Inhibitor Tranylcypromine Research Articles

Identification and Evaluation of Olive Phenolics in the Context of Amine Oxidase Enzyme Inhibition and Depression: In Silico Modelling and In Vitro Validation.

The Mediterranean diet well known for its beneficial health effects, including mood enhancement, is characterised by the relatively high consumption of extra virgin olive oil (EVOO), which is rich in bioactive phenolic compounds. Over 200 phenolic compounds have been associated with Olea europaea, and of these, only a relatively small fraction have been characterised. Utilising the OliveNetTM library, phenolic compounds were investigated as potential inhibitors of the epigenetic modifier lysine-specific demethylase 1 (LSD1). Furthermore, the compounds were screened for inhibition of the structurally similar monoamine oxidases (MAOs) which are directly implicated in the pathophysiology of depression. Molecular docking highlighted that olive phenolics interact with the active site of LSD1 and MAOs. Protein-peptide docking was also performed to evaluate the interaction of the histone H3 peptide with LSD1, in the presence of ligands bound to the substrate-binding cavity. To validate the in silico studies, the inhibitory activity of phenolic compounds was compared to the clinically approved inhibitor tranylcypromine. Our findings indicate that olive phenolics inhibit LSD1 and the MAOs in vitro. Using a cell culture model system with corticosteroid-stimulated human BJ fibroblast cells, the results demonstrate the attenuation of dexamethasone- and hydrocortisone-induced MAO activity by phenolic compounds. The findings were further corroborated using human embryonic stem cell (hESC)-derived neurons stimulated with all-trans retinoic acid. Overall, the results indicate the inhibition of flavin adenine dinucleotide (FAD)-dependent amine oxidases by olive phenolics. More generally, our findings further support at least a partial mechanism accounting for the antidepressant effects associated with EVOO and the Mediterranean diet.

Beyond the Brain: Perinatal Exposure of Rats to Serotonin Enhancers Induces Long-Term Changes in the Jejunum and Liver.

Serotonin (5-hydroxytryptamine, 5HT) homeostasis is essential for many physiological processes in the central nervous system and peripheral tissues. Hyperserotonemia, a measurable sign of 5HT homeostasis disruption, can be caused by 5HT-directed treatment of psychiatric and gastrointestinal diseases. Its impact on the long-term balance and function of 5HT in the peripheral compartment remains unresolved and requires further research due to possible effects on human health. We explored the effects of perinatal 5HT imbalance on the peripheral organs responsible for serotonin metabolism-the jejunum, a synthesis site, and the liver, a catabolism site-in adult rats. Hyperserotonemia was induced by subchronic treatment with serotonin precursor 5-hydroxytryptophan (5HTP) or serotonin degradation inhibitor tranylcypromine (TCP). The jejunum and liver were collected on postnatal day 70 and analyzed histomorphometrically. Relative mRNA levels of 5HT-regulating proteins were determined using qRT-PCR. Compared to controls, 5HTP- and TCP-treated rats had a reduced number of 5HT-producing cells and expression of the 5HT-synthesising enzyme in the jejunum, and an increased expression of 5HT-transporter accompanied by karyomegaly in hepatocytes, with these differences being more pronounced in the TCP-treated animals. Here, we report that perinatal 5HT disbalance induced long-term cellular and molecular changes in organs regulating 5HT-metabolism, which may have a negative impact on 5HT availability and function in the periphery. Our rat model demonstrates a link between the developmental abnormalities of serotonin homeostasis and 5HT-related changes in adult life and may be suitable for exploring the neurobiological substrates of vulnerability to behavioral and metabolic disorders, as well as for modeling the adverse effects of the prenatal exposure to 5HT enhancers in the human population.

LSD1 inhibition by tranylcypromine hydrochloride reduces alkali burn-induced corneal neovascularization and ferroptosis by suppressing HIF-1α pathway.

