Cannabis constituents reduce seizure behavior in chemically-induced and scn1a-mutant zebrafish.

Current antiepileptic drugs (AEDs) are undesirable for many reasons including the inability to reduce seizures in certain types of epilepsy, such as Dravet syndrome (DS) where in one-third of patients does not respond to current AEDs, and severe adverse effects that are frequently experienced by patients. Epidiolex, a cannabidiol (CBD)-based drug, was recently approved for treatment of DS. While Epidiolex shows great promise in reducing seizures in patients with DS, it is used in conjunction with other AEDs and can cause liver toxicity. To investigate whether other cannabis-derived compounds could also reduce seizures, the antiepileptic effects of CBD, Δ9-tetrahydrocannabinol (THC), cannabidivarin (CBDV), cannabinol (CBN), and linalool (LN) were compared in both a chemically-induced (pentylenetetrazole, PTZ) and a DS (scn1Lab-/-) seizure models. Zebrafish (Danio rerio) that were either wild-type (Tupfel longfin) or scn1Lab-/- (DS) were exposed to CBD, THC, CBDV, CBN, or LN for 24 h from 5 to 6 days postfertilization. Following exposure, total distance traveled was measured in a ViewPoint Zebrabox to determine if these compounds reduced seizure-like activity. Cannabidiol (0.6 and 1 µM) and THC (1 and 4 µM) significantly reduced PTZ-induced total distance moved. At the highest THC concentration, the significant reduction in PTZ-induced behavior was likely the result of sedation as opposed to antiseizure activity. In the DS model, CBD (0.6 µM), THC (1 µM), CBN (0.6 and 1 µM), and LN (4 µM) significantly reduced total distance traveled. Cannabinol was the most effective at reducing total distance relative to controls. In addition to CBD, other cannabis-derived compounds showed promise in reducing seizure-like activity in zebrafish. Specifically, four of the five compounds were effective in the DS model, whereas in the PTZ model, only CBD and THC were, suggesting a divergence in the mode of action among the cannabis constituents.

Multigenerational consequences of early-life cannabinoid exposure in zebrafish.

While Δ9-tetrahydrocannabinol (THC) has been widely studied in the realm of developmental and reproductive toxicology, few studies have investigated potential toxicities from a second widely used cannabis constituent, cannabidiol (CBD). CBD is popularized for its therapeutic potential for reducing seizure frequencies in epilepsy. This study investigated developmental origins of health and disease (DOHaD) via multigenerational gene expression patterns, behavior phenotypes, and reproductive fitness of a subsequent F1 following an F0 developmental exposure of zebrafish (Danio rerio) to THC (0.024, 0.12, 0.6 mg/L; 0.08, 0.4, 2 µM) or CBD (0.006, 0.03, 0.15 mg/L; 0.02, 0.1, 0.5 µM). Embryonic exposure at these concentrations did not cause notable morphological abnormalities in either F0 or F1 generations. However, during key developmental stages (14, 24, 48, 72, and 96 h post fertilization) THC and CBD caused differential expression of c-fos, brain-derived neurotrophic factor (bdnf), and deleted-in-azoospermia like (dazl), while in F1 larvae only CBD differentially expressed dazl. Larval photomotor behavior was reduced (F0) or increased (F1) by THC exposure, while CBD had no effect on F0 larvae, but decreased activity in the unexposed F1 larvae. These results support our hypothesis of cannabinoid-related developmental neurotoxicity. As adults, F0 fecundity was reduced, but it was not in F1 adults. Conversely, in the adult open field test there were no significant effects in F0 fish, but a significant reduction in the time in periphery was seen in F1 fish from the highest THC exposure group. The results highlight the need to consider long-term ramifications of early-life exposure to cannabinoids.

Mercury concentrations in fish from three major lakes in north Mississippi: Spatial and temporal differences and human health risk assessment.

