Distribution of morphine and methadone to the brain in a developmental chicken embryo model.

The use and/or misuse of opioids by pregnant women would expose the fetuses to these drugs during critical stages of development with serious effects for the newborn, like the neonatal abstinence syndrome (NAS). We have revisited an established chicken model for NAS to describe the distribution of morphine and methadone to the brain and explore its validity as a valuable alternative to rodent models. For this purpose, chicken eggs were injected with a single dose of 10 mg/kg or 20 mg/kg morphine or 20 mg/kg methadone onto the chorioallantoic membrane (CAM) on embryonal day 13. Whole brains and lungs were harvested and the concentrations of morphine, methadone and their subsequent metabolites (morphine-3-glucuronide and EDDP, respectively) determined in the brain and lungs at different time points using LC-MS/MS. Morphine and methadone, as well as their metabolites, were detected both in the brain and lungs, with significantly higher concentrations in the lungs. Pharmacokinetic modelling showed that the distribution of morphine to the brain followed a first-order absorption with transit compartments and linear elimination, with concentrations linearly dependent on dose. Moreover, methadone, but not morphine, reduced µ receptor (the main morphine receptor) binding, which can be of relevance for opioid tolerance. The present study is the first to report the brain distribution of morphine, which can be described by standard pharmacokinetic processes, and methadone in the developing chicken embryo. The present findings supplement the already established model and support the use of this chicken model to study NAS.

ANDA: an open-source tool for automated image analysis of in vitro neuronal cells.

BACKGROUND: Imaging of in vitro neuronal differentiation and measurements of cell morphologies have led to novel insights into neuronal development. Live-cell imaging techniques and large datasets of images have increased the demand for automated pipelines for quantitative analysis of neuronal morphological metrics. RESULTS: ANDA is an analysis workflow that quantifies various aspects of neuronal morphology from high-throughput live-cell imaging screens of in vitro neuronal cell types. This tool automates the analysis of neuronal cell numbers, neurite lengths and neurite attachment points. We used chicken, rat, mouse, and human in vitro models for neuronal differentiation and have demonstrated the accuracy, versatility, and efficiency of the tool. CONCLUSIONS: ANDA is an open-source tool that is easy to use and capable of automated processing from time-course measurements of neuronal cells. The strength of this pipeline is the capability to analyse high-throughput imaging screens.

Antidepressants escitalopram and venlafaxine up-regulate BDNF promoter IV but down-regulate neurite outgrowth in differentiating SH-SY5Y neurons.

Antidepressants are used to treat depression and some anxiety disorders, including use in pregnant patients. The pharmacological actions of these drugs generally determine the uptake and metabolism of a series of neurotransmitters, such as serotonin, norepinephrine, or dopamine, along with an increase in BDNF expression. However, many aspects of antidepressant action remain unknown, particularly whether antidepressants interfere with normal neurodevelopment when taken by pregnant women. In order to reveal cellular and molecular implications crucial to the functioning of pathways related to antidepressant effects, we performed an investigation on neuronally differentiating human SH-SY5Y cells. To our knowledge, this is the first time human SH-SY5Y cells in cultures of purely neuronal cells induced by controlled differentiation with retinoic acid are followed by short-term 48-h exposure to 0.1-10 µM escitalopram or venlafaxine. Treatment with antidepressants (1 µM) did not affect the electrophysiological properties of SH-SY5Y cells. However, the percentage of mature neurons exhibiting voltage-gated sodium currents was substantially higher in cultures pre-treated with either antidepressant. After exposure to escitalopram or venlafaxine, we observed a concentration-dependent increase in activity-dependent BDNF promoter IV activation. The assessment of neurite metrics showed significant down-regulation of neurite outgrowth upon exposure to venlafaxine. Identified changes may represent links to molecular processes of importance to depression and be involved in neurodevelopmental alterations observed in postpartum children exposed to antidepressants antenatally.

ANDA: An open-source tool for automated image analysis of neuronal differentiation.

