Inhibition with simultaneous spontaneous reactivation and aging of acetylcholinesterase by organophosphorus compounds: Demeton-S-methyl as a model.

The kinetic analysis of esterase inhibition by acylating compounds (organophosphorus, carbamates and sulfonylfluorides) sometimes cannot yield consistent results by fitting simple inhibition kinetic models to experimental data of complex systems. In this work kinetic data were obtained for demeton-S-methyl (DSM) with human acetylcholinesterase in two kinds of experiments: (a) time progressive inhibition with a range of concentrations, (b) progressive spontaneous reactivation starting with pre-inhibited enzyme. DSM is an organophosphorus compound used as pesticide and considered a model for studying the dermal exposure of nerve agents such as VX gas. A kinetic model equation was deduced with four different molecular phenomena occurring simultaneously: (1) inhibition; (2) spontaneous reactivation; (3) aging; and (4) ongoing inhibition (inhibition during the substrate reaction). A 3D fit of the model was applied to analyze the inhibition experimental data. The best-fitting model is compatible with a sensitive enzymatic entity. The second-order rate constant of inhibition (ki = 0.0422 µM-1 min-1), the spontaneous reactivation constant (ks = 0.0202 min-1) and the aging constant (kg = 0.0043 min-1) were simultaneously estimated. As an example for testing the model and approach, it was tested also in the presence of 5 % ethanol (conditions as previously used in the literature), the best fitting model is compatible with two apparent sensitive enzymatic entities (17 % and 83 %) and only one spontaneously reactivates and ages. The corresponding second-order rate constants of inhibition (ki = 0.0354 and 0.0119 µM-1 min-1) and the spontaneous reactivation and aging constants for the less sensitive component (kr = 0.0203 min-1 and kg = 0.0088 min-1) were estimated. The results were also consistent with a significant ongoing inhibition. These parameters were similar to those deduced in spontaneous reactivation experiments of the pre-inhibited samples with DSM in the absence or presence of ethanol. The two apparent components fit was interpreted by an equilibrium between ethanol-free and ethanol-bound enzyme. The consistency of results in inhibition and in spontaneous reactivation experiments was considered an internal validation of the methodology and the conclusions.

Investigation of the Effects of Metallic Nanoparticles on Fertility Outcomes and Endocrine Modification of the Hypothalamic-Pituitary-Gonadal Axis.

Nanotechnology is a very disruptive twenty-first-century revolution that will allow social and economic welfare to increase although it also involves a significant human exposure to nanoparticles. The aim of the present study was to contribute to the elucidation on whether metallic nanoparticles have a potential to induce fertility impairments. Regulatory studies that observed official OECD guidelines 415, 416 and 422 have failed to detect any fertility alterations caused by nanoparticle exposure. However, the scientific literature provides evidence that some nanoparticles may cause gonad impairments although the actual impact on fertility remains uncertain. This aim of the present study is to revisit the previously published RNAseq studies by analyzing the effects of several nanoparticles on the transcriptome of T98G human glioblastoma cells given that glial cells are known to play a pivotal role in the regulation of gonadotropin releasing hormone neurons. We found evidence that nanoparticles impair the gonadotropin releasing hormone receptor pathway and several related biological process like, among others, the cellular response to follicular stimulating hormone, cellular response to gonadotropin stimulus, cellular response to hormone stimulus, response to steroid hormone, ovulation cycle and response to estradiol. We propose that nanoparticles interfere with the ability of glial cells to regulate gonadotropin-releasing hormone neurons and, subsequently, the hypothalamic-pituitary-gonadal axis, potentially leading to fertility impairments. To our knowledge, this is the first proposal of a mode of action based on endocrine disruption for explaining the possible effects of nanoparticles on fertility. Whether these finding can be extended to other types of nanoparticles requires further investigation.

Reference Values on Children's Hair for 28 Elements (Heavy Metals and Essential Elements) Based on a Pilot Study in a Representative Non-Contaminated Local Area.

