ABSTRACT
Organophosphates (OPs), a class of phosphorus-containing chemicals that act by disrupting cholinergic transmission, include both toxic and fast-acting chemical warfare agents as well as less toxic but more easily accessible OP pesticides. The classical atropine/2-PAM antidote fails to protect against long-term symptoms following acute intoxication with OPs at levels that trigger status epilepticus. Acute OP intoxication also causes a robust neuroinflammatory response, which is implicated in the pathogenesis of long-term effects. In this study, we characterized the profiles of lipid mediators, important players in neuroinflammation, in the rat model of acute DFP intoxication. The profiles of lipid mediators were monitored in three different regions of the brain (cortex, hippocampus, and cerebellum) at 0, 1, 3, 7, 14, and 28â¯days post-exposure. The distribution pattern of lipid mediators was distinct in the three brain regions. In the cerebellum, the profile is dominated by LOX metabolites, while the lipid mediator profiles in cortex and hippocampus are dominated by COX metabolites followed by LOX and CYP 450 metabolites. Following acute DFP intoxication, most of the pro-inflammatory lipid mediators (e.g., PGD2 and PGE2) increased rapidly from day 1, while the concentrations of some anti-inflammatory lipid mediators (e.g. 14,15 EpETrE) decreased after DFP intoxication but recovered by day 14 post-exposure. The lipidomics results suggest two potential treatment targets: blocking the formation of prostaglandins by inhibiting COX and stabilizing the anti-inflammatory lipid mediators containing epoxides by inhibiting the enzyme soluble epoxide hydrolase (sEH).
Subject(s)
Brain/drug effects , Brain/metabolism , Cholinesterase Inhibitors/toxicity , Isoflurophate/toxicity , Lipidomics/methods , Organophosphates/toxicity , Animals , Male , Rats , Rats, Sprague-DawleyABSTRACT
Despite intensive effort and resulting gains in understanding the mechanisms underlying neuropathic pain, limited success in therapeutic approaches have been attained. A recently identified, nonchannel, nonneurotransmitter therapeutic target for pain is the enzyme soluble epoxide hydrolase (sEH). The sEH degrades natural analgesic lipid mediators, epoxy fatty acids (EpFAs), therefore its inhibition stabilizes these bioactive mediators. Here we demonstrate the effects of EpFAs on diabetes induced neuropathic pain and define a previously unknown mechanism of pain, regulated by endoplasmic reticulum (ER) stress. The activation of ER stress is first quantified in the peripheral nervous system of type I diabetic rats. We demonstrate that both pain and markers of ER stress are reversed by a chemical chaperone. Next, we identify the EpFAs as upstream modulators of ER stress pathways. Chemical inducers of ER stress invariably lead to pain behavior that is reversed by a chemical chaperone and an inhibitor of sEH. The rapid occurrence of pain behavior with inducers, equally rapid reversal by blockers and natural incidence of ER stress in diabetic peripheral nervous system (PNS) argue for a major role of the ER stress pathways in regulating the excitability of the nociceptive system. Understanding the role of ER stress in generation and maintenance of pain opens routes to exploit this system for therapeutic purposes.
Subject(s)
Diabetic Neuropathies/pathology , Endoplasmic Reticulum Stress , Neuralgia/pathology , Peripheral Nervous System/pathology , Animals , Apoptosis/drug effects , Apoptosis/genetics , Autophagy/drug effects , Autophagy/genetics , Blood Glucose/metabolism , Blotting, Western , Diabetes Mellitus, Experimental/blood , Diabetes Mellitus, Experimental/cerebrospinal fluid , Diabetes Mellitus, Experimental/drug therapy , Diabetes Mellitus, Experimental/pathology , Diabetic Neuropathies/blood , Diabetic Neuropathies/cerebrospinal fluid , Diabetic Neuropathies/drug therapy , Endoplasmic Reticulum Stress/drug effects , Endoplasmic Reticulum Stress/genetics , Enzyme Inhibitors/pharmacology , Epoxide Hydrolases/antagonists & inhibitors , Epoxide Hydrolases/metabolism , Male , Neuralgia/blood , Neuralgia/cerebrospinal fluid , Neuralgia/drug therapy , Peripheral Nervous System/drug effects , Phenylbutyrates/pharmacology , Phenylurea Compounds/pharmacology , Piperidines/pharmacology , RNA, Messenger/genetics , RNA, Messenger/metabolism , Rats, Sprague-Dawley , Skin/pathology , Streptozocin , Tunicamycin/pharmacologyABSTRACT
Tetramethylenedisulfotetramine (TETS, tetramine) is a formerly used and highly neurotoxic rodenticide. Its lethality, recent history of intentional use for mass poisoning, and the absence of a known antidote raise public health concerns. Therefore, rapid, high throughput, and sensitive methods for detection and quantification of TETS are critical. Instrumental analysis method such as GC/MS is sensitive but not rapid or high throughput. Therefore, an immunoassay selective to TETS was developed. The assay shows an IC50 of 4.5 ± 1.2 ng/mL, with a limit of detection of 0.2 ng/mL, comparable to GC/MS. Performance of the immunoassay was demonstrated by a recovery study using known concentrations of TETS spiked into buffer and human and mouse serum matrices giving recoveries in the range of 80-120%. The assay demonstrated good correlation in TETS recovery with established GC/MS analysis. The immunoassay was then used to quantify TETS concentration in the serum of mice exposed to 2× LD50 dose of TETS and to monitor kinetics of TETS clearance from blood over a short period of time. TETS concentration in the serum reached 150 ng/mL without significant change over 4 h post-treatment. Results obtained with the immunoassay had good correlation with GC/MS analysis. Overall, this immunoassay is an important tool to rapidly detect and quantify levels of TETS from biological samples with high sensitivity. The assay can be adapted to multiple formats including field or hospital use.
