Reduction In Body Length Research Articles

Bromuconazole exposure induces cardiac dysfunction by upregulating the expression LEF1

With the wide application of bromuconazole (BRO), a kind of triazole fungicide, the environmental problems caused by BRO have been paid more and more attention. In this study, adult male zebrafish were exposed to environmental related concentration and the maximum non-lethal concentration for zebrafish larvae (0,50 ng/L and 7.5 mg/L) for 7 days, respectively. Zebrafish exposed to BRO exhibited a significant reduction in body length and an increase in fatness index, indicating adverse physiological changes. Notably, the exposed zebrafish showed enlarged heart ventricular volumes and thinner heart walls. Transcriptome analysis of heart samples showed that BRO exposure mainly affected pathways related to cardiac energy metabolism. In addition, the amount of ATP in the heart tissue was correspondingly reduced, and the expression levels of genes related to controlling ion balance and myosin synthesis in the heart were also altered. The study extended its findings to the rat cardiomyocytes (H9C2), where similar cardiotoxic effects including changes in transcription of genes related to energy metabolism and heart function were also observed, suggesting a potential universal mechanism of BRO-induced cardiotoxicity. In a doxorubicin (DOX) induced larval zebrafish heart failure model, the expression of lymphoid enhancer-binding factor 1(LEF1), a key gene in the Wnt/β-catenin signaling pathway, was significantly increased in larval zebrafish and adult fish heart tissues and cardiomyocytes, suggesting that LEF1 might play an important role in BRO-induced cardiotoxicity. Taken together, BRO exposure could interfere with cardiac function and metabolic capacity by abnormal activation the expression of LEF1. The study emphasized the urgent need for monitoring and regulating BRO due to its harmful effects on the hearts of aquatic organisms.

Gelsenicine disrupted the intestinal barrier of Caenorhabditis elegans

Gelsemium elegans Benth. (G. elegans) is a traditional medicinal herb that has anti-inflammatory, analgesic, sedative, and detumescence effects. However, it can also cause intestinal side effects such as abdominal pain and diarrhea. The toxicological mechanisms of gelsenicine are still unclear. The objective of this study was to assess enterotoxicity induced by gelsenicine in the nematodes Caenorhabditis elegans (C. elegans). The nematodes were treated with gelsenicine, and subsequently their growth, development, and locomotion behavior were evaluated. The targets of gelsenicine were predicted using PharmMapper. mRNA-seq was performed to verify the predicted targets. Intestinal permeability, ROS generation, and lipofuscin accumulation were measured. Additionally, the fluorescence intensities of GFP-labeled proteins involved in oxidative stress and unfolded protein response in endoplasmic reticulum (UPRER) were quantified. As a result, the treatment of gelsenicine resulted in the inhibition of nematode lifespan, as well as reductions in body length, width, and locomotion behavior. A total of 221 targets were predicted by PharmMapper, and 731 differentially expressed genes were screened out by mRNA-seq. GO and KEGG enrichment analysis revealed involvement in redox process and transmembrane transport. The permeability assay showed leakage of blue dye from the intestinal lumen into the body cavity. Abnormal mRNAs expression of gem-4, hmp-1, fil-2, and pho-1, which regulated intestinal development, absorption and catabolism, transmembrane transport, and apical junctions, was observed. Intestinal lipofuscin and ROS were increased, while sod-2 and isp-1 expressions were decreased. Multiple proteins in SKN-1/DAF-16 pathway were found to bind stably with gelsenicine in a predictive model. There was an up-regulation in the expression of SKN-1:GFP, while the nuclear translocation of DAF-16:GFP exhibited abnormality. The UPRER biomarker HSP-4:GFP was down-regulated. In conclusion, the treatment of gelsenicine resulted in the increase of nematode intestinal permeability. The toxicological mechanisms underlying this effect involved the disruption of intestinal barrier integrity, an imbalance between oxidative and antioxidant processes mediated by the SKN-1/DAF-16 pathway, and abnormal unfolded protein reaction.

