Developmental exposure to pesticides that disrupt retinoic acid signaling causes persistent retinoid and behavioral dysfunction in zebrafish.

Early developmental exposure to environmental toxicants may play a role in the risk for developing autism. A variety of pesticides have direct effects on retinoic acid (RA) signaling and as RA signaling has important roles in neurodevelopment, such compounds may cause developmental neurotoxicity through an overlapping adverse outcome pathway. It is hypothesized that a pesticide's embryonic effects on retinoid function may correspond with neurobehavioral disruption later in development. In the current studies, we determined the effects of RA-acting pesticides on neurobehavioral development in zebrafish. Buprofezin and imazalil caused generalized hypoactivity in the larval motility test, whereas chlorothalonil and endosulfan I led to selective hypoactivity and hyperactivity, respectively. With buprofezin, chlorothalonil, and imazalil, hypoactivity and/or novel anxiety-like behaviors persisted in adulthood and buprofezin additionally decreased social attraction responses in adulthood. Endosulfan I did not produce significant adult behavioral effects. Using qPCR analyses of adult brain tissue, we observed treatment-induced alterations in RA synthesis or catabolic genes, indicating persistent changes in RA homeostasis. These changes were compound-specific, with respect to expression directionality, and potential patterns of homeostatic disruption. Results suggest the likely persistence of disruptions in RA signaling well into adulthood and may represent compensatory mechanisms following early life stage exposures. This study demonstrates that early developmental exposure to environmental toxicants that interfere with RA signaling causes short as well as long-term behavioral disruption in a well-established zebrafish behavioral model and expand upon the meaning of the RA adverse outcome pathway, indicating that observed effects likely correspond with the nature of underlying homeostatic effects.

An Evaluation of Human Induced Pluripotent Stem Cells to Test for Cardiac Developmental Toxicity.

To prevent congenital defects arising from maternal exposure, safety regulations require pre-market developmental toxicity screens for industrial chemicals and pharmaceuticals. Traditional embryotoxicity approaches depend heavily on the use of low-throughput animal models which may not adequately predict human risk. The validated embryonic stem cell test (EST) developed in murine embryonic stem cells addressed the former problem over 15 years ago. Here, we present a proof-of-concept study to address the latter challenge by updating all three endpoints of the classic mouse EST with endpoints derived from human induced pluripotent stem cells (hiPSCs) and human fibroblasts. Exposure of hiPSCs to selected test chemicals inhibited differentiation at lower concentrations than observed in the mouse EST. The hiPSC-EST also discerned adverse developmental outcomes driven by novel environmental toxicants. Evaluation of the early cardiac gene TBX5 yielded similar toxicity patterns as the full-length hiPSC-EST. Together, these findings support the further development of hiPSCs and early molecular endpoints as a biologically relevant embryotoxicity screening approach for individual chemicals and mixtures.

The role of lncRNA Meg3 in the proliferation of all-trans retinoic acid-treated mouse embryonic palate mesenchymal cells involves the Smad pathway.

Mesenchymal cell proliferation is critical for the growth of the palate shelf. All-trans retinoic acid (atRA), as well as pathways associated with TGF-ß/Smad signaling, play crucial roles in the proliferation of mouse embryonic palate mesenchymal (MEPM) cells. We have found that MEPM-cell proliferation was regulated by atRA and exogenous TGF-ß3 could significantly antagonize the atRA-mediated suppression of MEPM cell proliferation, which is closely associated with the regulation of TGF-ß/Smad signaling pathway. The long non-coding RNA (lncRNA) MEG3 has been reported to activate TGF-ß/Smad signaling, thereby regulating cellular proliferation, differentiation, and related processes. Here, we found that Meg3 expression increased significantly in atRA-treated MEPM cells while TGF-ß3 treatment markedly inhibited Meg3 expression and antagonized the effect of atRA on Meg3. Moreover, Smad2 was found to interact directly with Meg3, and atRA treatment significantly enriched Meg3 in Smad2-immunoprecipitated samples. After Meg3 deletion, the effects of atRA on the proliferation of MEPM cells and TGF-ß3-dependent protein expression were lost. Hence, we speculate that Meg3 has a role in the RA-induced suppression of MEPM cell proliferation by targeting Smad2 and thereby mediating TGF-ß/Smad signaling inhibition.