Subject(s)
Bridged-Ring Compounds/analysis , Immunoassay/methods , Neurotoxins/analysis , Animals , Antibodies/immunology , Bridged-Ring Compounds/blood , Bridged-Ring Compounds/immunology , Haptens/chemistry , Haptens/immunology , Humans , Limit of Detection , Mice , Neurotoxins/blood , Neurotoxins/immunologyABSTRACT
Epoxyeicosatrienoic acids (EETs), metabolites of arachidonic acid derived from the cytochrome P450 enzymes, are mainly metabolized by soluble epoxide hydrolase (sEH) to their corresponding diols. EETs but not their diols, have anti-inflammatory properties, and inhibition of sEH might provide protective effects against inflammatory bone loss. Thus, in the present study, we tested the selective sEH inhibitor, 1-trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl) urea (TPPU), in a mouse model of periodontitis induced by infection with Aggregatibacter actinomycetemcomitans Oral treatment of wild-type mice with TPPU and sEH knockout (KO) animals showed reduced bone loss induced by A. actinomycetemcomitans This was associated with decreased expression of key osteoclastogenic molecules, receptor activator of nuclear factor-κB/RANK ligand/osteoprotegerin, and the chemokine monocyte chemotactic protein 1 in the gingival tissue without affecting bacterial counts. In addition, downstream kinases p38 and c-Jun N-terminal kinase known to be activated in response to inflammatory signals were abrogated after TPPU treatment or in sEH KO mice. Moreover, endoplasmic reticulum stress was elevated in periodontal disease but was abrogated after TPPU treatment and in sEH knockout mice. Together, these results demonstrated that sEH pharmacological inhibition may be of therapeutic value in periodontitis.
Subject(s)
Alveolar Bone Loss/metabolism , Apoptosis/physiology , Endoplasmic Reticulum Stress/physiology , Epoxide Hydrolases/antagonists & inhibitors , Epoxide Hydrolases/metabolism , Receptor Activator of Nuclear Factor-kappa B/metabolism , Alveolar Bone Loss/diagnostic imaging , Alveolar Bone Loss/drug therapy , Animals , Apoptosis/drug effects , Endoplasmic Reticulum Stress/drug effects , Inflammation/diagnostic imaging , Inflammation/drug therapy , Inflammation/metabolism , Inflammation Mediators/antagonists & inhibitors , Inflammation Mediators/metabolism , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Periodontitis/diagnostic imaging , Periodontitis/drug therapy , Periodontitis/metabolism , Phenylurea Compounds/pharmacology , Phenylurea Compounds/therapeutic use , Piperidines/pharmacology , Piperidines/therapeutic use , Signal Transduction/drug effects , Signal Transduction/physiologyABSTRACT
BACKGROUND: Diabetic nephropathy (DN) is the leading cause of renal failure, and podocyte dysfunction contributes to the pathogenesis of DN. Soluble epoxide hydrolase (sEH, encoded by Ephx2) is a conserved cytosolic enzyme whose inhibition has beneficial effects on renal function. The aim of this study is to investigate the contribution of sEH in podocytes to hyperglycemia-induced renal injury. MATERIALS AND METHODS: Mice with podocyte-specific sEH disruption (pod-sEHKO) were generated, and alterations in kidney function were determined under normoglycemia, and high-fat diet (HFD)- and streptozotocin (STZ)-induced hyperglycemia. RESULTS: sEH protein expression increased in murine kidneys under HFD- and STZ-induced hyperglycemia. sEH deficiency in podocytes preserved renal function and glucose control and mitigated hyperglycemia-induced renal injury. Also, podocyte sEH deficiency was associated with attenuated hyperglycemia-induced renal endoplasmic reticulum (ER) stress, inflammation and fibrosis, and enhanced autophagy. Moreover, these effects were recapitulated in immortalized murine podocytes treated with a selective sEH pharmacological inhibitor. Furthermore, pharmacological-induced elevation of ER stress or attenuation of autophagy in immortalized podocytes mitigated the protective effects of sEH inhibition. CONCLUSIONS: These findings establish sEH in podocytes as a significant contributor to renal function under hyperglycemia. GENERAL SIGNIFICANCE: These data suggest that sEH is a potential therapeutic target for podocytopathies.
