Neurodevelopmental and other phenotypes recurrently associated with heterozygous BAZ2B loss-of-function variants.

The bromodomain adjacent to zinc finger 2B (BAZ2B) gene encodes a chromatin remodeling protein that has been shown to perform a variety of regulatory functions. It has been proposed that loss of BAZ2B function is associated with neurodevelopmental phenotypes, and some recurrent structural birth defects and dysmorphic features have been documented among individuals carrying heterozygous loss-of-function BAZ2B variants. However, additional evidence is needed to confirm that these phenotypes are attributable to BAZ2B deficiency. Here, we report 10 unrelated individuals with heterozygous deletions, stop-gain, frameshift, missense, splice junction, indel, and start-loss variants affecting BAZ2B. These included a paternal intragenic deletion and a maternal frameshift variant that were inherited from mildly affected or asymptomatic parents. The analysis of molecular and clinical data from this cohort, and that of individuals previously reported, suggests that BAZ2B haploinsufficiency causes an autosomal dominant neurodevelopmental syndrome that is incompletely penetrant. The phenotypes most commonly seen in association with loss of BAZ2B function include developmental delay, intellectual disability, autism spectrum disorder, speech delay-with some affected individuals being non-verbal-behavioral abnormalities, seizures, vision-related issues, congenital heart defects, poor fetal growth, and an indistinct pattern of dysmorphic features in which epicanthal folds and small ears are particularly common.

Genetic syndromes are prevalent in patients with comorbid neurodevelopmental disorders and catatonia.

Catatonia occurs at high rates in idiopathic and syndromic neurodevelopmental disorders. At our institution's multidisciplinary catatonia clinic, clinical genetic testing (including microarray, fragile X PCR and methylation, autism/ID expanded panels, and exome sequencing) was commonly completed as part of clinical workup on patients with co-occurring neurodevelopmental disorders and catatonia (performed in 36/48 cases or 75%). This testing identified a pathogenic or likely pathogenic finding in 15/36 patients (42%). Testing identified a VUS (variant of uncertain significance) in 9/36 patients (25%). On review of the VUS findings, 4/9 were felt to be suspicious and potentially diagnostic. Testing was negative for 12/36 patients (33%). Many of the variants identified in this cohort were found in genes involved in gamma aminobutyric acid (GABA) and glutamatergic synaptic signaling; imbalances of these neurotransmitters are hypothesized to be drivers of catatonia. More work is needed to further characterize the molecular underpinnings of catatonia in the setting of neurodevelopmental disorders, including expanding genetic testing to larger cohorts in the future.

Safety and utility of acute electroconvulsive therapy for agitation and aggression in dementia.

OBJECTIVE: Agitation and aggression are among the most frequent and disruptive behavioral complications of dementia that contribute to increased cost of care, hospitalization, caregiver burden, and risk of premature institutionalization. This current study examined the safety and efficacy of electroconvulsive therapy (ECT) as a treatment for behavioral disturbances in dementia. We hypothesized that ECT would result in reduced agitated and aggressive behaviors between baseline and discharge. METHODS: Twenty-three participants admitted to McLean Hospital (Belmont, MA, USA) and Pine Rest Christian Mental Health Services (Grand Rapids, MI, USA), with a diagnosis of dementia who were referred for ECT to treat agitation and/or aggression, were enrolled in the study. We administered the Cohen-Mansfield Agitation Inventory-Short Form, Neuropsychiatric Inventory-Nursing Home Version, Cornell Scale for Depression in Dementia, and the Clinical Global Impression Scale at baseline, during, and after the ECT course. RESULTS: Regression analyses revealed a significant decrease from baseline to discharge on the Cohen-Mansfield Agitation Inventory (F(4,8) = 13.3; p = 0.006) and Neuropsychiatric Inventory (F(4,31) = 14.6; p < 0.001). There was no statistically significant change in scores on the Cornell Scale for Depression in Dementia. The Clinical Global Impression scores on average changed from a rating of "markedly agitated/aggressive" at baseline to "borderline agitated/aggressive" at discharge. Treatment with ECT was well tolerated by most participants; discontinuation of ECT occurred for two participants because of recurrence of agitation and for three participants because of adverse events. CONCLUSIONS: Electroconvulsive therapy may be a safe treatment option to reduce symptoms of agitation and aggression in patients with dementia whose behaviors are refractory to medication management.