Pharmacoepigenomics circuits induced by a novel retinoid-polyamine conjugate in human immortalized keratinocytes.

Retinoids are widely used in diseases spanning from dermatological lesions to cancer, but exhibit severe adverse effects. A novel all-trans-Retinoic Acid (atRA)-spermine conjugate (termed RASP) has shown previously optimal in vitro and in vivo anti-inflammatory and anticancer efficacy, with undetectable teratogenic and toxic side-effects. To get insights, we treated HaCaT cells which resemble human epidermis with IC50 concentration of RASP and analyzed their miRNA expression profile. Gene ontology analysis of their predicted targets indicated dynamic networks involved in cell proliferation, signal transduction and apoptosis. Furthermore, DNA microarrays analysis verified that RASP affects the expression of the same categories of genes. A protein-protein interaction map produced using the most significant common genes, revealed hub genes of nodal functions. We conclude that RASP is a synthetic retinoid derivative with improved properties, which possess the beneficial effects of retinoids without exhibiting side-effects and with potential beneficial effects against skin diseases including skin cancer.

Cytotoxic and Genotoxic Effects of Cyanobacterial and Algal Extracts-Microcystin and Retinoic Acid Content.

In the last decade, it has become evident that complex mixtures of cyanobacterial bioactive substances, simultaneously present in blooms, often exert adverse effects that are different from those of pure cyanotoxins, and awareness has been raised on the importance of studying complex mixtures and chemical interactions. We aimed to investigate cytotoxic and genotoxic effects of complex extracts from laboratory cultures of cyanobacterial species from different orders (Cylindrospermopsis raciborskii, Aphanizomenon gracile, Microcystis aeruginosa, M. viridis, M. ichtyoblabe, Planktothrix agardhii, Limnothrix redekei) and algae (Desmodesmus quadricauda), and examine possible relationships between the observed effects and toxin and retinoic acid (RA) content in the extracts. The cytotoxic and genotoxic effects of the extracts were studied in the human hepatocellular carcinoma HepG2 cell line, using the MTT assay, and the comet and cytokinesis-block micronucleus (cytome) assays, respectively. Liquid chromatography electrospray ionization mass spectrometry (LC/ESI-MS) was used to detect toxins (microcystins (MC-LR, MC-RR, MC-YR) and cylindrospermopsin) and RAs (ATRA and 9cis-RA) in the extracts. Six out of eight extracts were cytotoxic (0.04-2 mgDM/mL), and five induced DNA strand breaks at non-cytotoxic concentrations (0.2-2 mgDM/mL). The extracts with genotoxic activity also had the highest content of RAs and there was a linear association between RA content and genotoxicity, indicating their possible involvement; however further research is needed to identify and confirm the compounds involved and to elucidate possible genotoxic effects of RAs.

Refinement of a differentiation protocol using neuroblastoma SH-SY5Y cells for use in neurotoxicology research.

Since most models used to study neuronal dysfunction display disadvantages and ethical concerns, a fast and reproducible in vitro model to study mitochondria-related neurodegeneration is required. Here, we optimized and characterized a 3-day retinoic acid-based protocol to differentiate the SH-SY5Y cell line into a neuronal-like phenotype and investigated alterations in mitochondrial physiology and distribution. Differentiation was associated with p21-linked cell cycle arrest and an increase in cell mass and area, possibly associated with the development of neurite-like extensions. Notably, increased expression of mature neuronal markers (neuronal-specific nuclear protein, microtubule-associated protein 2, ßIII tubulin and enolase 2) was observed in differentiated cells. Moreover, increased mitochondrial content and maximal area per cell suggests mitochondrial remodeling. To demonstrate that this model is appropriate to study mitochondrial dysfunction, cells were treated for 6 h with mitochondrial toxicants (rotenone, antimycin A, carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP) and 6-hydroxydopamine (6-OHDA)). Differentiated cells were more susceptible to increasing concentrations of FCCP, antimycin A, and rotenone, while 6-OHDA showed a distinct dose-dependent neurotoxicity pattern. Even though differentiated cells did not exhibit a fully mature/differentiated neuronal phenotype, the protocol developed can be used to study neurotoxicity processes, mitochondrial dynamics, and bioenergetic impairment, representing an alternative to study mitochondrial impairment-related pathologies in vitro.