The goal of this study was to compare total mercury (Hg) concentrations in fish muscle tissue and assess consumption health risks of fish collected from three north Mississippi lakes (Sardis, Enid, and Grenada) that are extensively used for fishing and recreation. Largemouth bass (LMB; n = 64), channel catfish (CC; n = 72), and white crappie (WC; n = 100), which represent a range of trophic levels, were collected during spring 2013 and 2014. Creel data estimated that anglers harvested approximately 370,000 kg of WC, 27,000 kg of CC, and 15,000 kg of LMB from the lakes annually. Median Hg wet weight concentrations were highest in LMB (443 ng/g), followed by CC (211 ng/g) and WC (192 ng/g). Fish-Hg concentrations were lower than those reported in fish >10 years ago. There were significant differences between lakes consistent across species. Grenada length-normalized fish-Hg concentrations were higher than those from Enid and Sardis. Because existing consumption advisories for CC are length based, the lack of relationship between length and Hg concentration indicated that the recommendations may not be sufficiently protective. Further, five different risk assessment paradigms yielded hazard quotient (HQ) values suggesting that existing fish consumption advisories may be insufficient to protect adults and especially children from exposure to Hg.

Gill histopathologies following exposure to nanosilver or silver nitrate.

Fish gill is the site for many crucial physiological functions. It is among the first sites of xenobiotic exposure, and gill histopathological alterations may be detected soon after toxicant exposure. Silver (Ag) is one of the most toxic metals to aquatic organisms mainly due to its ability to disrupt ionic regulation. The goal of this study was to determine the effect of ionic and nanoscale Ag on fathead minnow gills by examining gill histology and Na(+)/K(+)-ATPase immunoreactivity. Fathead minnows were exposed to two measured concentrations of silver nitrate (AgNO3: 1.3 or 3.7 µg/L as Ag(+)), citrate silver nanoparticles (citrate-AgNP: 15 or 39 µg/L), and polyvinylpyrrolidone-AgNP (PVP-AgNP) (AgNP: 11 or 50 µg/L). Circulatory disturbances were the most prevalent gill alterations detected and were significantly increased in all Ag treatment groups compared to control. AgNO3 (1.3 µg/L) was the only treatment that significantly elevated the number of total mucous goblet cells present. In all other Ag treatments, the percent of degenerated goblet cells was significantly increased compared to control. When the sum of all histopathological abnormalities (weighted index) was calculated, all Ag groups displayed a significantly higher index, with citrate-AgNP having the highest toxicity (index of 10 ± 0.32 versus 2.4 ± 0.6 in controls). Gill Na(+)/K(+)-ATPase immunoreactivity was decreased by Ag. These results indicated that both AgNO3 and AgNP created similar disruptions in gill structure and ionic regulation, possibly due to the ionic Ag portion of each treatment.

Assimilation of oil-derived elements by oysters due to the Deepwater Horizon Oil Spill.

During and after the Deepwater Horizon Oil Spill (DWHOS), oysters (Crassostrea virginica) were exposed to oil and susceptible to incidental consumption of surface and subsurface oil materials. We determined the contribution of oil materials from the DWHOS to diet of oysters by comparing carbon (C) and nitrogen (N) stable isotope ratios in oyster shell to ratios in suspended particulate matter (SPM) and in fresh and weathered oil. Average δ(13)C and δ(15)N values in oyster shell (-21 ± 1‰ and 9-11‰, respectively) were consistent with consumption of naturally available SPM as opposed to values in oil (-27 ± 0.2‰, 1.6 ± 0.4‰). Stable isotope ratios in oyster adductor muscle were similar to shell for δ(15)N but not δ(13)C, suggesting either a recent shift in diet composition or differential assimilation of C between tissue types. We found no evidence of assimilation of oil-derived C and N and, therefore, no evidence of an oyster-based conduit to higher trophic levels. Trace elements in shell were inconclusive to corroborate oil exposure. These findings are not an indication that oysters were not exposed to oil; rather they imply oysters either did not consume oil-derived materials or consumed too little to be detectable compared to natural diet.

Marine proteomics: a critical assessment of an emerging technology.

The application of proteomics to marine sciences has increased in recent years because the proteome represents the interface between genotypic and phenotypic variability and, thus, corresponds to the broadest possible biomarker for eco-physiological responses and adaptations. Likewise, proteomics can provide important functional information regarding biosynthetic pathways, as well as insights into mechanism of action, of novel marine natural products. The goal of this review is to (1) explore the application of proteomics methodologies to marine systems, (2) assess the technical approaches that have been used, and (3) evaluate the pros and cons of this proteomic research, with the intent of providing a critical analysis of its future roles in marine sciences. To date, proteomics techniques have been utilized to investigate marine microbe, plant, invertebrate, and vertebrate physiology, developmental biology, seafood safety, susceptibility to disease, and responses to environmental change. However, marine proteomics studies often suffer from poor experimental design, sample processing/optimization difficulties, and data analysis/interpretation issues. Moreover, a major limitation is the lack of available annotated genomes and proteomes for most marine organisms, including several "model species". Even with these challenges in mind, there is no doubt that marine proteomics is a rapidly expanding and powerful integrative molecular research tool from which our knowledge of the marine environment, and the natural products from this resource, will be significantly expanded.

