Aldo Keto Reductase 1B7 and Prostaglandin F2α Are Regulators of Adrenal Endocrine Functions

Prostaglandin F2α (PGF2α), represses ovarian steroidogenesis and initiates parturition in mammals but its impact on adrenal gland is unknown. Prostaglandins biosynthesis depends on the sequential action of upstream cyclooxygenases (COX) and terminal synthases but no PGF2α synthases (PGFS) were functionally identified in mammalian cells. In vitro, the most efficient mammalian PGFS belong to aldo-keto reductase 1B (AKR1B) family. The adrenal gland is a major site of AKR1B expression in both human (AKR1B1) and mouse (AKR1B3, AKR1B7). Thus, we examined the PGF2α biosynthetic pathway and its functional impact on both cortical and medullary zones. Both compartments produced PGF2α but expressed different biosynthetic isozymes. In chromaffin cells, PGF2α secretion appeared constitutive and correlated to continuous expression of COX1 and AKR1B3. In steroidogenic cells, PGF2α secretion was stimulated by adrenocorticotropic hormone (ACTH) and correlated to ACTH-responsiveness of both COX2 and AKR1B7/B1. The pivotal role of AKR1B7 in ACTH-induced PGF2α release and functional coupling with COX2 was demonstrated using over- and down-expression in cell lines. PGF2α receptor was only detected in chromaffin cells, making medulla the primary target of PGF2α action. By comparing PGF2α-responsiveness of isolated cells and whole adrenal cultures, we demonstrated that PGF2α repressed glucocorticoid secretion by an indirect mechanism involving a decrease in catecholamine release which in turn decreased adrenal steroidogenesis. PGF2α may be regarded as a negative autocrine/paracrine regulator within a novel intra-adrenal feedback loop. The coordinated cell-specific regulation of COX2 and AKR1B7 ensures the generation of this stress-induced corticostatic signal

A rapid, automatic and accurate assay for quantifying temocillin in human serum and CSF using turbulent flow liquid chromatography coupled to high‐resolution mass spectrometry. Clinical application

International audienceTemocillin is a β-lactamase-resistant penicillin used for the treatment of multiple drug-resistant Gram-negative bacteria. To maximize efficacy and avoid adverse effects, the dose regimen has to be quickly adjusted to the clinical situations. This necessitates the development of a rapid, reliable and accurate analytical method. Temocillin and the stable isotopically labeled internal standard ([13 C6 ]-amoxicillin) were extracted from either serum or cerebrospinal fluid by a turbulent flow liquid chromatographic method and eluted onto an octadecyl-silica phase with polar endcapping. Mass spectrometry was conducted using an exact mass determination method by electrospray positive ionization high-resolution mass spectrometry. The LLOQ and ULOQ of the present method were determined to be 0.4 and 200 μg/ml for serum and cerebrospinal fluid samples, respectively. The total analysis time was <7 min. The recovery ranged from 87.7 to 120.8%. Intra- and inter-day precision and trueness were tested at four concentration levels: 0.4, 8, 40 and 160 μg/ml. Values were 6.33 ± 1.53, 8.8 ± 1.3, 8.8 ± 0.36 and 2.1 ± 0.76%, and 5.0 ± 0.54, 9.9 ± 1.0, 5.8 ± 1.6 and 0.1 ± 1.1%, for inter- and intra-day analysis, respectively. Temocillin was found to be stable under all relevant laboratory conditions. The method was cross-validated with a microbiological assay. This method is suitable for accurate measurement of temocillin concentration in small volumes of serum or cerebrospinal fluid. Thanks to the online extraction procedure, the overall analytical time is compatible with high-throughput analysis for clinical application

Electrochemical Skin Conductance as a Marker of Painful Oxaliplatin-Induced Peripheral Neuropathy

Purpose. Oxaliplatin is a platinum compound widely used in gastrointestinal cancer treatment but produces dose-limiting peripheral neuropathy. New insights into oxaliplatin-induced peripheral neuropathy (OIPN) assessment are needed to detect more effectively this condition. In this context, we conducted Canaloxa study, a prospective preliminary clinical trial that aimed to investigate how Electrochemical Skin Conductance (ESC), a parameter used in small fiber neuropathy assessment, could be helpful in OIPN diagnosis. Methods. Cancer patients treated for at least three months with oxaliplatin and suffering from clinically OIPN were included. Electrochemical Skin Conductance, thermal thresholds, and neuropathic pain were assessed in all included patients. Results. During one year, 36 patients were included. The main result was the correlation between ESC and Neuropathic Pain Symptom Inventory score for hands (rho value = -0.69, p < 0.0001) and feet (rho value = -0.79, p < 0.0001). ESC values were lower in neuropathic patients with painful symptoms than in ones without painful symptoms (p = 0.0003 and p < 0.0001 for hands and feet, respectively). No correlation was observed between ESC and thermal thresholds. Conclusion. These preliminary data suggest that ESC could be a useful objective marker of painful oxaliplatin-induced neuropathy and could complement the use of subjective clinical scales. This study was prospectively registered on clinicaltrials.gov (NCT02827916) before patient recruitment has begun

P2X7 Cell Death Receptor Activation and Mitochondrial Impairment in Oxaliplatin-Induced Apoptosis and Neuronal Injury: Cellular Mechanisms and In Vivo Approach.

