Exploring developmental MeHg impact on extraembryonic and cardiac vessels and its effect on cardiomyocyte contractility.

The toxicity of methylmercury (MeHg) during embryonic development is a relevant issue that remains unclear and deserves investigation. In this sense, there is evidence that links the intake of contaminated food with cardiovascular pathologies in human adults and children. Thus, this study aimed to verify the impact of MeHg on the structure and integrity of extraembryonic and cardiac blood vessels and the contractile function of cardiomyocytes, also evaluating embryonic weight and the cardiosomatic index (CSI). Thus, chicken embryos, used as an experimental model, were exposed to a single dose of 0.1 µg MeHg/50 µl saline at E1.5 and analyzed at E10. After exposure, an increase in the number of extraembryonic blood vessels and the veins of the cardiac tissue was observed. These increases were accompanied by a reduction in the content of VEGF and VCAM proteins related to vessel growth and adhesiveness. Together, these results were related to reduced nitrite (NOx) levels. Furthermore, MeHg reduces the number of sarcomeres and increases the content of cardiac troponin I (cTnI), a protein that regulates contraction. In general, exposure to MeHg affected the integrity of extraembryonic and cardiac vessels and the contractile function of cardiomyocytes, which had a systemic impact evidenced by the reduction in embryonic weight gain and CSI.

Assessing testicular morphofunctionality under Roundup WG® herbicide exposure in zebrafish.

Glyphosate-based herbicides, like Roundup WG® (RWG) used for a range of crops, such as corn, soybean, coffee, sugarcane, rice, apple, and citrus, can reach aquatic ecosystems and impact non-target organisms like fish. Thus, the fish were exposed to three RWG concentrations plus one negative control, which represents the concentration allowed for inland Brazilian waters and concentrations found in surface water worldwide (0.0, 0.065, 0.65, and 6.5 mg a.i./L) for 7 and 15 days. Morphological analysis revealed significant alterations in the testicular structure, particularly in Sertoli cell extensions and cytoplasmic bridges between germ cells. Subcellular compartments also displayed alterations, including dilated mitochondria and the loss of electron density and autophagic vesicles. Gene transcript levels related to autophagy and steroidogenic regulation were upregulated in exposed fish. Germ cell quality was also affected, increasing ROS (reactive oxygen species) production and DNA fragmentation. The study highlighted the RWG reproductive toxicity, providing valuable insights into understanding the morphofunctional alterations in somatic and germ cells of Danio rerio. In conclusion, the environmental relevant concentrations used in this study were toxic to male somatic and germ cells, which raises a concern about the concentrations considered safe for human and animal use.

Developmental toxicity of pyriproxyfen induces changes in the ultrastructure of neural cells and in the process of skull ossification.

Some studies relate the use of pyriproxyfen (PPF) in drinking water with damage to embryonic neurodevelopment, including a supposed association with cases of microcephaly. However, the effects on neural cells and skull ossification in embryos remain unclear. This study aims to investigate the effects of PPF on the structure and ultrastructure of brain cells and its influence on the skull ossification process during embryonic development. Chicken embryos, used as an experimental model, were exposed to concentrations of 0.01 and 10 mg/l PPF at E1. The findings demonstrated that PPF led to notable ultrastructural alterations such as reduced cilia and microvilli of ependymal cells and damage to mitochondria, endoplasmic reticulum, Golgi bodies, and cell membranes in neural cells. The frequency of changes and the degree of these cell damage between the forebrain and midbrain were similar. PPF induced a reduction in fox3 transcript levels, specific for differentiation of neurons, and a reduction in the NeuN protein content related to mature neurons and dendritic branches. PPF impacted the ossification process of the skull, as evidenced by the increase in the ossified area and the decrease in inter-bone spacing. In conclusion, this study highlights the ability of PPF to affect neurodevelopmental processes by inducing ultrastructural damage to neural cells, concomitant with a reduction in NeuN and fox3 expression. This detrimental impact coupled with deficiencies in skull ossification can prevent the proper growth and development of the brain.

Impact of exposure to glyphosate-based herbicide on morphological and physiological parameters in embryonic and larval development of zebrafish.