Neurobehavioral effects of cyanobacterial biomass field extracts on zebrafish embryos and potential role of retinoids.

Cyanobacteria are known for their ability to produce and release mixtures of up to thousands of compounds into the environment. Recently, the production of novel metabolites, retinoids, was reported for some cyanobacterial species along with teratogenic effects of samples containing these compounds. Retinoids are natural endogenous substances derived from vitamin A that play a crucial role in early vertebrate development. Disruption of retinoid signalling- especially during the early development of the nervous system- might lead to major malfunctions and malformations. In this study, the toxicity of cyanobacterial biomass samples from the field containing retinoids was characterized by in vivo and in vitro bioassays with a focus on the potential hazards towards nervous system development and function. Additionally, in order to identify the compounds responsible for the observed in vitro and in vivo effects the complex cyanobacterial extracts were fractionated (C18 column, water-methanol gradient) and the twelve obtained fractions were tested in bioassays. In all bioassays, all-trans retinoic acid (ATRA) was tested along with the environmental samples as a positive control. Retinoid-like activity (mediated via the retinoic acid receptor, RAR) was measured in the transgenic cell line p19/A15. The in vitro assay showed retinoid-like activity by specific interaction with RAR for the biomass samples. Neurotoxic effects of selected samples were studied on zebrafish (Danio rerio) embryos using the light/dark transition test (Viewpoint, ZebraLab system) with 120 hpf larvae. In the behavioural assay, the cyanobacterial extracts caused significant hyperactivity in zebrafish at 120 hpf after acute exposure (3 h prior to the measurement) at concentrations below the teratogenicity LOEC (0.2 g dw L-1). Similar effect was observed after exposure to fractions of the extracts with detected retinoid-like activity and additive effect was observed after combining the fractions. However, the effect on behaviour was not observed after exposure to ATRA only. To provide additional insight into the behavioural effects and describe the underlying mechanism gene expression of selected biomarkers was measured. We evaluated an array of 28 genes related to general toxicity, neurodevelopment, retinoid and thyroid signalling. We detected several affected genes, most notably, the Cyp26 enzymes that control endogenous ATRA concentration, which documents an effect on retinoid signalling.

RNA-seq analysis of palatal transcriptome changes in all-trans retinoic acid-induced cleft palate of mice.

Cleft palate is a common congenital maxillofacial malformation in newborns. All-trans retinoic acid (atRA) is an ideal exogenous stimulus to construct a mouse cleft palate model. However, the precise pathogenic mechanism remains to be elucidated. In our study, to explore the toxicity of atRA on palatal shelves during different stages of palate development, a total of 100 mg/kg atRA was administered to C57BL/6 mice at embryonic day 10.5 (E10.5). Mouse embryonic palatal shelves at E13.5, E14.5, E15.5, and E16.5 were collected for RNA extraction and histological treatment. Changes in gene expression were tested through RNA-seq. Selected differentially expressed genes (DEGs) related to metabolic pathways, such as Ptgds, Ttr, Cyp2g1, Ugt2a1 and Mgst3, were validated and analyzed by Quantitative real-time PCR (qRT-PCR). In addition, Gene Oncology analysis showed that transcriptional changes of genes from extracellular matrix (ECM) components, such as Spp1, and crystallin family might play important role in palatal shelves elevation (E13.5-E14.5). Therefore, the protein expression level of Ttr and Spp1 from E13.5 to E16.5 were tested by immunohistochemistry (IHC). Besides, the mRNA level of Spp1, were down-regulated at E16.5 and the protein were down-regulated at E15.5 and E16.5 in all-trans retinoic acid group, suggesting that atRA may involve in palatal bone formation by regulating Spp1. Overall, gene transcriptional profiles were obviously different at each time point of palate development. Thus, this study summarized some pathways and genes that may be related to palatogenesis and cleft palate through RNA-seq, to provide a direction for subsequent studies on the mechanism and targeted therapy of cleft palate.

