Guideline No. 425a: Cannabis Use Throughout Women's Lifespans - Part 1: Fertility, Contraception, Menopause, and Pelvic Pain.

OBJECTIVE: To provide health care providers with the best evidence on cannabis use with respect to women's health. Areas of focus include general patterns of cannabis use as well as safety of use; care for women who use cannabis; stigma; screening, brief intervention, and referral to treatment; impact on hormonal regulation; reproductive health, including contraception and fertility; sexual function; effects on perimenopausal and menopausal symptoms; and use in chronic pelvic pain syndromes. TARGET POPULATION: The target population includes all women currently using or contemplating using cannabis. OUTCOMES: Open, evidence-informed dialogue about cannabis use, which will lead to improvement in patient care. BENEFITS, HARMS, AND COSTS: Exploring cannabis use through a trauma-informed approach provides the health care provider and patient with an opportunity to build a strong, collaborative, therapeutic alliance. This alliance empowers women to make informed choices about their own care. It also allows for the diagnosis and possible treatment of cannabis use disorders. Use should not be stigmatized, as stigma leads to poor "partnered care" (i.e., the partnership between the patient and care provider). Multiple side effects of cannabis use may be mistaken for other disorders. Currently, use of cannabis to treat women's health issues is not covered by public funding; as a result, individual users must pay the direct cost. The indirect costs of cannabis use are unknown. Thus, health care providers and patients must understand the role of cannabis in women's health issues, so that women can make knowledgeable decisions. EVIDENCE: PubMed, EMBASE, and grey literature were searched to identify studies of "cannabis use and effect on infertility, contraception, perimenopause and menopausal symptoms, and pelvic pain" published between January 1, 2018 and February 18, 2021. All clinical trials, observational studies, reviews (including systematic reviews and meta-analyses), guidelines, and conference consensus statements were included. Publications were screened for relevance. The search terms were developed using the Medical Subject Headings (MeSH) terms and keywords (and variants), including cannabis, cannabinoids, marijuana, dexanabinol, dronabinol, tetrahydrocannabinol; the specific terms to capture women's health were estrogen, estradiol, medroxyprogesterone acetate, vaginal contraception, oral contraceptives, fertilization, amenorrhea, oligomenorrhea, pelvic pain, dysmenorrhea, endometriosis, interstitial cystitis, vulvodynia, and menopause. VALIDATION METHODS: The authors rated the quality of evidence and strength of recommendations using the Grading of Recommendations Assessment, Development and Evaluation (GRADE) approach. See online Appendix A (Tables A1 for definitions and A2 for interpretations of strong and weak recommendations). INTENDED AUDIENCE: All heath care providers who care for women. SUMMARY STATEMENTS: RECOMMENDATIONS.

Directive clinique no 425a : Le cannabis aux différentes périodes de la vie des femmes - Partie 1 : Fertilité, contraception, ménopause et douleur pelvienne.