Background: Imaging of in vitro neuronal differentiation and measurements of cell morphologies has led to novel insights into neuronal development. Live-cell imaging techniques and large datasets of images has increased the demand for automated pipelines for quantitative analysis of neuronal morphological metrics. Results: We present ANDA, an analysis workflow for quantification of various aspects of neuronal morphology from high-throughput live-cell imaging screens. This tool automates the analysis of neuronal cell numbers, neurite lengths and neurite attachment points. We used rat, chicken and human in vitro models for neuronal differentiation and have demonstrated the accuracy, versatility, and efficiency of the tool. Conclusions: ANDA is an open-source tool that is easy to use and capable of automated processing from time-course measurements of neuronal cells. The strength of this pipeline is the capability to analyse high-throughput imaging screens.

Exposure to a human relevant mixture of persistent organic pollutants or to perfluorooctane sulfonic acid alone dysregulates the developing cerebellum of chicken embryo.

Prenatal exposure to persistent organic pollutants (POPs) is associated with neurodevelopmental disorders. In the present study, we explored whether a human-relevant POP mixture affects the development of chicken embryo cerebellum. We used a defined mixture of 29 POPs, with chemical composition and concentrations based on blood levels in the Scandinavian population. We also evaluated exposure to a prominent compound in the mixture, perfluorooctane sulfonic acid (PFOS), alone. Embryos (n = 7-9 per exposure group) were exposed by injection directly into the allantois at embryonic day 13 (E13). Cerebella were isolated at E17 and subjected to morphological, RNA-seq and shot-gun proteomics analyses. There was a reduction in thickness of the molecular layer of cerebellar cortex in both exposure scenarios. Exposure to the POP mixture significantly affected expression of 65 of 13,800 transcripts, and 43 of 2,568 proteins, when compared to solvent control. PFOS alone affected expression of 80 of 13,859 transcripts, and 69 of 2,555 proteins. Twenty-five genes and 15 proteins were common for both exposure groups. These findings point to alterations in molecular events linked to retinoid X receptor (RXR) signalling, neuronal cell proliferation and migration, cellular stress responses including unfolded protein response, lipid metabolism, and myelination. Exposure to the POP mixture increased methionine oxidation, whereas PFOS decreased oxidation. Several of the altered genes and proteins are involved in a wide variety of neurological disorders. We conclude that POP exposure can interfere with fundamental aspects of neurodevelopment, altering molecular pathways that are associated with adverse neurocognitive and behavioural outcomes.

Developmental neurotoxicity of acrylamide and its metabolite glycidamide in a human mixed culture of neurons and astrocytes undergoing differentiation in concentrations relevant for human exposure.

Neural stem cells (NSCs) derived from human induced pluripotent stem cells were used to investigate effects of exposure to the food contaminant acrylamide (AA) and its main metabolite glycidamide (GA) on key neurodevelopmental processes. Diet is an important source of human AA exposure for pregnant women, and AA is known to pass the placenta and the newborn may also be exposed through breast feeding after birth. The NSCs were exposed to AA and GA (1 ×10-8 - 3 ×10-3 M) under 7 days of proliferation and up to 28 days of differentiation towards a mixed culture of neurons and astrocytes. Effects on cell viability was measured using Alamar Blue™ cell viability assay, alterations in gene expression were assessed using real time PCR and RNA sequencing, and protein levels were quantified using immunocytochemistry and high content imaging. Effects of AA and GA on neurodevelopmental processes were evaluated using endpoints linked to common key events identified in the existing developmental neurotoxicity adverse outcome pathways (AOPs). Our results suggest that AA and GA at low concentrations (1 ×10-7 - 1 ×10-8 M) increased cell viability and markers of proliferation both in proliferating NSCs (7 days) and in maturing neurons after 14-28 days of differentiation. IC50 for cell death of AA and GA was 5.2 × 10-3 M and 5.8 × 10-4 M, respectively, showing about ten times higher potency for GA. Increased expression of brain derived neurotrophic factor (BDNF) concomitant with decreased synaptogenesis were observed for GA exposure (10-7 M) only at later differentiation stages, and an increased number of astrocytes (up to 3-fold) at 14 and 21 days of differentiation. Also, AA exposure gave tendency towards decreased differentiation (increased percent Nestin positive cells). After 28 days, neurite branch points and number of neurites per neuron measured by microtubule-associated protein 2 (Map2) staining decreased, while the same neurite features measured by ßIII-Tubulin increased, indicating perturbation of neuronal differentiation and maturation.

Paracetamol perturbs neuronal arborization and disrupts the cytoskeletal proteins SPTBN1 and TUBB3 in both human and chicken in vitro models.

