Spinal cord NLRP1 inflammasome contributes to dry skin induced chronic itch in mice.

BACKGROUND: Dry skin itch is one of the most common skin diseases and elderly people are believed to be particularly prone to it. The inflammasome has been suggested to play an important role in chronic inflammatory disorders including inflammatory skin diseases such as psoriasis. However, little is known about the role of NLRP1 inflammasome in dry skin-induced chronic itch. METHODS: Dry skin-induced chronic itch model was established by acetone-ether-water (AEW) treatment. Spontaneous scratching behavior was recorded by video monitoring. The expression of nucleotide oligomerization domain (NOD)-like receptor protein 1 (NLRP1) inflammasome complexes, transient receptor potential vanilloid type 1 (TRPV1), and the level of inflammatory cytokines were determined by western blot, quantitative real-time PCR, and enzyme-linked immunosorbent assay (ELISA) kits. Nlrp1a knockdown was performed by an adeno-associated virus (AAV) vector containing Nlrp1a-shRNA-eGFP infusion. H.E. staining was used to evaluate skin lesion. RESULTS: AEW treatment triggers spontaneous scratching and significantly increases the expression of NLRP1, ASC, and caspase-1 and the levels of IL-1ß, IL-18, IL-6, and TNF-α in the spinal cord and the skin of mice. Spinal cord Nlrp1a knockdown prevents AEW-induced NLRP1 inflammasome assembly, TRPV1 channel activation, and spontaneous scratching behavior. Capsazepine, a specific antagonist of TRPV1, can also inhibit AEW-induced inflammatory response and scratching behavior. Furthermore, elderly mice and female mice exhibited more significant AEW-induced scratching behavior than young mice and male mice, respectively. Interestingly, AEW-induced increases in the expression of NLRP1 inflammasome complex and the levels of inflammatory cytokines were more remarkable in elderly mice and female mice than in young mice and male mice, respectively. CONCLUSIONS: Spinal cord NLRP1 inflammasome-mediated inflammatory response contributes to dry skin-induced chronic itch by TRPV1 channel, and it is also involved in age and sex differences of chronic itch. Inhibition of NLRP1 inflammasome may offer a new therapy for dry skin itch.

Occupational exposure to petroleum-based and oxygenated solvents and oral and oropharyngeal cancer risk in men: A population-based case-control study in France.

OBJECTIVE: To examine the association between occupational exposure to petroleum-based and oxygenated solvents and the risk of oral and oropharyngeal cancer. METHODS: The ICARE study is a large population-based case-control study conducted in France between 2001 and 2007. This present analysis was restricted to men and included 350 and 543 cases of squamous cell-carcinoma of the oral cavity and oropharynx, respectively, and 2780 controls. Lifetime tobacco, alcohol consumption and complete occupational history were assessed through detailed questionnaires. Job-exposure matrices allowed us to assess occupational exposure to five petroleum-based solvents (white spirits; diesel/fuel oils/kerosene; gasoline; benzene; special petroleum products) and five oxygenated solvents (diethyl ether; tetrahydrofuran; ketones and esters; alcohols; ethylene glycol). Odds-ratios (ORs), adjusted for age, smoking, alcohol consumption and socioeconomic status, and 95% confidence intervals (CI) were estimated using unconditional logistic models. RESULTS: Associations between oral cancer risk and exposure to white spirits and diesel/fuel oils/kerosene were suggested, but there was no exposure-response trend. Concerning exposure to oxygenated solvents, participants with the highest levels of cumulative exposure to diethyl ether had a significant excess risk of oropharyngeal cancer (OR = 7.78, 95%CI 1.42 to 42.59; p for trend = 0.04). Ever exposure to tetrahydrofuran was associated with a borderline significant increased risk of oral cancer (OR = 1.87, 95%CI 0.97 to 3.61), but no exposure-response trend was observed. Additional adjustments for exposure to other solvents did not substantially change the results. CONCLUSION: Our results do not provide evidence for a major role of petroleum-based and oxygenated solvents in the occurrence of oral and oropharyngeal cancers in men.

Isoflurane is a suitable alternative to ether for anesthetizing rats prior to euthanasia for gene expression analysis.