Studies have been published, and laboratories offer services of measuring elements in hair as biomarkers of environmental exposure and/or control of essential elements (trace or macro). These reported values can have only sense if compared with adopted reference values. In this work, we propose provisional reference values based on a pilot child population. The concentrations of 28 elements were measured in children's hair samples. An observational, descriptive, cross-sectional study was conducted in a typical child population in the Mediterranean region void of excessive pollution problems to analyze 419 hair samples of children aged 3-12 years. Children were selected by a simple random method from eight primary education schools in different municipal districts, which included urban, rural and industrial areas. Samples of around 100 mg were washed and acid digested by an optimized procedure. All measures were performed using ICP-MS with Sc, Y and Re as internal standards. The statistical analysis was performed by two approaches: (a) considering all the data and (b) without outliers (second-order atypical data) to compare them with other published studies. The distribution curves in all the elements studied were asymmetric and did not fit the theoretical normality distributions. Therefore, the analysis based on percentiles was more appropriate. In most elements, only slight differences were observed with sex or age, which did not justify proposing separate reference ranges. From the results of this study, provisional reference values are proposed following two criteria: (a) simple application of the table of percentiles built by removing outlier values and (b) values after a detailed analysis case-by-case, considering other data as the distribution profile and other published data of each element. Although the pilot sample was from a limited area, it was carefully selected to be representative of a general non-contaminated population. With this limitation, the proposed reference values might be useful for researchers and physicians until a wider geographical study is available for a large number of elements.

Human-Based New Approach Methodologies in Developmental Toxicity Testing: A Step Ahead from the State of the Art with a Feto-Placental Organ-on-Chip Platform.

Developmental toxicity testing urgently requires the implementation of human-relevant new approach methodologies (NAMs) that better recapitulate the peculiar nature of human physiology during pregnancy, especially the placenta and the maternal/fetal interface, which represent a key stage for human lifelong health. Fit-for-purpose NAMs for the placental-fetal interface are desirable to improve the biological knowledge of environmental exposure at the molecular level and to reduce the high cost, time and ethical impact of animal studies. This article reviews the state of the art on the available in vitro (placental, fetal and amniotic cell-based systems) and in silico NAMs of human relevance for developmental toxicity testing purposes; in addition, we considered available Adverse Outcome Pathways related to developmental toxicity. The OECD TG 414 for the identification and assessment of deleterious effects of prenatal exposure to chemicals on developing organisms will be discussed to delineate the regulatory context and to better debate what is missing and needed in the context of the Developmental Origins of Health and Disease hypothesis to significantly improve this sector. Starting from this analysis, the development of a novel human feto-placental organ-on-chip platform will be introduced as an innovative future alternative tool for developmental toxicity testing, considering possible implementation and validation strategies to overcome the limitation of the current animal studies and NAMs available in regulatory toxicology and in the biomedical field.

A Transcriptomic Analysis of T98G Human Glioblastoma Cells after Exposure to Cadmium-Selenium Quantum Dots Mainly Reveals Alterations in Neuroinflammation Processes and Hypothalamus Regulation.

Quantum dots are nanoparticles with very promising biomedical applications. However, before these applications can be authorized, a complete toxicological assessment of quantum dots toxicity is needed. This work studied the effects of cadmium-selenium quantum dots on the transcriptome of T98G human glioblastoma cells. It was found that 72-h exposure to 40 µg/mL (a dose that reduces cell viability by less than 10%) alters the transcriptome of these cells in biological processes and molecular pathways, which address mainly neuroinflammation and hormonal control of hypothalamus via the gonadotropin-releasing hormone receptor. The biological significance of neuroinflammation alterations is still to be determined because, unlike studies performed with other nanomaterials, the expression of the genes encoding pro-inflammatory interleukins is down-regulated rather than up-regulated. The hormonal control alterations of the hypothalamus pose a new concern about a potential adverse effect of quantum dots on fertility. In any case, more studies are needed to clarify the biological relevance of these findings, and especially to assess the real risk of toxicity derived from quantum dots exposure appearing in physiologically relevant scenarios.