OBJECTIF: Fournir aux fournisseurs de soins de santé les meilleures données probantes sur l'utilisation de cannabis et la santé des femmes. Les domaines d'intérêt sont : les profils généraux d'utilisation du cannabis ainsi que la sécurité de la consommation; les soins aux femmes qui utilisent le cannabis; la stigmatisation; le dépistage, l'intervention brève et l'orientation vers le traitement; les effets sur la régulation hormonale; la santé reproductive, y compris la contraception et la fertilité; la fonction sexuelle; les effets sur les symptômes périménopausiques et postménopausiques; et l'utilisation dans le traitement des syndromes de douleur pelvienne chronique. POPULATION CIBLE: La population cible comprend toutes les femmes qui consomment ou utilisent du cannabis ou qui envisagent de le faire. RéSULTATS: Un dialogue ouvert et fondé sur des données probantes relativement à l'utilisation et la consommation de cannabis, dialogue qui mènera à l'amélioration des soins aux patientes. BéNéFICES, RISQUES ET COûTS: L'exploration de l'utilisation et de la consommation de cannabis par une approche basée sur la connaissance des traumatismes donne l'occasion au fournisseur de soins et à la patiente de créer une solide alliance thérapeutique collaborative. Cette alliance permet aux femmes de faire des choix éclairés sur leurs propres soins. Elle facilite également le diagnostic et le traitement possible des troubles de l'usage du cannabis. Il ne faut pas stigmatiser la consommation, car la stigmatisation nuit à l'alliance thérapeutique (c'est-à-dire le partenariat entre la patiente et le fournisseur de soins). Plusieurs effets indésirables de la consommation de cannabis peuvent être confondus avec d'autres problèmes de santé. À l'heure actuelle, l'utilisation du cannabis pour traiter les problèmes de santé féminine n'est pas financée par le secteur public; par conséquent, les utilisatrices doivent assumer les coûts directs. Les coûts indirects de l'utilisation de cannabis sont inconnus. Ainsi, les fournisseurs de soins et les patientes doivent comprendre le rôle du cannabis dans les problèmes de santé féminine de sorte que les femmes puissent prendre des décisions éclairées. DONNéES PROBANTES: Des recherches ont été effectuées dans PubMed, Embase et la littérature grise pour recenser des études publiées entre le 1er janvier 2018 et le 18 février 2021 concernant l'utilisation du cannabis et ses effets sur l'infertilité, la contraception, les symptômes périménopausiques et postménopausiques et la douleur pelvienne. Toutes les publications des types suivants ont été incluses : essais cliniques, études observationnelles, revues (y compris les revues systématiques et les méta-analyses), directives cliniques et déclarations de conférences de consensus. Un survol des publications a été effectué pour en confirmer la pertinence. Les termes de recherche ont été définis à l'aide des termes MeSH (Medical Subject Headings) et mots clés (et variantes) suivants : cannabis, cannabinoids, marijuana, dexanabinol, dronabinol et tetrahydrocannabinol. À ces termes ont été combinés les termes suivants afin de cerner la santé des femmes : estrogen, estradiol, medroxyprogesterone acetate, vaginal contraception, oral contraceptives, fertilization, amenorrhea, oligomenorrhea, pelvic pain, dysmenorrhea, endometriosis, interstitial cystitis, vulvodynia et menopause. MéTHODES DE VALIDATION: Les auteurs ont évalué la qualité des données probantes et la force des recommandations en utilisant l'approche d'évaluation, de développement et d'évaluation (GRADE). Voir l'annexe A en ligne (tableau A1 pour les définitions et tableau A2 pour l'interprétation des recommandations fortes et faibles). PROFESSIONNELS CONCERNéS: Tous les fournisseurs de soins de santé qui prodiguent des soins aux femmes. DÉCLARATIONS SOMMAIRES: RECOMMANDATIONS.

Guideline No. 425b: Cannabis Use Throughout Women's Lifespans - Part 2: Pregnancy, the Postnatal Period, and Breastfeeding.

OBJECTIVE: To provide health care providers with the best evidence on cannabis use and women's health. Areas of focus include screening, dependence, and withdrawal; communication and documentation; pregnancy (including maternal and fetal outcomes); maternal pain control; postpartum care (including second-hand smoking and parenting); and breastfeeding. TARGET POPULATION: The target population includes women who are planning a pregnancy, pregnant, or breastfeeding. BENEFITS, HARMS, AND COSTS: Discussing cannabis use with women who are planning a pregnancy, pregnant, or breastfeeding allows them to make informed choices about their cannabis use. Based on the limited evidence, cannabis use in pregnancy or while breastfeeding should be avoided, or reduced as much as possible if abstaining is not feasible, given the absence of safety and long-term follow up data on cannabis-exposed pregnancies and infants. EVIDENCE: PubMed and Cochrane Library databases were searched for articles relevant to cannabis use during pregnancy and breastfeeding published between January 1, 2018, and February 5, 2021. The search terms were developed using the MeSH terms and keywords and their variants, including cannabis, cannabinoids, cannabidiol, CBD, THC, marijuana, edible, pregnancy, pregnant, prenatal, perinatal, postnatal, breastfeed, breastfed, lactation, nursing, fetus, fetal, neonatal, newborn, and child. In terms of publication type, all clinical trials, observational studies, reviews (including systematic reviews and meta-analyses), guidelines, and conference consensus statements were included. The main inclusion criteria were pregnant and breastfeeding women as the target population, and exposure to cannabis as the intervention of interest. VALIDATION METHODS: The authors rated the quality of evidence and strength of recommendations using the Grading of Recommendations Assessment, Development and Evaluation (GRADE) approach. See online Appendix A (Tables A1 for definitions and A2 for interpretations of strong and weak recommendations). INTENDED AUDIENCE: All health care providers who care for women of reproductive age. SUMMARY STATEMENTS: RECOMMENDATIONS.

Directive clinique no 425b : Le cannabis aux différentes périodes de la vie des femmes - Partie 2 : Grossesse, période postnatale et allaitement.