Glyphosate-based herbicides (GBH) have been commonly used in agriculture to inhibit weed growth and increase yields. However, due to the high solubility of these herbicides in water, they can reach aquatic environments, by infiltration, erosion, and/or lixiviation, affecting non target organisms. Thus, this study aimed to characterize the toxicity of GBH Roundup WG® (RWG®) during the embryonic and larval development of Danio rerio. Embryos (3 hours post fertilization, hpf-until hatching) and larvae (3 days post fertilization, dpf to 6 dpf) were exposed to concentrations of 0.065 and 6.5 mg L-1 . They were evaluated for survival, hatching, spontaneous movements, heartbeat, morphology, and morphometry by in vivo photographs in microscope, cell proliferation and apoptosis by immunohistochemistry, and exploratory behavior and phototropism by video recording. Our results showed an increase in embryo and larvae mortality in those exposed to 0.065 mg L-1 , as well as a reduction in spontaneous embryo movements. The larval heartbeats showed a decrease at 4 dpf in the group exposed to 0.065 mg L-1 and an increase at 5 and 6 dpf in both exposed groups. Cell proliferation was reduced in both groups exposed in embryos and only in the 0.065 mg L-1 group in larvae, while cell death increased in embryos exposed to 6.5 mg L-1 . These results demonstrated the toxic effect of low concentrations of the herbicide RWG® during embryonic and larval development of non target organisms, as well as the importance of constantly reviewing acceptable limits for exposure in natural environments.

Methylmercury Toxicity During Heart Development: A Combined Analysis of Morphological and Functional Parameters.

The heart of higher vertebrates develops early as a tubular structure, which requires cellular and molecular events for proliferation, differentiation and apoptosis for growth, and individualization of cardiac chambers. Exposure to different stressors can cause disturbances in the normal development and functionality of the cardiovascular system. This study aimed to characterize the impact of methylmercury (MeHg) on heart development, specifically related to tissue morphology and parameters of vascular integrity and contractility, also focusing on cell cycle and apoptosis, using Gallus domesticus embryos as a model. The results showed morphological alterations, reduction in the thickness of the ventricular walls, and trabeculae changes in the hearts of embryos exposed to 0.1 µg MeHg/50 µL saline solution. These impacts were associated with increased contents of proteins related to cell cycle arrest and reduced cardiomyocyte proliferation. In addition, the contents of endothelial mediators for contractility and vascular integrity were imbalanced. The quantity and morphology of mitochondria of cardiomyocytes were injured. Together, these negative measurements impacted the reduction of heartbeats. In general, the parameters identified here demonstrate the relevance of combined molecular cellular tissue and physiological diagnosis for a better understanding of the cardiotoxicity of MeHg during development.

Pyriproxyfen exposure induces DNA damage, cell proliferation impairments and apoptosis in the brain vesicles layers of chicken embryos.

Larvicide pyriproxyfen (PPF), used in drinking water reservoirs to control Aedes mosquitoes, has already been shown as a possible cause of congenital anomalies in the central nervous system. However, the neurotoxic effects of PPF on the development of vertebrate embryos are still underexplored. Thus, the aim of this study was to investigate the effects of PPF on the morphometric parameters of the head and brain, as well as on the cell layers of the forebrain and midbrain, using embryos of Gallus domesticus as a model. Two sublethal PPF concentrations (0.01 mg/L and 10 mg/L), as defined by a survival curve, were tested. Analysis of the biometry of embryos showed significant reduction in body and brain mass and also in measurements of the head and brain. A reduction in cell layer thickness of the forebrain and midbrain was observed, accompanied by a reduction in the numerical density of cells per area. Changes in brain and head sizes and in the thickness of the cell layers of the forebrain and midbrain were significant at 10 mg/L PPF. Notably, PPF caused DNA doublestrand breaks and induced apoptosis in embryos exposed to 10 mg/L, which were accompanied by a reduction in cell proliferation. Regarding neuronal and glial differentiation, no changes were observed in the number of neurons and glial cells on the analyzed layers. Furthermore, PPF did not impact the head ossification process. These findings reveal that PPF is a strong stressor for neurodevelopment, causing damage to the cell architecture of brain vesicles.

Reproductive toxicity of Roundup WG® herbicide: impairments in ovarian follicles of model organism Danio rerio.