Case study: risk associated to wearing silver or graphene nanoparticle-coated facemasks for protection against COVID-19.

The world is living a pandemic situation derived from the worldwide spreading of SARS-CoV-2 virus causing COVID-19. Facemasks have proven to be one of the most effective prophylactic measures to avoid the infection that has made that wearing of facemasks has become mandatory in most of the developed countries. Silver and graphene nanoparticles have proven to have antimicrobial properties and are used as coating of these facemasks to increase the effectivity of the textile fibres. In the case of silver nanoparticles, we have estimated that in a real scenario the systemic (internal) exposure derived from wearing these silver nanoparticle facemasks would be between 7.0 × 10-5 and 2.8 × 10-4 mg/kg bw/day. In addition, we estimated conservative systemic no effect levels between 0.075 and 0.01 mg/kg bw/day. Therefore, we estimate that the chronic exposure to silver nanoparticles derived form facemasks wearing is safe. In the case of graphene, we detected important gaps in the database, especially regarding toxicokinetics, which prevents the derivation of a systemic no effect level. Nevertheless, the qualitative approach suggests that the risk of dermal repeated exposure to graphene is very low, or even negligible. We estimated that for both nanomaterials, the risk of skin sensitisation and genotoxicity is also negligible.

Titanium Dioxide, but Not Zinc Oxide, Nanoparticles Cause Severe Transcriptomic Alterations in T98G Human Glioblastoma Cells.

Titanium dioxide and zinc oxide are two of the most widely used nanomaterials. We assessed the effects of noncytotoxic doses of both nanomaterials on T98G human glioblastoma cells by omic approaches. Surprisingly, no effects on the transcriptome of T98G cells was detected after exposure to 5 µg/mL of zinc oxide nanoparticles during 72 h. Conversely, the transcriptome of the cells exposed to 20 µg/mL of titanium dioxide nanoparticles during 72 h revealed alterations in lots of biological processes and molecular pathways. Alterations to the transcriptome suggests that exposure to titanium dioxide nanoparticles might, potentially, compromise the integrity of the blood brain barrier integrity and cause neuroinflammation. The latter issue was further confirmed phenotypically with a proteomic analysis and by recording the release of interleukin 8. Titanium dioxide also caused autophagy, which was demonstrated through the increase in the expression of the autophagy-related 3 and microtubule associated protein 1 light chain 3 alpha genes. The proteomic analysis revealed that titanium dioxide nanoparticles might have anticancerigen properties by downregulating genes involved in the detoxication of anthracyclines. A risk assessment resulting from titanium dioxide exposure, focusing on the central nervous system as a potential target of toxicity, is necessary.

Effects of silver nanoparticles on T98G human glioblastoma cells.

Nanotechnology has been well developed in recent decades because it provides social progress and welfare. Consequently, exposure of population is increasing and further increases in the near future are forecasted. Therefore, assessing the safety of applications involving nanoparticles is strongly advisable. We assessed the effects of silver nanoparticles at a non-cytotoxic concentration on the performance of T98G human glioblastoma cells mainly by an omic approach. We found that silver nanoparticles are able to alter several molecular pathways related to inflammation. Cellular repair and regeneration were also affected by alterations to the fibroblast growth factor pathways operating mainly via mitogen-activated protein kinase cascades. It was concluded that, given the relevant role of glia on central nervous system maintenance homeostasis, exposure to silver nanoparticles could eventually lead to severe toxicity in the central nervous system, although current exposure levels do not pose a significant risk.

Case study: Is bisphenol S safer than bisphenol A in thermal papers?