Subject(s)
Diabetes Mellitus, Experimental/genetics , Diabetic Nephropathies/genetics , Epoxide Hydrolases/genetics , Hyperglycemia/genetics , Animals , Apoptosis/genetics , Autophagy/genetics , Diabetes Mellitus, Experimental/enzymology , Diabetes Mellitus, Experimental/pathology , Diabetic Nephropathies/enzymology , Diabetic Nephropathies/pathology , Endoplasmic Reticulum Stress/genetics , Enzyme Inhibitors/administration & dosage , Epoxide Hydrolases/antagonists & inhibitors , Humans , Hyperglycemia/enzymology , Hyperglycemia/pathology , Kidney/enzymology , Kidney/pathology , Mice , Podocytes/enzymologyABSTRACT
The arachidonic acid cascade is arguably the most widely known biologic regulatory pathway. Decades after the seminal discoveries involving its cyclooxygenase and lipoxygenase branches, studies of this cascade remain an active area of research. The third and less widely known branch, the cytochrome P450 pathway leads to highly active oxygenated lipid mediators, epoxy fatty acids (EpFAs) and hydroxyeicosatetraenoic acids (HETEs), which are of similar potency to prostanoids and leukotrienes. Unlike the COX and LOX branches, no pharmaceuticals currently are marketed targeting the P450 branch. However, data support therapeutic benefits from modulating these regulatory lipid mediators. This is being approached by stabilizing or mimicking the EpFAs or even by altering the diet. These approaches lead to predominantly beneficial effects on a wide range of apparently unrelated states resulting in an enigma of how this small group of natural chemical mediators can have such diverse effects. EpFAs are degraded by soluble epoxide hydrolase (sEH) and stabilized by inhibiting this enzyme. In this review, we focus on interconnected aspects of reported mechanisms of action of EpFAs and inhibitors of soluble epoxide hydrolase (sEHI). The sEHI and EpFAs are commonly reported to maintain homeostasis under pathological conditions while remaining neutral under normal physiological conditions. Here we provide a conceptual framework for the unique and broad range of biological activities ascribed to epoxy fatty acids. We argue that their mechanism of action pivots on their ability to prevent mitochondrial dysfunction, to reduce subsequent ROS formation and to block resulting cellular signaling cascades, primarily the endoplasmic reticulum stress. By stabilizing the mitochondrial - ROS - ER stress axis, the range of activity of EpFAs and sEHI display an overlap with the disease conditions including diabetes, fibrosis, chronic pain, cardiovascular and neurodegenerative diseases, for which the above outlined mechanisms play key roles.
Subject(s)
Endoplasmic Reticulum Stress/drug effects , Enzyme Inhibitors/pharmacology , Epoxide Hydrolases/antagonists & inhibitors , Epoxide Hydrolases/chemistry , Fatty Acids/chemistry , Fatty Acids/pharmacology , Mitochondria/drug effects , Animals , Epoxide Hydrolases/metabolism , Humans , Mitochondria/metabolism , SolubilityABSTRACT
Proton pump inhibitors such as omeprazole (OME) reduce the severity of gastrointestinal (GI) ulcers induced by nonsteroidal anti-inflammatory drugs (NSAIDs) but can also increase the chance of dysbiosis. The aim of this study was to test the hypothesis that preventive use of a soluble epoxide hydrolase inhibitor (sEHI) such as TPPU can decrease NSAID-induced ulcers by increasing anti-inflammatory epoxyeicosatrienoic acids (EETs). Dose- [10, 30, and 100 mg/kg, by mouth (PO)] and time-dependent (6 and 18 hours) ulcerative effects of diclofenac sodium (DCF, an NSAID) were studied in the small intestine of Swiss Webster mice. Dose-dependent effects of TPPU (0.001-0.1 mg/kg per day for 7 days, in drinking water) were evaluated in DCF-induced intestinal toxicity and compared with OME (20 mg/kg, PO). In addition, the effect of treatment was studied on levels of Hb in blood, EETs in plasma, inflammatory markers such as myeloperoxidase (MPO) in intestinal tissue homogenates, and tissue necrosis factor-α (TNF-α) in serum. DCF dose dependently induced ulcers that were associated with both a significant (P < 0.05) loss of Hb and an increase in the level of MPO and TNF-α, with severity of ulceration highest at 18 hours. Pretreatment with TPPU dose dependently prevented ulcer formation by DCF, increased the levels of epoxy fatty acids, including EETs, and TPPU's efficacy was comparable to OME. TPPU significantly (P < 0.05) reversed the effect of DCF on the level of Hb, MPO, and TNF-α Thus sEHI might be useful in the management of NSAID-induced ulcers.