Diethyl ether (ether) had been widely used in Japan for anesthesia, despite its explosive properties and toxicity to both humans and animals. We also had used ether as an anesthetic for euthanizing rats for research in the Toxicogenomics Project (TGP). Because the use of ether for these purposes will likely cease, it is required to select an alternative anesthetic which is validated for consistency with existing TGP data acquired under ether anesthesia. We therefore compared two alternative anesthetic candidates, isoflurane and pentobarbital, with ether in terms of hematological findings, serum biochemical parameters, and gene expressions. As a result, few differences among the three agents were observed. In hematological and serum biochemistry analysis, no significant changes were found. In gene expression analysis, four known genes were extracted as differentially expressed genes in the liver of rats anesthetized with ether, isoflurane, or pentobarbital. However, no significant relationships were detected using gene ontology, pathway, or gene enrichment analyses by DAVID and TargetMine. Surprisingly, although it was expected that the lung would be affected by administration via inhalation, only one differentially expressed gene was extracted in the lung. Taken together, our data indicate that there are no significant differences among ether, isoflurane, and pentobarbital with respect to effects on hematological parameters, serum biochemistry parameters, and gene expression. Based on its smallest affect to existing data and its safety profile for humans and animals, we suggest isoflurane as a suitable alternative anesthetic for use in rat euthanasia in toxicogenomics analysis.

Selective cytotoxic activity of new lipophilic hydroxytyrosol alkyl ether derivatives.

Recent data suggest that hydroxytyrosol, a phenolic compound of virgin olive oils, has anticancer activity. This communication reports the synthesis of decyl and hexadecyl hydroxytyrosyl ethers, as well as the cytotoxic activity of hydroxytyrosol and a series of seven hydroxytyrosol alkyl ether derivatives against A549 lung cancer cells and MRC5 non-malignant lung fibroblasts. Hydroxytyrosyl dodecyl ether (HTDE) showed the highest selective cytotoxicity, and possible mechanisms of action were investigated; results suggest that HTDE can moderately inhibit glycolysis, induce oxidative stress, and cause DNA damage in A549 cells. The combination of HTDE with the anticancer drug 5-fluorouracil induced a synergistic cytotoxicity in A549 cancer cells but not in non-malignant MRC5 cells. HTDE also displayed selective cytotoxicity against MCF7 breast cancer cells versus MCF10 normal breast epithelial cells in the 1-30 µM range. These results suggest that the cytotoxicity of HTDE is more potent and selective than that of parent compound hydroxytyrosol.

Maternal anesthesia via isoflurane or ether differentially affects pre-and postnatal behavior in rat offspring.

Our understanding of prenatal behavior has been significantly advanced by techniques for direct observation and manipulation of unanesthetized, behaving rodent fetuses with intact umbilical connections to the mother. These techniques involve brief administration of an inhalant anesthesic, enabling spinal transection of the rat or mouse dam, after which procedures can continue with unanesthetized dams and fetuses. Because anesthetics administered to the mother can cross the placental barrier, it is possible that fetuses are anesthetized to varying degrees. We compared in perinatal rats the effects of prenatal maternal exposure to two inhalant anesthetics: ether and isoflurane. Fewer spontaneous fetal movements and first postpartum nipple attachments were observed following maternal exposure to ether as compared to isoflurane. Neonatal breathing frequencies and oxygenation did not account for group differences in nipple attachment. Our results provide evidence that the particular inhalant anesthetic employed in prenatal manipulation studies determines frequencies of perinatal behavior.

Ether stress-induced Alzheimer-like tau phosphorylation in the normal mouse brain.

Tau is reversibly hyperphosphorylated in the mouse brain by starvation or cold water swimming. Here, we report tau phosphorylation in the hippocampus of normal mouse after ether anesthesia, known to trigger typical stress reactions. Robust phosphorylation of tau was observed immediately and 10min after ether vapor exposure at Ser202/Thr205 and Thr231/Ser235, sites typically phosphorylated in Alzheimer brains. The phosphorylation levels returned to baseline by 1h. The most conspicuous and consistent change in the protein kinases studied was the inactivating phosphorylation of Ser9 of TPKI/GSK3beta in close correspondence with tau phosphorylation. These findings show that tau phosphorylation is a rapid physiological process integral to stress response system, and suggest involvement therein of TPKI/GSK3beta.