Electroconvulsive therapy for the acute management of severe agitation in dementia (ECT-AD): A modified study protocol.

OBJECTIVE: This study began as a single-blind randomized controlled trial (RCT) to investigate the efficacy and safety of electroconvulsive therapy (ECT) for severe treatment-refractory agitation in advanced dementia. The aims are to assess agitation reduction using the Cohen-Mansfield Agitation Inventory (CMAI), evaluate tolerability and safety outcomes, and explore the long-term stability of agitation reduction and global functioning. Due to challenges encountered during implementation, including recruitment obstacles and operational difficulties, the study design was modified to an open-label format and other protocol amendments were implemented. METHODS: Initially, the RCT randomized participants 1:1 to either ECT plus usual care or simulated ECT plus usual care (S-ECT) groups. As patients were enrolled, data were collected from both ECT and simulated ECT (S-ECT) patients. The study now continues in an open-label study design where all patients receive actual ECT, reducing the targeted sample size from 200 to 50 participants. RESULTS: Study is ongoing and open to enrollment. CONCLUSION: The transition of the ECT-AD study design from an RCT to open-label design exemplifies adaptive research methodologies in response to real-world challenges. Data from both the RCT and open-label phases of the study will provide a unique perspective on the role of ECT in managing severe treatment-refractory agitation in dementia, potentially influencing future clinical practices and research approaches.

Manipulation of the HIF-Vegf pathway rescues methyl tert-butyl ether (MTBE)-induced vascular lesions.

Methyl tert-butyl ether (MTBE) has been shown to be specifically anti-angiogenic in piscine and mammalian model systems at concentrations that appear non-toxic in other organ systems. The mechanism by which MTBE targets developing vascular structures is unknown. A global transcriptome analysis of zebrafish embryos developmentally exposed to 0.00625-5mM MTBE suggested that hypoxia inducible factor (HIF)-regulated pathways were affected. HIF-driven angiogenesis via vascular endothelial growth factor (vegf) is essential to the developing vasculature of an embryo. Three rescue studies were designed to rescue MTBE-induced vascular lesions: pooled blood in the common cardinal vein (CCV), cranial hemorrhages (CH), and abnormal intersegmental vessels (ISV), and test the hypothesis that MTBE toxicity was HIF-Vegf dependent. First, zebrafish vegf-a over-expression via plasmid injection, resulted in significantly fewer CH and ISV lesions, 46 and 35% respectively, in embryos exposed to 10mM MTBE. Then HIF degradation was inhibited in two ways. Chemical rescue by N-oxaloylglycine significantly reduced CCV and CH lesions by 30 and 32% in 10mM exposed embryos, and ISV lesions were reduced 24% in 5mM exposed zebrafish. Finally, a morpholino designed to knock-down ubiquitin associated von Hippel-Lindau protein, significantly reduced CCV lesions by 35% in 10mM exposed embryos. In addition, expression of some angiogenesis related genes altered by MTBE exposure were rescued. These studies demonstrated that MTBE vascular toxicity is mediated by a down regulation of HIF-Vegf driven angiogenesis. The selective toxicity of MTBE toward developing vasculature makes it a potentially useful chemical in the designing of new drugs or in elucidating roles for specific angiogenic proteins in future studies of vascular development.

Use of genomic data in risk assessment case study: II. Evaluation of the dibutyl phthalate toxicogenomic data set.