Subject(s)
Diclofenac/adverse effects , Epoxide Hydrolases/antagonists & inhibitors , Intestines/drug effects , Phenylurea Compounds/chemistry , Phenylurea Compounds/pharmacology , Piperidines/chemistry , Piperidines/pharmacology , Ulcer/chemically induced , Ulcer/drug therapy , Animals , Anti-Inflammatory Agents, Non-Steroidal/adverse effects , Cytoprotection/drug effects , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Enzyme Inhibitors/therapeutic use , Gene Knockout Techniques , Intestinal Mucosa/metabolism , Intestines/pathology , Male , Mice , Peroxidase/metabolism , Phenylurea Compounds/therapeutic use , Piperidines/therapeutic use , Solubility , Tumor Necrosis Factor-alpha/blood , Ulcer/metabolism , Ulcer/pathologyABSTRACT
Epoxyeicosatrienoic acids (EETs), lipid mediators produced by cytochrome P450 epoxygenases, regulate inflammation, angiogenesis, and vascular tone. Despite pleiotropic effects on cells, the role of these epoxyeicosanoids in normal organ and tissue regeneration remains unknown. EETs are produced predominantly in the endothelium. Normal organ and tissue regeneration require an active paracrine role of the microvascular endothelium, which in turn depends on angiogenic growth factors. Thus, we hypothesize that endothelial cells stimulate organ and tissue regeneration via production of bioactive EETs. To determine whether endothelial-derived EETs affect physiologic tissue growth in vivo, we used genetic and pharmacological tools to manipulate endogenous EET levels. We show that endothelial-derived EETs play a critical role in accelerating tissue growth in vivo, including liver regeneration, kidney compensatory growth, lung compensatory growth, wound healing, corneal neovascularization, and retinal vascularization. Administration of synthetic EETs recapitulated these results, whereas lowering EET levels, either genetically or pharmacologically, delayed tissue regeneration, demonstrating that pharmacological modulation of EETs can affect normal organ and tissue growth. We also show that soluble epoxide hydrolase inhibitors, which elevate endogenous EET levels, promote liver and lung regeneration. Thus, our observations indicate a central role for EETs in organ and tissue regeneration and their contribution to tissue homeostasis.
Subject(s)
Eicosanoids/pharmacology , Endothelial Cells/metabolism , Epoxy Compounds/pharmacology , Neovascularization, Physiologic/physiology , Regeneration/physiology , Animals , Chromatography, Liquid , Eicosanoids/metabolism , Epoxide Hydrolases/antagonists & inhibitors , Epoxy Compounds/metabolism , Eye/blood supply , Immunohistochemistry , Kidney/physiology , Liver/physiology , Lung/physiology , Mice , Mice, Transgenic , Neovascularization, Physiologic/drug effects , Receptor, TIE-2/genetics , Regeneration/drug effects , Tandem Mass SpectrometryABSTRACT
Epoxyeicosatrienoic acids (EETs) are potent endogenous analgesic metabolites produced from arachidonic acid by cytochrome P450s (P450s). Metabolism of EETs by soluble epoxide hydrolase (sEH) reduces their activity, while their stabilization by sEH inhibition decreases both inflammatory and neuropathic pain. Here, we tested the complementary hypothesis that increasing the level of EETs through induction of P450s by omeprazole (OME), can influence pain related signaling by itself, and potentiate the anti-hyperalgesic effect of sEH inhibitor. Rats were treated with OME (100mg/kg/day, p.o., 7 days), sEH inhibitor TPPU (3mg/kg/day, p.o.) and OME (100mg/kg/day, p.o., 7 days)+TPPU (3mg/kg/day, p.o., last 3 days of OME dose) dissolved in vehicle PEG400, and their effect on hyperalgesia (increased sensitivity to pain) induced by PGE2 was monitored. While OME treatment by itself exhibited variable effects on PGE2 induced hyperalgesia, it strongly potentiated the effect of TPPU in the same assay. The significant decrease in pain with OME+TPPU treatment correlated with the increased levels of EETs in plasma and increased activities of P450 1A1 and P450 1A2 in liver microsomes. The results show that reducing catabolism of EETs with a sEH inhibitor yielded a stronger analgesic effect than increasing generation of EETs by OME, and combination of both yielded the strongest pain reducing effect under the condition of this study.
Subject(s)
Analgesics/pharmacology , Dinoprostone/metabolism , Enzyme Inhibitors/pharmacology , Epoxide Hydrolases/antagonists & inhibitors , Epoxy Compounds/pharmacology , Omeprazole/pharmacology , Pain/drug therapy , Animals , Cytochrome P-450 Enzyme System/metabolism , Epoxide Hydrolases/metabolism , Hyperalgesia/metabolism , Male , Microsomes, Liver/enzymology , Microsomes, Liver/metabolism , Pain/metabolism , Phenylurea Compounds/pharmacology , Piperidines/pharmacology , Rats , Rats, Sprague-DawleyABSTRACT
We need better medicines to control acute and chronic pain. Fatty acid amide hydrolase (FAAH) and soluble epoxide hydrolase (sEH) catalyze the deactivating hydrolysis of two classes of bioactive lipid mediators--fatty acid ethanolamides (FAEs) and epoxidized fatty acids (EpFAs), respectively--which are biogenetically distinct but share the ability to attenuate pain responses and inflammation. In these experiments, we evaluated the antihyperalgesic activity of small-molecule inhibitors of FAAH and sEH, administered alone or in combination, in two pain models: carrageenan-induced hyperalgesia in mice and streptozocin-induced allodynia in rats. When administered separately, the sEH inhibitor 1-trifluoromethoxyphenyl-3-(1-propionylpiperidine-4-yl)urea (TPPU) and the peripherally restricted FAAH inhibitor URB937 were highly active in the two models. The combination TPPU plus URB937 was markedly synergistic, as assessed using isobolographic analyses. The results of these experiments reveal the existence of a possible functional crosstalk between FAEs and EpFAs in regulating pain responses. Additionally, the results suggest that combinations of sEH and FAAH inhibitors might be exploited therapeutically to achieve greater analgesic efficacy.
