Neuroprotective and neurotoxic effects of endocannabinoid-like compounds, N-arachidonoyl dopamine and N-docosahexaenoyl dopamine in differentiated cultures of induced pluripotent stem cells derived from patients with Parkinson's disease.

The prominent protective effects in diverse neuron injury paradigms exerted by cannabinoids and in particular their endogenously produced species render the endocannabinoid system a promising molecular target in the treatment of neurodegenerative diseases. However, the effects of individual endocannabinoids in human cells remain poorly investigated. Neural derivatives of human induced pluripotent stem cells (iPSC) offer unique opportunities for studying the neuroprotective compounds and development of patient-specific treatment. For the first time the cytotoxic and neuroprotective effects endocannabinoids N-arachidonoyl dopamine (N-ADA) and N-docosahexaenoyl dopamine (N-DDA) were assessed in human neural progenitors and dopamine neurons derived from iPSCs of healthy donors and patients with Parkinson's disease. While the short-term treatment with the investigated compounds in 0.1-10 µM concentration range exerted no toxicity in these cell types, the long-term exposure to 0.1-5 µM N-ADA or N-DDA reduced the survival of human neural progenitors. At the same time, both N-ADA and N-DDA protected neural progenitors and terminally differentiated neurons both from healthy donors and patients with Parkinson's disease against oxidative stress induced by hydrogen peroxide. The observed dramatic difference in the mode of action of N-acyl dopamines points on the possible existence of novel pathogenic mechanism of neurodegeneration induced by prolonged uncompensated production of these substances within neuronal tissue and should also be considered as a precaution in the future development of N-acyl dopamine-based therapeutic drugs.

Biophysical and biomechanical properties of neural progenitor cells as indicators of developmental neurotoxicity.

Conventional in vitro toxicity studies have focused on identifying IC50 and the underlying mechanisms, but how toxicants influence biophysical and biomechanical changes in human cells, especially during developmental stages, remain understudied. Here, using an atomic force microscope, we characterized changes in biophysical (cell area, actin organization) and biomechanical (Young's modulus, force of adhesion, tether force, membrane tension, tether radius) aspects of human fetal brain-derived neural progenitor cells (NPCs) induced by four classes of widely used toxic compounds, including rotenone, digoxin, N-arachidonoylethanolamide (AEA), and chlorpyrifos, under exposure up to 36 h. The sub-cellular mechanisms (apoptosis, mitochondria membrane potential, DNA damage, glutathione levels) by which these toxicants induced biochemical changes in NPCs were assessed. Results suggest a significant compromise in cell viability with increasing toxicant concentration (p < 0.01), and biophysical and biomechanical characteristics with increasing exposure time (p < 0.01) as well as toxicant concentration (p < 0.01). Impairment of mitochondrial membrane potential appears to be the most sensitive mechanism of neurotoxicity for rotenone, AEA and chlorpyrifos exposure, but compromise in plasma membrane integrity for digoxin exposure. The surviving NPCs remarkably retained stemness (SOX2 expression) even at high toxicant concentrations. A negative linear correlation (R2 = 0.92) exists between the elastic modulus of surviving cells and the number of living cells in that environment. We propose that even subtle compromise in cell mechanics could serve as a crucial marker of developmental neurotoxicity (mechanotoxicology) and therefore should be included as part of toxicology assessment repertoire to characterize as well as predict developmental outcomes.

2-Arachidonoylglycerol mobilizes myeloid cells and worsens heart function after acute myocardial infarction.