Trace element concentrations in surface estuarine and marine sediments along the Mississippi Gulf Coast following Hurricane Katrina.

Hurricanes are relatively frequent ecological disturbances that may cause potentially long-term impacts to the coastal environment. Hurricane Katrina hit the Mississippi Gulf Coast in August 2005, and caused a storm surge with the potential to change the trace element content of coastal surface sediments. In this study, surface estuarine and marine sediments were collected monthly following the storm from ten sites along the Mississippi Gulf Coast (Mobile Bay, Grand Bay Bayous Heron and Cumbest, Pascagoula, Ocean Springs, Biloxi Gulf, Back Biloxi Bay, Gulfport Gulf, Gulfport Courthouse Rd, and Gulfport Marina). Concentrations of V, Cr, Mn, Fe, Co, Ni, Zn, As, Cd, and Pb were measured by inductively coupled plasma-mass spectrometry to evaluate their temporal and spatial variations in the year following Hurricane Katrina. Sediments were characterized by pH, particle size distribution and total carbon and nitrogen content. Trace element contents of the sediments were determined in both <2 mm and <63 µm grain size fractions. Results revealed no significant temporal and spatial variability in trace element concentrations, in either size fraction. Potential ecological risk of the sediments was assessed by using NOAA SQuiRTs' guideline values; most concentrations remained below probable adverse effects guidelines to marine organisms suggesting that trace elements redistributed by Hurricane Katrina would not cause an adverse impact on resident organisms. Instead, the concentrations of trace elements were site-dependent, with specific contaminants relating to the use of the area prior to Hurricane Katrina.

Determination of total and partially extractable solid-bound element concentrations using collision/reaction cell inductively coupled plasma-mass spectrometry and their significance in environmental studies.

Determination of solid-bound element concentrations is an important initial step in environmental studies especially for assessment of contamination level, and of origin, relative mobility, and fate of contaminants. This study revealed that a relatively new collision/reaction cell inductively coupled plasma-mass spectrometry is a potent tool for determining total and partially extractable solid-bound element (V, Cr, Mn, Fe, Co, Ni, Cu, Zn, As, and Pb) concentrations in a complex matrix solution containing HF and/or HCl. Six different extraction methods commonly used for environmental monitoring studies were tested for their bias and variability using estuarine and marine standard reference materials. Microwave-assisted methods based on concentrated [HNO3] or [HNO3 + HF (4:1)] and [HNO3 + HF + HCl (10:3:2)] were applied for determining pseudo-total and total element concentrations, respectively. Dilute-acids (1 M HNO3, 1 M HCl, and 0.5 M HCl) were utilized in single-step partial extraction protocols. Except the 0.5 M HCl cold-extraction method which was performed at room temperature, other partial extraction protocols used microwave-digestion. This study demonstrated that the use of microwave-assisted methods in studies aimed at determining the non-residual, non-specific extractable fractions of elements in solid environmental samples may result in overestimation, and thus needs to be re-examined. We believe that the cold extraction method will play a significant role in future environmental monitoring studies. Nevertheless, results of the cold extraction method not accompanied with total element concentrations have limited value, as the amount of extraction may vary significantly with the nature (origin) of the elements, and with the types of the samples. Therefore, we suggest combining microwave-assisted total digestion and 0.5 M HCl cold-extraction methods as a relatively cost- and time-effective, environmentally sound screening procedure for routine environmental monitoring programs involving a large number of samples from diverse geological and anthropogenic settings.

Transcriptomic Changes and the Roles of Cannabinoid Receptors and PPARγ in Developmental Toxicities Following Exposure to Δ9-Tetrahydrocannabinol and Cannabidiol.