Effects of two alternative plasticizers on the growth hormone-related endocrine system, neurodevelopment, and oxidative stress of zebrafish larvae

In response to the restriction of phthalate plasticizers, acetyl tributyl citrate (ATBC) and acetyl triethyl citrate (ATEC) have been used in medical devices and food packaging. In the present study, the effects of ATBC and ATEC on the development, behavior, growth hormone (GH)-related endocrine system, neurotransmitters, and oxidative stress of zebrafish embryo or larvae were investigated. After exposure of zebrafish to ATBC and ATEC (0, 0.03, 0.3, 3, 30, and 300 μg/L) for 96 h, developmental toxicity, behavioral changes under light/dark condition, changes in hormones and genes involved in GH/insulin-like growth factors (IGFs) axis, changes in hormone, enzyme, and genes related to neurodevelopment, antioxidant enzymes activities were determined. Larvae exposed to 30 or 300 μg/L ATBC showed significant reductions in body length and moving distance and speed, whereas no significant effects on development and locomotor behavior were observed in larvae exposed to ATEC. The contents of GH and IGF-I were significantly reduced in larvae exposed to 3, 30, and 300 μg/L ATBC. Hormonal changes in fish exposed to ATBC are well supported by regulation of genes related to GH (gh1) and the activity of IGF-I (igf1). In fish exposed to ATBC, reduced acetylcholinesterase activity and down-regulation of genes related to the central nervous system development (ache, gap43, mbpa, and syn21) were observed. ATBC increased the production of reactive oxygen species and the levels of superoxide dismutase, catalase, and glutathione peroxidase. Notably, pre-treatment with the classic antioxidant N-acetylcysteine alleviated ATBC-induced GH-related endocrine disruption and neurotoxicity. Our observations showed that exposure to low levels of ATBC could disturb the regulatory systems of GH/IGFs axis and neurobehavior, ultimately leading to developmental inhibition and hypoactivity, and that increased oxidative stress plays a major role in these toxicities.

Protective efficacy of six recombinant proteins as vaccine candidates against Echinococcus granulosus in dogs.

Cystic echinococcosis (CE) is caused by the infection of Echinococcus granulosus sensu lato (E. granulosus s.l.), one of the most harmful zoonotic helminths worldwide. Infected dogs are the major source of CE transmission. While praziquantel-based deworming is a main measure employed to control dog infections, its efficacy is at times compromised by the persistent high rate of dog re-infection and the copious discharge of E. granulosus eggs into the environment. Therefore, the dog vaccine is a welcome development, as it offers a substantial reduction in the biomass of E. granulosus. This study aimed to use previous insights into E. granulosus functional genes to further assess the protective efficacy of six recombinant proteins in dogs using a two-time injection vaccination strategy. We expressed and combined recombinant E. granulosus triosephosphate isomerase (rEgTIM) with annexin B3 (rEgANXB3), adenylate kinase 1 (rEgADK1) with Echinococcus protoscolex calcium binding protein 1 (rEgEPC1), and fatty acid-binding protein (rEgFABP) with paramyosin (rEgA31). Beagle dogs received two subcutaneous vaccinations mixed with Quil-A adjuvant, and subsequently orally challenged with protoscoleces two weeks after booster vaccination. All dogs were sacrificed for counting and measuring E. granulosus tapeworms at 28 days post-infection, and the level of serum IgG was detected by ELISA. Dogs vaccinated with rEgTIM&rEgANXB3, rEgADK1&rEgEPC1, and rEgFABP-EgA31 protein groups exhibited significant protectiveness, with a worm reduction rate of 71%, 57%, and 67%, respectively, compared to the control group (P < 0.05). Additionally, the vaccinated groups exhibited an inhibition of worm growth, as evidenced by a reduction in body length and width (P < 0.05). Furthermore, the level of IgG in the vaccinated dogs was significantly higher than that of the control dogs (P < 0.05). These verified candidates may be promising vaccines for the prevention of E. granulosus infection in dogs following two injections. The rEgTIM&rEgANXB3 co-administrated vaccine underscored the potential for the highest protective efficacy and superior protection stability for controlling E. granulosus infections in dogs.

