Mechanistic screening of reproductive toxicity in a novel 3D testicular co-culture model shows significant impairments following exposure to low-dibutyl phthalate concentrations.

To improve the mechanistic screening of reproductive toxicants in  chemical-risk assessment and drug development, we have developed a three-dimensional (3D) heterogenous testicular co-culture model from neonatal mice. Di-n-butyl phthalate (DBP), an environmental contaminant that can affect reproductive health negatively, was used as a model compound to illustrate the utility of the in vitro model. The cells were treated with DBP (1 nM to 100 µM) for 7 days. Automated high-content imaging confirmed the presence of cell-specific markers of Leydig cells (CYP11A1 +), Sertoli cells (SOX9 +), and germ cells (DAZL +). Steroidogenic activity of Leydig cells was demonstrated by analyzing testosterone levels in the culture medium. DBP induced a concentration-dependent reduction in testosterone levels and decreased the number of Leydig cells compared to vehicle control. The levels of steroidogenic regulator StAR and the steroidogenic enzyme CYP11A1 were decreased already at the lowest DBP concentration (1 nM), demonstrating upstream effects in the testosterone biosynthesis pathway. Furthermore, exposure to 10 nM DBP decreased the levels of the germ cell-specific RNA binding protein DAZL, central for the spermatogenesis. The 3D model also captured the development of the Sertoli cell junction proteins, N-cadherin and Zonula occludens protein 1 (ZO-1), critical for the blood-testis barrier. However, DBP exposure did not significantly alter the cadherin and ZO-1 levels. Altogether, this 3D in vitro system models testicular cellular signaling and function, making it a powerful tool for mechanistic screening of developmental testicular toxicity. This can open a new avenue for high throughput screening of chemically-induced reproductive toxicity during sensitive developmental phases.

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.

Altered gut microbiota community structure and correlated immune system changes in dibutyl phthalate exposed mice.

Di-n-butyl phthalate (DBP) is a ubiquitous environmental contaminant linked with various adverse health effects, including immune system dysfunction. Gut microbial dysbiosis can contribute to a wide range of pathogenesis, particularly immune disease. Here, we investigated the impact of DBP on the gut microbiome and examined correlations with immune system changes after five weeks oral exposure (10 or 100 mg/kg/day) in adult male mice. The fecal microbiome composition was characterized using 16S rRNA sequencing. DBP-treated mice displayed a significantly distinct microbial community composition, indicated by Bray-Curtis distance. Numerous amplicon sequence variants (ASVs) at the genus level were altered. Compared to the vehicle control group, the 10 mg/kg/day DBP group had 63 more abundant and 65 less abundant ASVs, while 60 ASVs were increased and 76 ASVs were decreased in the 100 mg/kg/day DBP group. Both DBP treatment groups showed higher abundances of ASVs assigned to Desulfovibrio (Proteobacteria phylum) and Enterorhabdus genera, while ASVs belonging to Parabacteroides, Lachnospiraceae UCG-006 and Lachnoclostridium were less common compared to the control group. Interestingly, an ASV belonging to Rumniniclostridium 6, which was less abundant in DBP-treated mice, demonstrated a negative correlation with the increased number of non-classical monocytes observed in the blood of DBP-treated animals. In addition, an ASV from Lachnospiraceae UCG-001, which was more abundant in the DBP-treated animals, showed a positive correlation with the non-classical monocyte increase. This study shows that DBP exposure greatly modifies the gut bacterial microbiome and indicates a potential contribution of microbial dysbiosis to DBP-induced immune system impairment, illustrating the importance of investigating how interactions between exposome components can affect health.

Persistent immunosuppressive effects of dibutyl phthalate exposure in adult male mice.