Secretory phospholipase A2 as a tumor-specific trigger for targeted delivery of a novel class of liposomal prodrug anticancer etherlipids.

The use of many common clinically relevant chemotherapeutics is often limited due to insufficient delivery to the tumor and dose-limiting systemic toxicities. Therefore, therapeutics that specifically target tumor cells and are nontoxic to normal cells are required. Here, we report the development of a novel class of liposomes composed of lipid prodrugs, which use the increased secretory phospholipase A2 type IIA (sPLA2) activity of the tumor microenvironment as a trigger for the release of anticancer etherlipids (AEL). Treatment of sPLA2-secreting tumor cells in vitro with liposomes consisting of proAELs resulted in growth inhibition comparable with addition of the AELs alone. Using a specific sPLA2 inhibitor, we showed the low cytotoxicity of the nonhydrolyzed proAEL liposomes and have proven the sPLA2 dependency of the activation of proAELs to cytotoxic AELs. In addition, we showed that our proAEL liposomes circumvent the inherent hemolytic toxicities associated with the use of etherlipids, thereby allowing i.v. administration of such therapeutics as nontoxic prodrug liposomes. Furthermore, using a sPLA2-secreting human colon cancer xenograft model, we showed that the proAEL liposomes are capable of inducing a tumor growth delay in vivo. Taken together, these data support the validity of this novel tumor-selective liposomal prodrug delivery strategy. This new approach also provides a promising system for tumor-selective delivery and release of conventional chemotherapeutics encapsulated in the sPLA2-degradable prodrug liposomes.

Health effects of exposure to ethylene glycol monoethyl ether in female workers.

Ethylene glycol monoethyl ether (EGEE) is a solvent commonly used in industry. To find the health effect of the solvent exposure in women, we did an investigation on 32 female workers exposed to EGEE in factories manufacturing photopolymer sensitization plate, and 20 subjects working in the same companies without potential exposure to the solvent. The mean age was 35.0 and 33.9 yr in the two groups, respectively. The mean concentration of the urinary metabolite (ethoxyacetic acid) was 120.87 mg/g creatinine (geometric mean) in the exposed group, and 2.71 mg/ g creatinine in the control group. Average RBC count and hemoglobin levels were normal in both groups. However, there were 2 subjects in the exposed group with an RBC count and hemoglobin concentration slightly lower than the standard. Out of 20 controls, 5 subjects reported irregular menstruation, and in comparison, 4 out of 32 exposed females had the same complaint. The most common health complaints were dizziness and swelling of the legs, with the same frequencies seen in both groups. Overall, our study suggests that although female workers were exposed to high concentrations of EGEE, subsequent health problems possibly due to such exposure were not significant.

Comparative study of the effects of toluene, benzene, 1,1,1-trichloroethane, diethyl ether, and flurothyl on anxiety and nociception in mice.

The main purpose of this study was to compare the effects of solvents from different chemical classes on anxiety and nociception. Independent groups of mice were exposed to air (control group), toluene (1000-4000 ppm), benzene (1000-4000 ppm), 1,1,1-trichloroethane (TCE, 2000-12000 ppm), diethyl ether (10,000-30,000) or flurothyl (200-600 ppm). After a 30-min exposure, animals were tested either in the anxiety paradigm conditioned defensive burying (CDB) test or in the hot plate test. All solvents but flurothyl produced anxiolytic-like actions being the order of potency toluene > benzene > TCE > diethyl ether. When tested in the hot plate paradigm, toluene and TCE increased nociception, benzene and diethyl ether had no effects, and flurothyl decreased nociception Additional groups of mice were conditioned to recognize the aversive stimulus (electrified prod) prior to toluene exposure and then tested in the CDB test. In unconditioned animals, toluene increased the number of shocks that mice received; however, when mice had previous experience in the CDB test, toluene lacked this effect. Taken together, these results show that inhalants have different effects with different potencies both in the CDB and in the hot plate tests. Additionally, data suggest that acute administration of toluene could impair learning.

Acute stress increases calcium current amplitude in rat hippocampus: temporal changes in physiology and gene expression.

