Long-Term Effects of Perinatal Exposure to a Glyphosate-Based Herbicide on Melatonin Levels and Oxidative Brain Damage in Adult Male Rats.

Concerns have been raised regarding the potential adverse health effects of the ubiquitous herbicide glyphosate. Here, we investigated long-term effects of developmental exposure to a glyphosate-based herbicide (GBH) by analyzing serum melatonin levels and cellular changes in the striatum of adult male rats (90 days old). Pregnant and lactating rats were exposed to 3% GBH (0.36% glyphosate) through drinking water from gestational day 5 to postnatal day 15. The offspring showed reduced serum melatonin levels (43%) at the adult age compared with the control group. The perinatal exposure to GBH also induced long-term oxidative stress-related changes in the striatum demonstrated by increased lipid peroxidation (45%) and DNA/RNA oxidation (39%) together with increased protein levels of the antioxidant enzymes, superoxide dismutase (SOD1, 24%), glutamate-cysteine ligase (GCLC, 58%), and glutathione peroxidase 1 (GPx1, 31%). Moreover, perinatal GBH exposure significantly increased the total number of neurons (20%) and tyrosine hydroxylase (TH)-positive neurons (38%) in the adult striatum. Mechanistic in vitro studies with primary rat pinealocytes exposed to 50 µM glyphosate demonstrated a decreased melatonin secretion partially through activation of metabotropic glutamate receptor 3 (mGluR3), while higher glyphosate levels (100 or 500 µM) also reduced the pinealocyte viability. Since decreased levels of the important antioxidant and neuroprotector melatonin have been associated with an increased risk of developing neurodegenerative disorders, this demonstrates the need to consider the melatonin hormone system as a central endocrine-related target of glyphosate and other environmental contaminants.

Perinatal exposure to a glyphosate-based herbicide causes dysregulation of dynorphins and an increase of neural precursor cells in the brain of adult male rats.

Glyphosate, the most used herbicide worldwide, has been suggested to induce neurotoxicity and behavioral changes in rats after developmental exposure. Studies of human glyphosate intoxication have reported adverse effects on the nervous system, particularly in substantia nigra (SN). Here we used matrix-assisted laser desorption ionization (MALDI) imaging mass spectrometry (IMS) to study persistent changes in peptide expression in the SN of 90-day-old adult male Wistar rats. The animals were perinatally exposed to 3 % GBH (glyphosate-based herbicide) in drinking water (corresponding to 0.36 % of glyphosate) starting at gestational day 5 and continued up to postnatal day 15 (PND15). Peptides are present in the central nervous system before birth and play a critical role in the development and survival of neurons, therefore, observed neuropeptide changes could provide better understanding of the GBH-induced long term effects on SN. The results revealed 188 significantly altered mass peaks in SN of animals perinatally exposed to GBH. A significant reduction of the peak intensity (P < 0.05) of several peptides from the opioid-related dynorphin family such as dynorphin B (57 %), alpha-neoendorphin (50 %), and its endogenous metabolite des-tyrosine alpha-neoendorphin (39 %) was detected in the GBH group. Immunohistochemical analysis confirmed a decreased dynorphin expression and showed a reduction of the total area of dynorphin immunoreactive fibers in the SN of the GBH group. In addition, a small reduction of dynorphin immunoreactivity associated with non-neuronal cells was seen in the hilus of the hippocampal dentate gyrus. Perinatal exposure to GBH also induced an increase in the number of nestin-positive cells in the subgranular zone of the dentate gyrus. In conclusion, the results demonstrate long-term changes in the adult male rat SN and hippocampus following a perinatal GBH exposure suggesting that this glyphosate-based formulation may perturb critical neurodevelopmental processes.

The cyanobacterial neurotoxin ß-N-methylamino-L-alanine (BMAA) targets the olfactory bulb region.