Increased exposure to manmade chemicals may be linked to an increase in immune-related diseases in humans and immune system dysfunction in wildlife. Phthalates are a group of endocrine-disrupting chemicals (EDCs) suspected to influence the immune system. The aim of this study was to characterize the persistent effects on leukocytes in the blood and spleen, as well as plasma cytokine and growth factor levels, one week after the end of five weeks of oral treatment with dibutyl phthalate (DBP; 10 or 100 mg/kg/d) in adult male mice. Flow cytometry analysis of the blood revealed that DBP exposure decreased the total leukocyte count, classical monocyte and T helper (Th) populations, whereas it increased the non-classical monocyte population compared to the vehicle control (corn oil). Immunofluorescence analysis of the spleen showed increased CD11b+Ly6G+ (marker of polymorphonuclear myeloid-derived suppressor cells; PMN-MDSCs), and CD43+staining (marker of non-classical monocytes), whereas CD3+ (marker of total T cells) and CD4+ (marker of Th cells) staining decreased. To investigate the mechanisms of action, levels of plasma cytokines and chemokines were measured using multiplexed immunoassays and other key factors were analyzed using western blotting. The observed increase in M-CSF levels and the activation of STAT3 may promote PMN-MDSC expansion and activity. Increased ARG1, NOX2 (gp91phox), and protein nitrotyrosine levels, as well as GCN2 and phosphor-eIRFα, suggest that oxidative stress and lymphocyte arrest drive the lymphocyte suppression caused by PMN-MDSCs. The plasma levels of IL-21 (promotes the differentiation of Th cells) and MCP-1 (regulates migration and infiltration of monocytes/macrophages) also decreased. These findings show that adult DBP exposure can cause persistent immunosuppressive effects, which may increase susceptibility to infections, cancers, and immune diseases, and decrease vaccine efficacy.

High-content analysis shows synergistic effects of low perfluorooctanoic acid (PFOS) and perfluorooctane sulfonic acid (PFOA) mixture concentrations on human breast epithelial cell carcinogenesis.

Perfluoroalkyl substances (PFAS) have been associated with cancer, but the potential underlying mechanisms need to be further elucidated and include studies of PFAS mixtures. This mechanistic study revealed that very low concentrations (500 pM) of the binary PFOS and PFOA mixture induced synergistic effects on human epithelial breast cell (MCF-10A) proliferation. The cell proliferation was mediated by pregnane X receptor (PXR) activation, an increase in cyclin D1 and CDK6/4 levels, decrease in p21 and p53 levels, and by regulation of phosphor-Akt and ß-catenin. The PFAS mixture also altered histone modifications, epigenetic mechanisms implicated in tumorigenesis, and promoted cell migration and invasion by reducing the levels of occludin. High-content screening using the cell painting assay, revealed that hundreds of cell features were affected by the PFAS mixture even at the lowest concentration tested (100 pM). The detailed phenotype profiling further demonstrated that the PFAS mixture altered cell morphology, mostly in parameters related to intensity and texture associated with mitochondria, endoplasmic reticulum, and nucleoli. Exposure to higher concentrations (≥50 µM) of the PFOS and PFOA mixture caused cell death through synergistic interactions that induced oxidative stress, DNA/RNA damage, and lipid peroxidation, illustrating the complexity of mixture toxicology. Increased knowledge about mixture-induced effects is important for better understanding of PFAS' possible role in cancer etiology, and may impact the risk assessment of these and other compounds. This study shows the potential of image-based multiplexed fluorescence assays and high-content screening for development of new approach methodologies in toxicology.

Di-n-Butyl Phthalate and Its Monoester Metabolite Impairs Steroid Hormone Biosynthesis in Human Cells: Mechanistic In Vitro Studies.

The widespread environmental contaminant di-n-butyl phthalate (DBP) has been linked with reduced testosterone levels and adverse reproductive health outcomes in men. However, the underlying mechanisms of these anti-androgenic effects and the potential effects on other classes of steroid hormones remain to be elucidated. Here, we conducted mechanistic studies in human adrenocortical H295R cells exposed to 1-500 µM of DBP or its metabolite, mono-n-butyl phthalate (MBP), for 48 h. Quantification of steroid hormones in the cell medium by liquid chromatography-mass spectrometry revealed that both phthalates significantly decreased testosterone, androstenedione, corticosterone, and progesterone levels, in particular after dibutyryl-cyclic-AMP stimulation of steroidogenesis. Western blot analysis of key steroidogenic proteins showed that DBP induced a dose-dependent decrease of CYP11A1 and HSD3ß2 levels, while MBP only significantly decreased CYP17A1 levels, indicating that the compounds affect early steps of the steroidogenesis differently. Both DBP and MBP exposure also lead to a dose-related decrease in HSD17ß3, the enzyme which catalyzes the final step in the testosterone biosynthesis pathway, although these effects were not statistically significant. Interestingly, DBP increased the cortisol concentration, which may be due to the non-significant CYP11B1 increase in DBP-exposed cells. In contrast, MBP decreased cortisol concentration. Moreover, the analysis of superoxide generation and quantification of the protein oxidation marker nitrotyrosine demonstrated that DBP induced oxidative stress in H295R cells while MBP reduced protein nitrotyrosine levels. These findings confirm the anti-androgenic effects of DBP and MBP and reveal several differences in their toxicological mechanisms, with possible implications for future research on phthalate toxicity.