Epidemiological studies have linked long-term/high-dose usage of paracetamol (N-acetyl-para-aminophenol, APAP) during pregnancy to adverse neuropsychiatric outcomes, primarily attention-deficit hyperactive disorder (ADHD), in the offspring. Though variable, ADHD has been associated with phenotypic alterations characterized by reductions in grey matter densities and aberrations in structural connectivity, effects which are thought to originate in neurodevelopment. We used embryonic chicken cerebellar granule neurons (CGNs) and neuronally differentiating human NTERA2 cells (NT2Ns) to investigate the in vitro effects of APAP on cell viability, migration, neuritogenesis, and the intracellular levels of various proteins involved in neurodevelopment as well as in the maintenance of the structure and function of neurites. Exposure to APAP ranging from 100 to 1600 µM yielded concentration- and time-dependent reductions in cell viability and levels of neurite arborization, as well as reductions in the levels of the cytoskeletal protein ß2-spectrin, with the highest APAP concentration resulting in between 50 and 75% reductions in the aforementioned metrics over the course of 72 h. Exposure to APAP also reduced migration in the NT2Ns but not CGNs. Moreover, we found concentration- and time-dependent increases in punctate aggregation of the cytoskeletal protein ß3-tubulin following exposure to APAP in both cell model systems, with the highest APAP concentration approximately doubling the number of aggregates over 72-120 h. Our findings demonstrate that APAP negatively perturbs neurite arborization degree, with concurrent reductions in the protein levels of ß2-spectrin and disruption of the integrity of ß3-tubulin, both proteins of which play important roles in neuronal structure and function.

Peripherally administered persistent organic pollutants distribute to the brain of developing chicken embryo in concentrations relevant for human exposure.

Persistent organic pollutants (POPs) can reach the fetal brain and contribute to developmental neurotoxicity. To explore the distribution of POPs to the fetal brain, we exposed chicken embryos to a POP mixture, containing 29 different compounds with concentrations based on blood levels measured in the Scandinavian human population. The mixture was injected into the allantois at embryonic day 13 (E13), aiming at a theoretical concentration of 10 times human blood levels. POPs concentrations in the brain were measured at 0.5, 1, 2, 4, 6, 24, 48, and 72 h after administration. Twenty-seven of the individual compounds were detected during at least one of the time-points analyzed. Generally, the concentrations of most of the measured compounds were within the order of magnitude of those reported in human brain samples. Differences in the speed of distribution to the brain were observed between the per- and polyfluoroalkyl substances (PFASs), which have protein binding potential, and the lipophilic polychlorinated biphenyls (PCBs), organochlorine pesticides (OCPs) and brominated flame retardants (BFRs). Based on pharmacokinetic modeling, PFASs were best described by a one compartment model. PFASs displayed relatively slow elimination (Kel) and persisted at high levels in the brain. Lipophilic OCPs and PCBs could be fitted to a 2-compartment model. These showed high levels in the brain relative to the dose administrated as calculated by area under the curve (AUC)/Dose. Altogether, our study showed that chicken is a suitable model to explore the distribution of POPs into the developing brain at concentrations which are relevant for humans.

Antiepileptic drugs lamotrigine and valproate differentially affect neuronal maturation in the developing chick embryo, yet with PAX6 as a potential common mediator.

Exposing the immature nervous system to specific antiepileptic drugs (AEDs) during pregnancy is linked to neurodevelopmental disorders such as autism spectrum disorder (ASD). Newer AEDs like lamotrigine (LTG) are hailed as safer, but recent epidemiological data suggest that even LTG carries a risk, although much lower than that associated with valproic acid (VPA), an older AED, which is also known to cause morphological alterations in the developing brain. Increasing evidence highlights cerebellar abnormalities as important in ASD pathophysiology. Transcription factor PAX6 is a key activity-dependent mediator and regulates crucial processes during cerebellar development. The chicken cerebellum recapitulates important characteristics of human cerebellar development, and may thus be suitable for the assessment of interventions aiming to modify maturation and cerebellar development. In the present study, exposure of chicken on embryonic day 16 (E16) to LTG or VPA resulted in decreased cerebellar mass and level of proliferating nuclear cell antigen (PCNA) for clinically relevant concentrations of VPA. However, both AEDs reduced cerebellar protein levels of PAX6 and MMP-9 at E17. Furthermore, PAX6 immunohistochemical staining of coronal sections of chicken cerebellum showed a significant reduction in PAX6-positive cell density and changes in cerebellar cortex thickness, mostly caused by the change in IGL-layer thickness. In conclusion, prenatal exposure to LTG or VPA provoked differential maturational changes in the developing cerebellum that may reflect some of the underlying molecular mechanisms for the observed human ASD pathology after AEDs exposure during pregnancy.

