Heptafluoroisobutyronitrile (C4F7N), a gas used for insulating and arc quenching in electrical switchgear, is neurotoxic in the mouse brain.

Fluoronitrile gas (C4F7N, CAS number 42532-60-5) is one of the most promising candidates as insulating and/or breaking medium in high and medium voltage electrical equipment. Besides its promising properties, C4F7N gas is however not devoid of acute toxicity when used pure or in gas mixtures. The toxicity was not extensively analyzed and reported. The aim of the present study was to analyze in mice the consequences of a single exposure to C4F7N gas, at different concentrations and different timepoints after exposure. Male and female Swiss mice were exposed to breathable air or C4F7N gas, at 800 ppmv or 1500 ppmv, for 4 h on day 0. Behavioral tests (spontaneous alternation in the Y-maze and object recognition) were performed on days 1, 7 and 14 to assess memory alterations. The animals were then sacrificed and their brains dissected for biochemical analyses or fixed with paraformaldehyde for histology and immunohistochemistry. Results showed behavioral impairments and memory deficits, with impairments of alternation at days 1 and 7 and object recognition at day 14. Histological alterations of pyramidal neuronal layer in the hippocampus, neuroinflammatory astroglial reaction, and microglial alterations were observed, more marked in female than male mice. Moreover, the biochemical analyses done in the brain of 1500 ppmv exposed female mice showed a reductive stress with decreased lipid peroxidation and release of cytochrome c, leading to apoptosis with increases in caspase-9 cleavage and γ-H2AX/H2AX ratio. Finally, electrophysiological analyses using a multi-electrode array allowed the measure of the extracellular activity of pyramidal neurons in the CA2 area and revealed that exposure to the gas not only prevented the induction of long-term potentiation but even provoked an epileptoid-like activity in some neurons suggesting major alterations of synaptic plasticity. This study therefore showed that an acute exposure of mice to C4F7N gas provoked, particularly in female animals, memory alterations and brain toxicity characterized by a reductive stress, microglial toxicity, loss of synaptic plasticity and apoptosis. Its use in industrial installations must be done with extreme caution.

The ellagitannin metabolite urolithin C is a glucose-dependent regulator of insulin secretion through activation of L-type calcium channels.

BACKGROUND AND PURPOSE: The pharmacology of polyphenol metabolites on beta-cell function is largely undetermined. We sought to identify polyphenol metabolites that enhance the insulin-secreting function of beta-cells and to explore the underlying mechanisms. EXPERIMENTAL APPROACH: INS-1 beta-cells and rat isolated islets of Langerhans or perfused pancreas preparations were used for insulin secretion experiments. Molecular modelling, intracellular Ca2+ monitoring, and whole-cell patch-clamp recordings were used for mechanistic studies. KEY RESULTS: Among a set of polyphenol metabolites, we found that exposure of INS-1 beta-cells to urolithins A and C enhanced glucose-stimulated insulin secretion. We further characterized the activity of urolithin C and its pharmacological mechanism. Urolithin C glucose-dependently enhanced insulin secretion in isolated islets of Langerhans and perfused pancreas preparations. In the latter, enhancement was reversible when glucose was lowered from a stimulating to a non-stimulating concentration. Molecular modelling suggested that urolithin C could dock into the Cav 1.2 L-type Ca2+ channel. Calcium monitoring indicated that urolithin C had no effect on basal intracellular Ca2+ but enhanced depolarization-induced increase in intracellular Ca2+ in INS-1 cells and dispersed cells isolated from islets. Electrophysiology studies indicated that urolithin C dose-dependently enhanced the L-type Ca2+ current for levels of depolarization above threshold and shifted its voltage-dependent activation towards more negative potentials in INS-1 cells. CONCLUSION AND IMPLICATIONS: Urolithin C is a glucose-dependent activator of insulin secretion acting by facilitating L-type Ca2+ channel opening and Ca2+ influx into pancreatic beta-cells. Our work paves the way for the design of polyphenol metabolite-inspired compounds aimed at ameliorating beta-cell function.

MAP Kinase cross talks in oxidative stress-induced impairment of insulin secretion. Involvement in the protective activity of quercetin.

