Jean-Pierre Anger

Influence of fatty acid ethanolamides and Δ9-tetrahydrocannabinol on cytokine and arachidonate release by mononuclear cells

The effects of arachidonic acid ethanolamide (anandamide), palmitoylethanolamide and delta9-tetrahydrocannabinol on the production of tumor necrosis factor-alpha (TNF-alpha), interleukin-4, interleukin-6, interleukin-8, interleukin-10, interferon-gamma, p55 and p75 TNF-alpha soluble receptors by stimulated human peripheral blood mononuclear cells as well as [3H]arachidonic acid release by non-stimulated and N-formyl-Met-Leu-Phe (fMLP)-stimulated human monocytes were investigated. Anandamide was shown to diminish interleukin-6 and interleukin-8 production at low nanomolar concentrations (3-30 nM) but inhibited the production of TNF-alpha, interferon-gamma, interleukin-4 and p75 TNF-alpha soluble receptors at higher concentrations (0.3-3 microM). Palmitoylethanolamide inhibited interleukin-4, interleukin-6, interleukin-8 synthesis and the production of p75 TNF-alpha soluble receptors at concentrations similar to those of anandamide but failed to influence TNF-alpha and interferon-gamma production. The effect of both compounds on interleukin-6 and interleukin-8 production disappeared with an increase in the concentration used. Neither anandamide nor palmitoylethanolamide influenced interleukin-10 synthesis. delta9-Tetrahydrocannabinol exerted a biphasic action on pro-inflammatory cytokine production. TNF-alpha, interleukin-6 and interleukin-8 synthesis was maximally inhibited by 3 nM delta9-tetrahydrocannabinol but stimulated by 3 microM delta9-tetrahydrocannabinol, as was interleukin-8 and interferon-gamma synthesis. The level of interleukin-4, interleukin-10 and p75 TNF-alpha soluble receptors was diminished by 3 microM delta9-tetrahydrocannabinol. [3H]Arachidonate release was stimulated only by high delta9-tetrahydrocannabinol and anandamide concentrations (30 microM). These results suggest that the inhibitory properties of anandamide, palmitoylethanolamide and delta9-tetrahydrocannabinol are determined by the activation of the peripheral-type cannabinoid receptors, and that various endogenous fatty acid ethanolamides may participate in the regulation of the immune response.

OVEREXPRESSION OF THE MULTIDRUG RESISTANCE-ASSOCIATED PROTEIN (MRP1) IN HUMAN HEAVY METAL-SELECTED TUMOR CELLS

Cellular and molecular mechanisms involved in the resistance to cytotoxic heavy metals remain largely to be characterized in mammalian cells. To this end, we have analyzed a metal‐resistant variant of the human lung cancer GLC4 cell line that we have selected by a step‐wise procedure in potassium antimony tartrate. Antimony‐selected cells, termed GLC4/Sb30 cells, poorly accumulated antimony through an enhanced cellular efflux of metal, thus suggesting up‐regulation of a membrane export system in these cells. Indeed, GLC4/Sb30 cells were found to display a functional overexpression of the multidrug resistance‐associated protein MRP1, a drug export pump, as demonstrated by Western blotting, reverse transcriptase‐polymerase chain reaction and calcein accumulation assays. Moreover, MK571, a potent inhibitor of MRP1 activity, was found to markedly down‐modulate resistance of GLC4/Sb30 cells to antimony and to decrease cellular export of the metal. Taken together, our data support the conclusion that overexpression of functional MRP1 likely represents one major mechanism by which human cells can escape the cytotoxic effects of heavy metals.

Drug-facilitated robbery or sexual assault: problems associated with amnesia

Abstract: Amnesia following sedative-hypnotic drug exposure is discussed. Anterograde amnesia clearly occurs with many benzodiazepines. Several drugs are assessed: benzodiazepines and two hypnotics in particular that are structurally unrelated to the benzodiazepines but share some of their properties: zolpidem and zopiclone. The amnesic effects of these drugs are described, memory process, biology of memory, and memory process impairment documented. With these drugs anterograde amnesia has been demonstrated to be dose dependent. This effect is associated with hypnotic drugs, however, the receptors are different. As regards forensic medicine, a significant and specific type of amnesia should be considered: amnesia automatism or amnesic complex automatism. Also, several cases observed in our laboratory are presented to demonstrate the impact of amnesia.

Resistance of human multidrug resistance-associated protein 1-overexpressing lung tumor cells to the anticancer drug arsenic trioxide

The human multidrug-resistance protein (MRP1) confers resistance to some heavy metals such as arsenic and antimony, mainly through mediating an increased cellular efflux of metal. However, it was recently suggested that arsenic, used under its trioxide derivative form as anticancer drug, is not handled by MRP1. The aim of the present study was to test this hypothesis in MRP1-overexpressing human lung tumor GLC4/Sb30 cells. Using the cytotoxicity MTT assay, GLC4/Sb30 cells were found to be 10.8-fold more resistant to arsenic trioxide (As2O3) than parental GLC4 cells. MK571, a potent inhibitor of MRP1 activity, almost totally reversed resistance of GLC4/Sb30 cells, but did not alter the sensitivity of GLC4 cells. Moreover, As2O3-loaded GLC4/Sb30 cells poorly accumulated arsenic through an increased MK571-sensitive efflux of metal. Finally, depletion of cellular glutathione levels in buthionine sulfoximine-treated GLC4/Sb30 cells was found to result in increased accumulation and reduced efflux of arsenic in cells exposed to As2O3, outlining the glutathione-dependence of MRP1-mediated transport of the metal. These results indicate that MRP1 overexpression in human tumor cells can confer resistance to As2O3, which may limit the clinical use of this anticancer drug for treatment of MRP1-positive tumors.

