+ channels, NMDA receptors, and metabotropic glutamate receptors. Brief (1 s) tetanization of Schaffer collaterals produced increases in intracellular Ca2+ levels in glial cells that were dependent on the frequency of stimulation (> or =50 Hz) and on synaptic transmission (abolished by tetrodotoxin). These Ca2+ responses were also antagonized by the L-type Ca2+ channel blocker nimodipine and the metabotropic glutamate receptor antagonist MCPG. However, the non-NMDA receptor antagonist CNQX significantly reduced the Schaffer collateral-evoked Ca2+ responses, while the NMDA antagonist APV did not. Thus, these synaptically mediated Ca2+ responses in glial cells involve the activation of L-type Ca2+ channels, group I/II metabotropic glutamate receptors, and non-NMDA receptors. These findings indicate that increases in intracellular Ca2+ levels induced in glial cells by local glutamate application and by synaptic activity share similar mechanisms (activation of L-type Ca2+ channels and group I/II metabotropic glutamate receptors) but also have distinct components (NMDA vs. non-NMDA receptor activation, respectively). Therefore, neuron-glia interactions in rat hippocampus in situ involve multiple, complex Ca2+-mediated processes that may not be mimicked by local glutamate application.

Differences detected in vivo between samples of aflatoxin-contaminated peanut meal, following decontamination by two ammonia-based processes.

A sample of peanut meal, highly contaminated with aflatoxins, has been subjected to decontamination by two commercial ammonia-based processes. The original contaminated and the two decontaminated meals were fed to rats for 90 days. No lesions associated with aflatoxin-induced hepatocarcinogenesis were detected histologically following feeding with the two detoxified meals. There were, however, clear differences between the two meals in respect of growth rates of the rats. In addition, feeding one of the detoxified meals resulted in hepatic abnormalities detected using novel immunohistochemical reagents. Differences between the two detoxified meals were also indicated by the results of studies using meals 'spiked' with [14C]-aflatoxin B1 prior to being subjected to the detoxification processes. The meals differed in the bioavailability of the label. It was concluded that peanut meal where an initial, unacceptable level of contamination with aflatoxins had been reduced by two ammonia-based processes to comparable, acceptable levels, may still have different effects in vivo when incorporated into animal diets.

Activation of Poly(ADP-Ribose)Polymerase in rat hepatocytes does not contribute to their cell death by oxidative stress.

Oxidative stress induced by tert-butyl hydroperoxide (tBOOH) in freshly isolated rat hepatocytes caused DNA damage and loss of membrane integrity. Such DNA lesions are likely to be single strand breaks since neither caryolysis nor chromatine condensation was seen in electron micrographs from tBOOH-treated cells. In addition, pulsed field gel electrophoresis of genomic DNA from both control and tBOOH-treated hepatocytes showed similar profiles, indicating the absence of internucleosomal DNA cleavage, a classical reflection of apoptotic endonuclease activity. The activation of the repair enzyme poly(ADP-ribose)polymerase (PARP) following DNA damage by tBOOH induced a dramatic drop in both NAD(+) and ATP. The inhibition of PARP by 3-aminobenzamide enhanced DNA damage by tBOOH, restored NAD(+) and ATP levels, but did not result in better survival against cell killing by tBOOH. The lack of the protective effect of PARP inhibitor, therefore, does not implicate PARP in the mechanism of tBOOH-induced cytotoxicity. Electron micrographs also show no mitochondrial swelling in cells under oxidative stress, but such organelles were mainly located around the nucleus, a picture already observed in autoschizis, a new suggested kind of cell death which shows both apoptotic and necrotic morphological characteristics.

Protein S-thiolation can mediate the inhibition of protein synthesis induced by tert-butyl hydroperoxide in isolated rat hepatocytes.

A rapid inhibition of protein synthesis is observed when isolated rat hepatocytes are incubated in the presence of 0.25-0.5 mM of tert-butyl hydroperoxide (tBOOH). Such an inhibition occurs in the absence of a cytolytic effect by tBOOH. Iron chelators (o-phenanthroline and desferrioxiamine), protected against oxidative cell death, but they did not modify the inhibition of protein synthesis caused by tBOOH (0.5 mM), suggesting that free radicals are less implicated in such an impairment. Electron micrographs of hepatocytes under oxidative stress show disaggregation of polyribosomes but not oxidative alterations, such as blebs or mitochondrial swelling. Protein synthesis inhibition is accompanied by a decrease in reduced glutathione (GSH) and an increase in glutathione disulfide (GSSG) and the level of protein S-thiolation (protein mixed disulfides formation). Such an increase of GSSG appears as a critical event since diethylmaleate (DEM) at 0.2 mM reduced GSH content by more than 50% but did not affect either GSSG content or protein synthesis. The addition of exogenous GSH and N-acetylcysteine (NAC) to tBOOH-treated hepatocytes significantly reduced the formation of protein mixed disulfides and restored the depressed protein synthesis either completely or partially. We suggest that S-thiolation of some key proteins may be involved in protein synthesis inhibition by tBOOH.