Activation of hippocampal glucocorticoid receptors in vitro increases calcium current amplitude through a process requiring DNA binding of receptor homodimers. We here investigated (i). whether similar increased calcium currents also occur following in vivo glucocorticoid receptor activation due to stress and (ii). if so, whether this can be explained by increased expression of calcium channel subunits. Rats were exposed to a novelty stress; some of the animals were pretreated with a glucocorticoid receptor antagonist. In subsequently prepared hippocampal slices, calcium currents were recorded from identified CA1 pyramidal neurons, after which RNA was collected, linearly amplified and hybridized with cDNA clones. Glucocorticoid receptor activation due to novelty exposure was associated with large total peak calcium currents and high-threshold noninactivating currents. Low-threshold calcium currents were not affected. Large total peak and noninactivating current amplitudes were also seen when animals received a more severe stressor, i.e. additional ether exposure. In the stressed groups, the total peak and high-threshold calcium current gradually increased with time resulting in a significant enhancement at >or=3 h after stress exposure. In the same cells, the summated (relative) RNA expression of various alpha1 calcium channel subunits was only transiently enhanced, prior to the functional changes. These data indicate that in vivo activation of glucocorticoid receptors due to stress gradually increases specific calcium current components. Prior to the functional change, increased expression of calcium channel subunits was observed, suggesting that the enhanced function could be explained by transcriptional regulation of the channels.

Effect of non-peptide corticotropin-releasing factor receptor type 1 antagonist on adrenocorticotropic hormone release and interleukin-1 receptors followed by stress.

Previous studies demonstrated that ether-laparotomy significantly increased iodine-125-labeled interleukin-1alpha ([125I]IL-1alpha) binding in the mouse anterior pituitary at 2 h after the onset of stress. Corticotropin-releasing factor (CRF) receptor antagonist, D-Phe CRF (12-41), abolished ether-laparotomy-induced increase in [125I]IL-1alpha binding in the pituitary, showing that CRF plays a pivotal role in the regulation of IL-1 receptors under stress conditions. In an attempt to define the effect of CRA 1000 (2-(N-(2-methylthio-4-isopropylphenyl)-N-ethylamino-4-(4-(3-fluorophenyl)-1,2,3,6-tetrahydropyridin-1-yl)-6-methylpyrimidine), a non-peptide CRF receptor type 1 antagonist on the regulation of hypothalamic-pituitary-adrenal (HPA) axis and IL-1 receptors in the mouse, we measured plasma adrenocorticotropic hormone (ACTH) and corticosterone levels, [125I]IL-1alpha binding and the expression of transcripts for type 1 IL-1 receptor (IL-1R1 mRNA) in the pituitary at 2 h after endotoxin lipopolysaccharide (LPS) treatment or ether-laparotomy stress with or without CRA 1000 pretreatment. A single injection of LPS dramatically increased plasma ACTH and corticosterone levels compared with saline injection. In contrast, plasma ACTH levels were significantly attenuated in response to one LPS injection following oral CRA 1000 pretreatment. LPS-induced plasma corticosterone levels tended to be lower after CRA 1000 pretreatment but it did not reach statistical significance. Ether-laparotomy stress significantly increased plasma ACTH and corticosterone levels at 2 h after the onset of stress and CRA 1000 pretreatment did not affect the peak ACTH and corticosterone levels following stress. Ether-laparotomy stress resulted in a robust increase in [125I]IL-1alpha binding and IL-1R1 mRNA levels in the pituitary. CRA 1000 pretreatment significantly decreased ether-laparotomy stress-induced IL-1R1 mRNA levels but did not affect [125I]IL-1alpha binding. Pretreatment with CRA 1000 without stress significantly increased [125I]IL-1alpha binding and IL-1R1 mRNA levels compared with those in vehicle pretreatment. These data demonstrate differential effects of CRA 1000 in HPA axis following endotoxin and ether-laparotomy stress and complex interactions between CRF and IL-1 receptors during stress.

[The effect of electromagnetic radiation in the millimeter range on the development of disorders in the liver induced by ether anesthesia (experimental research)]. / Vliianie élektromagnitnogo izlucheniia millimetrovogo diapazona voln na razvitie narushenii v pecheni, vyzyvaemykh éfirnym narkozom (éksperimental'noe issledovanie).

Rat experiments with ether anaesthesia (EA) indicate that EA induces enlargement of hepatic sinusoids, stasis and perivascular edema of the triad vessels and other changes in the liver. Right hypochondrium exposure to electromagnetic waves (4.76-5.08 mm) up to 20 days in rats given simultaneously ether anaesthesia showed that livers from the irradiated rats did not differ from control. Thus, millimetric wave radiation may prevent pathological alterations arising in white rat liver after ether anaesthesia.