Olfactory dysfunction is implicated in neurodegenerative disorders and typically manifests years before other symptoms. The cyanobacterial neurotoxin ß-N-methylamino-L-alanine (BMAA) is suggested as a risk factor for neurodegenerative disease. Detection of BMAA in air filters has increased the concern that aerosolization may lead to human BMAA exposure through the air. The aim of this study was to determine if BMAA targets the olfactory system. Autoradiographic imaging showed a distinct localization of radioactivity in the right olfactory mucosa and bulb following a unilateral intranasal instillation of 3H-BMAA (0.018 µg) in mice, demonstrating a direct transfer of BMAA via the olfactory pathways to the brain circumventing the blood-brain barrier, which was confirmed by liquid scintillation. Treatment of mouse primary olfactory bulb cells with 100 µM BMAA for 24 h caused a disruption of the neurite network, formation of dendritic varicosities and reduced cell viability. The NMDA receptor antagonist MK-801 and the metabotropic glutamate receptor antagonist MCPG protected against the BMAA-induced alterations, demonstrating the importance of glutamatergic mechanisms. The ionotropic non-NMDA receptor antagonist CNQX prevented the BMAA-induced decrease of cell viability in mixed cultures containing both neuronal and glial cells, but not in cultures with neurons only, suggesting a role of neuron-glial interactions and glial AMPA receptors in the BMAA-induced toxicity. The results show that the olfactory region may be a target for BMAA following inhalation exposure. Further studies on the relations between environmental olfactory toxicants and neurodegenerative disorders are warranted.

ß-N-Methylamino-L-alanine (BMAA) perturbs alanine, aspartate and glutamate metabolism pathways in human neuroblastoma cells as determined by metabolic profiling.

ß-Methylamino-L-alanine (BMAA) is a non-proteinogenic amino acid that induces long-term cognitive deficits, as well as an increased neurodegeneration and intracellular fibril formation in the hippocampus of adult rodents following short-time neonatal exposure and in vervet monkey brain following long-term exposure. It has also been proposed to be involved in the etiology of neurodegenerative disease in humans. The aim of this study was to identify metabolic effects not related to excitotoxicity or oxidative stress in human neuroblastoma SH-SY5Y cells. The effects of BMAA (50, 250, 1000 µM) for 24 h on cells differentiated with retinoic acid were studied. Samples were analyzed using LC-MS and NMR spectroscopy to detect altered intracellular polar metabolites. The analysis performed, followed by multivariate pattern recognition techniques, revealed significant perturbations in protein biosynthesis, amino acid metabolism pathways and citrate cycle. Of specific interest were the BMAA-induced alterations in alanine, aspartate and glutamate metabolism and as well as alterations in various neurotransmitters/neuromodulators such as GABA and taurine. The results indicate that BMAA can interfere with metabolic pathways involved in neurotransmission in human neuroblastoma cells.

Low-dose exposure to bisphenol A in combination with fructose increases expression of genes regulating angiogenesis and vascular tone in juvenile Fischer 344 rat cardiac tissue.

OBJECTIVES: Epidemiological studies report associations between exposure to the high-volume chemical and endocrine disruptor bisphenol A (BPA) and cardiovascular disorders, but there is a lack of experimental studies addressing the mechanisms of action of BPA on the cardiovascular system. In the present study, effects on markers for cardiovascular function of exposure to BPA and fructose in vivo in rat cardiac tissues, and of BPA exposure in human cardiomyocytes in vitro, were investigated. MATERIALS: Juvenile female Fischer 344 rats were exposed to 5, 50, and 500 µg BPA/kg bodyweight/day in their drinking water from 5 to 15 weeks of age, in combination with 5% fructose. Further, cultured human cardiomyocytes were exposed to 10 nM BPA to 1 × 104 nM BPA for six hours. Expression of markers for cardiovascular function and BPA target receptors was investigated using qRT-PCR. RESULTS: Exposure to 5 µg BPA/kg bodyweight/day plus fructose increased mRNA expression of Vegf, Vegfr2, eNos, and Ace1 in rat heart. Exposure of human cardiomyocytes to 1 × 104 nM BPA increased mRNA expression of eNOS and ACE1, as well as IL-8 and NFκß known to regulate inflammatory response. CONCLUSIONS: . Low-dose exposure of juvenile rats to BPA and fructose induced up-regulation of expression of genes controlling angiogenesis and vascular tone in cardiac tissues. The observed effects of BPA in rat heart were in line with our present and previous studies of BPA in human endothelial cells and cardiomyocytes. These findings may aid in understanding the mechanisms of the association between BPA exposure and cardiovascular disorders reported in epidemiological studies.

Environmental neurotoxin interaction with proteins: Dose-dependent increase of free and protein-associated BMAA (ß-N-methylamino-L-alanine) in neonatal rat brain.