Insulin secretion preservation is a major issue for the prevention or treatment of type 2 diabetes. We previously showed on ß-cells that quercetin (Q), but not resveratrol (R) or N-acetyl cysteine (NAC), amplified glucose-induced insulin secretion in a calcium- and ERK1/2-dependent manner. Quercetin, but not resveratrol or NAC, also protected ß-cell function and hyperamplified ERK1/2 phosphorylation in oxidative stress conditions. As quercetin may interfere with other stress-activated protein kinases (JNK and p38 MAPK), we further explored MAPK cross talks and their relationships with the mechanism of the protective effect of quercetin against oxidative stress. In INS-1 insulin-secreting ß-cells, using pharmacological inhibitors of MAPK pathways, we found that under oxidative stress (50 µm H2O2) and glucose-stimulating insulin secretion conditions: (i) p38 MAPK phosphorylation was increased and regulated by ERK1/2 (positively) and JNK (negatively), although p38 MAPK activation did not seem to play any significant role in oxidative stress-induced insulin secretion impairment; (ii) the JNK pathway appeared to inhibit both ERK1/2 activation and insulin secretion, although JNK phosphorylation was not significantly changed in our experimental conditions; (iii) the functionality of ß-cell in the presence of oxidative stress was closely linked to the level of ERK1/2 activation, (iv) quercetin, resveratrol, or NAC inhibited H2O2 -induced p38 MAPK phosphorylation. The preservation of ß-cell function against oxidative stress appears dependent on the balance between ERK1/2 and JNK activation. The protecting effect of quercetin appears due to ERK1/2 hyperactivation, possibly induced by L-type calcium channel opening as we recently showed.

Chicoric acid is an antioxidant molecule that stimulates AMP kinase pathway in L6 myotubes and extends lifespan in Caenorhabditis elegans.

Chicoric acid (CA) is a caffeoyl derivative previously described as having potential anti-diabetic properties. As similarities in cellular mechanism similarities between diabetes and aging have been shown, we explored on L6 myotubes the effect of CA on the modulation of intracellular pathways involved in diabetes and aging. We also determined its influence on lifespan of Caenorhabditis elegans worm (C. elegans). In L6 myotubes, CA was a potent reactive oxygen species (ROS) scavenger, reducing ROS accumulation under basal as well as oxidative stress conditions. CA also stimulated the AMP-activated kinase (AMPK) pathway and displayed various features associated with AMPK activation: CA (a) enhanced oxidative enzymatic defences through increase in glutathion peroxidase (GPx) and superoxide dismutase (SOD) activities, (b) favoured mitochondria protection against oxidative damage through up-regulation of MnSOD protein expression, (c) increased mitochondrial biogenesis as suggested by increases in complex II and citrate synthase activities, along with up-regulation of PGC-1α mRNA expression and (d) inhibited the insulin/Akt/mTOR pathway. As AMPK stimulators (e.g. the anti-diabetic agent meformin or polyphenols such as epigallocatechingallate or quercetin) were shown to extend lifespan in C. elegans, we also determined the effect of CA on the same model. A concentration-dependant lifespan extension was observed with CA (5-100 µM). These data indicate that CA is a potent antioxidant compound activating the AMPK pathway in L6 myotubes. Similarly to other AMPK stimulators, CA is able to extend C. elegans lifespan, an effect measurable even at the micromolar range. Future studies will explore CA molecular targets and give new insights about its possible effects on metabolic and aging-related diseases.

Short-term intravenous insulin infusion is associated with reduced expression of NADPH oxidase p47(phox) subunit in monocytes from type 2 diabetes patients.

Hyperglycemia is a well-known inducing factor of oxidative stress through activation of NADPH oxidase. In addition to its plasma glucose lowering effect, insulin may also have antioxidant activity and was shown to downregulate NADPH oxidase expression in vitro. In this study, we show that a short-term (3-day) intravenous insulin infusion in patients with type 2 diabetes induces normalization of both glycemia and mRNA expression of circulating monocyte p47(phox) subunit.

Preventive effects of nutritional doses of polyphenolic molecules on cardiac fibrosis associated with metabolic syndrome: involvement of osteopontin and oxidative stress.

We previously showed that grape extracts enriched in different polyphenolic families were similarly able to prevent reactive oxygen species (ROS) production, although having differential effects on various features of metabolic syndrome when administered at a dose of 21 mg/kg to the fructose (60%)-fed rat (a model of metabolic syndrome). In the present work, we analyzed on the same model the effect of pure polyphenolic molecules (catechin, resveratrol, delphinidin, and gallic acid) administered at a dose of 2.1 mg/kg. Delphinidin and gallic acid prevented insulin resistance, while gallic acid prevented the elevation of blood pressure. All molecules prevented cardiac ROS overproduction and NADPH overexpression. We also showed that fructose feeding was associated with cardiac fibrosis (accumulation of collagen I) and expression of osteopontin, a factor induced by ROS and a collagen I expression inducer. Collagen I and osteopontin expressions were prevented by the administration of all polyphenolic molecules. The potential use of polyphenols in the prevention of cardiac fibrosis should be further explored.

Pygeum africanum extract inhibits proliferation of human cultured prostatic fibroblasts and myofibroblasts.