Flubendazole exposure disrupts neural development and function of zebrafish embryos (Danio rerio)

Flubendazole (FBZ) is a benzimidazole anthelmintic drug widely used for treating parasitic infections by disrupting microtubule formation and function through tubulin binding. Recently, its use has extended to include anticancer applications, leading to increased environmental exposure to benzimidazole drugs. However, the impact of FBZ on neural development in aquatic organisms, particularly in aquatic vertebrates, remains poorly understood. This study aimed to investigate the potential developmental toxicity of FBZ during neural development using zebrafish model. Various assessments, including analysis of overall developmental changes, morphological abnormalities, apoptosis, gene expression alterations, axon length measurements, and electrophysiological neural function, were performed. FBZ exposure resulted in concentration-dependent effects on survival rate, hatching rate, heartbeat, and the occurrence of developmental abnormalities. Notably, FBZ-induced changes included reductions in body length, head size, and eye size, as well as the detection of apoptotic cells in the central nervous system. Gene expression analysis revealed upregulation of apoptosis-related genes (p53, casp3, and casp8), downregulation of neural differentiation-related genes (shha, nrd, ngn1, and elavl3), and alterations in neural maturation and axon growth-related genes (gap43, mbp, and syn2a). Additionally, shortened motor neuron axon length and impaired electrophysiological neural function were observed. These findings provide novel insights into the potential risks of FBZ on the neural development of zebrafish embryos, emphasizing the need for risk prevention strategies and therapeutic approaches to address the environmental toxicity of benzimidazole anthelmintics.

An in vivo functional assay to characterize human STAT5B genetic variants during zebrafish development.

Growth hormone (GH) binding to GH receptor activates janus kinase 2 (JAK2)-signal transducer and activator of transcription 5b (STAT5b) pathway, which stimulates transcription of insulin-like growth factor-1 (IGF1), insulin-like growth factor binding protein 3 (IGFBP3) and insulin-like growth factor acid-labile subunit (IGFALS). Although STAT5B deficiency was established as an autosomal recessive disorder, heterozygous dominant-negative STAT5B variants have been reported in patients with less severe growth deficit and milder immune dysfunction. We developed an in vivo functional assay in zebrafish to characterize the pathogenicity of three human STAT5B variants (p.Ala630Pro, p.Gln474Arg and p.Lys632Asn). Overexpression of human wild-type (WT) STAT5B mRNA and its variants led to a significant reduction of body length together with developmental malformations in zebrafish embryos. Overexpression of p.Ala630Pro, p.Gln474Arg or p.Lys632Asn led to an increased number of embryos with pericardial edema, cyclopia and bent spine compared with WT STAT5B. Although co-injection of WT and p.Gln474Arg and WT and p.Lys632Asn STAT5B mRNA in zebrafish embryos partially or fully rescues the length and the developmental malformations in zebrafish embryos, co-injection of WT and p.Ala630Pro STAT5B mRNA leads to a greater number of embryos with developmental malformations and a reduction in body length of these embryos. These results suggest that these variants could interfere with endogenous stat5.1 signaling through different mechanisms. In situ hybridization of zebrafish embryos overexpressing p.Gln474Arg and p.Lys632Asn STAT5B mRNA shows a reduction in igf1 expression. In conclusion, our study reveals the pathogenicity of the STAT5B variants studied.

Differential sensitivity of aquatic life stages of Pelophylax perezi to an acidic metal-contaminated effluent.

Acid mine drainage (AMD) involves complex mixtures of metals and hydrogen ions that can be highly toxic to the biota. Assessing the effects of AMD to aquatic stages of amphibians is key, as this group constitutes the vertebrate class with the highest proportion of species considered as threatened. Thus, the present work aimed at assessing the sensitivity of two aquatic life stages of the green frog Pelophylax perezi to an AMD originated from a cupric-pyrite mine. Embryos (Gosner stages 10-11) and tadpoles (Gosner stages 20-21) of P. perezi were exposed, for 96 h, to six AMD dilutions (1.39% to 7.5%). Endpoints involving responses at different levels of biological organization were monitored: mortality, malformations, hatching rates, body length and growth rate, enzymatic activity related with detoxification metabolism (glutathione S-transferase), and histopathologies (anatomical structures of the digestive, respiratory, and excretory systems). Embryos presented high mortality and malformation rates at AMD levels equal or above 5.36%, as well as premature hatching at 1.95% of AMD or higher. A significant reduction in body length and growth rate occurred in embryos and tadpoles exposed to 1.95% or higher levels of AMD, respectively. At the histological level, several abnormalities were observed for AMD-exposed tadpoles in a variety of tissues. One of the most noticeable histological changes occurred in the intestine that exhibited papillary epithelial hyperplasia along with a yellowish content and was more pronounced in tadpoles exposed to higher AMD levels. FEmbryos were more sensitive to lethal levels of AMD than tadpoles, suggesting embryos as a useful model life stage when performing amphibian risk assessment of mine drainage. Furthermore, AMD was highly toxic for P. perezi aquatic life stages since levels as low as 1.95% induced lethal effects. These results emphasize the importance of implementing efficient remediation methodologies for AMD, given its high toxicity.