WIF1 Suppresses the Generation of Suprabasal Cells in Acanthotic Skin and Growth of Basal Cell Carcinomas upon Forced Overexpression.

We analyzed the role of WIF1 in normal and acanthotic epidermis of 12-O-tetradecanoylphorbol-13-acetate (TPA) or all-trans-retinoic acid (ATRA)-treated and basal cell carcinoma (BCC)-bearing mice. WIF1 protein is located in the follicular infundibulum and interfollicular epidermis (IFE) in murine back skin. Within the hyperplastic epidermis of TPA- or ATRA-treated or BCC-bearing murine skin, WIF1 and Keratin 10 overlap in Ki67⁻ suprabasal layers, while basal epidermal layers expressing Ki67, and BCCs expressing Wif1 mRNA, are free of WIF1 protein. This is similar in human skin, with the exception that WIF1 protein is found in single Ki67⁻ basal epidermal cells in normal skin and additionally in Ki67+ cells in acanthotic skin. Wif1-deficiency enhances acanthosis of the murine BCC-associated epidermis, which is accompanied by an increase of Ki67+ and of Sca-1+ basal cells. WIF1 overexpression in allografted BCC-derived keratinocytes prevents growth and keratinization, involving enhanced phosphorylation of protein kinase C and extracellular signal-regulated kinase 1 and arguably factors secreted by the in vivo environment. In summary, WIF1 protein marks suprabasal layers in the normal IFE. It is also present in the epidermis overlaying BCCs where it diminishes proliferation of basal cells and production of differentiating suprabasal cells. In addition, WIF1 can prevent proliferation and keratinization of BCC-related keratinocytes.

MiR-106a-5p modulates apoptosis and metabonomics changes by TGF-ß/Smad signaling pathway in cleft palate.

BACKGROUND: The molecular mechanisms of abnormal palatogenesis were investigated in this study. A key regulator, miR-106a-5p, and its target pathway were analyzed. OBJECTIVES: This research is trying to clarify the underlying mechanism of the modulation of miRNA transcription during the formation of cleft palate by 7T and 9.4T NMR metabolomic platforms. METHOD: Differentially expressed miRNAs and mRNAs were analyzed by microarray analysis and verified by qRT-PCR. The protein expression in TGFß signaling pathways were analyzed by Western Blotting. The relationship between miR-106a-5p and TGFß were analyzed by luciferase reporter assay. Cell apoptosis were analyzed by flow cytometer. And finally, the metabonomics were analyzed by NMR and multivariate data analysis models (MVDA). RESULTS: The expression of miR-106a-5p increased in cleft palatal tissue and negatively correlated with the protein level of Tgfbr2. The luciferase assay further proved that the tgfbr2 was a direct target of miR-106a-5p. In another aspect, miR-106a-5p increased apoptosis level in palatal mesenchymal cells, possibly because its inhibition of TGFß signaling pathway. Moreover, low cholesterol and choline levels with high citric acid and lipid levels were observed by 7T and 9.4T NMR metabonomic analysis, which inferred the disorder of cell membrane synthesis in cleft palate formation. Furthermore, transformation from choline to phosphatidylcholine regulated by miR-106a-5p was also disrupted, resulting in phosphatidic choline synthesis disorder and reduced cell membrane synthesis. CONCLUSIONS: The regulatory mechanism of cleft palate was studied at transcriptional and metabolomics levels, which may provide important information in understanding the primary cause of this abnormality.

Reduced H3K27me3 leads to abnormal Hox gene expression in neural tube defects.

