Sodium valproate exposure influences the expression of pparg in the zebrafish model.

Valproic acid (VPA) is an anti-epileptic drug used alone or in combination with other medications to treat seizures, mania, and bipolar disorder. VPA recognized as a teratogenic chemical can cause severe birth defects mainly affecting the brain and spinal cord when administered during pregnancy. However, the potential mechanisms of developmental toxicity are still less studied, and in the present study, the influence of VPA exposure was evaluated on zebrafish early-life stages. Zebrafish were exposed to two sublethal concentrations of sodium valproate (SV) (0.06 mM and 0.15 mM) from 24 hours post-fertilization (hpf) to 96 hpf and the SV teratogenic potential was investigated through morphometric analysis of zebrafish larvae combined with the evaluation of cartilage profile. Moreover, the effect of SV on the transcription level of pparg was also performed. The results of the study showed the teratogenic potential of SV, which disrupts the morphometric signature of the head and body. The marked distortion of cartilage structures was paralleled to a malformation of telencephalon and optic tectum in both concentrations suggesting a high teratogen effect of SV on the brain. These data were further confirmed by the increased expression of pparg in the zebrafish head. Overall, the present study confirms the teratogenic activity of SV in the zebrafish model and, for the first time, points out the potential protective role of pparg in the SV dose-dependent toxicity.

Caffeic acid selectively eliminates teratogenic human-induced pluripotent stem cells via apoptotic cell death.

BACKGROUND: Induced pluripotent stem cells (iPSCs) generated from reprogrammed adult somatic cells are considered as a promising cell source in cell-based regenerative medicine. To avoid teratoma formation, which is a safety issue in iPSC-based cell therapy, it is important to selectively remove undifferentiated iPSCs that remain in the differentiated cell product before in vivo transplantation. Caffeic acid (CAA, 3,4-dihydroxy-cinnamic acid) is a phenolic compound synthesized from various vegetables, fruits, and herbs; it has shown various pharmacological activities against inflammation, cancer, infection, diabetes, and neurodegenerative diseases. However, the beneficial effects of CAA in iPSC-based cell therapy, such as the selective elimination of iPSCs and anti-teratoma effects, have not yet been explored. RESULTS: Here, we found that CAA induced apoptotic cell death in iPSCs; this process did not occur in iPSC-derived mesenchymal progenitor cells (MPCs) or human dermal fibroblast (hDFs). Under co-culture conditions with MPCs and hDFs, CAA treatment selectively removed iPSCs. In addition, CAA treatment in mixed cell culture with iPSCs and MPCs prior to grafting markedly suppressed iPSC-derived teratoma formation. Finally, CAA did not induce DNA damage in MPCs or hDFs. CONCLUSION: Taken together, these results suggest that CAA is effective in preparing safe iPSC-based therapeutic cells without the risk of teratoma formation and DNA damage in normal cells and iPSC-derived differentiated cells.

A novel surgical toxicological-free model of diaphragmatic hernia in fetal rats.

BACKGROUND: Teratogen-induced congenital diaphragmatic hernia (CDH) rat models are commonly used to study the pathophysiology. We have created a new and reliable surgically induced diaphragmatic hernia (DH) model to obtain a purely mechanical DH rat model, and avoid the confounding teratogen-induced effects on the lung development. METHODS: Fetal DH was surgically created on fetuses at E18.5 and harvested at E21.5 in rats. Four groups were evaluated (n = 16): control (CONT), control exposed to Nitrofen (CONT NIT), DH surgically created (DH SURG), and CDH Nitrofen (CDH NIT). Body weight, total lung weights, and their ratio (BW, TLW, and TLBR) were compared. Air space (AS), parenchyma (PA), total protein, and DNA contents were measured to verify lung hypoplasia. Medial wall thickness (MWT) of pulmonary arterioles was also analyzed. RESULTS: DH SURG showed significant hypoplasia (decreased in total protein and DNA) vs CONT (p < 0.05); DH SURG vs CDH NIT were similar in TLW and TLBR. DH SURG has less AS than CONT (p < 0.05) and similar PA compared to CONT NIT and CDH NIT, MWT were similarly increased in CONT NIT, DH SURG, and CDH NIT. CONCLUSIONS: This novel surgical model generates fetal lung hypoplasia contributing to the study of the mechanical compression effect on fetal lung development in DH. IMPACT: There is a critical need to develop a surgical model in rat to complement the findings of the well-known Nitrofen-induced CDH model. This experimental study is pioneer and can help to understand better the CDH pathophysiological changes caused by herniated abdominal viscera compression against the lung during the final stage of gestation in CDH fetuses, and also to develop more efficient treatments in near future.

