Butyl Benzyl Phthalate (BBP) disrupts neuromast development in embryonic zebrafish.

Butyl benzyl phthalate (BBP) is found in common household and industrial products world-wide. Phthalates are not covalently bound to plastics and continuously leach into the soil, sediment and aquatic environments. The lateral line system of fish is a mechanosensory system composed of neuromasts essential for survival behaviors including rheotaxis, schooling and predator avoidance. Here, we investigated the developmental toxicity of BBP on the developing lateral line neuromasts in zebrafish. Embryos were treated at gastrula stage with BBP and analyzed by DASPEI staining at 4 days post fertilization. We find that BBP negatively affects neuromast development leading to loss of DASPEI signal in neuromasts in a concentration dependent manner.

Di-butyl phthalate disrupts muscle, motor and sensory neuron development in embryonic zebrafish.

Phthalates are added to plastics to enhance its flexibility, durability and transparency. Phthalates are not covalently bound to plastics and leach into the environment. Phthalates are now pervasive and ubiquitously present in the atmosphere, soil and sediment, surface and wastewater. Phthalates are known endocrine disruptors and their effects on male and female reproduction are well noted. However, studies on the developmental effects of di-butyl phthalate (DBP) are limited. Here we investigated the developmental toxicity of DBP on motor and sensory neuron populations and the muscle structures motor neurons innervate using the zebrafish vertebrate model system. We investigated these effects during the time window of development spanning the period where embryonic patterning determines adult structures. We found that treatment with 2.5 µM DBP from 6 h post-fertilization (hpf) until 72hpf induces loss and disorganization of primary motor neuron innervation of the somatic tissue with concomitant disruptions to muscle fiber organization. Furthermore, we found disruptions to sensory motor neuron development including defects in dorsal root ganglion and their peripherally extending axons. Rohon-Beard sensory neurons were also disrupted showing loss of the bilateral soma positioning along the length of the spinal cord and their afferent axonal projections to the epithelium. Thus, we concluded that DBP is toxic to developing motor and sensory neurons during embryonic development.

Hindbrain defects induced by Di-butyl phthalate (DBP) in developing zebrafish embryos.

Di-butyl phthalate (DBP) is a globally used plasticizer found in alarmingly high concentrations in soil and water ecosystems. As phthalates are non-covalently bound to plastic polymers, phthalates easily leach into the aquatic environment. The effects of DBP on aquatic organisms is concerning, most notably, studies have focused on the endocrine-disrupting effects. However, reports on the developmental neurotoxicity of DBP are rare. Using the zebrafish vertebrate model system, we treated pre-gastrulation staged embryos with 2.5 µM DBP, a concentration environmentally noted. We find that general hindbrain structure and rhombomere patterning is disrupted at 72 h post fertilization (hpf). We investigated hindbrain specific neural patterning of cranial motor neurons and find defects in branchiomotor neuron patterning and migration. Furthermore, defects in r4 specific Mauthner neuron development were also noted. Thus, we conclude that DBP exposure during embryonic development induces defects to the hindbrain and concomitantly the neurons that are born and differentiate there.

Di-butyl phthalate (DBP) induces defects during embryonic eye development in zebrafish.

Di-butyl phthalate (DBP) is a phthalate ester (PAEs) added during the manufacturing of plastics to make them stronger, yet more pliable. DBP is noncovalently bound to plastics resulting in leaching into the environment. Concerning concentrations of DBP have been noted in surface and groundwater, aquatic ecosystems, soil and atmospheric environments globally. Global production of phthalates and thus concomitant exposure has increased over the years making studies on the ecological and environmental safety needed. Most of the literature on DBP focuses on the endocrine disrupting properties of phthalate esters, but the developmental toxicity of DBP is an understudied area. Here, we treat gastrula staged zebrafish embryos with environmentally relevant concentrations of DBP (2.5 µM). We find defects in eye development at 96 h post fertilization including a decrease in the size of the lens and retina in DBP-treated embryos. Defects in eye vascularization as well as loss of the optic nerve and optic tectum were also noted. Here we conclude that exposure to environmentally relevant doses of DBP during early embryonic development is toxic to eye development.