BACKGROUND: Neural tube defects (NTDs) are severe, common birth defects that result from failure of normal neural tube closure during early embryogenesis. Accumulating strong evidence indicates that genetic factors contribute to NTDs etiology, among them, HOX genes play a key role in neural tube closure. Although abnormal HOX gene expression can lead to NTDs, the underlying pathological mechanisms have not fully been understood. METHOD: We detected that H3K27me3 and expression of the Hox genes in a retinoic acid (RA) induced mouse NTDs model on E8.5, E9.5 and E10.5 using RNA-sequencing and chromatin immunoprecipitation sequencing assays. Furthermore, we quantified 10 Hox genes using NanoString nCounter in brain tissue of fetuses with 39 NTDs patients including anencephaly, spina bifida, hydrocephaly and encephalocele. RESULTS: Here, our results showed differential expression in 26 genes with a > 20-fold change in the level of expression, including 10 upregulated Hox genes. RT-qPCR revealed that these 10 Hox genes were all upregulated in RA-induced mouse NTDs as well as RA-treated embryonic stem cells (ESCs). Using ChIP-seq assays, we demonstrate that a decrease in H3K27me3 level upregulates the expression of Hox cluster A-D in RA-induced mouse NTDs model on E10.5. Interestingly, RA treatment led to attenuation of H3K27me3 due to cooperate between UTX and Suz12, affecting Hox gene regulation. Further analysis, in human anencephaly cases, upregulation of 10 HOX genes was observed, along with aberrant levels of H3K27me3. Notably, HOXB4, HOXC4 and HOXD1 expression was negatively correlated with H3K27me3 levels. CONCLUSION: Our results indicate that abnormal HOX gene expression induced by aberrant H3K27me3 levels may be a risk factor for NTDs and highlight the need for further analysis of genome-wide epigenetic modification in NTDs.

In Utero Amniotic Fluid Stem Cell Therapy Protects Against Myelomeningocele via Spinal Cord Coverage and Hepatocyte Growth Factor Secretion.

Despite the poor prognosis associated with myelomeningocele (MMC), the options for prenatal treatments are still limited. Recently, fetal cellular therapy has become a new option for treating birth defects, although the therapeutic effects and mechanisms associated with such treatments remain unclear. The use of human amniotic fluid stem cells (hAFSCs) is ideal with respect to immunoreactivity and cell propagation. The prenatal diagnosis of MMC during early stages of pregnancy could allow for the ex vivo proliferation and modulation of autologous hAFSCs for use in utero stem cell therapy. Therefore, we investigated the therapeutic effects and mechanisms of hAFSCs-based treatment for fetal MMC. hAFSCs were isolated as CD117-positive cells from the amniotic fluid of 15- to 17-week pregnant women who underwent amniocentesis for prenatal diagnosis and consented to this study. Rat dams were exposed to retinoic acid to induce fetal MMC and were subsequently injected with hAFSCs in each amniotic cavity. We measured the exposed area of the spinal cord and hepatocyte growth factor (HGF) levels at the lesion. The exposed spinal area of the hAFSC-treated group was significantly smaller than that of the control group. Immunohistochemical analysis demonstrated a reduction in neuronal damage such as neurodegeneration and astrogliosis in the hAFSC-treated group. Additionally, in lesions of the hAFSC-treated group, HGF expression was upregulated and HGF-positive hAFSCs were identified, suggesting that these cells migrated to the lesion and secreted HGF to suppress neuronal damage and induce neurogenesis. Therefore, in utero hAFSC therapy could become a novel strategy for fetal MMC. Stem Cells Translational Medicine 2019;8:1170-1179.

Immunohistochemical profile of CD markers in experimental neural tube defect.

Neural tube defects (NTDs) are the second most common birth defects worldwide. Stem cells play a critical role in the mechanisms underlying NTDs. We established an experimental NTD model in rats using retinoic acid (RA). We used mesenchymal and hemopoietic stem cell markers to determine their distribution in the mesenchyme in and around the neuroepithelium during the embryonic and fetal periods in both cranial and caudal regions. Adult female rats were given RA on days 5 and 10 of gestation and olive oil was administered to the control group. On days 10.5 and 15.5, embryos in the experimental and control groups were removed from the uterus. Embryos were embedded in paraffin and serial sections of the cranial and caudal neural tube were examined. We found severe cranial and caudal defects including axial rotation in the experimental groups using histochemistry. We used CD44, CD56, CD73, CD90, CD105, CD271 antibodies as mesenchymal stem cell markers and CD14, CD45 as hemopoietic stem cell markers. More CD44, CD56, CD90, CD105 and CD14 were detected during the embryonic period than the fetal period. CD73 was more frequent during the fetal period, whereas CD271 and CD45 were not significantly different. When CD44, CD56, CD73, CD90, CD105, CD271 immunostaining was found, NTDs were decreased early and increased later. We found no significant difference between CD14 and CD45. Formation of NTDs was due to deterioration of the of the neuroepithelial and surrounding stem cells. One reason for the formation of NTDs is that stem cells may develop defective cell-cell or cell-matrix interactions.