Chicken embryo as animal model to study drug distribution to the developing brain.

INTRODUCTION: Rodent models are routinely used to assess the safety and developmental toxicity of pharmaceuticals, along with analysis of their distribution. These models require sacrifice of parent females, have challenges in the estimation of the number of embryos and stage of development, and are expensive and time-consuming. In this study, we used fertilized chicken eggs as an alternative model to address drug distribution to the developing brain of two antiepileptic drugs, valproic acid (VPA) and lamotrigine (LTG) at two developmental stages. METHODS: VPA or LTG was injected into the allantois of the egg on embryonic day 13 (E13) or E16. Whole chicken brains were harvested at time-points of 5 min to 24 h and the concentrations of the drugs determined using GC/MS and LC-MS/MS, for VPA and LTG, respectively. RESULTS: VPA and LTG had distinct absorption and elimination phases and were found in the brain as early as 5-15 min after injection. Both drugs reached the brain in clinically relevant concentrations, with Cmax 10-30% of the calculated concentration assuming uniform distribution throughout the egg. LTG concentrations were higher when injected at E13 compared to E16. CONCLUSION: The chicken embryo model may be a suitable alternative animal model for preclinical drug distribution studies. It enables to easily approach antenatal development on an individual level, with a precise number of experimental animals, high reproducibility and low time and cost. Knowledge of the concentrations reaching the brain at different developmental stages with different drugs is important for the planning and interpretation of neurodevelopmental toxicity studies.

A human relevant mixture of persistent organic pollutants (POPs) and perfluorooctane sulfonic acid (PFOS) differentially affect glutamate induced excitotoxic responses in chicken cerebellum granule neurons (CGNs) in vitro.

Primary cultures of cerebellar granule neurons (CGNs) derived from chicken embryos were used to explore the effects on developmental neurotoxicity by a complex defined mixture of persistent organic pollutants (POPs). Its chemical composition and concentrations were based on blood levels in the Norwegian/Scandinavian population. Perfluorooctane sulfonic acid (PFOS) alone, its most abundant compound was also evaluated. Different stages of CGNs maturation, between day in vitro (DIV) 1, 3, and 5 were exposed to the POP mixture, or PFOS alone. Their combination with glutamate, an excitatory endogenous neurotransmitter important in neurodevelopment, also known to cause excitotoxicity was evaluated. Outcomes with the mixture at 500x blood levels were compared to PFOS at its corresponding concentration of 20 µM. The POP mixture reduced tetrazolium salt (MTT) conversion at earlier stages of maturation, compared to PFOS alone. Glutamate-induced excitotoxicity was enhanced above the level of that induced by glutamate alone, especially in mature CGNs at DIV5. Glutathione (GSH) concentrations seemed to set the level of sensitivity for the toxic insults from exposures to the pollutants. The role of N-methyl-D-aspartate receptor (NMDA-R) mediated calcium influx in pollutant exposures was investigated using the non-competitive and competitive receptor antagonists MK-801 and CGP 39551. Observations indicate a calcium-independent, but still NMDA-R dependent mechanism in the absence of glutamate, and a calcium- and NMDA-R dependent one in the presence of glutamate. The outcomes for the POP mixture cannot be explained by PFOS alone, indicating that other chemicals in the mixture contribute its overall effect.

Effects of a human-based mixture of persistent organic pollutants on the in vivo exposed cerebellum and cerebellar neuronal cultures exposed in vitro.