Di-butyl phthalate (DBP) induces craniofacial defects during embryonic development in zebrafish.

Di-butyl phthalate (DBP) is commonly added to make plastics softer and more pliable and is found in a variety of consumer and industrial products. Alarmingly high levels of DBP have been detected in water and sediment as DBP leaches from products. These levels are concerning and have led the Environmental Protection Agency to label DBP as a priority environmental pollutant and the European Commission to label DBP as a priority substance. Given the ubiquitous presence of DBP globally and continuous exposure to DBP, studies on the developmental toxicity of DBP are needed. The endocrine disrupting effects of DBP are well documented, but developmental toxicity of DBP during critical developmental time windows is understudied. Here, we investigate the developmental effects of DBP exposure during early development. We find defects in craniofacial development including a decrease in overall cranial size in DBP treated embryos, but the intraocular distance was increased compared to controls. Further investigation of jawbone development demonstrated loss of and disorganization of cartilage development. Defects in vascular innervation and neuronal patterning were also noted. Here we conclude that exposure to DBP during crucial time windows of embryonic development is toxic to craniofacial development.

Butyl benzyl phthalate (BBP) induces caudal defects during embryonic development.

Butyl benzyl phthalate (BBP) is commonly added during the manufacturing of plastics to increase flexibility and elasticity. However, BBP leaches off of plastic and environment presence has been detected in soil, groundwater and sediment potentially effecting organisms in the environment. Given the widespread uses of BBP in household, consumer goods and the presence of BBP in the environment, studies on developmental toxicity are needed. Here, we use a zebrafish model to investigate the early developmental toxicity of BBP. We treated gastrula staged embryos with increasing concentrations of BBP and noted concentration-dependent defects in caudal tail development, but the effect was caudal specific with no other developmental defects noted. In situ hybridization studies using muscle and notochord markers show alterations in muscle development and non-linear, kinked notochord staining. A more detailed antibody staining using a myosin specific marker shows disorganized myofibrils and a loss of chevron shaped somites. Furthermore, vascular development in the tail was also disrupted in a concentration dependent manner. We conclude that BBP is toxic to caudal development in zebrafish. The sensitivity of zebrafish during development to environmental toxins and chemicals has been useful in assessing the health of the aquatic environment. The results presented here are a useful early warning system for contamination that could affect human health.

Using Photovoice and Asset Mapping to Inform a Community-Based Diabetes Intervention, Boston, Massachusetts, 2015.

INTRODUCTION: Diabetes self-management takes place within a complex social and environmental context.  This study's objective was to examine the perceived and actual presence of community assets that may aid in diabetes control. METHODS: We conducted one 6-hour photovoice session with 11 adults with poorly controlled diabetes in Boston, Massachusetts.  Participants were recruited from census tracts with high numbers of people with poorly controlled diabetes (diabetes "hot spots").  We coded the discussions and identified relevant themes.  We further explored themes related to the built environment through community asset mapping.  Through walking surveys, we evaluated 5 diabetes hot spots related to physical activity resources, walking environment, and availability of food choices in restaurants and food stores. RESULTS: Community themes from the photovoice session were access to healthy food, restaurants, and prepared foods; food assistance programs; exercise facilities; and church.  Asset mapping identified 114 community assets including 22 food stores, 22 restaurants, and 5 exercise facilities.  Each diabetes hot spot contained at least 1 food store with 5 to 9 varieties of fruits and vegetables.  Only 1 of the exercise facilities had signage regarding hours or services.  Memberships ranged from free to $9.95 per month.  Overall, these findings were inconsistent with participants' reports in the photovoice group. CONCLUSION: We identified a mismatch between perceptions of community assets and built environment and the objective reality of that environment. Incorporating photovoice and community asset mapping into a community-based diabetes intervention may bring awareness to underused neighborhood resources that can help people control their diabetes.

Glyphosate induces cardiovascular toxicity in Danio rerio.