Glyphosate-based herbicides are widely used in global agriculture, and their effects on different non-target animal organisms have been the focus of many toxicological studies. Regarding the potential role of glyphosate-based herbicides as an endocrine disruptor, the present study aims to investigate the effects of the herbicide Roundup WG® (RWG) on female reproduction, specifically on the ovarian maturation of Danio rerio. Adult females were exposed to low concentrations of RWG (0.065, 0.65, and 6.5 mg L-1) for 15 days, and then the ovaries were submitted to structural and morphometric procedures, accompanied by analysis of the vitellin protein content. Our results showed an increase of initial ovarian follicle numbers, decrease of late ovarian follicles, and smaller diameter of ovarian follicles in fish exposed to 0.065 and 6.5 mg L-1. The thickness of vitelline envelope was reduced, and the vitellin protein content was increased in the ovarian follicle in the two highest concentrations. Ultrastructural changes in the ovarian follicular component were evident and expressed by the cell index; vacuolization in follicular cells, increase of perivitelline space, and impaired mitochondria in oocytes were observed. Therefore, RWG adversely affects the ovarian maturation in D. rerio, and these changes can lead to reproductive toxicity, compromising population dynamics.

Roundup® disrupts chitinolytic enzyme activity and ecdysteroid concentration in Macrobrachium potiuna.

The endocrine system of crustaceans regulates the molt cycle with ecdysteroid hormones, mainly the 20-hydroxyecdysone (20-HE). Moreover, the molt process requires the action of chitinolytic enzymes (e.g., chitinase, chitobiase) to break down the old cuticle. However, endocrine disrupting compounds (EDC) are capable of altering their normal functioning. Glyphosate-based herbicides (GBH), such as Roundup®, the most widely used herbicides, are found in freshwater environments and have been considered EDC for many aquatic organisms. Therefore, this study examined the effects of environmentally relevant GBH concentrations (0.0065, 0.065, and 0.28 mg L-1) on the 20-HE concentration and chitobiase activity in the decapod prawn Macrobrachium potiuna exposed for 14 days. Additionally, lipid peroxidation, a biomarker of membrane lipid degradation, was evaluated in hepatopancreas to assess cellular damage. Results showed that GBH decreased the 20-HE concentration in females at the two highest concentrations tested, while an increase was observed in males exposed to the highest GBH concentration. In addition, GBH also decreased chitobiase activity in males (all concentrations) and females (the two highest concentrations). Finally, GBH caused increased lipid peroxidation in males, indicating cellular damage in the hepatopancreas. In conclusion, this work suggests that GBH is an EDC for crustaceans by disrupting molting, which could lead to altered reproduction and thus population dynamics. Graphical abstract Decrease in the 20-HE concentration and chitobiase activity in muscle of males and females of the freshwater prawn Macrobrachium potiuna exposed to the herbicide Roundup® for 14 days.

Ultraviolet B radiation affects epithelial cell morphology and ultrastructure in the hepatopancreas of the freshwater decapod Macrobrachium olfersii.

The hepatopancreas is the digestive organ of crustaceans, and plays important roles also in the synthesis and secretion of sexual hormones, immunological defenses and xenobiotic detoxification. Although the importance of this organ in crustaceans cannot be underestimated, the effects of ultraviolet B (UVB) radiation on hepatopancreas are poorly understood. Moreover, Macrobrachium prawns, have a transparent carapace, which make them more susceptible to UVB radiation, since their internal organs, such as hepatopancreas, are easily reached by solar radiation. Therefore, we aimed to evaluate UVB radiation toxicity on the morphology and morphometry of hepatopancreatic epithelial cells, and to investigate these UVB effects in subcellular compartments of the ecologically-important freshwater decapod, Macrobrachium olfersii. Hepatopancreas from the UVB-irradiated group showed a granular cytoplasm, with non-defined cell limits. Morphometric analyses revealed that the UVB-irradiated group exhibited a higher frequency of fibrillar (F-cell), resorptive (R-cell) and midget (M-cell), and decreased the blister-like (B-cell). It was also observed increased vacuole frequencies and increased F-, B- and R-cell volumes in the UVB-irradiated group. In addition, it was observed increased B-cell vacuolar volumes and decreased R-cell vacuolar volumes. Ultrastructural alterations occurred in subcellular compartments in F- and R-cells, e.g. loss of mitochondrial crests, morphologically compatible with mitochondrial fission, rough endoplasmic reticulum cisternae dilation, dilation of Golgi lamellar sacs, and increased vacuole and concentric membrane formation in the UVB-irradiated group. Our data showed that the hepatopancreas is an important target of UVB radiation, as demonstrated by a series of organ-specific morphological and morphometric impairments. Therefore, cell damage caused by UVB radiation can compromise metabolic functions in epithelial cells from the hepatopancreas, potentially affecting absorption, secretion and digestion processes, vitellogenin synthesis, immune responses and xenobiotic detoxification.