AIMS: Myocardial infarction (MI) leads to an enhanced release of endocannabinoids and a massive accumulation of neutrophils and monocytes within the ischaemic myocardium. These myeloid cells originate from haematopoietic precursors in the bone marrow and are rapidly mobilized in response to MI. We aimed to determine whether endocannabinoid signalling is involved in myeloid cell mobilization and cardiac recruitment after ischaemia onset. METHODS AND RESULTS: Intravenous administration of endocannabinoid 2-arachidonoylglycerol (2-AG) into wild type (WT) C57BL6 mice induced a rapid increase of blood neutrophil and monocyte counts as measured by flow cytometry. This effect was blunted when using cannabinoid receptor 2 knockout mice. In response to MI induced in WT mice, the lipidomic analysis revealed significantly elevated plasma and cardiac levels of the endocannabinoid 2-AG 24 h after infarction, but no changes in anandamide, palmitoylethanolamide, and oleoylethanolamide. This was a consequence of an increased expression of 2-AG synthesizing enzyme diacylglycerol lipase and a decrease of metabolizing enzyme monoacylglycerol lipase (MAGL) in infarcted hearts, as determined by quantitative RT-PCR analysis. The opposite mRNA expression pattern was observed in bone marrow. Pharmacological blockade of MAGL with JZL184 and thus increased systemic 2-AG levels in WT mice subjected to MI resulted in elevated cardiac CXCL1, CXCL2, and MMP9 protein levels as well as higher cardiac neutrophil and monocyte counts 24 h after infarction compared with vehicle-treated mice. Increased post-MI inflammation in these mice led to an increased infarct size, an impaired ventricular scar formation assessed by histology and a worsened cardiac function in echocardiography evaluations up to 21 days. Likewise, JZL184-administration in a myocardial ischaemia-reperfusion model increased cardiac myeloid cell recruitment and resulted in a larger fibrotic scar size. CONCLUSION: These findings suggest that changes in endocannabinoid gradients due to altered tissue levels contribute to myeloid cell recruitment from the bone marrow to the infarcted heart, with crucial consequences on cardiac healing and function.

Effect of Cannabinoid Receptor Agonists on Isolated Rat Atria.

Cannabinoid CB2 receptor agonists are under investigation for clinical use. At the same time, synthetic cannabinoids have been implicated in a number of deaths. One cause of death is thought to be cardiac arrest subsequent to extreme tachycardia. Central mechanisms are thought to play a role in this, with CB1 but not CB2 receptors thought to mediate central effects. However, the direct effects of cannabinoids on the heart are less well understood. We therefore tested the effects of cannabinoids on isolated rat atria to test whether activation of myocardial CB1 and CB2 receptors could contribute to tachycardia. Although we found a moderate effect that can be attributed to CB1 receptors, we did not find any evidence for chronotropic effects by a CB2 receptor activation. Our results indicate that cannabinoid cardiotoxicity may partially involve CB1 receptors in the myocardium, and that CB2 receptor agonists are unlikely to have significant effects on the heart.

Detrimental effects of 2-arachidonoylglycerol on whole blood platelet aggregation and on cerebral blood flow after a focal ischemic insult in rats.

2-Arachidonoylglycerol (2-AG) is a major modulator of blood flow and platelet aggregation and a potential neuroprotectant. The present study investigated, for the first time, the effects of 2-AG on cerebral blood flow (CBF) in the first critical hours during middle cerebral artery occlusion (MCAO) and on platelet aggregation in rats. Adult male Sprague-Dawley rats ( n = 30) underwent permanent MCAO under isoflurane anesthesia and were randomly assigned to receive either 2-AG (6 mg/kg iv), monoacylglycerol lipase inhibitor JZL-184 (10 mg/kg iv), or vehicle ( n = 6 rats/group) treatment. CBF and cardiovascular responses were measured, by a blinded investigator, for up to 4 h. In separate experiments, platelet aggregation by 2-AG (19-300 µM) was assessed by whole blood aggregometry ( n = 40). 2-AG and JZL-184 significantly increased the severity of the CBF deficit versus vehicle (20.2 ± 8.8% and 22.7 ± 6.4% vs. 56.4 ± 12.1% of pre-MCAO baseline, respectively, P < 0.05) but had no effect on blood pressure or heart rate. While JZL-184 significantly increased the number of thrombi after MCAO, this did not reach significance by 2-AG. 2-AG induced platelet aggregation in rat whole blood in a similar manner to arachidonic acid and was significantly reduced by the cyclooxygenase inhibitors indomethacin and flurbiprofen and the thromboxane receptor antagonist ICI 192,605 ( P < 0.05). This is the first study showing that 2-AG increases the severity of the CBF deficit during MCAO, and further interrogation confirmed 2-AG-induced platelet aggregation in rats. These findings are important because 2-AG had previously been shown to exert neuroprotective actions and therefore force us to reevaluate the circumstances under which 2-AG is beneficial. NEW & NOTEWORTHY 2-Arachidonoylglycerol (2-AG) has neuroprotective properties; however, the present study revealed that 2-AG increases the severity of the cerebral blood flow deficit during middle cerebral artery occlusion in rats. Further interrogation showed that 2-AG induces platelet aggregation in rats. These findings force us to reevaluate the circumstances under which 2-AG is beneficial.