ß-Methylamino-L-alanine (BMAA) is implicated in the aetiology of neurodegenerative disorders. Neonatal exposure to BMAA induces cognitive impairments and progressive neurodegenerative changes including intracellular fibril formation in the hippocampus of adult rats. It is unclear why the neonatal hippocampus is especially vulnerable and the critical cellular perturbations preceding BMAA-induced toxicity remains to be elucidated. The aim of this study was to compare the level of free and protein-associated BMAA in neonatal rat brain and peripheral tissues after different exposures to BMAA. Ultra-high performance liquid chromatography-tandem mass spectrometry analysis revealed that BMAA passed the neonatal blood-brain barrier and was distributed to all studied brain areas. BMAA was also associated to proteins in the brain, especially in the hippocampus. The level in the brain was, however, considerably lower compared to the liver that is not a target organ for BMAA. In contrast to the liver there was a significantly increased level of protein-association of BMAA in the hippocampus and other brain areas following repeated administration suggesting that the degradation of BMAA-associated proteins may be lower in neonatal brain than in the liver. Additional evidence is needed in support of a role for protein misincorporation in the neonatal hippocampus for long-term effects of BMAA.

Intracellular fibril formation, calcification, and enrichment of chaperones, cytoskeletal, and intermediate filament proteins in the adult hippocampus CA1 following neonatal exposure to the nonprotein amino acid BMAA.

The environmental neurotoxin ß-N-methylamino-L-alanine (BMAA) has been implicated in the etiology of neurodegenerative disease, and recent studies indicate that BMAA can be misincorporated into proteins. BMAA is a developmental neurotoxicant that can induce long-term learning and memory deficits, as well as regionally restricted neuronal degeneration and mineralization in the hippocampal CA1. The aim of the study was to characterize long-term changes (2 weeks to 6 months) further in the brain of adult rats treated neonatally (postnatal days 9-10) with BMAA (460 mg/kg) using immunohistochemistry (IHC), transmission electron microscopy, and laser capture microdissection followed by LC-MS/MS for proteomic analysis. The histological examination demonstrated progressive neurodegenerative changes, astrogliosis, microglial activation, and calcification in the hippocampal CA1 3-6 months after exposure. The IHC showed an increased staining for α-synuclein and ubiquitin in the area. The ultrastructural examination revealed intracellular deposition of abundant bundles of closely packed parallel fibrils in neurons, axons, and astrocytes of the CA1. Proteomic analysis of the affected site demonstrated an enrichment of chaperones (e.g., clusterin, GRP-78), cytoskeletal and intermediate filament proteins, and proteins involved in the antioxidant defense system. Several of the most enriched proteins (plectin, glial fibrillar acidic protein, vimentin, Hsp 27, and ubiquitin) are known to form complex astrocytic inclusions, so-called Rosenthal fibers, in the neurodegenerative disorder Alexander disease. In addition, TDP-43 and the negative regulator of autophagy, GLIPR-2, were exclusively detected. The present study demonstrates that neonatal exposure to BMAA may offer a novel model for the study of hippocampal fibril formation in vivo.

High resolution metabolite imaging in the hippocampus following neonatal exposure to the environmental toxin BMAA using ToF-SIMS.

The environmental neurotoxin ß-N-methylamino-L-alanine (BMAA) is suggested to be linked with neurodegenerative disease. In a rat model, neonatal exposure to BMAA induced selective uptake in the hippocampus and caused cell loss, mineralization and astrogliosis as well as learning and memory impairments in adulthood. Moreover, neonatal exposure resulted in increased protein ubiquitination in the cornus ammonis 1 (CA1) region of the adult hippocampus indicating that BMAA may induce protein aggregation. Time-of-flight secondary ion mass spectrometry (ToF-SIMS) based imaging is a powerful technology for spatial profiling of small molecular weight compounds in biological tissues with high chemical specificity and high spatial resolution. The aim of this study was to characterize neurochemical changes in the hippocampus of six month-old rats treated neonatally (postnatal days 9-10) with BMAA. Multivariate data analysis of whole section ToF-SIMS scans was performed to delineate anatomical regions of interest based on their chemical distribution pattern. Further analysis of spectral data obtained from the outlined anatomical regions, including CA1 and dentate gyrus (DG) revealed BMAA-induced long-term changes. Increased levels of phospholipids and protein fragments in the histopathologically altered CA1 region as well as phosphate depletion in the DG were observed. Moreover, high resolution SIMS imaging revealed a specific localization of phosphatidylcholine lipids, protein signals and potassium in the histopathologically altered CA1. These findings demonstrate that ToF-SIMS based imaging is a powerful approach for probing biochemical changes in situ and might serve as promising technique for investigating neurotoxin-induced brain pathology.