Human consumption of cannabinoid-containing products during early life or pregnancy is rising. However, information about the molecular mechanisms involved in early life stage Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD) toxicities is critically lacking. Here, larval zebrafish (Danio rerio) were used to measure THC- and CBD-mediated changes on transcriptome and the roles of cannabinoid receptors (Cnr) 1 and 2 and peroxisome proliferator activator receptor γ (PPARγ) in developmental toxicities. Transcriptomic profiling of 96-h postfertilization (hpf) cnr+/+ embryos exposed (6 - 96 hpf) to 4 µM THC or 0.5 µM CBD showed differential expression of 904 and 1095 genes for THC and CBD, respectively, with 360 in common. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways enriched in the THC and CBD datasets included those related to drug, retinol, and steroid metabolism and PPAR signaling. The THC exposure caused increased mortality and deformities (pericardial and yolk sac edemas, reduction in length) in cnr1-/- and cnr2-/- fish compared with cnr+/+ suggesting Cnr receptors are involved in protective pathways. Conversely, the cnr1-/- larvae were more resistant to CBD-induced malformations, mortality, and behavioral alteration implicating Cnr1 in CBD-mediated toxicity. Behavior (decreased distance travelled) was the most sensitive endpoint to THC and CBD exposure. Coexposure to the PPARγ inhibitor GW9662 and CBD in cnr+/+ and cnr2-/- strains caused more adverse outcomes compared with CBD alone, but not in the cnr1-/- fish, suggesting that PPARγ plays a role in CBD metabolism downstream of Cnr1. Collectively, PPARγ, Cnr1, and Cnr2 play important roles in the developmental toxicity of cannabinoids with Cnr1 being the most critical.

Functional differences in the cytochrome P450 1 family enzymes from zebrafish (Danio rerio) using heterologously expressed proteins.

Mammalian cytochrome P450 1 (CYP1) genes are well characterized, but in other vertebrates only the functions of CYP1A genes have been well studied. We determined the catalytic activity of zebrafish CYP1A, CYP1B1, CYP1C1, CYP1C2, and CYP1D1 proteins using 11 fluorometric substrates and benzo[a]pyrene (BaP). The resorufin-based substrates, 7-ethoxyresorufin, 7-methoxyresorufin, and 7-benzyloxyresorufin, were well metabolized by all CYP1s except CYP1D1. CYP1A metabolized nearly all substrates tested, although rates for non-resorufin substrates were typically lower than resorufin-based substrates. Zebrafish CYP1s did not metabolize 7-benzyloxyquinoline, 3-[2-(N,N-diethyl-N-methylamino)ethyl]-7-methoxy-4-methylcoumarin or 7-methoxy-4-(aminomethyl)-coumarin. CYP1B1 and CYP1C2 had the highest rates of BaP metabolism. 3-Hydroxy-BaP was a prominent metabolite for all but CYP1D1. CYP1A showed broad specificity and had the highest metabolic rates for nearly all substrates. CYP1C1 and CYP1C2 had similar substrate specificity. CYP1D1 had very low activities for all substrates except BaP, and a different regioselectivity for BaP, suggesting that CYP1D1 function may be different from other CYP1s.

Occurrence and distribution of steroids, hormones and selected pharmaceuticals in South Florida coastal environments.

The common occurrence of human derived contaminants like pharmaceuticals, steroids and hormones in surface waters has raised the awareness of the role played by the release of treated or untreated sewage in the water quality along sensitive coastal ecosystems. South Florida is home of many important protected environments ranging from wetlands to coral reefs which are in close proximity to large metropolitan cities. Because, large portions of South Florida and most of the Florida Keys population are not served by modern sewage treatment plants and rely heavily on the use of septic systems, a comprehensive survey of selected human waste contamination markers was conducted in three areas to assess water quality with respect to non-traditional micro-constituents. This study documents the occurrence and distribution of fifteen hormones and steroids and five commonly detected pharmaceuticals in surface water samples collected from different near shore environments along South Florida between 2004 and 2006. The compounds most frequently detected were: cholesterol, caffeine, estrone, DEET, coprostanol, biphenol-A, beta-estradiol, and triclosan. The concentration detected for estrone and beta-estradiol were up to 5.2 and 1.8 ng/L, respectively. Concentrations of caffeine (5.5-68 ng/L) and DEET (4.8-49 ng/L) were generally higher and more prevalent than were the steroids. Distribution of microconstituents was site specific likely reflecting a diversity of sources. In addition to chemical analysis, the yeast estrogen screen assay was used to screen the samples for estrogen equivalency. Overall, the results show that water collected from inland canals and restricted circulation water bodies adjacent to heavily populated areas had high concentrations of multiple steroids, pharmaceuticals, and personal care products while open bay waters were largely devoid of the target analytes.