Ultraviolet-B radiation induces transcriptional modulation of components associated with the extracellular matrix in embryos of decapod Macrobrachium olfersii.

The extracellular matrix (ECM) is a non-cellular and three-dimensional structure, constituted by a macromolecular dynamic network that involves the cells in all animal tissues, including embryonic ones. Several studies with vertebrates and cell cultures have reported deleterious effects of ultraviolet-B (UVB) radiation on the components associated with the ECM. However, studies focusing on the UVB radiation effects on ECM components of crustaceans during embryonic development are very scarce. Thus, the aim of this study was to identify the coding sequences of components associated with the ECM and to evaluate the effect of UVB radiation on embryos of the ecologically-important decapod Macrobrachium olfersii. To evaluate the modulation of these ECM components during embryonic development, the transcript levels of Col4α1, Itgß, Lamα, Mmp1 and Timp in M. olfersii embryos were analyzed at early developmental stages (E1, E3 and E4), intermediate developmental stage (E7) and late developmental stages (E10 and E14). In addition, embryos at E7, which correspond to a landmark of crustacean development, were analyzed after 12 h of UVB exposure to verify UVB effects on the ECM components. The ECM component sequences were similar to other decapods, suggesting conservation of these genes among crustaceans. The results showed modulations of the ECM components of M. olfersii embryos that reflect the need for each component in the cellular mechanisms, necessary for normal embryonic development. After UVB exposure, embryos showed opacity of embryonic tissues and it was found the overexpression of Col4α1, Itgß, Mmp1 and Timp transcript levels (1.82-, 1.52-, 2.34- and 6.27-fold, respectively). These impairments can compromise important events for normal embryonic development, such as growth of optic lobes, caudal papilla, ramification of appendages and differentiation of organic systems. The results presented here, together with the effects on morphology, cell proliferation, differentiation, and apoptosis demonstrated previously, strengthen the knowledge of the complex impacts of UVB radiation on freshwater embryos. Nevertheless, our results encourage further investigations focusing on the assessment of UVB effects on different organisms in order to better understand the myriad of UVB effects on ECM components.

Modulation of antioxidant gene expressions by Roundup® exposure in the decapod Macrobrachium potiuna.

Glyphosate-based herbicides (GBH), including Roundup®, are the most used herbicides in agricultural and non-agricultural areas, which can reach aquatic environments through drift during application or surface runoff. Some studies, mostly in fish, demonstrated that GBH caused oxidative stress in non-target animals. However, only few information is available on the GBH effects in the antioxidant and stress proteins of many other organisms, such as freshwater crustaceans. Thus, we aimed to investigate the effects of environmentally relevant GBH concentrations on the relative transcript expression (RTE) of the superoxide dismutase (sod1), catalase (cat), selenium-dependent glutathione peroxidase (gpx), glutathione-S-transferase (gst), thioredoxin (txn), heat shock protein (hsp70 and hsp90) in the hepatopancreas of the ecologically important freshwater prawn Macrobrachium potiuna. Moreover, this study aimed to assess the gender-differences responses to GBH exposure. Male and female prawns were exposed to three Roundup WG® concentrations (0.0065, 0.065 and 0.28 mg of glyphosate/L) and a control group (0.0 mg/L) for 7 and 14 days. In general, males had an under-expression of the studied genes, indicating an oxidative stress and possible accumulation of ROS in the hepatopancreas. In the opposite, females had an overexpression of the same genes, indicating a more robust antioxidant system, in order to cope with the possible ROS increase after Roundup WG® exposure. Therefore, results confirmed that gender could be a confounding factor in ecotoxicological assessment of GBH effects. Additionally, this work highlights that sod1, cat, gpx, gst, txn, hsp70 and hsp90 gene expressions seem to be useful biomarkers to investigate the oxidative stress caused by Roundup WG® in Macrobrachium sp.

Exposure to homocysteine leads to cell cycle damage and reactive gliosis in the developing brain.