An evaluation of the toxicogenomic data set for dibutyl phthalate (DBP) and male reproductive developmental effects was performed as part of a larger case study to test an approach for incorporating genomic data in risk assessment. The DBP toxicogenomic data set is composed of nine in vivo studies from the published literature that exposed rats to DBP during gestation and evaluated gene expression changes in testes or Wolffian ducts of male fetuses. The exercise focused on qualitative evaluation, based on a lack of available dose-response data, of the DBP toxicogenomic data set to postulate modes and mechanisms of action for the male reproductive developmental outcomes, which occur in the lower dose range. A weight-of-evidence evaluation was performed on the eight DBP toxicogenomic studies of the rat testis at the gene and pathway levels. The results showed relatively strong evidence of DBP-induced downregulation of genes in the steroidogenesis pathway and lipid/sterol/cholesterol transport pathway as well as effects on immediate early gene/growth/differentiation, transcription, peroxisome proliferator-activated receptor signaling and apoptosis pathways in the testis. Since two established modes of action (MOAs), reduced fetal testicular testosterone production and Insl3 gene expression, explain some but not all of the testis effects observed in rats after in utero DBP exposure, other MOAs are likely to be operative. A reanalysis of one DBP microarray study identified additional pathways within cell signaling, metabolism, hormone, disease, and cell adhesion biological processes. These putative new pathways may be associated with DBP effects on the testes that are currently unexplained. This case study on DBP identified data gaps and research needs for the use of toxicogenomic data in risk assessment. Furthermore, this study demonstrated an approach for evaluating toxicogenomic data in human health risk assessment that could be applied to future chemicals.

Microbially mediated O-methylation of bisphenol A results in metabolites with increased toxicity to the developing zebrafish (Danio rerio) embryo.

Bisphenol A (BPA) is used in the manufacture of plastics, and has been identified in various environmental matrices, including human serum and breast milk. The prevalence of BPA in the environment and the potential exposure to humans underscores the need to more fully understand the fate of BPA in the environment and the resulting effects and toxicity to humans and other organisms. Here we demonstrate that Mycobacterium species, including Mycobacterium vanbaalenii strain PYR-1, are able to O-methylate BPA to its mono- and dimethyl ether derivatives (BPA MME and BPA DME, respectively). The O-methylation of BPA results in metabolites with increased toxicity as shown from differences in survival and occurrence of developmental lesions in developing zebrafish embryos exposed to BPA, BPA MME, and BPA DME. The mono- and dimethyl ether derivatives were more toxic than BPA, resulting in increased mortality at 5 (LC(50) = 0.66 and 1.2 mg L(-1)) and 28 (LC(50) = 0.38, <0.5 mg L(-1)) days post fertilization. Furthermore, exposure to either of the O-methylated metabolites resulted in an increase in the incidence of developmental lesions as compared to BPA exposure. These data illustrate a new mechanism for microbial transformation of BPA, producing metabolites warranting further study to understand their prevalence and effects in the environment.

Challenges in Managing Pericardial Disease Related to Post Viral Syndrome After COVID-19 Infection.

Although primarily a respiratory illness, coronavirus disease 2019 (COVID-19) has been associated with cardiac involvement with reported cases of myocardial ischemia, arrhythmia, myocarditis, pericarditis, and pericardial effusion leading to cardiac tamponade. Most cases of pericardial disease in this setting have been during the acute infection. Here, we present a patient who developed pericarditis leading to cardiac tamponade after the resolution of the acute COVID-19 infection. Her course of illness was further complicated by poor response to initial medical therapy with non-steroidal anti-inflammatory drugs (NSAIDs) and colchicine which could possibly be related to early exposure to steroids. It is often difficult to establish an underlying etiology for acute pericarditis. Similarly, in our case, although there is no definitive test to prove the causal relationship, this effusion is highly suspicious of being secondary to post viral sequelae after COVID-19 infection when considering the clinical course. It is important to consider pericardial disease as a late complication of COVID-19 even after apparent resolution of the acute infection and be mindful of the therapeutic challenges that we might face while managing such patients.

The Children's Hospital of Philadelphia's experience with donation after cardiac death.