Differential sensitivities of MRP1-overexpressing lung tumor cells to cytotoxic metals

The human multidrug-resistance protein (MRP1), known to mediate cellular efflux of a wide range of xenobiotics, including anticancer drugs, has also been shown to transport antimony, thereby conferring resistance to this heavy metal. The aim of the present study was to investigate whether other cytotoxic metals could be handled by MRPI using MRP1-overexpressing lung tumor GLC4/Sb30 cells. Such cells were found to be 3.4-, 12.7- and 16.3-fold more resistant than parental GLC4 cells to mercuric ion, arsenite and arsenate, respectively, whereas they remained sensitive to other cytotoxic metals tested such as copper, chromium, cobalt or aluminium. MK571, a potent inhibitor of MRP1 activity, almost totally reversed resistance of GLC4/Sb30 cells to mercuric ions and arsenic while it did not significantly alter sensitivity of GLC4 cells to metals. Arsenate-treated GLC4/Sb30 cells were found to poorly accumulate arsenic through increased MK571-inhibitable efflux of the metal. Arsenate, however, failed to alter MRP1-mediated transport of known MRP1 substrates such as calcein and vincristine. In conclusion, these findings demonstrated that MRP1 likely handled some, but not all, cytotoxic metals such as arsenic and mercuric ions in addition to antimony, therefore resulting in reduced toxicity of these compounds towards MRP1-overexpressing cells.

Method for monitoring alginate released in biological fluids by high-performance anion-exchange chromatography with pulsed amperometric detection.

The use of alginate-entrapped cells in cell therapy requires a method for monitoring possible released compound within biological fluids following either their implantation or inoculation in artificial organs. Oligomannuronic and oligoguluronic acids were prepared by enzymatic depolymerization with alginate lyase from Pseudomonas alginovora, characterized by high-performance anion-exchange chromatography with pulsed amperometric detection and quantitated in human, pig and rabbit blood, urine and tissue samples. The method was tested for linearity and detection limit, accuracy, intra- and inter-day precision. The limit of detection was 3 microgram/ml in both urine and plasma and 5 mg/g of tissues. The relative standard deviations (RSDs) of intra-day precision were 6.0-16.6% and 4.8-8.7% in plasma and urine, respectively; the RSDs of inter-day precision were 5.1-14.4% and 5.0-11.6% in plasma and urine, respectively. Thus, this method appears suitable for the measurement of released alginate from entrapped cells used in cell therapy.

Stimulation of Rb+ influx by bradykinin through Na+/K+/Cl− cotransport and Na+/K+-atpase in NIH-3T3 fibroblasts

Bradykinin receptor stimulation results in G-protein-coupled phospholipase activation, initiating protein kinase C (PKC) stimulation and cytosolic free Ca2+ concentration ([Ca2+]i) rises as signalling pathways. Using Rb+ as a tracer for K+, we have studied the mechanisms involved in bradykinin-stimulated Rb+ influx in NIH-3T3 fibroblasts. The furosemide-sensitive Na+/K+/Cl- cotransport and the ouabain-sensitive Na+/K(+)-ATPase were both involved in Rb+ influx under resting conditions with a ratio Na+/K+/Cl- cotransport/Na+/K(+)-ATPase (r) = 0.73. Bradykinin stimulated Rb+ influx (+82.6%) through both systems without changing their ratio (r = 0.72). PKC stimulation by a 15-min-treatment with phorbol 12-myristate 13-acetate (PMA) (2x10(-7) M) increased Rb+ influx in resting cells by 75.7% without affecting r (0.75). PKC inhibition by H-7, and PKC down-regulation by 24-h PMA (10(-6) M) treatment decreased the bradykinin-induced stimulation of Rb+ influx (+31% and +14.9% above control, respectively). Both down-regulation and inhibition of PKC dramatically reduced the furosemide-sensitive Na+/K+/Cl- cotransport, as r fell to 0.239 and 0.032 in bradykinin-stimulated cells after H-7 and 24-h PMA treatments, respectively. BAPTA/AM pretreatment (10(-4) M, 60 min), which complexed with [Ca2+]i, not only prevented the bradykinin-induced [Ca2+]i raise, but also partially inhibited bradykinin-induced Rb+ influx stimulation (+39% above control), without modifying r (0.76). We conclude that stimulation of PKC is a major pathway involved in bradykinin stimulation of Rb+ influx in NIH-3T3 fibroblasts, and that rises in [Ca2+]i participate in bradykinin signalling, possibly through PKC activation. Our data also suggest that active PKC is required for basal and bradykinin-stimulated Na+/K+/Cl- cotransport activity in these cells.

Memory Impairment after Drug-Facilitated Crimes

Memory impairment is a major problem after drug facilitated crimes. This chapter describes the memory process mechanisms, as well as the recent trends in molecular biology of memory: the wide acceptance and extension of the mechanism known as “synaptic tagging and capture” to a variety of memory forms, the role of plasticity-related proteins in metaplasticity that governs synaptic tagging and capture, and the role of an atypical isoform of protein kinase C, protein kinase M-zeta, in memory consolidation and persistence in the hippocampal CA1 region. Long-term potentiation is indeed the basis of memory consolidation at the cellular level in the hippocampus. After memories are consolidated in the hippocampal CA1 region, they are stored in a long-lasting form (days, weeks, years) elsewhere. As the perpetrators commonly use pharmaceutical drugs, such as sedative-hypnotic drugs due to their amnesic properties, to facilitate their crimes, anterograde amnesia following drugs or drugs of abuse, involving various neuronal population (GABAergic, cholinergic, glutamatergic, serotonergic) exposures, is considered.