Corneal neovascularization (CNV) is a sight-threatening condition that necessitates epigenetic control. The role of lysine-specific demethylase 1 (LSD1) in CNV remains unclear, despite its established significance in tumor angiogenesis regulation. An alkali burn-induced CNV mouse model was used in vivo. The effects of LSD1 inhibitor tranylcypromine hydrochloride (TCP) were examined through slit lamp, histological staining, and immunofluorescence. The expression of malondialdehyde (MDA), superoxide dismutase (SOD), and glutathione (GSH) levels were assessed in corneal tissues. Oxidative stress and ferrous ion expression during CNV were determined using 4-HNE, GPX4, and FerroOrange staining. In vitro, a hypoxia-reoxygenation (H/R) model was established using human umbilical vein endothelial cells (HUVECs) to study LSD1 or hypoxia-inducible factor (HIF-1α) knockdown and lentiviral overexpression of HIF-1α. The effects on HUVECs migration, invasion, and angiogenesis were evaluated through cell scratching assay, transwell migration assay and tube formation assay. The role of ferroptosis was investigated using ROS staining, FerroOrange staining, and key ferroptosis proteins. Further, The JAK2/STAT3 pathway's involvement in CNV regulation was explored through in vivo experiments with subconjunctival injection of AG490. The results showed a substantial correlation between corneal damage and LSD1 levels. In addition, HIF-1α expression was also elevated after alkali burns, and subconjunctival injection of TCP reduced corneal inflammation and neovascularization. Corneal alkali burns increased ROS levels and reduced antioxidative stress indicators, accompanied by elevated ferrous ion levels, which were reversed by TCP injection. In vitro, TCP or siRNAs inhibited H/R-induced ferroptosis and angiogenesis in HUVECs by affecting specific protein expressions and MDA, SOD, and GSH levels. HIF-1α levels, associated with ROS production, ferroptosis, and angiogenesis, increased during H/R, but were reversed by TCP or siRNA administration. HIF-1α overexpression counteracted the effects of LSD1 inhibition. Additionally, AG490 injection effectively reduced HIF-1α and VEGFA expression in the CNV model. These findings suggest that LSD1 inhibition via the HIF-1α-driven pathway prevents angiogenesis, oxidative stress, and ferroptosis in corneal alkali burn-induced CNV, highlighting LSD1 as a potential therapeutic target.

Phase I Study of the LSD1 Inhibitor Tranylcypromine (TCP) in Combination with All-Trans Retinoic Acid (ATRA) and Low-Dose Cytarabine (LDAC) in Elderly, Medically Non-Fit Patients with AML or High-Risk MDS (TRANSATRA trial)

Phase I Study of the LSD1 Inhibitor Tranylcypromine (TCP) in Combination with All-Trans Retinoic Acid (ATRA) and Low-Dose Cytarabine (LDAC) in Elderly, Medically Non-Fit Patients with AML or High-Risk MDS (TRANSATRA trial)

Chronic SSRI Treatment, but Not Norepinephrine Reuptake Inhibitor Treatment, Increases Neurogenesis in Juvenile Rats

There has been growing recognition that major depressive disorder is a serious medical disorder that also affects children. This has been accompanied by an increased use of antidepressant drugs in adolescents; however, not all classes of antidepressants are effective in children and adolescents. There is an increasing need to understand the differences in antidepressant action in different developmental stages. There are some data indicating that the behavioral effect of chronic antidepressant treatment in adult rodents is dependent on hippocampal neurogenesis; however, it is not known which classes of antidepressant drugs induce hippocampal neurogenesis in adolescent rodents. Three classes of antidepressant drugs were tested in two age groups of Sprague Dawley rats, pre-adolescent (postnatal days 11–24) and adolescent (postnatal days 21–34): monoamine oxidase inhibitors (MAOIs); selective serotonin reuptake inhibitors (SSRIs); serotonin norepinephrine reuptake inhibitors (SNRIs); and tricyclic antidepressants (TCAs). To address which classes of antidepressant drugs might alter the rate of mitogenesis in neural progenitor cells in an adolescent rodent model, adolescent Sprague Dawley rats were treated with the thymidine analog 5-bromo-deoxy-2′-uridine (BrdU) on postnatal days 21 and 22 and antidepressant drugs or vehicle for 14 days (postnatal days 21–34). To address which classes of antidepressant drugs might alter the rate of neurogenesis, postnatal day-21 Sprague Dawley rats were treated with antidepressant drugs or vehicle for 14 days (postnatal days 21–34) and BrdU on postnatal days 33 and 34. In both experimental paradigms, BrdU-positive cells in the subgranular zone and the granule cell layer were counted. Newborn neurons were identified in the neurogenic paradigm by identifying cells expressing both the neuronal specific marker NeuN and BrdU using confocal microscopy. Only the SSRI fluoxetine significantly altered the basal mitogenic and neurogenic rates in adolescent rats. Treatment with the monoamine oxidase inhibitor (MAOI) tranylcypromine (TCP) and the TCA desipramine did not alter the rate of hippocampal neurogenesis in the adolescent rats. This is consistent with human clinical observations, where only SSRIs have efficacy for treatment of depression in patients under the age of 18. In pre-adolescent rats, postnatal days 11–24, none of the drugs tested significantly altered the basal mitogenic or neurogenic rates. All of the classes of antidepressant drugs are known to induce hippocampal neurogenesis in adult rats. The mechanisms of action underlying this developmental difference in antidepressant drug action between juveniles and adults are not known.

LSD1 Inhibition Enhances the Immunogenicity of Mesenchymal Stromal Cells by Eliciting a dsRNA Stress Response.