Human sperm motility, capacitation and acrosome reaction are impaired by 2-arachidonoylglycerol endocannabinoid.

The endocannabinoids are cannabinoids synthesized by mammalian tissues. These compounds are closely related to the regulation of the male reproductive system. However, little is known about the effects produced by 2-arachidonoylglycerol (2AG) on in vitro human sperm functions. This study was undertaken to determine the effects produced by 2AG on fresh human sperm and in the capacitation technique. Semen samples from healthy young men were exposed to different concentrations of 2AG before and during capacitation technique. In this work, we have demonstrated that 2AG induces the spontaneous acrosome reaction and reduces progressive motility in fresh human sperm. During the capacitation technique, sperm becomes more sensitive to low concentrations of 2AG, triggering the acrosome reaction and inhibiting protein phosphorylation. In summary, 2AG affects the in vitro functionality of human sperm by reducing motility, inhibiting capacitation and triggering the acrosome reaction.

Interplay between serotonin and cannabinoid function in the amygdala in fear conditioning.

The possible interactions between the cannabinoid and serotonin systems in the regions of the brain involved in emotional learning and memory formation have been studied by some researchers. In view of the key role of the amygdala in the acquisition and expression of fear memory, we investigated the involvement of basolateral amygdala (BLA) serotonin 5-HT4 receptors in arachidonylcyclopropylamide (ACPA; selective CB1 cannabinoid receptor agonist)-induced fear memory consolidation impairment. In our study, a context and tone fear conditioning apparatus was used for testing fear conditioning in adult male NMRI mice. The results showed that intraperitoneal administration of ACPA 0.5 or 0.05, 0.1 and 0.5mg/kg immediately after training decreased the percentage of freezing time in context or tone fear conditioning respectively, suggesting a context- or tone-dependent fear memory consolidation impairment. Post-training intra-BLA microinjections of RS67333, as 5-HT4 serotonin receptor agonist, at doses of 0.025 and 0.05 µg/mouse also impaired context or tone memory consolidation, while RS23597, as 5-HT4 serotonin receptor antagonist, did not produce a marked difference in both fear memories as compared with the control group. Moreover, a subthreshold dose of RS67333 did not alter ACPA response in both fear conditionings. Interestingly, a subthreshold dose of RS23597 potentiated or reversed ACPA response at the dose of 0.01 or 0.05 respectively. It is concluded that BLA serotonin 5-HT4 receptors are involved in tone-dependent fear memory consolidation impairment induced by CB1 activation using ACPA, suggesting a modulatory role for serotonin 5-HT4 receptor.

Synergistic effect between prelimbic 5-HT3 and CB1 receptors on memory consolidation deficit in adult male Sprague-Dawley rats: An isobologram analysis.