Dihydrolipoamide dehydrogenase, pyruvate oxidation, and acetylation-dependent mechanisms intersecting drug iatrogenesis.

In human metabolism, pyruvate dehydrogenase complex (PDC) is one of the most intricate and large multimeric protein systems representing a central hub for cellular homeostasis. The worldwide used antiepileptic drug valproic acid (VPA) may potentially induce teratogenicity or a mild to severe hepatic toxicity, where the underlying mechanisms are not completely understood. This work aims to clarify the mechanisms that intersect VPA-related iatrogenic effects to PDC-associated dihydrolipoamide dehydrogenase (DLD; E3) activity. DLD is also a key enzyme of α-ketoglutarate dehydrogenase, branched-chain α-keto acid dehydrogenase, α-ketoadipate dehydrogenase, and the glycine decarboxylase complexes. The molecular effects of VPA will be reviewed underlining the data that sustain a potential interaction with DLD. The drug-associated effects on lipoic acid-related complexes activity may induce alterations on the flux of metabolites through tricarboxylic acid cycle, branched-chain amino acid oxidation, glycine metabolism and other cellular acetyl-CoA-connected reactions. The biotransformation of VPA involves its complete ß-oxidation in mitochondria causing an imbalance on energy homeostasis. The drug consequences as histone deacetylase inhibitor and thus gene expression modulator have also been recognized. The mitochondrial localization of PDC is unequivocal, but its presence and function in the nucleus were also demonstrated, generating acetyl-CoA, crucial for histone acetylation. Bridging metabolism and epigenetics, this review gathers the evidence of VPA-induced interference with DLD or PDC functions, mainly in animal and cellular models, and highlights the uncharted in human. The consequences of this interaction may have significant impact either in mitochondrial or in nuclear acetyl-CoA-dependent processes.

Pharmacokinetics of primary oxidative metabolites of thalidomide in rats and in chimeric mice humanized with different human hepatocytes.

The approved drug thalidomide is teratogenic in humans, nonhuman primates, and rabbits but not in rodents. The extensive biotransformation of 5'-hydroxythalidomide after oral administration of thalidomide (250 mg/kg) in rats was investigated in detail using liquid chromatography-tandem mass spectrometry. Probable metabolites 5'-hydroxythalidomide sulfate and glucuronide were extensively formed, with approximately tenfold and onefold peak areas, respectively, to the primary 5'-hydroxythalidomide measured using authentic standards. As a minor metabolite, 5-hydroxythalidomide was also detected. The output of simplified physiologically based pharmacokinetic rat models was consistent with the observed in vivo data under a metabolic ratio of 0.05 for the hepatic intrinsic clearance of thalidomide to unconjugated 5'-hydroxythalidomide. The aggregate of unconjugated and sulfate/glucuronide conjugated 5'-hydroxythalidomide forms appear to be the predominant metabolites in rats. Two hours after oral administration of thalidomide (100 mg/kg) to chimeric mice humanized with four different batches of genotyped human hepatocytes, the plasma concentration ratios of 5-hydroxythalidomide to 5'-hydroxythalidomide were correlated with replacement indexes of human liver cells previously transplanted in immunodeficient mice. These results indicate that rodent livers mediate thalidomide primary oxidation, leading to extensive deactivation in vivo to unconjugated/conjugated 5'-hydroxythalidomide and suggest that thalidomide activation might be dependent on the humanized livers in mice transplanted with human hepatocytes.

The effects of in utero exposure to teratogens on organ size: a prospective paediatric study.