Adult Exposure to Di-N-Butyl Phthalate (DBP) Induces Persistent Effects on Testicular Cell Markers and Testosterone Biosynthesis in Mice.

Studies indicate that phthalates are endocrine disruptors affecting reproductive health. One of the most commonly used phthalates, di-n-butyl phthalate (DBP), has been linked with adverse reproductive health outcomes in men, but the mechanisms behind these effects are still poorly understood. Here, adult male mice were orally exposed to DBP (10 or 100 mg/kg/day) for five weeks, and the testis and adrenal glands were collected one week after the last dose, to examine more persistent effects. Quantification of testosterone, androstenedione, progesterone and corticosterone concentrations by liquid chromatography-mass spectrometry showed that testicular testosterone was significantly decreased in both DBP treatment groups, whereas the other steroids were not significantly altered. Western blot analysis of testis revealed that DBP exposure increased the levels of the steroidogenic enzymes CYP11A1, HSD3ß2, and CYP17A1, the oxidative stress marker nitrotyrosine, and the luteinizing hormone receptor (LHR). The analysis further demonstrated increased levels of the germ cell marker DAZL, the Sertoli cell markers vimentin and SOX9, and the Leydig cell marker SULT1E1. Overall, the present work provides more mechanistic understanding of how adult DBP exposure can induce effects on the male reproductive system by affecting several key cells and proteins important for testosterone biosynthesis and spermatogenesis, and for the first time shows that these effects persist at least one week after the last dose. It also demonstrates impairment of testosterone biosynthesis at a lower dose than previously reported.

Tumorigenic activity of alternative per- and polyfluoroalkyl substances (PFAS): Mechanistic in vitro studies.

Environmental contaminants including long-chain per- and polyfluoroalkyl substances (PFAS) have been linked to cancer, which is a central cause of mortality in humans and many wildlife species. Today shorter-chain PFAS are extensively used as replacement compounds and commonly found in the environment. Mechanistic studies are important for a better understanding of their toxicological potential and possible role in cancer etiology. Here, we treated normal human breast epithelial cells (MCF-10A) with 500 pM to 500 µM of perfluorohexane sulfonate (PFHxS), undecafluorohexanoic acid (PFHxA), hexafluoropropylene oxide-dimer acid (GenX), perfluoro 3,6 dioxaoctanoic acid (PFO2OA), heptafluorobutyric acid (HFBA) and perfluorobutanesulfonic acid (PFBS) for 72 h to investigate potential effects on cell proliferation and neoplastic transformation. PFHxA, GenX, PFO2OA, HFBA and PFBS induced no alterations compared to controls at any of the concentrations tested. Exposure to 100 µM PFHxS on the other hand was shown to affect important regulatory cell-cycle proteins (cyclin D1, CDK6, p27, p53 and ERK) and induced cell proliferation, at least in part through activation of the constitutive androstane receptor (CAR) and the peroxisome proliferator-activated receptor alpha (PPARα). PFHxS also altered histone modifications and induced cell malignance by reducing the levels of adhesion proteins (E-cadherin and ß-integrin) and promoting cell migration and invasion. These results demonstrate that five out of six alternative PFAS tested are clearly less harmful to MCF-10A cells than previously studied PFOS and PFOA, but raise concerns about PFHxS that also has been associated with breast cancer in epidemiological studies.

Differential susceptibility of rat primary neurons and neural stem cells to PFOS and PFOA toxicity.

Per and polifluorinated substances (PFAS) are ubiquitous and persistent contaminants. Studies have indicated that fetuses and infants can be exposed to these chemicals in utero and through breastfeeding. Despite this, limited data about their effects on brain development are available. Here, we compared the effects of perfluoroctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA) exposure in rat primary neurons and neural stem cells (NSC). Treatment with 1-250 µM of either of these compounds caused no effects on cell viability or proliferation in primary neurons, while PFOS exposure increased the NSC proliferation already at the lowest concentration tested (1-100 µM). Further analysis showed that both PFOS and PFOA caused morphological alterations of NSC-derived neurons. The neurons derived from NSC treated with either of the PFAS demonstrated a decrease in cell body area. Exposure to 1 and 10 µM PFOA also affected the neurite network and caused an increase in the number of processes and branches per cell. None of the PFAS caused morphological alterations in primary neurons. These data suggest that NSC, mimicking the immature brain, is clearly more susceptible to PFOS and PFOA exposure than the primary neurons. The PFAS-induced alterations in NSC may be related to neurobehavioral alterations observed in rodents developmentally exposed to these compounds, and show the importance to consider the effects of these compounds on human brain development and disease.