Exposure to persistent organic pollutants (POPs), encompassing chlorinated (Cl), brominated (Br) and perfluoroalkyl acid (PFAA) compounds is associated with adverse neurobehaviour in humans and animals, and is observed to cause adverse effects in nerve cell cultures. Most studies focus on single POPs, whereas studies on effects of complex mixtures are limited. We examined the effects of a mixture of 29 persistent compounds (Cl + Br + PFAA, named Total mixture), as well as 6 sub-mixtures on in vitro exposed rat cerebellar granule neurons (CGNs). Protein expression studies of cerebella from in vivo exposed mice offspring were also conducted. The selection of chemicals for the POP mixture was based on compounds being prominent in food, breast milk or blood from the Scandinavian human population. The Total mixture and sub-mixtures containing PFAAs caused greater toxicity in rat CGNs than the single or combined Cl/Br sub-mixtures, with significant impact on viability from 500x human blood levels. The potencies for these mixtures based on LC50 values were Br + PFAA mixture > Total mixture > Cl + PFAA mixture > PFAA mixture. These mixtures also accelerated induced lipid peroxidation. Protection by the competitive N-methyl-D-aspartate (NMDA) receptor antagonist 3-((R)-2-Carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP) indicated involvement of the NMDA receptor in PFAA and Total mixture-, but not Cl mixture-induced toxicity. Gene-expression studies in rat CGNs using a sub-toxic and marginally toxic concentration ((0.4 nM-5.5 µM) 333x and (1 nM-8.2 µM) 500x human blood levels) of the mixtures, revealed differential expression of genes involved in apoptosis, oxidative stress, neurotransmission and cerebellar development, with more genes affected at the marginally toxic concentration. The two important neurodevelopmental markers Pax6 and Grin2b were downregulated at 500x human blood levels, accompanied by decreases in PAX6 and GluN2B protein levels, in cerebellum of offspring mice from mothers exposed to the Total mixture throughout pregnancy and lactation. In rat CGNs, the glutathione peroxidase gene Prdx6 and the regulatory transmembrane glycoprotein gene Sirpa were highly upregulated at both concentrations. In conclusion, our results support that early-life exposure to mixtures of POPs can cause adverse neurodevelopmental effects.

A human relevant mixture of persistent organic pollutants (POPs) and perfluorooctane sulfonic acid (PFOS) enhance nerve growth factor (NGF)-induced neurite outgrowth in PC12 cells.

Disruption of neurite outgrowth is a marker for neurotoxicity. Persistent organic pollutants (POPs) are potential developmental neurotoxicants. We investigated their effect on neurite outgrowth in PC12 rat pheochromocytoma cells, in absence or presence of nerve growth factor (NGF), an inducer of neuronal differentiation. Cells were exposed for 72 h to a defined mixture of POPs with chemical composition and concentrations based on blood levels in the Scandinavian population. We also evaluated perfluorooctane sulfonic acid (PFOS) alone, the most abundant compound in the POP mixture. Only higher concentrations of POP mixture reduced tetrazolium salt (MTT) conversion. High-content analysis showed a decrease in cell number, but no changes for nuclear and mitochondrial cellular health parameters. Robust glutathione levels were observed in NGF-differentiated cells. Live imaging, using the IncuCyte ZOOM platform indicated ongoing cell proliferation over time, but slower in presence of NGF. The pollutants did not inhibit neuritogenesis, but rather increased NGF-induced neurite length. PFOS induced neurite outgrowth to about 50 % of the level seen with the POP mixture. Neither the POP mixture nor PFOS affected neurite length in the absence of NGF. Our observations indicate that realistic complex mixtures of environmental pollutants can affect neuronal connectivity via NGF-induced neurite outgrowth.

Perfluoroalkyl acids potentiate glutamate excitotoxicity in rat cerebellar granule neurons.