Mesenchymal stromal cells (MSCs) are commonly known for their immune-suppressive abilities. However, our group provided evidence that it is possible to convert MSCs into potent antigen presenting cells (APCs) using either genetic engineering or pharmacological means. Given the capacity of UM171a to trigger APC-like function in MSCs, and the recent finding that this drug may modulate the epigenome by inhibiting the lysine-specific demethylase 1 (LSD1), we explored whether the direct pharmacological inhibition of LSD1 could instill APC-like functions in MSCs akin to UM171a. The treatment of MSCs with the LSD1 inhibitor tranylcypromine (TC) elicits a double-stranded (ds)RNA stress response along with its associated responsive elements, including pattern recognition receptors (PRRs), Type-I interferon (IFN), and IFN-stimulated genes (ISGs). The net outcome culminates in the enhanced expression of H2-Kb, and an increased stability of the cell surface peptide: MHCI complexes. As a result, TC-treated MSCs stimulate CD8 T-cell activation efficiently, and elicit potent anti-tumoral responses against the EG.7 T-cell lymphoma in the context of prophylactic vaccination. Altogether, our findings reveal a new pharmacological protocol whereby targeting LSD1 in MSCs elicits APC-like capabilities that could be easily exploited in the design of future MSC-based anti-cancer vaccines.

Anti-cancer effect of LINC00478 in bladder cancer correlates with KDM1A-dependent MMP9 demethylation

Accumulating evidence has highlighted the important roles of long intergenic non-coding RNAs (lincRNAs) during cancer progression. However, the involvement of LINC00478 in bladder cancer remains largely unclear. Accordingly, the current study sought to investigate the function of LINC00478 on malignant phenotypes of bladder cancer cells as well as the underlying mechanism. By integrating data from in silico analysis, we uncovered that LINC00478 was differentially expressed in bladder cancer. We further analyzed the expression of LINC00478 and matrix metalloprotein 9 (MMP9) in bladder cancer tissues and cell lines and observed a significant decline in LINC00478 expression and an elevation in MMP9 expression. In addition, chromatin immunoprecipitation, RNA-binding protein immunoprecipitation, and RNA pull-down assays predicted and validated that LINC00478 targeted lysine-specific demethylase-1 (KDM1A) and down-regulated the expression of MMP9 by decreasing the monomethylation on lysine 4 of histone H3 (H3K4me1) of MMP9 promoter. Treatment with KDM1A inhibitor tranylcypromine (TCP) also led to an increase in the enrichment of H3K4me1 in the MMP9 promoter region. Through gain- and loss-of-function approaches, we found that LINC00478 up-regulation diminished the malignant phenotype of bladder cancer cells in vitro, and further inhibited xenograft tumor growth and metastasis in vivo by repressing MMP9. Collectively, our findings unraveled a LINC00478-mediated inhibitory mechanism in bladder cancer via the recruitment of histone demethylation transferase KDM1A to the MMP9 promoter region, which can provide potential implications for novel therapeutic targets against bladder cancer.

Insufficient recovery of monoamine oxidase in a bioequivalence study of the monoamine oxidase inhibitor tranylcypromine: Recommendation of the tranylcypromine enantiomer test.

To investigate the recovery of monoamine oxidase (MAO) activity in the liver and gut of healthy subjects after a dose of 10mg of the irreversible MAO inhibitor tranylcypromine (TCP). A bioequivalence study of TCP with a wash-out of 1 week between 2 doses of 10mg TCP was re-analyzed for changes of the plasma concentrations of TCP enantiomers. Plasma concentrations of (+)-TCP and the ratio of (+)-TCP and (-)-TCP plasma concentrations were used as a measure of MAO activity because (+)-TCP is a more effective suicide inhibitor of MAO than (-)-TCP and, therefore considerably more metabolized by MAO. The area under the curve from the first to the last measured concentration (AUCt) and the maximum plasma concentration (Cmax) of (+)-TCP increased significantly in the second dose (p<0.0001) by 43.1% (11.8%) and 66.5% (26.4%), respectively, (mean with 95%CI in each case). The ratios (+)-TCP/(-)-TCP of AUCt and Cmax also increased significantly (p < 0.0001) by 27.3% (6.4%) and 25.9% (6.2%), respectively. No changes were found for the half-lives (T1/2) of both enantiomers. For the first dose, MAO is the main drug-metabolizing enzyme of (+)-TCP. MAO activity in the liver and gut is not completely recovered within 1 week after 1 dose of TCP. One week of wash-out may be insufficient in bioequivalence studies of irreversible MAO inhibitors. Prolonged inhibition of MAO after the treatment with irreversible MAO inhibitors may explain drug interactions during the switch from another MAO inhibitor to TCP. Enantiomer plasma concentrations of TCP after a dose of racemic TCP may be used as a test for gastrointestinal and hepatic MAO activity.