OBJECTIF: Fournir aux fournisseurs de soins les meilleures données probantes sur l'utilisation de cannabis et la santé des femmes. Les domaines d'intérêt sont le dépistage, la dépendance et le sevrage; la communication et la tenue de dossier; la grossesse (y compris les issues fœtales et maternelles); la gestion de la douleur maternelle; les soins postnataux (y compris la fumée secondaire et la parentalité); et l'allaitement. POPULATION CIBLE: Femmes enceintes, allaitantes ou qui planifient une grossesse. BéNéFICES, RISQUES ET COûTS: Discuter de l'utilisation de cannabis avec les femmes enceintes, allaitantes ou qui planifient une grossesse les aide à faire des choix éclairés. D'après des données probantes limitées, il faut éviter l'utilisation de cannabis pendant la grossesse ou l'allaitement, ou réduire la consommation au maximum si l'abstention n'est pas un objectif atteignable, étant donné l'absence de données sur l'innocuité et le suivi à long terme des grossesses et nourrissons exposés au cannabis. DONNéES PROBANTES: Les auteurs ont interrogé les bases de données PubMed et Cochrane Library pour extraire des articles sur l'utilisation de cannabis pendant la grossesse et l'allaitement publiés entre le 1er janvier 2018 et le 5 février 2021. Les termes de recherche ont été déterminés à partir de termes de recherche MeSH, de mots clés et de leurs variantes : cannabis, cannabinoids, cannabidiol, CBD, THC, marijuana, edible, pregnancy, pregnant, prenatal, perinatal, postnatal, breastfeed, breastfed, lactation, nursing, fetus, fetal, neonatal, newborn et child. Les auteurs ont inclus toutes les publications des types suivants : essais cliniques, études observationnelles, revues (y compris les revues systématiques et les méta-analyses), directives cliniques et déclarations de conférences de consensus. Les principaux critères d'inclusion étaient les femmes enceintes et allaitantes, comme population cible, et l'exposition au cannabis, comme intervention d'intérêt. MéTHODES DE VALIDATION: Les auteurs ont évalué la qualité des données probantes et la force des recommandations en utilisant le cadre méthodologique d'évaluation, de développement et d'évaluation (GRADE). Voir l'annexe A en ligne (tableau A1 pour les définitions et tableau A2 pour l'interprétation des recommandations fortes et faibles). PROFESSIONNELS CONCERNéS: Tous les fournisseurs de soins de santé qui prodiguent des soins aux femmes en âge de procréer. DÉCLARATIONS SOMMAIRES: RECOMMANDATIONS.

Bioinformatics Resource Manager: a systems biology web tool for microRNA and omics data integration.

BACKGROUND: The Bioinformatics Resource Manager (BRM) is a web-based tool developed to facilitate identifier conversion and data integration for Homo sapiens (human), Mus musculus (mouse), Rattus norvegicus (rat), Danio rerio (zebrafish), and Macaca mulatta (macaque), as well as perform orthologous conversions among the supported species. In addition to providing a robust means of identifier conversion, BRM also incorporates a suite of microRNA (miRNA)-target databases upon which to query target genes or to perform reverse target lookups using gene identifiers. RESULTS: BRM has the capability to perform cross-species identifier lookups across common identifier types, directly integrate datasets across platform or species by performing identifier retrievals in the background, and retrieve miRNA targets from multiple databases simultaneously and integrate the resulting gene targets with experimental mRNA data. Here we use workflows provided in BRM to integrate RNA sequencing data across species to identify common biomarkers of exposure after treatment of human lung cells and zebrafish to benzo[a]pyrene (BAP). We further use the miRNA Target workflow to experimentally determine the role of miRNAs as regulators of BAP toxicity and identify the predicted functional consequences of miRNA-target regulation in our system. The output from BRM can easily and directly be uploaded to freely available visualization tools for further analysis. From these examples, we were able to identify an important role for several miRNAs as potential regulators of BAP toxicity in human lung cells associated with cell migration, cell communication, cell junction assembly and regulation of cell death. CONCLUSIONS: Overall, BRM provides bioinformatics tools to assist biologists having minimal programming skills with analysis and integration of high-content omics' data from various transcriptomic and proteomic platforms. BRM workflows were developed in Java and other open-source technologies and are served publicly using Apache Tomcat at https://cbb.pnnl.gov/brm/ .