تأثير التدخين أثناء الحمل على حجم الوليد والمشعرات الأنثروبومترية: دراسة حشدية في اللاذقية، سوريا

Objective: To investigate the effect of smoking during pregnancy on the anthropometric measurements of the newborn and the effect of stopping smoking during the first trimester of pregnancy. Patients and methods: Our study was a cohort-analytical study conducted at Tishreen University Hospital in Lattakia-Syria during the period from January 2020 to January 2021. The study included 180 pregnant women with a single live pregnancy who met the inclusion criteria. They were divided into non-smokers, smokers who stopped smoking during the first trimester of pregnancy (group 1), smokers who continued smoking until the end of pregnancy (group 2). After birth. Neonate identified with Small for gestational age and low birth weight. Results: The study was founded that smoking during pregnancy increases the risk of (low birth weight) LBW by 16.3% and the risk of SGA (small for gestational age) by 79%, and smoking was associated with the birth of children with lower anthropometric measurements compared to the children of non-smoking mothers. As for the Ponderal Index &gt;90th, A.OR was 4.9 for group (1) versus 5.8 for group (2) and Head to Length Ratio &gt;90th was 3.4 for group (1) versus 4.6 for group (2), as it was found that increasing the number of cigarettes smoked per day increases the risk of The occurrence of adverse birth outcomes Conclusion: Maternal smoking, regardless of duration of smoking during pregnancy, was associated with neonates abnormal body proportions resulting from greater reductions in body length and head circumference compared to weight, smoking during the first trimester of pregnancy was associated with adverse birth outcomes, but with a lower A.OR than group (2).

Estrogenic and growth inhibitory responses to organophosphorus flame retardant metabolites in zebrafish embryos

Recent evidence has revealed that organophosphorus flame retardants (OPFRs) elicit a variety of toxic effects, including endocrine disruption. The present study examined estrogenic and growth inhibitory responses to OPFR metabolites in comparison to their parent compounds using zebrafish eleutheroembryos.11Eleutheroembryos are hatched embryos that still rely on their yolk for nutrition. For simplicity, we will refer to them as “embryos” for the rest of this paper. Exposure to 4-hydroxylphenyl diphenyl phosphate (HO-p-TPHP) but not its parent compound triphenyl phosphate (TPHP) elicited upregulation of a marker gene of estrogenic responses, cytochrome P450 19A1b (CYP19A1b), and this upregulation was reversed by co-exposure to an estrogen receptor antagonist. Tris(1,3-dichloro-2-propyl) phosphate (TDCIPP) and bis(1,3-dichloro-2-propyl) phosphate (BDCIPP), as well as 3-hydroxylphenyl diphenyl phosphate (HO-m-TPHP) and diphenyl phosphate (DPHP), did not elicit significant changes in the CYP19A1b expression. Reduction in body length was induced by TPHP and to a lesser extent by its hydroxylated metabolites. Altered expression of genes involved in the synthesis and action of thyroid hormones, including iodothyronine deiodinases 1 and 2, thyroid hormone receptor alpha, and transthyretin, were commonly observed for TPHP and its hydroxylated metabolites. Reduction in the body length was also seen in embryos exposed to TDCIPP but not BDCIPP. The transcriptional effect of TDCIPP was largely different from that of TPHP, with decreased expression of growth hormone and prolactin observed only in TDCIPP-exposed embryos. Considering the concentration-response relationships for the growth retardation and gene expression changes, together with existing evidence from other researchers, it is likely that prolactin is in part involved in the growth inhibition caused by TDCIPP. The present study showed similarities and differences in the endocrine disruptive effects of OPFRs and their metabolites.

The Effects of Dietary Polybrominated Diphenyl Ether Exposure and Rearing Temperature on Tadpole Growth, Development, and Their Underlying Processes.