The Risk Assessment Committee of the European Chemical Agency released a scientific opinion alerting that the risk associated with dermal occupational exposure to bisphenol A (BPA) via thermal paper might not be adequately controlled because the estimated exposure was around twice the Derived No Effect Level (DNEL) and the European Commission will effectively restrict BPA in thermal paper as soon as 2020. Bisphenol S (BPS) is currently being used as a BPA surrogate and is already widespread in thermal paper receipts. Based on publically available information in the scientific literature, we assessed the risk associated with dermal BPS exposure via thermal paper for the general and occupational populations to compare with BPA situation. We developed two exposure scenarios; one based on the total excreted BPS and another on exposure estimations by transferring BPS from the thermal paper matrix to skin. Both scenarios yielded similar exposures for the general population (0.016-0.013 µg/kg bw/day), but the exposure estimated for the workers in the second scenario (0.96 µg/kg bw/day) was around 17-fold higher than that estimated for the workers in the first scenario. The systemic DNELs for the general and workers populations were 0.45 and 0.91 µg BPS/kg bw/day, respectively, which were 4.6- and 19-fold higher than the respective dermal DNELs. Risk Characterisation Ratio (RCR) (estimated exposure through urinary excretion compared with the systemic DNEL) in the first and most reliable scenario suggested that the risk was adequately controlled. In the second scenario, however, the RCR suggests that the risk might not be adequately controlled for both the general population and workers. This work raises the necessity of generate more toxicodynamic and toxicokinetic information, specially using dermal exposures, to properly assess the risk associated to dermal BPS exposure because the situation might presumably get worse after 2020.

Hydrolyzing activities of phenyl valerate sensitive to organophosphorus compounds paraoxon and mipafox in human neuroblastoma SH-SY5Y cells.

The molecular targets of best known neurotoxic effects associated to acute exposure to organophosphorus compounds (OPs) are serine esterases located in the nervous system, although there are other less known neurotoxic adverse effects associated with chronic exposure to OPs whose toxicity targets are still not identified. In this work we studied sensitivity to the non-neuropathic OP paraoxon and to the neuropathic OP mipafox of phenyl valerate esterases (PVases) in intact and lysed human neuroblastoma SH-SY5Y cells. The main objective was to discriminate different unknown pools of esterases that might be potential targets of chronic effects from those esterases already known and recognized as targets to these acute neurotoxicity effects. Two components of PVases of different sensitivities were discriminated for paraoxon in both intact and lysed cells; while the two components inhibitable by mipafox were found only for intact cells. A completely resistant component to paraoxon of around 30% was found in both intact and lysed cells; while a component of slightly lower amplitude (around 20%) completely resistant to mipafox was also found for both preparations (intact and lysed cells). The comparison of the results between the intact cells and the lysed cells suggests that the plasma membrane could act as a barrier that reduced the bioavailability of mipafox to PVases. This would imply that the discrimination of the different esterases should be made in lysed cells. However, those studies which aim to determine the physiological role of these esterases should be necessarily conducted in intact cultured cells.

Roles of NTE protein and encoding gene in development and neurodevelopmental toxicity.

Neuropathy Target Esterase (NTE) is a membrane protein codified by gene PNPLA6. NTE was initially discovered as a target of the so-called organophosphorus-induced delayed polyneuropathy triggered by the inhibition of the NTE-associated esterase center by neuropathic organophosphorus compounds (OPs). The physiological role of NTE might be related to membrane lipid homeostasis and seems to be involved in adult organisms in maintaining nervous system integrity. However, NTE is also involved in cell differentiation and embryonic development. NTE is expressed in embryonic and adult stem cells, and the silencing of Pnpla6 by interference RNA in D3 mouse cells causes significant alterations in several genetic pathways related to respiratory tube and nervous system formation, and in vasculogenesis and angiogenesis. The silencing of gene PNPLA6 in human NT2 cells at the beginning of neurodifferentiation causes severe phenotypic alterations in neuron-like differentiated cells; e.g. reduced electrical activity and the virtual disappearance of markers of neural tissue, synapsis and glia. These phenotypic effects were not reproduced when NTE esterase activity was inhibited by neuropathic OP mipafox instead of being silenced at the genetic level. Neuropathic OP chlorpyrifos seems able to induce neurodevelopmental alterations in animals. However, the effects of chlorpyrifos in the expression of biomarker genes of differentiation in D3 cells differ considerably from the effects induced by Pnpla6 silencing. In conclusion, available information suggests that PNPLA6 and/or the NTE protein play a role in early neurodifferentiation stages, although this role is not dependent upon the esterase NTE center. Therefore, impairments caused by OPs, such as chlorpyrifos, on neurodevelopment are not due to inhibition of NTE esterase enzymatic activity.