Improved in vivo targeting of BCL-2 phenotypic conversion through hollow gold nanoshell delivery.

Although new cancer therapeutics are discovered at a rapid pace, lack of effective means of delivery and cancer chemoresistance thwart many of the promising therapeutics. We demonstrate a method that confronts both of these issues with the light-activated delivery of a Bcl-2 functional converting peptide, NuBCP-9, using hollow gold nanoshells. This approach has shown not only to increase the efficacy of the peptide 30-fold in vitro but also has shown to reduce paclitaxel resistant H460 lung xenograft tumor growth by 56.4%.

Treatment with Nitrate, but Not Nitrite, Lowers the Oxygen Cost of Exercise and Decreases Glycolytic Intermediates While Increasing Fatty Acid Metabolites in Exercised Zebrafish.

BACKGROUND: Dietary nitrate improves exercise performance by reducing the oxygen cost of exercise, although the mechanisms responsible are not fully understood. OBJECTIVES: We tested the hypothesis that nitrate and nitrite treatment would lower the oxygen cost of exercise by improving mitochondrial function and stimulating changes in the availability of metabolic fuels for energy production. METHODS: We treated 9-mo-old zebrafish with nitrate (sodium nitrate, 606.9 mg/L), nitrite (sodium nitrite, 19.5 mg/L), or control (no treatment) water for 21 d. We measured oxygen consumption during a 2-h, strenuous exercise test; assessed the respiration of skeletal muscle mitochondria; and performed untargeted metabolomics on treated fish, with and without exercise. RESULTS: Nitrate and nitrite treatment increased blood nitrate and nitrite levels. Nitrate treatment significantly lowered the oxygen cost of exercise, as compared with pretreatment values. In contrast, nitrite treatment significantly increased oxygen consumption with exercise. Nitrate and nitrite treatments did not change mitochondrial function measured ex vivo, but significantly increased the abundances of ATP, ADP, lactate, glycolytic intermediates (e.g., fructose 1,6-bisphosphate), tricarboxylic acid (TCA) cycle intermediates (e.g., succinate), and ketone bodies (e.g., ß-hydroxybutyrate) by 1.8- to 3.8-fold, relative to controls. Exercise significantly depleted glycolytic and TCA intermediates in nitrate- and nitrite-treated fish, as compared with their rested counterparts, while exercise did not change, or increased, these metabolites in control fish. There was a significant net depletion of fatty acids, acyl carnitines, and ketone bodies in exercised, nitrite-treated fish (2- to 4-fold), while exercise increased net fatty acids and acyl carnitines in nitrate-treated fish (1.5- to 12-fold), relative to their treated and rested counterparts. CONCLUSIONS: Nitrate and nitrite treatment increased the availability of metabolic fuels (ATP, glycolytic and TCA intermediates, lactate, and ketone bodies) in rested zebrafish. Nitrate treatment may improve exercise performance, in part, by stimulating the preferential use of fuels that require less oxygen for energy production.

PM2.5 Filter Extraction Methods: Implications for Chemical and Toxicological Analyses.

Toxicology research into the global public health burden of fine particulate matter (PM2.5) exposures frequently requires extraction of PM2.5 from filters. A standardized method for these extractions does not exist, leading to inaccurate interlaboratory comparisons. It is largely unknown how different filter extraction methods might impact the composition and bioactivity of the resulting samples. We characterized the variation in these metrics by using equal portions of a single PM2.5 filter, with each portion undergoing a different extraction method. Significant differences were observed between extraction methods for concentrations of elements and polycyclic aromatic hydrocarbons (PAHs) for the PM2.5 tested following its preparation for biological response studies. Importantly, the chemical profiles differed from those observed when we used standard protocols for chemical characterization of the ambient sample, demonstrating that extraction can alter both chemical component amounts and species profiles of the extracts. The impact of these chemical differences on sensitive end points of zebrafish development was investigated. Significant differences in the percent incidence and timing of mortality were associated with the PM2.5 extraction method. This research highlights the importance of and rationale for considering the extraction method when interlaboratory comparisons of PM2.5 toxicology research are made.

Formation of PAH Derivatives and Increased Developmental Toxicity during Steam Enhanced Extraction Remediation of Creosote Contaminated Superfund Soil.