OBJECTIVE: To describe our experience with pediatric donation after cardiac death. DESIGN: Retrospective chart review of all cases of donation after cardiac death from 1995 to 2005. SETTING: The Children's Hospital of Philadelphia pediatric intensive care unit. PATIENTS: Twelve patients who were pediatric organ donors after cardiac death. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Charts for 12 patients were located, and donation after cardiac death was confirmed. There were two females and ten males. Patient age ranged from 1 to 17 yrs (mean 8 yrs). Four patients had severe traumatic brain injury, and eight patients had hypoxic ischemic encephalopathy. The organs procured were 24 kidneys, eight livers, four lungs, and one pancreas. The organs transplanted were 23 kidneys, four livers, and one pancreas. Ten of 12 cases of withdrawal of life-sustaining support occurred in the operating room area; the other two occurred in the holding area and the postanesthesia care unit. Children received a wide range of medications at the time of extubation. No neuromuscular blockers were used. The time of extubation to time of death ranged from 4 mins to 30 mins, with a mean of 14.5 mins. Death was declared based on cardiac asystole confirmed by auscultation and transthoracic impedance, with organ procurement initiated 5 mins later. Regarding who initiated conversation about donation after cardiac death, nine cases were family initiated, one case was physician initiated, and in two there was a collaborative approach with the physician and representative from the organ procurement organization. Of the organs transplanted, all organs other than one kidney and one split liver graft were functioning at 1 yr post-transplant. CONCLUSIONS: Pediatric donation after cardiac death can be performed successfully; its impact on end-of-life care and bereavement needs further investigation.

Metabolism of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) by human CYP1B1 genetic variants.

Human cytochrome P450 1B1 (CYP1B1) plays a critical role in the metabolic activation of a variety of procarcinogens, including 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). The existence of human CYP1B1 missense genetic variants has been demonstrated, but their activities in metabolizing PhIP are unknown. In this study, we expressed 15 naturally occurring CYP1B1 variants (with either single or multiple amino acid substitutions) and determined their activity changes in metabolizing PhIP to its two major metabolites, 2-hydroxyamino-PhIP and 4'-hydroxy-PhIP. Although the PhIP-metabolizing activities of four variants (Ala(119)Ser, Pro(379)Leu, Ala(443)Gly, Arg(48)Gly/Leu(432)Val) were comparable with that of the expressed wild-type CYP1B1, five variants (Trp(57)Cys, Gly(61)Glu, Arg(48)Gly/Ala(119)Ser, Arg(48)Gly/Ala(119)Ser/Leu(432)Val, Arg(48)Gly/Ala(119)Ser/Leu(432)Val/Ala(443)Gly) exhibited more than 2-fold decrease in activity and a reduction in the catalytic efficiency (V(max)/K(m)) for both N- and 4-hydroxylation of PhIP. Six variants (Gly(365)Trp, Glu(387)Lys, Arg(390)His, Pro(437)Leu, Asn(453)Ser, Arg(469)Trp) showed little activity in PhIP metabolism, but the molecular mechanisms involved are apparently different. The microsomal CYP1B1 protein level was significantly decreased for the Trp(365), Lys(387), and His(390) variants and was not detectable for the Ser(453) variant. In contrast, there was no difference between the Trp(469) variant and the wild-type in the microsomal CYP1B1 protein level and P450 content but the Trp(469) variant totally lost its metabolic activity toward PhIP. The Leu(437) variant also had a substantial amount of CYP1B1 protein in the microsomes, but there was a lack of detectable P450 peak and activity. Our results should be useful in selecting appropriate CYP1B1 variants as cancer susceptibility biomarkers for human population studies related to PhIP exposure.

Matrix metalloproteinase-13 is required for zebra fish (Danio rerio) development and is a target for glucocorticoids.