Limited information is available regarding the cellular mechanisms of oxaliplatin-induced painful neuropathy during exposure of patients to this drug. We therefore determined oxidative stress in cultured cells and evaluated its occurrence in C57BL/6 mice. Using both cultured neuroblastoma (SH-SY5Y) and macrophage (RAW 264.7) cell lines and also brain tissues of oxaliplatin-treated mice, we investigated whether oxaliplatin (OXA) induces oxidative stress and apoptosis. Cultured cells were treated with 2-200 µM OXA for 24 h. The effects of pharmacological inhibitors of oxidative stress or inflammation (N-acetyl cysteine, ibuprofen, acetaminophen) were also tested. Inhibitors were added 30 min before OXA treatment and then in combination with OXA for 24 h. In SH-SY5Y cells, OXA caused a significant dose-dependent decrease in viability, a large increase in ROS and NO production, lipid peroxidation and mitochondrial impairment as assessed by a drop in mitochondrial membrane potential, which are deleterious for the cell. An increase in levels of negatively charged phospholipids such as cardiolipin but also phosphatidylserine and phosphatidylinositol, was also observed. Additionally, OXA caused concentration-dependent P2X7 receptor activation, increased chromatin condensation and caspase-3 activation associated with TNF-α and IL-6 release. The majority of these toxic effects were equally observed in Raw 264.7 which also presented high levels of PGE2. Pretreatment of SH-SY5Y cells with pharmacological inhibitors significantly reduced or blocked all the neurotoxic OXA effects. In OXA-treated mice (28 mg/kg cumulated dose) significant cold hyperalgesia and oxidative stress in the tested brain areas were shown. Our study suggests that targeting P2X7 receptor activation and mitochondrial impairment might be a potential therapeutic strategy against OXA-induced neuropathic pain

Chromatin condensation, P2X7 receptor activation and caspase-3 activity in oxaliplatin-treated C57BL/6 mice.

<p>Mice were repeatedly injected i.p. with 7 mg/kg oxaliplatin (OXA) at days 1, 2, 5 and 6 (28 mg/kg cumulated dose; n = 10). Mitochondrial activity was evaluated by determining mitochondrial membrane potential (A) using JC-1 test and mitochondrial levels of negatively charged phospholipids (B) using nonyl acridine orange test. Chromatin condensation (C) was evaluated using Hoechst 33342 test and P2X7 receptor activation (D) using YOPRO-1 test. The apoTarget^TM Caspase-3 Protease assay was used for the <i>in vitro</i> determination of caspase-3 proteolytic activity (E) in lysates of brain mitochondrial homogenates as described by the manufacturer's instructions. Values are the mean ± S.E.M. expressed as percentage of the control (n = 8). *: statistically different (p<0.05) from the mean values in control mice.</p

Chromatin condensation, P2X7R activation and caspase-3 activity in oxaliplatin-treated SH-SY5Y cells pre-treated by protective drugs.

<p>Cells (2×10⁵ cells/well) were exposed for 24 h to oxaliplatin (OXA) (50, 100 or 200 µM) after a 30-min pre-treatment either with acetaminophen (AAP, 50 µM), ibuprofen (IBU, 1 µM) or N-acetyl cysteine (NAC, 1 mM). Chromatin condensation (A) was evaluated using Hoechst 33342 test and P2X7 receptor (P2X7R) activation (B) using YOPRO-1 test. The apoTarget^TM Caspase-3 Protease assay was used for the <i>in vitro</i> determination of caspase-3 proteolytic activity (C) in lysates of SH-SY5Y cells as described by the manufacturer's instructions. Values are the mean ± S.E.M. expressed as percentage of the control, five different assays per group. Significance of differences: OXA alone versus control, *p<0.05; AAP, or IBU, or NAC versus OXA alone: $ p<0.05.</p

P2X7 receptor activation, chromatin condensation and caspase-3 activity in SH-SY5Y cells exposed to oxaliplatin.

<p>Cells were exposed 24 h to oxaliplatin (OXA) (2–200 µM). P2X7 receptor activation (A) was evaluated using YOPRO-1 test. The resulting fluorescence was viewed with a microscope (B). Chromatin condensation (C) was evaluated using Hoechst 33342 test. Caspase-3 proteolytic activity (D) in lysates of cells was evaluated using the apoTarget^TM Caspase-3 Protease assay. In these tests, cells were exposed or not to a 30-min pre-treatment with the specific P2X7 receptor antagonist Brilliant Blue G (BBG; 10 µM) prior to OXA for an additional 24 h. Values are the mean ± S.E.M. expressed as percentage of the control, five different assays per group. *: statistically different (p<0.05) from the mean values in control cells.</p

Oxidative stress and mitochondrial activity in oxaliplatin-treated C57BL/6 mice.

<p>Mice were repeatedly injected i.p. with 7 mg/kg oxaliplatin (OXA) at days 1, 2, 5 and 6 (28 mg/kg cumulated dose; n = 10). Oxidative stress was evaluated by ROS production using dihydroethidium (A) and DCF-DA (B) tests and NO content (C) by the Griess reaction. Values are the mean ± S.E.M. expressed as percentage of the control (n = 8). *: statistically different (p<0.05) from the mean values in control mice.</p