Studies that investigate the cellular effects of homocysteine (Hcy) on the differentiation of neural cells, and their involvement in establishment of cell layers in the developing brain are scarce. This study evaluated how Hcy affects the neural cell cycle and proteins involved in neuronal differentiation in the telencephalon and mesencephalon using the chicken embryo as a model. Embryos at embryonic day 2 (E2) received 20 µmol D-L Hcy/50 µl saline and analyzed at E6. The Hcy treatment induced an increase in the ventricular length of the telencephalon and also a reduction of the mantle layer thickness. We observed that Hcy induced impairments to the neural cell cycle and differentiation, which compromised the cell layers establishment in the developing brain. Hcy treatment also induced changes in gene and protein expression of astrocytes, characteristic of reactive gliosis. Our results point to new perspectives of evaluation of cellular targets of Hcy toxicity.

Effects of low glyphosate-based herbicide concentrations on endocrine-related gene expression in the decapoda Macrobrachium potiuna.

Glyphosate-based herbicides (GBH) are the most used herbicides worldwide and are considered as endocrine-disrupting compounds (EDC) for non-target organisms. However, effects of GBH on their endocrine systems remain poorly understood. Thus, the aim of this study was to assess the effects of low concentrations of Roundup WG® on growth and reproduction process molecules in both males and females of the decapod crustacean Macrobrachium potiuna, by the relative transcript expression levels of the ecdysteroid receptor (EcR), the molt-inhibiting hormone (MIH), and the vitellogenin (Vg) genes. Prawns were exposed to three concentrations of GBH (0.0065, 0.065, and 0.28 mg L-1) for 7 and 14 days. The results revealed that only in males the three genes transcript levels were influenced by the GBH concentration, time of exposure, and the interaction between the concentrations and time of exposure, suggesting that males were more sensitive to GBH than females. For males, after 7 days of exposure at 0.065 mg L-1, EcR and MIH were over-expressed, while the Vg expression was only over-expressed after 14 days. The present study highlighted that GBH impacted endocrine systems of M. potiuna. Moreover, EcR and MIH gene expressions could be promising EDC biomarkers of exposure in crustaceans. These results also indicate that GBH concentrations, considered secure by regulatory agencies, should be reviewed to minimize the effects on non-target organisms. Potential effects of glyphosate-based herbicides on the endocrine system of decapods Macrobrachium sp.

Histopathological and ultrastructural indices for the assessment of glyphosate-based herbicide cytotoxicity in decapod crustacean hepatopancreas.

Glyphosate-based herbicides (GBH), including Roundup, are the most widely used pesticides in the world. Glyphosate residues have been detected in surface and groundwater, in food, and in human blood and urine. The effects of this herbicide on different levels of biological organization are an important concern that needs to be investigated. In general, the toxicity of GBH in invertebrates is poorly understood, and it is the motivation of this study. Thus, the aim of this study was to evaluate cellular responses of the hepatopancreas, an organ involved in the detoxification process in invertebrates, after exposure to environmentally relevant concentrations of GBH, using prawn Macrobrachium potiuna as a model. Prawns were exposed to three concentrations of GBH (0.0065, 0.065 and 0.28 mg L-1) for 7 or 14 days. Alterations in the morphology of the hepatopancreas and in subcellular components of R cells, which are responsible for the detoxification process, were analyzed, and an index for subcellular alterations was standardized. GBH exposure induced tissue commitments on the hepatopancreas, as well as important impairments of R cells that could compromise the normal functioning of the cells, especially in the detoxification processes. The major cellular impairments were intense vacuolization, dilatation of the cisterns of the rough endoplasmic reticulum and Golgi bodies, increase of perinuclear space, necrosis, concentric membrane formation and mitochondria crest loss. Our data contribute to the knowledge of the cytotoxic effects of low GBH concentrations on aquatic invertebrates, specifically their effects on the hepatopancreas, an important organ for the metabolism of crustaceans. These results also indicate that concentrations considered safe by regulatory agencies should be reviewed to minimize the effects on non-target organisms. This study also contributes to the standardization of an ultrastructure index for the assessment of GBH in palaemonids, which could be used for the assessment of contaminants in crustaceans and other species with hepatopancreas.

Impact of homocysteine on vasculogenic factors and bone formation in chicken embryos.