Subject(s)
Amidohydrolases/antagonists & inhibitors , Analgesics/pharmacology , Enzyme Inhibitors/pharmacology , Epoxide Hydrolases/antagonists & inhibitors , Animals , Cannabinoids/therapeutic use , Carrageenan , Diabetic Neuropathies/complications , Diabetic Neuropathies/drug therapy , Drug Synergism , Hyperalgesia/chemically induced , Hyperalgesia/drug therapy , Male , Mice , Pain Measurement/drug effects , Phenylurea Compounds/therapeutic use , Piperidines/therapeutic use , Rats , Rats, Sprague-Dawley , Small Molecule Libraries , StreptozocinABSTRACT
Niacin is effective in treating dyslipidemias but causes cutaneous vasodilation or flushing, a side effect that limits its clinical use. Blocking prostaglandins in humans reduces but does not consistently eliminate flushing, indicating additional mechanisms may contribute to flushing. The transient receptor potential vanilloid 1 (TRPV1) channel, when activated, causes cutaneous vasodilation and undergoes tachyphylaxis similar to that seen with niacin. Using a murine model, early phase niacin-induced flushing was examined and TRPV1 channel involvement demonstrated using pharmacologic blockade, desensitization, and genetic knockouts (TRPV1 KO). The TRPV1 antagonist AMG9810 reduced the magnitude of the initial and secondary peaks and the rapidity of the vasodilatory response (slope). TRPV1 desensitization by chronic capsaicin reduced the initial peak and slope. TRPV1 KO mice had a lower initial peak, secondary peak, and slope compared with wild-type mice. Chronic niacin reduced the initial peak, secondary peak, and slope in wild-type mice but had no effect in knockout mice. Furthermore, chronic niacin diminished the response to capsaicin in wild-type mice. Overall, these data demonstrate an important role for TRPV1 channels in niacin-induced flushing, both in the acute response and with chronic administration. That niacin-induced flushing is a complex cascade of events, which should inform pharmacological intervention against this side effect.
Subject(s)
Flushing , Niacin/pharmacology , TRPV Cation Channels/metabolism , Vasodilation/drug effects , Acrylamides/pharmacology , Animals , Bridged Bicyclo Compounds, Heterocyclic/pharmacology , Capsaicin/pharmacology , Disease Models, Animal , Flushing/chemically induced , Flushing/metabolism , Mice , Mice, Knockout , Sensory System Agents/pharmacology , TRPV Cation Channels/antagonists & inhibitors , Vasodilator Agents/pharmacologyABSTRACT
The nerve damage occurring as a consequence of glucose toxicity in diabetes leads to neuropathic pain, among other problems. This pain dramatically reduces the quality of life in afflicted patients. The progressive damage to the peripheral nervous system is irreversible although strict control of hyperglycemia may prevent further damage. Current treatments include tricyclic antidepressants, anticonvulsants, and opioids, depending on the severity of the pain state. However, available therapeutics have drawbacks, arguing for the need to better understand the pathophysiology of neuropathic pain and develop novel treatments. Here we demonstrate that stabilization of a class of bioactive lipids, epoxygenated fatty acids (EpFAs), greatly reduces allodynia in rats caused by streptozocin-induced type I diabetes. Inhibitors of the soluble epoxide hydrolase (sEHI) elevated and stabilized the levels of plasma and spinal EpFAs, respectively, and generated dose-dependent antiallodynic effects more potently and efficaciously than gabapentin. In acute experiments, positive modulation of EpFAs did not display differences in insulin sensitivity, glucose tolerance, or insulin secretion, indicating the efficacy of sEHIs are not related to the glycemic status. Quantitative metabolomic analysis of a panel of 26 bioactive lipids demonstrated that sEHI-mediated antiallodynic effects coincided with a selective elevation of the levels of EpFAs in the plasma, and a decrease in degradation products coincided with the dihydroxy fatty acids in the spinal cord. Overall, these results argue that further efforts in understanding the spectrum of effects of EpFAs will yield novel opportunities in treating neuropathic pain.