Steam enhanced extraction (SEE) is an in situ thermal remediation technique used to remove and recover polycyclic aromatic hydrocarbons (PAHs) from contaminated soils. However, limited studies have been conducted on the formation of PAH derivatives during and after SEE of PAH contaminated soils. Creosote contaminated soil samples collected from the Wyckoff-Eagle Harbor Superfund site were remediated with laboratory scale SEE. The samples were quantified for unsubstituted PAHs and their derivatives and assessed for developmental toxicity, pre- and post-SEE. Following SEE, unsubstituted PAH concentrations decreased, while oxygenated PAH concentrations increased in soil and aqueous extracts. Differences in developmental toxicity were also measured and linked to the formation of PAH derivatives. Additive toxicity was measured when comparing unfractionated extracts to fractionated extracts in pre- and post-SEE samples. SEE is effective in removing unsubstituted PAHs from contaminated soil, but other, potentially more toxic, PAH derivatives are formed.

Establishing structure-property-hazard relationships for multi-walled carbon nanotubes: the role of aggregation, surface charge, and oxidative stress on embryonic zebrafish mortality.

Increasing use of carbon nanotubes (CNTs) in consumer and industrials goods increases their potential release, and subsequent risks to environmental and human health. Therefore, it is becoming ever more important that CNTs are designed to reduce or eliminate hazards and that hazard assessment methodologies are robust. Here, oxygen-functionalized multi-walled CNTs (O-MWCNTs), modified under varying redox conditions, were assessed for toxic potential using the zebrafish (Danio rerio) embryo model. Multiple physicochemical properties (e.g., MWCNT aggregate size, morphology, and rate; surface charge and oxygen concentration; and reactive oxygen species (ROS) generation) were characterized and related to zebrafish embryo mortality through the use of multivariate statistical methods. Of these properties, surface charge and aggregate morphology emerged as the greatest predictors of embryo mortality. Interestingly, ROS generation was not significantly correlated to observed mortality, contrary to prior predictions by nanotoxicology researchers. This suggests that the mechanism of MWCNT-induced mortality of embryonic zebrafish is physical, driven by electrostatic and shape effects, both of which are related to nanomaterial aggregation. This raises the importance of rigorously considering aggregation during aqueous-based nanotoxicology assays as nanomaterial aggregation can affect perceived nanomaterial toxicity. As such, future nanotoxicity studies relying on aqueous media must sufficiently consider nanomaterial aggregation.

Tris(1,3-dichloro-2-propyl)phosphate (TDCIPP) disrupts zebrafish tail fin development.

The flame retardant, tris(1,3-dichloro-2-propyl) phosphate (TDCIPP), is one of the most developmentally toxic organophosphate flame retardants (OPFRs). However, few mechanistic studies on phenotypic malformation caused by TDCIPP have been conducted. This study investigates the molecular mechanism underlying abnormal tail fin development consistently observed in zebrafish embryos exposed to TDCIPP. The results show that the defects in the tail fin (e.g., bent spine, defective caudal fin, and damaged tip) were associated with altered expression of transcription factors. The significant up-regulation of mmp9 and, among insulin-growth factor (IGF) families, igfbp-1a and igfbp1b was observed, whereas alterations in the expression of cdx4, igf1a, ifg1b, igf2b, and vegaa regulating tail development were dependent on time points. In accordance with changes in mRNA gene expression, TDCIPP impaired vessel formation and disorganized muscle in transgenic Tg(fli-GFP) zebrafish larvae. Furthermore, we found that the overexpression of mmp9 caused by TDCIPP was not linked to igfbp-1. Overall, these findings demonstrate that TDCIPP disrupts the progression of tail fin development, accompanied by defects in vessel and muscle formation in developing zebrafish embryos.

Coupling Genome-wide Transcriptomics and Developmental Toxicity Profiles in Zebrafish to Characterize Polycyclic Aromatic Hydrocarbon (PAH) Hazard.