Matrix metalloproteinases (MMPs) are endopeptidases that degrade the proteins of the extracellular matrix (ECM). Expression and activity of the MMPs are essential for embryogenesis, where MMPs participate in the normal ECM remodeling that occurs during tissue morphogenesis and development. Studies have demonstrated that MMP gene expression is inhibited by glucocorticoids in mammalian cell culture systems and that exposure to glucocorticoids causes developmental abnormalities in several species. Therefore, we proposed that glucocorticoids impede normal development through alteration of MMP expression. Zebra fish (Danio rerio) were used as a model to study MMP-13 expression both during normal embryogenesis and following acute exposure to two glucocorticoids, dexamethasone, and hydrocortisone. MMP-13 is one of three collagenases identified in vertebrates that catalyzes the degradation of type I collagens at neutral pH. MMP-13 expression varied during zebra fish development, with peak expression at 48 h post-fertilization (hpf). Morpholino knockdown studies showed that MMP-13 expression is necessary for normal zebra fish embryogenesis. Acute exposure to dexamethasone and hydrocortisone resulted in abnormal zebra fish development including craniofacial abnormalities, altered somitogenesis, blood pooling and pericardial and yolk sac edema as well as increased MMP-13 mRNA and activity at 72 hpf. In situ hybridization experiments were used to confirm the increase in MMP-13 expression following glucocorticoid treatment and showed elevated MMP-13 expression in the rostral trunk, brain, eye, heart, and anterior kidney of treated embryos. These data demonstrate that normal zebra fish embryogenesis requires MMP-13 and that dexamethasone and hydrocortisone modulate the expression of this gene, leading to increased activity and potentially contributing to subsequent dysmorphogenesis.

Perceived hearing status and attitudes toward noise in young adults.

PURPOSE: To estimate the prevalence of perceived hearing loss, tinnitus, and temporary threshold shift (TTS) in community college students and to see whether those students' attitudes toward noise affected their perception of their own possible hearing loss, tinnitus, and TTS. METHOD: Young adults (N = 245; age 18-27) completed 3 questionnaires: the Hearing Symptom Description, Youth Attitude to Noise Scale, and Adolescents' Habits and Hearing Protection Use. RESULTS: Perceived TTS and pain associated with loud noise were the most common hearing related factors, followed by perceived tinnitus and hearing loss. The students' attitudes toward noise in their daily environment showed the most negative response, whereas attitudes toward noise and concentration indicated a more positive, or less harmful, response. Chi-square analysis indicated a significant correlation between perceived hearing loss and respondents' overall attitudes toward noise exposure. Hearing protection use was limited for all participants, with the majority reporting never having used hearing protection. CONCLUSION: Approximately 6% of respondents reported perceived hearing loss, and 13.5% reported prolonged tinnitus. In general, participants had neutral attitudes toward noise. Over 20% of participants reported ear pain, tinnitus, and/or TTS after noise exposure at least sometimes. Coincidentally, few participants reported consistent use of hearing protection.

Developmental Deltamethrin Exposure Causes Persistent Changes in Dopaminergic Gene Expression, Neurochemistry, and Locomotor Activity in Zebrafish.

Pyrethroids are commonly used insecticides that are considered to pose little risk to human health. However, there is an increasing concern that children are more susceptible to the adverse effects of pesticides. We used the zebrafish model to test the hypothesis that developmental exposure to low doses of the pyrethroid deltamethrin results in persistent alterations in dopaminergic gene expression, neurochemistry, and locomotor activity. Zebrafish embryos were treated with deltamethrin (0.25-0.50 µg/l), at concentrations below the LOAEL, during the embryonic period [3-72 h postfertilization (hpf)], after which transferred to fresh water until the larval stage (2-weeks postfertilization). Deltamethrin exposure resulted in decreased transcript levels of the D1 dopamine (DA) receptor (drd1) and increased levels of tyrosine hydroxylase at 72 hpf. The reduction in drd1 transcripts persisted to the larval stage and was associated with decreased D2 dopamine receptor transcripts. Larval fish, exposed developmentally to deltamethrin, had increased levels of homovanillic acid, a DA metabolite. Since the DA system is involved in locomotor activity, we measured the swim activity of larval fish following a transition to darkness. Developmental exposure to deltamethrin significantly increased larval swim activity which was attenuated by concomitant knockdown of the DA transporter. Acute exposure to methylphenidate, a DA transporter inhibitor, increased swim activity in control larva, while reducing swim activity in larva developmentally exposed to deltamethrin. Developmental exposure to deltamethrin causes locomotor deficits in larval zebrafish, which is likely mediated by dopaminergic dysfunction. This highlights the need to understand the persistent effects of low-dose neurotoxicant exposure during development.

Ontogeny of voltage-sensitive calcium channel alpha(1A) and alpha(1E) subunit expression and synaptic function in rat central nervous system.