In low-income countries, prospective data on combined effects of in utero teratogen exposure are lacking and necessitates new research. The aim of the present study was to explore the effect of in utero teratogen exposure on the size of the kidneys and pancreas 5 years after birth in a low-income paediatric population. Data was collected from 500 mother-child pairs from a low-income setting. Anthropometric measurements included body weight, (BW) body height, mid-upper arm and waist circumference (WC). Clinical measurements included blood pressure (BP), mean arterial pressure and heart rate. Ultrasound measurements included pancreas, and kidney measurements at age 5 years. The main outcome of interest was the effect of maternal smoking and alcohol consumption on ultrasound measurements of organ size at age 5 years. Left and right kidney length measurements were significantly lower in smoking exposed children compared to controls (p = 0.04 and p = 0.03). Pancreas body measurements were significantly lower in smoking exposed children (p = 0.04). Multiple regression analyses were used to examine the associations between the independent variables (IDVs), maternal age, body mass index (BMI), mid-upper arm circumference (MUAC) and BW of the child, on the dependent variables (DVs) kidney lengths and kidney volumes. Also, the association between in utero exposure to alcohol and nicotine and pancreas size. WC was strongest (r = 0.28; p < 0.01) associated with pancreas head [F (4, 454) = 13.44; R2 = 0.11; p < 0.01] and tail (r = 0.30; p < 0.01) measurements at age 5 years, with in utero exposure, sex of the child and BMI as covariates. Kidney length and pancreas body measurements are affected by in utero exposure to nicotine at age 5 years and might contribute to cardiometabolic risk in later life. Also, findings from this study report on ultrasound reference values for kidney and pancreas measurements of children at age 5 years from a low-income setting.

Hypnotic effect of thalidomide is independent of teratogenic ubiquitin/proteasome pathway.

Thalidomide exerts its teratogenic and immunomodulatory effects by binding to cereblon (CRBN) and thereby inhibiting/modifying the CRBN-mediated ubiquitination pathway consisting of the Cullin4-DDB1-ROC1 E3 ligase complex. The mechanism of thalidomide's classical hypnotic effect remains largely unexplored, however. Here we examined whether CRBN is involved in the hypnotic effect of thalidomide by generating mice harboring a thalidomide-resistant mutant allele of Crbn (Crbn YW/AA knock-in mice). Thalidomide increased non-REM sleep time in Crbn YW/AA knock-in homozygotes and heterozygotes to a similar degree as seen in wild-type littermates. Thalidomide similarly depressed excitatory synaptic transmission in the cortical slices obtained from wild-type and Crbn YW/AA homozygous knock-in mice without affecting GABAergic inhibition. Thalidomide induced Fos expression in vasopressin-containing neurons of the supraoptic nucleus and reduced Fos expression in the tuberomammillary nuclei. Thus, thalidomide's hypnotic effect seems to share some downstream mechanisms with general anesthetics and GABAA-activating sedatives but does not involve the teratogenic CRBN-mediated ubiquitin/proteasome pathway.

Comparative Analysis of the Developmental Toxicity in Xenopus laevis and Danio rerio Induced by Al2 O3 Nanoparticle Exposure.

Engineered aluminum oxide nanoparticles (Al2 O3 NPs) having high-grade thermal stability and water-dispersion properties are extensively used in different industries and personal care products. Toxicological response evaluation of these NPs is indispensable in assessing the health risks and exposure limits because of their industrial disposal into the aquatic environment. We assessed and compared the developmental toxicity of Al2 O3 NPs in Xenopus laevis and Danio rerio over a period of 96 h using the frog embryo teratogenic assay Xenopus and a fish embryo toxicity assay. Engineered Al2 O3 NP exposure produced dose-dependent embryonic mortality and decreased the embryo length, indicating a negative effect on growth. Moreover, Al2 O3 NPs induced various malformations, such as small head size, a bent/deformed axis, edema, and gut malformation, dose-dependently and altered the expression of heart- and liver-specific genes in both X. laevis and D. rerio, as revealed by whole-mount in-situ hybridization and reverse transcriptase polymerase chain reaction. In conclusion, the toxicological data suggest that Al2 O3 NPs are developmentally toxic and teratogenic and negatively affect the embryonic development of X. laevis and D. rerio. Our study can serve as a model for the toxicological evaluation of nanomaterial exposure on vertebrate development that is critical to ensure human and environmental safety. Environ Toxicol Chem 2019;38:2672-2681. © 2019 SETAC.

An Evaluation of Hair, Oral Fluid, Earwax, and Nasal Mucus as Noninvasive Specimens to Determine Livestock Exposure to Teratogenic Lupine Species.