Glyphosate is a broad spectrum herbicide used aggressively in agricultural practices as well as home garden care. Although labeled "safe" by the chemical industry, doses tested by industry do not mimic chronic exposures to sublethal doses that organisms in the environment are exposed to over long periods of time. Given the widespread uses of and exposure to glyphosate, studies on developmental toxicity are needed. Here we utilize the zebrafish vertebrate model system to study early effects of glyphosate on the developing heart. Treatment by embryo soaking with 50µg/ml glyphosate starting at gastrulation results in structural abnormalities in the atrium and ventricle, irregular heart looping, situs inversus as well as decreased heartbeats by 48h as determined by live imaging and immunohistochemistry. Vasculature in the body was also affected as determined using fli-1 transgenic embryos. To determine if the effects noted at 48h post fertilization are due to early stage alterations in myocardial precursors, we also investigate cardiomyocyte development with a Mef2 antibody and by mef2ca in situ hybridization and find alterations in the Mef2/mef2ca staining patterns during early cardiac patterning stages. We conclude that glyphosate is developmentally toxic to the zebrafish heart.

Glyphosate induces neurotoxicity in zebrafish.

Glyphosate based herbicides (GBH) like Roundup(®) are used extensively in agriculture as well as in urban and rural settings as a broad spectrum herbicide. Its mechanism of action was thought to be specific only to plants and thus considered safe and non-toxic. However, mounting evidence suggests that GBHs may not be as safe as once thought as initial studies in frogs suggest that GBHs may be teratogenic. Here we utilize the zebrafish vertebrate model system to study early effects of glyphosate exposure using technical grade glyphosate and the Roundup(®) Classic formulation. We find morphological abnormalities including cephalic and eye reductions and a loss of delineated brain ventricles. Concomitant with structural changes in the developing brain, using in situ hybridization analysis, we detect decreases in genes expressed in the eye, fore and midbrain regions of the brain including pax2, pax6, otx2 and ephA4. However, we do not detect changes in hindbrain expression domains of ephA4 nor exclusive hindbrain markers krox-20 and hoxb1a. Additionally, using a Retinoic Acid (RA) mediated reporter transgenic, we detect no alterations in the RA expression domains in the hindbrain and spinal cord, but do detect a loss of expression in the retina. We conclude that glyphosate and the Roundup(®) formulation is developmentally toxic to the forebrain and midbrain but does not affect the hindbrain after 24 h exposure.

Evaluation of the developmental toxicity of lead in the Danio rerio body.

Lead has been utilized throughout history and is widely distributed and mobilized globally. Although lead in the environment has been somewhat mitigated, the nature of lead and its extensive uses in the past prohibit it from being completely absent from our environment and exposure to lead is still a public health concern. Most studies regarding lead toxicity have focused on the brain. However, little is found in the literature on the effects of lead in other tissues. Here, we utilize the zebrafish model system to investigate effects of lead exposure during early developmental time windows at 24, 48 and 72 h post fertilization in the body. We analyze whole body, notochord and somatic muscle changes, vascular changes of the body, as well as motor neuron alterations. We find lead exposure induces a curved body phenotype with concomitant changes in somite length, decreased notochord staining and abnormal muscle staining using live and in situ approaches. Furthermore, altered vasculature within the somatic regions, loss and/or alternations of motor neuron extension both dorsally and ventrally from the spinal cord, loss of Rohon-Beard sensory neurons, and increased areas of apoptosis were found. We conclude that lead is developmentally toxic to other areas of the developing embryo, not just the brain.

Neural alterations from lead exposure in zebrafish.