Subject(s)
Diabetes Mellitus, Experimental/drug therapy , Diabetes Mellitus, Type 1/drug therapy , Fatty Acids/metabolism , Hyperalgesia/drug therapy , Pain/drug therapy , Amines/pharmacology , Animals , Behavior, Animal , Cyclohexanecarboxylic Acids/pharmacology , Diabetes Mellitus, Experimental/metabolism , Diabetes Mellitus, Type 1/metabolism , Disease Models, Animal , Drug Design , Gabapentin , Glucose/metabolism , Insulin/metabolism , Lipids/therapeutic use , Male , Models, Chemical , Pain/metabolism , Rats , Rats, Sprague-Dawley , Spinal Cord/metabolism , gamma-Aminobutyric Acid/pharmacologyABSTRACT
Tetramethylenedisulfotetramine (TETS) is a potent convulsant poison for which there is currently no approved antidote. The convulsant action of TETS is thought to be mediated by inhibition of type A gamma-aminobutyric acid receptor (GABAAR) function. We, therefore, investigated the effects of post-exposure administration of diazepam, a GABAAR positive allosteric modulator, on seizure activity, death and neuroinflammation in adult male Swiss mice injected with a lethal dose of TETS (0.15mg/kg, ip). Administration of a high dose of diazepam (5mg/kg, ip) immediately following the second clonic seizure (approximately 20min post-TETS injection) effectively prevented progression to tonic seizures and death. However, this treatment did not prevent persistent reactive astrogliosis and microglial activation, as determined by GFAP and Iba-1 immunoreactivity and microglial cell morphology. Inhibition of soluble epoxide hydrolase (sEH) has been shown to exert potent anti-inflammatory effects and to increase survival in mice intoxicated with other GABAAR antagonists. The sEH inhibitor TUPS (1mg/kg, ip) administered immediately after the second clonic seizure did not protect TETS-intoxicated animals from tonic seizures or death. Combined administration of diazepam (5mg/kg, ip) and TUPS (1mg/kg, ip, starting 1h after diazepam and repeated every 24h) prevented TETS-induced lethality and influenced signs of neuroinflammation in some brain regions. Significantly decreased microglial activation and enhanced reactive astrogliosis were observed in the hippocampus, with no changes in the cortex. Combining an agent that targets specific anti-inflammatory mechanisms with a traditional antiseizure drug may enhance treatment outcome in TETS intoxication.
Subject(s)
Anti-Inflammatory Agents/administration & dosage , Anticonvulsants/administration & dosage , Brain/drug effects , Bridged-Ring Compounds , Diazepam/administration & dosage , Encephalitis/prevention & control , Enzyme Inhibitors/administration & dosage , Epoxide Hydrolases/antagonists & inhibitors , GABA Modulators/administration & dosage , Phenylurea Compounds/administration & dosage , Piperidines/administration & dosage , Seizures/prevention & control , Animals , Brain/enzymology , Brain/physiopathology , Brain Waves/drug effects , Disease Models, Animal , Drug Administration Schedule , Drug Therapy, Combination , Electroencephalography , Encephalitis/chemically induced , Encephalitis/enzymology , Encephalitis/physiopathology , Epoxide Hydrolases/metabolism , Male , Mice , Seizures/chemically induced , Seizures/enzymology , Seizures/physiopathology , Time FactorsABSTRACT
Lipid derived mediators contribute to inflammation and the sensing of pain. The contributions of omega-6 derived prostanoids in enhancing inflammation and pain sensation are well known. Less well explored are the opposing anti-inflammatory and analgesic effects of the omega-6 derived epoxyeicosatrienoic acids. Far less has been described about the epoxidized metabolites derived from omega-3 long chain fatty acids. The epoxide metabolites are turned over rapidly with enzymatic hydrolysis by the soluble epoxide hydrolase being the major elimination pathway. Despite this, the overall understanding of the role of lipid mediators in the pathology of chronic pain is growing. Here, we review the role of long chain fatty acids and their metabolites in alleviating both acute and chronic pain conditions. We focus specifically on the epoxidized metabolites of omega-6 and omega-3 long chain fatty acids as well as a novel strategy to modulate their activity in vivo.
Subject(s)
Epoxy Compounds/metabolism , Fatty Acids, Omega-3/metabolism , Fatty Acids, Omega-6/metabolism , Inflammation/metabolism , Nociception/physiology , Nociceptive Pain/metabolism , Anti-Inflammatory Agents/metabolism , Epoxide Hydrolases/metabolism , Humans , Signal TransductionABSTRACT
Pain is a major health concern even though numerous analgesic agents are available. Side effects and lack of wide-spectrum efficacy of current drugs justify efforts to better understand pain mechanisms. Stabilization of natural epoxy-fatty acids (EFAs) through inhibition of the soluble epoxide hydrolase (sEH) reduces pain. However, in the absence of an underlying painful state, inhibition of sEH is ineffective. Surprisingly, a pain-mediating second messenger, cAMP, interacts with natural EFAs and regulates the analgesic activity of sEH inhibitors. Concurrent inhibition of sEH and phosphodiesterase (PDE) dramatically reduced acute pain in rodents. Our findings demonstrate a mechanism of action of cAMP and EFAs in the pathophysiology of pain. Furthermore, we demonstrate that inhibition of various PDE isozymes, including PDE4, lead to significant increases in EFA levels through a mechanism independent of sEH, suggesting that the efficacy of commercial PDE inhibitors could result in part from increasing EFAs. The cross-talk between the two major pathways-one mediated by cAMP and the other by EFAs-paves the way to new approaches to understand and control pain.