Effects of mipafox, paraoxon, chlorpyrifos and its metabolite chlorpyrifos-oxon on the expression of biomarker genes of differentiation in D3 mouse embryonic stem cells.

Chlorpyrifos (CPS) is an organophosphorus compound (OP) capable of causing well-known cholinergic and delayed syndromes through the inhibition of acetylcholinesterase and Neuropathy Target Esterase (NTE), respectively. CPS is also able to induce neurodevelopmental toxicity in animals. NTE is codified by the Pnpla6 gene and plays a central role in differentiation and neurodifferentiation. We tested, in D3 mouse embryonic stem cells under differentiation, the effects of the NTE inhibition by the OPs mipafox, CPS and its main active metabolite chlorpyrifos-oxon (CPO) on the expression of genes Vegfa, Bcl2, Amot, Nes and Jun, previously reported to be under- or overexpressed after Pnpla6 silencing in this same cellular model. Mipafox did not significantly alter the expression of such genes at concentrations that significantly inhibited NTE. However, CPS and CPO at concentrations that caused NTE inhibition at similar levels to mipafox statistically and significantly altered the expression of most of these genes. Paraoxon (another OP with capability to inhibit esterases but not NTE) caused similar effects to CPS and CPO. These findings suggest that the molecular mechanism for the neurodevelopmental toxicity induced by CPS is not based on NTE inhibition, and that other unknown esterases might be potential targets of neurodevelopmental toxicity.

Expression of biomarker genes of differentiation in D3 mouse embryonic stem cells after exposure to different embryotoxicant and non-embryotoxicant model chemicals.

There is a necessity to develop in vitro methods for testing embryotoxicity (Romero et al., 2015) [1]. We studied the progress of D3 mouse embryonic stem cells differentiation exposed to model embryotoxicants and non-embryotoxicants chemicals through the expression of biomarker genes. We studied a set of 16 different genes biomarkers of general cellular processes (Cdk1, Myc, Jun, Mixl, Cer and Wnt3), ectoderm formation (Nrcam, Nes, Shh and Pnpla6), mesoderm formation (Mesp1, Vegfa, Myo1e and Hdac7) and endoderm formation (Flk1 and Afp). We offer dose response in order to derive the concentration causing either 50% or 200% of expression of the biomarker gene. These records revealed to be a valuable end-point to predict in vitro the embryotoxicity of chemicals (Romero et al., 2015) [1].

RNA transcripts for the quantification of differentiation allow marked improvements in the performance of embryonic stem cell test (EST).

Embryonic stem cell test (EST) is an in vitro validated assay for testing embryotoxicity. The EST needs improvements before being used for regulatory purposes, but also needs technical simplification for use in high throughput screenings. We propose the quantification in alterations of the differentiation of D3 monolayer cells cultures through the expression of biomarker genes in a shorter (5-day) and technically simpler (we use only monolayer cultures) test. We have defined a set of sixteen different genes biomarkers of ectoderm (Nrcam, Nes, Shh and Pnpla6), endoderm formation (Flk1 and Afp), mesoderm formation (Mesp1, Vegfa, Myo1e and Hdac7) and general cellular processes (Cdk1, Myc, Jun, Mixl, Cer and Wnt3). These, together with alterations in the viability of D3 and 3T3 cells and the prediction model of a classic EST, enhance the features of EST determinations to 100% concordance between in vivo-in vitro predictions with a set of seven different chemicals used in the validation of a classic EST. In conclusion, the proposed changes implemented in the classic EST confer it more reliability, speed and technical simplicity, which brings the EST closer to high throughput processes and regulatory purposes.

Organophosphorus pesticide chlorpyrifos and its metabolites alter the expression of biomarker genes of differentiation in D3 mouse embryonic stem cells in a comparable way to other model neurodevelopmental toxicants.