The serotonergic system has often been defined as a neuromodulator system, and is specifically involved in learning and memory via its various receptors. Serotonin is involved in many of the same processes affected by cannabinoids. The present study investigated the influence of bilateral post-training intra-prelimbic (PL) administrations of serotonergic 5-hydroxytryptamine type-3 (5-HT3) receptor agents on arachidonylcyclopropylamide (ACPA) (cannabinoid CB1 receptor agonist)-induced amnesia, using the step-through inhibitory avoidance (IA) task to assess memory in adult male Sprague-Dawley rats. The results indicated that sole intra-PL microinjection of ACPA (0.1 and 0.5 µg/rat) and 5-HT3 serotonin receptor agonist (m-Chlorophenylbiguanide hydrochloride, m-CPBG; 0.001, 0.01 and 0.1 µg/rat) impaired, whereas Y-25130 (a selective 5-HT3 serotonin receptor antagonist; 0.001 and 0.01 and 0.1 µg/rat) did not alter IA memory consolidation, by itself. Moreover, intra-PL administration of subthreshold dose of m-CPBG (0.0005 µg/rat) potentiated, while Y-25130 (0. 1 µg/rat) restored ACPA-induced memory consolidation deficit. The isobologram analysis showed that there is a synergistic effect between ACPA and m-CPBG on memory consolidation deficit. These findings suggest that 5-HT3 receptor mechanism(s), at least partly, play(s) a role in modulating the effect of ACPA on memory consolidation in the PL area.

Involvement of the serotonergic system of the ventral hippocampus (CA3) on amnesia induced by ACPA in mice.

Interactions between the cannabinoid and serotonin systems have been reported in many studies. In the present study, we investigated the influence of the serotonergic receptor agents on amnesia induced by the cannabinoid CB1 receptor agonist, arachydonilcyclopropylamide (ACPA). Bilateral guide-cannulae were implanted to allow intra-CA3 microinjection of the drugs. The results showed that the intra-peritoneal (i.p.) injection of ACPA induce amnesia but did not alter head dip latency, head dip counts, and locomotion. Moreover, intra-CA3 injection of M-Chlorophenylbiguanide (M-CHL, a 5-HT3 serotonin receptor agonist), Y-25130 (a 5-HT3 serotonin receptor antagonist), RS67333 (a 5-HT4 serotonin receptor agonist), and RS23597-190 (a 5-HT4 serotonin receptor antagonist) impaired memory but have no effect on head dip latency and locomotor activity. In addition, intra-CA3 injection of Y-25130, RS67333, and RS23597-190 heighten the ACPA-induced amnesia and head dip counts while did not alter head dip latency and locomotor activity. On the other hand, intra-CA3 microinjection of M-CHL could not modify the ACPA-induced amnesia, head dip latency and locomotor activity whereas increased head dip counts. It can be concluded that the amnesia induced by i.p. administration of ACPA is at least partly mediated through the serotonergic receptor mechanism in the CA3 area.

The dual effect of CA1 NMDA receptor modulation on ACPA-induced amnesia in step-down passive avoidance learning task.

It is well documented that cannabinoids play an important role in certain hippocampal memory processes in rodents. On the other hand, N-Methyl-d-aspartate receptors (NMDARs) mediate the synaptic plasticity related to learning and memory processes which take place in the hippocampus. Such insights prompted us to investigate the influence of dorsal hippocampal (CA1) NMDA receptor agents on amnesia induced by cannabinoid CB1 receptor agonist, arachidonylcyclopropylamide (ACPA) in male mice. One-trial step-down passive avoidance and hole-board apparatuses were used to examine the memory retrieval and exploratory behaviors, respectively. Based on our findings, pre-training intraperitoneal (i.p.) administration of ACPA (0.01mg/kg) decreased memory acquisition. Moreover, pre-training intra-CA1 infusion of NMDA (0.001, 0.0125, 0.025 and 0.2µg/mouse), d-AP7 (0.5 and 1µg/mouse) or AM251 (50ng/mouse) impaired the memory acquisition. Meanwhile, NMDA-treated animals at the doses of 0.0005, 0.05 and 0.1µg/mouse acquired memory formation. In addition, intra-CA1 microinjection of NMDA (0.0005) plus different doses of ACPA potentiated the ACPA response, while NMDA (0.1) plus the lower or the higher dose of ACPA potentiated or restored the ACPA response, respectively. Further investigation revealed that a subthreshold dose of d-AP7 could potentiate the memory acquisition impairment induced by ACPA. Moreover, the subthreshold dose of AM251 did not alter the ACPA response, while the effective dose of the drug restored the memory acquisition impairment induced by ACPA. According to these results, we concluded that activation of the NMDA receptors in the CA1 mediates a dual effect on ACPA-induced amnesia in step-down passive avoidance learning task.