Effect of the NAMPT Activator P7C3-A20 on γ-Globin Expression in Baboon CD34+ Erythroid Cell Cultures

Increased levels of Fetal Hemoglobin (HbF) reduce the symptoms of sickle cell disease (SCD) and lengthen the life span of patients. New, more effective pharmacological agents that can be safely administered long term to increase HbF levels in SCD patients are highly sought. Expression of the γ-globin gene in adult erythroid cells is normally repressed by the recruitment of multi-protein co-repressor complexes to the γ-globin promoter by sequence-specific DNA binding proteins including BCL11A, LRF1 and TR2/TR4. Enzymes contained within these co-repressor complexes, such as DNMT1, LSD1, G9A, and HDACs, modify the chromatin surrounding the γ-globin promoter by catalyzing repressive epigenetic modifications to both histones and DNA. Small molecule pharmacological inhibitors of these enzymes are potent inducers of HbF in various in cell culture and animal models and in SCD patients, but the use of these drugs in patients has been hindered by their dose-dependent effects on hematopoietic differentiation. An alternative strategy to the use of these pharmacological inhibitors to increase HbF would be to employ pharmacological activators that increase the activity of proteins that positively promote γ-globin expression. Previous studies have shown that pharmacological activators of the Sirtuin 1 protein deacetylase increased γ-globin expression in cultured human CD34+ erythroid progenitor cell cultures (Dai et al; Am J Hematol 92:1177-1186, 2017). Because Sirtuin deacetylase activity is dependent upon nicotinamide adenine dinucleotide (NAD) as a co-factor, we tested the hypothesis that increased concentrations of nicotinamide, an NAD precursor, would also increase γ-globin expression. Baboon bone marrow derived CD34+ erythroid progenitor cells from 4 individual baboons were cultured on AFT024 monolayers for 14 days in the presence and absence of varying concentrations of nicotinamide. Globin chain expression was measured in cell lysates by high performance liquid chromatography (HPLC). Nicotinamide (500μM) appeared to increase γ-globin 2 fold (0.015±0.098 γ/γ+β) compared to untreated controls (0.072±0.04 γ/γ+β; n=4; p<0.08). Because the nicotinamide levels used in this experiments are higher than can be easily achieved by dietary supplementation, additional experiments were performed to test the effect of P7C3-A20, an allosteric activator of nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme in the NAD synthesis, on γ-globin expression. Addition of P7C3-A20 (2.5μM) to CD34+ erythroid progenitor cultures on d1, 4, 7, and 10 increased γ-globin 2.7 fold (0.247±0.10 γ/γ+β) compared to vehicle-treated controls (0.090±0.06 γ/γ+β; n=5; p<0.01). P7C3-A20 treatment did not affect cell viability or growth at concentration< 2.5μM and dose-response experiments showed increased γ-globin in cultures treated with submicromolar concentrations of the drug. Addition of P7C3-A20 to cultures on days 1 and 4 resulted in near maximal stimulation of γ-globin expression with lesser effects when the drug was added on later days (d4 and7 or d7 and 10) strongly suggesting that the drug targets cells at an early stage of differentiation. Additional experiments showed that the effect of P7C3-A20 (2.5μM) in combination with either the DNMT1 inhibitor decitabine (DAC) or the LSD1 inhibitor tranylcypromine (TCP) resulted in a greater than additive effects on γ-globin expression in the absence of cytotoxicity (Figure 1). In conclusion, the NAMPT activator P7C3-A20 increased γ-globin expression in baboon CD34+ erythroid progenitor cells with greater than additive effects in combination with DAC or TCP. P7C3-A20 has potent in vivo effects as a neuroprotective drug in mouse models and non-human primates. Therefore, the potential of this drug for in vivo HbF induction warrants further investigation. [Display omitted] DisclosuresSaunthararajah: EpiDestiny: Consultancy, Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.

Pharmacological Rescue of the Brain Cortex Phenotype of Tbx1 Mouse Mutants: Significance for 22q11.2 Deletion Syndrome.