There are discrepancies about whether chlorpyrifos is able to induce neurodevelopmental toxicity or not. We previously reported alterations in the pattern of expression of biomarker genes of differentiation in D3 mouse embryonic stem cells caused by chlorpyrifos and its metabolites chlorpyrifos-oxon and 3,5,6-trichloro-2-pyridinol. Now, we reanalyze these data comparing the effects on these genes with those caused in the same genes by retinoic acid, valproic acid, and penicillin-G (model compounds considered as strong, weak, and non-neurodevelopmental toxicants, respectively). We also compare the effects of chlorpyrifos and its metabolites on the cell viability of D3 cells and 3T3 mouse fibroblasts with the effects caused in the same cells by the three model compounds. We conclude that chlorpyrifos and its metabolites act, regarding these end-points, as the weak neurodevelopmental toxicant valproic acid, and consequently, a principle of caution should be applied avoiding occupational exposures in pregnant women. A second independent experiment run with different cellular batches coming from the same clone obtained the same result as the first one.

An integrated approach for detecting embryotoxicity and developmental toxicity of environmental contaminants using in vitro alternative methods.

The main available alternatives for testing embryotoxicity are cellular tests with stem cells and in vitro-ex vivo tests with embryos. In cellular tests, the most developed alternative is the embryonic stem cell test, while the most developed tests involving embryos are the zebrafish and whole embryo culture test. They are technically more complex than cellular tests, but offer the advantage of determining the expectable phenotypic alteration caused by the exposure. Many efforts are currently being made, basically through proteomic and genomic approaches, in order to obtain improvements in predictivity of these tests. Development is a very complex process, and it is highly unlikely that a single alternative test can yield satisfactory performance with all types of chemicals. We propose a step-wise approach where model complexity, and consequently technical skills and economical costs, gradually increase if needed. The first level would be run short cellular assays to detect effects in early differentiation stages. The second level would involve longer cellular embryotoxicity tests to search embryotoxicants that have an effect on late differentiation stages. The third stage would consider tests with embryos because they allow the determination of hazards based on molecular and morphological alterations, and not only on differentiating cells.

Genomic and phenotypic alterations of the neuronal-like cells derived from human embryonal carcinoma stem cells (NT2) caused by exposure to organophosphorus compounds paraoxon and mipafox.

Historically, only few chemicals have been identified as neurodevelopmental toxicants, however, concern remains, and has recently increased, based upon the association between chemical exposures and increased developmental disorders. Diminution in motor speed and latency has been reported in preschool children from agricultural communities. Organophosphorus compounds (OPs) are pesticides due to their acute insecticidal effects mediated by the inhibition of acetylcholinesterase, although other esterases as neuropathy target esterase (NTE) can also be inhibited. Other neurological and neurodevelopmental toxic effects with unknown targets have been reported after chronic exposure to OPs in vivo. We studied the initial stages of retinoic acid acid-triggered differentiation of pluripotent cells towards neural progenitors derived from human embryonal carcinoma stem cells to determine if neuropathic OP, mipafox, and non-neuropathic OP, paraoxon, are able to alter differentiation of neural precursor cells in vitro. Exposure to 1 µM paraoxon (non-cytotoxic concentrations) altered the expression of different genes involved in signaling pathways related to chromatin assembly and nucleosome integrity. Conversely, exposure to 5 µM mipafox, a known inhibitor of NTE activity, showed no significant changes on gene expression. We conclude that 1 µM paraoxon could affect the initial stage of in vitro neurodifferentiation possibly due to a teratogenic effect, while the absence of transcriptional alterations by mipafox exposure did not allow us to conclude a possible effect on neurodifferentiation pathways at the tested concentration.

Functional pathways altered after silencing Pnpla6 (the codifying gene of neuropathy target esterase) in mouse embryonic stem cells under differentiation.