Hippocampal neural stem cells are more susceptible to the neurotoxin BMAA than primary neurons: effects on apoptosis, cellular differentiation, neurite outgrowth, and DNA methylation.

Developmental exposure to the environmental neurotoxin ß-N-methylamino-L-alanine (BMAA), a proposed risk factor for neurodegenerative disease, can induce long-term cognitive impairments and neurodegeneration in rats. While rodent studies have demonstrated a low transfer of BMAA to the adult brain, this toxin is capable to cross the placental barrier and accumulate in the fetal brain. Here, we investigated the differential susceptibility of primary neuronal cells and neural stem cells from fetal rat hippocampus to BMAA toxicity. Exposure to 250 µM BMAA induced cell death in neural stem cells through caspase-independent apoptosis, while the proliferation of primary neurons was reduced only at 3 mM BMAA. At the lowest concentrations tested (50 and 100 µM), BMAA disrupted neural stem cell differentiation and impaired neurite development in neural stem cell-derived neurons (e.g., reduced neurite length, the number of processes and branches per cell). BMAA induced no alterations of the neurite outgrowth in primary neurons. This demonstrates that neural stem cells are more susceptible to BMAA exposure than primary neurons. Importantly, the changes induced by BMAA in neural stem cells were mitotically inherited to daughter cells. The persistent nature of the BMAA-induced effects may be related to epigenetic alterations that interfere with the neural stem cell programming, as BMAA exposure reduced the global DNA methylation in the cells. These findings provide mechanistic understanding of how early-life exposure to BMAA may lead to adverse long-term consequences, and potentially predispose for neurodevelopmental disorders or neurodegenerative disease later in life.

Correction to: Perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA) induce epigenetic alterations and promote human breast cell carcinogenesis in vitro.

The original article can be found online.

Perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA) induce epigenetic alterations and promote human breast cell carcinogenesis in vitro.

Gene-environment interactions are involved in the development of breast cancer, the tumor type that accounts for the majority of the cancer-related deaths among women. Here, we demonstrate that exposure to PFOS (10 µM) and PFOA (100 µM)-two contaminants ubiquitously found in human blood-for 72 h induced breast epithelial cell (MCF-10A cell line) proliferation and alteration of regulatory cell-cycle proteins (cyclin D1, CDK6, p21, p53, p27, ERK 1/2 and p38) that persisted after a multitude of cell divisions. The contaminants also promoted cell migration and invasion by reducing the levels of E-cadherin, occludin and ß-integrin in the unexposed daughter cells. The compounds further induced an increase in global DNA methylation and differentially altered histone modifications, epigenetic mechanisms implicated in tumorigenesis. This mechanistic evidence for PFOS- and PFOA-induced malignant transformation of human breast cells supports a role of these abundant contaminants in the development and progression of breast cancer. Increased knowledge of contaminant-induced effects and their contribution to breast tumorigenesis is important for a better understanding of gene-environment interactions in the etiology of breast cancer.

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.

Mitotically heritable effects of BMAA on striatal neural stem cell proliferation and differentiation.

The widespread environmental contaminant ß-methylamino-L-alanine (BMAA) is a developmental neurotoxicant that can induce long-term learning and memory deficits. Studies have shown high transplacental transfer of 3H-BMAA and a significant uptake in fetal brain. Therefore, more information on how BMAA may influence growth and differentiation of neural stem cells is required for assessment of the risk to the developing brain. The aim of this study was to investigate direct and mitotically inherited effects of BMAA exposure using primary striatal neurons and embryonic neural stem cells. The neural stem cells were shown to be clearly more susceptible to BMAA exposure than primary neurons. Exposure to 250 µM BMAA reduced neural stem cell proliferation through apoptosis and G2/M arrest. At lower concentrations (50-100 µM), not affecting cell proliferation, BMAA reduced the differentiation of neural stem cells into astrocytes, oligodendrocytes, and neurons through glutamatergic mechanisms. Neurons that were derived from the BMAA-treated neuronal stem cells demonstrated morphological alterations including reduced neurite length, and decreased number of processes and branches per cell. Interestingly, the BMAA-induced changes were mitotically heritable to daughter cells. The results suggest that early-life exposure to BMAA impairs neuronal stem cell programming, which is vital for development of the nervous system and may result in long-term consequences predisposing for both neurodevelopmental disorders and neurodegenerative disease later in life. More attention should be given to the potential adverse effects of BMAA exposure on brain development.