ObjectivesTbx1 mutant mice are a widely used model of 22q11.2 deletion syndrome (22q11.2DS) because they manifest a broad spectrum of physical and behavioral abnormalities that is similar to that found in 22q11.2DS patients. In Tbx1 mutants, brain abnormalities include changes in cortical cytoarchitecture, hypothesized to be caused by the precocious differentiation of cortical progenitors. The objectives of this research are to identify drugs that have efficacy against the brain phenotype, and through a phenotypic rescue approach, gain insights into the pathogenetic mechanisms underlying Tbx1 haploinsufficiency.Experimental ApproachDisease model: Tbx1 heterozygous and homozygous embryos. We tested the ability of two FDA-approved drugs, the LSD1 inhibitor Tranylcypromine and Vitamin B12, to rescue the Tbx1 mutant cortical phenotype. Both drugs have proven efficacy against the cardiovascular phenotype, albeit at a much reduced level compared to the rescue achieved in the brain.MethodsIn situ hybridization and immunostaining of histological brain sections using a subset of molecular markers that label specific cortical regions or cell types. Appropriate quantification and statistical analysis of gene and protein expression were applied to identify cortical abnormalities and to determine the level of phenotypic rescue achieved.ResultsCortical abnormalities observed in Tbx1 mutant embryos were fully rescued by both drugs. Intriguingly, rescue was obtained with both drugs in Tbx1 homozygous mutants, indicating that they function through mechanisms that do not depend upon Tbx1 function. This was particularly surprising for Vitamin B12, which was identified through its ability to increase Tbx1 gene expression.ConclusionTo our knowledge, this is only the second example of drugs to be identified that ameliorate phenotypes caused by the mutation of a single gene from the 22q11.2 homologous region of the mouse genome. This one drug-one gene approach might be important because there is evidence that the brain phenotype in 22q11.2DS patients is multigenic in origin, unlike the physical phenotypes, which are overwhelmingly attributable to Tbx1 haploinsufficiency. Therefore, effective treatments will likely involve the use of multiple drugs that are targeted to the function of specific genes within the deleted region.

Cardiovascular Effects of Combining Subcutaneous or Intravenous Esketamine and the MAO Inhibitor Tranylcypromine for the Treatment of Depression: A Retrospective Cohort Study.

Background(Es)ketamine and monoamine oxidase inhibitors (MAOIs), e.g., tranylcypromine, are therapeutic options for treatment-resistant major depression. Simultaneous administration is currently not recommended because of concern about hypertensive crises.ObjectiveOur objective was to evaluate whether changes in systolic blood pressure (SBP), diastolic blood pressure (DBP), and heart rate (HR) during esketamine administration differed between patients who concomitantly received tranylcypromine and those who did not.MethodsThis was a retrospective cohort study utilizing cardiovascular monitoring data from inpatients treated for severe depression in unipolar, bipolar, and schizoaffective disorder. Primary outcomes were change in mean BP and HR during the first hour after intravenous or subcutaneous esketamine administration compared with baseline, controlled for confounders. Secondary analyses quantify differences in absolute BP during esketamine treatment and comparisons of BP peaks, temporal effects, and intraindividual comparisons before and after tranylcypromine initiation.ResultsOur analysis included 509 esketamine administrations in 43 patients, 14 of whom concomitantly received tranylcypromine. Controlling for creatinine and age, mean ± standard deviation (SD) BP changes were significantly increased by concomitant tranylcypromine treatment (ΔSBP: F[1,503] = 86.73, p < 0.001; ΔDBP: F[1,503] = 55.71, p < 0.001), but HR remained unaffected. Mean SBP change during esketamine administration was 2.96 ± 18.11 mmHg in patients receiving tranylcypromine (TCP+) and −8.84 ± 11.31 mmHg in those who did not (TCP−). Changes in DBP were −2.81 ± 11.20 mmHg for TCP+ and −10.77 ± 9.13 mmHg for TCP−. Moreover, we found a significant dose–response relationship between tranylcypromine dose and BP (SBP: B = 0.35, standard error [SE] = 0.12, 95% confidence interval [CI] 0.12–0.60, p = 0.004; adjusted R2 = 0.11, p = 0.008; DBP: B = 0.21, SE = 0.08, 95% CI 0.06–0.36, p = 0.007; adjusted R2 = 0.08; p = 0.023).ConclusionsAlthough statistically significant changes in BP were identified in patients receiving tranylcypromine and esketamine, these changes were clinically insignificant. Thus, combining esketamine and this MAOI appears to be safe at standard doses. The dose–response relationship calls for caution with higher doses of tranylcypromine.Supplementary InformationThe online version contains supplementary material available at 10.1007/s40263-021-00837-6.

Clinical Responsiveness to All-trans Retinoic Acid Is Potentiated by LSD1 Inhibition and Associated with a Quiescent Transcriptome in Myeloid Malignancies