Developmental exposure to Δ9-tetrahydrocannabinol (THC) causes biphasic effects on longevity, inflammation, and reproduction in aged zebrafish (Danio rerio).

Increased availability of cannabis and cannabinoid-containing products necessitates the need for an understanding of how these substances influence aging. In this study, zebrafish (Danio rerio) were exposed to different concentrations of THC (0.08, 0.4, 2 µM) during embryonic-larval development and the effects on aging were measured 30 months later and in the offspring of the exposed fish (F1 generation). Exposure to 0.08 µM THC resulted in increased male survival at 30 months of age. As the concentration of THC increased, this protective effect was lost. Treatment with the lowest concentration of THC also significantly increased egg production, while higher concentrations resulted in impaired fecundity. Treatment with the lowest dose of THC significantly reduced wet weight, the incidence of kyphosis, and the expression of several senescence and inflammatory markers (p16ink4ab, tnfα, il-1ß, il-6, pparα and pparγ) in the liver, but not at higher doses indicating a biphasic or hormetic effect. Exposure to THC did not affect the age-related reductions in locomotor behavior. Within the F1 generation, many of these changes were not observed. However, the reduction in fecundity due to THC exposure was worse in the F1 generation because offspring whose parents received high dose of THC were completely unable to reproduce. Together, our results demonstrate that a developmental exposure to THC can cause significant effects on longevity and healthspan of zebrafish in a biphasic manner.

Developmental exposure to cannabidiol (CBD) alters longevity and health span of zebrafish (Danio rerio).

Consumption of cannabinoid-containing products is on the rise, even during pregnancy. Unfortunately, the long-term, age-related consequences of developmental cannabidiol (CBD) exposure remain largely unknown. This is a critical gap given the established Developmental Origins of Health and Disease (DOHaD) paradigm which emphasizes that stressors, like drug exposure, early in life can instigate molecular and cellular changes that ultimately lead to adverse outcomes later in life. Thus, we exposed zebrafish (Danio rerio) to varying concentrations of CBD (0.02, 0.1, 0.5 µM) during larval development and assessed aging in both the F0 (exposed generation) and their F1 offspring 30 months later. F0 exposure to CBD significantly increased survival (~ 20%) and reduced size (wet weight and length) of female fish. While survival was increased, the age-related loss of locomotor function was unaffected and the effects on fecundity varied by sex and dose. Treatment with 0.5 µM CBD significantly reduced sperm concentration in males, but 0.1 µM increased egg production in females. Similar to other model systems, control aged zebrafish exhibited increased kyphosis as well as increased expression markers of senescence, and inflammation (p16ink4ab, tnfα, il1b, il6, and pparγ) in the liver. Exposure to CBD significantly reduced the expression of several of these genes in a dose-dependent manner relative to the age-matched controls. The effects of CBD on size, gene expression, and reproduction were not reproduced in the F1 generation, suggesting the influence on aging was not cross-generational. Together, our results demonstrate that developmental exposure to CBD causes significant effects on the health and longevity of zebrafish.

Combined and independent effects of hypoxia and tributyltin on mRNA expression and physiology of the Eastern oyster (Crassostrea virginica).

Oyster reefs are vital to estuarine health, but they experience multiple stressors and globally declining populations. This study examined effects of hypoxia and tributyltin (TBT) on adult Eastern oysters (Crassostrea virginica) exposed either in the laboratory or the field following a natural hypoxic event. In the laboratory, oysters were exposed to either hypoxia followed by a recovery period, or to hypoxia combined with TBT. mRNA expression of HIF1-α and Tß-4 along with hemocyte counts, biomarkers of hypoxic stress and immune health, respectively, were measured. In field-deployed oysters, HIF1-α and Tß-4 expression increased, while no effect on hemocytes was observed. In contrast, after 6 and 8 days of laboratory-based hypoxia exposure, both Tß-4 expression and hemocyte counts declined. After 8 days of exposure to hypoxia + TBT, oysters substantially up-regulated HIF1-α and down-regulated Tß-4, although hemocyte counts were unaffected. Results suggest that hypoxic exposure induces immunosuppression which could increase vulnerability to pathogens.