The environmental neurotoxin ß-N-methylamino-L-alanine inhibits melatonin synthesis in primary pinealocytes and a rat model.

The environmental neurotoxin ß-N-methylamino-L-alanine (BMAA) is a glutamate receptor agonist that can induce oxidative stress and has been implicated as a possible risk factor for neurodegenerative disease. Detection of BMAA in mussels, crustaceans, and fish illustrates that the sources of human exposure to this toxin are more abundant than previously anticipated. The aim of this study was to determine uptake of BMAA in the pineal gland and subsequent effects on melatonin production in primary pinealocyte cultures and a rat model. Autoradiographic imaging of 10-day-old male rats revealed a high and selective uptake in the pineal gland at 30 minutes to 24 hours after 14 C-L-BMAA administration (0.68 mg/kg). Primary pinealocyte cultures exposed to 0.05-3 mmol/L BMAA showed a 57%-93% decrease in melatonin synthesis in vitro. Both the metabotropic glutamate receptor 3 (mGluR3) antagonist Ly341495 and the protein kinase C (PKC) activator phorbol-12-myristate-13-acetate prevented the decrease in melatonin secretion, suggesting that BMAA inhibits melatonin synthesis by mGluR3 activation and PKC inhibition. Serum analysis revealed a 45% decrease in melatonin concentration in neonatal rats assessed 2 weeks after BMAA administration (460 mg/kg) and confirmed an inhibition of melatonin synthesis in vivo. Given that melatonin is a most important neuroprotective molecule in the brain, the etiology of BMAA-induced neurodegeneration may include mechanisms beyond direct excitotoxicity and oxidative stress.

Perfluorooctanoic acid (PFOA) exposure promotes proliferation, migration and invasion potential in human breast epithelial cells.

Despite significant advances in early detection and treatment, breast cancer remains a major cause of morbidity and mortality. Perfluorooctanoic acid (PFOA) is a suspected endocrine disruptor and a common environmental pollutant associated with various diseases including cancer. However, the effects of PFOA and its mechanisms of action on hormone-responsive cells remain unclear. Here, we explored the potential tumorigenic activity of PFOA (100 nM-1 mM) in human breast epithelial cells (MCF-10A). MCF-10A cells exposed to 50 and 100 µM PFOA demonstrated a higher growth rate compared to controls. The compound promoted MCF-10A proliferation by accelerating G0/G1 to S phase transition of the cell cycle. PFOA increased cyclin D1 and CDK4/6 levels, concomitant with a decrease in p27. In contrast to previous studies of perfluorooctane sulfate (PFOS), the estrogen receptor antagonist ICI 182,780 had no effect on PFOA-induced cell proliferation, whereas the PPARα antagonist GW 6471 was able to prevent the MCF-10A proliferation, indicating that the underlying mechanisms involve PPARα-dependent pathways. Interestingly, we also showed that PFOA is able to stimulate cell migration and invasion, demonstrating its potential to induce neoplastic transformation of human breast epithelial cells. These results suggest that more attention should be paid to the roles of PFOA in the development and progression of breast cancer.

Kynurenic Acid Restores Nrf2 Levels and Prevents Quinolinic Acid-Induced Toxicity in Rat Striatal Slices.