In preclinical studies, the lysine-specific histone demethylase 1A (LSD1) inhibitor tranylcypromine (TCP) combined with all-trans retinoic acid (ATRA) induces differentiation and impairs survival of myeloid blasts in non-acute promyelocytic leukemia acute myeloid leukemia (AML). We conducted a phase I clinical trial (NCT02273102) to evaluate the safety and activity of ATRA plus TCP in patients with relapsed/refractory AML and myelodysplasia (MDS). Seventeen patients were treated with ATRA and TCP (three dose levels: 10 mg twice daily, 20 mg twice daily, and 30 mg twice daily). ATRA-TCP had an acceptable safety profile. The MTD of TCP was 20 mg twice daily. Best responses included one morphologic leukemia-free state, one marrow complete remission with hematologic improvement, two stable disease with hematologic improvement, and two stable disease. By intention to treat, the overall response rate was 23.5% and clinical benefit rate was 35.3%. Gene expression profiling of patient blasts showed that responding patients had a more quiescent CD34+ cell phenotype at baseline, including decreased MYC and RARA expression, compared with nonresponders that exhibited a more proliferative CD34+ phenotype, with gene expression enrichment for cell growth signaling. Upon ATRA-TCP treatment, we observed significant induction of retinoic acid-target genes in responders but not nonresponders. We corroborated this in AML cell lines, showing that ATRA-TCP synergistically increased differentiation capacity and cell death by regulating the expression of key gene sets that segregate patients by their clinical response. These data indicate that LSD1 inhibition sensitizes AML cells to ATRA and may restore ATRA responsiveness in subsets of patients with MDS and AML.

Emerging investigator series: human CYP2A6 catalyzes the oxidation of 6:2 fluorotelomer alcohol.

The biotransformation of 6:2 fluorotelomer alcohol (6:2 FTOH) results in the production of bioactive and persistent metabolites, including perfluorinated carboxylic acids (PFCAs). While the products of 6:2 FTOH metabolism have been elucidated in several animal models, the responsible cytochrome P450 (CYP) isoform(s) have not been reported. Here, we characterized the in vitro oxidation of 6:2 FTOH using human liver microsomes and recombinant human CYPs. Six major xenobiotic metabolizing CYPs were screened for their capacity to catalyze 6:2 FTOH oxidation using chemical inhibitors selective towards CYP isoforms. Of the CYP isoforms investigated, CYP2A6 was the only enzyme capable of catalyzing 6:2 FTOH in human liver microsomes, with KM and Vmax values of 4076 ng mL-1 and 69 ng mL-1 min-1, respectively. We further probed the metabolic mechanism by plotting the 6:2 FTOH kinetic profile and extrapolating data to several possible kinetic models. 6:2 FTOH oxidation followed the typical one-site Michaelis-Menten kinetic model. This study also reports that 6:2 FTOH loss is associated with active CYP2A6 by incubating microsomes with the selective CYP2A6 inhibitor tranylcypromine, which bound competitively to the enzyme as determined by an increased KM (8796 ng mL-1) but unchanged Vmax value. Collectively, these findings provide a mechanistic perspective on the potential importance of CYP2A6 in the metabolic activation and phase I elimination of 6:2 FTOH and indirect human exposure to PFCAs.

Transgenerational effect of drug-mediated inhibition of LSD1 on eye pigment expression in Drosophila

BackgroundThe Drosophila melanogaster mutant white-mottled is a well-established model for position-effect variegation (PEV). Transposition of the euchromatic white gene into the vicinity of the pericentric heterochromatin caused variegated expression of white due to heterochromatin spreading. The establishment of the euchromatin-heterochromatin boundary and spreading of silencing is regulated by mutually exclusive histone modifications, i.e. the methylations of histone H3 at lysine 9 and lysine 4. Demethylation of H3K4, catalysed by lysine-specific demethylase LSD1, is required for subsequent methylation of H3K9 to establish heterochromatin. LSD1 is therefore essential for heterochromatin formation and spreading. We asked whether drug-mediated inhibition of LSD affects the expression of white and if this induced change can be transmitted to those generations that have never been exposed to the triggering signal, i.e. transgenerational epigenetic inheritance.ResultsWe used the lysine-specific demethylase 1 (LSD1)-inhibitor Tranylcypromine to investigate its effect on eye colour expression in consecutive generations by feeding the parental and F1 generations of the Drosophila melanogaster mutant white-mottled. Quantitative Western blotting revealed that Tranylcypromine inhibits H3K4-demethylation both in vitro in S2 cells as well as in embryos when used as feeding additive. Eye colour expression in male flies was determined by optical measurement of pigment extracts and qRT-PCR of white gene expression. Flies raised in the presence of Tranylcypromine and its solvent DMSO showed increased eye pigment expression. Beyond that, eye pigment expression was also affected in consecutive generations including F3, which is the first generation without contact with the inhibitor.ConclusionsOur results show that feeding of Tranylcypromine and DMSO caused desilencing of white in treated flies of generation F1. Consecutive generations, raised on standard food without further supplements, are also affected by the drug-induced alteration of histone modifications. Although eye pigment expression eventually returned to the basal state, the observed long-lasting effect points to a memory capacity of previous epigenomes. Furthermore, our results indicate that food compounds potentially affect chromatin modification and hence gene expression and that the alteration is putatively inherited not only parentally but transgenerationally.

Prostaglandin I2 mediates weak vasodilatation in human placental microvessels.