Inhibition of NRF2 signaling and increased reactive oxygen species during embryogenesis in a rat model of retinoic acid-induced neural tube defects.

Exposure to retinoic acid (RA) during pregnancy increases the risk of serious neural tube defects (NTDs) in the developing fetus. The precise molecular mechanism for this process is unclear; however, RA is associated with oxidative stress mediated by reactive oxygen species. Nuclear factor erythroid 2-related factor 2 (NRF2) is a master regulator of oxidative stress that directs the expression of antioxidant genes and detoxifying proteins to maintain redox homeostasis. We established a rat model of NTDs in which pregnant dams were administered all-trans (at)RA on gestational day 10, and oxidative stress levels and the spatiotemporal expression of NRF2 and its downstream targets were examined in the resulting embryos and in maternal blood. In the NTD group, total antioxidative capacity decreased and 8-hydroxy-2'-deoxyguanosine increased in maternal serum and fetal spinal cord tissues. Plasma GSH content, the GSH/GSSG ratio, and glutathione peroxidase activity in fetal spinal cords were lower in the NTD group relative to controls. We detected NRF2 protein reduction and concomitant upregulation of Kelch-like ECH-associated protein 1 (KEAP1) - a cytoplasmic inhibitor of NRF2 - in the NTD group. The mRNA and protein levels of downstream targets of NRF2 were downregulated in the spinal cords of NTD embryos. These data demonstrate substantial oxidative stress and NRF2 signaling pathway disruption in a model of NTDs induced by atRA. The inhibitory effects of atRA on NRF2 signaling may lower cellular defenses against RA-induced oxidative stress and could play important roles in NTD occurrence during embryonic development.

CD200-CD200R imbalance correlates with microglia and pro-inflammatory activation in rat spinal cords exposed to amniotic fluid in retinoic acid-induced spina bifida.

Spina bifida aperta is a congenital malformation characterized by the failure of neural tube closure resulting in an unprotected fetal spinal cord. The spinal cord then undergoes progressive damage, likely due to chemical and mechanical factors related to exposure to the intrauterine environment. Astrogliosis in exposed spinal cords has been described in animal models of spina bifida during embryonic life but its relationship with neuroinflammatory processes are completely unknown. Using a retinoic acid-induced rat model of spina bifida we demonstrated that, when exposed to amniotic fluid, fetal spinal cords showed progressive astrogliosis with neuronal loss at mid-gestation (E15) compared to unexposed spinal cords. The number of microglial cells with a reactive phenotype and activation marker expression increased during gestation and exhibited progressive disruption in the inhibitory immune ligand-receptor system. Specifically we demonstrate down-regulation of CD200 expression and up-regulation of CD200R. Exposed spinal cords demonstrated neuroinflammation with increased tissue water content and cytokine production by the end of gestation (E20), which correlated with active Caspase3 expression in the exposed layers. Our findings provide new evidence that microglia activation, including the disruption of the endogenous inhibitory system (CD200-CD200R), may participate in the pathogenesis of spina bifida through late gestation.

Mitochondrial dysfunction is implicated in retinoic acid-induced spina bifida aperta in rat fetuses.

Neural tube defects (NTDs) are the most common and severe congenital malformations, which result from failure of the neural tube to close during embryonic development. The etiology of NTDs is complex, caused by interactions between genetic defects and environmental factors, but the exact mechanisms of this disease are still not fully understood. We herein employ a Seahorse Bioscience microplate-based extracellular flux (XF) analyzer to determine mitochondrial function and quantify respiratory coupling to various bioenergetic functions using specific pharmacological inhibitors of bioenergetic pathways. We demonstrate that changes in coupling between ATP turnover and proton leak are correlated with NTDs. Further, we determined that the ATP content and oxidative stress levels in posterior spinal cords of rat embryos with NTDs between E11 and E14 was lower than that of normal controls. The present study reveals that mitochondrial dysfunction is associated with all-trans retinoic acid (atRA)-induced NTDs in rat embryos. Oxidative stress results from decreased antioxidant enzyme activity. This study provides a novel viewpoint for exploring the embryonic pathogenesis of atRA-induced NTDs.