Polycyclic Aromatic Hydrocarbons (PAHs) are diverse environmental pollutants associated with adverse human health effects. Many studies focus on the carcinogenic effects of a limited number of PAHs and there is an increasing need to understand mechanisms of developmental toxicity of more varied yet environmentally relevant PAHs. A previous study characterized the developmental toxicity of 123 PAHs in zebrafish. Based on phenotypic responses ranging from complete inactivity to acute mortality, we classified these PAHs into eight bins, selected 16 representative PAHs, and exposed developing zebrafish to the concentration of each PAH that induced 80% phenotypic effect. We conducted RNA sequencing at 48 h post fertilization to identify gene expression changes as a result of PAH exposure. Using the Context Likelihood of Relatedness algorithm, we inferred a network that links the PAHs based on coordinated gene responses to PAH exposure. The 16 PAHs formed two broad clusters: Cluster A was transcriptionally more similar to the controls, while Cluster B consisted of PAHs that were generally more developmentally toxic, significantly elevated cyp1a transcript levels, and induced Ahr2-dependent Cyp1a protein expression in the skin confirmed by gene-silencing studies. We found that cyp1a transcript levels were associated with transcriptomic response, but not with PAH developmental toxicity. While all cluster B PAHs predominantly activated Ahr2, they also each enriched unique pathways like ion transport signaling, which likely points to differing molecular events between the PAHs downstream of Ahr2. Thus, using a systems biology approach, we have begun to evaluate, classify, and define mechanisms of PAH toxicity.

Population genetic diversity in zebrafish lines.

Toxicological and pharmacological researchers have seized upon the many benefits of zebrafish, including the short generation time, well-characterized development, and early maturation as clear embryos. A major difference from many model organisms is that standard husbandry practices in zebrafish are designed to maintain population diversity. While this diversity is attractive for translational applications in human and ecological health, it raises critical questions on how interindividual genetic variation might contribute to chemical exposure or disease susceptibility differences. Findings from pooled samples of zebrafish support this supposition of diversity yet cannot directly measure allele frequencies for reference versus alternate alleles. Using the Tanguay lab Tropical 5D zebrafish line (T5D), we performed whole genome sequencing on a large group (n = 276) of individual zebrafish embryos. Paired-end reads were collected on an Illumina 3000HT, then aligned to the most recent zebrafish reference genome (GRCz10). These data were used to compare observed population genetic variation across species (humans, mice, zebrafish), then across lines within zebrafish. We found more single nucleotide polymorphisms (SNPs) in T5D than have been reported in SNP databases for any of the WIK, TU, TL, or AB lines. We theorize that some subset of the novel SNPs may be shared with other zebrafish lines but have not been identified in other studies due to the limitations of capturing population diversity in pooled sequencing strategies. We establish T5D as a model that is representative of diversity levels within laboratory zebrafish lines and demonstrate that experimental design and analysis can exert major effects when characterizing genetic diversity in heterogeneous populations.

Multidimensional chemobehavior analysis of flavonoids and neuroactive compounds in zebrafish.

The comparative analysis of complex behavioral phenotypes is valuable as a reductionist tool for both drug discovery and defining chemical bioactivity. Flavonoids are a diverse class of chemicals that elicit robust neuroactive and hormonal actions, though bioactivity information is limited for many, particularly for neurobehavioral endpoints. Here, we used a zebrafish larval chemomotor response (LCR) bioassay to comparatively evaluate a suite of 24 flavonoids, and in addition a panel of 30 model neuroactive compounds representing diverse modes of action (e.g. caffeine, chlorpyrifos, methamphetamine, nicotine, picrotoxin). Naïve larval zebrafish were exposed to concentration ranges of each compound at 120 hour post-fertilization (hpf) and locomotor activity measured for 5 h. The model neuroactive compounds were largely behaviorally bioactive (20 of 30) with most effects phenotypic of their known modes of action. Flavonoids rapidly and broadly elicited hyperactive locomotor effects (22 of 24). Multidimensional analyses compared responses over time and identified three distinct bioactive groups of flavonoids based on efficacy and potency. Using GABAergics to modulate hyperactive responses, two flavonoids, (S)-equol and kaempferol were tested for GABAA receptor antagonism, as well as a known GABAA receptor antagonist, picrotoxin. Pharmacological intervention with positive allosteric modulators of the GABAA receptor, alfaxalone and chlormethiazole, ameliorated the hyperactive response to picrotoxin, but not for (S)-equol or kaempferol. Taken together, these studies demonstrate that flavonoids are differentially bioactive and that the chemobehavioral effects likely do not involve a GABAA receptor mediated mode of action. Overall, the integrative zebrafish platform provides a useful framework for comparatively evaluating high-content chemobehavioral data for sets of structurally- and mechanistically-related flavonoids and neuroactive compounds.

Systematic developmental neurotoxicity assessment of a representative PAH Superfund mixture using zebrafish.