Human placental vessels (HPVs) play important roles in the exchange of metabolites and oxygen in maternal-fetal circulation. Endothelial-derived prostacyclin (prostaglandin I2, PGI2) is a critical endothelial vasodilator in the body. However, the physiological and pharmacological functions of endothelial PGI2 in the human placenta are still unclear. Human, sheep, and rat blood vessels were used in this study. Unlike non-placental vessels (non-PVs), the PGI2 synthesis inhibitor tranylcypromine (TCP) did not modify 5-hydroxytryptamine (5-HT)-induced vascular contraction, indicating that endothelial-derived PGI2 was weak in PVs. Vascular responses to exogenous PGI2 showed slight relaxation followed by a significant contraction at a higher concentration in HPV, which was inhibited by the thromboxane-prostanoid (TP) receptors antagonist SQ-29,548. Testing PVs and non-PVs from sheep also showed similar functional results. More TP receptors than PGI2 (IP) receptors were observed in HPVs. The whole-cell K+ current density of HPVs was significantly weaker than that of non-PVs. This study demonstrated the specific characteristics of the placental endogenous endothelial PGI2 system and the patterns of placental vascular physiological/pharmacological response to exogenous PGI2, showing that placental endothelial PGI2 does not markedly contribute to vascular dilation in the human placenta, in notable contrast to non-PVs. The results provide crucial information for understanding the endothelial roles of HPVs, which may be helpful for further investigations of potential targets in the treatment of diseases such as preeclampsia.

The MAO Inhibitor Tranylcypromine Alters LPS- and Aβ-Mediated Neuroinflammatory Responses in Wild-type Mice and a Mouse Model of AD.

Monoamine oxidase (MAO) has been implicated in neuroinflammation, and therapies targeting MAO are of interest for neurodegenerative diseases. The small-molecule drug tranylcypromine, an inhibitor of MAO, is currently used as an antidepressant and in the treatment of cancer. However, whether tranylcypromine can regulate LPS- and/or Aβ-induced neuroinflammation in the brain has not been well-studied. In the present study, we found that tranylcypromine selectively altered LPS-induced proinflammatory cytokine levels in BV2 microglial cells but not primary astrocytes. In addition, tranylcypromine modulated LPS-mediated TLR4/ERK/STAT3 signaling to alter neuroinflammatory responses in BV2 microglial cells. Importantly, tranylcypromine significantly reduced microglial activation as well as proinflammatory cytokine levels in LPS-injected wild-type mice. Moreover, injection of tranylcypromine in 5xFAD mice (a mouse model of AD) significantly decreased microglial activation but had smaller effects on astrocyte activation. Taken together, our results suggest that tranylcypromine can suppress LPS- and Aβ-induced neuroinflammatory responses in vitro and in vivo.

The Characterization of Ground Raspberry Seeds and the Physiological Response to Supplementation in Hypertensive and Normotensive Rats.

This study aimed to evaluate the protective role of ground raspberry seeds (RBS) as a source of polyphenols and essential fatty acids on blood plasma enzymatic antioxidant status, lipid profile, and endothelium-intact vasodilation during physiological and pathological conditions. Young normotensive Wistar–Kyoto rats (WKYs) and spontaneously hypertensive rats (SHRs) at ten weeks of age were fed with either a control diet or were supplemented with added 7% RBS for six weeks (n = 6). The main component of RBS was dietary fiber (64%) and the main polyphenols were ellagitannins (1.2%) and flavan-3-ols (0.45%). Irrespective of the rat model, ground RBS decreased liver enzyme aspartate aminotransferase (0.9-fold) and hydrogen peroxide scavenging capacity (Catalase, 0.9-fold). In supplemented SHRs, preincubation with inducible nitric oxide synthase (iNOS) inhibitor 1400W, nonselective cyclooxygenase (COX) inhibitor indomethacin, selective COX-2 inhibitor NS-398, prostacyclin (PGI2) synthesis inhibitor tranylcypromine (TCP), thromboxane receptor (TP) antagonist SQ-29548, thromboxane synthesis inhibitor furegrelate, and 20-HETE synthesis inhibitor HET0016 induced the same relaxant response to acetylcholine as in the nonsupplemented control group. In supplemented WKYs, atherogenic index was decreased (0.8-fold), while iNOS and COX-2-derived PGI2 increased acetylcholine-induced vasodilation. These effects of ground RBS may constitute a potential mechanism for preventing cardiovascular diseases.