The livestock industry in the western United States loses an estimated $500 million annually from livestock production losses due to poisonous plants. Poisoning of livestock by plants often goes undiagnosed because there is a lack of appropriate or available specimens for analysis. The Lupinus species represent an important toxic plant in western North America that can be toxic and/or teratogenic to livestock species due to the quinolizidine alkaloids. The objective of this study was to evaluate the potential of using earwax, hair, oral fluid, and nasal mucus as noninvasive specimens to determine livestock exposure to the teratogenic Lupinus species. Quinolizidine alkaloids were detected in these four matrices in cattle that were administered a single dose of Lupinus leucophyllus. In addition, quinolizidine alkaloids from lupine were detected in the earwax of cattle that grazed on lupine-infested rangelands. This study demonstrates the potential of earwax, hair, oral fluid, and nasal mucus as noninvasive specimens for chemical analyses to aid in the diagnosis of livestock that may have been exposed to and poisoned by plants.

Teratogenicity of valproic acid and its constitutional isomer, amide derivative valnoctamide in mice.

OBJECTIVES: The anticonvulsant valproic acid (VPA) has a known teratogenic effect capable of inducing major congenital malformations and developmental disorders. A comparative teratogenicity study of VPA and its analog valnoctamide (VCD), which is a new generation candidate antiepileptic drug, was carried out using Swiss Vancouver (SWV) mice. METHODS: Pregnant SWV dams were treated with either a single intraperitoneal injection of VPA (1.8 and 2.7 mmol/kg), VCD (1.8 and 2.7 mmol/kg), or vehicle on E8:12 (gestational day:hour). The numbers of implantation and resorption, viable and dead fetuses, and the presence of gross fetal visceral and skeletal abnormalities were determined (E18). Real-time Polymerase chain reaction (RT-PCR) arrays were used to analyze the expression of 84 genes related to the processes of neurogenesis and neural stem cell differentiation. RESULTS: Significant decreases in pregnancy weight gain and the number of live fetuses were observed when VPA was administered at the high dose, whereas the percentage of exencephalic fetuses was significantly increased in VPA treated compared with an equivalent VCD dosage group. There was a dose-related increase in visceral defects in the VPA-exposed fetuses. Missing skull bones and fused vertebrae in fetuses occurred at the high dose of VPA. Three genes (Mtap2, Bmp8b, and Stat3) were significantly upregulated and one (Heyl) was downregulated in samples from VPA-treated dams. CONCLUSIONS: The study demonstrates that the teratogenicity of VPA was significantly greater than that of an equimolar dose of VCD. Four genes (Mtap2, Bmp8b, Stat3, and Heyl) represent candidate target genes for the underlying teratogenic mechanism responsible for VPA-induced malformations.

AHR gene-dioxin interactions and birthweight in the Seveso Second Generation Health Study.

Background: 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD) is proposed to interfere with fetal growth via altered activity of the aryl hydrocarbon receptor (protein: AHR; gene: AHR) pathway which regulates diverse biological and developmental processes including xenobiotic metabolism. Genetic variation in AHR is an important driver of susceptibility to low birthweight in children exposed to prenatal smoking, but less is known about these genetic interactions with TCDD, AHR's most potent xenobiotic ligand. Methods: The Seveso Women's Health Study (SWHS), initiated in 1996, is a cohort of 981 Italian women exposed to TCDD from an industrial explosion in July 1976. We measured TCDD concentrations in maternal serum collected close to the time of the accident. In 2008 and 2014, we followed up the SWHS cohort and collected data on birth outcomes of SWHS women with post-accident pregnancies. We genotyped 19 single nucleotide polymorphisms (SNPs) in AHR among the 574 SWHS mothers. Results: Among 901 singleton births, neither SNPs nor TCDD exposure alone were significantly associated with birthweight. However, we found six individual SNPs in AHR which adversely modified the association between maternal TCDD and birthweight, implicating gene-environment interaction. We saw an even stronger susceptibility to TCDD due to interaction when we examined the joint contribution of these SNPs in a risk allele score. These SNPs were all located in noncoding regions of AHR, particularly in proximity to the promoter. Conclusions: This is the first study to demonstrate that genetic variation across the maternal AHR gene may shape fetal susceptibilities to TCDD exposure.

Acetaldehyde inhibits retinoic acid biosynthesis to mediate alcohol teratogenicity.