Kynurenic acid (KYNA) and quinolinic acid (QUIN) are metabolites produced in the degradation of tryptophan and have important neurological activities. KYNA/QUIN ratio changes are known to be associated with central nervous system disorders, such Alzheimer, Parkinson, and Huntington diseases. In the present study, we investigate the ability of KYNA in prevent the first events preceding QUIN-induced neurodegeneration in striatal slices of rat. We evaluated the protective effect of KYNA on oxidative status (reactive oxygen species production, antioxidant enzymes activities, lipid peroxidation, nitrite levels, protein and DNA damage, and iNOS immunocontent), mitochondrial function (mitochondrial mass, membrane potential, and respiratory chain enzymes), and Na+,K+-ATPase in striatal slices of rats treated with QUIN. Since QUIN alters the levels of Nrf2, we evaluated the influence of KYNA protection on this parameter. Striatal slices from 30-day-old Wistar rats were preincubated with KYNA (100 µM) for 15 min, followed by incubation with 100-µM QUIN for 30 min. Results showed that KYNA prevented the increase of ROS production caused by QUIN and restored antioxidant enzyme activities and the protein and lipid damage, as well as the Nrf2 levels. KYNA also prevented the effects of QUIN on mitochondrial mass and mitochondrial membrane potential, as well as the decrease in the activities of complex II, SDH, and Na+,K+-ATPase. We suggest that KYNA prevents changes in Nrf2 levels, oxidative imbalance, and mitochondrial dysfunction caused by QUIN in striatal slices. This study elucidates some of the protective effects of KYNA against the damage caused by QUIN toxicity.

Toxic Synergism Between Quinolinic Acid and Glutaric Acid in Neuronal Cells Is Mediated by Oxidative Stress: Insights to a New Toxic Model.

It has been shown that synergistic toxic effects of quinolinic acid (QUIN) and glutaric acid (GA), both in isolated nerve endings and in vivo conditions, suggest the contribution of these metabolites to neurodegeneration. However, this synergism still requires a detailed characterization of the mechanisms involved in cell damage during its occurrence. In this study, the effects of subtoxic concentrations of QUIN and/or GA were tested in neuronal cultures, co-cultures (neuronal cells + astrocytes), and mixed cultures (neuronal cells + astrocytes + microglia) from rat cortex and striatum. The exposure of different cortical and striatal cell cultures to QUIN + GA resulted in cell death and stimulated different markers of oxidative stress, including reactive oxygen species (ROS) formation; changes in the activity of antioxidant enzymes such as superoxide dismutase, catalase, and glutathione peroxidase; and depletion of endogenous antioxidants such as -SH groups and glutathione. The co-incubation of neuronal cultures with QUIN + GA plus the N-methyl-D-aspartate antagonist MK-801 prevented cell death but not ROS formation, whereas the antioxidant melatonin reduced both parameters. Our results demonstrated that QUIN and GA can create synergistic scenarios, inducing toxic effects on some parameters of cell viability via the stimulation of oxidative damage. Therefore, it is likely that oxidative stress may play a major causative role in the synergistic actions exerted by QUIN + GA in a variety of cell culture conditions involving the interaction of different neural types.

Kynurenic Acid Prevents Cytoskeletal Disorganization Induced by Quinolinic Acid in Mixed Cultures of Rat Striatum.

Kynurenic acid (KYNA) is a neuroactive metabolite of tryptophan known to modulate a number of mechanisms involved in neural dysfunction. Although its activity in the brain has been widely studied, the effect of KYNA counteracting the actions of quinolinic acid (QUIN) remains unknown. The present study aims at describing the ability of 100 µM KYNA preventing cytoskeletal disruption provoked by QUIN in astrocyte/neuron/microglia mixed culture. KYNA totally preserved cytoskeletal organization, cell morphology, and redox imbalance in mixed cultures exposed to QUIN. However, KYNA partially prevented morphological alteration in isolated primary astrocytes and failed to protect the morphological alterations of neurons caused by QUIN exposure. Moreover, KYNA prevented QUIN-induced microglial activation and upregulation of ionized calcium-binding adapter molecule 1 (Iba-1) and partially preserved tumor necrosis factor-α (TNF-α) level in mixed cultures. TNF-α level was also partially preserved in astrocytes. In addition to the mechanisms dependent on redox imbalance and microglial activation, KYNA prevented downregulation of connexin-43 and the loss of functionality of gap junctions (GJs), preserving cell-cell contact, cytoskeletal organization, and cell morphology in QUIN-treated cells. Furthermore, the toxicity of QUIN targeting the cytoskeleton of mixed cultures was not prevented by the N-methyl-D-aspartate (NMDA) antagonist MK-801. We suggest that KYNA protects the integrity of the cytoskeleton of mixed cultures by complex mechanisms including modulating microglial activation preventing oxidative imbalance and misregulated GJs leading to disrupted cytoskeleton in QUIN-treated cells. This study contributed to elucidate the molecular basis of KYNA protection against QUIN toxicity.