OBJECTIVE: To investigate the effect of Pygeum africanum (PA) extract on the proliferation of cultured human prostatic myofibroblasts and fibroblasts; this extract is used for treating urinary disorders associated with benign prostatic hyperplasia (BPH). MATERIALS AND METHODS: Primary cultures of prostatic stromal cells were obtained from histologically confirmed human BPH by enzymatic digestion. Cell proliferation was measured by 5-bromo2'-deoxy-uridine (BrdU) incorporation assays, and cytotoxicity by luminescent quantification of adenylate kinase activity. RESULTS: Cultured cells were labelled by an anti-vimentin antibody, and most of them by an alpha-smooth-muscle-actin antibody, revealing the presence of fibroblasts and myofibroblasts. BrdU incorporation tests showed that proliferation of cultured human stromal cells, stimulated by fetal calf serum, by basic fibroblast growth factor and by epidermal growth factor, was dose-dependently inhibited by PA extract (5-100 microg/mL). Except at 100 microg/mL, no acute cytotoxicity of the extract was detected after 24 h of culture. Similarly, the extract dose-dependently inhibited the proliferation of Madin-Darby canine kidney epithelial cells, but to a lesser extent; whatever the dose of extract, no acute toxicity was evident on this cell line. CONCLUSION: PA extract inhibits the proliferation of cultured human prostatic myofibroblasts and fibroblasts. We propose that cultured human prostatic cells offer a reliable model for preclinical screening of therapeutic agents, and to study the mechanisms underlying the inhibition of proliferation.

Differentiated rabbit prostatic stromal cells in primary culture display functional alpha1A-adrenoceptors.

AIMS: BPH is characterized by uncontrolled proliferation and increased contractility of prostatic smooth muscle cells. The activation of alpha1-adrenoceptors (alpha1-AR) seems involved in the latter event, but the lack of in vitro models expressing these receptors has hampered a more specific characterization of their role. In order to do so, we attempted to develop a new model of rabbit cultured prostatic stromal cells (PSC) in a non-proliferative and differentiated state. METHODS: The expression of cytoskeletal and stromal markers was confirmed by immunohistochemistry on primary cultured PSC. Alpha1-AR subtype expression was assessed by RT-PCR, while receptor coupling to the ERK1/ERK2 and calcium pathways was studied by Western Blot and Fura-2 calcium imaging, respectively. RESULTS: Cells grown under non-proliferative conditions displayed a differentiated phenotype, with expression of contractile cytoskeletal and stromal proteins. Furthermore, the alpha1A-AR was shown to activate ERK1/ERK2 as well as calcium signaling. CONCLUSION: These results emphasize the interest of this model for the characterization of PSC adrenergic regulation, in particular through the little-known alpha1A-AR.

Involvement of oxidative stress and NADPH oxidase activation in the development of cardiovascular complications in a model of insulin resistance, the fructose-fed rat.

Growing evidences suggest a role of oxidative stress in hypertension and cardiac hypertrophy. The fructose (60%)-fed rat represents a model of metabolic syndrome, associating insulin resistance and high blood pressure. In this model, hypertension, cardiac and vessels hypertrophy and markers of oxidative stress were determined. In addition, the production of reactive oxygen species (ROS) was evaluated at different times after the initiation of fructose-enriched diet in aorta, heart and polymorphonuclear cells. High fructose feeding was associated with an early (1-week) increase in ROS production by aorta, heart and circulatory polymorphonuclear cells, in association with enhanced markers of oxidative stress. Vascular and cardiac hypertrophy was also rapidly observed, while the rise in blood pressure was significant only after 3 weeks. In summary, our study suggests that the production of reactive oxygen species can be a key-event in the initiation and development of cardiovascular complications associated with insulin resistance.

Cultured gastrointestinal smooth muscle cells: cell response to contractile agonists depends on their phenotypic state.

In the digestive tract, the transit of ingested food induces a local contraction-relaxation reflex of which the smooth muscle cell (SMC) represents the functional unit. Although freshly isolated SMCs have been extensively used for in vitro studies, in specific cases cultured cells appear necessary. Because conventionally cultured SMCs lose their contractile properties, we have developed: (1) differentiated, contractile rabbit gastric SMCs (D-stim cells), cultured in a medium supplemented with insulin, and (2) proliferative, dedifferentiated rabbit gastric SMCs (P-stim cells), cultured in a medium supplemented with insulin, fetal serum, EGF and b-FGF. The proliferative index was 5 +/- 4% and 82 +/- 10%, respectively, for D-stim and P-stim cells. Expression of SM-myosin heavy chain was observed in 90% of D-stim cells, whereas it was progressively lost in P-stim cells. Carbachol (1-100 nM), glicentin (2 nM) and gastrin-17 (100 nM) induced contraction of D-stim cells cultured for 3 or 6 days, whereas they did not induce the contraction of P-stim cells; in contrast, gastrin-17 (10 nM) was able to stimulate DNA synthesis (1.86 +/- 0.09-fold increase) in P-stim cells. The coupling of muscarinic receptors to intracellular transduction pathways was evaluated in D-stim cells: at day 3, carbachol (100 nM) induced a twofold increase in the production of inositol tri-tetra-phosphates; in parallel, a phosphorylation of ERK MAP kinases occurred within 1 min of carbachol stimulation. In conclusion, cultured functional myocytes derived from mature tissue may be used for long-term studies concerning the events coupled either to proliferation or to motility regulation of differentiated SMCs due to the activation of G-protein-coupled receptors.