Perfluoroalkyl acids (PFAAs) are persistent man-made chemicals, ubiquitous in nature and present in human samples. Although restrictions are being introduced, they are still used in industrial processes as well as in consumer products. PFAAs cross the blood-brain-barrier and have been observed to induce adverse neurobehavioural effects in humans and animals as well as adverse effects in neuronal in vitro studies. The sulfonated PFAA perfluorooctane sulfonic acid (PFOS), has been shown to induce excitotoxicity via the N-methyl-D-aspartate receptor (NMDA-R) in cultures of rat cerebellar granule neurons (CGNs). In the present study the aim was to further characterise PFOS-induced toxicity (1-60 µM) in rat CGNs, by examining interactions between PFOS and elements of glutamatergic signalling and excitotoxicity. Effects of the carboxylated PFAA, perfluorooctanoic acid (PFOA, 300-500 µM) on the same endpoints were also examined. During experiments in immature cultures at days in vitro (DIV) 8, PFOS increased both the potency and efficacy of glutamate, whereas in mature cultures at DIV 14 only increased potency was observed. PFOA also increased potency at DIV 14. PFOS-enhanced glutamate toxicity was further antagonised by the competitive NMDA-R antagonist 3-((R)-2-Carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP) at DIV 8. At DIV 8, PFOS also induced glutamate release (9-13 fold increase vs DMSO control) after 1-3 and 24 h exposure, whereas for PFOA a large (80 fold) increase was observed, but only after 24 h. PFOS and PFOA both also increased alanine and decreased serine levels after 24 h exposure. In conclusion, our results indicate that PFOS at concentrations relevant in an occupational setting, may be inducing excitotoxicity, and potentiation of glutamate signalling, via an allosteric action on the NMDA-R or by actions on other elements regulating glutamate release or NMDA-R function. Our results further support our previous findings that PFOS and PFOA at equipotent concentrations induce toxicity via different mechanisms of action.

Opioid receptor-mediated changes in the NMDA receptor in developing rat and chicken.

The use of opioids during pregnancy has been associated with neurodevelopmental toxicity in exposed children, leading to cognitive and behavioural deficits later in life. The N-methyl-D-aspartate receptor (NMDAR) subunit GluN2B plays critical roles in cerebellar development, and methadone has been shown to possess NMDAR antagonist effect. Consequently, we wanted to explore if prenatal opioid exposure affected GluN2B subunit expression and NMDAR function in rat and chicken cerebellum. Pregnant rats were exposed to methadone (10 mg/kg/day) or buprenorphine (1 mg/kg/day) for the whole period of gestation, using an osmotic minipump. To further examine potential effects of prenatal opioid exposure in a limited time window, chicken embryos were exposed to a 20 mg/kg dose of methadone or morphine on embryonic days 13 and 14. Western blot analysis of cerebella isolated from 14 days old rat pups exposed to buprenorphine showed significantly lower level of the GluN2B subunit, while the opioid exposed chicken embryo cerebellar GluN2B expression remained unaffected at embryonic day 17. However, we observed increased NMDA/glycine-induced calcium influx in cerebellar granule neurone cultures from opioid exposed chicken embryos. We conclude that prenatal opioid exposure leads to opioid receptor-dependent reduction in the postnatal expression of GluN2B in rat cerebella, and increase in NMDA-induced calcium influx in chicken embryo cerebella.

PFOS-induced excitotoxicity is dependent on Ca2+ influx via NMDA receptors in rat cerebellar granule neurons.

Perfluoroalkyl acids (PFAAs) are persistent compounds used in many industrial as well as consumer products. Despite restrictions, these compounds are found at measurable concentrations in samples of human and animal origin. In the present study we examined whether the effects on cell viability of two sulfonated and four carboxylated PFAAs in cultures of cerebellar granule neurons (CGNs), could be associated with deleterious activation of the N-methyl-d-aspartate receptor (NMDA-R). PFAA-induced effects on viability in rat CGNs and unstimulated PC12 cells were examined using the MTT assay. Cells from the PC12 rat pheochromocytoma cell line lack the expression of functional NMDA-Rs and were used to verify lower toxicity of perfluorooctanesulfonic acid (PFOS) in cells not expressing NMDA-Rs. Protective effects of NMDA-R antagonists, and extracellular as well as intracellular Ca2+ chelators were investigated. Cytosolic Ca2+ ([Ca2+]i) was measured using Fura-2. In rat CGNs the effects of the NMDA-R antagonists MK-801, memantine and CPP indicated involvement of the NMDA-R in the decreased viability induced by PFOS and perfluorohexanesulfonic acid (PFHxS). No effects were associated with the four carboxylated PFAAs studied. Further, EGTA and CPP protected against PFOS-induced decreases in cell viability, whereas no protection was afforded by BAPTA-AM. [Ca2+]i significantly increased after exposure to PFOS, and this increase was completely blocked by MK-801. In PC12 cells a higher concentration of PFOS was required to induce equivalent levels of toxicity as compared to in rat CGNs. PFOS-induced toxicity in PC12 cells was not affected by CPP. In conclusion, PFOS at the tested concentrations induces excitotoxicity in rat CGNs, which likely involves influx of extracellular Ca2+ via the NMDA-R. This effect can be blocked by specific NMDA-R antagonists.