Immunohistochemical expression in the neocortex, hippocampus and cerebellum of the alpha(1A) or alpha(1E) subunit of the voltage-sensitive Ca(2+) channel was examined in Long-Evans hooded rats on gestational day 18 and postnatal days 1, 4, 7, 10, 14, 21, 90, 360 and 720. On gestational day 18 and postnatal day 1, alpha(1A) immunoreactivity was more dense in the neocortex and hippocampus than the cerebellum. By postnatal day 7, levels of alpha(1A) immunoreactivity increased dramatically in the cerebellum, while in neocortex, alpha(1A) immunoreactivity became more sparse, which approached the more diffuse pattern of cellular staining in the mature brain. Expression of alpha(1E) in the neocortex, hippocampus and cerebellum was much less dense than alpha(1A) between gestational day 18 and postnatal day 4. There was also significant alpha(1E) immunoreactivity in the mossy fibers of the hippocampus and in dendrites of Purkinje cells of the cerebellum. Depolarization-dependent 45Ca(2+) influx was examined in rat brain synaptosomes on postnatal days 4, 7, 10, 14, 21 and >60. In neocortical and hippocampal synaptosomes, 45Ca(2+) influx increased steadily with age and reached adult levels by postnatal day 10. In cerebellar synaptosomes, 45Ca(2+) influx was constant across all ages, except for a spike in activity which was observed on postnatal day 21. In neocortical and hippocampal synaptosomes, 100 nM omega-conotoxin MVIIC significantly inhibited 45Ca(2+) influx on postnatal day 10 and 14, respectively, or after. In cerebellar synaptosomes, influx was inhibited by omega-conotoxin MVIIC only on postnatal day 10 or prior. On postnatal day 7, 45Ca(2+) influx was not inhibited in neocortical and hippocampal synaptosomes by a combination of 10 microM nifedipine, 1 microM omega-conotoxin GVIA and 1 microM omega-conotoxin MVIIC, suggesting that an 'insensitive' flux predominates at this age. Overall, the results suggest that expression of voltage-sensitive Ca(2+) channels during development is dynamic and is important in central nervous system development.

Chronic exposure of killifish to a highly polluted environment desensitizes estrogen-responsive reproductive and biomarker genes.

Reproductive and endocrine disruption is commonly reported in aquatic species exposed to complex contaminant mixtures. We previously reported that Atlantic killifish (Fundulus heteroclitus) from the chronically contaminated Newark Bay, NJ, exhibit multiple endocrine disrupting effects, including inhibition of vitellogenesis (yolk protein synthesis) in females and false negative vitellogenin biomarker responses in males. Here, we characterized the effects on estrogen signaling and the transcriptional regulation of estrogen-responsive genes in this model population. First, a dose-response study tested the hypothesis that reproductive biomarkers (vtg1, vtg2, chg H, chg Hm, chg L) in Newark Bay killifish are relatively less sensitive to 17ß-estradiol at the transcriptional level, relative to a reference (Tuckerton, NJ) population. The second study assessed expression for various metabolism (cyp1a, cyp3a30, mdr) and estrogen receptor (ER α, ER ßa, ER ßb) genes under basal and estrogen treatment conditions in both populations. Hepatic metabolism of 17ß-estradiol was also evaluated in vitro as an integrated endpoint for adverse effects on metabolism. In the third study, gene methylation was evaluated for promoters of vtg1 (8 CpGs) and vtg2 (10 CpGs) in both populations, and vtg1 promoter sequences were examined for single nucleotide polymorphism (SNPs). Overall, these studies show that multi-chemical exposures at Newark Bay have desensitized all reproductive biomarkers tested to estrogen. For example, at 10ng/g 17ß-estradiol, inhibition of gene induction ranged from 62% to 97% for all genes tested in the Newark Bay population, relative to induction levels in the reference population. The basis for this recalcitrant phenotype could not be explained by a change in 17ß-estradiol metabolism, nuclear estrogen receptor expression, promoter methylation (gene silencing) or SNPs, all of which were unaltered and normal in the Newark Bay population. The decreased transcriptional sensitivity of estrogen-responsive genes is suggestive of a broad effect on estrogen receptor pathway signaling, and provides insight into the mechanisms of the endocrine disrupting effects in the Newark Bay population.