Superfund sites often consist of complex mixtures of polycyclic aromatic hydrocarbons (PAHs). It is widely recognized that PAHs pose risks to human and environmental health, but the risks posed by exposure to PAH mixtures are unclear. We constructed an environmentally relevant PAH mixture with the top 10 most prevalent PAHs (SM10) from a Superfund site derived from environmental passive sampling data. Using the zebrafish model, we measured body burden at 48 hours post fertilization (hpf) and evaluated the developmental and neurotoxicity of SM10 and the 10 individual constituents at 24 hours post fertilization (hpf) and 5 days post fertilization (dpf). Zebrafish embryos were exposed from 6 to 120 hpf to (1) the SM10 mixture, (2) a variety of individual PAHs: pyrene, fluoranthene, retene, benzo[a]anthracene, chrysene, naphthalene, acenaphthene, phenanthrene, fluorene, and 2-methylnaphthalene. We demonstrated that SM10 and only 3 of the individual PAHs were developmentally toxic. Subsequently, we constructed and exposed developing zebrafish to two sub-mixtures: SM3 (comprised of 3 of the developmentally toxicity PAHs) and SM7 (7 non-developmentally toxic PAHs). We found that the SM3 toxicity profile was similar to SM10, and SM7 unexpectedly elicited developmental toxicity unlike that seen with its individual components. The results demonstrated that the overall developmental toxicity in the mixtures could be explained using the general concentration addition model. To determine if exposures activated the AHR pathway, spatial expression of CYP1A was evaluated in the 10 individual PAHs and the 3 mixtures at 5 dpf. Results showed activation of AHR in the liver and vasculature for the mixtures and some individual PAHs. Embryos exposed to SM10 during development and raised in chemical-free water into adulthood exhibited decreased learning and responses to startle stimulus indicating that developmental SM10 exposures affect neurobehavior. Collectively, these results exemplify the utility of zebrafish to investigate the developmental and neurotoxicity of complex mixtures.

Comparative developmental toxicity of a comprehensive suite of polycyclic aromatic hydrocarbons.

Polycyclic aromatic hydrocarbons (PAHs) are widespread environmental contaminants that occur in complex mixtures. Several PAHs are known or suspected mutagens and/or carcinogens, but developmental toxicity data is lacking for PAHs, particularly their oxygenated and nitrated derivatives. Such data are necessary to understand and predict the toxicity of environmental mixtures. 123 PAHs were assessed for morphological and neurobehavioral effects for a range of concentrations between 0.1 and 50 µM, using a high throughput early-life stage zebrafish assay, including 33 parent, 22 nitrated, 17 oxygenated, 19 hydroxylated, 14 methylated, 16 heterocyclic, and 2 aminated PAHs. Additionally, each PAH was evaluated for AHR activation, by assessing CYP1A protein expression using whole animal immunohistochemistry (IHC). Responses to PAHs varied in a structurally dependent manner. High-molecular weight PAHs were significantly more developmentally toxic than the low-molecular weight PAHs, and CYP1A expression was detected in five distinct tissues, including vasculature, liver, skin, neuromasts and yolk.

In Vivo Characterization of an AHR-Dependent Long Noncoding RNA Required for Proper Sox9b Expression.

Xenobiotic activation of the aryl hydrocarbon receptor (AHR) by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) prevents the proper formation of craniofacial cartilage and the heart in developing zebrafish. Downstream molecular targets responsible for AHR-dependent adverse effects remain largely unknown; however, in zebrafish sox9b has been identified as one of the most-reduced transcripts in several target organs and is hypothesized to have a causal role in TCDD-induced toxicity. The reduction of sox9b expression in TCDD-exposed zebrafish embryos has been shown to contribute to heart and jaw malformation phenotypes. The mechanisms by which AHR2 (functional ortholog of mammalian AHR) activation leads to reduced sox9b expression levels and subsequent target organ toxicity are unknown. We have identified a novel long noncoding RNA (slincR) that is upregulated by strong AHR ligands and is located adjacent to the sox9b gene. We hypothesize that slincR is regulated by AHR2 and transcriptionally represses sox9b. The slincR transcript functions as an RNA macromolecule, and slincR expression is AHR2 dependent. Antisense knockdown of slincR results in an increase in sox9b expression during both normal development and AHR2 activation, which suggests relief in repression. During development, slincR was expressed in tissues with sox9 essential functions, including the jaw/snout region, otic vesicle, eye, and brain. Reducing the levels of slincR resulted in altered neurologic and/or locomotor behavioral responses. Our results place slincR as an intermediate between AHR2 activation and the reduction of sox9b mRNA in the AHR2 signaling pathway.

A data-driven weighting scheme for multivariate phenotypic endpoints recapitulates zebrafish developmental cascades.