Depression of growth rate due to polybrominated diphenyl ethers (PBDEs) has been documented in birds, mammals, amphibians, and fish at single temperatures. However, the underlying energetic mechanism for this effect and how it might change in relation to changing environmental temperature remain unstudied. We used a simple energy budget to address hypotheses regarding effects of PBDEs on tadpole (Lithobates pipiens) growth: that reductions in growth are linked to increased respiratory costs, reductions in digestive performance, differences in body composition, reductions in food intake, or a combination of these factors. From 18 days postfertilization (dpf) until 42 dpf, tadpoles were exposed dietarily to a pentabromodiphenyl ether mixture (DE-71TM ) at a concentration of 100 ng DE-71/g wet mass under a rearing temperature of either 22 or 27 °C. After 20 days of PBDE exposure, total PBDEs in tadpoles averaged 148.4 ng/g wet mass, with no differences by rearing temperature and approximately 50% higher than in their diet; controls not fed PBDE had levels <1 ng/g. Exposure to PBDE resulted in reductions in body length, mass, and development compared to controls, independent of rearing temperature; PBDE had no effect on measures of body composition, dry matter digestibility, or oxygen consumption. A simple energy budget using data from the present study revealed that a 10% decrease in feeding rate could explain the lower mass gain of tadpoles exposed to PBDE. Growth depression by PBDE could be due to (1) direct inhibition of growth processes by PBDE that indirectly decreases total energy demand and food intake, and (2) direct inhibition of food intake. Future studies to disentangle these possible pathways of PBDE effects are warranted. Environ Toxicol Chem 2021;40:3181-3192. © 2021 SETAC.

A new species of Orobdella (Hirudinida, Arhynchobdellida, Orobdellidae) from the Tsukuba Mountains in Japan

A small-type quadrannulate leech species, Orobdella montipumila sp. nov., from the Tsukuba Mountains in central Honshu island, Japan, is described. Phylogenetic analyses using nuclear 18S rRNA, 28S rRNA, histone H3, mitochondrial cytochrome c oxidase subunit I, tRNACys, tRNAMet, 12S rRNA, tRNAVal, 16S rRNA, tRNALeu and NADH dehydrogenase subunit 1 markers revealed that O. montipumila formed a monophyletic lineage with seven congeners that include the small-type quadrannulate O. masaakikuroiwai Nakano, 2014, which is endemic to montane regions of central Honshu. However, the present phylogenies showed that O. montipumila and O. masaakikuroiwai are not sister species, which confirms that reduction of body length has arisen independently in Orobdella leeches indigenous to central Honshu.

Long-term exposure of Daphnia magna to polystyrene microplastic (PS-MP) leads to alterations of the proteome, morphology and life-history

In the past years, the research focus on the effects of MP on aquatic organisms extended from marine systems towards freshwater systems. An important freshwater model organism in the MP field is the cladoceran Daphnia, which plays a central role in lacustrine ecosystems and has been established as a test organism in ecotoxicology. To investigate the effects of MP on Daphnia magna, we performed a chronic exposure experiment with polystyrene MP under strictly standardized conditions. Chronic exposure of D. magna to PS microparticles led to a significant reduction in body length and number of offspring. To shed light on underlying molecular mechanisms induced by microplastic ingestion in D. magna, we assessed the effects of PS-MP at the proteomic level, as proteins, e.g., enzymes, are especially relevant for an organism's physiology. Using a state-of-the-art mass spectrometry based approach, we were able to identify 28,696 different peptides, which could be assigned to 3784 different proteins. Using a customized bioinformatic workflow, we identified 41 proteins significantly altered in abundance (q-value <0.05) in the PS exposed D. magna. Among the proteins increased in the PS treated group were several sulfotransferases, involved in basic biochemical pathways, as well as GABA transaminase catalyzing the degradation of the neurotransmitter GABA. In the abundance decreased group, we found essential proteins such as the DNA-directed RNA polymerase subunit and other proteins connected to biotic and inorganic stress and reproduction. Strikingly, we further identified several digestive enzymes that are significantly downregulated in the PS treated animals, which could have interfered with the affected animal's nutrient supply. This may explain the altered morphological and life history traits of the PS exposed daphnids. Our results indicate that long-term exposure to PS microplastics, which are frequently detected in environmental samples, may affect the fitness of daphnids.

Evaluation of transgenerational effects caused by metals as environmental pollutants in Daphnia magna.