Subject(s)
Analgesia , Analgesics/pharmacology , Cyclic AMP/metabolism , Enzyme Inhibitors/pharmacology , Epoxide Hydrolases/antagonists & inhibitors , Pain , Second Messenger Systems/drug effects , Animals , Cyclic Nucleotide Phosphodiesterases, Type 4/metabolism , Epoxy Compounds/metabolism , Male , Pain/drug therapy , Pain/metabolism , Pain/physiopathology , Rats , Rats, Sprague-DawleyABSTRACT
Tetramethylenedisulfotetramine (tetramine; TETS) is a potent convulsant poison that is considered to be a chemical threat agent. To provide a basis for the investigation of antidotes for TETS-induced seizures, we characterized the convulsant activity of TETS in mice and rats when administered by the intraperitoneal, intravenous, oral, and intraventricular routes as a single acute dose and with repeated sublethal doses. In mice, parenteral and oral TETS caused immobility, myoclonic body jerks, clonic seizures of the forelimbs and/or hindlimbs, tonic seizures, and death. The CD50 values for clonic and tonic seizures after oral administration were 0.11 and 0.22 mg/kg, respectively. Intraventricular administration of TETS (5-100 µg) in rats also caused clonic-tonic seizures and death. In mice, repeated sublethal doses of TETS at intervals of 2, 24, and 48 h failed to result in the development of persistent enhanced seizure responsivity ("kindling") as was observed with repeated pentylenetetrazol treatment. In mice, sublethal doses of TETS that produced clonic seizures did not cause observable structural brain damage as assessed with routine histology and Fluoro-Jade B staining 7 days after treatment. However, 1 to 3 days after a single convulsant dose of TETS the expression of glial fibrillary acidic protein, an astrocyte marker, and ionized calcium binding adaptor molecule 1, a microglia marker, were markedly increased in cortex and hippocampus. Although TETS doses that are compatible with survival are not associated with overt evidence of cellular injury or neurodegeneration, there is transient reactive astrocytosis and microglial activation, indicating that brain inflammatory responses are provoked.
Subject(s)
Bridged-Ring Compounds/toxicity , Convulsants/toxicity , Seizures/chemically induced , Seizures/metabolism , Animals , Astrocytes/drug effects , Astrocytes/metabolism , Calcium-Binding Proteins/metabolism , Cerebral Cortex/drug effects , Cerebral Cortex/metabolism , Extremities , Glial Fibrillary Acidic Protein/metabolism , Gliosis/chemically induced , Gliosis/metabolism , Hippocampus/drug effects , Hippocampus/metabolism , Male , Mice , Microfilament Proteins/metabolism , Microglia/drug effects , Microglia/metabolism , Pentylenetetrazole/pharmacology , Picrotoxin/adverse effects , Rats , Rats, Sprague-DawleyABSTRACT
It has recently been reported that soluble epoxide hydrolase (sEH), the major enzyme that metabolizes epoxyeicosatrienoic acids (EETs), is expressed in axons of cortical neurons; however, the functional relevance of axonal sEH localization is unknown. Immunocytochemical analyses demonstrate predominant axonal localization of sEH in primary cultures of not only cortical but also sympathetic and sensory neurons. Morphometric analyses of cultured sensory neurons indicate that exposure to a regioisomeric mixture of EETs (0.01-1.0 µM) causes a concentration-dependent increase in axon outgrowth. This axon promoting activity is not a generalized property of all regioisomers of EETs as axonal growth is enhanced in sensory neurons exposed to 14,15-EET but not 8,9- or 11,12-EET. 14,15-EET also promotes axon outgrowth in cultured cortical neurons. Co-exposure to EETs and either of two structurally diverse pharmacological inhibitors of sEH potentiates the axon-enhancing activity of EETs in sensory and cortical neurons. Mass spectrometry indicates that sEH inhibition significantly increases EETs and significantly decreases dihydroxyeicosatrienoic acid metabolites in neuronal cell cultures. These data indicate that EETs enhance axon outgrowth and suggest that axonal sEH activity regulates EETs-induced axon outgrowth. These findings suggest a novel therapeutic use of sEH inhibitors in promoting nerve regeneration.
Subject(s)
Axons/physiology , Cerebral Cortex/physiology , Eicosanoic Acids/pharmacology , Sensory Receptor Cells/physiology , Animals , Cell Count , Cell Survival/drug effects , Cells, Cultured , Cerebral Cortex/cytology , Cerebral Cortex/drug effects , Dose-Response Relationship, Drug , Epoxide Hydrolases/metabolism , Female , Ganglia, Spinal/cytology , Ganglia, Spinal/drug effects , Immunohistochemistry , Neurons/drug effects , Pregnancy , Rats , Rats, Sprague-Dawley , Sensory Receptor Cells/drug effects , Subcellular Fractions/metabolism , Sympathetic Nervous System/cytology , Sympathetic Nervous System/drug effectsABSTRACT
The soluble epoxide hydrolase (sEH) enzyme regulates the levels of endogenous epoxygenated fatty acid (EFA) lipid metabolites by rapidly degrading these molecules. The EFAs have pleiotropic biological activities including the modulation of nociceptive signaling. Recent findings indicate that the EFAs, in particular the arachidonic acid (AA) derived epoxyeicosatrienoic acids (EETs), the docosahexaenoic acid (DHA) derived epoxydocosapentaenoic acids (EpDPEs) and eicosapentaenoic acid (EPA) derived epoxyeicosatetraenoic acids (EpETEs) are natural signaling molecules. The tight regulation of these metabolites speaks to their importance in regulating biological functions. In the past several years work on EFAs in regard to their activities in the nervous system evolved to demonstrate that these molecules are anti-inflammatory and anti-nociceptive. Here we focus on the recent advances in understanding the effects of sEH inhibition and increased EFAs on the nociceptive system and their ability to reduce pain. Evidence of their role in modulating pain signaling is given by their direct application and by inhibiting their degradation in various models of pain. Moreover, there is mounting evidence of EFAs role in the crosstalk between major nociceptive and anti-nociceptive systems which is reviewed herein. Overall the fundamental knowledge generated within the past decade indicates that orally bioavailable small molecule inhibitors of sEH may find a place in the treatment of a number of diverse painful conditions including inflammatory and neuropathic pain.