Intranasal delivery of central nervous system-retargeted human mesenchymal stromal cells prolongs treatment efficacy of experimental autoimmune encephalomyelitis.

Treatment with mesenchymal stromal cells (MSCs) is currently of interest for a number of diseases including multiple sclerosis. MSCs are known to target inflamed tissues, but in a therapeutic setting their systemic administration will lead to few cells reaching the brain. We hypothesized that MSCs may target the brain upon intranasal administration and persist in central nervous system (CNS) tissue if expressing a CNS-targeting receptor. To demonstrate proof of concept, MSCs were genetically engineered to express a myelin oligodendrocyte glycoprotein-specific receptor. Engineered MSCs retained their immunosuppressive capacity, infiltrated into the brain upon intranasal cell administration, and were able to significantly reduce disease symptoms of experimental autoimmune encephalomyelitis (EAE). Mice treated with CNS-targeting MSCs were resistant to further EAE induction whereas non-targeted MSCs did not give such persistent effects. Histological analysis revealed increased brain restoration in engineered MSC-treated mice. In conclusion, MSCs can be genetically engineered to target the brain and prolong therapeutic efficacy in an EAE model.

Protein association of the neurotoxin and non-protein amino acid BMAA (ß-N-methylamino-L-alanine) in the liver and brain following neonatal administration in rats.

The environmental neurotoxin ß-N-methylamino-L-alanine (BMAA) is not an amino acid that is normally found in proteins. Our previous autoradiographic study of (3)H-labeled BMAA in adult mice unexpectedly revealed a tissue distribution similar to that of protein amino acids. The aim of this study was to characterize the distribution of free and protein-bound BMAA in neonatal rat tissues following a short exposure using autoradiographic imaging and ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). The autoradiographic imaging of (14)C-L-BMAA demonstrated a distinct uptake of radioactivity that was retained following acid extraction in tissues with a high rate of cell turnover and/or protein synthesis. The UHPLC-MS/MS analysis conclusively demonstrated a dose-dependent increase of protein-associated BMAA in neonatal rat tissues. The level of protein-associated BMAA in the liver was more than 10 times higher than that in brain regions not fully protected by the blood-brain barrier which may be due to the higher rate of protein synthesis in the liver. In conclusion, this study demonstrated that BMAA was associated with rat proteins suggesting that BMAA may be misincorporated into proteins. However, protein-associated BMAA seemed to be cleared over time, as none of the samples from adult rats had any detectable free or protein-associated BMAA.

Probing the lipid chemistry of neurotoxin-induced hippocampal lesions using multimodal imaging mass spectrometry.

The environmental toxin ß-N-methylamino-L-alanine (BMAA) has been causatively linked to neurodegenerative disease pathology. In a rat model, neonatal BMAA exposure resulted in selective uptake in the hippocampal formation and caused learning and memory impairments in adult animals. Moreover, high dose neonatal BMAA exposure resulted in formation of protein inclusions in the CA1 region of the adult hippocampus. However the mechanism underlying BMAA induced neuropathology remains elusive. Imaging mass spectrometry is a powerful method for spatial interrogation of biochemical distribution in biological tissue with high chemical specificity. The aim of this study was to therefore characterize the lipid microenvironment of BMAA-induced hippocampal lesions in adult rats using matrix-assisted laser desorption/ionization (MALDI) and time-of-flight SIMS (ToF-SIMS imaging). Multimodal imaging was carried out by ToF-SIMS scans of the hippocampal formation followed by whole tissue scans using MALDI imaging. Multivariate analysis was performed on the SIMS data in order to delineate the spatial biochemistry surrounding the lesions. The data show lesion-specific localization of phosphatidylcholine fragments, suggesting neuroinflammatory glial cell activation. Complementary MALDI imaging data showed increased levels of phosphoethanolamines colocalizing with the proteopathic lesions pointing to macroautophagic mechanisms associated with neurotoxin-induced protein accumulation. Multimodal IMS by means of ToF-SIMS and MALDI mass spectrometry proved to be a powerful technique for neurotoxicological research.