The present study aimed to evaluate the acute and chronic toxicity of environmentally relevant concentrations of metals (Mn, Al, Fe, and Pb) in Daphnia magna and the generational transposition of reproductive and morphological damages. The effective concentration for 10% of the organisms from each metal was obtained by the acute toxicity test (96 hours); then, another five concentrations lower than this one were defined for the chronic experimentation (21days), in which the number of neonates generated by each individual was checked daily. At the end of the exposition, the lengths and number of morphological damages were recorded in each adult daphnid. During this, the molt generated on the 14th and 21st days were collected and cultivated for posterior evaluation of the same parameters. Alterations in the reproductive performance were observed in the organisms exposed to manganese and aluminum (4.0 and 0.5mgL-1, respectively). Organisms exposed to aluminum (0.05mgL-1) and iron (0.27mgL-1) showed a reduction in body length. It is also noteworthy that the molt of these adults and their respective offspring also presented reproductive alterations, especially the molt from the 14th day of lead exposure (0.02mgL-1) and the 21st day of manganese exposure (4.0mgL-1). Such effects allow us to conclude that environments polluted by metals can reduce the ability of the species to maintain themselves in the ecosystem. In addition, there is a need to increase the control and monitoring of metals, such as aluminum, which present risks even in low concentrations.

Using deep learning to track 3D kinematics

In recent years methamphetamine (MA) use has become more prevalent, and of particular concern is its growing popularity of MA among women of childbearing age. However, to date, studies examining MA effects on the developing offspring in laboratory animals are limited. Thus, the aim of this study was to determine if in utero MA exposure in rats at pharmacological doses can have a negative impact on neonatal neurodevelopment and behaviour. Pregnant Sprague-Dawley dams (n = 10 dams/group) received MA (0, 0.625, 1.25, 2.5 mg/kg) once daily via oral gavage from gestational day 7 to 21. Maternal body weight, food and water consumption were recorded daily. A range of standard neurodevelopment parameters was examined in the offspring during the neonatal period. There were no neurodevelopmental deficits observed with offspring exposed to 0.625 mg/kg MA, in fact, there were enhancements of neurodevelopment in some parameters at this low dose. However, exposure to the 1.25 mg/kg MA dose resulted in significant impairments in surface righting reflex and forelimb grip in both sexes. Exposure to the 2.5 mg/kg MA dose resulted in a significant reduction in ano-genital distance in males, and in both sexes resulted in delayed fur appearance and eye opening, impairments in surface righting reflex and negative geotaxis, and a reduction in body length. In conclusion, this study demonstrates that pharmacologically relevant doses of MA can have profound dose-related effects on neonatal outcome. If extrapolated to the clinical scenario this will give cause for concern regarding the risks associated with this drug of abuse at relatively low doses.

Developmental neurotoxicity of low concentrations of bisphenol A and S exposure in zebrafish

The vulnerability to environmental insults is heightened at early stages of development. However, the neurotoxic potential of bisphenol A (BPA) and bisphenol S (BPS) at developmental windows remains unclear. To investigate the mechanisms mediating the developmental neurotoxicity, zebrafish embryos were treated with 0.01, 0.03, 0.01, 0.3, 1 μM BPA/BPS. Also, we used Tg(HuC:GFP) zebrafish to investigate whether BPA/BPS could induce neuron development. The reduction in body length, and increased heart rate were significant in 0.3 and 1 μM BPA/BPS groups. The green fluorescence protein (GFP) intensity increased at 72 hpf and 120 hpf in Tg(HuC:GFP) larvae which was consistent with the increased mRNA expression of elval3 following BPS treatments, an indication of the plausible effect of BPS on embryonic neuron development. Additionally, BPA/BPS treatments elicited hyperactivity and reduced static time in zebrafish larvae, suggesting behavioral alterations. Moreover, qRT-PCR results showed that BPA and BPS could interfere with the normal expression of development-related genes vegfa, wnt8a, and mstn1 at the developmental stages. The expression of neurodevelopment-related genes (ngn1, elavl3, gfap, α1-tubulin, mbp, and gap43) were significantly upregulated in BPA and BPS treatments, except for the remarkable downregulation of mbp and gfap elicited by BPA at 48 (0.03 μM) and 120 hpf (0.3 μM) respectively; ngn1 at 48 hpf for 0.1 μM BPS. Overall, our results highlighted that embryonic exposure to low concentrations of BPA/BPS could be deleterious to the central nervous system development and elicit behavioral abnormalities in zebrafish at developmental stages.