Characteristics of the proopiomelanocortin system in the outdoor-bred domestic gander. II. Seasonal and circadian rhythmicity; effect of ether stress and lipopolysaccharide administration.

The responsiveness of the POMC system to exogenous stimuli and the diurnal and seasonal rhythmicity of ACTH and beta-endorphin (beta E) in plasma were studied in outdoor-reared domestic ganders. Plasma levels of ACTH- and beta E-like immunoreactivities were determined by direct and specific radioimmunoassays. In the first series of experiments immunoreactive (ir) ACTH and beta E were measured in the plasma of male domestic geese after 5 min of ether stress and after administration of 2 micrograms/kg lipopolysaccharide (LPS). Both ir-ACTH and ir-beta E levels increased 5 and 10 min after ether inhalation, but the increase in the ir-beta E concentration was only half that of the ir-ACTH. The plasma ir-ACTH levels were elevated after 60 and 120 min but not after 90 min of LPS administration: ir-beta E levels were unchanged at all time points. In a second series of experiments blood samples were taken on 30 March. 16 June, 4 August, and 27 October. On these days diurnal samplings were performed at 02:00, 06:00, 10:00, 14:00, 18:00, and 22:00 h. A two-way analysis of variance showed significant diurnal and seasonal changes for both hormones and significant interaction between the diurnal and seasonal levels. The highest daily mean values of the plasma ir-ACTH and ir-beta E concentrations were measured in June. The maximum of the ir-ACTH level was at 10:00 h in March and August, but at 22:00 h in June and October. The changes in ir-beta E concentrations paralleled those of ir-ACTH, but the changes did not reach statistical significance in every case.

Functional observational battery comparing effects of ethanol, 1,1,1-trichloroethane, ether, and flurothyl.

Several recent reports have demonstrated that acute solvent exposure in animals produces a profile of neurobehavioral effects similar to that of classical CNS depressant drugs such as the barbiturates and ethanol. The present investigation further delineated the behavioral pharmacology of three solvents [1,1,1-trichloroethane (TCE), ether, and flurothyl] using a functional observational battery (FOB) composed of 21 qualitative and quantitative measures of behavior. The profiles of acute effects produced by TCE and ether were similar to one another and similar to the profile of effects produced by the IP administration of ethanol. This profile of depressant effects included changes in posture, decreased arousal, disturbances in gait, decreased forelimb grip strength, increased landing foot splay, and impaired psychomotor coordination. Flurothyl exposure also produced dose-related effects on many of the measures in the FOB; however, unlike the depressant vapors, flurothyl did not affect measures of muscle tone and equilibrium such as forelimb grip strength and landing foot splay, or measures of sensorimotor reactivity, including the touch response and tail pinch response. In addition, flurothyl produced handling-induced convulsions in some mice. Recovery from the acute effects of these vapors was rapid and began within minutes of removal from the exposure chamber. These results provide further evidence that exposure to certain solvents produces a profile of reversible effects qualitatively similar to that produced by depressant drugs and alcohol, and that the FOB can be used to compare and contrast profiles of depressant and excitatory effects of inhalants.

Stress through handling for vaginal screening, serotonin, and ACTH response to ether.

The effect of duration of handling for vaginal smear screening on the adrenal weight and acute ACTH response to ether were examined in 4-day-cycling female rats, sacrificed at 97-103 days of age on diestrus-2 after evaluation of resistance to handling, thymus weight, and hypothalamic serotonin (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA). Prolonged handling paralleled increased resistance (behavioral response) to handling and adrenal weight but was inversely related to thymus weight. The hypothalamic 5-HT, 5-HIAA, and 5-HIAA/5-HT ratio, compared to controls with similar conditions of handling, were not modified after 2.5 min of ether despite the ACTH rise. In ether-stressed rats, the ACTH response to ether was lower after prolonged handling compared to short handling paralleling decreased thymus weight. In contrast, 5-HT, 5-HIAA, and the 5-HIAA/5-HT ratio were higher, paralleling increased resistance and adrenal weight. The results suggest chronic activation of the hypothalamo-pituitary-adrenal axis with positive serotonergic involvement after prolonged handling and resistance during vaginal screening and a negative implication of this activation on the acute ACTH response to ether.