Protective effect of quercetin on histomorphometric changes in kidney of retinoid acid-treated rat fetuses / Efecto protector de la quercetina sobre los cambios histomorfométricos en el riñón de fetos de rata tratados con ácido retinoico

SUMMARY: Retinoic acid, an active metabolite of vitamin A, plays essential signaling roles in mammalian embryogenesis. Prenatal rat fetuse exposure to retinoid induces some malformations in various organs, the most active and teratogenic metablolite is all-transretinoic acid (atRA). The teratogenic effects of some drugs can be prevented by the application of antioxidant drugs and stimulation of the maternal immune system. Also, quercetin, a naturally occurring flavonoid has excellent antioxidant properties. Therefore, the aim of this study was assess the protective effects of quercetin against atRA in fetuses of rat's kidney tissue. This study was performed on 40 pregnant rats that were divided into seven groups. Control group received normal saline and test groups received DMSO, quercetin (75 mg/kg), quercetin (200 mg/kg), atRA (25 mg/kg), atRA (25 mg/kg) plus quercetin (75 mg/kg) and atRA (25 mg/kg) plus quercetin (200 mg/kg), intraperitoneally at 8-10th days of gestation. Fetuses were collected at 20th day of gestation. Kidneys were collected and placed in 10 % buffered formalin solution. Then, kidneys were sectioned by routine method and stained by H&E and examined histologically. On histomorphomertrical examination, it was observed the priglomerular space and diameter of renal corpuscle in group which received only atRA were significantly (p≤0.05) greater than those received normal saline, dimethyl sulfoxide and quercetin, while these two indexes in group which received atRA plus quercetin significantly (p≤0.05) decreased by quercetin as dose dependent manner. Number of renal corpuscles were significantly (p≤0.05) decreased by atRA, but the quercetin could not affect the glomerular numbers. It is concluded that quercetin can protect fetuses against atRA damages and prevent their incidence probably via its antioxidant effect.

RESUMEN: El ácido retinoico, un metabolito activo de la vitamina A, desempeña un papel esencial de señalización en la embriogénesis de mamíferos. La exposición al ácido retinoico en fetos de ratas prenatales induce malformaciones en varios órganos, siendo el metabolito más activo y teratogénico el ácido transretinoico (ATRA). Los efectos teratogénicos de algunos medicamentos se pueden prevenir mediante la aplicación de medicamentos antioxidantes y la estimulación del sistema inmune materno. Además, la quercetina, un flavonoide de origen natural, tiene excelentes propiedades antioxidantes. Por lo tanto, el objetivo de este estudio fue evaluar los efectos protectores de quercetina contra ATRA en fetos de tejido de riñón de rata. Este estudio se realizó en 40 ratas preñadas que se dividieron en siete grupos. El grupo control recibió solución salina normal y los grupos de prueba recibieron DMSO, quercetina (75 mg / kg), quercetina (200 mg / kg), ATRA (25 mg / kg), ATRA (25 mg / kg) más quercetina (75 mg / kg) y ATRA (25 mg / kg) más quercetina (200 mg / kg), por vía intraperitoneal a los 8-10 días de gestación. Los fetos se recolectaron a los 20 días de gestación. Los riñones se recogieron y se colocaron en solución de formalina tamponada al 10 %. Luego, los riñones se seccionaron por método de rutina y se tiñeron con H & E y se examinaron histológicamente. En el examen histomorfométrico, se observó que el espacio periglomerular y el diámetro del corpúsculo renal en el grupo que recibió solo ATRA fueron significativamente (p≤0.05) mayores que los que recibieron solución salina normal, dimetilsulfóxido y quercetina, mientras que estos dos índices, en el grupo que recibió ATRA más quercetina, disminuyó significativamente (p≤0.05) en forma dependiente de la dosis. El número de corpúsculos renales disminuyó significativamente (p≤0.05) por el ATRA, pero la quercetina no pudo afectar el número de glomérulos. Se concluye que la quercetina puede proteger a los fetos contra daños de ATRA y prevenir su incidencia, probablemente, a través de su efecto antioxidante.