Lead was used extensively as a gas additive and pesticide, in paints, batteries, lead shot, pipes, canning and toy manufacturing. Although uses of lead have been restricted, lead persists in our environment especially in older homes, and generally in soil and water. Although extensive studies have determined that fetal and childhood exposures to lead have been associated with childhood and adolescent memory impairments and learning disabilities, there are limited studies investigating early neural and morphological effects that may lead to these behavioral and learning abnormalities. Here we utilize the zebrafish vertebrate model system to study early effects of lead exposure on the brain. We treat embryos with 0.2mM lead for 24, 48 and 72 h and analyze neural structures through live imagery and transgenic approaches. We find structural abnormalities in the hindbrain region as well as changes in branchiomotor neuron development and altered neural vasculature. Additionally, we find areas of increased apoptosis. We conclude that lead is developmentally neurotoxic to a specific region of the brain, the hindbrain and is toxic to branchiomotor neurons residing in rhombomeres 2 through 7 of the hindbrain and hindbrain central artery vasculature.

Brief embryonic strychnine exposure in zebrafish causes long-term adult behavioral impairment with indications of embryonic synaptic changes.

Zebrafish provide a powerful model of the impacts of embryonic toxicant exposure on neural development that may result in long-term behavioral dysfunction. In this study, zebrafish embryos were treated with 1.5mM strychnine for short embryonic time windows to induce transient changes in inhibitory neural signaling, and were subsequently raised in untreated water until adulthood. PCR analysis showed indications that strychnine exposure altered expression of some genes related to glycinergic, GABAergic and glutamatergic neuronal synapses during embryonic development. In adulthood, treated fish showed significant changes in swimming speed and tank diving behavior compared to controls. Taken together, these data show that a short embryonic exposure to a neurotoxicant can alter development of neural synapses and lead to changes in adult behavior.

An early Fgf signal required for gene expression in the zebrafish hindbrain primordium.

We have explored the role of fibroblast growth factor (Fgf) signaling in regulating gene expression in the early zebrafish hindbrain primordium. We demonstrate that a dominant negative Fgf receptor (FgfR) construct disrupts gene expression along the entire rostrocaudal axis of the hindbrain primordium and, using an FgfR antagonist, we find that this Fgf signal is required at early gastrula stages. This effect cannot be mimicked by morpholino antisense oligos to Fgf3, Fgf8 or Fgf24--three Fgf family members known to be secreted from signaling centers at the midbrain-hindbrain boundary (MHB), in rhombomere 4 and in caudal mesoderm at gastrula stages. We propose that an Fgf signal is required in the early gastrula to initiate hindbrain gene expression and that this is distinct from the later roles of Fgfs in patterning the hindbrain during late gastrula/early segmentation stages. We also find that blocking either retinoic acid (RA) or Fgf signaling disrupts hindbrain gene expression at gastrula stages, suggesting that both pathways are essential at this stage. However, both pathways must be blocked simultaneously to disrupt hindbrain gene expression at segmentation stages, indicating that these signaling pathways become redundant at later stages. Furthermore, exogenous application of RA or Fgf alone is sufficient to induce hindbrain genes in gastrula stage tissues, suggesting that the two-signal requirement can be overcome under some conditions. Our results demonstrate an early role for Fgf signaling and reveal a dynamic relationship between the RA and Fgf signaling pathways during hindbrain development.

Dynamic expression and regulation by Fgf8 and Pou2 of the zebrafish LIM-only gene, lmo4.

We report the expression of zebrafish lmo4 during the first 48 h of development. Like its murine ortholog, lmo4 is expressed in somitic mesoderm, branchial arches, otic vesicles, and limb (pectoral fin) buds. In addition, however, we report zebrafish lmo4 expression in the developing eye, cardiovascular tissue, and the neural plate and telencephalon. We demonstrate that expression in the rostral hindbrain requires acerebellar (ace/fgf8) and spiel ohne grenzen (spg/pou2) activity.

Dynamic expression and regulation by Fgf8 and Pou2 of the zebrafish LIM-only gene, lmo4.

We report the expression of zebrafish lmo4 during the first 48 h of development. Like its murine ortholog, lmo4 is expressed in somitic mesoderm, branchial arches, otic vesicles, and limb (pectoral fin) buds. In addition, however, we report zebrafish lmo4 expression in the developing eye, cardiovascular tissue, and the neural plate and telencephalon. We demonstrate that expression in the rostral hindbrain requires acerebellar (ace/fgf8) and spiel ohne grenzen (spg/pou2) activity.