MON-716 A Novel Human Heterozygous STAT5B Variant Leads to Impaired Growth and Developmental Defects in Zebrafish Embryos

Signal transducer and activator of transcription 5b (STAT5b) has been identified as a key downstream mediator of GH signaling in somatic growth. Autosomic recessive human mutations in STAT5B lead to severe growth retardation associated to immune dysregulation. On the other hand, some heterozygous STAT5B mutations have been associated to a milder form of the disease. We have identified a heterozygous novel STAT5B mutation by Whole Exome Sequencing (WES) in a 2.2-year-old boy who presented proportionate short stature (height -2.77 SDS) with mild immune dysregulation. He also had normal GH response to provocative tests, low IGF-I levels, and a limited response to IGF generation test. This variant is located within the highly conserved SH2 domain responsible for recognizing and interacting with tyrosine-phosphorylated target peptides. The aim of our study was to evaluate the functional consequences of this novel heterozygous human STAT5B variant (K632N), using the zebrafish as a biosensor system, to determine its pathogenicity. To do this, we performed overexpression experiments microinjecting construct-derived mRNA for the wildtype (WT) and mutant variant into zebrafish embryos at the 1-cell stage and assessed the consequences at 72 hours post fertilization (hpf). The missense variant was introduced into the full length STAT5B cDNA clone (Origene) by site-directed mutagenesis. To generate mRNA, WT and mutant forms of STAT5B cDNAs were linearized by digestion with XhoI, purified and subsequently transcribed with Mmessage Mmachine T7 Transcription Kit. Zebrafish embryos microinjected with 100 and 200 pg of mutant mRNA show a dose dependent significant reduction of body length at 72 hpf compared to those microinjected with the same dose of WT mRNA (p<0.001). Body length reduction with 100 pg of mutant mRNA was 4%, while with 200 pg was 12.7% (p<0.001). In addition, a significant number of embryos injected with mutant mRNA show developmental defects including pericardial edema, bent spine, and cyclopia compared to those injected with WT mRNA (p<0.001). In the case of pericardial edema, the number of affected embryos increased significantly with the mutant mRNA dose (p<0.005). In conclusion, our study was able to evidence the pathogenic nature of the STAT5B K632N variant since it leads to growth and developmental defects in zebrafish embryos. The zebrafish, and its conserved GH-IGF-I axis, constitutes an ideal in vivo model for characterizing the functional effect of genetic variants in ortholog human genes.

Impact of long-term parental exposure to Tamiflu metabolites on the development medaka offspring (Oryzias latipes)

Despite the widespread use of the antiviral drug, Tamiflu®, little is known about the long-term toxic effects of drug or its metabolites in an aquatic ecosystem. This study integrated epidemiological and ecotoxicological methods to determine environmentally relevant concentrations of Tamiflu. A model based on the species medaka (Oryzias latipes) was then used to determine the health status and reproductivity of adults exposed to the drug as well as the embryonic development of offspring. The proposed ecotoxicological model was also used to quantitatively and qualitatively evaluate the toxicodynamic parameters related to egg production, hatchability, and development. Our results revealed that at an environmentally relevant exposure, Tamiflu and its metabolites had no adverse effects on growth, survival, or fecundity of adult medaka. Nonetheless, we observed a reduction in hatchability under exposure to 300 μg L−1 and a reduction in body length under exposure exceeding 90 μg L−1. Under exposure to 300 μg L−1, the estimated spawning time to reach 50% of the maximum percentage of cumulative egg production (ET50) far exceeded that of the control group (without exposure to Tamiflu). We also observed a ∼ 3-fold decrease in maximum egg hatching (Emax). Based on an integrated epidemiological and ecotoxicological model, predictions of environmental concentrations of Tamiflu and its metabolites revealed that the influenza subtypes associated with increases in environmental concentrations: A(H3N2) > A(H1N1) > type B (in order of their effects). We also determined that A(H3N2) posed a potential risk to hatchability and development. Note however, the environmental concentrations of Tamiflu and its metabolites in most countries are lower than the effect concentrations derived in this study, indicating no hazards for aquatic environments. We recommend the use of hatchability and embryonic development as indicators in assessing the effects of long-term parental exposure to Tamiflu metabolites.

Low IGF-I Bioavailability Impairs Growth and Glucose Metabolism in a Mouse Model of Human PAPPA2 p.Ala1033Val Mutation.