Regulation of stress-induced transcriptional changes in the hypothalamic neurosecretory neurons.

Transcriptional changes in corticotropin-releasing factor (CRF) and arginine vasopressin (AVP) gene expression were studied by in situ hybridization histochemistry using cRNA probes directed against intronic sequences. Acute ether stress resulted in a rapid induction of CRF and a delayed activation of vasopressin heteronuclear (hn)RNA in the parvocellular neurosecretory neurons within the paraventricular nucleus (PVN) of the hypothalamus. To explore possible molecular mechanisms regulating stress-related neuropeptide expression in vivo, the time-courses of stress-induced activation of different transcription factor classes were compared to that of changes in neuropeptide transcription. The peak of CRF transcription was parallel to that of cAMP response-element binding protein (CREB) phosphorylation but preceded the induction of c-fos and NGFI-B mRNAs and Fos protein. In contrast, AVP expression occurred in step with immediate-early gene (IEG) responses, suggesting involvement of different mechanisms underlying stress-induced neuropeptide responses. The interference of glucocorticoid hormones with stress-induced neuropeptide and transcription-factor responses has also been revealed in rats acutely or chronically pretreated with glucocorticoids. Acute dexamethasone injection did not prevent neuropeptide and transcription factor responses to either inhalation, whereas chronic corticosterone administration completely blocked IEG and neuropeptide induction in the stress-related neurosecretory neurons.

Effects of antidepressants on thyroid stimulating hormone release in rats under ether stress.

We found inhibitory effects of antidepressants (clomipramine, maprotyline, mianserin and zimelidine) and 5-hydroxytryptophan (5-HTP) on thyroid stimulating hormone (TSH) release induced by ether stress in freely moving rats. We confirmed that ether stress suppressed the plasma TSH levels after 30 min. We then injected intravenously 250 ng thyrotropin releasing hormone (TRH), 0.1 mg/kg clomipramine, 2.5 mg/kg maprotyline, 2.5 mg/kg mianserin, 0.5 mg/kg zimelidine and 25 mg/kg 5-HTP simultaneously. These materials blocked the influences on plasma TSH levels by the ether stress. Serotonergic antidepressants (clomipramine, zimelidine) and 5-HTP (precursor of serotonin) had a higher potency against the ether stress. These results suggest that antagonizing effects against the ether stress may involve the serotonergic system in the pituitary gland.

Serum prolactin response to ether stress in diabetic rats: opiate system contribution.

Diabetes in streptozotocin-treated rats is associated with alterations in various neuroendocrine systems, including endogenous opioids. These changes are suggested to be responsible for the significant reduction in serum prolactin (PRL) response to a brief restraint stress in diabetic male rats, as compared with normoglycemic controls. The present study examines serum PRL response to ether exposure in diabetic male rats. The animals' response to ether stress, which is known to be related to the opioid system, was examined twice in each rat: shortly after cannula insertion (Day 1), and seven days later. In order to evaluate the opiate system involvement, the experiment was repeated on Day 1 and 7 after surgery in a group of rats which were pretreated with naltrexone (Nalt), an opioid receptor antagonist. Opioid receptor sensitization was also performed by prior acute morphine administration on Day 7 after cannulation surgery. Following adaptation to the cannulation, no difference in serum PRL response to ether stress was found between diabetic and normoglycemic rats. However, on Day 1 after surgery, a significant difference was found between the diabetic and control groups: the normoglycemic (control) group exposed to ether responded to the surgical stress by augmented serum PRL levels. This response was not recorded in the diabetic rats. Opioid receptor blockade by Nalt administration 30 min before ether exposure eliminated this difference. Opioid receptor sensitization by morphine pretreatment facilitated PRL secretion in normoglycemic rats exposed to either, while no effect could be distinguished in the diabetic group. It is therefore concluded that the streptozotocin-induced diabetic rats do not differ from normoglycemic ones in their ability to respond to acute ether stress by itself. However, enhanced PRL secretion induced by ether exposure under additional surgical stress, or by presensitization of the opioid receptors by morphine, is prevented in diabetic rats, probably due to diminished opioid receptor response.