Differential role of the basolateral amygdala 5-HT3 and 5-HT4 serotonin receptors upon ACPA-induced anxiolytic-like behaviors and emotional memory deficit in mice.

BACKGROUND AND AIM: The critical role of cannabinoidergic and serotonergic systems of the amygdala in modulation of anxiety-like behaviors and emotional memory has already been demonstrated. The present study aimed to investigate the possible role of the basolateral amygdala (BLA) 5-HT3 and 5-HT4 serotonergic systems upon ACPA (CB1 cannabinoid receptor agonist)-induced anxiolytic-like behaviors and emotional memory impairment using the elevated plus-maze (EPM) test-retest paradigm in male mice. METHOD: bilateral guide-cannulae were implanted to allow intra-BLA microinjection of serotonergic agents. RESULTS: the intraperitoneal injection of ACPA could induce anxiolytic-like behaviors and reduce the emotional memory formation. Intra-BLA injection of M-Chlorophenylbiguanide (M-Chl, a 5-HT3 serotonin receptor agonist) neither altered the anxiety-like behaviors nor the emotional memory formation by itself, while the higher dose of Y-25130 (a 5-HT3 serotonin receptor antagonist) reduced the emotional memory formation and locomotor activity but not the anxiety-like behaviors. Furthermore, injection of a higher dose of RS67333 and RS23597 (as 5-HT4 serotonin receptor agonist and antagonist, respectively) did not alter the anxiety-like behaviors, while reduced the emotional memory formation. In addition, the intra-BLA injection of M-Chl but not Y-25130 and RS67333 restored the ACPA-induced anxiolytic-like behaviors and emotional memory deficit, while a higher dose of RS67333 decreased the locomotor activity. Moreover, the intra-BLA microinjection of RS23597 could restore the ACPA-induced anxiolytic-like behaviors but not the emotional memory deficit. CONCLUSION: based on our findings, ACPA seems to induce its anxiolytic-like behaviors and emotional memory formation deficits via activation and deactivation of the BLA 5-HT4 and 5-HT3 serotonin receptors.

2-hydroxy arachidonic acid: a new non-steroidal anti-inflammatory drug.

BACKGROUND: Nonsteroidal anti-inflammatory drugs (NSAIDs) are a family of COX1 and COX2 inhibitors used to reduce the synthesis of pro-inflammatory mediators. In addition, inflammation often leads to a harmful generation of nitric oxide. Efforts are being done in discovering safer NSAIDs molecules capable of inhibiting the synthesis of pro-inflammatory lipid mediators and nitric oxide to reduce the side effects associated with long term therapies. METHODOLOGY/PRINCIPAL FINDINGS: The analogue of arachidonic acid (AA), 2-hydroxy-arachidonic acid (2OAA), was designed to inhibit the activities of COX1 and COX2 and it was predicted to have similar binding energies as AA for the catalytic sites of COX1 and COX2. The interaction of AA and 2OAA with COX1 and COX2 was investigated calculating the free energy of binding and the Fukui function. Toxicity was determined in mouse microglial BV-2 cells. COX1 and COX2 (PGH2 production) activities were measured in vitro. COX1 and COX2 expression in human macrophage-like U937 cells were carried out by Western blot, immunocytochemistry and RT-PCR analysis. NO production (Griess method) and iNOS (Western blot) were determined in mouse microglial BV-2 cells. The comparative efficacy of 2OAA, ibuprofen and cortisone in lowering TNF-α serum levels was determined in C57BL6/J mice challenged with LPS. We show that the presence of the -OH group reduces the likelihood of 2OAA being subjected to H* abstraction in COX, without altering significantly the free energy of binding. The 2OAA inhibited COX1 and COX2 activities and the expression of COX2 in human U937 derived macrophages challenged with LPS. In addition, 2OAA inhibited iNOS expression and the production of NO in BV-2 microglial cells. Finally, oral administration of 2OAA decreased the plasma TNF-α levels in vivo. CONCLUSION/SIGNIFICANCE: These findings demonstrate the potential of 2OAA as a NSAID.