CANNABINOID CONUNDRUM:: A STUDY OF MARIJUANA AND HEMP LEGALITY IN THE UNITED STATES.

Marijuana is the most commonly used, cultivated, and trafficked illicit drug worldwide. In the United States, the use and acceptance of marijuana is evolving rapidly as indicated by the volume of new state cannabis legislation. However, marijuana is still a Schedule I drug under the federal Controlled Substances Act (CSA). Further complicating the matter, the 2018 Farm Bill removed hemp from the list of controlled substances under the CSA, resulting in a market flooded with cannabidiol (CBD) products that have not been approved by the Food and Drug Administration (FDA). Many of the changes in state laws have occurred without significant input from medical or scientific communities. The status of marijuana and, until recently, hemp as Schedule I drugs under the CSA creates numerous restrictions which ultimately impact the industry as a whole. The central issues facing marijuana legality in the U.S. are: convoluted state and federal law, adverse health effects of cannabis use, research restrictions that produce knowledge gaps, and inconsistency between the U.S. Food and Drug Administration (FDA) and the U.S. Environmental Protection Agency (EPA) regulations. Marijuana policy must evolve to protect and inform both the general public and individuals involved in the cannabis industry. Potential reform options include: federal exemptions following state compliance, rescheduling marijuana, or complete removal from the CSA. The most vital step in the federal legalization process needs to be less restrictive research opportunities for marijuana.

Simultaneous determination of benzo[a]pyrene and eight of its metabolites in Fundulus heteroclitus bile using ultra-performance liquid chromatography with mass spectrometry.

A sensitive and fast method was developed to quantitate the carcinogenic polycyclic aromatic hydrocarbon benzo[a]pyrene (BaP) and eight of its oxidized metabolites by ultra-performance liquid chromatography (UPLC) coupling with mass spectrometry (MS). The UPLC method, using an acetonitrile:water gradient as a mobile phase, provided baseline separation of the BaP metabolites including three BaP diones. Linearity of detection was in the range of 0.2-5.0ng/microL, and limits of detection (LOD) were lower than 0.01ng/microL for BaP and all of the metabolites except BaP tetrol. In order to test this method in environmentally relevant samples, we exposed the small fish Fundulus heteroclitus to BaP and quantitated biliary BaP metabolites. Extraction recovery of all compounds varied from 65.4+/-21.3% to 92.4+/-3.0%. In exposed fish bile, the BaP diones, BaP-7,8-dihydrodiol, and 3-hydroxy BaP metabolites predominated, existing mainly as glucuronic acid conjugates. This UPLC-MS method will be useful for further defining the roles of cytochrome P450s with both in vivo and in vitro models in the understanding of the mechanisms of metabolic activation and detoxification of BaP.

Benzo(a)pyrene decreases brain and ovarian aromatase mRNA expression in Fundulus heteroclitus.

The higher molecular weight polycyclic aromatic hydrocarbons (PAHs) such as benzo(a)pyrene (BaP) are typically associated with genotoxicity, however, newer evidence suggests that these compounds may also act as endocrine system disruptors. We hypothesized that altered expression of the P450 enzyme aromatase genes could be a target for reproductive or developmental dysfunction caused by BaP exposure. Aromatase is at least partially responsible for estrogen homeostasis by converting androgens into estrogens. In fish, there are two isoforms of aromatase, a predominantly ovarian form, CYP19A1, and a brain form, CYP19A2. CYP19 mRNA expression was measured following BaP exposure (0, 10, 100 microg/L waterborne for 10 or 15 days) in Fundulus adults, juveniles and embryos by in situ hybridization. The CYP19A1 expression was significantly decreased after BaP exposure in the 3-month-old Fundulus immature oocytes, but BaP did not affect CYP19A1 expression at any stage in adult oocytes. In embryo brains, BaP significantly decreased CYP19A2 compared to controls by 3.6-fold at 14 days post-fertilization. In adults, CYP19A2 expression was decreased significantly in the pituitary and hypothalamus (81% and 85% of controls, respectively). Promoter regions of Fundulus CYP19s were cloned, and putative response elements in the CYP19A1 and CYP19A2 promoters such as CRE, AhR and ERE may be involved in BaP-mediated changes in CYP19 expression. In order to compare the mechanism of BaP-mediated inhibition with that of a known aromatase inhibitor, fish were also exposed to fadrozole (20 and 100 microg/L). Fadrozole did not significantly decrease the mRNA expression in embryos or adult Fundulus. However, aromatase enzyme activity was significantly decreased in adult ovary and brain tissues. These studies provide a greater molecular understanding of the mechanisms of action of BaP and its potential to impact reproduction or development.