Cracking the Egg: Potential of the Developing Chicken as a Model System for Nonclinical Safety Studies of Pharmaceuticals.

The advance of perinatal medicine has improved the survival of extremely premature babies, thereby creating a new and heterogeneous patient group with limited information on appropriate treatment regimens. The developing fetus and neonate have traditionally been ignored populations with regard to safety studies of drugs, making medication during pregnancy and in newborns a significant safety concern. Recent initiatives of the Food and Drug Administration and European Medicines Agency have been passed with the objective of expanding the safe pharmacological treatment options in these patients. There is a consensus that neonates should be included in clinical trials. Prior to these trials, drug leads are tested in toxicity and pharmacology studies, as governed by several guidelines summarized in the multidisciplinary International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use M3 (R2). Pharmacology studies must be performed in the major organ systems: cardiovascular, respiratory, and central nervous system. The chicken embryo and fetus have features that make the chicken a convenient animal model for nonclinical safety studies in which effects on all of these organ systems can be tested. The developing chicken is inexpensive, accessible, and nutritionally self-sufficient with a short incubation time and is ideal for drug-screening purposes. Other high-throughput models have been implemented. However, many of these have limitations, including difficulty in mimicking natural tissue architecture and function (human stem cells) and obvious differences from mammals regarding the respiratory organ system and certain aspects of central nervous system development (Caenorhabditis elegans, zebrafish).This minireview outlines the potential and limitations of the developing chicken as an additional model for the early exploratory phase of development of new pharmaceuticals.

Reactive oxygen species and cytotoxicity in rainbow trout hepatocytes: effects of medium and incubation time.

This study evaluated the effects of exposure medium and culture age on intracellular reactive oxygen species (ROS) development and cytotoxicity in fish hepatocytes following exposure to copper (Cu). ROS was quantified using the fluorescent probes DHR 123 and CM-H2DCFDA following exposure to Cu in Leibovitz' medium (L-15) or Tris-buffered saline (TBS). Similarly, culture age effects were investigated using 1-, 2- and 4-day-old cultured hepatocytes by exposing them to Cu in TBS. The exposure in L-15 resulted in significantly higher ROS compared to TBS using CM-H2DCFDA, but not DHR 123. The age of the primary cultures significantly affected the development of ROS for both probes. None of the exposures caused cytotoxicity in the hepatocytes. The results showed that both factors may affect responses to stressors, and suggested that the use of a simple medium such as TBS may be preferable for some applications. It is also preferable to use 1-day-old primary hepatocyte cultures.

High molecular weight DNA fragments are processed by caspase sensitive or caspase independent pathways in cultures of cerebellar granule neurons.

Many recent reports on internucleosomal DNA fragments have appeared, however, little is known about the mechanisms of the generation of their upstream high molecular weight (HMW) fragments. Caspases are a family of proteases with important functions in the execution of apoptotic cell death. The caspase-sensitivity of the formation of HMW fragments was therefore investigated using a specific caspase-3 inhibitor (Ac-DEVD-cmk) and a general caspase inhibitor (boc-D-fmk). Apoptosis inducing factor (AIF) can translocate to the nucleus and generate HMW fragments independently of caspase. Cultures of cerebellar granule neurons (CGNs) were therefore exposed to glutamate (100 micro M) or deprived of potassium and serum to induce apoptosis, or treated with a high concentration of calcium ionophore A23187 (1 micro M) to induce necrosis. Fragmentation of DNA into two classes of HMW fragments (>680 and 50-300 kbp) was observed after treatment with glutamate or A23187. Traces of approximately 50-kbp fragments were detectable after the K(+)/serum-deprivation. The amount of >680-kbp HMW fragments increased (i.e. their further degradation was inhibited) and cell death was reduced in the presence of Ac-DEVD-cmk or boc-D-fmk following glutamate treatment. Only boc-D-fmk treatment resulted in a similar accumulation of >680-kbp HMW fragments and reduced cell death after K(+)/serum-deprivation. No such changes were observed with caspase inhibitors after A23187 treatment. AIF redistribution was observed following glutamate treatment and K(+)/serum-deprivation. Thus, even in a simple cell culture of CGNs, HMW fragments are formed by diverse mechanisms: the degradation of DNA may be sensitive to different caspases or be caspase and AIF independent.