Modulation of anxiety-like behavior by the endocannabinoid 2-arachidonoylglycerol (2-AG) in the dorsolateral periaqueductal gray.

Anandamide and 2-arachidonoylglycerol (2-AG) are the two main endocannabinoids, exerting their effects by activating type 1 (CB1r) and type 2 (CB2r) cannabinoid receptors. Anandamide inhibits anxiety-like responses through the activation of CB1r in certain brain regions, including the dorsolateral periaqueductal gray (dlPAG). 2-AG also attenuates anxiety-like responses, although the neuroanatomical sites for these effects remained unclear. Here, we tested the hypothesis that enhancing 2-AG signaling in the dlPAG would induce anxiolytic-like effects. The mechanisms involved were also investigated. Male Wistar rats received intra-dlPAG injections of 2-AG, URB602 (inhibitor of the 2-AG hydrolyzing enzyme, mono-acylglycerol lipase--MGL), AM251 (CB1r antagonist) and AM630 (CB2r antagonist). The behavior was analyzed in the elevated plus maze after the following treatments. Exp. 1: vehicle (veh) or 2-AG (5 pmol, 50 pmol, and 500 pmol). Exp. 2: veh or URB602 (30 pmol, 100 pmol or 300 pmol). Exp. 3: veh or AM251 (100 pmol) followed by veh or 2-AG (50 pmol). Exp. 4: veh or AM630 (1000 pmol) followed by veh or 2-AG. Exp. 5: veh or AM251 followed by veh or URB602 (100 pmol). Exp. 6: veh or AM630 followed by veh or URB602. 2-AG (50 pmol) and URB602 (100 pmol) significantly increased the exploration of the open arms of the apparatus, indicating an anxiolytic-like effect. These behavioral responses were prevented by CB1r (AM251) or CB2r (AM630) antagonists. Our results showed that the augmentation of 2-AG levels in the dlPAG induces anxiolytic-like effects. The mechanism seems to involve both CB1r and CB2r receptors.

Influence of serum and albumin on the in vitro anandamide cytotoxicity toward C6 glioma cells assessed by the MTT cell viability assay: implications for the methodology of the MTT tests.

Anandamide (AEA), an endogenous ligand of cannabinoid CB1 and CB2 receptors, which also binds transient receptor potential vanilloid type 1 receptor (TRPV1), has been shown to display substantial selective cytotoxicity toward some cancer cell lines in vitro, although the relevant data are not consistent. In the present study, we employed the MTT test to assess short-term cytotoxicity of AEA on C6 rat glioma cell culture. When anandamide was administered to the culture medium with foetal bovine serum (FBS), no cytotoxic effect was observed following 24 h exposure of the glioma cells to micromolar concentrations of AEA. However, if no serum was present in the medium, micro-to-submicromolar concentrations of AEA induced dose-dependent cytotoxicity clearly detectable after 24 h. Control experiments made it possible to exclude significant interference of serum with the MTT test per se. Bovine serum albumin mimicked the effect of FBS. We conclude that the apparent inhibition of short-term cytotoxicity of AEA toward C6 rat glioma cells in vitro is caused by binding AEA to serum proteins such as albumin. Taking into account that blood serum or albumin is practically always present in cell culture media, we discuss implications of binding substances to serum proteins for methodology and interpretation of in vitro cytotoxicity testing.

Involvement of transient receptor potential vanilloid type 1 channels in the pro-convulsant effect of anandamide in pentylenetetrazole-induced seizures.