Inhibition of vitellogenin gene induction by 2,3,7,8-tetrachlorodibenzo-p-dioxin is mediated by aryl hydrocarbon receptor 2 (AHR2) in zebrafish (Danio rerio).

Vitellogenins are hepatically derived yolk-protein precursors required for oogenesis in all oviparous teleosts. Altered gene-regulation of vitellogenesis by environmental contaminants can have profound effects on reproductive success, and ultimately population sustainability. To better understand chemical effects on vitellogenin gene regulation, we tested the hypothesis that activation of the aryl hydrocarbon receptor 2 (AHR2) by dioxin inhibits the estrogen receptor pathway regulation of 3 vitellogenin genes (vtg1-3) in vivo, using zebrafish (Danio rerio) as a model teleost. Using an embryo-larval bioassay, embryos were either treated with 1000 pptr (parts-per-trillion, pg/mL) 17α-ethynylestradiol (EE2) alone from 6h post fertilization (hpf) to 4 days post fertilization (dpf), or pre-treated with dioxin (4-5 hpf) prior to EE2. Pre-treatment with 400 pptr 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD) or 1,2,3,7,8-pentachlorodibenzo-p-dioxin inhibited the EE2 induction of vtg1, vtg2 and vtg3 by >95% (p≤0.05). In comparison, a splice-blocking AHR2 morpholino used to down-regulate ahr2 expression significantly reduced the inhibition of vtg1, vtg2 and vtg3 by 400 pptr 2,3,7,8-TCDD (20.7-27.4% rescue). These studies demonstrate that 2,3,7,8-TCDD directly inhibits the vitellogenin pathway in vivo through activation of the AHR2. This work provides evidence for AHR2 dependent cross-talk inhibition of vitellogenin genes and offers insight into anti-estrogenic reproductive effects observed in oviparous species exposed to AHR agonist contaminants.

Craniofacial abnormalities and altered wnt and mmp mRNA expression in zebrafish embryos exposed to gasoline oxygenates ETBE and TAME.

Gasoline additives ethyl tert butyl ether (ETBE) and tertiary amyl methyl ether (TAME) are used world wide, but the consequence of developmental exposure to these hydrophilic chemicals is unknown for aquatic vertebrates. The effect of ETBE and TAME on zebrafish embryos was determined following OECD 212 guidelines, and their toxicity was compared to structurally related methyl tert-butyl ether (MTBE), which is known to target developing vasculature. LC50s for ETBE and TAME were 14 mM [95% CI=10-20] and 10 mM [CI=8-12.5], respectively. Both chemicals caused dose dependent developmental lesions (0.625-10 mM), which included pericardial edema, abnormal vascular development, whole body edema, and craniofacial abnormalities. The lesions were suggestive of a dysregulation of WNT ligands and matrix metalloproteinase (MMP) protein families based on their roles in development. Exposure to 5 mM ETBE significantly (p≤0.05) decreased relative mRNA transcript levels of mmp-9 and wnt3a, while 2.5 and 5 mM TAME significantly decreased wnt3a, and wnt8a. TAME also significantly decreased mmp-2 and -9 mRNA levels at 5mM. ETBE and TAME were less effective in altering the expression of vascular endothelial growth factor-a and -c, which were the only genes tested that were significantly decreased by MTBE. This is the first study to characterize the aquatic developmental toxicity following embryonic exposure to ETBE and TAME. Unlike MTBE, which specifically targets angiogenesis, ETBE and TAME disrupt multiple organ systems and significantly alter the mRNA transcript levels of genes required for general development.

Decreased vitellogenin inducibility and 17ß-estradiol levels correlated with reduced egg production in killifish (Fundulus heteroclitus) from Newark Bay, NJ.