Hepatic monoamine oxidase B is involved in endogenous geranylgeranoic acid synthesis in mammalian liver cells

Geranylgeranoic acid (GGA) originally was identified in some animals and has been developed as an agent for preventing second primary hepatoma. We previously have also identified GGA as an acyclic diterpenoid in some medicinal herbs. Recently, we reported that in human hepatoma-derived HuH-7 cells, GGA is metabolically labeled from 13C-mevalonate. Several cell-free experiments have demonstrated that GGA is synthesized through geranylgeranial by oxygen-dependent oxidation of geranylgeraniol (GGOH), but the exact biochemical events giving rise to GGA in hepatoma cells remain unclear. Monoamine oxidase B (MOAB) has been suggested to be involved in GGOH oxidation. Here, using two human hepatoma cell lines, we investigated whether MAOB contributes to GGA biosynthesis. Using either HuH-7 cell lysates or recombinant human MAOB, we found that: 1) the MAO inhibitor tranylcypromine dose-dependently downregulates endogenous GGA levels in HuH-7 cells; and 2) siRNA-mediated MAOB silencing reduces intracellular GGA levels in HuH-7 and Hep3B cells. Unexpectedly, however, CRISPR/Cas9-generated MAOB-KO human hepatoma Hep3B cells had GGA levels similar to those in MAOB-WT cells. A sensitivity of GGA levels to siRNA-mediated MAOB downregulation was recovered when the MAOB-KO cells were transfected with a MAOB-expression plasmid, suggesting that MAOB is the enzyme primarily responsible for GGOH oxidation and that some other latent metabolic pathways may maintain endogenous GGA levels in the MAOB-KO hepatoma cells. Along with the previous findings, these results provide critical insights into the biological roles of human MAOB and provide evidence that hepatic MAOB is involved in endogenous GGA biosynthesis via GGOH oxidation.

LSD1 inhibition yields functional insulin-producing cells from human embryonic stem cells

BackgroundHuman embryonic stem cells represent a potentially unlimited source of insulin-producing cells for diabetes therapy. While tremendous progress has been made in directed differentiation of human embryonic stem cells into IPCs in vitro, the mechanisms controlling its differentiation and function are not fully understood. Previous studies revealed that lysine-specific demethylase 1(LSD1) balanced the self-renewal and differentiation in human induced pluripotent stem cells and human embryonic stem cells. This study aims to explore the role of LSD1 in directed differentiation of human embryonic stem cells into insulin-producing cells.MethodsHuman embryonic stem cell line H9 was induced into insulin-producing cells by a four-step differentiation protocol. Lentivirus transfection was applied to knockdown LSD1 expression. Immunofluorescence assay and flow cytometry were utilized to check differentiation efficiency. Western blot was used to examine signaling pathway proteins and differentiation-associated proteins. Insulin/C-peptide release was assayed by ELISA. Statistical analysis between groups was carried out with one-way ANOVA tests or a student’s t test when appropriate.ResultsInhibition or silencing LSD1 promotes the specification of pancreatic progenitors and finally the commitment of functional insulin-producing β cells; Moreover, inhibition or silencing LSD1 activated ERK signaling and upregulated pancreatic progenitor associated genes, accelerating pre-maturation of pancreatic progenitors, and conferred the NKX6.1+ population with better proliferation ability. IPCs with LSD1 inhibitor tranylcypromine treatment displayed enhanced insulin secretion in response to glucose stimulation.ConclusionsWe identify a novel role of LSD1 inhibition in promoting IPCs differentiation from hESCs, which would be emerged as potential intervention for generation of functional pancreatic β cells to cure diabetes.

Increased expression and catalytic activity of prostacyclin synthase after simvastatin application to human umbilical vein endothelial cells

In addition to lowering blood cholesterol levels, statins are known to exert antiplatelet effects. One of the key factors contributing to the antiplatelet effects of statins includes the upregulation of prostacyclin (PGI2) level. The present study was undertaken to determine the effects of statins on prostacyclin synthase (PGIS, CYP8A1) and PGI2 synthesis at the molecular level. Human umbilical vein endothelial cells (HUVEC) were exposed to five structurally different statins (atorvastatin, simvastatin, pravastatin, lovastatin, and rosuvastatin) and changes in CYP8A1 expression levels and the metabolic activities of CYP8A1 were investigated. Among the tested statins, simvastatin induced significant PGIS expression at both transcriptional (2.9-fold, P&lt;0.05) and translational (1.8-fold, P&lt;0.05) levels. Treatment with a constitutive androstane receptor (CAR) agonist, phenobarbital, significantly increased CYP8A1 mRNA expression (3-fold, P&lt;0.01). A metabolite of prostacyclin, 6-keto prostaglandin F1?, was significantly increased by treatment with simvastatin (P&lt;0.01) and markedly repressed by the CYP8A1 inhibitor tranylcypromine (P&lt;0.01) and the CAR antagonist clotrimazole (P&lt;0.01) in HUVEC. The results of this study improve our understanding of the inter-individual variations in PGI2 levels. Clinical studies in humans are necessary to confirm the present in vitro results.