Anandamide, an endogenous agonist of CB(1) receptors, also activates TRPV1 but at a higher concentration. Studies demonstrate the anticonvulsant activity of anandamide via CB(1) receptors, while its action through TRPV1 is still ambiguous. Thus, the present study investigated the influence of anandamide on pentylenetetrazole-induced seizures in mice pretreated with TRPV1 or CB(1) receptor antagonists. Acute intracerebroventricular administration of low doses of anandamide (10, 20, or 40µg/mouse) produced anticonvulsant effect, while the pro-convulsant effect was evident at high doses (80 or 100µg/mouse). Interestingly, AM251 (2µg/mouse), a CB(1) antagonist pretreatment blocked the anticonvulsant effect, but augmented the pro-convulsant effect. Conversely, in the presence of inactive dose of capsazepine (1µg/mouse), a TRPV1 antagonist, anandamide exhibited significant anticonvulsant effect even at high doses with no change in its anticonvulsant effect. Moreover, mice treated with capsaicin, a TRPV1 agonist (10, or 100µg/mouse) exhibited pro-convulsant activity that was blocked by capsazepine pretreatment. However, capsazepine, per se at doses 10 or 100µg/mouse exhibited anticonvulsant effect. Like anandamide, the agents (AM404 and URB597), which increase its synaptic concentrations produced similar biphasic effects. Thus, these results indicate that anandamide exhibits both pro- and anticonvulsant activities by activating TRPV1 and CB(1) receptor respectively.

11-Oxoeicosatetraenoic acid is a cyclooxygenase-2/15-hydroxyprostaglandin dehydrogenase-derived antiproliferative eicosanoid.

Previously, we established that 11(R)-hydroxy-5,8,12,14-(Z,Z,E,Z)-eicosatetraenoic acid (HETE) was a significant cyclooxygenase (COX)-2-derived arachidonic acid (AA) metabolite in epithelial cells. Stable isotope dilution chiral liquid chromatography (LC)-electron capture atmospheric pressure chemical ionization (ECAPCI)/mass spectrometry (MS) was used to quantify COX-2-derived eicosanoids in the human colorectal adenocarcinoma (LoVo) epithelial cell line, which expresses both COX-2 and 15-hydroxyprostaglandin dehydrogenase (15-PGDH). 11(R)-HETE secretion reached peak concentrations within minutes after AA addition before rapidly diminishing, suggesting further metabolism had occurred. Surprisingly, recombinant 15-PGDH, which is normally specific for oxidation of eicosanoid 15(S)-hydroxyl groups, was found to convert 11(R)-HETE to 11-oxo-5,8,12,14-(Z,Z,E,Z)-eicosatetraenoic acid (ETE). Furthermore, LoVo cell lysates converted 11(R)-HETE to 11-oxo-ETE and inhibition of 15-PGDH with 5-[[4-(ethoxycarbonyl)phenyl]azo]-2-hydroxy-benzeneacetic acid (CAY10397) (50 µM) significantly suppressed endogenous 11-oxo-ETE production with a corresponding increase in 11(R)-HETE. These data confirmed COX-2 and 15-PGDH as enzymes responsible for 11-oxo-ETE biosynthesis. Finally, addition of AA to the LoVo cells resulted in rapid secretion of 11-oxo-ETE into the media, reaching peak levels within 20 min of starting the incubation. This was followed by a sharp decrease in 11-oxo-ETE levels. Glutathione (GSH) S-transferase (GST) was found to metabolize 11-oxo-ETE to the 11-oxo-ETE-GSH (OEG)-adduct in LoVo cells, as confirmed by LC-MS/MS analysis. Bromodeoxyuridine (BrdU)-based cell proliferation assays in human umbilical vein endothelial cells (HUVECs) revealed that the half-maximal inhibitory concentration (IC(50)) of 11-oxo-ETE for inhibition of HUVEC proliferation was 2.1 µM. These results show that 11-oxo-ETE is a novel COX-2/15-PGDH-derived eicosanoid, which inhibits endothelial cell proliferation with a potency that is similar to that observed for 15d-PGJ(2).

Safety assessment of EPA-rich triglyceride oil produced from yeast: genotoxicity and 28-day oral toxicity in rats.