HOXA9 is critical in the proliferation, differentiation, and malignancy of leukaemia cells both in vitro and in vivo.

Progress in the understanding of the molecular mechanism for acute myeloid leukaemia is of great significance to generate new potential targets for treatment. Recent studies showed that HOXA9, a homeodomain-containing transcription factor, is commonly deregulated in acute leukaemia. In this study, we elucidated the direct correlation between HoxA9 expression and progression of leukaemia using 2 different types of leukaemia cells HL-60 and MOLT-3. The HoxA9 expression level was decreased in leukaemia cells with the treatment of all-trans retinoic acid or arsenic trioxide (As2 O3 ). Downregulation of HoxA9 could impair the proliferation and promote the leukaemia cell death. HoxA9 silencing also potentiated the differentiation of leukaemia cells, and in vivo studies demonstrated that HoxA9 downregulation could interfere the tumour growth. Interestingly, HoxA9 silencing also led to the alteration in miRNA expression, mediating the promoting effect on the leukaemia cell differentiation. Therefore, this work provided a promising and potentially efficient target to leukaemia treatment, indicating that HoxA9 is likely to be an ideal candidate in the gene therapy against acute myeloid leukaemia. In this study, we elucidated the critical role of HoxA9 in the proliferation and differentiation of leukaemia cells both in vitro and in vivo. The effect of HoxA9 modulation was correlated with the clinical effect of all-trans retinoic acid and As2 O3 . Furthermore, HoxA9 also regulated the miRNA expression, controlling the leukaemia cell differentiation. Therefore, this work provided new insights into molecular mechanism underlying the leukaemia treatment, potentially putting forward a brand new target to the gene therapy against leukaemia.

A Protocol for In Vitro High-Throughput Chemical Susceptibility Screening in Differentiating NT2 Stem Cells.

The incidence of neurological diseases including learning and developmental disorders has increased in recent years. Concurrently, the number and volume of worldwide registered and traded chemicals have also increased. There is a broad consensus that the developing brain is particularly sensitive to damage by chemicals and that evaluation of chemicals for developmental toxicity or neurotoxicity is critical to human health. Human pluripotent embryonal carcinoma (NTERA-2 or NT2) cells are increasingly considered as a suitable model for in vitro developmental toxicity and neurotoxicity (DT/DNT) studies as they undergo neuronal differentiation upon stimulation with retinoic acid (RA) and allow toxicity assessment at different stages of maturation. Here we describe a protocol for cell fitness screening in differentiating NT2 cells based on the analysis of intracellular ATP levels allowing for the identification of chemicals which are potentially harmful to the developing brain. The described method is suitable to be adapted to low-, medium-, and high-throughput screening and allows multiplexing with other cell fitness indicators. While the presented protocol focuses on cell fitness screening in human pluripotent stem cells it may also be applied to other in vitro models.

NPM and BRG1 Mediate Transcriptional Resistance to Retinoic Acid in Acute Promyelocytic Leukemia.

Perturbation in the transcriptional control of genes driving differentiation is an established paradigm whereby oncogenic fusion proteins promote leukemia. From a retinoic acid (RA)-sensitive acute promyelocytic leukemia (APL) cell line, we derived an RA-resistant clone characterized by a block in transcription initiation, despite maintaining wild-type PML/RARA expression. We uncovered an aberrant interaction among PML/RARA, nucleophosmin (NPM), and topoisomerase II beta (TOP2B). Surprisingly, RA stimulation in these cells results in enhanced chromatin association of the nucleosome remodeler BRG1. Inhibition of NPM or TOP2B abrogated BRG1 recruitment. Furthermore, NPM inhibition and targeting BRG1 restored differentiation when combined with RA. Here, we demonstrate a role for NPM and BRG1 in obstructing RA differentiation and implicate chromatin remodeling in mediating therapeutic resistance in malignancies. NPM mutations are the most common genetic change in patients with acute leukemia (AML); therefore, our model may be applicable to other more common leukemias driven by NPM.