Developmental Effects of Cannabidiol and Δ9-Tetrahydrocannabinol in Zebrafish.

Cannabidiol (CBD) has gained much attention in the past several years for its therapeutic potential in the treatment of drug-resistant epilepsy, such as Dravet syndrome. Although CBD has shown anecdotal efficacy in reducing seizure frequency, little is known regarding the potential adverse side effects of CBD on physiology, development, organogenesis, or behavior. The goal of this project was to compare the relative morphological, behavioral, and gene expression phenotypes resulting after a developmental exposure to Δ9-tetrahydrocannabinol (THC) or CBD. Zebrafish were exposed from blastula through larval stage (96 h postfertilization [hpf]) to 0.3, 0.6, 1.25, 2.5, 5 mg/l (1, 2, 4, 8, 16 µM) THC or 0.07, 0.1, 0.3, 0.6, 1.25 mg/l CBD (0.25, 0.5, 1, 2, 4 µM). Despite the similarity in THC and CBD dysmorphologies, ie, edemas, curved axis, eye/snout/jaw/trunk/fin deformities, swim bladder distention, and behavioral abnormalities, the LC50 for CBD (0.53 mg/l) was nearly 7 times lower than THC (3.65 mg/l). At 96 hpf, c-fos, dazl, and vasa were differentially expressed following THC exposure, but only c-fos expression was significantly increased by CBD. Cannabidiol was more bioconcentrated compared with THC despite higher THC water concentrations. This work supports the potential for persistent developmental impacts of cannabinoid exposure, but more studies are needed to assess latent effects and their molecular mechanisms of toxicity.

Mechanistic Evaluation of Benzo[a]pyrene's Developmental Toxicities Mediated by Reduced Cyp19a1b Activity.

Benzo[a]pyrene (BaP) is a ubiquitous environmental contaminant that is both an endocrine disruptor and a carcinogen. Aromatase (CYP19) is a key enzyme in steroidogenesis that is responsible for conversion of androgens to estrogens and thus plays a key role in steroid homeostasis. We hypothesized that some of the adverse outcomes of early developmental exposure to BaP are the result of reduced Cyp19a1b activity. Our goal was to investigate the role of estrogen homeostasis during early development and determine the role of aromatase inhibition as a relevant mechanism in BaP's developmental toxicities. One-cell zebrafish embryos were injected with a Cyp19a1b-morpholino (MO) or control-MO. Other non-injected embryos were exposed to waterborne BaP, fadrozole (a Cyp19 inhibitor), estradiol (E2), BaP + E2, Cyp19a1b MO + E2, or fadrozole + E2 for 96 hours post-fertilization (hpf). Adverse outcomes were compared between treatments, and the ability of E2 co-exposure to rescue each observed dysmorphology was assessed. BaP significantly decreased cyp19a1b gene expression in 96 hpf zebrafish larvae homogenates. Concentrations of E2 in 48 hpf larvae were significantly decreased by BaP, fadrozole and Cyp19a1b-MO. Cumulative mortality of zebrafish larvae was significantly increased following BaP or fadrozole exposure or Cyp19a1b knockdown compared to controls. E2 co-treatment rescued mortality caused by 10 µg/L BaP, 10 µg/L fadrozole, or Cyp19a1b-MO. In a treatment-blinded morphological assessment of larvae at 96 hpf, several phenotypes were negatively impacted by BaP, fadrozole, or Cyp19a1b knockdown and rescued by exogenous E2 co-treatment; these included body length, optic vesicle size, swim bladder inflation, pericardial and abdominal edema, and incidence of normal larval tail shape. Abnormal pectoral fins were caused by BaP exposure only. Uninflated swim bladders were caused by all treatments including E2 alone. Our results indicate that certain BaP-mediated adverse developmental outcomes were mechanistically in accordance with BaP-mediated Cyp19a1b inhibition.