Alcohol consumption during pregnancy induces Fetal Alcohol Spectrum Disorder (FASD), which has been proposed to arise from competitive inhibition of retinoic acid (RA) biosynthesis. We provide biochemical and developmental evidence identifying acetaldehyde as responsible for this inhibition. In the embryo, RA production by RALDH2 (ALDH1A2), the main retinaldehyde dehydrogenase expressed at that stage, is inhibited by ethanol exposure. Pharmacological inhibition of the embryonic alcohol dehydrogenase activity, prevents the oxidation of ethanol to acetaldehyde that in turn functions as a RALDH2 inhibitor. Acetaldehyde-mediated reduction of RA can be rescued by RALDH2 or retinaldehyde supplementation. Enzymatic kinetic analysis of human RALDH2 shows a preference for acetaldehyde as a substrate over retinaldehyde. RA production by hRALDH2 is efficiently inhibited by acetaldehyde but not by ethanol itself. We conclude that acetaldehyde is the teratogenic derivative of ethanol responsible for the reduction in RA signaling and induction of the developmental malformations characteristic of FASD. This competitive mechanism will affect tissues requiring RA signaling when exposed to ethanol throughout life.

Structural basis of thalidomide enantiomer binding to cereblon.

Thalidomide possesses two optical isomers which have been reported to exhibit different pharmacological and toxicological activities. However, the precise mechanism by which the two isomers exert their different activities remains poorly understood. Here, we present structural and biochemical studies of (S)- and (R)-enantiomers bound to the primary target of thalidomide, cereblon (CRBN). Our biochemical studies employed deuterium-substituted thalidomides to suppress optical isomer conversion, and established that the (S)-enantiomer exhibited ~10-fold stronger binding to CRBN and inhibition of self-ubiquitylation compared to the (R)-enantiomer. The crystal structures of the thalidomide-binding domain of CRBN bound to each enantiomer show that both enantiomers bind the tri-Trp pocket, although the bound form of the (S)-enantiomer exhibited a more relaxed glutarimide ring conformation. The (S)-enantiomer induced greater teratogenic effects on fins of zebrafish compared to the (R)-enantiomer. This study has established a mechanism by which thalidomide exerts its effects in a stereospecific manner at the atomic level.

Species Differences in Renal Development and Associated Developmental Nephrotoxicity.

The developing kidney is sensitive to both morphological and functional disturbances during the gestational and postnatal phases of growth and differentiation. Exposure to drugs or chemicals during these critical windows of renal development can result in aplasia, dysplasia, polycystic kidney disease, hydronephrosis, or other features characteristic of nephrotoxicity, including tubule dilation, necrosis, or mineralization. Functional effects can occur without associated morphological abnormalities. Differences in the timing of nephrogenesis and morphologic renal development among species help to explain specific phenotypes of various gestational and postnatal teratogens and nephrotoxins. Functional maturation follows anatomical maturation, but important differences in maximally achieved glomerular filtration rate, concentrating ability and acid-base equilibrium between species makes comparison of these timings critical for accurate and consistent translation of laboratory animal toxicity data to the human clinical experience. Species and age dependent differences in the maturation of kidney transporters, renal xenobiotic metabolism and renal blood flow can have a profound effect on the toxicity profiles of agents and marked differences in the tolerability based on age. Advances in the understanding of the genetics of inherited renal diseases and the underlying cellular and molecular pathogenesis of renal developmental anomalies has helped provide mechanistic understanding of many teratogenic and perinatal nephrotoxic agents. Investigative studies have provided important translational and mechanistic information for assessing human pediatric nephrotoxic potential. Birth Defects Research 109:1243-1256, 2017. © 2017 Wiley Periodicals, Inc.

In Vitro Micropatterned Human Pluripotent Stem Cell Test (µP-hPST) for Morphometric-Based Teratogen Screening.