Zebrafish have become a key alternative model for studying health effects of environmental stressors, partly due to their genetic similarity to humans, fast generation time, and the efficiency of generating high-dimensional systematic data. Studies aiming to characterize adverse health effects in zebrafish typically include several phenotypic measurements (endpoints). While there is a solid biomedical basis for capturing a comprehensive set of endpoints, making summary judgments regarding health effects requires thoughtful integration across endpoints. Here, we introduce a Bayesian method to quantify the informativeness of 17 distinct zebrafish endpoints as a data-driven weighting scheme for a multi-endpoint summary measure, called weighted Aggregate Entropy (wAggE). We implement wAggE using high-throughput screening (HTS) data from zebrafish exposed to five concentrations of all 1060 ToxCast chemicals. Our results show that our empirical weighting scheme provides better performance in terms of the Receiver Operating Characteristic (ROC) curve for identifying significant morphological effects and improves robustness over traditional curve-fitting approaches. From a biological perspective, our results suggest that developmental cascade effects triggered by chemical exposure can be recapitulated by analyzing the relationships among endpoints. Thus, wAggE offers a powerful approach for analysis of multivariate phenotypes that can reveal underlying etiological processes.

Transgenerational inheritance of neurobehavioral and physiological deficits from developmental exposure to benzo[a]pyrene in zebrafish.

Benzo[a]pyrene (B[a]P) is a well-known genotoxic polycylic aromatic compound whose toxicity is dependent on signaling via the aryl hydrocarbon receptor (AHR). It is unclear to what extent detrimental effects of B[a]P exposures might impact future generations and whether transgenerational effects might be AHR-dependent. This study examined the effects of developmental B[a]P exposure on 3 generations of zebrafish. Zebrafish embryos were exposed from 6 to 120h post fertilization (hpf) to 5 and 10µM B[a]P and raised in chemical-free water until adulthood (F0). Two generations were raised from F0 fish to evaluate transgenerational inheritance. Morphological, physiological and neurobehavioral parameters were measured at two life stages. Juveniles of the F0 and F2 exhibited hyper locomotor activity, decreased heartbeat and mitochondrial function. B[a]P exposure during development resulted in decreased global DNA methylation levels and generally reduced expression of DNA methyltransferases in wild type zebrafish, with the latter effect largely reversed in an AHR2-null background. Adults from the F0 B[a]P exposed lineage displayed social anxiety-like behavior. Adults in the F2 transgeneration manifested gender-specific increased body mass index (BMI), increased oxygen consumption and hyper-avoidance behavior. Exposure to benzo[a]pyrene during development resulted in transgenerational inheritance of neurobehavioral and physiological deficiencies. Indirect evidence suggested the potential for an AHR2-dependent epigenetic route.

Comparative Toxicogenomic Responses to the Flame Retardant mITP in Developing Zebrafish.

Monosubstituted isopropylated triaryl phosphate (mITP) is a major component of Firemaster 550, an additive flame retardant mixture commonly used in polyurethane foams. Developmental toxicity studies in zebrafish established mITP as the most toxic component of FM 550, which causes pericardial edema and heart looping failure. Mechanistic studies showed that mITP is an aryl hydrocarbon receptor (AhR) ligand; however, the cardiotoxic effects of mITP were independent of the AhR. We performed comparative whole genome transcriptomics in wild-type and ahr2hu3335 zebrafish, which lack functional ahr2, to identify transcriptional signatures causally involved in the mechanism of mITP-induced cardiotoxicity. Regardless of ahr2 status, mITP exposure resulted in decreased expression of transcripts related to the synthesis of all-trans-retinoic acid and a host of Hox genes. Clustered gene ontology enrichment analysis showed unique enrichment in biological processes related to xenobiotic metabolism and response to external stimuli in wild-type samples. Transcript enrichments overlapping both genotypes involved the retinoid metabolic process and sensory/visual perception biological processes. Examination of the gene-gene interaction network of the differentially expressed transcripts in both genetic backgrounds demonstrated a strong AhR interaction network specific to wild-type samples, with overlapping genes regulated by retinoic acid receptors (RARs). A transcriptome analysis of control ahr2-null zebrafish identified potential cross-talk among AhR, Nrf2, and Hif1α. Collectively, we confirmed that mITP is an AhR ligand and present evidence in support of our hypothesis that mITP's developmental cardiotoxic effects are mediated by inhibition at the RAR level.