Developmental endochondral ossification requires constant blood supply, which is provided by the embryonic vascular network. High levels of homocysteine (Hcy) have vasculotoxic properties, but it remains unclear how Hcy disrupts blood vessel formation in endochondral ossification. Thus, we investigated the toxicity of Hcy on contents of vasculogenic factors (VEGF, VCAM-1, NOS3) and osteocalcin, using developing limbs as model. Chicken embryos were submitted to treatment with 20 µmol D-L Hcy at 12H&H and the analyses occur at 29H&H and 36H&H. We did not identify differences in the area of limb ossification in Hcy-treated (7.5 × 105 µm2 ± 3.9 × 104) and untreated embryos (7.6 × 105 µm2 ± 3.3 × 104) at 36H&H. In Hcy-treated embryos, we observed a significantly decrease of 46.8% at 29H&H and 26.0% at 36H&H in the number of VEGF-reactive cells. Also, treated embryos showed decrease of 98.7% in VCAM-1-reactive cells at 29H&H and 34.6% at 36H&H. The number of NOS3-reactive cells was reduced 54.0% at 29H&H and 91.5% at 36H&H, in the limbs of Hcy-treated embryos. Finally, in Hcy-treated embryos at 36H&H, we observed a reduction of 58.86% in the number of osteocalcin-reactive cells. Here, we demonstrated for the first time that the toxicity of Hcy is associated with a reduction in the contents of proteins involved in blood vessel formation and bone mineralization, which interferes with endochondral ossification of the limb during embryonic development. Graphical abstract.

Homocysteine-induced changes in cell proliferation and differentiation in the chick embryo spinal cord: implications for mechanisms of neural tube defects (NTD).

Maternal hyperhomocysteinemia during pregnancy is associated with increased risk of NTD in the offspring. Our study investigated the effects of homocysteine (Hcy) on proliferation and neuronal differentiation of the spinal cord cells in a chick embryo model. Embryos were treated with 20µmol D-L Hcy/50µL saline solution at embryonic day 2 (E2) and analyzed at embryonic days 4 (E4) and 6 (E6). Control embryos received exclusively 50µL saline solution. We performed immunolocalization and flow cytometry analyses using antibodies anti-phosphohistone H3 (pH3), anti-proliferating cell nuclear antigen (PCNA), anti-ß-tubulin III and anti-p53. Our results revealed that Hcy interferes in the proliferation of the neural cells, and that this effect is age-dependent and differed between Hcy-treated embryos with and without NTD. Also, Hcy induced a decrease of neuronal differentiation in the spinal cord at both embryonic ages. These findings contribute to clarifying the cellular bases of NTD genesis, under experimental hiperhomocysteinemia.

Light-mediated DNA Repair Prevents UVB-induced Cell Cycle Arrest in Embryos of the Crustacean Macrobrachium olfersi.

High levels of ultraviolet-B (UVB) radiation can negatively affect aquatic animals. Macrobrachium olfersi is a prawn that lives in clear freshwaters and during the breeding season, females carry eggs in an external brood pouch. Therefore, we hypothesize that eggs are also exposed to environmental UVB radiation. The aim of this study was to investigate whether UVB radiation induces DNA damage and compromises cell cycle in embryos of M. olfersi. In laboratory, UVB irradiance (310 mW. cm(-2) ) that embryos receive in the natural environment was simulated. After irradiation, embryos were kept under different light conditions in order to recognize the presence of cell repair. UVB radiation induces DNA damage, specifically thymine dimers. After 48 h of UVB exposure, a significant decrease in the level of these dimers was observed in embryos kept under visible light while it remained constant in the dark. Moreover, under visible light and darkness, a decrease in proliferation was observed after 48 h of irradiation. An increase in PCNA expression and decrease in p53 expression were observed after, respectively, 1 and 48 h of exposure. Our results showed that UVB radiation disturbs the cell cycle and induces DNA damage in M. olfersi embryos. However, under visible light these embryos showed successful DNA repair.

Effects of homocysteine on mesenchymal cell proliferation and differentiation during chondrogenesis on limb development.