Exposure to teratogenic chemicals during pregnancy may cause severe birth defects. Due to high inter-species variation of drug responses as well as financial and ethical burdens, despite the widely use of in vivo animal tests, it's crucial to develop highly predictive human pluripotent stem cell (hPSC)-based in vitro assays to identify potential teratogens. Previously we have shown that the morphological disruption of mesoendoderm patterns formed by geometrically-confined cell differentiation and migration using hPSCs could potentially serve as a sensitive morphological marker in teratogen detection. Here, a micropatterned human pluripotent stem cell test (µP-hPST) assay was developed using 30 pharmaceutical compounds. A simplified morphometric readout was developed to quantify the mesoendoderm pattern changes and a two-step classification rule was generated to identify teratogens. The optimized µP-hPST could classify the 30 compounds with 97% accuracy, 100% specificity and 93% sensitivity. Compared with metabolic biomarker-based hPSC assay by Stemina, the µP-hPST could successfully identify misclassified drugs Bosentan, Diphenylhydantoin and Lovastatin, and show a higher accuracy and sensitivity. This scalable µP-hPST may serve as either an independent assay or a complement assay for existing assays to reduce animal use, accelerate early discovery-phase drug screening and help general chemical screening of human teratogens.

The Dihydroxy Metabolite of the Teratogen Thalidomide Causes Oxidative DNA Damage.

Thalidomide [α-(N-phthalimido)glutarimide] (1) is a sedative and antiemetic drug originally introduced into the clinic in the 1950s for the treatment of morning sickness. Although marketed as entirely safe, more than 10 000 babies were born with severe birth defects. Thalidomide was banned and subsequently approved for the treatment of multiple myeloma and complications associated with leprosy. Although known for more than 5 decades, the mechanism of teratogenicity remains to be conclusively understood. Various theories have been proposed in the literature including DNA damage and ROS and inhibition of angiogenesis and cereblon. All of the theories have their merits and limitations. Although the recently proposed cereblon theory has gained wide acceptance, it fails to explain the metabolism and low-dose requirement reported by a number of groups. Recently, we have provided convincing structural evidence in support of the presence of arene oxide and the quinone-reactive intermediates. However, the ability of these reactive intermediates to impart toxicity/teratogenicity needs investigation. Herein we report that the oxidative metabolite of thalidomide, dihydroxythalidomide, is responsible for generating ROS and causing DNA damage. We show, using cell lines, the formation of comet (DNA damage) and ROS. Using DNA-cleavage assays, we also show that catalase, radical scavengers, and desferal are capable of inhibiting DNA damage. A mechanism of teratogenicity is proposed that not only explains the DNA-damaging property but also the metabolism, low concentration, and species-specificity requirements of thalidomide.

Assessing neurodevelopmental effects of arsenolipids in pre-differentiated human neurons.

SCOPE: In the general population exposure to arsenic occurs mainly via diet. Highest arsenic concentrations are found in seafood, where arsenic is present predominantly in its organic forms including arsenolipids. Since recent studies have provided evidence that arsenolipids could reach the brain of an organism and exert toxicity in fully differentiated human neurons, this work aims to assess the neurodevelopmental toxicity of arsenolipids. METHODS AND RESULTS: Neurodevelopmental effects of three arsenic-containing hydrocarbons (AsHC), two arsenic-containing fatty acids (AsFA), arsenite and dimethylarsinic acid (DMAV ) were characterized in pre-differentiated human neurons. AsHCs and arsenite caused substantial cytotoxicity in a similar, low concentration range, whereas AsFAs and DMAV were less toxic. AsHCs were highly accessible for cells and exerted pronounced neurodevelopmental effects, with neurite outgrowth and the mitochondrial membrane potential being sensitive endpoints; arsenite did not substantially decrease those two endpoints. In fully differentiated neurons, arsenite and AsHCs caused neurite toxicity. CONCLUSION: These results indicate for a neurodevelopmental potential of AsHCs. Taken into account the possibility that AsHCs might easily reach the developing brain when exposed during early life, neurotoxicity and neurodevelopmental toxicity cannot be excluded. Further studies are needed in order to progress the urgently needed risk assessment.

Anagyrine desensitization of peripheral nicotinic acetylcholine receptors. A potential biomarker of quinolizidine alkaloid teratogenesis in cattle.