Bioactive free IGF-I is critically important for growth. The bioavailability of IGF-I is modulated by the IGF-binding proteins (IGFBPs) and their proteases, such as pregnancy-associated plasma protein-A2 (PAPP-A2). We have created a mouse model with a specific mutation in PAPPA2 identified in a human with PAPP-A2 deficiency. The human mutation was introduced to the mouse genome via a knock-in strategy, creating knock-in mice with detectable protein levels of Papp-a2 but without protease activities. We found that the Pappa2 mutation led to significant reductions in body length (10%), body weight (10% and 20% in males and females, respectively), and relative lean mass in mice. Micro-CT analyses of Pappa2 knock-in femurs from adult mice showed inhibited periosteal bone expansion leading to more slender bones in both male and female mice. Furthermore, in the Pappa2 knock-in mice, insulin resistance correlated with decreased serum free IGF-I and increased intact IGFBP-3 concentrations. Interestingly, mice heterozygous for the knock-in mutation demonstrated a growth rate for body weight and length as well as a biochemical phenotype that was intermediate between wild-type and homozygous mice. This study models a human PAPPA2 mutation in mice. The mouse phenotype closely resembles that of the human patients, and it provides further evidence that the regulation of IGF-I bioavailability by PAPP-A2 is critical for human growth and for glucose and bone metabolism.

Effect of phthalates on development, reproduction, fat metabolism and lifespan in Daphnia magna

Phthalates are used as plasticizers to increase durability, resistivity and flexibility of plastic materials. The commonly used phthalate, diethylhexyl phthalate (DEHP) is used in different plastic materials like food packaging, toys and medical devices. DEHP has been linked to different toxicities in humans as well as in animals, and as a consequence other phthalates, including dibutyl phthalate (DBP) and diethyl phthalate (DEP) are being introduced. The increased use of phthalates has resulted in contamination of aquatic ecosystem and it directly threatens the aquatic life. In this study, we analyzed the effects of three phthalates DEHP, DEP and DBP using freshwater organism Daphnia magna. Although, exposure of the three phthalates at 1 and 10 μM did not result any lethality and hatching delay, the chronic exposure for 14 days resulted in reduction of body length. There was enhanced fat accumulation on exposure to all the phthalates, as indicated by oil red O staining. qRT-PCR analysis of genes involved in fat metabolism suggests that the increase in fat content could be due to inhibition of absorption and catabolism of fatty acids. Reproduction analysis showed that DBP and DEP did not alter fecundity but surprisingly, DEHP at 1 μM increased reproduction by 1.5 fold compared to control group. Phthalates also showed negative effect on lifespan as DEP at 10 μM and DBP at both 1 and 10 μM significantly reduced the lifespan. Our data indicates that along with the banned phthalate DEHP, the other substitute phthalates DEP and DBP could also have detrimental effect on aquatic organisms.

Effects of phenanthrene on different levels of biological organization in larvae of the sediment-dwelling invertebrate Chironomus sancticaroli (Diptera: Chironomidae)

The hydrocarbon phenanthrene is an organic compound commonly found in the environment. In aquatic ecosystems, it is highly toxic to organisms, although little is known about its effects on sediment-dwelling organisms. The purpose of this study was to evaluate phenanthrene effects on biochemical, histological, and ontogenetic levels in larvae of the sediment-dwelling invertebrate Chironomus sancticaroli at acute and chronic exposure. Lethal concentrations were estimated and toxicity (acute-96 h and chronic- 8 d) tests were performed at phenanthrene concentrations from 0.12 to 1.2 mg L−1. At acute and chronic exposure, we evaluated acetylcholinesterase (AChE), alpha esterase (EST-α), and beta esterase (EST-β) activities as well as histological alterations. In the assays with chronic exposure, effects on larval development were estimated using antennae length (instar estimative) and body length (growth estimative). The EST-α showed a significantly increased activity after 48 h at acute exposure to high concentrations of phenanthrene, while EST-β activity was increased after 48 and 72 h at acute exposure at higher concentrations and at 0.12 mg L−1 at chronic exposure. At acute exposure, the midgut showed alterations such as brush border disruption, gastric caeca regression, and lumen area reduction; the fat body showed nuclear alteration in the trophocytes, while the Malpighian tubules showed brush border reduction and the salivary glands were subject to cytoplasm vacuolation. At chronic exposure, the same alterations were observed, in addition to vacuolar coalescence in the trophocytes of the fat body. Regarding larval development, a reduction of body length was observed with increasing phenanthrene concentrations. Similarly, molting was delayed; in the control group, all larvae were in the fourth instar, while at higher phenanthrene concentrations, larvae were predominantly in the third instar. Phenanthrene had toxic effects on this chironomid, indicating risks for natural populations.