Maternal transfer of the cyanobacterial neurotoxin ß-N-methylamino-L-alanine (BMAA) via milk to suckling offspring.

The cyanobacterial neurotoxin ß-N-methylamino-L-alanine (BMAA) has been implicated in the etiology of neurodegenerative disease and proposed to be biomagnified in terrestrial and aquatic food chains. We have previously shown that the neonatal period in rats, which in humans corresponds to the last trimester of pregnancy and the first few years of age, is a particularly sensitive period for exposure to BMAA. The present study aimed to examine the secretion of (14)C-labeled L- and D-BMAA into milk in lactating mice and the subsequent transfer of BMAA into the developing brain. The results suggest that secretion into milk is an important elimination pathway of BMAA in lactating mothers and an efficient exposure route predominantly for L-BMAA but also for D-BMAA in suckling mice. Following secretion of [(14)C]L-BMAA into milk, the levels of [(14)C]L-BMAA in the brains of the suckling neonatal mice significantly exceeded the levels in the maternal brains. In vitro studies using the mouse mammary epithelial HC11 cell line confirmed a more efficient influx and efflux of L-BMAA than of D-BMAA in cells, suggesting enantiomer-selective transport. Competition experiments with other amino acids and a low sodium dependency of the influx suggests that the amino acid transporters LAT1 and LAT2 are involved in the transport of L-BMAA into milk. Given the persistent neurodevelopmental toxicity following injection of L-BMAA to neonatal rodent pups, the current results highlight the need to determine whether BMAA is enriched mother's and cow's milk.

The cyanobacterial amino acid ß-N-methylamino-l-alanine perturbs the intermediary metabolism in neonatal rats.

The neurotoxic amino acid ß-N-methylamino-l-alanine (BMAA) is produced by most cyanobacteria. BMAA is considered as a potential health threat because of its putative role in neurodegenerative diseases. We have previously observed cognitive disturbances and morphological brain changes in adult rodents exposed to BMAA during the development. The aim of this study was to characterize changes of major intermediary metabolites in serum following neonatal exposure to BMAA using a non-targeted metabolomic approach. NMR spectroscopy was used to obtain serum metabolic profiles from neonatal rats exposed to BMAA (40, 150, 460mg/kg) or vehicle on postnatal days 9-10. Multivariate data analysis of binned NMR data indicated metabolic pattern differences between the different treatment groups. In particular five metabolites, d-glucose, lactate, 3-hydroxybutyrate, creatine and acetate, were changed in serum of BMAA-treated neonatal rats. These metabolites are associated with changes in energy metabolism and amino acid metabolism. Further statistical analysis disclosed that all the identified serum metabolites in the lowest dose group were significantly (p<0.05) decreased. The neonatal rat model used in this study is so far the only animal model that displays significant biochemical and behavioral effects after a low short-term dose of BMAA. The demonstrated perturbation of intermediary metabolism may contribute to BMAA-induced developmental changes that result in long-term effects on adult brain function.

Neurotoxin-induced neuropeptide perturbations in striatum of neonatal rats.

The cyanobacterial toxin ß-N-methylamino-l-alanine (BMAA) is suggested to play a role in neurodegenerative disease. We have previously shown that although the selective uptake of BMAA in the rodent neonatal striatum does not cause neuronal cell death, exposure during the neonatal development leads to cognitive impairments in adult rats. The aim of the present study was to characterize the changes in the striatal neuropeptide systems of male and female rat pups treated neonatally (postnatal days 9-10) with BMAA (40-460 mg/kg). The label-free quantification of the relative levels of endogenous neuropeptides using mass spectrometry revealed that 25 peptides from 13 neuropeptide precursors were significantly changed in the rat neonatal striatum. The exposure to noncytotoxic doses of BMAA induced a dose-dependent increase of neurosecretory protein VGF-derived peptides, and changes in the relative levels of cholecystokinin, chromogranin, secretogranin, MCH, somatostatin and cortistatin-derived peptides were observed at the highest dose. In addition, the results revealed a sex-dependent increase in the relative level of peptides derived from the proenkephalin-A and protachykinin-1 precursors, including substance P and neurokinin A, in female pups. Because several of these peptides play a critical role in the development and survival of neurons, the observed neuropeptide changes might be possible mediators of BMAA-induced behavioral changes. Moreover, some neuropeptide changes suggest potential sex-related differences in susceptibility toward this neurotoxin. The present study also suggests that neuropeptide profiling might provide a sensitive characterization of the BMAA-induced noncytotoxic effects on the developing brain.