Anagyrine, a teratogenic quinolizidine alkaloid found in Lupinus spp., is proposed to undergo metabolism by pregnant cattle to a piperidine alkaloid which inhibits fetal movement, the putative mechanism behind crooked calf syndrome. The objective of this study was to test the hypothesis that anagyrine but not lupanine or sparteine can directly, without metabolism, desensitize nicotinic acetylcholine receptors (nAChR) in a cell culture model. SH-SY5Y cells expressing autonomic nAChR, and TE-671 cells expressing fetal muscle-type nAChR were exposed to lupine alkaloids or Dimethylphenylpiperazinium (DMPP) in log10 molar increments from 10nM to 100µM and then to a fixed concentration of acetylcholine (ACh) (10µM for SH-SY5Y cells and 1µM for TE-671 cells) and the responses measured with a membrane potential sensing dye to assess nAChR activation and desensitization. The selective ganglionic nAChR agonist DMPP used as a positive control, was a potent activator and desensitizer of nAChR expressed by SH-SY5Y cells. Lupanine was a weak agonist and desensitizer in SH-SY5Y cells and sparteine was without effect. Anagyrine acted as a partial agonist in both cell lines with EC50 values of 4.2 and 231µM in SH-SY5Y and TE-671 cells, respectively. Anagyrine was a desensitizer of nAChR with DC50 values of 6.9 and 139µM in SH-SY5Y and TE-671 cells, respectively. These results confirm the hypothesis that anagyrine is a potent and effective desensitizer of nAChR, and that anagyrine can directly, without metabolism, desensitize nAChR. Moreover, serum anagyrine concentrations may be a potential biomarker for lupine teratogenicity in cattle.

In vitro acute and developmental neurotoxicity screening: an overview of cellular platforms and high-throughput technical possibilities.

Neurotoxicity and developmental neurotoxicity are important issues of chemical hazard assessment. Since the interpretation of animal data and their extrapolation to man is challenging, and the amount of substances with information gaps exceeds present animal testing capacities, there is a big demand for in vitro tests to provide initial information and to prioritize for further evaluation. During the last decade, many in vitro tests emerged. These are based on animal cells, human tumour cell lines, primary cells, immortalized cell lines, embryonic stem cells, or induced pluripotent stem cells. They differ in their read-outs and range from simple viability assays to complex functional endpoints such as neural crest cell migration. Monitoring of toxicological effects on differentiation often requires multiomics approaches, while the acute disturbance of neuronal functions may be analysed by assessing electrophysiological features. Extrapolation from in vitro data to humans requires a deep understanding of the test system biology, of the endpoints used, and of the applicability domains of the tests. Moreover, it is important that these be combined in the right way to assess toxicity. Therefore, knowledge on the advantages and disadvantages of all cellular platforms, endpoints, and analytical methods is essential when establishing in vitro test systems for different aspects of neurotoxicity. The elements of a test, and their evaluation, are discussed here in the context of comprehensive prediction of potential hazardous effects of a compound. We summarize the main cellular characteristics underlying neurotoxicity, present an overview of cellular platforms and read-out combinations assessing distinct parts of acute and developmental neurotoxicology, and highlight especially the use of stem cell-based test systems to close gaps in the available battery of tests.

Gestational di-n-butyl phthalate exposure induced developmental and teratogenic anomalies in rats: a multigenerational assessment.

With the limited but ongoing usage of di-n-butyl phthalate (DBP) as plasticizer, the health effects of both phthalate and its alternatives are far from being understood. Multigenerational effects of phthalates were evaluated in rats upon exposure to DBP, aiming to provide some evidences about its potential in causing developmental teratogenicity. Gestational rats were exposed to DBP (500 mg/kg bw/day) and control groups with olive oil. On the 18th day of gestation, fetuses (F1) isolated from a few dams were subjected to prenatal screening, and the other rats were allowed to litter, and later postnatal screening was made. DBP-toxicated (F1) rats were crossed and reared up to three generations (F2 and F3) by adopting the same experimental design. A considerable decrease in the weight of placenta, low number of corpora lutea and increased resorptions, and pre- and postimplantation loss were observed in F1, F2, and F3 generations. Further, there was a decrease in the number of live births and fetal body weight with high mortality, the developmental indices showed reduction in litter size and sex ratio, and a considerable incidence of skeletal and malformation complex involving face and eye was observed in later generations compared to the first. The pre-weaning indices in neonates showed a considerable delay in physical growth milestones and poor scores in sensory motor development. Alterations noticed in the levels of thyroid profile and testosterone found to have a role in sensory motor, craniofacial development, and eye formation. In brief, results confirm multigenerational and fetotoxic effects of DBP; thereby, findings imply that developing tissues are the targets and endocrine disruption appears to be the underlying mechanism of phthalate action.