The 28-day repeat-dose oral and genetic toxicity of eicosapentaenoic acid triglyceride oil (EPA oil) produced from genetically modified Yarrowia lipolytica yeast were assessed. Groups of rats received 0 (olive oil), 940, 1880, or 2820 mg EPA oil/kg/day, or fish oil (sardine/anchovy source) by oral gavage. Lower total serum cholesterol was seen in all EPA and fish oil groups. Liver weights were increased in the medium and high-dose EPA (male only), and fish oil groups but were considered non-adverse physiologically adaptive responses. Increased thyroid follicular cell hypertrophy was observed in male high-dose EPA and fish oil groups, and was considered to be an adaptive response to high levels of polyunsaturated fatty acids. No adverse test substance-related effects were observed on body weight, nutritional, or other clinical or anatomic pathology parameters. The oil was not mutagenic in the in vitro Ames or mouse lymphoma assay, and was not clastogenic in the in vivo mouse micronucleus test. In conclusion, exposure for 28 days to EPA oil derived from yeast did not produce adverse effects at doses up to 2820 mg/kg/day and was not genotoxic. The safety profile of the EPA oil in these tests was comparable to a commercial fish oil.

Cannabinoid receptor 1 and 2 agonists increase lipid accumulation in hepatocytes.

BACKGROUND: Cannabinoid receptors CB1 and CB2 are expressed in the liver, but their regulation in fatty hepatocytes is poorly documented. The aim of this study was to investigate the effects of selective CB1 or CB2 agonists on the expression of key regulators of lipid metabolism. METHODS: We used an in vitro model of fatty liver by treating immortalized human hepatocytes and HepG2 cells with oleic acid and the selective agonists arachidonyl-2-chloroethylamide (ACEA) (CB1, 12 nM) and (2-iodo-5-nitrophenyl)-[1-(1-methylpiperidin-2-ylmethyl)-1H-indol-3-yl]-methanone (AM1241) (CB2, 16 nM). The quantity of intracellular lipids was assessed using Oil-Red-O and a biochemical triglyceride assay. The expression of several proteins regulating endocannabinoid signalling and lipid metabolism was quantified by real-time polymerase chain reaction and by Western blot. RESULTS: Both CB1 and CB2 agonists dose-dependently increased the degree of steatosis of oleic acid-treated fatty hepatocytes. Cannabinoid receptors were downregulated in the presence of steatosis, and treatment with a CB2 agonist increased the expression of CB1. Carnitine palmitoyltransferase 1 was significantly overexpressed and sterol response element-binding protein (SREBP)-1c, fatty acid synthase and lecithin-cholesterol acetyltransferase (LCAT) were downregulated in fatty immortalized human hepatocytes. Treatment with the CB agonists ACEA and AM1241 partially reversed these changes, except for SREBP-1c. CB2, but not CB1, agonism decreased the expression of apolipoprotein B. In HepG2 cells, only LCAT resulted increased after treatment with CB agonists. CONCLUSIONS: Not only CB1 but also CB2 participated in the regulation of lipid metabolism in human-derived immortalized hepatocytes by regulating the expression of key enzymes of lipid synthesis and transport.

Activity-based anorexia in C57/BL6 mice: effects of the phytocannabinoid, Delta9-tetrahydrocannabinol (THC) and the anandamide analogue, OMDM-2.

The activity-based anorexia (ABA) paradigm is one of the few animal models of human anorexia nervosa. We present here the translation of this approach to C57/BL6 mice, a common background for genetically modified mice, and investigate the effects of the cannabinoid agonist, Delta(9)-tetrahydrocannabinol (THC) and the endocannabinoid uptake inhibitor, OMDM-2 in this model. The ABA paradigm was optimised so that food-restricted wheel-running mice displayed anorexia, reduced body weight and disrupted activity and circadian cycles. These conditions produced a murine ABA model with a defined stage and stability to allow for pharmacological intervention. Daily Delta(9)-THC (0.5 mg/kg) decreased survival in the ABA animals but increased feeding in the survivors, OMDM-2 (3 mg/kg) increased food intake, but not sufficiently to reverse weight loss. The effects of this model on endocannabinoid tone in the brain remain to be determined. Since the endocannabinoid system may be implicated in anorexia nervosa and in view of the positive modulation by cannabinoids of some aspects of ABA in this study, further investigation of the effects of cannabinoids in ABA is warranted.