Neonatal exposure to the cyanobacterial toxin BMAA induces changes in protein expression and neurodegeneration in adult hippocampus.

The cyanobacterial toxin ß-N-methylamino-L-alanine (BMAA) has been proposed to contribute to neurodegenerative disease. We have previously reported a selective uptake of BMAA in the mouse neonatal hippocampus and that exposure during the neonatal period causes learning and memory impairments in adult rats. The aim of this study was to characterize effects in the brain of 6-month-old rats treated neonatally (postnatal days 9-10) with the glutamatergic BMAA. Protein changes were examined using the novel technique Matrix-Assisted Laser Desorption Ionization (MALDI) imaging mass spectrometry (IMS) for direct imaging of proteins in brain cryosections, and histological changes were examined using immunohistochemistry and histopathology. The results showed long-term changes including a decreased expression of proteins involved in energy metabolism and intracellular signaling in the adult hippocampus at a dose (150 mg/kg) that gave no histopathological lesions in this brain region. Developmental exposure to a higher dose (460 mg/kg) also induced changes in the expression of S100ß, histones, calcium- and calmodulin-binding proteins, and guanine nucleotide-binding proteins. At this dose, severe lesions in the adult hippocampus including neuronal degeneration, cell loss, calcium deposits, and astrogliosis were evident. The data demonstrate subtle, sometimes dose-dependent, but permanent effects of a lower neonatal dose of BMAA in the adult hippocampus suggesting that BMAA could potentially disturb many processes during the development. The detection of BMAA in seafood stresses the importance of evaluating the magnitude of human exposure to this neurotoxin.

CAR/FoxP3-engineered T regulatory cells target the CNS and suppress EAE upon intranasal delivery.

BACKGROUND: Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS). In the murine experimental autoimmune encephalomyelitis (EAE) model of MS, T regulatory (Treg) cell therapy has proved to be beneficial, but generation of stable CNS-targeting Tregs needs further development. Here, we propose gene engineering to achieve CNS-targeting Tregs from naïve CD4 cells and demonstrate their efficacy in the EAE model. METHODS: CD4+ T cells were modified utilizing a lentiviral vector system to express a chimeric antigen receptor (CAR) targeting myelin oligodendrocyte glycoprotein (MOG) in trans with the murine FoxP3 gene that drives Treg differentiation. The cells were evaluated in vitro for suppressive capacity and in C57BL/6 mice to treat EAE. Cells were administered by intranasal (i.n.) cell delivery. RESULTS: The engineered Tregs demonstrated suppressive capacity in vitro and could efficiently access various regions in the brain via i.n cell delivery. Clinical score 3 EAE mice were treated and the engineered Tregs suppressed ongoing encephalomyelitis as demonstrated by reduced disease symptoms as well as decreased IL-12 and IFNgamma mRNAs in brain tissue. Immunohistochemical markers for myelination (MBP) and reactive astrogliosis (GFAP) confirmed recovery in mice treated with engineered Tregs compared to controls. Symptom-free mice were rechallenged with a second EAE-inducing inoculum but remained healthy, demonstrating the sustained effect of engineered Tregs. CONCLUSION: CNS-targeting Tregs delivered i.n. localized to the CNS and efficiently suppressed ongoing inflammation leading to diminished disease symptoms.

Tamoxifen modulates cell migration and expression of angiogenesis-related genes in human endometrial endothelial cells.