Subject(s)
Analgesics, Non-Narcotic/pharmacology , Anti-Inflammatory Agents/pharmacology , Epoxide Hydrolases/antagonists & inhibitors , Hyperalgesia/drug therapy , Hyperalgesia/metabolism , Inflammation/drug therapy , Inflammation/metabolism , Pain/drug therapy , 8,11,14-Eicosatrienoic Acid/analogs & derivatives , 8,11,14-Eicosatrienoic Acid/metabolism , 8,11,14-Eicosatrienoic Acid/pharmacology , Analgesics, Non-Narcotic/metabolism , Animals , Anti-Inflammatory Agents/metabolism , Arachidonic Acid/metabolism , Cyclooxygenase 2/metabolism , Docosahexaenoic Acids/metabolism , Docosahexaenoic Acids/pharmacology , Eicosapentaenoic Acid/metabolism , Eicosapentaenoic Acid/pharmacology , Enzyme Inhibitors/pharmacology , Epoxide Hydrolases/metabolism , Humans , Hyperalgesia/physiopathology , Inflammation/physiopathology , Mice , Mice, Knockout , Nervous System/drug effects , Nervous System/metabolism , Nervous System/physiopathology , Nociception/drug effects , Pain/metabolism , Pain/physiopathology , Rats , Rats, Sprague-Dawley , Signal Transduction/drug effectsABSTRACT
The antibacterial soap additive triclocarban (TCC) is widely used in personal care products. TCC has a high environmental persistence. We developed and validated a sensitive online solid-phase extraction-LC-MS/MS method to rapidly analyze TCC and its major metabolites in urine and other biological samples to assess human exposure. We measured human urine concentrations 0-72 h after showering with a commercial bar soap containing 0.6% TCC. The major route of renal elimination was excretion as N-glucuronides. The absorption was estimated at 0.6% of the 70±15 mg of TCC in the soap used. The TCC-N-glucuronide urine concentration varied widely among the subjects, and continuous daily use of the soap led to steady state levels of excretion. In order to assess potential biological effects arising from this exposure, we screened TCC for the inhibition of human enzymes in vitro. We demonstrate that TCC is a potent inhibitor of the enzyme soluble epoxide hydrolase (sEH), whereas TCC's major metabolites lack strong inhibitory activity. Topical administration of TCC at similar levels to rats in a preliminary in vivo study, however, failed to alter plasma biomarkers of sEH activity. Overall the analytical strategy described here revealed that use of TCC soap causes exposure levels that warrant further evaluation.
Subject(s)
Anti-Infective Agents, Local/analysis , Baths/statistics & numerical data , Carbanilides/analysis , Environmental Exposure/statistics & numerical data , Water Pollutants, Chemical/analysis , Animals , Anti-Infective Agents, Local/metabolism , Anti-Infective Agents, Local/toxicity , Carbanilides/metabolism , Carbanilides/toxicity , Chromatography, Liquid , Environmental Exposure/analysis , Humans , Male , Rats , Solid Phase Extraction , Tandem Mass Spectrometry , Water Pollutants, Chemical/metabolism , Water Pollutants, Chemical/toxicityABSTRACT
During inflammation, a large amount of arachidonic acid (AA) is released into the cellular milieu and cyclooxygenase enzymes convert this AA to prostaglandins that in turn sensitize pain pathways. However, AA is also converted to natural epoxyeicosatrienoic acids (EETs) by cytochrome P450 enzymes. EET levels are typically regulated by soluble epoxide hydrolase (sEH), the major enzyme degrading EETs. Here we demonstrate that EETs or inhibition of sEH lead to antihyperalgesia by at least 2 spinal mechanisms, first by repressing the induction of the COX2 gene and second by rapidly up-regulating an acute neurosteroid-producing gene, StARD1, which requires the synchronized presence of elevated cAMP and EET levels. The analgesic activities of neurosteroids are well known; however, here we describe a clear course toward augmenting the levels of these molecules. Redirecting the flow of pronociceptive intracellular cAMP toward up-regulation of StARD1 mRNA by concomitantly elevating EETs is a novel path to accomplish pain relief in both inflammatory and neuropathic pain states.