High levels of homocysteine (Hcy) are related to an increased risk of the occurrence of congenital anomalies, including limb defects. However, few evaluations about how toxic levels of Hcy affect limb development have been reported. We investigated whether Hcy can affect the cell cycle proteins and proteins involved in mesenchymal cell differentiation during limb development, in a chicken embryo model. Embryos were treated with 20 µmol d-l Hcy/50 µl saline at embryonic day 2 and analyzed at embryonic day 6. Untreated control embryos received exclusively 50 µl saline solution. To identify cells in proliferation and cell cycle proteins, as well as Pax1/9 and Sox9 proteins, we performed immunolocalization and flow cytometry analyses using the antibodies anti-phosphohistone H3, anti-p53, anti-p21, anti-proliferating cell nuclear antigen, anti-Pax1, anti-Pax9 and anti-Sox9. No significant differences in cell proliferation were observed between Hcy-treated and untreated embryos. We observed a decrease of the proliferating cell nuclear antigen and p21 proteins, both involved in the G1 phase of cell cycle progression. On the other hand, in mesenchymal cells of the limbs, Hcy induces an increase of p53 protein, which can be activated by DNA damage. In cell differentiation, Hcy induced an increase mainly of Pax9 and Sox9 proteins. Our data indicate that the treatment with Hcy changes the mesenchymal cell dynamics during limb development, but does not change the morphology of the cartilage molds. These findings provide information to understand better the cellular basis of the toxicity of Hcy on chondrogenesis during limb development.

Protective effects of diphenyl diselenide in a mouse model of brain toxicity.

Interest in organoselenide chemistry and biochemistry has increased in the past three decades, mainly due to their chemical and biological activities. Here, we investigated the protective effect of the organic selenium compound diphenyl diselenide (PhSe)2 (5 µmol/kg), in a mouse model of methylmercury (MeHg)-induced brain toxicity. Our group has previously demonstrated that the oral and repeated administration (21 days) of MeHg (40 mg/L) induced MeHg brain accumulation at toxic concentrations, and a pattern of severe cortical and cerebellar biochemical and behavioral. In order to assess neurotoxicity, the neurochemical parameters, namely, mitochondrial complexes I, II, II-III and IV, glutathione peroxidase (GPx) and glutathione reductase (GR) activities, the content of thiobarbituric acid-reactive substances (TBA-RS), 8-hydroxy-2'-deoxyguanosine (8-OHdG), and brain-derived neurotrophic factor (BDNF), as well as, metal deposition were investigated in mouse cerebral cortex. Cortical neurotoxicity induced by brain MeHg deposition was characterized by the reduction of complexes I, II, and IV activities, reduction of GPx and increased GR activities, increased TBA-RS and 8-OHdG content, and reduced BDNF levels. The daily treatment with (PhSe)2 was able to counteract the inhibitory effect of MeHg on mitochondrial activities, the increased oxidative stress parameters, TBA-RS and 8-OHdG levels, and the reduction of BDNF content. The observed protective (PhSe)2 effect could be linked to its antioxidant properties and/or its ability to reduce MeHg deposition in brain, which was here histochemically corroborated. Altogether, these data indicate that (PhSe)2 could be consider as a neuroprotectant compound to be tested under neurotoxicity.

Homocysteine causes disruptions in spinal cord morphology and changes the expression of Pax 1/9 and Sox 9 gene products in the axial mesenchyme.

BACKGROUND: Neural tube defects (NTD) involve disruptions in the axial mesenchyme, and are related to an imbalance between folic acid (FA) and homocysteine (Hcy). This study evaluated the effects of FA/Hcy imbalance on cell proliferation and expression of the Pax 1/9 and Sox 9 gene products in the axial mesenchyme of chickens. METHODS: Embryos were incubated (38°C) and pretreated at 24 h and treated at 46 h of incubation. The experimental groups were: FA-pretreated with saline and treated with 0.5 µg FA/saline; Hcy-pretreated with 50 µl saline and treated with 20 µmol D,L-Hcy/50 µl saline; FA+Hcy-pretreated with 0.5 µg FA/50 µl saline and treated with 20 µmol D,L-Hcy/50 µl saline; and the control embryos were pretreated and treated with saline. Embryos were analyzed at E4 and E6. Immunohistochemistry was performed to identify proliferating cells and the expression of the gene products of Pax 1/9 and Sox 9. Total RNA of the E4 embryos was extracted and a RT-qPCR assay was performed to quantify Pax 1/9 mRNA expression. RESULTS: Hcy treatment caused spinal NTD and abnormalities in axial mesenchyme development, affecting the distribution of sclerotomal cells and chondrification. Hcy also reduced cell proliferation and changed the expression of Pax 1/9 and Sox 9 in the mesenchyme. CONCLUSIONS: Our data clarified the relationship between spinal NTD genesis and disruptions of Pax 1/9 and Sox 9 gene products in the axial mesenchyme caused by the FA/Hcy imbalance.