Targeting of drug to the hepatocytes by fatty acids. Influence of the carrier (albumin or galactosylated albumin) on the fate of the fatty acids and their analogs.

PURPOSE: The aim of this study was to evaluate the potential of fatty acids as shuttles to deliver xenobiotic inside the hepatocytes as well as to study the mechanism of incorporation into isolated hepatocytes when bound to native albumin or galactosylated albumin. Theoretically, they can enter into the hepatocytes after recognition of the Fatty Acid Binding Protein (FABPPM), or remain bound to galactosylated proteins and enter into these cells by a process known as receptor mediated endocytosis after selective recognition of the asialoglycoprotein receptor (ASGPR). METHODS: We synthesized a 3H-benzoyl adduct of lauric acid (BLA) (benzoyl adduct chosen to mimic any low molecular weight drug or contrast agent), and compared the behavior of BLA with oleic acid for their binding properties to carrier-proteins and the uptake mechanism by isolated hepatocytes. RESULTS: No significant difference was found in the binding properties of BLA for albumin and galactosylated albumin. The incorporation into the hepatocytes was found essentially depending on the FABPPM transport system whenever BLA was bound to albumin or to galactosylated albumin in the incubation medium: indeed, the transport was inhibited by phloretin (inhibitor of sodium dependent transport), increased when the free part of BLA was higher, and BLA was recovered in the cytosolic fraction of the hepatocytes. CONCLUSIONS: This study showed the convenience in using fatty acids as drug carriers possessing tropism for the hepatocytes.

Oxidative DNA damage by t-butyl hydroperoxide causes DNA single strand breaks which is not linked to cell lysis. A mechanistic study in freshly isolated rat hepatocytes.

In rat hepatocytes, DNA damage by t-butyl hydroperoxide (tBOOH) was measured by using the fluorimetric analysis of alkaline DNA unwinding. The electrophoretic profile of genomic DNA suggests single rather than double DNA strand breaks formation. Oxidative DNA modifications, measured as increased 8-hydroxy-deoxyguanosine content, were not detected. Lysis of hepatocytes and DNA strand breaks induced by tBOOH did not correlate, indicating that both processes are not interconnected. Since o-phenanthroline prevents against tBOOH-mediated effects on both DNA and membrane integrity, we discussed about a putative role of iron.

Cell death and lipid peroxidation in isolated hepatocytes incubated in the presence of hydrogen peroxide and iron salts.

The incubation of isolated hepatocytes in the presence of glucose plus glucose oxidase, a H2O2-generating system, resulted in extensive loss of cell viability, as expressed by the release of lactate dehydrogenase (LDH). Disturbance of metabolic functions such as glycogen and protein synthesis was also caused by H2O2, but in no case was malondialdehyde (MDA)-like products detected. The lytic effect of H2O2 was significantly enhanced by incubating hepatocytes in the presence of iron salts. Under these conditions, MDA-like products were detected, but lipid peroxidation and cell injury did not correlate. Iron chelators modulated the cytotoxicity of H2O2 in different (and opposite) ways: when iron was complexed with ADP, increased cell lysis was observed compared to uncomplexed iron plus H2O2. Iron-DTPA, on the contrary, decreased such a lytic effect. The preincubation of hepatocytes with desferrioxamine mesylate (Desferal; a strong iron chelator) abolished the cytolytic effects produced by the association of iron salts and H2O2, as well as the membrane oxidative injury due to H2O2 alone, thus suggesting the existence of an intracellular source of iron. This kind of mechanism (metal chelation rather than radical scavenging) is supported by the absence of any protective effect by some free radical scavengers against the oxidative injury induced by the association iron H2O2. Nevertheless, the glycogenolytic effects observed in the presence of H2O2 were not modified by Desferal. In our opinion, the cytotoxicity of the association H2O2 plus iron salts involves at least two different and independent mechanisms.

Biochemical changes in isolated hepatocytes exposed to tert-butyl hydroperoxide. Implications for its cytotoxicity.

When isolated hepatocytes were exposed to tert-butyl hydroperoxide (tBOOH) they lost their cellular membrane integrity. Decreased levels of GSH, increased phosphorylase a activity (an indirect index of the amount of free cytosolic Ca2+), and increase in the formation of malondialdehyde (MDA)-like products (an index of lipid peroxidation) preceded the release into the culture medium of the cytosolic enzyme lactate dehydrogenase (LDH), indicating that this later process was the consequence of the former intracellular events. While ATP levels were not modified during the incubation of cells with increasing concentrations of tBOOH, protein synthesis was decreased in a concentration-dependent manner. The glycogen content decreased at the same time as the increase in LDH leakage. The addition of promethazine (PMZ) an antioxidant molecule, prevented the lipid peroxidation, but did not protect cells against the oxidative effects of tBOOH, including loss of membrane integrity. Nevertheless, the addition of GSH to cell suspensions incubated with tBOOH, decreased the formation of MDA-like products, restored the protein synthesis rate, prevented partially the activation of phosphorylase a and preserved cell viability. On the basis of these results, we postulate that both GSH depletion and modification in phosphorylase a activity (Ca2+ levels) were the most relevant intracellular events to explain the cytotoxicity of tBOOH.