The selective estrogen receptor modulator tamoxifen is used for the prevention and treatment of breast cancer. The adverse effects of tamoxifen include vaginal endometrial bleeding, endometrial hyperplasia, and cancer, conditions associated with angiogenesis. The aim of this study was to examine the effects of tamoxifen on cell migration and angiogenesis-related gene expression in human endometrial endothelial cells (HEECs). The regulatory effects of tamoxifen on endometrial stromal cells and HEECs were also examined. HEECs and stromal cells were isolated and grown in monocultures or co-cultures, and incubated with 0.1 to 100 µmol/L tamoxifen for 48 hours. Quantitative PCR demonstrated that tamoxifen decreased the mRNA expression of vascular endothelial growth factor-A (VEGF-A) and increased the mRNA expression of VEGF receptor-1 and placental growth factor (PLGF) in HEECs. Tamoxifen's effects on VEGF-A were inhibited when HEECs were co-cultured with stromal cells. In addition, tamoxifen reduced VEGF-induced HEEC migration. The tamoxifen-metabolizing enzymes CYP1A1 and CYP1B1 were detected by immunohistochemistry in and around endometrial blood vessels and by quantitative PCR in HEECs. Our data suggest that tamoxifen changes the regulation of angiogenesis in the endometrium, likely by reducing angiogenic activity. The results also indicate that endometrial stromal cells regulate some of tamoxifen's effects in HEECs, and the presence of tamoxifen-metabolizing enzymes suggests tamoxifen bioactivation in the endometrial vasculature in vivo. These findings may help to elucidate the mechanism of the bleeding disturbances associated with tamoxifen treatment.

Proangiogenic effects of environmentally relevant levels of bisphenol A in human primary endothelial cells.

Bisphenol A (BPA) is widely used in the manufacturing of consumer products such as plastic food containers and food cans. Experimental studies suggest a relationship between exposure to BPA and changes in metabolic processes and reproductive organs. Also, epidemiological studies report an association between elevated exposure to BPA and cardiovascular disease and diabetes. Although alterations in the vascular endothelium are implicated in pathological conditions associated with BPA, little is known about the effects of BPA in the human endothelium. This study aimed to investigate the effects of 0.1 nM-1 µM of BPA on selected biomarkers of endothelial dysfunction, inflammation, and angiogenesis in human umbilical vein endothelial cells (HUVEC). The mRNA expression of biomarkers was assayed using qRT-PCR, and the production of nitric oxide and reactive oxygen species was measured using the H(2)DCFDA and the DAF-FM assays. The effect of BPA on phosphorylated eNOS was examined using Western blot and immunofluorescence, and the endothelial tube formation assay was used to investigate in vitro angiogenesis. BPA (≤1 µM) increased the mRNA expression of the proangiogenic genes VEGFR-2, VEGF-A, eNOS, and Cx43 and increased the production of nitric oxide in HUVEC. Furthermore, BPA increased the expression of phosphorylated eNOS and endothelial tube formation in HUVEC. These studies demonstrate that environmentally relevant levels of BPA have direct proangiogenic effects on human primary endothelial cells in vitro suggesting that the human endothelium may be an important target for BPA.

Effects of PCB126 and 17ß-oestradiol on endothelium-derived vasoactive factors in human endothelial cells.

Epidemiological and experimental studies suggest an association between elevated serum levels of co-planar PCBs and hypertension, and one study indicate that this effect is dependent on the level of oestrogen. This study investigated the effects of 3,3',4,4',5-pentachlorobiphenyl (PCB126) and 17ß-oestradiol (E2) on vasoactive factors in human umbilical vein endothelial cells (HUVEC). The results reveal that PCB126 stimulated the vasoconstriction factors COX-2 and PGF(2α) in HUVEC. An up-regulation of COX-2 expression was demonstrated using qRT-PCR, western blot and immunofluorescence and increased production of PGF(2α) was demonstrated using LC/MS² and enzyme immunoassay. Also, PCB126 slightly increased ROS production and decreased NO production in HUVEC. The addition of E2 enhanced PCB126-induced transcription of CYP1A1, CYP1B1 and COX-2 in HUVEC whereas an increased transcription of eNOS only occurred following combined treatment with E2 and PCB126. Immunofluorescence demonstrated that HUVEC expressed AHR and ERß but lacked ERα and the involvement of AHR and ERß on the effects of PCB126 was examined by the addition of AHR and ER antagonists. The binding of PCB126 to AHR was critical for the effects of PCB126 whereas the role of ERß was equivocal. In conclusion, these studies suggest that PCB126 induced changes in human endothelial cells that are characteristic for endothelial dysfunction in human hypertension and that PCB126-induced transcription of genes important for vascular function in human endothelial cells can be elevated by increased oestrogen levels. These findings may help understanding the mechanism for the association between PCB126 exposure and hypertension reported in human subjects and experimental animals.