Aquatic species inhabiting polluted estuaries are exposed to complex mixtures of xenobiotics which can alter normal reproduction. We previously reported that female Atlantic killifish (Fundulus heteroclitus) from the highly contaminated Newark Bay, NJ (USA) exhibited an inhibition of oocyte development due to reduced vitellogenin (egg-yolk precursor) levels. Our hypothesis was that the inhibition of oocyte development in Newark Bay killifish is due to (1) deficient levels of circulating 17ß-estradiol, and (2) a decreased sensitivity of the vitellogenin pathway to physiological doses of 17ß-estradiol. In the first study, adult naïve killifish from Tuckerton, NJ (reference) were caged at Tuckerton and Newark Bay. After 1 month, males caged at Newark Bay exhibited inductions of hepatic vitellogenin and estrogen receptor α, which were transient and returned to basal levels after 2 months (p≤0.05). In the second study, fecundity and 17ß-estradiol levels were measured in reproductively active adult females from Tuckerton and Newark Bay. Tuckerton females produced 140 eggs per female and Newark Bay females produced 11 eggs per female. Embryos from Newark Bay had 34% greater mortality and 28% less hatch, relative to Tuckerton. In addition, embryo mass and yolk-volume of Newark Bay embryos compared to Tuckerton embryos was 16% and 25% lower, respectively. Circulating 17ß-estradiol levels in Newark Bay females (0.26 ng/mL) were measured to be 8-fold lower than Tuckerton females (2.25 ng/mL). In the third study, adult killifish from both sites were dosed with 17ß-estradiol to assess the sensitivity of the vitellogenin pathway. At doses of 0.01, 0.1, 1 and 10 ng/g body weight, induction levels of circulating vitellogenin in Newark Bay males were significantly inhibited by 97, 99, 98 and 44%, respectively, compared to Tuckerton males. At doses of 0.01, 0.1, 1, 10 and 100 ng/g body weight, induction levels of circulating vitellogenin in Newark Bay females were inhibited by 89, 79, 61, 40 and 30%, respectively, compared to Tuckerton females. These differences in inducibility could not be explained by altered hepatic expression of estrogen receptors α, ßa or ßb. Based on the caged and dose-response studies, contaminants that down-regulate vitellogenin would interfere with its ability to be used as a biomarker for xeno-estrogen exposures. These studies demonstrate that contaminants within Newark Bay exert both estrogenic and anti-estrogenic responses which results in an overtly anti-estrogenic phenotype (reduced egg production due to inhibition of vitellogenesis).

Methyl tert butyl ether targets developing vasculature in zebrafish (Danio rerio) embryos.

Disruption of vascular endothelial growth factor (VEGF) signaling during early development results in abnormal angiogenesis and increased vascular lesions. Embryonic exposure to 0.625-10mM methyl tert butyl ether (MTBE), a highly water soluble gasoline additive, resulted in a dose dependent increase in pooled blood in the common cardinal vein (CCV), cranial hemorrhages and abnormal intersegmental vessels (ISVs). The EC50s for the lesions ranked in terms of likelihood to occur with MTBE exposure were: pooled blood in the CCV, 3.2 mM [95% CI: 2.2-4.7]>cranial hemorrhage, 11 mM [5.9-20.5]>abnormal ISV, 14.5 mM [6.5-32.4]. Organ systems other than the vascular system appear to develop normally, which suggests MTBE toxicity targets developing blood vessels. Equal molar concentrations (0.625-10mM) of the primary metabolites, tertiary butyl alcohol (TBA) and formaldehyde, did not result in vascular lesions, which suggested that the parent compound is responsible for the toxicity. Stage specific exposures were carried out to determine the developmental period most sensitive to MTBE vascular disruption. Embryos treated until 6-somites or treated after Prim-5 stages did not exhibit a significant increase in lesions, while embryos treated between 6-somites and Prim-5 had a significant increase in vascular lesions (p≤0.05). During the critical window for MTBE-induced vascular toxicity, expression of vegfa, vegfc, and flk1/kdr were significantly decreased 50, 70 and 40%, respectively. This is the first study to characterize disruption in vascular development following embryonic exposure to MTBE. The unique specificity of MTBE to disrupt angiogenesis may be mediated by the down regulation of critical genes in the VEGF pathway.