Neuropathy target esterase (NTE) is involved in several disorders in adult organisms and embryos. A relationship between NTE and nervous system integrity and maintenance in adult systems has been suggested. NTE-related motor neuron disease is associated with the expression of a mutant form of NTE and the inhibition and further modification of NTE by organophosphorus compounds is the trigger of a delayed neurodegenerative neuropathy. Homozygotic NTE knockout mice embryos are not viable, while heterozygotic NTE knockout mice embryos yields mice with neurological disorders, which suggest that this protein plays a critical role in embryonic development. The present study used D3 mouse embryonic stem cells with the aim of gaining mechanistic insights on the role of Pnpla6 (NTE gene encoding) in the developmental process. D3 cells were silenced by lipofectamine transfection with a specific interference RNA for Pnpla6. Silencing Pnpla6 in D3 monolayer cultures reduced NTE enzymatic activity to 50% 20 h post-treatment, while the maximum loss of Pnpla6 expression reached 80% 48 h postsilencing. Pnpla6 was silenced in embryoid bodies and 545 genes were differentially expressed regarding the control 96 h after silencing, which revealed alterations in multiple genetic pathways, such as cell motion and cell migration, vesicle regulation, and cell adhesion. These findings also allow considering that these altered pathways would impair the formation of respiratory, neural, and vascular tubes causing the deficiencies observed in the in vivo development of nervous and vascular systems. Our findings, therefore, support the previous observations made in vivo concerning lack of viability of mice embryos not expressing NTE and help to understand the biology of several neurological and developmental disorders in which NTE is involved.

Chlorpyrifos and its metabolites alter gene expression at non-cytotoxic concentrations in D3 mouse embryonic stem cells under in vitro differentiation: considerations for embryotoxic risk assessment.

The effects of organophosphate insecticide chlorpyrifos (CPF) on development are currently under discussion. CPF and its metabolites, chlorpyrifos-oxon (CPO) and 3,5,6-trichloro-2-pyridinol (TClP), were more cytotoxic for D3 mouse embryonic stem cells than for differentiated fibroblasts 3T3 cells. Exposure to 10 µM CPF and TClP and 100 µM CPO for 12 h significantly altered the in vitro expression of biomarkers of differentiation in D3 cells. Similarly, exposure to 20 µM CPF and 25 µM CPO and TClP for 3 days also altered the expression of the biomarkers in the same model. These exposures caused no significant reduction in D3 viability with mild inhibition of acetylcholinesterase and neuropathy target esterase by CPF and severe inhibition by CPO. We conclude that certain in vivo exposure scenarios are possible, which cause inhibition of acetylcholinesterase but without clinical symptoms that reach high enough systemic CPF concentrations able to alter the expression of genes involved in cellular differentiation with potentially hazard effects on development. Conversely, the risk for embryotoxicity by CPO and TClP was very low because the required exposure would induce severe cholinergic syndrome.

Shortening and Improving the Embryonic Stem Cell Test through the Use of Gene Biomarkers of Differentiation.

The embryonic Stem cell Test (EST) is a validated assay for testing embryotoxicity in vitro. The total duration of this protocol is 10 days, and its main end-point is based on histological determinations. It is suggested that improvements on EST must be focused toward molecular end-points and, if possible, to reduce the total assay duration. Five days of exposure of D3 cells in monolayers under spontaneous differentiation to 50 ng/mL of the strong embryotoxic 5-fluorouracil or to 75 µg/mL of the weak embryotoxic 5,5-diphenylhydeantoin caused between 20 and 74% of reductions in the expression of the following genes: Pnpla6, Afp, Hdac7, Vegfa, and Nes. The exposure to 1 mg/mL of nonembryotoxic saccharin only caused statistically significant reductions in the expression of Nes. These exposures reduced cell viability of D3 cells by 15, 28, and 34%. We applied these records to the mathematical discriminating function of the EST method to find that this approach is able to correctly predict the embryotoxicity of all three above-mentioned chemicals. Therefore, this work proposes the possibility of improve EST by reducing its total duration and by introducing gene expression as biomarker of differentiation, which might be very interesting for in vitro risk assessment embryotoxicity.